Isolation and characterization of intact vacuoles from suspension-cultured Arabidopsis cells

Abstract

It is well known that vacuole accumulates inorganic ions and metabolites to produce the turgor pressure. It is also known that the vacuole contributes to maintain the homeostasis of plant cells, for example the maintenance of metabolite levels or the degradation of intracellular components. But even now it is not enough to analyze these mechanisms. Isolation of vacuoles in bulk which are structually and functionally intact is needed for further analysis. In this study we established the isolation method of intact vacuoles from suspension-cultured Arabidopsis cells and characterized the samples. Intact vacuoles are prepared by a Percoll-density gradient after the disruption of protoplasts by osmotic shock. Western blotting with antibodies and measurement of activities of marker enzymes revealed that the isolated vacuoles have high purity and membrane integrity. By using these vacuoles, we are going to identify membrane transporters in tonoplasts and enzymes in vacuoles, and measure levels of metabolites.