Quantitation of Rates of Transport, Metabolic Fluxes, and Cytoplasmic Levels of Inorganic Carbon in Maize Root Tips during K+ Ion Uptake

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Abstract
Our aim was to determine whether fixation of inorganic carbon (C(i)), due to phosphoenolpyruvate carboxylase activity, is limited by the availability of C(i) in the cytoplasm of maize (Zea mays L.) root tips. Rates of C(i) uptake and metabolism were measured during K(2)SO(4) treatment, which stimulates dark C(i) fixation. (13)C(i) uptake was followed by (13)C-nuclear magnetic resonance (NMR); 5 millimolar K(2)SO(4) had no significant effect on (13)C(i) influx. The contribution of respiratory CO(2) production to cytoplasmic HCO(3) (-) was measured using in vivo(13)C-NMR and (1)H-NMR of cell extracts; K(2)SO(4) treatment had no effect on respiratory CO(2) production. The concentration of cytoplasmic HCO(3) (-) was estimated to be approximately 11 millimolar, again with K(2)SO(4) having no significant effect. These experiments allowed us to determine the extent to which extracellularly supplied (14)C(i) was diluted in the cytoplasm by respiratory CO(2) and thereby measure phosphoenolpyruvate (PEP) carboxylase activity in vivo using (14)C(i). PEP carboxylase activity in root tips was enhanced approximately 70% over controls within 12 minutes of the addition of 5 millimolar K(2)SO(4). The activity of carbonic anhydrase, which provides PEP carboxylase with C(i), was determined by saturation transfer (13)C-NMR to be more than 200 times that of PEP carboxylase in vivo. The regulation of PEP carboxylase in K(2)SO(4)-treated roots is discussed.