Plant and Cell Physiology Advance Access originally published online on November 16, 2008
Plant and Cell Physiology 2008 49(12):1839-1850; doi:10.1093/pcp/pcn165
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Involvement of Arabidopsis Clock-Associated Pseudo-Response Regulators in Diurnal Oscillations of Gene Expression in the Presence of Environmental Time Cues
Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Chikusa-ku, Nagoya, 464-8601 Japan
*Corresponding author: E-mail, yamasino{at}agr.nagoya-u.ac.jp; Fax, +81-52-789–4091.
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In plants, the circadian clock is implicated in the biological system that generates diurnal oscillations in cellular and physiological activities. The circadian clock must be synchronized (or entrained) to local time by environmental time cues, such as light/dark and/or hot/cold cycles. In Arabidopsis thaliana, although a number of clock-associated components have been uncovered over the last decade, the clock-associated elements that are involved in entrainment to environmental time cues are largely unknown. In this regard, we have been characterizing one core group of clock components that together control the pace of the central oscillator, including PSEUDO-RESPONSE REGULATOR9 (PRR9), PRR7, PRR5 and TIMING OF CAB2 EXPRESSION 1 (TOC1; or PRR1). The primary aim of this study is to clarify whether these PRR members are implicated in entrainment of the circadian clock to environmental time cues. For this purpose, the diurnal oscillation profiles of clock-controlled genes in the presence of environmental time cues were determined in a set of prr mutants, including a prr9 prr7 prr5 toc1 quadruple mutant. As an extreme phenotype, the prr9-10 prr7-11 prr5-11 toc1-2 quadruple mutant showed an arrhythmia phenotype even under light/dark and hot/cold cycles. In contrast, a cca1-1 lhy-11 toc1-2 triple mutant maintained robust oscillations in the presence of these environmental time cues, although their phases were markedly affected. Based on these results, we propose that the clock components PRR9, PRR7 and PRR5 together might represent elements necessary for the circadian clock to entrain properly to local time in response to light/dark and hot/cold cycles in natural habitats.
Keywords: Diurnal circadian rhythm — Light and temperature entrainment — Pseudo-response regulator family
Abbreviations: CCA1, CIRCADIAN CLOCK-ASSOCIATED 1; CO, CONSTANS; Col, Columbia-0; DD, continuous dark; ELF, EARLY FLOWERING; FFT-NLLS, fast Fourier transform non-linear least square; FKF1, FLAVIN-BINDING KELCH REPEAT F-BOX 1; GI, GIGANTEA; LHY, LATE ELONGATED HYPOCOTYL; LL, continuous light; LUX, LUX ARRHYTHMO; PCL1, PHYTOCLOCK 1; PIF, PHYTOCHROME INTERACTING FACTOR; PRR, PSEUDO-RESPONSE REGULATOR; qRT–PCR, quantitative reverse transcriptase-aided real-time PCR; STO, SALT TOLERANCE; TOC1, TIMING OF CAB2 EXPRESSION 1
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Introduction |
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During the last decade, there have been remarkable advances in understanding the plant circadian system (Gardner et al. 2006
, McClung 2006, Yakir et al. 2006, Harrisingh and Nitabach 2008, Mas 2008, McClung 2008). In Arabidopsis thaliana, intensive studies have uncovered a number of clock-associated components, which are considered to constitute the central oscillator that principally functions even in the absence of environmental time cues. An initial clock model has been proposed in which two types of clock-associated components generate transcriptional rhythms through a negative feedback loop (Alabadi et al. 2001, Alabadi et al. 2002). Proposed clock components include the MYB-related transcription factors CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and the homologous LATE ELONGATED HYPOCOTYL (LHY) (Schaffer et al. 1998, Wang and Tobin 1998, Mizoguchi et al. 2002). Another component, TIMING OF CAB2 EXPRESSION 1 (TOC1; also known as PRR1), is the founding member of the PSEUDO-RESPONSE REGULATOR (PRR) family (Makino et al. 2000, Matsushika et al. 2000, Strayer et al. 2000, Mas et al. 2003). In this prototype clock model, CCA1 and LHY directly repress transcription of the evening-expressed TOC1 gene, the product of which in turn indirectly activates the morning-expressed CCA1/LHY genes. In addition to TOC1, four other members of the PRR family (PRR9, PRR7, PRR5 and PRR3) are also currently believed to be clock-associated components (Mizuno and Nakamichi 2005). Furthermore, a number of other clock-associated components have been identified and characterized, and they include: EARLY FLOWERING 3 and 4 (ELF3 and ELF4), LUX ARRHYTHMO/PHYTOCLOCK 1 (LUX/PCL1) and several others. These clock-associated components have also been used to develop interlocking multiloop clock models to explain their complex roles in the clock system (McClung 2006, Mas 2008, McClung 2008, and references therein). Computational clock models have also been used to simulate the circadian profiles of CCA1/LHY, TOC1 and PRR9/PRR7 (Locke et al. 2006, Zeilinger et al. 2006). Regardless of whether current models are realistic, further studies are needed to provide a more detailed molecular picture of the Arabidopsis circadian clock.
Phenotypes of most Arabidopsis clock mutants were examined under either continuous light or dark conditions at a constant temperature, in order to analyze characteristics of the central oscillator per se, which is capable of running with a pace of about 24 h without any external time cue. The real world has a day/night cycle, however, and the circadian clock is implicated in a wide range of biological diurnal oscillations in natural habitats, including the movement of leaves and petals, stomatal opening and diurnal changes in photosynthetic activities (Mas 2005, Yakir et al. 2006, Hotta et al. 2007). Therefore, the circadian clock must be synchronized (or entrained) to local time in response to environmental time cues. Further refinement of the circadian clock model, therefore, will require a detailed examination of gene expression in some clock mutants throughout the day/night cycle (Millar 2004).
We have been characterizing one core group of clock components, including PRR9, PRR7, PRR5 and TOC1 (or PRR1) (Mizuno and Nakamichi 2005). In this study, we have established a set of prr loss-of-function mutants, including a prr9 prr7 prr5 toc1 quadruple mutant. Taking this advantage, here we attempted to address the issue of whether these PRR members are important for entrainment of the circadian clock to environmental time cues. For this purpose, clock-dependent gene expression under the day/night cycle was examined by employing a large set of pseudo-response regulator mutants, all of which carry a CCA1::LUC bioluminescence reporter. As a result, it was found that the prr9-10 prr7-11 prr5-11 toc1 quadruple loss-of-function mutant showed an arrhythmia phenotype even under light/dark or hot/cold cycles. Based on this and other results, we propose that the clock components PRR9, PRR7 and PRR5 together might represent elements necessary for the circadian clock to entrain properly to local time in response to light/dark and hot/cold cycles in natural habitats.
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Results and Discussion |
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Characterization of a clock-defective cca1 lhy toc1 triple loss-of-function mutant under continuous dark and light/dark cycle conditions
The cca1-1 lhy-11 toc1-2 triple mutant was recently characterized to confirm that all of these clock genes are necessary for maintaining circadian rhythms in constant light conditions (Table 1, Ding et al. 2007
, Niwa et al. 2007). However, the diurnal expression profiles of clock-controlled genes in this clock-defective triple mutant have not yet been fully examined. To address the main issue of this study the triple mutant plants were grown under a 12 h light/12 h dark cycle, and expression of the CCA1::LUC bioluminescence reporter was monitored (Fig. 1A, and Supplementary Fig. S1). To gain further insight, the plants were then transferred to continuous dark (DD) conditions without any external light stimulus. In this study, we used the free-running DD conditions in preference because we have previously characterized a set of prr mutants mainly in the presence of continuous light (LL) (Mizuno and Nakamichi 2005).
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As observed here for the reference wild type (Col) in DD, clock-controlled CCA1::LUC rhythms persisted with a period close to 24 h in the absence of environmental time cues (Fig. 1A, see Col, 72–168 h). In sharp contrast, transfer of the cca1 lhy toc1 triple mutants to DD resulted in the rapid loss of CCA1::LUC rhythm, as reported previously (Ding et al. 2007
, Niwa et al. 2007). The cca1-1 lhy-11 double mutant showed a similar profile, while the phenotype of the toc1-2 single mutant in DD was relatively subtle (Supplementary Fig. S2). In any case, these results confirm the current view that CCA1, LHY and TOC1 play crucial clock-associated roles.
Interestingly, the light/dark cycle could drive the CCA1::LUC diurnal oscillation in both the double and triple mutants, as well as in the wild type (Fig. 1A, 0 h to 72 h), and the rhythms disappeared in the mutants only after the point of subjective dark in DD. Such observations were often the case with many other clock-defective mutants (Mizoguchi et al. 2002, Hazen et al. 2005). In addition to some clock-defective mutants, it was also reported that a transgenic line constitutively expressing CCA1 (CCA1-ox) is arrhythmic under LL or DD conditions, whereas it exhibits robust diurnal oscillations of clock-controlled genes under a light/dark cycle (Green et al. 2002). The same was true for PRR1-ox (or TOC1-ox) (Makino et al. 2002). There are several possible explanations for these observations (Mizoguchi et al. 2002, Hazen et al. 2005): (i) activation of transcription of CCA1::LUC (or other clock-controlled genes) by transition from dark to light could generate such diurnal oscillations; (ii) the light/dark cycle could induce the expression of as yet unknown genes, the products of which could compensate the loss of CCA1, LHY and TOC1 functions in the circadian clock system; or (iii) the intrinsic clock could be perturbed in the mutants in a manner that is not readily detected by roughly measuring the expression profiles of clock-controlled genes. The last idea is relevant to the well-documented coincident model underlying the photoperiodic control of flowering time. The photoperiodic control of flowering time is best explained by the stable accumulation of CONSTANS (CO) during daytime, due to coincidence between the internal (circadian rhythm) and external (photoperiod) time cues (Imaizumi and Kay, 2006). In fact, the cca1 lhy toc1 mutant exhibits an early flowering phenotype even under a light/dark cycle (or short days), implying the defectiveness of its clock. Thus we favored the last possibility, as further examined in the next section.
The clock-defective cca1 lhy toc1 mutant shows an altered oscillation phase under a light/dark cycle
A close inspection of the diurnal oscillation profiles of CCA1::LUC in the cca1 lhy toc1 mutant revealed that the peak of CCA1::LUC within a given day appeared at a considerably advanced position, as compared with that of the wild type (Fig. 1A, lower part). The same was true for the double mutant (Mizoguchi et al. 2002). To examine whether other clock-controlled genes are affected in a similar manner, the cca1 lhy toc1 mutant was grown under a light/dark cycle, and then expression of the clock-associated PRR5 gene was examined by Northern blot hybridization analyses (Fig. 1B). Peaks of the evening-expressed PRR5 gene were detected in both the double and triple mutants at a strikingly advanced timing corresponding to dawn. The same relative phase advance was observed for other evening-expressed genes, including GIGANTEA (GI) and FLAVIN-BINDING KELCH REPEAT F-BOX 1 (FKF1) (data not shown, Niwa et al. 2007). To confirm this further, diurnal oscillation profiles in the triple mutant were further examined for the PHYTOCHROME INTERACTING FACTOR 5 (PIF5) and SALT TOLERANCE (STO) genes by quantitative reverse transcriptase-aided real-time PCR (qRT–PCR) analyses (Fig. 1C). In the mutant, the peaks of both these morning-expressed genes were expressed during the dark, thus out of phase and advanced relative to the wild-type peak.
Taking these results together, it is likely that the internal clock of plants lacking the core components CCA1, LHY and TOC1 is perturbed such that the expression peaks of a number of clock-controlled genes are advanced out of phase with the wild type. However, the triple mutant is capable of generating diurnal oscillations of certain clock-controlled genes, if not relatively punctual, suggesting that the clock lacking CCA1, LHY and TOC1 is still somehow capable of responding to external light/dark time cues.
Isolation of a large set of clock-defective prr loss-of-function mutants and characterization under a light/dark cycle
To address the main issue of this study further (see Introduction), we generated a set of mutants with the prr9-10, prr7-11, prr5-11 and toc1-2 loss-of-function (or hypomorphic) alleles in all combinations, including a prr9-10 prr7-11 prr5-11 toc1-2 quadruple mutant (Table 1). These prr mutants are all in the unified Columbia (Col) background (note that the allele numbers are omitted in the text hereafter for clarity). Most of these mutants have already been characterized with regard to the clock-associated phenotypes, mostly in the continuous light (Ito et al. 2003, Yamamoto et al. 2003, Nakamichi et al. 2005a, Nakamichi et al. 2005b, Ito et al. 2007, Nakamichi et al. 2007, Ito et al. 2008, Niwa et al. 2007, for a review, see Mizuno and Nakamichi 2005). A few others will be described elsewhere (see also Table 1). As has been documented extensively (Strayer et al. 2000, Eriksson et al. 2003, Kaczorowski et al. 2003, Michael et al. 2003, Farre et al. 2005, Nakamichi et al. 2005a, Nakamichi et al. 2005b, Salome and McClung 2005), the loss of function of any of the PRR genes results in a characteristic clock-associated phenotype. In the most extreme case, the prr9 prr7 prr5 toc1 quadruple mutant shows the following severe phenotypes: (i) arrhythmia in continuous light or dark conditions; (ii) photoperiod-insensitive late flowering; and (iii) extremely long hypocotyls of seedlings in red light. These clock-associated phenotypes collectively implicate the PRR proteins as the core of the circadian clock system. In this study, we attempted to characterize these mutants with special reference to their phenotypes with respect to diurnal oscillations of gene expression under a light/dark cycle, as rationalized above.
Hence bioluminescence of the CCA1::LUC reporter was monitored in the set of prr mutants grown under a light/dark cycle, followed by transfer to DD. First, the prr9 prr7 double mutant was compared with the prr7 prr5 double mutant (Fig. 2A) to confirm the fundamental and reference results that have been reported previously (Nakamichi et al. 2005a, Nakamichi et al. 2005b). The prr9 prr7 mutant showed a long period phenotype in DD, whereas the prr7 prr5 mutant had a short period phenotype with low amplitude under the same conditions. These results were consistent with those reported previously (Table 1). We examined for the first time the prr9 prr7 toc1 triple mutant in comparison with the prr7 prr5 toc1 triple mutant (Fig. 2B). An additive impact of the toc1 mutation on the free-running rhythms was also evident, as follows. The prr7 prr5 toc1 triple mutant had at most negligible free-running rhythm, and the long period phenotype of prr9 prr7 was in part compensated for by the toc1 mutation. More detailed examinations with regard to the clock-associated phenotypes of the prr9 prr7 toc1 triple mutant will be described elsewhere (Ito et al. unpublished data, see Table 1).
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Here we focused on diurnal oscillations of the CCA1::LUC expression in these clock-defective prr mutants under a light/dark cycle. Although diurnal oscillations of CCA1::LUC were observed in the prr mutants, it was evident that their amplitudes were considerably reduced, as compared with that of the cca1 lhy toc1 mutant (Figs. 1A, 2B). In this connection, the CCA1::LUC profiles in the toc1 single and prr9 prr5 toc1 triple mutants were also examined (Supplementary Fig. S2). Their phenotypes were rather subtle in that both of these mutants showed robust oscillation profiles of CCA1::LUC under a light/dark cycle. Taken together, it was suggested that the circadian clock system, lacking either of the pairs PRR9/PRR7 or PRR7/PRR5, is more severely defective than that lacking the CCA1/LHY pair, as judged by the fact that the diurnal oscillations of CCA1::LUC in both the prr9 prr7 and prr7 prr5 mutants are markedly dampened with a low amplitude even under a light/dark cycle. To support this critical notion further, the cca1 lhy prr7 prr5 quadruple mutant was examined in comparison with the cca1 lhy double mutant (Fig. 2C). As expected, the CCA1::LUC profile in the cca1 lhy prr7 prr5 mutant had much lower amplitudes than the cca1 lhy mutant under a light/dark cycle. It should be noted that both the mutants showed severely dampened free-running rhythms in DD.
The clock-defective prr mutants have altered oscillation phases under light/dark cycle
As mentioned earlier (Fig. 1), a phase alteration of the oscillation profile under a light/dark cycle was observed for certain clock-controlled genes in the cca1 lhy mutant. We previously observed similar events for a set of prr mutants (Nakamichi et al. 2005b). By employing a new set of prr multiple mutants (Table 1), they were further examined in this respect in a quantitative manner. Indeed, the prr mutants had altered phases which varied considerably from one mutant to another (Fig. 3, upper panel), and this was determined quantitatively (Fig. 3, lower part, for the original data set, see Supplementary Figs. S3, S4). The generalized view, deduced from these results, is that a mutant that results in a shorter period in its free-running rhythm exhibits a more advanced phase in its diurnal oscillation under a light/dark cycle, while a mutant with a longer period shows a more delayed phase (see also Table 1). To support this view further, the phase alterations in some other clock-defective mutants were also examined, including the toc1, cca1 lhy, cca1 lhy toc1 and cca1 lhy prr7 prr5 mutants (Supplementary Fig. S5). These results further supported the view that PRRs including TOC1 serve as important components for the circadian clock to work properly, not only under free-running conditions, but also even in the presence of an external light/dark time cue.
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The prr9 prr7 prr5 mutant is arrhythmic even under a light/dark cycle
Notably in the prr9 prr7 prr5 triple mutant, the diurnal oscillation profile of CCA1::LUC was severely dampened even under a light/dark cycle (Fig. 4A). To confirm this event further, the expression of intrinsic CCA1 mRNA itself was analyzed by Northern blot hybridization (Fig. 4B), and also by qRT–PCR (Fig. 4C), using independently prepared samples from the wild type and the prr triple mutant plants. Furthermore, this striking event needed to be confirmed by examining other appropriate clock-controlled gene probes, because it was recently suggested that degradation of CCA1 mRNA is also diurnally regulated (Yakir et al. 2007
). To this end, the expression of the clock-associated gene GI and the clock-controlled CCA1-like output gene (At5g17300) was also analyzed (Fig. 4D, E). These results together indicate that the circadian clock system is severely impaired in the prr9 prr7 prr5 triple mutant in such a way that the diurnal oscillation of some clock-controlled genes (at least CCA1, GI and At5g17300) became arrhythmic (or constitutive) even in the presence of an environmental light/dark time cue. The preliminary and similar results have been reported with regard to the expression profiles of TOC1 and PRR3 in the prr triple mutant, showing that the expression of TOC1 was constitutively low even under a light/dark cycle, whereas the expression of PRR3 was constitutively high (Nakamichi et al. 2005b). Notably, these results from the prr triple mutant are quite in contrast to those of the cca1 lhy toc1 mutant, in which robust oscillation was observed under light/dark conditions, as confirmed concomitantly (Fig. 4D, E, right-hand panels).
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Characterization of the prr9 prr7 prr5 toc1 quadruple mutant under a light/dark cycle
To confirm these ideas solidly, the prr9 prr7 prr5 toc1 mutant was also examined (Fig. 5A). The CCA1::LUC rhythm in the quadruple mutant disappeared almost completely even under a light/dark cycle, as compared with the prr9 prr7 prr5 triple mutant (Supplementary Fig. S6). In addition to this, qRT–PCR analysis was also performed by directly probing the expression profiles of CCA1, GI and At5g17300 in both the prr9 prr7 prr5 toc1 quadruple and cca1 lhy toc1 triple mutants (Fig. 5B–D). Taken together, we concluded that not only a free-running circadian clock but also a light/dark cycle-driven oscillator is impaired in plants lacking the multiple PRR clock-associated components. It was thought that the impact of the toc1 (or prr1) lesion appears to be rather subtle, as compared with other prr lesions (compare Figs. 4A and 5A).
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Characterization of the prr mutants under a hot/cold cycle
The critical question then arose as to whether or not the PRR components are also important for the circadian clock to be entrained to the hot/cold temperature cycle. To answer this question, oscillation profiles of certain clock-controlled genes were examined in the prr9 prr7 prr5 toc1 mutant, grown at a 22°C/12°C temperature cycle for 20 d in continuous light. As shown in Fig. 6, robust oscillation profiles were observed for CCA1, GI and At5g17300 in the temperature-entrained wild-type plants, as expected (Fig. 6). In contrast, such oscillations were markedly perturbed in the prr9 prr7 prr5 toc1mutant, as observed earlier for the mutant entrained to the light/dark cycle (see Fig. 5). Provided that a temperature cycle-driven oscillator system is present as assumed a priori (for an example, see Salome and McClung 2005
), such a clock system was also perturbed in the plant lacking the multiple PRR components. In contrast, robust oscillation profiles of the same genes were evident in the cca1 lhy toc1 mutant, concomitantly entrained to the temperature cycle (Fig. 6B, C, right-hand panels). Interestingly, the phases of oscillation profiles appeared at advanced positions in the temperature-entrained cca1 lhy toc1 mutant, as observed in the same mutant entrained under a light/dark cycle.
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It was previously reported that both the prr9 prr7 and prr7 prr5 double mutants do not respond properly to the temperature cycle (Nakamichi et al. 2005a
, Salome and McClung 2005). Taken altogether, we propose that the core oscillator components PRR9, PRR7 and PRR5 together represent elements necessary for the circadian clock to respond properly to both the environmental light/dark and hot/cold cycles. Also, these results suggested the general concept that both the photo-driven and thermo-driven circadian oscillators contain the common components PRRs and CCA1/LHY, at least in part.
Implications and conclusions
In this study, we characterized the diurnal oscillation of gene expression of a set of prr mutants (Table 1), including the prr9 prr7 prr5 toc1 quadruple mutant, in response to light/dark and hot/cold cycles. The perception and integration of these environmental time cues enable plants to synchronize the internal circadian clock to local time in natural habitats. In this respect, the so-called input pathway has been characterized to some extent in A. thaliana (Millar 2004, McClung 2006). Nevertheless, the molecular determinants that function within the interface of input pathway and central oscillator remain elusive. The results of this study provide us with a new insight into this longstanding issue, as discussed below.
Some prr multiple mutants (e.g. prr7 prr5 and prr9 prr7) show markedly altered rhythms with a shorter or longer period in free-running conditions (Nakamichi et al. 2005a). The results of this study showed that these prr multiple mutants exhibit a clock-associated phenotype even under a light/dark cycle (Figs. 2, 3). The general concept was revealed that a given mutant with a shorter period in the free-running rhythm exhibits a more advanced phase in the diurnal oscillation under a light/dark cycle, whereas a mutant with a longer period shows a more delayed phase (Fig. 3 and Table 1). This is consistent with the general assumption that altered circadian rhythms with shorter/longer periods under free-running conditions would (or should) result in advanced/delayed phases under diurnal entrainment conditions. Hence, the results of this study further support the current view that PRRs are important clock components that control the pace of the central oscillator.
More importantly, the results of this study demonstrated that the clock-defective phenotype of the prr9 prr7 prr5 triple mutant is striking in that the mutant is arrhythmic, not only under free-running conditions but also even in the presence of external light/dark time cues (Fig. 4). The same phenotypic lesion was also observed in the prr9 prr7 prr5 toc1 mutant (Fig. 5), but not in the cca1 lhy toc1 mutant (Fig. 1). In this respect, we previously reported that the prr9 prr7 prr5 triple mutant seedling is insensitive to light (particularly red light) signals as judged by inhibition of hypocotyl elongation in the presence of light (see Table 1), suggesting that a light signaling pathway is severely attenuated in this clock-defective mutant (Ito et al. 2007). These results consistently suggest that PRR9, PRR7 and PRR5 together are necessary for the clock to respond properly to the light signal. However, they must play much wider roles, because it was also found that the prr9 prr7 prr5 toc1 mutant could not entrain to the temperature cycle (Fig. 6). Taken together, these results strongly support the novel view that PRR9, PRR7 and PRR5 together constitute a part of the core circadian oscillator, and also they represent elements (or parts) necessary for the clock to respond properly to both the environmental time cues.
To consider the proposed view in this study further, it is worth mentioning the following points. (i) Both the prr9 prr7 prr5 triple and prr9 prr7 prr5 toc1 quadruple mutant plants could develop normally throughout the life cycle, and they could set flowers and seeds normally. However, they showed a phenotype of extremely late flowering (Table 1), and their final height was very tall (Supplementary Fig. S7), as has been reported for certain clock-defective gi mutants (Araki and Komeda 1993). Nevertheless, these phenotypes were assumed to have nothing to do with the defect of oscillation under a light/dark cycle, because certain gi loss-of-function mutants showed robust rhythmic oscillations of clock-controlled genes under a light/dark cycle (Fowler et al. 1999, Park et al. 1999). (ii) It should be emphasized once again that the plants expressing the CCA1 gene highly and constitutively (i.e. CCA1-ox transgenic plants) showed robust rhythmic oscillations of clock-controlled genes under a light/dark cycle (Matsushika et al. 2002). The same was true for TOC1-ox transgenic lines (Makino et al. 2002). Hence, it is unlikely that the defects of the prr9 prr7 prr5 triple and prr9 prr7 prr5 toc1 quadruple mutant plants in the presence of environmental time cues are an indirect consequence of blocking of clock functions due to, for instance, unusually high levels of expression of the core clock CCA1 and/or TOC1 genes.
Finally, the results of this study highlighted the difference between PRR9/PRR7/PRR5 and CCA1/LHY in their clock-associated roles. The mutant oscillator lacking both CCA1 and LHY can still respond and entrain to the environmental time cues, if not perfectly (Fig. 1), while the oscillator lacking PRR9, PRR7 and PRR5 could not do so (Fig. 4). In this context, another core clock component TOC1 (or PRR1) also appears to be not primarily responsible for responsiveness to the external time cues, because the impact of the toc1 lesion on diurnal oscillations under a light/dark cycle is less severe (compare Figs. 4A and 5A, and see also Supplementary Fig. S2). We previously characterized the genetic linkages between the PRR7/PRR5 and CCA1/LHY genes by establishing the cca1-1 lhy-11 prr7-11 prr5-11 quadruple mutant (Nakamichi et al. 2005b). The revealed genetic linkages between the prr7/prr5 and cca1/lhy alleles suggested that PRR7/PRR5 and CCA1/LHY function in a manner relatively independent from, or parallel to, each other. Such non-epistatic genetic linkages between PRR9/PRR7/PRR5 and CCA1/LHY in the clock-associated functions still hold even with regard to the diurnal oscillator functions under a light/dark cycle, as demonstrated in this study (Fig. 2C). Taken together, the intriguing message of this study is that the clock-associated functions of PRR9, PRR7 and PRR5 might be more independent from those of CCA1 and LHY than generally thought in the several current clock models, in which CCA1/LHY and PRR9/PRR7 are postulated to form an intimate transcriptional feedback loop (Mizuno and Nakamichi 2005, Locke et al. 2006). Clarification of this problem must await further experimentation to improve the current clock model(s) further.
During the last decade, many clock-defective mutants have been reported in A. thaliana. Nevertheless, it has long been puzzling that most of them exhibited robust oscillation profiles of clock-controlled genes in the presence of external time cues. In this context, we showed that certain multiple prr loss-of-function mutants are arrhythmic even in the presence of light/dark or hot/cold cycles. The results of this study suggest that the clock-associated components PRR9, PRR7 and PRR5 together might represent elements necessary for the circadian clock to entrain properly to local time in natural habitats.
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Materials and Methods |
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Plant lines and growth conditions
Arabidopsis thaliana plants used in this study are listed in Table 1. They are all in the Col background and, except for a few, carry the CCA1::LUC reporter gene (Nakamichi et al. 2004
). Plants were grown as described previously, unless otherwise noted (Matushika et al. 2000).
Preparation of RNA, Northern blot hybridization analysis and qRT–PCR analysis
Plants were grown on MS plates containing 1% sucrose under a 12 h light/12 h dark cycle conditions for 20 d and harvested at intervals for preparation of total RNA. RNA was purified with an RNeasy plant kit (QIAGEN, Valencia, CA, USA) and subjected to Northern blot hybridization analyses with the appropriate probes, as described previously (Matsushika et al. 2000, Makino et al. 2002). cDNA was generated from 3 µg of each RNA sample digested by RNase-free DNase I (QIAGEN) with ReverTra Ace (TOYOBO, Osaka, Japan) and oligo(dT) primer. The synthesized cDNAs were amplified with SYBR Premix Ex Taq II (TAKARA SHUZO CO., LTD., Kyoto, Japan) and the following primer set, using the StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA, USA). Primer sets used in this study were: CCA1, 5'-GGTGGACTGAGGAAGAAC-3' and 5'-GGAGAAAAATTTCTGAGCGTGAC-3'; GI, 5'-GGGTAAATATGCTGCTGGAGA-3' and 5'-CAGTATGACACCAGCTCCATT-3'; STO, 5'-ATGTTTCACACAAGAAGCCG-3' and 5'-CATTAACTCCACACATGCAG-3'; At5g17300, 5'-GGGGGAAATGACTATGCAC-3' and 5'-CTTCGAATCTGAACTGCGG-3'; and APX3, 5'-CTCCGTTCTCTCATCGC-3' and 5'-CAGAGATCGAGAGCGATC-3'. The APX3 encoding an ascorbate peroxidase isozyme was used an internal reference (Hazen et al. 2005). A standard thermal cycling program was used for all PCRs: 95°C for 60 s, 40 cycles of 95°C for 5 s, and 60°C for 30 s.
Measurement of bioluminescence, and data analyses
Plants used in this study contain a homozygous or heterozygous CCA1::LUC gene on the chromosome (Nakamichi et al. 2004, Nakamichi et al. 2005b). Bioluminescence assays were carried out, as described previously. Briefly, the luminescence-sensing equipment was established previously with a photomultiplier tube (Hamamatsu Photonics, Hamamatsu, Japan). Individual plants were grown for 20 d under 12 h light/12 h dark cycle conditions with white light (70 µmol s–1 m–2) on MS agar plates containing 5 µMÿ luciferin. If necessary, the plates were moved to continuous light or dark conditions. Bioluminescence was measured with a real-time monitoring and an auto-calculating system (Nakamichi et al. 2004). In every case, several samples were assayed independently to obtain consistent and reproducible results (see Supplementary Figs. S1, S3, S4). To normalize each bioluminescence profile of CCA1::LUC in a given mutant, the following practical formula was used. For each profile, bioluminescence values of every data point were divided by the lowest value detected within the 72 h light/dark cycles, and then subtracted by the mean value from the 24–48 h period. This normalization algorithm allowed amplitude and phase comparisons of CCA1::LUC expression profiles. Profiles were further analyzed by the fast Fourier transform non-linear least square (FFT-NLLS) algorism to calculate amplitudes, phases and relative amplitude errors.
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TopAbstractIntroductionResults and DiscussionMaterials and MethodsSupplementary dataFundingReferences |
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Supplementary data are available at PCP online.
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TopAbstractIntroductionResults and DiscussionMaterials and MethodsSupplementary dataFundingReferences |
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The Ministry of Ministry of Education, Culture, Sports, Science, and Technology of Japan Grants-in-Aid for Scientific Research on Priority Areas (No. 20061016 to T.Y.) and Grant-in-Aid for the GCOE Programs (Systems Biology).
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Alabadi D, Oyama T, Yanovsky MJ, Harmon FG, Mas P, Kay SA. Reciprocal regulation between TOC1 and LHY/CCA1 within the Arabidopsis circadian clock. Science (2001) 293:880–883.
Alabadi D, Yanovsky MJ, Mas P, Harmer SL, Kay SA. Critical role for CCA1 and LHY in maintaining circadian rhythmicity in Arabidopsis. Curr. Biol. (2002) 12:757–761.[CrossRef][Web of Science][Medline]
Araki T, Komeda Y. Analysis of the role of the late-flowering locus, GI, in the flowering of Arabidopsis thaliana. Plant J. (1993) 3:231–239.[Web of Science]
Eriksson ME, Millar AJ. The circadian clock. A plant's best friend in a spinning world. Plant Physiol. (2003) 132:732–738.
Ding Z, Doyle MR, Amashino RM, Davis SJ. A complex genetic interaction between Arabidopsis thaliana TOC1 and CCA1/LHY in driving the circadian clock and in output regulation. Genetics (2007) 176:1501–1510.
Farre ME, Harmer SL, Harmon FG, Yanovsky MJ, Kay SA. Overlapping and distinct roles of PRR7 and PRR9 in the Arabidopsis circadian clock. Curr. Biol. (2005) 15:47–54.[CrossRef][Web of Science][Medline]
Fowler S, Lee K, Onouchi H, Samach A, Richardson K, Morris B, et al. GIGANTEA: a circadian clock-controlled gene that regulates photoperiodic flowering in Arabidopsis and encodes a protein with several possible membrane-spanning domains. EMBO J. (1999) 18:4679–4688.[CrossRef][Web of Science][Medline]
Gardner MJ, Hubbard KE, Hotta CT, Dodd AN, Webb AA. How plants tell the time. Biochem. J. (2006) 397:15–24.[CrossRef][Web of Science][Medline]
Green RM, Tobin EM. Loss of the circadian clock-associated protein 1 in Arabidopsis results in altered clock-regulated gene expression. Proc. Natl Acad. Sci. USA (1999) 96:4176–4179.
Green RM, Tingay S, Wang ZY, Tobin EM. Circadian rhythms confer a higher level of fitness to Arabidopsis plants. Plant Physiol. (2002) 129:576–584.
Harrisingh MC, Nitabach MN. Circadian rhythms. Integrating circadian timekeeping with cellular physiology. Science (2008) 320:879–880.
Hazen SP, Schultz TF, Pruneda-Paz JL, Borevitz JO, Ecker JR, Kay SA. LUX ARRHYTHMO encodes a Myb domain protein essential for circadian rhythms. Proc. Natl Acad. Sci. USA (2005) 102:10387–10392.
Hotta CT, Gardner MJ, Hubbard KE, Baek SJ, Dalchau N, Suhita D, et al. Modulation of environmental responses of plants by circadian clocks. Plant Cell Environ. (2007) 30:333–349.[CrossRef][Medline]
Imaizumi T, Kay SA. Photoperiodic control of flowering: not only by coincidence. Trends Plant Sci. (2006) 11:550–558.[CrossRef][Web of Science][Medline]
Ito S, Matsushika A, Yamada H, Sato S, Kato T, Tabata S, et al. Characterization of the APRR9 pseudo-response regulator belonging to the APRR1/TOC1 quintet in Arabidopsis thaliana. Plant Cell Physiol. (2003) 44:1237–1245.
Ito S, Nakamichi N, Nakamura Y, Niwa Y, Kato T, Murakami M, et al. Genetic linkages between circadian clock-associated components and phytochrome-dependent red light-signal transduction in Arabidopsis thaliana. Plant Cell Physiol. (2007) 48:971–983.
Ito S, Niwa Y, Nakamichi N, Kawamura H, Yamashino T, Mizuno T. Insight into missing genetic links between two evening-expressed pseudo-response regulator genes TOC1 and PRR5 in the circadian clock-controlled circuitry in Arabidopsis thaliana. Plant Cell Physiol. (2008) 49:201–213.
Kaczorowski KA, Quail PH. Arabidopsis PSEUDO-RESPONSE REGULATOR7 is a signaling intermediate in phytochrome-regulated seedling deetiolation and phasing of the circadian clock. Plant Cell (2003) 15:2654–2665.
Kumagai T, Ito S, Nakamichi N, Niwa Y, Murakami M, Yamashino T, et al. The common function of a novel subfamily of B-Box zinc finger proteins with reference to circadian-associated events in Arabidopsis thaliana. Biosci. Biotechnol. Biochem (2008) 72(6):1539–49.[CrossRef]
Locke JC, Kozma-Bognar L, Gould PD, Feher B, Kevei E, Nagy F, et al. Experimental validation of a predicted feedback loop in the multi-oscillator clock of Arabidopsis thaliana. Mol. Syst. Biol. (2006) 2:59.[Medline]
Makino S, Kiba T, Imamura A, Hanaki N, Nakamura A, Suzuki T, et al. Genes encoding pseudo-response regulators: insight into His-to-Asp phosphorelay and circadian rhythm in Arabidopsis thaliana. Plant Cell Physiol. (2000) 41:791–803.[Web of Science][Medline]
Makino S, Matsushika A, Kojima M, Yamashino T, Mizuno T. The APRR/TOC1 quintet implicated in circadian rhythms of Arabidopsis thaliana: I. characterization with APRR1-overexpressing plants. Plant Cell Physiol. (2002) 43:58–69.
Mas P, Alabadi D, Yanovsky MJ, Oyama T, Kay SA. Dual role of TOC1 in the control of circadian and photomorphogenic responses in Arabidopsis. Plant Cell (2003) 15:223–236.
Mas P. Circadian clock signaling in Arabidopsis thaliana: from gene expression to physiology and development. Int. J. Dev. Biol. (2005) 49:491–500.[CrossRef][Web of Science][Medline]
Mas P. Circadian clock function in Arabidopsis thaliana: time beyond transcription. Trends Cell Biol. (2008) 18:966–977.
Matsushika A, Makino S, Kojima M, Mizuno T. Circadian waves of expression of the APRR1/TOC1 family of pseudo-response regulators: insight into the plant circadian clock in Arabidopsis thaliana. Plant Cell Physiol. (2000) 41:1002–1012.
Matsushika A, Makino S, Kojima M, Yamashino T, Mizuno T. The APRR1/TOC1 quintet implicated in circadian rhythms of Arabidopsis thaliana: II. Characterization with CCA1-overexpressing plants. Plant Cell Physiol. (2002) 43:118–122.
McClung CR. Plant circadian rhythms. Plant Cell (2006) 18:792–803.
McClung CR. Comes a time. Curr. Opin. Plant Biol. (2008) 11:1–7.[Medline]
Michael TP, Salome PA, Yu HJ, Spencer TR, Sharp EL, McPeek MA, et al. Enhanced fitness conferred by naturally occurring variation in the circadian clock. Science (2003) 302:1049–1053.
Millar AJ. Input signals to the plant circadian clock. J. Exp. Bot. (2004) 55:277–283.
Mizoguchi T, Wheatley K, Hanzawa Y, Wright L, Mizoguchi M, Song HR, et al. LHY and CCA1 are partially redundant genes required to maintain circadian rhythms in Arabidopsis. Dev. Cell (2002) 2:629–641.[CrossRef][Web of Science][Medline]
Mizuno T, Nakamichi N. Pseudo-response regulators (PRRs) or true oscillator components (TOCs). Plant Cell Physiol. (2005) 46:677–685.
Nakamichi N, Ito S, Oyama T, Yamashino T, Kondo T, Mizuno T. Characterization of plant circadian rhythms by employing Arabidopsis cultured cells with bioluminescence reporters. Plant Cell Physiol. (2004) 45:57–67.
Nakamichi N, Kita M, Ito S, Sato E, Yamashino T, Mizuno T. The Arabidopsis pseudo-response regulators, PRR5 and PRR7, coordinately play essential roles for circadian clock function. Plant Cell Physiol. (2005a) 46:609–619.
Nakamichi N, Kita M, Ito S, Yamashino T, Mizuno T. Three pseudo-response regulators, PRR9, PRR7, and PRR5, play together an essential role close to the circadian clock of Arabidopsis thaliana. Plant Cell Physiol. (2005b) 46:686–698.
Nakamichi N, Kita M, Niimura K, Ito S, Yamashino T, Mizoguchi T, et al. Arabidopsis clock-associated pseudo-response regulators PRR9, PRR7 and PRR5 coordinately and positively regulate flowering time through the canonical CONSTANS-dependent photoperiodic pathway. Plant Cell Physiol. (2007) 48:822–832.
Niwa Y, Ito S, Nakamichi N, Mizoguchi T, Niinuma K, Yamashino T, et al. Genetic linkages of the circadian clock-associated genes, TOC1, CCA1 and LHY, in the photoperiodic control of flowering time in Arabidopsis thaliana. Plant Cell Physiol. (2007) 48:925–937.
Park DH, Somers DE, Kim YS, Choy YH, Lim HK, Soh MS, et al. Control of circadian rhythms and photoperiodic flowering by the Arabidopsis GIGANTEA gene. Science (1999) 285:1579–1582.
Salome PA, McClung CR. Pseudo-response regulator 7 and 9 are partially redundant genes essential for the temperature responsiveness of the Arabidopsis circadian clock. Plant Cell (2005) 17:791–803.
Schaffer R, Ramsay N, Samach A, Corden S, Putterill J, Carre IA, et al. The late elongated hypocotyl mutation of Arabidopsis disrupts circadian rhythms and the photoperiodic control of flowering. Cell (1998) 93:1219–1229.[CrossRef][Web of Science][Medline]
Strayer C, Oyama T, Schultz TF, Raman R, Somers DE, Mas P, et al. Cloning of the Arabidopsis clock gene TOC1, an autoregulatory response regulator homolog. Science (2000) 289:768–771.
Wang ZY, Tobin EM. Constitutive expression of the CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) gene disrupts circadian rhythms and suppresses its own expression. Cell (1998) 93:1207–1217.[CrossRef][Web of Science][Medline]
Yakir E, Hilman D, Harir Y, Green RM. Regulation of output from the plant circadian clock. FEBS J. (2006) 274:335–345.[CrossRef]
Yakir E, Hilman D, Hassidim M, Green RM. CIRCADIAN CLOCK ASSOCIATED1 transcript stability and the entrainment of the circadian clock in Arabidopsis. Plant Physiol. (2007) 145:925–932.
Yamamoto Y, Sato E, Shimizu T, Nakamich N, Sato S, Kato T, et al. Comparative genetic studies on the APRR5 and APRR7 genes belonging to the APRR1/TOC1 quintet implicated in circadian rhythm, control of flowering time, and early photomorphogenesis. Plant Cell Physiol. (2003) 44:1119–1130.
Zeilinger MN, Farre EM, Taylor SR, Kay SA, Doyle FJ III. A novel computational model of the circadian clock in Arabidopsis that incorporates PRR7 and PRR9. Mol. Syst. Biol (2006) 2:58–64.[Medline]
(Received August 20, 2008; Accepted October 27, 2008)
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