Much of the work on freezing tolerance in the last several years has focused on the CBF pathway. Despite many studies on these genes, it still remains to be determined how their expression is regulated and what the precise contribution of each individual gene to freezing tolerance and cold acclimation is. The general objective of this proposal is twofold: 1) to better understand the regulation of CBF gene expression and the involvement of the CBF cold response pathway in freezing tolerance and 2) to examine other pathways involved in freezing tolerance. We will use Arabidopsis and a commercially important tree species, Eucalyptus. For the first objective, we propose to use three complementary approaches: i) the study of natural variation, ii) the use of reporter-genes as well as RNAi and over-expressing (OE) lines, and iii) the study of mutants affected in CBF regulation. In Arabidopsis, we will identify more efficient natural variants of CBF genes (coding sequences and promoters), as well as new regulators of CBF expression. In Eucalyptus, the project aims to characterize CBF gene expression, to identify the CBF regulon(s), and to obtain molecular markers for EguCBF genes. In a second part, we will use recombinant inbred lines (RILs) of Arabidopsis that are available at the SGAP for phenotyping with respect to freezing tolerance in order to begin to examine non-CBF pathways. The results we expect to obtain from this project will constitute an important step for better applying our knowledge of the CBF genes, and perhaps others, to improve the freezing tolerance of many crop plants.