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Antioxidant Activity in Salt-Stressed Barley Leaves: Evaluating Time- and

Age-Dependence and Suitability for the Use as a Biochemical Marker in
Breeding Programs

Article  in  Journal of Agronomy and Crop Science · April 2014

DOI: 10.1111/jac.12068

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 J Agro Crop Sci (2014) ISSN 0931-2250

SALINITY STRESS

Antioxidant Activity in Salt-Stressed Barley Leaves:

Evaluating Time- and Age-Dependence and Suitability for
the Use as a Biochemical Marker in Breeding Programs
Y. Fan1, M. Zhu1, S. Shabala1, C. D. Li2, P. Johnson1 & M. X. Zhou1
1 University of Tasmania, Kings Meadows, TAS, Australia
2 Department of Agriculture and Food Western Australia, South Perth, WA, Australia

Keywords Abstract
antioxidant enzyme; barley; reactive oxygen
species; salt tolerance Soil salinity disturbs the equilibrium between reactive oxygen species (ROS) pro-
duction and removal, leading to a dramatic increase in ROS concentration and
Correspondence oxidative damage. Enzymatic scavenging is one of the two main mechanisms
M. X. Zhou involved in ROS detoxification in plants. This study has investigated the role of
University of Tasmania
major antioxidant (AO) enzymes in mitigating salinity-induced oxidative stress in
P.O. Box 46
plant shoots. Firstly, two barley varieties were used to evaluate the activity of major
Kings Meadows
TAS 7249 AO enzymes in different leaves and at different times after salt treatment. Our
Australia results showed that AO enzyme activities had strong tissue and time specificity. A
Tel.: +61 363 365 204 further study was conducted using six barley varieties contrasting in salinity toler-
Fax: +61 363 365 395 ance. AO enzyme activities and proline contents were measured in the third leaves
Email: Meixue.Zhou@utas.edu.au of seedlings after plants were treated with 240 mM NaCl for 10 days. No significant
correlation was revealed between leaf AO activity and either plant grain yield or
Accepted March 26, 2014
plant survival rate under salt stress. In contrast, a significant increase in leaf proline
doi:10.1111/jac.12068 content under salt stress was found in all sensitive varieties, while in most tolerant
varieties, salt stress did not change leaf proline level. It is concluded that although
salinity induces changes in leaf AO enzyme activities, the changes cannot be used
as biochemical indicators in breeding for salinity tolerance.

to a lack of understanding of salt tolerance mechanisms

Introduction
and reliable, rapid, inexpensive and convenient screening
As one of the major abiotic stresses, soil salinity severely techniques (Zhu 2000, Munns and James 2003, Chen et al.
affects agricultural productivity. It is estimated that more 2005).
than 20 % of global irrigated land is affected by salinity Soil salinity affects plants in two phases: rapid osmotic
(Yeo 1999) and around two million hectares of broadacre stress that reduces shoot growth and slower ionic stress
farmland is affected by dry land salinity, with a further six which hastens senescence of older leaves due to elevated
million hectares at risk. Thus, the need to breed salt-toler- leaf Na+ content (Munns and Tester 2008). Osmotic
ant crops is evident. Two main approaches are used for stress impacts plant growth by reducing cell expansion
improving plant salt tolerance: (i) traditional breeding to and elongation rates, which leads to smaller and thicker
explore natural genetic variations through direct selection leaves, and down-regulating photosynthesis by immedi-
under stress or through mapping quantitative trait loci ately reducing stomatal aperture (Bradford 1976). The
(QTL) and a subsequent marker-assisted selection (MAS; main site of Na+ toxicity in most plants is the leaf blade
Flowers 2004, Lindsay et al. 2004) and (ii) genetic manipu- (Munns 2002). Plants response to ionic stress through
lation techniques to produce transgenic plants with new Na+ exclusion, which requires a good control of net
genes or different expression levels of existing genes to delivery of Na+ from root to shoot, and through tissue
improve plant salt tolerance (Cuin and Shabala 2007). In tolerance. Tissue tolerance is achieved by increasing
recent decades, plant genotyping has progressed rapidly, sequestration of Na+ into leaf vacuoles and accumulating
while phenotyping remains a bottleneck for breeding due K+ and compatible solutes (such as proline, sucrose,

© 2014 Blackwell Verlag GmbH 1

Fan et al.

glycine betaine, mannitol) in the cytosol and organelles In this work, we hypothesized that the possible answer to
to balance osmotic pressure of ions in vacuoles (Wang the above question may be a high tissue and time specificity
et al. 2005, Young-Pyo et al. 2007). of ROS production. Accordingly, this issue was addressed
Osmatic stress reduces the stomatal aperture restricting by elucidating kinetics of AO activity in leaves of various
the exchange of CO2 and O2, leading to rapid reactive oxy- physiological age/position exposed to different periods of
gen species (ROS) accumulation and oxidative stress (Rox- salinity stress. Then, the selected ‘optimal treatments’ were
as et al. 1997). ROS are partially reduced or activated used to compare AO profiles of leaves of barley varieties
derivatives of oxygen, including singlet oxygen (1O2), contrasting in salt tolerance (three salt-tolerant and three
superoxide anion (O2 ), hydrogen peroxide (H2O2) and salt-sensitive genotypes). We concluded that although
hydroxyl radical (HO●). They are highly reactive, toxic and salinity-induced changes of leaf AO enzymes activities, they
may cause DNA or RNA damage, protein oxidation and still cannot be used as biochemical indicators in breeding
lipid peroxidation (Bates et al. 1973). In general, the equi- for salinity tolerance.
librium between ROS production and removal by antioxi-
dant (AO) defence components is strictly controlled.
Materials and Methods
Under salinity stress, when CO2 availability is restricted,
this balance will be disturbed, leading to a remarkable
Plant materials and growth conditions
increase in ROS concentration, namely an oxidative burst
(Apostol et al. 1989). Apart from being highly reactive with Six barley (Hordeum vulgare L.) varieties were used in this
numerous biomolecules and causing irreversible damage to work. Among them, TX9425 (TX), YuYaoXiangTianErLeng
plant cells, ROS can also activate a range of Na+ and K+ (YYXT) and CM72 showed better tolerance to salt stress,
permeable ion channels (Demidchik et al. 2003) which dis- while Naso Nijo (NN), Franklin and Gairdner were sensi-
turb the cytosolic K+/Na+ ratio and lead to programmed tive (Zhou et al. 2012). Seeds of all varieties were provided
cell death (PCD; Shabala et al. 2007, Demidchik et al. by the Tasmanian Barley Breeding Program. Seeds were
2010), as well as participate in signal transduction pathways sown in 2-l pots filled with potting mixture. Six pots, each
and affect gene expression (Bradford 1976, Girotti 2001). contained a single variety, were placed in a 40-l bin, repre-
ROS detoxification in plants mainly involves two mecha- senting one replication of a treatment. Seedlings were
nisms: enzymatic- and non-enzymatic-scavenging mecha- grown in a glasshouse with controlled temperature (day/
nisms. Major non-enzymatic AOs include ascorbate, night, 28/16  2 °C) under natural sunlight at Mt Pleasant
glutathione (GSH), a-tocopherol, carotenoids, while ROS- Laboratories in Launceston. Three replications were
scavenging enzymes are superoxide dismutase (SOD), applied for all the treatments.
ascorbate peroxidase (APX), catalase (CAT), glutathione
peroxidase (GPX) and peroxidase (POD), monodehydro-
Treatments and sample collection
ascorbate reductase (MDAR), dehydroascorbate reductase
(DHAR) and glutathione reductase (GR). In addition, osm- Salt stress was started at the four leaf stage (15-day-old
olytes, such as proline, mannitol, glycinebetaine, ectoine, seedlings). Salt solution (240 mM NaCl) was used to wash
can be active in scavenging ROS and act through oxidative through the pots several times until the solution drained
detoxification (Roxas et al. 1997, Shen et al. 1997). out from the pots had consistent salt concentration. The
Changes in AO activities under salt stress were observed treatment was repeated every 3 days. Two contrasting vari-
in both roots (Bandeoglu et al. 2004) and leaves (Ben eties, TX and NN, were used for time- and tissue-depen-
Hamed et al. 2007). The lack of significant correlation dent experiments (so-called ‘Experiment I’). The second
between salinity stress tolerance and AO activity in roots and third leaves from the bottom were collected for enzyme
(Panda and Khan 2009, Chen et al. 2011, Maksimovic et al. measurements after 1, 2, 5, 10 days of salt treatment. In
2013) suggests that AO ROS detoxification does not make Experiment II, the third leaves from the bottom of six vari-
a major contribution to salt tolerance in this tissue. As for eties were collected 10 days after NaCl treatment for the
leaves, the reported results are variable. While some measurement of AO enzyme activities, MDA and proline
researchers reported a positive association between AO content. Chlorophyll and Na+, K+ contents were measured
production in leaves and plant salinity tolerance (Kim et al. in the first fully expanded leaves.
2005, Moradi and Ismail 2007, Jin et al. 2009), others
showed no or negative correlation between leaf AO activity
Grain yield and plant survival under salt stress
and salinity stress tolerance (Abogadallah et al. 2010,
Noreen et al. 2010, Parida and Jha 2010, Sabra et al. 2012). Grain yield
The possible mechanisms for such differences in AO Varieties were sown in large tanks (160 by 120 by 60 cm)
activity remain unclear. filled with potting mixture and located in a glasshouse.

2 © 2014 Blackwell Verlag GmbH

 Antioxidant Activity and Barley Salt Tolerance

Each variety contained 25–30 plants in a 70 cm – long enzyme activities which were determined by a spectropho-
row, with 15 cm gap between rows. After germination, tometer (Genesys10S UV-VIS; Thermo Fisher Scientific,
two replications were treated with 240 mM NaCl and the Scoresby, Australia).
other two replications were used as a control. All con- SOD activity was evaluated by the ability to inhibit pho-
tainers were linked with a drainage system connected to toreduction of nitroblue tetrazolium (NBT; Beyer and
the bottom of each container and an application system Fridovich 1987). CAT activity was determined by monitor-
across the top of each container. A 160-l sump was ing the disappearance rate of H2O2 at 240 nm according to
placed on the floor 1.5 m below the top of the containers the method of Aebi (1984). APX activity was assayed by
and connected to a pump capable of lifting water from following the rate of H2O2-dependent oxidation of ascorbic
the sump to the top of the containers. After germination, acid (AsA) according to the method of Nakano and Asada
240 mM NaCl solution was pumped from the sump (1981). POD activity was assessed by recording the
through the application system to the top of the contain- increased absorbance at 470 nm due to the oxidation of
ers which were used for salt treatment. The drainage sys- guaiacol according to Chance and Maehly (1955).
tem was kept closed until solution accumulated to a Protein content was determined according to the
depth of 10 mm over the surface of potting mixture. The method described by Bradford (1976) at 595 nm using
pump was then switched off, and the containers were bovine serum albumin as a standard.
allowed to remain soaked with the solution for 10–
15 min. The drainage system was then reopened and the
Determination of Na+ and K+ in leaves
salt solution returned to the sump. More salt was added
to adjust the solution to 240 mM. This procedure was To determine the sodium and potassium ions content, leaf
repeated several times until the solution drained from the sap of first fully expanded leaves were extracted and centri-
containers reached a consistent salt concentration (Zhou fuged at 2655 g for 10 min essentially as described else-
et al. 2012). This process was repeated at weekly intervals. where (Cuin et al. 2009). The supernatants were collected
After maturity, grain yield of both salt treated and to evaluate Na+ and K+ content using a flame photometer.
controls were recorded.
Estimation of lipid peroxidation and proline content
Plant survival
Plant survival rate has been used by many researchers as a The level of lipid peroxidation was determined in terms of
reliable indication of plant salt tolerance. A higher salt MDA content, a product of lipid peroxidation, following a
stress (320 mM NaCl) was also applied in this experiment. modified method of Heath and Packer (1968).
Most of the very sensitive varieties will not be able to sur- Proline content was estimated according to the method
vive to maturity under this concentration. A similar treat- of Bates et al. (1973) and Sayed et al. (2012). Leaf samples
ment system as described previously was applied but with were collected and ground to fine power. Proline content
no controls. Plant survival was scored according to leaf was determined by a standard curve from known concen-
chlorosis and plant healthiness (0 = all dead and 10 = no trations of L-proline.
damage).
Results
Measurement of leaf chlorophyll content
Growth and agronomical characters of barley varieties
Leaf chlorophyll content was tested in first fully expanded
leaves using SPAD-502 chlorophyll meter (Minolta, Tokyo, Salinity significantly impacted plant growth and yield. The
Japan). yield of all the varieties showed a significant (P < 0.05)
decrease, being more severe for sensitive varieties under
240 mM NaCl stress (Fig. 1a). Tolerant varieties (TX,
Determination of antioxidant enzyme activity and protein
CM72, YYXT) had greater plant survival under high salt
contents
stress than sensitive ones (NN, Gairdner, Franklin; Fig. 1b).
For the extraction of AO enzymes, 0.5 g of leaf samples was Figure 1c shows a typical difference between a tolerant
homogenized in mortars with 7 ml phosphate buffer solu- variety (TX) and a sensitive one (NN). After 10 days of
tion (PBS). Fresh leaf samples were used in both experi- 320 mM NaCl treatment starting from germination, no
ments. The extraction buffer was 50 mM PBS (pH 7.8) obvious symptom of salt stress was noticed in TX, while
containing 0.1 mM EDTA and 2 % PVP. The homogenates numerous yellow or dead leaves were found in NN. Plant
were centrifuged at 15 294 g for 30 min at 4°C, and the su- survival under high salt stress was consistent with relative
pernatants were collected (stored in 4°C) to evaluate the yields at lower salt stress (240 mM NaCl).

© 2014 Blackwell Verlag GmbH 3

Fan et al.

1
(a) (c) TX9425 Naso Nijo

GRAIN YIELD (% control)

0.8

0.6

0.4

0.2

0
(b)
10
PLANT SURVIVAL

in
72

er
XT
TX

kl
n
M

YY

an
rd
C

ai

Fr
Tolerant G
Sensitive

Fig. 1 Growth, survival and grain yield of barley varieties under salt stress. (a) Relative grain yield of six barley varieties under 240 mM NaCl stress; (b)
plant survival score of six barley varieties under 320 mM NaCl stress (0 = all dead; 10 = no obvious symptom); (c) plants treated with 320 mM NaCl
after germination growth with TX9425 showing much better salt tolerance than Naso Nijo.

increase (relative to control) in tolerant varieties and

Activities of antioxidant enzymes
decrease in sensitive varieties (Fig. 3b). APX activities of all
In Experiment I, two contrasting varieties – TX (tolerant) varieties were enhanced with no obvious differences
and NN (sensitive) – were selected to investigate the effect between tolerant and sensitive varieties (Fig. 3c). POD
of leaf age and duration of salt exposure on AO enzyme activity of all tolerant varieties showed a significant increase
activity. After 1, 2, 5, 10 days of 240 mM NaCl treatment, after salt treatment with only one sensitive variety (NN)
enzyme activities in second and third leaves of both varie- showing significant increase in POD activity (Fig. 3d).
ties were measured. SOD activity in both second and third Again, no significant correlation between POD activity and
leaves increased in both varieties after salt treatment plant salt stress tolerance was found (Table 2).
(Fig. 2a,b). No significant difference was found for either
CAT or APX activity in the two leaves of both varieties
Na+ and K+ content in leaves
under salt treatment (Fig. 2c–f, Table 1). Under salt treat-
ment, TX did not show significant changes in POD activity, Na+ exclusion and K+ retention are considered to be key
while POD activity was enhanced in NN, especially 2 and mechanisms for plant tolerance to salinity (Shabala and
10 days after the treatment (Fig. 2g,h, Table 1). Younger Cuin 2008). In Experiment I, Na+ content in leaves of both
leaves showed much lower POD activity, but the changes varieties increased significantly after salt treatment. The
caused by salt treatment were similar in both varieties. As longer the treatment, the higher the Na+ content (Fig. 4a).
shown in Figure 2, treatment times had little effect on Na+/K+ ratios showed a trend similar to one for Na+ con-
changes in the activity of different enzymes. In general, tent (Fig. 4c). The tolerant variety showed consistently
slightly larger difference was found between varieties at the lower Na+ contents and lower Na+/K+ ratios. No significant
10th day after treatment. For example, POD activity of NN changes were found in the K+ content of all except NN on
increased in both younger and older leaves after salt treat- the 10th day (not shown in the figure).
ment, while there was no significant change in POD activity In Experiment II, higher Na+ contents and Na+/K+ ratios
in the leaves of TX. Thus, further measurements (Experi- could be seen in salt-sensitive genotypes except for Gaird-
ment II) of different enzyme activities on other varieties ner (Fig. 4b,d). Both traits had negative correlation with
were conducted using the third leaves and after 10 days of plant survival (Table 2).
salt treatment.
Similar to the results in Experiment I, SOD activity of all
Chlorophyll content in leaves
varieties but Franklin increased under salt stress, with no
obvious correlation with salt tolerance of the varieties Chlorophyll contents of the first fully expanded leaves were
(Fig. 3a). Under salt stress, CAT activities showed trends of measured in TX and NN at 1, 2, 5 and 10 days after salt

4 © 2014 Blackwell Verlag GmbH

 Antioxidant Activity and Barley Salt Tolerance

250 250
(a) (b)
200 200

[U · mg–1 prot]
[U · mg–1 prot]
150 150

SOD
SOD
100 100

50 50

0 0
(c) (d)

[U · mg–1 prot · min–1]

[U · mg–1 prot · min–1]
600 600

CAT
CAT
400 400

200 200

0 0

[μmol · mg–1 prot · min–1]

[μmol · mg–1 prot · min–1]
(e) (f)
3 3

APX
APX

Fig. 2 Antioxidant enzyme activities of second 2 2

leaves (a, c, e, g) and third leaves (b, d, f, h)
1 1
from the bottom in TX9425 (TX) and Naso Nijo
(NN) at 1, 2, 5, 10 days after 240 mM NaCl
0 0
treatment. Older leaves (second from bottom) (g) (h) TX-control
[U · mg–1 prot · min–1]

[U · mg–1 prot · min–1]

had higher enzyme activities (especially for 3000 TX-salinity 3000
peroxidase (POD)). The differences among NN-control
POD

POD
2000 NN-salinity 2000
varieties, treatment times were more pro-
nounced in the third leaves at 10th day after
1000 1000
salt treatment. (a, b) Superoxide dismutase
activity; (c, d) catalase activity; (e, f) ascorbate 0 0
peroxidase activity; (g, h) POD activity. 1 2 5 10 1 2 5 10
Treatment time (days)
Mean  S.E. (n = 3, each sample contained
leaves from at least three plants). Enzyme activities in older leaves Enzyme activities in younger leaves

Table 1 ANOVA analysis of antioxidant enzyme activities (SOD, CAT, APX, POD) in TX and NN after 1, 2, 5, 10 days of 240 mM NaCl treatment

SOD CAT APX POD

Source
of D1 D2 D5 D10 D1 D2 D5 D10 D1 D2 D5 D10 D1 D2 D5 D10
variance F F F F F F F F F F F F F F F F

V 1.9 5.2 0.2 8.5* 24.6** 5.6* 4.0 5.0* 1.0 0.0 9.2** 0.3 1.9 0.7 1.1 0.8
T 5.3 4.1 21.0** 85.9** 1.1 0.6 6.7* 0.2 6.0* 4.8 8.4* 2.4 1.1 11.9* 0.5 5.8*
L 1.3 0.9 0.2 0.3 4.1 9.9* 24.7** 0.7 0.2 1.3 7.4* 0.3 24.4** 114.3** 22.0** 43.1**
V-T 0.0 0.7 0.1 2.7 0.3 6.8* 1.0 5.9* 1.6 0.1 0.9 5.9* 0.2 7.2* 0.8 7.8*
V-L 0.2 0.1 0.0 0.1 1.4 0.0 4.6 5.8* 0.8 41.9** 2.7 0.3 0.0 0.1 2.1 9.1**
T-L 0.0 0.1 0.1 4.8* 3.8 4.1 0.1 0.2 1.3 0.0 0.0 4.1 0.8 8.0* 0.5 1.5
V-T-L 1.0 1.9 0.5 0.1 6.3* 0.8 0.3 0.0 0.7 0.4 0.3 0.2 0.1 0.7 0.1 0.1

*P < 0.05; **P < 0.01.

APX, ascorbate peroxidase; CAT, catalase; MDA, monodehydroascorbate; NN, Naso Nijo; POD, peroxidase; SOD, superoxide dismutase; V, variety; T,
treatment; L, leaf age; D1-10, days after salt treatment.

treatment. Salt treatment showed no significant effects on between relative chlorophyll content and plant survival or
chlorophyll contents of TX genotype, while in NN plants, it relative grain yield (Table 2).
showed a continuous decrease with extended treatment
times (Fig. 4e). Similar results were found for other toler-
Lipid peroxidation
ant varieties which showed no changes or even an increase
in leaf chlorophyll content. However, chlorophyll contents As a product of lipid peroxidation, MDA is generally used
of two other sensitive varieties were not affected by salt as an indicator of levels of lipid peroxidation. As can be
treatment (Fig. 4f). Positive correlations were found seen from Figure 5a, MDA contents increased in all

© 2014 Blackwell Verlag GmbH 5

Fan et al.

Table 2 Correlations between antioxidant enzyme activities (SOD, CAT, APX, POD), MDA, Proline, Na+, K+, Na+/K+ ratio, chlorophyll content and
grain yield, plant survival in six barley varieties contrasting in salt tolerance

SOD CAT APX POD MDA Proline Na K Na/K Chl Grain yield Plant Survival

SOD 1
CAT 0.231 1
APX 0.176 0.384 1
POD 0.634 0.475 0.445 1
MDA 0.599 0.292 0.346 0.783 1
Proline 0.608 0.325 0.131 0.656 0.596 1
Na 0.387 0.355 0.436 0.481 0.554 0.870* 1
K 0.660 0.020 0.124 0.272 0.623 0.468 0.631 1
Na/K 0.047 0.616 0.283 0.322 0.309 0.819* 0.844 0.173 1
Chl 0.478 0.544 0.428 0.326 0.202 0.792 0.456 0.082 0.637 1
Grain yield 0.136 0.804 0.467 0.256 0.451 0.359 0.164 0.358 0.546 0.740 1
Plant Survival 0.363 0.674 0.093 0.239 0.127 0.862* 0.766 0.277 0.839* 0.844* 0.723 1

Higher plant survival score meant better surviving ability (The plant survival scores: 0 = all dead; 10 = no obvious symptom). *P < 0.05.
APX, ascorbate peroxidase; CAT, catalase; MDA, monodehydroascorbate; POD, peroxidase; SOD, superoxide dismutase.

200
(a) (b)

CAT [U · mg–1 prot · min–1]

300
SOD [U · mg–1 prot]

150

200
100

50 100

0 0
Control
APX [μmol · mg–1 prot · min–1]

(c) (d)
POD [U · mg–1 prot · min–1]

Fig. 3 Antioxidant enzyme activities of third

2.5 Salinity
1500 leaves (at four leaves stage) in six barley varie-
2 ties contrasting in salt tolerance (Tolerant: TX,
CM72, YuYaoXiangTianErLeng; Sensitive:
1.5 1000
Naso Nijo, Gairdner, Franklin) after 10 days
1 240 mM NaCl treatment. (a) Superoxide
500 dismutase activity; (b) catalase activity;
0.5 (c) ascorbate peroxidase activity; (d) peroxi-
dase activity. No evident differences of
0 0
enzyme activities were observed between tol-
XT

XT
TX

72

lin

TX

72

in
r

r
ne

ne
N

kl
nk

erant and sensitive varieties. Mean  S.E.

M

M
YY

YY
rd

rd

an
C

C
a
ai

ai
Fr

Fr
G

(n = 3, each sample contained leaves from at

Tolerant Sensitive Tolerant Sensitive least three plants).

varieties 10 days after salt treatment. No obvious patterns found in other two tolerant varieties CM72 and YYXT
were revealed for MDA levels between salt-tolerant and (Fig. 5b). Proline content under salt stress exhibited signifi-
salt-sensitive genotypes (Fig. 5a), reflected by the results of cant negative correlation with plant survival (Table 2).
correlation analysis (Table 2).
Discussion
Proline content
Leaf antioxidant enzyme activities do not correlate with
Plants need compatible solutes, such as proline in cytosol
salinity tolerance
and organelles, to balance the osmotic pressure of ions in
vacuoles caused by salinity. Higher proline contents were Contrasting salt concentrations has been widely used to
induced under salt stress in all sensitive varieties and one of investigate the change of various AOs under salt stress in
the tolerant varieties TX, while no obvious changes were different species (Ben Amor et al. 2006, Parida and Jha

6 © 2014 Blackwell Verlag GmbH

 Antioxidant Activity and Barley Salt Tolerance

300 500
(a) TX-control (b) Control
250 TX-salinity 400
Salinity
NN-control
200
NN-salinity 300

Na+ [mM]
Na+ [mM]
150
200
100
100
50

0 0
(c) (d)
0.8
1.5

Na+/K+ RATIO
Na+/K+ RATIO 0.6
1
0.4

0.5
0.2

0 0
CHLOROPHYLL (SPAD VALUE)

CHLOROPHYLL (SPAD VALUE)

(e) (f)
+ + +
Fig. 4 Na , Na /K and chlorophyll content (in 60
40
first fully expanded leaves). (a) Na+ content, (c) 50
Na+/K+ ratio and e, chlorophyll content (SPAD
40 30
value) of TX and Naso Nijo at 1, 2, 5, 10 days
after 240 mM NaCl treatment; (b) Na+ con- 30 20
tent, (d) Na+/K+ ratio and f, chlorophyll con-
20
tent of six barley varieties contrasting in salt 10
tolerance after 10 days 240 mM NaCl treat- 10
ment. Salinity-induced changes of Na+, Na+/K+ 0 0
and chlorophyll content (a, c, e), and obvious 1 2 5 10

XT
TX

72

in
r
ne
N

kl
difference (P < 0.05) between tolerant and Treatment Ɵme (days)
M

YY

rd

an
C

ai

Fr
G
sensitive varieties (b, d, f). Mean  S.E.
(n = 5). Tolerant Sensitive

2010, Sabra et al. 2012, Sergio et al. 2012). However, there changes in leaf AO enzyme activity in different leaves after
were very few reports on influences of treatment times and different times of salt treatment. Salt stress caused signifi-
leaf ages (e.g. younger and older leaves) on AOs activities, cant changes in activities of most of the enzymes, but treat-
which may be one of the reasons for the inconsistency of ment times showed little effect on enzyme activities
results from different reports. Salinity-specific induction of (Fig. 2). Leaf ages (younger and older) had significant
SOD isoforms in barley was reported by Kim et al. (2005). effects on POD activity, with the activities from younger
The differences in enzyme activities came from different leaves being much lower.
experimental conditions such as exposure time, salt level Oxidative stress can cause major damage to plants, and
and genotypes (Kim et al. 2005, Jin et al. 2009). Growing thus, the detoxification ability of plants becomes very
seasons also affected AO enzyme activity as plants grew important. However, it is still not clear whether higher lev-
slower in winter and photosynthesis rate differed if plants els AOs represent greater plant salt tolerance. A large num-
were grown in the field or in sites without consistent light ber of researchers proved positive association between AO
and temperature conditions (Ben Ahmed et al. 2009). ROS production and salt tolerance of plants including barley
are reduced or activated derivatives of oxygen produced by (Jin et al. 2009), rice (Moradi and Ismail 2007), Arabidopsis
aerobic metabolism such as photosynthesis and respiration, (Attia et al. 2008) and halophyte Cakile maritime (Ben
and they are highly reactive (Bates et al. 1973). As ROS Amor et al. 2006). Transgenic plants overexpressing AO
production and AO enzymes are highly unstable and time- genes also exhibited better salt tolerance (Sreenivasulu
dependent, higher AO activities at one particular ‘snapshot’ et al. 2004, Wang et al. 2004, Ushimaru et al. 2006, Eltayeb
(a measurement at one specific time) do not represent et al. 2007). In contrast, there are also numerous reports
higher plant salt tolerance. In this study, two varieties dif- that activities of AOs declined under salt stress (Abogadallah
fering in their salt tolerance were used to investigate et al. 2010, Hafsi et al. 2010, Noreen et al. 2010, Yang et al.

© 2014 Blackwell Verlag GmbH 7

Fan et al.

4 60
(a) (b) Control
3.5 Salinity
50
3

PROLINE [μg · g–1 FW]

MDA [μmol · g–1 FW]
40
2.5

2 30

1.5
20
1
10
0.5

0 0

XT

XT
TX

72

lin

TX

72

lin
r

r
ne

ne
N

N
nk

nk
M

M
YY

YY
rd

rd
C

C
a

a
ai

ai
Fr

Fr
G

G
Tolerant Sensitive Tolerant Sensitive

Fig. 5 Monodehydroascorbate (MDA) and proline content (in third leaves from bottom) of six barley varieties at 10 days after 240 mM NaCl treat-
ment. (a) MDA content; (b) proline content. Under salinity stress, MDA increased in all while proline showed significant difference (P < 0.05) between
tolerant and sensitive varieties. Mean  S.E. (n = 3, each sample contained leaves from at least three plants).

2010). Munns and Tester (2008) suggested that genetic dif- tents and lower Na+/K+ ratios in leaves which was consis-
ferences of salinity tolerance does not necessarily attribute tent with previous reports (Munns and James 2003,
to differences of ROS detoxifying ability. A similar conclu- Garthwaite et al. 2005, Chen et al. 2007b, Abraham and
sion was made by Maksimovic et al. (2013), pointing out Dhar 2010). Leaf chlorosis was considered as an adaptation
that higher AO activities at one particular time did not rep- by retaining internal water for transpiration demands vari-
resent higher salt tolerance as higher AO activities were ous stresses, for example drought stress (Champoux et al.
observed in sensitive plants and no correlation between 1995). Positive correlations between relative chlorophyll
SOD activity and salt tolerance was found under a large- contents under salt stress and plant survival were also found
scale screening. In this experiment, salt treatment caused a in this study (Fig. 4e,f; Table 2). The relatively higher chlo-
significant increase in SOD activity of all varieties but rophyll contents in salt-tolerant varieties was partly due to
Franklin (Fig. 3a). APX activities of almost all varieties less leaf chlorosis under salinity of the tolerant genotypes
increased and no significant decreases in POD activity were (Munns and James 2003, El-Tayeb 2005, Panda and Khan
found in any of six varieties (Fig. 3c,d). Overall, the activi- 2009, Wu et al. 2013). Leaf chlorosis or leaf senescence are
ties of the three AOs (SOD, APX and POD) showed no recognized as examples of PCD which could be triggered
correlations with salt tolerance (Table 2) and thus cannot under salinity due to Na+ accumulation or K+ loss (Shabala
be used as selection criteria for salt tolerance. Interestingly, et al. 2007, Shabala 2009).
CAT activities of all tolerant varieties tended to increase
while those of all sensitive varieties tended to decrease
Salt-stress increases lipid peroxidation in all varieties
under salt stress (Fig. 3b). However, the difference was small
and could not reliably be used in selecting salt-tolerant ROS-scavenging mechanisms mainly include SOD, CAT,
varieties. ascorbate/glutathione cycle and GPX cycle. In the ascor-
bate/glutathione cycle, APX detoxifies H2O2 to H2O and
simultaneously ascorbate is oxidated to MDA. After that,
Tolerant varieties have lower Na+/K+ ratio and higher
MDA could be reduced to ascorbate by MDAR with the
chlorophyll content in leaves
help of NADPH (Bradford 1976). Therefore, MDA is usu-
Higher K+/Na+ ratio (resulting from either better K+ ally deemed as an indicator of lipid peroxidation. In this
retention or Na+ exclusion, or both) is considered as a key experiment, MDA content increased under salt stress for all
determinant of salt tolerance (Chen et al. 2007a,b, Shabala varieties (Fig. 5a), which is due to more ROS production
and Cuin 2008). Higher Na+ content and Na+/K+ ratio in under salt stress (El-Tayeb 2005, Abogadallah et al. 2010,
sensitive varieties were observed in this experiment Chen et al. 2011). No obvious correlation was found
(Fig. 4b,d); both showed highly significant negative corre- between MDA contents and plant survival or grain yield
lations with plant survival (Table 2). These results indi- under salt stress (Fig. 5a; Table 2), and thus, the produc-
cated that tolerant genotypes used in this study had a tion of MDA may not be used as a physiological marker for
better ability for Na+ exclusion, leading to lower Na+ con- evaluating the extent of plant salt tolerance.

8 © 2014 Blackwell Verlag GmbH

 Antioxidant Activity and Barley Salt Tolerance

In conclusion, changes in the level of AO enzyme activity

Proline accumulates more in sensitive varieties and
and lipid peroxidation were induced by salt stress and
positively correlates with Na+ content and Na+/K+ ratio in
activities of leaf AO enzymes were influenced by leaf age,
leaves
salt concentration, time of treatment and genotype. How-
Apart from being an osmolyte to balance osmotic pres- ever, no significant correlation between plant salt tolerance
sure in cells, proline also acts as a ROS scavenger and and AO enzyme activity or MDA content was observed.
plays an important role in reducing oxidative stress Chlorophyll and proline contents and Na+/K+ ratio may be
induced by osmotic stress (Hong et al. 2000, Matysik used as possible criteria for selecting salt-tolerant varieties.
et al. 2002, Cuin and Shabala 2007, Kaul et al. 2008,
Szekely et al. 2008). During stress, the reduced rate of
Acknowledgements
the Calvin cycle causes insufficient electron acceptor
NADP+ and leads to ROS accumulation in green leaves This study was supported by Grains Research and Develop-
(Chaves et al. 2009). Proline biosynthesis in chloroplast ment Corporation of Australia and National Natural
maintains low NADPH/NADP+ ratios to sustain electron Science Foundation of China (31128014).
flow, thus reducing the extent of photoinhibition and
ROS production. Proline degradation in mitochondrion
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