Identification and characterization of nuclear genes involved in plastid genome maintenance in tobacco

• Main Author: Huang, Xun
• Other Authors: Day, Anil
• Published: University of Manchester 2013
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.740243

Plastids are plant cellular organelles derived from photosynthetic prokaryotes by endosymbiosis. Plastids are comprised of a family of intracellular organelles with roles in biosynthesis and storage of metabolic intermediates as well as photosynthesis. Plastids contain a genome required for phototrophic growth. Plastid DNA is present as DNA-protein complexes and is highly polyploid and homogeneous. It exists in both linear and circular forms, ranging from the monomer up to the octamer. To maintain the stability of plastid DNA, DNA replication, recombination and repair (DNA-RRR) pathways are required. In this work, six proteins involved in plastid DNA-RRR pathways were studied, namely MSH1, Whirly1, pTAC2, MutS2B, RecQsim and prokaryotic type DNA topoisomerase I (TopoIp). The roles of the proteins on tobacco chloroplast DNA were studied using: 1) over-expression and RNAi mediated down-regulation of gene expression to identify molecular and visible phenotypes, 2) GFP-fusions with N-terminal targeting sequences to identify sub-cellular locations. By constitutively over-expressing the selected DNA-RRR genes in tobacco, the results showed that the transcript levels of target genes could be increased from 2-fold higher for muts2B RNA to 172-fold higher for whirly1 RNA, whereas RNAi-mediated down-regulation decreased the transcript levels of target genes from 65% for topoIp RNA to 5% for whirly1 RNA compared to wild type plants. Over-expression of msh1 and whirly1 led to albino cotyledons. Down-regulation of msh1 and whirly1 gave rise to variegated phenotypes. Depletion of pTAC2 gave rise to pale green plants that showed severe growth retardation. Reduced level of TopoIp led to green/brown variegation that triggered premature death of plants. With the aid of GFP-fusion studies, MSH1 was found to be dual targeted to chloroplasts and mitochondria, while Whirly1, pTAC2, MutS2B, RecQsim and TopoIp were localized to chloroplasts. Recombination between direct repeats larger than 600 bp in plastids was shown to be stimulated by pTAC2 and MutS2B and inhibited by MSH1 and TopoIp. RecQsim appeared to play a dual role in this recombination event, whereas Whirly1 did not appear to play a role in this process. Recombination between repeats of less than 40 bp seemed to be inhibited by MSH1 and RecQsim. pTAC2 and TopoIp did not appear to participate in this process. Whirly1 seemed to inhibit recombination between 40 bp direct repeats. MutS2B appeared to stimulate recombination between 19 bp inverted repeats. Models for the mode action of each protein in the plastid DNA recombination pathway are discussed in this thesis. The thesis results provide a better understanding of DNA-RRR pathways in plastids. This may in future allow the manipulation of one or more of the pathways to provide improved techniques for transgene integration and marker gene excision, as well as to increase the copy number of plastid DNA and to regulate the level of homoplasmy.