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The selection and firing of DNA replication origins play key roles in ensuring that eukaryotes accurately replicate their genomes. This process is not well documented in plants due in large measure to difficulties in working with plant systems. We developed a new functional assay to label and map very early replicating loci that must, by definition, include at least a subset of replication origins. Arabidopsis (Arabidopsis thaliana) cells were briefly labeled with 5-ethynyl-2′-deoxy-uridine, and nuclei were subjected to two-parameter flow sorting. We identified more than 5500 loci as initiation regions (IRs), the first regions to replicate in very early S phase. These were classified as strong or weak IRs based on the strength of their replication signals. Strong initiation regions were evenly spaced along chromosomal arms and depleted in centromeres, while weak initiation regions were enriched in centromeric regions. IRs are AT-rich sequences flanked by more GC-rich regions and located predominantly in intergenic regions. Nuclease sensitivity assays indicated that IRs are associated with accessible chromatin. Based on these observations, initiation of plant DNA replication shows some similarity to, but is also distinct from, initiation in other well-studied eukaryotic systems.

DNA replication during S phase in eukaryotes is a highly regulated process that ensures the accurate transmission of genetic material to daughter cells during cell division. Replication follows a well-defined temporal program, which has been studied extensively in humans, Drosophila, and yeast, where it is clear that the replication process is both temporally and spatially ordered. The replication timing (RT) program is increasingly considered to be a functional readout of genomic features and chromatin organization. Although there is increasing evidence that plants display important differences in their DNA replication process compared to animals, RT programs in plants have not been extensively studied. To address this deficiency, we developed an improved protocol for the genome-wide RT analysis by sequencing newly replicated DNA ("Repli-seq") and applied it to the characterization of RT in maize root tips. Our protocol uses 5-ethynyl-2'-deoxyuridine (EdU) to label replicating DNA in vivo in intact roots. Our protocol also eliminates the need for synchronization and frequently associated chemical perturbations as well as the need for cell cultures, which can accumulate genetic and epigenetic differences over time. EdU can be fluorescently labeled under mild conditions and does not degrade subnuclear structure, allowing for the differentiation of labeled and unlabeled nuclei by flow sorting, effectively eliminating contamination issues that can result from sorting on DNA content alone. We also developed an analysis pipeline for analyzing and classifying regions of replication and present it in a point-and-click application called Repliscan that eliminates the need for command line programming.

DNA replication during S phase in eukaryotes is a highly regulated process that ensures the accurate transmission of genetic material to daughter cells during cell division. Replication follows a well-defined temporal program, which has been studied extensively in humans, Drosophila, and yeast, where it is clear that the replication process is both temporally and spatially ordered. The replication timing (RT) program is increasingly considered to be a functional readout of genomic features and chromatin organization. Although there is increasing evidence that plants display important differences in their DNA replication process compared to animals, RT programs in plants have not been extensively studied. To address this deficiency, we developed an improved protocol for the genome-wide RT analysis by sequencing newly replicated DNA ("Repli-seq") and applied it to the characterization of RT in maize root tips. Our protocol uses 5-ethynyl-2'-deoxyuridine (EdU) to label replicating DNA in vivo in intact roots. Our protocol also eliminates the need for synchronization and frequently associated chemical perturbations as well as the need for cell cultures, which can accumulate genetic and epigenetic differences over time. EdU can be fluorescently labeled under mild conditions and does not degrade subnuclear structure, allowing for the differentiation of labeled and unlabeled nuclei by flow sorting, effectively eliminating contamination issues that can result from sorting on DNA content alone. We also developed an analysis pipeline for analyzing and classifying regions of replication and present it in a point-and-click application called Repliscan that eliminates the need for command line programming.

Eukaryotes use a temporally regulated process, known as the replication timing program, to ensure that their genomes are fully and accurately duplicated during S phase. Replication timing programs are predictive of genomic features and activity and are considered to be functional readouts of chromatin organization. Although replication timing programs have been described for yeast and animal systems, much less is known about the temporal regulation of plant DNA replication or its relationship to genome sequence and chromatin structure. We used the thymidine analog, 5-ethynyl-2'-deoxyuridine, in combination with flow sorting and Repli-Seq to describe, at high-resolution, the genome-wide replication timing program for Arabidopsis (Arabidopsis thaliana) Col-0 suspension cells. We identified genomic regions that replicate predominantly during early, mid, and late S phase, and correlated these regions with genomic features and with data for chromatin state, accessibility, and long-distance interaction. Arabidopsis chromosome arms tend to replicate early while pericentromeric regions replicate late. Early and mid-replicating regions are gene-rich and predominantly euchromatic, while late regions are rich in transposable elements and primarily heterochromatic. However, the distribution of chromatin states across the different times is complex, with each replication time corresponding to a mixture of states. Early and mid-replicating sequences interact with each other and not with late sequences, but early regions are more accessible than mid regions. The replication timing program in Arabidopsis reflects a bipartite genomic organization with early/mid-replicating regions and late regions forming separate, noninteracting compartments. The temporal order of DNA replication within the early/mid compartment may be modulated largely by chromatin accessibility.

Plant cells undergo two types of cell cycles–the mitotic cycle in which DNA replication is coupled to mitosis, and the endocycle in which DNA replication occurs in the absence of cell division. To investigate DNA replication programs in these two types of cell cycles, we pulse labeled intact root tips of maize (Zea mays) with 5-ethynyl-2’-deoxyuridine (EdU) and used flow sorting of nuclei to examine DNA replication timing (RT) during the transition from a mitotic cycle to an endocycle. Comparison of the sequence-based RT profiles showed that most regions of the maize genome replicate at the same time during S phase in mitotic and endocycling cells, despite the need to replicate twice as much DNA in the endocycle and the fact that endocycling is typically associated with cell differentiation. However, regions collectively corresponding to 2% of the genome displayed significant changes in timing between the two types of cell cycles. The majority of these regions are small with a median size of 135 kb, shift to a later RT in the endocycle, and are enriched for genes expressed in the root tip. We found larger regions that shifted RT in centromeres of seven of the ten maize chromosomes. These regions covered the majority of the previously defined functional centromere, which ranged between 1 and 2 Mb in size in the reference genome. They replicate mainly during mid S phase in mitotic cells but primarily in late S phase of the endocycle. In contrast, the immediately adjacent pericentromere sequences are primarily late replicating in both cell cycles. Analysis of CENH3 enrichment levels in 8C vs 2C nuclei suggested that there is only a partial replacement of CENH3 nucleosomes after endocycle replication is complete. The shift to later replication of centromeres and possible reduction in CENH3 enrichment after endocycle replication is consistent with a hypothesis that centromeres are inactivated when their function is no longer needed., Author summary In traditional cell division, or mitosis, a cell’s genetic material is duplicated and then split between two daughter cells. In contrast, in some specialized cell types, the DNA is duplicated a second time without an intervening division step, resulting in cells that carry twice as much DNA. This phenomenon, which is called the endocycle, is common during plant development. At each step, DNA replication follows an ordered program in which highly compacted DNA is unraveled and replicated in sections at different times during the synthesis (S) phase. In plants, it is unclear whether traditional and endocycle programs are the same, especially since endocycling cells are typically in the process of differentiation. Using root tips of maize, we found that in comparison to replication in the mitotic cell cycle, there is a small portion of the genome whose replication in the endocycle is shifted in time, usually to later in S phase. Some of these regions are scattered around the genome and mostly coincide with active genes. However, the most prominent shifts occur in centromeres. The shift to later replication in centromeres is noteworthy because they orchestrate the process of separating duplicated chromosomes into daughter cells, a function that is not needed in the endocycle.

International audience; 5-methyl cytosine is widespread in plant genomes in both CG and non-CG contexts. During replication, hemi-methylation on parental DNA strands guides symmetric CG methylation on nascent strands, but non-CG methylation requires modified histones and small RNA guides. Here, we used immortalized Arabidopsis cell suspensions to sort replicating nuclei and determine genome-wide cytosine methylation dynamics during the plant cell cycle. We find that symmetric mCG and mCHG are selectively retained in actively dividing cells in culture, whereas mCHH is depleted. mCG becomes transiently asymmetric during S phase but is rapidly restored in G2, whereas mCHG remains asymmetric throughout the cell cycle. Hundreds of loci gain ectopic CHG methylation, as well as 24-nt small interfering RNAs (siRNAs) and histone H3 lysine dimethylation (H3K9me2), without gaining CHH methylation. This suggests that spontaneous epialleles that arise in plant cell cultures are stably maintained by siRNA and H3K9me2 independent of the canonical RNA-directed DNA methylation (RdDM) pathway. In contrast, loci that fail to produce siRNA may be targeted for demethylation when the cell cycle arrests. Comparative analysis with methylomes of various tissues and cell types suggests that loss of small-RNA-directed non-CG methylation during DNA replication promotes germline reprogramming and epigenetic variation in plants propagated as clones.

All plants and animals must replicate their DNA, using a regulated process to ensure that their genomes are completely and accurately replicated. DNA replication timing programs have been extensively studied in yeast and animal systems, but much less is known about the replication programs of plants. We report a novel adaptation of the "Repli-seq" assay for use in intact root tips of maize (Zea mays) that include several different cell lineages, and present replication profiles from cells in early, mid, and late S phase of the mitotic cell cycle. This represents the first genomic analysis of replication timing in a crop species, and the first in any plant species to characterize replication at the whole-genome level. Maize root tips have a complex replication timing program, including regions of distinct early, mid and late-S replication that each constitute between 20-24% of the genome, as well as other loci corresponding to ~32% of the genome that exhibit replication activity in two different time windows. Analyses of genomic, transcriptional, and chromatin features of the euchromatic portion of the maize genome provide evidence for a gradient of early replicating, open chromatin that transitions gradually to less open and less transcriptionally active chromatin replicating in mid S phase. Our genomic level analysis also demonstrated that the centromere core replicates in mid S, before heavily compacted classical heterochromatin, including pericentromeres and knobs, which replicate during late S phase.

Highlight We estimated S-phase duration for several plant species by following EdU-labeled nuclei from G1 to G2 using bivariate flow cytometry. S-phase duration is relatively consistent over a range of genome sizes., 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 G1 and G2 DNA 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.

5-Ethynyl-2'-deoxyuridine (EdU) is a nucleoside analog of thymidine that can be rapidly incorporated into replicating DNA in vivo and, subsequently, detected by using "click" chemistry to couple its terminal alkyne group to fluorescent azides such as Alexa Fluor 488. Recently, EdU incorporation followed by coupling with a fluorophore has been used to visualize newly synthesized DNA in a wide range of plant species. One particularly useful application is in flow cytometry, where two-parameter sorting can be employed to analyze different phases of the cell cycle, as defined both by total DNA content and the amount of EdU pulse-labeled DNA. This approach allows analysis of the cell cycle without the need for synchronous cell populations, which can be difficult to obtain in many plant systems. The approach presented here, which was developed for fixed, EdU-labeled nuclei, can be used to prepare analytical profiles as well as to make highly purified preparations of G1, S, or G2/M phase nuclei for molecular or biochemical analysis. We present protocols for EdU pulse labeling, tissue fixation and harvesting, nuclei preparation, and flow sorting. Although developed for Arabidopsis suspension cells and maize root tips, these protocols should be modifiable to many other plant systems.

The progress of nuclear DNA replication is complex in both time and space, and may reflect several levels of chro-matin structure and 3-dimensional organization within the nucleus. To understand the relationship between DNA rep-lication 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 thymi-dine 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.Key words

Spatiotemporal patterns of DNA replication have been described for yeast and many types of cultured animal cells, frequently after cell cycle arrest to aid in synchronization. However, patterns of DNA replication in nuclei from plants or naturally developing organs remain largely uncharacterized. Here we report findings from 3D quantitative analysis of DNA replication and endoreduplication in nuclei from pulse-labeled developing maize root tips. In both early and middle S phase nuclei, flow-sorted on the basis of DNA content, replicative labeling was widely distributed across euchromatic regions of the nucleoplasm. We did not observe the perinuclear or perinucleolar replicative labeling patterns characteristic of middle S phase in mammals. Instead, the early versus middle S phase patterns in maize could be distinguished cytologically by correlating two quantitative, continuous variables, replicative labeling and DAPI staining. Early S nuclei exhibited widely distributed euchromatic labeling preferentially localized to regions with weak DAPI signals. Middle S nuclei also exhibited widely distributed euchromatic labeling, but the label was preferentially localized to regions with strong DAPI signals. Highly condensed heterochromatin, including knobs, replicated during late S phase as previously reported. Similar spatiotemporal replication patterns were observed for both mitotic and endocycling maize nuclei. These results revealed that maize euchromatin exists as an intermingled mixture of two components distinguished by their condensation state and replication timing. These different patterns might reflect a previously described genome organization pattern, with “gene islands” mostly replicating during early S phase followed by most of the intergenic repetitive regions replicating during middle S phase. Electronic supplementary material The online version of this article (doi:10.1007/s11103-015-0364-4) contains supplementary material, which is available to authorized users.