Effects of two free radical scavengers and intermittent warming on chilling injury and polar lipid composition of cucumber and sweet pepper fruits

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1 Plant & Cell Physiol. (): - (979) Effects of two free radical scavengers and intermittent warming on chilling injury and polar lipid composition of cucumber and sweet pepper fruits Chien Yi Wang and James E. Baker Horticultural Crops Marketing and Postharvest Plant Physiology Laboratories, Federal Research, Science and Education Administration, U.S. Department of Agriculture, Beltsville, Maryland 7, U.S.A. (Received August, 978) Cucumber (Cucumis sativus L.) and sweet pepper {Capsicum annuum L.) fruits were chilled at. C for different periods and then transferred to C and subsequently evaluated for chilling injury. Sodium benzoate at nw or ethoxyquin at 9. mm, applied as a -min dip before chilling, increased the degree of unsaturation of 8-carbon fatty acids in the polar lipids and reduced the severity of chilling injury. Intermittent warming to C for hr at -day intervals also alleviated the chilling symptoms and increased fatty acid unsaturation of the polar lipids in cucumber and pepper fruits. Key words: Chilling injury Cucumber Free radical scavenger Intermittent warming Pepper Polar lipid. Chilling injury is one of the most important problems in the handling of certain fruits and vegetables (, 6, 7). The temperature for cold storage is often limited to C or above because some crops are susceptible to chilling injury. The chilled products develop surface pitting or other skin blemishes or internal browning, or they fail to ripen (6). Fresh fruits and vegetables exposed to chilling temperatures are particularly vulnerable to attack by decay organisms (-). The symptoms of injury become especially prominent after transfer to warmer temperatures. Numerous studies have been carried out in an attempt to alleviate and to elucidate the syndrome of chilling injury. Many biochemical and physiological changes were correlated with the symptoms of chilling injury, leading to the development of various concepts for the chilling phenomenon and different treatments for ameliorating the chilling injury (7). One method that ameliorates chilling injury is interruption of cold storage with one or more short periods of warm temperature. Increase in storage life and resistance to chilling associated with brief or intermittent warming periods during cold storage were reported in apples (), plums (6), peaches and nectarines (), citrus (, ), cranberries (8) and potatoes (9). Interruption of cold storage by a warming period also resulted in the arrest of chlorogenic acid accumulation and ascorbic acid decline due to low temperature (). Lyons and coworkers (8, 9) postulated that suceptibility to chilling might be related to the flexibility of mitochondrial membranes and that the difference in flexibility might be associated with the relative proportion of saturated and unsatu-

2 C. Y. Wang and J. E. Baker rated fatty acids of the membrane lipids. St. John and Christiansen (8) demonstrated that the ratio of linoleic acid (C8 : ) to linolenic acid (C8 : ) in polar lipids in root tips of cotton seedlings could be increased by treatment with -chloro- -(dimethyiamino)--phenyl-(//)-pyridazinone, a pyridazinone analog found to inhibit the production of linolenic acid by Hilton et al. (7). The chemical treatment also led to a decrease of chilling resistance in cotton seedlings. Additional evidence of the chemical regulation of linolenic acid synthesis and frost hardening was reported by Willemot () in winter wheat. On the other hand, Yamaki and Uritani () found no significant change in the ratio of unsaturated fatty acids to total fatty acids in the purified mitochondrial fraction of sweet potatoes after low temperature storage. In the presence of a free radical initiator and oxygen, unsaturated fatty acids undergo oxidative deterioration () leading to lipid peroxidation and an increased degree of saturation. The present study was undertaken to determine whether the free radical scavengers sodium benzoate and ethoxyquin could inhibit the oxidative destruction of unsaturated fatty acids and therefore modify the composition of polar lipids in the membrane and the susceptibility to chilling injury; and if intermittent warming could affect the ratio of saturated to unsaturated fatty acids in polar lipids and improve the resistance of cucumber and sweet pepper fruits to chilling. Materials and methods Samples: Cucumber (Cucumis sativus L. cv. Poinsett) and sweet pepper (Capsicum annuum L. cv. Keystone Resistant Giant No. ) fruits grown at Beltsville and on the Eastern Shore of Maryland were harvested either in the morning of the experiment or the day before. Fruits that were uniform in size and free from blemishes were chosen for the experiment. Chilling and intermittent warming: Cucumber fruits were stored at. C and 9-9% relative humidity for or 7 days. Sweet pepper fruits were stored under the same conditions for days or days. After the chilling period, 6 fruits from each experimental series were transferred to storage at C. Chilling injury was evaluated daily but reported only on the fifth, seventh and tenth days of storage at C. The severity of the injury was rated on a scale of to, with =no abnormality, =one to ten tiny pittings (trace), =more than ten tiny pittings (slight), enlarged depressions and browning of skin (moderate), and =injured areas overtaken by decay (severe). The extent of chilling injury was expressed as the percent of total fruits in each severity category. Each treatment was given a severity obtained by multiplying the extent of injury by the severity rating and adding the values for each rating. Samples for intermittent warming treatment were stored at. C for days, then transferred to C for hr and then returned to. C. The warming treatment was repeated at -day intervals until the samples received same lengths of chilling exposure as the controls. The severity of chilling injury for various treatments was compared in fruits kept for the same number of days at. C and at C for each treatment.

3 Chilling and polar lipids of cucumber and pepper Application of free radical scavengers: Sodium benzoate or ethoxyquin (6-ethoxy-l,-dihydro-,,-trimethylquinoline) was dissolved in. M potassium citrate at a concentration of HIM or 9. DIM, respectively. The ph was adjusted to.6 and a surfactant, Tween-, was added to give a final concentration of.%. Fruits of cucumber and sweet pepper were dipped in the above solutions for min before chilling. Higher concentrations of these chemicals used in preliminary experiments injured the surface of the fruits. Ten fruits were used for each chemical treatment in each chilling period. samples were dipped in. % Tween- and. M potassium citrate (ph.6) solution. The experiment was replicated twice. Polar lipid analysis: After chilling injury was rated, the outer cell layers ( to mm) from fruits per treatment were peeled and immediately dipped in liquid nitrogen and then freezedried. The dried samples were stored under nitrogen at C until analyzed. Polar lipids were extracted and quantitatively analyzed according to procedures described by St. John and Christiansen (8). The esterified fatty acids were analyzed using a Hewlett Packard 7A gasliquid chromatograph equipped with aflameionization detector and a.8-m stainless steel packed with % SP-6-PS on /-mesh Supelcoport. Temperature of the oven was 6 C; temperatures of the injector and detector were C; and the flow rate of carrier gas was ml/min. A known amount of n-heptadecanoic acid was included in all samples as an internal standard, and methyl heptadecanoate was used as an external standard. Identification of individual fatty acid methyl esters was made by using authentic standards. Peak area was measured with a Hewlett Packard 8A reporting integrator. The final concentration of each fatty acid was expressed as micrograms per gram of dry weight. The ratios of C8 : (oleic acid) + C8 : (linoleic acid) + C8 : (linolenic acid) to C8 : (stearic acid), C8 : + C8 : + C8 : to C8: + C6 : (palmitic acid), C8 : to C8 : were used to indicate the degree of unsaturation of the polar lipids. Results and discussion Cucumber fruits showed very little abnormality when kept continuously at. C for days. However, symptoms of chilling injury developed rapidly after the fruits were transferred to storage at C. Surface depressions and pitting appeared on the skin after one day at C. By the fifth day at C, % of the control fruits exhibited either moderate or severe symptoms of chilling injury (Table ). Ethoxyquin and sodium benzoate delayed the appearance of chilling injury. Samples treated with these chemicals did not develop symptoms of severe chilling injury until the seventh day at C. The intermittent wanning treatment also reduced chilling injury. Samples which had been warmed intermittently at -day intervals never developed any moderate or severe sympton of chilling injury throughout a -day holding period at C. The severity increased with increasing length of chilling. Cucumbers

4 6 C. Y. Wang and J. E. Baker Table Effect of free radical scavengers and intermittent warming on chilling injury in cucumber fruits stored at. Cfor days and then stored at C Days at C Treatment c Ethoxyquin c Sodium benzoate c Intermittent warming d Severity" rating / /o 7 Severity * cd 9 8 ef 6 6 7ef 7 f o/ /o 7 7 Severity 6 8 ab 8 de 8 ' de 7 f / la 6 6 Severity a 8 8 cd 6 be ef " Severity was rated on a scale of to with l=no abnormality, = trace, =slight, = moderate, and =severe injury. * Rating was obtained by multiplying percentage in the severity category with its numerical value. Mean separation was done by Duncan's multiple range test at % level. c Samples from these treatments were transferred to C after -day chilling period at. C. d Samples in this treatment were warmed to C for hr at -day intervals and received similar length of chilling exposure as control. Comparison of the severity of chilling injury for various treatments was made at a comparable chronological age. exposed to 7 days of chilling at. C had higher percentages of fruits with a severe rating than those exposed to days of chilling (data not shown). However, the beneficial effects of sodium benzoate, ethoxyquin, and intermittent warming were consistent for both lengths of chilling. At the end of days at C, 9% of the control fruits that were previously chilled for 7 days at. C displayed severe chilling injury, whereas only, and % of samples treated with ethoxyquin, sodium benzoate or intermittent warming, respectively, had severe injury.

5 Chilling and polar lipids of cucumber and pepper 7 Table Effect of free radical scavengers and intermittent warming on chilling injury in sweet pepper fruits stored at. Cfor JO days and then stored at C Days at C Treatment Severity" rating ' Ethoxyquin c Sodium benzoate' Intermittent warming d o/ 6 6 Severity * 8 8 cd 9 f h 9 g / /o 6 7 Severity 9 b 6 cd de 8 7 e / /o 8 6 Severity. 8 a 7 b c 6 9 d " Severity was rated on a scale of to with l=no abnormality, = trace, =slight, =moderate, and =severe injury. Rating was obtained by multiplying percentage in the severity category with its numerical value. Mean separation was done by Duncan's multiple range test at % level. ' Samples from these treatments were transferred to C after -day chilling period at. C. d Samples in this treatment were warmed to C for hr at -day intervals and received similar length of chilling exposure as control. Comparison of the severity of chilling injury for various treatments was made at comparable chronological age. Sweet peppers were also susceptible to chilling injury, but to a lesser extent than cucumbers. Trace to moderate chilling injury was seen in sweet pepper fruits exposed to. C for days (Table ). Although severe chilling injury did not develop in the control fruits, resistance to chilling was improved after treatment with ethoxyquin, sodium benzoate, or intermittent warming. The degree of unsaturation of 8-carbon fatty acids in the polar lipids of cucumber and pepper fruit tissues increased following exposure to chilling at. C (Tables

6 8 C. Y. Wang and J. E. Baker Table Effect of chilling temperature and free radical scavengers on fatty acid composition of polar lipids in 'Poinsett' cucumber fruit tissue Days at Fatty acid content (/«g/g dry wt) Degree of unsaturation ". C Treatment C6: C8: C8: C8: C8: A B C Ethoxyquin Sodium benzoate c 7.b.9 a. a. a.a. a.8 a.7 a.6 b. c. c Mean separation in columns by Duncan's multiple range test at % level. A=Ratio of C8:l + C8: + C8: to C8:; B=ratio of C8:l+C8: + C8: to C8: + C6:; C=ratio of C8: toc8:. and ) and was further enhanced by treatment with ethoxyquin or sodium benzoate. This enhancement was a result of the increased content of the more unsaturated fatty acids. The chilling induced increase in linolenic acid may reflect some shift in lipid metabolism similar to that occurring in the hardening process of plants. Similar increases in the content of unsaturated fatty acids after exposure to low temperatures found in cotton plants {8), winter wheat seedlings (), alfalfa roots and leaves (6, ), and peach trees () were associated with increased chilling resistance or cold hardiness. Treatment with the two free radical scavengers also increased the level of palmitic acid (C6 : ) in pepper and cucumber tissues. The reason for this increase is not known. When C6 : was included in the calculation for the degree of unsaturation, no significant differences in the ratios were found between different treatments (Tables and ). The ratio of C8 : /C8 : has been used to gauge chilling sensitivity by St. John and Christiansen (8). A low ratio would indicate chilling tolerance. There were significant decreases in this ratio in cucumber tissue treated with ethoxyquin and sodium benzoate. The ratios were also lower for treated pepper tissues than for control tissues, but the differences were not statistically significant. The inconsistency in the ratio of C8 :/C8 : indicates that free radical scavengers do not specifically influence the synthesis of linolenic acid as did the pyridazinone analog used by St. John and Christiansen (8). The double bond (DBI) has also been used to indicate the degree of unsaturation in membrane lipids (9). However, it is questionable whether or not the DBI accurately reflects Table Effect of chilling temperature and free radical scavengers on fatty acid composition of polar lipids in 'Keystone' pepper fruit tissue Days at. C Treatment Ethoxyquin Sodium benzoate As in legend of Table. Fatty acid content (/tg/g dry wt) C6: C8: C8: C8: C8: Degree of unsaturation " A 6. c 9.b.8a. a B.7b.6 a.9 a.7 a C.8 a.7 b.6b.9 b

7 Chilling and polar lipids of cucumber and pepper 9 the degree of unsaturation in membrane lipids (, 9). Our data expressed as DBI, showed a trend towards higher values for treated fruit than control fruit, but the differences were not very large (data not shown). The ratio that correlated best with observed increases in chilling resistance was the ratio of unsaturated C8 to saturated C8 fatty acids in the polar lipid fraction. Further experimentation is necessary to determine whether or not this ratio accurately reflects the degree of chilling resistance. Biological membranes containing abundant unsaturated lipids are often bathed in an oxygen-rich, metal-containing fluid and are susceptible to peroxidative attack involving free radicals (). Many free radicals are highly reactive chemically and can induce oxidative breakdown of the double bonds in the fatty acids of membrane lipids. This process can be inhibited by scavengers of free radicals, which, by removing free radicals, may retard the loss of unsaturated fatty acids in the membrane lipids. Ethoxyquin is known to be an antiodxiant () and is an effective inhibitor of superficial scald in apples (7). Scald is believed to be a form of chilling injury. The accumulation of toxic substances such as acetaldehyde or triene hydroperoxide after prolonged storage at chilling temperature has been proposed as the cause of scald (, ). Our data suggest that ethoxyquin may act as a free radical scavenger and prevent the oxidative destruction of the unsaturated bonds of the 8-carbon fatty acids in the membrane polar lipids. However, there is no direct proof for the mechanism by which ethoxyquin or sodium bezoate act to increase unsaturation of C8 fatty acids of polar lipids and chilling resistance. The fact that C6 : also increased after treatment with these compounds indicate that their effects may be more complicated than just an inhibition of fatty acid peroxidation. Further experiments to elucidate the nature of ethoxyquin and benzoate effects are under way. Prolonged chilling has been postulated to cause an imbalance in metabolism that leads to the accumulation of toxic metabolites in cells (6). These abnormal products may be metabolized by brief exposure to warmer temperatures and the injury may be reversed if the changes are still in the reversible stage. An optimum warming cycle for chilled cucumbers or sweet peppers was not determined in our study. Although intermittent warming treatment greatly reduced physiological Table Influence of intermittent warming on fatly acid composition of polar lipids in chilled cucumber and sweet pepperfruit tissue Fatty acid content (^g/g dry wt) Degree of unsaturation Treatment" C6: C8: C8: C8: C8: A B C Cucumber: Intermittent warming Sweet pepper: Intermittent warming b 7.8 a.b.a. b.a.8 b. a.9 a. a.6 a. b " cucumbers were exposed to. C for days and sweet peppers for days; intermittently warmed fruits were warmed to C for hr at -day intervals. * As in legend of Table.

8 C. Y. Wang and J. E. Baker breakdown caused by chilling (Tables and ), sweet pepper fruits were softened excessively by our warming treatment. A better combination of temperatures, length, and frequency of warming may further improve the keeping quality of pepper and cucumber fruits and enhance their capacity to withstand chilling temperatures. There was a marked increase in the levels of unsaturated fatty acids in the intermittently warmed fruits of cucumber and sweet pepper (Table ). While this increase might have affected chilling resistance, the reason for the increase is not clear. It is possible that chilling stress induces a rapid adjustment of metabolism upon return to C that includes increased synthesis of unsaturated fatty acids. Chilling affects several metabolic processes {7). All of these changes may be related to the degree of chilling injury and the capacity of plant to withstand stress conditions. Changes in fatty acid composition are only part of the overall reaction. However, our data strengthen the hypothesis that changes of fatty acid composition in membrane polar lipids may be an important factor affecting chilling resistance. The authors are grateful for the advice of Dr. Judith St. John on methods of fatty acid analysis and for the technical assistance of Mr. Irving Newman. ' References ( ) Anderson, R. E. and R. W. Penney: Intermittent warming of peaches and nectarines stored in a controlled atmosphere or air. J. Amer. Soc. Hort. Sci. : - (97). ( ) Brooks, C. and L. P. McColloch: Some storage diseases of grapefruit. J. Agr. Res. : 9- (96). () Davis, P. L. and R. C. Hofmann: Reduction of chilling injury of citrus fruits in cold storage by intermittent warming. J. Food Sci. 8: (97). () De La Roche, I. A., C.J. Andrews, M. K. Pomeroy, P. Weinberg and M. Kates: Lipid changes in winter wheat seedlings (Triticum aestivum L.) at temperatures inducing cold hardiness. Can. J. Bot. : -9 (97). ( ) Dogras, C. C, D. R. Dilley and R. C. Herner: Phospholipid biosynthesis and fatty acid content in relation to chilling injury during germination of seeds. Plant Physiol. 6: (977). ( 6) GerlofT, E. D., T. Richardson and M. A. Stahmann: Changes in fatty acids of alfalfa roots during cold hardening. Plant Physiol. : 8-8 (966). (7) Hilton, J. L., J. B. St. John, M. N. Christiansen and K. H. Norris: Interactions of lipoidal materials and a pyridazinone inhibitor of chloroplast development. Plant Physiol. 8: 7-77 (97). ( 8) Hruschka, H. W.: Physiological breakdown in cranberries Inhibition by intermittent warming during cold storage. Plant Dis. Rpt. : 9- (97). ( 9 ) Hruschka, H. W., W. L. Smidi and J. E. Baker: Reducing chilling of potatoes by intermittent warming. Amer. Potato J. 6: 8- (969). () Huelin, F. E. and I. M. Coggiola: Superficial scald, a functional disorder of stored apples. V. Oxidation of ci-farnesene and its inhibition by diphenylamine. J. Sci. Food Agr. : 8 (97). (//) Ketchie, D. O.: Fatty acids in the bark of Haleheaven peach as associated with hardiness. Proc. Amer. Soc. Hort. Sci. 88: -7 (966). () Kidd, F. and C. West: The cause and control of superficial scald of apples. Gt. Brit. Dept. Sci. and Ind. Res. Food Invest. Rept. 9: -7 (9). () Kuiper, P. J. C.: Lipids in alfalfa leaves in relation to cold hardiness. Plant Physiol. : (97).

9 Chilling and polar lipids of cucumber and pepper () Levitt, J.: The hardiness of plants. Academic Press, Inc., New York, N.Y. 78 p. 96. () Lieberman, M., C. C. Craft and M. S. Wilcox: Effect of chilling on the chlorogenic acid and ascorbic acid content of Porto Rico sweetpotatoes. Proc. Amer. Soc. Hort. Sci. 7: 6-68 (99). (76") Lutz, J. M. and R. E. Hardenburg: The commercial storage of fruits, vegetables, and florist and nursery stocks. U.S. Depl. Agr., Agr. Hdbk. No. 66, 9 p. (968). (7) Lyons, J. M.: Chilling injury in plants. Ann. Rev. Plant Physiol. : -66 (97). (8) Lyons, J. M. and J. K. Raison: Oxidative activity of mitochondria isolated from plant tissues sensitive and resistant to chilling injury. Plant Physiol. : (97). (79) Lyons, J. M., T. A. Wheaton and H. K. Pratt: Relationship between the physical nature of mitochondrial membranes and chilling sensitivity in plants. Plant Physiol. 9: 6-68 (96). () McColloch, L. P.: Alternaria rot following chilling injury of acorn squashes. U.S. Dept. Agr., Mkt. Res. Rpt. 8, 9 p. 96. () McColloch, L. P.: Chilling injury and alternaria rot of bell peppers, ibid. 6, 6p. 96. () McColloch, L. P.: Chilling injury of eggplant fruits, ibid. 79, p () McColloch, L. P. and J. T. Worthington: Low temperature as a factor in the susceptibility of mature-green tomatoes to alternaria rot. Phytopathology : -7 (9). () Pantastico, E. B., W. Grierson and J. Soule: Chilling injury in tropical and subtropical fruits. II. Limes and grapefruit. Proc. Amer. Soc. Hort. Sci. Trap. Reg. : 7-8 (968). () Slater, T. F.: Free radical mechanisms in tissue injury. Pion Limited, London, 8 p. 97. (6) Smith, W. H.: of low-temperature injury in the Victoria plum. Nature 9: - (97). (7) Smock, R. M.: A comparison of treatments for control of the apple scald disease. Proc. Amer. Soc. Hort. Sci. 69: 9- (97). (8) St. John, J. B. and M. N. Christiansen: Inhibition of linolenic acid synthesis and modification of chilling resistance in cotton seedlings. Plant Physiol. 7: 7-9 (976). (9) Wheaton, T. A.: Physiological comparisons of plants sensitive and insensitive to chilling temperatures. Doctoral Dissertation, Univ. of California, Davis (96). () Willemot, C: Simultaneous inhibition of linolenic acid synthesis in winter wheat roots and frost hardening by BASF -8, a derivative of pyridazinone. Plant Physiol. 6: - (977). (/) Yamaki, S. and I. Uritani: Mechanism of chilling injury in sweet potatoes. Part V. Biochemical mechanism of chilling injury with special reference to mitochondrial lipid components. Agr. Biol. Chem. 6: 7- (97).

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