Vacuolar pyrophosphatases confer anoxia tolerance in rice by pumping protons across the tonoplast

Vacuolar pyrophosphatase (V-PPase) is induced by a number of abiotic stresses and is thus thought to play a role in plant adaptation. This thesis reports on the regulation of six V-PPase genes in rice (Oryza sativa L.) coleoptiles under anoxia, using cultivars with varying flood tolerance at the germination stage to test this hypothesis. Quantitative PCR time courses showed that one vacuolar pyrophosphatase (OVP3) was consistently induced by anoxia, particularly in the flood-tolerant cultivar Amaroo where it rose 20-fold in 2 h. Regulation of OVP3 expression under anoxia was investigated by analysis of putative OVP promoters. The putative OVP3 promoter contained more previously identified anoxia-inducible motifs than the promoters of the other five OVP genes. GUS activity in transgenic rice plants transformed with the OVP3 promoter region linked to the GUS reporter gene was induced by anoxia but not by salt or cold: GUS staining was visible mainly in the stele of seminal roots. Transgenic Arabidopsis plants overexpressing AVP1 (an Arabidopsis vacuolar pyrophosphatase) showed increased anoxia resistance as measured by survival and growth rate during the recovery period in air after anoxic treatment. Transgenic rice plants overexpressing OVP3 had higher anoxia tolerance which was supported by reduced solute leakage, more polarised membrane potentials and higher cytosolic pH under anoxia, compared to wild type roots. Membrane potentials of knockouts of the OVP gene family were depolarised under anoxia compared to wild type plants. I conclude that OVP3 is a key gene determining anoxia tolerance in rice seedlings via its effect on membrane properties. The importance of OVP3 is significant as a single gene determinant of anoxia tolerance.