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In Situ/Subcellular Localization of Arabinogalactan Protein Expression by Fluorescent In Situ Hybridization (FISH)

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The Plant Cell Wall

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2149))

Abstract

The arabinogalactan proteins are highly glycosylated and ubiquitous in plants. They are involved in several aspects of plant development and reproduction; however, the mechanics behind their function remains for the most part unclear, as the carbohydrate moiety, covering the most part of the protein core, is poorly characterized at the individual protein level. Traditional immunolocalization using antibodies that recognize the glycosidic moiety of the protein cannot be used to elucidate individual proteins’ distribution, function, or interactors. Indirect approaches are typically used to study these proteins, relying on reverse genetic analysis of null mutants or using a reporter fusion system. In the method presented here, we propose the use of RNA probes to assist in the localization of individual AGPs expression/mRNAs in tissues of Arabidopsis by fluorescent in situ hybridization, FISH. An extensive description of all aspects of this technique is provided, from RNA probe synthesis to the hybridization, trying to overcome the lack of specific antibodies for the protein core of AGPs.

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Acknowledgments

This work was financed by FEDER through the COMPETE program, and by Portuguese National funds through FCT, Fundação para a Ciência eTecnologia (Project PTDC/AGR-GPL/115358/2009 and FCT - 02-SAICT-2017 – POCI-01-0145-FEDER-027839) and PhD grant SFRH/BD/111781/2015), and received support from Spanish–Portuguese Joint Project Nº E 30/12. EU project 690946 ‘SexSeed’ (Sexual Plant Reproduction – Seed Formation) funded by H2020-MSCA-RISE-2015.

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Authors and Affiliations

  1. Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Porto, Portugal

    Mário Luís da Costa

  2. Requimte- Associated Laboratory for Green Chemistry, Porto, Portugal

    Mário Luís da Costa & Sílvia Coimbra

  3. Biological Research Center, CIB-CSIC, Madrid, Spain

    María-Teresa Solís, María-Teresa Solís & Pilar S. Testillano

  4. Department of Genetics, Microbiology and Physiology, Complutense University of Madrid, Madrid, Spain

    Pilar S. Testillano

  5. Biosystems and Integrative Sciences Institute (BioISI), Porto, Portugal

    Sílvia Coimbra

Authors
  1. Mário Luís da Costa
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  2. María-Teresa Solís
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  3. Pilar S. Testillano
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  4. Sílvia Coimbra
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Corresponding author

Correspondence to Sílvia Coimbra .

Editor information

Editors and Affiliations

  1. Botany and Plant Science School of Natural Sciences, Ryan Institute for Environmental Marine and Energy Research School of Natural Sciences, The National University of Ireland Galway, Galway, Ireland

    Zoë A. Popper

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da Costa, M.L., Solís, MT., Testillano, P.S., Coimbra, S. (2020). In Situ/Subcellular Localization of Arabinogalactan Protein Expression by Fluorescent In Situ Hybridization (FISH). In: Popper, Z. (eds) The Plant Cell Wall. Methods in Molecular Biology, vol 2149. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0621-6_23

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  • DOI: https://doi.org/10.1007/978-1-0716-0621-6_23

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0619-3

  • Online ISBN: 978-1-0716-0621-6

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