Doctor of Philosophy (PhD)
The green alga Chlamydomonas reinhardtii possesses a CO2 concentrating mechanism (CCM) which helps in successful acclimation to low CO2 conditions. One of the main aspects of the CCM is bringing in inorganic carbon (Ci) into the cell as bicarbonate using Ci transporters. Current models of the CCM postulate that a series of ion transporters bring HCO3- from outside the cell to the thylakoid lumen where the carbonic anhydrase, CAH3, dehydrates accumulated HCO3- to CO2, raising the CO2 concentration for Rubisco. Previously, HCO3- transporters have been identified at both the plasma membrane (HLA3 and LCI1) and the chloroplast envelope (LCIA/NAR1.2), but the transporter thought to be on the thylakoid membrane has not been identified. The goal of this thesis has been to find the putative thylakoid transporter using a bioinformatics approach to identify candidate proteins followed by characterization of the interesting transporters using RNAi.
Nine candidate proteins were identified from the Chlamydomonas proteome which had transporter like domains and were upregulated in low CO2. Three of the candidates are the paralogous genes (BST1, BST2, BST3) belonging to the bestrophin family, which are not only upregulated in low CO2 conditions but their expression is controlled by CIA5, a transcription factor known to control Ci transporters of the CCM. YFP fusions demonstrate that all three proteins are located on the thylakoid membrane and interactome studies indicate that they might associate with other chloroplast CCM components. A single mutant defective in BST3, still grows normally on low CO2, indicating that the three bestrophin-like proteins may have redundant functions. Therefore, an RNAi approach was adopted to reduce the expression of all three genes at once. RNAi mutants with reduced expression of BST1-3, were unable to grow at low CO2 concentrations, exhibited a reduced affinity for inorganic carbon compared to the wild type cells, and exhibited reduced inorganic carbon uptake. We propose that these bestrophin-like proteins are essential components of the CCM and deliver HCO3- accumulated in the chloroplast stroma to CAH3 inside the thylakoid lumen
Another mutant , A144 was discovered to be a part of a large-scale mutagenesis project to select for mutants that have been transformed with a paromomycin cassette and grow slowly under low CO2 growth conditions. Using a modified form of the adapter PCR method, the location of the insert in A144 was found to be in a gene that codes for a protein with homology of fungal translational elongation factor (eEF3). This mutant cannot adapt to light after prolonged exposure to darkness and has a significantly lower rate of respiration than wild type. Thus we propose that EF3 putatively aids in the synthesis of stress proteins for dark adaptation.
Mukherjee, Ananya, "Identification and Characterization of Novel Transporters Involved in the CO2 Concentrating Mechanism of Chlamydomonas reinhardtii" (2019). LSU Doctoral Dissertations. 4886.
Moroney, James V.