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23 Οκτ 2013 (πριν από 3 χρόνια και 10 μήνες)

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J. Paul Davis
a
,

Netrphan Supatcharee
a,b,c
,

Ramji L. Khandelwal
c
,

Ravindra N. Chibbar
a

a
Plant Biotechnology Insti
tute, National Research Council of Canada, Plant Biotechnology
Institute, Saskatoon, Canada
b

National Center for Genetic Engineering and B
iotech
nology
(BIOTEC), Klong Luang, Pathumthani, Thailand

c
Department of Biochem
istry, College of Medicine, University of Sas
katchewan, Saskatoon,
Canada

Synthesis of Novel Starches
in Planta:
Opportunities and Challenges
1

In addition to being the main

source of energy in the human diet, starch is also used for a wide
variety of industrial processes. Potato, maize, wheat and cassava are the ma
jor sources for industrial
starch. While there are significant species
-
dependent differ
ences among these starc
hes, in most
cases it is necessary to chemically or physically modify the starches in order to meet the various
industrial needs. The advent of mo
lecular biology and genetic engineering has provided the
requisite technologies to pro
duce starches
in plant
a
with improved properties, which may reduce or
eliminate the need for
in vitro
starch modification. In the last decade significant progress in our un
-
derstanding of starch biosynthesis has been achieved through the use of these tech
nologies. In this
revi
ew, starch structure, functionality and use in industry are dis
cussed. The recent discoveries in
the area of starch biosynthesis are reviewed and several potential plant breeding and/or genetic
engineering strategies for the modifica
tion of starch synthe
sis
in planta
are presented.

Keywords:
Starch synthesis; Starch granule; Starch transformation; Starch modifica
tion

1 Introduction

Starch is the major reserve carbohydrate in plants. The starch stored in the seeds and tubers of
various agricul
tural crops

including maize, wheat, rice, barley, potato and cassava provides the
main source of energy in the human diet. In the developed world, starch is also utilized in the
production of food and beverages, as well as the manufacturing of adhesives, cosmetics, d
etergents,
pa
per and textiles [1]. Starch is currently being used in the production of biodegradable packing
materials [2] and the development of biodegradable plastics is becoming an in
creasingly attractive
alternative to petroleum
-
based prod
ucts. Expa
nsion of dietary and industrial uses of starch is
creating an increased demand for starch in the market place. However, native starches from the
various plant species have limited physicochemical properties, and thus are directly suitable for
relatively fe
w specific end us
es. For the majority of industrial uses, enzymatic and/or chemical
treatments are necessary to improve the starch functionality.

With the advancements in genetic engineering technolo
gies, it is now possible to modify starch
biosynthesis
in planta.
However, starch biosynthesis is a very complicat
ed and incompletely
characterized process. Furthermore,

Correspondence:
Ravindra N. Chibbar,
National Research Council of Canada, Plant
Biotechnology Institute, 110 Gymnasium Place, Saskatoon, SK,

Canada, S7N 0W9. Phone: +1
(0)306 975 5574, Fax: +(1)306 975 4839, e
-
mail: Ravi.Chibbar@NRC.CA.

the understanding of the relationships between starch composition, starch granule structure and the
functional properties of starch is incomplete. So far, the
use of mo
lecular biological techniques has
provided significant con
tributions to our understanding of starch biosynthesis. In the future, there is
a tremendous potential for transgene technology to modify starch synthesis and produce novel
starches that
will reduce or eliminate the need for costly and environmentally unfriendly post
-
harvest enzymatic and chemical modifications. In this review, various appli
cations of starch, the
biochemical characteristics of starch, and an update on the starch biosynthe
sis pathway will be
presented. In addition, several possible strategies for the modification of starch synthesis
in planta
that could be considered feasible in the near future will be dis
cussed.

2 The Chemical Composition of Starch

Starch, which is stored

in the form of water insoluble gran
ules, is composed primarily of two types
of glucose poly
mers: amylose and amylopectin. Amylose is a predomi
nantly linear chain of a
-
1,4
-
linked glucose residues that is sparsely branched with a
-
1,6
-
linkages (approximat
ely one branch per
1,000 glucose residues) [3, 4]. The de
gree of polymerization (dp) of amylose molecules is species
dependent, ranging from around 800 in maize and wheat to approximately 4500 in potato [5, 6].


















































In contrast, amylopectin is a much larger (dp 10
5

to 10
7
) and more complex polysaccharide. It is
composed of hun
dreds of short α
-
1,4 glucan chains joined together by α
-
1,6
-
linkages, with
approximately 5 % of the residues having both α
-
1,4 and α
-
1,6
-
l
inkages [7]. Studies have shown
that both the pattern of branching and the length of the glucan branch chains are non
-
random. The
ordered nature of the α
-
1,6
-
linkages indicates that the branches in the amylopectin molecule are
clustered. The branch length
distributions of amylopectins from a variety of dif
ferent plants were
shown to be polymodal, with peaks at approximately dp 15, and dp 45 [8]. The unique and high
ly
-
ordered structure of the amylopectin molecule, in terms of both branch location and branc
h length,
is essential to the formation of the starch granule (for recent reviews see [7, 9]).

Most wild type plants starches are composed of 15
-
30% amylose and 70
-
85% amylopectin. Several
starch synthe
sis mutants with significantly different amylose/amy
-
lopectin ratios have been
characterized.
Waxy
mutants, which contain >95% amylopectin, have been identified in maize [10],
rice [11], barley [12], wheat [13], potato [14], and cassava [15], while high amylose starches (50
-
85%) have been reported in
amylose

extender
mutants in maize [16], rice [17] and pea [18] and
maize
dull
mutants [16]. The physicochemical properties of starch are greatly affected by the
relative proportions of amylose and amy
lopectin. In addition to the amylose/amylopectin ratio, the
de
gree of polymerization of the glucan chains in both amylopectin [19] and amylose [6] has also
been shown to influence the functional properties of starch.

Other quantitatively minor components of starch include proteins, lipids and minerals. Starch
protein
s can be clas
sified as surface proteins, which can be readily extracted in aqueous solutions,
or integral proteins, which are ex
-
tractable only when a starch solution is heated to temper
atures
near or above the gelatinisation temperature [5]. In cereals,

starch protein levels range from 0.25
-
0.5%, while in potato and cassava, protein levels are generally <0.1% [1,20].

Differences between cereal and tuber starches also exist with respect to lipid content and
distribution (for a review see [21]). Lipid leve
ls are lower in tuber starch than cere
al starches.
Furthermore, in tuber starches, lipids are on
ly found on the granule surface, while starches from
cere
al endosperm have surface and integral lipids. The inte
gral lipids of cereal starches are
composed
of lysophos
-
pholipids (LPL) and free fatty acids (FFA). In barley and wheat the majority
of the starch lipids are LPL, while in maize and rice FFA predominate. The LPL and FFA have
been postulated to form a complex with amylose, and thus the lipid concentr
ation in starch is
positively correlat
-

ed with amylose content. Waxy mutant starches have much lower lipid contents, while high
amylose maize starches have increased lipid concentrations [21].

Starch contains several different minerals in small amounts, b
ut the most important mineral is
phosphorus. In cereals, which have lower total phosphorus contents than tubers, essentially all of
the phosphorus is present in the LPL. In tubers, the phosphorus is present as phos
phate groups
covalently bound to the C3 a
nd C6 carbons in the glucose monomers [22, 23].

Although the proportion of these minor components in starch is very small, they can have a
significant influence on the properties, and therefore the functionality of the starch.

3 Starch Granule Structure

Am
ylose and amylopectin are arranged into water insolu
ble, three
-
dimensional, semicrystalline
structures called starch granules. While many questions about the starch granule remain unresolved,
analysis has revealed that their structure is highly
-
ordered wi
th alternating layers of crystalline and
amorphous regions (for a review see [9]). Starch granules from
waxy
mutants, which contain less
than 2% amylose, are relatively normal in structure, while granules from some high amylose (low
amylopectin) maize muta
nts have an irregular structure [9, 24]. Based on these observations, and
the fact that no amylopectin
-
free mutants have been identified, it has been suggested that
amylopectin is a key factor in the formation of the crystalline regions, and therefore the
overall
structure of the starch granule [25].

There is substantial variation in the size and shape of starch granules among different plant species
(Tab. 1), but the reasons for these differences are not understood. The diameter of the granules can
range f
rom 1 to 100
\
xm,
while different shapes include polygonal, round/spherical, lenticular and
oval. In oat and rice there is a higher level of organization as many small, individual granules are
co
hesively bound together to form a compound granule [26]. Whe
at, barley, rye and triticale have
two different types of starch granules [27]. In wheat, A
-
type granules are lentic
ular in shape and
have diameters greater than 10 (j.m, while B
-
type granules are spherical with diameters less than 10
|im [28]. The A
-

and

B
-
type granules have also been shown to possess different chemical
compositions and different functional properties [29
-
32].
















































Tab.
1. Starch granule characteristics from important crop species.


4 Determinan
ts of Starch Physicochemical Properties

The chemical composition of starch, which includes the amylose/amylopectin content [33, 34],
amylopectin fine structure (including branching and branch chain length) [19, 35], and other minor
starch constituents such

as lipids [21] and phosphorus [36], has been shown to influ
ence the
physicochemical properties of starch. However, it is important to recognize that the functional
properties of a starch are not governed simply by the chemical compo
sition. As mentioned
previously, the different components of starch are organized in specific ways within the granule and
the association between the various components within the granule is a key determinant of the
physico
-
chemical properties of a starch. Currently the unders
tand
ing of the physical organization of
the starch constituents within the granule is incomplete, and therefore the rela
tionships between
starch chemical composition, organi
zation of the components within the granule and the prop
erties
of starch are no
t well defined.

5 Current Uses of Starch

As previously mentioned, in addition to being an impor
tant source of energy in the human diet,
starch is also uti
-

lized in many different industrial processes (see Tab. 2). Intact starch granules, i.e. in their na
tive
form, are used in the production of several different products. They provide a smooth sensation
when rubbed on the skin and thus are used in facial and talcum powders [37]. Native wheat granules
are also used to physically separate the sheets of carbo
nless copy paper to prevent premature
rupturing of ink microcapsules on the bottom sheet [38, 39]. Native granules have also been used as
filler material in the pro
duction of thin plastic films [40] and molded plastic prod
ucts [41].
However, the majority

of the industrial process
es and applications use starch after some or all of the
granule structure has been destroyed [1].

The utility of starch in most industrial applications resides in the ability of starch to form
viscoelastic pastes when granules ar
e heated in the presence of water. When cooled starch pastes
may remain as a viscous solution or form a gel. The term retrogradation is used to describe the
reassociation and crystallization of the glucan poly
mer molecules in the starch suspension that
oc
curs dur
ing cooling. This molecular reassociation causes effects such as precipitation, gelation
and changes in consisten
cy and opacity [42]. Retrogradation is largely dependent on the amylose
content of the starch. Generally, when compared to cereal sta
rches, potato and cassava starch
-

Tab.
2. Industrial uses of starch.




es produce relatively bland pastes with higher viscosities, better clarity and lower retrogradation
rates [1, 6, 20]. Pasting behavior is also influenced by many other factors associa
ted with the
pasting process, including the amount of water used, the heating regime, the pH of the solution and
shear stress induced by mechanical mixers and pumps [20].

markets. For example, in the United States, starch
-
based materials have captured abou
t 25% of the
loose
-
fill pack
aging market from the expanded polystyrene
-
type materials [48]. With the growing
global concern for the en
vironment, the use of starch in biodegradable plastics is an area that is
expected to show tremendous future growth.

In
the food industry, the largest industrial user of starch, the ability of a starch to change the
viscosity of other so
lutions is the most important functional property. Paste producing starches are
used to thicken products such as soups, sauces, gravies an
d dairy products, while starches that
readily retrograde (high amylose) are used to make gum candies and coatings for other
confectionary prod
ucts [1, 20, 39]. Starches can contribute to a wide range of other food quality
traits in addition to viscosity (
for a re
view see [20]). For example, in baked goods, starch com
-
position affects texture, moisture retention and shelf life. High amylose starches are important in
batters as they facilitate batter adhesion, even browning during cooking, and reduced oil u
ptake by
the product. Starches are also used as an emulsion stabilizer in salad dressings and to improve the
freeze
-
thaw stability of several products.

Recently, in the developed world, the adoption of health
ier lifestyles has become more popular.
This ha
s resulted in a marked increase in the amount of low
-

and no
-
fat food products available to
the consumer. In many cases, the appearance, texture, flavor and mouthfeel of the low
-
and no
-
fat
products have been maintained through the use of starch
-
based fat r
eplacers [20]. Furthermore,
tremendous interest currently surrounds the health bene
fits of ,,resistant starch" present in
retrograded high amylose starches. Resistant starches are not readily digestible in the small intestine
and it has been postulated th
at they promote the growth of microorganisms that are beneficial to the
gastrointestinal tract [43]. The de
mand for starch in the food industry is expected to increase as the
types of food products available to the consumer, including those with health be
nefits, continue to
grow.

In addition to the food industry, there are many other in
dustrial uses of starch (Tab. 2). For
example, starches with high levels of amylopectin are used in paper coatings and adhesives [44]. In
oil exploration, starches are used

as viscosity modifying agents in drilling muds [39, 45], while in
the construction industry, significant amounts of starch are used in joint compounds for finishing
gypsum panel walls [39]. Both granular starches [46] and gela
tinized starches [47] have b
een used
as fillers to make biodegradable plastics. While this market is still in its in
fancy, significant
progress has been made in some niche

The pastes and gels of native starches do not possess the physicochemical attributes necessary to
meet the very

diverse range of functional properties required by in
dustry. Therefore, in most cases,
it is necessary to chem
ically or physically modify the starch (see [20] for a re
view). The various
starch modifications, the details of which are beyond the scope of

this article, enhance the
functionality of the starch in many ways including: chang
ing the paste viscosity, stabilization of
paste viscosity, im
proving the clarity, water retention, freeze
-
thaw stability and adhesive properties
of the paste. The chemica
l and physical treatments used to modify starch are costly and in some
cases, environmentally unfriendly. With the de
velopment of molecular biology and genetic
engineering technologies, the challenge for the future is to modify starch biosynthesis
in plan
ta
in
order to produce novel starches. While the process of starch biosynthesis is complex and
incompletely understood, significant progress in this area has been made in the last decade [49].

6 An Update on Starch Biosynthesis Pathway

During photosynthesi
s transitory starch is synthesized in chloroplasts. During the night the
transitory starch is de
graded and transported as sucrose to amyloplasts of stor
age organs, where it is
incorporated into storage starch. Despite several decades of intensive researc
h on storage starch
biosynthesis in higher plants, the process is still in
completely understood. Progress in this area has
largely been acquired through the analysis of natural mutants of maize, rice, barley, potato and pea.
In addition, the study of the
monoceiiular alga
Chlamydomonas reinhardtii
has also provided
significant contributions in this regard. Un
der specific growth conditions,
Chlamydomonas
accumu
-
lates a polysaccharide that bears strong structural resem
blance to maize endosperm storage star
ch
[50]. Further
more, mutations affecting starch synthetic enzyme activi
ties in
Chlamydomonas
parallel those observed in maize [51]. Through studies in
Chlamydomonas
and higher plants, the
involvement of four different types of enzymes (ADP
-
glucose pyrop
hosphorylases, starch
synthases, branching enzymes and debranching enzymes) in starch biosynthesis has been well
documented [7, 9, 52].













































6.1

ADP
-
Glucose pyrophosphorylase

ADP
-
glucose pyrophosphorylase (AGPase) ca
talyzes the initial step in plant starch synthesis, the
production of ADP
-
glucose from glucose
-
1
-
phosphate and adenosine triphosphate. The relationship
between the level or activi
ty of AGPase and starch content has indicated that AG
Pase catalyzes the
rat
e
-
limiting step in starch biosynthe
sis for both photosynthetic and non
-
photosynthetic tis
sues [53].
AGPase exists as a tetrameric protein com
posed of two small and two large subunits, which are en
-
coded by distinct genes [54, 55]. It is believed that on
ly the small subunits possess catalytic activity,
whereas the large subunits may have a regulatory function [56]. Most plant AGPases are activated
by 3
-
phosphoglycerate (3
-
PGA) and inhibited by inorganic phosphate (Pj) [57]. How
ever, AGPases
from the endo
sperm of barley [54] and wheat [58] show no or insignificant responses to 3
-
PGA or
P|. In maize [59] and barley [60], plastidal and cytosolic isoforms of AGPases have been identified,
with the cy
tosolic activity predominating in the endosperm. Given the i
mportance of AGPase in
starch synthesis, it is a logi
cal target for manipulation by genetic engineering. The variation in
AGPase subcellular distribution and the regu
lation of AGPase activity indicate that different,
species
-
specific strategies will have

to be employed to manipulate starch biosynthesis through this
enzyme.

6.2

Starch synthases

Starch synthases (SS) catalyze the elongation of glucan chains through the introduction of an α
-
1,4
-
glucosidic linkages between the incoming glucose residues of AD
P
-
glucose and the growing glucan
chains. To date, four classes of SS have been identified in higher plants. One class is exclusively
granule bound (GBSS
-
types I and II), while the three other classes of starch synthases (I, II and
III)
may be located parti
ally or entirely in the soluble phase [7].

The role of GBSSI in starch synthesis has been well char
acterized as a result of studies using
amylose free
(waxy)
mutants.
Waxy
mutants, which have been identified in many plant species, lack
the 58
-
60 kDa GBSSI

{waxy
protein). Thus, GBSSI has been shown to be responsible for amylose
synthesis in plant storage organs [61]. Two models for amylose synthesis have been proposed [62].
In
the
first model, small
-
sized malto
-
oligosaccharides are used by GBSSI as a primer

for chain
elongation. In the second model, GBSSI extends a long outer chain of amy
-
lopectin
within the
starch granule, which is eventually
fol
lowed
by a cleavage step to form a free amylose mole
cule.
The latter model is supported by the observations
tha
t
in
Chlamydomonas,
GBSSI can influence
amy
-
lopectin
synthesis [63]. Recently, another isoform of

GBSS, GBSSII, was found in the pericarp, aleurone layer and embryos of both wild type and
waxy
wheats [64, 65]. Therefore, GBSSII is thought to be involved

in amylose synthesis in tissues other
than the endosperm.

SSI, SSII and SSI
II
are involved in amylopectin synthesis, although the roles for each enzyme class
have not been clearly established. SSI and SSII have been shown to be found in both the soluble
p
hase and as starch granule bound proteins in a number of different species [66
-
72]. SSIII is
exclusively soluble in maize [73] and wheat [74], but is both soluble and granule bound in potato
[75]. In contrast to GBSSI, which is functional only when bound t
o a starch granule, it is not clear
whether SSI, SSII and SSIII are functional enzymes when bound to the starch granule, or simply
become trapped inside the granule as it grows.

Studies of SS mutants and transgenic plants carrying an
-
tisense SS constructs
have demonstrated
the key role of these enzymes in the determination of the structure of amylopectin, and therefore
starch granules. To date no SSI mutants have been found. However, analyses of SSII mutants in pea
[67] and wheat [76], as well as the trans
genic antisense SSII potato [77] have demonstrated a
common pattern of changes in the starch from the mu
tants. Amylopectin branch length distributions
are altered with increased numbers of short chains (dp<10) and re
duced numbers of intermediate
-
sized ch
ains (dp 15
-
25). The significance of the changes in starch biosynthesis were reflected by
decreased starch content of the grains [67, 76], deformation of the starch granules [67, 76, 77], and
altered physicochemical properties of the starch [76, 77]. Throu
gh studies on the maize mutant,
dulH,
and transgenic potato carrying an antisense SSIII construct, it has also been shown that SSIII
contributes to amy
lopectin branch length determination [77, 78]. This effect results in alterations in
starch content, gra
nule morpholo
gy and starch physicochemical properties [77
-
79]. It has been
postulated that the different classes of SS make dif
ferent contributions to amylopectin synthesis [71,
72, 76, 80]. In order to effectively manipulate starch biosynthesis through
SS, it is first necessary to
clarify the respective contributions with additional research in this area.

6.3 Starch branching enzymes

Starch branching enzyme (SBE) introduces
α
-
1,6
-
gluco
-
sidic bonds to form branched
polysaccharides. Several isoforms of SBE have been identified in developing stor
age tissues of
higher plants [81]. SBE isoforms are clas
sified into two major groups based on their amino acid se
-
quences and the
in
vitro
catalytic properties of purified en
zymes. Type I SBE, which has been
identified in maize [82], wheat [83
-
85], potato [86], rice [87], pea [88] and











































cassava [89], has a higher affinity for amylose and trans
fe
rs longer glucan chains [85, 90, 91]. Type
II SBE prefers amylopectin as a substrate and transfers short chains [85, 90, 91]. SBEII has been
identified in maize [92], wheat [93], barley [94], potato [95], rice [96], and pea [18]. In de
veloping
pea embryos
, and kernels of wheat and maize, the
Sbe1
and
Sbe2
genes are differentially expressed.
Sbe2
expression begins at an early stage of development and continues throughout development,
while
Sbe1
ex
pression peaks at a later stage of development [84
-
86, 97].

The importance of SBEII in amylopectin synthesis is re
flected in the altered starch of
amylose
extender
mutants in maize, rice and pea [16
-
18]. These mutants, which lack SBEII, have reduced
total starch content, irregular gran
ule shape and size, and incr
eases in both the amylose content and
the average amylopectin branch length. Fur
thermore, the starch of these mutants also contains sig
-
nificant amounts of a glucan polymer termed Jntermedi
-
ate material", which has properties distinct
from amylose and amy
lopectin. The first SBE1 mutant was recently identified in maize [98]. There
was no apparent change in the leaf or endosperm starch in the SBEI mutants. In transgenic potato
tubers carrying antisense SBEI con
structs, SBE activity was reduced to approximat
ely 5% of
control. There was no difference in the proportions of amylose and amylopectin [99] or the
amylopectin branch length distribution [99, 100], but there was an increase in the average granule
size [100] and changes in the physi
-
cochemical propertie
s of the transgenic starches [99, 100].
Thus, SBEI may have more subtle effects on starch synthesis than SBEII. Based on the
in vitro
enzymatic properties, differential expression patterns and the inabil
ity of SBEI to compensate for
SBEII activity in the
amy
lose extender
mutants, it has been postulated that SBEI and SBEII have
different roles in starch biosynthesis [85, 90, 97].

Recently, a novel SBEI cDNA,
Sbeic,
was isolated from developing endosperm of wheat [101].
Sbeic
codes for a protein that is imm
unogenically related to, but much larg
er than previously
identified SBEI (152 kDa vs 80
-
100 kDa). It is an exclusively granule bound protein that is
preferentially associated with A
-
type (large) starch gran
ules. Furthermore, SBEIc is found only in
plant
species exhibiting a bimodal starch granule size distribution, which suggests that it may have a
role in the determina
tion of starch granule size and morphology [102].

6.4 Starch debranching enzymes

Starch debranching enzymes (DBE) cleave α
-
1,6 gluco
-
sidi
c bonds of a branched α
-
glucan in one
single step re
sulting in the liberation of the entire α
-
1,4
-
glucan chain.

Based on their amino acid sequences and substrate specificities, DBE are classified into two groups:
limit dex
-
trinase (also known as pullulana
se or R
-
enzyme) and isoamylase [103, 104]. Both classes
of DBE are soluble enzymes. While limit dextrinases are thought to exist as monomeric proteins,
reports indicate that isoamylase ex
ists as a multimeric protein in potato, rice and
Chlamy
-
domonas
[105
-
107].

It was originally thought that DBE are only involved in degradation of amylopectin in storage
starch during ger
mination or in transitory leaf starch. However, recent dis
coveries indicate that the
DBE are also involved in starch biosynthesis. Altho
ugh no limit dextrinase mutants have been
identified, a role for limit dextrinases in starch biosynthesis is suggested by their presence in
developing endosperm [108]. Evidence supporting the participation of isoamylases in amylopectin
synthesis has come f
rom studies on
sugary
-
1
mutants in maize and rice, as well as the STA7 mutant
of
Chlamydomonas.
In the
sugary
-
1
and STA7 mutants, amylopectin is replaced by a very highly
branched, water soluble polysaccharide (WSP) called phytoglycogen. Initially, reducti
ons in limit
-
dextrinase ac
tivity were associated with the
sugaryA
mutants of maize [109] and rice [110]. It was
later shown that the primary defect in the
sugary
-
1
mutants was in the isoamylase gene [111], and
that the reduction in limit dextrinase activ
ity was secondary to that defect [104]. Phytoglycogen ac
-
cumulation has also been observed in the
Chlamy
domonas Sta7
mutant, which lacks isoamylase
activity [112]. Based on these findings, two models have been de
veloped that postulate the role of
DBE in
amylopectin syn
thesis [7]. In the WSP clearing model, SS and SBE are proposed to
function both at the granule surface produc
ing amylopectin, and in the amyloplast stroma producing
WSP. In this model, the role of DBE is to degrade the WSP, which prevents
the diversion of SS and
SBE from the granule surface [113, 114]. In the second model, the glucan trimming model, DBE
cleave improperly posi
tioned
α
-
1,6 branches in a amylopectin precursor called "pre
-
amylopectin" to
allow for the correct alignment and growth of α
-
1,4 chains in amylopectin. This trimming is
essential for the synthesis of the highly
-
ordered structure of the amylopectin molecule, which

in
turn is necessary for the proper growth of the starch granule [7, 25]. While the exact mechanism(s)
of DBE action has not been elu
cidated, it is clear from recent research that DBE play a key role in
the determination of starch structure.

6.5 Other im
portant enzymes

As previously mentioned, both the phosphate content and chemical form of phosphorus differ in
tuber and cereal starches. In tuber starch the phosphate is covalently bonded to C3 or C6 positions
of the glucose monomers. A











































recent study has shown that an enzyme called the R1 protein is involved in phosphorylation of tuber
starch [36]. The R1 protein was subsequently shown to be an α
-
glu
-
can kinase [115]. Interestingly,
the binding of the R1
-
pro
tein to
transitory starch granules in potato leaves has been shown to be
reversible and influenced by unknown factors on the surface of the granule [116]. Further re
search
is required to identify the other enzymes and fac
tors involved in starch phosphorylation.

Several other enzymes with diverse catalytic activities may also participate in starch synthesis.
Disproportionat
-
ing enzyme (D
-
enzyme) was initially postulated to facili
tate starch degradation
through phosphorylases and hy
-
drolases by catalyzing the cond
ensation of short oligosaccharides
[117], as has been shown in
E. coli
[118]. In
Chlamydomonas,
observations suggest that D
-
enzyme
may be involved in starch synthesis and granule formation [119, 120]. However, starch content and
struc
ture was not signific
antly altered in transgenic potato tu
bers transformed with antisense D
-
enzyme constructs [121]. Additional research on the D
-
enzyme is required to determine its role in
starch synthesis in other crop species.

Through genetic engineering, it has also been
shown that starch quality and quantity can be affected
by manipulat
ing enzyme systems that are indirectly associated with starch synthesis. In dicots,
AGPase is only found within the plastids [122] and thus ATP, one of the substrates for AGPase,
must be i
mported into the plastids. Recently,
Tjaden
and associates [123] demonstrated that potato
tu
ber starch production and composition was correlated with the level of plastidial ATP/ADP
transporter activity. However, in maize [59] and barley [60] it has been
shown that AGPase is
primarily a cytosolic enzyme, and thus the cytosolic ADP
-
glucose must be translocated into the
amy
-
loplast for starch synthesis. In maize, this function is achieved by an adenylate transporter
called brittle
-
1 (bt
-
1) [124]. The importa
nce of bt
-
1 in starch synthesis in maize is reflected by the
decreased starch accumulation in
bt
-
1
mutants [79].

6.6 The complexity of starch biosynthesis

The complexity of starch biosynthesis is reflected by the participation of many different enzymes
and

their respec
tive isoforms, differences in the subcellular distribution of the enzymes and also by
temporal differences in the ex
pression of the genes involved. Determining the contribu
tions of each
enzyme to amylose, amylopectin and gran
ular structure

has been made more difficult, because there
is also a strong interdependence between the en
zymes involved. Mutations in one enzyme often
produce pleiotropic effects. Some examples include: the
dull1
mu
-

tant of maize in which a defect in the gene coding

for SSIII results in reduced activity of both SSIII
and SBEIIa [125]; the
rug5(SSW)
mutant in pea in which GBSSI activity is in
creased in the absence
of SSI I [67]; and the
sugary
-
1
mu
tant in maize in which a defect in the isoamylase gene re
sults in
re
duced expression of both isoamylase and limit dextrinase [104]. With the advent of molecular
biology techniques, significant progress in the understanding of starch biosynthesis has been
achieved in the last decade. In the future, the main challenge for re
searchers in this area is to
develop new technologies, that will be able to find relationships between starch biosynthesis gene
ex
pression and the structure of amylose, amylopectin and the starch granule. Efficient manipulation
of starch biosynthesis thro
ugh traditional plant breeding and genet
ic engineering will be possible
only when a more thorough understanding of the process has been achieved.

7
In Planta
Modification of Starch Synthesis

7.1 Increased starch quantity

To date, manipulation of two diffe
rent metabolic pathways has produced plants with increased
starch content. AG
Pase, the rate limiting enzyme in starch biosynthesis, has been the focus of
investigations to increase starch pro
duction and/or content in potato and maize. A 30% in
crease in
starch content was observed in transgenic po
tato expressing an £
coli
AGPase that is insensitive to
regulation by P
i

an inhibitor of potato AGPase [53]. Simi
-
larily, site
-
specific mutagenesis was used
in maize to pro
duce a mutant AGPase large subunit tha
t was also insen
sitive to P
i
. In this mutant,
seed weight was increased 11
-
18% without increasing or decreasing the percentage of starch [127].
Increased starch content in potato was also achieved through the manipulation of a different
enzyme, inorganic

pyrophosphatase (PP
i
). Expression of an
E. coli
PPase in transgenic potato
increased starch content by 20
-
30% [128]. A third approach to increase starch content in cereals that
is worthy of consideration is the manipula
tion of Bt
-
1, the plastidial adenyl
ate transporter [124]. Bt
-
1 mutants in maize have markedly reduced starch con
tents [79], and thus over
-
expression of Bt
-
1
may lead to increased starch content.

Johnson
et al. suggested that increasing the quantity of native starch produced in maize was no
t a
economically viable alteration in starch metabolism [129]. However, in some cases the
augmentation of starch synthesis may be necessary in order to make the production of unique
starches or other traits economically viable. Starch syn
thesis mutants of
ten have significantly
reduced starch contents associated with qualitative changes to the starch [75, 79, 126]. Therefore,
increasing the starch content in












































mutants may be necessary to increase the yields of the mu
tant starches.

Increasing starch quantity through genetic engineering may also be important in the development of
low phytate grains. Significant nutritional and environmental advan
tages are associated with the
reduction of phytate levels in grains [130].

However, in the low phytate maize mu
tants
characterized to date, there is a significant reduction in seed dry weight [131]. Increasing starch
synthesis through genetic engineering may be necessary to confer agronomic viability to this
potentially valuabl
e trait.

7.2 High phytoglycogen starches

Phytoglycogen is a highly
-
branched, water
-
soluble poly
-
saccharide that is produced in addition to
starch in the
sugary
-
1
mutants of maize [79] and rice [110], and the
Sta7
mutant in
Chlamydomonas
[112]. In both the
maize and rice mutants, the phytoglycogen content is approxi
mately 25
-
30% of
total carbohydrates. Increasing the phytoglycogen content of maize to levels >30% could pro
duce a
polysaccharide with unique functional properties including reduced viscosity, g
el formation and
retrograda
-
tion rate with increased water holding capacity [129]. In addition, it is speculated that
such a starch would have in
creased digestibility as a livestock feed, which could po
tentially have an
enormous economic impact [129, 132
]. Researchers have attempted to increase phytoglycogen
levels by stacking different mutant alleles in maize but they have not been able to produce a marked
increase in phytoglycogen content [78].

To date, no limit dextrinase mutants have been identified.
However,
in vivo
site
-
directed
mutagenesis systems are now available and the creation of a limit dextrinase mu
tant may prove
valuable in this regard.

There are other factors that must be taken into consider
ation with respect to the development of a
high
phyto
glycogen phenotype. First of all, the presence of phyto
glycogen in the
sugary
-
1
mutant is
associated with a re
duction in dry seed weight [79]. Increasing starch quanti
ty through genetic
engineering, as discussed in the previ
ous section, is one ap
proach that may be necessary for the
development of plants with a high phytoglycogen phe
notype. Secondly, the physiological role of
the starch granule as a non
-
hygroscopic, non
-
osmotic energy store must also be considered. A
significant reduction in starc
h levels as a result of increased phytoglycogen content may produce
problems with germination and therefore agro
nomic viability. Thus, there may be a limit in the
degree that phytoglycogen content may be increased.

7.3

Phosphorylated cereal starches

The
increased quantity and the chemical nature of the phosphorus in tuber starch contribute to its
superior func
tional qualities over cereal starch for many applications [6]. Using currently available
transformation techniques, it may be possible to produce p
hosphorylated starches in wheat, barley
and maize with endosperm
-
specific expres
sion of the R1 protein [36]. Down
-
regulation of R1
protein expression in potato and the corresponding reduction in starch phosphate content produced
starches with re
duced pea
k viscosity and elevated setback viscosity [41, 133]. The opposite
changes, increased peak viscosities and reduced setback, would therefore be anticipated in
phosphorylated cereal starches. Such changes would en
hance the utility of cereal starches for
ind
ustrial purposes. The exact nature of the changes to the cereal starch that would result from
phosphorlyation cannot be predicted as there are other differences between cereal and potato starch,
such as amylose content, the average size of amylose molecule
s, phospholipids content and granule
size. In addition to producing starches with novel proper
ties, the development of phosphorylated
cereal starches would create an additional system for researchers to in
vestigate R1 protein
-
mediated
amylopectin phospho
ryla
-
tion and the role of covalently bound phosphate in starch functionality.

7.4

Starch with intermediate amylose levels

"Partially
waxy
starch, i.e. starch with amylose levels be
tween normal and
waxy
starch, was
identified as a poten
tially valuable qu
ality trait in maize [129]. While this goal may be difficult to
achieve in maize because of its diploid genome, this trait is currently being developed in hexa
-
ploid
wheat (AABBDD,
2n=6x=42)
through traditional plant breeding. Screening of wheat germplasm
has iden
tified GBSSI mutants in each of the three wheat genomes [13, 134]. Recombination of
these mutant alleles has re
sulted in endosperm starch with intermediate amylose levels. For
example, a partially
waxy
wheat line with mu
tations in GBSSI B and GB
SSI D loci was shown to
have an amylose content of approximately 12.5 %, which was associated with unique crystallization
patterns and gela
-
tinization properties [135]. The properties of and uses for the partially waxy starch
are currently under investigat
ion.

7.5

Large or small granule starches

Starch granule size is an important factor in several in
dustrial processes including the production of
thin films [40], paper coatings, cosmetic products [1, 39], and car
bonless copy paper [38].
Furthermore, star
ch granule size is important in the brewing process. A significant por
tion of the
small B
-
type granules from barley is not com
-













































pletely gelatinized in the mash and the undegraded residue causes mechanical proble
ms during
subsequent processing, e.g. filtration [136]. There are significant addi
tional processing costs
required for the isolation of large or small granules. Thus, the
in planta
production of starches with
predominately large or small granules would be

very desirable.

Very little is known about the processes determining starch granule size. A few studies have
indicated that SBEI may be involved. Studies using antisense SBEI constructs have reported
increased granule size in trans
-
genic potato [100], and

an increased proportion of large A
-
type
granules in transgenic wheat [137]. It has also been suggested that SBEIc may play a role in the
deter
mination of starch granule size and morphology in wheat and barley, based on the observations
that: i) it is onl
y present in starches from plants with bimodal granule size distributions, and ii) it is
preferentially associated with A
-
type granules [102]. If SBEI or SBEIc are key factors in the
bimodal starch granule size distribution in wheat and barley, then down
-
r
egulation or a mutation in
either pro
tein should alter the proportion of A
-

and B
-
type granules. Thus, both transgenic antisense
SBEI and/or SBEIc ap
proach or traditional plant breeding techniques with muta
-
genesis could be
taken to investigate this poss
ibility. Giv
en the diploid and hexaploid genomes of barley and wheat
respectively, barley would be the plant of choice for initial investigations.

7.6 Starches with altered chain lengths and branching patterns

The molecular structures of amylose, amylopec
tin and the starch granule are important determinants
of the func
tional properties of a starch. There is potential to produce a wide range of new starches
through the alteration of the glucan chain lengths, branching patterns and granule crystallinity
[12
9]. Mutations in several different starch synthetic enzymes have been shown to affect the branch
length and degree of branching of amylopectin [67, 76, 77] resulting in altered physicochemical
properties of the starch [76, 77]. Given the many different enz
ymes in
volved in amylopectin
biosynthesis, various modifications to amylopectin structure and a diverse range of concomi
tant
functional changes are possible. Some functional changes in maize, including improved gel
formation and stabilized viscosity have

been postulated to have a signif
icant economic value in
maize [129].

In the future, through genetic engineering it will be possi
ble to manipulate the properties of
amylose. In wheat and barley phospholipid
-
complexed amylose affects several aspects of st
arch
quality including gelatinization tempera
ture and granule swelling [138], peak viscosity [139] and

paste clarity [42]. Amylose synthesis is controlled almost exclusively by GBSSI [62]. Mutant
GBSS1 isoforms with novel enzymatic properties could be dev
eloped
ex vivo
and expressed in
waxy
mutants of wheat and barley in or
der to produce amylose with modified structures (branch length,
degree of branching, etc.). The association of phospholipids with the mutant amylose molecules
may be altered, which in t
urn could produce starches with novel physicochemical properties.

7.7 Development of starches with β
-
linkages

The introduction of p
-
linkages has been identified as a po
tentially valuable modification to maize
starch [129]. The presence of intermittent p
-
l
inkages in amylose or amy
lopectin could result in
reduced digestibility, and thus be valuable as a source of resistant starch. A cDNA for cellu
lose
synthase (CS), the enzyme which assembles the cel
lulose molecule from UDP
-
glucose, has been
cloned [140].

However, altering amyloplast metabolism to intro
duce p
-
linkages into starch or
produce cellulose
-
like ma
terial will not be achieved simply by transforming plants with the CS
cDNA driven by an amyloplast specific pro
moter. Several other factors must be
modified, all of
which could conceivably be achieved with current molecular bi
ological techniques. For example,
CS is an integral plas
ma membrane protein that has an extra
-
cellular domain which attaches to the
cellulose polymer [141]. Therefore, a CS tra
nsgene would have to be structured so that the protein
would be targeted to the amyloplast membrane. Furthermore, for the introduction of p
-
linkages, the
cellu
lose binding domain of CS would have to be modified to use amylose or amylopectin as the
elongat
ion primer. Analysis of genes coding for other amyloplast membrane proteins could provide
some information with regards to the targeting of the transgenic CS, and with modern mol
ecular
biological techniques, it is possible that a recombi
-
nant CS gene with

the ability to elongate
amylose or amy
lopectin chains could be created. While there are un
doubtedly several other
metabolic obstacles that would have to be overcome for the possibility of p
-
linkages in starch to be
realized, the costs for developing thi
s type of starch are small compared to the potential value of
prod
ucts [129].

8 Conclusion

Starch is currently used in a wide range of different prod
ucts and the list of starch uses is expected
to increase. At present, the versatility of starch for these

industrial pur
poses is achieved largely
through chemical or physical modification of the starch. In the future, it will be possible to alter
starch synthesis
in planta
to produce improved starches, which may reduce or eliminate the need for













































post
-
harvest modification. Such starches would benefit industry by reducing processing costs, and
the environ
ment by reducing the need for the chemical treatments.

There are numerous reports of
in planta
starch modifica
tion

in the literature that have increased our
knowledge of starch biosynthesis. It is hoped that some novel, econom
ically valuable starches will
be produced in the near future through further investigations that modify starch synthesis
in planta.
In most cas
es however, the value of these in
vestigations will be in the development of well
-
defined
links between the genes controlling starch biosynthesis, the chemical composition of the starch, the
association of all the components of starch within the granules a
nd the physicochemical properties
the starch. Once these links have been established the production of tailor
-
made starches for
specific industrial needs will become a realis
tic possibility.

Acknowledgements

S.
Netrphan
gratefully thanks The Royal Thai Go
vern
ment for the graduate scholarship. Ms.
Karen
Caswell
is acknowledged for her help in the preparation of this man
uscript.

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(Received: June 11, 2002)

(Revised: October 21, 2002)

(Accepted: October 21, 2002
)