Copyright © Physiologia Plantarum 2001PHYSIOLOGIA PLANTARUM 112: 171–175. 2001ISSN 0031-9317Printed in Ireland—all rights reser6ed

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Role of the Arabidopsis CBF transcriptional activators in coldacclimation

Michael F. Thomashow*, Sarah J. Gilmour, Eric J. Stockinger1, Kirsten R. Jaglo-Ottosen and Daniel G. Zarka

Department of Crop and Soil Sciences, Michigan State Uni6ersity, East Lansing, MI 48824, USA1Present address: Department of Horticulture and Crop Science, The Ohio State Uni6ersity/OARDC, Wooster, OH 44691, USA*Corresponding author, e-mail: thomash6@msu.edu

Received 16 June 2000; revised 13 November 2000

Many plants increase in freezing tolerance upon exposure to COR genes and increases freezing tolerance without a lowlow nonfreezing temperatures, a phenomenon known as cold temperature stimulus. We have, therefore, proposed that the

CBF genes are ‘master switches’ that activate a regulon ofacclimation. A fundamental goal of cold acclimation researchgenes involved in cold acclimation. Significantly, the CBFis to identify genes with key roles in this response. Here we

review results from our laboratory regarding the discovery of genes themselves are responsive to low temperature; the levelsa family of transcriptional activators in Arabidopsis (Ara- of CBF transcripts begin increasing within 15 min of transfer-bidopsis thaliana) that regulates the expression of freezing ring plants to low temperature followed by accumulation oftolerance genes. Specifically, we have identified 3 genes that COR gene transcripts at 2–4 h. The CBF genes do not appear

to be subject to autoregulation as the promoter regions haveencode nearly identical transcriptional activators that bind tono evident CRT/DRE elements and overexpression of CBF1the CRT (C-repeat)/DRE (dehydration responsive element)does not induce expression of CBF3. Thus, we have suggestedDNA regulatory element present in the promoters of manythat COR gene induction involves a two-step cascade ofcold- and drought-inducible genes, including those designatedtranscriptional activators: the first step, CBF induction, in-COR (cold-regulated). These regulatory genes, CBF1, CBF2

and CBF3 (C6 RT/DRE b6 inding f6 actor), are located in tandem volving an unknown activator present at normal growth tem-array on chromosome 4. Overexpression of the CBF genes in perature and the second step, COR gene induction, involvingArabidopsis induces expression of the entire battery of known the action of the CBF activators.

and Dunn 1996, Thomashow 1999). The notion has beenthat some cold-induced changes in gene expression areprobably involved in adapting the plant to growth anddevelopment at low temperature while others might havedirect roles in the enhancement of freezing tolerance. Herewe review results from our laboratory regarding the discov-ery of a family of transcriptional activators in Arabidopsisthaliana, the CBF proteins, that control the expression of aregulon of low temperature-regulated genes that contributeto an increase in freezing tolerance.

Arabidopsis COR15a encodes a protein involved infreezing tolerance

Our early studies on cold acclimation in Arabidopsis re-sulted in the identification of 4 COR (cold-regulated) genes,

Many plants increase in freezing tolerance in response tolow temperature, a phenomenon known as cold acclimation(Sakai and Larcher 1987). Over the years, considerableeffort has been directed at determining the molecular basisof this response. These efforts have established that a com-plex array of biochemical and physiological changes occurwith cold acclimation ranging from alterations in lipid com-position to accumulation of sugars (Steponkus et al. 1993,Hughes and Dunn 1996, Thomashow 1999). Consistent withthis complexity are the results of genetic analysis indicatingthat the ability to cold acclimate is a quantitative genetictrait (see Thomashow 1990).

A central goal in cold acclimation research is to identifygenes with roles in freezing tolerance. One approach hascentered on the isolation and characterization of genes thatare induced during the cold acclimation response (Hughes

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COR6.6, COR15a, COR47 and COR78 (Hajela et al. 1990),each of which has subsequently been shown (from work inour laboratory and others) to be a member of a gene pair(Thomashow 1998). DNA sequence analysis indicated thatCOR6.6, COR15a and COR78 encode novel hydrophilicpolypeptides (Thomashow 1998) and that COR47 encodes amember of the dehydrin (or LEA II) protein family(Thomashow 1998), a group of proteins that have beenproposed to have a role in dehydration tolerance (Close1997). Interestingly, at least one member of each COR genepair is induced in response to dehydration stress (Hajela etal. 1990). This is significant as freeze-induced injury resultslargely from the severe cellular dehydration associated withfreezing (Levitt 1980). Thus, it seemed possible that theCOR genes might have roles in dehydration tolerance andtherefore be involved in both freezing and drought tolerance(Hajela et al. 1990). The first evidence in support of thishypothesis came from our work with the COR15a gene.

The COR15a gene encodes a 15-kDa polypeptide,COR15a, that is targeted to the chloroplasts (Lin andThomashow 1992). During import, COR15a is processed toa 9.4-kDa polypeptide, designated COR15am, which be-comes localized in the stroma (Lin and Thomashow 1992, S.J. Gilmour unpublished results). To determine whetherCOR15a might have a role in freezing tolerance, Artus et al.(1996) made transgenic Arabidopsis plants that expressedCOR15a at normal growth temperatures and compared thein vivo freezing tolerance of the chloroplasts in nonaccli-mated transgenic and wild-type plants. The results indicatedthat the chloroplasts of the transgenic plants were 1–2°Cmore freezing tolerant than the chloroplasts in wild-typeplants.

How does the COR15am protein function to enhancefreezing tolerance? Experiments conducted in collaborationwith Peter Steponkus and colleagues at Cornell Universityhave indicated that COR15am helps stabilize membranesagainst freeze-induced injury (Artus et al. 1996, Steponkuset al. 1998). Specifically, COR15a expression in nonaccli-mated plants was found to decrease the incidence of freeze-induced lamellar-to-hexagonal II phase transitions, a type ofmembrane lesion that commonly occurs in nonacclimatedplants. In addition, purified preparations of COR15am werefound to increase the lamellar-to-hexagonal II phase transi-tion temperature of dioleoylphosphatidylethanolamine andpromote formation of the lamellar phase in a lipid mixturecomposed of the major lipid species that comprise thechloroplast envelope. From these results, it was proposedthat COR15am acts to defer freeze-induced formation of thehexagonal II phase to lower temperatures.

While expression of COR15a in Arabidopsis resulted inan increase in chloroplast freezing tolerance, the effect wassmall. Moreover, no effect on whole plant survival could bedemonstrated (Jaglo-Ottosen et al. 1998, S. J. Gilmourunpublished results). This finding was not surprising giventhe quantitative nature of the freezing tolerance trait. Itseemed likely that during cold acclimation, the COR15agene product would act in concert with proteins encoded bythe other known (and yet to be discovered) COR genes. Thequestion raised was whether expression of the entire batteryof COR genes would result in an increase in freezing toler-

ance at a whole plant level. To address this question, weneeded to be able to coordinately induce COR genes atnormal growth temperatures. The ability to do this wasmade possible by our discovery of the CBF family oftranscriptional activators.

Arabidopsis COR genes are regulated by the CBFtranscriptional activators

Our early studies on COR15a and COR78 indicated that thepromoters of these genes were induced in response to lowtemperature and dehydration stress (Horvath et al. 1993,Baker et al. 1994) and that an element, referred to as theCRT (C-repeat), might be involved in cold responsiveness(Baker et al. 1994). Concurrently, Yamaguchi-Shinozakiand Shinozaki (1994), working with RD29a (an alternativedesignation for COR78), defined a regulatory element thatimparted both dehydration- and cold-responsive gene ex-pression. The element, designated the DRE (dehydrationresponsive element), has the same 5-bp core sequence foundin the CRT, CCGAC. The challenge then was to identify theregulatory protein(s) that presumably bound to this element.We accomplished this (Stockinger et al. 1997) using theyeast one-hybrid screen (Li and Herskowitz 1993) that wasdesigned to identify DNA binding proteins. These effortsresulted in the isolation of a cDNA for the first CRT/DRE-binding factor which we designated CBF1 (CRT/DRE-bind-ing factor 1) (Stockinger et al. 1997). CBF1 has a mass of 24kDa, an AP2 DNA-binding domain (Riechmann andMeyerowitz 1998) and an acidic C-terminal region that wehave recently shown acts as an activation domain (E. J.Stockinger unpublished results).

In our initial experiments, we demonstrated that expres-sion of CBF1 in yeast induces the expression of reportergenes that carry the CRT/DRE as an upstream activationsequence (Stockinger et al. 1997). These results establishedthat CBF1 is a transcriptional activator. Subsequent studiesdemonstrated that CBF1 is a member of a family of tran-scriptional activators that includes CBF2 and CBF3(Gilmour et al. 1998). The 3 proteins, which are encoded bygenes present in tandem array on chromosome 4, are greaterthan 85% identical in amino acid sequence. Like CBF1,CBF2 and CBF3 also induce the expression of reportergenes in yeast that carry the CRT/DRE as an upstreamactivator (Gilmour et al. 1998). The CBF1, CBF2 and CBF3genes have independently been isolated by the Shinozakiand Yamaguchi-Shinozaki laboratories (Liu et al. 1998,Shinwari et al. 1998) and are referred to as DREB1b,DREB1c and DREB1a, respectively.

Do the CBF transcription factors activate expression ofthe COR genes in Arabidopsis? Indeed, they do. We initiallydemonstrated this with CBF1 (Jaglo-Ottosen et al. 1998).The CBF1 gene was placed under control of the strongconstitutive CaMV 35S promoter, transformed into Ara-bidopsis and the transgenic plants were examined for CORgene expression. The results indicated that constitutive over-expression of CBF1 induced expression of the COR6.6,COR15a, COR47 and COR78 genes without a low tempera-ture stimulus (Jaglo-Ottosen et al. 1998) (Fig. 1). Subse-

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quent studies showed that overexpression of CBF2 andCBF3 also results in constitutive expression of the CORgenes at normal growth temperature (Gilmour et al. 2000, S.J. Gilmour, A. Sebolt unpublished results). Liu et al. (1998)have reported similar results with overexpression of CBF3(DREB1a). Taken together, the transgenic plant experi-ments indicate that the CBF genes are ‘master switches’ thatcontrol the expression of a ‘regulon’ of CRT/DRE-con-trolled genes that include COR6.6, COR15a, COR47 andCOR78. In wheat, a regulatory ‘master switch’ gene(s) thatcontrols expression of cold-regulated genes has beenmapped to chromosome 5D (Limin et al. 1997). How thisregulatory gene(s) relates to the CBF genes remains to bedetermined.

What effect does activating the CBF regulon of genes atnormal temperatures have on plant growth and develop-ment? The answer appears to be that it depends on the levelof expression of the CBF regulon. The initial CBF1-express-ing transgenic plants that we made (Jaglo-Ottosen et al.1998) did not exhibit any obvious effects on plant growth.In sharp contrast, the CBF3 (DREB1a)-expressing plantsdescribed by Liu et al. (1998) had a striking ‘dwarf’ pheno-type. Similarly, we have made transgenic plants that overex-

press CBF3 and have also found that they have a stuntedphenotype (Gilmour et al. 2000). The possibility that thisdifference is due to differences in the regulon of genesactivated by CBF1 and CBF3 has not been ruled out.However, the more likely explanation would seem to lie inthe fact that the level of COR gene expression is higher inour CBF3-expressing plants than in our CBF1-expressingplants, i.e., the higher the level of CBF regulon expression,the greater the effect on growth.

Expression of the CBF gene regulon increasesArabidopsis freezing tolerance

Does activation of the CBF regulon of genes affect thefreezing tolerance of whole plants? Our results indicate thatit does. Using an electrolyte leakage test to assess thefreezing tolerance of detached leaves, we found that CBF1overexpression resulted in an increase in freezing tolerancein nonacclimated plants; in one transgenic line, the increasewas about 1°C and in the other, the increase was about 3°C(Jaglo-Ottosen et al. 1998). Moreover, an increase in freez-ing tolerance of the more tolerant line could be detected atthe whole plant level; whereas most nonacclimated controlplants did not survive freezing at −5°C for 2 days, mostnonacclimated CBF1-expressing plants did. More recently(Gilmour et al. 2000), we have tested the freezing toleranceof nonacclimated CBF3-expressing transgenic plants thatconstitutively express the COR15am and COR6.6 proteinsat about 3–5-fold higher levels than those observed incold-acclimated control plants. The results indicate thatthese lines are about 4°C more freezing tolerant than nonac-climated control plants. Moreover, cold-acclimated CBF3-expressing plants were found to be more freezing tolerantthan cold-acclimated control plants.

Independently from us, Shinozaki, Yamaguchi-Shinozakiand colleagues have shown that expression of CBF3(DREB1a) increases the freezing tolerance of Arabidopsisplants (Liu et al. 1998, Kasuga et al. 1999). Moreover, theyhave shown that the CBF3-expressing plants are more toler-ant of drought and high salinity stress (Liu et al. 1998,Kasuga et al. 1999).

Fig. 1. Overexpression of CBF1 induces COR gene expressionwithout a low temperature stimulus. Arabidopsis thaliana wild-typeRLD plants (WT) and transgenic RLD plants (A6) that constitu-tively express CBF1 under control of the CaMV 35S promoter weregrown at 22°C (warm) or grown at 22°C and then transferred to4°C for 3 days (cold). Total RNA was prepared from plant tissueand analyzed for CBF1 and COR gene transcripts by northernanalysis using 32P-labeled probes. eIF4A (e6 ukaryotic i6 nitiationf6 actor 4A) is a constitutively expressed gene that is not responsiveto low temperature.

Fig. 2. CBF1 transcripts accumulate rapidly in response to lowtemperature. Wild-type Arabidopsis thaliana RLD plants weregrown at 22°C and then transferred to 4°C for the indicated times.Total RNA was prepared from plant tissue and analyzed for CBF1and COR gene transcripts by northern analysis using 32P-labeledprobes.

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Fig. 3. Model for CBF regulation of theCOR genes. This two-step transcriptionalactivator model is based on thatoriginally proposed by Gilmour et al.(1998). The proposed ICE transcriptionfactor (or protein it interacts with)becomes activated in response to lowtemperature, induces expression of theCBF transcriptional activators, which inturn active expression of the CBFregulon of genes (those controlled by theCRT/DRE-regulatory sequence). See textfor details.

Regulation of the CBF genes by low temperature

The results presented above indicate that expression of theCBF genes at normal growth temperatures results in theinduction of the COR genes. The question then is why arethe COR genes not normally expressed at normal growthtemperatures? The reason is that the CBF genes them-selves are cold-regulated. Specifically, we have found thatthe transcript levels for all 3 CBF genes increase dramati-cally within 15 min of transferring plants to low tempera-ture followed by accumulation of COR gene transcripts at2–4 h (Gilmour et al. 1998) (Fig. 2). Given the rapidity atwhich the CBF transcripts accumulate in response to lowtemperature, we have proposed that there is a transcrip-tion factor present at warm temperature that recognizesthe CBF promoters. This factor would not appear to bethe CBF proteins themselves as the promoters of the CBFgenes lack the CRT/DRE sequence and overexpression ofCBF1 does not cause accumulation of CBF3 transcripts(Gilmour et al. 1998). Thus, we have hypothesized thatCOR gene induction involves a two-step cascade of tran-scriptional activators in which the first step, CBF induc-tion, involves an unknown activator that we havetentatively designated ‘ICE’ (inducer of CBF expression)(see Fig. 3). ICE presumably recognizes a cold-regulatoryelement, the ‘ICE box’, present in the promoters of eachCBF gene. At warm temperature, ICE (or a factor that itinteracts with) is in an ‘inactive’ state, perhaps because itis sequestered in the cytoplasm by a negative regulatoryprotein or is in a form that does not bind to DNA ordoes not activate transcription effectively. Upon exposinga plant to low temperature, however, we envision a signaltransduction pathway becoming activated which results inmodification of ICE (or an associated factor), which inturn allows ICE to induce CBF gene expression. We arecurrently testing the various components of the ‘ICEmodel’. Efforts to date have resulted in the identificationof a 155-bp region of one CBF promoter that can impartcold-regulated gene expression (D. Zarka unpublished re-sults).

Use of the CBF genes to improve freezing tolerance

Freezing temperatures limit the geographical regions whereagronomic plants can be grown and contribute to the an-nual shortfalls between potential and actual crop yields.Improving the freezing tolerance of agronomic plants has,therefore, been a common goal of breeding programs. Un-fortunately, these efforts have met with limited success. Thefreezing tolerance of the most hardy wheat varieties today,for instance, is little better than that of varieties developedin the early 1900s (Sarhan and Danyluk 1998). This lack ofprogress is due largely to the quantitative nature of thefreezing tolerance trait. Additionally, the relative freezingtolerance of plant lines cannot be judged from the analysisof single plants, populations of plants must be tested andmethods to test for small differences in freezing toleranceare not amenable to high-throughput analysis. Thus, thediscovery of the CBF regulatory genes is of potential agro-nomic importance. Can the CBF genes be used to improvethe freezing (and drought) tolerance of plants without caus-ing significant adverse effects on growth and development?One indication that they may comes from the work ofKasuga et al. (1999) demonstrating that expression of CBF3(DREB1a) under control of the RD29a cold- and dehydra-tion-inducible promoter results in an enhancement of Ara-bidopsis freezing (and drought) tolerance while causingminimal obvious effects on plant growth. In addition, asearch of the protein and nucleic acid databases indicatesthat there are potential homologs of the CBF genes in awide range of plants. Research over the next few yearsshould provide important new information regarding the useof CBF genes to improve the stress tolerance of agronomicplants.

Acknowledgements – This work was supported by grants from theUSDA-NRICGP, the National Science Foundation and the Michi-gan Agricultural Experiment Station.

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