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1 Max Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, D-50829 Köln, Germany
2 Institute of Plant Biology, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary
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Abstract |
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Introduction |
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In the model plant Arabidopsis thaliana (Arabidopsis), several clock genes have been isolated and analyzed via molecular-genetic approaches (Eriksson & Millar 2003). These studies led to the first proposed mechanisms for the molecular basis for daily time keeping in plants. In this model, two morning-expressed Myb-transcription factors, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), and an evening-expressed pseudoresponse regulator TIMING OF CAB2 EXPRESSION 1 (TOC1) are believed to be positive/negative feedback components of the circadian oscillator (Eriksson & Millar 2003). More recently, mathematical approaches extended this model to interlocked feedback loops, and the additional clock components GIGANTEA (GI) was a proposed candidate for this interconnection (Locke et al. 2005). A molecular model is thus emerging regarding the components of the time-keeping pacemaker. It is less clear how this timer is modulated by external cues.
Various exogenous growth conditions, such as varying light, temperature, abiotic stress and disease, regulate a set of endogenous plant hormones. In turn, these hormones regulate an extensive array of physiological processes to ensure maximal fitness over the whole plant life cycle, including the response to varying environmental conditions (Srivastava 2002). These compounds have been collectively termed the phytohormones and include cytokinin, auxin, brassinosteroid (BR), abscisic acid (ABA), gibberellin (GA), ethylene and salicylic acid (SA) (for a review see Srivastava 2002; Bishop & Koncz 2002; Fedoroff 2002; Guo & Ecker 2004; Martínez et al. 2004). A single phytohormone can act on a diverse range of biological processes. Moreover, several phytohormones often function with interactive effects. For example, alternation of light signals change the phytohormone levels of cytokinin, auxin, BR, ABA and GA. These changes lead to an altered choice in various developmental decisions. This regulation level functions from seed germination to flowering reproduction. The general notion is that phytohormones are essential for plant to sense their exogenous and endogenous conditions.
As described above, both phytohormones and the clock play important roles in integrating environmental signals and in regulating plant development and metabolism. Reports on connections between these two systems has been limited. For example, it has been reported that the clock modulates ethylene synthesis, and auxin trafficking and responsiveness (Jouve et al. 1999; Thain et al. 2004). Recently, the phytohormones cytokinin, auxin and ABA have been shown to accumulate in diurnal patterns (Nováková et al. 2005). Thus, aspects of phytohormone biology are under clock control. However, systematic descriptions regarding the integration of a hormonal signal into the plant-circadian system have been lacking.
Here, we investigated whether there is a hormone-clock connection in plants, as seen in the pineal hormone melatonin-clock feedback in mammals (Reppert & Weaver 2002). This is noteworthy as phytohormone signaling and the plant circadian system are generally described as distinct pathways (Blázquez et al. 2002). In this report, we report that a variety of phytohormones regulate distinct rhythmic parameters of the clock. These defined parameters are periodicity, phase, amplitude and clock precision (Supplementary Fig. S1 provides a brief introduction into the nature of these parameters). We further derive a molecular-genetic model for cytokinin modulation of circadian phase. Collectively, we suggest that daily physiologic responses in plants are balanced with multiple feedbacks contributed to in part by the phytohormones. In contrast to the melatonin-circadian connection present in animal systems, plants have a circadian system unexpectedly coordinated by multiple hormonal feedbacks.
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Results |
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We directly investigated whether phytohormones regulate the circadian clock in Arabiodopsis. Classical phytohormones were exogenously added in separate experiments to unravel whether any of these compounds influence circadian rhythms, as assayed via the promoter: luciferase (LUC) system (Eriksson et al. 2003; Hall et al. 2003). Seedlings harboring a promoter:LUC marker were entrained under 12-h light/12-h dark cycles, and then transferred into imaging plates containing growth medium and the test phytohormone. We followed the circadian rhythms of transcription rates from the well-characterized marker genes CHLOROPHYLL A/B-BINDING PROTEIN (CAB2, also termed LHCB1*1) and COLD- AND CIRCADIAN-REGULATED 2 (CCR2, also termed AtGRP7) and the putative core-oscillator gene CCA1. These experiments were typically under free-running constant-light conditions (LL) or in constant darkness (DD) (Fig. 1, Supplementary Figs S2–S5) (Eriksson et al. 2003; Hall et al. 2003). Phytohormone effects on the CCR2 rhythm under free-running constant light conditions (LL) are illustrated in Fig. 1; constant darkness (DD) and the CCA1 and CAB rhythms are shown in supporting information (Fig. S2–S5).
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Cytokinin has a defined role in regulating developmental events in plant growth. We tested whether cytokinins modulate clock parameters (Fig. 1A,B; Supplementary Fig. S2). In our observations, the cytokinins 6-benylaminopurine (BA), and trans-zeatin delayed by 0.8–3 h the circadian phase of marker expressions both under LL and in DD (Fig. 1A,B; Supplementary Fig. S2A–F). The effects were more subtle under LL than that seen in DD (e.g. for CCR2 rhythm with t-zeatin, 01.54 h in DD vs 00.86 h under LL). Cytokinin shifted both the phase of the first peak of CCR2 and CAB2 rhythms and the second peak of CCA1 in DD (Supplementary Fig. S2B,D,E). The amplitude of gene expression was also altered. Strikingly, overt CAB2 rhythms were observed in plants in DD that had been exposed to exogenous cytokinin, whereas the CAB2 rhythm in wild-type plants normally damps in DD; R.A.E = 0.36 ± 0.05 with t-zeatin treatment, whereas R.A.E = 0.64 ± 0.03 without this cytokinin. The variance on periodicity of CAB2 in DD was significantly smaller with cytokinin treatment than without it, as demonstrated by F statistic (P < 0.001) (Fig. 5A,B; Supplementary Fig. S2F) (Millar et al. 1995). Cytokinin also had subtle effects on periodicity of CCR2 in DD (CCR2 in DD: control = 27.44 ± 00.09 h; t-zeatin = 26.77 ± 00.18 h) (Fig. 2). Complicated dose–responses of periodicity in response to cytokinin application were observed (Supplementary Fig. S6). Collectively, cytokinin can have tri-functional effects on the circadian system: to shorten periodicity, to delay phase and to support rhythmicity in the dark.
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Brassinosteroids (BRs) are known to regulate a diverse array of photomorphogenic behaviors. We investigated whether BRs affect the circadian system (Fig. 1E; Supplementary Fig. S4A–F). As a result of BR homobrassinolide (hBR) application, circadian periodicity was shortened for CCR2, CAB2 and CCA1 (1.0–2.7 h) rhythms under both LL and in DD (Fig. 2). The effects of hBR appeared stronger in DD than under LL (e.g. for CCR2 rhythm with hBR, 01.70 h shortened in DD vs 01.37 h under LL). Furthermore, as was seen with cytokinin application, robust CAB2 rhythms were observed in DD in BR-treated plants (Supplementary Fig. S4E,F); R.A.E = 0.27 ± 0.02 with hBR treatment, but R.A.E = 0.58 ± 0.04 without BR application. The variance on periodicity of CAB2 in DD was significantly lower after BR treatment than without it, as demonstrated by an F statistic (P < 0.001). Thus, BRs play an important role in promoting periodicity.
ABA controls a diverse range of biological processes from seed dormancy to senescence, and often is a responder to various stresses such as drought, salinity, cold and biotic stress. We therefore wondered if ABA would also integrate external signals to the circadian system (Fig. 1F; Supplementary Fig. S5). With regard to the clock, ABA application lengthened circadian periodicity under LL (e.g. the CCR2 rhythm was a 2.2 h longer period than the control) (Fig. 2). Though, the ABA effect on periodicity was not significant in DD; P = 0.03 and 0.1 for CCR2 and CCA1 in DD, respectively (Fig. 2). Thus, ABA lengthens the circadian periodicity, and the effect appears to be light dependent.
Gibberellins (GA) and ethylene are often known as a component of light signals. We therefore test applied GA and ethylene to the circadian system. Gibberellin A4 (GA4) did not noticeably affect CCR2 expression rhythm; however, GA4 shortened the period length and increased the amplitude of CAB and CCA1 rhythms (Fig. 2). The effect of GA4 is subtle and depends on the output measured. When we applied the ethylene-synthesis precursor, 1-aminocyclopropane-1-carboxylicacid (ACC) and looked for responses on the circadian system, we found modestly shortened periodicity (0.9 h) of CCR2 rhythm, but only in DD (Fig. 2). Furthermore, we found no significant effects on the other markers measured. Thus, GA and ethylene had subtle effects on the circadian system, but the effects depend on the output measured.
The circadian system influences photoperiodic flowering induction (Hayama & Coupland 2004). Recently, the phytohormones salicylic acid (SA) and nitric oxide (NO) were reported to modulate this transition from vegetate stage to reproduction (Martínez et al. 2004; He et al. 2004). This knowledge led us to test whether these compounds also modulate circadian parameters. We tested circadian effects in response to treatment with either SA or sodium nitroprusside (SNP), a donor for NO. These compounds in our experiments did not significantly influence circadian parameters (Fig. 2). We conclude that SA and NO are not phytohormones in the tuning of the circadian system.
Auxin affects circadian precision, as described above. Further, auxin is structurally similar to the pineal hormone melatonin, as both are indolamines (Kolár & Machácková 2005). To exclude the possibility that auxin structurally mimics a melatonin-like reaction, we also tested the consequence of melatonin addition on the plant-circadian system. Here we found no significant plant responses for any circadian parameters (data not shown). This confirms other's findings that melatonin is not a circadian-acting hormone in plants (Kolár & Machácková 2005).
In summary, the phytohormones described here pharmacologically affect the circadian system in distinct ways and change the rhythmic expression of marker genes, including one of the core oscillator genes CCA1.
Many phytohormone mutants exhibit aberrant clock phenotype
To confirm the above hormone pharmacology, we assayed rhythmic expression in hormone synthesis and perception mutants that harbor an introduced CCR2:LUC reporter; tested lines included cytokinin hypersensitive (ckh1), constitutive and photomorphogenesis and dwarfism (cpd), and ABA-deficient 2 (aba2) (Fig. 3) (Kubo & Kakimoto 2000; Bishop & Koncz 2002; Fedoroff 2002). The ckh1 mutant exhibited a short-period phenotype in DD (Fig. 3A). The cpd mutation displayed a 3 h long-period phenotype under LL (Fig. 3A). The aba2 mutant exhibited the predicted shortening-period phenotype in DD (01.55 h in DD) (Fig. 3B).
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Collectively, we found that many of the hormone mutants tested exhibited predictable clock phenotypes. A variety of ethylene mutants also exhibited conditional phenotypes in a light-dependent manner, and these phenotypes simulated cytokinin changes. We confirmed the pharmacology with these genetic data that phytohormone signals indeed modulate various parameters of the circadian clock.
Integration of a hormone cytokinin to the clock
To genetically define a mechanism for one of the hormone-clock integrations, we focused on the cytokinin signal. In plants, light inputs shorten period length and mutations in photoreceptors alter circadian rhythms (Millar et al. 1995; Somers et al. 1998; Salomé
et al. 2002). Recently, the cytokinin-activated ARABIDOPSIS RESPONSE REGULATOR 4 (ARR4) has been reported to modulate phytochrome B (phyB)-mediated red-light signaling (Sweere et al. 2001; To et al. 2004) (Fig. 7B). The phyB mutant has a short-period phenotype under LL and in DD, a long-period phenotype under continuous red (RR), and an out of phase phenotype with the rhythms of leaf movement (Somers et al. 1998; Salomé
et al. 2002). Therefore, if ARR4 and phyB contribute to the hormonal integration within the circadian system, the clock sensitivity to cytokinin should be altered in ARR4 over-producing plants (ARR4-ox) and in phyB mutants. To test our hypothesis, we generated ARR4-ox lines, harboring the CCR2:LUC transgene and assayed cytokinin responses of clock parameters under constant red light (RR) (Fig. 4, Supplementary Fig. S7). Before cytokinin application, we found no strong differences between control and ARR4-ox lines with regard to phase positions. After cytokinin application, the ARR4-ox plants exhibited an 
4 h delayed-peak phenotype under RR, whereas wild-type delayed the peak by only 2 h (Fig. 4C). Thus, ARR4-ox lines are hypersensitive to a cytokinin input towards circadian phase. We also investigated cytokinin effects on CCR2 and CAB rhythms in the phyB mutant (Figs 4 and 5). No significant change was detected in the peak position of the CCR2 rhythm in the phyB mutant after cytokinin application (Fig. 4). Furthermore, phyB failed to recover clock precision of the CAB rhythm in DD after cytokinin application (Fig. 5A,C); R.A.E. = 0.36 ± 0.05 in control with t-zeatin treatment, whereas R.A.E. = 0.67 ± 0.05 in phyB with cytokinin treatment: the variance of period in phyB was not significantly different upon cytokinin treatment, as demonstrated by F statistic (P = 0.37; P < 0.001 in wild-type). Thus, the phyB mutant is resistant to cytokinin inputs. Furthermore, we assayed CCR2 rhythms in plants altered for both ARR4-ox and phyB. This double "mutant" had a phenotype similar to the phyB single mutant (Fig. 4; Supplementary Fig. S7). Mathematical analyses confirmed that, in addition to peak-position changes, the CT phase delay was cytokinin dependent: the CT phase delays were 01.43 h in control, 02.35 h in ARR4-ox, and were not significant in phyB or ARR4-ox phyB. phyB is thus epistatic to ARR4-mediated cytokinin input to circadian phase. With regard to the phase of circadian rhythm, it is clear that ARR4-ox exhibited a cytokinin hypersensitive phenotype and the phyB mutation eliminated this sensitivity.
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Discussion |
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In several animal systems, classes of hormones that are rhythmically secreted from the suprachiasmatic nuclei (SCN) affect the circadian system to organize their oscillations with the various cells (Reppert & Weaver 2002). However, in contrast to the SCN seen in animal systems, there is no evidence for such a pacemaker or a master clock tissue in plants. Further, the rhythms of one plant organ have been reported to be independent from others, suggesting that the plant circadian clock is coupled more weakly than that of animal systems (Thain et al. 2000, 2002). These "uncoupled" clocks could be coupled through hormonal mediation, as phytohormones are trafficked molecules throughout the plant. This could suggest that hormone-clock connections function not only to synchronize oscillators in different tissues, but also functions to integrate external signals ensuring robust maintenance of the clock thought the whole organism. Remarkably, phytohormones can influence each other, and sometimes antagonize each other's function in plant development (Bishop & Koncz 2002; Fedoroff 2002; Srivastava 2002; Guo & Ecker 2004; Martínez et al. 2004). These actions are differential and cannot solely be via light signaling.
In our observation, cytokinin mediates circadian parameters via light signaling through ARR4 and phyB. Based on our results, we propose a genetic mechanism that describes the cytokinin-input to the clock (Fig. 7B): (i) cytokinin activates ARR4 (and perhaps, other response regulators), (ii) ARR4 alters phyB Pfr-stability, which is required for phyB-mediated light signaling (Sweere et al. 2001) and (iii) activated phyB changes the gene expression of the core-clock genes in a phase-dependent manner. A further effect of cytokinin on periodicity and phase responses were recently described (Salomé et al. 2006). In our observations, cytokinin alters phase more dominantly over periodicity. Perhaps, the high concentration of cytokinin used in the Salomé et al. (2006) report continuously delayed phase, and this manifested as lengthened-periodicity. This is exactly what we found in our detailed dose–response analysis (Supplementary Fig. S6). Similar complex cytokinin responses have been genetically observed (Smalle et al. 2002). Furthermore, the arr4 loss-of-function mutant was described as a long-period mutant only in the context of an arr3 mutation; however, over-expression of ARRs did not affect circadian rhythms in the previous report (Salomé et al. 2006). In our experiments, consistent with the report by Salomé et al. (2006), we found no strong changes of circadian parameters in ARR4-ox lines that were not treated with cytokinin, ARR4-ox plants exhibit delayed-phase phenotype only upon cytokinin treatment. A percieved inconsistency between the phenotype of the arr4 loss-of-function mutant and the over-expression lines has also been reported in light-related growth (Sweere et al. 2001; To et al. 2004). Moreover, we noticed a natural-variation difference of ecotypes used between our study and that of Salomé et al. (2006). In particular, the Col-0 ecotype used by Salomé et al. (2006) has an atypical response to the Ws ecotype used in our study (data not shown) These natural-variation differences are currently not understood. Additionally, we also report cytokinin-induced expression of the clock genes CCA1, GI and TOC1. It has been reported that other pseudoresponse regulator genes, PRR9 and PRR5, were also regulated by the hormone cytokinin (Brenner et al. 2005). These genes also play roles for circadian system and red light response (Eriksson et al. 2003; Mizuno 2004).
Auxin application eventually breaks clock precision under LL. To our knowledge, this is the first finding of phytohormone that disrupts circadian oscillation. Similar effects on clock precision have been observed in the circadian-gating mutants elf3 and elf4 (McWatters et al. 2000; Doyle et al. 2002). Auxin thus phenocopies the lack of precise and circadian maintenance seen in these gating mutants. Interestingly, auxin application in the morning repressed CCA1 gene expression, a molecular phenotype seen in elf3 and elf4. It is plausible that auxin modulates the circadian-gating mechanism.
Here, we showed that BR shortened clock periodicity on CCR2, CCA1, and CAB rhythms, and the BR-defect cpd mutants exhibited lengthened-period phenotype on CCR2 rhythms. Curiously, another BR-deficient mutant det2 was reported to shorten the period of CAB rhythm in DD (Millar et al. 1995). We confirmed this short-period phenotype in cpd harboring the CAB:LUC marker (data not shown). Moreover, though cytokinin antagonizes the BR function in hypocotyl elongation, both phytohormones mimic light signals in distinct but not opposite way with regard to the clock. Our findings imply new interactions among the hormone signaling. Thus, BRs indeed play an important role in clock periodicity, but understanding the detailed functions remains.
In classical studies, ABA application was reported to decrease circadian amplitude and to change phase, but not to alter the circadian periodicity in the leaf movement rhythm in Oxalis regnellii (Skrove et al. 1982). Here, in our precise molecular analysis, we found that ABA clearly lengthens periodicity. In the ABA treatment of leaf movement rhythms in Oxalis regnelli, the decreased amplitude might have masked periodicity changes. Another point of interest is the currently unclear mechanism by which ABA regulates circadian periodicity. Aspects of this mechanism might include the ABA signaling factor ABI3. It was previously shown to bind to the clock component TOC1 (Kurup et al. 2000). It is interesting that ABA reduced CCA1 mRNA. The interaction between ABA signal and ABI3–TOC1 interactions would be worthy of investigation.
Previous studies reported that ethylene does not affect circadian parameters, even though ethylene levels are rhythmic (Thain et al. 2004). Although these studies were clearly confirmed in our experiments, we did find that mutations altering ethylene synthesis and signaling can exhibit aberrant, albeit conditional, clock phenotypes. The phenotype of ethylene signaling mutants could exhibit cytokinin-like phenotypes. We conclude that, although ethylene itself is not a key compound for regulation of the circadian system, ethylene signaling can influence circadian processes.
It is notable that not all phytohormones have strong input responses to the clock. This supports the notion that different hormone-signaling pathways have alternative inputs to circadian parameters. For example, both GA treatments and an analysis of ga1 mutation did not reveal a strong effect of GA on the circadian system. This is in contrast with the genetic work on the SPY locus, a negative regulator of GA signaling. The spy mutant was previously shown to have a lengthen-periodicity phenotype (Tseng et al. 2004). These differences highlight overlapping and convergent phytohormone signals on the circadian system, as seen in the cytokinin-ethylene connection (Hass et al. 2004). GA signaling may have more than two pathways, and some of these pathways may mask the SPY effect. Alternatively, SPY might be a pleiotropic clock regulator and its effect on the clock is independent of GA action. With regard to hormones without an apparent clock-response, both SA and NO belong to this category. This is of interest, as these hormones regulate flowering time in Arabidopsis (He et al. 2004; Martínez et al. 2004). Perhaps, the SA and NO effects on flowering time are independent of the circadian system. In addition, application of the mammalian clock-related hormone melatonin caused no significant changes in circadian parameters. Plants thus integrate external hormonal signals to the clock in a different fashion (and use different hormones).
Unexpectedly, we found that many phytohormones control various aspects of the plant circadian system. The clock system is doubtless balanced amongst these hormones, whose levels change in response to altering environmental conditions (Srivastava 2002). Furthermore, in addition to the pseudo-response regulator TOC1/PRR family (Mizuno 2004), an authentic response regulator(s) is also an element in the circadian system. The two-component system of plants might be one of the integrators that connects environmental signals to the circadian clock. Collectively, when compared to the clock systems of cyanobacteria, fungi and animals, plants appear to have a flexible clock system(s), fine-tuned by multiple hormonal feedbacks. This is an assistant of clock flexibility to coordinate plant development and metabolism with ever changing environmental conditions. It will be of great interest to see how widely these fine-tuning mechanisms and multiple feedbacks are conserved among other organisms.
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Experimental procedures |
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All experiments were carried out in Arabidopsis thaliana ecotype Wassilewskija (Ws), except for the mutant tests and their respective wild-type backgrounds. Most mutant seed was provided by the Arabidopsis Biological Resource Center (ABRC, USA) and the Nottingham Arabidopsis Stock Center (NASC, UK). phyB-464–19 is in the Ws background (Reed et al. 1993), Agrobacterium tumefaciens and transgenic plants harboring promoter; luciferase (LUC) constructs were kindly provided by Prof Andrew Millar (University of Warwick, UK). The luciferase constructs reporting CCR2 rhythms were introduced into various mutants by fertilization. cpd mutants in Ler background were crossed to Ws plants harboring CCR2:LUC reporter genes 3 times. To generate ARR4-ox constructs, the ARR4 coding region was amplified by polymerase chain reaction (PCR), using the primer sequences 5'-GGGG-attB1-CTATGGCCAGAGACGGTGGTG-3' and 5'-GGGG-attB2-CTAATCTAATCCGGGACTCCTCA-3', and this was subcloned into pDONR207 (Invitrogen, Karlsruhe, Germany). The ARR4 coding region in pDONR207 was transferred into the pJAN33 vector, kindly provided by Dr Marc Jacoby and Prof Bernd Weisshaar (MPIZ, Germany). The pJAN33 vector harbors the CaMV 35S promoter to drive in plants high-level expression of the introduced gene. The ARR4-ox construct was introduced into Arabidopsis harboring the CCR2:LUC reporter transgene by the floral-dip method (Clough & Bent 1998). ARR4 transcript elevation was confirmed by RT-PCR analysis of the ARR4 mRNA in these over-expression lines (data not shown). The ARR4-ox plant harboring CCR2:LUC reporter transgene was crossed with the phyB mutant to generate the double "mutant" of this genotype. All chemicals and phytohormones were purchased from Sigma (Germany), except for homobrassinolide (Rose Scientific Ltd. Canada). Most hormones were dissolved into a stock solution using a dimethyl sulfoxide (DMSO) solvent.
Luminescence assays
Prior to measurements, seedlings were entrained at 22 °C under 12 h white light/12 h dark cycles (LD) while growing on Murashige-Skoog (MS) 3% sucrose–1% agar plates (pH 5.7) without phytohormone under cool-white light, 10 µmol m–2 s–1 for 7 days. Each seedling was transferred into imaging microtiter plates (Perkin Elmer, Juegesheim, Germany) containing MS 3% sucrose–1% agar media, pH 5.7, with 20 µM phytohormones or 0.01% DMSO as a control solvent for untreated plants. Such a hormone concentration is within experimental ranges commonly used pharmacologically for a given phytohormone, and is probably saturating, but within a physiologic (non-toxic) range. 0.01% DMSO had no significant effects on any parameter of the circadian clock (data not shown). Auxins were dissolved in dH2O. Afterwards, 5 mM luciferin was added on to the plants, and then the seedlings were entrained to another LD cycle. The luminescence rhythms were monitored using a luminescence scintillation counter, TOPCount NXT (Perkin Elmer), with or without custom constructed red and blue LEDs (2 µmol m–2 s–1) (Southern & Millar 2005). The luminescence rhythms, containing 3–4 cycles, were mathematically analyzed by the Microsoft Excel macro BRASS, as previously described (Southern & Millar 2005). Statistical tests of multiple independent replicates were carried out with one-factor ANOVA followed by Bonferroni multiple t-test.
RNA isolation and reverse transcriptase-PCR
Seedlings grown for 1 week under LD cycles were transferred to MS plates containing 20 µM of the phytohormone indicated in Fig. 6, or 0.01% DMSO as a control, at zeitgeber time (ZT) = 2 or ZT = 10, were incubated for 1 h, and then were harvested at ZT = 3 or ZT = 11. Total RNA was isolated from the seedlings using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The total RNA was treated with DNase I before reverse transcription. Reverse transcription was performed on 1.0 µg of total RNA with SuperscriptII (Invitrogen). Quantitative PCR were performed with iQ5 real-time PCR system (BIO-LAD). Gene-specific primers were previously described: CCA1 and TOC1 (Hall et al. 2003), GI (Mizoguchi et al. 2005), and UBQ10 (Blázquez & Weigel 1999).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: davis{at}mpiz-koeln.mpg.de
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References |
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0.6), whereas for this circadian parameter, most of the phyB mutants were out of the range of detection (90% 
0.6, 70% > 1.0). This indicated that phyB was arrhythmic in DD, with or without cytokinin.
etr1; 
ein4; 
ein5. The error bars indicate the S.E.M. *P < 0.005. Note that period phenotype affects the CT phase. Thus, the phase phenotypes of ein4 and ein5 under LL and of etr1 in DD (P < 0.05) represented the period changes.
