Role of FHY3 and FAR1 in transcriptional regulation of circadian clock resetting by red light

A host of biological process in organisms are regulated with an approximately 24 hour rhythm in tune with the external day / night cycle. These rhythms are not merely a response to external signals like dawn and dusk but are generated by an internal 'circadian' clock that continues to drive these rhythms even in constant environmental conditions. An internal rhythm allows an advantage in anticipating changes occurring over the day and in synchronising processes to occur in harmony with the day / night cycle. However, the circadian clock does not operate in isolation from the world / it is reset slightly every day in response to light signals in order to ensure that it is set to the correct time. The phenomenon of jetlag is a classical example where our internal clock must be reset by light, a process that occurs gradually over the course of a few days. Plants must, likewise, be able to entrain their circadian clock as the timing of dawn and dusk varies over the seasons. The clock in plants regulates a rhythm of leaf movement and the timing of production of the photosynthetic machinery amongst other processes, but the clock is also vital to the measurement of daylength in determining time of flowering. Both red and blue wavelengths are effective in clock resetting. Our aim here is to understand the mechanism by which red light resets the plant clock. We have previously demonstrated the involvement of a protein called FHY3 that is essential for the action of red light in resetting the clock. We have now identified a potential mechanism by which this acts in the nucleus of the plant cell to switch on a gene called CCA1, involved in the clock mechanism. Switching on CCA1 at a time when it is normally inactive has the result of resetting the clock. On the basis of new evidence, we propose a model for the way in which FHY3 acts in response to light along with two other proteins, FAR1 and ELF4. We will confirm this model experimentally and will carry out a search for other components also predicted to be part of this mechanism in order to synthesise a comprehensive model of red light input to the clock. An understanding of clock resetting may be particularly beneficial to agriculture where the timing of flowering is a critical determination of yield in many crops and even regulates the possible latitudes at which some crops can grow. In addition, overlap in the ways in which plant and animals generate their internal circadian rhythm may mean that this research could have implications in the study of human circadian defects.

No complete signalling pathway has been established to explain how the plant circadian clock is reset by red light. We recently demonstrated that FHY3 is essential for normal clock resetting by red light downstream of multiple phytochrome photoreceptors. FHY3 and its homologue FAR1 were recently demonstrated to be transcription factors binding to a FHY3/FAR1 binding site (FBS) to activate transcription. We have identified the FBS in promoters of CCA1 and ELF4, both key components in clock resetting in response to red light. CCA1 is part of a transcriptional feedback loop that comprises the central oscillator in plants. It accumulates with a circadian rhythm, peaking around dawn but its expression can also be strongly induced by red light. Up-regulation of CCA1 expression would have the effect of shifting the phase of the clock to a point at which CCA1 is high, thus resetting the clock. ELF4 has been shown to be essential for red light mediated clock resetting by regulating activation of CCA1. We have confirmed that FHY3 and FAR1 can bind to the ELF4 and CCA1 FBS in vitro and we aim to test the hypothesis that FHY3 and FAR1 act via the FBS to directly regulate ELF4 and CCA1 expression during clock resetting. We will confirm the importance of both FHY3 and FAR1 in red light activation of ELF4 and CCA1 and we will look for direct binding and activation in vivo. We will test a model whereby FHY3 and FAR1 act both directly at the CCA1 promoter and via activation of ELF4 to regulate CCA1. As FHY is constitutively nuclear and has been shown to constitutively activate transcription from the FBS, we will also dissect the CCA1 and ELF4 promoters to identify regions important in conferring red light-specificity. In parallel, we will also look for proteins directly interacting with FHY3 and FAR1 that may do this. Ultimately, we will construct and test a comprehensive model for red light input to the circadian clock via activation of CCA1.