Closing the protein gap in plant chronobiology

Abstract

Our modern understanding of diel cell regulation in plants stems from foundational work in the late 1990s that analysed the dynamics of selected genes and mutants in Arabidopsis thaliana. The subsequent rise of transcriptomics technologies such as microarrays and RNA sequencing has substantially increased our understanding of anticipatory (circadian) and reactive (light-or dark-triggered) diel events in plants. However, it is also becoming clear that gene expression data fail to capture critical events in diel regulation that can only be explained by studying protein-level dynamics. Over the past decade, mass spectrometry technologies and quantitative proteomic workflows have significantly advanced, finally allowing scientists to characterise diel protein regulation at high throughput. Initial proteomic investigations suggest that the diel transcriptome and proteome generally lack synchrony and that the timing of daily regulatory events in plants is impacted by multiple levels of protein regulation (e.g., post-translational modifications [PTMs] and protein-protein interactions [PPIs]). Here, we highlight and summarise how the use of quantitative proteomics to elucidate diel plant cell regulation has advanced our understanding of these processes. We argue that this new understanding, coupled with the extraordinary developments in mass spectrometry technologies, demands greater focus on protein-level regulation of, and by, the circadian clock. This includes hitherto unexplored diel dynamics of protein turnover, PTMs, protein subcellular localisation and PPIs that can be masked by simple transcript-and protein-level changes. Finally, we propose new directions for how the latest advancements in quantitative proteomics can be utilised to answer outstanding questions in plant chronobiology.

Multi-omic analysis of the Arabidopsis clock activator mutantrve 4 6 8reveals connections to carbohydrate metabolism and proteasome regulation

“…Ultimately, a connection between the circadian clock and diel protein turnover is logical given the consistent lack of coordinately fluctuating diel transcripts and proteins in Arabidopsis across multiple experiments (Baerenfaller et al, 2012;Graf et al, 2017;Mehta et al, 2021;Uhrig et al, 2021). Overall, our results implicate RVE8-like proteins as being involved in diel protein turnover, adding important context to the well-known disconnect between circadian transcript and protein abundancies.…”

“…Ontology Enrichments were performed using TheOntologizer (http://ontologizer.de/) using all identified proteins as the background for enrichment analyses (q-value ≤ 0.05), while phosphorylation motif enrichment analysis was performed using Motif-X (Chou and Schwartz, 2011) on the MoMo MEME suite (https://meme-suite.org/meme/) as previously described (Mehta et al, 2021). Subcellular localization information for proteins was obtained from SUBA4 (https://suba.live/).…”

“…This extensive body of work has revealed a number of roles for the circadian clock in plants, including in the timing of flowering (Shim et al, 2017), disease resistance (Lu et al, 2017) and mitigation of abiotic stress (Simon et al, 2020;Kidokoro et al, 2021) amongst others. However, despite the extensive use of genetic and transcriptomic technologies to understand the molecular impacts of core circadian clock components in Arabidopsis, much less is known about how, and/or if, these transcriptional changes manifest at the protein-level (Choudhary et al, 2015;Choudhary et al, 2016;Krahmer et al, 2022;Uhrig et al, 2019;Mehta et al, 2021;Uhrig et al, 2021).…”

Day and night isotope labelling reveal metabolic pathway specific regulation of protein synthesis rates in Arabidopsis

“…The outputs of this clock have been captured by multiple discrete measurements of transcript abundance and/or ribosome association (Missra et al., 2015), which have resulted in estimates that the clock controls the expression of one‐third of all genes encoded in the Arabidopsis genome (Covington et al., 2008). Abundance‐based proteomics and phosphoproteomics studies however suggest that the transcript abundance and ribosome loading variation only reveals part of a notably more complex picture (Mehta et al., 2021). Transcript abundances and protein abundances correlate poorly over the diurnal cycle (Mockler et al., 2007; Uhrig et al., 2021).…”

“…Without direct measurements of the biomolecules involved, they remain theoretical. Either way, a key gap in the knowledge available is what role diel/circadian controlled protein synthesis and degradation play in conferring the selective advantage gained through clock function (Mehta et al., 2021).…”