Abstract
5-Ethynyl-2′-deoxyuridine (EdU) is a nucleoside analog of thymidine that can be rapidly incorporated into replicating DNA in vivo and, subsequently, detected by using “click” chemistry to couple its terminal alkyne group to fluorescent azides such as Alexa Fluor 488. Recently, EdU incorporation followed by coupling with a fluorophore has been used to visualize newly synthesized DNA in a wide range of plant species. One particularly useful application is in flow cytometry, where two-parameter sorting can be employed to analyze different phases of the cell cycle, as defined both by total DNA content and the amount of EdU pulse-labeled DNA. This approach allows analysis of the cell cycle without the need for synchronous cell populations, which can be difficult to obtain in many plant systems. The approach presented here, which was developed for fixed, EdU-labeled nuclei, can be used to prepare analytical profiles as well as to make highly purified preparations of G1, S, or G2/M phase nuclei for molecular or biochemical analysis. We present protocols for EdU pulse labeling, tissue fixation and harvesting, nuclei preparation, and flow sorting. Although developed for Arabidopsis suspension cells and maize root tips, these protocols should be modifiable to many other plant systems.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bass HW, Wear EE, Lee T-J, Hoffman GG, Gumber HK, Allen GC, Thompson WF, Hanley-Bowdoin L (2014) A maize root tip system to study DNA replication programmes in somatic and endocycling nuclei during plant development. J Exp Bot 65:2747–2756
Darzynkiewicz Z, Traganos F, Zhao H, Halicka HD, Li JW (2011) Cytometry of DNA replication and RNA synthesis: historical perspective and recent advances based on “click chemistry”. Cytometry A 79A:328–337
Salic A, Mitchison TJ (2008) A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci U S A 105:2415–2420
Rostovtsev VV, Green LG, Fokin VV, Sharpless KB (2002) A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew Chem Int Ed Engl 41:2596–2599
Kotogany E, Dudits D, Horvath GV, Ayaydin F (2010) A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine. Plant Methods 6:5–19
Cooper S (2003) Rethinking synchronization of mammalian cells for cell cycle analysis. Cell Mol Life Sci 60:1099–1106
Galbraith DW, Harkins KR, Maddox JM, Ayres NM, Sharma DP, Firoozabady E (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220:1049–1051
Lee TJ, Pascuzzi PE, Settlage SB, Shultz RW, Tanurdzic M, Rabinowicz PD, Menges M, Zheng P, Main D, Murray JAH, Sosinski B, Allen GC, Martienssen RA, Hanley-Bowdoin L, Vaughn MW, Thompson WF (2010) Arabidopsis thaliana chromosome 4 replicates in two phases that correlate with chromatin state. Plos Genet 6:e1000982
Onelli E, Citterio S, OConnor JE, Levi M, Sgorbati S (1997) Flow cytometry, sorting and immunocharacterization with proliferating cell nuclear antigen of cycling and non-cycling cells in synchronized pea root tips. Planta 202:188–195
Zhang CQ, Barthelson RA, Lambert GM, Galbraith DW (2008) Global characterization of cell-specific gene expression through fluorescence-activated sorting of nuclei. Plant Physiol 147:30–40
Birnbaum K, Shasha DE, Wang JY, Jung JW, Lambert GM, Galbraith DW, Benfey PN (2003) A gene expression map of the Arabidopsis root. Science 302:1956–1960
Galbraith DW (2014) Flow cytometry and sorting in Arabidopsis. Methods Mol Biol 1062:509–537
Iyer-Pascuzzi AS, Benfey PN (2010) Fluorescence-activated cell sorting in plant developmental biology. Methods Mol Biol 655:313–319
Sheen J, Hwang SB, Niwa Y, Kobayashi H, Galbraith DW (1995) Green-fluorescent protein as a new vital marker in plant-cells. Plant J 8:777–784
Dolezel J, Greilhuber J, Suda J (2007) Estimation of nuclear DNA content in plants using flow cytometry. Nat Protocol 2:2233–2244
Folta KM, Kaufman LS (2006) Isolation of Arabidopsis nuclei and measurement of gene transcription rates using nuclear run-on assays. Nat Protocol 1:3094–3100
Galbraith DW, Lambert GM (2012) High-throughput monitoring of plant nuclear DNA contents via flow cytometry. Methods Mol Biol 918:311–325
Woodfine K, Fiegler H, Beare DM, Collins JE, McCann OT, Young BD, Debernardi S, Mott R, Dunham I, Carter NP (2004) Replication timing of the human genome. Hum Mol Genet 13:191–202
Gendrel AV, Lippman Z, Martienssen R, Colot V (2005) Profiling histone modification patterns in plants using genomic tiling microarrays. Nat Methods 2:213–218
Acknowledgements
This work was supported by grants from the National Science Foundation (IOS-1025830 to L.H.-B., W.F.T., and G.C.A.).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Wear, E.E. et al. (2016). Isolation of Plant Nuclei at Defined Cell Cycle Stages Using EdU Labeling and Flow Cytometry. In: Caillaud, MC. (eds) Plant Cell Division. Methods in Molecular Biology, vol 1370. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3142-2_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3142-2_6
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3141-5
Online ISBN: 978-1-4939-3142-2
eBook Packages: Springer Protocols