Download A role for `futile cycles` involving invertase and sucrose

Journal of Experimental Botany, Vol. 52, No. 358, pp. 881±889, May 2001
REVIEW ARTICLE
A role for `futile cycles' involving invertase and sucrose
synthase in sucrose metabolism of tomato fruit
Binh Nguyen-Quoc1,3 and Christine H. Foyer2
1
Centre de Recherche en Horticulture, Pavillon de l'Envirotron, FSAA, Universite Laval, QueÂbec,
Canada G1K 7P4
2
Department of Biochemistry and Physiology, IACR±Rothamsted, Harpenden, Herts AL5 2JQ, UK
Received 23 October 2000; Accepted 7 November 2000
Abstract
Current concepts of the factors determining sink
strength and the subsequent regulation of carbohydrate metabolism in tomato fruit are based upon
an understanding of the relative roles of sucrose
synthase, sucrose phosphate synthase and invertase,
derived from studies in mutants and transformed
plants. These enzymes participate in at least four
futile cycles that involve sugar transport between
the cytosol, vacuole and apoplast. Key reactions are
(1) the continuous rapid degradation of sucrose in
the cytosol by sucrose synthase (SuSy), (2) sucrose
re-synthesis via either SuSy or sucrose phosphate
synthase (SPS), (3) sucrose hydrolysis in the vacuole or apoplast by acid invertase, (4) subsequent
transport of hexoses to the cytosol where they are
once more converted into sucrose, and (5) rapid
synthesis and breakdown of starch in the amyloplast.
In this way futile cycles of sucroseuhexose interchange govern fruit sugar content and composition.
The major function of the high and constant invertase
activity in red tomato fruit is, therefore, to maintain
high cellular hexose concentrations, the hydrolysis
of sucrose in the vacuole and in the intercellular
space allowing more efficient storage of sugar in
these compartments. Vacuolar sugar storage may be
important in sustaining fruit cell growth at times
when less sucrose is available for the sink organs
because of exhaustion of the carbohydrate pools in
source leaves.
Key words: Futile cycles, sucrose metabolism, tomato fruit,
invertase, sucrose synthase.
3
Introduction
Tomato fruit are an excellent model for the investigation
of the regulation of sink activity and strength (Ho, 1996).
They are simultaneously utilization-sinks, sugar-storage
sinks and starch-storage sinks. Attempts to enhance crop
yield through genetic manipulation of sink activity have
thus far met with little success largely because of the
plasticity of metabolism (for review see Koûmann et al.,
1996; Sonnewald et al., 1994). Studies on the metabolism,
growth and development of `fruit' models such as tomato,
therefore, remain indispensable for the development of
strategies for the improvement of harvest index.
Tomato leaves, in common with most higher plants,
use sucrose as the major form of transported carbon.
Utilization of sucrose, a disaccharide composed of glucose and fructose, depends on cleavage into these hexose
units. This is catalysed by one of two enzymes, SuSy or
invertase. SuSy is a glycosyl transferase which, in the
presence of UDP, converts sucrose into UDP-glucose and
fructose. In contrast, invertase is a hydrolase cleaving
sucrose into the two monosaccharides. SuSy is a cytosolic
enzyme which is crucial to sucrose utilization in fruit
development (Sun et al., 1992; Wang et al., 1993). There is
a strong correlation between SuSy activity, the rate of
growth and amount of starch accumulated in tomato fruit
(Yelle et al., 1988).
Extracellular and vacuolar invertases link the transport
of sucrose into the apoplast and vacuole to hexose transport across the plasmalemma and tonoplast, respectively.
Different extracellular and vacuolar acid invertase isoenzymes have been puri®ed and cloned from tomato and
are characterized by highly differential sink tissue-speci®c
To whom correspondence should be addressed. Fax: q1 418 656 7871. E-mail: [email protected]
Abbreviations: ADPglc ppase, ADP-glucose pyrophosphorylase; BR, brassinosteroids; DAA, days after anthesis; F, fructose; F6P, fructose
6-phosphate; G, glucose; G1P, glucose 1-phosphate; G6P, glucose 6-phosphate; SPS, sucrose phosphate synthase; SuSy, sucrose synthase;
UDPG, UDP glucose.
ß Society for Experimental Biology 2001
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Nguyen-Quoc and Foyer
expression patterns (Konno et al., 1993; Sato et al., 1993;
N'tchobo, 1998; Godt and Roitsch, 1997). Tomato
invertases are encoded by a small gene family that
respond differently to environmental and metabolic cues
(Godt and Roitsch, 1997). One of the four extracellular
invertase genes of tomato (Lin 6), for example, has been
found to be induced by brassinosteroids (BR) in autotropic tomato cell suspension cultures linking BR-induced
growth responses to localized regulation of the source±
sink relationship (Prols et al., 2000). The role of each of
the extracellular and vacuolar invertase isoenzyme forms
in fruit development remains to be elucidated.
The correlation between the activity of ADP-glucose
pyrophosphorylase (ADPglc ppase) and the amount of
starch in tomato fruit (Yelle et al., 1988) and in other
plants (Okita, 1992; Preiss, 1988, 1991), has led to the
conclusion that this enzyme controls starch accumulation.
This conclusion appears to be convincing in certain cases,
but not at all in the other cases. The questions as to what
limits starch accumulation and which enzymes are the
most important in the regulation of starch synthesis
therefore remain unresolved.
The aim of the present review is to discuss recent
evidence that clari®es these crucial issues and to present
an integrating hypothesis of the mechanisms exerting
control of carbohydrate metabolism in tomato fruit.
Evidence is presented in support of an hypothesis involving four futile cycles of degradation and synthesis of
sucrose in the cytosol, in the vacuole and in the apoplast.
The data supporting the existence of each of these cycles
will now be discussed.
A `futile cycle' of degradation and
synthesis of sucrose in the cytosol
Sucrose unloading in tomato fruit
Recent advances in the analysis of sucrose transporters
(Gahrtz et al., 1996; Riesmeier et al., 1992, 1993; Sauer
and Stolz, 1994; Sauer and Tanner, 1989; Lemoine et al.,
1996; Weise et al., 2000) and of symplast connections and
paths of phloem unloading in plants (Patrick 1990; Ruan
and Patrick, 1995; Yin et al., 1995) have failed to resolve
key issues regarding sucrose unloading in sink organs. As
in other sink organs, the pattern of sucrose unloading
is not constant during tomato fruit development. The
unloading process consists of two phases: (i) a period of
rapid fruit growth occurring from 0 to 30±35 d after
anthesis (DAA), and (ii) a phase after rapid growth has
ceased. This lasts from about 35 DAA (depending on the
variety) to maturity and is characterized by slow growth
and very little additional accumulation of dry matter.
Two distinct modes of phloem unloading appear to
operate in each of these two stages. In the ®rst, sucrose is
unloaded intact, i.e. it is not degraded to its component
sugars prior to unloading (Damon et al., 1988; Dali et al.,
1992; N'tchobo et al., 1999). Similarly, experiments
investigating the unloading of asymmetrically-labelled
sucrose have shown that sucrose is not hydrolysed prior
to unloading (Dali et al., 1992; N'tchobo et al., 1999).
Despite the presence of acid invertase in the cell wall and
intercellular spaces, it suggests that in the ®rst phase of
fruit development sucrose is unloaded into the tomato
fruit via the symplast. This conclusion is consistent with
the observed continuity of cell connections, and numerous plasmodesmata, early in tomato fruit development.
Later in tomato fruit development these symplastic
connections are lost and sucrose is rapidly hydrolysed
during unloading (Ruan and Patrick, 1995). Similarly,
different patterns of glucose and sucrose uptake were
observed in protoplasts, isolated from fruit at different
development stages (Brown et al., 1997). The uptake of
glucose (but interestingly, not sucrose) was similar to that
observed in the intact fruit (Brown et al., 1997). The small
amount of sucrose that is unloaded by the apoplastic
pathway is hydrolysed by invertase and imported into the
fruit cells by hexose transporters. Later in fruit development the apoplastic pathway becomes the predominant.
The following discussion concerns the mechanisms of sucrose metabolism found principally in the ®rst phase of
fruit development when SuSy is highest and its activity
determines sink strength. Most of the data discussed below
were obtained with whole tomato fruit including the
seeds. However, since the parenchyma cells of the pericarp
represent the main part of the tomato fruit, the discussion
largely concerns sucrose metabolism in this tissue.
Sucrose unloading in tomato fruit is
a strictly controlled process
The amount of sucrose unloaded into tomato differs with
the age (Walker and Ho, 1977) and developmental stage
of the fruit. The two tomato lines possess similar fruit
SuSy activities, but have very different invertase activities
and show very similar patterns of sucrose unloading
(N'tchobo et al., 1999). In other experiments, in which the
activities of SuSy and SPS were increased in transgenic
plants, the rate of sucrose unloading into fruit was
proportionally increased (Nguyen-Quoc et al., 1999).
Sucrose unloading capacity was substantially decreased
in 7-d-old fruit from transgenic plants where SuSy activity
was decreased to 2% of control values. However, the
capacity of sucrose unloading in 23 DAA fruit, a point at
which SuSy activity is maximal in untransformed fruit,
was not signi®cantly decreased in the antisense SuSy
plants compared to controls (D'Aoust et al., 1999). These
observations provide evidence that invertase exerts little
or no control over sucrose unloading during the rapid
growth phase. While SuSy activity is the limiting factor in
the control of sucrose unloading in fruit for up to the ®rst
Sucrose metabolism in tomato fruit 883
10±15 d after anthesis, these data do not preclude a role
for invertase in the regulation of assimilate import into
older tomato fruit (N'tchobo, 1998).
The close correlation observed between the amount
of sucrose unloaded from the phloem and fruit SuSy
activity does not allow us to distinguish between (1) the
rate of sucrose unloading and (2) SuSy activity as the
major driving force determining sink strength. Moreover,
sucrose unloading is maintained, albeit at a low level,
during the later phase of slow growth (Walker and Ho,
1977), when SuSy activity is largely absent. Sucrose
unloading may be controlled, at least in part, by the
activity of the sugar transporters. These are regulated by
feedback controls related to the rate of sucrose hydrolysis
by SuSy.
Re-synthesis of sucrose
Tomato fruit contain two enzymes that are capable of
catalysing sucrose synthesis. These are SuSy and sucrose
phosphate synthase (SPS). The rate of sucrose synthesis
varies markedly with fruit age, a good correlation being
observed between sucrose synthesis and SuSy activity
(N'tchobo et al., 1999). It is important to consider
whether SuSy can catalyse sucrose synthesis in vivo.
When hexoses are the sole carbon source in the unloading medium, SuSy has been shown to catalyse sucrose
synthesis (Geigenberger and Stitt, 1993; Viola, 1996).
Although SuSy preferentially functions in the direction
of sucrose cleavage under in vivo conditions where sucrose
is unloaded, it is suggested that SuSy is also capable
of catalysing the synthetic reaction (Geigenberger and
Stitt, 1993; Viola, 1996). There are good reasons for
believing that SuSy does indeed catalyse sucrose synthesis
in vivo, in tomato fruit. Firstly, tomato fruit accumulate
high concentrations of hexoses (100 mmol g 1 FW) in
vacuoles and equal amounts of glucose and fructose are
present in the tissues. The vacuole represents a large
and possibly important source of fructose, which could
drive the reaction in the synthetic direction. Secondly,
while the rate of sucrose unloading into tomato fruit
varies markedly during fruit development, the turnover
rate remains constant. This implies that, for a given unit
of time, the proportion of unloaded sucrose that is again
converted into sucrose, does not change with fruit age.
This means that the absolute amount of re-synthesized
sucrose ¯uctuates considerably with fruit age, by as much
as a factor of 3 (N'tchobo et al., 1999). This would require
considerable variations in the activities of the enzymes
catalysing sucrose synthesis. SPS activity, however,
remains low throughout fruit development (Dali et al.,
1992; Miron and Schaffer, 1991; Klann et al., 1993).
Considered together these features suggest that sucrose
synthesis is catalysed by both SPS and SuSy in the rapid
growth phase of tomato fruit development.
Rapid turnover of sucrose unloaded in tomato fruit
Once unloaded into the tomato fruit, sucrose is broken
down and rapidly re-synthesized. Using sucrose labelled
asymmetrically, it was found that only 5±10% of the
sucrose molecules were labelled symmetrically after a 1 h
pulse of sucrose unloading in vitro. After a 2 h of subsequent chase, 60±90% of the sucrose was symmetrically labelled (N'tchobo et al., 1999; Nguyen-Quoc et al.,
1999). It was concluded, therefore, that almost all the
sucrose unloaded was re-synthesized within 2 h.
It is surprising that the turnover rate of sucrose was
virtually identical in control fruit and fruit from a mutant
lacking invertase (N'tchobo et al., 1999), whether measured at 10, 20 or 40 DAA. In contrast, sucrose turnover
in plants overexpressing SPS was increased by 60%
compared to untransformed controls. In the SPS overexpressors, fruit SuSy activity was also 25% higher than
in controls. This ®nding, together with the observed
absence (or very low activity) of alkaline invertase in
young tomato fruits and the symplastic pathway of
sucrose unloading, supports the hypothesis that the
cleavage of unloaded sucrose is catalysed by SuSy in the
cytosol.
Acid invertase is localized in the vacuole and extracellular compartments of the fruit. Therefore, invertase
can only hydrolyse sucrose when it is transported into
these compartments. A small amount of the sucrose
unloaded from the phloem into the fruit cells is exported
rapidly from the cytosol to the apoplast where it is
hydrolysed by apoplastic invertases (N'tchobo, 1998).
The rate of sucrose hydrolysis in the apoplast is small
compared to that in the cytosol catalysed by SuSy, which
is suf®cient to explain the constant sucrose turnover rate.
Similar mechanisms have been described in other sink
organs, such as developing potato tubers (Geigenberger
and Stitt, 1993; Viola, 1996) and Ricinum communis cotyledons (Geigenberger and Stitt, 1993) (Fig. 1).
A `futile cycle' of degradation and
synthesis of sucrose via the vacuole
and apoplast implicating a transient
storage role for acid invertase
Localization of acid invertase in the tomato fruit
In tomato, most of the invertase activity is attributable to
soluble acid invertase, the other alkaline and insoluble
invertase isoforms being either absent or present in
negligible amounts (Husain, 1999; Husain et al., 1999).
Tomato acid invertase isoforms have been puri®ed and
cloned (Yelle et al., 1991; Klann et al., 1992; Konno et al.,
1993; Sato et al., 1993) as have invertase inhibitor
proteins (Greiner et al., 1998; Sander et al., 1996). The
tomato apoplastic invertases are encoded by at least four
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Nguyen-Quoc and Foyer
Fig. 1. Model of the four principal `futile cycles' involved in sucrose metabolism in tomato fruit. The ®rst `futile cycle' involves degradation of sucrose
in the cytosol via sucrose synthase and sucrose re-synthesis of sucrose by SPS. The second `futile cycle' involves degradation of sucrose in vacuole by
invertase and re-synthesis in the cytosol catalysed by SPS or SuSy. The third futile cycle involves sucrose degradation in the apoplast by invertase
followed by sucrose synthesis in the cytosol catalysed by SPS or SuSy. The fourth cycle involved simultaneous synthesis and degradation of starch on
amyloplasts and connection to the cytosol via G6P. Enzymes: (1) UPD-glucose pyrophosphorylase, (2) phosphoglucomutase (3) phosphoglucose
isomerase, (4) fructokinase and hexokinase, (5) ADP-glucose pyrophosphorylase, (6) starch synthase, (7) starch phosphorylase and amylase,
(8) glucokinase and hexokinase.
different genes that are characterized by highly speci®c
tissue expression (Godt and Roitsch, 1997). Investigations to determine the activity and role of each acid
invertase isoform are complicated by dif®culties in developing unambiguous techniques for the study of compartmentation and the complex regulation of invertase by
inhibitors.
Immunogold labelling has showed that invertase
protein is present in the cell wall and vacuole compartments of tomato fruit cells (Husain et al., 2000a). The
activity of invertase in each cellular compartment may be
greatly decreased, however, by interaction a speci®c inhibitor protein (Greiner et al., 1998, 1999; Husain et al.,
2000a; Sander et al., 1996).
Correlations between fruit invertase activity and
hexose accumulation suggest that most acid invertase
activity is located in the vacuole, an hypothesis supported by the localization of invertase in tomato fruit
protoplasts (Konno et al., 1993). Conversely, Sato and
co-workers (Sato et al., 1993) have convincingly argued
that activity is also found in the intercellular space. In
the pericap of 20 and 50 DAA tomato fruits about 10%
of acid invertase activity was found in the apoplast
(N'tchobo, 1998).
Synthesis of sucrose in the cytosol and
degradation in the vacuole
In photosynthetic cells where the rate of sucrose synthesis
is high, there is only a very limited amount of futile
cycling between sucrose and hexoses (Foyer 1986, 1987;
Geigenberger and Stitt, 1991; Huber, 1989; Wendler et al.,
1990), even though sucrose in the cytosol is transferred
to the vacuole or apoplast where it is degraded. In
tomato fruit, high vacuolar hexose concentrations and
rapid turnover of sucrose in the cytosol support the
operation of a much more active futile cycle involving
inter-conversions between sucrose and hexoses.
The major function of the high and constant invertase
activity in red tomato fruit is to maintain the cellular
hexose concentrations. The amount of hexose found
in tomato leaves and fruit is determined by the activity of vacuolar acid invertase. A decreased invertase
activity would lead to increases in sucroseuhexose ratios
Sucrose metabolism in tomato fruit 885
(Yelle et al., 1988; Klann et al., 1993; Scholes et al., 1996;
Ohyama et al., 1995). This emphasizes not only the
continuous hydrolysis of sucrose catalysed by invertase,
but also the possibility of continuous sugar exchange
between cytosol and vacuole (sucrose in¯ux and hexose
ef¯ux). The possible physiological roles of acid invertase
and the function of this futile cycle in sink organ
development must now be considered.
A futile cycle involving the apoplast
In tomato fruits, apoplastic invertase is present at all
stages of fruit development (N'tchobo, 1998; Sato et al.,
1993; Godt and Roitsch, 1997). In the second stage of
fruit development during which sucrose is unloaded via
the apoplast, acid invertase is considered to be the main
enzyme of sucrose hydrolysis (Walker et al., 1978).
In young tomato fruit in which sucrose is unloaded via
the symplast, the role of apoplastic invertase is not
known. Only a small proportion of the sucrose unloaded
from the phloem in young fruit is exported from the
cytosol (N'tchobo, 1998). Hexoses produced from sucrose
in the apoplast are then returned to the cytosol for
sucrose re-synthesis forming a different futile cycle of
sucrose degradation and synthesis. The action of apoplastic invertase probably is essentially similar to that of
vacuolar invertase. However, the vacuole is a storage
organ for one single cell while the apoplast is a storage
organ for several cells, because the apoplast space connects cell to cell. The similarity between these futile cycles
of sucrose synthesis and degradation via vacuole or apoplast could explain the similar effect of high hexose accumulation observed in the plant over-expressing yeast
invertase in the apoplast or in the vacuole (Sonnewald
et al., 1991; Weber et al., 1998). The concentration of
hexoses in the cells could be dependent just on invertase
activity, regardless of its localization because of the
interconnection between these futile cycles.
Physiological role of invertase
The major function of invertase is to catalyse sucrose
hydrolysis. Other minor catalytic activities are known,
for example, invertase-mediated hydrolysis of raf®nose,
hydrolysis and synthesis of fructan (Pollock, 1986), or
synthesis of sucrose from raf®nose and glucose (Nadkarni
et al., 1992). If the vacuole is considered to be a storage
organelle, then vacuolar invertase would be expected
to exercise a storage function. However, data con®rming
this storage function are lacking and the physiological
role of acid invertase in tomato fruit remains somewhat
debatable. Tomato plants possessing low invertase
activity, as a result of either antisense transformation or
classical breeding (Ohyama et al., 1995; Scholes et al.,
1996; Yelle et al., 1988), had similar rates of fruit growth
to control plants. In this case the fruit had an altered
sugar composition. Transformed tomatoes with low
invertase activity accumulate more sucrose than hexose
and the fruit are slightly smaller than those of control
plants (Klann et al., 1996). This would suggest that
sucrose only accumulates in tomato fruit in the absence
of high soluble acid invertase activity. Fruit from
L. pimpinellifolium have higher invertase activities, greater
hexose contents and lower sucrose accumulation than
L. esculentum fruit (Husain et al., 2000b). Invertase activity is present even in young green L. pimpinellifolium
PI 126436 fruit and increases throughout development.
Introgression of the acid invertase gene on chromosome
3 from Lycopersicon pimpinellifolium PI 126436 into
L. esculentum FM 6203 modi®ed fruit sugar composition
but not the amount of sugar accumulated (Husain et al.,
2000b). The traits of high invertase activity and high
hexoses were lost in fruit of progeny containing the
L. pimpinellifolium invertase gene, suggesting that posttranscriptional controls are important in determining
overall fruit invertase activity.
Hexoses and not sucrose accumulate in the vacuoles
of most plants (Heineke et al., 1996). It is interesting
to speculate why this is the case. Considering that the
vacuole is a storage organelle, it is possible that sucrose
hydrolysis in the vacuole increases the storage rate (or
retention rate) of sugar in this compartment. The calculated fruit hexose content during the period of rapid
growth (10±25 DAA) is 100±150 mmol g 1 FW. In the
low-invertase mutants, the sucrose content is about
30 mmol g 1 FW, a value similar to that of the hexose
pool. Sucrose loading into the vacuole by the sucrose
antiport-transporter has been demonstrated in various
species (Andreev et al., 1990; Briskin et al., 1985; Getz
and Klein, 1994; Getz et al., 1991; Niland and Schmitz,
1995) and there is no requirement for sucrose hydrolysis
to allow vacuolar loading or unloading. Taking this into
account, the following hypothesis is proposed: the hydrolysis of sucrose in the vacuole and in the intercellular
space allows more ef®cient storage of sugar in these compartments. It is assumed, for example, that the storage
rate (the storage rate equals the import rate minus the
export rate) of sugar could be doubled when export rates
of sucrose and hexose are equal. In this scenario sucrose
is imported into vacuoles while hexoses are exported.
Every molecule of sucrose hydrolysed in the vacuole gives
two molecules of hexose. Hence, twice as many sugar
molecules are exported as sucrose molecules imported
and hydrolsed in the vacuole. Recent data on sugar
uptake into isolated tonoplast vesicles in vitro, showed
that net hexose uptake was more ef®cient than that of
sucrose (Milner et al., 1995). The term `sugar uptake' in
this experiment corresponds to the net sugar uptake rate
(sugar imported minus sugar exported), that is equivalent
to the sugar storage rate. The effect of accumulation of
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Nguyen-Quoc and Foyer
storage sugars in the vacuole through sucrose±hexose
conversion would be basically analogous to the effect of
conversion of sucrose to fructan or other polysaccharides.
The conversion of sucrose to hexoses may have other
bene®ts. For example, hexose accumulation may function to increase the osmotic potential of the tissues and
allow improved nutrient uptake. Hexoses play a role in
regulating gene expression and therefore, accumulation in
the vacuole would permit hexose storage without adverse
effects on gene expression since the hexoses would be
effectively excluded from the cytosol.
The hypothetical effect of vacuolar sugar storage
discussed above may be important for cell growth,
especially towards the end of the dark period, when less
sucrose is available for unloading into sink organs owing
to exhaustion of stocks in source leaves. This effect is all
the more marked when plants are grown under a regime
of short photoperiods or at low temperatures or under
weak illumination. In these conditions, the low-invertase
mutants produce relatively few fruit (Klann et al., 1996),
even though fruit production is normal if they are grown
with suf®cient light and at long photoperiods.
`Futile cycle' of synthesis and degradation
of starch in the amyloplast
Starch accumulation is high during the ®rst stage of fruit
development, that corresponds to the period of rapid
fruit growth. The kinetics of starch accumulation can be
divided into two phases. Net starch accumulation occurs
in the ®rst phase (0±20 DAA) while in the second phase
(20±35 DAA) net starch degradation occurs. This pattern
of starch accumulation correlates well with measured
changes in ADPglc ppase activity (Yelle et al., 1988).
Similar relationships have often been found in starch
storage organs from other plants. This implies that the
quantity of accumulated starch is related to the quantity
of product (ADP-glucose) supplied through the reaction
catalysed by ADPglc ppase. Since all the enzymes necessary for starch synthesis and degradation are present
throughout fruit development, it may be inferred that
some capacity for starch synthesis is always present. In
order to test this hypothesis, the amount of starch
synthesized from radioactively-labelled sucrose unloaded
into tomato fruit was studied in vitro (N'tchobo et al.,
1999; Nguyen-Quoc et al., 1999). These studies demonstrated that synthesis of starch could be observed at all
developmental stages. Surprisingly, the amount of newly
synthesized starch was proportional to the amount of
starch already accumulated in the fruit, while the
proportion of unloaded sucrose used for synthesis of
starch was always constant about 25% (N'tchobo et al.,
1999; Nguyen-Quoc et al., 1999; D'Aoust et al., 1999).
These results show clearly that there is continuous starch
synthesis and breakdown in the tomato fruit. The extent
of starch accumulation will depend on the relative rates
of synthesis and degradation. In tomato fruits, the most
important enzyme in starch breakdown is starch phosphorylase, which degrades starch to produce G1P. The
amylase activity is very low and the activities of both
starch phosphorylase and amylase change little during
fruit development (Yelle et al., 1988). The rate of starch
synthesis is variable whereas the rate of breakdown
appears to be relatively constant. The starch amount
accumulated decreases when the quantity of starch newly
synthesized is lower than the starch amount degraded.
The phenomenon of simultaneous starch synthesis
and degradation in amyloplasts has also been described
for other species such as pea (Hargreaves and ap Rees,
1988), banana (Hill and ap Rees, 1994) and potato
(Geigenberger and Stitt, 1991; Viola, 1996; Sweetlove
et al., 1996, 1999), constitutes an other example of `futile
cycle'.
The futile cycle of starch synthesis and degradation
presents two potential points for rigorous control. The
®rst resides over the enzyme ADPglc ppase and the
second over the point of entry (from cytosol to amyloplast) and exit (from amyloplast to cytosol) of glucose
6-P. The studies on starch synthesis in sink organs (maize,
pea, Arabidopsis, potato) where ADPglc ppase activity is
diminished via mutation or antisense transformation,
substantiate the regulatory role of this enzyme in the
direction of synthesis, i.e. limited enzyme activity limits
the synthesis of starch (Tsai and Nelson, 1966; Lin et al.,
1988; Neuhaus and Stitt, 1990; MuÈller-RoÈber et al., 1992;
Denyer et al., 1995). Conversely, there is no evidence to
suggest that augmentation of ADPglc ppase activity
causes a marked enhancement of starch content. The
increase of starch content in potato plants over-expressing
an E. coli gene for ADPglc ppase, was disproportionate
to the increase in activity (Stark et al., 1992). In a similar
experiment, Sweetlove and co-workers showed no effect
on starch accumulation (Sweetlove et al., 1996). All these
®ndings indicate that another control point over starch
synthesis, apart from ADPglc ppase, must exist. One
candidate is the concentration of hexose phosphates in
the amyloplasts, or the rate of exchange of hexose
phosphates between the cytosol and the amyloplast.
In tomato, the principal hexose-P which crosses the
amyloplast membrane is G6P (N'tchobo, 1998) which is
also the case in other dicot species (Hill and Smitt, 1991;
Neuhaus et al., 1993; Viola et al., 1991).
All the `futile cycles' are interconnected and
co-ordinated by sucrose synthase to form
a regulatory cycle
SuSy is strongly expressed in most plant storage organs
and its activity is often found to correlate with starch
accumulation or with organ growth. A recent study
Sucrose metabolism in tomato fruit 887
(Zrenner et al., 1995), in which SuSy expression in potato
tubers was repressed, demonstrates the crucial role of this
enzyme in sink organs. Nevertheless, current understanding of how SuSy activity regulates starch synthesis,
sucrose accumulation, or growth remains far from complete. From a consideration of other hypotheses (Dancer
et al., 1990; Geigenberger and Stitt, 1991, 1993; Hill and
ap Rees, 1994; Huber, 1989; Viola, 1996), as well as the
authors' own results, an explanation is offered here which,
it is hoped, will prove fruitful in provoking further
discussion and experimental work.
Sucrose unloading in sink organs takes place primarily
via the symplast and the quantity unloaded is determined
by SuSy activity. The concept that sink strength is
determined by SuSy activity is, thus, widely accepted. In
the main futile cycle, sucrose degradation is catalysed by
SuSy while sucrose synthesis is catalysed by SPS or SuSy.
This reaction sequence effectively controls the concentration of several major metabolites including UDPG, G1P,
G6P, F6P, F, G, and sucrose itself. These metabolites and
their products regulate cell wall synthesis, the respiration
rate, starch synthesis, storage in the vacuoles and other
product synthesis. For example, the amount of accumulated or newly-synthesized starch will depend on the
cytosolic concentration of G6P. In the same way, the rate
of G6P utilization will be related to the concentration
of F6P, which will in turn depend on rates of synthesis
and breakdown of sucrose. An increase in alkaline
invertase and SPS activities would be capable, in theory,
of compensating for a decrease in SuSy activity.
Conclusions
The key features of the regulation of sugar metabolism
in tomato fruit involves four principal futile cycles that
are all strictly connected. These are (i) rapid, continuous
degradation and re-synthesis of sucrose in the cytosol. In
this cycle, sucrose degradation is catalysed by SuSy and
sucrose synthesis by SuSy or SPS. It is proposed that
SuSy has the major regulatory role in this cycle. (ii)
Hydrolysis of sucrose in the vacuole, catalysed by acid
invertase. Most of the hexose is sequestered in the vacuole
but some is re-converted into sucrose in the cytosol. These
two opposing reactions form the second `futile cycle',
which functions to increase the ef®ciency of sugar storage
in these compartments. It is suggested that the physiological role of acid invertase is to provide a store for sucrose
equivalents (hexoses). (iii) Hydrolysis of unloaded sucrose
in the apoplast catalysed by apoplastic invertase. Most
of the hexoses formed in the apoplast is re-converted
to sucrose in the cytosol forming the third futile cycle.
(iv) Rapid ongoing synthesis and breakdown of starch in
the amyloplast. This constitutes the fourth `futile cycle'.
The relative rates of synthesis and breakdown determine
the quantity of starch accumulated. The overall rate of
starch synthesis is controlled by ADPglc ppase, however,
the relative rates of synthesis and degradation are controlled by the concentration of G6P. This, in turn,
depends on the relative ¯ux of this metabolite across
the amyloplast membrane. The G6P concentration in the
amyloplast will hence also depend, to some degree, on
the SuSy activity in the cytosol. The possibility cannot be
excluded that differential activity of the tonoplast-bound
sugar transporters (hexose and sucrose-transporters)
plays a regulatory role in determining the direction of
the SuSy reaction by control of the cytosolic sucrose and
hexoses pools (Getz and Klein, 1995).
Acknowledgements
We are thankful to Dr HP Getz, Dr S Huber and Dr A
Lecharny for their discussions on the preparation of this paper.
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