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' Crystalline Lamella J- Amorphous Lamella

Figure 1. Schematic representation of the starch metabolic pathway in sink cells of plants. Sucrose is unloaded apoplastically from the phloem to the cytosol of the sink cell, and symplastically between sink cells [7]. Carbon is then transported to the plastid either as a hexose phosphate or as ADPglucose by hexose phosphate transporter or ADPglucose transporter, respectively. Major enzymes involved in the pathway are: i, sucrose synthase; ii, UDPglucose pyrophosphorylase; iii, phosphoglucomutase; iv, ADPglucose pyrophosphorylase (AGPase); v, granule bound starch synthase (GBSS); vi, soluble starch synthase (SS); vii, starch-branching enzyme (SBE); viii, debranching enzyme (DBE); and ix, disproportionating enzyme (D-enzyme). PPi: inorganic pyrophosphate.

' Crystalline Lamella J- Amorphous Lamella

Figure 1. Schematic representation of the starch metabolic pathway in sink cells of plants. Sucrose is unloaded apoplastically from the phloem to the cytosol of the sink cell, and symplastically between sink cells [7]. Carbon is then transported to the plastid either as a hexose phosphate or as ADPglucose by hexose phosphate transporter or ADPglucose transporter, respectively. Major enzymes involved in the pathway are: i, sucrose synthase; ii, UDPglucose pyrophosphorylase; iii, phosphoglucomutase; iv, ADPglucose pyrophosphorylase (AGPase); v, granule bound starch synthase (GBSS); vi, soluble starch synthase (SS); vii, starch-branching enzyme (SBE); viii, debranching enzyme (DBE); and ix, disproportionating enzyme (D-enzyme). PPi: inorganic pyrophosphate.

2.1. Manipulation of AGPase in Transgenic Plants

To determine the role of AGPase towards starch synthesis during development of potato tubers, transgenic potato lines were developed in which the activity of AGPase was greatly reduced by expressing a cDNA encoding the small subunit of AGPase in the antisense orientation under the constitutive CaMV 35 S promoter [17]. As expected, AGPase activity was mostly reduced along with dramatically reduced starch content. However, because of the global nature in the reduction of the activity of AGPase, the experiment did not address the issue of flux control by AGPase on starch synthesis in non-photosynthetic tissues. Secondary effects on tuber starch metabolism caused by effects in leaves could not be excluded with this approach.

Transgenic potato plants over-expressing a bacterial form of AGPase (a homotetramer similar to the small subunit of the plant AGPase) driven by the tuber specific patatin promoter and possessing plastid targeting sequence resulted in an increase in starch synthetic rates [18-20], The measurements of flux gave a response coefficient close to 1 for the activity of pyrophosphorylase in respect to starch synthesis. Pulse-chase experiments with [u"14 C] sucrose showed that the increased flux into starch in the transformed tubers was accompanied by an increased rate of starch turnover. Ensuing evidence suggested that the increased turnover is associated with an increase in the capacity of the tubers to degrade starch [21]. Because these experiments were performed with a bacterial form of AGPase, which is less sensitive to the allosteric regulations in potato, it could not be concluded conclusively if AGPase has a regulatory role in the sink tissue as well.

To gain further insight, the antisense approach was utilized with a variation once again. Transgenic potato lines were developed by expressing the cDNA for the small subunit of AGPase in the antisense orientation under the control of the tuber specific patatin promoter [22]. Reduced AGPase activities lead to diversion of carbon from starch synthesis and toward sucrose synthesis. The estimated flux coefficient of AGPase over starch synthesis was 0.55, suggesting that in potato tubers there is a pivotal role for the 3-PGA-mediated regulation of AGPase in the co-ordination of starch metabolism.

2.2 Variations in AGPase activity that need to be resolved

The above experimental results do not explain the lack of sensitivity to 3-PGA and Pi by barley AGPase [13, 14], Also, a variant line (Rev6) that was generated through transposon-induced mutagenesis of the maize mutant SHRUNKEN2, exhibits reduction of AGPase activity by half, but with a concurrent increase in the amount of starch per seed [22], The large subunit of the heterotetrameric AGPase is mutated in this line. This altered form of AGPase was insensitive to Pi but was activated by 3-PGA. The obscure behavior of AGPase observed in maize and barley has not been observed in other cereals studied thus far [8].

Unlike other plants, developing maize [23] and barley [24] endosperms exhibit AGPase activity both in the cytoplasm and in the plastids, which is the usual site for plant starch synthesis. Investigations using maize [25] and barley [26] endosperms revealed abundant transcripts of the cytosolic form of AGPase, with negligible levels of the leaf plastidal form. The variations in the subunit composition, and consequently in properties of AGPase within and between plant organs, reflect variations in the regulation of flux through the pathway of starch synthesis [8], To understand the role of different AGPases it is important that the role of cytosolic AGPase be carefully considered too, by either down regulating the transcripts or by expressing them in plants where they have no known counterpart. Such experiments are underway and should provide more insight into control and optimal manipulation of starch biosynthetic pathways within the next few years.

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