To investigate the effect of water stress on carbon metabolism in growing potato tubers (Solanum tuberosum L.), freshly cut and washed discs were incubated in a range of mannitol concentrations corresponding to external water potential between 0 and )1.2 MPa. (i) Incorporation of [ 14 C]glucose into starch was inhibited in water-stressed discs, and labeling of sucrose was increased. High glucose overrode the changes at low water stress (up to )0.5 MPa) but not at high water stress. (ii) Although [ 14 C]sucrose uptake increased in water-stressed discs, less of the absorbed [ 14 C]sucrose was metabolised. (iii) Analysis of the sucrose content of the discs confirmed that increasing water deficit leads to a switch, from net sucrose degradation to net sucrose synthesis. (iv) In parallel incubations containing identical concentrations of sugars but differing in which sugar was labeled, degradation of [ 14 C]sucrose and labeling of sucrose from [ 14 C]glucose and fructose was found at each mannitol concentration. This shows that there is a cycle of sucrose degradation and resynthesis in these tuber discs. Increasing the extent of water stress changed the relation between sucrose breakdown and sucrose synthesis, in favour of synthesis. (v) Analysis of metabolites showed a biphasic response to increasing water deficit. Moderate water stress (0-200 mM mannitol) led to a decrease of the phosphorylated intermediates, especially 3-phosphoglycerate (3PGA). The decrease of metabolites at moderate water stress was not seen when high concentrations of glucose were supplied to the discs. More extreme water stress (300-500 mM mannitol) was accompanied by an accumulation of metabolites at low and high glucose. (vi) Moderate water stress led to an activation of sucrose phosphate synthase (SPS) in discs, and in intact tubers. The stimulation involved a change in the kinetic properties of SPS, and was blocked by protein phosphatase inhibitors. (vii) The amount of ADP-glucose (ADPGlc) decreased when discs were incubated on 100 or 200 mM mannitol. There was a strong correlation between the in vivo levels of ADPGlc and 3PGA when discs were subjected to moderate water stress, and when the sugar supply was varied. (viii) The level of ADPGlc increased and starch synthesis was further inhibited when discs were incubated in 300-500 mM mannitol. (ix) It is proposed that moderate water stress leads to an activation of SPS and stimulates sucrose synthesis. The resulting decline of 3PGA leads to a partial inhibition of ADP-glucose pyrophosphorylase and starch synthesis. More-extreme water stress leads to a further alteration of partitioning, because it inhibits the activities of one or more of the enzymes involved in the terminal reactions of starch synthesis. Abbreviations: ADPGlc = ADP-glucose; AGPase = ADP-glucose pyrophosphorylase; Fru6P = fructose-6-phosphate; Glc6P = glucose-6-phosphate; 3PGA = 3-phosphoglycerate; P i = inorganic phosphate; SPS = sucrose-phosphate synthase; UDPGlc = uridine-5′-diphosphoglucose; V max = activity of SPS assaye...

Abstract. (i) Sucrose-phosphate synthase (SPS) was purified 40-fold from stored potato (Solanum tuberosum L.) tubers to a final specific activity of 33-70 nkat.(mg protein) -~ via batch elution from diethylaminoethyl (DEAE)-sephacel, polyethylene glycol (PEG) precipitation and Mono Q anion-exchange chromatography. (ii) Immunoblotting revealed a major and a minor band with molecular weights of 124.8 kDa and 133.5 kDa, respectively. Both bands were also present in extracts prepared in boiling SDS to exclude proteolysis. No smaller polypeptides were seen, except when the preparations were incubated before application on a polyacrylamide gel. (iii) The enzyme preparation was activated by glucose-6-phosphate and inhibited by inorganic phosphate. Both effectors had a large effect on the K m (fructose-6-phosphate) and the K m (uridine-5-diphosphoglucose) with phosphate acting antagonistically to glucose-6-phosphate. (iv) Preincubation of potato slices with low concentrations of okadaic acid or microcystin resulted in a three-to fourfold decrease in the activity of SPS when the tissue was subsequently extracted and assayed. The decrease was especially marked when the assay contained low concentrations of substrates and glucose-6-phosphate, and inorganic phosphate was included. Preincubation with mannose or in high osmoticum resulted in an increase of SPS activity. (v) Analogous changes were observed in germinating Ricinus communis L. seedlings. After preincubation of the cotyledons in glucose, high SPS activity could be measured, whereas okadaic acid, omission of glucose, or addition of phosphate or sucrose led to a large decrease of SPS activity in the "selective" assay. (vi) It is argued that SPS from non-photosynthetic tissues * Present address as 2 Abbreviations: Fru6P=fructose-6-phosphate; Glc6P=glucose-6-phosphate; Pi --inorganic phosphate; PGI = phosphoglucose isomerase; PP2A = phosphoprotein phosphatase 2A; PEG = polyethyleneglycol; SPS =sucrose-phosphate synthase; UDPGIc= uridine-5-diphosphoglucose Correspondence to: R. Reimholz: FAX : 49 (6221) 56 5859 is regulated by metabolites and by protein phosphorylation in an analogous manner to the leaf enzyme.

These experiments investigate events involved in triggering sugar accumulation in the cold in tubers of Solanum tuberosum L. cv. Desirée. Sugar content, 14C‐glucose metabolism, metabolite levels and activities of sucrose phosphate synthase (SPS) and starch‐degrading enzymes were followed after transfer to 4°C. (i) Net sucrose accumulation began between 2 and 4 d. By 10 d, reducing sugars were also increasing. From 20 d onwards, sugar accumulation slowed. Sucrose fell, but reducing sugars continued to increase. (ii) To measure unidirectional sucrose synthesis, U‐[14C]glucose was injected into tubers after various times at 4°C. The tubers were then incubated for 6 h. After 1 d at 4°C, both the absolute and the relative (expressed as a percentage of the metabolized label) rates of sucrose synthesis decreased compared to those at 20°C. Between 2 and 4 d at 4°C, labelling of sucrose increased 3‐fold, to over 60% of the metabolized label. This high rate was maintained for up to 50 d in cold storage. When tissue slices were incubated with 2.5 mol m−3 U‐[14C]glucose, the rate of labelling of sucrose in slices from 6 d cold‐stored material was higher than in slices from warm‐stored material, irrespective of whether the incubation occurred at 4°C or at 20°C. (iii) Hexose‐phosphates increased during the first day after transfer to 4°C. Their levels fell during the next 3 d, as sucrose synthesis increased. They then rose (until 20 d) and fell, in parallel with the rise and decline of sucrose levels. UDPglucose remained unaltered during the first 4 d, and then increased and decreased in parallel with sucrose. (iv) SPS activity assayed in optimal conditions and the total amount of SPS protein did not change. However, when assayed in the presence of phosphate and limiting substrate concentrations, activity rose 3–5‐fold between 2 and 4 d. (v) Amylases and phosphorylases were investigated using zymograms to separate isoforms. Phosphorylases did not change. Between 2 and 4 d at 4°C, a new amylolytic activity appeared. (vi) Estimates of the specific activity of the phosphorylated intermediates and the absolute rate of sucrose synthesis (calculated from the 14C‐labelling data and metabolite analysis) showed that changed kinetic properties of SPS and decreased levels of hexose‐phosphate are accompanied by a 6–8‐fold stimulation of sucrose synthesis. They also show that the final level of sugar is partly determined by a cycle of sugar synthesis and degradation. (vii) It is concluded that the onset of sugar accumulation in cold‐stored tubers is initiated by a change in the kinetic properties of SPS and the appearance of a new amylolytic activity. It is discussed how other factors, including hexose‐phosphate levels and subcellular compartmentalization, could also influence the final levels of sugars by altering the balance of sugar synthesis and remobilization.

Antibodies raised against a peptide fragment (residues 60-456) of potato sucrose phosphate synthase (SPS) were used to investigate whether potato plants contain multiple forms of SPS. When a partially purified preparation of SPS from cold-stored potato tubers was separated on 5 % polyacrylamide gel electrophoresis (PAGE), four immunopositive bands were found with estimated molecular weights of 125, 127, 135 and 145 kDa. These bands were also found in rapidly prepared extracts and were termed SPS-la, SPS-lb, SPS-2 and SPS-3, respectively. Direct evidence that SPS-la and SPS-lb represent active SPS was provided by the finding that both are greatly reduced in plants expressing an antisense sequence derived from the potato leaf SPS gene. SPS-2 was not decreased in the antisense plants, indicating that it has a significantly different sequence. Evidence that SPS-2 represents active SPS was obtained by showing that the amount of SPS-la and SPS-lb protein remaining in the leaves and tubers of antisense potato plants was too low to account for the remaining SPS activity. The four immunopositive SPS forms had different tissue distributions. SPS-la was the major form in all tissues except petals, sepals and stamens. SPS-lb and SPS-2 were absent in very young growing tissues hut were present as minor forms in source leaves and sprouting tubers. The SPS-lh level was especially high in petals and sepals, and the SPS-

Abstract.Labelling experiments in which high-specificactivity [U-14C]sucrose or [u-lac]hexoses were injected into potato (Solanum tuberosum L. cv. Desiree) tubers showed that within 1 d of detaching growing tubers from their mother plant, there is an inhibition of starch synthesis, a stimulation of the synthesis of other major cell components, and rapid resynthesis of sucrose. This is accompanied by a general increase in phosphorylated intermediates, an increase in UDP-glucose, and a dramatic decrease of ADP-glucose. No significant decline in the extracted activity of enzymes for sucrose degradation or synthesis, or starch synthesis is seen within 1 d, nor is there a significant decrease in sucrose, amino acids, or fresh weight. Over the next 7 d, soluble carbohydrates decline. This is accompanied by a decline in sucrose-synthase activity, hexose-phosphate levels, and the synthesis of structural cell components. It is argued that a previously unknown mechanism acting at ADP-glucose pyrophosphorylase allows sucrose-starch interconversions to be regulated independently of the use of sucrose for cell growth.