Review
Blackwell Publishing, Ltd.
Tansley review
The breakdown of starch in leaves
Author for correspondence:
Samuel C. Zeeman
Tel: +41 31 6315222
Fax: +41 31 6314942
Email: [email protected]
Samuel C. Zeeman1, Steven M. Smith2 and Alison M. Smith3
Institute of Plant Sciences, University of Bern, Altenbergrain 21, CH−3013 Bern, Switzerland; 2Institute
1
of Cell and Molecular Biology, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JH, UK;
3
Department of Metabolic Biology, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK
Received: 13 January 2004
Accepted: 29 March 2004
doi: 10.1111/j.1469-8137.2004.01101.x
Contents
Summary
247
VI.
Export of starch catabolites
254
I.
Introduction
247
VII. Metabolism of glucose and maltose
255
II.
Structure of the starch granule
248
VIII. The emerging pathway of starch breakdown and
its regulation
256
III.
Initial attack on the granule and the role of glucan,
water dikinase
249
Acknowledgements
258
IV.
Debranching of branched glucans
250
References
258
V.
The metabolism of linear glucans
251
Summary
Key words: starch, amylase, α-glucan
phosphorylase, glucan, water dikinase,
glucanotransferase, Arabidopsis.
This review describes recent progress in discovering the pathway of starch breakdown in leaves. The synthesis of starch from photo-assimilated carbon is one of the
major biochemical fluxes in plants. Despite this, the pathway through which this
starch is remobilized has not been defined. Numerous enzymes that could participate in starch breakdown are present in leaves, but until recently, the relative importance of each had not been determined. Through studies using model species such
as Arabidopsis and potato, significant progress has now been made in determining
the roles of known enzymes, and in the discovery of novel proteins necessary for
breakdown. These data allow a tentative pathway for starch breakdown to be
mapped out, involving hydrolysis primarily to maltose and subsequent maltose
export to the cytosol. This provides a framework for complete discovery of the pathway and for the analysis of its regulation.
© New Phytologist (2004) 163: 247–261
I. Introduction
Transitory starch is a primary product of photosynthesis in
higher plants. It serves as a store of carbohydrate, which
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supports metabolism and growth during the dark when
photosynthesis is not possible. In some plants, up to half the
photo-assimilated carbon is stored as starch, to be remobilized
later. Considering this important function, it is not surprising
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that the metabolism of transitory starch is influenced by key
environmental factors such as daylength, temperature and
nutrient availability (Chatterton & Silvius, 1981; Grange,
1985; Fredeen et al., 1989; Qui & Israel, 1992; Paul & Stitt,
1993; Martindale & Leegood, 1997; Strand et al., 1997). It is
also not surprising that perturbations of starch metabolism
can have far-reaching consequences, reducing plant growth and
affecting development (Caspar et al., 1985, 1991; Schulze et al.,
1991; Huber & Hanson, 1992; Eimert et al., 1995; Corbesier
et al., 1998). What is surprising is our lack of understanding
of how starch is remobilized, and of how both synthetic and
degradative pathways are controlled and integrated with other
pathways of metabolism.
In most plants, starch is made in chloroplasts at the same
time as sucrose is made in the cytosol. Partitioning between
the two products is regulated in response to a number of
external and internal stimuli. The paradigm for this regulatory
mechanism was derived mainly from work on spinach leaves
in the 1980s (Stitt & Quick, 1989). It involves the integration
of feed-forward and feedback metabolic signals from photosynthesis and sucrose synthesis, respectively. This results in the
progressive increase in flux into the starch synthetic pathway
as the demand for sucrose is exceeded by the supply of photoassimilates. The reciprocal fluxes into starch and sucrose have
led to the notion that starch serves as an overflow for photosynthesis (Stitt & Quick, 1989). However, it is clear that not
all plants partition photoassimilates in exactly the same
manner as spinach, as the extent and pattern of starch accumulation vary considerably between species (Caspar et al., 1985;
Fondy et al., 1989; Servaites et al., 1989a; Scott & Kruger,
1994; Trevanion, 2002). Similarly, there is appreciable variation in both the timing and the degree of starch mobilization
at night. Starch degradation may begin soon after darkening
a plant (Stitt et al., 1978; Stitt & Heldt, 1981), but in several
species it commences only after an appreciable lag during
which other leaf carbohydrates are depleted (Gordon et al.,
1980; Fondy & Geiger, 1982; Zeeman & ap Rees, 1999).
Under simulated daylight conditions, starch degradation
can also commence before the onset of darkness or extend into
the light (Fondy et al., 1989; Servaites et al., 1989a). At these
times the level of irradiance (and hence the rate of photosynthesis) is low and carbohydrate released by starch degradation
can sustain a high rate of sucrose synthesis (Servaites et al.,
1989b). Darkening plants during the day also triggers degradation (Zeeman et al., 2002a). These observations illustrate
that leaf starch represents a dynamic pool of carbohydrate, the
breakdown of which is integrated with and regulated by other
metabolic pathways.
Knowledge of the pathway of starch degradation is a
prerequisite to understanding its regulation. Many enzymes
capable of participating in starch degradation can be measured
in extracts of leaves. These include hydrolases (e.g. amylases and
debranching enzymes), phosphorylases and glucanotransferases.
The participation of all the known enzymes would result in a
complex web of reactions, leading from a starch granule to
metabolites that could be exported from the chloroplast to the
cytosol. This contrasts with the pathway of starch synthesis (a
linear sequence of reactions leading from Calvin cycle intermediates to an α-1,4-glucan) and may be one reason why
relatively little progress has been made in evaluating the relative
importance of each enzymatic step in vivo. A more significant
complication, however, is the existence of multiple isoforms
of many glucan-metabolizing enzymes and the localization
of some of these isoforms outside the chloroplast. For example,
most of the amylolytic activity and phosphorolytic activity
in Arabidopsis leaves is extraplastidial (Lin et al., 1988a). The
function of these extraplastidial enzymes is unknown.
Recently, creative molecular biological approaches, together
with genome sequence information and postgenomic technologies, have allowed significant progress in understanding
starch degradation. These approaches have first resulted
in the discovery of a previously unknown protein essential for
degradation, second forced the reconsideration of supposedly
‘key’ enzymes and third, revealed a novel pathway for the utilization of degradation products in the cytosol. In the following
sections we describe our current understanding of starch
degradation in leaves, highlighting these recent discoveries.
II. Structure of the starch granule
Starch is a remarkable substance in that it consists of simple
polymers of glucose organized to form semicrystalline, insoluble
granules with an internal lamellar structure (Buléon et al., 1998).
This granular structure is relevant when considering the mechanism of starch degradation, as many glucan-metabolizing
enzymes appear to be unable to act upon intact granules as a
substrate. Most information on the structure of starch comes
from studies of the starch-storing organs of crop species rather
than leaves, but recent work has shown that leaf starch is
similar in many respects to storage starches (Fig. 1; Matheson,
1996; Zeeman et al., 2002b).
Starch is composed of the two glucan polymers, amylopectin and amylose. Amylopectin, by far the major component in
leaf starch, is a large molecule with a branched structure and
is responsible for the granular nature of starch. Amylose is
smaller, essentially linear and synthesized within the matrix
formed by amylopectin (Buléon et al., 1998). Glucosyl residues in both polymers are linked by α-1,4-bonds to form
chains of varying lengths. In amylopectin, these are linked by
α-1,6-bonds, forming branch points (Fig. 1a). In basic terms,
amylopectin is similar to glycogen, the polyglucan accumulated in animals, fungi and bacteria. However, amylopectin
differs from glycogen in two important ways: first, it has fewer
branch points and second, the branch points are arranged in
a discontinuous pattern that results in clusters of unbranched
chains (Fig. 1b). Within the granule, adjacent chains in the
clusters form double helices, which pack in ordered arrays
to form semicrystalline layers (Fig. 1c–e). This structure has
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Fig. 1 The structure and appearance of starch granules from leaves. (a–e) Illustration of the structure of amylopectin and its organization within
the starch granule. (a) Chains of α-1,4- and α-1,6-linked glucosyl residues within amylopectin. (b) Schematic drawing of the cluster structure
of amylopectin. (c) Cartoon of the double helices formed by neighbouring chains and their ordered packing. (d) Formation of semicrystalline
lamellae (containing ordered double helices) and amorphous lamellae (containing the branched regions), which alternate with 9-nm periodicity.
(e) ‘Growth ring’ structure of a starch granule (see j). Semicrystalline and amorphous lamellae make up ‘resistant’ semicrystalline layers between
which the glucan structure is less ordered. (f–j) Micrographs of starch granules from Arabidopsis leaves (bars, 2 µm). (f) Transmission electron
micrograph of a leaf mesophyll chloroplast containing starch granules (S). Leaves were harvested at the end of the photoperiod. Inset, similar
section pretreated using the silver proteinate method to stain glucan polymers. Note that the space around the granule does not contain glucan.
(g,h) Scanning electron micrographs of starch granules isolated at the end of the photoperiod from leaves of the wild type and the starch-excess
mutant sex4, respectively. (i) Wild-type starch granule viewed under polarized light. Note the ‘Maltese cross’ pattern of birefringence indicating
radial orientation of the constituent polymers. (j) Internal structure of a single starch granule from sex4 reminiscent of ‘Growth rings’. Granules
are cracked open and the exposed surface is partly degraded with α-amylase to reveal ‘resistant’ layers. For further details, see Zeeman et al.
(1998a, 2002b).
been likened to that of side-chain liquid crystal polymers
(Waigh et al., 2000). Interestingly, the establishment of this
pattern may itself require the participation of glucandegrading enzymes (debranching enzyme – see Section IV ),
but once formed, the semicrystalline structure adopted by
amylopectin is very stable and relatively resistant to the
actions of many enzymes.
It was suggested that transitory starch granules are composed of a central crystalline core surrounded by a ‘pasty
mantle’ (Beck, 1985). This idea was based on the presence of
an electron-transparent region surrounding granules in transmission electron micrographs, and the pasty appearance of
granules viewed by scanning electron microscopy. However,
sections treated with silver proteinate (the Thiery method,
which renders glucans electron-dense; Robertson et al., 1975)
show that the space surrounding the starch granules does not
contain glucans and is most likely an artefact of tissue fixation
and dehydration (Fig. 1f; Zeeman et al., 1998a). Furthermore,
granules prepared for scanning electron microscopy using gentle,
aqueous extraction techniques do not exhibit the reported
pasty appearance (Fig. 1g,h; Zeeman et al., 1998a, 2002b).
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III. Initial attack on the granule and the role of
glucan, water dikinase
The enzyme most frequently credited with the initial attack
on starch granules is endoamylase (α-amylase EC 3.2.1.1;
Preiss, 1982; Steup, 1988; Beck & Ziegler, 1989; Trethewey
& Smith, 2000). This enzyme is responsible for initiating the
mobilization of starch in the endosperm of germinating cereal
seeds (Fincher, 1989). However, the mobilization of endosperm
reserves represents an unusual situation in plants because,
at the stage of germination, the endosperm is nonliving
tissue. α-Amylase is secreted from the living aleurone cells
into the endosperm, where it attacks the starch at specific sites
(possibly at pores or channels in the granule surface) causing
a well-described ‘pitting’ of the granule. The soluble glucans
released by α-amylase serve as substrates for other hydrolytic
enzymes (Maeda et al., 1978; Fincher, 1989; Sun & Henson,
1990).
In most plant tissues, including leaves, starch is degraded
inside the plastid in which it was synthesized. Biochemical
studies, and more recently analyses of genome sequences, reveal
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that α-amylase is present inside chloroplasts (Okita et al.,
1979; Okita & Preiss, 1980; Lin et al., 1988a; Ziegler, 1988;
Li et al., 1992a; Stanley et al., 2002). In Arabidopsis, three genes
encode α-amylase-like proteins, one of which has a putative
transit peptide for chloroplast localization (Stanley et al., 2002).
It has often been suggested that – as in cereal endosperm – it
is responsible for the initial attack on starch granules in leaves.
This view was initially supported by analyses of an Arabidopsis
mutant that has a starch-excess phenotype (sex4 ). This mutant
has a reduction in chloroplastic α-amylase activity and a
reduced rate of starch breakdown at night, leading to the
gradual accretion of starch as the leaves age (Zeeman et al., 1998b;
Zeeman & ap Rees, 1999). The sex4 phenotype suggested
that chloroplastic α-amylase was required for normal rates of
starch degradation. (Zeeman et al., 1998b). However, the SEX4
locus lies on chromosome 3, which does not encode any of the
three α-amylase-like proteins. Furthermore, subsequent work
has demonstrated that removal of the chloroplastic α-amylase
using reverse genetics eliminates the same activity that is
reduced in sex4, but does not lead to a starch-excess phenotype
(S. M. Smith, unpubl. data). Thus, it seems unlikely that the
loss of this α-amylase can be the cause of the high starch in
sex4. As there are no other plastid-targeted α-amylases
annotated in the Arabidopsis genome, it seems possible, at
this stage, that starch breakdown in chloroplasts can proceed
without it.
There has been debate about whether α-glucan phosphorylase (EC 2.4.1.1) is also able to act upon isolated, intact starch
granules, by liberating glucose-1-phosphate from the ends of
α-1,4-linked chains (Kruger & ap Rees, 1983a; Steup et al.,
1983). The low affinity of the plastidial form of phosphorylase for large branched substrates is not consistent with such
a role (Steup & Schächtele, 1981; Shimomura et al., 1982).
Even if phosphorolysis of the granule surface does occur, it is
unlikely to have a significant impact without the additional
action of other enzymes such as α-amylase or debranching
enzyme. The action of phosphorylase (an exo-acting enzyme)
on external linear chains would rapidly uncover α-1,6-branch
points, past which the enzyme would be unable to act. It is
possible that hydrolytic enzymes ‘contaminate’ preparations
of isolated starch granules, which could result in an apparent
phosphorolytic activity. Malto-oligosaccharides released by
hydrolysis could be subsequently metabolized to glucose-1phosphate. Alternatively, granules might be damaged by harsh
extraction conditions, allowing access by enzymes that could
not attack the granule in vivo. Later studies of potato and
Arabidopsis have demonstrated that removal of the plastidial
phosphorylase does not prevent starch breakdown, illustrating that it is not essential for the degradation of starch
(Sonnewald et al., 1995; Zeeman et al., 2004).
Important recent work has revealed that a previously unknown
enzyme, glucan, water dikinase (GWD; EC 2.7.9.4) may regulate the extent to which other enzymes attack the starch granule.
Glucan, water dikinase catalyses the transfer of the β-phosphate
of ATP to either the C6 or C3 positions of the glucosyl residues
of amylopectin (Ritte et al., 2002). The frequency of phosphorylation of amylopectin is variable, depending on the
botanical source of the starch. In potato starch, for example,
approximately one in every 200 glucosyl residues is phosphorylated (Nielsen et al., 1994) whereas in Arabidopsis leaf
starch, the frequency is 1 in 2000 residues (Yu et al., 2001).
Removal of GWD via gene silencing in potato or mutation in
Arabidopsis causes a decrease in starch-bound phosphate and
leads to greatly increased starch levels in leaves (Lorberth
et al., 1998; Yu et al., 2001). The inverse correlation between
the degree of phosphorylation and the extent of starch
accumulation has led to the suggestion that the presence
of the phosphate residues is required for degradation to
proceed. One possible explanation is that charged phosphate
groups might disturb the packing of double helices within
the amylopectin molecule, creating hydrated clefts in the
semicrystalline lamellae. Alternatively, phosphorylated residues
could serve as specific binding sites or targets for degradative
enzymes.
It is not yet known whether it is the starch-bound phosphate or the GWD protein itself that is important in mediating
starch breakdown. Although the phosphorylation of amylopectin does occur during net starch synthesis (Nielsen et al.,
1994), GWD becomes bound to leaf starch granules during
starch breakdown (Ritte et al., 2000a) and therefore may be
more active at this time. Alternatively, there may be an additional function of the enzyme during starch breakdown.
However GWD functions, it clearly has a major influence on
starch metabolism and the fact that covalently bound phosphate has been reported for starches from many sources (Lim
et al., 1994; Blennow et al., 2000; Ritte et al., 2000b) suggests
a widely conserved mechanism. Interestingly, the starch from
cereal endosperm contains little or no phosphate (Lim et al.,
1994; Blennow et al., 2000; Ritte et al., 2000b). It is tempting
to speculate that this reflects different mechanisms for the
initiation and control of starch breakdown inside plastids of
living cells and in nonliving parts of plants.
IV. Debranching of branched glucans
Between 4% and 5% of the linkages in amylopectin are
α-1,6-branch points. The involvement of a debranching enzyme
is therefore essential for its complete breakdown. Two classes
of debranching enzyme, isoamylase (EC 3.2.1.68) and limit
dextrinase (pullulanase-type or R-enzyme; EC 3.2.1.142),
have been identified in plants (Ishizaki et al., 1983; Doehlert
& Knutson, 1991; Zhu et al., 1998) and localized in
chloroplasts (Okita et al., 1979; Ludwig et al., 1984; Kakefuda
et al., 1986; Li et al., 1992a; Zeeman et al., 1998a). Both are
hydrolases and, while similar in amino acid sequence and
predicted structure, differ in their substrate preference. The
defining difference is the ability of limit dextrinase to hydrolyse
the α-1,6-linkages in the secreted yeast polysaccharide, pullulan
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(sequential maltotriosyl units linked together with α-1,6bonds), although this is clearly not its substrate in vivo.
Interest in the role of debranching enzymes in starch degradation has been somewhat overshadowed by the discovery
that they play a key role in starch granule biosynthesis.
Mutants that lack isoamylase have decreased starch contents,
but accumulate an abnormal soluble polysaccharide similar
to glycogen (phytoglycogen). This remarkable phenotype has
been observed in species as diverse as cereals, Arabidopsis
and Chlamydomonas (James et al., 1995; Mouille et al., 1996;
Nakamura et al., 1997; Zeeman et al., 1998a; Burton et al.,
2002). Several hypotheses have been proposed that seek to
explain how a debranching step may be involved in the
normal biosynthesis of amylopectin, and why phytoglycogen
accumulates when debranching activity is absent. These
hypotheses have been recently reviewed elsewhere (Myers
et al., 2000; Ball & Morell, 2003).
There is good reason to suppose that debranching of
glucans during starch degradation is not carried out by the
isoamylase involved in starch synthesis. In the Arabidopsis dbe1
mutant, all the starch and phytoglycogen that accumulates in
leaves during the day is remobilized during the degradative
phase at night (Zeeman et al., 1998a). This phenomenon is
also observed in the sta7 mutant of Chlamydomonas (Dauvillée
et al., 2001). Such results indicate that another enzyme is
present which can degrade α-1,6-linkages, even though it
cannot compensate for the missing isoamylase in the synthetic
phase. Analysis of the Arabidopsis genome reveals that there
are three genes encoding isoamylase-like proteins (ISA1, ISA2
and ISA3) and a single gene encoding limit dextrinase (LDA).
The three isoamylase genes are conserved in divergent plant
species and there is evidence from potato and Arabidopsis that
the proteins encoded by ISA1 and ISA2 are subunits of one
heteromultimeric isoamylase protein in vivo (Hussain et al.,
2003; T. Delatte and S. C. Zeeman, unpubl. data). Loss of
either ISA1 or ISA2 in Arabidopsis leads to the phytoglycogen
accumulating phenotype described above. This leaves ISA3
and LDA as candidates for the debranching enzyme(s) with a
specific role in degradation. There is some evidence to suggest
that both may be involved. First, analysis of the activities of
the three potato isoamylase proteins in vitro revealed that
ISA3 has a remarkably high activity on β-limit amylopectin
(amylopectin digested with β-amylase, so that all chains external to the α-1,6-branch points are reduced to stubs of two or
three glucosyl residues in length)(Hussain et al., 2003). It
seems likely that glucan structures similar to those found in
β-limit amylopectin would be generated in vivo as the external
α-1,4-chains of amylopectin are degraded. Second, a recent
study of a maize mutant (zpu1) lacking LDA revealed that it
has a reduced rate of starch degradation in leaves during the
night, although most of the starch was still degraded (Dinges
et al., 2003). Furthermore, this line displayed a reduced rate
of starch mobilization in the endosperm of germinating grains
and a reduced rate of seedling growth.
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Review
It seems plausible that together ISA3 and limit dextrinase
may possess the range of substrate specificities required to
debranch glucan structures that arise during starch degradation, while ISA1 and ISA2 fulfil a more specialized role
in facilitating amylopectin synthesis. Alternatively, other
enzymes may be capable of hydrolysing the branch points. For
example, some isoforms of α-glucosidase are able to hydrolyse
α-1,6-linkages, although generally with low efficiencies (Sun
et al., 1995; Frandsen & Svensson, 1998).
V. The metabolism of linear glucans
Linear α-1,4-chains may be exposed to the action of
degradative enzymes either at the surface of the starch granule,
or when released from the granule into the chloroplast stroma
as oligosaccharides or branched glucans. Five enzymes could
be involved in the metabolism of such linear chains: αamylase, β-amylase, disproportionating enzyme (-enzyme),
α-glucosidase (maltase) and α-glucan phosphorylase. In the
past, emphasis has been placed on the contributions of
α-amylase and α-glucan phosphorylase (Preiss, 1982; Steup,
1988; Beck & Ziegler, 1989), but their importance may have
been overstated. There is growing evidence to suggest that
the combined actions of β-amylase and -enzyme are predominantly responsible for degrading linear glucans in Arabidopsis
leaves.
α-Glucan phosphorylase releases glucose-1-phosphate
from the nonreducing ends of linear chains. It has been extensively studied in leaves and frequently cited as an important
enzyme in starch breakdown. Several lines of circumstantial
evidence have supported this view. First, phosphorylase is
widespread in both eukaryotes and prokaryotes and its amino
acid sequence is highly conserved (Newgard et al., 1989). In
plants, distinct isoforms are present in the plastidial and
cytosolic compartments (Schächtele & Steup, 1986; Steup,
1988). Second, experiments with isolated spinach and pea
chloroplasts have indicated that phosphorylase can play a
significant role. When incubated in the dark in the presence
of inorganic phosphate, starch is broken down, and phosphorylated compounds formed (Levi & Gibbs, 1976; Levi &
Preiss, 1978; Stitt & ap Rees, 1980; Stitt & Heldt, 1981; Kruger
& ap Rees, 1983b). Third, the plastidial isoform has a high
affinity for linear glucans (Steup & Schächtele, 1981;
Shimomura et al., 1982), suggesting that it could utilize
the products of other enzymes such as debranching enzyme
or α-amylase. By contrast, the cytosolic isoform has a high
affinity for glycogen-like substrates (Steup & Schächtele, 1981;
Shimomura et al., 1982).
Experiments conducted to evaluate the contribution of
phosphorylase to starch degradation in vivo indicate that,
contrary to earlier suggestions, this enzyme has a minor role.
Removal of the plastidial isoform of phosphorylase in Arabidopsis by reverse genetics has no effect on the overall rate of
starch degradation in leaves (Zeeman et al., 2004). Similarly,
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Fig. 2 Malto-oligosaccharides in wild-type
Arabidopsis plants and three mutant lines
impaired in starch breakdown. (a) The maltose
content of wild-type Arabidopsis leaves
during the diurnal cycle. See Chia et al. (2004)
for details. (b) The maltose and maltotriose
contents of the wild type (wt) and the mutants
dpe1 (lacking chloroplastic D-enzyme;
Critchley et al., 2001), mex1 (lacking the
chloroplast envelope maltose transporter;
Niittylä et al., 2004) and dpe2 (lacking
cytosolic glucosyltransferase; Chia et al.,
2004). Note the change in scale on the y-axis.
repression of plastidial phosphorylase below the level of detection in potato leaves by gene silencing also has no apparent
effect on leaf starch content (Sonnewald et al., 1995). These
results show that phosphorylase is not required for starch degradation. However, an intriguing phenotype of the Arabidopsis
mutant lacking plastidial phosphorylase (Atphs1) suggests
that the enzyme has a more specific function. Under standard
growth conditions, Atphs1 plants develop small lesions on
their leaves, which are bordered by cells that accumulate an
excess of starch. An abrupt change in environmental conditions (e.g. a sudden shift from still, humid air to circulating,
dryer air) greatly increases the development of lesions, indicating
that the plants may be unable to endure transient periods of
water stress (Zeeman et al., 2004). Precisely what causes this
susceptibility is not yet understood. One view is that the
hexose-phosphates liberated by phosphorylase serve as substrates
specifically for chloroplast metabolism because they cannot be
directly exported to the cytosol (see Section VI). In some situations, such as the containment of a sudden, stress-induced
increase in reactive oxygen species, this supply of substrates
may be crucial (e.g. for the generation of reluctant via the oxidative pentose phosphate pathway; Zeeman et al., 2004).
There is both direct and indirect evidence that β-amylase
plays a significant role in metabolizing linear glucans
inside chloroplasts. β-Amylase (EC 3.2.1.2) is an exo-amylase,
which releases maltose from the nonreducing ends of
α-1,4-glucan chains. It cannot act on residues close to an
α-1,6-branch point, neither can it hydrolyse the branch points
themselves. The shortest α-1,4-linked glucan on which βamylase can act is maltotetraose (Chapman et al., 1972; Steup
& Schächtele, 1981). The enzyme is known to play an important function in the hydrolysis of starch reserves in germinating cereal endosperm and has been extensively studied in this
context (Ziegler, 1999). High activities of β-amylase are also
found in other plant tissues, including leaves (Doehlert &
Duke, 1983). In pea and Arabidopsis for example, the activity
of β-amylase exceeds the activities of other glucan-metabolising
enzymes by about an order of magnitude (Stitt et al., 1978;
Zeeman et al., 1998b).
There have been several reports, based on cell fractionation
techniques, that the β-amylase activity in leaves is present
both inside and outside the chloroplast (Stitt et al., 1978;
Kakefuda et al., 1986; Ziegler & Beck, 1986; Lin et al.,
1988a). Analysis of the Arabidopsis genome also indicates that
this is the case. Nine genes encoding β-amylase-like proteins
are annotated, four of which possess putative transit peptides
that would target them to the chloroplast. In one case, chloroplastic localization has been confirmed through production of a β-amylase–green flourescent protein (GFP) fusion
protein (Lao et al., 1999). There is circumstantial evidence
that chloroplastic β-amylase metabolizes glucans during
starch degradation. First, its product, maltose, increases at
the onset of starch breakdown during the night suggesting
that it may be an intermediate of starch catabolism (Fig. 2a;
Critchley et al., 2001; Chia et al., 2004; Weise et al., 2004).
Second, several studies have shown that appreciable amounts
of maltose are produced (Levi & Gibbs, 1976; Stitt & ap Rees,
1980; Stitt & Heldt, 1981; Kruger & ap Rees, 1983b) and
exported from isolated chloroplasts that are degrading starch
(Neuhaus & Schulte, 1996; Servaites & Geiger, 2002; Wiese
et al., 2004). Indeed, mutation of the recently discovered gene
encoding the chloroplast envelope maltose transporter (Niittylä
et al., 2004) causes maltose to accumulate at night to concentrations 40-times higher than that of the wild type (Fig. 2b; also,
see section VI). Furthermore, loss of a cytosolic enzyme
capable of metabolizing maltose (DPE2; Chia et al., 2004; Lu
& Sharkey, 2004) leads to the accumulation of even greater
levels of maltose (Fig. 2B; also see section VII). Together, these
results favour the idea that maltose produced by β-amylase is
a major product of starch degradation, and that it is exported
from the plastid and metabolized further in the cytosol.
Direct evidence for β-amylase function in transitory
starch breakdown was provided by antisense repression of a
chloroplast-targeted isoform in potato (Scheidig et al., 2002). The
leaves of transformed plants have reduced β-amylase activity
and exhibit a decrease in the amount of starch metabolized
during the night, compared with wild-type plants. Consequently, the leaf starch contents at the end of the night are
significantly increased. Similar observations have been made
in Arabidopsis mutants in which specific β-amylase isoforms
have been eliminated by insertional mutagenesis (S. M. Smith,
unpubl. data). At present, the significance of the multiple
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isoforms of chloroplastic β-amylase in Arabidopsis is not
clear. It is possible that functional redundancy of individual
isoforms may have arisen through gene duplication. Alternatively, each gene may have a distinct, tissue-specific expression
pattern, or encode a protein with distinct catalytic and/or
regulatory properties. The evaluation of single- and multiplegene knockouts should resolve these questions.
Most of the β-amylase activity in Arabidopsis leaves is
extraplastidial and its function is not known. The majority of
the extraplastidial activity is attributable to a single isoform
encoded at the RAM1 locus (reduced amylase; Laby et al.,
2001). This isoform has been well characterized (Lin et al.,
1988a; Caspar et al., 1989; Monroe & Preiss, 1990) and is
reportedly localized in phloem sieve elements (Wang et al.,
1995). Mutation of the RAM1 gene eliminates 90–95% of
the total β-amylase activity in the leaf, but has no apparent
effect on starch metabolism or phloem function, or any
other phenotypic consequences (Laby et al., 2001). In soybean,
mutants have also been reported in which β-amylase activity
is much reduced without any observable effects on starch
metabolism or growth (Hildebrand & Hymowitz, 1981). It is
possible that these mutants are also deficient in extraplastidial
isoforms of β-amylase. Cell fractionation studies on protoplasts from pea or wheat leaves have also indicated the
presence of β-amylase in the vacuole (Ziegler & Beck, 1986).
Again, the function of these β-amylase isoforms remains to be
established.
There is good evidence that disproportionating enzyme
(-enzyme) is localized in chloroplasts (Okita et al., 1979; Lin
et al., 1988a) and acts during starch degradation in a role
complementary to that of β-amylase. -enzyme is a 1,4-α-glucan:1,4-α--glucan, 4-α--glucanotransferase (EC 2.4.1.25)
which can catalyse a wide range of reactions, transferring
part of one glucan molecule (donor) to another (acceptor).
The glucan fragment transferred can be a maltosyl residue
or larger, maltotriose being the smallest donor species. The
acceptor can be a malto-oligosaccharide, a polyglucan or
even glucose (Lin & Preiss, 1988; Kakefuda & Duke, 1989;
Takaha et al., 1993). This range of possible reactions led to
some doubt over the function of -enzyme. However, the fact
that it exhibits its highest activity when supplied with small
linear malto-oligosaccharides such as maltotriose as substrate
supports the view that the enzyme has a role in starch breakdown. Its action on small glucans would release glucose
and simultaneously create larger molecules that would serve
as substrates for other glucan-degrading enzymes (such as
β-amylase or phosphorylase; Lin & Preiss, 1988; Kakefuda
& Duke, 1989; Takaha et al., 1993).
In Arabidopsis, a single gene encodes chloroplastic enzyme. Disruption of this gene via insertional mutagenesis
results in a phenotype that is entirely consistent with the proposed function of -enzyme in starch degradation (Critchley
et al., 2001). During the night, the rate of starch degradation
in the mutant is reduced compared with the wild type and
© New Phytologist (2004) 163: 247–261 www.newphytologist.org
Review
appreciable amounts of malto-oligosaccharide accumulate in
the mutants leaves. The accumulated malto-oligosaccharide is
almost exclusively maltotriose (Fig. 2b; Critchley et al., 2001).
This is significant in two ways. First, it provides another
line of evidence that β-amylase rather than phosphorylase is
responsible for the metabolism of linear glucans. β-Amylase
can hydrolyse maltotetraose (producing two maltose molecules) and maltopentaose (producing maltose and maltotriose) but cannot then act on the remaining maltotriose
(Chapman et al., 1972). Phosphorylase, on the other hand,
can metabolize maltopentaose to maltotetraose and glucose1-phosphate, but maltotetraose itself is a poor substrate
(Steup & Schächtele, 1981). Thus, in mutants lacking enzyme, accumulation of maltotriose rather than maltotetraose is indicative of glucan degradation via β-amylase rather
than phosphorylase. Second, -enzyme has its highest activity
with maltotriose as a substrate (Lin & Preiss, 1988; Kakefuda
& Duke, 1989), consistent with the idea that this is its substrate in vivo. Thus, it seems very likely that -enzyme serves
during starch degradation both to regenerate substrates for
further β-amylolysis and to release glucose.
The concerted actions of β-amylase and -enzyme would
result in the production of small amounts of glucose, and
large amounts of maltose. Both of these products can be
exported to the cytosol (see Section VI). However, there are
two possibilities for further metabolism of maltose in the
chloroplast: via α-glucosidase (maltase, EC 3.2.1.20) or
maltose phosphorylase (EC 2.4.1.8). Maltose phosphorylase
catalyses the phosphorolysis of maltose into glucose and
glucose-1-phosphate, and has been extensively described in
prokaryotes (Boos & Shuman, 1998; Ehrmann & Vogel, 1998).
Few data are available on the occurrence and function of maltose
phosphorylase in plants. It has been detected in the leaves of
pea seedlings, where it was localized to the chloroplast (Levi
& Preiss, 1978; Kruger & ap Rees, 1983b), and in poplar (Witt
& Sauter, 1994). There are, as yet, no genes described in plants
that encode proteins resembling the prokaryotic enzyme.
Many different types of α-glucosidase have been described
in plants. They are exo-acting enzymes and typically exhibit
broad substrate specificities (Frandsen & Svensson, 1998).
Acting on maltose, α-glucosidase catalyses its hydrolysis into
two glucose monomers. Only one study on pea seedlings has
reported an isoform of α-glucosidase localized in chloroplasts
(Beers et al., 1990). Purification and analysis of this isoform
showed that it is able to hydrolyse maltose and larger maltooligosaccharides (up to seven glucosyl residues length) with
equal efficiency, albeit with low affinity. The enzyme is also
capable of limited hydrolysis of larger glucans (Sun et al.,
1995). However, despite the presence of this enzyme, maltose
appears to be a major product of starch degradation in isolated
pea chloroplasts (Kruger & ap Rees, 1983b). In Arabidopsis,
there is no evidence for a chloroplastic α-glucosidase. None of
the five genes encoding proteins with sequence similarity to
known α-glucosidases are predicted to possess transit peptides
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for chloroplast localization. Furthermore, mutation of the
chloroplast maltose transporter results in a massive accumulation of maltose (Fig. 2b; Niittylä et al., 2004), presumably in
the chloroplast, suggesting that maltose is normally exported.
VI. Export of starch catabolites
Recently, significant progress has been made in understanding
how the products of starch breakdown are exported from the
chloroplast at night. Early work suggested that breakdown
was primarily phosphorolytic and that export occurred via the
triose-phosphate/phosphate translocator, as during photosynthesis. This now seems somewhat unlikely and, at least in
Arabidopsis, the evidence points firmly in favour of hydrolysis
and the export of neutral compounds.
Studies with isolated chloroplasts have provided valuable
insight but, together, have not yielded a clear picture. This is
because of variations in experimental design, the use of different species, and the responses of chloroplast metabolism to
the different conditions imposed (most notably the provision
of phosphate). In the absence of phosphate, maltose and
glucose are the predominant breakdown products (Stitt &
Heldt, 1981; Kruger & ap Rees, 1983b) and are exported
(Neuhaus & Schulte, 1996; Servaites & Geiger, 2002; Weise
et al., 2004). However, inclusion of phosphate in the incubation medium results in the production of increased amounts
of phosphorylated compounds (hexose-phosphates, 3phosphoglycerate (3-PGA) and triose-phosphates), presumably
through the stimulation of α-glucan phosphorylase. These
can accumulate in addition to, or partly in place of the neutral
ones (Stitt & Heldt, 1981; Kruger & ap Rees, 1983b). Thus,
uncertainty remained over the predominant form of carbohydrate exported in vivo. Most phosphorylated compounds
are exported as triose-phosphates or 3-PGA via the triosephosphate/phosphate translocator, rather than as hexose
phosphates. This is consistent with the inability of mesophyll
cell chloroplasts to translocate hexose phosphates under normal
circumstances (Flügge & Heldt, 1991; Quick et al., 1995).
Indirect evidence for the export of starch degradation
products by a route other than the triose-phosphate/phosphate
translocator in vivo was provided by the analysis of tobacco,
potato and Arabidopsis plants with a reduced capacity of the
triose-phosphate/phosphate translocator. These plants exhibit
a significant shift in photosynthetic partitioning away from
sucrose in favour of starch compared with the respective wild
types (Riesmeier et al., 1993; Heineke et al., 1994; Schneider
et al., 2002). In all cases, plant growth is normal or only
slightly reduced. The increased starch accumulation during
the day is matched by an increase in starch breakdown at night
or even starch turnover during the day. This suggests that
carbohydrate derived from starch breakdown is exported from
the chloroplast in a different form, such as hexose units or as
larger glucans, thus bypassing the restriction on triosephosphate export. Consistent with this, Häusler et al. (1998)
observed an increase in the glucose transport capacity of the
chloroplast envelope in the triose-phosphate/phosphate
translocator-deficient plants, compared with wild-type plants.
Similar conclusions were drawn from other studies in which
the synthesis of sucrose during photosynthesis is restricted
through a reduction of cytosolic fructose-1,6-bisphosphatase
(involved in the conversion of triose-phosphates to hexosephosphates). Metabolic changes were similar to those observed
with inhibition of triose-phosphate/phosphate translocator
(Sharkey et al., 1992; Zrenner et al., 1996).
Support for the idea that the products of starch degradation are exported as hexose or larger glucans was provided by
an independent approach using nuclear magnetic resonance.
Schleucher et al. (1998) studied the pattern of deuterium
incorporation (from deuterium-enriched water) into the
glucosyl fraction of sucrose made in tomato and bean leaves
during the night. The uneven distribution of label between
specific carbon atoms of the glucosyl moiety implied that the
carbohydrate used for sucrose synthesis does not pass through
the triose-phosphate pool, as this would involve equilibration
of the label between these carbon atoms.
The conclusion that can be drawn from the studies described
in this section is that neutral compounds released by hydrolysis
of starch (glucose, maltose or larger malto-oligosaccharides)
are exported from the plastid in vivo to provide substrates for
sucrose synthesis. It remains possible that some carbohydrate
(i.e. the products of phosphorolysis) may exit via the triosephosphate/phosphate translocator, but given the arguments
in Section V, it seems likely that this is a minor flux.
Schäfer et al. (1977) first described glucose transport across
the chloroplast envelope using uptake experiments with
isolated spinach chloroplasts. At physiological concentrations,
the glucose inside the chloroplasts equilibrates very rapidly
with that of the external medium. This indicates a carrier
with a high capacity that most likely facilitates bidirectional
diffusion (Schäfer et al., 1977; Servaites & Geiger, 2002).
Trethewey and ap Rees (1994a) reported that a starch-excess
mutant of Arabidopsis (sex1; Caspar et al., 1991) is deficient in
the ability to transport glucose across the chloroplast envelope
and argued that this provided strong support for the role
of the transporter during starch breakdown (Trethewey & ap
Rees, 1994b). However, it has subsequently been discovered
that the mutation in sex1 lies in a gene encoding GWD (Yu
et al., 2001; see Section III). Rescue of the sex1 phenotype
through transformation with the wild-type GWD gene (Yu
et al., 2001), but not through transformation with the gene
that putatively encodes the glucose transporter (Weber et al.,
2000) shows that the apparent deficiency in chloroplast
glucose transport in sex1 is not the cause of the excess starch.
Thus, the consequence of mutation of the chloroplast glucose
transporter remains unknown and there is no direct evidence
that it is essential for normal starch degradation.
A maltose transporter has also been characterized biochemically, and the gene has recently been identified (Herold et al.,
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1981; Rost et al., 1996; Niittylä et al., 2004). The transporter
cannot translocate malto-oligosaccharides longer than maltose,
although they competitively inhibit maltose transport. Like
glucose, maltose inside the chloroplasts rapidly equilibrates
to the concentration in the medium, suggesting facilitated
diffusion across the membrane. However, glucose does not
inhibit maltose transport, indicating that the glucose and
maltose transporters are distinct proteins (Rost et al., 1996).
Discovery of the MEX1 gene (maltose excess; Niittylä et al.,
2004), which encodes the maltose transporter, revealed it to
be a remarkable, plant-specific protein with a structure unlike
any other sugar transporter characterized to date. Genome
sequences and expressed sequence tags (ESTs) reveal that
MEX1 homologues are present in angiosperms, gymnosperms
and mosses, and are expressed in both photosynthetic and
nonphotosynthetic tissues. Therefore, maltose export may be
a common feature of starch-storing plastids.
Mutation of MEX1 has established that this transporter
plays a major role in normal starch degradation. During
periods of starch breakdown, maltose accumulates to very high
levels in the mutant (Fig. 2b), presumably because it is confined to the chloroplast where it is not efficiently metabolized.
Starch degradation is reduced, leading to an excess of starch
and a slow-growing phenotype (Niittylä et al., 2004). Further
evidence that the maltose that accumulates in the mutant
derives from starch breakdown comes from examination of
double mutants lacking both MEX1 and the ability to make
starch (due to loss of plastidial phosphoglucomutase). The double
mutant plants are starchless and have very low maltose levels.
The mex1 mutant plants also have a pale green appearance
(Fig. 3). The fact that the starchless mex1 double mutants are
green suggests that the pale phenotype may be a consequence
of maltose accumulation in the chloroplast (Niittylä et al., 2004).
Taken together, recent work indicates that, at least in
Arabidopsis, most of the carbon from starch degradation is
exported as maltose (Niittylä et al., 2004; Weise et al., 2004).
A smaller fraction of the carbohydrate is probably exported in
the form of glucose, produced by the action of -enzyme on
maltotriose (see Section V). The phenotype of mex1/dpe1
Review
double mutants supports this conclusion. These plants accumulate both maltose and maltotriose and are very severely
impaired in their growth and development (Fig. 3; Niittylä
et al., 2004). It will be important to establish whether this
picture can be extended to the leaves of other plants. It is
possible that the pattern of starch degradation and the nature
of the exported metabolites varies from one species to another,
or even with developmental stage or environmental conditions (Zeeman et al., 2004).
VII. Metabolism of glucose and maltose
The products of starch degradation exported from the chloroplast are used for the synthesis of sucrose for export from the
leaf, and for cellular metabolism. Both of these fates presumably
require that the exported products be converted first to hexosephosphates. Glucose exported from the chloroplast is almost
certainly phosphorylated by hexokinase to form glucose-6phosphate. Multiple isoforms of hexokinase exist in plants
(Arabidopsis, for example, has six genes encoding hexokinaselike proteins). These are either soluble in the cytosol or associated
with different subcellular organelles, including chloroplasts.
In pea and spinach, most of the chloroplast-associated activity
is localized to the cytosolic face of outer envelope, via the
presence of an N-terminal hydrophobic membrane anchor
(Stitt et al., 1978; Wiese et al., 1999). This localization prompted
the suggestion that glucose, released from the chloroplast
via the glucose transporter, is immediately phosphorylated
by the outer-envelope-bound hexokinase, thereby maintaining
a concentration gradient and thus glucose efflux (Wiese et al.,
1999). However, it has also been reported recently that in the
moss Physcomitrella patens, most of the chloroplast-associated
hexokinase is soluble in the stroma. DNA sequence information indicates that higher plants may also contain plastidtargeted hexokinases, which lack N-terminal membrane anchors
and are encoded by a distinct gene family (Olsson et al., 2003).
The possible existence of stromal hexokinase in chloroplasts has
obvious implications for the metabolism of glucose produced
during starch degradation. If glucose were phosphorylated
Fig. 3 The pale, slow-growing phenotype of the maltose transporter mutant mex1 and the severe phenotype of the double mutant mex1/dpe1,
which also lacks D-enzyme. Wild-type (left), mex1 (centre) and dpe1/mex1 (right) plants were grown in long-day conditions (16 h light, 8 h
dark) and photographed at the same age and at the same scale. Bar, 1 cm. For further details, see Niittylä et al. (2004).
© New Phytologist (2004) 163: 247–261 www.newphytologist.org
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inside the plastid, it would have to be converted to triosephosphates and then exported on the triose-phosphate/
phosphate translocator as, under normal circumstances, there
is no hexose phosphate transporter on the chloroplast envelope
(see Section VI). Microarray data (http://nasc.nott.ac.uk/)
indicate that the two Arabidopsis hexokinase genes of this type
(Olsson et al., 2003) are expressed either at very low levels
or not at all in leaves. Nevertheless, it will be important to
establish whether any glucose produced from starch degradation is metabolized via this route.
The fate of maltose in the cytosol was, until recently,
completely unknown. Hydrolytic cleavage via the action of
α-glucosidase would be an obvious next step. However, results
from two independent teams studying Arabidopsis have
suggested that this is not the case and have revealed instead a
novel mechanism for maltose utilization involving a cytosolic
glucanotransferase (Chia et al., 2004; Lu & Sharkey, 2004).
The Arabidopsis genome contains two genes, DPE1 and DPE2,
encoding glucanotransferase-like proteins. DPE1 encodes
-enzyme, which is present in the chloroplast and metabolizes
maltotriose (see Section V; Critchley et al., 2001). The protein encoded by DPE2 lacks a chloroplast transit peptide and
is more similar in sequence to the bacterial enzyme amylomaltase which, in Escherichia coli, is required for the metabolism
of imported maltose (Boos & Shuman, 1998). Mutations in
the DPE2 gene in Arabidopsis lead to a massive accumulation
of maltose, which reaches levels up to 100 times that of the
wild type (Fig. 2b). Furthermore, dpe2 mutants accumulate
excess starch, have a reduced rate of growth and are slightly
pale in appearance (Chia et al., 2004; Lu & Sharkey, 2004).
The dpe2 phenotype is thus very similar to that of the mex1
mutant, which lacks the chloroplast envelope maltose transporter (see Section VI; Niittylä et al., 2004), except that the
amount of maltose accumulated in dpe2 is higher than in
mex1. Chia et al. (2004) demonstrated that the DPE2 protein
is cytosolic. This suggests strongly that DPE2 is responsible
for the metabolism of maltose exported from the chloroplast
via MEX1. It seems likely that in dpe2, maltose accumulates
in both the cytosol and the chloroplast, because maltose transport across the chloroplast envelope is bidirectional (Herold
et al., 1981; Rost et al., 1996). In mex1, however, maltose is most
likely confined to the chloroplast. This might explain the
observed differences between the two mutants in the degree of
maltose accumulation.
Experiments in vitro indicate that the DPE2 protein catalyses the transfer of a single glucosyl residue from maltose onto
a polyglucan acceptor (Chia et al., 2004), releasing the second
glucose. The enzyme is specific for maltose as a donor and can
use large polyglucans such as glycogen (or to a lesser extent
amylopectin) as acceptors for the transferred glucose. Neither
maltose nor other small linear oligosaccharides were suitable
acceptors. These results indicate that, in the cytosol, half of
the glucosyl units derived from maltose are released as glucose
but the other half are transferred to some form of acceptor.
The nature of the acceptor, and the way in which the glucosyl
units are subsequently released from it into the hexose
phosphate pool, are currently unknown. It is possible that a
glycogen-like glucan may exist in the cytosol, although there is
no experimental evidence for this. Alternatively, a heteroglycan
composed of several sugars, such as that described by Yang
and Steup (1990), could fulfil this acceptor role. Interestingly,
this polymer is known to be a good substrate for the cytosolic
isoform of α-glucan phosphorylase. Hypothetically, phosphorylase could liberate as glucose-1-phosphate the glucosyl
residues transferred from maltose to the acceptor glycan by
DPE2. It seems likely that these exciting questions will be
answered in the near future.
VIII. The emerging pathway of starch breakdown
and its regulation
Based on the arguments presented in this review, we propose
that the pathway of starch degradation in Arabidopsis leaves
may resemble that shown in Fig. 4. It should be emphasized
that the pathway might be different in other species, and might
also change in response to environmental or developmental
cues. Thus, the major flux may differ from that indicated, or
additional enzymes may be present or be induced. For example,
some of the data obtained from pea suggest that both αglucosidase and maltose phosphorylase are present in chloroplasts
(Kruger & ap Rees, 1983b; Sun et al., 1995), whereas in
Arabidopsis, there is no evidence for either their presence or
their involvement in starch breakdown (Chia et al., 2004; Lu
& Sharkey, 2004; Niittylä et al., 2004). Several areas require
further work to resolve outstanding questions. First, which
enzymes liberate glucans from the granule surface and exactly
how is GWD involved. It is possible that the synergistic action
of more than one type enzyme is required, as suggested for
granule degradation in cereal endosperm (Sun & Henson,
1990). Second, the significance and function of the multiple
isoforms of some enzymes, such as β-amylase and debranching
enzyme need to be clarified. Third, the cytosolic acceptor for
the maltose-metabolising glucosyl transferase (DPE2) needs
to be characterized, as do the enzymes involved in its further
metabolism.
Starch breakdown is controlled in a way that integrates the
release of carbohydrate with its subsequent utilization, principally for sucrose synthesis and respiration (Fondy & Geiger,
1982; Servaites et al., 1989b; Zeeman & ap Rees, 1999).
Evidence for this control comes from several sources, but very
little is known about the regulatory mechanisms themselves.
First, after the light to dark transition, an appreciable lag is
frequently observed before the onset of starch breakdown
(Gordon et al., 1980; Fondy & Geiger, 1982; Zeeman & ap
Rees, 1999). It has been suggested that depletion of leaf
sugars, rather than darkness, may be a significant factor in
triggering starch breakdown (Gordon et al., 1980; Zeeman &
ap Rees, 1999). If this is the case, sugar-sensing mechanisms
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Review
Fig. 4 Proposed pathway of starch breakdown in Arabidopsis leaves. The sizes of the arrows and of the metabolite names indicate our estimates
of the respective fluxes. Hatched arrows and/or question marks indicate steps where considerable uncertainty remains. The proteins represented
by the italic numbers are as follows: 1, glucan, water dikinase; 2, α-amylase; 3, isoamylase; 4, limit dextrinase; 5, chloroplastic α-glucan
phosphorylase; 6, β-amylase; 7, D-enzyme; 8, glucose transporter; 9, maltose transporter; 10, triose-phosphate/phosphate translocator;
11, cytosolic glucosyltransferase; 12, hexokinase; 13, cytosolic α-glucan phosphorylase.
(e.g. hexokinase-mediated signalling; Rolland et al., 2002;
Moore et al., 2003) may have a role to play. Second, during
periods of net starch synthesis, no breakdown is detectable
despite the presence of glucan-degrading enzymes (Kruger
et al., 1983; Li et al., 1992b; Zeeman et al., 2002b). This
implies that the degradative enzymes are regulated, although
there is little direct evidence to support this. However, the
induction of degradation in the light in some circumstances
indicates that the normal regulation can be overridden
(Kruger et al., 1983; Häusler et al., 1998). Third, in Arabidopsis,
the starch accumulated during the day is degraded at an
almost constant rate, such that it lasts the night. This implies
a mechanism that integrates both partitioning into starch and
the rate of its subsequent remobilization. Remarkably, this
appears to be true even in mutants that accumulate reduced
amounts of starch owing to a reduction in ADPglucose
pyrophosphorylase activity (Lin et al., 1988b). However, in
mutants that accumulate soluble glucan (phytoglycogen)
rather than starch this control of degradation is not observed
and phytoglycogen is depleted before the end of the night
(Zeeman et al., 1998a; T. Delatte and S. C. Zeeman, unpubl.
data). This suggests a regulatory mechanism that is dependent
on the presence of granular starch. For example, regulation
might be applied to the enzyme(s) that release soluble glucans
© New Phytologist (2004) 163: 247–261 www.newphytologist.org
from the starch granule, while the enzymes that subsequently
metabolize these glucans are essentially unregulated. Alternatively, factors intrinsic to the granule itself, such as the amount
of covalently bound phosphate, may dictate the rate of
degradation. Thus, the regulation of GWD activity could
control the overall rate of starch breakdown during the dark.
Fourth, it has been suggested that an accumulation of maltooligosaccharides might feedback on the release of glucans
from the granule because mutants with elevated maltooligosaccharides levels have a slower rate of starch breakdown.
However, it is not clear whether such a feedback mechanism
would be significant in controlling the fluxes in a wild-type
plant, because malto-oligosaccharides are usually present in
very small amounts.
There are indications that starch metabolism is entrained to
the circadian rhythm of the plant. Measurement of the starch
contents in the leaves of plants transferred from a diurnal
regime to continuous light have shown that starch synthesis
ceases or slows during the subjective night (Kerr et al., 1985;
Li et al., 1992b), and in one case, starch was even degraded
(Kruger et al., 1983). In Arabidopsis, neither of these patterns
was observed and starch accumulation proceeded at the same
rate as during the day (Zeeman et al., 2002a). However, microarray studies have shown that several Arabidopsis genes encoding
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starch-degrading enzymes (e.g. chloroplastic isoforms of
α-amylase, β-amylase and GWD) are under the influence of
the circadian clock. Gene expression peaks at the end of the
day, as might be expected for a protein involved in night-time
metabolism (Harmer et al., 2000; Schaffer et al., 2001). Yu
et al. (2001) showed that the amount of GWD protein (the
only one of the three so far shown to be required for starch
degradation) does not fluctuate appreciably. In the case of
α- and β-amylases it is not known whether the amounts of
protein or enzymatic activities change in the same way as
their transcripts. A diurnally fluctuating α-amylase activity
has been reported for Arabidopsis leaves (Kakefuda & Preiss,
1997), but it is not yet known which gene encodes it or where
it is localized. Entrainment to the circadian clock may serve to
prime the pathway of starch degradation, but it seems likely
that other post-transcriptional mechanisms such as allosteric
control, protein phosphorylation or redox-regulation may
initiate and control precisely the flux through the pathway.
Although little is known about this topic, the recent progress
in elucidating the pathway of starch degradation undoubtedly
provides an important basis from which to study the regulatory mechanisms that control it.
Acknowledgements
We are indebted to numerous research workers in our own
laboratories whose efforts have contributed much to our
current understanding. We gratefully acknowledge support
from the Swiss National Science Foundation (NCCR – Plant
Survival and Grant no. 3100-067312.01/1), the Biotechnology
and Biological Science Research Council (BBSRC) of the UK
(Grant Nos D11089 and D11090 and a core strategic grant
to the John Innes Centre), the Roche Research Foundation
and the Gatsby Charitable Foundation.
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