Investigating cell type specific metabolism using GFP as a reporter protein

Thesis

Metabolic flux analysis (MFA) is a powerful technique for quantifying the intracellular fluxes in central carbon metabolism. It relies on detection of stable isotope labelling from metabolites such as amino acids derived from protein. Current standard techniques are, however, unable to distinguish between different cell types in heterogeneous tissue. The aim of the thesis was to address this problem by developing and validating a strategy using green fluorescent protein (GFP) with cell type specific expression as a reporter protein for investigating the fluxes in specific cell types in the Arabidopsis thaliana root. The fundamental difficulty in applying a reporter protein strategy in a multicellular organism arises from the limited amount of recombinant protein expressed by the cells.

The main novel contributions of the work in this thesis are threefold. First, a robust protocol for purification of GFP from the roots of Arabidopsis seedlings and for detection of reliable mass isotopomer distributions from the amino acids derived from GFP are described. Secondly, the reporter protein strategy is validated in this biological system with a focus on showing the data obtained by the use of the reporter protein is equal to that normally obtained from the total protein fraction. To expand on this, stable isotope labelling in isolated root hair cells is explored. These cells are easily isolated and show potential as a model system for cell type specific metabolism. Finally, the experimental data provide evidence for the feasibility of measuring data from specific cell types with appropriate mass spectrometric techniques. Analysis of cell type specific gene expression in this system suggests differences in the primary metabolism of different cell types cannot be ruled out without further investigation. Based on small scale in silico modelling described in this thesis, new solutions capable of providing data on sub-populations of cells are required, if central metabolism of the cell types differs significantly.

Authors: Rossi, M; Ratcliffe, R; Kruger, N

• DOI: 10.5287/ora-dezv7dzyb
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford