Determination of Auxin Transport Parameters on the Cellular Level

  • Jan Petrášek
  • Martina Laňková
  • Eva Zažímalová
Part of the Methods in Molecular Biology book series (MIMB, volume 1056)


The accumulation of radioactively labelled compounds in cells is frequently used for the determination of activities of various transport systems located at the plasma membrane, including the system for carrier-mediated transport of plant hormone auxin. The measurements of auxin transport could be performed on the tissue level as well, but for more precise quantitative analysis of activity of individual auxin carriers the model of plant cell cultures represents an invaluable tool. Here, we describe the method for the determination of the activities of auxin influx and efflux carriers in plant cells grown in a suspension using radiolabelled synthetic auxins 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (NAA). By making use of specific inhibitors of active auxin influx and efflux, as well as cell lines overexpressing or silencing particular auxin carriers, this method allows the determination of kinetic parameters of auxin flow across the plasma membrane and the activity of those carriers.

Key words

Auxin transport assay Plant cell cultures Auxin influx Auxin efflux Radiolabelled auxin 



This work was supported by the Czech Science Foundation, projects GAP305/11/2476 (JP) and GAP305/11/0797 (EZ).


  1. 1.
    Vanneste S, Friml J (2009) Auxin: a trigger for change in plant development. Cell 136:1005–1016PubMedCrossRefGoogle Scholar
  2. 2.
    Petrášek J, Friml J (2009) Auxin transport routes in plant development. Development 136:2675–2688PubMedCrossRefGoogle Scholar
  3. 3.
    Lewis DR, Muday GK (2009) Measurement of auxin transport in Arabidopsis thaliana. Nat Protoc 4:437–451PubMedCrossRefGoogle Scholar
  4. 4.
    Goldsmith MHM (1977) Polar transport of auxin. Annu Rev Plant Physiol Plant Mol Biol 28:439–478Google Scholar
  5. 5.
    Peer WA, Murphy AS (2007) Flavonoids and auxin transport: modulators or regulators? Trends Plant Sci 12:556–563PubMedCrossRefGoogle Scholar
  6. 6.
    Mancuso S, Marras AM, Magnus V, Baluska F (2005) Noninvasive and continuous recordings of auxin fluxes in intact root apex with a carbon nanotube-modified and self-referencing microelectrode. Anal Biochem 341:344–351PubMedCrossRefGoogle Scholar
  7. 7.
    Rubery PH, Sheldrake AR (1974) Carrier-mediated auxin transport. Planta 118: 101–121CrossRefGoogle Scholar
  8. 8.
    Nagata T, Nemoto Y, Hasezawa S (1992) Tobacco BY-2 cell-line as the Hela-cell in the cell biology of higher-plants. Int Rev Cytol 132:1–30Google Scholar
  9. 9.
    May M, Leaver C (1993) Oxidative stimulation of glutathione synthesis in Arabidopsis thaliana suspension cultures. Plant Physiol 103:621–627PubMedGoogle Scholar
  10. 10.
    Delbarre A, Muller P, Imhoff V, Guern J (1996) Comparison of mechanisms controlling uptake and accumulation of 2,4-dichlorophenoxy acetic acid, naphthalene-1-acetic acid, and indole-3-acetic acid in suspension-cultured tobacco cells. Planta 198:532–541CrossRefGoogle Scholar
  11. 11.
    Petrášek J, Elčkner M, Morris DA, Zažímalová E (2002) Auxin efflux carrier activity and auxin accumulation regulate cell division and polarity in tobacco cells. Planta 216:302–308PubMedCrossRefGoogle Scholar
  12. 12.
    Petrášek J, Zažímalová E (2006) The BY-2 cell line as a tool to study auxin transport. In: Biotechnology in Agriculture and Forestry. Tobacco BY-2 Cells: From Cellular Dynamics to Omics, Nagata, T., Matsuoka, K., Inzé, D. (eds.), Springer-Verlag, Berlin Heidelberg, 58:107–115Google Scholar
  13. 13.
    Petrášek J, Mravec J, Bouchard R, Blakeslee JJ, Abas M, Seifertová D et al (2006) PIN proteins perform a rate-limiting function in cellular auxin efflux. Science 312:914–918PubMedCrossRefGoogle Scholar
  14. 14.
    Parry G, Delbarre A, Marchant A, Swarup R, Napier R, Perrot-Rechenmann C et al (2001) Novel auxin transport inhibitors phenocopy the auxin influx carrier mutation aux1. Plant J 25:399–406PubMedCrossRefGoogle Scholar
  15. 15.
    Laňková M, Smith R, Pešek B, Kubeš M, Zažímalová E, Petrášek J et al (2010) Auxin influx inhibitors 1-NOA, 2-NOA, and CHPAA interfere with membrane dynamics in tobacco cells. J Exp Bot 61:3589–3598PubMedCrossRefGoogle Scholar
  16. 16.
    Rubery PH (1990) Phytotropins-receptors and endogenous ligands. Symp Soc Exp Biol 44:119–146PubMedGoogle Scholar
  17. 17.
    Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15: 473–497CrossRefGoogle Scholar
  18. 18.
    Petrášek J, Černá A, Schwarzerová K, Elčkner M, Morris DA, Zažímalová E (2003) Do phytotropins inhibit auxin efflux by impairing vesicle traffic? Plant Physiol 131:254–263PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Jan Petrášek
    • 1
  • Martina Laňková
    • 1
  • Eva Zažímalová
    • 1
  1. 1.Institute of Experimental Botany, ASCPragueCzech Republic

Personalised recommendations