PSEUDO-RESPONSE REGULATOR 7 and 9 Are Partially
Redundant Genes Essential for the Temperature
Responsiveness of the Arabidopsis Circadian Clock
Patrice A. Salome ´ and C. Robertson McClung1
Dartmouth College, Department of Biological Sciences, Hanover, New Hampshire 03755-3576
Environmental time cues, such as photocycles (light/dark) and thermocycles (warm/cold), synchronize (entrain) endoge-
nous biological clocks to local time. Although much is known about entrainment of the Arabidopsis thaliana clock to
photocycles, the determinants of thermoperception and entrainment to thermocycles are not known. The Arabidopsis
PSEUDO-RESPONSE REGULATOR (PRR) genes, including the clock component TIMING OF CAB EXPRESSION 1/PRR1, are
related to bacterial, fungal, and plant response regulators but lack the conserved Asp that is normally phosphorylated by an
upstream sensory kinase. Here, we show that two PRR family members, PRR7 and PRR9, are partially redundant; single
prr7-3 or prr9-1 mutants exhibit modest period lengthening, but the prr7-3 prr9-1 double mutant shows dramatic and more
than additive period lengthening in the light and becomes arrhythmic in constant darkness. The prr7-3 prr9-1 mutant fails
both to maintain an oscillation after entrainment to thermocycles and to reset its clock in response to cold pulses and thus
represents an important mutant strongly affected in temperature entrainment in higher plants. We conclude that PRR7 and
PRR9 are critical components of a temperature-sensitive circadian system. PRR7 and PRR9 could function in temperature
and light input pathways or they could represent elements of an oscillator necessary for the clock to respond to
Most organisms on the planet live under the daily cycles of night
and day, a consequence of the rotation of the earth on its axis.
Among these organisms, biological rhythmswith a period of 24 h
are widespread. Circadian rhythms are endogenously generated
and maintain a period close to 24 h even in the absence of the
entrainment provided by the daily cycles. These oscillations and
their synchronization with the environment are under genetic
control, and many mutants affecting one or a combination of
these important aspects of circadian rhythmicity have been
isolated in model systems, including Drosophila melanogaster,
and mice (Bell-Pedersen, 2002; Panda et al., 2002a; Golden and
Canales, 2004). Because expression of many clock components
is rhythmic, a coupled transcription/translation feedback loop
was proposed to comprise the central oscillator. The identifica-
tion and characterization of additional mutants has complicated
the simple feedback loop, and current models incorporate two
interconnected feedback loops as the core of the oscillatory
mechanism in fungi and animals (Loros and Dunlap, 2001;
Williams and Sehgal, 2001; Albrecht and Eichele, 2003).
In Arabidopsis thaliana, the current model proposes a single
feedback loop composed of the proteins CIRCADIAN CLOCK
ASSOCIATED 1 (CCA1), LATE AND ELONGATED HYPOCOTYL
(LHY), andTIMING OF CAB
RESPONSE REGULATOR 1 (TOC1/PRR1) (Alabadı ´ et al., 2001).
CCA1 and LHY are single-Myb domain transcription factors that
show dawn peaks in transcription as well as mRNA and protein
abundance (Schaffer et al., 1998; Wang and Tobin, 1998). CCA1
and LHY repress the expression of TOC1 through direct binding
to the TOC1 promoter (Alabadı ´ et al., 2001). Upon turnover
of CCA1 and LHY by an as yet unidentified mechanism, TOC1
repression is alleviated and TOC1 accumulates to peak levels
near dusk. Directly or indirectly, TOC1 closes the loop by
upregulating CCA1 and LHY. TOC1 is degraded via the protea-
some, mediated through SCFZTL, an SCF complex including the
F-box protein ZEITLUPE (ZTL) (Ma ´s et al., 2003a; Han et al.,
2004). CCA1 and LHY are partially redundant, because either
single mutant displays a short period, but the double mutant
becomes arrhythmic after 2 d in continuous conditions (Alabadı ´
et al., 2002; Mizoguchi et al., 2002). Plants carrying the strong
loss of function toc1-2 allele retain rhythmicity, albeit with short
period, under someconditions (in bluelightand in whitelight) but
fail to sustain an oscillation when assayed in red light and in the
dark (Ma ´s et al., 2003b). This suggests that other genes in
addition to TOC1 participate in the generation of the oscillation
and compensate for the loss of TOC1 under some conditions.
Taking the CCA1/LHY redundancy asamodel, onemight expect
that TOC1-related genes may function within or close to the
oscillator itself and show partial functional overlap with TOC1.
TOC1 is a member of the Arabidopsis PRR family, composed of
the rhythmically expressed genes PRR9, PRR7, PRR5, PRR3,
1To whom correspondence should be addressed. E-mail mcclung@
dartmouth.edu; fax 603-646-1347.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: C. Robertson
Article, publication date, and citation information can be found at
The Plant Cell, Vol. 17, 791–803, March 2005, www.plantcell.org ª 2005 American Society of Plant Biologists
and TOC1/PRR1 (Matsushika et al., 2000). PRRs lack the
conserved Asp that in typical response regulators is phosphor-
ylated by the upstream kinase of the two-component cascade
(Hwang et al., 2002). We and others have shown that loss-of-
function alleles in any of the five PRR genes yield a range of
circadian defects, including period and phase phenotypes,
although no single prr mutation confers arrhythmicity (Eriksson
et al., 2003; Kaczorowski and Quail, 2003; Ma ´s et al., 2003b;
Michael et al., 2003b; Mizuno, 2004). The PRRs can be divided
into three groups based on sequence similarity: PRR3/PRR7,
PRR5/PRR9, and TOC1. However, sequence similarity alone is
not an accurate predictor of functional overlap because loss-of-
function alleles of PRR5 and PRR9 display additive phenotypes
in the double mutant, implying distinct functions (Eriksson et al.,
We reasoned that mutants involved in the same clock function
(warm/cool) or photocycles (light/dark) and constant conditions
in which the clock is predicted to free-run. We therefore charac-
short period mutants prr3-1 and prr5-3 affect period length
modestly, indicating that PRR3 and PRR5 are not necessary for
the plant response to thermocycles. By contrast, prr7-3 and
prr9-1 double mutant exhibits a much stronger clock defect than
prr9-1 double mutant fails to reset in response to temperature
that the phenotypes of prr7-3 prr9-1 are not all dependent on
light. This mutant with insensitivity to temperature signals should
of plant circadian rhythms.
Effect of Thermocycles on Mutants Carrying
Loss-of-Function lhy, ztl, and prr Alleles
h at 228C followed by 12 h at 128C) or 22 to 188C thermocycles
(McClung et al., 2002). We analyzed cotyledon movement
rhythms for the representative short and long period clock
mutants, lhy-20 and ztl-4 (Michael et al., 2003b), and for prr
mutants. To facilitate the comparison of the timing of the
acrophase (peak) of the cotyledon rhythms on successive
days, data were presented as double plots, wherein the first
line shows the peak in cotyledon position of day 1 and day 2, the
to 22 to 128C cycles in constant light and released into free-run
(continuous conditions) at 228C. All mutant seedlings are en-
trained by thermocycles because the mutants share the same
phase as the wild type on the first day after release from
entrainment. Loss-of-function alleles of each of the PRRs, of
the clock components LHY and ZTL (Figure 1), as well as CCA1
and TOC1 (data not shown) yield circadian defects after either
light or temperature entrainment. Their cotyledon movement
phenotypes after thermocycles are also identical to their de-
scribed phenotypes after entrainment to photocycles (Figures
1A, 1D, 1G, 1J, 1M, and 1P). By contrast, loss-of-function alleles
of the PHYTOCHROME B red light photoreceptor, which func-
tions in light input but not in oscillator function, alter the phase of
expression of the clock output gene LIGHT HARVESTING
COMPLEX B (LHCB) after photocycles but not thermocycles
(Salome ´ et al., 2002). This suggests that varying entraining
conditions may prove useful in determining the respective
contribution of putative components of the clock and suggest
a role for the PRRs in temperature response either proximal to or
within the Arabidopsis oscillator.
Another approach to test the response of a mutant to thermo-
cycles is to characterize its circadian phenotype during entrain-
ment. We first tested the ability of mutants in the clock genes
to five photocycles at 228C and then released to 22 to 188C
thermocycles, provided in phase with the light–dark entrainment
(228C replaces light, 188C replaces dark), in continuous light. As
expected for short period mutants that are reset to an exact 24-h
period by the entraining cycles, lhy-20 (Figures 1B and 1C) and
toc1-2 (data not shown; Strayer et al., 2000) display a leading
circadian phase in cotyledon movement. The long period mutant
ztl-4 exhibits the expected lagging phase (Figures 1E and 1F).
thermocycles, we conclude that mutations in the clock genes
LHY, TOC1, and ZTL do not compromise entrainment to thermo-
response to thermocycles.
Mutations in PRR3 do not cause a leading phase during
rhythm is always in phase with the wild type, indicating that it is
effectively entrained by the thermocycles (Figures 1H and 1I).
response to thermocycles.
wild type, but the synchrony between the two genotypes is lost
after 3 to 4 d in the new entraining routine. By the end of the
experiment, prr5-3 mutant seedlings display a phaseopposite to
the wild type (Figures 1K and 1L). The double plot of acrophase
(phase of the peak) during thermocycles suggests that prr5-3
short period seen in the prr5-3 mutant is always modest
(;1 h shorter than the wild type; Michael et al., 2003b) and is
insufficient to readily explain the observed change in phase.
This phenomenon is not seen when the mutant is assayed in
T-cycles with T ¼ 28, or with T ¼ 12 (data not shown), indicating
normal entrainment to these thermocycles. T-cycles are entrain-
in a 28-h T-cycle, the plants are exposed to cycles of 14 h warm
(or light) and 14 h cool (or dark). Although we do not understand
the basis of this phase alteration of prr5-3 during entrainment to
thermocycles, we believe that prr5-3 mutants retain the ability to
entrain to thermocycles because the initial phase of cotyledon
movement is normal after thermocycles (Figure 1J) and during
T-cycles (Figure 1K; data not shown). Nonetheless, the delayed
achievement of a stable phase relationship seen in prr5-3 during
thermocycles suggests that PRR5 plays a role in temperature
entrainment of cotyledon movement.
792The Plant Cell
Figure 1. Cotyledon Movement during and after Temperature Entrainment Allows the Assignment of Clock Function.
Temperature-Insensitive Circadian Mutant793
During thermocycles, prr7-3 mutants initially fail to entrain to
the thermocycles and instead appear to free-run for the first 4 d.
The peak in cotyledonmovement occurs progressively laterthan
the wild type (Figures 1N and 1O) and follows the same slope
seen in the acrophase of cotyledon movement after thermo-
cycles (Figure 1M). This suggests that during this time, prr7-3
plants are not responsive to the new resetting cues provided by
lagging phase relative to the wild type, consistent with that
predicted for a long period mutant. Similar results have been
recorded for activity rhythms of many animals (DeCoursey,
1990). This initial failure to follow the entrainment has been at-
tributed to the circadian clock gating its own responsiveness to
environmental stimuli (Johnson, 1992) and suggests that the
show a weaker amplitude in prr7-3 than in the wild type. prr7-3 is
able to entrain to large amplitude (22 to 128C) cycles (see Figure
fail to entrain to 18 to 228C thermocycles administered with
T ¼ 20, 22, or 28 h (data not shown), demonstrating a partial
loss of circadian responsiveness to temperature.
cycles as seen after photocycles (Figures 1P and 1Q). prr9-1
mutants, like prr7-3 mutants, fail to entrain to 18 to 228C
thermocycles using T-cycles of 20, 22, and 28 h (data not
shown). Therefore, PRR7 and PRR9 functions are important for
proper entrainment of the cotyledon movement rhythm to both
photocycles and thermocycles.
PRR3 and PRR5 Are Required for Proper
To better define the roles, if any, of PRR3 and PRR5 in the clock,
we introduced translational fusions of clock gene promoters to
the noninvasive reporter gene LUCIFERASE (LUC) (Millar et al.,
1992) into prr mutants. Mutations of components of input path-
ways or in the clock itself are expected to cause a change in
the period and/or the phase of expression of the clock genes,
whereas mutation of a component of an output pathway from
the clock should not. CCA1:LUC, LHY:LUC, and TOC1:LUC
were introduced into theprr single mutants,and multiple (atleast
four) T2 lines for each combination of prr mutant and LUC fusion
were entrained to light–dark cycles or to 22 to 128C cycles for
10 d. Individual seedlings were transferred to 96-well plates and
released into continuous conditions, during which luciferase
activity was recorded. For each combination of transgene and
genotype, we did not observe any differences among lines for
period length or phase (data not shown). Although entrainment
by temperature cycles has been reported for LHCB and CAT3
transcription (Somers et al., 1998; Michael and McClung, 2002),
as well as for cotyledon movement (McClung et al., 2002), the
entrainment of the clock genes had not previously been directly
demonstrated. Figures 2A and 2B clearly show that the expres-
sion of CCA1, LHY, TOC1, and PRR7, as well as PRR9 (data not
shown), are entrained to the proper phase by thermocycles.
After entrainment to photocycles or thermocycles and release
into constant temperature and continuous white light, the prr3-1
andprr5-3mutantsdisplaya slightly(by;1h)short periodin the
Figure 1. (continued).
(A) to (C) Cotyledon movement for the short period mutant lhy-20.
(A) Double plot of the acrophase (peak) in cotyledon position for wild-type and mutant seedlings after thermocycles. Seedlings were grown for 7 d in
continuous light under a temperature entraining regime consisting of 12 h at 228C, followed by 12 h at 128C (HC, LL) and then released into continuous
conditions (HH, LL). ZT ¼ 0 corresponds to the cold-to-warm transition.
(B) Double plot of the wild type and lhy-20 during thermocycles. Seedlings were grown in photocycles for 5 d at 228C before being released into an in-
phase thermocycle regime (HH, LD into HC, LL).
(C) Mean (6SE, n ¼ 12) cotyledon movement traces for wild-type and lhy-20 seedlings during thermocycles as in (B). Black bars represent the cold part
of the cycle. Open squares, lhy-20; closed circles, wild-type Columbia (Col).
(D) and (E) Cotyledon movement for the long period mutant ztl-4. Double plot of cotyledon position after (D) or during (E) thermocycles as described in
(A) and (B).
(F) Mean (6SE, n ¼ 12) cotyledon movement traces of wild-type Col and ztl-4 mutant seedlings during thermocycles as described in (C). Open squares,
ztl-4; closed circles: wild-type Col.
(G) to (I) Cotyledon movement for the short period mutant prr3-1.
(G) and (H) Double plot of cotyledon position after (G) or during (H) thermocycles as described in (A) and (B).
(I) Mean (6SE,n ¼ 12)cotyledon movement traces ofwild-type Col and prr3-1 mutant seedlings during thermocycles as described in (C). Open squares,
prr3-1; closed circles, wild-type Col.
(J) to (L) Cotyledon movement for the short period mutant prr5-3.
(J) and (K) Double plot of the cotyledon position after (J) or during (K) thermocycles as described in (A) and (B).
(L) Mean (6SE, n ¼ 12) cotyledon movement traces for wild-type and prr5-3 seedlings during thermocycles as described in (C). Open squares, prr5-3;
closed circles, wild-type Col.
(M) to (O) Cotyledon movement for the long period mutant prr7-3.
(M) and (N) Double plot of the cotyledon position after (M) or during (N) thermocycles as described in (A) and (B).
(O) Mean (6SE, n ¼ 12) cotyledon movement traces for wild-type and prr7-3 seedlings during thermocycles as described in (C). Open squares, prr7-3;
closed circles, wild-type Col.
(P) to (R) Cotyledon movement for the lagging phase mutant prr9-1.
(P) and (Q) Double plot of the cotyledon position after (P) or during (Q) thermocycles as described in (A) and (B).
(R) Mean (6SE, n ¼ 12) cotyledon movement traces for wild-type and prr9-1 seedlings during thermocycles as described in (C). Open squares, prr9-1;
closed circles, wild-type Col.
794The Plant Cell
expression of CCA1, LHY, and TOC1 (Figures 2C to 2F, Table 1).
and the mutants seen on the first day upon release from
entrainment. Mutations in the clock components CCA1, LHY,
and TOC1 cause a much stronger effect on the expression of
clock-regulated genes, shortening their period by at least 3 h
(Alabadı ´ et al., 2002; Ma ´s et al., 2003b). These results show that
either PRR3 and PRR5 only contribute weakly to clock function
after photocycles and thermocycles or that their contribution is
redundantly specified, perhaps by TOC1, which also confers
a short period mutant phenotype. In particular, neither loss-of-
function seedling displayed the leading phase in cotyledon
movement during thermocycles that might have been expected
of mutants affecting a clock-associated gene or a clock com-
PRR7 and PRR9 Are Important for Clock Function
Transcription of the clock genes is affected in the prr7-3 and
prr9-1 mutants. After photocycles or thermocycles and release
into continuous conditions, prr7-3 seedlings display a long
period for all three clock genes (Figures 3A and 3B, Table 1).
Under the same conditions, prr9-1 mutants exhibit a long period
phenotype as well, consistent with Eriksson et al. (2003), but in
contrast with the lagging phase observed for cotyledon move-
ment(Figures 3C and3D;Michael etal.,2003b).Thegi-2 allele of
GIGANTEA displays distinct period phenotypes for cotyledon
movement and LHCB transcription (Park et al., 1999) and pro-
vides precedent for our results with prr9-1.
We conclude that loss of any member of the PRR family alters
the period ofCCA1,LHY, andTOC1 expression.Inparticular, we
note that loss-of-function alleles of TOC1 (Ma ´s et al., 2003b),
PRR3, and PRR5 (Figure 2) all shorten the period of the circadian
clock, although the most striking shortening is seen for TOC1
mutants. By contrast, loss-of-function alleles of PRR7 and PRR9
(Figure 3) lengthen the period of the clock. That loss of either
PRR7 or PRR9 does not result in arrhythmicity and that both
single loss-of-function mutants display a similar period length-
ening of the clock suggest that the two genes might be partially
Conditional Arrhythmicity of prr7-3 prr9-1 Double Mutants
We generated the prr7-3 prr9-1 double mutant for further
in cotyledon movement rhythms of >32 h (34.2 6 0.8 h, n ¼ 18),
much longer than seen in either single mutant (Figure 4A).
Therefore, PRR7 and PRR9 have partially overlapping functions
in the control of cotyledon movement in white light. The period of
cotyledon movement progressively lengthens as the number of
functional copies of PRR7 and PRR9 is reduced (Figure 4B). The
free-running period of prr7-3 heterozygotes is long (26.4 6 0.3 h,
n ¼ 8) and that of prr7-3 homozygotes is slightly longer (27.1 6
0.2 h, n ¼ 13). In seedlings homozygous for the prr7-3 allele and
heterozygous for the prr9-1 allele, the period of cotyledon
movement is ;28 h (28.1 6 0.2 h, n ¼ 12). The most dramatic
lengthening in period occurs upon loss of the final functional
allele (cf. prr7-3/prr7-3 prr9-1/PRR9 with prr7-3/prr7-3 prr9-1/
prr9-1; Figure 4B), supporting functional overlap between
PRR7 and PRR9 in the control of period length for cotyledon
Figure 2. PRR3 and PRR5 Only Weakly Contribute to Proper Clock
(A) and (B) Wild-type Col seedlings were grown for 10 d in a temperature
entraining regime consisting of 12 h at 228C, followed by 12 h at 128C, in
continuous light. At the end of the 10th day, seedlings were released into
continuous light and temperature of 228C (HC, LL into HH, LL). Mean
(6SE, n ¼ 24) traces are shown for CCA1:LUC and TOC1:LUC (A) and
LHY:LUC and PRR7:LUC (B). Hatched bars represent subjective cold
(C) Mean traces 6SE for TOC1:LUC activity in Col (n¼ 24) and prr3-1 (n ¼
12) in continuous light after photocycles (HH, LD into HH, LL). Hatched
bars represent subjective night.
(D) Mean period lengths in Col and prr3-1 seedlings for the LHY and
TOC1 reporter constructs. See Table 1 for mean values.
(E) Mean traces 6SE for TOC1:LUC activity in Col (n ¼ 24) and prr5-3 (n ¼
12) in continuous light after photocycles as in (C). Hatched bars
represent subjective night.
(F) Mean period lengths in Col and prr5-3 seedlings for the LHY and
TOC1 reporter constructs. See Table 1 for mean values.
Temperature-Insensitive Circadian Mutant795
movement. In addition, this demonstrates that one copy of
PRR9 is sufficient to compensate for more than half of the
period lengthening caused by loss of both PRR7 and PRR9.
Such dosage-dependent variation in period length is expected
for bona fide clock components; indeed, most circadian clock
mutants in animals are semidominant (Ralph and Menaker,
1988; Williams and Sehgal, 2001).
toc1-2 is rhythmic during and after thermocycles (data not
shown), suggesting that other gene(s) may compensate for the
loss of TOC1 under these conditions or that TOC1 is not
primarily responsible for responsiveness to temperature signal-
ing. We therefore tested the ability of prr7-3 prr9-1 seedlings
entrained to photocycles to remain entrained to subsequent
thermocycles. Wild-type and prr7-3 prr9-1 mutant seedlings
are rhythmic immediately after the photocycles. Upon transfer
to thermocycles, prr7-3 prr9-1 seedlings fail to remain en-
trained to a 24-h period and instead display a long period,
indicating an inability to entrain (Figure 4C). In addition, the
strength of the rhythm dampens after a few days and seedlings
become arrhythmic by the end of the experiment. In some
experiments the double mutant becomes arrhythmic immedi-
ately after release into thermocycles, as when the prr7-3 prr9-1
double mutant has been entrained only to thermocycles (Figure
4D). These results demonstrate that the prr7-3 prr9-1 mutant is
compromised in its ability to entrain to thermocycles. When
entrained exclusively to thermocycles, the prr7-3 prr9-1 double
mutant does not display rhythm either during or after entrain-
ment (Figure 4D). We conclude that PRR7 and PRR9 function in
the emergence of a rhythm in cotyledon movement in response
The prr7-3 prr9-1 Double Mutant Is Altered in Clock
The strong circadian defect seen in prr7-3 prr9-1 plants could be
limited to cotyledon movement or could have a more pervasive
effect on the circadian clock itself. Clock gene promoter:LUC
fusions were therefore used to directly measure the effect of loss
of both PRR7 and PRR9 on the clock. When released in
continuous light after photocycles, a strong defect in the ex-
pression of clock genes is evident (Figure 5A). The period of the
rhythm in white light in the prr7-3 prr9-1 double mutant is 8 h
longer than in the wild type (Figure 5A), which makes this mutant
an extreme long period mutant in Arabidopsis. The timing of the
first peak of CCA1 and LHY is not affected in the double mutant;
the timing of TOC1, however, is much delayed compared with
the wild type, peaking close to dawn of the second day. A
conversion of the phase value to circadian time (CT ¼ sidereal
phase 3 24/period) indicates that the lagging phase is a conse-
16.5 6 0.8 h, n ¼ 12; CTCOL¼ 16.5 6 0.7 h, n ¼ 12). A similar
effect was observed in the cca1 lhy double mutant early into
continuous light, where the acrophase of TOC1 expression was
shifted earlier in the day, while the acrophase of CCA1 and LHY
remained close to their normal time (Mizoguchi et al., 2002).
constant red or blue light (Figure 5B). Interestingly, the length-
ening in red or blue light was weaker than in continuous white
light (Figure 5B), suggesting that the white light phenotype
represents the additive effects of red and blue light defects.
Table 1. Circadian Parameters of prr Single and Double Mutants Described in This Study
Genotype Rhythm Period (6SE)n P Value
24.2 6 0.1
24.5 6 0.2
24.5 6 0.1
23.9 6 0.1
24.1 6 0.2
24.4 6 0.1
23.1 6 0.1
23.3 6 0.3
23.9 6 0.2
25.0 6 0.1
25.8 6 0.2
26.0 6 0.2
25.3 6 0.1
26.2 6 0.4
25.7 6 0.2
32.7 6 1.1
32.1 6 0.7
32.5 6 0.4
prr7-3 prr9-1 10
Luciferase activity after entrainment to photocycles was determined in seedlings grown under white light (15 to 25 mmol/m2/s1).
aP value from Student’s two-tailed homoscedastic t test comparing the mutant value with Columbia.
bP value from Student’s two-tailed heteroscedastic t test comparing the mutant value with Columbia.
796The Plant Cell
These results indicate that the prr7-3 prr9-1 double mutant is
impaired in both red and blue light signaling to the clock but may
also suggest that the presence of light is actively lengthening
period in the prr7-3 prr9-1 double mutant, which would be in
contrast with the normal period-shortening effect of light.
Defects in the expression of the clock genes are also apparent
in the absence of light. Oscillations of the transcription of a clock
gene in the dark have only been reported for CCA1 (Eriksson
et al., 2003), although oscillations in CCA1, LHY, and PRR1/
TOC1 mRNAs in the dark have been described (Nakamichi et al.,
2003). We therefore characterized the expression pattern of our
LUC fusions after release into darkness. As seen in Figures 5C
in the dark, with a period close to 26 h. Other genes maintaining
oscillations in the dark are CCR2 (Strayer et al., 2000), CAT3
(Michael and McClung, 2002), and PRR7 (Figure 5D). Although
wild-type plants show rhythmic expression from the CCA1 and
LHY promoters, the prr7-3 prr9-1 double mutant plants do not.
Instead, theyshow onepeak ofLUC activityforCCA1(Figure 5E)
and LHY (Figure 5F) on the first day in the dark, with a slightly
lagging phase relative to the wild type. LUC activity in the prr7-3
prr9-1 mutant then decreases and remains nonoscillating and
low. PRR7 and PRR9 are therefore crucial for clock function in
Welast wished to assessthe ability of the prr7-3 prr9-1 double
mutant to entrain to temperature cycles. We entrained wild-type
and prr7-3 prr9-1 plants to thermocycles (12 h at 228C followed
by12hat128C)for10dbefore transfertoconstant conditions.In
three of five trials, TOC1 expression in the double mutant
exhibited a peak during the first day in constant conditions that
prr7-3 prr9-1 seedlings then dampened rapidly. In the other two
(Figure 5H). These results suggest that thermocycles are not
effective in generating an oscillation in prr7-3 prr9-1 seedlings.
This is consistent with our results with cotyledon movement
during temperature entrainment (Figure 4).
The prr7-3 prr9-1 Double Mutant Does Not
Respond to Temperature
To directly test the response of the prr7-3 prr9-1 mutant to
temperature entrainment, we first entrained wild-type and mu-
tant plants to light–dark cycles, which are able to entrain the
prr7-3 prr9-1 mutant (Figure 5A). After 10 d, the seedlings were
transferred to 96-well plates and transferred to an in-phase
CCA1:LUC, LHY:LUC, and TOC1:LUC transgenes was recorded
from the beginning of the temperature entrainment (Figure 6).
Oscillations are evident for all three genes, but the observed
phases are different from wild-type seedlings. The morning
genes, CCA1 and LHY, peak later in prr7-3 prr9-1 double mutant
plants than in the wild type (Figures 6A and 6B). The evening
gene, TOC1, shows a peak accumulation at dawn, 12 h out of
phase with its wild-type expression peak (Figure 6C). The re-
suggests that it is driven in response to the changes in environ-
mental conditions rather than entrained. Consistent with this, in
the prr7-3 prr9-1 double mutant, TOC1 expression also shows
no anticipation of the cold-to-warm or warm-to-cold transitions,
in contrast with the wild type. The expression of CCA1 and LHY
displays some anticipation of the temperature transitions. How-
ever, unlike in the wild type, in prr7-3 prr9-1 their expression
levels remain elevated during the warm part of the entraining
cyclesand reachmuchlower levels duringthecoldportion of the
One consequence of the dawn-specific expression of TOC1 in
the prr7-3 prr9-1 double mutant is that the phase angle between
CCA1, LHY, and TOC1 is greatly altered. In wild-type seedlings,
the peak of CCA1 and TOC1 is temporally separated by 12 h
of TOC1 and CCA1 are brought much more closely together,
CCA1 peaking only 4 to 6 h after TOC1 (Figures 6F and 6G). In
addition, we noticed that CCA1 transcription now starts rising
PRR7 and PRR9, the positive action of TOC1 on CCA1 expres-
sion is manifested much earlier than in the wild type.
To further define the extent of temperature responses in the
double mutant, we performed PRC experiments to tempera-
ture pulses. Seedlings carrying the TOC1:LUC reporter were
Figure 3. PRR7 and PRR9 Are Important for Proper Period Length of the
(A) Mean (6SE) traces for TOC1:LUC activity in Col (n ¼ 24) and prr7-3 (n
¼ 12) in continuous light after photocycles (HH, LD into HH, LL). Hatched
bars represent subjective night.
(B) Mean period lengths in Col and prr7-3 seedlings for LHY:LUC and
TOC1:LUC. See Table 1 for mean values.
(C) Mean (6SE) traces for TOC1:LUC activity in Col (n ¼ 24) and prr9-1 (n
¼ 12) in continuous light after photocycles as in (A). Hatched bars
represent subjective night.
(D) Mean period lengths in Col and prr9-1 seedlings for LHY:LUC and
TOC1:LUC. See Table 1 for mean values.
Temperature-Insensitive Circadian Mutant 797
entrained to light–dark cycles for 10 d in a constant temperature
of 228C. At dawn of the 11th day, seedlings were transferred
into continuous light and temperature. Groups of seedlings were
then placed at 128C for 4 h before being returned to 228C. The
new phase was normalizedtothe free-runningperiod ofwild-type
and prr7-3 prr9-1 seedlings and plotted in Figure 6H. In the wild
type, temperature pulses induce phase delays in TOC1 expres-
day. By sharp contrast, similar temperature pulses given to
prr7-3 prr9-1 seedlings cause strong delays immediately after
release into continuous conditions, but the responsiveness
rapidly diminishes and the overall shape of the PRC is quite dis-
similar to that of the wild type. Our results demonstrate that PRR7
and PRR9 are important for proper clock function in the light.
However, the roles of PRR7 and PRR9 are not limited to light
because mutant plants become arrhythmic in the dark and after
entrainment to temperature. prr7-3 prr9-1 fails to reset to a
temperature entraining stimulus and represents a higher plant
mutant that is insensitive to temperature signals.
The rotating environment of the earth provides two sets of
stimuli, photocycles and thermocycles, for organisms to syn-
chronize their internal clock with their surroundings. Light en-
trainment is the best characterized in any organism (van der
Horst et al., 1999; Krishnan et al., 2001; Froehlich et al., 2002,
2003; Panda et al., 2002b; Ruby et al., 2002). Temperature input
to the clock has been much more elusive in most systems. In
Drosophila, Pittendrigh postulated ;50 years ago the existence
sensitive totemperature (Pittendrigh etal.,1958).Recent studies
on the Drosophila mutants per, tim, dclk, and cyc during
temperature entrainment have supported the existence of a sec-
ond temperature-sensitive mechanism with some character-
istics of a circadian oscillator (Yoshii et al., 2002). However, the
not known. Temperature cycles are effective entraining stimuli
in Neurospora, although the details of the mechanism of
Figure 4. Conditional Arrhythmicity of Cotyledon Movement in the prr7-3 prr9-1 Double Mutant.
(A) Mean (6SE, n ¼ 18) cotyledon movement traces of the prr7-3 prr9-1 double mutant in white light after entrainment to photocycles (HH, LD into HH,
LL). Hatched bars represent subjective night. Open squares, prr7-3 prr9-1; closed circles, wild-type Col.
(B) Mean (6SE, n ¼ 8 to 18) period length of plants carrying decreasing number of functional copies of PRR7 and PRR9.
(C) Mean (6SE, n ¼ 12) cotyledon movement traces of the prr7-3 prr9-1 double mutant during thermocycles (22 to 188C) after entrainment to
photocycles (HH, LD into HC, LL). Hatched bars represent subjective night. Open squares, prr7-3 prr9-1; closed circles, wild-type Col.
(D) Mean (6SE, n ¼ 12) cotyledon movement traces of the prr7-3 prr9-1 double mutant in constant conditions after entrainment to thermocycles (HC, LL
into HH, LL). Hatched bars represent subjective cool night. Open squares, prr7-3 prr9-1; closed circles, wild-type Col.
798The Plant Cell
temperature entrainment are incompletely defined. Many of the
known clock-regulated genes can be properly entrained by
thermocycles, but mutations in the clock component FRE-
QUENCY (FRQ) abolish temperature entrainment as measured
by gene expression (Nowrousian et al., 2003). The conidiation
rhythm also is entrainable by thermocycles. Temperature en-
trainment has been suggested to persist in the absence of FRQ
(Merrow et al., 1999), but, more recently, it has been argued that
FRQ is essential for proper temperature entrainment of the
conidiation rhythm (Pregueiro et al., 2005).
In plants, temperature entrainment has been extensively de-
scribed (Rensing and Ruoff, 2002). Studies on the CO2assim-
ilation rhythm of Kalanchoe ¨ plants demonstrated that very small
temperature steps, as little as 0.58C, could entrain the rhythm.
We and others have shown that cotyledon movement (McClung
et al., 2002) as well as the transcription rate of the output genes
LHCB (Somers et al., 1998) and CAT3 (Michael and McClung,
2002) could be entrained by thermocycles. Their thermophases
(or phase taken after entrainment to thermocycles) are identical
to their photophases (or phase taken after entrainment to photo-
cycles). But despite this breadth of knowledge, the effect of
temperature entrainment directly on clock components had
not been established. We show that, in addition to the number
of output rhythms that are set by thermocycles, the clock
genes CCA1, LHY, TOC1, PRR7, and PRR9 can be set to their
correct phase by thermocycles (Figures 4A and 4B; data not
to sense and respond to temperature changes (Eriksson et al.,
2002), but none can readily explain the exquisite sensitivity
to temperature changes exhibited by circadian systems. In S.
elongatus, a mutant defective in cikA, which encodes a kinase
and temperature signals (Schmitz et al., 2000). In addition, the
cikA mutant displays altered period, phase, or amplitude in the
circadian expression of many genes. Because oscillations per-
sist in the cikA mutant, it is not thought to play a role within the
clock per se but is critical for relaying environmental signals like
light and temperature to the clock. In this study, we provide
evidence that the two PRRs, PRR7 and PRR9, are essential for
temperature entrainment of the Arabidopsis clock. Uniquely
among Arabidopsis mutants, the prr7-3 prr9-1 double mutant
does not entrain to thermocycles.
Figure 5. PRR7 and PRR9 Are Critical for Rhythmicity in Complete
Darkness and after Thermocycles.
Plants were grown under the indicated conditions for 10 d before being
released into the appropriate free-running conditions.
(A) Mean (6SE, n ¼ 12 to 24) luciferase activity from TOC1:LUC in
continuous white light after entrainment to photocycles (HH, LD into HH,
LL). Open squares, prr7-3 prr9-1; closed circles, wild-type Col.
(B) Mean (6SE, n ¼ 12 to 24) period lengths in Col, prr7-3 and prr9-1
single mutants, and prr7-3 prr9-1 double mutants for TOC1:LUC activity.
Rhythmicity was assayed in continuous red (20 mmol m?2s?1), blue (10
mmol m?2s?1), or white (25 mmol m?2s?1) light after photocycles (12 h in
50 mmol m?2s?1white light, 12 h dark).
(C) Mean (6SE, n ¼ 12) luciferase activity from CCA1:LUC (open squares)
and TOC1:LUC (closed circles) in wild-type Col seedlings entrained by
photocycles for 10 d and released into continuous dark (HH, LD into HH,
(D) Mean (6SE, n ¼ 12) luciferase activity from LHY:LUC (open squares)
and PRR7:LUC (closed circles) in wild-type Col seedlings entrained by
photocycles for 10 d and released into continuous dark as in (C).
(E) and (F) Mean (6SE, n ¼ 3-4) CCA1:LUC (E) and LHY:LUC (F)
luciferase activity in Col (closed circles) and prr7-3 prr9-1 (open squares)
seedlings that were grown in photocycles for 10 d, then released into
continuous dark as in (C).
(G) and (H) Mean (6SE, n ¼ 12-24) TOC1:LUC luciferase activity in Col
(closed circles) and prr7-3 prr9-1 (open squares) seedlings that were
grown in thermocycles for 10 d, then released into continuous light and
constant temperature (228C) (HC, LL into HH, LL). In 3/5 experiments (G),
prr7-3 prr9-1 seedlings showed one first peak in TOC1:LUC activity,
antiphase with the wild type, before damping to a very low amplitude. In
the other (2/5) experiments (H), prr7-3 prr9-1 seedlings displayed no
oscillations in TOC1:LUC.
Temperature-Insensitive Circadian Mutant799
Figure 6. The prr7-3 prr9-1 Double Mutant Fails to Entrain to Temperature Cycles.
(A) to (C) Expression of the clock genes CCA1, LHY, and TOC1 during thermocycles. Seedlings were grown for 10 d under photocycles at 228C. At the
beginning of the 11th day, the plates were released into continuous light with an in-phase temperature entraining regime, where light is replaced by 12 h
at 228C, and dark is replaced by 12 h at 128C (HH, LD into HC, LL). Mean (6SE, n ¼ 36) luciferase activity for CCA1:LUC (A), LHY:LUC (B), and
TOC1:LUC (C) recorded for 4 d during entrainment for Col (closed circles) and prr7-3 prr9-1 double mutant (open squares) seedlings. Hatched bars
represent the cold night.
(D) and (E) Expression of CCA1 and TOC1 in wild-type Col seedlings during entrainment to thermocycles (HH, LD into HC, LL). In (D), the traces shown
in (A) and (C) for CCA1:LUC and TOC1:LUC are replotted for direct comparison of the respective phases of the two genes. Hatched bars represent
subjective night. In (E), the phase angles of the acrophases, normalized to a 24-h cycle, in CCA1:LUC (open squares) and TOC1:LUC (closed circles) in
wild-type Col seedlings during entrainment to thermocycles are plotted to illustrate the phase angle relationship in wild-type Col seedlings.
(F) and (G) Expression of CCA1 and TOC1 in prr7-3 prr9-1 double mutant seedlings during entrainment to thermocycles (HH, LD into HC, LL). In (F), the
traces shown in (A) and (C) for CCA1:LUC and TOC1:LUC are replotted for direct comparison of the respective phases of the two genes. Hatched bars
represent subjective night. In (G), the phase angles of the acrophases, normalized to a 24-h cycle, in CCA1:LUC (open squares) and TOC1:LUC (closed
circles) during entrainment to thermocycles are plotted to illustrate the phase angle relationship in prr7-3 prr9-1 seedlings.
(H) PRC of TOC1:LUC expression in response to cold temperature pulses. Seedlings entrained to photocycles and released into constant conditions
were subjected to 4-h cold pulses at intervals across the circadian cycle. TOC1:LUC activity was recorded for 7 d after the last pulse, and the extent of
phase shifts were determined as described (Michael et al., 2003a). Closed circles, the wild type; open squares, prr7-3 prr9-1 mutant.
800The Plant Cell
The role of PRR7 and PRR9 is not limited to temperature
entrainment because profound effects are seen in the prr7-3
period lengthening was more pronounced than has been seen in
any other long period mutant identified to date. Therefore, PRR7
and PRR9 are important for both red and blue light signaling to
the absence of light signaling, as demonstrated by the rapid loss
of rhythmicity in CCA1 and LHY expression in the dark.
We cannot conclude that PRR7 and PRR9 are required for
a temperature signaling cascade that provides input to the clock
because impaired clock function could also explain the loss
of temperature sensitivity in the prr7-3 prr9-1 double mutant.
Generally, thermocycles are weaker synchronizers than photo-
cycles (Rensing and Ruoff, 2002; Ashmore and Sehgal, 2003;
Liu, 2003). A strong circadian phenotype is evident in the prr7-3
prr9-1 double mutant under all conditions tested, which argues
against a place for PRR7 and PRR9 solely in a temperature
respond to resetting signals. Thus, the prr7-3 prr9-1 double
mutant might appear insensitive to temperature not because
temperature signaling is attenuated but rather because the
mutant clock in unable to fully respond to thermocycles, which
are relatively weak clock synchronizers.
for the integration of light and temperature signaling. Based on
their temporal expression pattern, one might expect PRR7 and
PRR9 to lie between CCA1/LHY and TOC1 on an oscillator loop.
This would posit that the PRR7/PRR9 pair plays an intermediary
role in the regulation of TOC1 expression by CCA1 and LHY.
However, the binding of CCA1 and LHY directly to the TOC1
promoter (Alabadı ´ et al., 2001) argues for a direct role of CCA1
and LHY in TOC1 regulation. In addition, mutations in CCA1,
LHY, or TOC1 alone do not cause the drastic phenotypes seen in
prr7-3 prr9-1 during and after thermocycles. Therefore, PRR7
and PRR9 cannot solely function between CCA1/LHY and TOC1
in a simple linear pathway. Nonetheless, there is clearly interac-
tion between PRR7/PRR9 and CCA1/LHY and TOC1. Mutations
in PRR7 and PRR9 strongly affect the expression pattern of
the clock genes CCA1, LHY, and TOC1. In addition, overexpres-
sion of TOC1 represses transcription of PRR9 (Mizuno, 2004),
indicating that TOC1 acts, either directly or indirectly, as a re-
pressor of PRR9.
Constitutive expression of a clock component can lead to
1998; Wang and Tobin, 1998; Ma ´s et al., 2003b), FRQ in
Neurospora (Aronson et al., 1994), and PER and TIM in Dro-
sophila (Zeng et al., 1994; Suri et al., 1999). That overexpression
of TOC1 results in arrhythmicity (Ma ´s et al., 2003b) is consistent
with its ascribed role in the oscillator. By contrast, overexpres-
sion of PRR3, PRR5, or PRR9 does not lead to arrhythmicity
(Mizuno, 2004). However, overexpression of the mouse CLOCK
gene does not lead to arrhythmicity but rather shortens the
period of the clock. Similarly, high constitutive levels of PRR9
shorten the period of the Arabidopsis clock (Ito et al., 2003).
required to induce arrhythmicity in plants, although potential
posttranslational modification required to activate either protein
might hinder this overexpression strategy.
Wepropose that PRR7 and PRR9 represent anentry point into
the clock for the temperature entrainment signaling cascade.
Analogously, TOC1 was proposed as an integrator of light
signaling because rhythmicity is compromised in the strong
toc1-2 mutant in red light (Ma ´s et al., 2003b). PRR7 and PRR9
could function in a temperature input pathway or they could
represent elements of the oscillator that, when lost, impair the
clock’s ability to respond to temperature signals. We prefer the
latter interpretation because the prr7-3 prr9-1 double mutant
signaling is not active. It remains possible, however, that PRR7
(e.g., light) input pathways and are not themselves elements of
a central oscillator. Nonetheless, the establishment of PRR7 and
PRR9 as essential for temperature entrainment represents an
important step in our understanding of temperature sensing and
signaling in plants.
Plant Growth and Genotypes
The T-DNA insertion alleles prr3-1 (Salk 090261), prr5-3 (Salk 064538),
prr7-3 (Salk 03430), prr9-1 (Salk 007551), lhy-20 (Salk 031092), and ztl-4
(Salk 012440) were described previously (Michael et al., 2003b). Early
characterization of leaf movement in response to temperature was
performed using the isogenic wild-type siblings from each allele (Michael
et al., 2003b) and the wild-type Columbia-2 (Col-2) (CS933). Because
Col-2 and the isogenic siblings behaved identically, subsequent analysis
has used Col-2 as the wild type. The prr7-3 prr9-1 double mutant
was constructed by standard genetic crossing and confirmed by PCR
Generation of Constructs and Transgenic Arabidopsis Plants
A new luciferase binary vector was derived from pZPVLUCþ (Schultz
et al., 2001) by replacement of the gentamicin resistance cassette with
the BASTA resistance gene from 35SpBarn (LeClerc and Bartel, 2001).
The resulting vector, pZPBAR, was then made Gateway compatible
(Invitrogen, Carlsbad, CA) by inserting the PCR-amplified attR-flanked
destination cassette from pK7WG2D (Karimi et al., 2002) at the BamHI
and HindIII sites upstream of LUC to create pZPBAR-DONR.
colony-PCR from the BACs and P1 clones F9D11, T25K16, and MBF13,
respectively, and cloned into the Gateway entry vector pENTR11. The
recombination reaction was performed using 1 mL of LR clonase mix and
2 mL of 53 buffer in a 10-mL reaction; the reaction was then transformed
into Escherichia coli DH5a and selected on 100 mg/mL of spectinomy-
cin. Clones were confirmed by colony-PCR using primers specific for the
recombined attB sites and transformed into plants via Agrobacterium
tumefaciens transformation (strain ASE) by vacuum infiltration (Bechtold
et al., 1993). Plants homozygous for prr3-1, prr5-3, prr7-3, and prr9-1
T-DNA insertions (Michael et al., 2003b) and the wild-type Col were
transformed by vacuum infiltration via Agrobacterium (Bechtold et al.,
1993). T1 seeds were collected and selected on 12.5 mg/mL of BASTA.
Resistant seedlings were allowed to self, and T2 seeds were collected.
Several lines for each reporter and genetic background were analyzed.
Temperature-Insensitive Circadian Mutant 801
Leaf Movement Assays, Luciferase Measurement,
and Data Analysis
Leaf movement was measured as described previously (Millar et al.,
1995; Salome ´ et al., 2002). All manipulations, LUC activity measurements
of seedlings entrained to photocycles and thermocycles, and data
analysis were performed as described (Michael and McClung, 2002).
For LUC measurement in the dark, adult prr7-3 prr9-1 plants were
sprayed with 2.5 mM luciferin and 0.05% Triton X-100 the day before the
start of imaging. Plants were transferred back into the long-day cycle
for 1 d. LUC activity in the wild type was measured on 10- to 14-d-old
seedlings transferred to 96-well plates. At zeitgeber time 12, plants were
transferred into a light-tight chamber, and LUC activity was recorded
every 2 h with a Hamamatsu digital CCD camera (C4742-98 ERG;
Hamamatsu Photonics, Hamamatsu City, Japan) using MetaMorph
software. All circadian data were analyzed by fast Fourier transform
nonlinear least squares (Plautz et al., 1997) and by Chrono (Roenneberg
and Taylor, 2000).
We thank Monika Swiatecka for help with luciferase imaging and Jay
Dunlap for helpful discussions. This work was supported by grants from
the National Science Foundation (MCB-0091008 and MCB-0343887).
Received November 20, 2004; accepted December 23, 2004.
Alabadı ´, D., Oyama, T., Yanovsky, M.J., Harmon, F.G., Ma ´s, P., and
Kay, S.A. (2001). Reciprocal regulation between TOC1 and LHY/
CCA1 within the Arabidopsis circadian clock. Science 293, 880–883.
Alabadı ´, D., Yanovsky, M.J., Ma ´s, P., Harmer, S.L., and Kay, S.A.
(2002). Critical role for CCA1 and LHY in maintaining circadian
rhythmicity in Arabidopsis. Curr. Biol. 12, 757–761.
Albrecht, U., and Eichele, G. (2003). The mammalian circadian clock.
Curr. Opin. Genet. Dev. 13, 271–277.
Aronson, B.D., Johnson, K.A., Loros, J.J., and Dunlap, J.C. (1994).
Negative feedback defining a circadian clock: Autoregulation of the
clock gene frequency. Science 263, 1578–1584.
Ashmore, L.J., and Sehgal, A. (2003). A fly’s eye view of circadian
entrainment. J. Biol. Rhythms 18, 206–216.
Bechtold, N., Ellis, J., and Pelletier, G. (1993). In planta Agrobacterium
mediated gene transfer by infiltration of adult Arabidopsis thaliana
plants. C.R. Acad. Sci. Paris, Life Sciences 316, 1194–1199.
Bell-Pedersen, D. (2002). Circadian rhythms in Neurospora crassa.
Mycol. Ser. 15, 187–214.
DeCoursey, P.J. (1990). Circadian photoentrainment in nocturnal mam-
mals: Ecological overtones. Biol. Behav. 15, 213–238.
Eriksson, M.E., Hanano, S., Southern, M.M., Hall, A., and Millar, A.J.
(2003). Response regulator homologues have complementary, light-
dependent functions in the Arabidopsis circadian clock. Planta 218,
Eriksson, S., Hurme, R., and Rhen, M. (2002). Low-temperature
sensors in bacteria. Philos. Trans. R. Soc. Lond. B Biol. Sci. 357,
Froehlich, A.C., Liu, Y., Loros, J.J., and Dunlap, J.C. (2002). White
Collar-1, a circadian blue light photoreceptor, binding to the fre-
quency promoter. Science 297, 815–819.
Froehlich, A.C., Loros, J.J., and Dunlap, J.C. (2003). Rhythmic binding
of a WHITE COLLAR-containing complex to the frequency promoter is
inhibited by FREQUENCY. Proc. Natl. Acad. Sci. USA 100, 5914–
Golden, S.S., and Canales, S.R. (2004). Cyanobacterial circadian
clocks—Timing is everything. Nat. Rev. Microbiol. 1, 181–190.
Han, L., Mason, M., Risseeuw, E.P., Crosby, W.L., and Somers, D.E.
(2004). Formation of an SCFZTLcomplex is required for proper
regulation of circadian timing. Plant J. 40, 291–301.
Hwang, I., Chen, H.-C., and Sheen, J. (2002). Two-component signal
transduction pathways in Arabidopsis. Plant Physiol. 129, 500–515.
Ito, S., Matsushika, A., Yamada, H., Sato, S., Kato, T., Tabata, S.,
Yamashino, T., and Mizuno, T. (2003). Characterization of the
APRR9 Pseudo-Response Regulator belonging to the APRR1/TOC1
quintet in Arabidopsis thaliana. Plant Cell Physiol. 44, 1237–1245.
Johnson, C.H. (1992). Phase response curves: What can they tell us
about circadian clocks? In Circadian Clocks from Cell to Human, T.
Hiroshige and K. Honma, eds (Sapporo, Japan: Hokkaido University
Press), pp. 209–249.
Kaczorowski, K.A., and Quail, P.H. (2003). Arabidopsis PSEUDO-
RESPONSE REGULATOR 7 is a signaling intermediate in phyto-
chrome-regulated seedling deetiolation and phasing of the circadian
clock. Plant Cell 15, 2654–2665.
Karimi, M., Inze ´, D., and Depicker, A. (2002). GATEWAY vectors for
Agrobacterium-mediated plant transformation. Trends Plant Sci. 7,
Krishnan, B., Levine, J.D., Lynch, M.K.S., Dowse, H.B., Funes,
P., Hall, J.C., Hardin, P.E., and Dryer, S.E. (2001). A new role
for cryptochrome in a Drosophila circadian oscillator. Nature 411,
LeClerc, S., and Bartel, B. (2001). A library of Arabidopsis 35S-cDNA
lines for identifying novel mutants. Plant Mol. Biol. 46, 695–703.
Liu, Y. (2003). Molecular mechanisms of entrainment in the Neurospora
circadian clock. J. Biol. Rhythms 18, 195–205.
Loros, J.J., and Dunlap, J.C. (2001). Genetic and molecular analy-
sis of circadian rhythms in Neurospora. Annu. Rev. Physiol. 63,
Ma ´s, P., Alabadı ´, D., Yanovsky, M.J., Oyama, T., and Kay, S.A.
(2003b). Dual role of TOC1 in the control of circadian and photomor-
phogenic responses in Arabidopsis. Plant Cell 15, 223–236.
Ma ´s, P., Kim, W.-Y., Somers, D.E., and Kay, S.A. (2003a). Targeted
degradation of TOC1 by ZTL modulates circadian function in Arabi-
dopsis thaliana. Nature 426, 567–570.
Matsushika, A., Makino, S., Kojima, M., and Mizuno, T. (2000).
Circadian waves of expression of the APRR1/TOC1 family of pseudo-
response regulators in Arabidopsis thaliana: Insight into the plant
circadian clock. Plant Cell Physiol. 41, 1002–1012.
McClung, C.R., Salome ´, P.A., and Michael, T.P. (2002). The Arabi-
dopsis circadian system. In The Arabidopsis Book, C.R. Somerville
and E.M. Meyerowitz, eds (Rockville, MD: American Society of Plant
Biologists), doi/10.1199/tab.0044, http://www.aspb.org/publications/
Merrow, M., Brunner, M., and Roenneberg, T. (1999). Assignment of
circadian function for the Neurospora clock gene frequency. Nature
Michael, T.P., and McClung, C.R. (2002). Phase-specific circadian
clock regulatory elements in Arabidopsis thaliana. Plant Physiol. 130,
Michael, T.P., Salome ´, P.A., and McClung, C.R. (2003a). Two Arabi-
dopsis circadian oscillators can be distinguished by differential
temperature sensitivity. Proc. Natl. Acad. Sci. USA 100, 6878–6883.
Michael, T.P., Salome ´, P.A., Yu, H.J., Spencer, T.R., Sharp, E.L.,
Alonso, J.M., Ecker, J.R., and McClung, C.R. (2003b). Enhanced
fitness conferred by naturally occurring variation in the circadian
clock. Science 302, 1049–1053.
802 The Plant Cell
Millar, A.J., Carre ´, I.A., Strayer, C.A., Chua, N.-H., and Kay, S.A.
(1995). Circadian clock mutants in Arabidopsis identified by luciferase
imaging. Science 267, 1161–1163.
Millar, A.J., Short, S.R., Hiratsuka, K., Chua, N.-H., and Kay, S.A.
(1992). Firefly luciferase as a reporter of regulated gene expression in
higher plants. Plant Mol. Biol. Rep. 10, 324–337.
Mizoguchi, T., Wheatley, K., Hanzawa, Y., Wright, L., Mizoguchi, M.,
Song, H.-R., Carre ´, I.A., and Coupland, G. (2002). LHY and CCA1
are partially redundant genes required to maintain circadian rhythms
in Arabidopsis. Dev. Cell 2, 629–641.
Mizuno, T. (2004). Plant response regulators implicated in signal trans-
duction and circadian rhythm. Curr. Opin. Plant Biol. 7, 499–505.
Nakamichi, N., Matsushika, A., Yamashino, T., and Mizuno, T.
(2003). Cell autonomous circadian waves of the APRR1/TOC1 quintet
in an established cell line of Arabidopsis thaliana. Plant Cell Physiol.
Nowrousian, M., Duffield, G.E., Loros, J.J., and Dunlap, J.C. (2003).
The frequency gene is required for temperature-dependent regulation
of many clock-controlled genes in Neurospora crassa. Genetics 164,
Panda, S., Hogenesch, J.B., and Kay, S.A. (2002a). Circadian rhythms
from flies to human. Nature 417, 329–335.
Panda, S., Sato, T.K., Castrucci, A.M., Rollag, M.D., DeGrip, W.J.,
Hogenesch, J.B., Provencio, I., and Kay, S.A. (2002b). Melanopsin
(Opn4) requirement for normal light-induced circadian phase shifting.
Science 298, 2213–2215.
Park, D.H., Somers, D.E., Kim, Y.S., Choy, Y.H., Lim, H.K., Soh, M.S.,
Kim, H.J., Kay, S.A., and Nam, H.G. (1999). Control of circadian
rhythms and photoperiodic flowering by the Arabidopsis GIGANTEA
gene. Science 285, 1579–1582.
Pittendrigh, C., Bruce, V., and Kaus, P. (1958). On the significance of
transients in daily rhythms. Proc. Natl. Acad. Sci. USA 44, 965–973.
Plautz, J.D., Straume, M., Stanewsky, R., Jamison, C.F., Brandes,
C., Dowse, H.B., Hall, J.C., and Kay, S.A. (1997). Quantitative
analysis of Drosophila period gene transcription in living animals.
J. Biol. Rhythms 12, 204–217.
Pregueiro, A.M., Price-Lloyd, N., Bell-Pedersen, D., Heintzen, C.,
Loros, J.J., and Dunlap, J.C. (2005). An essential role for the
Neurospora frequency gene in circadian entrainment to temperature
cycles. Proc. Natl. Acad. Sci. USA 102, 2210–2215.
Ralph, M.R., and Menaker, M. (1988). A mutation of the circadian
system in golden hamsters. Science 241, 1225–1227.
Rensing, L., and Ruoff, P. (2002). Temperature effect on entrainment,
phase shifting, and amplitude of circadian clocks and its molecular
bases. Chronobiol. Int. 19, 807–864.
Roenneberg, T., and Taylor, W. (2000). Automated recordings of
bioluminescence with special reference to the analysis of circadian
rhythms. Methods Enzymol. 305, 104–119.
Ruby, N.F., Brennan, T.J., Xie, X., Cao, V., Franken, P., Heller, H.C.,
and O’Hara, B.F. (2002). Role of melanopsin in circadian responses
to light. Science 298, 2211–2212.
Salome ´, P.A., Michael, T.P., Kearns, E.V., Fett-Neto, A.G., Sharrock,
R.A., and McClung, C.R. (2002). The out of phase 1 mutant defines
a role for PHYB in circadian phase control in Arabidopsis. Plant
Physiol. 129, 1674–1685.
Schaffer, R., Ramsay, N., Samach, A., Corden, S., Putterill, J., Carre ´,
I.A., and Coupland, G. (1998). LATE ELONGATED HYPOCOTYL, an
Arabidopsis gene encoding a MYB transcription factor, regulates
circadian rhythmicity and photoperiodic responses. Cell 93, 1219–
Schmitz, O., Katayama, M., Williams, S.B., Kondo, T., and Golden,
S.S. (2000). CikA, a bacteriophytochrome that resets the cyanobac-
terial circadian clock. Science 289, 765–768.
Schultz, T.F., Kiyosue, T., Yanovsky, M., Wada, M., and Kay, S.A.
(2001). A role for LKP2 in the circadian clock of Arabidopsis. Plant Cell
Somers, D.E., Webb, A.A.R., Pearson, M., and Kay, S.A. (1998). The
short-period mutant, toc1–1, alters circadian clock regulation of
multiple outputs throughout development in Arabidopsis thaliana.
Development 125, 485–494.
Strayer, C., Oyama, T., Schultz, T.F., Raman, R., Somers, D.E., Ma ´s,
P., Panda, S., Kreps, J.A., and Kay, S.A. (2000). Cloning of the
Arabidopsis clock gene TOC1, an autoregulatory response regulator
homolog. Science 289, 768–771.
Suri, V., Lanjuin, A., and Rosbash, M. (1999). TIMELESS-dependent
positive and negative autoregulation in the Drosophila circadan clock.
EMBO J. 18, 675–686.
van der Horst, G.T.J., et al. (1999). Mammalian Cry1 and Cry2 are
essential for maintenance of circadian rhythms. Nature 398, 627–630.
Wang, Z.-Y., and Tobin, E.M. (1998). Constitutive expression of the
CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) gene disrupts circadian
rhythms and suppresses its own expression. Cell 93, 1207–1217.
Williams, J.A., and Sehgal, A. (2001). Molecular components of the
circadian system in Drosophila. Annu. Rev. Physiol. 63, 729–755.
Yoshii, T., Sakamoto, M., and Tomioka, K. (2002). A temperature-
dependent timing mechanism is involved in the circadian system that
drives locomotor rhythms in the fruit fly Drosophila melanogaster.
Zoolog. Sci. 19, 841–850.
Zeng, H., Hardin, P.E., and Rosbash, M. (1994). Constitutive over-
expression of the Drosophila period protein inhibits period mRNA
cycling. EMBO J. 13, 3590–3598.
NOTE ADDED IN PROOF
While this manuscript was in press, an article by Farre ´ et al. also
described the role of PRR7 and PRR9 in the Arabidopsis clock.
Farre ´, E.M., Harmer, S.L., Harmon, F.G., Yanovsky, M.J., and Kay,
S.A. (2005). Overlapping and distinct roles of PRR7 and PRR9 in the
Arabidopsis circadian clock. Curr. Biol. 15, 47–54.
Temperature-Insensitive Circadian Mutant803