title: A flow cytometric method for estimating S-phase duration in plants.
abstract: The duration of the DNA synthesis stage (S phase) of the cell cycle is fundamental in our understanding of cell cycle kinetics, cell proliferation, and DNA replication timing programs. Most S-phase duration estimates that exist for plants are based on indirect measurements. We present a method for directly estimating S-phase duration by pulse-labeling root tips or actively dividing suspension cells with the halogenated thymidine analog 5-ethynl-2'-deoxyuridine (EdU) and analyzing the time course of replication with bivariate flow cytometry. The transition between G1and G2DNA contents can be followed by measuring the mean DNA content of EdU-labeled S-phase nuclei as a function of time after the labeling pulse. We applied this technique to intact root tips of maize (Zea mays L.), rice (Oryza sativa L.), barley (Hordeum vulgare L.), and wheat (Triticum aestivum L.), and to actively dividing cell cultures of Arabidopsis (Arabidopsis thaliana (L.) Heynh.) and rice. Estimates of S-phase duration in root tips were remarkably consistent, varying only by ~3-fold, although the genome sizes of the species analyzed varied >40-fold.
authors.affiliation: Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC 27695, USA lamickel@ncsu.edu.
authors.forename: Leigh
authors.initials: L
authors.lastname: Mickelson-Young
authors.affiliation: Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Emily
authors.initials: E
authors.lastname: Wear
authors.affiliation: Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Patrick
authors.initials: P
authors.lastname: Mulvaney
authors.affiliation: Department of Horticultural Science, North Carolina State University, Raleigh, NC 27695, USA.|Present address: Syngenta Crop Protection, LLC, Research Triangle Park, NC 27709, USA.
authors.forename: Tae-Jin
authors.initials: TJ
authors.lastname: Lee
authors.affiliation: Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC 27695, USA.|Present address: Department of Biochemistry, Duke University, Durham, NC 27710, USA.
authors.forename: Eric S
authors.initials: ES
authors.lastname: Szymanski
authors.affiliation: Department of Horticultural Science, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: George
authors.initials: G
authors.lastname: Allen
authors.affiliation: Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Linda
authors.initials: L
authors.lastname: Hanley-Bowdoin
authors.affiliation: Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: William
authors.initials: W
authors.lastname: Thompson
mesh_terms: D017360:Arabidopsis / Q000166:cytology / Q000254:growth & development; D018744:DNA, Plant / Q000378:metabolism; D003857:Deoxyuridine / Q000031:analogs & derivatives / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D016193:G1 Phase / Q000502:physiology; D016195:G2 Phase / Q000502:physiology; D001467:Hordeum / Q000166:cytology / Q000254:growth & development; D018519:Meristem / Q000166:cytology / Q000254:growth & development; D012275:Oryza / Q000166:cytology / Q000254:growth & development; D016196:S Phase / Q000502:physiology; D014908:Triticum / Q000166:cytology / Q000254:growth & development; D003313:Zea mays / Q000166:cytology / Q000254:growth & development
chemical_list: D018744:DNA, Plant; C031086:5-ethynyl-2'-deoxyuridine; D003857:Deoxyuridine
keywords: Arabidopsis; EdU; S-phase duration; barley; flow cytometry; maize; replication timing; rice; wheat.