Identification and functional role of the carbonic anhydrase Cah3 in thylakoid membranes of pyrenoid of Chlamydomonas reinhardtii

The distribution of the luminal carbonic anhydrase Cah3 associated with thylakoid membranes in the chloroplast and pyrenoid was studied in wild-type cells of Chlamydomonas reinhardtii and in its cia3 mutant deficient in the activity of the Cah3 protein. In addition, the effect of CO₂ concentration on fatty acid composition of photosynthetic membranes was examined in wild-type cells and in the cia3 mutant. In the cia3 mutant, the rate of growth was lower as compared to wild-type, especially in the cells grown at 0.03% CO₂. This might indicate a participation of thylakoid Cah3 in the CO₂-concentrating mechanism (CCM) of chloroplast and reflect the dysfunction of the CCM in the cia3 mutant. In both strains, a decrease in the CO₂ concentration from 2% to 0.03% caused an increase in the content of polyunsaturated fatty acids in membrane lipids. At the same time, in the cia3 mutant, the increase in the majority of polyunsaturated fatty acids was less pronounced as compared to wild-type cells, whereas the amount of 16:4ω3 did not increase at all. Immunoelectron microscopy demonstrated that luminal Cah3 is mostly located in the thylakoid membranes that pass through the pyrenoid. In the cells of CCM-mutant, cia3, the Cah3 protein was much less abundant, and it was evenly distributed throughout the pyrenoid matrix. The results support our hypothesis that CO₂ might be generated from HCO₃^- by Cah3 in the thylakoid lumen with the following CO₂ diffusion into the pyrenoid, where the CO₂ fixing Rubisco is located. This ensures the maintenance of active photosynthesis under CO₂-limiting conditions, and, as a result, the active growth of cells. The relationships between the induction of CCM and restructuring of the photosynthetic membranes, as well as the involvement of the Cah3 of the pyrenoid in these events, are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.