Starch Biosynthesis in Higher Plants

Starch Biosynthesis in Higher Plants

4.04 Starch Biosynthesis in Higher Plants: The Starch Granule IJ Tetlow and MJ Emes, University of Guelph, Guelph, ON, Canada © 2011 Elsevier B.V. A...

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4.04

Starch Biosynthesis in Higher Plants: The Starch Granule

IJ Tetlow and MJ Emes, University of Guelph, Guelph, ON, Canada © 2011 Elsevier B.V. All rights reserved.

4.04.1 4.04.2 4.04.3 4.04.4 4.04.5 4.04.6 4.04.7 4.04.8 4.04.9 References

Introduction Overview of Starch Structure Granule Initiation Control of Starch Granule Size Starches with Improved Functionalities Amylose-Free Starches High-Amylose Starches Prospects for Altering Amylopectin Structure Other Functional Properties

Glossary ADP-glucose pyrophosphorylase (AGPase) Enzyme that catalyzes an important step in the pathway of starch biosynthesis by converting glucose 1-phosphate and ATP into ADP-glucose (the soluble precursor for starch synthesis) and inorganic pyrophosphate. AGPase is exclusively plastidial in dicots but is located in plastids and cytosol in the monocots. amyloplasts Specialized nonphotosynthetic plastids whose primary role is in storage starch biosynthesis; found in storage tissues such as cereal endosperms and underground tubers. amylose extender A mutant of maize (Zea mays L.) which lacks starch branching enzyme IIb activity resulting in starch with altered properties. debranching enzymes (DBEs) Enzymes that include isoamylases and pullulanase. Isoamylases directly hydrolyze (debranch) α-(1→6)-linked branch points in amylopectin. Isoamylases can hydrolyze loosely branched water-soluble glucans (termed water-soluble polysaccharides or WSPs) such as glycogen and phytoglycogen but are less catalytically active toward more densely branched glucan substrates such as amylopectin clusters and pullulan. Pullulanase can debranch more densely branched glucan polymers, including the fungal polyglucan, pullulan, but are less effective at debranching more openly branched polyglucans such as glycogen. granule-associated proteins A designation that is arbitrary and is generally based on the ability of a protein to be retained within the starch fraction following vigorous aqueous (buffers, SDS, and proteinase treatments) and nonaqueous (acetone or ethanol treatments) extraction and washing techniques. Other proteins probably have a functional role at or inside the granule matrix but are far more loosely associated with the

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granules than those normally designated as ‘granule-associated’. granule-bound starch synthase (GBSS) A form of starch synthase found almost exclusively as a granule-associated protein and is the most abundant granule-associated protein. GBSS is responsible for amylose biosynthesis and is phylogenetically related to the soluble starch synthases involved in amylopectin biosynthesis. malto-oligosaccharides (MOS) Linear, unbranched α-(1→4)-O-linked malto-oligosaccharides of varying degree of polymerization (DP), usually less than 20. soluble starch synthase (SS) A phylogenetically related group of glucosyl transferases which transfer the glucosyl moiety of ADP-glucose to the reducing end of a preexisting α-(1→4)-O-linked glucan primer of variable length to synthesize amylopectin. Some eukaryotic SSs related to glycogen synthases utilize UDP-glucose as a substrate. starch branching enzyme (SBE) An enzyme that generates α-(1→6) linkages by cleaving internal α-(1→4) bonds of glucan chains and transferring the released reducing ends to C6 hydroxyls to form the branched structure of amylopectin. SBEs are related to the α-amylase super-family of enzymes and are able to generate α-(1→6) linkages on linear and branched glucan substrates. starch phosphorylase (SP) An enzyme that catalyzes a reversible reaction; it degrades linear glucan polymers (minimum of four residues) in the presence of inorganic phosphate (Pi) to yield glucose 1-phosphate, or synthesizes α-(1→4) linkages using glucose 1-phosphate. SP is able to work on the outer glucose residues on MOS and WSPs. SPs are found as plastidial (proposed role in starch synthesis) and cytosolic forms (involved in glucan degradation).

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4.04.1 Introduction Starch is a polyglucan produced by plants, occurring in nature as water-insoluble granules and functioning as a storage carbohydrate. The storage starches produced in cereal endosperms account for over 90% of the world market for starch, the majority of which is consumed directly as food or used as animal feed. There is an increased demand for starches in nonfood industries where variations in starch structure and physicochemical properties are exploited. Starch is a cheap, naturally renew­ able raw material utilized in the agrifood sector, in many different industrial applications, and as a source of energy after conversion to ethanol [1, 2], making it a versatile and highly useful commodity. Many of the desired physicochemical properties underpinning the different uses of starches can be produced and altered through chemical and enzyme modification and/or physical treatment [3]. The characterization of natural mutants through plant breeding and the identification and isolation of the major genes encoding the core components of the starch biosynthetic pathway have provided a basic understanding of starch granule formation (see review by keeling any Myers [4]). In addition to natural mutants, knowledge of the basic pathway has led to the production of a number of functional starches based upon predictions of starch structures resulting from various mutations/ gene deletions (see below). However, there still remain significant gaps in our understanding of the pathway, in particular, how the growth of starch granules is initiated and terminated and important details regarding the regulation and coordination of the many enzyme activities involved in polymer synthesis. Such information is a necessary prerequisite for the rational production of ‘designer’ starches in planta, which will reduce the need for costly and environmentally hazardous postharvest chemical treatments. This article presents a current overview of our understanding of starch structure and granule initiation and reviews efforts to modify granule structure and composition with the aim of producing functional starches for food and nonfood applications. The preceding chapter (Chapter 4.02) deals with our current knowledge of the enzymes involved in starch biosynthesis and how the various enzyme classes are regulated and coordinated in the plastid.

4.04.2 Overview of Starch Structure The starch granule is a complex structure with a hierarchical order composed of two distinct types of glucose polymer: amylose, a polymer of 100–10 000 glucosyl units comprising sparsely branched α-(1→4)-linked glucan chains; and amylopectin, a larger, highly and regularly branched glucan polymer typically constituting about 75% of the granule mass, produced by the formation of α-(1→6) linkages between adjoining straight glucan chains. There is approximately one branching point for every 20 glucose residues in amylopectin, which is approximately half the value normally found in glycogen. Amylopectin has an estimated molecular mass of 107–109 Da [5]. Over the years, many models have been proposed for the structure of amylopectin and its organization into granules; however, the cluster model has emerged as the most likely structure (see Figure 1 for an overview of the hierarchical structures that make up the starch granule). The polymodal distribution of glucan chain lengths and branch point clustering within amylopectin allow the short linear chains to pack together efficiently as parallel left-handed double helices. These are assembled in organized arrays, forming the basis of the semicrystalline nature of much of the matrix of the starch granule and resulting in the water-insoluble nature of starch. Chains within the clusters average approximately 12–15 glucosyl residues, while longer chains of 35–40 residues connect clusters [5, 8]. Granule formation is therefore driven by both the semicrystalline properties of amylopectin, as determined by the length of the linear chains of amylopectin, and the clustering and frequency of α-(1→6) linkages [6–9]. The products of partial α-amylolysis of various cereal starches, resulting from the nonrandom hydrolysis of glucosidic bonds between unit clusters (known as long B chains, or B2 and B3 chains, depending on the number of clusters interconnected), are fully consistent with the cluster model of amylopectin [10]. Pairs of adjacent chains within the clusters form double helices that help pack the glucan chains into crystalline layers (lamellae). At a higher order of organization, the lamellae form repeat units with a periodicity of approximately 9 nm. Groups of these semicrystalline and amorphous lamellae associate into units termed blocklets [11], which in turn associate into large crystalline structures several hundred nanometers wide termed growth rings. Growth rings are visible by light and scanning electron microscopy following treatment of broken granules with acid or amylolytic enzymes [12–14]. The crystalline structure of starch granules is highly conserved in higher plants at the molecular level [14, 15], as well as at the microscopic level, where the alternating regions of semicrystalline and amorphous material (growth rings) are present in all the higher plant starches studied to date [13, 16]. For more detailed reviews of starch structure models, see References 5 and 17–19. By contrast, glycogen produced by animals and bacteria, including many cyanobacteria, has a more homogeneously and highly branched (approximately 10%) open structure which expands in a globular fashion [20] resulting in a water-soluble polymer. The branch point distribution in glycogen is uniform, as opposed to discontinuous as found in starch, and is thought to limit glycogen particle size to a range of 10–50 nm (12 tiers) [21–23]. However, certain cyanobacteria synthesize a polyglucan, termed semiamy­ lopectin, which is an intermediate between amylopectin and glycogen in terms of the α-(1→4)-chain-length distribution [24]. The crystalline packing of glucan chains into water-insoluble granules enables carbohydrate to be stored at much higher densities than is possible for glycogen and other water-soluble polyglucans. The precise location of amylose within the granule is a matter of much debate, but it is thought to be found predominantly interspersed in a single-helical or random-coil form in the amorphous, less-crystalline regions [25, 26] and synthesized within a

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(a) (d)

(b) (c) 3 nm

Amorphous lamella

6–7.5 nm

Crystalline lamella

CH2OH O

CH2OH O OH

OH

O

OH

CH2OH O

O OH

OH

O OH

CH2OH

OH

OH

(e)

CH2OH O

O O

HO OH

α-(1→6) bond

O OH

CH2OH

O

OH

OH

OH CH2OH O

O

O

O OH

OH OH

α-(1→4) bond Figure 1 Schematic cross-sectional view of a starch granule showing concentric growth rings which are observed by microscopy when broken starch granules are etched by acids or partial amylolysis to reveal semicrystalline growth rings. The diagram illustrates the various levels of order of the structure of the starch granule. (a) Hilum; (b) amorphous growth rings/background glucan; (c) semicrystalline growth ring; (d) detailed schematic view of alternating regions of amorphous and crystalline lamellae with a repeat unit of approximately 9 nm which form clusters and make up the semicrystalline growth rings (which have periodicities of several hundreds of nm). The crystalline and amorphous lamellae arrange to form spherical ‘blocklets’ within the growth ring. Lamellar structures shown are based on the cluster models proposed by Robin et al. [6], French [7], and Hizukuri [8]. The arrow indicates direction of radial growth of the polymer from the nonreducing end of the glucan. (e) Orientation of glucose molecules in amylopectin showing α-(1→4) O-glycosidic linkages (formed by starch synthases) which are regularly branched in the α-(1→6) position by starch branching enzymes. The glucan chains which form clusters in the crystalline lamellae form double helices with neighboring chains.

preexisting amylopectin matrix. Amylose is readily leached from starch granules suggesting weak associations with the amylopectin granule matrix [27]. A common feature of all starches is that at some point in time they must be degraded (e.g., leaf starch at the end of the light period or storage starches during germination), and hence the granule structure must have built-in entry points (either channels, cavities, or zones of attack) for the enzymes involved in the degradation process [28–31]. Channels have been observed in A-type and B-type cereal starches [32]. The synthesis of this architecturally complex polymer assembly is achieved through the coordinated interactions of a suite of starch biosynthetic enzymes, including some that had traditionally been associated with starch degradation (for details see chapter). The complement of these starch metabolic enzymes is well conserved between plastids and tissues which make different types of starches, for example, transitory starch (made in chloroplasts) and storage starch (made in amyloplasts). With few exceptions, the various isoforms of the many starch metabolic enzymes can be found in both chloroplasts and amyloplasts, and the amino acid sequences of the various enzymes involved in starch metabolism are highly conserved [33–35]. In addition, mutations in analogous starch biosynthetic and degradative genes in higher plants show consistent trends, which illustrates conservation of their biological

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roles, although their impact varies depending upon the genetic background. Models have been proposed, which attempt to explain how the observed structure of starch is synthesized based upon knowledge of the enzymes of the core pathway, essentially derived from in vitro experimental evidence and analysis of mutants [36–39].

4.04.3 Granule Initiation Despite the considerable advances in our knowledge of the pathway of starch biosynthesis, the factors controlling starch granule initiation and granule size remain unclear. For a detailed current overview of the priming of storage glucan synthesis the reader is referred to a recent review by D’Hulst and Mérida [40]. The locus of granule initiation is termed the hilum, located in the center of the growing granule and thought to be essential for granule initiation/priming [41]. The exact composition of the hilum in terms of glucan structure and possible protein composition is at present unknown. It has been suggested that the granule forms from the hilum, from which radially oriented microtubules emanate which become the channels that terminate as openings (pores) on the granule surface [30]. In glycogen biosynthesis, a proteinaceous primer, termed glycogenin (UDP-α-D-glucose:glycogenin α-D-glucosyltransferase, EC 2.4.1. 186), was found to be covalently bound to glycogen [42, 43]. Glycogenin has also been shown to be involved in the priming of yeast glycogen synthesis [44]. Glycogenin is about 37 kDa in size, existing as a dimer [45] and uses UDP-glucose to form α-(1→4)-linked glucan which is covalently attached to a tyrosine residue (Tyr194) on the protein [45, 46]. The autoglucosylation reaction performed by glycogenin has an absolute requirement for Mg2+ or Mn2+, and about 7–11 glucosyl units can be attached to the glucosyl–tyrosine residue and then further elongated by glycogen synthase (GS, EC 2.4.1.11). Isoamylase (EC 3.2.1.41) (but not pullulanase) can remove the malto-oligosaccharide (MOS) chain from Tyr194 altering the rate of UDPglucose incorporation onto the protein [47]. There is some evidence suggesting that a similar proteinaceous primer in bacteria is able to utilize both UDP-glucose and ADP-glucose [48, 49]. However, some studies have shown that in prokaryotes the GS itself acts as the priming site for glycogen synthesis, undergoing autoglucosylation resulting in an MOS linked to the primer GS, which is then elongated [50]. Parallel systems of glucan polymer (starch) initiation have been proposed in higher plants with the discovery of a glycogenin-like starch initiation protein in Arabidopsis termed PGSIP1 [51]. Loss of isoamylase activity in barley, rice, and potato led to an increase in granule initiation [52–54], consistent with the idea that isoamylases suppress the sites of new granule initiation, although debranching enzymes (DBEs) are probably not directly involved in priming granule synthesis [55–57]. Pullulanase-type DBE (limit dextrinase) activity may also play a role in determining granule size. Downregulation of a pullulanase-type DBE inhibitor activity in barley caused a decrease in the number of small (B-type) granules, reduced amylose content, altered amylopectin glucan chain length distribution, and caused a reduction in starch content [58]. Recent evidence suggests the involvement of specific classes of starch synthase (SS, EC 2.4.1.21) in starch granule initiation. Mutants of Arabidopsis lacking SSIV were shown to be incapable of synthesizing more than one starch granule per chloroplast (the single granule has a distinct glucan organization in the hilum), indicating its involvement in granule initiation [59]. Recent data by Szydlowski et al. [60] suggest that the role of SSIV in granule initiation can be replaced by the phylogenetically related SSIII, and that elimination of both SSIII and SSIV prevents starch synthesis altogether in Arabidopsis indicating a dual role for these proteins in granule initiation. Glycogen-like structures have been associated with the priming of insoluble starch-like polyglucans [61], leading Szydlowski et al. [60] to speculate upon a role for SSIV in the interaction and formation of these polyglucans in the seeding of the starch granule. To date, there is no evidence for autoglucosylation activity by SSIII or SSIV acting in a priming role analogous to glycogenin or prokaryotic GS. An interesting study by Ral et al. [62] showed that the small spherical starch granule inside the chloroplast of the tiny picophytoplanktonic alga Ostreococcus tauri was capable of dividing synchronously with algal cell division resulting in a starch granule in the chloroplast of each daughter cell. Such a mechanism would not require the priming steps described above and beg many interesting questions surrounding the control of the division of the starch granule.

4.04.4 Control of Starch Granule Size There is a huge variation in the size and morphology of starch granules of different species and genotypes within a species, although in many species, granule size is homogeneous for a given developmental stage [63]. Populations of different granule size show different physicochemical characteristics (for a review, see References 64 and 65). Granule size and morphology range from <0.5 μm diameter for the near-spherical granules found in cells of the picophytoplanktonic green alga Ostreococcus tauri [62], approximately 5 μm for the round, flattened granules of Arabidopsis [66, 67], 2–30 μm for polyhedral granules of maize [5], to 20–100 μm for the large oval granules found in potato and pea (Pisum sativum L.) [5]. Cereals belonging to the Festucoid family of grasses such as wheat (Triticum aestivum), barley (Hordeum vulgare), oats (Avena sativa), and rye (Secale cereale) are characterized by a bimodal or trimodal distribution of starch granules in storage tissues [68–70]. Large lenticular-shaped A-type granules (15–30 μm diameter) are formed early in endosperm development, while the smaller spherical B-type granules (averaging 5–9 μm in diameter) are formed later in development [71]. Recent observations suggest a third, smaller class of starch granules (<5 μm), termed C-type granules, in cereals such as wheat [72]. The selective advantage conferred upon plants displaying a bimodal distribution of starch granules in storage tissues is at present unclear. It has been proposed that the different classes of starch granules in wheat endosperm (the large A-type, and

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smaller B- and C-type granules) may be produced in different cell types at different stages of endosperm development [71, 73, 74,]. Other studies have shown that the smaller B-type granules present in wheat and barley form inside protrusions from the amyloplast termed stromules (stroma containing tubules) [75–78]. The specific factors controlling the size of starch granules in all species are as yet unknown. Analysis of the physicochemical properties of A- and B-type starches in wheat endosperm reveals alterations in crystallinity and swelling properties, and a broader range of gelatinization temperature associated with smaller starch granules [65]. Granule size is an important factor for many applications. For example, in the production of carbonless copy papers and in the production of starch-based coatings and films starch granules of uniform size (spherical B-type granules, usually from wheat) are mixed with similarly sized encapsulated ink particles, and in the production of starch-based coatings and films [79, 80]. Reduction in granule size of potato tuber starch was achieved through expression of a starch-binding domain derived from Bacillus circulans cyclodextrin glycosyltransferase which was thought to interfere with glucan chain elongation by SSs [81].

4.04.5 Starches with Improved Functionalities The proportion of the two major components of the starch granule, amylose and amylopectin, varies between species, as does the size of the granules. In addition, starch also contains small amounts of lipid and phosphate [82]. The diversity of both composition and physicochemical properties gives rise to diverse processing properties, and therefore a multitude of end-uses for starch (for an overview, see Reference 83, and for a detailed recent review of starches in relation to food processing see Reference 84). A number of postharvest chemical modifications and treatments are currently used to modify starch structures to improve functionality [3]. For example, acid treatment or cross-linking of α-glucan chains within granules causes resistance to swelling when products are heated, making the starches tolerant to extremes of heat and cold and wide variations in pH, allowing such products to be used in a wide variety of processing conditions [85]. Chemical modifications such as cross-linking result in starches which are more resistant to amylolysis [86]. Recently, novel starches with improved functionalities have been produced in planta through the use of mutants and genetic modification. In the future, increased use of modified starches produced in planta will have the benefits of lower processing costs and a concomitant reduction in the production of environmentally hazardous waste materials. An increased understanding of the regulatory factors governing storage starch biosynthesis will allow a more targeted approach to the alteration and manipulation of starch structures produced in crop plants. Current capacities for biotechnological modification of starch quality in cereals have been discussed in Reference 87. The following section shows examples of functional starches, highlighting the extremes of structural modification: waxy (amylose-free) starches, high-amylose (resistant) starches, and starches with varying levels of phosphate.

4.04.6 Amylose-Free Starches Amylose-free starches, also called waxy starches, are produced by mutation of the waxy locus, encoding granule-bound starch synthase (GBSS) [88]. The physical properties peculiar to waxy starches include rapid gelatinization, yielding clear pastes as well as improved freeze–thaw stability compared to normal starches [89]. Consequently, waxy starches derived mainly from maize have found application in many areas of the food industry, such as stabilizers and thickeners, and as emulsifiers for salad dressings. Modified waxy maize starch is also important in processed meat products where its gelling properties are useful as a binder to maintain texture and stability of the processed product. Waxy potato starches have been produced by antisense downregulation of the GBSS gene [90]. Improvements in the freeze–thaw stability of waxy starches is usually accomplished by the use of chemical crosslinking; however, in waxy potato starch, this has been achieved by the simultaneous downregulation of three SS genes (GBSS, SSII, and SSIII) to produce a waxy starch with short-chain amylopectin [91]. This latter example shows how knowledge of the individual components of the pathway and their properties can be used to engineer improved functional starches, and how biotechnology may be useful in producing amylose-free (waxy) starches in other crop varieties.

4.04.7 High-Amylose Starches High-amylose starches have been produced by manipulating the degree of branching of amylopectin, as well as altering the proportion of amylose to amylopectin. In cereals, a high-amylose phenotype can be produced by a mutation in starch branching enzyme (SBE, EC 2.4.1.18) II genes. For example, the amylose extender (ae−) mutant of maize lacks activity of the major form of SBEII, termed SBEIIb [92, 93]. In wheat, suppression of SBEIIa and SBEIIb by RNA interference led to starch with >70% amylose levels (apparent anylase, see below) showing improvements in nutritional benefits [94]. High-amylose potato starch has also been produced by the downregulation of the corresponding SBEII gene, with improvements being made (amylose levels of more than 60%) by the additional inhibition of SBEI activity [95, 96]. In addition, the average chain length of potato amylose is much greater than that of cereal amylose [97, 98], making potato potentially a very useful source of high-amylose starch. High-amylose starches have many useful properties, particularly within the food industry (see the review by Klucinec and Keeling [99]). High-amylose starches are valued in the food industry because they display improved texture and reduced lipid absorption and can be processed into ‘resistant starch’. A characteristic structural feature of resistant starches is their high apparent amylose content (a combination of

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a higher proportion of amylose, and amylopectin with decreased branching and relatively longer glucan chains) [100]. The ae− mutant of maize forms the basis for the resistant corn starches currently available. Although they are invariably lower yielding than conventional genotypes, farmers are paid a premium to grow these added-value crops. High-amylose starches have also been produced recently in a barley mutant lacking SSIIa (the barley sex6 mutant [101]), which caused a reduction in amylopectin biosynthesis. The high-amylose starch produced in the barley amo mutants shows alterations in SBE activity [102], although the gene(s) responsible for this mutation remains to be identified. Resistant starches have been championed for human health with respect to a number of key physiological responses due to their low susceptibility to enzymic (amylolytic) hydrolysis [103–105] resulting from altered amylopectin structure [106]. Resistant starch shows reduced digestion in the small intestine compared with normal starches, with a higher proportion passing into the large intestine for fermentation by gut bacteria, producing short-chain fatty acids such as butyrate and acetate that are good for colon health [107, 108]. In addition, consumption of resistant starches results in a low glycemic index, reducing blood glucose surges, and helping to reduce the likelihood of type-2 diabetes [109–112]. Other physicochemical properties associated with high-amylose starches are exploited in the food industry. For example, highamylose starches have improved film-forming abilities compared with conventional starches, and are used as coatings on fried products, where they reduce fat uptake during cooking. High-amylose starches have a high gelling strength and are particularly suitable for the manufacture of boiled sweets/candies.

4.04.8 Prospects for Altering Amylopectin Structure Amylopectin essentially defines the structure of the starch granule. Consequently, much of the research effort directed toward modifying the structure of starch has been based upon an understanding of amylopectin biosynthesis. Unlike amylose biosynthesis, which seemingly requires just one enzyme (GBSS), amylopectin synthesis requires the coordinated action of a number of groups of enzymes (see the article on the enzymes of starch synthesis). Such complexity probably explains the difficulties encountered in the many attempts at targeted modification of starch structure by downregulating single enzymatic steps. Further, analysis of knockout mutations in certain genes, for example, SBEI, reveals different effects in different species, resulting in no demonstrable phenotype in maize [130] but causing alterations in starch structure in rice [131]. More dramatic alterations in starch structure have been achieved by simultaneous downregulation of multiple reactions, such as those seen in potato following reduction of SSII and SSIII activities [132, 133] and both SBEII forms in wheat [94]. Many of the key activities in the amylopectin biosynthetic pathway are coordinated through multienzyme complexes [134–135] and are controlled at the posttranslational level [93, 135] (see following chapter). It follows, therefore, that factors which control protein–protein interactions and individual enzyme activities will clearly be important targets for future strategies to modify starch structure.

4.04.9 Other Functional Properties The presence of phosphate groups on glucose residues of starch markedly alters the physicochemical properties, and hence the potential uses, of a given source of starch [113, 114]. High-phosphate starches (e.g., potato; see below) have fast hydration and swelling times and have high viscosity during heating [115]. There are two enzymes responsible for the covalent attachment of phosphate groups to starch (via C3 and C6 glucose residues): a granule-associated α-glucan water dikinase (ATP: α-1,4-glucan, water phosphotransferase (GWD), EC 2.7.9.4, previously designated R1), which is also found in the plastid stroma and is probably responsible for priming the starch for degradation by phosphorylating amylopectin on C6 of glucose residues [116–118] and phosphoglucan water dikinase (ATP: phospho-α-1,4-glucan, water phosphotransferase (PWD), EC 2.7.9.5), which phosphorylates glucan on the C3 position of glucose in amylopectin previously phosphorylated by GWD [119, 120]. The process of phosphoryla­ tion of glucose residues on starch (which in turn regulates starch turnover) is also regulated by granule-associated amylopectin phosphatases [121–125]. The process of starch degradation has been reviewed recently by Zeeman et al. [126]. Research suggests this is a promising target for manipulation of starch phosphate levels [116]. The phosphate content in potato starch is particularly high (0.089%) compared with text of cereals, with the highest levels of phosphate found in the hight-amylose types. Studies with transgenic potato have shown that the phosphate content of tuber starch can be altered in plants which show varied levels of SS and SBE activities. A study by Kossmann et al. [127] showed no effect on starch phosphate content when expression of SSI was suppressed in potato. However, plants with reduced levels of SSII showed a 50% reduction in starch phosphate [127]. Potato plants showing reduced expression of SSIII, however, displayed a 70% increase in starch phosphate [128]. This suggests that the SSII isoform of SS plays an important role in the incorporation of phosphorylated residues into the growing α-(1→4)-glucan chain. Antisense inhibition of SBE in potato caused elevated phosphate levels in the tuber starch [129].

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