ArticlePDF Available

The transcriptional factor LcDREB2 cooperates with LcSAMDC2 to contribute to salt tolerance in Leymus chinensis

Authors:

Abstract

S-Adenosyl-methionine decarboxylase (SAMDC) and dehydration responsive element-binding proteins (DREBs) can improve plant resistance to abiotic stresses. These proteins have been extensively studied, but the mechanism for transcriptional regulation of SAMDC remains unclear. In this paper, the LcSAMDC2 gene and its promoter were isolated from Leymus chinensis. Two DRE cis-elements were identified from the promoter of LcSAMDC2 and shown to bind with LcDREB2. Subcellular localization and yeast one-hybrid assay revealed that LcDREB2 is a transcription factor. An electrophoretic mobility shift assay (EMSA) showed that LcDREB2 can bind to the LcSAMDC2 promoter probe containing a DRE element. Over-expression of LcDREB2 in L. chinensis callus increased expression of LcSAMDC2. Co-expression of LcDREB2 and the promoter of LcSAMDC2 fused with GUS in tobacco activated GUS activity. These results indicate that LcSAMDC2 is the downstream gene of LcDREB2. In addition, transgenic expression of LcDREB2 and LcSAMDC2 in Arabidopsis can improve the salt stress tolerance of transgenic lines. These results indicate that LcDREB2 cooperating with LcSAMDC2 contributes to resistance to abiotic stress.
1 23
Plant Cell, Tissue and Organ Culture
(PCTOC)
Journal of Plant Biotechnology
ISSN 0167-6857
Volume 113
Number 2
Plant Cell Tiss Organ Cult (2013)
113:245-256
DOI 10.1007/s11240-012-0264-0
The transcriptional factor LcDREB2
cooperates with LcSAMDC2 to contribute
to salt tolerance in Leymus chinensis
Xianjun Peng, Lexin Zhang, Lixing
Zhang, Zhujiang Liu, Liqin Cheng, Ying
Yang, Shihua Shen, Shuangyan Chen &
Gongshe Liu
1 23
Your article is protected by copyright and all
rights are held exclusively by Springer Science
+Business Media Dordrecht. This e-offprint
is for personal use only and shall not be self-
archived in electronic repositories. If you wish
to self-archive your article, please use the
accepted manuscript version for posting on
your own website. You may further deposit
the accepted manuscript version in any
repository, provided it is only made publicly
available 12 months after official publication
or later and provided acknowledgement is
given to the original source of publication
and a link is inserted to the published article
on Springer's website. The link must be
accompanied by the following text: "The final
publication is available at link.springer.com”.
ORIGINAL PAPER
The transcriptional factor LcDREB2 cooperates with LcSAMDC2
to contribute to salt tolerance in Leymus chinensis
Xianjun Peng Lexin Zhang Lixing Zhang
Zhujiang Liu Liqin Cheng Ying Yang
Shihua Shen Shuangyan Chen Gongshe Liu
Received: 9 July 2012 / Accepted: 14 November 2012 / Published online: 25 November 2012
ÓSpringer Science+Business Media Dordrecht 2012
Abstract S-Adenosyl-methionine decarboxylase (SAMDC)
and dehydration responsive element-binding proteins (DREBs)
can improve plant resistance to abiotic stresses. These proteins
have been extensively studied, but the mechanism for tran-
scriptional regulation of SAMDC remains unclear. In this
paper, the LcSAMDC2 gene and its promoter were isolated
from Leymus chinensis. Two DRE cis-elements were identified
from the promoter of LcSAMDC2 and shown to bind with
LcDREB2. Subcellular localization and yeast one-hybrid assay
revealed that LcDREB2 is a transcription factor. An electro-
phoretic mobility shift assay (EMSA) showed that LcDREB2
canbindtotheLcSAMDC2 promoter probe containing a DRE
element. Over-expression of LcDREB2 in L. chinensis callus
increased expression of LcSAMDC2. Co-expression of
LcDREB2 and the promoter of LcSAMDC2 fused with GUS in
tobacco activated GUS activity. These results indicate that
LcSAMDC2 is the downstream gene of LcDREB2. In addition,
transgenic expression of LcDREB2 and LcSAMDC2 in
Arabidopsis can improve the salt stress tolerance of transgenic
lines. These results indicate that LcDREB2 cooperating with
LcSAMDC2 contributes to resistance to abiotic stress.
Keywords Abiotic stress DREB EMSA Promoter
SAMDC
Abbreviations
ABA Abscisic acid
MeJA Jasmonic acid methyl ester
DRE Dehydration responsive element
Spd Spermidine
Spm Spermine
SAMDC S-adenosylmethionine decarboxylase
EMSA Electrophoretic mobility shift assay
Introduction
In plants, polyamines (PAs) have been shown to play
important roles in various developmental processes,
including morphogenesis, growth, differentiation and
senescence (Pal Bais and Ravishankar 2002; Kusano et al.
2008). Moreover, they are implicated in defense responses
to various biotic and abiotic stresses (Alvarez et al. 2003;
Kusano et al. 2008; Takahashi and Kakehi 2010).
S-Adenosyl-methionine decarboxylase (SAMDC) is a key
enzyme in PA biosynthesis and catalyzes the conversion of
S-adenosyl-methionine (SAM) to decarboxylated S-aden-
osyl-methionine (dcSAM). The product dcSAM acts as an
aminopropyl donor for the biosynthesis of spermidine (spd)
and spermine (spm), and SAM is used in key metabolic
pathways such as ethylene biosynthesis. SAMDC is also
responsible for PA and ethylene balance in plants (Richard
et al. 1997; Petri et al. 2005).
Electronic supplementary material The online version of this
article (doi:10.1007/s11240-012-0264-0) contains supplementary
material, which is available to authorized users.
X. Peng L. Zhang L. Zhang Z. Liu L. Cheng S. Shen
S. Chen (&)G. Liu (&)
Institute of Botany, the Chinese Academy of Sciences,
Beijing, People’s Republic of China
e-mail: sychen@ibcas.ac.cn
G. Liu
e-mail: liugs@ibcas.ac.cn
L. Zhang Z. Liu
Graduate University of the Chinese Academy of Sciences,
Beijing, People’s Republic of China
Y. Yang
Beijing Command College of the Chinese People’s Armed
Police Force, Beijing, People’s Republic of China
123
Plant Cell Tiss Organ Cult (2013) 113:245–256
DOI 10.1007/s11240-012-0264-0
Author's personal copy
SAMDCs are highly conserved in many organisms and are
expressed throughout the cell. Previous studies indicated that
SAMDC was involved in diverse growth and developmental
processes, including cell division, cell proliferation and differ-
entiation, morphological development, and response to biotic or
abiotic stresses (Pal Bais and Ravishankar 2002;Kusanoetal.
2008). The expression of SAMDC genes was regulated at
multiple levels, including transcriptionally, translationally, and
posttranslationally (Hanfrey et al. 2002;Huetal.2005). At the
translational level, Hanfrey et al. (2002) revealed that the
expression level of SAMDC genes in plants was regulated by
negative feedback through the content of spd and spm and
involves a small uORF. The identification of the PEST sequence
and the enzyme auto-activation site indicated a role in post-
translational regulation (Tian et al. 2004;Huetal.2005). Barley
SAMDC gene expression was regulated by light and has a
diurnal rhythm (Dresselhaus et al. 1996). In soybeans, SAMDC2
gene expression was induced by low temperature, salinity,
drought and extraneous ABA, but not by injuries (Tian et al.
2004). In spite of the above studies, the regulatory mechanism of
SAMDC at the transcriptional level has not been fully explored.
Dehydration responsive element-binding proteins (DREBs)
exert protective effects under stress by activating the expression
of downstream genes, which contain dehydration responsive
elements (DREs) in their promoters (Sakuma et al. 2002;Jin
et al. 2010;Zhouetal.2012). The DREB binding specificity to
the cis-acting element DRE was studied by gel mobility shift
assay. It was found that in AtDREB2A, OsDREB2A and At-
DREB1A proteins the 14th valine and 19th glutamic acid are
conserved in the ERF/AP2 domain. The conserved nature of
DREB2-type protein suggests that these have similar binding
specificity in different plants (Agarwal et al. 2006).
Over-expression of DREBs in plants improves stress
resistance to salinity and drought due to increased expression
of stress response genes under normal and stress conditions
(Hirayama and Shinozaki 2007; Abdel Hamid et al. 2011).
Despite understanding the roles of DREB and SAMDC in
response to abiotic stress, it remains unclear whether SAM-
DC is the downstream gene of DREB and regulated by DREB
under abiotic stress. To address this question, L. chinensis
was used as a study system. L. chinensis is a perennial rhi-
zome grass distributed in the eastern Eurasian steppe
including the outer Baikal area of Russia, Korea, Mongolia,
the northern Plain, and the Inner Mongolian Plateau of
China. It plays important roles in environmental protection
and livestock development; In addition, it exhibits especially
strong resistance to various environmental stresses such as
cold, drought and salinity (Liu et al. 2008; Peng et al. 2011).
In the present study, LcDREB2 and LcSAMDC2 were
isolated from L. chinensis. Our results showed that LcDREB2
is a typical transcription factor and can bind to two DRE cis-
elements located in the promoter of LcSAMDC2. Transgenic
plants of LcDREB2 and LcSAMDC2 of Arabidopsis exhibited
significantly improved salt stress tolerance. The interaction
between LcDREB2 and LcSAMDC2 in resistance to salt stress
was confirmed by electrophoretic mobility shift assay
(EMSA), over-expression of LcDREB2 in L. chinensis and
co-expression of LcDREB2 and the promoter of LcSAMDC2
fused with GUS in tobacco. These experiments demonstrated
that LcSAMDC2 is the downstream gene of LcDREB2 and is
responsible for stress resistance.
Materials and methods
Plant material treatments and DNA and RNA extraction
For salt, ABA, MeJA and drought stress treatments, 8-week-
old seedlings of L. chinensis were irrigated with 400 mM
NaCl, 100 lMABA,100lM MeJA and 20 % PEG6000,
respectively. For the cold treatment, the seedlings were put in a
4°C incubator. The treated materials were collected at 0, 1, 3,
6, 12 and 24 h, immediately dropped in liquid nitrogen and
stored at -80 °C. Total RNA of the whole seedlings were
isolated using a Trizol Kit (Invitrogen, USA) according to the
manufacturer’s protocol and digested by RNase-free DNase I
(TaKaRa, Japan) to remove the genomic DNA. The genomic
DNA of L. chinensis was extracted using a DNAsecure Plant
Kit (TIANGEN BIOTECH, Beijing). The concentrations of
DNA and total RNA were determined using a NanoDrop 2000
(Thermo Fisher, USA).
cDNA synthesis and cloning of the gene and promoter
cDNA for amplification of the 50and 30ends of LcDREB2 and
LcSAMDC2 was prepared using a SMART
TM
RACE cDNA
Amplification Kit (Clontech, Japan). The RACE primers of
LcDREB2 and LcSAMDC2 were designed based on the results
of 454 high-throughput sequencing reactions (Table S1). The
50and 30ends of LcDREB2 and LcSAMDC2 were cloned
following the manufacturer’s instructions. The reaction con-
ditions were as follows: 94 °Cfor4min;36cyclesof94 °C
for 30 s, 68 °C for 30 s, 72 °C for 50 s; followed by 72 °Cfor
10 min. Full-length LcDREB2 and LcSAMDC2 cDNA were
amplified using cDNA for the 30end of RACE as a template.
The genomic sequences of LcDREB2 and LcSAMDC2 were
cloned using the same primers. The promoter of LcSAMDC2
was cloned with a TAIL PCR Kit (TaKaRa, Japan). All the
PCR products werecloned into the pMD19-Tvector (TaKaRa,
Japan) and sequenced by Sangon Biotech (Shanghai).
Transcriptional activity assay and DRE-binding
specificity analysis of LcDREB2
The entire coding region of LcDREB2 was inserted into the
BamHI and SalI sites of pBridge to yield a fusion protein in
246 Plant Cell Tiss Organ Cult (2013) 113:245–256
123
Author's personal copy
frame with GAL4 BD. The primers used for PCR are listed
in Table S1. The recombinant plasmid was sequenced to
ensure that it could express proteins accurately. The
recombinant plasmid was transformed into yeast AH109
with the reporter genes His3 and LacZ according to the
manufacturer’s instructions (Clontech, CA, Palo Alto).
pBridge-AtDREB2A was used as a positive control. The
pBridge vector served as a negative control. Transformed
yeasts were cultured on SD medium without His and Trp
(SD/–His–Trp).
The LcDREB2 coding region was fused behind the GAL4
activation domain (AD) in the yeast expression vector
pADgal2.1, and pAD-AtDREB2A served as the positive
control. The recombinant plasmids containing the reporter
genes were introduced into yeast strains Ym4271-DRE and
Ym4271-mDRE, respectively. Transformed yeasts were
cultured on SD medium without His (SD/–His). In addition,
10 mM 3-aminotriazole (3-AT) was added into the medium.
Yeast transformation, a colony-lift filter assay and a
b-galactosidase assay were performed according to the Yea st
Protocols Handbook (Clontech Laboratories, Inc.).
Semi-quantitative PCR and real-time PCR analysis
Reverse transcription was performed using a PrimeScript
TM
RT Reagent Kit (TaKaRa, Japan). Real-time PCR was
conducted with an MX3000p instrument (Strategene, USA)
and a SYBR
Ò
PrimeScript
TM
PCR Kit (TaKaRa, Japan).
The reaction conditions were as follows: 95 °C for 10 s and
40 cycles of 95 °C for 5 s and 60 °C for 20 s. The PCR
primers are listed in Table S1. The constitutive expression
of L. chinensis actin was monitored as a positive control.
The expression profiles of LcDREB2 and its splicing iso-
forms under stress treatments were detected by real-time
and semi-quantitative PCR.
In transgenic Arabidopsis, the expression levels of the
tolerance gene AtRD29A were determined by real-time
PCR. The expression level of AtRD29A was evaluated by
comparison with that of Arabidopsis actin. The expression
level of LcSAMDC2 was tested by semi-quantitative PCR
in L. chinensis transgenic callus.
The activity analysis of LcSAMDC2 promoter
The coding sequences of LcDREB2a and LcDREB2c were
inserted into the BamHI and SalI sites of the pET28a (?)
vector and then transformed into E. coli BL21 (DE3) cells
for prokaryotic expression. The promoter region (30 bp
length) containing the DRE element of LcSAMDC2 was
designed as the probe for EMSA. EMSA was performed
according to the Non-Radioactive EMSA User Manual
(Viagene, USA).
To determine the activity of the LcSAMDC2 promoter,
the CaMV 35S promoter of pCAMBIA1301 was replaced
by the promoter of LcSAMDC2 to promote the expression
of GUS. Semi-quantitative PCR and GUS histochemical
staining were used to test the activity of the LcSAMDC2
promoter.
Leymus chinensis, tobacco and Arabidopsis
transformation
The entire coding region of LcDREB2a was inserted into
the BamHI and KpnI sites of the pUN1301 vector (modified
from the pCAMBIA1301 vector and donated by the Chong
lab). The recombinant plasmid was transformed into
Agrobacterium tumefaciens EHA105 and transformation of
L. chinensis callus was performed as the protocol (Wang
et al. 2009).
To detect the subcellular localization of LcDREB2, the
entire coding region of LcDREB2a or LcDREB2c was
inserted into the NcoI and SpeI sites of the pCAMBIA1302
vector. The target gene was inserted in frame with green
fluorescent protein (GFP) to yield a fusion protein. The
recombinant plasmid was transformed into A. tumefaciens
EHA105. Transformation, selection and identification of
A. thaliana were performed according to Peng et al. (2011).
Stem and root cells of transformed A. thaliana were
observed under a laser confocal scanning microscope (Bio-
Rad MRC 1024).
Full-length LcDREB2a and LcSAMDC2 were ligated
into the pSN1301 vector (modified from the pCAM-
BIA1301 vector and donated by the Chong lab) under the
control of the CaMV 35S promoter. The pSN1301 vector
was used as a control. A. thaliana transformation was
carried out by the same method described above. T3 gen-
eration transgenic plants were used for stress tolerance
analysis.
To examine the salt tolerance of LcDREB2a transgenic
Arabidopsis, 1-week-old seedlings were transferred to MS
medium containing 175 mM NaCl. Their phenotype was
photographed after culturing 10 days. As for the
LcSAMDC2 transgenic Arabidopsis, 1-week-old seedlings
were transferred to flowerpots for recovery growth for
1 week and then irrigated with 300 mM NaCl. Stress tol-
erance experiments were repeated at least three times and
their survival rates were calculated. In addition, several
concentrations of salinity were tested to ensure that the
proper concentration was used for the final treatments.
To confirm the interaction of LcSAMDC2 and LcDREB2,
the CaMV 35S promoter of GUS in pSN1301-LcDREB2a
was replaced by the promoter of LcSAMDC2 to promote
expression of GUS. The recombinant plasmid pSN1301-
LcDREB2a-pro::LcSAMDC-GUS was introduced into
EHA105 and transformed tobacco. Transformation of
Plant Cell Tiss Organ Cult (2013) 113:245–256 247
123
Author's personal copy
tobacco was performed according to Hu et al. (2010).
pCAMBIA1301 and pro::LcSAMDC-GUS were used as
positive and negative controls, respectively.
Results
Gene cloning and expression profile of LcDREB2
and LcSAMDC2
Based on the results of 454 high-throughput sequencing
reactions, the cDNAs of LcDREB2 and LcSAMDC2 were
isolated from L. chinensis by RACE-PCR. The geno-
mic sequences of LcDREB2 and LcSAMDC2 were also
cloned. These sequences have been deposited to GenBank
(GenBank ID: JF754582, JF754583, JF754584, JF754585
and GU580936). Three alternative splicing products
(LcDREB2a,LcDREB2b and LcDREB2c)ofLcDREB2
were found by semi-quantitative PCR and sequencing. The
gene structures of LcDREB2 and LcSAMDC2 are shown in
Fig. 1A, B. Because the orthologous genes of LcDREB2b in
H. vulgare (Xue and Loveridge 2004), Z. mays (Qin et al.
2007) and O. sativa (Satoko et al. 2010) were considered as
non-functional isoforms, we just choosed LcDREB2a and
LcDREB2c for the following analysis. Phylogenetic analy-
sis of the DREBs including LcDREB2 and other DREB
proteins were conducted with MEGA 5.0 (Tamura et al.
2011). LcDREB2a and LcDREB2c were classified into the
A-2 group (Fig. 1C).
Semi-quantitative PCR and real-time PCR showed that
the LcDREB2a transcripts accumulated under various
stress and phytohormone treatments such as ABA, salinity,
PEG and MeJA. LcDREB2a presented different expression
pattern which implied that the response mechanism was
different under different abiotic stress (Fig. 2A). Under
normal conditions, the expression of LcDREB2b was
higher than that of LcDREB2a and LcDREB2c, and the
expression of LcDREB2c was much lower than that of
LcDREB2a and LcDREB2b. However, the three transcripts
of LcDREB2 were all induced under various stress treat-
ments (Fig. 2B). The semi-quantitative PCR results sug-
gested that LcSAMDC2 was significantly induced by salt,
drought and cold (Fig. 2C–E).
Subcellular localization and transcriptional activity
of LcDREB2
LcDREB2a and LcDREB2c respectively fused with the
N-terminal of GFP were transformed into Arabidopsis. The
CaMV35S::LcDREB2a-GFP and CaMV35S:: LcDREB2c-
GFP fusion proteins were targeted to the nucleus (Fig. 3A
1–6). By contrast, GFP fluorescence of control plasmid
CaMV35S::GFP was detected in the whole cell (Fig. 3A
7–9). These results suggested that both LcDREB2a and
LcDREB2c are nucleoproteins.
The recombinant plasmids were transformed into yeast
strain AH109 to test the transcriptional activation ability of
LcDREB2. The results showed that LcDREB2a or
LcDREB2c both could activate expression of the reporter
gene (Fig. 3B).
LcSAMDC2 promoter activity analysis
The promoter sequence of LcSAMDC2 was cloned using
TAIL-PCR (GenBank ID: JF923574). The GUS staining
analysis of transgenic Arabidopsis harboring Pro-
LcSAMDC2:: GUS revealed that the LcSAMDC2 promoter
was active in germinating seeds, 3 to 15 day-old seedlings
and florescences (Fig. 4A 2–6). Salt, drought and ABA
could induce GUS expression in seedlings (Fig. 4A 7–9).
The gene expression analysis (Fig. 4B) were consistent
with GUS staining, indicating strong induction of GUS in
germinating seeds and under stress treatments of salt,
drought and ABA.
DRE binding specificity of LcDREB2
Whether LCDREB2 specifically binds to DRE elements
were determined in vivo and in vitro experiments. Yeast
Ym4271-DRE with pAD-LcDREB2a,pAD-LcDREB2c and
pAD-AtDREB2A (as a positive control) grew well on
selective medium (Fig. 5A), but Ym4271-mDRE harboring
pAD-LcDREB2a,pAD-LcDREB2c and pAD-AtDREB2A
did not grow (Fig. 5A). In b-galactosidase activity assays,
the transformed yeast Ym4271-DRE exhibited a blue color.
These results indicated that LcDREB2a and LcDREB2c
specifically bind to the DRE element and activate expres-
sion of the reporter gene. The EMSA results suggested that
LcDREB2a and LcDREB2c bind to the LcSAMDC2 pro-
moter probe with the DRE element (Fig. 5B).
Regulation of LcSAMDC2 under LcDREB2
To address whether LcSAMDC2 is the downstream gene of
LcDREB2, transgenic experiments were performed in
tobacco and L. chinensis. The positive transformation was
confirmed by GUS staining (Fig. 5E, F and Fig. S1). Over-
expression of LcDREB2a in L. chinensis callus increased
the expression level of LcSAMDC2 (Fig. 5C).
A binary vector of pSN1301-LcDREB2a-ProLcSAM
DC2::GUS and a control vector of pProLcSAMDC2:GUS
were transformed into tobacco. Transgenic tobacco leaves
were cultivated on MS media for 14 days and collected for
GUS staining. A constitutive expression pattern was observed
in transgenic leaves containing pSN1301-LcDREB2a-ProLc
SAMDC2::GUS without dehydration stress but not in leaves
248 Plant Cell Tiss Organ Cult (2013) 113:245–256
123
Author's personal copy
containing pProLcSAMDC2::GUS (Fig. 5D–F). These results
demonstrated that ectopic expression of LcDREB2 is able to
activate expression of LcSAMDC2.
LcDREB2 and LcSAMDC2 enhance the salt tolerance
of over-expressed transgenic Arabidopsis
Expression of LcDREB2a induced the expression of Rd29A
(Fig. 6A), which was previously identified as a down-
stream gene of DREBs in Arabidopsis, and enhanced the
salt tolerance of transgenic Arabidopsis compared with the
control (Fig. 6B). In this study, an improved salt tolerance
was also observed when just the main ORF of LcSAMDC2
was transformed into Arabidopsis (Fig. 6C).
Discussion
High-throughput sequencing technology has recently been
applied to genome sequencing and to transcriptome and
Fig. 1 A The genomicseque nce structure and alternative splicing model of
LcDREB2. The difference among LcDREB2a,LcDREB2b and LcDREB2c
was the existence or lack of extron 2 or extron 3. LcDREB2a, containing
extron 1 and 4, was 1,288 bp in length with an ORF of 328 AA. LcDREB2b,
consisting of extron 1, 2 and 4, was 1,341 bp in length. LcDREB2c, including
all four extrons, was 1,429 bp and contained an ORF of 375 AA. BGenomic
sequence structure of LcSAMDC2. The gene of LcSAMDC2 had 3 exons and
2 introns, it only contained 3 exons and it belonged to constitutive splicing.
The main ORF is located at exon 3. CPhylogenetic analysis of LcDREB2
with other AP2/ERF transcription factors. The phylogenetic analyses were
conducted with MEGA5.0. The appended proteins are as follow s:T aDREB3
(ABC86564), GhDREB1L (ABD65473), AtTINY (NP_17271), GsDREB1
(ABX58043), GhDBP1 (AAQ08000), HhDREB2 (ACJ66376), TaDREB2
(ABB90544), ZmDBF1 (ACG35323), AtDRF1.1 (ABY66036), AtDRF1.3
(ABY66038), TaDREB5A (AAX13286), TaDREB5B (AAX13287), TaD-
REB3A (AAX13277), TaDREB3B (AAX13278), AsDRF1.1
(ACO35589), AsDRF1.3 (ACO35591), CdDREB1 (AAS46284),
CdDREB2 (AAS46285), SbDREB2 (ACA79916), FaDREB2
(CAG30547), PpDREB2 (AAS59530), TaDREB1 (AAL01124), As-
DREB2 (ABS11171), HbDREB2 (AAU29412), HvDREB1
(AAY25517), AtDREB2C (NP_565929), AtDREB2B (NP_187713),
LcDREB3a (EU999998), LcDREB3c (JF915847), GhDBP2 (AY619718),
ZmABI4 (AY125490), GhDBP1 (AY174160), OsDREB1A (AF300970),
TaDREB4B (AAX13283), AtDREB2A (NP_196160), GhDBP2
(AY619718), HvDRF1.1 (AAO38209), OsDREB2A (AAN02487),
GmDREB2 (ABB36645), GhDBP3a (DQ224382), GhDBP3b
(DQ224383), AtRAP2 (NP_564496), HvCBF (AF239616), OsDREB3
(XM_467836), ScCBF (AAF88096). ZmDREB2a (JF915836),
ZmDREB2c (JF915835), PjDREB2a (JF766082), PjDREB2c (JF766084),
OsDREB2Ba (JF915843), OsDREB2Bc (JF915845), TaDREB4A
(AAX13282), HvDRF1.3 (AAO38211), NtEREBP2 (Q40479) and NtERF1
(Q40476). It turned out that LcDREB2a and LcDREB2c were classifie d into
the A-2 group. DPhylogenetic analysis of LcSAMDC2 with other
SAMDCs. The phylogenetic analyses were conducted with MEGA 5.0.
The appended proteins are as follows: ApSAMDC (NP_001119611),
AtSAMDC (CAA69073), BjSAMDC (CAA65044), BtSAMDC
(AAA30359), CrSAMDC (AAC48989), CsSAMDC (ACQ91107),
DcSAMDC1 (AAG61146), DcSAMDC2 (AAD09839), DmSAMDC
(NP_477223), DsSAMDC (CAA69076), GmSAMDC (AAL89723),
LcSAMDC2 (ADD70015), OsSAMDC (CAA69074), SoSAMDC1
(ACT53876), SoSAMDC2 (CAA57170), StSAMDC (AAB32507), TaS-
AMDC (AAD17232), TmSAMDC (ABJ15728), TtSAMDC (CAA58762),
VvSAMDC (CAD98785), ZmSAMDC1 (ACF87816) and ZmSAMDC2
(CAA69075). The results show that LcSAMDC2 has higher identity with the
SAMDCs from Poaceae
Plant Cell Tiss Organ Cult (2013) 113:245–256 249
123
Author's personal copy
microRNA expression profiling (Sun et al. 2011). Based on
the results of 454 high-throughput sequencing reactions,
the full-length cDNA and genomic sequences of LcDREB2
and LcSAMDC2 were obtained from L. chinensis.
LcDREB2a and LcDREB2c both contain the complete
ORF. LcDREB2b had only one short ORF (66 AA) that did
not contain an AP2 or activation domain. This splicing
mode has been described as exon skipping by Reddy
(2007). Therefore, the orthologous genes of LcDREB2b in
H. vulgare (Xue and Loveridge 2004), Z. mays (Qin et al.
2007) and O. sativa (Satoko et al. 2010) were considered
non-functional isoforms. Based on the phylogenetic analy-
sis of the DREBs including LcDREB2 and other DREB
proteins with MEGA 5.0 (Tamura et al. 2011), LcDREB2a
and LcDREB2c were classified into the A-2 group of DREB
(Fig. 1C). So far, no alternative splicing or introns in the
DREB family have been reported except for the DREBs
from Poaceae plants such as Z. mays (Qin et al. 2007)This
study show that DREB2 alternative splicing also existed in
L. chinensis.
LcDREB2b was expressed under normal conditions and
induced by abiotic stresses while LcDREB2a and
LcDREB2c were only induced by abiotic stresses (Fig. 2B),
indicating that LcDREB2b might not be functional.
Therefore, the induction of LcDREB2a or LcDREB2c
expression might both be primarily responsible for the
increased stress resistance of L.chinensis.
The subcellular localization results suggested that both
LcDREB2a and LcDREB2c are nucleoproteins (Fig. 3A
1–9). A transcriptional activity assay showed that
Fig. 2 A Expression patterns of LcDREB2a in response to various
stresses by real-time PCR. Parallel reactions using actin primers were
performed to normalize the amounts of the added template. The x-axis
show the time of treatment. The y-axis show the relative expression
profile of LcDREB2a, which was normalized by the expression level
of actin. As for the salt treatment, the expression reached to the max
level at 6 h which was early than other treatments. This indicated that
LcDREB2a was very sensitive to salt stress. BExpression patterns of
three splicing isoforms of LcDREB2 (LcDREB2a,LcDREB2b and
LcDREB2c) in response to various stresses were detected by semi-
quantitative PCR. CK is the control plants with no treatments. The
treated seedlings collected at 6 h were used in this RT-PCR. Parallel
reactions using actin primers were performed to normalize the
amounts of added template. In the figure, there is no LcDREB2a or
LcDREB2c expressed in CK. While in other treatments, LcDREB2a
and LcDREB2c both have varying degrees expression. It suggested
that the expression of LcDREB2a or LcDREB2c was inducible under
abiotic stress. CEExpression patterns of LcSAMDC2 under salt,
drought and cold treatments. In the treatment of salt and drought,
LcSAMDC2 expressed higher as time goes on. LcSAMDC2 expressed
low at the time of 6 h by the treatment of cold, but the monolithic
expression was high. It shows that LcSAMDC2 was significantly
induced by salt, drought and cold
250 Plant Cell Tiss Organ Cult (2013) 113:245–256
123
Author's personal copy
LcDREB2a or LcDREB2c could activate expression of the
reporter gene (Fig. 3B). Although these transgenic plants
exhibited significantly enhanced salt tolerance compared
with control plants (Fig. 6B), over-expression of LcDREB2
caused no growth retardation in transgenic Arabidopsis
plants. Our results are different from previous reports that
Fig. 3 A Subcellular localization of LcDREB2. Arabidopsis was
transformed with CaMV35S::GFP (79) and CaMV35S::LcDREB2a-
GFP (13)orCaMV35S::LcDREB2c-GFP (46). The T1 transgenic
lines were observed under a confocal microscope. Photographs 1,4
and 7were taken under a dark field for green fluorescence.
Photographs 2,5and 8were taken under bright light. Photographs
3,6and 9were a combination of bright light and a dark field to
illustrate the morphology of the cells. The bar represents 10 lm.
Photographs (16) show that the CaMV35S::LcDREB2a-GFP and
CaMV35S::LcDREB2c-GFP fusion proteins were targeted to the
nucleus, while photographs (79) shows a positive control plasmid
CaMV35S::GFP whose GFP signal was detected in the whole cell.
These results suggested that both LcDREB2a and LcDREB2c are
nucleoproteins. BTranscriptional activation of LcDREB2a and
LcDREB2c. 1:pBridge-AtDREB2A;2:pBridge-LcDREB2a;3:
pBridge-LcDREB2c;4:pBridge. The middle plate shows the growth
status of the transformed yeast cells on selective medium. The right
plate shows the results of the reporter activity assay. 1and 4were
stand for pBridge-AtDREB2A and pBridge, which were positive and
negative control, respectively. 1,2and 3show transcriptional
activation, while 4had no signal. The results showed that LcDREB2a
or LcDREB2c both could activate expression of the reporter gene
Plant Cell Tiss Organ Cult (2013) 113:245–256 251
123
Author's personal copy
the over-expression of the OsDREB2A and AtDREB2A
genes resulted in only little phenotypic changes (Maruyama
et al. 2004).
SAMDC is the rate-limiting enzyme in the synthesis of
spm and spd, which are involved in cell division,
embryogenesis, fruit ripening and stress responses
(Hirasawa and Shimada 1994; Yamaguchi et al. 2006).
Over-expression of SAMDC in yeast greatly improved the
tomato fruit quality and its commercial value because fruit
maturity was delayed and the lycopene content of the fruit
was increased 2.5 fold (Mehtal et al. 2002). In addition,
over-expression of SAMDC also increased the stress
resistance of transgenic plants. Transgenic rice expressing
SAMDC from Tritordeum displayed normal growth under
Fig. 4 A Detection of LcSAMDC2 promoter activity by GUS
staining. The following photograph show the different phage or under
different treatment of the transgenic plants. 1CK, 2germination, 3
cotyledon, 4two species of true leaf, 5flowering period, 6stigma, 7
seedlings under salt treatment, 8seedlings under drought treatment
(treated with 20 % PEG for 3 h), 9seedlings under ABA treatment
(treated with 100 lM ABA for 3 h). 2,3,4,5and 6show GUS
staining signal except for CK. The GUS staining analysis of
transgenic Arabidopsis harboring Pro-LcSAMDC2:: GUS revealed
that the LcSAMDC2 promoter was active in germinating seeds,
3–15 day-old seedlings and florescences. 7,8and 9display GUS
staining signal which prove that salt, drought and ABA could induce
GUS expression in seedlings. The photographs of Brevealed
LcSAMDC2 promoter activity by semi-quantitative PCR. The signals
indicate strong induction of GUS in germinating seeds and under
stress treatments of salt, drought and ABA
252 Plant Cell Tiss Organ Cult (2013) 113:245–256
123
Author's personal copy
high salt stress (Roy and Wu 2002). Heterologous
expression of the human SAMDC gene led to stronger
salinity and drought resistance of tobacco (Waie and Rajam
2003). Accordingly, the over-expressed carnation SAMDC
gene strengthened tobacco resistance to abiotic stress. In
this study, improved salt tolerance was observed when the
Fig. 5 A DRE binding activities of LcDREB2a and LcDREB2c.
1and 6 pAD-AtDREB2A;2and 5 pAD-LcDREB2a;3and 4 pAD-
LcDREB2c;1,2and 3Ym4271 (DRE); 4,5and 6Ym4271 (mDRE).
The middle plate show the growth status of the transformed yeast cell
and the right plate shows the reporter activity assay. Ym4271-DRE
with pAD-LcDREB2a,pAD-LcDREB2c and pAD-AtDREB2A all
show GUS signals, while Ym4271-mDRE harboring pAD-
LcDREB2a,pAD-LcDREB2c and pAD-AtDREB2A had no such
signals. These results indicated that LcDREB2a and LcDREB2c
specifically bind to the DRE element and activate expression of the
reporter gene. BEMSA results. Shift banding which protein with its
special-binding DNA sequences indicated that LcDREB2a or
LcDREB2c bound to the probe of the LcSAMDC2 promoter. The
empty vector CK meant that the protein transformed into E. coli BL21
(DE3) contained only the pET28a (?) vector. The 1009cold
competitive CK meant that 100 times competitive probe without label
were added into the reaction of this lane. The results suggested that
LcDREB2a or LcDREB2c could bind to the LcSAMDC2 promoter
probe via the DRE element, which reduced the shift of the labeled
probe. CExpression patterns of LcDREB2a and LcSAMDC2 in
control (CK) and 35S::LcDREB2a transgenic callus of L. chinensis
(lines 1 and 2) under normal growth conditions. Parallel reactions
using actin primers were performed to normalize the amounts of
added template. CK means empty vector control. The quantity of
LcSAMDC2 increased with the expression of LcDREB2a which meant
that over-expression of LcDREB2a in L. chinensis calluse increased
the expression level of LcSAMDC2. DFLcDREB2a regulated the
expression of ProLcSAMDC2::GUS in transgenic tobacco. DWT, it
meant the wild type plants without any treatments. ETransgenic
tobacco with ProLcSAMDC2::GUS, and FpSN1301-LcDREB2a-
pro::LcSAMDC2::GUS were stained directly. Bars indicated 5 mm.
A constitutive expression pattern was observed in transgenic leaves
containing pSN1301-LcDREB2a-ProLcSAMDC2::GUS without
dehydration stress but not in leaves containing pProLcSAMDC2::-
GUS. These results demonstrated that ectopic expression of LcDREB2
was able to activate expression of LcSAMDC2
Plant Cell Tiss Organ Cult (2013) 113:245–256 253
123
Author's personal copy
main ORF of LcSAMDC was transformed into Arabidopsis
(Fig. 6C). A double mutant of the SAMDC gene in Ara-
bidopsis led to death of the mutant (Ge et al. 2006). These
results revealed that SAMDC plays significant and com-
plex roles in plant development and stress resistance.
The gene of LcSAMDC2 also had three exons and two
introns (Fig. 1B). There was no alternative splicing form
and only one mRNA product. Phylogenetic analysis of the
SAMDCs indicated that LcSAMDC2 has higher identity
with the SAMDCs from Poaceae (Fig. 1D). Protein
sequence alignment suggested that, there are three ORFs in
LcSAMDC2, as like SAMDCs cloned from other plants.
The small ORFs play an important regulatory role in the
translation of the main ORF (Ge et al. 2006). Removal of
the small uORF of SAMDC in transgenic plants led to a
relatively high translation efficiency. This activity resulted
in abnormal polyamine levels, such as the absence of
putrescine, reduced spm content and increased SAM levels
by more than 400 times (Hanfrey et al. 2002). Hu et al.
(2012) showed that when the endogenous level of spd was
low, expression of SAMDC was not up-regulated without
the intron in the leader sequence. These studies suggested
that the uORF-mediated translational regulation of SAMDC
was essential for polyamine homeostasis and normal
growth and development of plants.
Compared with investigations on translational regula-
tion, few studies have focused on transcriptional regulation
and no reports are available on the transcription factors
regulating the expression of LcSAMDC2. A previous study
show that the DREB2A protein specifically binds to the
Fig. 6 A Expression patterns of LcDREB2a and AtRD29A in control
(CK) and transgenic Arabidopsis plants (TL6 and TL10) under
normal growth conditions. CK means transgenic plants harboring
empty vector control. When there was LcDREB2a, the quantity of
AtRD29A obviously increased. Because AtRD29A was a downstream
gene of LcDREB2, and its expression was induced by LcDREB2a.
BSalt tolerance analysis of transgenic Arabidopsis plants with
LcDREB2a. 1-week-old control (CK) and transgenic Arabidopsis
seedlings grown on MS medium were transferred to MS medium
containing 175 mM NaCl and cultured for 2 weeks in a growth
chamber under 16 h light (22 °C) and 8 h dark (20 °C). CK meant
empty vector control. TL6 and TL10 grow strong, while CK were
nearly died. That meant LcDREB2a enhanced the salt tolerance of
transgenic Arabidopsis.CSalt tolerance analysis of transgenic
Arabidopsis plants with the main ORF of LcSAMDC2. 1-week-old
control (CK) and transgenic Arabidopsis seedlings grown on MS
medium were transferred to soil and cultured in the growth chamber
under 16 h light (22 °C) and 8 h dark (20 °C). After 1 week, they
were irrigated with 300 mM NaCl. CK meant empty vector control.
Before treatment, all samples exhibited similar phonotype. After the
same treatment, some plants of strains TL5, TL8 and TL10 were still
alive while all other plants died. It suggested that salt tolerance of
Arabidopsis was improved when just the main ORF of LcSAMDC2
was transformed
254 Plant Cell Tiss Organ Cult (2013) 113:245–256
123
Author's personal copy
DRE core motif (Sakuma et al. 2002). One DRE element
allows the DREB to bind and activate downstream gene
expression (Huang et al. 2008). In our study, two core
motif of DRE element (CCGAC) in the promoter of
LcSAMDC2 were found, suggesting a possible interaction
of the DREB with the LcSAMDC2 promoter. The results of
yeast one-hybrid assays indicated that LcDREB2a or
LcDREB2c could specifically bind the DRE element and
activates the expression of reporter gene. The EMSA
results suggested that LcDREB2a or LcDREB2c could
bind to the LcSAMDC2 promoter probe via the DRE ele-
ment (Fig. 5B). In addition, the expression level of
LcSAMDC2 was increased in LcDREB2a over-expressed in
L. chinensis (Fig. 5C and Fig.S1). Compared with the WT
(wild type, Fig. 5D), transgenic tobacco with pro::Lc-
SAMDC2::GUS exhibited slightly GUS expression
(Fig. 5E). However, when pSN1301-LcDREB2a-pro::Lc-
SAMDC2::GUS was introduced into tobacco, very high
GUS expression was detected (Fig. 5F). Over-expression
of LcSAMDC2 or LcDREB2a both increased the salt
resistance of transgenic Arabidopsis plants. These results
implied that LcDREB2 in cooperation with LcSAMDC2
might contribute to the resistance of plants to abiotic stress
(Fig. 7).
In summary, a novel DREB gene with alternative
splicing was identified from L. chinensis. In addition,
LcDREB2 can interact with the promoter via a DRE ele-
ment and activate expression of LcSAMDC2. To our
knowledge, this is the first report of a DREB’s involvement
in an ABA-dependent signaling pathway to regulate
SAMDC gene expression, especially in response to abiotic
stress.
Acknowledgments This study was funded by the National Natural
Science Foundation of China (30970291) and the National Basic
Research Program of China (‘‘973’’, 2007CB108905).
References
Abdel Hamid AK, Mamdouh SS, Mamdouh MN et al (2011) A
DREB gene from the xero-halophyte Atriplex halimus is induced
by osmotic but not ionic stress and shows distinct differences
from glycophytic homologues. Plant Cell, Tissue Organ Cult
106:191–206
Agarwal PK, Agarwal P, Reddy MK, Sopory SK (2006) Role of
DREB transcription factors in abiotic and biotic stress tolerance
in plants. Plant Cell Rep 25:1263–1274
Alvarez I, Tomaro ML, Benavides MP (2003) Changes in poly-
amines, proline and ethylene in sunflower calluses treated with
NaCl. Plant Cell Tiss Organ Cult 74:51–59
Dresselhaus T, Barcelo P, Hagel C, Lorz H, Humbeck K (1996)
Isolation and characterization of a Tritordeum cDNA encoding
S-adenosylmethionine decarboxylase that is circadian-clock-
regulated. Plant Mol Biol 30:1021–1033
Ge CM, Cui X, Wang YH, Hu YX, Fu ZM, Zhang DF, Cheng ZK,
Li JY (2006) BUD2, encoding an S-adenosylmethionine decar-
boxylase, is required for Arabidopsis growth and development.
Cell Res 16:446–456
Hanfrey C, Franceschetti M, Mayer MJ, Illingworth C, Michael AJ
(2002) Abrogation of upstream open reading frame-mediated
translational control of a plant S-adenosylmethionine decarbox-
ylase results in polyamine disruption and growth perturbations.
J Biol Chem 277:44131–44139
Hirasawa E, Shimada A (1994) The photoresponse of S-adenosyl-
methionine decarboxylase activity in leaves of Pharbitis nil.
Plant Cell Physiol 35:505–508
Hirayama K, Shinozaki K (2007) Perception and transduction of
abscisic acid signals, keys to the function of the versatile plant
hormone ABA. Trends Plant Sci 12:343–351
Hu WW, Gong H, Pua EC (2005) The pivotal roles of plant
S-adenosylmethionine decarboxylase untranslated leader
sequence in regulation of gene expression at the transcriptional
and post-transcriptional levels. Plant Physiol 138:276–286
Hu Y, Su Q, Zu Y, Liu J (2010) Co-expression of genes related to
glycine betaine biosynthesis from Salicornia europaea increase
salt tolerance of transgenic tobacco. Chin Agric Sci Bull
26:55–59
Hu YZ, Zeng YL, Guan B, Zhang FC (2012) Overexpression of a
vacuolar H
?
-pyrophosphatase and a B subunit of H
?
-ATPase
cloned from the halophyte Halostachys caspica improves salt
tolerance in Arabidopsis thaliana. Plant Cell Tissue Organ Cult
108:63–71
Huang B, Jin LG, Liu JY (2008) Identification and characterization of
the novel gene GhDBP2 encoding a DRE-binding protein from
cotton (Gossypium hirsutum). J Plant Physiol 165:214–223
Jin TC, Chang Q, Li WF, Yin DX et al (2010) Stress-inducible
expression of GmDREB1 conferred salt tolerance in transgenic
alfalfa. Plant Cell Tissue Organ Cult 100:219–227
Kusano TT, Berberich C, Takahashi Y (2008) Polyamines: essential
factors for growth and survival. Planta 228:367–381
Liu ZP, Chen ZY, Pan J, Li XF, Su M, Wang LJ, Li HJ, Liu GS
(2008) Phylogenetic relationships in Leymus (Poaceae: Triticeae)
revealed by the nuclear ribosomal internal transcribed spacer and
chloroplast trnL-F sequences. Mol Phylogenet Evol 46:278–289
Maruyama K, Sakuma Y, Kasuga M (2004) Identification of cold-
inducible downstream genes of the Arabidopsis DREB1A/CBF3
Fig. 7 The proposed signaling pathway of DREB and SAMDC in
L. chinensis under stress. The LcDREB2 protein is expressed in plant
cells under drought, grazing and salt stresses. These stresses
upregulate the LcSAMDC2 to produce more spd or spm to ensure
plant resistance to stress
Plant Cell Tiss Organ Cult (2013) 113:245–256 255
123
Author's personal copy
transcriptional factor using two microarray systems. Plant J
38:982–993
Mehtal RA, Cassol T, Li N, Ali N, Handa AK, Mattoo AK (2002)
Engineered polyamine accumulation in tomato enhances phy-
tonutrient content, juice quality, and vine life. Nat Biotechnol
20:613–618
Pal Bais H, Ravishankar G (2002) Role of polyamines in the ontogeny
of plants and their biotechnological applications. Plant Cell
Tissue Organ Cult 69:1–34
Peng XJ, Ma XY, Fan WH, Su M, Cheng LQ, Iftekhar A, Qi DM,
Byung-Hyun L, Shen SH, Liu GS (2011) Improved drought and
salt tolerance of Arabidopsis thaliana by transgenic expression
of a novel DREB gene from Leymus chinensis. Plant Cell Rep
30:1–10
Petri C, Alburquerque N, Pe
´rez-Tornero O, Burgos L (2005) Auxin
pulses and a synergistic interaction between polyamines and
ethylene inhibitors improve adventitious regeneration from
apricot leaves and Agrobacterium-mediated transformation of
leaf tissues. Plant Cell Tissue Organ Cult 82:105–111
Qin F, Kakimoto M, Sakuma Y, Maruyama K, Osakabe Y, Tran LSP,
Shinozaki K, Yamaguchi-Shinozaki K (2007) Regulation and
functional analysis of ZmDREB2A in response to drought and
heat stresses in Zea mays L. Plant J 50:54–69
Reddy AS (2007) Alternative splicing of pre-messenger RNAs in
plants in the genomic era. Annu Rev Plant Biol 58:267–294
Richard W, Alexandra C, Tiburcio AF (1997) Polyamines: small
molecules triggering pathways in plant growth and development.
Plant Physiol 113:1009–1013
Roy M, Wu R (2002) Over-expression of S-adenosylmethionine
decarboxylase gene in rice increases polyamine level and
enhances sodium chloride-stress tolerance. Plant Sci 5:987–992
Sakuma Y, Liu Q, Dubouzet JG, Abe H, Shinozaki K, Yamaguchi-
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
Biophy Res Co 290:998–1009
Satoko M, Junya M, Yoshida T, Todaka D, Ito Y, Maruyama K,
Shinozaki K, Yamaguchi-Shinozaki K (2010) Comprehensive
analysis of rice DREB2-type genes that encode transcription
factors involved in the expression of abiotic stress-responsive
genes. Mol Genet Genomics 283:185–196
Sun Y, Luo H, Li Y, Sun C, Song J, Niu Y, Zhu Y, Dong L, Lv A,
Tramontano E (2011) Pyrosequencing of the Camptotheca
acuminata transcriptome reveals putative genes involved in
camptothecin biosynthesis and transport. BMC Genomics 12:533
Takahashi T, Kakehi JI (2010) Polyamines: ubiquitous polycations with
unique roles in growth and stress responses. Ann Bot 105:1–6
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S
(2011) MEGA5: molecular Evolutionary genetics analysis using
maximum likelihood, evolutionary distance, and maximum
parsimony methods. Mol Biol Evol 28:2731–2739
Tian AG, Zhao JY, Zhang JS, Gai JY, Chen SY (2004) Genomic
characterization of the S-adenosylmethionine decarboxylase
genes from soybean. Theore Appl Genet 108:842–850
Waie B, Rajam MV (2003) Effect of increased polyamine biosyn-
thesis on stress responses in transgenic tobacco by introduction
of human S-adenosylmethionine gene. Plant Sci 164:727–734
Wang LJ, Li XF, Chen SY, Liu GS (2009) Enhanced drought
tolerance in transgenic Leymus chinensis plants with constitu-
tively expressed wheat TaLEA3. Biotechnol Lett 31:313–319
Xue GP, Loveridge CW (2004) HvDRF1 is involved in abscisic acid-
mediated gene regulation in barley and produces two forms of
AP2 transcriptional activators, interacting preferably with a CT-
rich element. Plant J 37:326–339
Yamaguchi K, Takahashi Y, Berberich T, Imai A, Miyazaki A,
Takahashi T, Michael A, Kusano T (2006) The polyamine
spermine protects against high salt stress in Arabidopsis thaliana.
FEBS Lett 580:6783–6788
Zhou C, Guo JS, Feng ZH, Cui XH, Zhu J (2012) Molecular
characterization of a novel AP2 transcription factor ThWIND1-
Lfrom Thellungiella halophila. Plant Cell Tissue Organ Cult
10:423–433
256 Plant Cell Tiss Organ Cult (2013) 113:245–256
123
Author's personal copy

Supplementary resources (2)

... Sheepgrass, which is widely distributed in Eurasia, adapts well to drought, cold, saline and alkaline conditions [32,33]. To explore the mechanism that underlies the abiotic stress tolerance of sheepgrass, several transcriptome analyses have been performed in the past few years [32][33][34][35], and several genes identified by transcriptome analyses have actually enhanced the abiotic stress tolerance of transgenic plants [36][37][38][39][40][41][42]. Although MYB transcription factors play pivotal roles in drought responses, there is still no report on MYB proteins from sheepgrass that elucidates their contributions to sheepgrass drought tolerance. ...
... Previous studies have proposed that DREB proteins, such as DREB1 and DREB2, regulate low-temperature and droughtresponsive genes by binding to the DRE/CTR elements through ABA-independent pathway [62,63]. In sheepgrass, the highest transcript level of LcDREB2a occurs at the 12th hour under 20% PEG6000 treatment [42], whereas LcMYB2 transcript accumulation reaches the highest point at 8 h after 300 Mm mannitol treatment (Fig. 1a). Expression profile sequence analysis showed that both contig62249 (LcDREB2C/LcDREB2B/LcDREB2A) and contig41859 (LcMYB2) were up-regulated by drought stress and returned to basal levels after rewatering; however, the fold change of contig41859 was larger than that of contig62249 in response to drought stress (Additional file 1: S1). ...
Article
Full-text available
Background: Drought is one of the most serious factors limiting plant growth and production. Sheepgrass can adapt well to various adverse conditions, including drought. However, during germination, sheepgrass young seedlings are sensitive to these adverse conditions. Therefore, the adaptability of seedlings is very important for plant survival, especially in plants that inhabit grasslands or the construction of artificial grassland. Results: In this study, we found a sheepgrass MYB-related transcription factor, LcMYB2 that is up-regulated by drought stress and returns to a basal level after rewatering. The expression of LcMYB2 was mainly induced by osmotic stress and was localized to the nucleus. Furthermore, we demonstrate that LcMYB2 promoted seed germination and root growth under drought and ABA treatments. Additionally, we confirmed that LcMYB2 can regulate LcDREB2 expression in sheepgrass by binding to its promoter, and it activates the expression of the osmotic stress marker genes AtDREB2A, AtLEA14 and AtP5CS1 by directly binding to their promoters in transgenic Arabidopsis. Conclusions: Based on these results, we propose that LcMYB2 improves plant drought stress tolerance by increasing the accumulation of osmoprotectants and promoting root growth. Therefore, LcMYB2 plays pivotal roles in plant responses to drought stress and is an important candidate for genetic manipulation to create drought-resistant crops, especially during seed germination.
... The TFs played an important role in activating multiple biological processes to insulate plant cells from cold. They functioned as a pivotal regulator for adaption of the plant through the binding of TFs to cognate cis-acting elements present in the promoter region of their target genes (Zou et al., 2010;Yang et al., 2012;Peng et al., 2013). The downstreamrelated genes were regulated by TFs to adapt to external environmental changes. ...
Article
Full-text available
Cold stress poses a serious threat to the survival and bloom of Verbena bonariensis. The enhancement of the cold tolerance of V. bonariensis is the central concern of our research. The WRKY transcription factor (TF) family was paid great attention to in the field of abiotic stress. The VbWRKY32 gene was obtained from V. bonariensis. The VbWRKY32 predicted protein contained two typical WRKY domains and two C2H2 zinc-finger motifs. Under cold stress, VbWRKY32 in leaves was more greatly induced than that in stems and roots. The overexpression (OE) in V. bonariensis increased cold tolerance compared with wild-type (WT). Under cold stress, the OE lines possessed showed greater recovery after cold-treatment restoration ratios, proline content, soluble sugar content, and activities of antioxidant enzymes than WT; the relative electrolyte conductivity (EL), the accumulation of malondialdehyde (MDA), hydrogen peroxide (H2O2), and superoxide anion (O2−) are lower in OE lines than that in WT. In addition, a series of cold-response genes of OE lines were compared with WT. The results revealed that VbWRKY32 worked as a positive regulator by up-regulating transcription levels of cold-responsive genes. The genes above can contribute to the elevation of antioxidant activities, maintain the membrane stability, and raise osmotic regulation ability, leading to the enhancement of the survival capacity under cold stress. According to this work, VbWRKY32 could serve as an essential gene to confer enhanced cold tolerance in plants.
... The DREB and ERF subfamilies have been thoroughly investigated for their role in plant stress responses. Overexpression of DREB2A from mung bean resulted in improved tolerance to drought and high-salt in transgenic Arabidopsis , while DREB TFs from other species like Suaeda, potato and sheep-grass conferred enhanced salinity and oxidative-stress tolerance in transgenic tobacco, potato and Arabidopsis, respectively, without hampering normal growth and development of the transgenic plants (Zhang et al. 2015a;Bouaziz et al. 2015;Peng et al. 2013). Recently, the ERF TFs from wheat and Lotus have been shown to promote salt and drought stress tolerance when overexpressed in transgenic wheat and Arabidopsis (Rong et al. 2014;Sun et al. 2014). ...
Chapter
Full-text available
Worldwide food security and sustainable development are alarming issues with respect to climate change. Abiotic stresses negatively influence plant growth and development thus reducing the crop yield and productivity. Amidst the abiotic stresses, salinity stress is the major problem in agriculture lands. There is a rapid increase in salt-affected areas due to insufficient rainfall, imperfect irrigation system and water contamination, resulting in entry of salts in the soil. While traditional approaches used to handle the situations have limitations, current agricultural practices must seek tailored solutions to meet the demands of growing population. To generate climate-smart crops, genetic engineering is an important tool that allows to introduce distinct genetic changes without abolishing native traits, is faster, more effective and applicable to a wide range of species. It has been proved that expression of foreign gene(s) promotes a higher level of salt-tolerance in heterologous plant systems. Till date, several genes have been transferred in plants to increase salinity-stress tolerance, which are involved in synthesis of stress-mitigating compounds, antioxidant enzymes, regulatory proteins and signaling pathways proteins, ion transporters, etc. However, our knowledge about regulatory mechanisms of the salinity tolerance is still enigmatic. In the present chapter, current progress in transgenic approaches and the potential of transgenic plants for enhancement of salinity stress tolerance are reviewed and summarized.
Article
Full-text available
Leymus chinensis is an important crop that can be fed to ruminants. The purpose of this study was to investigate the roles of Lactiplantibacillus plantarum and Lentilactobacillus buchneri in fermentation quality, aerobic stability, and dynamics of wilted L. chinensis silage microorganisms. Wilted L. chinensis silages were ensiled with/without L. plantarum and L. buchneri. After 14 and 56 days of ensiling, the silos were opened and subjected to a 7-day aerobic deterioration test. This study looked at the composition of fermentation products as well as the microbial communities in silage. Silage inoculated with L. plantarum and L. buchneri had an increased lactic acid content as well as lactic acid bacterial (LAB) quantity, but a decrease in pH and levels of butyric acid, 2,3-butanediol, and ethanol was observed during ensiling. Non-treated and L. plantarum-treated silages deteriorated in the 7-day spoilage test after opening day-14 silos, whereas L. buchneri-inoculated silage showed no signs of deterioration. Lactobacillus abundance increased in the 7-day spoilage test after opening day-56 silos, while undesirable microorganisms such as Acetobacter, Bacillus, and molds, namely, Aspergillus and Penicillium were inhibited within L. plantarum- and L. buchneri-inoculated silages. The composition of fermentation products was related to the bacterial community, particularly Lactobacillus, Enterococcus, and Acetobacter. To summarize, L. plantarum- and L. buchneri-inoculated silage enhanced fermentation quality during ensiling and inhibited aerobic spoilage in a 7-day spoilage test of 56 days ensiling within wilted L. chinensis silage.
Article
Even under optimal conditions many metabolic processes produce ROS like superoxide anion (O2-), hydrogen peroxide (H2O2) and hydroxyl radicals (OH⁻), particularly in chloroplast and mitochondria. The overproduction of ROS (O2⁻, H2O2, OH⁻, RCO etc.) results from the exposure to various environmental conditions like dehydration, heat, salinity and biotic stresses. All biomolecules like lipids, proteins and DNA are extensively damaged by the reactive oxygen species which disrupts the cell integrity further leading to its death. Plants possess both enzymic and non-enzymatic mechanism for scavenging ROS. The enzymic mechanisms are designed to minimize the concentration of O2 and H2O2. The overproduced enzymes are superoxide dismutase (SOD), peroxidase (POX), catalase (CAT), glutathione reductase (GR) and glutathione-synthesizing enzymes. Several evidences have shown that although oxidative stress is a lethal situation for cell by ROS (especially H2O2 and O2), it may be involved in cellular signaling procedure as second messenger to induce a large number of genes and produce proteins and osmoprotectant involved in salt stress defenses. This review gives an insight into the recent advancements on how antioxidant defense machinery, the antioxidant enzymes and the non-antioxidant metabolites work together to alleviate the negative effects of ROS and cross-talk with reactive sulphur nitrogen and carbomyl species which also act as an important signal molecule. This comprehensive knowledge about ROS action, their regulation through antioxidant machinery, interactions with RNS, RSS and RCS and in signal transduction will empower us in the development of salinity tolerant plants.
Article
Full-text available
Approximately 10% of agricultural land is subject to periodic flooding, which reduces the growth, survivorship, and yield of most crops, reinforcing the need to understand and enhance flooding resistance in our crops. Here, we generated RNA-Seq data from leaf and root tissue of domesticated sunflower to explore differences in gene expression and alternative splicing (AS) between a resistant and susceptible cultivar under both flooding and control conditions and at three time points. Using a combination of mixed model and gene co-expression analyses, we were able to separate general responses of sunflower to flooding stress from those that contribute to the greater tolerance of the resistant line. Both cultivars responded to flooding stress by upregulating expression levels of known submergence responsive genes, such as alcohol dehydrogenases, and slowing metabolism-related activities. Differential AS reinforced expression differences, with reduced AS frequencies typically observed for genes with upregulated expression. Significant differences were found between the genotypes, including earlier and stronger upregulation of the alcohol fermentation pathway and a more rapid return to pre-flooding gene expression levels in the resistant genotype. Our results show how changes in the timing of gene expression following both the induction of flooding and release from flooding stress contribute to increased flooding tolerance.
Chapter
Full-text available
In last decades, plants were increasingly subjected to multiple environmental abiotic stress factors as never before due to their stationary nature. Excess urbanization following the intense industrial applications introduced combinations of abiotic stresses as heat, drought, salinity, heavy metals etc. to plants in various intensities. Technological advancements brought novel biotechnological tools to the abiotic stress tolerance area as an alternative to time and money consuming traditional crop breeding activities as well as they brought vast majority of the problem themselves. Discoveries of single gene (as osmoprotectant, detoxyfying enzyme, transporter protein genes etc.) and multi gene (biomolecule synthesis, heat shock protein, regulatory transcription factor and signal transduction genes etc.) targets through functional genomic approaches identified abiotic stress responsive genes through EST based cDNA micro and macro arrays. In nowadays, genetic engineering and genome editing tools are present to transfer genes among different species and modify these target genes in site specific, even single nuclotide specific manner. This present chapter will evaluate genomic engineering approaches and applications targeting these abiotic stress tolerance responsive mechanisms as well as future prospects of genome editing applications in this field.
Article
Full-text available
The objective of this experiment was to evaluate the effects of the chopping length and additive on the fermentation characteristics and aerobic stability in silage of Leymus chinensis. L. chinensis was chopped to 1–2 cm and 4–5 cm, and immediately ensiled with the three treatments, i.e., 2% sucrose (fresh weight basis; SU), 1 × 105 cfu/g Lactobacillus plantarum (LP) or 1 × 105 cfu/g LP plus 2% sucrose (SU+LP). Silage treated with distilled water served as the control. After silage processing for 30 and 90 d, the fermentation quality of L. chinensis silage was evaluated. The composition of the fermentation products and the pH value in the silage were determined at 1, 3, 5 and 7 d after opening the silo. The results showed that in L. chinensis silage there was a lower pH value, higher lactic acid content and better aerobic stability at the 1–2 cm length than those at the 4–5 cm (p < 0.001). When the chopping length was 4–5 cm, the addition of either LP or SU+LP increased the content of lactic acid and acetic acid, and decreased the pH value and butyric acid content, compared to those of the control and SU treatment (p < 0.001). Furthermore, combination treatment of SU+LP performed better than LP alone, and the aerobic stability time of L. chinensis silage at 4–5 cm without any additives was the worst. In conclusion, enhanced fermentation quality and aerobic stability can be obtained by processing L. chinensis silage with the shorter length. When the L. chinensis is cut longer, e.g., 4–5 cm in this study, LP or SU+LP could be used as an effective method to improve the fermentation quality and aerobic stability of L. chinensis silage.
Chapter
In different regions around the world, abiotic stresses, including cold, drought, nutrient deficiency, toxicity, salinity, and flooding, minimize the rate of crop production. Abiotic stress problem has become an issue mainly in developing countries where they reason the large population food security, poverty, especially in rural areas. In environmental field conditions, plants experience various environmental stresses at once. According to research plant response to different stresses are varies from in comparison to individual stresses which produce non-additive effects. To understand abiotic stress on plants is a crucial topic in the field of plant research. Molecular biology and physiological analysis have been helped out to draw a map line to understand abiotic stresses in different plants and also Arabidopsis thaliana determination of its genome sequence had quite a great effect on plant research. A. thaliana research has been furnishing a useful understanding of all aspects of modern molecular biology. Complete genome sequences availability made it easy to access necessary information required for all genes, for example, including transcripts level, gene products, and their function, alternative patterns of splicing and putative cis-regulatory elements. Moreover, in natural stress response research in multiple plants instead of A. thaliana give rise to our knowledge related to the plant stress tolerance mechanisms. Based on this knowledge, progress in stress tolerance in crops has been strived by the meaning of marker-assisted breeding and gene transfer. In this chapter, we have summarized current progress in abiotic stress studies and have discussed new range perspectives in new directions for the future in a model plant.
Article
This study investigated the degradability of corn silage (CS) and Leymus chinensis silage (LS) in vitro, and evaluated the effect of various ratios on growth performance, digestion and serum parameters in beef cattle. A 72‐hr bath culture trial was performed to evaluate degradability and rumen fermentation characteristics of CS, LS and their combinations [67:33, 33:67, dry matter (DM) basis]. Forty Simmental steers, averaging 441.46 ± 4.45 kg of body weight (BW), were randomly allocated into four dietary treatments for 120‐d period. Diets were given as total mixed rations with a forage‐to‐concentrate ratio of 60:40 and CS:LS ratios of 100:0, 67:33, 33:67 and 0:100 (DM basis). The in vitro trial showed that DM and neutral detergent fibre (NDF) degradability decreased linearly as LS proportion increased, whereas CP degradability increased linearly. Additionally, increased acid detergent fibre (ADF) degradability was detected at 48 hr of incubation. Increasing the proportion of LS increased rumen liquor pH and decreased volatile fatty acid linearly including acetate, propionate and butyrate, whereas the ammonia‐N increased linearly at 12 and 72 hr of incubation. With increasing LS ratio, final BW, average daily gain and feed conversion ratio of steers decreased linearly, whereas DMI was not affected. Additionally, apparent digestibility of DM, organic matter, NDF and ADF linearly and quadratically decreased while ether extract apparent digestibility decreased linearly, and CP apparent digestibility was not affected. Serum glucose and urea nitrogen linearly and quadratically decreased while glutamic‐pyruvic transaminase activity linearly decreased as the proportion of LS increased. Other serum parameters including total triglycerides, total cholesterol, total protein, albumin and glutamic‐oxalacetic transaminease were not affected. Overall, enhancing ratio of LS caused inferior DM and NDF degradability but improved CP degradability in the combinations of LS and CS. A CS:LS ratio of 67:33 resulted in the best growth performance and nutrient utilization in steers.
Article
Full-text available
Responses of sunflower tissues to NaCl stress were studied in control (C), salt-stressed (S) and salt-adapted (T) calluses in terms of proline, polyamines and ethylene content for a period of 21 days. Salt-adapted calluses showed their adaptation to salinity by growing in the medium with 175 mM NaCl, at a similar rate than C calluses on medium without salt. Proline concentration was 27 times higher in salt-adapted calluses compared to control calluses at time 0, but salt stressed calluses (S calluses) were able to increase proline by day 21, demonstrating that proline was not just an osmoregulator but might be involved in other responses in sunflower salt-stressed calluses. Putrescine (Put) was the most abundant polyamine in C calluses at time 0, while spermidine (Spd) was the main polyamine in salt tolerant (T) calluses. Ethylene increased in C calluses until day 14, decreasing thereafter. In salt-adapted calluses, ethylene increased significantly over the concentration in C and S calluses by the end of the experiment. In control calluses, the highest level of total polyamines and the lowest of ethylene was found on day 21, while T calluses synthesized the highest ethylene level and had the lower polyamines level by this time. It seems that in salt-adapted calluses ethylene was related to stress tolerance and in salt sensitive tissues (S calluses), ethylene formation was related to senescence. The present data suggests a close relationship between proline, polyamines, ethylene and salt-stress tolerance in sunflower dedifferentiated tissues.
Article
Full-text available
Studying the regulation of stress inducible genes can lead to understanding of the mechanisms by which halophytes maintain growth and thrive under abiotic stress. Verifying whether ionic or osmotic components of salt stress control the regulation of Dehydration Responsive Element- binding transcription factor (DREB) was investigated in the present study. DREB belongs to AP2/EREB group that is induced under abiotic stress and regulates many stress inducible genes that contain DRE binding sites in their promoters. DREB in Atriplex halimus was regulated by the osmotic component but not by the ionic one of salt stress. It seemed that DREB was not involved in the regulation of sodium manipulating genes like NHX1, SOS1 or H +-PPase. Moreover, DREB could be involved directly of indirectly in CMO regulation because of timing of induction. Also, DREB was the most upregulated gene under salt (fivefold) and drought (twofold) conditions, which reinforced the importance of this gene in A. halimus tolerance to stress. Moreover, its constitutive expression under normal conditions also indicated its involvement in other growth and developmental programs. The tolerance of A. halimus and of halophytes, in general, could be attributed to the constitutive expression of its genes and differences in protein structures between halophytes and glycophytes. Keywords Atriplex halimus – DREB –Protein content–Gene expression–Salinity–Osmotic and ionic stress
Article
S-Adenosylmethionine decarboxylase activity in leaves of Pharbitis nil increased dramatically at lights-on and then gradually decreased in the light. The enzymatic activity fell dramatically at lights-off then increased slightly in darkness. Photoinduction was prevented by cycloheximide and the enzymatic activity fell dramatically upon treatment with cycloheximide in the light.
Article
Pi -nt biologists fall into two categories: those who believy polyamines play an important role in plant growth and development, and those who are skeptical. The latter group by far exceeds the former. Why is this? Over the years polyamines have been implicated in being involved in a wide array of processes in plants, ranging from triggering organogenesis to protecting against stress. However, the problem has been that a particular response or a developmental event has generally been correlated only with changes in polyamine levels and spectra. The question of how direct or indirect the effect of polyamines is has remained open to debate. Although parallels are often drawn with animal systems in which polyamines have been linked with cell proliferation, their exact role has yet to be firmly established. It is no surprise that a formative review with the provocative title "Do Polyamines Have Roles in Plant Development?" concluded that although this was indeed likely, there were no definitive supporting conclusions (Evans and Malmberg, 1989). Since 1989, however, several important advances have been made in plant polyamine research. Most of the genes encoding polyamine biosynthetic enzymes have been isolated, and antibodies
Article
Several AP2/ERF transcription factors are wound-induced regulators, which play key roles in controlling cell dedifferentiation. In this study, a novel AP2/ERF transcription factor, the WIND1-like gene (ThWIND1-L), was isolated and characterized from Thellungiella halophila. ThWIND1-L cDNA clone contained a full open reading frame (ORF) of 1,068 bp encoding 356 amino acids. The predicted amino acid sequence of ThWIND1-L cDNA shared remarkably high degree of identity with the Arabidopsis RAP2.4 (WIND1). The expression of ThWIND1-L rapidly responded to the wound signals, ectopic expression of ThWIND1-L in Arabidopsis plants promoted callus tissue formation without the presence of exogenous hormones. Furthermore, overexpression of ThWIND1-L influenced some primary transcription factors (TFs), which have been shown to be involved in cell dedifferentiation and shoot regeneration. These results indicated ThWIND1-L might be a wound-induced regulator, which played an important role in the control of cell dedifferentiation.
Article
The effect, on adventitious regeneration from apricot leaf explants and transformation of leaf tissues, of auxins pulses with NAA and 2, 4-D was tested. Addition of the polyamines putrescine and spermidine to the regeneration medium, alone or in combination with the ethylene inhibitors silver thiosulphate and aminoethoxyvinylglycine, were also tested to design a procedure that improved transformation efficiency. Spermidine at 2mM in combination with 0.5 M aminoethoxyvinylglycine and four-day pulses with two different concentrations of 2, 4-D increased significantly shoot regeneration. Spermidine at the same concentration but in combination with 60 M silver thiosulphate and four-day pulses with 9 M 2, 4-D also increased stable transformation events and GFP-expressing calluses probably by inducing a larger amount of dividing cells where Agrobacterium transferred its T-DNA. Since regeneration from apricot leaves occurs mostly from developing calluses, it is important to obtain many GFP-expressing calluses and, given that transformation efficiencies (number of transformed shoots per total number of explants) in woody plants are generally very low, approaches that allow the optimization of T-DNA transfer and total number of transformed cells obtained, will improve probabilities of obtaining transformed shoots.
Article
Recent developments in the metabolism and function of polyamines in plants is presented. Polyamines appear to be involved in a wide range of plant processes, however their exact role is not completely understood. In this review, the metabolic pathways involved in polyamine biosynthesis and degradation are explained, along with the transport and conjugation of these compounds. The studies involved in the understanding of function(s) of polyamines using metabolic inhibitors, as well as genetic and molecular approaches are described. Polyamine metabolism and profound changes in polyamine titres in response to infection by pathogens has been presented. Its role in adaptation of plants to stress is also presented. Molecular understanding of polyamines and their modulation in transgenics is also discussed. Further line of work in the understanding of the role of polyamines has also been focussed.
Article
To avoid the effects of Na+ toxicity, plants have developed mechanisms to sequester Na+ in vacuoles. This sequestration process is catalyzed by a vacuolar Na+/H+ antiporter, with the transmembrane electrochemical potential initially established by the tonoplast H+-ATPase and H+-pyrophosphatase (H+-PPase). In this study, we cloned HcVP1 and HcVHA-B encoding a vacuolar H+-PPase and a B subunit of H+-ATPase, respectively, from Halostachys caspica, a succulent shrub that is highly salt-tolerant and widely distributed in Central Asia. The cDNA of HcVP1 is 2,295bp and contains an open reading frame (ORF) of 764 amino acids and the HcVHA-B cDNA is 1,467bp with an ORF of 488 amino acids. Semi-quantitative PCR revealed that transcription of both genes in H. caspica is induced by salt stress. Additionally, increased seed germination and improved plant growth were observed in transgenic Arabidopsis thaliana plants heterologously expressing HcVP1 or HcVHA-B, relative to wild-type plants when grown in the presence of NaCl. Specifically, Na+ content in leaves of transgenic Arabidopsis plants was higher than in wild-type leaves. These results demonstrate that overexpression of a vacuolar H+-PPase and a B subunit of H+-ATPase from H. caspica may enhance salt tolerance in transgenic Arabidopsis through increased accumulation of Na+ in vacuoles. Keywords Halostachys caspica –H + -PPase–B subunit of H + -ATPase–Salt tolerance– Arabidopsis thaliana
Article
In attempt to improve salt tolerance of alfalfa (Medicago sativa L.) plants, a soybean DREB orthologue, GmDREB1, was introduced into alfalfa plants under the control of Arabidopsis Rd29A promoter. Its incorporation and expression in transgenic plants were confirmed by DNA and RNA gel-blot analyses. The level of salt tolerance of transgenic lines was significantly higher than that of wild-type control plants as measured by ion leakage, chlorophyll fluorescence value, contents of free proline and total soluble sugars. Moreover, northern blot analysis revealed that the transcript level of Δ1-pyrroline-5-carboxylate synthase (P5CS) was up-regulated in the transgenic plants. These results suggested that the stress-inducible expression of GmDREB1 conferred salt tolerance in transgenic alfalfa plants. In addition, no visible phenotypic alternations were observed in the transgenic plants due to the use of stress-inducible Rd29A promoter. It was the first to describe transgenic expression of DREB genes in alfalfa and these transgenic lines with high salt-tolerance are of significance in the forage breeding.