Skip to main content
SpringerLink
Log in

Plant Metabolomics: From Experimental Design to Knowledge Extraction

  • Protocol
  • First Online:
Legume Genomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1069))

Abstract

Metabolomics is one of the most recent additions to the functional genomics approaches. It involves the use of analytical chemistry techniques to provide high-density data of metabolic profiles. Data is then analyzed using advanced statistics and databases to extract biological information, thus providing the metabolic phenotype of an organism. Large variety of metabolites produced by plants through the complex metabolic networks and their dynamic changes in response to various perturbations can be studied using metabolomics. Here, we describe the basic features of plant metabolic diversity and analytical methods to describe this diversity, which includes experimental workflows starting from experimental design, sample preparation, hardware and software choices, combined with knowledge extraction methods. Finally, we describe a scenario for using these workflows to identify differential metabolites and their pathways from complex biological samples.

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

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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

Similar content being viewed by others

References

  1. Hartmann T (2007) From waste products to ecochemicals: fifty years research of plant secondary metabolism. Phytochemistry 68:2831–2846

    Article  PubMed  CAS  Google Scholar 

  2. Vranova E, Coman D, Gruissem W (2012) Structure and dynamics of the isoprenoid pathway network. Mol Plant 5:318–333

    Article  PubMed  CAS  Google Scholar 

  3. Zhang P, Foerster H, Tissier CP, Mueller L, Paley S, Karp PD, Rhee SY (2005) MetaCyc and AraCyc. Metabolic pathway databases for plant research. Plant Physiol 138:27–37

    Article  PubMed  CAS  Google Scholar 

  4. Plant Metabolic Network. http://www.plantcyc.org/release_notes/content_statistics.faces. Accessed 02-Feb-2013

  5. Wink M (2010) Introduction: biochemistry, physiology and ecological functions of secondary metabolites. Annu Plant Rev 40: Biochemistry of Plant Secondary Metabolism

    Google Scholar 

  6. Rhodes M (1994) Physiological roles for secondary metabolites in plants: some progress, many outstanding problems. Plant Mol Biol 24:1–20

    Article  PubMed  CAS  Google Scholar 

  7. Sakakibara H (2006) Cytokinins: activity, biosynthesis, and translocation. Annu Rev Plant Biol 57:431–449

    Article  PubMed  CAS  Google Scholar 

  8. Fluck RA, Leber PA, Lieser JD, Szczerbicki SK, Varnes JG, Vitale MA, Wolfe EE (2000) Choline conjugates of auxins. I. Direct evidence for the hydrolysis of choline-auxin conjugates by pea cholinesterase. Plant Physiol Biochem 38:301–308

    Article  CAS  Google Scholar 

  9. Winkel-Shirley B (2001) Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol 126:485–493

    Article  PubMed  CAS  Google Scholar 

  10. Winkel-Shirley B (2002) Biosynthesis of flavonoids and effects of stress. Curr Opin Plant Biol 5:218–223

    Article  PubMed  CAS  Google Scholar 

  11. Winkel-Shirley B (2001) It takes a garden. How work on diverse plant species has contributed to an understanding of flavonoid metabolism. Plant Physiol 127:1399–1404

    Article  PubMed  CAS  Google Scholar 

  12. Narasimhan K, Basheer C, Bajic VB, Swarup S (2003) Enhancement of plant-microbe interactions using a rhizosphere metabolomics-driven approach and its application in the removal of polychlorinated biphenyls. Plant Physiol 132:146–153

    Article  PubMed  CAS  Google Scholar 

  13. Rasmussen S, Parsons AJ, Jones CS (2012) Metabolomics of forage plants: a review. Ann Bot 110:1281–1290

    Article  PubMed  CAS  Google Scholar 

  14. Reuben S, Bhinu VS, Swarup S (2008) Rhizosphere metabolomics: methods and applications. In: Karlovsky P (ed) Secondary metabolites in soil ecology. Springer, Berlin, pp 37–68

    Chapter  Google Scholar 

  15. Schenk PM, Kazan K, Wilson I, Anderson JP, Richmond T, Somerville SC, Manners JM (2000) Coordinated plant defense responses in Arabidopsis revealed by microarray analysis. Proc Natl Acad Sci U S A 97:11655–11660

    Article  PubMed  CAS  Google Scholar 

  16. Dalmolin RJ, Castro MA, Rybarczyk Filho JL, Souza LH, de Almeida RM, Moreira JC (2011) Evolutionary plasticity determination by orthologous groups distribution. Biol Direct 6:22

    Article  PubMed  Google Scholar 

  17. Hart Y, Mayo AE, Milo R, Alon U (2011) Robust control of PEP formation rate in the carbon fixation pathway of C(4) plants by a bi-functional enzyme. BMC Syst Biol 5:171

    Article  PubMed  CAS  Google Scholar 

  18. Angelovici R, Fait A, Zhu X, Szymanski J, Feldmesser E, Fernie AR, Galili G (2009) Deciphering transcriptional and metabolic networks associated with lysine metabolism during Arabidopsis seed development. Plant Physiol 151:2058–2072

    Article  PubMed  CAS  Google Scholar 

  19. Brown PD, Tokuhisa JG, Reichelt M, Gershenzon J (2003) Variation of glucosinolate accumulation among different organs and developmental stages of Arabidopsis thaliana. Phytochemistry 62:471–481

    Article  PubMed  CAS  Google Scholar 

  20. Desbrosses GG, Kopka J, Udvardi MK (2005) Lotus japonicus metabolic profiling. Development of gas chromatography-mass spectrometry resources for the study of plant-microbe interactions. Plant Physiol 137: 1302–1318

    Article  PubMed  CAS  Google Scholar 

  21. Bhalla R, Narasimhan K, Swarup S (2005) Metabolomics and its role in understanding cellular responses in plants. Plant Cell Rep 24:562–571

    Article  PubMed  CAS  Google Scholar 

  22. Alisdair RF, Richard NT, Arno JK, Lothar W (2004) Innovation: metabolite profiling: from diagnostics to systems biology. Nat Rev Mol Cell Biol 5

    Google Scholar 

  23. De Vos R, Moco S, Lommen A, Keurentjes J, Bino R, Hall R (2007) Untargeted large-scale plant metabolomics using liquid chromatography coupled to mass spectrometry. Nat Protoc 2:778–791

    Article  PubMed  Google Scholar 

  24. Hall R (2006) Plant metabolomics: from holistic hope, to hype, to hot topic. New Phytol 169:453–468

    Article  PubMed  CAS  Google Scholar 

  25. Glinski M, Weckwerth W (2006) The role of mass spectrometry in plant systems biology. Mass Spectrom Rev 25:173–214

    Article  PubMed  CAS  Google Scholar 

  26. Patti G (2011) Separation strategies for untargeted metabolomics. J Sep Sci 34:3460–3469

    Article  PubMed  CAS  Google Scholar 

  27. Werner E, Croixmarie V, Umbdenstock T, Ezan E, Chaminade P, Tabet JC, Junot C (2008) Mass spectrometry-based metabolomics: accelerating the characterization of discriminating signals by combining statistical correlations and ultrahigh resolution. Anal Chem 80:4918–4932

    Article  PubMed  CAS  Google Scholar 

  28. Kueger S, Steinhauser D, Willmitzer L, Giavalisco P (2012) High-resolution plant metabolomics: from mass spectral features to metabolites and from whole-cell analysis to subcellular metabolite distributions. Plant J 70:39–50

    Article  PubMed  CAS  Google Scholar 

  29. Kim HK, Choi YH, Verpoorte R (2011) NMR-based plant metabolomics: where do we stand, where do we go? Trends Biotechnol 29:267–275

    Article  PubMed  CAS  Google Scholar 

  30. Dunn WB, Bailey NJ, Johnson HE (2005) Measuring the metabolome: current analytical technologies. Analyst 130:606–625

    Article  PubMed  CAS  Google Scholar 

  31. Morita A, Horie H, Fujii Y, Takatsu S, Watanabe N, Yagi A, Yokota H (2004) Chemical forms of aluminum in xylem sap of tea plants (Camellia sinensis L.). Phytochemistry 65:2775–2780

    Article  PubMed  CAS  Google Scholar 

  32. Veenstra T (2012) Metabolomics: the final frontier? Genome Med 4:40

    Article  PubMed  Google Scholar 

  33. David SW (2008) Quantitative metabolomics using NMR. TrAC Trends Anal Chem 27

    Google Scholar 

  34. Bedair M, Sumner LW (2008) Current and emerging mass-spectrometry technologies for metabolomics. Trac-Trend Anal Chem 27:238–250

    Article  CAS  Google Scholar 

  35. Dettmer K, Aronov P, Hammock B (2007) Mass spectrometry-based metabolomics. Mass Spectrom Rev 26:51–78

    Article  PubMed  CAS  Google Scholar 

  36. Parab GS, Rao R, Lakshminarayanan S, Bing YV, Moochhala SM, Swarup S (2009) Data-driven optimization of metabolomics methods using rat liver samples. Anal Chem 81:1315–1323

    Article  PubMed  CAS  Google Scholar 

  37. Broeckling CD, Huhman DV, Farag MA, Smith JT, May GD, Mendes P, Dixon RA, Sumner LW (2005) Metabolic profiling of Medicago truncatula cell cultures reveals the effects of biotic and abiotic elicitors on metabolism. J Exp Bot 56:323–336

    Article  PubMed  CAS  Google Scholar 

  38. Hall R, Beale M, Fiehn O, Hardy N, Sumner L, Bino R (2002) Plant metabolomics: the missing link in functional genomics strategies. Plant Cell 14:1437–1440

    Article  PubMed  CAS  Google Scholar 

  39. Bleeker PM, Diergaarde PJ, Ament K, Guerra J, Weidner M, Schutz S, de Both MT, Haring MA, Schuurink RC (2009) The role of specific tomato volatiles in tomato-whitefly interaction. Plant Physiol 151:925–935

    Article  PubMed  CAS  Google Scholar 

  40. Mayer F, Takeoka GR, Buttery RG, Whitehand LC, Naim M, Rabinowitch HD (2008) Studies on the aroma of five fresh tomato cultivars and the precursors of cis- and trans-4,5-epoxy-(E)-2-decenals and methional. J Agric Food Chem 56:3749–3757

    Article  PubMed  CAS  Google Scholar 

  41. Shuman JL, Cortes DF, Armenta JM, Pokrzywa RM, Mendes P, Shulaev V (2011) Plant metabolomics by GC-MS and differential analysis. Methods Mol Biol 678:229–246

    Article  PubMed  CAS  Google Scholar 

  42. Allwood J, Goodacre R (2010) An introduction to liquid chromatography-mass spectrometry instrumentation applied in plant metabolomic analyses. Phytochem Anal 21:33–47

    Article  PubMed  CAS  Google Scholar 

  43. Nordstrom A, Want E, Northen T, Lehtio J, Siuzdak G (2008) Multiple ionization mass spectrometry strategy used to reveal the complexity of metabolomics. Anal Chem 80:421–429

    Article  PubMed  Google Scholar 

  44. Bin Z, Jun Feng X, Leepika T, Habtom WR (2012) LC-MS-based metabolomics. Mol Biosyst 8

    Google Scholar 

  45. Obata T, Fernie A (2012) The use of metabolomics to dissect plant responses to abiotic stresses. Cell Mol Life Sci 69:3225–3243

    Article  PubMed  CAS  Google Scholar 

  46. Lei Z, Huhman D, Sumner L (2011) Mass spectrometry strategies in metabolomics. J Biol Chem 286:25435–25442

    Article  PubMed  CAS  Google Scholar 

  47. Gibon Y, Rolin D (2012) Aspects of experimental design for plant metabolomics experiments and guidelines for growth of plant material. Methods Mol Biol (Clifton, NJ) 860: 13–30

    Article  CAS  Google Scholar 

  48. Leek JT, Scharpf RB, Bravo HC, Simcha D, Langmead B, Johnson WE, Geman D, Baggerly K, Irizarry RA (2010) Tackling the widespread and critical impact of batch effects in high-throughput data. Nat Rev Genet 11:733–739

    Article  PubMed  CAS  Google Scholar 

  49. Fiehn O, Wohlgemuth G, Scholz M, Kind T, Lee do Y, Lu Y, Moon S, Nikolau B (2008) Quality control for plant metabolomics: reporting MSI-compliant studies. Plant J 53:691–704

    Article  PubMed  CAS  Google Scholar 

  50. Members MSIB, Sansone SA, Fan T, Goodacre R, Griffin JL, Hardy NW, Kaddurah-Daouk R, Kristal BS, Lindon J, Mendes P, Morrison N, Nikolau B, Robertson D, Sumner LW, Taylor C, van der Werf M, van Ommen B, Fiehn O (2007) The metabolomics standards initiative. Nat Biotechnol 25:846–848

    Google Scholar 

  51. Boccard J, Veuthey JL, Rudaz S (2010) Knowledge discovery in metabolomics: an overview of MS data handling. J Sep Sci 33:290–304

    Article  PubMed  CAS  Google Scholar 

  52. Hendriks MMWB, van Eeuwijk FA, Jellema RH, Westerhuis JA, Reijmers TH, Hoefsloot HCJ, Smilde AK (2011) Data-processing strategies for metabolomics studies. Trac-Trend Anal Chem 30:1685–1698

    Article  CAS  Google Scholar 

  53. Eliasson M, Rannar S, Trygg J (2011) From data processing to multivariate validation–essential steps in extracting interpretable information from metabolomics data. Curr Pharm Biotechnol 12:996–1004

    Article  PubMed  CAS  Google Scholar 

  54. Biswas A, Mynampati KC, Umashankar S, Reuben S, Parab G, Rao R, Kannan VS, Swarup S (2010) MetDAT: a modular and workflow-based free online pipeline for mass spectrometry data processing, analysis and interpretation. Bioinformatics 26:2639–2640

    Article  PubMed  CAS  Google Scholar 

  55. Smith CA, Want EJ, O'Maille G, Abagyan R, Siuzdak G (2006) XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Anal Chem 78:779–787

    Article  PubMed  CAS  Google Scholar 

  56. Pluskal T, Castillo S, Villar-Briones A, Oresic M (2010) MZmine 2: modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data. BMC Bioinformatics 11:395

    Article  PubMed  Google Scholar 

  57. Xia J, Mandal R, Sinelnikov IV, Broadhurst D, Wishart DS (2012) MetaboAnalyst 2.0—a comprehensive server for metabolomic data analysis. Nucleic Acids Res 40:W127–W133

    Article  PubMed  CAS  Google Scholar 

  58. Hegeman A (2010) Plant metabolomics—meeting the analytical challenges of comprehensive metabolite analysis. Brief Funct Genomics 9:139–148

    Article  PubMed  CAS  Google Scholar 

  59. Fan TW-M (2012) Considerations of sample preparation for metabolomics investigation. In: Fan TW-M, Lane AN, Higashi RM (eds) The handbook of metabolomics, vol 17, Methods in pharmacology and toxicology. Humana, Totowa, NJ, pp 7–27

    Chapter  Google Scholar 

  60. American Society for Mass Spectrometry (2009) Metabolomics ASMS Workshop Survey 2009. http://fiehnlab.ucdavis.edu/staff/kind/Metabolomics-Survey-2009

  61. Kind T, Fiehn O (2007) Seven Golden Rules for heuristic filtering of molecular formulas obtained by accurate mass spectrometry. BMC Bioinformatics 8:105

    Article  PubMed  Google Scholar 

  62. Biswas A, Rao R, Umashankar S, Mynampati KC, Reuben S, Parab G, Swarup S (2011) datPAV—an online processing, analysis and visualization tool for exploratory investigation of experimental data. Bioinformatics 27:1585–1586

    Article  PubMed  CAS  Google Scholar 

  63. Kanehisa M, Goto S (2000) KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 28:27–30

    Article  PubMed  CAS  Google Scholar 

  64. Kanehisa M, Goto S, Sato Y, Furumichi M, Tanabe M (2012) KEGG for integration and interpretation of large-scale molecular data sets. Nucleic Acids Res 40:D109–D114

    Article  PubMed  CAS  Google Scholar 

  65. Okuda S, Yamada T, Hamajima M, Itoh M, Katayama T, Bork P, Goto S, Kanehisa M (2008) KEGG Atlas mapping for global analysis of metabolic pathways. Nucleic Acids Res 36:W423–W426

    Article  PubMed  CAS  Google Scholar 

  66. Sana TR, Roark JC, Li X, Waddell K, Fischer SM (2008) Molecular formula and METLIN Personal Metabolite Database matching applied to the identification of compounds generated by LC/TOF-MS. J Biomol Technol 19:258–266

    Google Scholar 

  67. ChemSpider. http://www.chemspider.com/

  68. Horai H, Arita M, Kanaya S, Nihei Y, Ikeda T, Suwa K, Ojima Y, Tanaka K, Tanaka S, Aoshima K, Oda Y, Kakazu Y, Kusano M, Tohge T, Matsuda F, Sawada Y, Hirai MY, Nakanishi H, Ikeda K, Akimoto N, Maoka T, Takahashi H, Ara T, Sakurai N, Suzuki H, Shibata D, Neumann S, Iida T, Tanaka K, Funatsu K, Matsuura F, Soga T, Taguchi R, Saito K, Nishioka T (2010) MassBank: a public repository for sharing mass spectral data for life sciences. J Mass Spectrom 45:703–714

    Article  PubMed  CAS  Google Scholar 

  69. Kessner D, Chambers M, Burke R, Agus D, Mallick P (2008) ProteoWizard: open source software for rapid proteomics tools development. Bioinformatics 24:2534–2536

    Article  PubMed  CAS  Google Scholar 

  70. R Core Team (2012) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria

    Google Scholar 

  71. Sugimoto M, Kawakami M, Robert M, Soga T, Tomita M (2012) Bioinformatics tools for mass spectroscopy-based metabolomic data processing and analysis. Curr Bioinformatics 7:96–108

    Article  CAS  Google Scholar 

  72. Mochida K, Shinozaki K (2010) Genomics and bioinformatics resources for crop improvement. Plant Cell Physiol 51:497–523

    Article  PubMed  CAS  Google Scholar 

  73. Chagoyen M, Pazos F (2012) Tools for the functional interpretation of metabolomic experiments. Brief Bioinformatics. doi: 10.1093 /bib/bbs055

    Google Scholar 

  74. Tokimatsu T, Sakurai N, Suzuki H, Ohta H, Nishitani K, Koyama T, Umezawa T, Misawa N, Saito K, Shibata D (2005) KaPPA-view: a web-based analysis tool for integration of transcript and metabolite data on plant metabolic pathway maps. Plant Physiol 138:1289–1300

    Article  PubMed  CAS  Google Scholar 

  75. Junker BH, Klukas C, Schreiber F (2006) VANTED: a system for advanced data analysis and visualization in the context of biological networks. BMC Bioinformatics 7:109

    Article  PubMed  Google Scholar 

  76. Letunic I, Yamada T, Kanehisa M, Bork P (2008) iPath: interactive exploration of biochemical pathways and networks. Trends Biochem Sci 33:101–103

    Article  PubMed  CAS  Google Scholar 

  77. Xia J, Wishart DS (2010) MetPA: a web-based metabolomics tool for pathway analysis and visualization. Bioinformatics 26:2342–2344

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We acknowledge the financial support from the Singapore-Peking-Oxford Research Enterprise, COY-15-EWI-RCFSA/N197-1. We gratefully acknowledge Agilent Technologies, Singapore for their support in acquiring and analyzing the mass spectrometry data for the differential analysis of metabolites.

Author information

Authors and Affiliations

  1. Metabolites Biology Lab, Department of Biological Sciences, National University of Singapore, Singapore, Singapore

    Amit Rai

  2. Metabolites Biology Lab, Department of Biological Sciences, NUS Environmental Research Institute, National University of Singapore, Singapore, Singapore

    Shivshankar Umashankar

  3. Metabolites Biology Lab, Department of Biological Sciences, NUS Environmental Research Institute, Singapore Centre for Environmental Life Sciences Engineering, National University of Singapore, Nanyang Technological University, Singapore, Singapore

    Sanjay Swarup

Authors
  1. Amit Rai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shivshankar Umashankar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sanjay Swarup
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. CILR, The University of Newcastle Environmental & Life Sciences, Callaghan, New South Wales, Australia

    Ray J. Rose

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Rai, A., Umashankar, S., Swarup, S. (2013). Plant Metabolomics: From Experimental Design to Knowledge Extraction. In: Rose, R. (eds) Legume Genomics. Methods in Molecular Biology, vol 1069. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-613-9_19

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-613-9_19

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-612-2

  • Online ISBN: 978-1-62703-613-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation