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. , 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 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 Department of Biological Sciences, University of Warwick, Gibbet Hill reporter (luc). They found that the period of the Road, Coventry CV4 7AL, UK CAB2::luc activity rhythm is shortened under constant *e-mail: pavi@.bio.warwick.ac.uk 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 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 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 . CAB 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 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). The third QTL was iden- oscillator [18••], as do the white collar (wc) genes in tified independently in two populations and was Neurospora [19]. The genes wc-1 and wc-2 are zinc-finger identified as FLOWERING LOCUS C (FLC), which has transcription factors that activate the expression of one of recently been shown to encode a putative MADS-box the fungal clock genes, frequency, as well as other light- transcription factor [26]. This class of genes has not been responsive genes [19]. CCA1 could therefore be a associated with circadian timing previously. The QTL component of a light signalling pathway, providing the approach may allow rapid progress in gene identification; molecular link between the phytochromes and the time- it coincides with the development of genomic sequenc- keeper, either as part of an output pathway that feeds ing techniques; and the characterisation of many back to the clock or as part of the central oscillator. The time-to-flowering and light-signalling genes, the func- characterisation of the lhy null mutants and the lhy ; cca1 tions of which often overlap with circadian regulation. It double mutant will certainly help to position CCA1 and will also provide insight into the evolutionary adaptation LHY in the Arabidopsis circadian system. of the circadian system. How plants tell the time Murtas and Millar 45

Are we on time? Conclusions Many physiological processes in plants are known to be The diversity of circadian photoreceptors in plants synchronized with the daily cycle of the environment by demonstrates the potential importance of perceiving circadian rhythms [21,27,28]. Several plant species grow many wavelengths in order to run the daily timekeepers. poorly without environmental time cues [29], indicating It will be interesting to discover whether additional pho- that even plants, like humans, need to be able to answer toreceptors, such as phytochromes C, D and E, are also the question, ‘’Are we on time?’’ There has recently been involved in circadian entrainment. LHY and CCA1 are a boom in the discovery of circadian-regulated processes in certainly two good candidates for circadian clock compo- plants, including Arabidopsis. For example, Dowson-Day nents, and their functions in circadian timing, and Millar [30•] have shown that the circadian clock, as light-signalling and in the control of flowering may soon well as an already known wide range of endogenous and be clarified with the isolation and characterisation of sev- environmental factors [6,31,32], regulates the elongation of eral other clock genes. Newly-identified rhythmic the seedling hypocotyl. Our video-imaging experiments processes suggest the importance of time management show that hypocotyls elongate rhythmically under constant in plant development; it remains to be determined light, with maximum growth at subjective dusk and a whether a plant cell’s circadian schedule includes reset- growth arrest at subjective dawn. The rhythm is entrained ting the timekeepers of neighbouring cells. by light–dark cycles that are applied to the imbibed seed, and, as are other circadian rhythms, the cycle is shortened Acknowledgements in the mutant toc1-1 [24]. The mutant early flowering 3 () We are very grateful to those colleagues who communicated results prior to publication. Our research on circadian clocks is supported by grants from and overexpressors of CCA1 and LHY, all of which are pho- the Biotechnology and Biological Sciences Research Council, the Human toperiod-insensitive and arhythmic in continuous light, Science Frontiers Program Organisation and the Gatsby Charitable also have a distinct long-hypocotyl phenotype Foundation. [15,16,33,34]. Hypocotyl elongation in elf3 and the LHY overexpressor lacks the daily growth arrests (and is there- References and recommended reading fore arhythmic), suggesting that the long-hypocotyl Papers of particular interest, published within the annual period of review, phenotype of elf3 results from a defect in the circadian sys- have been highlighted as: tem [30•]. These experiments suggest how disruptive a • of special interest timing defect can be for plant physiology. •• of outstanding interest 1. Dunlap JC: Molecular bases for circadian clocks. Cell 1999, 96:271-290. Another gene that plays an important role in the photope- 2. Halaban R: Effects of light quality on the circadian rhythm of leaf riodic control of flowering in Arabidopsis [35] has recently movement of a short day plant. Plant Physiol 1969, 44:973-977. been identified and linked to the circadian clock. GIGAN- 3. Millar AJ, Straume M, Chory J, Chua N-H, Kay SA: The regulation of TEA (GI), isolated and characterised by the joint efforts of circadian period by phototransduction pathways in Arabidopsis. the Putterill and Coupland groups [36••], and indepen- Science 1995, 267:1163-1166. dently by the Nam and Kay groups [37••], encodes a novel, 4. Somers DE, Devlin PF, Kay AS: Phytochromes and cryptochromes putative membrane protein. GI gene expression is circadi- •• in the entrainment of the Arabidopsis circadian clock. Science1998, 282:1488-1490. an-controlled in both light and dark conditions. Some gi The authors measured the effect of light fluence rate on the circadian period mutations affect the circadian period [37••], and GI inter- of Arabidopsis photoreceptor-deficient mutants. Distinct functions of single photoreceptor genes in circadian light input are defined for the first time. The acts with other clock-associated genes that are thought to data presented confirm that plants use a combination of photopigments to control flowering in a common pathway [36••]. The sense the spectrum under different light conditions. absence of ELF3 causes arhythmic and upregulated 5. Guo HW, Yang WY, Mockler TC, Lin CT: Regulation of flowering expression of GI in elf3 mutant plants under continuous time by Arabidopsis photoreceptors. Science 1998, 279:1360-1363. light and, though to different extents, in long and short 6. Cashmore AR, Jarillo JA, Wu YJ, Liu D: Cryptochromes: blue light photoperiods. The increased expression of GI may explain receptors for plants and animals. Science 1999, 284:760-765. the early flowering of elf3. In the LHY and CCA1 overex- 7. Stanewsky R, Kaneko M, Emery P, Beretta B, Wager-Smith K, Kay SA, • Rosbash M, Hall JC: The cryb mutation identifies cryptochrome as pression lines, the circadian rhythm of GI is disrupted; a circadian photoreceptor in Drosophila. Cell 1998, 95:681-692. more interestingly, the absence of GI causes a dramatic The authors identified a cry gene, which is homologous to the plant blue light receptor cryptochrome, by mutation in a novel genetic screen that uses real- reduction in the expression of the endogenous CCA1 and time monitoring of clock gene expression. The cry mutation has defined LHY genes. Clearly, the identification of the proteins that effects on molecules that function within the circadian clock and affects the synchronisation of the fly’s behavioural rhythm. This paper presents the first interact with GI may help to clarify its function in this data suggesting that a cryptochrome molecule in animals is involved, as in complex regulatory network. It is unclear what function GI plants, in circadian entrainment. may have as a plasma membrane protein — could it be that 8. Hsu DS, Zhao XD, Zhao SY, Kasantsev A, Wang RP, Todo T, Wei YF, GI affects the cell-to-cell transmission of circadian rhythm Sancar A: Putative human blue light photoreceptors hCRY1 and hCRY2 are flavoproteins. 35 signals? Circadian organisation at the cellular level is now Biochemistry 1996, :13871-13877. particularly interesting. Recent studies indicate that, at the 9. Kume K, Zylka MJ, Sathyanarayanan S, Shearman PL, Weaver DR, Jin X, • Maywood ES, Hastings MH, Reppert SM: mCRY1 and mCRY2 are larger scale of entire plant organs, a functionally indepen- essential components of the negative limb of the circadian clock dent circadian system is present in each organ (SC Thain, feedback loop. Cell 1999, 98:193-205. At the opposite end of a growing spectrum of references from [7•], transient AJ Millar, unpublished data). expression experiments in tissue culture cells demonstrate that mouse pro- 46 Growth and development

teins related to cryptochrome are important in the nuclear localisation of cir- outputs throughout development in Arabidopsis. Development cadian clock proteins. The mouse CRYs are thus connected to oscillator 1998, 125:485-494. function rather than to light input. 25. Swarup K, Alonso-Blanco C, Lynn JR, Michaels SD, Amasino RM, 10. Kolar C, Adam E, Schafer E, Nagy F: Expression of tobacco genes for •• Koornneef M, Millar AJ: Natural allelic variation identifies new light-harvesting chlorophyll a/b binding-proteins of photosystem-II genes in the Arabidopsis circadian system. Plant J 1999, 20:67-77. is controlled by two circadian oscillators in a developmentally- Presents the results of the first QTL analysis of circadian rhythms that has iden- regulated fashion. Proc Natl Acad Sci USA 1995, 92:2174-2178. tified a candidate clock gene. This method of analysis proved to be efficient; evolutionary adaptations of the circadian system may now be uncovered. 11. McClung CR: The regulation of catalase in Arabidopsis. Free Radic Biol Med 1997, 23:489-496. 26. Michaels S, Amasino R: Flowering Locus C encodes a novel MADS domain protein that acts as a of flowering. Plant Cell 12. Heintzen C, Fischer R, Melzer S, Kappeler S, Apel K, Staiger D: 1999, 11:949-956. Circadian oscillations of a transcript encoding a germin-like protein that is associated with cell walls in young leaves of the 27. Sweeney BM: Rhythmic Phenomena in Plants. San Diego, California: long-days plant Sinapis alba L. Plant Physiol 1994, 106:905-915. Academic Press; 1987. 13. Boldt R, Scandalios JG: Circadian regulation of the Cat3 catalase 28. Webb AAR: Stomatal rhythms.InBiological Rhythms and gene in maize (Zea mays L.): entrainment of the circadian rhythm Photoperiodism in Plants. Edited by Lumsden PJ, Millar AJ. Oxford: of Cat3 by different light treatments. Plant J 1995, 7:989-999. BIOS Scientific; 1998:69-80. 14. Zhong NH, Painter JE, Salome PA, Straume M, McClung CR: 29. Highkin HR, Hanson JB: Possible interactions between light-dark Imbibition, but not release from stratification, sets the circadian cycles and endogenous daily rhythms on the growth of clock in Arabidopsis seedlings. Plant Cell 1998, 10:2005-2017. plants. Plant Physiol 1954, 29:301-302. 15. Schaffer R, Ramsay N, Samach A, Corden S, Putterill J, Carré IA, 30. Dowson-Day MJ, Millar AJ: Circadian dysfunction causes aberrant Coupland G: The late elongate hypocotyl mutation of Arabidopsis • hypocotyl elongation patterns in Arabidopsis. Plant J 1999, disrupts circadian rhythms and the photoperiodic control of 17:63-71. flowering. Cell 1998, 93:1219-1229. An automated video imaging system is used to show that the circadian clock controls hypocotyl elongation in Arabidopsis seedlings, starting as soon as Constitutive expression of the CIRCADIAN 16. Wang ZY, Tobin EM: the seed germinates. Mutations that affect the circadian system cause aber- CLOCK ASSOCIATED 1 (CCA1) gene disrupts circadian rhythms rant hypocotyl elongation patterns, some of which affect the final morpholo- and suppresses its own expression. Cell 1998, 93:1207-1217. gy of the plant. This suggests an explanation for the long hypocotyl 17. Thomas B, Vince-Prue D: Photoperiodism in Plants. San Diego: phenotype of arhythmic mutants such as elf3. Academic Press; 1997. 31. Jensen PJ, Hangarter RP, Estelle M: Auxin transport is required for 18. Green RM, Tobin EM: Loss of the circadian clock-associated hypocotyl elongation in light-grown but not dark-grown •• protein 1 in Arabidopsis results in altered clock-regulated gene Arabidopsis. Plant Physiol 1998, 116:455-462. expression. Proc Natl Acad Sci USA 1999, 96:4176-4179. 32. Chory J: Gibberellins, brassinosteroids and light-regulated A loss-of-function mutation in the CCA1 gene provides further information development. Plant Cell Environ 1997, 20:801-806. about CCA1 function. The cca1 mutation shortens the period of the circadi- an rhythms in four clock-controlled genes and also affects light-activated 33. Zagotta MT, Hicks KA, Jacobs CI, Hangarter RP, Meeks-Wagner DR: gene expression. CCA1 cannot therefore be completely redundant with its The Arabidopsis ELF3 gene regulates vegetative homologue LHY and might be involved in both the input and the output photomorphogenesis and the photoperiodic induction of domains of the circadian system and/or in the oscillator. flowering. Plant J 1996, 10:691-702. 19. Crosthwaite SK, Dunlap JC, Loros JJ: Neurospora wc-1 and wc-2: 34. Hicks KA, Millar AJ, Carré IA, Somers DE, Straume M, Meeks-Wagner DR, transcription, photoresponses, and the origins of circadian Kay SA: Conditional circadian dysfunction of the Arabidopsis rhythmicity. Science 1997, 276:763-769. early-flowering 3 mutant. Science 1996, 274:790-792. 20. Sugano S, Andronis C, Green RM, Wang ZY, Tobin EM: Protein 35. Koorneef M, Alonso-Blanco C, Peeters AJM, Soppe W: Genetic • kinase CK2 interacts with and phosphorylates the Arabidopsis control of flowering time in Arabidopsis. Annu Rev Plant Physiol circadian clock-associated1 protein. Proc Natl Acad Sci USA Plant Mol Biol 1998, 49:345-370. 1998, 95:11020-11025. The authors used a yeast two-hybrid screen to identify a regulatory subunit of the 36. Fowler S, Lee K, Onouchi H, Samach A, Richardson K, Morris B, protein kinase CK2 by its interaction with CCA1. Biochemical experiments indi- •• Coupland G, Putterill J: GIGANTEA: a circadian clock-controlled cate that the protein kinase can modulate CCA1 activity in vitro both by direct gene that regulates photoperiodic flowering in Arabidopsis and interaction and by phosphorylation of the CCA1 protein. These findings open up encodes a protein with several possible membrane-spanning the possibility that CK2 functions in the plant clock or in light regulation. domains. EMBO J 1999, 18:4679-4688. Another component of the photoperiodic regulatory network is identified by 21. McClung CR, Kay SA: Circadian rhythms in the higher plant, cloning a gene that is involved in the regulation of flowering time. Uniquely, it Arabidopsis thaliana. In Arabidopsis thaliana. Edited by Sommerville encodes a membrane protein, which suggests a possible role for GI in inter- CS, Meyerowitz E. Cold Spring Harbor, New York: Cold Spring cellular signalling. GI expression is studied in detail and shown to be con- Harbor Press; 1994:615-637. trolled by light and the circadian clock in a manner consistent with its function 22. Kloss B, Price JL, Saez L, Blau J, Rothenfluh A, Wesley CS, Young MW: in photoperiodic regulation. One interesting point highlighted in this paper is The Drosophila clock gene double-time encodes a protein closely the mutual control of GI and the clock-associated genes LHY and CCA1. related to human casein Ie. Cell 1998, 94:97-107. 37. Park DH, Somers DE, Kim YS, Choy YH, Lim HK, Soh MS, Kim HJ, •• 23. Fankhauser C, Yeh KC, Lagarias JC, Zhang H, Elich TD, Chory J: Kay SA, Nam HG: Control of circadian rhythms and photoperiodic PKS1, a substrate phosphorylated by phytochrome that modulates flowering by the Arabidopsis GIGANTEA gene. Science 1999, light signaling in Arabidopsis. Science 1999, 284:1539-1541. 285:1579-1582. Presents a contemporary cloning of GI and characterisation of the complex 24. Somers DE, Webb AAR, Pearson M, Kay SA: The short-period effects of mutant alleles upon the Arabidopsis circadian system. Importantly, mutant toc1-1, alters circadian clock regulation of multiple this paper suggests that gi mutations affect the input pathway to the clock.