The clock gene circuit in Arabidopsis includes a repressilator with additional feedback loops.

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.

Extension of a genetic network model by iterative experimentation and mathematical analysis.

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.

Luciferases as reporter genes.

Luciferase is the ideal reporter gene to provide temporal and spatial information on promoter activity in Arabidopsis and other eukaryotes; the noninvasive detection of luminescence and short half-life of luciferase activity allow repeated measurements of individual seedlings over several days to assay dynamic changes in gene expression. Transgenic or transiently transformed plants with a luciferase gene under the control of a promoter of interest are required. Detection of the low level of luminescence produced by the luciferase gene in Arabidopsis requires the use of low light detecting charge-coupled device (CCD) cameras or scintillation counters. This chapter contains protocols on assaying and imaging luciferase in vivo and the automation for high-resolution timecourses.

Defining the robust behaviour of the plant clock gene circuit with absolute RNA timeseries and open infrastructure.

Our understanding of the complex, transcriptional feedback loops in the circadian clock mechanism has depended upon quantitative, timeseries data from disparate sources. We measure clock gene RNA profiles in Arabidopsis thaliana seedlings, grown with or without exogenous sucrose, or in soil-grown plants and in wild-type and mutant backgrounds. The RNA profiles were strikingly robust across the experimental conditions, so current mathematical models are likely to be broadly applicable in leaf tissue. In addition to providing reference data, unexpected behaviours included co-expression of PRR9 and ELF4, and regulation of PRR5 by GI. Absolute RNA quantification revealed low levels of PRR9 transcripts (peak approx. 50 copies cell(-1)) compared with other clock genes, and threefold higher levels of LHY RNA (more than 1500 copies cell(-1)) than of its close relative CCA1. The data are disseminated from BioDare, an online repository for focused timeseries data, which is expected to benefit mechanistic modelling. One data subset successfully constrained clock gene expression in a complex model, using publicly available software on parallel computers, without expert tuning or programming. We outline the empirical and mathematical justification for data aggregation in understanding highly interconnected, dynamic networks such as the clock, and the observed design constraints on the resources required to make this approach widely accessible.

Temporal repression of core circadian genes is mediated through EARLY FLOWERING 3 in Arabidopsis.

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.

The molecular basis of temperature compensation in the Arabidopsis circadian clock.

Circadian clocks maintain robust and accurate timing over a broad range of physiological temperatures, a characteristic termed temperature compensation. In Arabidopsis thaliana, ambient temperature affects the rhythmic accumulation of transcripts encoding the clock components TIMING OF CAB EXPRESSION1 (TOC1), GIGANTEA (GI), and the partially redundant genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). The amplitude and peak levels increase for TOC1 and GI RNA rhythms as the temperature increases (from 17 to 27 degrees C), whereas they decrease for LHY. However, as temperatures decrease (from 17 to 12 degrees C), CCA1 and LHY RNA rhythms increase in amplitude and peak expression level. At 27 degrees C, a dynamic balance between GI and LHY allows temperature compensation in wild-type plants, but circadian function is impaired in lhy and gi mutant plants. However, at 12 degrees C, CCA1 has more effect on the buffering mechanism than LHY, as the cca1 and gi mutations impair circadian rhythms more than lhy at the lower temperature. At 17 degrees C, GI is apparently dispensable for free-running circadian rhythms, although partial GI function can affect circadian period. Numerical simulations using the interlocking-loop model show that balancing LHY/CCA1 function against GI and other evening-expressed genes can largely account for temperature compensation in wild-type plants and the temperature-specific phenotypes of gi mutants.

Forward genetic analysis of the circadian clock separates the multiple functions of ZEITLUPE.

The circadian system of Arabidopsis (Arabidopsis thaliana) includes feedback loops of gene regulation that generate 24-h oscillations. Components of these loops remain to be identified; none of the known components is completely understood, including ZEITLUPE (ZTL), a gene implicated in regulated protein degradation. ztl mutations affect both circadian and developmental responses to red light, possibly through ZTL interaction with PHYTOCHROME B (PHYB). We conducted a large-scale genetic screen that identified additional clock-affecting loci. Other mutants recovered include 11 new ztl alleles encompassing mutations in each of the ZTL protein domains. Each mutation lengthened the circadian period, even in dark-grown seedlings entrained to temperature cycles. A mutation of the LIGHT, OXYGEN, VOLTAGE (LOV)/Period-ARNT-Sim (PAS) domain was unique in retaining wild-type responses to red light both for the circadian period and for control of hypocotyl elongation. This uncoupling of ztl phenotypes indicates that interactions of ZTL protein with multiple factors must be disrupted to generate the full ztl mutant phenotype. Protein interaction assays showed that the ztl mutant phenotypes were not fully explained by impaired interactions with previously described partner proteins Arabidopsis S-phase kinase-related protein 1, TIMING OF CAB EXPRESSION 1, and PHYB. Interaction with PHYB was unaffected by mutation of any ZTL domain. Mutation of the kelch repeat domain affected protein binding at both the LOV/PAS and the F-box domains, indicating that interaction among ZTL domains leads to the strong phenotypes of kelch mutations. Forward genetics continues to provide insight regarding both known and newly discovered components of the circadian system, although current approaches have saturated mutations at some loci.

Circadian genetics in the model higher plant, Arabidopsis thaliana.

In recent decades, most research on the circadian rhythms of higher plants has been driven by molecular genetics. A wide variety of experimental approaches have discovered mutants in the plant circadian clock, yet the screens are far from saturated and there must still be important clock-related genes to identify. Direct methods to screen for circadian mutants include the original assay of rhythmic luminescence from promoter:luciferase constructs in planta or a recently developed assay based on stomatal rhythms. Mutants found through simpler screens of processes only partially controlled by the clock are still identifying novel and interesting circadian phenotypes when their rhythms are tested, while the sequenced genome and the large range of mutant stocks available have made reverse genetics increasingly powerful.

Response regulator homologues have complementary, light-dependent functions in the Arabidopsis circadian clock.

TIMING OF CAB EXPRESSION 1 ( TOC1) functions with CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) in a transcriptional feedback loop that is important for the circadian clock in Arabidopsis thaliana (L.) Heynh. TOC1 and its four paralogues, the Arabidopsis PSEUDO-RESPONSE REGULATOR (PRR) genes, are expressed in an intriguing daily sequence. This was proposed to form a second feedback loop, similar to the interlocking clock gene circuits in other taxa. We show that prr9 and prr5 null mutants have reciprocal period defects for multiple circadian rhythms, consistent with subtly altered expression patterns of CCA1 and TOC1. The period defects are conditional on light quality and combine additively in double-mutant plants. Thus PRR9 and PRR5 modulate light input to the circadian clock but are neither uniquely required for rhythm generation nor form a linear series of mutual PRR gene regulation.