Induction and Regulation of Ethylene Biosynthesis by Pectic Oligomers in Cultured Pear Cells1

Pectic oligomers induced a rapid, transient increase in ethylene biosynthesis when added to pear cells in suspension culture. The rate of ethylene biosynthesis increased within 30 to 40 minutes after oligomer addition, reached a maximum between 90 and 120 minutes after addition, and then decreased to basal rates of synthesis. Both the rapid increase and decrease in biosynthesis appear to be precisely regulated components of the ethylene response to oligomers. Induction of ethylene biosynthesis by pectic oligomers resulted in a reduced sensitivity of cells to further ethylene induction. This reduction in sensitivity occurred within 90 minutes after an oligomer treatment, slightly preceding the decline in ethylene synthesis. The degree of insensitivity induced was proportional to the concentration of oligomer in the first treatment. Induced insensitivity to elicitors appears to represent a novel mechanism which may limit continued ethylene biosynthesis after ethylene induction. Ethylene was produced by pear cells throughout the cell growth cycle, as cells increased in density over a 6 day period. Endogenous ethylene biosynthesis was at a maximum during the first 4 days of rapid cell growth, then declined to half the peak rate through day 10. Pectic oligomers could induce an increase in ethylene biosynthesis above this background rate only after day 5, as endogenous biosynthesis declined. Changes in sensitivity to added oligomer during the growth cycle may result from insensitivity to elicitors induced by growth processes. Many environmental responses and developmental processes in plants are accompanied by changes in the rate of ethylene biosynthesis. Ethylene synthesis is rapidly induced by a variety of environmental events, including wounding, mechanical perturbation, pathogen attack, elevated UV and CO2, and reduced oxygen (2, 9, 12, 22, 28). The rate of ethylene biosynthesis also changes rapidly during many developmental processes, such as germination, growth, elongation, abscission, and ripening (3, 10, 12, 28). Little is known of the physiological or molecular mechanisms by which ethylene synthesis is regulated in these instances (6, 9, 22). Less yet is known of the means or the extent to which evolved ethylene acts to regulate other temporally associated processes (7, 8, 14, 26). However, ethylene's rapid induction, moveThis research was supported by the Regional Research U.S. Department of Agriculture Project N.E. 87. 2Current address: Plant Gene Expression Center, ARS-USDA, 800 Buchanan St., Albany, CA 94710, and Department of Plant Biology, University of California at Berkeley, Berkeley, CA 94720. ment, and dissipation would make it an ideal signal to coordinate cellular responses to significant changes in a plant's internal or external environment. Of the various aspects of ethylene induction and action, regulation of the pathway of ethylene biosynthesis is best understood, and is best characterized in response to wounding and climacteric ripening. Ethylene is produced via a short biosynthetic pathway: SAM3 (or AdoMet) is converted to ACC by ACC synthase, and ACC to EFE (28). Ethylene biosynthesis in response to wounding follows a consistent pattern in most plants. The local rate of ethylene biosynthesis begins to increase rapidly 20 to 30 minutes after wounding, reaches a peak at 40 to 60 min, then declines (2, 9, 21). The rise in wound-induced ethylene biosynthesis is limited by ACC synthase activity, rather than EFE activity; the pattern of biosynthesis reflects the de novo synthesis, accumulation, and activity of ACC synthase in the induced cells (2, 9). Several mechanisms have been identified which may limit continued ethylene synthesis after induction (10, 28). For example, ethylene is known to act as a feedback regulator of its own biosynthesis. In response to wounding, this feedback is negative, and ethylene biosynthesis is self-limiting and transient (10). In climacteric ripening, this feedback is apparently positive, and ethylene biosynthesis is autocatalytic and persistent (15). McMurchie et al. (15) proposed the existence of two systems for regulating ethylene biosynthesis: system I controlling most developmental and wound ethylene, system II responsible for climacteric ethylene. It has been suggested that oligosaccharides released from cell walls by wounding may play a role in regulating various plant responses, including ethylene biosynthesis (19, 22, 27). Pectic oligomers, in particular, are known to induce wound and defense responses in many plants (16, 20, 27) and to induce transient ethylene biosynthesis in preclimacteric tomato pericarp discs (5). Oligosaccharides are also released from cell walls during fruit ripening and have been proposed as regulators of the ripening process (3, 4, 24). Pectic oligomers, in particular, have been shown to increase climacteric ethylene biosynthesis and tissue reddening in whole tomato fruit and tomato pericarp discs (4, 5). Cells in suspension cultures have been used as model systems for the investigation of both wound responses and rip3Abbreviations: SAM, S-adenosylmethionine; ACC, 1 -aminocyclopropane-l-carboxylic acid; EFE, etheylene-forming ezyme; PCV, packed cell volume; DP, degree of polymerization; G7, mixture of smaller pectic oligomers; G 12, mixture of larger pectic oligomers.