In storage tissues, starch is synthesized within amyloplasts, which are derived as are chloroplasts from proplastids. Starch is also synthesized diur-nally for transient assimilate storage in leaf chloroplasts. Starch biosynthesis is part of the complex process of tuberization, conversion of a stem into storage tissue, in potato and other crops producing storage tubers. In the cereals, starch is deposited in the starchy endosperm, whereas in most dicotyledonous plants it accumulates in fleshy cotyledons. Within developing endosperm, starch granules appear within a day of the onset of cellulariza-tion and continue until the grain dries (21-23). Tubers, however, have no sharp end point for starch biosynthesis.
Starch biosynthesis with its key enzymes and metabolites is diagrammed in Fig. 2. Photosynthate is generally supplied as sucrose via the phloem of the maternal tissues. Both source and sink strength are critical to starch yield in storage organs. In some plants, breakdown and resynthesis of sucrose appear necessary to maintain a sucrose gradient and thus sink strength, although this is not the case in others such as barley. The sucrose taken into the endosperm is subsequently converted into UDPglucose by sucrose synthase (UDPglucose:D-fructose-2-glucosyltransferase, EC 188.8.131.52):
This is a reversible reaction but, under the conditions found in storage tissues, the breakdown of sucrose is favored.
In many plants, sucrose synthase activity appears to be important to overall sink strength and hence yield. Antisense-mediated reductions in sucrose synthase levels in transgenic tomato (24) and potato (25) reduce overall starch biosynthesis, as do mutations to the sucrose synthase genes such as found in the maize shl and susl mutants (26). The UDPglucose product of sucrose synthase is then converted to glucose-1-phosphate by UDPglucose pyrophosphorylase (UTPglucose-1-phosphate uridylyltransferase, EC 2.7. 7.9). The UDPglucose pyrophosphorylase enzyme has been purified (27) and the gene encoding it cloned (28) from barley as well as from other plants. Glucose-1-phosphate is further processed to ADPglucose, the specific nu-
cleotide sugar that serves as the substrate for the starch synthases. This is catalyzed by the enzyme ADPglucose pyrophosphorylase (AGP, glucose-1-phosphate adenylyltransferase, EC 184.108.40.206) in the reaction
ATP + a-D-glucose-1-phosphate —> pyrophosphate + ADPglucose
The conversion of glucose-1-phosphate to ADPglucose by AGP is considered the first specific, or committed, step in starch biosynthesis. The AGP enzyme has been extensively studied and reviewed since the 1960s (29,30) and also is the target for engineering of the pathway as discussed in the following. The enzyme in all tissues is a heterotretramer of two regulatory (small) and two catalytic (large) subunits (31). In most tissues, it is allo-sterically regulated, activated by 3-phosphoglycerate but inhibited by orthophosphate. Due in part to its regulation and also to the severely shrunken phenotypes of mutants of AGP (32), it has been seen as the major control point for the flow of carbon into starch. Flux analyses, however, contradict this interpretation (33,34).
Until recently, it was universally held that AGP is nuclear encoded but localized in the plastids in all tissues, photosynthetic and storage. However, at least for maize and barley endosperm (30), a combination of investigations on the bt-1 mutant (35,36), studies of isolated amyloplasts (37), and messenger RNA (mRNA) transcriptional analyses (38,39) has shown that up to 95% of the cereal AGP is cytosolic (Fig. 2). A reasonable explanation for the difference between chloroplasts and amyloplasts regarding AGP localization rests on chloroplasts being sources of energy whereas amyloplasts are sinks. If AGP were restricted to amyloplasts, the ATP would have to be
Figure 1 Current view of starch structure and its successive stages of organization within the granule, (a) Segment of amylopectin indicating the two bond types, (b) An amylopectin cluster showing the double helices formed between adjacent chains, (c) Helices are packed into crystalline lamellae spaced at intervals of 9 nm, interspersed with amorphous regions containing the parallel branch points, (d) The interspersed crystalline and amorphous lamellae form concentric semicrystalline zones several hundreds of nanometers wide. These zones are separated by amorphous zones lacking orderly packing of amylopectin helices. A pair of semicrystalline and amorphous zones form a growth ring in the granule, (e) Scanning electron micrograph of a potato starch granule showing the growth rings. The granule has been digested with «-amylase to remove partially the amorphous zones, which are more easily hydrolyzed. (From Ref. 45.)
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