Abstract
Our understanding of the circadian clock function in plants has been markedly assisted by studies with the model species Arabidopsis thaliana. Molecular and genetics approaches have delivered a comprehensive view of the transcriptional regulatory networks underlying the Arabidopsis circadian system. The use of the luciferase as a reporter allowed the precise in vivo determination of circadian periods, phases, and amplitudes of clock promoter activities with unprecedented temporal resolution. An increasing repertoire of fine-tuned luciferases together with additional applications such as translational fusions or bioluminescence molecular complementation assays have considerably expanded our view of circadian protein expression and activity, far beyond transcriptional regulation. Further applications have focused on the in vivo simultaneous examination of rhythms in different parts of the plant. The use of intact versus excised plant organs has also provided a glimpse on both the organ-specific and autonomy of the clocks and the importance of long distance communication for circadian function. This chapter provides a basic protocol for in vivo high-throughput monitoring of circadian rhythms in Arabidopsis seedlings using bioluminescent reporters and a microplate luminometer.
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References
Young MW, Kay SA (2001) Time zones: a comparative genetics of circadian clocks. Nat Rev Genet 2:702–715
McClung CR (2006) Plant circadian rhythms. Plant Cell 18:792–803
Nagel DH, Kay SA (2012) Complexity in the wiring and regulation of plant circadian networks. Curr Biol 22:R648–R657
McClung CR (2013) Beyond Arabidopsis: the circadian clock in non-model plant species. Semin Cell Dev Biol 24:430–436
Wang ZY, Tobin EM (1998) Constitutive expression of the CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) gene disrupts circadian rhythms and suppresses its own expression. Cell 93:1207–1217
Matsushika A, Makino S, Kojima M, Mizuno T (2000) Circadian waves of expression of the APRR1/TOC1 family of pseudo-response regulators in Arabidopsis thaliana: insight into the plant circadian clock. Plant Cell Physiol 41:1002–1012
Nusinow DA, Helfer A, Hamilton EE, King JJ, Imaizumi T, Schultz TF, Farré EM, Kay SA (2011) The ELF4-ELF3-LUX complex links the circadian clock to diurnal control of hypocotyl growth. Nature 475:398–402
Haydon MJ, Mielczarek O, Robertson FC, Hubbard KE, Webb AAR (2013) Photosynthetic entrainment of the Arabidopsis thaliana circadian clock. Nature 502:689–692
Mizuno T, Nomoto Y, Oka H, Kitayama M, Takeuchi A, Tsubouchi M, Yamashino T (2014) Ambient temperature signal feeds into the circadian clock transcriptional circuitry through the EC night-time repressor in Arabidopsis thaliana. Plant Cell Physiol 55:958–976
Chen WW, Takahashi N, Hirata Y, Ronald J, Porco S, Davis SJ, Nusinow DA, Kay SA, Mas P (2020) A mobile ELF4 delivers circadian temperature information from shoots to roots. Nat Plants 6:416–426
Shaw SL, Ehrhardt DW (2013) Smaller, faster, brighter: advances in optical imaging of living plant cells. Annu Rev Plant Biol 64:351–375
Kirkpatrick A, Xu T, Ripp S, Sayler G, Close D (2019) Biotechnological advances in luciferase enzymes. In: Bioluminescence - analytical applications and basic biology. IntechOpen, Rijeka, Croatia, p 13
Uehara TN, Mizutani Y, Kuwata K, Hirota T, Sato A, Mizoi J, Takao S, Matsuo H, Suzuki T, Ito S et al (2019) Casein kinase 1 family regulates PRR5 and TOC1 in the Arabidopsis circadian clock. Proc Natl Acad Sci U S A 116:11528–11536
Millar AJ, Short SR, Chua NH, Kay SA (1992) A novel circadian phenotype based on firefly luciferase expression in transgenic plants. Plant Cell 4:1075–1087
JoVE Science Education Database (2020) Basic methods in cellular and molecular biology. Molecular cloning. JoVE, Cambridge, MA. https://www.jove.com/science-education/5074/molecular-cloning. Accessed 6 May 2020
Urquiza-García U, Millar AJ (2019) Expanding the bioluminescent reporter toolkit for plant science with NanoLUC. Plant Methods 15:68
Hall A, Brown P (2007) Monitoring circadian rhythms in Arabidopsis thaliana using luciferase reporter genes. In: Circadian rhythms. Humana Press, Totowa, NJ, pp 143–152
Mattanovich D, Rüker F, da Cämara Machado A, Laimer M, Regner F, Steinkeliner H, Himmler G, Katinger H (1989) Efficient transformation of Agrobacterium spp. by eletroporation. Nucleic Acids Res 17:6747–6747
Logemann E, Birkenbihl RP, Ülker B, Somssich IE (2006) An improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transformation protocol. Plant Methods 2(1):16
Kim H, Kim Y, Yeom M, Lim J, Nam HG (2016) Age-associated circadian period changes in Arabidopsis leaves. J Exp Bot 67:2665–2673
Takahashi N, Hirata Y, Aihara K, Mas P (2015) A hierarchical multi-oscillator network orchestrates the Arabidopsis circadian system. Cell 163:148–159
Moore A, Zielinski T, Millar AJ (2014) Online period estimation and determination of rhythmicity in circadian data, using the BioDare data infrastructure. In: Staiger D (ed) . Springer, New York, pp 13–44
Ogura R, Matsuo N, Wako N, Tanaka T, Ono S, Hiratsuka K (2005) Multi-color luciferases as reporters for monitoring transient gene expression in higher plants. Plant Biotechnol 22:151–155
Endo M, Shimizu H, Nohales MA, Araki T, Kay SA (2014) Tissue-specific clocks in Arabidopsis show asymmetric coupling. Nature 515:419–422
Xie Q, Wang P, Liu X, Yuan L, Wang L, Zhang C, Li Y, Xing H, Zhi L, Yue Z et al (2014) LNK1 and LNK2 are transcriptional coactivators in the Arabidopsis circadian oscillator. Plant Cell 26:2843–2857
Kanesaka Y, Okada M, Ito S, Oyama T (2019) Monitoring single-cell bioluminescence of Arabidopsis leaves to quantitatively evaluate the efficiency of a transiently introduced CRISPR/Cas9 system targeting the circadian clock gene ELF3. Plant Biotechnol 36:187–193
Kim J, Somers DE (2010) Rapid assessment of gene function in the circadian clock using artificial microRNA in Arabidopsis mesophyll protoplasts. Plant Physiol 154:611–621
Muranaka T, Oyama T (2016) Heterogeneity of cellular circadian clocks in intact plants and its correction under light-dark cycles. Sci Adv 2:e1600500
Okada M, Muranaka T, Ito S, Oyama T (2017) Synchrony of plant cellular circadian clocks with heterogeneous properties under light/dark cycles. Sci Rep 7:317
Lindsey BE, Rivero L, Calhoun CS, Grotewold E, Brkljacic J (2017) Standardized method for high-throughput sterilization of Arabidopsis seeds. J Vis Exp 2017:1–7
Wenden B, Toner DLK, Hodge SK, Grima R, Millar AJ (2012) Spontaneous spatiotemporal waves of gene expression from biological clocks in the leaf. Proc Natl Acad Sci U S A 109:6757–6762
Fukuda H, Ukai K, Oyama T (2012) Self-arrangement of cellular circadian rhythms through phase-resetting in plant roots. Phys Rev E 86:041917
Muranaka T, Kubota S, Oyama T (2013) A single-cell bioluminescence imaging system for monitoring cellular gene expression in a plant body. Plant Cell Physiol 54:2085–2093
England CG, Ehlerding EB, Cai W (2016) NanoLuc: a small luciferase is brightening up the field of bioluminescence. Bioconjug Chem 27:1175–1187
Acknowledgments
The Mas laboratory is funded with a research grant (PID2019-106653GB-I00) from the MCIN/AEI/10.13039/501100011033, from the Ramon Areces Foundation and from the Generalitat de Catalunya (AGAUR). P.M. laboratory also acknowledges financial support from the CERCA Program/Generalitat de Catalunya and by the Spanish Ministry of Economy and Competitiveness through the “Severo Ochoa Program for Centers of Excellence in R & D” 2016–2019 (CEX2019-000902-S). M.O. is funded with a “Severo Ochoa” Internationalization Postdoctoral Program.
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Okada, M., Mas, P. (2022). In Vivo Bioluminescence Analyses of Circadian Rhythms in Arabidopsis thaliana Using a Microplate Luminometer. In: Solanas, G., Welz, P.S. (eds) Circadian Regulation. Methods in Molecular Biology, vol 2482. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2249-0_27
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DOI: https://doi.org/10.1007/978-1-0716-2249-0_27
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