Putative Components of an Arabidopsis Circadian Clock

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synthase; this provided confirmation that sor- active discussions, and fulfilled one of the aims Sally Smith* bitol is involved in the mobility of boron of the symposium series: to ensure effective The Centre for Plant Root Symbiosis and Dept (Patrick H. Brown, UC Davis, USA). communication between pure and applied plant of Soil Science, The University of Adelaide, Several papers described screens to identify nutritionists and plant breeders. In essence, SA 5064, Australia traits relating to plant nutrition in wild acces- plant breeders like it ‘simple, crude and cheap’, sions, old cultivars, land races and modern with an emphasis on field selection, particu- Andrew Smith genotypes. The development of markers for larly in relation to yield. By contrast, plant physi- The Centre for Plant Root Symbiosis and Dept these traits and the usefulness of marker- ologists are reductionists, i.e. more interested of Botany, The University of Adelaide, assisted breeding were also discussed. Differ- in mechanistic details of the processes. More SA 5064, Australia ences in the approach used by physiologists meetings of this kind, in particular involving and plant breeders surfaced repeatedly and led large numbers of geneticists and plant breeders, *Author for correspondence to vigorous discussions. The convivial atmos- will be vital to bring the two camps together and (tel ϩ61 8 8303 7210; fax ϩ61 8 8303 6511; phere played a large part in generating these to optimize nutrition for plant yield and quality. e-mail [email protected])

It’s about time: putative components of an Arabidopsis circadian clock

Circadian rhythms are a widespread biologi- The plant clock ASSOCIATED 1 (CCA1) was first identified cal phenomenon and have been described for What then of plant clock components? In spite as an MYB-related transcription factor that both prokaryotes and eukaryotes. The rhythms of the historical importance of plants to the binds to a region of the Arabidopsis CAB1 are characterized by period lengths of ap- scientific study of circadian rhythms5, the (Lhcb1*3) promoter necessary for phytochrome proximately 24 h, and the normal environ- molecular components of plant clocks remain responsiveness11. The binding target of CCA1 mental cycles of light and temperature provide unknown and we cannot yet predict the extent (consensus AA[A/C]AATCT) in the CAB1 pro- temporal information that entrains (resets) the to which plant clocks will resemble the fun- moter is closely related to a region of the CAB2 biological clock. Within the laboratory it is gal, animal or cyanobacterial equivalents. In promoter that is sufficient to confer circadian possible to deprive organisms of environmen- Arabidopsis, a genetic screen based on alter- transcription on a luc reporter gene12, and CCA1 tal time cues, and it is the persistence of cir- ations in rhythmic expression of a luciferase also binds to this 36 bp clock regulatory re- cadian rhythms under these constant conditions (luc) transgene driven by regulatory elements gion11. If CCA1 really is the transcriptional acti- that demonstrates the endogenous nature of of a light harvesting chlorophyll a/b binding vator responsible for circadian transcription of the biological clock. protein gene (CAB2, also known as Lhcb1*1) the CAB genes, then one might expect CCA1 has revealed a series of timing of CAB (toc) abundance to oscillate also. Indeed, both CCA1 The central oscillator mutations that disrupt clock function6, but as mRNA and protein exhibit circadian oscil- For many years, the goal of research in this yet, none of the genes responsible have been lations in abundance, and the peak in CCA1 pro- field has been to identify components of the cloned. The early flowering 3 (elf3) mutant tein concentration precedes the peak in CAB circadian central oscillator. Mutant alleles was identified on the basis of a daylength- transcription10. A circadian oscillation in CCA1 of the period locus of Drosophila were first insensitive early flowering phenotype. The mu- binding-activity might also be predicted, and identified in 1971, and additional mutations tant elf3 exhibits conditional arrhythmicity of data addressing this issue are eagerly awaited. that affect fundamental properties of the clock both leaf movement and CAB gene expression and confer altered period length or arrhyth- in continuous light, but shows normal clock Manipulating the expression of CCA1 micity have since been isolated from a diverse function in continuous dark7. This has been Elaine Tobin’s lab sought to perturb CAB gene range of organisms, including cyanobac- interpreted as evidence that ELF3 encodes a transcription in transgenic plants by either teria, Neurospora and, of course, Arabidopsis1. component of a light input pathway as opposed under- or over-expressing CCA1. Expression Mutational analyses are most advanced in to a component of a central oscillator. Over- of antisense CCA1 mRNA in transgenic plants Drosophila and Neurospora, where the char- expression in transgenic Arabidopsis of a reduces phytochrome-mediated induction of acterization of the genes identified by these clock-regulated glycine-rich RNA-binding pro- CAB1, confirming the in vivo role of CCA1 as mutations has yielded a model of the central tein, GRP7 (also known as CCR2), blocks the a transcription activator11. The effects of under- oscillator as a negative feedback loop in which oscillation in mRNA abundance of GRP7 and expression of CCA1 on circadian rhythms have rhythmic transcription of key clock genes is the closely related GRP8 (also known as not been determined. However, transgenic lines inhibited by the nuclear accumulation of the CCR1), but does not affect other circadian overexpressing CCA1 (CCA1-ox) are arrhyth- protein products of these genes1,2. A spate of oscillations. This suggests that GRP7 is a key mic in CAB transcription, indicating that CCA1 recent publications has filled in critical gaps component of a slave (non-self-sustaining) abundance limits CAB transcription and that in the description of this negative feedback oscillator, but not of a central oscillator8. constitutive CCA1 expression is sufficient to loop (Fig. 1) and has illustrated striking con- However, the long wait for plant central oscil- yield constitutive CAB transcription. This is a servation, both at the level of loop function lator components may finally be over. Two re- satisfying result, indicating that CCA1 is im- and primary protein sequence motifs, of clock cent studies published in Cell, describe a pair of portant for circadian regulation of CAB tran- components from Drosophila, Neurospora putative components of an Arabidopsis clock9,10. scription. The phenotype of the CCA1-ox plants and mammals3,4. In Elaine Tobin’s lab, CIRCADIAN CLOCK is pleiotropic: CCA1-ox plants lose circadian

(a)PP (b) P P PER Photoreceptors TIM (mPer) (phytochromes DBT cryptochromes) (casein kinase) P P Other rhythms P P TOC ELF3 CCA1 CAB PER CYC dCLK LHY TIM (mPer) Other rhythms (BMAL1) (CLOCK)

mRNA GRP7 E-Box

Transcription of period, timeless (mPer1, mPer2, mPer3) Fig. 1. Models of the central circadian oscillator. (a) The model for animals. Drosophila components are indicated, with mammalian equivalents indicated in parentheses. P indicates period and timeless phosphorylation. (b) A model illustrating the conceptual framework of a plant central oscillator, and input and output pathways. The positions of the various components are speculative. Single arrows can represent multiple steps in a pathway. The GRP7 slave oscillator is indicated as a non-self-sustaining loop dependent upon input from the central oscillator for continued oscillation. Abbreviations: TIM, timeless; PER, period; DBT, Drosophila double-time; dCLK, Drosophila clock; ELF3, early flowering 3; LHY, late elongated hypocotyl; CCA1, circadian clock associated 1; CYC, cycle; BMAL1, brain and muscle arnt-like protein 1; CAB, chlorophyll a/b binding protein; TOC, timing of CAB; GRP7, glycine-rich RNA-binding protein 7. regulation of other mRNAs including CAT3 at least 80% identity. Members of the MYB construction of a mutant plant carrying loss- and GRP7, which oscillate 180Њ out of phase superfamily are found in all eukaryotes, and of-function alleles for both genes. Nonethe- with CAB genes; the plants are also arrhyth- animal haploid genomes typically include 1Ð3 less, it seems unlikely that CCA1 and LHY are mic in leaf movement and late flowering. MYB genes. In plants, the MYB family has ex- completely redundant, as there are subtle dif- Clearly, CCA1 does much more than simply panded dramatically: the Arabidopsis haploid ferences between the LHY overexpressor and activate CAB gene transcription. Critically, genome includes over 80 known MYB genes CCA1-ox phenotypes. In lhy mutants, en- CCA1 overexpression suppresses expression that are involved in many distinct processes13. dogenous LHY transcript abundance is arrhyth- of the endogenous CCA1 gene, indicating that However, the MYB domain is repeated in al- mic but at a level intermediate between peak CCA1 negatively autoregulates. Taken to- most all MYB superfamily members, whereas and trough levels of the wild-type oscillation. gether, these results implicate CCA1 as a key CCA1 and LHY have only a single copy of the However, CCA1 overexpression almost totally component of a circadian oscillator. MYB motif. represses endogenous CCA1 transcript accumulation and also represses LHY transcript Late flowering mutants Are CCA1 and LHY accumulation to trough levels. It will be inter- In an independent approach, George Coupland’s functionally redundant? esting to assess CCA1 transcript oscillations lab used a screen for late flowering mutations The phenotype of plants carrying the domi- in lhy mutants. (in an Arabidopsis population carrying the nant lhy allele is pleiotropic and similar to the maize AcÐDs transposon system) to identify phenotype of CCA1-ox plants. In addition to Role of phosphorylation late elongated hypocotyl (lhy)9. The mutant the delay in flowering, both exhibit elongated The key clock components period (PER) and phenotype co-segregates with a single Ds hypocotyls, and as with CCA1, LHY mRNA timeless (TIM) in Drosophila and frequency insertion; LHY was cloned by virtue of the abundance oscillates with a circadian rhythm (FRQ) in Neurospora, are differentially phos- molecular Ds tag. The Ds element employed in wild-type plants, but is arrhythmic in lhy phorylated throughout the circadian cycle2Ð4, carries an outwardly directed promoter (CaMV mutants. Expression of other clock-regulated and a mutation in the Drosophila double-time 35S) that is known to induce dominant mu- genes, including a cab:luc fusion transgene (dbt) gene, which encodes a casein kinase I tations through activation of adjacent genes. and grp7 is arrhythmic in lhy mutants. Simi- activity responsible for the phosphorylation Indeed, lhy is inherited as a dominant mutation larly, the rhythm in leaf movement is lost. of period, results in arrhythmicity15,16. Is there in which the Ds element is inserted upstream Thus, constitutive overexpression of LHY, as a role for phosphorylation in the activity of of LHY, resulting in constitutive high-level of CCA1, disrupts multiple rhythms. The fact CCA1 or LHY? Recently, Elaine Tobin’s expression9. Thus, the Ds-tagged lhy mutants that overexpression of either CCA1 or LHY lab performed a yeast two-hybrid screen for are functionally LHY overexpressors. simultaneously disrupts circadian clock func- Arabidopsis proteins that interact with CCA1 The most prominent feature of the deduced tion and delays flowering, provides genetic and identified a regulatory ␤ subunit of casein amino acid sequence of LHY is a single 47- confirmation of the critical role of the circa- kinase II (CK2), CKB3 (Ref. 17). Further in residue region related to the helixÐloopÐhelix dian clock in the photoperiodic determination vitro experiments showed that CCA1 interacts DNA-binding domain of the MYB family9. of flower initiation that has long been inferred with two other CK2 ␤ subunits, CKB1 and This region is 87% identical to the correspond- from physiological experiments14. CKB2, and with two CK2 catalytic ␣ subunits, ing region in CCA1, and is essential for DNA- Overexpression of either CCA1 or LHY CKA1 and CKA2. In addition, CK2, as well as binding in CCA1 (Ref. 11). The similarity confers similar phenotypes: are CCA1 and a CK2-like activity from Arabidopsis whole- between LHY and CCA1 extends beyond the LHY functionally redundant? This can best be cell extracts, phosphorylates CCA1 in vitro, MYB domain, and there are three other re- addressed through analysis of loss-of-function although this phosphorylation did not affect gions, each of 20Ð25 amino acids, which have alleles for each gene and, particularly, through the ability of recombinant CCA1 to bind to its

December 1998, Vol. 3, No. 12 455 trends in plant science research news

DNA target in electrophoretic mobility shift and LHY, and what are the regulators of CCA1 7 Hicks, K.A. et al. (1996) Conditional circadian assays. However, the treatment of plant extracts and LHY mRNAs that confer their circadian dysfunction of the Arabidopsis early-flowering 3 with protein phosphatase abolished the for- oscillations? mutant, Science 274, 790Ð792 mation of the major CCA1ÐDNA complex, as 8 Heintzen, C. et al. (1997) AtGRP7, a nuclear did treatment of the extracts with CK2 inhibi- Conclusion RNA-binding protein as a component of a tors. This clearly implicates phosphorylation These recent studies illustrate a marvelous circadian-regulated negative feedback loop in of CCA1 by the serine-threonine kinase activ- convergence. Elaine Tobin’s lab set out to Arabidopsis thaliana, Proc. Natl. Acad. Sci. ity of CK2 in the in vivo regulation of CCA1 study light regulation of gene expression and U. S. A. 94, 8515Ð8520 activity17. It will be important to monitor CK2 George Coupland’s lab set out to study photo- 9 Schaffer, R. et al. (1998) LATE ELONGATED activity as well as the phosphorylation state of periodic initiation of flowering, yet both have HYPOCOTYL, an Arabidopsis gene encoding a CCA1 throughout the circadian cycle and in converged on the circadian clock and, quite MYB transcription factor, regulates circadian response to phase-shifting stimuli; and the possibly, have identified components of a cen- rhythmicity and photoperiodic responses, Cell 93, potential effects of phosphorylation on LHY. tral oscillator. These reports were published 1219Ð1229 269 years after de Mairan first established that 10 Wang, Z-Y. and Tobin, E.M. (1998) Constitutive Future prospects a circadian rhythm (leaf movement in Mimosa) expression of the CIRCADIAN CLOCK Are CCA1 and LHY genuine clock compo- was endogenous19, and provide a first glimpse ASSOCIATED 1 (CCA1) gene disrupts circadian nents? Quite possibly, although several experi- into the molecular mechanism of a plant cir- rhythms and suppresses its own expression, Cell mental predictions must be fulfilled before cadian oscillator. One might say, it’s about 93, 1207Ð1217 CCA1 and LHY are elevated to that pantheon. time. 11 Wang, Z-Y. et al. (1997) A MYB-related Overexpression of either CCA1 or LHY results transcription factor is involved in the in arrhythmicity, satisfying one important cri- Acknowledgements phytochrome regulation of an Arabidopsis Lhcb terion. Loss-of-function alleles of clock com- My thanks to Tom Jack and Mary Lou Guerinot gene, Plant Cell 9, 491Ð507 ponents from Drosophila and Neurospora stop for comments on the manuscript. Work in my 12 Carré, I.A. and Kay, S.A. (1995) Multiple the clock1,2, making the analysis of loss of lab is supported by a National Science Foun- DNA-protein complexes at a circadian- function a critical test for both CCA1 and LHY. dation grant (MCB 9723482). regulated promoter element, Plant Cell 7, Pulses of CCA1 or LHY should shift the phase 2039Ð2051 of the clock to that normally specified by the in- C. Robertson McClung 13 Romero, I. et al. (1998) More than 80 R2R3-MYB duced level of CCA1 or LHY during their nor- Dept Biological Sciences, Dartmouth College, regulatory genes in the genome of Arabidopsis mal daily oscillations. However, it is known that Hanover, NH 03755-3576, USA thaliana, Plant J. 14, 273Ð284 output pathways can provide input to oscilla- (tel ϩ1 603 646 3940; fax ϩ1 603 646 1347; 14 Lumsden, P.J. and Millar, A.J., eds (1998) tors, and that input pathways may themselves e-mail [email protected]) Biological Rhythms and Photoperiodism in be under circadian control. Consequently, it is Plants, Bios Scientific Publishers quite difficult to establish unequivocally that References 15 Kloss, B. et al. (1998) The Drosophila clock a particular gene encodes a component of a 1 Dunlap, J.C. (1996) Genetic and molecular gene double-time encodes a protein closely central oscillator (for detailed discussion, see analysis of circadian rhythms, Annu. Rev. Genet. related to human casein kinase Ie, Cell 97, Ref. 18). Certainly CCA1 is a critical player 30, 579Ð601 97Ð107 in the output pathway that governs circadian 2 Young, M.W. (1998) The molecular control of 16 Price, J.L. et al. (1998) double-time is a regulation of CAB genes. If CCA1 also proves circadian behavioral rhythms and their entrainment novel Drosophila clock gene that to be a component of the central oscillator, it in Drosophila, Annu. Rev. Biochem. 67, 135Ð152 regulates PERIOD protein accumulation, will provide a dramatic example of multiple 3 Dunlap, J. (1998) An end in the beginning, Cell 94, 83Ð95 tasking within the circadian system. Science 280, 1548Ð1549 17 Sugano, S. et al. (1998) Protein kinase CK2 The properties of CCA1 and LHY make 4 Reppert, S.M. (1998) A clockwork explosion! interacts with and phosphorylates the them serious contenders as plant clock com- Neuron 21, 1Ð4 Arabidopsis circadian clock-associated 1 ponents. Could it be that MYB proteins will be 5 Bünning, E. (1960) Opening address: biological protein, Proc. Natl. Acad. Sci. U. S. A. 95, the plant functional analogs of the ubiquitous clocks, in Cold Spring Harbor Symposia on 11020Ð11025 PAS proteins [e.g. TIM, clock and cycle (brain Quantitative Biology XXV, pp. 1Ð9, Cold Spring 18 Roenneberg, T. and Merrow, M. (1998) and muscle arnt-like protein 1)] in animal and Harbor Laboratory Press Molecular circadian oscillators: an alternative fungal clocks1Ð4? Ultimately, we may need to 6 Millar, A.J. et al. (1995) Circadian clock mutants hypothesis, J. Biol. Rhythms 13, 167Ð179 wait for a fuller description of the complete in Arabidopsis identified by luciferase imaging, 19 de Mairan, J. (1729) Observation botanique, Hist. negative-feedback loop in plants before we Science 267, 1161Ð1163 Acad. Roy. Sci. 35Ð36 can be certain. Many key questions remain to be answered. For example, what is the effect of LHY overexpression on the oscillation in CCA1? The elf3 mutation eliminates LHY 4 easy ways to subscribe to Trends in Plant Science mRNA oscillations in continuous light9. Is the arrhythmicity of elf3 (Ref. 7) entirely due to this 1 Tel ϩ44 (0) 1444 475651 effect on LHY? If so, this implies that lhy loss- 2 Fax ϩ44 (0) 1444 445423 of-function alleles should confer arrhythmicity 3 E-mail [email protected] which argues against redundancy between LHY and CCA1. What are the phenotypes con- 4 Post the bound-in subscription card ferred by lhy or cca1 loss-of-function mu- All you need to provide is your name, address, credit card details and the date from tations and what is the phenotype of the double which you would like your subscription to start. (Please do not send credit card details mutant homozygous for loss of function of lhy by e-mail.) Please quote customer reference TM8F1C on all correspondence. and cca1? Also, what are other targets of CCA1

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