International Journal of Scientific Research in Environmental Sciences, 2(6), pp.

209-219, 2014
Available online at http://www.ijsrpub.com/ijsres
ISSN: 2322-4983; 2014 IJSRPUB
http://dx.doi.org/10.12983/ijsres-2014-p0209-0219


209
Full Length Research Paper

Using Physiological Traits to Evaluating Resistance of Different Barley Promising
Lines to Water Deficit Stress

Soleyman Mohammadi
1
, Behzad Sorkhi Lalehloo
2
, Mahdi Bayat
3
, Soran Sharafi
4
, Farshad Habibi
*5



1
West Azerbaijan Agricultural and Natural Resources Research Center, Miyandoab, West Azerbaijan, Iran
2
Seed and Plant Improvement Institute, Cereal Department, Karaj, Albors, Iran
3
Islamic Azad University, Mashhad Branch, Mashhad, Razavi Khorasan, Iran
4
Islamic Azad University, Mahabad Branch, Mahabad
,
West Azerbaijan, Iran
5
Islamic Azad University, Miandoab branch, Miandoab,

West Azerbaijan, Iran
*Corresponding author: E-mail: f.h1356@gmail.com

Received 23 February 2014; Accepted 07 May 2014

Abstract. To study waterstress-tolerant barley varieties, 20 barley lines were cultivated under full irrigation and limited
irrigation conditions where irrigation was stopped at anthesis stage in two separate field experiments during the 2007-2009
growing seasons at the Saatloo Research Farm, Azerbaijan, Iran. The experiments were laid out using RCBD with three
replications. The results from combined analysis of variance in both normal and stress conditions indicated that there were
significant differences among genotypes with regard to all studied traits which were due to high variation among the genotypes.
It was found that the activity of enzymes including SOD, GPX and CAT were increased under drought stress conditions, so
that tolerant genotypes had more changes in enzyme activity. On the other hand, MDA, Dityrosine and 8-oHdg were increased
under stress conditions where sensitive genotypes had stronger enzyme activity. Calculations of the correlation coefficients
among the studied traits under both stress and normal conditions also indicated that there were negative and significant
differences between antioxidant activity, lipid, protein, and DNA decadence. Finally, with regard to all traits, it was revealed
that in normal conditions genotypes 18 and 19 were the best performing lines, whereas the genotype 14 was least adapted line.
Therefore, genotypes 18 and 19 showed higher levels of resistance to water stress and the genotype 15 was more sensitive to
the drought conditions. The results also indicated that selecting more tolerant genotypes under stress conditions was the way to
overcome water deficit stress under terminal drought conditions.

Keywords: Antioxidant enzymes, Waterstress, Barley, Terminal drought conditions.

Abbreviations: CAT- Catalase; GPX- Glutathione Peroxidase; MDA- Malondialdehyde; RCBD- Randomized Complete
Block Design; ROS- Reactive Oxygen Species; SOD- Superoxide Dismutase; 8-oHdg- 8-hydroxy-2'-deoxyguanosine.

1. INTRODUCTION

One of the most Environment restricting factors
negatively affecting plant growth in the majority of
the worlds agricultural lands could be defined as a
drought stress (Tas and Tas, 2007), and greatly limits
crop production worldwide (Zhang et al., 2010). As a
common consequence of drought stress, the
production of reactive oxygen species (ROS), such as
superoxide radical (O
2-
), hydrogen peroxide (H
2
O
2
),
and hydroxyl radical (OH) is increased (Valentovic et
al., 2006) which is due to the enhanced leakage of
electrons to molecular oxygen (Arora et al., 2002).
Plant cell mitochondria and chloroplast are the two
major intracellular generators of reactive oxygen
species (Pan et al., 2006). These ROS are among
reactive cytotoxic for cells (Movahhedy-Dehnavy et
al., 2009) which could cause severe damage to DNA,
proteins, and lipids, and also alteration of stop natural
metabolism of plants (Clement et al., 2008). Plants are
immobile and unable to escape stressful environments
(Chai et al., 2005). Hence, they use some strategies
(including enzymatic and non-enzymatic antioxidant
defense systems) to eliminate or reduce the toxicity of
ROS (Creissen and Mullineaux, 2002). Some
antioxidant enzymes, including SOD (Nayyar and
Gupta, 2006), CAT (Zhenmei et al., 2009), GPX
(Zhen et al., 2009), and GRD (Demiral et al., 2005)
also play key roles in the formation and degradation
of H
2
O
2
. Levels of damage may become limited by
enzymatic and non-enzymatic scavengers of free
radicals (Aroca et al., 2003).
Mohammadi et al.
Using Physiological Traits to Evaluating Resistance of Different Barley Promising Lines to Water Deficit Stress
210
Due to low annual precipitation, many regions of
Iran suffer from water deficit. Therefore, due to
drought stress, barley production is decreased.
Understanding the physiological and biochemical
mechanisms conferring drought tolerance; thus, could
play a crucial role in developing appropriate selection
methods and breeding strategies.
The aim of this study was to investigate the effect
of drought stress on the activities of 20 barley
promising line/ cultivar antioxidant enzymes (CAT,
GPX, and SOD), lipid, protein, and DNA oxidation
(MDA, Dityrosine and 8-oHdg content).

2. MATERIALS AND METHODS

To evaluating resistance of twenty barley promising
lines/cultivars (Table 1) to water deficit stress with
physiological traits such as enzymes up regulated or
down regulated, lipid and protein concentration two
separate field experiments (normal and drought stress
conditions) were conducted in the 2007-2009
cropping seasons at the Saatloo Agricultural Research
Station (1338 m altitude, 35N, 45E), West Azerbaijan,
Iran. Saatloo has a 375mm annual rainfall on a long-
term average with a soil texture of clay-loom (30%
clay, 53% silt, and 17% sand), 1.27% organic matter,
pH of 7.5, and an EC of 2.5dS/m. The experiments
were conducted using a randomized complete block
design (RCBD) with three replications. Control plots
were watered at stem elongation, flowering, and
grain-filling stages whereas irrigation of stress plots
were stopped before occurring the flowering phase.
Before planting, the soil tillage was practiced based
on the research station/s routine. Fertilizers were
applied before sowing (100kg ha
-1
P
2
O
3
and 50kg ha
-1

N) in October, and at stem elongation (50kg ha
-1
N) in
March.

Table 1: Codes and Parentage of the studied barley lines/cultivars
Code Genotpye Code Genotpye
1 Rhn-03//L.527/NK1272 11 Mnitou//Alanda/Zafraa
2 Manitou//Alanda/Zafraa 12 Kny/K-273
3 Pamir-149/ Victoria 13 Pamir-065
4 AcuarioT75/Azaf 14 Pamir-168
5 Pamir-146//EA389-3/EA475-4 15 Prodcutiv/3/Rono//Alger/Ceres362-1-1
6 Alpha/Durra/Pamir-160 16 Belt67-1608/Slr/3/Dicktoo/Cascade//Hip/4/CWB117-77-9-7
7 Pamir-013/Sonata 17 Belt67-1608/Slr/3/Dicktoo/Cas
8 Robur/WA2196-68//Wysor 18 U.Sask.1766/Api//Cel/3Weeah/4/Lignee527/NK1272/5/Express
9 Bugar/DZ48-232 19 TWWd85-37/Kavir
10 Rhn-03//Lignee527/NK1272/5/Lignee527/Chn-
01/4/Lignee527/
20 Bahman

Table 2: Analysis of variance for contents of 8-hydroxy-2-deoxyguanosine (8-oHdg), Dityrosine, Malondialdehyde (MDA),
Glutathione Peroxidase (GPX), Catalase (CAT) and Superoxide Dismutase (SOD) measured under normal conditions
Mean
Square

Df GPX
(u mg
-1

protein)
CAT
(u mg
-1

protein)
SOD
(u mg
-1

protein)
8-oHdg
(nmol mg
protein)
Dityrosine
(nmol mg
protein)
MDA
(nmol mg
protein)
Year 1 429.4 * 630.2 35811.1 61.6 598.5 154.1
Ea 4 40.0 811.7 6790.2 12.3 112.9 51.8
Genotype 19 27497.4 ** 27349.5 ** 4869926.9
**
226.9 ** 4253.0 ** 3537.7 **
Genotype Year 19 47.9 96.2 18301.8 1.6 32.4 14.8
Eb 76 72.6 285.5 14080.4 3.3 52.3 23.7
CV (%) 5.7 5.7 5.9 10.9 4.8 5.2
* and **: significant at the 5% and 1% levels of probability, respectively. Mean squares without symbols are non-significant

To quantify antioxidant enzymatic activity in 20
barley genotype/ line, fifteen leaf samples were taken
randomly from each plot and were placed in liquid
nitrogen and then stored at -80C pending
biochemical analysis. In order to prepare samples for
the enzyme assays and protein measurement, the
leaves from each plant were washed with distilled
water and homogenized in a 0.16M Tris buffer
(pH=7.5) at 4C. Then, 0.5 mL of total homogenized
solution was used for protein determination using the
Lowry et al. (1951) method. Based on the amount of
protein per volume of homogenized solution, the
following enzymes were assayed in the volume
containing a known protein concentration in order to
calculate the specific activities of the enzymes. In this
study, chemical and biochemical characteristics were
measured based on blow methods:

International Journal of Scientific Research in Environmental Sciences, 2(6), pp. 209-219, 2014
211
Catalase (CAT) activity Paglia and Valentine
(1987)
(1)
Glutathion peroxidase (GPX) activity Paglia and Valentine
(1987)
(1)
Superoxide dismutase (SOD) activity Dhindsa et al. (1980) (2)
Lipid peroxidation (malondialdehyde Content) Sairam et al. (1998) (3)
Protein Damage (dityrosine content) Amado et al. (1984) (4)
Determination of 8-Hydroxy-2-Deoxyguanosine (8-oHdg) Bogdanov et al. (1999) (5)

Table 3: Analysis of variance for contents of 8-hydroxy-2-deoxyguanosine (8-oHdg), Dityrosine, Malondialdehyde (MDA),
Glutathione Peroxidase (GPX), Catalase (CAT) and Superoxide Dismutase (SOD) measured under normal conditions
Mean Square
Df
GPX
(u mg
-1

protein)
CAT
(u mg
-1

protein)
SOD
(u mg
-1

protein)
8-oHdg
(nmol mg
protein)
Dityrosine
(nmol mg
protein)
MDA
(nmol mg
protein)
Year 1 644.0 806.0 11388.0 9.6 29.0 385.2
Ea 4 164.4 974.4 26298.0 0.8 98.6 36.2
Genotype 19 34074.8 ** 29755.4 ** 6670689 ** 364.6 ** 5172.3 ** 3999.7 **
Genotype Year 19 159.0 280.7 6608.8 6.1 * 11.1 77.7
Eb 76 166.1 312.8 14995.0 3.5 30.1 112.1
CV (%) 7.2 5.2 5.1 9.9 3.2 9.0
* and **: significant at the 5% and 1% levels of probability, respectively. Mean squares without symbols are non-significant.

2.1. Statistical analysis

Main and interaction effects of experimental factors
were determined using analysis of variance (ANOVA)
in SAS software Ver. 9.12. The assumptions of
variance analysis were tested by ensuring
homogeneity of the residuals. The LSMEANS
command was used to compare means at a P<0.05
probability. To determine the relationship between
measured parameters and the seed yield, correlation
analyses were applied to the data using PROC CORR
in SAS.

3. RESULTS AND DISCUSSIONS

According to the results obtained, the combined
analyses of variance confirmed significant differences
among antioxidant activity and macromolecule
decadence of the genotypes under both normal and
stress conditions (Tables 2 and 3). These results
suggested that the genotypes studied show genetic
variations in terms of their adaption and reactions
under water stress conditions. Misra and Gupta (2006)
reported that production and activation of antioxidant
enzymes vary in different genotypes widely and effect
by stress regimes, growth stages, and environmental
conditions extremely. The results also indicated that
the year and year genotype effects had no
significant difference either under normal or stress
conditions. Thus it was revealed that the studied
genotypes could perform similarly regardless of the
year during which they have been investigated.
Sairam et al. (2002) stated that plants under stress
conditions (either drought or salinity) produce a
collection of antioxidant enzymes such as SOD, CAT,
GRD, Ascorbat Peroxidase, Glutathione transferase,
and Peroxidase to protect themselves from damage
caused by reactive oxygen species (Hernandez et al.,
2000). Johnson et al. (2003) believed that the
activities of antioxidant enzymes and the amounts of
antioxidants elevated under drought stress vary among
several plant species and even between two cultivars
of the same species (Selote and Khanna-Chopra,
2004). Furthermore, the reactions of the plants to
water stress could differ significantly at various
organizational levels depending upon the intensity and
duration of stress as well as plant species and
development stages (Dacosta and Huang, 2007).

3.1. Glutathione Peroxidaes (GPX)

Means comparison of enzyme GPX (Tables 4 and 5)
indicated that in normal conditions genotypes 1, 11,
18, 19 and genotypes 14, 15, 20 had maximum and
minimum enzyme content, respectively; according to
Aronachalam and Bandyopandyay rankings (1984),
these are situated in the upper and lower positions,
respectively. Means comparison of traits in stress
conditions also indicated that genotypes 1, 11, 18, 19
and genotypes 14, 15 had minimum and maximum
enzyme content, respectively; so according to
Aronachalam and Bandyopandyay rankings (1984),
these are situated in the upper and lower positions,
respectively. It can be concluded that the amounts of
GPX production follow a pattern among studied
genotypes, such that the genotypes with more GPX in
normal conditions had more GPX in stress conditions
as compared with other genotypes. Also, the average
and standard deviations of GPX content were 148.24
and 67.70 in normal conditions and 178.22 and 5.36 in
Mohammadi et al.
Using Physiological Traits to Evaluating Resistance of Different Barley Promising Lines to Water Deficit Stress
212
stress conditions, respectively (Table 6). So it can be
stated that although the GPX production varies from
one genotype to another, it was higher under stress
conditions in all studied genotypes. Bybordi et al.,
(2010) stated that, due to increasing ROS and
macromolecule decadence in plants, the levels of
antioxidant enzymes in stress conditions increase
significantly in environmental stresses such as drought,
salinity, and heat. The ranges of antioxidant levels in
normal and stress conditions were 201.00 and 222.17,
respectively; hence, it can be said that the level of
GPX in stress conditions was higher than in normal
conditions. These results had enough correlation with
the studies of Ananeiva et al. (2002). The results from
correlation coefficients between both traits of GPX
production and other traits in both normal and stress
conditions (Table 7) indicated that there was a
positive and significant correlation between GPX
levels and CAT and SOD levels, yet a negative and
significant correlation with MDA, Dityrosine, and 8-
oHdg. These results were logical and predictable,
because of reverse relations between antioxidant
enzyme contents and the decadence of
macromolecules due to the free radicals scavenging
effects of GPX. Therefore, genotypes with high
antioxidant enzyme content are the best volunteer
genotypes to cultivate in stress conditions because of
their higher resistance to drought conditions. Dat et al.
(2000) believed that plants with higher levels of
antioxidant enzymes (GPX and CAT) had more
ability to scavenge ROS in stress conditions, which
makes them more resistant to stress conditions. Thus,
these genotypes can be used for cultivation in regions
with a high potential for the occurrence of stress.

3.2. Catalase enzyme (CAT)

Means comparison of CAT levels indicated that in
normal conditions genotypes 11, 18, 19 and genotype
14 had maximum and minimum levels, respectively
(Table 4), so by the Aronachalam and Bandyopandyay
rankings method, these are situated in the upper and
lower positions, respectively. In drought conditions
genotypes 18, 19 and genotype 14 had maximum and
minimum CAT levels, respectively (Table 5), so
according to Aronachalam and Bandyopandyay
rankings, these are situated in the upper and lower
positions, respectively. Calculations of statistical
parameters indicated that average and standard
deviations were 296.64 and 67.51 in normal
conditions, and 337.36 and 70.42 in stress conditions,
respectively (Table 6). Therefore, according to these
results, the levels of CAT as for GPX were not only
different in all studied traits but were also higher in
stress conditions. On the other hand, the ranges of
change of CAT were 226.17 and 234.17 in normal and
stress conditions, respectively, which showed that the
amounts of enzyme were decrease in stress conditions.
The results from the correlation coefficient analysis of
CAT production and other traits in both normal and
stress conditions (Table 7) indicated that these had a
positive and significant correlation with contents of
GPX and SOD, yet a negative and significant
correlation with MDA, Dityrosine, and 8-oHdg. CAT
is one of the most important antioxidant enzymes
which plays a key role in decreasing damage from
peroxides. The increase of this enzyme under stress
conditions, therefore, causes a decrease in damage to
plasma membrane lipids, proteins, DNA, and RNA
within plant cells. An increase in antioxidant
production leads to a decrease in the decadence of
lipids, proteins, DNA, and RNA, so there is a negative
relation between antioxidant content and
macromolecule decadence. These results correspond
with the results from Prasad (2003). In another work,
Noctor and Foyer (1998) reported a positive
correlation in the activity of antioxidant enzymes.
Acclimation of plants to drought is considered to
promote antioxidant defense systems to face the
increased levels of activated oxygen species (AOS),
which in turn, causes membrane damage through lipid
peroxidation and indicated by malondialdehyde
(MDA) content, which is a main parameter for
evaluating membrane oxidation extent and is toxic for
cells (Shao et al., 2005). The same results were
reported by Dolatabadian et al. (2008), who showed
that salt stress increased lipid peroxidation (MDA
content) in canola cultivars.

3.3. Superoxide Dismutase enzyme (SOD)

SOD is one antioxidant enzyme that has a key role in
the process of scavenging hydrogen peroxides.
Therefore, this enzyme is an important criterion for
detecting barley genotypes resistant to drought
stresses. Superoxide dismutases (SODs), a group of
metalloenzymes, are considered the first defense
against ROS (Gratao et al., 2005). The results from
means comparison for SOD indicated that the contents
of SOD in genotypes 11, 18, 19 and genotypes 14, 15,
20 had maximum and minimum levels of SOD,
respectively (Tables 4 and 5), so according to
Aronachalam and Bandyopandyay rankings (1984),
these are situated in the upper and lower positions,
respectively. Calculations of statistical parameters
also indicated that average and standard deviations
were 1984.28 and 900.92 in normal conditions and
2401.46 and 1054.41 in stress conditions, respectively
(Table 6). According to these results, the levels of
SOD as for CAT and GPX were not only different in
all studied traits but were also higher in stress
conditions. Jin et al. (2006) stated that an increase in
International Journal of Scientific Research in Environmental Sciences, 2(6), pp. 209-219, 2014
213
drought stress causes an increase in activity of SOD
after 24 hours. It can be concluded that this rise is a
consequence of stress conditions to scavenging free
radicals. This enzyme is known as a main component
of the plant protective mechanism to overcome
environmental stresses. The ranges of SOD changes
in both normal and stress conditions were 2610.17 and
3370.67, respectively, which reveals that the ranges of
change in stress conditions are wider than in normal
conditions. The results from correlation coefficients
between SOD production and other traits in both
conditions indicated that there is a positive and
significant correlation between GPX and CAT and a
negative and significant one with MDA, Dityrosine,
and 8-oHdg (Table 7). Shan and Guo (2009) stated
that environmental stresses increased activity of
almost all antioxidant enzymes with a positive
correlation as well as a negative correlation with
macromolecule decadence. They also stated that
increased levels of antioxidant enzymes caused an
increase in plant resistance to environmental stresses
(Mirnoff, 1998).

Table 4: Means comparison of the different traits of barley genotypes and Aronachalam and Pandyopandyay Ranking in two
years under normal condition

3.4. Hydroxyl-2-deoxy guanosine-8 (8-oHdg)

Means comparison of 8-oHdg indicated that
genotypes 11 and genotypes 18, 19 had maximum
contents in both normal and stress conditions (Tables
4 and 5), so according to Aronachalam and
Bandyopandyay rankings (1984), these are situated in
the upper and lower positions, while genotypes 14, 15
and 20 had minimum 8-oHdg contents in both normal
and stress conditions, so according to Aronachalam
and Bandyopandyay rankings (1984), these are
situated in the upper and lower positions. Also,
calculations of statistical parameters indicated that
average and standard deviations were 16.52 and 6.15
in normal conditions, while they were 19.02 and 7.79
in stress conditions, respectively (Table 6). Overall, it
can be concluded that the content of 8-oHdg among
all studied genotypes are very different and
particularly more in stress conditions. The important
point in this study was the lesser amounts of 8-oHdg
Code GPX Rank CAT Rank SOD Rank
8-
oHdg
Rank Dityrosine Rank MDA Rank
Total
Rank
1 255.5
a
8
358.0
b
5
2711.0 b
4
12.3 ed
4.5
140.2 ef
5.5
86.3 ed
4.5
31.5
2 192.0
b
7
325 bc
4.5
2640.2 b
4
12.3 ed
4.5
137.7 ef
5.5
82.5 e
5
30.5
3 108.7
e
4
297.8
c
4
2126.8 c
3
13.7 d
4
146.0 df
5
91.0 ce
4
24
4 183.0
bc
6.5
291.7
c
4
2579.8 b
4
12.7 d
4
136.8 ef
5.5
84.7 de
4.5
28.5
5 86.7 fg 2.5 236 ed 2.5 1118.3 d 2 20.3 b 2 168.7 b 2 111.2 b 2 13
6 103.5
ef
3.5
230 ed
2.5
994.3 d
2
19.5 bc
2.5
167.8 b
2
109.7 b
2
14.5
7 127.0
d
5
299.0
c
4
2110.8 c
3
15.7 cd
3.5
159.5 bd
3
97.7 c
3
21.5
8 104.8
e
4
243.5
d
3
1137.0 d
2
21.3 b
2
162.2 bc
2.5
108.7 b
2
15.5
9 187.2
bc
6.5
345.0
b
5
2549.5 b
4
15.5 d
4
140.0 ef
5.5
87.8 ce
4
29
10 84.0 g 2 236 ed 2.5 962.5 d 2 21.8 b 2 166.0 bc 2.5 110.0 b 2 13
11 243.0
a
8
398 a
6
3260.8 a
5
8.0 f
6
109.2 g
7
54.2 f
6
38
12 176.8
bc
6.5
325 bc
4.5
2550.5 b
4
12.0 de
4.5
133.8 f
6
83.2 e
5
30.5
13 129.7
d
5
290.8
c
4
2123.7 c
3
15.8 cd
3.5
161.0 bd
3
84.0 de
4.5
23
14 61.8 h 1 185.2 f 1 695.0 e 1 27.7 a 1 195.2 a 1 138.3 a 1 6
15
54.5 h
1
205.5
ef
1.5
650.7 e
1
26.8 a
1
193.5 a
1
137.5 a
1
6.5
16 172.3
c
6
344.5
b
5
2461.5 b
4
14.3 d
4
134.0 f
6
82.3 e
5
30
17 131.0
d
5
292.8
c
4
2000.8 c
3
15.5 d
4
152.2 ce
4
94.0 cd
3.5
23.5
18 255.2
a
8
402.2
a
6
3127.0 a
5
8.5 fe
5.5
109.8 g
7
57.2 f
6
37.5
19 249.8
a
8
411.3
a
6
3229.7a
5
8.7 fe
5.5
111.2 g
7
57.0 f
6
37.5
20
58.3 h
1
214.8
ef
1.5
655.5 e
1
27.8 a
1
199.3 a
1
130.5 a
1
6.5
Means within each column with common letter(s) are not significantly different at 5% of probability.
Mohammadi et al.
Using Physiological Traits to Evaluating Resistance of Different Barley Promising Lines to Water Deficit Stress
214
in variants resistant to drought stress in comparison
with genotypes sensitive to drought stresses, so it
would be as a result of more production of antioxidant
enzymes (GPX, CAT, and SOD) in genotypes
resistant to drought stress. Manavalan et al. (2009)
showed that enzymatic antioxidant content played an
important role in scavenging harmful oxygen species.
The activities of antioxidant enzymes were altered
when plants were subjected to stress. Previously, an
increase in the level of antioxidants was reported with
an increase in stress intensity in maize and soybean by
Vasconcelos et al., (2009). Also, the ranges of trait
changes were 19.83 and 24.83 in both normal and
stress conditions; it suggests that the change of 8-
oHdg content in stress conditions was higher than in
normal conditions. On the other hand, the results from
correlation coefficients between 8-oHdg production
and other traits in both normal and drought conditions
(Table 7) revealed that there is a negative and
significant correlation between 8-oHdg content and
other traits such as GPX, CAT, and SOD, yet a
positive and significant correlation with Dityrosine
and MDA. Moreover, Lee et al. (2009) reported a
positive and significant correlation between CAT,
SOD, and Ascorbate Peroxidase (APX) under both
well-irrigated and water-deficit-stress conditions.
Furthermore, Lobato et al. (2008) also found a
positive and significant correlation between contents
of antioxidants.

Table 5: Means comparison of the different traits of barley genotypes and Aronachalam and Pandyopandyay Ranking in two
years under stress condition

3.5. Dityrosine

The protein content of plants is a fraction that is most
sensitive to oxidative stress due to high trends of ROS
to the amino acids of proteins for deactivating them.
Tompson et al. (1987) introduced Dityrosine as a
criterion for determining intracellular proteins. Means
comparison of Dityrosine revealed that genotypes 11,
Code GPX Rank CAT Rank SOD Rank
8-
oHdg
Rank Dityrosine Rank MDA Rank
Total
Rank
1 296.3
a
8
409 bc
5.5
2912 b
5
12.7 gi
8
156.2 ij
9.5
117.7
bd
3
39
2 222.2
b
7
352.3
d
4
2932 b
5
13.3 fh
8
154.2 ij
9.5
108.5
cd
3.5
37
3 126.7
ef
3.5
348.7
d
4
2353 c
4
15.8 eg
6
168.0 gh
7.5
109.0
cd
3.5
28.5
4 211.5
b
7
360.7
d
4
3036 b
5
14.7 eg
6
159.8 hi
8.5
116.0
bd
3
33.5
5
104.0 f
3
273.2
ef
2.5
1662 d
3
26.5 b
2
197.2 c
3
127.5
bc
2.5
16
6 135.7
e
4
273.0
ef
2.5
1284e
2
22.5 cd
3.5
194.5 cd
3.5
127.7
bc
2.5
18
7 174.8
d
5
353.2
d
4
2536 c
4
17.5 e
5
172.7 fg
6.5
118.0
bd
3
27.5
8 124.0
ef
3.5
264.7
ef
2.5
1338 e
2
22.3 d
4
193.0 ce
4
130.3
bc
2.5
18.5
9 206.3
b
7
369.0
d
4
2927 b
5
15.7 eg
6
155.2 ij
9.5
109.8
bd
3
34.5
10 102.7
fg
2.5
278.0
e
3
1621 d
3
26.3 bc
2.5
183.7 df
5
132.2 b
2
18
11 289.5
a
8
422 ab
6.5
4243 a
6
9.8 hi
8.5
125.8 k
11
81.3 e
5
45
12 206.2
b
7
351.5
d
4
2986 b
5
14.8
efg
6
156.2 ij
9.5
104.2 d
4
35.5
13 174.5
d
5
365.0
d
4
2479 c
4
16.8 ef
5.5
181.5 ef
5.5
115.5
bd
3
27
14
74.3 h
1
215.7
g
1
871.8 f
1
33.7 a
1
211.0 b
2
158.7 a
1
7
15
74.2 h
1
239.5
fg
1.5
908.2 f
1
32.8 a
1
222.8 a
1
169.0 a
1
6.5
16
205 bc
6.5
374 cd
4.5
2448 c
4
17.2 ef
5.5
145.8 j
10
108.0
cd
3.5
34
17
177 cd
5.5
362.3
d
4
2430 c
4
17.7 e
5
179.7 f
6
113.0
bd
3
27.5
18 294.7
a
8
449.8
a
7
4037 a
6
8.8 i
9
128.8 k
11
72.8 e
5
46
19 288.3
a
8
448.7
a
7
4147 a
6
8.8 i
9
128.7 k
11
69.8 e
5
46
20 76.7
gh
1.5
236.2
fg
1.5
880.3 f
1
32.5 a
1
222.8 a
1
158.2 a
1
7
Means within each column with common letter(s) are not significantly different at 5% of probability.
International Journal of Scientific Research in Environmental Sciences, 2(6), pp. 209-219, 2014
215
18, 19 had maximum levels of Dityrosine in both
normal and stress conditions (Tables 4 and 5), so
according to Aronachalam and Bandyopandyay
(1984) rankings, these are situated in the upper and
lower positions. Genotypes 14, 15, 20 in normal
conditions (Tables 4) and genotypes 15, 20 (Table 5)
in stress conditions had minimum levels of Dityrosine,
so according to Aronachalam and Bandyopandyay
rankings (1984), these are situated in the upper and
lower positions. Also, calculations of statistical
parameters indicated that average and standard
deviations were 151.20 and 26.62 in normal
conditions and 171.88 and 29.36 in stress conditions,
respectively (Table 6). Overall, it can be concluded
that the contents of Dityrosine among studied
genotypes are very different. Nevertheless, the
amounts of Dityrosine were higher in stress conditions
in all studied traits. Thus, an important point is that
there are lesser amounts of Dityrosine in genotypes
resistant to drought stresses than those that are
sensitive, due to the production of more antioxidant
enzymes (GPX, CAT, and SOD) in resistant
genotypes. The ranges of trait change were 90.17 and
97.00 in both normal and stress conditions (Table 6);
it suggests that the changes of Dityrosine content in
stress conditions were more than in normal conditions.
On the other hand, the results from the correlation
coefficient between Dityrosine production and other
traits in both normal and drought conditions (Table 7)
revealed that there is a negative and significant
correlation between 8-oHdg contents and other traits
such as GPX, CAT, and SOD, while there is a positive
and significant correlation with 8-oHdg and MDA.

Table 6: Univariate statistics for the traits studied under normal and stress conditions
Statistics Condition
GPX
(u mg
-1

protein)
CAT
(u mg
-1

protein)
SOD
(u mg
-1

protein)
8-oHdg
(nmol mg
protein)
Dityrosine
(nmol mg
protein)
MDA
(nmol mg
protein)
Maximum
Normal 255.50 411.33 3260.83 27.83 199.33 138.33
Drought Stress 296.33 449.83 4242.50 33.67 222.83 169.00
Minimum
Normal 54.50 185.17 650.67 8.00 109.17 54.17
Drought Stress 74.17 215.67 871.83 8.83 125.83 69.83
Range
Normal 201.00 226.17 2610.17 19.83 90.17 84.17
Drought Stress 222.17 234.17 3370.67 24.83 97.00 99.17
Average
Normal 148.24 296.64 1984.28 16.52 151.20 94.38
Drought Stress 178.22 337.36 2401.46 19.02 171.88 117.36
Standard
Deviation
Normal 67.70 67.51 900.92 6.15 26.62 24.28
Drought Stress 75.36 70.42 1054.41 7.79 29.36 25.82

Table 7: Correlation coefficient for the content of 8-hydroxy-2-deoxyguanosine (8-oHdg), Dityrosine, Malondialdehyde
(MDA), Glutathione Peroxidase (GPX), Catalase (CAT) and Superoxide Dismutase (SOD) studied under normal (above
diagonal) and stress (below diagonal) conditions

3.6. Malondialdehyde (MDA)

Fatty acids and lipids are more sensitive to ROS and
fall under oxidation rapidly. The studies revealed that
cellular and organellar membranes are the first parts
damaged in stress conditions affected by reactive
oxygen species (Candan and Tarhan, 2003) because of
the emission of cellular electrolytes following the
degradation of membranes and the increase in MDA
contents (Demiral and Turkan, 2005). So it can be
said that MDA is an appropriate candidate for
determining the levels of plant response to
environmental stresses (Bandeoglu et al., 2004). We
used MDA as a lipid peroxidation index. Means
comparison of results indicated that genotypes 11, 18,
19 and genotypes 14, 15, 20 had maximum and
minimum MDA content in both normal and drought
stress conditions, respectively (Tables 4 and 5), so
according to Aronachalam and Bandyopandyay
rankings (1984), these are situated in the upper and
lower positions, respectively, Also, calculations of
statistical parameters indicated that the average and
standard deviations were 94.38 and 24.28 in normal
conditions and 117.36 and 25.82 in stress conditions,
respectively (Table 6). Overall, it can be concluded
that the content of MDA among the studied genotypes
is very different. Nevertheless, the amounts of
Dityrosine were more in stress conditions in all
Traits GPX CAT SOD 8-oHdg Dityrosine MDA
GPX 0.95
**
0.94
**
-0.91
**
-0.92
**
-0.91
**

CAT 0.95
**
0.97
**
-0.93
**
-0.95
**
-0.96
**

SOD 0.94
**
0.96
**
-0.97
**
-0.95
**
-0.96
**

8-oHdg -0.93
**
-0.96
**
-0.94
**
0.97
**
0.97
**

Dityrosine -0.92
**
-0.94
**
-0.96
**
0.94
**
0.97
**

MDA -0.87
**
-0.92
**
-0.94
**
0.94
**
0.95
**

**: significant at 1% level of probability
Mohammadi et al.
Using Physiological Traits to Evaluating Resistance of Different Barley Promising Lines to Water Deficit Stress
216
studied traits. Thus, an important point is that there is
a lesser amount of MDA in genotypes resistant to
drought stresses than in those sensitive due to the
production of more antioxidant enzymes (GPX, CAT,
and SOD) in resistant genotypes in comparison with
sensitive genotypes. Bhattacharjee and Mukherjee
(2002) believed that the content of MDA in plant
tissue is a representation of the levels of a membrane
lipids decadence, because of their release following
lipid peroxidation and degradation. They stated that
levels of MDA in plants under stress are higher than
those in normal conditions. Also, the ranges of trait
change were 99.17 and 84.17 in both normal and
stress conditions; this suggests that the changes of
MDA content in stress conditions were more than
those in normal conditions. On the other hand, the
results from correlation coefficients between MDA
production and other traits in both normal and drought
conditions (Table 7) revealed that there is a negative
and significant correlation between MDA content and
other traits such as GPX, CAT, and SOD, while a
positive and significant correlation exists with 8-oHdg
and Dityrisine. Meloni et al. (2003) reported a
negative correlation between the increasing activity of
GPX with a decrease in lipid peroxidation. Also,
Shalata and Neumann (2001) believed that antioxidant
enzymes lead to a decrease in lipid peroxidation and
MDA contents due to scavenging ROS, so that an
increase in the activity of antioxidant enzymes leads
to a decrease in lipid degradation, and in turn that
caused an increase in plant resistance to oxidative
stresses (Sreenivasulu et al., 2000).

4. CONCLUSION

The production of reactive oxygen species (ROS)
upon occurrence of environmental stresses such as
salinity, drought, and cold is one of the substantial
factors damaging plants. In such conditions plants
respond to stress through enzymatic and non-
enzymatic systems in order to either neutralize or
decrease the levels of damage due to ROS. According
to results, there were significant differences among
traits studied under both normal and stress conditions
for deter mine the resistant barley cultivar/line. The
results demonstrated that the traits were significantly
different for the genotypes with regard to their levels
of adaptation and resistance against drought stress. It
was also revealed that the antioxidant levels varied in
different genotypes and were higher under stress
conditions. Additionally, a negative and significant
correlation between antioxidant production and
macromolecule damage was found, so that the
production of more antioxidant enzymes led to the
production of less macromolecular damage increasing
plant tolerance to stress conditions due to the
protective effects of cell membranes on lipids. Overall,
it can be concluded that genotypes 18 and 19 were the
best performers and acquired acceptable levels of
tolerance under stress conditions. Once under drought
conditions, these genotypes were capable of the
production of more antioxidant enzymes offering less
reduction of damage to the macromolecules. The
results of this study also revealed that SOD, CAT, and
GPX respectively could play key roles in plant
tolerance to drought stress. Lipids, proteins, and DNA
are more sensitive to drought stress conditions,
respectively. Therefore, determination of SOD
activity and lipid peroxidation are useful indices to
select the best genotypes for cultivating in regions
with high risk of drought conditions.

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International Journal of Scientific Research in Environmental Sciences, 2(6), pp. 209-219, 2014
219



Assistant Professor Dr. Soleiman Mohammadi obtained his first degree from Tabriz University in
Agronomy and Plant Breeding in 1992. He later pursued master in Agronomy in Tabriz University and
graduated in 1995. Dr. Soleiman Mohammadi received his doctorate from Tehran Azad University of
Science and Research branch in 2003 with major in Crop Physiology. He has published over 100
scientific articles in professional journals/proceeding and sits as the Editorial Member for two Iranian
journals. At present, Dr. Mohammadi works in West Azerbaijan Agricultural research and Natural
Recourses Center as a Cereal Researcher.





Mahdi Bayat is a Ph.D candidate in Department of Agronomy, Faculty of Agriculture, Urmia University,
Urmia, Iran. He received his first degree from guilan University in 2005 awarded with Master of Science
in agricultural science, plant breeding. His current research is focuses on saffron (Crocus sativus L.) in
respect of agronomy and breeding. To date, he has published several scientific articles in ISI and Iranian
journals, also has published 4 books, in Iran, related to apply SAS, MINITAB, SPSS and MSTAT-C
softwars in agricultural researches.






Dr. Soran Sharafi obtained his PhD in Agronomy at the Islamic Azad University of Science and
Research of Tehran Iran in 2011. He is currently Assistant Professor in Agronomy at the Islamic Azad
University of Mahabad Iran teaching Agroecology & Agronomy. His research has focused on Plant
Stress & Plant Nutrition. He has published several scientific articles in professional journals and
conference proceedings. He has also participated in various conferences, talks and seminars.







Assistant Professor Dr. Farshad Habibi obtained his PhD in Agronomy at the Islamic Azad University
of Science and Research of Tehran Iran in 2011. He has published several scientific articles in
professional ISI journals and Iranian journals. He is currently Assistant Professor in Agronomy at the
Islamic Azad University of Miandoab Iran, teaching plant physiology & Agronomy. He has also
participated in various conferences, talks and seminars. His research has focused on cereal Stress &
Nutrition also has published 1 book, in Iran, related to application of Information Technology in
agricultural (ISBN: 978-964-10-0848-4).