Rubinelli P¹, Patel S¹, Malek L², and Sayre RT¹

¹ Department of Plant Biology, Ohio State University, Columbus, OH 43210

² Department of Biology, Lakehead University, Thunder Bay, Ontario, Canada

The market for phytoremediation of heavy metal and organic waste pollution is expected to grow from a present estimate of $ 30 million to about $ 300 million by the year 2005. In this study, we expand on previous work done to investigate the heavy metal binding capacity of the wild type Chlamydomonas reinhardtii cell wall and to investigate the intracellular mechanisms of detoxification in this organism. Both heavy metal and organic toxins act as electrophiles, binding to nucleophilic centers in proteins and nucleic acids (often sulfhydryls and secondary amino groups). There they interfere with catalytic sites, destabilize protein structure, or cause genetic mutation. Both animals and plants have evolved a similar, three-phase mechanism for removal of electrophilic toxins. A number of heavy metal sensitive mutants of fission yeast have shed light on the molecules involved, as have biochemical studies of the glutathione polymer, phytochelatin and studies of the import of glutathione conjugates into the plant vacuole. In contrast to these studies, relatively little is known about these mechanisms in algae, despite both the important role of algae in ecosystems and advantages of using the unicellular alga Chlamydomonas for bioremediation (i.e., high surface area to volume ratio, phototropism, containment, heavy metal binding by cell wall polypeptides, and ease of transformation). We have taken advantage of the facile genetic analysis and physiology in this organism to isolate and characterize a number of cadmium-sensitive insertion-tagged mutants. Due to the lack of a large storage vacuole in this organism, we hypothesized that C. reinhardtii differs from other plants in the fate of xenobiotics, in that C. reinhardtii probably exports xenobiotics out of the cell into the surrounding medium as opposed to intracellular sequestration in the tonoplast. This hypothesis is supported by recent studies of marine phytoplankton showing that cadmium is exported from these cells as a phytochelatin complex. Our results suggest the following: 1) Export of the model xenobiotics monochlorobimane and rhodamine 123 from the cell into the surrounding medium occurs via glutathione conjugation and efflux through ATP-Binding-Cassette (ABC) transporters, as well as through an additional transporter(s) that lacks sensitivity to the ABC transporter inhibitor verapamil. 2) Several cadmium-sensitive insertional mutants have been identified that lack the characteristic response of wild type cells to verapamil inhibition of model xenobiotic efflux, consistent with insertional mutation of verapamil-sensitive transporter-encoding genes. 3) Molecular analysis by Southern blotting indicates the presence of ARG7 transgene selectable marker loci in the genomic DNA of these mutants, consistent with insertional tagging of the mutations conferring cadmium sensitivity.