Contribution of a low-CO2-inducible plasma membrane protein, LCI1, to the CO2 concentrating mechanism in Chlamydomonas reinhardtii
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In response to high CO2 variability in the environment, green algae, such as Chlamydomonas reinhardtii, have evolved multiple physiological states dictated by external CO2 concentration. Genetic and physiological studies demonstrated that at least three CO2 physiological states, a high CO2 (0.5–5% CO2), a low CO2 (0.03–0.4% CO2) and a very low CO2 (<0.02% CO2) state, exist in Chlamydomonas. To acclimate in the low and very low CO2 states, Chlamydomonas induces a sophisticated strategy known as a CO2 concentrating mechanism (CCM) that enables proliferation and survival in these unfavorable CO2 environments. CCM induction promotes high intracellular inorganic carbon (Ci) accumulation that subsequently increases local CO2 concentration at the site of ribulose-1,5 bisphosphate carboxylase-oxygenase (Rubisco), a central enzyme in CO2 assimilation, which minimizes photorespiration and increases CO2 fixation via the Calvin cycle. Active uptake of Ci from the environment is one fundamental aspect in the Chlamydomonas CCM and is comprised of CO2 and HCO3– uptake systems that play distinct roles in low and very low CO2 acclimation states. LCI1, a plasma membrane Ci transporter, has been linked through conditional overexpression to active Ci uptake. However, both the role of LCI1 in various CO2 acclimation states and the species of Ci, HCO3– or CO2, that LCI1 transports remain obscure. Thus, my research project aims to examine the roles of LCI1 in low and very low CO2 and to shed light on its preferred Ci species (HCO3–, CO2). In chapter 2, characterization of an LCI1 single mutant (lci1), I report on the impact of LCI1 absence on growth and photosynthesis, as well as on genetic crosses to test inheritance and linkage of the LCI1 mutation with observed phenotype in lci1. In chapter 3, investigation of physiological responses in double mutants LCI1-LCIB and LCI1-LCIA are reported. These double mutants’ studies uncovered the roles of LCI1 in low and very low CO2 acclimation states and also revealed functional relationships between LCI1-mediated Ci uptake and two known Ci uptake systems in Chlamydomonas, LCIB-mediated CO2 uptake and LCIA-associated HCO3– transport. Furthermore, comprehensive analyses of total Ci-dependent O2 evolution profiles and LCI1 uncompensated contributions in three, genetic and mutational backgrounds, at pH 6, 7.3, and 9 showed that CO2 is the Ci species preferred by LCI1. The research presented in this dissertation advances our understanding of LCI1 functional roles in the Chlamydomonas CCM.