Logo of plntcellLink to Publisher's site
Plant Cell. Mar 2005; 17(3): 791–803.
PMCID: PMC1069699

PSEUDO-RESPONSE REGULATOR 7 and 9 Are Partially Redundant Genes Essential for the Temperature Responsiveness of the Arabidopsis Circadian Clock

Abstract

Environmental time cues, such as photocycles (light/dark) and thermocycles (warm/cold), synchronize (entrain) endogenous 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 temperature signals.

INTRODUCTION

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 rhythms with 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, Neurospora crassa, Synechoccocus elongatus, golden hamsters, 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 identification 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), and TIMING OF CAB EXPRESSION 1/PSEUDO-RESPONSE REGULATOR 1 (TOC1/PRR1) (Alabadí 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 (Alabadí 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 proteasome, mediated through SCFZTL, an SCF complex including the F-box protein ZEITLUPE (ZTL) (Más 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 (Alabadí et al., 2002; Mizoguchi et al., 2002). Plants carrying the strong loss of function toc1-2 allele retain rhythmicity, albeit with short period, under some conditions (in blue light and in white light) but fail to sustain an oscillation when assayed in red light and in the dark (Más 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 as a model, one might 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, and TOC1/PRR1 (Matsushika et al., 2000). PRRs lack the conserved Asp that in typical response regulators is phosphorylated 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; Más 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., 2003).

We reasoned that mutants involved in the same clock function should behave similarly under a range of entraining thermocycles (warm/cool) or photocycles (light/dark) and constant conditions in which the clock is predicted to free-run. We therefore characterized the response of each prr mutant to thermocycles. The two 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 mutations impair the response to thermocycles. The prr7-3 prr9-1 double mutant exhibits a much stronger clock defect than either single mutant, indicating functional redundancy. The prr7-3 prr9-1 double mutant fails to reset in response to temperature pulses and also fails to maintain rhythmicity in the dark, indicating that the phenotypes of prr7-3 prr9-1 are not all dependent on light. This mutant with insensitivity to temperature signals should provide insight into a very important but thus far neglected aspect of plant circadian rhythms.

RESULTS

Effect of Thermocycles on Mutants Carrying Loss-of-Function lhy, ztl, and prr Alleles

Cotyledon movement rhythms can be entrained by 22 to 12°C (12 h at 22°C followed by 12 h at 12°C) or 22 to 18°C 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 second line day 2 and day 3, and so on. Seedlings were entrained to 22 to 12°C cycles in constant light and released into free-run (continuous conditions) at 22°C. All mutant seedlings are entrained 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 described 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 functions 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 (Salomé 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.

Figure 1.
Cotyledon Movement during and after Temperature Entrainment Allows the Assignment of Clock Function.

Another approach to test the response of a mutant to thermocycles is to characterize its circadian phenotype during entrainment. We first tested the ability of mutants in the clock genes LHY, TOC1, and ZTL to maintain entrainment to 22 to 18°C cycles after initial entrainment to photocycles. Seedlings were entrained to five photocycles at 22°C and then released to 22 to 18°C thermocycles, provided in phase with the light–dark entrainment (22°C replaces light, 18°C 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). Because none of the mutants go into free-run when transferred to thermocycles, we conclude that mutations in the clock genes LHY, TOC1, and ZTL do not compromise entrainment to thermocycles, indicating that these genes are not directly involved in the response to thermocycles.

Mutations in PRR3 do not cause a leading phase during thermocycles. Instead, the acrophase (peak) of the prr3-1 mutant rhythm is always in phase with the wild type, indicating that it is effectively entrained by the thermocycles (Figures 1H and 1I). Thus, under our conditions, PRR3 is dispensable for proper clock response to thermocycles.

In the same assay, the initial phase of prr5-3 is identical with the 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 phase opposite to the wild type (Figures 1K and 1L). The double plot of acrophase (phase of the peak) during thermocycles suggests that prr5-3 displays a short period and is in free-run (Figure 1K). However, the 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 = 28, or with T = 12 (data not shown), indicating normal entrainment to these thermocycles. T-cycles are entraining cycles in which the cycle length differs from 24 h. For example, 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.

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 cotyledon movement occurs progressively later than the wild type (Figures 1N and 1O) and follows the same slope seen in the acrophase of cotyledon movement after thermocycles (Figure 1M). This suggests that during this time, prr7-3 plants are not responsive to the new resetting cues provided by the transitions from cold to warm. However, starting on the fourth day, prr7-3 adopts and maintains, for at least four cycles, a stable 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 attributed to the circadian clock gating its own responsiveness to environmental stimuli (Johnson, 1992) and suggests that the phase response curve (PRC) to 4°C cold temperature pulses may show a weaker amplitude in prr7-3 than in the wild type. prr7-3 is able to entrain to large amplitude (22 to 12°C) cycles (see Figure 4A). prr7-3 mutants, unlike prr5-3 mutants, remain in free-run and fail to entrain to 18 to 22°C thermocycles administered with T = 20, 22, or 28 h (data not shown), demonstrating a partial loss of circadian responsiveness to temperature.

Figure 4.
Conditional Arrhythmicity of Cotyledon Movement in the prr7-3 prr9-1 Double Mutant.

prr9-1 mutants display the same lagging phase during thermocycles as seen after photocycles (Figures 1P and 1Q). prr9-1 mutants, like prr7-3 mutants, fail to entrain to 18 to 22°C 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 Period Determination

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 pathways 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 the prr single mutants, and multiple (at least four) T2 lines for each combination of prr mutant and LUC fusion were entrained to light–dark cycles or to 22 to 12°C 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 expression of CCA1, LHY, TOC1, and PRR7, as well as PRR9 (data not shown), are entrained to the proper phase by thermocycles.

Figure 2.
PRR3 and PRR5 Only Weakly Contribute to Proper Clock Function.

After entrainment to photocycles or thermocycles and release into constant temperature and continuous white light, the prr3-1 and prr5-3 mutants display a slightly (by ~1 h) short period in the expression of CCA1, LHY, and TOC1 (Figures 2C to 2F, Table 1). Both mutants are properly entrained by photocycles and thermocycles, as demonstrated by the synchrony between the wild type 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 (Alabadí et al., 2002; Más 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 component.

Table 1.
Circadian Parameters of prr Single and Double Mutants Described in This Study

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 movement (Figures 3C and 3D; Michael et al., 2003b). The gi-2 allele of GIGANTEA displays distinct period phenotypes for cotyledon movement and LHCB transcription (Park et al., 1999) and provides precedent for our results with prr9-1.

Figure 3.
PRR7 and PRR9 Are Important for Proper Period Length of the Clock.

We conclude that loss of any member of the PRR family alters the period of CCA1, LHY, and TOC1 expression. In particular, we note that loss-of-function alleles of TOC1 (Más 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 lengthening of the clock suggest that the two genes might be partially redundant.

Conditional Arrhythmicity of prr7-3 prr9-1 Double Mutants

We generated the prr7-3 prr9-1 double mutant for further analysis of the roles of these two genes in the control of circadian rhythmicity. Plants lacking both PRR7 and PRR9 display a period in cotyledon movement rhythms of >32 h (34.2 ± 0.8 h, n = 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 ± 0.3 h, n = 8) and that of prr7-3 homozygotes is slightly longer (27.1 ± 0.2 h, n = 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 ± 0.2 h, n = 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 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 signaling. 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 entrained 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 immediately 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 entrainment (Figure 4D). We conclude that PRR7 and PRR9 function in the emergence of a rhythm in cotyledon movement in response to thermocycles.

The prr7-3 prr9-1 Double Mutant Is Altered in Clock Gene Expression

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 expression 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 = sidereal phase × 24/period) indicates that the lagging phase is a consequence of the very long period because CT is now identical to the normal timing of TOC1 acrophase in the wild type (CTprr7-3 prr9-1 = 16.5 ± 0.8 h, n = 12; CTCOL = 16.5 ± 0.7 h, n = 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).

Figure 5.
PRR7 and PRR9 Are Critical for Rhythmicity in Complete Darkness and after Thermocycles.

prr7-3 prr9-1 plants also display a long period when released in constant red or blue light (Figure 5B). Interestingly, the lengthening 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. 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 and 5D, the circadian expression of all three clock genes persists 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, they show one peak of LUC activity for CCA1 (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 the dark.

We last wished to assess the 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 22°C followed by 12 h at 12°C) for 10 d before transfer to constant conditions. In three of five trials, TOC1 expression in the double mutant exhibited a peak during the first day in constant conditions that was out of phase with the wild type (Figure 5G). The rhythm of the prr7-3 prr9-1 seedlings then dampened rapidly. In the other two trials, no obvious rhythm could be observed in the double mutant (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 mutant 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 thermocycle (22 to 12°C) entraining regime. LUC activity from the 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 response of TOC1:LUC in the double mutant to temperature cycles suggests that it is driven in response to the changes in environmental 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. However, unlike in the wild type, in prr7-3 prr9-1 their expression levels remain elevated during the warm part of the entraining cycles and reach much lower levels during the cold portion of the thermocycles.

Figure 6.
The prr7-3 prr9-1 Double Mutant Fails to Entrain to Temperature Cycles.

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 (Figures 6D and 6E). In the prr7-3 prr9-1 double mutant, the peaks 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 before TOC1 reaches its trough, indicating that, in the absence of PRR7 and PRR9, the positive action of TOC1 on CCA1 expression 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 temperature pulses. Seedlings carrying the TOC1:LUC reporter were entrained to light–dark cycles for 10 d in a constant temperature of 22°C. At dawn of the 11th day, seedlings were transferred into continuous light and temperature. Groups of seedlings were then placed at 12°C for 4 h before being returned to 22°C. The new phase was normalized to the free-running period of wild-type and prr7-3 prr9-1 seedlings and plotted in Figure 6H. In the wild type, temperature pulses induce phase delays in TOC1 expression in the subjective morning and phase advances later during the 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 dissimilar 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.

DISCUSSION

The rotating environment of the earth provides two sets of stimuli, photocycles and thermocycles, for organisms to synchronize their internal clock with their surroundings. Light entrainment 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 of two coupled oscillators, one sensitive to light and the other one sensitive to temperature (Pittendrigh et al., 1958). Recent studies on the Drosophila mutants per, tim, dclk, and cyc during temperature entrainment have supported the existence of a second temperature-sensitive mechanism with some characteristics of a circadian oscillator (Yoshii et al., 2002). However, the exact nature of this temperature-dependent timing mechanism is not known. Temperature cycles are effective entraining stimuli in Neurospora, although the details of the mechanism of temperature entrainment are incompletely defined. Many of the known clock-regulated genes can be properly entrained by thermocycles, but mutations in the clock component FREQUENCY (FRQ) abolish temperature entrainment as measured by gene expression (Nowrousian et al., 2003). The conidiation rhythm also is entrainable by thermocycles. Temperature entrainment 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 described (Rensing and Ruoff, 2002). Studies on the CO2 assimilation rhythm of Kalanchoë plants demonstrated that very small temperature steps, as little as 0.5°C, 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 photocycles). 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 shown).

Several strategies have been described that allow an organism 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 that also contains a pseudoreceiver domain, is insensitive to light 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 persist 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.

The role of PRR7 and PRR9 is not limited to temperature entrainment because profound effects are seen in the prr7-3 prr9-1 mutant after entrainment to photocycles. In white light, the 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 clock as well. They are also crucial for proper clock function in 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 photocycles (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 signaling cascade. Rather, the oscillator mechanism remaining in the prr7-3 prr9-1 double mutant may be impaired and less able to 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.

Because of the strong effects of the loss of PRR7 and PRR9 on the clock, we suggest that they are clock components necessary 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 (Alabadí 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 interaction 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, overexpression of TOC1 represses transcription of PRR9 (Mizuno, 2004), indicating that TOC1 acts, either directly or indirectly, as a repressor of PRR9.

Constitutive expression of a clock component can lead to arrhythmicity, as seen with TOC1, CCA1, and LHY (Schaffer et al., 1998; Wang and Tobin, 1998; Más et al., 2003b), FRQ in Neurospora (Aronson et al., 1994), and PER and TIM in Drosophila (Zeng et al., 1994; Suri et al., 1999). That overexpression of TOC1 results in arrhythmicity (Más et al., 2003b) is consistent with its ascribed role in the oscillator. By contrast, overexpression 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). Simultaneous constitutive expression of PRR7 and PRR9 may be required to induce arrhythmicity in plants, although potential posttranslational modification required to activate either protein might hinder this overexpression strategy.

We propose that PRR7 and PRR9 represent an entry 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 (Más 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 exhibits circadian defects under conditions in which temperature signaling is not active. It remains possible, however, that PRR7 and PRR9 form essential elements of both temperature and other (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.

METHODS

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 analysis.

Generation of Constructs and Transgenic Arabidopsis Plants

A new luciferase binary vector was derived from pZPΩLUC+ (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.

The promoters from the genes CCA1, LHY, and TOC1 were amplified by 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 μL of LR clonase mix and 2 μL of 5× buffer in a 10-μL reaction; the reaction was then transformed into Escherichia coli DH5α and selected on 100 μg/mL of spectinomycin. 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 μg/mL of BASTA. Resistant seedlings were allowed to self, and T2 seeds were collected. Several lines for each reporter and genetic background were analyzed.

Leaf Movement Assays, Luciferase Measurement, and Data Analysis

Leaf movement was measured as described previously (Millar et al., 1995; Salomé 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).

Acknowledgments

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).

Notes

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 McClung (ude.htuomtrad@gnulccm).

Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.104.029504.

References

  • Alabadí, D., Oyama, T., Yanovsky, M.J., Harmon, F.G., Más, P., and Kay, S.A. (2001). Reciprocal regulation between TOC1 and LHY/CCA1 within the Arabidopsis circadian clock. Science 293, 880–883. [PubMed]
  • Alabadí, D., Yanovsky, M.J., Más, 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. [PubMed]
  • Albrecht, U., and Eichele, G. (2003). The mammalian circadian clock. Curr. Opin. Genet. Dev. 13, 271–277. [PubMed]
  • 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. [PubMed]
  • Ashmore, L.J., and Sehgal, A. (2003). A fly's eye view of circadian entrainment. J. Biol. Rhythms 18, 206–216. [PubMed]
  • 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 mammals: 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, 159–162. [PubMed]
  • Eriksson, S., Hurme, R., and Rhen, M. (2002). Low-temperature sensors in bacteria. Philos. Trans. R. Soc. Lond. B Biol. Sci. 357, 887–893. [PMC free article] [PubMed]
  • Froehlich, A.C., Liu, Y., Loros, J.J., and Dunlap, J.C. (2002). White Collar-1, a circadian blue light photoreceptor, binding to the frequency promoter. Science 297, 815–819. [PubMed]
  • 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, 5914–5919. [PMC free article] [PubMed]
  • Golden, S.S., and Canales, S.R. (2004). Cyanobacterial circadian clocks—Timing is everything. Nat. Rev. Microbiol. 1, 181–190. [PubMed]
  • Han, L., Mason, M., Risseeuw, E.P., Crosby, W.L., and Somers, D.E. (2004). Formation of an SCFZTL complex is required for proper regulation of circadian timing. Plant J. 40, 291–301. [PubMed]
  • Hwang, I., Chen, H.-C., and Sheen, J. (2002). Two-component signal transduction pathways in Arabidopsis. Plant Physiol. 129, 500–515. [PMC free article] [PubMed]
  • 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. [PubMed]
  • 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 phytochrome-regulated seedling deetiolation and phasing of the circadian clock. Plant Cell 15, 2654–2665. [PMC free article] [PubMed]
  • Karimi, M., Inzé, D., and Depicker, A. (2002). GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci. 7, 193–195. [PubMed]
  • 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, 313–317. [PubMed]
  • LeClerc, S., and Bartel, B. (2001). A library of Arabidopsis 35S-cDNA lines for identifying novel mutants. Plant Mol. Biol. 46, 695–703. [PubMed]
  • Liu, Y. (2003). Molecular mechanisms of entrainment in the Neurospora circadian clock. J. Biol. Rhythms 18, 195–205. [PubMed]
  • Loros, J.J., and Dunlap, J.C. (2001). Genetic and molecular analysis of circadian rhythms in Neurospora. Annu. Rev. Physiol. 63, 757–794. [PubMed]
  • Más, P., Alabadí, D., Yanovsky, M.J., Oyama, T., and Kay, S.A. (2003. b). Dual role of TOC1 in the control of circadian and photomorphogenic responses in Arabidopsis. Plant Cell 15, 223–236. [PMC free article] [PubMed]
  • Más, P., Kim, W.-Y., Somers, D.E., and Kay, S.A. (2003. a). Targeted degradation of TOC1 by ZTL modulates circadian function in Arabidopsis thaliana. Nature 426, 567–570. [PubMed]
  • 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. [PubMed]
  • McClung, C.R., Salomé, P.A., and Michael, T.P. (2002). The Arabidopsis 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/arabidopsis/.
  • Merrow, M., Brunner, M., and Roenneberg, T. (1999). Assignment of circadian function for the Neurospora clock gene frequency. Nature 399, 584–586. [PubMed]
  • Michael, T.P., and McClung, C.R. (2002). Phase-specific circadian clock regulatory elements in Arabidopsis thaliana. Plant Physiol. 130, 627–638. [PMC free article] [PubMed]
  • Michael, T.P., Salomé, P.A., and McClung, C.R. (2003. a). Two Arabidopsis circadian oscillators can be distinguished by differential temperature sensitivity. Proc. Natl. Acad. Sci. USA 100, 6878–6883. [PMC free article] [PubMed]
  • Michael, T.P., Salomé, P.A., Yu, H.J., Spencer, T.R., Sharp, E.L., Alonso, J.M., Ecker, J.R., and McClung, C.R. (2003. b). Enhanced fitness conferred by naturally occurring variation in the circadian clock. Science 302, 1049–1053. [PubMed]
  • Millar, A.J., Carré, 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. [PubMed]
  • 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., Carré, 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. [PubMed]
  • Mizuno, T. (2004). Plant response regulators implicated in signal transduction and circadian rhythm. Curr. Opin. Plant Biol. 7, 499–505. [PubMed]
  • 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. 44, 360–365. [PubMed]
  • 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, 923–933. [PMC free article] [PubMed]
  • Panda, S., Hogenesch, J.B., and Kay, S.A. (2002. a). Circadian rhythms from flies to human. Nature 417, 329–335. [PubMed]
  • Panda, S., Sato, T.K., Castrucci, A.M., Rollag, M.D., DeGrip, W.J., Hogenesch, J.B., Provencio, I., and Kay, S.A. (2002. b). Melanopsin (Opn4) requirement for normal light-induced circadian phase shifting. Science 298, 2213–2215. [PubMed]
  • 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. [PubMed]
  • Pittendrigh, C., Bruce, V., and Kaus, P. (1958). On the significance of transients in daily rhythms. Proc. Natl. Acad. Sci. USA 44, 965–973. [PMC free article] [PubMed]
  • 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. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Ralph, M.R., and Menaker, M. (1988). A mutation of the circadian system in golden hamsters. Science 241, 1225–1227. [PubMed]
  • 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. [PubMed]
  • Roenneberg, T., and Taylor, W. (2000). Automated recordings of bioluminescence with special reference to the analysis of circadian rhythms. Methods Enzymol. 305, 104–119. [PubMed]
  • 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. [PubMed]
  • Salomé, 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. [PMC free article] [PubMed]
  • Schaffer, R., Ramsay, N., Samach, A., Corden, S., Putterill, J., Carré, 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, 1219–1229. [PubMed]
  • Schmitz, O., Katayama, M., Williams, S.B., Kondo, T., and Golden, S.S. (2000). CikA, a bacteriophytochrome that resets the cyanobacterial circadian clock. Science 289, 765–768. [PubMed]
  • 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 13, 2659–2670. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Strayer, C., Oyama, T., Schultz, T.F., Raman, R., Somers, D.E., Más, 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. [PubMed]
  • Suri, V., Lanjuin, A., and Rosbash, M. (1999). TIMELESS-dependent positive and negative autoregulation in the Drosophila circadan clock. EMBO J. 18, 675–686. [PMC free article] [PubMed]
  • van der Horst, G.T.J., et al. (1999). Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms. Nature 398, 627–630. [PubMed]
  • 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. [PubMed]
  • Williams, J.A., and Sehgal, A. (2001). Molecular components of the circadian system in Drosophila. Annu. Rev. Physiol. 63, 729–755. [PubMed]
  • 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. [PubMed]
  • Zeng, H., Hardin, P.E., and Rosbash, M. (1994). Constitutive overexpression of the Drosophila period protein inhibits period mRNA cycling. EMBO J. 13, 3590–3598. [PMC free article] [PubMed]

NOTE ADDED IN PROOF

  • While this manuscript was in press, an article by Farré et al. also described the role of PRR7 and PRR9 in the Arabidopsis clock.
  • Farré, 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. [PubMed]

Articles from The Plant Cell are provided here courtesy of American Society of Plant Biologists
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...