Components of the CO2 concentrating mechanism of Chlamydomonas reinhardtii

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Aquatic photosynthetic organisms account for 50% of the Earth's CO2 fixation and O2 evolution. However, since the diffusion of CO2 is almost 10,000 times slower in water than in air, almost all phytoplankton and macroalgae have a mechanism that concentrates CO2 from the environment for photosynthesis. The uptake of inorganic carbon by aquatic photosynthetic organisms is a major nutrient flux and plays a significant role in the global carbon cycle. The CO2 concentrating mechanism is essential for photosynthesis at atmospheric levels of CO2 and for the survival of prokaryotic and eukaryotic algae. In cyanobacteria, the CO2 concentrating mechanism is known to include transporters for acquiring HCO3- from the medium and a carbonic anhydrase that converts the accumulated HCO3- to CO2 for fixation by Rubisco. The CO2 concentrating mechanism in eukaryotic algae is less well understood but involves inorganic carbon transporters and carbonic anhydrases. The goal of this proposal is to characterize a CO2 concentrating mechanism from a eukaryotic alga.For this work, the model organism Chlamydomonas reinhardtii will be used to identify components of the CO2 concentrating mechanism. C. reinhardtii is a unicellular, eukaryotic green alga which possesses a robust CO2 concentrating system. In addition, the C. reinhardtii genome has been sequenced and over 10,000 genes have been identified. The specific aim of this proposal is to identify new components of the CO2 concentrating mechanism in C. reinhardtii and to further characterize the transporters and carbonic anhydrases that have already been identified. Four major experimental initiatives are proposed. The first aim of the proposal is to identify new genes that increase in expression after a switch from high to low CO2 conditions. It has previously been found that the synthesis of the CO2 concentrating mechanism occurs only under low CO2 conditions. For these experiments the newly constructed macroarray containing more than 10,000 genes will be used. Any gene that increases in expression under low CO2 conditions should be detected using this macroarray. The second experiment is to screen for and characterize insertional mutants that have defects in the CO2 concentrating mechanism. These two proposed experiments, the microarray analyses and insertional mutagenesis, are designed to identify genes that potentially encode components of the CO2 concentrating mechanism. The third set of experiments will further characterize the carbonic anhydrase gene family of C. reinhardtii. Eight different carbonic anhydrase genes designated Cah1-Cah8 have been discovered and at least some of these carbonic anhydrases play important roles in the CO2 concentrating mechanism. The proposed work will focus on Cah7 and Cah8 the last two carbonic anhydrase genes found. This project has been ongoing for a number of years. In the final set of experiments, RNA interference or RNAi will be used to decrease the expression of genes that potentially encode transporters involved in the CO2 concentrating mechanism. If a gene is important in the CO2 concentrating mechanism, then reducing the expression of that gene may render the cell unable to survive on air levels of CO2. The newly released Chlamydomonas genome will significantly facilitate the proposed research. The proposed work will also play a role in the education of graduate students, undergraduates and high school students from a wide diversity of backgrounds. At Louisiana State University, there are many programs to foster participation of a diverse group of undergraduates in research (NSF REU, HHMI, etc), and students from many of these programs have done research in the PI's laboratory. The proposed research will provide a framework for these educational programs.