Evolution of Rab GTPase functions and endomembrane compartments in land plants

The majority of the humal population relies on plants for their food, fuel, clothing, building material, and medicines (timber, oil, cotton, grain, pulses, flax, etc etc). With increased understanding of plant biology, they are likley 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 revealled. 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. While it would be disingenuous to claim that the research we propse will directly improve any commercial product or process, the knowldge we gain may help ratuional design of plant-based materials in the future. We also have curiosity-driven reasons for wanting to perform thsi 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, how these systems evolved, and how they compare to the systems that perform analogous functions in other compex organisms such as humans. Plants, animals, and fungi shared a common ancestor approximately a one and half billion years ago. That ancester was probaby a single cell that had the basic elements shared by all cells of modern day descendants. While much of the what occurs in plant cells shares 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 inceasing 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 an make. We focus specifially on one family of proteins (the 'Rab proteins') which have a key role in defining the internal compartents of plant cells and directing material between them. It is essntial 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 collonisation 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 an shoot thsi compartment is uniquely localised along the edges of the cells and pertubing teh 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 leran more about the compostion and function of this compartment and how the evolution of new types of Rab protein jas facilitated this.

This proposal will test the hypothesis that diversification of the Rab11-related Rab-A GTPase family in land plants has facilitated the evolution of distinct secretory or endosomal compartments in cells of developing lateral organs. Circumstantial evidence suggests that membrane trafficking pathways have diversified independently in multicellular plants and animals but documented examples are rare. To investigate this process 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, has diversified greatly during land plant evolution, comprising 26 genes in six provisional subclasses (Rab-A1 to Rab-A6) in dicots. Previous work has shown that several of these proteins, including the entire Rab-A2 and -A3 subclasses associate with post-Golgi early-endosomal compartments. We have found that a Rab-A5 protein, in contrast, defines an independent and previously undescribed compartment that adopts a striking arrangement in cells of young organ primordia where it lies along the 12 edges of the cuboid cells. Perturbing the function of this protein disrupts the spatial control of cell expansion in root tips and leaves of transgenic plants. We now wish to: (i) test the hypotheses that Rab-A2 and -A5 GTPases define biochemically and functionally distinct compartments (ii) to understand how Rab-A2 and -A5 proteins are targeted to their respective compartments in cells of young lateral roots. Working entirely with transgenic organisms we aim to answer the following questions: 1. What features of RAB-A5c are responsible for its distinctive localisation at the edges of cells? 2. Do RAB-A2 and RAB-A5c have different interactors? 3. Do the Rab-A2 and Rab-A5c compartments contain distinct protein complements? 4. Do RAB-A2 and RAB-A5 mutants affect different aspects of biosynthetic or endocytic protein trafficking?