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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Van Buskirk EK, Decker PV, Chen M (2012) Photobodies in light signaling. Plant Physiol 158(1):52–60. https://doi.org/10.1104/pp.111.186411
Kaiserli E, Perrella G, Davidson ML (2018) Light and temperature shape nuclear architecture and gene expression. Curr Opin Plant Biol 45(Pt A):103–111. https://doi.org/10.1016/j.pbi.2018.05.018
Rausenberger J, Hussong A, Kircher S, Kirchenbauer D, Timmer J, Nagy F, Schafer E, Fleck C (2010) An integrative model for phytochrome B mediated photomorphogenesis: from protein dynamics to physiology. PLoS One 5(5):e10721. https://doi.org/10.1371/journal.pone.0010721
Kircher S, Gil P, Kozma-Bognar L, Fejes E, Speth V, Husselstein-Muller T, Bauer D, Adam E, Schafer E, Nagy F (2002) Nucleocytoplasmic partitioning of the plant photoreceptors phytochrome A, B, C, D, and E is regulated differentially by light and exhibits a diurnal rhythm. Plant Cell 14(7):1541–1555
Kircher S, Kozma-Bognar L, Kim L, Adam E, Harter K, Schafer E, Nagy F (1999) Light quality-dependent nuclear import of the plant photoreceptors phytochrome A and B. Plant Cell 11(8):1445–1456
Yu X, Sayegh R, Maymon M, Warpeha K, Klejnot J, Yang H, Huang J, Lee J, Kaufman L, Lin C (2009) Formation of nuclear bodies of Arabidopsis CRY2 in response to blue light is associated with its blue light-dependent degradation. Plant Cell 21(1):118–130. https://doi.org/10.1105/tpc.108.061663
Chen M, Galvao RM, Li M, Burger B, Bugea J, Bolado J, Chory J (2010) Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes. Cell 141(7):1230–1240. https://doi.org/10.1016/j.cell.2010.05.007
Kaiserli E, Paldi K, O'Donnell L, Batalov O, Pedmale UV, Nusinow DA, Kay SA, Chory J (2015) Integration of light and photoperiodic signaling in transcriptional nuclear foci. Dev Cell 35(3):311–321. https://doi.org/10.1016/j.devcel.2015.10.008
Perrella G, Kaiserli E (2016) Light behind the curtain: photoregulation of nuclear architecture and chromatin dynamics in plants. New Phytol 212:908. https://doi.org/10.1111/nph.14269
Feng CM, Qiu Y, Van Buskirk EK, Yang EJ, Chen M (2014) Light-regulated gene repositioning in Arabidopsis. Nat Commun 5:3027. https://doi.org/10.1038/ncomms4027
Legris M, Klose C, Burgie ES, Rojas CC, Neme M, Hiltbrunner A, Wigge PA, Schafer E, Vierstra RD, Casal JJ (2016) Phytochrome B integrates light and temperature signals in Arabidopsis. Science 354(6314):897–900. https://doi.org/10.1126/science.aaf5656
Huang H, Yoo CY, Bindbeutel R, Goldsworthy J, Tielking A, Alvarez S, Naldrett MJ, Evans BS, Chen M, Nusinow DA (2016) PCH1 integrates circadian and light-signaling pathways to control photoperiod-responsive growth in Arabidopsis. elife 5:e13292. https://doi.org/10.7554/eLife.13292
Huang H, Alvarez S, Bindbeutel R, Shen Z, Naldrett MJ, Evans BS, Briggs SP, Hicks LM, Kay SA, Nusinow DA (2016) Identification of evening complex associated proteins in Arabidopsis by affinity purification and mass spectrometry. Mol Cell Proteomics 15(1):201–217. https://doi.org/10.1074/mcp.M115.054064
Hattersley N, Shen L, Jaffray EG, Hay RT (2011) The SUMO protease SENP6 is a direct regulator of PML nuclear bodies. Mol Biol Cell 22(1):78–90. https://doi.org/10.1091/mbc.E10-06-0504
Posch M, Khoudoli GA, Swift S, King EM, Deluca JG, Swedlow JR (2010) Sds22 regulates aurora B activity and microtubule-kinetochore interactions at mitosis. J Cell Biol 191(1):61–74. https://doi.org/10.1083/jcb.200912046
Hands KJ, Cuchet-Lourenco D, Everett RD, Hay RT (2014) PML isoforms in response to arsenic: high-resolution analysis of PML body structure and degradation. J Cell Sci 127(Pt 2):365–375. https://doi.org/10.1242/jcs.132290
Karimi M, Inze D, Depicker A (2002) GATEWAY vectors for agrobacterium-mediated plant transformation. Trends Plant Sci 7(5):193–195
Pontes O, Li CF, Costa Nunes P, Haag J, Ream T, Vitins A, Jacobsen SE, Pikaard CS (2006) The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA processing center. Cell 126(1):79–92. https://doi.org/10.1016/j.cell.2006.05.031
Li CF, Pontes O, El-Shami M, Henderson IR, Bernatavichute YV, Chan SW, Lagrange T, Pikaard CS, Jacobsen SE (2006) An ARGONAUTE4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis thaliana. Cell 126(1):93–106. https://doi.org/10.1016/j.cell.2006.05.032
Oka Y, Matsushita T, Mochizuki N, Suzuki T, Tokutomi S, Nagatani A (2004) Functional analysis of a 450-amino acid N-terminal fragment of phytochrome B in Arabidopsis. Plant Cell 16(8):2104–2116. https://doi.org/10.1105/tpc.104.022350
Huang S, Deerinck TJ, Ellisman MH, Spector DL (1994) In vivo analysis of the stability and transport of nuclear poly(A)+ RNA. J Cell Biol 126(4):877–899
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1370-2_2
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1369-6
Online ISBN: 978-1-0716-1370-2
eBook Packages: Springer Protocols