High carbon dioxide requiring mutants of Chlamydomonas reinhardtti

Abstract

Chlamydomonas reinhardtii is a photosynthetic alga that has the ability to concentrate CO2 around Rubisco to achieve enhanced rates of photosynthesis in a low CO2 environment. This dissertation presents results obtained from the generation and analysis of four high CO2 requiring mutants of C. reinhardtii. The use of reverse genetics is a very powerful tool to dissect out the individual components of metabolic pathways. Two reverse genetics methods were utilized in this study: a random insertional mutagenesis method to discover genes that are required for growth in a low CO2 environment, and a directed mutagenesis approach, RNA interference, to determine the role of two low CO2 inducible genes in the carbon concentrating mechanism. The first high CO2 requiring mutant was determined to be defective at the Rubisco activase locus. The second mutant, cia6, had an insertion in a SET domain containing protein that may be involved in the regulation of the carbon concentrating mechanism. The third mutant, slc23, had an insertion in a gene that encodes for multiple splice variants that encode for at least four distinct WD40 repeat proteins that vary in their number of WD40 repeats. A targeted mutagenesis approach was utilized to silence the expression of the two low CO2 inducible, nearly identical genes, Ccp1 and Ccp2. RNA interference was successfully used to reduce the expression of Ccp1 and Ccp2 mRNAs and proteins to undetectable levels. Results suggest that the Ccp1 and Ccp2 proteins are required for growth in a low CO2 environment, but that these two proteins are not required for efficient photosynthesis at low levels of CO2.