P1: SFK/UKS BLBS102-c32 P2: SFK BLBS102-Simpson 618 March 21, 2012 14:2 Trim: 276mm X 219mm Printer Name: Yet to Come Part 5: Fruits, Vegetables, and Cereals Figure 32.5 Outline of the pathway of plastidial starch degradation The scheme is mainly based on recent molecular studies in Arabidopsis leaves (Ritte et al 2002, Smith et al 2003, Chia et al 2004) After phosphorylation of glucans on the surface of the starch granule via glucan water dikinase (GWD; R1-protein), starch granules are attacked most probably by α-amylase and the resulting branched glucans subsequently converted to unbranched α-1,4-glucans via debranching enzymes (isoamylase and pullulanase) Linear glucans are metabolized by the concerted action of β-amylase and disproportionating enzyme (D-enzyme, glucan transferase) to maltose and glucose The phosphorolytic degradation of linear glucans to Glc-1-P (glucose-1-phosphate) by α-1-4-glucan phosphorylase (α-1-4-GP) is also possible, but seems of minor importance under normal conditions Most of the carbon resulting from starch degradation leaves the chloroplast via a maltose transporter in the inner membrane Subsequent cytosolic metabolism of maltose involves the combined action of glucan transferase (D-enzyme), α-1-4-glucan phosphorylase (α-1-4-GP), and hexokinase (HK) Glucose transporter (GTP) and triose-P/Pi -translocator (TPT) are also shown be very stable and relatively resistant against enzymatic action in vitro More recently, molecular and genetic approaches have allowed rapid progress in clarifying the route of starch degradation in leaves of the model species Arabidopsis thaliana (Fig 32.5) The phosphorylation of starch granules by glucan water dikinase (GWD, R1) has been found to be essential for the initiation of starch degradation in leaves and tubers (Ritte et al 2002, Ritte et al 2004) Transgenic potato plants (Lorberth et al 1998) and Arabidopsis mutants (Yu et al 2001) with decreased GWD activity showed a decrease in starch-bound phosphate and were severely restricted in their ability to degrade starch, leading to increased starch accumulation in leaves The underlying mechanisms are still unknown Phosphorylation of starch may change the structure of the granule surface to make it more susceptible to enzymatic attack or may regulate the extent to which degradative enzymes can attack the granule It is generally assumed that the initial attack on the starch granule is catalyzed by α-amylase (endoamylase) This enzyme catalyzes the internal cleavage of glucan chains from amylose or amylopectin, yielding branched and unbranched α-1,4-glucans, which are then subject to further digestion (Fig 32.5) Debranching enzymes (isoamylase and pullulanase) are needed to convert branched glucans into linear glucans by cleaving the α-1,6 branch points Further metabolism of linear glucans could involve phosphorolytic or hydrolytic routes In the first case, α-glucan phosphorylase leads to the phosphorolytic release of Glc1-P, which can be further metabolized to triose-P within the chloroplast and subsequently exported to the cytosol via the triose-P/Pi translocator Recent results show that the contribution of α-glucan phosphorylase to plastidial starch degradation is relatively small Removal of the plastidial form of phosphorylase in Arabidopsis did not affect starch degradation in leaves of Arabidopsis (Zeeman et al 2004) and potato (Sonnewald et al 1995) It has been suggested that the phosphorolytic pathway could be more important to degrading starch under certain stress conditions (i.e., water stress; Zeeman et al 2004) However, no regulatory properties have been described for glucan 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 phosphorylase in plants, other than the effect of changes in the concentrations of inorganic phosphate on the activity of the enzyme (Stitt and Steup 1985) In the second case, hydrolytic degradation of linear glucans in the plastid can involve the combined four action enzymes: α-amylase, β-amylase, α-glucosidase and disproportionating enzyme (D-enzyme, glucan transferase) There is now direct molecular evidence that β-amylase (exoamylase) plays a significant role in this process (Scheidig et al 2002) This enzyme catalyzes the hydrolytic cleavage of maltose from the nonreducing end of a linear glucan polymer that is larger than maltotriose Maltotriose is further metabolized by D-enzyme, producing new substrate for β-amylase and releasing glucose (Fig 32.5) Recent studies document that most of the carbon that results from starch degradation leaves the chloroplast in the form of maltose, providing evidence that hydrolytic degradation is the major pathway for mobilization of transitory starch (Weise et al 2004) Elegant studies with Arabidopsis mutants confirmed this interpretation and identified a maltose transporter in the chloroplast envelope that is essential for starch degradation in leaves (Niittylăa et al 2004) The further metabolism of maltose to hexose-phosphates is then performed in the cytosol and is proposed to involve cytosolic forms of glycosyltransferase (D-enzyme; Lu and Sharkey 2004, Chia et al 2004), α-glucan phosphorylase (Duwenig et al 1997), and hexokinase, similar to maltose metabolism in the cytoplasm of E coli (Boos and Shuman 1998) It will be interesting to find the potato homolog of the maltose transporter and to investigate its role during starch degradation in tubers Despite recent progress in clarifying the route of starch degradation in Arabidopsis leaves and potato tubers, the regulation of this pathway still remains an open question More information is available concerning cereal seeds, where the enzymes involved in starch hydrolysis have been found to be especially active during seed germination, when starch is mobilized within the endosperm, which at this stage of development represents a nonliving tissue The most studied enzyme in this specialized system is α-amylase, which is synthesized in the surrounding aleurone layer and secreted into the endosperm This activity and that of α-glucosidase increase in response to the high levels of gibberellins present at germination A further level of control of the amylolytic pathway is achieved by the action of specific disulphide proteins that inhibit both α-amylase and debranching enzyme Thioredoxin h reduces and thereby inactivates these inhibitor proteins early in germination Glucose liberated from starch in this manner is phosphorylated by a hexokinase, before conversion to sucrose and subsequent transport to the developing embryo (Beck and Ziegler 1989) MANIPULATION OF STARCH YIELD In potato tubers, like all crop species, there has been considerable interest to increase the efficiency of sucrose to starch conversion and thus to increase starch accumulation by both conventional plant breeding and genetic manipulation strategies Traditional methodology based on the crossing of haploid potato lines and the establishment of a high density genetic map have allowed the 619 identification of quantitative trait loci (QTL) for starch content (Schăafer-Pregl et al 1998); however, this is outside the scope of this chapter, and the interested reader is referred to Fernie and Willmitzer (2001) Transgenic approaches in potato have focused primarily on the modulation of sucrose import (Leggewie et al 2003) and sucrose mobilization (Trethewey et al 1998) or the plastidial starch biosynthetic pathway (see Table 32.2); however, recently more indirect targets have been tested, which are mostly linked to the supply of energy for starch synthesis (see Tjaden et al 1998, Jenner et al 2001, Regierer et al 2002) To date, the most successful transgenic approaches have resulted from the overexpression of a bacterial AGPase (Stark et al 1991) and the Arabidopsis plastidial ATP/ADP translocator (Tjaden et al 1998), and the antisense inhibition of a plastidial adenylate kinase (Regierer et al 2002) in potato tubers The majority of previous attempts to improve the starch yield of potato tubers concentrated on the expression of a more efficient pathway of sucrose degradation, consisting of a yeast invertase, a bacterial glucokinase, and a sucrose phosphorylase (Trethewey et al 1998, 2001) However, although the transgenics exhibited decreased levels of sucrose and elevated hexose phosphates and 3-PGA with respect to wild type, these attempts failed Tubers of these plants even contained less starch than the wild type, but showed higher respiration rates Recent studies have shown that as a consequence of the high rates of oxygen consumption, oxygen tensions fall to almost zero within growing tubers of these transformants, possibly as a consequence of the high energy demand of the introduced pathway, and this results in a dramatic decrease in the cellular energy state (Bologa et al 2003, Geigenberger 2003b) This decrease is probably the major reason for the unexpected observation that starch synthesis decreases in these lines In general, oxygen can fall to very low concentrations in developing sink organs like potato tubers and seeds, even under normal environmental conditions (Geigenberger 2003b, Vigeolas et al 2003, van Dongen et al 2004) The consequences of these low internal oxygen concentrations for metabolic events during storage product formation have been ignored in metabolic engineering strategies Molecular approaches to increase internal oxygen concentrations could provide a novel and exiting route for crop improvement Another failed attempt at increasing tuber starch accumulation was the overexpression of a heterologous sucrose transporter from spinach under the control of the CaMV 35S promoter (Leggewie et al 2003) The rationale behind this attempt was that it would increase carbon partitioning toward the tuber; however, in the absence of improved photosynthetic efficiency this was not the case With respect to the plastidial pathway for starch synthesis, much attention has been focused on AGPase Analysis of potato lines exhibiting different levels of reduction of AGPase due to antisense inhibition have been used to estimate flux control coefficients for starch synthesis of between 0.3 and 0.55 for this enzyme (Geigenberger et al 1999a, Sweetlove et al 1999), showing that AGPase is collimating for starch accumulation in potato tubers In addition to having significantly reduced starch content in the tubers, these lines also exhibit very high tuber sucrose content, produce more but smaller tubers per plant, and Antisense inhibition Overexpression of AATP Antisense inhibition Antisense inhibition of AGPB Overexpression of glgC Antisense inhibition of GBSSI Plastidial ATP/ADP translocator Plastidial ATP/ADP translocator Plastidial adenylate kinase ADPGlc pyrophosphorylase ADPGlc pyrophosphorylase 620 Granule bound starch synthase Starch is free of amylose No effect Smaller grains, smaller tubers, increased tuber number Not determined Amylose content decreased by 40% Not determined Not determined Grains size decreased to 50%, strange tuber shape Grains with angular shape Grain/Tuber Morphology Not determined More amylose Less amylose Starch Structure Amylose free starch High starch — High starch High starch with more amylose — Application Kuipers et al 1994 Stark et al 1991 Măuller-Răober et al 1992, Lloyd et al 1999b Tjaden et al 1998, Geigenberger et al 2001 Regierer et al 2003 Tjaden et al 1998 Reference Trim: 276mm X 219mm Starch content increased by up to 30% No effect Starch content increased by up to 30% Starch content and yield increased by up to 60% Starch content decreased down to 10% of WT-level Starch content decreased to 20% Starch Content/ Yield 14:2 Transgenic Approach March 21, 2012 Target P2: SFK BLBS102-Simpson Table 32.2 Summary of Transgenic Approaches to Manipulate Starch Structure and Yield in Potato Tubers P1: SFK/UKS BLBS102-c32 Printer Name: Yet to Come Antisense inhibition of GBSS I, SS II & SS III Antisense inhibition of SBE I and SBE II Antisense inhibition of R1 Soluble and granulebound starch synthases Starch branching enzymes Starch water dikinase (R1) 621 No effect Less amylose, shorter amylopectin chains Amylose increased to >70%, low amylopectin, five-fold more P in starch Less P in starch Not determined Granule morphology altered Low P starch High amylose and high P starch Freeze/thaw- stable starch — Granules cracked with deep fissures Concentric granules — No effect Lorberth et al 1998 Schwall et al 2000 Abel et al 1996, Marshall et al 1996; Fulton et al 2002 Jobling et al 2002, Fulton et al 2002 Kuipers et al 1994 Trim: 276mm X 219mm Decreased yield No effect No effect Amylose increased up to 25.5% Amylopectin chain length altered, more P in starch 14:2 No effect March 21, 2012 Overexpression of GBSS I Antisense inhibition of SS III P2: SFK BLBS102-Simpson Granule-bound starch synthase Soluble starch synthase P1: SFK/UKS BLBS102-c32 Printer Name: Yet to Come P1: SFK/UKS BLBS102-c32 P2: SFK BLBS102-Simpson 622 March 21, 2012 14:2 Trim: 276mm X 219mm Printer Name: Yet to Come Part 5: Fruits, Vegetables, and Cereals produce smaller starch grains than the wild type (Măuller-Răober et al 1992; see also later discussion and Table 32.2) To date, one of the most successful approaches for elevating starch accumulation in tubers was that of Stark et al (1991), who overexpressed an unregulated bacterial AGPase This manipulation resulted in up to a 30% increase in tuber starch content However, it should be noted that the expression of exactly the same enzyme within a second potato cultivar did not significantly affect starch levels (Sweetlove et al 1996), indicating that these results might be highly context dependent Another more promising route to increase starch yield would be to manipulate the regulatory network leading to posttranslational redox activation of AGPase (see Fig 32.4) Based on future progress in this field, direct strategies can be taken to modify the regulatory components leading to redox regulation of starch synthesis in potato tubers More recent studies have shown that the adenylate supply to the plastid is of fundamental importance to starch biosynthesis in potato tubers (Loef et al 2001, Tjaden et al 1998) Overexpression of the plastidial ATP/ADP translocator resulted in increased tuber starch content, whereas antisense inhibition of the same protein resulted in reduced starch yield, modified tuber morphology, and altered starch structure (Tjaden et al 1998) Furthermore, incubation of tuber discs in adenine resulted in a considerable increase in cellular adenylate pool sizes and a consequent increase in the rate of starch synthesis (Loef et al 2001) The enzyme adenylate kinase (EC 2.7.4.3) interconverts ATP and AMP into ADP Because adenylate kinase is involved in maintaining the levels of the various adenylates at equilibrium, it represents an interesting target for modulating the adenylate pools in plants For this reason, a molecular approach was taken to downregulate the plastidial isoform of this enzyme by the antisense technique (Regierer et al 2002) This manipulation led to a substantial increase in the levels of all adenylate pools (including ATP) and, most importantly, to a record increase in tuber starch content up to 60% above wild type These results are particularly striking because this genetic manipulation also resulted in a dramatic increase in tuber yield during several field trials of approximately 40% higher than that of the wild type When taken in tandem, these results suggest a doubling of starch yield per plant In addition to the changes described above, more moderate increases in starch yield were previously obtained by targeting enzymes esoteric to the pathway of starch synthesis, for example, plants impaired in their expression of the sucrose synthetic enzyme, sucrose phosphate synthase (Geigenberger et al 1999b) This enzyme exerts negative control on starch synthesis since it is involved in a futile cycle of sucrose synthesis and degradation and leads to a decrease in the net rate of sucrose degradation in potato tubers (Geigenberger et al 1997) Although these results are exciting from a biotechnological perspective and they give clear hints as to how starch synthesis is coordinated in vivo, they not currently allow us to establish the mechanisms by which they operate It is also clear that these results, while promising, are unlikely to be the only way to achieve increases in starch yield Recent advances in transgenic technologies now allow the manipulation of multiple targets in tandem (Fernie et al 2001), and given that several of the successful manipulations described above were somewhat unexpected, the possibility that further such examples will be uncovered in the future cannot be excluded One obvious future target would be to reduce the expression levels of the starch degradative pathway since, as described above, starch content is clearly a function of the relative activities of the synthetic and degradative pathways Despite the fact that a large number of Arabidopsis mutants has now been generated that are deficient in the pathway of starch degradation, the consequence of such deficiencies has not been investigated in a crop such as potato tubers Furthermore, there are no reports to date of increases in starch yield in heterotrophic tissues displaying mutations in the starch degradative pathway A further phenomenon in potatoes that relates to starch metabolism is that of cold-induced sweetening, where the rate of degradation of starch to reducing sugars is accelerated As raw potatoes are sliced and cooked in oil at high temperature, the accumulated reducing sugars react with free amino acids in the potato cell, forming unacceptably brown- to black-pigmented chips or fries via a nonenzymatic, Maillard-type reaction Potatoes yielding these unacceptably colored products are generally rejected for purchase by the processing plant If a “coldprocessing potato” (i.e., one which has low sugar content even in the cold) were available, energy savings would be realized in potato-growing regions where outside storage temperatures are cool In regions where outside temperatures are moderately high, increased refrigeration costs may occur This expense would be offset, however, by removal of the need to purchase dormancyprolonging chemicals, by a decreased need for disease control, and by improvement of long-term tuber quality Although such a cold-processing potato is not yet on the market, several manipulations potentially fulfill this criterion, perhaps most impressively the antisense inhibition of GWD, which is involved in the initiation of starch degradation (Lorberth et al 1998) Further examples on this subject are excellently reviewed in a recent paper by Sowokinos (2001) While only a limited number of successful manipulations of starch yield have been reported to date, far more successful manipulations have been reported with respect to engineering starch structure These will be reviewed in Section “Manipulation of Starch Structure.” MANIPULATION OF STARCH STRUCTURE In addition to attempting to increase starch yield, there have been many, arguably more, successful attempts to manipulate its structural properties Considerable natural variation exists between the starch structures of crop species, with potato starch having larger granules, less amylose, a higher proportion of covalently bound phosphate, and less protein and lipid content than cereal starches The level of phosphorylation strongly influences the physical properties of starch, and granule size is another important factor for many applications; for example, determining starch noodle processing and quality (Jobling et al 2004) In addition, the ratio between the different polymer types can affect the functionality of different starches (Slattery et al 1998) High amylose starches are used in fried snack products to create crisp ... bacterial glucokinase, and a sucrose phosphorylase (Trethewey et al 19 98, 2001) However, although the transgenics exhibited decreased levels of sucrose and elevated hexose phosphates and 3-PGA with respect... in the cytosol and is proposed to involve cytosolic forms of glycosyltransferase (D-enzyme; Lu and Sharkey 2004, Chia et al 2004), α-glucan phosphorylase (Duwenig et al 1997), and hexokinase,... maltose metabolism in the cytoplasm of E coli (Boos and Shuman 19 98) It will be interesting to find the potato homolog of the maltose transporter and to investigate its role during starch degradation