ECOBIOS, Vol.

7 (1&2), 2014 ISSN: 0972-6446

(International Journal for Biology, Ecology and Allied Sciences)

Biochemical aspect of as toxicity in developing

wheat (Triticum aestivum) seedlings
S. Nath1*, H. Upadhyay2, M. Dey3, S. K. Panda4
1
Department of Microbiology, Assam University, Silchar – 788 011, Assam, India
2
Department of Botany, Karimganj College, Karimganj-788710, Assam, India
3
Department of Biotechnology, Indian Institute of Technology, Guwahati, Assam
4
Department of Life Sciences and Bioinformatics, Assam University, Silchar – 788 011, Assam

ABSTRACT
Arsenic stress is important abiotic stress that affects growth and productivity in crop plant.
In the present study, Wheat (Triticum aestivum) seedlings were exposed to different
concentrations of Arsenic (As) (V)[0, 50, 100 and 500µM] toxicity. Significant decrease in
growth in terms of root and shoot length was recorded. Fresh and dry weight of root and
shoot also showed significant decreased due to As stress. The content of H2O2 and proline
increased many fold when compared with control. Different types of scavenging enzymes
like ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), peroxidase
(POX), superoxide dismutase (SOD) also altered significantly. Thus, the current study
revealed that all these five stress marker enzymes enhanced many fold to cope up with the
toxicity imposed by As (V) in Triticum aestivum.

Key words: Antioxidants (APX, CAT, GR, POX, SOD); arsenate; arsenite; oxidative stress;
reactive oxygen species; wheat.

*E-mail - sheto_14@rediffmail.com

Published by
The Society for Biometry, Ecology & Econometrics (BEES), Karimganj, Assam, India 60
ECOBIOS, Vol. 7 (1&2), 2014
INTRODUCTION
Arsenic (As), a metalloid belongs to
group I carcinogen (http:// www.epa.gov /
ttn/ atw / hlthef / arsenic.html)1. Human
activities have caused an accumulation of
arsenic in soils mainly through the
production or use of arsenical pesticides,
manufacture of arsenic based compounds,
smelting of arsenic ores, mining processes
and fuel utilization2. The inorganic
arsenate [As (V)] and arsenite [As(III)]
are the main phytoavailable forms of
arsenic in soil solution3. In 2002, the
Environmental Protection Agency low-
ered the maximum contaminant level of
As in drinking water from 50 μg/L to 10
4
μg/L . No limit of total As or inorganic
As ingestion through food has yet been
5
given .
Arsenic concentration in terrestrial plants
under normal conditions are usually less
than 10 mg kg-1 though an average
threshold of 40 mg kg-1 was established
for crop plants6. However, due to it’s
presence in terrestrial and aquatic
environment, it affects the growth of
plants. Due to their sessile nature plants
cannot escape the stress conditions;
therefore, to cope–up with the toxicity
induced by As, plants have acquired
ECOBIOS, Vol. VII. (I&II), 2014
ISSN: 0972-6446
certain protective mechanisms which
usually activate in response to cell
signaling during stress condition7. Plants
might respond via increased expression of
reaction oxygen species (ROS), enzyme
co-factors, antioxidative enzymes (APX,
CAT, GR, POX, SOD), cellular redox
imbalance, oxidation of proteins, DNA
damage8 etc. According to Aksakal and
Esim 9, arsenic trioxide (As2O3) induced
oxidative stress in wheat (Triticum
aestivum) seedlings, which in turn
activated antioxidative mechanisms by
enhancing the activities of CAT, POX,
and SOD. Also, in other plant systems,
for example, bean and rice seedlings, As
induces oxidative stress 9, 10. Again, in
Brassica juncea As (V) significantly
hampered the growth and triggered
oxidative stress by enhancing ROS and
antioxidants 11.
The present study has been carried out
focusing on physiological and bioche-
mical response of wheat seedlings under
As (V) stress, and, also to have better
understanding of defense mechanism
played by wheat seedlings against As (V)
– toxicity.
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ECOBIOS, Vol. 7 (1&2), 2014
MATERIALS AND METHODS
Plant material and growth conditions
Fresh wheat (Triticum aestivum) seeds
collected from Flour Mill, Silchar, India,
were surface sterilizedwith 0.1% HgCl2
solution, rinsed with distilled water and
then transferred in Petridishes for
germination. The germinated seeds (after
two days of germination) were transferred
to the plastic pots containing Hoagland
nutrient medium (pH=6.8). The seedlings
were grown in a growth chamber under
continuous white light provided with
cool, fluorescent white tubes (Philips
20W TLD, India), with a photon flux
–2 –1
density of 52 E m s (PAR) and kept
at 25 2°C for 5 days. On the 5th day of
growth condition, arsenic treatment was
given in the form of sodium arsenate
(Na2HAsO4.7H2O). The concentrations
were 0, 50, 100 and 500µM. On 48th hour
that is on 2nd day after treatment, shoot
and root samples were harvested for the
analysis of growth and biochemical
parameters. The growth was determined
in terms of length (cm), fresh and dry
biomass (g) of root and shoot. The
experiment was repeated on 4th, 6th and 8th
day after treatment.
ECOBIOS, Vol. VII. (I&II), 2014
ISSN: 0972-6446
Biochemical analysis
Extract preparation
The wheat root and shoot tissues were
homogenized separately with 0.1 M
phosphate buffer (pH=6.8) in pre-chilled
mortar and pastle. The extract was
centrifuged at 17000 rpm for 15 mins at
40C in cooling centrifuge. The supernatant
was used for the assay of CAT, POX,
SOD and GR as per the methods of
Maehly and Chance, Chance and Maehly,
Giannopolitis and Ries and Halliwell and
12-15
Foyer respectively. The method of
16
Nakano and Asada was followed for the
extraction of APX
Hydrogen peroxide (H2O2)
For H2O2 content, 0.2 g tissue was
homogenized with 10% TCA and made
the volume 10 ml, centrifuged at 17000
rpm at 00C for 15 min. The supernatant
was immediately used for the estimation
of total peroxide content by the
17
ferrithiocyanate method . The assay
contained 1.6ml metabolite extract, 50%
TCA, 10mM ferrous ammonium sulphate
(Fe (NH4)2 (SO4)2. 6H2O), potassium thio-
cyanide (KSCN). Absorbance was taken
at 480nm. However, H2O2 content was
observed only in case of interaction study.
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ECOBIOS, Vol. 7 (1&2), 2014
Proline content
Proline was extracted and assayed as per
the method of18. 0.2 g tissue was
homogenized with 3% aqueous sulfo-
salicyclic acid and made the volume upto
5 ml, centrifuged at 3000 rpm at room
temperature for 10 min. The assay
contained 2ml extract, 2ml acid
ninhydrin, 2ml glacial acetic acid and
heated in boiling water bath for 60mins.
After cooling 4ml toluene was added and
mixed well by using cyclomixture.
Absorbance of the upper layer formed
was taken at 520nm for proline content
determination.
Antioxidants enzyme assay
Ascorbate peroxidase (APX)
For APX (1.11.1.11) extraction, the tissue
sample was homogenized with 0.1M
phosphate buffer (pH 6.8) containing
1mM ascorbate, centrifuged at 17000rpm
for 15 mins at 40C. The supernatant was
used as enzyme extract for assay mixture.
The assay mixture was prepared with
10mM H2O2, 0.5mM ascorbate, 0.1M
phosphate buffer (pH 6.8) and enzyme
extract. The absorbancy was taken at
290nm. With extinction co-efficient 2.8,
the APX activity was expressed as ΔA290
g-1 (fr.wt.) min-1 .
ECOBIOS, Vol. VII. (I&II), 2014
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Catalase (CAT) and peroxidase (POD)
assay
The reaction mixture for CAT (1.11.1.6)
contained 2ml (0.1M) phosphate buffer,
0.5ml 30mM H2O2, and 0.5ml distilled
water, incubated for 1 min. The
absorbancy was recorded at 240nm. With
extinction co-efficient = 43.6, the CAT
activity was expressed as unit min-1 g-
1
(fr.wt). Reaction mixture for POX
(1.11.1.7) contained 2.1ml (0.1M)
phosphate buffer, 0.3ml 1% guaicol,
0.3ml 1% H2O2, 0.3ml enzyme extract
and incubated for 5 minutes. Absorbancy
was taken at 470nm (extinction co-
efficient, ε = 26.6). The activity was
expressed as ΔA470g-1 (fr.wt.) min-1.
Superoxide dismutase (SOD) and
glutathione reductase (GR)
The assay mixture for SOD (1.15.1.1)
contained 79.2mM Tris-HCl buffer (pH
6.8), containing 0.12 mM ethylenediami-
notetracetic acid (EDTA) and 10.8 mM
tetraethylenediamine, bovine serum
albumin (0.0033%),6 mM nitroblue-
tetrazolium (NBT), 600 µM riboflavin in
5mM KOH and 0.2 ml enzyme extract.
Reaction was initiated by placing the
glass test tubes in between two
fluorescent tubes (Philips 20 W). By
switching the light on and off, the reaction
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ECOBIOS, Vol. 7 (1&2), 2014
was started and terminated, respectively.
The increase in absorbance due to
formazan formation was read at 560 nm.
The activity was expressed as ΔA560g-1
(fr.wt.)10min-1. The reaction mixture of
GR (1.6.4.2) contained 1.2ml (0.1M)
phosphate buffer, 0.1ml 5mM oxidized
glutathione, 0.1ml 3.5mM NADPH, 0.1ml
of 1% (m/v) albumin, 0.1ml enzyme
extract and incubated for 1 min. The
decrease in absorbance was measured at
340nm (co-eff. of absorbance=16.2mM-
1
cm-1). One unit of GR was defined as 0.1
µmol (NADPH oxidized) min-1 at 300C.
Statistical analysis
Each experiment was repeated three times
and the data presented are means ±
standard errors (SE). The results were
subjected to ANOVA and Tukey-test was
used for comparison between pairs of
treatments. The data analysis were carried
out using statistical package SPSS
(Statistical Package for Social Sciences)
10.0.
RESULT AND DISCUSSION
Length and biomass
According to the results obtained,
exposure of As (V) affected growth of
wheat seedlings. In case of root on 6th and
8th day, 1-fold decrease in length (in ‘cm’)
ECOBIOS, Vol. VII. (I&II), 2014
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was observed. In shoot also, significant (p
≤ 0.05) inhibition of growth was seen (fig.
1a & 1b). Further, on 8th day, we observed
significant (p ≤ 0.05) decrease in fresh
and dry biomass (g) of shoot (fig. 1c &
1d). Similarly, fresh and dry biomass of
root showed significant (p ≤ 0.05) growth
inhibition under all increasing concen-
trations (50, 100, 500µM) of As(V).
Maximum inhibition was observed under
500µM of As(V) treatment (fig. 1e & 1f).
Arsenic being an environmental pollutant
causes wilting, discoloration, and
plasmolysis in root and leaf cells and thus
affects root and leaf growth of plants. The
inhibitory effect of As(V) on growth have
also been reported in other plant species
like rice (Oryza sativa)19, mung bean
(Phaseolus aureus)20etc. Similar types of
results were reported by Aksakal and
Esim9 in wheat seedlings growing in
arsenic trioxide.
H2O2 content
H2O2 is produced predominantly in plant
cells during photosynthesis and photo-
respiration, and to a lesser extent in
respiration processes. It is the most stable
form of ROS, and therefore plays a
crucial role as a signalling molecule in
various physiological processes, and
whose activity thus greatly increases in
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ECOBIOS, Vol. 7 (1&2), 2014
response to abiotic stress21. In case of
both shoot and root tissue, H2O2 content
increased significantly (p ≤ 0.05). When
compared with control, H2O2 showed 2 –
3 fold increase in its content (fig. 2) on 6th
and 8th day under 500µM concentration.
On 2nd and 4th day, H2O2 accummulated
significantly with increasing
concentration of As(V). According to
Mittler22 higher concentration of H2O2
cause membrane damage by forming
hydroxyl radical and as such results into
lipid peroxidation. Increased production
of H2O2 under As (V) stress has been
10
reported in rice seedlings . The rate of
H2O2 production depends on the strength
and duration of the imposed stress 23. At
low concentrations, H2O2 helps in stress
acclimation by providing tolerance; for
example, an increase in activity of SOD in
response to As treatment indicates a rapid
dismutation of O2- into H2O2 in mung
24
bean , whereas at high concentrations it
causes cellular damage leading to cell
25
death . The high production of H2O2 in
wheat seedlings during As (V) stress
indicates that it impart greater affect by
causing strong oxidative damage. Further,
it possibly resulted in growth inhibition,
leaf chlorosis and reduction of plant
ECOBIOS, Vol. VII. (I&II), 2014
ISSN: 0972-6446
biomass which are evident in the present
investigation.
Proline content
Proline accumulation is an important
index for studying stress tolerance
capacity of the plants26. It acts as an
osmoprotectant. With increasing concen-
trations of As(V), proline increased
significantly. In shoot and root tissue,
under 500µM As(V) many fold increase
in proline’s content was observed (fig. 3).
On 8th day, 4–5 fold increase in it’s
accumulation was recorded. In the present
experiment, proline accumulation might
have helped in maintaining water relation,
prevented membrane distortion and acted
as a hydroxyl radical scavenger27.
Antioxidants enzymes
An increasesd generation of ROS due to
As treatment in plants is associated with
enhanced activities of scavenging
enzymes like APX, CAT, GR, POX and
SOD 2824. All these scavenging enzymes
are more or less present in the cells. APX
and POX are important components of the
ascorbate-glutathione cycle responsible
for the removal of H2O2 in different
cellular compartments29. We investigated
responses of these five antioxidant
enzymes in wheat seedlings under As(V)
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ECOBIOS, Vol. 7 (1&2), 2014
stress. The results indicated that 2 nd and
4th day APX activity in shoot tissue
enhanced under 50 and 500 µM As(V).
But with increase in duration APX
activity decreased. Whereas in 2nd day
root, 2.5 fold increased activity of APX
was observed (fig-4). APX has a higher
affinity for H2O2 than CAT and POX; it
might have more crucial role in the
management of ROS during any stress or
it might be responsible for the fine
modulation of ROS for signaling .
30
According to Asada31, the H2O2
scavenging system represented by APX
and CAT are more important in
partitioning tolerance than SOD as
reported in oxidative stressed wheat
varieties. APX is mainly located in
chloroplasts and any damage to
chloroplast is likely to affect its activity24.
So, a decrease in APX activity as obtained
in this study is not surprising. CAT
participates in the main defense system
against accumulation and toxicity of H2O2
and can play the role in controlling H2O2
level in plant cells. CAT showed
enhanced activity under various concen-
trations of As(V). In both shoot and root
tissue maximum increased activity was
observed under 500 µM As(V). However,
on 8th day CAT showed decreased activity
ECOBIOS, Vol. VII. (I&II), 2014
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in shoot (fig 5). The decreased activities
of APX and CAT in this study indicate
their inactivation or degeneration due to
As induced oxidative stress. Reduction in
CAT content in response to As stress has
also been reported in Taxithelium
nepalense32. In 2007, Singh et al.,24 also
reported decrease in CAT activity in
mung bean. In case of GR (shoot and
root), 4 fold increase in it’s activity was
recorded under 500 µM As(V) on 6th and
8th day (fig 6). The increase in activity of
POX and GR in response to As is
associated with excess of H2O2
detoxification, as POX acts upon H2O2
and forms GSSG which is further reduced
to GSH by GR. Increased activity of GR
results in higher amount of GSH which
has been shown to be associated with an
increase in ascorbate content and thus
provide better protection against oxidative
33
stress . POX belongs to enzymes that are
involved in development, growth, and
senescence processes of plants and
resistance against pathogens, wound
healing, synthesis of lignin and ethylene,
and decomposition of IAA34. This
enzyme catalyzes H2O2-dependent oxi-
dation of substrate and could play a role
in alleviating oxidative stress induced by
toxic metals9. In present experiment, POX
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ECOBIOS, Vol. 7 (1&2), 2014
showed less significant activity in both
shoot and root tissue; though on 2nd day
under 100 µM As(V) it showed 2.5 fold
increase in it’s activity (fig 7). The
increase in POX has also been reported in
24
bean due to As stress . Besides,
enhanced POX activity can be correlated
with higher lignification and consequent
stunted growth, as an acclimation to
stress35. In the present experimental
conditions, CAT activities on 6th day
displayed an increasing trend with
increasing concentration range, and then a
decreasing trend on 8th day. This situation
may be due to ineffective enzyme
synthesis or change in assembly of
enzyme subunits. Besides, a decline in
both CAT and POX activities in the
highest concentration suggests a possible
delay in the removal of H2O2 and toxic
peroxides mediated by CAT and POX,
and an enhancement of lipid peroxi-
dation9. On the other hand, the decline in
CAT and POX activities in higher
concentrations could be reasoned by
photoinactivation as suggested for plants
under heavy metals9. At lower arsenate
concentrations, activity of antioxidant
enzymes increased, whereas at higher
concentrations, POX and CAT activities
decreased in Vicia faba roots. The
ECOBIOS, Vol. VII. (I&II), 2014
ISSN: 0972-6446
increase in antioxidant activities could
represent an appropriate protection
against overproduction of peroxides when
As2O3 is taken by wheat9On the other
hand, the rate of activities of POX
decreased at the maximum arsenic
concentration. This situation may be
caused by the potential damages that
occur in the antioxidant defense system.
Also, the declines in the activities of CAT
and POX may be due to the formation of
protein complex with As, as to change the
structure or integrity of proteins36. The
result of SOD indicated that As(V)
induced significant increase in it’s (SOD)
activity under various treatments. In case
of both shoot and root tissue, SOD
showed many fold increased activity.
Maximum induction of SOD was seen
under 500 µM As (V) on 6th day shoot.
When compared with other sets of
experiments, it was observed that
maximum activity of SOD was recorded
on 6th day root; whereas, on 2nd day SOD
showed least activity (fig 8). SOD is
known as a first line defence mechanism
generated by plant cell against ROS37. An
increase in SOD activity indicates higher
production of H2O2, which has been seen
in the case of an increase in endogenous
peroxide accumulation. It has been
demonstrated that SOD in Zea mays was
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ECOBIOS, Vol. 7 (1&2), 2014
stimulated upon exposure to both arsenate
and arsenite38. It is well known that SOD
.-
dismutase superoxide anion (O2 ) to H2O2
and O2 in the cytosol, mitochondria and
chloroplast39. The increase in activity of
SOD has also been noticed in rice under
As (V) stress 10 and in wheat seedlings
under arsenic trioxide stress9. The
increase might be due to the protection
given by SOD to cell for combating stress
imposed by ROS. The variation in SOD
induction in the present study might be
due to the presence of different isoforms
of SOD in cellular compartments40.
Arsenic in it’s pentavalent (V) form
influenced the growth of wheat seedling
in terms of length and biomass. With
increasing concentrations of As(V),
activities of antioxidants (APX, CAT,
GR, POX and SOD) also enhanced. This
enhanced level of antioxidants indicated
their protective role against oxidative
damage imposed by arsenic toxicity.
From this experiment it is evident that
physiological and biochemical effect due
to As(V) stress is concentration and time
dependent.
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Potential phytotoxicty of lead and
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43.
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ECOBIOS, Vol. 7 (1&2), 2014 ISSN: 0972-6446

(a)

(b)

(c)

(d)

(e)

(f)

Fig. 1 : - Effect of As (V) on growth of wheat (Triticum aestivum) seedlings (on 2nd, 4th, 6th and
8th day of treatment) in terms of root length (a) and shoot length (b); fresh weight of shoot (c)
and dry weight of shoot (d); fresh weight of root (e) and dry weight of root (f). Values are
means ± S.E. based on three independent experimental data

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ECOBIOS, Vol. 7 (1&2), 2014 ISSN: 0972-6446

z
20000 H2O2 (s) H2O2 (r)
0µM 25000
15000

µM g-1 fr. wt.

µM g-1 fr. wt.

50µM 20000
10000 100µM 15000
500µM 10000
5000
5000
0 0
2nd day 4th day 6th day 8th day 2nd day 4th day 6th day 8th day

Duration Duration

Fig. 2 : - Showing H2O2 accumulation under different concentrations of arsenic(As +5) stress
in shoot (s) and root (r) tissue of wheat seedling. Others same as Fig.1.

0µM Proline (r)

Proline(s)
50µM 2.5
6 2
unit g-1 fr. wt.

5 100µ
unit g-1 fr. wt

4 M 1.5
3 1
2 *
0.5
1 *
0 0
2nd day 4th day 6th day 8th day 2nd day 4th day 6th day 8th day
Duration Duration

+5
Fig. 3 : - Showing proline content under different concentrations of arsenic (As ) stress in
shoot (s) and root (r) tissue of wheat seedling. Others same as Fig.1.

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ECOBIOS, Vol. 7 (1&2), 2014 ISSN: 0972-6446

APX (S)

15
0μM
unit g-1 fr wt.

10 50μM
5 100μM
500μM
0
2nd day 4th day 6th day 8th day
Duration

APX (R)

10
unit g-1 fr wt.

8
6
4
2
0
2nd day 4th day 6th day 8th day
Duration

Fig. 4 : - Activity of APX under different concentrations of arsenic (As +5)stress in shoot (S)
and root (R) tissue of wheat seedling. Others same as Fig.1.

CAT (S)

0.5
unit g-1 fr wt.

0.4
0.3
0.2
0.1
0
2nd day 4th day 6th day 8th day
Duration

CAT (R)

1.5
unit g-1 fr wt.

1
0.5
0
2nd day 4th day 6th day 8th day
Duartion

Fig. 5 : - Activity of CAT under different concentrations of arsenic (As +5)stress in shoot (S)
and root (R) tissue of wheat seedling. Others same as Fig.1.

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ECOBIOS, Vol. 7 (1&2), 2014 ISSN: 0972-6446

GR (S)

80
unit g-1 fr wt.

60
0μM
40
50μM
20
100μM
0
2nd day 4th day 6th day 8th day 500μM

Duration

GR (R)

80
unit g-1 fr wt.

60
40
20
0
2nd day 4th day 6th day 8th day
Duration

+5
Fig. 6 : - Activity of GR under different concentrations of arsenic (As ) stress in shoot (S)
and root (R) tissue of wheat seedling. Others same as Fig.1.

POX (S)

400
unit g-1 fr wt.

300
200
100
0
2nd day 4th day 6th day 8th day
Duration

POX (R)

400
unit g-1 fr wt.

300
200
100
0
2nd day 4th day 6th day 8th day
Duration

+5
Fig. 7 : - Activity of POX under different concentrations of arsenic (As ) stress in shoot (S)
and root (R) tissue of wheat seedling. Others same as Fig.1.

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ECOBIOS, Vol. 7 (1&2), 2014 ISSN: 0972-6446

SOD (S)

0.3
unit g-1 fr wt.

0.25
0μM
0.2
0.15 50μM
0.1 100μM
0.05
500μM
0
-0.05 2nd day 4th day 6th day 8th day

Duration

SOD (R)

0.4
unit g-1 fr wt.

0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
2nd day 4th day 6th day 8th day

Duartion

Fig. 8: - Activity of SOD under different concentrations of arsenic (As +5) stress in shoot (S)
and root (R) tissue of wheat seedling. Others same as Fig.1.

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