Plant Cell Physiol. 42(12): 1311–1320 (2001)
JSPP © 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 the Chloroplast
Edurne Baroja-Fernández, Francisco José Muñoz, Takashi Akazawa 1 and Javier Pozueta-Romero 2
Instituto de Agrobiotecnología y Recursos Naturales, Universidad Pública de Navarra/Consejo Superior de Investigaciones Científicas, Ctra. de
Mutilva s/n, Mutilva Baja, 31192 Navarra, Spain
A vast amount of information has accumulated which
supports the view that sucrose and starch are end-products
of two segregated, yet highly interconnected, gluconeogenic
pathways taking place in the cytosol and chloroplast,
respectively. However, several lines of experimental evidences indicate that, essentially identical to the case of heterotrophic tissues, starch formation in the photosynthetic
tissues may involve the direct import to the chloroplast of
cytosolic hexose (C6) units derived from the sucrose breakdown. This evidence is consistent with the idea that synthesis of a sizable pool of ADP-glucose takes place in the cytosol by means of sucrose synthase whereas, basically in
agreement with recent investigations dealing with glycogen
biosynthesis in bacteria and animals, chloroplastic phosphoglucomutase and ADP-glucose pyrophosphorylase are
most likely playing a role in channelling of glucose units
derived from the starch breakdown in the chloroplast, thus
making up a regulatory starch turnover cycle.
According to this new view, we propose that starch
production in the chloroplast is the result of a flexible and
dynamic mechanism wherein both catabolic and anabolic
reactions take place simultaneously in a highly interactive
manner. Starch is seen as an intermediate component of a
cyclic gluconeogenic pathway which, in turn, is connected
with other metabolic pathways. The possible importance of
metabolic turnover as a way to control starch production is
exemplified with the recently discovered ADP-glucose pyrophosphatase, an enzyme likely having a dual role in controlling levels of ADP-glucose linked to starch biosynthesis
and diverting carbon flow towards other metabolic pathways.
Key words: ADP-glucose — ADP-glucose pyrophosphatase
— Chloroplast — Gluconeogenesis — Sucrose synthase.
Abbreviations: ADPG, ADP-glucose; AGPase, ADPG pyrophosphorylase; AGPPase, ADPG pyrophosphatase; cpFBP aldolase, chloroplastic fructose 1,6-bisphosphate aldolase; cpFBPase, chloroplastic
1
2
;
fructose 1,6-bisphosphatase; cpPGM, chloroplastic phosphoglucomutase; G6P, glucose-6-P; SS, sucrose synthase; UDPG, UDP-glucose;
UGPase, UGPG pyrophosphorylase.
Introduction
Leaves represent the major site for photosynthesis in
higher plants. The sucrose formed there is exported to photosynthetically inactive parts such as roots, seeds, tubers and
fruits, frequently referred to as sink tissues, where it is converted to reserve polysaccharides, typically starch.
Unlike in other organisms, carbohydrate metabolism in
plants is a typical example of integrated control which is compartmented between the cytosol and the plastid as illustrated in
Fig. 1 (ap Rees 1980). Although gluconeogenesis occurs in
both compartments, the major storage carbohydrate, starch, is
restricted to the plastids, whereas the major transport carbohydrate, sucrose, is metabolized in the cytosol. Carbon fluxes
through these two pathways are highly regulated and coordinated with the rate of CO2 fixation to avoid inhibition of photosynthesis resulting from the depletion of the Calvin-Benson
cycle intermediates or accumulation of phosphorylated intermediates and depletion of Pi. The formation of both sucrose
and transitory starch in photosynthetic tissues is biochemically
regulated by events involving interactions between metabolites
and enzymes existing in both the cytosol and the chloroplast
(Walker 1970, Walker 1992). Thus, a thorough understanding
of the mechanisms underlying starch and sucrose metabolism
in the photosynthetic tissues requires knowledge of the
enzymes and transport systems operating therein.
Based on the capacity of chloroplasts to produce starch
from some type of hexose (C6) molecules imported from the
cytosol, different models seeking to clarify still-unsolved questions on starch metabolism will be presented and discussed in
this review. This paper will also try to develop new perspectives in relation to some enzyme activities which have escaped
the attention of plant biologists in recent years. Several reviews
Present address: 2-278 Kamenoi, Meito-ku, Nagoya, 465-0094 Japan.
Corresponding author: E-mail, [email protected]; Fax, +34-94-823-2191.
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Sucrose-starch conversion in leaves
Fig. 1 Pathways of sucrose and starch synthesis in photosynthetic cells. The enzymes are numbered as follows: 1, 1¢ FBP aldolase; 2, 2¢ FBPase;
3, PPi:fructose-6-P 1-phosphotransferase; 4, 4¢ phosphoglucoisomerase; 5, 5¢ PGM; 6, UGPase; 7, sucrose phosphate synthase; 8, sucrose phosphate phosphatase; 9, AGPase; 10, starch synthase.
covering the control of starch metabolism in both autotrophic and heterotrophic tissues have been published (Stitt and
Sonnewald 1995, Emes and Neuhaus 1997, Smith et al. 1997,
Neuhaus and Emes 2000, Kossmann and Lloyd 2000, Winter
and Huber 2000) to which readers are referred for complementary information. Previously we have written a review article
specifically focused on sucrose–starch transition in the heterotrophic tissues (Pozueta-Romero et al. 1999), which provides
an introduction to developments which will be discussed in
more detail here.
The overlooked pitfalls in the classical model of starch
biosynthesis in photosynthetic tissues
A vast amount of information has been obtained from various sources which appear to support the popular mechanistic
model illustrated in Fig. 1, according to which starch is categorized as the end-product of a gluconeogenic process exclusively taking place in the chloroplast. This view, basically
consistent with the conceptual idea that the chloroplast is a
complete photosynthetic unit (Arnon 1955), is also supported
by in organello experiments demonstrating that CO2-dependent
formation of starch can take place in the isolated chloroplast
(Gibbs and Gynkin 1958, Heldt et al. 1977). However, several
enigmatic observations have been reported which, needing special consideration as well as careful analysis, indicate the possible operation of multiple or alternative ways of starch biosynthesis in photosynthetic tissues.
Stability of ADP-glucose (ADPG) in the chloroplast during
starch synthesis—As discussed by ap Rees (1995), one of the
most intriguing questions in the field of starch biosynthesis is
related to the subcellular localization of ADPG. It has been
assumed for a long time that the synthesis of ADPG catalyzed
by ADPG pyrophosphorylase (AGPase) takes place exclusively
in the plastid. However, whilst this is thought to apply to leaves
(Okita et al. 1979), there is now an increasing body of experimental evidence showing that the actual location is significantly different in heterotrophic tissues (Kleczkowski 1996,
Denyer et al. 1996, Thorbjornsen et al. 1996, Chen et al. 1998,
Baroja-Fernández et al. 2000, Beckles et al. 2001). Thus, the
overall picture strongly suggests that, at least in some plant
organs, ADPG can be synthesized outside the plastid.
It is our belief that regardless of the fact that AGPase is
located in the cytosol or in the plastid, it seems obvious that the
sine qua non requirement for the topography of the reactions
leading to the synthesis of ADPG is that they take place in a
subcellular compartment where both gluconeogenic intermediates and enzymes remain stable. In this context, it is well established that during active starch biosynthesis in the illuminated
chloroplast there occurs an alkalization of the stroma (Werdan
et al. 1975, Flügge et al. 1980, Enser and Heber 1980, Oja et al.
1986, Heineke and Heldt 1988, Hauser et al. 1995a, Hauser et
al. 1995b) with a concurrent increase in Mg2+ concentration
from 3 mM to 6 mM (Portis 1981). Both factors are considered to play an important role in regulating CO2 fixation and
starch biosynthesis by modulating activities of several gluconeogenic and Calvin-Benson cycle enzymes. AGPase, the enzyme
catalyzing the synthesis of ADPG in the chloroplast, has an
absolute requirement for Mg2+ (Singh et al. 1984, Spilatro and
Preiss 1987) and is fully active at pH values ranging from 7.0
(stromal pH in the non-illuminated chloroplast) to 8.2 (stromal
Sucrose-starch conversion in leaves
Fig. 2 pH-stability curve of ADPG. Three mM ADPG was incubated
at 25°C for 30 min at different pH regimes. The remaining ADPG was
immediately analyzed by HPLC as described by Rodríguez-López et
al. (2000). The buffers used were 50 mM sodium acetate-acetic acid
(pH 4.0–5.5), 50 mM MES/NaOH (pH 5.5–6.5), 50 mM HEPES/
NaOH (pH 6.5–8.0) and 50 mM Tris-HCl (pH 8.0–10.0). Each buffer
contained 3 mM MgCl2.
pH during active CO2 fixation and starch synthesis in the
illuminated chloroplast). This means that, as pointed out by
Kaiser and Bassham (1979) and Kleczkowski (Kleczkowski
1999, Kleczkowski 2000), a possible involvement of stromal
pH changes in regulating ADPG formation seems unlikely.
Remarkably, however, some classic investigations concerning the biochemical characterization of ADPG pointed out
that this nucleotide sugar is unstable at basic pH conditions,
leading to the spontaneous hydrolytic breakdown producing
glucose-1,2-monophosphate, a scarcely metabolizable compound, and AMP (Murata et al. 1963, Murata et al. 1964,
Recondo et al. 1963). Furthermore, Zervosen et al. (1998) have
recently reported that ADPG is chemically unstable in a solution containing Mg2+. In an attempt to further characterize the
pH- and Mg2+-dependent stability of ADPG, preparations of
this nucleotide sugar were incubated at different pH conditions
in buffer solutions containing 3 mM MgCl2 and immediately
subjected to HPLC analysis. As shown in Fig. 2, ADPG was
found to be unstable at pH values higher than 7.5, less than
50% of the ADPG remaining in the assay cuvette after 30 min
incubation at 25°C and pH ranging from 8.0 to 8.5. These
results strongly indicate that, unless chloroplastic ADPG produced by AGPase is rapidly coupled with starch synthase to
produce starch, it will be spontaneously hydrolyzed in the illuminated chloroplast. Under such circumstances, starch formation will be prevented and the hydrolysis of ADPG will evoke a
non-coordinated action between CO2 fixation and carbon flow
through starch, thus leading to inhibition of photosynthesis. In
order to avoid the detrimental effect of basic pH on the stability of the nucleotide sugar, it is possible that, essentially analo-
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gous to the topography of the reactions leading to the synthesis
of cell wall polysaccharides inside the Golgi apparatus (Muñoz
et al. 1996, Neckelmann and Orellana 1998), extraplastidially
synthesized ADPG will be transported into the chloroplast to
be instantaneously utilized as the precursor of starch synthesis
(see below).
Starch formation in chloroplastic fructose 1,6bisphosphate aldolase (cpFBP aldolase) and chloroplastic
fructose 1,6-bisphosphatase (cpFBPase) deficient plants—Further contradictory evidence against the widely accepted
mechanism of starch biosynthesis illustrated in Fig. 1 is connected to the non-correlated relationship between activities of
some gluconeogenic enzymes and starch production. The
nuclear gene albostrians in barley (Hordeum vulgare) induces
mutations which are phenotypically expressed as white leaves
in otherwise green leaves. Plastids of white tissue have no
or almost undetectable activities of some enzymes such as
glyceraldehyde-3-phosphate-dehydrogenase, phosphoribulokinase, 3-phosphoglycerate kinase, triose-P isomerase, and
cpFBP aldolase (Boldt et al. 1992). These plastids pose an
enigmatic problem as they are capable of forming starch molecules at normal levels. The lack of enzymes involved in the
conversion of triose-P into hexose-P raises the question of how
gluconeogenesis may take place in these plastids.
Willmitzer and associates investigated the role of the
cpFBPase with respect to starch formation in leaves of potato
plants. The authors observed that elevation of sucrose concentrations in the culture medium, under conditions of both
light and darkness, led to declined levels of cpFBPase transcripts irrespective of the fact that starch normally accumulated (Kossmann et al. 1992). Furthermore they also observed
that transgenic plants with reduced cpFBPase activity produced starch at normal level when they were incubated in
sucrose-containing medium, whereas they produced less starch
comparing to the wild-type plants under fully autotrophic conditions (Kossmann et al. 1994). A question then arises as to
how leaves placed in a sucrose-containing media can produce
starch in the absence of cpFBPase which, as shown in Fig. 1, is
believed to play a key regulatory role in the carbon flux
through starch.
Starch formation in chloroplastic phosphoglucomutase
(cpPGM) and AGPase deficient mutants—If gluconeogenesis
were to take place exclusively as illustrated in Fig. 1, starch
biosynthesis would be completely blocked in mutants totally
lacking cpPGM or AGPase. Against this prediction, however,
Saether and Iversen (1991) and Fritzius et al. (2001) have demonstrated that Arabidopsis thaliana plants with no cpPGM and
AGPase accumulate quantifiable and readily detectable
amounts of starch in their chloroplasts. In addition, Harrison et
al. (1998) have shown that leaves from pea plants with no
cpPGM accumulate as much as 15% of the normal starch content. Furthermore, plants of A. thaliana and Vicia narbonensis
containing 5% of the normal AGPase are able to accumulate
40% and 75% of the normal starch content, respectively (Lin et
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Sucrose-starch conversion in leaves
Fig. 3 Amino acid sequence alignments of the maize Brittle-1 ADPG transporter (EMBL accession number: M79333) and proteins predicted
from nucleotide sequences existing in (a) dicotyledonous species, i.e. Solanum tuberosum and Arabidopsis thaliana (accession numbers X98474
and AL034567, respectively) and (b) monocotyledonous species, i.e. Triticum aestivum and Hordeum vulgare (accession numbers AW448477 and
AY033629, respectively).
al. 1988b, Weber et al. 2000a).
Incorporation of intact glucose skeletons into starch in
leaves fed with [1-14C]glucose—Mature leaves can produce
starch from exogenously added glucose. According to the
model illustrated in Fig. 1, one can predict that leaves fed for a
short time with [14C]glucose labelled in position 1 will show a
redistribution of the radiolabelled carbon in the glucose moiety
of the starch molecules produced; triose-P derived from the
cytosolic metabolism of [1-14C]glucose would be imported into
chloroplasts and subsequently transformed to hexose-P and
starch molecules whose glucose moiety will be labeled predominantly in the C3 and C4 positions. Against this prediction, however, results obtained by McLachlan and Porter
(1959) using source tobacco leaves fed with [1-14C]glucose
showed that the glucose moiety in the starch molecules is still
primarily labeled in position 1.
Alternative models of starch formation in photosynthetic tissues
Altogether, the above experimental evidence appears to
indicate that, under certain circumstances and essentially identical to the case of amyloplasts from heterotrophic tissues, import
of some hexose (C6) unit(s) from the cytosol may entail a crucially important role in the synthesis of starch in the chloroplast.
The inner envelope membranes of the fully autotrophic
chloroplast are shown to be impermeable to hexose-Ps (Fliege
et al. 1978, Douce and Joyard 1990) and therefore transport of
hexose-Ps cannot be linked to starch biosynthesis in the chloroplast (Heldt et al. 1977, Kammerer et al. 1998, Flügge 1999).
Exceptionally, leaves incubated for a long time with glucose
contain chloroplasts exhibiting hexose-P transport capacities
(Quick et al. 1995). On the other hand, although the chloroplast membranes are known to be equipped with a glucose
transporter (Schäfer et al. 1977), its function is to export the
Sucrose-starch conversion in leaves
glucose molecules formed during starch breakdown at night
(Trethewey and ap Rees 1994a, Trethewey and ap Rees 1994b,
Schleucher et al. 1998, Wiese et al. 1999, Weber et al. 2000b)
and to act as a bypass under conditions of limiting triose-P
export (Heineke et al. 1994, Häusler et al. 1998).
As will be discussed later, the existence of a machinery
transporting ADPG, another type of hexose (C6) unit, in the
inner envelope membranes of chloroplasts (Pozueta-Romero et
al. 1991a) may provide a possible clue to clarify the above
mentioned enigmatic observations. Although there are a
number of reports demonstrating the operation in amyloplasts
of a machinery importing ADPG which is then utilized for
starch synthesis (Pozueta-Romero et al. 1991b, PozuetaRomero et al. 1999), our knowledge about the nature of this
transporter in the chloroplast is still scanty. Interestingly
enough, however, both monocotyledonous and dicotyledonous
plant species contain sequences in their genomes encoding proteins sharing high homology to Brittle-1 (Fig. 3), a well known
ADPG transporter existing in the envelope membranes of
maize amyloplasts (Cao and Shannon 1997, Shannon et al.
1998). Most importantly, computer assisted amino-acid
sequence analyses of these proteins using the Predotar and
TargetP programs (Emanuelsson et al. 2000, Nielsen et al.
1997) revealed that they have characteristics of typical chloroplastic proteins. Needless to say, further investigations will be
necessary to demonstrate their involvement in the ADPG transport to the stromal phase of the chloroplast.
Starch formation in chloroplasts of developing green
fruits—Developing fruits are strong carbohydrate sinks and
utilize the newly imported sucrose as precursor for starch biosynthesis in their chloroplasts (Guan and Janes 1991). The
sucrose molecules enter the metabolic route in the fruit via
either invertase or sucrose synthase (SS) and, coupled with
other enzymatic reactions, are eventually converted to hexosePs and nucleotide sugars in the cytosol (Robinson et al. 1988,
Wang et al. 1993). The question then arises as to what is the
starch-precursor molecule entering the chloroplast in developing green fruits. In this sense, it is of great importance to point
out that, in contrast to chloroplasts in source leaves, chloroplasts from developing fruits possess a glucose-6-P (G6P)
importing machinery analogous to that reported in achlorophyllous plastids such as amyloplasts (Emes and Neuhaus 1997) as
well as in the guard-cell chloroplasts from pea leaflets (Overlach et al. 1993). Based on such information, a mechanism of
sucrose–starch conversion has been proposed wherein cytosolic
G6P and ATP entering the chloroplast are utilized as precursors of starch biosynthesis (Fig. 4a).
Another possible mechanism of starch synthesis occurring in fruit tissues is based on the existence of cytosolic
AGPase in pericarp cells from developing tomato fruits as well
as an ADPG transporting machinery (see above). According to
Chen et al. (1998), starch biosynthesis in developing fruits
takes place through the coupled actions of the cytosolic SS,
UDP-glucose pyrophosphorylase (UGPase), AGPase and the
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Fig. 4 Models of starch synthesis in green fruits. (a) Involvement of
a chloroplastic hexose-P transporter and a ATP/ADP transporter (Batz
et al. 1995); (b) Sucrose–starch conversion involving a cytosolic
AGPase and a chloroplastic ADPG translocator (Chen et al. 1998).
ADPG translocator existing in the envelope membranes of the
chloroplast (Fig. 4b).
Although the metabolic models illustrated in Fig. 4a and
Fig. 4b are consistent with the currently prevailing view that
SS plays a prime role in converting the newly imported sucrose
into UDP-glucose (UDPG) necessary for starch biosynthesis,
this idea is in conflict with recent reports showing that genetically engineered tomato plants with decreased SS activities
accumulate normal levels of starch (Chengappa et al. 1999,
D’Aoust et al. 1999).
Synthesis of starch in green leaves: cytosolic production of
ADPG by SS and cyclic turnover of starch in the chloroplast—
Sucrose produced in green leaves can be either transported to
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Sucrose-starch conversion in leaves
other parts of the plant, accumulated in the vacuole, or metabolized in the cytosolic compartment of the photosynthetic cell. A
first step in the metabolic breakdown of sucrose is the cleavage of the glycosidic bond by the following readily reversible
reaction:
Sucrose+NDP¨NDPG+fructose
which is catalyzed by SS in the cytosol by a near-equilibrium
reaction in vivo (Geigenberger and Stitt 1993).
In heterotrophic tissues SS produces UDPG which is then
utilized as precursor for multiple glycosylation reactions
involved in the synthesis of glycoproteins, glycolipids, cell
wall polysaccharides, and several others. UDPG can also be
converted to ADPG by the coupled reactions of UGPase and
AGPase (Kleczkowski 1996, Smith et al. 1997, Kossmann and
Lloyd 2000, Neuhaus and Emes 2000) to be subsequently utilized as substrate for starch biosynthesis (Fig. 4).
Generally speaking, SS activity is highest in sink tissues
that are importing the sucrose necessary to produce starch.
Young (sink) leaves show high SS activity (Larsen et al. 1985,
Nguyen-Quoc et al. 1990), whereas mature (source) leaves do
not. In some plant species, however, SS is nearly constant
throughout leaf development (Pollock 1976, Giaquinta 1978,
Claussen et al. 1985, Chan et al. 1990). Although the bulk of
SS in leaves is located in the companion and phloem parenchyma cells to energize sucrose loading (Nolte and Koch 1993,
Martin et al. 1993, Lerchl et al. 1995) several reports have
shown that SS is constitutively expressed in all cell types of the
plant (McCarty et al. 1986, Maraña et al. 1990), including mesophyll cells (Fu et al. 1995).
Although UDP is generally considered to be the preferred
nucleoside diphosphate for SS, numerous studies have demonstrated that ADP serves as an effective acceptor molecule to
produce ADPG (Delmer 1972, Silvius and Snyder 1979,
Porchia et al. 1999, Tanase and Yamaki 2000, for a review see
Pozueta-Romero et al. 1999). Furthermore, our presently ongoing investigations have revealed that ADPG synthesizing activities of SS in barley seeds are found to be many fold higher
than those of AGPase (Baroja-Fernández et al. submitted) the
overall results thus strongly indicating that direct production of
ADPG by SS plays a crucially important role in the sucrose–
starch conversion process.
In addition to the ability of converting ADPG to starch,
plastids are equipped with the enzymes capable of degrading
starch molecules (Preiss 1982, Steup 1988). Pulse chase as well
as starch pre-loading experiments using isolated chloroplasts
(Heldt et al. 1977, Stitt and ap Rees 1980, Stitt and Heldt
1981a, Stitt and Heldt 1981b) and intact leaves (Dickson and
Larson 1975, Scott and Kruger 1995, Häusler et al. 1998) have
provided evidence that, essentially in agreement with previous
reports dealing with gluconeogenic processes in bacteria (Gaudet et al. 1992, Belanger and Hatfull 1999, Guedon et al. 2000),
animals (Massillon et al. 1995, Bollen et al. 1998) and hetero-
Fig. 5 Mechanistic model scheme of starch biosynthesis in green
leaves involving the coordinated actions of SS and ADPG translocator
(Pozueta-Romero et al. 1999). The hydrolytic breakdown of starch is
metabolically connected to the intrachloroplastic synthesis of ADPG
by the coupled reactions involving hexokinase (Peavey et al. 1977,
Heldt et al. 1977, Stitt and ap Rees 1980, Stitt and Heldt 1981b),
cpPGM and AGPase. The enzymes are numbered as follows: 1, SS; 2,
starch synthase; 3, amylase; 4, hexokinase; 5, cpPGM; 6, AGPase.
trophic tissues of plants (Pozueta-Romero and Akazawa 1993,
Neuhaus et al. 1995, Sweetlove et al. 1996) chloroplasts can
synthesize and mobilize starch simultaneously, and both
cpPGM and AGPase may have a role in the scavenging of glucose molecules derived from starch breakdown.
Further experimental evidence showing that chloroplasts
can simultaneously synthesize and mobilize starch during the
light period can be found in the starch-accumulating mutants of
A. thaliana deficient in endo-amylase activity (Zeeman et al.
1998). These mutants have a higher starch content in their
leaves than the wild-type plants, which is mostly due to an
imbalance between the rates of starch synthesis and degradation.
Taking into account the capacities of SS to produce ADPG
directly from sucrose and ADP, and considering the occurrence of cyclic turnover of starch in the chloroplast, an alternative model of starch biosynthesis has been proposed wherein
SS catalyzes the de novo synthesis of ADPG in the cytosol
(Fig. 5) (Pozueta-Romero et al. 1991a). According to this
mechanistic model, alternative to that illustrated in Fig. 1 but
consistent with the previously discussed experimental evidences indicating that some form of C6 molecule enters the
chloroplast to be utilized as precursor for starch biosynthesis,
the net rate of starch biosynthesis in photosynthetic tissues
might be determined by the rate of import of ADPG synthesized by SS in the cytosol and the efficiency with which starch
Sucrose-starch conversion in leaves
breakdown products can be recycled to produce starch via the
coupled reactions catalyzed by cpPGM and AGPase (PozuetaRomero 1992, Pozueta-Romero and Akazawa 1993, cf. Fig. 6
Akazawa 1994). Under conditions of high light intensity, when
the rate of photosynthate supply by the chloroplast exceeds that
of export by the cell, sucrose will accumulate and SS will
actively produce the ADPG necessary for starch biosynthesis.
On the other hand, at night, when light intensity declines, both
the supply of photosynthate and the rate of sucrose synthesis
will be reduced. Under such circumstances, the production of
ADPG catalyzed by SS will be reduced and starch biosynthesis will be suppressed.
The most critical objection to the model illustrated in Fig.
5 involving cytosolic production of ADPG and subsequent
transport into the chloroplast is related to the existence of
starchless mutants deficient in cpPGM and AGPase (Caspar et
al. 1985, Lin et al. 1988a, Lin et al. 1988b, Müller-Röber et al.
1992, Okita 1992). However, it must be pointed out that the
gluconeogenic model illustrated in Fig. 5 predicts that, under
circumstances in which cpPGM and AGPase are blocked, the
recovery towards starch biosynthesis of the hexose units
derived from starch breakdown will also be blocked. Consequently, the carbon flux towards sucrose and soluble carbohydrates in the extraplastidic compartment will be enhanced,
accompanying a parallel decline of starch accumulation
(Pozueta-Romero et al. 1999).
Transgenic plants with reduced Pi/triose-P translocating
activity are found to accumulate more starch than wild plants
(Riesmeier et al. 1993, Heineke et al. 1994). According to the
mechanistic model presented in Fig. 5, however, these observations are not surprising, because the chloroplastic 3PGA/Pi
ratio in the transgenic plants is shown to be higher than in the
wild-type plants. Accordingly, AGPase is more active in the
transgenic plants than in the wild-type plants (Heineke et al.
1994) and the efficiency by which the starch breakdown products are recycled to produce starch will also be higher in the
transgenic plants comparing to the wild-type plants.
As to the lowered production of starch in the transgenic
plants with reduced activities of the Calvin-Benson cycle
enzymes such as cpFBP aldolase and cpFBPase (Kossmann et
al. 1994, Haake et al. 1998), these plants showed a reduction of
their photosynthetic activities accompanying a reduction of the
total carbohydrate content. It is thus quite predictable that
plants deficient in Calvin-Benson cycle enzymes also accumulate less starch than the wild-type plants.
Control of intracellular levels of ADPG linked to starch
biosynthesis
Although there is an abundant information on the synthetic reaction catalyzed by AGPase leading to the production
of ADPG, plant enzymes catalyzing the breakdown of this
nucleotide sugar have been poorly described. Dankert et al.
(1963) and Murata (1977) identified an ADPG phosphorylase
widely distributed in plants which is responsible for the phos-
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phorolytic breakdown of ADPG. On the other hand, PozuetaRomero and co-workers have recently discovered a widely distributed enzymatic activity designated as ADPG pyrophosphatase (AGPPase), which catalyzes the conversion of ADPG
to G1P and AMP (Rodríguez-López et al. 2000). Experiments
carried out to identify possible different isoforms of AGPPase
have demonstrated the occurrence of starch granule-bound and
soluble isoforms, the latter being located both inside and
outside the plastid (Rodríguez-López et al. 2000, BarojaFernández et al. 2000). Therefore, AGPPase is present in the
same compartment of the cell as are the established processes
of ADPG synthesis and utilization.
Interestingly enough, AGPPase activities are notably high
in organs such as leaves which are not specialized in accumulating reserve starch, but rather they accumulate starch in a
transitory form. This may indicate that AGPPase, which competes with starch synthase for the same substrate, ADPG, may
operate specially in organs where a dynamic turnover of starch
molecules occurs, having a dual role in controlling the synthesis of transitory starch in the chloroplast and in diverting excess
ADPG from the starch biosynthetic pathway to alternative metabolic routes in response to biochemical need (Fig. 6). This
hypothetical mechanism has been reinforced by the recent finding of an AGPPase in Escherichia coli whose activities have
been shown to regulate the levels of ADPG linked to glycogen
biosynthesis (Moreno-Bruna et al. 2001).
The levels of the mRNA encoding an AGPPase isoform
oscillate with a circadian rhythm in photosynthetically active
barley leaves, the maximum abundance being present during
dark periods coinciding with low or nule starch production
(Rodríguez-López et al. in preparation). On the other hand, the
existence of a plastidial AGPPase isoform which is fully active
at pH values occurring in the non-illuminated chloroplast (i.e.
pH 7.0) and almost totally inactive at pH values occurring in
the illuminated chloroplast during active starch biosynthesis
(pH 8.0 or higher) (Baroja-Fernández et al. 2000) indicate that
diurnal fluctuations of the pH in chloroplast may result in the
regulation of AGPPase activity, preventing starch biosynthesis
during darkness and allowing the transitory starch accumulation during illumination of the plant.
Plants exposed to high temperatures are shown to exhibit
perturbation of starch synthesis, declined levels of ADPG and
increased respiratory rates (Geigenberger et al. 1998). In this
context, it must be emphasized that we have observed that
AGPPase activities in plants grown at high temperatures are
several fold higher than those of plants grown at optimum temperature conditions for starch biosynthesis (Rodríguez-López et
al. in preparation). These observations further strengthen our
view that, under certain circumstances, AGPPase may play an
important role in blocking starch production and diverting carbon flow toward other metabolic pathways (Fig. 6).
Overall, information gained on AGPPase have lead us to
propose that the intracellular levels of ADPG linked to starch
biosynthesis can be determined by the balance between ADPG-
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Sucrose-starch conversion in leaves
Fig. 6 Control of intracellular levels of ADPG linked to starch biosynthesis by AGPPase. Under conditions of high ADPG production and low
starch synthesis, over-accumulation of ADPG will be prevented by the action of AGPPase and carbon flow will be diverted toward other metabolic pathways in response to biochemical needs.
synthesizing enzyme activities (i.e. SS and AGPase) and those
of ADPG utilization such as that catalyzed by starch synthase
and AGPPase (Baroja-Fernández et al. 2000). Further investigations using transgenic plants with altered AGPPase activities
will lead us to validate the importance of this enzyme in controlling starch production as well as in connecting gluconeogenesis with other metabolic pathways as illustrated in Fig. 6.
Concluding remarks and future prospect
Taken together with our previous review paper dealing
with the mechanism of starch formation in heterotrophic tissues
(Pozueta-Romero et al. 1999), this review does not intend to
draw a definitive picture of starch biosynthesis in plant cells.
Historically speaking, the original design of the central
dogma presented in Fig. 1 likely goes back to the time of
Arnon (1955) who performed a series of pioneering biochemical studies on photosynthesis and carbon metabolism using isolated chloroplasts. These and subsequent studies by a number
of successors have firmly established a conceptual view that
the chloroplast is a self-sustaining photosynthetic unit wherein
the CO2-dependent starch formation takes place. However,
based on experimental grounds herein discussed, it is our belief
that, essentially identical to the case of heterotrophic tissues,
there exists an active flow of ADPG between the cytosol and
the chloroplast in photosynthetic tissues as schematically illustrated in Fig. 5. According to this new mechanistic viewpoint,
gluconeogenic pathways leading to the production of sucrose
and starch are highly interconnected to each other, and they are
not segregated as illustrated in Fig. 1.
The active turnover of gluconeogenic molecules such as
ADPG and starch will be worthy of serious consideration for
future investigation. Studies on both animals and bacteria have
clarified the mechanism(s) controlling the levels of endogenous nucleotide sugars (Moreno-Bruna et al. 2001, Gasmi et
al. 1999). The rapid turnover of nucleotide sugars by nucleotide pyrophosphatases has been discussed to be an important
mechanism controlling the levels of substrates for glycosylation reactions leading to the production of glycoproteins and
glycolipids (Hickman et al. 1985). Although most of these
reactions take place in the Golgi apparatus and endoplasmic
reticulum, it is notable that control of the intracellular levels of
the nucleotide sugars takes place mostly in the cytosol. Will it
be too speculative to surmise that similar kind of mechanisms
occur in plants to control the levels of ADPG linked to the synthesis of saccharides such as starch? Will it be too naive to propose that the de novo synthesis of ADPG takes place in a compartment where this nucleotide sugar is chemically stable
during active starch synthesis, i.e. the cytosol, to be subsequently transported into the plastid? Finally, taking into
account the existence of photosynthetically active chloroplasts
in developing green fruits which produce starch from some
hexose (C6) unit imported from the cytosol, is it premature to
speculate on a possibility that chloroplasts from green leaves
operate in the same manner?
Sucrose-starch conversion in leaves
Acknowledgements
T. Akazawa wishes to record his most sincere appreciation for
the financial support from the Spanish Ministry of Culture and Education. We thank David Ndlovu for critical examination of the text.
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(Received June 27, 2001; Accepted October 3, 2001)