Following the uptake of carbon into the amyloplast, starch synthesis proceeds variously via (i) plastidial phosphoglucomutase and plastidial ADPglucose pyrophosphorylase, (ii) only via plastidial ADPglucose or (iii) via no intermediate steps prior to the polymerising reactions of starch synthases and branching enzymes (Smith et al., 1997). The exact route depends on the nature of the imported carbon source (see Figure 25.3). The first reaction of plastidial starch metabolism both in the potato tuber (Tauberger et al., 2000) and in the pea embryo (Hill and Smith, 1991) is the interconversion of glucose-6- and glucose-1-phosphate catalysed by plastidial phosphoglucomutase. Compelling evidence for the involvement of this enzyme in pea starch synthesis was provided by studies on the rug3 mutant which revealed that this locus encodes a plastidial phosphoglucomutase and that mutation at this locus results in a severe depletion of starch levels in pea embryos (Harrisson et al., 1998). In addition the depleted starch accumulation in transgenic potato plants exhibiting reduced levels of phosphoglucomutase (Tauberger et al., 2000) and the reduced gravitropic response of roots of the TC7 mutant of Arabidopsis (also deficient in this enzyme; Kiss et al., 1996) highlight its involvement in starch synthesis in other species. Moreover, transgenic potato plants with a reduction in the cytosolic isoform of phosphoglucomutase also exhibited reduced levels of starch, most probably due to a reduction in the available glucose 6-phosphate for uptake into the plastid (Fernie et al., 2001b).
The next reaction on the path to starch synthesis, which is catalysed by plastidial AGPase, has received much attention for a number of years. This reaction is often considered to be the first committed step of starch synthesis. AGPase utilises ATP and produces pyrophosphate, which is then hydrolysed by pyrophosphatase to yield 2Pi. The hydrolysis of PPi serves to remove the ADPglucose pyrophosphorylase reaction away from equilibrium. A cDNA encoding a soluble inorganic pyrophosphatase has been cloned from potato (Du Jardin et al., 1995), however, a functional assessment of the in vivo role of this protein is yet to be performed. Some evidence of a role for a pyrophosphatase activity was provided in experiments in which potato tuber discs were treated with fluoride (Viola and Davies, 1991), however, caution is required while interpreting these data as fluoride is a relatively promiscuous inhibitor. In many species including pea embryos, soybean cell suspension cultures and cauliflower buds, AGPase appears to be located exclusively in the plastid (MacDonald and ap Rees, 1983; Journet and Douce, 1985; Smith, 1988) and this isoform thus plays an important role in mediating the flux of carbon to starch. On removal or severe reduction of the AGPase activity in Arabidopsis or potato the level of starch was found to be dramatically reduced in all tissues (Lin et al., 1988a, 1988b; Müller-Röber et al., 1992). In direct contrast, when a non-regulated bacterial AGPase was expressed in various plant tissues the starch levels were dramatically increased (Stark et al., 1991). Plant AGPases are multisubunit proteins and expression studies in which the potato tuber enzyme way expressed in E. coli revealed that maximal activity can only be achieved on expression of both the large and the small subunit (Iglesias et al., 1993). Moreover, they are also allosterically regulated, being activated by 3-PGA and inhibited by Pi (Preiss, 1988; Sowokinos and Preiss 1992; Ballicora et al., 1995), and there is a clear evidence that changes in these metabolites are involved in the regulation of starch synthesis within leaves allowing the coordination of carbon assimilation, sucrose synthesis and starch synthesis (Stitt, 1997). There is also increasing evidence of a strong correlation between the 3PGA and ADPglucose levels and the rate of starch synthesis in potato tubers under a wide range of conditions (Geigenberger et al., 1997; 1998a). It is worth noting that using mutagenesis, 3PGA insensitive forms of the plant enzyme have also been created (Greene et al., 1996). However, since there are also several reports in the literature that the plant plastidial AGPase is clearly regulated, at least in vitro, by redox status (Fu et al., 1998; Ballicora et al., 2000), it is probable that this should also be taken into account in the design of future strategies intent on increasing starch content.
Whilst there is a wealth of information on the regulation of the plastidial isoform of AGPase detailed knowledge of the cytosolic isoform is very much limited. Several import studies provide evidence that the ADPglucose produced in the cytosol can be taken up by the plastid (Pozueta-Romera et al., 1991a, 1991b; Tetlow et al., 1994; Möhlmann et al., 1997), a process most probably mediated by the Brittle 1 protein (Sullivan et al., 1991). From these studies and from characterisation of maize Brittle 1 mutants which accumulated ADPglucose to 13 times the level found in wild-type plants (Shannon et al., 1996) it would seem that the Brittle 1 gene encodes for an amyloplastidial ADPglucose transporter. Despite these findings the physiological significance of cytosolic ADPglucose production remains unclear for a range of species. Thus, calculations of Denyer et al. (1996) demonstrate that the AGPase activity of the plastid is insufficient to account for measured rates of starch synthesis in barley endosperm, suggesting that at least some of the ADPglucose required for this process is provided by cytosolic production.
The recent purification of an ADPglucose pyrophosphatase from a range of plant species (Rodriguez et al., 2000) complicates matters further. This enzyme is believed to be co-localised with AGPase and to compete with starch synthase thus markedly blocking starch synthesis. Moreover, in studies in E. coli it was found that when this protein was reduced by insertional mutagenesis the level of glycogen marginally increased, suggesting this protein could play an important role in the regulation of carbohydrate storage (Moreno-Bruna et al., 2001). Furthermore, recent studies of 14-3-3 proteins within starch granules of Arabidopsis chloroplasts (Sehnke et al., 2001) indicate that these proteins may also be involved in the regulation of starch metabolism. However, to date no substantial data have been reported to characterise the physiological role of these proteins in plants and it is therefore unclear how important these proteins are in the regulation of starch metabolism. Despite this note of caution, it is clear that such regulatory genes could represent important future strategies. This is especially true when it is considered that the modification of pathway enzymes often has less than the desired effect.
Whilst the involvement of the above enzymes in starch biosynthesis are strictly species dependent, the starch polymerising activities are ever present and responsible for the formation of the two different macromolecular forms of starch, amylose and amylopectin. Starch synthases catalyse the transfer of the glucosyl moiety from ADPglucose to the non-linear end of an αα-1,4 glucan. The various starch synthases are able to extend 1, 4-glucans in both amylose and amylopectin. At least four different classes of starch synthases exist, designated as GBSS (granule-bound starch synthase), SSI, SSII and SSIII, which vary greatly in molecular weight, need for primers, substrate affinities and antigenic properties (for a review see Sivak and Preiss, 1998). It seems likely that most plant species contain the four different classes of starch synthase; however, the extent to which they contribute in vivo probably differs considerably between species (Denyer et al., 2001). Starch branching enzymes (SBE) are responsible for the formation of αα-1,6 branch points within amylopectin. Although there are more than two isoforms present in most plant species, all isoforms can be separated into two classes—most simply designated as A and B forms (Burton et al., 1995). The precise mechanism by which this is achieved is unknown, however it is thought to involve cleavage of a linear αα-1,4 linked glucose chain and reattachment of the chain to form an αα-1,6 linkage (Kossmann and Lloyd, 2000). The combined action of starch synthases and branching enzymes play an important role in determining the structure of starch which will be described in detail below. Other enzymes of starch synthesis and degradation are less well understood. Disproportionating enzyme (D-enzyme) is able to synthesise αα-1,4 glucans from maltose and has been suggested to be a candidate as a source of the malto-oligosaccharide primers required for starch synthesis. However, several lines of evidence suggest that this is unlikely to play a major role in starch synthesis in vivo. The maltose present in plant tissues is almost exclusively derived from starch (Kossmann and Lloyd, 2000) and transgenic plants exhibiting reduced D-enzyme expression had no effect on starch content (Takaha et al., 1998). Recent studies on an Arabidopsis mutant deficient in D-enzyme reveal a minor decrease in starch under certain conditions. However, they indicate that this enzyme primarily plays a role in the removal of malto-oligosaccharides during starch degradation (Critchley et al., 2001).
A further protein with a possible role in starch synthesis is the R1 protein. This protein has long been thought to be involved in the phosphorylation of starch since starch isolated from transgenic potato plants in which the R1 protein was reduced by antisense repression displayed only 10% of the phosphate content in wild-type potatoes (Lorbeth et al., 1998). Consistent with this proposal the enzymatic function of the protein was recently proved to be a starch water dikinase (Ritte et al., 2002).
To fully comprehend factors that determine starch biosynthesis knowledge of both synthetic and degradative functions is required. Currently, understanding of the roles of the starch degradative enzymes is relatively rudimentary. However, on the basis of several recent studies it has been proposed that several of the enzymes once regarded as operating exclusively in starch degradation also have a role in the synthesis of starch. The proposed catabolic and anabolic roles of debranching enzymes (isoamylases and pullanases), starch phosphorylase and αα- and ββ-amylases will be discussed below and their role in starch structure will be covered in greater detail in the later sections.
The endosperm from the Sugary-1 (Su-1) mutant of maize contains a second type of branched glucan other than amylopectin that is known as phytoglycogen. This mutant was shown to be deficient in an isoform of debranching enzyme (Pan and Nelson, 1984). This has since been confirmed when the Su-1 gene was cloned and found to encode an isoamylase-type enzyme (James et al., 1995). A similar phytoglycogen-accumulating Su-1 mutant has also been found in rice which exhibits changes in the activities of several enzymes of starch metabolism. The most dramatic of these by far was a 90% reduction in total debranching enzyme activity (Nakamara et al., 1996). That this reduction was specific to pullulanase was demonstrated immunologically, and pullulanase activity was found to correlate closely to phytoglycogen accumulation across rice lines producing different concentrations of soluble sugars (Nakamura et al., 1997). The sta-7 mutant of the monocellular green algae Chlamydomonas rheinhardtii has been found to contain no starch but a small amount of phytoglycogen (Mouille et al., 1996). Studies of this mutant revealed that the only activity of starch metabolism missing was that of a debranching enzyme. In combination these data present compelling evidence of a role of debranching enzymes in starch synthesis. However, the exact mechanism for this remains controversial. In the last few years two models have been proposed for amylopectin synthesis (summarised in Figure 25.4). In the model of Ball et al. (1996) glucans are synthesised within amylopectin until they reach a certain regular length which allows branching enzymes to act on them. Branching enzymes subsequently produce an uncrystalline glycogen-like polysaccharide (preamylopectin) on the outside of the linear chains. Debranching enzymes are then proposed to trim back the preamylopectin to leave amylopectin and in the process regenerate primer molecules to trigger a further cycle of synthesis and degradation. The second model of Zeeman et al. (1998a) suggests that starch is made exclusively by starch synthases and SBE and phytoglycogen is made as a byproduct of this process . They argue that phytoglycogen is subsequently degraded by a suite of enzymes including debranching enzymes and the products of this degradation can be used to support starch synthesis. Whichever of these models is correct it is clear that debranching enzymes play an important, albeit perhaps indirect, role in the process of starch biosynthesis.
The role of starch phosphorylase is less clear, since it catalyses a reversible reaction whereby glucose 1-phosphate is liberated from or incorporated into the non-reducing end of a glucan chain. Previously, based on the assumption that there was only a small amount of glucose 1-phosphate in the amyloplast, it was thought that the degradative reaction was favoured in vivo (Preiss and Levi, 1980; Steup, 1988). Plants contain both plastidial and cytosolic isoforms of this enzyme. On germination of pea embryos the plastidial isoform decreases 10-fold in activity whilst the cytosolic isoform remains unchanged (van Berkel et al., 1991). These data seem to preclude a major degradative role for the plastidial isoform and suggest that it is possible that the cytosolic isoform gains access to the starch as the amyloplastid membrane degrades. However, when the cytosolic isoform or either of the plastidial isoforms of this enzyme in potato is reduced by antisense repression no changes are observed in the rate of starch degradation (Duwenig et al., 1997a).
The degradation of starch by αα- and ββ-amylases is perhaps better characterised. Alpha-amylases are endoamylolytic, being able to break αα-1,4 bonds in amylose and amylopectin. These enzymes have been studied extensively, particularly in cereal endosperm, and are very varied with respect to both degradative ability and the extent of post-translational modification that they are subjected to (see Kossmann and Lloyd, 2000). Furthermore, in many species they appear to be gene families of αα-amylases with at least 10 in rice (Huang et al., 1990) and five in potato (Gausing and Kreiberg, 1989). It is thought that this great diversity may reflect the different roles for these enzymes in different tissues. Alpha-amylases appear to play a role in starch mobilisation during seed germination especially in cereals. In these crop plants it is thought that only hydrolytic enzymes have a role in the degradation of starch since large amounts of maltose and glucose accumulate and these substances are not known to be produced by the other degradative enzymes (Beck and Ziegler, 1989). Although the effects of αα-amylase during cereal seed germination has been well characterised for other plants its role is less clear. Studies on an Arabidopsis mutant with a reduced capacity to degrade leaf starch demonstrated that several by αα-amylases were extra-plastidial (Zeeman et al., 1998b). However, in this mutant it was one of the plastidial located αα-amylases that had a greatly reduced activity indicating that this isoform is responsible for starch degradation in Arabidopsis leaves. The role of αα-amylases in potato is currently unknown. However, in vitro studies of an αα-amylase purified from the potato starch granule revealed that at least one potato isoform is able, and correctly located, to degrade starch (Witt and Sauter, 1996).
In contrast to the αα-amylases, the ββ-amylases are exoamylolytic and liberate maltose residues progressively from the non-reducing ends of amylose and amylopectin until they react an αα-1,6 branch point. Their role in starch degradation is unclear as many isoforms have been found to be located in the vacuole. However, their activity increases during germination of seeds of mustard, maize and rice (Okomoto and Akazawa, 1980; Subbaramaiah and Sharma, 1989; Wang et al., 1997) and also on cold sweetening in potatoes (Hill et al., 1996; Nielsen et al., 1997). Since both these processes are associated with a time of active starch breakdown, it follows that there is at least some role for ββ-amylases during starch mobilisation.
The above studies have largely focussed on pathways as individual linear entities, however metabolism is in fact highly branched and better represented as a network since many pathways are linked by common metabolites and co-factors. In plant systems most prominent amongst such molecules are pyrophosphate and the adenylate and uridinylate pools. In addition other important factors that influence the regulation of carbohydrate metabolism should be considered before we begin discussing biotechnological strategies in earnest.
Pyrophosphate is an essential co-factor in starch storing organs since following SuSy-dependent sucrose cleavage, sucrose mobilisation requires the reaction catalysed by the pyrophosphate-dependent UDPglucose pyrophosphorylase (Zrenner et al., 1993). Since one molecule of pyrophosphate is required for each sucrose cleaved by the SuSy-dependent pathway and given that, in many heterotrophic cells the majority of the incoming sucrose is converted to starch, there are only two possible sources for the necessary pyrophosphate to fuel this reaction. It is either recycled across the amyloplast membrane from the starch biosynthetic pathway or it is produced by a cycling process involving pyrophosphate: fructose 6-phosphate, 1-phosphotransferase (PFP) or the tonoplast pyrophosphatase (Stitt, 1998). Correlative evidence for the former proposal includes (i) the observation that pyrophosphatase activity does not increase during the developmental switch from elongating stolon to growing tuber (Appeldoorn et al., 1999), (ii) the description of a pyrophosphate transporter in chloroplasts (Lunn and Douce, 1993) and (iii) measurements of pyrophosphate contents and the rates of sucrose degradation and starch synthesis in a range of transgenic lines exhibiting altered levels of pyrophosphate (Farre et al., 2000). Furthermore, analysis of metabolite levels in transgenic lines exhibiting reduced levels of PFP suggest that this enzyme is operating in the glycolytic direction and thus consuming pyrophosphate in vivo (Hajirezaei et al., 1994) and, therefore, is incapable of supporting sucrose degradation. Moreover, there was no difference in the rate of sucrose degradation (or resynthesis) in heterotrophic transgenic tobacco cells exhibiting increased levels of fructose 2,6-bisphosphate and therefore elevated in vivo activity of PFP (Fernie et al., 2001c). Taken together these data indicate that it is unlikely that PFP supplies pyrophosphate for sucrose degradation and it seems possible that the pyrophosphate level provides an important link between the catabolic reactions of the cytosol and the anabolic reactions of the plastid.
Direct evidence to support this model is however unfortunately lacking. When pyrophosphate levels were depressed by the expression of a bacterial pyrophosphatase, initial experiments revealed an inhibition of sucrose breakdown and a reduction in starch accumulation (Jelitto et al., 1992), whereas subsequent experimentation carried out with plants at a different developmental stage showed the exact opposite (Geigenberger et al., 1998b). A further discrepancy in the behaviour of plants exhibiting low levels of pyrophosphate is that they have been variously reported to be characterised by accelerated (Farre et al., 2001a) and delayed (Hajirezaei and Sonneewald, 1999) sprouting. However, different independent transgenic lines were used in these two studies and it is possible that different pyrophosphate levels trigger different effects. Despite these discrepancies, the combination of results to date suggest that pyrophosphate is clearly capable of effecting a variety of processes and, therefore, has a central, if somewhat enigmatic, role in the regulation of the sucrose to starch transition in heterotrophic tissues.
Studies on the effects of increasing the levels of adenylates (Tjaden et al., 1998; Loef et al., 2000; Geigenberger et al., 2001) and uridinylates (Loef et al., 2001) on biosynthesis, although limited to date, have revealed that these compounds are also important in the regulation of the starch biosynthetic pathway, with increases in cellular ATP (Loef et al., 2001) or of ATP supply to the plastid (Tjaden et al., 1998; Geigenberger et al., 2001) stimulating starch synthesis. Similarly, feeding potato tuber slices with precursors of uridinylate synthesis resulted in increase of uridinylates which stimulated starch synthesis and resulted in an increased partitioning of carbon towards starch.
In addition to their role as intermediates in metabolic pathways recently much attention has focused on the potential role of sugars as regulatory signals (Koch et al., 1990; Purcell et al., 1998). Driven by compelling evidence from the yeast system the primary candidate for a signal is glucose acting via sensing mechanisms involving either hexokinase or SNF1. In potato tubers the expression of invertase in the cytosol resulted in an accumulation of glucose and a resultant shift in partitioning from starch towards glycolysis (Trethewey et al., 1998). A similar metabolic shift was observed following the incubation of wild-type tuber discs in high-glucose concentrations (Geiger et al., 1998). However, the expression of a bacterial sucrose phosphorylase and the supertransformation of invertase expressing tubers with a bacterial glucokinase (Trethewey et al., 1998; Trethewey et al., 2001) displayed essentially the same metabolic phenotype despite the fact that they were not characterised by an increased glucose concentration. Furthermore, modulation in the activities of either isoform of potato tuber hexokinase had no major impact on either tuber morphology or on metabolism (Veramendi et al., 1999; 2001) thereby strongly arguing against a role for glucose-dependent signalling processes in the tuber.
Thus, the subject of sugar sensing remains highly controversial and there is reasonable evidence that sugar carriers or other factors at the plasma membrane may play a role in the regulation of heteotrophic metabolism (Sonnewald et al., 1997; Lalonde et al., 1999; Fernie et al., 2000, 2001a; Roitsch et al., 2000). Firstly, potato tubers expressing invertase in the apoplast are characterised by an increased rate of cell division and display a different metabolism to those expressing the invertase at a cytosolic location (Sonnewald et al., 1997) despite the fact that the hexoses released at both sites are able to enter metabolism (Fernie et al., 2000). Secondly, the rate of sucrose degradation and starch synthesis is stimulated when the unmetabolisable sucrose analogue palatinose is supplied to isolated tuber discs despite the fact that the uptake of palatinose into the tuber parenchyma is negligible (Fernie et al., 2001a). Whilst these studies allow us to speculate that these responses are mediated by a factor(s) localised in the plasma membrane (Lalonde et al., 1999), a lot of work is needed using a variety of approaches before the precise nature and role of sugar sensing in heterotrophic tissues can be defined and usefully manipulated. They do, however, remain an attractive alternative approach for metabolic manipulation since if such regulatory processes can be harnessed by the metabolic engineer as an opportunity to orchestrate rather than merely modify metabolism may arise.
Diseases of
Plants, deviations from the normal growth and development of plants
incited by microorganisms, parasitic flowering plants, nematodes, viruses, or
adverse environmental conditions. In the United States alone, known plant
diseases attributable to these causes are estimated to number more than 25,000;
the estimated annual losses therefrom add up to several billion dollars.
Injuries to plant life due primarily to insects, mites, or animals other than
nematodes are not regarded as plant diseases.
