Protein function underlying plasticity of the plant circadian clock

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, e.g. 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. Overall our data shows that the plant clock machinery is 'plastic' (i.e. it depends on conditions such as the organ being studied and the genetic makeup of the plant) rather than 'hard-wired'. The aim of this grant application is to extend our work by defining the causes and roles of this plasticity. The work will be carried out with the model plant Arabidopsis but in the longer term the data and ideas will be transferred to crop species, for example in relation to the control of tuber formation and tuber metabolism in potatoes.

The circadian clock controls many aspects of plant behaviour. We have recently made a major advance by showing that the clock in roots is a simplified slave version of the clock in shoots (James et al., Science 322 1832-1835, 2008). This has important implications for our understanding of the operation and importance of the clock. In particular, the machinery of the clock is much more plastic than had been appreciated, and depends on both conditions and genotype. The aim of this grant application is to extend our work by defining the causes and roles of this plasticity, focusing on the function of clock proteins. We will address the following questions using the organ specificity of the clock as one of our tools: (a) Do the pseudo-response regulators (PRRs) involved in the clock associate with specific regions of DNA? We will mainly use chromatin immunoprecipitation and high coverage sequencing of recovered DNA. (b) Is there any difference in the biochemical properties of PRR7 and PRR9 between shoots and roots? We will test for differences such as in pI, Mr and association with other proteins. (c) What can we learn about the arrangement of clock components from studies of organ specificity and synchronisation? We will probe the positioning of several components in the clock mechanism, and also test whether two new transcription factors are involved in the central clock machinery or are merely controlled by the clock. (d) How is GI expression controlled? We will probe the GI promoter using both in vitro and in vivo approaches. (e) How is plasticity of the clock affected by temperature? We will follow up preliminary evidence which indicates that the degree of plasticity of the clock may be affected by temperature.