Many plants increase in freezing tolerance in response to low non-freezing temperature through a process known as cold-acclimation. In Arabidopsis, cold acclimation is associated with the induction or repression of over a thousand genes. The CBF cold response pathway has a central role in these changes. Within minutes of transfer to low temperature, genes encoding three closely related transcription factors, CBF1-3, are induced and alter expression of more than one hundred target genes, which go on to impart freezing tolerance. An abiotic stress such as freezing can limit the productivity and relative fitness of a plant. Consequently, there is considerable interest in finding upstream regulators of the CBF pathway in hopes of expanding the geographical range and yield of important food and biofuel crops. Previous studies have shown that CBF1-3 are subject to circadian regulation and that cold induction of CBF1-3 is gated by the circadian clock. We identify specific clock components involved in the circadian oscillations, and thus, upstream regulation of CBF genes. We also demonstrate the involvement of these identified clock components in gating cold-regulated expression of CBF1-3 and CBF-target-genes. Furthermore, we show that these clock components affect plant freezing tolerance. Investigating natural variation of cold response pathways in ecotypes of Arabidopsis provides another means of distinguishing genes important for freezing tolerance. In previous studies, two Arabidopsis populations collected from Sweden (SW) and Italy (IT) were tested for fitness (survival and seed set) in reciprocal transplant experiments. Reciprocal transplant experiments revealed that home accessions at both sites had a strong advantage in terms of seed-set and survival. SW and IT recombinant inbred lines (RILs) were used to define fitness QTL in both locations. Since there is substantial variation in temperature across latitudes, genes associated with freezing tolerance may potentially underlie identified fitness QTL. This study shows that there are differences in the cold-acclimated freezing tolerance of SW and IT ecotypes under laboratory conditions. Through RNA-seq experiments, a set of genes that may contribute to differences in freezing tolerance between SW and IT is defined. RILs are used to map expression QTL (eQTL) for a subset of these low-temperature associated genes. Some of the eQTL mapped for these low-temperature associated genes overlap with previously identified fitness QTL and this study offers hypotheses as to the genes underlying these eQTL.
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