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O"; 0".' ‘>>-"~‘ 0 ' '.""",. ,.,'.‘..’.f ...4o-o'n O. ,4 0 " ’ ' . )0. -v«-" ’ l' ..: ' .u . . ..‘n ‘.-. . a .‘ 4 -" ‘7’..... .... .I...-ooo.o-o, "'.' ' .. ‘.'..o—. 4'v‘00'od ‘I r ' ‘ "' ' ' ° . "' °""'.‘ ‘(ooo n..4o 0' ' “ ' "' I. O ' n. )'t"""“" ' - , . . .-ot. 90 -v1 -‘ " ’\ ‘. 0.. o..', --" ' "0 ‘f' .'.. '-Jop0"Q/." ‘ ‘ --l° . '0 3'4-"' -‘ ‘ ' ."" ' .’ '.K. 0-»II 'l""-..“"'. ‘ ."-.—-o- 4 "“ I' . 0- .‘. z- 'v“' ‘2‘ ' ,. .' ,',', , . r . .10 05-. .0 "_ "3:. ,'.'..-J a. . '1 -0 HHHHHHHHHH [23:35; a) I 3900 ‘ . ‘ 3129310446 Uancmgy ‘ ’m“? __ “WM- ABSTRACT MORPHOLOGICAL AND PHYSIOLOGICAL ASPECTS OF CHERRY FRUIT ABSCISSION WITH REFERENCE TO 2-CHLOROETHYLPHOSPHONIC ACID BY Vernon Arie Wittenbach Morphological and histochemical changes, occurring in the transition zone between the pedicel and fruit of sour (Prunus cerasu: L. cv. Montmorency) and sweet (Prunus avium L. cv. Windsor) cherry during maturity, were followed in the absence and presence of 2-chloroethylphosphonic acid (CEPA). A natural weakening of the tissue at this zone was observed to begin near the start of the third growth phase. However, no visible signs of abscission were observed until 2-3 weeks prior to maturity. Changes in pectins, cellulose, and polysaccharides in the walls of cells of the abscission layer preceded separation in the sour cherry. Comparable changes and a well—defined abscission layer were not observed in sweet cherry. CEPA accelerated abscission by apparently increasing the rate of develOpment of the natur- ally occurring processes. An increase in ethylene evolution from cherry fruits near the onset of the third growth phase was correlated with the simultaneous decline in fruit Vernon Arie Wittenbach removal force, however, the level of ethylene was extremely low. Physiological aspects of cherry fruit abscission were established using an excised fruit technique. Stage of fruit development had a pronounced effect on the sensitiv- ity of fruit explants to CEPA. 3-Indoleacetic acid, CEPA, gibberellin A3 and abscisic acid hastened abscission in sour cherry. Only CEPA had a similar accelerating effect on sweet cherry. Cycloheximide applied to cherry fruit explants inhibited abscission and also negated the effect of CEPA. This could indicate that protein synthesis is necessary for cherry fruit abscission to occur. MORPHOLOGICAL AND PHYSIOLOGICAL ASPECTS OF CHERRY FRUIT ABSCISSION WITH REFERENCE TO 2-CHLOROETHYLPHOSPHONIC ACID BY Vernon Arie Wittenbach A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 1970 ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Dr. M. J. Bukovac for his assistance and encouragement during the course of this program of study. Appreciation is also extended to Dr. H. P. Rasmussen and Dr. C. E. Cress, members of my guidance committee, for their suggestions and for reading the manuscript. I would like to thank Dr. D. R. Dilley for his assistance and the use of his laboratory equipment and Mr. V. Shull for the use of the electron microprobe. The suggestions and help of fellow laboratory workers were also appreciated. A Special thanks is extended to my wife Pam for her continuous encouragement, patience and assistance in the preparation of this dissertation. ii TABLE OF CONTENTS ACKNOWLEDGMENTS . . . . . . . . . LIST OF TABLES . . . . . . . . . . LIST OF FIGURES. . . . . . . . . . LIST OF APPENDICES. . . . . . . . . INTRODUCTION . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . Leaf Abscission . . . . . . . . Fruit Abscission. . . . . . . . Cell Wall Changes and Enzymes in Abscission Protein Synthesis in Abscission. . . Effect of Growth Regulators on Abscission Auxins . . . . . . . . . . Ethylene. . . . . . . . . . Gibberellins . . . . . . . . Kinins . . . . . . . . . . Abscisic Acid . . . . . . . . Other Factors Influencing Abscission . CEPA. . . . . . . . . . . . MATERIALS AND METHODS. . . . . . . . Characterization of the Natural Fruit Abscission Process . . . . . . General Methods . . . . . . . Cherry Fruit DevelOpment . . . . CEPA Modification of FRF at Maturity Sour Cherry . . . . . . . . Sweet Cherry. . . . . . . . Upper vs. Lower Zone . . . . . Morphology of Abscission . . . . Histochemical Changes in the Abscission Zone During Fruit Separation . . Ethylene Evolution . . . . Characterization of the Abscission Process Detached Fruit Explants. . . . . iii Page ii vi viii 25 25 26 26 26 27 27 28 29 31 32 General Methods . . . . . . . Effect of Age on Fruit Abscission. . ReSponse of Mature Fruit Explants to Growth Regulators . . . . . . Role of Protein Synthesis . . . . RESULTS 0 O O O O O O O O I O O 0 Characterization of the Natural Fruit Abscission Process . . . Change in FRF with Fruit Development . Sour Cherry . . . . . . . . . Sweet Cherry. . . . . . . . . CEPA Promotion of Fruit Abscission . . Sour Cherry . . . . . . . . . Sweet Cherry. . . . . . CEPA Effect on Upper vs. Lower Zone. . Morphology of Abscission . . . . . Sour Cherry . . . . . . . . . Sweet Cherry. . . . . . . . . Histochemical Changes During Abscission Ethylene Evolution from Cherry Fruits During Development. . . . . . . Characterization of Fruit Abscission in Detached Fruits . . . FRF as Effected by CEPA and Stage of Fruit Development . . . . . . ReSponse of Mature Fruit Explants to Growth Regulators . . Effect of a Protein Synthesis Inhibitor on Abscission . . . . . . . . DISCUSSION 0 O O O O O O O O O O O Abscission Process . . . . . . . . Comparative ReSponse of Sour and Sweet Cherries to Plant Growth Substances Active in Leaf Abscission . . . . . Role of CEPA in Natural Fruit Abscission. SUMMARY 0 O O O O O O O O O O O 0 LITERATURE CITED . . . . . . . . . . APPENDIX 0 O O O O O O O O O O O 0 iv Page 32 33 33 36 37 37 37 37 38 41 41 41 41 45 45 48 58 68 74 74 77 80 83 83 86 91 95 98 111 LIST OF TABLES TABLE Page 1. Effect of CEPA on FRF at the upper and lower abscission zones of sweet cherry . . . . . 44 2. An estimate of changes in cell-wall consti- tuents of the abscission zone immediately prior to cell separation . . . . . . . . 64 3. Effect of CH and CEPA on abscission of sour cherry fruit explants . . . . . . . . . 81 4. Effect of CH and CEPA on abscission of sweet cherry fruit explants . . . . . . . . . 82 FIGURE 10. 11. 12. 13. 14. LIST OF FIGURES Page Bioassay used to study abscission in detaChed fruits O O O O O O O O O O O 34 Sour cherry fruit growth and FRF of the upper and lower abscission zones . . . . . 39 Sweet cherry fruit growth and FRF of the upper and lower abscission zones . . . . . 39 Effect of CEPA on fruit removal force of sour cherry . . . . . . . . . . . . 42 Effect of CEPA on fruit removal force of sweet cherry. . . . . . . . . . . . 42 The two potential abscission zones of cherry fruits at the time of CEPA application . . . 46 Development of the separation layer at the lower abscission zone of sour cherry. . . . 49 Detailed development of the separation layer in the cortical tissue at the lower abscission zone of sour cherry. . . . . . 51 Changes occurring in the lower abscission zone of sweet cherry . . . . . . . . . 53 Detailed development of abscission in the cortex at the lower abscission zone of sweet cherry. . . . . . . . . . . . 55 Sections from the lower abscission zone stained for polysaccharides. . . . . . . 60 Sections from the lower abscission zone stained for pectins . . . . . . . . . 62 Localization of calcium in the lower abscission zone. . . . . . . . . . . 66 Ethylene evolution from sour cherry fruits . . 69 vi FIGURE Page 15. Ethylene evolution from sweet cherry fruits . . 71 16. Change in FRF of CEPA-treated sour cherry fruit explants as effected by stage of fruit development . . . . . . . . . . 75 17. Change in FRF of CEPA-treated sweet cherry fruit explants as effected by stage of fruit development . . . . . . . . . . 75 18. Effect of growth regulators on FRF of sweet and sour cherry fruit explants. . . . 78 vii LIST OF APPENDICES APPENDIX Page Al. Number of replications and fruit per replication from which the data on ethylene evolution was obtained . . . . 112 A2. Standard deviations of the FRF values presented in Figure 2 for 'Montmor- ency' sour cherry . . . . . . . . . 113 A3. Standard deviations of the FRF values presented in Figure 3 for 'Windsor' sweet cherry. . . . . . . . . . . 114 viii INTRODUCTION Mechanical harvesters are revolutionizing the fruit industry. Blueberries and sour cherries are two crops where mechanical harvesting has become a standard practice. In addition, attempts are being made to mechanically harvest grapes, apples, pears, plums, oranges, sweet cherries, and olives, as well as several other fruit crops. One of the most serious problems to be overcome in harvesting these crops mechanically is the high retention force with which the fruit is held to the pedicel (Lamouria and Hartmann, 1959; Hendershott, 1965; Cooper et. a1., 1968; Bukovac, 1969). Cain (1967) has shown a high correlation between the fruit removal force and percentage of sour cher- ries harvested mechanically. In addition to lowering the per cent of fruit removal, high retention forces result in greater fruit injury (COOper g£_al., 1968). Therefore, recent work has been directed toward the use of chemicals to lower the fruit removal force. Chemi- cals have the advantage of being easily manipulated and of inducing the desired reSponse in a short period of time. Earlier studies have shown that a wide variety of compounds were capable of causing abscission of leaves (Weintraub et_al., 1952) and immature fruit (Batjer, 1954). Unfortun- ately, few of these chemicals have shown promise in hastening abscission of mature fruits, since they either cause exces- sive defoliation, injury to fruit and/or tree, or exhibit too wide a variation in response. New and different compounds are continually being tested. 2-Chloroethylph05phonic acid (CEPA, Ethrel), which was recently introduced, has been shown to promote abscission of peaches, apples, plums, and cherries (Edgerton and Green- halgh, 1969; Bukovac gt_al., 1969). However, much more research needs to be carried out with this chemical before it is released for general use. Control of abscission has several important practical advantages (Bukovac, 1969). First, a higher percentage of fruit can be removed by the harvester without damaging the tree. Second, a lower fruit removal force would result in less bruising and tearing of the fruit, thereby providing a higher quality product. Third, the availability of efficient chemicals would give growers the opportunity to program their harvests. Despite the need to control fruit abscission, few studies have been carried out to further our understanding of the chemical and physiological aSpects of this process. Most work in this area has been directed toward the phenomenon of leaf fall. Although fruits may have evolved from leaves there is little doubt that the fruit represents a much more complicated system (Jacobs, 1962). For instance, the fruit encloses one or more seeds with a very complex development of their own. In addition, the fruit is capable of very limited photosynthesis, and therefore, most of the organic material required for growth must come from other plant organs. Due to these differences in subtending organs, there exists a need to further identify the fruit abscission pro- cess. The present study was undertaken with three main objectives: 1) to compare the natural abscission process in maturing sweet and sour cherry fruits, 2) to study the changes associated with the promotion of cherry fruit abscis- sion by CEPA, and 3) to define the physiological parameters of fruit abscission through the use of fruit explants. LITERATURE REVIEW Leaf Abscission The phenomenon of leaf fall has intrigued man for thousands of years. Records from as far back as 285 B.C. include reference to leaf abscission (cited by Addicott, 1965). Several detailed anatomical studies of leaf fall were conducted prior to the beginning of this century. Lee (1911) summarized some of these earlier works and presented an excellent description of the morphological changes accom- panying leaf abscission in 45 dicot species. Shortly after this extensive study, Sampson (1918), concentrating on Coleus blumei Benth., investigated the microchemical and cellular changes occurring during leaf abscission. He concluded that separation resulted from the conversion of cellulose into pectose, which was further trans- formed to pectin. The accumulation of pectic acid weakened the middle lamella, since there was an insufficient amount of calcium to maintain solidity. However, more recent investigators (Facey, 1950; Valdovinos and Jensen, 1968) have suggested that separation is due to a breakdown of the pectic substances of the middle lamella. These authors indicate no conversion or only secondary changes in cellulose. Cell division has been shown to be associated with abscission (Lee, 1911; Sampson, 1918; Brown and Addicott, 1950; Facey, 1950). However, these workers could not agree as to whether it was directly involved with separation or only indirectly associated through the formation of a pro- tective layer. Gawadi and Avery (1950) studied secondary cell division in several plant Species and concluded that its only role was in the formation of a protective tissue forming the leaf scar. Brown and Addicott (1950) greatly simplified and advanced the research on abscission by introducing the use of explants. Their work indicated that explants exhibited the normal processes associated with leaf abscission. Many more studies on leaf abscission have been reported since 1950. Several have dealt with the cellular and chemical changes accompanying abscission (Scott and Leo- pold, 1966; Osborne, 1968b; Webster, 1968, 1970; Rasmussen and Bukovac, 1969; Bostrack and Daniels, 1969; Choudhuri and Chatterjee, 1970), while others have been concerned more with the ultrastructural changes (Bornman, Spurr, and Addicott, 1967; Morré, 1968; Jensen and Valdovinos, 1967, 1968; Valdov- inos and Jensen, 1968; Bednarz, 1970). In addition, the reader is referred to several excellent reviews (Addicott, 1954; Addicott and Lynch, 1955; Addicott, 1961, 1965, 1968; Jacobs, 1962, 1968; Rasmussen, 1965; Carns, 1966; COOper et al., 1968; Bednarz, 1970). Fruit Abscission Heinicke (1917) was one of the first investigators to study fruit abscission. He observed that the fall of flowers and immature apple fruit was preceded by the forma- tion of a layer of cells "similar to that which precedes leaf-fall." Separation occurred at the junction of the fruit stalk and spur, which appeared to be a naturally weak zone. In addition to describing the anatomy of the abscission zone, Heinicke (1917, 1919) determined histochemically the changes occurring during the formation of the abscission layer. The abscission zone had a lower affinity for cell wall stains, and in most cases a lignified layer was formed in the abscis- sion zone just prior to separation. The fall of immature apples has since been studied in greater detail (MacDaniels, 1936; McCown, 1938). Sec- ondary cell division was found to precede the formation of a well-defined abscission layer 6-8 cells in width. Separation resulted from the breakdown of the pectic compounds of the middle lamella and occurred within the limits of or distal to the abscission layer (McCown, 1938). However, apples which survived the June drop showed no indication of sec- ondary cell division or the formation of an abscission layer (McCown, 1938). In mature fruits separation resulted from the disintegration of walls of pre-existing cells, and even the abscission zone, 20-30 cells in width, did not determine the path of separation (MacDaniels, 1936; McCown, 1938). Therefore, even in apples, where separation of mature and immature fruit occurs at the same abscission zone, the two processes appear to be quite different. McCown (1943) in further studies concluded that depending on variety abscission was initiated independently in the cortex and pith. In the pith, separation was pre- ceded by swelling and extension of walls which was accom- panied by a physical change in the cellulose. Next the sec- ondary cellulose wall gradually disentegrated with concominant changes in the middle lamella. The dissolution of the pectic compounds of the middle lamella then allowed separation of cells. Final separation of the fruit was due to the mechani- cal tearing of the outer cortex and epidermal tissue and rup- turing of the vessels and fibers across the path of separa- tion. Soon after separation a protective layer formed leav- ing a fruit scar. Another early study of abscission in mature fruits was carried out by Barnell (1939) on avocado and mango fruits. In both there are two potential "abscission zones". The upper zone, at the junction of the pedicel and spur, is activated prior to the fall of immature fruits and after the separation of mature fruits. The lower zone at the transi- tion region between the fruit and pedicel, is involved in the separation of fruit at maturity. Barnell followed the development of abscission of the lower zone at fruit maturity and of the upper zone after fruit separation had occurred. Fruit abscission resulted from cleavage occurring along the middle lamellae of the cells. The cell walls and contents remained undamaged after separation. There was no evidence of the formation of an abscission cambium following fruit abscission. Further studies on fruit abscission have only been conducted within the last few years. Stosser (1967) inves- tigated the anatomical changes associated with the fall of immature sweet cherries. This separation occurred at the upper zone (between the pedicel and spur). Several recent studies have been concerned with the abscission of mature oranges (Hendershott, 1965; Wilson and Hendershott, 1967, 1968; Rasmussen and Cooper, 1968; Cooper, Rasmussen and Hutchinson, 1969) but only one dealt with the anatomy and histochemistry of this process (Wilson and Hender- shott, 1968). These authors described the abscission zone as being made up of slightly smaller cells and compressed tracheary elements. The protoplasm of cells in this zone became granular near fruit maturity, and there occurred a concominant accumulation of starch. Just prior to separation there appeared a distinct band of cells low in methylated pectins, which traversed the abscission zone. Separation took place through this band of cells and involved a lysigen- ous dissolution of cell walls. No cell division was observed during or prior to abscission. Also no suberin was deposited during this period, but with the beginning of separation, lignification occurred distal to the separation layer (in the fruit). A similar investigation has also been carried out on the abscission of mature sour cherries (Stdsser, Rasmussen, and Bukovac, 1969a, 1969b). Here too the abscission zone represented an area of structural weakness. Prior to separ- ation the walls of cells in the abscission zone showed a par- tial degradation of cellulose, pectins, and non-cellulosic polysaccharides. Calcium and magnesium were lost from these cell walls prior to and/or during cell separation. The separation of cells occurred without rupturing of cell walls. Final separation of the fruit was brought about by mechani— cal breaking of the vascular strands. Fruit "explants" have been used in several labora- tories to study the abscission process and to evaluate chemi- cals for their effect on abscission (Wilson and Hendershott, 1967, 1968; Rasmussen and Cooper, 1968; Stosser et al., 1969a; Zucconi, Stosser, and Bukovac, 1969). They have become an important tool for studying fruit abscission, since the abscission process is apparently identical to that observed in separation of mature fruit (Wilson and Hendershott, 1968; Stosser et al., 1969a). Cell Wall Changes and Enzymes in Abscission Cells of abscission zones, whether in leaves or fruits, are generally characterized by their smaller size and thinner walls (Addicott, 1965; Stosser et al., 1969a). 10 However, deSpite these anatomical differences the cells appear to have the same structural components and chemistry as other cells (Valdovinos and Jensen, 1968; Jensen and Valdovinos, 1968; Bednarz, 1970). The primary cell wall is composed of densely packed cellulose fibrils in an essentially crystalline array. These microfibrils are embedded in a matrix of usually amorphous and highly swollen material, consisting of hemicelluloses, pectic substances, and proteins (Setterfield and Bayley, 1961; Preston, 1964; Roélofsen, 1965; White, Handler, and Smith, 1968). The composition of the primary cell wall and middle lamellae has been well established except for the presence of proteins. The major uncertainty is the arrangement of this material. The timing of abscission, the highly localized region to which separation is confined, and the Specificity of break— down material, suggested the functioning of enzymes in abscis- sion. Hence, Bonner (1936) hypothesized that leaf abscission was due to a middle lamellae protopectinase and the enzyme polygalacturonase. Protopectinase was a logical choice, since pectic substances had been known to comprise the middle lamellae (Sampson, 1918). Pectin methylesterase (PME) was the first enzyme to be studied in detail. Osborne (1958) found PME activity to be higher in the pulvini of non—senescent bean leaves. There 11 appeared to be a decreasing gradient of activity from the pulvinus to the petiole. During senescence of the leaf this gradient fell and was generally reversed at the time of abscission. These results have since been confirmed by LaMotte et_al. (1969). Yager (1960a) added more evidence to the possible role of pectic enzymes. He found that it was possible to duplicate the separation of cells which normally occurs in abscission by incubating tissue from the abscission zone with various pectic enzymes. ‘It also was noted that factors which normally influence abscission, such as auxin, had a similar effect on these enzyme preparations. More recently evidence has been presented for the involvement of polygalacturonase (PG) in abscission (Ras- mussen, 1965). This enzyme mas found to be active in the abscission zone, petiole, and stem of the bean during abscission layer development. Both PME and PG are believed to function in the breakdown of pectins. Cellulase has also been indicated as possibly having a role in the formation of the abscission layer. Abeles (1969) found an increase in cellulase activity during abscis- sion in explants of bean, cotton and Coleus. Cellulase activity was localized in the cell separation layer and the increase in activity preceded the loss of break strength in the abscission layer (Craker and Abeles, 1969). l2 Ethylene, which hastens the abscission process, enhanced the activity of cellulase in bean explant abscission zones (Horton and Osborne, 1967). 2, 4, 5-Trichlorophenoxya- cetic acid, which delays abscission, was demonstrated to have the opposite effect. In addition, cellulase activity has been shown to increase during tomato fruit ripening (Dickin- son and McCollum, 1964) thereby being present at the time of fruit abscission. Indoleacetic acid oxidase has also been suggested as having a role in abscission (Hall and Morgan, 1963). Work with the IAA oxidase system in intact cotton plants revealed that certain phenols were cofactors while others were inhib— itors of the system 12.!1EE2 (Morgan, 1964). Therefore, using these phenols, further research was conducted to establish their effect on abscission of cotyledonary explants of cotton (Schwertner and Morgan, 1966). Results demon— strated that the cofactor phenols accelerated abscission while the inhibitory phenols delayed abscission, thereby suggesting a possible function for this enzyme in abscission. Protein Synthesis in Abscission Several authors have recently suggested that protein synthesis was essential for the development of the abscission layer (Lewis and Bakhshi, 1968; Abeles, 1968; Morré, 1968). Abeles and Holm (1966, 1967) showed RNA and protein synthe- sis inhibitors could greatly delay abscission in explants of bean, Coleus, and cotton depending on the concentration 13 used and the time of application. They also demonstrated that ethylene which accelerated abscission, caused an enhanced synthesis of RNA and protein in the separation layer. Abeles (1968) later hypothesized that RNA and protein synthe- sis were required for the formation of enzymes involved in the separation of cells during abscission. Other evidence, however, indicated the enzymes in abscission may already be present in the zone prior to the induction of abscission (Valdovinos and Ernest, 1967). The enzymes may be attached to the walls of cells in the poten- tial separation layer or be held in various unknown struc- tures observed in these cells (Jensen and Valdovinos, 1967). Thus, the initiation of abscission would simply involve the activation or release of these enzymes. Secondary cell division has often been proposed as being necessary for organ detachment (Addicott and Lynch, 1955; Webster, 1970). If cell division was necessary, it would explain the need for RNA and protein synthesis in abscission. However, Bednarz (1970) has obtained evidence indicating that cell division is not necessary for abscission to occur. Effect of Growth Regulators on Abscission Auxins Laibach (1933) was the first to demonstrate that abscission, induced by deblading, could be delayed by placing 14 orchid pollinia (a source of auxin) on the cut surface. Soon afterwards LaRue (1936) using synthetic IAA found it to be more effective in delaying abscission in debladed Coleus than a variety of auxin-containing substances. Although applied auxin was shown to have an influence on abscission, there was no evidence that auxin present in the leaf blade had a similar effect. This was, however, demonstrated by a later study in which the auxin content of bean leaves was followed during development (Shoji, Addicott, and Swets, 1951). Results of this work showed a normally high auxin content in the leaf, but as the leaflet yellowed and approached abscission, there occurred a rapid fall in auxin level. Several investigators have since shown that auxin applied distal to the abscission zone delayed abscission in direct relation to the concentration applied (Addicott and Lynch, 1951; Gaur and Leopold, 1955; Biggs and LeOpold, 1958). However, proximal applications hastened abscission. In reSponse to these opposite effects of auxin, Addicott, Lynch, and Carns (1955) proposed the auxin gradient theory to explain the control of abscission. According to this theory abscission is initiated by a fall in the ratio of distal to proximal auxin levels. Further work designed to test this hypothesis through the use of labeled auxin has failed to show the presence of such a gradient across the abscission zone 15 (Rubinstein and LeOpold, 1963; Rasmussen and Bukovac, 1966). Other interpretations, suggesting a quantitative rather than a qualitative reSponse to auxin, have been proposed (Gaur and Leopold, 1955; Biggs and Leopold, 1957). However, evi— dence obtained from the study by Rasmussen and Bukovac (1966) using autoradiographic techniques also argues against these explanations. Rubinstein and Leopold (1963) demonstrated a two- stage response of abscission to auxin--an induction stage during which auxin delayed abscission and a later stage where abscission was stimulated by auxin. In addition, they observed a two-phase concentration effect of auxin on abscis- sion. Low concentrations (10-6M) exhibited a slight acceler- ation of abscission while high concentrations (10-3M) delayed abscission. The authors explained these effects as the con- sequence of actions on the two separate stages. Low auxin levels and proximal application, due to their lower activity and slower transport, would actually be acting on the second stage and thereby exhibiting the prOper, accelerating reSponse. Both the gradient theory and the two phase action of auxin imply a direct effect of auxin on the abscission zone. However, other investigators have suggested an indirect role for auxin (Sacher, 1957; Jacobs, Kaushik, and Rochmis, 1964). Sacher (1957) concluded that auxin delayed abscission by maintaining membrane—integrity of tissue in the abscission 16 zone. Further support for this role of auxin was provided in a later study (Sacher, 1959). Jacobs et_al. (1964) put forth the hypothesis that auxin delayed abscission by main- taining petiole elongation. Although almost all the work designed to discover the effect of auxin on abscission has been carried out on leaf tissue, there is evidence to indicate that auxin may also play an important role in flower and fruit abscission (Gardner, Marth, and Batjer, 1939; Luckwill, 1948; Batjer, 1954; Yager, 1960b). Luckwill (1948) found a correlation between low levels of auxin in apple seeds and the fall of immature and mature fruits. Yager (1960b) observed that abscission of unpollinated tobacco flowers could be hastened by removing the younger leaves (source of auxin) and greatly delayed by application of IAA to the cut petioles of the removed leaves. Ethylene Ethylene has long been known to hasten abscission (Sampson, 1918). Gawadi and Avery (1950) and Hall (1952) suggested that abscission may be controlled by the ratio of auxin to ethylene. More studies implicate ethylene as a primary endogenous regulator of abscission and senescence processes (Abeles, 1966, 1967; Abeles and Holm, 1966; Burg, 1968). Because it is a gas and, hence, is freely diffusable in plant tissue, it hastens abscission regardless of the site of application. 17 Several investigations have demonstrated that auxin applications of greater than 10—6M will induce ethylene forma- tion in plant tissue (Morgan and Hall, 1964; Abeles and Rubinstein, 1964; Burg and Burg, 1966; Abeles, 1968; Halla- way and Osborne, 1969). Osborne (1968a) concluded that defoliation brought about by auxin was due to an accelerated senescence induced by ethylene, which was produced in the area of auxin application. Therefore, Abeles (1967) has proposed the difference in response observed for distal and proximal applications of auxin as being due to differences in tranSport. Distally applied auxin inhibits abscission regardless of the ethylene produced, since it is rapidly transported to the abscission zone. Conversely, auxin applied proximally stimulates abscis- sion because movement is slow and the ethylene produced dominates the reSponse. Although this explanation is attractive it fails to account for the acceleration of abscission obtained from distally applied auxin at low concentration. However, this acceleration is small and might be a response to stress induced by the application of the low level of auxin, since stress resulting from numerous sources has been shown to cause ethylene production in plant tissue (Nichols, 1966; ‘Jines, Grierson, and Edwards, 1968; Pratt and Goeschl, 1969). Other works have explained the acceleration of éibscission by ethylene as resulting from its ability to 18 reduce the amount of diffusable auxin in the plant (Valdov- inos, Ernest, and Henry, 1967; Burg, 1968; Goldsmith, 1968; Beyer and Morgan, 1969). Beyer and Morgan (1969) and Gold- smith (1968) found that prolonged treatment with ethylene resulted in an inhibition of basipetal auxin tranSport in peas and cotton. Other studies using ethylene indicated an increased destruction of auxin brought about by enhanced IAA oxidase activity and a decrease in the rate of auxin synthe- sis (Hall and Morgan, 1963; Valdovinos et_al., 1967). There is also evidence suggesting that ethylene may function by increasing membrane permeability (Von Abrams and Pratt, 1967). Such changes in membrane-integrity could result in the release of enzymes involved in abscission or the release of phenols and other substances which enhance IAA oxidase activity. However, recently Sacher and Salminen (1969) concluded that ethylene had no effect on membrane permeability of tissue sections from several plant species. Abeles and Holm (1969) also found evidence for the stimulation of RNA and protein synthesis by ethylene in bean abscission zones. Abeles (1968) later suggested that ethyl- ene hastened abscission by enhancing the synthesis of cell ‘wall degrading enzymes in the abscission zone. But dela Fuente and Leopold (1969) argued against this concept on the Ioasis of the short persistence of the ethylene stimulus (approximately one hour) after removing the gas. 19 Gibberellins Distal applications of gibberellin (GA) have been shown to slightly hasten abscission in bean and cotton explants (Chatterjee and LeOpold, 1964; Devlin and McIntyre, 1966; Bornman et_al., 1967). Chatterjee and Leopold (1964) 3M to 10'7M demonstrated that concentrations of GA from 10- all promoted abscission. They also suggested that this effect was principally on the first stage of the abscission process. However, Berman (1969) applied GA proximally to cotton and observed a delay in abscission. Muir and Valdovinos (1970) have also shown that GA applied to debladed Coleus plants had no effect on the abscission process. But, apply- ing GA to the stem apex resulted in a hastening of abscission in direct proportion to the concentration applied. In addi- tion, such treatments of GA caused an increase in the level of endogenous auxin. Therefore, the authors concluded that GA accelerated abscission by increasing the level of auxin proximal to the abscission zone. Kinins Kinins, like gibberellins, produce variable reSponses on abscission. Osborne and Moss (1963) observed distal applications of kinetin to accelerate abscission of bean explants, while applications directly to the abscission zone delayed abscission. 20 Other studies using bean explants and debladed bean plants with the Opposite leaf intact have indicated a two- phase reSponse to kinetin (Chatterjee and Leopold, 1964; Rasmussen, 1965). Low concentrations applied distally showed some promotion while higher concentrations delayed abscission. Rasmussen (1965) explained these findings on the basis of directed transport induced by the applied kinetin. High levels caused a mobilization of materials from the opposite leaf to the abscission zone, while low levels were only capa- ble of mobilizing substances from nearby to the point of application, thereby depleting the abscission zone of meta- bolites and hastening abscission. Recently, proximally applied kinins were shown to hasten abscission of cotton explants (Berman, 1969). This too might be due to a mobilization of materials out of the abscission zone. Abscisic Acid The role of abscisic acid (ABA) in abscission and other plant processes has been thoroughly covered in a recent review (Addicott and Lyon, 1969). Levels of ABA in cotton fruits have been correlated with natural fruit abscission (Davis, 1968). ABA has further been shown to hasten abscission of attached cherry fruits and cherry fruit explants (Zucconi et al., 1969). In the latter study abscis- sion layer development in the treated fruits was found to be quantitatively identical to the controls. 21 In addition, ABA has been demonstrated to be a potent accelerator of abscission in cotton explants (Bornman et_al., 1967). Craker and Abeles (1969) observed a two-fold effect of ABA on cotton and bean explants. It caused an increased production of ethylene and also increased the activity of cellulase. However, Weaver and Pool (1969) found that ABA was relatively ineffective compared to other compounds in pro- moting the abscission of grape berries and flowers. Other studies have suggested that some plants may rapidly inac- tivate ABA and thereby seriously limit its potential useful- ness (Addicott and Lyon, 1969). Other Factors Influencing Abscission Environmental factors such as light, temperature, water, minerals, oxygen, and other gases can greatly alter the time and rate of abscission (Carns, Addicott, and Lynch, 1951; Addicott, 1954, 1965, 1968; Addicott and Lynch, 1955; Rosen and Siegel, 1963; Simons, 1963). Sucrose and amino acids have also been demonstrated to influence abscission (Brown and Addicott, 1950; Biggs and Leopold, 1957; Hall, Herrero, and Katterman, 1962; Rubinstein and Leopold, 1962; Addicott, 1965, 1968; Devlin and McIntyre, 1966; Berman, 1969). Furthermore, work by Osborne (1955) has indicated the presence of an abscission promoting factor produced in sen- escing leaves. More recent studies have confirmed this 22 finding (Hall gt_al., 1962; Jacobs, Shield and Osborne, 1962). In addition, several workers have shown that aging of plants may modify the abscission process in still other important ways (Leinweber and Hall, 1959; Jacobs, McCready, and Osborne, 1966). Therefore, although explants and debladed petioles appear to undergo the normal abscission process, it is still unknown whether identical internal factors are involved. CEPA The introduction of 2—chloroethylphosphonic acid (CEPA, Ethrel) as a growth regulating compound has provided a simple means for studying the effect of ethylene. The first reported study of this compound was chemical in nature (Kabachnik and RosiIskaya, 1946). Later Maynard and Swan (1963) provided important information on the chemical sta- bility of this compound, but they did not realize the impor- tant growth regulating properties which it possessed. The list of growth regulating effects induced by CEPA is rapidly expanding. They include root initiation, auxillary bud stimulation, growth retardation, stimulation of fruit maturity, promotion of flowering, regulation of sex expression in cucumbers, leaf epinasty, defoliation, release of mature fruits, and a number of other reSponses which closely parallel those obtained with ethylene (Amchem Prod- ucts Inc., 1967; Warner and LeOpold, 1967; Russo, Dostal, and Leopold, 1968; Edgerton and Greenhalgh, 1969; Cooke and 23 Randall, 1968; McMurray and Miller, 1969; Iwahori, Ben Yehoshua, and Lyons, 1969; Byers, Dostal, and Emerson, 1969). The induction of pineapple flowering by CEPA was one of the early observations and has since become economically important (Amchem Products Inc., 1967; Cooke and Randall, 1968). Since ethylene was important in fruit ripening (Hansen, 1966), Russo and coworkers (1968) applied CEPA to banana fruits. Russo gt_al. (1968) observed CEPA to have the same effect on fruit ripening as ethylene gas. These ripening reSponses were also observed for pears and apples (Edgerton and Blanpied, 1968). In a more recent study CEPA was shown to have the same growth stimulating effect on fig fruit in growth stage II as ethylene (Crane et_al., 1970). This same kind of growth acceleration and ripening brought about by CEPA has also been shown for peaches and tomatoes (Byers et_al., 1969; Iwahori gt_al., 1969; Iwahori and Lyons, 1970). These studies all indicate a potential use of the chemical in regu- lating fruit ripening in the field. Warner and Leopold (1967) concluded that CEPA was effective in regulating growth responses through the stimu- lation of ethylene production in the plant tissue. In a later study they demonstrated that CEPA itself broke down in the plant with the release of ethylene (Warner and Leopold, 1969). They also presented a possible reaction for this breakdown. This mechanism, however, was shown to be 24 incorrect by Yang (1969). He concluded that the following reaction represented the breakdown of CEPA. C1-CH2-CH2-P\ - + H20-—9C1-CH2-CH2-P\\ :—9C1 + CH2=CH2 + H2P04 O + O = OH 0 HPO ( > C \ < 4) H H CEPA Ethylene Morgan (1969) found that CEPA hastened the abscis- sion of leaves, debladed petioles, and flower buds of cotton. He also presented evidence to indicate the accelerated abscission was due to ethylene "produced from or by the active ingredients in the formulation". Bukovac and coworkers (1969) have shown a potential use of CEPA as an aid to mechanical harvesting of sweet and sour cherries and plums. They observed a hastening of abscission within 4 days after application as measured by fruit removal force. At lower concentrations (500 ppm and lower) it was found that the fruit could be loosened sig- nificantly without any observable defoliation or other phytotoxic response. Other studies have since confirmed these findings (Anderson, 1969; Bukovac gt_al. unpublished data). In addition, CEPA has been observed to promote abscission of mature and immature apples and grapes (Edger- ton and Greenhalgh, 1969; Weaver and Pool, 1969). Therefore, data obtained with CEPA further supports the findings that ethylene may play an important role in the control of abscission. MATERIALS AND METHODS Characterization of the Natural Fruit Abscission Process General Methods The abscission of maturing sour (Prunus cerasus L., 'Montmorency') and sweet cherry (Prunus avium L., 'Windsor') fruits was followed morphologically during the 1968 and 1969 growing seasons. Similar results were obtained for both years, hence only the results obtained in 1969 will be given. A detailed development of abscission was followed for both control and 2-chloroethylphosphonic acid (CEPA) treated fruits. Fruit removal force measurements were collected throughout the growing season so that the progression of abscission could be related to the stage of fruit growth, ethylene evolution from the developing fruit, and anatomical and histochemical changes occurring in the abscission zone. The terms used in this dissertation to describe cherry fruit abscission are defined below: Abscission-—Separation of the fruit at the fruit: pedicel or P€dicel=spur junction brought about by physio- logical and/or mechanical processes. Synonym--Separation. Abscission zone--Transition zone between the fruit and pedicel (upper abscission zone) and/or the pedicel and spur (lower abscission zone). 25 26 Abscission 1ayer--A well-defined layer of cells in the abscission zone which undergo physiological changes leading to abscission. Synonym--Separation layer. Cherry Fruit Development Fruit growth and FRF measurements were made through- out the growing season. Fresh weight was determined twice a week from random samples of 20 or more fruit. All samples were taken in the morning at approximately the same time. FRF measurements at the upper and lower abscission zones were taken once a week with a Hunter Mechanical Force Gauge (Hunter Springs, Hatfield, Pa.) fitted with a claw. The procedure used to determine FRF at the lower abscission zone was similar to that described by Cain (1967). The fruit was pulled from its pedicel in line with the long axis imme- diately after picking the fruit with stems attached. FRF values at the upper zone were obtained by holding the Spur in the claw at its point of attachment to the branch and pulling the pedicel from the Spur, again in line with the long axis. A random sample of 20 fruits was used for FRF measurements at each zone for each date. CEPA Modification of FRF at Maturity Sour Cherry,--Three large branches on each of two uniform trees were selected and treated with CEPA at 0, 500, or 1000 ppm approximately 10 days prior to maturity. CEPA, acid-anhydride formulation (Amchem 66-329), was applied as a 27 foliar spray using 0.1% Tween 20 (polyoxyethylene—ZO-Sorbitan monolaurate) as a surfactant. FRF was determined on a random sample of 20 fruits from each branch at 0, 4, 8, and 12 days after application. Sweet Cherry.--Nine 7-year-old trees were selected for uniform vigor and fruit load. CEPA, acid formulation (Amchem 68-240), at 0, 500, and 1000 ppm was applied 5-7 days prior to optimum brining maturity (12-14 days before fresh market maturity) to single trees assigned to a randomized block design with three trees in each block. Application was made in the evening with a high pressure Sprayer using a single nozzle gun, and trees were sprayed to the drip point. No wetting agent was used. FRF measurements were taken on a random sample of 25 fruits from each tree at time of treat- ment and thereafter every other day for 14 days. Upper vs. Lower Zone.--To determine if CEPA influenced the pedicelzspur (upper) and fruit:pedicel (lower) abscission zones similarly, six pairs of branches with uniform fruit load were selected for treatment. One branch of each pair was assigned as the control and received only 0.1% Tween 20. The other branch was sprayed with CEPA at 1000 ppm and 0.1% Tween 20. The spray was applied to the drip point two weeks prior to optimum maturity. FRF measurements were made on 10 randomly selected fruits from each branch 12 days after treatment. The pedicel 28 was cut in two with a pair of scissors, thereby making it possible to record the pull force on both abscission zones from a single fruit. Morphology of Abscission Samples of fruit for the morphology study were obtained from the same CEPA treatments as the fruit retention force measurements. A random sample of 15 fruit was col- lected from each of the 0, 500, and 1000 ppm treated branches of sour cherry. These collections were made at time of treat- ment and thereafter every other day for 14 days. For sweet cherries, 10 fruits representative of that) date were selected from each tree. Samples were collected daily for the first 10 days and every other day thereafter for the next 6 days. Therefore, a total of 30 fruits for each treatment was gathered for both sweet and sour cherry at each date. Fruits were detached from the tree by cutting the pedicel above the upper abscission zone with a razor blade. The detached fruits were immediately killed and fixed in FAA (Formalin-acetic acid-alcohol, formulation from Jensen, 1962). Later, blocks of tissue containing the upper or lower abscis- sion zone were cut from the Spur and pedicel or fruit and pedicel respectively, dehydrated in a gradient series of t-butyl alcohol (Sass, 1958), embedded in Fisher Tissuemat (melting range 56—580C) and mounted on wooden blocks. The tissue was cut at 12 um and affixed to glass Slides with 29 Haupts adhesive, using 4% formalin to flatten and expand the sections (Jensen, 1962). The sections were passed through an alcohol series to water, stained with iron hematoxylin, dehydrated to xylene, and permanently mounted in Lipshaw mounting medium (Lipshaw Manufacturing Company, Detroit). Fifteen fruits were sectioned for each treatment and collection date. Since 500 ppm CEPA was nearly as effective as 1000 ppm, only photomicrographs of sections from fruit treated with the lower concentration are presented. Histochemical Changes in the Abscission Zone During Fruit Separation Changes in pectin, lignin, cellulose, polysaccharides, and starch were followed histochemically in the abscission zone during fruit maturation in both the sour and sweet cherry. FAA fixed tissue, representative of the various stages of abscission, was embedded in paraffin and sectioned at 15 um. All experiments were performed twice with similar results. The following staining reactions, as outlined by Jensen (1962), were used to localize the various constitu- ents: Constituents Test Used Pectin Ruthenium Red Pectin Hydroxylamine-Ferric Chloride Lignin Phloroglucinol-HCl Polysaccharides Periodic Acid-Schiff's Reagent Starch Iodine Potassium Iodide 30 Changes occurring in cellulose orientation of cell walls in the abscission zone were studied using plane- polarized light. Paraffin was removed from the sections, and they were mounted directly in Lipshaw mounting medium prior to observation. The distribution of water insoluble calcium and mag- nesium across the abscission zone was followed using an electron microprobe (Applied Research Laboratories Model EMX - SM). Paraffin sections cut at 15 um were mounted directly on quartz slides using only 4% formalin to flatten the sections on a warming plate. The sections were then incinerated by increasing the temperature from 210C to 3500C over a period of 5 hours then held at 3500C for 6 hours in a muffle furnace. The gradual increase in temperature pre- vented the paraffin from spattering (Bednarz, 1970). After incineration, the remaining stable white ash was coated with a conducting layer of carbon (approximately 2008 thick) and the specimen was examined with the electron microprobe. Operating conditions were 25 kilovolts electron accelerating potential and a sample current of 0.05 micro- amperes. The tissue scanned was the abscission zone just below the epidermal indentation. The distribution of Ca and Mg was obtained using two procedures. First, a scanning x-ray micrograph was obtained for the entire area. Then a 50 um wide beam was centered on the X axis and the section was moved under the beam, thereby providing a qualitative 31 line scan of Ca and Mg. Similar patterns of distribution for both Ca and Mg were obtained, and since the level of Mg was far below that of Ca, only the results for Ca are pre- sented. Ethylene Evolution Ethylene evolution from sweet and sour cherry fruit was measured twice a week from early develOpment to past maturity. The fruit was carefully detached from the tree at the upper abscission zone, immediately transferred to the laboratory and the pedicel was cut with a razor blade 3 to 4 mm from the fruit. Fruits during growth stages I and II (Tukey, 1934) were weighed, placed in 30 ml flasks, and sealed with a rub- ber vaccine cap. Larger fruits, later in the season were sealed in 264 ml glass jars with vaccine caps fitted in the covers (see Appendix, Table A1 for number of fruits per con- tainer and number of replications). Filter paper wicks saturated with 10% NaOH were sealed in the containers above the fruit to remove carbon dioxide. I The sealed containers, including appropriate controls (lacking only the fruit), were then transferred to a circu- lating water bath at 250C 7 10. Oxygen was added with a syringe every hour to maintain the level near normal. The level of oxygen and carbon dioxide was checked after 4 and 8 hours using a Vapor Fractometer Model 154 B. 32 Ethylene determinations were made after 0, 1, 2, 4, and 8 hr. The data obtained from the 8 hr determinations are presented in the results. Samples of 100 ml were taken from the 30 ml flasks and 1.0 m1 samples from the large con- tainers. These collections were made with gas—tight Hamilton syringes using the technique described by Lyons, McGlasson, and Pratt (1962). The samples were injected into a Varian Aerograph Series 1200 gas chromatograph system, using a 30 inch column containing Poropak Type R. Amounts of ethylene were determined by comparing peak heights to that of a known standard. Values of ethylene were converted to n1 fruit.l hr-1 and ul kg fresh weight-1 hr_l. In these determinations cor- rection was made for the volume diSplaced by the fruit. Characterization of the Abscission Process in Detached Fruit Explants General Methods Explants made up of the fruit and pedicel of Prunus cerasus L. cv. Montmorency (sour cherry) and Prunus avium L. cv. Windsor (sweet cherry) were used to study the physiology of fruit abscission. Uniform fruits (based on size and color) were selected, detached from the tree at the upper abscission zone, and transferred immediately to the labora- tory. The pedicels were then trimmed under water to a uni- form length of about 2.5 cm and the explants were positioned in 4 m1 test tubes containing 3.7 ml of the designated 33 treating solution (Figure 1). Two to four fruits were used per tube, depending on the stage of development, and each tube represented one replication of a treatment. The tubes were arranged in a randomized block design with 8-12 repli— cations. Explants were held in the dark for 80 hours at 250C. The volume of test solution was maintained by addi- tion of distilled water as needed. At the end of 80 hours 1 the FRF was measured. Effect of Age on Fruit Abscission.—-This experiment was conducted to establish the effect of stage of fruit development on abscission and sensitivity to ethylene (CEPA). Fruit explants, collected weekly from the end of growth stage I to maturity, were assayed as described above using CEPA at 0, 10‘5, 10‘4, and 10'3M. Response of Mature Fruit Explants to Growth Regu— lators.--Sweet and sour cherry fruit explants were obtained approximately 2-4 days prior to the first histochemical signs of cell separation in the abscission zone (10-12 days prior to maturity). The following growth regulators and concentra- tions were used to establish the abscission process: Compound Concentrations Used (M) 3-Indoleacetic acid (IAA) 0, 10'7, 10’6, 10'5, 10’4, 10’3 2-Chloroethylph05phonic acid 0, 10‘6, 10'5, 10'4, 10’3 (CEPA) (RS)-Abscisic acid (ABA) 0, 10‘7, 10‘6, 10'5, 10'4, 10'3 -6 -5 —4 -3 Gibberellin A (GA) 0, 10 , 10 , 10 , 10 3 Figure l. 34 Photograph illustrating the bioassay used to study abscission in detached fruits. 35 36 The effect of these compounds on abscission was determined by measuring the FRF after 80 hr and comparing the values with those of the controls. Role of Protein Synthesis.--Cycloheximide (CH) was used to indicate if protein synthesis was involved in fruit abscission. Cycloheximide resembles transfer RNA, therefore, the peptide chain being formed readily attaches to it. But unlike transfer RNA, cycloheximide remains bound to messen- ger RNA, and hence, terminates the formation of new protein molecules. Cycloheximide was either applied directly to the abscission zone (0.25 pg) or used as the treating solution for the fruit explants (10 ppm). In addition, it was used in combination with CEPA to establish if it would inhibit the CEPA-enhanced abscission. RESULTS Characterization of the Natural Fruit Abscission Process Change in FRF with Fruit Development Sour Cherry.--During the first few weeks of fruit development, the pedicel represented the weakest connection between the fruit and branch (Figure 2). Hence, when a force was applied to the fruit, separation occurred near the middle of the pedicel generally leaving a section of exposed con— ducting tissue, which did not separate along the same plane. Toward the end of stage I of fruit growth the Spur region (between the pedicel and Spur and the Spur and branch) became the weakest point of attachment, resulting initially in a sharp decline in FRF (standard deviations for the FRF values are given in Table A2-Appendix). Another decrease in FRF at the Spur occurred just prior to the start of the third growth phase. After this reduction there was a strengthening of the tissue and by fruit maturity the FRF at this zone had reached a maximum. The lower abscission zone Showed a very different pattern of development, in that, this zone continued to strengthen after anthesis until the start of the second growth phase, at which time it had reached a maximum value. 37 38 With the beginning of growth stage II there was a gradual decline in FRF, which became more pronounced just prior to the start of the third growth phase. This rapid decline in FRF continued through to maturity resulting in the lower zone becoming the weakest point of fruit attachment. Sweet Cherry.--In sweet cherries the FRF of the upper and lower abscission zones never reached as high a value in mid-season as was observed for sour cherries (Figure 3). During the first few weeks of fruit development the pedicel represented the weakest region. Then just prior to the start of growth stage II, there was a weakening of tissue at the upper zone (standard deviations for the FRF values are given in Table A3-Appendix). Another decrease in FRF at the upper zone appeared to be associated with early stage III of fruit growth and then, as the fruit approached maturity there was an apparent strengthening of the tissue resulting in a rise in FRF. The FRF at the lower abscission zone was observed to increase steadily during stage I of fruit growth, reaching a maximum value with the start of stage II. During the second growth phase there was only a Slight decline in FRF, but as the fruit entered stage III this reduction became pronounced. About two weeks prior to maturity the lower zone represented the weakest region of fruit attachment. However, even at maturity there was only about half as great a difference in FRF between the upper and lower zone as observed for the sour cherry. 39 Figure 2. A comparison of sour cherry fruit growth and FRF of the upper and lower abscission zones from full bloom to maturity. Figure 3. A comparison of sweet cherry fruit growth and FRF of the upper and lower abscission zones from early development to maturity. 5.00 § 0 O 300 2.00 FRESH WEIGHT- (9) + l.00 0.00 FRESH WEIGHT - (qI-o— 40 _ SOUR CHERRY 'Montmorancy' '1 2000 Point at Separation Hid-padicol j, s Spur : Fruit 2) I. , .4 I600 ' Loam} . AZ . , . )- -¢ 4 l200 Uppor ’6' \ " - Pad' I i ' j 800 ::°/ y / Growth I." c (v. I "' 400 . l L l 1 l 1 I I2 20 28 3 I3 2| 29 7 I3 May Juno July SAMPLING DATE 5_oor_ SWEET CHERRY 'Windsor' T2000 Point of Separation 3 4.00 — e 5"" % Fm" 1 I600 3 I UJ Lower AZ 0 3 0° ' l200 g . )— __, u- .1 - < . > 2.00 - 1000 g m Growth Curve 0: ' t- I.OO - u 1 400 S I! u. 0.00 l 1 l L I; J 1 O 20 26 3 II IS 27 5 I3 May June July SAMPLING DATE FRUIT REMOVAL FORCE 49)“ 41 CEPA Promotion of Fruit Abscission Sour Cherry.--CEPA at 500 and 1000 ppm, applied 10 days prior to maturity, caused a 50% reduction in FRF (Fig- ure 4). No significant difference was observed between con- centrations, however, both caused a significant reduction from the control. Maximum reSponse to the chemical was observed between 4 and 8 days after treatment. At 1000 ppm some leaf yellowing and abscission was evident (about 5%), but at 500 ppm only a few yellow leaves were observed (1%). No other injury was apparent on the tree, and the fruit showed no phytotoxicity. Sweet Cherry.--A similar response to CEPA was observed for sweet cherries (Figure 5). Both 500 and 1000 ppm caused a significant reduction in the force required to separate the fruit from the pedicel. Maximum reduction over the controls occurred between 6 and 10 days following appli- cation. Again, some leaf abscission and yellowing was observed at 1000 ppm (5%) but this was evident only on the lower, weaker limbs and Spurs. Little to no injury was apparent at 500 ppm. Treated fruits appeared to be slightly advanced in maturity as noted by color and firmness. CEPA Effect on Upper vs. Lower Zone CEPA did not promote the abscission of sweet cherries at the upper zone at a level (1000 ppm) which markedly reduced the FRF at the lower zone (Table 1). 2 4 .ucflom Loom How mSOHDMH>m© photomum oumo Iflocfi muoxomub Hmofluum> .soflumoflammm HMHHOM Hmpmm mxcc 4H ccc NH .OH .m .c .v .N muumno ummBm .HOm nocflz. mo mouom Hm>OEmH uflsum co «Emu mo uoowwm .m muomflm .ucfiom 30mm now msoHDMH>mo cumpcmum mumoapcfl mumxomub Havauum> .coflpmoflammm HMHHOM umumm mast NH pom w .v wuuono usom .mocmHOE lucoz. mo mouom Hm>oEoH uflsuw co dmmo mo pommmm .v musmflm 43 :8 - 1.53.35 5:? us... a. o. a c c m c H H 4 q q - 1 a Son 000. .228 . - I /,, (Emu I 13053. >¢¢wx0 hmw3m I 00. 00w 00m 00¢ 00m. 000 00h 000 000 000. (5) -3080.-.I 'IVAOWBH land cant I Plug—Ruck. mwhmd No.3... N. o v 0 H 4 _ w“ .. sad coo. . - can 000 c .3200 L Emu 30:35.82. 55:0 «:8 l 00. 00w 00m 00¢ com 000 005 000 000 000. (5) - 30305 WAorIz-Iu 1Ina.-I 44 TABLE l.--Effect of CEPA (1000 ppm) on fruit removal force (FRF) at the upper and lower abscission zones of 'Windsor' sweet cherry 12 days after foliar application. FRF Concentration Upper zone Lower zone (ppm) (9) o 946 a1 391 b 1000 1011 a 204 c lMeans followed by unlike letters are significantly differ— ent at P=0.05 (Tukey's w test). 45 Morphology of Abscission Both sour and sweet cherry fruits exhibit two abscission zones during deve1Opment (Figure 6). The upper zone is located at the junction of the pedicel and spur and is denoted by the constriction of tissue and the smaller isodiametric cells which comprise the zone (Figure 6A and C). Abscission at this zone occurs at the time of June drop and after detachment of the fruit, either at harvest or earlier in development. The other transition zone is at the junction of the fruit and pedicel (Figure 6B and D). This lower zone is also characterized by constriction of tissue and the pres- ence of small cells. Abscission is initiated in this region just prior to fruit maturity, although the degree of cell separation is different for 'Montmorency' than for 'Windsor'. Figure 6 denotes the state of abscission in sweet and sour cherries for the two zones at the time of CEPA application. Since no change was observed in the absence or presence of CEPA in the upper abscission zone from the time of treatment to maturity, the remainder of the data will deal entirely with the lower abscission zone. Sour Cherry.--Abscission in maturing sour cherries has already been described (Stosser et al., 1969a, 1969b). Separation occurs at the transition zone between the pedicel and fruit. The cells of the abscission zone are small and round compared to the larger cells of the pedicel and the more elongated cells of the fruit. Figure 6. 46 Photomicrographs illustrating the two abscission zones of cherry fruits at the time of CEPA application. (A) upper zone, sour cherry; (B) lower zone, sour cherry; (C) upper zone, sweet cherry; (D) lower zone, sweet cherry. az and arrows indicate abscission zone. p-pedicel, f-fruit, s-Spur 47 «UV '1‘ ' £333.51" M (I , 48 The effect of CEPA appears to be a hastening of the normal abscission process (Figures 7 and 8). The separation layer was first identified by a loss in affinity for haema- toxylin (Figure 7A and 8B—SL). Then cells all through the crescent shaped abscission layer began to separate (Figure 7B and E). No separation or other change, however, was evident across the vascular bundles (Figure 7D and H). The separation of cells began above the stony pericarp and pro- ceeded up to the natural indentation of the pedicel (Figure 7C and F). Generally there remains a few layers of cells next to the indentation where no cell separation occurs (Figure7). Cell separation appears to occur in the same manner for CEPA-treated and non-treated fruits (Figure 8). Gener- ally the cells along the separation layer pull apart leaving the cell walls intact (Figure 8C, D and F), however, separa- tion also occurs through cells leaving cell fragments behind (Figure 8D, G and H). CEPA, in addition to accelerating abscission, also hastens the degradation of cells in the abscission zone (Figure 8G and H). Cells adjacent to the separation layer in treated fruits exhibit greater distortion of cytoplasm and cell structure than observed in control fruits. Sweet Cherry.--Separation in the sweet cherry (Fig- ures 9 and 10) is less well-defined than in the sour cherry. Cells in the abscission zone are readily apparent due to Figure 7. 49 Photomicrographs showing development of the separation layer at the lower ab— scission zone of 'Montmorency' sour cherry. Control (A—D) and CEPA-treated (500 ppm) fruits (E—H) 2, 4, 6 and 8 days after treatment. Arrows indicate separation layer. p-pedicel, f-fruit. L... 1“ ° ‘ ‘ J, (‘0‘ V ‘ I ‘ ~- . ~~«.-.::1~ 7" .6)?” "1’0‘ '8“ I 1" ’a-- Figure 8. 51 PhotomicrOgraphs Showing a detailed develop- ment of the separation layer in the cortical tissue at the lower abscission zone of 'Mont- morency' sour cherry. Control (A—D) and CEPA- treated (500 ppm) fruits (E—H) 0, 2, 4 and 6 days after treatment. sl-separation layer or potential separation layer, p-pedicel, f-fruit. 52 Figure 9. 53 Photomicrographs illustrating changes occurring in the lower abscission zone of 'Windsor' sweet cherry during fruit maturation. Control (A-D) and CEPA-treated (500 ppm) fruits (E—H) 3, 6, 9 and 12 days after treatment. Arrows indicate abscission. p-pedicel, f—fruit. Figure 10. 55 Photomicrographs illustrating a detailed development of abscission in the cortex at the lower abscission zone of 'Windsor' sweet cherry. Control (A-D) and CEPA-treated (500 ppm) fruits (E-H) 0, 4, 8 and 12 days after treatment. az-abscission zone, p-pedicel, f-fruit. 56 57 their small Size compared to the larger cells of the adjacent pedicel and fruit tissues. Still these cells are more sim- ilar to those in the pedicel due to their thick cell walls in comparison to the thin-walled cells of the fruit (Figure 9A). Fruit abscission is characterized by the formation of a cavity just above the stony pericarp (Figure 9A, B and E). With the enlargement of this cavity, there appears to be a mechanical stress on the adjacent fruit tissue. Hence, the cells adjacent to the cavity collapse and are pushed in toward the cavity (Figure 9C and F). This movement of cells, which is enhanced by CEPA, later results in the collapse of the fruit lobe which surrounds the pedicel (Figure 9H). Mechanical stress, prior to the collapse of the lobe, causes the cells at the indentation to pull apart (Figure 9D, F and G). Cell separation as observed in the sour cherry is not evident except in the formation of the cavity above the pit, nor is there any evidence of a well- defined separation layer as seen in the sour cherry. There is no evidence of cell separation in the vascular bundles but they probably break in response to mechanical force. The structure of cells in the abscission zone appear much different from those in the sour cherry (Figures 8A and 10A). The cells are smaller and more compact in the sweet cherry, and in addition, they appear to be thicker-walled and contain more cementing material than those of the sour 58 cherry. Separation seldom proceeds in far from the indenta- tion point (Figure 8D and 9D) except in fruits treated with CEPA (Figures 9H and 10G and H) or in fruits left on the tree past maturity. When cell separation is observed in the cortex, it occurs through cell layers in the distal region of the abscission zone-—next to the fruit tissue (Figure 9D and H and Figure 10G and H). If only the latter stages of abscis- sion in CEPA-treated fruits are observed, the cells appear to have been cleanly separated along the middle lamella (Figure 9H and 10H). However, during cell separation in both treated and control fruits, it appears that the cells are generally being torn apart by mechanical force rather than through dissolution along cell walls (Figure 10D and G). It was also found that when control fruits were protected and allowed to become over mature (7-10 days past maturity) that abscission development advanced to the same degree as observed for CEPA-treated fruits 8-10 days after application. From these observations CEPA appears to function sim- ilarly in sweet and sour cherries. In both Species CEPA resulted in an apparent hastening of the normal abscission process. No cell enlargement or division was observed in the abscission zone of either the sweet or sour cherry. Histochemical Changes During Abscission Prior to cell separation (approximately 4 days), the walls of cells comprising the separation layer in the 59 abscission zone of sour cherries showed a loss of cell wall constituents. Such changes were not observed in the abscis- sion zone of sweet cherries, indicating differences in the nature of the separation process for the two species. Therefore, changes in cell wall constituents of the abscission zone and adjacent fruit and pedicel tissue were followed histochemically during abscission. No changes in the cell wall composition of either the fruit or pedicel tissue adjacent to the abscission zone were observed during abscission of either sweet or sour cherry. However, in sour cherries there was a loss of polysaccharides, cellulose, and pectins from the walls of cells of the separation layer in the distal portion of the abscission zone (Table 2). The loss of polysaccharides from the separation layer in sour cherry was readily apparent (Figure 11), whereas the loss of pectins (Figure 12) and cellulose was less pronounced. The 5-8 layers of cells composing the separation layer and undergoing these changes in composition were the cells which later separated. In sweet cherries Similar changes in polysaccharides (Figure 11), pectins (Figure 12), and cellulose did not pre- cede cell separation or even occur later in abscission (Table 2). Instead, separation occurred where the cellulose content per unit area of tissue was naturally lower due to a Figure 11. 60 Photomicrographs of sections from the lower abscission zone stained for polysaccharides with periodic acid--Schiff's reagent. (A) Sour cherry fruit prior to cell separation. (B) Sweet cherry just prior to the start of cell separation. (C) Sour cherry during cell separation. (D) Sweet cherry during localized cell separation. 61 Figure 12. 62 Photomicrographs of sections from the lower abscission zone stained for pectins with ruthenium red (A, B) and hydroxylamine ferric chloride (C, D). Sour cherry just prior to cell separation (A, C). Sweet cherry during localized cell separation (B, D). 63 a v . O O A... .. .4: ‘0‘”...9' » .9. o. v . s #00. O. of. a.‘ a .p 64 TABLE 2.--An estimate of changes in cell wall constituents of the abscission zone immediately prior to cell separation. Abscission zone Compound-test Sour Sweet Pectin-Ruthenium red -1 O Pectin-Hydroxylamine ferric chloride - O Cellulose-Polarized light - O Polysaccharides-PASzreagent — 0 10 (no change); - (decrease) 2Periodic Acid - Schiff's Reaction 65 decrease in cell number per unit area going from the distal region of the abscission zone to the adjacent fruit tissue. No change was observed in the starch content of the separation layer or abscission zone in either the sweet or sour cherry. However, some build up of starch was observed in a few Specialized cells proximal to the abscission zone. Also, no lignification of cells on either Side of the separa— tion layer was observed. Only fiber and vascular bundle cells along with cells of the epidermis of the fruit gave a positive reaction. Similar results were found for both the sweet and sour cherry. Localization of water insoluble calcium showed that even after the initial histochemical changes in the abscis— sion layer of sour cherry are observed, these cell walls still appear to contain as much Ca as walls of cells in the pedicel region (Figure 13A and B). Although more Ca was observed in the abscission layer than in the walls of cells of the fruit, this was to be expected since the fruit cells are larger and have thinner walls. In treated fruits of the same date, where the cells had already separated there was a loss of Ca from the abscission layer (Figure 13E and F). How- ever, this loss was probably associated with the absence of cell walls in this area (Figure 13E). Therefore, the Ca con- tent appears to be almost completely associated with the cell walls, and as these walls are degraded the Ca is lost. Similar results were observed for the sweet cherry (Figure 13C, D, G, and H). However, in the sweet cherry a Figure 13. 66 Localization of calcium in the lower abscission zone of sour and sweet cherry during fruit abscission. (A, C, E, G) Secondary electron micrographs of the abscission zone. The line graph indicates the distribution of calcium for a 50 um wide band centered on the x-axis. (B, D, F, H) Calcium x-ray micrographs of A, C, E, and G respectively. A. Sour cherry at the start of cell separ- ation. C. Sweet cherry just prior to cell separation. E. Sour cherry treated with CEPA Showing complete cell separation in the separation layer. G. Sweet cherry treated with CEPA showing localized cell separation. p-petiole, f—fruit, sl-separation layer, az-abscission zone. 68 higher level of Ca is apparently associated with the cells proximal to the abscission layer (Figure 13C and G). This may reflect more cementing material between these cells and represent the thicker-walled cells in this region as was mentioned earlier. Although a comparison between treated and non-treated cherries is presented, control fruits showing cell separation gave identical results to the treated fruits where the cell walls were completely separated. Ethylene Evolution from Cherry Fruits During Development Sweet and sour cherries exhibited a similar pattern of ethylene evolution throughout fruit development (Figures 14 and 15). However, the level of ethylene evolved was low for both Species. Sour cherries, on a per fruit basis (Figure 14 - n1 fruit-l hr—l), showed a decline in the level of ethylene during degradation of the flower parts. Just prior to the start of pit hardening there was a Sharp increase in ethylene evolution. The level then fell off during the remainder of growth stage I and stayed relatively constant during most of stage II. However, just before the start of stage III another increase in ethylene evolution was observed. 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