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

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 (Ci; CO 2 and HCO 3 − ) transporters; however, specific knowledge of analogous transporters has remained elusive in eukaryotic microalgae such as C. reinhardtii . Here we investigated whether the limiting-CO 2 –inducible, putative ABC-type transporter HLA3 might function as a HCO 3 − transporter by evaluating the effect of pH on growth, photosynthetic Ci affinity, and [ 14 C]-Ci uptake in very low CO 2 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 2 conditions) and/or simultaneous, apparently off-target knockdown of LCIA mRNA (which encodes limiting-Ci–inducible plastid envelope protein reported to transport HCO 3 − ) resulted in dramatic decreases in growth, Ci uptake, and photosynthetic Ci affinity, especially at pH 9, at which HCO 3 − is the predominant form of available Ci. Collectively, the data presented here provide compelling evidence that HLA3 is directly or indirectly involved in HCO 3 − transport, along with additional evidence supporting a role for LCIA in chloroplast envelope HCO 3 − transport and a role for LCIB in chloroplast Ci accumulation.