Circadian clocks regulate many cellular/physiological processes allowing the organisms to adapt to the predictable daily changes in the environment. At the core of the clock, components of the central oscillator mutually regulate their expression/activity via multiple feedback loops that results in an autonomous, self-sustained ~24h oscillation.
As the period of the core oscillation deviates from 24h, the clock must be synchronised to the environmental cycles in order to provide precise temporal information. In nature, this resetting occurs daily in response to periodic environmental cues (e.g. temperature, light). Light is absorbed by specialised photoreceptors and signals are forwarded to the oscillator where they cause an acute change in the level/activity of certain clock components that eventually results in a phase shift of the oscillation. By using luciferase reporters in different fusion constructs, the transcription/translation rates and protein abundance of the clock components will be measured in wild type transgenic plants before and after light pulses given at different times of the day.
The results will indicate the clock components that are first affected by the light pulse and therefore could be the primary targets for resetting signals, and the sequence of the molecular events resulting in a phase shift. A novel transient UV-B-specific inducible expression system will be used to replicate the effect of light induction of the clock components and corroborate the previous results. Next, a series of truncated phytochrome B (PHYB) photoreceptor proteins will be used to complement the phyb null mutant to identify the minimal fragment of PHYB that is sufficient to mediate red light specific resetting. This shortest fragment will also be tested for its potential role in the cross-talk with other signalling systems (photomorphogenesis, blue light receptors) by physiological tests, yeast two-hybrid assays and analysis of combined mutant backgrounds.
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