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1.  Data assimilation constrains new connections and components in a complex, eukaryotic circadian clock model 
Integrating molecular time-series data resulted in a more robust model of the plant clock, which predicts that a wave of inhibitory PRR proteins controls the morning genes LHY and CCA1.PRR5 is experimentally validated as a late-acting component of this wave.The family of sequentially expressed PRR proteins allows flexible entrainment of the clock, whereas a single protein could not, suggesting that the duplication of clock genes might confer this generic, functional advantage.The observed post-translational regulation of the evening protein TOC1 by interaction with ZTL and GI remains consistent with an indirect activation of TOC1 mRNA expression by GI, which was previously postulated from modelling.
Circadian rhythms are present in most eukaryotic organisms including plants. The core genes of the circadian clock are very important for plant physiology as they drive the rhythmic expression of around 30% of Arabidopsis genes (Edwards et al, 2006; Michael et al, 2008). The clock is normally entrained by daily environmental changes in light and temperature. Oscillations also persist under constant environmental conditions in a laboratory. The clock gene circuit in Arabidopsis is based on multiple interlocked feedback loops, which are typical of circadian genetic networks in other organisms (Dunlap and Loros, 2004; Bell-Pedersen et al, 2005). Mechanistic, mathematical models are increasingly useful in analysing and understanding how the observed molecular components give rise to the rhythmic behaviour of this dynamic, non-linear system.
Our previous model of Arabidopsis circadian clock (Locke et al, 2006) presented the core, three-loop structure of the clock, which comprised morning and evening oscillators and coupling between them (Figure 1). The morning loop included the dawn-expressed LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) genes, which negatively regulate their expression through activation of the inhibitor proteins, PSEUDO-RESPONSE REGULATOR 9 (PRR9) and PRR7. These were described by a single, combined model component, PRR9/7. The evening loop included the dusk-expressed gene TIMING OF CAB EXPRESSION 1 (TOC1), which negatively regulates itself through inhibition of a hypothetical activator, gene Y. The evening-expressed gene GIGANTEA (GI) contributes to Y function. The morning and evening loops were connected through inhibition of the evening genes by LHY/CCA1 and activation of LHY/CCA1 expression by a hypothetical evening gene X. Here, we extend the previous model of circadian gene expression (Locke et al, 2006) based on recently published data (Figure 1). The new model retains the good match of our previous model to the large volume of molecular time-series data, and improves the behaviour of the model clock system under a range of light conditions and in a wider range of mutants.
The morning loop was extended by adding a hypothetical clock component, the night inhibitor (NI), which acts together with PRR9 and PRR7 to keep the expression of LHY and CCA1 at low levels over a broad interval spanning dusk. This regulation is important to set the phase of LHY/CCA1 expression at dawn. Data from the literature suggested that the PRR5 gene was a candidate for NI, leading us to predict that the sequentially expressed PRR9, PRR7 and PRR5 proteins together formed a wave of inhibitors of LHY and CCA1. This hypothesis was tested under discriminating light conditions, in which the light interval is replaced with the dawn and dusk pulses of light to form a ‘skeleton photoperiod'. Combining this protocol with mutation of the PRR7 and/or PRR5 genes, our new experimental results validated the model predictions and confirmed that PRR5 contributes to the function that we modelled as NI. During revision of this paper, that result received further experimental support (Nakamichi et al, 2010).
Model simulations revealed the functional importance of the inhibitor wave in entraining the clock to the light/dark cycle. Separating PRR9 from the other inhibitors in the model showed how the strong light activation observed for this gene contributes to more rapid entrainment. The observed, post-translation regulation of all three inhibitor proteins by light (Farre and Kay, 2007; Ito et al, 2007; Kiba et al, 2007) was also included in the model. Light-regulated degradation provides a molecular mechanism to explain the later phase of LHY and CCA1 expression under long photoperiods compared with short photoperiods, in line with experimental observations.
The connection between evening and morning loops was revised by including the inhibition of the morning gene PRR9 by the evening component TOC1, based on the data on TOC1-overexpressing plants (Makino et al, 2002; Ito et al, 2005). This inhibition causes a delay of PRR9 expression relative to LHY/CCA1, which allows LHY/CCA1 to reach a higher expression level at dawn. Our simulations showed that a partial mutant that lacks this inhibition of PRR9 by TOC1 is sufficient to cause the higher level of PRR9 and the short circadian period observed in toc1 mutant plants.
The evening loop was extended by introducing the observed, post-translational regulation of the TOC1 protein by the F-box protein ZEITLUPE (ZTL) and stabilization of ZTL by its interaction with GI in the presence of light (Kim et al, 2007). GI's function in the clock model has thus been revised according to the data: GI promotes an inhibition of TOC1 protein function through positive regulation of ZTL. This results, together with negative regulation of Y by TOC1, in indirect activation of TOC1 mRNA expression by GI, which agrees with our earlier experimental data (Locke et al, 2006). Simulations showed that the post-translational regulation of TOC1 by ZTL and GI results in the observed long period of the ztl mutant and fast dampening of rhythms in the lhy/cca1/gi triple mutant.
This is the first mathematical model that incorporates the observed post-translational regulation into the genetic network of the Arabidopsis clock. In addition to specific, mechanistic insights, the model shows a generic advantage from the duplication of clock genes and their expression at different phases. Such clock gene duplications are observed in eukaryotes with larger genomes, such as the mouse. Analogous, functional duplication can be achieved by differential regulation of a single clock gene in distinct cells, as in Drosophila.
Circadian clocks generate 24-h rhythms that are entrained by the day/night cycle. Clock circuits include several light inputs and interlocked feedback loops, with complex dynamics. Multiple biological components can contribute to each part of the circuit in higher organisms. Mechanistic models with morning, evening and central feedback loops have provided a heuristic framework for the clock in plants, but were based on transcriptional control. Here, we model observed, post-transcriptional and post-translational regulation and constrain many parameter values based on experimental data. The model's feedback circuit is revised and now includes PSEUDO-RESPONSE REGULATOR 7 (PRR7) and ZEITLUPE. The revised model matches data in varying environments and mutants, and gains robustness to parameter variation. Our results suggest that the activation of important morning-expressed genes follows their release from a night inhibitor (NI). Experiments inspired by the new model support the predicted NI function and show that the PRR5 gene contributes to the NI. The multiple PRR genes of Arabidopsis uncouple events in the late night from light-driven responses in the day, increasing the flexibility of rhythmic regulation.
PMCID: PMC2964123  PMID: 20865009
Arabidopsis thaliana; biological clocks; circadian rhythms; mathematical model; systems biology
2.  Quantitative analysis of regulatory flexibility under changing environmental conditions 
Day length changes with the seasons in temperate latitudes, affecting the many biological rhythms that entrain to the day/night cycle: we measure these effects on the expression of Arabidopsis clock genes, using RNA and reporter gene readouts, with a new method of phase analysis.Dusk sensitivity is proposed as a simple, natural and general mathematical measure to analyse and manipulate the changing phase of a clock output relative to the change in the day/night cycle.Dusk sensitivity shows how increasing the numbers of feedback loops in the Arabidopsis clock models allows more flexible regulation, consistent with a previously-proposed, general operating principle of biological networks.The Arabidopsis clock genes show flexibility of regulation that is characteristic of a three-loop clock model, validating aspects of the model and the operating principle, but some clock output genes show greater flexibility arising from direct light regulation.
The analysis of dynamic, non-linear regulation with the aid of mechanistic models is central to Systems Biology. This study compares the predictions of mechanistic, mathematical models of the circadian clock with molecular time-series data on rhythmic gene expression in the higher plant Arabidopsis thaliana. Analysis of the models helps us to understand (explain and predict) how the clock gene circuit balances regulation by external and endogenous factors to achieve particular behaviours. Such multi-factorial regulation is ubiquitous in, and characteristic of, living systems.
The Earth's rotation causes predictable changes in the environment, notably in the availability of sunlight for photosynthesis. Many biological processes are driven by the environmental input via sensory pathways, for example, from photoreceptors. Circadian clocks provide an alternative strategy. These endogenous, 24-h rhythms can drive biological processes that anticipate the regular environmental changes, rather than merely responding. Many rhythmic processes have both light and clock control. Indeed, the clock components themselves must balance internal timing with external inputs, because circadian clocks are reset daily through light regulation of one or more clock components. This process of entrainment is complicated by the change in day length. When the times of dawn and dusk move apart in summer, and closer together in winter, does the clock track dawn, track dusk or interpolate between them?
In plants, the clock controls leaf and petal movements, the opening and closing of stomatal pores, the discharge of floral fragrances, and many metabolic activities, especially those associated with photosynthesis. Centuries of physiological studies have shown that these rhythms can behave differently. Flowering in Ipomoea nil (Pharbitis nil, Japanese morning glory) is controlled by a rhythm that tracks the time of dusk, to give a classic example. We showed that two other rhythms associated with vegetative growth track dawn in this species (Figure 5A), so the clock system allows flexible regulation.
The relatively small number of components involved in the circadian clockwork makes it an ideal candidate for mathematical modelling. Molecular genetic studies in a variety of model eukaryotes have shown that the circadian rhythm is generated by a network of 6–20 genes. These genes form feedback loops generating a rhythm in mRNA production. A single negative feedback loop in which a gene encodes a protein that, after several hours, turns off transcription is capable of generating a circadian rhythm, in principle. A single light input can entrain the clock to ‘local time', synchronised with a light–dark cycle. However, real circadian clocks have proven to be more complicated than this, with multiple light inputs and interlocked feedback loops.
We have previously argued from mathematical analysis that multi-loop networks increase the flexibility of regulation (Rand et al, 2004) and have shown that appropriately deployed flexibility can confer functional robustness (Akman et al, 2010). Here we test whether that flexibility can be demonstrated in vivo, in the model plant, A. thaliana. The Arabidopsis clock mechanism comprises a feedback loop in which two partially redundant, myb transcription factors, LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), repress the expression of their activator, TIMING OF CAB EXPRESSION 1 (TOC1). We previously modelled this single-loop circuit and showed that it was not capable of recreating important data (Locke et al, 2005a). An extended, two-loop model was developed to match observed behaviours, incorporating a hypothetical gene Y, for which the best identified candidate was the GIGANTEA gene (GI) (Locke et al, 2005b). Two further models incorporated the TOC1 homologues PSEUDO-RESPONSE REGULATOR (PRR) 9 and PRR7 (Locke et al, 2006; Zeilinger et al, 2006). In these circuits, a morning oscillator (LHY/CCA1–PRR9/7) is coupled to an evening oscillator (Y/GI–TOC1) via the original LHY/CCA1–TOC1 loop.
These clock models, like those for all other organisms, were developed using data from simple conditions of constant light, darkness or 12-h light–12-h dark cycles. We therefore tested how the clock genes in Arabidopsis responded to light–dark cycles with different photoperiods, from 3 h light to 18 h light per 24-h cycle (Edinburgh, 56° North latitude, has 17.5 h light in midsummer). The time-series assays of mRNA and in vivo reporter gene images showed a range of peak times for different genes, depending on the photoperiod (Figure 5C). A new data analysis method, mFourfit, was introduced to measure the peak times, in the Biological Rhythms Analysis Software Suite (BRASS v3.0). None of the genes showed the dusk-tracking behaviour characteristic of the Ipomoea flowering rhythm. The one-, two- and three-loop models were analysed to understand the observed patterns. A new mathematical measure, dusk sensitivity, was introduced to measure the change in timing of a model component versus a change in the time of dusk. The one- and two-loop models tracked dawn and dusk, respectively, under all conditions. Only the three-loop model (Figure 5B) had the flexibility required to match the photoperiod-dependent changes that we found in vivo, and in particular the unexpected, V-shaped pattern in the peak time of TOC1 expression. This pattern of regulation depends on the structure and light inputs to the model's evening oscillator, so the in vivo data supported this aspect of the model. LHY and CCA1 gene expression under short photoperiods showed greater dusk sensitivity, in the interval 2–6 h before dawn, than the three-loop model predicted, so these data will help to constrain future models.
The approach described here could act as a template for experimental biologists seeking to understand biological regulation using dynamic, experimental perturbations and time-series data. Simulation of mathematical models (despite known imperfections) can provide contrasting hypotheses that guide understanding. The system's detailed behaviour is complex, so a natural and general measure such as dusk sensitivity is helpful to focus on one property of the system. We used the measure to compare models, and to predict how this property could be manipulated. To enable additional analysis of this system, we provide the time-series data and experimental metadata online.
The circadian clock controls 24-h rhythms in many biological processes, allowing appropriate timing of biological rhythms relative to dawn and dusk. Known clock circuits include multiple, interlocked feedback loops. Theory suggested that multiple loops contribute the flexibility for molecular rhythms to track multiple phases of the external cycle. Clear dawn- and dusk-tracking rhythms illustrate the flexibility of timing in Ipomoea nil. Molecular clock components in Arabidopsis thaliana showed complex, photoperiod-dependent regulation, which was analysed by comparison with three contrasting models. A simple, quantitative measure, Dusk Sensitivity, was introduced to compare the behaviour of clock models with varying loop complexity. Evening-expressed clock genes showed photoperiod-dependent dusk sensitivity, as predicted by the three-loop model, whereas the one- and two-loop models tracked dawn and dusk, respectively. Output genes for starch degradation achieved dusk-tracking expression through light regulation, rather than a dusk-tracking rhythm. Model analysis predicted which biochemical processes could be manipulated to extend dusk tracking. Our results reveal how an operating principle of biological regulators applies specifically to the plant circadian clock.
PMCID: PMC3010117  PMID: 21045818
Arabidopsis thaliana; biological clocks; dynamical systems; gene regulatory networks; mathematical models; photoperiodism
3.  A novel computational model of the circadian clock in Arabidopsis that incorporates PRR7 and PRR9 
We developed a mathematical model of the Arabidopsis circadian clock, including PRR7 and PRR9, which is able to predict several single, double and triple mutant phenotypes.Sensitivity Analysis was used to identify the properties and time sensing mechanisms of model structures.PRR7 and CCA1/LHY were identified as weak points of the mathematical model indicating where more experimental data is needed for further model development.Detailed dynamical studies showed that the timing of an evening light sensing element is essential for day length responsiveness
In recent years, molecular genetic techniques have revealed a complex network of components in the Arabidopsis circadian clock. Mathematical models allow for a detailed study of the dynamics and architecture of such complex gene networks leading to a better understanding of the genetic interactions. It is important to maintain a constant iteration with experimentation, to include novel components as they are discovered and use the updated model to design new experiments. This study develops a framework to introduce new components into the mathematical model of the Arabidopsis circadian clock accelerating the iterative model development process and gaining insight into the system's properties.
We used the interlocked feedback loop model published in Locke et al (2005) as the base model. In Arabidopsis, the first suggested regulatory loop involves the morning expressed transcription factors CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), and the evening expressed pseudo-response regulator TIMING OF CAB EXPRESSION (TOC1). The hypothetical component X had been introduced to realize a longer delay between gene expression of CCA1/LHY and TOC1. The introduction of Y was motivated by the need for a mechanism to reproduce the dampening short period rhythms of the cca1/lhy double mutant and to include an additional light input at the end of the day.
In this study, the new components pseudo-response regulators PRR7 and PRR9 were added in negative feedback loops based on the biological hypothesis that they are activated by LHY and in turn repress LHY transcription (Farré et al, 2005; Figure 1). We present three iterations steps of model development (Figure 1A–C).
A wide range of tools was used to establish and analyze new model structures. One of the challenges facing mathematical modeling of biological processes is parameter identification; they are notoriously difficult to determine experimentally. We established an optimization procedure based on an evolutionary strategy with a cost function mainly derived from wild-type characteristics. This ensured that the model was not restricted by a specific set of parameters and enabled us to use a large set of biological mutant information to assess the predictive capability of the model structure. Models were evaluated by means of an extended phenotype catalogue, allowing for an easy and fair comparison of the structures. We also carried out detailed simulation analysis of component interactions to identify weak points in the structure and suggest further modifications. Finally, we applied sensitivity analysis in a novel manner, using it to direct the model development. Sensitivity analysis provides quantitative measures of robustness; the two measures in this study were the traces of component concentrations over time (classical state sensitivities) and phase behavior (measured by the phase response curve). Three major results emerged from the model development process.
First, the iteration process helped us to learn about general characteristics of the system. We observed that the timing of Y expression is critical for evening light entrainment, which enables the system to respond to changes in day length. This is important for our understanding of the mechanism of light input to the clock and will add in the identification of biological candidates for this function. In addition, our results suggest that a detailed description of the mechanisms of genetic interactions is important for the systems behavior. We observed that the introduction of an experimentally based precise light regulation mechanism on PRR9 expression had a significant effect on the systems behavior.
Second, the final model structure (Figure 1C) was capable of predicting a wide range of mutant phenotypes, such as a reduction of TOC1 expression by RNAi (toc1RNAi), mutations in PRR7 and PRR9 and the novel mutant combinations prr9toc1RNAi and prr7prr9toc1RNAi. However, it was unable to predict the mutations in CCA1 and LHY.
Finally, sensitivity analysis identified the weak points of the system. The developed model structure was heavily based on the TOC1/Y feedback loop. This could explain the model's failure to predict the cca1lhy double mutant phenotype. More detailed information on the regulation of CCA1 and LHY expression will be important to achieve the right balance between the different regulatory loops in the mathematical model. This is in accordance with genetic studies that have identified several genes involved in the regulation of LHY and CCA1 expression. The identification of their mechanism of action will be necessary for the next model development.
In plants, as in animals, the core mechanism to retain rhythmic gene expression relies on the interaction of multiple feedback loops. In recent years, molecular genetic techniques have revealed a complex network of clock components in Arabidopsis. To gain insight into the dynamics of these interactions, new components need to be integrated into the mathematical model of the plant clock. Our approach accelerates the iterative process of model identification, to incorporate new components, and to systematically test different proposed structural hypotheses. Recent studies indicate that the pseudo-response regulators PRR7 and PRR9 play a key role in the core clock of Arabidopsis. We incorporate PRR7 and PRR9 into an existing model involving the transcription factors TIMING OF CAB (TOC1), LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED (CCA1). We propose candidate models based on experimental hypotheses and identify the computational models with the application of an optimization routine. Validation is accomplished through systematic analysis of various mutant phenotypes. We introduce and apply sensitivity analysis as a novel tool for analyzing and distinguishing the characteristics of proposed architectures, which also allows for further validation of the hypothesized structures.
PMCID: PMC1682023  PMID: 17102803
Arabidopsis; circadian rhythms; mathematical modeling; parameter optimization; sensitivity analysis
4.  LATE ELONGATED HYPOCOTYL regulates photoperiodic flowering via the circadian clock in Arabidopsis 
BMC Plant Biology  2016;16:114.
Plants constantly monitor changes in photoperiod or day length to trigger the flowering cycle at the most appropriate time of the year. It is well established that photoperiodic flowering is intimately associated with the circadian clock in Arabidopsis. In support of this notion, many clock-defective mutants exhibit altered photoperiodic sensitivity in inducing flowering. LATE ELONGATED HYPOCOTYL (LHY) and its functional paralogue CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) constitute the core of the circadian clock together with TIMING OF CAB EXPRSSION 1 (TOC1). While it is known that TOC1 contributes to the timing of flowering entirely by modulating the clock function, molecular mechanisms by which LHY and CCA1 regulate flowering time have not been explored.
We investigated how LHY and CCA1 regulate photoperiodic flowering through molecular genetic and biochemical studies. It was found that LHY-defective mutants (lhy-7 and lhy-20) exhibit accelerated flowering under both long days (LDs) and short days (SDs). Consistent with the accelerated flowering phenotypes, gene expression analysis revealed that expression of the floral integrator FLOWERING LOCUS T (FT) is up-regulated in the lhy mutants. In addition, the expression peaks of GIGANTEA (GI) and FLAVIN-BINDING, KELCH REPEAT, F-BOX PROTEIN 1 (FKF1) genes, which constitute the clock output pathway that is linked with photoperiodic flowering, were advanced by approximately 4 h in the mutants. Furthermore, the up-regulation of FT disappeared when the endogenous circadian period is matched to the external light/dark cycles in the lhy-7 mutant. Notably, whereas CCA1 binds strongly to FT gene promoter, LHY does not show such DNA-binding activity.
Our data indicate that the advanced expression phases of photoperiodic flowering genes are associated with the clock defects in the lhy mutants and responsible for the reduced photoperiodic sensitivity of the mutant flowering, demonstrating that LHY regulates photoperiodic flowering via the circadian clock, similar to what has been shown with TOC1. It is notable that while LHY regulates photoperiodic flowering in a similar manner as with TOC1, the underlying molecular mechanism would be somewhat distinct from that exerted by CCA1 in Arabidopsis.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-016-0810-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4875590  PMID: 27207270
Arabidopsis thaliana; Circadian clock; Flowering time; LHY; Photoperiod
5.  A Genetic Screen for Leaf Movement Mutants Identifies a Potential Role for AGAMOUS-LIKE 6 (AGL6) in Circadian-Clock Control 
Molecules and Cells  2011;31(3):281-287.
The circadian clock in plants regulates many important physiological and biological processes, including leaf movement. We have used an imaging system to genetically screen Arabidopsis seedlings for altered leaf movement with the aim of identifying a circadian clock gene. A total of 285 genes were selected from publicly available microarrays that showed an expression pattern similar to those of the Arabidopsis core oscillator genes. We subsequently isolated 42 homozygous recessive mutants and analyzed their leaf movements. We also analyzed leaf movements of activation tagging mutants that showed altered flowering time. We found that agl6-1D plants, in which AGAMOUS-LIKE 6 (AGL6) was activated by the 35S enhancer, showed a shortened period of leaf movement as well as a high level of ZEITLUPE (ZTL) expression, reduced amplitude of LATE ELONGATED HYPOCOTYL (LHY) expression, and arrhythmic TIMING OF CAB EXPRESSION1 (TOC1)/CIRCADIAN CLOCK ASSOCIATED1 (CCA1) expression. A shortened period of leaf movement was also seen in 35S-AGL6-myc plants, although 35S-amiRAGL6 plants, transgenic plants overexpressing an artificial miRNA (amiR) targeting AGL6, showed unaltered leaf movement. The amplitude of CHLOROPHYLL A/B BINDING PROTEIN 2 (CAB2) expression, a circadian output gene, was also reduced in agl6-1D plants. Taken together, these results suggest that AGL6 plays a potential role in the regulation of the circadian clock by regulating ZTL mRNA level in Arabidopsis.
PMCID: PMC3932699  PMID: 21331777
AGL6; CCA1; circadian clock; leaf movement; ZTL
6.  Extension of a genetic network model by iterative experimentation and mathematical analysis 
Molecular Systems Biology  2005;1:2005.0013.
We extend the current model of the plant circadian clock, in order to accommodate new and published data. Throughout our model development we use a global parameter search to ensure that any limitations we find are due to the network architecture and not to our selection of the parameter values, which have not been determined experimentally. Our final model includes two, interlocked loops of gene regulation and is reminiscent of the circuit structures previously identified by experiments on insect and fungal clocks. It is the first Arabidopsis clock model to show such good correspondence to experimental data.Our interlocked feedback loop model predicts the regulation of two unknown components. Experiments motivated by these predictions identify the GIGANTEA gene as a strong candidate for one component, with an unexpected pattern of light regulation.*
This study involves an iterative approach of mathematical modelling and experiment to develop an accurate mathematical model of the circadian clock in the higher plant Arabidopsis thaliana. Our approach is central to systems biology and should lead to a greater, quantitative understanding of the circadian clock, as well as being more widely relevant to research into genetic networks.
The day–night cycle caused by the Earth's rotation affects most organisms, and has resulted in the evolution of the circadian clock. The circadian clock controls 24-h rhythms in processes from metabolism to behaviour; in higher eukaryotes, the circadian clock controls the rhythmic expression of 5–10% of genes. In plants, the clock controls leaf and petal movements, the opening and closing of stomatal pores, the discharge of floral fragrances and many metabolic activities, especially those associated with photosynthesis.
The relatively small number of components involved in the central circadian network makes it an ideal candidate for mathematical modelling of complex biological regulation. Genetic studies in a variety of model organisms have shown that the circadian rhythm is generated by a central network of between 6 and 12 genes. These genes form feedback loops generating a rhythm in mRNA production. One negative feedback loop in which a gene encodes a protein that, after several hours, turns off transcription is, in principle, capable of creating a circadian rhythm. However, real circadian clocks have proven to be more complicated than this, with interlocked feedback loops. Networks of this complexity are more easily understood through mathematical modelling.
The clock mechanism in the model plant, A. thaliana, was first proposed to comprise a feedback loop in which two partially redundant genes, LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), repress the expression of their activator, TIMING OF CAB EXPRESSION 1 (TOC1). We previously modelled this preliminary network and showed that it was not capable of recreating several important pieces of experimental data (Locke et al, 2005). Here, we extend the LHY/CCA1–TOC1 network in new mathematical models. To check the effects of each addition to the network, the outputs of the extended models are compared to published data and to new experiments.
As is the case for most biological networks, the parameter values in our model, such as the translation rate of TOC1 protein, are unknown. We employ here an optimisation method, which works well with noisy and varied data and allows a global search of parameter space. This should ensure that the limitations we find in our networks are due to the network structure, and not to our parameter choices.
Our final interlocked feedback loop model requires two hypothetical components, genes X and Y (Figure 4), but is the first Arabidopsis clock model to exhibit such a good correspondence with experimental data. The model simulates a residual short-period oscillation in the cca1;lhy mutant, as characterised by our experiments. No single-loop model is able to do this. Our model also matches experimental data under constant light (LL) conditions and correctly senses photoperiod. The model predicts an interlocked feedback loop structure similar to that seen in the circadian clock mechanisms of other organisms.
The interlocked feedback loop model predicts a distinctive pattern of Y mRNA accumulation in the wild type (WT) and in the cca1;lhy double mutant, with Y mRNA levels increasing transiently at dawn. We designed an experiment to identify Y based on this prediction. GIGANTEA (GI) mRNA levels fit very well to our predicted profile for Y (Figure 6), identifying GI as a strong candidate for Y.
The approach described here could act as a template for experimental biologists seeking to extend models of small genetic networks. Our results illustrate the usefulness of mathematical modelling in guiding experiments, even if the models are based on limited data. Our method provides a way of identifying suitable candidate networks and quantifying how these networks better describe a wide variety of experimental measurements. The characteristics of new putative genes are thereby obtained, facilitating the experimental search for new components. To facilitate future experimental design, we provide user-friendly software that is specifically designed for numerical simulation of circadian experiments using models for several species (Brown, 2004b).
*Footnote: Synopsis highlights were added on 5 July 2005.
Circadian clocks involve feedback loops that generate rhythmic expression of key genes. Molecular genetic studies in the higher plant Arabidopsis thaliana have revealed a complex clock network. The first part of the network to be identified, a transcriptional feedback loop comprising TIMING OF CAB EXPRESSION 1 (TOC1), LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), fails to account for significant experimental data. We develop an extended model that is based upon a wider range of data and accurately predicts additional experimental results. The model comprises interlocking feedback loops comparable to those identified experimentally in other circadian systems. We propose that each loop receives input signals from light, and that each loop includes a hypothetical component that had not been explicitly identified. Analysis of the model predicted the properties of these components, including an acute light induction at dawn that is rapidly repressed by LHY and CCA1. We found this unexpected regulation in RNA levels of the evening-expressed gene GIGANTEA (GI), supporting our proposed network and making GI a strong candidate for this component.
PMCID: PMC1681447  PMID: 16729048
biological rhythms; gene network; mathematical modelling; parameter estimation
7.  Modelling the widespread effects of TOC1 signalling on the plant circadian clock and its outputs 
BMC Systems Biology  2013;7:23.
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.
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.
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.
PMCID: PMC3614443  PMID: 23506153
Circadian rhythms; Biological clocks; Gene regulatory networks; Mathematical model; Systems biology
8.  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.
PMCID: PMC3321525  PMID: 22395476
biological clocks; circadian rhythms; gene regulatory networks; mathematical model; systems biology
9.  Conserved Function of Core Clock Proteins in the Gymnosperm Norway Spruce (Picea abies L. Karst) 
PLoS ONE  2013;8(3):e60110.
From studies of the circadian clock in the plant model species Arabidopsis (Arabidopsis thaliana), a number of important properties and components have emerged. These include the genes CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), GIGANTEA (GI), ZEITLUPE (ZTL) and TIMING OF CAB EXPRESSION 1 (TOC1 also known as PSEUDO-RESPONSE REGULATOR 1 (PRR1)) that via gene expression feedback loops participate in the circadian clock. Here, we present results from ectopic expression of four Norway spruce (Picea abies) putative homologs (PaCCA1, PaGI, PaZTL and PaPRR1) in Arabidopsis, their flowering time, circadian period length, red light response phenotypes and their effect on endogenous clock genes were assessed. For PaCCA1-ox and PaZTL-ox the results were consistent with Arabidopsis lines overexpressing the corresponding Arabidopsis genes. For PaGI consistent results were obtained when expressed in the gi2 mutant, while PaGI and PaPRR1 expressed in wild type did not display the expected phenotypes. These results suggest that protein function of PaCCA1, PaGI and PaZTL are at least partly conserved compared to Arabidopsis homologs, however further studies are needed to reveal the protein function of PaPRR1. Our data suggest that components of the three-loop network typical of the circadian clock in angiosperms were present before the split of gymnosperms and angiosperms.
PMCID: PMC3610754  PMID: 23555899
10.  Altered circadian rhythms regulate growth vigor in hybrids and allopolyploids 
Nature  2008;457(7227):327-331.
Segregating hybrids and stable allopolyploids display morphological vigor1,2,3, and Arabidopsis allotetraploids are larger than the parents Arabidopsis thaliana and A. arenosa1,4. The mechanisms are unknown. Circadian clocks mediate metabolic pathways and increase fitness in animals and plants5,6,7,8. Here we report that epigenetic modifications of the circadian clock genes CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY)9,10 and their reciprocal regulators TIMING OF CAB EXPRESSION 1 (TOC1) and GIGANTEA (GI)10,11,12 mediate expression changes in downstream genes and pathways. During the day, epigenetic repression of CCA1 and LHY induced expression of TOC1, GI and downstream genes that contain CCA1 binding site (CBS)13 in chlorophyll and starch metabolic pathways in allotetraploids and F1 hybrids, which produced more chlorophyll and starch than the parents in the same environment. Mutations in cca1 and cca1 lhy and daily repression of cca1 in TOC1:cca1-RNAi transgenic plants increased expression of downstream genes and chlorophyll and starch content, whereas constitutively expressing CCA1 or ectopically expressing TOC1:CCA1 had the opposite effects. The causal effects of CCA1 on output traits suggest that hybrids and allopolyploids gain advantages from the control of circadian-mediated physiological and metabolic pathways, leading to growth vigor and increased biomass.
PMCID: PMC2679702  PMID: 19029881
circadian clock; polyploidy; hybrid vigor; epigenetics; gene expression; biomass
11.  Temporal Repression of Core Circadian Genes Is Mediated through EARLY FLOWERING 3 in Arabidopsis 
Current Biology  2011;21(2):120-125.
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.
► 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
PMCID: PMC3028277  PMID: 21236675
12.  Circadian Rhythms in Floral Scent Emission 
To successfully recruit pollinators, plants often release attractive floral scents at specific times of day to coincide with pollinator foraging. This timing of scent emission is thought to be evolutionarily beneficial to maximize resource efficiency while attracting only useful pollinators. Temporal regulation of scent emission is tied to the activity of the specific metabolic pathways responsible for scent production. Although floral volatile profiling in various plants indicated a contribution by the circadian clock, the mechanisms by which the circadian clock regulates timing of floral scent emission remained elusive. Recent studies using two species in the Solanaceae family provided initial insight into molecular clock regulation of scent emission timing. In Petunia hybrida, the floral volatile benzenoid/phenylpropanoid (FVBP) pathway is the major metabolic pathway that produces floral volatiles. Three MYB-type transcription factors, ODORANT 1 (ODO1), EMISSION OF BENZENOIDS I (EOBI), and EOBII, all of which show diurnal rhythms in mRNA expression, act as positive regulators for several enzyme genes in the FVBP pathway. Recently, in P. hybrida and Nicotiana attenuata, homologs of the Arabidopsis clock gene LATE ELONGATED HYPOCOTYL (LHY) have been shown to have a similar role in the circadian clock in these plants, and to also determine the timing of scent emission. In addition, in P. hybrida, PhLHY directly represses ODO1 and several enzyme genes in the FVBP pathway during the morning as an important negative regulator of scent emission. These findings facilitate our understanding of the relationship between a molecular timekeeper and the timing of scent emission, which may influence reproductive success.
PMCID: PMC4829607  PMID: 27148293
floral volatile; circadian clock; pollinator; metabolic pathway; petunia; tobacco; Solanaceae
13.  Circadian Clock Regulates Dynamic Chromatin Modifications Associated with Arabidopsis CCA1/LHY and TOC1 Transcriptional Rhythms 
Plant and Cell Physiology  2012;53(12):2016-2029.
Circadian clocks enable organisms to adapt to a 24 h diurnal cycle and anticipate rhythmic changes in the environment. The Arabidopsis central oscillator contains three genes encoding core clock components. CIRCADIAN CLOCK ASSOCIATED 1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY) and TIMING OF CAB EXPRESSION 1 (TOC1) reciprocally repress genes encoding each other and are critical for the generation of circadian rhythms controlling many clock outputs. A precise regulation of transcriptional events is, therefore, essential for proper circadian function. Here, we investigated histone 3 (H3) tail modifications of CCA1, LHY and TOC1 under various conditions. We found specific association of only H3K4Me3 and H3K9/14Ac with the translational start site of these three genes. These H3 marks were enriched at circadian time points of their increased transcription at different photoperiods and under free-running conditions, suggesting circadian regulation of H3 modifications. Analysis of clock-compromised CCA1-overexpressing lines provided evidence that light/dark photoperiods signal the establishment of these chromatin changes which are gated by the clock.
PMCID: PMC3516852  PMID: 23128602
Arabidopsis thaliana; CCA1; Chromatin modifications; Circadian clock; LHY; TOC1
14.  CCA1 alternative splicing as a way of linking the circadian clock to temperature response in Arabidopsis 
Plant Signaling & Behavior  2012;7(9):1194-1196.
Most living organisms on the earth have the circadian clock to synchronize their biochemical processes and physiological activities with environmental changes to optimize their propagation and survival. CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) is one of the core clock components in Arabidopsis. Notably, it is also associated with cold acclimation. However, it is largely unknown how CCA1 activity is modulated by low temperatures. We found that the CCA1 activity is self-regulated by a splice variant CCA1β and the CCA1β production is modulated by low temperatures, linking the circadian clock with cold acclimation. CCA1β competitively inhibits the activities of functional CCA1α and LATE ELONGATED HYPOCOTYL (LHY) transcription factors by forming nonfunctional CCA1α-CCA1β and LHY-CCA1β heterodimers. Consequently, CCA1β-overexpressing plants (35S:CCA1β) exhibit shortened circadian periods as observed in cca1 lhy double mutants. In addition, elongated hypocotyls and petioles and delayed flowering of CCA1α-overexpressing plants (35S:CCA1α) were rescued by coexpression of CCA1β. Interestingly, low temperatures suppress CCA1 alternative splicing and thus derepress the CCA1α activity in inducing cold tolerance. These observations indicate that a cold-responsive self-regulatory circuit of CCA1 plays a role in plant responses to low temperatures.
PMCID: PMC3489659  PMID: 22899064
alternative splicing; Arabidopsis; CCA1; circadian clock; cold acclimation; freezing tolerance
15.  Alternative splicing and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis 
BMC Plant Biology  2014;14:136.
The circadian clock enables living organisms to anticipate recurring daily and seasonal fluctuations in their growth habitats and synchronize their biology to the environmental cycle. The plant circadian clock consists of multiple transcription-translation feedback loops that are entrained by environmental signals, such as light and temperature. In recent years, alternative splicing emerges as an important molecular mechanism that modulates the clock function in plants. Several clock genes are known to undergo alternative splicing in response to changes in environmental conditions, suggesting that the clock function is intimately associated with environmental responses via the alternative splicing of the clock genes. However, the alternative splicing events of the clock genes have not been studied at the molecular level.
We systematically examined whether major clock genes undergo alternative splicing under various environmental conditions in Arabidopsis. We also investigated the fates of the RNA splice variants of the clock genes. It was found that the clock genes, including EARLY FLOWERING 3 (ELF3) and ZEITLUPE (ZTL) that have not been studied in terms of alternative splicing, undergo extensive alternative splicing through diverse modes of splicing events, such as intron retention, exon skipping, and selection of alternative 5′ splice site. Their alternative splicing patterns were differentially influenced by changes in photoperiod, temperature extremes, and salt stress. Notably, the RNA splice variants of TIMING OF CAB EXPRESSION 1 (TOC1) and ELF3 were degraded through the nonsense-mediated decay (NMD) pathway, whereas those of other clock genes were insensitive to NMD.
Taken together, our observations demonstrate that the major clock genes examined undergo extensive alternative splicing under various environmental conditions, suggesting that alternative splicing is a molecular scheme that underlies the linkage between the clock and environmental stress adaptation in plants. It is also envisioned that alternative splicing of the clock genes plays more complex roles than previously expected.
PMCID: PMC4035800  PMID: 24885185
Arabidopsis thaliana; Circadian clock; Transcription factor; Alternative splicing; Nonsense-mediated decay (NMD); Environmental stress
16.  CUL1 regulates TOC1 protein stability in the Arabidopsis circadian clock 
The circadian clock is the endogenous timer that coordinates physiological processes with daily and seasonal environmental changes. In Arabidopsis thaliana, establishment of the circadian period relies on targeted degradation of TIMING OF CAB EXPRESSION 1 (TOC1) by the 26S proteasome. ZEITLUPE (ZTL) is the F-box protein that associates with the SCF (Skp/Cullin/F-box) E3 ubiquitin ligase that is responsible for marking TOC1 for turnover. CULLIN1 (CUL1) is a core component of SCF complexes and is involved in multiple signaling pathways. To assess the contribution of CUL1-containing SCF complexes to signaling within the plant oscillator, circadian rhythms were examined in the recessive, temperature-sensitive CUL1 allele axr6-3. The activity of CUL1 in this mutant declines progressively with increasing ambient temperature, resulting in more severe defects in CUL1-dependent activities at elevated temperature. Examination of circadian rhythms in axr6-3 revealed circadian phenotypes comparable to those observed in ztl null mutants; namely, lengthened circadian period, altered expression of core oscillator genes, and limited degradation of TOC1. In addition, treatment of seedlings with exogenous auxin did not alter TOC1 stability. These results demonstrate that CUL1 is required for TOC1 degradation and further suggest that this protein is the functional cullin for the SCFZTL complex.
PMCID: PMC2976475  PMID: 18433436
circadian rhythms; CUL1; post-translational regulation; ubiquitin ligase; proteasome; Arabidopsis
17.  Circadian Oscillation of the Lettuce Transcriptome under Constant Light and Light–Dark Conditions 
Although, the circadian clock is a universal biological system in plants and it orchestrates important role of plant production such as photosynthesis, floral induction and growth, there are few such studies on cultivated species. Lettuce is one major cultivated species for both open culture and plant factories and there is little information concerning its circadian clock system. In addition, most of the relevant genes have not been identified. In this study, we detected circadian oscillation in the lettuce transcriptome using time-course RNA sequencing (RNA-Seq) data. Constant light (LL) and light–dark (LD) conditions were used to detect circadian oscillation because the circadian clock has some basic properties: one is self-sustaining oscillation under constant light and another is entrainment to environmental cycles such as light and temperature. In the results, 215 contigs were detected as common oscillating contigs under both LL and LD conditions. The 215 common oscillating contigs included clock gene-like contigs CCA1 (CIRCADIAN CLOCK ASSOCIATED 1)-like, TOC1 (TIMING OF CAB EXPRESSION 1)-like and LHY (LATE ELONGATED HYPOCOTYL)-like, and their expression patterns were similar to those of Arabidopsis. Functional enrichment analysis by GO (gene ontology) Slim and GO Fat showed that the GO terms of response to light stimulus, response to stress, photosynthesis and circadian rhythms were enriched in the 215 common oscillating contigs and these terms were actually regulated by circadian clocks in plants. The 215 common oscillating contigs can be used to evaluate whether the gene expression pattern related to photosynthesis and optical response performs normally in lettuce.
PMCID: PMC4961695  PMID: 27512400
circadian clocks; enrichment; lettuce; oscillations; plant factories; transcriptome
18.  Experimental validation of a predicted feedback loop in the multi-oscillator clock of Arabidopsis thaliana 
Our computational model of the circadian clock comprised the feedback loop between LATE ELONGATED HYPOCOTYL (LHY), CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and TIMING OF CAB EXPRESSION 1 (TOC1), and a predicted, interlocking feedback loop involving TOC1 and a hypothetical component Y. Experiments based on model predictions suggested GIGANTEA (GI) as a candidate for Y. We now extend the model to include a recently demonstrated feedback loop between the TOC1 homologues PSEUDO-RESPONSE REGULATOR 7 (PRR7), PRR9 and LHY and CCA1. This three-loop network explains the rhythmic phenotype of toc1 mutant alleles. Model predictions fit closely to new data on the gi;lhy;cca1 mutant, which confirm that GI is a major contributor to Y function. Analysis of the three-loop network suggests that the plant clock consists of morning and evening oscillators, coupled intracellularly, which may be analogous to coupled, morning and evening clock cells in Drosophila and the mouse.
PMCID: PMC1682024  PMID: 17102804
circadian rhythm; genetic network; photoperiod; mathematical model; systems biology
19.  Rhythmic Oscillation of Histone Acetylation and Methylation at the Arabidopsis Central Clock Loci 
Molecules and Cells  2012;34(3):279-287.
Circadian clock genes are regulated by a transcriptional-translational feedback loop. In Arabidopsis, LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) transcripts are highly expressed in the morning. Translated LHY and CCA1 proteins repress the expression of TIMING OF CAB EXPRESSION 1 (TOC1), which peaks in the evening. TOC1 protein induces expression of LHY and CCA1, forming a negative feedback loop which is believed to constitute the oscillatory mechanism of the clock. The rhythmic oscillation of mouse clock genes mPERIOD 1 (mPER1) and mPER2 has been correlated with regular alteration of chromatin structure through histone acetylation/deacetylation. However, little is known about the relationship between the transcriptional activity of Arabidopsis clock genes and their chromatin status. Here, we report that histone H3 acetylation (H3Ac) and H3 lysine 4 tri-methylation (H3K4me3) levels at LHY, CCA1, and TOC1 are positively correlated with the rhythmic transcript levels of these genes, whereas H3K36me2 level shows a negative correlation. Thus, our study suggests rhythmic transcription of Arabidopsis clock genes might be regulated by rhythmic histone modification, and it provides a platform for future identification of clock-controlling histone modifiers.
PMCID: PMC3887839  PMID: 22878891
Arabidopsis; chromatin; circadian clock; circadian rhythm; histone modification
20.  Testing Time: Can Ethanol-Induced Pulses of Proposed Oscillator Components Phase Shift Rhythms in Arabidopsis? 
Journal of biological rhythms  2008;23(6):463-471.
Circadian rhythms are generated by endogenous central oscillators that respond to input from the environment and regulate rhythmic outputs. In Arabidopsis, more than a dozen components that affect rhythms have been identified and used to propose models of the central oscillator. However, none has been shown to fulfill one of the expected characteristics of an oscillator component: that a pulse of its expression shifts the phase of circadian rhythms. Here we show that a pulse of the proposed oscillator components CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) causes dramatic phase shifts in rhythms of expression of the circadian reporter CAB2∷LUC, as well as of the clock-associated genes TIMING OF CAB EXPRESSION 1 (TOC1) and GIGANTEA (GI). These results demonstrate that pulses of either CCA1 or LHY are capable of resetting the circadian clock. In contrast, a pulse of TOC1 expression did not elicit phase shifts. Control of TOC1 protein level is in part posttranscriptional; thus a pulse of TOC1 protein could be induced only at times when it is already high. Our work also shows that the ethanol-inducible system can be useful for achieving relatively short (<8 h) pulses of gene expression in seedlings.
PMCID: PMC2652257  PMID: 19060255
Arabidopsis thaliana; circadian rhythm; central oscillator; phase shift; clock gene; ethanol-inducible system
21.  Wheels within wheels: new transcriptional feedback loops in the Arabidopsis circadian clock 
F1000Prime Reports  2014;6:2.
The circadian clock allows organisms to temporally coordinate their biology with the diurnal oscillation of the environment, which enhances plant performance. Accordingly, a fuller understanding of the circadian clock mechanism may contribute to efforts to optimize plant performance. One recurring theme in clock mechanism is coupled transcription-translation feedback loops. To date, the majority of plant transcription factors constituting these loops, including the central oscillator components CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), and TIMING OF CAB2 EXPRESSION 1 (TOC1), and the related PSEUDO-RESPONSE REGULATORS (PRRs), are transcriptional repressors, leading to a model of the clock emphasizing repressive interactions. Recent work, however, has revealed that a subset of the REVEILLE (RVE) family of Myb transcription factors closely related to CCA1 and LHY are transcriptional activators in novel feedback transcription-translation feedback loops. Other recently identified transcriptional activators that contribute to clock function include LIGHT-REGULATED WD 1 (LWD1) and LWD2 and night light-inducible and clock-regulated transcription factors NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED1 (LNK1) and LNK2. Collectively, these advances permit a substantial reconfiguration of the clock model.
PMCID: PMC3883422  PMID: 24592314
22.  Crosstalk between the Circadian Clock and Innate Immunity in Arabidopsis 
PLoS Pathogens  2013;9(6):e1003370.
The circadian clock integrates temporal information with environmental cues in regulating plant development and physiology. Recently, the circadian clock has been shown to affect plant responses to biotic cues. To further examine this role of the circadian clock, we tested disease resistance in mutants disrupted in CCA1 and LHY, which act synergistically to regulate clock activity. We found that cca1 and lhy mutants also synergistically affect basal and resistance gene-mediated defense against Pseudomonas syringae and Hyaloperonospora arabidopsidis. Disrupting the circadian clock caused by overexpression of CCA1 or LHY also resulted in severe susceptibility to P. syringae. We identified a downstream target of CCA1 and LHY, GRP7, a key constituent of a slave oscillator regulated by the circadian clock and previously shown to influence plant defense and stomatal activity. We show that the defense role of CCA1 and LHY against P. syringae is at least partially through circadian control of stomatal aperture but is independent of defense mediated by salicylic acid. Furthermore, we found defense activation by P. syringae infection and treatment with the elicitor flg22 can feedback-regulate clock activity. Together this data strongly supports a direct role of the circadian clock in defense control and reveal for the first time crosstalk between the circadian clock and plant innate immunity.
Author Summary
Plants are frequently challenged by various pathogens. The circadian clock, which is the internal time measuring machinery, has been implicated in regulating plant responses to biotic cues. To better understand the role of the circadian clock in defense control, we tested disease resistance with Arabidopsis mutants disrupted in CCA1 and LHY, two key components of the circadian clock. We found that consistent with their contributions to the circadian clock, cca1 and lhy mutants synergistically affect resistance to both bacterial and oomycete pathogens. Disrupting the circadian clock caused by overexpression of CCA1 or LHY also results in severe disease susceptibility. Thus, our data further demonstrate a direct role of the circadian clock mediated by CCA1 and LHY in defense regulation. We also found that CCA1 and LHY act independently of salicylic acid mediated defense but at least through the downstream target gene GRP7 to regulate both stomata-dependent and -independent pathways. We further show that defense activation by bacterial infection and the treatment with the elicitor flg22 can also feed back to regulate clock activity. Together our study reveals for the first time reciprocal regulation of the circadian clock and plant innate immunity, significantly expanding our view of complex gene networks regulating plant defense responses and development.
PMCID: PMC3675028  PMID: 23754942
23.  REVEILLE8 and PSEUDO-REPONSE REGULATOR5 Form a Negative Feedback Loop within the Arabidopsis Circadian Clock 
PLoS Genetics  2011;7(3):e1001350.
Circadian rhythms provide organisms with an adaptive advantage, allowing them to regulate physiological and developmental events so that they occur at the most appropriate time of day. In plants, as in other eukaryotes, multiple transcriptional feedback loops are central to clock function. In one such feedback loop, the Myb-like transcription factors CCA1 and LHY directly repress expression of the pseudoresponse regulator TOC1 by binding to an evening element (EE) in the TOC1 promoter. Another key regulatory circuit involves CCA1 and LHY and the TOC1 homologs PRR5, PRR7, and PRR9. Purification of EE–binding proteins from plant extracts followed by mass spectrometry led to the identification of RVE8, a homolog of CCA1 and LHY. Similar to these well-known clock genes, expression of RVE8 is circadian-regulated with a dawn phase of expression, and RVE8 binds specifically to the EE. However, whereas cca1 and lhy mutants have short period phenotypes and overexpression of either gene causes arrhythmia, rve8 mutants have long-period and RVE8-OX plants have short-period phenotypes. Light input to the clock is normal in rve8, but temperature compensation (a hallmark of circadian rhythms) is perturbed. RVE8 binds to the promoters of both TOC1 and PRR5 in the subjective afternoon, but surprisingly only PRR5 expression is perturbed by overexpression of RVE8. Together, our data indicate that RVE8 promotes expression of a subset of EE–containing clock genes towards the end of the subjective day and forms a negative feedback loop with PRR5. Thus RVE8 and its homologs CCA1 and LHY function close to the circadian oscillator but act via distinct molecular mechanisms.
Author Summary
Circadian clocks help organize 24-hour rhythms in physiology and behavior so that critical organismal functions are optimally timed relative to highly predictable daily changes in the environment. Circadian clocks run at approximately the same pace across a wide range of temperatures, ensuring accurate timekeeping in all seasons. Although molecular components of the circadian clock are not conserved across higher taxa, eukaryotic circadian clocks are composed of analogous interlocked transcriptional feedback loops. In this study, we report the isolation and characterization of a new component of the plant circadian system, REVEILLE 8 (RVE8). RVE8 is a clock-regulated Myb-like transcription factor that binds with high affinity to the evening element (EE) promoter motif and helps to set the pace of the clock in a light- and temperature-dependent manner. RVE8 promotes expression of the clock component PSEUDO-RESPONSE REGULATOR 5 (PRR5), likely via direct action at the PRR5 promoter. RVE8 expression is in turn repressed by PRR5. Thus, RVE8 is a new component of the plant circadian oscillator that takes part in a novel transcriptional feedback loop.
PMCID: PMC3069099  PMID: 21483796
24.  Does the core circadian clock in the moss Physcomitrella patens (Bryophyta) comprise a single loop? 
BMC Plant Biology  2010;10:109.
The endogenous circadian clock allows the organism to synchronize processes both to daily and seasonal changes. In plants, many metabolic processes such as photosynthesis, as well as photoperiodic responses, are under the control of a circadian clock. Comparative studies with the moss Physcomitrella patens provide the opportunity to study many aspects of land plant evolution. Here we present a comparative overview of clock-associated components and the circadian network in the moss P. patens.
The moss P. patens has a set of conserved circadian core components that share genetic relationship and gene expression patterns with clock genes of vascular plants. These genes include Myb-like transcription factors PpCCA1a and PpCCA1b, pseudo-response regulators PpPRR1-4, and regulatory elements PpELF3, PpLUX and possibly PpELF4. However, the moss lacks homologs of AtTOC1, AtGI and the AtZTL-family of genes, which can be found in all vascular plants studied here. These three genes constitute essential components of two of the three integrated feed-back loops in the current model of the Arabidopsis circadian clock mechanism. Consequently, our results suggest instead a single loop circadian clock in the moss. Possibly as a result of this, temperature compensation of core clock gene expression appears to be decreased in P. patens.
This study is the first comparative overview of the circadian clock mechanism in a basal land plant, the moss P. patens. Our results indicate that the moss clock mechanism may represent an ancestral state in contrast to the more complex and partly duplicated structure of subsequent land plants. These findings may provide insights into the understanding of the evolution of circadian network topology.
PMCID: PMC3017809  PMID: 20550695
25.  Gene Expression in Plant Lipid Metabolism in Arabidopsis Seedlings 
PLoS ONE  2014;9(9):e107372.
Events in plant lipid metabolism are important during seedling establishment. As it has not been experimentally verified whether lipid metabolism in 2- and 5-day-old Arabidopsis thaliana seedlings is diurnally-controlled, quantitative real-time PCR analysis was used to investigate the expression of target genes in acyl-lipid transfer, β-oxidation and triacylglycerol (TAG) synthesis and hydrolysis in wild-type Arabidopsis WS and Col-0. In both WS and Col-0, ACYL-COA-BINDING PROTEIN3 (ACBP3), DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1) and DGAT3 showed diurnal control in 2- and 5-day-old seedlings. Also, COMATOSE (CTS) was diurnally regulated in 2-day-old seedlings and LONG-CHAIN ACYL-COA SYNTHETASE6 (LACS6) in 5-day-old seedlings in both WS and Col-0. Subsequently, the effect of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) from the core clock system was examined using the cca1lhy mutant and CCA1-overexpressing (CCA1-OX) lines versus wild-type WS and Col-0, respectively. Results revealed differential gene expression in lipid metabolism between 2- and 5-day-old mutant and wild-type WS seedlings, as well as between CCA1-OX and wild-type Col-0. Of the ACBPs, ACBP3 displayed the most significant changes between cca1lhy and WS and between CCA1-OX and Col-0, consistent with previous reports that ACBP3 is greatly affected by light/dark cycling. Evidence of oil body retention in 4- and 5-day-old seedlings of the cca1lhy mutant in comparison to WS indicated the effect of cca1lhy on storage lipid reserve mobilization. Lipid profiling revealed differences in primary lipid metabolism, namely in TAG, fatty acid methyl ester and acyl-CoA contents amongst cca1lhy, CCA1-OX, and wild-type seedlings. Taken together, this study demonstrates that lipid metabolism is subject to diurnal regulation in the early stages of seedling development in Arabidopsis.
PMCID: PMC4180049  PMID: 25264899

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