The Carbon Dioxide Concentrating Mechanism of Chlamydomonas Reinhardtii

Louisiana State University, Baton Rouge LA

Investigators

Abstract

Phytoplankton and algae in aquatic environments account for almost 50% of the carbon dioxide (CO2) fixed by photosynthesis on the Earth. Because the diffusion of CO2 is almost 10,000 times slower in water than in air, almost all of these phytoplankton and algae have a mechanism that concentrates CO2 from the environment for photosynthesis. This CO2 concentrating mechanism is essential for photosynthesis and the survival of algae. In photosynthetic cyanobacteria, the CO2 concentrating mechanism includes membrane proteins that transport bicarbonate (HCO3-) from the medium into the cell. A carbonic anhydrase enzyme then converts the HCO3- to CO2 for fixation by Rubisco, the enzyme that fixes CO2. The CO2 concentrating mechanism in eukaryotic algae is less well understood but is known to involve transport proteins and multiple carbonic anhydrases. The goal of this project is to characterize the CO2 concentrating mechanism present in the eukaryotic green alga Chlamydomonas reinhardtii. The model unicellular organism, C. reinhardtii, is ideal for studying the CO2 concentrating mechanism because the genome is sequenced, it has a robust CO2 concentrating mechanism and mutant strains that have defects in the CO2 concentrating mechanism have been identified. These are important tools that will be used to determine how the CO2 concentrating mechanism functions. Three major experimental initiatives are proposed. The first set of experiments builds on previous studies to further characterize the carbonic anhydrase gene family of C. reinhardtii. Through bioinformatics and classical biochemical studies, nine different carbonic anhydrase genes have been discovered in C. reinhardtii. The expression and the intracellular location of these proteins will be determined. RNA interference (RNAi) will be used to reduce the expression of specific carbonic anhydrase genes to determine whether or not they are involved in the CO2 concentrating mechanism. The second aim of the proposal is to identify and characterize transport proteins that carry HCO3-. For these studies, the genes encoding proteins thought to be bicarbonate transporters will be introduced into a cyanobacterial strain that is missing all known bicarbonate transporters. The third aim is to identify transport proteins on both the chloroplast thylakoid and chloroplast envelope membranes. For these studies proteomics will be used to identify proteins likely to be components of the CO2 concentrating mechanism. This work will increase the understanding of how the CO2 concentrating mechanism contributes to the global carbon cycle and will facilitate the prediction of algal responses to rising atmospheric CO2 levels. 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). The proposed research will provide a framework for these educational programs.