Biochemical and Biophysical Research Communications 463 (2015) 1225e1229

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

Overexpression of Arabidopsis NADPH-dependent thioredoxin

reductase C (AtNTRC) confers freezing and cold shock tolerance to
plants
Jeong Chan Moon a, 1, Sangmin Lee b, 1, Su Young Shin a, 1, Ho Byoung Chae c,
Young Jun Jung c, Hyun Suk Jung b, Kyun Oh Lee c, Jung Ro Lee a, d, *, Sang Yeol Lee c, **
a

National Institute of Ecology, 1210 Geumgang-ro, Maseo-myeon, Seocheon-gun 325-813, Republic of Korea
Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon, Republic of Korea
Division of Applied Life Science (BK21 program), PMBBRC, Gyeongsang National University, Jinju, Republic of Korea
d
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, United States
b
c

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 10 June 2015
Accepted 12 June 2015
Available online 15 June 2015

Overexpression of AtNTRC (AtNTRCOE) in Arabidopsis thaliana led to a freezing and cold stress tolerance,
whereas a knockout mutant (atntrc) showed a stress-sensitive phenotype. Biochemical analyses showed
that the recombinant AtNTRC proteins exhibited a cryoprotective activity for malate dehydrogenase and
lactic dehydrogenase. Furthermore, conclusive evidence of its interaction with nucleic acids in vitro is
provided here on the basis of gel shift and electron microscopy analysis. Recombinant AtNTRC efciently
protected RNA and DNA from RNase A and metal catalyzed oxidation damage, respectively. The C-terminal thioredoxin domain is required for the nucleic acideprotein complex formation. From these results, it can be hypothesized that AtNTRC, which is known to be an electron donor of peroxiredoxin,
contributes the stability of macromolecules under cold stress.
2015 Elsevier Inc. All rights reserved.

Keywords:
NADPH-dependent thioredoxin reductase
DNA binding
Thioredoxin
Peroxiredoxin
Cryoprotective activity

1. Introduction
Because of their sessile nature, the challenge of reactive oxygen
species (ROS) in plants is even greater under various stress conditions [1]. ROS are toxic molecules capable of causing oxidative
damage to proteins, DNA, and lipids [2]. ROS generated by temperature stress such as heat, cold or freezing have been shown to
injure cell membranes and proteins [3e5]. However, the most
stable form of ROS, hydrogen peroxide (H2O2), plays a crucial role as
a signaling molecule in various physiological processes [6]. Nevertheless, to minimize oxidative stress, plant cells are also equipped
with a number of antioxidant molecules and proteins in various
subcelluar organelles. Among them, peroxiredoxin (Prx) is a new
family of peroxidase using thiol (-SH) as an electron donor. Based
on the amino acid sequence similarity, plant Prxs can be divided

* Corresponding author. National Institute of Ecology, 1210 Geumgang-ro, Maseomyeon, Seocheon-gun 325-813, Republic of Korea.
** Corresponding author.
E-mail addresses: leejr73@nie.re.kr (J.R. Lee), sylee@gnu.ac.kr (S.Y. Lee).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bbrc.2015.06.089
0006-291X/ 2015 Elsevier Inc. All rights reserved.

into three classes: typical 2-Cys Prx, atypical 2-Cys Prx, and 1-Cys
Prx [7]. The typical 2-Cys Prx is found in plastids, activated by
chloroplast thioredoxins, and involved in the protection of the
photosynthetic apparatus against oxidative damage.
Recently, a chloroplastic hybrid protein with NADPHthioredoxin reductase (NTR) and thioredoxin domain was identied in Arabidopsis, rice and cyanobacteria and designated NTRC
[8,9]. NTRC is enzymatically active as if the separate components
NTR and thioredoxin are used together. An Arabidopsis knockout
mutant for NTRC showed growth retardation with pale-green
leaves and was sensitive to abiotic stresses such as methyl viologen, drought, and high salinity. Since NTRC efciently transferred
electrons to 2-Cys Prx, it was proposed to be involved in plant
protection against oxidative stress [9e11]. Recently, we showed
that AtNTRC acts as a disulde reductase, a foldase chaperone, and a
holdase chaperone as well [12]. The multiple functions of AtNTRC
depend on their oligomeric status. Higher molecular weight
(HMW) complexes of AtNTRC showed stronger activity as a holdase
chaperone, while low molecular weight (LMW) complexes
exhibited weaker holdase chaperone activity but stronger disulde
reductase and foldase chaperone activities. In addition, the

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AtNTRCOE in Arabidopsis conferred an enhanced thermotolerance

to plants [12]. Chlorella vulgaris NTRC (CvNTRC) showed lowtemperature inducible expression [13]. Furthermore, mRNA level
of AtNTRC was also induced in response to cold stress [12]. The
precise roles of NTRC under cold stress have not been elucidated
yet. The aim of this work was therefore to study the role of AtNTRC
under cold stress.
2. Materials and Methods
2.1. Plant materials
Arabidopsis thaliana wild-type (ecotype Columbia), AtNTRCOE,
and the T-DNA insertion mutants (SALK_012208) seeds were sterilized and sown on solid medium (1  Murashge and Skoog containing 0.25% phytagel and 3% Sucrose) in a petri dish (90  15 mm)
or on soil and incubated for 3 d at 4  C for synchronized germination. The seeds were grown under the conditions of
100e120 mmol m2s1 photosynthetic ux at 22  C, 70% humidity
and 16 h light/8 h dark cycles.
2.2. Cloning and expression of the AtNTRC protein in Escherichia
coli
The full-length and deletion mutant of AtNTRC gene were
cloned as previously described [10]. Expressions of all proteins
were performed according to Fu [14] in E. coli BL21(DE3) using the
pGEX2T and pET28a expression vector in MTB medium. Puried
protein was dialyzed into 20 mM TriseHCl (pH 8.0) containing
150 mM NaCl and then used for biochemical analysis.

pryuvate is oxidized to NAD. The oxidation of NADH results in a

decrease in absorbance to 340 nm. The freeze-labile enzyme LDH
(25 mg/ml) was dialyzed in 10 mM KPO4, pH 7.5 at 4  C for at least
3 h before use. Fifty microliters of this solution was mixed with an
equal volume of 2  cryoprotectant solution. Before freezethawing, 10 ml of LDH solution was used to measure the enzyme
activity before freezing. The remaining 90 ml of LDH solution was
frozen by immersing the closed Eppendorf microcentrifuge tubes in
liquid nitrogen for 5 min. The samples were thawed slowly in the
air at room temperature and assayed for residual enzyme activity
using a spectrophotometer (Beckman).
2.5. Nucleic acid binding analysis
Gel retardation assay with ds/ssDNA substrates was performed
as previously described [17], with minor modications. Singlestranded (M13mp18) or double-stranded (M13mp18 RF I) DNA
(New England Biolabs) was incubated with AtNTRC or GST in 100 ml
of binding buffer (10 mM TriseHCl, pH 7.5) and was maintained on
ice for 20 min. The samples were then separated on a 0.8% agarose
gel and stained with ethidium bromide for visualization of gel
shifts. The gel shift with mRNA substrates was performed as previously described [18]. Luciferase mRNA was in vitro transcribed
with the RiboMAX kit (Promega). For the RNA protection assay,
luciferase mRNA was incubated with 15 mg of AtNTRC or GST in the
binding buffer for 20 min on ice. RNase A was added to the RNAprotein mixtures, and the mixture was kept on ice for 15 min.
The mixture was then separated on 0.8% agarose gel and stained
with ethidium bromide for visualization.
2.6. DNA protection assay

2.3. Chilling and freezing-stress tolerance assays

After 3-d at 4  C, transgenic and wild-type seeds were germinated at 22  C on MS nutrient medium with 3% sucrose and 1.2%
agar. Chilling stress was imposed by incubating the seedlings at
12  C with 30 mmol m2s1 photosynthetic ux. Root length was
measured using pictures of seedlings and the software ImageJ
(National Institutes of Health). Freezing tolerance assays were
carried out in a temperature-programmable freezer. Nonacclimated or cold-acclimated (2 d, 4  C) plants were exposed
to 5  C for 5 h in darkness. After thawing at 4  C for 4 h in the dark,
plants were returned to their original growth conditions. Tolerance
to freezing was determined as the capacity of plants to resume
growth after 7 d of recovery under control conditions. Ion leakage
test after freezing was carried out essentially as described by Ishitani et al. [15]. Excised leaf was placed in a test tube containing
200 ml of deionized H2O, and the tube was placed in a temperatureprogrammable freezer. After incubation for indicated times, tubes
were removed and placed immediately on ice to allow gradual
thawing. The leaets then were transferred carefully to another
tube containing 25 ml of deionized water and shaken overnight,
followed by measurement of conductivity. The tubes with the
leaves were then autoclaved. After cooling down to room temperature, conductivities of the solutions were measured again. The
percentage of electrolyte leakage was calculated as the percentage
of the conductivity before autoclaving over that after autoclaving.
The ion leakage experiment was repeated twice with three replicates in each experiment.

DNA protection from oxidative damage was assessed in vitro

using pGEM-T plasmid DNA (Promega). Different amounts of
AtNTRC and plasmid DNA (Promega) were incubated in 50 ml of
binding buffer (10 mM TriseHCl, pH 7.5) for 20 min at ice. Then
100 mM FeSO4 and 10 mM H2O2 were sequentially added at 5 min
interval. After 5 min incubation, the reaction was terminated by the
addition of 8 ml of 6X stop buffer (20 mM TriseHCl pH 7.5, 10 mM
EDTA, and 1 mM thiolurea). The samples were resolved on a 0.8%
agarose gel and visualized with ethidium bromide.
2.7. Electron microscopy
The mixture of DNA and puried AtNTRC from the section
Nucleic acid binding analysis was diluted 50-fold with a solution
containing 10 mM TriseHCl, pH 7.5. Treated mixture samples (5 ml)
were immediately applied to carbon-coated grids that had been
glow-discharged (Harrick Plasma, Ithaca, NY) for 3 min in air, and
then grids were negatively stained using 1% uranyl acetate. These
grids were examined in a Technai G2 Spirit Twin transmission
electron microscope tted with anti-contaminator (FEI, U.S) operated at 120 kV (used instrumentation at Korea Basic Science Institute). Images were recorded on a 1 K  1 K CCD, multiscan camera
model 794 (Gatan, U.S) at a magnication of 45,000 (0.37 nm/
pixel).
3. Results and discussion

2.4. Cryoprotection assay

3.1. Overexpression of AtNTRC confers increased cold stress

tolerance to Arabidopsis

The cryoprotective activity of AtNTRC was assayed as described

previously [16]. The assay is based on the conversion of pyruvate to
lactate. During the reaction, an equimolar amount of NADH and

In a previous report, we showed that AtNTRCOE gave an

enchanced thermotolerance to Arabidopsis by its redox-dependent
holdase chaperone function [12]. A cytosolic small heat shock

J.C. Moon et al. / Biochemical and Biophysical Research Communications 463 (2015) 1225e1229

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Fig. 1. AtNTRC overexpressing plant had increased freezing and cold shock tolerance. (A) 3-d-old seedlings were directly frozen at 5  C for indicated time, and photographs are of
plants 7 d after freezing treatment. (B) Germinated plants were placed vertically at 12  C or 22  C for indicated time. (C) Electrolyte leakage from nonacclimated or acclimated wild
type and transgenic plants. The average of three independent experiments SD is shown.

protein (sHsp) up-regulated by low and high temperatures showed

cryoprotection activity for protein [19], and overexpression of a
yeast heat-shock protein gene, Hsp26, enhanced freezing tolerance
in Arabidopsis [20]. Cold induced expression of AtNTRC and implication of the sHsps in freezing tolerance urged us to examine
whether AtNTRCOE exhibits a freezing tolerance and/or atntrc
shows a freezing sensitivity phenotype. To examine the basal
freezing tolerance of AtNTRCOE and atntrc, 3-d-old Arabidopsis
seedlings grown on soil were incubated at 5  C for 5 h and
recovered for 7 d at 22  C. While AtNTRCOE plants exhibited a
relatively higher freezing tolerance, atntrc plants exhibited a
freezing sensitive phenotype with a decreased survival rate
compared to WT,(Fig. 1A). Cold stress tolerance of the plants was
also compared by measuring root lengths of the plants. When the

Fig. 2. Cold shock-induced protein aggregation was protected by AtNTRC. (A) Aggregation of MDH on repeated freezing in liquid N2 and thawing at ambient temperature
was tested with or without AtNTRC. After 10 freeze-thaw cycles, samples were separated into soluble (S) and pellet (P) fractions, and equal volumes of the fractions were
analyzed by SDS-PAGE. GST was used as negative control. (B) An LDH solution was
frozen with varying concentrations of AtNTRC, BSA or sucrose in liquid nitrogen for
5 min. The samples were thawed at room temperature and the LDH activity was
measured as described in Materials and Methods. The relative activity represents the
amount of LDH activity remaining after a freeze-thaw treatment as a percentage of the
pretreatment enzyme activity.

plants were grown at 22  C for 20 d, the root lengths of AtNTRCOE

and atntrc plants were similar with those of WT plants. When the
plants were grown at 14  C for 45d, however, the root lengths of
AtNTRCOE plants was slightly increased but that of atntrc plants was
signicantly decreased compared with those of WT plants (Fig. 1B).
30-d-old plants were subjected to basal and acquired freezing
shock and their ion leakages were analyzed. Consistent with the
freezing tolerance results, ion leakage in atntrc plants was higher,
whereas the AtNTRCOE was slightly lower than those of WT
(Fig. 1C). These results indicate that AtNTRC not only plays an

Fig. 3. Nucleic acid binding activity of AtNTRC. (A) AtNTRC proteins were incubated
with either ssDNA (upper panel) or dsDNA (lower panel) to analyze DNA binding activity. A range of AtNTRC proteins from 0 to 15 mg was used for analyses, and GST was
used as a negative control. (B) For analysis of RNA-binding activity of AtNTRC, AtNTRC
proteins were incubated with in vitro transcribed luciferase mRNA. (C) The complex of
DNA and AtNTRC were monitored by using negative staining electron microscopy.
Arrows (left panel, eld of DNA fragments) and arrow heads (right panel, eld of DNA
fragments associated with AtNTRC proteins) indicate the appearance of each molecule,
respectively. 100 nm scale bar applies to elds.

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J.C. Moon et al. / Biochemical and Biophysical Research Communications 463 (2015) 1225e1229

Fig. 4. Trx domain of AtNTRC is important for nucleic acid binding and protects nucleic acids from RNase A and ROS. TR- (A), and Trx-domain (B) of AtNTRC was subjected to RNA
binding assay. Various concentrations of AtNTRC proteins were incubated with luciferase mRNA, and GST was used as a negative control. RNA (C) and DNA (D) protection assays
were carried out as described under Materials and Methods. Arrow shows non-degraded super-shift RNA band caused by the nucleic acideprotein complex.

important role in thermotolerance in Arabidopsis but also exhibits

a function in cold stress tolerance.
3.2. AtNTRC inhibits protein aggregation under freezing stress
in vitro
Because AtNTRC functions as molecular chaperone, we tested
whether AtNTRC can protect protein aggregation under freezing
stress in vitro. First, aggregation of MDH on repeated freezing in
liquid N2 and thawing at ambient temperature was tested with or
without AtNTRC. MDH aggregation was efciently protected by
AtNTRC (Fig. 2A). Consistent with these results, AtNTRC also
showed a concentration-dependent cryoprotection activity for
lactate dehydrogenase (LDH) after the repeated freeze-thawing
cycles in liquid N2 (Fig. 2B). These results suggest AtNTRC can
efciently protect protein aggregation from freezing stress.
3.3. AtNTRC binds and protects nucleic acids in vitro
Nucleic acid binding proteins such as cold shock domain proteins (CSDPs) and glycine-rich RNA-binding proteins (GRPs) are
implicated in cold adaptation processes in plants [21e23]. They are
thought to facilitate protein translation at low temperature by
unwinding RNA secondary structures through RNA chaperone activity. Recently, some antioxidant proteins, which can bind to
nucleic acids and function as RNA chaperones, were reported
[24e26]. So, we examined whether AtNTRC can bind nucleic acids.
DNA binding activity of AtNTRC was determined by an electrophoretic mobility shift assay using single- or double-stranded
M13mp18 phage DNA. There was no difference in the DNA
mobility when the DNA was incubated with GST. However, a
distinctive mobility shift was observed when AtNTRC was added
(Fig. 3A) RNA binding of AtNTRC was examined by using in vitrotranscribed luciferase mRNA. An apparent mobility shift caused by
AtNTRC was also observed (Fig. 3B). The binding of AtNTRC to
nucleic acids was conrmed by electron microscopy analysis
(Fig. 3C). Compared to the general appearance of DNA fragments
under TEM images (Fig. 3C in left panel), wider shaped molecules
were observed in the presence AtNTRC, demonstrating that the
interaction of AtNTRC proteins with DNA fragments is the main
contributor to the additional densities on the fragments (Fig. 3C in
right panel). This observation conrmed the presence of AtNTRC
binding to nucleic acids.
The DNA binding activity of protein disulde isomerase, ERp57,
is associated with its thioredoxin like domain [27,28]. Thus, we

wondered which domain of AtNTRC is important for nucleic acid

binding. When the TR and Trx domains of AtNTRC were incubated
with luciferase mRNA, the TR domain did not change the RNA
mobility, however, an apparent mobility shift was detected with
the addition of the Trx domain (Fig. 4B). These data indicate that Trx
domain of AtNTRC is important for nucleic acid binding activity.
The formation of unfavorable secondary structures of mRNA at
lower temperatures prevents efcient initiation of translation and
affects mRNA stability [29,30], so we tested the effect of AtNTRC
binding on mRNA degradation caused by RNase A. An RNase A
resistant band was observed at the supershifted position in the
presence of AtNTRC (Fig. 4C). However, GST could not prevent the
mRNA degradation by RNase A. Because cold and freezing stress
generates ROS which can affect DNA stability, we also examined the
DNA protection ability of AtNTRC by using an in vitro DNA damage
assay. When the plasmid DNA was treated with 0.1 mM FeSO4 and
10 mM H2O2, most of the supercoiled DNA band disappeared
(Fig. 4D). However, a considerable amount of the supercoiled DNA
band was protected by the addition of AtNTRC.
From these results, we suggest that AtNTRC functions as a molecular chaperone for protein as well as a protector for nucleic acids
against RNase and oxidative stress. Further research is needed to
elucidate the exact underlying molecular mechanism of the
freezing tolerance of AtNTRCOE.
Acknowledgments
This work was carried out with the support of Cooperative
Research Program for Agriculture Science & Technology Development (grant#: PJ007850) RDA, Korea.
Transparency document
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.bbrc.2015.06.089.
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