Determination of malate levels during the swelling of vacuoles isolated from guard-cell protoplasts

A method for the preparation of vacuoles from guard cells ofVicia faba L. is described. Vacuoles were released from guard-cell protoplasts by osmotic shock and purified on a Ficoll gradient. Contamination of the vacuoles was examined by assaying marker enzymes, such as fumarase, glucose-6-phosphate dehydrogenase, phosphofructokinase, acid phosphatase and mannosidase. Potassium ions in the incubation medium caused increases in the volume of the vacuoles by a factor of about 2.6, while the malate level remained unchanged. In contrast, malate synthesis was stimulated during the swelling phase when complete guard-cell protoplasts were exposed to K⁺. The possible role of K⁺ as an efficient osmotic effector is discussed.Abbreviations DEAE diethylaminoethyl - GCP guard-cell protoplast(s) - GCV guard-cell vacuoles(s) - MCP mesophyll cell protoplast(s) - MCV mesophyll cell vacuole(s)