Circadian abundance and modification of proteins in arabidopsis

Abstract

Circadian clocks are endogenous pacemakers found in many organisms including plants, generating approximately 24h rhythms. Knowledge about the plant circadian clock plays a role for crop improvement. The plant circadian clock and its downstream outputs have been studied in detail by transcriptomics, however post-transcriptional and post-translational aspects are still to be researched. In addition, it has recently been shown that a protein modification remains rhythmic when rhythmic transcription is absent. This gives evidence for the existence of two oscillators: a transcription-translation feedback loop and a non-transcriptional oscillator. The aim of this PhD is to gain knowledge about circadian changes in abundance and phosphorylation of proteins as well as protein-protein interaction using the model plant Arabidopsis thaliana. I used high-throughput proteomics and phosphoproteomics methods to identify hundreds of phosposites that change in abundance in WT plants as well as dozens of proteins that exhibit circadian changes in their abundance. I also found significant temporal changes in protein phosphorylation in the transcriptionally arrhythmic mutant CCA1-Ox, albeit with dynamics different from the WT, demonstrating that without transcriptional rhythms, protein modification can still undergo rhythmic changes to some extent. In addition, I found reproducibly that the majority of changing phosphopeptides are most abundant at dawn and this is independent of the presence of a functional transcriptional oscillator. Roles of different kinases and affected phosphoproteins are discussed. I chose one of the rhythmically phosphorylated proteins, the bifunctional enzyme F2KP, for further functional experiments. In vitro experiments demonstrate that the rhythmic phosphosite is important for the activity of the enzyme. This is discussed in the light of circadian regulation of carbon metabolism. In addition to these studies on circadian protein abundance and modification, I investigated time-of-day dependent protein-protein interaction of the clock protein GIGANTEA (GI). Using an interaction proteomics timecourse, I identified about 100 potential new interactors of GI, some of which are candidates for links between diel timing and carbon metabolism. These results will help to generate hypotheses for explaining the surprising pleiotrophy of gi mutants.