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

Abstract.

Fluxes were investigated in growing tubers from wild-type potato (Solanum tuberosum L. cv. Desiree) and from transformants expressing a yeast invertase in the cytosol under the control of the tuber-specific patatin promoter either alone (EC 3.2.1.26; U-IN2-30) or in combination with a Zymomonas mobilis glucokinase (EC 2.7.1.2; GK3-38) by supplying radiolabelled [¹⁴C]sucrose, [¹⁴C]glucose or [¹⁴C]fructose to tuber discs for a 90-min pulse and subsequent chase incubations of 4 and 12 h, and by supplying [¹⁴C]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. Labelling kinetics did not reveal whether this was due to changes in the fluxes into or out of starch. However, they demonstrated that glycolysis is enhanced in the transgenic lines in comparison to the wild type. There was also a significant stimulation of sucrose synthesis, leading to a rapid cycle of sucrose degradation and resynthesis. The labelling pattern indicated that sucrose phosphate synthase (SPS; EC 2.4.1.14) was responsible for the enhanced 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, respectively, and experiments with protein phosphatase inhibitors indicated that this activation involves enhanced dephosphorylation of SPS. It is proposed that this activation of SPS is promoted by the elevated glucose 6-phosphate levels in the transgenic tubers. These results indicate the pitfalls of metabolic engineering without a full appreciation of the metabolic system and regulatory circuits present in the tissue under investigation.