Cell and tissue-specific metabolic flux analysis

Metabolic networks supply the precursors, energy and reducing power required for the synthesis, functioning and turnover of cellular components. Moreover the flows of material through the network – the metabolic fluxes – are crucial in determining the performance and productivity of an organism. In an agricultural context, the production of harvestable end-products of plant metabolism, such as starch in potato tubers or storage lipid in oilseeds, is entirely dependent on the flux phenotype of the plant; and in biotechnology, the exploitation of micro-organisms and plants hinges on an ability to reconfigure the metabolic network to favour a flux distribution that leads to the preferential synthesis of useful products.

Metabolic flux phenotypes can be determined from stable isotope-labelling experiments using the tools of metabolic flux analysis (MFA), but established techniques ignore the impact of cellular differentiation on the metabolic activity of multicellular organisms and generate flux maps that conceal the true contribution of the different cell types. The problem arises because the relationship between the fluxes and the labelling patterns is non-linear, and so averaged labelling data do not provide a true representation of the averaged fluxes. Cell-specific labelling information is required to solve this problem and we are developing a method for cell-specific MFA via the expression and purification of cell-specific proteins.

The project will exploit the cell-specific expression of green fluorescent protein (GFP) in Arabidopsis roots. GFP will be immunopurified after stable isotope labelling experiments, and the label distribution in peptides and amino acids derived from the GFP will be analysed by mass spectrometry. A critical question is the extent to which reliable flux maps can be deduced from cell-specific labelling data with only limited information about the cell-specific metabolic inputs and outputs that are usually used to constrain the flux solution, and so the project will begin with a systematic in silico investigation of this question to establish the consequences of limited cell-specific inputs and outputs on flux determination. Ultimately applications are envisaged in several areas of contemporary biological importance, including plant–pathogen interactions and the symbioses formed between plants and nitrogen-fixing bacteria.

Student profile

This project requires a strong background in biochemistry, including metabolism, an interest in metabolic analysis, and an aptitude for computational methods.