Gerben van Ooijen

Peroxiredoxins are conserved markers of circadian rhythms

Cellular life emerged ∼3.7 billion years ago. With scant exception, terrestrial organisms have evolved under predictable daily cycles owing to the Earth’s rotation. The advantage conferred on organisms that anticipate such environmental cycles has driven the evolution of endogenous circadian rhythms that tune internal physiology to external conditions. The molecular phylogeny of mechanisms driving these rhythms has been difficult to dissect because identified clock genes and proteins are not conserved across the domains of life: Bacteria, Archaea and Eukaryota. Here we show that oxidation–reduction cycles of peroxiredoxin proteins constitute a universal marker for circadian rhythms in all domains of life, by characterizing their oscillations in a variety of model organisms. Furthermore, we explore the interconnectivity between these metabolic cycles and transcription–translation feedback loops of the clockwork in each system. Our results suggest an intimate co-evolution of cellular timekeeping with redox homeostatic mechanisms after the Great Oxidation Event ∼2.5 billion years ago.

Circadian rhythms persist without transcription in a eukaryote

Circadian rhythms are ubiquitous in eukaryotes, and coordinate numerous aspects of behaviour, physiology and metabolism, from sleep/wake cycles in mammals to growth and photosynthesis in plants. This daily timekeeping is thought to be driven by transcriptional–translational feedback loops, whereby rhythmic expression of ‘clock’ gene products regulates the expression of associated genes in approximately 24-hour cycles. The specific transcriptional components differ between phylogenetic kingdoms. The unicellular pico-eukaryotic alga Ostreococcus tauri possesses a naturally minimized clock, which includes many features that are shared with plants, such as a central negative feedback loop that involves the morning-expressed CCA1 and evening-expressed TOC1 genes. Given that recent observations in animals and plants have revealed prominent post-translational contributions to timekeeping, a reappraisal of the transcriptional contribution to oscillator function is overdue. Here we show that non-transcriptional mechanisms are sufficient to sustain circadian timekeeping in the eukaryotic lineage, although they normally function in conjunction with transcriptional components. We identify oxidation of peroxiredoxin proteins as a transcription-independent rhythmic biomarker, which is also rhythmic in mammals. Moreover we show that pharmacological modulators of the mammalian clock mechanism have the same effects on rhythms in Ostreococcus. Post-translational mechanisms, and at least one rhythmic marker, seem to be better conserved than transcriptional clock regulators. It is plausible that the oldest oscillator components are non-transcriptional in nature, as in cyanobacteria, and are conserved across kingdoms.

Structure–function analysis of the NB-ARC domain of plant disease resistance proteins

Resistance (R) proteins in plants are involved in pathogen recognition and subsequent activation of innate immune responses. Most resistance proteins contain a central nucleotide-binding domain. This so-called NB-ARC domain consists of three subdomains: NB, ARC1, and ARC2. The NB-ARC domain is a functional ATPase domain, and its nucleotide-binding state is proposed to regulate activity of the R protein. A highly conserved methionine-histidine-aspartate (MHD) motif is present at the carboxy-terminus of ARC2. An extensive mutational analysis of the MHD motif in the R proteins I-2 and Mi-1 is reported. Several novel autoactivating mutations of the MHD invariant histidine and conserved aspartate were identified. The combination of MHD mutants with autoactivating hydrolysis mutants in the NB subdomain showed that the autoactivation phenotypes are not additive. This finding indicates an important regulatory role for the MHD motif in the control of R protein activity. To explain these observations, a three-dimensional model of the NB-ARC domain of I-2 was built, based on the APAF-1 template structure. The model was used to identify residues important for I-2 function. Substitution of the selected residues resulted in the expected distinct phenotypes. Based on the model, it is proposed that the MHD motif fulfils the same function as the sensor II motif found in AAA+ proteins (ATPases associated with diverse cellular activities)-co-ordination of the nucleotide and control of subdomain interactions. The presented 3D model provides a framework for the formulation of hypotheses on how mutations in the NB-ARC exert their effects.

The effector protein Avr2 of the xylem-colonizing fungus Fusarium oxysporum activates the tomato resistance protein I-2 intracellularly

To promote host colonization, many plant pathogens secrete effector proteins that either suppress or counteract host defences. However, when these effectors are recognized by the hosts innate immune system, they trigger resistance rather than promoting virulence. Effectors are therefore key molecules in determining disease susceptibility or resistance. We show here that Avr2, secreted by the vascular wilt fungus Fusarium oxysporum f. sp. lycopersici (Fol), shows both activities: it is required for full virulence in a susceptible host and also triggers resistance in tomato plants carrying the resistance gene I-2. Point mutations in AVR2, causing single amino acid changes, are associated with I-2-breakingFol strains. These point mutations prevent recognition by I-2, both in tomato and when both genes are co-expressed in leaves of Nicotiana benthamiana. Fol strains carrying the Avr2 variants are equally virulent, showing that virulence and avirulence functions can be uncoupled. Although Avr2 is secreted into the xylem sap when Fol colonizes tomato, the Avr2 protein can be recognized intracellularly by I-2, implying uptake by host cells.

Daily magnesium fluxes regulate cellular timekeeping and energy balance

Circadian clocks are fundamental to the biology of most eukaryotes, coordinating behaviour and physiology to resonate with the environmental cycle of day and night through complex networks of clock-controlled genes. A fundamental knowledge gap exists, however, between circadian gene expression cycles and the biochemical mechanisms that ultimately facilitate circadian regulation of cell biology. Here we report circadian rhythms in the intracellular concentration of magnesium ions, [Mg2+]i, which act as a cell-autonomous timekeeping component to determine key clock properties both in a human cell line and in a unicellular alga that diverged from each other more than 1 billion years ago. Given the essential role of Mg2+ as a cofactor for ATP, a functional consequence of [Mg2+]i oscillations is dynamic regulation of cellular energy expenditure over the daily cycle. Mechanistically, we find that these rhythms provide bilateral feedback linking rhythmic metabolism to clock-controlled gene expression. The global regulation of nucleotide triphosphate turnover by intracellular Mg2+ availability has potential to impact upon many of the cell’s more than 600 MgATP-dependent enzymes and every cellular system where MgNTP hydrolysis becomes rate limiting. Indeed, we find that circadian control of translation by mTOR is regulated through [Mg2+]i oscillations. It will now be important to identify which additional biological processes are subject to this form of regulation in tissues of multicellular organisms such as plants and humans, in the context of health and disease.

Multiple light inputs to a simple clock circuit allow complex biological rhythms

Circadian clocks are biological timekeepers that allow living cells to time their activity in anticipation of predictable environmental changes. Detailed understanding of the circadian network of higher plants, such as Arabidopsis thaliana, is hampered by the high number of partially redundant genes. However, the picoeukaryotic alga Ostreococcus tauri, which was recently shown to possess a small number of non-redundant clock genes, presents an attractive alternative target for detailed modelling of circadian clocks in the green lineage. Based on extensive time-series data from in vivo reporter gene assays, we developed a model of the Ostreococcus clock as a feedback loop between the genes TOC1 and CCA1. The model reproduces the dynamics of the transcriptional and translational reporters over a range of photoperiods. Surprisingly, the model is also able to predict the transient behaviour of the clock when the light conditions are altered. Despite the apparent simplicity of the clock circuit, it displays considerable complexity in its response to changing light conditions. Systematic screening of the effects of altered day length revealed a complex relationship between phase and photoperiod, which is also captured by the model. The complex light response is shown to stem from circadian gating of light-dependent mechanisms. This study provides insights into the contributions of light inputs to the Ostreococcus clock. The model suggests that a high number of light-dependent reactions are important for flexible timing in a circadian clock with only one feedback loop.

Transcomplementation, but not physical association of the CC-NB-ARC and LRR domains of tomato R protein Mi-1.2 is altered by mutations in the ARC2 subdomain

Race-specific disease resistance in plants is mediated by Resistance (R) proteins that recognize pathogen attack and initiate defence responses. Most R proteins contain a central NB-ARC domain and a C-terminal leucine-rich repeat (LRR) domain. We analyzed the intramolecular interaction of the LRR domain of tomato R protein Mi-1.2 with its N-terminus. We expressed the CC-NB-ARC and LRR parts in trans and analyzed functional transcomplementation and physical interactions. We show that these domains functionally transcomplement when expressed in trans. Known autoactivating LRR domain swaps were found to induce a hypersensitive response (HR) upon co-expression. Likewise, autoactivating mutants in the NB subdomain transcomplemented to induce HR. Point mutations in the ARC2 subdomain that induce strong autoactivation in the full-length Mi-1.2 protein, however, fail to induce HR in the transcomplementation assay. These data indicate distinct functions for the NB-ARC subdomains in induction of HR signalling. Furthermore, dissociation of the LRR is not required to release its negative regulation, as in all combinations of CC-NB-ARC and LRR domains tested, a physical interaction was observed.

Non-transcriptional oscillators in circadian timekeeping

Circadian clocks have evolved as an adaptation to life on a rotating planet, and orchestrate rhythmic changes in physiology to match the time of day. For decades, cellular circadian rhythms were considered to solely result from feedback between the products of rhythmically expressed genes. These transcriptional/translational feedback loops (TTFLs) have been ubiquitously studied, and explain the majority of circadian outputs. In recent years, however, non-transcriptional processes were shown to be major contributors to circadian rhythmicity. These key findings have profound implications on our understanding of the evolution and mechanistic basis of cellular circadian timekeeping. This review summarises and discusses these results and the experimental and theoretical evidence of a possible relation between non-transcriptional oscillator (NTO) mechanisms and TTFL oscillations.

Shotgun proteomic analysis of the unicellular alga Ostreococcus tauri

Ostreococcus tauri is a unicellular green alga and amongst the smallest and simplest free-living eukaryotes. The O. tauri genome sequence was determined in 2006. Molecular, physiological and taxonomic data that has been generated since then highlight its potential as a simple model species for algae and plants. However, its proteome remains largely unexplored. This paper describes the global proteomic study of O. tauri, using mass spectrometry-based approaches: phosphopeptide enrichment, cellular fractionation, label-free quantification and (15)N metabolic labeling. The O. tauri proteome was analyzed under the following conditions: sampling at different times during the circadian cycle, after 24h of illumination, after 24h of darkness and under various nitrogen source supply levels. Cell cycle related proteins such as dynamin and kinesin were significantly up-regulated during the daylight-to-darkness transition. This is reflected by their higher intensity at ZT13 and this transition phase coincides with the end of mitosis. Proteins involved in several metabolic mechanisms were found to be up-regulated under low nitrogen conditions, including carbon storage pathways, glycolysis, phosphate transport, and the synthesis of inorganic polyphosphates. Ostreococcus tauri responds to low nitrogen conditions by reducing its nitrogen assimilation machinery which suggests an atypical adaptation mechanism for coping with a nutrient-limited environment.

Proteasome Function Is Required for Biological Timing throughout the Twenty-Four Hour Cycle

Summary Circadian clocks were, until recently, seen as a consequence of rhythmic transcription of clock components, directed by transcriptional/translational feedback loops (TTFLs). Oscillations of protein modification were then discovered in cyanobacteria [1, 2]. Canonical posttranslational signaling processes have known importance for clocks across taxa [3–11]. More recently, evidence from the unicellular eukaryote Ostreococcus tauri revealed a transcription-independent, rhythmic protein modification [12] shared in anucleate human cells [13]. In this study, the Ostreococcus system reveals a central role for targeted protein degradation in the mechanism of circadian timing. The Ostreococcus clockwork contains a TTFL involving the morning-expressed CCA1 and evening-expressed TOC1 proteins [14]. Cellular CCA1 and TOC1 protein content and degradation rates are analyzed qualitatively and quantitatively using luciferase reporter fusion proteins. CCA1 protein degradation rates, measured in high time resolution, feature a sharp clock-regulated peak under constant conditions. TOC1 degradation peaks in response to darkness. Targeted protein degradation, unlike transcription and translation, is shown to be essential to sustain TTFL rhythmicity throughout the circadian cycle. Although proteasomal degradation is not necessary for sustained posttranslational oscillations in transcriptionally inactive cells, TTFL and posttranslational oscillators are normally coupled, and proteasome function is crucial to sustain both.