Experimental validation of a predicted feedback loop in the multi-oscillator clock of Arabidopsis thaliana

Abstract

Our computational model of the circadian clock comprised the feedback loop between LATE ELONGATED HYPOCOTYL (LHY), CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and TIMING OF CAB EXPRESSION 1 (TOC1), and a predicted, interlocking feedback loop involving TOC1 and a hypothetical component Y. Experiments based on model predictions suggested GIGANTEA (GI) as a candidate for Y. We now extend the model to include a recently demonstrated feedback loop between the TOC1 homologues PSEUDO-RESPONSE REGULATOR 7 (PRR7), PRR9 and LHY and CCA1. This three-loop network explains the rhythmic phenotype of toc1 mutant alleles. Model predictions fit closely to new data on the gi;lhy;cca1 mutant, which confirm that GI is a major contributor to Y function. Analysis of the three-loop network suggests that the plant clock consists of morning and evening oscillators, coupled intracellularly, which may be analogous to coupled, morning and evening clock cells in Drosophila and the mouse.

Figure 1

The three-loop Arabidopsis clock model accounts for 20 h rhythms in toc1 mutants. (A) Summary of the three-loop network, showing only genes (boxed), regulatory interactions (arrows) and the locations of light input (flashes). Two-component oscillators are distinguished by shading the gene names in yellow or blue. (B) CAB:LUCIFERASE (CAB:LUC) rhythms in WT (filled squares), toc1-9 (open squares) and toc1-10 (open diamonds) under constant red light (10 μmol m⁻² s⁻¹). Luminescence values were normalised to the average over the whole time course. Time zero is the onset of constant light (LL). (C) Simulated expression levels of LHY mRNA in the WT (black solid line) and toc1 backgrounds (green dotted line) in LL. Expression levels were normalised to the average level of expression. Translation rate of TOC1 mRNA in the simulated mutant is 1/1000 WT value.

Figure 2

GI acts as Y in a feedback loop with TOC1. (A) Simulation of LHY mRNA levels in the WT (black solid line) and gi backgrounds (Y translation rate reduced by 70%, red dotted line) under constant light (LL). (B) Corresponding experimental data assaying circadian control of WT CAB:LUC expression by video imaging. (C) Simulation of LHY mRNA under LL in lhy;cca1 (translation rate of LHY mRNA in simulated mutant is 1/1000 WT value, black line) and gi;lhy;cca1 mutants (red dotted line). (D) Corresponding experimental data assaying CAB:LUC expression. The gi;lhy;cca1 mutant is severely damped (only four out of 23 plants gave a period estimate within the circadian range, and those estimates had an average relative amplitude error of 0.86). All data were normalised to the average level of expression.

Figure 3

Three-loop network can track dawn and dusk. Simulations of TOC1 mRNA (black solid line) and LHY mRNA (red dotted line) using the three-loop network under photoperiods of (A) LD8:16 and (B) LD16:8. The vertical dotted line highlights the shift in the peak phase of TOC1 mRNA levels from LD8:16 to LD16:8. The peak phase of LHY mRNA is not shifted.