Skip to main content
SpringerLink
Log in

Comparison of Quantitative Metabolite Imaging Tools and Carbon-13 Techniques for Fluxomics

  • Protocol
  • First Online:
Book cover Plant Systems Biology

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 553))

Abstract

The recent development of analytic technologies allows fast analysis of metabolism in real time. Fluxomics aims to define the genes involved in regulation of flux through a metabolic or signaling pathway. Flux through a metabolic or signaling pathway is determined by the activity of its individual components; regulation can occur at many levels, including transcriptional, posttranslational, and allosteric levels. Currently two technologies are used to monitor fluxes. The first is pulse labeling of the organism with a tracer such as C13, followed by mass spectrometric analysis of the partitioning of label into different compounds. The second approach is based on the use of flux sensors, proteins that respond with a conformational change to ligand binding. Fluorescence resonance energy transfer (FRET) detects the conformational change and serves as a proxy for ligand concentration. Both methods provide high time resolution. In contrast to mass spectrometry assays, FRET nanosensors monitor only a single compound, but the advantage of FRET nanosensors is that they yield data with cellular and subcellular resolution.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Okumoto, S., Takanaga, H., and Frommer, W.B. (2008) Tansley review: quantitative imaging for discovery and assembly of the metabo-regulome. New Phytol. 180, 271–295.

    Google Scholar 

  2. Aronoff, S., Benson, A., Hassid, W.Z., and Calvin, M. (1947) Distribution of C14 in photosynthesizing barley seedlings. Science 105, 664–665.

    Article  PubMed  CAS  Google Scholar 

  3. Benson, A. and Calvin, M. (1947) The dark reductions of photosynthesis. Science 105, 648–649.

    Article  PubMed  CAS  Google Scholar 

  4. Nanchen, A., Fuhrer, T., and Sauer, U. (2007) Determination of metabolic flux ratios from 13C-experiments and gas chromatography-mass spectrometry data: protocol and principles. Methods Mol. Biol. 358, 177–197.

    Article  PubMed  CAS  Google Scholar 

  5. Sauer, U. (2006) Metabolic networks in motion: 13C-based flux analysis. Mol. Syst. Biol. 2, 62.

    Article  PubMed  Google Scholar 

  6. Schuetz, R., Kuepfer, L., and Sauer, U. (2007) Systematic evaluation of objective functions for predicting intracellular fluxes in Escherichia coli. Mol. Syst. Biol. 3, 119.

    Article  PubMed  Google Scholar 

  7. Kruger, N.J. and Ratcliffe, R.G. (2007) Dynamic metabolic networks: going with the flow. Phytochemistry 68, 2136–2138.

    Article  PubMed  CAS  Google Scholar 

  8. Ratcliffe, R.G. and Shachar-Hill, Y. (2006) Measuring multiple fluxes through plant metabolic networks. Plant J. 45, 490–511.

    Article  PubMed  CAS  Google Scholar 

  9. Wiechert, W., Schweissgut, O., Takanaga, H., and Frommer, W.B. (2007) Fluxomics: mass spectrometry versus quantitative imaging. Curr. Opin. Plant Biol. 10, 323–330.

    Article  PubMed  CAS  Google Scholar 

  10. Schwender, J., Ohlrogge, J., and Shachar-Hill, Y. (2004) Understanding flux in plant metabolic networks. Curr. Opin. Plant Biol. 7, 309–317.

    Article  PubMed  CAS  Google Scholar 

  11. Schwender, J., Ohlrogge, J.B., and 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.

    Article  PubMed  CAS  Google Scholar 

  12. Schwender, J., Goffman, F., Ohlrogge, J.B., and Shachar-Hill, Y. (2004) Rubisco without the Calvin cycle improves the carbon efficiency of developing green seeds. Nature 432, 779–782.

    Article  PubMed  CAS  Google Scholar 

  13. Boatright, J., Negre, F., Chen, X., Kish, C.M., Wood, B., Peel, G., Orlova, I., Gang, D., Rhodes, D., and Dudareva, N. (2004) Understanding in vivo benzenoid metabolism in petunia petal tissue. Plant Physiol. 135, 1993–2011.

    Article  PubMed  CAS  Google Scholar 

  14. Matsuda, F., Morino, K., Ano, R., Kuzawa, M., Wakasa, K., and Miyagawa, H. (2005) Metabolic flux analysis of the phenylpropanoid pathway in elicitor-treated potato tuber tissue. Plant Cell Physiol. 46, 454–466.

    Article  PubMed  CAS  Google Scholar 

  15. Wiechert, W. and de Graaf, A.A. (1996) In vivo stationary flux analysis by 13C labeling experiments. Adv. Biochem. Eng. Biotechnol. 54, 109–154.

    PubMed  CAS  Google Scholar 

  16. Chia, T., Thorneycroft, D., Chapple, A., Messerli, G., Chen, J., Zeeman, S. C., Smith, S.M., and Smith, A.M. (2004) A cytosolic glucosyltransferase is required for conversion of starch to sucrose in Arabidopsis leaves at night. Plant J. 37, 853–863.

    Article  PubMed  CAS  Google Scholar 

  17. Niittyla, T., Messerli, G., Trevisan, M., Chen, J., Smith, A.M., and Zeeman, S.C. (2004) A previously unknown maltose transporter essential for starch degradation in leaves. Science 303, 87–89.

    Article  PubMed  Google Scholar 

  18. Zeeman, S.C., Smith, S.M., and Smith, A.M. (2007) The diurnal metabolism of leaf starch. Biochem. J. 401, 13–28.

    Article  PubMed  CAS  Google Scholar 

  19. Neuhaus, H.E. (2007) Transport of primary metabolites across the plant vacuolar membrane. FEBS Lett. 581, 2223–2226.

    Article  PubMed  CAS  Google Scholar 

  20. Uys, L., Botha, F.C., Hofmeyr, J.H., and Rohwer, J.M. (2007) Kinetic model of sucrose accumulation in maturing sugarcane culm tissue. Phytochemistry 68, 2375–2392.

    Article  PubMed  CAS  Google Scholar 

  21. Endler, A., Meyer, S., Schelbert, S., Schneider, T., Weschke, W., Peters, S.W., Keller, F., Baginsky, S., Martinoia, E., and Schmidt, U.G. (2006) Identification of a vacuolar sucrose transporter in barley and Arabidopsis mesophyll cells by a tonoplast proteomic approach. Plant Physiol. 141, 196–207.

    Article  PubMed  CAS  Google Scholar 

  22. Junker, B.H., Lonien, J., Heady, L.E., Rogers, A., and Schwender, J. (2007) Parallel determination of enzyme activities and in vivo fluxes in Brassica napus embryos grown on organic or inorganic nitrogen source. Phytochemistry 68, 2232–2242.

    Article  PubMed  CAS  Google Scholar 

  23. Förster, T. (1948) Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann. Physik 6, 55.

    Article  Google Scholar 

  24. Looger, L.L., Lalonde, S., and Frommer, W.B. (2005) Genetically encoded FRET sensors for visualizing metabolites with subcellular resolution in living cells. Plant Physiol. 138, 555–557.

    Article  PubMed  CAS  Google Scholar 

  25. Miyawaki, A., Llopis, J., Heim, R., McCaffery, J.M., Adams, J.A., Ikura, M., and Tsien, R.Y. (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887.

    Article  PubMed  CAS  Google Scholar 

  26. Romoser, V.A., Hinkle, P.M., and Persechini, A. (1997) Detection in living cells of Ca2+-dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. A new class of fluorescent indicators. J. Biol. Chem. 272, 13270–13274.

    Article  PubMed  CAS  Google Scholar 

  27. Deuschle, K., Okumoto, S., Fehr, M., Looger, L.L., Kozhukh, L., and Frommer, W.B. (2005) Construction and optimization of a family of genetically encoded metabolite sensors by semirational protein engineering. Protein Sci. 14, 2304–2314.

    Article  PubMed  CAS  Google Scholar 

  28. Fehr, M., Frommer, W.B., and Lalonde, S. (2002) Visualization of maltose uptake in living yeast cells by fluorescent nanosensors. Proc. Natl. Acad. Sci. USA 99, 9846–9851.

    Article  PubMed  CAS  Google Scholar 

  29. Fehr, M., Lalonde, S., Ehrhardt, D.W., and Frommer, W.B. (2004) Live imaging of glucose homeostasis in nuclei of COS-7 cells. J. Fluoresc. 14, 603–609.

    Article  PubMed  CAS  Google Scholar 

  30. Fehr, M., Lalonde, S., Lager, I., Wolff, M.W., and Frommer, W.B. (2003) In vivo imaging of the dynamics of glucose uptake in the cytosol of COS-7 cells by fluorescent nanosensors. J. Biol. Chem. 278, 19127–19133.

    Article  PubMed  CAS  Google Scholar 

  31. Fehr, M., Takanaga, H., Ehrhardt, D.W., and Frommer, W.B. (2005) Evidence for high-capacity bidirectional glucose transport across the endoplasmic reticulum membrane by genetically encoded fluorescence resonance energy transfer nanosensors. Mol. Cell Biol. 25, 11102–11112.

    Article  PubMed  CAS  Google Scholar 

  32. Gu, H., Lalonde, S., Okumoto, S., Looger, L.L., Scharff-Poulsen, A.M., Grossman, A.R., Kossmann, J., Jakobsen, I., and Frommer, W.B. (2006) A novel analytical method for in vivo phosphate tracking. FEBS Lett. 580, 5885–5893.

    Article  PubMed  CAS  Google Scholar 

  33. Kaper, T., Looger, L.L., Takanaga, H., Platten, M., Steinman, L., and Frommer, W.B. (2007) Nanosensor detection of an immunoregulatory tryptophan influx/kynurenine efflux cycle. PLoS Biol. 5, e257.

    Article  PubMed  Google Scholar 

  34. Lager, I., Fehr, M., Frommer, W.B., and Lalonde, S. (2003) Development of a fluorescent nanosensor for ribose. FEBS Lett. 553, 85–89.

    Article  PubMed  CAS  Google Scholar 

  35. Okumoto, S., Looger, L.L., Micheva, K.D., Reimer, R.J., Smith, S.J., and Frommer, W.B. (2005) Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors. Proc. Natl. Acad. Sci. USA 102, 8740–8745.

    Article  PubMed  CAS  Google Scholar 

  36. Wiechert, W. (2001) 13C metabolic flux analysis. Metab. Eng. 3, 195–206.

    Article  PubMed  CAS  Google Scholar 

  37. Blank, L.M., Kuepfer, L., and Sauer, U. (2005) Large-scale 13C-flux analysis reveals mechanistic principles of metabolic network robustness to null mutations in yeast. Genome Biol. 6, R49.

    Article  PubMed  Google Scholar 

  38. Fischer, E. and Sauer, U. (2005) Large-scale in vivo flux analysis shows rigidity and sub-optimal performance of Bacillus subtilis metabolism. Nat. Genet. 37, 636–640.

    Article  PubMed  CAS  Google Scholar 

  39. Nöh, K., Gronke, K., Luo, B., Takors, R., Oldiges, M., and Wiechert, W. (2007) Metabolic flux analysis at ultra short time scale: isotopically non-stationary 13C labeling experiments. J. Biotechnol. 129, 249–267.

    Article  PubMed  Google Scholar 

  40. Nöh, K., Wahl, A., and Wiechert, W. (2006) Computational tools for isotopically instationary 13C labeling experiments under metabolic steady state conditions. Metab. Eng. 8, 554–577.

    Article  PubMed  Google Scholar 

  41. Deuschle, K., Chaudhuri, B., Okumoto, S., Lager, I., Lalonde, S., and Frommer, W.B. (2006) Rapid metabolism of glucose detected with FRET glucose nanosensors in epidermal cells and intact roots of Arabidopsis RNA-silencing mutants. Plant Cell 18, 2314–2325.

    Article  PubMed  CAS  Google Scholar 

  42. Dulla, C., Tani, H., Okumoto, S., Frommer, W.B., Reimer, R.J., and Huguenard, J.R. (2008) Imaging of glutamate in brain slices using FRET sensors. J. Neurosci. Methods 168, 306–319.

    Article  PubMed  CAS  Google Scholar 

  43. Kaper, T., Lager, I., Looger, L.L., Chermak, D., and Frommer, W.B. (2008) FRET sensors for quantitative monitoring of pentose and disaccharide accumulation in bacteria. Biotechnol. Biofuels. 1,11.

    Google Scholar 

  44. Farré, E.M., Tiessen, A., Roessner, U., Geigenberger, P., Trethewey, R.N., and Willmitzer, L. (2001) Analysis of the compartmentation of glycolytic intermediates, nucleotides, sugars, organic acids, amino acids, and sugar alcohols in potato tubers using a nonaqueous fractionation method. Plant Physiol. 127, 685–700.

    Article  PubMed  Google Scholar 

  45. Chaudhuri, B., Hörmann, F., Lalonde, S., Brady, S.D.O., Benfey, P., and Frommer, W.B. (2008) Protonophore- and pH-insensitive glucose and sucrose accumulation detected by FRET nanosensors in Arabidopsis root tips Plant J. 56, 948–962.

    Google Scholar 

  46. Allen, G.J., Chu, S.P., Harrington, C.L., Schumacher, K., Hoffmann, T., Tang, Y.Y., Grill, E., and Schroeder, J.I. (2001) A defined range of guard cell calcium oscillation parameters encodes stomatal movements. Nature 411, 1053–1057.

    Article  PubMed  CAS  Google Scholar 

  47. Lalonde, S., Ehrhardt, D.W., and Frommer, W.B. (2005) Shining light on signaling and metabolic networks by genetically encoded biosensors. Curr. Opin. Plant Biol. 8, 574–581.

    Article  PubMed  CAS  Google Scholar 

  48. Miyawaki, A. (2003) Visualization of the spatial and temporal dynamics of intracellular signaling. Dev. Cell 4, 295–305.

    Article  PubMed  CAS  Google Scholar 

  49. Tsien, R.Y. (2006) Breeding and building molecules to spy on cells and tumors. Keio J. Med. 55, 127–140.

    Article  PubMed  CAS  Google Scholar 

  50. Vogel, S.S., Thaler, C., and Koushik, S.V. (2006) Fanciful FRET. Sci. STKE 2006, re2.

    Google Scholar 

  51. Fiala, A. and Spall, T. (2003) In vivo calcium imaging of brain activity in Drosophila by transgenic cameleon expression. Sci. STKE 2003, PL6.

    Google Scholar 

  52. Palmer, A.E. and Tsien, R.Y. (2006) Measuring calcium signaling using genetically targetable fluorescent indicators. Nat. Protoc. 1, 1057–1065.

    Article  PubMed  CAS  Google Scholar 

  53. Roe, M.W., Fiekers, J.F., Philipson, L.H., and Bindokas, V.P. (2006) Visualizing calcium signaling in cells by digitized wide-field and confocal fluorescent microscopy. Methods Mol. Biol. 319, 37–66.

    Article  PubMed  CAS  Google Scholar 

  54. Iwano, M., Shiba, H., Miwa, T., Che, F.S., Takayama, S., Nagai, T., Miyawaki, A., and Isogai, A. (2004) Ca2+ dynamics in a pollen grain and papilla cell during pollination of Arabidopsis. Plant Physiol. 136, 3562–3571.

    Article  PubMed  CAS  Google Scholar 

  55. Monshausen, G.B., Bibikova, T.N., Messerli, M.A., Shi, C., and Gilroy, S. (2007) Oscillations in extracellular pH and reactive oxygen species modulate tip growth of Arabidopsis root hairs. Proc. Natl. Acad. Sci. USA 104, 20996–21001.

    Article  PubMed  CAS  Google Scholar 

  56. Schulte, A., Lorenzen, I., Bottcher, M., and Plieth, C. (2006) A novel fluorescent pH probe for expression in plants. Plant Methods 2, 7.

    Article  PubMed  Google Scholar 

  57. Young, J.J., Mehta, S., Israelsson, M., Godoski, J., Grill, E., and Schroeder, J.I. (2006) CO(2) signaling in guard cells: calcium sensitivity response modulation, a Ca(2+)-independent phase, and CO(2) insensitivity of the gca2 mutant. Proc. Natl. Acad. Sci. USA 103, 7506–7511.

    Article  PubMed  CAS  Google Scholar 

  58. Oparka, K.J. and Roberts, A.G. (2001) Plasmodesmata. A not so open-and-shut case. Plant Physiol. 125, 123–126.

    Article  PubMed  CAS  Google Scholar 

  59. Zal, T. and Gascoigne, N.R. (2004) Photobleaching-corrected FRET efficiency imaging of live cells. Biophys. J. 86, 3923–3939.

    Article  PubMed  CAS  Google Scholar 

  60. Takanaga, H., Chaudhuri, B., and Frommer, W.B. (2008) GLUT1 and GLUT9 as major contributors to glucose influx in HepG2 cells identified by a high sensitivity intramolecular FRET glucose sensor. Biochim. Biophys. Acta. 1778, 1091–1099.

    Google Scholar 

  61. Zimmermann, T., Rietdorf, J., Girod, A., Georget, V., and Pepperkok, R. (2002) Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair. FEBS Lett. 531, 245–249.

    Article  PubMed  CAS  Google Scholar 

  62. Garaschuk, O., Griesbeck, O., and Konnerth, A. (2007) Troponin C-based biosensors: a new family of genetically encoded indicators for in vivo calcium imaging in the nervous system. Cell Calcium 42, 351–361.

    Article  PubMed  CAS  Google Scholar 

  63. Takanaga, H., Chaudhuri, B., and Frommer, W.B. (2008) GLUT1 and GLUT9 as major contributors to glucose influx in HepG2 cells identified by a high sensitivity intramolecular FRET glucose sensor. Biochim. Biophys. Acta 1778, 1091–1099.

    Article  PubMed  CAS  Google Scholar 

  64. Lager, I., Looger, L.L., Hilpert, M., Lalonde, S., and Frommer, W.B. (2006) Conversion of a putative Agrobacterium sugar-binding protein into a FRET sensor with high selectivity for sucrose. J. Biol. Chem. 281, 30875–30883.

    Article  PubMed  CAS  Google Scholar 

  65. Bogner, M. and Ludewig, U. (2007) Visualization of arginine influx into plant cells using a specific FRET-sensor. J. Fluoresc. 17, 350–360.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by grants to WBF from NIH (NIDDK, 1RO1DK079109-01) and DOE (DE-FG02-04ER15542). TN was supported in part by an HFSP fellowship.

Author information

Authors and Affiliations

  1. Department of Plant Biology, Carnegie Institution for Science, Stanford, CA, USA

    Totte Niittylae, Bhavna Chaudhuri & Wolf B. Frommer

  2. Institute for Molecular Systems Biology, ETH Zürich, Zürich, Switzerland

    Uwe Sauer

Authors
  1. Totte Niittylae
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bhavna Chaudhuri
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Uwe Sauer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wolf B. Frommer
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

    Rights and permissions

    Reprints and permissions

    Copyright information

    © 2009 Humana Press, a part of Springer Science+Business Media, LLC

    About this protocol

    Cite this protocol

    Niittylae, T., Chaudhuri, B., Sauer, U., Frommer, W.B. (2009). Comparison of Quantitative Metabolite Imaging Tools and Carbon-13 Techniques for Fluxomics. In: Belostotsky, D. (eds) Plant Systems Biology. Methods in Molecular Biology™, vol 553. Humana Press. https://doi.org/10.1007/978-1-60327-563-7_19

    Download citation

    • DOI: https://doi.org/10.1007/978-1-60327-563-7_19

    • Published:

    • Publisher Name: Humana Press

    • Print ISBN: 978-1-60327-562-0

    • Online ISBN: 978-1-60327-563-7

    • eBook Packages: Springer Protocols

    Publish with us

    Policies and ethics

    Search

    Navigation