Recent efforts have defined a cis-acting DNA regulatory element in plants, the C-repeat͞dehydration 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-repeat͞DRE binding factor, CBF1 (C-repeat͞DRE 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-repeat͞DRE 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-repeat͞DRE 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-repeat͞DRE DNA regulatory element and, thus, is likely to have a role in cold-and dehydrationregulated gene expression in Arabidopsis.

Many plants, including Arabidopsis, show increased resistance to freezing after they have been exposed to low nonfreezing temperatures. This response, termed cold acclimation, is associated with the induction of COR (cold-regulated) genes mediated by the C-repeat/drought-responsive element (CRT/DRE) DNA regulatory element. Increased expression of Arabidopsis CBF1, a transcriptional activator that binds to the CRT/DRE sequence, induced COR gene expression and increased the freezing tolerance of nonacclimated Arabidopsis plants. We conclude that CBF1 is a likely regulator of the cold acclimation response, controlling the level of COR gene expression, which in turn promotes tolerance to freezing.

We further investigated the role of the Arabidopsis CBF regulatory genes in cold acclimation, the process whereby certain plants increase in freezing tolerance upon exposure to low temperature. The CBF genes, which are rapidly induced in response to low temperature, encode transcriptional activators that control the expression of genes containing the C-repeat/ dehydration responsive element DNA regulatory element in their promoters. Constitutive expression of either CBF1 or CBF3 (also known as DREB1b and DREB1a, respectively) in transgenic Arabidopsis plants has been shown to induce the expression of target COR (cold-regulated) genes and to enhance freezing tolerance in nonacclimated plants. Here we demonstrate that overexpression of CBF3 in Arabidopsis also increases the freezing tolerance of cold-acclimated plants. Moreover, we show that it results in multiple biochemical changes associated with cold acclimation: CBF3-expressing plants had elevated levels of proline (Pro) and total soluble sugars, including sucrose, raffinose, glucose, and fructose. Plants overexpressing CBF3 also had elevated P5CS transcript levels suggesting that the increase in Pro levels resulted, at least in part, from increased expression of the key Pro biosynthetic enzyme ⌬ 1 -pyrroline-5-carboxylate synthase. These results lead us to propose that CBF3 integrates the activation of multiple components of the cold acclimation response.

SummaryCold-induced expression of the Arabidopsis COR (coldregulated) genes is mediated by a DNA regulatory element termed the CRT (C-repeat)/DRE (dehydrationresponsive element). Recently, we identified a transcriptional activator, CBF1, that binds to the CRT/DRE and demonstrated that its overexpression in transgenic Arabidopsis plants at non-acclimating temperatures induces COR gene expression and increases plant freezing tolerance. Here we report that CBF1 belongs to a small family of closely related proteins which includes CBF2 and CBF3. DNA sequencing of an 8.7 kb region of the Arabidopsis genome along with genetic mapping experiments indicated that the three CBF genes are organized in direct repeat on chromosome 4 at 72.8 cM, closely linked to molecular markers PG11 and m600. Like CBF1, both CBF2 and CBF3 activated expression of reporter genes in yeast that contained the CRT/DRE as an upstream activator sequence. The transcript levels for all three CBF genes increased within 15 min of transferring plants to low temperature, followed by accumulation of COR gene transcripts at about 2 h. CBF transcripts also accumulated rapidly in response to mechanical agitation. The promoter regions of the CBF genes do not contain the CRT sequence, CCGAC, and overexpression of CBF1 did not have a detectable effect on CBF3 transcript levels, suggesting that the CBF gene family is not subject to autoregulation. We propose that cold-induced expression of CRT/DREcontaining COR genes involves a low temperaturestimulated signalling cascade in which CBF gene induction is an early event.

When Arabidopsis is exposed to low temperature a small gene family encoding transcription factors known as CBF1, CBF2, and CBF3 (also referred to as DREB1b, DREB1c, and DREB1a, respectively) is rapidly induced followed by expression of CBF-targeted genes, the CBF regulon, which act to bring about an increase in freezing tolerance. The CBF1, 2 and 3 proteins, though highly similar in amino acid sequence, are not identical, raising the question of whether the proteins have the same functions. Here we explored this issue by comparing the effects that overexpression of each CBF gene had on Arabidopsis growth and development, proline and sugar composition, freezing tolerance and gene expression. Taken together, the results support the conclusion that the CBF1, 2 and 3 transcriptional activators have redundant functional activities.