Cellular and biochemical context of Arabidopsis circadian-clock components

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.

Until now there is no mechanism described for how plant photoreceptors signal to the oscillator. Our previous identification of the key complex required for the oscillator to cycle and the genetic finding that this complex is the target of phytochromeB entry to the clock-setting process sets up a series of technical platforms to resolve such a mechanism and this answers in plants one of the longest standing questions in circadian chronobiology, whereby increases in light leads to periodicity acceleration.

We will use molecular-genetic approaches to improve the understanding of the structure-function relationship of ELF4 activation of ELF3. From there, new cell biological approaches will be employed to study the kinetics of when ELF3 is where. Building on our previous findings that ELF4 re-localises ELF3, we will characterise in diurnal-timed microscopy the details of phyB association to ELF3 in the nucleus and how and when ELF4 disrupts that interaction. Finally, system-genomics approach to define when and where the components of the repressive evening complex target transcriptional repression, the roles of timing and light in modulating targets will be found. New approaches to find chromatin targets of multiple components in one sample preparation facilitate future cost reductions to these sorts of experiments.

Together a series of technical innovations is brought together to solve how a clock can function in a varying light environment. This defines clock setting by light.

Numerous individuals and organizations benefit from the impact of this work. Our work on the evening-complex is known to control growth and the timing of development. As this programme here proposes to show that evening-complex function is under environmental modulation by phytochrome, a wide range of physiologists and developmental biologist should be engaged by this work. Our past efforts to successfully create one of the most modern mathematical models of the angiosperm clock is enhanced by the system overview of transcriptional occupancy we will define in this proposed work. The systems-biology community profits greatly from having data sets, such as what we have and will generate, that should link our current mathematical models of gene action to the informatically defined genomic targets of their regulation. Finally within the agricultural community, we have had great success in immediately transferring our model-system knowledge to the cereal barley, to be of immediate benefit to individuals involved in pre-breeding.
Our technical innovations to foster our science of this programme should have immediate impact on a range of plant molecular biologists, geneticists and cell biologists. Starting from our mathematical and biochemical placement of ELF3 in the clock, our work on chromatin associations of this transcriptional co-repressor links the positioning of this component to its action in the oscillator, and more broadly, to its output targets of control. The identity of those targets allows systems biologists to interact with informaticians in creating placement methods of regulators within statistical thresholds. As this in a context that is meaningful to plant geneticists, the pipeline we outline creates a work-space for understanding how multiple components of transcriptional complexes cooperatively verses disparately regulate targets. From this, several protocol innovations are to be developed that allow for ChIP-seq approaches to be more within an ease of use and this is at lower costs. The vector constructions we will we generate for cell biological studies of clock-protein migration over the day in a phytochrome-dependent manner will be donated to appropriate stock centres to foster new approaches in monitoring localisation dynamics, as related to complex assembly and conformational state. This is not limited to clock-cellular experiments and could have wide application.
We have current contact with Keygene corporation in the Netherlands to develop our Arabidopsis results for barley improvement. An on-going cooperation exists between us to explore evening complex and phytochrome components for utility in stress-relief under non-favourable conditions.
The connection to Keygene of course has strategic interest in translating our findings towards barley genotypes of immediate agricultural benefit. This translational interest extends to political and security issues as we partner with the University An-Najah National University in Nablus, The Palestinian territories, with the University of Jordon in Amman, Jordan, and with the breeding institutes of ICARDA. With our publication that barley ELF3 is the EAM8 locus, we have now been carefully working with these groups to exploit the role of the clock in drought tolerance. As dry areas are often bright areas, one can easily imagine that the work we propose here to link light input to the clock will similarly benefit stress-signaling efforts in plant breeding.