Cellular and biochemical context of Arabidopsis circadian-clock components

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The Arabidopsis circadian clock drives transcriptional rhythms of about 10,000 transcripts and this coordinates most aspects of plant growth and development. EARLY FLOWERING 3 is the key hub for diurnal signalling and it is required for the clock to work. We recently showed that ELF3 acts as a physical integrator for the ELF4 ligand and the LUX DNA-binding protein, together termed the Evening Complex (EC), and that this is required for the oscillator to cycle and to mediate repression of transcriptional targets. The EC was thus hypothesized to be a global transcriptional co-repressor. As the ELF3 gene encodes a protein of unknown biochemical and cellular activity, it has remained enigmatic as to how it performs its key role in clock maintenance. What we have shown is that light signalling can repress ELF3 function, and this has a crucial role in determining oscillator speed in response to the brightness of light. ELF3 binds the phytochrome B photoreceptor and we showed that this acts as an inactivation event. The mechanistic bases by which light signals through phyB inactivate ELF3 repression to accelerate circadian timing are the goals of this work. Integrated technical and thematic approaches would be used to tackle these questions.We propose a set of experiments to unravel the clock-resetting mechanism through ELF3 in response to ambient light in order to understand its mode of action. Using a combination of biochemical, cellular and systems biology experiments, we will examine the spatial-temporal function of ELF3 with a comparison to that of other components it requires for function. We will examine the localisation context of where ELF4 activates ELF3 and how phytochrome represses this. New genetic tests of existing models will be performed to probe the existing explicit hypotheses that exist within the mathematical equations that generate the current circadian-systems models. As genetic tests of EC components revealed a non-redundant action in their regulation of targets and growth and development, we will monitor the global, genome-wide binding of these chromatin-associated factors to define their non-overlapping targets to unravel the basis for their capacity to cooperatively versus differentially regulate transcriptional target genes. Taken together we envisage that the efforts of this proposal will provide the mechanistic basis of a long-standing problem in the clock community where light perception leads to acceleration of periodicity. This is a clock-resetting mechanism and its understanding will help us tailor crops for new latitudes and altitudes of growth.