Analysis of the circadian clock at the single cell level in a multi-cellular context.

One of the most important adaptations to living on the Earth is to the daily fluctuation in light. To cope with this, from simple prokaryotes to complex multicellular life like ourselves, organisms have evolved an endogenous timer, the circadian clock. Circadian clocks allow an organism to time activities and processes so that they occur at the optimum time of day. The clock also allows an organism to measure day length, and thus the seasons and to alter its biology accordingly. It is not surprising, therefore, that the circadian clock affects a huge range of biological processes. In plants, this includes key agricultural traits such as flowering time, dormancy, water use efficiency, nitrogen metabolism and yield. In the long term our work on the circadian clock is likely to inform and impact the development of new crop cultivars.

Our current understanding of the circadian clock is largely based on experiments on whole plants or groups of plants. Over the years, a combination of mathematical modelling and whole plant experiments have led to striking breakthroughs in our understanding of the clock components and how they fit together. Mathematical models provide a conceptual framework allowing us to test ideas and understand how different components of the circadian clock interact. However, this body of work has a critical problem. It assumes that every cell in a plant has a clock that behaves identically. Experimental evidence contradicts this idea, but until recently we have not had the tools to analyse the clock in single plant cells.

At Liverpool University, using live-cell imaging approaches pioneered for mammalian cell work, we have recently developed a method to monitor the clock in single cells across a living seedling. In this project we will survey the circadian clock with cellular resolution. Then, in collaboration with experts in circadian modelling and single cell analysis at the Sainsbury Laboratory, Cambridge, we will extract important information from this single cell data and build cell-type specific mathematical models. These models will be tested with further experiments, and will answer long-standing questions in circadian biology, such as how do clock networks vary between cell-types? And are there multiple oscillators in a single cell? Finally we will extend our models into a multicellular context, and use them to direct experiments to investigate how networks are coupling between cells, and how this affects rhythms in the whole plant.

Models of complex biological systems often make the assumption that one network structure is sufficient to explain a biological process across an organism. This is unlikely to be the case, with many systems having tissue specific network structures. The plant circadian clock is one example where both molecular and genetic analysis points towards tissue specific network structures. Current models fail to accommodate this complexity. This proposal aims to rectify this by using a combination of time-lapse microscopy and mathematical modelling to study the clock at the single-cell level.

We will survey with cellular resolution the plant circadian clock using a recently developed assay that utilises rhythmic changes in nuclear fluorescence of clock protein YFP/GFP fusions. Our preliminary data has already demonstrated the feasibility of this approach, as we have measured single cell rhythms in seedlings for 96 hours. Our data reveals a great deal of heterogeneity between different cells missed by previous whole plant assays.

We will survey the circadian clock across multiple tissue types, using multiple promoter:protein YFP/GFP fusions. Resultant datasets will form the basis for the construction of cell-type specific clock models. This work will allow us to examine how clock network topology varies across a seedling. Validation will come through the analysis of specific marker/mutant combinations.

The dataset will also inform the construction of multi-cellular models. These models will be used to direct experiments to examine the extent, range, and mechanism of cell-cell clock coupling. Using these approaches the project will provide new insight into the structure and complexity of the plant circadian clock.

This project will survey the plant circadian clock at the cellular level across a plant seedling. The data will be used to construct cell-type specific and multi-cellular models of the plant circadian clock. This is a basic science project addressing fundamental questions about the structure and function of the circadian clock in plants. As such, the immediate audience for this work will be the scientific community. However, the microscopy and analytic methods used and computational modelling approaches developed will be of interest to the biomedical industry, specifically the multi-cellular modelling. Moreover, the circadian clock affects many important agricultural traits with a clock gene homolog responsible for the photoperiodic insensitivity of Northern European cultivars of wheat and barley. Therefore, in the long term, plant breeders will be interested in this work.

1. Microscope manufacturers and image analysis software developers. Dr Marcello has a long term working relationship with Zeiss, Leica and Hamamatsu. He will use these contacts to discuss developments within this project. We will test new instruments and assist in future instrument and software development.

2. Biotechnology Industry. Both Cambridge Sainsbury laboratory and University of Liverpool actively engage with industry. Dr Locke already has collaborative projects with Microsoft and through his fellowship, Dr Hall annually meets with the plant research group at Unilever. Dr Hall will take part in 1 day meetings and a networking evening organized by Liverpool Business Gateway and use these events to discuss this work. Dr. Hall will also attend Bioscience KTN events. Industrial links will also be facilitated by the invitation of industry representatives to "Imaging days" organized by the Liverpool Center for Cell Imaging

3. UK breeders. Through Dr Hall's wheat work, he has found the best way to engage with the UK breeding community has been through regularly attending Monogram and UK Plant Science Networking meetings. Dr Hall will use these meetings to highlight why breeders should be interested in the approaches and output of this fundamental work.

4. Intellectual property. It is possible that IP may arise from the methodological and analytical approaches developed during this project. We will liaise with Liverpool Business Gateway to ensure the timely protection of IP during the project.

5. Scientific community. We will ensure the academic impact of this work through timely seminars and publications. We will aim to present outputs at workshops and international plant and chronobiology meetings. We will also present the work at the UK Monogram network and plant science meetings.

6. Outreach. Dr Hall has an active collaboration with the Liverpool World Museum and Science and Plants for Schools. He will use these contacts to showcase this work, announce discoveries and develop teaching resources that can be used in the classroom. Dr Hall will also provide placements for Nuffield sixth form students. Dr Locke will work with Elisabeth Burmeister, the Cambridge Sainsbury Laboratory outreach officer and take part in the Cambridge Science Festival.