Abstract
The green alga Chlamydomonas reinhardtii 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 (BST1, BST2, and BST3) 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 BST3 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 BST1-3 were unable to grow at low CO2 concentrations, exhibited a reduced affinity to inorganic carbon (Ci) compared with the wild-type cells, and showed reduced Ci 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.
Highlights
The green alga Chlamydomonas reinhardtii possesses a CO2 concentrating mechanism (CCM) that helps in successful acclimation to low CO2 conditions
We present evidence here that BST1–3 are chloroplast thylakoid localized anion transporters that are important components of the Chlamydomonas CCM
Cells that have reduced BST1–3 transcript levels fail to grow on low CO2 (Fig. 4), have a lower affinity for Ci (Fig. 5), and have a reduced ability to accumulate aCdCdMedm1o4Cdei l(sFisigt.ha6t).acAcukmeuylaatsepdecHt CoOf 3c−uirsrecnotnvCehrtleadmtyodoCmOo2nbays carbonic anhydrase 3 (CAH3), a carbonic anhydrase located in the thylakoid lumen [11,12,13]
Summary
The green alga Chlamydomonas reinhardtii possesses a CO2 concentrating mechanism (CCM) that helps in successful acclimation to low CO2 conditions. RNAi mutants with reduced expression of BST1–3 were unable to grow at low CO2 concentrations, exhibited a reduced affinity to inorganic carbon (Ci) compared with the wild-type cells, and showed reduced Ci 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. Chlamydomonas CCM models propose that mutants missing CAH3 accumulate the HCO3− brought into the chloroplast by the transport proteins but cannot convert that HCO3− to CO2, the actual substrate of Rubisco [12, 13] These CCM models postulate that the pH gradient across the thylakoid membrane in the light helps drive the conversion of HCO3− to constant (pKa) of the iCntOer2c.oTnvheersaiopnpaorfenHt CaOci3d−.
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