Altered metabolic fluxes result from shifts in metabolite levels in sucrose phosphorylase-expressing potato tubers

As reported in a previous paper (Plant, Cell and Environment 24, 357-365, 2001), introduction of sucrose phosphorylase into the cytosol of potato results in increased respiration, an inhibition of starch accumulation and decreased tuber yield. Herein a more detailed investigation into the effect of sucrose phosphorylase expression on tuber metabolism, in order to understand why storage and growth are impaired is described. (1) Although the activity of the introduced sucrose phosphorylase was low and accounted for less than 10% of that of sucrose synthase its expression led to a decrease in the activities of enzymes of starch synthesis relative to enzymes of glycolysis and relative to total amylolytic activity. (2) Incubation of tuber discs in [C-14]glucose revealed that the transformants display a two-fold increase of the unidirectional rate of sucrose breakdown. However this was largely compensated by a large stimulation of sucrose re-synthesis and therefore the net rate of sucrose breakdown was not greatly affected. Despite this fact major shifts in tuber metabolism, including depletion of sucrose to very low levels, higher rates of glycolysis, and larger pools of amino acids were observed in these lines. (3) Expression of sucrose phosphorylase led to a decrease of the cellular ATP/ADP ratio and energy charge in intact growing tubers. It was estimated that at least 30% of the ATP formed during respiration is consumed as a result of the large acceleration of the cycle of sucrose breakdown and re-synthesis in the transformants. Although the absolute rate of starch synthesis in short-term labelling experiments with discs rose, starch synthesis fell relative to other fluxes including respiration, and the overall starch content of the tubers was lower than in wild-type tubers. (4) External supply of amino acids to replace sucrose as an osmoticum led to a feed-back inhibition of glycolysis, but did not restore allocation to starch. (5) However, an external supply of the non-metabolizable sucrose analogue palatinose - but not sucrose itself - stimulated flux to starch in the transformants. (6) The results indicate that the impaired performance of sucrose phosphorylase-expressing tubers is attributable to decreased levels of sucrose and increased energy consumption during sucrose futile cycling, and imply that sucrose degradation via sucrose synthase is important to maintain a relatively large sucrose pool and to minimize the ATP consumption required for normal metabolic function in the wild type.