Closing the protein gap in plant chronobiology.

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.