Research ArticleCELL BIOLOGY

Heterogeneity of cellular circadian clocks in intact plants and its correction under light-dark cycles

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Science Advances  15 Jul 2016:
Vol. 2, no. 7, e1600500
DOI: 10.1126/sciadv.1600500
  • Fig. 1 Cellular rhythms were desynchronized after being released into constant conditions.

    Plants grown under LL were subjected to particle bombardment and subsequently entrained in an LD cycle before starting bioluminescence monitoring. (A) Bioluminescence (top) and bright-field (bottom) images of a colony of L. gibba with four fronds. Scale bar, 2 mm. (B) Mean luminescence (black circles) and luminescence traces (other colored solid lines) of 85 cells in the frond (LDtoLL1, table S1) (left) and 73 cells in the frond (LDtoDD1, table S1) (right). The number of cells peaking at every hour is shown at the top. (C) FFT-NLLS analysis for cellular rhythms under LL (LDtoLL1, days 1 to 16) (left) or DD (LDtoDD1, days 1 to 9) (right). RAEs of rhythm-sustained cells (closed circles) and other cells that did not meet the criteria for circadian rhythms (red crosses) were plotted against estimated FRPs (see Materials and Methods). (D) Temporal changes in SIs during the three experiments. Open and black bars indicate light and dark conditions, respectively.

  • Fig. 2 Asynchronous cellular rhythms under LL.

    Plants grown under LL were subjected to particle bombardment and subsequently precultured under LL for 24 hours before starting bioluminescence monitoring. (A) Mean luminescence (black circles) and luminescence traces (other colored solid lines) of 89 cells in the frond (LLtoLL1, table S1). The number of cells peaking at every hour is shown at the top. (B) Temporal changes of SIs of nine experiments. (C) Relationship between phase differences between cellular rhythms and cell-to-cell distances. For all pairs of cellular rhythms in each experiment (LLtoLL1 to LLtoLL9, table S1), phase differences on day 1.5 and cell-to-cell distances were both calculated. Statistics (mean and quartiles) for the phase differences measured were plotted at every 0.05-mm interval of cell-to-cell distances. Blue symbols at each cell-to-cell distance interval represent 95% confidence limits of the median, assuming that samples in each interval have the same median value. The confidence limits were calculated on the basis of resampling (10,000 repeats) from all samples. (D) Relationship between FRP differences between cellular rhythms and cell-to-cell distances. The FRP of each cellular rhythm during each experiment (LLtoLL1 to LLtoLL9) was estimated by FFT-NLLS and analyzed in the same manner as in (C). (E) Plot of coefficient of variation (CV) of peak-to-peak intervals (PPIs) against the mean of PPIs and their histograms for three experiments (LLtoLL1 to LLtoLL3).

  • Fig. 3 Synchronization of cellular rhythms to LD cycles and the formation of a centrifugal pattern of their locked phases within a frond.

    Plants grown under LL were subjected to particle bombardment and precultured under LL for 24 hours before starting bioluminescence monitoring. (A) Mean luminescence (black circles) and luminescence traces (other colored solid lines) of 90 cells in the frond (LLtoLD1, table S1). The number of cells peaking at every hour is shown at the top. (B) Cell-autonomous entrainment manner of cellular clocks. Time series of three experiments (LLtoLD1 to LLtoLD3, table S1) were sorted into eight groups according to the last TTs before the first dark signal. Each group has a 3-hour window. Means of PTs (red circles) and TTs (black circles) of each group were plotted (bar indicates SD). (C) Correlation between PLPTs under LD and FRPs under LL of three experiments (LLtoLD1 to LLtoLD3). (D) Bioluminescence image (upper left), bright-field image (bottom left), and spatial distribution of PLPTs (right) of the frond (LLtoLD1). PLPT is represented as the mean of PTs during days 5.5 to 8.5. The center of the spatial pattern was estimated by quadratic surface fitting and represented as a white square. Scale bars, 1 mm. (E) Correlation between PLPTs of cellular rhythms under LD and distances of cells from the center (LLtoLD1 to LLtoLD3). Open and black bars indicate light and dark conditions, respectively.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/7/e1600500/DC1

    fig. S1. Outline of the quantitative analysis of cellular circadian rhythms.

    fig. S2. Temporal changes in amplitudes of cellular luminescence rhythms after release into LL or DD from LD.

    fig. S3. Results of FFT-NLLS analysis for cellular rhythms under LL.

    fig. S4. Time evolution of the spatial distribution of cellular circadian phases after release into LL from LD.

    fig. S5. Characteristics of asynchronous cellular rhythms under LL.

    fig. S6. Time evolution of the spatial distribution of cellular circadian phases under LL.

    fig. S7. Relationship between phase differences between cellular rhythms and cell-to-cell distances.

    fig. S8. Population-level dynamics of cellular clocks explained by an ANOVA model.

    fig. S9. Correlation between characteristic parameters for cellular bioluminescence rhythms under LL.

    fig. S10. Cellular rhythms were synchronized to LD cycles within 2 days.

    fig. S11. Cellular clocks respond to the first dark signal in a phase-dependent manner.

    fig. S12. Correlation between FRPs during the first 3 days and during the subsequent 5 days.

    fig. S13. Locked phases in LD were significantly different among cells in a frond.

    fig. S14. Spatial distribution of PLPTs under LD.

    fig. S15. FRPs of cellular clocks showed no clear spatial patterns as PLPTs did.

    fig. S16. Spatial distribution of PLPTs after repeated LD cycles.

    fig. S17. Spatial distribution of TTs of delayed fluorescence rhythms.

    table S1. Summary of the quantitative analysis of cellular luminescence rhythms.

    movie S1. Desynchronization of cellular luminescence rhythms on a frond under LL.

    movie S2. Desynchronization and damping of cellular luminescence rhythms on a frond under DD.

    movie S3. Asynchronous cellular luminescence rhythms on a frond under LL and their synchronization to LD cycles.

    Reference (33)

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Outline of the quantitative analysis of cellular circadian rhythms.
    • fig. S2. Temporal changes in amplitudes of cellular luminescence rhythms after release into LL or DD from LD.
    • fig. S3. Results of FFT-NLLS analysis for cellular rhythms under LL.
    • fig. S4. Time evolution of the spatial distribution of cellular circadian phases after release into LL from LD.
    • fig. S5. Characteristics of asynchronous cellular rhythms under LL.
    • fig. S6. Time evolution of the spatial distribution of cellular circadian phases under LL.
    • fig. S7. Relationship between phase differences between cellular rhythms and cell-to-cell distances.
    • fig. S8. Population-level dynamics of cellular clocks explained by an ANOVA model.
    • fig. S9. Correlation between characteristic parameters for cellular bioluminescence rhythms under LL.
    • fig. S10. Cellular rhythms were synchronized to LD cycles within 2 days.
    • fig. S11. Cellular clocks respond to the first dark signal in a phase-dependent manner.
    • fig. S12. Correlation between FRPs during the first 3 days and during the subsequent 5 days.
    • fig. S13. Locked phases in LD were significantly different among cells in a frond.
    • fig. S14. Spatial distribution of PLPTs under LD.
    • fig. S15. FRPs of cellular clocks showed no clear spatial patterns as PLPTs did.
    • fig. S16. Spatial distribution of PLPTs after repeated LD cycles.
    • fig. S17. Spatial distribution of TTs of delayed fluorescence rhythms.
    • table S1. Summary of the quantitative analysis of cellular luminescence rhythms.
    • Legends for movies S1 to S3
    • Reference (33)

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.mp4 format). Desynchronization of cellular luminescence rhythms on a frond under LL.
    • movie S2 (.mp4 format). Desynchronization and damping of cellular luminescence rhythms on a frond under DD.
    • movie S3 (.mp4 format). Asynchronous cellular luminescence rhythms on a frond under LL and their synchronization to LD cycles.

    Files in this Data Supplement:

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