The Plasma Membrane of Avena Coleoptile

Treatment of living protoplasts from the Avenia coleoptile with enzymes and chemicals has provided new information about the external surface of the plasma membrane. Treatments with selected detergents and polyene antibiotics indicate that little sterol is present. The lysis of protoplasts in carboxymethyl-RNase which is enzymatically almost inactive provides strong evidence that the lysis previously observed in RNase is not an indication of RNA in the membrane. Divalent cations inhibit the RNase-induced lysis, indicating that such lysis involves the interaction of RNase with negatively charged sites on the plasma membrane surface. Tyrosinase treatment gives no lysis, showing that tyrosine does not play the role in these plasma membranes attributed to it in some animal cells. Peroxidase does not harm coleoptile protoplasts. For physiological studies of the plasma membrane of plant cells, the cell wall is sometimes a major impediment. Work can be done more easily upon the plasma membrane in situ if the walls are first removed enzymatically, leaving plant protoplasts (4, 5, 26, 27). During the treatment of fragile protoplasts with various chemicals and enzymes, the maintenance of spherical shape and of vigorous cyclosis becomes an easily observed bioassay for the lack of deleterious changes in the structure and function of the plasma membrane of living cells. Information on the nature of the plasma membrane surface can assist in the interpretation of such varied experimental results as (a) the effects of RNase upon wall-membrane interactions (19), (b) the location of the plasma membrane in cell homogenates (13, 30), and (c) the fusion or lack of fusion of protoplasmic surfaces under different experimental conditions (23). The data on protoplast treatments that are included in this paper provide prima facie evidence for the nonexistence in the plant plasma membrane of several molecules-sterols, RNA, and critical tyrosine residues. In addition, the plasma membrane surface is shown to bear negative charges. These indirect data are important because it has so far not proven possible to isolate plant plasma membranes for direct analyses. MATERIALS AND METHODS Protoplasts of coleoptiles of Avena sativa, cv. Victory, were prepared and used as described previously (26). In brief, diced, 5-mm subapical segments of 72-hr-old coleoptiles, from which I The National Science Foundation provided support through several fellowships and Grant GB 8006. the primary leaves had been removed, were placed for 1 to 1.5 hr in 0.50 M mannitol or 0.14 M KCl and 0.10 M CaCl2 (27) containing 3% of a partially purified polysaccharidase (predominantly cellulase) prepared from the fungus Myrothecium verrucaria. The enzyme was then washed out by two 15-fold dilutions with the osmoticum alone. Thirty to 300 protoplasts (10 ful) were transferred to depression slides with Lang-Levy micropipettes and counted by systematic scanning of the slides with a phase-contrast microscope. Next 10 ,AI of the treatment solution were distributed evenly over the slide. For all experiments reported here, the treatment time was 1 hr. The number of intact protoplasts then remaining on the slide was determined and divided by the initial number to give the "fraction surviving." Twenty-five representative protoplasts were inspected for the presence of cytoplasmic streaming, giving the value for "fraction streaming," a measure of the condition of the cytoplasm independent of the resistance of the plasma membrane to damage. Further information on the preparation and handling of protoplasts will be published elsewhere (Ruesink, in preparation). The antibiotics nystatin and amphotericin B (Squibb, Paris) were dissolved in DMSO2 because of solubility problems (33). The enzyme polyphenol oxidase (tyrosinase) (Worthington Biochemical Co.) and horseradish peroxidase (Worthington Biochemical Co., HPOF, activity 3900 units/mg) were used without further purification. MES was procured from Calbiochem. The ribonuclease was bovine pancreatic RNase, type I-A or II-A (Sigma). Carboxymethyl-RNase carboxymethylated at the I position of histidine-1 19 (7) was a gift from W. H. Stein.