Available online at www.sciencedirect.com
Regulation of starch metabolism: the age of enlightenment?
Oliver Kötting1, Jens Kossmann2, Samuel C Zeeman1 and James R Lloyd2
Starch and sucrose are the primary products of photosynthesis
in the leaves of most plants. Starch represents the major plant
storage carbohydrate providing energy during the times of
heterotrophic growth. Starch metabolism has been studied
extensively, leading to a good knowledge of the numerous
enzymes involved. In contrast, understanding of the regulation
of starch metabolism is fragmentary. This review summarises
briefly the known steps in starch metabolism, highlighting
recent discoveries. We also focus on evidence for potential
regulatory mechanisms of the enzymes involved. These
mechanisms include allosteric regulation by metabolites, redox
regulation, protein–protein interactions and reversible protein
phosphorylation. Modern systems biology and bioinformatic
approaches are uncovering evidence for extensive posttranslational protein modifications that may underlie enzyme
regulation and identify novel proteins which may be involved in
starch metabolism.
cluding soluble and granule-bound starch synthases
(STS and GBS, respectively), branching enzymes
(SBE), the debranching enzymes isoamylase and limit
dextrinase (ISA and LDA, respectively), a-glucan phosphorylases (PHS), disproportionating enzymes (DPE),
a-amylases (AMY) and b-amylases (BAM). In most
cases, multiple genes encode different isoforms of each
enzyme, which may have slightly different roles
depending on plant species and tissue. We illustrated
this complex interplay in our model (Figure 1) which
summarises previously published concepts (for review
see [1–6]) and includes the concepts discussed in the
present review. Despite our present knowledge of the
core enzymatic reactions, our understanding of the
regulation of the biosynthetic and degradative pathways
is far from complete.
Addresses
1
Institute of Plant Sciences, ETH Zurich, Universitaetsstr. 2, CH-8092
Zurich, Switzerland
2
Institute of Plant Biotechnology, Dept. Genetics, University of
Stellenbosch, Private Bag X1, Matieland 7602, South Africa
Here we focus on recent discoveries of the regulation of
starch synthesis and breakdown. We will summarise
current evidence for the control of starch metabolism
and then discuss mechanisms that were recently
suggested to be involved in the regulation of the pathway
including reversible starch phosphorylation, redox regulation and protein complex formation initiated by protein
phosphorylation.
Corresponding author: Kötting, Oliver ([email protected]), Kossmann,
Jens ([email protected]), Zeeman, Samuel C ([email protected])
and Lloyd, James R ([email protected])
Current Opinion in Plant Biology 2010, 13:321–329
This review comes from a themed issue on
Physiology and metabolism
Edited by Uwe Sonnewald and Wolf B. Frommer
Available online 18th February 2010
1369-5266/$ – see front matter
# 2010 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.pbi.2010.01.003
Introduction
Starch is the major higher plant storage carbohydrate and is
made up of the glucose polymers amylose and amylopectin. Plants use starch as an energy store when they cannot
generate enough energy through photosynthesis, such as in
leaves during the dark period. Moreover, starch represents
a cornerstone for human and animal nutrition and a feedstock for many industrial applications, including bioethanol production. Despite its simple composition, starch
forms complex semi-crystalline structures, starch granules,
which accumulate in plastids.
Starch metabolism in higher plants involves the concerted and controlled actions of many enzymes inwww.sciencedirect.com
Evidence for the control of starch metabolism
In plant storage organs, starch synthesis and degradation
can be developmentally separated (e.g. in the developing
and germinating cereal grain). In other tissues, such as
leaves, or in unicellular algae, these processes happen on a
diurnal basis and in a regulated manner. The amount of
photoassimilate partitioned into leaf starch varies between plant species. Whilst in some plants, like Arabidopsis, it is the major storage form [7–9], others
accumulate sucrose [7,10], raffinose-family oligosaccharides [11] or fructans [12] in addition to starch and sometimes to an even greater extent. In Arabidopsis, around
half of the photoassimilate is partitioned at a linear rate
into starch during the day [9]. Starch degradation in the
following night provides carbon for respiration and also
occurs at a nearly linear rate. Partitioning is affected by
developmental and source/sink effects, as well as by
environmental factors including light intensity, CO2 concentrations and photoperiod [13–17,18]. The effect of
photoperiod on the diurnal pattern of starch accumulation
has been particularly well studied ([18] and references
therein). Arabidopsis plants in a short photoperiod have a
higher rate of starch synthesis and a decreased rate of
starch degradation compared with plants grown in a long
photoperiod, presumably to adapt to the prolonged dark
period [18].
Current Opinion in Plant Biology 2010, 13:321–329
322 Physiology and metabolism
Figure 1
mensurate with the altered rate of starch degradation [17].
In contrast, most transcripts encoding starch synthesising
enzymes did not show a diurnal pattern except for two
starch synthase genes (GBS1 and STS2) [20]. Although
these studies clearly demonstrate the transcriptional
regulation of genes involved in starch metabolism, in
the cases where protein levels have also been studied,
they did not change in the same way, thus suggesting
additional regulatory mechanisms at the post-transcriptional level.
Reversible glucan phosphorylation
It has recently been shown that reversible phosphorylation
of the starch granule is essential for its subsequent degradation ([21–23,24], reviewed in [2,3,5,6]). Within starch
granules, glucan chains of the amylopectin fraction form
double helices which pack to form crystalline lamellae. It is
thought that during starch degradation, the lamellae and
the double helices at the granule surface become disrupted
by the addition of phosphate groups at the C6-position and
C3-position of individual glucosyl residues. Phosphate
addition is mediated by two enzymes, glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD),
respectively [25–28] (Figure 1b).
Evidence for the importance of starch phosphorylation in
vivo comes from the analysis of gwd1 (sex1) mutant plants.
These plants exhibit strongly reduced growth and
accumulate nearly phosphate-free starch to very high
levels [22,29]. The phenotype of pwd null mutants is
similar, but less severe, and pwd starch only lacks C3bound phosphate. These observations are consistent
with the idea that PWD acts downstream of GWD
[26–28].
Model for the regulation of starch synthesis and degradation in
Arabidopsis. Simplified cartoon of the pathways of starch synthesis (a)
and degradation (b) in Arabidopsis chloroplasts based on previously
published concepts [1–6]. Evidence for the (potential) regulatory
mechanisms described in this review is depicted with superscript red
letters: R, redox; C, complex formation; P, protein phosphorylation.
Regulation by metabolites is indicated as green dotted arrows
(stimulation) and magenta dotted lines (inhibition). Protein names are
shown in blue, metabolites in black and cellular compartments and
constituents in grey. ADG, AGPase; Fru-6-P, fructose-6-phosphate; Glc1-P, glucose-1-phosphate; Glc-6-P, glucose-6-phosphate; GLT,
glucose transporter; MEX, maltose transporter; PGI,
phosphoglucoisomerase; PGM, phosphoglucomutase. Note that the
pathways in other species and tissues might be different. For example,
in cereal endosperm AGPase activity is mostly localised to the cytosol,
and ADP-Glc is transported to the amyloplast by Brittle-1-like
transporters.
It was reported that transcript levels of many enzymes
involved in starch mobilisation show a diurnal pattern in
leaves [17,19,20], and that the expression of some of these
transcripts was under light-dependent circadian control
[17]. Alteration of the photoperiod led to rapid changes in
the transcript levels of starch degradative enzymes comCurrent Opinion in Plant Biology 2010, 13:321–329
The proposed disruption of the granule surface by phosphate esters would allow access by enzymes of starch
degradation such as b-amylase (BAM1 and BAM3) and
isoamylase (ISA3) [24,30] (Figure 1b). Hansen et al.
[31] used molecular dynamics simulations and NMR
spectroscopy to show that phosphate (particularly C3bound) induces structural changes, potentially disrupting
the amylopectin double helices. In addition, two in vitro
studies using crystalline maltodextrins as models for
starch granules revealed that GWD-mediated glucan
phosphorylation caused substantial solubilisation of the
constituent glucan chains [32,33]. The importance of the
disruption for starch degradation is highlighted by experiments in which phosphate-free starch granules isolated
from gwd1 mutants were incubated with recombinant
enzymes. Glucan release from the granules by b-amylase
(either BAM1 or BAM3) and isoamylase (ISA3) was
strongly increased upon simultaneous starch phosphorylation by GWD [30].
Many metabolic pathways are characterised by highly
controlled committed steps. Thus, the starch degradation
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Regulation of starch metabolism Kötting et al. 323
Table 1
Starch-related proteins with potential redox regulation
Enzymes with experimental evidence for redox regulation
Enzyme name
Gene name
ADP-Glc pyrophosphorylase
ADP-Glc pyrophosphorylase
ADP-Glc pyrophosphorylase
AGPB
AGPB
AGPB
Glucan, water dikinase
Phosphoglucan phosphatase
b-Amylase 1
GWD
SEX4, DSP4
BAM1
Species
Tissue/source
Reference
Solanum tuberosum
Solanum tuberosum
Arabidopsis thaliana,
Spinacia oleracea,
Solanum tuberosum
Solanum tuberosum
Arabidopsis thaliana
Arabidopsis thaliana
Heterologous expression (E. coli)
Tubers
Leaves
[41]
[43,44]
[45]
Heterologous expression (E. coli)
Heterologous expression (E. coli)
Heterologous expression (E. coli)
[47]
[35]
[49]
Proteins identified as potential Trx targets in proteomics screens
Protein name
b-Amylase
b-Amylase
a-Glucan phosphorylase (plastidial)
ADP-Glc translocator (Brittle-1)
Starch branching enzyme IIa
Species
Tissue/source
Reference
Spinacia oleracea
Hordeum vulgare
Triticum aestivum
Triticum aestivum
Triticum aestivum
Chloroplast stroma
Germinated seed embryos
Amyloplast
Amyloplast
Amyloplast
[73]
[74]
[75]
[75]
[75]
pathway may be controlled at the attack at the granule
surface, potentially through the regulation of GWD and
PWD. Whilst there is currently no evidence for the
regulation of PWD activity, several post-translational
mechanisms might regulate GWD as it was shown to
be redox-regulated (discussed below; Table 1) and phosphorylated (Table 2).
Recent work showed that the phosphorylation of starch is
reversible, and that removal of the phosphate groups
introduced by GWD and PWD is also essential for normal
starch breakdown. Arabidopsis plants lacking the plastidial, glucan-binding, phosphoglucan phosphatase SEX4
(also designated as PTPKIS1 [34] and DSP4 [35]) exhibit
reduced rates of starch degradation [24,35–37] and
accumulate soluble phospho-oligosaccharides during
the night [24]. In vitro studies showed that recombinant
SEX4 protein removes phosphate from different phosphoglucans including solubilised amylopectin [38], crystalline maltodextrins [78], native starch granules and
phospho-oligosaccharides [24]. No preference for the
removal of C6-bound or C3-bound phosphate could be
observed (M Hejazi et al., unpublished). The importance
of phosphate removal most likely stems from the fact that
BAMs cannot degrade phosphoglucans completely —
they are exo-acting enzymes that release maltose from
the non-reducing ends of glucan chains, but stop one or
two residues before a phosphate group [24,39]. After
phosphatase treatment, the soluble phospho-oligosaccharides found in sex4 mutants were completely degradable by BAM [24]. Intriguingly, a recent modelling
study suggested that phosphorylation of malto-oligosaccharides at the C3-position may increase binding affinity
to a potato b-amylase (PCT-BMYI), potentially inhibiting the enzyme [40]. In Arabidopsis wild-type plants,
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however, soluble phospho-oligosaccharides were not
detectable, indicating that steady-state levels are very
low [24]. Thus, a regulatory role in vivo remains questionable. There is preliminary evidence for the regulation
of SEX4 by product inhibition. Recombinant SEX4
activity was inhibited in vitro by non-phosphorylated
soluble malto-oligosaccharides at sub-millimolar concentrations (M Hejazi et al., unpublished). In addition, SEX4
was reported to be influenced by the redox status [35] (see
below).
Recently, a second putative phosphatase, LSF1 (for Like
SEX Four 1; At3g01510), has been shown to be required
for starch degradation in Arabidopsis [79]. Mutants lacking LSF1 have a starch-excess phenotype. However, it
has not yet been shown that LSF1 is an active phosphatase and, unlike sex4 plants, lsf1 plants do not accumulate
phospho-oligosaccharides. Interestingly, recent data
indicated that LSF1 interacts with BAM1 in Arabidopsis
leaves (M Umhang et al., unpublished). Although the
significance is not yet clear, this physical interaction
could potentially indicate that LSF1 acts in a regulatory
manner.
Redox regulation
The activities of several enzymes involved in starch
metabolism have been demonstrated to be affected by
reducing or oxidising conditions, indicating that they may
be regulated by the redox potential of the plastid stroma
[35,41–49,50]. A summary of the knowledge gained from
these studies is shown in Table 1. Much research in this
area has focused on the control of ADP-glucose pyrophosphorylase (AGPase), because this is the first enzyme on
the committed pathway of starch synthesis (Figure 1a).
Higher plants AGPase is a heterotetramer consisting of
Current Opinion in Plant Biology 2010, 13:321–329
324 Physiology and metabolism
Table 2
Phosphoproteins involved in starch metabolism
Arabidopsis proteins involved in starch metabolism that are phosphorylateda,b
Enzyme
AGI
Phosphorylated peptide c
Reference
VLIAEGNCG(pS)PR
ANGGFIM(s)A(s)HNPGGPE(y)DWGIK
VG(pS)NVQLK
IYVLTQFN(pS)A(pS)LNR
1. AED(pT)PKLYYNK
2. MEATDDE(pS)(pS)HVK(pT)TAK
TPFVKSGGN(pS)HLK
AVHSGADLESAIDTFL(pS)P(s)K
1. EL(pS)LHSIG(pS)K
2. FNIED(t)(s)(s)FQDLDDH(pS)K
SVGV(pS)SMNK
1. (s)GE(oxM)(t)D(s)(s)LL(s)I(pS)PP(s)AR
2. AHG(t)DP(pS)PPM(pS)PILGA(t)R
3. (pT)(pY)REGGIGGK
YDS(pS)AFGQVVATNR
ATIVNAK(pS)AIG(pS)LR
AQAS(pS)DGDEEEAIPLRSEGK
SINSE(s)D(s)DSDFPHENQQGNPGLGK
[69]
[69]
[69]
[70]
[69]
Gene name
Phosphoglucoisomerase
Phosphoglucomutase
AGPase (large subunit)
AGPase (small subunit)
Starch synthase III
At4g24620
At5g51820
At5g19220
At5g48300
At1g11720
PGI1
PGM1
APL1
APS1
STS3, SSIII
Glucan, water dikinase 1
Glucan, water dikinase 2
Transglucosidase (DPE2)
At1g10760
At4g24450
At2g40840
GWD1, SEX1
GWD2
DPE2
a-Amylase 3
b-Amylase 1
At1g69830
At3g23920
AMY3
BAM1, BMY7
b-Amylase 3
Limit dextrinase
Glucose transporter
Maltose transporter
At4g17090
At5g04360
At5g16150
At5g17520
BAM3, BMY8, ctBMY
LDA1
GLT1
MEX1
[69]
[69]
[69]
[69]
[69]
[70]
[70]
[69]
[69]
Proteins involved in starch metabolism from other species known to be phosphorylated
Protein
Phosphoglucomutase (plastidial isoform)
Starch branching enzyme I
Starch synthase II
Starch branching enzymes II and IIb
Granule-bound starch synthase
a-Glucan phosphorylase (plastidial isoform)
Starch branching enzyme IIb
Species
Reference
Pisum sativum
Triticum aestivum
Triticum aestivum
Triticum aestivum
Zea mays
Zea mays
Zea mays
[76]
[63]
[62]
[62,63]
[77]
[77]
[77]
a
The PhosPhAt database (phosphat.mpimp-golm.mpg.de, [69]) was searched for phosphorylated peptides from 43 Arabidopsis proteins either
known, or thought to be involved in starch metabolism. Data were also added from a recent study [70] not present in the PhosPhAt database to date.
b
29 of the 43 Arabidopsis proteins were not shown to be phosphorylated: AGPase large subunits 2 (At1g27680, APL2), 3 (At4g39210, APL3) and 4
(At2g21590, APL4); AGPase small subunit-like (At1g05610, APS2); starch synthases I (At5g24300, STS1, SSI), II (At3g01180, STS2, SSII) and IV
(At4g18240, STS4, SSIV); granule-bound starch synthase (At1g32900, GBS1, GBSS); starch branching enzymes I (At3g20440, SBE1), II (At5g03650,
SBE2) and III (At2g36390, SBE3); isoamylases I (At2g39930, ISA1), II (At1g03310, ISA2, DBE1) and III (At4g09020, ISA3); phosphoglucan, water
dikinase (At5g26570, PWD, GWD3); glucanotransferase (At5g64860, DPE1); a-glucan phosphorylase (plastidial, At3g29320, PHS1); a-amylases I
(At4g25000, AMY1) and II (At1g76130, AMY2); b-amylases 2 (At4g00490, BAM2, BMY9), 4 (At5g55700, BAM4), 5 (At4g15210, BAM5, BMY1, RAM1),
6 (At2g45880, BAM6), 7 (At2g45880, BAM7), 8 (At5g45300, BAM8) and 9 (At5g18670, BAM9, BMY3); SEX4 (At3g52180, SEX4); Like Sex Four 1
(At3g01510, LSF1); starch binding coiled-coil protein (At5g39790, COC).
c
The nomenclature for the phosphorylated peptides is as follows: confirmed or validated phosphorylated amino acids are capitalised, delineated by
brackets and the amino acid is preceded with a lower case p, for example (pS). Amino acids where the phosphorylation is ambiguous are enclosed in
brackets and are in lower case. All potential candidates are shown, for example (t)(s). Where the same peptide was examined several times all
identified sites are shown and where there was a conflict between ambiguous and validated amino acids it is shown as being validated.
two large and two small subunits. It has long been known
that AGPase is allosterically activated by 3-phosphoglycerate (3-PGA) and other glycolytic intermediates, and
inhibited by orthophosphate (Pi) and ADP [51,52]. Regulation by redox affects the small subunits which, under
oxidising conditions, becomes covalently linked via an
intermolecular disulfide bridge, thus forming a stable
dimer within the heterotetramer ([41,43–45], for review
see [53]). Dimerisation markedly decreases the activity of
the enzyme and alters its kinetic properties, making it less
sensitive to activation by 3-PGA and increasing its KM for
ATP [44,45]. Reductive activation monomerises the small
subunits, activating the enzyme, which is then sensitive
to fine control through its aforementioned allosteric reguCurrent Opinion in Plant Biology 2010, 13:321–329
lation. The result of this redox sensitivity in chloroplasts
is to increase the activity of the AGPase during the light
period (when starch is made) and decrease it during the
dark period (when starch is degraded). There is also some
evidence for redox affecting AGPase activity in potato
tuber amyloplasts. It has been demonstrated that when
potato tuber slices were incubated with dithiothreitol the
AGPase became monomerised, and when tubers were
detached from the plant they stopped synthesising starch
because of redox inactivation of AGPase [44]. It is unclear
though whether redox plays any role in determining the
rate of starch synthesis in amyloplasts under normal
growing conditions. The redox activation was suggested
to be mediated by thioredoxin (Trx), itself reduced
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Regulation of starch metabolism Kötting et al. 325
through photosynthesis, or by NADP-thioredoxin
reductase [43,50]. Interestingly, other proteins involved
in starch metabolism have been identified as potential
Trx targets in recent proteomic screens (Table 1). It has
also been shown that redox activation can occur in
response to sugars, and that trehalose 6-phosphate may
serve as an intermediate in the sucrose-dependent pathway [46,48].
Enzymes involved in starch phosphate metabolism are
affected by the redox potential. Both GWD and SEX4
proteins are activated under reducing conditions [35,47].
In addition, an isoform of b-amylase (BAM1) has also
been demonstrated to be active when reduced, but not
when oxidised [49]. At one level, such regulation is
counter-intuitive because the chloroplast stroma is generally considered to be a more reducing environment when
plants photosynthesise. This implies that these enzymes
would be more active during periods of starch synthesis
and not degradation. As such, this conflicts with data
showing that the rate of starch phosphorylation is greater
during periods of net starch degradation than synthesis in
chloroplasts [54], and physiological experiments that provide good evidence that SEX4 is active during the dark
period [24].
Protein complex formation and protein
phosphorylation
The roles of the many proteins known to be involved in
synthesising and degrading starch have often been considered in isolation. A recent paper, however, has demonstrated that two STS isoforms in Arabidopsis are probably
responsible for initiating starch granule synthesis as when
both are mutated starch synthesis is eliminated [55],
when a double mutant lacking two SBE isoforms was
produced starch accumulation was also eliminated [56].
To understand starch metabolism fully, therefore, the
physical and biochemical interactions between isoforms
need to be assessed. This has become especially important with the discovery that at least some of these proteins
form complexes with each other (both multimeric
enzymes and multi-enzyme complexes). The roles of
such complexes are beginning to be assessed. In Arabidopsis and potato, for example, two isoamylase isoforms
(ISA1 and ISA2) form a complex with each other. When
either isoform is mutated the complex cannot form and
the other subunit is also decreased in abundance, or
absent. Plants mutated in either gene synthesise aberrant
glucans; soluble phytoglycogen and reduced amounts of
insoluble starch [57–59].
Multi-enzyme complexes have been identified in wheat
and maize endosperm during the period of grain filling
(Table 3). These complexes contain known starch biosynthetic enzymes such as starch synthase and branching
enzyme isoforms as well as enzymes previously unknown
to be involved in starch synthesis (Table 3) [60,61–63].
Interestingly, some of the subunits have been demonstrated to be phosphorylated [60,62,63] and complex
formation has been proposed to be dependent on protein
phosphorylation (mediated by an as-yet unidentified
protein kinase). Dephosphorylation of complex components by the addition of alkaline phosphatase results
in subunit disassociation. It is tempting to believe that the
formation of such complexes is functionally significant. It
is plausible that different combinations of physically
associated enzymes could produce specific structures
within the starch granule that would not be produced
by the free, soluble enzymes. However, such functional
interactions have not yet been demonstrated, so their
roles remain to be assessed.
A number of enzymes identified in multi-enzyme complexes do not have defined roles in starch synthesis. For
example, a-glucan phosphorylase was found in a complex
in wheat and maize [63,64] together with SBE isoforms.
Although this enzyme has often been considered a starch
degradative enzyme, a phosphorylase deficient mutant in
rice has altered starch structure, indicating that it is
involved in synthesis. Nevertheless, its precise role in
the putative complexes — and in starch metabolism in
general — requires further study [65]. Pyruvate, phosphate dikinase (PPDK) and sucrose synthase (SuSy) were
also identified in a complex from maize [60] and it has
been speculated that they could influence starch synthesis by affecting substrate supply. However, there is as
yet no evidence to suggest that SuSy is located in the
amyloplast stroma, and its association with the starch
biosynthetic enzymes may occur during the extraction
procedure. PPDK does occur in the plastid stroma, however and, like AGPase, produces PPi. The presence of PPi
would thermodynamically inhibit AGPase and, although
Table 3
Starch enzyme complex formation in cereal endosperm
Species
Approximate size of protein complex (kDa)
Proteins present in complex a
Triticum aestivum
Triticum aestivum
Zea mays
Zea mays
Zea mays
Not determined
260
600
300
300
BEI, BEIIb, a-glucan phosphorylase
SSI, SSIIa, BEIIa or BEIIb
SSIIa, SSIII, BEIIa, BEIIb, PPDK, AGPase
SSIIa, BEIIa, BEIIb, a-glucan phosphorylase
SSI, SSIIa, BEI, BEIIa, a-glucan phosphorylase
a
Reference
[63]
[62]
[60,61]
[61]
[64]
BE, starch branching enzyme; SS, starch synthase.
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Current Opinion in Plant Biology 2010, 13:321–329
326 Physiology and metabolism
a plastidially localised pyrophosphatase is known to
degrade most PPi in that compartment, it was speculated
that PPDK might influence AGPase activity by metabolic
channelling of PPi [60].
The complexes found in cereal endosperm often contain
the starch synthase isoform SSIII (Table 3). This starch
synthase contains a coiled-coil protein domain [60], which
is known to be involved in protein–protein interactions. A
recent study that examined the presence of other coiledcoil domains in Arabidopsis proteins and identified a protein
(At5g39790) that is expressed in a diurnal manner at both
the mRNA and protein levels [66]. This protein also
contains a carbohydrate-binding module and was shown
to bind to starch in vitro [66]. The same study identified
several other starch metabolic enzymes that have predicted
coiled-coil domains (GBS1, STS1, STS2, STS3, STS4,
AMY3 and GWD1). It was hypothesised that the
At5g39790 protein might act as a regulatory scaffold for
the formation of enzyme complexes involving other coiledcoil domain proteins [66].
Another starch binding protein recently proposed to have
a regulatory role in starch metabolism is the b-amylaselike protein, BAM4. Mutations in this protein repress
starch degradation, yet BAM4 has no measurable catalytic
activity [67]. It was originally proposed that this effect
was caused by the interactions with other starch degradative enzymes, although attempts to isolate interacting
partners have not yet been successful [68]. As BAM4
binds to starch granules [68], it can be speculated that it is
this property, which is somehow important for its role in
starch degradation.
of that work tested these predictions using GFP fusions on
a subset of the proteins and found that less than 50% of
these actually targeted to the chloroplast. This demonstrates the need for more information about the subcellular
localisation of these proteins. Nevertheless, it is likely that
the analysis of Arabidopsis mutants lacking bona fide plastidial protein kinases and phosphatases will enable identification of specific interactions between them and starch
metabolic enzymes. One such protein kinase thought to be
involved in post-translational modifications of starch metabolic enzymes is MsK4 from Medicago sativa, the primary
protein sequence of which shows similarity to mammalian
glycogen synthase kinases. When this protein was
expressed in Arabidopsis it led to increased starch and
maltose accumulation, and rendered the plants more resistant to salt stress [72].
Conclusions
In this review we have examined recent advances in our
understanding of the regulation of starch metabolism.
These data are beginning to enlighten us, but much still
needs to be understood. For example, it is unclear for
many of the mechanisms discussed whether they play a
role in vivo, so more work is needed to confirm their
significance. If these mechanisms do play a role, then they
have to be integrated with variables known to influence
starch metabolism, such as sugar supply.
Acknowledgements
The authors would like to acknowledge the funding from the Swiss-South
African Joint Research Programme 08 IZ LS Z3122916 and the ETH
Zurich. We also thank Martin Umhang (ETH Zurich) for providing
unpublished data.
References and recommended reading
As mentioned, some of the protein complexes described
above are reliant on protein phosphorylation for their
assembly. Furthermore, phosphorylation is a common
means for the post-translational regulation of protein
activity. In a systems biology approach, several studies
have identified Arabidopsis proteins that are phosphorylated and the data from these have been curated in the
PhosPhAt database (http://phosphat.mpimp-golm.mpg.de)
[69]. We surveyed this database and identified 12 proteins
involved in starch metabolism that are phosphoproteins. A
further three were recently identified in a study on plastidial phosphoproteins (Table 2) [70]. Table 2 also contains
data on phosphoproteins involved in starch metabolism
identified in other species. Although the consequences of
phosphorylation on protein function are not known in most
of these cases, it is reasonable to suppose that some of these
modifications are regulatory in nature. This suggests that
plastid-localised protein kinases and protein phosphatases
may have a significant role in controlling starch metabolism. A survey of Arabidopsis protein kinases and phosphatases using computer programmes to identify transit
peptides indicated 45 kinases and 21 phosphatases as
potentially being localised in the plastids [71]. The authors
Current Opinion in Plant Biology 2010, 13:321–329
Papers of particular interest, published within the period of review,
have been highlighted as:
of special interest
of outstanding interest
1.
Ball SG, Morell MK: From bacterial glycogen to starch:
understanding the biogenesis of the plant starch granule.
Annu Rev Plant Biol 2003, 54:207-233.
2.
Lloyd JR, Kossmann J, Ritte G: Leaf starch degradation comes
out of the shadows. Trends Plant Sci 2005, 10:130-137.
3.
Fettke J, Eckermann N, Kötting O, Ritte G, Steup M: Novel starchrelated enzymes and carbohydrates. Cell Mol Biol 2006,
52(Suppl):OL883-OL904.
4.
Lu Y, Sharkey T: The importance of maltose in transitory starch
breakdown. Plant Cell Environ 2006, 29:353-366.
5.
Zeeman SC, Delatte T, Messerli G, Umhang M, Stettler M,
Mettler T, Streb S, Reinhold H, Kötting O: Starch breakdown:
recent discoveries suggest distinct pathways and novel
mechanisms. Funct Plant Biol 2007, 34:465-473.
6.
Zeeman SC, Kossmann J, Smith AM: Starch; its metabolism,
evolution and biotechnological modification in plants. Annu
Rev Plant Biol 2010, in press, doi:10.1146/annurev-arplant042809-112301.
7.
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Phaseolus vulgaris L. during square and sinusoidal light
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Regulation of starch metabolism Kötting et al. 327
8.
Upmeyer DJ, Koller HR: Diurnal trends in net photosynthetic
rate and carbohydrate levels of soybean leaves. Plant Physiol
1973, 51:871-874.
9.
Zeeman SC, ap Rees T: Changes in carbohydrate metabolism
and assimilate export in starch-excess mutants of
Arabidopsis. Plant Cell Environ 1999, 22:1445-1453.
excess 4 is a laforin-like phosphoglucan phosphatase
required for starch degradation in Arabidopsis thaliana. Plant
Cell 2009, 21:334-346.
This study shows for the first time that glucan dephosphorylation is crucial
for starch metabolism. Arabidopsis mutants lacking the phosphoglucan
phosphatase SEX4 accumulate phosphorylated intermediates of starch
breakdown and are impaired in starch degradation.
10. Stitt M, Gerhardt R, Kürzel B, Heldt HW: A role for fructose 2,6bisphosphate in the regulation of sucrose synthesis in spinach
leaves. Plant Physiol 1983, 72:1139-1141.
25. Ritte G, Lloyd JR, Eckermann N, Rottmann A, Kossmann J,
Steup M: The starch-related R1 protein is an a-glucan, water
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11. Bachmann M, Matile P, Keller F: Metabolism of the raffinose
family oligosaccharides in leaves of Ajuga reptans L. Coldacclimation, translocation, and sink to source transition:
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26. Baunsgaard L, Lütken H, Mikkelsen R, Glaring MA, Pham TT,
Blennow A: A novel isoform of glucan, water dikinase
phosphorylates pre-phosphorylated a-glucans and is
involved in starch degradation in Arabidopsis. Plant J 2005,
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12. Pollock CJ, Cairns AJ: Fructan metabolism in grasses and
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27. Kötting O, Pusch K, Tiessen A, Geigenberger P, Steup M, Ritte G:
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16. Gibon Y, Bläsing OE, Palacios-Rojas N, Pankovic D, Hendriks JH,
Fisahn J, Höhne M, Günther M, Stitt M: Adjustment of diurnal
starch turnover to short days: depletion of sugar during the
night leads to a temporary inhibition of carbohydrate
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activation of ADP-glucose pyrophosphorylase in the following
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17. Lu Y, Gehan J, Sharkey T: Daylength and circadian effects on
starch degradation and maltose metabolism. Plant Physiol
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18. Gibon Y, Pyl ET, Sulpice R, Lunn JE, Höhne M, Günther M, Stitt M:
Adjustment of growth, starch turnover, protein content and
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Extensive metabolomics analyses reveal the importance of the photoperiod for the regulation of central metabolism. When grown in very short
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19. Harmer S, Hogenesch J, Straume M, Chang H, Han B, Zhu T,
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20. Smith SM, Fulton DC, Chia T, Thorneycroft D, Chapple A,
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28. Ritte G, Heydenreich M, Mahlow S, Haebel S, Kötting O, Steup M:
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29. Smith AM, Stitt M: Coordination of carbon supply and plant
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30. Edner C, Li J, Albrecht T, Mahlow S, Hejazi M, Hussain H, Kaplan F,
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The authors describe a stimulatory effect of starch phosphorylation on
starch degradation by b-amylases. This effect is mutualistic as the activity
of b-amylases also stimulates starch phosphorylation by glucan, water
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31. Hansen PI, Spraul M, Dvortsak P, Larsen FH, Blennow A,
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32. Hejazi M, Fettke J, Haebel S, Edner C, Paris O, Frohberg C,
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crystalline maltodextrins and thereby initiates solubilization.
Plant J 2008, 55:323-334.
This work is essentially a characterization of crystalline maltodextrins, a
potent artificial substrate to study starch degradation in vitro. Moreover,
the authors show that phosphorylation of the crystalline maltodextrins
leads to the solubilisation of phosphorylated as well as non-phosphorylated glucan chains.
33. Hejazi M, Fettke J, Paris O, Steup M: The two plastidial starchrelated dikinases sequentially phosphorylate glucosyl
residues at the surface of both the A- and B-allomorph of
crystallized maltodextrins but the mode of action differs. Plant
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34. Fordham-Skelton AP, Chilley P, Lumbreras V, Reignoux S,
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plant protein tyrosine phosphatase interacts with SNF1related protein kinases via a KIS (kinase interaction sequence)
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21. Lorberth R, Ritte G, Willmitzer L, Kossmann J: Inhibition of a
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35. Sokolov LN, Dominguez-Solis JR, Allary AL, Buchanan BB,
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22. Yu TS, Kofler H, Häusler RE, Hille D, Flügge U-I, Zeeman SC,
Smith AM, Kossmann J, Lloyd J, Ritte G et al.: The Arabidopsis
sex1 mutant is defective in the R1 protein, a general regulator
of starch degradation in plants, and not in the chloroplast
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36. Zeeman SC, Northrop F, Smith AM, ap Rees T: A starchaccumulating mutant of Arabidopsis thaliana deficient in a
chloroplastic starch-hydrolysing enzyme. Plant J
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23. Nashilevitz S, Melamed-Bessudo C, Aharoni A, Kossmann J,
Wolf S, Levy AA: The legwd mutant uncovers the role of starch
phosphorylation in pollen development and germination in
tomato. Plant J 2009, 57:1-13.
37. Niittylä T, Comparot-Moss S, Lue W-L, Messerli G, Trevisan M,
Seymour MDJ, Gatehouse JA, Villadsen D, Smith SM, Chen J
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in plants and glycogen metabolism in mammals. J Biol Chem
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24. Kötting O, Santelia D, Edner C, Eicke S, Marthaler T, Gentry MS,
Comparot-Moss S, Chen J, Smith AM, Steup M et al.: Starch-
38. Gentry MS, Dowen RH, Worby CA, Mattoo S, Ecker JR, Dixon JE:
The phosphatase laforin crosses evolutionary boundaries and
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Current Opinion in Plant Biology 2010, 13:321–329
328 Physiology and metabolism
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39. Takeda Y, Hizukuri S: Studies on starch phosphate. Part 5. Reexamination of the action of sweet-potato beta-amylase on
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40. Dudkiewicz M, Siminska J, Pawlowski K, Orzechowski S:
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41. Fu YB, Ballicora MA, Leykam JF, Preiss J: Mechanism of
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42. Cho MJ, Wong JH, Marx C, Jiang W, Lemaux PG, Buchanan BB:
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43. Ballicora MA, Frueauf JB, Fu YB, Schurmann P, Preiss J:
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44. Tiessen A, Hendriks JH, Stitt M, Branscheid A, Gibon Y, Farré EM,
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45. Hendriks JH, Kolbe A, Gibon Y, Stitt M, Geigenberger P: ADPglucose pyrophosphorylase is activated by posttranslational
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46. Kolbe A, Tiessen A, Schluepmann H, Paul M, Ulrich S,
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47. Mikkelsen R, Mutenda KE, Mant A, Schürmann P, Blennow A: aGlucan, water dikinase (GWD): a plastidic enzyme with redoxregulated and coordinated catalytic activity and binding
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48. Lunn JE, Feil R, Hendriks JH, Gibon Y, Morcuende R, Osuna D,
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increases in trehalose 6-phosphate are correlated with redox
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49. Sparla F, Costa A, Lo Schiavo F, Pupillo P, Trost P: Redox
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50. Michalska J, Zauber H, Buchanan BB, Cejudo FJ, Geigenberger P:
NTRC links built-in thioredoxin to light and sucrose in
regulating starch synthesis in chloroplasts and
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9908-9913.
The authors show that NADP-thioredoxin reductase C (NTRC) activates
AGPase in response to light and sucrose in planta. In non-photosynthetic
tissue of mutant plants starch synthesis in response to light or sucrose is
drastically decreased, indicating an important role for NTRC in regulating
AGPase activity particularly in amyloplasts.
51. Ghosh HP, Preiss J: Adenosine diphosphate glucose
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Current Opinion in Plant Biology 2010, 13:321–329
54. Ritte G, Scharf A, Eckermann N, Haebel S, Steup M:
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55. Szydlowski N, Ragel P, Raynaud S, Lucas MM, Roldan I,
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of either class IV or class III starch synthases. Plant Cell 2009,
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This study describes the production of a mutant lacking two starch
synthase isoforms. Plants lacking both do not accumulate starch indicating that the two isoforms are responsible for granule initiation.
56. Dumez S, Wattebled F, Dauvillee D, Delvalle D, Planchot V,
Ball SG, D’Hulst C: Mutants of Arabidopsis lacking starch
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by cytoplasmic maltose accumulation. Plant Cell 2006,
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57. Bustos R, Fahy B, Hylton CM, Seale R, Nebane NM, Edwards A,
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58. Delatte T, Trevisan M, Parker ML, Zeeman SC: Arabidopsis
mutants Atisa1 and Atisa2 have identical phenotypes and lack
the same multimeric isoamylase, which influences the branch
point distribution of amylopectin during starch synthesis. Plant
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59. Wattebled F, Dong Y, Dumez S, Delvallé D, Planchot V, Berbezy P,
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Arabidopsis lacking a chloroplastic isoamylase accumulate
phytoglycogen and an abnormal form of amylopectin. Plant
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60. Hennen-Bierwagen TA, Lin Q, Grimaud F, Planchot V, Keeling PL,
James MG, Myers AM: Proteins from multiple metabolic
pathways associate with starch biosynthetic enzymes in
high molecular weight complexes: a model for regulation of
carbon allocation in maize amyloplasts. Plant Physiol 2009,
149:1541-1559.
Enzyme complexes containing starch metabolising enzymes were examined in maize endosperm. One enzyme not previously thought to be
involved in starch metabolism (pyruvate phosphate dikinase) was identified within one of the complexes. Although this result is very interesting,
further research is needed to evaluate its significance.
61. Hennen-Bierwagen TA, Liu F, Marsh RS, Kim S, Gan Q,
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62. Tetlow IJ, Beisel KG, Cameron S, Makhmoudova A, Liu F,
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63. Tetlow IJ, Wait R, Lu Z, Akkasaeng R, Bowsher CG, Esposito S,
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65. Satoh H, Shibahara K, Tokunaga T, Nishi A, Tasaki M, Hwang SK,
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A starch phosphorylase mutant was studied in rice. The starch in the
mutant had different physical properties than that found in the control,
indicating that starch phosphorylase plays a role in starch synthesis.
66. Lohmeier-Vogel EM, Kerk D, Nimick M, Wrobel S, Vickerman L,
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Regulation of starch metabolism Kötting et al. 329
This paper describes the analysis of a coiled-coil protein from Arabidopsis
and demonstrates that it binds to starch granules. The authors hypothesise that it may also bind to starch metabolic proteins by its coiled-coil
domain, and thereby could act as a regulatory scaffold protein.
73. Balmer Y, Koller A, del Val G, Manieri W, Schurmann P,
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67. Fulton DC, Stettler M, Mettler T, Vaughan CK, Li J, Francisco P,
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b-Amylase mutants were isolated in Arabidopsis. A mutation in the BAM4
isoform leads to a repression of starch degradation, even though no
catalytic fucntion for BAM4 could be demonstrated.
74. Hägglund P, Bunkenborg J, Maeda K, Svensson B: Identification
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69. Heazlewood JL, Durek P, Hummel J, Selbig J, Weckwerth W,
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70. Lohrig K, Muller B, Davydova J, Leister D, Wolters DA:
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71. Schliebner I, Pribil M, Zühlke J, Dietzmann A, Leister D: A survey of
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72. Kempa S, Rozhon W, Samaj J, Erban A, Baluska F, Becker T,
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76. Salvucci ME, Drake RR, Broadbent KP, Haley BE, Hanson KR,
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77. Grimaud F, Rogniaux H, James MG, Myers AM, Planchot V:
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78. Hejazi M, Fettke J, Kötting O, Zeeman SC, Steup M: The LaforinLike Dual-Specificity Phosphatase SEX4 from Arabidopsis
Hydrolyzes Both C6- and C3-Phosphate Esters Introduced by
Starch-Related Dikinases and Thereby Affects Phase
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79. Sylviane Comparot-Moss, Oliver Kötting, Michaela Stettler,
Christoph Edner, Alexander Graf, Weise SE, Sebastian Streb,
Wei-Ling Lue, Daniel MacLean, Sebastian Mahlow, Gerhard
Ritte, Martin Steup, Jychian Chen, Zeeman SC, Smith AM: A
Putative Phosphatase, LSF1, Is Required for Normal
Starch Turnover in Arabidopsis Leaves. Plant Physiol. 2010,
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Current Opinion in Plant Biology 2010, 13:321–329