The Generation of Non-Spherical Oat Protoplasts Indicates that Hyperpolarization is both Necessary and Sufficient for Stimulating Membrane Incorporation into the Plasma Membrane

We have devised conditions which produced isolated protoplastsof non-spherical shape and which, therefore, affected the mechanismsthat control the exchange of membrane material between the plasmamembrane and an intracellular membrane reservoir. Non-sphericalprotoplasts of Avena sativa were obtained if protoplasts weretreated with hypertonic shock in the presence of 1.0 mol m^–3LaCl₃ at pH 8.3. This indicated that their ability to removeplasma membrane material via endocytotic vesiculation was suppressed.Non-spherical protoplasts were obtained under isotonic conditionsif protoplasts were incubated with 1.0 mol m^-3 LaCl₃ at pH 8.3and the proton carrier CCCP (12 mmol m^–3) was added. Thenon-spherical protoplasts had intact membranes as judged bystaining with fluorescein diacetate. The loss of the sphericalshape was reversible. On addition of EDTA protoplasts resphericulatedimmediately. Incubation in isotonic solution at pH 8.3 containingeither only 1.0 mol m^–3 LaCl₃ or only CCCP did not influencethe protoplast shape. We conclude that the membrane hyperpolarizationinduced by CCCP at high pH acted to stimulate the incorporationof membrane material into the plasma membrane and, subsequently,produced nonspherical protoplasts if the removal of membranematerial was simultaneously suppressed. This demonstrates thatmembrane incorporation and removal are two largely independentprocesses.