Analysis of the Arabidopsis Circadian Signalling Network

Most living things are able to measure time. We are familiar with our human 'body clock' which acts as an internal watch because we experience the physiological effects of disruption of the clock when we cross time zones and get jet-lag. Plants also are able to measure time, they measure the length of the seasons to control the time to flower and many flowers open at specific times of the day, for example water lily flowers are only open in the middle of the day and close long before dusk. It is important to understand the plant clock because we have recently found that the circadian clock of plants helps make them grow twice as fast as plants that do not have a working clock. The time-keeper in plants is present in every cell and is known as a circadian clock. We have identified a new 'cog' in the circadian clock of plants. We think this cog is made up of the gas nitric oxide, a small sugar containing molecule called cyclic ADPR and the metal calcium. We will use new biological techniques that allow us to measure changes in these compounds in living cells and measure daily changes in the activity of all the genes in the plant to identify other 'cogs' of the clock. To do this we will use mathematical tools originally developed by Engineers to understand complex machines.

We have new data that describe a circadian signalling network with properties that suggests it forms a novel post-translational negative feedback loop in the Arabidopsis circadian clock. This feedback loop is postulated to include circadian oscillations of NO, cyclic ADPR and calcium. We propose to identify targets and regulators of the circadian signalling network and to test our hypotheses about its function. The first goal is to identify the nature of the oscillator regulating the circadian signalling network. We have an urgent need to identify the genetic controllers of the circadian signalling network because our preliminary data suggest that this pathway is not regulated by TOC1, a clock gene thought to regulate all circadian rhythms in Arabidopsis. This calls in to question the consensus model of circadian oscillator structure in plants. We propose to measure the effect of a range of mutants on circadian oscillations of nitric oxide production using an NO meter, cADPR production and ADPR cyclase activity using fluorescent assays optimized in our labs and changes in the concentration of free calcium ions using automated imaging of aequorin bioluminescence. Our second aim is to identify transcripts that are co-regulated with calcium with the eventual goal of identify regulators and targets for circadian oscillations of calcium. This is now possible because we have identified 2 experimental conditions in which calcium behaves differently to other circadian outputs. This presents the opportunity to use circadian analysis of transcript abundance in different experimental conditions, coupled with systems analysis tools to identify those transcripts which are closely associated with circadian oscillations of calcium. The data will be modelled to develop and test hypotheses about the identity and function of the events regulated by the circadian signalling network.