Electrophysiology of plant protoplasts in comparison of their intact cells.
By Dr. Shunnosuke Abe.
1 Isolation of protoplasts from plant cells suitable for electrophysiological studies.
We have enzymatically isolated giant protoplasts from Nitella expansa Allen.
These protoplasts posses intrinsic cytoplasmic membranes and all of the contents within the cytoplasm including the complete array of chloroplasts and ectoplasm, and showed active cytoplasmic streaming at about the speed as in the intact cells. Since cells underwent a conversion of shape from cylinder into sphere, the protoplasts have the north and south poles, where no streaming is observed, and the equator, where the fastest streaming is observed.
Materials and Methods
Plant material and Isolation of protoplasts
An actively growing terminal bud of Nitella expansa Allen was used as a source of protoplasts. Its unopened leaves were used for enzymatic digestion of the cell wall to isolate their protoplasts.
Enzyme treatment for digestion of cell wall.
Protoplasts were isolated as follows using a separating mesh, because any mechanical stresses such as shaking, filtering, and even gently swirling in ordinary isolation protocols should be avoided.
.A. A terminal bud was placed in an enzyme solution consisting of 1% Cellulase Onozuka R-10, 0.05% Macerozyme Onozuka R-10, 1mM CaCl2, 1 mM NaCl, 0.1 mM KCl, and an appropriate osmotic concentration of sorbitol (pH 5.5).
B. 1 hr after incubation with the enzyme solution.
C. 2 hr after incubation with the enzyme solution. Only debris was left over the mesh, and isolated protoplasts were on the bottom of the vessel (D). These freshly isolated protoplasts often have irregular shapes, and the cytoplasmic streaming has generally ceased.
Click the above picture to get a magnified image!
Next fig shows several isolated giant protoplasts above. Generally, after 10 to 30 min protoplasts recover their active cytoplasmic streaming and become reasonably good spheres.These spherical protoplasts are suitable for electrophysiology.
<----Click the image to magnify!
Next figure shows protoplasts isolated from carrot root cells by a similar method as Nitella. These are also large enough and active enough for electrophysiology.
2. Insertion of electrodes and Results.--Insertion of microelectrodes into Nitella protoplasts was performed with a electrical pulse insertion method developed by us and some results for the membrane potentials and action potentials are described
3. More results on membrane potentials
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