Isoelectric Focusing of Plant Cell Protoplasts

Abstract

ABSTRACIThe surface charge of plant protoplasts has been measured by a new technique, isoelectric focusing. The protoplasts were loaded in a dextran density gradient over which a pH gradient was superimposed. When voltage was applied, protoplasts moved to a point in the gradient corresponding to their isoelectric point (pl). The pI of the protoplasts varied with the compounds used for pH gradient generation. Using commercial ampholytes for pH gradient formation, the pI of all protoplasts tested was 4.4 ± 0.2, and viability following electrophoresis was low. Using an acetate/acetic acid mixture to generate the pH gradient, the pI of protoplasts varied from 3.7 to 5.3 depending on the species and tissue type of the parental cells. Postelectrophoresis viability was high. Using isoelectric focusing techniques, it was possible to separate mixtures of protoplasts derived from different species of plants.The zeta potential (a quantity related to surface charge) of plant protoplasts has been determined by measurements of the speed of protoplast movement in an electric field, their EPM' (7,15,18). Protoplasts derived from different tissue types and species were found to have different zeta potentials at constant pH, ranging (at pH 6) between -10 mv for mesophyll protoplasts from Vigna unguiictulata leaves to -36 mv for cortical protoplasts from Zea mays roots (15). Cell zeta potential has been correlated with several parameters such as viability (16) The IEF technique used here to separate plant cell protoplasts is based on that developed by Griffing and Quatrano (6) to determine the surface charge on plant organelles. The distinction between EPM techniques and IEF techniques is that while EPM is the rate of movement of the cells in an electric field at a constant pH, the pl is the pH at which the cells in an electric field no longer move. MATERIALS AND METHODSProtoplasts. Protoplasts were isolated from the leaves of periwinkle (Catharanthus roseus) plants, suspension culture cells of periwinkle cell line 916, and soybean (Glycine max) cell line SB-1 as described by Constabel (3).Material. The apparatus was described by Griffing and Quatrano (6). Clinical grade dextran (average mol wt 80,000) was obtained from Sigma Chemical Co. BioLyte ampholytes, pH range 3 to 5, 4 to 6, and 5 to 7, were obtained from BioRad Laboratories. All other chemicals were of the best grade commercially available.Electrofocusing Conditions. All gradients were prepared using equal volumes of solutions to be mixed, in a 50 ml gradient maker (Metaloglass Inc., Boston, MA). Low ionic strength acetate-containing gradients were prepared by mixing a high density (10% w/w) dextran solution containing 10 mm sodium acetate, 5 mm acetic acid, and 370 mm sorbitol with a low density (1% w/w) dextran solution containing 10 mM sodium acetate, 30 mM acetic acid, and 320 mm sorbitol. High ionic strength acetate-containing gradients were made up from a low density (1% dextran w/w) solution containing 50 mM sodium acetate, 100 mm acetic acid, and 100 mM sorbitol a...