The Role and Function of Cation Chloride Cotransporters in Plants

Ion transport across cellular membranes is essential for the viability of all organisms. This includes both ion transport into and out of cells, as well as across the membranes of intracellular organelles. One such organelle is the Trans-Golgi Network (TGN/EE), which is vital for the sorting and delivery of proteins in cells. Ion transport is typically mediated by membranespanning ion pumps, channels and transporters. Together with a proton pump, ion transporters maintain the low luminal pH of the TGN/EE. The regulation of this pH is an important aspect for TGN/EE function and therefore endomembrane trafficking. In this thesis, a new regulator of TGN/EE pH is identified and characterised, the cation chloride cotransporter (AtCCC1), which is the first pH regulatory component in the TGN/EE that does not transport protons itself. AtCCC1 is ubiquitously expressed and the transporter localises to the TGN/EE. Arabidopsis Atccc1 knockouts have a higher TGN/EE luminal pH and the mutants show defects in osmoregulatory capacity of the TGN/EE, which might indicate alterations in ion efflux. These defects in ion and pH regulation were found to impact TGN/EE function with Atccc1 exhibiting reduced rates of both endo- and exocytosis. This is accompanied by a severe and extensive mutant phenotype with reduced cell elongation, stunted growth, and altered root and shoot morphology. Interestingly, a reduction in the osmotic stress induced internalisation of protein was observed in Atccc1 in addition to a reduction in plasmolysis. The potential connection between TGN/EE pH and osmotic stress was assayed by measuring TGN/EE pH in response to salt and osmotic stress. Stress treatments resulted in an increased pH, revealing that the TGN/EE pH is dynamic in wildtype plants. In contrast, TGN/EE pH did not change in Atccc1. In conclusion, these results demonstrate that AtCCC1 contributes to regulation of ion fluxes and pH in the TGN/EE, which is important for endomembrane trafficking and for response to stresses.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 202