targeted to the plastid. Significantly, no phenotype was observed when the native AGPase gene (glgC) was introduced in potato (cv Russet Burbank) under a constitutive 35S CaMV promoter. However, a different response was obtained when the plant was transformed with a mutant gene {glgC16) which encodes an AGPase much less sensitive to the allosteric activator fructose 1,6 bisphosphate (Frul,6P2) and to the allosteric inhibitor Pi. Expression of this gene in potato, tomato and tobacco calli resulted in massive hyperaccumulation of starch leading to the conclusion that the AGPase reaction is a major rate limiting reaction in these systems. Indeed, the accumulation of starch in the calli and in the leaves of transplants was so massive as to be detrimental to the subsequent regeneration of plants. When the gene was introduced into potato plants using a tuber-specific patatin promoter, the tubers contained on average 35% more starch than the control tubers. Field-trials in USA with various cultivars expressing the bacterial gene showed increased dry matter content in the tubers (Table 2).

In parallel studies, Sweetlove et al. (121) found that the introduction of the mutated E. coli AGPase gene (glgC16) into cultivar Prairie had no effect on tuber starch content. They observed that in these tubers the increased starch biosynthetic rate was apparently offset by an increase in starch turnover rate. These results underline the importance of the parameters used for determining flux control coefficients. In this particular case, it is clear that increasing AGPase activity will increase the rate of starch synthesis but it may not lead to increase in starch content. Furthermore, the observation of Stark et al. (120) that up-regulation of starch synthesis in potato tubers occurred only when a de-regulated AGPase was expressed implies that a main factor involved in the control of flux is the regulation of AGPase activity in vivo. Strict adherence to the flux control analysis approach prevents the extension of this approach to regulated enzymes. Indeed, theoretical analyses show that changes in kinetic properties of enzymes may have larger effects on flux than does a change in the amount of enzyme protein (D. Fell, personal communication to Stitt and Sonnewald, 65).

5.3. Is AGPase regulated in vivo?

Apart from the results of Stark et al. (120) there are other data suggesting that regulation of AGPase in vivo affects starch synthesis. Incubation of potato tuber discs with mannose results in stimulation of starch synthesis (122). This has been attributed to Pi sequestration in the cytosol as phosphorylated mannose and is consistent with AGPase activation as a result of decreased Pi concentration in the amyloplast. There is also a reported large increase in starch content in transgenic potato tubers with decreased NAD-malic enzyme activity (H.L. Jenner, B.M. Winning, C.J. Leaver and S.A. Hill, unpublished data, cited in 123). The unexpected impact of this manipulation on starch synthesis has been explained in terms of stimulation of AGPase activity as a result of 3-PGA accumulation in the transgenic tubers, although this remains to be demonstrated. Another example is mutations in a gene (STA1) encoding one subunit of AGPase in Chlamydomonas reinhardtii which leads to marked reduction in starch content by apparently reducing the sensitivity of the enzyme to 3-PGA without affecting the activity of the enzyme measured in the absence of 3-PGA (124). Recently, germinal revertants of maize Sh2 mutants (the shrunken 2 gene coding for the large subunit of the endosperm AGPase) were isolated by means of /fc-mediated transposition of the element dissociation (Ds) (125). All revertants restored the wild-type open reading frame distal to the Ds insertion with some displaying imprecise excisions involving the insertion of one or two amino acids. One revertant allele, which contains an additional tyrosine and serine, conditioned an 11-18% increase in seed weight, which is associated with a decreased sensitivity to Pi inhibition in the altered AGPase. These results would imply that AGPase regulation in vivo exerts control not only on starch synthesis but also on general sink activity in maize.

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