43 How plants tell the time Giovanni Murtas* and Andrew J Millar† The components of the circadian system that have recently match biological time with solar time. As light is an impor- been discovered in plants share some characteristics with tant environmental cue for the entrainment of the those from cyanobacterial, fungal and animal circadian clocks. circadian clock, a long-standing goal has been the identifi- Light input signals to the clock are contributed by multiple cation of the specific photoreceptors that are responsible photoreceptors: some of these have now been shown to for resetting the oscillator [2,3]. The Kay team [4••] has function specifically in response to light of defined wavelength now reported that the phytochromes A and B (phyA and and fluence rate. New reports of clock-controlled processes phyB), and cryptochrome 1 (cry1) are circadian input pho- and genes are highlighting the importance of time management toreceptors. They tested the circadian regulation of the for plant development. clock-responsive CHLOROPHYLL A/B-BINDING PRO- TEIN 2 (CAB2) promoter in Arabidopsis plants carrying Addresses photoreceptor mutations using the firefly luciferase Department of Biological Sciences, University of Warwick, Gibbet Hill reporter gene (luc). They found that the period of the Road, Coventry CV4 7AL, UK CAB2::luc activity rhythm is shortened under constant *e-mail: [email protected] light — a response that is mediated by the photoreceptor †e-mail: [email protected] classes that are sensitive to red and blue light [3]. Current Opinion in Plant Biology 2000, 3:43–46 Measurements of periodicity under a range of light inten- 1369-5266/00/$ — see front matter © 2000 Elsevier Science Ltd. sities in plants that lack a single photoreceptor species (e.g. All rights reserved. phyA, phyB, cry1 or cry2) have now allowed the unique circadian input roles of the individual photoreceptors to be Abbreviations CAB2 CHLOROPHYLL A/B-BINDING PROTEIN 2 characterised. Both phyA and phyB are required for red CAT CATALASE light signalling to the clock at low fluence rates and high CCA1 CIRCADIAN CLOCK ASSOCIATED 1 fluence rates, respectively; whereas both cry1 and phyA CK casein kinase mediate light signalling under blue light of low fluence cry cryptochrome rate. cry1 is also active at high fluence rates of blue light. ELF3 EARLY FLOWERING 3 GI GIGANTEA No single photoreceptor mutant altered the period under LHY LATE ELONGATED HYPOCOTYL intermediate fluence rates, presumably because other phy phytochrome photoreceptors or redundant combinations of the photo- QTL quantitative trait loci receptors tested are functioning in these conditions: TOC TIMING OF CAB EXPRESSION wc white collar 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 Introduction insensitivity of the cry2 mutant is more likely to be caused Time is closely monitored in nature, as many biological by an alteration in cry2-controlled signalling to floral pro- processes are co-ordinated both within each organism and moters such as CONSTANS [4••,5], which might be in relation to the environment. Biological rhythms with modulated by the circadian clock. These results confirm diverse time-scales allow organisms to keep time. The bio- that plants use several photopigments to sense the spec- logical rhythms that are best understood occur with a period trum under different light conditions, such as twilight, of approximately one day and are known as ‘circadian midday sun or deep shade in the evening. Interestingly, rhythms’. These rhythms represent nature’s adaptation to proteins with properties similar to the Arabidopsis cryp- the earth’s 24 h rotation and its associated rhythms of light tochromes have been identified in Drosophila, humans and and temperature. Most species, from cyanobacteria to mice [6]: the Drosophila cryptochrome (dCRY) affects cir- humans, circadian clocks share fundamental properties: a cadian entrainment [7•]; the function of the human self-sustaining oscillator that generates the 24 h rhythm, cryptochrome remains unclear [8]; and recent evidence input pathways through which light signals reset or entrain suggests a role for the mouse cryptochrome as a central the oscillator and output pathways that connect the oscilla- component of the clock mechanism [9•]. tor to the clock-regulated processes in the cell [1]. In this review, we will focus on progress in research on plant circa- A variety of non-photic signals are known to entrain cir- dian rhythms achieved over the past year, all of which was cadian rhythms in many species, including plants: for made possible by molecular and genetic experiments using example, seed germination sets the phase of rhythmic the plant model system Arabidopsis thaliana. CAB gene expression in dark-grown seedlings [10]. In contrast, the expression of other genes, such as CATA- Matching local time LASE (CAT3) [11], was not rhythmic in plants grown To function as a circadian clock, the oscillator must be under constant conditions, until a light/dark or tempera- entrained to daily light and temperature cycles so as to ture stimulus was applied [12,13]. A tentative synthesis of 44 Growth and development these results from different species suggested that a cir- The next step in characterising CCA1 has been taken by cadian system was probably functioning in such plants; the Tobin group [20•]; using a yeast two-hybrid interac- for reasons that are not yet clear, the rhythmic regulation tion screen, they found a regulatory β subunit (CKB3) of did not extend to all possible targets. McClung and co- the protein kinase casein kinase 2 (CK2) that interacts workers have now demonstrated both rhythmic and with, and stimulates, the phosphorylation of the CCA1 arhythmic patterns the transcription of the catalase multi- protein in vitro [20•]. They found that the CCA1–CKB3 gene family of Arabidopsis [14]. Although CAT3 RNA interaction stimulated the binding of recombinant CCA1 accumulated arhythmically in dark-grown seedlings, the to DNA in vitro, but phosphorylation did not. They also circadian clock rhythmically gated the induction of CAT2 reported that CK2-like activity promotes the formation mRNA by light, indicating that a functional circadian sys- of a CCA1–DNA complex within a plant extract, howev- tem was present at this stage of development. After er, suggesting that it might modulate CCA1 function in varying the time at which seeds were released from strat- vivo. Rhythmic phosphorylation is part of at least one cir- ification (i.e. a shift from 4°C to 22°C), the authors cadian output pathway in plants that mediates the concluded that seed imbibition was the non-photic stim- circadian control of certain enzyme activities [21], but ulus that synchronised the circadian rhythms of seedlings there has been no evidence for protein kinase involve- [14]. Imbibition is probably equivalent to the ‘big bang’ ment in the oscillator mechanism. In contrast, in for plant rhythm research, as reliable circadian studies of Drosophila, the phosphorylation of circadian clock com- seeds before imbibition will be difficult. ponents has already been shown to play an important role. The fly gene doubletime, which is required for circa- The timekeepers dian rhythmicity, encodes a protein that is related to a Little is known about the molecular basis of plant circadi- human protein kinase, though it is not a CK2 [22]. an oscillators; however, both the Coupland group [15], Physiologically, the CCA1–CK2 interaction might either studying flowering time, and the Tobin group [16], focus- permit a kinase–substrate interaction or sequester one of ing on light-regulated gene expression, have recently the partners. Recent studies of phyA provide an inter- come very close to characterizing the clock mechanism. esting analogy: a two-hybrid interaction partner of phyA They have identified the genes LATE ELONGATED the cytoplasmic protein, PKS1 is phosphorylated by HYPOCOTYL (LHY) and CCA1 (CIRCARDIAN CLOCK phyA kinase and may affect the subcellular localisation ASSOCIATED 1), respectively, which encode homologous of the photoreceptor [23]. proteins that share a region of similarity with the c-myb transcription factor family. Both proteins show several fea- The detailed characterisation of TIMING OF CAB tures that are typical of clock components, including the EXPRESSION1-1 (toc1-1), the first circadian rhythm abolition of circadian rhythms when constitutively overex- mutant identified in Arabidopsis, suggests that it func- pressed [15–17]. The strong similarity between LHY and tions in the central oscillator [24]. The cloning of TOC1 CCA1 has, however, raised the possibility that these genes may soon clarify its precise role and its interaction, if any, are functionally redundant. A loss-of-function mutation, with CCA1 and LHY. A new approach to identifying clock cca1, has recently been shown to retain many features of genes in Arabidopsis relies on the natural allelic variation circadian control [18••], albeit with a shortened cycle. The among the many accessions (ecotypes) in this species period alteration demonstrates that CCA1 is not simply an [25••]. Recombinant inbred lines were used to map output signalling component that is downstream of the quantitative trait loci (QTLs) with small effects (up to oscillator but also important for controlling the rate of the 1.2 h) on the circadian period of leaf movement. Two of oscillator, either directly or indirectly. The maintenance of the three major QTLs were isolated in introgression rhythmicity in the cca1 mutant indicates that CCA1 func- lines, confirming their effects and interesting chromoso- tion is not essential for the circadian oscillator, possibly mal locations: one (RALENTANDO) is located just above because other proteins have partially redundant functions toc1 but represents a distinct gene; the location of the (LHY is one candidate). CCA1 functions in the light regu- other (ESPRESSO) overlaps with the clock-regulated lation of gene expression as well as affecting the circadian gene GIGANTEA (see below).