Thylakoid Lumen Carbonic Anhydrase (CAH3) Mutation Suppresses Air-Dier Phenotype of LCIB Mutant in Chlamydomonas reinhardtii

Abstract

An active CO 2 -concentrating mechanism is induced when Chlamydomonas reinhardtii acclimates to limiting inorganic carbon (Ci), either low-CO 2 (L-CO 2 ; air level; approximately 0.04% CO 2 ) or very low-CO 2 (VL-CO 2 ; approximately 0.01% CO 2 ) conditions. A mutant, ad1, which is defective in the limiting-CO 2 -inducible, plastid-localized LCIB, can grow in high-CO 2 or VL-CO 2 conditions but dies in L-CO 2 , indicating a deficiency in a L-CO 2 -specific Ci uptake and accumulation system. In this study, we identified two ad1 suppressors that can grow in L-CO 2 but die in VL-CO 2 . Molecular analyses revealed that both suppressors have mutations in the CAH3 gene, which encodes a thylakoid lumen localized carbonic anhydrase. Photosynthetic rates of L-CO 2 -acclimated suppressors under acclimation CO 2 concentrations were more than 2-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. However, photosynthetic rates of VL-CO 2 -acclimated cells under acclimation CO 2 concentrations were too low to support growth in spite of a significantly elevated intracellular Ci concentration. We conclude that LCIB functions downstream of CAH3 in the CO 2 -concentrating mechanism and probably plays a role in trapping CO 2 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 CO 2 to support growth in L-CO 2 -acclimated cells, but not in VL-CO 2 -acclimated cells, even in the absence of LCIB.CO 2 serves both as the substrate for photosynthesis and as an important signal to regulate plant growth and development, so variable CO 2 concentrations can impact photosynthesis, growth, and productivity of plants. Terrestrial C 4 plants have developed a CO 2 -concentrating mechanism (CCM) involving anatomical and biochemical adaptations to accumulate a higher concentration of CO 2 as substrate Rubisco and to suppress oxygenation of ribulose-1,5-bisP, a wasteful side reaction. In contrast, a different type of CCM is induced in the unicellular green microalga Chlamydomonas reinhardtii when the supply of dissolved inorganic carbon (Ci; CO 2 and HCO 3 2

Acclimation to Very Low CO2: Contribution of Limiting CO2 Inducible Proteins, LCIB and LCIA, to Inorganic Carbon Uptake in Chlamydomonas reinhardtii

“…of LCIB in CO 2 uptake into the accumulated HCO 3 2 pool is consistent with and can explain the observed physiological characteristics of LCIB mutants, LCIB/CAH3 double mutants (Duanmu et al, 2009b), and LCIB/LCIA double mutants, it is still speculative and needs to be rigorously tested at the biochemical level.…”

“…We recently proposed that LCIB may function similarly to the cyanobacterial ChpX/Y proteins in converting CO 2 to HCO 3 2 (Duanmu et al, 2009b;Wang and Spalding, 2014). ChpX/Y proteins in cyanobacteria are proposed to convert CO 2 to HCO 3 2 unidirectionally, in contrast with the bidirectional activity of CAs, via their linkage to NADH dehydrogenase complexes (Price et al, 2002).…”

“…and/or the thylakoid lumen, or physically preventing CO 2 escape from pyrenoids (Spalding et al, 1983a;Miura et al, 2004;Spalding, 2006, 2014;Duanmu et al, 2009b;Yamano et al, 2010). Here we demonstrate that the absence of LCIB causes photosynthetic defects under low Ci conditions that correlate with CO 2 concentrations.…”

“…It is unclear how LCIB is involved in Ci accumulation, largely because of its unknown biochemical characteristics and the lack of homologs of known function in other organisms. Genetic analysis of LCIB/CAH3 double mutants indicated that LCIB functions downstream of CAH3, a thylakoid lumen CA (Duanmu et al, 2009b). Because CAH3 catalyzes dehydration of accumulated HCO 3 2 to provide CO 2 for Rubisco inside the pyrenoid (Spalding et al, 1983b;Moroney et al, 2011), it has been hypothesized that LCIB captures CO 2 leaked from the pyrenoid, possibly by unidirectionally hydrating CO 2 back to HCO 3 2 (Duanmu et al, 2009b).…”

“…Genetic analysis of LCIB/CAH3 double mutants indicated that LCIB functions downstream of CAH3, a thylakoid lumen CA (Duanmu et al, 2009b). Because CAH3 catalyzes dehydration of accumulated HCO 3 2 to provide CO 2 for Rubisco inside the pyrenoid (Spalding et al, 1983b;Moroney et al, 2011), it has been hypothesized that LCIB captures CO 2 leaked from the pyrenoid, possibly by unidirectionally hydrating CO 2 back to HCO 3 2 (Duanmu et al, 2009b). If this hypothesis is correct, it also implies that LCIB may also function in active Ci accumulation by unidirectionally hydrating externally diffused CO 2 into the stromal HCO 3 2 pool (Wang and Spalding, 2014).…”