A Rapid and Robust Assay for Detection of S-phase Cell Cycle Progression in Plant Cells and Tissues by Using Ethynyl Deoxyuridine

Overview

Journal Plant Methods

Publisher Biomed Central

Specialty Biology

Date 2010 Feb 26

PMID 20181034

Citations 86

Authors

Edit Kotogany

Denes Dudits

Gabor V Horvath

Ferhan Ayaydin

Affiliations

Soon will be listed here.

Abstract

Background: Progress in plant cell cycle research is highly dependent on reliable methods for detection of cells replicating DNA. Frequency of S-phase cells (cells in DNA synthesis phase) is a basic parameter in studies on the control of cell division cycle and the developmental events of plant cells. Here we extend the microscopy and flow cytometry applications of the recently developed EdU (5-ethynyl-2'-deoxyuridine)-based S-phase assay to various plant species and tissues. We demonstrate that the presented protocols insure the improved preservation of cell and tissue structure and allow significant reduction in assay duration. In comparison with the frequently used detection of bromodeoxyuridine (BrdU) and tritiated-thymidine incorporation, this new methodology offers several advantages as we discuss here.

Results: Applications of EdU-based S-phase assay in microscopy and flow cytometry are presented by using cultured cells of alfalfa, Arabidopsis, grape, maize, rice and tobacco. We present the advantages of EdU assay as compared to BrdU-based replication assay and demonstrate that EdU assay -which does not require plant cell wall digestion or DNA denaturation steps, offers reduced assay duration and better preservation of cellular, nuclear and chromosomal morphologies. We have also shown that fast and efficient EdU assay can also be an efficient tool for dual parameter flow cytometry analysis and for quantitative assessment of replication in thick root samples of rice.

Conclusions: In plant cell cycle studies, EdU-based S-phase detection offers a superior alternative to the existing S-phase assays. EdU method is reliable, versatile, fast, simple and non-radioactive and it can be readily applied to many different plant systems.

Citing Articles

Ethylene-independent modulation of root development by ACC via downregulation of WOX5 and group I CLE peptide expression.

Mou W, Khare R, Polko J, Taylor I, Xu J, Xue D Proc Natl Acad Sci U S A. 2025; 122(6):e2417735122.

PMID: 39908106 PMC: 11831204. DOI: 10.1073/pnas.2417735122.

Establishing of 3D-FISH on frozen section and its applying in chromosome territories analysis in Populus trichocarpa.

Ning Y, Shang D, Xin H, Ni R, Wang Z, Zhen Y Plant Cell Rep. 2024; 43(11):255.

PMID: 39375198 DOI: 10.1007/s00299-024-03342-4.

A conserved GRAS-domain transcriptional regulator links meristem indeterminacy to sex determination in Ceratopteris gametophytes.

Geng Y, Xie C, Yan A, Yang X, Lai D, Liu X Curr Biol. 2024; 34(15):3454-3472.e7.

PMID: 39059395 PMC: 11364212. DOI: 10.1016/j.cub.2024.06.064.

CELLULOSE SYNTHASE-LIKE C proteins modulate cell wall establishment during ethylene-mediated root growth inhibition in rice.

Zhou Y, Gao Y, Zhang B, Yang H, Tian Y, Huang Y Plant Cell. 2024; 36(9):3751-3769.

PMID: 38943676 PMC: 11371184. DOI: 10.1093/plcell/koae195.

ARGONAUTE10 controls cell fate specification and formative cell divisions in the Arabidopsis root.

El Arbi N, Schurholz A, Handl M, Schiffner A, Hidalgo Prados I, Schnurbusch L EMBO J. 2024; 43(9):1822-1842.

PMID: 38565947 PMC: 11066080. DOI: 10.1038/s44318-024-00072-x.

References

Tsukaya H . Determination of the unequal fate of cotyledons of a one-leaf plant, Monophyllaea. Development. 1997; 124(7):1275-80. DOI: 10.1242/dev.124.7.1275. View

Diermeier-Daucher S, Clarke S, Hill D, Vollmann-Zwerenz A, Bradford J, Brockhoff G . Cell type specific applicability of 5-ethynyl-2'-deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry. Cytometry A. 2009; 75(6):535-46. DOI: 10.1002/cyto.a.20712. View

Gratzner H . Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: A new reagent for detection of DNA replication. Science. 1982; 218(4571):474-5. DOI: 10.1126/science.7123245. View

Galbraith D, Harkins K, Maddox J, Ayres N, Sharma D, Firoozabady E . Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science. 1983; 220(4601):1049-51. DOI: 10.1126/science.220.4601.1049. View

Warren M, Puskarczyk K, Chapman S . Chick embryo proliferation studies using EdU labeling. Dev Dyn. 2009; 238(4):944-9. PMC: 2664394. DOI: 10.1002/dvdy.21895. View

Kinoshita I, Sanbe A, Yokomura E . Difference in light-induced increase in ploidy level and cell size between adaxial and abaxial epidermal pavement cells of Phaseolus vulgaris primary leaves. J Exp Bot. 2008; 59(6):1419-30. DOI: 10.1093/jxb/ern055. View

Yokochi T, Gilbert D . Replication labeling with halogenated thymidine analogs. Curr Protoc Cell Biol. 2008; Chapter 22:Unit 22.10. DOI: 10.1002/0471143030.cb2210s35. View

Rodionov V, Fokin V, Finn M . Mechanism of the ligand-free CuI-catalyzed azide-alkyne cycloaddition reaction. Angew Chem Int Ed Engl. 2005; 44(15):2210-5. DOI: 10.1002/anie.200461496. View

Buck S, Bradford J, Gee K, Agnew B, Clarke S, Salic A . Detection of S-phase cell cycle progression using 5-ethynyl-2'-deoxyuridine incorporation with click chemistry, an alternative to using 5-bromo-2'-deoxyuridine antibodies. Biotechniques. 2008; 44(7):927-9. DOI: 10.2144/000112812. View

10.

Castro R, Van Lammeren A, Groot S, Bino R, Hilhorst H . Cell division and subsequent radicle protrusion in tomato seeds are inhibited by osmotic stress but DNA synthesis and formation of microtubular cytoskeleton are not. Plant Physiol. 2000; 122(2):327-36. PMC: 58870. DOI: 10.1104/pp.122.2.327. View

11.

Lucretti S, Nardi L, Nisini P, Moretti F, Gualberti G, Dolezel J . Bivariate flow cytometry DNA/BrdUrd analysis of plant cell cycle. Methods Cell Sci. 2000; 21(2-3):155-66. DOI: 10.1023/a:1009893008892. View

12.

Taylor J, Woods P, HUGHES W . THE ORGANIZATION AND DUPLICATION OF CHROMOSOMES AS REVEALED BY AUTORADIOGRAPHIC STUDIES USING TRITIUM-LABELED THYMIDINEE. Proc Natl Acad Sci U S A. 1957; 43(1):122-8. PMC: 528395. DOI: 10.1073/pnas.43.1.122. View

13.

Kolb H, Sharpless K . The growing impact of click chemistry on drug discovery. Drug Discov Today. 2003; 8(24):1128-37. DOI: 10.1016/s1359-6446(03)02933-7. View

14.

Morocz S, Donn G, Nerneth J, Dudits D . An improved system to obtain fertile regenerants via maize protoplasts isolated from a highly embryogenic suspension culture. Theor Appl Genet. 2013; 80(6):721-6. DOI: 10.1007/BF00224183. View

15.

STROOBANTS C, Sossountzov L, Miginiac E . DNA synthesis in excised tobacco leaves after bromodeoxyuridine incorporation: immunohistochemical detection in semi-thin spurr sections. J Histochem Cytochem. 1990; 38(5):641-7. DOI: 10.1177/38.5.2185311. View

16.

Vanstraelen M, Baloban M, Da Ines O, Cultrone A, Lammens T, Boudolf V . APC/C-CCS52A complexes control meristem maintenance in the Arabidopsis root. Proc Natl Acad Sci U S A. 2009; 106(28):11806-11. PMC: 2710644. DOI: 10.1073/pnas.0901193106. View

17.

Chapman S, Oparka K, Roberts A . New tools for in vivo fluorescence tagging. Curr Opin Plant Biol. 2005; 8(6):565-73. DOI: 10.1016/j.pbi.2005.09.011. View

18.

Kaiser C, Kamien A, Shah P, Chapman B, Cotanche D . 5-Ethynyl-2'-deoxyuridine labeling detects proliferating cells in the regenerating avian cochlea. Laryngoscope. 2009; 119(9):1770-5. PMC: 2737075. DOI: 10.1002/lary.20557. View

19.

Sasaki K, Adachi S, Yamamoto T, Murakami T, Tanaka K, Takahashi M . Effects of denaturation with HCl on the immunological staining of bromodeoxyuridine incorporated into DNA. Cytometry. 1988; 9(1):93-6. DOI: 10.1002/cyto.990090115. View

20.

Zhang C, Gong F, Lambert G, Galbraith D . Cell type-specific characterization of nuclear DNA contents within complex tissues and organs. Plant Methods. 2005; 1(1):7. PMC: 1277020. DOI: 10.1186/1746-4811-1-7. View