Journal of Experimental Botany, Vol. 50, No. 338, pp. 1457–1463, September 1999
Starch synthesis in tomato remains constant throughout
fruit development and is dependent on sucrose supply
and sucrose synthase activity
Hyacinthe N’tchobo1, Najeh Dali1, Binh Nguyen-Quoc1,3, Christine H. Foyer2 and Serge Yelle1
1 Centre de Recherche en Horticulture, FSAA, Université Laval, Quebec, Canada G1K 7P4
2 Department of Biochemistry and Physiology, IACR-Rothamsted, Harpenden, Herts AL5 2JQ, UK
Received 15 February 1999; Accepted 17 May 1999
Abstract
Studies designed to investigate the cellular pathway
of phloem unloading were conducted on two tomato
lines with either high or low fruit invertase activities.
Experiments were based on determination of the
degree to which 3H label from [3H]-(fructosyl)-sucrose
was randomized between fructose and glucose following exposure of excised fruit to a pulse of labelled
sucrose delivered through the pedicels. Fruit from the
low invertase line harvested 10, 20 and 40 d after
anthesis had similar sucrose uptake kinetics to the
high invertase line. A positive correlation was found
between sucrose synthase activity and sucrose uptake
in both low and high invertase lines. In contrast, no
correlation was observed between acid or neutral
invertase activities and sucrose uptake. Within the
putative apoplasmic sap collected from fruit, label in
[3H]-(fructosyl)-sucrose was randomized between the
free hexoses and sucrose hexose moieties. Label
asymmetry was retained in sucrose on arrival within
the tissues. Randomization patterns were similar in
both the low and high acid invertase lines. These data
support the view that sucrose imported into the fruit
was not exposed to extracellular hydrolysis. This suggests that movement from the phloem is likely to occur
predominantly through a symplastic pathway. About
25% of the sucrose taken up by the fruit was converted
into starch regardless of fruit age, suggesting that
starch turnover remains constant throughout fruit
development and that starch synthesis was dependent
on sucrose supply.
Key words: Invertase, sucrose synthase, sucrose turnover,
sucrose unloading, tomato fruit.
Introduction
Sucrose is the major sugar form in which carbohydrate
is transported in the phloem of tomato ( Walker and Ho,
1977). Sucrose concentration differences between the
source leaves and developing fruit determine both the
rate of translocation and the direction of carbon flow
( Walker et al., 1978). Sucrose hydrolysis is considered to
be the key mediator between carbohydrate unloading and
carbohydrate metabolism in developing fruit (Ho et al.,
1987). This relationship determines fruit establishment
and sink strength (Ho, 1988; Wang et al., 1993). The
activities of acid invertase (AI ), sucrose synthase (SuSy)
and sucrose phosphate synthase (SPS) may all contribute
to sink strength.
AI exists in at least two forms in tomato fruit, a cell
wall bound form located in the apoplast and a soluble
form found largely in the vacuole ( Konno et al., 1993;
Sato et al., 1993). The role of each AI form in fruit
carbohydrate metabolism will depend on the nature of
the pathway of sucrose unloading into the fruit. Two
pathways of sucrose unloading have been described. In
young fruit, unloading has been suggested to occur by a
symplastic route while in mature fruit sucrose unloading
is considered to be predominantly apoplastic (Damon
et al., 1988; Ruan and Patrick, 1995). Sucrose, unloaded
into mature fruit via the apoplast, is hydrolysed by AI
and the resulting hexoses are actively taken up into the
3 To whom correspondence should be addressed: Fax: +1 418 656 7871. E-mail: [email protected]
Abbreviations: ADPglc ppase, ADP-glucose pyrophosphorylase; AI, acid invertase; DAA, days after anthesis; DPM, disintegrations min−1; DTT,
dithiothreitol; FW, fresh weight; PMSF, phenylmethylsulphonyl fluoride; PVP, polyvinylpyrrolidone; SPS, sucrose phosphate synthase; SuSy,
sucrose synthase.
© Oxford University Press 1999
1458 N’tchobo et al.
cytoplasm (Damon et al., 1988; Brown et al., 1997; Ruan
et al., 1997). In young tomato fruit, AI is considered to
be predominantly vacuolar ( Klann et al., 1993; Konno
et al., 1993), but AI activity has been found in the
apoplastic sap, suggesting that it may also be present in
the intercellular spaces of the fruit (Damon et al., 1988;
Sato et al., 1993).
SuSy is considered to be a key enzyme of sucrose
cleavage in sink tissues. Transformed potato plants,
expressing SuSy in the antisense orientation, showed a
net decrease in total tuber dry weight ( Zrenner et al.,
1995). The absence of the major isoform of SuSy (SSI )
in the kernels of a maize mutant (bzm4) resulted in a
60% decrease in starch accumulation compared to wildtype plants (Chourey and Nelson, 1976). SuSy activity
has been correlated with the amount of sugar imported
into tomato fruit (Sun et al., 1992; Wang et al., 1993).
SPS might also be involved in sucrose turnover in tomato
fruit. A positive relationship between SPS activity and
sucrose storage was observed in late tomato fruit development (Miron and Schaffer, 1991). SPS activity is low in
young fruit (Dali et al., 1992) but it clearly increases
during development in both hexose and sucrose accumulators and, hence, may be sufficient to sustain measured
rates of sucrose synthesis.
Sucrose turnover, which is considered to regulate
carbon partitioning between different sugar pools in cells
(Geigenberger and Stitt, 1991), has been studied in sink
tissues (Dancer et al., 1990; Geigenberger and Stitt, 1991),
but has not been fully explored in tomato fruit. Direct
links between sucrose unloading and the activities of
enzymes involved in sucrose metabolism in tomato fruit
remain to be demonstrated. The present study was conducted in order to determine the relationships between
sucrose unloading and carbohydrate metabolism in
tomato fruit. The experiments were designed to investigate
the roles of SuSy and AI immediately after sucrose
unloading during the rapid growth phase of tomato fruit
development using two tomato lines with similar SuSy
but different AI activities.
Materials and methods
Plant material
Two tomato lines, having high and low fruit invertase activities,
were derived from the genetic cross between Lycopersicon
esculentum and L. chmielewskii (BC2F2 population; Accession
number LA1028; Yelle et al., 1988). Plants were grown in a
greenhouse with a 14 h photoperiod (at 400 mmol m−2 s−1),
25/22 °C day/night. Individual flowers from both lines were
tagged at anthesis to accurately follow fruit age and
development.
[3H]-(fructosyl)-sucrose uptake and starch synthesis in detached
fruits
Ten, 20 and 40 d after anthesis (DAA) fruits with the pedicel
attached were harvested from the invertase mutant and high
invertase lines. The fruit was placed in a tray containing moist
blotting paper to prevent excessive evaporation and maintained
at 21 °C in a greenhouse. The pedicels were placed in
microcentrifuge tubes containing 1 ml 60 mM sucrose and 3 mCi
of [3H ]-(fructosyl )-sucrose for 60 min (pulse). The radioactive
solutions were then removed and the pedicels were washed and
placed in 59 mM unlabelled sucrose for either 0, 60 and 120 min
(chase). At each time-point, the pedicels were removed and the
fruit were frozen and ground in liquid nitrogen and homogenized
in 80% (v/v) ethanol. An aliquot of the homogenate was used
to measure total radioactivity in the fruit. To determine the
rate of starch synthesis from labelled sucrose, 18 fruit per line
were given a 90 min radiolabel pulse as described above. The
time-course of starch synthesis from labelled sucrose was
measured after 0, 30, 60, 120 min, 24 h, and 48 h chase periods.
Analysis of labelled sugars
To determine the quantities of radiolabelled fructose, glucose
and sucrose, fruit were ground to homogeneity in liquid
nitrogen and suspended in 80% ethanol as described previously.
Ethanolic extracts were then incubated at 80 °C for 15 min and
centrifuged at 15 000 g for 10 min. The pellet fraction was
washed four times with 80% ethanol. Supernatant fractions
were pooled, lyophilized and resuspended in H O. Sugars
2
were separated by HPLC ( WATO44355, Waters column,
4.6×250 mm) and the radioactivity in each fraction was
determined in a liquid scintillation counter ( Wallac 1409,
Turku, Finland). Sucrose was hydrolysed by incubation with
yeast invertase for 30 min at 25 °C. The glucose and fructose
formed was estimated by HPLC and incorporation of label was
measured by liquid scintillation counting.
Analysis of labelled starch
The ethanol-insoluble fractions were evaporated to dryness by
heating at 60 °C. To solubilize starch, 5 vols (per initial fresh
weight) of 0.02 N NaOH were added to each sample, and the
mixtures heated at 100 °C for 15 min. The samples were cooled,
and 1 vol. of 1.5 M citrate buffer (pH 4.6) containing
100 U ml−1 of amyloglucosidase (Sigma Chemical Co, St Louis,
USA) was added to each sample. The mixtures were incubated
overnight at 55 °C and centrifuged at 15 000 g for 10 min.
Glucose, formed during the reaction, was measured using an
oxidase electrode analyser (commercial YSI model select 2700,
Yellow Springs Instrument Co. Inc., OH, USA) with a ‘glucose
membrane’ where glucose oxidase is immobilized. In this system,
glucose oxidase converts glucose to -glucono-lactone and
H O in the presence of O . H O produced during the reaction
2 2
2 2 2
was measured at a platinum anode. The radioactivity in the
solubilized starch fractions was determined by liquid scintillation
counting.
Extraction of apoplastic fluid
Apoplastic fluid was extracted from fruit pericarp tissue from
both tomato lines harvested at 20 DAA, using the infiltration
techniques described (Sato et al., 1993). The 20 DAA fruit
were cut into 5 mm discs which were first washed with 250 ml
cold water (4 °C ) and were then vacuum-infiltrated with 10 ml
buffer containing 20 mM potassium phosphate (pH 5.5),
0.5 mM b-mercaptoethanol and 1 M NaCl at 4 °C. The collected
extracellular washing fluid (EWF ) was collected by centrifugation and filtered through two layers of Miracloth. This
procedure was repeated three times. Extracellular proteins were
concentrated by centrifugation on a Centricon-10 (Amicon,
Beverly, USA) membrane at 4 °C. EWF fractions were used for
Starch synthesis in tomato fruits 1459
the analysis of sugars and determination of a-mannosidase (a
vacuolar marker enzyme) and polygalacturonase (an apoplastic
marker enzyme) activities. Cytoplasmic contamination was
determined via the activity of glucose 6-phosphate dehydrogenase as described previously ( Vanacker et al., 1998).
Enzyme activity assays
All enzymes were extracted according to the procedures
described as follows (Sun et al., 1992): 1 g fresh tissue was
homogenized in 5 ml of 200 mM HEPES-KOH buffer (pH 7.5)
containing 0.5 mM EDTA, 3 mM Mg-acetate, 1 mM MnCl ,
2
2 mM EDTA, 0.5 mM PMSF, 5 mM DTT, 20 mM
b-mercaptoethanol, 5% (v/v) glycerol, 1% (w/v) insoluble PVP,
and 1% (w/v) Dowex-1 (chloride form). After 5 min incubation
in ice, the homogenate was centrifuged at 15 000 g for 20 min
at 4 °C. The extracts were then desalted on columns of
Sephadex G-25.
Sucrose cleavage by neutral invertase and SuSy was assayed
essentially as described (Huber and Akazawa, 1986). The
reaction mixtures consisted of 100 mM HEPES-KOH buffer
(pH 7.5) containing 3 mM Mg-acetate, 0.5 mM EDTA, 1 mM
DTT, 0.4 mM NAD, 1 mM ATP, 100 mM sucrose, and 10 ml
of a mixture of coupling enzymes consisting of 4 units of
hexokinase, 4 units of glucoisomerase, and 2 units of NADspecific glucose-6-P dehydrogenase in a total volume of 1 ml.
SuSy activity was determined by the addition of 1 mM of UDP.
AI was assayed in citrate/phosphate buffer (pH 4.5) (according
to Yelle et al., 1991).
as the import from leaves or labelled sucrose import into
discs cut from the fruit (Damon et al., 1988; Yelle et al.,
1988). Randomization patterns were similar in both the
low and high invertase lines, but the total amount of
labelled sucrose taken up was 15% less in the former than
the latter (Fig. 1A). Sucrose uptake was higher in 20 DAA
fruit compared to those harvested at 10 DAA or 40 DAA.
(b) Activities of sucrose-degrading enzymes: SuSy activity
was higher in fruit harvested 20 DAA than in those
harvested at either 10 DAA or 40 DAA ( Fig. 1B). AI
activity increased from 10 to 20 DAA in the high invertase
line and then remained stable for up to 40 DAA. Maximal
extractable AI activity was significantly lower in the low
Results
Sucrose uptake and activities of sucrose-degrading
enzymes
(a) Sucrose uptake: Preliminary experiments to assess the
distribution of label derived from sucrose in the different
parts of the tomato fruit showed that similar amounts of
[3H ] were present in each section of the fruit ( Table 1).
Sucrose was therefore uniformly unloaded into the fruit
and a 1 h pulse used in these in vitro loading experiments
was sufficient to allow uniform sucrose uptake. Label
asymmetry was retained in sucrose on arrival within the
tissues showing that sucrose degradation and isomerization prior to import were minimal (data not shown).
The methods used in the present study therefore compare
favourably with the methods used in other studies, such
Table 1. Partitioning of radioactivity (DPM g−1 FW) in different
sections of 40 DAA tomato fruit fed with 1 ml of 60 mM sucrose
containing 2 mCi [3H]-(fructosyl)-sucrose for 1 h (pulse)
Subsequently fruit were sectioned into three parts and the radioactivity
in each part measured. Values represent the mean±SD of three separate
experiments.
Longitudinal sections
Transverse sections
A
B
C
1
2
3
2415±350
2665±560
2314±280
2627±460
2879±450
2440±256
2256±339
2504±235
2051±245
Fig. 1. Sucrose import (A) and the activities of sucrose-degrading
enzymes (B–D) during the development of low (%) and high (&)
invertase containing fruit. Each point represents the mean of three
separate determinations ±SD.
1460 N’tchobo et al.
invertase line than in the high invertase line (Fig. 1C ).
Invertase activity measured at neutral pH was low and
constant throughout fruit development in both tomato
lines (Fig. 1D). A strong correlation between sucrose
uptake into the fruit and SuSy activities was observed in
both tomato lines with an overall correlation coefficient
of 0.91 (Fig. 2A). In contrast, no correlation was found
between either acid or neutral invertases and sucrose
uptake. SPS activity was low and constant throughout
the early stages of fruit development (data not shown),
as has been observed previously (Dali et al., 1992; Klann
et al., 1993).
Distribution of radioactivity
The distribution of radioactivity was measured after 1 h
labelling with [3H ]-(fructosyl )-sucrose pulse followed by
transfer of labelled fruit to unlabelled sucrose for 2 h
chase (Fig. 3). The zero and 2 h chase measurements
reveal that sucrose metabolism was different at the two
time-points as indicated by the pattern of distribution of
radioactivity ( Table 2). Despite the large difference in
invertase activities between the two lines and between the
stages of fruit development ( Fig. 1C ), the distribution of
radioactivity was similar in all the fruit (Fig. 3).
Immediately after the pulse, about 80% of the radioactivity was found in sucrose, 5% in hexoses and 8% in starch,
while after the 2 h chase 60% of the label was found in
Fig. 3. The conversion of labelled sucrose into starch in low invertase
(A) and high invertase (B) lines.
sucrose, 10% in hexoses and 25% in starch at all fruit
ages. Major differences between the low and high invertase
lines were observed in the partitioning of radioactivity
between free hexoses. Although the high invertase line
accumulates hexoses, the percentage of free labelled
hexose was generally lower than that in the low invertase
line ( Table 2). This is particularly evident in the 40 DAA
fruit ( Table 2). The quantity of radioactivity in free
hexoses calculated as the percentage of radioactivity in
hexose fraction ( Table 1), multiplied by the amount of
total radioactivity taken up g−1 FW (Fig. 1), was similar
(about 850 DPM g−1 FW ) in both lines after the 1 h
pulse. Similar patterns of partitioning of radioactivity
into sucrose, glucose and fructose were found in the
apoplast of fruit from both lines with, for example, 43.6%
and 49% of the radioactivity found in the sucrose fractions, 24.6% and 24.5% in free glucose and 26.5% and
31.8% in the free fructose fraction of the low and high
invertase lines, respectively ( Table 3). The percentage of
radioactivity in the apoplast was 6.8% and 6.7% of total
labelled sucrose taken up in the high and low invertase
lines, respectively.
Turnover of imported sucrose
Fig. 2. Correlations between sucrose uptake and sucrose synthase
activity (A) and de novo starch synthesis (B) during development of
the low invertase and high invertase lines. Lines were fitted by the least
square method (r2=determination coefficient). Each point represents
the mean of three separate determinations as shown in Figs 1 and 2
and Table 3.
The symmetry of label in sucrose and free hexoses was
measured immediately after the pulse and after a 2 h
chase ( Table 4). Immediately after the pulse, the symmetry of label in sucrose was between 11% and 26% but
after a 2 h chase this had increased to 73%. The conversion
of labelled fructose to labelled glucose was more rapid in
the free hexose fraction than in the sucrose fraction, but
Starch synthesis in tomato fruits 1461
Table 2. Percentage of radioactivity in the sucrose, hexose, and starch fractions extracted from 10, 20 and 40 DAA fruit of high
invertase and low invertase lines, which were fed with 1 ml of 60 mM sucrose containing 3 mCi [3H]-(fructosyl)-sucrose for 1 h pulse
(0 h) followed by a chase (2 h)
The values represent the mean of ±SD of three separate experiments.
Fruit age
(DAA)
Tomato line
Sucrose (%)
Hexoses (%)
0h
2h
0h
Starch (%)
2h
0h
2h
10
High invertase
Mutant
81.3±11.9
75.5±7.5
61.1±13.9
58.8±8.1
5.0±0.5
7.5±0.7
8.3±0.6
10.3±0.7
5.6±1.3
4.2±0.8
25.0±1.1
23.5±3.7
20
High invertase
Mutant
81.3±2.8
82.1±12.5
66.7±18.2
67.9±14.3
2.5±0.6
3.6±0.7
5.2±0.6
6.1±1.1
11.9±1.3
10.0±1.1
24.5±0.9
23.2±2.5
40
High invertase
Mutant
78.0±12.9
62.5±3.85
65.4±22.9
53.8±21.5
5.0±1.4
11.3±1.3
8.4±1.3
16.1±2.2
5.7±1.4
5.0±1.3
20.3±3.3
20.4±3.2
Table 3. Percentage of radioactivity in the sucrose, glucose and
fructose fractions extracted from the apoplast of 40 DAA tomato
fruit supplied with [3H]-(fructosyl)-sucrose for 1 h
Tomato lines
was similar to that of sucrose uptake ( Fig. 1; Table 2).
The ratio of radioactivity measured in the starch fraction
to total radioactivity present in the tissue provides an
indication of the amount of starch synthesized from
sucrose. The proportion of sucrose conversion into starch
increased from 5% to 25% in the 2 h chase period and
remained stable thereafter. The same proportion of the
sucrose taken up by the fruit was converted into starch
( Table 2; Fig. 3), irrespective of AI activity or stage of
fruit development.
Sucrose Glucose (G) Fructose (F ) G/Fa A/Tb
(%)
(%)
(%)
High invertase
43.6
Mutant invertase 49.0
24.6
24.5
31.8
26.5
0.77
0.92
6.8%
6.7%
aRatio of free glucose to free fructose.
bPercentage of radioactivity in apoplastic fraction/total fruit extract.
similar trends were observed. To compare labelling patterns a parameter termed ‘symmetry of labelling in free
hexose fraction’ was used. This was calculated by doubling the amount of radioactivity in the free glucose
fraction and dividing it by the total radioactivity in
glucose and fructose. Immediately after the pulse between
19–30% of the total hexose pool was present as labelled
glucose increasing to 41.5% after the 2 h chase period.
The symmetry of labelling of the free hexose fraction
increased from 38.6% to 60%, immediately after the pulse,
to 83.1% after the 2 h chase.
After the 2 h chase, about 25% of the label from sucrose
was found in starch in both lines, regardless of the age
of the fruit ( Table 2). The pattern of starch accumulation
Discussion
Sucrose uptake via the pedicel in this in vitro system
produced similar patterns of labelling to those observed
by Walker et al. who used an in vivo labelling system
( Walker et al., 1978), suggesting that sucrose uptake
occurred via the same pathway in both cases. While the
volume of solution taken up depended on fruit size (data
not shown) the amount of sucrose absorbed from the
solution was dependent on the stage of fruit development
( Fig. 1A), suggesting that sucrose uptake is a controlled
process.
The data obtained in this study support the conclusion
Table 4. Symmetry of radiolabelling in sucrose and in free hexose fractions after a 1 h pulse (0 h) and a 2 h chase (2 h)
Tomato fruit 10, 20 and 40 DAA were supplied with 58 mM sucrose containing 3 mCi [3H ]-(fructosyl )-sucrose.
Fruit ages
(DAA)
Invertase activity of fruit
Symmetry (G/F ratio) of radiolabelling (%) in
Sucrose
Free hexoses
0h
2h
0h
2h
10
High
Low
12.2±4.0
11.1±4.2
49.5±6.1
44.2±14.0
47.1±21.8
60.0±6.2
74.1±9.0
80.0±8.6
20
High
Low
26.5±9.4
18.3±4.1
61.8±5.4
58.8±8.3
42.1±3.7
57.1±4.9
72.6±13.1
79.0±17.8
40
High
Low
17.9±5.1
13.7±4.1
67.6±6.4
73.3±18.8
48.2±7.2
38.6±17.6
83.1±10.4
81.8±15.4
1462 N’tchobo et al.
that sucrose imported into the fruit was not exposed to
extracellular hydrolysis. Movement from the phloem is,
therefore, likely to occur predominantly through a symplastic pathway. This conclusion, however, is based on the
following assumptions that could not be fully tested
within the scope of the present study: (a) no hydrolysis
and resynthesis of sucrose took place prior to entry into
the fruit; (b) movement of labelled sucrose into the fruit
occurred via the phloem; (c) the contamination of the
apoplastic fluid by cytoplasmic components was minimal;
and (d ) that any putative apoplastic step would involve
the entire extracellular space. This does not exclude the
possibility of a very restricted apoplastic site, proximal
to the phloem, where unloading may occur followed by
re-loading into the fruit symplasm for delivery to the
storage parenchyma cells. The following paragraph
attempts to address some of the issues raised in (a) to
(d) above.
A high degree of asymmetrical labelling has been
detected in the apoplastic hexose fraction of 20 DAA
tomato fruit after supplying [3H ]-(fructosyl )-sucrose to
leaves (Damon et al., 1988). The results obtained in this
study suggest that labelling of the apoplastic hexose
fractions was approximately equal, similar to the observations of Yelle et al. ( Yelle et al., 1988). Different degrees
of randomization of asymmetrically-labelled sucrose have
been reported (Damon et al.. 1988; Yelle et al.. 1988;
Dali et al.. 1992). In the present study similar labelling
times and amounts of sucrose found in the apoplastic
fluid obtained from pericarp tissues, were comparable to
those reported by Damon et al. (Damon et al.. 1988).
Uptake of 3H-glucose yielded similar results to those
obtained with labelled sucrose (N’tchobo et al., unpublished results). It has also been suggested that sucrose
unloading occurred predominantly via an apoplastic route
in 23–25 DAA fruit (Ruan and Patrick, 1995). In contrast, the results obtained in the present study on sucrose
unloading in tomato fruit suggested the presence of a
symplastic pathway in fruit up to 40 DAA. The decrease
in symplastic cell connections in the fruit after 20 DAA,
observed in anatomical studies (Ruan and Patrick, 1995;
Johnson et al., 1988; Offler and Horder, 1992) may
provide an explanation for the observed decrease in
sucrose import into older tomato fruit.
The role of sucrose synthase in regulating sucrose uptake
SuSy activity was high (Fig. 1B) in the rapid growth
phase of fruit development. SuSy protein amounted to
some 7% of the total soluble protein (calculated on the
basis of the specific activity of purified tomato SuSy
14 U mg−1 protein. A positive correlation between the
amount of sucrose taken up and SuSy activity was
observed ( Fig. 3A). These results strongly suggest that
SuSy is involved in the control of sucrose import into
tomato fruit. The low percentage of radioactivity in the
hexose fractions following the short labelling times used
in these experiments or with longer labelling times in
other studies (Ho et al., 1987; Yelle et al., 1988) requires
that metabolism of unloaded sucrose occurs largely in the
cytosol where SuSy is localized.
After a 1 h pulse with asymmetrically-labelled sucrose
only 15% of the sucrose pool was found to be labelled
symmetrically. After the 2 h chase period 60% of the
sucrose was labelled symmetrically, suggesting that sucrose had been synthesized during the chase period.
Substantial sucrose turnover had, therefore, occurred in
the fruit. Sucrose degradation may be catalysed by either
(i) neutral cytosolic invertase, or (ii) apoplastic AI, or
(iii) vacuole AI, or (iv) SuSy. SuSy would appear to be
the most important enzyme catalysing sucrose degradation, since a strong correlation was found between SuSy
activity and the amount of sucrose taken up by the fruit
in both the low invertase and high invertase lines
( Fig. 1A). The rate of conversion from asymmetrical to
symmetrical sucrose labelling was related to SuSy activity
( Fig. 1; Table 3).
Transformed tomato fruit with increased SPS activity
showed increased sucrose turnover, suggesting that SPS
activity may be a limiting step in sucrose synthesis in
tomato fruit (Nguyen-Quoc et al., 1999). However, the
involvement of SuSy in sucrose synthesis cannot be
discounted (Dancer et al., 1990; Geigenberger and Stitt,
1993; Hill and ap Rees, 1994).
Starch accumulation occurs gradually following anthesis, reaching a maximum in 20 DAA fruit. This is
followed by a period when the amount of starch stored
in the fruit decreases until a minimum is reached at
40 DAA. Fruit ADPglc ppase activity shows a similar
pattern of change ( Yelle et al., 1988) to that of starch.
The relative rate of starch synthesis from labelled sucrose
remained constant even when the total amount of starch
decreased (Fig. 2). About 25% of the sucrose supplied to
the fruit was used for starch synthesis. These results
suggest that starch synthesis and degradation occur simultaneously in tomato and that the amount of starch
synthesized depends on the amount of sucrose unloaded
into the fruit. The positive correlation observed between
sucrose unloading and starch synthesis (r=0.97, P<0.01;
Fig. 3B) supports this conclusion. The percentage (25%)
of radioactivity in the starch fraction was constant over
the 2 h chase period, suggesting that starch synthesis and
degradation occurred simultaneously at the same sites on
the starch molecule.
The observations that starch synthesis depends on
sucrose supply and that starch accumulation depends on
the sum of starch synthesis and degradation rates suggests
that starch forms a transient carbohydrate reservoir for
fruit growth. At the end of the fruit development, starch
accounts for only a small portion of the dry matter (Ho
Starch synthesis in tomato fruits 1463
et al., 1983) and thus does not contribute to the soluble
sugar reservoir (Schaffer and Petreikov, 1997). The correlation between the activities of SuSy, fructose kinase,
ADPglc ppase, starch synthase, and starch accumulation
(Schaffer and Petreikov, 1997) may be explained if the
activities of these enzymes are regulated relative to the
quantity of imported sucrose.
Simultaneous starch synthesis and degradation in
amyloplasts has previously been described in species
such as banana (Hill and ap Rees, 1994) and potato
(Geigenberger and Stitt, 1991; Sweetlove et al., 1996).
Tomato fruit provide an interesting model for the study
of the role of starch turnover in sink tissues. Since ADPglc
ppase has been considered to be a controlling step in
starch accumulation the amount of sucrose imported into
sink tissues could be an important determinant of starch
biosynthesis and accumulation.
References
Brown MM, Hall LJ, Ho LC. 1997. Sugar uptake by protoplasts
isolated from tomato fruit tissues during various stages of
fruit growth. Physiologia Plantarum 101, 533–539.
Chourey PS, Nelson OE. 1976. The enzymatic deficiency
conditioned by the shrunken-1 mutations in maize.
Biochemistry and Genetics 14, 1041–1055.
Dali N, Michaud D, Yelle S. 1992. Evidence for the involvement
of sucrose phosphate synthase in the pathway of sucrose
accumulation in sucrose accumulating tomato fruits. Plant
Physiology 99, 434–438.
Damon S, Hewitt J, Nieder M, Bennett AB. 1988. Sink
metabolism in tomato fruit. II. Phloem unloading and sugar
intake. Plant Physiology 87, 731–736.
Dancer J, Hatzfeld WD, Stitt M. 1990. Cytosolic cycles regulate
the turnover of sucrose in heterotrophic cell-suspension
cultures of Chenopodium rubrum L. Planta 182, 223–231.
Geigenberger P, Stitt M. 1991. A futile cycle of sucrose synthesis
and degradation is involved in regulating partitioning between
sucrose, starch and respiration in cotyledons of germinating
Ricinus communis L. seedlings when phloem transport is
inhibited. Planta 185, 81–90.
Geigenberger P, Stitt M. 1993. Sucrose synthase catalyzes a
reversible reaction in vivo in developing potato tubers and
other plant tissues. Planta 189, 329–339.
Hill SA, ap Rees T. 1994. Fluxes of carbohydrate metabolism
in ripening bananas. Planta 192, 52–60.
Ho LC. 1988. Metabolism and compartmentation of imported
sugars in sink organs in relation to sink strength. Annual
Review of Plant Physiology 39, 355–378.
Ho LC, Grange RI, Picken AJ. 1987. An analysis of the tomato
accumulation of water and dry matter in tomato fruit. Plant.
Cell and Environment 10, 157–162.
Ho LC, Sjut V, Hoad GV. 1983. The effect of assimilate supply
on fruit growth and hormone levels in tomato plants. Plant
Growth Regulation 10, 157–162.
Huber SC, Akazawa T. 1986. A novel sucrose pathway for
sucrose degradation in cultured sycamore cells. Plant
Physiology 81, 1008–1013.
Johnson C, Hall JL, Ho LC. 1988. Pathways of uptake and
accumulation of sugars in tomato fruits. Annals of Botany
61, 593–603.
Klann HM, Chetelat RT, Bennet AB. 1993. Expression of acid
invertase gene controls sugar composition in tomato
(Lycopersicon) fruit. Plant Physiology 103, 863–870.
Konno Y, Vedvick T, Fitzmaurice L, Mirkov E. 1993. Purification,
characterization, and subcellular localization of soluble
invertase from tomato fruit. Journal of Plant Physiology
141, 385–392.
Miron D, Schaffer AA. 1991. Sucrose phosphate synthase,
sucrose synthase, and invertase activities in developing fruit
of Lycopersicon esculentum Mill. and the sucrose accumulating
Lycopersicon hirsutum Humb. and Bonpl. Plant Physiology
95, 623–627.
Nguyen-Quoc B, N’tchobo H, Foyer CH, Yelle S. 1999.
Overexpression of sucrose phosphate synthase increase sucrose unloading in transformed tomato fruit. Journal of
Experimental Botany 50, 785–791.
Offler CE, Horder B. 1992. The cellular pathway of short
distance transfer of photosynthates in developing tomato
fruit. Plant Physiology 99, (suppl.) 41.
Ruan YL, Patrick JW. 1995. The cellular pathway of postphloem sugar transport in developing tomato fruit. Planta
196, 434–444.
Ruan YL, Patrick JW, Brady C. 1997. Protoplast hexose carrier
activity is a determinate of genotypic difference in hexose
storage in tomato fruit. Plant. Cell and Environment 20,
341–349.
Sato T, Iwatsubo T, Takahashi M, Nakagawa H, Ogura O,
Mori H. 1993. Intracellular localization of acid invertase in
tomato fruit and molecular cloning of a cDNA for the
enzyme. Plant Cell Physiology 34, 263–269.
Schaffer AA, Petreikov M. 1997. Sucrose to starch metabolism
in tomato fruit undergoing transient starch accumulation.
Plant Physiology 113, 739–746.
Sun J, Loboda T, Sung S-JS, Black CCJ. 1992. Sucrose
synthase in wild tomato, Lycopersicon chmielewskii, and
tomato fruit sink strength. Plant Physiology 98, 1163–1169.
Sweetlove LJ, Burrell MM, ap Rees T. 1996. Starch metabolism
in tuber of transgenic potato (Solanum tuberosum) with
increased ADPglucose pyrophosphorylase. Biochemical
Journal 320, 493–498.
Vanacker H, Carver TLW, Foyer CH. 1998. Pathogen-induced
changed in the antioxidant status of the apoplast in barley
leaves. Plant Physiology 117, 1103–1114.
Walker AJ, Ho LC. 1977. Carbon translocation in the tomato:
Carbon import and fruit growth. Annals of Botany 41,
813–823.
Walker AJ, Ho LC, Baker DA. 1978. Carbon translocation in
the tomato: pathways of carbon metabolism in the fruit.
Annals of Botany 42, 901–909.
Wang F, Sanz A, Brenner ML, Smith A. 1993. Sucrose synthase,
starch accumulation, and tomato fruit sink strength. Plant
Physiology 101, 321–327.
Yelle S, Chetelat RT, Dorais M, Deverna JW, Bennett AB.
1991. Sink metabolism in tomato fruit. Genetic and biochemical analysis of sucrose accumulation. Plant Physiology 95,
1026–1036.
Yelle S, Hewitt JD, Robinson NL, Damon S, Bennet AB. 1988.
Sink metabolism in tomato fruit. III. Analysis of carbohydrate
assimilation in a wild species. Plant Physiology 87, 737–740.
Zrenner R, Salabounat M, Willmitzer L, Sonnewald U. 1995.
Evidence of the crucial role of sucrose synthase for sink
strength using transgenic plants (Solanum tuberosum L.). The
Plant Journal 709, 97–107.