Oligonucleotide and cDNA microarrays have been used to analyse the mRNA levels of 8,000 genes in Arabidopsis thaliana throughout the day/night cycle. Genes involved in signal transduction and in various metabolic pathways were found to be coordinately regulated by circadian rhythms and/or by light

Davis, Seth J.

Millar, A. J. (Andrew J.)

English

PubMed

Davis, S J

Millar, A J

Edinburgh Research Explorer

     Edinburgh Research Explorer                                      Watching the hands of the Arabidopsis biological clockCitation for published version:Davis, SJ & Millar, AJ 2001, 'Watching the hands of the Arabidopsis biological clock' Genome Biology, vol 2,no. 3, reviews1008-reviews1008.4 ., 10.1186/gb-2001-2-3-reviews1008Digital Object Identifier (DOI):10.1186/gb-2001-2-3-reviews1008Link:Link to publication record in Edinburgh Research ExplorerDocument Version:Publisher final version (usually the publisher pdf)Published In:Genome BiologyPublisher Rights Statement:Open AccessGeneral rightsCopyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)and / or other copyright owners and it is a condition of accessing these publications that users recognise andabide by the legal requirements associated with these rights.Take down policyThe University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorercontent complies with UK legislation. If you believe that the public display of this file breaches copyright pleasecontact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately andinvestigate your claim.Download date: 20. Feb. 2015http://genomebiology.com/2001/2/3/reviews/1008.1commentreviewsreportsdeposited researchinteractionsinformationrefereed  researchMinireviewWatching the hands of the Arabidopsis biological clock Seth J Davis and Andrew J MillarAddress: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.Correspondence: Andrew J Millar. E-mail: Andrew.Millar@warwick.ac.ukAbstractOligonucleotide and cDNA microarrays have been used to analyse the mRNA levels of 8,000 genes inArabidopsis thaliana throughout the day/night cycle. Genes involved in signal transduction and in variousmetabolic pathways were found to be coordinately regulated by circadian rhythms and/or by light. Published: 5 March 2001Genome Biology 2001, 2(3):reviews1008.1–1008.4The electronic version of this article is the complete one and can befound online at http://genomebiology.com/2001/2/3/reviews/1008© BioMed Central Ltd (Print ISSN 1465-6906; Online ISSN 1465-6914)Plants coordinate many biochemical and physiologicalprocesses with the Earth’s 24 hour rotation, presumablyimproving their fitness by anticipating the predictablecycles of light and temperature in the environment [1]. Tworecent papers have examined biological rhythms in plantsusing genome-wide transcript-analysis techniques. Theseapproaches have uncovered candidate regulatory genes andhave both confirmed and extended the range of physiologicalprocesses known to be under rhythmic control. The analysis of biological rhythms is complicated by the factthat many rhythmic processes are directly influenced bylight (Figure 1). In the natural day/night cycle, light regula-tion can lead to rhythmic changes that are referred to asdiurnal responses. Additionally, many physiologicalchanges anticipate the coming dawn or dusk and continueto show an oscillation of roughly 24 hours even in constantconditions; these are termed circadian rhythms. The manyovert morphological rhythms include leaf movements [2],the opening of stomatal pores [3] and the photoperiodicinduction of flowering [4]. In addition, biological rhythmshave been found to regulate the expression of genes encod-ing enzymes and regulatory and structural proteins [5].Daily cycling of these gene products appears to coordinatephysiological processes in the plant with the ambient envi-ronment [1]. Together, these developmental and enzymaticrhythms are thought to optimize photon capture and avoidconcurrent stresses, thus providing energy for both nitrogenand carbon uptake.There are two main technologies for array-based global gene-expression analysis; both are based on nucleic-acidhybridization but one relies on cDNAs (usually attached toglass) while the other uses synthetic oligonucleotides. In tworecent papers [6,7], each of these technologies was used toundertake the global analysis of steady-state mRNA levels inArabidopsis thaliana plants maintained under diurnaland/or circadian growth conditions. This is the first timeglobal transcript analysis has been used to uncover the net-works of rhythmic genes in any organism. Schaffer et al. [7]analyzed gene expression using cDNA arrays generated bythe Arabidopsis Functional Genomics Consortium (a consor-tium, funded by the US National Science Foundation, thatincludes Stanford, Wisconsin and Michigan State Universi-ties). The Arabidopsis cDNA array, which represents approx-imately 7,800 unique genes or around 30% of expressedsequences, was used to identify diurnal expression changes insamples harvested under a 12 hour light/12 hour dark cycle,and circadian regulation in plants transferred to constantlight or darkness. Schaffer et al. [7] analysed 13 slideshybridized to RNAs isolated after diurnal or circadian treat-ment. The Arabidopsis cDNA array results were quite repro-ducible, with fewer than 2% of transcripts measured ashaving a greater than two-fold difference in abundance inindependent, replicate RNA samples. Approximately 11% ofgenes showed differential expression at one or more of fourphases tested during the light/dark cycle (data available at[8]). About 25% of genes that showed diurnal regulation werealso circadian-regulated, as revealed by differential transcript2 Genome Biology Vol 2 No 3 Davis and Millarabundance after 12 hours versus 24 hours under constantconditions. In total, around 2% of the genes represented onthe array were scored as circadian-regulated. The same Arabidopsis cDNA array has been used to analyzetranscripts obtained under a wide range of physiological con-ditions, including auxin treatment, viral challenge and accli-mation to light [9]. Schaffer et al. [7] could therefore performcluster analysis using available published and unpublisheddata, in order to identify 33 genes regulated principally bythe circadian clock. Some of these genes will probably proveto be informative markers for future circadian studies; theymight also represent a functionally significant group. As thepublicly accessible expression databases grow, such clusteranalysis should prove its utility in unraveling gene regulatorynetworks in plants.Harmer et al. [6] used an oligonucleotide-based array(GeneChip, pioneered by Affymetrix; Santa Clara, CA) thatcontains information on approximately 8,200 genes. TheRNA samples analyzed were isolated from plants grown in alight/dark cycle but harvested following transfer to continu-ous light, so this experiment specifically tested circadian reg-ulation. The steady-state mRNA levels were measured inFigure 1Circadian rhythms in plant gene expression. (a) Simplified regulatory circuit for the circadian system. Light (represented by abulb) regulates some genes directly, leading to diurnal rhythms under cycles of light and dark. Light also resets the circadianoscillator, which in turn regulates the expression of clock-controlled genes. Some of the latter are also directly light-regulated. The circadian ‘gate’ rhythmically modulates light signaling. (b) Circadian-clock-controlled genes exhibit peakexpression levels at several phases with respect to the light/dark cycle (represented by hatched and white bars above thegraph). Examples of clock-regulated gene sets are given, with peak expression before subjective dawn (for example, thephenylpropanoid synthesis pathway; red) and early in the subjective day (for example, photosynthesis-related genes; green).0 24 48Time in constant light (hours)Expression level72GateOscillatorLight-regulated genesDual regulationClock-controlled genes(a)(b)commentreviewsreportsdeposited researchinteractionsinformationrefereed  researchhttp://genomebiology.com/2001/2/3/reviews/1008.3replicate hybridizations of 12 samples harvested every4 hours, covering two circadian cycles in constant light. Adedicated biomathematical software tool was developed todetect rhythmic patterns in these data. Here, around 6% ofgenes were found to be under circadian control. Interestingly,all phases of expression were well represented, suggestingthat the clock regulates gene expression throughout theday/night cycle, rather than clock-controlled genes beingexpressed only at a limited number of preferred phases. Several authors have noted that genes with functions in pho-tosynthesis are often maximally expressed in the earlymorning, presumably optimizing photosynthetic functionduring the daylight hours. Harmer et al. [6] propose severalstriking new examples in which rhythmic regulation appearsorchestrated such that genes that act coordinately areexpressed simultaneously. Some of these gene sets functionin concert with the daily environmental cycle, in so-calledexternal coordination. For example, 23 genes involved in thesynthesis of photoprotective phenylpropanoids are maxi-mally expressed before dawn, including CHALCONE SYN-THASE [10]. Other gene sets might be expressed in a fixedtemporal relationship to key metabolic processes (internalcoordination). Nine genes involved in the energeticallydemanding process of nitrogen assimilation were expressedearly in the day, for example, when cellular energy levels areincreased by light harvesting. The detection of such func-tional clustering is highly valuable, shedding new light onthe potential importance of biological rhythms for vegetativeplant growth. In such large data sets, however, this analysiscurrently requires either encyclopedic knowledge of plantbiochemistry or an extensive literature review. For many metabolic processes, diurnal regulation mightallow sufficient coordination with the environment, so circa-dian anticipation of dawn/dusk might add little advantage[11]. It might not be surprising, therefore, that Schaffer et al.[7] found many more genes under direct diurnal controlthan regulated by the circadian system. Is it significant thatgenes conferring metabolic function were the largest identi-fiable class of diurnal- and circadian-controlled genes?Harmer et al. [6] suggest that this finding reflects the betterunderstanding of enzymatic genes than those of other func-tions. For instance, about 25% of circadian-regulated genesencode proteins of unknown function and another 25% areuncharacterized genes containing limited sequence homol-ogy to known genes [6,7]. Many of these latter genes havesequence homology to kinases, phosphatases and transcrip-tion factors. A substantial number of rhythmic regulatoryfactors are therefore probably waiting to be discovered.Some of these presumably underlie the observed circadian‘gating’ of signal transduction, which fine tunes biologicalresponses over the day/night cycle [12].Patterns of coordinate gene expression might reflect regula-tion by common transcription factor(s) binding to similarpromoter element(s). Indeed, some transcripts encodingtranscription factors were found to oscillate in phase withtranscripts of their known target genes. Harmer et al. [6]identified a conserved sequence upstream of 31 genes thatwere expressed at the end of the subjective day. They con-firmed in transgenic plants that this ‘evening element’ wasrequired for robust circadian transcription from one suchpromoter. These results are profound, because bioinformaticapproaches alone accurately predicted a gene-regulatorysequence. Correlations between coordinate gene expressionand promoter sequences should be increasingly useful inuncovering regulatory mechanisms for organisms withsequenced genomes.The simultaneous application of the two microarray methodsallows some tentative technical comparisons that might high-light the importance of using both array technologies as wellas of the choice of genes to analyze. The overall number ofcircadian-controlled genes observed in constant light wasquite different: about 6% of genes for the oligonucleotidearray; about 0.5% for the cDNA array, which identified fewercycling genes in the light than in darkness. Setting aside dif-ferences in the numbers of RNA samples tested from circa-dian conditions and in the analytical methods, the identity ofthe genes assayed might also have contributed. Genes on theoligonucleotide-based chip were a relatively random samplecreated using genome sequence from the ArabidopsisGenome Initiative [13]. Genes on the cDNA array are not arandom sample, as these genes were isolated as part of theArabidopsis Expressed Sequence Tag collection [14], whichcontains virtually all highly expressed Arabidopsis genes andlacks many rare transcripts. Global expression profiling of theentire genome will be required to test the suggestion that asmaller percentage of abundant transcripts cycle, but this willbe possible now that the complete Arabidopsis genomesequence is publicly available [13].Global transcript analysis can potentially return four results:identifying new markers for research, revealing cis-acting ele-ments and trans-acting factors that cluster with a pattern ofregulation, identifying novel patterns of regulation, andfinding clusters of co-regulated genes with a functional con-nection that suggests the physiological importance of theirregulation. The two Arabidopsis circadian microarray paperswere successful by all these criteria. The current challenge is tofollow up some of these data with higher-resolution methods,before microarrays designed to sample the entire transcrip-tome of Arabidopsis uncover the complete complement ofclock-controlled genes. Then, the work will really begin.References1. Somers DE: The physiology and molecular bases of the plantcircadian clock. Plant Physiol 1999, 121:9-19.2. Engelmann W, Johnsson A: Rhythms in organ movement. In:Biological Rhythms and Photoperiodism in Plants. Edited by Lumsden PJ,Millar AJ. Oxford: BIOS Scientific; 1998.3. Webb AAR: Stomatal rhythms. In: Biological Rhythms and Photo-periodism in Plants. Edited by Lumsden PJ, Millar AJ. Oxford: BIOSScientific; 1998.4. Samach A, Coupland G: Time measurement and the control offlowering in plants. BioEssays 2000, 22:38-47.5. Kreps JA, Kay SA: Coordination of plant metabolism and devel-opment by the circadian clock. Plant Cell 1997, 9:1235-1244.6. Harmer SL, Hogenesch LB, Straume M, Chang HS, Han B, Zhu T,Wang X, Kreps JA, Kay SA: Orchestrated transcription of keypathways in Arabidopsis by the circadian clock. Science 2000,290:2110-2113.7. Schaffer R, Landgraf J, Monica A, Simon B, Larson M, Wisman E:Microarray analysis of diurnal and circadian-regulated genesin Arabidopsis. Plant Cell 2001, 13:113-123.8. Downloadable files of differentially expressed genes showingdiurnal or circadian rhythm [http://www.prl.msu.edu/circl]9. Arabidopsis Functional Genetics Consortium (StanfordUniversity) [http://AFGC.Stanford.EDU/afgc_html/site2.htm]10. Deikman J, Hammer PE: Induction of anthocyanin accumula-tion by cytokinins in Arabidopsis thaliana. Plant Physiol 1995,108:47-57.11. Pittendrigh CS: Temporal organization: reflections of aDarwinian clock-watcher. Annu Rev Physiol 1993, 55:17-54.12. Millar AJ, Kay SA: Integration of circadian and phototransduc-tion pathways in the network controlling CAB gene tran-scription in Arabidopsis. Proc Natl Acad Sci USA 1996,93:15491-15496.13. Arabidopsis Genome Initiative: Analysis of the genomesequence of the flowering plant Arabidopsis thaliana. Nature2000, 408:796-815.14. Newman T, De Bruijn FJ, Green P, Keegstra K, Kende H, McIntoshL, Ohlrogge J, Raikhel N, Somerville S, Thomashow M, et al.: Genesgalore: a summary of methods for accessing results fromlarge-scale partial sequencing of anonymous ArabidopsiscDNA clones. Plant Physiol 1994, 106:1241-1255.4 Genome Biology Vol 2 No 3 Davis and Millar

A: Rhythms in organ movement. In: Biological Rhythms and Photoperiodism in Plants. Edited by Lumsden PJ,

Arabidopsis Genome Initiative: Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature

Coordination of plant metabolism and development by the circadian clock. Plant Cell

Downloadable files of differentially expressed genes showing diurnal or circadian rhythm [http://www.prl.msu.edu/circl]

E: Microarray analysis of diurnal and circadian-regulated genes in Arabidopsis. Plant Cell

Genes galore: a summary of methods for accessing results from large-scale partial sequencing of anonymous Arabidopsis cDNA clones. Plant Physiol

Hammer PE: Induction of anthocyanin accumulation by cytokinins in Arabidopsis thaliana. Plant Physiol

Integration of circadian and phototransduction pathways in the network controlling CAB gene transcription in Arabidopsis.

SA: Orchestrated transcription of key pathways in Arabidopsis by the circadian clock. Science

Temporal organization: reflections of a Darwinian clock-watcher. Annu Rev Physiol

The physiology and molecular bases of the plant circadian clock. Plant Physiol

Time measurement and the control of flowering in plants. BioEssays

Watching the hands of the Arabidopsis biological clock

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  University of Warwick institutional repository: http://go.warwick.ac.uk/wrapThis paper is made available online in accordance with publisher policies. Please scroll down to view the document itself. Please refer to the repository record for this item and our policy information available from the repository home page for further information.  To see the final version of this paper please visit the publisher’s website. Access to the published version may require a subscription.   Author(s): Seth J Davis and Andrew J Millar Article Title: Watching the hands of the Arabidopsis biological clock Year of publication: 2001 Link to published version: http://dx.doi.org/ 10.1186/gb-2001-2-3-reviews1008Publisher statement: None   http://genomebiology.com/2001/2/3/reviews/1008.1commentreviewsreportsdeposited researchinteractionsinformationrefereed  researchMinireviewWatching the hands of the Arabidopsis biological clock Seth J Davis and Andrew J MillarAddress: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.Correspondence: Andrew J Millar. E-mail: Andrew.Millar@warwick.ac.ukAbstractOligonucleotide and cDNA microarrays have been used to analyse the mRNA levels of 8,000 genes inArabidopsis thaliana throughout the day/night cycle. Genes involved in signal transduction and in variousmetabolic pathways were found to be coordinately regulated by circadian rhythms and/or by light. Published: 5 March 2001Genome Biology 2001, 2(3):reviews1008.1–1008.4The electronic version of this article is the complete one and can befound online at http://genomebiology.com/2001/2/3/reviews/1008© BioMed Central Ltd (Print ISSN 1465-6906; Online ISSN 1465-6914)Plants coordinate many biochemical and physiologicalprocesses with the Earth’s 24 hour rotation, presumablyimproving their fitness by anticipating the predictablecycles of light and temperature in the environment [1]. Tworecent papers have examined biological rhythms in plantsusing genome-wide transcript-analysis techniques. Theseapproaches have uncovered candidate regulatory genes andhave both confirmed and extended the range of physiologicalprocesses known to be under rhythmic control. The analysis of biological rhythms is complicated by the factthat many rhythmic processes are directly influenced bylight (Figure 1). In the natural day/night cycle, light regula-tion can lead to rhythmic changes that are referred to asdiurnal responses. Additionally, many physiologicalchanges anticipate the coming dawn or dusk and continueto show an oscillation of roughly 24 hours even in constantconditions; these are termed circadian rhythms. The manyovert morphological rhythms include leaf movements [2],the opening of stomatal pores [3] and the photoperiodicinduction of flowering [4]. In addition, biological rhythmshave been found to regulate the expression of genes encod-ing enzymes and regulatory and structural proteins [5].Daily cycling of these gene products appears to coordinatephysiological processes in the plant with the ambient envi-ronment [1]. Together, these developmental and enzymaticrhythms are thought to optimize photon capture and avoidconcurrent stresses, thus providing energy for both nitrogenand carbon uptake.There are two main technologies for array-based global gene-expression analysis; both are based on nucleic-acidhybridization but one relies on cDNAs (usually attached toglass) while the other uses synthetic oligonucleotides. In tworecent papers [6,7], each of these technologies was used toundertake the global analysis of steady-state mRNA levels inArabidopsis thaliana plants maintained under diurnaland/or circadian growth conditions. This is the first timeglobal transcript analysis has been used to uncover the net-works of rhythmic genes in any organism. Schaffer et al. [7]analyzed gene expression using cDNA arrays generated bythe Arabidopsis Functional Genomics Consortium (a consor-tium, funded by the US National Science Foundation, thatincludes Stanford, Wisconsin and Michigan State Universi-ties). The Arabidopsis cDNA array, which represents approx-imately 7,800 unique genes or around 30% of expressedsequences, was used to identify diurnal expression changes insamples harvested under a 12 hour light/12 hour dark cycle,and circadian regulation in plants transferred to constantlight or darkness. Schaffer et al. [7] analysed 13 slideshybridized to RNAs isolated after diurnal or circadian treat-ment. The Arabidopsis cDNA array results were quite repro-ducible, with fewer than 2% of transcripts measured ashaving a greater than two-fold difference in abundance inindependent, replicate RNA samples. Approximately 11% ofgenes showed differential expression at one or more of fourphases tested during the light/dark cycle (data available at[8]). About 25% of genes that showed diurnal regulation werealso circadian-regulated, as revealed by differential transcript2 Genome Biology Vol 2 No 3 Davis and Millarabundance after 12 hours versus 24 hours under constantconditions. In total, around 2% of the genes represented onthe array were scored as circadian-regulated. The same Arabidopsis cDNA array has been used to analyzetranscripts obtained under a wide range of physiological con-ditions, including auxin treatment, viral challenge and accli-mation to light [9]. Schaffer et al. [7] could therefore performcluster analysis using available published and unpublisheddata, in order to identify 33 genes regulated principally bythe circadian clock. Some of these genes will probably proveto be informative markers for future circadian studies; theymight also represent a functionally significant group. As thepublicly accessible expression databases grow, such clusteranalysis should prove its utility in unraveling gene regulatorynetworks in plants.Harmer et al. [6] used an oligonucleotide-based array(GeneChip, pioneered by Affymetrix; Santa Clara, CA) thatcontains information on approximately 8,200 genes. TheRNA samples analyzed were isolated from plants grown in alight/dark cycle but harvested following transfer to continu-ous light, so this experiment specifically tested circadian reg-ulation. The steady-state mRNA levels were measured inFigure 1Circadian rhythms in plant gene expression. (a) Simplified regulatory circuit for the circadian system. Light (represented by abulb) regulates some genes directly, leading to diurnal rhythms under cycles of light and dark. Light also resets the circadianoscillator, which in turn regulates the expression of clock-controlled genes. Some of the latter are also directly light-regulated. The circadian ‘gate’ rhythmically modulates light signaling. (b) Circadian-clock-controlled genes exhibit peakexpression levels at several phases with respect to the light/dark cycle (represented by hatched and white bars above thegraph). Examples of clock-regulated gene sets are given, with peak expression before subjective dawn (for example, thephenylpropanoid synthesis pathway; red) and early in the subjective day (for example, photosynthesis-related genes; green).0 24 48Time in constant light (hours)Expression level72GateOscillatorLight-regulated genesDual regulationClock-controlled genes(a)(b)commentreviewsreportsdeposited researchinteractionsinformationrefereed  researchhttp://genomebiology.com/2001/2/3/reviews/1008.3replicate hybridizations of 12 samples harvested every4 hours, covering two circadian cycles in constant light. Adedicated biomathematical software tool was developed todetect rhythmic patterns in these data. Here, around 6% ofgenes were found to be under circadian control. Interestingly,all phases of expression were well represented, suggestingthat the clock regulates gene expression throughout theday/night cycle, rather than clock-controlled genes beingexpressed only at a limited number of preferred phases. Several authors have noted that genes with functions in pho-tosynthesis are often maximally expressed in the earlymorning, presumably optimizing photosynthetic functionduring the daylight hours. Harmer et al. [6] propose severalstriking new examples in which rhythmic regulation appearsorchestrated such that genes that act coordinately areexpressed simultaneously. Some of these gene sets functionin concert with the daily environmental cycle, in so-calledexternal coordination. For example, 23 genes involved in thesynthesis of photoprotective phenylpropanoids are maxi-mally expressed before dawn, including CHALCONE SYN-THASE [10]. Other gene sets might be expressed in a fixedtemporal relationship to key metabolic processes (internalcoordination). Nine genes involved in the energeticallydemanding process of nitrogen assimilation were expressedearly in the day, for example, when cellular energy levels areincreased by light harvesting. The detection of such func-tional clustering is highly valuable, shedding new light onthe potential importance of biological rhythms for vegetativeplant growth. In such large data sets, however, this analysiscurrently requires either encyclopedic knowledge of plantbiochemistry or an extensive literature review. For many metabolic processes, diurnal regulation mightallow sufficient coordination with the environment, so circa-dian anticipation of dawn/dusk might add little advantage[11]. It might not be surprising, therefore, that Schaffer et al.[7] found many more genes under direct diurnal controlthan regulated by the circadian system. Is it significant thatgenes conferring metabolic function were the largest identi-fiable class of diurnal- and circadian-controlled genes?Harmer et al. [6] suggest that this finding reflects the betterunderstanding of enzymatic genes than those of other func-tions. For instance, about 25% of circadian-regulated genesencode proteins of unknown function and another 25% areuncharacterized genes containing limited sequence homol-ogy to known genes [6,7]. Many of these latter genes havesequence homology to kinases, phosphatases and transcrip-tion factors. A substantial number of rhythmic regulatoryfactors are therefore probably waiting to be discovered.Some of these presumably underlie the observed circadian‘gating’ of signal transduction, which fine tunes biologicalresponses over the day/night cycle [12].Patterns of coordinate gene expression might reflect regula-tion by common transcription factor(s) binding to similarpromoter element(s). Indeed, some transcripts encodingtranscription factors were found to oscillate in phase withtranscripts of their known target genes. Harmer et al. [6]identified a conserved sequence upstream of 31 genes thatwere expressed at the end of the subjective day. They con-firmed in transgenic plants that this ‘evening element’ wasrequired for robust circadian transcription from one suchpromoter. These results are profound, because bioinformaticapproaches alone accurately predicted a gene-regulatorysequence. Correlations between coordinate gene expressionand promoter sequences should be increasingly useful inuncovering regulatory mechanisms for organisms withsequenced genomes.The simultaneous application of the two microarray methodsallows some tentative technical comparisons that might high-light the importance of using both array technologies as wellas of the choice of genes to analyze. The overall number ofcircadian-controlled genes observed in constant light wasquite different: about 6% of genes for the oligonucleotidearray; about 0.5% for the cDNA array, which identified fewercycling genes in the light than in darkness. Setting aside dif-ferences in the numbers of RNA samples tested from circa-dian conditions and in the analytical methods, the identity ofthe genes assayed might also have contributed. Genes on theoligonucleotide-based chip were a relatively random samplecreated using genome sequence from the ArabidopsisGenome Initiative [13]. Genes on the cDNA array are not arandom sample, as these genes were isolated as part of theArabidopsis Expressed Sequence Tag collection [14], whichcontains virtually all highly expressed Arabidopsis genes andlacks many rare transcripts. Global expression profiling of theentire genome will be required to test the suggestion that asmaller percentage of abundant transcripts cycle, but this willbe possible now that the complete Arabidopsis genomesequence is publicly available [13].Global transcript analysis can potentially return four results:identifying new markers for research, revealing cis-acting ele-ments and trans-acting factors that cluster with a pattern ofregulation, identifying novel patterns of regulation, andfinding clusters of co-regulated genes with a functional con-nection that suggests the physiological importance of theirregulation. The two Arabidopsis circadian microarray paperswere successful by all these criteria. The current challenge is tofollow up some of these data with higher-resolution methods,before microarrays designed to sample the entire transcrip-tome of Arabidopsis uncover the complete complement ofclock-controlled genes. Then, the work will really begin.References1. Somers DE: The physiology and molecular bases of the plantcircadian clock. Plant Physiol 1999, 121:9-19.2. Engelmann W, Johnsson A: Rhythms in organ movement. In:Biological Rhythms and Photoperiodism in Plants. Edited by Lumsden PJ,Millar AJ. Oxford: BIOS Scientific; 1998.3. Webb AAR: Stomatal rhythms. In: Biological Rhythms and Photo-periodism in Plants. Edited by Lumsden PJ, Millar AJ. Oxford: BIOSScientific; 1998.4. Samach A, Coupland G: Time measurement and the control offlowering in plants. BioEssays 2000, 22:38-47.5. Kreps JA, Kay SA: Coordination of plant metabolism and devel-opment by the circadian clock. Plant Cell 1997, 9:1235-1244.6. Harmer SL, Hogenesch LB, Straume M, Chang HS, Han B, Zhu T,Wang X, Kreps JA, Kay SA: Orchestrated transcription of keypathways in Arabidopsis by the circadian clock. Science 2000,290:2110-2113.7. Schaffer R, Landgraf J, Monica A, Simon B, Larson M, Wisman E:Microarray analysis of diurnal and circadian-regulated genesin Arabidopsis. Plant Cell 2001, 13:113-123.8. Downloadable files of differentially expressed genes showingdiurnal or circadian rhythm [http://www.prl.msu.edu/circl]9. Arabidopsis Functional Genetics Consortium (StanfordUniversity) [http://AFGC.Stanford.EDU/afgc_html/site2.htm]10. Deikman J, Hammer PE: Induction of anthocyanin accumula-tion by cytokinins in Arabidopsis thaliana. Plant Physiol 1995,108:47-57.11. Pittendrigh CS: Temporal organization: reflections of aDarwinian clock-watcher. Annu Rev Physiol 1993, 55:17-54.12. Millar AJ, Kay SA: Integration of circadian and phototransduc-tion pathways in the network controlling CAB gene tran-scription in Arabidopsis. Proc Natl Acad Sci USA 1996,93:15491-15496.13. Arabidopsis Genome Initiative: Analysis of the genomesequence of the flowering plant Arabidopsis thaliana. Nature2000, 408:796-815.14. Newman T, De Bruijn FJ, Green P, Keegstra K, Kende H, McIntoshL, Ohlrogge J, Raikhel N, Somerville S, Thomashow M, et al.: Genesgalore: a summary of methods for accessing results fromlarge-scale partial sequencing of anonymous ArabidopsiscDNA clones. Plant Physiol 1994, 106:1241-1255.4 Genome Biology Vol 2 No 3 Davis and Millar

     Edinburgh Research Explorer                                      Watching the hands of the Arabidopsis biological clockCitation for published version:Davis, SJ & Millar, AJ 2001, 'Watching the hands of the Arabidopsis biological clock', Genome Biology, vol.2, no. 3, reviews1008-reviews1008.4 . https://doi.org/10.1186/gb-2001-2-3-reviews1008Digital Object Identifier (DOI):10.1186/gb-2001-2-3-reviews1008Link:Link to publication record in Edinburgh Research ExplorerDocument Version:Publisher's PDF, also known as Version of recordPublished In:Genome BiologyPublisher Rights Statement:Open AccessGeneral rightsCopyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)and / or other copyright owners and it is a condition of accessing these publications that users recognise andabide by the legal requirements associated with these rights.Take down policyThe University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorercontent complies with UK legislation. If you believe that the public display of this file breaches copyright pleasecontact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately andinvestigate your claim.Download date: 10. Jan. 2026http://genomebiology.com/2001/2/3/reviews/1008.1commentreviewsreportsdeposited researchinteractionsinformationrefereed  researchMinireviewWatching the hands of the Arabidopsis biological clock Seth J Davis and Andrew J MillarAddress: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.Correspondence: Andrew J Millar. E-mail: Andrew.Millar@warwick.ac.ukAbstractOligonucleotide and cDNA microarrays have been used to analyse the mRNA levels of 8,000 genes inArabidopsis thaliana throughout the day/night cycle. Genes involved in signal transduction and in variousmetabolic pathways were found to be coordinately regulated by circadian rhythms and/or by light. Published: 5 March 2001Genome Biology 2001, 2(3):reviews1008.1–1008.4The electronic version of this article is the complete one and can befound online at http://genomebiology.com/2001/2/3/reviews/1008© BioMed Central Ltd (Print ISSN 1465-6906; Online ISSN 1465-6914)Plants coordinate many biochemical and physiologicalprocesses with the Earth’s 24 hour rotation, presumablyimproving their fitness by anticipating the predictablecycles of light and temperature in the environment [1]. Tworecent papers have examined biological rhythms in plantsusing genome-wide transcript-analysis techniques. Theseapproaches have uncovered candidate regulatory genes andhave both confirmed and extended the range of physiologicalprocesses known to be under rhythmic control. The analysis of biological rhythms is complicated by the factthat many rhythmic processes are directly influenced bylight (Figure 1). In the natural day/night cycle, light regula-tion can lead to rhythmic changes that are referred to asdiurnal responses. Additionally, many physiologicalchanges anticipate the coming dawn or dusk and continueto show an oscillation of roughly 24 hours even in constantconditions; these are termed circadian rhythms. The manyovert morphological rhythms include leaf movements [2],the opening of stomatal pores [3] and the photoperiodicinduction of flowering [4]. In addition, biological rhythmshave been found to regulate the expression of genes encod-ing enzymes and regulatory and structural proteins [5].Daily cycling of these gene products appears to coordinatephysiological processes in the plant with the ambient envi-ronment [1]. Together, these developmental and enzymaticrhythms are thought to optimize photon capture and avoidconcurrent stresses, thus providing energy for both nitrogenand carbon uptake.There are two main technologies for array-based global gene-expression analysis; both are based on nucleic-acidhybridization but one relies on cDNAs (usually attached toglass) while the other uses synthetic oligonucleotides. In tworecent papers [6,7], each of these technologies was used toundertake the global analysis of steady-state mRNA levels inArabidopsis thaliana plants maintained under diurnaland/or circadian growth conditions. This is the first timeglobal transcript analysis has been used to uncover the net-works of rhythmic genes in any organism. Schaffer et al. [7]analyzed gene expression using cDNA arrays generated bythe Arabidopsis Functional Genomics Consortium (a consor-tium, funded by the US National Science Foundation, thatincludes Stanford, Wisconsin and Michigan State Universi-ties). The Arabidopsis cDNA array, which represents approx-imately 7,800 unique genes or around 30% of expressedsequences, was used to identify diurnal expression changes insamples harvested under a 12 hour light/12 hour dark cycle,and circadian regulation in plants transferred to constantlight or darkness. Schaffer et al. [7] analysed 13 slideshybridized to RNAs isolated after diurnal or circadian treat-ment. The Arabidopsis cDNA array results were quite repro-ducible, with fewer than 2% of transcripts measured ashaving a greater than two-fold difference in abundance inindependent, replicate RNA samples. Approximately 11% ofgenes showed differential expression at one or more of fourphases tested during the light/dark cycle (data available at[8]). About 25% of genes that showed diurnal regulation werealso circadian-regulated, as revealed by differential transcript2 Genome Biology Vol 2 No 3 Davis and Millarabundance after 12 hours versus 24 hours under constantconditions. In total, around 2% of the genes represented onthe array were scored as circadian-regulated. The same Arabidopsis cDNA array has been used to analyzetranscripts obtained under a wide range of physiological con-ditions, including auxin treatment, viral challenge and accli-mation to light [9]. Schaffer et al. [7] could therefore performcluster analysis using available published and unpublisheddata, in order to identify 33 genes regulated principally bythe circadian clock. Some of these genes will probably proveto be informative markers for future circadian studies; theymight also represent a functionally significant group. As thepublicly accessible expression databases grow, such clusteranalysis should prove its utility in unraveling gene regulatorynetworks in plants.Harmer et al. [6] used an oligonucleotide-based array(GeneChip, pioneered by Affymetrix; Santa Clara, CA) thatcontains information on approximately 8,200 genes. TheRNA samples analyzed were isolated from plants grown in alight/dark cycle but harvested following transfer to continu-ous light, so this experiment specifically tested circadian reg-ulation. The steady-state mRNA levels were measured inFigure 1Circadian rhythms in plant gene expression. (a) Simplified regulatory circuit for the circadian system. Light (represented by abulb) regulates some genes directly, leading to diurnal rhythms under cycles of light and dark. Light also resets the circadianoscillator, which in turn regulates the expression of clock-controlled genes. Some of the latter are also directly light-regulated. The circadian ‘gate’ rhythmically modulates light signaling. (b) Circadian-clock-controlled genes exhibit peakexpression levels at several phases with respect to the light/dark cycle (represented by hatched and white bars above thegraph). Examples of clock-regulated gene sets are given, with peak expression before subjective dawn (for example, thephenylpropanoid synthesis pathway; red) and early in the subjective day (for example, photosynthesis-related genes; green).0 24 48Time in constant light (hours)Expression level72GateOscillatorLight-regulated genesDual regulationClock-controlled genes(a)(b)commentreviewsreportsdeposited researchinteractionsinformationrefereed  researchhttp://genomebiology.com/2001/2/3/reviews/1008.3replicate hybridizations of 12 samples harvested every4 hours, covering two circadian cycles in constant light. Adedicated biomathematical software tool was developed todetect rhythmic patterns in these data. Here, around 6% ofgenes were found to be under circadian control. Interestingly,all phases of expression were well represented, suggestingthat the clock regulates gene expression throughout theday/night cycle, rather than clock-controlled genes beingexpressed only at a limited number of preferred phases. Several authors have noted that genes with functions in pho-tosynthesis are often maximally expressed in the earlymorning, presumably optimizing photosynthetic functionduring the daylight hours. Harmer et al. [6] propose severalstriking new examples in which rhythmic regulation appearsorchestrated such that genes that act coordinately areexpressed simultaneously. Some of these gene sets functionin concert with the daily environmental cycle, in so-calledexternal coordination. For example, 23 genes involved in thesynthesis of photoprotective phenylpropanoids are maxi-mally expressed before dawn, including CHALCONE SYN-THASE [10]. Other gene sets might be expressed in a fixedtemporal relationship to key metabolic processes (internalcoordination). Nine genes involved in the energeticallydemanding process of nitrogen assimilation were expressedearly in the day, for example, when cellular energy levels areincreased by light harvesting. The detection of such func-tional clustering is highly valuable, shedding new light onthe potential importance of biological rhythms for vegetativeplant growth. In such large data sets, however, this analysiscurrently requires either encyclopedic knowledge of plantbiochemistry or an extensive literature review. For many metabolic processes, diurnal regulation mightallow sufficient coordination with the environment, so circa-dian anticipation of dawn/dusk might add little advantage[11]. It might not be surprising, therefore, that Schaffer et al.[7] found many more genes under direct diurnal controlthan regulated by the circadian system. Is it significant thatgenes conferring metabolic function were the largest identi-fiable class of diurnal- and circadian-controlled genes?Harmer et al. [6] suggest that this finding reflects the betterunderstanding of enzymatic genes than those of other func-tions. For instance, about 25% of circadian-regulated genesencode proteins of unknown function and another 25% areuncharacterized genes containing limited sequence homol-ogy to known genes [6,7]. Many of these latter genes havesequence homology to kinases, phosphatases and transcrip-tion factors. A substantial number of rhythmic regulatoryfactors are therefore probably waiting to be discovered.Some of these presumably underlie the observed circadian‘gating’ of signal transduction, which fine tunes biologicalresponses over the day/night cycle [12].Patterns of coordinate gene expression might reflect regula-tion by common transcription factor(s) binding to similarpromoter element(s). Indeed, some transcripts encodingtranscription factors were found to oscillate in phase withtranscripts of their known target genes. Harmer et al. [6]identified a conserved sequence upstream of 31 genes thatwere expressed at the end of the subjective day. They con-firmed in transgenic plants that this ‘evening element’ wasrequired for robust circadian transcription from one suchpromoter. These results are profound, because bioinformaticapproaches alone accurately predicted a gene-regulatorysequence. Correlations between coordinate gene expressionand promoter sequences should be increasingly useful inuncovering regulatory mechanisms for organisms withsequenced genomes.The simultaneous application of the two microarray methodsallows some tentative technical comparisons that might high-light the importance of using both array technologies as wellas of the choice of genes to analyze. The overall number ofcircadian-controlled genes observed in constant light wasquite different: about 6% of genes for the oligonucleotidearray; about 0.5% for the cDNA array, which identified fewercycling genes in the light than in darkness. Setting aside dif-ferences in the numbers of RNA samples tested from circa-dian conditions and in the analytical methods, the identity ofthe genes assayed might also have contributed. Genes on theoligonucleotide-based chip were a relatively random samplecreated using genome sequence from the ArabidopsisGenome Initiative [13]. Genes on the cDNA array are not arandom sample, as these genes were isolated as part of theArabidopsis Expressed Sequence Tag collection [14], whichcontains virtually all highly expressed Arabidopsis genes andlacks many rare transcripts. Global expression profiling of theentire genome will be required to test the suggestion that asmaller percentage of abundant transcripts cycle, but this willbe possible now that the complete Arabidopsis genomesequence is publicly available [13].Global transcript analysis can potentially return four results:identifying new markers for research, revealing cis-acting ele-ments and trans-acting factors that cluster with a pattern ofregulation, identifying novel patterns of regulation, andfinding clusters of co-regulated genes with a functional con-nection that suggests the physiological importance of theirregulation. The two Arabidopsis circadian microarray paperswere successful by all these criteria. The current challenge is tofollow up some of these data with higher-resolution methods,before microarrays designed to sample the entire transcrip-tome of Arabidopsis uncover the complete complement ofclock-controlled genes. Then, the work will really begin.References1. Somers DE: The physiology and molecular bases of the plantcircadian clock. Plant Physiol 1999, 121:9-19.2. Engelmann W, Johnsson A: Rhythms in organ movement. In:Biological Rhythms and Photoperiodism in Plants. Edited by Lumsden PJ,Millar AJ. Oxford: BIOS Scientific; 1998.3. Webb AAR: Stomatal rhythms. In: Biological Rhythms and Photo-periodism in Plants. Edited by Lumsden PJ, Millar AJ. Oxford: BIOSScientific; 1998.4. Samach A, Coupland G: Time measurement and the control offlowering in plants. BioEssays 2000, 22:38-47.5. Kreps JA, Kay SA: Coordination of plant metabolism and devel-opment by the circadian clock. Plant Cell 1997, 9:1235-1244.6. Harmer SL, Hogenesch LB, Straume M, Chang HS, Han B, Zhu T,Wang X, Kreps JA, Kay SA: Orchestrated transcription of keypathways in Arabidopsis by the circadian clock. Science 2000,290:2110-2113.7. Schaffer R, Landgraf J, Monica A, Simon B, Larson M, Wisman E:Microarray analysis of diurnal and circadian-regulated genesin Arabidopsis. Plant Cell 2001, 13:113-123.8. Downloadable files of differentially expressed genes showingdiurnal or circadian rhythm [http://www.prl.msu.edu/circl]9. Arabidopsis Functional Genetics Consortium (StanfordUniversity) [http://AFGC.Stanford.EDU/afgc_html/site2.htm]10. Deikman J, Hammer PE: Induction of anthocyanin accumula-tion by cytokinins in Arabidopsis thaliana. Plant Physiol 1995,108:47-57.11. Pittendrigh CS: Temporal organization: reflections of aDarwinian clock-watcher. Annu Rev Physiol 1993, 55:17-54.12. Millar AJ, Kay SA: Integration of circadian and phototransduc-tion pathways in the network controlling CAB gene tran-scription in Arabidopsis. Proc Natl Acad Sci USA 1996,93:15491-15496.13. Arabidopsis Genome Initiative: Analysis of the genomesequence of the flowering plant Arabidopsis thaliana. Nature2000, 408:796-815.14. Newman T, De Bruijn FJ, Green P, Keegstra K, Kende H, McIntoshL, Ohlrogge J, Raikhel N, Somerville S, Thomashow M, et al.: Genesgalore: a summary of methods for accessing results fromlarge-scale partial sequencing of anonymous ArabidopsiscDNA clones. Plant Physiol 1994, 106:1241-1255.4 Genome Biology Vol 2 No 3 Davis and Millar

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Watching the hands of the Arabidopsis biological clock

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