Download Overexpression of sucrose phosphate synthase

Journal of Experimental Botany, Vol. 50, No. 335, pp. 785–791, June 1999
Overexpression of sucrose phosphate synthase increases
sucrose unloading in transformed tomato fruit
Binh Nguyen-Quoc1, Hyacinthe N’Tchobo1, Christine H. Foyer2,3 and Serge Yelle1
1 Centre de Recherche en Horticulture, Pavillon de l’Envirotron, FSAA, Université Laval, Québec G1K 7P4,
Canada
2 Department of Biochemistry and Physiology, Institute of Arable Crops Research, Rothamsted, Harpenden,
Hertfordshire AL5 2JQ, UK
Received 12 October 1998; Accepted 12 February 1999
Abstract
Sucrose unloading and sink activity were examined in
tomato plants (Lycopersicon esculentum) overexpressing sucrose phosphate synthase (SPS; EC 2.3.1.14).
Like the leaves, the fruit of the transformed tomato
plants had elevated (2.4-fold) SPS activity. SPS overexpression in tomato fruit did not significantly change
acid invertase, and only slightly reduced ADPglc ppase
activity, but enhanced sucrose synthase activity by 27%. More importantly, the amount of sucrose
unloaded into the fruit was considerably increased.
Using [3H]- (fructosyl)-sucrose in in vitro unloading
experiments with harvested 20-d-old fruit, 70% more
sucrose was unloaded into the transformed fruits compared to the untransformed controls. Furthermore, the
turnover of the sucrose unloaded into the fruit of transformed plants was 60% higher than that observed in the untransformed controls. Taken together,
these results demonstrate that SPS overexpression
increases the sink strength of transformed tomato
fruit.
Key words: Sucrose phosphate synthase (SPS), starch,
sucrose unloading, tomato fruit, carbon metabolism.
Introduction
Sucrose phosphate synthase (SPS ) catalyses net sucrose
synthesis in plants. SPS activity is generally high in source
tissues and low in sink organs (Huber and Huber, 1992).
SPS activity can be a controlling factor for sucrose
synthesis and also for photosynthesis (Stitt et al., 1988).
However, in transformed potato plants, a decrease in
SPS activity of 60–70% caused no significant inhibition
of photosynthesis compared to the untransformed controls (Geigenberger et al., 1995).
To increase photosynthesis and sucrose synthesis,
Worrell et al. (1991) genetically manipulated tomatoes to
overexpress a maize SPS cDNA, under the control of the
promoter of the small subunit of Rubisco from tobacco
(rbcs). Several rbcs lines showed a higher SPS activity in
their leaves than untransformed controls ( Worrell et al.,
1991). These were characterized and plant growth evaluated under different conditions (Galtier et al., 1993, 1995;
Micallef et al., 1995; Laporte et al., 1997). The SPS
activity in the transformed leaves was six times greater
than that of the controls. At low irradiances no increase
in net photosynthesis was observed, but the light- and
CO -saturated rates of photosynthesis in transformed
2
leaves were increased by about 20% (Galtier et al., 1993,
1995; Micallef et al., 1995). Carbon allocation within the
plant was not modified except in very young plants where
an increase in root dry weight was observed (Galtier et
al., 1993; Laporte et al., 1997). Furthermore, SPS overexpression caused an increase in the sucrose/starch ratio
of leaves (Galtier et al., 1993, 1995; Signora et al., 1998),
but this was due more to increased turnover of starch
than to increased sucrose biosynthesis (Galtier et al.,
1993). Most interestingly, the total fruit number in the
SPS transformants was increased to 1.5 times that of the
control (Micallef et al., 1995). The fruit on the trans-
3 To whom correspondence should be addressed. Fax: +44 1582 763010. E-mail: [email protected]
Abbreviations: ADPglc ppase, ADP-glucose pyrophosphorylase; DAA, days after anthesis; DPM, disintegrations min−1; DDTT, dithiothreitol; EDTA,
ethylenediaminetetraacetic acid; FW, fresh weight; HEPES, (N-[2-hydroxyethyl]piperazine-N-[4-butanesulphonic acid]); PMSF, phenylmethylsulphonyl
fluoride; PVPP, polyvinylpolypyrrolidone; SPS, sucrose phosphate synthase; SuSy, sucrose synthase; TBS, TRIS-buffered saline; TCA, trichloroacetic acid.
© Oxford University Press 1999
786 Nguyen-Quoc et al.
formed plants matured earlier and total fruit dry weight
was increased by up to 32% when these plants were grown
in air and high light (1000 mmol m−2 s−1). However, no
increase in total fruit yield was observed in conditions of
CO enrichment (Micallef et al., 1995).
2
In sink tissues, unloaded sucrose has to be hydrolysed
for metabolism. Depending on the plant species unloaded
sucrose can be hydrolysed either by invertase or by
sucrose synthase. Several studies have shown that
unloaded sucrose can undergo a futile cycle of degradation and resynthesis in sink tissues (N’tchobo, 1998a, b;
ap Rees and Hill, 1994; Dancer et al., 1990; Geigenberger
and Stitt, 1993; Hargreaves and ap Rees, 1988; Viola,
1996; Wendler et al., 1990). In such a cycle, the nature
of the enzyme that catalyses the resynthesis of sucrose
remains to be elucidated. Geigenberger and Stitt (1993)
suggested that sucrose resynthesis in sink tissues is catalysed by both SuSy and SPS. In sink tissues, SPS is
present at low levels: 2.5–10% of SuSy activity in rapidly
growing-sinks (Dali et al., 1992; Klann et al., 1993; Weber
et al., 1996). Sucrose phosphate synthase has been considered to be the limiting factor in the regulation of sucrose
accumulation in tomato fruit (Dali et al., 1992; Miron
and Schaffer, 1991). However, this hypothesis was not
supported by later observations which suggest that the
sucrose concentration of tomato fruit was determined by
acid invertase activity ( Klann et al., 1993; Ohyama et al.,
1995; Scholes et al., 1996). Increased sucrose accumulation in transgenic potato tubers with low ADPglc ppase
activity, however, was correlated with increased SPS
activity (Geigenberger et al., 1995).
While the role of SPS in relation to putative futile
cycles has been studied in sink tissues (Geigenberger and
Stitt, 1993), the role of SPS in determining sink strength
and sucrose unloading remains to be elucidated. This
study has examined sucrose metabolism in tomato fruit
where SPS is overexpressed. It has shown that overexpression of SPS in young fruit favours increased sucrose
synthesis, resulting in increased sink cleavage and sink
activity.
Materials and methods
Plant material and growing conditions
Tomato (Lycopersicon esculentum var. UC82B) seed of untransformed and transformed (Line 3812–9) lines was obtained from
Calgene Inc ( USA). The transformed tomatoes used in the
present study are an homozygous line ( T4) with only one
transgene insertion (verified by Southern blot; data not shown).
This transgenic clone was used in previous studies for analysis
of photosynthesis capacity (Galtier et al., 1993) and fruit yield
(Micallef et al., 1995) under the name: SSU-9. Expression of
the maize SPS cDNA was under the control of the Rubisco
small subunit promoter. The plants were grown in a greenhouse
with a 14 h photoperiod, 25/22 °C day/night. Irradiance was
700 mmol m−1 s−1.
Enzyme extraction
Freshly harvested tomato fruits were ground in liquid nitrogen.
Proteins were extracted into buffer A consisting of 200 mM
HEPES (pH 7) 3 mM magnesium acetate, 0.5 mM EDTA,
1 mM PMSF, 5 mM DTT, 20 mM 2-mercaptoethanol, 10%
ethyleneglycol, 1% PVPP, and 1% Dowex-1 (chloride form) as
previously described (Sun et al., 1992). The ratio of fruit fresh
weight (g) to extraction volume (ml ) was 155. The homogenate
was incubated on ice for 5 min, passed through one layer of
Miracloth (InterScience, Markham, Ontario, Canada) and
centrifuged at 20 000 g for 10 min. The supernatant was desalted
through Sephadex G-25 superfine (Pharmacia Biotech, Baie
D’Urfé, Québec, Canada) with buffer B which consisted of
50 mM HEPES (pH 7.0), 1 mM EDTA, 1 mM DTT, 0.5 mM
PMSF, and 2 mM magnesium acetate.
Enzyme assays
Sucrose phosphate synthase activity was determined at limiting
substrate concentrations (2 mM UDP glucose, 2 mM F6P and
10 mM G6P) with 5 mM Pi, as described by Huber and Huber
(1991). Sucrose formation was determined by the modified
anthrone method ( Van Handel, 1968).
Sucrose synthase and neutral invertase activities were assayed
in the direction of sucrose breakdown by a coupled enzyme
assay as described previously (Huber and Akazawa, 1986). The
reaction mixture (1 ml total volume) consisted of buffer B
containing 100 mM sucrose, 1 mM ATP, 0.4 mM NAD, 4 units
hexokinase, 4 units glucose isomerase, 2 units 6-phosphoglucose
dehydrogenase (from Leucomostoc mesenteroides: NAD-dependent form without invertase contamination obtained from
Boehringer Mannheim, Laval, Québec, Canada), and the change
in absorbance at 340 nm at 25 °C was measured. This provided
an estimation of background invertase activity arising from
contamination of the coupling enzymes. After 2 min, 20 ml of
sample was added allowing estimation of the neutral invertase
activity. Sucrose synthase activity was measured by the
subsequent addition of 1 mM of UDP (final concentration).
Acid invertase was assayed in acetate buffer (pH 4.5). 100 ml
of sample was mixed with 500 ml of 100 mM acetate buffer
containing 3% sucrose. After 15 min incubation at 30 °C, the
reaction was neutralized with 50 ml of 1 M TRIS-HCl buffer
(pH 8.0). Further enzyme activity was prevented by boiling the
mixtures for 5 min. The hexose formed in the reaction was
measured in an oxygen electrode (commercial YSI model select
2700, Yellow Spring Instrument Co. Inc., Ohio, USA) with a
glucose membrane where glucose oxidase is immobilized. By
the action of glucose oxidase in the presence of O , glucose was
2
converted to -glucono-lactone and H O . The amount of H O
2 2
2 2
formed was measured using a platinum anode (see the YSI
manual for 2700 SELECT analyser).
ADPglc ppase activity was measured in the direction of G1P
formation in a coupling reaction as previously described by
Rocher et al. (1989). Desalted extract (40 ml ) was added to
450 ml HEPES-NaOH buffer (50 mM, pH 7.5) containing 3 mM
Mg-acetate, 0.1 mg BSA, 1 mM ADPglucose, 0.3 mM NAD, 1
unit phosphoglucose mutase, and 1 unit 6-phosphoglucose
dehydrogenase. The reaction was started by the addition of
1 mM PPi, and the formation of NADH was measured by the
change in absorbance at 340 at 25 °C.
Western blotting and tissue printing
For Western blot assay, the total protein in non-desalted extract
was immediately precipitated with 3% TCA. After centrifugation, the pellet was solubilized in the electrophoresis loading
buffer (pH 6.8). Five mg total protein from each sample were
Overexpression of SPS 787
separated on 10% SDS–PAGE as described by Laemmli (1970).
Proteins were electrotransferred to nitrocellulose membrane for
immunodetection with polyclonal antibodies raised against
maize SPS ( Worrell et al., 1991; 1/5000 dilution) or polyclonal
antibody against maize SuSy (Nguyen-Quoc et al., 1990; 1/5000
dilution) as primary antisera with alkaline phosphatase-coupled
secondary antibodies (Boehringer Mannheim, Laval, Québec,
Canada) using the NBT/BCIP substrate combination for colour
development (Promega, Madison, MN, USA). The density of
the Western blot band was determined by NIH image (version
1.61) application on the scan image. Tissue printing was as
described by Cassab and Varner (1987) with a slight modification as follows. The tomato fruit was cut in half along the
centre and proteins were printed onto a nitrocellulose membrane. The membrane was washed in the TBS buffer containing
0.1% SDS and boiled for 10 min in TBS buffer to denature the
endogenous phosphatase. The enzyme immunodetection protocol was identical to that used for Western blotting.
In vitro sucrose unloading and carbohydrate determination
Unloading was assayed by a modification of the technique
described by Dali et al. (1992). The tomato fruit with their
peduncles attached were harvested. The peduncles were plunged
immediately into 1 ml of 2% unlabelled sucrose and 3 mCi
(6.106 DPM ) [3H ]- (fructosyl )-sucrose (Dupont Canada Inc.,
Mississauga, Ontario, Canada). After a 1 h pulse, the fruits
were transferred to 2% unlabelled sucrose (chase). Whole fruit
samples were removed from the solution at different times (0,
30, 60, 90, 120 min). The peduncle was removed before analysis.
Fruits were ground in liquid N and then homogenized in 5
2
vols of 80% ethanol. The homogenate was boiled for 10 min at
80 °C. After 10 min centrifugation at 12 000 g, the supernatant
was collected and used for determination of sucrose and hexoses
and the pellet was retained for starch analysis. For the soluble
sugar determination, the supernatant was lyophilized, resuspended in water and purified by ion exchange chromatography
(cation and anion AG resin; Biorad, Mississauga, Ontario,
Canada). The sucrose and hexose fractions were separated on
carbohydrate HPLC column (Millipore, Ville Ste Laurent,
Québec, Canada). Sucrose, glucose and fructose fractions
corresponding to the standards were collected and the radioactivity was measured in a liquid scintillation counter ( Wallac,
Turku, Finland ). To determine the symmetrical labelling of the
sucrose molecule, the purified sucrose fraction was hydrolysed
by yeast invertase and re-fractioned by HPLC.
Data analysis
Statistical analyses were performed using analysis of variance
(ANOVA).
Results
Analysis of sucrose metabolising enzymes and
carbohydrate composition of fruit
Enzyme activity. Previous studies have shown that sink
activity in tomato fruits is maximum at 20 DAA (Ho
et al., 1987). In transformed tomato plants with altered
sucrose metabolism enzyme activities (SuSy, SPS,
invertase or ADPglc ppase), the timing of fruit development was not changed (data not shown). Therefore SPS,
SuSy, acid invertase and ADPglc ppase activities in 20-dold fruits from transformed and untransformed (control )
plants were analysed. Table 1 shows that the SPS activity
of fruit from transformed plants, measured with limiting
concentrations of substrate, was 2.4 times higher than
that of untransformed controls. This increase was accompanied by a significant increase in SuSy activity (27%;
P≤0.05). The increase in activity of these two enzymes
was comparable in terms of the capacity to cleave or
synthesize sucrose ( Table 1). Acid invertase and ADPglc
ppase activities were not significantly different from those
in untransformed controls.
The expression of transgene in the fruit was demonstrated by Western blot ( Fig. 1A, lane 2). One protein
band was detected with a molecular mass of 120 kDa,
equivalent to the size of the maize SPS peptide. This
result suggests that the increase in SPS activity in the
transformed fruits was due to over-expression of SPS.
Similarly, the increase in SuSy activity in transformed
tomato fruits correlated with increased enzyme protein
as determined by semi-quantitative scanning of the SuSy
peptide in Western blot ( Fig. 1B). The band intensity of
SuSy peptide in the transformed fruit ( Fig. 1B, lane 2)
was 57% higher than that in untransformed ( Fig. 1B, lane
1) controls.
The increases in SPS and SuSy activities in transformed
tomato fruits correlated with increased enzyme protein
as determined by Western blotting with maize SPS
( Fig. 1A) and SuSy (Fig. 1B) antibodies. Maize SPS
antibodies did not crossreact with endogenous tomato
SPS (Fig. 1A), while the antibody for SuSy from maize
cross-reacted with the native tomato SuSy isoforms. No
bands of native SPS protein were detected with the
antibodies to the maize enzyme ( Fig. 1A, track 1), but in
the transformed line (Fig. 1A, line 2) one band was
detected which corresponded to a molecular mass of
120 kDa which is equivalent to the size of the maize SPS
peptide. The density of staining of this SPS band was
equivalent to that detected in extracts from transformed
leaves when gels were loaded on an equal protein basis
(data not shown). Figure 1B shows the immunodetection
of SuSy in tomato fruits from untransformed (track 1)
and transformed plants (track 2). The SuSy band in the
transformed line was clearly much more intense, for an
equivalent protein concentration, than in untransformed
plants indicating that the quantity of this enzyme protein
was increased in the former compared to the latter. The
molecular mass of tomato SuSy was estimated to be
about 90 kDa by comparison with standard protein
molecular markers (BioRad, Massachusetts, USA).
The expression of SPS in the different tissues of the
tomato fruit from transformed and untransformed plants
was compared by tissue printing. The tissue prints from
transformed fruit obtained with maize SPS antibodies
( Fig. 2B) shows that the amounts of these enzyme proteins are relatively homogeneous in all types of tissue
within the fruit and are similar in both the external and
788 Nguyen-Quoc et al.
Table 1. Activities of SPS (measured at limiting substrate concentrations), sucrose synthase, acid invertase, and ADPglc ppase in 20
DAA fruits from untransformed and transformed tomato plants; values represent the mean±SE, n=3 plants
Untransformed
Transformed
SPS
(nmol min−1 g−1 FW )
SuSy
(nmol min−1 g−1 FW )
Acid invertase
(nmol min−1 g−1 FW )
ADPglc ppase
(nmol min−1 g−1 FW )
96±3
233±26
1254±35
1593±147
1397±335
1422±21
719±15
639±21
Rubisco promoter, the maize SPS protein was found to
be present in the whole fruit, as was SuSy.
Fig. 1. Western blot analysis of SPS (A) and SuSy (B) in control (1)
and transformed (2) tomato fruits. Five mg of total protein was applied
per track. Proteins extracted from 20 DAA fruits had been precipitated
with 3% of TCA and separated in 10% SDS-PAGE before transfer to
the nitrocellulose membrane. The blot was probed either with maize
polyclonal SPS antibodies (A) or SuSy polyclonal antibodies (B)
followed by secondary antibodies coupled to NBT/BCIP/alkaline
phosphatase detection. STD: prestained SDS-PAGE protein standards
(BioRad).
Carbohydrate analysis. When the carbohydrate composition (sucrose, hexose and starch) of 20 DAA fruit was
analysed ( Table 2), the soluble sugar concentrations and
the sucrose/hexose ratios in transformed fruit were found
to similar to those of the controls. However, the amount
of starch in the transformed fruit was significantly higher
than that in the controls ( Table 2; P≤0.05).
In vitro unloading of sucrose
In order to distinguish between effects due to SPS overexpression in the source leaves from those occurring in
the sink tissues alone, fruits from transformed and control
plants were detached and used for unloading experiments.
The detached 20 DAA fruits were supplied with labelled
sucrose. Figure 3 shows the unloading patterns of transformed and control fruit. After 1 h of labelling and
subsequent 30, 60, 90, or 120 min chase periods with
unlabelled sucrose, the total radioactivity (Fig. 3A)
unloaded in the transformed fruit was about 70% higher
than that found in the controls. In the shortest chase
times (up to 30 min) the majority of the radioactivity was
found in sucrose (data not shown). The differences
between the observed amount of radioactivity in transformed and control fruit was due to variations in radioactivity in soluble sugars (Fig. 3A, C ). No differences in
radioactivity were found in the starch fractions. With
longer chase times (from 60 min onwards), however, the
increase in radioactivity in the transformed fruit was
caused by differences in both the soluble sugar and starch
fractions. Moreover, as the period of the chase increased
the ratio of soluble sugar to starch became similar in
both lines.
Sucrose turnover and sucrose to starch conversion
Fig. 2. Tissue printing of tomato fruit with SPS (A, B) and SuSy (C,
D) antibodies. 20 DAA fruit from control (A, C ) and transformed (C,
D) plants are shown. Tomato fruit were cut in two and printed onto a
nitrocellulose membrane. One of the halves was used to detect SPS and
other to detect SuSy. All other details were as for Fig. 1.
internal parts of the fruit. A very low background was
observed in untransformed fruit probed with maize SPS
antibody ( Fig. 2A). In marked contrast, probing with
SuSy antibody yielded comparable prints in both types
of plant (Fig. 2C, D). These results demonstrate that,
even though SPS was placed under the control of the
SPS overexpression in the fruit may accelerate the rate of
sucrose synthesis or sucrose turnover. Since the unloaded
Table 2. Sucrose, hexose and starch contents of 20 DAA fruits
from transformed and untransformed tomato plants; values
represent the mean±SE, n=3 plants
Untransformed
Transformed
Sucrose
(mg g−1 FW )
Hexoses
(mg g−1 FW )
Starch
(mg g−1 FW )
1.76±0.13
1.88±0.05
13.03±0.67
12.15±0.30
13.04±0.54
14.82±0.45
Overexpression of SPS 789
Fig. 4. Symmetry of labelling in the sucrose molecule in control and
transgenic tomato fruits. [3H ]- (fructosyl )-sucrose was supplied to 20
DAA tomato fruits. After a 1 h pulse the fruits were transferred to
unlabelled sucrose and after 30, 60, 90, and 120 min of chase, the
symmetry of labelling of sucrose was analysed. Values are means±SD
for three independent fruits.
Fig. 3. Influence of SPS overexpression on sucrose uptake into detached
tomato fruit. Twenty DAA fruit were harvested with their peduncles
attached. The peduncles were immediately plunged into a solution
containing 1 ml of 2% sucrose and 6.106 DPM of [3H ]- (fructosyl )sucrose. After a 1 h pulse, the fruit were transferred to a 2% unlabelled
sucrose solution. At different chase times (0, 30, 60, 90, 120 min) the
fruits were frozen in liquid N and analysed for total radioactivity (A),
2
radioactivity found in the soluble sugar fraction (B) and radioactivity
in the starch fraction (C ). Values are means±SD of three
determinations.
sucrose is labelled on the fructose moiety only, the rate
of change in labelling symmetry within the sucrose molecules in the fruit may be used to estimate the rate of
sucrose resynthesis. The rate of conversion of label from
fructose to glucose within the sucrose molecule was more
rapid in transformed fruits than in controls ( Fig. 4). The
time required for conversion of 50% of the sucrose
molecules in the transformed fruit was 48 min compared
with 78.5 min in the untransformed fruit, representing a
60% higher rate in the transformed plants.
Sucrose turnover in transformed tomato fruits was
higher than in controls, whereas the rate of conversion
from sucrose to starch was essentially similar in both
lines ( Fig. 5). Although the conversion rate early in the
chase was slower in the transformed fruit, the sucrose to
starch conversion patterns were similar in fruit from both
lines. At zero time, after the 1 h pulse, only about 5% of
unloaded sucrose had been converted into starch, but 2 h
Fig. 5. Conversion of sucrose to starch in control and transgenic
tomato fruits. The ratio of starch to total radioactivity, expressed as a
percentage, was calculated from Fig. 3. Values are means±SD of three
determinations.
after the pulse about 25% of the unloaded sucrose had
been converted into starch.
Discussion and conclusions
In tomato fruit overexpression of SPS leads to an increase in
SuSy and sink activity
The rate of import of assimilate in the rapid growth phase
of tomato fruit development (about 20 DAA) has been
considered to be crucial for determining final fruit weight
(Grange and Andrews, 1993; Ho, 1996; Ho et al., 1987).
This period in fruit development is characterized by
maximum SuSy and ADPglc ppase activities. Moreover,
sucrose unloading also attains a maximum rate at this
point (Dali et al., 1992). The correlation observed
between the rate of sucrose breakdown and the quantity
of sucrose unloaded into the fruit has led to the hypothesis
that either invertase or sucrose synthase can limit sucrose
unloading and, in turn, determine yield (Ho, 1996). In
contrast to the original hypothesis that suggested that
invertase is the limiting step in the regulation of assimilate
790 Nguyen-Quoc et al.
import in tomato ( Walker et al., 1978), Sun et al. (1992)
have suggested that SuSy is one of the principal factors
regulating fruit growth. This hypothesis is supported by
the observations of Wang et al. (1993), who reported a
good correlation between SuSy activity and relative fruit
growth. Such observations suggest that the cleavage of
sucrose in the cytosol may be the limiting step for sucrose
import into tomato fruit. However, sucrose imported into
this sink organ may undergo rapid turnover involving
both degradation and resynthesis (Geigenberger and Stitt,
1993; N’tchobo, 1998a, b).
In young tomato fruits, where sucrose is unloaded
principally via the symplast (N’tchobo, 1998a, b; Ruan
and Patrick, 1995; Yelle et al., 1988; Yin et al., 1995),
SuSy and SPS could together catalyse a cycle of cleavage
and resynthesis in the cytosol (Geigenberger and Stitt,
1993). SPS overexpression resulted in a 2.4-fold increase
in SPS activity in the fruit. The cleavage of sucrose by
SuSy was also enhanced. In the transformed fruit, the
observed increase in SPS led to a 27% increase in SuSy
activity. The increase in maximal extractable SuSy activity
(339 nmol min−1 g−1FW ) was in the same range as that
of SPS activity measured in conditions of limiting substrates (137 nmol min−1 g−1 FW ). Hence, the increase in
the capacity for sucrose synthesis in the transformed fruit
led to an even greater increase in the capacity for sucrose
cleavage.
Increase in SPS and SuSy activities led to an increase in
sucrose turnover and sucrose import
Since the capacity of both sucrose synthesis and sucrose
degradation was increased in the fruits of the transformed
plants, it is pertinent to consider whether this leads to
increased sucrose turnover. Sucrose turnover in fruit from
the SPS transformants was found to be 60% higher than
that measured in the untransformed controls. The sucrose
used in these experiments was labelled in the fructose
moiety. The resynthesis of sucrose by SuSy or SPS must
lead to similar changes in the symmetry of labelling in
sucrose molecules in both transformed and untransformed
lines. Increased SPS activity would only result in enhanced
sucrose import if the rate of sucrose turnover was related
to the rate of import. This is indeed what was observed
since the increase in SuSy and SPS activities in transformed fruit led to an increase in the sucrose turnover
rate of about 60%. This, in turn, led to a calculated
increase in the rate of sucrose import of 70%.
The increase in sucrose import by the sink organs may
have been caused by a change in the rate of sucrose
export from source leaves (Ho, 1996; Micallef et al.,
1995). However, in the rbcS transformants SPS activity
was increased in both leaves and fruit. A change in sink
activity involving increased SPS or SuSy activity may be
sufficient to increase sink strength. The recent patent
application by Calgene, showing that the expression of
the maize SPS cDNA under the control of a fruit-specific
promoter in tomato plants leads to an increase in yield,
confirms this hypothesis.
Increased sucrose import in tomato fruit leads to an
increase in starch synthesis
ADPglc ppase activity was not increased in the transformed fruit, but the sucrose/starch ratio was changed by
SPS overexpression. The amount of starch and the rate
of starch synthesis were higher in transformed fruit than
in those from untransformed plants. Starch accumulation
is therefore not only dependent on ADPglc ppase activity,
but also on the amount of sucrose unloaded into the
fruit. The rate of sucrose resynthesis in tomato fruit may
therefore determine the extent of starch synthesis as it
does in leaves (Galtier et al., 1993, 1995; Signora et al.,
1998). However, overexpression of SPS in fruit increased
starch accumulation, while in the leaves the inverse was
true.
Acknowledgement
The authors are indebted to Calgene Inc., USA, for providing
the transformed tomato seed overexpressing sucrose phosphate
synthase under the control of the promoter of the small subunit
of ribulose-1,5-bisphosphate carboxylase-oxygenase.
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