P1: SFK/UKS BLBS102-c32 P2: SFK BLBS102-Simpson March 21, 2012 14:2 Trim: 276mm X 219mm Printer Name: Yet to Come 32 Starch Synthesis in the Potato Tuber P Geigenberger and A.R Fernie Introduction What Is Starch? Routes of Starch Synthesis and Degradation and Their Regulation Manipulation of Starch Yield Manipulation of Starch Structure Conclusions and Future Perspectives References INTRODUCTION Starch is the most important carbohydrate used for food and feed purposes and represents the major resource of our diet The total yield of starch in rice, corn, wheat, and potato exceeds 109 tons/year (Kossmann and Lloyd 2000, Slattery et al 2000) In addition to its use in a nonprocessed form, due to the low cost incurred, extracted starch is processed in many different ways Processed starch is subsequently used in multiple forms, for example in high-fructose syrup, as a food additive, or for various technical processes based on the fact that as a soluble macromolecule it exhibits high viscosity and adhesive properties (Table 32.1) The considerable importance of starch has made increasing the content and engineering the structural properties of plant starches major goals of both classical breeding and biotechnology over the last few decades (Smith et al 1997, Sonnewald et al 1997, Regierer et al 2002) Indeed, since the advent and widespread adoption of transgenic approaches some 15 years ago gave rise to the discipline of molecular plant physiology, much information has been obtained concerning the potential to manipulate plant metabolism For this chapter, we intend to review genetic manipulation of starch metabolism in potato (Solanum tuberosum) Potato is one of the most important crops worldwide, ranking fourth in annual production behind the cereal species rice (Oryza sativa), wheat (Triticum aestivum), and maize (Zea mais) Although in Europe and North America the consumption of potatoes is mainly in the form of processed foodstuffs such as fried potatoes and chips, in less developed countries it represents an important staple food and is grown by many subsistence farmers The main reasons for the increasing popularity of the potato in developing countries are the high nutritional value of the tubers combined with the simplicity of its propagation by vegetative amplification (Fernie and Willmitzer 2001) Since all potato varieties are true tetraploids and display a high degree of heterozygosity, genetics have played only a minor role in metabolic studies in this species However, because the potato is a member of the Solanaceae family, it was amongst the first crop plants to be accessible to transgenic approaches Furthermore, due to its relatively large size and metabolic homogeneity, the potato tuber represents a convenient experimental system for biochemical studies (Geigenberger 2003a) In this chapter we will describe transgenic attempts to modify starch content and structure in potato tubers that have been carried out in the last two decades In addition to describing biotechnologically significant results we will also detail fundamental research in this area that should enable future biotechnology strategies However, we will begin by briefly describing starch, its structure, and its synthesis WHAT IS STARCH? For many years, it has been recognized that the majority of starches consist of two different macromolecules, amylose and amylopectin (Fig 32.1), which are both polymers of glucose and are organized into grains that range in size from µm to more than 100 µm Amylose is classically regarded as an essentially linear polymer wherein the glucose units are linked through α-1-4-glucosidic bonds In contrast, although amylopectin contains α-1-4-glucosidic bonds, it also consists of a high proportion of α-1-6-glucosidic bonds This feature of amylopectin makes it a more branched, larger molecule than amylose, having a molecular weight of 107 –108 as opposed to × 105 –106 Food Biochemistry and Food Processing, Second Edition Edited by Benjamin K Simpson, Leo M.L Nollet, Fidel Toldr´a, Soottawat Benjakul, Gopinadhan Paliyath and Y.H Hui C 2012 John Wiley & Sons, Inc Published 2012 by John Wiley & Sons, Inc 613 P1: SFK/UKS BLBS102-c32 P2: SFK BLBS102-Simpson March 21, 2012 14:2 Trim: 276mm X 219mm 614 Printer Name: Yet to Come Part 5: Fruits, Vegetables, and Cereals Table 32.1 Industrial Uses of Starch Starch and Its Derivates Processing Industry Application Amylose and amylopectin (polymeric starch) Food Thickener, texturants, extenders, low-calorie snacks Beater sizing, surface sizing, coating Wrap sizing, finishing, printing Absorbents, adhesives, biodegradable plastics Sweeteners or stabilizing agents Products of starch hydrolysis, such as glucose, maltose, or dextrins Paper Textile Polymer Food Fermentation Pharmaceutical Chemical Feedstock to produce ethanol, liquors, spirits, beer, etc Feedstock to produce drugs and medicine Feedstock to produce organic solvents or acids Source: Adapted from Jansson et al 1997, with modification These molecules can thus be fractionated by utilizing differences in their molecular size as well as in their binding behavior Moreover, amylose is able to complex lipids, while amylopectin can contain covalently bound phosphate, adding further complexity to their structure In nature, amylose normally accounts between 20 and 30% of the total starch However, the percentage of amylose depends on the species and the organ used for starch storage The proportion of amylose to amylopectin and the size and structure of the starch grain give distinct properties to different extracted starches (properties important in food and industrial purposes; Dennis and Blakeley 2000) Starch grain size is also dependent on species and organ type It is well established that starch grains grow by adding layers, and growth rings within the grain may represent areas of fast and slower growth (Pilling and Smith 2003); however, very little is known about how these highly ordered structures are formed in vivo The interested reader is referred to articles by Buleon et al (1998) and Kossmann and Lloyd (2000) Figure 32.1 Starch structure: Amylose (A) is classically regarded as an essentially linear polymer wherein the glucose units are linked through α-1-4-glucosidic bonds In contrast, although amylopectin contains α-1-4-glucosidic bonds, it also consists of a high proportion of α-1-6-glucosidic bonds (B) P1: SFK/UKS BLBS102-c32 P2: SFK BLBS102-Simpson March 21, 2012 14:2 Trim: 276mm X 219mm Printer Name: Yet to Come 32 Starch Synthesis in the Potato Tuber ROUTES OF STARCH SYNTHESIS AND DEGRADATION AND THEIR REGULATION With the possible exception of sucrose, starch is the most important metabolite of plant carbohydrate metabolism It is by far the most dominant storage polysaccharide and is present in all major organs of most plants, in some instances at very high levels Due to the high likelihood of starch turnover, its metabolism is best considered as the balance between the antagonistic operation of pathways of synthesis and degradation To investigate the regulation of starch synthesis in more detail, growing potato tubers have been used as a model system Unlike many other tissues, the entry of sucrose into metabolism is relatively simple, in that it is unloaded symplasmically from the phloem, degraded via sucrose synthase to fructose and UDPglucose, which are converted to hexose monophosphates by fructokinase and UDPglucose pyrophosphorylase, respectively (Geigenberger 2003a) In contrast to sucrose degradation, which is localized in the cytosol, starch is synthesized predominantly, if not exclusively, in the plastid The precise pathway of starch synthesis depends on the form in which carbon crosses the amyloplast membrane (Fig 32.2) This varies between species and has been the subject of considerable debate (Keeling et al 1988, Hatzfeld and Stitt 1990, Tauberger et al 2000) Categorical evidence that carbon enters potato tuber, Chenopodium rubrum suspension cell, maize endosperm, and wheat endosperm amy- 615 loplasts in the form of hexose monophosphates rather than triose phosphates was provided by determination of the degree of randomization of radiolabel in glucose units isolated from starch following incubation of the various tissues with glucose labeled at the C1 or C6 positions (Keeling et al 1988, Hatzfeld and Stitt 1990) The cloning of a hexose monophosphate transporter from potato and the finding that the cauliflower homolog is highly specific for glucose-6-phosphate provides strong support for this theory (Kammerer et al 1998) Further evidence in support of glucose-6-phosphate import was provided by studies of transgenic potato lines in which the activity of the plastidial isoform of phosphoglucomutase was reduced by antisense inhibition, leading to a large reduction in starch content of the tubers (Tauberger et al 2000) These data are in agreement with the observations that heterotrophic tissues lack plastidial fructose1,6-bisphosphatase expression and activity (Entwistle and Rees 1990, Kossmann et al 1992) The results of recent transgenic and immunolocalization experiments have indicated that the substrate for uptake is most probably species specific, with clear evidence for the predominant route of uptake in the developing potato tuber being in the form of glucose-6-phosphate By contrast, in barley, wheat, oat, and possibly maize, the predominant form of uptake, at least during early stages of seed endosperm development, is as ADP-glucose (Neuhaus and Emes 2000) Irrespective of the route of carbon import, ADP-glucose pyrophosphorylase (AGPase, EC 2.7.7.27) plays an important role in starch synthesis, catalyzing the conversion of Figure 32.2 Pathways of sucrose to starch conversion in plants: 1, ADP-glucose transporter; 2, ATP/ADP translocator; 3, glucose-1-phoshate (Glc1P) translocator; 4, glucose-6-phosphate (Glc6P) translocator; 5, cytosolic ADP-glucose pyrophosphorylase (AGPase); 6, cytosolic phosphoglucomutase; 7, plastidial AGPase; 8, plastidial phosphoglucomutase In growing potato tubers, incoming sucrose is degraded by sucrose synthase to fructose and UDPglucose and subsequently converted to fructose-6-phosphate (Fru6P) and Glc1P by fructokinase and UDPglucose pyrophosphorylase, respectively (not shown in detail) The conversion of Fru6P to Glc6P in the cytosol is catalyzed by phosphoglucoisomerase, and the cleavage of PPi to Pi in the plastid is catalyzed by inorganic pyrophosphatase In potato tubers, there is now convincing evidence that carbon enters the plastid almost exclusively via the Glc6P translocator, whereas in cereal endosperm, the predominant form of uptake is as ADPglucose P1: SFK/UKS BLBS102-c32 P2: SFK BLBS102-Simpson 616 March 21, 2012 14:2 Trim: 276mm X 219mm Printer Name: Yet to Come Part 5: Fruits, Vegetables, and Cereals Figure 32.3 Routes of amylose and amylopectin synthesis within potato tuber amyloplasts For simplicity, the possible roles of starch-degrading enzymes in trimming amylopectin have been neglected in this scheme glucose-1-phosphate and ATP to ADP-glucose and inorganic pyrophosphate Inorganic pyrophosphate is subsequently metabolized to inorganic phosphate by a highly active inorganic pyrophosphatase within the plastid AGPase is generally considered as the first committed step of starch biosynthesis since it produces ADP-glucose, the direct precursor for the starch polymerizing reactions catalyzed by starch synthase (SS) (EC 2.4.1.21) and branching enzyme (EC 2.4.1.24; Fig 32.3) These three enzymes appear to be involved in starch synthesis in all species With the exceptions of the cereal species described above, which also have a cytosolic isoform of AGPase, these reactions are confined to the plastid The activities of AGPase and SS are sufficient to account for the rates of starch synthesis in a wide variety of photosynthetic and heterotrophic tissues (Smith et al 1997) Furthermore, changes in the activities of these enzymes correlate with changes in the accumulation of starch during development of storage organs (Smith et al 1995) AGPase from a range of photosynthetic and heterotrophic tissues is well established to be inhibited by phosphate, which induces sigmoidal kinetics, and to be allosterically activated by 3-phosphoglycerate (3PGA), which relieves phosphate inhibition (Preiss 1988; Fig 32.4) There is clear evidence that allosteric regulation of AGPase is important in vivo to adjust the rate of starch synthesis to changes in the rate of respiration that go along with changes in the levels of 3PGA, and an impressive body of evidence has been provided that there is a strong correlation between the 3PGA and ADP-glucose levels and the rate of starch synthesis under a wide variety of environmental conditions (Geigenberger et al 1998, Geigenberger 2003a) More recently, an important physiological role for posttranslational redox regulation of AGPase has been established (Tiessen et al 2002) In this case, reduction of an intermolecular cysteine bridge between the two small subunits of the heterotetrameric enzyme leads to a dramatic increase of activity, due to increased substrate affinities and sensitivity to allosteric activation by 3PGA (Fig 32.4) Redox activation of AGPase in planta correlated closely with the potato tuber sucrose content across a range of physiological and genetic manipulations (Tiessen et al 2002), indicating that redox modulation is part of a novel regulatory loop that directs incoming sucrose towards storage starch synthesis (Tiessen et al 2003) Crucially, it allows the rate of starch synthesis to be increased in response to sucrose supply and independently of any increase in metabolite levels (Fig 32.4), and it is therefore an interesting target for approaches to improving starch yield (see later) There are at least two separate sugar signaling pathways leading to posttranslational redox activation of AGPase, one involving an SNF1-like protein kinase (SnRK1), the other involving hexokinase (Tiessen et al 2003) Both hexokinase and SnRK1 have previously been shown to be involved in the transcriptional regulation of many plant genes Obviously, the transduction pathway that regulates the reductive activation of AGPase in plastids and the regulatory network that controls the expression of genes in the cytosol share some common components How the sugar signal is transferred into the plastid and leads to redox changes of AGPase is still unknown, but may involve interaction of specific thioredoxins with AGPase Subcellular analyses of metabolite levels in growing potato tubers have shown that the reaction catalyzed by AGPase is far from equilibrium in vivo (Tiessen et al 2002), and consequently, the flux through this enzyme is particularly sensitive to regulation by the above-mentioned factors It is interesting to note that plants contain multiple forms of AGPase, which is a tetrameric enzyme comprising two different polypeptides, a small subunit and a large subunit, both of which are required for full activity (Preiss 1988) Both subunits are encoded by multiple genes, which are differentially expressed in different tissues Although the precise function of this differential expression is currently unknown, it seems likely that these isoforms will differ in their capacity to bind allosteric regulators If this is indeed the case, then different combinations of small and large subunits should show different sensitivity to allosteric regulation—such as those observed in tissues from cereals While there is a wealth of information on the regulation of the plastidial isoforms of AGPase—the only isoform present in the potato tuber—very little is known about cytosolic isoforms in other species Several important studies provide evidence that the ADP-glucose produced in the cytosol can be taken up by the plastid (Sullivan et al 1991, Shannon et al 1998) From these studies and from characterization of mutants unable to transport ADP-glucose, it would appear that this is a predominant route for starch synthesis within maize Despite these findings, the physiological significance of cytosolic ADP-glucose production remains unclear for a range of species However, it has been calculated that the AGPase activity of the plastid is insufficient P1: SFK/UKS BLBS102-c32 P2: SFK BLBS102-Simpson March 21, 2012 14:2 Trim: 276mm X 219mm Printer Name: Yet to Come 32 Starch Synthesis in the Potato Tuber 617 Figure 32.4 Regulation of starch synthesis in potato tubers: ADPGlc-pyrophosphorylase (AGPase) is a key regulatory enzyme of starch biosynthesis It is regulated at different levels of control, involving allosteric regulation, regulation by posttranslational redox modification and transcriptional regulation Redox regulation of AGPase represents a novel mechanism regulating starch synthesis in response to changes in sucrose supply (Tiessen et al 2002) There are at least two separate sugar-sensing pathways leading to posttranslational redox activation of AGPase, one involving an SNF1-like protein kinase (SnRK1), the other involving hexokinase (HK) (Tiessen et al 2003) to account for the measured rates of starch synthesis in barley endosperm, suggesting that at least some of the ADP-glucose required for this process is provided by cytosolic production (Thorbjornsen et al 1996) It is interesting to note that the involvement of the various isoforms of AGPase in starch biosynthesis is strictly species dependent, whereas the various starch-polymerizing activities are ever present and responsible for the formation of the two different macromolecular forms of starch, amylose and amylopectin (Fig 32.3) SS catalyze the transfer of the glucosyl moiety from ADP-glucose to the nonreducing end of an α-1,4-glucan and are able to extend α-1,4-glucans in both amylose and amylopectin There are four different SS isozymes—three soluble and one that is bound to the starch granule Starch branching enzymes (SBE), meanwhile, are responsible for the formation of α-1,6 branch points within amylopectin (Fig 32.3) The precise mechanism by which this is achieved is unknown; however, it is thought to involve cleavage of a linear α-1,4-linked glucose chain and reattachment of the chain to form an α-1,6 linkage Two isozymes of starch branching enzyme, SBEI and SBEII, are present and differ in specificity The former preferentially branches unbranched starch (amylose), while the latter preferentially branches amylopectin Furthermore, in vitro studies indicate that SBEII transfers smaller glucan chains than does SBEI and would therefore be expected to create a more highly branched starch (Schwall et al 2000) The fact that the developmental expression of these isoforms correlates with the structural properties of starch during pea embryo development is in keeping with this suggestion (Smith et al 1997) Apart from that, isoforms of isoamylase (E.C 3.2.1.68) might be involved in debranching starch during its synthesis (Smith et al 2003) While the pathways governing starch synthesis are relatively clear, those associated with starch degradation remain somewhat controversial (Smith et al 2003) The degradation of plastidial starch can proceed via phosphorolytic or hydrolytic cleavage mechanisms involving α-1-4-glucan phosphorylases or amylases, respectively The relative importance of these different routes of starch degradation has been a matter of debate for many years The question is whether they are, in fact, independent pathways, since oligosaccharides released by hydrolysis can be further degraded by amylases or, alternatively, by phosphorylases In addition to this, the mechanisms responsible for the initiation of starch grain degradation in the plastid remain to be resolved, since starch grains have been found to ... important in food and industrial purposes; Dennis and Blakeley 2000) Starch grain size is also dependent on species and organ type It is well established that starch grains grow by adding layers, and. .. between 20 and 30% of the total starch However, the percentage of amylose depends on the species and the organ used for starch storage The proportion of amylose to amylopectin and the size and structure... 5: Fruits, Vegetables, and Cereals Table 32.1 Industrial Uses of Starch Starch and Its Derivates Processing Industry Application Amylose and amylopectin (polymeric starch) Food Thickener, texturants,