Effects of la on surface charges, dielectrophoresis, and electrofusion of barley protoplasts

When dielectrophoresis and electrofusion of barley (Hordeum vulgare var Moor) leaf protoplasts were assayed in the presence of 0.1 to 1 millimolar lanthanum ion (La(3+)) in the basal medium (0.7 molar mannitol, 1 millimolar piperazine-N, N-bis[2-ethanesulfonic acid]-Na [pH 6.7], 0.1 millimolar CaCl(2)), dielectrophoresis and induction of electrofusion were strongly inhibited. The latter remained inhibited and the former recovered by about 60% after washing the La(3+) -treated protoplasts without EDTA. These inhibitions were almost completely abolished by washing the La(3+) -treated protoplasts with 1 millimolar EDTA. Inductively coupled plasma atomic emission spectroscopic analysis revealed that protoplasts retained a considerable amount of La(3+) after washing without EDTA and released most of the bound La(3+) by washing with 1 millimolar EDTA. This tightly bound La(3+) seemed responsible for the inhibition of electrofusion and dielectrophoresis that was observed in the La(3+) -treated protoplasts after washing. zeta-potentials of protoplasts were -39.0+/-3.2 millivolts, -16.7 +/- 2.6 millivolts, and virtually zero in media containing 0, 0.1, and 0.3 millimolar La(3+) (I = 7.2 millimolar), respectively, and had a positive value (+ 14.2 +/- 2.2 millivolts) in the presence of 1 millimolar La(3+). These effects of La(3+) on zeta-potentials were easily abolished by washing without EDTA. This indicates that charged species located at the surface of plasma membrane of protoplasts cannot account for the sites at which La(3+) exerts its inhibition of dielectrophoresis and electrofusion. In contrast, the promotion of spherical fusion and the reduction of broken fusion products observed in the presence of La(3+) were almost completely abolished by washing without EDTA. Our results also indicate that the initial induction and development of electrofusion can be studied independently.