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Immunolocalization of PIN and ABCB Transporters in Plants

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

Abstract

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 

Notes

Acknowledgements

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

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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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