Upload
gengmao
View
215
Download
1
Embed Size (px)
Citation preview
ORIGINAL PAPER
Isolation and characterization of two DREB1 genes encodingdehydration-responsive element binding proteins in chicory(Cichorium intybus)
Mingxiang Liang • Dandan Chen • Manman Lin •
Qingsong Zheng • Zengrong Huang •
Zhongyuan Lin • Gengmao Zhao
Received: 4 June 2013 / Accepted: 12 October 2013 / Published online: 17 October 2013
� Springer Science+Business Media Dordrecht 2013
Abstract Two novel DREB (dehydration-responsive
element-binding protein) genes, designated as CiDREB1A
and CiDREB1B, were cloned from chicory (Cichorium
intybus). Both of these genes contained a conserved
EREBP/AP2 domain and were classified into the A-1
subgroup of the DREB subfamily based on phylogenetic
analysis. Quantitative real-time PCR analysis revealed that
low temperature, but not ABA, greatly induced the
expression of both CiDREB1 genes, suggesting that these
genes are involved in ABA-independent stress signaling
pathways. A subcellular localization assay revealed that
both CiDREBs localized to the nucleus. In addition, we
showed by yeast one-hybrid analysis that these two Ci-
DREB proteins bind to the DRE motif of RD19A. These
results suggest that CiDREB1A and CiDREB1B are
important regulators of stress-responsive signaling in
chicory.
Keywords Abiotic stresses � Subcellular
localization � Yeast one-hybrid
Introduction
Abiotic stresses, such as cold, salinity, and osmotic stress,
have adverse effects on plant growth and development
(Mahajan and Tuteja 2005). To protect cells and improve
the chance of survival under such harsh environments,
plants, as sessile organisms, regulate their physiological,
cellular, and molecular pathways (Huang et al. 2012). To
tolerate long-term external stresses, plants must adjust
various internal mechanisms, especially at the genetic
level. The stress response genes, particularly those encod-
ing transcription factors, are tightly regulated during the
stress response. Transcription factors, which bind to spe-
cific cis-acting elements in the promoters of downstream
genes, serve as master switches to affect the response to
abiotic stresses and finally impart tolerance in plants (Mi-
zoi et al. 2012).
To date, various transcription factors involved in plant
abiotic responses have been identified and classified into
Electronic supplementary material The online version of thisarticle (doi:10.1007/s10725-013-9866-8) contains supplementarymaterial, which is available to authorized users.
M. Liang (&) � D. Chen � M. Lin � Q. Zheng (&) � Z. Huang �Z. Lin � G. Zhao
College of Resources and Environmental Sciences, Nanjing
Agricultural University, Tongwei Road 1, Xuanwu District,
Nanjing 210095, Jiangsu Province, China
e-mail: [email protected]
Q. Zheng
e-mail: [email protected]
D. Chen
e-mail: [email protected]
M. Lin
e-mail: [email protected]
Z. Huang
e-mail: [email protected]
Z. Lin
e-mail: [email protected]
G. Zhao
e-mail: [email protected]
M. Liang � D. Chen � M. Lin � Q. Zheng � Z. Huang � Z. Lin �G. Zhao
Jiangsu Key Lab of Marine Biology, Nanjing, China
123
Plant Growth Regul (2014) 73:45–55
DOI 10.1007/s10725-013-9866-8
several families, such as bZIP (basic leucine zipper), NAC
(NAM/ATAF/CUC), and MYB (myb proto-oncogene
protein) (Wang et al. 2003). One such family, the dehy-
dration-responsive element-binding proteins (DREBs), has
received considerable attention. DREBs belong to the ERF
(ethylene-responsive element binding factor) family, which
is a subfamily of the APETLA2 (AP2)/ethylene-responsive
element-binding protein (EREBP) transcription factors.
DREBs, which contain a conserved 60-amino-acid DNA-
binding AP2/EREBP domain, bind to the downstream
C-repeat/dehydration responsive element (CRT/DRE).
Based on their gene structures in the model plant Arabi-
dopsis, DREB members can be divided into six subfami-
lies, A1-A6, with A1 and A2 constituting the two largest
DREB subfamilies. DREB1s, also referred to as CBF (C-
repeat binding factor) proteins, which belong to the A1
subgroup, are mainly involved in the cold stress response
(Agarwal et al. 2006).
The Arabidopsis genome contains six DREB1 members,
i.e., DREB1A/CBF3, DREB1B/CBF1, DREB1C/CBF2,
DREB1D/CBF4, DDF1/DREB1F, and DDF2/DREB1E
(Sakuma et al. 2002). DREB1s have two short sequences, a
nuclear localization signal consensus (NLS) and a DSAWR
motif downstream of the AP2 domain, which separate
DREB1s from the other AP2 proteins (Akhtar et al. 2012).
In the conserved AP2 domain, two amino acids, the 14th
valine (Val) residue and the 19th glutamic acid (Glu) res-
idue, are highly conserved and determine the DNA-binding
specificity. However, the 19th Glu residue was found to be
replaced by Val in some plant species (Cao et al. 2001).
DREB1s activate a downstream gene, such as RD29A/
COR78/LT178, by binding to the DRE element in its pro-
moter. The core DRE element contains a CCGAC
sequence, which is essential for the interaction with
DREBs (Yamaguchi-Shinozaki and Shinozaki 2009).
DREB1s are well represented in dicotyledonous species,
such as canola (Brassica napus) (Savitch et al. 2005),
tomato (Solanum lycopersicum) (Zhang et al. 2004),
chrysanthemum (Dendranthema grandiflorum) (Tong et al.
2009), and soybean (Glycine max) (Li et al. 2005), and also
in monocotyledonous species, such as barley (Hordeum
vulgare) (Choi et al. 2002), rice (Oryza sativa) (Dubouzet
et al. 2003), and rye (Secale cereale) (Xiong and Fei 2006),
which suggests that DREB1s may be ubiquitous in plants.
DREB1s are typically expressed at low levels in all plant
tissues in the absence of abiotic stress, and strongly
expressed under low temperatures. Three DREB1s in
Arabidopsis, DREB1A/B/C, were strongly induced under
cold stress (Gilmour et al. 1998; Liu et al. 1998). The
remaining three DREB1 genes were not induced by abiotic
stress conditions (Sakuma et al. 2002). Due to the crosstalk
between low temperature and other abiotic stresses, some
DREB1 s are also responsive to other external stimulations.
BrCBF from Chinese cabbage (Brassica campestris L.ssp.
Chinensis) (Jiang et al. 2011), MbDREB1 from apple
(Malus baccata (Lnn.) Borkh) (Yang et al. 2011), and
VviDREB1 from Vaccinium vitis-idaea (Wang et al. 2010)
were simultaneously responsive to cold, salt stress, osmotic
stress, and ABA treatment. OsDREB1B transcripts accu-
mulated to high levels under cold stress and high temper-
ature (Qin et al. 2007). Constitutive expression of DREB1
family members in transgenic plants consistently results in
the induction of stress-responsive genes and increases plant
freezing tolerance (Tong et al. 2009; Gilmour et al. 1998;
Ito et al. 2006). The heterologous expression of cotton
DREB1 driven by a constitutive or inducible promoter
increased the soluble sugar and chlorophyll content in
leaves and improved freezing tolerance in transgenic wheat
(Gao et al. 2009). Transgenic China Rose (Rosa chinensis
Jacq.) heterologously expressing Medicago truncatula
DREB1C driven by an inducible RD19A promoter exhib-
ited enhanced freezing ability (Chen et al. 2010). All of the
abovementioned evidence demonstrates that DREB1 pro-
teins have conserved roles in different plant species.
Chicory (Cichorium intybus L), a perennial herbaceous
plant with resistance to cold and osmotic stress, provides
various products (e.g., ground chicory root and endives) for
human consumption and is extensively planted as a forage
crop for livestock (Bais and Ravishankar 2001). The iso-
lation of DREB1 genes from chicory would not only pro-
vide insight into the mechanism of cold tolerance, but
would also have important agricultural applications. Here,
we isolated two DREB1 homologs from chicory, and
characterized their expression patterns, subcellular local-
izations, and DRE-binding activities. Our results provide
the basis for developing transgenic chicory with improved
abiotic tolerance.
Materials and methods
Plant materials, growth conditions, and stress
treatments
Chicory (Cichorium intybus L. Commander) seeds were
grown in plastic pots containing organic cultivation matrix
(organic matter content C50 %, nitrogen, phosphorus and
potassium content C2.5 %, Zhenjiang Xing Nong Organic
Fertilizer Co., Ltd. http://www.ding-ye.cn/product/xxyjzpjz)
in the greenhouse. The plants were watered daily with Hoa-
gland solution (Hewitt et al. 1966) and germinated under a
light intensity of 392–415 lmol m-2 s-1 during a 16-h light/
8-h dark regimen at 25 �C/18 �C for 2 weeks. For the stress
treatments, plants were further treated with 15 % PEG
(osmotic stress), 100 mM NaCl (salinity), or 100 lM ABA
for 0, 1, 3, 6, or 24 h. For the temperature treatments, plants
46 Plant Growth Regul (2014) 73:45–55
123
were transferred to a growth chamber at 4 or 36 �C for 0, 1, 3,
6, or 24 h, respectively. For the time course experiments in the
absence of abiotic stresses, plants were harvested at 1, 3, 5, and
6 months, respectively. Excised leaf samples and root sam-
ples of plants subjected to different treatments and used for
DNA or RNA extraction were harvested and immediately
frozen in liquid N2 and stored at -70 �C. These samples were
later used for DNA or RNA extraction. All experiments were
repeated in biological triplicate.
Genomic DNA extraction, total RNA isolation,
and cDNA synthesis
Prior to extraction of genomic DNA and total RNA, samples
were grounded in frozen mortars. DNA was extracted from
chicory tissues using the quick DNA miniprep for PCR
analysis of plant tissue from Rogers and Bendich’s CTAB-
based protocol (Rogers and Bendich 1985). Total RNA was
extracted from each sample using an E.Z.N.A. Plant RNA
Kit (Omega Biotek, Cat#R6827), according to the manu-
facturer’s instructions. The quality and quantity of extracted
DNA and RNA from all samples was confirmed by both
agarose gel visualization and spectrophotometry (Thermo
Scientific, NanoDropTM 1000). Prior to reverse transcrip-
tion, total RNA samples were pretreated with RNase-Free
DNase Set (Omega Biotek, Cat#E1091) at 25–30 �C for
15 min, to eliminate any contaminating genomic DNA.
Primary cDNA was synthesized using a PrimeScript� 1st
Strand cDNA Synthesis Kit (TaKaRa Code: D6110A),
according to the manufacturer’s instructions.
Cloning of CiDREB1 transcription factors
Chicory ESTs (expressed sequence tags) were downloaded
from the NCBI website (http://www.ncbi.nlm.nih.gov/).
More than 54,000 ESTs were retrieved and used to create a
database with Bioedit software (Tom Hall, 7.0.9). Known
DREB1 sequences from different plant species were used
to perform a local BLAST query of this EST database.
Highly similar EST sequences were identified and assem-
bled. To verify the assembled sequences, primers used to
amplify the entire open reading frames of those sequences
were designed using Primer Premier 5.0 software. PCR was
carried out using 1 ll cDNA as template, 1 ll primers
(10 lM), 0.4 ll dNTPs (10 mM), 2 ll buffer, and 0.2 ll
Taq DNA polymerase in 20-ll reaction. PCR conditions
were 94 �C, 5 min, 1 cycle; 94 �C, 1 min; 53 �C, 1 min,
and 72 �C, 1 min, 30 cycles; and 72 �C, 5 min, 1 cycle.
The amplification products were gel-purified and cloned
into a PMD-19T vector. The ligated vectors were trans-
formed into E. coli DH5a and plated on LB plates con-
taining 100 lg ll-1 of ampicillin. Each DREB1 was
further sequenced to confirm accuracy.
Bioinformatics analysis
The deduced proteins from the above sequenced DREB1 CDS
(coding sequences) were aligned using the ClustalW method
in MegAlign (DNASTAR, Inc., Madison, WI). A phyloge-
netic tree was generated based on the ClustalW protein
alignment analysis using the Neighbor-Joining method in
MEGA 4. The following sequences with their corresponding
accession numbers were used for the bioinformatics analysis:
AtDEEB1A (NM_118680), AtDEEB1B (NM_118681), At-
DEEB1C (NM_118679), AtDEEB2A (NM_120623), AtA-
BI4 (NM_129580), AtTINY (NM_122482), RAP2.10
(NM_119854), and AtRAP2.4(NM_102069) from Arabi-
dopsis; CaDEEBLP1 (AY496155) from hot pepper (Capsi-
cum annuum L.); DgDREB1A (DQ430764) and DgDREB1B
(DQ430763) from chrysanthemum (D. grandiflorum);
GhDBP1 (AY174160) from cotton (Gossypium hirsutum L.);
HvCBF1 (AF418204), HvCBF2 (AF442489), and HvCBF3
(AF239616) from barley (Hordeum vulgare subsp. Vulgare);
LpCBF3 (AY960831) from perennial ryegrass (Lolium per-
enne L.); MtDREB1C (DQ267620) from Barrel Medic
(Medicago truncatula); OsDREB1A (AF300970) and Os-
DREB1B (AF300972) from rice (Oryza sativa L.); and
ZmABI4 (AY125490), ZmDBF1 (AF493800), ZmDBF2
(AF493799), ZmDREB1A (AF450481), ZmDRE-
B2A(NP_001105876), and ZmDBP4 (FJ805752) from maize
(Zea mays).
Quantitative PCR (qPCR) assay and data analysis
Quantitative PCR was performed using SYBR Premix EX
Taq (Takara, DRR420A) in an ABI 7500 Real Time PCR
System, according to the manufacturer’s instructions. Pri-
mer pairs of two DREB1s were designed outside the AP2
domain to avoid interference. The PCR products of both
CiDREB fragments were further sequenced to confirm the
specificities of the primer-pairs. Each 20-ll reaction con-
tained 10 ll SYBR Premix Ex Taq, 0.4 ll each of 10 lM
primers, 0.4 ll ROX Reference DyeII (509), 2.0 ll DNA
template, and 6.8 ll dd H2O. The program consisted of an
initial denaturation stage at 95 �C for 30 s, followed by 40
cycles of 95 �C for 3 s, and annealing at 60 �C for 34 s.
The chicory 18S gene was used to normalize the amount of
cDNA in the qPCR reaction. Each sample was analyzed in
technical triplicate. Since the deviation error of the
amplification efficiency between target genes and the ref-
erence gene was less than 10 %, according to our trial
experiments, data were processed using the 2-DDCT
method, according to Livak et al. (Livak and Schmittgen
2001; Schmittgen and Livak 2008). The sample harvested
in the first month of the experiment was arbitrarily used as
the control for the growth experiments in the absence of
abiotic stresses. To analyze gene transcript levels in the
Plant Growth Regul (2014) 73:45–55 47
123
stress experiments, gene transcript levels in each treatment
sample were first normalized to the same mixed cDNA
sample, which included all samples subjected to abiotic
stress, and further compared to the corresponding time-
point control sample to avoid circadian rhythm interfer-
ence. Fold changes were used for quantitative analysis.
Subcellular localization of CiDREB1A and CiDREB1B
proteins
CiDREB1A and CiDREB1B were fused to the N-end of
green fluorescent protein (GFP) to yield a fusion protein.
The entire coding sequence of CiDREB1A or CiDREB1B
was individually inserted into the NcoI and SpeI sites of the
pCAMBIA 1302 vector under the control of the CaMV 35S
promoter. Approximately 2 lg of the plasmid construct
was used to coat gold particles, and a plasmid containing
AtDREB2C was used as the positive control. Inner epi-
dermal peels of onion were cultured in hypertonic � Mu-
rashige and Skoog (MS) medium supplemented with
0.6 mol/l sorbitol for 4 h and were then transformed with
plasmid-coated gold particles using the PDS-1000 bom-
bardment system (Bio-Rad, Canada) at 1,100 psi. The
bombarded peels were incubated in hypertonic medium at
25 �C for 10 h and then transferred to � MS and cultured
overnight. GFP fluorescence in the onion epidermal cells
was observed under a laser confocal scanning microscope
(Leica, Germany) at a wavelength of 488 nm.
DRE-binding activities of the CiDREB proteins
To analyze DRE-binding activity, the full-length CiDREB1s
were respectively cloned into the SmaI and BamHI sites of
the GAL4 activation vector (pGADT7-AD). Yeast one-
hybrid analysis was carried out according to the manufac-
turer’s instructions (Clontech, Cat. 630491). pAD-Ci-
DREB1A and pAD-CiDREB1B were transformed into yeast
strain Y187 carrying the reporter genes His3 and LacZ under
the control of the RD29A promoter containing three copies
of the DRE sequence (TACCGACAT) or mutated DRE
(mDRE) sequence (TATTTTCAT), respectively. The
growth status of the transformed yeast cells was compared
on selective synthetic dextrose (SD) medium without His
plus 10 mM 3-aminotriazole (SD/-his ? 10 mM 3-AT).
Results and discussion
Isolation and sequence analysis of CiDREB1A
and CiDREB1B
Using the homologous regions of previously reported CBF/
DREB1 genes from other plants, we identified seven ESTs
in a chicory EST database with the help of Bioedit soft-
ware, which we assembled into two full-length DREB1
genes, designated as CiDREB1A (accession number
KC801338) and CiDREB1B (KC811151). PCR amplifica-
tion and sequence verification confirmed that both
sequences were indeed CiDREBs. We also amplified the
genomic sequences of the CiDREBs. No introns were
present in either CiDREB gene. The CDSs (coding
sequences) of CiDREB1A and CiDREB1B were 699 and
669 bp long, encoding 232 and 222 amino acids,
respectively.
An alignment of CiDREB1A and CiDREB1B with other
EREBP/AP2 proteins indicated that both encode DREB1-
type proteins (Fig. 1). Further analysis of their deduced
amino acid sequences revealed that these proteins contain
one conserved AP2 domain, of 64 and 65 amino acids,
respectively. The secondary structures of these domains,
predicted using Predict Protein (http://www.predictprotein.
org/), consisted of three b-sheets and one a-helix, as is
typical for the AP2 domain. Furthermore, both CiDREB1s
contain a putative nuclear localization signal sequence
(NLS), RKKKAGRKKFRETRHP, in the N-terminal
region, and a DSAWR sequence downstream of the AP2
domain, which were often found in known DREB1 mem-
bers from various species. Conserved Val and Glu residues
were found at the 14th and 19th amino acid positions,
respectively, in the second b-sheet of the AP2 domain of
CiDREB1B. Previous studies suggested that the Val and
Glu residues at the 14th and 19th amino acid residues in the
AP2 domain are essential for specific binding to DRE.
Val14 is absolutely conserved in many plants and deter-
mines the protein’s specific binding activity, whereas the
amino acid present in the 19th position may vary slightly
(Cao et al. 2001). Surprisingly, the Val residue at the 14th
position in the AP2 domain is replaced with Ile in Ci-
DREB1A. Such a substitution was rarely reported before.
However, a similar result was found in Arabidopsis CBF4,
with Val14 being replaced with Tyr (Haake et al. 2002).
Occasionally, different amino acids were found at the 19th
position in some plant species (Cao et al. 2001). For
example, Glu19 was replaced with Leu in Arabidopsis
Rap2.4 (Okamuro et al. 1997), whereas a rice OsDREB1,
OsDREB1C, did not have a conserved Val residue at the
19th amino acid position of the AP2 domain (Dubouzet
et al. 2003). Although it is unclear whether this substitution
affects CiDREB1A function, our subcelluar localization
and yeast one-hybrid experiments showed that this
replacement did not change the cellular location of Ci-
DREB1A proteins or its DRE motif-binding capability. In
one study, Ala37, which resides in the a-helix of the AP2
domain, was demonstrated to be essential for binding with
DRE and the GCC box (Liu et al. 2006). This implies that
Ala37 plays a crucial role in the protein’s DNA-binding
48 Plant Growth Regul (2014) 73:45–55
123
activity or in the stability of the AP2 domain. Phylogenetic
analysis showed that both CiDREB1 proteins clustered into
the DREB1 A1 subgroups. It was found that these two
proteins shared the highest level of sequence identities with
DgDREB1A (accession number DQ430764), and
DgDREB1B (accession number DQ430763) in chrysan-
themum, which, like chicory, belongs to the composite
family. CiDREB1A shows 64 % identity with chrysan-
themum DgDREB1A, while CiDREB1B has 67 % identity
with DgDREB1B, based on the full-length deduced amino
acid sequence (Fig. 2). Similar results were observed when
the corresponding AP2 domains of plant DREB1s were
compared (Supplementary Fig. 1). Thus, DREB1 genes
from many plant species are highly conserved in gene
structure. An investigation of the genetic relationships
between DREB1 proteins from different plants may pro-
vide some clues about the relative evolutionary relationship
in various species.
Expression analysis of CiDREB1 genes by quantitative
real-time PCR
First, we analyzed the tissue-specific expression profiles of
the CiDREB1 genes at various points throughout the
growing season (Fig. 3). The expression level of Ci-
DREB1B was relatively higher than that of CiDREB1A in
the absence of external stresses. Both CiDREB1s, but
especially CiDREB1B, were expressed at relatively higher
levels in the leaves than in the roots. The expression levels
of both CiDREB1s were progressively down-regulated
during the growing season. These results suggest that Ci-
DREB1A and CiDREB1B are predominantly active in
young plants, which are particularly sensitive to external
conditions, and that the two CiDREB1s may have different
roles.
Next, we examined the expression patterns of the Ci-
DREB1 genes under various abiotic stresses (Fig. 4a, b, c, d
Fig. 1 Comparison of the deduced amino acid sequences of
CiDREB1A and CiDREB1B with other DREB1/CBF-like proteins.
The amino acid sequences are shown as follows: AtDEEB1A
(NM_118680); CaDEEBLP1 (AY496155); CiDREB1A
(KC801338); CiDREB1B (KC811151); DgDREB1A (DQ430764);
DgDREB1B (DQ430763); HvCBF1 (AF418204); HvCBF2
(AF442489); HvCBF3 (AF239616); LpCBF3 (AY960831); and
MtDREB1C (DQ267620); OsDREB1A (AF300970). Identical amino
acids are indicated by asterisks. The putative nuclear localization
signals (PKK/RPAGRxKFxETRHP and DSAWR) are boxed. The
AP2/EREBP DNA-binding domain is indicated by a black line. The
fourteenth and nineteenth amino acids are indicated by triangles
Plant Growth Regul (2014) 73:45–55 49
123
and e). The expression patterns of CiDREB1A and Ci-
DREB1B differed slightly under various conditions
(Fig. 4a). For cold stress (4 �C) treatment, the expression
level of CiDREB1A in roots increased dramatically at 6 h
and diminished after that. However, CiDREB1A expression
in leaves increased a little at 3 h and then dropped greatly.
The expression levels of CiDREB1B were higher in roots
than in leaves after a 24-h cold treatment. In chrysanthe-
mum, the expression of DgDREB1A, -B, and -C consis-
tently peaked 5 h after cold treatment, similar to the
CiDREB1 s (Tong et al. 2009). In general, the expression
profile of each CiDREB1 gene was similar to that of
Fig. 2 Phylogenetic relationship of DREB transcription factors based
on amino acid sequences comparison of the full length proteins.
CiDREB1A and CiDREB1B are framed. Multiple sequence align-
ments were performed by MEGA4.1. DREB sequences were retrieved
from GenBank. The accession numbers of the above proteins are as
follows: AtDEEB1A (NM_118680); AtDEEB1B (NM_118681);
AtDEEB1C (NM_118679); AtDEEB2A (NM_120623); AtABI4
(NM_129580); AtTINY (NM_122482); RAP2.10 (NM_119854); At
RAP2.4 (NM_102069);CiDREB1A (KC801338); CiDREB1B
(KC811151); CaDEEBLP1 (AY496155); DgDREB1A (DQ430764);
DgDREB1B (DQ430763); GhDBP1 (AY174160); HvCBF1
(AF418204); HvCBF2 (AF442489); HvCBF3 (AF239616); LpCBF3
(AY960831); MtDREB1C (DQ267620); OsDREB1A (AF300970);
OsDREB1B (AF300972); ZmABI4 (AY125490); ZmDBF1
(AF493800); ZmDBF2 (AF493799); ZmDREB1A (AF450481);
ZmDREB2A (NP_001105876); and ZmDBP4 (FJ805752)
Fig. 3 The expression levels of CiDREB1A and CiDREB1B in the
roots and leaves of unstressed plants at various points throughout the
growing season. The sample harvested in the first month of the
experiment was arbitrarily used as the control for the growth
experiments in the absence of abiotic stresses. Experiments were
repeated three times. Error bars represent the standard errors of three
biological replicates of a single treatment
50 Plant Growth Regul (2014) 73:45–55
123
Fig. 4 Quantitative analysis of
CiDREB1A and CiDREB1B
expression levels. a, b, c, d and
e The relative expression of
CiDREB1A and CiDREB1B
under low temperature (4 �C),
heat stress (36 �C), salinity
stress (100 mM NaCl), osmotic
stress (15 % PEG) and 100 lM
ABA, respectively. To analyze
gene transcript levels in the
stress experiments, gene
transcript levels in each
treatment sample were first
normalized to same mixed
cDNA sample which included
all samples under the abiotic
stress and further compared to
the corresponding time-point
control sample to avoid
circadian rhythm interference.
Total RNA was extracted from
the tender leaves and roots of
two-week-old chicory in the
same position after 0, 1, 3, 6,
and 24 h of exposure to various
stresses. Expression values were
normalized against translation
factor 18S levels. Experiments
were repeated three times. Error
bars represent the standard
errors of three biological
replicates of a single treatment
Plant Growth Regul (2014) 73:45–55 51
123
chrysanthemum DREB1A/B/C genes under cold treatment.
LpCBF3, from perennial ryegrass (Lolium perenne L.),
clustered into the DREB1A-like gene family and was also
induced by cold stress (Xiong and Fei 2006). Under heat
stress (36 �C) treatments, CiDREB1A expression peaked at
1 h in the roots, and gradually decreased after 3 h of
treatment. The expression levels of CiDREB1A in leaves
were almost unchanged by heat treatment (Fig. 4b). Ci-
DREB1B expression was down-regulated in both roots and
leaves during the 24-h heat stress test period.
Under salinity stress, the expression level of CiDREB1A
was generally lower than that of the control, but the leaves
had much lower transcript levels than did the roots at most
time points (Fig. 4c). The expression pattern of CiDREB1B
was similar to that observed for heat stress, with transcript
levels decreasing dramatically in the seedlings. CiDREB1s
were down-regulated in roots and leaves by salinity treat-
ment, whereas DgDREB1A was slightly up-regulated in
roots and unchanged in shoots. Generally, the two Ci-
DREB1s, similar to the DgDREB1s, exhibited a greater
response in root tissues than in leaves or shoots under stress
conditions (Tong et al. 2009). Previously, CaDREBLP1,
isolated from hot pepper (Capsicum annuum Linn.) and
defined as a DREB1-type gene, was also found to be
responsive to salt (Hong and Kim 2005).
CiDREB1A expression in roots was variable under
osmotic treatment, with expression being inhibited at 3 and
6 h and increasing at 24 h (Fig. 4d). Osmotic stress (PEG)
treatment sharply up-regulated the expression of Ci-
DREB1B in roots after 1 and 6 h, but only moderately
increased the transcript level at 3 and 24 h. Both Ci-
DREB1s were induced in roots by osmotic stress, while
only DgDREB1A was responsive to osmotic stress, which
implies that CiDREB1s and DgDREB1s have different
roles (Tong et al. 2009). CaDREBLP1 was also reported to
be responsive to osmotic stress (Hong and Kim 2005).
To explore the possible regulatory pathway underlying
the response of CiDREB1 to abiotic stresses, we evaluated
the effect of external ABA applications on CiDREB1
expression (Fig. 4e). The transcript levels of both Ci-
DREB1s were largely unchanged in leaves, whereas they
decreased slightly in roots, especially for CiDREB1A.
Plants generally simultaneously respond to environmental
stress through two pathways, the ABA-dependent and
AtDREB2C-
EGFP
(positive
control)
CiDREB1A-
EGFP
CiDREB1B-
EGFP
Fluorescence
images
DAPI
images
Brightfield
images
Overlaid
images
Fig. 5 Subcellular localization of the CiDREB1 proteins in onion
epidermal cells. Onion epidermal cells were transiently transformed
with CiDREB1-EGFPs. The subcellular localization of CiDREB1-
EGFP fusion proteins and AtDREB2C-EGFP were tracked by
fluorescence confocal microscopy 24 h after bombardment.
Fluorescence images, DAPI (4, 6-diamidino-2-phenylindole) images,
brightfield images, and corresponding overlaid images of represen-
tative cells expressing DREB2C-EGFP or CiDREB1stress treatments,
plants were further treated s-EGFP fusion proteins are shown.
AtDREB2C-EGFP was used as a positive control
52 Plant Growth Regul (2014) 73:45–55
123
ABA-independent pathway. ABA plays a crucial role in
inducing the expression of some stress-responsive genes
and in facilitating adaptation to different abiotic stresses. A
previous report showed that DREB1/CBF is independent of
the ABA response (Yamaguchi-Shinozaki and Shinozaki
2009). Our work shows that CiDREB1A expression was
almost unaltered by ABA treatment, suggesting that Ci-
DREB1A is mainly involved in ABA-independent signaling
pathways. However, CiDREB1B expression was reduced in
leaves subjected to ABA treatment. Interestingly, several
DREB1 proteins have been reported to be involved in the
ABA-dependent pathway. The expression of BjDREB1B
from Brassica juncea L was induced by osmotic stress,
salt, low temperature, and ABA, implying that BjDREB1B
may act as a cross-point between ABA-independent and
ABA-dependent stress signaling pathways (Cong et al.
2008). CBF4, a DREB1-related transcription factor in
Arabidopsis, is activated by osmotic stress and ABA, but
not by cold stress (Haake et al. 2002). This finding shows
that plant DREB1s may be responsive to abiotic stress
through ABA- dependent or ABA-independent pathways
and that cross-talk between the two pathways exists.
CiDREB1A and CiDREB1B are targeted to the nucleus
In a previous study, NLSs, which are required for nuclear
localization, were frequently identified in DREB tran-
scription factors (Sakuma et al. 2002; Agarwal et al. 2006;
Jaglo et al. 2001). Since the sequences of CiDREB1A and
CiDREB1B contained a typical DREB1-type nuclear
localization signal (NLS), PKKKAGRKKFRETRHP, we
expected the proteins to localize to the nucleus. To verify
their subcellular localization, we fused the two CiDREB1s
to the N-terminal of GFP and used empty GFP plasmid as a
negative control and then transferred the resulting con-
structs into onion epidermal cells by particle bombardment.
As shown in Fig. 5, the fusion proteins localized to the
nucleus of onion epidermal cells, as did the positive con-
trol, AtDREB2C-GFP. GFP alone (empty pCAMBIA 1302
vector) was observed throughout the cell (Supplementary
Fig 2). Similar results were previously reported for
LcDREB3a (Xianjun et al. 2011), CMe-DREB1 (Mizuno
et al. 2006), and HvDREB1 (Xu et al. 2009). These results
indicate that CiDREB1A and CiDREB1B are nuclear
proteins, with possible functions as transcription factors.
DRE-binding activities of the CiDREB proteins
As our sequence and expression analyses indicated that the
cloned genes are DREB1 homologs, we tested the DRE-
binding activities of the two CiDREB1 proteins using a
yeast one-hybrid assay. The full-length cDNAs of both
CiDREB1 s were individually inserted into a pGADT7-AD
expression vector with a GAL4 activation domain. The
recombinant plasmids were transformed into two Y187
yeast strains that carried the dual reporter genes His3 and
LacZ under the control of the DRE or mutated DRE motif,
respectively. Yeast cells harboring the wild-type DRE
motifs of either of the CiDREB1 s grew well on SD/-his
plus 10 mM 3-AT selective medium (Fig. 6, upper half).
However, yeast cells transformed with mutant DRE
(mDRE) motifs of the CiDREBs could not grow in the
same medium (Fig. 6, lower half). These results confirm
that both CiDREB1 proteins possess DNA-binding abilities
and can specifically bind to DRE, but not mDRE motifs.
In conclusion, we have cloned and studied the expres-
sion patterns of two CiDREBs from chicory, a cold- and
drought-resistant species. We confirmed that these proteins
are DREB1 members that localized to the nucleus. The two
CiDREBs show similar expression patterns throughout the
plant’s life cycle in the absence of abiotic stresses. Those
two DREB1s are both up-regulated by cold or osmotic
stress, but moderately inhibited by salinity. The change in
gene expression does not appear to be regulated by the
ABA pathway, since ABA treatment did not alter the
transcript levels. Further experiments are needed to further
explore the roles of these genes in abiotic resistance.
Acknowledgments We thank Kathleen Farquharson from Plante-
ditors Company for valuable comments on the manuscript revision.
This research was supported by grants from the National High
Technology Research and Development Program (‘‘863’’Program,
2011AA100209), National Natural Science Foundation of China
(31201842), the Doctoral Program of Higher Education of China
(20120097120015), Fundamental Research Funds for the Central
Universities (KYZ201206), the Priority Academic Program Devel-
opment of Jiangsu Higher Education Institutions (RAPD program,
Fig. 6 Analysis of DNA-binding activities of CiDREB1A and
CiDREB1B protein by the yeast one-hybrid assay in vivo. The
growth of transformed yeast cells (left) was examined on synthetic
dextrose (SD) medium without His plus 10 mM 3-aminotriazole
(SD/-his ? 10 mM 3-AT) at 30 �C. Empty pAD was used as a
negative control (left panel). mpAD represents mutant DRE motifs of
RD19A and was used to verify the DREB’s binding specificity. The
upper half was tested using the normal DRE motif and the down panel
was examined using the mutant DRE motif. The sketch (right)
indicates the location of different transformed yeast strains
Plant Growth Regul (2014) 73:45–55 53
123
809001), and the Scientific Research Foundation of the State Human
Resource Ministry.
References
Agarwal PK, Agarwal P, Reddy M, Sopory SK (2006) Role of DREB
transcription factors in abiotic and biotic stress tolerance in
plants. Plant Cell Rep 25(12):1263–1274
Akhtar M, Jaiswal A, Taj G, Jaiswal J, Qureshi M, Singh N (2012)
DREB1/CBF transcription factors: their structure, function and
role in abiotic stress tolerance in plants. J Genet 91(3):385–395
Bais HP, Ravishankar GA (2001) Cichorium intybus L.–cultivation,
processing, utility, value addition and biotechnology, with an
emphasis on current status and future prospects. J Sci Food Agric
81(5):467–484
Cao Z-F, Li J, Chen F, Li Y-Q, Zhou H-M, Liu Q (2001) Effect of two
conserved amino acid residues on DREB1A function. Biochem-
istry (Moscow) 66(6):623–627
Chen J-R, Lu J-J, Liu R, Xiong X-Y, Wang T-X, Chen S-Y, Guo L-B,
Wang H-F (2010) DREB1C from Medicago truncatula enhances
freezing tolerance in transgenic M. truncatula and China Rose
(Rosa chinensis Jacq.). Plant Growth Regul 60(3):199–211
Choi D-W, Rodriguez EM, Close TJ (2002) Barley Cbf3 gene
identification, expression pattern, and map location. Plant
Physiol 129(4):1781–1787
Cong L, Chai T-Y, Zhang Y-X (2008) Characterization of the novel
gene BjDREB1B encoding a DRE-binding transcription factor
from Brassica juncea L. Biochem Biophys Res Commun
371(4):702–706
Dubouzet JG, Sakuma Y, Ito Y, Kasuga M, Dubouzet EG, Miura S,
Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) OsDREB
genes in rice, Oryza sativa L., encode transcription activators
that function in drought-, high-salt-and cold-responsive gene
expression. Plant J 33(4):751–763
Gao S-Q, Chen M, Xia L-Q, Xiu H-J, Xu Z-S, Li L-C, Zhao C-P,
Cheng X-G, Ma Y-Z (2009) A cotton (Gossypium hirsutum)
DRE-binding transcription factor gene, GhDREB, confers
enhanced tolerance to drought, high salt, and freezing stresses
in transgenic wheat. Plant Cell Rep 28(2):301–311
Gilmour SJ, Zarka DG, Stockinger EJ, Salazar MP, Houghton JM,
Thomashow MF (1998) Low temperature regulation of the
Arabidopsis CBF family of AP2 transcriptional activators as an
early step in cold-induced COR gene expression. Plant J
16(4):433–442
Haake V, Cook D, Riechmann J, Pineda O, Thomashow MF, Zhang
JZ (2002) Transcription factor CBF4 is a regulator of drought
adaptation in Arabidopsis. Plant Physiol 130(2):639–648
Hewitt EJ, Bureaux CA, Royal F (1966) Sand and water culture
methods used in the study of plant nutrition, vol 431. Cambridge
Univ Press, Cambridge
Hong J-P, Kim WT (2005) Isolation and functional characterization
of the Ca-DREBLP1 gene encoding a dehydration-responsive
element binding-factor-like protein 1 in hot pepper (Capsicum
annuum L. cv. Pukang). Planta 220(6):875–888
Huang G-T, Ma S-L, Bai L-P, Zhang L, Ma H, Jia P, Liu J, Zhong M,
Guo Z-F (2012) Signal transduction during cold, salt and drought
stresses in plants. Mol Biol Rep 39(2):969–987
Ito Y, Katsura K, Maruyama K, Taji T, Kobayashi M, Seki M,
Shinozaki K, Yamaguchi-Shinozaki K (2006) Functional ana-
lysis of rice DREB1/CBF-type transcription factors involved in
cold-responsive gene expression in transgenic rice. Plant Cell
Physiol 47(1):141–153
Jaglo KR, Kleff S, Amundsen KL, Zhang X, Haake V, Zhang JZ,
Deits T, Thomashow MF (2001) Components of the Arabidopsis
C-repeat/dehydration-responsive element binding factor cold-
response pathway are conserved inbrassica napus and other plant
species. Plant Physiol 127(3):910–917
Jiang F, Wang F, Wu Z, Li Y, Shi G, Hu J, Hou X (2011)
Components of the Arabidopsis CBF cold-response pathway are
conserved in non-heading Chinese cabbage. Plant Mol Biol Rep
29(3):525–532
Li X-P, Tian A-G, Luo G-Z, Gong Z-Z, Zhang J-S, Chen S-Y (2005)
Soybean DRE-binding transcription factors that are responsive to
abiotic stresses. Theor Appl Genet 110(8):1355–1362
Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-
Shinozaki K, Shinozaki K (1998) Two transcription factors,
DREB1 and DREB2, with an EREBP/AP2 DNA binding
domain separate two cellular signal transduction pathways in
drought-and low-temperature-responsive gene expression,
respectively, in Arabidopsis. Plant Cell 10(8):1391–1406
Liu Y, Zhao T-J, Liu J-M, Liu W-Q, Liu Q, Yan Y-B, Zhou H-M
(2006) The conserved Ala37 in the ERF/AP2 domain is essential
for binding with the DRE element and the GCC box. FEBS Lett
580(5):1303–1308
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression
data using real-time quantitative PCR and the 2-DDCT method.
Methods 25(4):402–408
Mahajan S, Tuteja N (2005) Cold, salinity and drought stresses: an
overview. Arch Biochem Biophys 444(2):139–158
Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K (2012) AP2/ERF family
transcription factors in plant abiotic stress responses. Biochim
Biophys Acta (BBA) Gene Regul Mechan 1819(2):86–96
Mizuno S, Hirasawa Y, Sonoda M, Nakagawa H, Sato T (2006)
Isolation and characterization of three DREB/ERF-type tran-
scription factors from melon (Cucumis melo). Plant Sci
170(6):1156–1163
Okamuro JK, Caster B, Villarroel R, Van Montagu M, Jofuku KD
(1997) The AP2 domain of APETALA2 defines a large new
family of DNA binding proteins in Arabidopsis. Proc Natl Acad
Sci 94(13):7076–7081
Qin Q-L, Liu J-G, Zhang Z, Peng R-H, Xiong A-S, Yao Q-H, Chen
J-M (2007) Isolation, optimization, and functional analysis of the
cDNA encoding transcription factor OsDREB1B in Oryza Sativa
L. Mol Breeding 19(4):329–340
Rogers SO, Bendich AJ (1985) Extraction of DNA from milligram
amounts of fresh, herbarium and mummified plant tissues. Plant
Mol Biol 5(2):69–76
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 Bio-
phys Res Commun 290(3):998–1009
Savitch LV, Allard G, Seki M, Robert LS, Tinker NA, Huner NP,
Shinozaki K, Singh J (2005) The effect of overexpression of two
Brassica CBF/DREB1-like transcription factors on photosyn-
thetic capacity and freezing tolerance in Brassica napus. Plant
Cell Physiol 46(9):1525–1539
Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by
the comparative CT method. Nat Protoc 3(6):1101–1108
Tong Z, Hong B, Yang Y, Li Q, Ma N, Ma C, Gao J (2009)
Overexpression of two chrysanthemum DgDREB1 group genes
causing delayed flowering or dwarfism in Arabidopsis. Plant Mol
Biol 71(1–2):115–129
Wang W, Vinocur B, Altman A (2003) Plant responses to drought,
salinity and extreme temperatures: towards genetic engineering
for stress tolerance. Planta 218(1):1–14
Wang Q-J, Xu K-Y, Tong Z-G, Wang S-H, Gao Z-H, Zhang J-Y, Zong
C-W, Qiao Y-S, Zhang Z (2010) Characterization of a new
54 Plant Growth Regul (2014) 73:45–55
123
dehydration responsive element binding factor in central arctic
cowberry. Plant Cell Tissue Organ Cult (PCTOC) 101(2):211–219
Xianjun P, Xingyong M, Weihong F, Man S, Liqin C, Alam I, Lee
B-H, Dongmei Q, Shihua S, Gongshe L (2011) Improved
drought and salt tolerance of Arabidopsis thaliana by transgenic
expression of a novel DREB gene from Leymus chinensis. Plant
Cell Rep 30(8):1493–1502
Xiong Y, Fei S-Z (2006) Functional and phylogenetic analysis of a
DREB/CBF-like gene in perennial ryegrass (Lolium perenne L.).
Planta 224(4):878–888
Xu Z-S, Ni Z-Y, Li Z-Y, Li L-C, Chen M, Gao D-Y, Yu X-D, Liu P,
Ma Y-Z (2009) Isolation and functional characterization of
HvDREB1—a gene encoding a dehydration-responsive element
binding protein in Hordeum vulgare. J Plant Res 122(1):121–130
Yamaguchi-Shinozaki K, Shinozaki K (2009) DREB regulons in
abiotic-stress-responsive gene expression in plants. Molecular
Breeding of Forage and Turf. Springer, Berlin, pp 15–28
Yang W, Liu X-D, Chi X-J, Wu C-A, Li Y-Z, Song L-L, Liu X-M,
Wang Y-F, Wang F-W, Zhang C (2011) Dwarf apple MbDREB1
enhances plant tolerance to low temperature, drought, and salt
stress via both ABA-dependent and ABA-independent pathways.
Planta 233(2):219–229
Zhang X, Fowler SG, Cheng H, Lou Y, Rhee SY, Stockinger EJ,
Thomashow MF (2004) Freezing-sensitive tomato has a func-
tional CBF cold response pathway, but a CBF regulon that
differs from that of freezing-tolerant Arabidopsis. Plant J
39(6):905–919
Plant Growth Regul (2014) 73:45–55 55
123