The Components of the CO2 Concentrating Mechanism of Chlamydomonas reinhardtii

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9304662 Moroney Unicellular algae, including Chlamydomonas reinhardtii, have the capacity to concentrate carbon dioxide internally, allowing these organisms to grow photoautotrophically at carbon dioxide concentrations lower than most higher plants can tolerate. This carbon dioxide concentrating mechanism is inducible; it is present only in cells that have been grown with limiting carbon dioxide conditions. An important aspect of this Ci (Ci = CO2 + HCO3- + CO3-2) accumulation system is located in the chloroplasts. Intact chloroplasts isolated from low carbon dioxide-grown C. reinhardtii cells retain the ability to accumulate Ci to much higher levels than intact chloroplasts isolated from high carbon dioxide-grown cells. During the last grant period, Dr. Moroney has identified at least six polypeptides that are specifically induced by growth on low carbon dioxide and are missing in the high carbon dioxide- requiring strain cia-5. One of these inducible proteins is a carbonic anhydrase located in the periplasmic space and another is a 36 kDa polypeptide localized to the chloroplast envelope. The locations of other inducible proteins and their functions are unknown. In the past year, a cDNA library to C. reinhardtii cells adapting to low carbon dioxide conditions has been constructed. By differential hybridization, four classes of recombinant phages have been identified that contain genes that are preferentially expressed under four classes low carbon dioxide conditions. It is proposed to study the carbon dioxide concentrating mechanism in C. reinhardtii by characterizing the components of this transport system and by delineating the role of the chloroplast in carbon dioxide acquisition. Dr. Moroney will first characterize the low carbon dioxide-inducible genes recently identified and connect these genes with the polypeptides known to be induced by low carbon dioxide growth conditions. Antibodies will be raised against fusion proteins consisting of t he lacZ protein linked to the product of the inducible gene. These antibodies will then be used to localize the proteins within the C. reinhardtii cell using organelle preparations outlined within the proposal. Dr. Moroney will also generate mutants that require high carbon dioxide, focussing on strains that are missing only one of the inducible polypeptides. It is hoped to eventually link a specific gene with its gene product and with a strain deficient in that gene product. The characterization of the proteins of this transport system will increase our understanding of the low carbon dioxide adjustment in algae and the role of the chloroplast in this photosynthetic adaptation. ***