Use of non-aqueous fractionation and metabolomics to study chloroplast function in Arabidopsis

Chloroplasts are the chemical factories of plant cells because they are able to fix inorganic carbon and -convert it to a wide-range of photoassimilates that are exported to the cytosol and other sub-cellular compartments. If the regulation of these processes is to be understood, the in vivo concentrations of a large number of metabolites have to be measured in all of these compartments separately. Sophisticated analytical approaches and continued advances in the technology of mass spectrometry coupled to a variety of fractionation and separation techniques allow the reliable analysis of a comprehensive complement of metabolites in photosynthetic tissues. Metabolomic approaches allow the multi-parallel analysis of a wide-range of metabolic intermediates and have been used for rapid phenotyping of different genotypes and environmental effects in plants. In addition to this, methods have been developed to analyse metabolite levels in different sub-cellular compartments of plant cells. Here, we describe methods for sub-cellular fractionation of Arabidopsis leaves using a non-aqueous density gradient technique, sample preparation suitable for metabolite profiling using gas-chromatography-mass spectrometry, and calculation of sub-cellular metabolite concentrations.