Subcellular detection of lipase activity in plant protoplasts using fluorescence microscopy

Measurement of lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) activity in tissue extracts often poses problems due to the inhibitors released during homogenization. The present investigation provides a first report on the localization of lipase activity in viable plant protoplasts isolated from the cotyledons of germinating seeds of sunflower, cotton and peanut by fluorescence microscopy, using a lipase specific, 'glycerol-derived' synthetic substrate, i.e., 1,2-O-dilauryl-rac-3-glycero-glutaric acid-resorufin ester, commonly used for in vitro assays. Due to its lipophilicity, this chromogenic substrate readily permeates plasma membrane of plant protoplasts. Subsequent lipase action leads to cleavage of this substrate to release resorufin, which can be visualized due to emission of red fluorescence (lambda(max) excitation - 567 nm; lambda(max) emission - 584.6 nm) at its intracellular locations.