Diurnal changes in concerted plant protein phosphorylation and acetylation in Arabidopsis organs and seedlings

Abstract

Summary Protein phosphorylation and acetylation are the two most abundant post‐translational modifications (PTMs) that regulate protein functions in eukaryotes. In plants, these PTMs have been investigated individually; however, their co‐occurrence and dynamics on proteins is currently unknown. Using Arabidopsis thaliana, we quantified changes in protein phosphorylation, acetylation and protein abundance in leaf rosettes, roots, flowers, siliques and seedlings at the end of day (ED) and at the end of night (EN). This identified 2549 phosphorylated and 909 acetylated proteins, of which 1724 phosphorylated and 536 acetylated proteins were also quantified for changes in PTM abundance between ED and EN. Using a sequential dual‐PTM workflow, we identified significant PTM changes and intersections in these organs and plant developmental stages. In particular, cellular process‐, pathway‐ and protein‐level analyses reveal that the phosphoproteome and acetylome predominantly intersect at the pathway‐ and cellular process‐level at ED versus EN. We found 134 proteins involved in core plant cell processes, such as light harvesting and photosynthesis, translation, metabolism and cellular transport, that were both phosphorylated and acetylated. Our results establish connections between PTM motifs, PTM catalyzing enzymes and putative substrate networks. We also identified PTM motifs for further characterization of the regulatory mechanisms that control cellular processes during the diurnal cycle in different Arabidopsis organs and seedlings. The sequential dual‐PTM analysis expands our understanding of diurnal plant cell regulation by PTMs and provides a useful resource for future analyses, while emphasizing the importance of analyzing multiple PTMs simultaneously to elucidate when, where and how they are involved in plant cell regulation.

Diurnal dynamics of the Arabidopsis rosette proteome and phosphoproteome

“…We then generated a hierarchical heat map of the phosphopeptides to identify clusters of proteins at each light transition with similar phosphorylation dy-namics (Supplemental Figure 2 and 3). When compared to datasets of phosphorylated proteins previously identified in Arabidopsis growing under free-running cycle conditions (Choudhary et al, 2015;Krahmer et al, 2019), or at the ED and EN time-points of a 12-hour photoperiod (Reiland et al, 2009;Uhrig et al, 2019), our data reveals proteins that have diurnal changes in their phosphorylation status and also novel rate-of-change information for these phosphorylation events (Supplemental Figure 2 and 3). For example, the L-D cluster I has phosphoproteins involved in nitrogen metabolism and the cell cycle (AD10 and AD30) and the L-D cluster III (BD30) has phosphoproteins involved in plastid organization (Supplemental Figure 2).…”

“…Instead of regulating proteins by adjusting their abundance, plants might react to expected diurnal changes in conditions by regulating the activity of proteins. This would be supported by the fact that most circadian regulation in Arabidopsis involves post-translational modifications of proteins such as protein phosphorylation (Choudhary et al, 2015;Krahmer et al, 2019;Uhrig et al, 2019) independent of changes in mRNA and/or protein levels. Further studies will be necessary to understand which factors explain these findings.…”

“…More than 30% of all Arabidopsis genes are regulated by the circadian clock at the transcript level (Blasing et al, 2005;Covington, Maloof, Straume, Kay, & Harmer, 2008). However, less is known about how the resulting diurnal transcriptome relates to protein abundance (Abraham et al, 2016;Choudhary, Nomura, Wang, Nakagami, & Somers, 2015;Graf et al, 2017) and post-translational protein modifications (Choudhary et al, 2015;Uhrig, Schlapfer, Roschitzki, Hirsch-Hoffmann, & Gruissem, 2019), both of which may also affect protein function at light-dark transitions and throughout the diurnal cycle. Transcript and protein abundance changes are often disconnected because changes in transcript levels show no corresponding change in protein abundance (Baerenfaller et al, 2012;Seaton et al, 2018).…”

“…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 mutant 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). Similarly, studies quantifying changes in the phosphoproteome at ED and EN in Arabidopsis rosette leaves, roots, flowers, siliques and seedlings have revealed a large number of diurnally changing phosphorylation events corresponding to diverse protein kinase motifs (Reiland et al, 2009;Uhrig et al, 2019).…”