Ananya Mukherjee, Chun Lau, Charlotte Walker, Ashwani Rai, Camille Prejean, Gary Yates, Thomas Emrich-Mills, Spencer Lemoine, David Vinyard, Luke Mackinder, and James V. Moroney

The green alga Chlamydomonas reinhardtii possesses a CO₂ concentrating mechanism (CCM) which helps in successful acclimation to low CO₂ conditions. Current models of the CCM postulate that a series of ion transporters bring HCO₃^- from outside the cell to the thylakoid lumen, where the carbonic anhydrase CAH3 dehydrates accumulated HCO₃^- to CO₂, raising the CO₂ concentration for Rubisco. Previously, HCO₃^- transporters have been identified at both the plasma membrane and the chloroplast envelope, but the transporter thought to be on the thylakoid membrane has not been identified. Three paralogous genes (BST1, BST2, BST3) belonging to the bestrophin family have been found to be upregulated in low CO₂ conditions, and their expression is controlled by CIA5, a transcription factor that controls many CCM genes. YFP fusions demonstrate that all three proteins are located on the thylakoid membrane, and interactome studies indicate that they might associate with chloroplast CCM components. A single mutant defective in BST3 still grows nearly normally on low CO₂, indicating that the three bestrophin-like proteins may have redundant functions. Therefore, an RNAi approach was adopted to reduce the expression of all three genes at once. RNAi mutants with reduced expression of BST1-3 were unable to grow at low CO₂ concentrations, exhibited a reduced affinity to inorganic carbon compared to the wild type cells, and showed reduced inorganic carbon uptake We propose that these bestrophin-like proteins are essential components of the CCM that deliver HCO₃^- accumulated in the chloroplast stroma to CAH3 inside the thylakoid lumen.