Epsilon Open Archive

Abstract

This thesis covers the possibilities and limitations of studying primary metabolism in intact plants, with special focus on heavy isotope labelling and mass spectrometry methodology. In paper I, a series of Arabidopsis thaliana mutants lacking one or both genes of mitochondrial malate dehydrogenase (mMDH characterised) were characterised. We found that mMDH has a complex respiration-controlling role. In paper II, we continued to study one of the single mutants, mmdh1. We developed a method using ¹³CO₂ to label whole plants and analyse their metabolic profiles by gas and liquid chromatography coupled to mass spectrometry (GC- and LC-MS). The results show that mmdh1 had a metabolic phenotype that revealed an altered flux through photorespiration, especially under low CO₂ conditions. Combining incorporation data with metabolite pool size deepened our understanding regarding the role of mMDH in photorespiration, respiration, and cellular redox balance. The practical and theoretical aspects of ¹³C-labelling of plants learned from this study were used for designing the experimental setup of paper III, a labelling study of developing wood in hybrid aspen. The labelling strategy, time resolution, and sampling had to be adapted to suit woody plants.

Two months old trees were labelled with ¹³CO₂ in a 4 h burst and then sampling of source leaves, stem phloem, and developing wood continued over 24 hours. Since sucrose is the main carbon transporter the analysis was concentrated around this metabolite. We found previously unrecognised temporal patterns in wood biosynthesis and an indication that the diurnal cycle serves as a cue in the regulation of carbon allocation in developing wood.

To study systems with complex structures such as plants, it is informative to study specific cell types rather than whole plants or whole organs. In paper IV, a method was developed for analysis of metabolite profiles in cell type specific cells. Isolated protoplast from Arabidopsis roots were sorted by fluorescence-activated cell sorting (FACS) and their metabolite profiles were analysed by GC-MS. The method is fast, robust and reliable for analysis of cell type specific metabolic profiling and a beginning to meet the call for new metabolite analysis techniques with a high spatial resolution.