Characterization of the CO2 Concentrating Mechanism of Chlamydomonas reinhardtii

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Aquatic photosynthetic organisms account for almost 50% of the Earth's CO2 fixation and O2 evolution. The fixation of CO2 by algae plays an important role in the global carbon cycle, and most photosynthesis in the ocean is accomplished by phytoplankton, single-celled photosynthetic organisms. Carbon dioxide diffusion is almost 10,000 times faster in air than in water and almost all phytoplankton and macroalgae have a mechanism that concentrates CO2 from the environment for photosynthesis. The CO2 concentrating mechanism is essential for photosynthesis at atmospheric levels of CO2 and for the survival of phytoplankton. In this study, Chlamydomonas reinhardtii will be used to identify components of the CO2 concentrating mechanism. C. reinhardtii will be used as the experimental organism since it is a simple unicellular green alga with a single chloroplast. C. reinhardtii also possesses a robust CO2 concentrating system and can be grown photoautophically using CO2 as the carbon source or heterotrophically using acetate as the carbon source. The CO2 concentrating mechanism of C. reinahrdtii will be studied using an insertional mutagenesis approach. Insertional mutagenesis allows for the direct isolation of genes associated with a mutant phenotype, thus permitting a genetic and biochemical analysis of function. In previous work from our laboratory, a large number of mutants that cannot grow on low concentrations of CO2 were generated by transforming C. reinhardtii cells with BleR, a gene encoding resistance to the antibiotic Zeocin. In these mutants that cannot grow on low CO2 concentrations, we hypothesize that the inserted BleR gene has disrupted a gene essential to the CO2 concentrating mechanism. We now propose to characterize these mutants physiologically and genetically and identify the interrupted genes. Mutants exhibiting linkage of the BleR insert to poor growth on low CO2 will be tested for CO2 fixation and inorganic carbon accumulation. Once inserted into the C. reinhardtii genome, BleR offers unique sequences allowing the use of inverse PCR or TAIL-PCR and these methods will be used to identify the interrupted gene. It is expected that some of the insertions will be in genes encoding transport proteins or carbonic anhydrases required for the delivery of CO2 to the enzyme that fixes CO2, ribulose-1,5-bisphosphate carboxylase/oxygenase. It is also expected that genes encoding proteins involved in photorespiration and the low CO2 signal transduction pathway will be disrupted. The characterization of the proteins of the CO2 concentrating mechanism will add to our understanding of how algae have increased their efficiency of CO2 fixation. An understanding of this process will also help scientists predict how marine organisms might respond to increasing global CO2 concentrations. This research will also contribute to the training of students at the undergraduate and graduate level. Graduate students will learn molecular biology methods and apply these methods to the ecophysiological question of how algae acquire CO2 for photosynthesis. Undergraduates researchers will also contribute to this project, gaining valuable research experience. Our research program has attracted a number of undergraduates from underrepresented groups and we will continue to recruit from these groups.