Insertional suppressors of Chlamydomonas reinhardtii that restore growth of air-dier lcib mutants in low CO2

Chlamydomonas reinhardtii and other microalgae show adaptive changes to limiting CO2 conditions by induction of CO2-concentrating mechanisms. The limiting-CO2-inducible gene, LCIB, encodes a soluble plastid protein and is proposed to play a role in trapping CO2 released by CAH3 (thylakoid lumen carbonic anhydrase) catalyzed dehydration of accumulated Ci, especially in low CO2 (L-CO2; similar to 0.04% CO2) conditions. To gain further insight into the mechanisms of Ci uptake and accumulation in L-CO2 acclimated C. reinhardtii, we performed an insertional mutagenesis screen to isolate extragenic suppressors that restore the growth of lcib mutants (pmp1 and ad1) in L-CO2. Four independent suppressors are described here and classified by their photosynthetic affinities for Ci and expression patterns of known limiting-CO2-inducible transcripts. Genetic analysis of the four suppressors identified two allelic, dominant suppressors (su4 and su5), and two recessive suppressors (su1 and su8). Consistent with the suppression phenotype, both the relative affinities of photosynthetic O-2 evolution and internal Ci accumulation in all four suppressors were substantially increased relative to pmp1/ad1 in L-CO2 acclimated cells. The relative affinities of pmp-su1 and ad-su8 for Ci were nearly the same as wild type, but that of pmp-su4/su5 was intermediate between pmp-su1 and pmp1. Also, the interactions between lcib mutations and each of the three suppressors varied over the range of CO2 acclimation states. Our results suggest complex contributions of LCIB-dependent and independent active Ci uptake/accumulation systems in various CO2 acclimation states and therefore provide new clues about the roles played by LCIB in limiting Ci acclimation.