Cell cycle regulation in different leaves of Arabidopsis thaliana plants grown under control and cadmium-exposed conditions

Cadmium (Cd) is well known to inhibit vegetative plant growth. However, knowledge regarding its influence on the cell cycle is scarce and mainly limited to cell cultures and root tissue. Therefore, the main aim of this study was to investigate the effects of Cd exposure on cell division and endoreduplication in Arabidopsis thaliana leaves. In order to do so, we first investigated whether different leaves of the same rosette harvested at one time point (i.e. leaf position) could be used as an alternative for leaf age when investigating the cell cycle. To this end, wild-type A. thaliana plants were grown hydroponically with or without the addition of 5 μM CdSO4. Leaf growth and development, cell division, endoreduplication and the expression of cell cycle-related genes were investigated in separate leaves. The results show that different leaf positions constitute a developmental series that can be used to deduce the development of a single leaf over time. Furthermore, our data indicate that the concentration of nuclei in leaf extracts measured via flow cytometry can be used as a proxy to determine the effects of stress factors on the extent of cell division in A. thaliana leaves, reducing the need to perform time consuming microscopic analyses. Finally, we show that Cd exposure significantly reduces cell number, cell size and nuclear DNA content, implying an inhibition of both cell division and endoreduplication. These effects accumulate over time and contribute to the Cd-induced disturbance of leaf growth and development. At the molecular and cellular level, Cd increases hydrogen peroxide levels and induces the expression of marker genes for oxidative stress and DNA damage and genes encoding CDK inhibitors of the SIAMESE-RELATED family, suggesting that the Cd-induced inhibition of cell cycle progression is intertwined with oxidative stress and subsequent DNA damage.