Russet Spotting Caused by Ethylene1

Russet spotting (RS), consisting of numerous small brown spots on the midrib of head lettuce (Lactuca sativa), is a physiological disorder induced by exposure to ethylene. In leaves suffering RS, the increase in spotting was accompanied by a parallel increase in the amount of phenolic compounds. Of these, chlorogenic acid and isochlorogenic acid were identified. Ethylene induced high phenylalanine ammonia-lyase (PAL) activity and RS formation in the susceptible cultivar Salinas, but not in the resistant cultivar Calmar. In the absence of ethylene neither significant PAL induction nor RS occurred. No correlation was found between the increase in polyphenol oxidase or peroxidase and the development of RS. The increase in PAL activity, however, was closely correlated with the development of RS. The increase in PAL activity preceded the development of RS, and the extent of RS was directly related to the level of PAL. Three temperatures (0.5, 5.5, and 12.5 C) were compared on the basis of their influence on both RS and PAL induction. At the lowest temperature (0.5 C) neither PAL induction nor RS occurred to a significant extent. At the highest temperature (12.5 C) an initial rapid increase in PAL activity and an earlier development of spotting were observed, but subsequently there was a decrease in both PAL activity and the rate of development of RS. At the medium temperature (5.5 C) both PAL activity and RS increased progressively with time. The decline ofPAL activity at a higher temperature might be attributed to inactivation of the enzyme. Thus, a temperature favorable for induction of PAL activity by ethylene was also favorable for RS. These observations indicate a close interrelationship between the induction of PAL activity and the development of RS in response to ethylene, and suggest a causal relationship between the two events. PAL serves as a useful biochemical marker for the RS reaction. Russet spotting is one of the most common commercially important disorders of postharvest lettuce (Lactuca sativa). It is observed as numerous small brown spots along both sides of the midrib, and may spread over the leaf blade during senescence (16, 18). It is caused by exposure of the lettuce to ethylene (25). According to Morris et al. (19), the factors affecting RS4 are: 'This work was supported in part by National Science Foundation Grants PCM 75-14444 and OIP 74-24426. 2 Present address: Department of Horticulture, Faculty of Agriculture, Shizuoka University, Okya, Shizuoka 422, Japan. 3 Present address: Wood Research Inistitute, Kyoto University, Uji, Kyoto, Japan. 4 Abbreviations: RS: russet spotting; PAL: phenylalanine ammonialyase; CAH: cinnamic acid-4-hydroxylase; OMT: 0-methyltransferase; PPO: polyphenol oxidase; CHA: chlorogenic acid; ICHA: isochlorogenic acid. 31 temperature (maximum susceptibility at near 5 C), ethylene (0.1 Al of ethylene caused RS within 3 days and the maximum effect was apparent after 7 days), 02 (low 02 inhibited RS), CO2 (increasing concentrations inhibited RS), and maturity (firmer heads were more susceptible). Lipton (17) and Ilker et al. (10) examined russet spots microscopically, and noted that discoloration spread to several subepidermal mesophyll cells as well as to the vascular tissue, and that the collapse of mesophyll cells resulted in pit-like depressions. Although RS has been the subject of detailed examination in respect to the symptomology and the various factors which influence the development (16-19, 25), no biochemical studies have been reported. Since the metabolism of phenolic compounds has often been thought to be involved in necrogensis, we have examined activities of five enzymes (PAL, CAH, OMT, PPO, and peroxidase) involved in phenol metabolism as possible biochemical markers, and have investigated the relationship between the activation of these enzymes and the development of RS caused by ethylene. MATERIALS AND METHODS "Crisphead"-type lettuce was used for all experiments. Cultivar Salinas was harvested at Salinas, California, and cv. Calmar was obtained at a local wholesale market. Harvested lettuce heads were stored at 0.5 C until used. Wrapper and cap leaves were removed and the next six to seven leaves were taken for experimental use. Ten to 12 leaves were placed in a 8.7-liter glass jar with a metal lid. A continuous flow of humidified air (air control) or air containing 1.2 ,ul/l of ethylene (ethylene treatment) at a flow rate of 10 liter/hr was applied to the leaves. Ethylene concentration was monitored by gas chromatography. Leaves were kept at 5.5 C except for those otherwise specified. The small russet-brown spots appeared mostly on the midrib of ethylene-treated leaves, but were also found on the blade. To describe the extent of injury, a rating scale of I to 9 was used. A score of I on this scale indicates no injury, and a score of 9 extreme injury. After an appropriate period of incubation in either air or air plus ethylene, discs (9-mm diameter) were excised from the midribs of the leaves with a cork borer. For determination of phenolic content, the discs were blended in a Sorvall Omni-Mixer and extracted in 80o ethanol. The filtrate was assayed for phenolic content with a Folin-Ciocalteu phenol reagent (6) using p-coumaric acid as a standard. For the identification of CHA and ICHA the ethanol was concentrated under reduced pressure and the concentrate was analyzed by paper chromatography and TLC. Fluorescence of CHA, ICHA, and other authentic polyphenolic compounds was observed under an UV lamp providing a maximal wavelength of 366 nm. For isolation ofCHA and ICHA, ethylene-treated leaves showing severe symptoms of RS (score 7-9) were used. The www.plantphysiol.org on January 16, 2018 Published by Downloaded from Copyright © 1978 American Society of Plant Biologists. All rights reserved. HYODO, KURODA, AND YANG concentrated extract was streaked on Whatman 3MM paper and developed withlbutanol-acetic acid-water (4:1:5, v/v). The bands corresponding to CHA and ICHA were cut out and eluted with 40 and 80% ethanol, respectively. After concentration these eluates were subjected to paper electrophoresis at pH 7 or 10 at 36 v/cm for 40 min. Further purification ofCHA and ICHA fractions was carried out by paper chromatography developed with 5% acetic acid. TLC was carried out on cellulose plates with a solvent of ethyl acetate-acetic acid-water (9:2:2, v/v). CHA, ICHA, and caffeic acid were detected by fluorescence under UV light in the presence or absence of NH3 vapor, and by the Hoepfner reaction (24). Phenolic spots were revealed by spraying with a mixture of FeCl3 and K3Fe(CN)6. ICHA was a gift from J. Corse. Enzyme Assays. Eight to 10 midrib discs (2-3 g) were homogenized with mortar and pestle in ml of 0.1 m Tris-HCI buffer (pH 7.3) containing 5 mm 2-mercaptoethanol and 0.3 g of Polyclar AT. The homogenate was centrifuged at 12,000g for 20 min. Extraction and centrifugation were carried out in the cold (below 4 C). PAL activity of the supernatant was assayed by measuring the increase in absorption at 290 nm as described elsewhere (8); 1 unit is defined as the amount of PAL which produces ,umol of cinnamic acid in hr under the specified conditions (8). The supernatant was also used for CAH and OMT assay by the procedure previously reported (7, 13). For the assays of PPO and peroxidase, the supernatant was dialyzed three times against 2 liters of0.01 m K-phosphate for 8 hr to remove 2-mercaptoethanol. The PPO was assayed by following 02 consumption in the presence of caffeic acid at 30 C with an 02 electrode. The reaction mixture contained 7 ,umol of caffeic acid, 0.5 mmol ofK-phosphate (pH 6.5), and a suitable amount of enzyme (derived from less than 50 mg fresh wt of tissue) in a total volume of 3.1 ml. Peroxidase activity was measured by the rate of o-dianisidine oxidation, as described elsewhere (28). All experiments were repeated more than once, and the data reported here represent the mean of duplicate samples in a typical experiment. Although there were some variations in PAL activities, the trends were identical.