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.
This year the world’s human population reached seven billion. An important challenge will be how to raise enough food to feed this many people. Crop yield is directly linked to the plant’s ability to efficiently perform photosynthesis. In crop plants, such as rice or soy bean, photosynthesis can be limited by the amount of atmospheric carbon dioxide (CO2) that reaches the site of fixation in the leaf. The enzyme Rubisco, short for ribulose-1, 5-bisphosphate carboxylase/oxygenase, catalyzes the reaction of CO2 with the sugar ribulose-1, 5-bisphosphate. This is the first step in photosynthetic CO2 fixation into sugar. Many photosynthetic algae have developed ways to improve the delivery of CO2 to Rubisco. This process is called the CO2 concentrating mechanism or CCM. The goal of this research project was to discover how these algae accomplish this uptake of CO2 and how they enhance photosynthesis when the CO2 concentration is low. The long-term goal of this work is to identify components of the CCM that might be transferred from algae to crop plants to improve photosynthesis and crop yield. This project used the alga Chlamydomonas reinhardtii to study the CCM. C. reinhardtii is a simple green alga that has been used as a model organism in laboratories for many years. We have made tremendous strides towards understanding the CCM in C. reinhardtii. We have learned that Rubisco is located in a structure called the pyrenoid that is located inside the chloroplast of C. reinhardtii. We have also indentified nine carbonic anhydrases in C. reinhardtii. Carbonic anhydrases are proteins that catalyze the inter-conversion of CO2 and bicarbonate. In humans, carbonic anhydrases are important in getting CO2 made by muscles and tissues to the lungs to be exhaled. In plants, carbonic anhydrases have the opposite role and help bring CO2 into the plant for photosynthesis. We have learned where most of these carbonic anhydrases are located in C. reinhardtii and what their role is in photosynthesis. Finally, we have identified five transport proteins that might help bring CO2 to the chloroplast pyrenoid. We also localized some of these transport proteins within the C. reinhardtii cell to help to understand their role in photosynthesis. We have learned that disrupting the pyrenoid structure or some of the carbonic anhydrases also disrupts the CCM and reduces photosynthesis at low CO2 concentrations. The findings of this project not only increase our knowledge of photosynthesis, but also identify components of the CCM that might aid in improving photosynthesis in crop plants in the future. We have made this knowledge freely available to the scientific community through scientific publications and scientific presentations and our work is the basis for a number of projects designed to increase photosynthesis in plants.
