Susceptibility to chilling injury prevents the cultivation of many important crops and limits the extended storage of horticultural commodities. Although freezing tolerance is acquired through cold-induced gene expression changes mediated in part by the CBF family of transcriptional activators, whether plant chilling resistance or sensitivity involves the CBF genes is not known. We report here that an Arabidopsis thaliana mutant impaired in the coldregulated expression of CBF genes and their downstream target genes is sensitive to chilling stress. Expression of CBF3 under a strong constitutive promoter restores chilling resistance to the mutant plants. The mutated gene was cloned and found to encode a nuclear localized RNA helicase. Our results identify a regulator of CBF genes, and demonstrate the importance of gene regulation and the CBF transcriptional activators in plant chilling resistance. L ow temperature is an important environmental factor that greatly influences the growth, development, survival, and distribution of plants (1). Most plants from temperate regions can cold-acclimate, i.e., they show increased tolerance to freezing temperatures after an exposure to low, nonfreezing temperatures (2). At least part of the basis of cold acclimation is that exposure to low temperatures induces the expression of many plant genes (2, 3). Some of the cold-induced gene products such as COR15A have been shown to mitigate membrane damage caused by freezing stress (4, 5).The dehydration-responsive element (DRE)͞C-repeat (CRT) cis-element is present in the promoters of many of the coldresponsive genes such as RD29A (also known as COR78 or LTI78) and COR15A (6-8). In Arabidopsis thaliana, the CBF family of transcriptional activators, also known as DREB1s, bind to the DRE͞CRT element and activate the expression of COR͞RD genes (8, 9). Cold induces rapid and transient expression of CBF1, CBF2, and CBF3, which in turn activate the downstream COR genes (10). Ectopic expression of CBF1 or CBF3 in transgenic Arabidopsis plants leads to constitutive expression of COR genes and enhanced freezing tolerance without cold-acclimation treatment (9, 11). These studies demonstrate a critical role of the CBF regulon in the acquisition of freezing tolerance. An important challenge ahead is to identify regulators of the CBF genes.Recently, the Arabidopsis HOS1 protein was shown to be a negative regulator of CBF genes (12). CBFs and their downstream COR genes show enhanced cold induction in hos1 mutant plants (12, 13). The Arabidopsis esk1 mutants are constitutively freezing-tolerant, but are not affected in the expression of genes with the DRE͞CRT cis-element, suggesting that ESK1 may be involved in a CBF-independent cold-response pathway (14). The sfr6 mutation reduces cold and abscisic acid (ABA) induction of the CRT͞DRE genes but has no effect on the expression of CBF genes (15). Therefore, no positive regulator of CBFs has been identified to date.Many important crops and fruits that originated from the tropics or subtropics, such a...