Plant Cell Physiol. 44(5): 500–509 (2003)
JSPP © 2003
Sucrose Synthase Catalyzes the de novo Production of ADPglucose Linked to
Starch Biosynthesis in Heterotrophic Tissues of Plants
Edurne Baroja-Fernández 1, Francisco José Muñoz 1, Takayo Saikusa 2, Milagros Rodríguez-López 1,
Takashi Akazawa 3 and Javier Pozueta-Romero 1, 4
1
Instituto de Agrobiotecnología y Recursos Naturales, Universidad Pública de Navarra/Consejo Superior de Investigaciones Científicas, Ctra.
Mutilva s/n, 31192, Mutilva Baja, Navarra, Spain
2
Agricultural Research Center, Ministry of Agriculture and Fishery, Kannondai 3-1-1, Tsukuba, 305-8666 Japan
3
2-278 Kamenoi, Meito-ku, Nagoya, 465-0094 Japan
;
Introduction
By using barley seeds, developmental changes of
ADPglucose (ADPG)-producing sucrose synthase (SS) and
ADPG pyrophosphorylase (AGPase) have been compared
with those of UDPglucose (UDPG), ADPG, sucrose (Suc)
and starch contents. Both ADPG-synthesizing SS and
AGPase activity patterns were found to correlate well with
those of ADPG and starch contents. Remarkably, however,
maximal activities of ADPG-synthesizing SS were found to
be several fold higher than those of AGPase throughout
seed development, the highest rate of starch accumulation
being well accounted for by SS. Kinetic analyses of SS from
barley endosperms and potato tubers in the Suc cleavage
direction showed similar Km values for ADP and UDP,
whereas apparent affinity for Suc was shown to be higher
in the presence of UDP than with ADP. Moreover, measurements of transglucosylation activities in starch granules
incubated with purified SS, ADP and [U-14C]Suc revealed a
low inhibitory effect of UDP. The ADPG and UDPG
contents in the transgenic S-112 SS and starch deficient
potato mutant [Zrenner et al. (1995) Plant J. 7: 97] were
found to be 35% and 30% of those measured in wild-type
plants, whereas both glucose-1-phosphate and glucose-6phosphate contents were found to be normal as compared
with those of wild-type plants. The overall results thus
strongly support a novel gluconeogenic mechanism reported
previously [Pozueta-Romero et al. (1999) Crit. Rev. Plant Sci.
18: 489] wherein SS catalyses directly the de novo production of ADPG linked to starch biosynthesis in heterotrophic tissues of plants.
Sucrose synthase (SS) (EC 2.4.1.13) catalyzes the following reversible reaction
NDPglucose+fructose«sucrose (Suc)+NDP,
where N stands for uridine, adenosine, guanosine, cytidine,
thymidine or inosine.
Whilst the abundance of SS in the Golgi apparatus and
plasma membranes has recently evoked the idea that it is
involved in directing the carbon flow to cell wall synthesis
(Amor et al. 1995, Nakai et al. 1999) the major role commonly
attributed to this enzyme in sink organs is to convert the Suc
imported from leaves into UDPglucose (UDPG), which is then
transformed stepwise to glucose-1-phosphate (G1P), glucose-6phosphate (G6P) and ADPglucose (ADPG) necessary for
starch biosynthesis (Fig. 1a, b) (Müller-Röber et al. 1992, Okita
1992, ap Rees 1995, Villand and Kleczkowski 1994, Denyer et
al. 1996, Buchanan et al. 2000, Kossmann and Lloyd 2000,
Neuhaus and Emes 2000, Tauberger et al. 2000, Tiessen et al.
2002). Genetic evidence demonstrating the importance of SS in
starch production comes from studies of the sh1 starch deficient mutants of maize (Chourey and Nelson 1976) and the
reduction of the starch content in the genetically engineered
potato tubers and carrots that exhibited a decrease in SS activities (Zrenner et al. 1995, Tang and Sturm 1999).
The currently prevailing schemes of Suc–starch conversion in sink organs, involving the coupled reactions of SS,
UDPG pyrophosphorylase (UGPase), phosphoglucomutase
(PGM) and ADPG pyrophosphorylase (AGPase) (Fig. 1a, b),
assume that intracellular levels of ADPG linked to starch biosynthesis are exclusively controlled by AGPase and starch synthase. However, in recent years an increasing body of evidence
has appeared that points out inconsistencies with such mechanisms, and previews the likelihood of the operation of alternative gluconeogenic pathway(s).
By employing two-dimensional NMR, Roscher et al.
(1998) have recently demonstrated that, essentially identical to
the case of bacteria, yeast, animals and photosynthetic tissues
of plants, the function of UGPase in non-photosynthetic tis-
Keywords: Gluconeogenesis — Hordeum vulgare — Solanum
tuberosum — Sucrose Synthase (EC 2.4.1.13).
Abbreviations: ADPG, ADPglucose; AGPase, ADPG pyrophosphorylase; DAP, days after pollination; Glc, glucose; G1P, glucose-1phosphate; G6P, glucose-6-phosphate; HK, hexokinase; PGI, phosphoglucoisomerase; PGM, phosphoglucomutase; Suc, sucrose; SS,
sucrose synthase; U, unit; UDPG, UDPglucose; UGPase, UDPG pyrophosphorylase.
The nucleotide sequence of potato tuber SS encoding cDNA has
been submitted to the EMBL Nucleotide Sequence Database under the
accession number AJ537575.
4
Corresponding author: E-mail, [email protected]; Fax, +34-9-4823-2191.
500
Sucrose–starch conversion in heterotrophic tissues
sues of maize is to synthesize UDPG rather than to degrade it.
The fact that a dramatic reduction of UGPase activity in transgenic potato tubers does not affect the starch content (Zrenner
et al. 1993) but significantly reduces the conversion of G1P
into UDPG necessary for Suc production during storage
(Spychalla et al. 1994, Borovkov et al. 1996) further strengthens the view that, as against to the pathways represented in
Fig. 1a and b, UGPase is involved in the synthesis of UDPG in
starch-storing organs. It should also be recalled that the steady
state concentrations of UDPG, G1P and ADPG in developing
rice seeds appear to rule out the possibility that UGPase is
involved in the Suc–starch conversion process (Su 1995).
The “perplexing” reduction in starch content in transgenic
potato tubers with high glucokinase and invertase (Trethewey
et al. 1998) is not consistent with the metabolic scheme represented in Fig. 1a where a cytosolic hexose-P enters the amyloplast to be subsequently utilized as precursor for the synthesis
of ADPG and starch in dicotyledonous plants. Furthermore, the
gluconeogenic process illustrated in Fig. 1a is also inconsistent
with the presence of plants with null or severely reduced plastid PGM and AGPase activities accumulating almost normal or
readily detectable amounts of starch (Saether and Iversen 1991,
Harrison et al. 1998, Tauberger et al. 2000, Weber et al. 2000,
Fernie et al. 2002).
There is convincing evidence that most of ADPG in wheat
endosperms is synthesized primarily in the cytosol (Beckles et
al. 2001). This nucleotide-sugar is transported into the amyloplast by the action of brittle-1, an ADPG translocator occurring in the inner envelope membranes of the wheat amyloplasts (Tetlow et al. 1994, Shannon et al. 1998, Andon et al.
2002). However, as against to the pathway illustrated in Fig.
1b, AGPase is mostly, if not all, located in the amyloplast of
wheat endosperms (Entwistle and ap Rees 1988, Tetlow et al.
1994, ap Rees 1995, Ainsworth et al. 1995). The overall information thus strongly indicates the occurrence of cytosolic
enzyme(s) distinguishable from AGPase which catalyses the
production of ADPG.
Although UDP is generally considered to be the preferred
nucleoside diphosphate for SS, numerous studies have shown
that ADP serves as an effective acceptor molecule to produce
ADPG (Murata et al. 1966, Delmer 1972, Silvius and Snyder
1979, Ross and Davies 1992, Su 1995, Nakai et al. 1998,
Porchia et al. 1999, Tanase and Yamaki 2000). The successful
utilization of recombinant potato SS for the industrial type
large-scale production of ADPG (Zervosen et al. 1998) further
strengthens the view that SS can effectively utilize ADP.
Based on the capacities of SS to produce ADPG, an alternative model of Suc–starch conversion has been proposed to
occur in the heterotrophic tissues of both mono- and dicotyledonous plants wherein SS catalyses directly the de novo
production of ADPG in the cytosol (Pozueta-Romero 1992,
Pozueta-Romero et al. 1999), which is subsequently imported
501
into the stromal phase of the amyloplast by the action of an
ADPG translocator (Fig. 1c) (Pozueta-Romero et al. 1991,
Naeem et al. 1997, Shannon et al. 1998, Baroja-Fernández et
al. 2001). Essentially in line with previous investigations
describing the occurrence of cyclic gluconeogenic processes
in bacteria (Gaudet et al. 1992, Belanger and Hatfull 1999,
Guedon et al. 2000) and animals (David et al. 1990, Massillon
et al. 1995, Bollen et al. 1998) in which glycogen synthesis and
degradation take place simultaneously, active turnover of starch
has been shown to occur in non-photosynthetic tissues of plants
(Pozueta-Romero and Akazawa 1993, Neuhaus et al. 1995,
Sweetlove et al. 1996b). Accordingly, the newly proposed
mechanistic model of Suc–starch conversion shown in Fig. 1c
assumes that both PGM and AGPase play a role in synthesizing
ADPG from the glucose (Glc) units derived from the starch
breakdown (Pozueta-Romero and Akazawa 1993, PozuetaRomero et al. 1999, Baroja-Fernández et al. 2001). In addition,
this mechanistic model assumes that UGPase is not involved in
the gluconeogenic process but its role is to produce UDPG
rather than to degrade it.
To explore the extent to which SS is responsible for the
direct conversion of Suc to ADPG linked to starch biosynthesis in heterotrophic tissues, it is absolutely necessary to analyze the developmental pattern of ADPG-synthesizing activities of SS and compare it with those of AGPase activities and
the contents of Suc, ADPG as well as starch. Furthermore, it
will be necessary to analyze the contents of gluconeogenic
intermediates in plants with altered activities of SS. Enigmatically, in spite of the fact that the genetically engineered potato
plants bearing marginally low SS activities have been shown to
accumulate less starch than wild-type plants (Zrenner et al.
1995), so far contents of G6P, G1P, UDPG and ADPG have not
been investigated, which will be a matter of study in the
present investigation.
Results
Developmental patterns of ADPG, UDPG, Suc and starch in
ripening barley seeds
Consistent with the results reported by Weschke et al.
(2000), Suc content in barley seeds at their initial stage of
development was high (50 mmol (g FW)–1), reaching a maximum (70 mmol (g FW)–1) at nearly 10 d after pollination (DAP)
(Fig. 2a). ADPG content was markedly low during the first 10–
15 DAP and then rose dramatically to reach maximal levels of
150 nmol (g FW)–1 at 20–25 DAP (Fig. 2b). Matching well
with the ADPG accumulation pattern, starch content was low
during the initial 10–15 DAP and increased to values of
2.2 mmol of Glc units (g FW)–1 (400 mg (g FW)–1) at 20–
25 DAP (Fig. 2c). In a sharp contrast, UDPG content did not
fluctuate much throughout the whole seed development process (Fig. 2b).
502
Sucrose–starch conversion in heterotrophic tissues
Fig. 1 Schematic models of Suc–starch conversion in heterotrophic organs involving the coupled reactions of SS, UGPase, PGM and AGPase
(schemes “a” and “b”) and ADPG-synthesizing activities of SS (“c”, Pozueta-Romero et al. 1999). Both (a) and (b) imply that ADPG synthesis is
exclusively catalyzed by AGPase, whereas in (c) the de novo synthesis of ADPG is catalyzed by SS and AGPase entails a role in producing
ADPG from the Glc units derived from the starch breakdown thus making up a cyclic gluconeogenic pathway. The mechanism illustrated in (a) is
suggested to occur in dicotyledonous plants (Müller-Röber et al. 1992, Tauberger et al. 2000, Tiessen et al. 2002), whereas (b) represents the pathway occurring in developing seeds of monocotyledonous plants (Denyer et al. 1996, Kleczkowski 1996, Thorbjornsen et al. 1996a, Thorbjornsen
et al. 1996b). The pathway represented in (c) is suggested to occur in both monocotyledonous and dicotyledonous plants.
ADPG-synthesizing activities of SS are many fold higher than
those of AGPase and correlate well with both ADPG and starch
accumulation patterns in barley seeds
We have examined the activity patterns of ADPGproducing SS and AGPase, and compared them with those of
Suc, ADPG, UDPG and starch levels in developing barley seeds
(see Fig. 2). As shown in Fig. 3, both ADPG-synthesizing SS
and AGPase activity patterns are found to correlate well with
those of ADPG and starch accumulation (cf. Fig. 2b and c,
respectively). AGPase activities at optimal (Fig. 3a) and sub-
Sucrose–starch conversion in heterotrophic tissues
Fig. 2 Content of (a) Suc (filled square), (b) ADPG (filled circle) and
UDPG (filled triangle) and (c) starch (filled diamond) in developing
barley seeds. Data are represented as mean ± SD (n = 4).
optimal conditions (Fig. 3b), (4 and 1.5 units (U) (g FW)–1,
respectively) are found to be comparable to those reported previously in various Hordeum species and cultivars (Kleczkowski
et al. 1993, Thorbjornsen et al. 1996a, Igamberdiev and Kleczkowski 2000). However, it will be pointed out that specific
activities of ADPG-synthesizing SS are shown to be exceedingly higher than those of AGPase at any developmental stage,
the highest rate of starch accumulation in the endosperm taking place between 15 and 20 DAP (70 nmol of Glc transferred
to starch (g FW)–1 min–1, cf. Fig. 2c) being well accounted for
by the ADPG-producing SS activities existing at 15–20 DAP
with 500 mM Suc (Fig. 3a) and 70 mM Suc (Fig. 3b) (15 and
4.5 U (g FW)–1, respectively).
Kinetic properties of barley and potato SS
One of the most critical objections against the Suc–starch
conversion model involving direct ADPG formation conferred
by SS (see Fig. 1c) comes from the long standing belief that
UDP serves as the principal, if not the exclusive, form of Glc
acceptor in the reaction catalyzed by SS (ap Rees 1995). However, as reviewed by Pozueta-Romero et al. (1999), there are
503
Fig. 3 Activities of ADPG-synthesizing SS (open circle) and
AGPase (open square) in developing barley seeds. Measurements of
the enzymatic activities were performed as described in Materials and
Methods. In (a), SS and AGPase were measured in the presence of
500 mM Suc and 1 mM G1P/ATP, respectively. In (b), SS and AGPase
were measured in the presence of 70 mM Suc and 0.5 mM of both
G1P/ATP, respectively. Data are represented as mean ± SD (n = 4).
numerous reports demonstrating that ADP can indeed act as an
effective substrate. We have thus examined the affinity of barley and potato SS towards both UDP and ADP. As shown in
Table 1, the Km values for ADP and UDP were shown to be
nearly similar in the presence of saturating Suc, whereas the
Vmax was 1.5-fold higher with UDP compared with ADP. The
kinetic studies of the Suc cleavage reaction in the presence of
fixed nucleoside diphosphate revealed a notably higher affinity
for Suc in the presence of saturating UDP than in the presence
of saturating ADP.
Coupling of ADPG synthesis catalyzed by SS with starch synthesis
Towards the end of elucidating the role of ADPGsynthesizing capacities of SS in the overall mechanism of Suc–
starch conversion we have analyzed the rate of starch synthesis
in starch granules from barley endosperms incubated with
purified SS, 2 mM ADP and different concentrations of
[U-14C]Suc. Emulating the physiological conditions existing in
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Sucrose–starch conversion in heterotrophic tissues
Table 1
Kinetic parameters of barley and potato SS
Variable substrate
Fixed substrate
Km
(mM)
Barley
Vmax
(U(mg protein)–1)
0.15
0.22
190
290
Potato
Km
Vmax
(mM) (U (mg protein)–1)
ADP
UDP
Suc (1 M)
Suc (1 M)
Suc
ADP (2 mM)
210
190
220
65
Suc
UDP (2 mM)
30
290
35
80
the barley endosperm cells at 20 DAP, the balance between SS
activity and starch content in the assay mixture was adjusted to
40 U (g starch)–1 (cf. Fig. 2c and Fig. 3).
The results presented in Fig. 4 clearly show that starch
biosynthesis increases in parallel with an elevation of the Suc
concentration in the assay mixture, the highest rate of starch
accumulation in the endosperm taking place at 15–20 DAP
(0.18 nmol Glc transferred (mg starch)–1 min–1, cf. Fig. 2c)
being well accounted for by physiological Suc concentrations.
Since both ADP and UDP serve as substrates for SS it can
be predicted that UDP may inhibit the SS-dependent production of ADPG necessary for starch biosynthetic reaction (ap
Rees 1995). To test this possibility, a time course analysis of
the transglucosylation has been carried out by using starch
granules that have been incubated with SS, 2 mM ADP,
300 mM [U-14C]Suc in the absence or presence of 1–3 mM
UDP. As shown in Fig. 5, UDP does not exert a marked inhibitory effect on the Suc–starch conversion process.
Intracellular levels of G1P, G6P, UDPG and ADPG in genetically engineered S-112 SS deficient potato tubers are inconsistent with the classical view of Suc–starch conversion
The classical mechanisms of Suc–starch conversion illustrated in Fig. 1a and Fig. 1b predict that UDPG and ADPG are
exclusively produced by SS and AGPase, respectively. However, the newly proposed gluconeogenic mechanism illustrated
in Fig. 1c predicts that ADPG is produced by both AGPase and
SS. Furthermore, it predicts that UGPase is not involved in the
starch biosynthetic process but, together with SS, catalyzes the
production of UDPG necessary for other metabolic pathways.
To distinguish which one of the models illustrated in Fig.
1 may entail a predominant role in starch storing organs we
have determined the contents of G6P, G1P, UDPG and ADPG
in the tubers of genetically engineered S-112 potato plants
0.30
0.22
65
80
which, containing 4% of the normal SS activities, accumulate
less starch than wild-type plants (Zrenner et al. 1995). According to the classical mechanism of Suc–starch conversion
involving the stepwise conversion of UDPG produced by SS
into ADPG (Fig. 1a, b), levels of the four molecules in S-112
tubers should be drastically reduced as compared with the
wild-type plants. In clear contrast, it is expected from the
newly proposed mechanistic model (Fig. 1c) that only ADPG
and UDPG, but not G6P and G1P, should be reduced.
As presented in Table 2, UDPG and ADPG contents in the
S-112 tubers were found to be 35% and 30% of those measured in the wild-type plants, respectively, whereas both G1P
and G6P contents in the S-112 tubers did not differ much with
respect to those of wild-type plants. The overall results thus are
inconsistent with the classical mechanistic scheme involving
the stepwise conversion of UDPG produced by SS into starch
(Fig. 1a, b), and further confirm the mechanism shown in Fig.
1c wherein SS catalyses directly the de novo production of
ADPG linked to starch biosynthesis.
Discussion
The mechanism of Suc–starch conversion illustrated in
Fig. 1c predicts that ADPG can be synthesized by two different mechanisms: (1) de novo, catalyzed by SS using the newly
imported Suc and (2) by the scavenging of Glc units derived
from the starch breakdown, throughout the combined actions of
PGM and AGPase.
Since both UDP and ADP are known to compete for the
reaction catalyzed by SS, it has often been argued that this
enzymatic reaction is not the principal source of ADPG in the
heterotrophic cell (De Fekete and Cardini 1964, Okita 1992, ap
Rees 1995). However, the following observations strongly indicate that SS directly produces a sizable pool of ADPG linked to
Table 2 ADPG, UDPG, G1P and G6P contents in wild-type and transgenic S-112 SS deficient
potato tubers
UDPG
G1P
G6P
ADPG
(nmol (g FW)–1) (nmol (g FW)–1) (nmol (g FW)–1) (nmol (g FW)–1)
Wild type
Transgenic S112
5.9±0.77
2.0±0.32
94.3±9.46
24.2±4.45
13.81±1.93
15.08±1.88
110.83±9.27
110.93±13.71
Sucrose–starch conversion in heterotrophic tissues
Fig. 4 Coupling system of ADPG-producing SS and starch synthase.
Ten mg of starch granules isolated from barley amyloplasts were
resuspended in 800 ml of 50 mM HEPES pH 7.0/2 mM EDTA/2 mM
DTT and incubated under active shaking at 37°C with 0.4 U of SS,
2 mM ADP and [U-14C]Suc (5´105 dpm ml–1) at different concentrations. After 15 min incubation, samples were collected to measure the
incorporated radioactivities as described in Materials and Methods.
Data are represented as mean ± SD (n = 4).
starch biosynthesis in the heterotrophic tissues of plants:
(1) SS from both barely seeds and potato tubers has high affinity towards ADP under conditions of high Suc (Table 1);
(2) Suc contents in developing barley seeds and potato tubers
are high under conditions of active starch production (Fig.
2a, cf. Fig. 4 Zrenner et al. 1995);
(3) The highest rate of starch accumulation in the endosperm
taking place between 15 and 20 DAP can be well
accounted for by the ADPG-producing SS activities existing at 15–20 DAP (Fig. 2c, 3); and
(4) UDP does not exert a strong inhibitory effect on transglucosylation activities in starch granules incubated with purified SS, ADP and Suc (Fig. 5).
Taking into account all of the limitations inherent in basing conclusions on in vitro activities, developmental analyses
of the activities of gluconeogenic enzymes have been useful in
pointing to potentially limiting steps in the Suc–starch conversion process (Tsai et al. 1970, Sowokinos 1976, Nakamura and
Yuki 1992, Merlo et al. 1993). Thus, in attempting to clarify
which one of the three mechanistic pathways represented in
Fig. 1 entails a predominant role in the starch biosynthetic
process, we carried out analyses of ADPG-synthesizing SS
activities in developing barley seed. As presented in Fig. 3,
ADPG-synthesizing SS activities in developing barley seeds
were found to correlate well with both the ADPG and starch
accumulation patterns, but not with the UDPG content pattern
(Fig. 2, 3). Furthermore, ADPG-synthesizing SS activities were
shown to be several fold higher than those of AGPase. SS
505
Fig. 5 Interaction of UDP and ADP in the Glc transfer from Suc into
starch in the coupling system of ADPG-producing SS and starch synthase. Ten mg of starch granules isolated from barley amyloplasts were
resuspended in 800 ml of 50 mM HEPES pH 7.0/2 mM EDTA/2 mM
DTT, and incubated under active shaking at 37°C with 0.4 U of SS,
2 mM ADP, 300 mM [U-14C]Suc (5´105 dpm ml–1) and the indicated
concentrations of UDP. After 5 min incubation, the reaction was
stopped as described in Materials and Methods. Data are represented
as mean ± SD (n = 3).
activities exceedingly higher than those of AGPase have been
also reported in maize endosperms (Doehlert and Kuo 1990,
Singletary et al. 1997) and potato tubers (Sweetlove et al.
1996a, Tauberger et al. 2000), the overall information further
indicating that a sizable pool of ADPG linked to starch biosynthesis is directly produced by SS.
Results presented in Table 2 show that S-112 antisensed
transgenic potato tubers bearing only 4% of the normal SS
activity contain as much as 35% of the normal UDPG. This relatively high UDPG content is ascribable to the synthetic reaction of UGPase. In line with these observations we must
emphasize that the SS deficient Sh1 maize endosperms are
characterized by having levels of UDPG comparable to those
of wild-type endosperms (cf. Fig. 3 Shannon et al. 1996). The
overall results thus strongly indicate that a considerable amount
of the intracellular UDPG in heterotrophic plant tissues is produced by UGPase and, essentially identical to the case of bacteria, yeast, animals and photosynthetic tissues of plants, the
function of UGPase in heterotrophic tissues is to produce
UDPG rather than to degrade it (Roscher et al. 1998).
Investigations carried out by using the S-112 antisensed
potato plants deficient in both SS activity and starch (Zrenner
et al. 1995) have been critically important in making up the
classical model of Suc–starch conversion involving the stepwise transformation of UDPG produced by SS into starch (Fig.
506
Sucrose–starch conversion in heterotrophic tissues
1a, b). According to this model, either G6P and/or G1P play an
important rate-limiting role in the overall gluconeogenic process. Furthermore, this model predicts that the levels of both
molecules should be drastically reduced in the S-112 plants.
However, as shown in Table 2, pools of both hexose-phosphates in S-112 plants are found to be normal, as compared
with the wild-type plants; the overall information thus demonstrating that G6P and/or G1P are not gluconeogenic intermediates as illustrated in Fig. 1a and b. As to the source of G6P and
G1P it is highly conceivable that they are derived from the Suc
breakdown catalyzed by the coupled reactions of invertase and
hexokinase(s) which, as demonstrated by Trethewey et al.
(1998), are not involved in the Suc–starch conversion process.
Concerning the advantage(s) conferred by SS in directly
producing ADPG, we can put forward the following hypothetical implications:
(1) From the evolutionary viewpoint, it is conceivable that
ADPG-synthesizing SS may confer decisively important
advantages in vascular plants where carbon metabolism is
compartmentalized between cytosol and amyloplast, facilitating a rapid and efficient conversion of Suc into starch
when there is a plentiful supply of Suc into the cell.
(2) From the standpoint of energy consumption, the direct
conversion of Suc into ADPG in the cytosol as illustrated
in Fig. 1c, is probably more efficient than the mechanisms illustrated in Fig. 1a and b involving numerous
stepwise reactions requiring ATP as well as hexose-P and
ATP importing machineries.
(3) Finally, the cytosolic production of ADPG by SS may
subject a large amount of carbon to the starch synthesis
without depleting plastidial hexose-P pools which are
required for the oxidative pentose phosphate pathway and
for the synthesis of fatty acids and amino acids (Neuhaus
and Emes 2000).
Materials and Methods
Plant material
Barley (Hordeum vulgare cv. Scarlett) plants were grown in the
fields of the Public University of Navarra. Seeds at different developmental stages were harvested and stored at –80°C until needed. Wildtype potato plants (Solanum tuberosum L. cv Desirée), and S-112 SS
antisensed plants were cultivated in growth chambers in individual
pots under a 16 h light (21°C)/8 h dark (16°C) regime.
Extraction and purification of SS
Unless otherwise indicated, all steps were carried out at 4°C. For
small-scale extractions of barley SS, endosperms were quickly frozen
in liquid nitrogen, ground to a slurry in a mortar containing 3-fold
(v/w) of extraction buffer (50 mM HEPES, pH 7.0/2 mM EDTA/2 mM
DTT), filtered through four layers of Miracloth, and desalted by ultrafiltration on Centricon YM-10 (Amicon, Bedford, MA, U.S.A.).
Purification of barley SS2 (Martínez de Ilarduya et al. 1993,
Guerin and Carbonero 1997) was performed by using 30 DAP
endosperms as described by Rodríguez-López (2002). For potato SS
purification, a full SS4 encoding cDNA (Fu and Park 1995) was overexpressed in Escherichia coli as described by Barratt et al. (2001).
Crude homogenates from both barley endosperms and E. coli cells
were adjusted to 70% (NH4)2SO4. The precipitates obtained after
30 min of centrifugation at 30,000´g were resuspended in extraction
buffer. The samples were then subjected to gel filtration on a Superdex 200 column (Pharmacia LKB) pre-equilibrated with 50 mM
HEPES, pH 7.0/150 mM NaCl. The partially purified enzyme preparations were applied to a Mono-Q column (HR 5´5, Pharmacia) equilibrated with extraction buffer and eluted with a 45 ml gradient of 0–
0.8 M KCl in 40 mM Tris-HCl pH 8.0 at a flow rate of 1 ml min–1.
Enzymatically active fractions were then desalted and loaded onto a
uridine 5¢-diphosphoglucuronic acid-agarose affinity column (Sigma)
equilibrated with 50 mM HEPES pH 7.5/1 mM DTT/1 mM EDTA.
After washing the column, SS was eluted with 0.3 M KCl.
Protein content was determined by the Bradford method using a
Bio-Rad XL-100 prepared reagent (Bio-Rad, San Diego, CA, U.S.A.).
Enzyme assays
Unless otherwise indicated, all enzymatic reactions were performed at 37°C. Measurements of AGPase were performed in the
direction of ADPG synthesis as described by Kleczkowski et al.
(1993) but ADPG was determined by HPLC as described by
Rodríguez-López et al. (2000). The assay mixture contained 50 mM
HEPES (pH 8.0), 7 mM MgCl2, and the indicated amounts of ATP and
G1P. After 10 min at 37°C, the reactions were stopped by boiling the
assay mixture for 30 s.
SS activities were measured in the direction of Suc breakdown.
The reaction mixture for SS contained 50 mM HEPES, pH 7.0/1 mM
EDTA/20% polyethilenglycol/1 mM MgCl2/ 15 mM KCl/2 mM of
either ADP or UDP, the indicated amounts of Suc and desalted protein
extract in a total volume of 50 ml. After 5 min of incubation, the reaction was stopped by boiling in a dry bath for 1 min. The reaction products (i.e. ADPG, UDPG and fructose) were then measured by using
either one of the following assay systems.
Assay A: ADPG and UDPG were determined chromatographically as described by Rodríguez-López et al. (2000).
Assay B: Fructose content was determined spectrophotometrically employing the commonly used hexokinase/phosphoglucoisomerase/G6P dehydrogenase coupling method (Amor et al. 1995).
Assay C: ADPG content was determined spectrophotometrically
in a 300 ml cocktail containing 50 mM Tris-HCl, pH 7.5/10 mM
MgCl2/0.6 mM NAD+ and 1 U each of ADP-sugar pyrophosphatase
from E. coli (Moreno-Bruna et al. 2001), PGM and G6P dehydrogenase from Leuconostoc mesenteroides. After 20 min of incubation,
NADH production was monitored at 340 nm in a Multiskan EX spectrophotometer (Labsystems, Chicago, IL, U.S.A.). A negligible
amount of NADH was produced by any protein extract in the absence
of ADP in the assay mixture.
Kinetic parameters such as Km and Vmax were evaluated by
Lineweaver–Burk plots using the purified SS preparation. One U is
defined as the amount of enzyme that catalyzes the production of
1 mmol of product per min.
Determination of soluble sugars
Soluble sugars were extracted as described by Heim et al. (1993).
Suc was determined using a commercial invertase based test kit
(Boehringer Mannheim). Both G1P and G6P were determined as
reported by Zrenner et al. (1993). ADPG and UDPG were measured
by using one of the following assay systems.
Assay A: HPLC on Waters Associate’s system fitted with a Partisil10-SAX column as described by Rodríguez-López et al. (2000).
Recovery assay for ADPG and UDPG was done and calculated to be
96% and 98%, respectively. To further confirm that measurements of
these nucleotide-sugars were correct, ADPG and UDPG eluted from
Sucrose–starch conversion in heterotrophic tissues
the Partisil-10-SAX column were enzymatically hydrolyzed with E.
coli AGPPase (Moreno-Bruna et al. 2001) and with ushA encoded
UDPG hydrolase (Burns and Beacham 1986) respectively, and then
assayed enzymatically for G1P.
Assay B: HPLC with pulsed amperometric detection on a DX500 Dionex system fitted to a Carbo-Pac PA1 column (250 mm long)
as described by Rolletschek et al. (2002).
Starch synthesis
Starch granules were prepared from amyloplasts of 20 DAP barley endosperms according to the method of Thorbjornsen et al.
(1996a). Ten mg of starch granules were resuspended in 800 ml of
50 mM HEPES pH 7.0/2 mM EDTA/2 mM DTT, and were incubated
in Eppendorf tubes at 37°C with 0.4 U of SS, 3 mM ADP, and the indicated amount of [U-14C]Suc and UDP. At the indicated incubation
times, reactions were stopped by adding 1 ml of 75% methanol/1%
KCl to 50-ml aliquots of the starch granule suspension. Parallel experiments omitting ADP or SS in the assay mixture were carried out as
negative controls. Zero-time incubation was achieved by adding 1 ml
of methanol/KCl solution to the starch granule suspension before the
addition of [U-14C]Suc. The methanol/KCl-insoluble fractions containing starch were washed thoroughly, and radioactivities were measured
to estimate the starch formation as described by Rodríguez-López et
al. (2000).
Acknowledgments
We thank María José Villafranca, Concepción Ramírez and
Marta Gómez-Revuelto (I.A.N.R., Spain) for their expertise and technical support. T. Akazawa records his sincere appreciation for support
from the Spanish Ministry of Culture and Education. We are very
thankful to Dr. R. N. Trethewey (Metanomics GmbH, Berlin) who
kindly gifted to us the S-112 transgenic potato plants deficient in SS
(Zrenner et al. 1995). We wish to thank Dr. A. Viale (University of
Rosario, Argentina), Dr. E. Echeverria (University of Florida) and H.
Beevers (University of California, Santa Cruz) who critically examined the manuscript. This research was partially supported by the grant
BIO2001–1080 from the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain).
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(Received November 7, 2002; Accepted February 20, 2003)