Phototropism pp 165-172 | Cite as

Immunolocalization of IAA Using an Anti-IAA-C-Antibody Raised Against Carboxyl-Linked IAA

  • Takeshi NishimuraEmail author
  • Tomokazu Koshiba
Part of the Methods in Molecular Biology book series (MIMB, volume 1924)


Plant hormone indole-3-acetic acid (IAA) plays a crucial role in plant physiological events such as plant development, differentiation, and environmental responses. IAA is synthesized in specific focal cells and/or tissues such as the coleoptile tip in maize and the root tip and young leaf primordia in Arabidopsis thaliana. Recent studies have shown that formation of an IAA maxima or concentration gradient, created by the changing expression and cellular localization of IAA transport proteins, crucially controls plant physiological events. For this reason, visualization of IAA molecules at the cell and tissue levels is necessary to accurately determine the distribution of IAA in plants. Immunolocalization of IAA is a means to directly visualize IAA and observe its localization and distribution in plant cells and tissues. Here, we introduce an immunolocalization protocol to observe IAA distribution that uses a specific anti-IAA-C-antibody raised against carboxyl-linked IAA. This method is applicable for various plant samples and is reliable for specifically detecting IAA in plant tissues.

Key words

Arabidopsis 1-Ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride (EDAC) Indole-3-acetic acid (IAA) IAA-C-antibody IAA-N1-antibody Maize 


  1. 1.
    Petrasek J, Friml J (2009) Auxin transport routes in plant development. Development 136:2675–2688CrossRefGoogle Scholar
  2. 2.
    Linh NM, Verna C, Scarpella E (2017) Coordination of cell polarity and the patterning of leaf vein networks. Curr Opin Plant Biol 41:116–124CrossRefGoogle Scholar
  3. 3.
    Ulmasov T, Murfett J, Hagen G, Guilfoyle TJ (1997) Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements. Plant Cell 9:1963–1971CrossRefGoogle Scholar
  4. 4.
    Marchant A, Bhalerao R, Casimiro I, Kklof J, Casero PJ, Bennett M, Sandberg G (2002) AUX1 promotes lateral root formation by facilitating indole-3-acetic acid distribution between sink and source tissues in the Arabidopsis seedling. Plant Cell 14:589–597CrossRefGoogle Scholar
  5. 5.
    Brunoud G, Wells DM, Oliva M, Larrieu A, Mirabet V, Burrow AH et al (2012) A novel sensor to map auxin response and distribution at high spatio-temporal resolution. Nature 482:103–106CrossRefGoogle Scholar
  6. 6.
    Nakamura A, Higuchi K, Goda H, Fujiwara MT, Sawa S, Koshiba T et al (2003) Brassinolide induces IAA5, IAA19, and DR5, a synthetic auxin response element in Arabidopsis, implying a cross talk point of brassinosteroid and auxin signaling. Plant Physiol 133:1843–1853CrossRefGoogle Scholar
  7. 7.
    Nishimura T, Toyooka K, Sato M, Matsumoto S, Lucas MM, Strnad M, Baluska F, Koshiba T (2011) Immunohistochemical observation of indole-3-acetic acid at the IAA synthetic maize coleoptile tip. Plant Signal Behav 6:2013–2022CrossRefGoogle Scholar
  8. 8.
    Schlicht M, Strnad M, Scanlon MJ, Mancuso S, Hochholdinger F, Palme K et al (2006) Auxin immunolocalization implicates vesicular neurotransmitter-like model of polar auxin transport in root apices. Plant Signal Behav 1:122–133CrossRefGoogle Scholar
  9. 9.
    Shi L, Miller I, Moore R (1993) Immunocytochemical localization of indole-3-acetic acid in primary root of Zea mays. Plant Cell Environ 16:967–973CrossRefGoogle Scholar
  10. 10.
    Moctezuma E (1999) Change in auxin patterns in developing gynophores of the peanut plant (Arachis hypogaea L.). Ann Bot 83:235–242CrossRefGoogle Scholar
  11. 11.
    Fedorova E, Redondo FJ, Koshiba T, Pueyo JJ, de Felipe MR, Lucas MM (2005) Aldehyde oxidase (AO) in the root nodules of Lupinus albus and Medicago truncatula: identification of AO in meristematic and infection zones. Mol Plant-Microbe Interact 18:405–413CrossRefGoogle Scholar
  12. 12.
    Aloni R, Schwalm K, Langhans M, Ullrich CI (2003) Gradual shifts in sites of free-auxin production during leaf-primordium development and their role in vascular differentiation and leaf morphogenesis in Arabidopsis. Planta 216:841–853Google Scholar
  13. 13.
    Rodriguez-Sanz H, Manzanera JA, Solis MT, Gomez-Garay A, Pintos B, Risueno MC, Testillano PS (2014) Early markers are present in both embryogenesis pathway from microspores and immature zygotic embryos in cork oak, Quercus suber L. BMC Plant Biol 14:224CrossRefGoogle Scholar
  14. 14.
    Livanos P, Galatis B, Apostolakos P (2016) Deliberate ROS production and auxin synergistically trigger the asymmetrical division generating the subsidiary cells in Zea mays stomatal complexes. Protoplasma 253:1081–1099CrossRefGoogle Scholar
  15. 15.
    Woodward AW, Bartel B (2005) Auxin: regulation, action and interaction. Ann Bot 95:707–735CrossRefGoogle Scholar
  16. 16.
    Korasick DA, Enders TA, Strader LC (2013) Auxin biosynthesis and storage forms. J Exp Bot 64:2541–2555CrossRefGoogle Scholar
  17. 17.
    Mori Y, Nishimura T, Koshiba T (2005) Vigorous synthesis of indole-3-acetic acid in the apical very tip leads to a constant basipetal flow of the hormone in maize coleoptiles. Plant Sci 168:467–473CrossRefGoogle Scholar
  18. 18. Mishkind ML, FG Plumely, NV Raikhel (1987) Immunochemical analysis of plant tissue. In: KC Vaughn (ed) Other cytochemical staining procedures, CRC Press, Boca RatonGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Graduate School of Bioagricultural SciencesNagoya UniversityNagoyaJapan
  2. 2.Department of Biological SciencesGraduate School of Science, Tokyo Metropolitan UniversityHachiojiJapan

Personalised recommendations