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.)).
Competing Interest Statement
The authors have declared no competing interest.
