Expression patterns of the intracellular ion exchangers AtNHX5 and AtNHX6 and their role in subcellular trafficking to the vacuole

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Animal, Plant and Soil Science, School of Life Sciences, College of Science, Health and Engineering, AgriBio, the Centre for Agribiosciences, La Trobe University, Bundoora.

The family of sodium-hydrogen exchangers (NHX) are highly conserved across all taxa and function in cellular homeostasis via the antiport of a monovalent metal ion (Na+/K+/Li+) for a proton (H+) across a membrane. A sub-group of NHX proteins which localise to internal membranes have a role in lysosome or vacuolar trafficking and pH regulation in animal, plant and yeast cells, and are implicated in important human pathophysiological and neurological diseases. In plants and fungi, these antiporters are also important determinants of salt tolerance. The Arabidopsis thaliana intracellular sodium-proton antiporters NHX5 and NHX6 have a well-documented role in plant development, and have been used to improve salt tolerance in a variety of species. Recent evidence suggests that intracellular NHX proteins provide a “proton leak” to finely regulate the pH of intracellular compartments, and that maintenance of this pH is critical for vacuolar trafficking of soluble cargo. However, the expression patterns and regulatory elements of these antiporters, as well as their impacts on other subcellular trafficking machinery and processing enzymes, are largely unknown. This thesis first describes the investigation of the whole plant expression patterns of NHX5 and NHX6 using quantitative PCR and a promoter:GUS reporter construct. Secondly, the role of NHX5 and NHX6 in the processing of the predominant seed storage proteins (SSPs) and the impact on other members of the SSP processing and trafficking pathways is investigated. HPLC-MS and complimentary proteomic approaches were used to investigate the processing and accumulation of SSPs and the associated processing enzymes in the nhx5 nhx6 double mutant. Finally, the suite of known mis-trafficked vacuolar cargo was expanded using a novel methodology for identifying secreted proteins, and a bioinformatics comparison was conducted to identify common sorting determinants or other features.