Metabolic cycles in primary metabolism of cell suspensions of Daucus carota L. analysed by C-NMR

In the work described in this thesis, uptake and conversion of sugar by cells of batch-grown suspensions of Daucus carota L. were studied. Invasive techniques (measurements of enzyme activities and sugar and starch levels) and non-invasive techniques ( 13C-NMR) were used to follow the pathway of sugar molecules and of certain atoms within these molecules to analyze "futile" cycles between hexoses and sucrose, between hexoses and pentoses using the oxidative pentose phosphate pathway (OPPP) and between triose and hexose phosphates.The activities of the various metabolic cycles were analyzed in logarithmic phase cells in relation to sugar uptake and storage of carbohydrates and in stationary phase cells in relation to consumption of stored carbohydrates. Plant cell metabolism appeared to excel both in metabolic cycling and in substrate conversions by parallel enzymes catalyzing similar reactions like PP i - and ATP-dependent fructose-6-phosphate phosphotransferase (PFP/PFK) and invertase/sucrose synthase (SUSY).In the logarithmic growth phase sugar metabolism starts with uptake of hexoses, since high levels of cell wall-bound invertase hydrolyse all the external sucrose. It was concluded that at the start of the logarithmic growth phase sugars are present in excess resulting in a fully active glycolytic, respiratory and oxidative pentose phosphate pathway. More ATP and building blocks than necessary for maintenance of the cells give rise to synthesis of new enzymes and precursors necessary for growth. After a few days cells start dividing and enter the logarithmic growth phase. Production of biomass and synthesis of sucrose and starch are coupled processes.Fructose-6-phosphate is synthesized in the cytosol by soluble fructokinase activity, and acts as a substrate for the invertase-mediated sucrose cycle and the triose-hexose phosphate cycle in the cytosol. Via glucose-6-phosphate, a substantial part of the fructose is substrate for synthesis of UDP-glucose necessary for sucrose and cellulose synthesis in the cytosol and apoplast, respectively. Furthermore, glucose-6-phosphate is used in the OPPP-cycle in the cytosol and in the plastids, and for starch synthesis in the plastids. Glucose will be phosphorylated by the mitochondrial-associated hexokinases and supply substrates to the respiratory pathway. Exchange between glucose-6-phosphate in the respiratory "compartment" and structural component "compartment" also occurs, although limited.Due to the high hexose phosphorylating activity, the levels of hexoses are relatively low and the levels of hexose phosphates and UDPG are relatively high in the cytosol; as a result sucrose is synthesized and the sucrose cycle mediated by invertase is active, while the sucrose cycle mediated by SUSY is inactive. Excess sucrose will be taken up into the vacuole, where it is protected from hydrolysis as long as the levels of hexoses are high enough to inhibit acid invertase activity. Cycling of carbon between triose and hexose phosphates was observed by the occurrence of [6- 13C]-labelled sucrose and hexoses after feeding [1- 13C]-glucose or fructose.In the logarithmic growth phase high activity of the OPPP was observed, even when respiration was increased 4-fold by dilution of the cells. Therefore, it is concluded that OPPP activity was not subject to extensive metabolic regulation. It is suggested, that OPPP activity takes place in the cytosol during the whole culture period; the OPPP in the plastids probably was only active in the logarithmic growth phase.As soon as the external supply of carbon is exhausted, cytosolic carbohydrates and, via respiratory control, the resulting ATP decrease. Reverse reactions with respect to stored sucrose and starch occur. In the stationary growth phase, hexose units released from stored sucrose and starch are used in glycolysis to fulfil the demand of substrates for cell maintenance. Both the OPPP activity and the growth rate decline in this period because of the lack of carbon for the production of new cells.If external hexoses are supplied to these cells, low OPPP cycling and high sucrose cycling are observed, indicating that the OPPP activity is down-regulated by coarse control of its enzymes rather than by availability of sugar. Furthermore, (cytosolic) triose-hexose phosphate cycling was observed, which was related to the level of PP i -dependent fructose-6-phosphate phosphotransferase (PFP) which catalyzes the gluconeogenetic reaction from fructose-1,6-bisphosphate into fructose-6-phosphate. Hydrolysis of sucrose by invertase and SUSY in the cytosol and by acid invertase in the vacuole takes place in the stationary growth phase and result in activity of the "cytosolic" and the "vacuolar" sucrose cycles.The above described phenomena imply that plants have multiple ways to control carbon metabolism and carbon partitioning in cells and tissues. "Environmental" conditions in batch cultured cells with respect to humidity and temperature will be rather constant, and changes in availability of nutrients and oxygen will only occur gradually. The meaning of the described high activities of metabolic cycles might thus be redundant under the controlled conditions in the laboratory. However, in field-grown plants these properties might be essential to survive adverse environmental conditions.