Effects of La³⁺ on Surface Charges, Dielectrophoresis, and Electrofusion of Barley Protoplasts

Abstract

When dielectrophoresis and electrofusion of barley (Hordeum vulgare var Moor) leaf protoplasts were assayed in the presence of 0.1 to 1 mil-limolar lanthanum ion (La3+) in the basal medium (0.7 molar mannitol, 1 millimolar piperazine-N,N-bis[2-ethanesulfonic acid]-Na [pH 6.7], 0.1 millimolar CaCl2), dielectrophoresis and induction of electrofusion were strongly inhibited. The latter remained inhibited and the former recovered by about 60% after washing the La3+-treated protoplasts without EDTA. These inhibitions were almost completely abolished by washing the La3+-treated protoplasts with 1 millimolar EDTA. Inductively coupled plasma atomic emission spectroscopic analysis revealed that protoplasts retained a considerable amount of La3+ after washing without EDTA and released most of the bound La3+ by washing with 1 millimolar EDTA. This tightly bound La3+ seemed responsible for the inhibition of electrofusion and dielectrophoresis that was observed in the La3+-treated protoplasts after washing. {-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 La3+ (I= 7.2 millimolar), respectively, and had a positive value (+ 14.2 ± 2.2 millivolts) in the presence of 1 millimolar La3+. These effects of La3+ on {-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 La3+ 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 La3+ were almost completely abolished by washing without EDTA. Our results also indicate that the initial induction and development of electrofusion can be studied independently. In our earliest study of electrofusion (19) and in related studies (1-3), the importance of calcium ions and a change of the membrane state into a fusible state in induction of electrofusion have been pointed out. We used Ca2+ in order to stabilize and enhance electrofusion. The effectiveness of Ca2 + on electrofusion was recently confirmed with plant (7, 14, 17, 20, 21) and animal cells (15). On the other hand, a low fusion efficiency, compared with a weakly conductive pure osmoticum medium, has been reported because addition of Ca salts increases conductivity of media (25). In mesophyll protoplasts from peas, Ca2+ reduces the threshold for electrofusion (7). In protoplasts from tobacco leaf cells and cultured potato cells, fusion frequencies are increased by the addition of Ca2+ (20). An absolute requirement of Ca2 + in elec-trofusion is reported with animal cells (15). In some cases, Mg2+ I Supported by a grant from the Ministry of Education, Science and Culture of Japan. ions also facilitate electrofusion, in addition to or instead of Ca2 + (25). These stimulative effects of Ca2+ on electrofusion do not seem to be adequately explained by the reduction of the fluidity of the lipid...

La3+ uptake and its effect on the cytoskeleton in root protoplasts of Zea mays L.

“…Because growth and cytoskeletal responses showed different lag times, complex and multiple LAN effects are likely, such as ion signaling and homeostasis (Fig. 6) and interaction with ion channels (Osawa and Matsumoto 2002) or binding to aromatic rings, carboxyl group and phospholipids (Abe and Takeda 1988). The similarities between LAN and Al effects suggest effects on root growth through interactions with the CW, plasma membrane, or symplasm (Kochian 1995).…”

“…1991) and there has been some debate as to whether LAN can across the cell membrane at all (Lehmann et al 2000;Gao et al 2003). Although there is binding and electrostatic attraction of LAN to plasma membranes (Abe and Takeda 1988;Yermiyahu et al 1997), it is not clear whether LAN directly or indirectly affects a calciumdependent signal transduction pathway. Ultrastructural studies may not be sensitive enough to accurately depict the actual distribution of LAN in situ (Lehmann et al 2000), therefore a kinetic uptake study was performed.…”

Interactive Effects of Al3+, H+, and Other Cations on Root Elongation Considered in Terms of Cell-Surface Electrical Potential

“…PT was assigned half the value of 2.4 x 10-6 mol/m2 for phosphatidylcholine in pure vesicles (2). Our value for RT was selected to obtain a computed value for E. of -48.7 mV at pH 6.7 in a medium of 1 Once our parameters had been chosen, our model was checked against some additional published values. For example, raising Na+ to 6 mm from 1 mm reduced our computed E. from -48.7 to -39.1 mV compared with Abe and Takeda's change from -48.8 to -38.9 mV, and a reduction of the pH of that 6 mm Na+ solution to 3.6 changed our computed E, to -4.3 mV compared with Abe and Takeda's measured value of -2.1 mV.…”

“…Because of the binding of H+ with carboxylate, phosphate, and amino groups, charge reversal of the plasma membrane surface can occur as the pH there drops below 4 (22). Divalent and polyvalent cations can also bind, and the latter can cause charge reversals at the membrane surface (1,21). Charge screening occurs because coulombic attractions concentrate cations around the cell-surface negative charges.…”

Physicochemical techniques in description of interactions in model and native plant membranes under stressful conditions and in physiological processes

“…Although zeta potential is not equal to surface potential, it is often used for the characterization of electric properties of liposomes, as an analogue of resting potential of cell membrane (Kutschera et al 2010;Honary and Zahir 2013). The electric properties of some organelles (as plastids/chloroplasts) and protoplasts, with exposed lipid layers, can also be measured (Nagata and Melchers 1978;Abe and Takeda 1988;Obi et al 1989aObi et al , b, 1990Gimmler et al 1991).…”