Aging and cell death in an unicellular alga

In contrast to numerous data on aging and programmed cell death (PCD) in higher plants published in the last years, there is a tremendous lack of information on cellular and molecular pathways leading to cell death in unicellular plant organisms. The theoretically "immortal" unicellular green alga Micrasterias is an ideal model system to obtain new insight, as cell development, morphogenesis, ultrastructure, involvement of organelles in cell growth, cytoskeleton function, secretion and environmental impact on cell differentiation are very well known. The proposed project is intended to find out whether unfavourable environmental conditions lead to aging or cell death processes in Micrasterias, possibly functioning as survival strategy for the population. Based on recent new observations of organelle degradation processes in Micrasterias which differ completely from "autophagy" known from other plant and animal organisms, the following four aims shall be attained during the project: 1. to analyse organelle turnover during the normal cell cycle. Mostly by means of new energy filtering transmission electron microscopic (EFTEM) techniques combined with immuno methods and confocal scanning laser microscopy details of organelle degradation via lytic compartments and via release through the plasma membrane shall be analysed in dependence of the cell age. 2. to clarify whether PCD occurs in Micrasterias under normal conditions and upon experimental induction. Different PCD detection assays again in combination with EFTEM shall be used to characterize PCD hallmarks. 3. cellular and molecular cell death pathways shall be analyzed by studying morphological and physiological changes of organelles such as mitochondria, nucleus, chloroplasts, ER, dictyosomes and the cytoskeleton. Key regulators of PCD shall be identified and localized by using specific inhibitors and antibodies, as well as by molecular biological investigations. In collaboration with the Genetics Division of our Department a cDNA library shall be constructed to identify genes involved in cell death events and to study there up or down regulation. 4. As soon as cell death pathways in Micrasterias have been identified environmental impact on PCD shall be investigated. It shall be probed which cell death pathways are activated upon exposure of the cells to nutrient deprivation, low light, temperature stress or increased salt concentration. The proposed studies are supposed to yield valuable information on cellular and ecological aspects of cell death and aging in plant organisms in general which may have implications on agriculture and human nutrition.

"Programmed cell death" defined by active metabolic processes leading to a concerted dying of cells and tissues are not only important for human and animal organisms but play an essential role also in development, reproduction, stress resistance and aging of plants. In higher plants, programmed cell death and its intracellular regulation are well investigated, yet in unicellular organisms and particularly in algae there are only a few documents about its occurrence and almost nothing is known about the pathways involved. However, knowledge of these cell death events is essential for understanding the evolutionary origin of survival strategies in biology. Subject of the investigations of the present project was the unicellular peat-bog green alga Micrasterias, which has served as a cell biological model system since many years and numerous data on development and growth regulation are already available. The alga is theoretically immortal as it reforms only one of its two semicells during division. This makes it particularly interesting to study whether a kind of "suicide program" may be induced by certain environmental or experimental conditions. Directed elimination of cells leading to conservation of the species by sufficient supply of a few individuals could present an important survival strategy for the alga. Various light- and electron microscopic techniques, measurements of physiological functions such as photosynthesis and respiration as well as molecular biological methods have been employed during this project in order to determine whether programmed cell death takes place in Micrasterias and by which hallmarks it is characterized. The use of different cell death inductors known form other plant or animal organisms showed that Micrasterias is capable of performing programmed cell death and that several of the processes involved are similar to those in higher plants. One of the main results of this study was that the course of the cell death pathway depends on the kind of the inductor. This means that if a cell is exposed to e.g. toxic oxygen radicals it responses with another "suicide program" than if it faces incompatible high salt concentrations. In the case of high salinity Micrasterias additionally exhibited a kind of "self destruction" by directed dissolving of cell components. Programmed cell death can be induced in the alga also by different heavy metals. Regulators of acetylcholine which are important signal compounds in the nervous system but also exist in plants, and nitric oxide, however, may lead to a form cell death in Micrasterias which can not be regarded as programmed. The results of this project have led to 8 publications in international scientific journals and have been presented during various scientific meetings. One PhD thesis and three master theses were completed during the project.