Evaluation of UV damage at DNA level in Nicotiana plumbaginifolia protoplasts using single cell gel electrophoresis

The aim of the present study was to observe the induction and repair of single strand breaks (Ssbs) and double strand breaks (Dsbs) in mesophyll protoplasts of Nicotiana plumbaginifolia, irradiated with UV-C and cultured under light or dark conditions. DNA damage and repair was determined by the neutral and alkaline comet assay to reveal Dsbs and Ssbs respectively. Subculturing protoplasts for 4 h at low temperature was essential to reduce the amount of Dsbs to the detection limit of the assay procedure. Light-cultured protoplasts showed a significant increase of Ssbs and Dsbs compared to dark cultured protoplasts, in which the number of Ssbs and Dsbs remained very constant throughout the experiments. UV treatment significantly enhanced the levels of Ssbs and Dsbs in light and dark cultured protoplasts. On average, equal levels of DNA damage were observed under light or dark conditions. Formulations introduced to evaluate the contribution of UV-C or light treatment in repair kinetics of DNA damage, showed that the number of Ssbs, but not of Dsbs, evolved differently for light and dark cultured protoplasts. DNA repair was more rapidly observed under light conditions and occurred in different repair phases. Observations are discussed in relation to the involvement of chromatin remodelling, photosynthetic active radiation and DNA repair mechanisms.