Almost every living organism exposed to the day/night cycle on earth has evolved time-keeping mechanisms that exhibit approximately 24-hour periods. Circadian clocks are present in life forms ranging from bacteria to humans, and enable organisms to keep track of time in a robust manner, as exemplified by the persistence of circadian rhythms under constant conditions. The ability to anticipate changes and respond accordingly is believed to confer an adaptive advantage. In Arabidopsis thaliana, the circadian clock is involved in nearly all aspects of growth and development and the PSEUDO-RESPONSE REGULATOR (PRR) family of circadian clock components are master regulators of diverse biological processes. Mutations in the PRRs result in delayed flowering times, elongated hypocotyl lengths, and an increased tolerance to drought and cold as observed in the prr5prr7prr9 triple mutant. Up to 90% of expressed genes oscillate under diel conditions and approximately 30% of expressed genes are circadian regulated. However, not much is known regarding the exact processes that are regulated by specific clock components. To determine the outputs of the clock that are directly regulated by the PRRs, I identified the genome-wide targets of PRR9 and PRR7. I showed that PRR9 and PRR7 are directly involved in the repression of master regulators of plant growth, light signaling, and stress responses. A majority of PRR9 and PRR7 targets exhibited peak gene expression around dawn and were repressed by PRR9 and PRR7. These findings indicate that PRR9 and PRR7 are important for cyclic gene expression by repressing the transcription of morning-expressed genes. I found an enrichment of PRR7 target genes involved in abiotic stress responses and accordingly, we observed that PRR7 is involved in the oxidative stress response and the regulation of stomata conductance. PRR9 and PRR7 binding sites are located near transcriptional start sites showing low nucleosome occupancy and high DNase I hypersensitivity, indicating that PRR9 and PRR7 bind to regulatory regions of DNA. Furthermore, binding motif analyses reveal that PRR9 and PRR7 binding sites are located in noncoding regions conserved among at least nine Brassicaceae species and are enriched in G-box motifs, indicating that these sequences are functionally relevant. A comparison of binding site similarity among PRR9, PRR7, PRR5, and TOC1 provided insight on the overlapping and distinct roles of the PRRs.
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