Our long term goal is to understand in detail how the Arabidopsis circadian clock functions and how it interacts with the phytochrome system. Our proposed work is based on our discovery during the last grant period that CCA1, a transcription factor involved in phytochrome regulation of gene expression, is not just part of the phytochrome signal transduction chain, but is also a central player in circadian rhythms of plants. In the work proposed here we will investigate the details of how CCA1 and several closely related proteins act in the circadian system. We will use a genetic approach to identify other components involved in plant circadian rhythms. We will also explore the roles of a protein kinase (CK2) that we found interacts with CCA1, and then went on to demonstrate that it is involved in regulating circadian rhythms in vivo. Our specific experimental goals for the next five years are: 1. To identify components of the circadian oscillator in Arabidopsis. We will determine whether (or not) CCA1, LHY, and the APRR/TOC family comprise the oscillator system. In addition, we will isolate and characterize mutants that should identify other components important for clock function. 2. To examine the roles of transcription, RNA stability, translation and protein modification in regulation of the circadian system. We will carry out-experiments to establish the levels at which CCA1 and LHY are regulated and to elucidate the pathways that are important for their regulation. We will focus especially on the levels that are affected by light signals. 3. To further identify the roles of protein phosphorylation by CK2 in regulation of the circadian clock. We will a.) examine whether phosphorylation by CK2 is involved in the degradation of clock proteins in vivo b.) test whether CK2 is an essential regulator of the circadian oscillator and c.) test whether the CK2 subunits have unique functions in targeting specific substrates by isolating and characterizing lines of plants missing one or a combination of the CK2 subunits 4. To delineate any additional roles of CCA1 and LHY by using microarrays of Arabidopsis genes to investigate the network of genes CCA1 and LHY control. We will also examine their involvement alone and in conjunction with other transcription factors in the regulation of another putative oscillator gene, APRR9.
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