A Spatiotemporal Analysis of Plant DNA Replication in Developing Root Tips

During plant development, somatic 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. We compared the patterns of replicating plant chromosomal DNA in S-phase nuclei from mitotic and endocycling cells. Maize root tips were incubated with the thymidine analog EdU to pulse-label replicating DNA. After fixation, nuclei were isolated and the EdU-labeled DNA was conjugated to an Alexa-488 fluorochrome. Mitotic and endocycling nuclei were then sorted into early, mid or late S-phase fractions based on DNA content and EdU incorporation. The temporal and spatial patterns of DNA synthesis of the different S-phase populations were analyzed using 3D deconvolution microscopy. Maize chromosomal DNA synthesis exhibited distinct patterns in early, mid and late S phase. During the mitotic cell cycle (2C to 4C), DNA replicated during early S phase was distributed throughout the nucleus and was primarily in the non-centromeric hemisphere and outside of the DAPI-stained 300-nm fibers. In contrast, DNA replicated in mid S-phase largely co-localized with the 300 nm fibers, while DNA replicated in late S phase was limited to knob and centromere-associated heterochromatin. Surprisingly, these stage-specific, S-phase patterns were preserved in endocycling nuclei (4C to 8C), indicating that a similar replication program persists in the endocycle. The distinctive temporal DNA replication patterns established that the maize root tip system is a tractable model for elucidating functional and regulatory features of plant DNA replication.