Thylakoid localized bestrophin-like proteins are essential for the CO₂ concentrating mechanism of <i>Chlamydomonas reinhardtii</i>.

Abstract

The green alga <i>Chlamydomonas reinhardtii</i> possesses a CO₂ concentrating mechanism (CCM) that 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 3 (CAH3) dehydrates accumulated HCO₃^- to CO₂, raising the CO₂ concentration for Ribulose bisphosphate carboxylase/oxygenase (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 (<i>BST1</i>, <i>BST2</i>, and <i>BST3</i>) belonging to the bestrophin family have been found to be up-regulated in low CO₂ conditions, and their expression is controlled by CIA5, a transcription factor that controls many CCM genes. YFP fusions demonstrate that all 3 proteins are located on the thylakoid membrane, and interactome studies indicate that they might associate with chloroplast CCM components. A single mutant defective in <i>BST3</i> has near-normal growth on low CO₂, indicating that the 3 bestrophin-like proteins may have redundant functions. Therefore, an RNA interference (RNAi) approach was adopted to reduce the expression of all 3 genes at once. RNAi mutants with reduced expression of <i>BST1</i>-<i>3</i> were unable to grow at low CO₂ concentrations, exhibited a reduced affinity to inorganic carbon (C_i) compared with the wild-type cells, and showed reduced C_i 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.