Overexpression of EaDREB2 and pyramiding of EaDREB2 with the pea DNA helicase gene (PDH45) enhance...

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Plant Cell Reports ISSN 0721-7714Volume 34Number 2 Plant Cell Rep (2015) 34:247-263DOI 10.1007/s00299-014-1704-6

Overexpression of EaDREB2 andpyramiding of EaDREB2 with the peaDNA helicase gene (PDH45) enhancedrought and salinity tolerance in sugarcane(Saccharum spp. hybrid)Sruthy Maria Augustine, J. AshwinNarayan, Divya P. Syamaladevi,C. Appunu, M. Chakravarthi,V. Ravichandran, Narendra Tuteja, et al.

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ORIGINAL PAPER

Overexpression of EaDREB2 and pyramiding of EaDREB2with the pea DNA helicase gene (PDH45) enhance droughtand salinity tolerance in sugarcane (Saccharum spp. hybrid)

Sruthy Maria Augustine • J. Ashwin Narayan •

Divya P. Syamaladevi • C. Appunu • M. Chakravarthi •

V. Ravichandran • Narendra Tuteja • N. Subramonian

Received: 9 July 2014 / Revised: 16 October 2014 / Accepted: 5 November 2014 / Published online: 5 December 2014

� Springer-Verlag Berlin Heidelberg 2014

Abstract

Key message EaDREB2 overexpressed in sugarcane

enhanced tolerance to drought and salinity. When co-

transformed with plant DNA helicase gene, DREB2

showed greater level of salinity tolerance than in single-

gene transgenics.

Abstract Drought is one of the most challenging agri-

cultural issues limiting sustainable sugarcane production

and can potentially cause up to 50 % yield loss. DREB

proteins play a vital regulatory role in abiotic stress toler-

ance in plants. We previously reported that expression of

EaDREB2 is enhanced by drought stress in Erianthus ar-

undinaceus. In this study, we have isolated the DREB2

gene from E. arundinaceus, transformed one of the most

popular sugarcane variety Co 86032 in tropical India with

EaDREB2 through Agrobacterium-mediated transforma-

tion, pyramided the EaDREB2 gene with the gene coding

for PDH45 driven by Port Ubi 2.3 promoter through par-

ticle bombardment and evaluated the V1 transgenics for

soil deficit moisture and salinity stresses. Soil moisture

stress was imposed at the tillering phase by withholding

irrigation. Physiological, molecular and morphological

parameters were used to assess drought tolerance. Salinity

tolerance was assessed through leaf disc senescence and

bud sprout assays under salinity stress. Our results indicate

that overexpression of EaDREB2 in sugarcane enhances

drought and salinity tolerance to a greater extent than the

untransformed control plants. This is the first report of the

co-transformation of EaDREB2 and PDH45 which shows

higher salinity tolerance but lower drought tolerance than

EaDREB2 alone. The present study seems to suggest that,

for combining drought and salinity tolerance together, co-

transformation is a better approach.

Keywords Sugarcane transformation � DREB2 � PDH45 �Erianthus arundinaceus � Drought tolerance � Salinity

tolerance

Introduction

Sugarcane (Saccharum spp. hybrid) is an important com-

mercial crop in India with diverse uses. One of the major

limitations in sugarcane cultivation is the frequent occur-

rence of drought, salinity, extreme temperatures, etc.

Enhanced tolerance to multiple stresses is an important

target for improving the performance of the crop in the

field. DREB genes are the first discovered families of

transcription factors related to abiotic stress gene regula-

tion (Moran et al. 1994). DREB2 is normally induced by

Communicated by Prakash Lakshmanan.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00299-014-1704-6) contains supplementarymaterial, which is available to authorized users.

S. M. Augustine � J. Ashwin Narayan � C. Appunu �M. Chakravarthi � N. Subramonian (&)

Sugarcane Breeding Institute (ICAR), Coimbatore, Tamil Nadu,

India

e-mail: [email protected]

D. P. Syamaladevi

Indian Grass and Fodder Research Institute Regional Station,

Avikanagar, Rajasthan, India

V. Ravichandran

Department of Rice, Tamil Nadu Agricultural University,

Coimbatore, Tamil Nadu, India

N. Tuteja

International Centre for Genetic Engineering and Biotechnology,

New Delhi, India

123

Plant Cell Rep (2015) 34:247–263

DOI 10.1007/s00299-014-1704-6

Author's personal copy

http://dx.doi.org/10.1007/s00299-014-1704-6

water stress, high salinity and heat shock (Liu et al. 1998;

Kasuga et al. 1999; Nakashima et al. 2000; Sakuma et al.

2006) and its overexpression activates the expression of

genes possessing a CRT/DRE cis-element (Stockinger

et al. 1997; Liu et al. 1998). These genes play a crucial role

in providing tolerance to multiple stresses and display

overlapping responses to different stress conditions.

DREBs control the expression of stress-responsive genes

via the ABA-independent pathway in abiotic stress. In

Arabidopsis, overexpression of DREB2 driven by a con-

stitutive promoter improved tolerance to drought, salinity

and cold without growth retardation (Liu et al. 1998).

Recently, we reported a twofold increase in the relative

expression of DREB2 under water stress in Erianthus ar-

undinaceus compared to the moderately drought-tolerant

sugarcane cultivar Co 86032 (Augustine et al. 2014). Reis

et al. (2014) have recently reported the overexpression of

AtDREB2A in sugarcane imparting drought tolerance, and

the current paper is perhaps the first report on the salinity

tolerance of DREB2 in sugarcane.

Genetic engineering of plants for enhanced drought

tolerance is mostly based on the manipulation of both

transcription and signaling factors or genes that directly

protect the cells against water deficit (Valliyodan et al.

2006). The highly conserved domains in DREB2 proteins

are important for their specific biological functions, and

identifying such critical domains will help in achieving

efficient crop improvement strategies through genetic

engineering. The plants overexpressing DREBs are repor-

ted to impart tolerance to drought, salinity and/or cold in

combination with different promoters (Latha and Prasad

2011). DREB2 is the major transcription factor that binds

to the cis-acting elements of most of the osmotic-stress-

inducible genes responsible for osmotolerance to the plants

under stress conditions (Hussain et al. 2011). Transgenic

tobacco plants overexpressing PgDREB2 (Pennisetum

glaucum) showed enhanced tolerance to both hyperionic

and hyperosmotic stresses (Kasuga et al. 2004). Overex-

pression of PeDREB2 (Populus euphratica) in Arabidopsis

enhanced drought and salinity tolerance (Chen et al. 2009).

Similarly, overexpression of the PgDREB2 and GmDREB2

(Glycine max) separately in tobacco enhanced salt and

osmotic tolerance (Chen et al. 2007; Agarwal et al. 2010).

Rice plants transformed with SbDREB2 (Sorghum bicolor)

improved yield and drought tolerance under water limita-

tion (Bihani et al. 2011).

Helicases are a ubiquitous group of motor proteins

essential for all living organisms. The pea DNA helicase 45

(PDH45), the first plant nuclear DNA helicase, was over-

expressed and purified from a bacterial system (Pham et al.

2000). Studies have shown that PDH45 is upregulated in

pea seedlings under high salt (200 mM of NaCl), and

overexpression of the gene conferred salinity tolerance in

tobacco (Sanan Mishra et al. 2005). The PDH45 gene is

also upregulated by other abiotic stresses including dehy-

dration, wounding and low temperature (Sanan Mishra

et al. 2005; Manjulatha et al. 2014).

To restore cellular functions and induce stress tolerance

in plants, transfer of a single gene encoding a specific stress

protein may not be sufficient to achieve the required tol-

erance levels (Bohnert et al. 1995). This constraint can be

overcome by the two promising approaches, namely

overexpression of a stress inducible transcription factor

that regulates a number of other genes, and pyramiding of

one or more genes (Yamaguchi-Shinozaki and Shinozaki

1994; Chinnusamy et al. 2005). Hence, with a view to

assessing the expression of the abiotic stress tolerance in

the single-gene and double-gene transforments, we have

isolated the gene coding for the transcription factor DREB2

from E. arundinaceus (IK 76-81), transformed the sugar-

cane variety Co 86032 with DREB2 alone and pyramided it

with PDH45 gene and evaluated the transgenics for drought

and salinity tolerance through different morphological,

physiological and molecular parameters.

Materials and methods

Vector construction and generation of transgenics

The DREB2 gene was isolated from E. arundinaceus (IK

76-81) using gene-specific primers designed from Sorghum

bicolor (NCBI accession no. EU500654) and Zea mays

(NCBI accession no. NM 001158997) through polymerase

chain reaction (PCR) (1 cycle at 94 �C for 5 min, followed

by 35 cycles of 94 �C for 45 s, 61.5 �C for 30 s and 72 �C

for 2 min). The pCAMBIA1305.1 vector was restricted

with BamHI and NcoI restriction enzymes to release the

CaMV 35S promoter driving the GUS gene and to insert

Port Ubi 2.3 promoter (Phillip et al. 2013). Port Ubi 2.3

promoter was amplified from pPORT-Ubi-GFP (Phillip

et al. 2013) using two specific primers: forward, 50-GAT

CGGATCCACTATCACCCTCGAGGTG-30 (BamHI site

underlined) and reverse, 50-GATCCCATGGCTGCAAGA

AATAATCACCAA-30 (NcoI site underlined). The newly

constructed plasmid was named pPORT-UBI2.3-GUS.

After sequencing and confirmation, the full-length

sequence of EaDREB2 was amplified with two specific

primers: forward, 50-GATTACTAGTATGGAGCTGGGA

GACGC-30 (SpeI site underlined) and reverse, 50-GAT

TGCTAGCCTAATATGCAAAAAGGCTAAACCCA-30

(NheI site underlined), and cloned into pPORT-UBI2.3-

GUS in place of the GUS gene. The resultant construct

pSBI-DREB2 (Fig. 1a) was transferred to Agrobacterium

tumefaciens LBA4404 by the freeze–thaw method (Sam-

brook and Russel 1989). For Agrobacterium-mediated

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transformation in sugarcane variety Co 86032, the method

described by Arvinth et al. (2010) was followed, and

transgenic plants were selected on hygromycin (50 mg/l)

selection medium.

The full-length PDH45 gene was amplified from

pRT101 vector (Sanan Mishra et al. 2005) obtained from

Dr. Narendra Tuteja, ICGEB, New Delhi, India, using two

specific primers: forward, 50-GATTCCATGGATGGCGA

CAACTTCTGTGG-30 (NcoI site underlined) and reverse,

50-GGCCCGCTAGCTTATATAAGATCACCAATATTC-

30 (NheI site underlined), and cloned into pPORT-UBI2.3-

GUS in place of GUS and the resultant construct was

named pSBI-PDH45 (Fig. 1b). pSBI-PDH45 was co-

transferred with pSBI-DREB2 to sugarcane variety Co

Fig. 1 Schematic diagram of

the plant expression vectors

a pSBI-DREB2 and b pSBI-

PDH45

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86032 through particle bombardment using the method

described by Bower and Birch (1992). The transgenic

plants were selected on hygromycin (50 mg/l) selection

medium.

Molecular analyses and selection of transgenic events

Standard PCR technique was used to detect the presence of

the transgene in regenerated putative transgenic sugarcane

plantlets. The promoter-gene fusion primer sequences

50-GTAGCCTAGTTCTTGCTTGGCAT-30 and 50-GTG

TCGGAAACTACTCCTAGCC-30 for EaDREB2 and 50-GCATGTGTGAATGGTGCGATTTG-30 and 50-AGGAG

TTCCAGACACCACGTGAAC-30 for PDH45 were used

for PCR reactions. Plants with the expected size amplicon

were selected for further experiments. Further confirmation

was done with the marker gene (Hpt) using primers 50-GATCTCCAATCTGCGGGATC-30 and 50-ACTCACCG

CGACGTCTGTCG-30 to generate the 800 bp product.

Plant material and stress treatments

Twenty-five independent EaDREB2 transgenic events and

22 co-transformed independent transgenic events (EaD-

REB2 and PDH45) of sugarcane along with the wild-type

(WT-untransformed control sugarcane variety Co 86032)

plants were vegetatively multiplied from V0 plants, thus

taking the events to V1 stage. Five biological replicates for

each event along with the WT plants were planted in 16’’

pots (containing soil, sand and farmyard manure in 1:1:1

ratio) and maintained in a greenhouse for moisture-stress

screening. The pots were arranged in the greenhouse in a

completely randomized design and irrigated daily for

119 days.

Water stress was imposed on the plants at the tillering

phase (120 days after planting) by withholding irrigation

for 10 days and was released on the 11th day, and normal

irrigation was continued. Relative water content and cell

membrane thermostability tests were carried out on the

0th and 10th day of drought induction. Gene expression

and gas-exchange parameters were recorded on the

10th day after drought induction. Chlorophyll content and

photosynthetic efficiency (Fv/Fm) were recorded on the

0th and 10th day after withholding the irrigation and on

15th day after the release of drought (re-irrigation). Soil

samples were collected from five randomly selected pots

at 10, 20 and 30 cm depth using an auger, and moisture

was estimated by gravimetric method using a moisture

analyzer (A&D model Mx-50). Salinity tolerance was

assessed through leaf disc and bud germination assays.

All the observations were recorded from five biological

and three technical replications of both transgenics and

WT.

Visual scoring

The transgenic events along with the WT were visually

observed for wilting of leaves on the 5th and 10th day after

drought induction. Leaf wilting was scored on a scale of

1–4 modified from Engelbrecht et al. (2007) as: 1, no

wilting; 2, slightly wilting; 3, wilting, wherein the plant

showed leaf wilting only during hot hours from which the

leaves recovered; and 4, severe wilting, wherein wilted

leaves did not recover. The mean score was computed from

five plants from each event.

Cell-membrane thermostability

Cell-membrane injury is regarded as an indicatior of the

ability of the plant to tolerate drought. To estimate the cell-

membrane injury percentage, the cell-membrane thermo-

stability test was carried out in both V0 and V1 generations

following the method described by Martineau et al. (1979).

For V0 generation, third fully opened leaves of 120-day-old

plants grown under normal irrigated conditions were used,

and for V1 generation leaves were collected on 0th and

10th day after drought induction. Leaf discs (0.5 cm

diameter) were made up to 200 mg, washed thrice with

distilled water and finally 20 ml of distilled water added to

each control and treatment tubes (2.5 cm 9 15 cm). The

tubes were covered with aluminium foil, incubated at

60 �C in a thermostatically controlled water bath for

20 min and placed at 10 �C for 12 h to allow the diffusion

of electrolytes into the water. After recording the initial

conductance at 30 �C, the tubes were heated at 100 �C for

20 min and final conductance was recorded after cooling.

Membrane Injury percentage was computed using the fol-

lowing formula:

Membrane injury % ¼ 1� 1� T1=T2ð Þ = 1�C1=C2ð Þ½ �� 100

where T and C refer to the values for treatment and control

samples, and the subscripts 1 and 2 denote the initial and

final conductance, respectively.

Leaf water status or relative water content (RWC)

Fully opened third leaves in the whorl from 120-day-old

transgenics and WT plants were subjected to measurement

of leaf relative water content (RWC) on the 0th and

10th day after drought induction. Leaf RWC was calcu-

lated based on fresh (FW), turgid (TW) and dry weights of

0.2 g leaf samples. After recording the fresh weights of

excised leaves, sample leaf discs were soaked in de-ionized

water for 4 h at room temperature in a closed petri dish,

blot-dried and weighed for TW. Samples were then dried at

90 �C for 72 h and the dry weight was recorded. RWC was

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computed using the formula of Barrs and Weatherley

(1962): RWC ¼ FW�DWð Þ= TW�DWð Þ½ � � 100:

Gas-exchange parameters

Gas-exchange parameters, i.e., photosynthesis rate (A),

stomatal conductance (gs) and transpiration rate (E), were

recorded from the middle part of fully opened third leaf on

the 10th day of drought induction (soil moisture & 8.1 %)

using a portable photosynthesis system (Li-6400, Li-COR

Inc., Lincoln, Nebraska, USA) with a leaf chamber of

2 cm 9 3 cm and an integrated light source (LI-6400-

02B). Gas-exchange parameters were measured at

130 lmol m-2 s-1 [CO2], 40 �C (closed cabinet incubated

to 40 �C) and RH *60–70 %. The external CO2 concen-

tration in air was maintained at 380 lmol mol-1 in the

reference cuvette. All the gas-exchange parameters were

measured at a leaf temperature of 32 ± 2.0 �C at 30 s

intervals over a period of 2 h from 9.00 a.m. to 11.00 a.m.

Chlorophyll measurements

Total chlorophyll content was recorded on the 0th and

10th day after drought induction (soil moisture 25 and

8.1 %, respectively), and 15 days after re-irrigation (25 %

soil moisture) using a chlorophyll meter (SPAD-502, Ko-

nica Minolta, Japan).

Photosynthetic efficiency (Fv/Fm)

The maximum potential photochemical efficiency, defined

as the ratio of variable to maximum fluorescence emitted

by chlorophyll (Fv/Fm), was estimated using a portable

OS-30P chlorophyll fluorimeter (Optisciences, USA).

Photosynthetic efficiency (Fv/Fm) was recorded on the 0th

and 10th day after drought induction and 15 days after the

release of drought. The plants were dark-adapted for

20 min prior to measurement. Maximal fluorescence under

light exposure (Fm0) was obtained by imposing a 1 s sat-

urating flash to the leaf to reduce the entire PS II reaction

centre after attaining steady-state fluorescence (Ft). The

minimal fluorescence immediately after light exposure

(Fo0) was determined by imposing dark while a far red light

was simultaneously switched on to oxidize PS II rapidly by

drawing electrons from PS II to PS I.

Gene expression analysis using quantitative real-time

polymerase chain reaction (qPCR)

To elucidate the role of transgenes, the relative expression

of six abiotic stress (salt, cold and heat) responsive genes,

i.e. RD29 (response to dehydration), LEA (late embryo-

genesis abundant protein), ERF (ethylene responsive

factor), COR15 (cold regulated protein), ERD (early

responsive to dehydration) and BRICK/HSPC300, was

assessed along with the transgenes. Five transgenic events,

each selected from the EaDREB2 and co-transformed

events based on the physiological parameters, were used

for gene expression analysis. Total RNA was isolated from

the third fully opened leaf sample using Trizol reagent

(Sigma Chemicals, USA) following the method of Chom-

czynski and Mackey (1995). First-strand cDNAs were

synthesized from total RNA using the Fermentas first-

strand cDNA synthesis kit and oligo (dT) primers (Fer-

mentas International Inc, Ontario, Canada) following the

manufacturer’s instructions. The actin transcript was used

as an internal control to quantify the relative transcript

levels. To make sure that actin gene did not respond to

drought stress, the expression of the gene was determined

in both irrigated and drought-stressed samples. cDNA

fragments and actin gene were amplified with gene-specific

Table 1 Visual scoring of pSBI-DREB2 and co-transformed trans-

genic events for soil moisture stress

Event no. Score after stress induction

DREB2 transgenics Co-transformed events

5th day 10th day 5th day 10th day

1 1 3 1 3

2 1 2 1 2

3 1 1 1 1

4 1 1 1 2

5 2 3 2 2

6 2 1 1 2

7 1 1 1 3

8 1 1 1 1

9 1 2 1 3

10 1 4 1 2

11 2 2 1 3

12 1 2 1 3

13 1 2 1 3

14 1 1 2 2

15 1 3 1 3

16 1 2 2 2

17 1 1 1 1

18 2 2 2 1

19 2 2 2 1

20 1 3 1 1

21 1 2 1 1

22 1 2 1 1

23 1 3 – –

24 1 1 – –

25 1 4 – –

WT 3 4 3 4

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primers (Table 1, supplementary data). All PCRs were

performed for 40 cycles (1 cycle at 95 �C for 10 min fol-

lowed by 40 cycles of 95 �C for 15 s and 60 �C for 1 min).

qPCR was performed using MESAGREEN master mix and

StepOne real-time PCR system (Applied Biosystems,

Burlington, ON, Canada). The CT values for both the target

and internal control genes were used for the quantification

of transcripts by comparative CT method normalization.

Later, the products were analysed through a melt-curve

analysis to check the specificity of PCR amplification. Each

reaction was performed three times, and the expression of

target gene was calculated using the formula (Livak and

Schmittgen 2001): 2-DDCt, where DDCt = DCt sample-

DCt actin. DDCt values reflect the relative expression of

the target gene upon exposure to drought stress.

Leaf disc senescence assay for measuring salinity

tolerance

Leaf disc assay was carried out to evaluate the sensitivity

of the transformed and WT plants to sodium chloride

(NaCl) stress as described by Fan et al. (1997). Two grams

of fresh leaf sample (3.0 cm 9 2.0 cm) was excised from

healthy and fully expanded third leaves of 120-day-old

plants. The discs were floated in a 10 ml solution of

250 mM NaCl and experimental control (0 mM NaCl) for

72 h and then used for measuring chlorophyll content

spectrophotometrically after extraction in methanol

(Crafts-Brandner et al. 1984). The damage caused by stress

was reflected in the degree of bleaching observed in the

leaf tissue after 72 h. The treatment was carried out in

continuous white light at 25 ± 2 �C.

Bud germination assay

Ten independent transgenic events were selected for bud

germination assay based on the leaf disc senescence assay.

Single bud setts were planted in cavity trays and grown at

30 �C temperature and 60 % relative humidity. To evaluate

germination under salt stress, single bud setts were irri-

gated daily with 25 ml of 0 mM, 100 mM (5.844 g/l),

200 mM (11.688 g/l) and 300 mM NaCl (17.532 g/l)

solutions for a period of 26 days. The number of buds

germinated and their shoots height were recorded on the

26th day of planting.

Statistical analysis

For statistical analysis of the data, five biological and three

technical replicates from each of the transgenic events were

used. Student’s t test (P B 0.05) was conducted using

XLSTAT 2013.5 to analyse the data under normal and

stressed conditions also to compare the EaDREB2 trans-

genics with co-transformed events.

Results

Isolation of DREB2 gene from E. arundinaceus

and generation of transgenics

The DREB2 gene (1.5 Kb) was isolated from E. arundin-

aceus and the sequence was submitted to Gen Bank

(accession no.KJ670161) with the name EaDREB2. The

isolated DREB2 gene featured a 702-bp-long intron flanked

by a 70-bp-long exon sequence at the 50 end and a 722-bp-

long exon in the 30 end. The isolated gene sequence had

high sequence homology (98–100 %) with the reported

DREB2 gene sequences of Sorghum bicolor and Zea mays.

The translated amino acid sequence showed high homol-

ogy (95–98 %) with other reported DREB2 protein

sequences.

The hygromycin-resistant sugarcane transformants

were confirmed through PCR using primers of promoter-

gene fusion and Hpt marker gene. In promoter-gene

fusion primer screening, the expected amplicon size of

1 kb for DREB2 and 1.2 kb for PDH45 were obtained.

Twenty-five events in EaDREB2 and 22 events in co-

transformed events were positive for the transgene inte-

gration. The morphological and growth characteristics of

transgenic plants were similar to those of the WT plants

(Fig. 2a, c).

Cell-membrane thermostability in V0 transgenics

The standard test for cell-membrane thermostability was

performed in the V0 EaDREB2 and co-transformed trans-

genic events (Fig. 3a). Significantly lower membrane

injury, which indicates higher cell-membrane stability, was

observed in all the EaDREB2 transgenic events

(43–85.03 %) than in WT. As against 95.5 % injury in WT,

56 % of transgenic events (14 out of 25) showed injury

percentage of 50–60 % with the lowest (43.24 %) in the

eighth event and the highest (85.03 %) in the tenth event

(Fig. 3a). In co-transformed events, too, membrane ther-

mostability was significantly higher with a range of

42.37–95.45 %. Sixty-three percent (14 out of 22) of the

events had membrane injury of 50–75 % with the lowest

injury (42.37 %) in the 11th event. The lowest stability was

in the 15th event with 95.45 % membrane injury (Fig. 3b).

There was a significant difference in cell-membrane ther-

mostability between EaDREB2 transgenics and co-trans-

formed events.

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Morphological and physiological drought stress

characteristics in V1 plants

The wilting symptoms in each of the transgenics on 5th and

10th day (Fig. 2b, d) were recorded (Table 1). In EaD-

REB2 transgenics, 32 % of plants (8 out of 25) and in the

co-transformed events 36 % of plants (8 out of 22) were

almost normal, showing very little wilting which was rated

as 1, whereas in WT all the plants showed the extreme

wilting, which was scored as four.

There was a significant increase in the membrane sta-

bility of V1 EaDREB2 transgenics with decreasing mois-

ture levels (Fig. 3c), though it varied between events. The

membrane injury varied between 43.07 and 82.58 % at

25 % soil moisture (i.e. with normal irrigation). At 8.1 %

soil moisture, the injury percentage ranged between 26.35

Fig. 2 Screening of transgenic

events for soil moisture stress

and bud germination assay for

salinity stress. a EaDREB2 and

WT at 25 % soil moisture,

b EaDREB2 and WT at 8.1 %

soil moisture, c pSBI-PDH45

and WT at 25 % soil moisture,

d pSBI-PDH45 and WT at

8.1 % soil moisture along with

the WT for soil moisture stress.

Bud germination assay for

e WT, f pSBI-DREB2 and

g pSBI-PDH45

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and 57.14 %. Out of 25 transgenic events screened, 48 %

of the events (12 out of 25) showed lower levels of

membrane injury, i.e., 50–60 % at 25 % soil moisture,

whereas in 68 % of the events (17 out of 25) membrane

injury was between 30 and 50 % at 8.1 % soil moisture,

suggesting enhanced cell-membrane stability under stress

in the transgenic events with the lowest injury percentage

of 26.35 % in the 13th event. A significant increase in the

membrane stability was also observed in co-transformed

events under soil moisture stress. In the co-transformed

events, the membrane injury varied between 39.46 and

95.7 % at 25 % soil moisture (Fig. 3d) and between 24.8

and 84.06 % at 8.1 % soil moisture. Out of the 22 inde-

pendent transgenic events screened, 68 % of the events (15

out of 22) showed reduced levels of membrane injury

(50–80 %) at 25 % soil moisture and 77 % of the events

(17 out of 22) showed 30–50 % injury at 8.1 % soil

moisture. The lowest injury percentage (24.89 %) at 8.1 %

was in the eighth event. At 25 % soil moisture, 5 % of the

transgenic events had the cell-membrane injury close to

that of the WT plants. Cell-membrane injury showed a

decreasing trend with the increasing moisture stress in all

the events studied. However, in WT plants, the membrane

injury increased from 93.2 to 98.71 % when soil moisture

decreased from 25 to 8.1 %. There was no significant

difference in the membrane stability of EaDREB2 trans-

genics and co-transformed events under soil moisture

stress.

Higher relative water content in both EaDREB2 and co-

transformed transgenic events at V1 stage

In EaDREB2 transgenic events, a significantly higher

RWC was observed under moisture stress (Fig. 4a). Four

events exhibited a maximum of 3 % reduction in RWC at

8.1 % soil moisture from the normal irrigated condition.

Fifty-two percent (13 out of 25) of the transgenic events

had maximum of 10 % reduction in RWC, with three

events showing reduced RWC similar to that of the WT,

i.e. 15–30 % reduction from the normal irrigated condition.

In WT, the RWC decreased by 30 % when the soil mois-

ture was reduced from 25 to 8.1 %. In the co-transformed

events, out of 22 independent events screened, 50 % of the

transgenic events (11 out of 22) had a maximum of 10 %

reduction and five events showed a maximum 20 %

reduction in RWC (Fig. 4b). Three events (6, 8, and 9)

exhibited reduced RWC almost equal to that of the WT, i.e.

25–30 % reduction. There was no significant difference in

the RWC between EaDREB2 and co-transformed events

under soil moisture stress condition.

Fig. 3 Cell-membrane thermostability in EaDREB2 transgenics and

WT a V0 stage at 25 % soil moisture, b V1 stage at 8.1 and 25 % soil

moisture stress. All the transgenic events differ significantly from the

WT at P B 0.05, by Student’s t test. Cell-membrane thermostability

in co-transformed events and WT, c V0 stage at 25 % soil moisture

and d V1 stage at 8.1 and 25 % soil moisture stress. Data show a

significant difference in the CMS between transgenic events and WT,

ns- not significant; at P B 0.05, by Student’s t test. Data are presented

as mean ± SD (n = 5) and error bars represent SD

254 Plant Cell Rep (2015) 34:247–263

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Maintaining higher gas-exchange parameters,

chlorophyll content and photosynthetic efficiency

In EaDREB2 transgenic events, the gas-exchange param-

eters, such as stomatal conductance, transpiration rate and

photosynthesis rate, were significantly higher under soil

moisture stress (Fig. 5a–c). The eighth event showed a

10-fold increase in stomatal conductance and transpiration

rate. Sixty-eight percent of the events (17 out of 25) had

1–3 times higher stomatal conductance and transpiration

rate. All the events maintained 2–3 times higher photo-

synthetic rate than WT. In co-transformed events, out of

the 22 events, 18 were screened for stomatal conductance,

transpiration rate and photosynthetic rate at 8.1 % soil

moisture (Fig. 5d–f). Fifty-five percent of the events (10

out of 18) had higher stomatal conductance and transpira-

tion rate, which was two times higher than WT, and all the

other events had similar or lower stomatal conductance and

transpiration rate as that of WT. The highest photosynthetic

rate was in the sixth event, i.e. 11 % more than the WT,

and all the other events except the fifth event had higher

(2–3 times) photosynthetic rate than WT. The stomatal

conductance and transpiration rate showed a significant

difference between EaDREB2 and co-transformed events

but not for photosynthetic rate.

Both EaDREB2 and the co-transformed events had

significantly higher chlorophyll content (Fig. 6a, b). At the

beginning of the induction of stress, the chlorophyll content

(SPAD values) in transgenics varied between 35 and 45

SPAD units, whereas in WT it was around 35 SPAD units,

indicating not much variation under normal irrigated con-

dition. In nine EaDREB2 transgenic events, the maximum

of three SPAD units reduction was observed after the

induction of drought stress, indicating maintenance of a

higher level of chlorophyll content. Fifty-six percent of the

transgenic events (14 out of 25) had maximum of 5 SPAD

units reductions in chlorophyll content under stress, and it

varied between 30 and 40 SPAD units (Fig. 6a). In addi-

tion, an increased level of chlorophyll content (36–42

SPAD units) was observed after re-irrigation. In co-trans-

formed events, 77 % of the transgenics (17 out of 22) had a

maximum of seven SPAD units reduction in chlorophyll

Fig. 4 Relative water content

in transgenics and WT with and

without soil moisture stress

a pSBI-DREB2, b co-

transformed events. Data

followed by an (asterisk) show a

significant difference with

respect to control Co 86032

(P B 0.05; Student’s t test).

Data are presented as

mean ± SD (n = 5) and error

bars represent SD

Plant Cell Rep (2015) 34:247–263 255

123


content, and it varied between 26 and 38 SPAD units under

moisture stress, whereas in WT, 10 SPAD units reduction

(25 SPAD units) was observed on the 10th day of stress.

After re-irrigation for 15 days, the SPAD value was around

27 SPAD units in these plants (Fig. 6a, b). The chlorophyll

content (33–41 SPAD units) in all the transgenic events

showed a drastic increase after re-irrigation (Fig. 6b).

There was a significant difference in chlorophyll content

between the EaDREB2 and co-transformed events under

soil moisture stress.

The photosynthetic quantum efficiency was significantly

higher in both DREB2 and co-transformed events com-

pared to the WT (Fig. 6c, d). In EaDREB2 transgenics, the

Fv/Fm values varied between 0.62 and 0.76 under stress

(Fig. 6c). Three events (4, 8 and 14) recorded a maximum

Fv/Fm value of 0.76, and 52 % the events (13 out of 25)

showed a value of 0.60–0.70. After the release of stress, it

varied between 0.73 and 0.77, which was same as the 0th

day value. In co-transformed events, 22 transgenic events

were screened, and the Fv/Fm values varied between 0.59

Fig. 5 Gas exchange parameters in transgenics and WT at 8.1 % soil

moisture. In EaDREB2 transgenics a stomatal conductance, b tran-

spiration rate and c photosynthesis rate. In co-transformed events,

d stomatal conductance, e transpiration rate and f photosynthesis rate.

All the transgenic events differ significantly with respect to control

Co 86032 (P B 0.05; Student’s t test). Data are presented as

mean ± SD (n = 3) and error bars represent SD

256 Plant Cell Rep (2015) 34:247–263

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and 0.70 (Fig. 6d). In 81 % of the events (18 out of 22), it

varied between 0.60 and 0.70, and after the release of stress

the values increased to 0.72–0.76. Under moisture stress,

there was reduction (0.50) in WT, which increased to 0.73

after release of stress. In general, the transgenic events had

better photosynthetic efficiency than WT. There was a

significant difference in the Fv/Fm values between EaD-

REB2 and co-transformed events under soil moisture

stress.

Fig. 6 Chlorophyll content and photosynthetic efficiency in trans-

genics and WT with and without stress. a Chlorophyll content in

EaDREB2 transgenics along with WT, b chlorophyll content in co-

transformed events along with WT, c photosynthetic efficiency in

EaDREB2 transgenics along with WT, d photosynthetic efficiency in

co-transformed events along with WT. Data show a significant

difference in the photosynthetic efficiency between transgenics and

untransformed control, ns not significant at P B 0.05, by Student’s

t test. Data are presented as mean ± SD (n = 5) and error bars

represent standard deviation

Table 2 Bud germination percentage of pSBI-DREB2 transgenic

plants in pots supplied with 0 mM (water), 100 mM, 200 mM and

300 mM NaCl solution for 26 days

Event no. 0 mM (%) 100 mM (%) 200 mM (%) 300 mM (%)

1 100 80 80 60

2 80 100 60 20

4 100 100 60 60

8 100 80 80 40

14 100 100 80 20

15 80 80 0 0

18 100 60 40 0

20 100 60 20 20

22 100 80 80 80

24 100 60 20 20

WT 100 – – –

Table 3 Bud germination percentage of sugarcane co-transformed

with pSBI-DREB2 and pSBI-PDH45 in pots supplied with 0 mM

(water), 100 mM, 200 mM and 300 mM NaCl solution for 26 days

Event no. 0 mM (%) 100 mM (%) 200 mM (%) 300 mM (%)

1 100 40 0 0

2 100 100 80 60

4 100 80 60 0

6 100 80 0 0

9 80 20 20 20

11 100 100 80 60

13 80 40 40 40

15 100 80 80 80

16 100 20 20 20

18 80 40 0 0

WT 100 – – –

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The pooled data of all the physiological parameters,

together with the visual scoring of both EaDREB2 and co-

transformants along with WT, are given in Tables 2 and 3

(supplementary data).

Expression analysis of stress-inducible genes using

quantitative real-time PCR

In EaDREB2 events, the transgene was 2300-fold upreg-

ulated compared to WT under moisture stress. The relative

expression of HSP70, DNA helicase 45, ERD, RD29, LEA,

ERF, COR15 and BRICK were also upregulated 562-, 57-,

477-, 1075-, 1187-, 117-, 2036- and 1751-fold, respec-

tively, in the transgenic events (Fig. 7a). In the co-trans-

formed events, the EaDREB2 and DNA helicase 45 were

1683- and 526-fold upregulated, respectively, under

moisture stress. The relative expression of HSP70, ERD,

RD29, LEA, ERF, COR15 and BRICK were increased

590-, 226-, 1416-, 1203-, 399-, 325- and 166-fold,

respectively, in the transgenic events (Fig. 7b).

Overexpression of EaDREB2 and EaDREB2-PDH45

events results in tolerance to excess salinity

To understand tolerance level of the transgenic events to

salinity, leaf disc senescence assay was carried out. The

chlorophyll content in the EaDREB2 and co-transformed

events were significantly higher than in WT plants, indi-

cating tolerance to salinity (Fig. 8b, d). Measurement of

the chlorophyll content provided further support for a

positive relationship between the overexpression of the

transgenes and tolerance to excess salinity. In WT, there

was complete bleaching of the excised leaf under stress,

and the chlorophyll reduction was as high as 9 lg.

Salinity-induced chlorosis was less in EaDREB2 over-

expressing events (Fig. 8a). The minimum chlorosis was

observed in the 14th event with 0.03 lg reduction of

chlorophyll, while the maximum chlorosis was in the 16th

event with 6.48 lg reduction of chlorophyll (Fig. 8b). The

chlorophyll loss was significantly lower in co-transformed

DREB2-PDH45 overexpressing events than in WT

(Fig. 8c). The minimum chlorosis was observed in the

fourth event with 0.31 lg reduction of chlorophyll and

the maximum was in the 20th event with 7.75 lg

reduction (Fig. 8d). There was a significant difference in

the chlorophyll content between EaDREB2 and co-trans-

formed events.

Transgenic events had better bud germination

than the WT under salinity stress

Transgenic events and WT were morphologically similar

when grown without NaCl (Fig. 2e–g). Increasing the salt

concentrations significantly affected the germination per-

centage and shoot length of the transgenic events to vary-

ing degrees. Ten EaDREB2 independent events (1, 2, 4, 8,

14, 15, 18, 20, 22 and 24) were selected from the leaf disc

senescence assay and screened along with the WT. The

WT failed to germinate in all the three salt concentrations,

indicating susceptibility of this variety to excess salinity.

Three events (2, 4 and 14) had 100 %, four events (1, 8, 15

and 22) had 80 % and three events (18, 20 and 24) had

60 % germination in 100 mM NaCl. At 200 mM NaCl,

four events (1, 8, 14 and 22) had 80 %, two events (2 and

4) had 60 %, one event (18) had 40 % and two events (20

and 24) had 20 % germination. At 300 mM NaCl, one

event (22) had 80 %, two events (1 and 4) had 60 %, one

event (8) had 40 % and four events (2, 14, 20 and 24) had

20 % germination, suggesting its enhanced tolerance to

excess salinity (Fig. 2f; Table 2). In ten selected co-trans-

formed transgenic events (1, 2, 4, 6,9, 11, 13, 15, 16 and

18), two events (2 and 11) had 100 %, three events (4, 6

and 15) had 80 %, three events (1, 13 and 18) had 40 %

and two events (9 and 16) had 20 % germination in

100 mM NaCl. At 200 mM NaCl, three events (2, 11 and

15) had 80 %, one event (4) had 60 %, one event (13) had

40 % and two events (9 and 16) had 20 % germination, and

three events (1, 6 and 18) failed to germinate. At 300 mm

NaCl, one event (15) had 80 %, two events (2 and 11) had

60 %, one event (13) had 40 % and two events (9 and 16)

Fig. 7 Relative expression of the abiotic stress-responsive genes

a EaDREB2 transgenics and b co-transformed events

258 Plant Cell Rep (2015) 34:247–263

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Fig. 8 Leaf disc senescence

assay for salinity tolerance in

transgenic sugarcane

a representative picture to show

phenotypic differences in the

leaf segments of EaDREB2

transgenics (eighth event) and

WT after incubation in 0 and

250 mM NaCl solution for 72 h.

b Chlorophyll content from the

leaf discs of EaDREB2

transgenic events and WT after

incubation in 0 and 250 mM

NaCl solution for 72 h. Data

show a significant difference in

the chlorophyll content between

transgenics and untransformed

control at P B 0.05, by

Student’s t test, c representative

picture to show phenotypic

differences in the leaf of

sugarcane co-transformed with

EaDREB2 and pSBI-PDH45

(13th event) along with

untransformed control after

incubation in 0 and 250 mM

NaCl solution for 72 h,

d chlorophyll content from leaf

discs of sugarcane co-

transformed with EaDREB2 and

pSBI-PDH45 transgenic events

along with WT after incubation

in 0 and 250 mM NaCl solution

for 72 h. Data show a significant

difference in the chlorophyll

content between transgenics and

untransformed control at

P B 0.05, by Student’s t test.

Data are presented as

mean ± SD (n = 5) and error

bars represent SD

Plant Cell Rep (2015) 34:247–263 259

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had 20 % germination and four events (1, 4, 6 and 18)

failed to germinate (Fig. 2g; Table 3).

Shoot height is one of the critical growth parameters,

and was measured as an indicator of salinity tolerance in

transgenic events (Tables 4 and 5: supplementary data). As

there was no germination of the WT in any of the salt

treatments, shoot length measurements could not be

recorded. The shoot length of different transgenics varied

between events under different salt concentrations.

Discussion

Induction of transcription factors contributes to maintain-

ing the integrity of leaf and root cells in soybean during

water stress by triggering the expression of genes associ-

ated with the protection of cell structures against dehy-

dration (Li et al. 2005). DREB2 is the main drought-

responsive transcription factor determining the expression

levels of a number of downstream genes like RD29,

HSP70, etc. (Sakuma et al. 2006; Khan 2011; Mizoi et al.

2011; Qin et al. 2011). The higher transcript level of LEA,

RD29, ERD, ERF, COR15, HSP70 and BRICK genes in

the EaDREB2 overexpressed transgenic sugarcane would

have imparted a higher level of drought and salinity tol-

erance. In the present study the co-expression of the

transgenes resulted in the reduction of relative expression

of the individual transgenes. Reduced expression of the

individual transgenes in co-transformed events may be the

result of perturbation in the entire transcription process of

the plant due to the overexpression of multiple transgenes

under a strong constitutive promoter. Another explanation

for this reduction can be the probable crosstalk between

EaDREB2 and PDH45 pathways. Pyramiding of a tran-

scription factor along with a gene that is not regulated by

the transcription factor per se could not be found in the

literature. In co-transformed events, DREB2, HSP70,

RD29, LEA, ERD, ERF, COR15, BRICK and PDH45 were

significantly upregulated compared to WT but not as high

as in DREB2 transgenics. The screening for drought tol-

erance in these transgenic events has shown that overex-

pression of EaDREB2 led to higher tolerance to drought,

whereas the co-transformed events have shown an

increased tolerance to salinity stress.

It is interesting to note that the transgenics over-

expressing EaDREB2 and the co-transformed events were

found to have increased membrane stability under normal

irrigated condition. However, further increase in the

membrane stability was observed under moisture stress.

The constitutive overexpression of barley group-3 LEA

gene in rice displayed significantly increased tolerance to

water deficit and salinity stresses, which was associated

with higher growth rate, delayed onset of stress damage

symptoms and improved recovery following release of

stress (Xu et al. 1996). Overexpression of LEA in rice also

showed significantly higher RWC, improved turgor, less

reduction in shoot and root growth and improved cell-

membrane stability under prolonged drought conditions. It

was found that LEA did not function as an osmolyte but

contributed to the membrane protection and thus drought

tolerance in rice plants (Chandra Babu et al. 2004). In

EaDREB2 and co-transformed events, the LEA protein was

upregulated 500- and 216-fold, respectively, suggesting

that this might be one of the reasons for its enhanced

membrane stability. Both the EaDREB2 and the co-trans-

formed events showed significantly higher RWC under

moisture stress. This is in accordance with previous reports

on several other transgenic crop plants such as Cenchrus

spp. chickpea, groundnut, wheat, sorghum, and maize

under moisture stress (Nagy et al. 1995; El Hafid et al.

1998; Madhusudan et al. 2002; Bhushan et al. 2007;

Chandra and Dubey 2010).

Drought tolerance associated with stomatal control has

been reported in plants (Pinheiro et al. 2005). The trans-

genic events showed an increased photosynthetic rate than

WT, indicating a substantial protection of photosynthetic

machinery, particularly PS II, during drought stress. It was

found that HSP70 participates in the protection of PS II

against photoinhibition and in the repair of PS II in green

algae (Schroda et al. 1999; Yokthongwattana et al. 2001)

and plays a critical role in the assembly of new PS II core

in tobacco (Tan et al. 2011). This may explain one of the

reasons for the observed higher photosynthetic rate in the

transgenic events. As the main source of energy for plants

is photosynthesis, preservation of the photosynthetic

machinery and its protection contribute significantly to the

plant’s ability to withstand the stress (Mackova et al.

2013). The EaDREB2 and co-transformed events had sig-

nificantly higher chlorophyll content, thus contributing to

the maintenance of higher photosynthetic capacity under

moisture stress. Reduction or no change in chlorophyll

content under drought stress has been observed in different

plant species, and its intensity depends on stress rate and

duration (Rensburg van 1994; Kyparissis et al. 1995; Jag-

tap et al. 1998).

Photochemical quantum efficiency is measured by the

chlorophyll fluorescence parameter Fv/Fm, with maximum

photochemical efficiency of photosystem II in the dark-

adapted state, a trait positively correlated with the organi-

zation and vitality of photosystem II (Aharon et al. 2003).

In healthy leaves, the Fv/Fm value is usually close to 0.8 in

most plant species; therefore, a lower value indicates that a

proportion of PS II reaction centres are damaged or inac-

tivated, a phenomenon termed as photoinhibition and

commonly observed in plants under stress (Baker and

Horton 1987; Baker and Rosenqvist 2004; Zlatev 2009;

260 Plant Cell Rep (2015) 34:247–263

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Vaz and Sharma 2011). Fv/Fm ratios are significantly

higher in EaDREB2 and co-transformed events. The ratio

was not much reduced in transgenic events under moisture

stress, suggesting that the function of reaction centres was

fairly protected in transgenic plants relative to the WT. The

maize transgenic events over expressing PEPC was

reported to show better photosynthetic and water use effi-

ciency with improved dry matter production under soil

moisture stress (Jeanneau et al. 2002).

In WT, the chlorophyll content decreased significantly

compared to the EaDREB2 and co-transformed events

under salinity, suggesting the high salinity tolerance

potential of the transgenic events. Ben Saad et al. (2012)

reported higher stress tolerance in AlSAP (zinc finger

protein) overexpressing rice plants, which maintained the

photosynthetic apparatus integrity by stimulating an

endogenous adaptive potential. Cushman and Bohnert

(2000) suggested that the overexpression or upregulation of

LEA provides salinity tolerance in transgenic plants.

Generally, DREB2 genes are induced by dehydration, high

salinity and heat stress (Liu et al. 1998; Nakashima et al.

2000) and the activation of DREB2 leads to the upregu-

lation of downstream stress-responsive gene expression

and this may be one of the mechanisms for salinity toler-

ance. The mechanism of PDH45-mediated salinity stress

tolerance in transgenics is not clearly understood. How-

ever, based on the properties of PDH45 it was suggested

that there might be two sites of action: (1) it may act at the

translation level to enhance or stabilize protein synthesis,

or (2) it may associate with DNA multisubunit protein

complexes to alter gene expression (Sanan Mishra et al.

2005).

A significant tolerance against salinity stress in trans-

genic events was observed in leaf disc senescence and in

bud germination assays. In numerous crop plants, the

stages of seed germination and early seedling growth are

most susceptible to abiotic stresses (Mito et al. 2010; Se-

khar et al. 2010). However, we could not find any literature

on germination study for salinity tolerance in a vegetatively

propagated plant species. One transgenic event over-

expressing EaDREB2 and one event in the co-transformed

event have shown as high as 80 % germination under high

salt concentration of 300 mM NaCl, while WT failed to

germinate even at lower concentrations. This result agrees

with several previous studies in barley, tomato, Lactuca

sativa, rice and tobacco (Ungar 1978; Bliss et al. 1986;

Bradford 1990; Singla-Pareek et al. 2003; Sanan-Mishra

et al. 2005; Ray and Islam 2008; Dang et al. 2011; Amin

et al. 2012 and Singh et al. 2012), which showed that salt

stress is an important limiting factor for germination in

different crop species. Here, the transgenic plants that

overexpressed EaDREB2 and EaDREB2-PDH45 events

showed increased tolerance to salt stress during bud ger-

mination (Fig. 1e–g).

In conclusion, our results show that transformation of

sugarcane with the EaDREB2 gene under the control of the

Port Ubi 2.3 promoter enhanced the tolerance to water

deficit and salinity stress through improved physiological

adaptation and enhanced stress-related gene expression.

Improved salinity tolerance was observed in the co-trans-

formed events compared to that of EaDREB2. Thus, for

combining drought and salinity tolerance, pyramiding of

these two genes seems to be a better approach. Further

replicated field studies are needed to confirm their toler-

ance to drought and salinity, along with the yield potential

and water use efficiency.

Author contribution statement S. M. Augustine was

responsible for conception and design of experiments,

acquisition of data and analysis, interpretation of data and

drafting of the manuscript; acquisition of data was done by

Ashwin Narayanan, D. P. Syamaladevi, C. Appunu, V.

Ravichandran, and M. Chakravarthi; N. Tuteja provided

the PDH45 construct and N. Subramonian took care of

study conception and design, analysis and interpretation of

data and critical revision.

Acknowledgments This work was supported by the Department of

Biotechnology (DBT) (Grant No. 102/IFD/SAN/325/2013-2014),

Government of India. The authors are grateful to Dr. J. Srikanth,

Principal Scientist, Sugarcane Breeding Institute, Coimbatore, for

critical comments for the improvement of the manuscript.

Conflict of interest The authors declare that they have no conflict

of interest.

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Overexpression of EaDREB2 and pyramiding of EaDREB2 with the pea DNA helicase gene (PDH45) enhance...

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