325

J. Crop Sci. Biotech. 2012 (December) 15 (4) : 325 ~ 334

DOI No. 10.1007/s12892-012-0065-3

RESEARCH ARTICLE

Exogenous Calcium Alleviates the Impact of Cadmium-

Induced Oxidative Stress in Lens culinaris Medic. Seedlings
Through Modulation of Antioxidant Enzyme Activities
Dibyendu Talukdar*

Plant Cell and Stress Biology Laboratory, Department of Botany, R.P.M. College, University of Calcutta, Uttarpara, Hooghly
712258, India

Received: June 28, 2012 / Revised: August 14, 2012 / Accepted: August 24, 2012
Ⓒ Korean Society of Crop Science and Springer 2012

Abstract
The effect of calcium (Ca) on lentil (Lens culinaris Medic.) seedlings exposed to cadmium (Cd) stress was studied by investigat-
ing plant growth and antioxidant enzyme activities. Plants were grown for 14 days in full-strength Hoagland nutrient media supple-
mented with Cd concentrations of 0, 10, 20, and 40 µM, and on corresponding medium supplied with 5 mM Ca(NO3)2 prior to Cd
addition. Increasing Cd led to accumulation of metal and reduced the fresh weight of the shoots more strongly than that of the roots.
Cd concentrations of 20 and 40 µM were selected to study its toxic effect on seedlings. Activities of superoxide dismutase, ascorbate
peroxidase, catalase, dehydroascorbate reductase, and glutathione reductase decreased at much higher magnitude in the shoots than
those observed in the roots under Cd exposure. Failure of antioxidant defense in scavenging of reactive oxygen species was evi-
denced by abnormal rise in H2O2, resulting in enhancement of lipid peroxidation and membrane electrolyte leakage as the marks of
Cd-induced oxidative stress in lentil seedlings. Ca priming in the media significantly reduced the Cd accumulation and considerably
alleviated the adverse impact of Cd treatment by modulating the antioxidant enzyme activity. Mitigation of Cd-induced stress by Ca
application was strongly suggested by declining levels of H2O2 and consequent lowering of oxidative damage of membrane.
Consequently, this enhanced fresh mass of plant parts as the sign of Ca-mediated normal growth in Cd-treated lentil seedlings.

Key words : cadmium, calcium, growth, Lens culinaris, oxidative stress, reactive oxygen species

Introduction
Cadmium (Cd) is a non-essential element for plant metab-
olism even though it is rapidly taken up by plant roots and
can be loaded into the xylem for its transport into the leaves
(Sanita di Toppi and Gabrielli 1999; Wagner 1993). Cd accu-
mulation is very toxic to plants, causing poor growth and low
biomass accumulation through interference in several meta-
bolic processes, such as water and mineral uptake (Sandalio
et al. 2001), photosynthesis (Mobin and Khan 2007), enzyme
activity (Clemens 2006; Siddiqui et al. 2012), and cellular
redox homeostasis (Ortega-Villasante et al. 2005; Romero-
Dibyendu Talukdar ( ) enzymes, thus affecting cell viability (Sandalio et al. 2001).
E-mail: dibyendutalukdar9@gmail.com
Phone: 910923099774, 910332663-0191
Puertas et al. 2004). Moderate Cd contamination of arable
soils can result in considerable Cd accumulation in edible
parts of crops (Uraguchi and Fujiwara 2012). Cd functions
as a non-redox-active metal so it cannot generate free radi-
cals and reactive oxygen species (ROS) itself. However, Cd
can raise the basal ROS level either by inactivation or down-
regulation of ROS-scavenging antioxidant enzymes and
metabolites (Clemens 2006; Sanita di Toppi and Gabrielli
1999; Talukdar 2012c). These species react with cellular
lipid, proteins, pigments, and nucleic acids and cause lipid
peroxidation, membrane damage, and inactivation of
Normally, metabolism of ROS is under tight regulation with-
in an aerobic cell. However, when a plant experiences oxida-

The Korean Society of Crop Science

326 Calcium Alleviates the Cadmium-Induced Stress in Lentil
tive stress the critical balance between ROS generation and
its scavenging by antioxidant molecules is disturbed, result-
ing in disruption of redox homeostasis heavily in favor of
oxidative state (Ortega-Villasante et al. 2005; Romero-
Puertas et al. 2004). To withstand the stress, plants have
evolved a complex but well-coordinated antioxidant defense
system, comprised of both enzymatic (superoxide dismutase,
ascorbate peroxidase, dehydroascorbate reductase, glu-
tathione reductase, catalase, etc.) and non-enzymatic (ascor-
bate, glutathione, carotenoids, etc.) compounds (Noctor and
Foyer 1998). The response of these compounds, however,
largely depends on both external and internal stimuli and also
genetic backgrounds of the plants under stress (Talukdar
2011a, 2012a), and their modulation ultimately determines
plant growth and development (De Pinto and De Gara 2004).
Calcium (Ca) is involved in the regulation of plant cell
metabolism and signal transduction (Rentel and Knight 2004;
Yang and Poovaiah 2003) and modulates cellular processes
by binding proteins, such as calmodulin, which in turn regu-
lates the activity of target proteins (Hu et al. 2007). Recent
findings indicate reversing of Cd-induced oxidative stress by
exogenous application of Ca in Arabidopsis thaliana (Suzuki
2005), Sedum alfredii (Tian et al. 2011), and in leguminous
crops (Rodríguez-Serrano et al. 2009; Wang and Song 2009).
Thus, the impact of Ca added to growth medium was studied
as a protector against Cd toxicity in the present material.
Lentil (Lens culinaris Medic.) is one of the oldest known
protein-rich pulse crops and among the cool season legumes,
it is the richest in the important amino acids (lysine, arginine,
leucine, and sulphur containing amino acids) (Williams et al.
1994). More than 85% of the annual global production
occurs in four specific regions in which eastern half of the
Indo-Gangetic plain of South Asia including India, Nepal,
and Bangladesh occupies the major (32%) share (FAOSTAT
2008). However, the declining/unstable trend of its produc-
tion in many lentil-growing countries is a major cause of
concern for pulse food security throughout the world
(Akibode and Maredia 2011). One of the major causes of
dwindling lentil production is its sensitivity to diverse types
of abiotic stresses including cold, drought, heat, salinity, and
nutrient toxicity (Bandeoglu et al. 2004; Muehlbauer et al.
2006; Yau and Erskine 2000). Although lentil shows higher
sensitivity to abiotic stress than broadbean and soybean
(Muehlbauer et al. 2006) and large parts of lentil-cultivated
areas in several countries are metal contaminated, perusal of
literature cites only scanty information regarding response of
this legume crop to heavy metal induced oxidative stress
(Gautam and Pandey 2008; Kiran and Sahin 2006; Talukdar
2013). In view of the wide-scale contamination of agricultur-
al fields by Cd in India (Mandal and Bhattacharyya 2012;
Roy et al. 2010) and its toxicity on growth of many legumi-
nous crops (Anjum et al. 2011; Rodríguez-Serrano et al.
2009; Sandalio et al. 2001; Talukdar 2012c) the effect of this
heavy metal on response of antioxidant defense and the
process of amelioration needs urgent study. No reports
regarding the mitigation of metal stress by Ca application is
available in lentil. Thus, the present investigation was under-
taken to ascertain whether exogenous application of Ca pro-
tects L. culinaris seedlings against Cd stress.
Materials and Methods
Plant material and Cd treatments
Fresh and healthy seeds of lentil (Lens culnaris Medic. cv.
B 17) were allowed to germinate at dark in two separate sets
on moistened filter paper at 25°C. Germinated seedlings were
randomly placed in polythene pots (10 plants per pot) con-
taining 300 mL of Hoagland’s No. 2 nutrient media, and
were allowed to grow for 14 d. The media were then supple-
mented with four different concentrations (0, 10, 20, and 40
µM) of CdCl2 (Sigma-Aldrich Chemicals, Bangalore, India)
and were allowed to grow for another 14 d. Six replicates for
each treatment were prepared to give a total of 24 pots.
Nutrient solution was refreshed every alternate day to pre-
vent depletion of nutrients as well as Cd in the course of the
plant’s exposure to the metal. Seedlings and plants were
placed in a growing chamber with a 14-h photoperiod, at
25°C, humidity of 70%, and a photon flux density of 200
µmol m-2 s-1 (plant level). Untreated plants were used as con-
trol. To determine the effect of Ca, control, and Cd-treated
plants were supplemented with 5 mM (this concentration was
selected on the basis of preliminary experiments, data not
shown) Ca(NO3)2 one day before the addition of Cd in sepa-
rate experiments. Thus, at first set, there were three Cd con-
centrations (10, 20, and 40 µM) and one control treatment for
the fresh weight determination and cadmium accumulation,
and then in the second set a control and two Cd concentra-
tions (20 and 40 µ M) were selected where calcium was
added 1 day before Cd. The increase in fresh weight of the
roots and the shoots (stems + leaves) was measured after 2
weeks of cultivation.
Determination of Cd content
The plant samples were carefully washed with deionized
water and oven-dried at 105°C for 6 h followed by 60°C for
24 h. Dried samples were ground to a fine powder using a
porcelain mortar and pestle, and then (1 g per sample) digest-
ed by 4:1 (v/v) HNO3-HClO4 mixture in a microwave sys-
tem. Cd concentration was determined using an atomic
absorption spectrophotometer (Perkin-Elmer, Analyst 300)
following the manufacturer’s instructions. A standard refer-
ence material (SRM) of tomato leaves (Item number 1573a,
from National Institute of Standards and Technology, USA)
were analyzed in the same procedure at the start, during, and
at the end of the measurements as part of the quality assur-
ance/quality control protocol. The certified value of Cd con-
centration and our measured concentration (mg kg -1 dry
weight) of the dried tomato leaves were 1.52 ± 0.04 and 1.65
± 0.09 (n = 8), respectively, showing good correlation (r =
0.822) between certified (NIST®) and the measured value.

Antioxidant enzyme assays
Fresh tissue (250 mg) was homogenized in 1 mL of 50
mM potassium phosphate buffer (pH 7.8) containing 1 mM
EDTA, 1 mM dithiotreitol, and 2% (w/v) polyvinyl pyrroli-
done (PVP) using chilled mortar and pestle kept in ice bath.
The homogenate was centrifuged at 15,000 Xg at 4°C for 30
min. Clear supernatant was used for enzyme assays. For
measuring APX (ascorbate peroxidase) activity, the tissue
was separately ground in homogenizing medium containing
2.0 mM ascorbate in addition to the other ingredients. All
assays were done at 25°C. Soluble protein content was deter-
mined according to Bradford (1976) using Bovine Serum
Albumin (BSA) as a standard.
Superoxide dismutase (SOD, EC 1.15.1.1) activity was
determined by nitro blue tetrazolium (NBT) photochemical
assay according to Beyer and Fridovich (1987). In this
method, 1 mL of solution containing 50 mM potassium phos-
phate buffer (pH 7.8), 9.9 mM of L-methionine, 57 µM of
NBT, 0.025% triton-X-100 was added into small glass tubes,
followed by 20 µL of enzyme extract. Reaction was started
by adding 10 µL of riboflavin solution (0.044 mg mL-1) and
placing the tubes in an aluminium foil-lined box having two
20-W fluorescent lamps for 7 min. A parallel control was run
where buffer was used instead of the sample. After illumina-
tion, the absorbance of solution was measured at 560 nm. A
non-irradiated complete reaction mixture served as a blank.
SOD activity was expressed as U (unit) mg-1 protein. One
unit of SOD was equal to that amount which causes a 50%
decrease of SOD-inhibited NBT reduction.
Ascorbate peroxidase (APX, EC 1.11.1.11) activity was
assayed according to the method of Nakano and Asada
(1981). Three milliliter of the reaction mixture contained 50
mM potassium phosphate buffer (pH 7.0), 0.1 mM EDTA,
0.5 mM ascorbate, 0.1 mM H 2 O 2 , and 0.1 mL enzyme
extract. The hydrogen peroxide-dependent oxidation of
ascorbate was followed by a decrease in the absorbance at
290 nm (extinction coefficient 2.8 mM-1 cm-1). APX activity
was expressed as nmol ascorbate oxidized min-1 mg-1 protein.
Catalase (CAT, EC 1.11.1.6) activity was measured
according to Chance and Maehly (1955) with slight modifi-
cations. Enzymatic activity was initiated by adding 50 µL of
enzyme extract into the reaction mixture containing 500 µL
of potassium phosphate buffer (0.1 M, pH 6.5), 250 µL of
distilled water, and 200 µL of 75 mM H2O2. CAT activity
was monitored at 240 nm for 2 min at 25°C after initiation of
the reaction, and was measured against a blank reaction mix-
ture containing no enzyme extract. CAT specific activity
(nmol H2O2 degraded min-1 mg-1 protein) was calculated using
the molar absorptivity of 43.6 M-1 cm-1for H2O2 at 240 nm.
Dehydroascorbate reductase (DHAR, EC 1.8.5.1) enzyme
activity was measured following the protocol of Nakano and
Asada (1981). The complete reaction mixture contained 50
mM potassium phosphate buffer (pH 7.0), 2.5 mM GSH, 0.2
mM DHA, and 0.1 mM EDTA in a final volume of 1 mL.
Reaction was started by addition of suitable aliquots of
JCSB 2012 (December) 15 (4) : 325 ~ 334 327
enzyme extract and the increase in absorbance was recorded
at 30 s intervals for 3 min at 265 nm. Enzyme activity was
expressed as µmol ascorbate formed min-1 mg-1 protein.
Glutathione reductase (GR, EC 1.6.4.2) activity was deter-
mined by monitoring the glutathione dependant oxidation of
NADPH, as described by Carlberg and Mannervik (1985). In
a cuvette, 0.75 mL 0.2 M potassium phosphate buffer (pH
7.0) containing 2 mM EDTA, 75 µL NADPH (2 mM), and
75 µL oxidized glutathione (20 mM) were mixed. Reaction
was initiated by adding 0.1 mL enzyme extract to the cuvette
and the decrease in absorbance at 340 nm was monitored for
2 min. GR specific activity was expressed as nmol NADPH
oxidized min-1 mg-1 protein.
H2O2 content and lipid peroxidation assay
H2O2 was estimated following the methods of Wang et al.
(2007). Fresh tissue of 0.1 g was powdered and blended with
3 mL acetone for 30 min at 4°C. Then the sample was fil-
tered through eight layers of gauze cloth. After addition of
0.15 g active carbon, the sample was centrifuged twice at
3,000 Xg for 20 min at 4°C, then 0.2 mL 20% TiCl4 in HCl,
and 0.2 mL ammonia was added to 1 mL of the supernatant.
After reaction, the compound was centrifuged at 3,000 Xg
for 10 min, the supernatant was discarded, and the pellet was
dissolved in 3 mL of 1 M H2SO4 and spectrum measurement
was taken at 410 nm. H2O2 content was measured from the
absorbance at 410 nm using a standard curve. Lipid peroxi-
dation rates were determined by measuring the malondialde-
hyde (MDA) equivalents following the method of Hodges et
al. (1999). About 0.5 g of fresh tissue was homogenized in a
mortar with 80% ethanol. The homogenate was centrifuged
at 3,000 Xg for 12 min at 4°C. The pellet was extracted twice
with the same solvent. The supernatants were pooled and 1
mL of this sample was added to a test tube with an equal vol-
ume of either the solution comprised of 20% TCA and 0.01%
butylated hydroxy toluene (BHT) or a solution of 20% TCA,
0.01% BHT, and 0.65% TBA. Samples were heated at 95°C
for 25 min and cooled to room temperature. Absorbance was
measured at 450, 532, and 600 nm. Level of lipid peroxides
was calculated following Hodges et al. (1999) and expressed
as nmol MDA g-1 fresh weight.
Electrolyte leakage
Electrolyte leakage (EL) was assayed by measuring the
ions leaching from tissue into deionized water. Fresh samples
(100 mg) were cut into small pieces (about 5 mm segments)
and placed in test tubes containing 10 mL deionized water.
Tubes were kept in a water bath at 32°C for 2 h. After incu-
bation, electrical conductivity (EC1) of the bathing solution
was recorded with an electrical conductivity meter
(Systronics M-308, Kolkata, India). The samples were then
autoclaved at 121°C for 20 min to completely kill the tissues
and release all electrolytes. Samples were then cooled to
25°C and final electrical conductivity (EC2) was determined.
The EL was expressed as a percentage by the formula, EL
328 Calcium Alleviates the Cadmium-Induced Stress in Lentil
Fig. 1. Effect of Cd (A) and in combination with Ca (B) on the fresh weight of Lens culi-
naris Medic. seedlings grown on 14 days in nutrient media. Data are means ± SE (n=
6). Significant differences between control and Cd-treated seedlings, and between Cd
alone and Ca (5 mM) + Cd (20 and 40 µM) treated plants were indicated by single
asterisk (*) and double asterisk (**), respectively, at P < 0.05.
(%) = (EC1)/(EC2) X 100 (Dionisio-Sese and Tobita 1998).
Statistical analysis
The results presented are the mean values ± standard
errors obtained from at least six replicates. Statistical signifi-
cance of mean values between control and treated seedlings
was determined by student t-test (two tailed) using Microsoft
Excel software ‘data analysis’ 2007. Multiple comparisons
among treatments were performed by ANOVA using soft-
ware STATISTICA 6.0 (StatSoft, Tulsa, USA). A probability
of P < 0.05 was considered significant.
Results
Fresh weight
Fresh weight of shoot in 14-day-old lentil seedlings reduced
with increasing concentration of Cd in the media. However,
the differences between the control and the Cd-treated plants
for fresh weight became significant (P < 0.05) at 20 µM and
40 µM. Lowest mass value was recorded in the 40 µM Cd
(Fig. 1A) but no visible symptoms of toxicity in shoots were
observed. Likewise, a significant reduction in the root fresh
weight was observed with the 20 and 40 µM Cd concentra-
tions (Fig. 1A). Therefore, the 20 and 40 µM Cd were selected
to ascertain the protective role of Ca under Cd stress. Mass
weight became significant (F-test) among the treatments but
plant phenotype was quite normal (data not shown).
The application of Ca before addition of Cd in the media
Fig. 2. Accumulation of Cd in the plant parts of L. culinaris seedlings at control (0 µM),
selected toxic concentrations (20 and 40 µM) of Cd, and the effect of Ca application on
Cd content in control, Ca + Cd (20 and 40 µM) treated plants. Data are means ± SE
(n= 6). The differences of Cd accumulation between control and Cd treated plants were
significant (*) at P < 0.05. Cd content reduced significantly (**) in Ca + Cd treated
plants compared to Cd treatment alone at P < 0.05
significantly enhanced the fresh weight of both the shoots
and the roots of lentil seedlings (Fig. 1B). Comparing Cd
treatment alone (Fig. 1A), fresh weight of shoots increased
about 2-2.5-fold while that of roots enhanced about 1.5 - 1.7-
fold when 5 mM Ca was added prior to Cd treatment of 20
and 40 µM (Fig. 1B). Ca addition enhanced the fresh weight
of both the shoot and the roots close to control level (Fig.
1B). Comparing control treatment, no significant change in
shoot weight was noticed in plants grown in 0 + Ca media
(Fig. 1B). Also, no contrasting phenotypic changes in rela-
tion to control were observed in plants subjected to 0 + Ca
and Ca + Cd treatments (data not presented).
Cd content
Accumulation of Cd in lentil seedlings increased with ele-
vated concentration and the higher accumulation was esti-
mated in the shoot portions compared with the roots (Fig. 2).
Ca application reduced the Cd content by more than 2.5-fold
in the shoots and approximately 2-fold in the roots of 20 µM
Cd media. In the 40 µM Cd media, this reduction was about
2-fold in shoots and nearly 1.5-fold in the roots (Fig. 2). Ca-
treated plants still contained higher amounts of Cd than the
control. Cd accumulation level varied significantly (F-test, P
< 0.05) among the treatments (data not presented).
Antioxidant enzyme activity
The responses of five antioxidant enzymes, SOD, APX,
DHAR, GR, and CAT greatly differed in lentil seedlings sub-
jected to Cd treatment (particularly 20 and 40 µM) and in Ca
+ Cd-supplemented media. No significant change for enzyme
activity, however, was noticeable between control and 0 + Ca
media. For plants grown in the control medium (Fig. 3), SOD
activity was 1.5-fold higher in the shoot than in the roots.
However, the situation got reversed once the seedlings were
exposed to 20 µM Cd media. SOD level now declined more
than 2.5-fold in the shoots but increased by nearly 1.2-fold in
the roots (Fig. 3). The effect of Cd was more severe in 40
µM as the declining trend of SOD activity in the shoot accel-
erated further (Fig. 3). In the Ca-primed Cd media, SOD
activity was greatly enhanced in the upper part of both con-

Fig. 3. Effects of Cd alone and in combination with Ca on SOD activity of L. culinaris
seedlings. Data are means ± SE (n= 6). Significant differences between control and Cd
as well as Ca + Cd treated plants were indicated by asteisk (*) at P < 0.05. Significant
differences between the effects of Cd alone and Ca + Cd combination were marked by
double asterisk (**) at P < 0.05
Fig. 4. Effects of Cd alone and in combination with Ca on APX activity of L. culinaris
seedlings. Data are means ± SE (n= 6). Significant differences between control and Cd
as well as Ca + Cd treated plants were indicated by asteisk (*) at P < 0.05. Significant
differences between the effects of Cd alone and Ca + Cd combination were marked by
double asterisk (**) at P < 0.05
trol and treated seedlings (Fig. 3). Comparing Cd treatment
alone, prior addition of Ca in 20 and 40 µM Cd media result-
ed in increase of its activity > 2-fold in the shoots (Fig. 3). In
the roots, SOD activity was the same irrespective of the Ca
addition (Fig. 3).
Activities of both the APX and CAT enzymes decreased
significantly (P < 0.05) in the shoots exposed to 20 and 40
µM Cd media (Figs. 4 and 5). In comparison to Cd treatment
alone, Ca pre-treatment in the 20 and 40 Cd µ M media
enhanced both the APX and CAT activity close to the control
level in the shoots (Figs. 4 and 5). Root activities, however,
remained unchanged in both Cd-treated and Ca + Cd-treated
seedlings.
The DHAR activity in the shoots was significantly (P <
0.05) higher than in the roots of untreated lentil seedlings
(Fig. 6). The increased concentration of Cd led to significant
(P < 0.05) decrease of enzyme activity in both organs of
seedlings in the 20 and 40 µM Cd media (Fig. 6). Ca priming
in the Cd-supplemented medium remarkably enhanced
DHAR level in both the shoots and the roots, but the effect
was more pronounced in the shoots (Fig. 6). On the other
hand, GR activity was significantly (P < 0.05) higher in the
roots than in the shoots under un-stressed conditions (Fig. 7).
Imposition of 20 µM Cd in the media led to sharp decline
(nearly 2 - 4 fold) of GR level in both the organs, but the
effect was more severe in the roots than that in the shoots. In
JCSB 2012 (December) 15 (4) : 325 ~ 334 329
Fig. 5. Effects of Cd alone and in combination with Ca on CAT activity in L. culinaris
seedlings. Data are means ± SE (n= 6). Significant differences between control and Cd
as well as Ca + Cd treated plants were indicated by asteisk (*) at P < 0.05. Significant
differences between the effects of Cd alone and Ca + Cd combination were marked by
double asterisk (**) at P < 0.05
Fig. 6. Effects of Cd alone and in combination with Ca on DHAR activity of L. culinaris
seedlings. Data are means ± SE (n= 6). Significant differences between control and Cd
as well as Ca + Cd treated plants were indicated by asteisk (*) at P < 0.05. Significant
differences between the effects of Cd alone and Ca + Cd combination were marked by
double asterisk (**) at P < 0.05
the 40 µM Cd media, further reduction of GR activity was
noticed in both the organs, but this time the effect was more
pronounced in the shoots than in the roots (Fig. 7). Compared
with Cd treatment alone, prior application of Ca significantly
(P < 0.05) enhanced the GR activity in both the organs of the
lentil seedlings under Cd exposure (Fig. 7).
H2O2, MDA content, and electrolyte leakage (EL%)
H2O2 content exhibited an upward trend in both parts of
the Cd-treated seedlings (Fig. 8). Comparing the control, the
rise was about 1.8 - 2.5-fold in the shoots and nearly 1.5-fold
in the roots resulting in significant differences between con-
trol and treated (20 and 40 µM Cd) plants. Prior addition of
Ca in Cd-supplemented media considerably reduced H 2O2
level in both parts of the treated seedlings with a notable
effect on the shoot (Fig. 8).
The accumulation of MDA, a cytotoxic product of mem-
brane lipid peroxidation, increased in Cd-treated seedlings
and was more pronounced in the shoots than in the roots
(Fig. 9). Its highest increase of about 3-fold over the control
was noticed in the shoots of 40 µ M Cd-treated lentil
seedlings (Fig. 9). Prior application of Ca to Cd in the nutri-
ent media greatly reduced the MDA content both in the
shoots and in the roots (Fig. 9). For instance, ca application
reduced the MDA level by about 2.5-fold in shoots and about
1.5-fold in the roots of 40 µM Cd-treated plants (Fig. 9).
330 Calcium Alleviates the Cadmium-Induced Stress in Lentil
Fig. 7. Effects of Cd alone and in combination with Ca on GR activity of L. culinaris
seedlings. Data are means ± SE (n= 6). Significant differences between control and Cd
as well as Ca + Cd treated plants were indicated by asteisk (*) at P < 0.05. Significant
differences between the effects of Cd alone and Ca + Cd combination were marked by
double asterisk (**) at P < 0.05
Fig. 8. Effects of Cd alone and in combination with Ca on H2O2 accumulation of L.
culinaris seedlings. Data are means ± SE (n= 6). Significant differences between control
and Cd as well as Ca + Cd treated plants were indicated by asteisk (*) at P < 0.05.
Significant differences between the effects of Cd alone and Ca + Cd combination were
marked by double asterisk (**) at P < 0.05
Similarly, EL% enhanced significantly (P < 0.05) in the 20
and 40 µM Cd-treated plants but reduced close to control
level in Ca + Cd media (Fig. 10).
Discussion
Growth inhibition is a well-documented response of plants
to the toxic level of Cd ions (Schützendübel and Polle 2002).
The present results with lentil seedlings revealed that the
shoots were relatively more sensitive to the toxic concentra-
tions (20 and 40 µM) of Cd than the roots, although no visi-
ble symptoms of Cd toxicity like leaf chlorosis, yellow vein,
etc. were observed. Decrease of shoot mass in complete
absence of visible damage in upper part of the plants was
also observed in Cd-treated Pisum (Sandalio et al. 2001) and
Trifolium (Wang and Song 2009), while in other plant
species symptoms of leaf chlorosis have been described
under Cd treatment (Ouzounidou et al. 1997). The reduction
of shoot growth in the present lentil seedlings was signifi-
cantly alleviated by the exogenous application of 5 mM Ca
prior to the addition of Cd in the culture media. This is sug-
gested by significant increases of fresh weight almost to the
control level in the Ca + Cd-supplemented media with more
positive effect on the shoots. The outcome is more significant
as the shoots accumulated much higher Cd than the roots,
and priming of nutrient solution by Ca considerably prevent-
Fig. 9. Effects of Cd alone and in combination with Ca on malondealdehyde (MDA)
content of L. culinaris seedlings. Data are means ± SE (n= 6). Significant differences
between control and Cd as well as Ca + Cd treated plants were indicated by asteisk (*)
at P < 0.05. Significant differences between the effects of Cd alone and Ca + Cd com-
bination were marked by double asterisk (**) at P < 0.05
Fig. 10. Effects of Cd alone and in combination with Ca on membrane electrolyte leak-
age (EL%) of L. culinaris seedlings. Data are means ± SE (n= 6). Significant differences
between control and Cd as well as Ca+ Cd treated plants were indicated by asteisk (*)
at P < 0.05. Significant differences between the effects of Cd alone and Ca + Cd com-
bination were marked by double asterisk (**) at P < 0.05
ed the Cd accumulation in the upper part. The result clearly
demonstrates the competition between both elements not
only for the same transporters but also for intracellular Ca-
binding proteins and plasma membrane, as has been
explained earlier (Rivetta et al. 1997; Rodríguez-Serrano et
al. 2009). Obviously, significantly higher fresh weight in the
present lentil seedlings grown in Ca + Cd media is indicative
of the positive role Ca played in the prevention of Cd uptake,
as has also been reported earlier in other crops (Kim et al.
2002; Rodríguez-Serrano et al. 2009; Suzuki 2005).
Decrease in fresh weight of both organs by toxic concen-
tration (20 and 40 µM) of Cd in the media and its alleviation
by Ca pre-treatment is intimately associated with antioxidant
defense response of lentil seedlings to Cd exposure and its
modulation in the Ca-primed Cd media. When Cd was solely
added in the media, it led to significant decrease in activities
of SOD, APX, DHAR, and CAT, exhibiting more severe
effect in the shoots than in the roots. Significantly, the degree
of reduction of GR differed in two organs depending on Cd
concentrations; higher in the roots at 20 µM but at 40 µM Cd
in the shoots. The reduction of SOD, APX, and CAT activity
severely impeded the scavenging of superoxide radicals as
well as removal of H2O2 in the Cd-treated lentil seedlings.
Furthermore, low levels of DHAR and GR activity badly
affected the regeneration of reduced antioxidant molecules.
Together, this led to an increase in H2O2 content and a con-
comitant rise of lipid peroxidation and electrolyte leakage of

membranes with a more detrimental effect on the shoots than
on the roots. Growing evidence suggests that a correlation
exists between the rate of increase in H2O2 content and level
of membrane lipid peroxidation as well as electrolyte leak-
age, and therefore these are often used as biochemical mark-
ers to assess the extent of oxidative damage in plants under
stress (Bandeo lu et al. 2004; Talukdar 2011b, 2011c). The
present results strongly indicate onset of Cd-induced oxida-
tive stress manifested as oxidative damage to membranes in
the lentil seedlings due to declining levels of antioxidant
enzyme activities. This could explain the decrease of fresh
weight observed in lentil seedlings under Cd stress. The near
complete cease of root growth at higher Cd concentration
may be not only related to the failure of H2O2-scavengimg
machinery but is also due to a steep decline in GR levels. GR
is extremely important to maintain GSH redox in favor of a
reduced state, which plays a pivotal role in the maintenance
of root growth under stress conditions as has been reported
earlier in Arabidopsis (Vernoux et al. 2000) and in the legu-
minous plant, Lathyrus sativus L. (Talukdar 2011a, 2012b, c)
and Phaseolus vulgaris L. (Talukdar 2012d).
The remarkable enhancement in fresh weight of both the
organs of Cd-treated lentil seedlings was possibly due to a
significant increase of the antioxidant enzyme activities in Ca
+ Cd-supplemented media. This was more pronounced in the
shoots rather than in the roots, and it is noteworthy in this
aspect that shoots accumulated higher Cd with more toxic
effect than the roots. The modulation of all five enzyme
activities close to the control level, even at toxic concentra-
tions of Cd, ensured efficient ROS-scavenging. This resulted
in declining levels of both H 2 O 2 and MDA content in
response to Ca application. Consequently, this ensured recov-
ery of fresh weight by mitigating the oxidative damage
induced by Cd treatment alone in the lentil seedlings. The
result agreed with earlier reports in which Cd reduced SOD
activity and thus, exogenous supply of Ca reversed the Cd-
treated phenotype through up-regulation of antioxidant
capacity (Rodríguez-Serrano et al. 2009; Wang and Song
2009).
Possible mechanism of action of exogenous Ca on modu-
lation of five antioxidant enzyme activities under un-stressed
control and Cd treatment is worth mentioning. Compared to
the control, Ca-priming triggered no noticeable changes in
enzyme activity under un-stressed conditions but significant-
ly enhanced it during Cd exposure. This apparent conflicting
situation can be explained primarily by the fact that activities
of all five enzymes under study were not constitutively
expressed; rather it was possibly controlled by a complex but
well-integrated mechanistic network in which Ca-signaling
forms an important part. Although the detail mechanism is
still debatable, Ca can regulate the activity of target proteins
directly or via CaM, a ubiquitous calcium-binding protein,
and the Ca/CaM complex regulates activities of antioxidant
enzymes (Gong et al. 1997; Rentel and Knight 2004; Yang
and Poovaiah 2003) along with a number of protein kinases
and transcription factors (Rentel and Knight 2004).
JCSB 2012 (December) 15 (4) : 325 ~ 334 331
Accumulating evidence also strongly suggests a cross-talk
between Ca-CaM and H2O2, a predominant ROS, in regula-
tion of antioxidant enzymes activities (Hu et al. 2007; Jiang
and Huang 2001). The status of cellular H2O2 informs the
plant cell about the severity of the oxidative stress and hence
the appropriate level of antioxidant enzyme activities through
gene induction (Hu et al. 2007; Rentel and Knight 2004).
Studies involving Arabidopsis revealed that Ca influx within
the cell is controlled by cellular H2O2 level and that applica-
tion of H2O2 stimulated Ca influx through a hyperpolariza-
tion-activated Ca-permeable channel (Hu et al. 2007; Rentel
and Knight 2004). The H2O2 level was quite normal in the
present lentil seedlings under un-stressed conditions, and
although not measured, this possibly maintains intrinsic Ca
content in its normal level reflecting Ca-homeostasis in lentil
plants primed with Ca under un-stressed conditions. This
inhibited excess formation of Ca/CaM complex, which in
turn was perhaps instrumental in preventing an un-regulated
increase of enzyme activity beyond the normal level in lentil
plants subjected to 0 + Ca treatment. Excess Ca can induce
cytoxic damage to the plant cell (Jiang and Huang 2001), and
absence of any such symptoms and quite normal growth of
the present lentil plants exposed to 0 + Ca media indicated
some regulations on excess Ca influx.
This situation, however, had a significant twist once the
plants were subjected to Cd and Ca + Cd treatments. Studies
with Ca inhibitors and CaM antagonists revealed that Ca-
CaM is required for H 2O 2-induced up-regulation of the
expression and activities of antioxidant enzymes in the leaves
of maize plants (Hu et al. 2007). In the present study, a sig-
nificant increase in H 2 O 2 content over the control was
observed during Cd exposure, while both H2O2 and enzyme
activity levels were close to control values in Ca + Cd-treated
plants irrespective of Cd doses. Exogenous Ca/extracellular
CaM reportedly induced H2O2 production in guard cells of
Vicia faba (Chen et al. 2004), whereas H2O2 treatment can
induce the expression of the CaM gene (Hu et al. 2007) indi-
cating a cross-talk between CaM and ROS in response to
stresses or stimuli. In the present study, Ca-pri ming reduced
the accumulation of H2O2 from its toxic level in Cd-treated
lentil plants and stimulated antioxidant enzyme activities
especially in shoots possibly through enhanced production of
Ca/CaM complex to a certain magnitude. Obviously, normal
levels of H2O2 which were similar under 0 + Ca and Ca + Cd
treatments served as a critical control point for modulation of
antioxidant enzyme activities in shoots of lentil seedlings.
The H2O2 level crossed this limit during Cd exposure alone,
raised alarmingly to a level that became toxic to plants. The
results, thus, revealed dual roles of H2O2; it exacerbated
oxidative damage during Cd exposure in the absence of Ca
but was regulated to a level where it could serve as a signal
molecule for modulation of antioxidant enzyme activities in
the presence of Ca. The possibility of the involvement of Ca-
dependent signal transduction concomitant with the regula-
tion of antioxidant enzymes under stress has been reported in
cereals, legumes, and in other crops (Hu et al. 2007;
332 Calcium Alleviates the Cadmium-Induced Stress in Lentil
Rodríguez-Serrano et al. 2009; Yang and Poovaiah 2003).
Interestingly, Ca-priming in the Cd-treated present lentil
seedlings never stimulated antioxidant enzyme activities
beyond the control level, and also maintained H2O2 content
in its normal level. The necessity of this tight regulation can
be explained if we analyze simultaneous improvement of all
the five enzyme activities in Ca + Cd-treated plants. The
increase in SOD activity in shoots and hence, enhanced gen-
eration of H2O2 was counterbalanced by high APX and CAT
activity for efficient scavenging of H2O2 in presence of Ca.
High DHAR and GR activity, on the other hand, effectively
regenerated reduced AsA and GSH, respectively, from their
oxidized forms. The concerted rise of these enzyme activities
to normal level in Ca + Cd-treated lentil plants has thus facil-
itated favorable redox balance which may be linked to
changes at the levels of Ca, and in turn, to the induction of
antioxidant defense through activation of Ca-CaM kinase in
association with H2O2 sensing, as explained earlier (Mittler
2002; Rivetta et al. 1997). Ca reportedly reversed the down-
regulation of Cu/Zn-SOD isozymes in Cd-treated pea
(Rodríguez-Serrano et al. 2009) and up-regulated antioxidant
enzymes in alleviating Cd toxicity in other cases (Kim et al.
2002; Siddique et al. 2012; Suzuki 2005). It seems unlikely
in the present study that Ca directly regulated the activities of
antioxidant enzymes. Rather, exogenous Ca regulated H2O2
content at a critical level which then affected the internal Ca
status through controlled influx and concomitant formation
of the Ca-CaM complex in modulating the enzyme activities
under 0 + Ca, Cd alone, and Ca + Cd treatments. However,
all antioxidant enzymes and their isoforms may not be CaM-
binding proteins, and thus, further study is needed to deci-
pher the exact sequence of events orchestrated by Ca and
H2O2 in modulating antioxidant enzyme activities towards
the tolerance of lentil plants to Cd.
Along with modulating antioxidant enzyme activity
through balanced response of H2O2, Ca pre-treatment may
directly alleviate Cd-induced oxidative damage in lentil
seedlings by several other possible mechanisms. The inhibi-
tion of MDA content may be due to the role of Ca in control-
ling membrane structure and function by binding to phospho-
lipids that stabilizes lipid bilayers and thus provides structur-
al integrity to cellular membranes (Hirschi 2004). This was
evident from significant decline in percentage of membrane
electrolyte leakage in Ca + Cd-treated present lentil plants.
The ameliorative actions of exogenous Ca may also be
directly related to cytokinin action in promoting the Chl
biosynthesis (Lechowski and Bialczyk 1993), to the active
exclusion of toxic Cd by the formation and excretion of
Cd/Ca-containing crystals (Choi et al. 2001), ion transport,
apoptosis that coordinate, at least in part, the plants tolerance
to Cd (DalCorso et al. 2010; Yang and Poovaiah 2003).
Further study in this regard, however, is needed.
In conclusion, the current investigation for the first time
suggests that the external application of Ca prior to addition
of Cd in the nutrient media could alleviate oxidative stress of
lentil seedlings by improving its antioxidant enzyme activity
in Cd-stressed plants to a significant extent. Obviously, Ca
priming modulated the antioxidant defense response in favor
of plant growth under elevated Cd concentrations, and H2O2
level, presumably, played a pivotal role in this event in cross-
talk between Ca and ROS signaling. Further studies, howev-
er, are needed to ascertain this signaling event involved in
Ca-priming in Cd-stressed lentil plants.
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