The circadian clock gene circuit controls protein and phosphoprotein rhythms inArabidopsis thaliana

Abstract

The 24-hour rhythmicity in the levels of many eukaryotic mRNAs contrasts with the long half-lives of most detected proteins. Such stable molecular species are not expected to reflect the RNA rhythms, emphasizing the need to test the circadian regulation of protein abundance and modification. Here we present circadian proteomic and comparable phosphoproteomic time-series from Arabidopsis thaliana plants, estimating that 0.4% of quantified proteins and a much larger proportion of quantified phosphosites were rhythmic under constant light conditions. Approximately half of the quantified phospho-sites were most phosphorylated at subjective dawn, when SnRK1 protein kinase was a candidate regulator.A CCA1-over-expressing line that disables the plant clock gene circuit lacked most or all circadian protein phosphorylation, indicating that the phospho-dawn is regulated by the canonical clock mechanism. The few phospho-sites that fluctuated despite CCA1-over-expression still tended to peak in abundance close to subjective dawn, indicating that their temporal regulation was less dependent on the clock gene circuit. To test the potential functional relevance of our datasets, we conduct phosphomimetic experiments using the bifunctional enzyme fructose-6-phosphate-2-kinase / phosphatase (F2KP), as an example. The clock gene circuit controls diverse protein targets in metabolism and physiology via phosphorylation, including where the bulk abundance of the cognate proteins is arrhythmic.

Diurnal Dynamics of the Arabidopsis Rosette Proteome and Phosphoproteome

“…To determine if the significant changes we measured in the diurnal proteome could be controlled by the circadian clock, we next compared our data to a quantitative proteomics dataset acquired under free-running (continuous light) conditions (Krahmer et al, 2019). Our dataset of 4762 quantified proteins contains 1800 of the 2038 proteins (88%) reported by Krahmer et al (2019), allowing us to directly compare proteome results between studies (Supplemental Data 7).…”

“…To determine if the significant changes we measured in the diurnal proteome could be controlled by the circadian clock, we next compared our data to a quantitative proteomics dataset acquired under free-running (continuous light) conditions (Krahmer et al, 2019). Our dataset of 4762 quantified proteins contains 1800 of the 2038 proteins (88%) reported by Krahmer et al (2019), allowing us to directly compare proteome results between studies (Supplemental Data 7). To avoid identification of differences based on the fact that the quantitative proteome analysis described above and the JTK_cycle analysis used by Krahmer et al (2019) differ in their methods, we also performed a JTK_cycle analysis to identify proteins cycling with a 22 or 24 h period (Hughes et al, 2010).…”

“…Our dataset of 4762 quantified proteins contains 1800 of the 2038 proteins (88%) reported by Krahmer et al (2019), allowing us to directly compare proteome results between studies (Supplemental Data 7). To avoid identification of differences based on the fact that the quantitative proteome analysis described above and the JTK_cycle analysis used by Krahmer et al (2019) differ in their methods, we also performed a JTK_cycle analysis to identify proteins cycling with a 22 or 24 h period (Hughes et al, 2010). Unlike our previous analysis of diurnal proteome fluctuations, which identified 288 significantly changing proteins regardless of cycling preconditions, the JTK_cycle analysis approach aims to elucidate proteins exhibiting diurnal fluctuations in the form of a cosine function, and correspondingly evaluates how well these changes in abundance fit with this expected cosine behavior.…”

“…To compare diurnal protein fluctuations to free running circadian clock fluctuations published by Krahmer et al (2019; dataset PXD009230 available at ProteomeXchange) we performed an equivalent analysis using the JTK cycle to identify proteins cycling with 22 or 24 h period (Hughes, Hogenesch, & Kornacker, 2010). The exact loading script JTK_analysis.zip is available upon request.…”

“…In plants, diurnal protein phosphorylation is regulated either in response to light, by the circadian clock (Choudhary et al, 2015), or both (Uhrig et al, 2019), while the clock itself is regulated by phosphorylation (Kusakina & Dodd, 2012; Uehara et al, 2019). Recent studies of the circadian phosphoproteome combining the analysis of a free-running cycle and the circadian clock mutants elf4 (Choudhary et al, 2015) or CCA1-OX over-expression (Krahmer et al, 2019) have revealed temporally modified phosphorylation sites related to casein kinase II (CKII) and sucrose non-fermenting kinase 1 (SnRK1). SnRKs are likely involved in the regulation of the circadian phosphoproteome because the transcription of genes encoding multiple SnRK and calcinuerin B-like (CBL) interacting kinases (CIPK) was mis-regulated in the Arabidopsis circadian clock mutants cca1/lhy1, prr7prr9, toc1 and gi201 mutants at end-of-day (ED) and end-of-night (EN) (Graf et al, 2017).…”