Coordination of robust single cell rhythms in the Arabidopsis circadian clock via spatial waves of gene expression
- University of Liverpool, United Kingdom
- University of Cambridge, United Kingdom
- Ritsumeikan University, Japan
- Hungarian Academy of Sciences, Hungary
- University of Szeged, Hungary
- Norwich Research Park, United Kingdom
- Microsoft Research, United Kingdom
Figures
Figure 1 with 6 supplements
Quantitative time-lapse microscopy reveals single cell clock dynamics across the plant.
(a), Current models of the clock (Pokhilko et al., 2012) predict undamped oscillations. (b), CCA1:LUC expression bulk averaged over multiple seedlings shows damping clock oscillations (mean ± s.e.m.;…
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Figure 1—source data 1
The percentage of rhythmic cells for WT experiment.
Columns 2–4 identify rhythmic cells using three different methods described in BioDare. Column two uses FFT-NLLS (Fast Fourier Transform Non Linear Least Squares), with Goodness of Fit (GOF) parameter of 0.9. Column three uses Spectrum Resampling (SR) with GOF of 1 and Column four uses mFourFit with GOF of 1. See Materials and methods for details. Column five shows percentage of cell traces that were identified as rhythmic by all three methods and where periods from each method were within 2.5 hr of each other (as described in the Materials and methods). These data were taken forward for further analysis.
- https://doi.org/10.7554/eLife.31700.010
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Figure 1—source data 2
The percentage of rhythmic cells for repeat WT experiment.
Columns 2–4 identify rhythmic cells using three different methods described in BioDare. Column two uses FFT-NLLS (Fast Fourier Transform Non Linear Least Squares), with Goodness of Fit (GOF) parameter of 0.9. Column three uses Spectrum Resampling (SR) with GOF of 1 and Column four uses mFourFit with GOF of 1. See Materials and methods for details. Column five shows percentage of cell traces that were identified as rhythmic by all three methods and where periods from each method were within 2.5 hr of each other (as described in the Materials and methods). These data were taken forward for further analysis.
- https://doi.org/10.7554/eLife.31700.011
Figure 1—figure supplement 1
The CCA1-YFP protein is functional and rescues the cca1-11 mutation.
(a) The emission spectra from 450 to 650 nm for the CCA1-YFP in each tissue is consistent with the known YFP emission spectrum. The black line represents the root tip, green the root, grey the …
Figure 1—figure supplement 2
Confocal imaging and processing pipeline.
Blank images at the end of a time-lapse run are removed in ImageJ (NIH). Data are then split into brightfield, RFP and YFP wavelengths and unwanted wavelengths are cropped to reduce dimensionality. …
Figure 1—figure supplement 3
Mean and single cell oscillations of CCA1 nuclear localisation for repeat WT experiments.
(a), Mean traces of single cell CCA1-YFP for WT repeat seedling; root tip (grey), root (black), hypocotyl (blue) and cotyledon (red). (b), All individual cell traces in each tissue type from (a). (c,…
Figure 1—figure supplement 4
Mean and single cell oscillations of CCA1 nuclear localisation for CCA1-long experiments.
(a), Mean traces of single cell CCA1-YFP for CCA1-long seedling; root tip (grey), root (black), root/hypocotyl section (cyan), hypocotyl (blue) and cotyledon (red) (b), Individual cell traces in …
Figure 1—figure supplement 5
Individual cell oscillations of CCA1-YFP reveal tissue specificity in robustness of oscillations.
50 cells from the root tip (top left), root (bottom left), hypocotyl (top right), and cotyledon (bottom right) were randomly selected and plotted. Individual traces are background subtracted and …
Figure 1—figure supplement 6
Single cell rhythms have stable amplitudes across the plant.
(a), Amplitude of single cell rhythms for first oscillation after 72 hr (in constant light) compared to amplitude of single cell oscillations for last oscillation before end of movie (165 hr), for …
Figure 2 with 3 supplements
Single cell analysis reveals tissue level differences in robustness of the clock.
(a), Rhythmic cell amplitudes in the imaged sections normalised to the amplitude of the mean trace (green line). Whiskers represent 9th and 91st percentile. (b), Between-cell and within-cell period …
Figure 2—figure supplement 1
Tissue level differences in robustness synchronisation, and period of single cell clock oscillations in repeat WT experiment.
(a), Rhythmic cell amplitudes per section imaged with amplitude of the mean trace overlaid (green line). Whiskers represent 9th and 91st percentile. (b), Between-cell and within-cell period …
Figure 2—figure supplement 2
Tissue level differences in robustness, synchronisation, and period of single cell clock oscillations in CCA1-long experiment.
(a), Rhythmic cell amplitudes per section imaged with amplitude of the mean trace per section overlaid (green line). Whiskers represent 9th and 91st percentile. (b), Between-cell and within-cell …
Figure 2—figure supplement 3
Periods display no spatial structure in z direction for WT repeat and CCA1-long experiments.
(a–b), Scatterplot in the y-z direction of rhythmic cells for repeat WT experiment in all imaged plant sections (a) or in the root and root tip sections only (b). (c), Scatterplot in the y-z …
Figure 3 with 4 supplements
Single cell period differences and cell-to-cell coupling generate spatial waves of clock gene expression.
(a), Scatterplot of the rhythmic cells in all imaged plant sections stitched together in x-y direction. Colour indicates the oscillation period. (b), Period values vs. longitudinal position on the …
Figure 3—figure supplement 1
Spatial waves of clock gene expression are seen in clock luciferase reporter lines.
(a), Representative space-time plot of normalised CCA1:LUC (N = 2, n = 10) expression across longitudinal sections of the root. Seedlings were imaged under constant red and blue light. (b), …
Figure 3—figure supplement 2
Synchronisation analysis based on the order parameter.
(a–g), Time evolution of the order parameter R (Kuramoto, 1984) is plotted for the root tip (a), lower root (b), upper root (c), lower hypocotyl (d), upper hypocotyl (e), cotyledon (f). (g), Order …
Figure 3—figure supplement 3
Synchronisation analysis based on the synchronisation index.
(a–f), Time evolution of the synchronisation index (Garcia-Ojalvo et al., 2004) is plotted for the root tip (a), lower root (b), upper root (c), lower hypocotyl (d), upper hypocotyl (e), cotyledon (f…
Figure 3—figure supplement 4
Synchronisation analysis applied to CCA1-long dataset based on the order parameter.
(a–g), Time evolution of the order parameter R is plotted for the root tip (a), root (sect 1) (b), root (sect 2) (c), root/hypocotyl (d), hypocotyl (sect 1) (e), hypocotyl (sect 2) (f), cotyledon (g)…
Videos
Video 1
Peaks of CCA1-YFP expression in the lower hypocotyl and root region.
Video of CCA1-YFP raw (left panel) and normalised (right panel) expression in rhythmic cells imaged from 29 h (root section) or 29.5 h (lower hypocotyl) in LL. Both colour intensity and size of spot …
Video 2
Peaks of CCA1-YFP expression in the cotyledon region.
Video of CCA1-YFP raw (left panel) and normalised (right panel) expression in rhythmic cells imaged from in LL. Both colour intensity and size of spot indicate expression level. Frame number is …
Video 3
Peaks of CCA1-YFP expression in the upper hypocotyl region.
Video of CCA1-YFP raw (left panel) and normalised (right panel) expression in rhythmic cells imaged from 29 h in LL. Both colour intensity and size of spot indicate expression level. Frame number is …
Video 4
Waves of PRR9:LUC expression in the lower hypocotyl and root.
Video of PRR9:LUC luminescence in the hypocotyl and root of a single seedling from 8–144 h after transfer to constant light. Frame intervals are 1.5 h and scale bar shows 0.5 mm.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| gene (Arabidopsis thaliana) | CCA1 | PMID: 9657153 | TAIR:AT2G46830 | |
| gene (Arabidopsis thaliana) | PRR9 | PMID: 14634162 | TAIR:AT2G46790 | |
| gene (Arabidopsis thaliana) | GI | PMID: 17102804 | TAIR:AT1G22770 | |
| strain, strain background (Arabidopsis thaliana) | cca1-11 | PMID: 14555691 | TAIR:1008081946 | |
| strain, strain background (Agrobacterium tumefaciens) | GV3101 | doi:10.1007/BF00331014 | ||
| transfected construct | CCA1::CCA1-YFP | This paper | Ask for ‘CCY_pPB’ | promoter::protein construct |
| transfected construct | 35S::H2B-RFP | PMID: 22466793 | promoter::protein construct | |
| biological sample (Arabidopsis thaliana) | CCA1::LUC | PMID: 20530216 | Ask for ‘B8-5’ | promoter::luciferase construct; Col-0 background |
| biological sample (Arabidopsis thaliana) | GI::LUC | PMID: 20530216 | Ask for ‘A2-1-4’ | promoter::luciferase construct; Col-0 background |
| biological sample (Arabidopsis thaliana) | PRR9::LUC | PMID: 20530216 | Ask for ‘G8-5’ | promoter:Luciferase construct; Col-0 background |
| biological sample (Arabidopsis thaliana) | CCA1-YFP WT (Ws) | This paper | Ask for ‘1–1’ | CCA1::CCA1-YFP; 35S::H2B-RFP, WT clock period |
| biological sample (Arabidopsis thaliana) | CCA1-YFP long (Ws) | This paper | Ask for ‘3–1’ | CCA1::CCA1-YFP; 35S::H2B-RFP, long clock period |
| Recombinant DNA reagent | pPCV812 | PMID: 18980642 | ||
| sequence-based reagent | CCA1_CDS_Fwd | Sigma-Aldrich | 5’-AAAGGATCCATGGAGACAAATTCGTCTGGA-3’ | |
| sequence-based reagent | CCA1_CDS_Rev | Sigma-Aldrich | 5’-ATACCCGGGTGTGGAAGCTTGAGTTTCCAA-3’ | |
| sequence-based reagent | CCA1_prom_Fwd | Sigma-Aldrich | 5’-AAAGAATTCATTTAGTCTTCTACCCTTCATGC-3’ | |
| sequence-based reagent | CCA1_prom_Rev | Sigma-Aldrich | 5’-ATAGGATCCCACTAAGCTCCTCTACACAACTTC-3’ | |
| software | Imaris | BitPlane, Switzerland | version 7.0 | |
| software | ImageJ | National Institutes of Health, U.S.A. | public domain | |
| software | MATLAB | MathWorks, U.K. | version 2015b | |
| algorithm | MATLAB code | This paper | https://gitlab.com/slcu/teamJL/Gould_etal_2018 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.31700.025
