In vivo fluorescence labeling of plasma membrane from plant tissue and cells

Abstract

Eosin-5-maleimide (EMA) is a membrane impermeable thiol reagent and was used to label proteins of the outer surface of the plasma membrane from intact cells and plant tissue. The covalent reaction of the fluorescent dye with cysteine residues of proteins allowed the visualization of the labeled cell compartment by microscopy, as well as in membrane preparations of cell homogenates from corn, squash and carrots. Density gradients from a resuspended membrane pellet (1000–15 000 × g) showed a fluorescent band at 40–42% (w/w) sucrose in all specimens. This EMA-enriched zone was well separated from the protein maximum and from membranes, which could be classified as tonoplast, golgi apparatus and mitochondria by its marker enzymes. A conformity of the fluorescence maximum with maximum enzyme activity of the plasma membrane marker (UDPG)-steroyl-glucosyl transferase (SGT) could be observed in a few cases (e.g. corn), although SGT activity varied considerably (32–40% sucrose) according to the object and the tissue used. Microscopic analysis of the labeled cells detected a strong fluorescence in the region of the cell wall and the plasma membrane. The use of EMA as a tool for the labeling and identification of plant plasma membranes is discussed.