1. ORIGINAL PAPER
Cloning and expression analysis of MuNAC4 transcription factor
protein from horsegram (Macrotyloma uniflorum (Lam.) Verdc.)
conferred salt stress tolerance in Escherichia coli
Merum Pandurangaiah • K. Eswaranarayana Reddy • G. Lokanadha Rao •
M. Sivakumar • O. Sudhakarbabu • A. Nareshkumar • M. Ramya •
T. Kirankumar • G. Veeranagamallaiah • Chinta Sudhakar
Received: 25 January 2012 / Revised: 21 June 2012 / Accepted: 22 June 2012 / Published online: 12 July 2012
Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2012
Abstract The NAC gene family encodes plant-specific
transcription factors which play diverse roles in abiotic
stress responses of plants. NAC TFs reported to be
involved in the regulatory pathway of multiple abiotic
stresses. In the present study, a salt stress-inducible NAC
gene, named MuNAC4 (Macrotyloma uniflorum NAC4)
from horsegram was isolated, cloned, characterized and
studied its expression. Real-time PCR expression analysis
showed up-regulation of MuNAC4 in horsegram across
salinity, cold, drought and dehydration stress conditions.
However, salt stress resulted a sixfold increase in MuNAC4
transcript levels. The involvement of MuNAC4 in abiotic
stress tolerance was investigated by cloning MuNAC4 gene
in expression vector pET28a and transformation in Esch-
erichia coli BL21 (DH3). The apparent molecular weight
of the recombinant protein was found to be 38.3 kDa as
evident from SDS-PAGE analysis. The functional role of
MuNAC4 in Escherichia coli confers the tolerance against
salt (6 % NaCl), heavy metal (100 mM CuSO4) and water
stresses (6 % PEG). The E. coli cells transformed with
pET28a ? MuNAC4 was showed better survival ratio and
higher growth rates under NaCl stress than other abiotic
stress conditions when compared to control cells (BL21/
pET28a). This provides the experimental evidence that
MuNAC4 protein enhance salt stress tolerance of E. coli
cells suggesting their suitability as candidates for genetic
manipulations for enhanced crop tolerance to salt stress and
other abiotic stresses.
Keywords Abiotic stress Á Bacterial expression Á
Horsegram Á MuNAC4 (Macrotyloma uniflorum NAC4) Á
RT-PCR
Introduction
Plants are sessile organisms and cannot escape from
adverse environment conditions and are exposed to various
types of abiotic stresses, such as drought, cold, heat, and
high salinity during their life cycle. In response to stresses,
plants activate a number of defence mechanisms that
function to increase tolerance to the adverse conditions
imposed by stresses (Yamaguchi-Shinozaki and Shinozaki
2006). This inducible adaptation or acclimation process has
evolved throughout the plant life cycle and is critical for
the survival of all plants. A major event in response to
stresses is the perception and transduction of stress signals
through signalling components, which results in the acti-
vation of numerous stress-related genes (Shinozaki and
Yamaguchi-Shinozaki 2007). The expression of these
stress-related genes is largely regulated by specific tran-
scription factors. In plants, many families of transcription
factors, including AP2/ERF, MYB, MYC, bZIP, HSF, NAC,
WRKY and C2H2 zinc-finger transcription factors play
Communicated by Z.-L. Zhang.
M. Pandurangaiah, K. E. Reddy contributed equally to the work.
M. Pandurangaiah Á K. E. Reddy Á G. Lokanadha Rao Á
M. Sivakumar Á O. Sudhakarbabu Á A. Nareshkumar Á
M. Ramya Á T. Kirankumar Á G. Veeranagamallaiah Á
C. Sudhakar (&)
Department of Botany, Plant Molecular Biology Unit,
Sri Krishnadevaraya University, Anantapur 515 003, India
e-mail: chintasudhakar@yahoo.com
Present Address:
G. Lokanadha Rao
Microbe-Plant Interaction Group, School of Biology
and Environmental Studies, University College,
Dublin, Ireland
123
Acta Physiol Plant (2013) 35:139–146
DOI 10.1007/s11738-012-1056-1

2. important roles in eliciting stress response by regulating the
expression of stress-responsive genes under different stress
conditions (Singh et al. 2002; Vincour and Altman 2005;
Yamaguchi-Shinozaki and Shinozaki 2006). Among the
transcription factors, NAC (NAM, ATAF, and CUC) family
proteins are one of the largest families containing plant-
specific transcription factors. There are 105 NAC family
members in Arabidopsis, 140 members in rice, and 183
members in Glycine max, 142 members in Vitis vinifera,
125 members in Sorghum bicolour, 190 members in Zea
mays (http://www.planttfdb.cbi.pku.edu.cn/). Based on the
sequence similarities, it is found that highly conserved
N-terminal region of NAC domain proteins (DNA-binding
domain, DBD) and is further divided into five conserved
sub-domains (A–E). The C-terminal region of NAC domain
proteins found to be more diverged and serves as a potential
transcriptional activator (TAR) (Xie et al. 2000; Duval et al.
2002). Several NAC type proteins are involved in the
development of the shoot apical meristem, floral organs,
lateral root formation, senescence, flowering and secondary
wall formation (Takada et al. 2001; Hibara et al. 2003;
Vroemen et al. 2003; Xie et al. 2000; Duval et al. 2002;
Olsen et al. 2005). Some of NAC proteins are involved in
both abiotic and biotic stress tolerance of plants (Olsen et al.
2005). Membrane-associated NAC transcription factors that
are involved in stress responses and plant development have
also been found (Chen et al. 2008). NAC with transmem-
brane motif 1 (NTM1) mediates cytokinin signalling during
cell division flowering (Kim et al. 2006) and NTM-LIKE 8
regulates salt responsive flowering (Kim et al. 2007) and
gibberellic acid-mediated salt signalling in seed germination
(Kim et al. 2008). The evidences for the direct involvement
of NAC transcription factor genes under abiotic stresses are
very few and the regulatory roles of these transcription
factors in plant stress or defense responses are not clearly
understood. Therefore, it is important to determine the bio-
logical functions of novel NAC proteins required for
improvement of abiotic stress tolerance in crops.
Horsegram (Macrotyloma uniflorum (Lam.) Verdc.) is
an important dryland legume grain crop widely cultivated
in arid and semi arid regions. This crop comes up reason-
ably well in dryland areas with receding soil moisture
conditions and in poor soils where other crops fail to grow.
Depending on its growth conditions and life cycle, there is
high probability that this plant may contains a large number
of genes that can be used to provide stress tolerance to this
crop (Reddy et al. 2008). Owing to high genetic similarity
among legume genomes comprehension of the underlying
genetic mechanism of various stress-related genes from
this plant will be of great advantage to transfer these genes
in other legumes for improved stress tolerance. To this
direction, we have isolated a multiple-stress inducible NAC
(MuNAC4) gene from horsegram leaves, which shows
significant response to abiotic stresses. In the present study,
we further evaluated the MuNAC4 role in response to
abiotic stresses in prokaryotic system. In this direction, we
have constructed a recombinant plasmid, pET28a ? Mu-
NAC4 over expressing full-length MuNAC4 protein in E.
coli cells and confirmed by SDS-PAGE analysis. In addi-
tion, we carried out stress assays and survival ratios of the
recombinant cells to confirm the role of MuNAC4 gene in
tolerance to different abiotic stress conditions. We believe
that these findings could potentially be used to improve
plant tolerance to abiotic stresses using gene transfer
technology.
Materials and methods
Plant material and growth conditions
Seeds of horsegram (Macrotyloma uniflorum (Lam.) Ver-
dc.) were procured from Andhra Pradesh Agricultural
Experimental Station, Anantapur. The seeds were surface
sterilized with 0.1 % (w/v) sodium hypochlorite solution
for 5 min, rinsed thoroughly with distilled water and ger-
minated in plastic pots containing 2 kg of soil:sand (2:1)
mixture maintained under natural photoperiod (10–12 h;
temperature 28 ± 4 °C) in departmental botanic garden.
A 21-day-old horsegram plants were subjected to water
stress conditions by withholding supply of water. The plants
were supplied with water to reach soil moisture levels
(SML) to 25 % by measuring SML regularly by standard-
ized gravimetric method for 5 days. The leaf samples were
harvested, flash frozen and stored at -80 °C prior to use.
Isolation of MuNAC4 transcription factor gene
RNA extraction Total RNA was isolated by the RNeasy
Plant Mini Kit (Qiagen, USA) from frozen leaf samples
according to manufacturer’s protocol. The concentration of
RNA from each samples were determined by UV spec-
trophotometry at A260, while the quality of total RNAs
analyzed by 1 % ethidium-bromide agarose-gel electro-
phoresis. The contaminated genomic DNA was removed
by Turbo DNA-free (Ambion, USA) treatment. Then, 5 lg
of total RNA used as a template for cDNA synthesis using
MMLV transcriptase (Fermentas, Germany) with oligo (dT)
primer according to the protocol.
Primer design
The NAC4 sequences from Glycine max were aligned by
ClustalW and the forward 50
-ATGGGAGTTCCAGAGGA
AGAC-30
and reverse 50
-TCAATTCCTGAACCCGAAC
C-30
primers were designed for isolation of NAC4 cDNA
140 Acta Physiol Plant (2013) 35:139–146
123

3. from horsegram. The PCR was carried out using cDNA as
template. The optimized PCR conditions for isolation of
gene using cDNA as template are 200 ng of primers,
250 lM dNTP’s, 3 U of Taq DNA polymerase (Fermentas)
in 50 ll of reaction. PCR conditions followed are step 1:
95 °C, 5 min, 1 cycle; step 2: 95 °C, 1 min; 57 °C, 45 s;
72 °C, 2 min for 30 cycles. The final extension was carried
at 72 °C for 10 min, 1 cycle. The amplified product was gel
purified by GeneJet PCR purification kit (Fermentas, Ger-
many), subsequently cloned in pTZ57R/T (Fermentas,
Germany) and sequenced (Macrogen, Korea). The obtained
sequence was confirmed by BLAST search with other near
related NAC genes from Glycine max and other plants.
Expression analysis of MuNAC4 transcript
by real-time PCR (RT-PCR)
A 21-day old horsegram plants were subjected to (1) cold
stress by transferring plants to a growth chamber set at
10 °C and samples were collected after 8 h, (2) salt stress
was imposed by adding 250 mM NaCl to the pots and
samples were collected after 72 h, (3) dehydration stress
was imposed by allowing up-rooted plants to dry on filter
papers at ambient conditions and leaf samples were col-
lected after 8 h. (4) Drought stress by withholding water
and samples were collected at the time of visible leaf
wilting. Control plants (unstressed) were maintained at
100 % SML in parallel for all the treatments. After com-
pletion of stress treatments, stressed and unstressed leaf
samples were collected, flash frozen in liquid nitrogen and
stored at -80 °C prior to RNA isolation.
Total RNA was isolated from frozen samples by RNA
isolation kit (Qiagen) and quantified with UV–vis spectro-
photometer (Shimadzu Japan). cDNA was synthesized by
using cDNA synthesis kit (Fermentas). The cDNA was used
as template for RT-PCR analysis using MuNAC4 gene-
specific primers (IDT Primer designing tool) forward 50
-
TGGACCAACCCTTCGGTTCTGAA-30
, MuNAC4 reverse
50
-CATTGCACGCGTTGTAGTTCACC-30
, and actin gene
primers, forward 50
-TCTCCTTGTATGCAAGTGGTCG-30
,
reverse 50
-ACCAGCGAGATCCAAACGAAGG-30
were
used as a internal controls for the RT-PCR. The RT-PCR
was carried in StepOne RT-PCR machine (Applied Bio-
systems, USA) conditions followed were step 1:95 °C,
5 min, 1 cycle; step 2: 95 °C, 1 min; 57 °C, 45 s; 72 °C,
2 min for 40 cycles. The final extension was carried at 72 °C
for 10 min, 1 cycle. After 40 cycles, the specificity of the
amplifications was checked by heating from 60 to 95 °C
with a ramp speed of 1.9 °C min-1
, resulting in melting
curves. Triplicate measurements were carried out to deter-
mine the mRNA abundance of each gene in each sample.
Data analysis was performed using SDS 2.2.1 software
(Applied Biosystems, USA). Amplification curves were
analyzed with a normalized reporter (Rn: the ratio of the
fluorescence emission intensity of the SYBR Green to the
fluorescence signal of the passive reference dye) threshold of
0.2 to obtain the CT values (threshold cycle). Data were
normalized to reference gene Actin; DCT = CT (gene)
- CT (Actin). Its expression was measured with three rep-
licates in each PCR run, and the average CT was used for
relative expression analyses. The fold change value was
calculated using the expression 2-DD
CT, where DDCT rep-
resents, DCT treatment - DCT control. The results obtained
were transformed to log2 scale.
Cloning and over expression of MuNAC4 gene
in E. coli
The MuNAC4 cDNA (Accession: HS109648) was PCR
amplified using forward primer—AGC CAT ATG ATG
GGA GTT CCA GAG GAA GAC and reverse primer—
GCG AAG CTT TTC AAT TCC TGA ACC CGA ACC
with flaking restriction sites of Nde1 and HindIII, respec-
tively. The amplified product was digested with Nde1
and HindIII restriction endonucleases cloned into pET28a.
E. coli BL21 (DH3) cells (Novagen, USA) were trans-
formed with recombinant plasmid (pET28a ? MuNAC4)
and pET28a vector alone. The transformed cells were
screened and induced for the expression of recombinant
protein with 1 mM of IPTG (isopropyl b-D-1-thiogalacto-
pyranoside) for 8 h at 37 °C.
Expression analysis of MuNAC4 in E. coli
by SDS-PAGE (12.5 %)
For preparation of cell-free extracts bacteria were harvested
by centrifugation at 5,000 rpm for 10 min and resultant
pellet was washed twice with 0.5% NaCl. Total proteins
were extracted from the bacterial cells with extraction buffer
containing (500 mM Tris–HCl (pH 6.8), 10 % SDS, and
10 % glycerol). Protein concentrations were determined
using Lowry method (1951) with a bovine serum albumin as
a standard. The sample was quantified and stored in small
aliquots at -20 °C until further characterization. Prior to
electrophoresis, b-mercaptoethanol and bromophenol blue at
final concentrations of 10 % and 100 mg/ml were added to
the samples, and the samples were heated in boiling water
bath for 3 min. SDS-PAGE was carried out on 12.5 % (w/v)
polyacrylamide slab gel electrophoresis as per the method of
Sambrook and Russel (2001).
Abiotic stress tolerance assay in transformed E. coli
cells
To evaluate the protective abilities of MuNAC4 protein,
the effects of salt (6 % w/v NaCl), drought (6 % w/v PEG)
Acta Physiol Plant (2013) 35:139–146 141
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4. and heavy metal (100 mM CuSO4) on the growth of
transformed E. coli BL21 cells with pET28a (empty vector)
and pET28a ? MuNAC4 (recombinant plasmids) were
examined. After cultures were adjusted to OD600 0.6, IPTG
was added to final concentration of 1 mM to induce
expression of the inserted gene. After incubation for 8 h at
37 °C, the E. coli cells were added to fresh LB liquid
medium containing NaCl (6 %), PEG (6 %) and CuSO4
(100 mM) stress. The original OD600 values of all E. coli
groups were adjusted to same value, and then culture of
recombinant E. coli and control strains culture continued at
37 °C. OD600 values were recorded every hour for 10 h.
The experiments were repeated four times, and the mean
and standard deviation were calculated.
Survival ratio of E. coli under abiotic stress
To study the survival ratio of the transformed cells under
abiotic stress conditions, recombinant cells and control E. coli
were diluted serially to 10-6
. 10 ll from each sample spread
onto LB plates containing NaCl (6 %), PEG (6 %), and
CuSO4 (100 mM). After the plates were incubated for 12 h at
37 °C, the colony numbers appearing on the plates were
counted and used for calculating the survival ratios. The
survival ratio was calculated according to the formula: sur-
vival ratio = (mean number of colonies on stresses plate/
mean number of colonies on LB plate) 9 100. Experiments
were repeated four times with three replicate plates each time.
Mean survival ratios and standard deviations were calculated.
Results
Molecular cloning of MuNAC4 gene
The PCR amplification product (HS109648) was about
1,021 bp (Fig. 1), this gene was highly homologous to
NAC genes in other plant species. The purified PCR
product and pET28a were digested with Nde1 and HindIII,
then purified and ligated together. E. coli BL21 (DH3) was
transformed with the recombinant vector by heat shock and
cultured at 37 °C on LB agar with kanamycin (50 lg/ml)
for selection of transformed clones. The cloned gene was
confirmed by restriction digestion and DNA sequencing.
The open reading frame of 1,021 bp (MuNAC4) encodes
339 amino acids with a calculated molecular mass of the
MuNAC4 product is 38.3 kDa.
Differential expression of transcripts under different
abiotic stresses
For studying the expression analysis of MuNAC4, real-
time PCR was carried out using cDNA from cold, salt
(NaCl), dehydration, and drought-treated plants for differ-
ent time period as mentioned in experimental design ear-
lier. The drought and salt stress caused a significant
increase in the MuNAC4 transcript levels than dehydration
and cold stress conditions (Fig. 2). The transcript levels
increased by about 8-fold during drought, 5.5-fold by salt
stress. In dehydration stress and cold stress resulted in an
increase transcript levels by 2.0-fold and 1.6-fold, respec-
tively. These results strongly suggest that the MuNAC4
gene play an important role in the regulation of water stress
responses and also reveals the existence of a significant
cross talk in multiple stress tolerance.
Expressional analysis of MuNAC4 in E. coli
by SDS-PAGE
To examine the expression of the foreign gene in recom-
binant E. coli, total proteins of bacteria were electropho-
resed on 12 % SDS-PAGE. SDS-PAGE protein profile
clearly showed a specific band with apparent molecular
mass 38.3 kDa after 8 h of 1 mM IPTG induction (Fig. 3).
This further supports theoretical mass of the protein
encoded by MuNAC4 gene (38.3 kDa).
Effect of abiotic stresses on the growth of E. coli cells
having MuNAC4 gene: E. coli cell survival and growth
The recombinant (BL21/pET28a ? MuNAC4) and control
(BL21/pET28a) cells showed similar growth on LB med-
ium in overnight grown culture, whereas on NaCl (6 %),
PEG (6 %) and CuSO4 (100 mM) supplemented medium,
the recombinant cells (BL21/pET28a ? MuNAC4 ? stress
factor) showed increase in growth as compared to control
cells (BL21/pET28 ? stress factor) (Fig. 4a–c). These
results indicate that recombinant protein enhances growth
of bacteria in salt and heavy metal stress conditions.
However, there was a small increase in the growth of
bacteria under PEG stress conditions (Fig. 4b). The cells
with empty vector alone, showed slower growth as com-
pared to recombinant cells (Fig. 5).
Effect of abiotic stresses on survival ratio
of transformed E. coli cells
The recombinant cells were spotted (at serial dilution 10-6
)
on LB basal medium and medium supplemented with
NaCl, PEG and CuSO4 (Table. 1). The transformed E. coli
cells had more survival rate than un-transformed cells. The
highest improvement in the growth of transformed cells
was observed under copper stress conditions. Transformed
cells recorded almost 10 times higher growth than
untransformed cells. The number of colonies in general,
lesser in medium supplement with stress than in medium
142 Acta Physiol Plant (2013) 35:139–146
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5. with no supplement. In the medium supplemented with
NaCl, the number of recombinant bacterial colonies was
more than that of medium supplemented with PEG or
CuSO4.
Discussion
Generally model plants such as tobacco, Arabidopsis and
yeast mutants are being used widely for functional vali-
dation of genes and also for characterizing genes. Recently
many investigators have isolated plant genes responsive to
abiotic stress by functional expression screening in E. coli
(Hong et al. 2010; Gupta et al. 2010; Chaurasia et al. 2008).
Few recent publications have reported better growth of
bacteria by over expression of plant functional genes such
as DREB2A, pcs (Gupta et al. 2010; Chaurasia et al. 2008).
Many stress proteins are usually located in cytoplasm of
the plant cells. The principle of functional expression
screening is that due to expression of foreign plant genes
carried by plasmid DNA in prokaryotic cells, the host E.
coli cells acquire stress tolerance. The results show that
expression of plant gene directly contributes to increasing
stress tolerance of the prokaryotes and could share some
common protective mechanisms with higher plants under
stress conditions (Garay-Arroyo et al. 2000; Lan et al.
2005; Yamada et al. 2002).
NAC family, represents one of the largest plant tran-
scription factor families, is only found in plants to date
Fig.1 a cDNA reverse transcribed from leaf RNA isolated from
horsegram, used as template for amplification of MuNAC4 gene using
gene-specific primers. MuNAC4 was isolated and amplified as
described in ‘‘Materials and methods’’). DNA samples were run on
1 % agarose gel and visualized by ethidium bromide staining. Lane M
DNA ladder (1 kb, Fermentas). Lane 1 (L1) PCR product of
MuNAC4 (1,021 bp). b Agarose gel showing double digested
recombinant clones with Nde1 and HindIII showing the presence of
1,021 bp fragment and pET28a ? MuNAC4 with Nde1 and HindIII
showing release of (1,021 bp) fragment
0
1
2
3
4
5
6
7
8
9
Cold Salt Drought Dehydration
Foldincreaseoverthecontrol
Treatments
Fig. 2 MuNAC4 gene expression levels under different abiotic
stresses assayed using RT-PCR
Fig. 3 SDS-PAGE (12.5 %) analysis of MuNAC4 protein expression
in E. coli BL21 (Comassie blue staining). Lane M protein marker
(kDa), L1 whole cell lysate of BL21 E. coli cells containing the empty
vector pET28a without IPTG induction, L2 whole lysate of BL21
E. coli cells containing the empty vector pET28a obtained at 8 h post-
induction with 1 mM IPTG, L3 whole cell lysate of non-induced
BL21 E. coli cells containing the plasmid pET28a ? MuNAC4, L4
whole cell lysate of the same cells obtained at 8 h post-induced with
1 mM IPTG
Acta Physiol Plant (2013) 35:139–146 143
123

6. (Riechmann et al. 2000). NAC TFs are reported in plants
which regulate both ABA dependent and independent
genes. They are expressed in different tissues at various
developmental stages and are involved in many aspects of
plant growth and development (Olsen et al. 2005). RT-PCR
expression analysis revealed MuNAC4 increased in
horsegram subjected to different abiotic stresses and
showed higher expression under drought and salt than cold
and dehydration stress conditions (Fig. 2). In Arabidopsis
overexpression of three NAC genes ANAC019, ANAC055
and ANAC072 showed significant increase during drought
conditions (Fujita et al. 2004; Tran et al. 2004). The NTL8
gene belongs to the NAC family has shown to influence
flowering time under salt stress (Kim et al. 2007). Car-
NAC3 is a transcriptional activator showed to be involved
in the drought response (Hui Peng et al. 2009). GmNAC3
and GmNAC4 were showed to be induced with ABA, JA
and salinity (Guilherme et al. 2009). The expression of
SNAC group NAC proteins (ANAC019, ANAC055 and
ANAC072 (RD26) is induced by drought, high salinity and
by the phytohormones, ABA and methyl jasmonic acid
(Tran et al. 2004). Recently, Wu et al. (2009) reported that
Arabidopsis plants over-expressing the abiotic stress-
responsive gene ATAF1, which is homologous to RD26,
showed improved drought tolerance. However, these
reports confined to few NAC transcription factors and
indicated in general, that NAC factors have important roles
for the control of abiotic stress tolerance. NAC TF proteins
are considered to be plant specific, therefore, it is difficult
to discern as to how the NAC TFs transformed bacterial
cells were showing better tolerance under stress conditions.
When abiotic stress was applied to the bacterial culture,
abiotic stress would cause the intra-cellular dehydration
and damage of both proteins and cellular membrane (Wang
et al. 2003; Xiong and Zhu 2002; Lan et al. 2005). Our
results on growth rate and survival ratios indicated
improved stress tolerance of the recombinant bacteria cells
by overexpression of MuNAC4 gene conferring protective
function in host cells against damage caused by stress on
proteins and cellular membrane thereby imparting toler-
ance to bacteria. Gupta et al. (2010) have reported better
growth of E. coli cells by overexpression of plant stress
tolerant functional gene SbDREB2A. The PM2, a group 3
LEA protein from soybean showed salt stress tolerance in
E. coli (Liu and Zheng 2005). The expression of
A
B
C
0.5
0.7
0.9
1.1
1.3
1.5
1.7
0 1 2 3 4 5 6 7 8 9 10
0 1 2 3 4 5 6 7 8 9 10
0 1 2 3 4 5 6 7 8 9 10
O.D.at600nm
Time (in hours)
pET28a
pET28a+ stress
pET28a+NAC4+stress
0.5
0.7
0.9
1.1
1.3
1.5
1.7
1.9
O.D.at600nm
Time (in hours)
pET28a
pET28a+ stress
pET28a+NAC4+stress
0.5
0.7
0.9
1.1
1.3
1.5
1.7
1.9
O.D.at600nm
Time (in hours)
pET28a
pET28a+ stress
pET28a+NAC4+stress
Fig. 4 Growth analysis of E. coli carrying MuNAC4 gene was
carried on LB liquid medium with different supplements. a NaCl
(6 %), b PEG (6 %), c CuSO4(100 mM). OD600 was recorded at 1 h
interval up to 10 h and mean values are represented in graph. a NaCl
(6 %) stress: b PEG (6 %) stress: c CuSO4 (100 mM) stress
0
100
200
300
400
500
600
NaCl PEG CuSO4
Survivalratio
Treatments
MuNAC4
pET28a
Fig. 5 Survival ratio of recombinants under abiotic stress conditions.
The cultures of BL/pET28a, BL/pET28a ? MuNAC4 were spread on
LB plates and the plates with supplemented with NaCl (6 %), PEG
(6 %) and CuSO4 (100 mM). The colony numbers appearing on the
plates were counted and used for calculating the survival ratio
Table 1 Number of colonies of BL/pET28a and pET28a ? MuNAC4 on LB plates supplemented with serial dilution 10-6
, NaCl (6 %) PEG
(6 %) and CuSO4 (100 mM) (±SD, figures in parenthesis are percent decrease)
Strain Control (LB medium) NaCl PEG CuSO4
BL/pET28a 612 ± 18 (100) 278 ± 15 (45) 139 ± 11 (22) 10 ± 3 (1.6)
BL/pET28a ? MuNAC4 688 ± 26 (100) 546 ± 27 (79) 315 ± 17 (45) 90 ± 10 (13)
144 Acta Physiol Plant (2013) 35:139–146
123

7. phytochelatin synthase gene (pcs) in E. coli showed better
protection by heat, salt, carbofuron (pesticide), cadmium,
copper and UV stress (Chaurasia et al. 2008). In the present
study, E. coli (recombinant cells) showed better tolerance
to salt, PEG induced water and heavy metal stress. Further,
MuNAC4 showed to impart relatively higher tolerance of
E. coli cells to NaCl and desiccation stress as compared to
other abiotic stress assays supporting its salt stress
responsive nature. This study shows that novel MuNAC4
gene from horsegram could be better candidate for devel-
oping stress tolerant crops. In conclusion, the present data
predicts that novel MuNAC4 genes can serve as an
important genetic resource for abiotic stress tolerance.
Author contribution CS designed the experiment. MP,
KER and GLR carried out TA cloning and bacterial
expression studies. AN, MR and TK carried out SDS-PAGE
protein analysis. MS and OS carried out RT-PCR. MP, GV
and CS wrote paper. All co-authors provided inputs to
improve the manuscript.
Acknowledgments The financial support from DBT (BT/PR9609/
AGR/02/451/2007) and DST (SR/SO/PS-001/2011), GOI, New Delhi
in the form of research grants to Chinta Sudhakar is gratefully
acknowledged.
References
Chaurasia N, Mishra Y, Rai LC (2008) Cloning expression, analysis
of phytochelatin synthase (pcs) gene from Anabaena sp. PCC
7120 offering multiple stress tolerance in Escherichia coli.
Biochem Biophys Res Commun 376:225–230
Chen YN, Slabaugh E, Brandizzi F (2008) Membrane-tethered
transcription factors in Arabidopsis thaliana: novel regulators in
stress response and development. Curr Opin Plant Biol 11:695–701
Duval M, Hsieh TF, Kim SY, Thomas TL (2002) Molecular
characterization of AtNAM: a member of the Arabidopsis
NAC domain superfamily. Plant Mol Biol 50:237–248
Fujita M, Fujita Y, Maruyama K, Seki M, Tran LSP, Yamaguchi-
Shinozaki K, Shinozaki K (2004) A dehydration induced NAC
protein, RD26, is involved in a novel ABA-dependent stress
signaling pathway. Plant J 39:863–876
Garay-Arroyo A, Colmenero-Flores JM, Garciarrubio A, Covarrubias
AA (2000) High hydrophilic proteins in prokaryotes and
eukaryotes are common during conditions of water deficit.
J Biol Chem 275:5668–5674
Gupta K, Agarwal PK, Reddy MK, Jha B (2010) SbDREB2A, an A-2
type DREB transcription factor from extreme halophyte Sali-
cornia brachiata confers abiotic stress tolerance in Escherichia
coli. Plant Cell Rep 29:1131–1137
Hibara K, Takada S, Tasaka M (2003) CUC1 gene activates the
expression of SAM-related genes to induce adventitious shoot
formation. Plant J 36:687–696
Hong GX, Jiang J, Wang BC, Li HY, Wang YC, Yang CP, Liu GF
(2010) ThPOD3, a truncated polypeptide from Tamarix hispida,
conferred drought tolerance in Escherichia coli. Mol Biol Rep
37:1183–1190
Kim SG, Kim SY, Park CM (2007) A membrane-associated NAC
transcription factor regulates salt-responsive flowering via
flowering locust in Arabidopsis. Planta 226:647–654
Kim SG, Lee AK, Yoon HK, Park CM (2008) A membrane-bound
NAC transcription factor NTL8 regulates gibberellic acid-
mediated salt signaling in Arabidopsis seed germination. Plant
J 55:77–88
Kim YS, Kim SG, Park JE, Park HY, Lim MH, Chua NH, Park CM
(2006) A membrane- bound NAC transcription factor regulates
cell division in Arabidopsis. Plant Cell 18:3132–3144
Lan Y, Cai D, Zheng YZ (2005) Expression in Escherichia coli of
three different group soybean LEA genes to investigate
enhanced stress tolerance. Acta Bot Sin 47:5
Liu Y, Zheng Y (2005) PM2, a group 3 LEA protein from soybean,
and its 22-mer repeating region confer salt tolerance in
Escherichia coli. Biochem Biophys Res Commun 31:325–332
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein
measurement with foline phenol reagent. J Biol Chem 193:
265–275
Olsen AN, Ernst HA, Leggio LL, Skriver K (2005) NAC transcription
factors: structurally distinct, functionally diverse. Trends Plant
Sci 10:79–87
Peng H, Yu X, Cheng H, Shi Q, Zhang H, Li J, Ma H (2009) Cloning
and characterization of a novel NAC family gene CarNAC1
from chickpea (Cicer arietinum L.). Mol Biotech 4:814
Pinheiro GL, Marques CS, Costa MD, Reis PA, Alves MS, Carvalho
CM, Fietto LG, Fontes EP (2009) Complete inventory of soy
bean NAC transcription factors: sequence conservation and
expression analysis uncover their distinct roles in stress
response. Gene 444:10–23
Reddy PC, Sairanganayakulu G, Thippeswamy M, Sudhakar Reddy
P, Reddy MK, Sudhakar C (2008) Identification of stress-
induced genes from the drought tolerant semi-arid legume crop
horsegram (Macrotyloma uniflorum (Lam.) Verdc.) through
analysis of subtracted expressed sequence tags. Plant Sci 175:
372–384
Reddy PS, Mallikarjuna G, Kaul T, Chakradhar T, Mishra RN,
Sopory SK, Reddy MK (2010) Molecular cloning and charac-
terization of gene encoding for cytoplasmic Hsc70 from
Pennisetum glaucum may play a protective role against abiotic
stresses. Mol Gen Genet 283:243–254
Riechmann J, Heard G, Martin L, Reuber CZ, Jiang J, Keddie L,
Adam O, Pineda OJ, Ratcliffe RR, Samaha R, Creelman M,
Pilgrim P, Broun JZ, Zhang D, Ghandehari BK, Sherman G,
Yu L (2000) Arabidopsis transcription factors: genome wide
comparative analysis among eukaryotes. Science 290:2105–2110
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory
manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York
Shinozaki K, Yamaguchi-Shinozaki K (2007) Gene networks
involved in drought stress response and tolerance. J Exp Bot
58:221–227
Singh K, Foley RC, Onate-Sanchez L (2002) Transcriptional factors
in plant defense and stress responses. Curr Opin Plant Biol
5:430–436
Takada S, Hibara K, Ishida T, Tasaka M (2001) The CUP-SHAPED
COTYLEDON1 gene of Arabidopsis regulates shoot apical
meristem formation. Development 128:1127–1135
Tran LSP, Nakashima K, Simpson S, Seki M, Shinozaki K,
Yamaguchi-Shinozaki K (2004) Isolation and functional analysis
of Arabidopsis stress-inducible NAC transcription factors that
bind to a drought-responsive cis-element in the early responsive
to dehydration stress promoter. Plant Cell 16:2481–2498
Vincour B, Altman A (2005) Recent advances in engineering plant
tolerance to abiotic stress: achievements and limitations. Curr
Opin Plant Biol 16:123–132
Vroemen CW, Mordhorst AP, Albrecht C, Kwaaitaal MA, de Vries
SC (2003) The CUP-SHAPED COTYLEDON3 gene is required
for boundary and shoot meristem formation in Arabidopsis. Plant
Cell 15:1563–1577
Acta Physiol Plant (2013) 35:139–146 145
123

8. Wang W, Vinocur B, Altman A (2003) Plant responses to drought,
salinity and extreme temperatures: towards genetic engineering
for stress tolerance. Planta 218:1–14
Wu Y, Deng Z, Lai Y, Zhang Y, Yang C, Yin B, Zhao Q, Zhang L,
Li Y, Xie Q (2009) Dual function of Arabidopsis ATAF1 in
abiotic and biotic stress responses. Cell Res 19:1279–1290
Xie Q, Frugis G, Colgan D, Chua NH (2000) Arabidopsis NAC1
transduces auxin signal downstream of TIR1 to promote lateral
root development. Genes Dev 14:3024–3036
Xiong L, Zhu JK (2002) Molecular and genetic aspects of plant
responses to osmotic stress. Plant Cell Environ 25:131–139
Yamada A, Sekifuchi M, Mimura T, Ozeki Y (2002) The role of plant
CCTa in salt- and osmotic-stress tolerance. Plant Cell Physiol.
43:1043–1048
Yamaguchi-Shinozaki K, Shinozaki K (2006) Transcriptional regu-
latory networks in cellular responses and tolerance to dehydra-
tion and cold stress. Annu Rev Plant Biol 57:781–803
146 Acta Physiol Plant (2013) 35:139–146
123