Parallel analysis ofArabidopsiscircadian clock mutants reveals different scales of transcriptome and proteome regulation

Abstract

The circadian clock regulates physiological processes central to growth and survival. To date, most plant circadian clock studies have relied on diurnal transcriptome changes to elucidate molecular connections between the circadian clock and observable phenotypes in wild-type plants. Here, we have integrated RNA-sequencing and protein mass spectrometry data to comparatively analyse the lhycca1, prr7prr9, gi and toc1 circadian clock mutant rosette at the end of day and end of night. Each mutant affects specific sets of genes and proteins, suggesting that the circadian clock regulation is modular. Furthermore, each circadian clock mutant maintains its own dynamically fluctuating transcriptome and proteome profile specific to subcellular compartments. Most of the measured protein levels do not correlate with changes in their corresponding transcripts. Transcripts and proteins that have coordinated changes in abundance are enriched for carbohydrate- and cold-responsive genes. Transcriptome changes in all four circadian clock mutants also affect genes encoding starch degradation enzymes, transcription factors and protein kinases. The comprehensive transcriptome and proteome datasets demonstrate that future system-driven research of the circadian clock requires multi-level experimental approaches. Our work also shows that further work is needed to elucidate the roles of post-translational modifications and protein degradation in the regulation of clock-related processes.

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

“…Significant protein phosphorylation changes were most frequently found in flowers (ED) and in rosettes (EN), whereas significant protein acetylation changes were most abundant in roots (ED) and in seedlings (EN) (Data S2; Table ). In contrast, few proteins in the proteomes from the same organs and seedlings had a Log2‐fold abundance change of >1.0 at the corresponding time points (Data S3), consistent with previous reports (Baerenfaller et al ., ; Graf et al ., ; Seaton et al ., ). To determine whether the observed changes in PTM levels resulted from changes in protein abundance we compared the quantified PTMome and total proteome.…”

“…Our current understanding of circadian and light control is largely derived from genetic and transcriptomic studies, which have revealed the extent to which the expression of genes is regulated by the clock and the diurnal cycle (Endo et al, 2014;Greenham and McClung, 2015;Nagel et al, 2015;Nohales and Kay, 2016). More recent studies have shown that transcript and protein abundance may not always be correlated at the light-dark transitions during the diurnal cycle, indicating that transcriptional changes are not necessarily predictive for regulation at the protein level (Baerenfaller et al, 2012;Graf et al, 2017;Seaton et al, 2018). Moreover, it is currently unknown if this lack of coincident transcript and protein-level regulation also extends to proteins whose function is circadian clock-or light controlled by phosphorylation or acetylation, or both.…”

What makes ribosomes tick?

“…46 Analysis of the Arabidopsis proteome revealed that 30-40% of proteins with rhythmic protein levels do not have rhythmic transcripts, 70 implicating translation and turnover in generating these rhythms. Meanwhile, many rhythmic mRNAs have no detectable protein rhythm, 44,45 suggesting that Model 2 applies. Moreover, comparing between liver and kidney, ribosome occupancy is more similar than mRNA transcript levels for a large number of genes.…”

“…If our goal is to not just measure and understand, but also to predict and control 83 diel cycles of gene expression, then we require corresponding quantitative mathematical models of gene expression that are founded on a rigorous theoretical framework from molecular biochemistry. Specifically, diurnal protein levels 30,40,41,44,45,70,84 are determined by rates of mRNA synthesis and mRNA degradation, mRNA translation (protein synthesis), and protein turnover. Regarding these processes, the more widely available data are on mRNA transcript levels, 85,86 which are the dynamic balance between transcription and turnover, in the same way that protein levels are the dynamic balance of protein synthesis and protein turnover.…”

“…Initial studies in the mouse liver and Arabidopsis found rhythmic proteins without rhythmic mRNA expression, pointing to post-transcriptional diurnal or circadian control. [40][41][42][43] However, in Arabidopsis for instance, among hundreds of abundant proteins, nearly all are nonrhythmic, even though most are encoded by rhythmic mRNAs 40,44,45 suggesting that translational regulation or turnover may silence transcript-level rhythms. These considerations focus the lime light on a fundamental but long neglected question: how the clock and diurnal light dark and nutrient cycles 87 and the plant model from.…”