The CO ₂ -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- CO₂. 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.