Plasma membrane alteration during bacteria-induced hypersensitive reaction in tobacco suspension cells as monitored by intracellular accumulation of fluorescein

Alteration of plant cell plasma membrane lipid phase during bacteria-induced hypersensitive reaction has been reported. To improve the detection of membrane lipid phase alteration during bacteria-induced hypersensitive reaction we monitored the accumulation of the fluorescent molecule fluorescein, from added fluorescein diacetate. Cells exposed to fluorescein diacetate accumulate fluorescein by rapid diffusion of fluorescein diacetate through the lipid phase of the plasma membrane and intracellular release of the acetate moieties by enzymes having esterase activity. Therefore, alterations of the lipid phase which affect fluorescein accumulation by changing the diffusion rate of fluorescein diacetate through the membrane can be monitored. The hypersensitive reaction was induced in tobacco suspension cultured cells by addition of the pathogenic bacterium, Pseudomonas syringae pv. syringae. Dramatic reduction of fluorescein accumulation was observed after 2 h. Tobacco cells exposed to a Tn5 insertion mutant of the pathogen, which does not induce the hypersensitive reaction, or the compatible pathogen Pseudomonas syringae pv. tabaci, did not show a reduction in fluorescein accumulation. Evaluation of possible mechanisms indicated that decreased fluorescein accumulation resulted from decreased membrane permeability to fluorescein diacetate. Since fluorescein diacetate is a lipid-soluble, non-polar molecule, the lowered plasma membrane permeability to fluorescein diacetate implies that alteration of plasma membrane lipid phase occurs during the hypersensitive reaction of tobacco to P. syringae pv. syringae.