Measurement of Subcellular Metabolite Concentrations in Relation to Phloem Loading

The key step of carbon export from green leaves is the loading of sugars into the phloem. To fully understand and quantify this process, it is essential to know the concentration of sugars in the different compartments of the cells along the phloem loading pathway. However, determining subcellular metabolite concentrations has been technically challenging. This paper describes a technique to measure metabolite levels in the chloroplast, the cytosol, and the vacuole of mesophyll cells with high accuracy. The nonaqueous fractionation (NAF) technique is arguably the method of choice to analyze the subcellular metabolite distributions as it minimizes the risk of metabolite interconversions or redistribution during the process. The principle of NAF is the separation of small subcellular particles, which are obtained by homogenization, lyophilization, and sonication, in a nonaqueous density gradient. Due to the varying composition-dependent density of the fragments, their segregation reflects compartmental distributions throughout the gradient. By determining marker enzymes for chloroplast stroma, cytosol, and vacuole in gradient fractions the proportions of each subcellular compartment in each gradient fraction can be analyzed. The measured distribution of marker enzymes and of metabolites in each fraction of the gradient can be used to calculate the subcellular distribution of the metabolites.