Isolation and Transport Properties of Protoplasts from Cortical Cells of Corn Roots

Abstract

A procedure was developed for the enzymic isolation of large quantities of protoplasts from the cortex of Z. mays L. WF9 .times. MO 17 roots. Cortex was separated from the primary root, sectioned and the cell walls digested for 3.5 h in 2% (wt/vol) Cellulysin, 0.1% Pectolyase Y-23, 1 mM CaCl2, 0.05% bovine serum albumin, 0.5 mM dithiothreitol in 0.6 M mannitol (pH 5.6). Cortical cell protoplasts were collected by centrifugation and purified by flotation in a Ficoll step gradient. The yield of protoplasts was .apprx. 650 .times. 103/g fresh tissue. To obtain maximum yield it was essential to include an effective pectinase (Pectolyase Y-23) and protectants (bovine serum albumin and dithiothreitol) in the digestion medium. Cortical cell protoplasts exhibited energy-dependent uptake of K+ (86Rb), H232PO4- and 36Cl- as well as net H+ extrusion. Ion fluxes were sustained for at least 3 h. Influx of K+ was highest between pH 7.5 and 8.0, whereas the influx of H2PO4- was greatest between pH 4.0 and 5.0 K+ and H2PO4- influx and net H+ efflux were inhibited by respiratory poisons such as cyanide (0.1 mM) and oligomycin (5 .mu.g/ml) and by inhibitors of plasma membrane ATPase such as diethylstilbestrol (50 .mu.M). Calculated flux for Cl- was low, but not greatly different from that observed for other plant cells. K+ flux was somewhat high, probably because the K+ concentration in the cortical cells was below steady-state. Apparently, isolated cortical cell protoplasts retain transport properties which are similar to those of root tissue.