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RESEARCH
Overexpression of Horsegram (Macrotyloma uniflorumLam.Verdc.) NAC Transcriptional Factor (MuNAC4)in Groundnut Confers Enhanced Drought Tolerance
Merum Pandurangaiah • G. Lokanadha Rao • O. Sudhakarbabu •
A. Nareshkumar • K. Kiranmai • U. Lokesh • Ganesh Thapa •
Chinta Sudhakar
� Springer Science+Business Media New York 2014
Abstract The NAC family being the largest plant-specific
transcription factors functions in diverse and vital physio-
logical processes during development. NAC proteins are
known to be crucial in imparting tolerance to plants against
abiotic stresses, such as drought and salinity, but the func-
tions of most of them are still elusive. In this study, we report
for the first time expression of the MuNAC4, a member of
NAC transcription factor from horsegram (Macrotyloma
uniflorum) conferring drought tolerance. The groundnut
(Arachis hypogaea) transgenics were generated using
recombinant MuNAC4 binary vector transformation
approach. Molecular analysis of these transgenic lines
confirmed the stable gene integration and expression of the
MuNAC4 gene. Twelve lines of T5 generation exhibited
significantly enhanced tolerance to drought stress with
proliferated lateral root growth as compared to wild types.
Transgenics exposed to long-term desiccation stress assays
showed increased lateral roots and greenish growth. The
physiological parameters analysis also suggests that over-
expression of MuNAC4 plays a significant role in improving
the water stress tolerance of transgenic groundnut, reducing
the damage to membrane structures and enhancing osmotic
adjustment and antioxidative enzyme regulation under
stress. This study validates MuNAC4 as an important can-
didate gene for future phytoengineering approaches for
drought tolerance in crop plants.
Keywords Drought stress � Groundnut � Horsegram �MuNAC4 � Stress tolerance
Introduction
Drought stress negatively influences growth, survival and
productivity of rainfed crops. Drought tolerance is a
complex trait, the expression of which depends on action
and interaction of different morphological, physiological
and molecular traits [1, 2]. Plant responses to water deficit
can be analysed by systematically identifying and charac-
terizing the cellular, biochemical and molecular basis of
the genes (traits). The early events of the adaptation of
plants to drought stress include the perception of stress
signals and subsequent signal transduction, leading to the
activation of various physiological and biochemical
responses [3, 4]. Within the signal transduction networks
that are involved in the conversion of stress signal per-
ception to stress-responsive gene expression, various tran-
scription factors (TFs) and cis-acting elements present in
stress-responsive promoters function not only as molecular
switches for gene expression, but also as terminal points of
signal transduction in the signalling processes. The iden-
tification and molecular tailoring of novel TFs have the
potential to improve tolerance of crop plants under hostile
conditions. The most important step in plant stress toler-
ance is the activation of stress-related gene expression,
which is largely regulated by specific transcription factors.
Over 7 % of higher plant genomes encode for putative
transcriptional factors, about 45 % of which are from
families specific to plants. Several homologous families of
transcription factors have been reported to play roles in
eliciting stress responses [5]. Typically, the TFs contain a
distinct type of DNA-binding domain and transcriptional
M. Pandurangaiah � O. Sudhakarbabu � A. Nareshkumar �K. Kiranmai � U. Lokesh � C. Sudhakar (&)
Department of Botany, Sri Krishnadevaraya University,
Anantapuram 515003, India
e-mail: chintasudhakar@yahoo.com
G. Lokanadha Rao � G. Thapa
Microbe-Plant Interaction Group, School of Biology and
Environmental Studies, University College, Dublin, Ireland
123
Mol Biotechnol
DOI 10.1007/s12033-014-9754-0
regulation region (TRR) and are capable of activating or
repressing the transcription of multiple target genes [6].
In plants, the NAC gene family encodes a large number of
transcription factor genes. The NAC acronym was derived
from three genes that were initially discovered to contain
NAC domains: NAM (for no apical meristem), ATAF1 and -
2, and CUC2 (for cup-shaped cotyledon) [7]. The C-termi-
nal regions of NAC proteins are highly divergent [8] and are
responsible for the diversity of the transcriptional activities
exhibited by NAC proteins [9, 10]. The earliest reports of
NAC genes include the NAM from Petunia (Petunia
hybrid), which determines the position of the shoot apical
meristem [11], and CUC2 from Arabidopsis, which partic-
ipates in the development of embryos and flowers [7].
Many of the NAC family proteins have been identified
and are implicated in many diverse functions and cellular
processes, such as developmental [12], hormonal functions
and signal transduction [13] in various plant species. At-
NAC072 (RD29), AtNAC019, AtNAC055 and ANAC102
from Arabidopsis [14, 15], SNAC1, SNAC2/OsNAC6,
OsNAC5 and OsNAC10 from rice [8, 16–19] and BnNAC
from Brassica napus [20] were shown to be involved in
responses to various environmental stresses. The level of
OsNAC19 transcription was elevated by infection with the
fungus Magnaporthe grisea [21] suggesting that OsNAC19
is involved in the defence response of rice to M. grisea
infection. AtNAC2, another stress-related NAC gene in
Arabidopsis, functions downstream of the ethylene and
auxin signal pathways and enhances salt tolerance and lat-
eral root development when overexpressed [22]. NAM-B1,
an NAC gene found in wheat, is involved in nutrient
remobilization from the leaves to the developing grains [23],
whereas GRAB1 and GRAB2 were found to interact with
the dwarf geminivirus RepA protein to control geminivirus
DNA replication associated with plant growth and devel-
opment in wheat [24]. NAC proteins generally function as
transcriptional activators, and AtNAM, ATAF1, AtNAC2
and AtNAC3 have been shown to act as transcriptional
activators in a yeast assay system [22].
Drought is one of the main limitations of growth and
yield components in groundnut (Arachis hypogaea L.).
Since 80 % of the world’s groundnut production is under
rainfed agriculture system with limited inputs, the
increasing frequency of drought constitutes a major abiotic
stress in groundnut. We have isolated a stress-responsive
NAC4, a member of NAC family from a drought tolerant
horsegram (Macrotyloma uniflorum; MuNAC4) which
shows a typical significant response to abiotic stresses. In
the present investigation, we further confirmed the func-
tional role of the MuNAC4 gene in drought tolerance in
MuNAC4-transformed groundnut. This study provides new
insight into the role and function of MuNAC4 in drought
stress tolerance in an important oil crop species.
Materials and Methods
Construction of Plant Expression Vector
The full-length MuNAC4 gene was PCR amplified from
cDNA with primer sets having NdeI and HindIII sites in the
forward and reverse primers, respectively, and sequence
validated. The isolated MuNAC4 gene was cloned to con-
struct recombinant pC2301 vector (designated pC2301-
MuNAC4) driven by Cauliflower mosaic virus (CaMV)
35S constitutive promoter having the screenable marker
gene nptII. The construct pC2301-MuNAC4 was trans-
formed into Agrobacterium tumefaciens strain EHA105
(designated EHA105-MuNAC4) for Agrobacterium-medi-
ated transformation.
Agrobacterium-Mediated Transformation of Groundnut
EHA105-MuNAC4 was used for Agrobacterium-mediated
transformation in 2-day-old seedlings of groundnut (variety
Narayani). Two-day-old groundnut seedlings were
immersed in the bacterial suspension for 3 days at 28 �C.
For co-cultivation, Murashige and Skoog (MS) solid
medium was supplemented with 3 mg/L 6-BA, incubated
at 25 ± 2 �C for 3 days in the dark. For selective culti-
vation, MS medium was supplemented with 1, 100 and
200 mg/L Kanamycin (Kan) incubated under a 16 h/
8 h day/night cycle. After 8 days, the survived groundnut
seedlings were transferred to plastic pots for hardening and
root development. After 10 days, the established seedlings
were moved to earthen pots filled with 5 kg of a soil and
sand mix (2 parts soil: 1 part sand). The plants were ade-
quately fertilized and grown under controlled conditions
and advanced for next generations.
Identification of Putative Transformants by PCR
and qRT-PCR Analysis
The genomic DNA was extracted from putative transgenic
plants, and transformation was confirmed by PCR analysis
using nptII and GUS gene-specific primers. To compare the
PCR-identified transgenics and wild-type lines at tran-
scripts expression level, equal concentration of cDNA was
used to perform qRT-PCR using gene-specific MuNAC4
and actin primers. The cDNA was made using oligoDT
primers according to manufacturer’s instructions (Fer-
mentas). The gene-specific MuNAC4 primers were
designed and comparative CT method of quantitation
(DDCT method) was done using actin gene as a reference.
The primers for the MuNAC4 gene were: forward 50-TGG
ACC AAC CCT TCG GTT CTG AA-30 and reverse 50-CAT TGC ACG CGT TGT AGT TCA CC-30. Reactions
were performed in triplicate, containing 100 ng of cDNA,
Mol Biotechnol
123
0.4 lL of each primer (5 pmol), 10 lL Power SYBR
Green Master Mix (Applied Biosystems, USA) and sterile
Milli-Q water for a final volume of 15 lL. The PCRs were
performed in Applied-Biosystems Step-One Real-time
PCR system (USA). The RT-PCR setup was followed by
40 cycles of 1 min at 95 �C, 1 min at 57 �C and 1 min at
72 �C. Relative fold change for MuNAC4 gene expression
was quantified using the comparative methods Ct: 2-DCt
and 2-DDCt, with data obtained from a pool of at least three
biological replicates that were individually validated.
Plant Growth Conditions and Stress Assay
for Transgenic Groundnut Plants
Initially, the experimental pots were maintained in glass-
house and moved to natural conditions (photoperiod
10–12 h; temperature 28 ± 3 �C) before imposing the
drought stress. The pots were watered twice daily to keep
the soil saturated. Simultaneously, a set of wild-type plants
was also maintained under similar conditions. Forty-day-
old plants in both sets were subjected to drought stress by
withholding water supply for 14 days. After 14 days of
drought stress, leaf relative water content (RWC), cell
membrane stability (CMS), malondialdehyde (MDA), total
chlorophyll content, total soluble sugar, free proline con-
tent and antioxidative enzyme activities were measured in
both wild-type and transgenic groundnut plants. To test the
drought tolerance of transgenic groundnut plants over-
expressing MuNAC4, a set of wild and transgenic plants
was continued under drought stress conditions for three
successive weeks and recoded the lateral root growth and
biomass.
Physiological Analysis of Transgenic Groundnut
Exposed to Drought Stress
Leaf samples were collected and experiments were done in
three replicates for all the cases, representing mean values
were used for statistical analysis by DMR test.
Determination of Relative Water Content
Leaf RWC was measured according to Barrs and Weath-
erly [25]. For this, leaf discs of 1-cm diameter were pre-
pared from leaf samples and fresh weight of 25 leaf discs,
in three replicates, was recorded. Then, the leaf discs were
hydrated for 6 h in water and their turgid weights were
recorded. The samples were then dried in an oven at 80 �C
for 24 h and weighed.
The RWC was determined as follows:
RWC ¼ FW � DMð Þ = TM � DMð Þ � 100
Estimation of Cell Membrane Stability
Leaf CMS was determined according to Leopold et al. [26].
Leaf discs of 1-cm diameter were prepared from leaf
samples and incubated in 10 mL of double distilled water
for 2 h. The solution was filtered, and OD (optical density)
was examined at 273 nm (Initial OD). Subsequently, leaf
discs were boiled in the distilled water for 30 min, cooled
and filtered and OD was examined at 273 nm (Final OD).
Percent leakage was calculated using the following for-
mula: Percent leakage = (Initial OD/Final OD) 9 100. The
results were calculated as the average of the percent
leakage of 150 leaf discs taken from three replicates.
Malondialdehyde (MDA) Content
The levels of MDA content were determined by the thio-
barbituric acid (TBA) reaction as described by Peever and
Higgins [27]. One gram of tissue (FW) was homogenized
in 5 mL of 0.1 % (w/v) TCA. The homogenate was cen-
trifuged at 10,0009g for 5 min, and 4 mL of 20 % TCA
containing 0.5 % (w/v) TBA was added to 1 mL of the
supernatant. The mixture was heated at 95 �C for 30 min
and then quickly cooled on ice. The contents were centri-
fuged at 10,000 g for 15 min, and the absorbance was
measured at 532 and 600 nm in Shimadzu 1801 UV
spectrophotometer after subtracting the non-specific
absorbance at 600 nm. The concentration of MDA was
determined by its extinction coefficient of 155 mM/cm,
MDA content expressed as l/mol/g/FW.
Estimation of Total Chlorophyll Content
Total chlorophyll content was estimated in the leaves
according to Arnon [28]. Fresh leaves were taken, washed
and homogenized using 80 % cold acetone. The homoge-
nate was centrifuged at 3,000 rpm for 30 min, and the
supernatant was collected. The sediment was re-extracted
with 80 % cold acetone. All the supernatants collected
were pooled and made to known volume with 80 % ace-
tone. The OD of the acetone extract was measured at 645
and 663 nm against 80 % acetone in a UV–Vis spectro-
photometer (Shimadzu 1800, Japan). Total chlorophyll
content was calculated by employing the following for-
mula: TCC = 20.2 9 OD (at 645 nm) ? 8.02 9 OD (at
663 nm).
Estimation of Soluble Sugars and Free Proline Content
Soluble sugars were determined [29] based on the method
of phenolsulphuric acid. 0.5 g fresh weight of leaf tissue
was homogenized with deionized water, filtered and then
treated with 5 % phenol and 98 % sulphuric acid, incu-
bated for 1 h, and absorbance was measured at 485 nm by
spectrophotometer (Varian). Contents of soluble sugar
Mol Biotechnol
123
were expressed as mg/g/FW, whereas for free proline
determination, fresh leaf samples were extracted with 3 %
aqueous sulfosalicylic acid [30] and filtered through four-
layered muslin cloth and the filtrate was collected. Two
millilitres of filtrate was taken into a test tube and to this,
2 mL of acid ninhydrin and 2 mL of glacial acetic acid
were added. The tubes were incubated at 100 �C for 1 h in
a boiling water bath and were then transferred to an ice
bath to terminate the reaction. 4 mL of toluene was added
to the tubes and mixed thoroughly using a test tube stirrer
for 15 s. The toluene layer containing the chromophore
was separated from the aqueous phase by aspiration. Then,
the absorbance of the solution was measured at 520 nm in a
UV–Vis spectrophotometer (Shimadzu 1800, Japan)
against toluene. Proline was measured from the standard
curve prepared with authentic proline, and its amount was
calculated on dry weight basis.
Superoxide Dismutase (SOD) Enzyme Assay
Superoxide dismutase activity was determined as described
[31], and the reaction mixture (1.5 mL) contained 50 mM
phosphate buffer (pH 7.8), 0.1 lM EDTA, 13 lM NBT,
2 lM riboflavin and enzyme extract. Riboflavin was added
last, and tubes were shaken and illuminated with two 20-W
fluorescent tubes. The reaction was allowed to proceed for
15 min after which the lights were switched off and the
tubes covered with a black cloth. Absorbance of the reac-
tion mixture was read at 560 nm. One unit of SOD activity
was defined as the amount of enzyme required to cause
50 % inhibition of NBT photoreduction rate.
Ascorbate Peroxidase (APX) Enzyme Assay
The ascorbate peroxidase (APX, E.C. 1.11.1.11) was
assayed [32], and the reaction mixture for measuring APX
activity contained 50 mM sodium phosphate buffer (pH
7.0), 0.2 mM EDTA, 0.5 mM Ascorbic acid, 250 mM
H2O2 and 50 lg of protein. The activity was recorded as
decrease in absorbance at 290 nm for 1 min, and the
amount of ascorbate oxidized was calculated from the
extinction coefficient 2.6 mM/cm.
Guaiacol Peroxidase (GPX) Enzyme Assay
Guaiacol peroxidase activity was determined as described
earlier [33] using a reaction mixture containing 30 mM
2-methoxyphenol (guaiacol) and 4 mM H2O2 in 0.2 M
sodium acetate buffer (pH 6.0). Enzymatic activity was
defined as the consumption of 1 lmol of guaiacol min/cm3
at room temperature using a coefficient of absorbance for
tetraguaiacol of 26.6 mM/cm.
Results
Structure and Sequence Analysis of MuNAC4 Gene
MuNAC4 gene was isolated by PCR amplification of
cDNA using the gene-specific primers flanked with Nde1
and HindIII sites. The PCR product was purified, ligated in
the T/A vector pTZ57/R (Fermentas) and transformed into
E. coli DH5a strain. The positive clones were screened and
sequenced. The full-length cDNA of MuNAC4 consists of
1,024 bp nucleotides (Accession # HS109648). The
sequence has open reading of 1,021 bp encoding MuNAC4
of 339 amino acids with an initiation codon of ATG. The
deduced aminoacid sequence comprises 339 aminoacid
residues with a calculated molecular mass of 38.23 kDa
with isoelectric point 6.40. Phylogenetic analysis revealed
that the MuNAC4 protein belongs to the GmNAC4 sub-
group (Fig. 1a). It was found that all subgroups of NAC
proteins were sharing a highly conserved sequence at the
NAC domain in the N-terminal region and some unknown
motifs in the C-terminal region.
Generation of Transgenic Plants
The binary vector (pC2301:35S: nptII: MuNAC4: GUS)
construct (Fig. 1b) was initially confirmed by restriction
analysis. Then, the binary vector plasmid was mobilized
into Agrobacterium strain EHA105 by electroporation and
use for MuNAC4 gene transformation into groundnut
seedlings by Agrobacterium-mediated method. Primary
transformants of groundnut were obtained using the tissue
culture-independent in planta transformation procedure
[34]. The transformants of groundnut T1 seeds were har-
vested from T0, plants were screened for kanamycin
resistance, and we confirmed the transformants. Initially,
fifty T1 seeds were tested for the typical kanamycin resis-
tance symptoms i.e. mainly, leaf chlorosis. Leaves showing
chlorotic symptoms (yellowing) were grouped into resis-
tant, moderately resistant and susceptible. Putative trans-
formants showed normal growth and resistance against
kanamycin and no symptoms of leaf chlorosis were
observed at 200 mg/L kanamycin, while wild type showed
no growth in the presence of kanamycin (200 mg/L)
(Fig. 2). Only resistant and moderately resistant healthy
plants were selected and advanced for next generation.
Molecular Analysis of the Putative MuNAC4
Transformants
The genomic DNA was extracted from both wild-type
plants and putative MuNAC4 transformants (T5 genera-
tion) by CTAB method. Using the genomic DNA as tem-
plate, PCR was performed for nptII (selectable marker) and
Mol Biotechnol
123
GUS. The DNA of wild-type groundnut lines did not give
any amplification for nptII and GUS primers, suggesting
that the wild-type groundnut plants did not carry any nptII
and GUS gene encoding for kanamycin resistance, and the
putative transformants showed that the amplifications of
both (nptII and GUS) genes are 500 bp in size (Fig. 3a, b).
RT-PCR was used to analyse the expression MuNAC4
transcript levels in both wild and transgenics using cDNA
as template. The qRT-PCR result shows that expression of
MuNAC4 transcripts was upregulated in transgenic
groundnut plants as compared to wild-type plants (Fig. 4).
These results suggest the overexpression of MuNAC4 gene
in transgenic groundnut lines.
Overexpression of MuNAC4 in Groundnut Enhances
Tolerance to Drought Stress
In T5 generation, twelve transgenic groundnut plants were
confirmed by PCR analysis and were found to have
enhanced expression of MuNAC4 in all independent
transgenic groundnut plants (Fig. 3a, b). All of the
transgenic groundnut plants had phenotypes similar to
those of wild groundnut plants at different development
stages (data not shown). Forty-day-old wild-type and
transgenic groundnut plants overexpressing MuNAC4 were
exposed to drought stress conditions by withholding water
for 14 days. At the end of second week of drought treat-
ment, all of the wild-type pants displayed severe leaf-
wilting symptoms, whereas the transgenic lines showed
better vigour and greenish growth (Fig. 5) even after 3
weeks of drought stress. To test the drought tolerance of
transgenic groundnut plants overexpressing MuNAC4, we
withheld water from transgenic and wild-type plants for
three successive weeks. We also compared the growth
status of transgenic and wild-type plants under drought
conditions (Table 1). After this drought treatment, the
transgenic seedlings were taller and had better root growth
than the wild-type plants, which displayed an overall
general inhibition of growth. After drought stress, the
transgenic plants had increased lateral roots and growth
than the wild type (Fig. 6), and the transgenic plants
showed more root biomass than wild-type plants under
Fig. 1 a Phylogenetic relationship of MuNAC4 protein. Phylogenetic
tree was derived for MuNAC4 and NAC family protein sequences
using the programme phylogeny.fr tool. The MuNAC4 shows high
homology with GmNAC4 and cluster in other NACs. b The
schematic diagram of MuNAC4 overexpressing vector with the npt
II gene as a screening marker. The MuNAC4 coding region also
includes a gene expression cassette which contains the 35S promoter,
the npt II gene coding region and the 35S polyA region. The entire
expression cassette is flanked by the left border (LB) and right border
(RB) sequences in the pCAMBIA2301 binary vector
Fig. 2 Wild-type and T1 groundnut seeds on MS half-strength medium with kanamycin (200 mg/L). a No germination and growth were
observed in wild-type seeds, b Healthy seedlings with greenish leaves were observed in groundnut T1 transformants
Mol Biotechnol
123
normal conditions and stress treatments. These results
indicate that MuNAC4 may be able to confer transgenic
groundnut plants with an enhanced tolerance to drought
stress.
Overexpressing MuNAC4 Displayed Better Leaf
Relative Water Content, Cell Membrane Stability,
MDA and Total Chlorophyll Content in Transgenic
Groundnut Plants
The relative water contents (RWC) of drought-stressed
plants were measured in wild-type and transgenic plants
grown under normal and drought-stressed conditions
(Fig. 7). We noted a significant reduction in the water
contents of plants, and the reduction became more under
drought stress in wild-type plants. Compared to the wild-
type plants, the transgenic plants had maintained signifi-
cantly higher water content than the wild-type plants under
drought stress conditions. The result suggested that the
water-holding ability of transgenic plant was higher than
that of wild-type plants. Disruption of the membrane
integrity caused by stress can be estimated by measuring
the leakage of cytoplasmic solutes from leaf discs. Under
well-watered conditions, there were no differences in
electrolyte leakage between wild-type and transgenic
plants (Fig. 7). MDA, as the final product of cellular
membrane lipid peroxidation, is a key parameter for eval-
uating the extent of damage in plants. The level of MDA
was increased dramatically by severe drought condition
(Fig. 7). The level of MDA was ranged between 3 to 4
Fig. 3 a : Molecular analysis of
wild-type and T5 transgenic
plants. lane 1 wild-type plant
genomic DNA; lane 3 1 kb
molecular marker; lane 2 and 4–
12: Amplification PCR products
of nptII with genomic DNA
template from T5 transgenic
lines. b: Molecular analysis of
wild-type and T5 transgenic
plants. Lane 1 wild-type plant;
lane 3 100 bp molecular
marker; lanes 2 and 4–12:
Amplification PCR products of
GUS with genomic DNA
template from T5 transgenic
plants
Fig. 4 qRT-PCR expression analysis of MuNAC4 in response to
drought stress at different time intervals. Forty-day-old plants of both
sets were subjected to drought stress by withholding water supply.
Total RNA was isolated from leaves collected at different time points
(0, 6, 12, 24 and 48 h). The 2-DDCt method was used to measure the
relative expression levels of the MuNAC4 gene in wild-type and
transgenic lines under drought-stressed conditions. All RT-PCR
expression assays were performed and analysed at three times in
independent biological experiments. Error bars represent standard
error of the mean. Actin transcripts used as internal control
Mol Biotechnol
123
times higher of control in transgenic plants under drought
stress, whereas in the same condition, wild-type plants
were about 5 times higher of control conditions. There
were no significant differences in the CMS and MDA
levels between wild-type and transgenic plants under nor-
mal conditions. These results indicated that MuNAC4
overexpression in groundnut could alleviate cell membrane
injury caused under drought stress. Similarly, the total
chlorophyll content was measured in leaves of both wild-
type and transgenic plants under normal and drought con-
ditions (Fig. 7). In general, the chlorophyll content
decreased both in wild-type and transgenic plants due to
drought treatments. However, the total chlorophyll content
was significantly higher in transgenic plants than the wild-
type plants under drought stress conditions.
Overexpressing MuNAC4 in Groundnut Increases
Accumulation of Soluble Sugars and Proline Under
Drought Stress
It has been reported that osmolytes play crucial role in
maintaining osmotic homoeostasis in plants and adaptation
enhancing plant tolerance to diverse environmental stresses
and well established that osmolytes such as soluble sugars
and proline are involved in osmotic adjustment in plants as
crucial factors, playing adaptive roles in enhancing plant
tolerance to a wide range of abiotic stresses [35]. Our data
reveal that transgenic groundnut carrying MuNAC4
enhanced the tolerance to drought stress, suggesting that
osmolyte accumulation may participate in enhancing the
stress tolerance of transgenic groundnut. To address this
hypothesis, we determined the soluble sugars and free
proline in wild-type and transgenic groundnut plants under
normal and stressed conditions. Our results show that the
soluble sugar contents in transgenic lines were about 1.5-
fold higher than those in wild-type plants under normal
conditions (Fig. 7). Under drought stress conditions, the
soluble sugar contents were increased by 0.5- and 3.5-fold
in wild-type and transgenic lines, respectively, compared to
their respective plants grown under normal conditions
(Fig. 3a), suggesting that MuNAC4 can participate in
regulating the accumulation of soluble sugars in transgenic
groundnut under both drought and normal conditions. In
contrast to the changes in soluble sugar, there was no
Fig. 5 Overexpression of MuNAC4 enhanced drought stress toler-
ance in groundnut. Severe leaf wilting was seen in wild-type plants
and stay-greenish leaves in transgenic groundnut plants
Table 1 Shoot growth, lateral root growth and root biomass of wild-type and transgenic plants (mean of 6 plants) under normal and drought
conditions
Parameters Wild-type plants Transgenic plants
Normal Drought stressed Normal Drought stressed
Shoot growth (cm) 18.07a ± 1.2 17.03a ± 1.4 28.06b ± 2.3 35.32c ± 3.5
Lateral root growth (cm) 27.54a ± 1.62 38.27b ± 1.8 34.39c ± 3.1 68.25d ± 3.6
Root biomass (g) 0.857a ± 0.2 0.923a ± 0.25 1.046b ± 0.5 1.825c ± 0.4
Data are expressed as means of three independent experiments with 6 plants each, and error bars indicate SD. The mean values in a row followed
by a different letter for each treatment are significantly different (P B 0.05) according to Duncan’s multiple range test
Fig. 6 Variation of root growth between wild-type and transgenic
groundnut plants. Profuse lateral root growth in transgenic groundnut
compared to wild-type plants
Mol Biotechnol
123
significant difference in the contents of proline between
wild-type and MuNAC4 transgenic plant under normal
conditions. Under drought stress conditions, the levels of
proline in the transgenic lines were approximately 5.5-fold
higher than those in wild-type plants, whereas it was 1.5-
fold higher in the wild-type plants compared to plants
grown under normal conditions (Fig. 7), implying that
MuNAC4 may promote proline biosynthesis in transgenic
groundnut under drought conditions.
Overexpressing MuNAC4 in Groundnut Increases
Antioxidative Enzyme Efficacy Under Drought Stress
Antioxidant defence systems are well known for scaveng-
ing ROS produced in different stressful conditions, such as
activation of the antioxidant enzymes superoxide dismu-
tase (SOD), ascorbate peroxidase (APX), catalase (CAT),
guaiacol peroxidase (G-POD) and peroxidase (POD) [36,
37]. The activities of APX, SOD and guaiacol peroxidase
(G-POD) significantly increased in groundnut transgenic
plants after exposure to drought stress (Fig. 8). There was
no significant difference in antioxidative enzyme activities
between transgenic and wild-type plants grown under
normal conditions. In general, the activities of SOD, APX
and G-POD increased in both transgenic lines and wild-
type plants. However, drought stress increased the activi-
ties of SOD, APX and G-POD by 2.0-, 4.0- and 2.5-fold,
respectively, in transgenic groundnut plants compared to
wild-type plants. These results indicated that overexpres-
sion of SOD, APX and G-POD in transgenic groundnut
plants enhanced antioxidant enzyme activities and con-
ferred better tolerance to drought stress than wild-type
plants. Therefore, our results indicate that MuNAC4 may
function as a transcriptional regulator and tune the network
Fig. 7 Comparison of physiological indices between transgenics and
wild-type groundnut plants under normal and drought stress condi-
tions. a RWC—relative water content, b CMS—cell membrane
stability, c TCC—total chlorophyll content, d proline content,
e MDA—malondialdehyde levels, and f soluble sugar content
Mol Biotechnol
123
controlling stress-responsive traits. In general, overex-
pression of MuNAC4 in groundnut significantly improved
abiotic stress tolerance with increased root biomass, higher
osmolyte levels, better water retaining capacity, membrane
stability and antioxidant efficacy.
Discussion
Drought stress is one of the most limiting environmental
factors and adversely affects all aspects of plant phenology
and metabolism leading to yield loss. To overcome this
enormous issue, many research efforts have been made to
enhance the drought tolerance of crop varieties through
both plant breeding and biotechnological approaches.
Transcription factors are powerful tools in the engineering
of crop plants with enhanced tolerance to environmental
stresses, as their transgenic application can lead to the up-
regulation of a series of stress-related genes [38]. The NAC
transcription factors have been characterized for their roles
in plant growth, development and stress tolerance [39–41].
Several investigators reported that a subfamily of NAC
transcription factors played a pivotal role in various abiotic
stresses including salinity, drought and low temperature
[17, 42]. In earlier studies, we reported that the overex-
pression of a NAC family transcription factor from
horsegram, MuNAC4 gene, enhances tolerance to multiple
stresses, such as PEG, NaCl and CuSO4 in bacterial cells
[43]. In the study reported here, we demonstrated that
ectopic expression of MuNAC4 gene enhances groundnut
tolerance to drought stress by improved lateral root growth,
accumulation of osmolytes and better antioxidative enzy-
matic defence. These results highlight the potential of
MuNAC4 as a useful transcriptional regulator in engi-
neering plant tolerance to abiotic stresses. Recent reports
[44] on OsNAC5 and OsNAC10 overexpressing in rice
caused enlarged roots and enhanced drought tolerance
under field drought conditions. These results suggested that
the OsNAC5 and OsNAC10 may confer drought resistance
through the altered root architecture. In our study, the lat-
eral root growth and root biomass of transgenic lines were
higher than that of the wild type under drought stress. A
longer root system should have facilitated water absorption
from deeper soils and thus strengthened drought tolerance
and increased biomass under water-deficit conditions as
evidenced earlier [45]. The overexpression of MuNAC4
in groundnut significantly increased root development
(Fig. 6), which suggested that the development of larger
roots should be favourable for drought resistance breeding.
Several reports revealed that the plant NAC family plays
critical role in responses to abiotic stresses [13, 46–48].
ANAC019, ANAC055 and ANAC072 confer enhanced
tolerance to drought stress [6]. SNAC1, OsNAC10 and
OsNAC5 confer significant enhancement of drought and
ABA tolerance [8, 16, 49]. Overexpression of SNAC2,
OsNAC6, OsNAC045 and OsNAC063 results in enhanced
tolerance to multiple abiotic stresses [17–19]. An AtNAC2
gene confers enhanced tolerance to auxin, ethylene [22]
conditions. Overexpression of the GmNAC11 and
GmNAC20 genes enhances transgenic soybean tolerance to
drought, salinity and freezing stress [50]. In this study, the
expression of MuNAC4 under prolonged drought stress
was assessed, and overexpression led to enhanced tolerance
to drought stress groundnut. Both morpho-physiological
evidences strongly demonstrated that the transgenic
Fig. 8 Analysis of antioxidant enzyme activities between wild type
(WL) and transgenics (TGL1–TGL12) under normal and drought
stress conditions. a Ascorbate peroxidase (APX) enzyme activity
assay. b Superoxidase dismutase (SOD) enzyme activity assay.
c Guaiacol peroxidase (G-POD) enzyme activity assay. Data indicate
means and standard errors of three biological replicates
Mol Biotechnol
123
groundnut lines were more tolerant to drought stress than
wild-type plants. Groundnut transgenics overexpressing
MuNAC4 plants performed better than wild-type plants
under drought stress. More importantly, transgenic
MuNAC4 groundnut showed no significant differences
from the wild-type plants in all parameters studied under
normal condition, which is in accordance with the results in
transgenic rice [16]. Transgenic groundnut plants delayed
leaf wilting and maintained growth compared to wild-type
plants during the drought stress. After 3 weeks of drought
stress, transgenic groundnut showed better vigour and
greenish growth, which implied MuNAC4 in improving
agronomic traits in groundnut (Figs. 5, 6). Several reports
have indicated that NAC TFs play an important role in
water stress responses [51, 52]. In Arabidopsis, overex-
pression of the dehydration-inducible genes ANAC019,
ANAC055 and ANAC072 improved water stress tolerance
[6]. The transgenic rice overexpressing a drought-inducible
SNAC1 gene could maintain leaf turgor and better spikelet
fertility under drought stress. Arabidopsis NAC1 and At-
NAC2 transcription factors are known to stimulate lateral
root development through auxin signalling [10, 24]. Simi-
larly, AtNAC2 was up-regulated by ABA and salinity
treatments, suggesting the interaction of root development
and osmotic stress. Recently, in Arabidopsis, it was
reported that a stress-inducible GmNAC3 and 4 stimulated
lateral root development and stress tolerance. In the present
study, we demonstrated that MuNAC4 was induced by
drought stress and suggest that MuNAC4 transcription
factor has a role in regulation of plant root development in
response to environmental stress.
Osmotic adjustment is a fundamental cellular tolerance
response to drought stress and could be linked to the
accumulation of osmoprotectants such as soluble sugars,
proline, glycine betaines, other QACs, polyamines, etc.
The osmotic potential is a direct reflection of the osmotic
adjustment capability at the physiological level and has
been used as an effective index for assessing crop geno-
types for osmotic stress tolerance. It has been reported that
soluble sugars and proline are involved in osmotic adjust-
ment in plants as crucial factors, playing adaptive roles in
enhancing plant tolerance to a wide range of environmental
stresses [35]. Proline contributes to osmotic adjustment and
the protection of macromolecules during dehydration [53]
and as a hydroxyl radical scavenger. Previous studies have
found that plants may enhance stress tolerance by accu-
mulating osmolytes to adjust the osmotic potential and
protect cell structures [35, 53]. Our results showed that the
contents of free proline and soluble sugars in the transgenic
lines were higher than that of wild-type groundnut plants
under drought stress conditions (Fig. 7), suggesting that
MuNAC4 can regulate free proline and soluble sugar bio-
synthesis. Proline has been reported to play important roles
in maintaining cellular osmotic adjustment and balancing
cell redox status, and scavenging reactive oxygen species
thereby confers stress tolerance [54, 55].
The relative water content of transgenic groundnut was
significantly higher than that of wild-type groundnut plants
under drought treatment, indicating that MuNAC4 could
help to prevent water loss in plants. Cell membrane sta-
bility, which is an indicator of the capacity to protect the
plasma membrane integrity under stress, was also signifi-
cantly lower in transgenic groundnut plants, demonstrating
that MuNAC4 gene reduced damage to cell membranes in
transgenic plants. In this study, we observed no significant
difference in the RWC in both normal and drought con-
ditions of transgenic and wild types. CMS is a major
component of abiotic stress tolerance in plants under stress
conditions. CMS was used for assessing tolerance to des-
iccation, temperature and freezing stresses [56]. In this
study, the CMS is significantly lower in transgenic plants
and wild-type plants under drought stress, demonstrating
less membrane damage caused by overexpression of
MuNAC4. Since CMS has a positive relationship with
several physiological and biochemical parameters [57], it
was predicted that MuNAC4 transgenic plants might have
strong capacities to tolerate abiotic stresses, as evidenced
in groundnut (Fig. 7). Both group plants subjected to
drought stress decreased with increasing water stress over
wild-type plants, but the percent decrease was higher in
wild-type plants than transgenic plants.
Drought stress induces oxidative stress through genera-
tion of ROS, and the ability of the plant to mobilize
enzymatic defence against uncontrolled production of ROS
may be an important facet of their drought tolerance [58].
Many previous works proved that transgenic plants with
elevated levels of chloroplast-targeted antioxidant genes
such as SOD, APX, CAT and GR exhibited enhanced
protection against oxidative stress [36]. In this work,
drought deficit stress significantly increased the activities
of all the scavenging enzymes (SOD, APX and G-POX) in
transgenic plants. The SOD and APX appeared to be
important enzymes for overcoming drought-induced oxi-
dative stress as these enzymes could be the first line of
defence during drought acclimation process. During
drought stress, a relatively less increase of MDA produc-
tion was observed in transgenic groundnut plants than wild-
type plants. Further, SOD, APX and G-POD activities
increased significantly in transgenic plants after drought
stress (Fig. 8). These results led us to hypothesize that
constitutive expression of MuNAC4 improves cell mem-
brane integrity in transgenic plants. In this study, we
obtained evidence that the overexpression of MuNAC4 in
groundnut was able to increase root biomass and the levels
of soluble sugars, free proline, RWC, CMS, TCC and
MDA coupled with antioxidant efficacy resulting in
Mol Biotechnol
123
transgenic groundnut with an enhanced tolerance to
drought stress conditions. These physiological alterations
may lead to the maintenance of normal osmotic regulation
or the balancing of cell redox status in transgenic
groundnut plants and, consequently, the enhanced toler-
ance to drought stress. A detail picture of the regulatory
mechanism of MuNAC4 in groundnut is not yet clear.
Consequently, it would be meaningful to explore more
targeted genes involved in the processes of stress response
in groundnut by the microarray and proteomic approaches.
In summary, our findings suggest that MuNAC4 is an
important transcriptional regulator in engineering crops to
improve tolerance to abiotic stresses.
Acknowledgments This research work was supported in a form of
research grant (SR/SO/PS/0001/2011) to CS by Department of Sci-
ence & Technology, SERB, Govt of India, New Delhi which is being
greatly acknowledged.
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