Abstract
Diel regulation of protein levels and protein modification had been less studied than transcript rhythms. Here, we compare transcriptome data under light-dark cycles to partial proteome and phosphoproteome data, assayed using shotgun mass-spectrometry, from the alga Ostreococcus tauri, the smallest free-living eukaryote. 10% of quantified proteins but two-thirds of phosphoproteins were rhythmic. Mathematical modelling showed that light-stimulated protein synthesis can account for the observed clustering of protein peaks in the daytime. Prompted by night-peaking and apparently dark-stable proteins, we also tested cultures under prolonged darkness, where the proteome changed less than under the diel cycle. The dark-stable, prasinophyte-specific proteins were also reported to accumulate when O. tauri formed lipid droplets. In the phosphoproteome, 39% of rhythmic phospho-sites reached peak levels just before dawn. This anticipatory phosphorylation suggests that a clock-regulated phospho-dawn prepares green cells for daytime functions. Acid-directed and proline-directed protein phosphorylation sites were regulated in antiphase, implicating the clock-related, casein kinases 1 and 2 in phase-specific regulation, alternating with the CMGC protein kinase family. Understanding the dynamic phosphoprotein network should be facilitated by the minimal kinome and proteome of O. tauri. The data are available from ProteomeXchange, with identifiers PXD001734, PXD001735 and PXD002909. This submission updates a previous version, posted on bioRxiv on 4th April 2018, as https://www.biorxiv.org/content/10.1101/287862v1
Highlight The phosphorylation of most protein sites was rhythmic under light-dark cycles, and suggested circadian control by particular kinases. Day-peaking, rhythmic proteins likely reflect light-stimulated protein synthesis in this microalga.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Zeenat Noordally; zeenat.noordallyed{at}gmail.com, Matthew Hindle; matthew.hindle{at}gmail.com, Sarah F. Martin; sarahfriedemartin{at}gmail.com, Daniel Seaton; daniel.d.seaton{at}gmail.com, Ian Simpson; Ian.Simpson{at}ed.ac.uk, Thierry Lebihan; tlebihan{at}gmail.com, Andrew Millar; andrew.millar{at}ed.ac.uk
We have clarified the main text, with a revised and updated Introduction, revised Results, and added Discussion section. In particular, overlapping proteomics results of Smallwood et al. bioRxiv 2018 are now discussed. Figure and Supplementary Figure panels are very similar or identical, with some rearrangements to fit the revised Results. The Methods section is little altered. Supplementary Tables are identical. Data Availability is updated.
Abbreviations
- PM
- phosphopeptide motif;
- LD
- light-dark cycles;
- ZT
- Zeitgeber Time;
- DA
- dark adaptation;
- PC
- principal component;
- CK1
- casein kinase 1;
- CK2
- casein kinase 2;
- GSK3
- Glycogen Synthase Kinase 3;
- CMGC
- Cyclin-dependent kinase, Mitogen-activated protein kinase, Glycogen synthase kinase, CDC-like kinase;
- CCA1
- Circadian Clock Associated 1 protein.