Abstract
Plant root development is a complex spatial-temporal process that originates in the root apical meristem (RAM). To shape the organ’s structure signaling within the different cells and the different cell files must be coordinated. Thereby, diverging kinetics of cell growth in these files needs to be integrated with differential cell growth and local differences in cell proliferation frequency. Understanding the local differences in cell cycle duration in the RAM is crucial to build a holistic view on the different regulatory processes and requires a quantitative estimation of the underlying mitotic cell cycle phases’ timing at every cell file and every position. Unfortunately, so far precise methods for such analysis are missing.
This study presents a robust and straightforward pipeline to determine simultaneously the duration of the cell cycle’s key stages in all cell layers of a plant’s root. The technique combines marker-free experimental techniques based on detection of incorporation of 5-ethynyl-2′-deoxyuridine (EdU) and mitosis with a deep-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. The subsequent G2-phase duration however 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 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 to be in the range of 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.
