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 Zea mays L. WF9 x MO 17 roots. Cortex was separated from the primary root, sectioned, and the cell walls digested for 3.5 hours in 2% (w/v) Cellulysin, 0.1% Pectolyase Y-23, 1 millimolar CaCl(2), 0.05% bovine serum albumin, 0.5 millimolar dithiothreitol in 0.6 molar 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 approximately 650 x 10(3)/gram 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(+) ((86)Rb), H(2) (32)PO(4) (-), and (36)Cl(-) as well as net H(+) extrusion. Ion fluxes were sustained for at least 3 hours. Influx of K(+) was highest between pH 7.5 and 8.0, whereas the influx of H(2)PO(4) (-) was greatest between pH 4.0 and 5.0. K(+) and H(2)PO(4) (-) influx and net H(+) efflux were inhibited by respiratory poisons such as cyanide (0.1 millimolar) and oligomycin (5 micrograms per milliliter), and by inhibitors of plasma membrane ATPase such as diethylstilbestrol (50 micromolar). 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. The results indicate that isolated cortical cell protoplasts retain transport properties which are similar to those of root tissue.