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,

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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

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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

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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

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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

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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

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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

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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

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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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