Many plants increase in freezing tolerance upon exposure to low nonfreezing temperatures, a phenomenon known as cold acclimation. In this review, recent advances in determining the nature and function of genes with roles in freezing tolerance and the mechanisms involved in low temperature gene regulation and signal transduction are described. One of the important conclusions to emerge from these studies is that cold acclimation includes the expression of certain cold-induced genes that function to stabilize membranes against freeze-induced injury. In addition, a family of Arabidopsis transcription factors, the CBF/DREB1 proteins, have been identified that control the expression of a regulon of cold-induced genes that increase plant freezing tolerance. These results along with many of the others summarized here further our understanding of the basic mechanisms that plants have evolved to survive freezing temperatures. In addition, the findings have potential practical applications as freezing temperatures are a major factor limiting the geographical locations suitable for growing crop and horticultural plants and periodically account for significant losses in plant productivity.

Many plants, including Arabidopsis, increase in freezing tolerance in response to low, nonfreezing temperatures, a phenomenon known as cold acclimation. Previous studies established that cold acclimation involves rapid expression of the CBF transcriptional activators (also known as DREB1 proteins) in response to low temperature followed by induction of the CBF regulon (CBF-targeted genes), which contributes to an increase in freezing tolerance. Here, we present the results of transcriptome-profiling experiments indicating the existence of multiple low-temperature regulatory pathways in addition to the CBF cold response pathway. The transcript levels of ‫ف‬ 8000 genes were determined at multiple times after plants were transferred from warm to cold temperature and in warm-grown plants that constitutively expressed CBF1, CBF2, or CBF3. A total of 306 genes were identified as being cold responsive, with transcripts for 218 genes increasing and those for 88 genes decreasing threefold or more at one or more time points during the 7-day experiment. These results indicate that extensive downregulation of gene expression occurs during cold acclimation. Of the cold-responsive genes, 48 encode known or putative transcription factors. Two of these, RAP2.1 and RAP2.6, were activated by CBF expression and thus presumably control subregulons of the CBF regulon. Transcriptome comparisons indicated that only 12% of the cold-responsive genes are certain members of the CBF regulon. Moreover, at least 28% of the cold-responsive genes were not regulated by the CBF transcription factors, including 15 encoding known or putative transcription factors, indicating that these cold-responsive genes are members of different low-temperature regulons. Significantly, CBF expression at warm temperatures repressed the expression of eight genes that also were downregulated by low temperature, indicating that in addition to gene induction, gene repression is likely to play an integral role in cold acclimation.

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.