Watching the hands of the Arabidopsis biological clock

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

Modelling the widespread effects of TOC1 signalling on the plant circadian clock and its outputs

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This work was supported by the European Commission FP7 Collaborative Project TiMet (project 245143). SynthSys is a Centre for Integrative and Systems Biology supported by BBSRC and EPSRC award D019621. Work in P.M. laboratory is supported by grants from the Ramón Areces Foundation, from the Spanish Ministry of Science and Innovation (MICINN) (BIO2010-16483) and from EUROHORCS (European Heads Of Research Councils) and the European Science Foundation (ESF) through the EURYI Award.Peer reviewedPublisher PD

Circadian rhythms: PASsing time

AbstractLinks are being discovered between the circadian clock mechanisms in different species. The Neurospora Frequency protein has a rhythm of abundance and phosphorylation similar to that of the Drosophila Period protein, and Neurospora and mouse clock components, like Period, have ‘PAS’ domains

Review: emerging concepts in the pathogenesis of tendinopathy

Tendinopathy is a common clinical problem and has a significant disease burden attached, not only in terms of health care costs, but also for patients directly in terms of time off work and impact upon quality of life. Controversy surrounds the pathogenesis of tendinopathy, however the recent systematic analysis of the evidence has demonstrated that many of the claims of an absence of inflammation in tendinopathy were more based around belief than robust scientific data. This review is a summary of the emerging research in this topical area, with a particular focus on the role of neuronal regulation and inflammation in tendinopathy

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 complexity(1,2), 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 timekeeping(3-6). Results from this model organism have impacted not only plant science, but also mammalian biology(7). 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. Genomic(1), Transcriptomic(8), and Proteomic(9) information for this species is freely available, whereas the previously reported methods(6,10) 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

Robustness from flexibility in the fungal circadian clock

Background Robustness is a central property of living systems, enabling function to be maintained against environmental perturbations. A key challenge is to identify the structures in biological circuits that confer system-level properties such as robustness. Circadian clocks allow organisms to adapt to the predictable changes of the 24-hour day/night cycle by generating endogenous rhythms that can be entrained to the external cycle. In all organisms, the clock circuits typically comprise multiple interlocked feedback loops controlling the rhythmic expression of key genes. Previously, we showed that such architectures increase the flexibility of the clock's rhythmic behaviour. We now test the relationship between flexibility and robustness, using a mathematical model of the circuit controlling conidiation in the fungus Neurospora crassa. Results The circuit modelled in this work consists of a central negative feedback loop, in which the frequency (frq) gene inhibits its transcriptional activator white collar-1 (wc-1), interlocked with a positive feedback loop in which FRQ protein upregulates WC-1 production. Importantly, our model reproduces the observed entrainment of this circuit under light/dark cycles with varying photoperiod and cycle duration. Our simulations show that whilst the level of frq mRNA is driven directly by the light input, the falling phase of FRQ protein, a molecular correlate of conidiation, maintains a constant phase that is uncoupled from the times of dawn and dusk. The model predicts the behaviour of mutants that uncouple WC-1 production from FRQ's positive feedback, and shows that the positive loop enhances the buffering of conidiation phase against seasonal photoperiod changes. This property is quantified using Kitano's measure for the overall robustness of a regulated system output. Further analysis demonstrates that this functional robustness is a consequence of the greater evolutionary flexibility conferred on the circuit by the interlocking loop structure. Conclusions Our model shows that the behaviour of the fungal clock in light-dark cycles can be accounted for by a transcription-translation feedback model of the central FRQ-WC oscillator. More generally, we provide an example of a biological circuit in which greater flexibility yields improved robustness, while also introducing novel sensitivity analysis techniques applicable to a broader range of cellular oscillators

Consistent Robustness Analysis (CRA) Identifies Biologically Relevant Properties of Regulatory Network Models

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.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.Consistent Robustness Analysis can indicate both the relative plausibility of different models and also the critical components and processes controlling each model

Clock proteins: Turned over after hours?

Classical scholars interested by the rhythmic movements of some plants' leaves made the first records of daily biological rhythms. The mechanism underlying their 24-hour timing is now known as the circadian system or circadian clock. Recent studies have uncovered three members of a small protein family that control development and may be novel components of the clock in the model plant Arabidopsis thaliana ([1,2] and M. Wada, personal communication in The circadian clocks of plants and almost all other eukaryotes behave very similarly: all have a rhythmic period close to 24 hours and can be reset by changes in ambient light. Studies of mutant insects, fungi, cyanobacteria and rodents with altered or absent timing have identified several genes involved in negative feedback loops, through which a few proteins rhythmically regulate the transcription of their cognate genes Routes around the clock Clock genetics in higher plants was uneventful until the 1990s. Then, staying up late in the lab, Nagy and Kay in Chua's group serendipitously found that the circadian clock controlled transcription from the chlorophyll a/b-binding protein (CAB) promoter, which was a workhorse of plant molecular biology. Kay proposed to find clock mutant plants, based upon their mis-timed CAB transcription. The other proposed ingredients were a bioluminescent firefly luciferase (luc) reporter gene, which might allow CAB:luc transgenic plants to glow rhythmically, and a photon-counting camera (initially located in Singapore). We identified our first circadian clock mutants in the latter half of 1992, by screening mutagenised CAB:luc plants for mis-timed luminescence Clock mutants of Arabidopsis are now arriving like buses. The circadian clock is a genome-wide regulator that regulates many processes, such as flowering and the growth of the seedling stem or hypocotyl. Genetic screens for mutants that mis-regulate any of these processes may therefore recover clock-associated genes. Targeted screens are not even required, because long-hypocotyl seedlings and plants with altered flowering time -like the fkf mutant [2] -stand out in any genetic screen in Arabidopsis. Biochemical or molecular screening for proteins that regulate gene expression can likewise uncover clock proteins, if the clock controls the gene of interest. The 'circadian clock associated' (CCA1) protein was identified by its binding to the CAB promoter, but itself turns out to be rhythmically expressed and involved in a clock-like negative feedback loop As the Arabidopsis genome sequence rolled out in 1999, the third member of the ZTL family, LKP2, was identified by its homology of part of its protein product to the LOV domain (M. Wada, personal communication in Sequence relationships The LOV sequence near the amino terminus of ZT