Structural and functional properties of a borate efflux transporter from barley

Plants differ in their abilities to utilise BA and to tolerate excess BA. Physiological and molecular studies have confirmed that much of this variability can be explained by differences in uptake and distribution of BA in plants, which is primarily controlled by a group of membrane transporters. Both BA-deficiency and toxicity are major management issues in agriculture, and there is a need for in-depth investigation to understand how BA transport can be best manipulated to optimise crop production. Gaining a basic insight into the structure and function of borate transporters localised in cell plasma membranes would contribute significantly to our understanding how BA uptake and its distribution fits into the metabolic network of ion balance and homeostasis in plants. The gene encoding a borate transporter in barley, designated HvBot1, was identified by Sutton and co-workers (2007). The authors found the increased copies of transcripts in the BA-tolerant barley variety Sahara 3771 (Sahara). High expression of this gene in the BA tolerant barley variety (Sahara) compared to a sensitive variety (Clipper) resulted in the reduced amount of net borate ions being moved into the shoots from roots. Another gene HvNIP2;1 identified in Sahara was found to be involved in the transport of neutral BA (Schnurbusch et al., 2010b). It was however found that under the high BA concentrations in soils there was reduced expression of this gene in tolerant varieties. Therefore the tolerance mechanism in Sahara was suggested to be mediated by a combination of reduced expression of HvNIP2;1 to limit BA uptake and high expression of HvBot1 to remove excess borate ions from roots (Schnurbusch et al., 2010a; Pallotta et al., 2014). Despite these findings, the exact molecular mechanisms underlying the transport of borate ions by HvBot1 have not been described so far. The work presented here aimed to characterise the HvBot1 transport protein in vitro by applying a combination of techniques involving molecular biology, protein biochemistry and biophysics, nanotechnology and molecular modelling. Answers were sought to the following questions: What are the key transport properties that underlie transport of borate ions and what are the kinetic and mechanistic properties that are intrinsic to the function of HvBot1? For example, are there any caton-binding sites that are involved in the regulation of the transport process by HvBot1?Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Agriculture, Food and Wine, 2015