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A phospho-dawn of protein modification anticipates light onset in the picoeukaryote Ostreococcus tauri - PubMed

  • ️Sun Jan 01 2023

A phospho-dawn of protein modification anticipates light onset in the picoeukaryote Ostreococcus tauri

Zeenat B Noordally et al. J Exp Bot. 2023.

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 with partial proteome and phosphoproteome data, assayed using shotgun MS, from the alga Ostreococcus tauri, the smallest free-living eukaryote. A total of 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. Among the dark-stable proteins were prasinophyte-specific sequences that 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.

Keywords: Light signalling; marine microalgae; phosphoproteomics; photoperiod; photosynthetic pico-eukaryotes; proteomics; systems biology.

© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1.
Fig. 1.

Daily variation in transcripts, proteins, and phosphopeptide motifs. (A) Workflow for proteomics in O. tauri under LD cycles. Overlap in (B) detected and quantified gene loci. (C) Significantly changing (filled circles) or not significantly changing (dashed circles) loci for transcripts (Monnier et al., 2010), proteins, and phosphopeptide motifs; genomic loci were excluded (square brackets). (D–I) Bi-plots of PCA for the time series of mean levels of each (D, E) transcript, (F, G) protein, and (H, I) phosphopeptide motif. The proportion of the variance for each PC is indicated. Dot locations show the weighting of each RNA/protein/phosphopeptide motif time series in each PC; colours show the assigned cluster (as in Supplementary Fig. S4A, B). Loading arrows show the magnitude (by length) and relative contribution (by direction) of data from each time point to the PCs that are plotted, hence the angles between loading arrows indicate correlation (0°) and anticorrelation (180°).

Fig. 2.
Fig. 2.

Distribution of rhythmic protein and phosphopeptide motif peaks, with examples. Temporal distribution of peaking profiles in (A) transcripts, (B) proteins, and (C) phosphopeptide motifs. (D, F) Simulated protein profiles from RNAs peaking at (D) ZT0 or (F) ZT16, with (red line) or without light-regulated translation (black line). (E) Predicted distribution of protein peak times, with light-regulated translation. Examples of genes with (G) high-amplitude and similar protein (solid line) and phosphopeptide motif profiles (coloured lines), or (H) phosphopeptide motif profiles that differ from the protein profile. (G, H) Protein and phosphopeptide motif, left axis; RNA profile (dashed line), right axis. Error bars, SE. Light/dark are indicated by white/black bars.

Fig. 3.
Fig. 3.

Regulation of dark-accumulating proteins. Protein abundance profiles (A) of rhythmic prasinophyte-specific proteins in cluster P6 in LD and DA conditions. (B) Optical density (OD600; line, right axis) and total protein per cell (columns, left axis) under LD and DA conditions. (C) Correlation of protein degradation rates (Martin et al., 2012) and relative protein levels after DA; chloroplast proteins (circles, chloroplast-encoded a have solid outline); mitochondrial proteins (triangles, mitochondria-encoded outlined); PLP-enzymes (squares, marked in key); prasinophyte-specific proteins (diamonds).

Fig. 4.
Fig. 4.

Protein and phosphopeptide motif regulation. Phosphomotif (coloured lines) and RNA profiles (Monnier et al., 2010) (dashed lines) of the photoreceptors, clock components, transcription factors. and kinases indicated, under LD cycles. Left axis range 26 (64-fold) except OtCCA1 (phosphopeptide motif changes 150-fold) and OtCOL2 (phosphopeptide motifs change up to 20-fold). Right (RNA) axis range 12, for log2 data (212=4096-fold in untransformed data). Error bars, SE. Light/dark indicated by white/black bars. PHOT, phototropin photoreceptor; LOV-HK, LOV domain-histidine kinase photoreceptor; COL, CONSTANS-like transcription factor.

Fig. 5.
Fig. 5.

Motif enrichment and rhythmic protein kinases and phosphatases under LD cycles. (A) Rhythmic phosphopeptide motifs peaking at each time point on protein kinase (black) and phosphatase (grey) proteins (numbers). (B) Enrichment of proline-directed motifs, for kinases shown in the key (dashed line, P-value=0.05). (C) pLogo sequence motifs of rhythmic phosphopeptide motifs peaking at each time point (foreground; fg), relative to all detected phosphopeptides (background; bg). ±3.80 indicates P-value=0.05; residues above and below the axis are over- and under-represented, respectively. (D) Rhythmic phosphopeptide motifs by kinase/phosphatase family, annotated with example proteins.

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