Expression of an alfalfa (Medicago sativa L.) ethylene responsefactor gene MsERF8 in tobacco plants enhances resistanceto salinity

Tingting Chen • Qingchuan Yang • Margaret Gruber •

Junmei Kang • Yan Sun • Wang Ding •

Tiejun Zhang • Xinquan Zhang

Received: 24 August 2011 / Accepted: 19 December 2011 / Published online: 31 December 2011

� Springer Science+Business Media B.V. 2011

Abstract Ethylene response factors (ERF) play crucial

roles in plant development and response to stresses. Here, a

novel cDNA fragment (MsERF8) encoding an ERF protein

with an AP2 domain was isolated and characterized from

alfalfa. The MsERF8 cDNA has an open reading frame of

603 bp and encodes a nuclear protein of 201 amino acids.

Q-RT-PCR analysis revealed that MsERF8 was strongly

enriched in roots and leaves compared with stems, flower

buds and flowers of mature alfalfa plants. Bioinformatic

analysis of the MsERF8 promoter indicated a number of

elements associated with stress-related responses, and

MsERF8 transcripts in alfalfa seedlings were induced by

NaCl, PEG6000, Al2(SO4)3 and five different hormones.

Expression of MsERF8 in transgenic tobacco plants

resulted in higher tolerance to salinity than with non-

transgenic plants. This data shows that MsERF8 is a gene

which prevents or alleviates salinity damage and has strong

potential to impart salt tolerance to other crop plants.

Keywords Alfalfa � Ethylene response factor gene �Expression analysis � Subcellular localization � Transgenic

tobacco � Salt stress

Abbreviations

IAA Indole-3-acetic acid

ABA Abscisic acid

GA Gibberellin acid

SA Salicylic acid

Eth Ethrel

MeJA Methyl jasmonate

MDA Malondialdehyde

Introduction

Ethylene is an important hormone with roles in plant

growth, development, and response to biotic and abiotic

stresses [1]. Early researches on its function were focused

on preservation of fruits and vegetables. However, recent

studies revealed that a variety of environmental stresses,

such as cold, salt, and drought, induce ethylene production

in plants [2].

Apetala 2/Ethylene Response Factors (AP2/ERF) are

transcription factors characterized by the presence of the

AP2/ERF DNA-binding domain and play significant roles

in regulating plant biotic and abiotic stress-responsive gene

expression [3, 4]. The AP2/ERF super-family of genes/

proteins can be divided into three sub-families according to

the number of AP2 domains, AP2 sub-family proteins with

two AP2 domains in tandem, RAV sub-family proteins

with a B3 domain and a single AP2 domain, and ERF sub-

family proteins with a single AP2 domain [5]. The ERF

sub-family members are sometimes further divided into

two major subfamilies, the CBF/DREB subfamily and the

T. Chen � Y. Sun (&)

Department of Grassland Science, College of Animal Science

and Technology, China Agricultural University, Beijing 100193,

People’s Republic of China

e-mail: ctsoffice@yahoo.com.cn

T. Chen � Q. Yang � J. Kang � W. Ding � T. Zhang

Institute of Animal Science, Chinese Academy of Agricultural

Sciences, Beijing 100193, People’s Republic of China

T. Chen � X. Zhang

Department of Grassland Science, Animal Science and

Technology College, Sichuan Agricultural University,

Ya’an 625014, People’s Republic of China

M. Gruber

Saskatoon Research Centre, Agriculture and Agri-Food Canada,

107 Science Cresc, Saskatoon, SK S7N0X2, Canada

123

Mol Biol Rep (2012) 39:6067–6075

DOI 10.1007/s11033-011-1421-y

ERF subfamily [5]. The CBF/DREB subfamily plays a

crucial role in the response of plants to abiotic stress by

recognizing the dehydration-responsive element (DRE),

which has a core motif of A/GCCGAC [6, 7], the ERF

subfamily genes are mainly involved in response to biotic

stresses such as pathogenesis by recognizing the cis-acting

element AGCCGCC, known as the GCC box [8]. However,

strong evidence suggests that the ERF subfamily proteins

also play significant roles in abiotic stress responses. For

instance, expression of the BrERF4 from Brassica rapa

increases tolerance to salt and drought in Arabidopsis

plants [9]. Expression of the tomato JERF3 gene leads to

increases in WCOR413-like, OsEnol, and OsSPDS2 gene

expression and enhances drought and osmotic stress tol-

erance in transgenic rice [10]. Transgenic rice expressing

JERF1 also shows resistance to drought stress [11]. In rice,

the expression of tERF2 is involved in the cold stress

response [12]. In tobacco, expression of the soybean

GmERF3 gene, an AP2/ERF type transcription factor,

increases tolerances to salt and drought [13]. In alfalfa,

overexpression a putative Medicago truncatula AP2

domain-containing transcription factor gene WXP1,

enhances drought tolerance [14].

Alfalfa is one of the most important leguminous forage

plant all over the world and has a wide distribution in irri-

gated arid and semi-arid regions [15]. Salinity has adverse

effects on alfalfa productivity and crop quality. In this paper,

we report the isolation and characterization of an ERF gene

MsERF8 from alfalfa and the ability of this gene to confer

salt stress tolerance in transgenic tobacco. These results

suggest that MsERF8 would be a useful tool for engineering

crop plants with improved tolerance to salt stress.

Materials and methods

Plant materials and growth conditions

Medicago sativa L. cv. ‘‘Zhongmu No. 1’’ seeds were

germinated on MS solid medium in the dark at 25�C for

1 day and then transferred to a 16 h light/8 h dark condi-

tion (LD). Nicotiana benthamiana was used as a recipient

host for plant transformation.

For hormone treatments, 2-week-old alfalfa seedlings

were transferred to half-strength MS liquid medium sup-

plemented with IAA (50 lM), ABA (100 lM), GA

(50 lM), SA (100 lM), Eth (40 lM) or MeJA (100 lM)

for 0,1 and 12 h. For abiotic stress treatments, 2-week-old

alfalfa seedlings were then transferred to half-strength MS

liquid medium supplemented with 200 mM NaCl, 15%

PEG6000 or 60 lM Al2(SO4)3 for 0, 1, 6, 12 and 24 h.

Aluminum toxicity is an important growth-limiting factor

for plants in acid soils, hence, the pH value of �-strength

MS liquid medium for Al2(SO4)3 stress was set at 4.5 using

1 mol/l HCl buffer. After each treatment, a mixture of

leaves, stems and roots from eight seedlings were harvested

and analyzed by Q-RT-PCR, with eight untreated alfalfa

seedlings as control plants.

For tissue specificity, roots, stems, leaves, flower buds

and flowers were harvested from a 3-year-old alfalfa plant

and quick-frozen in liquid nitrogen, then stored at -80�C

for total RNA extraction and Q-RT-PCR analysis.

Cloning, tissue specificity, and expression analysis

of MsERF8

Total alfalfa RNA was isolated with Trizol (Biomed Bio-

tech, Beijing, China) according to the manufacturer’s

instruction and reverse transcribed into cDNA with oligo

d(T)18 primer using M-MLV reverse transcriptase (Takara,

Dalian, China) in a reaction volume of 40 ll. Two gene-

specific alfalfa primers ME-F and ME-R (Table 1) were

designed with DNAMAN software (Lynnon Biosoft, USA)

from a M. truncatula protein sequence obtained by using

the AP2 domain as a probe to search the Medicago data-

base in NCBI. Thermo-cycling was performed with alfalfa

cDNA for 35 cycles (94�C for 30 s, 56�C for 30 s and 72�C

for 1 min), followed by an additional polymerization step

at 72�C for 5 min. The PCR product was then purified, sub-

cloned, sequenced, and a BLASTn (http://blast.ncbi.nlm.

nih.gov/Blast.cgi) analysis conducted. A phylogenetic tree

of AP2/ERF proteins was constructed by the neighbor-

joining method for alfalfa and other plant species using

MEGA4.1 software (http://www.megasoftware.net/).

To analyze MsERF8 expression in mature alfalfa tissues

and 2-week-old seedlings, total RNA was used to synthe-

size cDNA using a SuperScript III First-Strand Synthesis

SuperMix (Invitrogen, Carlsbad, CA, USA) according to

the manufacturer’s instruction. Transcripts were measured

in seedling aerial tissues grown in different hormone and

abiotic stress conditions or in individual mature plant tis-

sues in 20 ll Q-RT-PCR assays using a Premix� Ex TaqTM

(Perfect Real Time) kit (Takara, Dalian, China) and spe-

cific primers MsERF-Frt and MsERF-Rrt (Table 1)

designed with DMAMAN software. The alfalfa Actin gene

was used as an internal control and the primers (MsAC-

TIN-Frt and MsACTIN-Rrt) were listed in Table 1. Q-RT-

PCR conditions were set at 2 min at 50�C, followed by

2 min at 95�C, and 40 cycles of 95�C (15 s) and 60�C

(30 s) with ABI 7500 Real-Time PCR instrument (Applied

Biosystems, USA). For each pair of primers, gel electro-

phoresis and melting curve analyses were performed to

ensure that only a single PCR amplicon of the expected

length and melting temperature was generated. The level of

each mRNA was calculated using the mean threshold cycle

(Ct) value and normalized to that of the actin reference

6068 Mol Biol Rep (2012) 39:6067–6075

123

gene. All results were shown as means of at least three

independent RNA extractions (including three technical

replicates) with corresponding standard deviations (SD).

Transient expression of MsERF8 protein in onion

epidermal cells

The cDNA sequence of MsERF8 was subcloned as a green

fluorescent protein (GFP) fusion protein into the XhoI/SpeI-

digested pA7-GFP expression vector with two primers

MsERFXhoI and MsERFSpeI which included recognition

sites for XhoI and SpeI (Table 1). The fusion binary vector

was then used to transform onion epidermal cells using a

gene gun (GJ-1000, Scientz Biotechnology, China). Sub-

cellular localization of transiently expressed MsERF8–GFP

fusion protein was detected by a confocal laser scanning

microscope (Olympus FV500, Japan).

Isolation the MsERF8 promoter

The MsERF8 promoter was isolated from alfalfa genomic

DNA by chromosome walking using gene-specific SP

primers (Table 1, as sequence became available) and a

Genome Walking kit (Takara, Dalian, China) according to

the manufacturer’s instructions. The PLANTCARE data-

base software (http://www.bioinformatics.psb.ugent.be/

webtools/plantcare) was used to identify motifs within the

promoter region.

Construction of plant expression vector and generation

of transgenic tobacco

To obtain MsERF8 transgenic tobacco plants, the open

reading frame (ORF) of MsERF8 was sub-cloned into the

BamHI/XbaI-digested expression vector pBI121 (down-

stream of the CaMV 35S promoter) with two gene-specific

primers MsERFXbaI and MsERFBamHI, which included

recognition sites for XbaI and BamHI (Table 1). The

resulting binary vector was used to transform tobacco using

a standard protocol [16]. The transgenic tobacco plants

were screened using 200 mg/l kanamycin, and confirmed

further by PCR and RT-PCR. Q-RT-PCR for detect the

MsERF8 expression level in T1 transgenic tobacco plants

was performed using 2xSYBR Green qPCR Master Mix

(Invitrogen, USA) and an Opticon II system (Bio-Rad

Laboratories/MJ Research, Waltham, MA, USA). The

gene-specific primers and analysis methods was identical

to Q-RT-PCR analysis in alfalfa.

Salt-tolerance analysis of transgenic tobacco

For germination rates under saline conditions, non-trans-

genic seeds and 100 T1 seeds each for four independent

transgenic genotypes were placed on MS agar media with

0, 100, 200, 300 and 400 mM NaCl under 16 h light/8 h

dark condition (LD), 25�C conditions in a greenhouse and

Table 1 Primers used to clone and analyze MsERF8

Primer name Primer sequence Purpose

ME-F 50-CATGTTGTTACTTTTCCTGAGTTCC-30 Cloning of MsERF8

ME-R 50-AAACAAACTTGTACAGCATCACCAG-30 Cloning of MsERF8

MsERF-Frt 50-GTGACAACATCTCTGACCCGT-30 Q-RT-PCR of MsERF8

MsERF-Rrt 50-ACCCTGCTTCCTTCCCTTGAT-30 Q-RT-PCR of MsERF8

MsACT-Frt 50-GCATTGTAGGTCGTCCTCGTCAC-30 Q-RT-PCR of MsACTIN

MsACT-Rrt 50-GGAAGGGCATAACCCTCGTAGAT-30 Q-RT-PCR of MsACTIN

NbEF-Frt 50-CCTCAAGAAGGTTGGATACAAC-30 Q-RT-PCR of tobacco EF-1a gene

NbEF-Rrt 50-TCTTGGGCTCATTAATCTGGTC-30 Q-RT-PCR of tobacco EF-1a gene

MsERF-FXho1 50-CTCGAGATGACAACAACAAAAGAAACTTC-301 Sub-cloning MsERF8 in GFP fusion binary vector

for transient expression

MsERF-RSpe1 50-ACTAGTTGAACTTGAACTTGAACATCTTGT-302 Sub-cloning MsERF8 in GFP fusion binary vector

for transient expression

MsERF-FXba1 50-TCTAGAATGACAACAACAAAAGAAACTTC-303 Sub-cloning MSERF8 into binary vector for plant

transformation

MsERF-RBamH1 50-GGATCCTGAACTTGAACTTGAACATCTTGT-304 Sub-cloning MSERF8 into binary vector for plant

transformation

SP1 50-ACTTGTACAGCATCACCAGCAACTG-30 Chromosome walking

SP2 50-CTTAGGATCACAAACTCCAGCTTCC-30 Chromosome walking

SP3 50-TCTCAGCTGCAAATTTCCCCCATGG-30 Chromosome walking

SP4 50-GTTTAACAGGATGTAGAAACGGGTC-30 Chromosome walking

1,2,3,4 Underlined sequences, recognition sites for 1XhoI, 2SpeI, 3XbaI, and 4BamHI

Mol Biol Rep (2012) 39:6067–6075 6069

123

the germination rate was counted when there were no seeds

germinated any more.

To testify the salt-tolerance of transgenic plants, T1

selfed seeds were germinated in 16 h light/8 h dark at 25�C

on kanamycin-containing MS medium (200 mg/l). Kmr

seedlings (21-day-old) were transferred to earthenware pots

and grown in soil without selection in a controlled envi-

ronment chamber for an additional period of 3 weeks.

Well-watered five T1 plants per genotype were treated with

250 mM NaCl solution for 0 and 10 days and three plant

leaves from each plant were harvested, the relative water,

free proline and MDA transgenic and non-transgenic plants

were measured, and each experiment was performed at

least in triplicate, the data was analyzed for significant

differences of the means by analysis of variance (ANOVA)

using a LSD test in SAS (P \ 0.05).

Determination of leaf tissue water status was evaluated

by calculating the relative water content in fresh leaves of

MsERF8 transgenic tobacco lines and WT plants following

the method of Turner [17]. Proline content was assayed

using a colorimetric method [18] and pure proline as a

standard. Malondialdehyde (MDA) content was used to

measure lipid membrane peroxidative damage due to stress

according to Wang et al. [19].

Results

Cloning and bioinformatics analysis of the MsERF8

gene

A search of the M. truncatula protein database in NCBI

using an AP2 protein domain sequence [20] as a probe

revealed an unknown M. truncatula protein (GenBank ID:

ACJ85345.1) which contains a single AP2 domain

Fig. 1 Comparison of the amino acid sequence of MsERF8 with the

Medicago truncatula unknown protein ACJ85345.1 and other AP2/

ERF super-family proteins. a Alignment of MsERF8 with

ACJ85345.1. MSERF8 has only one amino acid difference from the

M. truncatula unknown protein, the sequence underlined was AP2

domain. b Phylogenetic tree analysis of MSERF8 and other ERF sub-

family members. Capsella rubella (CrAP2: AAR15465.1); Malus 9

domestica (MdAP2: ADE41112.1); Capsicum annuum (CaERF1:

AAX20035.1); Arabidopsis thaliana (AtERF5: BAA97157.1); Sola-num lycopersicum (SlERF5: AAS72389.1); Olimarabidopsis pumila(OpAP2: AAR15484.1); Capsicum annuum (CaERF2: AAX20037.1);

Arabidopsis arenosa (AaAP2: AAR15499.1); Glycine max (GmERF:

ADK22067.1); Brassica oleracea (BoERF: ABD65036.1); Medicagosativa (MsDREB1: ABY78835.1); Medicago sativa subsp. falcata(MfDREB1s: ABY78834.2); Medicago sativa subsp. falcata(MfDREB1: ABV22882.2); Oryza sativa Japonica Group (OsDREB:

AAO39764.1); Corylus heterophylla (ChDREB: AEF79999.1); Pru-nus persica (PpDREB: ABR19831.1); Glycine max (GmRAV-like:

AAZ66389.1); Capsicum annuum (CaRAV1: AAW83473.1); Camel-lia sinensis (CsRAV: ACT33043.1); Capsicum annuum (CaRav2:

AAQ05799.1); Solanum lycopersicum (SlRAV2; ABY57635.1);

Solanum lycopersicum (SlRAV1: ABY57634.1)

6070 Mol Biol Rep (2012) 39:6067–6075

123

(Fig. 1a). We designed a pair of primers based on this

sequence (Table 1) and used the first-strand cDNA of

alfalfa cv. ‘‘Zhongmu No. 1’’ as a template to obtain a

679 bp fragment with a predicted ORF of 603 bp, named

MsERF8. The alfalfa gene encoded a protein of 201 amino

acids (GenBank ID: JF965422) and had just one amino

acid difference (S ? L) compared with the M. truncatula

unknown protein (Fig. 1a). Phylogenetic tree analysis of

MsERF8 and other AP2/ERF proteins revealed that

MsERF8 belongs to the ERF sub-family (Fig. 1b).

In silico analysis of the MsERF8 promoter sequence

The 1,032 bp upstream flanking regions of MsERF8 were

isolated using genome walking and specific primers

(Table 1). The fragments were sequenced, aligned, and then

characterize by bioinformatics using the PLANTCARE

database to search for plant cis-acting regulatory elements.

Several putative regulatory cis-elements were identified

within the amplified fragments (Table 2). These included a

number of elements associated with stress-related and

hormone responses: ARE (anaerobic induction), HSE

(heat stress response), MBS (drought-inducibility), ERE

(ethylene-responsive) and a TATC-box (gibberellin-

responsiveness).

Expression of MsERF8 gene in different tissues

To clarify the pattern of MsERF8 expression in alfalfa

organs, Q-RT-PCR was carried out with cDNA from dif-

ferent tissues of a 3-year-old plant (Fig. 2a). Detectable

transcript levels for MsERF8 were recovered from leaves,

stems, roots, flowers and flower buds. Expression in roots

and leaves were noticeably higher than in other tissues.

Stress-induced transcription of MsERF8 in alfalfa

seedlings

Since upstream sequences in the MsERF8 gene showed

several motifs with putative function in abiotic stress and

hormone response, transcripts for MSERF8 were measured

by Q-RT-PCR in response to multiple abiotic stresses.

Under Al2(SO4)3 treatment, transcripts accumulated *3-

fold higher by 6 h and then declined back to a basal level

by 12 h (Fig. 2b). In response to PEG6000 treatment,

MsERF8 transcripts increased *2-fold by 6 h, then

reached a maximum by 12 h and declined by 24 h

(Fig. 2c). In the case of NaCl, the MsERF8 expression

level did not change obviously at 1 or 6 h after treatment,

but was substantially elevated by 12 h and then reached a

maximum by 24 h (Fig. 2d).

Applications of abscisic acid (ABA), giberellin (GA3),

salicylic acid (SA), ethryl (Eth) and methyl jasmonate

(MeJA) to alfalfa seedlings for 12 h induced the expres-

sion of MsERF8 transcripts (Fig. 2e). With ABA or MeJA

treatments, expression levels of MsERF8 peaked at 1 h.

Under GA or SA treatments, expression levels were ini-

tially static but then reached a maximum level at 12 h

after treatment. In response to Eth treatment, transcript

accumulation peaked immediately at 1 h and then main-

tained similar levels up to the end of the testing period

(12 h). However, expression of MsERF8 was not induced

by IAA.

Subcellular localization of the MsERF8 protein

To investigate the sub-cellular location of the MsERF8

protein, a GFP-coding sequence was fused in-frame to the

30 end of the MsERF8 gene under the control of the cau-

liflower mosaic virus (CaMV) 35S promoter. The

MsERF8–GFP gene fusion was transiently expressed in

onion epidermal cells using a gene gun and detected by

confocal microscopy. GFP signals were detected exclu-

sively in the nucleus in the transient expression assays

(Fig. 3), indicating that the MsERF8 protein is a nuclear

protein.

Generation of transgenic tobacco lines

Tobacco explants infected with Agrobacterium tumefaciens

containing a MsERF8 binary vector were selectively cul-

tured on kanamycin medium. Six independent transgenic

lines were detected by PCR, RT-PCR and designated as

lines 1–6. The results revealed that the MsERF8 gene was

Table 2 Putative cis-elements present in the promoter sequence of

MsERF8

Putative

cis-element

Sequence Function

ARE TGGTTT cis-Acting regulatory element

essential for anaerobic

induction

Box I TTTCAAA Light responsive element

CCAAT-box CAACGG MYBHv1 binding site

ERE ATTTCAAA Ethylene-response element

HSE AAAAAATTTC cis-Acting element in heat

stress response

MBS CGGTCA

CAACTG

MYB binding site involved

in drought-inducibility

O2-site GATGATGTGG cis-Acting regulatory element

in zein regulation

Skn-1_motif GTCAT cis-Acting regulatory element

required for endosperm

expression

TATC-box TATCCCA cis-Acting element in

gibberellin-response

Mol Biol Rep (2012) 39:6067–6075 6071

123

Fig. 2 Q-RT-PCR analysis of

MsERF8 in alfalfa. a Transcript

levels in mature (3-year-old)

plant. R roots, S stems, L leaves,

F flowers, FB flower buds.

Transcript abundance after a

24 h treatment of 2-week-old

seedlings by Al2(SO4)3 (b),

PEG6000 (c), and NaCl (d);

e transcript abundance after a

12 h hormone treatment with

SA, MeJA, GA3, Eth and ABA,

and IAA

Fig. 3 Subcellular localization of the MsERF8–GFP fusion and the

pA7-GFP control plasmid in onion epidermal cells. The fluorescence

signals were examined by a confocal laser scanning microscope. a–

c GFP fluorescence from cells expressing MsERF8–GFP fusion

protein. d–f GFP fluorescence from cells expressing a GFP non-fusion

empty vector. a, d Dark field vision, c, f bright field vision, b,

e superposition of the bright light vision

6072 Mol Biol Rep (2012) 39:6067–6075

123

inserted into the genome of transgenic tobacco and

MsERF8 could be transcribed into mRNA (Fig. 4a).

Expression levels in T1 plants of lines 1, 2, 4 and 6 were

higher than those of lines 3 and 5 (Fig. 4b), so these four

transgenic lines were characterized for tolerance to saline

conditions.

Expression of MsERF8 confers tolerance to salt stress

in transgenic tobacco

To investigate whether constitutive expression of the

MsERF8 gene in tobacco might provide protection against

salt stress. Relative water content, MDA content and pro-

line content were measured in four independent transgenic

lines and one non-transgenic line before and after treatment

with 250 mM NaCl for 10 days. In addition, the germi-

nation rates of transgenic and non-transgenic seeds were

measured under 0, 100, 200, 300 and 400 mM NaCl

treatments. Under non-saline conditions, no differences

were observed for germination among any of the genotypes

(Fig. 5a). However, with increasing salt concentration,

germination decreased in all genotypes, but most especially

in the non-transgenic genotype.

Relative water content in transgenic and WT plants was

analyzed after 10 days of growth in 250 mM NaCl (Fig. 5b).

Prior to salt treatment, no significant difference (P [ 0.05)

was observed for relative water content between the trans-

genic and WT plants. After treatment with NaCl, relative

water content decreased in all genotypes, but especially in

the non-transgenic plants (P \ 0.05). In contrast, the relative

water content of transgenic line 1, 2, 4 and 6 were 37.3, 40.8,

35.4 and 38.2% higher than non-transgenic plants respec-

tively (P \ 0.05). Prior to salt treatment, significant differ-

ences in proline levels were not observed between the

transgenic and WT plants (P [ 0.05). After salt stress was

initiated, the proline content in WT plants changed very little

while proline in transgenic lines was significantly raised

(P \ 0.05) compared with non-transgenic plants (Fig. 5c).

MDA was highly accumulated after salt treatment for

10 days in all transgenic and WT plants, indicating that

lipid peroxidation occurred in all plants due to salt stress.

However, MDA content of transgenic lines was signifi-

cantly lower than in non-transgenic plants (P \ 0.05). For

example, MDA contents of lines 2 and 6 were 31.2 and

12.9% lower than non-transgenic plants, respectively

(Fig. 5d).

Fig. 4 Molecular analysis of transgenic tobacco seedlings trans-

formed with MsERF8. a PCR and RT-PCR confirmation of transgenic

lines 1 through 6. CK-, non-transgenic plants. CK?, transgenic

plants containing T-DNA from binary plasmid MsERF8. b Q-PCR

analysis of MsERF8 in transgenic tobacco plants. Transgenic lines 1,

2, 4 and 6 were selected for salt resistance analysis due to their higher

expression levels

Fig. 5 Salinity tolerance of

MsERF8? transgenic tobacco.

a Germination rate of transgenic

seeds. b Relative water content.

c Proline content. d MDA

content. WT wide-type plants.

Transgenic lines were

significantly more tolerant

(P \ 0.05) than non-transgenic

plants under 10 days of salt

treatment

Mol Biol Rep (2012) 39:6067–6075 6073

123

Discussion

Growth and development of plants and productivity of crops

are affected by various abiotic stresses such as drought, cold

and high salinity. Under adverse conditions, plants will

signal and then activate the expression of regulatory genes to

ultimately induce resistance genes and improve plant stress

tolerance [21]. Alfalfa is one of the most important legumi-

nous forage plants, and salt stress is a major limiting factor

for its growth and crop yield. To address this limitation, we

isolated an alfalfa ERF gene MsERF8, which encodes a

nuclear protein of 201 amino acids and belongs to the ERF

subfamily of the AP2/ERF superfamily.

The expression of MsERF8 in alfalfa seedlings was

induced by salt, drought, and Al2(SO4)3, suggesting that

MsERF8 may be involved in various abiotic stress

responses in alfalfa. Previous study revealed that ERF

genes in plants were usually regulated by different phyto-

hormones. For example, the Tsi1 in tobacco is induced by

salt, SA, and MeJA, and regulates the expression of disease

resistance genes and osmotic stress resistance genes [22].

The GmERF3 in soybean is induced by SA, JA, ethylene

and ABA [13]. ABA plays a key role extensively involved

in response to abiotic stressors such as drought, low tem-

perature, and osmotic stress [23, 24]. In contrast, the

phytohormones SA, MeJA, and Ethylene are important

inducers of defence-related genes [13]. The expression of

MsERF8 in alfalfa also was induced by treatment with SA,

MeJA, GA, Eth and ABA. These results indicate that the

MsERF8 gene is likely to be regulated by signaling path-

ways that are activated by abiotic stress, ABA, SA, MeJA,

GA3 and ethylene.

Promoters are the primary regulators of gene expression

at the transcriptional level and are keys to controlling

transgenes in transgenic organisms [25]. In most transgenic

plants, the target gene is driven by a powerful constitutive

promoter, such as the CaMV 35S [26] and its derivatives

[27], and is expressed at high levels. However, continuous

strong accumulation of transgenic products, especially

toxins, could interfere with plant metabolic pathways and

lead to undesirable pleiotropic effects in transgenic plants.

Hence, the identification of gene promoters leading to tis-

sue-specific or developmental stage-specific expression of

transgenes is highly desirable for the development of new

genetically modified (GM) crops [28]. Likewise, analyses

of native promoters will most likely reveal a large variety

of heretofore undiscovered cis-regulatory elements, which

will increase our understanding of gene expression regu-

lation [29]. The promoter sequence obtained for MsERF8

codes for several recognized regulatory sequence motifs.

Future experiments with this gene should involve a

functional analysis of these individual sequences using

promoter deletion lines.

Many ERF transcription factors can activate the expres-

sion of downstream genes and also inhibit multiple plant

stress responses [30, 31]. Different ERF isoforms have been

expressed in various plant species and shown substantial

tolerance to salinity and drought. There is no clear difference

in efficiency for these different isoforms, nor any differences

for their origins from glycophytes or halophytes. In the

present study, transformation of tobacco with the MsERF8

gene resulted in salt tolerant tobacco plants. Similarly,

expression of the GmERF3 confers salt and drought tolerance

on transgenic tobacco [13]. Expression of tomato JERF1

enhances the drought and salt tolerance of transgenic tobacco

and rice [32, 33], while expression of JERF1 and JERF3 in

tobacco enhances tolerance to salt stress [34, 35]. Expression

of WXP gene in alfalfa plants showed reduced water loss and

decreased epidermal permeability, therefore the transgenic

plants were much more drought-tolerant than the control

plants [14]. In present study, after treatment with NaCl, rel-

ative water content decreased in all genotypes, but especially

in the non-transgenic lines, these result suggest that the

MsERF8 transgene product possibly prevents water loss

during salinity stress. Proline accumulation is a common

physiological indicator of the plant’s response to various

biotic and abiotic stresses and proline is important in plant

adaptation to stress conditions such as salt and drought [36].

We found that there was no significant difference in the

contents of proline between WT and MsERF8 transgenic

lines before NaCl treatment. But proline was increased in

MsERF8 transgenic lines compared with the WT plants under

NaCl treatment. Previous research indicates that MDA, an

end product of membrane lipid peroxidation, is indicator of

free-radical production and cellular membrane injury caused

by various stresses [37]. Our results revealed that MDA

content of transgenic lines was significantly lower than in

non-transgenic plants, suggesting that lipid peroxidation was

lower in MsERF8-enhanced plants. Most important, the

germination rates of MsERF8-enhanced lines were signifi-

cant higher than non-transgenic lines. All of these indicated

that expression MsERF8 in tobacco confer salt-tolerance to

transgenic plants. In the future, MsERF8 gene has the

potential to be used as a candidate gene for enhancing toler-

ance to salinity in breeding crops. It should also be tested for

its ability to enhance other forms of abiotic stress tolerance.

Acknowledgments This work was supported by the National

Natural Science Foundation of China (Grant no. 30871819) and the

earmarked fund for Modern Agro-industry Technology Research

System (No. CARS-35).

References

1. Chen YE, Schaller GE (2005) Ethylene signal transduction. Ann

Bot 95:901–915

6074 Mol Biol Rep (2012) 39:6067–6075

123

2. Morgan PW, Drew MC (1997) Ethylene and plant responses to

stress. Physiol Plant 100:620–630

3. Sakuma Y, Liu Q, Dubouzet JG, Abe H, Shinozaki K, Yamag-

uchi-Shinozaki K (2002) DNA-binding specificity of the ERF/

AP2 domain of Arabidopsis DREBs, transcription factors

involved in dehydration- and cold-inducible gene expression.

Biochem Biophys Res Commun 290:998–1009

4. Zhang GY, Chen M, Chen XP, Xu ZS, Guan S, Li LC, Li AL,

Guo JM, Mao L, Ma YZ (2008) Phylogeny, gene structures, and

expression patterns of the ERF gene family in soybean (Glycinemax L.). J Exp Bot 59(15):4095–4107

5. Nakano T, Suzuki K, Fujimura T, Shinshi H (2006) Genome-

wide analysis of the ERF gene family in Arabidopsis and rice.

Plant Physiol 140:411–432

6. Yamaguchi-Shinozaki K, Shinozaki K (1994) A novel cis-acting

element in an Arabidopsis gene is involved in responsiveness to

drought, low-temperature, or high-salt stress. Plant Cell

6:251–264

7. Thomashow MF (1999) Plant cold acclimation: freezing toler-

ance genes and regulatory mechanism. Annu Rev Plant Physiol

Plant Mol Biol 50:571–599

8. Hao DY, Ohme-Takagi M, Sarai A (1998) Unique mode of GCC

box recognition by the DNA-binding domain of ethylene-

responsive element-binding factor (ERF domain) in plant. J Biol

Chem 273(41):26857–26861

9. Seo YJ, Park JB, Cho YJ, Jung C, Seo HS, Park SK, Nahm BH,

Song JT (2010) Overexpression of the ethylene-responsive factor

gene BrERF4 from Brassica rapa increases tolerance to salt and

drought in Arabidopsis plants. Mol Cells 30:271–277

10. Zhang HW, Liu W, Wan LY, Li F, Dai LY, Li DJ, Zhang ZJ,

Huang RF (2010) Functional analyses of ethylene response factor

JERF3 with the aim of improving tolerance to drought and

osmotic stress in transgenic rice. Transgenic Res 19:809–818

11. Zhang ZJ, Li F, Li DJ, Zhang HW, Huang RF (2010) Expression

of ethylene response factor JERF1 in rice improves tolerance to

drought. Planta 232:765–774

12. Tian Y, Zhang HW, Pan XW, Chen XL, Zhang ZJ, Lu XY,

Huang RF (2011) Overexpression of ethylene response factor

TERF2 confers cold tolerance in rice seedlings. Transgenic Res

20:857–866

13. Zhang GY, Chen M, Li LC, Xu ZS, Chen XP, Guo JM, Ma YZ

(2009) Overexpression of the soybean GmERF3 gene, an AP2/

ERF type transcription factor for increased tolerances to salt,

drought, and diseases in transgenic tobacco. J Exp Bot

60(13):3781–3796

14. Zhang JY, Broeckling CD, Blancaflor EB, Sledge MK, Sumner

LW, Wang ZY (2005) Overexpression of WXP1, a putative

Medicago truncatula AP2 domain-containing transcription factor

gene, increases cuticular wax accumulation and enhances drought

tolerance in transgenic alfalfa (Medicago sativa). Plant J

42:689–707

15. Bnaebderrahim MA, Mansour H, Ali F (2009) Diversity of

lucerne (Medicago sativa L.) populations in south Tunisia. Pak J

Bot 41(6):2851–2861

16. Hofgen R, Willmitzer L (1988) Storage of competent cells for

agrobacterium transformation. Nucleic Acids Res 16:9877

17. Turner NC (1981) Techniques and experimental approaches for

the measurement of plant water status. Plant Soil 58:339–366

18. Bates LS, Waldren RP, Teare ID (1973) Rapid determination of

free proline for water stress studies. Plant Soil 39:205–207

19. Wang YC, Gao CQ, Liang YN, Wang C, Yang CP, Liu GF

(2010) A novel bZIP gene from Tamarix hispida mediates

physiological responses to salt stress in tobacco plants. J Plant

Physiol 167:222–230

20. Okamuro JK, Caster B, Villarroel R, Montagu MV, Jofuku KD

(1997) The AP2 domain of APETALA2 defines a large new

family of DNA binding proteins in Arabidopsis. Proc Natl Acad

Sci USA 94:7076–7081

21. Fujimoto SY, Ohta M, Usui A, Shinshi H, Ohme-Takagi M

(2000) Arabidopsis ethylene-responsive element binding factors

act as transcriptional activators or repressors of GCC box-medi-

ated gene expression. Plant Cell 2:393–404

22. Park JM, Park CJ, Lee SB, Ham BK, Shin R, Paek KH (2001)

Overexpression of the tobacco Tsi1 gene encoding an EREBP/

AP2-type transcription factor enhances resistance against pathogen

attack and osmotic stress in tobacco. Plant Cell 13:1035–1046

23. Mauch-Mani B, Mauch F (2005) The role of abscisic acid in

plant-pathogen interactions. Curr Opin Plant Biol 8:409–414

24. Shinozaki K, Yamaguchi-Shinozaki K, Seki M (2003) Regulatory

network of gene expression in the drought and cold stress

responses. Curr Opin Plant Biol 6:410–417

25. Potenza C, Aleman L, Sengupta-Gopalan C (2004) Targeting

transgene expression in research, agricultural, and environmental

applications: promoters used in plant transformation. In Vitro

Cell Dev Biol Plant 40:1–22

26. Covey SN, Lomonossoff GP, Hull R (1981) Characterisation of

cauliflower mosaic virus DNA sequences which encode major

polyadenylated transcripts. Nucleic Acids Res 9(24):6735–6747

27. Balazs E, Bouzoubaa S, Guilley H, Jonard G, Paszkowski J,

Richards K (1985) Chimeric vector construction for higher plant

transformation. Gene 40(2–3):343–348

28. Tu CH, Liu WP, Huang LL, Mo YQ, Yang DZ (2009) Cloning and

transcriptional activity of a novel ovarian-specific promoter from

rat retrovirus-like elements. Arch Biochem Biophys 485:24–29

29. Rushton PJ, Reinstadler A, Lipka V, Lippok B, Somssich IE

(2002) Synthetic plant promoters containing defined regulatory

elements provide novel insights into pathogen- and wound-

induced signaling. Plant Cell 14:749–762

30. Kitajima S, Koyama T, Ohme-Takagi M, Shinshi H, Sato F

(2000) Characterization of gene expression of NsERFs, tran-

scription factors of basic PR genes from Nicotiana sylvestris.

Plant Cell Physiol 41(6):817–824

31. Lorenzo O, Piqueras R, Sanchez-Serrano JJ, Solano S (2003)

Ethylene Response Factor 1 integrates signals from ethylene and

jasmonate pathways in plant defense. Plant Cell 15:165–178

32. Zhang XL, Zhang ZJ, Chen J, Chen Q, Wang XC, Huang RF

(2005) Expressing TERF1 in tobacco enhances drought tolerance

and abscisic acid sensitivity during seedling development. Planta

222:494–501

33. Gao SM, Zhang HW, Tian Y, Li F, Zhang ZJ, Lu XY, Chen XL,

Huang RF (2008) Expression of TERF1 in rice regulates

expression of stress-responsive genes and enhances tolerance to

drought and high-salinity. Plant Cell Rep 27:1787–1795

34. Zhang HW, Huang ZJ, Xie BY, Chen Q, Tian X, Zhang XL,

Zhang HB, Lu XY, Huang DF, Huang RF (2004) The ethylene-,

jasmonate-, abscisic acid- and NaCl-responsive tomato tran-

scription factor JERF1 modulates expression of GCC box-con-

taining genes and salt tolerance in tobacco. Planta 220:262–270

35. Wang H, Huang ZJ, Chen Q, Zhang ZJ, Zhang HB, Wu YM,

Huang DF, Huang RF (2004) Ectopic overexpression of tomato

JERF3 in tobacco activates downstream gene expression and

enhances salt tolerance. Plant Mol Biol 55:183–192

36. Verbruggen N, Hermans C (2008) Proline accumulation in plants:

a review. Amino Acids 35:753–759

37. Esfandiari E, Shekari F, Shekari F, Esfandiari M (2007) The

effect of salt stress on antioxidant enzymes, activity and lipid

peroxidation on the wheat seedling. Not Bot Hort Agrobot Cluj

35(1):48–56

Mol Biol Rep (2012) 39:6067–6075 6075

123