43
The components of the circadian system that have recently been discovered in plants share some characteristics with those from cyanobacterial, fungal and animal circadian clocks. Light input signals to the clock are contributed by multiple photoreceptors: some of these have now been shown to function specifically in response to light of defined wavelength and fluence rate. New reports of clock-controlled processes and genes are highlighting the importance of time management for plant development.
Addresses
Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK
*e-mail: [email protected]
†e-mail: [email protected]
Current Opinion in Plant Biology2000, 3:43–46
1369-5266/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved.
Abbreviations
CAB2 CHLOROPHYLL A/B-BINDING PROTEIN 2 CAT CATALASE
CCA1 CIRCADIAN CLOCK ASSOCIATED 1 CK casein kinase
cry cryptochrome
ELF3 EARLY FLOWERING 3
GI GIGANTEA
LHY LATE ELONGATED HYPOCOTYL phy phytochrome
QTL quantitative trait loci
TOC TIMING OF CAB EXPRESSION wc white collar
Introduction
Time is closely monitored in nature, as many biological processes are co-ordinated both within each organism and in relation to the environment. Biological rhythms with diverse time-scales allow organisms to keep time. The bio-logical rhythms that are best understood occur with a period of approximately one day and are known as ‘circadian rhythms’. These rhythms represent nature’s adaptation to the earth’s 24 h rotation and its associated rhythms of light and temperature. Most species, from cyanobacteria to humans, circadian clocks share fundamental properties: a self-sustaining oscillator that generates the 24 h rhythm, input pathways through which light signals reset or entrain the oscillator and output pathways that connect the oscilla-tor to the clock-regulated processes in the cell [1]. In this review, we will focus on progress in research on plant circa-dian rhythms achieved over the past year, all of which was made possible by molecular and genetic experiments using the plant model system Arabidopsis thaliana.
Matching local time
To function as a circadian clock, the oscillator must be entrained to daily light and temperature cycles so as to
match biological time with solar time. As light is an impor-tant environmental cue for the entrainment of the circadian clock, a long-standing goal has been the identifi-cation of the specific photoreceptors that are responsible for resetting the oscillator [2,3]. The Kay team [4••] has now reported that the phytochromes A and B (phyA and phyB), and cryptochrome 1 (cry1) are circadian input pho-toreceptors. They tested the circadian regulation of the clock-responsive CHLOROPHYLL A/B-BINDING PRO-TEIN 2 (CAB2) promoter in Arabidopsis plants carrying photoreceptor mutations using the firefly luciferase reporter gene (luc). They found that the period of the
CAB2::luc activity rhythm is shortened under constant light — a response that is mediated by the photoreceptor classes that are sensitive to red and blue light [3]. Measurements of periodicity under a range of light inten-sities in plants that lack a single photoreceptor species (e.g. phyA, phyB, cry1 or cry2) have now allowed the unique circadian input roles of the individual photoreceptors to be characterised. Both phyA and phyB are required for red light signalling to the clock at low fluence rates and high fluence rates, respectively; whereas both cry1 and phyA mediate light signalling under blue light of low fluence rate. cry1 is also active at high fluence rates of blue light. No single photoreceptor mutant altered the period under intermediate fluence rates, presumably because other photoreceptors or redundant combinations of the photo-receptors tested are functioning in these conditions: multiple mutations should ultimately recapitulate the peri-od of wild-type plants in darkness. The cry2 mutant had almost no effect on circadian period, so the photoperiod insensitivity of the cry2mutant is more likely to be caused by an alteration in cry2-controlled signalling to floral pro-moters such as CONSTANS [4••,5], which might be modulated by the circadian clock. These results confirm that plants use several photopigments to sense the spec-trum under different light conditions, such as twilight, midday sun or deep shade in the evening. Interestingly, proteins with properties similar to the Arabidopsis cryp-tochromes have been identified in Drosophila, humans and mice [6]: the Drosophila cryptochrome (dCRY) affects cir-cadian entrainment [7•]; the function of the human cryptochrome remains unclear [8]; and recent evidence suggests a role for the mouse cryptochrome as a central component of the clock mechanism [9•].
A variety of non-photic signals are known to entrain cir-cadian rhythms in many species, including plants: for example, seed germination sets the phase of rhythmic
CAB gene expression in dark-grown seedlings [10]. In contrast, the expression of other genes, such as CATA-LASE (CAT3) [11], was not rhythmic in plants grown under constant conditions, until a light/dark or tempera-ture stimulus was applied [12,13]. A tentative synthesis of
How plants tell the time
these results from different species suggested that a cir-cadian system was probably functioning in such plants; for reasons that are not yet clear, the rhythmic regulation did not extend to all possible targets. McClung and co-workers have now demonstrated both rhythmic and arhythmic patterns the transcription of the catalase multi-gene family of Arabidopsis [14]. Although CAT3 RNA accumulated arhythmically in dark-grown seedlings, the circadian clock rhythmically gated the induction of CAT2
mRNA by light, indicating that a functional circadian sys-tem was present at this stage of development. After varying the time at which seeds were released from strat-ification (i.e. a shift from 4°C to 22°C), the authors concluded that seed imbibition was the non-photic stim-ulus that synchronised the circadian rhythms of seedlings [14]. Imbibition is probably equivalent to the ‘big bang’ for plant rhythm research, as reliable circadian studies of seeds before imbibition will be difficult.
The timekeepers
Little is known about the molecular basis of plant circadi-an oscillators; however, both the Couplcircadi-and group [15], studying flowering time, and the Tobin group [16], focus-ing on light-regulated gene expression, have recently come very close to characterizing the clock mechanism. They have identified the genes LATE ELONGATED HYPOCOTYL (LHY) and CCA1 (CIRCARDIAN CLOCK ASSOCIATED 1), respectively, which encode homologous proteins that share a region of similarity with the c-myb
transcription factor family. Both proteins show several fea-tures that are typical of clock components, including the abolition of circadian rhythms when constitutively overex-pressed [15–17]. The strong similarity between LHY and
CCA1has, however, raised the possibility that these genes are functionally redundant. A loss-of-function mutation,
cca1, has recently been shown to retain many features of circadian control [18••], albeit with a shortened cycle. The period alteration demonstrates that CCA1 is not simply an output signalling component that is downstream of the oscillator but also important for controlling the rate of the oscillator, either directly or indirectly. The maintenance of rhythmicity in the cca1 mutant indicates that CCA1 func-tion is not essential for the circadian oscillator, possibly because other proteins have partially redundant functions (LHY is one candidate). CCA1 functions in the light regu-lation of gene expression as well as affecting the circadian oscillator [18••], as do the white collar (wc) genes in
Neurospora [19]. The genes wc-1and wc-2are zinc-finger transcription factors that activate the expression of one of the fungal clock genes, frequency, as well as other light-responsive genes [19]. CCA1 could therefore be a component of a light signalling pathway, providing the molecular link between the phytochromes and the time-keeper, either as part of an output pathway that feeds back to the clock or as part of the central oscillator. The characterisation of the lhy null mutants and the lhy;cca1
double mutant will certainly help to position CCA1 and
LHY in the Arabidopsis circadian system.
The next step in characterising CCA1 has been taken by the Tobin group [20•]; using a yeast two-hybrid interac-tion screen, they found a regulatory βsubunit (CKB3) of the protein kinase casein kinase 2 (CK2) that interacts with, and stimulates, the phosphorylation of the CCA1 protein in vitro [20•]. They found that the CCA1–CKB3 interaction stimulated the binding of recombinant CCA1 to DNA in vitro, but phosphorylation did not. They also reported that CK2-like activity promotes the formation of a CCA1–DNA complex within a plant extract, howev-er, suggesting that it might modulate CCA1 function in vivo. Rhythmic phosphorylation is part of at least one cir-cadian output pathway in plants that mediates the circadian control of certain enzyme activities [21], but there has been no evidence for protein kinase involve-ment in the oscillator mechanism. In contrast, in
Drosophila, the phosphorylation of circadian clock com-ponents has already been shown to play an important role. The fly gene doubletime, which is required for circa-dian rhythmicity, encodes a protein that is related to a human protein kinase, though it is not a CK2 [22]. Physiologically, the CCA1–CK2 interaction might either permit a kinase–substrate interaction or sequester one of the partners. Recent studies of phyA provide an inter-esting analogy: a two-hybrid interaction partner of phyA the cytoplasmic protein, PKS1 is phosphorylated by phyA kinase and may affect the subcellular localisation of the photoreceptor [23].
The detailed characterisation of TIMING OF CAB EXPRESSION1-1 (toc1-1), the first circadian rhythm mutant identified in Arabidopsis, suggests that it func-tions in the central oscillator [24]. The cloning of TOC1
may soon clarify its precise role and its interaction, if any, with CCA1and LHY. A new approach to identifying clock genes in Arabidopsisrelies on the natural allelic variation among the many accessions (ecotypes) in this species [25••]. Recombinant inbred lines were used to map quantitative trait loci (QTLs) with small effects (up to 1.2 h) on the circadian period of leaf movement. Two of the three major QTLs were isolated in introgression lines, confirming their effects and interesting chromoso-mal locations: one (RALENTANDO) is located just above
toc1 but represents a distinct gene; the location of the other (ESPRESSO) overlaps with the clock-regulated gene GIGANTEA (see below). The third QTL was iden-tified independently in two populations and was identified as FLOWERING LOCUS C (FLC), which has recently been shown to encode a putative MADS-box transcription factor [26]. This class of genes has not been associated with circadian timing previously. The QTL approach may allow rapid progress in gene identification; it coincides with the development of genomic sequenc-ing techniques; and the characterisation of many time-to-flowering and light-signalling genes, the func-tions of which often overlap with circadian regulation. It will also provide insight into the evolutionary adaptation of the circadian system.
Are we on time?
Many physiological processes in plants are known to be synchronized with the daily cycle of the environment by circadian rhythms [21,27,28]. Several plant species grow poorly without environmental time cues [29], indicating that even plants, like humans, need to be able to answer the question, ‘’Are we on time?’’ There has recently been a boom in the discovery of circadian-regulated processes in plants, including Arabidopsis. For example, Dowson-Day and Millar [30•] have shown that the circadian clock, as well as an already known wide range of endogenous and environmental factors [6,31,32], regulates the elongation of the seedling hypocotyl. Our video-imaging experiments show that hypocotyls elongate rhythmically under constant light, with maximum growth at subjective dusk and a growth arrest at subjective dawn. The rhythm is entrained by light–dark cycles that are applied to the imbibed seed, and, as are other circadian rhythms, the cycle is shortened in the mutant toc1-1[24]. The mutant early flowering 3 (elf3) and overexpressors of CCA1 and LHY, all of which are pho-toperiod-insensitive and arhythmic in continuous light, also have a distinct long-hypocotyl phenotype [15,16,33,34]. Hypocotyl elongation in elf3 and the LHY
overexpressor lacks the daily growth arrests (and is there-fore arhythmic), suggesting that the long-hypocotyl phenotype of elf3results from a defect in the circadian sys-tem [30•]. These experiments suggest how disruptive a timing defect can be for plant physiology.
Another gene that plays an important role in the photope-riodic control of flowering in Arabidopsis [35] has recently been identified and linked to the circadian clock. GIGAN-TEA (GI), isolated and characterised by the joint efforts of the Putterill and Coupland groups [36••], and indepen-dently by the Nam and Kay groups [37••], encodes a novel, putative membrane protein. GIgene expression is circadi-an-controlled in both light and dark conditions. Some gi
mutations affect the circadian period [37••], and GI inter-acts with other clock-associated genes that are thought to control flowering in a common pathway [36••]. The absence of ELF3 causes arhythmic and upregulated expression of GI in elf3 mutant plants under continuous light and, though to different extents, in long and short photoperiods. The increased expression of GImay explain the early flowering of elf3. In the LHY and CCA1 overex-pression lines, the circadian rhythm of GI is disrupted; more interestingly, the absence of GI causes a dramatic reduction in the expression of the endogenous CCA1and
LHY genes. Clearly, the identification of the proteins that interact with GI may help to clarify its function in this complex regulatory network. It is unclear what function GI may have as a plasma membrane protein — could it be that
GIaffects the cell-to-cell transmission of circadian rhythm signals? Circadian organisation at the cellular level is now particularly interesting. Recent studies indicate that, at the larger scale of entire plant organs, a functionally indepen-dent circadian system is present in each organ (SC Thain, AJ Millar, unpublished data).
Conclusions
The diversity of circadian photoreceptors in plants demonstrates the potential importance of perceiving many wavelengths in order to run the daily timekeepers. It will be interesting to discover whether additional pho-toreceptors, such as phytochromes C, D and E, are also involved in circadian entrainment. LHY and CCA1 are certainly two good candidates for circadian clock compo-nents, and their functions in circadian timing, light-signalling and in the control of flowering may soon be clarified with the isolation and characterisation of sev-eral other clock genes. Newly-identified rhythmic processes suggest the importance of time management in plant development; it remains to be determined whether a plant cell’s circadian schedule includes reset-ting the timekeepers of neighbouring cells.
Acknowledgements
We are very grateful to those colleagues who communicated results prior to publication. Our research on circadian clocks is supported by grants from the Biotechnology and Biological Sciences Research Council, the Human Science Frontiers Program Organisation and the Gatsby Charitable Foundation.
References and recommended reading
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