Organ communication in the Arabidopsis circadian clock

Lead Research Organisation: University of Glasgow
Department Name: School of Life Sciences

Abstract

Life has evolved on the planet Earth, which rotates on its axis, and therefore continuously undergoes a cycle of light and darkness lasting 24 h in total. As a result, most, perhaps all, organisms possess a 'circadian clock' that has a period of about 24 h and that determines the time at which various physiological processes occur. The clock does not keep exact time but is re-set each day by signals such as light. The 'circadian rhythm' most familiar to humans is our sleep/wake cycle, the cause of jetlag and problems associated with shiftwork. Others include locomotor activity in rodents (e.g. hamsters run on an activity wheel at night rather than in the day) and leaf movements in bean plants. The latter rhythm was first described nearly 300 years ago! The circadian clock provides organisms with a significant advantage because it allows them to anticipate light/dark changes and adjust their behaviour accordingly, not just react to the changes. Comparing the clock in animals, plants and fungi, it seems that the 'design principles' are basically the same but the machinery is quite different. Some bacteria contain another type of clock. This implies that a 'clock' has evolved separately at least four times during the history of life on earth, indicating the key nature of its role. It is important to understand the way that the circadian clock functions in plants, particularly because in many plants it interacts with daylength to control flowering time. With the advent of global warming and climate change, it is desirable to extend the latitude at which particular crops grow. But daylength changes with latitude, so crop plants grown for their seeds may not be productive at different latitudes even if they can grow well. If we understand how the clock works, we should be able to breed or select crop variants that can grow productively at different latitudes. There have been huge advances in the last ten years or so in our understanding of the mechanism of the circadian clock in plants. However most of these have come from experiments on whole seedlings grown on agar plates containing sugars, with their roots exposed to the prevailing light/dark cycle. Such conditions are clearly irrelevant to a mature plant with its roots in the dark without sugars! We have carried out experiments in a more realistic situation, using mature plants with their roots in constant darkness while their leaves are exposed to the light/dark cycle. We have made two findings that radically affect the way we think the plant clock works. First, the clock is organ-specific, i.e. the machinery in the root is not the same as in the shoot. Secondly, the shoot is able to send a signal to the root that re-sets the root clock each day. Neither of these properties had been suspected before our work. The aim of this grant application is to extend our work and define the machinery and the functions of the circadian clock in different organs of mature plants.

Technical Summary

The circadian clock controls many aspects of plant behaviour. The current consensus is that the clock comprises three interlocking negative feedback loops, and that it is cell-autonomous. Our recent data presents a very different perspective. First, clock machinery is organ-specific; the root clock seems to involve only one feedback loop, so oscillations in the expression of TOC1 and other components are not needed for rhythmicity. Secondly the deduced functions of the transcription factors CCA1 and LHY are quite different between roots and shoots. In shoots they can inhibit the expression of some genes and activate that of others: in roots, they can only activate. Thirdly, the clock is not organ-autonomous; the root clock can be re-set daily by a signal from the shoots, but this can be disrupted by altering the availability of sugars. The objectives of this application are to extend our work and define the machinery and the functions of the circadian clock in different organs of mature plants. We will address the following questions: (a) How do CCA1 and/or LHY differ between shoots and roots? We will test for changes in phosphorylation state and complex formation, and use chromatin immunoprecipitation to address directly the question of whether CCA1 and LHY bind to evening elements in roots. (b) Which root transcripts are driven to oscillate in light/dark cycles and are they affected by sucrose? These questions will be answered though the use of microarrays. (c) How do shoots communicate with roots to control gene expression? We will use several approaches - use of sugar analogues and selective pathway inhibitors, use of signalling and metabolic mutants, and measurement of metabolite levels. (d) What is the underlying cause of the difference in the clock between shoots and roots? We will explore this simply by studying the roots of seedlings on plates and of mature plants, both exposed to light/dark cycles and in constant darkness.

Publications

10 25 50
 
Description We have characterised the major differences between the circadian clocks of plant shoots and roots. Using improved qPCR procedures we found that components of the clock evening loop do oscillate in roots in constant conditions, though with low amplitude over a high background. We studied the phosphorylation of CCA1 and LHY in vitro, and found that the main protein kinase involved is not a member of the casein kinase family; this is contrary to previous reports based on other methods. However we were not able to assess the phosphorylation state of the proteins in vivo because their abundance is too low and we could not obtain sufficiently clean immunoprecipitates. Experiments on metabolite control of the root circadian clock proved negative; we found no clear evidence to support the view that sucrose synchronises the root clock with the shoot clock. (In subsequent work, not on this grant, we found that the root clock is entrained by light piping.) We made one completely unexpected but very important finding, that alternative splicing is one of the mechanisms used to control the circadian clock in response to temperature changes. We developed a collaboration with John Brown's group at Dundee University, defined the changes in splicing of many clock genes during cooling and showed that splicing of LHY and PRR7 transcripts is important in control of the clock.
Exploitation Route In the future, potentially by the agrochemical industry to improve crop productivity through better understanding of root biology
Sectors Agriculture, Food and Drink

 
Title Imaging mature roots 
Description We can image luciferase activity in mature roots and shoots independently 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2013 
Provided To Others? Yes  
Impact A major paper has been submitted 
 
Description School information 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact I regularly help biology pupils at 2 local schools with university applications, particularly to Oxbridge, and also host visits to my lab to give brief work experience.

I have contributed to students applying to and being offered places at Oxbridge
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,2010,2011,2012,2013,2014,2015