Download Analysis of the Compartmentation of Glycolytic

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 Tiessen2, Ute Roessner, Peter Geigenberger2, Richard N. Trethewey3, 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.
Compartmentation is one of the distinguishing
characteristics of plant metabolism (ap Rees, 1987). A
true understanding of the nature and regulation of
plant metabolic networks can only be achieved when
the metabolic interactions between subcellular compartments have been charted and subjected to analysis through experimental procedures. Because of the
profound difficulties associated with measuring enzymes, metabolites, and fluxes in specific subcellular
compartments, our understanding of plant metabolism has lagged far behind that of animal and microbial systems.
Although methods have been developed for the
assay of subcellular metabolite levels in leaf tissue
(Stitt et al., 1989), and the interactions between plas1
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 [email protected]; fax
44 –331–567– 8408.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.010280.
tidial and cytosolic metabolism during photosynthesis have been partially characterized (Stitt, 1997), little is known about the metabolic networks in
heterotrophic cells. There are two main reasons for
this. First, there is a lack of suitable methods for
organelle isolation, which is a particularly difficult
problem in heterotrophic cells because these often
contain large starch grains that cause extra damage to
the organelles during fractionation. Second, although
leaf metabolism is highly conserved between different species (Heineke et al., 1997), heterotrophic tissues usually form differentiated organs with specific
functions and therefore studies can be extrapolated
between organs only with extreme caution.
Advances in plant molecular biology have allowed
components of specific subcellular compartments to
be rapidly cloned and characterized. The nowroutine tools and procedures for the genetic manipulation of plants have also allowed the precise manipulation of the activity of proteins or enzymes
associated with particular subcellular compartments.
However, the extent to which transgenic approaches have been able to deepen understanding of
metabolism, particularly in heterotrophic tissues,
have been severely limited by the ability to measure
metabolism at the subcellular level.
Plant Physiology, October 2001, Vol.
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685
Farré et al.
The work presented here focuses on potato (Solanum tuberosum L. cv Desiree) tubers. The subcellular
organization of tubers is poorly understood particularly in comparison with other heterotrophic tissues
such as pea (Pisum sativum) roots, maize (Zea mays)
roots, or cauliflower (Brassica oleracea var. botrytis)
buds (Neuhaus and Emes, 2000). Import studies on
isolated potato tuber amyloplasts have led to inconclusive results on the nature of the fluxes across the
amyloplast membrane, probably due to the extreme
experimental difficulty (Schott et al., 1995; Naeem et
al., 1997; Wischmann et al., 1999). Further, due to the
bulkiness of tuber tissue, NMR methods are also
difficult to apply. Because of these restrictions, most
of the information on subcellular metabolism in tubers has come indirectly from the use of transgenic
plants (e.g. Müller-Röber et al., 1992; Stark et al.,
1992; Sweetlove et al., 1996; Tjaden et al., 1998; Trethewey et al., 1999; Tauberger et al., 2000).
In this study, we addressed three major open questions related to subcellular metabolism in potato tuber cells: the location of hexoses and Suc (Trethewey
et al., 1998, 1999), the availability of substrates for
starch biosynthesis in the amyloplasts (Tjaden et al.,
1998; Tauberger et al., 2000), and the distribution of
pyrophosphate (PPi; Farré et al., 2000). A nonaqueous fractionation method, based upon the procedure
that has been successfully applied to the study of
subcellular leaf metabolites (Stitt et al., 1989; Heineke
et al., 1997), was selected and adapted for the fractionation of tuber tissue. With this method, enzyme
and metabolite stability during the fractionation procedure is achieved by maintaining a water-free and
nonpolar environment. Metabolites in the different
subcellular fractions were measured in part with a
recently established gas chromatography-mass spectrometry (GC-MS) technique (Roessner et al., 2000).
To calculate subcellular metabolite concentrations,
the volume of the specific compartment must be
known. We have determined the volumes of subcellular compartments of growing potato tubers using
electron microscopy techniques.
tionation gradient ideally should be as small as possible to reduce the possibility of including material
from different organelles. The use of a ball mill followed by 2 min of sonication led to an average
particle size of 2 to 3 ␮m, as estimated by light
microscopy (data not shown). ADP-Glc pyrophosphorylase (AGPase) (E.C. 2.2.7.27; Kim et al., 1989)
and pyrophosphatase (E.C. 3.6.1.1; Weiner et al.,
1987) were chosen as markers for the plastid, and
␣-mannosidase was selected for the vacuolar compartment (E.C. 3.2.1.24; Boller and Kende, 1979). PPidependent phosphofructokinase (PFP; E.C. 2.7.1.90;
MacDonald and Preiss, 1986) and UDP-Glc pyrophosphorylase (UGPase; E.C. 2.7.7.9; Kleckowski,
1994, and references therein) were both used as
cytosolic markers. Phosphoenolpyruvate (PEP)-carboxylase (E.C. 4.1.1.31) has often been used as a
marker for the cytosol (Stitt et al., 1978); however,
activity is low in tubers and its measurement is therefore highly error prone. Initial experiments showed
that the distribution of PEP-carboxylase correlated
exactly with the activities of PFP and UGPase (data
not shown). Because the latter two enzymes are
highly active in growing tubers and therefore can be
easily measured, we decided to use them as routine
markers.
Figure 1 shows the marker enzyme distributions in
the fractionated material. The separation of compartments is comparable with many other published examples of nonaqueous fractionation (see Fig. 1, Gerhardt and Heldt, 1984; see Tables II and III, Weiner et
RESULTS
Separation of Tuber Material into
Subcellular Compartments
The most important requirements for a fractionation procedure to study subcellular metabolite distributions are the fast quenching and inactivation of
any biological activity of the plant material and the
avoidance of metabolite redistribution during the
separation procedure. The nonaqueous fractionation
method described here meets both of these criteria.
The principle of this method is the separation of
lyophilized tissue particles in a nonaqueous medium.
The central assumption made is that the metabolites
and proteins in a particular region of the cell aggregate together as the plant material is lyophilized. The
size of the particles applied to the nonaqueous frac686
Figure 1. Typical marker enzyme distribution of the fractionated
material from developing tubers. Developing tuber samples were
taken from 10-week-old plants grown in 2.5-L pots in the greenhouse. The tissue was fractionated using a nonaqueous fractionation
procedure and the activities of marker enzymes were determined in
the different fractions, four in A and five in B. Data represent the
percentage of activity in each fraction.
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Plant Physiol. Vol. 127, 2001
Nonaqueous Fractionation of Potato Tubers
Table I. Subcellular distributions and concentrations of phosphorylated intermediates in potato
tubers
Developing tuber samples were taken from 10-week-old plants grown in 2-L pots in the greenhouse.
The tissue was fractionated using a nonaqueous procedure. Metabolites in each fraction were measured
in trichoracetic acid (TCA) extracts by an enzyme-coupled test or by HPLC. The subcellular distributions
were calculated by comparing the metabolite and marker enzyme distributions using a threecompartment calculation program. The results represent the means ⫾ SE of measurements on four
different fractionations with different tuber samples. Concentrations were calculated using the estimation of subcellular volumes shown in Fig. 3. PPi was determined in a separate set of plants than the other
metabolites and its total tissue content was estimated from Farré et al. (2000). n.d., Not detected.
Total Tissue
Content
Metabolite
% of Total Tissue Content (mM)
Amyloplast
Cytosol
Vacuole
65.5 ⫾ 4.4 (0.46)
72.0 ⫾ 2.9 (0.122)
90.5 ⫾ 6.6 (0.038)
77.8 ⫾ 5.2 (0.97)
65.0 ⫾ 10.2 (0.26)
43.5 ⫾ 14.8 (0.08)
57.3 ⫾ 3.5 (0.901)
19.8 ⫾ 7.6 (0.084)
40.0 ⫾ 5.3 (0.031)
74.3 ⫾ 2.8 (0.495)
94.3 ⫾ 2.9 (0.24)
14.3 ⫾ 12.0 (0.003)
34.5 ⫾ 4.4 (0.30)
27.5 ⫾ 2.6 (0.057)
6.8 ⫾ 4.3 (0.004)
22.3 ⫾ 5.2 (0.34)
35.0 ⫾ 10.2 (0.17)
22.5 ⫾ 11.4 (0.051)
42.8 ⫾ 3.5 (0.83)
76.8 ⫾ 6.6 (0.40)
60.0 ⫾ 5.3 (0.058)
25.8 ⫾ 2.8 (0.21)
5.8 ⫾ 2.9 (0.018)
79.0 ⫾ 10.1 (0.023)
n.d.
0.8 ⫾ 0.4 (⬍0.001)
2.8 ⫾ 2.4 (⬍0.001)
n.d.
n.d.
34.0 ⫾ 10.7 (0.014)
n.d.
3.5 ⫾ 2.0 (0.003)
n.d.
n.d.
n.d.
6.7 ⫾ 5.4 (⬍0.001)
nmol g
fresh wt⫺1
3-P-Glycerate
PEP
Pyruvate
Glc-6-P
Fru-6-P
Glc-1-P
UDP-Glc
UTP
UDP
ATP
ADP
PPi
82
20
5
140
46
21
180
49
9
77
29
3
al., 1987; see Fig. 1, Sharkey and Vanderweer, 1989;
see Table III, Dancer et al., 1990; see Fig. 1, Riens et
al., 1991). The separation of the organelle fractions is
not complete, but is sufficent for calculating the metabolite distributions in the three main compartments
(cytosol, plastid, and vacuole) using the deconvolution approach described by Riens et al. (1991). We
chose to take four to five fractions instead of the six
to seven taken by Riens et al. (1991) to have enough
material in each fraction to be able to measure the
marker enzyme distribution and metabolites as accurately as possible. We used the mean percentage
distribution of pyrophosphatase and AGPase activities as plastidial marker and the mean distribution of
UGPase and PFP as cytosolic marker to reduce the
error due to the variability of single enzyme measurements in each fraction.
The marker enzyme distribution along the gradient
is similar to that found in leaves (Gerhardt and
Heldt, 1984). The vacuolar material is found in the
more dense fractions and the cytosolic material is
equally distributed along the gradient, but with a
relative enrichment in the higher part of the gradient.
Plastidial material was mainly found in the second
fraction as a narrow band at a density of approximately 1.54 g cm⫺3.
When analyzing the distribution of citrate synthase
(E.C. 4.1.3.7), a marker enzyme for the mitochondrial
matrix (Stitt et al., 1989), an analogous distribution to
the cytosolic markers in wild-type tuber was observed (data not shown). Therefore, for the following
calculations, the “cytosol” represents a “cytosolic ⫹
mitochondrial” compartment. This is similar to the
Plant Physiol. Vol. 127, 2001
situation with leaf tissue, where no appropriate separation of the mitochondrial compartment has been
achieved using a nonaqueous fractionation method
(Gerhardt and Heldt, 1984).
Stability of Metabolites during the
Fractionation Procedure
In the protocol upon which this work is based (Stitt
et al., 1989), the majority of the fractionation procedure was carried out at 4°C. Because it is difficult to
reliably meet this condition while keeping the material water free during the whole experimental procedure, we decided to test whether or not metabolites
were stable during fractionation at room temperature, where water-free handling can be more precisely guaranteed. To this end, metabolite levels per
milligram protein of frozen tuber discs were compared with levels found after fractionation at room
temperature. Even notoriously unstable metabolites
such as pyruvate showed no difference between the
two procedures (data not shown).
Variability in the Determination of
Metabolite Distributions
Three sets of plants were used for the analysis of
different metabolites. Plants were grown under the
same conditions in all three experiments and the
tubers used were of the same developmental stage.
The first set was used to measure phosphorylated
intermediates and nucleotides (Table I). GC-MS measurements were performed with the second set (Ta-
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687
Farré et al.
ble II) and PPi was determined in the third set of
plants (Table I). This experimental setup was necessary because it was impossible to complete all measurements with the same set of plants, given the
amount of material required for each analysis
procedure.
Two ways of calculating subcellular metabolite distributions from nonaqueous fractionation data have
been used: a two-compartment analysis as described
by Gerhardt and Heldt (1984), and a threecompartment analysis; for example, the one described by Riens et al. (1991). The first method is
based on correlation curves between marker enzymes and metabolite distribution in the different
fractions, and makes the assumption that the metabolite studied is found exclusively in two compartments. The second assumes a distribution between
three compartments. Because most of the metabolites
we have analyzed are predicted to be distributed
between three compartments, the second analysis
was preferred, and the calculations of the subcellular
distributions of metabolites were carried out as described by Riens et al. (1991). This calculation essentially follows a deconvolution approach. It is based
on the assumption that the metabolites are confined
to the plastidial, cytosolic, and vacuolar compartment as designated by the corresponding marker
enzymes. The evaluation is done by a computer program testing all possible cases for the distribution of
a certain metabolite between the three compartments
at intervals of 1%; for example: (a) plastid 100%,
cytosol 0%, and vacuole 0%; (b) plastid 99%, cytosol
1%, and vacuole 0%; and (c) plastid 99%, cytosol 0%,
and vacuole 1%.
There are 5,151 possibilities for the distribution of a
metabolite between the three compartments, and this
procedure calculates which of the possibilities agrees
most closely with the experimental results.
The data represent the mean values based upon
three to four independent fractionations each with
different tuber samples. As already observed for leaf
tissue, this method gives highly reproducible results
for metabolites almost exclusively located in one
compartment (e.g. malic acid or hexoses that are
predominantly located in the vacuole). A higher variation is found when metabolites are located in more
than one organelle. The variability is greatest when
the proportion found in a particular compartment is
low (less than 20% of the total). Given this variability,
we estimate that the limit of detection of a compound
in a particular organelle is around 5% of the total
amount in the tissue.
The Combination of GC-MS and Nonaqueous
Fractionation Allows the Measurement of the
Subcellular Distribution of a Large
Number of Compounds
A GC-MS based method that allows the analysis of
a large number of metabolites in parallel was devel688
oped recently in our laboratory (Roessner et al.,
2000). This method was combined with the nonaqueous fractionation technique to study metabolite compartmentation. To calculate their subcellular distributions, detectable amounts of the compounds in
each fraction are needed. Therefore, the total number
of compounds that can be analyzed is reduced in
comparison to the high number that can be identified
in a tuber total extract. Due to the large amount of data
generated, hierarchical cluster analysis was used to
group metabolites that showed a similar fractionation
pattern along the gradient. The mean fractionation
pattern (percentage distribution in the fractions) of all
the compounds belonging to one cluster was used to
calculate the subcellular distribution of that cluster in
a particular gradient. Two main clusters, A and B, of
seven and 13 metabolites, respectively, were identified
(Fig. 2, Table II). The sd from the mean between different compounds belonging to one cluster was lower
than 10% in most fractions. Only fractions that contained less than 10% of the total content in the gradient
had higher deviations (Table III). Apart from the two
main clusters A and B, two further small clusters were
repeatedly detected. Cluster C contained Glc-6-P and
Fru-6- and Cluster D contained the amino acids Glu
and Asp (Fig. 2, Table II). The remaining compounds
did not have distributions similar enough to be
grouped within separate clusters.
Table II. Subcellular distribution of metabolites in potato tubers
Developing tuber samples were taken from 10-week-old plants
grown in 2-L pots in the greenhouse. The tissue was fractionated
using a nonaqueous procedure. Metabolites in each fraction were
measured in methanol extracts using GC-MS. Cluster analysis was
performed on the percentage distribution in the fractions of the
gradient. Cluster A, Quinate, Lys, isocitrate, fumarate, malate, Man,
and citrate. Cluster B, Ala, Gly, Ser, Thr, Tyr, Phe, Val, 5-oxo-Pro,
Orn, mannitol, inositol, shikimate, and succinate. Cluster C, Fru-6-P
and Glu-6-P. Cluster D, Asp and Glu. The subcellular distributions
were calculated by comparing the metabolite and marker enzyme
distributions using a three-compartment calculation program. The
results represent the means ⫾ SE of measurements on three different
fractionations with different tuber samples. n.d., Not detected.
Metabolite
Cluster A
Cluster B
Cluster C
Cluster D
Suc
Glc
Fru
Pro
Leu
Iso-Leu
Met
Tyr
Asn
Gln
Homo-Ser
Trp
% of Total Tissue Content
Amyloplast
Cytosol
Vacuole
3.8 ⫾ 1.9
16.5 ⫾ 6.1
58.8 ⫾ 9.3
46.0 ⫾ 9.0
5.0 ⫾ 3.3
8.7 ⫾ 3.7
15.7 ⫾ 6.7
11.3 ⫾ 6.9
12.0 ⫾ 5.7
12.7 ⫾ 5.4
12.3 ⫾ 2.8
18.3 ⫾ 2.6
34.7 ⫾ 6.0
19.5 ⫾ 11.3
26.7 ⫾ 8.0
37.0 ⫾ 16.8
1.3 ⫾ 0.6
8.3 ⫾ 3.7
39.3 ⫾ 7.8
32.3 ⫾ 11.7
17.7 ⫾ 3.1
14.3 ⫾ 5.9
n.d.
39.3 ⫾ 6.2
35.7 ⫾ 3.4
20.3 ⫾ 8.6
3.3 ⫾ 2.7
1.3 ⫾ 1.1
1.7 ⫾ 1.0
32.0 ⫾ 18.5
5.3 ⫾ 4.4
n.d.
95.0 ⫾ 2.5
75.3 ⫾ 7.7
2.0 ⫾ 1.7
21.7 ⫾ 7.3
77.3 ⫾ 0.7
77.0 ⫾ 5.7
84.3 ⫾ 6.7
49.3 ⫾ 6.7
52.3 ⫾ 3.0
67.0 ⫾ 7.6
84.3 ⫾ 2.3
80.3 ⫾ 1.8
63.7 ⫾ 5.5
48.5 ⫾ 7.2
68.0 ⫾ 3.7
63.0 ⫾ 16.8
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Plant Physiol. Vol. 127, 2001
Nonaqueous Fractionation of Potato Tubers
Table III. Distribution of metabolites belonging to cluster A in the different fractions of one gradient
Developing tuber samples were taken from 10-week-old plants grown in 2-L pots in the greenhouse.
The tissue was fractionated using a nonaqueous procedure. Metabolites in each fraction were measured
in methanol extracts using GC-MS. The data represent the percentage of each metabolite in the different
fractions of a gradient.
Fraction
1
2
3
4
% in Fraction
Lys
Man
Citrate
Malate
Fumarate
Quinate
Isocitrate
46.5
38.3
10.9
4.3
52.1
36.6
8.4
3.0
49.8
42.3
0.1
7.9
51.6
36.5
8.5
3.4
52.2
34.1
9.5
4.2
48.1
39.1
8.8
4.0
50.2
35.8
9.6
4.4
Determination of Subcellular Volumes
To determine the volumes of the subcellular compartments, over 60 electron micrographs were taken
from representative regions of the potato tissue. From
these photographs, the relative volumes (percent of
total) were calculated according to the principle of
Delesse (1847). For the calculation of the mitochondrial volume, the relative volume of the mitochondria
as percentage of the cytosol in the high magnification
photographs was multiplied by the mean volume of
the cytosol determined in the low magnification
photographs.
The mean volume (percent of total) of the different
compartments of potato tuber tissue is shown in
Figure 3. The histograms of volume distributions
show the range of volumes obtained for each compartment. The relative volumes (percent of total)
were converted to absolute volumes per unit of mass
by taking into account that under our growth conditions the specific density of the tubers was 1.16 g
fresh weight mL⫺1.
Most compartments show a gaussian distribution
with a clear single maximum. The values for the
cytosol show a broad bimodal distribution as would
be expected from two different types of tissue: a
small number of meristematic cells with a higher
percentage, and a high number of differentiated storage cells with a lower percentage of cytosol. It is
unfortunate that with this method it is not possible to
separate the meristematic from storage cells. In potato tuber, the major compartment was the vacuole,
with 67% of the cell volume (0.58 mL g fresh
weight⫺1), followed by the plastid (15%, 0.13 mL g
fresh weight⫺1) and cytosol (12%, 0.1 mL g fresh
weight⫺1), whereas other compartments had a much
smaller volume (Fig. 3).
The estimation of the actual aqueous volume of
amyloplasts is problematic because the starch granule fills most of the plastid volume in potato tubers.
Little is known about the in vivo water content of
starch granules, the characteristics of the granule
water, and the diffusion capacity of metabolites
through the starch granule (Tang et al., 2000). Only
the extreme situations can be estimated; the actual in
vivo situation will lie somewhere between these two
estimates. If we assume that metabolites can diffuse
Plant Physiol. Vol. 127, 2001
Mean ⫾
SD
50.1 ⫾ 2.0
37.5 ⫾ 2.5
8.0 ⫾ 3.3
4.5 ⫾ 1.5
freely through the starch granule, and that plastids
occupy about 15% of the cell volume, the absolute
volume would be 0.127 mL g fresh weight⫺1. If it is
assumed that metabolites are restricted to the free
diffusible water space, which does not seem to be
higher than 10% of the total amyloplast volume (Kosegarten et al., 1995), the volume estimations in this
case would be 0.013 mL g fresh weight⫺1.
Calculation of the Subcellular Concentrations
Tables I and II show the percentage distribution of
metabolites between different subcellular compartments. These data, together with measurements of
Figure 2. Dendogram obtained following hierarchical cluster analysis of metabolite distributions along a density gradient. Only metabolites are shown that have a similar clustering behavior in at least
three out of four gradients. The complete linkage method was used in
the assignment of clusters.
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689
Farré et al.
Figure 3. Volume of subcellular compartments of growing potato tuber tissue. Potato tuber tissue was visualized through
transmission electron microscopy. From these photographs, the relative volumes (percent of total) were calculated according
to the principle of Delesse (1847). The histograms of volume distributions show the range of volumes obtained for each
compartment. The relative volumes (percent of total) were converted to absolute volumes per unit of mass by taking into
account that under our growth conditions the density of 1.16 g fresh weight mL⫺1.
the total tissue contents and the calculation of the
subcellular volumes described in the previous section, were used to estimate the metabolite concentrations in the amyloplast, cytosol, and vacuole (Tables
I and IV). Because we did not achieve a separation
between the mitochondria and the cytosol, the volume of the cytosolic compartment used for the calculations was 0.11 mL g fresh weight⫺1.
The calculations of plastidial concentrations in Tables I and IV were done assuming unrestricted diffusion of metabolites in the starch granule. However,
in “Results” and “Discussion,” the plastidial concentrations given in parentheses represent the values
calculated assuming the case in which diffusion
across the granule is restricted.
690
Phosphorylated Intermediates Are Partitioned
between the Cytosol and the Plastid
More than one-half of the Glc-6-P and Fru-6-P was
located within the plastid (Table I). These results
were confirmed in the second experiment (Table II).
From these values, the estimated concentrations
were: 1 mm (10 mm) for Glc-6-P in the plastid, 0.34
mm for Glc-6-P in the cytosol, 0.26 mm (2.6 mm) for
Fru-6-P in the plastid, and 0.17 mm for Fru-6-P in the
cytosol (Table I). The distribution of Glc-1-P along
the gradient was unexpected. In general, Glc-1-P was
present at a higher percentage than the other hexose
phosphates in fraction 1 and a lower percentage in
fractions 3 and 4; therefore, up to 34% correlated with
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Plant Physiol. Vol. 127, 2001
Nonaqueous Fractionation of Potato Tubers
Table IV. Estimation of subcellular metabolite concentrations in
developing potato tubers
Calculations were done using data from Table II and the estimated
subcellular volumes shown in Figure 3. n.d., Not detected.
Metabolite
Total Tissue Content
nmol g fresh wt
Citratea
Fumaratea
Isocitratea
Malatea
Quinatea
Shikimatea
Succinatea
Frub
Glca
Mana
Suca
Fru-6-Pb
Glc-6-Pa
Inositola
Mannitola
Alaa
Aspa
Glub
Glya
Iso-Leua
Leua
Lysa
Meta
Phea
Proa
Sera
Thrb
Tyrb
Vala
Amyloplast
⫺1
18,860
200
170
5,390
15,670
370
970
900
23,840
140
25,910
40
210
60
60
1,680
1,270
1,800
230
940
430
1,060
650
590
180
1,340
1,100
900
2,510
Cytosol
Vacuole
mM
5.6
0.059
0.050
1.6
4.6
0.48
1.26
1.11
16
0.04
10
0.19
0.97
0.078
0.078
2.2
4.6
6.5
0.30
0.94
0.41
0.31
0.63
0.77
0.16
1.7
1.4
1.17
3.3
2.1
0.022
0.019
0.6
1.7
0.27
1.27
n.d.
31
0.02
41
0.14
0.74
0.044
0.044
1.2
3.7
5.2
0.17
1.71
1.37
0.12
0.19
0.43
0.63
1.0
0.8
0.66
1.8
31.1
0.329
0.280
8.9
25.8
0.48
1.27
1.32
32
0.23
35
0.001
0.01
0.078
0.078
2.2
0.5
0.7
0.30
1.09
0.39
1.75
0.95
0.77
0.15
1.7
1.4
1.17
3.3
a
Calculations were done using the measurements of the total
b
tissue content from Roessner et al. (2000).
Calculations were
done using the measurements of the total tissue content from Trethewey et al. (1998).
the vacuolar marker (Table I). This particularly high
correlation with the vacuole was also found in other
experiments (data not shown).
The glycolytic intermediates 3-P-glycerate, PEP,
and pyruvate were located mainly in the plastid (Table I). The concentration of 3-P-glycerate was estimated to be 0.30 mm in the cytosol and 0.46 mm (4.6
mm) within the plastid (Table I). These concentrations are lower than those in potato leaves. Leidreiter
et al. (1995) estimated a concentration of 2 mm for
3-P-glycerate in the stroma and 1.7 mm for 3-Pglycerate in the cytosol.
Adenine and Uridine Nucleotides Show Different
Partitioning between the Cytosol and the Plastid
The majority of the uridine nucleotides were found
in the cytosol (Table I), at similar levels to those
found in leaves (Dancer et al., 1990). More than 70%
of the UTP and 60% of the UDP were found in the
cytosol. This leads to estimated cytosolic concentraPlant Physiol. Vol. 127, 2001
tions for UTP of 0.40 mm and for UDP of 0.058 mm,
which are very similar to the concentrations calculated for leaf tissue (Dancer et al., 1990). However, we
found an unexpected subcellular distribution for
UDP-Glc, where only 42% was located in the cytosol,
with an estimated concentration of 0.83 mm (Table I)
and a significant amount found in the plastid, in
disagreement with the distribution found in leaf tissue where the location was almost exclusively cytosolic (Dancer et al., 1990).
On the contrary, adenine nucleotides were found
mainly associated with the plastidial marker (Table
I). The estimated concentrations for ATP in the plastid and in the cytosol were 0.49 (4.9 mm) and 0.2 mm,
respectively, and for ADP 0.24 mm (2.4 mm) and
0.018 mm (Table I), respectively. The ADP-Glc content in potato tubers is very low; in our experiment,
we measured 3.6 ⫾ 0.1 nmol g fresh weight⫺1, which
is in agreement with previous studies (Geigenberger
et al., 1994; Farré et al., 2000). We could only detect
ADP-Glc in the fraction enriched for amyloplasts
(data not shown) and therefore were not able to
calculate the exact metabolite distribution. However,
this indicates that in potato tubers, ADP-Glc is probably exclusively located in the plastid with a concentration of approximately 28 ␮m.
PPi Is Almost Exclusively Present in the Cytosol
As shown in Figure 1, alkaline pyrophosphatase
activity appeared to correlate with AGPase activity
and therefore was exclusively located in the plastid.
On the other hand, PPi was mainly located in the
cytosol, with an estimated concentration of about 23
␮m (Table I). Although this concentration is lower
than that estimated for leaves, the distributions of
soluble inorganic pyrophosphatase activity and PPi
between the cytosolic and plastidial compartments
are identical to the distribution in green tissues
(Weiner et al., 1987).
Glc, Fru, and Suc Accumulate in the Vacuole
Most of the sugars were found in the vacuole.
About 77% of both Suc and Glc were located in this
compartment (Table II), giving an estimated concentration of 35 mm for Suc and 32 mm for Glc (Table
IV). Seventeen percent of Suc was found to be located
in the cytosol (Table II). Other experiments that we
have performed (E.M. Farré and L. Willmitzer, unpublished data) corroborate this finding. Therefore,
we estimated that the cytosolic concentration of Suc
is around 40 mm (Table IV). In the case of Glc, 8.7%
was found in the plastid (Table II), with an estimated
concentration of about 16 mm (160 mm; Table IV). In
the case of Fru, 84% was found in the vacuole and
15% in the plastid, whereas the amount of Fru
present in the cytosol was below the detection limit
(Table II).
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691
Farré et al.
The Vacuole Contains a Large Pool of Different
Organic Acids, Amino Acids, and Sugar Alcohols
Most amino acids, organic acids, and sugar alcohols were grouped in cluster A and cluster B and
were mainly located in the vacuole (Table II). When
the estimates of subcellular volumes were taken into
consideration, all compounds in cluster A had higher
vacuolar than cytosolic concentrations and compounds in cluster B had similar concentrations in the
vacuole and the cytosol (Table IV). A high percentage
of most amino acids were found associated with the
vacuole. The exceptions were Asp and Glu (Cluster
D), which were mainly located in the plastid and the
cytosol. This distribution is similar to that found for
leaves, where a high percentage of these two amino
acids was found in the stroma (Leidreiter et al., 1995).
A substantial amount of Asn was also associated
with the plastidial markers. The amino acids Pro,
Leu, and iso-Leu displayed a higher amount that
appeared correlated with the cytosolic compartment.
Estimated Mass Action Ratios for Key Enzymes in
Potato Tuber Carbohydrate Metabolism
From data on the subcellular distributions of sugars, hexose phosphates, nucleotides, and PPi shown
in Tables I and IV, it is possible to calculate the mass
action ratios of different cytosolic and plastidial reactions involved in carbon metabolism. These ratios
are independent of the actual volume of a particular
compartment. For the calculation of the mass action
ratio of the AGPase, we made the assumption that
ADP-Glc is exclusively located in the amyloplast because we could only detect it in those fractions enriched for plastidial markers. The mass action ratios
of phosphoglucoisomerase, phosphoglucomutase,
and UGPase are close to their Keq, whereas the mass
action ratio of the AGPase reaction is 500 times lower
than the Keq (Table V).
The level of Fru in the cytosol is below the limit of
detection of our method, which we have estimated to
be 5% of the total content of a compound. In Table V,
we calculated the mass action ratio of Suc synthase
assuming 5% of Fru is located in the cytosol, which
corresponds to a cytosolic concentration of 0.5 mm.
DISCUSSION
Despite the central importance of the compartmentation of metabolic pathways in plant cells, until now
there have been few studies exploring this issue and
most of these studies deal exclusively with photosynthetically active tissue. We have used a nonaqueous
fractionation method to study subcellular metabolite
distributions in potato tuber. To the best of our
knowledge, this is the first report on metabolite distributions between the vacuolar, cytosolic, and plastidial compartments of a heterotrophic tissue. Although Liu and Shannon described a nonaqueous
fractionation method for the isolation of maize endosperm starch granules and their associated metabolites, their method used glycerol and 3-chloro-1,2propandiol, which they later showed to lead to the
inactivation of enzyme activities, thus preventing a
comparison with the distribution of marker enzymes
(Liu and Shannon, 1981a; Shannon et al., 1998). We
have chosen a method that uses nonpolar solvents
(heptane and tetrachlorethylene) because the stability
of both metabolites and enzymes in these media allows the measurement of marker enzyme activities
and therefore the identification of the isolated
fractions.
Table V. Calculation of the molar mass action ratio of different reactions in the cytosol and plastid
of potato tubers
The values of the mass action ratios were calculated using the metabolite concentrations in Tables
I and IV. Cytosolic Fru content is assumed to be 5% of the total. ADP-Glc is assumed to be located
exclusively in the amyloplast and with a tissue content of 3.6 nmol g fresh wt⫺1.
Reaction
In Vivo Molar Mass
Action Ratio
Cytosol
Plastid
Theoretical Keq
Phosphoglucoisomerase
关Fru-6-P]
[Glc-6-P]
0.5
0.26
0.51
Phosphoglucomutase
[Glc-6-P]
[Glc-1-P]
6.8
12.0
19
Suc synthase
关Fru] [UDP-Glc]
[Suc] [UDP]
0.18
–
0.15– 0.56
UGPase
关Glc-1-P] [UTP]
[UDP-Glc] [PPi]
0.96
–
3.2
AGPase
关ADP-Glc] [PPi]
[Glc-1-P] [ATP]
–
0.002
1
a
692
Equation Used to Estimate the
Molar Mass Action Ratio
Geigenberger et al. (1993).
b
a
a
a
a
b
Keq for AGPase was calculated with AGo⬘ ⫽ 2.88 (Kruger, 1997).
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Plant Physiol. Vol. 127, 2001
Nonaqueous Fractionation of Potato Tubers
The Relative Subcellular Volumes in Growing Potato
Tubers Are Similar to the Ones in Leaves
Cell and amyloplast sizes change during tuber development as shown in the work of Tauberger et al. (1999)
and therefore we determined the subcellular volumes
in growing tubers of the same developmental stage as
the ones used for biochemical analysis. The subcellular
volumes of source leaves from different species have
been determined already (for review, see Heineke et al.,
1997). In mesophyll cells, although the absolute volumes show considerable differences between the species, the relative compartmentation is rather similar
(Heineke et al., 1997). In leaves, the percent of total cell
volume taken up by various organelles ranges from
73% to 79% for vacuoles, 16% to 19% for chloroplasts,
3.8% to 7% for cytoplasm, and 0.5% to 1% mitochondria. These values are similar to the values determined
for growing potato tubers (Fig. 3).
Hexose Phosphates and Glycolytic Intermediates
Are Distributed Differently between the
Cytosol and the Amyloplast
Most of the phosphorylated intermediates analyzed were distributed between the cytosol and the
plastid and were absent from the vacuole. Liu and
Shannon (1981b) also found many different phosphorylated intermediates associated with starch
granules in maize endosperm.
The cloning of a Glc-6-P/phosphate translocator
that is highly expressed in potato tubers (Kammerer
et al., 1998) and the reduction of starch synthesis due
to the specific inhibition of a plastidial phosphoglucomutase (Tauberger et al., 2000) has led to the conclusion that Glc-6-P is taken up by the plastids as
substrate for starch synthesis in potato tubers. Both
Glc-6-P and Fru-6-P were located in the amyloplast in
significant amounts. The estimated cytosolic concentration of Glc-6-P is 0.34 to 0.8 mm (Tables I and IV),
which is similar to the apparent Km of the plastidial
Glc-6-P/phosphate translocator from pea roots (0.7
mm, Kammerer et al., 1998), and also to the concentration of 1 mm at which Hill and Smith (1991) observed saturation of the rate of starch synthesis from
Glc-6-P in isolated amyloplasts from developing pea
embryos. The phosphoglucoisomerase mass action
ratio does not differ significantly between the cytosol
and the plastid, although the cytosolic reaction seems
to be slightly closer to the theoretical equilibrium
constant (Keq; Table V). Therefore, it is apparent that
the phosphoglucoisomerase reaction is close to equilibrium in both compartments in potato tubers. In
leaves, however, the chloroplastic mass action ratio
differs from the cytosolic one, the latter being closer
to equilibrium (Gerhardt et al., 1987; Schleucher et
al., 1999).
Glc-1-P shows an unexpected distribution: A significant percentage was found associated with the vacuolar marker. Because vacuoles have a high unspecific
Plant Physiol. Vol. 127, 2001
phosphatase activity (De, 2000), it is unlikely that Glc1-P is actually located in this organelle. It is possible
that the Glc-1-P is associated with a further compartment that cofractionates with the vacuole; for example, the Golgi or endoplasmatic reticulum system. The
Glc-6-P/Glc-1-P ratio in the cytosol (6.8) was lower
than the one in the plastid (12.0), although due to the
high variability in the Glc-1-P measurements, these
values are not significantly different. The in vivo molar mass action ratio of the phosphoglucomutase reaction therefore is close to the Keq (Table V).
The 3-P-glycerate distribution in tubers resembles
the distribution found in leaves, in which a high
percentage of this compound was associated with the
chloroplast (Heineke et al., 1997). The plastidial 3-Pglycerate concentration ranged between 0.46 mm (assuming unrestricted diffusion) and 4.6 mm (if diffusion is restricted) and thus lies in the range of the A0.5
of AGPase from potato tubers, which has been determined to be 0.4 mm (Sowokinos and Preiss, 1982).
However, due to the lack of information about the
concentration of the AGPase inhibitor orthophosphate (Sowokinos and Preiss, 1982), it is difficult to
draw any conclusions concerning the in vivo activity
of this enzyme in potato tubers. The different pools of
3-P-glycerate, cytosolic and plastidial, may change
independently as they have been shown to do in
leaves (Gerhardt et al., 1987). This could explain why
correlations between 3-P-glycerate and starch synthesis have only been shown in some cases (Hajirezaei et
al., 1994; Geigenberger et al., 1997; Preiss, 1997; Farré
et al., 2001), and not in others (Geigenberger et al.,
1994; Geiger et al., 1998; Trethewey et al., 1998, 1999;
Fernie et al., 2001).
The high percentage of PEP and pyruvate associated with the amyloplast leading to similar concentrations in the plastid and the cytosol is surprising.
PEP is needed in the plastid for the shikimic acid
pathway leading to aromatic amino acids and other
secondary metabolites, and pyruvate is used for fatty
acid biosynthesis. It is still unclear whether potato
tuber amyloplasts have a complete sequence of glycolytic enzymes and are able to synthesize PEP and
pyruvate from hexose phosphates. Although a PEP/
phosphate translocator has recently been found in
several photosynthetic and non-photosynthetic plastids (Fischer et al., 1997), its presence in potato tubers
has not yet been shown.
Adenine and Uridine Nucleotides, with the
Exception of UDP-Glc, Have a Similar Distribution to
Those Found in Leaves
Estimated cytosolic ATP and ADP concentrations
(0.21 and 0.018 mm, respectively) were similar to the
concentrations found in Ricinus communis phloem
sap, which is free from organelles and despite its
specific transport function is often considered as cytosolic (Geigenberger et al., 1993), and in darkened
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693
Farré et al.
spinach (Spinacia oleracea) leaf cytosol (Heineke et al.,
1991), but were lower than concentrations found for
leaves in the light (Heineke et al., 1991). The cytosolic
ADP/ATP ratio (0.08) was lower than the plastidial
ratio (0.49). This situation is analogous to that observed in photosynthetic tissue (wheat [Triticum aestivum] leaf protoplasts, Stitt et al., 1982; spinach
leaves, Heineke et al., 1991). The amyloplast membrane in potato tubers contains an ATP/ADP transporter with a Km for both nucleotides of around 20
␮m (Tjaden et al., 1998). This transporter probably
catalyzes a counterexchange of ATP and/or ADP
(Trentmann et al., 2000). The differences in the ratio
of both nucleotides might favor a net import of ATP,
which in turn could secure the supply of ATP for
starch biosynthesis in potato amyloplasts.
We could only detect ADP-Glc in the fractions
enriched for amyloplasts and therefore were not able
to calculate the subcellular distribution of this compound. However, this finding agrees with the exclusive location of ADP-Glc in the plastid of potato
tubers.
The similarity between the cytosolic ADP/ATP
(0.08) and UDP/UTP (0.15) ratios supports the hypothesis formulated by Dancer et al. (1990) that the
ATP and UTP energy systems are equilibrated via a
cytosolic nucleotide-diphosphate kinase. Uridine nucleotides (UTP and UDP) are almost exclusively located in the cytosol in tubers, similar to the situation
found in leaf tissue (Dancer et al., 1990). Low plastidial concentrations of UTP and UDP are, perhaps, a
general characteristic. Previous studies have concluded that UDP-Glc is absent from chloroplasts
(Gerhardt et al., 1987; Bligny et al., 1990; Dancer et al.,
1990). Therefore, it is surprising that a significant
amount of UDP-Glc was found to be associated with
plastidial markers in potato tubers. Based on the
current knowledge, it can be excluded that UDP-Glc
is a major substrate for starch synthesis in potato
tubers (Kossman and Lloyd, 2000, and references
therein). UDP-Glc had been shown to act as substrate
of amylogenin a protein that was thought to act as
primer for starch synthesis. However, it seems that
amylogenin is not located in the amyloplasts but
rather in the Golgi apparatus and has been proposed
to be involved in cell wall biosynthesis (Dhugga et
al., 1997; Bocca et al., 1999). Because UDP-Glc is the
substrate for cellulose synthesis, the high amount of
UDP-Glc colocalized with the amyloplast might also
be interpreted as indicating the presence of cell wall
fractions comigrating with this compartment. UDPGlc in the plastid might be involved in the biosynthesis of sulfolipids. The enzyme SQD1, which catalyzes the transfer of SO3⫺ to UDP-Glc, is thought to
be involved in the biosynthesis of sulfoquinovosyl
headgroup in plant sulfolipids, and is localized in the
plastids (Essigmann et al., 1998).
694
Although More Than 75% of the Suc Is Located in the
Vacuole, the Concentration of Suc in the
Cytosol and the Vacuole Is Similar
Most of the Suc, Glc, and Fru are located in the
vacuole. However, due to the small volume of the
cytosol relative to the vacuole, cytosolic concentrations (approximately 40 mm for Suc and 33 mm for
Glc) are similar to those in the vacuole (approximately 35 mm for Suc and 32 mm for Glc; Table IV).
Therefore, it appears that sugars do not accumulate
against a concentration gradient in potato tuber
vacuoles. The same distribution has been reported
for leaves of different species (Heineke et al., 1997;
De, 2000). This agrees with experiments showing
facilitated diffusion of Suc across the tonoplast (for
review, see Martinoia et al., 2000). Until now, only
one putative tonoplast Suc transporter has been
cloned, that from sugar beet (Beta vulgaris; Chiou and
Bush, 1996).
Potato tubers are characterized by low invertase
activities during the starch accumulation phase. It is
still unclear which proportion of the Suc is cleaved by
the acid or the alkaline invertases and where this
cleavage occurs. Isla et al. (1998) showed that cleavage of Suc occurs in isolated vacuoles from potato
tubers. However, the antisense repression of a soluble acid invertase in potato did not lead to a change
in the Suc/hexose ratio in growing tubers, only in
cold-stored tubers (Zrenner et al., 1996). Because the
apoplast probably contains significant amounts of
Suc (Trethewey et al., 1999; Fernie et al., 2000), it is
also possible that cleavage occurs via acidic invertases in the extracellular space. The resulting hexoses
may be taken into intracellular compartments via
membrane or vesicular transport. We found a high
vacuolar Glc to Fru ratio and a cytosolic concentration of Fru that was below the level of detection (less
than 5% of the total amount). Because potato tubers
are characterized by low hexokinase and high fructokinase activities (Renz and Stitt, 1993), the Fru produced either in the cytosol or vacuole must in any
case be accessible to the cytosolic fructokinase.
We reproducibly found a low amount of Suc (10
mm [100 mm], Table IV) associated with the plastid.
This is not in agreement with the current textbook
position of Suc being absent from plastids. However,
the possibility of Suc accumulating in the plastid has
been previously described. Chloroplasts from coldhardened cabbage (Brassica oleracea var. capitata) contained up to 20% of the cellular Suc (Santarius and
Milde, 1977) and tobacco (Nicotiana tabacum) plants
expressing a cytosolic invertase accumulate Suc in
the chloroplast up to a concentration of 20 mm (Heineke et al., 1994), similar to the concentration that we
estimate in tubers. Furthermore, Liu and Shannon
(1981b) found Suc associated with maize endosperm
starch granules. The production of fructans in plastids (Gerrits et al., 2001) can be seen as strong indirect
evidence for the presence of Suc in this organelle.
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Plant Physiol. Vol. 127, 2001
Nonaqueous Fractionation of Potato Tubers
Further studies are needed to analyze if plastids have
the capacity to import or synthesize Suc.
The AGPase Reaction Is Irreversible in Vivo, Whereas
Suc Synthase and UGPase Reactions Are Near
Equilibrium But Slightly Biased toward Suc Breakdown
We found that the distributions of PPi and soluble
inorganic pyrophosphatase resemble the distributions found in photosynthetic tissues. Therefore, the
plastidial PPi concentration is very low, 3 ␮m (30 ␮m;
Table I). The calculation of the mass action ratio of
the AGPase reaction shows that it differs significantly from the theoretical equilibrium constant of 1
(Table V). Therefore, this reaction is likely to be effectively irreversible in vivo, supporting a long
standing view of the role of AGPase as a central
regulatory enzyme in starch biosynthesis (Preiss,
1997). The presence of detectable quantities of PPi in
the plastid, even given the large variability observed
in its concentration, leaves open the possibility that
PPi is rapidly recycled from the plastid to the cytosol
to support Suc breakdown (Farré et al., 2000).
Suc synthase and UGPase represent the first two
steps of Suc metabolism in potato tubers. When their
mass action ratios are compared with the Keq (Table
V), it is clear that both reactions are close to equilibrium, although slightly biased toward Suc and UDPGlc breakdown, which is in close agreement with the
data of Geigenberger and Stitt (1993).
We were not able to detect Fru in the cytosol in
either of our experiments. Therefore, for the calculation of the mass action ratio of Suc synthase, we had
to estimate the cytosolic concentration of Fru. We
have assumed it to be 5% of the total Fru content,
which is the estimated limit of detection of our
method (see comments in “Results”). Even if this
assumption would disagree with the real value by a
factor of 5, our estimate of the mass action ratio for
Suc synthase would still be close to its Keq. The
calculated concentrations of Suc (40 mm), UDP (0.06
mm), and UDP-Glc (0.83 mm) are similar to the Kms
of the Suc synthase reaction, which are 50 to 100, 0.1
to 0.7, and 1 to 2 mm, respectively (Avigad, 1982).
Vacuoles Accumulate a Large Range of Sugars, Sugar
Alcohols, Organic Acids, and Amino Acids
The GC-MS measurements show that the vacuole
accumulates a large variety of different compounds:
sugars, sugar alcohols, organic acids, and amino
acids.
Most of the organic acids measured had higher
vacuolar than cytosolic concentrations (they were
grouped in cluster A, Table II). High amounts of
organic acids in vacuoles have been reported from
several species (De, 2000; Martinoia et al., 2000). It
seems that there is a relatively small active organic
Plant Physiol. Vol. 127, 2001
acid pool located in the cytosol, mitochondria and/or
amyloplast, and a large pool in the vacuole. It is
interesting that not only do organic acids that are
TCA cycle intermediates accumulate in vacuoles, but
also shikimic acid and quinic acid, which are involved in the shikimate pathway.
Like the organic acids, most amino acids accumulate in the vacuole. However, for most amino acids,
the vacuolar concentrations were similar to or lower
than the cytosolic concentrations. As found in potato
leaves (Leidreiter et al., 1995) and in other species
(De, 2000), the total amino acid concentration in the
vacuole is lower than in the cytosol although the total
amino acid content is higher. An active extrusion of
amino acids from vacuoles by translocators has been
proposed (Winter et al., 1993; Martinoia et al., 2000).
Little is known about amino acid synthesis in tubers.
It is still unclear to what extent de novo synthesis
occurs as compared with amino acids imported from
the phloem or derived from lytic breakdown of proteins inside the vacuole. The large pool of amino
acids in the vacuole could have two functions: storage of nitrogen and homeostasis of amino acid metabolism (De, 2000).
CONCLUSION
The development and application of a method with
which to study subcellular metabolite distributions
in potato tubers is described. The determination of a
large number of compounds by combining traditional enzymatic measurements with GC-MS measurements reveals that many metabolites (including
amino acids, organic acids, and uridine nucleotides)
have a distribution similar to those in leaf tissue.
Vacuoles contain most of the sugars, sugar alcohols,
and organic and amino acids. The substrates for
starch biosynthesis, hexose phosphates, and ATP
were found in significant amounts in the amyloplast.
Analogous to the leaf situation, soluble inorganic
pyrophosphatase activity was exclusively associated
with the plastid and PPi was mainly located in the
cytosol.
MATERIALS AND METHODS
Materials
Potato (Solanum tuberosum L. cv Desiree) was supplied
by Saatzucht Lange AG (Bad Schwartau, Germany). Plants
were grown from stem cuttings. The plants used for biochemical analysis were raised in the greenhouse in 2-L pots
under a 16-h-light, 8-h-dark regime at 22°C with supplementary light to ensure a minimum of 250 ␮mol photons
m⫺2 s⫺1; plants were 10 weeks old and completely green
when the tubers were harvested. Tubers (20–40 g fresh
weight) were still growing when harvested. This developmental stage of tubers (cv Desiree) is commonly used for
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695
Farré et al.
the study of growing tuber metabolism and comparison of
metabolite data between tubers harvested from plants between 8 and 12 weeks old is readily possible (Trethewey et
al., 1999; Farré et al., 2000, 2001; Roessner et al., 2000;
Tauberger et al., 2000). To sample tuber material, a cylinder
(12-mm diameter) was cut perpendicular to the stolon-apex
axis in the middle of the tuber (Merlo et al., 1993). For
biochemical analysis, tuber slices 1 mm thick were cut from
the cylinder and immediately frozen in liquid nitrogen and
stored at ⫺80°C until use. All enzymes were purchased
from Boehringer Mannheim (Mannheim, Germany), with
the exception of PFP from Propionibacterium freudenreichii
shermanii, which was purchased from Sigma (Deisenhofen,
Germany). All chemicals were obtained from either Sigma
or Merck (Darmstadt, Germany).
Nonaqueous Fractionation of Tuber Tissue
The method described here was originally based upon
the procedure of Stitt et al. (1989) for the analysis of leaf
subcellular metabolism. The material (approximately 4 g
fresh weight per gradient) was homogenized using a ball
mill precooled with liquid nitrogen. The frozen powder
was resuspended in liquid nitrogen, placed in a plastic
beaker, and then dried at 4 Pa for 48 h in a lyophilizer,
which had been precooled to ⫺30°C. The temperature in
the lyophilizer was left to rise to room temperature after
the vacuum had been reached. The lyophilizer was ventilated with dry N2 gas and the plastic beakers were quickly
closed, placed in boxes containing silica gel, and stored in
plastic bags at ⫺20°C. The dry tuber powder in the plastic
beaker was resuspended in 20 mL of a tetrachlorethyleneheptane mixture (66:34 [v/v]; density ⫽ 1.3 g cm ⫺3; the
solvents were dried and stored over molecule sieve beads,
all from Merck) and ultrasonicated for a total of 120 s, with
10-s pulses and 10-s breaks (Bandolin Sonoplus HD 200,
MS 73/D, Bandolin, Berlin). To prevent overheating, the
plastic beaker was placed on ice and closed with a foam
seal. The suspension was then poured through a polyester
monolen sieve with a pore size ⬍30 ␮m, diluted 3-fold with
heptane, and centrifuged for 10 min at 2,200g (CS 6KR,
Beckmann, Munich). The clear supernatant was discarded
and the sediment was resuspended in 3 mL of a
tetrachlorethylene-heptane mixture (1.3 g cm⫺3). Two
200-␮L aliquots were withdrawn (for determination of enzyme activity and metabolites in the unfractionated material), and the remaining material was transferred to a
30-mL Teflon centrifuge tube (Nalge Nunc Int., Hereford,
UK). The gradient was underlayed using an 11-cm-long
needle. A linear gradient (20 mL between 1.43 and 1.62 g
cm⫺3) was made using a gradient former connected to a
peristaltic pump (Econo Pump, Bio-Rad, Munich) followed
by a 5-mL cushion of tetrachlorethylene (1.62 g cm⫺3). The
gradients were centrifuged for 1 h at 10,000g at 7°C (swingout rotor AS4.13, ultracentrifuge Centrikon T-124, Kendro,
Berlin). The centrifuge tube contents were removed from
the bottom in four or five fractions (1–2 mL for fraction 2
and 2–5 mL for other fractions) using the needle. Each of
these fractions was divided into two unequal portions,
696
consisting of one-third of the volume for assay of marker
enzymes and two-thirds of the volume for assay of metabolites. The divided portions and the two aliquots taken
from the material applied to the gradient were all diluted
3-fold with heptane and centrifuged for 10 min at 2,200g
(Beckmann CS 6KR). After discarding the supernatant, the
samples were dried in a desiccator containing a silica gel
drying agent for 12 h, and then extracted for assay of
enzymes or metabolites.
Enzyme Assays
Extracts were prepared as described by Geigenberger
and Stitt (1993) with the exception that bovine serum albumin was not added to the extraction buffer. Enzymes were
assayed according to the following references: ADP-Glcpyrophosphorylase (AGPase; E.C. 2.2.7.27) activity was
measured as described by Müller-Röber et al. (1992); inorganic alkaline pyrophosphatase (E.C.3.6.11) was measured
using the assay described by Jelitto et al. (1992) except that
the reaction buffer contained 20 mm MgCl2, and termination and detection of phosphate was carried out as described by Gross and ap Rees (1986); UGPase (E.C. 2.7.7.9)
was measured as described by Zrenner et al. (1993); PFP
(E.C. 2.7.1.90) was assayed as described by Burrell et al.
(1994); ␣-mannosidase (E.C. 3.2.1.24) was determined as
detailed by Stitt et al. (1989) with the exception that the
reaction was stopped with 1 m NaCO3; and citrate synthase
(E.C. 4.1.3.7) was measured as described by Stitt et al.
(1989). Total protein was determined by the method of
Bradford (1976).
Determination of Metabolic Intermediates
TCA extracts were prepared as described by Trethewey
et al. (1998). Carbohydrates were measured as detailed by
Trethewey et al. (1998) and hexose phosphates, 3-Pglycerate, PEP, and pyruvate were determined in the extracts photometrically as described by Stitt et al. (1989). PPi
was measured according to Farré et al. (2000). Pseudoextracts (without tissue) were also prepared to confirm the
absence of significant PPi contamination in all the solutions
and vessels used in the procedure.
Nucleotides were measured in the same TCA extracts
using an HPLC method (Fernie et al., 2001). The reliability
of the TCA extraction and assay protocol has been confirmed previously (e.g. Trethewey et al., 1998; Veramendi
et al., 1999; Farré et al., 2000).
GC-MS analysis was carried out with methanol extracts
as described by Roessner et al. (2000).
Data Analysis
A three-compartment calculation program (Bestfit) that
has been described in detail by Riens et al. (1991) was used
to evaluate the subcellular metabolite distribution. The
results in Tables I and II represent the means ⫾ se of
measurements on four (Table I) or three (Table II) different
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Copyright © 2001 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 127, 2001
Nonaqueous Fractionation of Potato Tubers
fractionations each from a different tuber sample. Hierarchical cluster analysis was performed with the software
Pirouette 2.6 (Infometrix, Woodinville, WA). The complete
linkage method was then used in the assignment of clusters. The hierarchical cluster analysis uses the Euclidean
distance matrix.
duction to electron microscopy, and Prof. Mark Stitt for
critical comments on the manuscript. The work of P.G. and
A.T. was supported by DFG grant Ge 878/1–1.
Electron Microscopy
LITERATURE CITED
Tuber tissue was fixed in 2% (w/v) glutaraldehyde (in
phosphate buffer, pH 7.4) for 1 h, washed five times in
phosphate buffer, and then contrasted in 2% (w/v) osmium tetraoxide (OsO4) for 1 h and in 1:10 diluted osmium
solution overnight. The samples were washed five times in
double distilled water and dehydrated by subsequent incubation in higher concentrations of acetone (30%, 50%,
70%, 90%, and 100% [w/v]). The samples were then incubated in an acetone/Spurr mix (Spurr, 1969) (increasing
concentrations of Spurr 33%, 66%, and 100% [w/v] for 1 h
per concentration, and overnight at 100% [w/v]). The samples were then placed in Spurr and dried for a week at
60°C. Sample blocks were then trimmed with a razor and
sectioned in an ultra-microtome (Leica Microsystems, Wetzler, Germany) with a glass knife. The slices (50–80 nm)
were placed onto copper grids and dried. Grids were then
stained by incubation in uranil-acetate solution for 6 min
and 6 min in lead-citrate solution. The samples were visualized with a transmission electron microscope (Carl Zeiss,
Göttingen, Germany).
ap Rees T (1987) Compartmentation of plant metabolism.
In DD Davies, ed, The Biochemistry of Plants, Vol 12.
Academic Press, New York, pp 87–115
Avigad G (1982) Sucrose and other disaccharides. In FA
Loewus, W Tanner, eds, Encyclopedia of Plant Physiology. Springer-Verlag, Berlin, pp 217–347
Bligny R, Gardestrom P, Roby C, Douce R (1990) 31P NMR
studies of spinach leaves and their chloroplasts. J Biol
Chem 265: 1319–132
Bocca SN, Kissen R, Rojas-Beltran JA, Noel F, Gebhardt
C, Moreno S, du Jardin P, Tandecarz JS (1999) Molecular cloning and characterization of the enzyme UDPglucose: protein transglucosylase from potato. Plant
Physiol Biochem 37: 809–819
Boller T, Kende H (1979) Hydrolytic enzymes in the central vacuole of plant cells. Plant Physiol 63: 1123–1132
Bradford MM (1976) A rapid and sensitive method for the
quantification of microgram quantities of protein using
the principle of protein dye binding. Anal Biochem 72:
248–254
Burrell MM, Mooney PS, Blundy MDC, Wilson F, Green
J, Blundy KS, ap Rees T (1994) Genetic manipulation of
6-phosphofructokinase in potato tubers. Planta 194:
95–101
Chiou T-J, Bush DR (1996) Molecular cloning, immunochemical localization to the vacuole, and expression in transgenic yeast and tobacco of a putative
sugar transporter from sugar beet. Plant Physiol 110:
511–520
Dancer J, Neuhaus HK, Stitt M (1990) Subcellular compartmentation of uridine nucleotides and nucleoside-5⬘diphosphate kinase in leaves. Plant Physiol 92: 637–641
De DN (2000) Plant Cell Vacuoles: An Introduction. CSIRO
Publishing, Collingwood, Australia
Delesse MA (1847) Procédé mecanique pour determiner la
composition des roches. C R Acad Sci (Paris) 25: 544
Dhugga KS, Tiwari SC, Ray PM (1997) A reversibly glycosylated polypetide (RGP1) possibly involved in plant
cell wall synthesis: purification, gene cloning, and transGolgi localization. Proc Natl Acad Sci USA 94: 7679–7684
Essigmann B, Güler S, Narang RA, Linke D, Benning C
(1998) Phosphate availability affects the thylakoid lipid
composition and the expression of SQD1, a gene required for sulfolipid biosynthesis in Arabidopsis thaliana.
Proc Natl Acad Sci USA 95: 1950–1955
Farré EM, Bachmann A, Willmitzer L, Trethewey RN
(2001) Acceleration of potato tuber sprouting by the expression of a bacterial pyrophosphatase. Nature Biotechnol 19: 268–272
Farré EM, Geigenberger P, Willmitzer L, Trethewey RN
(2000) A possible role for pyrophosphate in the coordi-
Determination of Subcellular Volumes
Electron micrographs of thin sections from fixed material were used for the evaluation of subcelluar volumes
according the principle of Delesse (1847): “the areal density
of profiles on sections is an unbiased estimate of the volume density of structures” (Weibel and Bolender, 1973;
Winter et al., 1993). Over 60 electron micrographs were
taken from material from four independent fixation procedures and from eight different tubers. Low magnification
pictures (⫻700) in which several cells could be visualized
were preferentially used to determine the volumes of most
organelles. High magnification pictures (⬎1,000⫻) were
used to determine the volumes of mitochondria that were
not always visible in the low magnification pictures.
The relative volumes (percent of total) were converted to
absolute volumes per unit of mass by taking into account
that under our growth conditions a tuber slice of 130 mg
fresh weight contained 1 mg protein and had a volume of
113 ␮L (0.86 mL g fresh weight⫺1).
ACKNOWLEDGMENTS
We would like to thank Frank Huhn for careful supervision of greenhouse plants, Cornelia Wagner for the introduction to GC-MS, Doreen Brust for the help with the
cluster analysis, Megan McKenzie for careful editing of the
manuscript, Prof. Dieter Heineke for the Bestfit software
and helpful discussion, Prof. Werner Herth for the introPlant Physiol. Vol. 127, 2001
Received March 21, 2001; returned for revision May 29,
2001; accepted June 22, 2001.
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Copyright © 2001 American Society of Plant Biologists. All rights reserved.
697
Farré et al.
nation of cytosolic and plastidial carbon metabolism
within the potato tuber. Plant Physiol 123: 681–688
Fernie AR, Riesmeier JW, Martini A, Ramalingan S,
Willmitzer L, Trethewey RN (2000) Consequences of the
expression of a bacterial glucokinase in potato tubers,
both in combination with and independently of a yeastderived invertase. Aust J Plant Physiol 27: 827–833
Fernie AR, Roscher A, Ratcliffe RG, Kruger NJ (2001)
Fructose 2,6-bisphosphate activates pyrophosphate:
fructose-6-phosphate 1-phosphotransferase and increase
triose phosphate to hexose phosphate cycling in heterotrophic cells. Planta 212: 250–263
Fischer K, Kammerer B, Gutensohn M, Arbinger B, Weber A, Häusler RE, Flügge UI (1997) A new class of
plastidic phosphate translocators: a putative link between primary and secondary metabolism by the phosphoenolpyruvate/phosphate antiporter. Plant Cell 9:
453–462
Geigenberger P, Langenberger S, Wilke I, Heineke D,
Heldt HW, Stitt M (1993) Sucrose is metabolised by sucrose synthase and glycolysis within the phloem complex
of Ricinus communis L. seedlings. Planta 190: 446–453
Geigenberger P, Merlo L, Reimholz R, Stitt M (1994)
When growing potato tubers are detached from their
mother plant there is a rapid inhibition of starch synthesis, involving inhibition of ADP-glucose pyrophosphorylase. Planta 193: 486–493
Geigenberger P, Reimholz R, Geiger M, Merlo L, Canale
V, Stitt M (1997) Regulation of sucrose and starch metabolism in potato tubers in response to short-term water
deficit. Planta 201: 502–518
Geigenberger P, Stitt M (1993) Sucrose synthase catalyzes
a readily reversible reaction in vivo in developing potato
tubers and other plant tissues. Planta 190: 440–450
Geiger M, Stitt M, Geigenberger P (1998) Metabolism in
potato tuber slices responds differently after addition of
sucrose and glucose. Planta 206: 245–252
Gerhardt R, Heldt HW (1984) Measurement of subcellular
metabolite levels in leaves by fractionation of freezestopped material in nonaqueous media. Plant Physiol 75:
542–547
Gerhardt R, Stitt M, Heldt HW (1987) Subcellular metabolite levels in spinach leaves. Plant Physiol 83: 399–407
Gerrits N, Turk SCHJ, van Dun KPM, Hulleman SHD,
Visser RGF, Weisbeek PJ, Smeekens SCM (2001) Sucrose metabolism in plastids. Plant Physiol 125: 926–934
Gross P, ap Rees T (1986) Alkaline inorganic pyrophosphatase and starch synthesis in amyloplasts. Planta 167:
140–145
Hajirezaei M, Sonnewald U, Viola R, Carlisle S, Dennis
D, Stitt M (1994) Transgenic potato plants with strongly
decreased expression of pyrophosphate:fructose-6phosphate phosphotransferase show no visible phenotype and only minor changes in metabolic fluxes in their
tubers. Planta 192: 16–30
Heineke D, Lohaus G, Winter H (1997) Compartmentation
of C/N Metabolism. In CH Foyer, WP Quick, eds, A
Molecular Approach to Primary Metabolism in Higher
Plants. Tailor & Francis Ltd, London, pp 205–217
698
Heineke D, Riens B, Grosse H, Hoferichter P, Peter U,
Flügge UI, Heldt HW (1991) Redox transfer across the
inner chloroplast envelope membrane. Plant Physiol 95:
1131–1137
Heineke D, Willdenberger K, Sonnewald U, Willmitzer
L, Heldt HW (1994) Accumulation of hexoses in leaf
vacuoles: studies with transgenic tobacco plants expressing yeast-derived invertase in the cytosol, vacuole or
apoplasm. Planta 194: 29–33
Hill LM, Smith AM (1991) Evidence that glucose
6-phosphate is imported as the substrate for starch synthesis by the plastids of developing pea embryos. Planta
185: 91–96
Isla MI, Vattuone MA, Sampietro AR (1998) Hydrolysis of
sucrose within isolated vacuoles from Solanum tuberosum
L. tubers. Planta 205: 601–605
Jelitto T, Sonnewald U, Willmitzer L, Hajirezeai M, Stitt
M (1992) Inorganic pyrophosphate content and metabolites in potato and tobacco plants expressing E.coli pyrophosphate in their cytosol. Planta 188: 238–244
Kammerer B, Fischer K, Hilpert B, Schubert S, Gutesohn
M, Weber A, Flügge UI (1998) Molecular characterization of a carbon transporter in plastids from heterotrophic tissues: the glucose 6-phosphate/phosphate antiporter. Plant Cell 10: 105–117
Kim WT, Franceschi VR, Okita TW, Robinson NL, Morell
M, Preiss J (1989) Immunocytochemical localization of
ADPglucose pyrophosphorylase in developing potato
tuber cells. Plant Physiol 91: 217–220
Kleckowski L (1994) Glucose activation and metabolism
through UDP-glucose pyrophosphorylase in plants. Phytochemistry 37: 1507–1515
Kosegarten H, Zetsche K, Mengel K (1995) Isolation of
intact storage tissue amyloplasts from suspensioncultured potato cells (Solanum tuberosum) and determination of their intermembrane and stroma volumes.
J Appl Bot 69: 211–214
Kruger NJ (1997) Carbohydrate synthesis and degradation.
In D Dennis, DH Turpin, DD Lefebvre, DB Layzell, eds,
Plant Metabolism. Addison Wesley Longman, Harlow,
pp 83–104
Kossman J, Lloyd J (2000) Understanding and influencing
starch biochemistry. Crit Rev Plant Sci 19: 171–226
Leidreiter K, Kruse A, Heineke D, Robinson DG, Heldt
HW (1995) Subcellular volumes and metabolic concentrations in potato (Solanum tuberosum cv. Désirée) leaves.
Bot Acta 108: 439–444
Liu TL, Shannon JC (1981a) A nonaqueous procedure for
isolating starch granules with associated metabolites
from maize (Zea mays L.) endosperm. Plant Physiol 67:
518–524
Liu T-TY, Shannon JC (1981b) Measurement of metabolites associated with nonaqueously isolated starch granules from immature Zea mays L. endosperm. Plant
Physiol 67: 525–529
MacDonald FD, Preiss J (1986) The subcellular location and
characteristics of pyrophosphate-fructose-6-phosphate
phosphotransferase from suspension-cultured cells of
soybean. Planta 167: 240–245
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2001 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 127, 2001
Nonaqueous Fractionation of Potato Tubers
Martinoia E, Massonneau A, Frangne N (2000) Transport
processes of solutes across the vacuolar membrane of
higher plants. Plant Cell Physiol 41: 1175–1186
Merlo L, Geigenberger P, Hajirezaei M, Stitt M (1993)
Changes in carbohydrates metabolites and enzyme activities in potato tubers during development, and within a
single tuber along a stolon-apex gradient. J Plant Physiol
142: 392–402
Müller-Röber B, Sonnewald U, Willmitzer L (1992) Inhibition of the ADP-pyrophosphorylase in transgenic portatoes leads to sugar-storing tubers and influences tuber
formation and expression of tuber storage protein genes.
EMBO J 11: 1229–1238
Naeem M, Tetlow IJ, Emes MJ (1997) Starch synthesis in
amyloplasts purified from developing potato tubers.
Plant J 11: 1095–1103
Neuhaus HE, Emes MJ (2000) Nonphotosynthetic metabolism in plastids. Annu Rev Plant Physiol Plant Mol Biol
51: 111–140
Preiss J (1997) Modulation of starch synthesis. In CH Foyer,
WP Quick, eds, A Molecular Approach to Primary Metabolism in Higher Plants. Tailor & Francis Ltd, London,
pp 81–104
Renz A, Stitt M (1993) Substrate specificity and product inhibition of different forms of fructokinase and
hexokinases in developing potato tubers. Planta 190:
166–175
Riens B, Lohaus G, Heineke D, Heldt HW (1991) Aminoacid and sucrose content determined in the cytosolic,
chloroplastic, and vacuolar compartments and in the
phloem sap of spinach leaves. Plant Physiol 97: 227–233
Roessner U, Wagner C, Kopka J, Trethewey RN,
Willmitzer L (2000) Simultaneous analysis of metabolites
in potato tuber by gas chromatography-mass spectrometry. Plant J 23: 131–142
Santarius KA, Milde H (1977) Sugar compartmentation in
frost-hardy and partially deharded cabbage leaf cells.
Planta 136: 163–166
Schleucher J, Vanderveer P, Markley JL, Sharkey TD
(1999) Intramolecular deuterium distributions reveal disequilibrium of chloroplast phosphoglucose isomerase.
Plant Cell Environ 22: 525–533
Schott K, Borchert S, Müller-Röber B, Heldt HW (1995)
Transport of inorganic phosphate and C3- and C4-sugar
phosphates across the envelope membranes of potato
tuber amyloplasts. Planta 196: 647–652
Shannon J, Pien F, Cao H, Liu K (1998) Brittle-1, and
adenylate translocator facilitates transfer of extraplastidial synthesized ADP-glucose into amyloplasts of
maize endosperms. Plant Physiol 117: 1235–1252
Sharkey TD, Vanderveer PJ (1989) Stromal phosphate concentration is low during feedback limited photosynthesis. Plant Physiol 91: 679–684
Sowokinos JR, Preiss J (1982) Pyrophosphorylases in Solanum tuberosum: III. Purification, physical, and catalytic
properties of ADP-glucose pyrophosphorylase in potatoes. Plant Physiol 69: 1459–1466
Spurr AR (1969) A low viscosity epoxy resin embedding
medium for electron microscopy. J Ultrastruct Res 26: 31
Plant Physiol. Vol. 127, 2001
Stark DM, Timmerman KP, Barry GF, Preiss J, Kishore
GM (1992) Regulation of the amount of starch in plant
tissues by ADP-glucose pyrophosphorylase. Science 258:
287–291
Stitt M, Bulpin PV, ap Rees T (1978) Pathway of starch
breakdown in photosynthetic tissue of Pisum sativum.
Biochim Biophys Acta 544: 200–214
Stitt M, Lilley RM, Heldt HW (1982) Adenine nucleotide
levels in the cytosol, chloroplasts, and mitochondria of
wheat leaf protoplasts. Plant Physiol 70: 971–977
Stitt M, Lilley RM, Gerhardt R, Heldt HW (1989) Metabolite levels in specific cells and subcellular comparments
of plant leaves. Methods Enzymol 174: 518–550
Stitt M (1997) The flux of carbon between the chloroplast
and cytoplasm. In DT Dennis, DB Layzell, DD Lefebvre,
DH Turpin, eds, Plant Metabolism. Longman Singapore
Publishers (Pte) Ltd, Singapore, pp 382–400
Sweetlove LJ, Burrell MM, ap Rees T (1996) Starch metabolism in tubers of transgenic potato (Solanum tuberosum) with increased ADPglucose pyrophosphorylase.
Biochem J 320: 493–498
Tang H-R, Godward J, Hills B (2000) The distribution of
water in native starch granules: a multinuclear NMR
study. Carbohydr Polymers 43: 375–387
Tauberger E, Fernie AR, Emmermann M, Renz A, Kossman J, Willmitzer L, Trethewey RN (2000) Antisense
inhibition of plastidial phosphoglucomutase provides
compelling evidence that potato tuber amyloplasts import carbon from the cytosol in the form of glucose-6phosphate. Plant J 23: 43–53
Tauberger E, Hoffmann-Benning S, Fleisher-Notter H,
Willmitzer L, Fisahn J (1999) Impact of invertase overexpression on cell size, starch granule formation and cell
wall properties during tuber development in potatoes
with modified carbon allocation patterns. J Exper Bot 50:
477–486
Tjaden J, Möhlman T, Kampfenkel K, Henrichs G, Neuhaus HE (1998) Altered plastidic ATP/ADP-transporter
activity influences potato (Solanum tuberosum L.) tuber
morphology, yield and composition of tuber starch.
Plant J 16: 531–540
Trentmann O, Decker C, Winkler HH, Neuhaus HE (2000)
Charged amino-acid residues in transmembrane domains of the plastidic ATP/ADP transporter from Arabidopsis are important for transport efficiency, substrate
specificity, and counter exchange properties. Eur J Biochem 267: 4098–4105
Trethewey RN, Geigenberger P, Riedel K, Hajirezaei
M-R, Sonnewald U, Stitt M, Riesmeier JW, Willmitzer
L (1998) Combined expression of glucokinase and invertase in potato tubers leads to a dramatic reduction in
starch accumulation and a stimulation of glycolysis.
Plant J 15: 109–118
Trethewey RN, Geigenberger P, Sonnewald U, Hennig A,
Müller-Röber B, Willmitzer L (1999) Induction of the
activity of glycolytic enzymes correlates with enhanced
hydrolysis of sucrose in the cytosol of transgenic potato
tubers. Plant Cell Environ 22: 71–79
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2001 American Society of Plant Biologists. All rights reserved.
699
Farré et al.
Veramendi J, Roessner U, Renz A, Willmitzer L, Trethewey RN (1999) Antisense repression of hexokinase
1leads to an overaccumulation of starch in leaves of
trans-genic potato plants but not to significant changes in
tuber carbohydrate metabolism. Plant Physiol 121: 1–11
Weibel ER, Bolender RP (1973) Stereological techniques
for electron microscopic morphometry. In MA Hayat, ed,
Principles and Techniques of Electron Microscopy, Biological Applications. Van Nostrand Reinhold Company,
New York, pp 237–296
Weiner H, Stitt M, Heldt HW (1987) Subcellular compartmentation of pyrophosphate and alkaline pyrophosphatase in leaves. Biochim Biophys Acta 893: 13–21
700
Winter H, Robinson DG, Heldt HW (1993) Subcellularvolumes and metabolite concentrations in barley leaves.
Planta 191: 180–190
Wischmann B, Nielsen TH, Moller BL (1999) In vitro
biosynthesis of phosphorylated starch in intact potato
amyloplasts. Plant Physiol 119: 455–462
Zrenner R, Schüler K, Sonnewald U (1996) Soluble acid
invertase determines the hexose-to-sucrose ratio in coldstored potato tubers. Planta 198: 246–252
Zrenner R, Willmitzer L, Sonnewald U (1993) Analysis of
the expression of potato uridinediphosphate-glucose pyrophosphorylase and its inhibition by antisense RNA.
Planta 190: 247–252
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
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