THESE LE” "'7 :"f- v 7- g -. . - 3;. , 3 f". fij’? “99" ‘ , 1 :; “.31.. 2P .1) ‘F'flm .a~ . ‘I-¢‘-"Lv ~53? I: _' 4 k)‘ w..,. L-L. J {i K ‘ This is to certify that the dissertation entitled PHYSIOLOGICAL CAUSES FOR CHANGES IN CARBON DIOXIDE AND ETHYLENE PRODUCTION BY BRUISED APPLE FRUIT TISSUES presented by Joshua D. Klein has been accepted towards fulfillment of the requirements for Ph.D. Horticulture degree in M/m Major professor Date June 7, 1983 MSU is an Affirmative Action/Equal Opportunity Institulion 0-12771 MSU LIBRARIES BEIURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. 1 PHYSIOLOGICAL CAUSES FOR CHANGES IN CARBON DIOXIDE AND ETHYLENE PRODUCTION BY BRUISED APPLE FRUIT TISSUES By Joshua D. Klein A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1983 ABSTRACT PHYSIOLOGICAL CAUSES FOR CHANGES IN CARBON DIOXIDE AND ETHYLENE PRODUCTION BY BRUISED APPLE FRUIT TISSUES By Joshua D. Klein Apples (Malus domestica Borkh., cv. 'Empire') dropped onto a hard, smooth surface so as to receive an impact bruise midway between stem and calyx rapidly evolved C02 in proportion to drop height and number. The response occurred for fruits ranging in maturity from the preclimacteric stage of endogenous enthylene production to the postclimacteric stage. Enhanced €02 production, which continued for 12 to 24 hours after damage, may serve as a good index for damage that occurs to fruit during handling and transport. Neither aerobic nor anaerobic respiratory increases were responsible for the enhanced C02 output. Rather, the increased C02 production was due to decarboxylation of vacuolar malic acid by cyto- plasmic malic enzyme in the 0.5 cm of cortical tissues below the fruit surface at the bruise site, as shown by a decrease in titratable acidity of extracts from bruised tissues compared to nonbruised begin- ning 1 to 3 hours post-impact. Further evidence for the enzymatic Joshua D. Klein nature of the C02 response is that the optimal temperature range for simultaneously increased C02 production and decreased malate concen- tration in bruised tissues was 20 to 30°C. Bruising of preclimacteric fruit caused an increase in ethylene evolution, whereas bruising of fruits that had begun to ripen caused a decrease in ethylene evolution. Concentrations of 1-aminocyclo- propane-l-carboxylic acid (ACC) were similar in bruised and nonbruised tissues during the initial 24 hours after impact-bruising, but by 48 hours the ACC in bruised tissues had declined to 60% of the levels in nonbruised tissues. Although ethylene production by both bruised and nonbruised tissues increased with temperature increase from 0 to 30°C, the relative decrease in ethylene evolution due to bruising remained consistent at 50%. This was also true at 40°C despite the overall marked decrease in ethylene production by both bruised and nonbruised tissues. The similar ability of bruised and nonbruised apple tissues to convert exogenously supplied ACC to ethylene together with the consistent percentage decrease in ethylene production by bruised tissues at 0 to 40°C, indicates that the decrease in ethylene pro- duction upon bruising is due to physical destruction of the cells. rather than to a physiological reaction as found for the CO2 response. To Adina, of course. b~n ivy nix: nizn .nxba by n~5y nxi 03:85 item Many have done valiantly, But you exceed them all. Proverbs 31:29 ii ACKNOWLEDGMENTS I wish to thank my major professor, Dr. D. H. Dewey, for his steady guidance, encouragement, and support during the entire course of this study. Drs. D. R. Dilley and A. C. Cameron were very helpful during discussions of some of this work. I thank them for serving on my advisory committee and for their critical reading of the disserta- tion. I also wish to thank Drs. F. G. Dennis, P. Markakis, and J. Lee for serving on my committee. The good-humored and dedicated technical assistance of Mike Parker was greatly appreciated. Thanks are also due to the denizens of rooms 104 and 106, who provided welcome relief from apples with discussions of beans, grapes, tulips, and even less pertinent sub- jects. Dr. Miklos Faust of the USDA Fruit Lab, Beltsville, MD, first taught me that horticultural research is not only its own reward, but can be fun, too. In the 11 years since then (and certainly before), my parents and grandparents have always supported my academic and other pursuits, and for this I am profoundly grateful. Portions of this research were sponsored by American-Israel Binational Agricultural Research Development Fund Project No. 1-113-80, administered jointly by Drs. D. H. Dewey and K. Peleg. iii TABLE OF CONTENTS LIST OF TABLES . LIST OF FIGURES INTRODUCTION LITERATURE REVIEW Introduction . . Nature of the C0 response by injured fruits . . C02 evolution ans malate decarboxylation in fruits . Wound ethylene production in fruits . Literature Cited . SECTION I Carbon dioxide and theylene production by bruised 'Empire' apples . Abstract. Introduction . Materials and methods Results . Discussion Conclusion . Literature Cited SECTION II Levels of l-aminocyclopropane-l-carboxylic acid and ethylene in bruised apple tissue Abstract. Introduction Materials and methods Results and discussion Literature cited CONCLUSIONS . APPENDIX . iv Page vii Heads-boo w 16 17 17 20 24 48 53 54 Table LIST OF TABLES Section I Effect of 0, 1, and 3 drop-impacts from a height of 1 m on C02 evolution by 'Empire' apples . . Effect of fruit age on C02 production by tissues excised from impact-damaged 'Empire' apples Effect of fruit age on ethylene evolution from tissues excised from impact-damaged 'Empire' apples . Effect of atmospheric composition on volatile evolution by bruised and nonbruised 'Empire' apple tissues Effect of vacuum infiltration with 0.4 ll mannitol and impact damage on volatile evolution by 'Empire' apple tissues . . . . . . . . . . Effect of fruit age on malic acid concentration in tissue extracts from impact-damaged 'Empire' apples Section II Ethylene and ACC concentrations in bruised 'Empire' apple fruit tissues . . . . . . . Appendix Effect of 0, 1, and 3 drop-impacts from a height of 1 m on internal ethylene concentrationirlpreclimacteric 'Empire' apples Ethylene evolution from preclimacteric 'Empire' apples after impact drops from various heights . . . Oxygen uptake by bruised and nonbruised 'Empire' apple tissues as affected by exogenous malic acid . Page 26 29 36 4O 41 43 63 74 74 75 Table Page Ethylene evolution from bruised and nonbruised 'Empire' apple tissues as affected by exogenous malic acid . . 75 Ethylene evolution from bruised and nonbruised 'Empire' apple tissues as affected by incubation with or without 0.4 mM ACC after heating in a microwave oven for 4 minutes . . . . . . . . . . . . . . . . 76 vi Figure 10. LIST OF FIGURES Section'I Relationship between C0 production 4 hours post-impact and drop height of 'Empire' apples . . . . C02 production by 'Empire' apple fruits following har- vest at 21 days after full bloom and bruising by 6 kg compression for 10 second Relationship between C02 production by excised bruised tissue and drop height of 'Empire' apples 3 hours post-impact. . . . . . C0 production by 'Empire' apples bruised at 0 hours, Eh bruises excised or occluded 2.2 hours post-impact. C02 production by bruised and nonbruised excised 'Empire' apple fruit tissues at successive intervals below fruit surface, 4 hours post-imact . . Relationship between ethylene evolution 27 hours post- impact and drop height of preclimacteric 'Empire' apples harvested August 31 . . . . . Relationship between internal ethylene concentration 24 hours post-impact and dr0p height of 'Empire' apples harvested August 26 . . . . . . . Ethylene evolution by bruised and nonbruised excised 'Empire' apple fruit tissues at successive intervals below fruit surface, 4 hours post-impact Oxygen uptake by excised bruised and nonbruised 'Empire' apple flesh tissue . . . . Chromatograms of extracts from bruised and nonbruised 'Empire' apple fruit tissues at 6 and 72 hours post- impact . . . . . . . . . . . . . vii Page 25 27 3O 31 32 34 35 37 39 44 Figure Page 11. C02 production and malate concentration in excised bruised 'Empire' apple tissues from fruit harvested 45 and stored at 0°C for various times . 12. Effect of temperature on 002 production, (A) eth lene evolution, (B), and malic acid concentration; (C of bruised and nonbruised excised 'Empire' apple fruit tissues; (0) 002, malate, and ethylene concentrations in bruised tissues as affected by temperature.. . . 47 Section II 1. Ethylene (A) and ACC (B) concentrations in bruised and nonbruised 'Empire' apple fruit tissues excised at indicated times after bruising . . . . . . . 62 2. Ethylene evolution from bruised and nonbruised 'Empire' apple fruit tissues excised at indicated times after bruising and incubated for 1 hour with or without 0.4 mM ACC . . . . . . . . . . . 64 3. Ethylene evolution from bruised 'Empire' apple fruit tissues excised at indicated times after bruising and incubated for 1 hour with or without 0.4 mM ACC . . . . . . . . . . . . . . 65 viii INTRODUCTION Mechanical damage occurring during the handling, storage, and shipping of fresh produce is a major cause of postharvest losses. The damage is time-consuming and difficult to detect and quantify, since each fruit must be individually inspected. Frequently, the damage is not immediately apparent, so that numerous examinations may be required as the produce moves through marketing channels. A nondestructive system to detect and quantify damage to large quantities of produce would be invaluable in designing or comparing shipping and marketing containers that would afford maximum protection from postharvest injury. Basic requirements for such a system would include ease, reproducibility, and rapidity of measure- ments. The system should also eliminate the need for visual inspec- tion of produce, since it is a subjective and lengthy process that requires the package to be opened and each item inside to be examined for evidence of injury. Since many crops respond to injury by changes in carbon dioxide or ethylene production, it is possible that atmos- pheric composition changes within a sealed container of produce may provide a useful, rapid, and accurate index of produce damage. Since little is known about the effect of physical damage on physiological functions of apple fruit tissue, the purpose of the research reported here was (1) to examine the effects of fruit maturity and physical injury on ethylene and C02 production by bruised apples and to determine if there is a correlation between degree of injury and amount of volatile evolution and (2) to determine the physiologi- cal bases for increased C02 production, and to a lesser extent for the ethylene response by bruised apple fruits. LITERATURE REVIEW Introduction Postharvest losses of fresh produce at the consumer level range from 10 to 30%, mostly due to mechanical damage (20). Research- ers attempting to quantify and develop indices of injuries that occur as crops move from growers to consumers have measured energy absorbed by test packages (22,23,45,46,47), number of bruises (35), and such physiological parameters as percent soluble solids, titratable acid- ity, and respiratory activity (26,37,39). Many studies have focused on the relationship between increased CO2 production by fruits and mechanical damage caused by impact1 (6,11,14,25,26,28,29,42,50), com- pression (1,30,31), or vibration (37,38,39) during harvesting, hand- ling, and transport. Crops showing damage response include apple1 (10, 48), avocado (6,56), banana (34), cantaloupe (36), cherry (30,31,37,41), Cranberry (32), grapefruit (14,50), lemon (14), orange (11,14,25,26),and tomato (1,29,38,39). In all cases, an increased output of C02 from damaged fruits compared to nondamaged fruits usually occurred imme- diately following injury and declined to basal (control) levels within 2 to 7 days. Increases or decreases in ethylene production also occur in response to injury (2,17,28,32,42). 1Massey, jr., L.M. 1982. »NE-103 Annual report (Jan. 1 to I)ec. 31, 1982) Postharvest physiology of fruits. 13 p. Nature of the 002 response by ‘ifijuredifruits Although many of the reports of increased C02 prdduction in response to injury refer to enhancement of respiratory activity, there has been limited research on the actual effect of damage on oxygen uptake to verify it as a respiration response. Avocados that were shaken or dropped were advanced by as much as 10 days in respira- tory climacteric as measured by 02 uptake, compared to undamaged fruit (6). There was an immediate increase in respiration upon damage, followed by a decrease, with a subsequent strong increase in 02 uptake leading to the early climacteric rise. However, the response was erratic, was not consistently observed, and was not compared with 002 production. Other researchers (5,56) found no advance in the onset of either the CD2 or the ethylene climacteric at 20°C for avocados wounded by removal of a plug of pulp. Damaged fruit held at 14°C, however, showed a two-day advance in onset of both 002 and ethylene climacteric (56). Burg and Thimann (10) noted increased 02 uptake as well as enhanced C02 production in cut plugs and slices of apple fruits. Robitaille and Janick (42), however, pointed out that 02 uptake should decrease rather than increase in destroyed apple tissue cells, and suggested therefore that increased C02 production by bruised apples was not a result of normal respiration. Carbon dioxide output by bruised Satsuma mandarin orange ‘fruits increased preferentially over 02 uptake during the first 5 l1ours after injury (25). The respiratory quotient (R0 = C02 output rate/02 uptake rate of tissue) during this period changed from a basal level of 1.26 to 1.99, indicating enhanced C02 production. In the next 4 hours, however, 02 uptake was markedly stimulated and the RQ fell to 1.00. This fall reflected both a decrease in the enhanced C02 output and an increase in 02 uptake. C02 output still exceeded basal levels 9 hours after bruising. The respiratory quotient in this case (25) does not seem to reflect actual mitochondrial respiratory activity, since changes in C02 output and 02 uptake were not temporally linked. Further evidence that the 02 uptake and C02 output were not related is that similar increases in C02 production were noted in bruised Satsumas held under either aerobic or anaerobic conditions (25). In addition, although bruised tomatoes emitted more C02 than nonbruised fruit at comparable stages of maturity (1), their mitochondria phosphorylated at equal rates and had similar 002 (N). When respiratory activity was measured on individual tart cherry fruits before and after bruising 02 consumption due to bruis- ing rose 50%, while C02 production increased 126% (41). The RQ increased from a basal level of 1.8 to 2.47 after bruising. Since respiratory quotients that approximate unity are generally considered to occur in systems that utilize carbohydrates as a substrate, while RQs less than 1.0 derive from systems utilizing lipids and R05 greater than 1.0 indicate acid substrates (19), the source of the 'extra' C02 in damaged tissues in the above-cited studies (25,41) was likely acidic. Further evidence for an acid substrate comes from studies of bruised tomatoes (29,39) and Satsuma mandarins (26), in which acids decreased in bruised fruits compared to nonbruised, while soluble solids remained constant and C02 production increased. 002 evolution and malate decarbo- xylation in fruits Marks and Varner (31) suggested that the additional C02 pro- duced following bruising of fruit is due to successive decarboxyla- tion of malate and pyruvate. When they labelled the organic acid fraction of 'Royal Anne' cherries by exposing the fruits to 14 C02. they found more than 95% of the total radioactivity in malic acid. Subsequent paper chromatographic and autoradiographic comparisons of extracts of bruised and nonbruised fruit indicated that malate was the only radioactive constituent to disappear more rapidly from the bruised fruit than from the controls. Neal and Hulme (40) demonstrated that the ability of apple peel discs to decarboxylate malate increased as fruits matured (the 'malate effect'). They found that malate loss was precisely balanced by increased production of C02 and acetaldehyde, in a ratio of 2:1:1 of C02 produced:malate utilized:acetaldehyde formed. This is in agreement with the pathway: malate ——————»vpyruvate + C02-——————+ acetaldehyde + C02. Dilley (13) found that the synthesis and specific activity of malic enzyme, which catalyzes malic acid decarboxylation, increased as apple fruits matured and proceeded through the climacteric rise in C02 evolution. Similar results were noted for pyruvate carboxylase (24), which catalyzes formation of acetaldehyde from pyruvic acid. These changes may explain the observed loss of acidity in apples as they ripen (15), since the major acid in apple fruits is malate (49, 51). Although malic enzyme seems to have an obligate requirement for oxygen (40), no increased 02 uptake was noted during presumed enzyme activity when malate was added to peel discs from mature apples (18,40). In contrast, Fidler (15) found decreased titratable acidity of apples, presumably due to malic enzyme activity, under both aerobic and anaerobic conditions as fruit went through the cli- macteric. He also found a metabolic pathway for acetaldehyde meta- bolism which involved formation of both ethanol and 002 regardless of oxygen tension (16). Interestingly, Thomas (48) reported as early as 1931 an increased 'zymasis' in bruised applies that resulted in increased ethanol and acetaldehyde accumulation in the fruit. This accumulation was not caused by anaerobic fermentation at the bruise site, since it proceeded under conditions of 100% oxygen. 'Zymasis' thus seemingly corresponds to coupled malate/pyruvate decarboxyla- tion. Brusing of apple fruits leads to cell rupture (22), which in turn can result in mixing malic acid from the vacuole with cyto- plasmic contents. Malic enzyme, which is associated with the cyto- plasm rather than with the mitochondria (7), could then decarboxylate malic acid, resulting in an increase in C02 production by the damaged tissue. Formation of 'excess' 002 would decrease as the malate sub- strate is depleted by enzyme activity. 8 Enhanced C02 production by bruised tomatoes and oranges may be similar to that of apples. The titratable acidity of tomatoes (12,27,43) and oranges (44) decreased simultaneously with a decrease in malate concentration as the fruits ripen. The soluble fractions of these fruits also possess malic enzyme and pyruvate carboxylase, which increase in activity as fruits mature (7,21). The decreased acidity (26,38) and increased C02 production (25,26,29,37,38) noted in bruised tomatoes and citrus may thus be due to the linked activi- ties of the above-mentioned enzymes, similar to the apparent case in apples. Wound ethylene production in fruits Various types of stress or damage can induce increased ethylene production by plants (2). The increased ethylene may, in turn, induce an early CD2 or ethylene climacteric in wounded tissues (35,56), or may subsequently decline to basal levels (34). The ethylene-forming system involves the production of S-adenosylmethionine (SAM) from methionine. SAM, in turn, is converted enzymatically to 1-aminocyclopropane-l-carboxylic acid (ACC), with subsequent forma- tion of ethylene from ACC (3,8). Increased ethylene production rates reported for cut tomato (9,55), orange albedo (55), and mung bean hypocotyl (55) 'tissues were due to enhanced ACC synthase activity and the consequently increased production of ACC and ethylene. The conversion of ACC to ethylene is inhibited by anaerobiosis (52), by excessively high or low temperatures (4,54), and by osmotic shock (4). These findings, plus discontinuities in Arrhenius plots for ethylene production over the temperature range 0 to 30°C (4,33) suggest that membrane integrity is essential for the formation of ethylene from ACC. Robitaille and Janick (42), upon observing that ethylene production decreased in apples bruised six weeks after harvest, hypothe- sized that cell disruption by bruising destroys the sites of ethylene synthesis. Lougheed and Franklin (28), however, noted increased internal ethylene concentrations in bruised preclimacteric apples. They proposed that bruising preclimacteric fruits induces ethylene production in immature, nonethylene-producing tissue, whereas damage to ripened fruit which are already forming ethylene endogenously 1 harvested and destroys the ethylene productive tissue. Massey bruised 'Idared' apples at intervals over a four-week period in the fall and found that internal ethylene concentrations of preclimacteric fruit increased proportionally to the number of impacts. The response was not evident once the fruit entered the climacteric rise in ethylene production. Burg and Thimann (10) reported no increase in ethylene evolution from cut postclimacteric apple tissues. Bruising preclimacteric apples probably leads to a stimula- tion of ACC synthase activity, followed by increased ACC and ethylene concentrations in the fruit (55). In contrast to preclimacteric fruit, cell walls and membranes of fruit that have begun endogenous ethylene production and ripening are likely more susceptible to rup- ture upon bruising (22,46). This disruption of the site of ACC con- version to ethylene (4,33) would lead to an accumulation of ACC and an 1Massey, NE-103 Annual report, 13 p. 10 inhibition, rather than a stimulation, of ethylene production in bruised tissues. 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Grierson, and G.J. Edwards. 1968. Respiration, internal atmosphere, and ethylene evolution of citrus fruit. Proc. Amer. Soc. Hort. Sci. 92:227-232. 51. 52. 53. 54. 55. 56. 15 Wills, R.B.H. and W.B. McGlasson. 1968. Changes in the organic acids of Jonathan apples during cool storage in relation to the development of breakdown. Phytoch. 7:733-739. Yang, S.F. 1981. Biosynthesis of ethylene and its regulation. In Friend, J. and M.J.C. Rhodes, eds. Recent advances in the 'ETOChemistry of fruits and vegetables. Academic Press, London and New York. PP. 89-106. Yu, Y.B., 0.0. Adams, and S.F. Yang. 1979. 1-aminocyclopropane- carboxylate synthase, a key enzyme in ethylene biosynthesis. Archiv. Bioch. Biophys. 198:280-186. Yu, Y.B., 0.0. Adams, and S.F. Yang. 1980. Inhibition of ethy- lene production by 2,4-dinitr0phenol and high temperature. Plant Physiol. 66:286-290. Yu, Y.B. and S.F. Yang. 1980. Biosynthesis of wound ethylene. Plant Physiol. 66:281-28. Zauberman, G. and Y. Fuchs. 1981. Effect of wounding on 'Fuerte' avocado ripening. HortScience 16:496-497. Section I Carbon dioxide and ethylene production by bruised 'Empire' apples 16 ABSTRACT Apples (Malus domestica Borkh., cv. 'Empire') dropped onto a hard, smooth surface so as to receive an impact bruise midway between stem and calyx rapidly evolved C02 in proportion to the drop height and number. The increase in C02 evolution was not due to enhanced respiratory activity, since there was no concomitant increase in 02 uptake by excised bruised tissues, nor was it due to localized anaero- bic fermentaion at the bruise site. Rather, the increased C02 was due to decarboxylation of vacuolar malic acid by cytoplasmic malic enzyme in the 0.5 cm of cortical tissues below the fruit surface at the bruise site, as shown by a decrease in the titratable acidity of extracts from bruised tissues compared to controls. The CD2 response occurred in fruits of all maturities. Bruising resulted in increased ethylene evolution from preclimacteric fruits, but caused a 50% decrease in ethylene evolution from fruits that had begun to ripen. Enhanced C02 evolution from bruised tissues is temperature-dependent, while the percentage decrease in ethylene is consistent over the temperature range of 0° to 40°C and is due to physical destruction of cells at the bruise site. Keywords: apple--fruit damage--carbon dioxide-~ethylene--malic acid Introduction Mechanical injury to fruit by physical forces causes changes in carbon dioxide (C02) and ethylene evolution by the fruit. Enhanced 17 18 C02 production (compared to basal control levels) resulted from impact damage to apple (8),1 cranberry (21), grapefruit (11,35), orange (11), Satsuma mandarin (9,14,15), and tomato (18). Compression and vibration damage resulted in elevated C02 levels in treated cherries (19,20,23) and tomatoes (24,25). In all cases, C02 evolution rates returned to basal levels within 2 to 7 days following damage. Increasing the degree of damage by increasing the force of impact, number of impacts, force of compression, or time period and frequency of vibration resulted in proportionally enhanced C02 production in many fruits. Interest has focused on the measurement of C02 as an index of damage to fruit during harvesting, handling, and shipping. The ethylene response of damaged fruit generally parallels that of 002 (2,12). Robitaille and Janick (25), however, observed a decrease in ethylene production by apples bruised 6 weeks after harvest, while Lougheed and Franklin (12) noted increase internal ethylene concentrations in bruised preclimacteric apples. To recon- cile the differences in these observations, Lougheed and Franklin (12) proposed that bruising preclimacteric fruits induces ethylene produc- tion in non-ethylene-producing tissue, whereas damage to ripened fruits that are already producing ethylene destroys productive tissue. Internal ethylene concentrations of preclimacteric 'Idared' apples increased proportionally with the number of impacts, but the response 1Massey, jr., L.M. 1982. NE-103 Annual Report (Jan 1 to Dec. 31, 1982). Postharvest physiology of fruits. 19 was not evident once the fruit entered the climacteric rise in ethylene production.1 Yu et al. (37) have shown that wound ethylene may be due to enhanced activity of 1-aminocyclopropane-l-carboxylic acid (ACC) synthase, with consequently increased production of ACC and ethylene. Although reports of enhanced C02 production following injury frequently refer to "enhanced respiratory activity," only a few studies have considered both 02 uptake and C02 evolution in damaged fruit. C02 production by bruised Satsuma mandarins increased compared to 02 uptake during the first 5 hours after injury (14). Subsequently, how- ever, 02 uptake was highly stimulated and there was a decrease in the enhanced C02 output. Oxygen consumption by bruised sweet cherries rose 50%, while C02 production increased 126% (27). In neither case were increases in both 02 uptake and C02 output closely enough linked to indicate that the effect of bruising took place at the level of mitochondrial respiratory activity. Mitochondria isolated from bruised or unbruised tomatoes phosphorylated at equal rates and had similar 002 (1). Marks and Varner (19) found that labelled malic acid was the only radioactive constituent to disappear more rapidly from bruised cherries than from control fruit and concluded that the additional C02 produced following bruising is due to successive decarboxylation of malate and pyruvate. Similar mechanisms may explain the simultane- ous increase in C02 production levels and decrease in titratable acidity noted in bruised tomatoes (18,24) and Satsuma mandarins (9,15). 11m. 20 The purpose of this investigation was to examine the effects of fruit maturity and physical damage by impact on ethylene and C02 production by bruised apple fruits, and to determine the physiological basis for enhanced C02 production by the damaged tissues. Materials and methods Fruit treatment. Apples (Malus domestica Borkh., cv. 'Empire'), selected for uniformity of size and freedom from defects, were care- fully. harvested from experimental plantings and used inmediately or stored under refrigeration in air or controlled atmosphere conditions for later use. Fruit were equilibrated at 20°C for 12 to 18 hours before bruise treatment, which consisted of dropping individual fruit from a height of 1 m, except where otherwise indicated, onto a hard, smooth surface. Apples were dropped so as to receive an impact bruise approximately midway between the stem and calyx. Chalk dust applied to the impact surface identified the impact sites on fruit dropped from a height of 25 cm or less. Compression bruises were administered only to apples harvested 21 days after full bloom by means of a plastic disc (75 mm diam x 3 mm thick) attached to the penetration head of an Effige penetrometer mounted on a modified drill press. A compression force of 6 kg was applied to each fruit for 10 seconds. Individual mature fruits were placed in 500 ml respiration containers and either attached to a supply of ethylene-free air at ca. 25 ml/min flow rate or sealed for 1 hr to allow fruit emanations to accumulate in the headspace. In the latter case, sealed jars were 21 flushed with air between measurements before being resealed at the indicated times. In experiments with small immature fruits (fruit- lets), two each were placed in 70 ml test tubes fitted with serum caps and constantly aerated at ca. 10 ml/min. Tissue discs of bruised and unbruised tissue measuring 25 mm x 5 mm and weighing ca. 3 g were excised from a given apple with a cork borer, halved, and incubated at 20°C in 12 ml centifuge tubes sealed with serum caps. When C02 evolution from the tissue and malate concen- tration of the juice were determined for the same fruit, one-half of a disc was used for each analysis. Since each fruit provided its own control tissue, the effect of bruising on ethylene, C02, or malate concentrations in an individual fruit was expressed as a percent of control. Gas and malic acid determination. Carbon dioxide concentrations were determined with a Carle 8700 (804-8) Basic gas chromatograph equipped with silica gel-molecular sieve columns mounted in parallel and a differential thermal conductivity detector, using He carrier gas at ca. 70°C. Acetaldehyde and ethanol headspace concentrations were determined with a BioGas model 12-110 gas chromatograph equipped with a Porapak-P column and a metal oxide semiconductor detector and using air carrier gas at ambient temperature. Ethylene was analyzed with a Varian Series 1700 gas chromatograph equipped with an activated alumina column and a flame ionization detector, and utilizing N2 carrier gas at 70°C. Internal ethylene samples were withdrawn directly from the central cavity by inserting an 18 gauge x 4 cm needle through the fruit calyx according to the procedure of Saltveit (30). 22 Oxygen was determined with a Gilson differential respirometer, using manometric techniques (33). Tissues were placed in ca. 20 ml Warburg flasks containing 2.5 ml 0.05 M phosphate buffer.(pH 4.5) at 20°C, with 5 N KOH and a paper wick in the center well as a CD2 adsorbent. In some cases, malate (adjusted to pH 4.5 with KOH) was added to the buffer from the sidearm of the flask to yield a final concentration of 0.l M malic acid. Flasks were allowed to equilibrate for 15 to 30 minutes prior to taking readings. A 0.5 ml aliquot of expressed juice from tissue half-discs was added to 50 ml of distilled COZ-free water for malic acid determina- tions (3). Diluted samples were titrated to pH 8.1 with 0.03 or 0.06 N NaOH and the results expressed as mg malic acid/100 ml extract (33, 36), after calculation as follows: mg malic acid/100 ml extract = ml N30“ x mTNZgUpTe67 x 100 (3). Qualitative analysis of malic acid was done by paper chromato- graphy (3). N-butanol (100 ml), water (100 ml), and formic acid (10.7 ml) were mixed in a separatory funnel with 15 ml of a 1% aqueous bromocresol green solution. After thorough shaking, the lower, aqueous layer was discarded and the upper layer placed in a chromato- graphy developing tank. Aliquots (5 ul) of juice from bruised and unbruised tissues, as well as 0.3% (w/v) standard malate solution, were spotted onto Whatman No. 1 paper, which was then placed in the tank for approximately 6 hours. Upon removal from the tank, yellow spots corresponding to malate developed at an Rf of approximately 0.5. 23 Other experimentalgprocedures. The effect of anaerobiosis on bruised and unbruised tissue discs was determined by twice evacuating tubes containing half-discs of tissue to 200 torr and restoring to ambient pressure conditions with M2 or air. Anaerobiosis was also induced by vacuum-infiltrating unbruised tissues with a solution of 0.4 M mannitol (pH 4.0) in order to flood intercellular tissue airspaces. Headspace analyses of flooded tissues were statistically compared with untreated unbruised and bruised tissues of a given fruit for ethylene, C02, acetaldehyde, and ethanol concentrations. The effect of the bruised tissue upon the C02 production by the whole fruit two hours after impact was determined by lightly covering the epidermis at the bruised area with a petrolatum/lanolin mix (1:1, v/v) or by excising the bruised tissue and covering the resultant cut surface with petrolatum/lanolin prior to sealing the fruit in con— tainers. C02 evolution rates of unbruised fruit and fruit with intact bruises were compared with those of treated fruit. The site of C02 production within bruised tissue was identified by taking a 12 mm diam. cork borer plug through the center of the bruised tissue and dividing it into 4 discs, each 5 mm thick. Similar plugs and discs were cut from the unbruised side of a given fruit 4 hours after bruising. Discs were sealed in tubes as described earlier and monitored for headspace accumulations of C02 and ethylene. Since the epidermis was retained on outermost discs, the rates of C02 evolu- tion and 02 uptake for epidermal tissues and the subjacent 0.5 cm of cortex were determined by a similar experiment in which the two tissues were cut as above and separated by a razor blade. 24 Fruit were equilibrated for 12-18 hours at 0°, 10°, 20°, 30°, and 40°C prior to determining the effect of temperature on CO2 and ethylene evolved by bruised tissues. Following impact, fruit were returned to the appropriate temperature-controlled chamber for 2 hours prior to the removal of bruised and unbruised tissues for analyses of gas and malate concentrations. Means were based on 4 to 6 replications in all experiments. Results The C02 evolution from mature whole apples dropped onto a hard surface was positively correlated to drop height at the 1% level of significance (Fig. 1). The number of drop impacts also affected CO2 output (Table 1), with significant differences between fruit with 3 impacts and controls (0 impacts) for at least 24 hours after bruising. Between 3 and 12 hours after bruising, C02 production by fruits with 3 impacts was also significantly greater than that of fruit with 1 impact, which, in turn, exceeded controls during that time. Fruitlets bruised by compression when harvested 21 days after full bloom responded immediately with a significant increase in C02 output (Fig. 2). This increase was not as marked when the fruits were rebruised 22.3 hours after the initial treatment. As with mature, impact-damaged fruit, fruitlets subjected to compression ultimately returned to C02 production rates similar to controls. The increase in CO2 production by excised tissues from damaged mature and ripening fruit was immediate and was invariably sustained 25 Whole fruit f: 30- 4 hours post-impact E T ,L I 5» 25 - 3 CH 8 O y: 0.66 x + 20.9 20 r= 0.66" O 20 4O 60 80 1 00 Drap. height (cm) Figure 1.--Relationship between C0 production 4 hours post-impact and drop height of 'Empire' apples. 26 Table 1. Effect of 0, 1, and 3 drop-impacts from a height of 1 m on C02 evolution by 'Empire' apples.z C02 evolution (pl-g'l-hr’l) No. of Impacts Hours post-impact 1 3 6 12 24 48 0 20a 20a 23a 22a 21a 22a 1 23ab 27b 28b 27ab 22ab 24a 3 26b 34c 34c 31c 26b 24a ZMeans are from 2 pooled experiments, and are separated within columns by Duncan's multiple range test, 5% level. 27 .zpm>wuomammg Fo>mp mocmuwmwcmwm RH so am an we?» sumo um mpcmsummgu cmmzpmn mmucmcmwwwoul «e.« .ucoumm oH Lo» cowmmmgasoo ox m an mcwmwzcn ecu soopn F 3* Lance mxmu Hm um umo>cms mcwzo—Fom muwagm «Page .mgqum. An cowpuauoga ou .N «gnaw; cofiuoafiocuuaon 952.. on ov on on or o . . . . . . . . . . . Remap r . 03 T . WNP o N 0 Z r l... . . ocu m... i a ... r _ . mum M UGO—am lol no 02%. In.» .. 3229.02 1.1 . 0mm .0 1 5.393600 9. m ._ mum Eco... =3 .33 230 a f. n n n . n P p n n n .0 4 COO 28 for at least 12 hours after impact, regardless of fruit age (Table 2). C02 production by bruised tissues remained higher than that of unbruised for at least 24 hours post-impact only in fruit that were preclimacteric or just at the climacteric rise in internal ethylene concentration. Several experiments show that excess C02 output by bruised fruit came exclusively from the bruised tissue. The correlation between C02 evolution and height to impact in excised bruised tissue (Fig. 3) was similar to that of whole fruit as shown in Fig. 1, but the response was more pronounced and of greater statistical signifi- cance (r = 0.66,significant at 1% level for whole fruit; r = 0.75, significant at the 0.l% level for excised bruise). Damaged fruits in which the bruised tissue was excised or covered with petrolatum/ lanolin initially produced significantly less C02 than fruit with intact bruises (Fig. 4). Fruit with excised or covered bruises pro- duced C02 at the same rate as nonbruised fruit when measured 9 houurs post-impact. After 24 hours, however, fruits from all treatments were similar 'h1 C02 production. Browning of apple cortex tissues due to bruising extended 13 to 15 mm below the fruit surface in fruit dropped 1 m. Figure 5 shows that the tissues at the fruit surface (and therefore nearest the point of impact) produced significantly more C02 than nonbruised tissues at 4 hours post impact. Deeper tissues showed no increases in C02 output. In a separate experiment, excised epidermal tissues from both bruised and unbruised sites on the apple 3 hours post impact produced 160 pl -1 COZ-g'loh However, cortical tissues 0.5 cm below the epidermis 29 .pm>mp um as» an acmuwmpcmpm use nouns? 1pmoq Lao; can we.» mappasom some sysop: muucogowmwu mammvu .m: umauu+u=_ mums: uamuxu» .po>mp um .uumu omen; opavupas m.=mu::o an mzog c.5uwx mcovuagunum comz~ 11 11 -1 was emu nag” n-~ can“ coca uumwagacoz a 11 - - an me «e um mm as uemaecm 11 1- 11 mm on em mm ~o Km umm_:gn:oz as mH\oH 1- own emu «nu «ma aufia emu" uumfi now“ vom_:gncoz a 11 cm ac so an em Hm No“ mm convasm m: -1 em mm No em co no mo so ummpagncoz om m~\o~ .- - eNm can -a ne_~ ecu” emNH emu“ eem_=ceeez a -1 11 mm mm mm co mm om om camwagm m: m: 11 11 do co #5 cm an HR co vomwacacoz m“ wfl\o~ mum nawm own" own“ onefia 11 can“ vampagncoz a -1 -1 mm mm mm Nu mm 11 mm vme=gm .- 1- me me am mm H“ 1- mm semaaceeoz o ~H\o~ «Hm namofi use“ naNHH been” cam“ uuHeH uu_eH come" cem_=caeoz a ¢¢ we om we cog ofiu mm mo" em uwmwngm m: m: n: ma cc ~¢ do an on on as me vmm_=ca=oz o ~H\m cw” mm Nu we cm N" m m fl 1 omugoum ounce. anon mgzoz mama?» upou umo>cez . . n N A~1ce H-a _ V cu mxmo unopnno .mstEN. ummuEeu-uuoae_ scum vomwuxm «mama?» an cowauavoca mou go one uwsgw mo gummmw .N o—nop 3O 150 ' '1 C I E 100 '1' 9’ y = 0.63 x + 63.6 3 r = 0.70"" U Excised tissue 3 hours post-impact O 20 4O 6O 60 ‘l 00 Drop height (cm) Figure 3. Relationship between C02 production by excised bruised tissue and drop height of 'Empire' apples 3 hours post-impact. 31 .Fm>wp um .umma mace; mPQVupae m.cmoc=o An we?“ gone we umumcmnmm memo: .uumaEw-umoa mesa; ~.~ umuapouo go umm_oxm mmmwzcn suwz .mgao; c an commaen mmpgqm .mgwasm. An cowuoaoogn moo .e mezmvd . -ll “MG-.QHWI-HMOQ ”530—..— eu on or up w e o 033.: monik— ow , O «N O 79 ea m4- 6 . mu u. 03.6.6 355 114.1- N. coca-coo 03:5 1.1 mm 1...» «03:. 03:5 Ial. now-55:02 11.1 cm 32 I] Nonbruised Bruised 100 I I 25' - 0.0-0.5 0.5-1.0 1.0-1.5 1.5-2.0 Depth below fruit surface (cm) Figure 5. C02 production by bruised and nonbruised excised 'Empire' apple fruit tissues at successive intervals below fruit surface, 4 hours post-impact. Mean separations at each depth by Duncan's multiple range test, 5% level. 33 produced significantly more C02 at the bruise site (77 ul-g'lohr-l) 1~hr'1). than at a control site (35 ul-g' Preliminary studies (16) showed that fruit which were pre- climacteric in ethylene production (<0.1 ul-l'1 internal ethylene) had no consistent significant increases in external ethylene production or internal ethylene concentration due to bruising until approximately 24 hours after impact. After that time, ethylene evolution was posi- tively correlated with drop height to impact (Fig. 6). A similar, although considerably less significant, response was measured separately for internal ethylene concentration (Fig. 7). In studies with excised tissues, once fruit entered the cli- macteric rise in ethylene production (> 0.1 ul-l'1 internal ethylene), less ethylene was produced by bruised tissue than by controls (Table 3). Ethylene evolution from bruised tissues declined to 50% of control values by 6 hours after impact, with the exception of fruit from the Sept. 12 harvest. The value for ethylene evolution from bruised tissues of this harvest, expressed as a percentage of controls, fluctuated during the period after bruising, but always exceeded 60. There was occasionally considerable variation in ethylene evolution between fruits of the first two sampling dates at several times of sampling after impact. As a result, the overall mean ethylene values for control and bruise tissues in some instances do not correspond to the overall percentage increase or decrease in ethylene evolution due to damage, although the percentages were consistent within sampling times. The decrease in ethylene evolution was confined to the outer- most section of tissue at the bruise site (Fig. 8). 34 20 Whole fruit, preclimacteric 1:: 27 hours post-impact ° 2 ,_° 15 I m a: 3 2 10 y=1.13x+7.1 2 .111- >~ o f==<7f745 J: in" o 5 O 20 40 60 80 100 Drop height (cm) Figure 6. Relationship between ethylene evolution 27 hours post-impact and drop height of preclimacteric 'Empire' apples harvested August 31. 35 internal ethylene 24 hours post-impact 4O ‘1' 3 30 o C: 2 E o 11‘- 2o _ y: 0.26x + 19.3 0 r I 0.46' 10 O 20 4O 6O 80 1 00 Drop height (cm) Figure 7. Relationship between internal ethylene concentration 24 hours post-impact and drop height of 'Empire' apples harvested August 26. I36 ._o>ep um ago on beeo_e_=m_m men uooaew1umon one; zone cvgovz one we.“ ucwpnsem coco cwguwx moocosouwvu mama.» .m: nouuorvcp egos: unooxua .po>o_ am .omo» omen» opavopas m.=noc=o a; use; :_=u_x vouosonom menus compagaco: “N 1- 11 1- umm ammm ammo oowe oawm oom vom_:gncoz a 11 1- 11 on am so mm «5 mm vomwagm 11 1- 1- gm Hm co“ as "ea ma” vomwagncoz an“ as w~\o~ 1- 11 ammm own umm comm once omm oame vompsgncoz a 11 11 o.am m.me m.wm ~.~m ¢.we m.wo o.~m vom_:gm 1- 11 m.~o~ ~.mo~ o.w- o.mm ~.oo~ o.-~ ~.mm vomvagncoz mom om mH\oH 11 - comm new emu cmm onom cum one vomvasacoz a 11 - ~.- m.o~ —.o~ o.m~ ~.- m.m~ ".Nm vomwagm 1- 1- ~.m~ m.mw o.o~ m.~e m.m~ o.m~ o.m~ com—=uacoz mo" ma mflxofi - - new new poem gnaw can 1- one vomvagacoz a 11 11 m.- o.e~ m.~ o.m o.m~ - m.¢m convaom m: 11 11 o.- o.mm m.e~ m.m~ e.- 11 ~.m~ vompagncoz o~.o o ~H\o~ comm nmcfiw ammo 11 11 ammfifi gums" amp 1- convagacoz u m~.o m~.o m~.o 11 11 eH.o om.o mm.o - vomvagm m: m: m: m: -.o o~.o -.o 11 11 o~.o m~.c ~m.o -1 vompagacoz eo.o o Nfixm o~_ ca Nu as «N Na m m H AH-F.an oco » um ouogoom poeasp1umoq nose: 03mm?» P ; upou umo>guz pucsou=~ «zoo Afi-c;.31m.pev seepage“ .e.o.e~ Nmopaau .oLwQEm. nomuEuu1uouasw seem vomvoxo mozmmwo soc; covaapo>o ocopaguo so one u_:gm mo uoomwu .n epoch 3O 20 Ethylene (nl - 9'1 - hr“) 10 Figure 8. 37 l1 [:1 Nonbruised Bruised . 0.0-0.5 0.5- 1 .0 1 .O- 1 .5 1 .5-210 Depth below fruit surface (cm) Ethylene evolution by bruised and nonbruised excised 'Empire' apple fruit tissues at successive intervals below fruit surface, 4 hours post-impact. Mean separa- tions at each depth by Duncan's multiple range test, 5% eVel. 38 Oxygen uptake rates of bruised and unbruised tissues were similar when followed for 48 hours after impact (Fig. 9), with the only significant difference occurring at 10 hours post-impact. Addi- tion of 0.1 M malate to the incubation medium had no effect on 02 uptake (16). Measurement of volatile evolution by excised tissues in air and in N2 (Table 4) showed significantly decreased evolution of ethylene and CO2 for both bruised and control tissues in N2. Although ethylene evolution rates for damaged and non-damaged tissues were similar in N2, a significantly greater amount of C02 was evolved by the bruised tissues in this atmosphere. Acetaldehyde evolution rates were significantly greater for bruised than nonbruised tissues only in N2. Bruised tissues likewise produced significantly more ethanol than unbruised tissues in N2. N0 ethanol was detected under aerobic conditions. Unbruised tissue plugs, when vacuum-infiltrated with 0.4 M mannitol to serve as a possible model for impact-damaged tissues, produced ethylene and acetaldehyde in amounts equivalent to those produced by bruised tissues (Table 5). Tissues which had been infil- trated or bruised produced significantly less ethylene and significantly more acetaldehyde than untreated tissue. Bruised tissue produced significantly more C02 than nonbruised controls, with or without mannitol infiltration. Only infiltrated tissue produced ethanol. The malic acid concentration in extracts taken periodically from bruised tissues decreased immediately and continually compared to 39 I T 1 I T 1 q W 1 120 5‘\ €100 \ =7 '," 80 a: 3 0 60 a: :3 l 9 ON 40 Excised tissue 20 -- Nonbruised —o— Bruised 0 10 20 30 4O 50 Hours post-impact Figure 9. Oxygen uptake by excised bruised and nonbruised 'Empire' apple flesh tissue. 40 Table 4. Effect of atmospheric composition on volatile evolution by bruised and nonbruised 'Empire' apple tissues.y1z Ethylene C02 Acetaldehyde Ethanbl (nl-g'l-hr'l) (ul-g-l-hr'l) (nl-g'lohr'l) (nl-g'l-hrTI) Nitrogen Nonbruised 4.6a 37.3a 6.9a 25.4a Bruised 5.9a 51.1b 10.3b 40.7b 5.1:: Nonbruised 18.8C 4315b 6.8a < 5 Bruised 10.8b 70.ac 8.3ab < 5 ySamples taken 3 hours post-impact. zMean separation by Duncan's multiple range test, 5% level. 41 Table 5. Effect of vacuum infiltration with 0.4 M mannitol and impact damage on volatile evolution by 'Empire' apple tissues.Y-Z Ethylene C02 Acetaldehyde Ethanol (nl-g'lohr'l) (ul-g'l-hr'l) (nl-g'I-hr’l) (nl-g'l-hr'l) Nonbruised 33.4b 55.38 7.68 < 5 Bruised 16.2a 76.3b 19.4b < 5 Infiltrated 14.2a 56.6a 15.8b 65.5 J; ySamples taken 3 hours post-impact or post-infiltration. zMean separation by Duncan's multiple range test, 5% level. 42 controls following impact damage (Table 6). The decline is clearly reflected in the ratio of bruised to control tissue malate concen- tration, which decreased to as low as 18 for apples tested 79 days after harvest. As was the case for ethylene, in some instances mean malate concentrations for bruised and nonbruised tissues do not correspond to the overall percentage decrease because of variability between fruits within sampling times. The percentage decrease among fruits, however, was consistent within sampling time. The decline in malate concentration was confirmed qualitatively by paper chromato- grams. As illustrated in Figure 10, extracts from older bruised tis- sues (72 hours post-impact) showed smaller spots corresponding to malate than extracts taken from tissues 6 hours after bruising. A comparison of the malic acid and C02 concentrations in bruised tissue, expressed as percent of control (Fig. 11) shows that the initial decline in malic acid concentration due to bruising is inversely proportional to the rise in C02 evolution. It was not possible, however, to calculate a precise stoichiometric relation- ship for these two tissue constituents. The evolution rates of both CD2 and ethylene for bruised and nonbruised tissues increased with temperature increases from 0 to 30°C (Fig. 12, A & B). Regardlesszof temperature, the ratio of ethylene evolution from bruised tissues to that of control was always approximately 0.5 (Fig. 12, D). The C02 output varied with temperature, with the maximum differential between bruised and control tissues occurring at 20 and 30°C. These temperatures likewise 43 pcnomwpcemm men oonaee1umon .—o>mp we on» an Lao; ecu use» ace—asam some :.eu+: moocogmmepe mane.» .m: eoeoo.ec. mums: unooxe» .Po>o_ we .omoa mucus opavupas m.:ooc=o me «so; c.ga.3 mco.eaguaom cuuz~ 11 een an“ enc ocu one emm eom_=gecoz u 11 oe_ ee NnH emm ~n~ wen eomvagm c 1- ooe oe~ eon een ¢~n em" eomF=gecoz an m~\oH eonv emu 11 oefle oeme oom on“ eom_=ge:oz u eefi cg“ 11 cc" nu“ eon em“ eomvsgn Nan own 11 com new new eew eomeagecoz on n~\o~ oHn nnn can eefie eomm ones ewe“ vomeagecoz a em— «an no“ me_ can emm men voovage m: cue eon eNN men men enn wen nonpagecoz e_ m~\oH on~ -1 ace eoe oeeu 11 one nonessecoz a new - eoN new can 11 een oompagn m: eme 11 nen wen ene - can eon—:gecoz o ~H\oH nun men mun ooe oes 11 enm vomeagecoz a we" an" eeH can «en 11 Nne convage m: Nne own eon wee New 11 «we eom_:gecoz o ~H\m us we em N“ e n u ounuoun uoanw-umon meno: msmmvh epou umo>gmz name 365-... E 8:95 32 3:: ~mopnna .os_g5e. eonnEne-uooaep sage muoegoxm mammeo :_ cowuugocoocoo upon o_poe so one owagv ea uooemu .e opeop 44 HOURS POST-IMPACT Figure 10. Chromatograms of extracts from bruised and nonbruised 'Empire' apple fruit tissues at 6 and 72 hours post- impact. N = nonbruised; B = bruised; M = malate standard (0.3%, w/v). ' 45 Figure 11. C02 production and malate concentration in excised bruised 'Empire' apple tissues from fruit harvested and stored at 0°C for various times. Percent of nonbruised 46 mom/\j 1 ' f 1 Harvest 9/ 12 + 0 days " 100 r «Y 50 ' Melate ' L e 1 J J a j J J J J __A l 1 50 1 1 i I r 100 150 . T ‘I 1 Harvest 10/18 0 15 days 50 \ Malate ‘ 'F 150 1 W W r Harvest 10/18 e 79 days 100 60 70 Hours post-impact 47 150» A '1 - '1" -.- 11 2100* LSDDSI 1 7. 7 Mahmud! - O . q 3 3- 8““ 1 II o l L 400 7 E ' i 2... g E a i ’ "' :00» “SI ' ‘-——~ g; A A 4 f l J l J L O 10 2O 30 40 30 4O TEMPERATURE (°C) 10 20 Figure 12. Effect of tem erature on C02 production, (A), ethylene evolution, (B , and malic acid concentration; (C) of bruised and nonbruised excised 'Empire' apple fruit tissues; (0) C02, malate, and ethylene concentrations in bruised tissues as affected by temperature. 48 resulted in the greatest decrease in malate concentration in damaged tissue (Fig. 12,C&D). Ethylene evolution was greatly depressed at 40°C (Fig. 1218). Discussion C02 evolution from impact-damaged whole apples increased during the initial 2 to 3 hours following damage, then declined to levels equivalent to those of nondamaged fruit 6 to 12 hours after impact. This effect due to damage was similar in excised bruised tissues, although the C02 response was more immediate, was sustained for a longer period of time, and was greater in magnitude when measured for bruised tissue alone than for whole fruit. Since non- damaged tissues comprise at least 90% of the weight of impact-damaged apples (M. Parker, unpublished observation), the lesser magnitude and shorter period of response of whole bruised fruit is probably due to the confounding presence of nondamaged tissues within the fruit, which dilute the overall increase in C02 emanating from the bruise. Fruit with excised or occluded bruises generally evolved C02 in amounts similar to intact, nondamaged fruit, thereby demonstrating that the increased yield of C02 from impact-damaged whole fruit is localized at the site of the bruise. An exception to this was petrolatum/lanolin-treated fruit, which evolved C02 at an intermediate level between intact bruised and unbruised fruit 9 hours post-impact. This exception may have been due to a lag in diffusion of excess C02 from the bruise site through the relatively permeable cortex to an unoccluded site on the fruit before passing into the jar headspace (7). Within the bruise itself, the C02 increase was confined to the 49 cortical tissues closest to the site of impact, where damaged cells were probably greatest in number. The increase in CO2 evolution as a result of bruising does not appear to be a result of aerobic respiration, since there was no con- comitant increase in 02 uptake by damaged tissues. Oxygen uptake by both bruised and nonbruised tissues remained steady during the initial 6 hours of incubation in phosphate buffer, but subsequently increased dramatically before decreasing again. Burg and Thimann (8) found that soaking apple tissues in water had no effect on 02 uptake, but only reported results for up to 90 minutes of soaking. Van Stevenink (34), however, cited several studies in which soaking tissues in vari- ous osmotica for greater lengths of time led to increased 02 uptake due to induced activity of any of several enzyme system? Incubation in buffer for more than 6 hours may therefore be responsible for the increased 02 uptake by both bruised and nonbruised apple tissues. Nonetheless, the increases are not related to enhanced C02 production by the bruised tissue. The possibility that rupture of the cells upon bruising caused localized flooding of interEBllular spaces and a resultant enhanced production of CO2 by anaerobic fermentaion was not substan- tiated when near-anaerobiosis was induced in a N2 atmosphere or by mannitol-infiltration of tissue intercellular spaces. Neither pro- cedure increased C02 evolution to levels comparable to those of bruised tissues. The increase in acetaldehyde concentrations in aerobic bruised apple tissues noted in these experiments was also reported by Thomas (31). 50 Impact damage to fruit probably ruptures the vacuolar mem- brane as well as the cell wall (14), resulting in a release of vacuolar malic acid into the cytoplasm, where it can be decarboxylated by cytoplasmic malic enzyme (10). This was reflected by a decrease in titratable acidity of bruise tissue extracts (expressed as malic acid) that was paralleled by an increase in C02 evolution from the bruise. Once the malate substrate is decarboxylated, C02 output by bruised tissues falls below that of intact tissue, since ruptured cells are unable to carry on normal respiratory activity. Neal and Hulme (26) demonstrated a 1:2:1 stoichiometric rela- tionship for the decarboxylation of malate to C02 and acetaldehyde in apple peel discs. They further showed that this enzymatic activity did not involve 02 uptake. My findings support these conclusions, even though a stoichiometric relationship could not be demonstrated. The malic acid concentration in bruised tissues declined simultane- ously with increased C02 and acetaldehyde evolution. The rate of decline in malate concentration was more rapid in older fruit, in keeping with the increased specific activity of malic enzyme as fruit ripen (10). Temperature dependency of the increased rate of C02 production also indicates the enzymatic nature of the response. The decreased C02 evolution. by bruised tissues compared to control at 40°C may have been due to temperature inhibition of enzymatic decarboxylase , activity. The greater malic acid concentration in bruised tissues as a percent of control at 40°C compared to that at 20 or 30° is also indicative of less decarboxylation at higher temperatures. 51 Fruits dropped from greater heights are more severely damaged upon impact, and thus have more ruptured cells and vacuoles at the bruise site (13), with increased C02 evolution in proportion to the number of damaged cells. On this basis, C02 production could be used as a measure of the degree of injury sustained by the fruit and expressed as an index relative to the basal rate of 002 production by undamaged fruit. This technique could be particularly useful in determining the protection from damage afforded by different types of containers used in storage and transport of fruit. Unlike the C02 response, the increase in ethylene production by impact-damaged preclimacteric apples is not due exclusively to increased production at the bruise site. The increased internal ethylene concentrations of bruised fruit over controls indicates that the entire fruit is affected rather than just the bruised tissue. Although the major barrier to gas diffusion in apples is the epidermis (7), gas diffusion from the cortex to the core is thought to be partially limited by vascular tissues (5). There is the possibility that enough impact energy was transmitted to the core tissues, which are metabolically more active than cortical tissues (28) to effect an increase in ethylene production. Energy transmission by vibration caused an increase in ethylene production by tomatoes (24,25). Once fruit ripened enough to produce a quantity of endogenous ethylene, impact damage resulted in a 50% decrease in ethylene evolu- tion from bruised tissues compared to controls. This percentage reduc- tion remained consistent, although the 010 for ethylene production by 52 both bruised and unbruised tissues (as calculated from data in Fig. 12 B) averaged 1.7 in the range of 10 to 30°C. I suggest that the similarity of 010 in the two tissues is due to the presence of numer- ous intact cells within the bruise, whereas the overall reduction in ethylene evolution is due to actual physical damage to cells, rather than to a physiological response induced by the energy of impact. The reduction in ethylene evolution at 40°C noted here has been reported previously (22,36). Increased ethylene production upon bruising of apples not yet producing endogenous ethylene could have been due to the stimulation of ACC synthase activity, followed by increased ACC and ethylene concentrations in the fruit, as demonstrated by Yu and Yang (38) in other injured plant tissues. However, as fruit gain the capacity to produce ethylene and proceed through ripening, the cell walls and membranes could be more susceptible to rupture upon impact (13,29). Disruption of the membranes, which are the site of ACC conversion to ethylene (4,22), would lead to an increase in ACC concentration at the bruise site, since ACC synthesis is not membrane bound (6), while ethylene production would be inhibited in the affected tissues. 1 and The increased ethylene evolution upon bruising noted by Massey Lougheed and Franklin (17) with preclimacteric apples, and the decrease in ethylene evolution from mature bruised apples out of storage found by Robitaille and Janick (28), lend support to the above hypothesis. 11bid. 53 Conclusion The increase in C02 evolution from apples after bruising is not due to enhanced aerobic or anaerobic respiratory activity, but rather is due to decarboxylation of malic acid in cortical tissues at the bruise site. Since the amount of C02 evolved due to bruising is proportional to the degree of damage to the fruit, it can perhaps be used as an index for injury that occurs during fruit handling and transport. In contrast to the enzymatic nature of the CO2 response, the decrease in ethylene evolution from mature bruised apple tissues is due to physical disruption of the cells at the bruise site. Literature cited 1. 10. 11. Abdul-baki, A.C. 1964. Respiratory changes in normal and bruised tomatoes during ripening. Ph.D. thesis. University of Illinois, Champaign. Dis. Abst. 25:6138. Abeles, F. B. 1973. Ethylene in Plant Biology. Academic Press, New York, p. 87-102. Amerine, M.A. and C.S. Ough. 1980. Methods for analysis of musts and wines. Wiley-Interscience. New York, p. 45-73. Apelbaum, C., A.C. Burgoon, J.A. Anderson, T. Solomos, and M. Lieberman. 1981. Some characteristics of the system converting 1-aminocyclopropane-I-carboxylic acid to ethylene. Plant Physiol. 67:80-84. Brandle, R. 1968. Die Verteilung der Sauerstoffkonzenirationen in fleischigen Speicherorganen (Apfel, Bananen, und Kartoffelk- nollen). Berichte der Schweizerische Botanische Gesselschaft, 78:330-364. (English Summary.) Boller, T., R.C. Herner, and H. Kende. 1979. Assay for and enzymatic formation of an ethylene precursor, l-aminocyclo- propane-l-carboxylic acid. Planta 145:293-303. Burg, S.P. and E.A. Burg. 1965. Gas exchange in fruits. Physiol. Plant. 18:870-884. Burg, S.P. and K.V. Thimann. 1960. Studies in ethylene produc- tion of apple tissue. Plant Physiol. 35:24-35. Chuma, Y., H. Izumi, and T. Matsuoka. 1967. Bruise and respiration characteristics of citrus unshiu as related to material handling and in-transit injury. J. Soc. Agr. Machin. Japan 29:104-109. Dilley, D.R. 1962. Malic enzyme activity in apple fruit. Nature 196:387-388. Eaks, I.L. 1961. Techniques to evaluate injury to citrus fruit from handling practices. Proc. Amer. Soc. Hort. Sci. 78:190- 196. 54 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 55 Fleuriet, A. and J.J. Marchieux. 1977. Effets des blessures sur les composes phenoliques des fruits de tomato "cerise" (Lyc0persicum esculentum var. cerasiforme). Physiol. Veg. 15:239-250. Holt, J.E. and 0. Schoorl. 1977. Bruising and energy dissipa- tion in apples. J. Text. Stud. 7:421-432. Hyodo, H., Y. Hasegawa, Y. Iba, and M. Manago. 1979. Enhance- ment of C0 evolution by Satsuma mandarin (Citrus unshiu, Marc.) fruit by dropping. J. Japan. Soc. Hort. Sci. 48: 353-358. Iwamoto, M., T. Shiga, and Y. Chuma. 1976. Effects of dropping and waxing practices in the packing-house line on the quality of Satsuma mandarin (Citrus unshiu, Marc.) J. Japan Soc. Hort. Sci. 45:203-209. Klein, J.D. 1983. Physiological causes for changes in carbon dioxide and ethylene production by bruised apple fruit tissues. Ph.D. dissertation, Michigan State University, East Lansing. Lougheed, E.C. and E. W. Franklin. 1974. Ethylene production increased by bruising of apples. HortScience 9:192-193. MacLeod, R.F., A.A. Kader, and L.L. Morris. 1976. Stimulation of ethylene and C02 production of mature-green tomatoes by impact bruising. HortScience 11:604-606. Marks, J.D.K. 1957. Metabolism of aging cells. Ph.D. disser- tation, The Ohio State University, Columbus. Diss. Abst. 18: 1968. Marks, J.D. and J.E. Varner. 1957. The effects of bruising injury on the matabolism of fruit. Plant Physiol. 32:xlv (supplement). Massey, jr., L.M., B.R. Chase, and M.S. Starr. 1982. Effect of rough handling on C02 evolution from 'Hawes' cranberries. HortScience 17:57-58. Mattoo, A.K., J.E. Baker, E. Chalutz, and M. Lieberman. 1977. ‘ Effect of temperature on the ethylene-synthesizing systems in apple, tomato, and Penicillium digitatum. Plant and Cell Physiol. 18:715-719. Mitchell, F.G., G. Mayer, and A.A. Kader. 1980. Injuries cause deterioration in sweet cherries. Calif. Agric. 34:14-15. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 35. 56 Nakamura, R. and T. Ito. 1976. The effect of vibration on the respiration of fruit. 1. Changes in the expiration rate of tomato fruit during and after vibration. J. Japan Soc. Hort. Sci. 45:313-319. Nakamura, R., T. Ito, and A. Inaba. 1977. The effect of vibra- tion on the respiration rate of fruit. 11. Effects of vibra- tion on the respiration rate and quality of tomato fruit gagiggoripening after vibration. J. Japan Soc. Hort. Sci. 46: Neal, G.E. and A. C. Hulme. 1958. The organic acid metabolism of Bramley's Seeling apple peel. J. Exp. Bot. 9:142-157. Pollack, R.L. and C.H. Hills. 1956. Respiratory activity of normal and bruised red tart cherry (Prunus cerasus). Federation Proceedings 15:328. Robitaille, H.A. and J. Janick. 1973. Ehtylene production and bruise injury in apple. J. Amer. Soc. Hort. Sci. 98:411-413. Saltveit, jr., M.E. 1982. Procedures for extracting and analyz- ing internal gas samples from plant tissues by gas chromato- graphy. HortScience 17:878-881. Schoorl, D. and J.E. Holt. 1977. The effects of storage time and temperature on the bruising of Jonathan, Delicious, and Granny Smith apples. J. Text. Stud. 8:409-41. Thomas, M. 1931. The production of ethyl alcohol and acetalde- hyde by fruits in relation to the injuries occurring in stor- age. 11. Injuries to apples and pears occurring in the presence of oxygen and in the absence of accumulations of carbon dioxide in the storage atmosphere. Ann. App. Biol. 18:60-74. Ulrich, R. 1970. Organic Acids, pp. 89-118. jg; Hulme, A.C., ed. The biochemistry of fruits and their products. Academic Press, London. ‘ Umbreit, W.W., R.H. Burris, and J.E. Stauffer. 1972. Mano- metric techniques, fifth edition. Burgess Publ. Co., Minneapolis, Minnesota. PP; 1-109. Van Stevenink, R.F.M. 1975. The ”washing" or "aging" phenomenon in plant tissues. Ann. Rev. Plant Physiol. 26:237-258. Vines, H.M., W. Grierson, and G.J. Edwards. 1968. Respiration, internal atmosphere, and ethylene evolution of citrus fruit. Proc. Am. Soc. Hort. Sci. 92:227-232. 57 36. Wills, R.B.H. and W. B. McGlasson. 1968. Changes in the organic acids of Jonathan apples during cool storage in relation to the development of breakdown. Phytochemistry 7:733-739. 37. Yu, Y.B., 0.0. Adams, and S.F. Yang. 1980. Inhibition of ethylene production by 2,4-dinitrophenol and high temperature. Plant Physiol. 66:286-290. 38. Yu, Y.B. and S.F. Yang. 1980. Biosynthesis of wound ethylene. Plant Physiol. 66:281-285. SECTION II LEVELS OF 1-AMINOCYCLOPROPANE-1—CARBOXYLIC ACID AND ETHYLENE IN BRUISED APPLE TISSUE 58 Abstract Concentrations of l-aminocyclopropane-I-carboxylic acid (ACC) in bruised and nonbruised apple (Malus domestica Borkh., cv. 'Empire') fruit tissues were similar for 24 hours after impact-bruising, while there was a simultaneous 65% decrease in ethylene production by bruised tissues compared to nonbruised. ACC concentrations in bruised tissues declined to 60% of control levels by 48 hours post-impact. Bruised and nonbruised tissues were similar in relative ability to convert exogenously supplied ACC to ethylene, indicating a physical rather than a physiological cause for decreased ethylene production by bruised apple tissue. Introduction Wounding or stress leads to increased ethylene production by many plant tissues (1,10,14,15). Apple fruits that have begun pro- ducing endogenous ethylene, however, show a decrease in ethylene evolution as a result of bruising (11,14,17). The increase in ethylene production by wounded plant tissues is due to an induction of the synthesis of ACC synthase (6,20), which catalyzes the formation of ACC (5,6), which in turn is enzymatically converted to ethylene (2). Excessively high or low temperatures (5,18,19), osmotic shock (4), or anaerobiosis (18) lead to an accumulation of ACC in the tissue and an inhibition of ethylene production due to interference in the conversion of ACC to ethylene. Bruising causes the rupture of apple 59 60 cell walls and associated membranes (9), which are the presumed sites of ethylene synthesis from ACC (16). The purpose of this investiga- tion was to determine if the reduction in ethylene production by bruised tissues of post-climacteric apples is caused by a change in ACC con- tent or metabolism of these tissues. Materials and methods Plant material. Post-climacteric apples (average internal ethylene concentration of 180 ul-I'l), which do not form wound ethylene upon being cut (12), were taken from 0°C storage, equilibrated to 20°C in 15 hours, and dropped l m onto a hard, smooth surface to create an impact bruise approximately midway between the stem and the calyx. Fruits were selected randomly from the population of bruised apples at specific times poSt-impact. Discs of bruised and nonbruised tissue measuring 25 mm x 5 mm and weighing 3 g were then excised from a given apple with a cork borer. Determination of ethylene and ACC. One-half of each tissue discs was incubated at 20°C in a 12 ml centrifuge tube sealed with a serum cap. At 1 hour, a 1-ml gas sample was taken with a syringe for ethylene analysis by gas chromatography. A 0.3 ml aliquot of juice expressed from the corresponding half of the tissue disc was used for determina- tion of ACC by the method of Lizada and Yang (13). Unfiltered juice converted 60 to 80% of exogenously supplied ACC to ethylene. Tissue capability to convert ACC to ethylene was determined with half-discs of bruised and nonbruised tissue excised at various 61 times after bruising and incubated for 1 hour at 20°C in 25 ml Erlenmyer flasks that were sealed with serum caps. Ethylene evolution was determined after 1 hour of incubation in 2.5 ml distilled deionized water with or without 0.4 mM ACC. Results and Discussion Ethylene production by bruised apple tissues decreased to 50% of the control by 1 hour post-impact (Fig. 1-A and Table 1) and even- tually declined to 30% at the end of the experiment. Differences between ACC concentrations in bruised and nonbruised tissues were not significant in the first 24 hours after impact (Fig. I-B and Table 1). Levels of ACC in bruised tissues decreased to 60% of nonbruised by 48 hours after bruising, although the corresponding ethylene production rates remained steady. The conversion of ACC to ethylene is a tightly coupled reaction with a rapid turnover rate (19). Thus, bruised apple tissues continued to produce ethylene at a rate of 1.8 nmole-g'lohr'l, even while containing only 0.6 nmole-g'1 ACC. The decline in ACC content of bruised tissues between 24 and 48 hours post-impact may have been due to decreased production of ACC from S-adenosylmethionine (SAM). An alternative possibility is that ACC was converted to its conjugate N-malonyl-ACC (MACC) (3,8) in cells no longer capable of converting ACC to ethylene. MACC, a poor source of ethylene compared to ACC (7), is not measured with 'free' ACC in the Lizada and Yang assay (13) unless previously hydrolyzed in hot HCl (8,7). 62 7.0 ~ 5.0 . 4.0 . 3.0 ~ Ethylene (nmole- g '1- hr") 20 L {\I— Bruised Nonbruised s-I-I s-r-t 1.6 - 1.4 - 12 ACC (nmole- 9") T .l—Pl \} 0.6 i . % J l l l J j #1 l I 0 1O 2O 30 4O 50 Hours post-impact Figure 1. Ethylene (A) and ACC (8) concentrations in bruised and non- bruised 'Empire' apple fruit tissues excised at indicated times after bruising. Means are from 2 pooled experiments, i S.E. 63 Table 1. Ethylene and ACC concentrations in bruised 'Empire' apple fruit tissuesz Ethylene ACC Hours post-impact (nmole-g'l-hr'l) (nmole-g‘l) % of nonbruised 1 53a 125a 3 49a 124a 6 47a 117ab 12 35b 94b 24 33b 97ab 48 30b 63c 2Data are from 2 pooled experiments; mean separations within columns by DMRT, 5% level. 64 14o .. l l l l l l l T 1 20 . Nonbruised (+ACC) . .s (D C) 1 00 CD ' Nonbruised (-ACC) Bruised (0- ACC) . . ' ‘mm Ethylene (nl g'l-I'l) a s ' f“? l l N C) 1 Hours post-impact Figure 2. Ethylene evolution from bruised and nonbruised 'Empire' apple fruit tissues excised at indicated times after bruising and incubated for 1 hour with or without 0.4 mM ACC 65 I t 1 i I I 1 1 (ND - . s ./ 3; 5K) . 4 5 . I: e O 2'; . 530- / 'I E : !' -ACC O > 0201- ' O C 3 310- . Ill 1 l j I 1 l n l 1 I e l J I e l 0 20 4O 60 80 Hours post-impact Figure 3. Ethylene evolution from bruised 'Empire' apple fruit tissues excised at indicated times after bruising and incubated for 1 hour with or without 0.4 mM ACC 66 Bruised and nonbruised tissues bathed in 0.4 mM ACC produced 20 to 90% more ethylene than tissues without exogenous ACC (Fig. 2). Both bruised and nonbruised tissues produced equal amounts of ethylene when cooked 4 minutes in a microwave oven prior to incubation without exogenously supplied ACC (11), thus eliminating the possibility of nonenzymatic conversion of ACC to ethylene. Ethylene production ratios of bruised to nonbruised tissues were not affected by addition of ACC (Fig. 3), indicating that both tissues metabolized exogenous and endogenous ACC in a similar manner. Therefore, the 50% decrease in ethylene production by bruised tissues compared to controls must result from the physical destruction of cells within the bruised area rather than from differences in ethylene biosynthesis. Further evidence that the reduction is physical rather than physiological 'hi nature is that the ratio of bruised to non- bruised ethylene production rates remains constant at 0.5 over a temperature range of 0° to 40°C (11). Although epidermal tissues from both nonbruised and bruised apples produce similar amounts of ethylene, there is a 50% reduction in ethylene production by the subjacent corti- cal tissues as a result of bruising (11,17). I conclude that the reduction in ethylene production by bruised cortical tissues of post- climacteric apple fruit is a consequence of cell destruction within the bruised area, since the surviving cells readily metabolize ACC to ethylene. Literature cited 10. 11. Abeles, F8 1973 Ethylene in plant biology. Academic Press, New York. pp. 87-102 Adams, 00 and SF Yang 1979 Ethylene biosynthesis: identification of 1-aminocycloprOpane-I-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc Nat Acad Sci USA 76:170-174 Amrhein, N, D Schneebeck, H Skorupka, S Tophof, J Stockigt. 1981 Identification of a major metabolite of the ethylene precursor I-aminocyclopropane-l-carboxylic acid in higher plants. Naturwissenschaften 68:19-620 Apelbaum, A, AC Burgoon, JD Andersen, T Solomos, M Lieberman 1981 Some characteristics of the system converting l-aminocyclopropane- l-carboxylic acid to ethylene. Plant Physiol 67:80-84 Boller, T, RC Herner, H Kende 1979 Assay for and enzymatic forma- tion of an ethylene precursor, 1-aminocyclopropane-I-carboxylic acid. Planta 145:293-303 Boller, T and H Kende 1980 Regulation of wound ethylene synthesis in plants. Nature 286:259-260 Hoffman, NE, JR Fu, and SF Yang 1983 Identification and metabolism of 1-(malonylamino)cyclopropane-I-carboxylic acid in germinating peanut seeds. Plant Physiol. 71:197-199 Hoffman, NE, SF Yang, T McKeon 1982 Identification of 1-(malony- lamino)cyclopropane-l-carboxylic acid as a major conjugate of 1-aminocylcopropane-l-carboxylic acid, an ethylene precursor in higher plants. Biochem Biophys Res Commun 104:765-770 Holt, JE and D Schoorl 1977 Brusing and energy dissipation in apples. J Texture Stud 7:421-432 Hyodo, H 1978 Ethylene production by wounded tissue of citrus fruit. Plant Cell Physiol 19:545-551 Klein, JD 1983 Physiological causes of changes in carbon dioxide and ethylene production by bruised apple fruit tissue. Ph.D. thesis. Michigan State University, East Lansing. 67 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 68 Lieberman, M and SY Wang 1982 Influence of calcium and magnesium on ethylene production by apple tissue slices. Plant Physiol 69:1150-1155 Lizada, MCC and SF Yang 1979 A simple and sensitive assay for 1-aminocyclopropane-l-carboxylic acid Anal Biochem 100:140-145 Lougheed, EC and EW Franklin 1974 Ethylene production increased by bruising of apples. HortScience 9:192-193 MacLeod, RF, AA Kader, LL Morris 1976 Stimulation of ethylene and C02 production of mature-green tomatoes by impact bruis- ing. HortScience 11:604-606. Mattoo, AK, JE Baker, E Chalutz, M Lieberman 1977 Effect of temperature on the ethylene-synthesizing systems in apple, tomato, and Penicillium digitatum. Plant and Cell Physiol 18: 175-179 Robitaille, HA and J Janick 1973 Ethylene production and bruise injury in apple. J Amer Soc Hort Sci 98:411-413. Wang, CY and 00 Adams 1982 Chilling-induced ethylene production in cucumbers (Cucumis sativus L.) Plant Physiol 69:424-427 Yang, S F 1980 Regulation of ethylene biosynthesis HortScience 15:238-243 Yu Y8 and SF Yang 1980 Biosynthesis of wound ethylene. Plant Physiol 66:281-285 Yu, YB, 00 Adams, SF Yang 1980 Inhibition of ethylene production by 2,4-dinitrophenol and high temperature. Plant Physiol 66: 286-290 . CONCLUSIONS 1. Both the C02 and the ethylene response in bruised apples are due to disruption of the cells at the bruise site. The increase in C02 evolution from apples after bruising is not due to enhanced aerobic or anaerobic respiratory activity, but rather is due to decarboxylation of malic acid in the 0.5 cm or cortical tissue near the fruit surface at the point of impact. 2. The amount of C02 evolved due to bruising is proportional to the degree of damage and could be useful as an index for assessing physical injury that occurs during fruit handling and transport. 3. In contrast to the enzymatic nature of the CO2 response, the decrease in ethylene evolution from mature bruised apple tissues is due to physical disruption of cells in the bruised areas. Intact cells in bruised tissues from mature apples produce ethylene from exogenously supplied ACC and over a range of temperatures in the same manner as nonbruised tissues. 69 APPENDIX 70 Appendix Some experimental data, unnecessary for support of the pro- posed hypotheses, were omitted in the two journal manuscripts that comprise the majority of this thesis. They concern the ethylene pro- duction of preclimacteric fruit, the effect of exogenous malate on 02 uptake and ethylene production by bruised and nonbruised tissues, and the lack of conversion of exogenous ACC to ethylene in dead tissues and are presented here with brief discussions to provide background material that supplements the manuscripts. Fruit that were preclimacteric in ethylene production (<0.1 ul-l'”1 internal ethylene) did not have a consistent ethylene evolution response to bruising until approximately 24 hours after impact. The positive correlation of internal ethylene concentration with number of impact-drops shown in Table 1 occurred only 24 hours after bruising. Similarly, the linear regression of ethylene evolu- tion on height to drop (Table 2) was not significant (r - 0.14), nor were there any significant differences between treatment means, when fruit were sealed 16 hours post-impact. When the sealed containers were opened, aerated thoroughly, and resealed with the same fruit 27 hours post-impact, the resulting regression of ethylene evolution on dr0p height was highly significant (r = 0.75, significant at the 0.1% level), as shown in Fig. 6 in the first manuscript. The 71 72 physiological basis for the development of the ethylene response over time in bruised preclimacteric apples bears further investigation. Oxygen uptake rates of bruised and nonbruised tissues were similar when followed over 48 hours post-impact. The addition of 0.1 M malic acid to the incubation medium of nonbruised tissues 6 hours after initial measurements had no effect on 02 uptake rate, compared to tissues without exogenous malate (Table 3). The statis- tically significant difference in 02 uptake rate of bruised tissues with exogenous malate compared to other tissue treatments also existed befgrg_malate was added. Exogenous malate was not likely a factor in the lower rate of 02 uptake in this case, as noted in the last 14 hours of this experiment, when there were no statistical dif- ferences between treatments. As noted in the first manuscript, these data support the concept that the enzymatic decarboxylation of malate by mature apple tissues does not require 02 uptake. Neither the decrease in ethylene evolution by bruised apple tissues compared to controls nor the overall ethylene output of both tissues were affected by exogenous malate (Table 4). Both bruised and nonbruised tissue samples heated 4 minutes at ‘high' setting in a microwave oven had only residual ethylene produc- tion capacity, and no ability to convert exogenous ACC to ethylene (Table 5). Since there is evidently no system capable of converting exogenous ACC to ethylene in cooked ("dead") tissue pieces, the ability of noncooked bruised tissues to effect the conversion indicates that at least some cells in those tissues were "alive". I have no 73 explanation for the apparently greater production of ethylene by cooked bruised tissues compared to cooked nonbruised. 74 Table 1. Effect of 0, 1, and 3 drop-impacts from a height of 1 m on internal ethylene concentration in preclimacteric ‘Empire' apples)“z Internal ethylene concentration (nl-l‘l) Number of impacts Hours post-impact 1 3 5 12 24 0 19a -- 23 ns 15a 20a 1 18a 21 28 31b 30b 3 - 29b 20 26 24ab 74c yFruit harvested Aug. 31, bruised Sept. 2. Different lots of bruised fruit sampled at a given time. zMean separations within column by Duncan's multiple range test, 5% level. Table 2. Ethylene evolution from preclimacteric 'Empire' apples after impact drops from various heightsY’Z Drop Height (cm) 0 12.5 25 50 100 1 Ethylene (nl-kg' -hr'1) 11.0 10.0 13.6 14.6 14.1 ns yFruit harvested Aug. 31, bruised Sept. 1 2N0 significant difference between means by Duncan's multiple range test, 5% level 75 Table 3. Oxygen uptake by bruised and nonbruised 'Empire' apple tissues as affected by exogenous malic acid (0.1 M)y,z Oxygen uptake (pl-QTI-hr'l) Tissue Hours post-impact 2 4 6 8 10 24 30 36 48 Bruised 23ns 26a 27a 37ab 36b 128a 111ns 90 ns 50ns Nonbruised 32 28a 30a 47a 70a 115a 117 103 77 Bruised + malate 29 19b 166 17b 13b 49b 99 106 70 Nonbruised + malate 28 36a 42a 55a 65a 117a 122 104 84 yMalate added to incubation medium 6.25 hours post-impact ZMean separations within columns by Duncan's multiple range test, 5% level Table 4. Ethylene evolution from bruised and nonbruised 'Empire' apple tissues as affected by exogenous malic acid (0.1M)¥12 Ethylene evolution (nl+g'1-hr'1) Tissue Hours post-impact 2 4 6 8 10 24 30 36 48 Bruised 45ns 45a 45a 49a 47a 52a 47a 43a 36a Nonbruised 68 86b 92b 97b 139b 228b 153b 213b 186b Bruised + malate 40 50ab 51a 50ab 56a 59a 53a 49a 45a Nonbruised + malate 55 72a 80b 80ab 125b 186b 156b l61b 130b yMalate added to incubation medium 6.25 hours post-impact zMean separations within columns by Duncan's multiple range test, 5% level 76 Table 5. Ethylene evolution from bruised and nonbruised 'Empire' apple tissues as affected by incubation with or without 0.4 mM ACC after heating in a microwave oven for 4 minutes Tissue Treatment Nonbruised Bruised Ethylene (nl-g’lohr'l) -ACC 0.44 1.50 +ACC 0.47 1.30 nIcwIcAN STATE UNIV. LIBRARIES lli‘INIIIWIIWI"inIiillillllmll|ll“WNW 31293000804975