Plant Physiology and Biochemistry 70 (2013) 354e359

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Plant Physiology and Biochemistry

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Research article

Overexpression of Arachis hypogaea NAC3 in tobacco enhances

dehydration and drought tolerance by increasing superoxide
scavenging
Xu Liu b,1, Shuai Liu a,1, Jiali Wu a, Biyu Zhang a, Xiaoyun Li a, Youchen Yan a, Ling Li a, *
a
b

Guangdong Provincial Key Lab of Biotechnology for Plant Development, College of Life Science, South China Normal University, Guangzhou 510631, China
Molecular Analysis and Genetic Improvement Center, South China Botanical Garden, Chinese Academy of Science, Guangzhou 510650, China

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 24 April 2013
Accepted 13 May 2013
Available online 5 June 2013

Drought stress can severely affect plant growth and substantially diminish crop yields. We previously
isolated Arachis hypogaea NAC3 (AhNAC3), a dehydration-induced NAM/ATAF/CUC (NAC) gene from peanut. In this study, to examine the role of AhNAC3 in stress tolerance, we constructed transgenic tobacco
lines overexpressing AhNAC3. The transgenic plants showed hyper-resistance to dehydration and drought
stresses and accumulated more proline and less superoxide anion (O

2 ) than wild type under dehydration
and drought conditions. Moreover, the transgenic plants showed upregulation of four functional genes,
superoxide dismutase (SOD), pyrroline-5-carboxylate synthetase (P5SC), late embryogenic abundant proteins
(LEA), and early response to drought 10 (ERD10C). Protein localization and transactivation analysis suggested that AhNAC3 activates its specic targets in the nucleus. These results suggest that AhNAC3 is a
dehydration-induced transcription factor that improves water stress tolerance by increasing superoxide
scavenging and promoting the accumulation of various protective molecules.
Crown Copyright 2013 Published by Elsevier Masson SAS. All rights reserved.

Keywords:
NAC transcription factor
Dehydration
Drought
Superoxide anion
Arachis hypogaea

1. Introduction
Drought stress can severely affect plant growth and development and therefore substantially affect yields of important crops.
To deal with water decits, plants have developed a variety of
physiological and biochemical strategies. One of the most important steps in these defense strategies is the activation of the
expression of drought-resistance genes, a process largely regulated
by specic TFs (Transcription Factors) [1]. Several TFs, such as
DREB2A [2], ABF2 [3], and AtMYB44 [4], have been well characterized for key roles in regulating plant stress responses and
improving drought tolerance.
The NAC (NAM, ATAF, CUC) proteins constitute one of the largest
families of plant-specic TFs. NAC family members generally
contain a conserved NAC domain with a DNA-binding and nuclear
localization signal sequence in their N-terminal region [5,6]. The Cterminal region of NAC proteins is highly variable, and has been
shown to function in transcriptional activation [7]. More than 200
NAC family members have been predicted and classied in the
Arabidopsis thaliana and Oryza sativa genomes [7,8]. NAC TFs play
important roles in plant development, such as formation of organs
* Corresponding author. Tel.: 86 020 85211378; fax: 86 020 85215535.
E-mail address: liling@scnu.edu.cn (L. Li).
1
These authors are equal to this work.

[9,10], secondary walls [11,12], and lateral roots [13,14]. Furthermore, some NAC family genes are involved in tolerance to abiotic
and/or biotic stress; these include Arabidopsis thaliana AtNAC072/
RD26, AtNAC019, and AtNAC055 [15,16], and ATAF1, ATAF2 [17,18],
and Oryza sativa SNAC1, SNAC2/OsNAC6, and ONAC045 [19e21].
These stress-induced NAC TFs mainly belong to the SNAC (Stressresponsive NAC) group [22]. Recently, GmNAC20 from soybean
(Glycine max) and TaNAC2 from wheat (Triticum aestivum) also have
been found to respond to abiotic stresses [23,24], but mechanisms
of action of the stress-responsive NACs remain largely unknown.
Peanuts, or groundnuts (Arachis hypogaea), are an important
global crop, but regional drought stress can severely limit the yield
of peanuts [25]. Therefore, improvement of drought resistance in
peanuts is necessary and important. To this end, we previously
isolated AhNAC3 (Arachis hypogaea NAC3), a dehydration-inducible
NAC-like gene, from peanut. AhNAC3 is specically induced by
drought, high salt, and ABA (Abscisic acid) treatment [Figure S1],
but its specic function remains to be examined.
In this study, we characterized the AhNAC3 gene, examining its
stress-responsive expression prole, cellular localization, and
transcriptional activity. Overexpression of AhNAC3 in transgenic
tobacco resulted in signicantly improved tolerance to dehydration
and drought stresses. Unlike AhNAC2 gene [26], AhNAC3 did not
directly change ABA responses in stress tolerance, but rather mainly
increased mesenchymal antioxidant and protective molecules by

0981-9428/$ e see front matter Crown Copyright 2013 Published by Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.plaphy.2013.05.018

X. Liu et al. / Plant Physiology and Biochemistry 70 (2013) 354e359

modulating expression of NtSOD, NtLEAs, NtERD10C, and NtP5CS.

These results will contribute to improving our understanding of the
molecular basis of enhanced dehydration and drought tolerances
conferred by stress-responsive NAC genes and to the possibility of
improving drought resistance in economically important corps.
2. Result
2.1. Expression of AhNAC3 during development and stress
Expression levels of AhNAC3 in young leaves or mature leaves
were higher than those in seeds, roots, stems, and owers [Fig. 1A].
AhNAC3 expression and RWC (relative water content) were also
measured in leaves under various water stresses. In dehydration
stress, the AhNAC3 transcript levels rapidly increased by 0.5 h of
dehydration, reaching maximum levels at 2 h and remaining
elevated until 10 h, when the leaves had wilted more severely (RWC:
22.7  3.7%) than at 0 h. In osmotic stress, the AhNAC3 transcript
levels gradually increased after PEG treatment for 2 h, reaching a
peak at 10 h. In soil drought treatment, the AhNAC3 transcript levels
initially remained steady, then increased at 10 d and 14 d as the RWC
decreased (71.7  4.6% and 57.5  5.8%, respectively). The RWCs in
different treatments are shown at each time point [Fig. 1B].
2.2. AhNAC3 activates transcription and localizes to the nucleus
To examine the function of AhNAC3 in activation of transcription, we rst examined its subcellular localization using a fusion to

355

the GFP (green uorescent protein) marker. Protoplast cells transformed with the GFP control displayed uorescence throughout the
cell [Fig. 1C]. By contrast, in the protoplast cells transformed with
AhNAC3-GFP, uorescence was detected exclusively in the nucleus
[Fig. 1C], indicating that AhNAC3 localizes to the nucleus.
We next examined whether AhNAC3 could activate transcription. To this end, we used a yeast transactivation assay with
AhNAC3 fused to a DNA-binding domain (BD) and tested for activation of b-galactosidase and of a marker conferring histidine
auxotrophy. The transformants grew well on non-selective SD
medium with L-histidine (His). On L-histidine dropout medium
(His
), recombinant transformants (BD-AhNAC3) grew and showed
b-galactosidase activity while the control transformants (BD only)
did not [Fig. 1D]. These results showed that AhNAC3 functions as a
transcriptional activator.

2.3. Overexpression of AhNAC3 in tobacco enhances tolerance to

dehydration and drought
To examine the function of AhNAC3 gene in stress tolerance, we
made transgenic tobacco plants overexpressing AhNAC3 and tested
the transgenic plants for stress tolerance. Ten kanamycin-screened
transgenic lines were obtained by PCR with primers specic to
AhNAC3 and NPT2, and six transgenic lines with single copy insertions were selected by Southern blot [Figure S2]. The overexpression of AhNAC3 in three transgenic lines (T4, T5, and T9) was
conrmed by RT-PCR [Figure S2]. During dehydration stress, the
water loss in three transgenic lines and WT (wild type) seedlings

Fig. 1. Expression pattern, subcellular localization and transactivation activity of AhNAC3. (A) The tissue specic expression of AhNAC3 in seeds, roots, young leaves, mature leaves,
owers and stems, as measured by quantitative RT-PCR. Data represent means of three duplicate data and vertical columns are means  SD. (B) Expression of AhNAC3 in response to
dehydration, PEG (30%, w/v), and drought treatments in young leaves. The RWC (%) of different treatments are shown below each bar. (C) Nuclear localization of AhNAC3. p35S::GFP
(as a control) and p35S::AhNAC3-GFP plasmids were transiently expressed in tobacco mesophyll protoplast cells. Dark eld uorescence images show GFP uorescence and bright
eld images show cell morphology for the GFP control and AhNAC3-GFP. Merged images are shown at right. (D) Transactivation activity of AhNAC3. Fusion proteins of pGBKT7AhNAC3 (BD-AhNAC3) and pGBKT7 (BD, as a negative control) were expressed in yeast strain AH109. The transformants were incubated on SD/Trp
(His) and SD/Trp
/His
/
Ade
(His
) to examine their growth and were tested for b-galactosidase activity.

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X. Liu et al. / Plant Physiology and Biochemistry 70 (2013) 354e359

gradually increased, but higher water losses were detected in WT

than in the AhNAC3-overexpressing plants [Fig. 2D]. At 3 h of
dehydration, WT leaves exhibited much more serious wilting than
the three transgenic lines [Fig. 2C]. Morphological differences
became apparent (WT leaves started to wither and yellow) after
drought treatment for 10 d. When the drought stress was extended
to 14 d, the leaves of three transgenic lines remained green, but WT
became withered [Fig. 1B]. Following drought stress, more than 50%
of the transgenic plants survived after 2 d recovery, but only 33.3%
of WT plants survived. The survival of three transgenic lines was
signicantly higher than WT [Fig. 1A]. The above results indicated
that AhNAC3-overexpressing plants were more tolerant than WT to
either dehydration or drought water stress.
2.4. Improved oxidative tolerance and proline accumulation in
AhNAC3-overexpressing tobacco under water stress
To examine the mechanism by which AhNAC3 increases water
stress tolerance in tobacco, we visualized O

2 accumulation,
measured SOD activity, and measured proline levels. Histochemical
staining showed that there was no difference in O

2 levels between
three transgenic lines and WT seedlings under control conditions

[Fig. 3A]. Dehydration for 2 h resulted in increased O

2 levels in both
WT and transgenic plants, but WT accumulated more O

2 than
transgenic plants [Fig. 3A]. Similarly, under drought for 7 d, the
mature leaves of three transgenic lines accumulated less O

2 than
WT [Figure S3]. In control conditions, there was no signicant
difference in SOD activities between transgenic and WT seedlings,
except the T4 lines were higher than WT. After dehydration for 2 h,
SOD activity was signicantly higher in transgenic plants than in
WT [Fig. 3B]. SOD activities decreased in the three transgenic lines
and WT after 7 d of drought, but SOD activity remained higher than
WT in leaves of T4, T5 and T9 under control or drought treatments
[Figure S3]. Also, the proline content was signicantly higher in T4,
T5 and T9 plants than WT after drought [Fig. 3C].
Before water stress, transcript levels of NtSOD and NtERD10C
were obviously enhanced in three transgenic lines compared with
those of WT, whereas those of NtLEA and NtP5CS only showed a
minor change [Fig. 4]. Water stress caused upregulation of all four
genes in WT and transgenic plants, and the mRNA levels of the four
genes were higher in the three transgenic lines than in WT [Fig. 4].
These results indicated that overexpression of AhNAC3 in tobacco
led to a change in the transcript levels of superoxide-related and
resistance-related genes.

Fig. 2. Overexpression of AhNAC2 enhances drought and dehydration tolerance in tobacco. (A) The survival rates of independent AhNAC3-overexpressing lines (T4, T5 and T9) and
wild-type (WT) tobacco plants under drought treatment. Columns are means  SD (n 20), and asterisks (*) indicate signicant differences between the WT and AhNAC3overexpressing lines (p < 0.05). (B) The phenotypes of transgenic and WT tobacco plants under drought stress. Two-week-old plants of AhNAC3 overexpression lines (T4, T5 and T9)
and WT plants were grown for 14 d without watering and then watered for 2 d. (C) The phenotypes of transgenic and WT tobacco plants under dehydration for 2 h. (D) The water
loss of AhNAC3 overexpression lines (T4, T5 and T9) and WT tobacco plants under dehydration treatment. Columns are means  SD (n 20).

X. Liu et al. / Plant Physiology and Biochemistry 70 (2013) 354e359

357

Fig. 3. SOD enzyme activity and O2
accumulation in AhNAC3 transgenic lines under dehydration treatment. (A) Histochemical staining by nitro blue tetrazolium (NBT) to reveal
accumulation of O2
in whole plants of wild type (WT) and transgenic lines (T4, T5, and T9) subjected to dehydration for 2 h. (B) Activity of SOD enzyme in WT, T4, T5 and T9
seedlings under control and 2 h of dehydration conditions. Enzymes were extracted and assayed as described in Materials and methods. (C) The proline accumulation of WT, T4, T5
and T9 plants under control and drought for 7 d. * indicates that the values of the transgenic lines are signicantly different from those of WT (p < 0.05).

3. Discussion
The AhNAC3 had been characterized as a cDNA sequence (GenBank locus: EU755022) by our previous cloning and sequencing, its
function remained unknown. AhNAC2 was the rst gene isolated
and functionally characterized as a stress-responsive NAC gene in

peanut, and is involved in improving drought and salt tolerances.

Also, overexpression of AhNAC2 resulted in hyper-sensitivity to ABA
[26]. AhNAC3, a novel member of the peanut NAC family, contains
an ORF of 1008 bp and encodes 335 amino acids with 73% sequence
similarity to AhNAC2 (1050 bp, 349 amino acids). This raises the
possibility that AhNAC2 and AhNAC3 may function redundantly.

Fig. 4. The expression levels of stress-related genes in AhNAC3 transgenic lines before and after dehydration treatment. Expression levels of the genes (NtSOD: AB093097, NtLEA:
AF053076, NtERD10C: AB049337.1, NtP5CS: HM854026.1) were measured by quantitative RT-PCR before and after dehydration for 2 h.

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X. Liu et al. / Plant Physiology and Biochemistry 70 (2013) 354e359

Our results showed that transgenic tobacco plants overexpressing

AhNAC3 are more dehydration- and drought-resistant than WT.
However, overexpression of AhNAC3 in transgenic plants did not
affect ABA sensitivity, as no differences were observed between
transgenic line and WT in germination rate and root growth under
ABA treatment [Figure S4]. This indicates that AhNAC3 probably
plays a specic role in the regulation of drought tolerances,
different from AhNAC2 function in ABA signaling. This implies that
these two stress-responsive NAC genes do not have completely
redundant functions even though they are all involved in stress
responses.
Accumulation of superoxide anion (O

2 ) has been used as an
effective index to assess resistance to drought stress of different
crop genotypes, because it directly reects the capability to resist
oxidative stress [27,28]. Here, the AhNAC3-overexpressing transgenic lines had enhanced antioxidative capacity, reecting by
decreased accumulation of O

2 compared to WT under water stress
[Fig. 3]. It has been reported that SOD activity plays important roles
in enhancing tolerance to oxidative stress in tobacco, and that
increasing SOD activity may result in reduced O

2 levels [29,30]. As
expected, increased SOD activity was detected in transgenic plants,
suggesting that overexpression of AhNAC3 might cause the reduction in O

2 , or AhNAC3 might act as a positive regulator of ROS
metabolism. Low levels of O

2 commonly indicate high resistance to
abiotic stresses in transgenic plants, which is consistent with the
above physiological results under water stress, and may also
explain the enhanced tolerances to dehydration and drought in
transgenic plants.
Our results also showed that AhNAC3 protein has transcription
activation activity and nuclear localization [Fig. 1], indicating that
AhNAC3 acts in transcriptional regulation of target genes. Expression of NtSOD, NtLEA5, NtERD10C, NtP5CS genes was up-regulated
by dehydration and was even higher in the AhNAC3-overexpressing transgenic lines than in WT [Fig. 4], indicating that these
genes were more intensely induced in transgenic plants. The rapidresponding mRNA levels of NtSOD and NtP5CS in the transgenic
lines can explain the lower O

2 and higher proline levels compared
to WT. NtLEA and NtERD10C encode hydrophilic LEA proteins that
are assumed to play critical roles in combating cellular dehydration
[31,32]. Differential expression of NtLEA and NtERD10C in the
transgenic lines suggested that the transgenic plants might synthesize more protective chaperones for protein stabilization and
efciently binding water, thus providing better defense against
water loss in dehydration or drought stress [33,34].
In summary, we functionally characterized the stressresponsive AhNAC3 gene from peanut. AhNAC3 specically
responded to water stresses, and its overexpression improved
dehydration and drought tolerance in transgenic tobacco by
increasing O

2 scavenging and up-regulating various stress-related
genes. Thus, AhNAC3 may serve as a candidate gene for molecular
breeding of drought-tolerant peanut varieties, and it has potential
biotechnological applications to improvement drought tolerance.

4. Materials and methods

4.1. Plant materials
South China peanut cultivars (A. hypogaea L. cv. Shanyou523)
were used in this study. All peanut plants were grown in controlled
growth chambers at 25  C, 60% relative humidity, with 16 h light/
8 h dark photoperiod (light intensity: 200 mol m
2 s
1) described
previously [25]. Tobacco cultivars (Nicotiana tabacum L. cv. NC90)
were grown and maintained under the same conditions described
above.

4.2. Stress treatments for peanut

For the investigation of AhNAC3 expression, the young leaves
and roots from 2-week-old peanut, mature leaves, stems, and
owers from 5-week-old peanut plants were collected for RNA
preparation. For dehydration treatment, two-week-old peanut
seedlings were taken out of soil and dehydrated on 3 mm lter
paper for 0.5, 1, 2, 5, or 10 h. For osmotic stress treatments, the
seedlings were cultured in a hydroponic system contained 30% PEG
(polyethylene glycol) for 1, 2, 5, 10, or 24 h. Leaves were collected
after treatment. Drought treatments were performed as described
previously [26]. After withholding water, we harvested the leaves at
1, 7, 10, or 14 d. Samples were frozen immediately in liquid nitrogen
for RNA extraction and stored at
80  C until further use.
4.3. Quantitative RT-PCR
Total RNA was isolated with TRIzol reagent (Invitrogen), and
about 1 mg of DNaseI-treated total RNA was reverse transcribed with
SUPERSCRIPT III Reverse Transcriptase (Invitrogen) in a reaction
volume of 20 mL to generate the rst-strand cDNA according to the
manufacturers instructions. Quantitative PCR was performed using
an Optical 96-well Fast Thermal Cycling Plate with ABI 7500 realtime PCR system. Each reaction contained 10 mL of 2 SYBR Premix
Ex Taq (Takara), 20 ng of cDNA, and 0.1 mmol L
1 gene-specic
primers in a nal volume of 20 mL. The thermal cycle used was:
95  C for 30 s, then 40 cycles of 95  C for 5 s, 60  C for 34 s. The AhNAC3
GenBank accession number is EU755022 and the AhNAC3 target
genes NtSOD (AB093097), NtLEA (AF053076), NtERD10C
(AB049337.1), NtP5CS (HM854026.1) and an internal control gene
NtActin (U60490), were used for quantitative tests (Primer sequences
are provided in Table S1). The relative expression levels were calculated using the relative 2
DDCt method as described previously [26].
4.4. Proline analysis
Proline contents in tobacco plants 10 d after the start of drought
treatment were measured according to the method described by
Shan et al. [35].
4.5. Relative water content determination in peanut
Leaves were removed from 2-week-old peanut plants, and
treated with dehydration for various times, then measured for fresh
weight (FW) according to the method described by Yang et al. [36].
Turgid weight (TW) was determined after rehydration for 12 h at
4  C. To determine the dry weight (DW), the samples were ovendried at 80  C for 24 h. The RWC was calculated according to the
following equation: RWC (%) [(FW
DW)/(TW
DW)]  100%.
4.6. Drought stress tolerance assays and water-loss measurements
in tobacco
Three-week-old tobacco plants were subjected to drought
stress. Water was withheld completely for 14 d in wild type (WT)
and transgenic lines, and then the treated plants were continuously
watered for 2 d for recovery. Control plants were continuously
watered for 16 d. For water-loss measurements, 2-week-old tobacco plants were placed on 3 mm lter paper in weighing dishes,
and the plants were allowed to dehydrate in the environment
chambers as described above. Weights of the plants were recorded
at regular intervals. The water-loss is a percentage ratio of the
difference between W0 (the weight of the 0 h treatment) and Wi
(the weight at each time period, i 0.5, 1, 1.5, 2, 2.5 or 3 h) relative
to the difference between W0 and W3.

X. Liu et al. / Plant Physiology and Biochemistry 70 (2013) 354e359

4.7. NBT staining and SOD activity measurement

In situ accumulation of superoxide anion (O

2 ) was examined by
histochemical staining with nitro blue tetrazolium (NBT). For O

2
detection, the samples from dehydration (2-week-old whole tobacco plants before and after dehydration 2 h) or drought (leaves
from 4-week-old tobacco after drought 10 d) treatments were
immersed in 1 mg mL
1 fresh NBT solution (prepared in
10 mmol L
1 phosphate buffer, pH 7.8) and incubated under light at
25  C until dark spots were observed. The stained samples were
then cleared and kept in 70% ethanol. For extraction of superoxide
dismutase (SOD, EC 1.15.1.1), about 0.5 g of tissue was ground in
liquid nitrogen with a pre-cooled pestle and mortar, and homogenized in 5 mL of extraction buffer containing 50 mmol L
1 phosphate buffer (pH7.8) and 1% polyvinylpyrrolidone (PVP). The
homogenate was centrifuged at 10,000 g for 20 min at 4  C and the
resulting supernatant was collected for enzyme activity measurement. SOD activities, expressed as units (U mL
1), were spectrophotometrically measured according to the method described by
Shi et al. [37].
Acknowledgements
We are grateful to Dr. Zhang-Ming Wang for technical support
and critical reading of the manuscript. This work was supported by
National Natural Science Foundation of China (31171468), Guangdong Natural Science Foundation (9151063101000001 and
10251063101000010), and The Science and Technology Project of
Guangzhou City (2012J4100115).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.plaphy.2013.05.018.
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