Circadian Proteomics Reassesses the Temporal Regulation of Metabolic Rhythms by Chlamydomonas Clock

Circadian clocks execute temporal regulation of metabolism by modulating the timely expression of genes. Clock regulation of mRNA synthesis was envisioned as the primary driver of these daily rhythms. mRNA oscillations often do not concur with the downstream protein oscillations, revealing the importance to study protein oscillations. Chlamydomonas reinhardtii is a well-studied miniature plant model. We quantitatively probed the Chlamydomonas proteome for two subsequent circadian cycles using high throughput SWATH-DIA mass spectrometry. We quantified > 1000 proteins, half of which demonstrate circadian rhythms. Among these rhythmic proteins, > 90% peak around subjective midday or midnight. We uncovered key enzymes involved in Box C/D pathway, amino acid biosynthesis, fatty acid (FA) biosynthesis and peroxisomal β-oxidation of FAs are driven by the clock, which were undocumented from earlier transcriptomic studies. Proteins associated with key biological processes such as photosynthesis, redox, carbon fixation, glycolysis and TCA cycle show extreme temporal regulation. We conclude that circadian proteomics is required to complement transcriptomic studies to understand the complex clock regulation of organismal biology. We believe our study will not only refine and enrich the evaluation of temporal metabolic processes in C. reinhardtii but also provide a novel understanding of clock regulation across species.