Abstract
Phytochromes are red/far-red light receptors that perceive and transduce light signals to regulate various physiological responses. A large number of phytochrome interacting proteins have been identified using genetic and biochemical approaches. A direct interaction between phytochrome and its interacting proteins, therefore, defines one of the critical steps to initiate light signaling cascades in plants. Thus it is important to thoroughly examine the light-dependent interaction between phytochromes and putative phytochrome-interacting proteins. In this chapter, a protocol for rapid and simple light dependent in vitro coimmunoprecipitation between phytochromes and phytochrome interacting factors is described. In principle, this protocol can be adapted for other putative phytochrome interacting proteins.
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References
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
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
Bae G, Choi G (2008) Decoding of light signals by plant phytochromes and their interacting proteins. Annu Rev Plant Biol 59:281–311
Paik I, Yang S, Choi G (2012) Phytochrome regulates translation of mRNA in the cytosol. Proc Natl Acad Sci U S A 109:1335–1340
Ni W, Xu S-L, González-Grandío E, Chalkley RJ, Huhmer AFR, Burlingame AL, Wang Z-Y, Quail PH (2017) PPKs mediate direct signal transfer from phytochrome photoreceptors to transcription factor PIF3. Nat Commun 8:15236
Ni W, Xu S-L, Tepperman JM, Stanley DJ, Maltby DA, Gross JD, Burlingame AL, Wang Z-Y, Quail PH (2014) A mutually assured destruction mechanism attenuates light signaling in Arabidopsis. Science 344:1160–1164
Shi H, Shen X, Liu R, Xue C, Wei N, Deng XW, Zhong S (2016) The red light receptor phytochrome B directly enhances substrate-E3 ligase interactions to attenuate ethylene responses. Dev Cell 39:597–610
Sheerin DJ, Menon C, zur Oven-Krockhaus S, Enderle B, Zhu L, Johnen P, Schleifenbaum F, Stierhof Y-D, Huq E, Hiltbrunner A (2015) Light-activated phytochrome A and B interact with members of the SPA family to promote photomorphogenesis in Arabidopsis by reorganizing the COP1/SPA complex. Plant Cell 27:189–201
Lu X-D, Zhou C-M, Xu P-B, Luo Q, Lian H-L, Yang H-Q (2015) Red-light-dependent interaction of phyB with SPA1 promotes COP1–SPA1 dissociation and photomorphogenic development in Arabidopsis. Mol Plant 8:467–478
Hughes RM, Vrana JD, Song J, Tucker CL (2012) Light-dependent, dark-promoted interaction between Arabidopsis cryptochrome 1 and phytochrome B proteins. J Biol Chem 287:22165–22172
Huq E, Al-Sady B, Hudson M, Kim C, Apel K, Quail PH (2004) Phytochrome-interacting factor 1 is a critical bHLH regulator of chlorophyll biosynthesis. Science 305:1937–1941
Oh E, Kim J, Park E, Kim J-I, Kang C, Choi G (2004) PIL5, a phytochrome-interacting basic helix-loop-helix protein, is a key negative regulator of seed germination in Arabidopsis thaliana. Plant Cell 16:3045–3058
Ni M, Tepperman JM, Quail PH (1999) Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. Nature 400:781–784
Huq E, Quail PH (2002) PIF4, a phytochrome-interacting bHLH factor, functions as a negative regulator of phytochrome B signaling in Arabidopsis. EMBO J 21:2441–2450
Paik I, Kathare PK, Kim J-I, Huq E (2017) Expanding roles of PIFs in signal integration from multiple processes. Mol Plant 10:1035–1046
Luo Q, Lian H-L, He S-B, Li L, Jia K-P, Yang H-Q (2014) COP1 and phyB physically interact with PIL1 to regulate its stability and photomorphogenic development in Arabidopsis. Plant Cell 26:2441–2456
Park E, Park J, Kim J, Nagatani A, Lagarias JC, Choi G (2012) Phytochrome B inhibits binding of PHYTOCHROME INTERACTING FACTORs to their target promoters. Plant J 72:537–546
Shen H, Moon J, Huq E (2005) PIF1 is regulated by light-mediated degradation through the ubiquitin-26S proteasome pathway to optimize photomorphogenesis of seedlings in Arabidopsis. Plant J 44:1023–1035
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
Acknowledgments
We thank members of the Huq laboratory for critical reading of the manuscript. This work was supported by grants from the National Institute of Health (NIH) (GM-114297) and National Science Foundation (MCB-1543813) to E.H.
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Paik, I., Huq, E. (2019). Rapid Examination of Phytochrome–Phytochrome Interacting Factor (PIF) Interaction by In Vitro Coimmunoprecipitation Assay. 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_2
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DOI: https://doi.org/10.1007/978-1-4939-9612-4_2
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