International Journal of Agriculture and Crop Sciences.
Available online at www.ijagcs.com
IJACS/2013/5-4/401-409
ISSN 2227-670X ©2013 IJACS Journal
Evaluation of total soluble protein and antioxidant
activities in two spring cultivars of canola (Brassica
napus L.) in response to low temperature
Zeinab Moieni-Korbekandi1*, Ghasem Karimzadeh1, Mozafar Sharifi2
1. Plant Breeding and Biotechnology Department, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran.
2. Department of Plant Biology, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran.
*
Corresponding author email: [email protected]
ABSTRACT: Canola (Brassica napus L.), as the third oil seed worldwide, is an important resource in
providing raw oil in Iran. The expansion of spring annual production is limited by high temperature during
the flowering. In Iran where high temperature in summer appears to be premature, canola is cultivated in
autumn or winter. However, spring cultivars are perceived to be low resistant to low-temperatures
particularly in the early stages. In efficient physiological processes, they are induced in cold-tolerance.
The present work aims at assessing the physiological traits of two spring canola cultivars: cold-sensitive
(Option 500, cv.1) and cold-resistant (Zarfam, cv. 2). They responded to cold treatment at a four-leaf
stage in terms of the amount of total protein and SOD (superoxide dismutase), POX (peroxidase) and
CAT (catalase) antioxidant enzymes by a treatment comprising shifts from 22 °C to 10 °C. Variance
analyses showed significant differences between sampling times, cultivars and temperature treatments as
a result of low temperature (LT) on SOD, CAT, and POX. As a result of LT on protein content, there were
significant differences between all sources of variation except between-temperature treatments. In both
cultivars, antioxidant activity was changed differently, increasing significantly under low-temperature, in
comparison with the controls but in Zarfam cv. cultivar, it was more. In cold-resistant the highest increase
in protein content and SOD activity under low-temperature was observed in the second day. It was in the
fourth day of POD and CAT activity that the features of cold hardening in Zarfam cv. were found. It shows
the importance of cellular antioxidant machinery for protection against low-temperature.
Keywords: Brassica napus L., Canola, Protein content, Cold stress, Antioxidant enzymes.
Abbreviation: SOD- Superoxide dismutase; POX- Peroxidase; CAT- Catalase; LT- Low Temperature;
ROS-reactive oxygen species; ANOVA-Analysis of variance
INTRODUCTION
The genus Brassica includes a number of important crop species used for various purposes, such as
producing oil seeds, vegetables, fodders and condiments (Gustafsson et al., 1983). Oil seeds, along with cereals,
are the second global food resources. One of the most important oil seed crops is canola (B. napus L.), which is
the third oil seed in the world among annual oil seeds by yields >14% (Weiss, 2000). In Iran, Brassica napus L. is
an important resource in providing raw oil due to its cultivation capability in various regions, high percentage and
desired quality of oil. Therefore, the most important prerequisite in developing this crop is adapting varieties in
different conditions (Naseri, 1991). Low temperatures cause more crop losses worldwide and may be a significant
factor influencing plant distribution (Burke et al., 1976). During the last decade, many efforts have been made to
understand the biology of Brassica species (Janda et al., 2003; Karimzadeh et al., 2003; Wang-hao et al., 2007;
Intl J Agri Crop Sci. Vol., 5 (4), 401-409, 2013
Fahimirad, 2010), as well as collecting and conserving the Brassica germplasm (Gustafsson et al., 1983). They
have also recognized that Brassica species are valuable in investigating important key areas of resistance,
especially cold resistance. Also, great advances have been made in terms of cold-induced genes and cytological
mechanisms in the shape of cold resistance (White et al., 1994; Jiang et al., 1996; Diaz et al., 1997; Mieczyslaw,
1999; Rapacz, 2002). Exposure of plants to unfavorable environmental conditions such as temperature extremes,
including low-temperature stress, can increase the production of ROS, e.g., 1O2, O2•-, H2O2 and OH•. As a result of
protecting themselves against these toxic oxygen intermediates, plant cells and their organelles like chloroplast,
mitochondria and peroxisomes employ antioxidant defense systems (Gill and Tuteja, 2010). The optimum
temperature for maximal growth and development of spring-type oilseed rape is just over 20°C, and it is best grown
between 12°C and 30°C. After the emergency, seedlings prefer relatively cool temperatures up to flowering. High
temperatures for flowering will hasten the plant’s development, reducing the period of flowering to maturity (The
Organisation for Economic Co-operation and Development, OECD, 1997). In other words, the expansion of spring
annual production is limited by high temperature stress during the flowering and seed development stages (Raymer
et al., 1990). Therefore, in Iran, where the high temperature in spring and summer appears to be premature,
different cultivars of oilseed rape are cultivated in autumn and winter (Madani et al., 2005). However, spring
cultivars are perceived to have low resistance level in low temperatures. This is consistent with field observations
(Fowler and Carles, 1979) and laboratory studies. Canola tolerates cold at rosette stage (six to eight leaves) better
than other stages. But before one call cold acclimation, efficient physiological processes are induced in coldtolerance, such as the concentration of cell extract, enzymatic and protein changes (Madani et al., 2005). This
study aimed at examining the physiological traits of two spring canola cultivars, cold-sensitive (Option 500, cv.1)
and cold-resistant (Zarfam, cv. 2), which responded to cold treatment in early stages (four leaves) in terms of the
amount of total protein and antioxidant enzymes in leaves and stems by a treatment comprising shifts from 22 °C to
10 °C.
MATERIALS AND METHODS
Plant materials and treatments
The seeds of two Canola (Brassica napus L. 4x = 2n = 38) cultivars, Zarfam as a spring cold-tolerant and
Option 500 as a spring cold sensitive cultivar were supplied from the Seed and Plant Improvement Institute (SPII),
Karaj, Iran. The seeds were grown in plastic pots (150 mm diameter × 150 mm deep) filled with a mixture of five
parts of soft mold, two parts of sands, two parts of clay and two parts of loamy soil (garden soil). The seedlings
were grown in a controlled growth chamber at a constant air temperature of 22/16 °C (day/night) with an
illumination provided by white fluorescent tubes at a fluency rate of 300 µmol m-2 s-1 at soil level for 16 h d-1 until
the four-leaf developmental stage. In this time, half of the pots were either maintained continuously in this condition
(control treatment), or transferred to a cold growth chamber at 10/3 °C at the same fluency rate and photoperiod as
above for seven days (cold treatment).
The seedlings (airy parts) were sampled on a day when half of the pots were transferred to the cold growth
chamber (the zero time). It was randomly repeated on the second, fourth, and seventh days over a 7-day
experimental period. At the same sampling times, the samples were also taken from the control plants (those kept
at 22 °C). The harvested samples were frozen in liquid nitrogen and kept at -80 °C for assaying protein and
antioxidant enzymes.
Protein extraction and quantification
Total soluble proteins were extracted from the leaves and stems for assaying protein and Enzymes by a
modification of the method described by Ausubel et al. (1987). This consisted of homogenization with a chilled
mortar and pestle using a buffer containing ice-cold 50 mM Tris-HCl, pH 7.5; 2 mM EDTA and 0.01% (v/v) 2mercaptoethanol. The homogenate was centrifuged at 11000 rpm for 30 min at 4 °C. Supernatant was recentrifuged at 4000 rpm for 20 min and stored at –20°C for analysis (Hames & Rickwood, 1990).
Protein extracts were thawed and their concentration was determined by a colorimetric method, as described
by Bradford (1976) using a commercially available reagent (Bio-Rad protein assay dye reagent). In the Bradford
assay, protein concentration is determined by quantifying the binding of the dye, Coomassie Brilliant Blue G-250, to
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Intl J Agri Crop Sci. Vol., 5 (4), 401-409, 2013
the unknown protein solution, as compared to known standards. The tubes containing 100 l aliquots of known
concentrations of Bovine Serum Albumin (BSA; 0.156 mg l-1 to 10 mg l-1 in 0.15 M NaCl) were prepared. Blank
tubes containing 100 l of 0.15 M NaCl were also prepared. One ml Coomassie Brilliant Blue solution was added to
each tube and stirred on a vortex. The reactions were left at room temperature for 2 min. The absorbance at
wavelength of 595 nm was determined against the blank and the standard curve of absorbance versus protein
concentration plotted (Copeland, 1994). The reactions containing dilutions of the soluble protein extracts (unknown
concentrations) were set up as above and the absorbance at 595 nm was determined. The protein concentration of
the extracts was determined from the standard curve using Hitachi, U-2800 (Japan) Spectrophotometer.
Superoxide dismutase (SOD) activity assay
The activity of Superoxide dismutase (SOD) was measured using the method of Giannopolitis and Ries
(1997) by monitoring the inhibition of nitroblue tetrazolioum (NBT) reduction at 560 nm. The reaction mixture (3 ml)
contained 50 mM phosphate buffer (pH 7.5), 50 mM carbonate sodium (pH 10.2), 0.1 mM Na-EDTA, 1 mM
riboflavin, 12 mM L-methionine, 75 mM NBT and 50 l enzyme extract. The reaction was carried out in test tubes at
25°C under the illumination of a fluorescent lamp (40-W). The reaction was allowed to run for 10 min and stopped
by switching the light off. Blanks and controls were run in the same manner without illumination and enzyme,
respectively. Under the experimental condition, the initial rate of reaction, as measured by the difference in the
increase of absorbance at 560 nm (Hitachi Spectrophotometer, U-2800, Japan) in the presence and absence of
extract, was proportional to the amount of enzyme. The unit of SOD activity was defined as the amount of enzyme
that inhibits the NBT photoreduction by 50%. SOD activity values are given in units per mg of protein.
Peroxidase (POX) and catalase (CAT) activity assay
According to Kar and Mishra (1976), Peroxidase (POX) activity was assayed with some modifications and the
activities of CAT were measured using the modified method of Arrigoni (1992). The POX reaction solution (3 ml)
contained 60 mM potassium acetate buffer (pH 6.1), 5 mM 2 2, 28 mM guaiacol and the CAT reaction solution (3
ml) contained 50 mM potassium phosphate buffer (pH 7.0), hydrogen peroxide 3%, and 100 ml enzyme extract.
The reactions were initiated by adding enzyme extract (50 l). Changes in the absorbance at 470 and 240 nm were
read every 15 sec over one minute using Hitachi, U-2800 (Japan) spectrophotometer for POX and CAT,
respectively. Enzyme activities were calculated based on absorbance changes per minute per mg protein.
Data analysis
The data were analyzed using 3_factorial balanced ANOVA on the basis of randomized complete design
(RCD) with 3 replications. Cultivars, temperature treatments, and sampling times were considered as factors with
2, 2, and 4 levels, respectively (Table 1). Means and standard errors (SE) were used to compare the effects of
temperature treatment within each cultivar at each sampling time. Moreover, to validate any correlation, all
physiological variables were correlated vs. sampling times and where they were significant, linear regression
analyses were calculated. To prove the differences between two temperatures, their slopes (b values and SE) were
also statistically analyzed using t-test (Table 2). Analysis of variance, using Multi-Factorial Balanced Model, and ttest were conducted in Minitab Statistical Software (Minitab Inc., State College, PA, USA; Fry, 1993; Ryan & Joiner,
2001).
RESULTS AND DISCUSSION
The Effect of cold on the amount of total soluble protein
Low temperature is an important environmental factor that limits plant distribution, survival and crop yield.
Exposure to a low non-lethal temperature usually induces a variety of biochemical, physiological and molecular
changes in plants which can result in an acclimation response (Levitt, 1980). It can be involved in the synthesis and
accumulation of low molecular weight (Hansen et al. 1997, Nanjo et al. 1999) and high molecular weight
(Steponkus et al., 1998) proteins, and changes in protein content and enzyme activities (Roberts, 1982; Guy and
Carter, 1984; Cloutier, 1984; Calderon and Pontis, 1985; Li, 1985). Low temperature could result in the synthesis of
cold shock proteins (Hughes & Dunn, 1996). In our study, based on ANOVA results (Table1), significant differences
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Intl J Agri Crop Sci. Vol., 5 (4), 401-409, 2013
were found between temperature treatments, sampling times and cultivars in the amount of protein (***P <0.001) in
response to low temperature, whereas based on the results of t-test (Fig. 1), there was no significant difference
between temperature treatments in the amount of protein in cold-sensitive, (Option 500, cv.1) in any of sampling
times. The difference in the amount of protein in cold-resistant cultivar (Zarfam, cv. 2) was significant in all
sampling times (***P <0.001). It was the most on the second day in comparison with control samples (29%) (Fig.1).
In addition, there was the same linear pattern of increased protein content with sampling times in both cultivars
during low temperature (***P <0.001), but a significant difference between the two temperature regression slopes
was revealed only in cold-resistant cultivar (Zarfam, cv. 2) (**P <0.01) (Table 2). In some species, cold treatment
had no effect on protein synthesis. For example, in Lolium temulentum, the pattern of polypeptide synthesis in the
leaf base was not altered significantly in response to an abrupt drop in temperature from 20 to 5°C (Thomas, 1983;
Thomas and Stoddart, 1984; Stoddart et al. 1986; Ougham, 1987), but in our experiment, in agreement with
findings of Sangwan et al. (2001), Lee et al. (2002), Karimzadeh et al. (2003) and Fahimirad (2010), low
temperature stress changed the amount and pattern of proteins in Brassica napus. The same results were obtained
in other plant species such as Medicago sativa (Mohapatra et al., 1987), Triticum aestivum and Secale cereale
(Howarth & Ougham, 1993; Hughes & Dunn, 1996; Sarhan et al., 1997; Kolesnichenko et al., 1997, 2000; Javadian
et al. (2010) and Hordeum vulgare (Crosatti et al., 1996, 1999; Cattivelli et al., 1997; Bravo et al., 1999). In winter
cereals, the expression of cold-induced genes is virtually associated with their potential for improved resistance to
low temperature. Among these, wheat wcs120 gene family which encodes a group of proteins with high frequency
range of 12 to 200 kDa can be a useful model to understand the molecular genetics of freezing tolerance (Houde et
al., 1995; sarhan et al., 1997 cited in Fowler et al., 2001). Therefore, accumulation in soluble protein content can be
one feature of cold hardening in Zarfam cv1., as compared with Option 500 cv2.
(mg/g FW)
Total soluble protein
1.0
1.0
cv. 1
0.8
0.8
0.6
0.6
0.4
cv. 2
0.4
0
2
4
6
0
2
4
6
Sampling time (day)
Figue1. Changes in total soluble proteins in cold-sensitive cultivar Option-500 (cv.1) and cold-resistant cultivar Zarfam
(cv.2) in control (contined line- 22/ 6 ºC) and Low-temperature (spotted line- 10/3 ºC) seedlings in different sampling times.
Values are means (n = 3) ± SE, but where bars are absent, the variation about the mean was less than the diameter of the
symbol
Effect of cold on the antioxidant enzyme activity
Various abiotic stresses such as temperature extremes, heavy metals, drought, water availability, air
pollutants and nutrient deficiency lead to the overproduction of reactive oxygen species (ROS) in plants. They
could be highly reactive and toxic, and cause damage to proteins, lipids, carbohydrates and DNA, ultimately
resulting in oxidative stress (Gill and Tuteja, 2010). Plants have non-enzymatic and enzymatic antioxidant systems
to protect cells from oxidative damage (Mittler, 2002). The non-enzymatic mechanisms consist of antioxidants
(such as ascorbate, glutathione, tocopherol and carotenoids), proteins (such as chaperones, dehydrins and HSPs)
and osmolytes (such as proline, glycine betaine and raphinose) (Sairam and Tyagi, 2004; Ashraf and Foolad,
2007).The enzymatic components include super oxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC
1.11.1.6), peroxidase (POX; EC 1.11.1.7), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR),
monodehydroascorbate reductase (MDAR) and glutathione reductase (GR; EC1.6.4.2) (Olmos and Hellin, 1997;
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Scheidig et al, 2002; Shao et al., 2008). In this study, the effect of cold on the activity of SOD, POX and CAT in
Brassica napus was investigated.
Superoxide dismutase activity (unit/mg
Protein)
Superoxide dismutase (SOD)
The SODs remove O2- by catalyzing its dismutation, one O2- being reduced to H2O2 and the other one being
oxidized to O2 (Mittler, 2002). Gill and Tuteja (2010) suggested that metallo-enzyme SOD is the most effective
intracellular enzymatic antioxidant which is ubiquitous in all aerobic organisms and in all subcellular compartments,
it is prone to ROS mediated oxidative stress. In the current study, due to the effect of low temperature on SOD
activity, significant differences were found between temperature treatments, sampling times and cultivars (***P
<0.001) (Table 1). Significant differences between temperature treatments in SOD activity of cold-sensitive cultivar
(Option 500, cv. 1) were distinguished on the second (**P <0.01), fourth, and seventh (***P <0.001) days, and the
maximum difference was observed on the fourth day of low temperature in comparison with the control sample
(55%) (Fig. 2). In cold-resistant cultivar, Zarfam (cv. 2), a significant difference between temperature treatments in
SOD activity was observed in all sampling times (***P <0.001) and the highest increase in SOD activity was on the
second day in comparison with the control sample (126%) (Fig. 2). Moreover, there was a linear relationship
between increased SOD activity and time during low-temperature (***P <0.001) with significantly different
regression slopes in comparison to the control (***P <0.001) in each cultivar (Table 2). Similarity, the expression of
SOD different isoforms was found under low temperature stress (4 °C) in two canola cultivars (Wang-hao et al.,
2007).
60
60
cv. 1
cv. 2
38
38
15
15
0
2
4
6
0
2
4
6
Sampling time (day)
Figure2. Changes in superoxide dimutase activity in cold-sensitive cultivar Option-500 (cv.1) and cold-resistant cultivar Zarfam
(cv.2) in control (continued line- 22/ 6 ºC) and Low-temperature (spotted line- 10/3 ºC) seedlings in different sampling times.
Values are means (n = 3) ± SE, but where bars are absent, the variation about the mean was less than the diameter of the
symbol
In our study, SOD activity was encouraged under low temperature with a little different pattern and a different
intensity between the two cultivars. The highest level of SOD activity was on the second day in zarfam cv. and was
more intense than Option 500 cv. As McCord (1988) mentioned, the high importance of this enzyme against
oxidative enzyme well established that various environmental stresses such as low temperature lead to the
increased generation of ROS, where SOD was proposed to be important in plant stress tolerance.
Peroxidase (POX)
The observed changes in POX activity under cold treatment included significant differences between
temperature treatments, sampling times (***P <0.001) and cultivars (*P <0.05) based on ANOVA results (Table 1).
The results of t-test showed significant differences between temperature treatments in POX activity on the second
(**P <0.01), fourth, and seventh (*P <0.05) days in cold-sensitive cultivar, Option 500 (cv.1), and on the second
(**P <0.01), fourth, and seventh (***P <0.001) days in cold-resistant cultivar, Zarfam (cv.2). The most increased
POX activity under low temperature was on the fourth day in both cultivars (354% and 916%, respectively in
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Intl J Agri Crop Sci. Vol., 5 (4), 401-409, 2013
comparison with the control) (Fig. 3). There was also a linear relationship between the increase of POX activity with
time in the period of low temperature in cold-sensitive cultivar, Option 500 (cv. 1), (**P <0.01) and in cold-resistant
cultivar, Zarfam (cv. 2), (***P <0.001). Along with a significant difference between control and treatment regression
slopes, there was a significant difference between control and treatment regression slopes in both cultivars (***P
<0.001) (Table 2). Therefore, POX activity was increased under low temperature with nearly the same pattern but
higher intensity in Zarfam cv.
Peroxidase activity ( A490 /
min/mg protein)
7
7
cv. 1
4
cv. 2
4
0
0
0
2
4
6
0
2
4
6
Sampling time (day)
Figure3. Changes in Peroxidase activity in cold-sensitive cultivar Option-500 (cv.1) and cold-resistant cultivar Zarfam (cv.2) in
control (continued line- 22/ 6 ºC) and Low-temperature (spotted line- 10/3 ºC) seedlings in different sampling times. Values are
means (n = 3) ± SE, but where bars are absent, the variation about the mean was less than the diameter of the symbol
Our results were not in agreement with Fahimirad (2010) findings. In that study, POX activity in cold-sensitive
canola cultivar was more than cold-resistant cultivar. Scebba et al. (1998), Janda et al. (2003) and Javadian et al.,
(2010) supported our results. There was increased peroxidase activity in corn (Zea mays) (Prasad, 1996) and
tobacco (Nicotiana tabacum) (Parvanova et al., 2004) under cold-stress condition. Therefore, POX enzyme can
contribute to the tolerance of plants including spring canola under cold stress.
( A240 / Catalase activity
min/mg protein)
2
2
cv. 1
cv. 2
1
1
0
0
0
2
4
6
0
2
4
6
Sampling time (day)
Figure 4. Changes in Catalase activity in cold-sensitive cultivar Option-500 (cv.1) and cold-resistant cultivar Zarfam (cv.2) in
control (continued line- 22/ 6 ºC) and Low-temperature (spotted line- 10/3 ºC) seedlings in different sampling times. Values are
means (n = 3) ± SE, but where bars are absent, the variation about the mean was less than the diameter of the symbol
Catalase (CAT)
Regarding catalase activity, cold treatment caused significant differences between temperature treatments,
sampling times and cultivars according to ANOVA (***P <0.001) (Table 1). The results of t-test showed significant
differences between temperature treatments in cold-sensitive cultivar in CAT activity on the second (***P <0.001),
fourth, and seventh (**P <0.01) days, and maximum increased CAT activity under low temperature was on the
second day in comparison with the control sample (101%). In cold-resistant cultivar, Zarfam (cv. 2), there were
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Intl J Agri Crop Sci. Vol., 5 (4), 401-409, 2013
significant differences between temperature treatments in CAT activity on the second, fourth, seventh days (***P
<0.001), and the largest increased activity was on the fourth day in comparison with the control sample (420%)
(Fig. 4). After the second day, CAT activity (the highest level) was constant in Option 500 cv. while CAT activity in
zarfam cv., after the fourth day, fluctuated (Fig. 4). In other words, the relationship between the increased CAT
activity and the time was linear only in cold-sensitive cultivar Option 500 (cv. 1) (*P <0.05) with a significant
difference between control and treatment regression slopes (***P <0.001) (Table 2). According to the results, the
CAT activity was increased under low temperature in both cultivars but with a more intensity in zarfam cv. and an
absolutely different pattern relative to Option 500 cv. These findings were confirmed by Prasad et al. (1996) and
Fahimirad (2010), who found that cold stress treatment could increase catalase activity.
Table 1. Mean squares (MS) of the three_balanced factorial ANOVA on the basis of completely randomized design (CRD)
for four physiological characters in two Canola cultivars
Degrees of
freedom
Temperature (T) 1
Cultivar (Cv.)
1
Sampling time (S) 3
T × Cv.
1
T×S
3
Cv. S
3
T × Cv. × S
3
Error
32
Total
47
CV%
S.O.V
ns
MS
Protein
***
1.891
***
21.160
***
4.953
*
0.737
***
0.457
*
0.105
ns
0.139
0.124
Superoxide dismutase
***
17.493
***
5.88
***
3.127
***
1.410
***
0.023
***
0.557
*
0.268
0.0772
Peroxidase
***
19.865
*
0.832
***
2.718
**
1.214
***
2.410
**
0.808
11.7%
9.26%
12.6%
0.144
Catalase
***
20.474
***
3.853
***
1.177
***
3.508
***
2.571
*
0.359
ns
**
0.298 0.598
0.102
11%
—nonsignificant (P > 0.05). *, **, *** Significant at P < 0.05, P < 0.01, and P < 0.001, respectively.
2
Table 2. Linear coefficient of determination (R ) and slope (b) comparisons of 4 physiological parameters in: cold-sensitive
cultivar Option-500 (cv.1) and cold-resistant cultivar Zarfam or at control (22/ 6 ºC) or at 20°C followed by transfer to Lowtemperature (10 /3 ºC) on day 12 for 7 d (2) vs. sampling times. SE = Standard error
Cultivars
t-value
22 /16 ºC
2
R (%)
b±SE
10 /3 ºC
2
R ( %)
b±SE
48.70
87.6
0.035 ± 0.011
b
0.060 ± 0.007
a
80.8
81.7
0.045 ± 0.007
a
0.108 ± 0.016
2.8
53.4
0.311 ± 0.584
b
2.399 ± 0.708
b
71.9
88
5.313 ±1.049
a
9.604 ±1.119
12.80
34.1
-0.097 ± 0.080
b
0.139 ± 0.061
b
58.1
82
0.702 ± 0.188
a
1.532 ± 0.227
2.80
29.70
-0.008 ± 0.016
a
-0.040 ± 0.020
b
44.1
10
0.147 ± 0.052
a
0.124 ± 0.117
1) Protein
ns
Cv.1
Cv.2
-0.71
**
-2.71
2) Superoxide
dismutase
Cv.1
Cv.2
13.14
***
-5.44
3) Peroxidase
Cv.1
Cv.2
-3.9
***
-5.93
4) Catalase
Cv.1
Cv.2
-2.84
ns
-1.37
ns
***
***
***
a
a
a
a
—nonsignificant (P > 0.05). *, **, *** Significant at P < 0.05, P < 0.01, and P < 0.001, respectively.
CONCLUSION
A significant increase in the amount of total soluble protein and the specific activity of antioxidant enzymes as
a result of low temperature in all sampling times was expected in comparison with the control in cold-resistant
cultivar, Zarfam cv. However, in cold-sensitive cultivar, option 500 (cv. 1), the increased antioxidant activity was
only observed under low temperature and there was no increase in total soluble protein content. Perhaps, the
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Intl J Agri Crop Sci. Vol., 5 (4), 401-409, 2013
increase in antioxidant enzymes was not high enough to lead to the increased total protein content. Moreover,
different proteins are involved in plants. So, the decrease or no change in the amount of some other protein under
cold stress is possible. On the other hand, there may be no correlation between antioxidant activity and the amount
of protein. Therefore, more explanation depends on the study of the quantity and quality of soluble total proteins as
well as correlation between protein and antioxidant enzymes.
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