Planta (2002) 216: 1–16
DOI 10.1007/s00425-002-0831-4
R EV IE W
Christian Fankhauser Æ Dorothee Staiger
Photoreceptors in Arabidopsis thaliana : light perception, signal
transduction and entrainment of the endogenous clock
Received: 2 May 2002 / Accepted: 4 June 2002
Ó Springer-Verlag 2002
Abstract To keep track of fluctuations in spectral
composition and intensity of incoming sunlight, plants
engage a plethora of photosensory pigments. Absorption of light by these photoreceptors sets in motion
signaling cascades that ultimately influence the plant’s
physiology. Many light-controlled processes are based
on modulation of gene activity in response to changes in
irradiation. The molecular basis of this regulation and
the downstream components transducing signals from
the photoreceptors are not fully understood yet, but
recent evidence suggests that some of those routes are
rather short. The phytochrome photoreceptors have
been found to influence light-responsive promoters by
direct contact with transcription factors. Additionally,
the cryptochrome blue-light receptors directly interact
with a key repressor of photomorphogenesis, suggesting
that light activation of photoreceptors could initiate
photomorphogenesis through posttranslational regulation. This review focuses on recent insights into photosensory transduction mechanisms as well as on our
current understanding of light entrainment of the endogenous clock.
Keywords Arabidopsis Æ Circadian rhythm Æ
Cryptochrome Æ Endogenous clock Æ
Photoreceptor Æ Phytochrome
Dedicated to Prof. Nikolaus Amrhein, Zürich, on the occasion of
his 60th birthday.
C. Fankhauser
Department of Molecular Biology,
30 quai E. Ansermet,
1211 Genève 4, Switzerland
D. Staiger (&)
Institute for Plant Sciences,
ETH, 8092 Zürich, Switzerland
E-mail: dorothee.staiger@ipw.biol.ethz.ch
Fax: +41-1-6321081
Introduction
During their entire life cycle plants are very sensitive to
their light environment. Light is a key factor influencing
all major developmental transitions such as seed germination or flowering, for example. Plants accurately
perceive fluctuations in the intensity, spectral quality,
directionality, and periodicity (day length) of the
incoming light. More than 80 years ago Garner and
Allard (1920) demonstrated that the pigments necessary
for such photomorphogenic responses were distinct
from the pigments required for photosynthesis. These
early photobiological experiments led to the discovery of
the phytochromes, the first plant photoreceptors to be
identified. Over the next few decades it became apparent
that plants possess photoreceptors monitoring: UVB,
UVA, blue, red and far-red light (Kendrick and
Kronenberg 1994). The molecular nature of the UVB
photoreceptors is still unknown, but three families of
plant photoreceptors have now been identified: the
phototropins, cryptochromes and phytochromes
(Cashmore et al. 1999; Casal 2000; Christie and Briggs
2001; Nagy and Schaefer 2002; Quail 2002a, b).
Upon light perception these photoreceptors initiate
signaling cascades. Early steps in these processes are
probably specific to a given photoreceptor, but there is
clearly interaction and integration of the signals generated by the different photosensory pigments (Casal 2000;
Quail 2002a). These same light signaling events also
serve as a resetting cue for the circadian clock and there
is increasing evidence suggesting that the light input
pathways to the circadian clock and general light perception are closely linked (Devlin and Kay 2001). It is,
however, premature to exclude the possible existence of
a photosensory system dedicated to the circadian clock.
We will focus on recent data concerning light perception
in Arabidopsis as well as signal transduction during
photomorphogenesis and resetting of the circadian
clock by light. For specific details we suggest that the
following reviews be consulted (Cashmore et al. 1999;
2
Fankhauser 2000; Hudson 2000; Lin 2000b; Smith 2000;
Christie and Briggs 2001; Devlin and Kay 2001;
McWatters et al. 2001; Roden and Carré 2001; Parks
et al. 2001; Nagy and Schaefer 2002; Quail 2002a, b).
Photoreceptors
Phototropins
The response of plants to unidirectional light had already been described by Charles Darwin and the first
action spectrum was recorded by Julius von Sachs
(Darwin 1881; Briggs and Huala 1999). Light-driven
tropic growth is primarily induced by the blue region of
the visible spectrum. The model plant Arabidopsis thaliana possesses two photoreceptors for this specific light
response: phot1, formerly known as nph1 (non-phototropic hypocotyl 1), and phot2 (formerly npl1, NPH-1
like 1; Briggs et al. 2001). Phot1 and phot2 code for Ser/
Thr protein kinases with amino-terminal chromophorebinding domains. Two FMN molecules bind to so-called
LOV (light, oxygen, voltage) domains. LOV domains
are a subset of PAS (Per/Arnt/Sim) domains that are
often found in proteins that sense environmental stimuli.
Phot1 is associated with the plasma membrane, whereas
the localization of phot2 is currently not known
(Christie and Briggs 2001).
We now know that in addition to their function in
phototropic hypocotyl growth phot1 and phot2 are also
essential for other blue-light responses. Their role has
been demonstrated for the inhibition of hypocotyl
growth during the very first minutes of blue light irradiation, for light-regulated ion fluxes, for chloroplast
movement and for stomata opening (Folta and Spalding
2001a; Jarillo et al. 2001a; Kagawa et al. 2001; Kinoshita
et al. 2001; Sakai et al. 2001; Babourina et al. 2002 ).
Interestingly, the requirement for these two photoreceptors depends on the light intensity and the physiological response. For stomatal opening they have
redundant functions whereas for positive phototropism
of the hypocotyl phot1 clearly plays the predominant
role in low light intensities, and both phototropins have
a redundant function to detect high light intensities
(Kinoshita et al. 2001; Sakai et al. 2001).
Since phot1 and phot2 are light-regulated protein
kinases, we have some clear ideas about the early steps
in this signaling cascade (Christie et al. 1998; Sakai et al.
2001). Nuclear magnetic resonance (NMR) studies
suggest that light induces a conformational change in
the photoreceptor that modulates the protein kinase
activity (Salomon et al. 2001).
Genetic studies have identified a few additional elements important for phot1-mediated signaling
(Motchoulski and Liscum 1999; Harper et al. 2000;
Sakai et al. 2000). Our current knowledge indicates that
phot1/phot2 are responsible for a number of specific
blue-light responses for which the other photoreceptors
are dispensable (Christie and Briggs 2001). In contrast,
other light responses such as de-etiolation, transition to
flowering or photic entrainment of the circadian clock
require the cryptochromes and the phytochromes but
not the phototropins (Casal 2000; Devlin and Kay 2001;
Quail 2002b). It would, however, be premature to conclude that there is no interaction between the phototropins and the other photoreceptors (Folta and Spalding
2001a). Phototropism is not induced by red light, but a
red light pretreatment enhances the effect of a subsequent blue light treatment. This enhancement of the
phototropic curvature is mediated by the phytochromes,
clearly suggesting a possible interaction between the two
families of photoreceptors (Stowe-Evans et al. 2001).
Cryptochromes
The first blue-light photoreceptor to be identified at the
molecular level was a cryptochrome (Ahmad and
Cashmore 1993). Cryptochromes are completely unrelated in structure to phototropins; they are composed of
an amino-terminal domain with homology to DNA
photolyases and a carboxy-terminal domain of unknown
biochemical function. This family of photoreceptors has
probably evolved from an ancestral DNA photolyase,
but has lost the photolyase activity; however, cryptochromes like DNA photolyases still bind to two chromophores, a Pterin and FAD (Cashmore et al. 1999;
Christie and Briggs 2001; Lin 2000a).
In Arabidopsis there are two cryptochromes, cry1 and
cry2. Their carboxy terminal domains are highly divergent, but appear to have related functions (Guo et al.
1998; Yang et al. 2000). Since their discovery in Arabidopsis, cryptochromes have been encountered in numerous other plant species and also in animals
(Cashmore et al. 1999). In animals they play a clear
function in the circadian system, either as a photoreceptor to entrain the central oscillator or as a component
of the oscillator itself, depending on the species
(Stanewsky et al. 1998; van der Horst et al. 1999; Young
and Kay 2001). Arabidopsis cryptochromes have
important functions during de-etiolation and for the
transition from vegetative to reproductive growth
(Batschauer 1999; Cashmore et al. 1999; Lin 2000a;
Quail 2002b). During de-etiolation, cry2 is particularly
important in response to low blue light intensities in
contrast to cry1, which has a prevalent role in response
to strong blue light (Lin 2000b). Interestingly, in contrast to cry2, which is strongly down-regulated in response to blue light, cry1 protein levels are not lightregulated. This presumably explains why cry2 plays a
major role in low light and cry1 in high light (Cashmore
et al. 1999; Lin 2000a; Quail 2002b).
Genome-wide analysis of light-regulated gene expression has demonstrated that about 15% of all transcripts are light-modulated (Ma et al. 2001; Tepperman
et al. 2001). Interestingly, the subsets of genes regulated
by different monochromatic lights appear to be very
similar, but not identical (Kuno and Furuya 2000;
3
Ma et al. 2001). Such studies have also highlighted the
importance of cry1 and cry2 in blue-light-modulated
gene expression. It must, however, be pointed out that
cry1cry2 double mutants still display blue-light-regulated gene expression, reinforcing the notion of co-action
of multiple photoreceptors in blue light (Casal 2000; Ma
et al. 2001).
Cryptochromes are found in the nucleus of Arabidopsis plants, both in the light and in the dark (Cashmore
et al. 1999; Kleiner et al. 1999). However a fusion protein between b-glucuronidase (GUS) and the carboxyterminal domain of CRY1 is cytosolic in the light and
nuclear in the dark, raising the possibility for action in
both sub-cellular compartments (Yang et al. 2000). Recent experiments strongly suggest that light activation
exposes the carboxy-terminal portion of the protein,
which then triggers the light-response (Yang et al. 2000).
This de-etiolation response appears to require the
physical interaction between the carboxy-terminal domain of the cryptochromes and the COP1 protein
(Wang et al. 2001; Yang et al. 2001).
COP1 was identified in a mutant displaying constitutive photomorphogenesis or de-etiolation (Deng et al.
1992). The recessive nature of cop1 alleles suggests that
COP1 acts as a repressor of photomorphogenesis in the
dark (Osterlund et al. 1999). One of the modes of action
of COP1 is to inhibit an activator of light-regulated
genes, HY5, presumably by targeting it to the proteasome (Osterlund et al. 2000). The direct interaction between COP1 and the cryptochromes suggests that upon
light activation their carboxy terminus inhibits COP1
function. This may in turn allow HY5 to accumulate
and promote de-etiolation.
In keeping with such a model of a short signaling
route downstream of the photoreceptors, only a limited
number of mutants have been described that affect deetiolation in blue light specifically. The SUB1 (short
under blue light) locus is clearly necessary for normal deetiolation in blue light; however, its role is not restricted
to blue light since sub1 seedlings also show altered deetiolation in far-red light (Guo et al. 2001). SUB1 is a
Ca2+-binding protein mainly localized in the cytoplasm.
We currently do not know by what mechanism SUB1
modulates phytochrome and cryptochrome signaling
(Guo et al. 2001). However, genetic data indicate that
SUB1 also inhibits the bZIP transcription factor HY5
that works downstream of both the phytochromes and
the cryptochromes (Guo et al. 2001).
Phytochromes
Phytochromes were originally defined as the photoreceptors mediating physiological responses that are induced by red light and, most importantly, that can be
inhibited if a pulse of far-red light follows the inductive
red light pulse (Kendrick and Kronenberg 1994). Based
on these photobiological assays it was postulated
that this photoreceptor must exist in two spectrally
interchangeable forms: an inactive red-light-absorbing
conformation (Pr) and an active far-red light-absorbing
form (Pfr). This prediction was confirmed in 1959 when
phytochrome was purified from etiolated oat seedlings
by Butler and colleagues (Butler et al. 1959).
We now distinguish between different modes of
phytochrome-mediated light perception: low-fluence responses (LFRs) which obey the above description (red/
far-red reversibility), very low-fluence responses
(VLFRs) which are most sensitive to red light, but also
occur in response to a broad light spectrum and are not
reversible, and high-irradiance responses (HIRs) which
require prolonged illumination (or light pulses with a
high frequency) (Shinomura et al. 2000; Smith 2000;
Nagy and Schaefer 2002; Quail 2002b). In higher plants,
phytochromes are encoded by a small gene family, designated PHYA to PHYE in Arabidopsis (Quail 2002b).
In addition to the genes encoding the apoproteins, HY1
and HY2 are required to synthesize the chromophore
that is covalently attached to the phytochrome (Davis
et al. 1999; Muramoto et al. 1999; Kohchi et al. 2001).
These light sensors are classified into type-I, or light
labile, and type-II, or light stable, phytochromes (Quail
2002b). In Arabidopsis, phyA is the only type-I phytochrome. Genetic studies have indicated that phyA is
responsible for the VLFR and the FR-HIR (Quail
2002b). Mutant analysis has shown that among the typeII phytochromes, phyB plays a predominant role (Quail
2002b). Phenotypes for single phyD and phyE mutants
are generally only uncovered in double mutant combinations (Whitelam and Devlin 1997; Quail 2002b). Despite the apparently distinct modes of action of type-I
and type-II phytochromes there are also a number of
well-documented cases where they act additively, synergistically and sometimes even antagonistically (Casal
2000). A detailed list of all the light responses mediated
by this family of photoreceptors is beyond the scope of
this review. As an example, Fig. 1 illustrates the phenotypes of a phyB mutant during de-etiolation, vegetative growth and the transition to flowering. The most
striking phenotypes of this mutant are the reduced inhibition of hypocotyl elongation by light, the reduced
chlorophyll accumulation, the longer petioles and the
early transition to flowering.
Phytochromes are synthesized in their inactive Pr
form in the dark. This light sensor is composed of two
major domains separated by a small hinge region. The
amino-terminal portion has the photosensory function
and binds to the linear tetrapyrrole chromophore. Upon
Pr-to-Pfr phototransformation, large structural changes
occur in the protein (Quail 2002b). Those changes are
postulated to activate the carboxy-terminal domain,
which is composed of a PAS-related domain and a histidine-kinase-related domain. The exact nature of this
‘‘activation’’ is still under intense scrutiny but a lot of
progress has been made on this topic recently.
We now know that upon light perception
phytochromes translocate from the cytosol to the
nucleus (Nagy and Schaefer 2000, 2002). The subcellular
4
Fig. 1. Phenotypes of Columbia wild type and phyB mutants
of Arabidopsis thaliana (Reed
et al. 1993) throughout the life
cycle. Left Wild-type seedlings
and phyB-9 mutants grown for
6 days in white light. Right, top
Wild type and phyB-9 mutants
grown for 20 short days (8 h
white light per day). Right,
bottom After 5 weeks in short
days, wild-type plants remain
vegetative, whereas phyB-9 mutants have undergone floral
transition
localization of phytochrome had long been a subject of
controversy. In fact, an effect of light on the subcellular
distribution of phytochrome had already been reported
in 1975 (Mackenzie et al. 1975), but these findings were
largely ignored until this issue was conclusively settled in
1996 (Sakamoto and Nagatani 1996). In the nucleus
phytochrome is found in discrete speckles. The functional significance of this re-localization is still not fully
understood, but the very good correlation between the
light requirements for nuclear translocation and defined
phytochrome-mediated responses clearly indicates that it
is an important regulatory step (Nagy and Schaefer
2000, 2002). We should, however, not conclude that only
nuclear phytochrome has a function (Mustilli and
Bowler 1997; Nagy and Schaefer 2002).
A second important breakthrough in the field was the
discovery of prokaryotic phytochromes (Vierstra and
Davis 2000). Some of these photoreceptors have been
studied in quite some detail and an important conclusion
that can be drawn is that bacteriophytochromes are
light-regulated histidine kinases (Yeh et al. 1997;
Vierstra and Davis 2000; Bhoo et al. 2001). It is therefore clear that plant phytochromes are descendants of a
bacterial light sensor that translates the light signal into
a protein kinase activity. At this point it is worth
pointing out that phototropins are also light-modulated
protein kinases. This finding revived an old theory
according to which plant phytochromes could be lightregulated enzymes. In fact there is good evidence that
oat phytochrome A is a light-regulated Ser/Thr kinase
(Yeh and Lagarias 1998; Fankhauser 2000). This might
sound surprising since Ser/Thr kinases and His kinases
belong to different protein families, and plant phytochromes posses a histidine-kinase-related domain rather
than a Ser/Thr protein kinase domain. However, autophosphorylation activity has been demonstrated for
oat phyA purified from both plant and recombinant
sources (Yeh and Lagarias 1998; Fankhauser 2000).
Interestingly, oat phytochrome A is phosphorylated in
vivo and some of the mapped sites correspond to sites of
in vitro autophosphorylation (Fankhauser 2000).
Moreover, recombinant oat phyA also phosphorylates
several substrates in vitro that might be relevant for
photomorphogenesis in vivo (Ahmad et al. 1998;
Fankhauser et al. 1999; Colon-Carmona et al. 2000). A
recent publication suggests that this activity is actually
important for phyA-mediated signaling in Arabidopsis
(Maloof et al. 2001).
Phytochrome signaling
A large number of mutants deficient in light perception
during de-etiolation have been identified since the
pioneering screen of Maarten Koornneef in 1980
(Koornneef et al. 1980). We will not present a detailed
description of all the identified mutants but try to
highlight a few trends that emerge from these studies.
5
The mutants have been classified according to the
color of light they are unable to perceive: blue, red and/
or far-red (Quail 2002a). The major photoreceptors
sensing these light qualities during de-etiolation are the
cryptochromes, phyB and phyA, respectively, and the
signaling mutants are classified into pathways triggered
by a particular photoreceptor (Quail 2002b). As anticipated from photobiological studies, some loci act specifically downstream of a single photoreceptor whereas
others presumably integrate information from multiple
light sensors (Casal 2000; Quail 2002a).
Light does not only affect de-etiolation of seedlings.
A number of more challenging screens using, for example, the shade-avoidance response or the transition to
flowering in adult plants, have also uncovered components of these light-perception pathways (Devlin et al.
1998; Guo et al. 1998; Fowler et al. 1999; Park et al.
1999). We have currently very little understanding of the
order of action of these numerous components. The
analysis of a number of mutants suggests early branching in these signaling cascades so that it is probably
much more accurate to describe them as signaling webs
(Casal 2000; Quail 2002b; Wang and Deng 2002; Zhou
et al. 2002). However, the analysis of mutants with a deetiolation (det) or constitutive photomorphogenic (cop)
phenotype and mutants with reduced light sensitivity
over the whole light spectrum suggests that all these
branches finally converge (Quail 2002b). This conclusion
is very much in line with global gene expression analysis
revealing that the sets of genes regulated by different
light qualities largely overlap (Ma et al. 2001).
The molecular identification of numerous phytochrome-signaling components suggests that there are
cytosolic, nuclear and actually also chloroplastic events
(Moller et al. 2001; Quail 2002b). Yeast two-hybrid assays have identified several proteins that interact directly
with the photoreceptor (Ni et al. 1998; Choi et al. 1999;
Fankhauser et al. 1999; Jarillo et al. 2001b; Sweere et al.
2001). The importance of these proteins in phytochrome-mediated signaling has been confirmed using
reverse genetic approaches. The best-understood part of
phytochrome signaling is the ‘‘nuclear branch’’. The first
identified component of this pathway is PIF3 (phytochrome interacting factor), a predicted transcription
factor with a bHLH (basic helix-loop-helix) domain (Ni
et al. 1998). Notably, PIF3 binds to phytochrome in a
conformation-specific manner with Pfr having much
greater affinity than Pr (Ni et al. 1999). Genetic and
molecular data suggest that PIF3 is primarily involved in
phytochrome B signaling (Ni et al. 1998; Halliday et al.
1999; Zhu et al. 2000). In vitro, phyB in its Pfr form
makes a complex with PIF3 when it is bound to its DNA
target site (Martinez-Garcia et al. 2000). Indeed, redlight induction of CCA1 (circadian clock associated) and
LHY (late elongated hypocotyl) genes is attenuated in
antisense plants with reduced PIF3 levels (MartinezGarcia et al. 2000). Taken together, these data establish
a very short signaling cascade through which phytochromes following their light-triggered translocation
into the nucleus influence gene activity by direct contacts
with transcription factors already bound to their cognate target sequence. Remarkably, a direct effect of
phytochrome on transcription was already proposed in
1987 when it was discovered that purified oat phyA
augments the in vitro transcription rate of CAB genes in
isolated barley nuclei (Mösinger et al. 1987).
Genome-based expression studies have indicated that
a large fraction of genes that are rapidly light induced in
a phyA-dependent manner encode transcription factors
(Tepperman et al. 2001). This suggests a mechanism by
which phytochromes directly activate key transcriptional regulators such as PIF3, which subsequently upregulate a second wave of transcription factors such as
CCA1 and LHY (Quail 2002b). It is very unlikely that
PIF3 is the only transcription factor in the first class for
several reasons. First of all, plants with reduced PIF3
levels have modest phenotypes compared with photoreceptor mutants. It must, however, be said that the
phenotype of a true loss-of-function pif3 allele has not
yet been reported. Moreover, another bHLH protein
with significant similarity to PIF3 was genetically identified as a phyA-signaling component; however, RSF1/
HFR1/REP1 does not appear to physically interact with
phyA (Fairchild et al. 2000; Soh et al. 2000; Spiegelman
et al. 2000).
In conclusion, these studies raise a number of important questions: How does phytochrome promote the
transcriptional activity of PIF3? Are there many other
transcription factors with similar roles to PIF3? Does
this nuclear branch explain the whole story? The answer
to this last question is most probably no. One could
imagine that the cytosolic and chloroplastic events are
just modulators of the really important nuclear branch.
However, there are phytochrome responses that only
require minutes and are therefore unlikely to require de
novo protein synthesis (Folta and Spalding 2001b).
Moreover, it is quite striking to compare the relatively
strong morphological phenotype of phyB mutants
(Fig. 1) with the considerably more modest impact of
this photoreceptor on light-regulated gene expression
(Ma et al. 2001). These data suggest additional phyB
functions that do not require transcriptional regulation.
The identification of PIF3 as a central regulator also
implies a direct signaling route from phytochrome to the
endogenous clock system, since two of its immediate
targets, CCA1 and LHY, are important constituents of
this system (see below).
The endogenous clock
Plants as photosynthetic organisms are particularly dependent on sunlight which is restricted to a limited time
window of the day–night cycle. Thus, a day in the life of
a plant resembles a day in an industrial enterprise in
some aspects: exact timing of individual processes is
required to optimally deploy limited resources and to
coordinate the overall process in the most efficient way.
6
Like workers that obey a strict schedule, plants time
their physiology and behavior. To do so, they have
evolved an internal timekeeper, the endogenous clock. It
allows the anticipation of regular fluctuations in the
availability of the most important resource to plants,
sunlight.
The clock imposes a 24-hour rhythm on certain
physiological processes so that they always occur at the
optimal phase of the light–dark cycle. These ‘‘circadian’’
rhythms are the external manifestation of the internal
clock, the so-called output. They range from leaf
movement, growth processes, flower opening and fragrance emission to photosynthesis and carbon metabolism (Somers 1999; Staiger and Heintzen 1999; Barak
et al. 2000; Harmer et al. 2001; McClung 2001). Underlying many of these physiological rhythms are endogenous rhythms in gene activity. They were first
discovered for light-induced transcripts that continued
to cycle between low and high levels even in continuous
illumination (Kloppstech 1985; Nagy et al. 1988). Regulation is mostly exerted at the transcriptional level
through clock-response elements in the promoters and
cognate trans-acting factors (Carré and Kay 1995; Nagy
et al. 1988; Wang and Tobin 1998; Staiger and Apel
1999; Staiger et al. 1999).
A global view of endogenous rhythms in transcript
abundance has been obtained through microarray analysis with probes sampled around the clock (Harmer et al.
2000; Schaffer et al. 2001). Clusters of genes operating in
defined metabolic pathways, developmental processes
and light responses have been identified that peak at
similar times of the day. Among those, the well-studied
phenylpropanoid pathway turned out to be under clock
control. Twenty-three genes encoding biosynthetic enzymes are coordinately activated at the end of the night.
This has been suggested to serve the production of UVprotective compounds in anticipation of dawn (Harmer
et al. 2000). Notably, the very same screen uncovered
circadian regulation of an MYB-type factor, PAP1
(production of anthocyanin pigment), whose overexpression causes activation of phenylpropanoid biosynthetic genes and enhanced accumulation of lignin,
hydroxycinnamic acid esters, and flavonoids (Borevitz
et al. 2000; Harmer et al. 2000). Clock-regulation of this
key transcription factor thus may serve to synchronously
activate the entire phenylpropanoid pathway.
A similar microarray analysis of circadian gene expression in Drosophila melanogaster identified more than
100 cycling genes, many of which, surprisingly, map to
the same chromosomal region and appear to be transcriptionally co-regulated (McDonald and Rosbash
2001; Ueda et al. 2002).
Components of the clock system
The persistence of circadian rhythms even in the absence
of environmental timing cues indicates that they are
driven by a self-sustaining oscillator. Integral to the
oscillator are clock proteins that oscillate with a 24-h
rhythm: clock genes are rhythmically transcribed and
after a certain delay clock proteins feed back to inhibit
transcriptional activity of their own genes (Dunlap 1999;
Allada et al. 2001; Young and Kay 2001). Additionally,
posttranscriptional regulation of the clock transcripts as
well as posttranslational modifications of the clock
proteins contribute to the maintenance of the 24-h periodicity (Edery 1999; Allada et al. 2001). The rhythmically produced clock proteins in turn translate
temporal information into rhythmic physiology via
output-signal transduction chains (Brown and Schibler
1999). Thus, the phases of clock gene mRNA and clock
protein oscillations are indicators of internal time.
While this picture has been firmly established for
D. melanogaster, mammals and Neurospora crassa, the
players in higher plants have not been unambiguously
identified. Three proteins are candidates to form part of
the central clock mechanism in A. thaliana (Yanovsky
and Kay 2001).
TOC1 (timing of CAB expression) has been identified
by the Kay laboratory in a screen for mutants with aberrant output rhythms by monitoring the noninvasive
luciferase (luc) marker under control of the cycling
CAB2 promoter (Millar et al. 1995b). The toc1 mutation
shortens the period of several rhythms, including gene
expression, leaf movement and stomatal movement, and
influences photoperiodic flower induction (Somers et al.
1998b). TOC1 is a nuclear protein with an N-terminal
receiver domain known from response regulators of the
His-to-Asp phosphorelay system (Strayer et al. 2000).
Indeed, TOC1 has also been identified in a search for
response regulators. Since it lacks two phospho-accepting aspartate residues, it presumably does not function
via a classical phosphorelay mechanism. Accordingly it
has been designated APRR1, Arabidopsis pseudo response regulator (Matsushika et al. 2000).
Furthermore, two transcription factors with a single
MYB-like domain, CCA1 and LHY, have been implicated in the generation of rhythms, as their constitutive
overexpression disrupts several rhythms, including leaf
movement and transcript oscillations (Schaffer et al.
1998; Wang and Tobin 1998). CCA1 overexpression also
strongly represses the endogenous CCA1 transcript oscillations. Thus the CCA1 protein and CCA1 transcript
are part of a negative autoregulatory circuit. Moreover,
CCA1 and LHY negatively regulate each other’s transcripts, indicative of close interconnection between the
feedback loops (Wang and Tobin 1998; Fowler et al.
1999). Based on the identification of these autoregulatory circuits it can be deduced that transcriptional
feedback loops also play an important role in rhythm
genesis in higher plants, although the transcription factors do not share sequence homology with known clock
components from mammals, flies and fungi.
Lately, a model has emerged showing that reciprocal
regulation between the CCA1/LHY loops on the one
hand and TOC1 on the other hand may constitute the
core of an oscillator in Arabidopsis (Alabadi et al. 2001).
7
Negative interaction between CCA1/LHY and TOC1
was inferred from the opposite phases of the TOC1 and
CCA1/LHY mRNAs that cycle with an evening peak
and a peak around dawn, respectively (Strayer et al.
2000). Indeed, in plants constitutively producing LHY
or CCA1, TOC1 mRNA oscillations are depressed to
trough levels, implicating LHY and CCA1 as negative
regulators of TOC1. In turn, LHY and CCA1 oscillations have a shorter period in toc1 mutant plants and
peak levels are strongly reduced in the presumptive lossof-function mutant toc1-2, indicating a positive action of
TOC1 on CCA1 and LHY (Alabadi et al. 2001). Since
CCA1 and LHY are not arrhythmic in the absence of
functional TOC1, one may envisage factors acting redundantly with TOC1 (see below).
CCA1 was identified through its interaction with the
promoter of the LHCB1*1 (CAB2) gene that cycles with
a morning peak. CCA1 binds an AAA/CAATCT motif
found within a 36-bp element mediating circadian regulation (Carré and Kay 1995; Wang and Tobin 1998).
CCA1 and LHY also influence transcripts with an evening phase, as constitutive expression of CCA1 or LHY
causes arrhythmic expression of AtGRP7/CCR2 encoding a glycine-rich RNA-binding protein that itself is part
of a subordinated negative autoregulatory circuit
(Heintzen et al. 1997; Schaffer et al. 1998; Wang and
Tobin 1998). This regulation has been predicted to occur
through putative CCA1-binding sites within a minimal
clock response element of the AtGRP7/CCR2 promoter
(Staiger and Apel 1999; Alabadi et al. 2001; Quail
2002b).
An AAAATATCT element with strikingly similarity
to the CCA1-binding site has been discovered in genes
cycling with an evening phase, including TOC1, and
CCA1 and LHY indeed bind to this element of the
TOC1 promoter (Harmer et al. 2000; Alabadi et al.
2001). As TOC1 cycles in antiphase to CAB2, binding of
CCA and LHY to the same element in either the TOC1
or CAB2 promoter has opposite effects on promoter
activity. Thus, promoter architecture presumably plays
an important role in determining the response to these
regulators.
Additional layers of complexity may have to be
added to this outline, since CCA1 and LHY, as well as
TOC1/APRR1, turned out to be members of multigene
families (Matsushika et al. 2000). The ‘‘APRR1/TOC1
quintet’’ family is under co-ordinate clock control.
APRR9 is induced by light and appears shortly after
dawn. APRR7, APRR5, APRR3 and APRR1/TOC1
subsequently appear sequentially at 2- to 3-h intervals.
Their free-running rhythms are differentially affected by
overexpression of APRR1/TOC1 and CCA1. Moreover,
light induction of APRR9 is absent in APRR1-ox plants.
These observations suggest that in addition to the mutual interaction between CCA1 and TOC1/APRR1 each
member of the ‘‘APRR1/TOC1 quintet’’ is under exquisite control of CCA1 and APRR1/TOC1, although
the molecular underpinnings remain to be resolved
(Makino et al. 2002; Matsushika et al. 2002).
Furthermore, several CCA1 and LHY homologues
have been identified that oscillate with a peak mostly
around dawn and thus have been designated the Reveille
(RVE) family (Andersson et al. 1999). This opens the
possibility that homo- and heterotypic interactions
within the families may contribute to the core oscillatory
loop.
Light input to the clock
A prime function of the endogenous clock is to provide
an endogenous estimate of the environmental time. In
constant conditions in the laboratory, the endogenous
clock runs with a period of about, but not exactly, 24 h
whereas in nature, the period is exactly 24 h. In other
words, to be in synchrony with the outside world, plants
have to monitor the 24-h geophysical cycle and adjust
their endogenous clock on a daily basis. Periodic
changes between dawn and dusk – with light being the
principle ‘‘zeitgeber’’ – serve to set the phase of the endogenous clock. As phase is determined by the level of a
clock component, entrainment of the oscillator requires
that a clock component reacts to incoming light in a
manner dependent on the time of day.
Recent data obtained in several laboratories begin to
shed some light on the components of the Arabidopsis
oscillator system that may be a target for this
‘‘entrainment’’, the photoreceptors that provide light
information as well as the intermediates of the respective
input signaling cascades.
Photoreceptors
The available Arabidopsis photoreceptor mutants have
allowed their roles in light input to the clock to be tested.
Broadly speaking, no single photoreceptor has been
identified as having an essential function to reset the
circadian clock. This was illustrated very clearly for a
phyAphyBcry1cry2 quadruple mutant that is severely
impaired for de-etiolation but retains leaf movement
rhythms that can entrain to different photoperiods
(Yanovsky et al. 2000a).
The importance of those photoreceptors could,
however, be uncovered by testing the period length of
circadian-regulated transcripts during constant illumination with monochromatic light. In extended darkness,
the free-running period of CAB2::LUC rhythms
lengthens to 30–36 h, a much longer value than observed
for free-running periods in other organisms (Millar et al.
1995a). The period becomes shorter at increasing light
intensities. This inverse relationship between period
length and light intensity in plants and light-active animals is known as Aschoff’s rule (Aschoff 1979). Both
blue light and red light contribute to this period shortening (Millar et al. 1995a). Mutants deficient in specific
photoreceptors revealed that phyA, phyB, phyD and
phyE mediate red-light effects on the pace of the clock,
8
and cry1 and cry2 mediate blue light input (Somers et al.
1998a; Devlin and Kay 2000). cry1cry2 double mutants
retain rhythmicity of CAB2::LUC expression, in contrast to mice that upon knockout of both cry1 and cry2
become completely arrhythmic (Somers et al. 1998a; van
der Horst et al. 1999; Devlin and Kay 2000). Cryptochromes in plants are thus not integral components of
the clockwork in contrast to their mammalian counterpart.
Co-action between cry2 and phytochromes to control
the pace of the clock is demonstrated in cry2 mutants
that have a longer period of CAB2::LUC mostly in
white light, indicating requirement for simultaneous
activation of cry and phy (Mas et al. 2000). Indeed, cry2
physically interacts with phyB in discrete regions of the
nucleus (Mas et al. 2000).
Cry1 mutants show deficiency in entrainment of
CAB2::LUC rhythms also in low-fluence-rate red light
(Devlin and Kay 2000). As period lengthening in cry1phyA double mutants is similar to that in the individual
mutants and cry1 does not significantly absorb red light,
cry1 presumably acts as a signaling intermediate downstream of phyA. In fact, a physical interaction between
phyA and cry1 has previously been demonstrated: cry1
undergoes light-dependent phosphorylation by phyA
(Ahmad et al. 1998).
Evidence for an impact of phyA and cry1 on circadian rhythms was also obtained through an intriguing
observation made for the catalase 3 transcript that cycles with an evening peak. In prolonged darkness, the
transcript attains a high constitutive level, in contrast to
many oscillating transcripts in plants that rapidly damp
to undetectable levels in constant darkness. This damping to high levels is phyA and cry1 dependent but the
mechanism involved is so far not understood (Zhong
et al. 1997).
Mutants affected in phot1 show no defect in signaling
to the clock (Devlin and Kay 2001). The role of phot2
has not yet been tested. Nevertheless, the repertoire of
clock photoreceptive molecules may still expand. Good
candidates are members of the ZEITLUPE/FKF/LKP
family. The ztl mutant has a period-lengthening phenotype of multiple rhythms that is strongly dependent
on light intensity, and in addition it has a late-flowering
phenotype (Kiyosue and Wada 2000; Somers et al.
2000). Overexpression of LKP2 (LOV Kelch protein 2)
causes arrhythmicity of transcript oscillations both in
constant light and darkness, as well as delayed photoperiodic flower induction (Schultz et al. 2001). A third
member, FKF1 (flavin-binding, kelch repeat, F box)
was also identified on the basis of a late-flowering phenotype of the fkf1 null mutation (Nelson et al. 2000).
Deletion of FKF1 influences the waveform of circadian
oscillation and FKF1 itself cycles, in contrast to ZTL
and LKP2. Although based on these observations it is
difficult to assign an exact role to this trio, these proteins
feature three interesting motifs suggestive of a possible
biochemical function (Kiyosue and Wada 2000; Nelson
et al. 2000; Somers et al. 2000; Schultz et al. 2001). By
analogy with phototropins, their N-terminal LOV/PAS
domain could bind a chromophore. However, the effects
of ZTL on period length are observed both in red and
blue light, suggesting that in red light ZTL might act as a
signaling intermediate downstream of a phytochrome (a
situation analogous to the cry1/phyA interaction in red
light, see above). This suggests that the LOV/PAS domain may mediate protein–protein interaction and any
light-dependent activity of ZTL may result, directly or
indirectly, from stimulation by other photoreceptors. A
recent study indeed shows interaction of ZTL/ADAGIO
with both cry1 and phyB (Jarillo et al. 2001b). ZTL/
FKF1/LKP2 also have F-box motifs that in other
proteins serve to target them for ubiquitin-dependent
proteolysis, as well as kelch domains mediating protein–
protein interaction. Based on the predicted functions of
the domains, the ZTL family members may interact in a
light-dependent manner with proteins affecting period
length. Through targeting these molecules for proteolytic degradation, they may influence the length of the
circadian cycle. Potential targets include clock-associated molecules, including ZTL itself, as well as photoreceptors.
Expression of photoreceptors is under control
of the clock
A recent investigation shows that the genes encoding the
major photoreceptors are not uniformly active
throughout the day. Rather, the promoter activity of
phytochromes and cryptochromes is diurnally regulated
(Bognar et al. 1999; Toth et al. 2001). Transcripts encoding the light-stable proteins phyB, phyC, phyD,
phyE and cry1 peak during the early hours of the daily
light phase whereas those encoding light-labile phyA
and cry2 reach their highest level close to dusk (Fig. 2).
This may reflect the importance of the newly synthesized
proteins, e.g. for detection of photoperiod extension and
end-of-day response to far-red.
The mRNA oscillations persist in constant light and
in constant darkness, indicative of endogenous control.
Thus, photoreceptors, on the one hand, transduce light
signals to the clock and thus are part of the input
pathway and, on the other hand, have to be considered
part of the clock output. The rhythmically produced
photoreceptors can, by feedback, temporally restrict
light input to the clock so that resetting cues are most
efficiently perceived at the appropriate times of day.
Fig. 2. Time-of-day specific expression of Arabidopsis photoreceptors. Depicted is the relative phase of promoter activity as
determined by measuring luciferase activity in transgenic Arabidopsis plants. zt Zeitgeber time, hours after lights on. Modified from
Toth et al. (2001)
9
Reduced synthesis of photoreceptive molecules during
the night may serve to buffer the circadian clock against
moonlight, for example, which does not signal dawn or
dusk and thus should not reset the clock (Bünning and
Moser 1969).
To exert this type of regulation, protein levels of the
photoreceptors should also change systematically over
the course of the day. This has so far only been demonstrated for CRY2 (Bognar et al. 1999; El-Din El-Assal
et al. 2001). However, accumulation of phytochromes in
the nucleus appears to vary over the course of the light–
dark cycle. Moreover, extensive interactions both
among photoreceptors and with signaling components
provide a basis for additional layers of posttranslational
control of activity that we are only beginning to appreciate (Nagy and Schaefer 2002; Quail 2002a, b).
Light input transduction
As described in the previous section, partially overlapping functions and extensive cross-talk between multiple
photoreceptors are apparent for the light input to the
clock and general photomorphogenesis. We still know
little about the extent to which the corresponding
downstream signaling cascades are shared.
ELF3 (early flowering) is among the first components
with a demonstrated role in light signaling to the clock.
Elf3 mutants flower as early in short days as in long
days. Leaf movement and LHCb1*1 (CAB2) promoter
activity are arrhythmic in continuous light whereas
rhythmicity is retained in continuous darkness, suggesting that the defect may affect light input (Hicks et al.
1996).
In wild-type plants, phytochrome induction of CAB2
is rhythmically repressed by the clock, allowing a higher
inducibility during the day than during the night (Millar
and Kay 1996). This circadian ‘‘gating’’ of the acute
light response is lost in elf3, leading to constitutive CAB
activation (McWatters et al. 2000). Thus, the apparent
arrhythmicity in constant light at least in part results
from an aberrant reaction to light. In a complementary
approach, ELF3 signaling to the clock was tested by
assaying phase shifts elicited by light pulses administered
at different times. Phase shifts were reduced in plants
constitutively overproducing ELF3 during the night,
again indicating that ELF3 then negatively modulates
light input (Covington et al. 2001). Elf3 transcript and
protein oscillate, reaching their highest concentration at
dusk, i.e. around the time they are predicted to be required to antagonize light input to the clock (McWatters
et al. 2000; Covington et al. 2001; Hicks et al. 2001; X.L.
Liu et al. 2001).
ELF3 has been proposed to be part of an additional loop through which it feeds back to periodically
restrict light input to the clock (McWatters et al.
2000). Similar to the rhythmically produced photoreceptors that feed back onto light input (Fig. 3),
feedback by an oscillating signaling intermediate
Fig. 3. The Arabidopsis circadian system. Photoreceptors are
under clock control and feed back to modulate light input to the
clock
allows rhythmic input to the oscillator under otherwise
constant conditions.
The exact role of ELF3 in general light signaling is
not understood. The phenotype of elf3 mutants that is
most apparent in red light suggests a role downstream of
the phyB photoreceptor, and a physical interaction between phyB and ELF3 has been shown (Reed et al. 2000;
X.L. Liu et al. 2001). However, double mutants between
elf3 and phyB are additive under certain light conditions,
clearly indicating that ELF3 does not exclusively work
downstream of the phyB photoreceptor.
A role in light signaling has also been demonstrated
for GIGANTEA, originally identified in a mutant with
extremely delayed onset of flowering in long days. The gi
mutation subsequently was found to affect leaf movement rhythms and transcript oscillations (Fowler et al.
1999; Park et al. 1999). The period-shortening phenotype of the gi-1 allele is dependent on fluence rate, suggesting that GI mediates light input to the clock. On the
other hand, GI itself undergoes circadian oscillation.
Rhythmic expression of GI as well as of several other
cyclic transcripts is impaired in gi-2 mutants that lack
functional GI protein. Therefore, a model has been
proposed according to which GI is part of a feedback
loop that is required for a central oscillator to maintain
period and amplitude (Fowler et al. 1999; Park et al.
1999).
A further gi allele was recovered in a screen for defects in the inhibition of hypocotyl elongation in red
light (Huq et al. 2000). GI therefore functions in phyBmediated photomorphogenesis and affects the light input to the clock.
Other light signaling intermediates that are cyclically
expressed include SPA1 and RPT2, but a potential
function in clock regulation has not yet been reported
(Harmer et al. 2000).
Of the numerous other light signaling components
operating downstream of the photoreceptors only very
few have been tested for a direct role in light input to the
clock. Cop1 and det1 mutants with a de-etiolated phenotype in the absence of light shorten the free-running
period of the CAB2::LUC rhythm in the dark although
the mechanism has not been investigated (Millar et al.
1995a). The action of both FHY1 (far-red elongated
hypocotyl) and FHY3, two phyA signaling components,
10
is required for phase shifting of leaf movement rhythms
in response to far-red light. This is interesting since
FHY3 is only required for a subset of phyA-mediated
responses and suggests that phyA in its HIR signaling
mode is necessary for resetting the clock in response to
far-red light (Yanovsky et al. 2000b, 2001).
Oscillator components responding to light input
Some of the clock components that are directly influenced by light have been identified. Whereas TOC1
mRNA is not directly light-responsive, CCA1 mRNA is
rapidly and transiently induced by red light in etiolated
seedlings (Wang and Tobin 1998). By analogy, rapid
induction of CCA1 and LHY following phytochrome
activation has been implicated in clock resetting by light
at dawn: This presumably is mediated by the bHLH
factor PIF3 through the same short transcriptional
cascade as described above (Martinez-Garcia et al.
2000).
Figure 4 summarizes a current model (Alabadi et al.
2001; Quail 2002b). Upon light activation, phytochrome
migrates to the nucleus and forms complexes with PIF3
bound to the G box of CCA1 (and, by inference, LHY).
Activation at dawn leads to increased CCA1 and LHY
transcript and protein levels. Perturbation of CCA1 and
LHY steady-state levels then affects TOC1 levels
through reciprocal regulation, as LHY and CCA1 coordinately repress TOC1. Progressive decline in CCA1
and LHY levels over the course of the day subsequently
allows TOC1 to accumulate which then, directly or
Fig. 4. Model of how light input may influence a core oscillator in
Arabidopsis. Red-light activation of phytochrome leads to translocation into the nucleus where Phy interacts with promoter-bound
PIF3. This leads to rapid activation of CCA1 and, perhaps, LHY.
LHY and CCA1 coordinately repress TOC1. Progressive decline in
CCA1 and LHY levels in the course of the day allows TOC1 to
accumulate which then, directly or indirectly, stimulates production
of CCA1 and LHY. In addition to these interactions within the
presumptive core oscillator, high CCA1 and LHY levels in the
morning activate CAB2 and likely other morning genes, and may
repress evening genes like AtGRP7/CCR2. Elevated TOC1 levels
later in the day then allow expression of evening genes. Modified
according to Alabadi et al. (2001) and Quail (2002b)
indirectly, stimulates production of CCA1 and LHY. In
addition to these interactions within the presumptive
core oscillator, high CCA1 and LHY levels in the
morning act downstream to activate CAB2 and likely
other morning genes and possibly repress evening genes,
for example AtGRP7/CCR2. Decreasing levels of
CCA1/LHY and increasing levels of TOC1 later in the
day then allow expression of evening genes. According
to this model, PIF3 may be a key player, integrating
phytochrome regulation of the circadian system as well
as of photomorphogenesis.
One should be aware that this may not yet be the
complete picture. APRR1/TOC1 has been shown to
associate with PIF3 as well as with a PIF3 like protein,
PFL1 (Makino et al. 2002). If these interactions prove to
occur in planta, they may add another twist.
It remains to be determined whether CCA1 induction
depends on time-of-day, a prerequisite for a stimulus
that resets the clock. Furthermore, it needs to be established whether CCA1 and LHY would also serve as
targets for clock resetting by blue or far-red light. As
PIF3 has been implicated in phytochrome signaling
specifically, at least blue light may be read by other
components.
Additional ‘‘zeitgebers’’
Although light represents the most prominent ‘‘zeitgeber’’ in higher plants, regular temperature changes can
entrain the oscillator in the absence of periodic light
cues. In mustard and Arabidopsis plants grown from
seed in continuous light at a constant temperature,
transcripts encoding the chlorophyll-binding proteins or
glycine-rich RNA-binding proteins do not undergo circadian oscillations. If plants are exposed to regular
temperature cycles, oscillations with a 24-h period are
synchronized and/or initiated (Heintzen et al. 1994a, b;
Somers et al. 1998b).
In seedlings of Lycopersicum esculentum, temperature
cycles are even dominant over light programs in the
control of rhythms in polyamine content (N’Doye et al.
1994). At present the way in which plants perceive
temperature cues is not understood.
The large temperature step Arabidopsis plants encounter upon transfer from stratification conditions at
4 °C to regular growth conditions at ambient temperature does not reset the phase (Zhong et al. 1998). This
has been taken to suggest that during an initial phase the
oscillator is unable to respond to temperature. In contrast, imbibition of dried seeds can act to entrain circadian rhythms in Arabidopsis (Zhong et al. 1998).
Photoperiodism
The endogenous clock not only tells time of the day, but
also provides an internal estimate of the season of the
year. Resetting the clock by light enables plants to detect
11
the gradual shift in the time of sunrise during the seasonal progression. Monitoring the seasons offers a selective advantage to plants by allowing appropriate
reactions in order to prevent damage by unfavorable
environments. For example, at high latitudes, short days
in autumn lead to induction of bud dormancy and cold
hardiness in anticipation of adverse winters. Most importantly, the transition from vegetative to reproductive
growth in many plant species is controlled by annual
modulation of photoperiod and thermoperiod (Simpson
and Dean 2002). This ensures that onset of flowering
and seed set terminate in a favorable season. Short-day
induction, for example, would allow plants to flower
before a dense leaf canopy develops. In out-crossing
species, coordination of flowering within the population
by way of photoperiodic control increases the possibility
for genetic recombination.
In the 1930s, Erwin Bünning had suggested that
plants rely on their endogenous timekeeper – the circadian clock – to measure daylength (Bünning 1936). Experimental support now comes from the discovery of
mutants in the facultative long-day plant Arabidopsis
that are disrupted in both circadian rhythmicity and
flowering time: Lhy mutant plants with disrupted circadian rhythms flower late in long days and the lateflowering mutant gi additionally affects leaf movement
rhythms and LHC transcript oscillations (Schaffer et al.
1998; Fowler et al. 1999; Park et al. 1999). The elf3
mutant is insensitive to daylength, flowering as early in
short days as in long days, and affects light input to the
circadian clock (Hicks et al. 1996; McWatters et al. 2000;
Covington et al. 2001; X.L. Liu et al. 2001).
Two related views of how clock function may translate into photoperiodic flower induction have been
proposed (Fig. 5). According to the model of ‘‘external
coincidence’’, environmental light impinging on the
plant in a light-sensitive phase of an endogenous circadian rhythm would promote transition in flowering in
long-day plants, for example Arabidopsis, and retard it
in short-day plants, for example rice. The ‘‘internal
Fig. 5. Model of photoperiodic control by rhythmically expressed
flowering time genes. In short days (SD), high expression of a
regulatory gene (such as CO) would be largely confined to the dark
phase (solid line). In long days (LD), light would modify the
rhythms such that high-level expression would occur concomitant
with environmental light (external coincidence) and/or that highlevel expression of one regulator (solid line) would occur concomitantly with that of another regulator (dashed line; internal
coincidence)
coincidence’’ model, in contrast, posits that two co-existing endogenous rhythms would fall into phase under
flower-promoting photoperiods (Wagner et al. 1998).
CONSTANS (CO) is a flowering-time gene that
promotes floral transition specifically in long days. It has
recently been found that CO mRNA levels are under
clock control (Samach et al. 2000; Suarez-Lopez et al.
2001). The observation that these oscillations are modified according to the photoperiod suggests that CO
levels may be one of the endogenous rhythms postulated
by these models: under short days, the CO peak is
largely confined to darkness, whereas under inductive
long days, high CO levels coincide with light (SuarezLopez et al. 2001). Furthermore, CO rhythms are affected in mutants with altered photoperiodic response:
CO oscillates at a higher level in the early flowering elf3
mutant. In contrast, CO oscillates with lower amplitude
in the late-flowering gi-3 mutant, and in the lhy mutant,
CO is expressed at a reduced level with a different phase.
The late-flowering phenotypes are corrected by CO
overexpression. As LHY, GI and ELF3 all are part of
the circadian system, the clock presumably influences
the photoperiodic response by setting the CO rhythm.
CO then promotes flowering via downstream genes
(Samach et al. 2000). Indeed, it causes an immediate
target, FT, to oscillate with a similar phase (SuarezLopez et al. 2001).
A QTL (quantitative trait locus) controlling photoperiodic flowering in rice, a short-day plant, turned out
to be related to CO, and a CO ortholog from another
short-day plant, Pharbitis nil, corrects late flowering of
the Arabidopsis co mutant (Yano et al. 2000; J. Liu et al.
2001). This raises the intriguing possibility that related
proteins may participate in photoperiodic timekeeping
in both long-day and short-day plants, although the
exact mode of action remains to be resolved.
Although a detailed analysis of CO protein levels
under the different photoperiods has not yet been reported, initial experiments point to CO being unstable,
providing an additional possibility for regulation of its
activity by light (Suarez-Lopez et al. 2001). Interestingly,
the steady-state abundance of the CRY2 blue-light receptor has been found to play a crucial role in determining floral transition. QTL analysis has identified cry2
as a trait distinguishing the flowering time behavior of
two Arabidopsis ecotypes, demonstrating the importance
of this locus for adaptation of Arabidopsis in the natural
environment (El-Din El-Assal et al. 2001). cry2 mutants
are late flowering in long days specifically, pointing to a
role of this photoreceptor to detect daylength extension
(Guo et al. 1998). Indeed, CRY2 levels may themselves
be a marker for photoperiod: whereas in long days,
CRY2 levels do not notably vary in the light, CRY2 is
rapidly down-regulated in the light during short days
and only re-accumulates at dusk. Moreover, a single
amino acid substitution in CRY2 of the EDI accession
leads to a substantial increase in CRY2 protein during
short days and confers an early flowering phenotype.
Rhythmicity of the CAB2::LUC marker and leaf
12
movement is not affected by this mutation, suggesting
that the effect of cry2 on photoperiodic flowering may
not be exerted via cry2 signaling through the clock but
may rather affect flowering-time genes directly (El-Din
El-Assal et al. 2001).
Current efforts focus on unraveling the interaction
between the multiple pathways mediating transition
to flowering in response to light and photoperiod, to
endogenous developmental cues, to gibberellins or
vernalization. Interestingly, two of the key factors integrating floral pathways are immediate targets of CO
(Samach et al. 2000; Simpson and Dean 2002).
Conclusion
Over recent years, more and more evidence has accumulated showing overlapping functions between individual photoreceptors during photomorphogenesis.
Furthermore, in addition to transduction chains triggered specifically by one photoreceptor, extensive crosstalk between signaling cascades downstream of multiple
photoreceptors has become apparent. Thus, it may be
more appropriate to view the downstream events in the
light of an entire signaling network (Casal 2000; Quail
2002a).
Entrainment of the endogenous clock by environmental light appears to use, at least partly, the welldescribed photosensory pigments as well as many of the
signaling intermediates also involved in photomorphogenetic regulation, pointing to extensive cross-talk. In
addition, previously undescribed molecules have been
found to play a prominent, although as yet not fully
understood, role in light regulation of the circadian
system, as for example the ZTL family. Also, phyD and
phyE play only a minor role during de-etiolation but
clearly contribute to entrainment of the clock (Aukerman et al. 1997; Devlin et al. 1998; Devlin and Kay
2000). Most of the photoreceptors, as well as some
components of the light input pathway, are themselves
under circadian control, suggesting that these components feed back to regulate the plant’s light responses in
a time-of-day dependent manner.
In summary, despite the fact that our current understanding is far from complete it appears that it will not
be easy to tease apart the function of light perception
from its specific role to entrain the circadian clock.
Acknowledgements We would like to express our sincere gratitude
to Nick Amrhein, a dedicated teacher and good colleague and
mentor, for his help, support and encouragement over the years.Due to space constraints, we have attempted to highlight a few
examples of recent progress in unraveling light signaling and have
had to use reviews rather than original publications on a number of
occasions. We apologize to those colleagues whose contributions
could not be included. We thank Dr. Dieter Rubli and Nicolas
Roggli for help with the figures. Work in our laboratories has been
supported by the Swiss National Foundation (631-058151.99 for
C.F. and 31-052475.97 for D.S.), the EMBO Young Investigator
Program (C.F.) and the ETH Research Commission (D.S.).
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