Isolation and expression characterization of CBF2 in <i>vitis amurensis</i> with stress

doi:10.4236/as.2013.49062

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Vol.4, No.9, 466-472 (2013) Agricultural Sciences

http://dx.doi.org/10.4236/as.2013.49062

Isolation and expression characterization of CBF2 in

vitis amurensis with stress

Chang Dong1,2, Jianmin Tao1, Meng Zhang1, Yang Qin2, Zhiying Yu1, Bailin Wang2,

Binhua Cai1, Zhen Zhang1*

1College of Horticulture, Nanjing Agricultural University, Nanjing, China; *Corresponding Author: [email protected],

[email protected], [email protected]

2Department of Horticulture, Heilongjiang Academy of Agricultural Science, Harbin, China

Received 5 June 2013; revised 5 July 2013; accepted 1 August 2013

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

The transcription factor VaCBF2, which interacts

with C-repeat/DRE and its promoter, was iso-

lated from Vitis amurensis. The VaCBF2 amino

acid sequence contained a conserved AP2 do-

main of 56 amino acids and a potential nuclear

localization sequence. The sequence of VaCBF2

showed a high level of homology with other

CBF2 family members. Phylogenetic analysis

showed that the amino acid sequences may be

CBF2 proteins with evolutionary relationship.

Quantitative reverse-transcription polymerase

chain reaction analysis indicated that the ex-

pression of VaCBF2 gene in tissues (roots, stems,

leaves, and petioles) was induced by low tem-

perature, high salinity, and application of ab-

scisic acid and salicylic acid in a time-depen-

dent manner but to different extents in the cold-

hardy V. amurensis and the less cold-hardy Vitis

vinifera. The presence of cis-elements such as

MYC and ABRE in VaCBF2 promoter further con-

firmed that this promoter was a component of

the CBF transduction pathway involved in plant

response to multiple stresses.

Keywords: Vitis amurensis ; Stress; CBF2;

Expression

1. INTRODUCTION

Abiotic stresses such as low temperature, drought, and

salinity can adversely affect crop growth and develop-

ment. Thus, improving stress tolerance in breeding is

important. Plants respond and acclimate to environmen-

tal stresses by activating network events from stress per-

ception to effector gene expression [1]. Over the past

years, the C-repeat element-binding factor (CBF)/dehy-

dration responsive element binding (DREB) cold re-

sponse pathway is the most recognized freezing tolerance

pathway in plants [2]. All CBF proteins specifically bind

to the C-repeat element (CRT)/dehydration responsive ele-

ment (DRE) in the promoter region of downstream genes

[3,4]. These proteins regulate their downstream gene

expression and enhance the tolerance of plants to low

temperature, drought, and high salinity. Thus, CBF regu-

lation has a fundamental function in cold acclimation [5].

Arabidopsis CBF1, CBF2, and CBF3 are major tran-

scriptional factors affecting cold-inducible gene expres-

sion [6,7]. CBF4 from Arabidopsis is weakly induced by

cold stress [8], whereas DREB2A is induced by drought

and salinity [9]. These transcript profiles suggest that

CBFs have different expression levels in plants and that a

degree of cross-talk exists between these pathways [10].

Furthermore, CBFs widely exist in herbaceous plants

that are responsive to cold [11]. Puhakainen et al. [12]

demonstrated that the CBF pathway also exists in woody

trees. Since then, CBF orthologs have been reported in

various woody plants, including Vitaceae [13], poplar

[14], blueberry [15], peach [5], and apple [4]. Among

woody species, a positive correlation also exists between

the cold tolerance and CBF transcript level of woody

species [4,5,14,16]. Therefore, the CBF cold pathway

appears to be conserved in woody plants.

Vitis amurensis is one of the most widely used species

for freeze-tolerant rootstock and winemaking in grape

cultivation. In this study, the VaCBF2 gene was isolated

from V. amurensis. We also compared CBF2 gene expres-

sion in the leaves of cold-tolerant wild V. amurensis and

cold-sensitive Vitis vinifera “Manicure Finger” at different

times of cold treatment. Results confirmed the V. amu-

rensis transcript patterns in roots, stems, leaves, and peti-

oles at different times of low temperature, high salinity,

C. Dong et al. / Agricultural Sciences 4 (2013) 466-472 467

and abscisic acid (ABA) and salicylic acid (SA) treatments.

2. MATERIAL AND TREATMENTS

Sixty-day-old in vitro samples of wild V. amurensis

and V. vinifera “Manicure Finger” grown in Murashige

and Skoog (MS) medium under a 16 h light/8 h dark re-

gime at 25˚C were subjected to the following treatments

for stress-responsive gene expression: cold stress by

transferring to 4˚C, salinity stress by supplying 200 mM

NaCl, and hormonal treatments by directly supplying

ABA (10 μM) or SA (5 μM) to plants at different times.

2.1. Isolation of the VaCBF2 Gene

Sixty-day-old in vitro samples of wild V. amurensis

were grown in MS medium under a 16 h/8 h dark regime

at 25˚C. Total RNA was extracted from non-stressed or

cold-stressed plants in vitro using the Lagonigro’s me-

thod [17]. First-strand cDNA was synthesized using Pri-

mer Mix according to the instructions of ReverTra Ace

RT Kit (TOYOBO, Japan). The complete coding se-

quence was amplified using the gene-specific primers

C2F (5’-TCATCAACATCCTCTCCTTCTCG-3’) and C2R

(5’-GCTAAAAGTGTGTATGGCAGTGA-3’). The prim-

ers were designed based on the genome of Vitis

(http://www.vitisgenome.it). Amplification was carried

out under the following conditions: 94˚C for 4 min; 36

cycles of 94˚C for 30 s, 60˚C for 45 s, and 72˚C for 60 s;

and a final extension at 72˚C for 10 min. The polymerase

chain reaction (PCR) products were cloned and sequenc-

ed by pMD19-T vector. Sequence alignments were con-

ducted using DNAMAN software (Version 5.2.2, Lynnon

Biosoft).

2.2. Promoter Isolation of VaCBF2

The isolation of the promoter sequence was carried out

using a Universal Genome Walker Kit (Clontech, USA)

on 10 ng of V. amurensis genomic DNA digested by four

blunt-end-generating restriction enzymes (EcoRI, DraI,

SacI, and StuI). After purification, the restriction frag-

ments were ligated with Genome Walker adaptors. PCR

was amplified on each restriction fragment set using the

primers C2-R1 (5’-GCTAAAAGTGTGTATGGCAG-

TGA-3’) and C2-R2 (5’-CTTCACTCACCCATTTGT-

TCTCATT-3’) for first- and second-step PCRs, respec-

tively. Finally, the PCR products were cloned into pMD

19-T simple vector and sequenced. The sequences were

analyzed online by Plant CARE.

2.3. Expression Analysis of VaCBF2 by

Real-Time Quantitative Reverse

Transcription (qRT)-PCR

RNA and cDNA were obtained as described above.

qRT-PCR was performed in 20 mL volumes containing

10 mL of SYBR Premix Ex Taq mix (TaKaRa), 0.2 mM

forward primer (5’-TGAGAACAAATGGGTGAGTG-

3’), 0.2 mM reverse primer (5’-TGATGGAGGTTGCT-

GAAAA-3’), and 1 mL of diluted (1:10 v/v) cDNA. The

PCR regime consisted of denaturation at 94˚C for 4 min,

followed by 40 cycles of 95˚C for 10 s, 60˚C for 20 s,

and 72˚C for 20 s. Melting curve analysis was performed

from 80˚C to 95˚C at 0.5˚C intervals. VaCBF2 transcript

levels were normalized with grape malate dehydrogenase

gene as an internal control using the forward primer

5’-GCATCTGTGGTTCTTGCAGG-3’ and reverse pri-

mer 5’-CCCTTTGAGTCCACAAGCCAA-3’. The data

of 0.5 h of V. vinifera in leaves at 4˚C were used as basis

for relative comparison. Relative expression was calcu-

lated relative to a calibrator using the formula 2−ΔΔCt. Two

independent replicates were performed per experiment.

3. RESULTS

3.1. Identification of VaCBF2 Gene from V.

amurensis

The sequence of the V. amurensis cDNA clone includ-

ed one open reading frame (ORF). Compared with the

GenBank database, this sequence resulted in an iden-

tical coding sequence to the CBF2 cDNA from V. vinif-

era and V. riparia. This result clearly indicated that the

gene in V. amurensis did not have introns interrupting

its ORF. The coding regions encoded polypeptides of

250 amino acids with a molecular mass of 27.55 kD and

a theoretical isoelectric point of 10.23. The amino acid

alignment of the sequence revealed that 97% and 96%

of the residues were identical to the CBF2 of V. riparia

and V. vinifera, respectively. Moreover, the degrees of

similarity to the CBF2 of Parthenocissus inserta and

Eucalyptus grandis were high (94% and 73%, respec-

tively). Sequence alignment against various CBF pro-

teins suggested that this cDNA encoded a CBF-type pro-

tein in V. a mure n sis (Figure 1). Therefore, the isolated

cDNA was named VaCBF2. In other plants, basic resi-

dues similar to CBF2 were included in their N-terminal

regions. These residues potentially represented an un-

clear localization signal (NLS) and putative APETALA

2/ethylene response factor (AP2/ERF) DNA-binding do-

main. VaCBF2 also contained acidic C-terminal frag-

ments that can act as the transcriptional activation do-

main, but the LWSY signature was modiﬁed from LW-

SY to LWNHDFL in the grape protein. In addition, po-

tential recognition motifs for CBF protein were de-

tected, such as DSAWR, Domain III, and Domain IV

(Figure 1). These results indicated that VaCBF2 from V.

amurensis was a CBF2 or tholog of other plants but with

unique features.

C. Dong et al. / Agricultural Sciences 4 (2013) 466-472

468

Figure 1. Multiple alignment of VaCBF2 with the CBF2 proteins of Vitaceae, Parthenocissus,

and Arabidopsis. The amino acid sequences are from NCBI [AtCBF2 (ABV27118), PtCBF2

(CBF94893), VrCBF (AAR28674), and VvCBF (AAR28677)]. The nuclear localization sig-

nal, AP2, DSAWR, and LWNH domains are indicated by bars.

3.2. Phylogenetic Analysis of VaCBF2

A BLAST search was performed using the deduced

amino acid sequence of VaCBF2. Several homologous

sequences of VaCBF2 in higher plants were found in the

GenBank database. Alignment analysis and domain

comparison indicated that VaCBF2 shared high homol-

ogy with other proteins in the AP2/ERF domain (Figures

1 and 2). By systematic phylogenetic analysis, AP2/ERF

proteins were classified into 17 plant CBF proteins using

the neighbor-joining phylogenetic method (Figure 2).

The results showed that the sequence from V. amurensis

belonged to the CBF2 cluster of CBF proteins.

3.3. Sequence Analy sis of VaCBF2 Promoter

VaCBF2 promoter was obtained using the UGWK me-

thod (Figure 3). The result revealed that a typical TATA-

box was located at −82 bp upstream of the ATG transla-

tional initiation codon. Furthermore, cis-acting elements

involved in stress-related responses were found in VaCBF2

promoter. These elements including ABRE, MYC (MYC

recognition site), TC-rich repeats, and CGTCA-motif were

necessary for stress-responsive expression under abiotic

stress.

Figure 2. Bootstrap phylogenetic analysis of the selection of

plant CBF proteins from NCBI. The amino acid sequences are

VrCBF1 (AAR28671), AgCBF1 (ABU55659), VaCBF1

(ADY17818), VvCBF1 (AAR28673), PtCBF1 (ABU55661),

PiCBF2 (ABU55670), CaCBF2 (ABU55671), VaCBF2

(ADY17812), VrCBF2 (AAR28674), VvCBF2 (AAR28677),

VaCBF3 (ADY17813), VIVvCBF3 (ACT97164), AcCBF4

(ABU55676), PiCBF4 (ABU55674), VaCBF4 (ADY17814),

VvCBF4 (XP_002280097), and CvCBF4 (ABU55679).

3.4. Accumulation of CBF2 in Vitaceae

under Low-Temperature Stress

expression in vitro in the relatively cold-hardy wild V.

CBF2 gene expression in leaves was compared with

OPEN ACCESS

C. Dong et al. / Agricultural Sciences 4 (2013) 466-472 469

Figure 3. Distribution of putative regulatory elements in VaCBF2 promoter from V. amurensis.

amurensis and the relatively cold-sensitive V. vinifera

cultivar “Manicure Finger” (Figure 4). V. amurensis rea-

ched the maximum midwinter cold hardiness level at

<−40˚C, whereas “Manicure Finger” was cold hardy at

approximately −7˚C. The expression levels of CBF2 in

the cold-hardy V. amurensis and the cold-sensitive V. vi-

nifera were not detected under normal conditions (Fig-

ure 4(a)). In both samples, expression slightly increased

and peaked at 48 h under cold treatment. However, the

expression level of VaCBF2 in the cold-hardy V. amuren-

sis was higher than that of VvCBF2 in the cold-sensitive

V. vinifera at a low temperature (Figure 4(b)). This find-

ing was further confirmed by qRT-PCR.

3.5. Expression Pattern of VaCBF2 at Low

Temperature

Accumulations of each transcript in the roots, stems,

leaves, and petioles were analyzed by qRT-PCR to fur-

ther determine the acquisition mechanisms of cold ac-

climation in relation to VaCBF2 gene expression. Accu-

mulations of VaCBF2 transcript in all organs were acti-

vated and exhibited different expression patterns at a low

temperature (Figure 5). During the subsequent chilling

period, the transcript level of VaCBF2 in the leaves

slightly increased at the early stage of treatment and then

peaked on days 2 or 3. By contrast, the transcript level of

VaCBF2 in roots and stems sharply increased, reached

the peak at 8 and 48 h, respectively, and then gradually

decreased. However, mRNA expression in the petioles

was wavy and ebbed at 1 and 24 h of treatment at 4˚C.

3.6. Expression Pattern of VaCBF2 in

Salinity Stress

We analyzed the expression of VaCBF2 gene under

high salinity stress to obtain more information about its

expression under stress. Results showed that VaCBF2

expression was induced by salinity throughout the treat-

ment period (Figure 6). However, distinct organs dis-

played marked differences in gene expression levels

throughout the treatment period. The expression of

VaCBF2 in petioles, stems and leave speaked at approxi-

mately 6 and 8 h of treatment, respectively. The peak

transcript accumulation in petioles was observed at 2 h,

whereas that in roots was wavy.

3.7. ABA and SA Are Regulatory Factors

Affecting VaCBF2

ABA and SA have important functions in physiologi-

cal and genetic regulation during stress. We hypothesized

that the expression pattern of VaCBF2 may be affected

by ABA and SA. Therefore, we measured the transcript

level of organs treated with ABA and SA and then com-

pared their expression patterns. The expression of

VaCBF2 was induced by exogenous ABA and SA treat-

ments (Figure 7). However, treatments with ABA and

C. Dong et al. / Agricultural Sciences 4 (2013) 466-472

470

Figure 4. Transcript accumulation of Vitis CBF2 under low temperature stresses. (a) An expres-

sion difference of CBF2 in the cold-hardy V. amurensis (Va) and the less cold-hardy V. vinifera

“Manicure Finger” (Vv). (b) Expression of organism in Vitaceae at low temperature by

qRT-PCR.

Figure 5. Changes in the mRNA accumulation of VaCBF2 in

roots, stems, leaves, and petioles under 4˚C treatment.

SA exerted significantly different effects on gene expres-

sion. Under ABA treatment, the peak pattern was found

at 6 h of treatment in stems, at the early and late stages in

roots, and at 48 h in leaves. Under SA treatment, the

transcript levels of VaCBF2 were higher. The relative

transcript levels in organs under SA were several times

higher than those under ABA, and this high level was

maintained in all tissues throughout entire treatment

stages.

4. DISCUSSION

We isolated and characterized a complete cDNA se-

quence of CBF2 from V. am ur e nsis. The N-terminal re-

gion of VaCBF2 was found to share the conserved CBF2

domains with that of other known CBF2 (Figure 1). Vitis

CBFs have been proven to be a nuclear protein [18], and

VaCBF2 was found to be mapped in CBF/DREB sub-

family of the CBF2 group through phylogenetic analysis

(Figure 2). The conservation of these sequences in evo-

lutionarily diverse plant species suggested that they

played an important functional role. These data indicated

that VaCBF2 was a CBF protein that may function as a

Figure 6. Changes in the mRNA accumulation of VaCBF2 in

roots, stems, leaves, and petioles under 200 mM NaCl treat-

ment.

transcription factor in V. amurensis.

As one of the important environmental factors affec-

ting abiotic stress signal pathway, low temperature in-

duced and regulated expression of many genes, and this

regulation occurred at the transcript level. Cold report-

edly induces the expression of most reported CBF genes,

which is important for the expression of cold acclimation

and cold tolerance [6,16]. In addition, the expression of

cor 6.6 and kin 1 depended on the induction of CBFs. In

the present study, we demonstrated that VaCBF2 tran-

scription was induced by cold.

CBF2 transcripts were detectable in V. amurensis and V.

vinifera in different tissues (roots, stems, leaves, and

petioles) under low temperature (Figures 4 and 5). These

transcript accumulations remained for a long period, with

lower expression in leaves than in roots and stems. In

agreement with the high identity between their amino

acid domains, the expression patterns showed almost the

same changes between V. amurensis and V. vinifera

C. Dong et al. / Agricultural Sciences 4 (2013) 466-472 471

(a) (b)

Figure 7. Changes in the mRNA accumulation of VaCBF2 in roots, stems, and leaves under ABA and SA treatments.

“Manicure Finger” under low-temperature treatment, ex-

cept in V. amurensis in which the level was high. Inter-

estingly, CBF2 expression in V. vinifera “Chardonnay”

was visible in tissues (leaves, tips, buds, and stems) un-

der natural conditions [18], whereas CBF2 expression in

V. amurensis was not detectable in leaves, stems, roots,

and petioles. The same results were observed in leaves of

V. vinifera “Manicure Finger”. This finding was further

confirmed by qRT-PCR. The important role of CBF2

proteins in the freezing tolerance of V. amurensis can be

attributed to the difference in regulon size between V.

vinifera and V. a mur e nsis because of the high similarity

between VaCBF2 and VvCBF2 (Figure 1). Liu et al. [19]

showed that rice CBF2 expression is induced by CaCl2

and MeJA, cold, dry, and NaCl treatments but not by

ABA and SA under experimental conditions. At the same

time, rice CBF2 was expressed in shoots and seeds but

not in roots and leaves. Novillo et al. [7,20] also reported

that CBF2 is induced later than CBF1 and CBF3 during

cold acclimation. Novillo et al. [20] confirmed that non-

acclimated and cold-acclimated CBF2 mutants have

higher capacity for tolerating freezing and dehydration

than the corresponding WT plants. Over expression of

Arabidopsis CBF2 increases plant freezing tolerance

through the proline and sugar pathways and by inducing

the expression of similar gene sets. All these results sug-

gest that the three CBFs may not have an equivalent

function with other CBFs.

Thomashow [1] suggested that a low-temperature sig-

nal recipient element, inducer of CBF expression (ICE),

can induce the expression of CBF at normal temperature.

However, ICE exists in the non-activated status at normal

temperature. Chinnusamy et al. [21] found that the tran-

scription factor ICE1 is expressed at a base level in Ara-

bidopsis. ICE is a MYC-like bHLH transcription factor

which specially binds to the MYC recognition site in the

CBF promoter region and then activates CBF gen-

erating a low temperature. After analyzing promoter of

VaCBF2 gene, we identified several MYC sites upstream

of the initiation transcription site to which MYC tran-

scription factor can specifically bind (Figure 3). The pro-

moter sequences of this gene were also found to contain

several cis-regulatory elements, such as ABRE, TC-rich

repeat, and CGTCA motif. These elements were related

to stress response, specifically to cold/dehydration. These

cis-regulatory elements were conserved in several plant

species. The presence of these conserved motifs con-

firmed that VaCBF2 gene was regulated by cold, high

salinity, and application of hormones of ABA and SA.

The multiple stress response of the VaCBF2 gene also

suggested that V. amurensis can be used for further re-

search on molecular mechanism and stress breeding.

5. ACKNOWLEDGEMENTS

This work was supported by the National Technology System for

Grape Industry (No.CARS-30-zp-4) and “948” Key Project (No.

2011-G28).

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