J Agro Crop Sci (2014) ISSN 0931-2250

SALINITY STRESS

Overexpression of Dehydroascorbate Reductase Confers

Enhanced Tolerance to Salt Stress in Rice Plants (Oryza sativa
L. japonica)
Y. S. Kim1, I. S. Kim1, S. Y. Shin1, T. H. Park2, H. M. Park3, Y. H. Kim3, G. S. Lee3, H. G. Kang4,
S. H. Lee5 & H. S. Yoon1
1 Department of Biology, Kyungpook National University, Daegu, South Korea
2 Department of Horticulture, Daegu University, Gyeongsan, South Korea
3 Genomics Division, National Academy of Agricultural Science, RDA, Suwon, South Korea
4 Subtropical Horticulture Research Institute, Jeju National University, Jeju, South Korea
5 Department of Biology, Gyeongsang National University, Jinju, South Korea

Keywords Abstract
ascorbate recycling; dehydroascorbate
reductase; ion leakage; redox state; salinity; Dehydroascorbate reductase (DHAR, EC 1.8.5.1) helps to maintain redox pools
transgenic rice of ascorbate (AsA) by recycling dehydroascorbate (DHA) to AsA. To investigate
whether DHAR influences the acquired tolerance of rice plants to abiotic stresses,
Correspondence cDNA encoding DHAR (OsDHAR1) was isolated from rice and used to develop
I. S. Kim and H. S. Yoon
OsDHAR1-overexpressing transgenic rice plants regulated by a maize ubiquitin
Department of Biology
promoter. The incorporation and expression of the transgene was confirmed by
College of Natural Sciences
Kyungpook National University polymerase chain reaction (PCR) and semi-quantitative reverse transcription
Daegu 702-701 PCR, real-time PCR, Western blot and enzyme activity. The overexpression of
South Korea OsDHAR1 greatly increased the DHAR activity and the AsA/DHA ratio, follow-
Tel.: +82 53 950 5348 ing increase in AsA content and decrease in DHA content. In addition, the
Fax: +82 53 952 5348 enzyme activity of monodehydroascorbate reductase, glutathione reductase and
Email: hsy@knu.ac.kr; 92kis@hanmail.net
ascorbate peroxidase, which are related to the ascorbate–glutathione systems, was
enhanced in the presence and the absence of salt stress in homozygous transgenic
Accepted April 22, 2014
rice plants (OsDHAR1-OX1, -OX2) harbouring Ubi::OsDHAR1. In addition,
doi:10.1111/jac.12078 OsDHAR1-expressing transgenic rice plants enhanced the redox state by reducing
both hydroperoxide and malondialdehyde levels under salt and methyl viologen
(MV) stress conditions, which led to better plant growth, ion leakage and quan-
tum yield (Fv/Fm). Therefore, our results show that the overexpression of
OsDHAR1 increases the adaptation of rice plants to salt stress, by maintaining the
AsA pool, ion homoeostasis and redox homoeostasis. Finally, the findings of
this study indicate that OsDHAR1 plays an important role in attenuating the
deleterious effects of various abiotic stresses.

factors and a complex phenotypic and physiological trait in

Introduction
In agricultural systems, crop plants are frequently exposed
to a variety of conditions (high salinity, nutrient excess or
depletion, desiccation, flooding, extreme temperature,
ozone, herbicides and photochemical cycling) that affect
their growth, development and productivity (Dhaliwal
et al. 1999, Bajguz and Hayat 2009, Wang et al. 2010,
Burkart et al. 2013, Ionov et al. 2013). Among these stres-
sors, high salinity is one of the most brutal environmental
plants. It can often lead to ion disequilibrium following
Na+ and Cl
accumulation as well as hyperionic and hyper-
osmotic stress, disrupting overall metabolic activities such
as the reduction in leaf expansion, stomata closure and
reduced photosynthesis, thus limiting the biomass, produc-
tivity and geographical distribution of crop plants (Zhang
et al. 2012, Kumar et al. 2013, Zhang and Shi 2013). In
particular, elevated Na+ concentrations can induce defi-
ciency of the essential element, potassium, impose toxic

444 © 2014 Blackwell Verlag GmbH, 200 (2014) 444–456


effects by perturbing K+-dependent processes and induce
deleterious changes in protein conformation (Zhang et al.
2012, Zhang and Shi 2013). To overcome the unfavourable
saline environment, plants maintain homoeostasis by oper-
ating a rapid osmotic and ionic signalling programme. For
example, diverse proteins involved in carriers and pumps
such as Na+/H+ antiporters and Na+/K+ symporters are
working towards ion homoeostasis in the cell by control-
ling Na+ uptake, Na+ exclusion from xylem, Na+ efflux
from roots, Na+ compartmentation into vacuoles and con-
trol of apoplastic Na+ transport through bypass flow under
salt stress (Zhang et al. 2012, Kumar et al. 2013). Some
crops are likely to use more than one mechanism to cope
with saline environments (Zhang et al. 2012).
On the other hand, an unfortunate consequence of
high soil salinity in crop plants is the excessive generation
of reactive oxygen species (ROS) intermediates, such as
superoxide radicals (O
2 ), hydrogen peroxide (H2O2) and
hydroxyl radicals (OH
) (Mittler 2002, Sairam et al. 2005).
ROS production affects membrane potential, along with
other essential macromolecules, resulting in the inhibition
of photosynthetic capacity, photosynthetic pigments, pro-
teins, DNA and lipids (Demiral and Turkan 2005). To pro-
tect themselves from ROS-induced oxidative damage, crop
plants have evolved a wide range of defence systems that
could be enhanced to resist unfavourable abiotic stress
conditions, including antioxidant molecules and antioxida-
tive enzymes (Tari et al. 2013). Among these defence sys-
tems, ascorbate (AsA) is a major antioxidant molecule that
is primarily localised in known sources of ROS generation,
including chloroplasts, cytosol and mitochondria (Potters
et al. 2002, Frei et al. 2012), where it contributes to various
aspects of their life cycles (Gallie 2013). Moreover, AsA
plays a role in protection from photochemical cycling, is
involved in stress resistance, regulates several aspects of
plant development and serves as a signal molecule (Kerk
and Feldman 1995, Noctor et al. 2000, Garcia et al. 2009).
AsA also acts as a major antioxidant molecule in stress
resistance (Pignocchi and Foyer 2003, Eltayeb et al. 2006),
as well as a cofactor of several antioxidant enzymes
involved in the AsA recycling pathway (Pignocchi et al.
2003). The AsA pool in plants is maintained by the major
AsA biosynthesis pathway, which includes GDP-mannose
30 ,50 -epimerase (GME) and L-galactono-1,4-lactone dehy-
drogenase (GalDH). This pool is also maintained by AsA
recycling enzymes, such as monodehydroascorbate reduc-
tase (MDHAR) and dehydroascorbate reductase (DHAR)
within the cell or between organs (Garcia et al. 2009,
Haroldsen et al. 2011, Qin et al. 2011). The AsA recycling
pathway plays a particularly important role in the response
and adaptation to stress in plants. During cellular pro-
cesses, AsA is oxidised into a short-lived monodehydro-
ascorbate (MDHA) radical in response to excess ROS
© 2014 Blackwell Verlag GmbH, 200 (2014) 444–456
OsDHAR1-Mediated Stress Response to Salt
during stress and senescence (Yin et al. 2010, Gallie 2013),
after which it is reduced back to AsA by MDHAR, while
the remainder of the antioxidant molecule degrades to AsA
and dehydroascorbate (DHA) (Noctor and Foyer 1998, Le
Martret et al. 2011). DHA then undergoes irreversible
spontaneous hydrolysis to 2,3-diketogulonic acid or is reor-
ganised to AsA by DHAR via glutathione (GSH) (Deutsch
2000). DHAR enables crop plants to convert DHA and
hence recover AsA before it is lost. Therefore, DHAR repre-
sents a key factor involved in the maintenance of the
reduced pool of AsA and is of great importance to abiotic
stress resistance in plants (Eltayeb et al. 2007, Gallie 2013).
To date, many DHAR cDNAs have been obtained from
wheat (Chen et al. 2003b), tomatoes (Zou et al. 2006),
spinach (Shimaoka et al. 2000), rice (Urano et al. 2000)
and Arabidopsis (Yoshida et al. 2006). In addition, the
proteins of many DHAR cDNAs have been purified and
characterised from spinach leaves (Hossain and Asada
1984), rice (Kato et al. 1997) and potato tubers (Dipierro
and Borraccino 1991). Transgenic Arabidopsis thaliana
(Ushimaru et al. 2006) and Escherichia coli (Amako et al.
2006, Shin et al. 2008) that overexpress rice DHAR have
been shown to enhance the ability of plants to adapt to salt
and oxidative stress, respectively. However, the stress
response of transgenic rice expressing OsDHAR1 under
high salinity has not been previously reported. These physi-
ological investigations contribute towards improving our
understanding about the effects of abiotic stress on plants.
In addition, genes with functional regeneration, such as
DHAR, have been the focus of much interest by crop plant
physiologists and agronomists.
Rice (Oryza sativa) is a staple food of Koreans and has
extremely high economic value in Korea. The recent
increase in obesity prevalence and other diseases has raised
interest in rice-based diets and has prompted breeding
programmes for stress-resistant rice and research studies
on enhancing rice productivity. Studies utilising stress-
resistant gene complexes in rice are envisioned to circum-
vent problems such as a decline in productivity, limited
acreage and extended cultivation periods (Kumar et al.
2013). These studies are also geared towards the reunifica-
tion with and future distribution of foods to North Korea.
Rice has been studied as an important monocot model
system for molecular and genetic studies in plant science
research because rice has the smallest genome among crop
plants and shares substantial co-linearity with membranes
of the grass family, including all the important cereals
(Xing and Zhang 2010). Based on the above results, we
cloned OsDHAR1 cDNA from the leaf tissue of rice,
designed a gene construct to be controlled by the maize
ubiquitin promoter (Ubi-p) as a constitutive promoter and
transformed the gene construct into rice plants. We then
investigated whether the overexpression of OsDHAR1 led
445
Kim et al.

(a) (b)

(c) (d)

Fig. 4 Analysis of cellular damage in salt and MV-treated leaf discs of transgenic and control WT rice plants. (a) Relative ion leakage was measured
from the leaf discs of OsDHAR1-OX1, OsDHAR1-OX2 and WT rice plants floating in 10 lM MV solution. Leaf discs were treated with 10 lM MV at
25 °C and at a light intensity of 150 lmol photons m
2 s
1. The percentages of ion leakage were calculated based on 100 % of the values obtained
after autoclaving. Ion leakage photograph from the leaf discs of transgenic and WT rice plants floated on 10 lM MV solution was determined based
on the measurement of electrolyte leakage over time post-treatment (0 and 72 h) (boxed panel). (b) Changes in Fv/Fm in transgenic and WT rice
plants under salt stress. During exposure to salt stress, the leaf discs were pre-incubated in darkness overnight before Fv/Fm was measured under
500 mM NaCl conditions at 25 °C and at a light intensity of 150 lmol photons m
2 s
1. Square, WT plants; circle, OsDHAR-OX1 plants; triangle,
OsDHAR-OX1 plants. Effect of salt on the H2O2 (c) and MDA (d) content in the leaves of the OsDHAR1-OX1, OsDHAR1-OX2 and WT rice plants after
exposure to 100 mM NaCl for 12 days. White bars, salt-free conditions; grey bars, salt-treated conditions. Data are the means  S.D. of 3 replicates,
with each replicate containing 10 leaf discs. Bars labelled with asterisks show significant differences between transgenic and WT rice plants, in which
P < 0.05 as determined by Origin.

H2O2 production under salt stress conditions (Fig. 4c),
whereas H2O2 levels decreased by about 1.6-fold in trans-
genic rice plants under salt stress compared with control
WT rice plants. The increase in MDA content was more
pronounced in control WT rice plants compared with
that in OsDHAR1-OX1 and OsDHAR1-OX2 rice plants
(Fig. 4d). Our results indicate that the overexpression of
OsDHAR1 in transgenic rice plants protects plant cells from
salt-induced oxidative damage by improving redox homo-
eostasis, ion homoeostasis and photosynthetic capacity
compared with that in WT rice plants.
Discussion
Plants cells use their available machinery to combat various
abiotic stresses such as oxidative stress by scavenging excess
ROS, with several previous studies clearly demonstrating
that enhanced DHAR gene expression and AsA content are
enhanced in the presence of high salinity, low temperature,
H2O2 and drought stress (Urano et al. 2000, Jiang and
Zhang 2002, Kwon et al. 2003, Eltayeb et al. 2007, Gill and
Tuteja 2010). ROS generated as a result of stress may be
scavenged by non-enzymatic antioxidants, including AsA
and GSH, or by enzymatic activity, such as DHAR (Noctor
and Foyer 1998). To increase AsA content through regener-
ation from DHAR, we developed transgenic rice plants
overexpressing the OsDHAR1 gene. The OsDHAR1 gene
was found to be significantly overexpressed in OsDHAR1-
OX1 and OsDHAR1-OX2 rice plants in the presence and
absence of high salinity when compared to WT rice plants;
However, the OsDHAR1 gene was also slightly upregulated
in non-transgenic WT rice plants under salt stress. AsA
redox status (AsA/DHA ratio) increased to a similar extent
in OsDHAR1-OX1 and OsDHAR1-OX2 rice plants under
the same stress conditions when compared to WT rice
plants. Therefore, the higher expression of the OsDHAR1
gene improved the acquired tolerance of transgenic rice
plants to salt stress, as well as ROS-induced oxidative stress
caused by H2O2, cold and drought polyethylene glycol
(PEG). Similar resistance in response to DHAR expression

452 © 2014 Blackwell Verlag GmbH, 200 (2014) 444–456

Kim et al.
Development Administration and funded by the Priority
Research Centers Program (2011-0030748) through the
National Research Foundation (NRF) of the Ministry of
Education, Science and Technology, Korea.
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Kim et al.
Protein extraction, sodium dodecyl sulphate polyacrylamide
gel electrophoresis and Western blot analysis
Rice leaf tissue (0.2 g) was ground in liquid nitrogen and
homogenised for 20 min at 4 °C in extraction buffer con-
taining 50 mM Tris-HCl (pH 7.5), 10 mM EDTA, 0.7 M
sucrose, 0.1 M KCl, 2 mM phenylmethylsulfonyl fluoride
(PMSF) and protease inhibitor cocktails. The crude protein
was then identified by centrifugation at 18 000 g for
20 min at 4 °C, after which the crude protein concentra-
tion was determined using a protein assay reagent (Bio-
Rad, Hercules, CA, USA), with bovine serum albumin
being used as a standard. For Western blot analysis, protein
(20 lg) was separated by 12 % sodium dodecyl sulphate
polyacrylamide gel electrotransfer (SDS-PAGE) and trans-
ferred by electrotransfer to a polyvinylidene difluoride
membrane (Millipore, Billerica, MA, USA). The mem-
branes were then blocked for 1.5 h at room temperature
with blocking solution (5 % non-fat skim milk in Tris-
HCl-buffered saline containing 0.05 % Tween 20 (TBST)
plus 0.02 % sodium azide) and incubated overnight at
4 °C with each primary antibody that was diluted properly
in blocking solution. Polyclonal antisera against OsDHAR1
were raised in rabbits after DHAR protein purification in
E. coli BL21. Each blot was washed four times for 40 min
(10 min per wash) with TBST and then incubated for 1.5 h
at room temperature with a secondary antibody. After fur-
ther four washes (10 min each) with TBST, the immunore-
active proteins were visualised using ECL Western blotting
detection reagent (GE Healthcare, Uppsala, Sweden).
Biochemical analysis
Crude protein extract from rice leaves was used for ascorbate
peroxidase (APX), MDHAR, DAHR and glutathione reduc-
tase (GR) activity assays. The extraction buffers were as fol-
lows: 50 mM potassium phosphate (pH 7.5), 3 mM MgCl2,
1 mM EDTA, 1 mM PMSF and protease inhibitor cocktails
in MDHAR and DHAR, and 1 ml extraction buffer includ-
ing 50 mM potassium phosphate buffer (pH 7.0), 1 % Tri-
ton X-100 and 7 mM 2-mercaptoethanol and protease
inhibitor cocktails in GR and APX. The assay for MDHAR
activity was performed at room temperature in a 1 ml reac-
tion mixture containing 50 mM potassium phosphate (pH
7.2), 0.2 mM NADH, 2 mM AsA, one unit of ascorbate oxi-
dase and crude extract. The absorbance was measured at
340 nm, and the activity was calculated using an absorbance
coefficient of 6.2 mM
1 cm
1. One unit of MDHAR activity
was defined as the amount of enzyme that oxidises 1 nmol
of NADH per min at 25 °C (Drew et al. 2007, Eltayeb et al.
2007). The assay for DHAR activity was performed at room
temperature in a 1 ml reaction mixture containing 50 mM
potassium phosphate (pH 7.0), 0.1 mM EDTA, 0.5 mM
448
DHA, 2.5 mM GSH and crude extract. The absorbance was
measured at 265 nm, and the activity was calculated using
an absorbance coefficient of 14.6 mM
1 cm
1 (Nakano and
Asada 1981). One unit of DHAR activity was defined as the
amount of enzyme that produces 1 nmol of AsA per min at
25 °C. The assay for GR activity was performed at room
temperature in a 1 ml reaction mixture containing 50 mM
phosphate buffer (pH 7.6), 1.7 mM EDTA, 1.0 mM GSSG,
0.1 mM NADH and crude extract, with the absorbance being
measured at 340 nm (Foyer and Halliwell 1976). The assay
for APX activity was performed according to Nakano and
Asada (1981), by measuring the decrease in absorbance at
290 nm as AsA was oxidised. The specific enzyme activity
was expressed as units per milligram of protein. The reaction
mixtures with the homogenate were extracted, and the AsA
was analysed according to the method developed by Ahn
et al. (1999), with minor modifications. The AsA content
was determined by spectrophotometric analysis, as described
by Gillespie and Emerson (2007).
Measurement of malondialdehyde and H2O2 levels
The level of lipid peroxidation was determined by measuring
malondialdehyde (MDA) through a thiobarbituric acid assay
(Heath and Packer 1968). In brief, 1.0 ml rice sample
(0.1 mg of crude protein) (Chen and Gallie 2006) was
combined with 2.0 ml TCA-TBA-HCl reagent (15 % w/v
trichloroacetic acid, 0.375 % w/v thiobarbituric acid and
0.25 N hydrochloric acid) and mixed thoroughly. The solu-
tion was then heated in a boiling water bath for 15 min.
After cooling, the flocculent precipitate was removed by cen-
trifugation at 13 500 g for 10 min, and the absorbance of
the sample was determined at 535 nm against a blank that
contained all of the reagents minus the lipid. The MDA
concentration of the sample was calculated using an extinc-
tion coefficient of 1.56 9 105 M
1 cm
1 (Hodges et al.
1999). The H2O2 content was measured using the method
described by Ma et al. (2012), with minor modifications.
MV treatment and ion leakage analysis
Methyl viologen damage was measured using rice leaf discs,
according to the method described by Lee et al. (2007),
with minor modifications. In brief, 10 leaf discs (1 cm in
diameter) from the fully expanded leaves of three different
rice plants grown in a greenhouse for 8 weeks were floated
on a solution containing 0.4 % (w/v) sorbitol and MV at
concentrations ranging from 0 to 10 lM. The leaf discs
were then placed in separate petri dishes and incubated
for 12 h at 25 °C in the dark to allow the MV to diffuse
into the leaves. After pre-incubation, the leaf discs were illu-
minated (150 lM m
2 s
1) until use. The conductivity of
the decanted MV solution was determined using an ion
© 2014 Blackwell Verlag GmbH, 200 (2014) 444–456

conductivity metre (Istek, Seoul, Korea). The concentrated
solution was then recovered from the conductivity metre cell
and autoclaved with the damaged leaf discs for 15 min at
121 °C, to release all of the solutes, after which the conduc-
tivity of the solution was measured again. The per cent elec-
trolyte leakage attributable to MV stress was then determined
by dividing the conductivity value of the sample before auto-
claving by that after autoclaving (100 % electrolyte leakage).
The tests measuring the visible damage caused by MV appli-
cation were repeated in triplicate. The percentage of leaf
damage that appeared on the leaves after MV treatment was
evaluated 6 days after treatment (0 % indicated no damage
to the leaves; 100 % indicated fully damaged leaves).
Chlorophyll fluorescence
To infer the functional damage to photosynthesis in the
plant leaves, chlorophyll fluorescence determination was
estimated based on the photochemical yield (Fv/Fm),
which represents the maximum quantum yield of photo-
system II, using a chlorophyll fluorescence metre (Handy
PEA; Hansatech, Kings Lynn, UK) after dark adaptation
overnight. Net photosynthesis was measured by O2 evolu-
tion from leaf discs. Salt-induced damage was analysed
using leaf discs, as described by Oh et al. (2005), with
minor modifications. Leaf discs (diameter, 1 cm) from the
three leaves of two different plants grown in a glasshouse
for 8 weeks were transferred to 5.0-cm petri dishes, each
containing 5 ml NaCl solution. Ten leaf discs were placed
in each petri dish and then incubated for 20 h at 25 °C in
darkness to allow for salt to diffuse into the leaves, after
which the Fv/Fm value was measured at six time intervals
(at 0, 4, 8, 12, 16 and 20 h) at 25 °C under continuous light
intensity photons (150 lmol m
2 s
1).
Statistical analysis
Significant differences in the measured parameters between
transgenic and WT rice plants were identified by ORIGIN
PRO 8.0. Means were considered to be significantly different
at P < 0.05. All experiments were carried out at least in
triplicate, and the results are expressed as the mean  the
standard deviation (S.D.).
Results
Development of transgenic rice plants
To assess the stress tolerance of rice plants overexpressing
OsDHAR1 encoding DHAR (EC 1.8.5.1), we produced 19
independent event lines (T0 generation) of transgenic rice
plants that carried an OsDHAR1 gene construct regulated by
the maize ubiquitin promoter and the nos terminator
© 2014 Blackwell Verlag GmbH, 200 (2014) 444–456
OsDHAR1-Mediated Stress Response to Salt
(Fig. 1a, upper panel). The primary (T0) transgenic rice
plants were screened in the presence of DL-phosphinothricin
encoded by the bar gene. Seeds of the next generation (T1)
were harvested separately from each independent line, after
which 20 plants per line were grown in a paddy field during
the farming season, and their progeny (T2 seeds) were har-
vested. Because the overexpression of OsDHAR1 in trans-
genic A. thaliana has been reported to increase plant
tolerance to salt stress (Ushimaru et al. 2006), we first evalu-
ated the salt tolerance of transgenic rice plants. To screen for
transgenic lines that were tolerant to salt stress, a total of
1900 (T2) rice plants (19 independent rice lines 9 100 per
seed subline) and 100 non-transgenic WT rice plants were
grown on separate lines in 20 small pots containing a
100 mM NaCl solution under greenhouse conditions at 28–
32 °C. Seven transgenic lines showed significantly increased
tolerance to salt stress compared with WT rice plants (Figure
S1). Finally, (T3) subline plants that originated from the two
independent lines, OsDHAR1-OX1 and OsDHAR1-OX2,
were selected for subsequent physiological and biochemical
experiments. These plants were also identified as putative
homozygous transgenic lines through PCR, using Ubi-F and
OsDHAR-R2 with the Ubi promoter (Fig. 1a, lower panel).
All of the WT, OsDHAR1-OX1 and OsDHAR1-OX2 rice
plants were healthy when grown under normal conditions
(Fig. 1b, left panel; Figure S2a), but displayed clear visual
differences when grown under high salt conditions (Fig. 1b,
right panel). Specifically, almost all of the control WT rice
plants withered at 25 days after salt treatment, whereas the
OsDHAR1-OX1 and OsDHAR1-OX2 rice plants appeared to
endure the treatment well (Fig. 1b). To evaluate the capacity
of the plants to recover from salt damage, OsDHAR1-OX1,
OsDHAR1-OX2 and control WT rice plants were grown
under stress conditions at 100 mM NaCl for 30 days and
then irrigated for a further 14 days with non-saline water.
OsDHAR1-OX1 and OsDHAR1-OX2 rice plants rapidly
resumed vegetative growth, whereas non-transgenic WT rice
plants did not (Fig. 1c). The fresh weight of the recovered
transgenic rice plants was also threefold higher compared
with that of the control WT rice plants (Fig. 1d). In addi-
tion, OsDHAR1 overexpression in transgenic rice plants
showed increased adaptation to other abiotic stresses,
including low temperature, drought and hydrogen peroxide
(Figure S2b). Thus, our results show that the OsDHAR1
transgene is integrated into the rice genome and that the
epistatic overexpression of the OsDHAR1 gene enhances the
tolerance of transgenic rice plants to salt stress.
Expression and structure of the OsDHAR1 transgene in
transgenic rice plants
To examine the constitutive expression of the OsDHAR1
gene under the regulation of the ubiquitin promoter in
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salt-tolerant transgenic rice lines exposed to salt, rice plants
grown for about 4 weeks after germination were exposed
to 100 mM NaCl and sampled at that time point. Overex-
pression of the OsDHAR1 gene was established by detecting
the levels of mRNA, protein and enzyme activity. In control
WT rice plants, OsDHAR1 mRNA was slightly increased by
salt stress compared with the steady state. However, the
OsDHAR1-OX1 and OsDHAR1-OX2 rice plants accumu-
lated a greater amount of OsDHAR1 mRNA under salt stress
compared with control WT rice plants (Fig. 2a, upper
panel). Real-time PCR was performed to confirm OsDHAR1
expression and produced similar results to semi-quantitative
RT-PCR (Fig. 2a, lower panel). Western blot analysis
showed that the transgenic rice plants produced high levels
of OsDHAR1 protein in a pattern similar to that of mRNA
under normal and salt stress conditions (Fig. 2b). The com-
plete nucleotide and amino acid (A.A.) sequence of the
OsDHAR1 gene were composed of 642 bp and 214 A.A,
respectively. The predicted molecular weight was approxi-
mately 23.5 kDa. The AsA/DHA ratio increased from a ratio
of 1.6 in the control WT rice plants to more than 3.2 in the
transgenic rice plants in the presence of salt when compared
to normal conditions (Fig. 2c). An increase in the AsA/DHA
ratio of OsDHAR1-expressing transgenic rice plants resulted
from an increase in AsA content and a reduction in DHA
content in the absence and presence of salinity, as compared
to WT rice plants. In particular, the ratio increase was more
pronounced under stress conditions compared with normal

(a)
(c)

(b)

Fig. 2 Analysis of OsDHAR1 expression and ascorbate (AsA) redox status in transgenic and WT rice plants after salt treatment. (a) Semi-quantitative
reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR analyses of OsDHAR1 gene expression of OsDHAR1-OX1, OsDHAR1-OX2
and WT rice plants subjected to 100 mM NaCl treatment for 12 days. The gene for tubulin was utilised as an internal control. White bars, salt-free
conditions; black bars, salt-treated conditions. (b) Western blot analysis of transgenic rice plants with enhanced tolerance to salt stress stably express-
ing the OsDHAR1 transgene, and post-transcriptional processes in OsDHAR1-OX1 and OsDHAR1-OX2 rice plants following treatment with 100 mM
NaCl for 12 days. Tubulin protein was utilised as a protein loading control. OsDHAR1-OX1 and OsDHAR1-OX2, independent transgenic rice plants;
WT, wild-type rice plants. (c) Measurements of total AsA, AsA, dehydroascorbate and AsA/DHA ratio in leaves of OsDHAR1-OX1, OsDHAR1-OX2 and
WT rice plants after exposure to 100 mM NaCl for 12 days. White bars, normal conditions; grey bars, salt-treated conditions. Data are expressed as
the means  S.D. of at least three replications from three independent experiments. Bars labelled with asterisks show significant differences between
transgenic and WT rice plants, in which P < 0.05 as determined by Origin.

450 © 2014 Blackwell Verlag GmbH, 200 (2014) 444–456


conditions. Therefore, these results show that OsDHAR1
overexpression in transgenic plants improves AsA redox
pool by reducing DHA in the presence of high salinity.
Enzyme activity of DHAR, MDHAR, APX and GR in
transgenic rice plants
To elucidate the mechanism by which OsDHAR1-OX1 and
OsDHAR1-OX2 rice plants tolerate salt stress, antioxidant
enzyme activity related to the AsA-GSH system was investi-
gated in the presence of salt stress. DHAR enzyme activity
was also measured in rice plants that were of the same age
as those evaluated in the semi-quantitative RT-PCR and
Western blot analyses using leaves from the control WT,
OsDHAR1-OX1 and OsDHAR1-OX2 rice plants. The
results indicated that the DHAR activity of OsDHAR1-OX1
and OsDHAR1-OX2 rice plants increased approximately
2.0-fold under normal and salt stress conditions (Fig. 3a).
Moreover, the antioxidant activity of MDHAR, APX and
GR enzymes increased approximately 1.5-fold or more in
the OsDHAR1-OX1 and OsDHAR1-OX2 rice plants com-
pared with the control WT rice plants under normal and
salt stress conditions, respectively (Fig. 3b–d). These results
indicate that OsDHAR1 expression co-activates cell rescue
systems to generate high salt tolerance.
Enhanced tolerance of transgenic rice plants to abiotic
stress
We further evaluated whether rice plants overexpressing
OsDHAR1 exhibited enhanced tolerance to other abiotic
(a)
Fig. 3 Assay of enzyme activity in transgenic
and WT rice plants. The activity of DHAR (a),
MDHAR (b), APX (c) and GR (d) isolated from
the leaves of OsDHAR1-OX1, OsDHAR1-OX2 (c)
and WT rice plants was measured after treat-
ment with water containing 100 mM NaCl for
12 days. White bars, normal conditions; grey
bars, salt-treated conditions. Data are
expressed as the means  S.D. of at least
three replications from three independent
experiments. Bars labelled with asterisks show
significant differences between transgenic and
WT rice plants, in which P < 0.05 as
determined by Origin.
© 2014 Blackwell Verlag GmbH, 200 (2014) 444–456
OsDHAR1-Mediated Stress Response to Salt
stresses such as MV, which cause oxidative damage to
plants. Specifically, we examined ion leakage, because the
disruption of the membrane by ROS-induced stress leads
to the release of cytoplasmic solutes from leaf discs. There
was no difference in electrolyte leakage between transgenic
and WT rice plants under normal conditions, whereas Os-
DHAR1-OX1 and OsDHAR1-OX2 rice plants incurred sig-
nificantly less damage compared with control WT rice
plants at each time interval after MV treatment (Fig. 4a).
Chlorophyll content did not change under normal condi-
tions. OsDHAR1-OX1 and OsDHAR1-OX2 rice plants
exhibited noticeably higher chlorophyll content in the pres-
ence of MV as compared to WT rice plants, even though
chlorophyll content noticeably decreased in both transgenic
and WT rice plants under the same conditions (Fig. 4a,
boxed panel). We also examined chlorophyll fluorescence,
because the effects of high salinity exposure to leaf discs
have been used to investigate salt stress tolerance. Photo-
chemical yield (Fv/Fm) is the most common chlorophyll
fluorescence test used to determine the effects of stress on
photosystem II. To measure the contribution of OsDHAR1
overexpression to chlorophyll fluorescence, transgenic and
control WT rice plants were subjected to 500 mM NaCl and
evaluated at various time points. As shown in Fig. 4b, all of
the rice plants showed an initial Fv/Fm value that was very
close to 0.8, which is the approximate optimal value for
many plant species. However, the Fv/Fm value decreased
with time after NaCl treatment, occurring at a slower rate
in OsDHAR1-OX1 and OsDHAR1-OX2 rice plants com-
pared with control WT rice plants. Moreover, transgenic
and control WT rice plants exhibited significantly higher
(b)
(d)
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(a) (b)

(c) (d)

Fig. 4 Analysis of cellular damage in salt and MV-treated leaf discs of transgenic and control WT rice plants. (a) Relative ion leakage was measured
from the leaf discs of OsDHAR1-OX1, OsDHAR1-OX2 and WT rice plants floating in 10 lM MV solution. Leaf discs were treated with 10 lM MV at
25 °C and at a light intensity of 150 lmol photons m
2 s
1. The percentages of ion leakage were calculated based on 100 % of the values obtained
after autoclaving. Ion leakage photograph from the leaf discs of transgenic and WT rice plants floated on 10 lM MV solution was determined based
on the measurement of electrolyte leakage over time post-treatment (0 and 72 h) (boxed panel). (b) Changes in Fv/Fm in transgenic and WT rice
plants under salt stress. During exposure to salt stress, the leaf discs were pre-incubated in darkness overnight before Fv/Fm was measured under
500 mM NaCl conditions at 25 °C and at a light intensity of 150 lmol photons m
2 s
1. Square, WT plants; circle, OsDHAR-OX1 plants; triangle,
OsDHAR-OX1 plants. Effect of salt on the H2O2 (c) and MDA (d) content in the leaves of the OsDHAR1-OX1, OsDHAR1-OX2 and WT rice plants after
exposure to 100 mM NaCl for 12 days. White bars, salt-free conditions; grey bars, salt-treated conditions. Data are the means  S.D. of 3 replicates,
with each replicate containing 10 leaf discs. Bars labelled with asterisks show significant differences between transgenic and WT rice plants, in which
P < 0.05 as determined by Origin.

H2O2 production under salt stress conditions (Fig. 4c),
whereas H2O2 levels decreased by about 1.6-fold in trans-
genic rice plants under salt stress compared with control
WT rice plants. The increase in MDA content was more
pronounced in control WT rice plants compared with
that in OsDHAR1-OX1 and OsDHAR1-OX2 rice plants
(Fig. 4d). Our results indicate that the overexpression of
OsDHAR1 in transgenic rice plants protects plant cells from
salt-induced oxidative damage by improving redox homo-
eostasis, ion homoeostasis and photosynthetic capacity
compared with that in WT rice plants.
Discussion
Plants cells use their available machinery to combat various
abiotic stresses such as oxidative stress by scavenging excess
ROS, with several previous studies clearly demonstrating
that enhanced DHAR gene expression and AsA content are
enhanced in the presence of high salinity, low temperature,
H2O2 and drought stress (Urano et al. 2000, Jiang and
Zhang 2002, Kwon et al. 2003, Eltayeb et al. 2007, Gill and
Tuteja 2010). ROS generated as a result of stress may be
scavenged by non-enzymatic antioxidants, including AsA
and GSH, or by enzymatic activity, such as DHAR (Noctor
and Foyer 1998). To increase AsA content through regener-
ation from DHAR, we developed transgenic rice plants
overexpressing the OsDHAR1 gene. The OsDHAR1 gene
was found to be significantly overexpressed in OsDHAR1-
OX1 and OsDHAR1-OX2 rice plants in the presence and
absence of high salinity when compared to WT rice plants;
However, the OsDHAR1 gene was also slightly upregulated
in non-transgenic WT rice plants under salt stress. AsA
redox status (AsA/DHA ratio) increased to a similar extent
in OsDHAR1-OX1 and OsDHAR1-OX2 rice plants under
the same stress conditions when compared to WT rice
plants. Therefore, the higher expression of the OsDHAR1
gene improved the acquired tolerance of transgenic rice
plants to salt stress, as well as ROS-induced oxidative stress
caused by H2O2, cold and drought polyethylene glycol
(PEG). Similar resistance in response to DHAR expression

452 © 2014 Blackwell Verlag GmbH, 200 (2014) 444–456


has been observed in Arabidopsis DHAR-overexpressing
transgenic tobacco plants (Eltayeb et al. 2006). Our results
show that a constitutive maize ubiquitin promoter effec-
tively regulates the transcriptional and translational levels
of transgenic rice plants and confers increased tolerance to
salt stress. As shown in transgenic rice plants, this ubiquitin
promoter is generally available and active in all or most cell
types of monocotyledonous plants and could prove useful
for a variety of applications in gene transfer studies of plant
groups (McElroy et al. 1990, McElroy and Brettell 1994).
In a previous study, various abiotic stresses were found
to be correlated with AsA levels in plants (Conklin and
Barth 2004), which are regulated by higher DHAR activity
(Chen and Gallie 2005). The tolerance effect of DHAR
overexpression has also been reported for some transgenic
plants, such as tobacco and potato (Kwon et al. 2001, Chen
and Gallie 2005, 2006, Qin et al. 2011), as well as for E. coli
(Shin et al. 2008). Increased AsA content and AsA redox
state (AsA/DHA ratio) confer salt tolerance (Ushimaru
et al. 2006) to transgenic tobacco plants overexpressing the
human DHAR gene (Kwon et al. 2003). On the exposure
to salt stress, plant cells operate a range of defence systems,
including the AsA-GSH cycle (Ahmad et al. 2010).
OsDHAR1-OX1 and OsDHAR1-OX2 rice plants showed
markedly increased antioxidant enzyme activity, including
DHAR, MDHAR, GR and APX activity, under salt stress
when compared to WT rice plants. In antioxidant systems,
DHAR, MDHAR, GR and APX are well known to function
in the AsA-GSH cycle of plants. MDHAR contributes to
the continuous regeneration of AsA to scavenge toxic H2O2
via the AsA-GSH cycle and is composed of AsA and GSH
cofactors in transgenic rice plants. AsA is oxidised into an
unstable short-lived monodehydroascorbate (MDHA) rad-
ical in response to the production of excess ROS under a
range of stress conditions. Moreover, because DHA under-
goes irreversible spontaneous hydrolysis to 2,3-diketogu-
lonic acid, it must be converted to AsA by DHAR in the
presence of GSH as a reducing agent (Noctor and Foyer
1998, Deutsch 2000). Therefore, DHAR is the most impor-
tant factor involved in maintaining a reduced pool of AsA
and is a key factor in the adaptation of plants to various
abiotic stress conditions (Eltayeb et al. 2007). Higher AsA
level contributes to the maintenance of higher APX activity,
because AsA is used as an electron donor by APX to cata-
lyse the reduction of H2O2. A significant difference in GR
activity was observed between transgenic and WT rice
plants following NaCl challenge. Specifically, enzyme activ-
ity was approximately 1.5-fold higher in OsDHAR1-OX1
and OsDHAR1-OX2 rice plants compared with in WT rice
plants in the presence of NaCl. This finding indicates that
the activation of MDHAR and GR by AsA and GSH recy-
cling through the AsA-GSH cycle was more efficient at con-
verting oxidised AsA and GSH to reduced AsA and GSH.
© 2014 Blackwell Verlag GmbH, 200 (2014) 444–456
OsDHAR1-Mediated Stress Response to Salt
To date, many studies have shown that the overexpression
of antioxidant genes (such as SOD, CAT, APX, DHAR,
guaiacol peroxidase (GPX) and GR) enhances salt stress
tolerance in transgenic wheat, maize, potato, cotton,
Brassica spp., canola, tomato, strawberry and rice (Washko
et al. 1992, Asada 1997, Pasqualini et al. 2001, Chen et al.
2003b, Chen and Gallie 2005, Eltayeb et al. 2007, Ahmad
et al. 2010, Qin et al. 2011). The high AsA/DHA ratio in
OsDHAR1-OX1 and OsDHAR1-OX2 rice plants might
provide enough substrate to operate an AsA-GSH cycle
containing DHAR, MDHAR, APX and GR at levels suffi-
cient to detoxify ROS and enhance redox homoeostasis,
ion homoeostasis and photochemical yield (Fv/Fm), while
decreasing H2O2 content and lipid peroxidation products
by minimising oxidative damage through the buffering of
ROS caused by abiotic stresses. Overall, these results indi-
cate that the enhanced levels of antioxidant enzymes
involved in the AsA-GSH cycle were caused by the intro-
duction of the OsDHAR1 transgene to transgenic rice
plants, which improved the redox state reduction in ROS
accumulation by exogenous stimuli. However, the precise
co-regulation defence mechanism employed by the
OsDHAR1 and AsA-GSH systems has yet to be elucidated.
Thus, we demonstrated that OsDHAR1 overexpression is
critical for transgenic rice plants to tolerate to tolerance
ROS-induced abiotic stresses.
In conclusion, in this study, we present the further analy-
sis of the physiological role of DHAR in the abiotic stress
adaptation of crop plants. Our results indicate that the ele-
vation of antioxidants and antioxidant enzymes through
the overexpression of the DHAR gene might significantly
contribute towards enhancing the enhanced stress tolerance
of plants subjected to salt stress. Specifically, we demon-
strated that the function of the OsDHAR1 gene in trans-
genic rice plants conferred a significantly high AsA/DHA
ratio of stress tolerance. Therefore, we assume that
OsDHAR1-OX1 and OsDHAR1-OX2 rice plants might
significantly increase stress resistance by maintaining the
AsA redox state and redox homoeostasis through high AsA
redox accumulation in response to salt stress conditions.
Additional studies are required to establish rational pro-
duction of a stress tolerance mechanism that is applicable
to redox homoeostasis, ion homoeostasis and photochemi-
cal yield of all crops with enhanced tolerance to various
abiotic stresses. Furthermore, the effects of the overexpres-
sion of the OsDHAR1 gene on the redox state of GSH and
the expression of multiple antioxidant enzymes require
investigation.
Acknowledgements
This work was supported by a grant from the Next-
Generation BioGreen 21 Program (No. PJ008115), Rural
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Kim et al.
Development Administration and funded by the Priority
Research Centers Program (2011-0030748) through the
National Research Foundation (NRF) of the Ministry of
Education, Science and Technology, Korea.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Figure S1 Selection of salt-tolerant homozygous trans-
genic rice plants.
Figure S2 Effects of abiotic stresses on OsDHAR1-OX1,
-OX2, and WT rice plants.
© 2014 Blackwell Verlag GmbH, 200 (2014) 444–456