Evolution and diversity of secretory pathways in land plants.

The majority of the human population relies on plants for their food, fuel, clothing, building material, and medicines (timber, oil, cotton, grain, pulses, flax, etc). With increased understanding of plant biology, plants are likely in the near future to be capable of producing novel types of fuel and medicine and also to deliver traditional products with lower inputs or on more marginal land. We would like to understand more about how plants grow and produce these valuable products. In particular plant cell walls provide a variety of important commodities from dietary fibre to potential new biofuels, but the complexity and diversity of these structures is still being revealed. We have very little understanding of how the components on the inside of the cells assemble and organise the diverse components of the wall on the outside of the cell. Plants, animals, and fungi shared a common ancestor approximately a one and half billion years ago. That ancestor was probaby a single cell that had the basic elements shared by all cells of modern day descendants. While many of the processes that occur in plant cells share similarity with processes in animals and fungi it is also clear that during evolution, each group of organisms has elaborated on the original mechanisms to meet the specific demands imposed by their increasing complexity in the sea and on land. Part of this evidence comes from genome sequencing projects which reveals the complexity of the proteins that an organism can make. We focus specifically on one family of proteins (the 'Rab proteins') which have a key role in defining the internal compartments of plant cells and directing material between them. It is essential that newly made molecules are directed to the appropriate place if plant cells are to undergo organised growth. We can see that some branches of the Rab protein family have become much more diverse in plants during their colonisation of land over the last 450 million years. Our recent work has shown that one such branch defines a new internal compartment not previously recognised. Furthermore in the cells at the growing tips of the root and shoot this compartment is uniquely localised along the edges of the cells and pertubing the function of the protein causes the cells to lose control of their shape as they grow. We would now like to use genetic and biochemical methods to learn more about the composition and function of this compartment and how the evolution of new types of Rab protein has facilitated the acquisition of land plant-specific traits that contribute to growth and survival in terrestrial environments. While it would be disingenuous to claim that the research we propose will directly improve any commercial product or process, the knowledge we gain may help rational design of plant-based materials in the future. We also have curiosity-driven reasons for wanting to perform this research. Plants are composed of cells that each grow to adopt a specific size and shape that contribute to the overall form and function of the organism. We are trying to understanding some of the mechanisms that allow cells to do this. Specifically we want to understand how the diverse internal compartments of the cells contribute to cell growth and shape, and to dynamic processes such as defence against disease-causing organisms. We also want to understand how these systems evolved, and how they compare to the systems that perform analogous functions in other complex organisms such as humans.

This proposal will test the hypothesis that plant-specific Rab GTPases and one of their novel interactors each define distinct membrane trafficking pathways to the plasma membrane in Arabidopsis Circumstantial evidence suggests that membrane trafficking pathways to the plasma membrane have diversified independently in multicellular plants and animals. To investigate this we are using Arabidopsis to study the Rab family of regulatory GTPases that contribute to the specification of membrane identity and membrane targeting. One branch of the family, the Rab-A branch, which is implicated in post-Golgi trafficking has diversified greatly during land plant evolution, comprising 26 genes in six provisional subclasses (Rab-A1 to Rab-A6) in dicots. We have shown that the Rab-A2 subclass associates with a post-Golgi early-endosomal compartment while a Rab-A5 protein defines an independent and previously undescribed compartment that lies along the edges of meristematic cells. Perturbing the function of the Rab-A2 or Rab-A5 proteins disrupts the spatial control of cell expansion and cell plate assembly but has little or no effect on numerous secreted or plasma membrane markers. This suggests that they may act in specialised secretory or recycling pathways. We have also recently shown that the Rab-E subclass, which associates with the Golgi and plasma membrane exhibits a novel and plant-specific interaction with a phosphatidylinositol-(4)-5 Kinase (PIP5K2) but the functional significance of this is still unclear. Using marker proteins, proteomics and glycomics in transgenic organisms we aim to answer the following questions: 1. Are both Golgi- and TGN-localised Rabs involved in the default pathway? 2. Other than PIN2, what else traffics via the RAB-A2a sensitive pathway(s)? 3. What traffics through the RAB-A5c sensitive pathway(s)? 4. What traffics through the Rab-A4 sensitive pathway(s)? 5. What traffics through the Rab-E and PIP5K2 sensitive pathway(s)?

General. The work proposed here is at the core of the BBSRC mission of providing basic plant bioscience that underpins agriculture, biotechnology, and responding to environmental change. It impacts therefore in areas where BBSRC contributes most to scientific, economic, and cultural development in the UK and elsewhere. as apart from an internationally competitive research programme at a leading UK University department it also contributes to the competetiveness and attractiveness of the UK as a place for investment in contemporary bioindustry. Specific. The biological processes we will investigate determine how plant cells interact and develop, how roots and shoots interact with their environment for mineral acquistion and defence and how they assemble and modify the cell wall. This represents the most abundant renewable resource on the planet. The 150-200 billion tonnes of cell wall material fixed by land plants alone represents 70% of the CO2 that they fix. Cell walls provide numerous fuels, building timber, dietary fibres and textiles. It is increasingly recognised that improved knowledge of plant cell wall structure and assembly will provide opportunities to use cell walls more efficiently and sustainably than at present. Indeed it is estimated that only 2% of the cell wall biomass of crop plants is used by current processes. More understanding of the basic biology will be needed if rational improvements are to be made and the full potential of plant biomass is to be realised by industry. Public Awareness. This proposal addresses four research topics that are of public interest: - Plant evolution - Plant architecture - Plant defence - Plant products including biofuels This has been and will be developed through talks to local societies and schools, popular science publications, and collaboration with broadcasters. Career Development. The multidisciplinary nature of the research will provide the PDRA and associate research students with a broad, thorough, and valuable training in contemporary plant cell biology and biochemistry.