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

Abstract

BackgroundProgress 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.ResultsApplications 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.ConclusionsIn 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.

Biological differences that distinguish the 2 major stages of wound healing in potato tubers

“…10 Important and surprisingly, sensitive 5-ethynyl-2 0 -deoxyuridine (EdU) labeling for detection of tuber nuclei that were wound-induced to synthesize DNA showed that de novo DNA synthesis occurred with a maximum number of nuclei induced into the S-phase of the cell cycle by 18 h after wounding (Table 1). 11,12 The number of nuclei in the S-phase rapidly decreased and was near zero by 48 h after wounding; this was roughly 5 d prior to the creation of a nascent phellogen and induction of cell division as a distinguishing part of wound periderm biosynthesis. This period of DNA biosynthesis translates to about 30 h in which induced cells progress through the S-phase with cessation of DNA biosynthesis about 4 d prior to completion of CLF and about 5 d prior to initiation of cell division and associated WPF.…”

A Secreted Effector Protein ofUstilago maydisGuides Maize Leaf Cells to Form Tumors

“…Triton X-100 treatment results in a nuclear stain and is compatible with EdU costaining. Moreover, it prevents the cell shrinkage and quick penetration of the fixer and allows better preservation of mitotic stages (Kotogány et al, 2010).…”

Genomic Analysis of the DNA Replication Timing Program during Mitotic S Phase in Maize (Zea mays) Root Tips

“…A key benefit of using EdU is that a heat or acid denaturation step is not required for immunoprecipitation (reviewed in Darzynkiewicz et al, 2011). Hence, it is possible to visualize EdU-labeled DNA while maintaining native subnuclear structure (Kotogany et al, 2010;Bass et al, 2014) and to separate labeled from unlabeled nuclei by flow cytometry prior to DNA isolation . We previously used the EdU labeling system to investigate the spatio-temporal aspects of DNA replication in proliferating root tip cells (Bass et al, 2015).…”