Facilitation of electrofusion of plant protoplasts by membrane-active agents

Abstract

Protoplasts isolated from a suspension culture of Daucus carota were subjected to electrofusion by means of a combination of dielectrophoresis to align the cells and brief d.c. shocks to induce fusion. The protoplasts were treated with agents known to alter membrane structure and function in order to define the factors that limit electrofusion. Lysophosphatidylcholine, dimethylsulfoxide and calcium chloride enhanced electrofusion; low temperature reduced fusion. However, lysophosphatidylcholine, calcium chloride, and low temperature all decreased membrane fluidity as measured by electron spin resonance spectroscopy of intact carrot protoplasts labeled with 5-doxylstearic acid. These spectra were taken in the presence of Fe(CN) 6 3− and thus largely reflect the fluidity of the plasma membrane. Treating the protoplasts with pronase or proteinase K also facilitated electrofusion. The proteinase K effect was largely reversed by the specific protease inhibitor phenylmethylsulfonyl fluoride. Protoplasts labeled with fluorescein isothiocyanate-concanavalin A did not exhibit any perceptible capping or clustering of membrane proteins in response to protease treatment. Enhancement of protoplast electrofusion by proteases may be due to improvement of cell-cell contacts through the removal of membrane surface determinants. However, the possibility that proteases enhance electrofusion by causing aggregation of membrane proteins cannot be eliminated.