Metabolic flux analysis in complex isotopolog space. Recycling of glucose in tobacco plants

Tobacco plants grown in vitro were supplied with a mixture of [U-C-13(6)]glucose and unlabelled sucrose via the root system. After 20 days, leaves were harvested and extracted with water. Glucose was isolated from the extract and was analysed by C-13 NMR spectroscopy. All C-13 signals appeared as complex multiplets due to C-13-C-13 coupling. The abundance of 21 isotopologous glucose species was determined from the C-13 NMR signal integrals by numerical deconvolution using a genetic algorithm. The relative fractions of specific isotopologs in the overall excess of C-13-labelled specimens establish flux contributions via glycolysis/glucogenesis, pentose phosphate pathway, citric acid cycle and Calvin cycle including (CO2)-C-13 refixation. The fluxes were modelled and reconstructed in silico by a novel rule-based approach yielding the contributions of circular pathways and the degree of multiple cycling events. The data indicate that the vast majority of the proffered [U-C-13(6)]glucose molecules had been modified by catabolism and subsequent glucogenesis from catabolic fragments, predominantly via passage through the citric acid cycle and the pentose phosphate pathway. (C) 2004 Elsevier Ltd. All rights reserved.