Modulation of various physio-chemical changes in Vigna unguiculata l. var. KBc iV subjected to nacl and Peg stress

Abstract

Abiotic stresses are the unavoidable great threat for crop production. Crop health can be predicted on the basis of various parameters, which includes the accumulation of photosynthetic pigments, metabolites, reactive oxygen species (ROS) and antioxidants etc. The present study was carried out to compare the tolerance mechanisms operated in V. unguiculata var. KBC-IV subjected to NaCl and PEG stresses and assessed through morphological, physiological and biochemical changes. For this particular study, V. unguiculata was subjected to different concentrations of NaCl and PEG for 8 d. Based on this preliminary study, NaCl (75mM) and PEG (15%) was fixed as the concentration, which had the potential to impart 50% growth retardation. PEG treated plants had decreased photosynthetic pigments, protein, proline, total soluble sugar, and membrane stability index relative to control, whereas carotenoid, MDA, superoxide and total free amino acids content increased significantly, indicating that cell homeostasis was severely hampered under PEG stress. When compared to PEG (15mM) stress, exposure of NaCl (75mM) led to increased accumulation of plant osmolytes, chlorophyll contents and antioxidative enzymes activities. This coordinated response ensured the maintenance of steady state level of ROS in seedlings so as to cope up with NaCl stress in a better way than PEG stress.

Full text

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Modulation of various physio-chemical changes
in Vigna unguiculata L. var. KBC IV subjected
to NaCl and PEG stress
Riya Johnson and Jos T Puthur*
Plant Physiology and Biochemistry Division, Department of Botany,
University of Calicut, C.U. Campus P.O., Kerala-673635, India
Email: jtputhur@yahoo.com
Abstract
Abiotic stresses are the unavoidable great threat for crop production. Crop health can
be predicted on the basis of various parameters, which includes the accumulation of
photosynthetic pigments, metabolites, reactive oxygen species (ROS) and antioxidants
etc. The present study was carried out to compare the tolerance mechanisms operated
in V. unguiculata var. KBC-IV subjected to NaCl and PEG stresses and assessed
through morphological, physiological and biochemical changes. For this particular
study, V. unguiculata was subjected to different concentrations of NaCl and PEG
for 8 d. Based on this preliminary study, NaCl (75mM) and PEG (15%) was fixed as
the concentration, which had the potential to impart 50% growth retardation. PEG
treated plants had decreased photosynthetic pigments, protein, proline, total soluble
sugar, and membrane stability index relative to control, whereas carotenoid, MDA,
superoxide and total free amino acids content increased significantly, indicating
that cell homeostasis was severely hampered under PEG stress. When compared
to PEG (15mM) stress, exposure of NaCl (75mM) led to increased accumulation
of plant osmolytes, chlorophyll contents and antioxidative enzymes activities. This
coordinated response ensured the maintenance of steady state level of ROS in
seedlings so as to cope up with NaCl stress in a better way than PEG stress.
Key words: antioxidants, PEG, ROS, Vigna unguiculata.
Introduction
Global climate change and increasing human population is a big challenge to the global
food production systems (Rosenzweig and Parry, 1994). Arable land is limited, so
increased productivity has to come from existing land, which necessitates minimizing
losses resulting from abiotic and biotic stresses along with efficient harvesting and

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storage techniques. As an outcome of global climate change, environmental stress
factors might induce several damages to crop plants in the coming decades. Abiotic stress
is defined as environmental conditions that reduce growth and yield of plants below
optimum levels. Salinity stress is a severe factor limiting agricultural crop production
in arid and semi-arid regions. Furthermore, increasing salinity is threatening the soil
resources. It has been reported that due to salt stress up to 20% of the irrigated land have
been affected (Abogadallah, 2010). Drought stress is also another abiotic stress limiting
crop growth and productivity in many regions of the world, the loss of which is more
than any other single environmental factor (Farooq et al. 2009a, b). Polyethylene glycol
(PEG) compounds have been used to simulate water stress effects in plants.
The response of plants towards stress differs significantly at various organizational
levels, depending upon intensity and duration of stress as well as plant species and its
stage of development. The genotypic response to osmotic stress have been identified
to influence a range of morphological and physiological characteristics, including
root development, stomatal activity, osmotic adjustment, abscisic acid and proline
levels in cowpea. Osmotic stress results in the increased generation of reactive
oxygen species (ROS) (Munne-Bosch., 2005). Osmotic stress inhibits or slows down
photosynthetic carbon fixation mainly through limiting the entry of CO2 into the leaf
or directly inhibiting metabolism (Smirnoff, 1993; Loggini et al., 1999; Apel and Hirt,
2004). To counteract the toxicity of ROS, plant cells have developed an antioxidative
system, consisting of low-molecular weight antioxidants like ascorbate, tocopherol,
glutathione, and carotenoids, as well as protective enzymes (Vinocur et al., 2005).
Both salinity and drought stress have profound effects on plant physiology. Each of
these stresses induces stomatal closure; decrease chlorophyll contents and reduce
photosynthesis (Nemeth et al., 2002; Ma et al., 2012). It also disrupts cell membrane
function and elicits lipid peroxidation, protein degradation, and disturbs redox
homeostasis by forming reactive oxygen species (ROS) (Mühling and Läuchli, 2003).
Cowpea (Vigna ungiculata L.) belongs to family fabaceae, also known as black eye pea,
is an important legume grown as cash crop throughout the world. Cow pea (Vigna
ungiculata L.) is the world’s most important legume crop, plays huge role in food chain
and has immense importance because of its high protein content, and hence referred to
as “poor man’s meat”(Diouf and Hilu, 2005). Cowpea is rich in protein and is normally
cultivated for green pods and dry beans (Abeer et al., 2015). The legumes are very
important both ecologically and agriculturally because of their ability to fix nitrogen
in the root nodules in a symbiotic interaction with soil rhizobia. Legumes have a vital
role in the world cropping pattern and form a major constituent of food and both biotic
and abiotic stress factors significantly affect yield and productivity of leguminous crops
worldwide. It is well adapted to different environmental conditions and could be used

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as an alternative crop for salt affected soils (Serraj and Sinclair, 2002). Cowpea seeds
were subjected to salt (75 mM NaCl) and iso-osmotic level of PEG (15% - which would
impose only osmotic stress) and, a comparative analyses on changes in the growth,
accumulation of osmolytes, and activities of antioxidant enzymes were carried out.
2. Materials and methods
2.1 Plant material and growth condition
The study was carried out in Vigna unguiculata var. KBC-IV. Seeds were collected
from Regional Agricultural Research Station (RARS), Pattambi, Kerala, India.
Seeds were surface sterilized with 0.1% HgCl2 solution for 5 min and further seeds
were washed thoroughly with distilled water. Further, these seeds were subjected
to NaCl (75 mM) and PEG (15%) stress. Seedlings were raised in culture bottles (11
x 22 cm) containing absorbent cotton soaked with double distilled water (control),
NaCl (75 mM) and PEG (15%) incubated in a plant growth chamber under controlled
conditions of temperature (24±20C), light intensity (300 μmolm-2s-1) and relative
humidity (55±5%) with a 14/10 h photoperiod. The growth and biochemical traits of
all the seedlings were recorded on 8 d after germination. For each experiment, there
were three treatments: C (Control), NaCl stress (75 mM) and PEG (15%).
2.2 Photosynthetic pigments
Chlorophyll and carotenoid content of the seedlings was estimated by the method of
Arnon(1949).
2.3 Estimation of reactive oxygen species and membrane stability index
The malondialdehyde content (MDA) estimation was done according to the method
of Heath and Packer (1968). Membrane stability index was measured according to
Sairam et al., (1997).
2.4 Determination of metabolites and nonenzymatic antioxidants contents
The proline content in seedlings was analysed as per the method of Bates et al. (1973)
using 3% sulfosalicylic acid as the extraction medium; a standard curve was prepared
with L-proline. Total soluble sugar content was extracted with 80% ethanol from
fresh cowpea seedlings and measured according to the protocol of Dubois et al. (1956);
the standard curve was prepared with D-glucose. Total free amino acids content in
seedlings was analysed as per the method of Moore and Stein (1948) using 80% ethanol
as extraction medium; a standard curve was prepared with leucine. Soluble protein
content was estimated according to Bradford (1976). The total phenolic content of
seedlings was estimated by the method of Folin and Denis (1915) and the standard
curve was prepared with catechol.

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2.5 Determination of enzymatic antioxidants
Crude enzyme extract was prepared from fresh seedlings by the protocol of Yin et al.
(2009). Protein estimation in the enzyme extract was done as described in the method
of Bradford (1976); BSA was used as the standard. Ascorbate peroxidase (APX, EC
1.11.1.11) activity was measured as described by Nakano and Asada (1981) and one
unit of APX was defined as mmoles of ascorbate oxidised per minute per mg protein.
Superoxide dismutase (SOD, EC 1.15.1.1) activity was assayed by monitoring its ability
to inhibit the photochemical reduction of nitrobluetetrazolium(NBT) according to
Giannopolitis and Ries (1977). The activity of catalase (CAT, EC 1.11.1.6) in the fresh
samples was determined by following the method of Kar and Mishra (1976). It was
measured by analysing the decomposition of H2O2 and one unit of the enzyme was
described in terms of mmoles H2O2 decomposed per minute per mg protein.
2.6 Statistical analysis
Statistical analysis of different parameters was carried out according to Duncan’s
multiple range tests at 5% probability level. One-way ANOVA was applied using the
SPSS software 16.0, SPSS Inc., Chicago, USA. The data is an average observation
from three independent experiments, each with three replicates. The data represent
mean ± standard error.
3. Results
3.1 Photosynthetic pigments
Total chlorophyll content in the leaves of NaCl (75mM) treated plants, showed a
significant increase of 60% than the control .Whereas the plant subjected to PEG
(15%) showed only 24% increase (fig.1A)
3.2 MDA content and Membrane stability index
The MDA accumulation was enhanced in seedlings under NaCl stress (43%) whereas
in PEG treatment the increase was 62% (Fig.1B). Similarly the membrane stability
index significantly increased in seedlings emerged under NaCl (24%) and PEG (45%)
stress as compared to control (Fig.1C).

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51
3.2 MDA content and Membrane stability index
The MDA accumulation was enhanced in seedlings under NaCl stress (43%) whereas
in PEG treatment the increase was 62% (Fig.1B). Similarly the membrane stability index
significantly increased in seedlings emerged under NaCl (24%) and PEG (45%) stress as
compared to control (Fig.1C).
Fig. 1 Total chlorophyll (A), MDA (B) and Membrane stability index (C) content in seedlings
exposed to NaCl and PEG. C (Control), NaCl (75 mM)-seeds subjected to salinity stress and PEG
(15%)-seeds subjected to polyethylene glycol stress).
3.3 Osmolytes and Non- enzymatic antioxidants
Treatment with NaCl and PEG induced significant increase of proline content. However
compared to PEG, NaCl treatment induced accumulation of higher proline content in cowpea
seedlings. In cowpea treated wth NaCl, maximum proline accumulation was observed and the
increase was 289% whereas the plants treated with PEG recorded an increase of 45% only over
the control plant (fig.2A). Increase in the accumulation of total soluble sugar was observed in
cowpea treated with NaCl (47%) whereas under PEG treatment 25% increase was recorded over
the control (fig.2B).
0
200
400
600
800
1000
1200
1400
1600
1800
control NaCl PEG
Total chlorophyll (µg/DW)
control NaCl PEG
AAA
0
10
20
30
40
50
60
70
control NaCl PEG
MDA content
(µmol/g DW)
control NaCl PEG
B
0
2
4
6
8
10
12
Control NaCl PEG
Membrane stability index %
Control NaCl PEG
C
Fig. 1 Total chlorophyll (A), MDA (B) and Membrane stability index (C) content in seedlings
exposed to NaCl and PEG. C (Control), NaCl (75 mM)-seeds subjected to salinity stress and PEG
(15%)-seeds subjected to polyethylene glycol stress).
Osmolytes and Non- enzymatic antioxidants
Treatment with NaCl and PEG induced significant increase of proline content. However
compared to PEG, NaCl treatment induced accumulation of higher proline content in
cowpea seedlings. In cowpea treated wth NaCl, maximum proline accumulation was
observed and the increase was 289% whereas the plants treated with PEG recorded
an increase of 45% only over the control plant (fig.2A). Increase in the accumulation
of total soluble sugar was observed in cowpea treated with NaCl (47%) whereas under
PEG treatment 25% increase was recorded over the control (fig.2B).

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52
Fig. 2 Proline (A), Total soluble sugar (B) and Total phenolic (C) content in seedlings exposed to
NaCl and PEG. C (Control), NaCl (75 mM)-seeds subjected to salinity stress and PEG (15%)-seeds
subjected to polyethylene glycol induced drought stress).
In cowpea, the accumulation of protein content was higher in seedlings exposed to NaCl
and PEG stress over control plants. A gradual increasing trend of total protein content was
recorded in NaCl treated plants (65%) as compared to control. However, when cowpea was
treated with PEG, it recorded a sudden increase as compared to that of control but lesser content
than that recorded under NaCl stress. But there was decreasing trend of total free amino acids
content in seedlings subjected to both NaCl and PEG stresses.
Phenolics content of NaCl treated cowpea showed a significant increase when compared
to PEG. In cowpea treated with NaCl, 75 mM induced maximum accumulation of phenolics
content (38%) whereas in seedling treated with PEG observed a negligible increase of only 6%
in phenolic content accumulation as compared to control.
3.4 Enzymatic antioxidants
NaCl induced a significant increase in the SOD activity in cowpea seedlings than that
exposed to PEG. The cowpea treated with NaCl induced an increase in SOD activity (59%)
0
500
1000
1500
2000
2500
control NaCl PEG
Proline (µg/g DW)
control NaCl PEG
A
0
20
40
60
80
100
120
control NaCl PEG
Total soluble sugar content
(mg/g DW)
control NaCl PEG
B
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
control NaCl PEG
Total Phenolic content (mg/g
DW)
control NaCl PEG
C
Fig. 2 Proline (A), Total soluble sugar (B) and Total phenolic (C) content in seedlings exposed to
NaCl and PEG. C (Control), NaCl (75 mM)-seeds subjected to salinity stress and PEG (15%)-seeds
subjected to polyethylene glycol induced drought stress).
In cowpea, the accumulation of protein content was higher in seedlings exposed to
NaCl and PEG stress over control plants. A gradual increasing trend of total protein
content was recorded in NaCl treated plants (65%) as compared to control. However,
when cowpea was treated with PEG, it recorded a sudden increase as compared to
that of control but lesser content than that recorded under NaCl stress. But there was
decreasing trend of total free amino acids content in seedlings subjected to both NaCl
and PEG stresses.
Phenolics content of NaCl treated cowpea showed a significant increase when compared
to PEG. In cowpea treated with NaCl, 75 mM induced maximum accumulation of
phenolics content (38%) whereas in seedling treated with PEG observed a negligible
increase of only 6% in phenolic content accumulation as compared to control.
Enzymatic antioxidants
NaCl induced a significant increase in the SOD activity in cowpea seedlings than that
exposed to PEG. The cowpea treated with NaCl induced an increase in SOD activity
(59%) whereas the seedlings treated with PEG recorded an increase of 24% over
the control plants. Similar trend was observed in catalase and APX activities.

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53
whereas the seedlings treated with PEG recorded an increase of 24% over the control plants.
Similar trend was observed in catalase and APX activities.
Fig. 3 SOD (A), CAT (B), APX (C) and Total soluble protein (D) content in seedlings exposed to
NaCl and PEG stress. C (Control), NaCl (75 mM)-seeds subjected to salinity stress and PEG (15%)-
seeds subjected to polyethylene glycol induced drought stress).
4. Discussion
The immediate effects of salt and PEG stresses are osmotic, but their long-term effects
differ. PEG-mediated stress continues to exert osmotic effects on plants, while salt stress effects
are largely due to ion toxicity and metabolic imbalance (Munns and Tester 2008). In the present
work, using PEG and NaCl, the mechanisms adopted by plants to avoid or tolerate the respective
stress conditions were studied. Cowpea tissues showed a more severe reduction in growth on
being subjected to PEG stress, indicating sensitivity of the actively growing seedlings towards
stress condition. Whereas salt stress did not cause such severe growth retardation, which
indicates differences in the response to these two stress factors at cellular level of organization.
Cowpea seedlings subjected to NaCl and PEG stress results in the accumulation of ROS
and oxidative stress leads to the membrane damage through increased rate of lipid peroxidation
0
0.5
1
1.5
2
2.5
3
control NaCl PEG
SOD activity
(U/mg protein)
control NaCl PEG
A
0
0.5
1
1.5
2
2.5
3
control NaCl PEG
CAT activity
(U/mg protein)
control NaCl PEG
B
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
control NaCl PEG
APX activity
(U/mg protein)
control NaCl PEG
C
0
100
200
300
400
500
600
control NaCl PEG
Total soluble protein content
(mg/g DW)
control NaCl PEG
D
Fig. 3 SOD (A), CAT (B), APX (C) and Total soluble protein (D) content in seedlings exposed to NaCl
and PEG stress. C (Control), NaCl (75 mM)-seeds subjected to salinity stress and PEG (15%)-seeds
subjected to polyethylene glycol induced drought stress).
4. Discussion
The immediate effects of salt and PEG stresses are osmotic, but their long-term
effects differ. PEG-mediated stress continues to exert osmotic effects on plants, while
salt stress effects are largely due to ion toxicity and metabolic imbalance (Munns and
Tester 2008). In the present work, using PEG and NaCl, the mechanisms adopted
by plants to avoid or tolerate the respective stress conditions were studied. Cowpea
tissues showed a more severe reduction in growth on being subjected to PEG stress,
indicating sensitivity of the actively growing seedlings towards stress condition.
Whereas salt stress did not cause such severe growth retardation, which indicates
differences in the response to these two stress factors at cellular level of organization.
Cowpea seedlings subjected to NaCl and PEG stress results in the accumulation of
ROS and oxidative stress leads to the membrane damage through increased rate of
lipid peroxidation and thus decreased the membrane stability index in seedlings. In
response to stress treatments the plants enhances the synthesis of varius osmolytes
such as sugar, proline, glycine betaine, amino acid. Phenolics accumulate because of
their functions as intermediate in lignin biosynthesis and thus preserves the plant
cells by building a physical barrier. Moreover, phenolics accomplish the task of

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neutralizing the ROS and therefore the induction of phenolic accumulation observed
in this study, proves that the plant are encountered with oxidative stress.
Overall, the seedlings were able to avoid salt stress in a much better than PEG stress.
Salt is known to cause ionic imbalance and toxicity due to accumulation of Na+ ions.
It is possible that tissues could have used the mineral components of the nutrient
medium to attain a level of ion homeostasis, but unable to cope with dehydration stress
imposed by PEG. When stress avoidance mechanisms are insucient, stress tolerance
mechanisms are required to prevent cellular damage arising from dehydration or ion
toxicity (Verslues et al. 2006). Cowpea seeds subjected to salt stress showed higher
accumulation of SOD, indicating that stress tolerance mechanisms were eective in
preventing membrane damage at the cellular level. Both the stresses led to signicant
increase in the activity of CAT, which probably were induced in response to ROS but
did not play a signicant role in stress tolerance. In PEG treated seeds, APX activity
was signicantly reduced and SOD activity was at par with control. However, in
NaCl treated seeds, APX and SOD activities were signicantly increased and could
be the major reason for tolerance response of seedlings towards this stress.
5. Conclusion
The coordinated efforts of tissues at plant level was required to cope with both abiotic
stresses. The cowpea seedlings were able to better cope up with ion toxicity imparted
by NaCl stress , but it was not the same withPEG stress.
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