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Killing of Plant Cells by Pectic Enzymes: the Lack of Direct Injurious Interaction Between Pectic Enzymes or Their Soluble Reaction Products and Plant Cells. H. G. Basham, Former Liberty Hyde Bailey Research Assistant, Department of Plant Pathology, Cornell University, Ithaca, New York 14850, Present address of senior author: Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina 27607; D. F. Bateman, Professor and Chairman, Department of Plant Pathology, Cornell University, Ithaca, New York 14850. Phytopathology 65:141-153. Accepted for publication 25 July 1974. DOI: 10.1094/Phyto-65-141.

An isozyme of endopectate lyase (EC ef1 pl 9.4) produced by Erwinia chrysanthemi was purified by adsorption to CM Sephadex, DEAE cellulose “batch preparation”, CM Sephadex column chromatography, and isoelectric focusing. Disc gel electrophoresis of purified EC ef1 lyase yielded a single protein band that caused lyitic degradation of sodium polypectate, as well as maceration and cell death of potato and tobacco pith tissue. Cell injury in tissue treated with EC ef1 lyase was characterized by a rapid loss of ions and water from cytoplasm (ca. 50% in 1 hour), indicating damage to the plasmalemma of cells. Tissue plasmolysed to the point of incipient plasmolysis in the presence of enzyme was protected from ion loss, but not from cell wall breakdown. Several endopectic enzymes differing in pH optima, isoelectric points, and mechanisms of substrate cleavage (lyitic or hydrolytic), macerated and killed tissue. There was no evidence for a direct interaction between pectic enzyme and the plasmalemma. When isolated tobacco protoplasts were subjected to osmotic stretching in the presence of EC ef1 lyase, protoplast (plasmalemma) injury was not enhanced. When tissue or isolated cell walls were treated with EC ef1 lyase, no soluble toxic products or by-products were detected in supernatants of the digests. Upon fractionation of the enzymatic digests of isolated cell walls or tissue, the fractions toxic to tissues contained residual EC ef1 lyase activity. Beet tissue, resistant to wall breakdown and cell injury by EC ef1 lyase, was not injured by enzyme digests of tobacco tissue. Hydrogen peroxide (100mM) did not cause electrolyte loss from potato tissue, nor did it accumulate in tissue treated with EC ef1 lyase. Loss of electrolytes (cell injury) in tobacco and potato tissue paralleled the course of lyitic degradation of plant cell walls. All enzymes which macerated tissue caused cell death, but an exopolygalacturonase from Verticillium caused no significant maceration or cell death in potato tissue. The results of this study are consistent with the hypothesis that membrane damage in cells treated with endopectic enzymes results from a loss in the ability of enzymatically degraded plant cell walls to support the limiting membrane of the cell.

Additional keywords: soft rot, membrane permeability, Verticillium albo-atrum, Rhizopus stolonifer, phytotoxin.