ORIGINAL ARTICLE

Functional profiling of EcaICE1 transcription factorgene from Eucalyptus camaldulensis involved in cold responsein tobacco plants

Yuanzhen Lin & Huiquan Zheng & Qian Zhang & Chunxin Liu & Zhiyi Zhang

Received: 31 July 2012 /Accepted: 17 January 2013# Society for Plant Biochemistry and Biotechnology 2013

Abstract A full-length cDNA designated as the EcaICE1gene was isolated from Eucalyptus camaldulensis. TheEcaICE1 gene was predicted to encode a MYC-like proteinof 523 amino acids presenting a considerable degree ofhomology to currently identified plant inducer of CBF ex-pression 1 (ICE1) proteins. RT-PCR assays indicated thatEcaICE1 gene was expressed in a constitutive pattern inintact E. camaldulensis plants or the cold-treated (4 °C)plantlets. Strikingly, over-expression of the EcaICE1 genein transgenic tobacco plants could enhance the tolerance tocold stress. While over-expression of the EcaICE1 generesulted in a profound change of transcript level of C-repeat Binding Factor 1 (CBF1), CBF3 and group 2 LEAprotein NtERD10C genes in transgenic tobacco plants. Fur-ther analysis revealed that EcaICE1 could function as atranscription activator in planta. Briefly, our results stronglysuggest that the EcaICE1 gene is involved in the plantdefense responses to low temperature.

Keywords Eucalyptus camaldulensis . Cold stresses .

Transcription factor . ICE1

AbbreviationsICE1 Inducer of CBF expression 1CBF C-repeat/dehydration-responsive element Binding

Factor

LEA Late embryogenesis abundant proteinCORs Cold-regulated genesbHLH Basic helix-loop-helix

Introduction

Growth and distribution of plants could be limited by lowtemperature. Many plant species also developed an acquiredfreezing tolerance known as cold acclimation (CA) for adap-tion to low temperature. To date, there has been great progresstowards identification of genes involved in cold acclimationand freezing tolerance of plants. These genes could be classi-fied into two major groups (Pearce 1999; Chinnusamy et al.2007): one group encoded products that directly protectedplant cells against frost stress, such as the enzymes requiredfor the biosynthesis of various somoprotectants, and the lateembryogenesis abundant (LEA) proteins, and antifreeze pro-teins, chaperones and detoxification enzymes; the secondgroup was responsible for gene expression regulation andsignal transduction during cold stress response, including thatof some transcription factor genes and protein kinases genes.C-repeat Binding Factors (CBFs) genes encoded proteins thatcould bind to the C-repeat/dehydration-responsive element(CRT/DRE) and activate cold-responsive gene expression(Gilmour et al. 1998). Several CBF genes, such as CBF1,CBF2 andCBF3, had been identified inArabidopsis (Gilmouret al. 1998), and it was observed that over-expression of theCBF genes in transgenic Arabidopsis (Jaglo et al. 1998;Kasuga et al. 1999) and tobacco plants (Kasuga et al. 2004)was able to enhance freezing tolerance. Intriguingly, the ex-pression of CBF genes was also induced by low temperature(Gilmour et al. 1998), suggesting an up-regulation mechanismfor this type of genes. ICE1 belonged to the MYC-like basichelix-loop-helix (bHLH) transcription factor family that

Y. Lin (*) :C. LiuCollege of Forestry, South China Agricultural University,Guangzhou 510642, Chinae-mail: yzhlin@scau.edu.cn

Y. Lin : Z. ZhangNational Engineering Laboratory for Tree Breeding,Beijing Forestry University, Beijing 100083, China

H. Zheng :Q. ZhangGuangdong Academy of Forestry, Guangzhou 510520, China

J. Plant Biochem. Biotechnol.DOI 10.1007/s13562-013-0192-z

activated CBF expression under low temperature (Chinnusamyet al. 2003). The ICE1 gene and/or its homologues had beenfound in a set of plant species, including Arabidopsis thaliana(Chinnusamy et al. 2003; Fursova et al. 2009), Capsella bur-sapastoris (Wang et al. 2005), Populus suaveolens (Lin et al.2007), Triticum aestivum (Badawi et al. 2008), Camellia sinen-sis (Wang et al. 2012), and apple (Malus×domestica) (Feng etal. 2012) plants. Notably, it was evidenced that over-expression of the ICE1 genes could improve the freezingor chilling tolerance of transgenic plants (Badawi et al.2008; Liu et al. 2010).

Eucalyptus species are the important commercial treegroup commonly regarded as the source of wood pulp.The functionality of the Eucalyptus CBF genes were alsowell studied to date (Kayal et al. 2006; Gamboa et al. 2007;Navarro et al. 2009, 2011), but the knowledge about its up-stream regulator component typically for that of the ICE1 orICE1-like modules is still rather limited. Herein, wereported an ICE-like transcription factor gene (EcaICE1,Accession: HQ891008) from Eucalyptus camaldulensisand its transcription pattern in intact or cold-treated (4 °C)plantlets. Furthermore, the functionality of EcaICE1 wasprofiled in transformed tobacco plants by transient andstable expression assays.

Materials and methods

Plant materials and growth conditions

Tissue culture plantlets of Eucalyptus camaldulensis cv. 103were raised and synchronized (using vegetative stem cuttingcontaining an axillary bud) on 1/2 Murashige-Skoog (MS)medium (Murashige and Skoog 1962) supplemented with20 g/l sucrose, 5.5 g/l agar, 0.5 mg/l IBA and 0.1 mg/l NAA,adjusted to pH5.8, in a growth chamber with a 16/8 h light/darkphotoperiod at 25 °C. The 30-day-old rooting plantlets werethen used for tissue collection or subjected to the cold treatment.

Tissue culture plantlets of tobacco (Nicotiana tabacumcv. W38) were raised on MS medium supplemented with30 g/l sucrose, 5.5 g/l agar, 0.1 mg/l NAA and adjusted topH5.8. The plantlets were maintained in a growth chamberwith a 16/8 h light/dark photoperiod at 25 °C. The fullydeveloped tobacco leaves were then used for genetic trans-formation experiments.

Plant treatment

The 30-day-old rooting plantlets of E. camaldulensis weretransferred to a chamber at 4 °C with a 16/8 h light/darkphotoperiod for cold treatment. The E. camaldulensis leaveswere then harvested at time points 0, 0.5, 1, 2, 4, 8 and 24 hpost-treatment. For cold tolerance assay of transgenic

tobacco plants, the 2-month growing plantlets of tobaccowere treated directly from 25 °C to 0 °C for 24 h withoutcold acclimation so as to investigate plant phenotypechange. The E. camaldulensis or tobacco plantlets grownat 25 °C with a 16/8 h light/dark photoperiod were used ascontrols.

Total DNA and RNA extraction and first-strand cDNAsynthesis

Genomic DNA was extracted from the mature leaves oftobacco with a Plant Genomic DNA Kit (TIANGEN,Beijing, P.R. China). Total RNA was extracted from theE. camaldulensis or tobacco sample using the RNAprepPure Plant Kit (Promega, Madison, WI, USA), andtreated with an RNAse-free DNAseI to eliminate resid-ual genomic DNA according to the manufacturer’sinstructions (Promega, Madison, WI, USA). Both thegenomic DNA and total RNA were then evaluated fol-lowing agarose gel electrophoresis and spectrophotomet-rical analysis. Total RNA was employed to generate thefirst-strand cDNA using a PrimeScript RT Reagent Kitfollowing the manufacturer’s instructions (NEB Biolabs,Beijing, P. R. China).

Isolation of the Eucalyptus camaldulensis ICE1 geneEcaICE1

Full-length EcaICE1 cDNA sequence was obtained byPCR from the first-strand cDNA with primer pairs 5′-TTGGATCCATGGTTCTGGGTGGCGAGGAAGACG-3 ′ and 5 ′ -AAGAGCTCAGGAACATCGTCTTGCTCTCTAG-3′ (The added restriction sites BamH Iand Sac I were underlined, respectively), which werewell designed according to the TBLASTN resultsthrough the query of the highly conserved bHLH do-main and C-terminal amino acid sequence of plantICE1 proteins to GenBank EST database of Eucalyptusand E. grandis genome. The PCR products were clonedinto pGEM-T vector (Promega, Madison, WI, USA)and sequenced.

Computer-assisted analysis

The online program Compute pI⁄Mw (http://web.expasy.org/compute_pi/; Bjellqvist et al. 1993) was used to predict thetheoretical pI (isoelectric point) and MW (molecular weight)of the EcaICE1 protein. The EcaICE1 and plant ICE1 homo-logue amino acid sequences retrieved from NCBI (http://www.ncbi.nlm.nih.gov/) GenBank database were subjectedto CLUSTALX 1.83 software for multiple alignment analysis.The online bioinformatics server of the Swiss Institute ofBioinformatics (SIB) (http://cn.expasy.org/) was employed

J. Plant Biochem. Biotechnol.

Fig. 1 Multi-alignment and tertiary structure of EcaICE1 protein.a Amino acid alignment of the EcaICE1 protein with nine plant ICE1proteins. The predicted protein domains were shown. The Arabidopsisthaliana AtICE1 (Accession: AAP14668), Capsella bursa CbICE53(Accession: AAS7935), Camellia sinensis CsICE1 (Accession:GQ229032), E. camaldulensis EcaICE1 (Accession: HQ891008), Oryzasativa OsbHLH001 (Accession: AK102594), Ricinus communis RcICE1

(Accession: EQ973773), Populus suaveolens PsICE1 (Accession:ABF48720), P. trichocarpa PtICE1 (Accession: ABN58427), Triticumaestivum TaICE41 (Accession: EU562183) and Vitis vinifera VvCE1(Accession: XM_002284492) proteins are included. b The predictedtertiary structure of EcaICE1 protein. The tertiary structure was formedby “AKNLMAERRRRKKLNDRLYMLRSVVPRSARMDRASIFGEAI DYLKEV”

J. Plant Biochem. Biotechnol.

to identify conserved domains and functional domains andpredict tertiary structure. The phylogenetic trees were con-structed using the neighbor-joining method with 1,000 boot-strap replications by MEGA version 4.1(Tamura et al. 2007).

Semi-quantitative RT-PCR and Real-time quantitativeRT-PCR

Semi-quantitative RT-PCR and Real-time quantitative RT-PCR (qRT-PCR) assays were conducted according to Zhenget al. (2009). To qualify or quantify the expression of the targetgenes EcaICE1 (Accession: HQ891008), tobacco CBF1 (Ac-cession: EB439334) and CBF3 (Accession: GO308056) andNtERD10C (Accession: AB049337, a family member ofgenes encoding group 2 LEA proteins, Kasuga et al. 2004),the Eucalyptus ACTIN gene (Accession: CT983089) or tobac-co ACTIN gene (Accession: U60491) was used as anendogenous control gene. RT-PCR primers used were asfollows: EcaICE1 forward primer, 5′-CTGGGTGGCGAGGAAGACG-3′, and reverse primer, 5′-CCAAATTCCCCAATTTCTCAGC-3′; Eucalyptus EcaACTIN forward prim-er, 5′-GGCATCACACATTCTACAACG-3′, and reverseprimer, 5′-CCAGAACAATACCAGTTGTAC-3′. qRT-PCRprimers used were as follows: EcaICE1 forward primer, 5′-GAGAGCAGTGAACATCCACA-3′, and reverse primer, 5′-

GGACATCTTGACCTTCCCTA-3′; tobacco NtCBF1 for-ward primer, 5′-GGATGAGGAGACGCTATTCTG-3′, andreverse primer, 5′-TGTGAACACTGAGGTGGAGG-3′; to-bacco NtCBF3 forward primer, 5′-TATTCAGAAGGCGACCGT-3′, and reverse primer, 5′-CCTCCTCGTCCATAAACAA-3′; tobacco NtERD10C forward primer,5′-CTCATGCCCAAGAGGAACAT-3′, and reverse primer, 5′-GCCCGTCCTCTCCTATTTCT-3′; tobacco NtACTIN for-ward primer, 5′-CTGCTGGAATTCACGAAACA-3′, and re-verse primer, 5′-GCCACCACCTTGATCTTCAT-3′. EachPCR assay was carried out for three biological replicates andeach assay replicated two technological repeats in separateexperiments.

Vector construction

The full-length open reading frame (ORF) of the EcaICE1gene was PCR-amplified from an EcaICE1 cDNA clonewith a primer pairs as described previously (see the sectionof ‘Isolation of EcaICE1’). The resultant PCR product wasdigested with BamH I and Sac I (Promega, Madison, WI,USA) and purified with a QIAquick Gel Extraction Kit(Qiagen, Hilden, Germany). This purified product was thenfused to the CaMV 35S promoter sequence of the pBI121vector (Clontech, USA) that was previously digested withBamH I and Sac I to release the GUS reporter gene. Theresultant vector was designated as pBI121-EcaICE1. For theconstruction of vector AtCBF3p-GUS, the AtCBF3 promoter(Accession: EF523130) sequence was cloned from thegenomes of Arabidopsis thaliana plants by PCR with primerpairs 5′-TCATGGATCCACCATTTGTTAATGCATGATGG-3′, and reverse primer, 5′-GCTCAAGCTTTCTGTTCTAGTTCAGG-3′ (The added restriction sites BamH Iand Hind III were underlined, respectively). Theobtained AtCBF3 promoter was digested with BamH Iand Hind III (Promega, Madison, WI, USA) and puri-fied with purified with a QIAquick Gel Extraction Kit(Qiagen, Hilden, Germany), and then fused to the GUSreporter gene of the pBI121 vector (Clontech, USA) thatwas previously digested with BamH I and Hind III torelease the 35S promoter. Both the pBI121-EcaICE1 andAtCBF3p-GUS were confirmed by sequencing.

Plant transformation

Tobacco leaf disc transformation was performed using theAgrobacterium-mediated method (Horsch et al. 1985). Theputative transgenic plantlets were confirmed by PCR andRT-PCR. The verified transgenic tobaccos were then prop-agated and synchronized (using vegetative stem cuttingcontaining an axillary bud) from primary transformants inMS medium. The 30-day-old in-vitro-grown plantlets weretransferred to soil and cultivated in a growth chamber at 25 °C

Fig. 2 Phylogenetic tree of the plant ICE1 proteins and their homo-logs. The ICE1 proteins and their homologs employed were as follows:A. thaliana AtICE1 (Accession: AAP14668), C. bursa CbICE53 (Ac-cession: AY506804), C. sinensis CsICE1 (Accession: GQ229032), E.camaldulensis EcaICE1 (Accession: HQ891008), E. globules EgICE1(Accession: AEF33834), G. max GmICE1 (Accession: FJ393223),Hordeum vulgare HvICE2 (Accession: DQ151536), Lotus japonicasLjbHLH23 (Accession: FJ379764),Malus × domesticaMdbHLH (Ac-cession: EF495202), Medicago truncatula MtHLH (Accession:AC149131), O. sativa OsbHLH001 (Accession: AK102594), P. sua-veolens PsICE1 (ABF48720), P. trichocarpa PtrICE1 (ABN58427),Ricinus communis RcICE1 (Accession: EQ973773), T. aestivumTaICE41 (Accession: EU562183) and TaICE87 (EU562184), V. vinif-era VvICE1 (Accession: XM_002284492), and Zea mays ZmICE2(Accession: EU974475)

J. Plant Biochem. Biotechnol.

under a 16/8 h light/dark photoperiod for 1 month, and thenused for subsequent cold tolerance assay and gene expressionexperiments.

Electrolyte leakage assay

Electrolyte leakage assay was performed by the method (Linet al. 2009). After subjected to different cold temperatures(4 °C, 0 °C, −2 °C, −4 °C) for 2 h, 10 leaf discs samples (5mmin diameter by stiletto) for each group were washed andimmersed in 5 ml deionized water. Following vacuum infil-tration, the electrical conductivity of the supernatant (S1) wasmeasured by a SevenEasy-S30 detector (Mettler, Swiss). Thesamples were then terminally boiled to measure the ultimatemaximum conductivity (S2). The relative leakage degree (L)was assessed by the ratio S1/S2.

Agrobacterium-mediated effecter-reporter transientexpression assay

The two different Agrobacterium EHA105 cultures(OD600=0.8) carrying (pBI121-EcaICE1) or reporter(AtCBF3p-GUS) plasmid were mixed at a 1:1 ratio, andco-infiltrated into intact tobacco leaves for the co-expression

of the effecter and reporter by using an Agrobacterium-medi-ated transient expression assay as described by Yang et al.(2000). GUS activity was measured as described by Jeffersonet al. (1987). The intact tobacco leaves were ground in liquidnitrogen, homogenized in GUS extraction buffer (50 mMNaH2PO4, pH7.0, 10 mM EDTA, 0.1 % Triton X-100,0.1 % (w/v) SDS, 10 mM β-mercaptoethanol), and centri-fuged at 12,000 rpm at 4 °C for 5 min. The supernatant wasused to determine GUS activity. GUS activity was calculatedas nmol of MU per milligram of soluble protein per minute.The supernatant protein concentration was assessed by themethod described by Bradford (1976). Tobacco leaves infil-trated with plasmid (pBI121-GUS) were used as acomparison.

Results

Characterization of the Eucalyptus camaldulensis ICE1gene

The EcaICE1 cDNA (Accession No. HQ891008) was 1792-bp long and comprised a complete open reading frame(ORF) encoding a 523 amino acid peptide. Computer-

Fig. 3 Semi-quantitative RT-PCR analysis of EcaICE1 gene in Euca-lyptus camaldulensis. a Tissue expression pattern of EcaICE1. c Timecourse of EcaICE1 gene transcript level under the treatment of cold(4 °C). b and d Relative transcript level of the EcaICE1 gene. Expres-sion level of EcaICE1 gene in panel a and c was quantified by referringto the Eucalyptus ACTIN gene that was arbitrarily set to 1.0 forstandardization. The expression data calculated by TotalLab gel anal-ysis software version 12.1 (Nonlinear Dynamics, Newcastle uponTyne, UK) was corrected with the ratio 4 because the size of PCR

product of EcaICE1 gene was rather different from that of the ACTINgene. Eucalyptus ACTIN gene (Accession: CT983089) was used as areference gene. Each RT-PCR assay of the same tissue or cold treat-ment samples was carried out for three biological replicates and eachfor two technological repeats in separate experiments. The mean andstandard deviation of the relative EcaICE1 transcript level in respectivetissue or cold treatment samples were present. Significance wasassessed using the ANOVA Fisher’s LSD test (P<0.05)

J. Plant Biochem. Biotechnol.

assisted analysis revealed that the predicted amino acidsequence of the EcaICE1 ORF had an estimated proteinMWof approximately 56.6 kDa and a pI of 5.88. Moreover,the EcaICE1 protein was found to harbor several conserveddomains: a serine-rich region (S-rich), basic helix loop helixdomain (bHLH), zipper region (ZIP), and SUMO conjuga-tion motif (Fig. 1a). The S-rich and Boxes I–III showedhighly conserved fragments in eudicot plant ICE1 sequen-ces, while boxes i and ii showed sequences specificallyconserved in woody plants. Notably, box iii with a stretchof 13 amino acids (GKNGGNSSNANST) was only foundin woody plant ICE1 proteins. In addition, the predictedtertiary structure of the bHLH domain of EcaICE1

(Fig. 1b) consisted of two alpha-helices separated by a loopregion, which was highly conserved in plant ICE1 proteins(Fig. 1a). Based on the phylogenetic analysis, it was furtherrevealed that the EcaICE1 protein belonged to the eudicotgroup and presented a closely evolutionally relationshipwith EgICE1 from E. globulus (Fig. 2). These results indi-cated that EcaICE1 might have ICE1 functions in Eucalyp-tus cold stress responses.

The expression pattern of EcaICE1 gene

Semi-quantitative RT-PCR assays indicated that basal ex-pression of EcaICE1 gene occurred in all the tested tissues,

Fig. 4 Over-expression of theEcaICE1 gene enhanced coldtolerance of the transgenictobacco plants. a Schematicrepresentation of pBI121-EcaICE1 vector for genetictransform assay. NOS-T, Nosterminator. b Identification ofthe transgenic lines by genomicPCR. c RT-PCR analysis ofEcaICE1 gene in transgenictobacco plants harboringpBI121-EcaICE1. d Coldtolerance assay for transgenictobacco plants harboringpBI121-EcaICE1 at 0 °C for24 h without cold acclimation. eElectrolyte leakage assay at 25 °C,4 °C, 0 °C, −2 °C and −4 °C for2 h. Data was mean with standarddeviations obtained from threeindependent biologicalexperiments. The significance ofthe differences in gene expressionlevels between transgenic andwild-type tobacco plants wasassessed using Student’s t test(**P<0.01 and *P<0.05)

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including leaf, root and stem of E. camaldulensis plants(Fig. 3a). Strikingly, the EcaICE1 gene had a significantlyhigher level in leaves compared to stems and roots by usingthe ANOVA Fisher’s LSD test (*P<0.05) (Fig. 3b), similarto that of the AtICE1 gene in Arabidopsis (Chinnusamy etal. 2003). Additionally, it was observed that the EcaICE1gene expression could not be significantly altered by thetreatment of cold in the time range from 0.5 to 24 h (P>0.05) (Fig. 3c and d). These results suggested a constitutive

expression pattern of the EcaICE1 in E. camaldulensi undereither the intact or cold-treated conditions.

Over-expression of EcaICE1 gene enhanced the coldtolerance of the transgenic tobacco plants

In order to elucidate the functional role of EcaICE1 under thecold stress in plants, EcaICE1 was over-expressed in trans-genic tobaccos under the control of CaMV 35S promoter

Fig. 5 Transcript levels of EcaICE1 target genes (NtCBF1 (Acces-sion: EB439334), NtCBF3 (Accession: GO308056) and NtERD10c(Accession: AB049337, a family member of genes encoding group 2LEA proteins)) in transgenic tobacco plants during cold stresses. Thetransgenic tobacco lines included TG13, TG16 and TG20. WT, wild-type plants (tobacco). Tobacco ACTIN gene (Accession: U60491)

was used as a reference gene. Data was mean with standard devia-tions obtained from three independent biological experiments. Thesignificance of the differences in gene expression levels betweentransgenic and wild-type tobacco plants was assessed using Student’st test (**P<0.01 and *P<0.05)

Fig. 6 Agrobacterium-mediated transient expression of GUS reportergene in AtCBF3p-GUS vector regulated by EcaICE1. a Schematicrepresentation of the reporter and effector vectors. GUS, β-glucuroni-dase gene. NOS-T, Nos terminator. AtCBF3p, Arabidopsis AtCBF3gene promoter (Accession: EF523130). b GUS-reporter activities intransient transformed tobacco leaves. Data was mean with standard

deviations obtained from three independent biological experiments.The significance of the differences in gene expression levels wasassessed using Student’s t test (**P<0.01). Tobacco leaves infiltratedwith only the reporter plasmid (AtCBF3p-GUS) were used as a nega-tive control. Tobacco leaves infiltrated with only the reporter plasmid(pBI121-GUS) was used as a comparison

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(Fig. 4a). Independent transgenic tobacco lines were obtainedby kanamycin-resistance selection, and further confirmed bygenomic PCR and RT-PCR (Fig. 4b and c). A total of 40transgenic lines were obtained, and cold stress assay foundthat 11 transgenic lines did not show improvement of coldtolerance while the rest 29 transgenic lines improved coldtolerance. Three representative transgenic lines includingTG13, TG16 and TG20 were selected from above 40 trans-genic lines for further assays. The TG13 and TG16 linespresented a relatively high EcaICE1 level, while undetectablepattern of EcaICE1 was observed for TG20 line possibly dueto the silence effect in plants (Fig. 4c). As indicated in Fig. 4d,over-expression of EcaICE1 gene significantly increased thecold tolerance of TG13 and TG16 compared to that of the WTplants. WT and TG20 plants exhibited severe injury: the firstand second true leaves, and even some entire plants were dead.However in the transgenic lines TG13 and TG16, there wasslight injury in the second and third true leaves, even thoughsome of the first true leaves were severely injured. Addition-ally, electrolyte leakage analysis was presented in Fig. 4e.Among the temperature from −2 °C to −4 °C, the percentagesof electrolyte leakage of TG13 and TG16 lines increased fromapproximately 38.0 % to 50%, significantly lower than that ofWT and TG20 lines (increased from approximately 68 % to90 %) (t test, *P<0.05 and **P<0.01), indicating that theleaves of tobacco plants over-expressing EcaICE1 were lesssusceptible to cold stress, which was consistent with the resultsof cold tolerance assay. These results demonstrated theEcaICE1 gene could enhance the cold resistance of the trans-genic plants under low temperature stresses.

EcaICE1 over-expression increased the expression of coldresponsive genes in tobacco plants

Since ICE1 had been found to induce expression of CBFs andsome cold responsive genes, the transcript levels of these cold-regulated genes was monitored by qRT-PCR analysis. Over-expression of the EcaICE1 gene could cause an up-regulationof the cold responsive modules including NtCBF1, NtCBF3and NtERD10C genes in transgenic tobacco plants, comparedto WT plants (Fig. 5). Although EcaICE1 was expressedunder normal temperature, accumulation of NtCBFs and coldresponsive gene NtERD10C was greatly induced after coldstress, indicating that EcaICE1 would be activated throughcold treatment. These results revealed the EcaICE1 gene wasresponsible for the defense responses of the transgenic plantsto low temperature.

The transactivation activity analysis of EcaICE1

To access the regulatory effect of EcaICE1 in planta, anAgrobacterium-mediated effector-reporter transient expres-sion assay was employed. As shown in Fig. 6, expression of

the EcaICE1 gene could significantly augment the GUSactivity by 10.4 folds in transient transformed tobaccoleaves(**P<0.01), compared to that of control, indicatinga positive effect of the EcaICE1 gene on the function ofCBF promoter.

Discussion

In the present study, we isolated and characterized a newICE1, EcaICE1, form Eucalyptus camaldulensi. The pre-dicted protein of EcaICE1 showed high conserved domainsin the Ser 403 site and Box II, besides in the bHLH domain(Fig. 1a). Ser 403 site was fully identical in all plant ICE1proteins, which was found important for the transactivationactivity and stabilization of ICE1 in planta (Miura et al.2011). Chinnusamy et al. (2003) evidenced that a substitu-tion of R236 of Box II with H236 of Arabidopsis ice1mutant resulted in the loss of ICE1 function suggesting apivotal role of intact sequence of Box II for the functionalactivity of ICE1. Moreover, one of the most importantfunction domains of plant ICE1 proteins was the bHLHdomain. The bHLH genes played important roles in plantgrowth and development, such as signal transduction, abi-otic stress responses and flower development (Carretero-Paulet et al. 2010). Plant bHLH proteins were classified into26 subfamlies by evolutionary relationships and differentfunctions (Pires and Dolan 2010). Arabidopsis ICE1(Chinnusamy et al. 2003), wheat ICE1 and ICE2 (Badawiet al. 2008), and tea CsICE1(Wang et al. 2012), involved inplant cold responses, were all belonging to the SubfamilyIIIb group that was characterized by the highly conserved C-terminal regions besides the bHLH domain (Pires and Dolan2010). Similarly, EcaICE1 possessed the typical conservedC-terminal region and it belonged to the Subfamily IIIb,implying that EcaICE1 might have key functions in Euca-lyptus cold stress responses.

The expression patterns of the EcaICE1 in E. camaldu-lensi were constitutive under either the intact or cold-treatedconditions (Fig. 3b and d). Indeed, constitutive expressionof ICE1 protein genes was also found in other plant speciesincluding Arabidopsis (Chinnusamy et al. 2003), wheat(Badawi et al. 2008), tea (Wang et al. 2012) and apple (Fenget al. 2012). Constitutive expression of ICE1 protein genesmight allow a pre-deposition of constitutive ability of theplants in response to certain abiotic stresses, e.g. cold.

Previous studies showed that over-expression of ICE1could enhance the cold tolerance and expression of itsdownstream cold responsive genes in transgenic plants.Chinnusamy et al. (2003) reported that over-expression ofAtICE1 could only increase the expression of AtCBF2,AtCBF3 and cold-regulated genes (CORs) under cold stress,and it was evidenced freezing tolerance of transgenic plants

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was improved after cold acclimation. Fursova et al. (2009)found that over-expression of another Arabidopsis ICE1-like protein only resulted in dominant changes of AtCBF1transcription level and increased freezing resistance in trans-genic Arabidopsis after cold acclimation. While Badawi etal. (2008) discovered that over-expression of the wheat ICEgenes in Arabidopsis could increase freezing tolerance ofArabidopsis, and induced a higher expression of AtCBF2,AtCBF3 and some CORs only after cold acclimation. Fur-thermore, they suggested that other factors induced by lowtemperature were required for wheat ICE activity. Otherstudies, such as that from Liu et al. (2010), it was revealedthat over-expressing AtICE1 in cucumber was able to raisethe chilling tolerance, and simultaneously changed the lev-els of several cold-responsive associated contents (e.g. sol-uble sugar, free proline and malondialdehyde (MDA)) in thetransgenic plants. While Feng et al. (2012) uncovered thatectopic expression of apple ICE1-like gene inhibited theroot growth of transgenic Arabidopsis plants but promotedthe root growth and chilling tolerance and remarkably in-duced AtCBFs expression under cold stress. Compared tothese studies, herein, it was also found that over-expressionof the EcaICE1 gene could enhance the tolerance of thetransgenic plants to cold stresses (Fig. 4d and e), and theexpression of cold-responsive components (Fig. 5), reveal-ing that EcaICE1 might also function as a key transcriptionfactor in plants during cold stresses.

In Arabidopsis, AtICE1 could bind to MYC recognitionelements (CANNTG) in the AtCBF3 promoter by AtICE1bHLH domain, to induce expression of AtCBF3 but little effecton the expression of AtCBF1 and AtCBF2 (Chinnusamy et al.2003). While Badawi et al. (2008) reported that both wheatICE1 and ICE2 genes could bind to different MYC elements ofthe wheat CBF1 promoter. Feng et al. (2012) found that ICE1-like protein (MdCIbHLH) function especially with MdCBF2promoter to activate downstream target genes under cold stress.Since over-expression of the EcaICE1 gene strikingly en-hanced expression of CBF1 and CBF3 in the transgenic tobac-co plants, and that EcaICE1 could have a positive effect on thefunction of AtCBF3 promoter (Fig. 6), it was suspected thatEcaICE1 might bind to the CBF gene promoter sequence toactivate the CBF gene expression in planta.

In this work, we had isolated and characterized an ICE-like transcription factor gene (EcaICE1) from E. camaldu-lensis. The constitutive expression pattern was revealed inthe native plantlets. Moreover, it was demonstrated thatexpression of the EcaICE1 gene could increase the toleranceof transgenic plants (e.g. tobacco) upon the challenge ofcold, possibly due to its regulatory ability on down-streamcold/low temperature responsive gene (e.g. CBF gene) ex-pression in planta. The present data furthered our under-standing of the functional role of ICE-like transcriptionfactor gene in E. camaldulensis.

Acknowledgements This work was supported by Open ResearchProject of National Engineering Laboratory for Tree Breeding, BeijingForestry University, China (FOP2010-4), Foundation for DistinguishedYoung Talents in Higher Education of Guangdong, China(LYM10040), and Key Laboratory of Biomass Energy of GuangdongHigher Education Institutes,South China Agricultural University, Chi-na (BOP2012-7). We thank Dr. Washington Gapare from CSIRO PlantIndustry in Australia for paper revision.

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