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A simple pipeline for cell cycle kinetic studies in the root apical meristem.

Root system architecture ultimately depends on precise signaling between different cells and tissues in the root apical meristem (RAM), and through its integration with environmental cues. This study describes a simple pipeline to simultaneously determine cellular parameters, nuclei geometry and cell cycle kinetics in the RAM. The method uses marker-free techniques for nucleus and cell boundaries detection, and 5-ethynyl-2'-deoxyuridine (EdU) staining for DNA replication quantification. Based on this approach, we characterized differences in cell volume, nucleus volume and nucleus shape across different domains of the Arabidopsis thaliana RAM. We found that DNA replication patterns were cell-layer and region dependent. G2 phase duration, which varied from 3.5 h in the pericycle to more than 4.5 h in the epidermis, was found associated with some nuclei geometry features. Endocycle duration was determined as the time required to achieve 100% EdU positive cells in the elongation zone and, as such, it was estimated to be in the range of 5 h for the epidermis and cortex. This experimental pipeline could be used to precisely map cell cycle duration in the RAM of mutants and in response to environmental stress in several plant species without the need for introgressing molecular cell cycle markers.