Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeatyDRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit (cold acclimationyCOR genesydrought stressysignal transductionyyeast)

Recent efforts have defined a cis-acting DNA regulatory element in plants, the C-repeatydehydration 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-repeatyDRE binding factor, CBF1 (C-repeatyDRE 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-repeatyDRE 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-repeatyDRE 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-repeatyDRE DNA regulatory element and, thus, is likely to have a role in coldand dehydrationregulated gene expression in Arabidopsis. Freezing temperatures and drought are two adverse environmental conditions that limit the geographical distribution of plants and account for significant reductions in the yields of agriculturally important crops. Consequently, considerable effort has been directed at determining the nature of freezing and drought injury and the protection mechanisms plants have evolved that increase tolerance for these environmental ‘‘stresses.’’ Significantly, these studies have revealed that freezing injury is largely a consequence of freeze-induced dehydration (1). Thus, freezing and drought tolerance might have certain protective mechanisms in common. Indeed, it has been observed that some plants increase in freezing tolerance in response to dehydration stress (2, 3). The molecular basis of this cross-protection is not fully understood but appears to include the expression of common genes. For instance, certain COR (cold-regulated) genes of Arabidopsis thaliana that encode novel hydrophilic polypeptides, such as COR15a and COR78, are induced during cold acclimation, the process whereby plants increase in freezing tolerance in response to low nonfreezing temperatures, and are up-regulated in response to water deficit (4). It has been hypothesized that these and other COR genes might help plant cells tolerate the potentially damaging effects of dehydration associated with freezing and drought (4). Indeed, recent studies indicate that constitutive expression of COR15a, which encodes a chloroplast-targeted polypeptide, enhances the freezing tolerance of chloroplasts and has multiple effects on the freezing tolerance of plasma membranes (5). A major goal now is to determine how plants sense low temperature and water deficit and process this information to alter gene expression. An important advance in this regard was the recent identification of the C-repeatydehydration responsive element (DRE) cis-acting DNA regulatory element (6–8). The element, which has a 5-bp core sequence of CCGAC, designated the C-repeat (7), is present in one tomultiple copies in the promoters of many cold-regulated plant genes, including the Arabidopsis genes COR15a (7) and COR78yRD29A (ref. 6; COR78 and RD29A are alternative designations for the same gene) and the Brassica napus gene BN115 (8). Deletion analysis of the Arabidopsis COR15a promoter indicated that the C-repeat might be part of a cis-acting cold-regulatory DNA element (7). This was first demonstrated by YamaguchiShinozaki and Shinozaki (6), who showed that two 9-bp DNA elements containing the C-repeat, present in the promoter of COR78yRD29A, induce cold-regulated gene expression when fused to a reporter gene. Moreover, they found that the two DNA elements also stimulated transcription in response to dehydration stress. These C-repeat-containing elements were, therefore, given the designation DRE. Many of the changes in gene expression that occur in response to low temperature and drought require the action of abscisic acid (ABA; refs. 9 and 10). However, studies employing the ABA-insensitive (abi) mutants of Arabidopsis indicate that there are also ABA-independent signal transduction pathways for coldand dehydration-regulated gene expression (11, 12). Indeed, the results of Yamaguchi-Shinozaki and Shinozaki (6) indicate that the C-repeatyDRE is not responsive to ABA levels and, thus, appears to impart coldand dehydration-regulated gene expression through an ABAindependent pathway. How does the C-repeatyDRE impart coldand dehydrationregulated gene expression? A critical step toward answering this key question is determining the nature of the protein The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA 0027-8424y97y941035-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: DRE, dehydration responsive element; ABA, abscisic acid; EREBP, ethylene response element binding protein; X-Gal, 5-bromo-4-chloro-3-indolyl b-D-galactoside. Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. U77378). ‡To whom reprint requests should be addressed.