| Many plants can sense low temperature and respond by altering gene expression. These changes in gene expression can ultimately lead to increased freezing tolerance as part of a dynamic physiological process known as cold acclimation. One well known pathway that contributes to cold acclimation is the CBF/DREB1 cold response pathway. This pathway includes the action of the transcriptional activators CBF1, 2, and 3 (also known as DREB1b, c, and a, respectively) that are rapidly induced in response to low temperature and activate the expression of a suite of target genes, known as the CBF regulon. The CBF regulon gene products act in concert to increase plant freezing tolerance. Work in this dissertation focused on gaining a better understanding of the changes in gene expression that occur in response to low temperature, the extent to which the CBF pathway contributes to these changes, and whether six other transcription factors that were coordinately regulated with CBF2 affected cold-responsive genes.; In this work, the Affymetrix GeneChip containing probe sets for approximately 24,000 genes was used to define a core set of cold-responsive transcripts. A total of 514 transcripts were found to have altered transcript accumulation, 302 of which were up-regulated and 212 down-regulated, in plants grown in two commonly used growth conditions (soil and solid media). These genes were termed the COS (cold s&barbelow;tandard) gene set. Hierarchical clustering assigned each transcript to one of seven expression classes and bioinformatic analysis revealed multiple novel potential cis-elements present in the promoters of genes in each expression class. Further bioinformatic analysis revealed additional motifs among one of these expression classes, cluster III, but experiments indicated no evidence that any of the novel motifs in clusters III or IV were cold-responsive. One element, GTGATCAC, conferred constitutive GUS activity when fused as a tetramer in front of the reporter. A functional analysis of the COS transcripts revealed how the plants might be globally altering their metabolism in order to cope with low temperature, which has generated a number of new hypotheses that can be tested.; Plants overexpressing CBF2 or one of six transcription factors that were coordinately regulated with CBF2 were also profiled using the GeneChips. The CBF2 regulon was comprised of 85 cold-induced and eight cold-repressed genes. Of the six genes that were induced in parallel with CBF2 only one, ZAT12, influenced cold-responsive genes when constitutively expressed. The ZAT12 regulon contained nine cold-induced genes and 15 cold-repressed genes. Constitutive expression of ZAT12 resulted in a small, but reproducible increase in freezing tolerance, indicating a role for ZAT12 in cold acclimation. ZAT12 also appeared to have a role in a negative regulatory circuit that dampened expression of the CBF genes. Constitutive expression of ZAT12 dampened CBF transcript accumulation in response to low temperature, while decreased levels of ZAT12 resulted in higher levels of CBF transcript accumulation.; The definition of the COS genes provides a framework on which the low temperature regulatory networks of Arabidopsis can be constructed and this dissertation begins this process by defining two regulons, the CBF2 and ZAT12 regulons. Future experiments will expand our knowledge of these and other networks and could eventually lead to novel strategies for engineering plants with increased stress tolerance. |
