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1.  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
2.  Photoperiodic flowering regulation in Arabidopsis thaliana 
Photoperiod, or the duration of light in a given day, is a critical cue that flowering plants utilize to effectively assess seasonal information and coordinate their reproductive development in synchrony with the external environment. The use of the model plant, Arabidopsis thaliana, has greatly improved our understanding of the molecular mechanisms that determine how plants process and utilize photoperiodic information to coordinate a flowering response. This mechanism is typified by the transcriptional activation of FLOWERING LOCUS T (FT) gene by the transcription factor CONSTANS (CO) under inductive long-day conditions in Arabidopsis. FT protein then moves from the leaves to the shoot apex, where floral meristem development can be initiated. As a point of integration from a variety of environmental factors in the context of a larger system of regulatory pathways that affect flowering, the importance of photoreceptors and the circadian clock in CO regulation throughout the day has been a key feature of the photoperiodic flowering pathway. In addition to these established mechanisms, the recent discovery of a photosynthate derivative trehalose-6-phosphate as an activator of FT in leaves has interesting implications for the involvement of photosynthesis in the photoperiodic flowering response that were suggested from previous physiological experiments in flowering induction.
PMCID: PMC4326075
Photoperiodism; Flowering; Phenology; Circadian Clock; Florigen
3.  Comparative genomic analysis of light-regulated transcripts in the Solanaceae 
BMC Genomics  2009;10:60.
Plants use different light signals to adjust their growth and development to the prevailing environmental conditions. Studies in the model species Arabidopsis thaliana and rice indicate that these adjustments are mediated by large changes in the transcriptome. Here we compared transcriptional responses to light in different species of the Solanaceae to investigate common as well as species-specific changes in gene expression.
cDNA microarrays were used to identify genes regulated by a transition from long days (LD) to short days (SD) in the leaves of potato and tobacco plants, and by phytochrome B (phyB), the photoreceptor that represses tuberization under LD in potato. We also compared transcriptional responses to photoperiod in Nicotiana tabacum Maryland Mammoth (MM), which flowers only under SD, with those of Nicotiana sylvestris, which flowers only under LD conditions. Finally, we identified genes regulated by red compared to far-red light treatments that promote germination in tomato.
Most of the genes up-regulated in LD were associated with photosynthesis, the synthesis of protective pigments and the maintenance of redox homeostasis, probably contributing to the acclimatization to seasonal changes in irradiance. Some of the photoperiodically regulated genes were the same in potato and tobacco. Others were different but belonged to similar functional categories, suggesting that conserved as well as convergent evolutionary processes are responsible for physiological adjustments to seasonal changes in the Solanaceae. A β-ZIP transcription factor whose expression correlated with the floral transition in Nicotiana species with contrasting photoperiodic responses was also regulated by photoperiod and phyB in potato, and is a candidate gene to act as a general regulator of photoperiodic responses. Finally, GIGANTEA, a gene that controls flowering time in Arabidopsis thaliana and rice, was regulated by photoperiod in the leaves of potato and tobacco and by red compared to far-light treatments that promote germination in tomato seeds, suggesting that a conserved light signaling cascade acts across developmental contexts and species.
PMCID: PMC2644711  PMID: 19192291
4.  Deletion of chloroplast NADPH-dependent thioredoxin reductase results in inability to regulate starch synthesis and causes stunted growth under short-day photoperiods 
Journal of Experimental Botany  2013;64(12):3843-3854.
Plastid-localized NADPH-dependent thioredoxin reductase C (NTRC) is a unique NTR enzyme containing both reductase and thioredoxin domains in a single polypeptide. Arabidopsis thaliana NTRC knockout lines (ntrc) show retarded growth, especially under short-day (SD) photoperiods. This study identified chloroplast processes that accounted for growth reduction in SD-acclimated ntrc. The strongest reduction in ntrc growth occurred under photoperiods with nights longer than 14h, whereas knockout of the NTRC gene did not alter the circadian-clock-controlled growth of Arabidopsis. Lack of NTRC modulated chloroplast reactive oxygen species (ROS) metabolism, but oxidative stress was not the primary cause of retarded growth of SD-acclimated ntrc. Scarcity of starch accumulation made ntrc leaves particularly vulnerable to photoperiods with long nights. Direct interaction of NTRC and ADP-glucose pyrophosphorylase, a key enzyme in starch synthesis, was confirmed by yeast two-hybrid analysis. The ntrc line was not able to maximize starch synthesis during the light period, which was particularly detrimental under SD conditions. Acclimation of Arabidopsis to SD conditions also involved an inductive rise of ROS production in illuminated chloroplasts that was not counterbalanced by the activation of plastidial anti-oxidative systems. It is proposed that knockout of NTRC challenges redox regulation of starch synthesis, resulting in stunted growth of the mutant lines acclimated to the SD photoperiod.
PMCID: PMC3745738  PMID: 23881397
ADP-glucose pyrophosphorylase; Arabidopsis thaliana; chloroplast; NTRC; ROS; starch; thioredoxins.
5.  SRR1 is essential to repress flowering in non-inductive conditions in Arabidopsis thaliana  
Journal of Experimental Botany  2014;65(20):5811-5822.
SRR1 regulates flowering time in Arabidopsis by integrating photoperiodic and photoperiod-independent signals. By promoting expression of several repressors of FT, SRR1 represses flowering in non-inductive conditions.
Timing of flowering is determined by environmental and developmental signals, leading to promotion or repression of key floral integrators. SENSITIVITY TO RED LIGHT REDUCED (SRR1) is a pioneer protein previously shown to be involved in regulation of the circadian clock and phytochrome B signalling in Arabidopsis thaliana. This report has examined the role of SRR1 in flowering time control. Loss-of-function srr1-1 plants flowered very early compared with the wild type under short-day conditions and had a weak flowering response to increasing daylength. Furthermore, FLOWERING LOCUS T (FT) transcript levels were elevated already in short days in srr1-1 compared with the wild type. This correlated with elevated end of day levels of CONSTANS (CO), whereas levels of CYCLING DOF FACTOR 1 (CDF1), a repressor of CO transcription, were reduced. srr1-1 gi-2 and srr1-1 co-9 double mutants showed that SRR1 can also repress flowering independently of the photoperiodic pathway. srr1-1 flowered consistently early between 16 °C and 27 °C, showing that SRR1 prevents premature flowering over a wide temperature range. SRR1 also promotes expression of the repressors TEMPRANILLO 1 (TEM1) and TEM2. Consequently their targets in the gibberellin biosynthesis pathway were elevated in srr1-1. SRR1 is thus an important focal point of both photoperiodic and photoperiod-independent regulation of flowering. By stimulating expression of the FT-binding repressors CDF1, TEM1 and TEM2, and FLC, flowering is inhibited in non-inductive conditions.
PMCID: PMC4203120  PMID: 25129129
Arabidopsis; circadian clock; flowering time control; photoperiod; repressors; SRR1.
6.  Expression conservation within the circadian clock of a monocot: natural variation at barley Ppd-H1 affects circadian expression of flowering time genes, but not clock orthologs 
BMC Plant Biology  2012;12:97.
The circadian clock is an endogenous mechanism that coordinates biological processes with daily changes in the environment. In plants, circadian rhythms contribute to both agricultural productivity and evolutionary fitness. In barley, the photoperiod response regulator and flowering-time gene Ppd-H1 is orthologous to the Arabidopsis core-clock gene PRR7. However, relatively little is known about the role of Ppd-H1 and other components of the circadian clock in temperate crop species. In this study, we identified barley clock orthologs and tested the effects of natural genetic variation at Ppd-H1 on diurnal and circadian expression of clock and output genes from the photoperiod-response pathway.
Barley clock orthologs HvCCA1, HvGI, HvPRR1, HvPRR37 (Ppd-H1), HvPRR73, HvPRR59 and HvPRR95 showed a high level of sequence similarity and conservation of diurnal and circadian expression patterns, when compared to Arabidopsis. The natural mutation at Ppd-H1 did not affect diurnal or circadian cycling of barley clock genes. However, the Ppd-H1 mutant was found to be arrhythmic under free-running conditions for the photoperiod-response genes HvCO1, HvCO2, and the MADS-box transcription factor and vernalization responsive gene Vrn-H1.
We suggest that the described eudicot clock is largely conserved in the monocot barley. However, genetic differentiation within gene families and differences in the function of Ppd-H1 suggest evolutionary modification in the angiosperm clock. Our data indicates that natural variation at Ppd-H1 does not affect the expression level of clock genes, but controls photoperiodic output genes. Circadian control of Vrn-H1 in barley suggests that this vernalization responsive gene is also controlled by the photoperiod-response pathway. Structural and functional characterization of the barley circadian clock will set the basis for future studies of the adaptive significance of the circadian clock in Triticeae species.
PMCID: PMC3478166  PMID: 22720803
Hordeum vulgare; Circadian Clock; Photoperiod; Flowering; Ppd-H1
7.  Evolutionarily conserved photoperiod mechanisms in plants 
Plant Signaling & Behavior  2009;4(7):642-644.
Day-length and the circadian clock control critical aspects of plant development such as the onset of reproduction by the photoperiodic pathway.1 CONSTANS (CO) regulates the expression of a florigenic mobile signal from leaves to the apical meristem and thus is central to the regulation of photoperiodic flowering.2 This regulatory control is present in all higher plants,3 but the time in evolution when it arose was unknown. We have shown that the genomes of green microalgae encode members of the CONSTANS-like (COL) protein family. One of these genes, the Chlamydomonas reinhardtii CO homolog (CrCO), can complement the co mutation in Arabidopsis.4 CrCO expression is controlled by the clock and photoperiod in Chlamydomonas and at the same time is involved in the correct timing of several circadian output processes such as the accumulation of starch or the coordination of cell growth and division. We have proposed that, since very early in the evolutionary lineage that gave rise to higher plants, CO homologs have been involved in the photoperiod control of important developmental processes, and that the recruitment of COL proteins in other roles may have been crucial for their evolutionary success.
PMCID: PMC2710563  PMID: 19820341
photoperiod; flowering; constans; signaling; Arabidopsis; Chlamydomomas
8.  Genetic variation in four maturity genes affects photoperiod insensitivity and PHYA-regulated post-flowering responses of soybean 
BMC Plant Biology  2013;13:91.
Absence of or low sensitivity to photoperiod is necessary for short-day crops, such as rice and soybean, to adapt to high latitudes. Photoperiod insensitivity in soybeans is controlled by two genetic systems and involves three important maturity genes: E1, a repressor for two soybean orthologs of Arabidopsis FLOWERING LOCUS T (GmFT2a and GmFT5a), and E3 and E4, which are phytochrome A genes. To elucidate the diverse mechanisms underlying photoperiod insensitivity in soybean, we assessed the genotypes of four maturity genes (E1 through E4) in early-flowering photoperiod-insensitive cultivars and their association with post-flowering responses.
We found two novel dysfunctional alleles in accessions originally considered to have a dominant E3 allele according to known DNA markers. The E3 locus, together with E1 and E4, contained multiple dysfunctional alleles. We identified 15 multi-locus genotypes, which we subdivided into 6 genotypic groups by classifying their alleles by function. Of these, the e1-as/e3/E4 genotypic group required an additional novel gene (different from E1, E3, and E4) to condition photoperiod insensitivity. Despite their common pre-flowering photoperiod insensitivity, accessions with different multi-locus genotypes responded differently to the post-flowering photoperiod. Cultivars carrying E3 or E4 were sensitive to photoperiod for post-flowering characteristics, such as reproductive period and stem growth after flowering. The phytochrome A–regulated expression of the determinate growth habit gene Dt1, an ortholog of Arabidopsis TERMINAL FLOWER1, was involved in the persistence of the vegetative activity at the stem apical meristem of flower-induced plants under long-day conditions.
Diverse genetic mechanisms underlie photoperiod insensitivity in soybean. At least three multi-locus genotypes consisting of various allelic combinations at E1, E3, and E4 conferred pre-flowering photoperiod insensitivity to soybean cultivars but led to different responses to photoperiod during post-flowering vegetative and reproductive development. The phyA genes E3 and E4 are major controllers underlying not only pre-flowering but also post-flowering photoperiod responses. The current findings improve our understanding of genetic diversity in pre-flowering photoperiod insensitivity and mechanisms of post-flowering photoperiod responses in soybean.
PMCID: PMC3698206  PMID: 23799885
Photoperiod; Soybean; Flowering; Determinate habit; Post-flowering; Genetic variation
9.  LOV Domain-Containing F-Box Proteins: Light-Dependent Protein Degradation Modules in Arabidopsis 
Molecular Plant  2012;5(3):47-56.
Plants constantly survey the surrounding environment using several sets of photoreceptors. They can sense changes in the quantity (=intensity) and quality (=wavelength) of light and use this information to adjust their physiological responses, growth, and developmental patterns. In addition to the classical photoreceptors, such as phytochromes, cryptochromes, and phototropins, ZEITLUPE (ZTL), FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1), and LOV KELCH PROTEIN 2 (LKP2) proteins have been recently identified as blue-light photoreceptors that are important for regulation of the circadian clock and photoperiodic flowering. The ZTL/FKF1/LKP2 protein family possesses a unique combination of domains: a blue-light-absorbing LOV (Light, Oxygen, or Voltage) domain along with domains involved in protein degradation. Here, we summarize recent advances in our understanding of the function of the Arabidopsis ZTL/FKF1/LKP2 proteins. We summarize the distinct photochemical properties of their LOV domains and discuss the molecular mechanisms by which the ZTL/FKF1/LKP2 proteins regulate the circadian clock and photoperiodic flowering by controlling blue-light-dependent protein degradation.
PMCID: PMC3355347  PMID: 22402262
blue light; LOV domain; ZTL; FKF1; LKP2; photoperiodic flowering; circadian clock; Arabidopsis
10.  Isolation and functional analysis of CONSTANS-LIKE genes suggests that a central role for CONSTANS in flowering time control is not evolutionarily conserved in Medicago truncatula 
The zinc finger transcription factor CONSTANS has a well-established central role in the mechanism for photoperiod sensing in Arabidopsis, integrating light and circadian clock signals to upregulate the florigen gene FT under long-day but not short-day conditions. Although CONSTANS-LIKE (COL) genes in other species have also been shown to regulate flowering time, it is not clear how widely this central role in photoperiod sensing is conserved. Legumes are a major plant group and various legume species show significant natural variation for photoperiod responsive flowering. Orthologs of several Arabidopsis genes have been shown to participate in photoperiodic flowering in legumes, but the possible function of COL genes as integrators of the photoperiod response has not yet been examined in detail. Here we characterize the COL family in the temperate long-day legume Medicago truncatula, using expression analyses, reverse genetics, transient activation assays and Arabidopsis transformation. Our results provide several lines of evidence suggesting that COL genes are unlikely to have a central role in the photoperiod response mechanism in this species.
PMCID: PMC4166892  PMID: 25278955
legume; flowering; photoperiod; Medicago; CONSTANS
11.  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
12.  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
13.  A Morning-Specific Phytohormone Gene Expression Program underlying Rhythmic Plant Growth 
PLoS Biology  2008;6(9):e225.
Most organisms use daily light/dark cycles as timing cues to control many essential physiological processes. In plants, growth rates of the embryonic stem (hypocotyl) are maximal at different times of day, depending on external photoperiod and the internal circadian clock. However, the interactions between light signaling, the circadian clock, and growth-promoting hormone pathways in growth control remain poorly understood. At the molecular level, such growth rhythms could be attributed to several different layers of time-specific control such as phasing of transcription, signaling, or protein abundance. To determine the transcriptional component associated with the rhythmic control of growth, we applied temporal analysis of the Arabidopsis thaliana seedling transcriptome under multiple growth conditions and mutant backgrounds using DNA microarrays. We show that a group of plant hormone-associated genes are coexpressed at the time of day when hypocotyl growth rate is maximal. This expression correlates with overrepresentation of a cis-acting element (CACATG) in phytohormone gene promoters, which is sufficient to confer the predicted diurnal and circadian expression patterns in vivo. Using circadian clock and light signaling mutants, we show that both internal coincidence of phytohormone signaling capacity and external coincidence with darkness are required to coordinate wild-type growth. From these data, we argue that the circadian clock indirectly controls growth by permissive gating of light-mediated phytohormone transcript levels to the proper time of day. This temporal integration of hormone pathways allows plants to fine tune phytohormone responses for seasonal and shade-appropriate growth regulation.
Author Summary
In plants, stems elongate faster at dawn. This time-of-day–specific growth is controlled by integration of environmental cues and the circadian clock. The specific effectors of growth in plants are the phytohormones: auxin, ethylene, gibberellins, abscisic acid, brassinosteroids, and cytokinins. Each phytohormone plays an independent as well as an overlapping role in growth, and understanding the interactions of the phytohormones has dominated plant research over the past century. The authors present a model in which the circadian clock coordinates growth by synchronizing phytohormone gene expression at dawn, allowing a plant to control growth in a condition-specific manner. Furthermore, the results presented provide a new framework for future experiments aimed at understanding the integration and crosstalk of the phytohormones.
Why do plants grow faster at dawn? New results suggest that light and the circadian clock coordinate growth by synchronizing the expression of plant hormone genes at dawn.
PMCID: PMC2535664  PMID: 18798691
14.  Circadian clock-regulated physiological outputs: dynamic responses in nature 
The plant circadian clock is involved in the regulation of numerous processes. It serves as a timekeeper to ensure that the onset of key developmental events coincides with the appropriate conditions. Although internal oscillating clock mechanisms likely evolved in response to the earth’s predictable day and night cycles, organisms must integrate a range of external and internal cues to adjust development and physiology. Here we introduce three different clock outputs to illustrate the complexity of clock control. Clock-regulated diurnal growth is altered by environmental stimuli. The complexity of the photoperiodic flowering pathway highlights numerous nodes through which plants may integrate information to modulate the timing of phenological outputs. Comparative analyses among ecotypes that differ in flowering response reveal additional environmental cues and molecular processes that have developed to influence flowering. We also explore the process of cold acclimation, where circadian inputs, light quality, and stress responses converge to improve freezing tolerance in anticipation of colder temperatures.
PMCID: PMC3742325  PMID: 23435352
circadian clock; photoperiodic flowering; phenology; diurnal growth; cold acclimation; adaptation
15.  The expression of VvPHYA and VvPHYB transcripts is differently regulated by photoperiod in leaves and buds of grapevines 
Plant Signaling & Behavior  2009;4(7):614-616.
Light signals perceived by phytochromes (Phys) and cryptochromes (Crys) play key roles in plant growth and development and in photoperiod dependant process such as flowering, tuberization, seasonal growth cessation and dormancy. The integration of the light signals with the endogenous circadian oscillator provides plants with a mechanism to monitor changes in photo-period or day-length. In a recent report, we established that in Vitis vinifera L. cv Thompson Seedless, photoperiod drives the entrance of buds into endodormancy (ED) and modifies the expression of VvPHYA and VvPHYB transcripts in grapevine leaves, suggesting that both VvPHYs could play crucial roles in SD-induced transition of bud into ED. Here, we aimed to establish whether the transition of grapevine buds into ED is a mere consequence of a decision taken in the leaf or whether the bud responds by itself to photoperiod. Results show that in defoliated grapevine canes, bud-ED development is delayed compared with non-defoliated control canes, and that under LD-photoperiod both VvPHYA and VvPHYB transcripts are highly expressed in grapevine buds, whilst under SD-photoperiod both VvPHYs are downregulated and expression can not be detected. Overall, the results suggest that grapevine bud behaves as semi-autonomous organ in sensing the photoperiod signal, and that VvPHYA and VvPHYB gene expression is differently regulated by photoperiod in leaf and bud of grapevines.
PMCID: PMC2710553  PMID: 19820336
photoperiod; phytochromes; Vitis vinifera
16.  Arabidopsis MSI1 functions in photoperiodic flowering time control 
Appropriate timing of flowering is crucial for crop yield and the reproductive success of plants. Flowering can be induced by a number of molecular pathways that respond to internal and external signals such as photoperiod, vernalization or light quality, ambient temperature and biotic as well as abiotic stresses. The key florigenic signal FLOWERING LOCUS T (FT) is regulated by several flowering activators, such as CONSTANS (CO), and repressors, such as FLOWERING LOCUS C (FLC). Chromatin modifications are essential for regulated gene expression, which often involves the well conserved MULTICOPY SUPRESSOR OF IRA 1 (MSI1)-like protein family. MSI1-like proteins are ubiquitous partners of various complexes, such as POLYCOMB REPRESSIVE COMPLEX2 or CHROMATIN ASSEMBLY FACTOR 1. In Arabidopsis, one of the functions of MSI1 is to control the switch to flowering. Arabidopsis MSI1 is needed for the correct expression of the floral integrator gene SUPPRESSOR OF CO 1 (SOC1). Here, we show that the histone-binding protein MSI1 acts in the photoperiod pathway to regulate normal expression of CO in long day (LD) photoperiods. Reduced expression of CO in msi1-mutants leads to failure of FT and SOC1 activation and to delayed flowering. MSI1 is needed for normal sensitivity of Arabidopsis to photoperiod, because msi1-mutants responded less than wild type to an intermittent LD treatment of plants grown in short days. Finally, genetic analysis demonstrated that MSI1 acts upstream of the CO-FT pathway to enable an efficient photoperiodic response and to induce flowering.
PMCID: PMC3945484  PMID: 24639681
Arabidopsis; flowering time; chromatin; MSI1; photoperiod; FLOWERING LOCUS T (FT); CONSTANS (CO)
17.  Role for Circadian Clock Genes in Seasonal Timing: Testing the Bünning Hypothesis 
PLoS Genetics  2014;10(9):e1004603.
A major question in chronobiology focuses around the “Bünning hypothesis” which implicates the circadian clock in photoperiodic (day-length) measurement and is supported in some systems (e.g. plants) but disputed in others. Here, we used the seasonally-regulated thermotolerance of Drosophila melanogaster to test the role of various clock genes in day-length measurement. In Drosophila, freezing temperatures induce reversible chill coma, a narcosis-like state. We have corroborated previous observations that wild-type flies developing under short photoperiods (winter-like) exhibit significantly shorter chill-coma recovery times (CCRt) than flies that were raised under long (summer-like) photoperiods. Here, we show that arrhythmic mutant strains, per01, tim01 and ClkJrk, as well as variants that speed up or slow down the circadian period, disrupt the photoperiodic component of CCRt. Our results support an underlying circadian function mediating seasonal daylength measurement and indicate that clock genes are tightly involved in photo- and thermo-periodic measurements.
Author Summary
The circadian clock consists of an extensive genetic network that drives daily rhythms of physiological, biochemical and behavioural processes. The network is evolutionary conserved and has been extensively studied in a broad range of organisms. Another genetic network constitutes the photoperiodic clock and monitors the seasonal change in day-length. Here, we address a major and long-standing question in chronobiology: whether the circadian clock is involved in photoperiodic timing, also known as the Bünning hypothesis. Drosophila, as with many other insects in temperate regions, exhibits a photoperiodic response that allows the insect to anticipate and survive the winter. Here we show that the cold-tolerance of the fly is regulated by the photoperiod. We use this phenotype to test day-length timing in various circadian clock mutants and observe that in null clock mutants, the photoperiodic response is abolished, whereas in mutants that exhibit short or long daily cycles, the photoperiodic response is modified, further supporting a circadian-clock function. Overall, these results provide the first evidence in Drosophila that support for the Bünning hypothesis, and pave the way for the genetic dissection of seasonal timing in Drosophila melanogaster.
PMCID: PMC4154681  PMID: 25188283
18.  Regulation of Stomatal Tropism and Infection by Light in Cercospora zeae-maydis: Evidence for Coordinated Host/Pathogen Responses to Photoperiod? 
PLoS Pathogens  2011;7(7):e1002113.
Cercospora zeae-maydis causes gray leaf spot of maize, which has become one of the most widespread and destructive diseases of maize in the world. C. zeae-maydis infects leaves through stomata, which is predicated on the ability of the pathogen to perceive stomata and reorient growth accordingly. In this study, the discovery that light was required for C. zeae-maydis to perceive stomata and infect leaves led to the identification of CRP1, a gene encoding a putative blue-light photoreceptor homologous to White Collar-1 (WC-1) of Neurospora crassa. Disrupting CRP1 via homologous recombination revealed roles in multiple aspects of pathogenesis, including tropism of hyphae to stomata, the formation of appressoria, conidiation, and the biosynthesis of cercosporin. CRP1 was also required for photoreactivation after lethal doses of UV exposure. Intriguingly, putative orthologs of CRP1 are central regulators of circadian clocks in other filamentous fungi, raising the possibility that C. zeae-maydis uses light as a key environmental input to coordinate pathogenesis with maize photoperiodic responses. This study identified a novel molecular mechanism underlying stomatal tropism in a foliar fungal pathogen, provides specific insight into how light regulates pathogenesis in C. zeae-maydis, and establishes a genetic framework for the molecular dissection of infection via stomata and the integration of host and pathogen responses to photoperiod.
Author Summary
Fungal diseases of crop plants are a significant threat to global food security. Improving host resistance is the most cost-effective and environmentally sound strategy for sustainable disease management. However, many devastating diseases of important crops have proven impossible to manage through genetic resistance alone, thus underscoring the need for new strategies to improve resistance. For example, although many fungal pathogens utilize natural openings in leaves to infect host plants, little is known about the underlying molecular dialogue between the host and the pathogen, or whether resistance during this stage of pathogenesis can be improved through breeding. In this study, we explored the ability of a common fungal foliar pathogen of maize to sense and penetrate pores in the leaf epidermis involved in gas exchange (stomata). We determined that light is required for the fungus to sense stomata and identified a blue-light photoreceptor in the fungus that mediates stomatal tropism and infection. Intriguingly, orthologous photoreceptors in other fungi entrain endogenous circadian rhythms, and plants are known to regulate stomatal aperture in response to daily cycles of light and darkness. This study raises the distinct possibility that plants and pathogens coordinate their responses to photoperiod, thus providing a novel insight into fungal pathogenesis.
PMCID: PMC3145785  PMID: 21829344
19.  The Influence of Light Quality, Circadian Rhythm, and Photoperiod on the CBF-Mediated Freezing Tolerance 
Low temperature adversely affects crop yields by restraining plant growth and productivity. Most temperate plants have the potential to increase their freezing tolerance upon exposure to low but nonfreezing temperatures, a process known as cold acclimation. Various physiological, molecular, and metabolic changes occur during cold acclimation, which suggests that the plant cold stress response is a complex, vital phenomenon that involves more than one pathway. The C-Repeat Binding Factor (CBF) pathway is the most important and well-studied cold regulatory pathway that imparts freezing tolerance to plants. The regulation of freezing tolerance involves the action of phytochromes, which play an important role in light-mediated signalling to activate cold-induced gene expression through the CBF pathway. Under normal temperature conditions, CBF expression is regulated by the circadian clock through the action of a central oscillator and also day length (photoperiod). The phytochrome and phytochrome interacting factor are involved in the repression of the CBF expression under long day (LD) conditions. Apart from the CBF regulon, a novel pathway involving the Z-box element also mediates the cold acclimation response in a light-dependent manner. This review provides insights into the progress of cold acclimation in relation to light quality, circadian regulation, and photoperiodic regulation and also explains the underlying molecular mechanisms of cold acclimation for introducing the engineering of economically important, cold-tolerant plants.
PMCID: PMC3709746  PMID: 23722661
CBF regulon; cold acclimation; circadian clock
20.  Circadian clock adjustment to plant iron status depends on chloroplast and phytochrome function 
The EMBO Journal  2012;32(4):511-523.
Plant chloroplasts are not only the main cellular location for storage of elemental iron (Fe), but also the main site for Fe, which is incorporated into chlorophyll, haem and the photosynthetic machinery. How plants measure internal Fe levels is unknown. We describe here a new Fe-dependent response, a change in the period of the circadian clock. In Arabidopsis, the period lengthens when Fe becomes limiting, and gradually shortens as external Fe levels increase. Etiolated seedlings or light-grown plants treated with plastid translation inhibitors do not respond to changes in Fe supply, pointing to developed chloroplasts as central hubs for circadian Fe sensing. Phytochrome-deficient mutants maintain a short period even under Fe deficiency, stressing the role of early light signalling in coupling the clock to Fe responses. Further mutant and pharmacological analyses suggest that known players in plastid-to-nucleus signalling do not directly participate in Fe sensing. We propose that the sensor governing circadian Fe responses defines a new retrograde pathway that involves a plastid-encoded protein that depends on phytochromes and the functional state of chloroplasts.
Circadian clock adjustment to plant iron status depends on chloroplast and phytochrome function
The circadian clock of Arabidopsis is found to be hardwired to cellular iron levels, with chloroplasts playing a central role in iron sensing.
PMCID: PMC3579136  PMID: 23241948
circadian clock; chloroplasts; iron; sensor
21.  HvLUX1 is a candidate gene underlying the early maturity 10 locus in barley: phylogeny, diversity, and interactions with the circadian clock and photoperiodic pathways 
The New Phytologist  2013;199(4):1045-1059.
Photoperiodic flowering is a major factor determining crop performance and is controlled by interactions between environmental signals and the circadian clock. We proposed Hvlux1, an ortholog of the Arabidopsis circadian gene LUX ARRHYTHMO, as a candidate underlying the early maturity 10 (eam10) locus in barley (Hordeum vulgare L.).The link between eam10 and Hvlux1 was discovered using high-throughput sequencing of enriched libraries and segregation analysis. We conducted functional, phylogenetic, and diversity studies of eam10 and HvLUX1 to understand the genetic control of photoperiod response in barley and to characterize the evolution of LUX-like genes within barley and across monocots and eudicots.We demonstrate that eam10 causes circadian defects and interacts with the photoperiod response gene Ppd-H1 to accelerate flowering under long and short days. The results of phylogenetic and diversity analyses indicate that HvLUX1 was under purifying selection, duplicated at the base of the grass clade, and diverged independently of LUX-like genes in other plant lineages.Taken together, these findings contribute to improved understanding of the barley circadian clock, its interaction with the photoperiod pathway, and evolution of circadian systems in barley and across monocots and eudicots.
PMCID: PMC3902989  PMID: 23731278
barley (Hordeum vulgare); circadian clock; early maturity 10; gene duplication; LUX ARRHYTHMO; photoperiodic flowering; Ppd-H1
22.  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
23.  Photoperiodic Regulation of Flowering Time through Periodic Histone Deacetylation of the Florigen Gene FT 
PLoS Biology  2013;11(9):e1001649.
The seasonal cue day length regulates the timing of the floral transition in plants through periodic histone modifications of the FT gene, which encodes a flowering signal in plants. These modifications dampen FT expression at dusk to prevent precocious flowering.
The developmental transition from a vegetative to a reproductive phase (i.e., flowering) is timed by the seasonal cue day length or photoperiod in many plant species. Through the photoperiod pathway, inductive day lengths trigger the production of a systemic flowering signal, florigen, to provoke the floral transition. FLOWERING LOCUS T (FT), widely conserved in angiosperms, is a major component of the mobile florigen. In the long-day plant Arabidopsis, FT expression is rhythmically activated by the output of the photoperiod pathway CONSTANS (CO), specifically at the end of long days. How FT expression is modulated at an adequate level in response to the long-day cue to set a proper flowering time remains unknown. Here, we report a periodic histone deacetylation mechanism for the photoperiodic modulation of FT expression. We have identified a plant-unique core structural component of an Arabidopsis histone deacetylase (HDAC) complex. In long days, this component accumulates at dusk, and is recruited by a MADS-domain transcription factor to the FT locus specifically at the end of the day, leading to periodic histone deacetylation of FT chromatin at dusk. Furthermore, we found that at the end of long days CO activity not only activates FT expression but also enables HDAC-activity recruitment to FT chromatin to dampen the level of FT expression, and so prevent precocious flowering in response to the inductive long-day cue. These results collectively reveal a periodic histone deacetylation mechanism for the day-length control of flowering time in higher plants.
Author Summary
The timing of the developmental transition from a vegetative to a reproductive phase is critically important for reproductive success in flowering plants. Plants synchronize the timing of their floral transition with the changing seasons to flower at a suitable time. The change in day length, or photoperiod, is a key seasonal cue, especially at high latitudes. It is through the photoperiod pathway that day lengths trigger the production of a systemic flowering signal, florigen, to induce the transition from vegetative to reproductive growth and flowering. The FT protein is a major component of the mobile florigen signal. In the model flowering plant Arabidopsis, FT mRNA expression is rhythmically activated by the gene CONSTANS, which is the output of the photoperiod pathway, specifically at the end of long days. In this study, we aimed to address how the level of FT expression is modulated in response to the long-day cue to set the appropriate flowering time. We show that on long days a transcription factor recruits histone deacetylase activity to remove acetyl marks from histones at the FT gene specifically at dusk, thereby dampening FT mRNA expression upon its transcriptional activation by CONSTANS. This sets the correct level of FT expression at the end of long days, and thus prevents precocious flowering in response to the long-day cue.
PMCID: PMC3760768  PMID: 24019760
24.  Potent Induction of Arabidopsis thaliana Flowering by Elevated Growth Temperature 
PLoS Genetics  2006;2(7):e106.
The transition to flowering is an important event in the plant life cycle and is modulated by several environmental factors including photoperiod, light quality, vernalization, and growth temperature, as well as biotic and abiotic stresses. In contrast to light and vernalization, little is known about the pathways that mediate the responses to other environmental variables. A mild increase in growth temperature, from 23 °C to 27 °C, is equally efficient in inducing flowering of Arabidopsis plants grown in 8-h short days as is transfer to 16-h long days. There is extensive natural variation in this response, and we identify strains with contrasting thermal reaction norms. Exploiting this natural variation, we show that FLOWERING LOCUS C potently suppresses thermal induction, and that the closely related floral repressor FLOWERING LOCUS M is a major-effect quantitative trait locus modulating thermosensitivity. Thermal induction does not require the photoperiod effector CONSTANS, acts upstream of the floral integrator FLOWERING LOCUS T, and depends on the hormone gibberellin. Analysis of mutants defective in salicylic acid biosynthesis suggests that thermal induction is independent of previously identified stress-signaling pathways. Microarray analyses confirm that the genomic responses to floral induction by photoperiod and temperature differ. Furthermore, we report that gene products that participate in RNA splicing are specifically affected by thermal induction. Above a critical threshold, even small changes in temperature can act as cues for the induction of flowering. This response has a genetic basis that is distinct from the known genetic pathways of floral transition, and appears to correlate with changes in RNA processing.
When to flower is an important decision in the life cycle of a plant, as it determines the plant's reproductive success. Not surprisingly, plants closely monitor the state of their life cycle along with the external environment in order to determine the onset of flowering. Several factors including light, temperature, and abiotic stress are known to affect the timing of flowering. The authors show that growth temperatures above a finely tuned threshold can rapidly trigger flowering, bypassing the need for other inductive stimuli such as day length. Exploiting a combination of Mendelian genetics, natural variation, and genomics, they show thermal induction of flowering to have a unique genetic basis. Genomic responses to temperature and light during floral induction differ, and temperature-specific changes include alterations in RNA processing.
PMCID: PMC1487179  PMID: 16839183
25.  Mapping-by-Sequencing Identifies HvPHYTOCHROME C as a Candidate Gene for the early maturity 5 Locus Modulating the Circadian Clock and Photoperiodic Flowering in Barley 
Genetics  2014;198(1):383-396.
Phytochromes play an important role in light signaling and photoperiodic control of flowering time in plants. Here we propose that the red/far-red light photoreceptor HvPHYTOCHROME C (HvPHYC), carrying a mutation in a conserved region of the GAF domain, is a candidate underlying the early maturity 5 locus in barley (Hordeum vulgare L.). We fine mapped the gene using a mapping-by-sequencing approach applied on the whole-exome capture data from bulked early flowering segregants derived from a backcross of the Bowman(eam5) introgression line. We demonstrate that eam5 disrupts circadian expression of clock genes. Moreover, it interacts with the major photoperiod response gene Ppd-H1 to accelerate flowering under noninductive short days. Our results suggest that HvPHYC participates in transmission of light signals to the circadian clock and thus modulates light-dependent processes such as photoperiodic regulation of flowering.
PMCID: PMC4174949  PMID: 24996910
barley; circadian clock; photoperiod; flowering; phytochrome

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