Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit

Abstract

Recent efforts have defined a cis-acting DNA regulatory element in plants, the C-repeat/dehydration responsive element (DRE), that stimulates transcription in response to low temperature and water deficit. Here we report the isolation of an Arabidopsis thaliana cDNA that encodes a C-repeat/DRE binding factor, CBF1 (C-repeat/DRE Binding Factor 1). Analysis of the deduced CBF1 amino acid sequence indicates that the protein has a molecular mass of 24 kDa, a potential nuclear localization sequence, and a possible acidic activation domain. CBF1 also has an AP2 domain, which is a DNA-binding motif of about 60 aa present in the Arabidopsis proteins APETALA2, AINTEGUMENTA, and TINY; the tobacco ethylene response element binding proteins; and numerous other plant proteins of unknown function. The transcript levels for CBF1, which appears to be a single or low copy number gene, did not change appreciably in plants exposed to low temperature or in detached leaves subjected to water deficit. Binding of CBF1 to the C-repeat/DRE was demonstrated in gel shift assays using recombinant CBF1 protein expressed in Escherichia coli. Moreover, expression of CBF1 in yeast was found to activate transcription of reporter genes containing the C-repeat/DRE as an upstream activator sequence but not mutant versions of the DNA element. We conclude that CBF1 can function as a transcriptional activator that binds to the C-repeat/DRE DNA regulatory element and, thus, is likely to have a role in cold- and dehydration-regulated gene expression in Arabidopsis.

Figure 1

Approach used to isolate cDNA inserts encoding a C-repeat/DRE binding domain. (A) Screening strategy (see text for details). (B) Activity of the “positive” plasmids in yeast reporter strains carrying the indicated upstream activator sequences. The oligonucleotides (oligo) used to make the upstream activator sequence elements (Table 1), the number of inserts, and their direction of insertion are indicated.

Figure 2

Analysis of the pACT-11 cDNA clone. (A) Schematic drawing of the pACT-11 cDNA insert. (B) DNA and amino acid sequence of the 24-kDa polypeptide. The AP2 domain is indicated by double underlines. The basic amino acids that potentially act as a nuclear localization signal are indicated with asterisks. The BclI site immediately upstream of the 24-kDa polypeptide used in subcloning the 24-kDa polypeptide and the EcoRV site used in subcloning the 3′ end of CBF1 are indicated by single underlines. (C) Schematic drawing showing locations of the potential nuclear localization signal (NLS), the AP2 domain, and the acidic region of the 24-kDa polypeptide. Numbers indicate amino acid residues. (D) Comparison of the AP2 domains from the 24-kDa polypeptide and the tobacco DNA binding protein EREBP2 (25). Identical amino acids are indicated with single vertical lines; similar amino acids are indicated by colons; amino acids that are invariant in AP2 domains (26) are indicated with asterisks; and the histidine residue present in CBF1 and TINY (27) that is a tyrosine residue in all other described AP2 domains is indicated with a caret. A single amino acid gap in the CBF1 sequence is indicated by a dot.

Figure 3

Activation of reporter genes in yeast by the 24-kDa polypeptide. “Expression constructs” were transformed into yeast strains carrying the lacZ reporter gene fused to direct repeat dimers of either the wild-type COR15a C-repeat/DRE (oligonucleotide MT50) or the mutant M3COR15a C-repeat/DRE (oligonucleotide MT125). β-Galactosidase activity (nanomoles of o-nitrophenol produced per minute × milligrams of protein) ± SD was determined from cultures grown in triplicate.

Figure 4

Gel shift assays demonstrating that CBF1 binds to the C-repeat/DRE. The C-repeat/DRE probe (1 ng) used in all reactions was a ³²P-labeled dimer of the oligonucleotide MT50, a wild-type C-repeat/DRE from COR15a (Table 1). Shown in the first four lanes are gel shift assays using either BSA or protein extracts prepared from E. coli harboring either the pET-28a(+) expression vector alone (vector) or the vector plus an insert encoding the 24-kDa polypeptide in sense (sense insert) or antisense (antisense insert) orientation. Shown in the next eight lanes are the E. coli protein extract prepared from the “sense insert” strain plus the indicated competitor C-repeat/DRE sequences (100 ng). The 1×, 2×, and 3× refer to whether the oligonucleotides were monomers, dimers, or trimers, respectively, of the indicated C-repeat/DRE sequences.

Figure 5

CBF1 is a unique or low copy number gene. Arabidopsis DNA (≈1 μg) was digested with the indicated restriction endonucleases, and Southern transfers were prepared and hybridized with a ³²P-labeled probe encoding the entire CBF1 polypeptide.

Figure 6

Effects of low temperature and water deficit on CBF1 and COR15a transcript levels. Northern transfers were prepared from total RNA (20 μg) isolated from Arabidopsis plants treated at low temperature for the indicated time or detached leaves that either were (+) or were not (−) subjected to a water deficit (WD) treatment. The transfers were hybridized with ³²P-labeled probes for either the entire CBF1 coding sequence or the COR15a gene. After autoradiography, the transfers were hybridized a second time with the constitutively expressed eIF-4A (eukaryotic Initiation Factor 4A) gene (32) to confirm that RNA loading and transfer were uniform between the samples (data not shown). The autoradiograms for the COR15a and CBF1 probes were exposed for 1 h and 5 days, respectively. Longer exposures for the COR15a probe indicated that COR15a transcript levels begin to increase by 2 h.