Putative inorganic carbon transport and accumulation systems associated with the carbon dioxide concentrating mechanism in Chlamydomonas reinhardtii

The CO2-concentrating mechanism (CCM) of Chlamydomonas reinhardtii and other microalgal species is essential for photosynthetic growth in most natural settings. A great deal has been learned regarding the CCM in cyanobacteria, including identification of inorganic carbon transporters, while specific knowledge of analogous transporters has remained elusive in eukaryotic microalgae such as C. reinhardtii. Here, we have investigated whether the limiting-CO2-inducible, putative ABC-type transporter, HLA3 might function as a HCO3- transporter by evaluating the effect of pH on growth, photosynthetic Ci affinity and [ 14C]-Ci uptake in very low CO2 conditions following RNA interference (RNAi) knockdown of HLA3 mRNA levels in wild-type and mutant cells. The data provide compelling evidence that HLA3 is directly or indirectly involved in HCO3- transport and provide additional evidence supporting a role for LCIA in chloroplast envelope HCO3 - transport and for LCIB in chloroplast Ci accumulation.;To further elucidate the function of LCIB, we identified two ad1 suppressors that can grow in low-CO2 but die in very low-CO 2. Molecular analyses revealed that both suppressors have mutations in the CAH3 gene, which encodes a thylakoid lumen localized carbonic anhydrase. Photosynthetic rates of low-CO2 acclimated suppressors under acclimation CO2 concentrations were more than two fold higher than ad1, apparently resulting from a more than 20 fold increase in the intracellular concentration of Ci as measured by direct Ci uptake. We conclude that LCIB functions downstream of CAH3 in the CCM and probably plays a role in trapping CO2 released by CAH3 dehydration of accumulated Ci. Apparently dehydration by the chloroplast stromal carbonic anhydrase CAH6 of the very high internal Ci caused by the defect in CAH3 provides Rubisco sufficient CO2 to support growth in low-CO2 acclimated cells, but not in very low-CO2 acclimated cells, even in the absence of LCIB.