Download New enzymes, new pathways and an alternative view

Biocatalysis and Biotransformation, Jan/Feb/March/April 2006; 24(1/2): 63 /76
ORIGINAL ARTICLE
New enzymes, new pathways and an alternative view on starch
biosynthesis in both photosynthetic and heterotrophic tissues of plants
FRANCISCO JOSÉ MUÑOZ1, MARIA TERESA MORÁN ZORZANO1,
NORA ALONSO-CASAJÚS1, EDURNE BAROJA-FERNÁNDEZ1, ED ETXEBERRIA2, &
JAVIER POZUETA-ROMERO1
1
Instituto de Agrobiotecnologı́a, Universidad Pública de Navarra/Consejo Superior de Investigaciones Cientı́ficas Ctra.
Mutilva s/n, 31192, Mutilva Baja, Navarra, Spain and 2University of Florida, IFAS, Department of Horticultural Sciences,
Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL, 33850, USA
(Received 16 May 2005; revised 26 August 2005)
Abstract
Since the initial discovery showing that ADPglucose (ADPG) serves as the universal glucosyl donor in the reaction catalyzed
by starch synthase, the mechanism of starch biosynthesis in both leaves and heterotrophic organs has generally been
considered to be an unidirectional process wherein ADPG pyrophosphorylase (AGPase) exclusively catalyzes the synthesis
of ADPG and acts as the major limiting step of the gluconeogenic process. There is however mounting evidence that ADPG
linked to starch biosynthesis is produced de novo in the cytosol by means of sucrose synthase (SuSy). In this review we show
and discuss the numerous pitfalls of the ‘classic’ view of starch biosynthesis. In addition, we describe many overlooked
aspects of both ADPG and starch metabolism. With the overall data we propose an ‘alternative’ model of starch
biosynthesis, applicable to both photosynthetic and heterotrophic tissues, according to which both sucrose and starch
biosynthetic processes are tightly interconnected by means of an ADPG synthesizing SuSy activity. According to this new
view, starch metabolism embodies catabolic and anabolic reactions taking place simultaneously in which AGPase plays a
vital role in the scavenging of starch breakdown products.
Keywords: ADPglucose, ADPglucose pyrophosphorylase, Calvin cycle, starch, starch turnover, sucrose, sucrose synthase
Abbreviations: ADPG, ADPglucose, AGPase, ADPG pyrophosphorylase, APPase, alkaline pyrophosphatase, Glc, glucose,
G1P, Glc-1-phosphate, G6P, Glc-6-phosphate, HK, hexokinase, Pi, orthophosphate, 3PGA, 3-phosphoglycerate, PGI,
phosphoglucoisomerase, PGM, phosphoglucomutase, PPi, pyrophosphate, Suc, sucrose, SPS, Suc phosphate synthase, SuSy,
Suc synthase, UDPG, UDPglucose, UGPase, UDPG pyrophosphorylase, WT, wild type
Introduction
Starch is the main storage carbohydrate in vascular
plants. Its abundance as a naturally occurring
compound is surpassed only by cellulose, and
represents the primary source of calories in the
human diet. Worldwide production of this polysaccharide amongst the main horticultural crops exceeds 109 tons/year. Because of its unique
physicochemical properties, the use of starch is of
utmost importance in practically every industry in
existence. Furthermore, its utilization as a renewable
polymer and clean energy source is becoming
increasingly attractive as a consequence of social
concerns about industrial wastes generated from
fossil fuels. Taking into account the very basic role
that starch plays in modern societies, a thorough
understanding of the mechanisms involved in its
synthesis will be critically important for the rational
design of experimental traits aimed at improving
yields in agriculture, and producing more and better
polymers that fit both industrial needs and social
demands.
Correspondence: Javier Pozueta-Romero, Instituto de Agrobiotecnologı́a, Universidad Pública de Navarra/Consejo Superior de
Investigaciones Cientı́ficas; Ctra. Mutilva s/n, 31192, Mutilva Baja, Navarra, Spain. Tel: (34) 948168009. Fax: (34) 948232191.
E-mail: [email protected]
ISSN 1024-2422 print/ISSN 1029-2446 online # 2006 Taylor & Francis
DOI: 10.1080/10242420500518839
64
F. J. Muñoz et al.
Starch is synthesized in the plastid
The most fundamental difference between plants
and other eukaryotic organisms is the possession of
an additional compartment inside the cell, the
plastid. Enclosed by two envelope membranes, the
inner one contains a variety of transporters that
mediate the exchange of metabolites between the
plastid and the surrounding cytosol.
Among different plastid types, chloroplasts of
photosynthetically active organs such as adult leaves
are bioenergetically autonomous (Arnon 1955).
Mature chloroplast possess the photochemical capacity to provide chemical energy (ATP) during
illumination, fix carbon, and eventually synthesize
starch that, owing to the diurnal rise and fall of its
levels, is designated as ‘transitory starch’. By contrast, production of reserve starch in non-photosynthetic amyloplasts of heterotrophic organs, i.e.
tubers and seed endosperms, depends upon the
incoming supply of carbon precursors and energy
from the cytosol. This fairly obvious digression
between transitory and long-term storage starch
leads to the conclusion that the gluconeogenic
pathway(s) leading to starch production in amyloplasts must be distinguishable from that of chloroplasts.
A classic view of the starch biosynthetic process
The synthesis of both transitory and reserve
starch are complex mechanisms biochemically regulated by events involving interactions between metabolites and enzymes present in both the cytosol
and plastids (Okita et al. 1992; Fernie et al. 2002c;
Geigenberger 2003; Tetlow et al. 2004; Smith et al.
2005).
Since the initial discovery showing that ADPglucose (ADPG) serves as the universal glucosyl donor
in the reaction catalyzed by starch synthase (Recondo et al. 1963; Murata et al. 1963), the mechanism of starch biosynthesis in chloroplasts and
amyloplasts has generally been considered to be an
unidirectional and vectorial process wherein ADPG
pyrophosphorylase (AGPase) exclusively catalyzes
the synthesis of ADPG and pyrophosphate (PPi),
and acts as the major limiting step of the gluconeogenic process (Caspar et al. 1985; Preiss 1988;
Müller-Röber et al. 1992; Okita 1992; Stark et al.
1992). AGPase is a reversible enzyme whose reaction is greatly displaced from equilibrium through
the rapid and irreversible hydrolysis of PPi mediated
by the plastidial alkaline pyrophosphatase (APPase)
(Gross & ap Rees 1986). In addition, AGPase is a
highly regulated enzyme, since it is subjected to
redox regulation (Hendriks et al. 2003), is allosteri-
cally activated by 3-phosphoglycerate (3PGA), and
inhibited by orthophosphate (Pi) (Kleczkowski
1999, 2000; Crevillén et al. 2003).
Plants defective in AGPase activity exhibit a
significant decrease in starch content (Tsai & Nelson
1966; Dickinson & Preiss 1969; Lin et al. 1988a,b;
Müller-Röber et al. 1992), whereas plants with
increased AGPase exhibit an increase in starch
content (Stark et al. 1992; Smidansky et al. 2002;
Sakulsingharoj et al. 2004). The combined data thus
unequivocally show that AGPase plays a pivotal role
in the starch biosynthetic process.
Transitory starch biosynthesis in photosynthetic tissues
Information gained from both biochemical and
genetic approaches strongly support the notion that
starch is the end-product of a pathway directly
linked to the Calvin cycle by means of plastid
phosphoglucose isomerase (PGI), and that exclusively takes place in the chloroplast (Figure 1)
(Kossmann et al. 1994; Haake et al. 1998). This
model, basically consistent with the conceptual
idea that the chloroplast is a self-sustaining
photosynthetic unit (Arnon 1955), appears to be
strongly supported by in organello experiments
demonstrating that CO2-dependent formation of
starch can take place in the isolated chloroplast
(Heldt et al. 1977).
Photosynthetically synthesized triose-P molecules
are exported to the cytosol and converted to sucrose
(Suc) that will be transported to heterotrophic parts
of the plant. According to the ‘classic model’, when
phloem loading capacity is saturated, Suc production in the cytosol is arrested and carbon flux
diverted towards starch production in the chloroplast. Thus, according to this view, Suc and transitory starch are end products of two segregated
gluconeogenic pathways taking place in the cytosol
and chloroplast, respectively.
Starch biosynthesis in heterotrophic tissues of
di-cotyledonous plants
Suc produced in photosynthetically active plant
parts is imported by the heterotrophic tissues and
used as carbon source for energy production and its
ultimate conversion to starch in the amyloplast.
Because both chloroplasts and amyloplasts are
ontogenically related, it was originally assumed that
analogous mechanisms of starch biosynthesis operate in both plastid types, i.e. a triose-P is the
precursor molecule of starch entering the amyloplast. However, NMR spectroscopy experiments of
starch biogenesis in wheat and maize grains using
13
C-NMR (Keeling et al. 1988; Hatzfeld & Stitt
Starch biosynthesis in both photosynthetic and heterotrophic tissues of plants
65
CO2
Triose-P
CalvinBenson
cycle
Triose-P
Pi
1´
Pi
1
FBP
FBP
2´
3
F6P
2
F6P
4´
4
G6P
5´
5
2Pi
G1P
Starch
Sucrose
G6P
8
G1P
11
6
9
PPi
10
7
UDPG
ADPG
Suc-P
F6P
Chloroplast
Cytosol
Figure 1. Classic view of Suc and starch synthesis in leaves. The enzymes are numbered as follows: 1, 1?, fructose-1,6-bisphosphate
aldolase; 2, 2?, fructose 1,6-bisphosphatase; 3, PPi: fructose-6-P 1- phosphotransferase;4, 4?, PGI; 5, 5? PGM; 6, UGPase; 7, SPS; 8, Suc
phosphate phosphatase; 9, AGPase; 10, starch synthase; 11, APPase. According to this view, the starch biosynthetic process takes place
exclusively in the chloroplast and segregated from the sucrose biosynthetic pathway taking place in the cytosol.
1990) were crucial in demonstrating that a C-6
molecule, not a triose-P, is the precursor of starch
biosynthesis in amyloplasts. Further investigations of
the enzyme capacities of amyloplasts (Entwistle & ap
Rees 1988; Frehner et al. 1990) supported the view
that C-6 molecules entering amyloplasts are the
precursors of starch biosynthesis.
The classic pathway of Suc-starch conversion in
heterotrophic tissues of dicotyledonous plants is
illustrated in Figure 2. Soon after Suc enters the
heterotrophic cell, it is broken down by Suc synthase
(SuSy) that catalyzes the reversible reaction whereby
Suc and UDP are converted to UDPglucose
Starch
(UDPG) and fructose. UDPG is then converted to
glucose-1-P (G1P) by UDPG pyrophosphorylase
(UGPase) and G1P subsequently metabolized to
glucose-6-phosphate (G6P) by means of the cytosolic phosphoglucomutase (PGM). G6P then enters
the amyloplast where it is converted to starch by the
sequential activities of plastid PGM, AGPase and
starch synthase (Kammerer et al. 1998; Tauberger et
al. 2000).
Unlike chloroplasts that can produce ADPG from
the photochemically produced ATP, amyloplasts are
unable to generate ATP and therefore, it is assumed
to be imported from the cytosol. In agreement with
ADPG
2Pi
APPase
ADP
ATP
ADP
PPi
AGPase
Sucrose
ATP
G1P
UDP
SuSy
Fru
PGM
G6P
G6P
PGM
G1P
UGP
UGPase
UTP
Amyloplastt
UDPG
PPi
Cytosoll
Figure 2. Schematic representation of the classic model of Suc-starch conversion in heterotrophic tissues of di-cotyledonous plants.
66
F. J. Muñoz et al.
studies that had shown that AGPase is largely
located in the amyloplast of cereal endosperms
(Entwistle & ap Rees 1988; ap Rees 1995; Miller
& Chourey 1995; Brangeon et al. 1997). Although
sequence analyses of some AGPase encoding
cDNAs appear to indicate the occurrence of extraplastidial AGPase isoform(s) (Thornbjørnsen et al.
1996b; Johnson et al. 2003) we must emphasize that
the idea that most of AGPase is extraplastidial in
cereal seeds is principally based on cell fractionation
studies performed by using soluble (stromal) fractions obtained from mechanically disrupted amyloplasts (Denyer et al. 1996; Burton et al. 2002;
Johnson et al. 2003). Most importantly, however,
immunolocalization and proteomic analyses using
whole amyloplast preparations have shown that most
of plastid AGPase is bound to starch granules and
envelope membranes (Miller & Chourey 1995;
Brangeon et al. 1997; Andon et al. 2002). The
overall results thus strongly indicate that, similar to
the case of heterotrophic organs of dicotyledonous
plants, plastid AGPase must be abundant in cereal
endosperms and likely plays an important role in the
control of starch biosynthesis.
this presumption, investigations carried out independently in several laboratories have firmly established that amyloplasts contain an ATP/ADP
translocator in their envelopes (Pozueta-Romero
et al. 1991b, Neuhaus et al. 1997). By using
genetically engineered plants, Neuhaus et al. reported that the rate of ATP import from the cytosol
controls the rate of starch synthesis and affects the
composition of starch in potato tubers (Geigenberger et al. 2001).
Starch biosynthesis in cereal endosperms
In contrast to the case of heterotrophic tissues of
dicotyledonous plants, most of AGPase is cytosolic
in cereal endosperms (Villand & Kleczkowski 1994;
Kleczkowski 1996; Thornbjørnsen et al. 1996a;
Denyer et al. 1996; Beckles et al. 2001; Johnson et
al. 2003), whereas plastid AGPase accounts for less
than 10% of the total activity and is not sufficient to
catalyze the normal rate of starch synthesis. Accordingly, a gluconeogenic model has been proposed
wherein the stepwise reactions of SuSy, UGPase and
AGPase take place in the cytosol to catalyze the
production of the ADPG necessary for starch
biosynthesis (Figure 3).
An intriguing aspect of this metabolic scheme is
connected to APPase. All APPase is considered to be
located in the plastids of both mono- and dicotyledonous plants (Gross & ap Rees 1986; Denyer
et al. 1996; Thornbjørnsen et al. 1996a; Beckles et
al. 2001; Tetlow et al. 2003) and plays an important
role in the starch biosynthetic process since it
displaces the AGPase reaction from equilibrium
through the rapid removal of PPi (see Figures 1
and 2). The arising question is: if most of AGPase is
located in the cytosol in cereal endosperms, what is
the physiological role of APPase?
The finding that most of AGPase in cereal
endosperms is extraplastidial conflicts with previous
Some inconsistencies of the classic view of
starch biosynthesis
A vast amount of information has been obtained
from various sources that appear to support the
models illustrated in Figures 1 /3, wherein AGPase
is the only enzyme synthesizing the ADPG linked to
starch biosynthesis (Müller-Röber et al. 1992; Okita
1992; ap Rees 1995). However, in recent years an
increasing body of experimental evidence points to
inconsistencies with such mechanisms. Some of
these inconsistencies have been recently discussed
(Pozueta-Romero et al. 1999; Baroja-Fernández et
al. 2001, 2003, 2005) and are listed below.
Starch
Sucrose
UDP
SuSy
Fru
ADPG
ADPG
AGPase
PPi
Amyloplast
G1P
ATP
UGPase
UTP
Cytosol
Figure 3. Scheme of the classic model of Suc-starch conversion in cereal endosperms.
PPi
UDPG
Starch biosynthesis in both photosynthetic and heterotrophic tissues of plants
Inconsistencies of the classic model of starch biosynthesis
in photosynthetic tissues
1. The gluconeogenic model illustrated in Figure
1 is not consistent with the presence of plants
with null or severely reduced chloroplastic
plastid PGM and AGPase activities accumulating readily detectable amounts of starch in their
leaves (Harrison et al. 1998; Weber et al. 2000).
In fact, we have observed that Arabidopsis
leaves totally lacking either plastid PGM or
AGPase (Caspar et al. 1985; Lin et al. 1988)
can accumulate as much as 15% of the normal
starch content when plants are cultured in the
presence of Suc (Figure 4). Moreover, leaves of
plants with a deficiency in either plastid PGM
or AGPase cultured in the presence of sucrose
can accumulate higher levels of starch than
Wild type (WT) leaves of plants cultured without Suc.
2. UGPase, cytosolic PGM and Suc phosphate
synthase (SPS) are involved in Suc biosynthesis. The classic view of starch biosynthesis
illustrated in Figure 1 predicts that, under
conditions that synthesis of Suc is prevented,
carbon flow will be diverted towards starch
biosynthesis. However, leaves of transgenic
plants with reduced UGPase, cytosolic PGM
and SPS are characterized by containing low
levels of both Suc and starch in their leaves
(Strand et al. 2000; Fernie et al. 2002a;
Kleczkowski et al. 2004).
3. According to the classic view of starch biosynthesis illustrated in Figure 1, the transitory
starch biosynthetic process is directly linked to
8,0
Starch (µmol/ g FW)
7,0
6,0
5,0
4,0
3,0
2,0
1,0
67
the Calvin cycle by means of plastid PGI.
However, Calvin cycle intermediates such as
erythrose-4-phosphate and 3PGA are potent
inhibitors of plastid PGI (Kelly et al. 1980;
Dietz 1985; Backhausen et al. 1997) and the
stromal concentrations of these compounds in
the illuminated chloroplast are considerably
higher than their Ki values for plastid PGI
(Bassham & Krause 1969; Dietz 1985).
Furthermore, the stromal G6P/fructose-6phosphate ratio is quite low and displaced
from equilibrium in the illuminated leaf (Dietz
1985), indicating that (a) plastid PGI is
strongly inhibited under conditions of active
CO2 fixation and starch production and (b) the
starch biosynthetic process is not directly linked
to the Calvin cycle in photosynthetic tissues.
4. ADPG accumulates in leaves (Smith et al.
1990; Baroja-Fernández et al. 2004). However,
this nucleotide-sugar spontaneously hydrolyzes
to AMP and the poorly metabolizable glucose1,2-monophosphate under conditions of alkaline pH and high Mg2 concentration occurring in the illuminated chloroplast (Zervosen et
al. 1998; Baroja-Fernández et al. 2001). Therefore, unless internal compartments occur inside
the chloroplast that prevent spontaneous hydrolytic breakdown of ADPG, this molecule
cannot accumulate in the chloroplast during
active starch biosynthesis in leaves.
5. Leaves can produce starch from exogenously
added glucose (Glc). According to the model
illustrated in Figure 1, one can predict that
leaves fed for a short time with [14C]Glc labelled
in position 1 will show a redistribution of the
radiolabelled carbon in the Glc moiety of the
newly synthesized starch molecule. Triose-P
derived from the cytosolic metabolism of
[1-14C]Glc would be imported into chloroplasts
and subsequently transformed to hexose-P and
starch molecules whose Glc moiety will be
labelled predominantly in the C1 and C6 positions. Against this prediction, however, results
obtained by McLachlan & Porter (1959) using
source tobacco leaves fed with [1-14C]Glc
showed that the Glc moiety in the starch
molecules is still primarily labelled in position 1.
0,0
WT
TL25
+ sucrose
TC7
WT
TL25
TC7
-sucrose
Figure 4. Both plastid PGM and AGPase lacking leaves of
Arabidopsis (TC7 and TL25, respectively) (Caspar et al. 1985;
Lin et al. 1988) accumulate readily detectable amounts of starch,
especially in the presence of sucrose. Plants cultured in the
presence of glucose were grown in MS medium supplemented
with 50 mM sucrose. Starch content was measured as described
by Baroja-Fernández et al. (2004).
Inconsistencies of the classic model of starch biosynthesis
in heterotrophic tissues
1. Two-dimensional NMR studies have demonstrated that, in contrast to the mechanistic
models illustrated in Figures 2 and 3, and
essentially identical to the situation in bacteria,
yeast, animals and photosynthetic tissues, the
68
2.
3.
4.
5.
6.
7.
F. J. Muñoz et al.
function of UGPase in heterotrophic tissues of
maize is to synthesize UDPG rather than to
degrade it (Roscher et al. 1998).
A dramatic reduction of UGPase activity in
transgenic potato tubers does not affect the
starch content (Zrenner et al. 1993) implying
that UGPase is not involved in the process of
starch biosynthesis.
The reduction in starch content and enhancement of both G6P and G1P levels in glucokinase and invertase overexpressing potato tubers
(Trethewey et al. 1998) is not consistent with
the metabolic scheme represented in Figure 2,
wherein a cytosolic hexose-P enters the amyloplast to be subsequently utilized as gluconeogenic precursor. According to the ‘classic’
model of starch biosynthesis, increasing invertase and glucokinase activities should be accompanied by a concomitant increase of both
hexose-Ps and starch.
The gluconeogenic models illustrated in Figures
2 and 3 are not consistent 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 & Iversen 1991; Harrison et al.
1998; Weber et al. 2000; Fritzius et al. 2001;
Fernie et al. 2002b; Johnson et al. 2003).
The gluconeogenic model illustrated in Figure
2 predicts that levels of the gluconeogenic
intermediates UDPG, G1P, G6P and ADPG
should be drastically reduced in the heterotrophic tissues of plants deficient in SuSy.
However, the S-112 antisensed potato plants
bearing only 4% of the normal SuSy activity
(Zrenner et al. 1995) contain normal levels of
both G1P and G6P (Baroja-Fernández et al.
2003), indicating that hexose-Ps do not serve as
precursors of the starch biosynthetic process.
The reduction of starch content and the
enhancement of levels of both G6P and G1P
in potato tubers expressing a bacterial Suc
phosphorylase (Trethewey et al. 2001) is not
consistent with the metabolic scheme represented in Figure 2 wherein a cytosolic hexose-P
enters the amyloplast to be subsequently utilized as gluconeogenic precursor. According to
the ‘classic’ model of starch biosynthesis, heterologous expression of Suc phosphorylase
should be accompanied by a concomitant
increase of both hexose-Ps and starch.
Changes in cytosolic fructokinase in transgenic
potato tubers are not accompanied by changes
on starch content (Davies et al. 2005) implying
that hexose-Ps are not directly involved in the
starch biosynthetic process.
Some overlooked aspects of both ADPG and
starch metabolism
Occurrence of an ADPG transport machinery in the
envelope membranes of plastids
While investigating the mechanism(s) of ATP entry
into the plastids, Pozueta-Romero et al. (1991a,b)
found that both chloroplasts and amyloplasts are able
to import ADPG from the cytosol. The estimated
rate of starch production in isolated chloroplasts
incubated with 0.2 mM ADPG was 5 nmol of Glc
transferred to starch (mg chlorophyll)1 min1,
which is comparable to values reported in the
literature (5 /15 nmol of Glc transferred to starch
(mg chlorophyll)1 min 1 (Lin et al. 1988; Strand et
al. 2000; Baroja-Fernández et al. 2004).
Although ADPG transport machineries have been
reported to occur in the plastid envelopes of both
mono- and di-cotyledonous plant species (Cao et al.
1995; Sullivan & Kaneko 1995; Shannon et al. 1996,
1998; Möhlmann et al. 1997; Naeem et al. 1997;
Patron et al. 2004), our knowledge about their
nature is still scanty. In this respect, plants contain
a large family of sequences in their genomes encoding putative chloroplast proteins sharing high similarity to Brittle-1 (http://plantst.sdsc.edu/plantst), a
well known ADPG transporter existing in amyloplasts from cereal endosperms (Shannon et al. 1998;
Patron et al. 2004). While some of them are known
to specifically transport AMP, ADP and ATP when
functionally integrated into the bacterial cytoplasmic
membrane (Leroch et al. 2005), others containing a
putative KKGGL ADPG-binding motif (BarojaFernández et al. 2001, 2004) need to be further
studied to understand their possible involvement in
ADPG transport.
SuSy: a source of extraplastidial ADPG in both sink and
source organs
The occurrence of ADPG transport machineries in
the inner envelope membrane of plastids evoked the
idea that a sizable pool of ADPG is produced outside
the plastid (Pozueta-Romero et al. 1991b, 1999). In
this context, it should be noted that ADP serves as an
effective acceptor molecule of SuSy producing
ADPG (Delmer 1972; Silvius & Snyder 1979; Nakai
et al. 1998; Porchia et al. 1999; Tanase & Yamaki
2000; Baroja-Fernández et al. 2003). SuSy is a highly
regulated enzyme (Koch et al. 1992; Asano et al.
2002; Pozueta-Romero et al. 2004) that is very active
in reserve organs, but it also occurs in the mesophyll
cells of source leaves (Fu et al. 1995; Wang et al.
1999). Evidence that SuSy is engaged in the Sucstarch conversion process comes from the study of
SuSy deficient plants that accumulate less starch
Starch biosynthesis in both photosynthetic and heterotrophic tissues of plants
than WT plants (Chourey & Nelson 1976; Zrenner et
al. 1995; Tang & Sturm 1999; Ruan et al. 2003)
which, according to Baroja-Fernández et al. (2003),
is ascribable to the reduced capacity of these plants to
produce ADPG in the cytosol.
Occurrence of starch turnover
In addition to the ability of converting ADPG to
starch, plastids are equipped with enzymes capable
of degrading starch (Steup 1988). Furthermore,
pulse chase, as well as starch pre-loading experiments using isolated plastids (Heldt et al. 1977; Stitt
& ap Rees 1980; Stitt & Heldt 1981a,b; PozuetaRomero & Akazawa 1993) and both autotrophic and
heterotrophic organs (Jones et al. 1959; Chan & Bird
1960; Scott & Kruger 1995; Sweetlove et al. 1996,
Häusler et al. 1998, Nguyen-Quoc & Foyer 2001,
Schneider et al. 2002), have provided evidence that,
essentially in agreement with reports describing
gluconeogenic processes in bacteria (Gaudet et al.
1992; Belanger & Hatfull 1999; Guedon et al. 2000)
and animals (David et al. 1990; Massillon et al.
1995), plastids are capable of synthesizing and
mobilizing starch concurrently, and both plastid
PGM and AGPase have a role in scavenging Glc
molecules derived from starch breakdown.
Against the idea that synthesis and breakdown of
starch occur simultaneously in leaves, pulse-chase
experiments on illuminated Arabidopsis leaves have
recently shown that none of the 14C incorporated
into starch during a short pulse of 14CO2 was
released during a subsequent chase (Zeeman et al.
2002). Based on these results, and assuming that
starch biosynthesis occurs following a ‘primer nonreducing-end’ mechanism of polysaccharide chain
elongation, it was concluded that starch synthesis is
not accompanied by significant turnover (Zeeman et
al. 2002). This conclusion however appears to be
based on erroneous grounds, since it has been
recently shown that starch biosynthesis occurs following a ‘nonprimer reducing-end’ insertion mechanism (Mukerjea et al. 2002; Mukerjea & Robyt
2004). Because (i) b-amylase appears to control
transitory starch degradation (Scheidig et al. 2002;
Niittylä et al. 2004; Lloyd et al. 2005) and (ii) this
enzyme liberates maltose from the non-reducing end
of starch chains, it is not surprising that illuminated
leaves exposed to a short pulse of 14CO2 do not
release any label during the subsequent chase.
Taking into account all the limitations in investigating starch turnover and that quantification of
starch turnover relies on both the method employed
to estimate starch cycling and on the validity of the
assumptions made with respect to the order in which
glucosyl units deposited in glycogen are removed
69
(Landau 2001), the combined evidence strongly
indicates that starch synthesis and breakdown takes
place concurrently during active starch biosynthesis.
An alternative model of starch biosynthesis in
both photosynthetic and heterotrophic tissues
involving the cytosolic production of ADPG by
SuSy and occurrence of cyclic turnover of
starch
Taking into account the capacity of SuSy to produce
ADPG directly from Suc and ADP, and considering
the occurrence of a cyclic turnover of starch, an
alternative model of starch biosynthesis in both sink
and source organs has been proposed wherein SuSy
catalyzes the de novo synthesis of ADPG in the
cytosol (Pozueta-Romero et al. 1991a,b; BarojaFernández et al. 2003) (Figure 5). According to
this model, the net rate of starch biosynthesis might
be determined by the rate of import of ADPG
synthesized by SuSy in the cytosol and the efficiency
with which starch breakdown products can be
recycled back to starch via the coupled reactions
catalyzed by plastid PGM and AGPase (PozuetaRomero & Akazawa 1993; Pozueta-Romero et al.
1999; Baroja-Fernández et al. 2001, 2003, 2004,
2005). Therefore, according to this new view, both
Suc and starch biosynthetic processes are intimately
connected by means of ADPG producing SuSy
activity in both photosynthetic and heterotrophic
organs.
The ‘alternative model’ postulates that the starch
biosynthetic pathway in source leaves is not directly
connected to the Calvin cycle by means of plastid
PGI (Figure 5A). This does not conflict with the
occurrence of starch deficient pPGI mutants (Jones
et al. 1986; Kruckeberg et al. 1989; Yu et al. 2000),
since leaves from these plants also have reduced
photosynthetic capacity that, in addition to reducing
starch biosynthesis, causes a reduced growth phenotype. Furthermore, the ‘alternative model’ postulates that UGPase is not involved in the Suc-starch
conversion process in heterotrophic organs and,
essentially identical to the situation in bacteria,
yeast, animals and photosynthetic tissues of plants,
its function is to synthesize UDPG rather than to
degrade it (Figure 5B).
The ‘alternative’ view is highly compatible with
most, if not all, starch mutant and transgenic plants
described to date. For example, the ‘alternative’ view
predicts that in the ‘low starch’ phenotype resulting
from deficiencies in either plastid PGM or AGPase
(Caspar et al. 1985; Lin et al. 1988; Müller-Röber et
al. 1992) the recovery of the Glc units derived from
the starch breakdown for starch biosynthesis will also
be deficient, resulting in a parallel decline of starch
70
F. J. Muñoz et al.
CO2
A
Triose-P
Calvin-Benson
cycle
Triose-P
Pi
PGM
G6P
G1P
Pi
Sucrose
ADP
AGPa se
Fru
ADPG
ADP
Starch
Chloroplast
ADPG
ADP
Cytosol
B
Starch
ADP
ADPG
ADP
ADPG
Fru
ADP
SuSy
Sucrose
AGPase
AGPase
G1P
PGM
G1P
G6P
G6P
PGM
Amyloplast
Cytosol
Figure 5. Model of starch biosynthesis in (A) photosynthetic and (B) heterotrophic tissues involving the cytosolic production of ADPG by
SuSy. A side product of the reaction catalyzed by SuSy is fructose, which can be diverted towards glycolysis or recycled towards sucrose
biosynthesis (Keeling et al. 1988; Geigenberger & Stitt 1991).
accumulation (Baroja-Fernández et al. 2001, 2004,
2005).
Leaves with a ‘high starch/Suc’ balance arising
when (a) export of triose phosphates from the
chloroplast is constrained (Riesmeier et al. 1993;
Heineke et al. 1994), (b) either cytosolic PGI or
fructose bisphosphatase activities are low (Neuhaus
et al. 1989; Zrenner et al. 1996; Strand et al. 2000),
and (c) fructose-2,6-bisphosphate levels are high
(Scott & Kruger 1995), are characterized by a high
3PGA/Pi ratio as compared with WT leaves. High
3PGA/Pi ratio activates AGPase. Under those circumstances, the ‘alternative’ view predicts that the
recovery of the Glc units derived from the starch
breakdown will also be enhanced, resulting in an
equilibrium favouring a high starch/Suc balance.
The ‘alternative’ model of starch biosynthesis in
leaves predicts that both Suc and starch biosynthetic
pathways are tightly connected by means of the
ADPG producing SuSy. In this respect, the occurrence of UGPase-, cytosolic PGM- and SPS-deficient plants (Strand et al. 2000; Fernie et al. 2002a;
Kleczkowski et al. 2004) is consistent with the
‘alternative’ view (but not with the ‘classical’ one),
since these plants are characterized by containing low
levels of both Suc and starch in their leaves as
compared with WT leaves. It is worth noting that
leaves from the latter two types of plants have normal
3PGA/Pi ratios as compared with WT leaves. In
addition, the occurrence of sedoheptulose-1,7-bisphosphatase overexpressing plants (Miyagawa et
al. 2001; Lefebvre et al. 2005) is also compatible
with the ‘alternative’ model of starch biosynthesis
since their leaves are characterized by having high
levels of both Suc and starch.
SuSy, but not AGPase, controls the
intracellular levels of ADPG linked to
transitory starch biosynthesis in source leaves
Using transgenic plants expressing a bacterial
ADPG hydrolase (Moreno-Bruna et al. 2001) in
either the cytosol or chloroplast, we recently provided definitive evidence demonstrating that most of
ADPG linked to starch biosynthesis accumulates in
the cytosol (Baroja-Fernández et al. 2004). Further-
Starch biosynthesis in both photosynthetic and heterotrophic tissues of plants
New players in the starch field
9,0
8,0
Starch (µmol/ g FW)
7,0
6,0
5,0
4,0
3,0
2,0
1,0
0,0
WT
35S-SuSy-NOS
WT
35S-SuSy-NOS
1,4
1,2
ADPG (nmol/ g FW)
more, we also demonstrated that extraplastidial
ADPG is intimately linked to starch biosynthesis.
The implications of these findings evidently raised
the question as to which of the two enzymes known
to synthesize ADPG, i.e. AGPase and SuSy, is the
predominant source of cytosolic ADPG accumulating in leaves.
To address this question, we measured the ADPG
content in the TL25 starch deficient Arabidopsis
thaliana plants lacking AGPase (Lin et al. 1988).
Furthermore, we produced and characterized SuSyoverexpressing leaves. As illustrated in Figure 6,
whereas measurements of starch content confirmed
previous studies showing that TL25 contain marginally lower levels of starch (cf. Figure 4), this
reduction in starch content was not accompanied
by any measurable reduction in intracellular ADPG
levels (Muñoz et al. 2005). In clear contrast, SuSy
overexpressing leaves were shown to accumulate
higher levels of both ADPG and transitory starch
than WT leaves (Figure 7). The combined data thus
strongly indicate that SuSy, but not AGPase, is the
predominant source of ADPG accumulating in
leaves.
71
1,0
0,8
0,6
0,4
0,2
The discrepancy between the ‘classic’ and ‘alternative’ mechanistic models of starch biosynthesis
indicates that our knowledge of the gluconeogenic
process is still at a rudimentary level. Better understanding of the regulation of starch synthesis will
require more research into new and fundamental
aspects of the gluconeogenic process, either at the
biochemical, cell biological and molecular levels. In
0,0
Figure 7. ADPG and starch levels in fully expanded source leaves
from WT and SuSy-overexpressing plants. Analyses were conducted as in Figure 6.
this respect, the following items will require special
attention:
ADP G (n m o l / g F W )
0,30
ADPG hydrolytic enzymes
0,25
0,20
0,15
0,10
0,05
0,00
TL25
WT
Figure 6. Source leaves of Arabidopsis of the starch deficient
AGPase TL25 mutant contain normal ADPG levels as compared
with WT leaves. ADPG analyses were conducted using fully
expanded leaves after 9 h illumination (300 mmol photons s 1
m 2). Both ADPG and starch content were measured as
described by Baroja-Fernández et al. (2004).
Rodrı́guez-López et al. (2000) demonstrated the
occurrence in plants of a widely distributed enzymatic activity, designated as ADPG pyrophosphatase, which catalyzes the hydrolytic breakdown of
ADPG to G1P and AMP. The finding of ADPG
pyrophosphatase in plants prompted us to examine
the possible occurrence of ADPG cleaving enzymes
in bacteria. These investigations revealed that a
member of the ‘Nudix’ family (Bessman et al.
1996) is present in Escherichia coli that regulates
the intracellular levels of ADPG linked to glycogen
biosynthesis.
This led us to propose that some ADPG cleaving
enzymes are involved in the control of intracellular
levels of ADPG linked to starch biosynthesis, and in
connecting gluconeogenesis with other metabolic
pathways (Baroja-Fernández et al. 2000, 2001).
72
F. J. Muñoz et al.
However, continued molecular genetic analysis will
be needed to determine whether or not plant ADPG
hydrolases play a crucial role in preventing metabolic
flux towards starch. With the completion of the
Arabidopsis and Rice Genome projects, numerous
candidates for ADPG cleaving enzymes can be
identified, and can now be used to establish their
biochemical and biological functions via over-expression or antisense approaches, isolation of true
mutants from tagged populations, or the expression
of coding regions in heterologous systems.
Endocytic uptake of Suc into the vacuole
Conversion of Suc to starch in heterotrophic plant
cells commences with Suc breakdown in the apoplast and/or cytosol, followed by a series of enzymatic pathways that have been described above.
Based on a comparison of transgenic potato plants
exhibiting heterologous expression of invertase in the
apoplast or cytosol, Willmitzer et al. have recently
suggested that the point of carbon entry into the
cell’s metabolism differs according to whether Suc
breakdown takes place in the apoplast or in the
cytosol (Sonnewald et al. 1997; Fernie et al. 2000;
Hajirezaei et al. 2000). Their observations also
implied an endocytic mechanism of transport into
the vacuole and subsequent release to the cytosol,
which gained considerable relevance after recent
demonstrations of the occurrence of endocytic
sucrose uptake into the vacuole of heterotrophic
cells (Etxeberria et al. 2005a,b) and previous demonstrations of a tonoplast-bound SuSy involved in
Suc mobilization from the vacuole (Etxeberria &
Gonzalez 2003; Pozueta-Romero et al. 2004).
To further our understanding of Suc-starch conversion in heterotrophic cells, and to know whether
endocytic uptake of Suc into the vacuole is required
prior to its conversion into starch, we should
investigate the starch production capacities of heterotrophic tissues exposed to various endocytic
inhibitors. Furthermore, we should characterize
mutants with altered endocytic capacities.
Acknowledgements
This research was partially supported by the grants
BIO2001-1080 and BIO2004-01922 from the Comisión Interministerial de Ciencia y Tecnologı́a and
Fondo Europeo de Desarrollo Regional (Spain).
M.T.M-Z. acknowledges the Spanish Ministry of
Culture and Education for a pre-doctoral fellowship.
We are indebted to Axel Tiessen (Max Planck
Institut, Golm, Germany), Eckehard Neuhaus (University of Kaiserslautern, Germany) and Kay Denyer
(John Innes Centre, Norwich, United Kingdom) for
critical discussions.
References
Andon NL, Hollingworth S, Koller A, Greenland AJ, Yates JR,
Haynes PA. 2002. Proteomic characterization of wheat amyloplasts using identification of proteins by tandem mass spectrometry. Proteomics 2:1156 /1168.
Asano T, Kunieda N, Omura Y, Ibe H, Kawasaki T, Takano M,
Sato M, Furuhashi H, Mujin T, Takaiwa F, Wu C-Y, Tada Y,
Satozawa T, Sakamoto M, Shimada H. 2002. Rice SPK, a
calmodulin-like domain protein kinase, is required for storage
product accumulation during seed development: phosphorylation of sucrose synthase is a possible factor. Plant Cell 14:619 /
628.
ap Rees T. (1995). In: Sucrose metabolism, biochemistry,
physiology and molecular biology. Pontis HG, Salerno GL,
Echeverria EJ, Editors. Rockville MD: American Society of
Plant Physiologists, 14, 143 /155
Arnon DI. 1955. The chloroplast as a complete photosynthetic
unit. Science 122:9 /16.
Backhausen JE, Jöstingmeyer P, Scheibe R. 1997. Competitive
inhibition of spinach leaf phosphoglucose isomerase isoenzymes by erythrose 4-phosphate. Plant Sci 130:121 /131.
Baroja-Fernández E, Muñoz FJ, Akazawa T, Pozueta-Romero J.
2001. Reappraisal of the currently prevailing model of starch
biosynthesis in photosynthetic tissues: A proposal involving the
cytosolic production of ADP-glucose by sucrose synthase and
occurrence of cyclic turnover of starch in chloroplast. Plant
Cell Physiol 42:1311 /1320.
Baroja-Fernández E, Muñoz FJ, Pozueta-Romero J. 2005. A
response to Neuhaus et al . Trends Plant Sci 10:156 /159.
Baroja-Fernández E, Muñoz FJ, Saikusa T, Rodrı́guez-López M,
Akazawa T, Pozueta-Romero J. 2003. Sucrose synthase catalyzes the de novo production of ADPglucose linked to starch
biosynthesis in heterotrophic tissues of plants. Plant Cell
Physiol 44:500 /509.
Baroja-Fernández E, Muñoz FJ, Zandueta-Criado A, MoránZorzano MT, Viale AM, Alonso-Casajús N, Pozueta-Romero J.
2004. Most of ADPglucose linked to starch biosynthesis occurs
outside the chloroplast in source leaves. Proc Natl Acad Sci
USA. 101:13080 /13085.
Baroja-Fernández E, Zandueta-Criado A, Rodrı́guez-López M,
Akazawa T, Pozueta-Romero J. 2000. Distinct isoforms of
ADPglucose pyrophosphatase and ADPglucose pyrophosphorylase occur in the suspension-cultured cells of sycamore (Acer
pseudoplatanus L.). FEBS Lett 480:277 /282.
Bassham JA, Krause GH. 1969. Free energy changes and
metabolic regulation in steady-state photosynthetic carbon
reduction. Biochim Biophys Acta 189:207 /221.
Beckles DM, Smith AM, ap Rees T. 2001. A cytosolic ADPglucose pyrophosphorylase is a feature of graminaceous endosperms. Plant Physiol 125:818 /827.
Belanger AE, Hatfull G. 1999. Exponential-phase glycogen
recycling is essential for growth of Mycobacterium smegmatis . J
Bacteriol 181:6670 /6678.
Bessman MJ, Frick DN, O?Handley SF. 1996. The MutT
proteins or ‘Nudix’ hydrolases, a family of versatile, widely
distributed, housecleaning enzymes. J Biol Chem 271:25059 /
25062.
Brangeon J, Reyss A, Prioul J-L. 1997. In situ detection of
ADPglucose pyrophosphorylase expression during maize endosperm development. Plant Physiol Biochem 35:847 /858.
Burton RA, Johnosn PE, Beckles DM, Fincher GB, Jenner HL,
Naldrett MJ, Denyer K. 2002. Characterization of the genes
encoding the cytosolic and plastidial isoforms of ADP-glucose
Starch biosynthesis in both photosynthetic and heterotrophic tissues of plants
pyrophosphorylase in wehat endosperm. Plant Physiol
130:1464 /1475.
Cao H, Sullivan TD, Boyer CD, Shannon JC. 1995. Bt1, a
structural gene for the major 30 /44 kDa amyloplast membrane
polypeptides. Physiol Plant 95:176 /186.
Caspar T, Huber SC, Somerville C. 1985. Alterations in growth,
photosynthesis, and respiration in a starchless mutant of
Arabidopsis thaliana (L.) deficient in chloroplast phosphoglucomutase activity. Plant Physiol 79:11 /17.
Chan T-T, Bird IF. 1960. Starch dissolution in tobacco leaves in
the light. J Exp Bot 11:335 /340.
Chourey PS, Nelson OE. 1976. The enzymatic deficiency
conditioned by the shrunken-1 mutations in maize. Biochem
Genet 14:1041 /1055.
Crevillén P, Ballicora MA, Mérida A, Preiss J, Romero JM. 2003.
The different large subunit isoforms of Arabidopsis thaliana
ADP-glucose pyrophosphorylase confer distinct kinetic and
regulatory properties to the heterotetrameric enzyme. J Biol
Chem 278:28508 /28515.
David M, Petit WA, Laughlin MR, Shulman RG, King JE, Barrett
EJ. 1990. Simultaneous synthesis and degradation of rat liver
glycogen. J Clin Invest 86:612 /617.
Davies Hv, Shepherd LVT, Burrell MM, Carrari F, UrbanczykWochniak E, Leisse A, Hancock RD, Taylor M, Viola R, Ros
H, McRae D, Willmitzer L, Fernie AR. 2005. Modulation of
fructokinase activity of potato (Solanum tuberosum ) results in
substantial shifts in tuber metabolism. Plant Cell Physiol
46:1103 /1115.
Delmer DP. 1972. The purification and properties of sucrose
synthetase from etiolated Phaseolus aureus seedlings. J Biol
Chem 247:3822 /3828.
Denyer K, Dunlap F, Thornbjørnsen T, Keeling P, Smith AM.
1996. The major form of ADP-glucose pyrophosphorylase in
maize (Zea mays L.) endosperm is extra-plastidial. Plant
Physiol 112:779 /785.
Dickinson DB, Preiss J. 1969. Presence of ADP-glucose pyrophosphorylase in shrunken-2 and brittle-2 mutants of maize
endosperm. Plant Physiol 44:1058 /1062.
Dietz K-J. 1985. A possible rate-limiting function of chloroplast
hexosemonophosphate isomerase in starch synthesis of leaves.
Biochim Biophys Acta 839:240 /248.
Entwistle G, ap Rees T. 1988. Enzymic capacities of amyloplasts
from wheat endosperm. Biochem J 255:391 /396.
Etxeberria E, Baroja-Fernandez E, Muñoz FJ, Pozueta-Romero J.
2005a. Sucrose inducible endocytosis as a mechanism for
nutrient uptake in heterotrophic plant cells. Plant Cell Physiol
46:474 /481.
Etxeberria E, Gonzalez P. 2003. Evidence for a tonoplastassociated form of sucrose synthase and its potential involvement in sucrose mobilization from the vacuole. J Exp Bot
54:1407 /1414.
Etxeberria E, Gonzalez P, Pozueta-Romero J. 2005b. Sucrose
transport in Citrus juice cells: Evidence for an endocytic
transport system. J Am Soc Hort Sci 130:269 /274.
Fernie AR, Riesmeier JW, Martini JW, Ramalingan S, Willmitzer
L, Trethewey RN. 2000. Consequences of the expression of a
bacterial glukokinase in potato tubers, both in combination
with and independently of a yeast derived invertases. Australian
J Plant Physiol 27:827 /833.
Fernie AR, Tauberger E, Lytovchenko A, Roessner U, Willmitzer
L, Trethewey RN. 2002a. Antisense repression of cytosolic
phosphoglucomutase in potato (Solanum tuberosum ) results in
severe growth retardation, reduction in tuber number and
altered carbon metabolism. Planta 214:510 /520.
Fernie AR, Swiedrych A, Tauberger E, Lytovchenko A, Trethewey RN, Willmitzer L. 2002b. Potato plants exhibiting
combined antisense repression of cytosolic and plastidial
73
isoforms of phosphoglucomutase surprisingly approximate
wild type with respect to the rate of starch synthesis. Plant
Physiol Biochem 40:921 /927.
Fernie AR, Willmitzer L, Trethewey RN. 2002c. Sucrose to
starch: a transition in molecular plant physiology. Trends Plant
Sci 7:35 /41.
Frehner M, Pozueta-Romero J, Akazawa T. 1990. Enzyme sets of
glycolysis, gluconeogenesis, and oxidative pentose phosphate
pathway are not complete in nongreen highly purified amyloplasts of sycamore (Acer pseudoplatanus L.) cells suspension
cultures. Plant Physiol 94:538 /544.
Fritzius T, Aeschbacher R, Wiemken A, Wingler A. 2001.
Induction of ApL3 expression by trehalose complements the
starch-deficient Arabidopsis mutant adg2-1 lacking ApL1, the
large subunit of ADP-glucose pyrophosphorylase. Plant Physiol
126:883 /889.
Fu H, Kim SY, Park WD. 1995. A potato Sus3 sucrose synthase
gene contains a context-dependent 3? element and a leader
intron with both positive and negative tissue-specific effects.
Plant Cell 7:1395 /1403.
Gaudet G, Forano E, Dauphin G, Delort A-M. 1992. Futile
cycling of glycogen in Fibrobacter succinogenes as shown by in
situ 1H-NMR and 13C-NMR investigation. Eur J Biochem
207:155 /162.
Geigenberger P. 2003. Regulation of sucrose to starch conversion
in growing potato tubers. J Exp Bot 54:457 /465.
Geigenberger P, Stamme C, Tjaden J, Schulz A, Quick PW,
Betsche T, Kersting HJ, Neuhaus E. 2001. Tuber physiology
and properties of starch from tubers of transgenic potato plants
with altered plastidic adenylate transporter activity. Plant
Physiol 125:1667 /1678.
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.
Gross P, ap Rees T. 1986. Alkaline inorganic pyrophosphatase and
starch synthesis in amyloplasts. Planta 167:140 /145.
Guedon E, Desvaux M, Petitdemange H. 2000. Kinetic analysis
of Clostridium cellulolyticum carbohydrate metabolism: importance of glucose 1-phosphate and glucose 6-phosphate branch
points for distribution of carbon fluxes inside and outside cells
as revealed by steady-state continuous culture. J Bacteriol
183:2010 /2017.
Haake V, Zrenner R, Sonnewald U, Stitt M. 1998. A moderate
decrease of plastid aldolase activity inhibits photosynthesis,
alters the levels of sugars and starch, and inhibits growth of
potato plants. Plant J 14:147 /158.
Hajirezaei MR, Takahata Y, Trethewey RN, Willmitzer L,
Sonnewald U. 2000. Impact of elevated cytosolic and apoplastic invertases activity on carbon metabolism during potato
tuber development. J Exp Bot 51:439 /445.
Harrison CJ, Hedley CL, Wang TL. 1998. Evidence that the rug3
locus of pea (Pisum sativum L.) encodes plastidial phosphoglucomutase confirms that the imported substrate for starch
synthesis in pea amyloplasts is glucose-6-phosphate. Plant J
13:753 /762.
Hatzfeld W-D, Stitt M. 1990. A study of the rate of recycling of
triose phosphates in heterotrophic Chenopodium rubrum cells,
potato tubers, and maize endosperm. Planta 180:198 /204.
Häusler RE, Schlieben NH, Schulz B, Flügge U-I. 1998.
Compensation of decreased triose phosphate/phosphate translocator activity by accelerated starch turnover and glucose
transport in transgenic tobacco. Planta 204:366 /376.
Heineke D, Kruse A, Flügge UI, Frommer WB, Riesmeier JW,
Willmitzer L, Heldt HW. 1994. Effect of antisense repression of
74
F. J. Muñoz et al.
the chloroplast triose-phosphate translocator on photosynthetic
metabolism in transgenic potato plants. Planta 193:174 /180.
Heldt HW, Chon CJ, Maronde D, Herold A, Stankovic ZS,
Walker DA, Kraminer A, Kirk MR, Heber U. 1977. Role of
orthophosphate and other factors in the regulation of starch
formation in leaves and isolated chloroplasts. Plant Physiol
59:1146 /1155.
Hendriks JHM, Kolbe A, Gibon Y, Stitt M, Geigenberger P. 2003.
ADP-glucose pyrophosphorylase is activated by posttranslational redox-modification in response to light and to sugars in
leaves of Arabidopsis and other plant species. Plant Physiol
133:838 /849.
Johnson PE, Patron NJ, Bottrill AR, Dinges JR, Fahy BF, Parker
ML, Waite DN, Denyer K. 2003. A low-starch barley mutant,
Riso 16, lacking the cytosolic small subunit of ADP-glucose
pyrophosphorylase, reveals the importance of the cytosolic
isoform and the identity of the plastidial small subunit. Plant
Physiol 131:684 /696.
Jones TWA, Gottlieb LD, Pichersky E. 1986. Reduced enzyme
activity and starch level in an induced mutant of chloroplast
phosphoglucose isomerase. Plant Physiol 81:367 /371.
Jones H, Martin RV, Porter HK. 1959. Translocation of 14Carbon
in tobacco following assimilation of 14Carbon dioxide by a
single leaf. Ann Bot 23:493 /508.
Kammerer B, Fischer K, Hilpert B, Schubert S, Gutensohn M,
Weber A, Flügge U-I. 1998. Molecular characterization of a
carbon transporter in plastids from heterotrophic tissues: the
glucose 6-phosphate/phosphate antiporter. Plant Cell 10:105 /
117.
Keeling PL, Wood JR, Tyson RH, Bridges IG. 1988. Starch
biosynthesis in developing wheat grain: evidence against the
direct involvement of triose phosphates in the metabolic
pathway. Plant Physiol 87:311 /319.
Kelly GJ, Latzko E. 1980. Oat leaf phosphoglucose isomerase:
competitive inhibition by erythrose-4-phosphate. Photosynth
Res 1:181 /187.
Kleczkowski LA. 1996. Back to the drawing board: redefining
starch synthesis in cereals. Trends Plant Sci 1:363 /364.
Kleczkowski LA. 1999. A phosphoglycerate to inorganic phosphate ratio is the major factor in controlling starch levels in
chloroplasts via ADP-glucose pyrophosphorylase regulation.
FEBS Lett 448:153 /156.
Kleczkowski LA. 2000. Is leaf ADP-glucose pyrophosphorylase an
allosteric enzyme. Biochim Biophys Acta 1476:103 /108.
Kleczkowski LA, Geisler M, Ciereszko I, Johansson H. 2004.
UDP-glucose pyrophosphorylase. An old protein with new
tricks. Plant Physiol 134:912 /918.
Koch KE, Nolte KD, Duke ER, McCarty DR, Avigne WT. 1992.
Sugar levels modulate differential expression of maize sucrose
synthase genes. Plant Cell 4:59 /69.
Kossmann J, Sonnewald U, Willmitzer L. 1994. Reduction of the
chloroplastic fructose-1,6-bisphosphatase in transgenic potato
plants impairs photosynthesis and plant growth. Plant J 6:637 /
650.
Kruckeberg L, Neuhaus HE, Feil R, Gottlieb LD, Stitt M. 1989.
Decreased-activity mutants of phosphoglucose isomerase in the
cytosol and chloroplast of Clarkia xantiana . Biochem J
261:457 /467.
Landau BR. 2001. Methods for measuring glycogen cycling. Am J
Physiol Endocrinol Metab 281:E413 /E419.
Lefebvre S, Lawson T, Fryer M, Zakhleniuk OV, Lloyd JC, Raines
CA. 2005. Increased sedoheptulose-1,7-bisphosphatase activity
in transgenic tobacco plants stimulates photosynthesis and
growth from an early stage in development. Plant Physiol
138:451 /460.
Leroch M, Kirchberger S, Haferkamp I, Wahl M, Neuhaus HE,
Tjaden J. 2005. Identification and characterization of a novel
plastidic adenine nucleotide uniporter from Solanum tuberosum .
J Biol Chem 280:17992 /18000.
Lin T-P, Caspar T, Somerville CR, Preiss J. 1988. Isolation and
characterization of a starchless mutant of Arabidopsis thaliana
(L.) Heynh lacking ADPglucose pyrophosphorylase activity.
Plant Physiol 86:1131 /1135.
Lloyd JR, Kossmann J, Ritte G. 2005. Leaf starch degradation
comes out of the shadows. Trends Plant Sci 10:130 /137.
Massillon D, Bollen M, De Wulf H, Overloop K, Vanstapel F, Van
Hecke P, Stalmans W. 1995. Demonstration of a glycogen/
glucose 1-phosphate cycle in hepatocytes from fasted rats. J
Biol Chem 270:19351 /19356.
McLachlan GA, Porter DA. 1959. Replacement of oxidation by
light as the energy source for glucose metabolism in tobacco
leaf. Proc R Soc Lond 150:460 /473.
Miller ME, Chourey PS. 1995. Intracellular immunolocalization
of adenosine 5?-diphosphoglucose pyrophosphorylase in developing endosperm cells of maize (Zea mays L.). Planta
197:522 /527.
Miyagawa Y, Tamoi M, Shigeoka S. 2001. Overexpression of a
cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase
in tobacco enhance photosynthesis and growth. Nat Biotechnol
19:965 /969.
Möhlmann T, Tjaden J, Henrichs G, Quick WP, Häusler R,
Neuhaus HE. 1997. ADPglucose drives starch synthesis in
isolated maize endosperm amyloplasts. Characterization of
starch synthesis and transport properties across the amyloplast
envelope. Biochem J 324:503 /509.
Moreno-Bruna B, Baroja-Fernández E, Muñoz FJ, BastarricaBerasategui A, Zandueta-Criado A, Rodrı́guez-López M, Lasa
I, Akazawa T, Pozueta-Romero J. 2001. Adenosine diphosphate sugar pyrophosphatase prevents glycogen biosynthesis in
Escherichia coli . Proc Natl Acad Sci USA 98:8128 /8132.
Mukerjea R, Robyt JF. 2004. Starch biosynthesis: the primer
nonreducing-end mechanism versus the nonprimer reducingend two-site insertion mechanism. Carbohydr Res 340:245 /
255.
Mukerjea R, Yu L, Robyt JF. 2002. Starch biosynthesis: mechanism for the elongation of starch chains. Carbohydr Res
337:1015 /1022.
Müller-Röber BT, Sonnewald U, Willmitzer L. 1992. Inhibition
of the ADP-glucose pyrophosphorylase in transgenic potatoes
leads to sugar-storing tubers and influences tuber formation
and expression of tuber storage protein genes. EMBO J
11:1229 /1238.
Muñoz FJ, Baroja-Fernández E, Morán-Zorzano MT, Viale AM,
Etxeberria E, Alonso-Casajús N, Pozueta-Romero J. (2005).
Sucrose synthase controls both intracellular ADPglucose levels
and transitory starch biosynthesis in source leaves. Plant Cell
Physiol (in press).
Murata T, Minamikawa T, Akazawa T. 1963. Adenosine diphosphate glucose in rice and its role in starch synthesis. Biochem
Biophys Res Commun 13:439 /443.
Naeem M, Tetlow IJ, Emes MJ. 1997. Starch synthesis in
amyloplasts purified from developing tubers. Plant J
11:1095 /1103.
Nakai T, Konishi T, Zhang X-Q, Chollet R, Tonouchi N,
Tsuchida T, Yoshinaga F, Mori H, Sakai F, Hayashi T. 1998.
An increase in apparent affinity for sucrose of mung bean
sucrose synthase is caused by in vitro phosphorylation or
directed mutagenesis of Ser11. Plant Cell Physiol 39:1337 /
1341.
Neuhaus HE, Kruckeberg AL, Feil R, Stitt M. 1989. Reducedactivity mutants of phosphoglucose isomerase in the cytosol
and chloroplast of Clarkia xantiana . Planta 178:110 /122.
Neuhaus HE, Thom E, Möhlmann T, Steup M, Kampfenkel K.
1997. Characterization of a novel eukaryotic ATP/ADP trans-
Starch biosynthesis in both photosynthetic and heterotrophic tissues of plants
locator located in the plastid envelope of Arabidopsis thaliana L.
Plant J 11:73 /82.
Nguyen-Quoc B, Foyer CH. 2001. A role for ‘futile cycles’
involving invertase and sucrose synthase in sucrose metabolism
of tomato fruit. J Exp Bot 52:881 /889.
Niittylä T, Messerli G, Trevisan M, Chen J, Smith AM, Zeeman
SC. 2004. A novel maltose transporter essential for starch
degradation in leaves. Science 303:881 /889.
Okita TW. 1992. Is there an alternative pathway for starch
synthesis? Plant Physiol 100:560 /564.
Patron NJ, Greber B, Fahy BF, Laurie DA, Parker ML, Denyer K.
2004. The lys5 mutations of barley reveal the nature and
importance of plastidial ADP-glc transporters for starch
synthesis in cereal endosperm. Plant Physiol 135:2088 /2097.
Porchia AC, Curatti L, Salerno GL. 1999. Sucrose metabolism in
cyanobacteria: sucrose synthase from Anabaena sp. strain PCC
7119 is remarkably different from the plant enzymes with
respect to substrate affinity and amino-terminal sequence.
Planta 210:34 /40.
Pozueta-Romero J, Akazawa T. 1993. Biochemical mechanism of
starch biosynthesis in amyloplasts from cultured cells of
sycamore (Acer pseudoplatanus ). J Exp Bot 44S:297 /306.
Pozueta-Romero J, Ardila F, Akazawa T. 1991a. ADP-glucose
transport by the chloroplast adenylate translocator is linked to
starch biosynthesis. Plant Physiol 97:1565 /1572.
Pozueta-Romero J, Frehner M, Viale A, Akazawa T. 1991b.
Direct transport of ADPglucose by an adenylate translocator is
linked to starch biosynthesis in amyloplasts. Proc Natl Acad Sci
USA 88:5769 /5773.
Pozueta-Romero J, Perata P, Akazawa T. 1999. Sucrose-starch
conversion in heterotrophic tissues of plants. Crit Rev Plant Sci
18:489 /525.
Pozueta-Romero J, Pozueta-Romero D, González P, Etxeberria E.
2004. Activity of membrane-associated sucrose synthase is
regulated by its phosphorylastion status in cultured cells of
sycamore (Acer pseudoplatanus ). Physiol Plantarum 122:275 /
280.
Preiss J. (1988). In: The biochemistry of plants. Preiss, J. editor.
Academic Press, NY. Vol. 14, pp. 181 /254.
Recondo E, Dankert M, Leloir LF. 1963. Adenosine diphosphate
glucose and starch synthesis. Biochem Biophys Res Commun
12:204 /207.
Riesmeier JW, Flügge UI, Schulz B, Heineke D, Heldt HW,
Willmitzer L, Frommer WB. 1993. Antisense repression of the
chloroplast triose phosphate translocator affects carbon partitioning in transgenic potato plants. Proc Natl Acad Sci USA
90:6160 /6164.
Rodrı́guez-López M, Baroja-Fernández E, Zandueta-Criado A,
Pozueta-Romero J. 2000. Adenosine diphosphate glucose
pyrophosphatase: a plastidial phosphodiesterase that prevents
starch biosynthesis. Proc Natl Acad Sci USA 97:8705 /8710.
Roscher A, Emsley L, Raymond P, Roby C. 1998. Unidirectional
steady state rates of central metabolism enzymes measured
simultaneously in a living plant tissue. J Biol Chem
273:25053 /25061.
Ruan Y-L, Llewellyn DJ, Furbank RT. 2003. A fiberless seed
mutation in cotton is associated with lack of fibre cell initiation
in ovule epidermis and alterations in sucrose synthase expression and carbon partitioning in developing seeds. Plant Cell
15:952 /964.
Saether N, Iversen T-H. 1991. Gravitropism and starch statoliths
in an Arabidopsis mutant. Planta 184:491 /497.
Sakulsingharoj C, Choi S-B, Hwang S-K, Edwards GE, Bork J,
Meyer CR, Preiss J, Okita TW. 2004. Engineering starch
biosynthesis for increasing rice seed weight: the role of the
cytoplasmic ADP-glucose pyrophosphorylase. Plant Sci
167:1323 /1333.
75
Scheidig A, Fröhlich A, Schulze S, Lloyd JR, Kossmann J. 2002.
Downregulation of a chloroplast-targeted b-amylase leads to a
starch-excess phenotype in leaves. Plant J 30:581 /591.
Schneider A, Häusler RE, Kolukisaoglu Ü, Kunze R, van der
Graaff E, Schwacke R, Catoni E, Desimone M, Flügge U-I.
2002. An Arabidopsis thaliana knock-out mutant of the
chloroplast triose phosphate/phosphate translocator is severely
compromised only when starch synthesis, but not starch
mobilisation is abolished. Plant J 32:685 /699.
Scott P, Kruger NJ. 1995. Influence of elevated fructose-2,6bisphosphate levels on starch mobilization in transgenic tobacco leaves in the dark. Plant Physiol 108:1569 /1577.
Shannon JC, Pien F-M, Liu K-C. 1996. Nucleotides and
nucleotide sugars in developing maize endosperms. Synthesis
of ADP-glucose in brittle 1 . Plant Physiol 110:835 /843.
Shannon JC, Pien F-M, Cao H, Liu K-C. 1998. Brittle-1, an
adenylate translocator, facilitates transfer of extraplastidial
synthesized ADP-glucose into amyloplasts of maize endosperms. Plant Physiol 117:1235 /1252.
Silvius JE, Snyder FW. 1979. Comparative enzymic studies of
sucrose metabolism in the taproots and fibrous roots of Beta
vulgaris L. Plant Physiol 64:1070 /1073.
Smidansky ED, Clancy M, Meyer FD, Lanning SP, Blake NK,
Talbert LE, Giroux MJ. 2002. Enhanced ADP-glucose pyrophosphorylase activity in wheat endosperm increases seed
yield. Proc Natl Acad Sci USA 99:1724 /1729.
Smith AM, Neuhaus HE, Stitt M. 1990. The impact of decreased
activity of starch-branching enzyme on photosynthetic starch
synthesis in leaves of wrinkled-seeded peas. Planta 181:310 /
315.
Smith AM, Zeeman SC, Smith SM. 2005. Starch degradation.
Ann Rev Plant Biol 56:73 /98.
Sonnewald U, Hajirezaei MR, Kossmann J, Heyer A, Trethewey
RN, Willmitzer L. 1997. Expression of a yeast invertase in the
apoplast of potato tubers increased tuber size. Nat Biotechnol
15:794 /797.
Stark DM, Timmerman KP, Barry GF, Preiss J, Kishore GM.
1992. Regulation of the amount of starch in plant tissues by
ADPglucose pyrophosphorylase. Science 258:287 /292.
Steup M. (1988). In: The biochemistry of plants. Stumpf PK,
Conn EE editors. Academic Press, NY. Vol. 14, pp. 255 /295.
Stitt M, ap Rees T. 1980. Carbohydrate breakdown by chloroplasts of Pisum sativum . Biochim Biophys Acta 627:131 /143.
Stitt M, Heldt HW. 1981a. Simultaneous synthesis and degradation of starch in spinach chloroplasts in the light. Biochim
Biophys Acta 638:1 /11.
Stitt M, Heldt HW. 1981b. Physiological rates of starch breakdown in isolated intact spinach chloroplasts. Plant Physiol
68:755 /761.
Strand A, Zrenner R, Trevanion S, Stitt M, Gustafsson P,
Gardeström P. 2000. Decreased expression of two key enzymes
in the sucrose biosynthesis pathway, cytosolic fructose-1,6bisphosphatase and sucrose phosphate synthase, has remarkably different consequences for photosynthetic carbon metabolism in transgenic Arabidopsis thaliana . Plant J 23:759 /770.
Sullivan TD, Kaneko Y. 1995. The maize brittle 1 gene encodes
amyloplast membrane polypeptides. Planta 196:477 /484.
Sweetlove LJ, Burrell MM, ap Rees T. 1996. Starch metabolism in
tubers of transgenic potato (Solanum tuberosum ) with increased
ADPglucose pyrophosphorylase. Biochem J 320:493 /498.
Tanase K, Yamaki S. 2000. Purification and characterization of
two sucrose synthase isoforms from japanese pear fruit. Plant
Cell Physiol 41:408 /414.
Tang G-Q, Sturm A. 1999. Antisense repression of sucrose
synthase in carrot (Daucus carota L.) affects growth rather
than sucrose partitioning. Plant Mol Biol 41:465 /479.
76
F. J. Muñoz et al.
Tauberger E, Fernie AR, Emmermann M, Renz A, Kossmann J,
Willmitzer L, Trethewey RN. 2000. Antisense inhibition of
plastidial phosphoglucomutase provides compelling evidence
that potato tuber amyloplasts import carbon from the cytosol in
the form of glucose-6-phosphate. Plant J 23:43 /53.
Tetlow IJ, Davies EJ, Vardy KA, Bowsher CG, Burrell MM, Emes
MJ. 2003. Starch synthesis and carbohydrate oxidation in
amyloplasts from developing wheat endosperm. J Exp Bot
54:715 /725.
Tetlow IJ, Morell MK, Emes MJ. 2004. Recent developments in
understanding the regulation of starch metabolism in higher
plants. J Exp Bot 55:2131 /2145.
Thornbjørnsen T, Villand P, Denyer K, Olsen O-A, Smith AM.
1996a. Distinct isoforms of ADPglucose pyrophosphorylase
occur inside and outside the amyloplast in barley endosperm.
Plant J 10:243 /250.
Thornbjørnsen. T, Villand P, Kleczkowski LA, Olsen OA. 1996b.
A single gene encodes two different transcripts of ADP-glucose
pyrophosphorylase small subunit from barley (Hordeum vulgare ). Biochem J 313:149 /154.
Trethewey RN, Fernie AR, Bachmann A, Fleischer-Notter H,
Geigenberger P, Willmitzer L. 2001. Expression of a bacterial
sucrose phosphorylase in potato tubers results in a glucoseindependent induction of glycolysis. Plant Cell Environ
24:357 /365.
Trethewey RN, Geigenberger P, Riedel K, Hajirezaei M-R,
Sonnewald U, Stitt M, Riesmeier JW, Willmitzer L. 1998.
Combined expression of glucokinase and invertase in potato
tubers leads to a dramatic reduction in starch accumulation and
a stimulation of glycolysis. Plant J 15:109 /118.
Tsai CY, Nelson OE. 1966. Starch-deficient maize mutant lacking
adenosine diphosphate glucose pyrophosphorylase activity.
Science 151:341 /343.
Villand P, Kleczkowski LA. 1994. Is there an alternative pathway
for starch biosynthesis in cereal seeds? Z Naturforsch 49:215 /
219.
Wang A-Y, Kao M-H, Yang W-H, Sayion Y, Liu L-F, Lee P-D, Su
J-C. 1999. Differentially and developmentally regulated expression of three rice sucrose synthase genes. Plant Cell Physiol
40:800 /807.
Weber H, Rolletschek H, Heim U, Golombek S, Gubatz S, Wobus
U. 2000. Antisense-inhibition of ADP-glucose pyrophosphorylase in developing seeds of Vicia narbonensis moderately
decreases starch but increases protein content and affects
seed maturation. Plant J 24:33 /43.
Yu T-S, Lue W-L, Wang S-M, Chen J. 2000. Mutation of
Arabidopsis plastid phosphoglucose isomerase affects leaf starch
synthesis and floral initiation. Plant Physiol 123:319 /325.
Zeeman SC, Tiessen A, Pilling E, Kato KL, Donald AM, Smith
AM. 2002. Starch synthesis in Arabidopsis. Granule synthesis,
composition, and structure. Plant Physiol 129:516 /529.
Zervosen A, Römer U, Elling L. 1998. Application of recombinant sucrose synthase-large scale synthesis of ADP-glucose. J
Mol Catal 5:25 /28.
Zrenner R, Krause KP, Apel P, Sonnewald U. 1996. Reduction of
the cytosolic fructose-1,6-bisphosphatase in transgenic potato
plants limits photosynthetic sucrose biosynthesis with no
impact on plant growth and tuber yield. Plant J 9:671 /681.
Zrenner R, Salanoubat M, Willmitzer L, Sonnewald U. 1995.
Evidence of the crucial role of sucrose synthase for sink
strength using transgenic potato plants (Solanum tuberosum
L.). Plant J 7:97 /107.
Zrenner R, Willmitzer L, Sonnewald U. 1993. Analysis of the
expression of potato uridinediphosphate-glucose pyrophosphorylase and its inhibition by antisense RNA. Planta
190:247 /252.