Light and circadian regulation of clock components aids flexible responses to environmental signals

Abstract

The circadian clock measures time across a 24 h period, increasing fitness by phasing biological processes to the most appropriate time of day. The interlocking feedback loop mechanism of the clock is conserved across species; however, the number of loops varies. Mathematical and computational analyses have suggested that loop complexity affects the overall flexibility of the oscillator, including its responses to entrainment signals. We used a discriminating experimental assay, at the transition between different photoperiods, in order to test this proposal in a minimal circadian network (in Ostreococcus tauri) and a more complex network (in Arabidopsis thaliana). Transcriptional and translational reporters in O. tauri primarily tracked dawn or dusk, whereas in A. thaliana, a wider range of responses were observed, consistent with its more flexible clock. Model analysis supported the requirement for this diversity of responses among the components of the more complex network. However, these and earlier data showed that the O. tauri network retains surprising flexibility, despite its simple circuit. We found that models constructed from experimental data can show flexibility either from multiple loops and/or from multiple light inputs. Our results suggest that O. tauri has adopted the latter strategy, possibly as a consequence of genomic reduction.

Keywords: biological clocks; flexibility; marine algae; mathematical analysis; nonlinear dynamics; photoperiod; systems biology.

Fig 1

Ostreococcus tauri core circadian markers respond quickly to photoperiod transitions. O. tauri cells expressing constructs of CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) or TIMING OF CAB 1 (TOC1) tagged with the marker gene LUCIFERASE (LUC) were entrained under 8 : 16 h, light : dark cycles (short days, SD) and imaged for 2 d: (a) CCA1::CCA1::LUC; (b) TOC1::TOC1::LUC. At 48 h, photoperiod conditions were switched to 16 : 8 h, light : dark cycles (long days, LD). All traces are normalized to the average of each time series and then averaged across eight replicate cultures. Red arrows highlight acute responses to light; black arrows denote circadian peaks. Interpeak differences between circadian peaks were calculated for SD (open squares), LD (open triangles) and SD to LD (closed diamonds) photoperiod conditions, for CCA1::CCA1::LUC (c) and TOC1::TOC1::LUC (d). White (day) and grey (night) shading represent the photoperiods in (a) and (b), respectively.

Fig 2

Modelling the effects of individual light inputs on Ostreococcus tauri phase in perturbed light conditions. The system was entrained in 12 : 12 h, light : dark cycles and released into continuous light (LL) after a single night of varying length (Skotoperiod; shaded area). Peaks (bright lines) and troughs (dark lines) are shown as functions of time and the timing of dawn. (a) The original model (blue), also included in all panels. (b–e) The effects of removing light inputs by fixing one light-dependent parameter to the value it would normally hold in the light (red) or dark (green): (b) CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) protein degradation rate (D_c,l and D_c,d in the equations); (c) activation of CCA1 transcription by (TIMING OF CAB 1) TOC1 (R_c,l/R_c,d); (d) TOC1 protein degradation rate (D_t,l/D_t,d); (e) activation rate of TOC1 protein (K_t,l/K_t,d). (f) Removing the ‘light accumulator’ effect on TOC1 transcription, so TOC1 is expressed as if in constant light (grey) or modulated directly by light without an accumulator (purple). The numbers above the panels show the impact of each light input on the overall model fit, expressed as the change in the cost function after model alteration and local parameter refitting, relative to the lowest cost obtained with all or no light inputs. The first number is the increase in cost if the light input is removed from the full model, and the second is the decrease in cost if the light input is added to the model with no light inputs.

Fig 3

Arabidopsis thaliana's response to the short day (SD) to long day (LD) transition. A. thaliana plants containing luciferase (LUC)-tagged clock gene constructs were entrained under white light for 6 d in SD (8 : 16 h blue and red light : dark cycles), and following 2 d of imaging conditions were switched to LD (16 : 8 h blue and red light : dark cycles) and imaged for a further 4 d (open squares). Controls were entrained and imaged under LD (closed triangles). Error bars on luminescence plots denote ± SE of the mean (n = 12–16). Interpeak differences were calculated from circadian peaks (black arrows); errors represent average interpeak difference from two biologically independent experiments; SD, open squares; LD, open triangles; SD to LD, closed diamonds. (a) CIRCADIAN CLOCK ASSOCIATED 1, CCA1::LUC; (b) CCA1::LUC interpeak difference; (c) TIMING OF CAB1, TOC1::LUC; (d) TOC1::LUC interpeak difference; (e) GIGANTEA, GI::LUC; (f) GI::LUC interpeak difference. (g) Individual day traces for GI::LUC: day 1, black squares; day 2, black triangles; day 3, red crosses; day 4, red dashes; day 5, white circles; day 6, white diamonds; day 7, white squares; day 8, white triangles. ZT, Zeitgeber Time (hours since lights-on).

Fig 4

Variations in clock flexibility with feedback loops and light inputs. Circles denote the flexibility dimension d of a model in 12 : 12 h, light : dark cycles, while triangles denote the flexibility under constant light conditions (continuous light for Arabidopsis and Ostreococcus; continuous dark for Neurospora). (a) Flexibility increases with loop number under constant conditions. In each case, the computed value of d is higher in light : dark cycles than in constant light conditions, showing that incorporating light entrainment yields a more flexible circuit. In particular, the single-loop O. tauri circuit (T2011) exhibits a significant increase in flexibility compared with the single-loop A. thaliana circuit (L2005A). (b) Plotting flexibility against the sum total of loops and light inputs for each model reveals a positive trend, implying that light inputs augment clock flexibility in a similar manner to feedback loops. (Note that in both plots, points with the same x-axis value have been slightly offset for clarity.) For all models, d is defined as the number of significant singular values of the matrix M* that maps parameter perturbations to perturbations of the corresponding free-running or entrained limit cycle (the singular value spectra {σ_k} of the entrained models are shown in Fig. S7).

Fig 5

Dusk sensitivities measure phase flexibility. Dusk sensitivities for the peak (upward triangle) and trough (downward triangle) times of mRNA (m) and bulk protein (P) variables of all genes in four of the most flexible models. These were calculated from simulations of the model in 12 : 12 h, light : dark cycles in each case. A dusk sensitivity close to 1 indicates dusk-dominant entrainment; one close to 0 indicates dawn-dominant entrainment. Intermediate values denote flexible entrainment, in which both dawn and dusk information is integrated. Where an expression profile has multiple peaks or troughs, dusk sensitivities are plotted from left to right, in chronological order after dawn, with the convention that in each peak/trough pair, the trough follows the peak in time. While the ArabidopsisL2005B circuit exhibits only dawn or dusk locking, the more flexible OstreococcusT2011 and Arabidopsis L2006 models have several components with intermediate light responses (e.g. the trough of TOC1 mRNA which tracks the middle of the night in both models). The most flexible circuit, ArabidopsisP2011, exhibits the broadest spectrum of dusk sensitivities. The positive correlation between network and phase flexibility is quantified further in Fig. S6.