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Acclimation of hydrogen peroxide enhances salt toleranceby activating defense-related proteins in Panax ginseng C.A.Meyer
Gayathri Sathiyaraj • Sathiyaraj Srinivasan • Yu-Jin Kim •
Ok Ran Lee • Shonana Parvin • Sri Renuka Devi Balusamy •
Atlanzul Khorolragchaa • Deok Chun Yang
Received: 25 April 2013 / Accepted: 6 February 2014
� Springer Science+Business Media Dordrecht 2014
Abstract The effect of exogenously applied hydrogen
peroxide on salt stress tolerance was investigated in Panax
ginseng. Pretreatment of ginseng seedlings with
100 lM H2O2 increased the physiological salt tolerance of
the ginseng plant and was used as the optimum concentration
to induce salt tolerance capacity. Treatment with exogenous
H2O2 for 2 days significantly enhanced salt stress tolerance
in ginseng seedlings by increasing the activities of ascorbate
peroxidase, catalase and guaiacol peroxidase and by
decreasing the concentrations of malondialdehyde (MDA)
and endogenous H2O2 as well as the production rate of
superoxide radical (O2-). There was a positive physiological
effect on the growth and development of salt-stressed seed-
lings by exogenous H2O2 as measured by ginseng dry weight
and both chlorophyll and carotenoid contents. Exogenous
H2O2 induced changes in MDA, O2-, antioxidant enzymes
and antioxidant compounds, which are responsible for
increases in salt stress tolerance. Salt treatment caused
drastic declines in ginseng growth and antioxidants levels;
whereas, acclimation treatment with H2O2 allowed the
ginseng seedlings to recover from salt stress by up-regulation
of defense-related proteins such as antioxidant enzymes and
antioxidant compounds.
Keywords Hydrogen peroxide � Ascorbate peroxide �Catalase � Malondialdehyde � Salt stress � Guaiacol
peroxidase � Proline
Introduction
Abiotic stresses cause broad losses in agricultural produc-
tion worldwide by interrupting cellular homeostasis and
changing physical and biological processes, leading to
decreased growth and subsequently decreased yield.
Among abiotic stresses, salinity is the major environmental
factor limiting plant growth and productivity [1]. When
plants are subjected to salinity, reactive oxygen species
(ROS) such as superoxide, hydrogen peroxide (H2O2),
singlet oxygen, and hydroxyl radicals rapidly accumulate
[2]. These free radicals disrupt normal metabolism by
reacting with a number of other molecules and metabolites
such as DNA, pigments, proteins, lipids, and other essential
cellular molecules, leading to a series of destructive pro-
cesses [3, 4]. To prevent or alleviate the effects of ROS and
to cope with the potential damage from salinity, plants
have evolved a variety of defense mechanisms. These
include accumulation of osmolytes such as proline, gly-
cine, betaine and sugars and the up-regulation of antioxi-
dant enzymes [5] including superoxide dismutase (SOD),
glutathione peroxidase (GSH-Px), ascorbate peroxidase
(APX), glutathione reductase (GR), dehydroascorbate
reductase (DHAR), and monodehydroascorbate reductase
(MDHAR) [6]. Salt stress alters the critical balance
between the production of ROS and the quenching
Gayathri Sathiyaraj and Sathiyaraj Srinivasan contributed equally to
this work.
G. Sathiyaraj � S. Srinivasan � Y.-J. Kim � O. R. Lee �S. Parvin � S. R. D. Balusamy � A. Khorolragchaa � D. C. Yang
Korean Ginseng Center for Most Valuable Products & Ginseng
Genetic Resource Bank, Kyung Hee University, Suwon 449-701,
South Korea
e-mail: [email protected]
D. C. Yang (&)
Department of Oriental Medicinal Material & Processing,
College of Life Science, Kyung Hee University, 1 Seocheon,
Kiheung Yongin, Kyunggi 449-701, South Korea
e-mail: [email protected]
123
Mol Biol Rep
DOI 10.1007/s11033-014-3241-3
activities of antioxidants. This instability in equilibrium
leads to sudden increases in intracellular levels of ROS and
can cause significant damage to cell structures [7].
Among the ROS, H2O2 is a non-radical ROS, being a
molecule that carries no net charge [8]. H2O2 has a longer
half-life (about 1 ms) than other ROS [9] and is more likely
to be a long distance signaling molecule [10]. Membrane
channels such as aquaporins and peroxiporin have been
reported to facilitate H2O2 trans-membrane transport in
combination with water [11]. Exogenously applied H2O2 has
been reported to overcome cellular injuries caused by vari-
ous stresses. H2O2 plays a dual role in plants: at low con-
centrations, it acts as an acclamatory signal, triggering
tolerance to various stresses [4, 12, 13], and at high con-
centrations, it organizes programmed cell death [14]. During
normal conditions, H2O2 is generated during the Mehler
reaction (chloroplast), electron transport (mitochondria), and
photorespiration (peroxisomes). Abiotic and biotic stresses
also enhance H2O2 generation via enzymatic sources such as
NADPH oxidases or cell wall peroxidases [15].
Exogenous H2O2 levels establish the metabolism of anti-
oxidant enzymes, and its signaling was shown to be signifi-
cant in several processes in plants such as stomatal closure,
senescence [16], photorespiration and photosynthesis [17],
stomatal movement [18], cell cycle [4], growth, gravitropism,
and regulation of basic acclimatory defense and develop-
mental processes in plants [13, 19]. Recent investigations
have revealed that H2O2 is a central component of the signal
transduction cascade involved in plant adaptation to a
changing environment [19]. Addition of H2O2 to a nutrient
solution induces chilling tolerance in mung bean seedlings
[20]. In maize, exogenously applied H2O2 increases salt tol-
erance by increasing the activities of antioxidants [21]. H2O2
contributes to the phenomenon known as ‘‘acclimation tol-
erance,’’ where exposure of plants to low levels of one stress
offers protection against another stress [22, 23].
Panax ginseng is an oriental medicinal plant belonging
to the Araliaceae family. Ginseng has numerous pharma-
cological effects involved in normalizing the human met-
abolic system and also has activity against headache,
fatigue, dizziness, nausea, and asthma [24]. Ginseng is a
long-term duration plant; during long cultivation periods,
much care is needed since plant growth is susceptible to
many environmental factors including abiotic stresses, such
as salinity and climate, and biotic stress.
Decreases in growth and stress due to salt have been
reported in P. ginseng [25]. The purpose of our study was
to develop mechanisms to protect ginseng from salinity
stress. The aim of the investigation was to evaluate whether
exogenous low levels of H2O2 could protect P. ginseng
seedlings from salt tolerance by examining physiological
and biochemical changes with and without H2O2 pretreat-
ment. Pretreatment with H2O2 provides an easy, low cost,
and effective strategy to overcome environmental stress
problems. Exogenous H2O2 application is a convenient and
effective approach for enhancing salinity tolerance of crops
and eventually improving crop productivity under high
salinity conditions.
Materials and methods
Plant material and pre-treatment
Panax ginseng seeds were collected from the Korean Ginseng
Research Center, South Korea. In vitro embryo cultures were
grown in bottles containing half-strength Moorashige and
Skoog (MS, Duchefa Biocheme, Netherland) media. After
three days, embryos were transferred to full strength MS
media in the growth chamber under the following conditions:
12/12 h photoperiod; light intensity 70 lmol m2 s-1; tem-
perature 23 ± 1� C. Preliminary studies were performed
using various concentrations of H2O2 ranging between 0.05
and 250 lM, and the best concentrations for growth were
determined. Plants were pre-treated with a nutrient solution
(MS media) containing 100 lM H2O2 for 2 days (accli-
mation treatment), while some plants remained in nutrient
solution which is used for control.
Salt stress treatment
H2O2 pre-treated plants were kept in MS nutrient solution
along with salt (150 mM). The plants were subjected to one
of four treatments: control (not pretreated with H2O2 and not
salt-stressed); unacclimated-stressed (not pre-treated with
H2O2 and salt-stressed); acclimated-unstressed (pre-treated
with H2O2 and not salt-stressed); and acclimated-stressed
(pre-treated with H2O2 and salt-stressed). All experiments
were carried out under green house conditions. The mean
values of temperature, relative air humidity and photosyn-
thetic active radiation (at noon) were 25 �C, 65 % and
1,200 mmol m2 s-1, respectively. Seedlings were harvested
just before pre-treatment (day 0), at the end of pre-treatment
(before the start of salt additions—day 2), and at 1 (day 6), 5
(day 10) and 10 (day 15) days after the end of salt treatment.
Plants from each treatment group were separated into leaf,
shoot and roots for dry mass (DM) determinations, as
described by Neto [21]. The first fully expanded leaf and the
younger third of the root system were frozen in liquid
nitrogen, lyophilized, ground to a powder and stored in a
freezer (-70 �C) for further biochemical analyses.
Chlorophyll and carotenoid estimation
The chlorophyll and carotenoid content in leaves provide
important internal information for predicting plant growth
Mol Biol Rep
123
status [26]. Plant tissue (10 mg) was homogenized with
80 % acetone in Eppendorf test tubes. Subsequently, the
tubes were incubated for 10 min in the dark and centri-
fuged at 2,5009g for 15 min. Supernatants were preserved
for spectrophotometry.
For chlorophyll: Chla = (11.93 * OD664) - (1.93 * OD647);
Chlb = (20.36 * OD647) - (5.5 * OD664).
For carotenoid: DA CAR480 = DA CAR480 ? 0.114
DA663 - 0.638 DA645.
Relative water content
RWC was measured in H2O2 acclimated, salt treated, H2O2
acclimated ? salt treated and control (medium only) groups.
RWC represents a useful indicator of the state of water balance
of a plant because it expresses the absolute amount of water
that the plant requires to reach artificial full saturation [27].
Fresh weight � dry weight � 100=dry weight
Growth parameters
Green plant parts and roots were oven dried at 65 �C until
constant weight and biomass (g) were determined using an
electronic scale. Plant height was also measured.
Extract preparation
Lyophilized leaf (0.20 g) and root (0.15 g) powders were
homogenized using a mortar and pestle with 4 ml of ice-
cold extraction buffer (100 mM potassium phosphate buf-
fer, pH 7.0, 0.1 mM EDTA). The homogenate was filtered
through muslin cloth and centrifuged at 16,0009g for
15 min. The supernatant fraction was used as a crude
extract for enzyme activity and lipid peroxidation assays.
All operations were carried out at 4 �C.
Total catalase assay
Total catalase (CAT, EC 1.11.1.6) activity was measured
according the method of Beers and Sizer [28]. Decreases in
H2O2 were monitored at 240 nm and quantified by the
molar extinction coefficient (36 M-1 cm-1). The results
are expressed as mmol H2O2 min-1 g-1 dry mass (DM).
Total ascorbate peroxide assay
Total ascorbate peroxide (APX, EC 1.11.1.1) activity was
assayed according to Nakano and Asada [29]. Enzyme
activity was quantified using the molar extinction coeffi-
cient for ascorbate (2.8 mM-1 cm-1). Results are expres-
sed in mmol H2O2 min-1 g-1 DM, taking into
consideration that 2 mol ascorbate are required for reduc-
tion of 1 mol H2O2 [30].
Total guaiacol peroxidase assay
Total guaiacol peroxidase (GPX, EC 1.11.1.7) activity was
determined as described by Urbanek et al. [31]. Enzyme
activity was quantified by the amount of tetraguaiacol formed
using its molar extinction coefficient (26.6 mM-1 cm-1).
Results are expressed as mmol H2O2 min-1 g-1 DM, taking
into consideration that 4 mol H2O2 are reduced to produce
one mol tetraguaiacol [32].
Proline content determination
Free proline (Pro) content was determined as previously
reported. Free Pro was extracted from salt-stressed ginseng
hairy roots. Acid-ninhydrin was prepared by warming
1.25 g ninhydrin in 30 ml glacial acetic acid and 20 ml of
6 M phosphoric acid with agitation until dissolved and was
stored at 4 �C. Plant material (0.5 g) was homogenized in
10 ml of 3 % aqueous sulfosalicylic acid, and the
homogenate was filtered through Whatman # 2 filter paper.
Two milliliters of filtrate was reacted with 2 ml acid nin-
hydrin and 2 ml of glacial acetic acid in a test tube for 1 h
at 100 �C. The reaction was terminated in an ice bath. The
reaction mixture was extracted with 4 ml toluene in a test
tube by mixing vigorously for 15–20 s. The chromophore
containing toluene was aspirated from the aqueous phase,
warmed at room temperature, and the absorbance was read
at 520 nm using toluene for a blank. The Pro concentration
was determined using a standard curve and calculated on a
fresh weight basis as follows:
l moles proline per gm of fresh weight material
¼
lg proline per ml � ml toluene
115:5lg
l molg sample
5
Hydrogen peroxide assay
H2O2 content was determined by measuring the absorbance
of titanium–hydroperoxide complex [33]. The complex
was dissolved in 1 M sulfuric acid, and absorbance of the
supernatant was measured at 415 nm against a blank. The
concentration of H2O2 was determined using the standard
curve plotted with known concentrations of H2O2.
Lipid peroxidation assay
Malondialdehyde (MDA) concentration was measured
according to Predieri et al. [34]. The MDA concentration
was determined by its molar extinction coefficient
(155 mM-1 cm-1), and the results were expressed as
l mol MDA g-1 FW.
Mol Biol Rep
123
Superoxide radical (O2-) determination
The production rate of O2- was measured by a modified
method as described by Elstner and Heupel [35]. The
specific absorbance at 530 nm was determined. Sodium
nitrite was used as a standard solution to calculate the
production rate of O2-.
Gene transcriptome analysis
RNA isolation and real-time quantitative RT-PCR
The RT-PCR was done to determine the transcript level of
ginseng defence marker genes such as polygalacturonase
inhibiting protein (PGIP) [36], ribonuclease-2 (PR-10)
[37], chitinase (PR-2) [38], calmodulin [39], sesquiterpene
synthase [40] and spermidine synthase [41]. RNA was
extracted from plants using an RNeasy kit (Qiagen,
Valencia, CA, USA) according to the manufacture’s
instructions. The quality and concentration of RNA was
measured using a spectrophotometer (GE Nanovalue,
USA). To obtain first strand cDNA, 5 lg of total RNA was
reverse transcribed using a Power cDNA kit (Invitrogen,
USA) as per the manufacturer’s instructions and diluted to
80 % using nuclease-free water. Real-time quantitative
PCR was performed using a real-time rotary analyzer
(Rotor-Gene 6000, Corbett Life Science, Sydney, Austra-
lia) with 10 lg of cDNA in a 10 ll reaction volume using a
SYBR� Green Sensimix Plus Master Mix (Quantace,
Watford, England). The housekeeping gene encoding the
actin protein was used as a control in the experiment and
was amplified with forward primers: 50-CGT GAT CTT
ACA GAT AGC TTG ATG A-30; reverse: 50-AGA GAA
GCT AAG ATT GAT CCT CC-30. PCR conditions for the
40 cycles were 95 �C for 10 s, 58 �C for 10 s, and 72 �C
20 s; then extension at 72 �C for 8 min. Fluorescence was
detected and measured in a real-time PCR thermocycler,
and the geometric increase in fluorescence corresponding
to an exponential increase in the product was used to
determine the threshold cycle (CT) for each reaction. All
real-time PCR reactions were performed in triplicate.
Three samples were analyzed for each treatment; values are
given as mean ± SD.
Results
Optimum pretreatment concentration of exogenous
H2O2
In order to determine the optimized concentration of H2O2
for the enhancement of ginseng seedling growth, 4-week-
old ginseng seedlings were treated with different
concentrations (0.05–250 lM) of H2O2 against 150 mM of
NaCl and their growth was monitored. Various concen-
trations of H2O2 treatment caused significant reductions in
plant height and plant pigment content in ginseng seedlings
(Fig. 1a). On average, a 40 % reduction was seen in plant
height at H2O2 concentrations of 200 and 250 lM. Pre-
treatment of ginseng seedlings with 100 lM H2O2
increased plant height (up to 8 cm) and dry weight
(0.24 g), as well as the levels of photosynthetic pigments
such as chlorophyll and carotenoid (Fig. 1b) compared
with the 150 mM salt-stressed seedlings (control). Finally
the dosage of H2O2 (0.05–50 lM) increased the dry
weight, plant height, chlorophyll and carotenoid contents
of the seedlings, among them 100 lM H2O2 showed
maximum of all against salt stress. However, the physio-
logical characteristics decreased by increasing concentra-
tion of H2O2 level ([100 lM). Therefore, 100 lM H2O2
was selected as the optimal concentration for the accli-
mation of ginseng seedlings for salt stress tolerance.
Effect of exogenous H2O2 pretreatment on plant growth
during salt stress
There were significant decreases in the total chlorophyll
and carotenoid contents in salt-stressed ginseng seedlings.
Exogenous H2O2 had no impact on plant pigments before
5 days of treatment, but were significantly increased at day
7 (Fig. 2a, b). The highest shoot and root dry-weight was
recorded from the seedlings receiving 100 lM H2O2, fol-
lowed by that of the control. The RWC also added support
to the above result; an increased RWC was observed in
H2O2-pretreated, salt-stressed seedlings compared to all of
the other seedlings (Fig. 2c). Acclimated-stressed plants
had their shoot, root and leaf dry-weight reduced compared
to that of the control (Fig. 2d). Salt-stressed ginseng
seedlings showed deleteriously effected growth parame-
ters. Comparing acclimated-stressed with un-acclimated-
stressed plants revealed that there were increases in growth,
pigment, and RWC in the acclimated-stressed group, which
reflects a recovery capacity against salt stress. The data
also suggest that H2O2 pre-treatment by itself does not
affect plant growth in relation to control treatment.
Effect of exogenous H2O2 pretreatment on antioxidant
activity
To examine whether exogenous H2O2 induces the antiox-
idant enzymes involved in the protection of seedlings under
salt stress, we measured the activities of the antioxidant
system including proline, CAT, APX and GPX. Free pro-
line content steadily increased in salt-stressed seedlings
compared to the control, but a significant increase in pro-
line content was detected in the H2O2-acclimated, salt-
Mol Biol Rep
123
stressed seedling compared to the un-acclimated, salt-
stressed seedlings at day 7. Only exogenous H2O2-treated
seedlings showed no difference from the control. H2O2
levels in both the acclimated-stressed and un-acclimated-
stressed seedlings were significantly increased compared to
the control and un-acclimated-stressed seedlings. During
days 1 and 3, the salt-stressed seedlings did not show any
increase in CAT activity (Fig. 3a); CAT activity was pro-
voked at day 5 and declined at day 7. The activity of CAT
was influenced by H2O2 treatment; however, in the variants
with salt application.
Both CAT and APX activities followed the same general
trend throughout the experiment for all treatments; i.e.,
they increased with time (Fig. 3b). The GPX activity
gradually increased, reaching a maximum at day 7. A more
remarkable increase was observed in the seedlings with
exogenous H2O2 combined with 150 mM NaCl (Fig. 3c).
The salt stress alone caused an increase in GPX activity
compared to the control and the H2O2-treated seedlings.
The H2O2-acclimated, salt-stressed seedlings showed more
antioxidant enzymes activities, which in turn helps the
plant tolerate salt stress. H2O2 pretreatment also increased
the oxidative stress marker proline rather than salt treated
seedlings (Fig. 3d).
MDA, superoxide and H2O2 contents
The MDA content was increased in salt-stressed seedlings
compared to both control and H2O2-acclimated seedlings
(Fig. 4a). H2O2 pretreatment did not increase the concen-
tration of MDA, thus protecting the seedlings from mem-
brane damage. Changes in the rate of superoxide
generation are shown in Fig. 4b. Compared to the control,
salt stress caused a significant increase in the concentration
Pla
nt h
eigh
t (cm
)
0
2
4
6
8
10
dry-
wei
ght (
g/pl
ant)
0.00
0.05
0.10
0.15
0.20
0.25
0.30Plant heightPlant dry-weight
H2O2 concentration (uM)
control 0.05 0.1 5 10 50 100 200 250
Chl
orop
hyll
(ug/
ml F
W)
0
2
4
6
8
10
20
30
40
Car
oten
oid
(ug/
ml F
W)
0.00
0.05
0.10
0.15
0.20
0.25
0.30ChlorophyllCarotenoid
(a)
(b)
Fig. 1 Effect of different
concentrations of H2O2 on
ginseng seedlings treated with
150 mM salt: a plant height and
dry weight and b chlorophyll
and carotenoid contents. Data
bar represents mean value with
±SD. Control represents
seedlings grown in MS media
only
Mol Biol Rep
123
of superoxide in ginseng seedlings. In contrast, H2O2 pre-
treatment in salt stressed seedlings showed a decrease in
superoxide content, demonstrating effective inhibition by
exogenous H2O2. Application of H2O2 did not markedly
influence endogenous H2O2 concentration. On the other
hand, salt treatment led to a increase in endogenous H2O2
in the seedlings (Fig. 4c). These results indicated that
exogenous H2O2 treatment protects ginseng seedlings from
damage by salt stress.
Effect of exogenous H2O2 on defense-related gene
expression
The relative mRNA concentrations of seedlings from all
treatment groups were studied after the application of salt
stress (day 1). Genes reported to be responsible for defense
mechanism in ginseng such as, PGIP, ribonuclease-2 (PR-
10), chitinase (PR-2), calmodulin, sesquiterpene synthase
and spermidine synthase showed significant up-regulation
in H2O2-acclimated salt-stressed seedlings compared to the
other treatment groups (Fig. 5). These data clearly indicate
that H2O2 is involved in signaling and the up-regulation of
defense genes, which in turn facilitate salt tolerance in
ginseng seedlings.
Discussion
In the present study, we observed that plant growth was
affected by salinity, but H2O2 pre-treatment decreased the
deleterious effects of salt stress. Significant weight gain per
day was seen in H2O2-treated seedlings (Fig. 1). After the
fifth day compared to the control, indicating that the use of
exogenous H2O2 at low concentrations increases the
physiological characteristic of ginseng seedlings by
increasing both photosynthetic pigments and growth. H2O2
enhances cell division and is involved in the differentiation
of the cell wall [42]. Similarly, growth stimulation by
exogenous H2O2 was demonstrated in barley [43], wheat
[44], pea [45], maize [21] and melon [46]. H2O2 is an
unstable molecule; when it breaks down, a single oxygen
atom and a molecule of water are released. The singlet
oxygen ion is extremely reactive and will attach itself to
either another O- atom to form a stable oxygen molecule
or may attack a nearby organic molecule. Both the stable
and O- forms increase the level of dissolved oxygen [47].
Therefore, low doses of H2O2 can increase the mass and
length of root [48]. Salinity causes significant effect on the
growth (plant height and plant dry-weight) and pigments
(chlorophyll and carotenoid) of ginseng seedlings (Fig. 2).
This reduction may be due to instability of pigment protein
complexes by ions during salt stress [49]. The decline in
photosynthesis observed in cases of salinity could be
attributed to stomata factors. During salt stress, the con-
centration of CO2 in chloroplasts decreases because of the
reduction in stomata conductance in spite of the apparent
stability of CO2 concentration in the intercellular spaces
[50]. However, it was observed that exogenous H2O2
acclimation decreased the deleterious effect of salinity on
0
5
10
15
20
25
30
35
40
MS H2O2 Salt H2O2 + Salt
1day3day5day7day
0
0.5
1
1.5
2
MS H2O2 Salt H2O2 + Salt
0
200
400
600
800
1000
1200
1400
1600
control H2O2 salt H2O2+salt
Rel
ativ
e w
ater
con
tent
(%
)
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
control H2O2 salt H2O2+salt
Leaf
Stem
Root
Chl
orop
hyll
(ug
/g F
W)
Car
oten
oid
( ug/
g F
W)
dry-
wei
ght (
g)(a)
(b)
(c)
(d)
Fig. 2 Physiological characterization of ginseng seedlings under salt
stress: a chlorophyll content b carotenoid content, c relative water
content and d dry-weight. Data represent significant value of
*p \ 0.005 and **p \ 0.01
Mol Biol Rep
123
the growth of ginseng. Therefore, both the photochemical
and biochemical aspects of photosynthesis are affected by
salinity [51].
Our results suggest that exogenously applied H2O2
increases the activities of CAT, APX and GPX, which in
turn further inactivate ROS production. The balance
between GPX and APX or CAT activities in cells is crucial
for determining the steady-state levels of superoxide radi-
cals and H2O2 [42]. This balance, together with the
sequestering of metal ions, is thought to be important for the
prevention of highly toxic hydroxyl radical formation via
the metal-dependent Haber–Weiss or Fenton reactions [21].
The different affinities of APX and CAT for H2O2 suggest
that they belong to two different classes of H2O2-scavenging
enzymes; APX might be responsible for the fine modulation
of ROS for signaling, whereas CAT might be responsible for
or the removal of excess ROIs during stress. The high
activation of antioxidant enzymes due to H2O2 has been
demonstrated in wheat to protect against drought stress [42]
and in maize to protect against salt stress [21].
Proline (Pro) accumulation is an essential indicator for
plant response to salt stress [52]. The ginseng seedlings
under salt stress showed greater increases in proline
content than the control. Therefore, salt-tolerant seedlings
accumulate more proline, which correlates with an
adaptation to salinity [53, 54]. The H2O2-pretreated, salt-
stressed ginseng seedlings showed a maximum amount
of proline than the salt-stressed ones. Higher concentra-
tions of proline are related to the osmotic potential of
the leaf and help in osmotic adjustment. In addition to
the role as an osmolyte, proline can also confer enzyme
protection and increase membrane stability under various
conditions [55]. Proline accumulation may also help in
non-enzymatic free radical detoxifications [56]. Thus, the
increase of proline may trigger tolerance to salt stress in
ginseng seedlings.
H2O2-pretreated seedlings demonstrated a significant
increase in growth under salt stress, which was concomi-
tant with the decreased production of MDA. The salt-
stressed seedlings showed an increase in MDA, resulting in
peroxidation of lipids and leading to loss of membrane
integrity [57]. As lipid peroxidation is the symptom most
ascribed to oxidative damage, it is often used as an indi-
cator of membrane damage [58, 59]. Exogenous H2O2
0
5
1
5
2
5
3
control H2O2 H2O2 +salt salt
1 day
3 day
5 day
7 day
Time interval
1 day 3 day 5 day 7 day
AP
X a
ctiv
ity (
Ug-1
prot
ein)
30
40
50
60
70
80
90
100controlH2O2H2O2+salt salt
Time interval
1 day 3 day 5 day 7 day
CA
T a
ctiv
ity (
U/g
-1pr
otei
n)
0
20
40
60
80
100
controlH2O2H2O2+saltsalt
Time interval
1 day 3 day 5 day 7 day
GP
X a
ctiv
ity (
Ug-1
pro
tein
)
0
10
20
30
40
50
60
Fre
e p
rolin
e(m
g/g
FW
)
Treatments
(a) (b)
(c)(d)
Fig. 3 Effect of H2O2 pretreatment on the activity of the antioxidant system in ginseng seedlings under salt stress. Control represents ginseng
seedlings without treatment. a CAT activity, b GPX activity, c APX activity and d proline content
Mol Biol Rep
123
treatment was able to prevent lipid peroxidation and thus
protect the cells from the damage of saline conditions.
In ginseng seedlings, increased salinity was directly
related to the level of endogenous H2O2 and superoxide
content. Excess H2O2 and superoxide lead to oxidative
stress and are capable of injuring cells. Exogenous H2O2
decreased the concentration of both endogenous H2O2 and
superoxide, which represents less damage to the cells and
may occur due to increases in H2O2 scavengers such as
CAT and APX. Our results suggest that the increase in
antioxidant enzyme activity plays a central protective role
in superoxide and H2O2 scavenging processes [41]. Wang
et al. [60] also suggested that exogenous H2O2 pretreat-
ment notably decreases the concentration of endogenous
H2O2 concentration in plants.
It is known that H2O2 participates in the physiological
metabolism of plants and activates defense responses to
various stresses [61]. In ginseng seedlings, pretreatment
with exogenous H2O2 led to elevated expressions of
defense-related genes. Genes such as spermidine synthase
[41], calmodulin [39], sesquiterpene [40] and PR genes
like PR-10 [37] and chitinase [62] are reported to be
expressed under salt stress in ginseng. Thus H2O2 acti-
vates both antioxidant and defensive genes in plants.
During abiotic stress, the ROS such as superoxide and
H2O2 are initially produced to activitate ROS scavengers
such as CAT and APX. At the same time H2O2 can
diffuse into cells and activates many of the gene
expression of plant defense proteins by triggering tran-
scription factors (TFs) [13]. Recently, it was suggested
that H2O2 is not only a defensive signaling molecule, but
that it also functions as a signaling molecule during
normal growth and development [19]. H2O2 can modu-
late the activities of many components in signaling, such
as protein phosphatases, protein kinases and TFs [63]. It
will be interesting to study H2O2 as a signaling regulator
of defense genes in plants, and more research is neces-
sary to understand its mechanism. Taken together, our
data support the results seen in wheat [60] and barley
[21] indicating that pretreatment with low concentrations
of H2O2 has a stimulative effect on the acclimation
process.
In conclusion, under salt stress, all major metabolic
processes including photosynthesis, protein synthesis, and
energy and lipid metabolism are affected. Ginseng
seedlings pretreated with low concentrations of H2O2
demonstrated increased growth rates and pigment con-
tents under salt stress. H2O2 acclimation under normal
growth conditions enhanced salt tolerance in ginseng
by enhancing antioxidant enzyme activities, thereby
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
control H2O2 H2O2 + salt salt
1 day
3 day
5 day
7 day
0
2
4
6
8
10
12
14
16
control H2O2 H2O2 + salt salt0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
control H2O2 H2O2 + salt saltH
2O2
(uM
/g F
W)
MD
A (
nmol
/g F
W)
Sup
erox
ide
( ug/
g F
W)
Treatments Treatments
Treatments
(a) (b)
(c)
Fig. 4 Effect of H2O2 pretreatment on a lipid peroxidation (MDA), b superoxide and c endogenous H2O2 level under salt stress. Data represent
significant value of *p \ 0.001 and **p \ 0.05
Mol Biol Rep
123
decreasing ROS production and lipid peroxidation.
Additional data provided here to suggest that H2O2
metabolism is involved in signaling processes for gin-
seng salt tolerance by elevating defense related genes.
This study gave preliminary idea of H2O2 acclimation
and their regulation on antioxidant; defense response
genes. So, the further study is to over express the
defense marker genes in Arabidopsis and to study their
mechanism against environmental stress in future.
Acknowledgments This research was supported by iPET (112142-
05-1-CG000), Korea Institute of Planning and Evaluation for
Technology in Food, Agriculture, Forestry and Fisheries, Republic of
Korea.
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