Russian Journal of Plant Physiology

, Volume 60, Issue 3, pp 431–436 | Cite as

Quantitative analysis of IAA in DR5::GUS transgenic arabidopsis plants

  • G. A. Pozhvanov
  • A. L. Shavarda
  • S. S. Medvedev
Methods

Abstract

In the study of auxin transport, transgenic constructs, including DR5::GUS, are widely used for visualization of phytohormone localization. Previously we proposed a method for quantitative evaluation of the IAA content by histochemical staining for glucuronidase activity. In this work, this method was complemented by quantitative data on the content of IAA in plants obtained by gas chromatography-mass spectrometry (GC/MS), which allowed more accurate characterization of the lateral IAA gradient arising at the Arabidopsis thaliana (L.) Heynh (ecotype Columbia 0) root gravistimulation. Applied method of IAA analysis, combining GC/MS and histochemistry, can be used for quantitatification of the other plant hormone distribution in transgenic plants with the GUS reporter.

Keywords

Arabidopsis thaliana IAA redistribution GUS reporter gas chromatography mass-spectrometry digital image analysis 

Abbreviations

GC/MS

gas chromatography with mass-selective detection

GUS

reporter enzyme β-D-glucuronidase from Escherichia coli

RGB

color model where the combination of red, green, and blue color (R, G, B) produces the final color of digital image pixel

TMS

trimethylsilyl fragment (Si(CH3)3)

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References

  1. 1.
    Medvedev, S.S., Mechanisms and Physiological Role of Polarity in Plants, Russ. J. Plant Physiol., 2012, vol. 59, pp. 502–514.CrossRefGoogle Scholar
  2. 2.
    Peer, W.A., Blakeslee, J.J., Yang, H., and Murphy, A.S., Seven Things We Think We Know about Auxin Transport, Mol. Plant, 2011, vol. 4, pp. 487–504.PubMedCrossRefGoogle Scholar
  3. 3.
    Nick, P., Han, M.J., and An, G., Auxin Stimulates Its Own Transport by Shaping Actin Filaments, Plant Physiol., 2009, vol. 151, pp. 155–167.PubMedCrossRefGoogle Scholar
  4. 4.
    Pozhvanov, G., Suslov, D., and Medvedev, S., Reorganization of Actin Cytoskeleton after Gravistimulation of Arabidopsis Roots, Tr. Tomskogo Gos. Univ., 2010, vol. 275, pp. 305–308.Google Scholar
  5. 5.
    Ulmasov, T., Murfett, J., Hagen, G., and Guilfoyle, T.J., Aux/IAA Proteins Repress Expression of Reporter Genes Containing Natural and Highly Active Synthetic Auxin Response Elements, Plant Cell, 1997, vol. 9, pp. 1963–1971.PubMedGoogle Scholar
  6. 6.
    Brunoud, G., Wells, D.M., Oliva, M., Larrieu, A., Mirabet, V., Burrow, A.H., Beeckman, T., Kepinski, S., Traas, J., Bennett, M.J., and Vernoux, T., A Novel Sensor to Map Auxin Response and Distribution at High Spatio-Temporal Resolution, Nature, 2012, vol. 482, pp. 103–106.PubMedCrossRefGoogle Scholar
  7. 7.
    Lewis, D. and Muday, G., Measurement of Auxin Transport in Arabidopsis thaliana, Nat. Protocols, 2009, vol. 4, pp. 437–451.CrossRefGoogle Scholar
  8. 8.
    Ribnicky, D., Cooke, T., and Cohen, J., A Microtechnique for the Analysis of Free and Conjugated Indole-3-Acetic Acid in Milligram Amounts of Plant Tissue Using a Benchtop Gas Chromatograph-Mass Spectrometer, Planta, 1997, vol. 204, pp. 1–7.CrossRefGoogle Scholar
  9. 9.
    Ljung, K., Bhalerao, R.P., and Sandberg, G., Sites and Homeostatic Control of Auxin Biosynthesis in Arabidopsis during Vegetative Growth, Plant J., 2001, vol. 28, pp. 465–474.PubMedCrossRefGoogle Scholar
  10. 10.
    Pozhvanov, G.A. and Medvedev, S.S., Auxin Quantification Based on Histochemical Staining of GUS under the Control of Auxin-Responsive Promoter, Russ. J. Plant Physiol., 2008, vol. 55, pp. 706–711.CrossRefGoogle Scholar
  11. 11.
    Kopka, J., Schauer, N., Krueger, S., Birkemeyer, C., Usadel, B., Bergmüller, E., Dörmann, P., Weckwerth, W., Gibon, Y., Stitt, M., Willmitzer, L., Fernie, A.R., and Steinhauser, D., GMD@CSB.DB: The Golm Metabolome Database, Bioinformatics, 2005, vol. 21, pp. 1635–1638, http://bioinformatics.oxfordjournals.org/content/21/8/1635.full PubMedCrossRefGoogle Scholar
  12. 12.
    Roessner, U. and Bowne, J., What Is Metabolomics All about? BioTechniques, 2009, vol. 46, pp. 363–365.PubMedCrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2013

Authors and Affiliations

  • G. A. Pozhvanov
    • 1
    • 2
  • A. L. Shavarda
    • 1
    • 2
  • S. S. Medvedev
    • 1
  1. 1.Department of Plant Physiology and BiochemistrySt. Petersburg State UniversitySt. PetersburgRussia
  2. 2.Komarov Institute of BotanyRussian Academy of SciencesSt. PetersburgRussia

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