Acta Physiol Plant (2013) 35:667–677
DOI 10.1007/s11738-012-1107-7
ORIGINAL PAPER
Effects of ascorbic acid and gibberellin A3 on alleviation of salt
stress in common bean (Phaseolus vulgaris L.) seedlings
Sakineh Saeidi-Sar • Hossein Abbaspour •
Hossein Afshari • Saeed Reza Yaghoobi
Received: 20 May 2012 / Revised: 8 September 2012 / Accepted: 11 September 2012 / Published online: 27 September 2012
Ó Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2012
Abstract Salinity, a severe environmental factor, has
limited the growth and productivity of crops. Many com-
pounds have been applied to minimize the harmful effects
of salt stress on plant growth. An experiment was con-
ducted to investigate the interactive effects of exogenous
ascorbic acid (AsA) and gibberellic acid (GA3) on common
bean (Phaseolus vulgaris L. cv. Naz) seedlings under salt
stress. The changes of growth parameters, photosynthetic
and non-photosynthetic pigments and potassium content
showed that the addition of 1 mM AsA and/or 0.05 mM
GA3 considerably decreased the oxidative damage in
common bean plants treated with 200 mM NaCl. The
NaCl-stressed seedlings exposed to AsA or GA3, specifi-
cally in their combination, exhibited an improvement in
sodium accumulation in both roots and shoots, as compared
to NaCl-treated plants. NaCl treatment increased hydrogen
peroxide (H2O2) content and lipid peroxidation indicated
by accumulation of malondialdehyde (MDA), whereas the
interaction of AsA with GA3 decreased the amounts of
MDA and H2O2. In the meantime, interactive effect of
these substances enhanced protein content and the activity
of the antioxidant enzyme, guaiacol peroxidase, in com-
mon bean plants under salt stress. It was concluded that
synergistic interaction between AsA and GA3 could
alleviate the adverse effects of salinity on P. vulgaris
seedlings.
Communicated by P. K. Nagar.
S. Saeidi-Sar (&)
H. Abbaspour
H. Afshari
S. R. Yaghoobi
Department of Biology, Faculty of Science, Damghan Branch,
Islamic Azad University, Damghan, Iran
e-mail: s_saeidisar@yahoo.com
Keywords Antioxidants
Ascorbic acid
Gibberellin

Phaseolus vulgaris L.
Salt stress
Abbreviations
ABA Abscisic acid
AsA Ascorbic acid
GA3 Gibberellic acid
GPOX Guaiacol peroxidase
IAA Indole-3-acetic acid
MDA Malondialdehyde
ROS Reactive oxygen species
Introduction
Salinity is one of the worldwide environmental constraints
to crop production. Seven percent of the land’s surface and
5 % of cultivated lands are affected by salinity, and it is an
important factor which can limit the growth and produc-
tivity of plants (Flowers et al. 1977). The inhibitory effect
of salinity stress is largely due to the ionic and osmotic
stress (Magome et al. 2008). Salt stress influences some
basic metabolic processes in plants such as photosynthesis,
lipid metabolism and protein synthesis (Parida and Das
2005). Decrease of photosynthetic rates in plants under salt
stress is mainly due to the reduction in photosynthetic
pigments (Dubey 2005), as well as limitations in photo-
synthetic electron transport and partial stomatal closure
(Zhang et al. 2010). Additionally, the production of reac-
tive oxygen species (ROS), including superoxide radical
(O2-), hydroxyl radical (OH
), singlet oxygen (1O2) and
hydrogen peroxide (H2O2), are the characteristics of bio-
chemical changes during salt stress. The excess production
of ROS during salinity stress results from impaired electron
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transport processes in chloroplast and mitochondria as well stress conditions can result from an altered hormonal bal-
as from pathways such as photorespiration (Zhu et al. ance, and hormone application provides an attractive
2004). When plants are subjected to stress, the balance approach to cope with stress (Saeidi-Sar et al. 2007).
between the production of ROS and the quenching activity Our previous studies revealed that supplying low levels
of the antioxidant becomes upset, often resulting in oxi- of AsA and gibberellic acid (GA3) could alleviate the
dative stress (Smirnoff 1993). A sudden and dramatic poisonous effects of Ni on soybean plants (Saeidi-Sar et al.
increase in cellular ROS production disrupts normal 2007). However, none of these studies have focused on the
metabolism through oxidative damage to photosynthetic plant species in the presence of NaCl stress. The major
pigments, proteins, nucleic acids and lipids (Zhu et al. objective of this study was to investigate the effects of AsA
2004). Accordingly, malondialdehyde (MDA) as a product and/or gibberellic acid (GA3) on the number of physio-
of lipid peroxidation is accumulated in tissues when plants logical aspects of common bean plants under saline con-
are exposed to salinity stress (Shalata and Neumann 2001). ditions, and to determine the extent to which synergistic
Scavenging of ROS in plant cells is occurred by effects of AsA and GA3 can ameliorate the adverse effects
endogenous protective mechanisms involving antioxidant of salt stress on this plant.
molecules and enzymes. Plants use a diverse array of
enzymes such as superoxide dismutases, catalases and
peroxidases as well as low molecular mass antioxidants
Materials and methods
such as ascorbate, carotenoids and reduced glutathione to
scavenge different types of ROS (Zhu et al. 2004). Plants
Plant materials and treatments
with high levels of antioxidants, both enzymatic and non-
enzymatic, either constitutive or induced, have greater
Seeds of common bean (Phaseolus vulgaris L. cv. Naz)
resistance to this oxidative damage in plant cells by
were surface-sterilized with 20 % NaClO3 for 5 min and
avoiding lipid and protein peroxidation (Younis et al.
then thoroughly rinsed with distilled water. Seeds were
2010).
germinated on several layers of wet tissue paper at 24 °C in
Ascorbic acid (AsA) is an important antioxidant in
darkness. Five-day-old seedlings were transferred to
plants which accumulates in plants as an adaptive mecha-
300 cm3 containers with half strength Hoagland’s solution
nism to environmental stress such as salinity. AsA regu-
(three seedlings per container). Then, 10-day-old seedlings
lates stress response as a result of a complex sequence of
were transferred to fresh half strength Hoagland’s solution,
biochemical reactions such as activation or suppression of
supplemented with sodium chloride (0, 200 mM) either
key enzymatic reactions, induction of stress responsive
with or without AsA (1 mM) or GA3 (0.05 mM), or AsA
proteins synthesis, and the production of various chemical
(1 mM) ? GA3 (0.05 mM). The solutions were continu-
defense compounds (Khan et al. 2011). The protective role
ously aerated and refreshed every day. Solution-pH was
of AsA in plant cells from the adverse effects of salt stress
daily adjusted to 6.5. Plants were grown in growth chamber
was described by Athar et al. (2009) and Younis et al.
with a 16/8 light/dark photoperiod at 175 lmol m-2 s-1
(2010).
PPFD, a day/night temperature cycle of 26/22 °C and
On the other hand, the role of plant hormones under
65 ± 5 % relative humidity. After 10 days, the plants were
salinity stress is critical in modulating physiological
removed and washed with deionized distilled water. The
responses that will eventually lead to adaptation to an
samples for estimation of plant dry matter and ion analysis
unfavorable environment. Abiotic stresses alter the levels
were dried at 70 °C for 48 h. Fresh plant materials were
of plant hormones and decrease plant growth. Additionally,
frozen in liquid nitrogen and stored at -70 °C.
the complex regulation of plant responses to stress implies
the existence of a special signal transduction chain between
Determination of pigments
stress signals and responses, in which plant hormones are
an integral part of the stress-controlling mechanism in
The levels of Chlorophyll a, Chlorophyll b and carotenoid
plants (Zeevaart and Creelman 1988). Gibberellins (GAs)
were measured in acetone extracts according to Arnon
are a class of phytohormones that control many aspects of
(1949). The concentrations of Chlorophyll a and Chloro-
plant growth and development, including seed germination,
phyll b were calculated from equations derived by Hendry
leaf expansion, stem elongation, flower initiation and
and Grime (1993).
development, sex determination, and fruit development (Li
et al. 2010). Previous studies have suggested the possible Chlorophyll a (mg g-1 FW) = [(12.7 A663 – 2.69 A645)/
involvement of GA in stress adaptation in some plants 1,000 9 FW] 9 V
(Achard et al. 2006; Rodriguez et al. 2006; Magome et al. Chlorophyll b (mg g-1 FW) = [(22.9 A645 – 4.68 A663)/
2008; Maggio et al. 2010). Reduced plant growth under 1,000 9 FW] 9 V

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Carotenoid content was determined by the equation of
Price and Hendry (1991)
Carotenoid (lmol g-1 FW) = [(A480 ? 0.114 A663)
- (0.638 A663) 9 V/112.5 9 FW]
where V is volume of the sample (mL), A is absorbance and
FW is fresh weight (g). Anthocyanin concentration of leaves
was determined by the method of Mancinelli et al. (1988) and
anthocyanin content was expressed to A530 g-1 FW.
Determination of K and Na
Root and shoot samples were wet digested with a HNO3
and HClO4 acid mixture (5:1, v/v), and analyzed by a flame
photometer (Flame Photometer, model 410, Sherwood
Company), as described by Williams and Twine (1960).
Determination of H2O2 content
Content of hydrogen peroxide was measured according to the
procedure of Velikova et al. (2000). Fresh samples (0.1 g)
were homogenized with 3 mL 0.1 % (w/v) trichloroacetic
acid (TCA) in ice bath and the homogenate was centrifuged at
12,000g for 15 min. Then 0.5 mL of 10 mM phosphate buffer
(pH 7.0) and 1 mL of 1 M KI were added to 0.5 mL of the
supernatant. The absorbance of supernatant was read at
390 nm. The amount of H2O2 was calculated using the
extinction coefficient and expressed as lmol g-1 FW.
Determination of MDA content
Lipid peroxidation was determined by estimating the
malondialdehyde (MDA) content according to Kramer
et al. (1991). Frozen samples (0.5 g) mixed with 5 mL
50 mM phosphate buffer (pH 7.8) was crushed into a fine
powder in a mortar and pestle under liquid nitrogen. The
homogenate was centrifuged at 10,000g for 20 min at 4 °C,
with the supernatant being used for MDA determination. A
mixture of 1 mL extracts (MDA) and 2 mL 0.6 % thio-
barbituric acid (TBA) was produced, boiled for 15 min,
cooled and centrifuged for 10 min (4,000g). The concen-
tration of MDA was calculated from the absorbance at 600,
532 and 450 nm, and MDA contents were determined
using the following formula:
MDA (lmol g-1 FW) = [6.45 9 (D532 - D600) - 0.56
D450] 9 V/W, where D532, D600 and D450 are the absorbance at
600, 532 and 450 nm, respectively, and V is the volume of
extraction, W is the fresh weight of sample.
Enzyme extraction and protein content
Frozen plant materials (roots or shoots) were ground in
100 mM ice-cold phosphate buffer (pH 7.0), containing
669
0.1 mM EDTA, 1 % (w/v) insoluble polyvinylpolypyrr-
olidone, and 0.2 mM AsA (Kang et al. 2003). The
homogenates were purified by centrifugation at 12,000g in
4 °C for 60 min. Protein content was measured by the
method of Bradford (1976), with BSA as a standard.
Determination of peroxidase activity
The activity of guaiacol peroxidase (GPOX, EC 1.11.1.7)
was determined in a 3-mL reaction mixture containing
50 mM potassium phosphate, pH 7.0, 0.1 mM Na2EDTA,
5 mM H2O2, and 30 mM guaiacol (Fielding and Hall
1978). The increase in absorbance, due to tetraguaiacol
formation, was recorded at 470 nm. The GPOX activity
was expressed in units per mg protein. One unit of enzyme
activity was defined as the amount necessary to decompose
1 lmol of substrate per min. All spectrophotometric anal-
yses were conducted at 25 °C with a Shimadzu UV-2 101
PC spectrophotometer (Japan).
Statistical analysis
All experimental data in the present study were subjected
to an analysis of variance (ANOVA) using the SAS sta-
tistical software. Furthermore, significant differences
among the mean values of treatments were compared by
the least significant difference (LSD) test method at
P B 0.05 using the MSTAT-C computer program.
Results
ANOVA results (Table 1) showed that the salinity stress
had significant effects on all parameters except root fresh
weight, root length, leaf area, root MDA content and root
H2O2 content. Interaction of NaCl with GA3 and/or AsA
was significant on some traits, but under salinity stress,
interactive effects of NaCl and GA3 were significant on
more parameters than influences of other combined-
treatments.
The effects of NaCl, AsA and GA3 on seedling growth
expressed as fresh and dry weights of roots and shoots,
plant height, root length, leaf area and leaf dry weight are
shown in Tables 2 and 3. Salinity caused a significant
reduction of 39 and 37 % in fresh and dry weight of shoot,
respectively, as compared to control plants. However, this
inhibition was alleviated in the presence of AsA ? GA3,
the fresh and dry weight of shoot in common bean plants in
combination treatment of NaCl, AsA and GA3 decreased to
17 and 8 %, respectively.
The supplied NaCl reduced fresh and dry weight of root,
plant height and root length by 18, 24, 30, and 7 %,
respectively, as compared with the controls. Plants treated
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Table 1 Analysis of variance

Dependent variable Independent variable
(ANOVA) about the influences
of NaCl, GA3, AsA and their NaCl GA3 AsA GA3 9 AsA NaCl 9 GA3 NaCl 9 AsA NaCl 9 GA3
interactions on growth 9 AsA
parameters, pigments, ions,
proteins, MDA and H2O2 Shoot fresh weight *** *** ns ns ns ns **
contents, and GPOX activity of Shoot dry weight *** ** * ns ** ** *
Phaseolus vulgaris L.
Root fresh weight ns ** ** ns ns ns *
Root dry weight *** *** *** ns *** ** **
Root length ns ns * ns ns ** ns
Plant height ** ** ns ns ns ns ns
Leaf area ns ns ns ns ns ns ns
Leaf dry weight * ** ns ** ** ** ns
Chlorophyll a content *** ** ns ns * ** ns
Chlorophyll b content *** ** * ns ns ns *
Carotenoid content *** *** * ns ** *** ns
Anthocyanin content *** *** *** ** ** *** ***
Root Na content *** *** *** *** *** ns **
Shoot Na content *** ** *** ** ** *** **
Root K content *** *** ** ns *** ns ***
Shoot K content *** *** *** *** *** *** ***
Root protein content *** *** *** ns * ** ***
Shoot protein content * *** ** ns *** ns ns
Root MDA content ns ns ns ns ns ns ns
Shoot MDA content *** *** *** *** ** *** ***
The denoted symbols indicate Root H2O2 content ns ns ns ns ns ns ns
significant difference at the Shoot H2O2 content ** ns * ns ns ns ns
0.001 (***), 0.01 (**), and 0.05 Root GPOX activity *** ns ** ns ** * ns
(*) levels
Shoot GPOX activity ** ns ** ns ** ns ns
ns Not significant

with AsA or GA3, specifically AsA ? GA3 ,were less
affected and almost showed no growth reduction.
The leaf area and leaf dry weight were reduced under
salinity stress, but the reduction of leaf area was not sig-
nificant. Under salinity stress, exogenous application of
AsA or GA3 increased leaf area. Also, the interaction of
AsA and GA3 had no significant effects on leaf area. In
saline condition, treatment with AsA and GA3, alone but
not combined, significantly increased leaf dry weight
(Table 3).
The results of photosynthetic and non-photosynthetic
pigments are presented in Table 4. Salinity stress led to a
significant decrease of chlorophylls (Chl a, Chl b) and
carotenoid contents, whereas plants treated with AsA and
GA3 were less affected. In the other hand, NaCl caused a
significant decrease in anthocyanin content. In saline and
non-saline conditions, exogenous application of GA3 and
AsA, alone or combined, significantly enhanced anthocy-
anin content (Table 4).
Treatment of the common bean seedlings with NaCl
resulted in significant accumulation of sodium in both
organs, however in the roots it was more pronounced
(Table 5). Both in the roots and shoots, Na content mark-
edly decreased in response to GA3 or AsA application. The
lowest content of sodium in aboveground parts of NaCl-
treated plants was observed in the simultaneous application
of GA3 and AsA (Table 5). In plants exposed to salinity
stress, potassium concentration strongly decreased in both
roots and shoots. Under the same conditions, exogenous
application of AsA or GA3 significantly enhanced potas-
sium concentration in both studied organs (Table 5).
As shown in Table 6, treatment of seedlings with
200 mM of NaCl, led to lowered content of protein in the
roots and shoots over the control. Otherwise in saline
condition, AsA or GA3 treatment exhibited a stimulatory
effect on the accumulation of proteins in both roots and
shoots. Under the same conditions, maximum protein
content was attained in plants treated with AsA ? GA3.
Common bean seedlings treated with 200 mM NaCl had
higher level of MDA in both roots and shoots than the
control or those with other treatment. The application of
AsA, compared with GA3, led to a higher decrease in the
shoot MDA content under normal and stress conditions. In
the presence of 200 mM NaCl, application of AsA ? GA3

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Table 2 Effects of sodium chloride, gibberellic acid and ascorbic acid on fresh weights of root (FWROOT) and shoot (FWSHOOT), dry weights of
root (DWROOT) and shoot (DWSHOOT), in Phaseolus vulgaris L.
NaCl (mM) AsA (mM) GA3 (mM) FWROOT (g plant-1) FWSHOOT (g plant-1) DWROOT (g plant-1) DWSHOOT (g plant-1)

0 0 0 0.57 ± 0.03cd 1.58 ± 0.04b 0.054 ± 0.001c 0.192 ± 0.007ab

cd c c
0 1 0 0.53 ± 0.06 1.35 ± 0.08 0.051 ± 0.002 0.172 ± 0.006cd
bc ab b
0 0 0.05 0.69 ± 0.04 1.77 ± 0.04 0.065 ± 0.002 0.183 ± 0.002abc
a a a
0 1 0.05 0.90 ± 0.01 1.87 ± 0.09 0.079 ± 0.004 0.194 ± 0.005a
d d d
200 0 0 0.47 ± 0.04 0.96 ± 0.09 0.041 ± 0.001 0.121 ± 0.006f
a c b
200 1 0 0.91 ± 0.07 1.27 ± 0.11 0.064 ± 0.002 0.152 ± 0.006e
200 0 0.05 0.66 ± 0.05bcd 1.33 ± 0.01c 0.051 ± 0.002c 0.160 ± 0.008de
200 1 0.05 0.85 ± 0.15ab 1.30 ± 0.03c 0.062 ± 0.002b 0.178 ± 0.003bc
LSD(0.05) 0.2048 0.2120 0.0054 0.0154
The values (mean ± SE) with different letter within columns are statistically different (P B 0.05) according to LSD test

Table 3 Effects of sodium chloride, gibberellic acid and ascorbic acid on root length (RL), plant height (PH), leaf area (LA) and leaf dry weight
(DWLEAF) in Phaseolus vulgaris L.
NaCl (mM) AsA (mM) GA3 (mM) RL (cm plant-1) PH (cm plant-1) LA (cm2 plant-1) DWLEAF (g plant-1)

0 0 0 10.03 ± 0.75cd 12.70 ± 1.10bc 28.10 ± 2.48a 0.076 ± 0.002a

0 1 0 9.50 ± 1.00d 11.20 ± 0.81cd 25.10 ± 3.02a 0.058 ± 0.002bc
abc a a
0 0 0.05 11.73 ± 0.43 15.74 ± 1.11 25.60 ± 0.90 0.063 ± 0.002abc
bcd ab a
0 1 0.05 10.67 ± 0.17 14.50 ± 0.76 28.00 ± 2.77 0.069 ± 0.003ab
d d a
200 0 0 9.40 ± 0.56 8.93 ± 0.75 23.20 ± 5.32 0.049 ± 0.001c
a cd a
200 1 0 13.40 ± 0.45 11.50 ± 0.80 26.20 ± 4.76 0.058 ± 0.003bc
200 0 0.05 9.67 ± 1.09cd 12.63 ± 0.91bc 25.10 ± 4.14a 0.057 ± 0.005bc
ab bc a
200 1 0.05 12.33 ± 0.73 11.83 ± 1.17 27.40 ± 1.66 0.079 ± 0.005a
LSD(0.05) 2.125 2.822 9.988 0.0173
The values (mean ± SE) with different letter within columns are statistically different (P B 0.05) according to LSD test

Table 4 Effects of sodium chloride, gibberellic acid and ascorbic acid on content of chlorophyll a (Chl a), chlorophyll b (Chl b), carotenoid
(Car) and anthocyanin (Ant) in leaves of Phaseolus vulgaris L.
NaCl (mM) AsA (mM) GA3 (mM) Chl a (mg g-1 FW) Chl b (mg g-1 FW) Car (lmol g-1 FW) Ant (Abs530 g-1 FW)

0 0 0 1.685 ± 0.123ab 0.578 ± 0.057a 0.157 ± 0.007a 0.0208 ± 0.0004fg

c cd cd
0 1 0 1.404 ± 0.049 0.412 ± 0.028 0.114 ± 0.004 0.0230 ± 0.0010ef
a a a
0 0 0.05 1.760 ± 0.020 0.558 ± 0.059 0.166 ± 0.013 0.0344 ± 0.0011d
abc ab bc
0 1 0.05 1.525 ± 0.119 0.534 ± 0.012 0.126 ± 0.004 0.0565 ± 0.0021a
200 0 0 0.856 ± 0.046e 0.249 ± 0.014e 0.077 ± 0.008e 0.0186 ± 0.0002 g

de e d e
200 1 0 1.050 ± 0.115 0.242 ± 0.029 0.103 ± 0.005 0.0245 ± 0.0003
200 0 0.05 1.267 ± 0.084cd 0.418 ± 0.025bc 0.123 ± 0.004bc 0.0406 ± 0.0004b
bc de b
200 1 0.05 1.423 ± 0.124 0.269 ± 0.053 0.138 ± 0.001 0.0374 ± 0.0013c
LSD(0.05) 0.2791 0.1161 0.0173 0.0029
The values (mean ± SE) with different letter within columns are statistically different (P B 0.05) according to LSD test

decreased MDA content in both roots and shoots. Also,
combination of AsA and GA3 induced maximum decrease
of MDA content in the roots under salinity stress (Table 6).
Our data in Table 7 showed that hydrogen peroxide
(H2O2) content was increased in common bean plants
which were treated with salinity, but this increment was
only significant in the aboveground parts. Also, H2O2
contents in the roots of the controlled and treated seedlings
were considerably higher when compared to those for the
shoots. In seedlings treated with 200 mM NaCl, the
application of AsA or AsA ? GA3 could decline concen-
tration of hydrogen peroxide in both organs, while inter-
active influences of NaCl and GA3 had no significant role
in reduction of H2O2 content (Table 7).

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Table 5 Effects of sodium chloride, gibberellic acid and ascorbic acid on content of sodium in root (NaROOT) and shoot (NaSHOOT), and
potassium in root (KROOT) and shoot (KSHOOT) in Phaseolus vulgaris L.
NaCl AsA GA3 NaROOT NaSHOOT KROOT KSHOOT
(mM) (mM) (mM) (lmol g-1 DW) (lmol g-1 DW) (lmol g-1 DW) (lmol g-1 DW)

0 0 0 73.33 ± 4.41e 36.11 ± 1.47d 247.22 ± 4.75a 196.67 ± 2.55c

g d b
0 1 0 49.44 ± 2 36.67 ± 0.96 234.44 ± 3.89 181.67 ± 2.89d
ef d c
0 0 0.05 62.22 ± 1.47 34.44 ± 1.47 191.11 ± 1.11 180.56 ± 1.47d
0 1 0.05 59.44 ± 2.42fg 34.44 ± 2.94d 226.67 ± 3.85b 189.44 ± 2cd
200 0 0 372.22 ± 7.35a 93.33 ± 2.89a 81.67 ± 3.85g 163.33 ± 2.89e
b bc d
200 1 0 324.44 ± 4.01 61.11 ± 3.64 122.78 ± 1.47 255.00 ± 6.74ab
d b e
200 0 0.05 295.00 ± 1.67 67.78 ± 2.94 109.44 ± 2.42 249.44 ± 2b
c c f
200 1 0.05 307.22 ± 3.09 57.78 ± 2 93.33 ± 5.09 263.33 ± 0.96a
LSD(0.05) 11.310 7.340 10.730 9.457
The values (mean ± SE) with different letter within columns are statistically different (P B 0.05) according to LSD test

Table 6 Effects of sodium chloride, gibberellic acid and ascorbic acid on content of protein in root (ProROOT) and shoot (ProSHOOT), and
malondialdehyde in root (MDAROOT) and shoot (MDASHOOT) in Phaseolus vulgaris L.
NaCl AsA GA3 ProROOT ProSHOOT MDAROOT MDASHOOT
(mM) (mM) (mM) (mg g-1 FW) (mg g-1 FW) (lmol g-1 FW) (lmol g-1 FW)

0 0 0 1.58 ± 0.01c 1.21 ± 0.13bc 19.33 ± 4.89a 4.73 ± 0.33bc

0 1 0 1.94 ± 0.05b 1.23 ± 0.07c 14.51 ± 2.13a 2.75 ± 0.21de
a d a
0 0 0.05 2.15 ± 0.11 0.94 ± 0.05 18.14 ± 6.36 3.14 ± 0.15de
ab bc a
0 1 0.05 2.09 ± 0.05 1.27 ± 0.01 15.90 ± 2.98 2.01 ± 0.14e
e e a
200 0 0 0.59 ± 0.06 0.62 ± 0.07 24.28 ± 7.43 13.41 ± 1.15a
d cd a
200 1 0 0.87 ± 0.01 1.06 ± 0.03 20.31 ± 10.46 3.29 ± 0.11cde
200 0 0.05 0.92 ± 0.04d 1.55 ± 0.06b 25.45 ± 6.87a 5.71 ± 0.59b
c a a
200 1 0.05 1.66 ± 0.05 2.02 ± 0.21 19.88 ± 4.80 3.68 ± 0.20cd
LSD(0.05) 0.1624 0.2984 18.740 1.4660
The values (mean ± SE) with different letter within columns are statistically different (P B 0.05) according to LSD test

The activity of antioxidant enzyme, GPOX, was always
lower in the shoots than in the roots in controls as well as in
other treatments (Table 7). The activity of this enzyme was
slightly increased in response to NaCl alone; but in the
combination with AsA or GA3, enzyme activity was much
higher than when NaCl was given alone. The plants treated
with NaCl and AsA ? GA3 exhibited the highest values
for GPOX activity in both roots and shoots (Table 7).
Discussion
In the present study, salt stress caused significant inhibition
in growth of P. vulgaris seedlings. Similar results were
reported by Tejera et al. (2004, 2005), who indicated that
growth of common bean plants considerably decreased by
salt stress. Salinity can inhibit plant growth by altering the
water potential, increasing the ion toxicity, inhibiting the
cell division and cell expansion, or causing an ion imbal-
ance (Arshi et al. 2005). In this context, Younis et al.
(2010) reported that the growth reduction caused by
salinity stress is due to inhibited apical growth in plants as
well as endogenous hormonal imbalance. In both cases,
reduction could have been caused by the toxic effects of
ions (Na? and Cl-) on metabolism or from adverse
water relations. In addition, a secondary aspect of salinity
stress in plants is the stress-induced production of ROS
(Manchanda and Garg 2008). The enhanced production of
ROS during salinity stress lead to the progressive oxidative
damage and ultimately cell death and growth suppression
(Ruiz-Lozano et al. 2012).
The growth characteristics of common bean seedlings
under salinity stress were effectively improved with
GA3 ? AsA supplement, implying that this treatment can
alleviate the deleterious effects of salt stress. Gibberellic
acid has been reported to be helpful in enhancing growth of
wheat, maize and tomato under saline conditions (Ashraf
et al. 2002; Kaya et al. 2006; Maggio et al. 2010). Salt
stress was strong enough to inhibit plant growth due to
reduction in gibberellin production (Kaya et al. 2006).

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Table 7 Effects of sodium chloride, gibberellic acid and ascorbic acid on content of hydrogen peroxide in root (H2O2 ROOT) and shoot (H2O2
SHOOT), and guaiacol peroxidase activity in root (GPOXROOT) and shoot (GPOXSHOOT) in Phaseolus vulgaris L.

NaCl AsA GA3 H2O2 ROOT H2O2 SHOOT GPOXROOT GPOXSHOOT

(mM) (mM) (mM) (lmol g-1 FW) (lmol g-1 FW) (U mg-1 pro) (U mg-1 pro)

0 0 0 7.31 ± 2.70a 2.09 ± 0.36bc 67.3 ± 5.9cd 41.6 ± 5.2bc

a c dc
0 1 0 8.98 ± 3.86 1.62 ± 0.19 68.5 ± 8.1 54.4 ± 8.1ab
a c d
0 0 0.05 7.42 ± 2.10 1.52 ± 0.35 56.7 ± 4.4 33.5 ± 3.8c
0 1 0.05 6.36 ± 1.73a 1.56 ± 0.29c 60.8 ± 5.2d 34.0 ± 4.6c
200 0 0 12.56 ± 4.81a 3.33 ± 0.60a 68.7 ± 2.6cd 42.0 ± 1.2bc
a ab b
200 1 0 10.94 ± 5.69 3.00 ± 0.58 89.3 ± 5.2 54.0 ± 3.8ab
a a bc
200 0 0.05 13.26 ± 5.84 3.53 ± 0.29 81.2 ± 6.7 46.3 ± 8.6bc
a c a
200 1 0.05 9.66 ± 3.48 1.78 ± 0.39 108.0 ± 6.0 68.0 ± 4.2a
LSD(0.05) 9.996 1.207 17.160 16.240
The values (mean ± SE) with different letter within columns are statistically different (P B 0.05) according to the LSD test

Therefore, addition of exogenous gibberellic acid might
increase seedling growth by enhancing the content of
endogenous gibberellin as that mentioned by Rodriguez
et al. (2006). Additionally, the enhancement of growth rate
by gibberellin might result in an enlargement of leaf area,
activation of cell division and/or cell elongation, stimula-
tion of photosynthetic rate, modified partitioning of pho-
tosynthates, or in their combination. The GA3-mediated
invertase activity in elongating shoots could result in a
significant accumulation of hexoses required for the pri-
mary cell wall biosynthesis, thus favoring seedling growth
under stress condition (Saeidi-Sar et al. 2007).
On the other hand, the beneficial effect of AsA on plant
growth may be attributed to the fact that AsA is involved in
the regulation of root elongation, cell vacuolation and cell
expansion (Smirnoff 1996). AsA-induced increase in
growth under non-saline conditions may have been due to a
double action of AsA on cell growth by modifying the cell
cycle and stimulating quiescent cells to divide, and by
accelerating cell elongation. In addition, ascorbate is a
co-factor for prolyl-hydroxylase that post-translationally
hydroxylates proline residues in cell wall hydroxyproline-
rich glycoproteins required for cell division and expansion
(Smirnoff and Wheeler 2000). Moreover, AsA increases
the content of IAA, which stimulates cell division and/or
cell enlargement and this, in turn, improves plant growth
(Khan et al. 2011).
Measurement of photosynthetic pigments under salinity
conditions in P. vulgaris seedlings explains that NaCl
salinity has the negative effects on these pigments. The
adverse effects of salt stress on chlorophylls and carote-
noids were counteracted by exogenous application of GA3
and AsA. The high salinity caused a disturbed chloroplast
structure, number and size, which affected chlorophyll
content and/or caused disruption of chloroplasts by oxi-
dative stress that causes a decrease in chlorophyll content
(Rahman et al. 2000). Also, reduction in chlorophyll con-
tent under salinity stress is attributed to salt-induced
acceleration of chlorophyll enzymes degradation and the
instability of pigment–protein complex (Hernandez and
Almansa 2002).
Among the positive effects of AsA in the counteraction
of the adverse effects of salt stress are the stabilization and
protection of the photosynthetic pigments and the photo-
synthetic apparatus from oxidization (Khan et al. 2011).
Ascorbic acid can mitigate the adverse effects of salinity
through increasing the content of IAA and GA3 and
decreasing ABA level (Khan et al. 2011), which may be
involved in protecting the photosynthetic apparatus and
consequently increasing the photosynthetic pigments. AsA
has a major role in photosynthesis, acting in the Mehler
peroxidase reaction with ascorbate peroxidase to regulate
the redox state of photosynthetic electron carriers and as a
co-factor for violaxanthin de-epoxidase, an enzyme
involved in xanthophyll cycle-mediated photoprotection
(Smirnoff and Wheeler 2000). Consequently, in AsA-
treated plants, high level of carotenoids can synergistically
function with ascorbic acid to provide an effective barrier
against oxidation under salinity stress.
On the other hand, GA3 also plays a vital role in toler-
ance to salt stress by improving plant growth and chloro-
phyll synthesis (Maggio et al. 2010). In addition, the
inhibitory effect of GA3 on chlorophyll catabolism might
be partly due to the down regulation of the activities of
enzymes involved in chlorophyll catabolism and the alle-
viation of oxidative chlorophyll bleaching (Li et al. 2010).
The concentration of anthocyanin was appreciably
increased due to exogenous application of GA3 and AsA
under both stressful and non-stressful conditions. AsA
functions as an enzyme co-factor for anthocyanins syn-
thesis (Smirnoff and Wheeler 2000). Zhang et al. (2012)
indicated that leaves containing anthocyanins had a greater

123
674
antioxidant potential than did green leaves, and anthocya-
nins can be used as antioxidants to extinguish ROS pro-
duced in either PSI or PSII under environmental stresses.
Accordingly, the powerful antioxidative capability of
anthocyanins in the presence of GA3 and AsA mitigated
the adverse effects of NaCl on common bean plants.
The present study also revealed that the imposition of
salt stress caused an increase in uptake and accumulation of
Na? and a decrease in K? concentration in both roots and
shoots of common bean plants, whereas improvements in
amounts of these ions were observed in application of GA3
and AsA to stressful media. Salt stress is known to enhance
the uptake and accumulation of toxic ions such as Na? in
plant species (Sibole et al. 1998; Ashraf et al. 2002; Kaya
et al. 2006; Nasir et al. 2010; Zhang et al. 2011). Higher
Na? uptake by roots of common bean seedlings under salt
stress conditions is possibly caused by inhibition of the
Na?/H?-antiporters operating normally at the plasma
membrane (Roslyakova et al. 2011). Sodium uptake is
mediated by both voltage-dependent and -independent
cation channels. Voltage-dependent cation channels such
as K? inward rectifiers (HKT, HAK and KUP) mediate
Na? uptake into root cells. Sodium competes with K?
uptake through Na?–K? co-transporters and may also
block the K?-specific transporters of root cells, thus high
levels of Na? can induce the conformational changes
in protein structure and membrane depolarization, and lead
to the perception of ion toxicity in saline conditions
(Manchanda and Garg 2008).
Exogenous supply of AsA enhanced potassium con-
centration in NaCl-stressed plants. This increase may be
attributed to the positive effect of AsA on the root growth,
which consequently increased the absorption of different
nutrients and alleviated the harmful effects of salinity. In
addition, AsA would inhibit a stress-induced increase in the
leakage of essential electrolytes following peroxidative
damage to plasma membranes (Khan et al. 2011). More-
over, the increase of transmembrane electron transport via
cytochrome b using ascorbic acid depolarizes the plasma
membrane, and activates the H?-ATPase resulting in
increased ion uptake such as K? (Smirnoff and Wheeler
2000). Almost all interactions between salinity and both
GA3 or AsA decreased Na? concentration and/or increased
K? concentration as compared to salinized plants. These
results agree with Aldesuquy (1995) who reported that GA3
reduced the accumulation of toxic ions in plant tissues
under saline conditions; and Athar et al. (2008) who sug-
gested that the protection of wheat plants against salt stress
by an exogenous supply of AsA is caused indirectly as a
result of its effect on K? uptake, which plays an essential
role in many metabolic processes. GA3 and/or AsA-treated
plants accumulated lower quantities of Na? in roots, which
were possibly related to mobilization of defense
123
Acta Physiol Plant (2013) 35:667–677
mechanisms that restricted Na? entry or promoted the
extrusion of Na? to the external medium. The preferential
accumulation of sodium in roots over the shoots may be
interpreted as a mechanism of tolerance in at least two
ways, firstly: maintenance of a substantial potential for
osmotic water uptake into the roots, secondly: restricting
the spread of Na? to the shoots (Renault et al. 2001).
We observed a great reduction in protein content of both
shoot and root of common bean plants under NaCl stress.
Metabolic toxicity of Na? is largely a result of its ability to
compete with K? for binding sites of multiple enzymes in
the cytoplasm that are required for cellular function. Pro-
tein synthesis also requires high concentrations of K?,
owing to the K? requirement for the binding of tRNA to
ribosomes and probably other aspects of ribosome function
(Tester and Davenport 2003). Thus, disruption of protein
synthesis by elevated concentrations of Na? appears to be
an important cause of damage by NaCl. Salt stress can also
increase the production of ROS and cause damage to
proteins. One possibility is that autophagy might be
responsible for degrading oxidized proteins under salt
stress (Liu et al. 2009). But in the presence of AsA, the
soluble protein content increased, indicating that it may
have an important role in plant adaptation to a high NaCl
content (Huang et al. 2011). Possibly, the protective effect
of AsA under salinity stress is more related to a reduction
in ROS damages to essential proteins and/or nucleic acids
(Noctor and Foyer 1998; Smirnoff and Wheeler 2000;
Khan et al. 2011).
It has been reported that ROS, including superoxide and
hydrogen peroxide, are elevated with increasing the salin-
ity, due to the imbalance in the production and destruction
of ROS (Harinasut et al. 2003). In the present study, the
P. vulgaris seedlings responded to NaCl treatment with
enhancement of H2O2 content, which was higher in the
roots than the shoots. Hydrogen peroxide has been shown
to negatively influence proliferation of cells (Santoro et al.
2005). Thus, H2O2 can play an important role in the inhi-
bition of growth of NaCl-stressed plants. Salt stress is also
known to result in extensive lipid peroxidation, which has
often been used as indicator of salt induced oxidative
damage in membranes. The observed enhanced value for
MDA content in the plants raised from treatment with NaCl
alone indicates that NaCl toxicity may be responsible for
increasing lipid peroxidation leading to cell damage.
Exogenous application of GA3, AsA and GA3 ? AsA
reduced NaCl-induced increase in the contents of H2O2 and
MDA. Gibberellins appear to play a key role against oxi-
dative stress by decreasing accumulation of ROS and
preventing lipid peroxidation, which were induced by
salinity. These alleviating effects of GA3 were highly
correlated with the increasing activities of antioxidant
enzymes (Maggio et al. 2010). However, in this study AsA
Acta Physiol Plant (2013) 35:667–677
was a more efficient antioxidant than GA3 against NaCl-
induced oxidative damage to P. vulgaris plants. Ascorbate is
involved in controlling the intracellular ROS level by direct
scavenging or via the ascorbate–glutathione cycle. This might
provide the means to protect the cell against uncontrolled
oxidation and improved growth (Noctor and Foyer 1998;
Smirnoff and Wheeler 2000; Bartoli et al. 2006).
Plants possess efficient systems for scavenging active
oxygen species that protect them from destructive oxida-
tive reactions (Zhu et al. 2004). As part of this system,
antioxidant enzymes are key elements in the defense
mechanisms. In our study, slight increase in GPOX activity
was observed in root and shoot of common bean seedlings
exposed to NaCl. These results suggest that this antioxidant
enzyme has possible defensive role in the removal of H2O2
under salinity stress. The increased total peroxidase activity
in response to salinity has been reported (Chen et al. 1993;
Sreenivasulu et al. 2000; Harinasut et al. 2003). The plants
treated with NaCl in combination with GA3 and AsA
exhibited much higher values for antioxidant enzyme
activity than when NaCl was given alone. GA3 and AsA
probably reverse the effect of NaCl stress in common bean
seedlings by enhancing peroxidase activity, as well as by
decreasing H2O2 production and lipid peroxidation.
Accordingly, improved activity of GPOX by the combined
application of GA3 and AsA resulted in an increase in the
capacity of detoxification mechanism and also in an
improvement in the capacity of tolerance to NaCl stress.
In conclusion, results of the present study suggest that
NaCl toxicity is associated with induction of oxidative
stress in P. vulgaris seedlings leading to increased gener-
ation of H2O2, elevated levels of lipid peroxidation and
protein oxidation, and a decline in ratios of carotenoids and
anthocyanins, but deleterious effect of salinity was coun-
teracted by ascorbic acid and gibberellin A3. However, the
mechanisms by which GA3 could induce salt tolerance in
plants are not yet clear. Salinity perturbs the hormonal
balance in plants. Therefore, hormonal homeostasis under
salt stress might be the possible mechanism of GA3-
induced plant salt tolerance. In this respect, the beneficial
effects of AsA might be attributed to the increase of pho-
tosynthesis activity, as well as to the role of AsA in several
defense mechanisms against oxidative stress under salinity
stress conditions. In order to reduce the oxidative damages
caused by NaCl stress, the protective role of AsA ? GA3
was often better than applying AsA or GA3 alone. The
increased tolerance to NaCl stress in the presence of AsA
and GA3 was manifested in terms of enhanced growth,
GPOX activity, chlorophylls and potassium contents. But
further researches are required to decipher the mechanisms
through which AsA in combined with GA3 acts, and how
synergistically effects of them might be connected with salt
tolerance.
675
Author contribution Dr. S. Saeidi-Sar designed the
experimental framework and supervised the whole work,
and contributed to all the experimental process, data
interpretation and discussion as well as paper writing.
Dr. H. Abbaspour and Dr. H. Afshari designed the exper-
iments and contributed to the experimental process and
interpretation of results, and also involved in preparation of
the manuscript. Dr. S.R. Yaghoobi performed the data
analysis and paper editing.
Acknowledgments This work was supported by a grant from the
research funds appropriated to Damghan Branch—Islamic Azad
University, Damghan, Iran (No. 51424881019003).
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