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1.  Light and circadian regulation of clock components aids flexible responses to environmental signals 
The New Phytologist  2014;203(2):568-577.
The circadian clock measures time across a 24 h period, increasing fitness by phasing biological processes to the most appropriate time of day. The interlocking feedback loop mechanism of the clock is conserved across species; however, the number of loops varies. Mathematical and computational analyses have suggested that loop complexity affects the overall flexibility of the oscillator, including its responses to entrainment signals.We used a discriminating experimental assay, at the transition between different photoperiods, in order to test this proposal in a minimal circadian network (in Ostreococcus tauri) and a more complex network (in Arabidopsis thaliana).Transcriptional and translational reporters in O. tauri primarily tracked dawn or dusk, whereas in A. thaliana, a wider range of responses were observed, consistent with its more flexible clock. Model analysis supported the requirement for this diversity of responses among the components of the more complex network. However, these and earlier data showed that the O. tauri network retains surprising flexibility, despite its simple circuit.We found that models constructed from experimental data can show flexibility either from multiple loops and/or from multiple light inputs. Our results suggest that O. tauri has adopted the latter strategy, possibly as a consequence of genomic reduction.
doi:10.1111/nph.12853
PMCID: PMC4286021  PMID: 24842166
biological clocks; flexibility; marine algae; mathematical analysis; nonlinear dynamics; photoperiod; systems biology
2.  Model selection reveals control of cold signalling by evening-phased components of the plant circadian clock 
The Plant Journal  2013;76(2):247-257.
Circadian clocks confer advantages by restricting biological processes to certain times of day through the control of specific phased outputs. Control of temperature signalling is an important function of the plant oscillator, but the architecture of the gene network controlling cold signalling by the clock is not well understood. Here we use a model ensemble fitted to time-series data and a corrected Akaike Information Criterion (AICc) analysis to extend a dynamic model to include the control of the key cold-regulated transcription factors C-REPEAT BINDING FACTORs 1–3 (CBF1, CBF2, CBF3). AICc was combined with in silico analysis of genetic perturbations in the model ensemble, and selected a model that predicted mutant phenotypes and connections between evening-phased circadian clock components and CBF3 transcriptional control, but these connections were not shared by CBF1 and CBF2. In addition, our model predicted the correct gating of CBF transcription by cold only when the cold signal originated from the clock mechanism itself, suggesting that the clock has an important role in temperature signal transduction. Our data shows that model selection could be a useful method for the expansion of gene network models.
doi:10.1111/tpj.12303
PMCID: PMC4278413  PMID: 23909712
Arabidopsis; circadian clock; cold acclimation; temperature; gene network model
3.  The reduced kinome of Ostreococcus tauri: core eukaryotic signalling components in a tractable model species 
BMC Genomics  2014;15(1):640.
Background
The current knowledge of eukaryote signalling originates from phenotypically diverse organisms. There is a pressing need to identify conserved signalling components among eukaryotes, which will lead to the transfer of knowledge across kingdoms. Two useful properties of a eukaryote model for signalling are (1) reduced signalling complexity, and (2) conservation of signalling components. The alga Ostreococcus tauri is described as the smallest free-living eukaryote. With less than 8,000 genes, it represents a highly constrained genomic palette.
Results
Our survey revealed 133 protein kinases and 34 protein phosphatases (1.7% and 0.4% of the proteome). We conducted phosphoproteomic experiments and constructed domain structures and phylogenies for the catalytic protein-kinases. For each of the major kinases families we review the completeness and divergence of O. tauri representatives in comparison to the well-studied kinomes of the laboratory models Arabidopsis thaliana and Saccharomyces cerevisiae, and of Homo sapiens. Many kinase clades in O. tauri were reduced to a single member, in preference to the loss of family diversity, whereas TKL and ABC1 clades were expanded. We also identified kinases that have been lost in A. thaliana but retained in O. tauri. For three, contrasting eukaryotic pathways – TOR, MAPK, and the circadian clock – we established the subset of conserved components and demonstrate conserved sites of substrate phosphorylation and kinase motifs.
Conclusions
We conclude that O. tauri satisfies our two central requirements. Several of its kinases are more closely related to H. sapiens orthologs than S. cerevisiae is to H. sapiens. The greatly reduced kinome of O. tauri is therefore a suitable model for signalling in free-living eukaryotes.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-640) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-640
PMCID: PMC4143559  PMID: 25085202
Conserved eukaryote signalling; Protein kinase phylogeny; Ostreococcus tauri; Model kinome; Phosphorylation; TOR signalling; MAPK cascade; Circadian clock
4.  Strengths and Limitations of Period Estimation Methods for Circadian Data 
PLoS ONE  2014;9(5):e96462.
A key step in the analysis of circadian data is to make an accurate estimate of the underlying period. There are many different techniques and algorithms for determining period, all with different assumptions and with differing levels of complexity. Choosing which algorithm, which implementation and which measures of accuracy to use can offer many pitfalls, especially for the non-expert. We have developed the BioDare system, an online service allowing data-sharing (including public dissemination), data-processing and analysis. Circadian experiments are the main focus of BioDare hence performing period analysis is a major feature of the system. Six methods have been incorporated into BioDare: Enright and Lomb-Scargle periodograms, FFT-NLLS, mFourfit, MESA and Spectrum Resampling. Here we review those six techniques, explain the principles behind each algorithm and evaluate their performance. In order to quantify the methods' accuracy, we examine the algorithms against artificial mathematical test signals and model-generated mRNA data. Our re-implementation of each method in Java allows meaningful comparisons of the computational complexity and computing time associated with each algorithm. Finally, we provide guidelines on which algorithms are most appropriate for which data types, and recommendations on experimental design to extract optimal data for analysis.
doi:10.1371/journal.pone.0096462
PMCID: PMC4014635  PMID: 24809473
5.  Regulatory principles and experimental approaches to the circadian control of starch turnover 
In many plants, starch is synthesized during the day and degraded during the night to avoid carbohydrate starvation in darkness. The circadian clock participates in a dynamic adjustment of starch turnover to changing environmental condition through unknown mechanisms. We used mathematical modelling to explore the possible scenarios for the control of starch turnover by the molecular components of the plant circadian clock. Several classes of plausible models were capable of describing the starch dynamics observed in a range of clock mutant plants and light conditions, including discriminating circadian protocols. Three example models of these classes are studied in detail, differing in several important ways. First, the clock components directly responsible for regulating starch degradation are different in each model. Second, the intermediate species in the pathway may play either an activating or inhibiting role on starch degradation. Third, the system may include a light-dependent interaction between the clock and downstream processes. Finally, the clock may be involved in the regulation of starch synthesis. We discuss the differences among the models’ predictions for diel starch profiles and the properties of the circadian regulators. These suggest additional experiments to elucidate the pathway structure, avoid confounding results and identify the molecular components involved.
doi:10.1098/rsif.2013.0979
PMCID: PMC3869173  PMID: 24335560
starch metabolism; circadian rhythms; Arabidopsis thaliana; mathematical modelling; systems biology; biological clocks
6.  Functional analysis of the rodent CK1tau mutation in the circadian clock of a marine unicellular alga 
BMC Cell Biology  2013;14:46.
Background
Casein Kinase 1 (CK1) is one of few proteins known to affect cellular timekeeping across metazoans, and the naturally occurring CK1tau mutation shortens circadian period in mammals. Functional conservation of a timekeeping function for CK1 in the green lineage was recently identified in the green marine unicell Ostreococcus tauri, in spite of the absence of CK1's transcriptional targets known from other species. The short-period phenotype of CK1tau mutant in mammals depends specifically on increased CK1 activity against PERIOD proteins. To understand how CK1 acts differently upon the algal clock, we analysed the cellular and proteomic effects of CK1tau overexpression in O. tauri.
Results
Overexpression of the CK1tau in O. tauri induces period lengthening identical to overexpression of wild-type CK1, in addition to resistance to CK1 inhibitor IC261. Label-free quantitative mass spectrometry of CK1tau overexpressing algae revealed a total of 58 unique phospho-sites that are differentially responsive to CK1tau. Combined with CK1 phosphorylation site prediction tools and previously published wild-type CK1-responsive peptides, this study results in a highly stringent list of upregulated phospho-sites, derived from proteins containing ankyrin repeats, kinase proteins, and phosphoinositide-binding proteins.
Conclusions
The identical phenotype for overexpression of wild-type CK1 and CK1tau is in line with the absence of critical targets for rodent CK1tau in O. tauri. Proteomic analyses reveal that two thirds of previously reported CK1 overexpression-responsive phospho-sites are shared with CK1tau. These results indicate that the two alleles are functionally indiscriminate in O. tauri, and verify the identified cellular CK1 target proteins in a minimal circadian model organism.
doi:10.1186/1471-2121-14-46
PMCID: PMC3852742  PMID: 24127907
Casein Kinase 1; Circadian clock; Minimal model; Ostreococcus tauri; Quantitative mass spectrometry; Phospho-proteomics; Bioinformatics
7.  FKF1 conveys crucial timing information for CONSTANS stabilization in the photoperiodic flowering 
Science (New York, N.Y.)  2012;336(6084):1045-1049.
Plants use day-length information to coordinate flowering time with the appropriate season to maximize reproduction. In Arabidopsis, the long-day specific expression of CONSTANS (CO) protein is crucial for flowering induction. Although light signaling regulates CO protein stability, the mechanism by which CO is stabilized in the long-day afternoon has remained elusive. Here we demonstrate that FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) protein stabilizes CO protein in the afternoon in long days. FKF1 interacts with CO through its LOV domain, and blue light enhances this interaction. In addition, FKF1 simultaneously removes CYCLING DOF FACTOR 1 (CDF1) that represses CO and FLOWERING LOCUS T (FT) transcription. Together with CO transcriptional regulation, FKF1 protein controls robust FT mRNA induction through multiple feedforward mechanisms that accurately control flowering timing.
doi:10.1126/science.1219644
PMCID: PMC3737243  PMID: 22628657
8.  Functional Analysis of Casein Kinase 1 in a Minimal Circadian System 
PLoS ONE  2013;8(7):e70021.
The Earth’s rotation has driven the evolution of cellular circadian clocks to facilitate anticipation of the solar cycle. Some evidence for timekeeping mechanism conserved from early unicellular life through to modern organisms was recently identified, but the components of this oscillator are currently unknown. Although very few clock components appear to be shared across higher species, Casein Kinase 1 (CK1) is known to affect timekeeping across metazoans and fungi, but has not previously been implicated in the circadian clock in the plant kingdom. We now show that modulation of CK1 function lengthens circadian rhythms in Ostreococcustauri, a unicellular marine algal species at the base of the green lineage, separated from humans by ~1.5 billion years of evolution. CK1 contributes to timekeeping in a phase-dependent manner, indicating clock-mediated gating of CK1 activity. Label-free proteomic analyses upon overexpression as well as inhibition revealed CK1-responsive phosphorylation events on a set of target proteins, including highly conserved potentially clock-relevant cellular regulator proteins. These results have major implications for our understanding of cellular timekeeping and can inform future studies in any circadian organism.
doi:10.1371/journal.pone.0070021
PMCID: PMC3723912  PMID: 23936135
9.  Stochastic properties of the plant circadian clock 
Circadian clocks are gene regulatory networks whose role is to help the organisms to cope with variations in environmental conditions such as the day/night cycle. In this work, we explored the effects of molecular noise in single cells on the behaviour of the circadian clock in the plant model species Arabidopsis thaliana. The computational modelling language Bio-PEPA enabled us to give a stochastic interpretation of an existing deterministic model of the clock, and to easily compare the results obtained via stochastic simulation and via numerical solution of the deterministic model. First, the introduction of stochasticity in the model allowed us to estimate the unknown size of the system. Moreover, stochasticity improved the description of the available experimental data in several light conditions: noise-induced fluctuations yield a faster entrainment of the plant clock under certain photoperiods and are able to explain the experimentally observed dampening of the oscillations in plants under constant light conditions. The model predicts that the desynchronization between noisy oscillations in single cells contributes to the observed damped oscillations at the level of the cell population. Analysis of the phase, period and amplitude distributions under various light conditions demonstrated robust entrainment of the plant clock to light/dark cycles which closely matched the available experimental data.
doi:10.1098/rsif.2011.0378
PMCID: PMC3284129  PMID: 21880617
circadian clock; Arabidopsis thaliana; discrete stochastic model; Bio-PEPA process algebra; oscillatory systems
10.  Modelling the widespread effects of TOC1 signalling on the plant circadian clock and its outputs 
BMC Systems Biology  2013;7:23.
Background
24-hour biological clocks are intimately connected to the cellular signalling network, which complicates the analysis of clock mechanisms. The transcriptional regulator TOC1 (TIMING OF CAB EXPRESSION 1) is a founding component of the gene circuit in the plant circadian clock. Recent results show that TOC1 suppresses transcription of multiple target genes within the clock circuit, far beyond its previously-described regulation of the morning transcription factors LHY (LATE ELONGATED HYPOCOTYL) and CCA1 (CIRCADIAN CLOCK ASSOCIATED 1). It is unclear how this pervasive effect of TOC1 affects the dynamics of the clock and its outputs. TOC1 also appears to function in a nested feedback loop that includes signalling by the plant hormone Abscisic Acid (ABA), which is upregulated by abiotic stresses, such as drought. ABA treatments both alter TOC1 levels and affect the clock’s timing behaviour. Conversely, the clock rhythmically modulates physiological processes induced by ABA, such as the closing of stomata in the leaf epidermis. In order to understand the dynamics of the clock and its outputs under changing environmental conditions, the reciprocal interactions between the clock and other signalling pathways must be integrated.
Results
We extended the mathematical model of the plant clock gene circuit by incorporating the repression of multiple clock genes by TOC1, observed experimentally. The revised model more accurately matches the data on the clock’s molecular profiles and timing behaviour, explaining the clock’s responses in TOC1 over-expression and toc1 mutant plants. A simplified representation of ABA signalling allowed us to investigate the interactions of ABA and circadian pathways. Increased ABA levels lengthen the free-running period of the clock, consistent with the experimental data. Adding stomatal closure to the model, as a key ABA- and clock-regulated downstream process allowed to describe TOC1 effects on the rhythmic gating of stomatal closure.
Conclusions
The integrated model of the circadian clock circuit and ABA-regulated environmental sensing allowed us to explain multiple experimental observations on the timing and stomatal responses to genetic and environmental perturbations. These results crystallise a new role of TOC1 as an environmental sensor, which both affects the pace of the central oscillator and modulates the kinetics of downstream processes.
doi:10.1186/1752-0509-7-23
PMCID: PMC3614443  PMID: 23506153
Circadian rhythms; Biological clocks; Gene regulatory networks; Mathematical model; Systems biology
11.  Network balance via CRY signalling controls the Arabidopsis circadian clock over ambient temperatures 
Temperature compensation of the Arabidopsis circadian clock is shown to be mediated by the interaction of light and temperature at the level of the crytochrome photoreceptors. These findings reveal that light and temperature share common input mechanisms to the circadian network.
We provide evidence that blue light signalling via the cryptochromes is important for the temperature-dependent control of circadian period in plants.Light and temperature converge upon common targets in the circadian network.We have constructed a temperature-compensated model of the plant circadian clock by adding a temperature effect to a subset of light-sensitive processes.The model matches experimental data and predicted a temperature-dependent change in the protein level of a key clock gene.
Circadian clocks exhibit ‘temperature compensation', meaning that they show only small changes in period over a broad temperature range. Several clock genes have been implicated in the temperature-dependent control of period in Arabidopsis. We show that blue light is essential for this, suggesting that the effects of light and temperature interact or converge upon common targets in the circadian clock. Our data demonstrate that two cryptochrome photoreceptors differentially control circadian period and sustain rhythmicity across the physiological temperature range. In order to test the hypothesis that the targets of light regulation are sufficient to mediate temperature compensation, we constructed a temperature-compensated clock model by adding passive temperature effects into only the light-sensitive processes in the model. Remarkably, this model was not only capable of full temperature compensation and consistent with mRNA profiles across a temperature range, but also predicted the temperature-dependent change in the level of LATE ELONGATED HYPOCOTYL, a key clock protein. Our analysis provides a systems-level understanding of period control in the plant circadian oscillator.
doi:10.1038/msb.2013.7
PMCID: PMC3619941  PMID: 23511208
circadian rhythm; genetic network; mathematical model; systems biology
12.  SBSI: an extensible distributed software infrastructure for parameter estimation in systems biology 
Bioinformatics  2013;29(5):664-665.
Summary: Complex computational experiments in Systems Biology, such as fitting model parameters to experimental data, can be challenging to perform. Not only do they frequently require a high level of computational power, but the software needed to run the experiment needs to be usable by scientists with varying levels of computational expertise, and modellers need to be able to obtain up-to-date experimental data resources easily. We have developed a software suite, the Systems Biology Software Infrastructure (SBSI), to facilitate the parameter-fitting process. SBSI is a modular software suite composed of three major components: SBSINumerics, a high-performance library containing parallelized algorithms for performing parameter fitting; SBSIDispatcher, a middleware application to track experiments and submit jobs to back-end servers; and SBSIVisual, an extensible client application used to configure optimization experiments and view results. Furthermore, we have created a plugin infrastructure to enable project-specific modules to be easily installed. Plugin developers can take advantage of the existing user-interface and application framework to customize SBSI for their own uses, facilitated by SBSI’s use of standard data formats.
Availability and implementation: All SBSI binaries and source-code are freely available from http://sourceforge.net/projects/sbsi under an Apache 2 open-source license. The server-side SBSINumerics runs on any Unix-based operating system; both SBSIVisual and SBSIDispatcher are written in Java and are platform independent, allowing use on Windows, Linux and Mac OS X. The SBSI project website at http://www.sbsi.ed.ac.uk provides documentation and tutorials.
Contact: stg@inf.ed.ac.uk
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btt023
PMCID: PMC3582266  PMID: 23329415
13.  Peroxiredoxins are conserved markers of circadian rhythms 
Nature  2012;485(7399):459-464.
Summary
Cellular life emerged ~3.7 billion years ago. With scant exception, terrestrial organisms have evolved under predictable daily cycles due to the Earth’s rotation. The advantage conferred upon 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 characterising 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 time-keeping with redox homeostatic mechanisms following the Great Oxidation Event ~2.5 billion years ago.
doi:10.1038/nature11088
PMCID: PMC3398137  PMID: 22622569
15.  Genomic Transformation of the Picoeukaryote Ostreococcus tauri 
Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.
doi:10.3791/4074
PMCID: PMC3476405  PMID: 22825291
Microbiology;  Issue 65;  Plant Biology;  Microbial Oceanography;  Marine Biology;  Genetics;  Transformation;  Electroporation;  Marine algae;  plankton;  Cell biology;  Ostreococcus tauri;  Plant Science;  Reduced complexity;  Circadian
16.  Digital clocks: simple Boolean models can quantitatively describe circadian systems 
The gene networks that comprise the circadian clock modulate biological function across a range of scales, from gene expression to performance and adaptive behaviour. The clock functions by generating endogenous rhythms that can be entrained to the external 24-h day–night cycle, enabling organisms to optimally time biochemical processes relative to dawn and dusk. In recent years, computational models based on differential equations have become useful tools for dissecting and quantifying the complex regulatory relationships underlying the clock's oscillatory dynamics. However, optimizing the large parameter sets characteristic of these models places intense demands on both computational and experimental resources, limiting the scope of in silico studies. Here, we develop an approach based on Boolean logic that dramatically reduces the parametrization, making the state and parameter spaces finite and tractable. We introduce efficient methods for fitting Boolean models to molecular data, successfully demonstrating their application to synthetic time courses generated by a number of established clock models, as well as experimental expression levels measured using luciferase imaging. Our results indicate that despite their relative simplicity, logic models can (i) simulate circadian oscillations with the correct, experimentally observed phase relationships among genes and (ii) flexibly entrain to light stimuli, reproducing the complex responses to variations in daylength generated by more detailed differential equation formulations. Our work also demonstrates that logic models have sufficient predictive power to identify optimal regulatory structures from experimental data. By presenting the first Boolean models of circadian circuits together with general techniques for their optimization, we hope to establish a new framework for the systematic modelling of more complex clocks, as well as other circuits with different qualitative dynamics. In particular, we anticipate that the ability of logic models to provide a computationally efficient representation of system behaviour could greatly facilitate the reverse-engineering of large-scale biochemical networks.
doi:10.1098/rsif.2012.0080
PMCID: PMC3405750  PMID: 22499125
systems biology; circadian gene networks; Boolean logic; photoperiodism; Arabidopsis thaliana
17.  The clock gene circuit in Arabidopsis includes a repressilator with additional feedback loops 
Recent findings are incorporated into a new mathematical model of the plant circadian clock, revealing a complex circuit structure comprised of multiple negative feedback loops, and predicting a repressive role for a key regulator, TOC1, which the authors confirm experimentally.
The feedback structure of the plant clock's evening loop was reconstructed based on multiple data, and is now represented by the evening complex (ELF3–ELF4–LUX), which represses transcription from the ELF4 and LUX promoters.Computational analysis of timeseries data from mutant plants predicts that TOC1 is a repressor of the key morning genes LHY and CCA1, not an activator. Analysis of LHY and CCA1 expression in TOC1 gain- and loss-of-function plants confirms this prediction.Light induction of LHY and CCA1 expression is predicted to determine the clock's response to brief light pulses, matching the observed phase-response curve.The evening complex controls LHY and CCA1 expression by a double-negative connection, rather than direct activation, forming part of a three-component repressilator circuit, which is itself only part of the more complex circuit of the clock system.
Circadian clocks synchronise biological processes with the day/night cycle, using molecular mechanisms that include interlocked, transcriptional feedback loops. Recent experiments identified the evening complex (EC) as a repressor that can be essential for gene expression rhythms in plants. Integrating the EC components in this role significantly alters our mechanistic, mathematical model of the clock gene circuit. Negative autoregulation of the EC genes constitutes the clock's evening loop, replacing the hypothetical component Y. The EC explains our earlier conjecture that the morning gene PSEUDO-RESPONSE REGULATOR 9 was repressed by an evening gene, previously identified with TIMING OF CAB EXPRESSION1 (TOC1). Our computational analysis suggests that TOC1 is a repressor of the morning genes LATE ELONGATED HYPOCOTYL and CIRCADIAN CLOCK ASSOCIATED1 rather than an activator as first conceived. This removes the necessity for the unknown component X (or TOC1mod) from previous clock models. As well as matching timeseries and phase-response data, the model provides a new conceptual framework for the plant clock that includes a three-component repressilator circuit in its complex structure.
doi:10.1038/msb.2012.6
PMCID: PMC3321525  PMID: 22395476
biological clocks; circadian rhythms; gene regulatory networks; mathematical model; systems biology
18.  Watching the hands of the Arabidopsis biological clock 
Genome Biology  2001;2(3):reviews1008.1-reviews1008.4.
Oligonucleotide and cDNA microarrays have been used to analyse the mRNA levels of 8,000 genes in Arabidopsis thaliana throughout the day/night cycle. Genes involved in signal transduction and in various metabolic pathways were found to be coordinately regulated by circadian rhythms and/or by light.
PMCID: PMC138914  PMID: 11276428
19.  Circadian rhythms persist without transcription in a eukaryote 
Nature  2011;469(7331):554-558.
Circadian rhythms are ubiquitous in eukaryotes, and co-ordinate numerous aspects of behaviour, physiology and metabolism, from sleep/wake cycles in mammals to growth and photosynthesis in plants1,2. This daily timekeeping is thought to be driven by transcriptional/translational feedback loops, whereby rhythmic expression of clock gene products regulates expression of associated genes in approximately 24-hour cycles. The specific transcriptional components differ between phylogenetic kingdoms3. The unicellular pico-eukaryotic alga, Ostreococcus tauri, possesses a naturally minimised clock, which includes many features that are shared with higher eukaryotes (plants), such as a central negative feedback loop that involves the morning-expressed CCA1 and evening-expressed TOC1 genes4. Given that recent observations in animals and plants have revealed prominent post-translational contributions to timekeeping5, 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, though 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 mammals6. Moreover we show that pharmacological modulators of the mammalian clockwork have the same effects on rhythms in Ostreococcus. Post-translational mechanisms, and at least one rhythmic marker, appear to be better conserved than transcriptional clock regulators. It is plausible that the oldest oscillator components are non-transcriptional in nature, as in cyanobacteria7, and are conserved across kingdoms.
doi:10.1038/nature09654
PMCID: PMC3040569  PMID: 21270895
20.  Proteasome Function Is Required for Biological Timing throughout the Twenty-Four Hour Cycle 
Current Biology  2011;21(10):869-875.
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.
Highlights
► CCA1 protein degradation rate is clock regulated ► Sensitivity of the circadian clock to proteasomal inhibition is phase independent ► Nontranscriptional rhythms only rely on the proteasome while coupled to the TTFL
doi:10.1016/j.cub.2011.03.060
PMCID: PMC3102177  PMID: 21530263
21.  Full genome re-sequencing reveals a novel circadian clock mutation in Arabidopsis 
Genome Biology  2011;12(3):R28.
Map based cloning in Arabidopsis thaliana can be a difficult and time-consuming process, specifically if the phenotype is subtle and scoring labour intensive. Here, we have re-sequenced the 120-Mb genome of a novel Arabidopsis clock mutant early bird (ebi-1) in Wassilewskija (Ws-2). We demonstrate the utility of sequencing a backcrossed line in limiting the number of SNPs considered. We identify a SNP in the gene AtNFXL-2 as the likely cause of the ebi-1 phenotype.
doi:10.1186/gb-2011-12-3-r28
PMCID: PMC3129678  PMID: 21429190
22.  Microarray data can predict diurnal changes of starch content in the picoalga Ostreococcus 
BMC Systems Biology  2011;5:36.
Background
The storage of photosynthetic carbohydrate products such as starch is subject to complex regulation, effected at both transcriptional and post-translational levels. The relevant genes in plants show pronounced daily regulation. Their temporal RNA expression profiles, however, do not predict the dynamics of metabolite levels, due to the divergence of enzyme activity from the RNA profiles.
Unicellular phytoplankton retains the complexity of plant carbohydrate metabolism, and recent transcriptomic profiling suggests a major input of transcriptional regulation.
Results
We used a quasi-steady-state, constraint-based modelling approach to infer the dynamics of starch content during the 12 h light/12 h dark cycle in the model alga Ostreococcus tauri. Measured RNA expression datasets from microarray analysis were integrated with a detailed stoichiometric reconstruction of starch metabolism in O. tauri in order to predict the optimal flux distribution and the dynamics of the starch content in the light/dark cycle. The predicted starch profile was validated by experimental data over the 24 h cycle. The main genetic regulatory targets within the pathway were predicted by in silico analysis.
Conclusions
A single-reaction description of starch production is not able to account for the observed variability of diurnal activity profiles of starch-related enzymes. We developed a detailed reaction model of starch metabolism, which, to our knowledge, is the first attempt to describe this polysaccharide polymerization while preserving the mass balance relationships. Our model and method demonstrate the utility of a quasi-steady-state approach for inferring dynamic metabolic information in O. tauri directly from time-series gene expression data.
doi:10.1186/1752-0509-5-36
PMCID: PMC3056741  PMID: 21352558
23.  Ubiquitin ligase switch in plant photomorphogenesis: A hypothesis 
Journal of Theoretical Biology  2011;270(1):31-41.
The E3 ubiquitin ligase COP1 (CONSTITUTIVE PHOTOMORPHOGENIC1) plays a key role in the repression of the plant photomorphogenic development in darkness. In the presence of light, COP1 is inactivated by a mechanism which is not completely understood. This leads to accumulation of COP1’s target transcription factors, which initiates photomorphogenesis, resulting in dramatic changes of the seedling’s physiology.
Here we use a mathematical model to explore the possible mechanism of COP1 modulation upon dark/light transition in Arabidopsis thaliana based upon data for two COP1 target proteins: HY5 and HFR1, which play critical roles in photomorphogenesis. The main reactions in our model are the inactivation of COP1 by a proposed photoreceptor-related inhibitor I and interactions between COP1 and a CUL4 (CULLIN4)-based ligase. For building and verification of the model, we used the available published and our new data on the kinetics of HY5 and HFR1 together with the data on COP1 abundance. HY5 has been shown to accumulate at a slower rate than HFR1. To describe the observed differences in the timecourses of the “slow” target HY5 and the “fast” target HFR1, we hypothesize a switch between the activities of COP1 and CUL4 ligases upon dark/light transition, with COP1 being active mostly in darkness and CUL4 in light. The model predicts a bi-phasic kinetics of COP1 activity upon the exposure of plants to light, with its restoration after the initial decline and the following slow depletion of the total COP1 content. CUL4 activity is predicted to increase in the presence of light. We propose that the ubiquitin ligase switch is important for the complex regulation of multiple transcription factors during plants development. In addition, this provides a new mechanism for sensing the duration of light period, which is important for seasonal changes in plant development.
doi:10.1016/j.jtbi.2010.11.021
PMCID: PMC3021735  PMID: 21093457
Arabidopsis thaliana; Mathematical model; Systems biology; HY5; HFR1
24.  Temporal Repression of Core Circadian Genes Is Mediated through EARLY FLOWERING 3 in Arabidopsis 
Current Biology  2011;21(2):120-125.
Summary
The circadian clock provides robust, ∼24 hr biological rhythms throughout the eukaryotes. The clock gene circuit in plants comprises interlocking transcriptional feedback loops, reviewed in [1], whereby the morning-expressed transcription factors CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) repress the expression of evening genes, notably TIMING OF CAB EXPRESSION 1 (TOC1). EARLY FLOWERING 3 (ELF3) has been implicated as a repressor of light signaling to the clock [2, 3] and, paradoxically, as an activator of the light-induced genes CCA1 and LHY [4, 5]. We use cca1-11 lhy-21 elf3-4 plants to separate the repressive function of ELF3 from its downstream targets CCA1 and LHY. We further demonstrate that ELF3 associates physically with the promoter of PSEUDO-RESPONSE REGULATOR 9 (PRR9), a repressor of CCA1 and LHY expression, in a time-dependent fashion. The repressive function of ELF3 is thus consistent with indirect activation of LHY and CCA1, in a double-negative connection via a direct ELF3 target, PRR9. This mechanism reconciles the functions of ELF3 in the clock network during the night and points to further effects of ELF3 during the day.
Highlights
► ELF3 is a regulator of TOC1, PRR9, GI, and PRR7 gene expression ► Repression by ELF3 is genetically separable from repression by LHY and CCA1 ► ELF3 physically associates with the promoter of PRR9 in a time-dependent manner
doi:10.1016/j.cub.2010.12.013
PMCID: PMC3028277  PMID: 21236675
25.  Consistent Robustness Analysis (CRA) Identifies Biologically Relevant Properties of Regulatory Network Models 
PLoS ONE  2010;5(12):e15589.
Background
A number of studies have previously demonstrated that “goodness of fit” is insufficient in reliably classifying the credibility of a biological model. Robustness and/or sensitivity analysis is commonly employed as a secondary method for evaluating the suitability of a particular model. The results of such analyses invariably depend on the particular parameter set tested, yet many parameter values for biological models are uncertain.
Results
Here, we propose a novel robustness analysis that aims to determine the “common robustness” of the model with multiple, biologically plausible parameter sets, rather than the local robustness for a particular parameter set. Our method is applied to two published models of the Arabidopsis circadian clock (the one-loop [1] and two-loop [2] models). The results reinforce current findings suggesting the greater reliability of the two-loop model and pinpoint the crucial role of TOC1 in the circadian network.
Conclusions
Consistent Robustness Analysis can indicate both the relative plausibility of different models and also the critical components and processes controlling each model.
doi:10.1371/journal.pone.0015589
PMCID: PMC3002950  PMID: 21179566

Results 1-25 (38)