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Pentatricopeptide proteins as tools for chloroplast engineering in Marchantia polymorpha https://doi.org/10.17863/CAM.123109 Boxall, WT Haseloff, James Marchantia polymorpha is a liverwort, one of the early-diverging groups of land plants, and has seen a renaissance as a model plant system in recent years as a result of access to a range of powerful tools for genome engineering both at the nuclear and chloroplast level. Along with rapid regeneration that enables the facile generation of large numbers of nuclear-transformants, and rapid progression to homoplasmy during chloroplast transformation. Additionally, its planar morphology is particularly suited to microscopic analysis, especially of subcellular compartments. In this thesis I validated a pipeline for genome assembly refinement using short-read sequencing data in combination with high-quality reference sequences, to produce an updated version of the nuclear genomes for the Cam accession of M. polymorpha. The Cam accession is particularly well suited to chloroplast transformation due to the ease with which spores can be generated to use in the biolistic delivery of DNA. I then demonstrated a substantial improvement in genome contiguity for the Cam nuclear genomes compared to previous versions, resulting in an increase in genome coverage of ~21.5% for both Cam-1 and Cam-2, particularly from a large increase in repeat content. The updated Cam genomes were then used to perform a large-scale analysis of pentatricopeptide repeat proteins (PPR) in M. polymorpha. The PPR proteins are a group of nuclear-encoded RNA-binding proteins that have seen a significant expansion in land plants, and which localise to the chloroplasts and mitochondria to facilitate organellar gene expression. The results indicated that M. polymorpha PPR genes are intron rich, containing on average 4 introns each, this was in contrast to the PPR genes of flowering plants which are predominantly intron-less as a result of recent expansion through retrotransposition, suggesting that the M. polymorpha PPR genes have an ancient origin. Analysis was performed on multiple RNA-seq data sets from different developmental stages and tissue types of M. polymorpha, highlighting patterns of regulation shared amongst putative chloroplast and mitochondrial PPR proteins respectively. Potential RNA-binding sequences for all of the M. polymorpha PPR proteins were then generated, and used to make more refined in vivo target predictions for a subset of the PPR proteins. New vectors for golden gate assembly were generated, one of which was used to verify the subcellular localisation of the selected subset of PPR proteins. Subsequently, improvements were made to the M. polymorpha chloroplast transformation protocol, which were used in combination with the new vectors to generate a range of transformants expressing mTurquoise2 from the chloroplast chromosome. An alternative approach for facilitating heterologous protein production in the chloroplast was then validated through retargeting the previously identified mitochondrial PPR proteins to drive mTurquoise2 expression in the chloroplast. Two approaches were then explored for the enhancement of growth in M. polymorpha. The first making use of the MpGLK transcription factor as a tool to accelerate chloroplast development, which was successful in generating transformants that outperformed the wild type, but produced unwanted phenotypes in some transformant lines. Supplementation with the MRL1 PPR protein resulted in a significant boost in growth in wild type plants. However, it caused a dramatic reduction in growth when added to a chloroplast transformant dependent upon MRL1 to drive mTurquoise2 expression, while also boosting the mTurquoise2 expression beyond previously attained levels. University of Cambridge 2025-03-31 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/392406 https://www.repository.cam.ac.uk/bitstreams/5369e108-06d3-477c-8516-bf7de85c68dd/download 0b48ad9fd87c0d14f9c4d14dc9df3c3c https://www.repository.cam.ac.uk/bitstreams/8be882b0-031c-4d35-8b27-5be98d117089/download 87eda9de84448d1f82354d60eee3eb5f http://purl.org/NET/rdflicense/allrightsreserved Biolistics Chloroplast Marchantia Marchantia polymorpha Pentatricopeptide Plastid PPR