Estimation of cell cycle kinetics in higher plant root meristem links organ position with cellular fate and chromatin structure

Abstract

Root development is a complex spatial-temporal process that originates in the root apical meristem (RAM). To keep the organ’s shape in harmony, the different cell files’ growth must be coordinated. Thereby, diverging kinetics of cell growth in these files may be obtained not only by differential cell growth but also by local differences in cell proliferation frequency. Understanding potential local differences in cell cycle duration in the RAM requires a quantitative estimation of the underlying mitotic cell cycle phases’ timing at every cell file and every position. However, so far, precise methods for such analysis are missing.This study presents a robust and straightforward method to determine the duration of the cell cycle’s key stages in all cell layers in a plant’s root simultaneously. The technique combines marker-free experimental techniques based on detection of incorporation of 5-ethynyl-2’-deoxyuridine (EdU) and mitosis with a high-resolution plant phenotyping platform to analyze all key cell cycle events’ kinetics.In the Arabidopsis thaliana L. RAM S-phase duration was found to be as short as 18-20 minutes in all cell files. But subsequent G2-phase duration depends on the cell type/position and varies from 3,5 hours in the pericycle to more than 4,5 hours in the epidermis. Overall, S+G2+M duration in Arabidopsis under our condition is 4 hours in the pericycle and up to 5,5 hours in the epidermis.Endocycle duration was determined as the time required to achieve 100% EdU index in the transition zone and estimated as 3-4 hours.Besides Arabidopsis, we show that the presented technique is applicable also to root tips of other dicot and monocot plants (tobacco (Nicotiana tabacum L.), tomato (Lycopersicon esculentum L.) and wheat (Triticum aestivum L.)).

Methods forin situquantitative root biology

“…In conclusion, module II allows us to quantify cell cycle events in situ for all cell types and calculate the kinetics of the cell cycle (Pasternak et al 2021a).…”

“…Van’t Hof (1967) further modified this method to accumulate cells in mitosis by additional colchicine treatment. Nowadays, 5-ethynyl-20-deoxyuridine (EdU) incorporation has been used to study cell cycle kinetics in the root (Buck et al 2008; Pasternak et al 2021a). A detailed study of the primary root (PR) meristem of the Arabidopsis plant model was published by Dolan et al (1993) one century after the pioneer work of Maxwell (1893).…”

A perspective on cell proliferation kinetics in the root apical meristem

“…More recently, continuous treatments with nucleoside analogues, e.g. 5-ethynyl-2′-deoxyuridine (EdU), more friendly than radioactive compounds, have become widespread and the average values obtained for Arabidopsis were ~17–20 h in some reports ( Hayashi et al , 2013 ; Eekhout et al , 2021 ) and much shorter, ~8–10 h, depending on the cell type, in others ( Pasternak et al , 2021 , Preprint). While these measurements provide an approximation to the average values of T and the cell division rates in different cell types, they fail to be sufficiently reliable when these parameters are to be determined at different positions along the RAM (for a detailed review, see Baskin, 2000 ).…”

Cyclin-Dependent Kinases and CTD Phosphatases in Cell Cycle Transcriptional Control: Conservation across Eukaryotic Kingdoms and Uniqueness to Plants

“…Similar to human cells, the cell cycle lengths of plant cells, with a size range of 10–100 µm, can also vary, depending on the cell types. Recent studies of Arabidopsis root cells found that their cell cycles are in the range of approximately 9–10 h, which is similar in tomato, tobacco, and other plant species with bigger genomes than Arabidopsis [ 8 ]. While humans have a relatively longer G1 phase (46% of the total cell cycle duration) than budding yeast (23% of the cycle duration), the absolute time for DNA duplication in yeast is understandably shorter than in humans, given that the yeast genome (12 Mb) is considerably smaller than that in humans (3 Gb).…”