We have investigated whether starch accumulation in heterotrophic cell-suspension cultures of Chenopodium rubrum L., developing potato (Solarium tuberosum L.) tubers or maize (Zea mays L.) endosperm involves import of triose phosphates or of hexose units into the plastid, and whether there is a rapid recycling of triose phosphates back to hexose phosphates in the cytosol of these tissues, (i) Cell suspensions, potato tubers or intact maize kernels were supplied with [1-14C] glucose or [6-14C]glucose. The glucosyl residues from starch were isolated and degraded by an enzymic procedure to determine how much radioactivity had been redistributed into the opposite half of the glucose molecule. Randomisation was incomplete, affecting only 18%–38% of the molecules in Chenopodium, 16%–26% of the molecules in potato, or 30% of the molecules in maize. It is concluded that the major route for starch synthesis involves import of hexose units, (ii) The glucosyl and fructosyl moieties of sucrose were isolated and degraded to determine the extent of recycling in the cytosol. There was significant randomisation, lying between 30%–40% in Chenopodium, 20%–26% in potato, and 8%–12% in maize. It is concluded that there is considerable recycling of triose phosphates in the cytosol. (iii). Sucrose from cells supplied with [1-14C]glucose was more randomised than sucrose from cells supplied with [6-14C]glucose. This is explained in terms of the oxidative pentose-phosphate pathway (iv). Equations are used to estimate the rate of recycling from triose phosphates to hexose phosphates in the cytosol. The estimated rate of recycling is considerably larger than the net glycolytic flux or the activity of the oxidative pentose-phosphate cycle.

The present experiments were carried out to investigate the effect of increased fluxes of H+ across the plasmalemma on glycolysis in heterotrophic cell suspension cultures of Chenopodium rubrum L. (1) Increased H+ influx was produced by adding glucose, 6‐deoxyglucose, 2‐deoxyglucose, or sodium fluoride. The net influx decreased to zero after 3 min. This recovery was accompanied by an increase in the rate of O2 uptake, but not of dark CO2 fixation. When glucose or fluoride were added, the increase of O2 uptake occurred without a decrease in the ATP/ADP ratio, and was large enough to provide the ATP that would be needed for compensatory H+ extrusion via the plasmalemma H+‐ATPase. When 2‐deoxyglucose was added, the rise of respiration was restricted by sequestration of phosphate and depletion of phosphorylated metabolites, the ATP/ADP ratio declined, and a slow net H+ influx started again after 4 min. (2) Alkalinisation of the medium to induce an H+ efflux resulted in rapid activation of dark CO2 fixation, but not of O2‐uptake. (3) A stimulation of respiration or dark CO2 fixation was always accompanied by a decrease of phosphoenolpyruvate. This shows that the primary sites for regulation of glycolysis are pyruvate kinase and phosphoenolpyruvate carboxylase, respectively. (4) There was no consistent relation between glycolytic flux and triose‐phosphates or hexose‐phosphates. This shows that the reactions involved in carbohydrate mobilisation and the conversion of hexose‐phosphates to triose‐phosphates only have a secondary role in stimulation of glycolysis. (5) Phosphofructokinase will be stimulated as a consequence of the decrease in phosphoenolpyruvate. (6) The increase in glycolytic flux occurred independently of (in the case of 2‐deoxyglucose and fluoride), or before (in the case of glucose), any increase of fructose‐2,6‐bisphosphate. When fructose‐2,6‐bisphosphate did increase (after supplying glucose), this was accompanied by an increase of triose‐phosphate and fructose‐1,6‐bisphosphate, which otherwise remained very low. It is argued that fructose‐2,6‐bisphosphate increases as a consequence of the decrease of glycerate‐3‐phosphate, a known inhibitor of the synthesis of this regulator metabolite. However, activation of pyrophosphate fructose‐6‐phosphate phosphotransferase by fructose‐2,6‐bisphosphate does not play an obligatory role in the stimulation of glycolysis.

Experiments were carried out to determine whether pyrophosphate: fructose-6-phosphate phosphotransferase (PFP) catalyses the rapid recycling of triose phosphates that is found in the cytosol of heterotrophic cell cultures of Chenopodium rubrum L. (W.-D. Hatzfeld, M. Stitt, 1990, Planta, 180, 198–204). Oxygen uptake, carbohydrate turnover, fructose 2,6-bisphosphate (Fru2,6bisP), glycolytic intermediates, adenine and uridine nucleotides, pyrophosphate and the activity of PFP and glycolytic enzymes were monitored for 48 h after subculturing carbohydrate-depleted cells onto glucose. Immediately after transfer there was an increase in the amount of Fru2,6bisP, and of the hexose phosphate. The triose phosphates, fructose-1,6-bisphosphate and inorganic pyrophosphate increased gradually over the next 24 h. This was accompanied by a tripling in the extractable activity of PFP, but not of phosphofructokinase. The activity of fructose-1,6-bisphosphatase was 20–50fold lower than that of PFP. It is calculated that the activity of PFP is high enough to catalyse the observed rate of cycling between the triose phosphates and the hexose phosphates, based on the measured Vmax capacity of the enzyme, the known kinetic properties, and the measured levels of its reactants and Fru2,6bisP. The changes in the levels of Fru2,6bisP were not correlated with the rate of respiration. Instead, the rate of O2 uptake was inversely related to the phosphoenolpyruvate level, showing that pyruvate kinase or phosphoenolpyruvate carboxylase are regulating the use of glucose for respiration. There was also no relation between Fru2,6bisP, and partitioning to sucrose or starch. It is proposed that the main function of the cycle in these cells is to maintain high levels of inorganic pyrophosphate and triose phosphates, which are necessary for the remobilisation of sucrose and for biosynthesis in the plastid, and that ‘coarse’ and ‘fine’ control of PFP play an important role in regulating this cycle.

We have investigated whether sucrose accumulation in heterotrophic cell-suspension cultures of Chenopodium rubrum L. is regulated by a cycle in which sucrose is simultaneously synthesised and degraded. Net sucrose accumulation was measured by monitoring the sucrose content, unidirectional synthesis was monitored by supplying pulses of [14C] glucose, and unidirectional degradation was estimated from the difference between unidirectional synthesis and net accumulation. When 50 mM glucose was supplied to carbohydrate-depleted cells there was a rapid net accumulation of sucrose, which stopped after 24 h. The incorporation of 14C into sucrose was similar to the initial rate of net sucrose accumulation, but rapid 14C incorporation continued after the cells had stopped accumulating sucrose. A method was developed to rapidly separate sucrose-phosphate synthase (SPS) from uridine-diphosphate-hydrolysing activities which interfered with the assay. The cells contained enough SPS activity to catalyse the observed rate of sucrose synthesis. SPS activity increased in cells which had stopped accumulating sucrose, and the enzyme became less sensitive to inhibition by inorganic phosphate. Sucrose synthase and alkaline invertase activity were four- and twofold higher than SPS activity, and both degradative enzymes increased in cells which had stopped accumulating sucrose. Sucrose synthase is strongly modulated by the concentration of sucrose and by competitive feedback regulation by fructose in these cells. It is concluded that sucrose accumulation ceases in these cells because the rate of degradation of sucrose increases until it matches the rate of synthesis. It is discussed how this cycle is regulated, and how it may interact with the substrate cycle between triose-phosphates and hexose-phosphates (Hatzfeld and Stitt, 1990, Planta 180, 198–204). These cycles allow sucrose turnover to respond in a highly sensitive manner to small changes in the balance between the supply of sucrose and the demand for carbon for respiration and biosynthesis in the cell.

Experiments were carried out to provide direct evidence that pyrophosphate: fructose‐6‐phosphate phosphotransferase (PFP) can operate as a glycolytic enzyme in some circumstances. A large increase in the rate of glycolysis was produced by adding uncoupler and alkalinising the medium of heterotrophic Chenopodium rubrum cells. Initially, a marked decrease in phosphoenolpyruvate, 3‐phosphoglycerate and hexose phosphates occurred, but no change in fructose 2,6‐bisphosphate or inorganic pyrophosphate was observed. However, a gradual increase in the rate of O2 uptake during the subsequent 5 min was accompanied by an increase in fructose 2,6‐bisphosphate and a decrease in pyrophosphate, providing evidence that activation of PFP contributes to the increase in rate of glycolysis. This was accompanied by partial recovery of the adenine nucleotide energy status.