Circadian abundance and modification of proteins in arabidopsis
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Date
28/06/2016Item status
Restricted AccessEmbargo end date
31/12/2100Author
Krahmer, Johanna
Metadata
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.