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

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.. In the first part

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 orgamecha-nisms 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

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.

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

TOC1promoter [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

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 CONSTANShomolog 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.

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

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

CO2 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

[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 timgat-ing 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-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

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 circir-cadian

system controls expression of the gene encoding

phos-phoenolpyruvate carboxylase kinase that regulates

phosophorylation of phosphoenolpyruvate carboxylase

to catalyse fixation of CO2 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 (Ca2+) is a second messenger in many differ-ent processes in the plant cell and there is evidence

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 Ca2+ 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 Ca2+; cytosolic oscillations of

Ca2+ continue in constant light, whereas chloroplastic

oscillations of Ca2+ continue only in constant dark

[103] Furthermore, circadian oscillations of cytosolic

Ca2+in tobacco seedlings have different phases in

dif-ferent tissues and in Arabidopsis the phase of circadian

oscillations of cytosolic Ca2+ 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 Ca2+ 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

Ca2+ is part of the output signaling from the clock

As yet, however, no molecular decoders of the

circa-dian Ca2+ oscillations, such as calcium-binding

pro-teins, have been experimentally proven in plants

although a number of potential Ca2+ 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)

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