Abstract
Photobodies are membraneless subnuclear organelles that contain the red and far-red photoreceptors, phytochromes. Photobody biogenesis has been postulated to play important roles in early light signaling events. The size and number of photobodies are highly dynamic in response to the quality and quantity of light and correlated tightly with phytochrome-mediated seedling morphogenesis. Here, we provide a detailed protocol for characterization of the three-dimensional morphology of photobodies, including sample preparation, fluorescence microscopy, and image analysis. Although this method was developed initially for characterizing photobodies, it can be adopted to analyze other membraneless or membrane-bound subcellular organelles.
Keywords
- Photobody
- Phytochrome
- Nuclear body
- Photomorphogenesis
- Subnuclear organization
This is a preview of subscription content, access via your institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Chen M, Chory J (2011) Phytochrome signaling mechanisms and the control of plant development. Trends Cell Biol 21:664–671
Yamaguchi R, Nakamura M, Mochizuki N, Kay SA, Nagatani A (1999) Light-dependent translocation of a phytochrome B-GFP fusion protein to the nucleus in transgenic Arabidopsis. J Cell Biol 145:437–445
Kircher S, Gil P, Kozma-Bognár L, Fejes E, Speth V, Husselstein-Müller T, Bauer D, Ádám É, Schäfer 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 14:1541–1555
Kleiner O, Kircher S, Harter K, Batschauer A (1999) Nuclear localization of the Arabidopsis blue light receptor cryptochrome 2. Plant J 19:289–296
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 21:118–130
Gu N-N, Zhang Y-C, Yang H-Q (2012) Substitution of a conserved glycine in the PHR domain of Arabidopsis CRYPTOCHROME 1 confers a constitutive light response. Mol Plant 5:85–97
Lian H-L, He S-B, Zhang Y-C, Zhu D-M, Zhang J-Y, Jia K-P, Sun S-X, Li L, Yang H-Q (2011) Blue-light-dependent interaction of cryptochrome 1 with SPA1 defines a dynamic signaling mechanism. Genes Dev 25:1023–1028
Liu B, Zuo Z, Liu H, Liu X, Lin C (2011) Arabidopsis cryptochrome 1 interacts with SPA1 to suppress COP1 activity in response to blue light. Genes Dev 25:1029–1034
Más P, Devlin PF, Panda S, Kay SA (2000) Functional interaction of phytochrome B and cryptochrome 2. Nature 408:207–211
Favory J-J, Stec A, Gruber H et al (2009) Interaction of COP1 and UVR8 regulates UV-B-induced photomorphogenesis and stress acclimation in Arabidopsis. EMBO J 28:591–601
Van Buskirk EK, Decker PV, Chen M (2012) Photobodies in light signaling. Plant Physiol 158:52–60
Kircher S, Kozma-Bognar L, Kim L, Adam E, Harter K, Schäfer E, Nagy F (1999) Light quality-dependent nuclear import of the plant photoreceptors phytochrome A and B. Plant Cell 11:1445–1456
Chen M, Schwab R, Chory J (2003) Characterization of the requirements for localization of phytochrome B to nuclear bodies. Proc Natl Acad Sci U S A 100:14493–14498
Rockwell NC, Su Y-S, Lagarias JC (2006) Phytochrome structure and signaling mechanisms. Annu Rev Plant Biol 57:837–858
Burgie ES, Vierstra RD (2014) Phytochromes: an atomic perspective on photoactivation and signaling. Plant Cell 26:4568–4583
Jung J-H, Domijan M, Klose C et al (2016) Phytochromes function as thermosensors in Arabidopsis. Science 354:886–889
Legris M, Klose C, Burgie ES, Rojas CCR, Neme M, Hiltbrunner A, Wigge PA, Schäfer E, Vierstra RD, Casal JJ (2016) Phytochrome B integrates light and temperature signals in Arabidopsis. Science 354:897–900
Klose C, Venezia F, Hussong A, Kircher S, Schäfer E, Fleck C (2015) Systematic analysis of how phytochrome B dimerization determines its specificity. Nat Plants 1:15090
Chen M, Galvão RM, Li M, Burger B, Bugea J, Bolado J, Chory J (2010) Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes. Cell 141:1230–1240
Galvão RM, Li M, Kothadia SM, Haskel JD, Decker PV, Van Buskirk EK, Chen M (2012) Photoactivated phytochromes interact with HEMERA and promote its accumulation to establish photomorphogenesis in Arabidopsis. Genes Dev 26:1851–1863
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
Van Buskirk EK, Reddy AK, Nagatani A, Chen M (2014) Photobody localization of phytochrome B is tightly correlated with prolonged and light-dependent inhibition of hypocotyl elongation in the dark. Plant Physiol 165:595–607
Qiu Y, Li M, Pasoreck EK et al (2015) HEMERA couples the proteolysis and transcriptional activity of PHYTOCHROME INTERACTING FACTORs in Arabidopsis photomorphogenesis. Plant Cell 27:1409–1427
Qiu Y, Pasoreck EK, Reddy AK, Nagatani A, Ma W, Chory J, Chen M (2017) Mechanism of early light signaling by the carboxy-terminal output module of Arabidopsis phytochrome B. Nat Commun 8:1905
Bauer D, Viczián A, Kircher S et al (2004) CONSTITUTIVE PHOTOMORPHOGENESIS 1 and multiple photoreceptors control degradation of PHYTOCHROME INTERACTING FACTOR 3, a transcription factor required for light signaling in Arabidopsis. Plant Cell 16:1433–1445
Al-Sady B, Ni W, Kircher S, Schäfer E, Quail PH (2006) Photoactivated phytochrome induces rapid PIF3 phosphorylation prior to proteasome-mediated degradation. Mol Cell 23:439–446
Gendreau E, Traas J, Desnos T, Grandjean O, Caboche M, Höfte H (1997) Cellular basis of hypocotyl growth in Arabidopsis thaliana. Plant Physiol 114:295–305
Wang Z-Y, Nakano T, Gendron J et al (2002) Nuclear-localized BZR1 mediates brassinosteroid-induced growth and feedback suppression of brassinosteroid biosynthesis. Dev Cell 2:505–513
Nybo K (2012) GFP imaging in fixed cells. BioTechniques 52:359–360
Acknowledgments
This work was supported by National Institute of Health grant R01GM087388 to M.C.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Yoo, C.Y., Williams, D., Chen, M. (2019). Quantitative Analysis of Photobodies. In: Hiltbrunner, A. (eds) Phytochromes. Methods in Molecular Biology, vol 2026. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9612-4_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9612-4_10
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9611-7
Online ISBN: 978-1-4939-9612-4
eBook Packages: Springer Protocols