Post-translational regulation of cell physiology by the circadian clock

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Plant cells contain a biological clock that is fundamental to plant growth and survival. I discovered that seedlings in which the clock is stopped are half the size of those with a normal clock, and that if the clock is not synchronized with the environment, seedlings are stunted with reduced photosynthesis (Dodd et al. 2005). Maximum seed production also depends on the biological clock. Since these findings are of enormous agricultural importance, understanding the functions of the circadian clock is a timely and essential part of ensuring sustainable and secure food production in the future. Critically, our knowledge of the mechanisms by which the circadian clock enhances plant performance is very limited. The experiments that we propose will provide new knowledge to address this deficit. They will investigate two unresolved questions in plant biology; (i) what aspects of cell function are controlled by the circadian clock? (ii) what processes communicate timing information from the clock to circadian-regulated aspects of cell function? (i) What aspects of cell function are controlled by the circadian clock? Our preliminary experiments indicate that current understanding of the circadian organization of plant cell function requires extensive revision with information concerning protein abundance. We will identify membrane and soluble proteins that have circadian rhythms of abundance to discover biochemical mechanisms that are optimized by circadian regulation. (ii) How does reversible phosphorylation communicate timing information from the clock to circadian-regulated proteins within the cell? Reversible phosphorylation of proteins forms an important part of cell signalling that regulates the activity of enzymes directly, controls gene expression, and controls protein degradation. Genes encoding a large number of protein kinases are circadian regulated, and examples of circadian-regulated protein kinases are present in both plants and animals. This suggests that reversible phosphorylation has the potential to signal circadian timing information within plant cells. We will use two strategies to investigate the involvement of reversible phosphorylation in circadian signalling. First, we will identify proteins that undergo circadian rhythms of phosphorylation using a technique called phosphoproteomics. Second, we will identify the protein kinases and phosphatases that underlie the rhythms of phosphorylation for the proteins we identify that are of known importance to the cell. This information will be used to understand the signalling pathway between the circadian clock and the protein that is regulated by reversible phosphorylation. By combining my expertise (PI, Dr Dodd) in the circadian regulation of cell physiology and signalling with (a) specialist technology, expertise and methods within the Proteomics Laboratory in the Department of Biology's Technology Facility and (b) expertise in discovering novel protein-protein interactions in Arabidopsis (Hybrigenics S.A.) and protein kinase signalling (Prof. Jörg Kudla, Universität Münster), this research will advance significantly our understanding of the integration of the circadian clock in plants with cell function.