title: Regulation of Primary Metabolism in Response to Low Oxygen Availability as Revealed by Carbon and Nitrogen Isotope Redistribution.
abstract: Based on enzyme activity assays and metabolic responses to waterlogging of the legume Lotus japonicus, it was previously suggested that, during hypoxia, the tricarboxylic acid cycle switches to a noncyclic operation mode. Hypotheses were postulated to explain the alternative metabolic pathways involved, but as yet, a direct analysis of the relative redistribution of label through the corresponding pathways was not made. Here, we describe the use of stable isotope-labeling experiments for studying metabolism under hypoxia using wild-type roots of the crop legume soybean (Glycine max). [(13)C]Pyruvate labeling was performed to compare metabolism through the tricarboxylic acid cycle, fermentation, alanine metabolism, and the γ-aminobutyric acid shunt, while [(13)C]glutamate and [(15)N]ammonium labeling were performed to address the metabolism via glutamate to succinate. Following these labelings, the time course for the redistribution of the (13)C/(15)N label throughout the metabolic network was evaluated with gas chromatography-time of flight-mass spectrometry. Our combined labeling data suggest the inhibition of the tricarboxylic acid cycle enzyme succinate dehydrogenase, also known as complex II of the mitochondrial electron transport chain, providing support for the bifurcation of the cycle and the down-regulation of the rate of respiration measured during hypoxic stress. Moreover, up-regulation of the γ-aminobutyric acid shunt and alanine metabolism explained the accumulation of succinate and alanine during hypoxia.
authors.affiliation: Energy Metabolism Group (C.A., C.P., M.R., J.T.v.D.) and Central Metabolism Group (T.O., A.R.F.), Max Planck Institute of Molecular Plant Physiology, D-14476 Potsdam-Golm, Germany;Plant Metabolomics Laboratory, Instituto de Tecnologia Química e Biológica António Xavier-Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal (C.A.);Departamento de Produção Animal e Vegetal, Faculdade de Ciências Agrárias, Universidade Federal do Amazonas, Manaus, Amazonas 69082-653, Brazil (M.R.);University of Angers (H.D., A.M.L.) and Institut National de la Recherche Agronomique (A.M.L.), Unité Mixte de Recherche 1345 IRHS, SFR 4207 QUASAV, F-49045 Angers, France; andInstitute for Biology I, RWTH Aachen University, D-52056 Aachen, Germany (J.T.v.D.) antonio@itqb.unl.pt.
authors.forename: Carla
authors.identifier: http://orcid.org/0000-0001-6747-1243
authors.initials: C
authors.lastname: António
authors.affiliation: Energy Metabolism Group (C.A., C.P., M.R., J.T.v.D.) and Central Metabolism Group (T.O., A.R.F.), Max Planck Institute of Molecular Plant Physiology, D-14476 Potsdam-Golm, Germany;Plant Metabolomics Laboratory, Instituto de Tecnologia Química e Biológica António Xavier-Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal (C.A.);Departamento de Produção Animal e Vegetal, Faculdade de Ciências Agrárias, Universidade Federal do Amazonas, Manaus, Amazonas 69082-653, Brazil (M.R.);University of Angers (H.D., A.M.L.) and Institut National de la Recherche Agronomique (A.M.L.), Unité Mixte de Recherche 1345 IRHS, SFR 4207 QUASAV, F-49045 Angers, France; andInstitute for Biology I, RWTH Aachen University, D-52056 Aachen, Germany (J.T.v.D.).
authors.forename: Carola
authors.initials: C
authors.lastname: Päpke
authors.affiliation: Energy Metabolism Group (C.A., C.P., M.R., J.T.v.D.) and Central Metabolism Group (T.O., A.R.F.), Max Planck Institute of Molecular Plant Physiology, D-14476 Potsdam-Golm, Germany;Plant Metabolomics Laboratory, Instituto de Tecnologia Química e Biológica António Xavier-Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal (C.A.);Departamento de Produção Animal e Vegetal, Faculdade de Ciências Agrárias, Universidade Federal do Amazonas, Manaus, Amazonas 69082-653, Brazil (M.R.);University of Angers (H.D., A.M.L.) and Institut National de la Recherche Agronomique (A.M.L.), Unité Mixte de Recherche 1345 IRHS, SFR 4207 QUASAV, F-49045 Angers, France; andInstitute for Biology I, RWTH Aachen University, D-52056 Aachen, Germany (J.T.v.D.).
authors.forename: Marcio
authors.identifier: http://orcid.org/0000-0003-4937-3855
authors.initials: M
authors.lastname: Rocha
authors.affiliation: Energy Metabolism Group (C.A., C.P., M.R., J.T.v.D.) and Central Metabolism Group (T.O., A.R.F.), Max Planck Institute of Molecular Plant Physiology, D-14476 Potsdam-Golm, Germany;Plant Metabolomics Laboratory, Instituto de Tecnologia Química e Biológica António Xavier-Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal (C.A.);Departamento de Produção Animal e Vegetal, Faculdade de Ciências Agrárias, Universidade Federal do Amazonas, Manaus, Amazonas 69082-653, Brazil (M.R.);University of Angers (H.D., A.M.L.) and Institut National de la Recherche Agronomique (A.M.L.), Unité Mixte de Recherche 1345 IRHS, SFR 4207 QUASAV, F-49045 Angers, France; andInstitute for Biology I, RWTH Aachen University, D-52056 Aachen, Germany (J.T.v.D.).
authors.forename: Houssein
authors.initials: H
authors.lastname: Diab
authors.affiliation: Energy Metabolism Group (C.A., C.P., M.R., J.T.v.D.) and Central Metabolism Group (T.O., A.R.F.), Max Planck Institute of Molecular Plant Physiology, D-14476 Potsdam-Golm, Germany;Plant Metabolomics Laboratory, Instituto de Tecnologia Química e Biológica António Xavier-Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal (C.A.);Departamento de Produção Animal e Vegetal, Faculdade de Ciências Agrárias, Universidade Federal do Amazonas, Manaus, Amazonas 69082-653, Brazil (M.R.);University of Angers (H.D., A.M.L.) and Institut National de la Recherche Agronomique (A.M.L.), Unité Mixte de Recherche 1345 IRHS, SFR 4207 QUASAV, F-49045 Angers, France; andInstitute for Biology I, RWTH Aachen University, D-52056 Aachen, Germany (J.T.v.D.).
authors.forename: Anis M
authors.identifier: http://orcid.org/0000-0002-9985-2363
authors.initials: AM
authors.lastname: Limami
authors.affiliation: Energy Metabolism Group (C.A., C.P., M.R., J.T.v.D.) and Central Metabolism Group (T.O., A.R.F.), Max Planck Institute of Molecular Plant Physiology, D-14476 Potsdam-Golm, Germany;Plant Metabolomics Laboratory, Instituto de Tecnologia Química e Biológica António Xavier-Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal (C.A.);Departamento de Produção Animal e Vegetal, Faculdade de Ciências Agrárias, Universidade Federal do Amazonas, Manaus, Amazonas 69082-653, Brazil (M.R.);University of Angers (H.D., A.M.L.) and Institut National de la Recherche Agronomique (A.M.L.), Unité Mixte de Recherche 1345 IRHS, SFR 4207 QUASAV, F-49045 Angers, France; andInstitute for Biology I, RWTH Aachen University, D-52056 Aachen, Germany (J.T.v.D.).
authors.forename: Toshihiro
authors.identifier: http://orcid.org/0000-0001-8931-7722
authors.initials: T
authors.lastname: Obata
authors.affiliation: Energy Metabolism Group (C.A., C.P., M.R., J.T.v.D.) and Central Metabolism Group (T.O., A.R.F.), Max Planck Institute of Molecular Plant Physiology, D-14476 Potsdam-Golm, Germany;Plant Metabolomics Laboratory, Instituto de Tecnologia Química e Biológica António Xavier-Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal (C.A.);Departamento de Produção Animal e Vegetal, Faculdade de Ciências Agrárias, Universidade Federal do Amazonas, Manaus, Amazonas 69082-653, Brazil (M.R.);University of Angers (H.D., A.M.L.) and Institut National de la Recherche Agronomique (A.M.L.), Unité Mixte de Recherche 1345 IRHS, SFR 4207 QUASAV, F-49045 Angers, France; andInstitute for Biology I, RWTH Aachen University, D-52056 Aachen, Germany (J.T.v.D.).
authors.forename: Alisdair R
authors.initials: AR
authors.lastname: Fernie
authors.affiliation: Energy Metabolism Group (C.A., C.P., M.R., J.T.v.D.) and Central Metabolism Group (T.O., A.R.F.), Max Planck Institute of Molecular Plant Physiology, D-14476 Potsdam-Golm, Germany;Plant Metabolomics Laboratory, Instituto de Tecnologia Química e Biológica António Xavier-Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal (C.A.);Departamento de Produção Animal e Vegetal, Faculdade de Ciências Agrárias, Universidade Federal do Amazonas, Manaus, Amazonas 69082-653, Brazil (M.R.);University of Angers (H.D., A.M.L.) and Institut National de la Recherche Agronomique (A.M.L.), Unité Mixte de Recherche 1345 IRHS, SFR 4207 QUASAV, F-49045 Angers, France; andInstitute for Biology I, RWTH Aachen University, D-52056 Aachen, Germany (J.T.v.D.).
authors.forename: Joost T
authors.identifier: http://orcid.org/0000-0001-7944-9289
authors.initials: JT
authors.lastname: van Dongen
mesh_terms: D002247:Carbon Isotopes / Q000378:metabolism*; D019069:Cell Respiration; D002952:Citric Acid Cycle; D008401:Gas Chromatography-Mass Spectrometry; D007553:Isotope Labeling / Q000379:methods; D009587:Nitrogen Isotopes / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism; D018517:Plant Roots / Q000378:metabolism; D019289:Pyruvic Acid / Q000737:chemistry / Q000378:metabolism; D013025:Glycine max / Q000378:metabolism* / Q000502:physiology; D013385:Succinate Dehydrogenase / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D010940:Plant Proteins; D005680:gamma-Aminobutyric Acid; D019289:Pyruvic Acid; D013385:Succinate Dehydrogenase; D010100:Oxygen