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Advances in Environmental Biology, 10(10) October 2016, Pages: 148-154

AENSI Journals

Advances in Environmental Biology

ISSN-1995-0756 EISSN-1998-1066
Journal home page: http://www.aensiweb.com/AEB/

Exogenous ascorbic acid and hydrogen peroxide

alleviates salt-induced oxidative stress in rice (Oryza
sativa L.) by enhancing antioxidant enzyme activities
and proline content

Rani Roy, 1M. Tahjib-Ul-Arif M, 1Tahmina Akter, 1Shuma Rani Ray, 2,3M. Abu Sayed

Department of Biochemistry and Molecular Biology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.
United Graduate School of Agricultural Science, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan.
Department of Biochemistry and Molecular Biology, Hajee Mohammad Danesh Science and Technology University, Dinajpur-5200,

Address For Correspondence:

M. Tahjib-Ul-Arif, Bangladesh Agricultural University, Department of Biochemistry and Molecular Biology, Faculty of Agriculture,
Mymensingh-2202, Bangladesh.
E-mail: arif1002215@gmail.com
This work is licensed under the Creative Commons Attribution International License (CC BY).

Received 28 August 2016; Accepted 18 October 2016; Available online 22 October 2016

Exogenous protectants such as ascorbic acid (AsA) and signaling molecules such as hydrogen peroxide (H 2O2) could be a promising
approach to mitigate salt-induced oxidative stress in rice. The present study was carried out to examine the effects of exogenous application
of hydrogen peroxide (H2O2) and ascorbic acid (AsA) on the growth, content of photosynthetic pigments, proline, ascorbic acid and
antioxidant enzymes of rice (Oryza sativa L.) cv. BRRI dhan56 seedlings grown in greenhouse hydroponically under 125mM NaCl. The
plants exposed to the NaCl stress exhibited a significant reduction in growth, leaf photosynthetic pigments, ascorbate peroxidase activity
(APX) as well as increased in proline, ascorbic acid content and catalase (CAT) activity. The AsA and H2O2 application not only mitigated
the inhibitory effects of salt stress but also induced a stimulatory effect on all the studied growth parameters. The activities of antioxidant
enzymes (CAT and APX) as well as proline contents of rice plants were significantly increased after AsA and H2O 2 application. The present
study; therefore, suggests that exogenous AsA and H2O2 confer tolerance to salt stress in rice by enhancing proline accumulation and
antioxidant defense systems, in addition exogenous H2O2 maintain higher endogenous ascorbate content.

KEYWORDS: Catalase, ascorbate peroxidase, proline, rice, salinity stress.

Rice (Oryza sativa L.) is one of the main staple food in Bangladesh. The total production of rice has
become static over the years with the gradual decrease of crop-growing areas. The static production of rice is
still attributable to the lack of suitable improved cultivars for different environmental conditions. Adverse
climatic changes pose a great threat for the agriculture and the future food security. Among the various abiotic
stresses salinity is one of the most threatening factors in coastal area in Bangladesh. Coastal areas constitute
about 2.5 million ha which amount to about 11% of the total cropland.
Recent report showed that salinity is the second most widespread soil problem next to drought in rice
growing countries and is considered as a serious constraint to increased rice production worldwide [1]. The
induction of salt tolerance in plants is crucial to maintain their economic yield. This can be achieved either
through genetic modifications or chemical treatments [2]. The strategies of plant breeding and genetic

Copyright 2016 by authors and Copyright, American-Eurasian Network for Scientific Information (AENSI Publication).


Popy Rani Roy et al, 2016

Advances in Environmental Biology, 10(10) October 2016, Pages: 148-154

engineering are long-term and complex endeavours to develop salt tolerance that have had limited success [3].
Alternatively, the exogenous application of plant growth regulating compounds is an efficient and technically
simpler approach to cope with the deleterious effects of salinity on plants [4, 5]. Exogenous application of
protectants, plant growth regulators, fertilizers, and non-enzymatic antioxidants has been successfully used to
minimize the adverse effects of salinity on plant growth and yield [6, 7].
Ascorbic acid is a naturally occurring compound with antioxidant activity that plays a pivotal role in plant
cell adaptation to salinity stress.[8]. It has been used to counteract the adverse effects of salt stress in many crop
plants [9, 10, 11] as well as functions in whole plant metabolism [12]. Furthermore, experimental studies on
different plants have shown that exogenous application of AsA may reduce salt-induced adverse effects and
results in a significant increment of growth and yield [10, 11, 13]. AsA is absorbed readily after exogenous
application [14, 15] and moves within the plant [16]. Therefore, foliar application of AsA improves salt
tolerance of crop plants in a number of ways [17, 18, 19].
In plants, hydrogen peroxide is one of the major and the most stable ROS and regulates basic processes,
such as acclimation, defense and development [20]. The generation of H2O2 is increased due to a wide variety of
stresses, and some authors have suggested that H2O2 is a key factor mediating the phenomena of acclimation
and cross-tolerance [21]. Recent investigations have revealed that H2O2 is a central component of the signal
transduction cascade involved in plant adaptation to a changing environment [21]. H2O2 plays a dual role in
plants: at low concentrations, it acts as an acclamatory signal, triggering tolerance to various stresses [22, 23],
and at high concentrations, it organizes programmed cell death [24]. Exogenous application of H2O2 at low
concentrations signals the induction of defense responses in plants against oxidative stresses and assists in
triggering stress resistance mechanism [25]. In maize, exogenously applied H2O2 increases salt tolerance by
increasing the activities of antioxidants [26].
The present study was carried out to evaluate the potential of exogenously applied AsA and H2O2 in
alleviation of salt stress in rice seedlings and study proline accumulation, antioxidant enzyme activities,
endogenous ascorbate content and growth under NaCl stress in the presence of AsA and H 2O2 treatments.
Experimental conditions:
The experiment was conducted at the laboratory of the Department of Biochemistry and Molecular Biology,
Bangladesh Agricultural University, Mymensingh, during January to March 2016. Plants were grown in
hydroponic solution using Peter 20:20:20 and FeSO4.5H2O solution for 21days.
Plant materials and Treatments:
BRRI dhan56 (High yielding rice variety) was used in this study. Six treatment combinations viz.
T0=control (no NaCl or no AsA or no H2O2), T1=125mM NaCl, T2= 150 mg/L AsA, T3= 125 mM NaCl+150
mg/L AsA, T4=1mM H2O2, T5=125 mM NaCl+1mM H2O2 were used. NaCl was used to develop salinity. The
experiment was laid out in a completely randomised block design with three replications. Initially, plants of T 2
and T3 treatment groups treated by exogenous 150 mg/L ASA and T4 and T5 treatment group were treated by
1mM H2O2 for 7 days with nutrient solution. After 7days, salinity was imposed in T 1, T3 and T5 treatment
Growth parameter:
Shoot length and root length were recorded after 21 days of treatments. Ten randomly selected plants of
each treatment were used for data recording. Root length was measured from the base to the top of root whereas
shoot length was measured from the base to the tip of the longest leaf of the shoot.
Biochemical and Enzymatic determination:
After 21 days, leaf sample was collected and further biochemical analyses were performed. Chlorophyll and
proline content were measured as previously described [27, 28]. Enzymes assays for catalase (EC: and
ascorbate peroxidase (EC were determined by the method as described previously [29]. AsA content
was determined by the titrimetric method using dye () solution.
Statistical analysis:
Data for all parameters were statistically analyzed using the MSTAT-C statistical package. Statistically
different groups were compared using an LSD test (P<0.01).
Protective effect of ascorbic acid and H2O2 on growth of rice against salt stress:


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Salt stress caused a significant decrease in growth (root length and shoot length) of rice when were exposed
to 125mM NaCl (Table 1). Exogenous application of AsA and H2O2 significantly increased root and shoot
growth while seedlings were under salinity stress.
Effect of exogenous AsA and H2O2 on chlorophyll, proline and AsA contents:
Our present data shows that a significant (P 0.01) reduction in chlorophyll-a, chlorophyll-b as well as
proline contents under salt stress. Intriguingly, there was no change in case of AsA contents. Interestingly,
exogenous application of AsA and H2O2significantly (P0.01) increased of Chlorophyll-a, chlorophyll-b,
proline and AsA contents (Table 1). But these exogenous protectants could not increase chlorophyll contents up
to the control both in non-saline and saline condition. On the other hand exogenous H2O2 highly increased
proline content and significantly increase AsA content compared to saline and non-saline condition.
Activity of antioxidant enzymes induced by exogenous AsA and H2O2 under salt stress:
To investigate whether AsA and H2O2 enhance antioxidant defense system of rice under salt stress,
activities of major ROS- scavenging antioxidants enzyme viz. CAT and APX were measured. Salt stress caused
significant increase in CAT and decrease in APX activities in rice. Exogenous application of AsA and H2O2
significantly increase CAT and APX activities compared to salt stress condition and non-stress condition (Table
Salt stress caused a remarkable reduction in all growth parameters measured in rice plants exposed to 125
mM saline stress. Exogenous application of AsA reduced the adverse effect of salt stress on different growth
parameters of rice seedlings (Table 1). Excess salt induced ionic and osmotic stresses that disturbed metabolism
and led to reduction of plant development [30]. AsA induced increase in growth of rice plants under saline
conditions might be the role of AsA in increasing cell division and/or cell enlargement [17]. Previous studied
revealed that AsA plays a multiple roles in plant growth, functioning in cell division, cell wall expansion, and
other development processes [31]. It has also been observed that exogenous application of AsA can also
counteract salt-induced growth inhibition in plants, e.g., brassica [11], durum wheat [32], barley [33], and
sunflower [34]. [17] observed that AsA applied at 100 mg/ L improved growth of a salt tolerant cultivar of
wheat under saline conditions and this AsA-induced enhancement of growth was found to be associated with
enhanced photosynthetic efficiency. Similar growth promoting effect of AsA was also observed in chickpea [9].
In our study, H2O2 application significantly increased root length under salinity stress. Similar result was found
by [35] that H2O2 significantly improved the growth of salt stressed plants.
Chlorophyll is one of the most important pigment components, providing photosynthetic ability of a plant.
Chlorophyll-a and chlorophyll-b contents decreased in rice cultivars due to salt stress (Table 1). In our study,
exogenous AsA and H2O2 alleviated the negative effect of salt stress and improved chlorophyll content (Table
1). These results are totally different to those of [36] in which a significant decrease in chlorophyll content has
been observed in salt stressed wheat plants due to exogenously applied ascorbic acid. However, in contrast, in
many reports an improvement in photosynthetic pigments has been reported due to exogenous application of
ascorbic acid, e.g., in Brassica campestris [11] and Chickpea [9]. On the other hand, exogenous H2O2
application could be associated with the H2O2-mediated increase in AsA and glutathione (GSH) concentrations,
which act as an antioxidants and photosynthetic machinery protector from salt-induced ROS. It has been shown
that pre-treatment of seeds with H2O2 increase the net photosynthetic rate in wheat seedlings [37, 38].
The activities of antioxidant enzymes (CAT and APX) in the rice plants were increased significantly in
exogenously applied AsA condition under the NaCl stress as well as only after the AsA application (Table 1).
These higher CAT and APX activities help plants in defense to possible oxidative damage. [39] suggested that
improved antioxidative defense system due to foliar application of ascorbic acid alleviates the detrimental
effects of salinity and increased resistance to salinity in the maize plants. Similarly to our results, [17, 13]
reported an increase in antioxidant enzyme activities in wheat plants after an AsA application.
As well as, salt stress increased CAT activities but decreased APX activity in plants and in exogenously
H2O2 treated plants under salt stress produced significantly increased CAT and APX antioxidant enzymes
(Table 1). [40] found that exogenous H2O2 is able to moderate activities of antioxidant enzymes reducing stress
intensity at low light. Recently, [41] found that Panax ginseng seedlings treated with 100 M H2O2 for 2 days
showed enhanced salinity tolerance and increased activities of CAT and APX.
Proline accumulation is often used as stress indicator in plants [42, 43]. Its content significantly increased in
the rice seedlings exposed to the salt stress (Table 1) and might serve as a compatible solute [44]. It has been
found that a salt stress up-regulated the enzymes involved in proline biosynthesis [45]. The proline content of
the rice leaves also shows an increasing trend after the AsA application under the NaCl stress (Table 1). Similar
results have been reported in maize [46], barley [33], where applications of different levels of AsA increase the
proline content of plants.


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Also, proline content increased with the H2O2 application under non saline and salt stress compared to
control. Maximum increase of about 3 times in proline content was noted with the 1mM H 2O2 under salt
stressed plants compared to control. Proline can protect plants from stress through different mechanisms,
including osmotic adjustment, detoxification of ROS, protection of membrane integrity, and stabilization of
proteins/enzymes [47]. Exogenous H2O2 may increase the proline content in Nitraria tangutorum, which could
be alleviated by H2O2 scavengers [48]. In the present study, accumulation of proline content under salt stress
was increased by the application of H2O2 and reversed the deleterious effects of salt stress. The reversal of
adverse effects of salt stress on photosynthesis by H2O2 may be attributed to the accumulation of proline which
maintained the cellular osmotic adjustments by increasing osmotic potential and water potential and protected
photosynthetic machinery from salt stress by acting as an oxygen radical scavenger. [49] suggested that H2O2
might be involved in signal transduction events, leading to proline accumulation in maize seedlings, and that the
H2O2-induced proline accumulation.
AA is a small soluble antioxidant molecule fulfils essential metabolic functions in plant cells [50].
According to [51], the ascorbic acid could react directly with hydroxyl radicals, superoxide, and singlet oxygen
and thus provides protection by scavenging free radicals. Although salinity had increased the leaf AsA contents,
it did not promote any significant alteration. Exogenous AsA and H2O2 increased endogenous AsA content.
Similar result was shown by [17] where exogenous application of AsA increased endogenous level of AsA of
wheat. Also, [35] have been reported that, H2O2 pre-treatment in maize plants increased AsA content.
Overall mechanism of AsA and H2O2 to bring salt tolerance in rice seedlings is shown in Figure.1.
Table 1: Effect of exogenous AsA and H2O2 on different morphological and biochemical parameters. Values represent the
replications. Same letter in a column represents insignificant difference at p 0.01.
Morphological parameters
Biochemical parameters
Treatments Shoot length Root length
(mM min-1 (M min-1
(mg g-1 F.
g F.W)
g F.W)
1.3660.03f 1.0960.04d 1.30.07e
1.3960.02e 0.760.01e
2.7250.04c 95.035.07b
53.110.78ab 15.820.72ab 1.4540.02d 1.3130.02c 1.5470.03d 100.661.72b 0.2720.01b
53.840.85ab 14.980.98bc 1.4970.01b 1.0590.09d 2.9170.03b 105.364.43b 0.2710.01b
T4 (1mM
1.4680.01c 1.9000.04a 4.2030.04a 152.467.0a
13.490.61bc 1.6130.02a 1.5890.05b 4.3120.03a 162.017.78a 0.2640.0c
CV (%)
Level of

Fig. 1: Overall mechanism of AsA and H2O2 to bring salt tolerance in rice seedlings.

mean of three

(mg g-1 F.






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The present study and the available literature reviewed in the discussion suggest that exogenous AsA and
H2O2 are effective in improving salt stress tolerance in rice seedlings. Enhancement of enzymatic antioxidants
and proline might be responsible for regulation of ROS during NaCl stress. The elevated activity of antioxidant
system, at least in part, protected the photosynthetic machinery and increased the tolerance of the rice plants to
the NaCl stress. However, still very little is known about the role of AsA and H 2O2 in plants. Although some
researchers have successfully used AsA and H2O2 in different plants for mitigating adverse effect of different
abiotic stresses; its roles in tolerances to diversified of stresses in relation to plant architecture, growth and
metabolism still demand huge researches.
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