applicationsof arabidopsis genome

10
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 elds 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 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 tuber s of potatoes) are staple food stuff s in the human diet, providing up to 80% of the daily caloric 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 t 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 ic 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 conrmed 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 of starch-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 of Arabidopsis 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 (7080%) and amyl ose (2030%), both of which are polymers of a-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 efciently 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 ne structure of amylopectin. These factors inuence 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 of water and subsequent solubilization of amylose and amy- www.sciencedirect.com Current Opinion in Biotechnology 2010, 22:110

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COBIOT-839; NO. OF PAGES 10

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

 Available online at www.sciencedirect.com

Progress in Arabidopsis starch research and potentialbiotechnological applicationsDiana 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 Biotechnology  2010, 22:1–10

This review comes from a themed issue onPlant 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

IntroductionStarch 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]. Plantsalso 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].

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 andinterplay 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 starchStarch 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 amylopectinpack 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 botanicalsources 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 

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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).

 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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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

group also confers a very high swelling power to starch

gels [16,17].

Chemical and physical modifications of starch are costlyand 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 andfreeze–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-

siblefor 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 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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have seen the study of starch synthesis in Arabidopsisenter 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 someof 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 starchbiosynthetic 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 differentamylopectin 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,

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

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Table 2

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

Mutation Starch content Phosphate

content

 Amylose

content

Starch granule

morphology

 Amylopectin chain length

distribution

Reference

 ss2 /  ss3 ## n.d. "" Larger, distorted """ short, ## intermediate [23] ss1 /  ss4 ## n.d. n.d. Single granule,

bigger

## short, " intermediate [24]

 ss2 /  ss4 ## n.d. n.d. Single granule,bigger

"" short, # intermediate [24]

 ss3 /  ss4 Not detected – – – – [24]

 ss1 /  ss2 /  ss3 ## n.d. "" Smaller """ short,# intermediate,# long

[24]

 ss1 /  ss2 /  ss4 ## n.d. "" Single granule,

bigger

# short, " intermediate [24]

 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

detected,

phytoglycogen

n.d. "" n.d. """ short, ## intermediate [37,38]

 isa1 /  isa2 /  lda ##, phytoglycogen n.d. n.d. Smaller, irregular "" short, ## int ermediat e [37] isa1 /  isa2 /  isa3 ###, phytoglycogen n.d. n.d. Smaller, cracked,

fissured

"" short, ## int ermediat e [37]

 isa1 /  isa2 /  isa3 /  lda Not detected,

phytoglycogen,

limit dextrins

n.d. n.d. Tiny spherical

particles (up to

100 nm diameter)

""" short, ## intermediate [37]

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.

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the presence of either SSIII or SSIV appears to beessential 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 amylopectinsynthesis 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 chloroplastica-

amylase, in addition to all four DBE genes, partiallyrestores starch synthesis (Figure 1g). Thus, the defect

in starch granule biosynthesis in the absence of DBEs is

nota 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 bythese Arabidopsis studies.

DBEs have also been implicated in the control of thecorrect 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 ectopicallyprime 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-

tiesof the endogenousstarch 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 phosphateMost native starches contain phosphate groups mono-

esterified to the glucose residues. The extent of phos-

phorylation varies from a relatively high level in potatotuber 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-hydrolyzingenzymes, such as exoamylases (b-amylases) and DBEs.

6 Plant biotechnology 

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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 S tarch EX cess 4)is

also required for proper starch metabolism. This suggests

an interdependencebetweenreversible 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 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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