Genetics, Development and Cell Biology Ph.D. candidate — Alfredo Kono

Major — Genetics

Major Professor — Distinguished Professor Steven Rodermel and Emeritus Professor Marty Spalding

Title — Contribution of a low-CO₂-inducible plasma membrane protein, LCI1, to the CO₂ concentrating mechanism in Chlamydomonas reinhardtii

Abstract — 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 (CMM) 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 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.