The Induction of Ethylene Production from Pear Cell Culture by Cell Wall Fragments

Abstract

Macerase, a pectinase-containing enzyme mixture, was used to digest cell walls isolated from cultured pear cells. Following digestion, the reaction mixture was boiled to inactivate enzymes. Addition of soluble aliquots of the mixture to suspension cultures of pear cells led to a rapid and transient production of ethylene by the cells. Mechanical injury due to causes such as wounding and infection by fungi can induce increased ethylene production by plants. Although the role of the enhanced ethylene production is still unknown, it may participate in increasing resistance to disease (14), perhaps by inducing the synthesis of phytoalexins (4, 8, 10). Alternatively, ethylene may induce symptoms of pathogen attack , such as the formation of vascular system-obstructing gels (13). Fungal elicitors, oligosaccharides, or glycopeptides, extracted from mycelia, hyphal cell walls, and culture filtrates of fungi, have been shown to induce ethylene production when added to plant tissues (4, 7, 12). Anderson et al. (2) have shown that Cellulysin, a cell wall digesting preparation derived from Trichoderma viride, can also induce increased ethylene production when added to tobacco leaf discs. One mechanism of Cellulysin-induced ethylene synthesis may be the production of cell wall fragments that themselves induce ethylene synthesis. In this work, we investigate the ethylene induction capability of Macerase, a pectinase-containing enzyme mixture derived from Rhizopus, and of the cell wall fragments that are produced when Macerase digest pear cell walls. MATERIALS AND METHODS Materials. Pear cells were provided by Dr. Roger Romani, and cultured as previously described (9). Cells were grown for 7 d before transfer to medium lacking 2,4-D for another 7 d. Estimations of viability (selective staining by Evan's blue) and cell count (packed cell volume method) were performed as described by Puschmann and Romani (9). Macerase and ACC were purchased from Calbiochem. Preparation of Cell Walls. Cell walls were isolated from pear cells cultured for 7 d in growth medium. The medium was removed by vacuum filtration and the cells were ground with a mortar and pestle in liquid N2 and then in liquid N2 plus 100 ' Supported by grant PCM 84-14971 from the National Science Foundation. w ,to p / R A S E lMARASE 0 c = ~~~~~~~CO/JTROL-0 A 0 2 4 HOUR AFTER ADDITION FIG. 1. Time course of ethylene production by 5 ml pear cell cultures following the addition of 250 Ml 0.2% Macerase or products of pear cell walls digested with Macerase for 3 h. In the control, pear cells were incubated with a Macerase-cell wall mixture that had been boiled immediately after being mixed. Duplicate samples were used for each treatment. mm K-phosphate (pH 7.0). The homogenate was centrifuged at 20,200g for 20 min, and the supernatant discarded. The pellet was washed successively with 200 ml each 100 mm K-phosphate buffer (pH 7.0), buffer containing 1 M NaCl, deionized H20, a chloroform:methanol mixture (1:1), and acetone. It was finally dried in a vacuum oven at 50°C. Cell wall fragments were generated by incubating cell walls with a 0.2% Macerase (w/v) solution at a concentration of 30 mg cell walls/ml Macerase at 25°C. At specified times, aliquots of the mixture were boiled for 5 min and then centrifuged with a Lourdes table top centrifuge at 1,080g for 5 min. Aliquots from the supernatant were used for ethylene induction experiments. Different concentrations of a 3 h digest were obtained by diluting it with water. Ethylene and ACC Determinations. Pear cells (1.5-2.0 x 106 cells) in medium lacking 2,4-D were placed in a 25 ml Erlen-meyer flask with the test materials and then incubated at 25°C on a rotary shaker at 150 rpm. After capping with a rubber serum stopper for 15 min, 1 ml air samples were withdrawn from the headspace ofthe flasks, and ethylene content was determined on a gas chromatograph equipped with an alumina column and a flame ionization detector. The ACC2 content in the pear cells was determined by centrifuging the cells, and extracting the pellet with 80% ethanol at 60°C for 20 min. The ethanol extract was dried under vacuum, dissolved in 2 ml water, and assayed for 2 Abbreviations: ACC, 1-aminocyclopropane-I-carboxylic acid; AVG, aminoethoxyvinylglycine. 929