Soil salinity is one of the major factors of soil degradation. It
has reached 19.5% of the irrigated land and 21% of the dry-land agriculture
existing on the globe (FAO, 2000). Salinity inhibition of plant growth
is the results of osmotic and ionic effects and the different plant species
have developed different mechanisms to cope with these affects (Munns,
High salt concentrations, usually sodium chloride, cause osmotic stress
by decreasing water potential within the cells and ionic stress due to
specific inhibition of metabolic processes. Plants respond to salinity
by sequestering toxic ions in the vacuoles and accumulation of compatible
solutes in the cytoplasm to balance the decrease of water potential (Di
Martino et al., 2003).
Biochemical studies have shown that plants under salinity stress accumulate
number of metabolites, which are termed compatible solutes because they
do not interfere with biochemical reactions. These metabolites include
carbohydrates, such as manitol, sucrose and raffinose oligosaccharides
and nitrogen-containing compounds, such as amino acids and polyamines
(Bohnert et al., 1995).
In many researches, salinity tolerance has been studied in relation to
regulatory mechanisms of osmotic and ionic homeostasis (Ashraf and Harris,
2004). Salt stress, like other abiotic stresses can also lead to oxidative
stress through the increase in Reactive Oxygen Species (ROS), such as
superoxide (O2–), hydrogen. peroxide (H2O2)
and hydroxyl radicals (OH), which are highly reactive and may cause cellular
damage through oxidation of lipids, proteins and nucleic acids (Apel and
Hirt, 2004). To minimize the effects of oxidative stress, plant cells
have evolved a complex antioxidant system, which is composed of low-molecular
mass antioxidants (glutathione, ascorbate and carotenoids) as well as
ROS-scavenging enzymes, such as: superoxide dismutase (SOD), catalase
(CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and glutathione
reductase (GR) (Alscher et al., 1997; Apel and Hirt, 2004).
The relationship between osmoregulation, salt tolerance and nutrient
uptake is not clear. Studies about the effects of salt stress on wheat
plant mostly concerned on the changes in growth parameter and osmoregulation
for breeding purpose. In this study, we aimed to provide an insight view
to salt stress and antioxidants enzymes system in wheat plant. In order
to understand the mechanisms relevant in salt. tolerance. Therefore, the
objective of this investigation was to evaluate the effects of salinity
stress on three wheat cultivars and correlate these effects to changes
in ionic, organic solute accumulation and antioxidants enzymes.
MATERIALS AND METHODS
This study was conducted in the university of Zabol, Iran during
April-June 2007. The experiment design was a completely randomized 3x4
factorial design with three replicates.
Seeds of three bread wheat cultivars (Kavir, Hamon and Pishtaz) were
sown in trays containing vermiculite, water daily with distilled water
and kept in a greenhouse.
Plant were grown under greenhouse conditions with a 13 h photoperiod
of natural daylight, maximum and minimum temperatures were 26 and 18°C,
respectively and relative humidity was 40% on average. Five days from
seedling. emergence they were transferred to trays containing half-strength
Hoagland`s nutrient solution and 10 days later they were transferred to
plastic plots containing 3 L of full strength nutrient solution.
Four salinity treatments were imposed by adding S0 = 0 (control),
S1 = 100, S2 = 200 89 and S3 = 300 mM
NaCl to the nutrient solution. 20 days after beginning of salt additions
the plants were harvested, organic and inorganic solutes were extracted
from mature leaf blades. In this extract, soluble carbohydrates (Horwitz,
1975) and proline (Bates et al., 1973) were determined. The contents
of Na+ and K+ were determined by using a Jemway
PFP7 Flame photometer.
Enzyme assays: The APX activity was determined according to Nakano
and Asada (1981). The assay mixture consisted of 50 μL of the enzyme
extract, 50 mM phosphate buffer (pH = 6.0), 0.1 μM EDTA, 0.5 mM ascorbate
and 1.0 mM H2O2 in a total volume of 1.5 mL. Ascorbate
oxidation was monitored by reading the absorbance at 290 nm at the moment
of H2O2 addition and 1 min later. The difference
in absorbance was divided by the ascorbate molar extinction coefficient
(2.8 mM-1 cm-1) and the enzyme activity expressed
as μmol of H2O2 min-1 mg-1
protein, taking into consideration that 1.0 mol of ascorbate is required
for the reduction of 1.0 mol of H2O2 (Mckersie and
Specific GPX and CAT activity were measured according to Urbanek et
al. (1991) and Beers and Sizer (1952), respectively.
Statistical analyses: All data were analyzed with SAS Institute
Inc 6.12. All data were first analyzed by ANOVA to determine significant
(p = 0.05) treatment effects. Significant differences between individual
means were determined using Fisher`s protected least significant difference.
Antioxidant enzyme activities: Results of this study showed,
the activity of antioxidant enzymes are elevated with increased salinity
from S0 = 0 (control) to S3 = 300 mM NaCl. Figure
1 shows that leaf-CAT level is increased with increasing salinity
meanly Pishtaz cultivar which had the highest CAT activity and 300 mM
NaCl induced 85.1% increase in the activity of this enzyme in plants than
As a result of salt stress, leaf-APX activity varied according the stressed
variety: in fact, we observed three different behaviours: Pishtaz showed
the same APX activity whatever the salt concentration and Kavir showed
an APX increase with increasing salt concentration. Hamon cultivar had
the highest APX activity and S2 salinity induced 65.8% increase
in the active of this enzyme than control treatment (Fig.
In addition to APX, the activities of GPX in Hamon cultivar increased
only until S1 (100 mM NaCl) after that, the leaf-GPX in Hamon
cultivar decreased. Leaf GPX-activity of other two cultivars (Kavir and
Pishtaz) increased with increasing salinity levels. Between Kavir
||Catalase (CAT) activity in leaves of three wheat cultivars
at salinity levels
||Ascorbate peroxidase (APX) activity in leaves of three
cultivars wheat at salinity levels
||Guaiacol peroxidase (GPX) activity in leaves of three
wheat at salinity levels
and Pishtaz cultivars, Pishtaz had the maximum GPX activity and 300 mM
NaCl induced 55.5% increase in the activity of this enzyme in the plants
Biochemical components and nutrient uptake: The results of this
study indicate, that application of NaCl in the growing medium significantly
(p<0.001) affected proline and carbohydrate concentration in the three
wheat cultivars. By increasing salinity from S0 = 0 to S3
= 300 mM NaCl proline and carbohydrate contents increased meanly at Hamon
and Kavir varieties.
Among the cultivars, Pishtaz cultivar had the maximum concentration of
proline and carbohydrate. In the S3 treatment Pishtaz had 76.1%
proline content of S0 in their green leaves tissue, but maximum
carbohydrate content in Pishtaz saw in S2 treatment after increasing
salinity, carbohydrate content in green leaves tissue of this cultivar
decreased (Fig. 4, 5).
Results of this study showed that considerable change in the plant`s
chemical composition. In general, potassium essential ion for plant growth
and by increasing salinity levels from S0 (control) to moderate
salinity (S1 = 100 mM NaCl) K+ content of Kavir
and Pishtaz cultivars decreased higher than Hamon cultivar. By increasing
salinity levels from S1 to S3, Hamon had the
||Carbohydrate concentration in leaves of three wheat
cultivars at salinity levels
||Proline concentration in leaves of three wheat cultivars
at salinity levels
||K+ content in leaves of three wheat cultivars
at salinity levels
highest decreasing of K+ content in its shoot. In S3
treatment Hamon had the lowest and Pishtaz had the highest K+
content among the cultivars (Fig. 6).
Compared to K+, sodium content in the shoot was increased
by each salinity level in the three cultivars. In the three cultivars
and in the S3 treatment, Na+ content in shoot was
2.5 times than S1. Among the cultivars, Hamon and Pishtaz had
the highest and lowest Na+ content in their shoots (Fig.
||Na+ content in leaves of three wheat cultivars
at salinity levels
Even under optimal condition, reactive oxygen species including
superoxide, hydrogen peroxide, hydroxyl radicals and single oxygen are
metabolic by products of plant cell. These reactive oxygen species affect
lipid peroxidation, protein denaturation and DNA mutation (Bowler et
al., 1992). To remove reactive oxygen species, plant cells possess
an antioxidant system consisting in low molecular weight antioxidants
such as ascorbate, α-tocopherol, glutathione and carotenoids, as
well as antioxidant enzymes. These include superoxide dismutase (SOD),
ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GPX)
(Nakano and Asada, 1981). In this study, present results showed the different
antioxidant enzymes in three wheat cultivars in the control treatment
which could be the result of genetic differences.
When the plants were subjected to salt stress, by increasing salinity
level from control to 200 mM NaCl the increases in CAT activity was higher
in the Pishtaz cultivar. After that in 300 mM NaCl, CAT activity was not
affected by the salinity stress (Figure 1). This result is similar to
reports of Sairam et al. (2002) in wheat.
In addition to CAT, the activities of the APX (Fig. 2) and GPX (Fig.
3) from three wheat cultivars were also affected by salinity levels. By
application NaCl in the growing medium from 0 too 300 mM, the activities
of leaf-APX and GPX in three wheat cultivars increased but among the cultivars
Pishtaz cultivar had the lowest and highest APX and GPX activities, respectively.
Badawi et al. (2004) found a strong correlation between salt tolerance
and APX activities in tobacco. However in this study Pishtaz had the lowest
CAT in its leaf, but this cultivar had the highest APX and GPX activities
in its leaf when subjected to salt stress.
A better understanding of physiological responses under drought and/or
salinity may help in programs in which the objective is to improve the
drought and/or salt tolerance of crop varieties. During the course of
these stresses, active solute accumulation of compatible solutes such
as amino acids, polyamines and carbohydrates is claimed to be an effective
stress tolerance mechanism (Rosa-Ibarra and Maiti, 1995).
In this study present results showed that salinity affected compatible
solutes and by increasing salinity levels from 0 to 300 mM NaCl proline
and carbohydrate concentration in leaf green tissue of three wheat cultivars
increased. Among the cultivars, Pishtaz cultivar had the highest carbohydrate
(until 200 mM NaCl level) and proline concentration (Fig. 4, 5).
we found a positive correlation between proline accumulation and Leaf-APX
(r2 = 0.56), Leaf-GPX (r2 = 0.63) and Leaf-CAT (r2
= 0.73). These results indicated which antioxidant enzymes and compatible
solutes help to plant adaptation when subjected to salt stress. Proline
plays a protective role of subcellular structures and scavenging free
radicals. Sucrose is accumulated in many plant tissues in response to
environmental stress, including salinity for playing an osmoregulation
role and cryoprotection (Balibrea et al., 1997). Under salinity
stress osmotic adjustment by accumulation of compatible solutes such as
amino acids and carbohydrate, can provide condition to continue water
and nutrient uptake by plant.
In general, the content of potassium, essential ion for plant growth.
In this study the K+ contents shoot showed a marked decreased
with the increase of salinity. Highly significant differences were absorbed
among treatments and cultivars. The Pishtaz showed the maximum accumulative
mean of four treatments and Kavir had the lowest K+ contents
in its shoot.
In the shoots of these cultivars, Na+ showed an increase in
concentration with salinity that approximately matches a decrease in K+
concentration. It is well documented that NaCl depresses the accumulation
of K+ in whole plants (Al-Rawahy et al., 1992). It seems
that Na+ concentrations in the shoot may have had a more significant
effect on plant antioxidant enzyme activities and compatible solutes such
as proline and carbohydrates.
In this study, we found a positive correlation between Na+
concentration in the shoots and the antioxidant enzyme activities and
compatible solutes in the leaves. The findings match those of Schachtman
et al. (1989) how showed that sensitivity to salt is correlated
to Na+ concentration in wheat shoots. The lower Na+
concentration in shoot of Pishtaz cultivar compared to other cultivars
indicated that Pishtaz cultivar can tolerant salinity stress better than
other cultivars (Kavir and Hamon).
Among the cultivars, Pishtaz cultivar had the best antioxidant enzyme
activities and accumulate compatible solutes such as proline and carbohydrate
in its shoot in all levels of salinity. This cultivar although had the
highest K+ and lowest Na+ concentration in its shoot
and it had the best salinity tolerance in this study.