Knockdown of limiting-CO^sub 2^-induced gene HLA3 decreases HCO^sub 3^-transport and photosynthetic Ci affinity in Chlamydomonas reinhardtii

The CO...-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 (Ci; CO... and HCO...) transporters; however, specific knowledge of analogous transporters has remained elusive in eukaryotic microalgae such as C. reinhardtii. Here we investigated whether the limiting-CO...-inducible, putative ABC-type transporter HLA3 might function as a HCO... transporter by evaluating the effect of pH on growth, photosynthetic Ci affinity, and [...C]-Ci uptake in very low CO... conditions following RNA interference (RNAi) knockdown of HLA3 mRNA levels in wild-type and mutant cells. Although knockdown of HLA3 mRNA alone resulted in only modest but high-pH-dependent decreases in photosynthetic Ci affinity and Ci uptake, the combination of nearly complete knockdown of HLA3 mRNA with mutations in LCIB (which encodes limiting-Ci-inducible plastid-localized protein required for normal Ci uptake or accumulation in low-CO... conditions) and/or simultaneous, apparently off-target knockdown of LCIA mRNA (which encodes limiting-Ci-inducible plastid envelope protein reported to transport HCO...) resulted in dramatic decreases in growth, Ci uptake, and photosynthetic Ci affinity, especially at pH 9, at which HCO... is the predominant form of available Ci. Collectively, the data presented here provide compelling evidence that HLA3 is directly or indirectly involved in HCO... transport, along with additional evidence supporting a role for LCIA in chloroplast envelope HCO... transport and a role for LCIB in chloroplast Ci accumulation. (ProQuest: ... denotes formulae/symbols omitted.)