Tuber-specific expression of a yeast invertase and a bacterial glucokinasein potato leads to an activation of sucrose phosphate synthase and the creation of a sucrose futile cycle

Fluxes were investigated in growing tubers from wild-type potato (Solanum t uberosum L. cv. Desiree) and from transformants expressing a yeast invertas e in the cytosol under the control of the tuber-specific patatin promoter e ither alone (EC 3.2.1.26; U-IN2-30) or in combination with a Zymomonas mobi lis glucokinase (EC 2.7.1.2; GK3-38) by supplying radiolabelled [C-14]sucro se, [C-14]glucose or [C-14]fructose to tuber discs for a 90-min pulse and s ubsequent chase incubations of 4 and 12 h, and by supplying [C-14]fructose for 2 h and 4 h to intact tubers attached to the mother plant. Contrary to the expectation that this novel route for sucrose degradation would promote starch synthesis, the starch content decreased in the transgenic lines. La belling kinetics did not reveal whether this was due to changes in the flux es into or out of starch. However, they demonstrated that glycolysis is enh anced in the transgenic lines in comparison to the wild type. There was als o a significant stimulation of sucrose synthesis, leading to a rapid cycle of sucrose degradation and resynthesis. The labelling pattern indicated tha t sucrose phosphate synthase (SPS; EC 2.4.1.14) was responsible for the enh anced recycling of label into sucrose. In agreement, there was a 4-fold and 6-fold increase in the activation status of SPS in U-IN2-30 and GK3-38, re spectively, and experiments with protein phosphatase inhibitors indicated t hat this activation involves enhanced dephosphorylation of SPS. It is propo sed that this activation of SPS is promoted by the elevated glucose 6-phosp hate levels in the transgenic tubers. These results indicate the pitfalls o f metabolic engineering without a full appreciation of the metabolic system and regulatory circuits present in the tissue under investigation.