COBIOT-839; NO. OF PAGES 10 Please cite this artic le in press as: Santelia D, Zeeman SC. Progress in Arab idops is starch research and potentia l biote chno logica l appl icatio ns, Curr Opin Biotec hnol (2010), doi:10.1016/ j.copbio.2010.11.014 Available online at www.sciencedirect.com 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 ([email protected]) Current Opinion in Biotechnology2010, 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 tuber s of potatoes) are staple food stuff s 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 pro duc e starches wit h nov el features that better fit industrial needs. A comprehensive understanding of starch biosynthetic pathways and struc- tural properties is fundamental to these aims [ 6,7]. In the past decade, the wealth of genetic and genomic resources in the model plant Arabidopsis thaliana has been used to tack le funda menta l scient ific quest ions about sta rch met abo lis m tha t cou ld not eas ily be add res sed using starch crops. Many genes encoding starch-rela ted enzymes are widel y con ser ved in hig her pla nts [ 8 ]. Comparison of the transitory leaf starch system with tuber and see d end osperm sys tems has confirmed tha t the enzymes have simil ar biological func tion s. This illus trat es the utility of the Arabidopsis genetic system. Transitory leaf starch is synthesized and then degraded during the course of a single diurn al cyc le, all owi ng the rol es ofstarch-me tab olizing enz ymes in bot h processes to be studied. Continued use of theArabid opsi s syst em is lik ely to grant further insights into the complex functions and inter play betwe en known starch biosy nthet ic enzyme s 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 ofArabidopsis research in improving our knowledge on the mecha nisms of starch granule initiat ion and assemb ly, and on elucid ati ng the rol e of glu can tra nsi ent pho s- phorylation in starch breakdown. Industrial uses for starch Starch consi sts of two major components, amylopecti n (70–80%) and amyl ose (20–30%), both of which are polymers ofa-D-glucose units. Amylose is an essentially linear a-1,4-l inked polyme r of up to several thousand glucose residues. Amylopectin is a large r a-1,4-linked polymer, regu larly branc hed 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 org ani zed int o hig her -or der structures tha t make up sta rch gra nul es [ 9]. Sta rch es fro m dif fer ent bot ani cal sources vary in size, composition, and fine structure ofamylopectin. These factors influence the physical proper- tie s and end -uses for the dif fer ent nat ura l sta rch es (further details about the structural variables that deter- mine starch properties and functionality are described in Box 1). The mos t important phy sical cha nge s tha t tak e place durin g industrial process ing of native starches are the swelling of the granules upon heating in an excess ofwater and subsequent solubilization of amylose and amy- www.sciencedirect.com Current Opinion in Biotechnology2010, 22:1–10
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Please cite this article in press as: Santelia D, Zeeman SC. Progress in Arabidopsis starch research and potential biotechnological applications, Curr Opin Biotechnol (2010), doi:10.1016/ j.copbio.2010.11.014
Figure 1
Amylose Amylopectin
ADPGlc
SSs
DBEsBEs
GBSS
Glc1P
ATP
PPiADG
ATP
AMP+ Pi
PWD
ATP
AMP+ Pi
GWD
P
P
Pi
SEX4
Pi
SEX4
(a)
pal
S
VV
S
2 µm 2 µm pal
pal
pal
epi epi
epi epi
S
S
S
S
(b) (c) (d) (e)
(g)(f)
(j)(i)(h)
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 amylopectinchains 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, waterdikinase (PWD) phosphorylate glucan chains (at the C6 and C3 positions, respectively), while SEX4 dephosphorylates them (see text for details).
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
Current Opinion in Biotechnology 2010, 22:1–10 www.sciencedirect.com
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 andpotato [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-
Progress in Arabidopsis starch research and potential biotechnological applications Santelia and Zeeman 3
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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 theend of the day, visualized by SEM. Bars = 2 mm, from Zeeman et al. [64].
Box 1 Relationship between starch structure and starch functionality
Variations in the amylose to amylopectin ratio, the amylopectin chainlength, the degree of phosphorylation and starch granule size and
shape are known to contribute to differences in the swelling behaviorof 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 viscoussolutions 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-amylosestarches are desirable inprocessed 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 alower 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 adhesiveproducts. Genetic engineeringof 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 tostarch granule hydration status and negatively correlated to its
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 starcheshave 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 tuberstarch is highly phosphorylated, as phosphorylation is integral to itsmetabolism [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 markedlyincrease 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 potatotubers have larger granules up to 100 mm in diameter. Size of starch
granule is particularly important in applications where starch is used asfiller, 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 themolecular 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, thereare considerable inconsistencies in the literature on this subject (see
[61] and references therein) and further investigations are required in
the future.
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sicochemical modifications in order to deliver optimal
functionality. In addition, crops with added-value
starches generally have a lower yield than the equiv-
alent wild-type crops producing normal starch. Thedevelopment of novel starches with further improved
functionality with no need for subsequent chemical
modifications, and the increase of starch yieldsrepresent obvious biotechnological targets.
Mechanisms of starch granule biosynthesisand the potential for crop improvementSome 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 donehave greatly accelerated progress. The past few years
4 Plant biotechnology
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Table 1
Summary of single mutants with altered starch content and changes in starch structure due to the mutation
Mutation Locus Enzyme Starch content Phosphatecontent
Amylosecontent
Starch granulemorphology
Amylopectinchain length
distribution
Reference
ss1 At5g24300 Starchsynthase I
(SSI)
# at ED Normal Normal Smaller,elongated
## short," intermediate
[65]
ss2 At3g01180 Starch
synthase II(SSII)
Normal # " Larger, distorted "" short,# intermediate
[23]
ss3 At1g11720 Starch
synthase III
(SSIII)
" at ED in LD "" Normal Normal Minor changes [66]
ss4 At4g18240 Starch
synthase IV
(SSIV)
# at ED in LD n.d. Normal Single granule,
bigger
Minor changes [25]
be1a At3g20440 Branchingenzyme I
(BEI)
Normalb n.d.b Normalb Normalb Normalb [27,67]
be2 At5g03650 Branching
enzyme II(BEII)
Normal n.d. Normal Slightly larger Minor changes [27]
be3 At2g36390 Branching
enzyme III(BEIII)
Normal n.d. Normal Slightly larger Minor changes [27]
isa1 At2g39930 Isoamylase 1(ISA1)
##,phytoglycogen
n.d. " Smaller,irregular
" short,# intermediate
[29,30]
isa2 At1g03310 Isoamylase 2
(ISA2)
##,
phytoglycogen
n.d. Normal Smaller,
irregular
" short,
# intermediate
[29,30]
isa3 At4g09020 Isoamylase 3(ISA3)
"" Normal " Normal "" short,# intermediate
[30,36,38]
lda At5g04360 Limit dextrinase
(LDA)
Normal n.d. Normal Normal Normal [30,36,38]
sex1 At1g10760 Glucan, waterdikinase 1
(GWD1)
""" Not detected "" Larger Normal [45,64]
pwd At5g26570 Phosphoglc.,water dikinase(PWD)
" # C3, " C6 n.d. n.d. Normal [42
,47,48]
sex4 At3g52180 Starch excess
four (SEX4)
"" """ (p-oligos) """ Larger,
fewer,
rounded,thicker
Normal [51,64]
lsf1 At3g01510 Like SEXFOUR 1
(LSF1)
"" " " Normal Normal [52]
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 phosphorylatedglucans.a BEI is notrelated to thestandard plant A-typeor B-typeSBE families butshowsmore similarityto theglycogen-branching enzymes from fungi and
animals [27].b Unconfirmed mutant data [67].
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ED, end of day; EN, end of night; SD, short day; LD, long day; #, reduced; ##, greatly reduced; ###, dramatically reduced; ", increased;"", greatlyincreased; """, dramatically increased; short chains, DP6–DP12; intermediate chains, DP13–DP28; long chains, DP29–DP40; and n.d., not
determined.
www.sciencedirect.com Current Opinion in Biotechnology 2010, 22:1–10
(for Like S ex F our), 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 phosphatecontent. 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 storedpotato 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.
ConclusionsThe improvement in our understanding of starch biosyn-thesis resulting from basic research in Arabidopsis creates
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 thisbasic 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.
References and recommended readingPapers of particular interest, published within the period of review,have been highlighted as:
of special interest of outstanding interest
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8 Plant biotechnology
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enzymes but restored by the subsequent removal of anendoamylase. Plant Cell 2008, 20:3448-3466.
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