Regulated cell death (RCD) is a gene-regulated process that allows plants to grow and develop and adapt to stressful conditions. It has recently been shown that even in the unicellular green alga Chlamydomonas reinhardtii, processes similar to regulated cell death in plants are observed under certain stress conditions.
Alga C. reinhardtii is a model organism for researching various physiological processes such as photosynthesis, respiration, adaptation to various radiations, osmotic and oxidative stress. In C. reinhardtii, regulated cell death may be observed under several stressful conditions, such as the addition of hydrogen peroxide or acetic acid, and the characteristics of RCD may also be observed after irradiation with UV light. Exposure of C. reinhardtii cells to various radiations, such as UV light, causes morphologically characteristic changes such as an increase in vacuole volume, DNA laddering, and the presence of phosphatidylserine on the outside of the cell membrane.
Since the mechanisms that lead to regulated cell death in this organism are not clear yet, we wanted to establish a new method for inducing oxidative stress that would facilitate our study of the RCD process in C. reinhardtii. C. reinhardtii cells were therefore irradiated with UV light for different time periods to determine the radiation exposure associated with RCD-related processes. The parameters monitored in cells irradiated with UV light are cell survival after irradiation and nuclease and proteolytic activity. After irradiation of the cells with UV light, morphological changes were observed with a light microscope. We observed an increase in the volume of vacuoles in cells that have been exposed to the stressor for a long time. Agarose gel electrophoresis allowed us to verify the activity of nucleases that degrade genomic DNA into ordered fragments of equal DNA sizes. The formation of ordered DNA fragments is a characteristic feature of cells in which RCD has occurred. By performing proteolytic assay, we monitored the activity of proteases with tripsin-like activity, which were presumed to be crucial in RCDs. Unfortunately, we chose a rather short irradiation time of cells with UV light and a nonspecific substrate (Z-FR-AMC), which coved be the reason why no difference in protease activity was observed.
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