Immunolocalization of PIN and ABCB Transporters in Plants

  • Nicola Carraro
  • Wendy Ann PeerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1398)


PIN auxin efflux carriers and ABCB auxin transporters are important for polar auxin transport, organogenesis and long distance auxin transport. Along with the auxin influx symporter AUX1, they are essential for tropic responses such as gravitropism and phototropism where lateral redistribution of auxin is required for the tropic response to occur. Immunolocalization of plant membrane transporters is an effective technique to determine the transporters’ subcellular localization patterns in the tissues of interest, especially when fluorescent protein fusions of the protein of interest are not available. Immunolocalization is also a valuable tool for validation of the localization of fluorescent protein fusions when the fusions are available. Here we describe the procedures to prepare plant tissue samples and fix them for whole mount or embedding and sectioning. We focus on immunolocalizations of PINs and ABCBs in Arabidopsis and maize tissues. In addition, we describe treatments of roots with inhibitors of cellular trafficking: brefeldin A (BFA), a fungal compound that blocks exocytosis; wortmannin, a fungal compound that inhibits phosphatidylinositol 3-kinase and induces fusion of pre-vacuolar compartments and multi-vascular bodies; and oryzalin, a fungal compound that depolymerizes microtubules. Inhibitor treatments are performed prior to fixation and affect the localization patterns of PINs and ABCBs, giving insight into cell type -specific trafficking mechanisms. The procedures described for Arabidopsis and maize can be easily adapted for other herbaceous plants.

Key words

ABCB Antibody Antisera Arabidopsis Fixation Maize Membrane proteins PIN Trafficking inhibitor Whole mount 



We thank the many lab members who modified techniques over the last 15 years. This work was supported by the Maryland Agriculture Experiment Station.


  1. 1.
    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 9:1963–1971PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Brunoud G, Wells DM, Oliva M, Larrieu A, Mirabet V, Burrow AH, Beeckman T, Kepinski S, Traas J, Bennett MJ, Vernoux T (2012) A novel sensor to map auxin response and distribution at high spatio-temporal resolution. Nature 482:103–106CrossRefPubMedGoogle Scholar
  3. 3.
    Truernit E, Bauby H, Dubreucq B, Grandjean O, Runions J, Barthélémy J, Palauqui J-C (2008) High-resolution whole-mount imaging of three-dimensional tissue organization and gene expression enables the study of phloem development and structure in Arabidopsis. Plant Cell 20:1494–1503PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Peer WA, Hosein FN, Bandyopadhyay A, Makam SN, Otegui MS, Lee GJ, Blakeslee JJ, Cheng Y, Titapiwatanakun B, Yakubov B, Bangari B, Murphy AS (2009) Mutation of the membrane-associated M1 protease APM1 results in distinct embryonic and seedling developmental defects in Arabidopsis. Plant Cell 21:1693–1721PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Brundrett MC, Enstone DE, Peterson CA (1988) A berberine-aniline blue fluorescent staining procedure for suberin, lignin, and callose in plant tissue. Protoplasma 146:133–142CrossRefGoogle Scholar
  6. 6.
    Murphy A, Taiz L (1997) Correlation between potassium efflux and copper sensitivity in ten Arabidopsis ecotypes. New Phytol 136:211–222CrossRefGoogle Scholar
  7. 7.
    Boutté Y, Crosnier M-T, Carraro N, Traas J, Satiat-Jeunemaitre B (2006) The plasma membrane recycling pathway and cell polarity in plants: studies on PIN proteins. J Cell Sci 119:1255–1265CrossRefPubMedGoogle Scholar
  8. 8.
    Carraro N (2002) Auxin transport and organs formation in Arabidopsis and maize. Dissertation, Università Degli Studi di Padova—Université Paris XIGoogle Scholar
  9. 9.
    Titapiwatanakun B, Blakeslee JJ, Bandyopadhyay A, Yang H, Mravec J, Sauer M, Cheng Y, Adamec J, Nagashima A, Geisler M, Sakai T, Friml J, Peer WA, Murphy AS (2009) ABCB19/PGP19 stabilises PIN1 in membrane microdomains in Arabidopsis. Plant J 57:27–44CrossRefPubMedGoogle Scholar
  10. 10.
    Yang H, Richter GL, Wang X, Młodzińska E, Carraro N, Ma G, Jenness M, Chao D, Peer WA, Murphy AS (2013) Sterols and sphingolipids differentially function in trafficking of the Arabidopsis ABCB19 auxin transporter. Plant J 74:37–47CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of AgronomyPurdue UniversityWest LafayetteUSA
  2. 2.Department of Environmental Science and TechnologyUniversity of MarylandCollege ParkUSA
  3. 3.Department of Plant Science and Landscape ArchitectureUniversity of MarylandCollege ParkUSA

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