Subcellular localization of heavy metals in bog algae

Fresh water algae may be exposed to various heavy metals due to agricultural practices, industrial waste waters and atmospheric pollution. Whereas numerous studies have investigated heavy metal impact on physiological and molecular responses of algae, or on biomass production, only little information is so far available on subcellular localization of heavy metals in intra- or extracellular compartments. This lack in data is at least in part due to limited employment of analytical transmission electron microscopy, like electron energy loss spectroscopy in biological sciences so far. It is the central aim of this project to localize heavy metals such as copper, cadmium, aluminium, zinc, iron, chromium and lead in fresh water green algae after experimental exposure, by modern energy filtering techniques in combination with advanced cryo-preservations such as high pressure freeze fixation. This method does not only allow assignment of heavy metals to ultrastructural details at high resolution but also provides information on chemical bonding respectively on complexation of the metals. In contrast to numerous investigations on algae which are capable of hyperaccumulating heavy metals and are thus of economic interest for phytoremidiation, the objects of our project are freshwater green algae growing exclusively in oligotrophic acid bog ponds. Their natural habitats belong to the last intact ecosystems in Europe and are extremely endangered by heavy metal impact. Accumulation and subcellular distribution of heavy metals will be compared in three different algal species Micrasterias denticulata, Netrium digitus and Desmidium swartzii revealing different cell walls and growth modes. Localization of heavy metal binding shall be correlated to the distribution of cell wall or mucilage polymers such as pectins, to obtain insight into binding capacities. Moreover, intracellular sequestration into cell compartments and binding to ligands shall be studied. Short- and long-term effects of heavy metals on cell growth, division rates, biomass production, changes in the ultrastructure of organelles and secretion pathways as well as on photosynthetic activity shall be analysed. It shall be determined in which way environmental factors such as pH, temperature and salt concentration of the surrounding medium influence uptake and sequestration of the single metals. Possible defence mechanisms against heavy metal toxication such as increase in production of phytochelatines known to bind heavy metals, or involvement of the amino acid prolin in detoxification shall be elucidated.

Increasing traffic, industrial production and agricultural practices may lead to enhanced release of heavy metals into air, soil and water. Particularly natural habitats of algae like wetlands or peat bogs with their high atmospheric humidity are endangered by entry of metals via aerosols. Whereas numerous studies have focused so far on physiological changes, alterations in biomass production or molecular mechanisms of plant heavy metal response, it was the central aim of this project to analyze intracellular distribution of heavy metals in fresh water algae in order to get insight into heavy metal uptake, complexation, storage and possible detoxification mechanisms. Modern energy filtering electron microscopic techniques such electron energy loss spectroscopy (EELS) have been employed together with confocal laser scanning microscopy and different light and fluorescence microscopic techniques, physiological tests and other analytical methods. In the well investigated model alga Micrasterias denticulata it was shown that copper, cadmium, aluminium, zinc, iron, chromium and lead are taken up into the cells and compartmentalized to different degrees. Al and Pb for example were sequestered in the cell wall whereas Cu, Zn, Fe and Cd were additionally found inside different organelles and compartments. This indicates that the cell wall of Micrasterias may act as a kind of filter preventing higher amounts of metals entering the cytoplasm. As another detoxification mechanism of the alga, the study provided evidence for storage of metals in vacuoles and binding to small peptides, the so called phytochelatins in the same way as in higher plants. Cd which was not compartmentalized in Micrasterias at all and was thus distributed equally throughout the cytoplasm, turned out to be the most toxic one among the metals tested. It evoked its toxic effects in Micrasterias by disturbing calcium homeostasis and by inducing severe morphological and functional disorder in particular organelles, the dictyosomes. This result led to the start of 3-D analyses by a new electron microscopic technique (focused-ion-beam scanning electron microscopy; FIB-FESEM) in cooperation with the University of Munich, which will be continued in a follow-up project. Pb generated its toxic effect on growth of Micrasterias by changing the physical properties of the cell wall and Cr was measured inside of abnormal bag-like structures underneath the cell wall together with iron, indicating an extrusion mechanism for Cr in form of a chromium-iron-oxide. It was also shown during this project that many of the negative effects of metals on cell physiology and ultrastructure in Micrasterias could be totally or partly ameliorated or prevented by pre-addition of divalent cations such as Fe, Ca and Zn to the culture medium. The results of this project were published in several internationally reviewed journals and were presented during numerous meetings or during invited lectures at different universities. 5 master theses and one doctor thesis were completed during this project.