Elucidating molecular level details of the plant Borate transporter structure and function.

All cells are surrounded by a membrane made up of fatty lipid molecules. This membrane acts as an effective barrier separating the contents of the cell from the external environment. The lipid membrane itself is impermeable to all but a limited number of molecules, however cells need to have a means of taking up key nutrients and removing waste products. The import and export of a wide range of molecules across the membrane is mediated via a system of specialized proteins called membrane transporters, which are embedded into the lipid layer. These transporter proteins bind a specific substrate or cargo on one side of the membrane, undergo a reconfiguration and then release the substrate on the other side of the membrane. The ability to transport key nutrients into and out of plant cells is fundamental to healthy plant growth and development. However, our current understanding of how plants take up minerals and precisely regulate their concentration inside cells is very limited due to a lack of detailed information on the transport proteins responsible for their uptake and distribution. One key nutrient is boron which is taken up from the soil by the roots and has essential roles in the formation and strength of the plant cell wall. Plants are tolerant of a very narrow range between deficient and toxic soil solution concentration. Boron deficiency causes reduced plant size and impaired seed production, both of which have a major impact on crop yield. Boron toxicity also results in compromised plant growth and development. Vast areas of agricultural land in China and West and Central Africa have poor fertility as a result of boron deficiency, while land in Australia, Turkey and Chile is affected by boron toxicity. BOR1 is a membrane transporter with roles in boron distribution from plant roots to other growing tissues. It is very important to understand precisely in what form and how boron is taken up into cells through BOR1. Central to increasing understanding of the operation of BOR1 is a technique called X-ray crystallography which allows us to obtain very detailed information on the arrangement of the atoms within a protein structure. Obtaining such detailed structures of membrane proteins such as transporters remains very challenging as it is necessary to remove the proteins from their membrane environment into a detergent containing solution so they can be isolated and crystallised. We have made substantial progress towards obtaining a detailed structure of BOR1 having obtained well diffracting crystals of BOR1 from both the model plant, mouse-ear cress, and the important crop, rice. We will additionally complement the structural studies with analysis of the protein in plant cells and whole plants. The techniques we will use allow us to study the protein in a native environment and also assess the functionality of a range of mutant BOR1s. Taken together with the structure, the plant based functional analysis will provide an opportunity to build a uniquely detailed picture of how BOR1 works. This should form the basis of future studies attempting to generate plants with improved tolerance to suboptimal soil boron concentrations.

Boron (B) is an essential plant nutrient with a key role in the generation of ester cross-linked rhamnogalacturonan II through the formation of apisoyl dimers, essential for structure and function of the extracellular matrix. B deficiency and toxicity are major barriers to efficient crop growth in many parts of the world. B uptake, efflux and distribution are mediated by a complex system of integral membrane channels and transporters, one of which, BOR1, is a membrane transporter with important roles in the active transport of B for xylem loading. Despite the importance of this protein in the effective delivery of B to developing and growing plant tissues, remarkably little is known about its mechanism of action. The precise chemical nature of the substrate and molecular basis of BOR1 energisation are key unknowns. In order to investigate these and other key questions we will employ a combination of high-resolution X-ray crystallographic and plant functional studies. The research leading to this proposal has made significant progress towards the objectives through growth of well diffracting crystals of BOR1 from both the model plant Arabidopsis thaliana, and rice, Oryza sativa. In addition, we have a complete native diffraction dataset for the Oryza sativa BOR1. We aim to optimize the current crystals and solve the high resolution structure of at least one plant BOR1. The structural studies will be complemented with plant cell and whole plant studies aiming to identify and characterize key functional regions of the protein. Taken together with the structure, the plant based functional analysis will provide an opportunity to build a uniquely detailed picture of how BOR1 works. This should form the basis of future studies attempting to generate plants with improved tolerance to suboptimal soil B concentrations.

The main objective of the proposal is to gain highly detailed insight into the mechanism of action of a plant transporter protein through a combination of high resolution structural studies and plant functional analysis. Given that this is very much a basic science project the immediate impact of the results will be in scientific advancement in the areas of membrane transporter biology, structural biology and plant mineral nutrition. However in the longer term the research findings should be of major interest for plant breeders and plant producers having implications for crop productivity. The research undertaken will also have significant impact through the strengthening of collaborative links between the applicants based at Imperial and Warwick. The results of our research will be disseminated to the wider scientific community through the publication of manuscripts in high impact journals and presentations at national and international meetings. Key findings will also be promoted through press releases to the media and through the Imperial College and University of Warwick websites. Where there is scope for further exploitation of the research findings, we will take advantage of the resources at both Imperial and Warwick to protect arising IP. We will additionally use the research findings to establish further collaborations with Industry. We will also undertake public engagement activities in a variety of formats and at both institutions with the aim of making our research findings available and accessible to the general public as well as inspiring the new generation of scientists. The PDRAs funded by the proposal will benefit by developing high quality research skills in world-class research environments as well as having the opportunity to build networks with complementary researchers. It is anticipated that they will also gain significant experience of presenting data at conferences and writing manuscripts. As a member of the Imperial College Post-doc Centre or the Warwick Learning and Development Centre they will also gain training in more generic professional development skills including CV preparation and interview training to facilitate their next step in academia, industry or other relevant careers.