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Progress in Arabidopsis starch research and potential
biotechnological applications
Diana Santelia and Samuel C Zeeman
For the past decade, Arabidopsis has been the model higher
plant of choice. Research into leaf starch metabolism has
demonstrated that Arabidopsis is a useful system in which to
make fundamental discoveries about both starch biosynthesis
and starch degradation. This review describes recent
discoveries in these fields and illustrates how such discoveries
might be applied in the green biotechnology sector to improve
and diversify our starch crops.
Address
Department of Biology, ETH Zurich, Universitaetsstr. 2, CH-8092 Zurich,
Switzerland
Corresponding author: Zeeman, Samuel C (szeeman@ethz.ch)
Current Opinion in Biotechnology 2010, 22:1–10
This review comes from a themed issue on
Plant biotechnology
Edited by Adi Avni and Miguel Blazquez
0958-1669/$ – see front matter
# 2010 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2010.11.014
Introduction
Starch is the major storage carbohydrate in higher plants
and is used to sustain metabolism, growth and develop-
ment at times when photosynthesis is not active. During
the day, plants store some photo-assimilates in leaves as
starch and remobilize it at night for respiration and to
produce sucrose for export to the sink tissues [1,2]. Plants
also accumulate starch in heterotrophic organs and use it
to fuel regrowth. Many of these starch-storing organs (e.g.
the seeds of cereal crops, the roots of cassava and the
tubers of potatoes) are staple foodstuffs in the human
diet, providing up to 80% of the daily calorific intake.
From an industrial perspective, starch represents a cheap,
renewable material, whose unique physicochemical prop-
erties are increasingly exploited in the agri-food sector
and in many manufacturing processes [3,4]. Starch is also
used as a feedstock for bio-ethanol production (e.g. corn
and cassava [5]). The use of major food crops for non-
food purposes has spurred on efforts to synthesize more
starch in plants, and to produce starches with novel
features that better fit industrial needs. A comprehensive
understanding of starch biosynthetic pathways and struc-
tural properties is fundamental to these aims [6,7].
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In the past decade, the wealth of genetic and genomic
resources in the model plant Arabidopsis thaliana has been
used to tackle fundamental scientific questions about
starch metabolism that could not easily be addressed
using starch crops. Many genes encoding starch-related
enzymes are widely conserved in higher plants [8].
Comparison of the transitory leaf starch system with tuber
and seed endosperm systems has confirmed that the
enzymes have similar biological functions. This illustrates
the utility of the Arabidopsis genetic system. Transitory
leaf starch is synthesized and then degraded during the
course of a single diurnal cycle, allowing the roles of
starch-metabolizing enzymes in both processes to be
studied. Continued use of the Arabidopsis system is likely
to grant further insights into the complex functions and
interplay between known starch biosynthetic enzymes
and facilitate the discovery of as-yet unknown enzymes
and regulatory factors. These discoveries will provide key
leads for the starch biotechnology sector.
In this review, we focus on the latest contributions of
Arabidopsis research in improving our knowledge on the
mechanisms of starch granule initiation and assembly,
and on elucidating the role of glucan transient phos-
phorylation in starch breakdown.
Industrial uses for starch
Starch consists of two major components, amylopectin
(70–80%) and amylose (20–30%), both of which are
polymers of a-D-glucose units. Amylose is an essentially
linear a-1,4-linked polymer of up to several thousand
glucose residues. Amylopectin is a larger a-1,4-linked
polymer, regularly branched with a-1,6-branch points.
Short, linear adjacent chain segments within amylopectin
pack efficiently into layers (crystalline lamellae) of paral-
lel double helices (Figure 1a). These crystalline lamellae
alternate with amorphous lamellae containing the branch
points. The resulting insoluble semi-crystalline matrix is
organized into higher-order structures that make up
starch granules [9]. Starches from different botanical
sources vary in size, composition, and fine structure of
amylopectin. These factors influence the physical proper-
ties and end-uses for the different natural starches
(further details about the structural variables that deter-
mine starch properties and functionality are described in
Box 1).
The most important physical changes that take place
during industrial processing of native starches are the
swelling of the granules upon heating in an excess of
water and subsequent solubilization of amylose and amy-
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2 Plant biotechnology

Figure 1

(a) Glc1P
ATP
ADG
PPi

ADPGlc
GBSS SSs
BEs
DBEs AMP
+ Pi

ATP
GWD
SEX4
P Pi

Pi
P
SEX4
AMP
PWD + Pi

ATP

Amylose Amylopectin

(b) (c) (d) epi (e) epi

S
S
pal
S

S
V 2 µm V
2 µm pal

(f) (g) pal

(h) (i) (j)
pal

S
epi epi

Current Opinion in Biotechnology

Starch granule synthesis, structure and morphology. (a) Simplified scheme of starch synthesis (left). The filled circles in the amylose and amylopectin
models represent individual glucosyl residues. The structural relationship between amylose and amylopectin (middle). Pairs of adjacent amylopectin
chains form double helices (depicted as cylinders) that pack in ordered semi-crystalline arrays. Amylose (blue) forms unordered structures within the
amorphous parts of the granule. Reversible phosphorylation of amylopectin chains (right): glucan, water dikinase (GWD) and phosphoglucan, water
dikinase (PWD) phosphorylate glucan chains (at the C6 and C3 positions, respectively), while SEX4 dephosphorylates them (see text for details).
Abbreviations: Glc1P, glucose 1-phosphate; ADG, ADPglucose pyrophosphorylase; GBSS, granule-bound starch synthase; SSs, starch synthases;
BEs, branching enzymes; SEX4, phosphoglucan phosphatase. (b–j) Starch granule morphology in Arabidopsis mutants, visualized by transmission
electron microscopy (TEM) or scanning electron microscopy (SEM). (b, c) Starch granules at the end of the day in leaf palisade cells of wild type (b) and
ss4 (c). S, starch; V, vacuole. Visualized by TEM, from Roldan et al. [25]. (d–g) Starch granules and/or soluble glucans (arrowheads) accumulating at

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Progress in Arabidopsis starch research and potential biotechnological applications Santelia and Zeeman 3
Box 1 Relationship between starch structure and starch functionality
Variations in the amylose to amylopectin ratio, the amylopectin chain
length, the degree of phosphorylation and starch granule size and
shape are known to contribute to differences in the swelling behavior
of granules and the functionality of starches from different origins.
Thus, knowledge of starch structural and compositional parameters is
vital when attempting to predict and improve starch functionality.
Amylose to amylopectin ratio and amylopectin structure
The amylose/amylopectin ratio affects starch gelatinization and recrys-
tallization properties. During processing, amylopectin forms viscous
solutions that are stable in water at room temperature for days. By
contrast, amylose forms a gel that is stable in solution at temperatures
greater than 60–70 8C, but on cooling it will rapidly aggregate or
crystallize (‘retrogradation’). Thus, low-amylose starches are desirable in
processed foods, as they confer freeze–thaw stability [57]. A major
achievement of starch genetic improvement was accomplished by the
simultaneous antisense down-regulation of three SS in potato tubers
(GBSS, SSII and SSIII), which resulted in the production of an amylose-
free, short-chain amylopectin starch with exceptional freeze–thaw
stability [58]. By contrast, high-amylose starches or starches that have a
lower degree of amylopectin branching are characterized by higher
gelatinization temperatures and a lower peak viscosities [16,59]. The
high gelling strength and the film-forming ability of these starches make
them useful in the production of corrugated board, paper and adhesive
products. Genetic engineering of potato tubers by antisense inhibition of
both branching enzyme isoforms resulted in the production of a very
high-amylose starch in potato [19].
Degree of phosphorylation
The amount of covalently bound phosphate is positively correlated to
starch granule hydration status and negatively correlated to its
lopectin (‘gelatinization’). Amylose diffuses out of the
swollen granule and, on cooling, forms a continuous gel
phase. Swollen amylopectin-enriched granules aggregate
into gel particles, generating a viscous solution. This two-
phase structure, called starch paste, is desired in many
applications where processed starches are used as thick-
eners or binders. Native starches have few other uses, as
the polymers are relatively inert.
Many industrial applications require the modification of
native starches, such as oxidation, esterification, hydro-
xymethylation, dextrinization and cross-linking. These
modifications overcome the limitations of native starch
properties (e.g. stabilize the polymers against severe
heating, shear, freezing or storage; [10,11]). Such modi-
fied starches find innumerable applications in food
industries, particularly in confectionery, bakery,
thickening and emulsification, and in non-food sectors
as adhesive gums, biodegradable materials, sizing agents
in textile and paper industry [3,12]. Starch phosphoryl-
ation is the only known in vivo modification of starch
[13]. The presence of phosphate induces structural
changes in amylopectin, promoting the solubility of
the glucan chains [14,15]. The presence of phosphate
the end of the day in leaf palisade (pal) and adjacent epidermal cell (epi) plastids of the wild type (d), isa1isa2 (e), isa1isa2isa3lda (f),
isa1isa2isa3ldaamy3 (g), visualized by TEM. Bars = 2 mm, from Streb et al. [37]. (h–j) Starch granules isolated from wild type (h), sex1 (i), sex4 (j) at the
end of the day, visualized by SEM. Bars = 2 mm, from Zeeman et al. [64].
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crystallinity [60]. The increased water binding-capacity of high-
phosphate starches, associated with a low swelling temperature,
renders them less prone to retrogradation. High-phosphate starches
have improved transparency, improved viscosity and freeze–thaw
stability [16]. Their charged nature also makes them particularly useful
as surface coatings in the paper-making industry [17]. Potato tuber
starch is highly phosphorylated, as phosphorylation is integral to its
metabolism [44]. By contrast, cereal starches are almost phosphate
free, as their degradation after seed germination proceeds via a
different enzymatic system than that in leaves of tubers. However, the
creation of highly phosphorylated cereal starches could markedly
increase their uses.
Granule size
Starches from cereals vary considerably in size (2–35 mm). In wheat,
starch granules exhibit a bimodal size distribution, with larger lenticular
starch granules coexisting with smaller spherical granules [61]. Rice
has a uniform distribution of small granules (5 mm) whereas potato
tubers have larger granules up to 100 mm in diameter. Size of starch
granule is particularly important in applications where starch is used as
filler, such as the paper-making industry [62]. While larger starch
granules confer a very high swelling power and high viscosity, small
granules are reported to have a lower gelatinization temperature and
give a smoother paste texture [61]. In some studies, differences in the
molecular structure of amylopectin and amylose have been correlated
with granule size [61]. In barley, for example, small granules have a
decreased degree of amylopectin polymerization [63]. However, there
are considerable inconsistencies in the literature on this subject (see
[61] and references therein) and further investigations are required in
the future.
group also confers a very high swelling power to starch
gels [16,17].
Chemical and physical modifications of starch are costly
and frequently employ treatments with hazardous
chemicals. Thus, research has focused on ways of produ-
cing starches with enhanced properties directly in planta
[6]. Naturally occurring ‘waxy’ mutations in maize result
in starch containing amylopectin but no amylose, which
has improved paste clarity and freeze–thaw stability after
processing compared to wild-type maize starch. The
waxy phenotype is caused by mutations affecting gran-
ule-bound starch synthase (GBSS), the enzyme respon-
sible for amylose synthesis (Figure 1a [18]), and waxy-like
starches have also been produced in other crops [6].
Another example of the genetic improvement of starch
quality is the high-amylose starch (e.g. from maize and
potato [19]). In contrast to the waxy starches, high-amy-
lose starches have a much higher gelatinization tempera-
ture conferring a better gel texture and adhesion
capacity.
Despite the improved functionality provided by
these novel starches, they still require additional phy-
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4 Plant biotechnology

Table 1

Summary of single mutants with altered starch content and changes in starch structure due to the mutation

Mutation Locus Enzyme Starch content Phosphate Amylose Starch granule Amylopectin Reference
content content morphology chain length
distribution
ss1 At5g24300 Starch # at ED Normal Normal Smaller, ## short, [65]
synthase I elongated " intermediate
(SSI)
ss2 At3g01180 Starch Normal # " Larger, distorted "" short, [23]
synthase II # intermediate
(SSII)
ss3 At1g11720 Starch " at ED in LD "" Normal Normal Minor changes [66]
synthase III
(SSIII)
ss4 At4g18240 Starch # at ED in LD n.d. Normal Single granule, Minor changes [25]
synthase IV bigger
(SSIV)
be1 a At3g20440 Branching Normal b n.d. b Normal b Normal b Normal b [27,67]
enzyme I
(BEI)
be2 At5g03650 Branching Normal n.d. Normal Slightly larger Minor changes [27]
enzyme II
(BEII)
be3 At2g36390 Branching Normal n.d. Normal Slightly larger Minor changes [27]
enzyme III
(BEIII)
isa1 At2g39930 Isoamylase 1 ##, n.d. " Smaller, " short, [29,30]
(ISA1) phytoglycogen irregular # intermediate
isa2 At1g03310 Isoamylase 2 ##, n.d. Normal Smaller, " short, [29,30]
(ISA2) phytoglycogen irregular # intermediate
isa3 At4g09020 Isoamylase 3 "" Normal " Normal "" short, [30,36,38]
(ISA3) # intermediate
lda At5g04360 Limit dextrinase Normal n.d. Normal Normal Normal [30,36,38]
(LDA)
sex1 At1g10760 Glucan, water """ Not detected "" Larger Normal [45,64]
dikinase 1
(GWD1)
pwd At5g26570 Phosphoglc., " # C3, " C6 n.d. n.d. Normal [42,47,48]
water dikinase
(PWD)
sex4 At3g52180 Starch excess "" """ (p-oligos) """ Larger, Normal [51,64]
four (SEX4) fewer,
rounded,
thicker
lsf1 At3g01510 Like SEX "" " " Normal Normal [52]
FOUR 1
(LSF1)

ED, end of day; EN, end of night; SD, short day; LD, long day; #, reduced; ##, greatly reduced; ###, dramatically reduced; ", increased; "", greatly
increased; """, dramatically increased; short chains, DP6–DP12; intermediate chains, DP13–DP28; long chains, DP29–DP40; n.d., not determined;
C3, glucosyl unit phosphorylated in the C3 position; C6, glucosyl unit phosphorylated in the C6 position; and p-oligos, soluble phosphorylated
glucans.
a
BEI is not related to the standard plant A-type or B-type SBE families but shows more similarity to the glycogen-branching enzymes from fungi and
animals [27].
b
Unconfirmed mutant data [67].

sicochemical modifications in order to deliver optimal Mechanisms of starch granule biosynthesis

functionality. In addition, crops with added-value
starches generally have a lower yield than the equiv-
alent wild-type crops producing normal starch. The
development of novel starches with further improved
functionality with no need for subsequent chemical
modifications, and the increase of starch yields
represent obvious biotechnological targets.
and the potential for crop improvement
Some of the fundamental discoveries on starch biosyn-
thesis were made in crop plants and pre-date the Arabi-
dopsis model system. However, the recent availability of
large mutant populations of Arabidopsis and the ease and
speed with which molecular genetic studies can be done
have greatly accelerated progress. The past few years

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Progress in Arabidopsis starch research and potential biotechnological applications Santelia and Zeeman 5

have seen the study of starch synthesis in Arabidopsis
enter an exciting phase, with the discovery of new protein
factors and the production of multiple mutant lines,
which shed new light on the mechanisms controlling
starch granule initiation and assembly.
ADPglucose (ADPGlc) is the substrate for starch biosyn-
thesis in higher plants. There are differences in the
biochemical pathway of ADPGlc production between
cell types and species [20]. However, the downstream
starch biosynthetic enzymes are remarkably similar in
isoform type and function [8]. Amylopectin is sufficient
to generate a semi-crystalline starch granule. Amylose is
deposited mainly as non-crystalline chains within the
granule [21]. The synthesis of amylopectin involves
the coordinated actions of at least three classes of enzyme
(Figure 1a [20,22]). Starch synthases (SSs) transfer the
glucosyl unit from ADPGlc to a growing glucan chain,
generating a new a-1,4-glycosidic bond. Branching
enzymes (BEs) introduce branch points (a-1,6-linkage)
via a glucanotransferase reaction, increasing the number
of non-reducing ends. The subsequent removal of some
of these branch points by debranching enzymes (DBEs)
also facilitates the formation of a proper crystalline starch
granule. The roles of these proteins are discussed briefly
here, and in greater detail elsewhere [22].
Plants contain multiple isoforms of each class of starch
biosynthetic enzymes. In general, mutations in a particu-
lar type of SSs, BEs or DBEs alter amylopectin structure
and, in some cases, change granule morphology and the
physical properties of the starch (Table 1). Each class of
SS is thought to be responsible for synthesizing different
amylopectin chain lengths. Yet a certain level of redun-
dancy exists. The preference/competition between
different SS isoforms for the ends of linear chains will
influence the structure of the growing amylopectin mol-
ecule. Simultaneous deficiency of more than one SS
isoform results either in a starch structural phenotype
equivalent to the sum of the phenotypes of the
corresponding single mutants (e.g. SSI and III), or to a
more severe alteration (e.g. SSII and III) indicative of
synergistic actions between the enzymes (Table 2
[23,24]).
Mutation in any one of the four soluble SSs does not
prevent starch granule formation (Table 1). However,
Arabidopsis mutants defective in SSIV usually contain
just one enlarged granule per chloroplast (Figure 1b and c
[25]), indicating a role for SSIV in the establishment of a
correct number of starch granules. Simultaneous loss of
SSIII and SSIV abolishes starch accumulation, despite
other SSs remaining enzymatically active [24]. Thus,

Table 2

Summary of multiple mutants with altered starch content and changes in starch structure due to the mutation

Mutation Starch content Phosphate Amylose Starch granule Amylopectin chain length Reference
content content morphology distribution
ss2/ss3 ## n.d. "" Larger, distorted """ short, ## intermediate [23]
ss1/ss4 ## n.d. n.d. Single granule, ## short, " intermediate [24]
bigger
ss2/ss4 ## n.d. n.d. Single granule, "" short, # intermediate [24]
bigger
ss3/ss4 Not detected – – – – [24]
ss1/ss2/ss3 ## n.d. "" Smaller """ short, [24]
# intermediate,# long
ss1/ss2/ss4 ## n.d. "" Single granule, # short, " intermediate [24]
bigger
be1/be2 Normal n.d. Normal n.d. Minor changes [27]
be1/be3 Normal n.d. normal n.d. Minor changes [27]
be2/be3 Not detected – – – – [27]
isa1/isa2 ##, phytoglycogen n.d. n.d. Smaller, irregular " short, # intermediate [29,37]
isa2/lda ##, phytoglycogen n.d. n.d. Smaller, irregular " short, # intermediate [30,37]
isa3/lda """, limit dextrins n.d. Normal Bigger, rounded "" short, # intermediate [36,37,38]
isa1/isa3 ##, phytoglycogen n.d. " Smaller, irregular "" short, # intermediate [37,38]
isa1/isa3/lda Little or none n.d. "" n.d. """ short, ## intermediate [37,38]
detected,
phytoglycogen
isa1/isa2/lda ##, phytoglycogen n.d. n.d. Smaller, irregular "" short, ## intermediate [37]
isa1/isa2/isa3 ###, phytoglycogen n.d. n.d. Smaller, cracked, "" short, ## intermediate [37]
fissured
isa1/isa2/isa3/lda Not detected, n.d. n.d. Tiny spherical """ short, ## intermediate [37]
phytoglycogen, particles (up to
limit dextrins 100 nm diameter)

ED, end of day; EN, end of night; SD, short day; LD, long day; #, reduced; ##, greatly reduced; ###, dramatically reduced; ", increased;"", greatly
increased; """, dramatically increased; short chains, DP6–DP12; intermediate chains, DP13–DP28; long chains, DP29–DP40; and n.d., not
determined.

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6 Plant biotechnology
the presence of either SSIII or SSIV appears to be
essential for granule initiation. Further investigations will
be necessary to explain their mode of action.
There are two classes of BEs (BEI and BEII). Class I
preferentially transfers longer chains than class II [26].
Reducing both isoforms simultaneously using an anti-
sense approach in potato leads to highly abnormal starch
granules, with longer chains and less branched amylopec-
tin [19]. In Arabidopsis, the situation is unusual because
there are two members of class II, but no enzyme that
shows similarity to class I [27]. No significant structural
changes are detected in the single mutants (Table 1),
whereas plants lacking both BEII isoforms are unable to
make starch (Table 2 [27]).
The role of DBEs in starch biosynthesis is more com-
plicated. Mutations that reduce or eliminate isoamylase
(ISA) activity of the ISA1 class in Chlamydomonas [28],
Arabidopsis (Figure 1e [29,30]) and cereal endosperms
[31,32] result in the replacement of some or all of the
starch with phytoglycogen, a soluble glycogen-like poly-
mer. In Arabidopsis and potato, ISA1 forms a hetero-
multimeric complex with ISA2, in which ISA1 represents
the catalytic subunit and ISA2 the non-catalytic subunit
[33]. Mutation of either gene (Table 1), or both simul-
taneously (Table 2), leads to a comparable phytoglyco-
gen-accumulating phenotype [29,30]. In the unicellular
alga Chlamydomonas, substitution of starch with phyto-
glycogen upon loss of ISA activity is essentially complete
[28]. Thus, a mandatory role for DBEs in starch granule
crystallization was initially proposed [34,35]. Overlap-
ping functions between DBE isoforms in amylopectin
synthesis was suggested as an explanation for the milder
phenotype of Arabidopsis and cereal mutants, where the
substitution of starch with phytoglycogen was only par-
tial (Figure 1d and e). Loss of the remaining DBEs in
Arabidopsis (ISA3 and LDA) does not lead to phytogly-
cogen production (Table 1) and these isoforms are
thought to be mainly involved in starch degradation
[36]. However, Arabidopsis mutants lacking all DBE
isoforms are devoid of starch (Figure 1f [37,38]). While
this result supported a mandatory role for DBEs in
granule biosynthesis, Streb et al. (2008) demonstrated
that mutating AMY3, which encodes the chloroplastic a-
amylase, in addition to all four DBE genes, partially
restores starch synthesis (Figure 1g). Thus, the defect
in starch granule biosynthesis in the absence of DBEs is
not a consequence of the loss of DBE activity per se. Streb
et al. (2008) proposed that the change in glucan structure
resulting from the loss of DBEs enables other enzymes
(e.g. AMY3) not normally involved in amylopectin bio-
synthesis to act on the aberrant glucans to influence their
final structure [37]. While this shows that DBEs are not
essential for starch granule formation, their role in facil-
itating the process is nevertheless strongly supported by
these Arabidopsis studies.
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DBEs have also been implicated in the control of the
correct number of starch granules because Arabidopsis
and other plant species lacking DBEs have numerous
smaller granules in their plastids [39,40]. This effect
could be indirect, since the accumulation of small, soluble
glucans observed in these mutants might ectopically
prime granules.
The goal of studies on the starch biosynthetic enzymes in
Arabidopsis is to unravel the complex genetic and bio-
chemical interactions between them and to develop struc-
tural models that can explain both the molecular
organization of amylopectin and the insoluble nature of
starch. Unfortunately, with the small amounts of starch that
can be extracted from Arabidopsis, it is difficult to assess the
relationship between the novel starch structures obtained
and their resultant physicochemical properties. Neverthe-
less, translational research to apply the knowledge gained
in Arabidopsis into agriculturally relevant plants may result
in the development of novel starches with useful function-
alities. By controlling the combinations and relative activi-
ties of the endogenous starch biosynthetic enzymes, a range
of starches with differences in composition, polymer struc-
ture, granule size and solubility should be possible. Intro-
duction of genes from other systems could further diversify
polymer structure and potentially introduce secondary
modifications to the glucan.
Factors controlling the amount of starch-
bound phosphate
Most native starches contain phosphate groups mono-
esterified to the glucose residues. The extent of phos-
phorylation varies from a relatively high level in potato
tuber starch (0.5% of glucosyl units) to almost undetect-
able amounts in the cereal starches [13]. Phosphate esters
are exclusively found on amylopectin, mostly at the C-6
and, to a lesser extent, the C-3 positions of the glucosyl
units [41,42]. Within the structure of amylopectin, the
phosphates are predominantly located on longer-than-
average chains in the amorphous regions [13]. The pre-
sence of phosphate significantly influences the molecular
structure, crystallinity, functional properties and potential
uses of starch (see Box 1 and references therein). The
presence of phosphate, particularly on the C3 position,
alters the geometry of the adjacent glucosidic linkages
[43], and is likely to disrupt the formation and packing of
glucan double helices [14].
Glucan phosphorylation is essential for the normal metab-
olism of leaf starch [44,45]. Glucan, water dikinase (SEX1/
GWD) phosphorylates the C6 position of glucosyl residues
[46], while phosphoglucan, water dikinase (PWD) phos-
phorylates the C3 position of pre-phosphorylated glucan
chains [42,47,48]. Starch phosphorylation is believed to
increase the hydration status of the granule–stroma inter-
face, facilitating the action of the glucan-hydrolyzing
enzymes, such as exoamylases (b-amylases) and DBEs.
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Progress in Arabidopsis starch research and potential biotechnological applications Santelia and Zeeman 7
These enzymes exhibit relatively little activity on native,
non-phosphorylated starch granules [49]. Removal of the
phosphate groups, at both the C3 and C6 positions, by
phosphoglucan phosphatase (SEX4, for Starch EXcess 4) is
also required for proper starch metabolism. This suggests
an interdependence between reversible starch phosphoryl-
ation and glucan hydrolysis [50,51].
Arabidopsis mutants lacking either of the glucan, water
dikinases or SEX4 accumulate starch to high levels. The
starch itself is altered in the amount or location of the
glucan-bound phosphate, in amylose content, and in
granule size (Table 1, Figure 1h–j). In sex4 mutants, most
of the glucan-bound phosphate is present as soluble
phospho-oligosaccharides, presumed intermediates of
starch breakdown, which are below the limit of detection
in wild-type plants [51]. Phospho-oligosaccharides are
released from starch granule surface by AMY3 and the
DBE isoamylase 3 (ISA3; [51]). Mutation of these two
enzymes in addition to SEX4 abolishes the accumulation
of phospho-oligosaccharides and leads to highly elevated
levels of starch, which is increased in phosphate com-
pared to the wild type (D. Santelia and S.C. Zeeman,
unpublished data). Furthermore, Arabidopsis contains
two glucan phosphatase homologues, LSF1 and LSF2
(for Like Sex Four), both of which are implicated in starch
metabolism ([52]; D. Santelia and S.C. Zeeman, unpub-
lished data) but whose precise functions remain to be
elucidated.
Interestingly, manipulation of enzymes directly involved
in the synthesis of amylopectin, such as SSIII [23], BEI
and BEII [19], also results in increased starch phosphate
content. This effect is correlated with an overall increase
of the average amylopectin chains length in these
mutants [41].
GWD is currently a target of the starch biotechnology
industry. Decreasing its activity can increase starch con-
tents and prevent unwanted starch degradation in stored
potato tubers, while increasing its activity can elevate
granule-bound phosphate content [22]. However, the
impact of manipulating PWD and SEX4 in starch crops
has yet to be determined. Starch phosphorylation occurs
during both starch synthesis and degradation, although at
different rates [53]. Given the antagonistic activities of
glucan, water dikinases and phosphoglucan phosphatases,
the level of phosphate on starch may be controlled by
both processes rather than by phosphorylation alone.
Hence, the coordinated modulation of GWD, PWD
and SEX4 in tissues such as cereal endosperm may further
increase the amount of starch-bound phosphate and alter
the ratio at the C3 and C6 positions.
Conclusions
The improvement in our understanding of starch biosyn-
thesis resulting from basic research in Arabidopsis creates
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new options for the rational design of novel starches.
However, testing their suitability for downstream appli-
cations is not trivial, since large amounts of starch are
needed. Improvements in our ability to predict starch
functionality from structural data or to evaluate starch
properties on a small scale will enhance the transfer of this
basic knowledge to crop plants.
Strategies for controlling starch yield will be more com-
plicated. Enhanced starch yields have been obtained by
increasing ADPglucose pyrophosphorylase activity (the
regulated step in the starch biosynthetic pathway; [54]),
increasing ATP supply to the plastid [55], and decreasing
plastidial adenylate kinase activity [56]. However, opti-
mizing assimilate partitioning between new plant bio-
mass and useful storage compounds such as starch will
require systems-level understanding of plant growth.
Knowledge of the factors controlling photosynthetic
capacity and resource allocation within the plant, and
of the metabolic networks in both source and sink tissues,
will be crucial. It remains a major challenge to interpret
the large molecular profiling datasets from transcriptomic,
proteomic and metabolomic experiments in such a way as
to rationally engineer plant metabolism. Arabidopsis is
the best higher-plant system to pioneer such systems
biology methods, but it remains to be seen how good a
model it will be for the control of resource allocation in
distantly related plant species, where distinct regulatory
mechanisms may have evolved.
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