Skip to main content

Comprehensive Metabolomics Studies of Plant Developmental Senescence

  • Protocol
  • First Online:
Plant Senescence

Abstract

Leaf senescence is an essential developmental process that involves diverse metabolic changes associated with degradation of macromolecules allowing nutrient recycling and remobilization. In contrast to the significant progress in transcriptomic analysis of leaf senescence, metabolomics analyses have been relatively limited. A broad overview of metabolic changes during leaf senescence including the interactions between various metabolic pathways is required to gain a better understanding of the leaf senescence allowing to link transcriptomics with metabolomics and physiology. In this chapter, we describe how to obtain comprehensive metabolite profiles and how to dissect metabolic shifts during leaf senescence in the model plant Arabidopsis thaliana. Unlike nucleic acid analysis for transcriptomics, a comprehensive metabolite profile can only be achieved by combining a suite of analytic tools. Here, information is provided for measurements of the contents of chlorophyll, soluble proteins, and starch by spectrophotometric methods, ions by ion chromatography, thiols and amino acids by HPLC, primary metabolites by GC/TOF-MS, and secondary metabolites and lipophilic metabolites by LC/ESI-MS. These metabolite profiles provide a rich catalogue of metabolic changes during leaf senescence, which is a helpful database and blueprint to be correlated to future studies such as transcriptome and proteome analyses, forward and reverse genetic studies, or stress-induced senescence studies.

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

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 79.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 99.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 129.99
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. Noodén L (1980) Senescence in whole plants. In: Thimann KV (ed) Senescence in plants. CRC Press, Boca Raton, FL, pp 219–258

    Google Scholar 

  2. Yoshida S (2003) Molecular regulation of leaf senescence. Curr Opin Plant Biol 6:79–84

    Article  CAS  PubMed  Google Scholar 

  3. van Doorn WG (2004) Is petal senescence due to sugar starvation? Plant Physiol 134:35–42

    Article  PubMed  PubMed Central  Google Scholar 

  4. Yang ZL, Ohlrogge JB (2009) Turnover of fatty acids during natural senescence of Arabidopsis, brachypodium, and switchgrass and in Arabidopsis beta-oxidation mutants. Plant Physiol 150:1981–1989

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Seltmann MA, Stingl NE, Lautenschlaeger JK et al (2010) Differential impact of lipoxygenase 2 and jasmonates on natural and stress-induced senescence in Arabidopsis. Plant Physiol 152:1940–1950

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Masclaux C, Valadier MH, Brugiere N et al (2000) Characterization of the sink/source transition in tobacco (Nicotiana tabacum L.) shoots in relation to nitrogen management and leaf senescence. Planta 211:510–518

    Article  CAS  PubMed  Google Scholar 

  7. Quirino BF, Reiter WD, Amasino RD (2001) One of two tandem Arabidopsis genes homologous to monosaccharide transporters is senescence-associated. Plant Mol Biol 46:447–457

    Article  CAS  PubMed  Google Scholar 

  8. Stessman D, Miller A, Spalding M et al (2002) Regulation of photosynthesis during Arabidopsis leaf development in continuous light. Photosynth Res 72:27–37

    Article  CAS  PubMed  Google Scholar 

  9. Diaz C, Purdy S, Christ A et al (2005) Characterization of markers to determine the extent and variability of leaf senescence in Arabidopsis. A metabolic profiling approach. Plant Physiol 138:898–908

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Masclaux-Daubresse C, Carrayol E, Valadier MH (2005) The two nitrogen mobilisation- and senescence-associated GS1 and GDH genes are controlled by C and N metabolites. Planta 221:580–588

    Article  CAS  PubMed  Google Scholar 

  11. Masclaux-Daubresse C, Purdy S, Lemaitre T et al (2007) Genetic variation suggests interaction between cold acclimation and metabolic regulation of leaf senescence. Plant Physiol 143:434–446

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Pourtau N, Jennings R, Pelzer E et al (2006) Effect of sugar-induced senescence on gene expression and implications for the regulation of senescence in Arabidopsis. Planta 224:556–568

    Article  CAS  PubMed  Google Scholar 

  13. Wingler A, Purdy S, MacLean JA et al (2006) The role of sugars in integrating environmental signals during the regulation of leaf senescence. J Exp Bot 57:391–399

    Article  CAS  PubMed  Google Scholar 

  14. Wingler A, Delatte TL, O'Hara LE et al (2012) Trehalose 6-phosphate is required for the onset of leaf senescence associated with high carbon availability. Plant Physiol 158:1241–1251

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Watanabe M, Balazadeh S, Tohge T et al (2013) Comprehensive dissection of spatiotemporal metabolic shifts in primary, secondary, and lipid metabolism during developmental senescence in Arabidopsis. Plant Physiol 162:1290–1310

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Giavalisco P, Li Y, Matthes A et al (2011) Elemental formula annotation of polar and lipophilic metabolites using C-13, N-15 and S-34 isotope labelling, in combination with high- resolution mass spectrometry. Plant J 68:364–376

    Article  CAS  PubMed  Google Scholar 

  17. Hummel J, Segu S, Li Y et al (2011) Ultra performance liquid chromatography and high resolution mass spectrometry for the analysis of plant lipids. Front Plant Sci 2:54

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Peoples MB, Dalling MJ (1978) Degradation of ribulose 1,5-bisphosphate carboxylase by proteolytic enzymes from crude extracts of wheat leaves. Planta 138:153–160

    Article  CAS  PubMed  Google Scholar 

  19. Bradford MM (1976) rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  20. Stitt M, Lilley RMC, Gerhardt R et al (1989) Determination of metabolite levels in specific cells and subcellular compartments of plant leaves. Methods Enzymol 174:518–552

    Article  CAS  Google Scholar 

  21. Gibon Y, Blaesing OE, Hannemann J et al (2004) A robot-based platform to measure multiple enzyme activities in Arabidopsis using a set of cycling assays: Comparison of changes of enzyme activities and transcript levels during diurnal cycles and in prolonged darkness. Plant Cell 16:3304–3325

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Noctor G, Foyer CH (1998) Ascorbate and glutathione: keeping active oxygen under control. Annu Rev Plant Physiol Plant Mol Biol 49:249–279

    Article  CAS  PubMed  Google Scholar 

  23. Zagorchev L, Seal CE, Kranner I et al (2013) A central role for thiols in plant tolerance to abiotic stress. Int J Mol Sci 14:7405–7432

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Anderson ME (1985) Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol 113:548–555

    Article  CAS  PubMed  Google Scholar 

  25. Fahey RC, Newton GL (1987) Determination of low-molecular-weight thiols using monobromobimane fluorescent labeling and high-performance liquid-chromatography. Methods Enzymol 143:85–96

    Article  CAS  PubMed  Google Scholar 

  26. Saito K, Kurosawa M, Tatsuguchi K et al (1994) Modulation of cysteine biosynthesis in chloroplasts of transgenic tobacco overexpressing cysteine synthase [O-acetylserine(thiol)-lyase]. Plant Physiol 106:887–895

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Lindroth P, Mopper K (1979) High-performance liquid-chromatographic determination of subpicomole amounts of amino-acids by precolumn fluorescence derivatization with ortho-phthaldialdehyde. Anal Chem 51:1667–1674

    Article  CAS  Google Scholar 

  28. Kim H, Awazuhara M, Hayashi H et al (1997) Analysis of O-acetyl-L-serine in in vitro cultured soybean cotyledons. In: Cram WJ, De Kok LJ, Stulen I et al (eds) Sulphur metabolism in higher plants: molecular, ecophysiological and nutrition aspects. Backhuys Publishers, Leiden, pp 307–309

    Google Scholar 

  29. Lisec J, Schauer N, Kopka J et al (2006) Gas chromatography mass spectrometry-based metabolite profiling in plants. Nat Protoc 1:387–396

    Article  CAS  PubMed  Google Scholar 

  30. Erban A, Schauer N, Fernie AR et al (2007) Nonsupervised construction and application of mass spectral and retention time index libraries from time-of-flight gas chromatography-mass spectrometry metabolite profiles. In: Weckwerth W (ed) Methods in molecular biology. Humana Press, Totowa, NJ, pp 19–38

    Google Scholar 

  31. Strehmel N, Hummel J, Erban A et al (2008) Retention index thresholds for compound matching in GC-MS metabolite profiling. J Chromatogr B Analyt Technol Biomed Life Sci 871:182–190

    Article  CAS  PubMed  Google Scholar 

  32. Allwood JW, Erban A, de Koning S et al (2009) Inter-laboratory reproducibility of fast gas chromatography-electron impact-time of flight mass spectrometry (GC-EI-TOF/MS) based plant metabolomics. Metabolomics 5:479–496

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Sanchez DH, Szymanski J, Erban A et al (2010) Mining for robust transcriptional and metabolic responses to long-term salt stress: a case study on the model legume Lotus japonicus. Plant Cell Environ 33:468–480

    Article  CAS  PubMed  Google Scholar 

  34. 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

    Article  CAS  PubMed  Google Scholar 

  35. Luedemann A, von 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

    Article  CAS  PubMed  Google Scholar 

  36. Dettmer K, Aronov PA, Hammock BD (2007) Mass spectrometry-based metabolomics. Mass Spectrom Rev 26:51–78

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Tohge T, Fernie AR (2010) Combining genetic diversity, informatics and metabolomics to facilitate annotation of plant gene function. Nat Protoc 5:1210–1227

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

B. M.-R. and S.B. thank the Deutsche Forschungsgemeinschaft (DFG) for funding (FOR 948; MU 1199/14-1 and 14-2, and BA4769/1-2). We thank the Max Planck Society (MPG) for funding, the Max Planck Institute of Molecular Plant Physiology (MPI-MP) for providing metabolomics and bioinformatics services, and the greenteam of the institute for growing plants.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Rainer Hoefgen .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Watanabe, M. et al. (2018). Comprehensive Metabolomics Studies of Plant Developmental Senescence. In: Guo, Y. (eds) Plant Senescence. Methods in Molecular Biology, vol 1744. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7672-0_28

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7672-0_28

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7670-6

  • Online ISBN: 978-1-4939-7672-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics