title: Novel Approach for High-Throughput Metabolic Screening of Whole Plants by Stable Isotopes.
abstract: Here, we demonstrate whole-plant metabolic profiling by stable isotope labeling and combustion isotope-ratio mass spectrometry for precise quantification of assimilation, translocation, and molecular reallocation of (13)CO2 and (15)NH4NO3 The technology was applied to rice (Oryza sativa) plants at different growth stages. For adult plants, (13)CO2 labeling revealed enhanced carbon assimilation of the flag leaf from flowering to late grain-filling stage, linked to efficient translocation into the panicle. Simultaneous (13)CO2 and (15)NH4NO3 labeling with hydroponically grown seedlings was used to quantify the relative distribution of carbon and nitrogen. Two hours after labeling, assimilated carbon was mainly retained in the shoot (69%), whereas 7% entered the root and 24% was respired. Nitrogen, taken up via the root, was largely translocated into the shoot (85%). Salt-stressed seedlings showed decreased uptake and translocation of nitrogen (69%), whereas carbon metabolism was unaffected. Coupled to a gas chromatograph, labeling analysis provided enrichment of proteinogenic amino acids. This revealed significant protein synthesis in the panicle of adult plants, whereas protein biosynthesis in adult leaves was 8-fold lower than that in seedling shoots. Generally, amino acid enrichment was similar among biosynthetic families and allowed us to infer labeling dynamics of their precursors. On this basis, early and strong (13)C enrichment of Embden-Meyerhof-Parnas pathway and pentose phosphate pathway intermediates indicated high activity of these routes. Applied to mode-of-action analysis of herbicides, the approach showed severe disturbance in the synthesis of branched-chain amino acids upon treatment with imazapyr. The established technology displays a breakthrough for quantitative high-throughput plant metabolic phenotyping.
authors.affiliation: Institute of Systems Biotechnology, Saarland University, 66123 Saarbrücken, Germany (L.M.D., V.B., C.W.);Institute of Biochemical Engineering, University of Technology Braunschweig, 38106 Braunschweig, Germany (D.R., G.M., C.B., K.K.);BASF SE, 67117 Limburgerhof, Germany (H.B.); andMetanomics GmbH, 10589 Berlin, Germany (O.E.B., R.F., T.E.).
authors.forename: Lisa Maria
authors.identifier: 0000-0001-5385-1739
authors.initials: LM
authors.lastname: Dersch
authors.affiliation: Institute of Systems Biotechnology, Saarland University, 66123 Saarbrücken, Germany (L.M.D., V.B., C.W.);Institute of Biochemical Engineering, University of Technology Braunschweig, 38106 Braunschweig, Germany (D.R., G.M., C.B., K.K.);BASF SE, 67117 Limburgerhof, Germany (H.B.); andMetanomics GmbH, 10589 Berlin, Germany (O.E.B., R.F., T.E.).
authors.forename: Veronique
authors.initials: V
authors.lastname: Beckers
authors.affiliation: Institute of Systems Biotechnology, Saarland University, 66123 Saarbrücken, Germany (L.M.D., V.B., C.W.);Institute of Biochemical Engineering, University of Technology Braunschweig, 38106 Braunschweig, Germany (D.R., G.M., C.B., K.K.);BASF SE, 67117 Limburgerhof, Germany (H.B.); andMetanomics GmbH, 10589 Berlin, Germany (O.E.B., R.F., T.E.).
authors.forename: Detlev
authors.identifier: 0000-0003-1928-5600
authors.initials: D
authors.lastname: Rasch
authors.affiliation: Institute of Systems Biotechnology, Saarland University, 66123 Saarbrücken, Germany (L.M.D., V.B., C.W.);Institute of Biochemical Engineering, University of Technology Braunschweig, 38106 Braunschweig, Germany (D.R., G.M., C.B., K.K.);BASF SE, 67117 Limburgerhof, Germany (H.B.); andMetanomics GmbH, 10589 Berlin, Germany (O.E.B., R.F., T.E.).
authors.forename: Guido
authors.identifier: 0000-0003-0081-5050
authors.initials: G
authors.lastname: Melzer
authors.affiliation: Institute of Systems Biotechnology, Saarland University, 66123 Saarbrücken, Germany (L.M.D., V.B., C.W.);Institute of Biochemical Engineering, University of Technology Braunschweig, 38106 Braunschweig, Germany (D.R., G.M., C.B., K.K.);BASF SE, 67117 Limburgerhof, Germany (H.B.); andMetanomics GmbH, 10589 Berlin, Germany (O.E.B., R.F., T.E.).
authors.forename: Christoph
authors.identifier: 0000-0003-0418-8939
authors.initials: C
authors.lastname: Bolten
authors.affiliation: Institute of Systems Biotechnology, Saarland University, 66123 Saarbrücken, Germany (L.M.D., V.B., C.W.);Institute of Biochemical Engineering, University of Technology Braunschweig, 38106 Braunschweig, Germany (D.R., G.M., C.B., K.K.);BASF SE, 67117 Limburgerhof, Germany (H.B.); andMetanomics GmbH, 10589 Berlin, Germany (O.E.B., R.F., T.E.).
authors.forename: Katina
authors.initials: K
authors.lastname: Kiep
authors.affiliation: Institute of Systems Biotechnology, Saarland University, 66123 Saarbrücken, Germany (L.M.D., V.B., C.W.);Institute of Biochemical Engineering, University of Technology Braunschweig, 38106 Braunschweig, Germany (D.R., G.M., C.B., K.K.);BASF SE, 67117 Limburgerhof, Germany (H.B.); andMetanomics GmbH, 10589 Berlin, Germany (O.E.B., R.F., T.E.).
authors.forename: Horst
authors.initials: H
authors.lastname: Becker
authors.affiliation: Institute of Systems Biotechnology, Saarland University, 66123 Saarbrücken, Germany (L.M.D., V.B., C.W.);Institute of Biochemical Engineering, University of Technology Braunschweig, 38106 Braunschweig, Germany (D.R., G.M., C.B., K.K.);BASF SE, 67117 Limburgerhof, Germany (H.B.); andMetanomics GmbH, 10589 Berlin, Germany (O.E.B., R.F., T.E.).
authors.forename: Oliver Ernst
authors.initials: OE
authors.lastname: Bläsing
authors.affiliation: Institute of Systems Biotechnology, Saarland University, 66123 Saarbrücken, Germany (L.M.D., V.B., C.W.);Institute of Biochemical Engineering, University of Technology Braunschweig, 38106 Braunschweig, Germany (D.R., G.M., C.B., K.K.);BASF SE, 67117 Limburgerhof, Germany (H.B.); andMetanomics GmbH, 10589 Berlin, Germany (O.E.B., R.F., T.E.).
authors.forename: Regine
authors.initials: R
authors.lastname: Fuchs
authors.affiliation: Institute of Systems Biotechnology, Saarland University, 66123 Saarbrücken, Germany (L.M.D., V.B., C.W.);Institute of Biochemical Engineering, University of Technology Braunschweig, 38106 Braunschweig, Germany (D.R., G.M., C.B., K.K.);BASF SE, 67117 Limburgerhof, Germany (H.B.); andMetanomics GmbH, 10589 Berlin, Germany (O.E.B., R.F., T.E.).
authors.forename: Thomas
authors.identifier: 0000-0002-3294-3666
authors.initials: T
authors.lastname: Ehrhardt
authors.affiliation: Institute of Systems Biotechnology, Saarland University, 66123 Saarbrücken, Germany (L.M.D., V.B., C.W.);Institute of Biochemical Engineering, University of Technology Braunschweig, 38106 Braunschweig, Germany (D.R., G.M., C.B., K.K.);BASF SE, 67117 Limburgerhof, Germany (H.B.); andMetanomics GmbH, 10589 Berlin, Germany (O.E.B., R.F., T.E.) christoph.wittmann@uni-saarland.de.
authors.forename: Christoph
authors.identifier: 0000-0002-7952-985X
authors.initials: C
authors.lastname: Wittmann
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000597:Amino Acids, Branched-Chain / Q000187:drug effects / Q000378:metabolism; D002244:Carbon / Q000737:chemistry / Q000378:metabolism; D002245:Carbon Dioxide / Q000737:chemistry; D002247:Carbon Isotopes / Q000737:chemistry / Q000378:metabolism; D019069:Cell Respiration; D002849:Chromatography, Gas; D006019:Glycolysis; D006540:Herbicides / Q000494:pharmacology; D057166:High-Throughput Screening Assays / Q000379:methods*; D018527:Hydroponics / Q000379:methods; D007093:Imidazoles / Q000494:pharmacology; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry; D053858:Metabolic Networks and Pathways / Q000187:drug effects / Q000502:physiology; D055442:Metabolome; D055432:Metabolomics; D009525:Niacin / Q000031:analogs & derivatives / Q000494:pharmacology; D009566:Nitrates / Q000737:chemistry; D009584:Nitrogen / Q000737:chemistry / Q000378:metabolism; D009587:Nitrogen Isotopes / Q000737:chemistry / Q000378:metabolism; D012275:Oryza / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism* / Q000502:physiology*; D010427:Pentose Phosphate Pathway; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000032:analysis / Q000378:metabolism; D018517:Plant Roots / Q000378:metabolism; D018520:Plant Shoots / Q000378:metabolism; D012492:Salts / Q000378:metabolism; D036226:Seedlings / Q000187:drug effects / Q000378:metabolism
chemical_list: D000596:Amino Acids; D000597:Amino Acids, Branched-Chain; D002247:Carbon Isotopes; D006540:Herbicides; D007093:Imidazoles; D009566:Nitrates; D009587:Nitrogen Isotopes; D010940:Plant Proteins; D012492:Salts; D002245:Carbon Dioxide; D009525:Niacin; D002244:Carbon; C076656:imazapyr; D009584:Nitrogen; C006568:ammonium nitrate