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