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Effect of sodium carbonate-induced
salinity–alkalinity on some key osmoprotectants,
protein profile, antioxidant enzymes, and lipid
peroxidation in two mulberry (Morus alba L.)
cultivars
ab
c
a
Parvaiz Ahmad , Munir Ozturk , Satyawati Sharma & Salih Gucel
a
d
Biochemistry Lab, Centre for Rural Development and Technology, IIT, New Delhi, India
b
Department of Botany, S. P. College, Srinagar, Jammu and Kashmir, India
c
Institute of Environmental Sciences, Near East University, Nicosia, North Cyprus
d
Department of Botany, Science Faculty, Ege University, Bornova, Izmir, Turkey
Accepted author version posted online: 22 Oct 2013.Published online: 13 Nov 2013.
To cite this article: Parvaiz Ahmad, Munir Ozturk, Satyawati Sharma & Salih Gucel (2014) Effect of sodium carbonateinduced salinity–alkalinity on some key osmoprotectants, protein profile, antioxidant enzymes, and lipid peroxidation in
two mulberry (Morus alba L.) cultivars, Journal of Plant Interactions, 9:1, 460-467, DOI: 10.1080/17429145.2013.855271
To link to this article: http://dx.doi.org/10.1080/17429145.2013.855271
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Journal of Plant Interactions, 2014
Vol. 9, No. 1, 460–467, http://dx.doi.org/10.1080/17429145.2013.855271
RESEARCH ARTICLE
Effect of sodium carbonate-induced salinity–alkalinity on some key osmoprotectants, protein
profile, antioxidant enzymes, and lipid peroxidation in two mulberry (Morus alba L.) cultivars
Parvaiz Ahmada,b*, Munir Ozturkc, Satyawati Sharmaa and Salih Guceld
a
Biochemistry Lab, Centre for Rural Development and Technology, IIT, New Delhi, India; bDepartment of Botany, S. P. College,
Srinagar, Jammu and Kashmir, India; cInstitute of Environmental Sciences, Near East University, Nicosia, North Cyprus;
d
Department of Botany, Science Faculty, Ege University, Bornova, Izmir, Turkey
Downloaded by [Near Eastern University], [Salih Gucel] at 01:51 07 November 2014
(Received 6 June 2013; final version received 10 October 2013)
The changes in accumulation of two potential osmoprotectants (proline and glycine betaine), lipid peroxidation
appraised as malondialdehyde (MDA) level, activities of key antioxidant enzymes such as superoxide dismutase (SOD:
EC 1.15.1.1), catalase (CAT: EC 1.11.1.6), peroxidase (POD: EC 1.11.1.7), and glutathione reductase (GR: EC 1.6.4.2),
and soluble protein profile in two cultivars of mulberry (S146 and Sujanpuri) differing in alkalinity (NaHCO3) tolerance
were investigated at 2-month intervals up to 6-month growth under stress conditions. Varying levels of salinity–
alkalinity developed in soil were 0, 30, 40, and 50 g of NaHCO3 kg−1 soil with pH 7.8, 9.1, 9.8, and 10.3, respectively.
Alkali stress led to a consistent accumulation of proline and glycine betaine in mulberry leaves with time. The activities
of leaf SOD, CAT, POD, and GR increased with increase in external salt concentration and pH. The increase in
antioxidant enzyme activities was higher in cv. S146 than cv. Sujanpuri, whereas rate of lipid peroxidation measured in
terms of MDA was higher in cv. Sujanpuri as compared to cv. S146. Protein profile revealed that some unknown
proteins of low molecular mass (10–32.5 kDa) were induced by NaHCO3 stress, but differently in two cultivars.
Keywords: alkalinity stress; mulberry; growth; proline; protein; antioxidants
Abbreviations: ROS, reactive oxygen species; GB, glycine betaine; MDA, malondialdehyde; SOD, superoxide
dismutase; CAT, catalase; POD, peroxidase; GR, glutathione reductase
1. Introduction
Salt affected soils have been categorized into three
different types such as saline, saline–alkali, and alkaline
soils depending on total concentration (electrical conductivity) of soluble salts they possess, pH of the soil
solution, and exchangeable sodium percentage of the soil
(Qadir et al. 2006; Rasool et al. 2012). Although NaCl is
the dominant salt in most salt affected soils, other salts
such as Na2SO4, MgCl2, KCl, NaHCO3 are also
frequently found in considerable amounts in such soils
(Bie et al. 2004). High levels of alkalinity in soil or
irrigation waters adversely affect seed germination and
crop growth and productivity (Wang et al. 2011; Lin
et al. 2012; Zhang et al. 2012). Much work has been
done on the salinity tolerance of various crops using
NaCl, whereas pH of the root-growing medium has been
maintained close to neutral. Alkalinity is associated with
the presence of sodium in soil such as Na2CO3 or
NaHCO3 which damages plants by sodium toxicity as
well as high pH (Chen et al. 2012). However, relatively
few studies have been undertaken on the effects of
alkaline soils on plant growth and productivity. The
investigations carried out on the effects of alkalinity
developed by NaHCO3 on two populations of Populus
cathayana (Lu et al. 2009) have revealed that increasing
pH of the saline growth medium causes more damage to
cell membranes. The alkalinity at the same time severely
upsets the mechanism of ion uptake in both populations.
*Corresponding author. Email: parvaizbot@yahoo.com
© 2013 Taylor & Francis
Not much work has been conducted on antioxidant
studies under alkalinity stress.
Mulberry is an economically important tree in the
global plant diversity. It is cultivated under both irrigated
and rain-fed conditions. Salt problems are generally
more pronounced in the areas of tropical and subtropical
regions (Agastian & Vivekananda 1997; Kumar et al.
1999; Lal et al. 2008; Jaleel et al. 2009; Vijayan et al.
2003, 2009), which hamper its fruit yield and leafy
biomass. Salinity tolerance of these trees has not been
investigated extensively at the physiological and biochemical levels. In view of this, the present study was
undertaken to examine the effects of saline–alkaline
medium created by NaHCO3 on two potential osmoprotectants such as proline and glycine betaine, some key
antioxidant enzymes, extent of salt-induced lipid peroxidation, and changes in soluble proteins in two mulberry
cultivars differing in tolerance to NaHCO3.
Soil is affected with different salts such as NaCl,
Na2SO4, NaHCO3, Na2CO3, etc. The impact of salt
stress generally contains osmotic and ionic stress effects.
However, alkali stress added the influence of high pH
which inhibits ion uptake and disrupts ionic balance of
plant cells. Alkalinity stress is more harmful than salinity
stress and is reported by many researchers (Guo et al.
2010; Wang et al. 2011; Javid et al. 2012; Lin et al.
2012). Maximum papers are published on salinity stress
on plants and the research on alkalinity stress is very
less. The biochemical and antioxidative responses in
Journal of Plant Interactions
plants during alkalinity stress is very less. Alkalinity
stress being the most crucial part which plants face in the
era of climate change, so need to be studied extensively
at par with salinity stress.
2. Materials and methods
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2.1. Plant material and NaHCO3 treatment
Nodal cuttings of 15 cm in length comprising at least 3–4
active buds were excised from field grown 2-year old
S146 and Sujanpuri cultivars of Morus alba L. These
were spontaneously planted in polythene bags containing
soil and sand (3:1 ratio). Before planting, the required
pH in the soil was attained by applying 0, 30, 40, and
50 g of NaHCO3 kg−1 soil. The bags were left under
natural climatic conditions with a mean photoperiod of
12–13 h and a temperature of 28 ± 4°C. These were
watered daily and pH of the soil extract was monitored,
and adjusted at regular intervals. Experiment was conducted using expanded third leaf from the top of the
plant. The data on growth and biomass yield, shoot
length, and fresh and dry weights were recorded after 2,
4, and 6 months of the application of NaHCO3 treatment.
Proline concentration was determined using the
method of Bates et al. (1973). Fresh leaves (300 mg)
were homogenized in 10 ml of 3% aqueous sulfosalicylic
acid. The homogenate was centrifuged at 9000 × g for
15 min. A 2-ml aliquot of the supernatant was mixed with
an equal volume of acetic acid and acidic ninhydrin, and
then incubated for 1 h at 100°C, and reaction was
terminated in an ice bath before extraction with 4 ml of
toluene. The extract was vortexed for 20 s, the chromatophore-containing toluene was aspirated from the aqueous phase, and absorbance was determined
photometrically at 520 nm (Beckman 640 D, USA) using
toluene for a blank.
Glycine betaine was measured according to Grieve
and Grattan (1983). Dried and finely ground plant
material (300 mg) was mechanically shaken with 20 ml
deionized water for 24 h at 25°C. The samples were then
filtered and the filtrates were diluted (1:1) with 2 N
H2SO4. Aliquots (0.5 ml) were taken into centrifuge
tubes and cooled in ice bath for 1 h. Cold KI-I2 reagent
(0.20 ml) was added and then reactants were gently
stirred. The tubes were stored at 4°C for 16 h and then
centrifuged at 10,000 rpm for 15 min at 0°C. The
supernatant was carefully aspirated with a fine tipped
glass tube. The periodide crystals were dissolved in 9.0
ml of 1,2-dichloroethane and mixed vigorously. After
2 h, the absorbance was measured at 365 nm using a
spectrophotometer. A reference standard of glycine
betaine (50–200 mg ml−1) was prepared in 1 N H2SO4.
Lipid peroxidation was determined by measuring
malondialdehyde (MDA) formation according to Madhava Rao and Sresty (2000). Fresh leaves of 300 mg
were homogenized with 2.5 ml of 0.1% trichloroacetic
acid (TCA). The homogenate was centrifuged for 10 min
at 10,000 rpm. For every 1 ml of the aliquot, 4 ml of
20% TCA containing 0.5% thiobarbituric acid was
461
added. The mixture was heated at 95°C for 30 min and
then cooled quickly on an ice bath. Afterwards, the
mixture was centrifuged for 15 min at 10,000 rpm and
the absorbance of the supernatant was measured at 532
nm. Measurements were corrected for unspecific turbidity by subtracting the absorbance at 600 nm. The
concentration of MDA was calculated using an extinction coefficient of 155 mM−1 cm−1.
The enzyme extraction was performed with fresh leaf
material (0.3 g) homogenized in 5.0 ml of extraction
mixture (100 mM potassium phosphate buffer of pH 7.0
and 0.1 mM ethylenediaminetetraacetic acid [EDTA]).
The process was carried out under cold conditions (4°C).
After filtering the sample, it was centrifuged at 10,000 × g
for 20 min and the supernatant so obtained was used for
enzyme.
Superoxide dismutase (SOD) activity was estimated
following the method of Dhindsa et al. (1981). The assay
mixture, consisting of 1.5 ml reaction buffer, 0.2 ml of
methionine, 0.1 ml enzyme extract with equal amount of
1 M Na2CO3, 2.25 mM nitroblue tetrazolium (NBT)
solution, 3 mM EDTA, 60 mM riboflavin, and 1.0 ml of
double distilled water, was incubated under a 15 W
inflorescent lamp for 10 min at 25/28°C. Blank A,
containing the same reaction mixture, was placed in the
dark. Blank B containing the same reaction mixture
except for the enzyme extract was placed in light along
with the sample. The reaction was terminated by switching off the light, and the tubes were covered with a blank
cloth. Absorbance at 560 nm of each sample along with
blank B was read against blank A and the difference in
percentage of color reduction between blank B and the
sample was calculated. Fifty percent of color reduction
was considered one unit of enzyme activity, and activity
was expressed as EU mg−1 protein.
Catalase (CAT) activity in leaves was estimated by
the method given by Aebi (1984). CAT activity was
determined by examining the disappearance of H2O2 by
measuring a dropping off in absorbance at 240 nm. The
reaction was carried out in a final volume of 2 ml of
reaction mixture containing reaction buffer with 0.1 ml
of 3 mM EDTA, 0.1 ml of enzyme extract, and 0.1 ml of
3 mM H2O2. The reaction was allowed to run for 10
min. Activity was calculated using extinction coefficient
(ε) 0.036/mM/cm and expressed in EU/mg protein. One
unit of enzyme determined the amount necessary to
decompose 1 µmol of H2O2 per min at 25°C.
For peroxidase (POD) activity, method given by Kar
and Mishra (1976) was used. Five milliliters of the assay
mixture for the POD activity comprised 125 µmoles
of phosphate buffer, pH 6.8, 50 µmoles of pyrogallol,
50 µmoles of H2O2, and 1 ml of the 20 times diluted
enzyme extract. This was incubated for 5 min at 25°C
after which the reaction was stopped by adding 0.5 ml
of 5% (v/v) H2SO4. The amount of purpurogallin formed
was determined by taking the absorbance at 420 nm.
Change in enzyme units mg−1 protein denoted the
enzyme activity.
Glutathione reductase (GR) activity was assayed as
per the method of Foster and Hess (1980). The reaction
462
P. Ahmad et al.
A
T1
T2
T3
200
T4
B
T1
T2
T3
T4
60
160
30
20
10
120
80
40
2nd
4th
6th
2nd
4th
S146
2nd
6th
T1
T2
T3
T4
2nd
4th
6th
40
30
20
10
0
0
0
C
50
Dry weight (gm)
40
Fresh weight (gm)
Shoot length (cm)
50
4th
6th
2nd
S146
Sujanpuri
4th
2nd
6th
4th
Number of months
Number of months
6th
S146
Sujanpuri
Sujanpuri
Number of months
software program (AgRes 3.01). Critical difference was
worked out at 5% level of probability.
mixture consists of enzyme extract, 100 mM potassium
phosphate buffer (pH 7.0) containing 1.0 mM EDTA,
150 mM nicotinamide adenine dinucleotide phosphate
(NADPH), and 500 mM oxidized glutathione. The
enzyme activity was measured at 340 nm. Activity was
calculated using the extinction coefficient for NADPH of
6.22 mM−1 cm−1 and expressed as mmol NADPH
oxidized mg−1 protein.
To determine the molecular weight of the protein
subsets, SDS-PAGE under denaturing conditions was
used as described by Laemmli (1970) using 12.5%
acrylamide concentration. Each sample (protein extract)
was subjected to 95°C for 2 min in 10 mM Tris–HCl
(pH 7) that contained 2% (w/v) SDS, 2% β-mercaptoethanol, and 5% (w/v) glycerol, before electrophoresis.
An aliquot (40 µl) of the prepared sample was loaded per
well after mixing with loading buffer bromophenol blue
and glycerine. The gel was removed from the apparatus,
immersed in staining solution containing coomassie
brilliant blue-R250. Broad range molecular weight
marker was used to estimate the molecular weights of
the sample proteins.
3. Results
3.1. Growth and biomass yield
The results related to the effect of NaHCO3 on growth
and biomass yield are depicted in Figure 1A–C. The
shoot length decreases in both cultivars at all stress
levels. The concentration of 50 g NaHCO3 kg−1 soil
decreases the shoot length to 53.4% in S146 and 69.7%
in Sujanpuri as compared to the control after 6 months
(Figure 1A). The fresh weight also decreased by 54.6%
in S146 and 62.6% in Sujanpuri as compared to the
control after 6 months. A decrease in dry weight in both
cultivars was observed as 55.2% and 67% in S146 and
Sujanpuri, respectively, as compared to the control after
6 months (Figure 1B and C).
3.2. Free proline content
The results related to the effect of NaHCO3 on proline
content are presented in Figure 2A. The proline content
increased to 82% in S146 and 77% in Sujanpuri at 50 g
NaHCO3 kg−1 soil after 2 months. Further increase of
87% and 79.7% in proline content was observed in S146
2.2. Statistical analysis of data
Data for each growth or biochemical parameter were
subjected to analysis of variance using Agris Statistical
A
T1
T2
T3
B
T4
160
120
80
40
0
2nd
4th
6th
S146
2nd
4th
6th
Sujanpuri
Number of months
T1
T2
T3
T4
35
30
25
20
15
10
5
0
16
MDA (µ mol/g f. wt)
200
GlB (µmol/g f. wt)
Proline (µmol/g f.wt.)
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Figure 1. Effect of different concentrations of NaHCO3 (g/kg soil) on shoot length (A), shoot fresh weight (B), and shoot dry weight
(C) of two cultivars of mulberry (means SE; n = 5). All treatments are significantly different at P ≤ 0.05 from control (Duncan’s
multiple range test [DMRT]).
Note: T1 = 0.0, pH 7.8; T2 = 30 g, pH 9.1; T3 = 40 g, pH 9.8; and T4 = 50 g, pH 10.3.
2nd
4th
6th
S146
2nd
4th
Sujanpuri
Number of months
6th
C
T1
T2
T3
T4
12
8
4
0
2nd
4th
6th
S146
2nd
4th
6th
Sujanpuri
Number of months
Figure 2. Effect of different concentrations of NaHCO3 on proline (µmol/g f.wt.) (A), glycine betaine (µmol/g f.wt.) (B), and
malondialdehyde (MDA) (C) contents in two cultivars of Morus alba L. at different time intervals (means SE; n = 5). All treatments
are significantly different at P ≤ 0.05 from control (DMRT).
Note: T1 = 0.0, pH 7.8; T2 = 30 g, pH 9.1; T3 = 40 g, pH 9.8; and T4 = 50 g, pH 10.3.
600
A
T1
T2
T3
T4
500
400
300
200
100
0
2nd
4th
6th
2nd
S146
4th
CAT (Unit mg–1 protein)
SOD (Unit mg–1 protein)
Journal of Plant Interactions
463
B
500
T1
100
0
6th
2nd
4th
Sujanpuri
6th
T2
T3
2nd
S146
4th
6th
Sujanpuri
Number of months
T4
80
60
40
20
GR (Unit mg–1 protein)
POD (Unit mg–1 protein)
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T1
T4
200
16
C
T3
300
Number of months
100
T2
400
D
T1
T2
T3
T4
14
12
10
8
6
4
2
0
0
2nd
4th
6th
S146
2nd
4th
6th
Sujanpuri
Number of months
2nd
4th
6th
S146
2nd
4th
6th
Sujanpuri
Number of months
Figure 3. Effect of different concentrations of NaHCO3 (g/kg soil) on superoxide dismutase (SOD) (A), peroxidase (POD)
(B), catalase (CAT) (C), and glutathione reductase (GR) (D) activities of two cultivars of mulberry (means SE; n = 5). All treatments
are significantly different at P ≤ 0.05 from control (DMRT).
Note: T1 = 0.0, pH 7.8; T2 = 30 g, pH 9.1; T3 = 40 g, pH 9.8; and T4 = 50 g, pH 10.3.
and Sujanpuri, respectively, at 50 g NaHCO3 kg−1 soil
after 6 months.
Sujanpuri after 2 months of application of NaHCO3
stress. However after 6 months of treatment, S146
showed an increase of 83.3% and Sujanpuri showed
69% as compared to control.
3.3. Glycine betaine
Glycine betaine content increased significantly in the salt
stressed leaves of both cultivars (Figure 2B). However,
the magnitude of increase was greater in the leaves of cv.
S146 than cv. Sujanpuri. The glycine betaine increased
by 59.5% in the stressed leaves of S146 and 32.9% in
3.4. Lipid peroxidation
Salt stress caused a significant increase in MDA content
in both the cultivars at all stress levels (Figure 2C). The
degree of accumulation was 29.3%, 40.6%, and 48.9%
Figure 4. Effect of different concentrations of NaHCO3 on protein profile of two cultivars of mulberry in vivo after (A) 2nd month
and (B) 6th month.
Note: M = marker, A = control, B = 30 g, C = 40 g, D = 50 g NaHCO3, A/ = control, B/ = 30 g, C/ = 40 g, and D/ = 50 g NaHCO3.
464
P. Ahmad et al.
in S146 after 2, 4, and 6 months, respectively, at 50 g
NaHCO3 kg−1 soil. However, Sujanpuri showed 49.1%
after 2 months, 51.8% after 4 months, and 69.8% after 6
months at 50 g NaHCO3 kg−1 soil.
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3.5. SOD activity
The activity of SOD increased in both mulberry cultivars
at all concentrations of NaHCO3 and the results are
presented in Figure 3A. After 2 months of NaHCO3
stress, the increase of SOD activity was 43.4% and
41.8% in S146 and Sujanpuri, respectively, at 50 g
NaHCO3 kg−1 soil. Further increase of 62.1% in S146
and 61.1% in Sujanpuri was observed after 6 months at
the same NaHCO3 level.
3.6. CAT activity
The results regarding effect of NaHCO3 on CAT activity
are presented in Figure 3B. The increase in CAT activity
was 51% in S146 and 49.9% in Sujanpuri after 2 months
of treatment with 50 g NaHCO3 kg−1 soil, and it further
increased to 67.3% and 63.4%, in S146 and Sujanpuri
cultivars, respectively, after 6 months. A significant
increase in CAT activity was observed only after 6
months and was concentration- and time-dependent.
3.7. POD activity
The results related to the effect of NaHCO3 on POD
activity are presented in Figure 3C. The POD activity
increased in both cultivars but the maximum increase was
observed in S146 than Sujanpuri. The POD activity
increased in the order of 54.7% < 66.5% < 72% in S146
and 52.5% < 62.4% < 69.2% in Sujanpuri after 2, 4, and 6
months of stress, respectively, at 50 g NaHCO3 kg−1 soil.
3.8. GR activity
The GR activity increased in both cultivars on exposure
to NaHCO3 and the results are depicted in Figure 3D.
After 6 months of treatment with 50 g NaHCO3 kg−1
soil, S146 cultivar showed an increase of 46.6% and
Sujanpuri showed 42.9% as compared to control.
3.9. Expression of proteins of different molecular mass
Highest NaHCO3 treatment for 2 months caused the
expression of new low molecular mass proteins (21.0,
22.5, and 25 kDa) in cv. S146 (Figure 4A, lane D) after 2
months, whereas the same proteins along with two other
protein molecules of 10 and 32.5 kDa appeared in cv.
Sujanpuri (Figure 4A, lane D/). However, after 6 months
of NaHCO3 treatment, the expression of protein of 22.5
kDa was masked in both cultivars and new protein
molecules of 24 and 57 kDa appeared in both cultivars
particularly at the treatment of 50 g NaHCO3 kg−1 soil
(Figure 4B, lanes D and D/).
4. Discussion
Alkalinity is known to suppress growth and yield of
most plants, but its effects due to high pH have been
reported to be more severe than those of salt stress (Lu
et al. 2009; Ahmad & Sharma 2010). In our studies, we
found out that sodium carbonate-induced alkalinity
caused a marked inhibition in the growth of two
mulberry cultivars appraised as shoot fresh and dry
weights and shoot length, but this reduction in growth
was more pronounced in cv. Sujanpuri than in cv. S146.
Such a NaHCO3-induced decrease in growth has earlier
been reported in two populations of a tree species,
P. cathayana (Lu et al. 2009). The growth inhibition
caused by salt stress has been reported in Triticum
aestivum (Guo et al. 2010), Leymus chinesis (Sun &
Hong et al. 2011), Medicago ruthenica (Yang et al.
2011), Brassica juncea (Javid et al. 2012), and variety of
tree species e.g. Salvadora persica (Ramoliya et al.
2004), Olea europea (Chartzoulakis et al. 2002), and
Simmondsia chinesis (Roussos et al. 2006). However,
growth inhibition in a plant due to salt or alkali stress
could occur due to regulation of a multitude of physiological and biochemical processes such as water relations
(Munns 2002; Meloni & Martinez 2009; Ahmad et al.
2012b), ion transport and accumulation (Hasegawa et al.
2000), photosynthesis (Dubey 2005; Ashraf et al. 2008),
accumulation of osmoprotectants (Ashraf & Foolad
2007; Koyro et al. 2012; Katare et al. 2012), synthesis
of antioxidant enzymatic and nonenzymatic molecules
(Mittler 2002; Ahmad et al. 2010; Patade et al. 2011;
Bano et al. 2013; Kaya et al. 2013; Rasool et al. 2013),
synthesis of plant growth regulators (Ashraf et al. 2010),
mineral nutrient metabolism (Marschner 1995), and
many others.
One of the most important biochemical responses of
plants to abiotic stresses is overproduction of different
types of compatible solutes such as proline, glycine
betaine, sugars, soluble proteins, amino acids, etc.
(Ahmad et al. 2007; Koyro et al. 2012). Of all
compatible solutes, proline and glycine betaine occur
widely in higher plants and accumulate in considerable
amounts in salt stressed plants (Ahmad et al. 2006;
Koyro et al. 2012; Ahmad et al. 2012b). In this study,
both proline and GB increased markedly with increase in
external NaHCO3 level and growth period. Relatively
NaHCO3 tolerant cv. S146 was superior to cv. Sujanpuri
in accumulating both osmoprotectants under alkaline
regimes. This pattern of accumulation of the two
osmoprotectants clearly shows that they could be used
as potential indicators of alkalinity tolerance in mulberry
as has earlier been reported in broad bean (Azooz et al.
2011), mustard (Ahmad et al. 2012a, 2012b), and
chickpea (Rasool et al. 2013).
The membrane damage is reported to be a primary
effect of salt stress on most plant species out of the
variety of salt-induced adverse effects (Ashraf et al.
2008; Ahmad et al. 2012a, 2012b). Most common way
of appraising membrane damage is the measurement of
amount of MDA, which accumulates in the cells as a
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Journal of Plant Interactions
result of lipid peroxidation (Mandhania et al. 2006;
Ahmad et al. 2012a, 2012b). It has been reported to be
one of the important selection criteria of salt tolerance in
most plants as salt sensitive plants generally accumulate
higher amounts of MDA than do salt tolerant ones
(Mandhania et al. 2006; Ahmad et al. 2012a, 2012b). In
the present study, MDA content increased in both
varieties with time as well as with increase in external
NaHCO3 level. The varietal difference was also quite
evident as S146 was relatively tolerant cultivar, accumulated markedly lower amounts of MDA as compared to
cv. Sujanpuri. Similar findings have been reported in two
diverse populations of P. cathayana (Lu et al. 2009).
Plants tend to counteract reactive oxygen species
produced by stress (Ahmad et al. 2010, 2011; Bano et al.
2013; Kaya et al. 2013). They synthesize both enzymatic
and nonenzymatic antioxidants in high amounts for this
purpose (Ahmad et al. 2010, 2011). Most important
enzymatic antioxidants are SOD, CAT, PODs, and GR.
In this study, the activities of all four enzymes increased
consistently with increase in NaHCO3 concentration of
the growth medium as well as with increase in growth
period. However, the activities were higher in cv. S146
than cv. Sujanpuri under alkaline regimes. A negative
association between the activities of these enzymes and
MDA content is evident from the results presented here,
which clearly reveals that the tolerant cultivar produces
excessive amounts of antioxidant enzymes to counteract
a variety of oxidants generated by salt stress, which may
have played an effective role in maintaining the stability
of membranes. This is manifested from the lower
accumulation of MDA in NaHCO3 tolerant cv. S146
than salt sensitive cv. Sujanpuri. Results showing
NaHCO3-induced enhanced activities of different antioxidant enzymes as observed in two mulberry cultivars
are in parallelity with those reported earlier in the same
species under salt stress (Sudhakar et al. 2001; Harinasut
et al. 2003; Ahmad et al. 2012b) and in P. cathayana
under NaHCO3 stress (Lu et al. 2009).
Salt stress also causes considerable changes in the
expression of soluble proteins or de novo synthesis of
proteins of different molecular mass (Thomas & Bohnert
1993). In the present investigation, de novo synthesis of
proteins of low molecular mass (21.0, 22.5, and 25.0
kDa) took place in both mulberry cultivars after 2
months of growth at the highest alkaline regime (50 g
NaHCO3 kg−1 soil). However, with prolonged exposure
(for 6 months) of cultivars, two new protein molecules of
24 and 57 kDa appeared in addition to the existing
proteins. An osmotin-like protein (MW= 24–26 kDa) in
salt stressed Mesembryanthemum crystallinum increased
relative to that in nonstressed plants (Thomas & Bohnert
1993). Changes in the gene activation, transcription, and
translation (protein synthesis) often occurs during the
acclimatization process under stressful environments and
thus are thought to be involved in the induction of
tolerance to salts (Moran et al. 2002).
465
5. Conclusion
Overall, tolerance of mulberry plants to NaHCO3 was
found to be associated with enhanced accumulation/
synthesis of the key osmoprotectants, proline and
glycine betaine, as well as upregulation of antioxidant
enzymes such as SOD, CAT, POD, and GR. These
biomolecules could act as potential biochemical selection criteria for tolerance in mulberry to NaHCO3.
Acknowledgments
This paper is part of PhD work of Dr Parvaiz Ahmad. S
Sharma was the PhD guide of Dr Parvaiz Ahmad. Prof. S
Gucel, cross checked the results which Dr Parvaiz got during
the work on this paper and helped him a lot in writing this
paper. Prof. Munir Ozturk, drafted the experimental design,
which cultivars we should use and also helped Dr Parvaiz in
statistical analysis of this paper. Authors are thankful to CSIRNew Delhi, India for providing financial assistance to PA.
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