Studies On Trans-Acting Factors Involved In Chloroplast Rna Editing In Arabidopsis Thaliana
RNA editing is a post-transcriptional process that changes specific cytidines to uridines in the organelles of most land plants. Although members of the editosome have begun to be elucidated, the identity of the deaminase is still unknown. Members of the pentatricopeptide repeat (PPR) protein family are known to be the site-recognition factors in RNA editing. Most PPR editing factors also carry extra C-terminal motifs called the E domain and the DYW domain, where the DYW domain features amino acids conserved with the active site in other cytidine deaminases. I examined the DYW motifs of two chloroplast PPR-DYW editing factors: REQUIRED FOR ACCD RNA EDITING 1 (RARE1), which is responsible for editing a single site in the accD transcript, and QUINTUPLE EDITING FACTOR 1 (QED1), which is responsible for the editing of five plastid sites. Truncation of the DYW motifs greatly decreased the editing efficiency of RARE1 and QED1 when transfected into protoplasts. Using sitedirected mutagenesis, the conserved deaminase residues were targeted along with a highly conserved potential second zinc binding site. The mutant proteins could not efficiently complement the RNA editing phenotype when expressed in mutant protoplasts, indicating that these deaminase residues may be important for RNA editing efficiency for certain PPR editing factors. Since QED1 affects five different chloroplast sites including sites that changes the coded protein sequences of matK, ndhB, and rpoB, I further examined the functional consequences of these sites not being edited in qed1 mutants. qed1 mutants feature a stunted growth phenotype, the presence of yellow patches that increases with age, a lack of NA(P)H dehydrogenase (NDH) activity, and an abnormal chloroplast morphology as well as a smaller average cell size which can be observed in mutant protoplasts. These phenotypes were found to be at least partially complemented using qed1 mutant lines which have a stably integrated QED1 with a 3xFLAG-StrepII tag attached to its N-terminus. The line carrying one stably integrated allele, though not able to complement the mutant phenotypes to wild-type levels, was able to partially complement the mutant phenotypes, likely due to the presence of only one allele rather than two.
RNA Editing; Chloroplast; Arabidopsis thaliana
Ph. D., Genetics
Doctor of Philosophy
dissertation or thesis