7 The synthesis of the starch granule Alison M. Smith, Kay Denyer, Samuel C. Zeeman, Anne Edwards and Cathie Martin 1. Introduction Starch is the main form in which higher plants store carbon, and by far the major com- ponent of the harvested parts of the world’s major crops. For example, starch con- tributes between 50 and 80% of the dry weight of mature cereal grains, potato tubers and pea seeds. This makes it the major carbohydrate of nutritional importance in the human diet. It is also of enormous value as a raw material for a wide range of indus- tries. When cooked in the presence of water, starch swells (gelatinizes) to form gels or pastes which are used as thickeners, texturizers, stabilizers and fat-replacements in the food industry and in the manufacture of materials including paints, glues, paper, tex- tiles, pharmaceuticals and biodegradable plastics. However, in spite of its importance, the way in which starch is synthesized in the plant is still poorly understood. In this chapter, we will describe briefly the structure of starch, then discuss recent progress in discovering how it is synthesized. 2. The structure of the granule 2.1 The polymers and their organization Starch occurs in plants as semi-crystalline granules of complex structure and organi- zation, Granules are made up of two types of glucose polymer: amylose, which con- sists of essentially linear chains of glucose units and contributes about 30% of storage starches, and amylopectin, a highly branched polymer which makes up the remaining 70% of storage starches. The amylopectin molecule contains regularly spaced clus ters of chains of 12~20 glucose units. These clusters of shorter branches are joined by longer chains (40 or more glucose units) which span two, three or more clusters. This distinctive, polymodal distribution of chain lengths within the amylopectin molecule allows the polymer to become organized to form a semi-crystalline granule (see Figure 1). Within the granule, adjacent short chains within clusters form double Plant Carbohydrate Biochemistry, edived by J.A. Bryant, M.M. Burrell and NJ. Kruger © 1999 BIOS Scientific Publishers Ltd, Oxford. 7980 CH. 7. THE SYNTHESIS OF THE STARCH GRANULE © ©) Crystalline lamella [Amorphous —| | _tameiia _| ‘Semi-crystalline Amorphous zone zone Figure 1. Organization of the starch granule. (a) The cluster structure of the amylopectin molecule, and formation of double helices between adjacent chains within the clusters. (b) The double helices become ordered in regular arrays, giving rise to crystalline lamellae. These alternate with amorphous lamellae in which the branch points of the amylopectin molecule are primarily located. The periodicity of this repeat structure is 9 nm. (c) Semi-crystalline zones of alternating crystalline and amorphous lamellae alternate with amorphous zones, with a periodicity of hundreds of nanometres. Reprinted from Photosynthesis: Physiology and Metabolism, R.C. Leegood, T-D. Sharkey and $. von Caemmerer (eds), 1999, Starch synthesis in leaves, R. Trethewey and A.M. Smith, Figure 1, with kind permission from Kluwer Academic Publishers. helices. These helices pack together in ordered arrays to give crystalline lamellae, Crystalline lamellae are separated by amorphous lamellae containing the branch points of the polymer, giving rise to semi-crystalline zones of concentrically orga- nized, alternating crystalline and amorphous lamellae. The semi-crystalline zones themselves alternate with a periodicity of hundreds of nanometres with amorphous zones in which the amylopectin is considerably less organized (French, 1984; Jenkins et al., 1993). Much of the amylose component of the granule is probably within the amorphous zone as single helices, interspersed with amylopectin molecules (Jane et al., 1992). A semi-crystalline-amorphous repeat is called a ‘growth ring’. New micro- scopic and physical techniques are revealing still further levels of organization within the granule. For example, it has been proposed that the semi-crystalline regions are organized into interdigitating suprahelical structures (Oostergetel and van Bruggen, 1993), and into discrete domains or ‘blocklets’ (Gallant et al., 1997). 2.2 Approaches to understanding granule synthesis ‘The complexity of the starch granule potentially presents a major challenge to the biologist trying to understand its synthesis. However, there is now reason to suppose that at least part of the organization of amylopectin to form a granule may be a process of self-assembly, rather than biologically catalysed. It is proposed that amy- lopectin has the properties of a side-chain liquid crystal polymer. Provided that an appropriate polymodal distribution of branch lengths is generated by enzymes at the outer edge of the growing granule, the newly synthesized material will form double helices and crystallize (Waigh et al., 1996, 1997, 1998). The main tasks of the biolo- gist are therefore to explain how a polymodal distribution of branch lengths may be generated at the outer edge of the granule, and how both a highly branched polymer,A.M. SMITH ET AL. 81 amylopectin, and an essentially unbranched polymer, amylose, may be synthesized at the same time. "A valuable approach to these problems is to examine the biochemical basis of the many mutations known to affect che structure and composition of starch in storage organs, Some such mutations apparently affect only one aspect of starch structure and composition, For example, mutations at the waxy loci of cereals, the amf locus of potato and the Jam locus of pea all eliminate the amylose component of starch and hove little effect on amylopectin or on granule organization (Denyer et al., 19955 Hovenkamp-Hermelink et al., 1987; Smith and Martin, 1993). However, most of these monations affect many aspects of the starch. The mutations at ther locus of pea and the sugary loci of maize and rice, for example, affect radically the whole structure and organization of the granule (Colonna and Mercier, 1984; Matsuo er al., 1987; Sumner and Somers, 1944). These are further discussed below. In spite of the complex effects of many of the mutations on granule structure and composition, almost all of those thus far characterized lie in genes encoding two enzymes: starch synthase and starch-branching enzyme. These two enzymes alone can potentially synthesize both amylose and amylopectin. Starch synthase adds a glucose nit from the sugar nucleotide ADPglucose to the non-reducing end of a glucose hain, Starch- branching enzyme cleaves a length from the end of 2 chain and transfers ‘rao the side of the same or an adjacent chain to forma branch. Both enzymes exist as multiple, distinct isoforms, each of which is encoded by a different gene. It seems rea- sonable to suggest that the presence in vivo of several different isoforms of the two enzymes, each with different specificities, locations on the granule and developmental timing of activity, might be very important in allowing amylopectin and amylose to be synthesized (Smith et al. 1997). We will examine first whether the existence of multi- ple isoforms of these two enzymes provides a sufficient explanation for amylopectin synthesis, then examine the role of distinct isoforms in amylose synthesis. 3. The synthesis of amylopectin 3.1 The role of starch-branching enzymes “The existence of multiple isoforms of starch-branching enzymes offers a possible explanation for the polymodal distribution of branch lengths of amylopectin. This type of distribution might result from the simultaneous activities of rwo or more ito- Foome with different preferences for the length of chain transferred. Support for this ‘den comes from examination of the isoforms of starch-branching enzyme for which primary amino acid sequences are available. These can be divided on the basis of. clear differevices in sequence into two classes, referred to as A and B (Burton et aly 1995). Storage organs of, for example, pea, wheat, maize, rice and potato, possess members of uth classes, Detailed studies of the A and B isoforms of maize endosperm in eitro and when expressed in Escherichia coli show that they do indeed differ in their preferences Yor the length of chain transferred (Guan and Preiss, 1993s Guan etal, 19955 Takeda et 12, 1993), and it seems likely that the differences between the maize isoforms reflect differences between the A and B classes generally. “The existence in several species of mutations which eliminate activity of the A iso form of starch branching enzyme has allowed us to test the idea that the differences in properties between the A and B isoforms can account for the polymodal distribution of82 CH. 7. THE SYNTHESIS OF THE STARCH GRANULE chain lengths of amylopectin. ‘The mutation at the r locus of pea lies in the gene encod ing the A isoform (Bhattacharyya et al., 1990). Activity of starch-branching enzyme in the embryo and leaf of the mutant pea is much lower than in the organs of the wild-type pea. All of the activity in the mutant pea is contributed by the B isoform, whereas in the wild-type pea the activity is contributed by both A and B isoforms (Smith, 1988). However, although the average chain length of amylopectin in the mutant pea is longer than that in the wild-type pea, the basic polymodal distribution of chain lengths is unaf- fected by the mutation (Lloyd et al., 1996; Tomlinson et al., 1997). The starch granules of the mutant embryo, although of unusual morphology, contain semicrystalline zones of the same basic structure as those in starch from wild-type embryos (Jenkins and Donald, 1995). We conclude that the synthesis of amylopectin with a structure appro- priate for organization to form a starch granule does not require two different isoforms of starch-branching enzyme. 3.2 The role of starch synthases As with starch-branching enzyme, the isoforms of starch synthase responsible for amylopectin synthesis can be divided into several classes on the basis of distinct dif- ferences in their primary sequences. At present three such classes, known as SSI, SII and SSIIT, can be defined, Many organs possess members of more than one class, and there is good evidence that the endosperm of maize and the tubers of potato possess all three (Abel et al., 1996; Gao et al., 1998; Harn et al., 1998; Knight et al., 1998; Marshall et al., 1996; Tomlinson et ai., 1998). Until recently, however, it was not known whether SSI, II and III played different roles in amylopectin synthesis, and what these roles might be. The discovery of a mutant pea specifically lacking the SSII isoform gave us the first evidence that different isoforms do indeed play different roles. A mutation at the rug5 locus of pea lies in the gene encoding SSII, an isoform which accounts for over half of the amylopectin-synthesizing starch synthase activity in the wild-type pea embryo (Craig et al., 1998, Denyer and Smith, 1992; Dry et al., 1992), In the absence of SSI, there are small increases in the activity of other isoforms of starch synthase so that total activity in the mutant is similar to that in wild-type embryos and the rate of starch synthesis is also similar throughout most of embryo development. In spite of this, the starch of the mutant is radically different from that of wild-type peas, The amylopectin is seriously deficient in chains of about 40-50 glucose units: the length required to span two clusters. Iris enriched in both very short chains and in extremely long chains. These changes xesult in highly contorted granules, presumably because the organization of the amylopectin has been affected by the changes in its branch length distribution (Craig et al., 1998). The phenotype of the rug6 mutant indicates that the SSII isoform of starch synthase plays some specific role in the synthesis of amylopectin, a role which other isoforms cannot replace, To provide further information about the extent to which individual isoforms have specific roles, and to discover whether particular classes of isoforms play particular, definable roles, we examined the roles of the two main isoforms responsible for amylopectin synthesis in the potato tuber. Isoforms of the SSIII and SSII class respectively account for about 80% and 10-15% of the soluble starch syn- thase activity in the tuber (Edwards et al., 1995; Marshall et al., 1996). Tubers of trans- genic plants expressing antisense RNA for one or both of these isoforms have theA.M. SMITH ET AL. 83 same starch contents as normal tubers, but have amylopectin with very different chain length profiles. The chain length profiles of amylopectin from lines with reductions in SSIf, lines with reductions in SSIIT, and lines with reductions in both SSII and STII all differ considerably and reproducibly from each other, and this is reflected in differ- ences between the lines in granule morphology and the gelatinization properties of the starch. The phenotype of the lines with reductions in both SSIL and SSIII is very dif- ferent from that which could be predicted from the phenotypes of lines in which either SSII or SSIII is reduced (Edwards et al., 1999). "Two important conclusions can be drawn from these analyses. Firstly, SSI and SSITT both make distinct and specific contributions to the synthesis of amylopectin. Secondly, their contributions depend not only upon their intrinsic properties but also upon whether the other isoform is present, Thus the contribution made by SSL when SSIII is present is probably different from that which it makes when SSUI is absent, and the same is true for SSIII in the presence and absence of SIT. This com- plexity is not surprising, The growing surface of the starch granule, which is the sub- Strate for an isoform of starch synthase in vivo, is a complex product of the actions of several different isoforms of starch synthase and starch-branching enzyme. The product of the isoform will depend both upon its intrinsic properties and upon the precise nature of the substrate with which it is presented. Its contribution will shus Gepend upon the complement of other isoforms of starch synthase and starch- branching enzyme present, and this in turn will depend on a host of genetic, devel opmental and environmental factors. Thus although apparently similar isoforms of starch synthase are present in many different starch-synthesizing organs, it is likely that their contributions to amylopectin synthesis will be different in each of these organs. 3.3 A possible role for debranching enzymes Although most of the previously described mutations with radical effects on granule structure and composition lie in genes encoding starch synthase and starch-branching enzyme, there are three notable exceptions. Mutations at the sugary! loci of maize and rice and the STA7 locus of the unicellular green alga Chlamydomonas rheinbardtit result in the replacement of some or all of the starch with a water-soluble glucan known as phytoglycogen (Matsuo et al., 19873 Mouille et al., 1996; Sumner and Somers, 1944). Phytogiycogen is more highly branched than amylopectin, and does not form organized, semi-crystalline granules. All three mutations affect the activity during the period of starch synthesis of a class of enzymes known as debranching enzyme (Mouille et al., 1996; Nakamura et: al., 1996; Pan and Nelson, 1984): in fact the mutation at the sugary! locus of maize has been shown to lie in a gene encoding one such enzyme, isoamylase (James et al., 1995). These enzymes are involved in starch degradation, but are also known to be present in starch-synthesizing organs. They hydrolyse the linkages which form the branch points in glucans. The phenotypes of the mutants have led to the development of a model in which debranching is an inte- gral part of the synthesis of amylopectin (Ball ez al., 1996). "The model proposes that in the first step in the synthesis of a cluster of branches at the surface of the growing granule, starch synthase elongates from a bed of short chains, When chains of sufficient length have been synthesized, branching enzyme is able to act. A highly branched, unorganized glucan (known as pre-amylopectin84 CH.7. THE SYNTHESIS OF THE STARCH GRANULE synthesized. This is in turn trimmed by debranching enzyme to give a bed of short chains from which the next round of chain elongation can occur. This ‘trimming’ model is appealing in that it integrates the synthesis of amylopectin with its organization to form a granule, and offers an explanation for the accumulation of phytoglycogen in mutants deficient in debranching enzyme (that is, phytoglycogen is an accumulation of pre-amylopectin). However, it is not known whether starch- branching enzyme and debranching enzymes have the rather specific properties required of them by the model. Interpretation of the phenotypes of the sugary mutants is also complicated by several major pleiotropic effects on enzymes other than debranching enzymes (Nakamura et al., 1996; Singletary et al, 1997). Thus although the mode! has been widely quoted, and has stimulated much new research into debranching enzymes, it has not yet proved possible to test it rigorously. We have recently discovered an Arabidopsis mutant (dbe1) which sheds new light on the validity of the model. This mutant specifically lacks an isoamylase present in wild- type chloroplasts, probably because of a mutation in a gene encoding this enzyme. Effects of the mutation on other enzymes of starch synthesis and degradation are minimal. During the day the chloroplasts of the mutant accumulate both starch and an abnormal, soluble, highly branched glucan similar to the phytoglycogen of the sugary mutants. Although the rate of starch synthesis is much lower than in wild-type leaves, the starch of che mutants is indistinguishable from that of the wild type with respect to granule morphology and the chain length distribution of its amylopectin (Zeeman etal., 1998) Iris not easy to reconcile che ‘trimming’ model with our observation that two very different branched glucans, amylopectin and phytoglycogen, accumulate in the same chloroplasts at the same time in the dbel mutant. The simple expectation from the model is that reductions in debranching enzyme activity would produce a single type of branched glucan, with a chain length distribution less like that of amylopectin and more like that of phytoglycogen as the extent of reduction of activity is increased. We Propose instead that debranching enzyme is not directly involved in amylopectin syn- thesis, and that it plays a role in recycling soluble products of starch synthases and branching enzymes, as follows (see Figure 2). Although the major substrate for starch synthases and starch-branching enzymes in the normal plastid is che surface of the granule, starch synthase can also potentially elongate small malto-oligosaccharides (e.g. maltose and maltotriose) which are likely to be present in the plastid stroma. The consequences of this activity would be dele terious for starch synthesis, Extensive elongation of the soluble glucans would allow branching by starch-branching enzyme, creating more reducing ends and allowing accelerated synthesis of branched, soluble ghucan at the expense of synthesis of starch at the granule surface. We argue that the accumulation of soluble glucans is normally Prevented by the presence in the stroma of a suite of enzymes capable of attacking soluble linear and branched glucans. There is good evidence for the presence of such enzymes in starch-synthesizing plastids; in addition to isoamylase many plastids ave Known to have high activities of, for example, phosphorylase and disproportionating enzyme, Deficiencies in this recycling mechanism, for example a lack of isoamylase, will allow soluble glucans to accumulate. The rate of starch synthesis will be reduced because the soluble glucan presents an alternative substrate for the starch synthases and starch-branching enzymes, but some normal amylopectin can continee to be synthesized.i | ! A.M. SMITH 67 AL. 85 Figure 2.4 model to explain the phytoglycoge-accumlating phenotype ofthe Arabidopsis eager Teck (a) A eloroplast from a wild-type lea. i which starch synthase (SS) and starch- Drancbing enzynae (SBE) are primarily involved in the synthess of starch from ADPetncate CADPGS at dhe surface ofthe starch granule, These enzymes may also elaborate small may Cosa heides present inthe stroma, but the products ofthis activity will be degraded (dashed Th) by stromal sarch-degrading enzymes, including isoamplase (b) A eloroplast from @ dbel leaf Lovsofnoamplase activity reduces the ate a which soluble product of starch synthase and wer bvanching enzyme can be degraded. Elaboration ofthese soluble glucans exceeds the rate of their degradation and they i cmmuate in the stroma as phytoglycogen. Te soluble glucans provide faage artounts of substrate for starch synthase and stareh-brancbing enzyme. This reluces the soe ir ofthese enzymes availabe for sarc synthesis, and thus reduces de rate of starch synthesis. We believe that this very indirect role for isoamylase provides a simpler explanation of the phenotype of the dbel mutant than does the trimming model, and that it may “iso offer an alnernative explanation of the sugary phenotypes. However, these ideas are at present very speculative, and this remains a field in which detailed and innovar tive experimentation is urgently required. 4. The synthesis of amylose "The synthesis of amylose is an exclusive function of a class of isoforms of starch syn- these bound to starch granules, knowa as granule-bound starch synthase T or GBSSI. The starch of mutants lacking GBSSI, and of transgenic plants in which GBSSI has been severely reduced by expression of antisense RNA, contains litle or no amylose (Denyer et ali, 1995; Hovenkamp-Hermelink et al., 1987s van der Leij et al., 1991s Sinith and Martin, 1993; Visser et al, 1991). The reasons why GBSSI, and not other Jeoforme of starch synthase, can synthesize amylose are not yet clear, but recent stud- see of GBSST are revealing unusual properties which point to an explanation. Early speculation about the mechanism of amylose synthesis was based on the fact that OBSEI is bound to the starch granule, Tewas argued that the product of GBSST, in contrast to the products of other, soluble isoforms of starch synthase, would be within the matrix of the granule, and hence inaccessible to starch-branching enzyme. The prod- uct would thus remain unbranched (Denyer et al., 1993). Detailed studies of granule- '86 CH. 7. THE SYNTHESIS OF THE STARCH GRANULE bound proteins have revealed this explanation to be inadequate. Isoforms of starch syn- thase other than GBSSI are also found tightly bound to the granule. These isoforms are also present in the soluble fraction of the stroma, and it is likely that they become incor- porated into the matrix of the granule as their amylopectin product crystallizes around them. Although they have been shown to be capable of activity within and after extrac- tion from the granule matrix, the fact that they are present in the starch of mutants lack- ing GBSSI reveals that they cannot synthesize amylose (Denyer et al., 1993, 1995; Edwards et al., 1996; Hylton et al., 1996). Thus features of GBSSI other than, or inaddi- tion to, its location on the granule must be required for amylose synth We have investigated these features using starch granules isolated from wild-type and mutant peas. Wild-type starch granules contain both the GBSSI and SSII isoforms of starch synthase. Granules of the Jam mutant contain only SSII (Denyer et al., 1995), and those of the rag5 mutant contain only GBSSI (Craig et al., 1998). When supplied with ADPglucose, the isoforms within the isolated granules incorporate the glicose unit into the starch, The use of ADP[C]glucose, followed by fractionation of the starch into amylose and amylopectin, can thus reveal which polymers are elongated by the two isoforms. We discovered that neither $SII nor GBSSI incorporated glucose into amylose in isolated granules: both elongated chains within the amylopectin frac- tion, This suggested that a soluble factor, which was lost during isolation of the gran- ules, was necessary for amylose synthesis, In agreement with this idea, addition of low concentrations of small, soluble malto-oligosaccharides such as maltose and malto- triose to the isolated granules allowed GBSSI, but not SIL, to synthesize amylose within the granule. GBSSI can use these malto-oligosaccharides as initial substrates (primers) for synthesis of considerably longer chains which are unable to diffuse out of the starch granule, whereas SSIT cannot (Denyer et al., 1996, and unpublished data). ‘We investigated whether the failure of SSII to synthesize amylose was because it cannot use malto-oligosaccharides as substrates, or because it cannot elongate them sufficiently to prevent their diffusion out of the granule. Products of about nine or more glucose units in length are likely to be trapped inside the granule. By examining the soluble products from granules containing only SSII, we found that this isoform can indeed use short malto-oligosaccharides as substrates, but that it adds only a single glucose unit before dis- sociating. Thus the only detectable product of elongation of maltotriose by SSII (chree glucose units) was maltotetraose (four glucose units). In contrast, GBSSI synthesized a whole range of soluble products from maltotriose, ranging from maltotetraose up to about nine glucose units in length (K. Denyer et al, unpublished data). This reveals that GBSST does not dissociate from its immediate glucan product, but uses it as a substrate for the addition of further glucose units from ADPglucose. This processive mode of action allows the synthesis of long chains even at high concentrations of malto-oligosac- charide primer. Our initial studies of GBSSI in a soluble form (from an E. coli expression system) suggest that its mode of action and other kinetic properties may be strongly influenced by its location within the amylopectin matrix of the gramule. A primary aim of further work will be to define the influence of amylopectin on the enzyme, and to iden- tify the regions of the protein responsible for its association with the granule matrix, The fundamental difference in reaction mechanism between SSII and GBSSI is probably central to the unique ability of GBSSI to synthesize amylose. We suggest that, in vivo, GBSSI within the granule processively elongates malto-oligosaccharides which diffuse in from the stroma. This produces chains too long to diffuse out of the granule, and these are subjected to further elongation to produce amylose, The factorsA.M. SMITH ET AL. 87 ‘which determine the amount of amylose synthesized and the size of the moiecules produced remain to be determined. References Abel, GiJ.W, Springer, E, Willmitzer, L. and Kossmann, J. (1996) Cloning and functional analysis of a CDNA encoding a novel 139 kDa starch synthase from potato (Solanum tubero- sum L). 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