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ISSN 10214437, Russian Journal of Plant Physiology, 2012, Vol. 59, No. 2, pp. 206–211. © Pleiades Publishing, Ltd., 2012.
Published in Fiziologiya Rastenii, 2012, Vol. 59, No. 2, pp. 232–237.
206
1
INTRODUCTION
Salinity is one of the major abiotic factors limiting
plants productivity. The total global area of salt
affected soils, including saline and sodic soils, is
831 million ha (6% of total land area of the world).
Apart from natural sodicity, 1500 million ha of land
farmed by dryland agriculture, 32 million ha (2%) are
affected by secondary salinity to a varying degree. In
India, more than 8.6 million ha of land is saltaffected,
which constitutes a major part of problem soil in India
[1]. Most crop plants are susceptible to salinity even
below 30 mM. Processes, such as seed germination,
seedling growth and vigor, vegetative growth, flower
ing, and fruit set, are adversely affected by high salt
concentrations, ultimately causing diminished eco
nomic yield and also quality of products [2]. It is
assumed that salt stress would cause an imbalance of
the cellular ions resulting in ion toxicity and osmotic
stress, thus affecting the plant growth, morphology,
and survival [2]. Three salt effects on plants can be dis
tinguished: (1) an osmotic effect, which makes water
1
This text was submitted by the authors in English.
uptake difficult [3]; (2) the toxic effect resulting from
the ability of sodium to compete with
K
+
for binding
sites essential for cell function [3]; and (3) a nutri
tional effect due to the limitation of nutrient uptake
and transport by antagonism between
Na
+
and essen
tial cations on the one hand and between
Cl
–
and
essential anions on the other hand [3].
In addition to ionic and osmotic components, salt
stress also leads to oxidative stress through an increase
in an amount of reactive oxygen species (ROS), such
as superoxide, hydrogen peroxide, and hydroxyl radi
cals [4]. It is suggested that excessive production of
ROS during salinity stress results from impaired elec
tron transport processes in chloroplasts and mito
chondria [5]. The ROS interact with a wide range of
molecules, causing pigment cooxidation, lipid per
oxidation, membrane destruction, protein denatur
ation, and DNA mutation [4]. Both D’Souza and
Devaraj [6] and Oueslati et al. [7] had reported that
H
2
O
2
and may play a leading role in the mecha
nism of salt injury in
Lablab purpureus
and
Mentha
pulegium
leaves. This evidence suggests that mem
branes are the primary sites of salt injury to cells and
organelles [8], because ROS can react with unsatur
ated fatty acids to cause peroxidation of essential
membrane lipids in the plasmalemma or intracellular
organelles, which finally leads to the leakage of cell
O2
•–
Physiological and Antioxidant Responses of Germinating
Cicer arietinum
Seeds to Salt Stress
1
S. Keshavkant, J. Padhan, S. Parkhey, and S. C. Naithani
Seed Biology Lab, School of Life Sciences, Pt. Ravishankar Shukla University, Raipur, 492 010 India;
fax: 917712262583; email: skeshavkant@gmail.com
Received August 3, 2010
Abstract
—
Cicer arietinum
(gram) is an important proteinrich pulse crop in Indian subcontinent, the Med
iterranean region, Ethiopia, and Mexico. We studied the effects of different salt concentrations on radicle
growth and different markers of oxidative stress, e.g., superoxide radical, MDA, protein carbonyls, as well as
antioxidant compounds. Physiological and biochemical parameters were assessed in the radicles of germinat
ing gram seeds after 1 and 7 days of treatments with 15, 30, 45, and 60 mM NaCl. The results showed that salt
exerted a stronger effect (17fold) on radicle length than on their dry weight (5fold). This growth decrease
was accompanied by an excessive (3fold) accumulation of ROS and resulting protein carbonyl and MDA
formation (3–6fold). As to the responses of antioxidant compounds to salinity of the growing medium, all
the enzymatic molecules (SOD, CAT, POX, and APX) showed significant (4–6fold) reductions in their
activities. Our results suggest that under salinity substantially higher amounts of oxidative stress markers
(superoxide, MDA, and protein carbonyls) in collaboration with suppression of the ROS detoxification sys
tem ultimately led to gram radicle growth inhibition and severe oxidative stress.
Keywords: Cicer arietinum
, antioxidant compounds, salt stress, oxidative stress, lipid peroxidation, superoxide
radical.
DOI:
10.1134/S1021443712010116
Abbreviations
: APX—ascorbate peroxidase; CAT—catalase;
DNP—2,4dinitrophenylhydrazine; PMSF—phenylmethylsul
fonyl fluoride; POX—guaiacol peroxidase; SOD—superoxide
dismutase; TBARS—thiobarbituric acidreactive substances.
RESEARCH
PAPERS
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 59 No. 2 2012
PHYSIOLOGICAL AND ANTIOXIDANT RESPONSES OF GERMINATING 207
contents and cell death [9]. MDA content, a product
of lipid peroxidation, is considered as an indicator of
oxidative damage [4].
Under stressful conditions, plants have evolved
complex mechanisms to struggle against these oxida
tive stresses by the synchronous action of various enzy
matic and nonenzymatic antioxidants. Of these,
superoxide dismutase (SOD), catalase (CAT), guaia
col peroxidase (POX), and ascorbate peroxidase
(APX) form the antioxidant enzymatic component
[10]. These antioxidants play a significant role in
detoxifying ROS [2]. SOD dismutates superoxide rad
icals to
H
2
O
2
, which is the initial reaction of ROS
detoxification and a key component of the ROSscav
enging system [10], whereas CAT, POX, and APX are
involved in converting
H
2
O
2
into water and oxygen.
Thus, antioxidants may provide for a strategy to
enhance plant salinity tolerance. There is enough evi
dence that alleviation of oxidative damage and
increased salinity tolerance are often correlated with
an efficient antioxidant defense system in plants [6].
Similarly, increased SOD, CAT, POX, and APX may
be correlated to salinity tolerance [5]. A number of
salttolerant transgenic plants overexpressing antioxi
dant compounds were reported [6].
Cicer arietinum
(gram) is an important pulse crop in
Indian subcontinent, the Mediterranean region, Ethi
opia, and Mexico. It is an excellent nitrogen fixer and
a proteinrich crop. Gram is largely grown on low
moisture soils where evaporation exceeds precipita
tion, resulting in salt accumulation on the soil surface
[11]. There are conflicting reports on the effects of
salinity on seed germination and growth of gram and
many other species. Lauter and Munns [11] classified
gram as a mild saltsensitive species, like other
legumes, and showed that gram seed germination is
relatively less affected by salinity than subsequent
seedling growth. Except for the general information
about gram mild tolerance to salt, there are no reports
indicating biochemical and physiological bases of its
response to salinity. Therefore, the objective of this
study was to evaluate the effects of salinity on antioxi
dant enzymes and other markers of abiotic stress in
germinating gram seeds.
MATERIALS AND METHODS
Germination and salinity treatment.
Gram (
Cicer
arietinum
) seeds were surfacesterilized with
1% sodium hypochlorite solution for 10–15 min,
thoroughly washed 4–5 times with distilled water, and
placed for germination in a plastic box (
30
×
15
×
5
cm)
on wet filter paper. These boxes were placed in darkness
at
30
−
33
°
C
until the radicle length reached 1 mm.
Distilled water was supplied to the germinating seeds
as and when necessary.
To study the effect of salinity on radicle growth, the
seeds having 1mm radicle were grown in 0 (control),
15, 30, 45, and 60 mM NaCl solutions. In each treat
ment 100 seeds were used. The seeds were harvested
after one and seven days of treatments to analyze the
change in the length and dry weight of radicles and
also for other biochemical investigations. All bio
chemical analysis were performed in five replicates
and repeated twice.
Determination of growth parameters.
Random
selection of ten replicates from each treatment was
done. The radicles were removed from the seeds and
gently blotted. The growth indices measured were
length and dry weight of the radicles.
Estimation of superoxide.
The five radicles were
homogenized in cold (
4
°
C
) sodium phosphate buffer
(0.2 M, pH 7.2) containing diethyldithiocarbamate
(
10
−
3
M) to inhibit SOD activity [12]. The homoge
nate was immediately centrifuged for 5 min at
8945
g
.
NaCl concentration, mM
90
75
60
45
30
15
0
1
2
(a)
Radicle length, mmRadicle dry weight, mg
12
9
6
3
0
604530150
1
2
(b)
604530150
Fig. 1.
Change in length (a) and dry weight (b) of gram
radicles after 1 and 7 days of growth in distilled water and
various concentrations of NaCl solutions.
(
1
) 1st day; (
2
) 7th days.
Measurements are means ± SD of 10 separate measure
ments.
208
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 59 No. 2 2012
KESHAVKANT
et al.
In the supernatant, superoxide anion was mea
sured by its capacity to reduce nitro blue tetrazolium
(
2.5
×
10
−
4
M). The absorbance of the end product was
measured at 540 nm. The formation was
expressed as mmol/radicle.
Lipid peroxidation
was measured as the concentra
tion of thiobarbituric acidreactive substances
(TBARS) equated with MDA [13] and expressed as
nmol/radicle.
Extraction of protein and enzymes.
The 50 radicles
were homogenized in Zivy’s buffer (0.03 M Tris base
(pH 8.5), 10 mM ascorbic acid, 1 mM EDTA,
5mMMgCl
2
, 1 mM DTT, 1 mM PMSF (dissolved in
isopropanol)) by using a cold mortar and pestle and a
pinch of sterilized silica. The homogenates were cen
trifuged at 14000
g
for 20 min at 4
°
C to get clear super
O2
•–
()
O2
•–
natant, which can be used as a source for estimation of
protein and enzymes.
Protein concentration
was determined by the
method of Bradford [14]. BSA was used as a standard,
and the content was expressed as
µ
g protein/radicle.
Protein carbonyl
contents were determined by the
reaction with 2,4dinitrophenylhydrazine (DNP) as
described by Levine et al. [15]. Protein (nearly 1 mg)
was incubated for 10 min with 10 mM DNP in 2 M
HCl. For each sample, a blank without DNP was run
in parallel. After precipitation with 10% TCA, the pel
let was washed three times with ethanol : ethyl acetate
(1 : 1, v/v). The carbonyl content was calculated from
the absorbance of the protein–2,4dinitrophenylhy
drazone derivative at 370 nm and expressed as mmol/g
protein.
Superoxide dismutase
(EC 1.15.1.1) activity was
determined by measuring the inhibition of pyrogallol
autooxidation at 420 nm and quantified by the method
of Marklund and Marklund [16]. The unit of SOD
activity was defined as the amount of enzyme that
inhibited the nitro blue tetrazolium photoreduction by
50%. SOD activity values is given in units/(mg protein
min).
Catalase
(EC 1.11.1.6) activity was assayed using
the method of Chance and Maehly [17], and activity
was expressed as
A
240
/(mg protein min).
Guaiacol peroxidase
(EC 1.11.1.7) activity was
assayed by the method of Chance and Maehly [17]
using the guaiacol test. The tetraguaiacol formed in
the reaction has a maximum absorption at 470 nm.
Thus, the reactions can be readily followed photomet
rically. The enzyme activity was expressed as
A
470
/(mg
protein min).
Ascorbate peroxidase
(EC 1.11.1.11) activity was
measured according to Nakano and Asada [18] by
monitoring the rate of ascorbate oxidation at 290 nm.
The activity of the enzyme was expressed as
A
290
/(mg
protein min).
Statistical analysis.
In general, mean values were
examined statistically by using the oneway analysis of
variance (ANOVA) at a significance level of
P
< 0.05
followed by the Turkey–Kramer multiple comparison
tests.
RESULTS AND DISCUSSION
Growth Analysis
All results showed that studied parameters were sig
nificantly affected by salt stress, which, like all other
abiotic stresses, slowed down radicle growth. It was
reported that the typical symptom of salt injury to the
plant is growth retardation due to the inhibition of cell
elongation [9]. When gram radicles were subjected to
various NaCl treatments, significant reductions (
P
<
0.001) in both length (17fold) and dry weight (5fold)
were observed (Figs. 1a, 1b), which confirms that radi
cle elongation was more seriously affected by salinity
NaCl concentration, mM
1
2
(a)
604530150
12
(b)
604530150
0.9
0.6
0.3
0
MDA content, nmol/radicle
160
120
80
40
0
Protein carbonyl level, mmol/g protein
Fig. 2.
MDA (a) and protein carbonyl (b) levels in radicles
of gram grown in distilled water and various concentra
tions of NaCl solutions for 1 and 7 days.
(
1
) 1st day; (
2
) 7th days.
Values are means ± SD of 5 separate measurements.
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 59 No. 2 2012
PHYSIOLOGICAL AND ANTIOXIDANT RESPONSES OF GERMINATING 209
stress than its biomass. Similar observations were also
noticed in sunflower [3], beans [6, 9], and mentha [7]
under salinity stress.
Superoxide, Lipid Peroxidation, and Protein
Carbonylation
Salt stress leads to oxidative stress, thus increasing
the likelihood of excessive ROS formation by impair
ing the electron transport chain in subcellular com
partments [4]. These ROS directly attack lipids and
proteins and cause lipid peroxidation as well as
destruction of lipids and protein moieties [7, 9]. They
generate changes in polyunsaturated fatty acids that
affect the structural and functional properties of cell
membranes, such as inactivation of membranebound
proteins and an increase in membrane permeability
[5]. In the present study, MDA and protein carbonyl
accumulation was increased with an increase in NaCl
concentration, indicating the higher rates of lipid perox
idation (6fold) as well as protein destruction (3fold)
(Figs. 2a, 2b) through salinityinduced overproduc
tion (3fold) of superoxides (Fig. 3). It means that
NaCl concentration was positively correlated with
superoxide, MDA, and carbonyl formation. Our
results are in agreement with observations reported by
Yasar et al. [9], Davenport et al. [3], and Ouslati et al.
[7] working on beans, sunflower, and mentha, respec
NaCl concentration, mM
604530150
20
16
12
8
4
0
12
Superoxide content, mmol/radicle
Fig. 3.
Content of superoxide radical in radicles of gram grown at various salt concentrations for 1 and 7 days.
(
1
) 1st day; (
2
) 7th days.
Values presented are means ± SD of 5 separate measurements.
9
6
3
0
Protein content,
µ
g/radicle
NaCl concentration, mM
604530150
2
1
Fig. 4.
Protein content in radicles of gram grown at various salt concentrations.
(
1
) 1st day; (
2
) 7th days.
Values are means ± SD of 5 separate measurements.
210
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 59 No. 2 2012
KESHAVKANT
et al.
tively, under salt stress, and the results showed that the
accumulation of superoxide, MDA, and protein car
bonyls was due to reduced antioxidant activities. Dav
enport et al. [3] concluded that the damage caused by
ROS to proteins had greater consequences for whole
cell inhibition than effects on other macromolecules.
These modified/carbonylated proteins can undergo a
change in their hydrophobicity, produce protein
aggregates, or give rise to the formation of peptide
fragments [19]. The authors also demonstrated that
these oxidatively modified proteins were selectively
used as an index of oxidative stress [19].
Soluble Protein
Salt stress produced a remarkable decrease in solu
ble proteins in the radicle extracts with increase in the
salinity level (Fig. 4). This drop was stronger (
P
< 0.01)
at 60 mM NaCl reaching to a 7fold decrease for
7 days of incubation. It was observed that salt stress
imposed significant reductions in the soluble protein
levels in mulberry, finger millet, and rice, and this
reduction was directly proportional to the salt concen
trations used [5, 20].
Antioxidant Compounds
In any biological system, the levels of ROS are reg
ulated by the rates of their generation, degradation,
and scavenging by antioxidant enzymes [21]. SOD is
the first defense enzyme, which converts superoxide to
H
2
O
2
that can be scavenged by different classes of per
oxidases, e.g., guaiacol and ascorbate peroxidase [10].
A close relation between antioxidant capacity and
NaCl tolerance was demonstrated for numerous plant
species such as
Oryza sativa
[20],
Phaseolus vulgaris
[9],
L. purpureus
[6],
M. pulegium
[7], etc. Moreover,
in recent studies it was reported varying responses of
plant antioxidants specific for species and tissues [5,
9]. In NaClstressed gram radicles, substantial reduc
tions (4–6fold) in the activities of SOD, CAT, POX,
and APX were noticed with increasing salinity, and, on
the other side, high activities were recorded in the
control radicles (Fig. 5). Salinity suppressed antioxi
dant enzyme activities may be due to prevention of
new compound synthesis or photoinactivation [22].
For these reasons, scavenging of these dangerous rad
icals was not done perfectly. Consequently, this radical
attacks to the vital biomolecules and damages mem
branes [5]. Candan and Tarhan [8] concluded that an
increase in antioxidant enzyme activities and a
decrease in oxidative damage were closely related to
12
(a)
(b)
(c)
(d)
SOD activity, unit/(min mg protein)
CAT activity,
A
240
/(min mg protein)POX activity,
A
470
/(min mg protein)APX activity,
A
290
/(min mg protein)
40
30
20
10
0
20
15
10
0
5
5
4
3
2
1
0
40
30
20
10
0
1
1
1
NaCl concentration, mM
604530150
2
2
2
Fig. 5.
Effec ts of vario us Na Cl co ncen trat ions on SO D (a) ,
CAT (b), POX (c), and APX (d) activities in radicles of
gram after 1 and 7 days of treatment.
(
1
) 1st day; (
2
) 7th days.
Values are means ± SD of 5 separate measurements.
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 59 No. 2 2012
PHYSIOLOGICAL AND ANTIOXIDANT RESPONSES OF GERMINATING 211
salt stress tolerance of any species. Thus, these decre
ments in antioxidant activities clearly spell out that the
failure of central protective mechanism is a major
cause of oxidative stress conditions in gram radicles
under salt stress.
In conclusion, the results show that salt stress is
accompanied by oxidative stress in germinating gram
seeds. The responses of seedlings to salinity include a
significant inhibition of the radicle length, more sub
stantial than a decrease in radicle biomass accumula
tion. Salt stress also imposes pronounced accumula
tion of superoxide, MDA, and protein carbonyls with
a concomitant loss in protein content and antioxidant
enzyme activities. Perhaps, this excessive ROS are the
foremost cause for destruction of biomolecules, like
lipids and proteins, in saltstressed gram radicles.
Another possible reason of this oxidative stress is the
failure of its ROS detoxification system (SOD, CAT,
POX, and APX), which is utmost required for scaveng
ing superoxide radicals.
ACKNOWLEDGMENTS
The authors thank to the Head of the School of Life
Sciences (Pt. Ravishankar Shukla University) Raipur
for providing the necessary facilities.
We also acknowledge the financial support given by
the University Grants Commission, New Delhi.
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