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Plant Physiol, October 2001, Vol. 127, pp. 685-700
Analysis of the Compartmentation of Glycolytic Intermediates,
Nucleotides, Sugars, Organic Acids, Amino Acids, and Sugar Alcohols in
Potato Tubers Using a Nonaqueous Fractionation
Method1
Eva M.
Farré,*
Axel
Tiessen,2
Ute
Roessner,
Peter
Geigenberger,2
Richard N.
Trethewey,3 and
Lothar
Willmitzer
Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am
Mühlenberg 1, 14476 Golm, Germany (E.M.F., U.R., R.N.T.,
L.W.); and Botanisches Institut, Universität Heidelberg, Im
Neuenheimer Feld 360, 69120 Heidelberg, Germany (A.T., P.G.)
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 suitable experimental methods with which
intracellular metabolism can be measured. Here, we describe a
nonaqueous fractionation method that allows the subcellular distributions of metabolites in developing potato (Solanum
tuberosum L. cv Desiree) tubers to be calculated. In addition,
we have coupled this fractionation method to a recently described gas
chromatography-mass spectrometry procedure that allows the measurement
of a wide range of small metabolites. To calculate the subcellular
metabolite concentrations, we have analyzed organelle volumes in
growing potato tubers using electron microscopy. The relative volume
distributions in tubers are very similar to the ones for source leaves.
More than 60% of most sugars, sugar alcohols, organic acids, and amino acids were found in the vacuole, although the concentrations of these
metabolites is often higher in the cytosol. Significant amounts of the
substrates for starch biosynthesis, hexose phosphates, and ATP were
found in the plastid. However, pyrophosphate was located almost
exclusively in the cytosol. Calculation of the mass action ratios of
sucrose synthase, UDP-glucose pyrophosphorylase, phosphoglucosisomerase, and phosphoglucomutase indicate that these enzymes are close to equilibrium in developing potato tubers. However,
due to the low plastidic pyrophosphate concentration, the reaction
catalyzed by ADP-glucose pyrophosphorylase was estimated to be
far removed from equilibrium.
1
This work was supported by the
Max-Planck-Gesellschaft (grant to E.M.F.).
2
Present address: Max-Planck-Institut für
Molekulare Pflanzenphysiologie, Am Mühlemberg 1, 14476 Golm, Germany.
3
Present address: Metanomics GmbH and Co. KGaA, Tegeler
Weg 33, 10589 Berlin, Germany.
*
Corresponding author; e-mail farre{at}mpimp-golm.mpg.de; fax
44-331-567-8408.
© 2001 American Society of Plant Physiologists
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