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Photobody Detection Using Immunofluorescence and Super-Resolution Imaging in Arabidopsis

Part of the Methods in Molecular Biology book series (MIMB,volume 2297)

Abstract

Light triggers changes in plant nuclear architecture to control differentiation, adaptation, and growth. A series of genetic, molecular, and imaging approaches have revealed that the nucleus forms a hub for photo-induced protein interactions and gene regulatory events. However, the mechanism and function of light-induced nuclear compartmentalization is still unclear. This chapter provides detailed experimental protocols for examining the morphology and potential functional significance of light signaling components that localize in light-induced subnuclear domains, also known as photobodies. We describe how immunolabeling of endogenous proteins and fluorescent in situ hybridization (FISH) could be combined with confocal imaging of fluorescently tagged proteins to assess co-localization in Arabidopsis nuclei. Furthermore, we employ a super-resolution imaging approach to study the morphology of photobodies at unprecedented detail.

Key words

  • Photobodies
  • Light signaling
  • Arabidopsis
  • Nuclear localization
  • Immunofluorescence
  • Super-resolution imaging

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Acknowledgments

Many thanks to Dr. Markus Posch (University of Dundee) for his help in performing super-resolution imaging on plant tissue. We are grateful to Prof. Akira Nagatani for providing us with an anti-phyB antibody . The authors would also like to thank the COST-INDEPTH action for organizing and funding G.P. to participate at the “3D FISH and Image Analysis” training workshop at Clermont-Ferrand, France. E.K. is grateful to the BBSRC for the New Investigator Grant Award BB/M023079/1 and the John Grieve Bequest for supporting her Lectureship. A.Z. is funded by a PhD studentship from the College of Medical, Veterinary and Life Sciences at the University of Glasgow.

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Correspondence to Eirini Kaiserli .

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Perrella, G., Zioutopoulou, A., Hamilton, A., Kaiserli, E. (2021). Photobody Detection Using Immunofluorescence and Super-Resolution Imaging in Arabidopsis . In: Yin, R., Li, L., Zuo, K. (eds) Plant Photomorphogenesis. Methods in Molecular Biology, vol 2297. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1370-2_2

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  • DOI: https://doi.org/10.1007/978-1-0716-1370-2_2

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1369-6

  • Online ISBN: 978-1-0716-1370-2

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