Effects of La3+ 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.