Characterization of cooperative bicarbonate uptake into chloroplast stroma in the green alga Chlamydomonas reinhardtii

The supply of inorganic carbon (Ci; CO(2) and HCO(3)(–)) is an environmental rate-limiting factor in aquatic photosynthetic organisms. To overcome the difficulty in acquiring Ci in limiting-CO(2) conditions, an active Ci uptake system called the CO(2)-concentrating mechanism (CCM) is induced to increase CO(2) concentrations in the chloroplast stroma. An ATP-binding cassette transporter, HLA3, and a formate/nitrite transporter homolog, LCIA, are reported to be associated with HCO(3)(–) uptake [Wang and Spalding (2014) Plant Physiol 166(4):2040–2050]. However, direct evidence of the route of HCO(3)(–) uptake from the outside of cells to the chloroplast stroma remains elusive owing to a lack of information on HLA3 localization and comparative analyses of the contribution of HLA3 and LCIA to the CCM. In this study, we revealed that HLA3 and LCIA are localized to the plasma membrane and chloroplast envelope, respectively. Insertion mutants of HLA3 and/or LCIA showed decreased Ci affinities/accumulation, especially in alkaline conditions where HCO(3)(–) is the predominant form of Ci. HLA3 and LCIA formed protein complexes independently, and the absence of LCIA decreased HLA3 mRNA accumulation, suggesting the presence of unidentified retrograde signals from the chloroplast to the nucleus to maintain HLA3 mRNA expression. Furthermore, although single overexpression of HLA3 or LCIA in high CO(2) conditions did not affect Ci affinity, simultaneous overexpression of HLA3 with LCIA significantly increased Ci affinity/accumulation. These results highlight the HLA3/LCIA-driven cooperative uptake of HCO(3)(–) and a key role of LCIA in the maintenance of HLA3 stability as well as Ci affinity/accumulation in the CCM.