11
ORIGINAL PAPER Isolation and characterization of two DREB1 genes encoding dehydration-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 Ó 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 Á Subcellular localization Á 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 this article (doi:10.1007/s10725-013-9866-8) contains supplementary material, which is available to authorized users. M. Liang (&) Á D. Chen Á M. Lin Á Q. Zheng (&) Á Z. Huang Á Z. Lin Á 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 Á D. Chen Á M. Lin Á Q. Zheng Á Z. Huang Á Z. Lin Á 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

Isolation and characterization of two DREB1 genes encoding dehydration-responsive element binding proteins in chicory (Cichorium intybus)

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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.

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