Glucose and Glycine Metabolism in Regenerating Tobacco Protoplasts

Abstract

The metabolic states and the uptake and metabolism of [1-13C]glucose, [2-13C]glycine and [15N]glycine in intact N. tabacum L. mesophyll protoplasts were measured by 13C and 15N NMR spectroscopy. Changes in the concentration of metabolites during the first 2 days of culture in darkness were followed. Protoplasts isolated in 0.55 M mannitol medium showed a drop in the concentration of all the intracellular metabolites during the first 28 h 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 NH4+ assimilation were observed. Glucose uptake and metabolism and cell division were inhibited by 3 mM glycine, suggesting that the accumulating serine or the release of ammonia during serine synthesis had toxic effects in this system.