Stress-Responsive Gene ICE1 From Vitis Amurensis Increases Cold

8/13/2019 Stress-Responsive Gene ICE1 From Vitis Amurensis Increases Cold

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Research article

Stress-responsive gene ICE1 from Vitis amurensis increases cold

tolerance in tobacco

Q2 Chang Donga,b, Zhen Zhang a,**, JunpengRen a, Yang Qin b, Jinfeng Huang a, Yan Wang a,

Binhua Cai a, Bailin Wang b, Jianmin Tao a,*

a College of Horticulture, Nanjing Agricultural University, Nanjing 210095, Chinab Department of Horticulture, Heilongjiang Academy of Agricultural Science, Harbin 150069, China

a r t i c l e i n f o

Article history:

Received 4 June 2013

Accepted 25 July 2013

Available online xxx

Keywords:

Vitis amurensis

ICE1

Tobacco

Osmotic substances

Cold tolerance

Chilling tolerance

a b s t r a c t

We report the identication of the inducer of CBF expression 1 (ICE1) from Vitis amurensis, an upstream

transcription factor that regulates the transcription of CBF-like genes. The structure of the basic helix-

loop-helix domain of VaICE1 is closely related to that of ICE1 in woody plants. This gene is strongly

induced in leaves, roots, stems, and petioles by cold temperature. With longer duration of exposure to

cold treatments, the expression patterns of organs exhibit differences, which are not observed in normal

condition. Transgenic tobacco over-expressing VaICE1 has higher chilling tolerance and survival ability

by improving the activities of superoxide dismutase, peroxidase, and catalase, as well as the chlorophyll

yield.

2013 Published by Elsevier Masson SAS.

1. Introduction

As one of the primary fruits consumed by human beings, grape

holds a worldwide importance. However, the damage caused by

low temperature to grapes is a difcult problem that hinders

growth and development, seriously affects production and quality,

and impedes the introduction of superior grape varieties with cold

sensitivity. Bio-breeding engineering is an effective and economical

approach to overcome these problems and obtain cold-tolerant

grape varieties.

Early studies have identied numerous genes in plants that

change gene expression, namely, CBF1, CBF2, and CBF3 under cold

stress [1e3]. The expression of CBFs activate the expression of

genes with the DRE/CRT promoter element at warm temperatures,

resulting in constitutive freezing tolerance[4,5]. The inducer of CBFexpression 1 (ICE1), which acts upstream of the CBFs in the cold-

response pathway, has been recently identied. Arabidopsis ICE1

binds to the CBF3 promoter and activates CBF3 expression during

cold treatment[6]. Subsequently, the activated CBF3 binds to the

CRT/DREcis-acting element (CCGAC) in the promoter regions and

induces the expression of downstream cold-responsive genes

(COR15A) and other cold acclimation genes, thereby improving

freezing tolerance[6].

Some cold-inducible genes have been isolated and identied

[7,8], but homologous genes ofArabidopsis ICE1 have not been re-

ported in grapes. Vitis amurensis is a freeze-tolerant wild grape

species that is native to Northern China. V. amurensisis one of the

most widely used species for rootstock and winemaking in grape

cultivation, thus providing a potential molecular biological resource

to improve cold tolerance in grapes by transforming V. amurensis

ICE1. In this study, we successfully isolated the cDNA sequences and

transferred VaICE1 into tobacco by the Agrobacterium-mediated

transformation method, and studied the effects of VaICE1 over-

expression on the cold tolerance of tobacco.

2. Results

2.1. Cloning and characterization of VaICE1

Based on the sequence analysis, we obtained a cDNA sequence

of 1609 bp consisting of a 1548 bp ORF. The ORF encodes a deduced

protein of 516 amino acids with a predicted molecular mass of

55.7 kDa and a pI of 5.42. The amino acid sequence analysis

revealed that the deduced protein contains a basic helix-loop-helix

(bHLH) domain[9]with 52 amino acids, and has high similarity to

Arabidopsis ICE1 (Fig. 1A). Therefore, the gene was designated as

* Corresponding author. Fax: 86 25 84396724.

** Corresponding author. Fax: 86 25 84395724.

E-mail addresses: [email protected] (C. Dong), [email protected]

(Z. Zhang),[email protected](J. Tao).

Contents lists available atScienceDirect

Plant Physiology and Biochemistry

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c om / l o c a t e / p l a p h y

0981-9428/$e see front matter 2013 Published by Elsevier Masson SAS.

http://dx.doi.org/10.1016/j.plaphy.2013.07.012

Plant Physiology and Biochemistry xxx (2013) 1e6

Please cite this article in press as: C. Dong, et al., Stress-responsive gene ICE1 from Vitis amurensisincreases cold tolerance in tobacco, PlantPhysiology and Biochemistry (2013), http://dx.doi.org/10.1016/j.plaphy.2013.07.012

PLAPHY3727_proof 7 August 2013 1/6
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VaICE1 and submitted to GenBank (GenBank: ADY17816). The

phylogenetic analysis for ICE1 of plants clearly divided data into

two groups (Fig. 1B). VaICE1 was assigned to the wood group

closest toEucalyptusICE1 (GenBank: AEF33833, ADY68776).

2.2. Expression of VaICE1 in different organs under cold stress

The transcripts of the VaICE1 gene in organs were distinct un-

der non-stress and low-temperature conditions. Transcript accu-

mulation was not observed under non-stress condition in roots,

leaves, stems, and petioles (Fig. 2A). This nding was further

conrmed by qRT-PCR. However, the transcripts of roots, stems,

leaves, and petioles were tremendously and rapidly induced under

low temperature. A sharp increase was observed at 0.5 he72 h at

4 C, and different expression patterns were observed in the organs

(Fig. 2B).

2.3. Chilling tolerance of transgenic tobacco

To explore the function of VaICE, the overexpressing vector,

including VaICE1, was transformed into tobacco and three

transgenic lines (4-1, 4-4, and 4-8) selected for further testing of

VaICE1. Wilting and ooding of wild-type leaves were surveyed

after storage at 4 C for 2 h. The survival rate reached 0%

(Fig. 3A). However, the overexpressing transgenic lines showed no

signicant morphological changes when stored at 4 C for 2 h,

and the survival rate reached 53% (4-1), 71% (4-4), and 71% (4-8),

respectively. The lines were placed in normal conditions for 12 d torecover. No vital changes were observed in the wild-type, whereas

the transgenic lines showed no effects of low temperature

(Fig. 3B).

To explore the chilling tolerance of VaICE1 overexpression on

owering time, wild-type and transgenic lines were exposed

to 4 C for 4 h and then returned tonormal conditions within 10 d.

The results show that all leaves were ooded and tip stems curled

after chilling treatment of all wild-type and transgenic lines.

However, after returning to normal growth conditions, the leaves of

the wild-type lines appeared dry. Some axillary buds at the foot of

the branches germinated, but were weak, curled, and had dry

leaves (Fig. 3C). For the transgenic lines, some ooded leaves

recovered their green color, and all axillary buds germinated with

strong, normal, and green leaves.

Fig. 1. Sequence alignment and phylogenic analysis of the ICE domain of VaICE1 and other ICE proteins. (A) Comparison of the deduced ICE domain of VaICE1 with other ICE

proteins from AtICE1 (NP_189309), BrICE1 (ADZ24264), CbICE1 (AAS79350), ChICE1 (ADZ48234), EcICE1 (ADY68776), EsICE1 (ACT68317), RsICE1 (ADY68771), MdbHLH (ADL36591),

BjICE (AEE00745), PtICE1 (ABN58427), and RcICE1 (XP_002511101). The putative bHLH domain is marked. The conserved amino acid residues are indicated in black. (B) The

phylogenetic tree was constructed using VaICE1, together with other closely related genes. The genes were from PtICE1 (ABN58427), RcICE1 (XP_002511101), ChICE (ADZ48234),

CsICE (ACT90640), GmICE (ACJ39211), VaICE1 (ADY17816), Vvinifera (XP_002274711), EgICE1 (AEF33833), EcICE1 (ADY68776), RsICE1 (ADY68771), BrICE1 (ACB70963), BnICE1

(AEL33687), EsICE (ACT68317), CbICE1 (AAS79350), and AtICE1 (NP_189309). Q3

C. Dong et al. / Plant Physiology and Biochemistry xxx (2013) 1e62


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Fig. 2. Expression patterns of the VaICE1 gene in various organs at 4 C. (A) RT-PCR analysis under non-stress condition; (B) qRT-PCR analysis at 4 C in organs. Bars indicate the

standard deviation.

Fig. 3. Morphological changes of transgenictobacco overexpressingVaICE1 underchilling treatments. (A)Morphological characteristics andsurvival ratesupon storageat 4 Cfor2h.(B)

Growth states after returning to normal condition for 12 d. (C) Germinating states of axillary bud after returning to normal condition within 10 d at

4

C for 4 h during

owering time.

C. Dong et al. / Plant Physiology and Biochemistry xxx (2013) 1e6 3




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2.4. Enzyme activities and chlorophyll yield of transgenic tobacco

The cold tolerance of VaICE1 in tobacco was further investigated

by analyzing enzyme activities involved in osmotic regulation

pathways in non-stress conditions and 4 C for 2 h. The results

show that VaICE1 overexpression signicantly increased the ac-

tivities of SOD, CAT, and POD under normal and cold conditions.

Sharp increases were observed under low temperature, with the

enzyme activities of transgenic lines being several times higher

than that of wild-type lines (Fig. 4). A signicant increase was

observed in the chlorophyll content under low temperature. These

results reveal that VaICE1 acts as a causal factor for increasing os-

motic accumulation and chloroplast protection in transgenic lines.

3. Discussion

Plants are confronted with numerous stresses that induce or

suppress the expression of a large number of genes. Low temper-

ature triggers the transcription of the CBF family of transcription

factors, which in turn activate the transcription of genes containing

the DRE/CRTpromoter element [1,4]. ICE1 is an important transcript

factor for the CBF3 gene inArabidopsisand has a critical function in

the CBF cold accumulation pathway in plants[6,10]. Therefore, coldsignaling for freezing tolerance requires a cascade of transcriptional

regulations. In this study, we isolated and analyzed ICE1 of

V. amurensis, an upstream transcription factor of this cascade. Ac-

cording to the genome ofVitisdatabase, VaICE1, which consists of

four exons and is located in chromosome 14, contains a sequence

encoding a transcription factor of the bHLH family.

Expression of the VaICE1 gene was strongly induced in leaves,

roots, stems, and petioles by low temperature, but not by normal

condition (Fig. 2). TheArabidopsisICE1 gene was regulated by low

temperature and participated in the CBF cold signal transduction

pathway [6]. Badawi et al. (2008) demonstrated that wheat ICE

gene is induced by low temperature and displays constitutive

expression [11]. However, in our study, we found that VaICE1

expression does not occur in normal condition (Fig. 2A). No reports

currently exist about that the ICE1 gene being non-constitutively

expressed. qRT-PCR was carried out in V. amurensis to conrm

this phenomenon. This phenomenon is possibly due to the genes of

different materials appearing different and the numbers of ICE1,

such as ICE1 and ICE2 inArabidopsis[6,12], and ICE41 and ICE87 in

Triticum [11]. These results need to be analyzed further for

conrmation.

Transgenic tobacco with VaICE1 overexpression displayed a

chilling-responsive phenotype, which became more severe with

higher survival rate (Fig. 3). A similar phenomenon was observed in

transgenic Arabidopsis overexpressing TaICE gene [16], indicating

that the biological function of this protein resembles that of

transgenic plants. This function further activated the accumulation

of osmotic enzyme and improved the chlorophyll yield of trans-

genic lines under chilling temperature (Fig. 4). Cold-responsive

genes encode a diverse array of proteins, such as enzymes

involved in respiration and metabolism of lipids, antioxidants,

antifreeze proteins, and similar substances [10,13,14]. CAT, POD, and

SOD are important osmotic substances that are synthesized or

accumulated to balance the osmotic pressure of the stress envi-

ronment[15]. The activities of SOD and POD enzymes increased in

transgenic tobacco lines under chilling temperature, as well as theactivity of CAT enzyme (Fig. 4). Antioxidant enzymes can reduce the

toxicity caused by O2 production under stress. Thus, the increased

activities of SOD, CAT, and POD can reduce the injury from cold

stress. These results are consistent with the results of Liu et al.[8].

Under cold conditions, plants grow more slowly, and some even

show growth defects or damage. Some of these cold-induced

growth changes are attributed to the slowing of photosynthesis

and generally low metabolic activities in the cold. Our data reveal

that overexpressing VaICE1 altered plant growth and development

at low temperatures. We also observed the increase of chlorophyll

yield. Therefore, tobacco with overexpressed VaICE1 possibly pro-

tects chloroplasts, which subsequently improves plant growth in

the cold.

Fig. 4. Enzyme activity and total chlorophyll content in transgenic tobacco. Fifty-day-old seedlings were withheld from below 4 C for 2 h before dates were taken. Bars indicate the

standard deviation. **P

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Stress-Responsive Gene ICE1 From Vitis Amurensis Increases Cold