Vacuolar acidification relies on the combined activity of endomembrane proton pumps

The vacuole is not only the largest organelle of mature plant cells, it is also vital for the plants life. Due to their sessile lifestyle plants accumulate essential ions and metabolites inside their vacuole in times of excess but they can also serve as a reservoir under starvation conditions. Moreover, harmful compounds can be sequestered inside vacuoles in order to detoxify plant cells. Vacuoles provide structural support for the plant and are required for growth since osmotically driven water uptake generates turgor pressure that leads to cell expansion. To enable all transport processes, two proton pumps exist at the vacuolar membrane (tonoplast), the vacuolar H+-ATPase (V-ATPase) and H+-pyrophosphatase (V-PPase) that catalyse energy-dependent proton translocation to build up a proton gradient that is the driving force for secondary active transport. In contrast to the V-PPase, the V-ATPase is a multisubunit enzyme complex that consists of a cytosolic V1-subcomplex where ATP hydrolysis takes place and a membrane-bound Vo-subcomplex that is responsible for H+ translocation. Depending on the VHA-a subunit isoform that is incorporated into the complex, the proton pump is localized at the TGN/EE (VHA-a1) or at the tonoplast (VHA-a2, VHA-a3). Vacuoles of the tonoplast V-ATPase double mutant vha-a2 vha-a3 are known to have an increased pH, however they remain acidic to a certain extent. Thus, in the first chapter we aimed to elucidate how the remaining H+ gradient is established. First, we overexpressed the V-PPase in vha-a2 vha-a3, however no rescue of the mutant phenotype was observed. To gain insights into the role of the V-PPase for vacuolar acidification we created triple mutants lacking the tonoplast V-ATPase and the V-PPase and found that those plants are viable and have a neutral pH in rosette leaves. Since root vacuoles of the triple mutant were still acidified, we suggest that TGN/EE localized V-ATPase complexes and additional transport processes are involved in vacuolar pH regulation. With the second chapter of this thesis we clarify contradictory results concerning V-PPase overexpression and mutant lines. We show that the increased biomass of the 35S:AVP1 line is not due to constitutive overexpression, since neither RNA nor protein level and enzyme activity were elevated. Moreover, we found that the mutant growth phenotype of avp1-1 is caused by a second site T-DNA insertion in GNOM.