Abstract
Plant metabolic pathways and the molecular and atomic fluxes through them can be deduced using stable isotopically labeled substrates. To this end one prerequisite is accurate measurement of the labeling pattern of targeted metabolites. Experiments are generally limited to the use of single-element isotopes, mainly 13C. Here, we summarize the application of gas chromatography-time of flight mass spectrometry (GC-TOF-MS) for metabolic studies using differently labeled elemental isotopes applied to both intact organelles and whole plant tissue. This method allows quantitative evaluation of a broad range of metabolic pathways without the need for laborious (and potentially inaccurate) chemical fractionation procedures commonly used in the estimation of fluxes following incubation in radiolabeled substrates. We focus herein on the determination of isotope labeling in organic and amino acids.
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References
Morgan MJ, Lehmann M, Schwarzlander M et al (2008) Decrease in manganese superoxide dismutase leads to reduced root growth and affects tricarboxylic acid cycle flux and mitochondrial redox homeostasis. Plant Physiol 147:101–114
Giegé P, Heazlewood JL, Roessner-Tunali U et al (2003) Enzymes of glycolysis are functionally associated with the mitochondrion in Arabidopsis cells. Plant Cell 15:2140–2151
Graham JWA, Williams TCR, Morgan M et al (2007) Glycolytic enzymes associate dynamically with mitochondria in response to respiratory demand and support substrate channeling. Plant Cell 19:3723–3738
Nunes-Nesi A, Carrari F, Lytovchenko A et al (2005) Enhanced photosynthetic performance and growth as a consequence of decreasing mitochondrial malate dehydrogenase activity in transgenic tomato plants. Plant Physiol 137:611–622
Araújo WL, Nunes-Nesi A, Osorio S et al (2011) Antisense inhibition of the iron-sulphur subunit of succinate dehydrogenase enhances photosynthesis and growth in tomato via an organic acid-mediated effect on stomatal aperture. Plant Cell 23:600–627
Araújo WL, Tohge T, Osorio S et al (2012) Antisense inhibition of the 2-oxoglutarate dehydrogenase complex in tomato demonstrates its importance for plant respiration and during leaf senescence and fruit maturation. Plant Cell 24:2328–2351
Schwender J, Shachar-Hill Y, Ohlrogge JB (2006) Mitochondrial metabolism in developing embryos of Brassica napus. J Biol Chem 281:34040–34047
Roessner-Tunali U, Liu J, Leisse A et al (2004) Kinetics of labelling of organic and amino acids in potato tubers by gas chromatography–mass spectrometry following incubation in 13C labelled isotopes. Plant J 39:668–679
Yuan J, Bennett BD, Rabinowitz JD (2008) Kinetic flux profiling for quantitation of cellular metabolic fluxes. Nat Protocols 3:1328–1340
Haverkorn van Rijsewijk BRB, Nanchen A, Nallet S et al (2011) Large-scale 13C-flux analysis reveals distinct transcriptional control of respiratory and fermentative metabolism in Escherichia coli. Mol Syst Biol 7
Birkemeyer C, Luedemann A, Wagner C et al (2005) Metabolome analysis: the potential of in vivo labeling with stable isotopes for metabolite profiling. Trends Biotechnol 23:28–33
Huege J, Goetze J, Schwarz D et al (2011) Modulation of the major paths of carbon in photorespiratory mutants of Synechocystis. Plos One 6:e16278
Huege J, Sulpice R, Gibon Y et al (2007) GC-EI-TOF-MS analysis of in vivo carbon-partitioning into soluble metabolite pools of higher plants by monitoring isotope dilution after 13CO2 labelling. Phytochemistry 68:2258–2272
Araújo WL, Nunes-Nesi A, Trenkamp S et al (2008) Inhibition of 2-oxoglutarate dehydrogenase in potato tuber suggests the enzyme is limiting for respiration and confirms its importance in nitrogen assimilation. Plant Physiol 148:1782–1796
Studart-Guimarães C, Fait A, Nunes-Nesi A et al (2007) Reduced expression of succinyl-coenzyme A ligase can be compensated for by up-regulation of the γ-aminobutyrate shunt in illuminated tomato leaves. Plant Physiol 145:626–639
Dal CV, Tieman DM, Tohge T et al (2011) Identification of genes in the phenylalanine metabolic pathway by ectopic expression of a MYB transcription factor in tomato fruit. Plant Cell 23:2738–2753
Araújo WL, Ishizaki K, Nunes-Nesi A et al (2010) Identification of the 2-hydroxyglutarate and isovaleryl-CoA dehydrogenases as alternative electron donors linking lysine catabolism to the electron transport chain of Arabidopsis mitochondria. Plant Cell 22:1549–1563
Kleessen S, Araújo WL, Fernie AR et al (2012) Model-based confirmation of alternative substrates of mitochondrial electron transport chain. J Biol Chem 287:11122–11131
Ratcliffe RG, Shachar-Hill Y (2006) Measuring multiple fluxes through plant metabolic networks. Plant J 45:490–511
Libourel IGL, Shachar-Hill Y (2008) Metabolic flux analysis in plants: from intelligent design to rational engineering. Annu Rev Plant Physiol Plant Mol Biol 59:625–650
Schwender J, Ohlrogge J, Shachar-Hill Y (2004) Understanding flux in plant metabolic networks. Curr Opin Plant Biol 7:309–317
Schwender J (2008) Metabolic flux analysis as a tool in metabolic engineering of plants. Curr Opin Biotechnol 19:131–137
O’Grady J, Schwender J, Shachar-Hill Y et al (2012) Metabolic cartography: experimental quantification of metabolic fluxes from isotopic labelling studies. J Exp Bot 63:2293–2308
Allen DK, Ohlrogge JB, Shachar-Hill Y (2009) The role of light in soybean seed filling metabolism. Plant J 58:220–234
Blank L, Kuepfer L, Sauer U (2005) Large-scale 13C-flux analysis reveals mechanistic principles of metabolic network robustness to null mutations in yeast. Genome Biol 6:R49
Fischer E, Sauer U (2005) Large-scale in vivo flux analysis shows rigidity and suboptimal performance of Bacillus subtilis metabolism. Nat Genet 37:636–640
Lonien J, Schwender J (2009) Analysis of metabolic flux phenotypes for two Arabidopsis mutants with severe impairment in seed storage lipid synthesis. Plant Physiol 151:1617–1634
Fernie AR, Roscher A, Ratcliffe RG et al (2001) Fructose 2,6-bisphosphate activates pyrophosphate: fructose-6-phosphate 1-phosphotransferase and increases triose phosphate to hexose phosphate cycling in heterotrophic cells. Planta 212:250–263
Schwender J, Ohlrogge JB, Shachar-Hill Y (2003) A flux model of glycolysis and the oxidative pentosephosphate pathway in developing Brassica napus embryos. J Biol Chem 278:29442–29453
Dieuaide-Noubhani M, Raffard G, Canioni P et al (1995) Quantification of compartmented metabolic fluxes in maize root tips using isotope distribution from 13C- or 14C-labeled glucose. J Biol Chem 270:13147–13159
Roscher A, Kruger NJ, Ratcliffe RG (2000) Strategies for metabolic flux analysis in plants using isotope labelling. J Biotechnol 77:81–102
Glawischnig E, Gierl A, Tomas A et al (2001) Retrobiosynthetic nuclear magnetic resonance analysis of amino acid biosynthesis and intermediary metabolism. Metabolic flux in developing maize kernels. Plant Physiol 125:1178–1186
Rontein D, Dieuaide-Noubhani M, Dufourc EJ et al (2002) The metabolic architecture of plant cells - Stability of central metabolism and flexibility of anabolic pathways during the growth cycle of tomato cells. J Biol Chem 277:43948–43960
Edwards S, Nguyen BT, Do B et al (1998) Contribution of malic enzyme, pyruvate kinase, phosphoenolpyruvate carboxylase, and the Krebs cycle to respiration and biosynthesis and to intracellular pH regulation during hypoxia in maize root tips observed by nuclear magnetic resonance imaging and gas chromatography mass spectrometry. Plant Physiol 116:1073–1081
Luedemann A, Malotky L, Erban A et al (2012) TagFinder: Preprocessing software for the fingerprinting and the profiling of gas chromatography–mass spectrometry based metabolome analyses. Methods Mol Biol 860:255–286
Lommen A (2012) Data (Pre-)processing of Nominal and Accurate Mass LC-MS or GC-MS Data Using MetAlign. Methods Mol Biol 860:229–253
Luedemann A, Strassburg K, Erban A et al (2008) TagFinder for the quantitative analysis of gas chromatography–mass spectrometry (GC-MS)-based metabolite profiling experiments. Bioinformatics 24:732–737
Van Winden WA, Wittmann C, Heinzle E et al (2002) Correcting mass isotopomer distributions for naturally occurring isotopes. Biotechnol Bioeng 80:477–479
Wittmann C, Heinzle E (1999) Mass spectrometry for metabolic flux analysis. Biotechnol Bioeng 62:739–750
Osorio S, Do PT, Fernie AR (2012) Profiling primary metabolites of tomato fruit with gas chromatography/mass spectrometry. Methods Mol Biol 860:101–109
Hasunuma T, Harada K, Miyazawa S-I et al (2010) Metabolic turnover analysis by a combination of in vivo 13C-labelling from 13CO2 and metabolic profiling with CE-MS/MS reveals rate-limiting steps of the C3 photosynthetic pathway in Nicotiana tabacum leaves. J Exp Bot 61:1041–1051
Acknowledgements
Financial support from the Max-Planck-Society (to WLA and ARF), the Deutsche Forschungsgemeinschaft (grant no. DFG-SFB429 to ARF), and the National Council for Scientific and Technological Development CNPq-Brazil (grant number 472787/2011-0 to WLA) is gratefully acknowledged.
Conflict of interest: The authors declare that they have no conflict of interest.
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Araújo, W.L., Tohge, T., Nunes-Nesi, A., Obata, T., Fernie, A.R. (2014). Analysis of Kinetic Labeling of Amino Acids and Organic Acids by GC-MS. In: Dieuaide-Noubhani, M., Alonso, A. (eds) Plant Metabolic Flux Analysis. Methods in Molecular Biology, vol 1090. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-688-7_7
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DOI: https://doi.org/10.1007/978-1-62703-688-7_7
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