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

Lead Research Organisation: Royal Holloway, University of London
Department Name: Biological Sciences

Abstract

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.

Technical Summary

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.

Publications

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McWatters HG (2011) Timing in plants--a rhythmic arrangement. in FEBS letters

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Siddiqui H (2016) FHY3 and FAR1 Act Downstream of Light Stable Phytochromes. in Frontiers in plant science

 
Description In almost all organisms there exists a daily "circadian" clock which regulates many metabolic, development and physiological processes. Rather than just responding to dawn or dusk, the circadian clock is an internal rhythm which allows anticipation of these changes.

We identified for the first time the mechanism that the plant clock uses to regulate rhythmic expression of a single gene. The mechanism is somewhat similar to that of a grandfather clock. The mechanism is driven by some force, a weight or a spring, that moves the hands forward while a pendulum forms a timekeeping mechanism to regulate that movement. In plants we showed that light provides that driving force. Light acts through a group of proteins including FHY3 which are normally degraded in the dark but which are stabilised by light and then directly bind to the ELF4 gene, activating it."

The "pendulum" is another of the genes in this clock loop, a negative-acting component called Circadian Clock Associated (CCA1). The CCA1 gene is activated during the night but activity declines again during the day. The product of this gene, the CCA1 protein, binds negatively to the ELF4 gene but also binds to FHY3 preventing its association with the ELF4 gene. Thus, the ELF4 gene is activated during the day and deactivated again during the night. We went on to confirm that this process can continue just a well in constant light as in day / night cycles, forming a genuinely internal timing mechanism.

In further research just published we demonstrate that the action of FHY3 and FAR1 in upregulation of ELF4 is light dependent. Furthermore, although FHY3 and FAR1 have been exclusively characterized as components of the phytochrome A signalling pathway because of their importance in regulating expression of phyA nuclear importers, we show that, as transcription factors in their own right, FHY3 and FAR1 act downstream of light stable phytochromes, phyB, phyD, and phyE. This is significant as it means they may be involved in processes like shade avoidance whereby phyB, phyD and phyE perceive changes in the ratio of red:far red wavelengths in light reflected from neighbouring vegetation. This triggers dramatic elongation growth to prevent overtopping by competitors but it is also a major limit on planting density in agriculture. We demonstrate that light stable phytochrome acts in a red/far-red reversible manner to regulate the level of FHY3 protein. We also observed that ELF4 shows specific FHY3 and FAR1- mediated light induction in the evening and we show that regulation by light stable phytochromes at this time is important as it allows the plant to maintain normal ELF4 expression beyond dusk when the day length shortens, something which would not be possible through light labile phytochrome action. Without FHY3 and FAR1, ELF4 expression falls rapidly at dusk and in short days this results in an early drop in ELF4 expression, accompanied by a de-repression of an ELF4 target gene which is involved in elongation growth.
Exploitation Route Our findings may ultimately allow agricultural developments as a result of improving our understanding of plant adaptation to the environment. We could, hopefully, really benefit from better understanding of the clock mechanism in terms of crop productivity. As an example of the importance of the clock it was recently revealed elsewhere that the clock also regulates how a plant invests its reserves. As a result, there is a strong link to crop productivity.
Sectors Agriculture, Food and Drink,Environment

 
Description Royal Holloway Reid Studentship
Amount £55,000 (GBP)
Organisation Royal Holloway, University of London 
Sector Academic/University
Country United Kingdom
Start 02/2012 
End 02/2016
 
Title Reporter lines 
Description Mutant plants which lack the light-regulated transcription factors investigated in this study and which contain bioluminescent reporter transgenes reporting activity of clock-regulated genes 
Type Of Material Biological samples 
Year Produced 2012 
Provided To Others? Yes  
Impact This was a key part of the Nature Cell Biology publication associated with this award. 
 
Title Microarray data 
Description Microarray (global gene expression) analysis under a range of conditions for mutants lacking the light-regulated transcription factors that are the focus of this study 
Type Of Material Database/Collection of data 
Year Produced 2012 
Provided To Others? Yes  
Impact The data has formed the basis of three current PhD projects in my lab 
 
Description Coordinated transcriptional regulation underlying the circadian clock in Arabidopsis 
Organisation Yale University
Country United States 
Sector Academic/University 
PI Contribution Staff time. Generation of reagents. Experiments contributing to publication.
Collaborator Contribution Staff time. Generation of reagents. Experiments contributing to publication.
Impact Li, G., Siddiqui, H., Teng, Y., Lin, R., Wan, X., Li, J., Lau, O., Ouyang, X., Dai, M., Wan, J., Devlin, P.F., Deng, X.W. and Wang, H. (2011) Coordinated Transcriptional Regulation Underlying the Circadian Clock in Arabidopsis. Nature Cell Biology. 13, 616-622
Start Year 2008
 
Description Media interest (plant clocks) 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact The press release was picked up by one or two online popular science magazines

n/a
Year(s) Of Engagement Activity 2011
URL https://www.royalholloway.ac.uk/research/news/newsarticles/grandfathertimekeepsplantstickingover.asp...
 
Description University Open Days 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact As admissions tutor for the last 4 years, I have spoken about this work at over 30 University Open Days. The subject frequently sparked questions and discussion afterwards

Individual undegraduate applicants to Royal Holloway used this as part of their decision on choice of university
Year(s) Of Engagement Activity 2011,2012,2013,2014,2015