Limitations of Deuterium-Labelled Substrates for Quantifying NADPH Metabolism in Heterotrophic Arabidopsis Cell Cultures.

NADPH is the primary source of cellular reductant for biosynthesis, and strategies for increasing productivity via metabolic engineering need to take account of the requirement for reducing power. In plants, while the oxidative pentose phosphate pathway is the most direct route for NADPH production in heterotrophic tissues, there is increasing evidence that other pathways make significant contributions to redox balance. Deuterium-based isotopic labelling strategies have recently been developed to quantify the relative production of NADPH from different pathways in mammalian cells, but the application of these methods to plants has not been critically evaluated. In this study, LC-MS was used to measure deuterium incorporation into metabolites extracted from heterotrophic Arabidopsis cell cultures grown on [1-2H]glucose or D2O. The results show that a high rate of flavin-enzyme-catalysed water exchange obscures labelling of NADPH from deuterated substrates and that this exchange cannot be accurately accounted for due to exchange between triose- and hexose-phosphates. In addition, the duplication of NADPH generating reactions between subcellular compartments can confound analysis based on whole cell extracts. Understanding how the structure of the metabolic network affects the applicability of deuterium labelling methods is a prerequisite for development of more effective flux determination strategies, ensuring data are both quantitative and representative of endogenous biological processes.