Download The control of amylose synthesis

J. Plant Physiol. 158. 479 – 487 (2001)
 Urban & Fischer Verlag
http://www.urbanfischer.de/journals/jpp
The control of amylose synthesis
Kay Denyer*, Philip Johnson, Samuel Zeeman, Alison M. Smith
John Innes Centre, Norwich Research Park, Colney, Norfolk NR4 7UH, UK
Received October 16, 2000 · Accepted January 12, 2001
Summary
Starch granules are composed of two types of glucose polymer, amylose and amylopectin, that differ
in size and structure. One of the most intriguing challenges in understanding starch synthesis is to
explain the apparently simultaneous synthesis of two such different polymers. One isoform of starch
synthase, GBSSI, is responsible for amylose synthesis but can also contribute to amylopectin synthesis. The factors which determine the partitioning of GBSSI activity between these two processes
are largely unknown. Understanding the properties of GBSSI and how these differ from the properties of the amylopectin-synthesising isoforms of starch synthase are important to the understanding
of the control of amylose synthesis. In this review, we will describe how the synthesis of amylose and
amylopectin are integrated and what factors may determine the relative amounts of these two polymers.
Key words: starch – amylose – amylopectin – starch synthase – GBSSI – malto-oligosaccharide
Abbreviations: GBSSI granule-bound starch synthase I. – SS starch synthase. – ADPG adenosine
5′-diphospho-glucose. – MOS malto-oligosaccharide. – dp degree of polymerization
Introduction
Starch is a major storage product of many of the seeds and
other plant storage organs that we are most familiar with,
those we eat. Yet despite the fact the starch is commonplace,
we still do not completely understand the structure of starch
or how it is synthesised. If we are to manipulate starch synthesis in crop plants to provide more starch or starches with
particular desirable qualities, we must first understand the
process of starch synthesis and how it is controlled. One of
the most intriguing challenges in understanding how starch is
made is to explain how the two very different polymers which
constitute starch, amylose and amylopectin, are synthesised
* E-mail corresponding author: [email protected]
in the same place at the same time. Although there are still
questions to be answered, some significant progress towards
this goal has been made recently. For recent reviews of the control of amylopectin synthesis see Myers et al. (2000) and Smith
et al. (1999). In this review, we will briefly describe the structure
and composition of starch and then describe in more detail
what is known about the mechanism of amylose synthesis and
how the amount of amylose in starch might be controlled.
The structure and composition of starch
The basic unit of the starch granule is a branched polymer of
glucose called amylopectin. Each molecule of amylopectin is
composed of many short chains of glucose molecules. These
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1,4-linked chains are joined together by 1,6 branch points
and the chains are arranged into clusters in which adjacent
chains form double helices (for review, see Thompson 2000).
Amylopectin is one of the largest natural polymers, the
molecular masses of amylopectin molecules from most plants
being greater than 108 (Banks and Greenwood 1975). Amylopectin molecules are arranged radially within the starch granule with the ends of the chains pointing towards the surface
(French 1984). The clusters of double-helical chains in adjacent amylopectin molecules can align themselves to form
semi-crystalline arrays with in the granule. However, not all of
the granule is crystalline. There are alternating concentric
layers of amorphous and semi-crystalline amylopectin which
are visible, in some circumstances, as growth rings within the
granules. We do not know the orientation or arrangement of
amylopectin with in the amorphous layers. For more details of
the organisation of amylopectin within starch granules, see
Gallant et al. (1997).
It is possible for a starch granule to be composed entirely
of amylopectin. However, most starches also contain amylose, another sort of glucose polymer. During storage organ
development, the amylose content usually increases with
age. For example in maize seeds, the proportion of the starch
that was amylose increased from 18 % to 26.5 % between 14
and 28 days after pollination (Tsai et al. 1970). Most seed
storage starches have a final amylose content of 25 – 30 %
(Deatherage et al. 1955).
Compared to amylopectin, amylose is a smaller molecule
with longer chains and a limited number of branch linkages.
The long chains of amylose have a high capacity to bind iodine in solution and this imparts a blue colour to amylosecontaining starches when these are stained with iodine. Amylopectin has a much lower iodine-binding capacity and stains
red-brown with iodine solution.
The amylose fraction of starch comprises both linear and
branched molecules (Takeda et al. 1987). The ratio of linear to
branched molecules varies according to the origin of the
amylose. For example, in rice starch, two thirds of the amylose is unbranched. The number of chains per branched
amylose molecule varies and is usually lower for cereal starches than for non-cereal starches (e.g. branched corn amylose
has on average 5.3 chains whilst that from tapioca has 17.1;
Takeda et al. 1993). For rice amylose, the average number of
glucose units (dp) in the branched molecules is 1180 whilst
that of the linear molecules is 740 (Takeda et al. 1993).
The location of amylose within the amylopectin matrix of
the granule is not entirely known. Amylose in solution very
readily crystallizes but in the starch granule, amylose is not
crystalline. It is supposed that amylose resides largely in the
amorphous regions of the granule. In support of this, bluestaining rings have been observed in the starch granules of
low-amylose potato tubers when stained with iodine (Kuipers
et al. 1994). However, some amylose may also be present
within the semi-crystalline areas of the starch granule (Jenkins
and Donald 1994). The distribution of amylose within starch
granules has been investigated by J-L Jane and colleagues.
Partial gelatinization of potato starch granules showed that
amylose at the periphery of the granule is more concentrated
and has a smaller molecular size than amylose at the core
(Jane and Shen 1993). Experiments with cross-linking agents
(Jane et al. 1992) have shown that in potato and corn starch,
individual amylose molecules are interspersed amongst the
amylopectin molecules rather than being grouped together.
When heated in water, starch forms a thick paste which
makes it a valuable ingredient in many processed foods. The
texture of cooked starch is influenced by its amylose content
(White 1994). Starches with a normal amylose content form a
paste which lacks clarity and tends to retrograde (recrystallize). This gives the starch paste very poor freeze – thaw stability. This retrogradation is due largely to the crystallization of
amylose molecules. Consequently, starches with no amylose
produce pastes with little tendency to retrograde. Also, amylose free starches are more easily gelatinized than starches
with a normal amylose content and form clearer pastes.
Starches from mutants with low starch-branching enzyme activity, such as the ae mutant of maize, have a higher than
normal amylose content (50 – 60 % of the starch). These highamylose starches gelatinize at higher temperatures than
normal starch and produce very viscous pastes with a strong
tendency to retrograde. Separated amylose can be used to
form strong transparent films and therefore high-amylose
starches are being investigated as a potential source of a
natural industrial polymer for the manufacture of, for example,
biodegradable plastics.
In the following sections we will describe in more detail the
synthesis of the amylose component of starch. However, it is
clear that the synthesis of amylose is intimately linked to and
dependent upon the synthesis of amylopectin.
The mechanism of amylose synthesis
Both amylose and amylopectin are synthesised by enzymes
called starch synthases. These add the glucose residue from
ADPglucose to the non-reducing end of a growing glucan
chain. Starch synthases can be divided into four classes on
the basis of their primary amino acid sequences (SSI, SSII,
SSIII and granule-bound SSI [GBSSI]). Accumulating evidence suggests that all four types of starch synthase are present in storage organs. The analysis of mutants and transgenics with reduced amounts of individual isoforms shows that
each plays a unique role in the synthesis of amylose and
amylopectin (Kossmann and Lloyd 2000, Smith et al. 1999).
Only one of the isoforms of starch synthase, GBSSI, is required for amylose synthesis. There are well-characterized
mutants of many species that lack amylose and all of these
specifically lack GBSSI activity. The other isoforms of starch
synthase can not substitute for GBSSI in the synthesis of
amylose. They must, therefore, be primarily or entirely in-
The control of amylose synthesis
Figure 1. Domain comparison of starch synthase sequences from
maize endosperm. Proteins sharing domains homologous to those
present in maize SSIII were identified and aligned using the ProDom
database of protein domain families (http://www.toulouse.inra.fr/
prodom.html). The four maize SS sequences shown are presented as
examples of this alignment. The SwissProt ID of these sequences are:
GBSSI, P04713; SSI, O49064; SSII, O48900; SSIII, O64923. Three
domains were identified within these SS sequences by ProDom. Domain I is shown in black, domain II in grey and domain III is hatched.
The length of the bars corresponds to the number of amino acids represented.
volved in amylopectin synthesis. So to understand amylose
synthesis we need to discover what is unique about GBSSI.
In amino acid sequence, GBSSI shows strong similarity to
the other isoforms of starch synthase (Ball et al. 1998, Kossman et al. 1999, Li et al. 1999). At present, there is no information to indicate which regions of GBSSI confer the unique
ability to synthesise amylose or which regions of the other
isoforms of starch synthase prevent this function. Sequence
analysis (ProDom; Corpet et al. 1998) shows that all isoforms
of starch synthase share three homologous domains (Fig. 1)
and vary most in the length of the sequence at the N-terminal
region, preceeding domain I. Domain I is found in no enzymes other than starch synthases. In contrast, domain II is
also found, for example, in yeast α-amylase and domain III,
which is a putative glucosyl transferase domain, is also found,
for example, in sucrose synthase and sucrose phosphate
synthase. Based on affinity labelling and site directed mutagenesis, it was shown that the lysine residue (lys 15) in a conserved motif (KTGGL) formed part of the ADPG binding site
of E.coli glycogen synthase (Furukawa et al. 1990, 1993).
ADPG is the glucosyl donor in the reaction catalysed by glycogen and starch synthases. This motif is conserved between
glycogen and starch synthases (e.g. KTGGL in domain I of
GBSSI from maize). Thus, it was assumed that this motif
could be the ADPG binding site in the plant enzymes also.
However, there is a very similar motif in domain III of the
starch synthases (e.g. STGGLV in GBSSI from maize). Site directed mutagenesis of the lysine in this motif in domain III (lys
497) of maize SSIIa suggests that it, rather than the motif in
domain I, may function as the ADPG binding site in plant
starch synthases (Zhong et al. 2000).
GBSSI is known to differ from the other starch synthase
isoforms in its location within the plastid. GBSSI is almost en-
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tirely starch granule-bound whereas other starch synthase
isoforms and also isoforms of starch-branching enzyme are
located both within the granules and in the surrounding
stroma (Denyer et al. 1997b). There is good evidence to suggest that GBSSI is distributed throughout the granule interior,
not just attached to the surface of the granule, and that at
least part of the buried GBSSI is active. For example, GBSSI
protein in isolated granules is inaccessible to proteases
(Rahman et al. 1995) and GBSSI activity is not removed by
treating granules with salts or detergents (Tanaka et al. 1967).
These results show that GBSSI is buried within the amylopectin matrix. GBSSI activity can be measured in isolated intact
granules showing that some of the buried GBSSI is active
and that ADPG can penetrate to the site of GBSSI within the
granule. However, the activity of GBSSI is increased if the
granules are broken open by grinding (Hylton et al. 1996) or
chemically gelatinised with urea (Frydman and Cardini 1967)
showing that not all of the GBSSI within intact starch granules
is active or accessible to ADPG.
Evidence that GBSSI synthesises amylose within the granule matrix in vivo came from studies of transgenic potatoes
(Visser et al. 1991, Kuipers et al. 1994, Tatge et al. 1999). In
potato tubers in which GBSSI was specifically reduced by
expression of antisense RNA, the amylose content of the
starch was much reduced. However, rather than an even distribution of the small amount of amylose within these granules, iodine staining revealed that most or all of the amylose
was confined to a central core region. Careful measurements
of the proportion of the granule occupied by the amylose in
tubers of different sizes from young and old plants showed
that the core size increased as the granule size increased
(Fig. 2). This showed that amylose is synthesised deep within
the granule as the granule grows by deposition of amylopectin at the surface. In the transgenics with very little GBSSI,
amylose synthesis is slow and lags behind amylopectin synthesis resulting in amylose being confined to the core regions
of granules. In wild type plants with a higher GBSSI activity,
the synthesis of amylose probably keeps pace with the syn-
Figure 2. The relationship between developmental age and the proportion of the granule containing amylose in transgenic potatoes with
reduced activity of GBSSI. The diagram is based on the work of
Tatge et al. (1999). The granules were stained with iodine solution.
This revealed a blue-staining core-region containing amylose (indicated in grey in the diagram) and a red-staining peripheral region (indicated in white) consisting of amylopectin only. The size of the blue
core increased as the volume of the granule increased indicating that
amylose is synthesised inside the amylopectin matrix of the granule.
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thesis of amylopectin so that the synthesis of the newest
amylose molecules occurs just under the granule surface
and no peripheral amylose-free zone is visible.
The fact that amylose synthesis occurs within the granule
whereas the bulk of the synthesis of amylopectin occurs at
the surface, could potentially explain aspects of the different
structures of these polymers. It is likely, for example, that glucans synthesised inside the granule are not as susceptible to
branching as glucans at the surface of the granule. However,
this alone can not explain the unique role of GBSSI. There are
isoforms of starch synthase other than GBSSI within starch
granules (Denyer et al. 1997a). Mutations that eliminate GBSSI
and amylose do not eliminate these isoforms from starch
granules. This shows that being buried inside the granule is
not the only requirement for an amylose-synthesising starch
synthase. GBSSI must have other unique properties that allow it alone to synthesise amylose.
The unique properties of GBSSI
To gather information about the unique properties of GBSSI,
we studied its activity in starch granules isolated from developing pea embryos (Denyer et al. 1996). Granules were supplied with radiolabelled ADPG then solubilized and the polymers were size-fractionated to separate the amylose and
amylopectin components. We found that rather than incorporating glucose into amylose as it would in the plant, GBSSI in
isolated granules incorporated glucose largely into the amylopectin fraction. We postulated that the reason for the lack of
amylose synthesis in vitro was that small soluble glucans
necessary for amylose synthesis had been washed out of the
granules during their isolation. Evidence to support this idea
came from experiments in which granules were prepared
without the usual extensive washing. These showed higher
rates of incorporation of glucose into amylose relative to amylopectin. Also when pure samples of malto-oligosaccharides
(MOS) consisting of 2–7 glucose units (dp 2–7) were added
to isolated granules, amylose synthesis was greatly stimulated. The stimulation of amylose synthesis by MOS is not
unique to isolated pea granules. MOS stimulates amylose
synthesis in granules isolated from potato (Denyer et al. 1996),
maize (Denyer and Keeling, unpublished results) Arabidopsis
(Zeeman and Smith, unpublished results) and the unicellular
alga, Chlamydomonas reinhardtii (van de Wal et al. 1998).
These experiments with isolated granules suggested a
possible mechanism for amylose synthesis in which GBSSI,
but not other isoforms of starch synthase within the granule,
synthesises amylose by elongating MOS primers. The MOS
must be able to diffuse into the granules to the site of GBSSI
activity. There are several pieces of evidence to support this
mechanism. First, granules containing GBSSI showed MOSdependent amylose synthesis whereas granules from the lam
pea mutant, which contain SSII but not GBSSI, showed no
amylose synthesis in the presence or absence of MOS (De-
nyer et al. 1996). Second, in experiments with radiolabelled
maltose (dp 2) and unlabelled ADPG, radiolabelled amylose
was formed showing that the maltose was elongated (Denyer
et al. 1999 a). Thus, MOS acts as a primer for amylose synthesis rather than as an activator of GBSSI which stimulates
the enzyme to elongate glucans already present in the granule. Third, it is known that granules are permeable to molecules up to approximately 1 kDa in size, roughly the size of
maltoheptaose (French 1984).
In these initial experiments, we looked at the insoluble
products of the granule-bound isoforms of starch synthase
only. In later experiments (Denyer et al. 1999 a), we also
looked at soluble products made by granule-bound starch
synthases during incubations of isolated granules. These soluble products consisted of elongated MOS. These experiments showed that MOS diffuses into the granules, is elongated and then some or all of the elongated MOS diffuses
back out of the granules.
In experiments with granules containing only SSII, the soluble products were always only one glucose unit longer than
the supplied MOS e.g. if we supplied maltotriose, the product
was maltotetraose. In experiments with granules containing
only GBSSI, products more than one glucose longer than the
supplied MOS were also observed. This suggests that the
mechanism of elongation by GBSSI is different from that of
SSII. When SSII binds to maltotriose, for example, it adds a
single glucose unit from ADPG to make maltotetraose and
then the enzyme and product dissociate. SSII must then bind
to another MOS, most likely another maltotriose molecule
(since the concentration of this substrate in our assays was
very much higher than the concentration of the product, maltotetraose) to make a second product molecule. In contrast,
when GBSSI binds to maltotriose and elongates this to give
maltotetraose, it does not always then dissociate from the
product. In some cases, GBSSI is able to add another glucose unit from ADPG to the maltotetraose to make maltopentaose and then another to make maltohexaose and so on.
Elongation in this manner is called processive elongation.
This processive reaction mechanism may, in part, explain
the unique ability of GBSSI to synthesise amylose. However,
there are probably other equally important factors that allow
amylose synthesis to take place. First, GBSSI within starch
granules has much higher affinity for MOS than does SSII (at
least 20-fold higher; Denyer et al. 1999 a). This means that at
the low concentrations of MOS likely to be present in vivo
(see later), GBSSI will elongate MOS much more actively
than SSII. Second, the limited permeability of the amylopectin matrix allows short MOS to diffuse readily in and out of the
granule but longer MOS synthesised by GBSSI can not escape from the granule. The trapping of long MOS within the
granule means that these molecules can be further elongated by GBSSI to eventually form mature amylose molecules.
In experiments in which GBSSI was expressed in soluble
form in E. coli, it was observed that, unlike the granule-bound
The control of amylose synthesis
483
switched from the A-type crystal structure normally found in
Chlamydomonas to the B-type.
This work on Chlamydomonas starch led to an alternative
model for amylose synthesis, not involving MOS (van de Wal
et al. 1998, Ball et al. 1998). In this model, GBSSI elongates
amylopectin to create extremely long chains. These amylosesized chains are then cleaved from the amylopectin by an as
yet unidentified hydrolyase activity. What prevents the putative hydrolase from cleaving glucan chains smaller than amylose from the amylopectin is not clear. In similar experiments
with starch from pea embryos, no transfer of radioactive glucose from the amylopectin to the amylose fraction was observed (Denyer et al. 1999 a). However, GBSSI in Chlamydomonas, as in higher plants, can elongate MOS in vitro to generate amylose. Thus, in Chlamydomonas, amylose can be
generated in vitro by two different mechanisms whilst in higher
plants, amylose synthesis from MOS only has been demonstrated. Which of these two mechanisms, if either, operates in
vivo is not yet known.
Figure 3. A model to explain the synthesis of amylose from malto-oligosaccharide primers. GBSSI binds tightly to the amylopectin matrix
of starch granules. In this location, it has a high affinity for MOS. It
elongates MOS processively to make long glucan chains. These long
glucans are trapped within the amylopectin matrix and are further
elongated to form amylose.
enzyme, soluble GBSSI has a low affinity for MOS and does
not elongate processively (Denyer et al. 1999 b). However,
when supplied with a small amount of amylopectin, the affinity of GBSSI for MOS increases and the enzyme becomes
processive. So these important and unique kinetic properties
of GBSSI may be conferred on the enzyme by its interaction
with the amylopectin matrix of the granule.
To summarise this work, we suggest a model to explain the
unique ability of GBSSI to make amylose (Fig. 3).
GBSSI can elongate amylopectin chains
A number of studies of isolated starch granules have shown
that GBSSI incorporates glucose into amylopectin as well as
into amylose. The glucose is incorporated specifically into the
longest amylopectin chains (Baba et al. 1987, Denyer et al.
1996, van de Wal et al. 1998) suggesting that, as well as synthesizing amylose, GBSSI in vivo may contribute to the determination of the structure of amylopectin. In experiments with
starch granules isolated from nitrogen-starved Chlamydomonas cells, van de Wal et al. (1998) showed that the radioactive glucose initially incorporated into amylopectin was, after
extended periods of incubation (6 h), transferred to a lower
molecular weight fraction similar in size and structure to amylose. After even longer periods of incubation (24 – 48 h), more
label was transferred from amylopectin to amylose and the
crystallinity of the starch decreased from 27 % to 16 % and
Are malto-oligosaccharides present in
starch synthesising cells?
For amylose synthesis to occur from a MOS primer in vivo,
there must be a sufficient concentration of MOS available to
GBSSI within the plastid (0.1mmol/L maltotriose gives half the
maximal rate of amylose synthesis for isolated pea starch
granules, Denyer et al. 1999 a). In isolated granules, the elongation of MOS occurs at the expense of amylopectin elongation. Thus, the extent to which GBSSI uses amylopectin or
MOS as a substrate may depend upon the availability of MOS.
There are two pieces of evidence that MOS is present in
plant cells. First, heat-treated, soluble extracts of potato tubers stimulated amylose synthesis when added to isolated
potato starch granules together with ADPG (Denyer et al.
1996). The stimulatory material in the soluble extracts was
destroyed by pre-incubation with amyloglucosidase indicating that it was a glucan. Second, direct measurements of
MOS have been made for several plant organs. For example,
there are low levels of MOS (0.05 – 0.10 mg glucose-equivalents g –1 fresh weight), mostly in the form of maltose, in the
leaves of Arabidopsis thaliana throughout the day – night cycle
(Critchley et al. 2000). If certain assumptions are made (that
all of the MOS is maltose, that the maltose is confined to the
plastid, that 1 g of tissue has a volume of 1 mL, and that the
stroma occupies 7 % of the total cell volume) then it is possible to estimate that concentration of MOS in the chloroplasts of Arabidopsis leaves is 2 – 4 mmol/L. This is probably
sufficient to allow amylose synthesis to occur by MOS elongation.
In a mutant of Arabidopsis which lacks disproportionating
enzyme, MOS accumulates to very high levels, particularly at
night (up to 15-times higher than the MOS content of the wild
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type). Interestingly, the mutant accumulates more amylose in
its starch than the wild type. This may indicate that the level
of MOS in wild type Arabidopsis leaves is limiting for amylose
synthesis. However, there are metabolic differences other
than the increase in MOS concentration between the mutant
and wild type leaves and these may also differentially influence amylose synthesis.
MOS will be generated in vivo when starch is degraded or
modified. In the plastids of starch-synthesising storage organs and in chloroplasts in the light period, significant activities of several starch-degradative enzymes are known to be
present. These include amylases, starch-debranching enzymes and disproportionating enzyme (Bulpin and ap Rees
1978, Doehlert and Knutson 1991, Duffus and Rosie 1973, Takaha et al. 1993). A limited amount of starch degradation during the phase of starch accumulation in storage organs can
not be ruled out and may be involved in the creation of the
ordered structure of the amylopectin molecule (for review see
Myers et al. 2000, Smith 1999). The trimming of branched
glucans, for example by isoamylase, could contribute some
of the MOS required for amylose synthesis. It is not known
whether enzymes with hydrolytic activity exist within the granule. If they do exist, they might also contribute to the pool of
MOS available to GBSSI.
What controls the amount of amylose?
In the previous section we indicated that, in some circumstances, the availability of MOS could limit amylose synthesis.
In this section, we will discuss other factors that could potentially control amylose synthesis. These include the existence
of different forms of GBSSI that could potentially have different properties and functions, the activity of GBSSI, the availability of the substrate ADPG and the properties of the amylopectin matrix of the starch granule.
There is increasing evidence that different GBSSI genes
are expressed in different parts of the plant. Examination of
the granule-bound proteins in different parts of lam mutant
and wild type peas plants showed that only lam embryos entirely lacked a GBSSI-like protein (Denyer et al. 1997a). In the
other parts of the plant, we found that a GBSSI-like protein
was present even in the mutant. This protein is the product of
a second GBSSI gene which we called GBSSIb. GBSSIb is
expressed in leaves, pods and nodules but not in pea embryos. In pods, both GBSSI and GBSSIb are present but
GBSSIb accounts for most of the granule-bound starch synthase activity. Studies of wheat have also shown that GBSSIlike proteins are differentially expressed in different parts of
the plant. The GBSSI in wheat endosperm is a different gene
product from that in the rest of the plant (Fujita and Taira
1998, Nakamura et al. 1998).
The amylose content of starches from different parts of the
plant varies, with storage starches tending to have more
amylose than transitory starches. For example, in pea plants,
we found that the starch of leaves, pods and nodules had far
less amylose than that of the embryos (Denyer et al. 1997 a).
The existence of different forms of GBSSI in the various parts
of a plant raises the possibility that there may be some differentiation of function within the GBSSI-class of starch synthases. However, the properties of GBSSI from pea embryo starch
and GBSSIb from pod starch of the lam pea mutant are very
similar. First, the enzymes are present in these starches in similar amounts and activities. Second, both GBSSI and GBSSIb
can synthesise amylose in vitro when isolated granules are
supplied with MOS. Thus, the differing amylose contents of
starches from different parts of the plant are not obviously related to the fact that there are different forms of GBSSI.
Studies of mutant and transgenic plants have shown that
the activity of GBSSI in the storage organs of wild type plants
does not limit amylose synthesis. For example, in maize
seeds, Tsai (1974) showed that GBSSI activity was linearly
proportional to the number of Wx alleles present in the endosperm. However, the amylose content was not linearly related
to the activity of GBSSI. Endosperms containing two thirds of
the normal GBSSI activity have only slightly lower amylose
content than normal endosperms. This implies that increasing the GBSSI content above normal would result in little, if
any, increase in amylose content (Fig. 4). Studies of potatoes
with reduced GBSSI activity have shown a similar picture
(Visser et al. 1990, Flipse et al. 1996). The GBSSI activity had
to be reduced considerably below the wild type level before
there was a significant effect on amylose content.
Another potential factor controlling amylose synthesis is
the availability of the substrate, ADPG. There is good theoretical and experimental evidence to show that the availability of
ADPG could limit amylose synthesis in vivo (Clarke et al.
1999). First, GBSSI has a low affinity for ADPG (e.g. pea
starch granules, apparent kM = 1.4 mmol/L) compared to that
of the starch synthase isoforms responsible for amylopectin
Figure 4. The dosage effect of Wx genes on the production of GBSSI
activity and amylose content. Data are taken from Tsai (1974). The Wx
gene encodes an active GBSSI. Wild type maize endosperm, being
triploid, has three copies of the Wx gene. A mutant maize that lacked
GBSSI activity (zero Wx genes) was crossed to wild-type maize to
create plants with one or two copies of the Wx gene. The GBSSI activity is indicated by the circles and the amylose content by squares.
The control of amylose synthesis
synthesis (kM = 0.06 – 0.6 mmol/L, data from many species).
Estimates of the concentration of ADPG in plastids of developing pea embryos show that the ADPG concentration
(0.9 mmol/L) is much lower than that required to saturate the
GBSSI reaction. However, the ADPG concentration in plastids
is close to that required to saturate the reaction catalysed by
the amylopectin-synthesising isoforms of starch synthase.
This means that if the ADPG concentration were to fall, amylose synthesis would be reduced to a greater extent than
would amylopectin synthesis.
Second, work on some low-starch mutants of pea shows
that reductions in ADPG content are accompanied by reductions in amylose content (Clarke et al. 1999). Mutations at the
rb, rug3 and rug4 loci of pea affect enzymes involved in
ADPG production and consequently, the ADPG content of
these embryos is lower than that of wild type embryos. These
mutations in pea do not affect enzymes directly involved in
starch synthesis and yet all cause a decrease in the proportion of amylose in the starch (Fig. 5). In Chlamydomonas, mutations at the STA1 and STA5 loci affect enzymes involved in
ADPG production and cause a reduction in the amylose content of the starch (van den Koornhuyse et al. 1996). Reduced
amylose contents were also observed in the starches of potato tubers in which the activity of AGPase (the enzyme responsible for the synthesis of ADPG) had been reduced by
the expression of antisense constructs (Lloyd et al. 1999). Although the ADPG contents of these Chlamydomonas mutants
and antisense potato tubers were not measured it is assumed
that the decrease in the rate of ADPG synthesis would result in
a decrease in the concentration of this metabolite. This supports the idea that, at least in some circumstances, the concentration of ADPG can influence the amylose content.
485
The nature of the amylopectin matrix could influence the
amylose content of the granule in several ways. We have
shown that amylopectin influences GBSSI activity directly,
conferring kinetic properties which favour the elongation of
MOS. The permeability of the matrix to the substrates, ADPG
and MOS may also influence GBSSI activity. For example, the
concentration of soluble substrates for GBSSI may decrease
with increasing distance from the surface of the granule limiting its activity particularly in the centre of the granule. The
space available within the amylopectin matrix may also be
important. This could set an upper limit on the amount of
amylose which can be synthesised. This may be the reason
why wild type storage starches all have an amylose content
of approximately 25 – 30 % and no higher (Flipse et al. 1996).
Conclusion
We have learnt much about the unique properties of GBSSI
that enable this enzyme to synthesise amylose. However,
there are still some remaining questions about the role of
GBSSI. It is clear that GBSSI can elongate amylopectin
chains within the granule as well as synthesising amylose.
Whether these amylopectin chains or soluble MOS derived
from them are the primers for GBSSI in vivo has still to be resolved. The study of starch synthesis in different plant organs
makes it clear that starch granules containing GBSSI activity
do not necessarily synthesize amylose. In granules that accumulate very little amylose, GBSSI may contribute primarily
to the synthesis of amylopectin or it may contribute little to the
synthesis of any component of the starch granule. Further
studies of the roles of GBSSI in different sorts of starch synthesising plant organs are required to answer these questions.
Acknowledgements. Financial support by the Agri-Food directorate
of the BBSRC, U.K. (SZ) and Dupont Agricultural products (PJ) is gratefully acknowledged.
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