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. Mol Syst Biol. 2: 59

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. ### Synopsis 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][1]). 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][2]), 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][3]), 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][4]). *Footnote: Synopsis highlights were added on 5 July 2005. Mol Syst Biol. 1: 2005.0013 [1]: #ref-24 [2]: #F4 [3]: #F6 [4]: #ref-5

The phytochrome family of plant photoreceptors has a central role in the adaptation of plant development to changes in ambient light conditions. The individual phytochrome species regulate different or partly overlapping physiological responses. We generated transgenic Arabidopsis plants expressing phytochrome A to E:green fluorescent protein (GFP) fusion proteins to assess the biological role of intracellular compartmentation of these photoreceptors in light-regulated signaling. We show that all phytochrome:GFP fusion proteins were imported into the nuclei. Translocation of these photoreceptors into the nuclei was regulated differentially by light. Light-induced accumulation of phytochrome species in the nuclei resulted in the formation of speckles. The appearance of these nuclear structures exhibited distinctly different kinetics, wavelengths, and fluence dependence and was regulated by a diurnal rhythm. Furthermore, we demonstrate that the import of mutant phytochrome B:GFP and phytochrome A:GFP fusion proteins, shown to be defective in signaling in vivo, is regulated by light but is not accompanied by the formation of speckles. These results suggest that (1) the differential regulation of the translocation of phytochrome A to E into nuclei plays a role in the specification of functions, and (2) the appearance of speckles is a functional feature of phytochrome-regulated signaling.

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.

In plants subjected to UV-B radiation, responses are activated that minimize damage caused by UV-B. The bZIP transcription factor ELONGATED HYPOCOTYL5 (HY5) acts downstream of the UV-B photoreceptor UV RESISTANCE LOCUS8 (UVR8) and promotes UV-B-induced photomorphogenesis and acclimation. Expression of HY5 is induced by UV-B; however, the transcription factor(s) that regulate HY5 transcription in response to UV-B and the impact of UV-B on the association of HY5 with its target promoters are currently unclear. Here, we show that HY5 binding to the promoters of UV-B-responsive genes is enhanced by UV-B in a UVR8-dependent manner in Arabidopsis thaliana. In agreement, overexpression of REPRESSOR OF UV-B PHOTOMORPHOGENESIS2, a negative regulator of UVR8 function, blocks UV-B-responsive HY5 enrichment at target promoters. Moreover, we have identified a T/G-box in the HY5 promoter that is required for its UV-B responsiveness. We show that HY5 and its homolog HYH bind to the T/G^HY5-box cis-acting element and that they act redundantly in the induction of HY5 expression upon UV-B exposure. Therefore, HY5 is enriched at target promoters in response to UV-B in a UVR8 photoreceptor-dependent manner, and HY5 and HYH interact directly with a T/G-box cis-acting element of the HY5 promoter, mediating the transcriptional activation of HY5 in response to UV-B.