Arabidopsis Circadian Clock Research Articles

Organ specificity and communication in the Arabidopsis circadian clock

Organ specificity and communication in the Arabidopsis circadian clock

Temperature compensation of the Arabidopsis circadian clock

Temperature compensation of the Arabidopsis circadian clock

Disruption of the Arabidopsis circadian clock is responsible for extensive variation in the cold-responsive transcriptome.

In plants, low temperature causes massive transcriptional changes, many of which are presumed to be involved in the process of cold acclimation. Given the diversity of developmental and environmental factors between experiments, it is surprising that their influence on the identification of cold-responsive genes is largely unknown. A systematic investigation of genes responding to 1 d of cold treatment revealed that diurnal- and circadian-regulated genes are responsible for the majority of the substantial variation between experiments. This is contrary to the widespread assumption that these effects are eliminated using paired diurnal controls. To identify the molecular basis for this variation, we performed targeted expression analyses of diurnal and circadian time courses in Arabidopsis (Arabidopsis thaliana). We show that, after a short initial cold response, in diurnal conditions cold reduces the amplitude of cycles for clock components and dampens or disrupts the cycles of output genes, while in continuous light all cycles become arrhythmic. This means that genes identified as cold-responsive are dependent on the time of day the experiment was performed and that a control at normal temperature will not correct for this effect, as was postulated up to now. Time of day also affects the number and strength of expression changes for a large number of transcription factors, and this likely further contributes to experimental differences. This reveals that interactions between cold and diurnal regulation are major factors in shaping the cold-responsive transcriptome and thus will be an important consideration in future experiments to dissect transcriptional regulatory networks controlling cold acclimation. In addition, our data revealed differential effects of cold on circadian output genes and a unique regulation of an oscillator component, suggesting that cold treatment could also be an important tool to probe circadian and diurnal regulatory mechanisms.

FIONA1 Is Essential for Regulating Period Length in theArabidopsisCircadian Clock

In plants, the circadian clock controls daily physiological cycles as well as daylength-dependent developmental processes such as photoperiodic flowering and seedling growth. Here, we report that FIONA1 (FIO1) is a genetic regulator of period length in the Arabidopsis thaliana circadian clock. FIO1 was identified by screening for a mutation in daylength-dependent flowering. The mutation designated fio1-1 also affects daylength-dependent seedling growth. fio1-1 causes lengthening of the free-running circadian period of leaf movement and the transcription of various genes, including the central oscillators CIRCADIAN CLOCK-ASSOCIATED1, LATE ELONGATED HYPOCOTYL, TIMING OF CAB EXPRESSION1, and LUX ARRHYTHMO. However, period lengthening is not dependent upon environmental light or temperature conditions, which suggests that FIO1 is not a simple input component of the circadian system. Interestingly, fio1-1 exerts a clear effect on the period length of circadian rhythm but has little effect on its amplitude and robustness. FIO1 encodes a novel nuclear protein that is highly conserved throughout the kingdoms. We propose that FIO1 regulates period length in the Arabidopsis circadian clock in a close association with the central oscillator and that the circadian period can be controlled separately from amplitude and robustness.

PRR3 Is a vascular regulator of TOC1 stability in the Arabidopsis circadian clock.

The pseudoresponse regulators (PRRs) participate in the progression of the circadian clock in Arabidopsis thaliana. The founding member of the family, TIMING OF CAB EXPRESSION1 (TOC1), is an essential component of the transcriptional network that constitutes the core mechanism of the circadian oscillator. Recent data suggest a role in circadian regulation for all five members of the PRR family; however, the molecular function of TOC1 or any other PRRs remains unknown. In this work, we present evidence for the involvement of PRR3 in the regulation of TOC1 protein stability. PRR3 was temporally coexpressed with TOC1 under different photoperiods, yet its tissue expression was only partially overlapping with that of TOC1, as PRR3 appeared restricted to the vasculature. Decreased expression of PRR3 resulted in reduced levels of TOC1 protein, while overexpression of PRR3 caused an increase in the levels of TOC1, all without affecting the amount of TOC1 transcript. PRR3 was able to bind to TOC1 in yeast and in plants and to perturb TOC1 interaction with ZEITLUPE (ZTL), which targets TOC1 for proteasome-dependent degradation. Together, our results indicate that PRR3 might function to modulate TOC1 stability by hindering ZTL-dependent TOC1 degradation, suggesting the existence of local regulators of clock activity and adding to the growing importance of posttranslational regulation in the design of circadian timing mechanisms in plants.

PRR7 protein levels are regulated by light and the circadian clock in Arabidopsis

Interlocking transcriptional loops and regulated protein degradation are the principal mechanisms involved in the generation of self-sustaining circadian rhythms in many organisms. In Arabidopsis the first proposed regulatory transcriptional loop involved the transcription factors CIRCADIAN CLOCK ASSOCIATED (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) and the pseudo-response regulator TIMING OF CHLOROPHYLL A/B BINDING PROTEIN (TOC1/PRR1). Recent findings indicate that the TOC1 homologues PRR7 and PRR9 might also be involved in transcriptional regulatory loops with CCA1 and LHY. In this study we show that the overexpression of PRR7 in Arabidopsis leads to severely compromised circadian rhythms. These transgenic lines display significantly reduced levels of CCA1 and LHY RNA, providing further evidence for a transcriptional feedback loop between PRR7 and these transcription factors. In addition, we show that the PRR7 protein is phosphorylated in a circadian regulated manner and that its levels are post-translationally regulated by both diurnal and circadian mechanisms. The Arabidopsis circadian oscillator is therefore likely to be entrained to light/dark cycles both through transcriptional and post-transcriptional mechanisms.

CIRCADIAN CLOCK ASSOCIATED1 Transcript Stability and the Entrainment of the Circadian Clock in Arabidopsis

The circadian clock is an endogenous mechanism that generates rhythms with an approximately 24-h period and enables plants to predict and adapt to daily and seasonal changes in their environment. These rhythms are generated by molecular oscillators that in Arabidopsis (Arabidopsis thaliana) have been shown to consist of interlocking feedback loops involving a number of elements. An important characteristic of circadian oscillators is that they can be entrained by daily environmental changes in light and temperature. Previous work has shown that one possible entrainment point for the Arabidopsis oscillator is the light-mediated regulation of expression of one of the oscillator genes, CIRCADIAN CLOCK ASSOCIATED1 (CCA1). In this article, we have used transgenic plants with constitutive CCA1 expression to show that light also regulates CCA1 transcript stability. Our experiments show that CCA1 messenger RNA is relatively stable in the dark and in far-red light but has a short half-life in red and blue light. Furthermore, using transgenic plants expressing chimeric CCA1 constructs, we demonstrate that the instability determinants in CCA1 transcripts are probably located in the coding region. We suggest that the combination of light regulation of CCA1 transcription and CCA1 messenger RNA degradation is important for ensuring that the Arabidopsis circadian oscillator is accurately entrained by environmental changes.

Mutations in CHLOROPLAST RNA BINDING provide evidence for the involvement of the chloroplast in the regulation of the circadian clock in Arabidopsis

The Arabidopsis circadian system regulates the expression of up to 36% of the nuclear genome, including many genes that encode photosynthetic proteins. The expression of nuclear-encoded photosynthesis genes is also regulated by signals from the chloroplasts, a process known as retrograde signaling. We have identified CHLOROPLAST RNA BINDING (CRB), a putative RNA-binding protein, and have shown that it is important for the proper functioning of the chloroplast. crb plants are smaller and paler than wild-type plants, and have altered chloroplast morphology and photosynthetic performance. Surprisingly, mutations in CRB also affect the circadian system, altering the expression of both oscillator and output genes. In order to determine whether the changes in circadian gene expression are specific to mutations in the CRB gene, or are more generally caused by the malfunctioning of the chloroplast, we also examined the circadian system in mutations affecting STN7, GUN1, and GUN5, unrelated nuclear-encoded chloroplast proteins known to be involved in retrograde signaling. Our results provide evidence that the functional state of the chloroplast may be an important factor that affects the circadian system.

Pea LATE BLOOMER1 is a GIGANTEA ortholog with roles in photoperiodic flowering, deetiolation, and transcriptional regulation of circadian clock gene homologs.

Genes controlling the transition to flowering have been studied in several species, including Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), but have not yet received much attention in legumes. Here, we describe a new allelic series of late-flowering, photoperiod-insensitive mutants in the pea (Pisum sativum) LATE BLOOMER1 (LATE1) gene and show that LATE1 is an ortholog of Arabidopsis GIGANTEA. Mutants display defects in phytochrome B-dependent deetiolation under red light and in the diurnal regulation of pea homologs of several Arabidopsis circadian clock genes, including TIMING OF CAB1, EARLY FLOWERING4, and CIRCADIAN CLOCK ASSOCIATED1/LATE ELONGATED HYPOCOTYL. LATE1 itself shows strongly rhythmic expression with a small but distinct acute peak following dark-to-light transfer. Mutations in LATE1 prevent the induction of a FLOWERING LOCUS T (FT) homolog FTL in long days but cause only minor alteration to the rhythmic expression pattern of the only known group Ia CONSTANS homolog COLa. The late-flowering phenotype of late1 mutants can be completely rescued by grafting to the wild type, but this rescue is not associated with a significant increase in FTL transcript level in shoot apices. Genetic interactions of late1 with the photoperiod-insensitive, early-flowering sterile nodes (sn) mutant and impairment of the LATE1 diurnal expression rhythm in sn plants suggest that SN may also affect the circadian clock. These results show that several functions of Arabidopsis GIGANTEA are conserved in its pea ortholog and demonstrate that genetic pathways for photoperiodic flowering are likely to be conserved between these two species. They also suggest that in addition to its role in the floral transition, LATE1 also acts throughout reproductive development.

In silico analysis of the endogenous time-keeping mechanism in citrus

The endogenous time-keeping mechanism is responsible for organizing plant physiology and metabolism according to periodic environmental changes, such as diurnal cycles of light and dark and seasonal progression throughout the year. In plants, circadian rhythms control gene expression, stomatal opening, and the timing component of the photoperiodic responses, leading to enhanced fitness due to increased photosynthetic rates and biomass production. We have investigated the citrus genome databases of expressed sequence tags (EST) in order to identify genes coding for functionally characterized proteins involved in the endogenous time-keeping mechanism in Arabidopsis thaliana. Approximately 180,000 EST sequences from 53 libraries were investigated and 81 orthologs of clock components were identified. We found that the vast majority of Arabidopsis circadian clock genes are present in citrus species, although some important components are absent such as SRR1 and PRR5. Based on the identified transcripts, a model for the endogenous oscillatory mechanism of citrus is proposed. These results demonstrate the power of comparative genomics between model systems and economically important crop species to elucidate several aspects of plant physiology and metabolism.

Systems biology flowering in the plant clock field

Mol Syst Biol. 2: 60 In the 18th Century, the Swedish botanist Karl von Linne designed a ‘Flower‐Clock’ by arranging a series of various plant species according to the respective time their flowers open or close every day. Watching this ‘Flower‐Clock’, one can then estimate the time of the day by noting the pattern of flower opening and closing. It has been a well‐known fact since Linne's early times that plants can open or close their flowers at a precise time of the day. However, we still do not fully understand the design principles of the molecular network that underlies the cellular circadian clock, which achieves to predict, often with exquisite precision, the cyclic changes in the environment due to the rotation of earth. In two articles currently published in Molecular Systems Biology , Millar and co‐workers (Locke et al , 2006) and Doyle and co‐workers (Zeilinger et al , 2006) propose a plausible design for the plant circadian clock. In previous work, Millar and co‐workers extended an initial ‘one‐loop model’ of the plant circadian clock into a ‘two‐loop model’ (Figure 1) (Locke et al , 2005). In the simple ‘one‐loop model’ (Figure 1 …

Arabidopsis Response Regulators ARR3 and ARR4 Play Cytokinin-Independent Roles in the Control of Circadian Period

Light and temperature are potent environmental signals used to synchronize the circadian oscillator with external time and photoperiod. Phytochrome and cryptochrome photoreceptors integrate light quantity and quality to modulate the pace and phase of the clock. PHYTOCHROME B (phyB) controls period length in red light as well as the phase of the clock in white light. phyB interacts with ARABIDOPSIS RESPONSE REGULATOR4 (ARR4) in a light-dependent manner. Accordingly, we tested ARR4 and other members of the type-A ARR family for roles in clock function and show that ARR4 and its closest relative, ARR3, act redundantly in the Arabidopsis thaliana circadian system. Loss of ARR3 and ARR4 lengthens the period of the clock even in the absence of light, demonstrating that they do so independently of active phyB. In addition, in white light, arr3,4 mutants show a leading phase similar to phyB mutants, suggesting that circadian light input is modulated by the interaction of phyB with ARR4. Although type-A ARRs are involved in cytokinin signaling, the circadian defects appear to be independent of cytokinin, as exogenous cytokinin affects the phase but not the period of the clock. Therefore, ARR3 and ARR4 are critical for proper circadian period and define an additional level of regulation of the circadian clock in Arabidopsis.

Functional Characterization of Phytochrome Interacting Factor 3 for the Arabidopsis thaliana Circadian Clockwork

Light, in a quality- and quantity-dependent fashion, induces nuclear import of the plant photoreceptors phytochromes and promotes interaction of these receptors with transcription factors including PHYTOCHROME INTERACTING FACTOR 3 (PIF3). PIF3 was shown to form in vitro a ternary complex with the G-box element of the promoters of LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and the Pfr conformer of phytochromes. CCA1 and LHY together with TIMING OF CAB EXPRESSION 1 (TOC1) constitute a transcriptional feed-back loop that is essential for a functional circadian clock in Arabidopsis. These findings led to the hypothesis that the PIF3-containing ternary complex regulates transcription of light-responsive genes and is involved in phototransduction to the central circadian clockwork. Here we report that (i) overexpression or lack of biologically functional PIF3 does not affect period length of rhythmic gene expression or red-light-induced resetting of the circadian clock and (ii) the transcription of PIF3 displays a low-amplitude circadian rhythm. We demonstrated previously that irradiation of etiolated seedlings induces rapid, phytochrome-controlled degradation of PIF3. Here we show that nuclear-localized PIF3 accumulates to relatively high levels by the end of the light phase in seedlings grown under diurnal conditions. Taken together, we show that (i) PIF3 does not play a significant role in controlling light input to and function of the circadian clockwork and (ii) a yet unknown mechanism limits phytochrome-induced degradation of PIF3 at the end of the day under diurnal conditions.

ELF4 is a phytochrome‐regulated component of a negative‐feedback loop involving the central oscillator components CCA1 and LHY

Evidence has been presented that a negative transcriptional feedback loop formed by the genes CIRCADIAN CLOCK ASSOCIATED (CCA1), LATE ELONGATED HYPOCOTYL (LHY) and TIMING OF CAB (TOC1) constitutes the core of the central oscillator of the circadian clock in Arabidopsis. Here we show that these genes are expressed at constant, basal levels in dark-grown seedlings. Transfer to constant red light (Rc) rapidly induces a biphasic pattern of CCA1 and LHY expression, and a reciprocal TOC1 expression pattern over the first 24 h, consistent with initial induction of this synchronous oscillation by the light signal. We have used this assay with wild-type and mutant seedlings to examine the role of these oscillator components, and to determine the function of ELF3 and ELF4 in their light-regulated expression. The data show that whereas TOC1 is necessary for light-induced CCA1/LHY expression, the combined absence of CCA1 and LHY has little effect on the pattern of light-induced TOC1 expression, indicating that the negative regulatory arm of the proposed oscillator is not fully functional during initial seedling de-etiolation. By contrast, ELF4 is necessary for light-induced expression of both CCA1 and LHY, and conversely, CCA1 and LHY act negatively on light-induced ELF4 expression. Together with the observation that the temporal light-induced expression profile of ELF4 is counter-phased to that of CCA1 and LHY and parallels that of TOC1, these data are consistent with a previously unrecognized negative-feedback loop formed by CCA1/LHY and ELF4 in a manner analogous to the proposed CCA1/LHY/TOC1 oscillator. ELF3 is also necessary for light-induced CCA1/LHY expression, but it is neither light-induced nor clock-regulated during de-etiolation. Taken together, the data suggest (a) that ELF3, ELF4, and TOC1 all function in the primary, phytochrome-mediated light-input pathway to the circadian oscillator in Arabidopsis; and (b) that this oscillator consists of two or more interlocking transcriptional feedback loops that may be differentially operative during initial light induction and under steady-state circadian conditions in entrained green plants.

Circadian Control of Messenger RNA Stability. Association with a Sequence-Specific Messenger RNA Decay Pathway

Transcriptional and posttranscriptional regulation are well-established mechanisms for circadian gene expression. Among the latter, differential messenger RNA (mRNA) stability has been hypothesized to control gene expression in response to the clock. However, direct proof that the rate of mRNA turnover can be regulated by the clock is lacking. Previous microarray expression data for unstable mRNAs in Arabidopsis (Arabidopsis thaliana) revealed that mRNA instability is associated with a group of genes controlled by the circadian clock. Here, we show that CCR-LIKE (CCL) and SENESCENCE ASSOCIATED GENE 1 transcripts are differentially regulated at the level of mRNA stability at different times of day. In addition, the changes in CCL mRNA stability continue under free-running conditions, indicating that it is controlled by the Arabidopsis circadian clock. Furthermore, we show that these mRNAs are targets of the mRNA degradation pathway mediated by the downstream (DST) instability determinant. Disruption of the DST-mediated decay pathway in the dst1 mutant leads to aberrant circadian mRNA oscillations that correlate with alterations of the half-life of CCL mRNA relative to parental plants in the morning and afternoon. That this is due to an effect on the circadian control is evidenced by mRNA decay experiments carried out in continuous light. Finally, we show that the defects exhibited by dst mutants are reflected by an impact on circadian regulation at the whole plant level. Together, these results demonstrate that regulation of mRNA stability is important for clock-controlled expression of specific genes in Arabidopsis. Moreover, these data uncover a connection between circadian rhythms and a sequence-specific mRNA decay pathway.

Temperature Entrainment of the Arabidopsis Circadian Clock

Most eukaryotic organisms as well as some prokaryotes (cyanobacteria) have an endogenous circadian clock that keeps track of the earth's 24-h day/night cycle and regulates the activity of numerous metabolic pathways. The existence of the circadian clock as an endogenous feature of organisms and not

DET1 regulates the proteasomal degradation of LHY, a component of the Arabidopsis circadian clock

Multiple photoreceptors contribute to the entrainment of the Arabidopsis circadian clock to daily cycles of light and darkness but little is known of the mechanisms by which these pathways affect the central oscillator. Here we investigate the epistatic interaction between DE-ETIOLATED 1 (DET1), a negative regulator of light-regulated gene expression, and LATE ELONGATED HYPOCOTYL (LHY), one of the core components of the circadian oscillator. The daily onset of LHY gene expression was advanced by approximately 4 h in det1-1 mutant plants, suggesting that the wild-type DET1 protein might function to repress its transcription during the subjective night. lhy-1 det1-1 double mutants exhibited arrhythmic expression of the CAB gene in constant light, similar to the lhy-1 mutant parent. However, additive effects of the lhy-1 and det1-1 mutations on CAB2 expression patterns were revealed under diurnal light-dark cycles. Since the lhy-1 mutation causes aberrant, constitutive transcription of LHY from a constitutive viral promoter, this observation indicated that effects of DET1 were not mediated through the regulation of LHY transcription. Furthermore, the light-driven, rhythmic accumulation of the LHY protein in the lhy-1 mutant was altered by the det1-1 mutation, suggesting that DET1 might regulate LHY expression at the post-transcriptional level. In vitro protein degradation assays demonstrated that the LHY protein is turned over rapidly through the proteasome pathway. Similar degradation was observed whether plant tissue was harvested during the light or dark portion of the diurnal cycle, but the process was significantly accelerated in det1-1 mutant extracts. These results indicate that the wild-type DET1 protein acts to inhibit the proteolytic turnover of the LHY protein, and suggest a mechanism for the period-shortening effect of the det1-1 mutation. These findings add to recent evidence suggesting a role for DET1 in a ubiquitination pathway and identify a substrate for DET1-regulated protein turn-over.

PSEUDO-RESPONSE REGULATOR 7 and 9 are partially redundant genes essential for the temperature responsiveness of the Arabidopsis circadian clock.

Environmental time cues, such as photocycles (light/dark) and thermocycles (warm/cold), synchronize (entrain) endogenous biological clocks to local time. Although much is known about entrainment of the Arabidopsis thaliana clock to photocycles, the determinants of thermoperception and entrainment to thermocycles are not known. The Arabidopsis PSEUDO-RESPONSE REGULATOR (PRR) genes, including the clock component TIMING OF CAB EXPRESSION 1/PRR1, are related to bacterial, fungal, and plant response regulators but lack the conserved Asp that is normally phosphorylated by an upstream sensory kinase. Here, we show that two PRR family members, PRR7 and PRR9, are partially redundant; single prr7-3 or prr9-1 mutants exhibit modest period lengthening, but the prr7-3 prr9-1 double mutant shows dramatic and more than additive period lengthening in the light and becomes arrhythmic in constant darkness. The prr7-3 prr9-1 mutant fails both to maintain an oscillation after entrainment to thermocycles and to reset its clock in response to cold pulses and thus represents an important mutant strongly affected in temperature entrainment in higher plants. We conclude that PRR7 and PRR9 are critical components of a temperature-sensitive circadian system. PRR7 and PRR9 could function in temperature and light input pathways or they could represent elements of an oscillator necessary for the clock to respond to temperature signals.

Is the role of the short-day solely to switch off the CONSTANS in Arabidopsis?

Plants in the genus Arabidopsis are facultative LD plants that flower much earlier under LD conditions than SD regimens, with the photoperiod (or LD) pathway contributing to floral acceleration. LHY and CCA1 genes, among other factors, have central roles in the circadian clock of Arabidopsis, which plays a key role in measuring day length. GI gene mediates the circadian clock and floral activator genes, CO and FT, to control photoperiodic flowering. GI is required to set the peak phase of CO expression at the end of the light period under LD conditions, so that the CO protein is stabilized and activated by light to increase FT expression. However, recent studies have demonstrated that the role of SDs is not solely to switch off CO activity. For example, GI interacts with SPY, a negative regulator of the GA signal. The flowering times of gi mutants were still significantly later under SD conditions than LD regimes, which suggests that GI has a potential role in accelerating the start of flowering, even under SDs. Over-expression of either FT or TSF genes caused early flowering, and the acceleration of flowering was enhanced under SDs, suggesting that SDs have an additional role to that in the LHY/CCA1-GI-CO-FT pathway. In this short review, we discuss the hidden roles of SDs in controlling flowering based on recent studies of the molecular genetics of flowering time in Arabidopsis.

Overlapping and Distinct Roles of PRR7 and PRR9 in the Arabidopsis Circadian Clock

The core mechanism of the circadian oscillators described to date rely on transcriptional negative feedback loops with a delay between the negative and the positive components . In plants, the first suggested regulatory loop involves the transcription factors CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) and the pseudo-response regulator TIMING OF CAB EXPRESSION 1 (TOC1/PRR1). TOC1 is a member of the Arabidopsis circadian-regulated PRR gene family . Analysis of single and double mutants in PRR7 and PRR9 indicates that these morning-expressed genes play a dual role in the circadian clock, being involved in the transmission of light signals to the clock and in the regulation of the central oscillator. Furthermore, CCA1 and LHY had a positive effect on PRR7 and PRR9 expression levels, indicating that they might form part of an additional regulatory feedback loop. We propose that the Arabidopsis circadian oscillator is composed of several interlocking positive and negative feedback loops, a feature of clock regulation that appears broadly conserved between plants, fungi, and animals.