Doctor of Philosophy (PhD)
The aim of this thesis is to investigate the CO2 concentrating mechanism (CCM) of Chlamydomonas reinhardtii and to develop a quick method for estimating the activity of carbonic anhydrases (CAs). The first project demonstrates that there are two almost identical mitochondrial CAs in C. reinhardtii, CAH4 and CAH5, that help to maintain photosynthesis and minimize the leak of CO2 generated by respiration and photorespiration. We used an RNAi approach to reduce the expression of CAH4 and CAH5 so that their physiological functions could be studied. RNAi mutants with low expression of CAH4 and CAH5 have impaired rates of photosynthesis under ambient air levels of CO2 (0.04% [v/v] CO2 in air). These strains are not able to grow in very low-CO2 conditions (2 in air) but grew normally on elevated CO2 (5% [v/v] CO2 in air). These RNAi mutants had a poor affinity for inorganic carbon (Ci = CO2 + HCO3-) and their ability to accumulate Ci is impaired. We hypothesize that CAH4 and CAH5 work in the mitochondria to convert the CO2 released from respiration and photorespiration and the CO2 leaked from the chloroplast to HCO3-, thus “recapturing” this potentially lost CO2.
In the second project, we developed a method to estimate CA activity using a mutant complementation approach in Saccharomyces cerevisiae (hereafter referred to as yeast) in order to study the role of CAs in photosynthetic organisms. In studies based on this method, we found that CAH4/5 and CAH3 from C. reinhardtii, βCA3 from Arabidopsis thaliana and different modified forms of Human CA II are all active CAs. We also determined that codon optimization can improve protein expression in the heterologous yeast system. It is important to know if CAs are active, as introducing CA activity in the correct location in a plant chloroplast could accelerate efforts to improve crop plant photosynthetic efficiency. It will also help to understand how CAs function across different locations in photosynthetic organisms.
Rai, Ashwani, "The Importance of CO2 Recapture in the CO2 Concentrating Mechanism of Chlamydomonas Reinhardtii" (2022). LSU Doctoral Dissertations. 5913.
Moroney, James V.