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Plant Physiology 132:2058-2072 (2003) © 2003 American Society of Plant Biologists A Bypass of Sucrose Synthase Leads to Low Internal Oxygen and Impaired Metabolic Performance in Growing Potato Tubers1Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Golm, Germany
Plants possess two alternative biochemical pathways for sucrose (Suc) degradation. One involves hydrolysis by invertase followed by phosphorylation via hexokinase and fructokinase, and the other route—which is unique to plants—involves a UDP-dependent cleavage of Suc that is catalyzed by Suc synthase (SuSy). In the present work, we tested directly whether a bypass of the endogenous SuSy route by ectopic overexpression of invertase or Suc phosphorylase affects internal oxygen levels in growing tubers and whether this is responsible for their decreased starch content. (a) Oxygen tensions were lower within transgenic tubers than in wild-type tubers. Oxygen tensions decreased within the first 10 mm of tuber tissue, and this gradient was steeper in transgenic tubers. (b) Invertase-overexpressing tubers had higher activities of glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase, and (c) higher levels of lactate. (d) Expression of a low-oxygen-sensitive Adh1-  -glucuronidase reporter gene
construct was more strongly induced in the invertase-overexpressing background
compared with wild-type background. (e) Intact transgenic tubers had lower ATP
to ADP ratios than the wild type. ATP to ADP ratio was restored to wild type,
when discs of transgenic tubers were incubated at 21% (v/v) oxygen. (f) Starch
decreased from the periphery to the center of the tuber. This decrease was
much steeper in the transgenic lines, leading to lower starch content
especially near the center of the tuber. (g) Metabolic fluxes (based on
redistribution of 14C-glucose) and ATP to ADP ratios were analyzed
in more detail, comparing discs incubated at various external oxygen tensions
(0%, 1%, 4%, 8%, 12%, and 21% [v/v]) with intact tubers. Discs of Suc
phosphorylase-expressing lines had similar ATP to ADP ratios and made starch
as fast as wild type in high oxygen but had lower ATP to ADP ratios and lower
rates of starch synthesis than wild type at low-oxygen tensions typical to
those found inside an intact tuber. (h) In discs of wild-type tubers,
subambient oxygen concentrations led to a selective increase in the mRNA
levels of specific SuSy genes, whereas the mRNA levels of genes encoding
vacuolar and apoplastic invertases decreased. (i) These results imply that
repression of invertase and mobilization of Suc via the energetically less
costly route provided by SuSy is important in growing tubers because it
conserves oxygen and allows higher internal oxygen tensions to be maintained
than would otherwise be possible.
Oxygen access to internal tissues can be a problem in plants. Oxygen falls to low levels within metabolically active, dense, or bulky plant tissues, even when external oxygen concentrations are high. Low internal oxygen concentrations have been reported in growing tubers (Geigenberger et al., 2000  ),
developing seeds (Quebedeaux and Hardy,
1976; Porterfield et al.,
1999; Gibon et al.,
2002; Rolletscheck et al.,
2002), fruits (Magness,
1920; Banks, 1983;
Ke et al., 1995), roots
(Lushuk and Salveit, 1991;
Thomson and Greenway, 1991),
and in phloem tissue (van Dongen et al.,
2003). Based on studies in growing wild-type potato (Solanum
tuberosum) tubers, Geigenberger et al.
(2000) concluded that falling
internal oxygen leads to: (a) a restriction of glycolysis and respiration and
a decrease in adenylate levels; (b) a widespread decrease in biosynthetic
activity, which decreases ATP consumption; and (c) a switch to pathways that
consume less ATP. They pointed out that saving ATP could be an important
metabolic adaptation to decrease oxygen consumption and prevent the tissue
from driving itself into anoxia.
There are two alternative routes of Suc degradation in plants. One involves
irreversible hydrolysis (
The two pathways of Suc degradation to hexose-phosphates differ in their
energy costs. Although breakdown of a molecule of Suc via invertase requires
two molecules of ATP, breakdown via SuSy and UGPase requires only one molecule
of PPi (Huber and Akazawa,
1986
In view of the possible implications for oxygen consumption, we decided to
investigate if Suc degradation via SuSy allows maintenance of increased
internal oxygen levels and improved storage metabolism in tubers. Ectopic
overexpression of invertase (Sonnewald et
al., 1997
Ectopic Expression of Invertase or Suc Phosphorylase Leads to Steeper Oxygen Gradients within Growing Tubers
To investigate whether the bypass of the endogenous SuSy route affects
internal oxygen tensions in growing tubers, oxygen concentrations were
measured within tubers of the transgenic plants expressing invertase or Suc
phosphorylase via the tuber-specific B33 patatin promoter
(Fig. 1). At 2 and 10 mm below
the periderm, oxygen concentrations were measured directly by inserting an
oxygen micro-electrode (tip diameter < 1 mm) along a transverse axis into
the tuber. In the wild type, oxygen levels showed large gradients from the
surface toward the center of the tuber. Oxygen fell from 21% (v/v) at the
tuber surface to 9.7% (v/v) at 2 mm and to 4.3% (v/v) at 10 mm below the
periderm (Fig. 1). Similar
gradients were seen in tubers of different sizes (approximately 10–30 g
fresh weight), but absolute values for oxygen concentrations were generally
lower when tuber size increased (data not shown). This is probably due to a
decrease in the surface to volume ratio under these conditions. The results
are similar to the oxygen gradients reported earlier in growing wild-type
tubers (Geigenberger et al.,
2000
Because oxygen concentrations fell to very low levels within transgenic
tubers, almost approaching zero, we investigated whether this was accompanied
by increased expression of fermentative enzymes or accumulation of the
respective fermentation products (Fig.
2). The activities of ADH (Fig.
2D) and LDH (Fig.
2E), representing enzymes involved in ethanolic and lactic
fermentation, respectively, were present in wild-type tubers, which confirms
previous findings (Geigenberger et al.,
2000
The induction of LDH was accompanied by an increase in lactate levels in the transgenic tubers, which was, however, only significant for line Inv2-30 (Fig. 2F). There were only slight changes in ethanol levels in the transformants; however, these were not consistent across lines—a fact that may be due to very large fluctuations in ethanol concentrations between individual tubers (data not shown).
Detailed studies demonstrate that Adh1 is progressively induced
when oxygen is decreased over a wide range of subambient concentrations in
Arabidopsis (Dolferus et al.,
1994 Figure 3 shows GUS staining in representative lines when smaller tubers (approximately 5–10 g fresh weight) were compared. In lines expressing GUS in the wild-type background, GUS activity was highest in the vascular bundles of stems and tubers, and much weaker staining was observed in tuber parenchyma tissue. This contrasts with lines expressing GUS in the Inv2-30 background, which showed strong GUS staining also in the tuber parenchyma tissue in addition to the vascular bundles. Similar results were obtained across all transformant lines. When tubers became larger (20–40 g fresh weight), GUS staining increased in the tuber parenchyma of wild-type GUS lines, whereas no further increase was observed in Inv-GUS transformants (data not shown). Independently of the genetic background, GUS staining was similar in the parenchyma and vascular tissues of the stems (Fig. 3). The specific induction of GUS expression in the parenchyma tissue of small tubers in lines with an Inv2-30 background is consistent with oxygen being decreased in these lines as a consequence of overexpression of invertase via the B33 promoter. Surprisingly, no clear gradient in GUS staining was observed within tuber transects; however, this could be due to bleeding of the product of the GUS reaction across cells.
At the moment, we cannot exclude that factors other than low oxygen led to
increased GUS expression in the transgenic lines. The Arabidopsis Adh1-gene is
also induced by low temperature, dehydration, and wounding
(Dolferus et al., 1994
To investigate whether the decrease in internal oxygen levels affects
energy metabolism in transgenic tubers, ATP to ADP ratios were analyzed in
tissue sampled rapidly (within 2 s) from the center of an intact tuber. The
ATP to ADP ratio was relatively low in growing wild-type tubers (see also
Geigenberger et al., 2000
The decrease in adenylate energy state could be reversed by incubating freshly cut discs from invertase (Fig. 4A) and Suc phosphorylase-expressing (Fig. 4B) tubers (1 mm thick, 8-mm diameter) in aerated buffer solutions for 2 h. Addition of 100 mM Suc had no substantial effect on ATP to ADP ratios. This confirms that the low cellular energy state in intact tubers and the additional decrease of the energy state in the transformant tubers is due to low internal oxygen levels. We used line SP-2 to investigate in more detail the short-term changes in ATP to ADP ratios after exposure of discs to air (results are means ± SE, n = 3): In discs frozen in liquid nitrogen within 2 s of harvesting, the ATP to ADP ratio was lower in the transformant (1.4 ± 0.1) than wild-type (2.1 ± 0.2) material. With 2 min of exposure to air after cutting the discs, the ATP to ADP ratio recovered, reaching higher values in SP-2 (3.8 ± 0.5) than in wild type (3.0 ± 0.3). After 10 min, ATP to ADP ratios increased further to 4.5 ± 0.5 in SP-2 and 4.2 ± 0.5 in the wild type. These results demonstrate that tissue has to be quenched immediately after sampling from potato tubers to allow accurate measurements of adenine nucleotide levels.
Previous studies showed that ectopic expression of invertase
(Trethewey et al., 1998
The above results show a striking similarity between internal oxygen and
starch gradients, which suggest a link between starch synthesis and oxygen
tension in tubers. We used line SP-2 to investigate in more detail whether the
decrease in starch levels in the transgenic tubers is an indirect effect due
to the lower oxygen concentrations. Thin tissue discs (1 mm thick, 8-mm
diameter) were prepared from the center of wild-type and Suc
phosphorylase-expressing tubers and incubated in 1 mM
[U-14C]Glc in the presence of 0%, 1%, 4%, 8%, 12%, or 21% (v/v)
oxygen by bubbling premixed gases through the medium. After 2 h,
redistribution of radiolabel into starch, phosphoester, organic acids, amino
acids, and Suc was analyzed, and starch synthetic, glycolytic, and Suc
synthetic fluxes calculated (Fig. 6,
A–J). For comparison, distribution of radiolabel and
metabolic fluxes was also investigated after injection of [U-14C]
Glc into intact tubers in planta. Previous studies have shown that this
approach cannot be used for invertase-expressing tubers because in this case,
incoming 14C-Glc is mixing with large endogenous pools, leading to
complex and massive isotopic dilution effects
(Trethewey et al., 1999
There were no significant changes in the uptake of [U-14C]Glc
between wild type and transformant or in response to changes in external
oxygen tension (data not shown). Figure 6,
A to E, show the percentage distribution of radiolabel that was
metabolized to other compounds. In wild-type discs incubated at 21% (v/v)
oxygen, the largest portion of label was converted to starch (approximately
60%; Fig. 6A), which is
consistent with earlier studies (see
Geigenberger et al., 1997
In discs of Suc phosphorylase-expressing tubers incubated at 21% (v/v)
oxygen, less of the label was distributed to starch
(Fig. 6A) and more of the label
was distributed to phosphoesters (Fig.
6B) and Suc (Fig.
6E) than in wild-type discs (see also
Fernie et al., 2002
In Figure 6, A to E, label
distribution in intact tubers also was analyzed. Expression of Suc
phosphorylase led to similar changes in label distribution between starch,
organic acids, and amino acids as in discs incubated at low (1%–4%
[v/v]) external oxygen. Labeling of phosphoesters and Suc was different in
discs compared with intact tubers. This could be due to changes in the
internal Suc and hexose-phosphate pools during incubation of discs, which have
been frequently observed in earlier experiments
(Geigenberger et al.,
1997
The absorbed [U-14C] Glc will mix with internal unlabeled pools,
so movement of label will not necessarily reflect fluxes into the various
pools (Geigenberger et al.,
1997 To estimate glycolytic flux (Fig. 6H), label in organic acids and amino acids was summed and divided by the specific activity of the hexose-phosphate pool. Compared with the wild type, Suc phosphorylase-expressing tuber discs had higher rates of glycolysis at 21% (v/v) external oxygen. Decreasing oxygen in the range between 20% and 1% (v/v) did not lead to a restriction of glycolysis in discs of the transformants. When oxygen was decreased to 1% (v/v) or below, there was a stronger increase of glycolytic flux in the transformant than in wild-type discs in absolute terms, probably reflecting increased fermentative activity. Again, the results obtained in intact tubers resembled those for discs incubated in low oxygen (Fig. 6H). Expression of Suc phosphorylase led to a dramatic increase in the rate of Suc synthesis in discs at high external oxygen levels, which was less marked at low oxygen (Fig. 6I). The ratio between the rate of starch synthesis and the rate of glycolysis is shown in Figure 6J. Expression of Suc phosphorylase led to a general decrease in starch synthesis relative to glycolysis, but the decrease was much stronger in discs incubated at low oxygen or in intact tubers. At 0% to 1% (v/v) external oxygen, the decrease was dramatic, leading to starch to glycolysis ratios of almost zero in the transformant.
The labeling studies presented in Figure 6, A to J, show that Suc phosphorylase expression leads to a decreased rate of starch synthesis in the presence of low-oxygen tensions but not at high oxygen. To investigate whether this could be due to changes in the adenylate energy state under these conditions, we analyzed ATP and ADP levels in samples taken in parallel (Fig. 6, K–N). Expression of Suc phosphorylase led to significantly higher ATP levels (Fig. 6K) and a higher ATP to ADP ratio in discs incubated at high external oxygen (12% and 21% [v/v]). However, when oxygen was decreased below 8% (v/v), ATP level and ATP to ADP ratio were more severely reduced in Suc phosphorylase-expressing tuber tissue than in the wild type, leading to a progressively lower adenylate energy state. Expression of Suc phosphorylase led to a similar decrease of the ATP to ADP ratio of intact tubers, which resembled discs incubated at 1% to 4% (v/v) external oxygen. These changes in adenylate energy state reflect the changes in the rate of starch synthesis (compare Fig. 6, G with M), indicating that the low-oxygen-induced inhibition of starch synthesis in the transformant is attributable to a decrease in the cellular energy state under these conditions.
The results presented so far suggest that a bypass of the endogenous SuSy route leads to impaired metabolic performance in hypoxic conditions. This occurs because: (a) Increased oxygen consumption leads to lower tissue oxygen levels, and (b) The transformants perform less effectively than wild-type tubers at low tissue oxygen tensions. The question is raised of whether expression of the endogenous genes encoding SuSy and invertase in tubers is regulated to allow SuSy to substitute for invertase when the oxygen concentration decreases.
Recent studies with maize roots demonstrate that specific SuSy genes are
induced and invertase gene repressed upon hypoxia
(Zeng et al., 1999
Low oxygen led to a selective increase in the mRNA levels of Sus2 and Sus3, which resembled the increase of Adh1 (Fig. 7, A, C, D, G). Sus1 mRNA levels were not substantially altered (Fig. 7, A, B), except an approximately 2-fold increase at 45 min in zero oxygen. Induction of Sus2 and Sus3 was already evident after 45 min of incubation and increased progressively with decreasing oxygen tensions. The mRNA levels of Sus2 and Sus3 were also high in intact tubers, resembling discs under low oxygen (Fig. 7A). Both Sus2 and Sus3 mRNA levels showed a further dramatic increase when intact tubers were submerged for 24 h (data not shown). We do not think that these changes in gene expression reflect wound responses because: (a) Expression of wound-inducible Sus1 was largely unchanged, and (b) Expression of Sus2 and Sus3 also increased in intact tubers under low oxygen. Low oxygen led to a reciprocal decrease in mRNA levels, down to the limits of detection, of genes encoding vacuolar and apoplastic invertase (Fig. 7, A, E, and F). Expression of invertase was not detectable in intact tubers and in discs incubated at low oxygen but was clearly induced upon incubation of discs in 8% to 21% (v/v) external oxygen.
Analysis of enzyme activities revealed a 2-fold decrease of invertase
activity in response to low oxygen after 8 h, whereas total SuSy activity was
unaltered (Fig. 8A). However,
there were changes in the subcellular distribution of SuSy activity. When
oxygen was decreased, less SuSy activity was found in the microsomal fraction,
whereas more SuSy activity was found in the soluble fraction
(Fig. 8B). Binding of SuSy to
the microsomal fraction has been found previously to be involved in the
channeling of carbon toward cell wall synthesis
(Winter et al., 1997
Our results show that overexpression of invertase or Suc phosphorylase to bypass the energetically less expensive SuSy route leads to a strong decrease in internal oxygen tensions in growing tubers, a marked decrease in their energy state, and an inhibition of starch synthesis. These marked changes in metabolism are due to: (a) increased oxygen consumption, leading to lower tissue oxygen levels; and (b) a less effective metabolic performance at low tissue oxygen tension. It implies an important role for the plant-specific SuSy pathway in reducing the degree to which internal oxygen falls and in allowing better maintenance of metabolism under the low-oxygen tensions present within plant tissues.
We provide several independent lines of evidence that overexpression of Suc
phosphorylase or invertase leads to lower oxygen tensions within growing
tubers. First, direct measurements of oxygen concentrations within growing
tubers reveal decreased oxygen levels compared with the wild type
(Fig. 1). Second, analyses of
enzyme activities reveal that enzymes that are known to be induced by
low-oxygen conditions (anaerobic proteins; see
Dennis et al., 2000
We propose that the decrease in internal oxygen levels is a direct
consequence of increased oxygen consumption within the transgenic tubers.
Expression of Suc phosphorylase or invertase leads to a 2- or 3-fold increase
in respiration rates, respectively (Trethewey et al.,
1998
A further interesting possibility is that the heterologous invertase and
Suc phosphorylase proteins are not susceptible to feedback mechanisms that
regulate the rate of Suc degradation in wild-type tubers, in particular,
mechanisms that decrease Suc degradation when oxygen levels fall. In wild-type
tubers, falling ATP and rising ADP may restrict fructokinase activity, leading
to an increase of Fru and feedback inhibition of SuSy when oxygen is low
(Geigenberger et al., 2000
There was a marked decrease in the adenylate energy state in transgenic
tubers compared with wild type (Figs.
4 and
6;
Fernie et al., 2002 There were steep starch gradients from the periphery toward the center of transgenic tubers (Fig. 5), similar to the gradients in oxygen concentration (Fig. 1). The spatial decrease in starch content was much steeper in the transformants than in wild-type tubers. The decrease in starch content in the transformants relative to the wild type was highest near the tuber center, where tissue oxygen levels fall below 2% (v/v). This is broadly in agreement with the measurements of the effect of low oxygen on the rate of starch synthesis in tuber discs, in which a marked inhibition was not found until oxygen dropped to about 4% and 1% (v/v) in discs from Suc phosphorylase and wild-type tubers, respectively (Fig. 6). Further, these labeling experiments showed that expression of Suc phosphorylase leads to a decreased rate of starch synthesis in intact tubers, which is reversed if the tuber discs are incubated at 21% (v/v) oxygen. This provides evidence that the inhibition of starch synthesis in intact transgenic tubers is attributable to decreased tissue oxygen tensions.
The immediate cause of the inhibition of starch synthesis in low oxygen is
probably the low adenylate energy state. It has been demonstrated recently
that antisense inhibition of the plastidic ATP to ADP translocator leads to an
inhibition of starch accumulation in potato tubers
(Tjaden et al., 1998
Previous studies show that low oxygen also leads to an increased
sensitivity of potato tubers toward pathogens
(Butler et al., 1990
In contrast to animals, plants lack specialized circulation systems, and
oxygen falls to low levels within many plant tissues (see above). Based on
studies in potato tubers, Geigenberger et al.
(2000
These results further imply a specific role of PPi in conserving oxygen.
The cytosol of the plant cell contains significant levels of PPi
(Weiner et al., 1987
Experiments with tuber slices show that the reductions in cellular energy
state and starch synthetic rates in response to Suc phosphorylase expression
are also present in isolated tuber discs incubated in low external oxygen (0%,
1%, or 4% [v/v]). In these instances, changes in energy state and starch flux
in transgenic versus wild-type tuber discs are unlikely to be due to
differences in tissue oxygen concentrations. This indicates that a bypass of
the SuSy pathway also leads to less effective metabolic performance at a given
low tissue oxygen level, probably due to a decrease in the energy efficiency
of Suc degradation (Fig. 6M).
Interestingly, this is accompanied by an increased glycolytic flux and
fermentative activity in zero oxygen (Fig.
6H) and is consistent with earlier studies on an
Sus1:Sh1 double mutant in maize and an Sus1
antisense line in potato, indicating that decreased SuSy leads to impaired
anoxic and postanoxic resistance in roots
(Ricard et al., 1998
Our results imply that a shift in the pathway of Suc degradation from
invertase to SuSy allows a higher cellular energy state to be established in
the presence of a lower respiratory or fermentative activity at a given
low-oxygen tension. A reduction of cellular energy requirements and a
concomitant suppression of ATP-generating pathways have been identified
previously as important adaptive responses to low oxygen, both in animals
(Hochachka et al., 1997
Plant Material
Potato plants (Solanum tuberosum L. cv Desirée, Saatzucht
Fritz Lange, Bad Schwartau, Germany) were grown in well-aerated soil (3-L
pots) supplemented with Hakaphos grün slow-release fertilizer (100 g per
230 L of soil; BASF, Ludwigshafen, Germany) in a growth chamber (350 µmol
photons m-2 s-1 irradiance, 14-h day/10-h night regime,
20°C, 50% relative humidity), or in a greenhouse during the summer (16 h
of light/8 h of dark, 20°C/18°C day/night, 60% relative humidity) with
supplementing light as in Tiessen et al.
(2002
Transformation of wild-type potato cv Desirée and the yeast
(Saccharomyces cerevisiae) invertase-expressing line U-INV2-30
(Sonnewald et al., 1997
A cork borer (8-mm diameter) was forced through the middle, removed, and
the tissue plug rapidly forced out and simultaneously sliced into
approximately 1-mm-thick discs, which fell directly into liquid nitrogen
(Geigenberger et al., 2000
Intact tubers growing near the surface of the pot (where the oxygen concentration of the soil was above 18% [v/v]; data not shown) were excavated. The internal oxygen tension was measured 1 to 2 min later by introducing an O2 microelectrode (diameter of the tip < 1 mm; Toepffer Lab Systems, Goeppingen, Germany) into the tuber tissue.
Tuber discs (8-mm diameter, 1-mm thickness) were cut directly from the
center of growing tubers attached to the fully photosynthesizing mother plant,
washed quickly with 10 mM MES (pH 6.5; KOH), pre-incubated for 45
min in buffer containing 2 mM Glc and 20 mM mannitol
using 50-mL Falcon tubes in a water bath at 20°C (approximately eight
discs in 20 mL), and [U-14C]Glc (final specific activity 18.5 KBq
µmol-1; Amersham-Buchler, Freiburg, Germany) was added, and
incubation was continued for another 2 h
(Geigenberger et al., 2000
Labeling experiments with intact tubers were performed as in Geigenberger
and Stitt (2000
Discs were extracted with 80% (v/v) ethanol at 80°C (1 mL per two
discs), re-extracted in two subsequent steps with 50% (v/v) ethanol (1 mL per
two discs for each step), the combined supernatants dried under an air stream
at 35°C, taken up in 1 mL of water ("soluble fraction"), and
separated into neutral, anionic, and basic fractions by ion-exchange
chromatography; the neutral fraction (2.5 mL) was freeze dried, taken up in
100 µL of water, and further analyzed by thin-layer chromatography
(Geigenberger et al., 1997
Tissue slices (30 discs in approximately 80 mL of medium) were incubated
using glass vessels allowing continuous aeration with premixed gases (see
above). Slices were harvested as in Geigenberger et al.
(2000
Total RNA was extracted from potato tubers according to Logemann et al.
(1987
Enzymes were extracted according to Geigenberger and Stitt
(1993
We wish to thank Mark Stitt (MPI Molecular Plant Physiology, Golm, Germany) for his support and interest in this work, stimulating discussions, and helpful comments on the manuscript. We are grateful to Lothar Willmitzer (MPI Molecular Plant Physiology, Golm, Germany) for support and providing the Suc phosphorylase-expressing plants, Uwe Sonnewald (IPK, Gatensleben, Germany) for providing the invertase-expressing lines, Liz Dennis (Commonwealth Scientific and Industrial Research Organization, Plant Industry, Canberra, Australia) for the gift of the Arabidopsis ADH1-GUS construct, Norman Brisson (Département de Biochemie, Université de Montreal) for providing the potato Adh1 cDNA, and Rita Zrenner (MPI Molecular Plant Physiology, Golm, Germany) for providing the potato invertase cDNAs. We are grateful to Ute Roessner (MPI Molecular Plant Physiology, Golm, Germany) for technical help during harvest of material, to Björn Junker (MPI Molecular Plant Physiology, Golm, Germany) for photographic work, and to John Lunn (MPI Molecular Plant Physiology, Golm, Germany) for critical reading of the manuscript. Received February 17, 2003; returned for revision March 6, 2003; accepted May 5, 2003.
1 This work was supported by the Deutsche Forschungsgemeinschaft (grant nos. Ge 878/1–1 and Ge 878/1–3 to K.L.B. and P.G.). 
2 These authors contributed equally to the paper.
3 Present address: Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328,
69120 Heidelberg, Germany.
4 Present address: Fisiologia Molecular de Plantas, Departamento de Biologia
Vegetal, Universidade Federal de Viçosa, Viçosa, Brazil. * Corresponding author; e-mail geigenberger{at}mpimp-golm.mpg.de; fax 49–331–567–8408.
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ABSTRACT
RESULTS
G0' = -29.3 kJ
mol-1) into Glc and Fru via invertase, with a low
Km for Suc (7–15 mM;
Avigad, 1982
