The Chlamydomonas reinhardtii chloroplast envelope protein LCIA transports bicarbonate in planta.

LCIA (low CO2-inducible protein A) is a chloroplast envelope protein associated with the CO2-concentrating mechanism of the green alga Chlamydomonas reinhardtii. LCIA is postulated to be a HCO3- channel, but previous studies were unable to show that LCIA was actively transporting bicarbonate in planta. Therefore, LCIA activity was investigated more directly in two heterologous systems: an Escherichia coli mutant (DCAKO) lacking both native carbonic anhydrases and an Arabidopsis mutant (ßca5) missing the plastid carbonic anhydrase ßCA5. Neither DCAKO nor ßca5 can grow in ambient CO2 conditions, as they lack carbonic anhydrase-catalyzed production of the necessary HCO3- concentration for lipid and nucleic acid biosynthesis. Expression of LCIA restored growth in both systems in ambient CO2 conditions, which strongly suggests that LCIA is facilitating HCO3- uptake in each system. To our knowledge, this is the first direct evidence that LCIA moves HCO3- across membranes in bacteria and plants. Furthermore, the ßca5 plant bioassay used in this study is the first system for testing HCO3- transport activity in planta, an experimental breakthrough that will be valuable for future studies aimed at improving the photosynthetic efficiency of crop plants using components from algal CO2-concentrating mechanisms.

LCIB functions as a carbonic anhydrase: evidence from yeast and Arabidopsis carbonic anhydrase knockout mutants.

Chlamydomonas reinhardtii evolved a CO2-concentrating mechanism (CCM) because of the limited CO2 in its natural environment. One critical component of the C. reinhardtii CCM is the limiting CO2 inducible B (LCIB) protein. LCIB is required for acclimation to air levels of CO2. C. reinhardtii cells with a mutated LCIB protein have an 'air-dier' phenotype when grown in low CO2 conditions, meaning they die in air levels of CO2 but can grow in high and very low CO2 conditions. The LCIB protein functions together with its close homolog in C. reinhardtii, limiting CO2 inducible C protein (LCIC), in a hexameric LCIB-LCIC complex. LCIB has been proposed to act as a vectoral carbonic anhydrase (CA) that helps to recapture CO2 that would otherwise leak out of the chloroplast. Although both LCIB and LCIC are structurally similar to ßCAs, their CA activity has not been demonstrated to date. We provide evidence that LCIB is an active CA using a Saccharomyces cerevisiae CA knockout mutant (∆NCE103) and an Arabidopsis thaliana ßCA5 knockout mutant (ßca5). We show that different truncated versions of the LCIB protein complement ∆NCE103, while the full length LCIB protein complements ßca5 plants, so that both the yeast and plant mutants can grow in low CO2 conditions.

A Rapid Method for Detecting Normal or Modified Plant and Algal Carbonic Anhydrase Activity Using Saccharomyces cerevisiae.

In recent years, researchers have attempted to improve photosynthesis by introducing components from cyanobacterial and algal CO2-concentrating mechanisms (CCMs) into terrestrial C3 plants. For these attempts to succeed, we need to understand the CCM components in more detail, especially carbonic anhydrase (CA) and bicarbonate (HCO3−) transporters. Heterologous complementation systems capable of detecting carbonic anhydrase activity (i.e., catalysis of the pH-dependent interconversion between CO2 and HCO3−) or active HCO3− transport can be of great value in the process of introducing CCM components into terrestrial C3 plants. In this study, we generated a Saccharomyces cerevisiae CA knock-out (ΔNCE103 or ΔCA) that has a high-CO2-dependent phenotype (5% (v/v) CO2 in air). CAs produce HCO3− for anaplerotic pathways in S. cerevisiae; therefore, the unavailability of HCO3− for neutral lipid biosynthesis is a limitation for the growth of ΔCA in ambient levels of CO2 (0.04% (v/v) CO2 in air). ΔCA can be complemented for growth at ambient levels of CO2 by expressing a CA from human red blood cells. ΔCA was also successfully complemented for growth at ambient levels of CO2 through the expression of CAs from Chlamydomonas reinhardtii and Arabidopsis thaliana. The ΔCA strain is also useful for investigating the activity of modified CAs, allowing for quick screening of modified CAs before putting them into the plants. CA activity in the complemented ΔCA strains can be probed using the Wilbur−Anderson assay and by isotope exchange membrane-inlet mass spectrometry (MIMS). Other potential uses for this new ΔCA-based screening system are also discussed.