Optimization of low-input flow cytometry protocols for Arabidopsis thaliana leaf nuclei

Introduction

Ionizing radiation is known to cause diverse types of damage in living cells, including DNA strand breaks and disruption of the cell cycle [1]. Flow cytometry is a key method to quantify nuclear DNA content and endoreduplication in plants, but standard protocols often require relatively large amounts of tissue and are not optimized for high-throughput screening of many genotypes. To be applicable to large-scale experiments, the protocol must be optimized to work with low amounts of sample material while still generating informative metrics that allow reliable statistical analyses.

Objectives

This project aims to develop and benchmark a robust, low-input flow cytometry protocol for Arabidopsis thaliana that can later be applied to large panels in our lab. Building on published methods for A. thaliana leaf ploidy analysis and plant DNA content measurement [2-4], the student will perform focused experiments to grow plant material and systematically test how buffer composition, mechanical disruption, tissue amount and sample handling affect data quality. Nuclei will be isolated from small leaf pieces, stained with DNA-binding dyes and analysed by flow cytometry to assess ploidy profiles. Data quality will be quantified using the coefficient of variation (CV) of the 2C peak, nuclei yield and debris levels. The optimized protocol will be validated on a small set of A. thaliana accessions or mutants as a pilot for future experiments. The project combines practical lab work, basic data analysis and protocol development, and should result in a “ready-to-use” standard operating procedure for low-input plant flow cytometry in our group.

References

[1] Duarte, G.T., Volkova, P.Y., Fiengo Perez, F., Horemans, N., 2023. Chronic ionizing radiation of plants: An evolutionary factor from direct damage to non-target effects. Plants 12(5), 1178.

[2] Yang, L., Wang, Z., Hua, J. 2019. Measuring Cell Ploidy Level in Arabidopsis thaliana by Flow Cytometry. In: Gassmann, W. (eds) Plant Innate Immunity. Methods in Molecular Biology, vol 1991. Humana, New York, NY.

[3] Cousin A, Heel K, Cowling WA, Nelson MN. 2009. An efficient high-throughput flow cytometric method for estimating DNA ploidy level in plants. Cytometry A. 75(12):1015-9.

[4] Loureiro J, Kron P, Temsch EM, et al. 2021. Isolation of plant nuclei for estimation of nuclear DNA content: Overview and best practices. Cytometry. 99: 318–327.