Download Impact of invertase overexpression on cell size

Journal of Experimental Botany, Vol. 50, No. 333, pp. 477–486, April 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
Eva Tauberger1, Susanne Hoffmann-Benning2, Helga Fleischer-Notter1, Lothar Willmitzer1 and
Joachim Fisahn1,3
1 Max Planck Institut für molekulare Pflanzenphysiologie, Karl Liebknechtstr. 25, D-14476 Golm, Germany
2 Institut für Genbiologische Forschung, i.L. Ihnestr. 63, D-14195 Berlin, Germany
Received 8 June 1998; Accepted 25 September 1998
Abstract
Introduction
Transgenic potato tubers that overexpressed either a
cytosolic or an apoplastic invertase in the wild type or
AGPase antisense background were used to analyse
the effect of invertase activity on cell expansion,
starch granule formation and turgor pressure during
tuber development. Although the transgenic plants did
not develop a visible phenotype in aerial regions the
size and number of tubers were significantly modified
in the various lines. Transmission electron and light
microscopy were performed to monitor starch grain
size and number, cell size and cell wall thickness.
Water potential, osmotic pressure, and, indirectly,
turgor pressure were determined during the final
stages of tuber development. Glucose levels were high
in transgenic tubers that overexpressed a yeastderived invertase. The number of starch grains per
cell was almost identical in all transgenic lines.
However, the amount of starch was modified in the
transgenics as compared to the wild type. As expected,
the size of starch grains was reduced in all lines that
expressed an AGPase antisense mRNA. These results
indicate that invertase activity and glucose levels do
not affect initiation of starch grain formation during
the early stages of tuber development, but growth of
starch corns in the later stages of tuber maturation.
Sucrose is the major form of translocated carbon in most
plant species. Therefore, it is a major substrate for plant
growth and generation of turgor pressure. In potato there
is strong evidence that sucrose is unloaded from the
phloem into tuber storage parenchyma cells through
plasmodesmata (symplastic unloading, Oparka and Prior,
1988; Oparka et al., 1992). The initial cleavage of sucrose
in potato tubers occurs mostly through sucrose synthase
and to a smaller extent through invertase (Pressey, 1969;
ap Rees and Morrell, 1990; Ross and Davies, 1992). It
was reported that the activity of sucrose synthase was
low in small tubers, but increased to a peak before
declining at the end of the growing season (Pressey, 1969;
Tsay and Kuo, 1980). To investigate the role of sucrose
cleavage during tuber development on a cellular basis,
transgenic potato plants were used which overexpressed
a yeast-derived invertase in the cytosol or the apoplast in
a wild-type (Büssis et al., 1997; Sonnewald et al., 1997)
or AGPase antisense background.
Invertase has also been discussed to be involved in the
control of phloem unloading and rapid growth of sink
organs. In terms of a model described by Roitsch and
Tanner (1996) sucrose would be released from the sieve
elements into the apoplast and subsequently split by
invertase. Uptake of hexoses into the sink cells would
then be mediated by hexose transport proteins. A number
of reports indicate that in organs which grow by cell
elongation, acid invertase is the dominant enzyme for
Key words: Potato, tuber, development, starch, osmotic
pressure, water potential.
3 To whom correspondence should be addressed. Fax: +49 331 977 2301. E-mail [email protected]
Abbreviations: AGPase, ADP-glucose-pyrophosphorylase; TEM, transmission electron microscopy.
© Oxford University Press 1999
478 Tauberger et al.
Fig. 1. Developmental stages of a potato tuber. Growth of potato
tubers was divided into six classes which are depicted in the photograph.
Stage one was the earliest stage of tuber development and characterized
by a thickening of the stolon. Mature tubers were obtained in stage 6.
sucrose breakdown, whereas in non-growing sink tissue
sucrose synthase predominates (Sung et al., 1989). In
dark-grown seedlings of sunflower as well as in bean
internodes and pea epicotyls the specific activities of
invertase were closely associated with the rate of cell
elongation (Maclachland et al., 1970; Morris and Arthur,
1985; Kutschera and Köhler, 1994). The potential importance of invertase during growth and development is also
indicated in transgenic plants overexpressing yeast
invertase in various compartments of Arabidopsis (von
Schaewen et al., 1990), tobacco ( Heinecke et al., 1994;
Hoffmann-Benning et al., 1997; Sonnewald et al., 1997),
potato (Büssis et al., 1997; Sonnewald et al., 1997), and
tomato (Dickinson et al., 1991).
Cleavage of sucrose can affect turgor pressure. This
was demonstrated in a recent study by Hoffmann-Benning
et al. (1997). In particular, transgenic tobacco plants that
overexpressed a yeast-derived invertase in the vacuole
exhibited a severe increase in turgor pressure within
epidermis and mesophyll cells. In contrast, turgor and
osmotic pressure remained largely constant during rapid
stem elongation in darkness ( Kutschera, 1991a, b). Since
stem elongation is accompanied by a corresponding
accumulation of soluble sugars, it was proposed that
uptake of hexoses into expanding cells may counter the
dilution of the vacuolar contents caused by water uptake.
Hence internal osmotic pressure and thus cell turgor
could be maintained by the uptake of sugars.
However, sugars not only function as substrates for
growth but affect sugar-sensing systems that initiate
changes in gene expression ( Koch, 1996). The realization
that these genes are also affected by exogenous stimuli
(Sturm and Chrispeels, 1990) and endogenous hormones
further complicates our understanding of the mechanisms
for the regulation of sugar metabolism, turgor and
growth. Recently it has become apparent that sugars
affect the expression of certain genes involved in many
essential processes such as photosynthesis, glycolysis,
sucrose and starch metabolism, defense mechanisms, cell
cycle regulation, and growth (Sheen, 1994; Koch, 1996).
The aim of the present investigation was to elucidate
the relationship between sucrose cleavage and cell size,
starch granule formation, and turgor pressure during
potato tuber development. Genetic engineering provides
a novel approach to modulate the degree of sucrose
cleavage and thus growth and development of specific
plant organs. Several lines of transgenic plants were used
that overexpressed an invertase which was targeted either
to the cytosol or to the apoplast in a wild-type or an
AGPase antisense background. All transgenic plants
showed no phenotype in aerial parts, but changes in
tuber development and size and in starch and sugar
accumulation.
Materials and methods
Plant material
Wild-type and transgenic plants were grown in the greenhouse
at a 25 °C day (14 h) and a 20 °C night temperature and watered
twice a day as described previously (Fisahn et al., 1995). The
relative humidity was 65%.
Wild type (Solanum tuberosum cv. Desirée) were obtained
from Saatzucht Fritz Lange (Bad Schwartau, Germany).
U-IN-1: transgenic plants expressed an apoplastic invertase
from Sacchoromyces cerevisiae (Sonnewald et al., 1991).
U-IN-2: transgenic lines expressed a cytosolic invertase from
Sacchoromyces cerevisiae (Sonnewald et al., 1997).
B-AGP-93: transgenic potato expressed ADP glucosepyrophosphorylase antisense mRNA (Müller-Röber et al.,
1992).
H-AI: AGPase antisense plant with a second transgene
coding for an invertase from Sacchoromyces cerevisiae that was
targeted to the apoplast (Trethewey et al., 1998).
H-IN: AGPase antisense plant with a second transgene
coding for an invertase from Sacchoromyces cerevisiae that was
targeted to the cytosol ( Trethewey et al., 1998).
The genes of the transgenic plants were under the transcriptional control of the class I patatin promotor (B33) which
shows tuber specific expression.
Tuber sample collection
Experiments were performed on the tuber developmental stages
shown in Fig. 1. Samples for microscopy and biochemical
analysis were taken in the morning. The protocol for sample
collection is depicted in Fig. 2. To reduce variations due to
different potato tuber areas equivalent samples were taken from
all stages of tuber development: In stages 1–3 cross-sections of
1 mm were taken and the outer half viewed in the microscope.
In stages 4–6 samples were taken with a cork-borer (5 mm
diameter), discarded the outermost mm and used the next 1 cm
of the resulting cylinder for biochemical and the next 1–2 mm
for microscopical analysis.
Microscopy
Samples were collected as described above. Quarters of the
resulting discs were fixed and embedded as described in
Hoffmann-Benning et al. (1994). Ultrathin sections (80 nm)
were stained with lead citrate and viewed in a Philips 400
transmission electron microscope (TEM ) at the Max Planck
Effects of invertase overexpression on tuber development 479
tubers than the wild type and U-IN-2 plants more but
smaller ones. The latter result has also been found by
Sonnewald et al. (1997). Supertransformants expressing
both invertase and an AGPase antisense construct had
tuber sizes and numbers intermediate between wild type
and AGPase antisense plants.
Tuber developmental stages
Tubers of different lines were analysed during several
stages of development ( Fig. 1). State 1 was characterized
by a thickening of the stolon. The later phases were
divided into five additional classes. Mature tubers were
developed in stadium 6.
Sugar analysis
Fig. 2. Schematic diagram of the protocol that was applied for sample
collection. For investigation of the tuber developmental stages 1–3,
cross-sections were used to measure parameters of interest. To obtain
samples from developmental stages 4–6, a cork-borer was radially
inserted into growing tubers.
Institute for Molecular Biology, Berlin. Measurements of cell
wall thickness were obtained from these micrographs.
Osmotic pressure
1 cm slices of all tuber stages were homogenized for 15–20 s
and centrifuged at 13 000 rpm for 1 min. The osmotic pressure
of 50 ml supernatant or appropriate dilutions was determined
using a microliter osmometer (Osmomat 030, Gonotech, Berlin,
Germany; Hoffmann-Benning et al., 1997).
Water potential measurements
1 mm slices of stages 4–6 were collected with a cork-borer with
opening diameter of 5 mm. Water potential was measured using
a Wescor-H33T dew point microvoltmeter and C52 sample
chamber (Logan, UT, USA).
Sucrose and hexose concentrations
Samples were immediately frozen, extracted in 80% ethanol at
80 °C for 1 h and sugars measured as described (Stitt et al.,
1989; Sonnewald et al., 1991).
All measurements are presented as mean values plus standard
deviation. Means were calculated from at least 20 repeats.
Results
Phenotypes and number of tubers
No visible phenotype evolved in the aerial regions of all
transgenic lines that were investigated in the present
study. They all resembled the wild type. However,
significant differences between the transgenic lines
emerged in the number and the size of tubers ( Fig. 3;
Table 1). AGPase antisense plants were characterized by
a higher amount of tubers, but, of smaller size. U-IN-1
plants (apoplastic localization) had fewer, but bigger
As expected, sugar analysis resulted in high levels of
glucose in transgenic plants that overexpressed a yeastderived invertase ( Fig. 4). This increase started to be
visible in stage 3 except for U-IN-2 plants where changes
were less obvious until stage 4. When the glucose-tosucrose ratio was calculated (not shown) a significant
increase was seen from stage 3 onwards when the invertase
was localized in the apoplast. When it was targeted to
the cytoplasm changes were seen starting in stage 4. Thus
the effect of invertase activity on glucose levels was
stronger and visible earlier when the enzyme was localized
in the apoplast. However, cytoplasmic targeting led to an
equally high or even higher glucose content and to
elevation in osmolality during the last developmental
stage and the harvest experiment.
Osmolarity
Osmolarity showed significant differences in late stages
of tuber development (Table 2). In particular, osmolality
was reduced in AGPase antisense plants and increased in
both cytoplasmic invertase lines in stage 6 of tuber
development. Apoplastic invertase plants showed significant differences compared to the wild type in the early
stages of tuber development, probably due to the different
compartmentation of the sugars or due to other osmotically active substances participating in the osmotic
adjustment (data not shown).
Microscopy
Cross-sections of comparable tuber regions of transgenic
and wild-type plants were made to analyse the effect of
invertase activity and glucose accumulation on cell and
starch grain size and growth in the various stages of tuber
development. One representative set of photographs is
shown in Fig. 5 (stage 5). A summary of the results
concerning cell size and starch grain size is shown in Figs
6 and 7. In the early stages of tuber development cells
remained small, indicating that cell division is one
important factor leading to tuber growth. During that
480 Tauberger et al.
Fig. 3. Characteristic display of the amounts and sizes of tubers that were obtained from the lines investigated in the present study. The highest
number of tubers was developed by the AGP-93 transgenic lines.
time the number of starch grains fluctuated (Fig. 8). In
stages 3–6 the number of starch grains was reduced in all
lines, while the cell diameter increased. This result indicated that cells located within the sampled regions ceased
to divide and tissue grew by cell expansion. The number
of starch grains remained almost constant after one last
division indicating that initiation of starch grains had
ceased.
Parallel to cell size, starch grain size and cell wall
thickness were monitored using electron micrographs
(Figs 7, 9). Size of starch grains increased continuously
during tuber development in almost all plant lines.
However, clear differences were visible in the various
lines. While starch grains of wild-type plants increased
continuously, those in AGPase antisense plants showed
no more enlargement from stage 3 onwards. Plants
expressing invertase in the wild-type background exhibited starch grain deposition similar to that of wild-type
plants. Plants with the invertase expressed in the AGPase
antisense background developed starch grains similar to
B-AGP plants. Cell wall diameter of all lines increased
during tuber maturation (Fig. 9). Significant increases in
cell wall diameter were developed in the transgenic lines
U-IN-1 and 2 as compared to the wild type in the final
stage 6 of tuber development.
Water relations
Water potential and turgor pressure fluctuated during the
last stages of tuber development ( Table 2). Turgor
pressure was calculated from these water potential and
osmotic pressure determinations according to Taiz and
Table 1. Average number of tubers per genotye
Genotype
Wild type
B-AGP-93
U-IN-1
U-IN-2
H-AI
H-IN
Number of tubers
8±2
28±4
7±2
9±2
12±2
15±2
Effects of invertase overexpression on tuber development 481
Fig. 4. Sugar distributions of all potato lines that were investigated, exhibited characteristic patterns in the individual developmental stages. Glucose
levels were high in the later stages of tuber development in plants with invertase overexpression. U-IN-1: transgenic plants expressed an apoplastic
invertase from Sacchoromyces cerevisiae. U-IN-2: transgenic lines expressed a cytosolic invertase from Sacchoromyces cerevisiae. B-AGP-93:
transgenic potato expressed ADP glucose-pyrophosphorylase antisense mRNA. H-AI: AGPase antisense plant with a second transgene coding for
an invertase from Sacchoromyces cerevisiae that was targeted to the apoplast. H-IN: AGPase antisense plant with a second transgene coding for an
invertase from Sacchoromyces cerevisiae that was targeted to the cytosol.
Table 2. Water potential and osmolality of transgenic plants in the developmental stages 4–6
Stage
Wild type
U-IN-1
U-IN-2
H-AI
H-IN
B-AGP
Water potential
(MPa)
4
5
6
−0.4±0.08
−0.4±0.1
−0.7±0.11
−0.4±0.1
−0.4±0.1
−0.5±0.08
−0.6±0.1
−0.7±0.08
−0.5±0.1
−0.5±0.1
−0.4±0.11
−0.6±0.08
−0.3±0.11
−1.0±0.1
−0.5±0.11
−0.2±0.1
−0.5±0.08
−0.5±0.1
Osmolality
(mOsmol kg−1 fw)
4
5
6
400±12
370±15
340±18
420±15
370±12
360±10
350±10
440±10
460±9
370±13
300±15
310±12
330±18
300±13
400±9
250±10
300±12
241±18
Zeiger (1991). Because of the fluctuations in water
potential and osmotic pressure turgor from stages 4 to 6
was averaged. As a results, apoplastic invertase lines
exhibited the highest turgor pressure values ( Table 3).
These values were significantly higher than those of the
wild type ( Table 3). All other lines were reduced in turgor.
for an individual developmental stage of a tuber existed,
this classification depended on the subjective estimation
of the experimenter. However, similar fluctuations in
morphometric parameters during tuber development were
reported by Sanz et al. (1996).
Analysis of scatter
Discussion
Most of the measurements exhibited a rather big scatter
between individual stages. Therefore, major sources of
errors are due to the classification of developing tubers
into individual categories. Since no objective parameters
The neccesity for assimilate partitioning during tuber
development has been well established (Oparka et al.,
1992). However, little attention has been paid to the
interaction between sugar availability, cell division and
482 Tauberger et al.
Fig. 5. Characteristic sample of cross-sections ( light microscopy) of
stage 5 of tuber development. (A) Wild type, (B) apoplastic invertase,
(C ) cytosolic invertase, (D) AGPase antisense, (E ) apoplastic invertase
in AGPase antisense background, (F ) cytosolic invertase in AGPase
antisense background.
expansion as well as cell wall properties during tuber
development ( Xu et al., 1998). A perfect tool to investigate the role of these parameters was provided through
the regeneration of transgenic plants affected in sucrose
partitioning (Sonnewald et al., 1997). Those plants
showed changes in final tuber size due to overexpression
of a yeast invertase in different cell compartments ( Fig. 3).
These changes were probably a consequence of modified
sugar distribution. The role of various cell wall and
osmotic parameters on starch grain and tuber development was investigated.
Sugar analysis
Fig. 6. Diameters of individual cells were obtained from light micrographs as shown in Fig. 4. Cell diameter increased from stage 3 onwards
in all lines indicating that tubers are mainly growing by cell expansion.
U-IN-1: transgenic plants expressed an apoplastic invertase from
Sacchoromyces cerevisiae. U-IN-2: transgenic lines expressed a cytosolic
invertase from Sacchoromyces cerevisiae. B-AGP-93: transgenic potato
expressed ADP glucose-pyrophosphorylase antisense mRNA. H-AI:
AGPase antisense plant with a second transgene coding for an invertase
from Sacchoromyces cerevisiae that was targeted to the apoplast. H-IN:
AGPase antisense plant with a second transgene coding for an invertase
from Sacchoromyces cerevisiae that was targeted to the cytosol.
Due to the correlation that emerges from the measurements depicted in Figs 1 and 4, initiation of tubers might
involve high levels of glucose. In all lines investigated the
glucose levels, even those of the wild type were significantly higher than fructose and sucrose (Ross et al., 1994;
Visser et al., 1994). Because of these high glucose levels
in the wild type it has to be assumed that an endogenous
invertase or sucrose synthase is highly active in this phase
of tuber initiation. Although the transgenic lines expressed
an additional invertase, no increase in glucose levels were
detected. These results suggest that either the B33
promotor is not active in the early stages of tuber
development or that sufficient, endogeneous invertase
activity is already present in this early stage of tuber
initiation ( Visser et al., 1994). High levels of glucose have
been reported to induce the expression of several genes
(Sheen, 1994; Koch, 1996). It might be speculated that a
defined level of glucose is required for the initiation of
tuber formation. Although other compounds have been
proposed to induce tuber initiation, as for example,
jasmonic acid or tuberonic acid ( Koda and Okazawa,
1988; Vreugdenhil and Struik, 1989; Yoshihara et al.,
1989), high glucose levels might addidionally be required
to induce tuberization.
Common to all lines that were investigated is a reduction of glucose in the second stage of tuber development
( Fig. 4). This reduction in glucose is paralleled by an
increase in sucrose (Ross et al., 1994). Therefore, it seems
most likely that the B33 promotor is not active in these
early stages of tuber development, as sucrose should be
cleaved more efficiently in the lines overexpressing
invertase ( Visser et al., 1994). In later stages of tuber
Effects of invertase overexpression on tuber development 483
Fig. 7. Diameter of starch grains. Starch grains of wild-type plants and
invertase lines in the wild-type background increased during the final
stages of tuber development. Starch grains of AGPase antisense lines
did not expand in these late stages of tuber maturation.
development (stages 4–6) a pronounced effect of the
transgenic invertase became apparent.
Invertase activity as reflected by the glucose-to-sucrose
ratio started to be obvious in tuber developmental stage
3. At this point the initiation of starch grains had already
started in all plants ( Visser et al., 1994). When comparing
Figs 4 and 7 it might be suggested that starch grain
growth could be modified by the kinds of sugars available.
Higher availability of glucose led to a reduced starch
grain size in some transgenic lines while their number
remained comparable to that of wild-type plants ( Fig. 7,
8).
Tuber growth
Tubers grew by cell division and cell expansion ( Xu et al.,
1998). This was confirmed by the fact that the cell size
did not increase even though tubers grew bigger (Fig. 6).
After stage 3 all sets of transgenic plants showed cell
enlargement at almost the same rate as wild-type plants
even though the sugar distribution and osmolarity
exhibited big variations. Tubers were growing in different
Fig. 8. Number of starch grains per cell. Almost identical amounts of
starch grains were observed in the cells of mature tubers.
numbers and to different sizes. The tuber size decreased
in plants with invertase overexpression when the glucoseto-sucrose ratio increased. This leads to the conclusion
that invertase activity and subsequent glucose accumulation affect cell expansion rather than cell division.
Cell wall
Cell wall thickness was affected to some degree by the
genetic manipulations ( Fig. 9). However, the increased
amount of glucose was possibly stored mainly in the
vacuole of transgenic lines, since synthesis of cell wall
constituents represents a minor pathway for carbon
allocation. Previous reports indicated that cell wall constituents amount to approximately 4% of the dry weight
(Hoff and Castro, 1969).
Similar to our results a significant increase in cell wall
diameter was described for the leaves of transgenic plants
that overexpressed a vacuolar invertase (HoffmannBenning et al., 1997). In addition, these vacuolar invertase
lines exhibited a strong elevation in turgor pressure.
484 Tauberger et al.
induce a pronounced effect on the water potential and
the osmolality. What is not detected is a clear correlation
between water potential and osmolality. It might be
expected that a reduction in osmolality corresponded with
an increase in water potential. Moreover, a positive
correlation between water potential and osmotic potential
was observed in the U-IN-2 line between stages 5 and 6.
Unloading and turgor
Fig. 9. Cell wall thickness increased in all plant lines. Increases were
more pronounced in the late stages of tuber development.
Water relations
Water potentials became more negative in the developmental stage 6 as compared to stage 4 in almost all lines.
Only the transgenic lines that overexpress a cytosolic
invertase ( U-IN-2) deviate from this pattern. During
tuber development from stages 4–6 the osmolality
declined in the wild type, the apoplastic invertase in the
AGPase antisense background and B-AGPase antisense
lines ( Table 2). In the final stage 6 of tuber development
the water potential of the wild type is more negative than
in all other transgenic lines ( Table 2). However, the
osmolality of wild-type tubers in this developmental stage
is intermediate to the osmolalities of the transgenic lines.
Therefore, our experiments on these transgenic plant lines
indicate that modifications in sugar allocation patterns
The observed levels of glucose are in line with a symplastic
unloading of sucrose from the phloem (Oparka et al.,
1992). Immediate conversion of sucrose in glucose
increases the sink strength of the growing stolon.
Apoplastic unloading seems unfavourable under these
conditions of high glucose, since the hexose carrier would
then have to transport hexoses against a very high
concentration gradient (Roitsch and Tanner, 1996).
An increase in sink strength by invertase overexpression
should enable an efficient unloading of sucrose into these
sinks. Therefore, it should be expected that cytoplasmic
invertase overexpressing tubers should have the highest
sink strength. However, the uncontrolled increase in
glucose due to an additional invertase could induce a
closure of plasmodesmata and thus inhibit further
unloading. This potential closure of plasmodesmata could
explain the size of tubers that expressed invertase in an
AGPase antisense background. High turgor pressure
would result in a closure of plasmodesmata and thus
inhibit further unloading. Experiments performed by
Oparka et al. (1991) provided direct evidence that
plasmodesmata function as pressure-sensitive valves.
These workers demonstrated that raising the turgor pressure between two cells resulted in a closure of
plasmodesmata.
Apoplastic invertase plants exhibited the highest turgor,
whereas cytosolic invertases had the lowest turgor with
the wild type in between ( Table 3). This finding might be
explained by the assumption of symplastic unloading and
leakage of sucrose into the apoplast as follows (Fig. 10).
Cleavage of sucrose in the apoplast increases the osmotic
potential of the apoplast. Therefore, plasmodesmata will
not close, even at high glucose concentration in the
cytoplasm, since they are turgor regulated. When finally
the level for closure is reached these cells contain much
more osmotic sugars than the wild type. The turgor is
higher since hexoses are subsequently taken up from the
apoplast due to hexose transport proteins. Because of
Table 3. Average of turgor pressure (MPa) in tuber developmental stages 4–6
Turgor pressure
(MPa)
Wild type
U-IN-1
U-IN-2
H-AI
H-IN
B-AGP
0.43±0.05
0.55±0.04
0.30±0.03
0.30±0.03
0.30±0.04
0.33±0.03
Effects of invertase overexpression on tuber development 485
thickness of the cell walls. Turgor pressure was highest
in the apoplastic invertase plants which developed the
biggest tubers.
Acknowledgements
We thank Professor Dr U Sonnewald (Gatersleben, Germany)
and Dr Müller-Röber (Golm, Germany) for the generous gift
of some of the transgenic potato plants.
References
Fig. 10. Schematic diagram of turgor pressure generation due to
invertase overexpression. Sucrose enters tuber parenchyma cells via
plasmodesmata. In the upper panel a leakage of sucrose into the
apoplast has been indicated. Apoplastic invertase will cleave sucrose
and thus increase the osmotic potential of the apoplast. In the lower
panel, leakage of sucrose into the apoplast is reduced as a consequence
of overexpression of cytosolic invertase.
this active uptake of hexoses into the cytoplasm the
turgor can gain high values since the amounts of apoplastic glucose are much smaller than in the cytoplasm
Cytosolic invertase plants have an additional mechanism to cleave sucrose ( Fig. 10). Therefore, the leakage of
sucrose into the apoplast is further reduced. The threshold
for plasmodesmatal closure will thus be obtained at
relatively low glucose concentrations in the cytoplasm.
The wild type would be intermediate, since small amounts
of sucrose can leak into the apoplast thus slightly increasing the osmotic potential of the apoplast. An AGPase
antisense background provides similar conditions as are
found in the cytosolic invertase plants. Elevated levels of
sugars are always present in the cytoplasm of these
transgenic plants resulting in an early closure of plasmodesmata. Therefore, AGPase antisense, cytosolic
invertase, and cytosolic and apoplastic invertase plants in
the AGPase antisense background exhibit almost identical
turgor pressures.
Summary
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gene was not active during the early stages of tuber
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stage 3 onwards and the subsequent invertase activity led
to an increase in the glucose-to-sucrose ratio and, as a
consequence to smaller starch grains. Final sizes of cells
reflected some variation, similar to variations in the
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