Localization of the Ethylene-synthesizing System in Apple Tissue

Abstract

Apple (Malus sp.) slices graduaDly lost the ability to synthesize ethylene when incubated with a mixture of enzymes that digest ceDl wails. The released protoplasts did not produce ethylene. The release of protoplasts was faster from climacteric fruit slices than from preclimacteric tissue. In protoplast suspension culture, as new cell wal was deposited (as judged by the intensity of fluorescence of regenerating protoplasts stained with Calcofluor White and the incorporation of labeled myo-inositol into their ethanol-insoluble residue), ethylene synthesis was graduaDly regained. Restored ethylene synthesis reached a maximum after 80 hours in protoplasts from preclimacteric fruit and in 120 hours in those from climacteric tissue. Addition of methionine (1 mM) to the culture medium was essential for appreciable synthesis of ethylene; and this synthesis was inhibited by the aminoethoxy analogue of rhizobitoxine and by propyl gallate, inhibitors of ethylene synthesis in higher plants. We suggest that the ethylene-synthesizing enzyme system is highly structured in the apple cell and is localized in a cell wall-cell membrane complex.The role of ethylene as a plant hormone regulating many aspects of growth and development has attracted considerable attention in recent years (16). With the discovery of methionine as the immediate precursor of ethylene in higher plants (17), it was hoped that an enzyme system converting methionine to ethylene would be isolated. So far, however, no such system has been demonstrated in vitro. Apparently this is due to something unique about the ethylene-synthesizing system in vivo, perhaps its structure, location, or mode of action. We recently reported (21) that the ethylene-synthesizing system in higher plants is influenced by its lipid environment and appears to be localized in the plasma membrane.Since the enzymic ethylene-synthesizing system could not survive the destruction of the cell, we thought that intact protoplasts might be useful for the study of ethylene biosynthesis. We therefore undertook to study the relation of the ethylene-synthesizing system of apple tissue to cell wall degradation and regeneration. From data presented we suggest that the enzymic system converting methionine to ethylene is highly structured and located in the cell membrane-cell wall complex of a higher plant cell. Glassware was autoclaved at 21 p.s.i. for 20 min. Preincubated apple slices were immersed in a lytic enzyme solution (5 ml/g of fresh tissue) of sorbitol (0.6 M), pectinase (0.2%), cellulase (0.5%), rhozyme HP-150 concentrate (0.5%), chloramphenicol (50 ,ug/ml), and fungizone (0.5 ,ug/ml) adjusted to pH 5.8. Control slices were immersed in a solution of sorbitol-chloramphenicol-fungizone only. Twenty-five-ml Erlenmeyer flasks containing the slices were incubated at 30 C on a shaker (shaking speed 42 strokes/min) until microscopic examination indicated the presence of protoplasts in the solution. The enzyme-protoplast mixture was passed through a sterile filter screen of stainless steel...

Regulation of Ethylene Biosynthesis in Virus-Infected Tobacco Leaves

“…Hence, a physical barrier for methionine between the intercellular space and the site of ethylene production is not apparent. This agrees with the possibility that (part of) the ethylene-producing enzyme system(s) is located at the plasmalemma (14,16). Since the specific radioactivity of the ethylene produced during the first 3 h after vacuum infiltration was already in accordance with that of the methionine within the leaf, [14Cimethionine supplied by vacuum infiltration is equilibrated very rapidly with any methionine pool within the leaf cells.…”

Polyamines Inhibit Biosynthesis of Ethylene in Higher Plant Tissue and Fruit Protoplasts

“…Polyamines may bind ionically to membrane targets, causing conformational changes that impair the functionality of the ethylene-synthesizing system. This hypothesis appears to be incompatible with a report (6) thesis, as suggested earlier (4,(9)(10)(11)(12)14), and if polyamines alter that microenvironment.…”

“…This step appears to be associated with a particulate fraction of the cell (7,11). The fact that ethylene production in situ (particularly the ACC-to-ethylene step) is susceptible to osmotic shock (4,12,14), cold shock (4,14), lipophilic membrane perturbants (4,(9)(10)(11)14), and ionic and nonionic detergents (4,1,12,14) indicates involvement of a membrane function in ethylene biosynthesis. Therefore, polyamines may interact with the plant membranes in a way that inhibits ethylene evolution by a physical mechanism.…”

Influence of Enol Ether Amino Acids, Inhibitors of Ethylene Biosynthesis, on Aminoacyl Transfer RNA Synthetases and Protein Synthesis

“…The inhibition by these compounds is probably due to their structural resemblance to methionine and leucine. One cause of rhizobial-induced chlorosis (8) in soybean by rhizobitoxine and the inhibition of growth of Bacillus sp. (13) by MVG, may, therefore be that it inhibits protein synthesis and charging of tRNAs in the host cells.…”

“…Rhizobitoxine was first isolated from root nodules produced by Rhizobium japonicum in soybean, Glycine max (L.) Merr., wherein it causes symptoms of rhizobial-induced chlorosis (8). The aminoethoxy analog (AVG) of rhizobitoxine was isolated from a Streptomyces strain and inhibited the growth of three Bacilli species and of S. cellulosae (10).…”