Cell-Type Specific Metabolic Flux Analysis: A Challenge for Metabolic Phenotyping and a Potential Solution in Plants

Abstract

Stable isotope labelling experiments are used routinely in metabolic flux analysis (MFA) to determine the metabolic phenotype of cells and tissues. A complication arises in multicellular systems because single cell measurements of transcriptomes, proteomes and metabolomes in multicellular organisms suggest that the metabolic phenotype will differ between cell types. In silico analysis of simulated metabolite isotopomer datasets shows that cellular heterogeneity confounds conventional MFA because labelling data averaged over multiple cell types does not necessarily yield averaged flux values. A potential solution to this problem—the use of cell-type specific reporter proteins as a source of cell-type specific labelling data—is proposed and the practicality of implementing this strategy in the roots of Arabidopsis thaliana seedlings is explored. A protocol for the immunopurification of ectopically expressed green fluorescent protein (GFP) from Arabidopsis thaliana seedlings using a GFP-binding nanobody is developed, and through GC-MS analysis of protein hydrolysates it is established that constitutively expressed GFP reports accurately on the labelling of total protein in root tissues. It is also demonstrated that the constitutive expression of GFP does not perturb metabolism. The principal obstacle to the implementation of the method in tissues with cell-type specific GFP expression is the sensitivity of the GC-MS system.

Keywords

• Arabidopsis thaliana
• cellular differentiation
• green fluorescent protein (GFP)
• metabolic flux analysis
• metabolic phenotype
• primary metabolism
• reporter protein
• systems biology