C 2 H 4 PRODUCTION BY CHILLED CUCUMBERS Chilled tissue produced more labeled

Chilling at 2.5 C accelerated the synthesis of 1-aminocyclopropane-lcarboxylic acid (ACC) and C2H. production in cucumber fruit. Skin tissue contained higher levels ofACC and was more sensitive to chilling than was cortex tissue. Accumulation of ACC in chilled tissue was detected after 1 day of chilling and remained elevated even after C2H4 production started to decline. These data suggest that ACC synthesis is readily stimulated by chilling, whereas the system that converts ACC to C2H4 is vulnerable to chilling injury. Chilling-induced C2H4 production was inhibited by aminoethoxyvinylglycine, sodium benzoate, propyl gallate, 2,4dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, and cycloheximide. The utilization of methionine for ACC formation and chilling-induced C2H1 biosynthesis was established using L-13,4-14Clmethionine. Chilled tissue had a higher capacity to convert L-13,4-14Clmethionine to ACC and C2H4 than did nonchilled tissue. Chilling temperatures have been observed to stimulate the production of C2H4 by tissues of a number of plants, including citrus, avocados, and pears (6, 13, 15). However, very little is known about the mechanism of this chilling-induced C2H4 production. It has recently been recognized that ACC2 is a precursor for C2H4 in many higher plant tissues (1, 4, 9). This unusual amino acid is produced from methionine via SAM. The proposed sequence for the pathway of C2H4 biosynthesis thus becomes: methionine -* SAM -ACC -* C2H4. The study presented here was initiated to determine if this pathway accounts for the production of C2H4 from chilled tissue and to identify the step(s) that are enhanced by chilling temperature. The C2H4 biosynthetic pathway is inhibited by AVG (7), uncouplers of oxidative phosphorylation (12), and free radical scavengers (2, 3). Therefore, the effect of these inhibitors on C2H4 production and ACC levels in chilled cucumbers has been examined. MATERIALS AND METHODS Chilling of Samples. Cucumbers (cv. Poinsett 76) grown in Beltsville, Md. , were used for all experiments. Freshly harvested 'This research was conducted while D. 0. Adams held a National Research Council Postdoctoral Research Associateship in the Postharvest Physiology Laboratory, Beltsville, Md. 2Abbreviations: ACC, I-aminocyclopropane-I-carboxylic acid; AVG, aminoethoxyvinylglycine 12-amino-4-(2'-aminoethoxy)-trans-3-butenoic acid]; CCCP, carbonyl cyanide m-chlorophenylhydrazone; DNP, 2,4-dinitrophenol; SAM, S-adenocylmethionine; PG: propyl gallate; SB, sodium benzoate. samples were divided into two lots and stored at 2.5 C or 13 C. Each day, development of chilling injury symptoms was evaluated in five cucumbers from each treatment group that were transferred to a room maintained at 20 C. Chilling injury was evaluated as previously described (14), and the severity of injury was rated on a scale of 1 to 5 (1, no abnormality; 2, trace; 3, slight; 4, moderate; and 5, severe injury). C2H4 and ACC Determination. C2H4 production by intact cucumbers stored at 2.5 C or 13 C was measured daily. Duplicate samples of two cucumbers each from each temperature treatment were transferred to a room maintained at 25 C and were allowed to warm to room temperature. Then they were placed in 1-liter chambers and sealed for 1 h. Gas samples then were taken for C2H4 determination by GC (11). After C2H4 determination, 3 g skin and 3 g cortex tissue were taken from each cucumber and assayed for ACC content by the method of Lizada and Yang (8). Incorporation of L-13,4-14CIMethionine. Three g skin tissue were placed in a 50-ml Erlenmeyer flask with 4 ml 10 mm Mes buffer (pH 6.0) containing 2% sucrose and 33.3 jg/ml chloramphenicol. After addition of 1 ml 2 ,uCi L[3,4-14C]methionine (52 ,uCi/4imol), the flasks were sealed with serum caps and incubated at 30 C for 6 h. At the end of the incubation period, gas samples were withdrawn and assayed for radioactive C2H4. For this assay, 12ml samples were injected into evacuated scintillation vials sealed with serum caps that contained 1 ml 100 mm mercuric acetate in glass-distilled methanol as absorbant. The vials were kept at 4 C for 3 h; then, 10 ml Aquasol scintillation fluid were added, and radioactivity then was determined by scintillation counting. Detection of 114CIACC. For chromatographic assay of radioactive ACC, the skin tissue was removed from the incubation flasks at the end of the incubation period, washed with H20, and homogenized in 70%0 ethanol. The residue after filtration was reextracted twice with 70%o ethanol, and the combined extracts were concentrated in vacuo at 40 C. The residue was taken up in 5 ml H20 and applied to a Dowex 50 column (H' form). The column was washed with H20, and the amino acids were eluted with 3 N NH40H. An aliquot of the concentrated eluate was chromatographed on Whatman 3MM paper along with 0.1 . tmol authentic ACC. Butanol-acetic acid-H20 (4:1:5, v/v) was used as developing solvent. Authentic ACC on the chromatogram was visualized by spraying with ninhydrin; radioactive ACC was located with a Baird-Atomic radiochromatogram scanner. A second aliquot of the concentrated amino acid fraction was taken for determination of labeled ACC by the method of Lizada and Yang (8). To determine the efficiency of the degradation of ACC to C2H4, 100 nmol unlabeled ACC was added to the sample as an internal standard, and the per cent recovery was calculated from the C2H4 produced after the reaction. Labeled C2H4 formed from [14C]ACC was absorbed in mercuric acetate and counted as described above. Testing of Possible Inhibitors of C2H. Production. Test solutions of AVG (0.005, 0.05, and 0.1 mM), SB (1 mm), PG (1 mM), DNP (0.05 mM), CCCP (0.05 mM) and cycloheximide (0.05 mM) were each made up in 10 mm Mes buffer (pH 6.0) and 2% sucrose. Each 25-ml Erlenmeyer flask contained 1 g skin tissue and 100