A genomics-based analysis of chloroplast replication/recombination/repair pathways using RNAi proteomics and transplastomic plants

Chloroplasts are microscopic compartments in plant cells and contain the green pigment chlorophyll. They harvest sunlight and capture carbon dioxide to produce living organic matter. These solar energy converters have considerable scope for bio-manufacturing useful products. Because the process requires little input and is energy-efficient and carbon-neutral it will allows sustainable production of bio-molecules important to maintain our quality of life. Chloroplasts are unique in containing their own blueprint comprised of a circle of ~100 genes. These genes were known to be very important but resisted our early attempts to study them using modern molecular tools. Transplastomics, is a new and exciting method, that allows the detailed study of chloroplast genes. Despite the importance of chloroplast genes to life on this planet and sustainable manufacture of high-value products, we know virtually nothing about the processes that maintain these genes in plants. This project will study these processes by identifying the proteins that interact with chloroplast genes. We will use advanced transplastomics to find out what effects these proteins have on chloroplast genes. This work will allow us to understand better how these genes are protected from the damaging effects of sunlight, and how they are maintained and inherited in plants. By studying the machinery responsible for maintaining chloroplast genes we will understand one of the key molecular processes required for chloroplasts to function and this underpins the healthy growth and development of plants.

An important set of extra-nuclear genes are located in the plastids of plant cells. Plastid genes are conserved, present in many copies per cell, inherited from the female parent in most angiosperms, and are essential for photosynthesis and plant development. Transgenes integrate by homologous recombination and are highly expressed in plastids. These features drive academic and industrial research on plastid genes. Despite the importance of plastid genes our knowledge of DNA-replication/recombination/repair (DNA-RRR) pathways in plastids is extremely poor. Proteins involved in these pathways are encoded by the nucleus. We have identified plastid targeted DNA-RRR proteins using bioinformatics, genome databases and published proteomic studies. Reverse genetics will be used to study the roles of these genes. Because knockouts of plastid DNA-RRR genes can be lethal we will use RNAi-mediated down-regulation to reduce protein levels. We will target 5 key genes involved in homologous recombination, repair and replication of plastid DNA. We will examine the effects of reduced protein levels on plastid DNA-RRR pathways using transplastomic plants, enabling recombination rates to be measured and plastid inheritance to be followed. We will determine the effects of reduced DNA-RRR proteins on overall phenotype, tolerance to DNA damaging agents, plastid mutation rate and plastid DNA levels. We will purify and compare plastid fractions enriched in DNA-RRR proteins from wild type and down-regulated lines, using an established method, to study interactions between DNA-RRR proteins and to identify new proteins in the pathway. This first detailed study of plastid DNA-RRR proteins will provide the first experimental results to test genomics-based models of plastid DNA-RRR pathways and will have a major impact on transplastomic research.