Manipulating plastid DNA replication/recombination/repair pathways to study plastid genome maintenance & improve transplastomic technologies in crops.

Chloroplasts contain the green pigment chlorophyll and are microscopic components of plant cells. They are responsible for photosynthesis and convert carbon dioxide into organic living material. These centres of photosynthesis carry out many vital plant functions and are essential for plant productivity and the performance of crops. Chloroplasts are especially interesting because they contain a small but very important set of genes that are very different from the set of genes organised as chromosomes in the nucleus of cells. These genes are essential for chloroplasts to function but were difficult to study until relatively recently. Transplastomics is a new field of research that allows the genes in chloroplasts to be studied directly and will allow improvements to chloroplast genes that enhance plant productivity in a changing environment. These transplastomic studies have shown that chloroplast genes have a number of desirable features. They code for important proteins that are amongst the most abundant proteins found in leaves and they are not spread through pollen. Moreover transplastomic technologies are extremely precise in enabling targeted improvements to chloroplast genes. Transplastomic technologies allow chloroplasts to be used as solar energy convertors which are a carbon efficient and sustainable manufacturing platform for producing useful biomolecules. Genes are made of DNA and they are maintained by molecular machines that replicate and repair them. The genes in chloroplasts are continually damaged by the action of sunlight. Efficient repair and replication is required to maintain functional chloroplast genes. We are studying the molecular machines responsible for maintaining chloroplast genes by removing specific components of the machines and then examining the impact of their removal on the maintenance of chloroplast genes. Our results have identified key components and molecular mechanisms required for maintaining chloroplast genes. Whilst much progress has been made using transplastomic technologies, a current limitation is their efficiencies particularly in important crops such as oil seed rape. In this research we will exploit our knowledge of the gene maintenance machinery in chloroplasts to improve the efficiency of transplastomic methods. This will allow the benefits of transplastomic research to be realised in important crops. This work will also provide new knowledge on the molecular processes that maintain the integrity of chloroplast genes. Because this process is important for plant growth and development it will improve our understanding of a key molecular process required for plant productivity.

Plastids contain a conserved genome that is essential for plant growth & development & is amenable to modification using transplastomic technologies. Precise targeted integration of transgenes, maternal inheritance of plastids & high yield expression of recombinant proteins drive applications of transplastomic plants in agriculture & industry. Conservation of the plastid genetic system means that the basic mechanism of foreign gene integration into plastid genomes is conserved in all plants in which plastid transformation has been established. Plastid genomes are present in multiple copies per plastid, the sequence is uniform within a plant (homoplasmic) & the copy number varies during plant development. Relatively little is known on the plastid DNA maintenance machinery responsible for these features. The DNA replication/recombination/repair (DNA-RRR) proteins involved in maintaining plastid genes are encoded by the nucleus. In this work we will exploit our knowledge of plastid DNA-RRR proteins & pathways to provide approaches to improve the overall efficiency of plastid transformation, which is particularly important for translating applications of transplastomic technologies to crops, which are recalcitrant to plastid transformation. The approaches will be developed in Nicotiana tabacum, the model for transplastomic research, & tested in Brassica napus. We will use inducible vectors to enable transient changes in the levels of native plastid DNA-RRR proteins as well as foreign DNA-RRR proteins. The impact of these changes on plastid genome maintenance, raising the efficiency of plastid transformation & the levels of recombinant protein expression in plastids will be determined. The work has applications in developing new tools for transplastomic technologies & will improve our fundamental understanding of the key processes & components responsible for conferring plastid genome stability, which is essential for chloroplast function & plant growth

Transplastomic research has considerable commercial applications and the results will benefit: Agribusiness- interested in chloroplast encoded traits Pharma companies- interested in manufacturing high value products in plants This will include small and medium sized enterprises and large multinational companies The plant breeding industry: plastids encode an important set of genes and the tools in the project will allow plant breeders to make changes in the plastid genomes of important crops Staff: These include the PDRA and technician employed on the grant who will gain new expertise and knowledge in an area of fundamental research that has potential commercial applications. The applicant will gain new contacts in academia and industry following dissemination of information in peer review journal and seminars. The applicant is often asked to provide expert opinion on plastid transformation to government agencies and industry and will be able to deliver this for the project. Through networks such as the synthetic plant products for industry network (SPPI-NET) the wider significance of this work relating to synthetic plastid genomes and synthetic biology will be communicated. The wider public will benefit from gaining a new understanding of an important process in plants. This Faculty has a dedicated public engagement office who provides support and advice for public engagement activities. Many of these activities are hosted by the Manchester Museum and involve staff & students from Plant Sciences explaining their work. Any IP filed by the University Technology Transfer Company (UMIP) and associated commercialization will benefit the UK's economic competitiveness