Báo cáo khoa học: Regulation of output from the plant circadian clock pot

REVIEW ARTICLE Regulation of output from the plant circadian clock Esther Yakir, Dror Hilman, Yael Harir and Rachel M. Green Department of Plant Sciences and the Environment, Institute for Life Sciences, Hebrew University, Jerusalem, Israel What is a circadian system? 2 Circadian systems are widespread endogenous mecha- nisms that allow organisms to time their physiological changes to predictable day ⁄ night cycles. They have evolved in a wide range of organisms, from cyano- bacteria to mammals, indicating their importance in life processes. Among an enormous variety of 24 h rhythms that are controlled by the circadian system are nitrogen-fixation in cyanobacteria, olfactory responses in Drosophila and sleep patterns in humans [1]. The basic oscillator mechanism that generates the rhythms is being elucidated in several model organisms [1] and consists of transcriptional–translational posit- ive ⁄ negative feedback loops involving a group of clock genes. The oscillator can be set (entrained) by signals from the environment, such as the daily changes in light and temperature, transduced via input pathways. Finally, output pathways link the oscillator to the var- ious biological processes whose rhythms it controls. The Arabidopsis circadian oscillator Most of the work on the circadian oscillator in plants has been carried out using the model plant Arabidopsis thaliana. The plant oscillator appears to be comprised Keywords circadian; Arabidopsis; plant; output; pathway; transcription; oscillator; hormone; calcium Correspondence R. M. Green, Department of Plant Sciences and the Environment, Institute for Life Sciences, Hebrew University, Givat Ram, Jerusalem 91904, Israel Fax: +972 2 658 4425 Tel. +972 2 658 5391 E-mail: rgreen@vms.huji.ac.il (Received 24 October 2006, accepted 23 November 2006) doi:10.1111/j.1742-4658.2006.05616.x Plants, like many other organisms, have endogenous biological clocks that enable them to organize their physiological, metabolic and developmental processes so that they occur at optimal times. The best studied of these biolo- gical clocks are the circadian systems that regulate daily ( 24 h) rhythms. At the core of the circadian system in every organism are oscillators respon- sible for generating circadian rhythms. These oscillators can be entrained (set) by cues from the environment, such as daily changes in light and tem- perature. Completing the circadian clock model are the output pathways that provide a link between the oscillator and the various biological processes whose rhythms it controls. Over the past few years there has been a tremen- dous increase in our understanding of the mechanisms of the oscillator and entrainment pathways in plants and many useful reviews on the subject. In this review we focus on the output pathways by which the oscillator regulates rhythmic plant processes. In the first part of the review we describe the role of the circadian system in regulation at all stages of a plant’s development, from germination and growth to reproductive development as well as in multiple cellular processes. Indeed, the importance of a circadian clock for plants can be gauged by the fact that so many facets of plant development are under its control. In the second part of the review we describe what is known about the mechanisms by which the circadian system regulates these output processes. Abbreviations APRR7, ARABIDOPSIS PSEUDORESPONSE REGULATOR 7; APRR9, ARABIDOPSIS PSEUDORESPONSE REGULATOR 9; CAT3, CATALASE 3; CBS, CCA1-binding site; CCA1, CIRCADIAN CLOCK ASSOCIATED 1 1 ; CCL, CCR-LIKE; CCR1, COLD CIRCADIAN RHYTHM RNA BINDING 1; CCR2, COLD CIRCADIAN RHYTHM RNA BINDING 2; CK, cytokinin; CO, CONSTANS; DST, downstream element; EE, evening element; FT, FLOWERING LOCUS T; GA, gibberellin; Hd1, heading date 1; Hd3A, heading date 3A; IAA, indole-3-acetic acid; LHY, LATE ELONGATED HYPOCOTYLS; LUX, LUX ARRYTHMO; RCA, RUBISCO ACTIVASE; SEN1, SENESCENCE-ASSOCIATED GENE 1; TOC1, TIMING OF CAB1. FEBS Journal 274 (2007) 335–345 ª 2006 The Authors Journal compilation ª 2006 FEBS 335 of components analogous to those described in other model organisms. Over the past decade, several puta- tive Arabidopsis clock components have been identified through mutational analysis and have been proposed to form a positive ⁄ negative feedback loop to generate circadian rhythms. Towards the end of the night, the positive element, TIMING OF CAB1 (TOC1), is involved in inducing the expression of CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELON- GATED HYPOCOTYL (LHY) [2]. CCA1 and LHY are known to encode transcription factors that, in vitro, can be phosphorylated [3,4] and can bind the TOC1 promoter [2]. Thus, during the day, CCA1 and LHY might directly repress the expression of TOC1. Towards evening, following a drop in levels of CCA1 and LHY, TOC1 expression increases, completing the feedback loop. More recently, additional genes, inclu- ding EARLY FLOWERING 4 (ELF4), GIGANTEA (GI) and LUX ARRYTHMO (LUX), and feedback loops have been identified suggesting that the oscillator is more complex and may be composed of several interlocking feedback loops [2,5–9]. Such an arrange- ment is likely to be important for conferring stability to the oscillator and is part of the mechanism ensuring that the circadian system is able to function accurately under a range of environmental conditions [10–12]. Clearly, to be of use to an organism, an oscillator needs to be entrained by environmental signals [13]. Light and temperature are the most important of such signals. Phytochromes, cryptochromes and members of the ZTL ⁄ FKF1 ⁄ LPK2 family of proteins [14–16] have all been shown to be light receptors for entrainment [17,18]. Several genes, including EARLY FLOWER- ING 3 (ELF3) and TIME FOR COFFEE (TIC) have also been implicated in the input signaling pathways from light to the clock [19,20]. There is not always, however, a clear distinction between oscillator and input elements in the circadian system. For example, the TOC1 paralogs, ARABI- DOPSIS PSEUDORESPONSE REGULATORS 7 and 9 (APRR7 and APRR9) both appear to function as part of the oscillatory mechanism, possibly forming an additional regulatory feedback loop similar to those found in other organisms [21,22]. At the same time, APRR7 and APRR9 also have a role in regulating light and temperature input to the oscillator [13,21]. Output processes regulated by the circadian oscillator Because there are already many excellent recent reviews on the mechanism of the oscillator and its entrainment [13,23,24], we focus on output from the oscillator. We start with an overview of the multiple roles that the circadian system has in regulation at all stages of a plant’s life before describing what is known about the mechanisms by which the circadian system regulates these output processes. The role of the circadian system during development The circadian clock controls many developmental pro- cesses throughout the life cycle of the plant. Some of these processes take place on a daily basis and are directly regulated by the circadian clock. Others occur annually and are controlled by changes in day-length (photoperiod) that are detected by the circadian system. Germination At the earliest stage of development the circadian sys- tem may regulate seed germination (Fig. 1A). In many species, including downy birch (Betula pubescens), Lap- land diapensia (Diapensia lapponica) and leatherleaf (Chamaedaphne calyculata), germination is controlled by day-length [25–28]. The existence of photoperiodic control of germination suggests that, at least in some plant species, the circadian system is functioning in seeds. Consistent with this idea, imbibition (the absorb- ance of water) by Arabidopsis seeds synchronizes circa- dian-controlled gene expression [29]. Furthermore, in dry (quiescent) onion (Allium cepa) seeds there is a circadian rhythm in gas exchange that continues in con- stant darkness [30], indicating that there may be a func- tioning oscillator in seeds even before germination. Growth The circadian system continues to regulate many devel- opmental processes that occur shortly after germina- tion. For example, Arabidopsis hypocotyls elongate with a circadian pattern immediately upon germination (Fig. 1B). The rate of hypocotyl growth is greatest in the evening and minimal in the morning, and can be entrained by light even before the cotyledons emerge from the seed coat [31]. A similar pattern of elongation has also been found in adult plants such as tomato (Lycopersicon esculentum) and red goosefoot (Chenopo- dium rubrum) [32–34]. Cotyledon and leaf movements are regulated by the circadian system in Arabidopsis and other species like legumes (Fig. 1C) [31,35]. How- ever, the mechanisms, for example, changes in cell turgor and differential cell growth, controlling the movements vary between species. The rate of Arabid- opsis stem circumnutations is also under circadian Circadian clock output in plants E. Yakir et al. 336 FEBS Journal 274 (2007) 335–345 ª 2006 The Authors Journal compilation ª 2006 FEBS control (Fig. 1D), and is greatest at dawn [36]. During the growth period, the circadian clock also regulates shade-avoidance responses (Fig. 1E) that enable plants to detect competition from other plants for light energy and react by enhancing stem and petiole growth [37]. Reproductive development The best-characterized developmental phenomenon regulated by the circadian clock is the transition from vegetative to reproductive development via the photo- periodic pathway (Fig. 1F). Some 70 years ago a model was proposed for photoperiodic sensing [38]. According to this model, called the external coinci- dence model, the circadian clock controls the expres- sion of a light-sensitive component. When there is a coincidence between light and sufficiently high levels of the light-sensitive component in the leaves, flowering is promoted. In recent years, research on Arabidopsis (a plant that flowers earlier under conditions of long days) has shown that the protein encoded by the CON- STANS (CO) gene is the light-sensitive component [39]. Briefly, the circadian clock controls CO mRNA levels so that under long days CO transcript levels start to rise well before sunset and stay high till the next morning. Under short days CO mRNA accumu- lates to significant levels only after sunset [40] and although CO translation occurs rapidly the protein is unstable in the dark. By contrast, during the day far- red and blue light stabilize CO protein, and CO accumulates in the nucleus [41]. CO then activates the transcription of the floral regulator FLOWERING LOCUS T (FT) [42]. FT mRNA, and possibly protein, moves from the leaf to the shoot apex and promotes flowering [43]. Interestingly, the components of the photoperiodic flowering pathway appear to be conserved even in plants that have a very different developmental response to increasing day-length. Thus, in rice (Oryza sativa), a plant that flowers early under short days, the CONSTANS homolog Hd1 also acts as the component integrating between the circadian clock and light sig- nal, however, instead of activating the FT homolog (Hd3a) under long days, Hd1 repress Hd3a under these conditions [44,45]. Pollination Following the transition to reproductive development, the circadian clock continues to control physiological events, such as pollination, that are important for suc- cessful seed formation. Many plants rely on pollinators that are active during a specific time of the day. In some species, in order to maximize the possibility of pollination and minimize the chances of damage, the circadian system regulates flower opening so that it occurs only during part of the day when potential poll- inators are most active (Fig. 1G). Thus Arabidopsis [46] petals open in the morning and close at midday, whereas night-blooming cestrum (Cestrum nocturnum) [47] petals open in the evening and close around dawn. A B C F M N G H I J K L D E Fig. 1. The circadian system has a regulatory role in nearly all aspects of a plant’s life. (A) Germination, (B) hypocotyl elongation, (C) leaf movements, (D) circumnutations, (E) shade avoidance, (F) flowering time, (G) flower opening, (H) scent production, (I) tuberization, (J) winter dormancy, (K) stomatal opening, (L) photosynthesis, (M) photoprotection, and (N) protection from temperature extremes. E. Yakir et al. Circadian clock output in plants FEBS Journal 274 (2007) 335–345 ª 2006 The Authors Journal compilation ª 2006 FEBS 337 Another important feature of the plant–pollinator relationship is the plant’s signature scent, which is a combination of volatile compounds unique for each plant species. Some volatiles are regulated by the circa- dian clock so that they are emitted in the correct phase with the plant’s pollinator activity (Fig. 1H) [47]. For example, in snapdragon (Antirrhinum majus) flowers which are pollinated by bees, the emission of methyl benzoate, myrcene and (E)-b-ocimene, is high during the day [48,49]. By contrast, methyl benzoate, is emit- ted at night by tobacco (Nicotiana suaveolens) and petunia flowers (Petunia · hybrida) in order to attract moths [49]. These rhythms are controlled by the clock and are probably a result of circadian changes in mRNA levels and enzyme activity in the biosynthesis pathway and in the levels of available substrate [48–50]. Nectar secretion is a further factor affecting success- ful pollination that may be timed to correspond with pollinator activity. In some species of the family Com- positae, nectar secretion is under diurnal control and very possibly also under circadian control [51]. Other photoperiod-regulated processes In addition to the transition from vegetative to repro- ductive development, several other processes in the plant’s life circle are controlled, at least in part, by photoperiod and thus, probably, the circadian system. One of these processes is the development of storage organs (Fig. 1I). In many cultivars of potatoes, inclu- ding Solanum tuberosum ssp. Andigena, tuberization depends on photoperiod and there is evidence that a potato ortholog of CONSTANS might be involved [52]. Another photoperiod-controlled process is the winter dormancy of temperate-zone woody plants (Fig. 1J). In chestnut (Castanea sativa) trees LHY and TOC1 orthologs might play a part in regulating the dormant state [53], and in aspen (Populus tremula) and black cottonwood (Populus trichocarpa) trees short- day-induced dormancy is controlled by CO and FT [54]. The role of the circadian system in the regulation of cellular processes Stomatal opening Circadian regulation can also be seen at the level of a single cell. One important example is the circadian rhythm observed in stomatal (leaf pore) opening (Fig. 1K). In Arabidopsis, stomatal conductance is higher during the day than at night [55], whereas in crassulacean acid metabolism 3 plants, stomatal opening has an opposite phase [56]. In addition the circadian clock gates sensitivity of stomata to extracellular sig- nals, such as light [57]. Photosynthesis and carbon dioxide fixation Photosynthesis and carbon fixation are two of the many important cellular processes that take place at a specific time of day (Fig. 1L). The expression of many Arabidopsis genes participating in the light-harvesting reactions of photosynthesis is under clock control [58,59]. Among them are the LHCA and LHCB gene families, which encode chlorophyll a ⁄ b binding poly- peptides for photosystems I and II, as well as genes that are involved in the biosynthesis of chlorophyll and RUBISCO SMALL SUBUNIT (RBCS) and RUBISCO ACTIVASE ( RCA) that participate in car- bon fixation [58,60]. It is not yet clear whether the circadian expression of mRNA in these pathways is always matched by circadian regulation at the level of protein synthesis. However, in some cases protein lev- els are under circadian control, for example, the syn- thesis of LHCB and RCA in tomato is regulated by the circadian system [61]. The rhythm of expression of photosynthesis genes appears to be correlated with the circadian rhythms observed in stomatal opening and CO 2 assimilation [62]. The circadian system also regu- lates post-translational modification of photosynthetic components such as phosphorylation of the D1 protein in duckweed (Spirodela oligorrhiza) [63]. Beside genes that encode proteins with a role in pho- tosynthesis reactions, some genes encode proteins that are involved in photorespiration, and sugar metabo- lism and transport are also under circadian control [58,64,65]. Furthermore, it has been suggested that there is a clock-controlled correlation between the energy-producing process of photosynthesis and the expression of genes involved in energy-consuming pro- cesses such as nitrogen assimilation [58]. Stress responses The circadian system appears to have a role in regula- ting responses to both abiotic and biotic stresses. For example, although plants need sun in order to pro- duce energy, high light levels can also be very dam- aging. Thus, before sunrise plants express genes encoding enzymes in the biosynthesis of photoprotect- ing pigments [58] and this expression is under circadian regulation (Fig. 1M). Similarly, the mRNA levels of some genes involved in cold protection are highest at dusk (Fig. 1N) [58]. Furthermore, the up- regulation by cold of some major genes is gated by the circadian clock to a specific time during the day Circadian clock output in plants E. Yakir et al. 338 FEBS Journal 274 (2007) 335–345 ª 2006 The Authors Journal compilation ª 2006 FEBS [66], as is sensitivity of the plants themselves to high and low temperatures. Thus cotton (Gossypium hirsu- tum) is most sensitive to cold at the beginning of the day and to high temperatures in the evening [67]. It has been suggested that this circadian-controlled gat- ing of the timing of sensitivity to extreme tempera- tures might be a way for the plant to distinguish between changes in temperatures during the course of the day and seasonal changes in temperature. In addi- tion, the circadian clock also regulates mRNA levels of some pathogen-related genes in Arabidopsis [59]. As an indication of the importance of the circadian sys- tem in regulating stress responses, microarray experi- ments have shown that around 70% of the known clock-controlled genes may also be regulated by cold, salt or drought stresses [68]. Mechanisms for regulating output In contrast with mammals, which have a central pace- maker in the brain to regulate the other oscillators in the body, plant circadian clocks appear to be auto- nomous. Thus, different plant organs can maintain rhythmic expression of genes with different phases [69]. Futhermore, genes can cycle with varying periods in different cells [70]. These differences in phase and per- iod may be a result either of tissue-specific changes in input pathways and ⁄ or of modifications in the oscilla- tor mechanism itself, although the available evidence suggests that the basic oscillator mechanism is funda- mentally conserved [70]. It seems unlikely, however, that tissue-specific differences in the oscillator mechan- ism are sufficient to regulate a wide range of output processes with different phases. In general, despite extensive evidence, gathered over the years, that the circadian system has a regulatory role in nearly all aspects of a plant’s life, remarkably little is known about the actual mechanisms by which the oscillator regulates these outputs. Indeed, one of the most intriguing, but least understood, questions is how the oscillator can regulate so many different plant processes, including gene expression, with a wide vari- ety of phases throughout the day. Transcriptional control Transcriptional control is probably one of the most important levels of regulation for controlling developmental, physiological and metabolic outputs. Research in mice, Drosophila and Neurospora has shown that a large percentage of the genome in a vari- ety of organisms is under clock control [71–73]. An extreme case of transcription clock control was found in the cyanobacterium Synechococcus elongates PCC 7942 which has most of its genome under clock control [74]. A two-component signaling system seems to be one of the mechanisms by which the cyanobacte- rial clock regulates transcription [75,76]. In Arabidopsis as much as 36% of the genome is controlled by the circadian system [58,59,77,78] and because at least two components of the circadian oscil- lator are transcription factors (CCA1 and LHY), an appealing idea is that plant oscillator components directly control the expression of some genes. Several motifs have been identified in the promoters of circa- dian-regulated genes and have been suggested as tar- gets for binding by CCA1 and LHY. One such motif, AAATATCT, also known as the Evening Element (EE), is over-represented in the promoters of circadian clock-controlled genes that show a peak of expression in the evening [58,77,78]. In vitro assays show that CCA1 and LHY can bind directly to the EE element [2,6,79]. Moreover, an artificial promoter containing four tandem repeats of the EE separated by 16 ran- dom nucleotides confer evening-phased gene expression to a luciferase reporter, confirming that the EE may be sufficient to determine the phase of gene expression [79]. Furthermore, mutations in the EE alter circadian rhythms of gene expression, demonstrating that this motif is necessary for evening phase of these genes [2,58,80]. By contrast, many of the circadian-regulated genes with an evening phase do not contain EEs in their promoters, whereas EEs have been found in the promoters of morning genes. The EE differs in only one base pair from another important circadian motif, the CCA1-binding site (CBS) sequence, AAAAATCT. Wang et al. [81] first characterized the CBS as the site for CCA1 binding in CAB1 promoter. However, the CBS does not appear to be over-represented in circadian gene promoters [78]. Furthermore, there is contradictory evidence regarding the role of the CBS in phase determination. Harmer and Kay [79] found that changing the EE to a CBS in a synthetic promoter based on the COLD CIRCADIAN RHYTHM RNA BINDING 2 (CCR2) promoter did not alter the peak expression phase of the gene. By contrast, Michael and McClung [80] showed that altering the EE motif in CATALASE 3 (CAT3) 4 promoter to a CBS could change the peak expression phase of the gene from evening to morning. Together, the results from these two groups suggest that other elements in CAT3 promoter are required for phase determination and that the context of the motif is important. Clearly, therefore, the EE and CBS are insufficient to explain circadian expression of all the clock- E. Yakir et al. Circadian clock output in plants FEBS Journal 274 (2007) 335–345 ª 2006 The Authors Journal compilation ª 2006 FEBS 339 controlled genes and combinations of known and unknown motifs are necessary for the correct phasing of circadian genes. Recently, Harmer and Kay [79] have identified a morning element (AACCACGA AAAT) sequence that might have a role in determining circadian expression and phase by binding of a yet unknown transcription activator. Other motifs might include the light-activation sequence G-box (CAC GTG) and the related Hex element, but these have not yet been tested experimentally [77,78,82]. Post-transcriptional control The oscillator also regulates post-transcriptional con- trol. For example, CCR2 mRNA accumulation is a result of both transcriptional and post-transcriptional regulation. CCR2 regulates the splicing of its own transcript and that of the closely related COLD CIR- CADIAN RHYTHM RNA BINDING 1 (CCR1), thus circadian-regulated changes in the levels of CCR2 pro- tein affect CCR1 and CCR2 mRNA accumulation [83– 86]. Another example is circadian control of the half- life of some transcripts. CCR-LIKE 5 (CCL) and SENESCENCE ASSOCIATED GENE 1 (SEN1 ) have a longer half-life in the morning than in the afternoon even under conditions of constant light and tempera- ture [87]. CCL and SEN1 have in their 3¢-UTRs a downstream element (DST) that can mediate transcript stability [88]. Furthermore, CCL and SEN1 mRNA decay is altered in a mutant that affects the DST decay pathway [87]. Thus, DST may be involved in circadian regulation of transcript stability. Included in the cohort of plant genes controlled by the circadian system are many genes that encode regu- latory proteins such as kinases and phosphatases. These proteins may act as secondary regulators of cir- cadian output pathways. For example, the circadian system controls expression of the gene encoding phos- phoenolpyruvate carboxylase kinase that regulates phosophorylation of phosphoenolpyruvate carboxylase to catalyse fixation of CO 2 in crassulacean acid meta- bolism plants [89]. The role of hormones in regulating circadian output Hormones affect most of the known circadian-con- trolled processes in plants and it is likely that, at least in some cases, the clock operates through changes in hormone levels or hormone perception. However, to date, there is only limited evidence for the role of hormones in delivering information from the clock to output processes. In many plant species, including Arabidopsis, barley (Hordeum distychum), wheat (Triticum aestivum), rye (Secale cereale), red goosefoot and cotton, ethylene production is under circadian control [90,91]. In Ara- bidopsis, ethylene levels peak in the middle of the sub- jective day. This pattern of ethylene accumulation is correlated with the circadian regulation of expression of ACC SYNTHASE which encodes the enzyme responsible for the synthesis of the ethylene precursor, 1-amino-cyclopropane-1-carboxilic acid. The fact that ethylene production is regulated by the circadian oscil- lator might suggest that ethylene has a role in regula- ting output processes. However, mutant plants that are affected in ethylene biosynthesis and signaling show no differences in rhythmic hypocotyl elongation or leaf movement, two rhythmic growth processes that circa- dian-controlled oscillations of ethylene might be expec- ted to regulate [92]. Thus the biological significance of rhythmic ethylene production is still unclear. Greater success has been achieved in connecting auxin to circadian regulation of growth. The levels of free indole-3-acetic acid (IAA) and its conjugated form, IAA–aspartate, were shown to cycle in continuous light both in floral stems and in rosette leaves of Arabidopsis [93]. There is also a circadian control of the expression of genes involved in auxin transport and auxin response [58,59]. More interestingly, the abolition of rhythmic stem elongation following removal of the floral stem, the endogenous source of auxin, can be res- cued by the application of exogenous auxin [93]. The relationship between the circadian clock and gibberellins (GAs) is more complicated. Both the clock, via the photoperiod pathway, and GAs affect flowering time in Arabidopsis, but most evidence reveals genetic differences between the photoperiod pathway and the GA pathway [94]. However in other plants, such as darnel ryegrass (Lolium temulentum), GAs may have a role in regulating photoperiodic flowering [95–98]. Thus, it is possible that GAs have a role in regulating circadian output. There is some circumstantial evidence that other hormones may be involved in regulating circadian out- put. In carrot (Daucus carota), the levels of cytokinins (CKs) are under circadian control [99]. While in tobacco, CKs, as well as IAA and abscisic acid, are rhythmic under diurnal condition [100]. It has been suggested that CKs may also be a part of an input pathway to the Arabidopsis clock [101,102]. The role of calcium in regulating circadian output Calcium (Ca 2+ ) is a second messenger in many differ- ent processes in the plant cell and there is evidence Circadian clock output in plants E. Yakir et al. 340 FEBS Journal 274 (2007) 335–345 ª 2006 The Authors Journal compilation ª 2006 FEBS that it may play this role in the regulation of output pathways from the circadian clock. Consistent with a potential role in regulating circadian output, circadian oscillations in free Ca 2+ have been demonstrated in the cytosol and chloroplast of tobacco (Nicotiana plumbaginifolia) and in the cytosol of Arabidopsis [103,104]. However, there are differences in the circa- dian oscillations of Ca 2+ ; cytosolic oscillations of Ca 2+ continue in constant light, whereas chloroplastic oscillations of Ca 2+ continue only in constant dark [103]. Furthermore, circadian oscillations of cytosolic Ca 2+ in tobacco seedlings have different phases in dif- ferent tissues and in Arabidopsis the phase of circadian oscillations of cytosolic Ca 2+ is modulated by the entraining photoperiod [104,105]. These results may reflect a possible role for calcium in mediating different clock controlled processes, including photoperiodism, in different cells. Finally there is evidence that Ca 2+ has a role in the circadian regulation of leaf movement in legumes, and in stomatal opening and photoperiod- controlled flowering in morning glory (Pharbitis nil) [35,106]. Together these results strongly suggest that Ca 2+ is part of the output signaling from the clock. As yet, however, no molecular decoders of the circa- dian Ca 2+ oscillations, such as calcium-binding pro- teins, have been experimentally proven in plants although a number of potential Ca 2+ decoders have been identified [106]. Conclusions The circadian system clearly plays an extremely important role in the life of plants, and indeed other organisms. However, despite this significance, and in spite of the considerable advances in our understand- ing of how the oscillator and input pathways func- tion, there is still much we do not understand of how the circadian system is able to accurately regulate so many output processes. Deciphering the mechanisms by which these output processes are controlled may allow us to modify specific pathways that are regula- ted by the circadian system. It will also give us a bet- ter understanding of this important aspect of the lives of plants. Acknowledgements The authors would like to thank Miri Hassidim, Shai Yerushalmi, Ido Kron and David Greenberg for their critical reading of the manuscript. Our apologies to the many researchers whose work was not cited due to limitation of space. This work was supported by ISF grants (0397232 and 0397386). References 1 Young MW & Kay SA (2001) Time zones: a compara- tive genetics of circadian clocks. Nat Rev Genet 2, 702– 715. 2 Alabadi D, Oyama T, Yanovsky MJ, Harmon FG, Mas P & Kay SA (2001) Reciprocal regulation between TOC1 and LHY ⁄ CCA1 within the Arabidopsis circadian clock. Science 293, 880–883. 3 Sugano S, Andronis C, Green RM, Wang Z-Y & Tobin EM (1998) Protein kinase CK2 interacts with and phosphorylates the Arabidopsis circadian clock- associated 1 protein. Proc Natl Acad Sci USA 95, 11020–11025. 4 Sugano S, Andronis C, Ong MS, Green RM & Tobin EM (1999) The protein kinase CK2 is involved in regu- lation of circadian rhythms in Arabidopsis. Proc Natl Acad Sci USA 96, 12362–12366. 5 Doyle MR, Davis SJ, Bastow RM, McWatters HG, Kozma-Bognar L, Nagy F, Millar AJ & Amasino RM (2002) The ELF4 gene controls circadian rhythms and flowering time in Arabidopsis thaliana. Nature 419, 74–77. 6 Hazen SP, Schultz TF, Pruneda-Paz JL, Borevitz JO, Ecker JR & Kay SA (2005) LUX ARRHYTHMO encodes a Myb domain protein essential for circadian rhythms. Proc Natl Acad Sci USA 102, 10387– 10392. 7 Kikis EA, Khanna R & Quail PH (2005) ELF4 is a phytochrome-regulated component of a negative-feed- back loop involving the central oscillator components CCA1 and LHY. Plant J 44, 300–313. 8 Mizoguchi T, Wright L, Fujiwara S, Cremer F, Lee K, Onouchi H, Mouradov A, Fowler S, Kamada H, Putterill J et al. (2005) Distinct roles of GIGANTEA in promoting flowering and regulating circadian rhythms in Arabidopsis. Plant Cell 17, 2255–2270. 9 Paltiel J, Amin R, Gover A, Ori N & Samach A (2006) Novel roles for GIGANTEA revealed under environmental conditions that modify its expression in Arabidopsis and Medicago truncatula. Planta 224, 1255–1268. 6 10 Locke JC, Millar AJ & Turner MS (2005) Modelling genetic networks with noisy and varied experimental data: the circadian clock in Arabidopsis thaliana. J Theoret Biol 234, 383–393. 11 Edwards KD, Lynn JR, Gyula P, Nagy F & Millar AJ (2005) Natural allelic variation in the temperature- compensation mechanisms of the Arabidopsis thaliana circadian clock. Genetics 170, 387–400. 12 Gould PD, Locke JC, Larue C, Southern MM, Davis SJ, Hanano S, Moyle R, Milich R, Putterill J, Millar AJ et al. (2006) The molecular basis of temperature compensation in the Arabidopsis circadian clock. Plant Cell 18, 1177–1187. E. Yakir et al. Circadian clock output in plants FEBS Journal 274 (2007) 335–345 ª 2006 The Authors Journal compilation ª 2006 FEBS 341 13 Salom AP & McClung CR (2005) What makes the Arabidopsis clock tick on time? A review on entrain- ment. Plant, Cell Environ 28, 21–38. 14 Nelson DC, Lasswell J, Rogg LE, Cohen MA & Bartel B (2000) FKF1, a clock-controlled gene that regulates the transition to flowering in Arabidopsis. Cell 101, 331–340. 15 Schultz TF, Kiyosue T, Yanovsky M, Wada M & Kay SA (2001) A role for LKP2 in the circadian clock of Arabidopsis. Plant Cell 13, 2659–2670. 16 Somers DE, Schultz TF, Milnamow M & Kay SA (2000) ZEITLUPE encodes a novel clock-associated PAS protein from Arabidopsis. Cell 101 , 319–329. 17 Devlin PF & Kay SA (2001) Circadian photopercep- tion. Annu Rev Physiol 63, 677–694. 18 Imaizumi T, Tran HG, Swartz TE, Briggs WR & Kay SA (2003) FKF1 is essential for photoperiodic-specific light signalling in Arabidopsis. Nature 426, 302–306. 19 Hall A, Bastow RM, Davis SJ, Hanano S, McWatters HG, Hibberd V, Doyle MR, Sung S, Halliday KJ, Amasino RM et al. (2003) The TIME FOR COFFEE gene maintains the amplitude and timing of Arabidop- sis circadian clocks. Plant Cell 15, 2719–2729. 20 McWatters HG, Bastow RM, Hall A & Millar AJ (2000) The ELF3 zeitnehmer regulates light signalling to the circadian clock. Nature 408, 716–720. 21 Farre EM, Harmer SL, Harmon FG, Yanovsky MJ & Kay SA (2005) Overlapping and distinct roles of PRR7 and PRR9 in the Arabidopsis circadian clock. Curr Biol 15, 47–54. 22 Yamamoto Y, Sato E, Shimizu T, Nakamich N, Sato S, Kato T, Tabata S, Nagatani A, Yamashino T & Mizuno T (2003) Comparative genetic studies on the APRR5 and APRR7 genes belonging to the APRR1 ⁄ TOC1 quintet implicated in circadian rhythm, control of flowering time, and early photomorphogenesis. Plant Cell Physiol 44, 1119–1130. 23 Gardner MJ, Hubbard KE, Hotta CT, Dodd AN & Webb AAR (2006) How plants tell the time. Biochem J 397, 15–24. 24 McClung CR (2006) Plant circadian rhythms. Plant Cell 18, 792–803. 25 Baskin JM & Baskin CC (1976) Effect of photoperiod on germination of Cyperus inflexus seeds. Bot Gaz 137, 269–273. 26 Black M & Wareing PF (1954) Photoperiodic control of germination in seed of birch (Betula pubescens Ehrh.). Nature 174, 705–706. 27 Black M & Wareing PF (1955) Growth studies in woody species VII. Photoperiodic control of germina- tion in Betula pudescens Ehrh. Physiologia Plantarum 8, 300–316. 28 Densmore RV (1997) Effect of day length on germina- tion of seeds collected in Alaska. Am J Bot 84, 274– 278. 29 Zhong HH, Painter JE, Salome PA, Straume M & McClung CR (1998) Imbibition, but not release from stratification, sets the circadian clock in Arabidopsis seedlings. Plant Cell 10 , 2005–2018. 30 Bryant TR (1972) Gas exchange in dry seeds: circadian rhythmicity in the absence of DNA replication, tran- scription, and translation. Science 178, 634–636. 31 Dowson-Day MJ & Millar AJ (1999) Circadian dys- function causes aberrant hypocotyl elongation patterns in Arabidopsis. Plant J 17, 63–71. 32 Fernandez SR & Wagner E (1994) New method of measurement and analysis of the stem extension growth rate to demonstrate complete synchronisation of Chenopodium rubrum plants by environmental con- ditions. J Plant Physiol 144, 362–369. 33 Lecharny A & Wagner E (1984) Stem extension rate in light-grown plants. Evidence for an endogenous circa- dian rhythm in Chenopodium rubrum. Physiologia Plan- tarum 60, 437–443. 34 Tukey HB & Ketellapper HJ Jr (1963) Length of the light–dark cycle and plant growth. Am J Bot 50, 110– 115. 35 Webb AAR (2003) The physiology of circadian rhythms in plants. New Phytologist 160, 281–303. 36 Niinuma K, Someya N, Kimura M, Yamaguchi I & Hamamoto H (2005) Circadian rhythm of circumnuta- tion in inflorescence stems of Arabidopsis. Plant Cell Physiol 46, 1423–1427. 37 Salter MG, Franklin KA & Whitelam GC (2003) Gat- ing of the rapid shade-avoidance response by the circa- dian clock in plants. Nature 426, 680–683. 38 Bu ¨ nning E (1936) Die endogene tagesrhythmik als grundlage der photoperiodischen reaktion. Ber Dtsch Bot Ges 54, 590–607. 39 Searle I & Coupland G (2004) Induction of flowering by seasonal changes in photoperiod. EMBO J 23, 1217–1222. 40 Suarez-Lopez P, Wheatley K, Robson F, Onouchi H, Valverde F & Coupland G (2001) CONSTANS med- iates between the circadian clock and the control of flowering in Arabidopsis. Nature 410, 1116–1120. 41 Valverde F, Mouradov A, Soppe W, Ravenscroft D, Samach A & Coupland G (2004) Photoreceptor regula- tion of CONSTANS protein in photoperiodic flower- ing. Science 303, 1003–1006. 42 Samach A, Onouchi H, Gold SE, Ditta GS, Schwarz- Sommer Z, Yanofsky MF & Coupland G (2000) Dis- tinct roles of CONSTANS target genes in reproductive development of Arabidopsis. Science 288, 1613–1616. 43 Huang T, Bohlenius H, Eriksson S, Parcy F & Nilsson O (2005) The mRNA of the Arabidopsis gene FT moves from leaf to shoot apex and induces flower- ing. Science 309, 1694–1696. 44 Hayama R, Yokoi S, Tamaki S, Yano M & Shima- moto K (2003) Adaptation of photoperiodic control Circadian clock output in plants E. Yakir et al. 342 FEBS Journal 274 (2007) 335–345 ª 2006 The Authors Journal compilation ª 2006 FEBS pathways produces short-day flowering in rice. Nature 422, 719–722. 45 Kojima S, Takahashi Y, Kobayashi Y, Monna L, Sasaki T, Araki T & Yano M (2002) Hd3a, a rice ortholog of the Arabidopsis FT gene, promotes transi- tion to flowering downstream of Hd1 under short-day conditions. Plant Cell Physiol 43, 1096–1105. 46 van Doorn WG & van Meeteren U (2003) Flower open- ing and closure: a review. J Exp Bot 54, 1801–1812. 47 Overland L (1960) Endogenous rhythm in opening and odor of flowers of Cestrum nocturnm. Am J Bot 47, 378–382. 48 Dudareva N, Martin D, Kish CM, Kolosova N, Gorenstein N, Faldt J, Miller B & Bohlmann J (2003) (E)-a-Ocimene and myrcene synthase genes of floral scent biosynthesis in snapdragon: function and expres- sion of three terpene synthase genes of a new terpene synthase subfamily. Plant Cell 15, 1227–1241. 49 Kolosova N, Gorenstein N, Kish CM & Dudareva N (2001) Regulation of circadian methyl benzoate emis- sion in diurnally and nocturnally emitting plants. Plant Cell 13, 2333–2347. 50 Verdonk JC, Ric de Vos CH, Verhoeven HA, Haring MA, van Tunen AJ & Schuurink RC (2003) Regula- tion of floral scent production in petunia revealed by targeted metabolomics. Phytochemistry 62, 997–1008. 51 Pesti J (1976) Daily fluctuations in the sugar content of nectar and periodicity of secretion in the Compositae. Acta Agron Acad Sci Hung 25, 5–17. 52 Martinez-Garcia JF, Virgos-Soler A & Prat S (2002) Control of photoperiod-regulated tuberization in potato by the Arabidopsis flowering-time gene CON- STANS. Proc Natl Acad Sci USA 99, 15211–15216. 53 Ramos A, Perez-Solis E, Ibanez C, Casado R, Collada C, Gomez L, Aragoncillo C & Allona I (2005) Winter disruption of the circadian clock in chestnut. Proc Natl Acad Sci USA 102, 7037–7042. 54 Bohlenius H, Huang T, Charbonnel-Campaa L, Brun- ner AM, Jansson S, Strauss SH & Nilsson O (2006) CO ⁄ FT regulatory module controls timing of flowering and seasonal growth cessation in trees. Science 312, 1040–1043. 55 Somers DE, Webb AAR, Pearson M & Kay SA (1998) The short-period mutant, toc1-1, alters circadian clock regulation of multiple outputs throughout development in Arabidopsis thaliana. Development 125, 485–494. 56 Thimann KV, Tan Z-Y & Park J (1992) Cycling of stomatal aperture in leaves of plants with crassulacean acid metabolism under constant conditions. Am J Bot 79, 23–27. 57 Gorton HL, Williams WE & Assmann SM (1993) Cir- cadian rhythms in stomatal responsiveness to red and blue light. Plant Physiol 103, 399–406. 58 Harmer SL, Hogenesch JB, Straume M, Chang H-S, Han B, Zhu T, Wang X, Kreps JA & Kay SA (2000) Orchestrated transcription of key pathways in Arabi- dopsis by the circadian clock. Science 290, 2110–2113. 59 Schaffer R, Landgraf J, Accerbi M, Simon V, Larson M & Wisman E (2001) Microarray analysis of diurnal and circadian-regulated genes in Arabidopsis. Plant Cell 13, 113–123. 60 Pilgrim ML & McClung CR (1993) Differential invol- vement of the circadian clock in the expression of genes required for ribulose-1,5-bisphosphate carboxylase ⁄ oxygenase synthesis, assembly, and activa- tion in Arabidopsis thaliana. Plant Physiol 103, 553– 564. 61 Martino-Catt S & Ort DR (1992) Low temperature interrupts circadian regulation of transcriptional activ- ity in chilling-sensitive plants. Proc Natl Acad Sci USA 89, 3731–3735. 62 Salome PA, Michael TP, Kearns EV, Fett-Neto AG, Sharrock RA & McClung CR (2002) The out of phase 1 mutant defines a role for PHYB in circadian phase control in Arabidopsis. Plant Physiol 129, 1674–1685. 63 Booij-James IS, Swegle WM, Edelman M & Mattoo AK (2002) Phosphorylation of the D1 photosystem II reaction center protein is controlled by an endogenous circadian rhythm. Plant Physiol 130, 2069–2075. 64 Lu Y, Gehan JP & Sharkey TD (2005) Daylength and circadian effects on starch degradation and maltose metabolism. Plant Physiol 138, 2280–2291. 65 McClung CR, Hsu M, Painter JE, Gagne JM, Karls- berg SD & Salome PA (2000) Integrated temporal reg- ulation of the photorespiratory pathway. Circadian regulation of two Arabidopsis genes encoding serine hydroxymethyltransferase. Plant Physiol 123, 381–392. 66 Fowler SG, Cook D & Thomashow MF (2005) Low temperature induction of Arabidopsis CBF1, 2, and 3 is gated by the circadian clock. Plant Physiol 137, 961– 968. 67 Rikin A, Dillwith JW & Bergman DK (1993) Correla- tion between the circadian rhythm of resistance to extreme temperatures and changes in fatty acid compo- sition in cotton seedlings. Plant Physiol 101, 31–36. 68 Kreps JA, Wu Y, Chang H-S, Zhu T, Wang X & Har- per JF (2002) Transcriptome changes for Arabidopsis in response to salt, osmotic, and cold stress. Plant Physiol 130, 2129–2141. 69 Thain SC, Hall A & Millar AJ (2000) Functional inde- pendence of circadian clocks that regulate plant gene expression. Curr Biol 10, 951–956. 70 Thain SC, Murtas G, Lynn JR, McGrath RB & Millar AJ (2002) The circadian clock that controls gene expression in Arabidopsis is tissue specific. Plant Phy- siol 130, 102–110. 71 Ceriani MF, Hogenesch JB, Yanovsky M, Panda S, Straume M & Kay SA (2002) Genome-wide expression analysis in Drosophila reveals genes controlling circa- dian behavior. J Neurosci 22, 9305–9319. E. Yakir et al. Circadian clock output in plants FEBS Journal 274 (2007) 335–345 ª 2006 The Authors Journal compilation ª 2006 FEBS 343 72 Nowrousian M, Duffield GE, Loros JJ & Dunlap JC (2003) The frequency gene is required for temperature- dependent regulation of many clock-controlled genes in Neurospora crassa. Genetics 164, 923–933. 73 Panda S, Antoch MP, Miller BH, Su AI, Schook AB, Straume M, Schultz PG, Kay SA, Takahashi JS & Hogenesch JB (2002) Coordinated Transcription of key pathways in the mouse by the circadian clock. Cell 109, 307–320. 74 Liu Y, Tsinoremas NF, Johnson CH, Lebedeva NV, Golden SS, Ishiura M & Kondo T (1995) Circadian orchestration of gene expression in cyanobacteria. Genes Dev 9, 1469–1478. 75 McClung CR (2006) Two-component signaling pro- vides the major output from the cyanobacterial circa- dian clock. Proc Natl Acad Sci USA 103, 11819–11820. 76 Takai N, Nakajima M, Oyama T, Kito R, Sugita C, Sugita M, Kondo T & Iwasaki H (2006) A KaiC-asso- ciating SasA–RpaA two-component regulatory system as a major circadian timing mediator in cyanobacteria. Proc Natl Acad Sci USA 103, 12109–12114. 77 Edwards KD, Anderson PE, Hall A, Salathia NS, Locke JCW, Lynn JR, Straume M, Smith JQ & Millar AJ (2006) FLOWERING LOCUS C mediates natural variation in the high-temperature response of the Arabidopsis circadian clock. Plant Cell 18, 639– 650. 78 Michael TP & McClung CR (2003) Enhancer trapping reveals widespread circadian clock transcriptional con- trol in Arabidopsis. Plant Physiol 132, 629–639. 79 Harmer SL & Kay SA (2005) Positive and negative factors confer phase-specific circadian regulation of transcription in Arabidopsis. Plant Cell 17, 1926–1940. 80 Michael TP & McClung CR (2002) Phase-specific cir- cadian clock regulatory elements in Arabidopsis. Plant Physiol 130, 627–638. 81 Wang ZY, Kenigsbuch D, Sun L, Harel E, Ong MS & Tobin EM (1997) A Myb-related transcription factor is involved in the phytochrome regulation of an Arabi- dopsis Lhcb gene. Plant Cell 9, 491–507. 82 Hudson ME & Quail PH (2003) Identification of pro- moter motifs involved in the network of phytochrome A-regulated gene expression by combined analysis of genomic sequence and microarray data. Plant Physiol 133, 1605–1616. 83 Heintzen C, Nater M, Apel K & Staiger D (1997) AtGRP7, a nuclear RNA-binding protein as a compo- nent of a circadian-regulated negative feedback loop in Arabidopsis thaliana. Proc Natl Acad Sci USA 94, 8515–8520. 84 Schoning JC & Staiger D (2005) At the pulse of time: protein interactions determine the pace of circadian clocks. FEBS Lett 579, 3246–3252. 85 Staiger D, Apel K & Trepp G (1999) The Atger3 pro- moter confers circadian clock-regulated transcription with peak expression at the beginning of the night. Plant Mol Biol 40, 873–882. 86 Staiger D, Zecca L, Kirk DAW, Apel K & Eckstein L (2003) The circadian clock regulated RNA-binding protein AtGRP7 autoregulates its expression by influ- encing alternative splicing of its own pre-mRNA. Plant J 33, 361–371. 87 Lidder P, Gutierrez RA, Salome PA, McClung CR & Green PJ (2005) Circadian control of messenger RNA stability. Association with a sequence-specific messenger RNA decay pathway. Plant Physiol 138, 2374–2385. 88 Newman TC, Ohme-Takagi M, Taylor CB & Green PJ (1993) DST sequences, highly conserved among plant SAUR genes, target reporter transcripts for rapid decay in tobacco. Plant Cell 5, 701–714. 89 Hartwell J, Gill A, Nimmo GA, Wilkins MB, Jenkins GI & Nimmo HG (1999) Phosphoenolpyruvate car- boxylase kinase is a novel protein kinase regulated at the level of expression. Plant J 20, 333–342. 90 Jasoni RL, Cothren T, Morgan PW & Sohan DE (2004) Circadian ethylene production in cotton. Plant Growth Regulation 36, 127–133. 91 McClung CR (2000) Circadian rhythms in plants: a millennial view. Physiologia Plantarum 109, 359–371. 92 Thain SC, Vandenbussche F, Laarhoven LJJ, Dowson- Day MJ, Wang Z-Y, Tobin EM, Harren FJM, Millar AJ & Van Der Straeten D (2004) Circadian rhythms of ethylene emission in Arabidopsis. Plant Physiol 136, 3751–3761. 93 Jouve L, Gaspar T, Kevers C, Greppin H & Degli Agosti R (1999) Involvement of indole-3-acetic acid in the circadian growth of the first internode of Arabidop- sis. Planta 209, 136–142. 94 Boss PK, Bastow RM, Mylne JS & Dean C (2004) Multiple pathways in the decision to flower: enabling, promoting, and resetting. Plant Cell 16, S18–S31. 95 King RW & Evans LT (2003) GIBBERELLINS and FLOWERING OF GRASSES and CEREALS: prizing open the lid of the ‘florigen’ black box. Annu Rev Plant Biol 54, 307–328. 96 King RW, Evans LT, Mander LN, Moritz T, Pharis R & Twitchin B (2003) Synthesis of gibberellin GA6 and its role in flowering of Lolium temulentum. Phytochem- istry 62, 77–82. 97 King RW, Moritz T, Evans LT, Martin J, Andersen CH, Blundell C, Kardailsky I & Chandler PM (2006) Regulation of flowering in the long-day grass Lolium temulentum by gibberellins and the FLOWERING LOCUS T cene. Plant Physiol 141, 498–507. 98 MacMillan CP, Blundell CA & King RW (2005) Flow- ering of the grass Lolium perenne. Effects of vernaliza- tion and long days on gibberellin biosynthesis and signaling. Plant Physiol 138, 1794–1806. 99 Stiebeling B & Neumann K-H (1986) Identification and concentation of endogenous cytokinins in carrots Circadian clock output in plants E. Yakir et al. 344 FEBS Journal 274 (2007) 335–345 ª 2006 The Authors Journal compilation ª 2006 FEBS [...]... (2001) The calcium rhythms of different cell types oscillate with different circadian phases Plant Physiol 125, 787–796 105 Love J, Dodd AN & Webb AAR (2004) Circadian and diurnal calcium oscillations encode photoperiodic information in Arabidopsis Plant Cell 16, 956–966 106 Dodd AN, Love J & Webb AAR (2005) The plant clock shows its metal: circadian regulation of cytosolic free Ca2+ Trends Plant Sci... Chua N-H & Zuo J (2006) Cytokinin affects circadianclock oscillation in a phytochrome B- and Arabidopsis response regulator 4-dependent manner Physiologia Plantarum 127, 277–292 Circadian clock output in plants 103 Johnson CH, Knight MR, Kondo T, Masson P, Sedbrook J, Haley A & Trewavas A (1995) Circadian oscillations of cytosolic and chloroplastic free calcium in plants Science 269, 1863–1865 104 Wood... a circadian rhythm J Plant Physiol 127, 111–121 100 Novakova M, Motyka V, Dobrev PI, Malbeck J, Gaudinova A & Vankova R (2005) Diurnal variation of cytokinin, auxin and abscisic acid levels in tobacco leaves J Exp Bot 56, 2877–2883 101 Salome PA, To JPC, Kieber JJ & McClung CR (2006) Arabidopsis response regulators ARR3 and ARR4 play cytokinin-independent roles in the control of circadian period Plant. .. Plant Cell 16, 956–966 106 Dodd AN, Love J & Webb AAR (2005) The plant clock shows its metal: circadian regulation of cytosolic free Ca2+ Trends Plant Sci 10, 15–21 FEBS Journal 274 (2007) 335–345 ª 2006 The Authors Journal compilation ª 2006 FEBS 345 . ARTICLE Regulation of output from the plant circadian clock Esther Yakir, Dror Hilman, Yael Harir and Rachel M. Green Department of Plant Sciences and the. Furthermore, the up- regulation by cold of some major genes is gated by the circadian clock to a specific time during the day Circadian clock output in plants