The Components 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. Due to the slow diffusion of CO2 in water, algae have developed methods of facilitating CO2 and HCO3- uptake to enhance photosynthesis. Unicellular algae, including Chlamydomonas reinhardtii, have the capacity to concentrate CO2 internally, allowing these organisms to grow photoautotrophically at very low external CO2 concentrations. This CO2 concentrating mechanism is inducible; it is present only in cells that have been grown with limiting CO2 conditions. A model has been proposed to explain how CO2 is concentrated in eukaryotic unicellular algae. In this model there is a mechanism for bicarbonate accumulation, a packaging of Rubisco and a generation of CO2 at the location of Rubisco. This model successfully addresses the question of how algae can concentrate CO2, a molecule that can readily diffuse through biological membranes. Essentially, HCO3- which does not cross membranes readily is first accumulated in the chloroplast and CO2 is generated at or near the enzyme that fixes CO2, ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco). This allows Rubisco to catalyze the carboxylation reaction before the CO2 can leak from the cell. In the past three years it has been demonstrated that Rubisco is specifically located in the chloroplast pyrenoid. The carbonic anhydrase that dehydrates HCO3-, generating CO2 in the chloroplast has also been identified. In addition, membrane proteins induced by growth on low CO2 have been discovered. The function of these membrane proteins remains to be proven but they might function as HCO3- transporters. In this proposal three approaches have been outlined that will identify the physiological role of these membrane proteins. In the first approach, a series of mutations will be generated by insertional mutagenesis. The transformants will then be screened for cells that cannot grow on low CO2 levels and are defective in the CO2 concentrating mechanism. In cases where the insertion is located within the gene this approach will allow for the cloning of a gene that plays a part in the CO2 concentrating mechanism, facilitating the identification of that gene. The second approach will be to constitutively express specific membrane proteins that are normally only expressed under low CO2 to see if they enhance CO2 uptake and fixation. In this case, the gene of interest will be fused with a constitutive promoter. The third approach is to knock out specific genes to see if they are required for the operation of the CO2 concentrating mechanism. This approach relies upon rare homologous recombination events which have been demonstrated to occur in C. reinhardtii. An approach has been outlined that employs a positive selectable marker and a linked negative selectable marker that will allow us to identify rare homologous recombinations. This approach has been successful with other eukaryotic organisms. 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 organism might respond to increasing global CO2 concentrations.