Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Smith EN

PROVIDER: S-EPMC6835633 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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

Smith Edward N EN McCullagh James S O JSO Ratcliffe R George RG Kruger Nicholas J NJ

Metabolites 20190928 10

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 t ...[more]

PMID: 31569392

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Project description:Interactions between photoautotrophic and heterotrophic microorganisms are central to the marine microbial ecosystem. Lab cultures of one of the dominant marine photoautotrophs, Synechococcus, have historically been difficult to render axenic, presumably because these bacteria depend upon other organisms to grow under these conditions. These tight associations between Synechococcus and heterotrophic bacteria represent a good relevant system to study interspecies interactions. Ten individual Synechococcus strains, isolated from eutrophic and oligotrophic waters, were chosen for investigation. Four to six dominant associated heterotrophic bacteria were detected in the liquid cultures of each Synechococcus isolate, comprising members of the Cytophaga-Flavobacteria-Bacteroides (CFB) group (mainly from Flavobacteriales and Cytophagales), Alphaproteobacteria (mainly from the Roseobacter clade), Gammaproteobacteria (mainly from the Alteromonadales and Pseudomonadales), and Actinobacteria The presence of the CFB group, Gammaproteobacteria, and Actinobacteria showed clear geographic patterns related to the isolation environments of the Synechococcus bacteria. An investigation of the population dynamics within a growing culture (XM-24) of one of the isolates, including an evaluation of the proportions of cells that were free-living versus aggregated/attached, revealed interesting patterns for different bacterial groups. In Synechococcus sp. strain XM-24 culture, flavobacteria, which was the most abundant group throughout the culture period, tended to be aggregated or attached to the Synechococcus cells, whereas the actinobacteria demonstrated a free-living lifestyle, and roseobacters displayed different patterns depending on the culture growth phase. Factors contributing to these succession patterns for the heterotrophs likely include interactions among the culture community members, their relative abilities to utilize different compounds produced by Synechococcus cells and changes in the compounds released as culture growth proceeds, and their responses to other changes in the environmental conditions throughout the culture period.IMPORTANCE Marine microbes exist within an interactive ecological network, and studying their interactions is an important part of understanding their roles in global biogeochemical cycling and the determinants of microbial diversity. In this study, the dynamic relationships between Synechococcus spp. and their associated heterotrophic bacteria were investigated. Synechococcus-associated heterotrophic bacteria had similar geographic distribution patterns as their "host" and displayed different lifestyles (free-living versus attached/aggregated) according to the Synechococcus culture growth phases. Combined organic carbon composition and bacterial lifestyle data indicated a potential for succession in carbon utilization patterns by the dominant associated heterotrophic bacteria. Comprehending the interactions between photoautotrophs and heterotrophs and the patterns of organic carbon excretion and utilization is critical to understanding their roles in oceanic biogeochemical cycling.

Dataset Information

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

Publications

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

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