Analysis of the compartmentation of glycolytic intermediates, nucleotides,sugars, organic acids, amino acids, and sugar alcohols in potato tubers using a nonaqueous fractionation method

The compartmentation of metabolism in heterotrophic plant tissues is poorly understood due to the lack of data on metabolite distributions and fluxes between subcellular organelles. The main reason for this is the lack of sui table experimental methods with which intracellular metabolism can be measu red. Here, we describe a nonaqueous fractionation method that allows the su bcellular distributions of metabolites in developing potato (Solanum tubero sum L. cv Desiree) tubers to be calculated. In addition, we have coupled th is fractionation method to a recently described gas chromatography-mass spe ctrometry procedure that allows the measurement of a wide range of small me tabolites. To calculate the subcellular metabolite concentrations, we have analyzed organelle volumes in growing potato tubers using electron microsco py. The relative volume distributions in tubers are very similar to the one s for source leaves. More than 60%. of most Sugars, sugar alcohols, organic acids, and amino acids were found in the vacuole, although the concentrati ons of these metabolites is often higher in the cytosol. Significant amount s of the substrates for starch biosynthesis, hexose phosphates, and ATP wer e found in the plastid. However, pyrophosphate was located almost exclusive ly in the cytosol. Calculation of the mass action ratios of sucrose synthas e, UDP-glucose pyrophosphorylase, phosphoglucosisomerase, and phosphoglucom utase indicate that these enzymes are close to equilibrium in developing po tato tubers. However, due to the low plastidic pyrophosphate concentration, the reaction catalyzed by ADP-glucose pyrophosphorylase was estimated to b e far removed from equilibrium.