The Biosynthesis of Starch Granules - Biomacromolecules (ACS

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Biomacromolecules 2001, 2, 335-341

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The Biosynthesis of Starch Granules Alison M. Smith* John Innes Centre,† Colney Lane, Norwich NR4 7UH, U.K. Received November 22, 2000; Revised Manuscript Received March 12, 2001

Although composed simply of glucose polymers, the starch granule is a complex, semicrystalline structure. Much of this complexity arises from the fact that the two primary enzymes of synthesis-starch synthase and starch-branching enzyme-exist as multiple isoforms. Each form has distinct properties and plays a unique role in the synthesis of the two starch polymers, amylose and amylopectin. The debranching enzyme isoamylase also has a profound influence on the synthesis of amylopectin. Despite much speculation, no acceptable model to explain the interactions of all of these enzymes to produce amylose and amylopectin has thus far emerged. The organization of newly synthesized amylopectin to form the semicrystalline matrix of the granule appears to be a physical process, implying the existence of complex interactions between biological and physical processes at the surface of the growing granule. The synthesis of the amylose component occurs within the amylopectin matrix. My aim in this article is to provide an overview of the synthesis of the starch polymers and the way in which they are organized to form the starch granule. My main focus will be on the nonphotosynthetic, starch storing organs of higher plants. I shall first describe the systemsthe pathway of supply of substrate, the starch granule itself, and the two enzymes necessary for the synthesis of glucose polymers. This forms a background for discussion of the models put forward for the synthesis of the polymers and the way in which they become organized during synthesis to form a granule. I shall highlight the problems that confront biologists in attempting to understand the way in which the starch granule is formed, a process which lies at the boundary between the disciplines of biology, chemistry, and physics. The Source of Carbon In the cells of major starch-storing organs such as the tubers of potato and the endosperms of cereal grains, starch is synthesized from sucrose imported from the leaves where it is made in photosynthesis (Figure 1). Although the initial metabolism of sucrose occurs in the cytosol, the synthesis of starch occurs exclusively inside an organelle, the plastid or amyloplast. In starch-storing organs such as potato tubers and legume seeds, sucrose is converted into glucose 6-phosphate, which enters the amyloplast via a transporter in the membrane.1-4 Once inside the amyloplast, glucose 6-phosphate is converted to glucose 1-phosphate and then, via the enzyme ADPglucose pyrophosphorylase, to the sugar nucleotide ADPglucose. ADPglucose is the substrate for the starch synthases which synthesize the starch polymers. The pathway of ADPglucose synthesis in the endosperm of developing cereal grain proceeds differently from that in other starch-synthesizing organs (Figure 1). Although some * Fax: +44 1603 450045. E-mail: [email protected]. † The John Innes Centre is supported by a Competitive Strategic Grant from the Biotechnology and Biological Sciences Research Council, U.K.

Figure 1. Basic pathways of synthesis of ADPglucose, the substrate for starch synthases, from sucrose in nonphotosynthetic cells. In all nonphotosynthetic starch-storing organs apart from endosperms of developing cereal grains, sucrose is converted to glucose 6-phosphate, or perhaps to glucose 1-phosphate, in the cytosol. This hexose phosphate enters the plastid via a transporter on the inner envelope and is converted to ADPglucose via ADPglucose pyrophosphorylase. In cells of developing cereal endosperms, ADPglucose is made in the cytosol via a cytosolic isoform of ADPglucose pyrophosphorylase and enters the plastid via a specific transporter. Cereal endosperms also retain the capacity for hexose phosphate transport and a plastidial ADPglucose pyrophosphorylase, but most of the flux from sucrose to ADPglucose probably proceeds via the cytosolic ADPglucose pyrophosphorylase.

import of glucose 6-phosphate occurs, as in other starchstoring organs, most of the ADPglucose for starch synthesis is actually made in the cytosol by a distinct cytosolic form of ADPglucose pyrophosphorylase. The ADPglucose enters the amyloplast via a specific transporter.5-8 The reasons why cereals and grasses have evolved a different route for ADPglucose synthesis in their major starch-storing organs remain the subject of speculation.

10.1021/bm000133c CCC: $20.00 © 2001 American Chemical Society Published on Web 04/26/2001

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The factors that control the flux of carbon into starch in starch-storing organs are poorly understood. Much attention has been focused on ADPglucose pyrophosphorylase because its activity is regulated by several metabolites and because it can be regarded as the first committed step on the pathway of starch synthesis. It is often referred to as the “rate-limiting step” in starch synthesis. In leaves, modulation of ADPglucose pyrophosphorylase activitysby the metabolites 3-phosphoglycerate and inorganic phosphate and perhaps by reductive activationsis undoubtedly important in controlling the overall rate of starch synthesis in the chloroplast during photosynthesis.9,10 However, in those storage organs where its flux control coefficient has been measured, it is clear that other steps on the pathway must also be of considerable importance in the control of flux. In pea and potato plants, two-thirds or more of the control of flux into starch in the storage organ (the embryo and the tuber, respectively) lies in steps other than ADPglucose pyrophosphorylase.11,12 Overall, it is very likely that the distribution of control of flux between steps on the pathway will vary considerably from one type of organ to another, and through organ development.

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Figure 2. The organization of amylopectin to form a starch granule. The shorter chains of the amylopectin molecule are clustered together at intervals of 9 nm along the axis of the molecules (A). Within a cluster, adjacent chains form double helices. These pack together in a regular manner to form crystalline lamellae, which alternate with amorphous lamellae where the branch points are located (B). Within the granule, zones of alternating amorphous and crystalline lamellae themselves alternate with amorphous zones, forming “growth rings” several hundreds of nanometers in width (C).

granule is thought to occur.15,17 One repeat on this scale is known as a “growth ring”. The Starch-Synthesizing Enzymes

The Starch Granule Starch occurs in plants as water-insoluble granules, which display an enormous diversity of size and shape. Granules in potato tuber and canna rhizomes may be well over 100 µm in length whereas those in taro (Colocasia antiquorum) roots are only around 1-3 µm. Many granules approximate to spheres or prolate spheroids, but some are extremely elongatedsfor example in the rhizomes of Dieffenbachias or possess more complex morphologysfor example the A-type granules of wheat, barley, and rye endosperm, which are flattened spheroids with an equatorial groove.13,14 Starch granules consist of two different sorts of glucose polymer, amylose and amylopectin. Amylose is a mainly linear polymer consisting of long chains of R1,4-linked glucose units. It makes up about 20-30% of the starch granule in starch-storing organs. The remainder of the granule consists of amylopectin, a branched polymer in which linear chains of R1,4-linked glucoses are joined together by R1,6 linkages. The branch points are arranged so that clusters of chains of about 12-20 glucose units occur at regular intervals of about 9 nm along the axis of the molecule. Chains of about 45 glucose units span two clusters, and chains of about 70 glucose units span three clusters.15-17 The polymodal distribution of chain lengths and their cluster arrangement are thought to underlie the packing of amylopectin molecules to form the semicrystalline matrix of the granule (Figure 2). Within clusters, adjacent chains form double helices which pack together in ordered arrays, giving rise to crystalline lamellae. The crystalline lamellae alternate with amorphous lamellae formed by the regions in which the branch points occur, with a repeat distance of 9 nm. The alternating lamellae form concentric, semicrystalline zones within the granule. These zones alternate, with a periodicity of a few hundreds of nanometers, with amorphous zones in which amylopectin molecules are in a less organized state, and in which much of the amylose component of the

In theory, the only enzymes required to synthesize the starch polymers are starch synthase and starch-branching enzyme. The former adds the glucosyl unit from ADPglucose to the nonreducing end of a glucose chain via an R1,4 linkage. The latter cleaves a linear glucose chain and transfers the cleaved portion to a glucose residue within an acceptor chain via an R1,6 linkage to form a branch. Support for the idea that these are the main enzymes involved in polymer synthesis comes from the analysis of mutant plants with altered starches. A host of such mutants have been described from a wide range of species. They include plants in which the starch granules have an altered amylose-to-amylopectin ratio or a distorted shape caused by abnormalities of the branching pattern of amylopectin. Almost all of the mutations causing these effects are in genes encoding starch synthase or starch-branching enzyme. Simply mixing starch synthase, starch-branching enzyme, and ADPglucose in vitro does not produce amylose and amylopectin, let alone an organized starch granule. The production of two different polymers and their assembly into a granule in vivo must require considerable additional complexity. One obvious source of such complexity is the existence of multiple isoforms of both starch synthase and starch-branching enzyme. There is increasing evidence that storage organs contain at least two different isoforms of starch synthase and as many as five isoforms of starch synthase.18 The isoforms thus far identified fall into discrete classes based on their primary amino acid sequences, and these classes show considerable conservation between species. For example, developing pea and maize seeds and potato tubers all possess starch-branching enzymes belonging to the A and B classes and starch synthases of the GBSS (granule-bound starch synthase), SSI, SSII, and SSIII classes.19-28 It seems possible that the synthesis of amylose and amylopectin in vivo might result from the interactions of multiple isoforms of starch synthase and starch-branching

Biosynthesis of Starch Granules

enzyme, each with distinct properties and a specific role in polymer synthesis. Evidence for and against this explanation of polymer synthesis is considered in the next two sections. Amylopectin Synthesis There is now compelling evidence that different isoforms of starch synthase and starch-branching enzyme play distinct roles in the synthesis of amylopectin. Most of the evidence comes from analysis of mutants of pea, maize, rice, and the unicellular green alga Chlamydomonas lacking single isoforms of these enzymes, and from studies of transgenic potatoes in which activities of one or more isoforms of the enzymes have been reduced by the expression of antisense RNA. I shall consider two examples: potato tubers in which the starch synthase isoforms SSII and SSIII have been reduced (SSII and SSIII antisense lines), and tubers in which the starch branching enzymes A and B have been reduced (SBEA and SBEB antisense lines). In both of these studies, lines of potato with reductions in either one or the other or both isoforms have been produced, and analyses of the chainlength distribution of their amylopectins (by fluorescenceassisted gel or capillary electrophoresis or by highperformance anion exchange chromatography with pulsed amperometric detection) have been used to deduce the roles and extent of interaction of the isoforms. Large reductions in activities of either SSII or SSIII in potato tubers alter the chain length distribution of amylopectin. Importantly, the effects of a reduction in SSII are qualitatively different from those of a reduction in SSIII. For example, the proportion of chains of 8 to 12 glucose units is strongly increased in SSII antisense plants but not in SSIII antisense plants. When both SSII and SSIII are reduced simultaneously, the effect on chain length distribution is different again and very different from that expected if SSII and SSIII act in an independent, additive way in the synthesis of amylopectin.23,29 Two important conclusions emerge from these analyses. First, SSII and SSIII make different contributions to the synthesis of amylopectin. Second, the actions of the two isoforms are not independent: the action of one influences the action of the other. This interaction may well reflect the fact that they both act on the same moleculesthe substrate of one is the product of the other. The picture for the two isoforms of starch-branching enzyme in potato tubers is remarkably similar to that for the starch synthases. Reductions in SBEA have very different effects from reductions in SBEB. Whereas starch from SBEB antisense plants appears almost normal, that from SBEA antisense plants is radically altered in amylopectin chainlength distribution. When both isoforms are reduced simultaneously, the effects on amylopectin chain-length distribution are quantitatively different from, and much more severe than, those observed in the SBEA antisense plants.26,30,31 Thus the two isoforms play distinct roles in amylopectin synthesis, and their actions are interdependent. Taken together, these experiments with potato are consistent with the idea that the existence of multiple, distinct, interacting forms of starch synthase and starch-branching enzyme can explain much of the complexity of branching

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of the amylopectin molecule. However, one major piece of evidence indicates that this is not a complete or sufficient explanation of amylopectin synthesis. Although most of the mutations which alter starch lie in genes encoding starch synthases and starch-branching enzyme, some lie in genes encoding a type of starch-debranching enzyme known as isoamylase. Debranching enzymes cleave R1,6 linkages and have traditionally been regarded as enzymes of starch degradation. The first evidence that they are involved in synthesis came from the sugary1 mutant of maize. This mutant accumulates some starch, but most of the glucan in the endosperm is in the form of phytoglycogen, an R1,4-, R1,6-linked glucose polymer which is more highly branched than amylopectin and has a different branching pattern. Molecular and biochemical analyses of the developing endosperm revealed that the mutation at the sugary1 locus lies in an isoamylase gene and that isoamylase activity is severely reduced or eliminated in the mutant.32-34 Since the identification of the mutation in sugary maize, apparently equivalent mutations have been identified in several other species of plant. The sugary1 mutant of rice accumulates a little starch and large amounts of phytoglycogen in the endosperm and is deficient in activity of debranching enzyme.35-37 The dbe1 mutant of Arabidopsis carries a mutation in an isoamylase gene and accumulates both phytoglycogen and starch in its chloroplasts during photosynthesis.38 The sta7 mutant of the unicellular green alga Chlamydomonassan organism which normally accumulates starch similar in structure to that of higher plantssalso lacks isoamylase and accumulates small amounts of phytoglycogen and no starch.39 The fact that lack of isoamylase causes similar phenotypes in such a wide range of species suggests strongly that this enzyme, as well as starch synthase and starch-branching enzyme, is necessary for normal starch synthesis. Two, very different models have been put forward to explain how isoamylase may be involved in starch synthesis. The first proposes a direct involvement in the synthesis of amylopectin (Figure 3A). In this model, starch synthase and starch-branching enzyme synthesize a polymer called preamylopectin at the granule surface. Preamylopectin does not have the appropriate branching structure to become organized and incorporated as part of the granule matrix. It is converted to amylopectin primarily through the action of isoamylase, which “trims” branches until the branching structure is appropriate for crystallization onto the granule surface. In the absence of isoamylase, much of the preamylopectin does not achieve an appropriate structure for crystallization. It is further acted on by synthases and branching enzyme and accumulates as phytoglycogen.40 The second model proposes that isoamylase is only indirectly involved in amylopectin synthesis, which is an exclusive function of starch synthases and starch-branching enzymes (Figure 2B). As well as elongating and branching chains at the surface of the growing granule, starch synthases and starch-branching enzymes can also potentially act on small malto-oligosaccharidessfor example maltose and maltotriosespresent in the soluble fraction of the amyloplast. This elaboration of small molecules is prevented by the

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Figure 3. Models proposed to explain the role of the debranching enzyme isoamylase in the synthesis of amylopectin. In scheme A, starch synthase and branching enzyme synthesize pre-amylopectin, a polymer more highly branched than amylopectin. “Trimming” of this polymer via debranching enzyme removes some of the branches and allows the polymer to crystallize onto the surface of the starch granule. The maltooligosaccharides released by the trimming may be converted back to ADPglucose or may be reincorporated onto chains within the preamylopectin via a disproportionating enzyme. In isoamylase-deficient mutants (dashed line), failure of the trimming process means that preamylopectin does not achieve an appropriate branching pattern for crystallization to occur. Further elaboration of the preamylopectin by starch synthase and starch-branching enzyme results in the accumulation of phytoglycogen.40 In scheme B, starch synthase and starch-branching enzyme synthesize amylopectin rather than a preamylopectin which requires trimming. These enzymes may also act on small malto-oligosaccharides present in the soluble fraction of the plastid. Elaboration of these malto-oligosaccharides is prevented by “scavenging” enzymes, including debranching enzyme, which degrade soluble glucans. In the absence of isoamylase (dashed lines), soluble glucans are elaborated by starch synthase and starchbranching enzyme and accumulate as phytoglycogen.38

actions of suite of “scavenging” enzymes which rapidly degrade soluble glucans. Isoamylase is seen as a critical component of this scavenging mechanism. In the absence of isoamylase, the soluble glucans elaborated by starch synthase and starch-branching enzyme tend to accumulate as phytoglycogen. The synthesis of these soluble glucans restricts the availability of enzyme and substrate for starch synthesis, reducing the rate of starch accumulation.38 Although these models differ radically in the roles they propose for isoamylase, it is very difficult to design experi-

ments which will distinguish unambiguously between them. It remains possible that neither is correct. Considerable effort is underway in several labs to resolve this issue, and answers should emerge in the next year or two. In the meantime the role of isoamylase remains one of the least understood aspects of the process of starch synthesis. Amylose Synthesis It has been known for many years that the synthesis of the essentially linear, amylose component of starch is a

Biosynthesis of Starch Granules

function of one particular class of isoform of starch synthase known as granule-bound starch synthase I or GBSSI. This understanding comes from study of mutants in many species in which no amylose is synthesized: the starch granules consist entirely of amylopectin. The best-known mutants of this type are the waxy mutants of cereals, and equivalent mutations also affect the starch of pea embryos, potato tubers, the perisperm of Amaranthus seeds, and Chlamydomonas cells. All of these mutants lack activity of GBSSI, and in those thus far studied at a molecular level, the mutations have been shown to lie in genes encoding GBSSI.41-47 The precise mechanism by which GBSSI synthesizes amylose remains unknown, but discoveries about the properties of the enzyme offer some important clues. Unlike other isoforms of the enzyme, GBSS is entirely associated with the starch granule.48 The tight association of GBSSI with the granule is confirmed by experiments in which isolated granules are incubated with reagents which remove or inactivate surface proteins. The GBSS protein and its activity are largely resistant to these treatments, suggesting that much of it is actually within the matrix.49-51 The location of GBSSI within the matrix of the granule formed the basis for early ideas about the mechanism of amylose synthesis. It was suggested that the products of GBSS remained unbranched because branching enzymes were not present, or not active, inside the matrix where GBSS is located. It is now apparent that this is not a sufficient explanation for the ability of GBSS to make amylose. Although the other isoforms of starch synthase are mainly located in the soluble fraction of the amyloplast, a significant proportion of the protein of some of these isoforms is located in the granule matrix and can be shown to retain its activity in this location.48,52,53 For example, although most of the measurable starch synthase activity of starch granules from pea embryos is attributable to GBSSI, up to 20% is attributable to the SSII isoform. At least three isoforms other than GBSSI are found in the matrix of starch granules from maize endosperm. Mutations which eliminate the activity of GBSS, and with it all of the amylose component of the starch granule, do not affect the amount or activity of these other, granule-bound starch synthases.42,53 This shows that simply being inside the matrix of the granule is not a sufficient qualification for making amylose: GBSSI must have some other, unique property which allows it to make long, linear polymers when other isoforms cannot. The properties of GBSSI are strongly dependent upon its close association with amylopectin. Addition of amylopectin to assays containing soluble GBSSI (synthesized in a bacterial expression system) strongly influences several of its kinetic properties.54,55 In a mutant of Chlamydomonas unable to synthesize amylopectin, GBSSI synthesizes only very small amounts of an insoluble, amylose-like glucan.56 Further insight into the properties of GBSSI has been gained from analysis of its activity inside isolated starch granules, monitored by supplying the granules with radioactive ADPglucose. In the absence of any other substrates, GBSSI within the granule incorporates glucose from ADPglucose into long chains of amylopectin rather than into amylose.57,58 However if small malto-oligosaccharides (mal-

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tose up to maltoheptaose) are also supplied, GBSS elongates these to produce amylose inside the matrix.58,59 The ability to elongate malto-oligosaccharides to make long chains of amylose is unique to GBSSI. Detailed study of the products made from malto-oligosaccharides by GBSSI, both inside the granule matrix and when expressed in a soluble form in a bacterial expression system, reveals that GBSSI acts processively.54,59 After addition of a glucose unit from ADPglucose to a malto-oligosaccharide, the enzyme does not necessarily dissociate from its product. Instead, it may use the product as a further substrate, adding further glucose units from ADPglucose and building up a chain of substantial length. In contrast, other isoforms of starch synthase act distributively on malto-oligosaccharides. After addition of a single glucosyl unit from ADPglucose, these isoforms dissociate from their product. The difference in mode of action between GBSSI and other isoforms of starch synthase may explain why GBSSI alone can synthesize amylose. Malto-oligosaccharides of less than about eight glucose units can diffuse into the matrix of the granule from the surrounding soluble fraction of the amyloplast.60 If they encounter an isoform of starch synthase other than GBSSI, the distributive mode of action of the isoform means that elongation is likely to be limited to one or a very few glucose units. If they encounter GBSSI, its processive mode of action means that long chains may be built up. Once the chain exceeds about eight glucose units, it is too large to diffuse freely within the matrix spaces and is thus trapped to become, after further elongation, the amylose component of the starch. Experiments with starch granules isolated from the unicellular alga Chlamydomonas suggest an alternative to malto-oligosaccharides as the glucan substrate for amylose synthesis. In these granules, chains within the amylopectin fraction which have been elongated by GBSSI are cleaved by an unknown mechanism to form amylose.61 There is at present no experimental evidence from intact plants to indicate whether synthesis of amylose proceeds via elongation of amylopectin chains followed by cleavage or by elongation of malto-oligosaccharides. It is entirely possible that both mechanisms may operate. Whatever its glucan substrate in vivo, the fact that GBSSI has a processive mode of elongation is probably critical in allowing it to synthesize the long, linear chains of amylose. The Assembly of the Granule Because the mechanisms of synthesis of the amylopectin and amylose polymers are not resolved, understanding of the assembly of polymers to form a granule is very limited. However, there is reason to believe that the organization of the newly synthesized amylopectin chains to form the semicrystalline matrix of the granule is primarily a physical rather than a biological process. Two lines of evidence support this view. First, all of the mutations reported to have major effects on the structure of starch lie in genes encoding starch synthase, starch-branching enzyme, and isoamylase. None lies in genes encoding proteins likely to be involved in organizing carbohydrate chains. Second, analyses of physical data (X-ray scatter and microdiffraction, neutron

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scatter, and 13C cross-polarization magic angle spinning NMR) reveal that amylopectin may have the structure of a side-chain liquid crystalline polymer.62,63 If this is the case, it is expected to self-assemble into ordered lamellae. A purely physical explanation for the organization of amylopectin molecules to form the granule matrix implies a complex interface between biological and physical processes at the surface of the growing granule. The factors which ensure that amylopectin is synthesized with an appropriate branching structure to allow self-assembly may be both biologicals the properties of the starch-synthesising enzymessand physicalsthe nature of the granule surface at which new synthesis must occur. There are two further problems in explaining how amylopectin is assembled to form a granule. First, nothing is known about the processes which initiate granule formation. This is not simply a physical process, since the number of granules which initiate per amyloplast is under genetic control. In some organs only one or a very few granules initiate (for example potato tuber), whereas in others, multiple, independent initiations occur (for example oat and rice endosperm). So far, however, mutational analysis has not identified any protein required for granule initiation. In mammals and yeast, a self-glucosylating protein called glycogenin is involved in the initiation of synthesis of molecules of glycogen, an R1,4-, R1,6-linked glucose polymer resembling phytoglycogen in structure.64 Self-glucosylating proteins with properties similar in some respects to those of glycogenin have been identified in plants, but evidence thus far suggests that they are involved in the synthesis of cell-wall polymers rather than starch.65 The synthesis of glycogen in bacteria does not require a glycogenin-like protein, and there is no a priori reason such a protein might be necessary for the initiation of synthesis of starch granules. The second major phenomenon in granule assembly which remains to be explained is the development of growth rings. It has been widely assumed that these alternating semicrystalline and amorphous zones form because starch laid down at night is different in structure from that laid down during the day. This assumption is based on the observation that growth rings are not present in the endosperm starch of wheat and barley plants grown under constant conditions of light and temperature.66,67 However, rings are still present in the starch of potato tubers that developed under constant conditions.68 It seems likely that several factorsswhich could be both biological and physicalsmay interact to control growth ring formation, and the relative importance of these factors may differ from one sort of starch-storing organ to another. Amylose is not necessary for the formation of a semicrystalline starch granule. The granules of amylose-free starches contain growth rings, and their degree of crystallinity is the same as or higher than that of normal, amylosecontaining starches.69,70 Evidence from NMR studies indicates that a considerable amount of the amylose in the granule is in a single-helical state and not part of the crystalline matrix.71 Leaching experiments, on the other hand, result in extraction of amylose which is initially water soluble but which precipitates in the course of time as a crystalline

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material. Amylose is only water soluble in its random coil form, from which it can be concluded that it is present in the granule in the random coil form if not complexed by lipids or fatty acids. Single helices are stabilized by lysophospholipids and fatty acids, present in some but not all starches.72 Amylose molecules appear to be dispersed among amylopectin molecules73 and may be located primarily in the amorphous zones of the growth rings. The fact that amylose does not form part of the crystalline matrix is consistent with the idea that it is synthesized in spaces inside the matrix, rather than at the surface in the same location as amylopectin synthesis. This idea is confirmed by analysis of starch from tubers of low-amylose potatoes (generated by the expression of antisense RNA for GBSSI). Throughout the development of these tubers amylose is located almost exclusively in the centers (cores) of granules, and the peripheral regions of the granules are amylose-free.74 The volume of the amylose-containing core increases as the overall volume of the granule increases.75 This distribution arises because amylose synthesis in the matrix spaces fails to keep pace with the creation of the matrix, giving rise to an amylose-free peripheral region. In a normal granule, matrix spaces are filled with amylose as soon as the matrix has been laid down, so that no amylose-free zone is apparent at the edge of the granule. Concluding Remarks I have emphasized in this review that much remains to be discovered about the synthesis of the starch granule. In most of the areas of ignorance, progress has until recently been impeded by lack of suitable techniques and experimental systems. The situation is now being changed dramatically by the advent of routine methods for plant transformation, better and much more comprehensive methods of identifying genes, and radically new ways of creating, selecting, and characterizing mutant plants. These methods are allowing biologists to make unprecedented progress in identifying the components necessary for starch synthesis and in defining their roles, importance, and interactions. This approach generates extremely well-defined mutant and transgenic plants containing starches with a wide range of altered structures, which are ideal material for chemists and physicists elucidating the relationship between starch structure and the physicochemical properties of interest to industry. Closer links between research on the biology of starch synthesis and the chemistry and physics of the material will allow the rational “design” of starches for industrial usesthe alteration in the plant of expression of particular enzymes to enable the synthesis of starch with particular, desirable physicochemical properties. Research at the interface between biology and physics also offers the exciting prospect of understanding the interplay between the synthesis of amylopectin and its crystallization to form a granule. Acknowledgment. I am most grateful to all of my colleagues at the John Innes Centre whose work and ideas are described in this review and to Athene Donald, Jay-lin Jane, and Mike Gidley for their patient and lucid explanations of the chemical and physical structure of starch.

Biosynthesis of Starch Granules

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