Photosynthetic entrainment of the Arabidopsis thaliana circadian clock

Abstract

Circadian clocks provide a competitive advantage in an environment that is heavily influenced by the rotation of the Earth1,2, by driving daily rhythms in behaviour, physiology and metabolism in bacteria, fungi, plants and animals3,4. Circadian clocks comprise transcription–translation feedback loops, which are entrained by environmental signals such as light and temperature to adjust the phase of rhythms to match the local environment3. The production of sugars by photosynthesis is a key metabolic output of the circadian clock in plants2,5. Here we show that these rhythmic, endogenous sugar signals can entrain circadian rhythms in Arabidopsis thaliana by regulating the gene expression of circadian clock components early in the photoperiod, thus defining a ‘metabolic dawn’. By inhibiting photosynthesis, we demonstrate that endogenous oscillations in sugar levels provide metabolic feedback to the circadian oscillator through the morning-expressed gene PSEUDO-RESPONSE REGULATOR 7 (PRR7), and we identify that prr7 mutants are insensitive to the effects of sucrose on the circadian period. Thus, photosynthesis has a marked effect on the entrainment and maintenance of robust circadian rhythms in A. thaliana, demonstrating that metabolism has a crucial role in regulation of the circadian clock.

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Figure 1: Photosynthetically derived sugars influence the circadian clock in A. thaliana.
Figure 2: Metabolically active sugar is a zeitgeber that acts differently from light.
Figure 3: Photosynthetically derived sugar represses PRR7 late in the photoperiod.
Figure 4: PRR7 contributes to circadian sugar signalling.

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Acknowledgements

This work was supported by BBSRC grant BB/H006826/1. We thank J. O’Neill, J. Davies and J. Hibberd for comments on the manuscript.

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M.J.H. and A.A.R.W. designed the research. M.J.H., O.M., F.C.R. and K.E.H. performed the experiments and analysed the data. M.J.H. and A.A.R.W. prepared the manuscript.

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Correspondence to Alex A. R. Webb.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 A model for entrainment of the A. thaliana circadian clock by photosynthetically derived sugars.

From dawn, light activates PRR7 and drives photosynthesis. The concentrations of simple sugars produced by photosynthesis accumulate within the plant during the day (red dashed line), peaking around 4–8 h after dawn. High endogenous sugar concentrations lead to suppression of the PRR7 promoter, contributing to the phase of PRR7 rhythms. PRR7 is a transcriptional repressor of the circadian clock component CCA1. Thus, the rhythms of endogenous sugars derived from photosynthesis contribute to circadian entrainment through PRR7. We propose that the timing of these events represents a ‘metabolic dawn’. Dawn is a time-dependent gradient of light intensity, whereas ‘metabolic dawn’ represents a gradient of increasing metabolite concentration. The metabolic gradient lags behind that of light and contributes to the setting of the circadian clock. In the model, previously established relationships are shown by black connectors, and novel relationships proposed in this study are shown by orange connectors.

Extended Data Figure 2 Effects of exogenous sucrose and photosynthesis inhibition on circadian rhythms.

a, b, Period estimates for the rhythms of the promoter:LUC reporters in continuous low light (a) or continuous light (b) in plants grown in media with or without sucrose (mean ± s.d.; n = 4). c, d, Promoter:LUC reporter rhythms (mean ± s.e.m.) and relative amplitude error versus period plots for seedlings in media in the presence or absence of DCMU in continuous low light (c) or continuous light (d) (n = 4). * P < 0.05; ** P < 0.01; *** P < 0.001; compared with untreated plants by two-tailed Student’s t-test. n refers to number of biological replicates.

Extended Data Figure 3 The rhythms of endogenous sugars peak in the morning and are reduced by inhibition of photosynthesis.

a, Leaf sucrose and glucose concentrations in 10-day-old seedlings growing in 12 h light and 12 h dark cycles (mean ± s.d.; n = 3). b, Glucose, fructose and sucrose concentrations 4 h after subjective dawn in 13-day-old seedlings grown in CO2-free air or ambient air in continuous low light for 5 days (mean ± s.d.; n = 3). c, Glucose concentration in 10-day-old seedlings growing in a light–dark cycle at 12–36 h after transfer to DCMU or control media at dusk (mean ± s.d.; n = 3). * P < 0.05; ** P < 0.01 by two-tailed Student’s t-test compared with ZT0 in a and compared with control conditions in b and c. n refers to number of biological replicates.

Extended Data Figure 4 Effects of DCMU, norflurazon or lincomycin on CCA1:LUC rhythms in the presence or absence of exogenous sucrose.

a, CCA1:LUC rhythms in continuous low light for seedlings transferred to media containing DCMU in the presence of the indicated exogenous sucrose concentrations compared with control media (mean ± s.e.m.; n = 4). b, CCA1:LUC rhythms in continuous light for seedlings transferred to media containing DCMU, norflurazon or lincomycin in the absence (left) or presence (right) of exogenous sucrose (mean ± s.e.m.; n = 4). n refers to number of biological replicates.

Extended Data Figure 5 Altering reactive oxygen species (ROS) production does not influence circadian rhythms.

a, CAB2:LUC rhythms in seedlings transferred to continuous light and treated with 1 mM glutathione or 5 mM ascorbate. The short-period mutant toc1-1 and long-period mutant ztl-1 were included as positive controls (means ± s.d.; n = 2–3). b, Relative amplitude error versus period plot for leaf movement rhythms in wild-type plants and NADPH oxidase rbohD,F mutants45 in continuous light (mean ± s.e.m.). c, Promoter:LUC rhythms and relative amplitude error versus period plots for seedlings grown in continuous light or continuous low light and treated with 10 µM diphenyleneiodonium (DPI) or 0.1% (v/v) dimethylsulphoxide (DMSO) at 0 h (mean ± s.e.m.; n = 4). n refers to number of biological replicates.

Extended Data Figure 6 Metabolically active sugars sustain circadian rhythms in darkness.

a, CCA1:LUC rhythms in continuous dark in seedlings grown in media containing the indicated sugars or control treatments (mean ± s.e.m.; n = 4). b, Promoter:LUC rhythms (mean ± s.e.m.; n = 4) and relative amplitude error versus period plots (n = 4–8) for seedlings grown in continuous dark in media with or without sucrose. Note that rhythms could not be detected in seedlings grown without sucrose for the morning-expressed CCA1:LUC or PRR9:LUC but could be detected for the evening-expressed GI:LUC and TOC1:LUC, despite the small amplitude. n refers to number of biological replicates.

Extended Data Figure 7 Exogenous sugar can set the circadian phase in dark-adapted seedlings.

a, Time to the first circadian peak of promoter:LUC reporters in seedlings treated with sucrose after 72 h (subjective dawn, CT0) or 84 h (subjective dusk, CT12) in continuous dark (mean ± s.d.; n = 4). b, Promoter:LUC rhythms of seedlings after sucrose or mannitol treatment as in a (mean ± s.e.m.; n = 4). c, CCA1 transcript level relative to UBQ10 in seedlings treated with sucrose or mannitol after 72 h in continuous dark (mean ± s.d.; n = 3). ** P < 0.01; *** P < 0.001 by two-tailed Student’s t-test. n refers to number of biological replicates.

Extended Data Figure 8 Phase setting by sugar and light.

a, Change in the period of CCA1:LUC after pulses of sucrose compared with control seedlings in continuous low light (mean ± s.d.; n = 8). b, Phase response of TOC1:LUC to pulses of sucrose for seedlings in continuous low light (mean ± s.d.; n = 8). c, Phase response of CCA1:LUC to pulses of mannitol (mean ± s.d.; n = 8). d, LUC reporter rhythms (mean ± s.e.m.), time to the circadian peak (mean ± s.d.) and period estimates (mean ± s.d.) in seedlings grown in continuous darkness for 72 h then transferred to continuous light or continuous low light (n = 4). e, CCA1:LUC rhythms (mean ± s.e.m.) and time to the circadian peak in seedlings following transfer to continuous light or continuous low light in control media, medium containing DCMU, or medium containing DCMU and sucrose after 72 h in continuous dark (n = 4). * P < 0.05; *** P < 0.001 by two-tailed Student’s t-test. n refers to number of biological replicates.

Extended Data Figure 9 Regulation of the circadian clock by sugar requires PRR7.

a, Change in PRR7:LUC luminescence after 3 h treatment with sucrose relative to untreated plants (mean ± s.d.; n = 4). The data were normalized across the time series, and the change relative to untreated plants was plotted. b, Change in CCA1:LUC luminescence in wild-type plants and prr7-11 mutants after 3 h treatment with sucrose relative to untreated plants (mean ± s.d.; n = 8). The data were normalized across the time series, and the change relative to untreated plants of the appropriate genotype was plotted. c, Period estimates of rhythms of delayed fluorescence in wild-type and mutant seedlings in continuous low light in media with or without exogenous sucrose (mean ± s.d.; n = 4). d, Phase response of CCA1:LUC to pulses of sucrose in prr7-11 seedlings in continuous low light (mean ± s.d.; n = 8). Compare this with the sucrose PRC for CCA1:LUC in wild-type seedlings in Fig. 2c. * P < 0.05; ** P < 0.01; *** P < 0.001 by Student’s two-tailed t-test compared with controls in a and c and compared with wild-type plants in b. n refers to number of biological replicates.

Extended Data Figure 10 Effect of exogenous sucrose on circadian period in circadian, sugar-insensitive and light-signalling mutants.

LUC reporter rhythms in circadian, sugar-insensitive and light-signalling mutants in continuous low light in media with or without exogenous sucrose (mean ± s.e.m.; n = 4). The reporter is CCA1:LUC in all lines except for Ws, cca1-11 (CAB2:LUC) and toc1-21 (CCR2:LUC). Period estimates are shown in blue (control) and red (sucrose) for each line (mean ± s.d.; n = 8). n refers to number of biological replicates.

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Haydon, M., Mielczarek, O., Robertson, F. et al. Photosynthetic entrainment of the Arabidopsis thaliana circadian clock. Nature 502, 689–692 (2013). https://doi.org/10.1038/nature12603

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