Plant Physiol. (1999) 119: 463-470

The Arabidopsis CBF Gene Family Is Composed of Three Genes Encoding AP2 Domain-Containing Proteins Whose Expression Is Regulated by Low Temperature but Not by Abscisic Acid or Dehydration¹

Joaquín Medina, Mónica Bargues, Javier Terol, Manuel Pérez-Alonso, and Julio Salinas^*

Departamento de Mejora Genética y Biotecnología, Instituto Nacional de Investigaciones Agrarias y Alimentarias, Carretera de la Coruña, Km. 7, 28040 Madrid, Spain (J.M., J.S.); and Departamento de Genética, Universidad de Valencia, 46100 Burjasot, Spain (M.B., J.T., M.P.-A.)

	ABSTRACT

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We have identified two genes from Arabidopsis that show high similarity with CBF1, a gene encoding an AP2 domain-containing transcriptional activator that binds to the low-temperature-responsive element CCGAC and induces the expression of some cold-regulated genes, increasing plant freezing tolerance. These two genes, which we have named CBF2 and CBF3, also encode proteins containing AP2 DNA-binding motifs. Furthermore, like CBF1, CBF2 and CBF3 proteins also include putative nuclear-localization signals and potential acidic activation domains. The CBF2 and CBF3 genes are linked to CBF1, constituting a cluster on the bottom arm of chromosome IV. The high level of similarity among the three CBF genes, their tandem organization, and the fact that they have the same transcriptional orientation all suggest a common origin. CBF1, CBF2, and CBF3 show identical expression patterns, being induced very rapidly by low-temperature treatment. However, in contrast to most of the cold-induced plant genes characterized, they are not responsive to abscisic acid or dehydration. Taken together, all of these data suggest that CBF2 and CBF3 may function as transcriptional activators, controlling the level of low-temperature gene expression and promoting freezing tolerance through an abscisic acid-independent pathway.

	INTRODUCTION

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Many plant species from temperate regions can increase their freezing tolerance in response to low, nonfreezing temperatures (Levitt, 1980; Sakai and Larcher, 1987). This process, called cold acclimation, involves several biochemical and physiological changes that seem to be regulated through changes in gene expression (Thomashow, 1994). Genetic analyses revealed that multiple genes are involved in cold acclimation (Thomashow, 1990), and a wide number of genes whose transcript levels accumulate in response to low temperatures have been isolated and characterized (Thomashow, 1994; Hughes and Dunn, 1996; Capel et al., 1997; Gana et al., 1997; Hong et al., 1997; Capel et al., 1998; Kiyosue et al., 1998; Urao et al., 1998). However, the precise role that these genes play in the process of cold acclimation remains to be determined.

During the past few years a major goal in the study of gene expression induced by low temperature has been to determine the specific cis-acting regulatory sequences. Yamaguchi-Shinozaki and Shinozaki (1994) first identified two 9-bp DNA elements in the promoter of the Arabidopsis RD29A gene that activated gene expression in response to low temperature and drought when fused to a reporter gene. The two elements contained the low-temperature DRE core sequence CCGAC, also named C-repeat by Thomashow and colleagues (Baker et al., 1994). This sequence, hereafter referred to as LTRE, has been found to be essential for the low-temperature responsiveness of other cold-induced plant genes, including the Arabidopsis gene COR15A (Baker et al., 1994), the Brassica napus gene BN115 (Jiang et al., 1996), and the wheat gene WCS120 (Ouellet et al., 1998). Although many of the changes in gene expression that occur during the process of cold acclimation are mediated by ABA (Bray, 1993; Welin et al., 1994), the results of Yamaguchi-Shinozaki and Shinozaki (1994) revealed that the LTRE is not responsive to ABA, suggesting that it imparts cold- and dehydration-regulated gene expression through an ABA-independent pathway.

A step toward increased understanding of the molecular mechanisms that control cold acclimation and, therefore, how low temperatures regulate gene expression was the isolation and characterization of a cDNA from Arabidopsis encoding a C-repeat/DRE/LTRE-binding protein, CBF1 (C-repeat/DRE-Binding Factor; Stockinger et al., 1997). CBF1 was described as a single- or low-copy-number gene, the expression levels of which did not change appreciably in plants exposed to low temperatures or water stress. The deduced CBF1 amino acid sequence indicated that the protein had in its N-terminal region a potential nuclear localization sequence followed by an AP2 DNA-binding motif and an acidic C-terminal half that might act as an activator domain (Stockinger et al., 1997). Furthermore, expression analyses in yeast demonstrated that CBF1 could function as a transcriptional activator, since it promoted the transcription of a reporter gene containing the LTRE as an activator sequence. Recently, Jagglo-Ottosen et al. (1998) showed that overexpression of CBF1 in Arabidopsis transgenic plants induced the expression of some cold-regulated genes and increased freezing tolerance of non-cold-acclimated, transformed plants. They concluded that CBF1 is a positive regulator of the cold-acclimation process, controlling gene expression and promoting freezing tolerance.

An interesting question raised from the results mentioned above is whether Arabidopsis contains homologs of CBF1 or other genes encoding LTRE-binding proteins. Here we describe the identification and characterization of two Arabidopsis genes homologous to CBF1. Both genes, which we have called CBF2 and CBF3, are organized in tandem with CBF1 on chromosome 4 of Arabidopsis and constitute a small gene family. Like CBF1, both genes encode proteins containing putative nuclear localization signals, AP2 DNA-binding motifs, and potential acidic activation domains. We also show that expression of CBF1, CBF2, and CBF3 is induced very early and in a transient manner during the process of cold acclimation but is not induced by ABA or dehydration treatments. Based on these results, we suggest a potential role for CBF2 and CBF3 in cold acclimation and freezing tolerance.

	MATERIALS AND METHODS

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Plant Material and Treatments

2

1

Molecular Biology Methods

For DNA- and RNA-blot hybridizations, DNA probes were radioactively labeled with [-³²P]dCTP using the Megaprime kit (Amersham). A 692-bp RsaI fragment from CBF2 and a 1124-bp HindIII fragment from CBF3 containing corresponding coding regions were cloned into pBluescript and used as probes to simultaneously detect all members of the CBF gene family. Alternatively, DNA fragments partially encompassing the 3-noncoding regions were used as gene-specific probes. The CBF1-specific probe consisted of a 181-bp PCR-amplified fragment containing 25 nucleotides of coding sequence and 156 nucleotides of 3-noncoding region that was obtained by using the primers 5-GTGAAGCAAAGAAGTAGAAAACG-3 and 5-GTGACGTGTCGCTTTGGAGTTAC-3 (Stockinger et al., 1997).

The specific probe for the CBF2 gene consisted of a 199-bp PCR-amplified fragment produced by using the primers 5-GCATTTAAGAATAGCCCACAC-3 and 5-CGACGGCGATGATGACGACGT-3. The fragment had 40 nucleotides of coding sequence and 159 nucleotides of 3-noncoding region. The CBF3-specific probe consisted of a 227-bp PCR-amplified fragment covering 38 nucleotides of coding sequence and 189 nucleotides of 3-noncoding region. The fragment was obtained by using the primers 5-TATTTTGATTTGTTGCTTATGG-3 and 5-TCGAGGGAGATGATGACGTGTCC-3. In addition, a 700-bp PCR-amplified fragment from the Arabidopsis KIN1 gene was also used as a probe (Kurkela and Frank, 1990). This fragment was produced by using the primers 5-CCCGGATCCGGCACCACCACTCCCTTTAG-3and 5-GGGGAATTCGAATATAAGTTTGGCTCGTC-3. Finally, as an RNA-loading control, a probe consisting of a 700-bp EcoRI-HindIII fragment corresponding to the RBP4-coding region (Kim et al., 1990) was used.

Databases were searched for sequence similarities using the Basic Local Alignment Search Tool (BLAST) program of the National Center for Biotechnology Information (Altschul et al., 1997). A comparison of the nucleotide and amino acid sequences was performed with the software package PC/Gene 6.5 (Intelligenetics, Mountain View, CA). Putative phosphorylation sites were identified by comparing the amino acid sequences against all phosphorylation sites stored in the PROSITE pattern database (Bairoch et al., 1997).

	RESULTS

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Identification and Characterization of CBF2 and CBF3, Two Genes Homologous to CBF1

View larger version (35K): [in this window] [in a new window]   Figure 1. Genomic organization of the CBF1, CBF2, and CBF3 genes. A, Physical map of CBF genes on chromosome IV of Arabidopsis. The ORFs are shown with open bars. The restriction sites are HindIII (H), XbaI (X), and ScaI (S). The direction of transcriptions is indicated by arrows. B, DNA-blot hybridizations of Arabidopsis genomic DNA (4 µg) digested with ScaI (S), NdeI (N), XbaI (X), BglII (B), and HindIII (H). The probes used were the 692-bp RsaI fragment from CBF2 and the 1124-bp HindIII fragment from CBF3, as described in ``Materials and Methods''. The positions of molecular size markers are in the center.

Analysis of the 5 regions of CBF1, CBF2, and CBF3 (Fig. 2) showed that these regions also have a moderate level of similarity. In addition, they showed the presence of sequences with similarity to known regulatory sequences identified in other plant genes. Thus, the core CANNTG consensus motif, as well as the CACGTC- and TACGTG-related sequences, which are present in the promoter region of many genes that are regulated by different environmental stresses and ABA (Guiltinan et al., 1990; Williams et al., 1992; Busk and Pages, 1998), were found in the promoters of the CBF genes. Furthermore, the pentamer CAGCC, which corresponds to the LTRE core sequence (CCGAC) in reverse orientation, was present in the CBF promoters. The sequence CCGTC, which differs in only one nucleotide from the LTRE motif, was also found in the 5 region of CBF1 (Fig. 2).

View larger version (73K): [in this window] [in a new window]   Figure 2. Alignment of the 5 upstream sequences of CBF1, CBF2, and CBF3. Six-hundred-eighty bases of the 5-untranslated region of CBF1 were aligned with 720 and 702 bases of the corresponding regions of CBF2 and CBF3, respectively. Asterisks indicate nucleotides identical to the CBF1 sequence. Hyphens indicate gaps inserted in the sequences for better alignment. The initiation ATGs and putative TATA boxes (TATAAA) are double underlined. CANNTG motifs and related sequences CACGTC and TACGTG are shown in gray boxes. Black boxes highlight the CAGCC pentamers. The CCGTC sequence is single underlined.

The coding regions of CBF2 and CBF3 encoded two polypeptides of 216 amino acids each, with a predicted molecular mass of 24 kD. The pIs for both polypeptides were low: 5.0 for CBF2 and 4.9 for CBF3. The amino acid alignment of CBF2 and CBF3 revealed that 76% of the residues were identical and 85% were similar (Fig. 3A). When compared with CBF1, CBF2 and CBF3 also showed a very high degree of similarity (84% and 86%, respectively; Fig. 3A). Like CBF1, CBF2 and CBF3 included in their N-terminal regions basic residues that potentially represent nuclear localization signals (Raikhel, 1992) and putative AP2 DNA-binding domains (Weigel, 1995; Ohme-Takagi and Shinshi, 1995). Comparison of the AP2 domains from the CBF proteins and the Arabidopsis DNA-binding protein AtEBP (Buttner and Singh, 1997) revealed high similarity (Fig. 3B). Furthermore, CBF2 and CBF3 also contained acidic C-terminal fragments (pIs of 3.8 and 3.6, respectively) that might act as transcriptional activation domains (Hahn, 1993). In addition, they showed potential recognition sites for protein kinase C and casein kinase II (Kennelly and Krebs, 1991). Some of these sites were conserved among the three CBF polypeptides as Ser-13, Ser-56, which is inside of the AP2 domain, and Thr-151 (Fig. 3A).

View larger version (72K): [in this window] [in a new window]   Figure 3. Amino acid sequences of CBF1, CBF2, and CBF3 proteins. A, Sequence alignment. Amino acid residues identical to the CBF1 sequence at a given position are indicated by asterisks. Points represent gaps inserted in the sequences for better alignment. Black box corresponds to the potential nuclear localization signals. Gray boxes highlight the AP2 domains. Underlined amino acids indicate potential recognition sites for protein kinase C () and casein kinase II (). B, Comparison of the AP2 domains from CBF1, CBF2, and CBF3 proteins and the Arabidopsis ethylene-responsive element-binding protein AtEBP (Bütner and Singh, 1997). Identical amino acids are highlighted in black boxes. Points represent gaps inserted in the sequences for better alignment. Residues belonging to the conserved AP2 domain elements YRG and RAYD (Okamuro et al., 1997) are indicated. Amino acids in the RAYD conserved element predicted to form an amphipatic -helix that might promote DNA binding (Okamuro et al., 1997) are underlined.

The Expression of CBF1, CBF2, and CBF3 Genes Is Specifically Induced by Low Temperature in Different Organs of Arabidopsis

To analyze whether the expression of CBF genes is induced by low temperature, total RNA was prepared from leaves of 4-week-old Arabidopsis plants exposed to 4°C for different periods. Considering the high similarity among the coding sequences of the three CBF genes analyzed, CBF1-, CBF2-, and CBF3-specific transcripts were identified by RNA-blot hybridizations with specific probes prepared from DNA fragments corresponding to their 3-untranslated regions (see ``Materials and Methods''). DNA-blot hybridizations showed that each probe hybridized with only a single restriction fragment in each digestion, demonstrating that it specifically recognized the corresponding gene (not shown). As shown in Figure 4, CBF1, CBF2, and CBF3 transcripts accumulated with very similar kinetics in response to low temperatures. An increase of CBF mRNA levels was already detectable after 30 min of exposure to low temperature, reaching maximal levels of accumulation after 1 h. This increase was transient, since transcript levels decreased thereafter.

View larger version (75K): [in this window] [in a new window]   Figure 4. CBF1, CBF2, and CBF3 transcripts accumulate in response to low temperature but not in response to ABA or water stress. RNA-blot hybridizations were performed with total RNA (10 µg per lane) isolated from leaves exposed to 4°C for the indicated time, sprayed with 100 µM ABA (A), sprayed with the ABA solvent (C), or dehydrated until losing 50% of their fresh weight (D). The probes used were the 3 fragments of CBF1, CBF2, and CBF3 and the fragments of the KIN1 and RBP4 genes described in ``Materials and Methods''.

To date, most characterized cold-regulated genes are also responsive to ABA and water stress (Thomashow, 1994; Hughess and Dunn, 1996; Rouse et al., 1996; Shinozaki and Yamaguchi-Shinozaki, 1996; Capel et al., 1997; Hong et al., 1997; Kirch et al., 1997). To determine whether the accumulation of CBF mRNAs was specifically regulated by low temperature or was also responsive to these treatments, Arabidopsis plants were either treated with 100 µM ABA or dehydrated until losing 50% of their initial fresh weight. Total RNAs isolated from these plants and from controls were used to perform RNA-blot hybridizations with the CBF-specific probes previously described. Figure 4 shows that CBF transcripts did not accumulate in response to ABA or dehydration, indicating that the expression of CBF genes is specifically regulated by low temperature. As positive controls for treatments and RNA loading, hybridizations with KIN1 and RBP4 probes were carried out, respectively (Fig. 4). The KIN1 probe recognizes a gene from Arabidopsis, the expression of which is induced by low temperature, ABA, and dehydration treatments (Kurkela and Franck, 1990), whereas the RBP4 probe recognizes an Arabidopsis gene that is expressed constitutively (Kim et al., 1990).

The accumulation of CBF mRNAs in different organs of Arabidopsis in response to low temperature was studied in total RNA isolated from leaves, stems, and roots of plants exposed to 4°C for 1 h. As controls, RNAs from the same organs of plants grown at 20°C were also isolated. RNA-blot hybridizations were performed with the CBF-specific probes used in previous experiments. The results obtained revealed that, in response to low temperature, CBF transcripts accumulated to similar levels in all organs analyzed (Fig. 5). To monitor RNA loading, hybridizations with the RBP4 probe (see above) were carried out with the same membranes.

View larger version (81K): [in this window] [in a new window]   Figure 5. Accumulation of CBF1, CBF2, and CBF3 transcripts in different organs of Arabidopsis in response to low temperature. RNA-blot hybridizations were carried out with total RNA (15 µg per lane) isolated from leaves, stems, and roots of plants grown at 22°C (C) or exposed to 4°C for 1 h (4°). The specific probes for the CBF genes and the probe used for RBP4 are described in ``Materials and Methods''.

	DISCUSSION

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A significant step toward understanding the molecular mechanisms that control the process of cold acclimation was the recent isolation of a cDNA corresponding to Arabidopsis CBF1, the first identified C-repeat/DRE/LTRE-binding factor (Stockinger et al., 1997). Here we present the identification and characterization of two novel Arabidopsis genes, CBF2 and CBF3, both of which are highly similar to CBF1. Furthermore, we demonstrate that the expression of all three CBF genes is regulated by low temperature.

Genomic sequence analyses indicated that CBF1, CBF2, and CBF3 do not show introns among their coding sequences and are organized in tandem on chromosome 4 of Arabidopsis. The high similarity existing among CBF1, CBF2, and CBF3, together with their close linkage and the fact that they have the same transcriptional orientation, clearly suggests a common origin, probably by two consecutive duplications of an ancestral gene and subsequent divergence through mutations. It is interesting that the same origin has been proposed for the members of five families of low-temperature-responsive genes from Arabidopsis, in which the homologous genes are arranged in tandem in the genome (Nordin et al., 1991; Gilmour et al., 1992; Kurkela and Borg-Franck, 1992; Horvath et al., 1993; Wilhelm and Thomashow, 1993; Yamaguchi-Shinozaki and Shinozaki, 1993; Welin et al., 1994; Capel et al., 1997). However, the reason that low-temperature-regulated genes are so frequently organized in tandem remains unknown.

The 5 regulatory sequences of CBF1, CBF2, and CBF3 have diverged more than the coding regions but still keep a high level of similarity, which may result in the identical expression patterns shown by these genes. The fact that CBF1, CBF2, and CBF3 are not responsive to ABA seems to indicate that the CANNTG sequence, repeated several times in their upstream regions, is not sufficient to confer ABA responsiveness in the context of CBF promoters. The consensus LTRE core sequence, CCGAC, which has been reported to be essential for the low-temperature responsiveness of several genes (Baker et al., 1994; Yamaguchi-Shinozaki and Shinozaki 1994; Jiang et al., 1996; Ouellet et al., 1998), is not found in the CBF promoters. Only the variant CCGTC, which differs in just one nucleotide from the LTRE, is present in the 5 region of CBF1. Whether this sequence can confer the low-temperature response remains to be seen. However, it is interesting to emphasize that the pentamer CAGCC, which is the LTRE core sequence in reverse orientation, is present in the promoters of all CBFs.

CBF2 and CBF3 polypeptides contain a 60-amino acid motif, the AP2 domain, that is evolutionarily conserved in plants (Okamuro et al., 1997) and has been described as a DNA-binding domain (Ohme-Takagi and Shinshi, 1995; Weigel, 1995). CBF2 and CBF3, like CBF1, also have potential nuclear localization sequences in their N-terminal regions and acidic C-terminal fragments. Moreover, CBF1, CBF2, and CBF3 show potential recognition sites for protein kinase C and casein kinase II. Some of these sites are conserved among the three proteins, and one of them, Ser-56, is located in the AP2 domain. Recently, Vazquez-Tello et al. (1998) proposed that the expression of WCS120, a low-temperature-inducible gene from wheat that contains two LTREs in its promoter region, may be regulated by nuclear factors whose binding activity is modulated by phosphorylation/dephosphorylation mechanisms. We speculate that the potential phosphorylation sites found in the CBF proteins may play an important role in their functions.

Expression analyses revealed that CBF2 and CBF3 are positively regulated by low temperature. Furthermore, in contrast to previous data (Stockinger et al., 1997), CBF1 transcripts also accumulated in response to low temperature in our experimental conditions. The cold-inducible expression of CBF genes does not show marked differences. It is not organ specific, since CBF mRNAs accumulate to similar levels in different organs of Arabidopsis, and is transiently regulated. The accumulation of CBF transcripts increases rapidly after plants are transferred to low-temperature conditions, reaching the highest levels after 1 h of exposure and decreasing thereafter. This expression pattern suggests that CBF genes should be involved in responses that are transiently produced when plants are exposed to low temperatures, and fits with the notion that their induction should be an early amplification event in the low-temperature-induced signaling cascade, preceding and prompting the accumulation of transcripts corresponding to CBF-regulated genes. On the other hand, the expression of CBF genes is not regulated by ABA treatment or water stress, two conditions that have been shown to increase plant freezing tolerance (Cloutier and Siminovitch, 1982; Chen and Gusta, 1983; Mäntylä et al., 1995) and induce the expression of most cold-inducible genes (Thomashow, 1994; Hughes and Dunn, 1996; Rouse et al., 1996; Shinozaki and Yamaguchi-Shinozaki, 1996; Capel et al., 1997; Hong et al., 1997; Kirch et al., 1997).

CBF1 has been described as a transcriptional activator that binds to the LTRE sequence, inducing the expression of some low-temperature genes and increasing freezing tolerance (Stockinger et al., 1997; Jagglo-Ottosen et al., 1998). Our data indicate that CBF2 and CBF3 have a high degree of homology with CBF1 (>84%) and show an identical expression pattern. This strongly suggests that they may fulfill a similar function as CBF1, controlling the level of low-temperature-regulated gene expression and promoting freezing tolerance. The fact that CBF genes are not responsive to ABA indicates that they should be involved in regulating the expression of low-temperature-inducible genes through an ABA-independent pathway. Moreover, taking into consideration that LTRE is able to confer response to dehydration (Yamaguchi-Shinozaki and Shinozaki, 1994), the fact that the expression of CBF genes is not induced by this stress suggests that LTRE-binding proteins other than CBFs should mediate dehydration-regulated gene expression through LTREs.

Recently, the isolation and characterization of five Arabidopsis cDNAs encoding LTRE-binding proteins have been reported (Liu et al., 1998). Three of these proteins, designated DREB1A, DREB1B, and DREB1C, correspond to CBF2, CBF1, and CBF3, respectively. The other two proteins, named DREB2A and DREB2B, also contain AP2 DNA-binding domains, basic residues in their N-terminal regions that might function as nuclear localization signals, and acidic C-terminal regions that might act as transcriptional activation domains. Transactivation experiments using Arabidopsis protoplasts from leaves revealed that CBF2 (DREB1A) and DREB2A proteins can function as transcriptional activators. Furthermore, overexpression of CBF2 (DREB1A) cDNA resulted in transgenic plants showing strong expression of the target genes under unstressed conditions, an increase in tolerance to freezing and dehydration, and a dwarfed phenotype. In contrast, transgenic plants overexpressing DREB2A cDNA revealed few phenotypic changes and showed weak expression of the target genes under unstressed conditions. It is interesting that the expression of the DREB2A and DREB2B genes seems to be strongly induced in leaves by dehydration and high-salt stress, but not by low temperature, as with the CBF (DREB1) genes. All of these results confirm that, like CBF1, CBF2 and CBF3 are trans-acting factors that regulate low-temperature-induced gene expression promoting freezing tolerance. The results also show that DREB2 is an independent family of LTRE-binding proteins that function in a separate signal transduction pathway under dehydration conditions.

Considering the large number of genes whose expression is induced in response to low temperature, we hypothesized that differences in the sequences of the CCGAC core element and/or in the sequences that surround it may result in the recruitment of distinct CBF proteins. A similar situation has been described for the G-box sequence CANNTG and the bZIP proteins (Williams et al., 1992). The availability of all CBF genes makes it possible to perform in vivo and in vitro experiments to test this hypothesis.

	FOOTNOTES

   Received July 29, 1998; accepted November 5, 1998.

	ABBREVIATIONS

Abbreviations: DRE, drought-responsive element. LTRE, low-temperature-responsive element. ORF, open reading frame.

	ACKNOWLEDGMENTS

We thank Dr. G. Salcedo and Dr. J.J. Sánchez-Serrano for critically reading the manuscript. We are grateful to E. Rodríguez and A. Redondo for their technical assistance with growing plants.

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