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. 2010 Jan 28;6(1):5.
doi: 10.1186/1746-4811-6-5.

A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine

Edit Kotogány  1 , Dénes Dudits, Gábor V Horváth, Ferhan Ayaydin
Affiliations

A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine

Edit Kotogány et al. Plant Methods. .

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.

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Figures

Figure 1
Figure 1
Comparison of EdU-based and BrdU-based replication assays on Arabidopsis suspension culture. (A) EdU click reaction-based replication assay. Mild detection conditions without cell wall digestion or DNA digestion led to a better preserved cellular and nuclear morphology. Following 2 h 10 μM EdU incubation on 2d-old Arabidopsis culture, incorporated EdU was detected with click reaction using Alexa Fluor 488 azide (green). (B) BrdU-based replication assay on 2d-old Arabidopsis culture following 2 h 10 μM BrdU incubation. Harsh detection conditions resulted in partial loss of cellular and nuclear integrity. Alexa Fluor 488 antibody was used for immunodetection of BrdU (green). Nuclei were counterstained with DAPI and pseudocolored red. Arrows indicate nuclei with weak DNA staining. Differential interference contrast (DIC) transmission images were overlaid onto fluorescence images at the last panel. Scalebar: 10 μm.
Figure 2
Figure 2
EdU-based replication assay on 2d-old monocot and dicot plant cultures. EdU (10 μM, 2 h) incorporated cells were detected with click reaction using Alexa Fluor 488 azide (green panel). DNA is counterstained with DAPI (red). Transmission images (DIC) were overlaid onto fluorescence images at the last panel. (A) Alfalfa (M. sativa ssp. varia A2), (B) Arabidopsis (A. thaliana ecotype Columbia), (C) Grape (V. berlandieri × V. rupestris cv. 'Richter 110'), (D) Maize (Z. mays, cv. H1233), (E) Rice (O. sativa ssp. japonica cv. 'Unggi 9'), (F) Tobacco (N. tabacum cv. Petit Havana SR1). Arrows in green panel indicate spotty labeled early S-phase cells. Arrowheads in red panel show cells in mitosis. Scalebar: 10 μm.
Figure 3
Figure 3
Ratio of EdU-labeled and mitotic cells as dual parameter proliferation analysis of 2d-old plant cultures. EdU-labeled S-phase cells (in addition to cells which were recently in S-phase) and mitotic cells were scored as frequencies for the indicated cultures using EdU assay (green) and DAPI labeling (red), respectively. EdU was used at 10 μM concentration for 2 h. Inset photo shows an EdU-labeled maize cell (green) next to a cell in telophase stage of mitosis (red). In two independent experiments, more than 500 cells were counted for each culture. Scalebar: 10 μm.
Figure 4
Figure 4
The effect of EdU concentration and incubation duration on EdU labeling index of 36 h-old Arabidopsis cultures. EdU-Alexa Fluor 488-labeled cells were scored as frequencies for the indicated EdU concentrations and incubation durations following EdU assay on 36 h-old Arabidopsis cultures. In two independent experiments, more than 500 cells were counted for each experimental condition.
Figure 5
Figure 5
Cell cycle analysis with EdU using flow cytometry. Formaldehyde fixed (A-D and G) or unfixed (E, F) nuclei isolated from EdU-treated and untreated rice (A, B, E, F), alfalfa (C, D) and Arabidopsis (G) cultures were analyzed by flow cytometry. Uniparametric DNA histograms of propidium iodide intensity (FL2-A) are shown at upper panels. Lower panels show biparametric dot-plot analysis of Alexa Fluor 488-EdU intensity (FL1-H, logarithmic scale) versus propidium iodide intensity (FL2-H). Braces in biparametric plots indicate the total EdU-labeled population or background green fluorescence above EdU threshold (see Methods). Inset photos show fluorescence images of isolated nuclei counterstained with DAPI (red) following EdU assay with Alexa Fluor 488 azide (green). Scalebars are 5 μm. An example of quadrant thresholds is shown for biparametric plot of Figure 5B. The ratios of nuclei in each quadrant are displayed as percentage on the lower right corners of each biparametric plot. (G) Ratio of indicated biparametric fractions (quadrants Q1-Q4) after EdU (15' and 30') and DMSO treatment of Arabidopsis suspension cultures.
Figure 6
Figure 6
Quantitative analysis of EdU incorporation on rice root meristems using EdU assay. Rice roots (O. sativa L. ssp. japonica 'Nipponbare') were incubated for 2, 4, 6 hours with 20 μM EdU which was detected using Alexa Fluor 488 azide (green). (A) Fluorescence stereo microscopy images of EdU-labeled root tips. Transmission images of roots were pseudocolored red and merged with green EdU signal. Scalebar 300 μm. (B) Laser scanning confocal microscopy images of single optical sections of 6 μm (optical depth) on the median plane of rice root tips. Cell wall autofluorescence (red pseudocolor) and transmission images were overlaid onto green EdU signal. Inset photo shows a close-up of a 4 h EdU-treated cell with EdU-Alexa Fluor 488 labeled segregating chromosomes. Scalebar 300 μm. (C) Confocal images of EdU-labeled and DAPI-stained (DNA) cells from the meristem region of 2 h EdU treated root tips. Arrowheads (from left to right) indicate an anaphase, a prophase and a metaphase cell with well-preserved morphology. Scalebar 10 μm. (D) Quantitation of average EdU signal intensity of the meristem region after 2, 4 and 6 hours EdU incubation.
Figure 7
Figure 7
The effect of EdU concentration and incubation time on rice root tip EdU assay. EdU assay on rice (O. sativa L. ssp. japonica 'Nipponbare') root tips were shown for the indicated EdU concentrations and incubation durations. Control root tips (0 μM) were treated with 0.1% DMSO (vehicle) and incubated with Alexa488-containing assay cocktail to assess the level of nonspecific background and tissue autofluorescence in green channel. Cell wall autofluorescence (red pseudocolor) and transmission images were overlaid onto green EdU-Alexa Fluor 488 signal. Average green signal intensities (intensity/μm2) of the meristem regions were shown at the lower right corners. Higher detector sensitivity was used for green channel detection as compared to Figure 6B. Scalebar: 150 μm.
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