Glucose and glycine metabolism in regenerating tobacco protoplasts: followed nondestructively by nuclear magnetic resonance spectroscopy.

The metabolic states and the uptake and metabolism of [1-(13)C]glucose, [2-(13)C]glycine, and [(15)N]glycine in intact Nicotiana tabacum L. (cv Xanthi) mesophyll protoplasts were measured by (13)C and (15)N nuclear magnetic resonance spectroscopy. Changes in the concentration of metabolites during the first two days of culture in darkness were followed. Protoplasts isolated in 0.55 molar mannitol medium showed a drop in the concentration of all the intracellular metabolites during the first 28 hours of culture. Uptake of glucose and synthesis of glucose-derived metabolites were observed, indicating activity of glycolysis and the tricarboxylic acid cycle. Addition of glycine caused the accumulation of serine in dark cultured protoplasts, via the photorespiratory pathway. Glutamate dehydrogenase and glutamine synthetase activities in photorespiratory NH(4) (+) assimilation were observed. Glucose uptake and metabolism and cell division were inhibited by 3 millimolar glycine, suggesting that the accumulating serine or the release of ammonia during serine synthesis had toxic effects in this system.