A maize root tip system to study DNA replication programmes in somatic and endocycling nuclei during plant development

Abstract

The progress of nuclear DNA replication is complex in both time and space, and may reflect several levels of chromatin structure and 3-dimensional organization within the nucleus. To understand the relationship between DNA replication and developmental programmes, it is important to examine replication and nuclear substructure in different developmental contexts including natural cell-cycle progressions in situ. Plant meristems offer an ideal opportunity to analyse such processes in the context of normal growth of an organism. Our current understanding of large-scale chromosomal DNA replication has been limited by the lack of appropriate tools to visualize DNA replication with high resolution at defined points within S phase. In this perspective, we discuss a promising new system that can be used to visualize DNA replication in isolated maize (Zea mays L.) root tip nuclei after in planta pulse labelling with the thymidine analogue, 5-ethynyl-2'-deoxyuridine (EdU). Mixed populations of EdU-labelled nuclei are then separated by flow cytometry into sequential stages of S phase and examined directly using 3-dimensional deconvolution microscopy to characterize spatial patterns of plant DNA replication. Combining spatiotemporal analyses with studies of replication and epigenetic inheritance at the molecular level enables an integrated experimental approach to problems of mitotic inheritance and cellular differentiation.

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

“…Most of the nuclei from the terminal 1-mm region had DNA contents ranging from 2C to 4C, characteristic of cells undergoing a mitotic cell cycle (Baluska, 1990). Approximately 20% of the nuclei had DNA contents above 4C, indicative of some cells transitioning into a programmed endocycle ( Figure 1C; Bass et al, 2014Bass et al, , 2015. In 1-mm root tips, nuclei from endocycling cells can be excluded based on DNA content as shown in Figure 1C and are not considered further in this article.…”

“…We developed a system (reviewed in Bass et al, 2014) to examine the spatial, temporal, and genomic patterns of DNA replication in maize, using rapidly cycling cells from root tips pulselabeled with the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU). EdU offers a substantial improvement over classical replication timing assays, which have typically used 5-bromo-2'-deoxyuridine (BrdU) to label and immunoprecipitate newly replicated DNA (Hiratani et al, 2008;Schwaiger et al, 2009;Chen et al, 2010;Hansen et al, 2010;Lee et al, 2010;Ryba et al, 2011).…”

“…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).…”

The Control of Arabidopsis thaliana Growth by Cell Proliferation and Endoreplication Requires the F-Box Protein FBL17

“…To detect cells actively replicating their DNA, we used 5-ethynyl-29-deoxyuridine (EdU), a nucleoside analog of thymidine (Bass et al, 2014). Strikingly, despite the fact that the fbl17 meristematic root zone appears short with fewer cells, quite a few of them showed EdU labeling, indicating that they entered into the S phase ( Figure 7A).…”

Development of novel dyes and fluorescence-imaging based approaches for cellular localization and dynamics of lipid droplets

“…The assay was also used to differentiate cells in early and late Sphase in root tips of Arabidopsis seedlings (Hayashi et al, 2013). The assay was successfully applied in alfalfa suspension cultured cells and root tips (Bazin et al, 2013), tomato root tips (Ron et al, 2013;Kuznetsova & Sheval', 2013), field bean root tips (Schubert et al, 2011), asparagus cladodes (Nakayama et al, 2012), tobacco suspension cultured cells (Tresch et al, 2011), rice suspension cultured cells (Dudits et al, 2011) and maize root tips (Bass et al, 2014). Further, this assay was used to…”