Phosphorus-31 nuclear magnetic resonance investigation of the in vivo regulation of intracellular pH in cell suspension cultures of Nicotiana tabacum: The effects of oxygen supply, nitrogen, and external pH change

Abstract

31P NMR was used to study the in vivo response of intracellular phosphorus-containing compounds of cell suspension cultures of Nicotiana tabacum to extracellular events. Limitation of the oxygen supply in a static system (sealed tube) caused a strong pH decrease of the cytoplasm and a smaller fall in the vacuolar pH. The rate of this process was independent of the age of the cells, except for those at the end of the growth cycle where either reserve supplies of inorganic phosphate have been depleted or metabolism has ceased. The cells can be returned quickly to their normal pH and metabolic status by reoxygenation after depletion times as long as 4.5 h. Regassing with N 2 after anaerobiosis also caused the return of the nearly normal pH status, which indicates that rapid cell acidification is caused almost entirely by CO 2 accumulation. In a second type of anaerobiosis, attained by continual passage of N 2, cytoplasmic pH fell only slowly to a constant value higher than in the static case. Here acidification appeared to arise, at least in part, from lactate accumulation. Provided aeration occurred, the cytoplasm was maintained at a constant pH of 7.5 ± 0.1 for changes in the medium pH value between 6.5 and 3. The biochemical and biotechnological implications of these results are discussed.