Molecular Basis of Plant Cold Acclimation: Insights Gained from Studying the CBF Cold Response Pathway

Abstract

For the 75th Anniversary Issue of Plant Physiology, I contributed an article (Thomashow, 2001) in which I highlighted recent advances in the identification of genes with roles in cold acclimation: the process whereby certain plants increase in freezing tolerance in response to low nonfreezing temperatures. At that time, about 20 Arabidopsis (Arabidopsis thaliana) genes had been identified as cold regulated, and it seemed that it might be relatively easy to model the low-temperature transcriptional regulatory network. Since then, largely through the development and use of microarrays and other genomic tools, we have learned that exposing plants to low temperature triggers a highly complex regulatory program that results in extensive reorganization of the transcriptome. These changes include up- and down-regulation of hundreds of genes occurring in waves with time of incubating plants at low temperature (Fowler and Thomashow, 2002; Maruyama et al., 2004; Hannah et al., 2005; Vogel et al., 2005; Oono et al., 2006; Kilian et al., 2007; Robinson and Parkin, 2008). A hierarchical cluster analysis of the transcriptome data of Kilian et al. (2007) clearly reveals these features (Fig. 1A) and provokes a number of fundamental questions. How do plants sense low temperature; that is, what is the nature of the low-temperature thermometer? How is this information processed to activate the first wave of cold-regulated genes? What is the regulatory logic that underlies the cascading pattern of the low-temperature gene network? And what biological functions can be ascribed to the genes that constitute the various circuits of the network? At present, the answers to these questions are far from complete, but significant insights have been gained. Here, I will highlight a few of the advances that speak to these issues, focusing on the CBF cold response pathway of Arabidopsis. Additional information on the topics covered here and other facets of cold acclimation can be found in other recent reviews (Chinnusamy et al., 2007; Guy et al., 2008; Penfield, 2008; Galiba et al., 2009; Hua, 2009). Waves of cold-regulated genes produced upon exposing Arabidopsis plants to low temperature. The AtGenExpress global stress expression data set (Kilian et al., 2007) was analyzed using the Bioconductor LIMMA package to identify genes that were up- or down-regulated at least 4-fold (false discovery rate < 0.01) at any time point of the cold treatment. This resulted in the identification of 1,335 up-regulated and 1,061 down-regulated genes. A, Genes were grouped using k-means cluster analysis. Log2-transformed expression levels are represented by the color gradient included at the bottom of the cluster diagram. The extreme values given are −3 to +3 or higher in expression level. B, The patterns for up-regulated genes encoding transcription factors (174 total) were identified according to Arabidopsis Gene Ontology (Berardini et al., 2004) and grouped using k-means cluster analysis. Determining the overall “wiring diagram” of the low-temperature regulatory network will not be a trivial matter. Indeed, among the more than 1,000 genes that are cold induced, there are more than 170 genes that encode transcription factors (Fig. 1B). Presumably, all of these transcription factors act in concert to reconfigure the transcriptome at low temperature. However, as has been learned in other systems, certain transcription factors can serve as major regulatory “hubs” controlling the expression of a large number of genes and contribute much to the biology under study. The Arabidopsis CBF transcription factors constitute such a regulatory hub for cold acclimation. Arabidopsis encodes three cold-inducible CBF genes, CBF1, CBF2, and CBF3 (Stockinger et al., 1997; Gilmour et al., 1998; Medina et al., 1999), also referred to as DREB1b, DREB1c, and DREB1a, respectively (Liu et al., 1998). These genes, which are located in tandem array in the genome, encode closely related transcription factors that are members of the AP2/ERF family of DNA-binding proteins. The CBF proteins bind to the CRT/DRE regulatory element present in the promoters of target genes, referred to as the CBF regulon, and stimulate their transcription. Induction of the CBF genes occurs within about 15 min of transferring plants to low temperature (4°C), followed by induction of the CBF target genes beginning at about 2 to 3 h. Vogel et al. (2005) found that 85 (28%) of the 302 genes that were determined to be cold induced in wild-type plants were also induced by constitutive overexpression of CBF2 in transgenic plants grown at warm temperature, indicating that the CBF cold response pathway plays a major role in configuring the low-temperature transcriptome. This may be an underestimate of the size of the CBF regulon, as it assumes that the CBF transcription factors by themselves are sufficient to induce the expression of target genes at warm temperature. This may not be the case, as combinatorial regul