Post-translational regulation of cell physiology by the circadian clock

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.

There is an urgent need to establish the cellular bases for the large increases in productivity that are conferred by the circadian clock. This requires knowledge of both the cellular targets of circadian regulation and of the signalling pathways that communicate timing information from the circadian oscillator to these mechanisms. Surprising differences exist between the circadian timing of transcript and protein abundance in the liver and SCN of the mouse. Our new data reveal this is also the case in Arabidopsis. Proteome information is therefore required to understand the integrated circadian regulation of cell physiology. I propose to (a) investigate the circadian organization of the abundance and phosphorylation state of the membrane and cytosolic sub-proteomes, and (b) test the hypothesis that reversible phosphorylation functions as a output timing signal from the clock. Inclusion of membrane sub-proteomes in these experiments is important because membrane transport is pivotal to abiotic stress tolerance, cell signalling, growth regulation and mineral nutrition, and these mechanisms are commonly regulated by reversible phosphorylation. We have developed strategies for quantitative label-free LC/MS-based analysis of plant sub-proteomes over circadian time-courses and request funds to capitalize on our exciting preliminary findings. We will quantify circadian variation in protein and phosphoprotein abundance within the a) cytosolic and b) plasma and vacuolar membrane-enriched sub-proteomes from Arabidopsis. Using defined criteria, we will select from these data a small number of proteins involved in core aspects of metabolism and stress tolerance and identify candidate interacting protein kinases and phosphatases using yeast-two-hybrid screening. Potential interactions will be investigated in vivo using bimolecular fluorescence complementation. Finally, we will investigate the functioning of newly-identified putative circadian signalling pathways.

Non-academic beneficiaries of this research will be (a) plant breeders and agricultural biotechnologists, (b) recipients of research training, (c) the public, (d) the UK economy overall. This research will provide information to address the large question of how the circadian clock benefits living organisms and how timing information is communicated within cells, and so has implications beyond the plant sciences. The agricultural biotechnology and plant breeding sectors will benefit from this research. A key challenge for 21st century plant sciences is to identify genetic targets that can be manipulated to optimize productivity and nutritional content. I previously showed that circadian regulation is essential for maximum productivity. My preliminary proteomics experiments have revealed circadian regulation of proteins required for the agronomically-essential mechanisms of photosynthesis, water use and stress tolerance. Our findings will therefore be important for plant breeders and agricultural biotechnologists developing high-performance plants because deliberate or inadvertent alterations to the circadian regulation of agronomically-important proteins is likely to have substantial productivity implications. However, we do not know the identity or understand the circadian regulation of these proteins. This research seeks to address this deficit. University-sector outputs that are most frequently considered to be important by commercial organizations are (in order of ranking) publications, informal interactions and interactions at public meetings/conferences (Cohen et al. 2002). These strategies form part of this proposal and we will use them to ensure that agricultural biotechnologists and plant breeders can benefit from our findings. Research training will be received by the PDRA employed on this grant. Training will cover experimental and analytical techniques in functional proteomics, bioluminescence and fluorescence imaging of gene expression and cell signalling, molecular cloning, analysis, presentation and sharing of large and complex data-sets, and communication of research by oral and written presentation. The training will therefore contribute to both capacity-building in specific research skills and also incorporate training in broad-based skills. A full-time Skills and Training Co-ordinator is employed by the Department of Biology, who facilitates career development of PDRAs and graduate students in order to maximise the impact of the key university-sector output of highly trained staff. Masters-level proteomics courses in the Centre for Excellence in Mass Spectrometry (Univ. of York) draw upon frontier technologies and methods in proteomics and so development and findings from this research will feed directly into this future training. The PI has a track record of participation in Science Week open days and will during 2010 tutor for the Gatsby Summer School, which is an important opportunity to enthuse the very best undergraduates with plant sciences and so retain the best researchers within the UK's knowledge-based economy. It is important that research findings are communicated to the public because curiosity-driven research has enriching quality of life benefits. To ensure this occurs, we will exploit the Departmental BioLog magazine, Biology Matters schools outreach magazine, University of York newsletter, Department of Biology Outreach Committee and Research Open Days, and press releases when appropriate. The presence of research of the highest international quality within the UK makes the UK an attractive host for knowledge-based and biotechnology companies. This is for two reasons: (a) the university sector output of highly informed, knowledgeable, bioscience literate and technically-skilled individuals; (b) the potential for interactions between industry and university research laboratories. This project incorporates training and conferences that will enable these benefits to be realized.