Biology subjects

Pasternak, T. P.

Publications and source records attributed to Pasternak, T. P..

3 recordsLinked to original sources

bioRxiv2021

Estimation of cell cycle kinetics in higher plant root meristem with cellular fate and positional resolution.

Plant root development is a complex spatial-temporal process that originates in the root apical meristem (RAM). To shape the organs structure signaling between the different cells and cell files must be highly coordinated. Thereby, diverging kinetics of chromatin remodeling and cell growth in these files need to be integrated and balanced by differential cell growth and local differences in cell proliferation frequency. Understanding the local differences in cell cycle duration in the RAM and its correlation with chromatin organization is crucial to build a holistic view on the different regulatory processes and requires a quantitative estimation of the chromatin geometry and 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 in parallel the duration of cell cycles key stages in all cell layers of a plants root and their nuclei organization. The methods combine marker-free techniques based on the detection of the nucleus, deep analysis of the chromatin phase transition, 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 20-30 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 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 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.).

cell biology↗

bioRxiv2020

Oxidative stress inducing agents copper and alloxan accelerate cell cycle re-entering of somatic plant cells in the presence of suboptimal exogenous auxin.

The physiological status of differentiated somatic plant cells and kinetics of re-entering in cell cycle were investigated in the case of Medicago sativa leaf protoplasts after the application of oxidative stress-inducing agents. Excess copper (30 M) and alloxan (0.5 mM) accelerated cell cycle re-entry at an exogenous auxin concentration that alone was insufficient to induce cell activation. Application of stress-inducing agents accelerated changes in the nuclei landscape with further faster re-entry in DNA replication and cytokinesis. This acceleration was accompanied by a lower level of reactive oxygen species (ROS) accumulations. At later stages, stress-agents treated cells resemble stem cells in planta with a smaller size, higher cell viability, lower ROS level, and lower activities of major ROS scavenging enzymes. A similar cellular response could be achieved by increasing the exogenous auxin concentration. Based on these experimental results, it is suggested that sub-lethal stress treatments evoke a transient cell state that accelerates cellular reprogramming. We also speculate that this transient cell state serves as an effective mechanism for protection against oxidative stress.

plant biology↗

bioRxiv2020

A simple high efficiency and low cost in vitro growth system for phenotypic characterization and seed propagation of Arabidopsis thaliana

BackgroundArabidopsis research relies extensively on the use of in vitro growth for phenotypic analysis of the seedlings and characterization of plant responses to intrinsic and extrinsic cues. For this purpose, stress-free optimal growth conditions should be set up and used as a reference especially in studies aimed at characterizing the plant responses to abiotic and biotic stresses. Currently used standard in vitro protocols for growth and characterization of Arabidopsis thaliana plants often suffer from sub-optimal composition due to an excessively high nutritional content which represents a stress per se and an experimental bias. ResultsWe describe a simple protocol for in vitro growth of Arabidopsis plants in which the phenotypic analysis is based on an optimized and nutritionally balanced culture medium. We show that the protocol is robustly applicable for growth of several Arabidopsis mutants, including mutants lacking the root system. This protocol enables rapid high scale seed production in vitro avoiding soil usage while saving space and time. The optimized in vitro protocol aims at: 1) making in vitro growth as close as possible to natural soil conditions by optimizing nutrient balance in the medium; 2) simplifying phenotypic and molecular investigation of individual plants by standardizing all steps of plant growth; 3) enabling seeds formation also in genotypes with severe defect in the root system; 4) minimizing the amount of waste and space for plant growth by avoiding soil usage. ConclusionsHere we report an optimized protocol for optimal growth of Arabidopsis thaliana plants to avoid biases in phenotypic observation of abiotic/biotic stress experiments. The protocol also enables the completion of the whole life cycle in vitro within 40-45 days and a satisfactory seed set for further propagation with no need for facilities for plant growth in soil and seed sterilisation.

plant biology↗