Journal of Research in Agricultural Science
Vol. 7, No. 1 (2011), pages: 49-57
Downloaded from journals.khuisf.ac.ir at 16:30 IRDT on Saturday June 17th 2017
Effect of Different NaCl Salinity on Antioxidant Enzyme Activity and
Relative Water in Winter Canola (Brassica. napus)
SEDGHALI ZAMANI1*, MOHAMMAD TAHER NEZAMI2, AHMAD BYBORDI1, MARYAM BEHDAD3,
MOHAMMAD BAGHER KHORSHIDI1
1-East Azarbaijan Agricultural and Natural Resources Research Center, East Azarbaijan, Iran
2-Karaj Branch, Islamic Azad University, Karaj, Iran
3-Department of Horticulture, Khorasgan (Isfahan)Branch, Islamic Azad University, Isfahan, Iran
Received: 21 December 2009
*
Accepted:17 October 2010
Corresponding author: Email: [email protected]
ABSTRACT
To evaluate the effect of different levels of salinity on antioxidant enzymes activity of leaves and plant
physiological characteristics of canola as a marker of resistance to salinity, an experiment was
conducted in greenhouse of Research Center of Agriculture and Natural resources in East Azarbaijan
in 2009. Treatments included combinations of five different levels of salinity (0, 50, 100, 150, and 200
mM) and four cultivars of brassica included (Elite, Licord, SLM046 and Okapi). With increasing
salinity, stomatal conductivity, transpiration and leaf relative water decreased in all cultivars. Effect of
salt stress on antioxidant enzymes of leaves was significant, and changes in the amount of super
oxided dismutase enzymes, catalase and glutathion reductase was observed. Enzyme activity in the
period of growth increased and in plant maturity decreased. Comparison between canola cultivars
showed the strong correlation between leaf relative water and enzyme super oxide dismutase activity.
SLM046 showed the least changes in leaf relative water level and the highest enzyme activity and was
resistant cultivar and Elite were the most sensitive cultivar to salinity.
Keywords: Antioxidant enzymes, Salinity, Relative water, Canola
INTRODUCTION
Ghasemi et al. (2002) reported that more
than 400 million hectares worldwide
affected by the influence of either salt or
sodium which was about 6% of the world's
lands. From 230 million ha of irrigated
land area in the world, 45 million hectares
(19.5%) and from 1,500 million ha of rain
fed lands under cultivation, 32 million ha
(2.1%) affected with different degrees of
salinity. From 15 million ha of cultivated
lands in Iran, 6 million ha is irrigated land
(30%) equivalent to 1.7 million ha affected
by salinity. This showed importance of
salinity as a serious factor in most parts of
49
the country. Oilseeds are the second world
food stocks. Additionally, these seeds have
a rich reserve of protein and fatty acids
(Sairam and Srivastava, 2001).
Ehsanfar et al. (2006) reported that in
poor and saline soils which other plants
cannot have good growth, Canola grew well
and produced seed. Bahizire and Frencois
(2007) reported that canola production
levels with the variability amount of salt
because of the effect of salinity, soil,
mineral elements and type on the
establishment of canola seedlings is
different. According to Corwin et al.
(1996), salinity is a worldwide problem in
all regions especially in irrigated lands of
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SEDGHALI ZAMANI ET AL./ JRAS Vol. 7, No. 1 (2011) 49-57
arid and semiarid regions. Salinity limits
soil fertility in irrigated regions of the
world. Due to low rainfall in these areas,
soil leaching does not occur. Therefore,
crops encountered with salinity problems.
Bohnet (1979) believes that salinity
increased concentration of sodium anions
and other toxic as HCO3-, CO3-- and SO4--in the soil. For obtained optimal
performance under salt stress, resistant
plant is required. On the other hand, high
diversity in plant species, each with
different hereditary traits and specific
mechanisms to maintain survival, it seems
that identification, modification and
selection of salinity resistant species can be
useful (Karimi, 1996). Generally, plants
understand a wide range of environmental
stresses that ultimately cause oxidative
stress in plants. Mechanism of resistance in
some internal tensions was a result of a
plant communication and coordinate
complex. In Stress condition, lack of
balance between absorb the energy and
consumption process caused active oxygen
(ROS:
Reactive
Oxygen
Species)
production by photosynthetic organs and its
inability to inhibit will eventually lead to
tension in the cell membrane and symptoms
caused by oxidative damage (Blokhina and
Virolainen, 2003). Increasing in active
oxygen radicals in plants causes reducing of
toxic effects of oxidative stresses induced
by salt stress, and activated a variety of
mechanisms in plant. In these conditions,
increased levels of Antioxidants and ROS
inhibitor enzymes reduced toxic effects of
oxidative stress. Sensitivity of enzymes
extracted from cultivars exposed to NaCl
salinity is similar to enzymes of salinity
sensitive cultivars (Kafi et al., 2003).
Antioxidant enzymes are the fastest units
checking against attack of active oxygen
(Dirk and Montago, 2002).
Physiological characteristics of plants,
including changes in stomata closing
pattern growth regulators and accumulation
of metabolites are important illustration of
compatibility to stress conditions (Khavari,
1996; Singh et al., 2004). Therefore,
salinity effects studying by help of
enzymes, can identify resistant rootstocks
more quickly, because there is a strong
correlation
between
tolerance
to
environmental stresses and changes in the
concentration of antioxidant enzymes in
photosynthetic plants and provided any
substance in favor of synthesis in cells
subject by genes, responsible genes can
identify for synthesis of this material and its
transfer to other plants to produce salinity
resistant
rootstocks.
For
optimal
performance in salt stress conditions, an
appropriate resistant plant is needed.
Therefore, to achieve new methods and
applications to increase production per area
in dry and semi dry climates, for using salt
water is necessary (Karimi, 1996; Weiss,
2000).
The main objectives of this study was
investigate the response of canola cultivars
to saline stress by assessment their
antioxidant enzymes in vegetative growth
stage to determine the most tolerant canola
cultivars.
MATERIALS AND METHODS
This study was conducted in greenhouse
of Agriculture and Natural Resources
Research Center of East Azarbaijan as a
factorial experiment based on randomized
complete blocks design with three
replications in 2009. Five salinity levels
combination (0, 50, 100, 150, and 200 mM)
and four cultivars of canola (Elite, Licord,
SLM046 and Okapi) were arranged as main
and subplot, respectively. Disinfectant after
sodium hypochlorite with 10 percent 3 to 5
times with distilled water and were washed
into plastic pots (30 cm diameter mouth and
35 cm height) containing a mixture of
perlite and vermiculite 1:1 ratio to the
number five seeds in each pot depth of 1 –
1.5 cm were planted. To prevent salt
accumulation in pots, two number of 1 cm
50
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SEDGHALI ZAMANI ET AL./ JRAS Vol. 7, No. 1 (2011) 49-57
diameter holes were placed at the bottom of
the pots as drainage.
During the growth period pots placed
among 50 cm in greenhouse with daily
temperature 25 ± 3 °C , and 18 ± 3 °C night
in normal light and after establishment of
seedlings in complete medium, two plants
per pot maintained and the rest were
removed. Salinity with Hoagland nutrient
solution gradually imposed through
irrigation in the four-leaf stage. Sampling
for Determine antioxidants, at three
vegetative growth stages, flowering, early
maturity and pod bulking, one third upper
leaves were done. Dyndsa method was used
to enzyme extracts preparation for
determination of antioxidant enzymes
activity (Sairam et al., 2002). For
determining the amount of SOD, Sairam et
al. (2001) method was used. For
determination of catalase enzyme in
extracts, Chns and Mhly (Sairam el at.,
2002) method was used. For determine the
amount of glutathione reductase, Smith and
colleagues (Arora et al., 2002) method was
used. To evaluate physiological traits
responses to salinity stress, every 10 days
interval, the value of stomatal conductivity
and the youngest developed leaf
transpiration rate during plant growth was
measured with LCi Portable Photosynthesis
System device.
For determination of leaf relative water
in pod filling phase, the youngest developed
leaf sampling from the upper third was used
(Sairam and Srivastava, 2001).
Analysis of variance was done by
MINITAB software and means compared
with Duncan multi-range test at 5 percent
probability level with software MSTATC
and plotting figures were done by EXCEL.
RESULTS AND DISCUSSION
Super oxide dismutase
Super oxide dismutase enzyme activity
showed significant difference among
cultivars so SLM046 showed highest and
Elite showed minimum SOD enzyme
activity (Figure 1). Increasing plant age
from vegetative stage to maturity stage
decreased SOD activity in SLM046 and
Licord. But the amount of this enzyme
activity in Elite in flowering stage was
higher than vegetative stage. While, the
highest enzyme amount during the growth
period belonged to SLM046. With
increasing salt in nutrient solution from 150
mM to 200 mM NaCl, the amount of
enzyme per leaf weight unit decreased
(Figure 2). This could be due to destruction
of SOD producer structures when plants
exposed to NaCl. Due to local reduction of
Na+ in the leaves, reduced enzyme activity
in resistant cultivars than susceptible
cultivars was seen (Vaidyanathan et al.,
2003). With increasing salinity, the plant
antioxidant system activated and increasing
enzyme super oxide dismutase activity is a
first defense barrier against attack of
oxygen radicals (Mirmohammadi Meybod
et al., 2002) and until plant can inhibit
produced
superoxide,
this
process
continues.
The results of researchers on other plants
also showed that the SOD activity in
resistant cultivars is higher than susceptible
cultivars to salinity.
The amount of enzyme increased with
increasing salinity and plant age (Reddy
and Srivastava, 2003). Results of study on
catalase showed that the highest amount of
enzyme belonged to Elite, where there was
no significant difference between SLM046
and Licord (Figure 3).
51
SEDGHALI ZAMANI ET AL./ JRAS Vol. 7, No. 1 (2011) 49-57
a
Activity SOD (Aunit
min. grf.wt)
b
b
15
c
10
5
0
Elite
Okapi
Slm046
cultivars
Licord
Figure 1. Average rate of superoxide dismutase activity in canola cultivars
Elite
SLM046
Elite
SLM046
Okapi
Licord
Elite
SLM046
Okapi
Licord
30
25
20
15
10
5
0
S1
S2
S3
S4
activity SOD(Aunit min.grf.wt)
35
35
30
25
20
15
10
5
0
S5
Okapi
Licord
40
40
activity SOD(Aunit min.grf.w t)
activity SOD(Aunit min.grf.wt)
40
S1
Flowering initial
S2
S3
S4
35
30
25
20
15
10
5
0
S1
S5
S2
S3
S4
S5
Maturity initial
Flowering initial
Figure 2 . Comparison the effect of salinity on SOD activity in the three growing stage S1, S2, S3, S4
and S5, respectively salt 0,50,100, 150, and 200 mM
2.5
a
b
2
2.5
b
c
c
Activity Catalase
(A240. min.
g.fr.wt)
Activity Catalase
(A240. min. g.fr.wt)
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20
1.5
1
0.5
0
S1
S2
S3
S4
a
b
2
c
1.5
c
1
0.5
0
S5
Elite
salinity
Slm 046
cultivars
Figure 3. Comparison of the mean catalase
activity in different levels of salinity
Figure 4. Changes in catalase activity of different
canola cultivars
52
Studies of Kafi et al. (2003) showed that
increasing salinity significantly decreased
catalase enzyme activity. Comparison of
catalase activity showed the highest
catalase activity in plant was at flowering
stage and minimum activity in the
vegetative growth stage. Meanwhile, the
maximum catalase activity in different
growth stages of canola plant was different.
Results of Sairam and Srivastava (2001)
also showed that with increasing plant age
up to stage 50 % of pollination, the amount
of catalase increased, the amount of
catalase in the salinity of 200 mM
significantly reduced (Figure 5). Catalase
converts hydrogen peroxide enzyme to
water and molecular oxygen and as a result
of salt stress, the rate of catalase decreases
in leaves and roots (Gramer 2002).
Similarly, comparison of enzyme activity at
different levels of salinity showed
increasing levels of salinity from 0 mM to
100 mM declined the catalase activity. But,
b
cd
c
d
1.5
1
Glutathione reductase
The lowest activity of Glutathione
reductase (GR) was observed in SLM046
but Okapi and Licord showed highest
enzyme activity (Figure 6). The results
showed that the lowest GR enzyme activity
related to the salinity level was 100 mM
(Figure 7).
The minimum GR activity was observed
in SLM046 at pod maturity stage, because
it can coordinate activities of catalase and
glutathione reductase and hydrogen
peroxide to inhibit the activity of SOD. In
all studied enzymes, the level of enzyme
activity increased with plant age (Figure.
8).
b
Flowe ring initial
a
2.5
c
2
Activity
Catalase
(A240. min.
2
2.5
a
Activity
Catalase
(A240. min.
2.5
with increasing salinity to 150 mM
increased the enzyme activity was observed
again (Figure 4).
Maturity initial
a
Ve ge tative stage
Activity
Catalase
(A240. min.
d
1.5
e
1
0.5
0.5
S1
S2
S3 S4
salinity
S1
S5
b
2
1.5
d
e
c
1
0.5
0
0
S2
S3 S4
salinity
0
S5
S1 S2 S3 S4 S5
salinity
Figure 5. Effect of salt stress on catalase activity balance the developmental period S1, S2, S3, S4 and
S5, respectively salt 0, 50, 100, 150, 200 mM
0.5
0.4
a
0.5
b
b
Reductase Glutathione
Activity (A 560. min.
gr.fr.wt)
Reductase Glutathione
Activity Catalase (A560
min. gr .fr.wt)
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SEDGHALI ZAMANI ET AL./ JRAS Vol. 7, No. 1 (2011) 49-57
bc
c
0.3
0.2
0.1
0
S1
S2
S3
S4
salinity
a
a
0.4
0.3
b
bc
0.2
0.1
0
S5
Elite
Figure 6. Comparison of reductase glutathione
activity in different salinity levels
53
Okapi Slm046 Licord
cultivars
Figure 7. The average activity of glutathione
reductase canola cultivars
SEDGHALI ZAMANI ET AL./ JRAS Vol. 7, No. 1 (2011) 49-57
Licord
Elite
0 .9
0 .8
0 .7
0 .6
0 .5
0 .4
0 .3
0 .2
0 .1
0
Okapi
SLM046
Licord
1
Elite
Glutathione Reductase
activity (A560 min.grf.wt)
SLM 046
Glutathione Reductase
activity (A560 min.grf.wt)
Okapi
0.8
0.6
0.4
0.2
0
S1
S2
S3
S4
S1
S5
S2
S3
S4
S5
Okapi
SLM046
Licord
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
S1
Maturity initial
Flowering initial
S2
S3
S4
S5
Flowering initial
Figure 8. The interaction of salinity and time on the level of glutathione reductase activity in four
cultivars of canola S1, S2, S3, S4, and S5, respectively salt 0,50,100, 150 200 mM
Leaf relative w ater Rate(%)
Transpiration Rate(mMol.s)
Licord
Licord
cultivars
Cultivars
Glutathione Reductase
activity (A560 min.grf.wt)
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Elite
SLM046
Okapi
SLM046
Okapi
Elite
Elite
0
0.1
0.2
50 mM
100mM
0.3
salinity
0 mM
150mM
0
0.4
200mM
Figure 9. Changes in leaf relative water level
increased salinity of canola cultivars
PHYSIOLOGICAL CHARACTERISTICS
Relative water content and transpiration
rate decreased in all cultivars as a result of
increasing salt (Figs 9 and 10).The results
of other researchers also confirmed this
(Mahmoud et al., 2003). With salinity
stress, leaf relative water content decreased
with increasing sodium chloride in Elite,
Okapi, SLM046 and Licord as 20.06,
20.41, 20.58 and 20.43 %, respectively.
Other studies also have shown that resistant
wheat to cultivars, resistant relative leaf,
less than half of resistant cultivars and plant
54
0 mM
200
50 mM
400
salinity
100 mM
150 mM
600
200 mM
Figure 10. Reduction of transpiration rate with
increasing salinity in canola cultivars
age difference between resistant cultivars
and half are less resistant, high salinity are
not significantly difference (Sairam el at.,
2001).
Transpiration rate with transition from 0
mM to 150 mM, decreased 65.49 %, and
further increase in salinity caused no
significant changes in transpiration rate.
Transpiration differences rate among
cultivars was no significant, but in all
varieties with increasing growth period,
after 25 days of implantation, a significant
change in transpiration rate was showed
(Figure 11).
SEDGHALI ZAMANI ET AL./ JRAS Vol. 7, No. 1 (2011) 49-57
Transpiraion Rate
(mM.s)
Elite
0.08
Okapi
0.06
SLM046
Licord
0.04
0.02
0
25
35
45
55
After days implant
Figure 11. Changes in transpiration rate with increasing plant age in different cultivars
of canola
Stomatal conductivity(Mmol m2.s)
100
50
0
0
mM
ty
ini
sal
50
mM
Elite
Elite
Okapi
SLM046
Licord
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0.1
100
mM
150
mM
200
mM
Okapi
SLM046
Licord
cul
tiva
rs
Figure 12. Interaction between salinity and the type of canola on the rate of stomatal
conductance S1, S2, S3, S4 and S5, respectively salt 0,50,100, 150 200 mM
to 150 mM has no specific reaction by
stomata (Figure 12).
Regression relationship between SOD
activity and leaf relative water content
among different cultivars was significant
and negative, relationship revealed that
SLM046 with highest super oxide
dismutase enzyme activity and the lowest
leaf relative water content was resistant and
Elite with the lowest enzyme activity and
the highest rate of leaf water content was
salt sensitive cultivars (Figure 13). The
results showed that, because the SOD
enzyme as the first barrier of defense
against
active
oxygens
attack
(Mirmohammadi Meybod el at., 2002), salt
stress increased cell ROS production and
SOD activity until 100 mM NaCl.
Stomata conductivity
Stomata
conductivity
affected
environmental effects such as light
intensity, concentration of CO2, and soil
environment moisture and genetic factors
influence intensity of photosynthesis and
growth via stomata conductivity (Mashuf
et al., 2003). In this experiment, with
increasing salinity, plants react quickly and
to reduce the effects of secondary salinity,
attempt to close the stomata and reduce
withdrawal of water from plant as
transpiration. Therefore, in all four varieties
stomatal conductance reduction at 50 mM
compared to control was observed and only
Licord showed that increasing salinity level
55
Therefore, produced SOD volume in this
level of salinity, can inhibit oxidative
agents. Simultaneous with SOD activity
and followed production of H2O2,
glutathione reductase also increased its
activity until 150 mM to inhibit produced
hydrogen peroxide in environment. With
increasing salinity level to 150 mM two
cases arise: First, parallel to the amount of
SOD activity, active catalase and
glutathione reductase unable to reduce
H2O2 level in medium to reduce partially
the activity of SOD in 150 mM. The
highest amount of SOD enzyme activity
and the lowest leaf content was observed in
SLM046, so it seems that SLM046 more
resistant to salinity among cultivars could
be as compatible plant with desired
performance
in
saline
land
and
recommended for field studies.
18
16
14
SOD enzyme activity
(Unit min. ig. Fr. Wt)
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SEDGHALI ZAMANI ET AL./ JRAS Vol. 7, No. 1 (2011) 49-57
12
10
8
y = - 1 . 2198 x + 127 . 76
R 2 = 0 . 9878
6
4
2
0
82
83
84
85
86
%
87
88
89
RWC
Figure 13. Regression between RWC and SOD enzyme activity between different rapeseed cultivars
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