Cytoplasm

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UDPgluc fnic

I fructans Amylose mffifr Ucuolej

Amylopfast A"ty1opectin

Figure 2 Schematic diagram of the currently accepted pathway for starch biosynthesis in storage organs. Photosynthate is transported as sucrose from source leaves through the phloem to the storage organ. It is then moved as sucrose into the storage tissues or cleaved in some plants by a cell wall invertase to glucose and fructose (not shown) to be resynthesized as sucrose by sucrose phosphate synthase in the cytoplasm. Storage as fructans in the vacuole represents an alternative to starch biosynthesis in temperate grasses. The key enzymes of starch biosynthesis are (1) sucrose synthase (SucSyn), (2) UDPglucose pyrophosphorylase (UGP), (3) ADPglucose pyrophosphory-lase (AGP), (4) phosphoglucomutase (PGM), (5) granule-bound starch synthase (GBSS), (6) soluble starch synthase (SS), (7) starch branching enzyme (SBE), and (8) debranching enzyme (DBE). Not all alternative shunts In the pathway are shown. Fructose can be converted to glucose-1-phosphate via fructokinase (FK), phosphoglucoisomerase (PGI), and phosphoglucomutase (PGM). The relative proportions of ADPglucose synthesized in the cytoplasm and amyloplast vary from species to species. Translocators are shown as ovals on the organelle membranes.

imported and then converted to PPi and AGPglucose. This is energetically less favorable than movement of ADPglucose into the plastid and transport of ADP outward in return. Nevertheless, some AGP is plastidic even in the cereals, where the majority is cytoplasmic; the relative roles of the two forms remain to be established. Furthermore, in potato tubers it appears that the majority of the carbon moves as glucose-6-phosphate into the amyloplasts

(40), where it is subsequently converted to glucose-1-phosphate and then to starch. The details of the pathway in any particular plant are important regarding the possibilities of modifying starch quantity or quality. Starch content as well as starch quality can be affected by modulating the activity and properties of the AGP present in storage tissues.

C. Synthesis of Amylose

Amylose consists of glucose subunits linked by a-1,4 bonds into linear chains, with occasional a-1,6 branch points connecting additional a-1,4-linked chains onto a backbone (41). In barley, the average chain length is 1800 glucose units (42), but it may vary in the cereals between 1000 and 4400 glucose moieties, yielding a molecular weight of between 1.6 X 105 and 7.1 X 105 (43). In most normal starches, amylose makes up 20-30% of the total by weight. This is reduced to virtually none in the waxy mutants. The general features of amylose and the other main component of starch, amylopectin, are well established (44,45). The a-1,4 links in both amylose and amylopectin (see later) are made by the starch synthases (EC 2.4.1.21). The enzyme occurs in multiple forms, but all forms use ADPglucose as the glucose donor to the growing chain. In the storage organs, namely endosperm, cotyledons, and tubers, amylose is synthesized by the form called granule-bound starch synthase I (GBSS or GBSSI).

The "waxy" starches, perhaps the most common example of a modified carbohydrate created through both breeding and transgenic biotechnology, virtually completely lack amylose because of the absence of the GBSSI (46-48). The other forms of starch synthase are unable to compensate in such mutants, called waxy ("glutinous" in rice) because of the resulting property of the starch (the gene for GBSSI thereby being wx). In nonstorage tissues, however, amylose continues to be synthesized in waxy mutants, a form of the starch synthase called GBSSII carrying out the task in the cases examined (49). The gene or transcript for GBSSI has been cloned from many sources; alignments of these sequences revealed that these are highly conserved (50). The a-1,6 branch points in amylose are not synthesized by GBSSI or GBSSII but may derive from the action of a starch branching enzyme (SBE, see next for amylopectin) or from a branched oligosaccharide as the starch-synthetic substrate, with the poorly branched product subsequently elongated by GBSS. The substrate for amylose biosynthesis remains controversial and in vivo may be either amylopectin chains or soluble malto-oligosaccharides, both, or neither (45).

D. Synthesis of Amylopectin

Amylopectin is considerably more complex as a molecule, and its biosynthesis is commensurately more intriguing. The linear, a-l,4-linked portion of the polymer is produced by the soluble starch synthases (EC 2.4.1.21), which catalyze growth of the a-1,4 glucan chain by addition of glucose residues from ADPglucose. Historically, these enzyme forms received their name because they are not bound tightly to the starch granule, in contrast to the granule-bound starch synthase or GBSS (51). More recently, it has become clear that all forms of starch synthase are to some extent partitioned onto the starch granules or somehow become trapped in the growing, insoluble granule, so the original distinction is not very useful (52).

The amylopectin-synthesizing starch synthases are found in multiple forms in virtually all plants examined (reviewed in Ref. 45). Alignment of the proteins encoded by the sequenced soluble synthase form divides them into three main groups: SSI, SSII, and SSIII (53,54). Investigations of mutants and transgenics lacking or reduced in the activity of one of the SS forms indicate that each plays a specific, or at least preferential, role in amylopectin synthesis (9,11,55,56). These efforts have been complemented by expression of specific forms in E. coli and analyses of the a-glucan products made in the bacteria (57). From such experiments, SSII appears to synthesize a-1,4 chains of intermediate length (11), whereas the SSI form in barley (J. Tanskanen and A. H. Schulman, unpublished) appears to be involved in initiation of new chains (56). Potatoes expressing antisense to SSII (58), consistent with this view, have reduced relative abundance of chains of DP 18-50. The complexity of amylose biosynthesis from the perspective of engineering the pathway lies not only in the multiplicity of forms but also in their overlapping roles. Although one form may, because of its kinetics, be responsible for producing chains of a certain size class, in a mutant or transgenic plant where this form is absent another form may substitute but only partially or with identical results. The combination of overlapping roles and pleiotropism can lead to novel or unpredicted amylopectin structures in engineered starches (58,59).

The soluble synthases cover half of the story of amylopectin biosynthesis, however. The starch branching enzymes (SBEs, a-1,4-glucan, a-1,4-glucan-6-glucosyl transferase, EC 2.4.1.18, Q-enzyme) are responsible for producing the a-1,6 branches on the amylopectin molecule, which can then be further extended by the soluble starch synthases (44,60). Because it is the branching of amylopectin that confers its specific functional properties and behavior in food and beverage production, the SBEs have attracted considerable interest for the genetic tailoring of starch. The SBEs are transferases rather than synthases, detaching an a-1,4 -linked oligoglucan from the end of an amylopectin chain and moving it into an a-1,6 position elsewhere in the molecule. Nevertheless, they stimulate soluble starch synthases by increasing the effective substrate concentration determined by the number of nonreducing a-glucan ends in the amylopectin. As with the starch syn-

thases, multiple isoforms have been identified that show organ (usually leaf or storage tissue) or temporal specificity in their expression patterns (61,62).

The various forms show differences as well in the length of chains transferred, which has implications for engineering of starch. These forms have been characterized as A or B types by their distinct properties (63,64). Antisense work in potato indicates that SBE A is responsible for transferring shorter chains than SBE B because average chain length increases in its absence (65). The well-known amylose-extender (ae) mutants illustrate the profound effect SBE has on starch properties. Rather than containing an increased amount of amylose as would be produced by the GBSS enzyme, these plants are in fact defective in amylopectin branching (47,66,67).

Over the last several years, a revolution in thinking about amylopectin biosynthesis has taken place with the introduction of the preamylopectin trimming model (5,6). The model addresses the question of how the non-random distribution of branch points typical of amylopectin may arise. It also helps to explain why mutants lacking a debranching enzyme such as the sugary 1 of maize (68) or a similar one in the alga Chlamydomonas (6) and Arabidopsis (69) contain a highly branched a-glucan referred to as phy-toglycogen. In the model, SBEs and debranching enzymes (DBEs) carry out discontinuous steps of synthesis and amylolysis so that excess branches added by the SBE are removed. Crystallization of the product removes it from the cycle and fixes the structure as, in essence, a partially debranched glycogen.

An alternative, the soluble glucan recycling model, has been proposed (69). In this hypothesis, DBE plays only a subsidiary role in forming amylopectin, helping to turn over branched, soluble oligoglucans. This hypothesis explains the occurrence of phytoglycogen in DBE mutants but does not take the clustered branching of amylopectin into account. The validity of the two models is currently difficult to test. Furthermore, the actual in vivo functions of the soluble starch synthases, SBEs, and debranching enzymes still remain to be disentangled from the pleiotropic effects seen in mutants and antisense experiments.

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