CBF͞DREB1 (C-repeat-binding factor͞dehydration responsive element-binding factor 1) genes encode a small family of transcriptional activators that have been described as playing an important role in freezing tolerance and cold acclimation in Arabidopsis. To specify this role, we used a reverse genetic approach and identified a mutant, cbf2, in which the CBF2͞DREB1C gene was disrupted. Here, we show that cbf2 plants have higher capacity to tolerate freezing than WT ones before and after cold acclimation and are more tolerant to dehydration and salt stress. All these phenotypes correlate with a stronger and more sustained expression of CBF͞DREB1-regulated genes, which results from an increased expression of CBF1͞DREB1B and CBF3͞DREB1A in the mutant. In addition, we show that the expression of CBF1͞DREB1B and CBF3͞DREB1A in response to low temperature precedes that of CBF2͞DREB1C. These results indicate that CBF2͞DREB1C negatively regulates CBF1͞DREB1B and CBF3͞ DREB1A, ensuring that their expression is transient and tightly controlled, which, in turn, guarantees the proper induction of downstream genes and the accurate development of Arabidopsis tolerance to freezing and related stresses.F reezing temperatures greatly limit the geographical distribution and growing season of plants and cause negative effects on crop quality and productivity. As a consequence, appreciable effort has been conducted to determine the adaptive mechanisms plants have evolved to survive this adverse environmental condition. Many plants, including Arabidopsis, increase their freezing tolerance in response to low, nonfreezing temperatures. This process, called cold acclimation (1), is complex and involves a number of biochemical and physiological changes, ranging from alterations in lipid composition to accumulation of sugars (2). Different studies have suggested that low-temperature-regulated gene expression is critical in plants for cold acclimation (2). Low-temperature-responsive genes encode a diverse number of proteins, including enzymes involved in respiration and metabolism of carbohydrates, lipids, phenylpropanoids and antioxidants, molecular chaperones, antifreeze proteins, among others, with a believed function in freezing tolerance (2).During the past few years, substantial progress has been made toward understanding how low temperatures regulate gene expression. In particular, a family of transcription factors in Arabidopsis known either as C-repeat-binding factor (CBF)1, CBF2, and CBF3 (3, 4) or dehydration-responsive elementbinding factor (DREB)1B, DREB1C, and DREB1A (5), respectively, has been identified. These factors belong to the Apetala 2͞ethylene-responsive element-binding protein (AP2͞ EREBP) family of DNA-binding proteins (6) and bind to the cold-and dehydration-responsive DNA regulatory element (DRE) (7), also termed C-repeat (CRT) (8). CRT͞DRE elements contain the conserved CCGAC core sequence, which is sufficient to induce gene transcription under cold stress (7,8) and is present in the promoters of many cold-induc...

The C-repeat-binding factor (CBF)/dehydration-responsive element-binding factor (DREB1) proteins constitute a small family of Arabidopsis transcriptional activators (CBF1/DREB1B, CBF2/ DREB1C, and CBF3/DREB1A) that play a prominent role in cold acclimation. A fundamental question about these factors that remains to be answered is whether they are functionally equivalent. Recently, we reported that CBF2 negatively regulates CBF1 and CBF3 expression, and that CBFs are subjected to different temporal regulation during cold acclimation, which suggested this might not be the case. In this study, we have analyzed the expression of CBF genes in different tissues of Arabidopsis, during development and in response to low temperature, and characterized RNA interference (RNAi) and antisense lines that fail to accumulate CBF1 or/and CBF3 mRNAs under cold conditions. We found that CBF1 and CBF3 are regulated in a different way than CBF2. Moreover, in contrast to CBF2, CBF1 and CBF3 are not involved in regulating other CBF genes and positively regulate cold acclimation by activating the same subset of CBF-target genes. All these results demonstrate that CBF1 and CBF3 have different functions than CBF2. We also found that the CBF regulon is composed of at least two different kind of genes, one of them requiring the simultaneous expression of both CBF1 and CBF3 to be properly induced. This indicates that CBF1 and CBF3 have a concerted additive effect to induce the whole CBF regulon and the complete development of cold acclimation.freezing tolerance ͉ low temperature ͉ DREB1 ͉ abiotic stress ͉ cold signaling T he identification of the C-repeat-binding factors (CBF1-3)(1-3), also named dehydration-responsive element-binding factors (DREB1B, -1C, and -1A, respectively) (4), represented a significant step toward the understanding of how gene expression is regulated during cold acclimation, the adaptive response whereby many plants increase their freezing tolerance in response to low nonfreezing temperatures (5). The CBFs/DREB1s belong to the AP2/EREBP family of transcription factors (6) and bind to the cold-and dehydration-responsive DNA regulatory element designated C-repeat (CRT)/dehydration response element (DRE) (7,8). CRT/DRE elements contain the conserved CCGAC core sequence, which is sufficient to activate gene transcription under cold stress (7,8) and is present in the promoters of many cold-inducible genes (9). The CBF/DREB1 genes do not contain the CCGAC sequence in their promoters but are also induced by low temperature. This induction is transient and precedes that of cold-inducible genes with the CRT cis element. The expression of CBF/DREB1 genes, however, is not activated by dehydration and salt stress (2-4).An important issue in the study of CBFs is their individual function. Despite extensive research carried out on these transcriptional activators, whether they have overlapping function (s) has not yet been conclusively established. Constitutive overexpression of each CBF in Arabidopsis results in similar express...

Arabidopsis CBF genes (CBF1–CBF3) encode transcription factors having a major role in cold acclimation, the adaptive process whereby certain plants increase their freezing tolerance in response to low non-freezing temperatures. Under these conditions, the CBF genes are induced and their corresponding proteins stimulate the expression of target genes configuring low-temperature transcriptome and conditioning Arabidopsis freezing tolerance. CBF2 seems to be the most determinant of the CBFs since it also regulates CBF1 and CBF3 expression. Despite the relevance of CBF genes in cold acclimation, little is known about the molecular components that control their expression. To uncover factors acting upstream of CBF2, mutagenized Arabidopsis containing the luciferase reporter gene under the control of the CBF2 promoter were screened for plants with de-regulated CBF2 expression. Here, the identification and characterization of five of these mutants, named acex (altered CBF2 expression), is presented. Three mutants show increased levels of cold-induced CBF2 transcripts compared with wild-type plants, the other two exhibiting reduced levels. Some mutants are also affected in cold induction of CBF1 and CBF3. Furthermore, the mutants characterized display unique phenotypes for tolerance to abiotic stresses, including freezing, dehydration, and high salt. These results demonstrate that cold induction of CBF2 is subjected to both positive and negative regulation through different signal transduction pathways, some of them also mediating the expression of other CBF genes as well as Arabidopsis responses to abiotic stresses.

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