Analyzing the In Vivo Status of Exogenously Applied Auxins: A HPLC-Based Method to Characterize the Intracellularly Localized Auxin Transporters

  • Sibu Simon
  • Petr Skůpa
  • Petre I. Dobrev
  • Jan Petrášek
  • Eva Zažímalová
  • Jiří Friml
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1056)

Abstract

Exogenous application of biologically important molecules for plant growth promotion and/or regulation is very common both in plant research and horticulture. Plant hormones such as auxins and cytokinins are classes of compounds which are often applied exogenously. Nevertheless, plants possess a well-established machinery to regulate the active pool of exogenously applied compounds by converting them to metabolites and conjugates. Consequently, it is often very useful to know the in vivo status of applied compounds to connect them with some of the regulatory events in plant developmental processes. The in vivo status of applied compounds can be measured by incubating plants with radiolabeled compounds, followed by extraction, purification, and HPLC metabolic profiling of plant extracts. Recently we have used this method to characterize the intracellularly localized PIN protein, PIN5. Here we explain the method in detail, with a focus on general application.

Key words

Auxin Auxin metabolism BY-2 tobacco cells PIN proteins HPLC Plant cell cultures 

Notes

Acknowledgements

This work was supported by grants from the Czech Ministry of Education (MSM0021622415), the Grant Agency of the Academy of Sciences of the Czech Republic (IAA601630703), and the Odysseus program of Research Foundation-Flanders (JF), Grant Agency of the Czech Republic, projects n. P305/11/0797 (JP), and P305/11/0797 (EZ). We thank Annick Bleys for critical reading of this chapter.

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Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Sibu Simon
    • 1
  • Petr Skůpa
    • 2
  • Petre I. Dobrev
    • 2
  • Jan Petrášek
    • 3
  • Eva Zažímalová
    • 3
  • Jiří Friml
    • 1
  1. 1.Institute of Science and Technology Austria (IST Austria)KlosterneuburgAustria
  2. 2.Institute of Experimental Botany, Academy of Sciences of the Czech RepublicPragueCzech Republic
  3. 3.Institute of Experimental Botany, ASCPragueCzech Republic

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