PHYSTOLOGY 0F CHERRY FRtfiT ABSCISSTON. Dissertation for the Degree of Ph.‘ D. MICHEGAN STATE UNIVERSITY VERNON ARTE WITTENW 1974 j LIBRARY “ Michigan State University This is to certify that the thesis entitled Physiology of Cherry Fruit Abscission presented by Vernon Arie Wittenbach has been accepted towards fulfillment of the requirements for Eh- D- degree in Horticulture //7/, 2/2i g/g/ucd Myer professor /* f 7 , . / DateK+-( — ma 47 2,4/ / 7 /§ 74;, / v / .2/ c/ 0-7639 h ‘E ‘ amoms av ‘g HDAG & SIM 800K BINDERY INC. LIBRARY amocns I :Pnllsl’ogt, memor- ABSTRACT PHYSIOLOGY OF CHERRY FRUIT.ABSCISSION By Vernon Arie Wittenbach SECTION I. EVIDENCE FOR A TWO-PHASE PROCESS AND THE INVOLVEMENT OF ETHYLENE Abscission in the sour cherry (£52223 cerasus L., cv. Ment- morency) can be separated into two distinct phases. Phase I, a pre-separation phase, is characterized by increasing break- force, and explants prepared from fruits during this phase ex- hibit no potential for abscission. Ethylene and 2-(chloroethyl)- phosphonic acid (ethephon) have no effect on abscission when applied early in Phase 1, whereas later applications shorten the phase. The separation phase, Phase II, is characterized by a declining break-force, and explants have a potential for abscis- sion. Exogenous ethylene hastens absciasion during this phase, while lowering the level of endogenous ethylene delays abscission. Neither rates of ethylene evolution nor levels of endogenous ethylene in the fruit were correlated with abscission. Cyclohex- imide inhibited abscission while promoting ethylene production. The possible roles of ethylene and protein synthesis in abscis- sion are discussed. Vernon Arie Wittenbach SECTION II. A ROLE FOR ETHYLENE IN MECHANICALLY-INDUCED ABSCISSION OF IMMATURE FRUITS Injury-induced abscission of sour cherry fruit (Prunus cerasus L., cv. Montmorency) was correlated.with a marked in- crease in ethylene evolution from the seed following treatment. Ethylene evolution subsequent to injury of the seed at various stages of development suggested that the induced ethylene pro- duction was associated with the nucellar tissue. Ethephon also induced immature fruit abscission; however, abscission followed seed abortion. Although exogenous ethylene appeared to directly influence abscission at the pedunclezpedicel zone, injury- induced ethylene did not appear to act directly at this zone. The mechanisms involved in abscission of immature and mature fruit, although associated with different zones, appear to be similar. The possible role of ethylene in immature cherry fruit abscission is discussed. SECTION III. PEROXIDASE ACTIVITY IN THE ABSCISSION ZONE IN RELATION TO SEPARATION Peroxidase activity was demonstrated in the abscission zone and adjacent tissues of sour cherry fruit (Prunus cerasus L., cv. Montmorency) from Stage I of fruit growth to maturity. Activity was greatest in the receptacle and abscission zone tissues, with only a low level in the fruit.. A histochemical difference was observed in the peroxidase of the abscission layer from that of the adjacent tissues. Moreover, peroxidase activity in the abscission zone increased to a maximum at a stage of development coinciding with the initiation of the separation phase. This Vernon Arie Wittenbach increase in activity was accompanied by an increase in two of the major isoenzymes and the appearance of a third. The relation- ship between changes in peroxidase and abscission in fruit ex- plants was less clear. Ethylene and ethephon had no significant effect on total peroxidase activity; however, ethylene appeared to increase the activity of a basic isoenzyme. Cycloheximide treatment decreased total and isoenzyme activity. Lowering the endogenous ethylene level did not reduce total activity, although the activity of a basic isoenzyme was decreased. PHYSIOLOGY OF CHERRY FRUIT ABSCISSION By Vernon Arie Wittenbach A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 197h ACKNOWLEDGMENTS The author would like to express his appreciation to Dr. M. J. Bukovac for his counsel and guidance during the course of this study and in preparing the manuscripts. Apprec- iation is also extended to the members of my Guidance Committee: Drs. F. G. Dennis, D. R. Dilley, P. Markakis, and M. J. Zabik. I am also grateful to Dr. Dilley for allowing me the use of his laboratory equipment. A very special thanks is extended to my wife Pam for her love, encouragement, and assistance during this program of study. ii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . GENERAL INTRODUCTION . . . . . . . . . . SECTION I: EVIDENCE FOR A.TWO-PHASE PROCESS AND THE INVOLVEMENT OF ETHYLENE INTRODUCTION . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . RESIJLTS . O O O O O O O O O C O O O DISCIJSSION . O O O O O O O O O O O O O LITERAME CITED 0 O 0 O O O O O O O O 0 SECTION II: A ROLE FOR ETHYLENE IN MECHANICALLY-INDUCED ABSCISSION OF IMMATURE FRUITS I MRODUCT ION O O I O O O O O O O O O 0 MATERIALS AND METHODS . . . . . . . . . RESULTS I O O O O O O O O O O O O O O DIS CUSS ION C O O O O O O O O O O O 0 0 LITERATURE CITED. . . . . . . . . . . . SECTION III: PEROXIDASE ACTIVITY IN THE ABSCISSION IN RELATION TO SEPARATION INTRODUCTION . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . RESULTS. . . . . . . . . . . . . DISCUSSION. . . . . . . . . . . . LITERATURE CITED. . Page iv vi 18 23 27 28 33 52 56 59 6O 63 78 83 #v— -.. Table LIST OF TABLES Page Section I Abscission Potential of Fruit Explants . . . . . . . 9 Effect of Ethephon and Cycloheximide on Fruit Removal Force of Explants Prepared from Fruit at Different Stages of Development. . . . . . . . . . . . . 10 Effect of Cycloheximide on Ethylene Production by Mint Explants O O I O O O O O O O O O O O 0 11 Effect of Exogenous and Endogenous Ethylene on Abscission of Explants Prepared from Fruits at Different Stages of mve loPme nt 0 O O O O O O O O I O O O O O O 1.2 Influence of Ethephon and Cycloheximide on Fruit Enlargement and Abscission . . . . . . . . . . . l3 Ethylene Concentration in the Internal Atmosphere of Cherry Fruit at Different Stages of Development . . . 17 Section II Effect of the tissue injured on ethylene evolution and fruit abscission early in Stage II . . . . . . . . 38 Effect of applied and injuryainduced ethylene on fruit removal force (FRF) at the peduncle:pedicel zone and on seed abortion late in Stage I . . . . . . 50 Effect of ethylene pretreatment on the subsequent evolution of ethylene by detached sour cherry fruit at various stages of development . . . . . . . . . 51 Section III Peroxidase activity in the abscission zone and associated tissues as related to stage of fruit develop- mnt o a o o o a a a o o o o o a a o o o 67 iv Table Page Peroxidase activity in the abscission zone as related to fruit removal force (FRF) and the stage of fruit development . . . . . . . . . . . 68 Effect of cycloheximide (CH) and ethephon on peroxidase activity and fruit removal force (FRF) in the abscission zone of fruit explants. . . . . . . . . 69 Effect of ethylene and reduced pressure on peroxidase activity and fruit removal force (FRF) in the abscission zone of fruit explants. . . . . . . . . 71 Figure LIST OF FIGURES Section I Fruit removal force (FRF) at the pedicelzfruit abscission zone in relation to stage of fruit development . . . . . . . . . . . . Ethylene evolution from sour cherry fruits during development. . . . . . . . . . . . Section II Diagram of the sour cherry fruit in Stage II showing four positions of drilling to induce injury of the developing fruit and seed. . . . . Fruit removal force (FRF) at the peduncle:pedicel abscission zone in relation to stage of fruit de ve lopme nt 0 0 O O O O O O O O O O O O Photomicrographs of longitudinal sections through the fruit illustrating development of the seed at those times when injury treatments were made . . Photomicrographs showing the appearance of the seed h days after drilling into the seed through the micropylar or chalazal end . . . . . . . . . Ethylene evolution (I) and fruit removal force (FRF) at the pedunclezpedicel (II) and pedicelzfruit (III) abscission zones of injured sour cherry fruit .. a. Ethylene evolution (I) and fruit removal force (FRF) at the peduncle:pedicel (II) and pedicel:fruit (III) abscission zones of immature sour cherry fruit as a result of ethephon treatment. . . . . . Photomicrographs depicting ethephon-induced seed abort ion 0 O O O O O O O O O 0 O O O 0 vi Page 15 30 3h 36 ho N2 MS h? Figure Page Section III 1. Localization of peroxidase in the abscission layer and associated tissues . . . . . . . . . . . . 6h 2. Zymograms of acidic and basic peroxidase isoenzymes extracted from the pedicel:fruit abscission zone at various stages of fruit development . . . . . . . 72 3. Zymograms of acidic peroxidase isoenzymes extracted from the pedicel°fruit abscission zone of fruit explants treate with 10' M cycloheximide, distilled water, or 10' M ethephon. . . . . . . . . . . . . . 7h h. Zymograms of acidic and basic peroxidase isoenzymes extracted from the pedicelzfruit abscission zone of fruit explants subjected to oxygen at 0.2 atm, air at 1 atm, or 10 ,n/I ethylene at 1 atm pressure. . . 76 5. Degradation of IAA by isoenzymes of peroxidase extracted from the pedicel:fruit abscission zone of sour cherry fruit . . . . . . . . . . . 79 vii Guidance Committee: The Paper-Format was adopted for this dissertation in accordance with departmental and university regulations. The dissertation body was separated into three sections. The first section was prepared for publication in Plant Physiology. The second and third sections were styled for publication in the Journal of the American Society for Hert- icultural Science. viii GENERAL INTRODUCTION The process of fruit abscission has not been extensively studied. Auxin sprays promote abscission of immature fruit and delay abscission of mature fruit, while ethylene and ethylene producing chemicals promote abscission of both immature and mature fruit. Work in this laboratory has been concerned with sour cherry fruit abscission. Immature and mature fruit abscission can be studied independently in the cherry, as separation of immature fruit occurs at the peduncle:pedicel abscission zone, while mature fruit abscise at the pedicel:fruit zone. Special emphasis has been directed toward the process of abscission in mature fruit, since control of this process would provide a basis for improved hand and machine harvest as well as programed harvesting. Maturation of sour cherry fruit is accompanied by a decline in break-force at the pedicel:fruit zone. This decline has been associated histochemically with the loss of pectic substances and polysaccharides and a change in cellulose orientation in the ‘walls of cells of the abscission layer. These results suggest the involvement of celldwall degrading enzymes in cherry fruit abscission. In addition, enzymes not involved in cell wall degradation have also been localized in the abscission layer. Furthermore, the rate of abscission can be altered by exogenous application of chemicals. Cycloheximide will inhibit abscission, ix 'while ethephon, an ethylene releasing chemical, will promote separation. These studies have provided us with the means to investigate the process of cherry fruit abscission and to answer some of the physiological questions relating to abscission of immature and mature fruit. The present investigation was undertaken with three main objectives: 1) to examine the influence of ethylene on abscission of mature fruit, 2) to establish the role of the seed and ethylene in mechanically induced abscission of immature fruit, and 3) to study the relationship between peroxidase and mature fruit abscission. SECTION I EVIDENCE FOR A TWO-PHASE PROCESS AND THE INVOLVEMENT OF ETHYLENE INTRODUCTION Auxin and ethylene have both been implicated as natural regu- lators of leaf abscission (5, 20). Early workers in this field suggested that abscission might be controlled by a balance between these two hormones (15, 17). Evidence favoring such a balance 'was provided indirectly by the separation of the leaf abscission process into two distinct phases based on sensitivity to auxins and ethylene (l, 28), i.e., an induction phase during which auxin delayed abscission and ethylene had no effect, and a later phase when both auxin and ethylene stimulated abscission. Recently, the length of phase I in bean leaf explants has been altered by supplying or removing ethylene (3, 21). The latter study (21) demonstrated the importance of endogenous ethylene, since removing ethylene from the system significantly delayed abscission. The process of fruit abscission has not been extensively studied. Applications of ethylene and ethephon, an ethylene releasing chemical, will enhance fruit abscission in a wide range of plant species (10, ll, 13, 1h, 16, 18, 19, 23). Moreover, ethylene treatment increases the level of cell wall degrading enzymes in fruit abscission zones (19, 21, 26, 27) and regulates enzyme secretion into the cell wall (26). Endogenous ethylene also appears to have an important role, since reducing the in- ternal level markedly delays abscission in young cotton fruit (23). Earlier, we presented evidence which suggested the presence 2 3 of two phases in cherry fruit abscission (33). We not describe a series of experiments designed to more critically define these phases in relation to fruit development, to determine the effects of both exogenous and endogenous ethylene on abscission, and to further elucidate the mechanism involved in abscission. MATERIALS AND METHODS General Methods. Sour cherry fruit (Prgggg cerasus L., cv. Montmorency) in various stages of development, were collected as needed just prior to initiating an experiment. Separation was followed at the abscission zone between the pedicel and fruit, the point of natural separation at maturity. Procedures used to quantitatively determine the fruit removal force (FRF) at this zone and in conducting the fruit explant bioaasay have been pre- viously described (11, 33). Change in Abscission Potential with Development. Changes in PR? were determined on a unifbrm sample of 20 fruit at weekly intervals from the end of Stage I of fruit growth through maturity. Abscission potential, the capacity of explants to exhibit abscis- sion, was determined in explants prepared from comparable fruit at the same time. FRF was measured after 80 hr with explants held in distilled water at 232 2 c (33). Stage of fruit development ‘was monitored by obtaining fresh weight measurements twice weekly utilizing a representative sample of 20 fruits. Effect of Exogenous Ethylene. Ethephon [2-(chloroethyl)- phosphonic acid (Amchem Products, Inc)] and ethylene gas were used to determine the effect of exogenous ethylene on the two h phases of abscission. Ethephon was supplied in the explant treating solution at 10'3 M. The effects of cycloheximide (lO’h M) on ethylene evolution and abscission of explants prepared from fruits in different stages of development was also established. Control explants were held in distilled water. Explants were treated with 10 Pl/l ethylene gas in 10 liter desiccators. A flow-through system was employed in which ethylene was premixed with compressed air and allowed to bleed into the desiccators through a vacuum regulator (Matheson model 19) at a rate of 8.5 liters/hr controlled by a needle valve to the vacuum line. Control explants were held under the same conditions in ethylene-free air. To make certain that the influence of cycloheximide and ethylene on abscission was not simply an indirect effect due to altered fruit enlargement, freshyweight of the fruit was recorded before and after treatment. Effect of Endogenous Ethylene. If ethylene is involved in abscission, then reducing the endogenous level early in the separation phase should delay abscission. Endogenous levels of ethylene were reduced to one-fifth by subjecting explants in desiccators to 0.2 atm pressure. The same flow-through system was used as described earlier except pure oxygen was allowed to bleed into the desiccators, thereby maintaining its partial pressure at atmospheric level. As a control for the reduced pressure, an additional treatment containing 50 Pl/l ethylene in pure oxygen was supplied to explants at 0.2 atm and compared to 10 Pl/l ethylene in compressed air at one atm. 5 Ethylene Evolution and Internal Concentrations. Ethylene evolution was determined twice a week from full bloom until after maturity. Fruits were detached from the tree and the pedicel was cut 3 to h mm above the fruit. Ten fruits were sealed in 25 ml flasks or 10 to 30 fruits in 265 ml glass containers, depending on the stage of fruit development. A filter paper wick saturated with 10% KOH was sealed in each container to absorb C02. Two to h replications were used. Sealed containers, including appropriate controls (lacking only fruit) were held in a water bath at 25: l C for 8 hr. CO2 and 0 levels were 2 monitored using a Perkin-Elmer vapor Fractometer, Model l5hB, and 02 was supplied to maintain the atmosphere at 211 3%. Ethylene evolution was determined by assaying 1.0 ml of the gas phase by gas chromatography (29). The concentration of endogenous ethylene was determined using a medification of the procedure of Beyer and Morgan (8). Twenty five to 50 fruit were subjected to a vacuum of 200 mm for 30 sec. The extracted gas was then assayed for ethylene by gas chromatography. RESULTS Change in Abscission Potential with Development. FRF in- creased early in fruit development (Figure 1) indicating a strengthening of the abscission zone tissue. Then at the begin- ning of Stage III a perceptible decline in FRF began, which increased in rate and reached its lowest value at fruit maturity. The abscission potential of explants prepared from fruits Figure l.--Fruit removal force (FRF) at the pedicel:fruit abscission zone in relation to stage of fruit development. FRF and fresh weight measurements were made on a sample of 20 fruit. -o-( 5) 39303 WAowau lines CON 00¢ 00m 00m 000. CON. 00¢. 00m. 00m. r I fil n. - v.20 0275533 3:... h n mm mm _N t mass m. m n q u n q q u u u u 1 mm 1 ans. em ow o. q N. nonononono eennns44cc‘ (5)1H9I3M Hsasa + o. no 8 in different stages of development is shown in Table 1. Abscis- sion potential becomes apparent for the first time in explants harvested June 26, i.e., the beginning of Stage II of fruit growth, which corresponds to the first evidence of a decrease in FRF in attached fruits (Figure 1). Influence of Exogenous Ethylene. Ethephon did not promote abscission in explants prepared from fruits in mid-Stage II of development (Table 2). However, it markedly enhanced abscission in explants from fruits collected June 18 (late Stage II)-- one week before control (non-ethylene treated)exp1ants demonp strated a potential to abscise--and in explants from fruits in Stage III of development. Cycloheximide inhibited abscission in explants obtained from fruit in late Stage III of development; however, it significantly reduced the FRF of explants from fruits (late Stage II) harvested June 18 (Table 2). Cycloheximide caused a rapid and marked enhancement of ethylene production in explants from fruits har- vested June 22 (Table 3). Explants prepared at different stages of fruit development responded similarly to both ethylene gas at 10 Hl/l and to ethephon (compare Table 2 and Table A). Because of the close correlation between fruit enlargement in Stage III and the onset of the decline in FRF, the effects of ethephon and cycloheximide on these two processes were assessed at an early stage of fruit development. There was no significant effect of either ethephon or cycloheximide on fresh weight; however, both significantly reduced the FRF (Table 5). Similar TABLE 1.--Absciasion Potential of Fruit Explants. Explants were prepared at weekly intervals from the end of Stage I of fruit growth to maturity. The explants were posi- tioned with the cut end of the pedicel in distilled water and held in the dark at 23t 2 C. At the end of 80 hr the fruit re- moval force (FRF) was recorded and used as an index of the abscis- sion potential. Each treatment included 20 single fruit replications. FRF Date explants Initial (8) After 80 hr (g) “L of Initial prepared May 27 1167 a1 1269 a 108.7 June h 1&85 a lh28 a 96.2 June 11 1530 a 1509 a 98.6 June 18 1550 a 15h2 a 99.5 June 25 1512 a 1260 b 83.3 July 2 998 a 1:08 b l+0.9 July 8 398 a 321 b 80.7 1 Mean separation (in rows) by Tukey's to test, P - 0.01. 10 TABLE 2.--Effect of;Ethephon and Cycloheximide on Fruit Removal Force of Explants Prepared from Fruit at Different Stages of Development. Sour cherry fruit explants were prepared at weekly intervals from mid-Stage II to mid-Stage III of fruit growth. Explants were positioned with the cut end of the pedicel in test tubes fiontaining distilled water, ethephon (10"3 M), or cycloheximide (10' M) and held in the dark at 233 2 C. After 80 hr the fruit removal force (FRF) was recorded and used as a measure of abscission. Each treatment included 20 single fruit replications. FRF (a) Date explants Control Ethephon Cycloheximide _prepared June 11 1502 a1 lh52 a 1hs6 a June 18 151+5 a 936 c lid-+0 b June 25 1281 a 33h b 1262 a July 2 h08 b 218 c 771 a 1Mean separation (in rows) by Tukey's to test, P = 0.05. 11 TABLE 3.--Effect of Cycloheximide on Ethylene Production 13! Fruit Explants. Explants prepared from fruits harvested June 22 were positioned with the cut end of the pedicel in small beakers of distilled water or cycloheximide (10" M) and sealed in 26h ml glass containers. The gas phase was assayed after 1 and ’4 hr for ethylene by gas chromatography. Ethylene (pl kg"1 hr'l) Time of sampling Control Cycloheximide 1 hr 0.021 h1 1.179 a h hr 0.016 b 11.099 a lMean separation (in rows) by Tukey's cutest, P z 0.01. 12 TABLE h.--Effect of Exogenous and Endogenous Ethylene on Abscission of Explants Prepared from Fruits at Different Stages of Development. Fruit explants were subjected to the indicated treatments in desiccators using a flow-through system. Explants were positioned ‘with the cut end of the pedicel in distilled water in the dark at 23: 2 C. Fruit removal force (FRF) was measured after 72 hr. FBI“ (8) Date Air + 10 ,41/1 02 + 50 ,41/1 explants Air, at C Hh, at 02, at CQHh, at prepared 760 mm 760 mm 150 mm 150 mm May 27 1292 a1 1118 b 1395 a 1185 ab June h lh33 b 1373 b 1537 a 1396 b June 11 1531 a 1358 b 151,4 a 1386 b June 18 15% a 1291 b 15h8 a 1302 b June 25 1278 b 383 c 138A a 362 c July 2 A92 b 320 c 673 a 302 c 1 Mean separation (in rows) by Tukey's to test, P=0‘.01. TABLE 5.--Inf1uence of Ethephon and Cycloheximide on Fruit Enlargement and Abscission. Explants were prepared June 18 and positioned with the cut end of the pe icel in test tubes contaifling distilled water, ethephon (10" M) or cycloheximide (10' M) and held in the dark at 231 2 c. After 80 hr the fresh weight and fruit removal force (FRF) were recorded. Measurement Control Ethephon Cycloheximide Fresh wt (g) 0.90 a1 0.89 a 0.86 a FRF (a) 15145 a 936 c 1th b lMean separation (in rows) by Tukey's a) test, P = 0.05. (114 observations have been made with ethephon on intact fruit in the field and with ethylene on fruit explants in the laboratory (unpublished data). Influence of Endogenous Ethylene. Reducing the endogenous level of ethylene to one-fifth by reduced pressure significantly inhibited abscission of explants prepared from fruits in Stage III of development, June 25 and July 2 (Table h). Adding ethylene (50 1.41/1) at 0.2 atm to provide a level equivalent to 10 Pl/l ethylene at one atm (column 3 vs 5, Table it) resulted in an identical response and confirmed that reduced pressure had no significant effect on abscission other than indirectly by re- dicing the endogenous ethylene level. Ethylene EVolution and Concentrations in the Internal Atmosphere. The rate of ethylene evolution with cherry fruit development (Figure 2) was similar whether determined on a per fruit (n1 fruit'l hr'l) or fresh weight (”1 kg'1 hr'l) basis. There was almost a steady decline in evolution with fruit de- velopment. The level of ethylene decreased rapidly during Stage I, except for an increase just prior to pit hardening. The level then declined during the remainder of Stage I and remained relatively constant during most of Stage II. Just before the start of Stage III another increase was observed. Thereafter, the level declined and remained nearly constant until near maturity when another small peak was observed. Ethylene concentrations in the internal atmosphere of cherry fruit were also low for the period monitored (from mid-Stage II to 1 week prior to maturity--Table 6). The concentration. was 15 Figure 2.-~Ethylene evolution from sour cherry fruits dur- ing development. Fruits were sealed in contain- ers with KOH (lO%) wicks to absorb C02. Ethylene evolution was determined by assaying 1.0 ml of the gas phase by gas chromatography. These values were obtained for a different season than the rest of the studies, and therefore, the dates cannot be compared. However, the values are related to stage of fruit development, which should be com- parable between years. l6 -°-(.-m.-6n m- Monmoaa ”H 30 oo _. cow con. 00¢. l‘ I ‘V Good 0006 l .llli 2:202 335: wh22 0. __ h n mm nm .N t n. m n _ mm em ON 0. N. W}. . . - . . . q . . . q . . All HH 32m IV All H 32m - b )- 35 2.2. 1 1 1 1 1 1 1 1 1 o. ‘0. 0. '0. o. '0. 0. '0. 0. <1- ro 10 N N — — o o 4 D. ¢ + (5) 111913111 Hsaad 36 Figure 3.--Photomicrographs of longitudinal sections through the fruit illustrating development of the seed at those times when injury treatments were made. A, late Stage I; B, early Stage II; C, mid-Stage II: D, late Stage II; E, early Stage III. 37 38 TABLE l.--Effect of the tissue injured on ethylene evolution and fruit abscission early in Stage II. Tissue injured 0211,, (,.1 kg'1 hr'l) it Abscission Control (non injured) 0.211 b2 h b Mesocarp 0.116 b 5 b Mesocarp + endocarp 0.36 b 11 b Seed (micropylar end) 10.33 a 88 a zMean separation (in columns) by Tukey's 00 test, P = 0.01. 39 injured. Fruit abscission was induced only following injury to the seed, the same treatment that caused a marked increase in ethylene evolution (Table 1). V Iiury of the seed at various stages of development. Dam- aging the seed at either the micropylar or chalazal end during Stage I resulted in degradation of the nucellar tissue and shrivel- ing of the integuments (Figure 11, A and D). In addition, ethylene evolution shortly after injury was markedly increased in comparison with uninjured control fruit (Figure 5-I), and the FRF at the pedunclezpedicel zone was greatly reduced after 10 days. All injured fruits abscised (Figure 5-III) within 2 wk. Similar results were obtained for the early and mid-Stage II treatments (Figure 5, I and II, B and C). However, neither the endosperm tissue nor embryo appeared to undergo degradation like the nucellar tissue (Figure 11, B and E). The endosperm only appeared to shrivel later, while the embryo showed only the scar of injury from the micropylar end and retarded growth corresponding to the time of treatment. Furthermore, the rate of shriveling of the integuments appeared to be dependent on the degradation of the underlying tissue, i.e. whether it was the endosperm or nucellus. In late Stage II of development, injury-induced ethylene evolution was greatly reduced (Figure 5-I, D). Correlated with this low rate of ethylene evolution was a slight decrease in FRF at the peduncle:pedicel zone, and the number of fruits which abscised due to injury was considerably less than when injury was induced early in Stage II (Figure 5-II). With the onset of 110 Figure 11.--Photomicrographs showing the appearance of the seed h days after drilling into the seed through the micropylar (A-C) or chalazal (D-F) end. A and D, late Stage I; B and E, mid-Stage II; C and F, early Stage III. hl 1‘1 h2 Figure 5.--Ethylene evolution (1) and fruit removal force (FRF) at the peduncle:pedicel (II) and pedicel:fruit (III) abscission zones of immature sour cherry fruit. Ethylene determinations were made during the first h hr and FRF 10 days after treatment. Control, no injury; micropyle, injury to the seed at the micropylar end; chalaza, injury to the seed at the chalazal end. Insert (I) indicates time of treatment in relation to fruit development (A, May 29; B, June 5: C, June 12; D, June 17; E, June 22). Vertical brackets indicate standard deviations. +, injured fruits abscised; 1, approximately one-half of the injured fruits abscised. 1&3 Bl. Al we. mam mmt sol... mum C1 CMC BBQ .71 e o e e e 00,. o e e e.o.o.e.e.a-e.o.e.. e. 304040430?wewowewewemowevweweweeoe. ”villi/1.. 10000000... waeeeeeuoeeueueueueuoueneneneuen. 301010.010. 0000000 000 0 0 0 0 0 0 0 0 0 0 . .l A, l . h _ p p b a 6 4 2 O 8 .7... 79. .1 a 295.6%. erao 1000 .. n ® FRUIT ABSCISED 87 3. more... .3529. tam... - J 300- .P_ mmm 654 p— 00 mm ' III f I600“ I400- IZOO- IOOO - _ O O 8 m 400 r- L O 200 F .3 moron 252%. tax... TIME of TREATMENT 111: Stage III of fruit growth, damage to the seed resulted in no appreciable ethylene evolution (Figure 5-I, E). Moreover, the fruit did not abscise (Figure 5-II) but persisted with no visible effects other than the scar left by the drill (Figure h, C and F). Injury to the seed at the end of Stage II or start of Stage III resulted in no measurable acceleration of maturity. Although the abscising fruit separated at the peduncle: pedicel zone, there was a significant reduction in FRF at the pedicel:fruit zone in response to seed injury (Figure 5-III). Furthermore, the magnitude of reduction in FRF increased with subsequent treatments until late in Stage II (Figure 5-III, D) ‘when the injury-induced ethylene was greatly reduced. Thus in the last two treatments (Figure 5-III, D and E) the FRF at the pedicel:fruit zone of the injured fruit was only slightly lower than that of the control fruit. Influence of exogenous ethylene. Ethephon caused a marked increase in the rate of ethylene evolution shortly after treatment at all stages (Figure 6-I). Fruit treated‘with either concn at the end of Stage I abscised at the peduncle:pedice1 zone (Figure 6-II, A). Furthermore, at this early data both concn caused seed abortion (Figure 7A). However, only the higher concn induced seed abortion and abscission during early and mid-Stage II (Figure 6-II and 7B), even though both concn caused a significant increase in ethylene evolution (Figure 6-I). By the onset of Stage III, neither concn induced seed abortion or abscission (Figure 6-II), although both markedly enhanced ethylene evolution (Figure 6-I). Ethephon in late Stage II slightly accelerated 85 Figure 6.--Ethy1ene evolution (I) and fruit removal force (FRF) at the peduncle: pedicel (II) and pedicel:fruit (III) abscission zones of immature sour cherry fruit. Ethylene determinations were made during the first h hr and FRF 10 days after treatment. Control (0 ppm) and ethephon (500 and 1000 ppm) treated fruits. See insert Figure 5-I for time of treatment. Vertical brackets indicate standard deviations. +, injured fruits abscised; 3, approx- imately one-half of the injured fruits abscised. 000000.0‘000000000 1000000000} H0M000 v 0 0 0 0 0 0 0 0 0 0000000000 0 0 0 0000000000W0Mo g comma F. . ..._..._.. .3.3.3.“.u.u.u.n.u.u.u.u.n.u.n. ”cue.wenw"wneueweheueneweneneneuewenenenmnews. Q0“‘“.“““‘...““.“““ snowencwewewowewewewewe.ewewewewewewewewe.ewewewewewewewem ..0..I..........0...0.... . 0....0..00I00.000.000....0.........00 0.00.0...00 w>000000000000000000 va010.000.000.00000000000... .00.. O, E 500 ppm E53 1000 ppm D 09911: 5m. .u.u.u.u.n.n.u.u.u.u.mum.”.”.n.u.u.u.u.n.u.n.n.n.... go... new: n.”.n.u.u.n.n.n.u.n.u.n.n.u.u.u.n.n.n.u.n.u.n.u.u.u.”.n.n.u.u.u.u.n.u.u.n.u.303." g; .’.’.’.’ . . . . ‘ ‘ ‘ ‘s ................ ‘ ‘. ‘ ‘ ‘ ‘ ‘ ‘ ‘ ‘ ' ‘ ‘ . ‘ ‘ ‘ ‘ ‘ ‘ . ‘ ‘ ‘s a!!!" noueneueeobevenouens swans».wowewewowewowowoweuemowewew newewewew new.» V4. wewowewewewewewewewewewewewowewewewewewew04s C 2.5 .u.u."meanness.".3.HAAAAAAAAAA. .u.u.u.u.n.u.u."A.n.u.u.u.u.n.u.n.u.n.u.u. 010 0 0 0 0 ”000000000. ”Huh“ wwmwwmwwwu.wwwwwwmwwmwwe.tween-M...w. New.when.nowouomouwmouowomoucmowomemenoneuomou 0. TIME of TREATMENT ® FRUIT ABSCISED I.‘0..........O...‘........ .‘0000...0........0...I.. I...0......0.........0.... ”an.mmmwmwmwmww.wmwmwwwwwwwwwwwwwuwu y. _ u 0 O O 6 . _ O O O O 4 2 h - p m 0 0 O O 5 4 8 600 #- IGOO ~ I400 - l200 - I000 .72 79. .1 a 29.-30>“. £8 13 more... 232% tags .3 moron 25:3 :2: In Figure 7.--Photomicrographs depicting ethephon-induced seed abortion. A, six days after treatment with 500 ppm in late Stage I; B, eight days after treatment with 1000 ppm in early Stag II. 1&9 fruit coloring. Ethephon also caused a reduction in FRF at the pedicel:fruit zone (Figure 6-III). The lower concn was effective during Stages I and III, but only the higher concn caused a significant re- duction in FRF during Stage II. Treatment of detached fruiting branches with 10 ,11/1 ethylene caused a significant reduction in FRF at the upper zone after 80 hr (Table 2). However, no apparent damage to the seed was observed at the end of the experiment other than a slight brown discoloration at the chalazal end of the nucellus. Ethylene applied to the fruits during mid-Stage II induced an enhanced rate of ethylene evolution (Table 3). But, if ethylene ‘was applied later in development no increase in ethylene evo- lution was observed. Influence of injury-induced ethylene. Injuring the seeds of fruits on detached, defoliated branches at the micropylar end late in Stage I failed to cause a reduction in FRF after 80 hr (Table 2). Similar results were obtained with a second experiment run for 200 hr (unpublished data). Ethylene at either lolnl/l or 50 )41/1 enhanced abscission. Reducing the level of injury- induced ethylene to one-fifth had no significant effect on abscis- sion (Table 2). Injury to the seeds did, however, result in seed abortion. 50 TABLE 2.--Effect of applied and injury-induced ethylene on fruit removal force (FRF) at the pedunclezpedicel zone and on seed abortion late in Stage I. Treatmentz FRF (g) Seed abortion Air, at 760 mm 768 ay _x 10 pl/l cam, in air, at 760 mm 358 b s 50 ,41/1 0211;, in 02, at 150 mm 372 b * Injured seed in air, at 760 mm 73% a + Injured seed in 02, at 150 mm 717 a + zBranches with treated fruit were held in desiccators at the indicated pressure and gas phase conditions for 80 hr. yMean separation by Tukey's “test, P = 0.01. xSeed healthy (-), seed aborted (+), seed healthy except for a small brown discoloration at chalazal end of nucellus (*). 51 TABLE 3.--Effect of ethylene pretreatment on the subsequent evolution of ethylene by detached sour cherry fruit at various stages of development. Pretreatmentz Date Stage of Air 10 nl/l 021-11., development ‘fll kg’1 hr'”1 June 9 Mid-Stage II 0.15 by 1.83 a June 18 Late Stage II 0.11 a 0.10 a July 3 Mid-Stage III 0.0h a 0.05 a 212 hr pretreatment followed by 2 hr in ethylene-free air prior to enclosure and sampling. yMean separation (in rows) by Tukey's uitest, P = 0.01. 52 DISCUSSION A correlation appears to exist between ethylene evolution and abscission. Injury to the ovary wall tissue (mesocarp and endocarp) did not significantly increase ethylene evolution or fruit abscission (Table 1). However, damage to the seed markedly enhanced the rate of ethylene evolution and resulted in abscission of immature fruit. Fruit in Stage I to mid-Stage II of growth showed a marked increase in ethylene production and abscised as a result of injury to the seed, regardless of the site of injury (Figure 5, I and II). Damaging the seed in Stage III, however, failed to induce ethylene evolution or abscission. Comparing these results on ethylene evolved.with the injury to the seed (Figure 5-I vs. Figure h), it would appear that the nucellus is the primary tissue responsible for the high rate of ethylene evol- ved. The integuments are probably not important since little or no ethylene was evolved (Figure 5-I) when the seed was damaged in Stage III (Figure h). The endosperm and embryo apparently are not involved, since neither begins to enlarge until the onset of Stage II and yet a high rate of ethylene evolution was ob- served in Stage I. Furthermore, injury to the fully-enlarged embryo did not significantly increase ethylene evolution. Ethephon caused immature fruits to abscise (Figure 6-II). Ethephon at 500 ppm was effective during Stage I, while 1000 ppm 'was effective during most of Stage II. Moreover, there appeared to be a correlation between ethephon induced seed abortion and fruit abscission. 53 On the other hand, fruit abscission was induced on isolated branches at the peduncle:pedicel zone in late Stage I with ethylene without evidence of seed abortion (Table 2). This suggests that ethylene can induce abscission at this zone provided I it can be supplied to the zone from the fruit or adjacent tissue at an effective level. Perhaps ethylene produced in the nucellus during seed abortion can directly influence abscission of immature fruit, since the vascular system may provide a direct path of low diffusion resistance from the seed to the peduncle:pedicel zone. However, it is also possible that ethylene may influence abscission indirectly by reducing the trasnport of auxin (l) or some other juvenility factor to the zone. The ability of ethylene treatment to induce ethylene pro- duction in fruit during Stage II but not later in development (Table 3), suggests that the ethylene produced is derived from the nucellus. Since little or no injury of the seed‘was observed following similar treatments (Table 2), the nucellus may produce ethylene autocatalytically rather than simply releasing it upon injury. Therefore, any treatment which will trigger this mechan- ism, i.e. mechanical, chemical or environmental injury of the seed, may cause a high rate of ethylene production. The reason for the failure of fruits on detached branches to abscise in response to seed injury is unclear (Table 2). The results suggest, however, that injury-induced ethylene is not the sole factor controlling abscission at the peduncle:pedicel zone. Crane and Nelson (5) showed that apricots with maleic hydrazide-induced aborted seeds persisted as healthy fruit so 5h long as competition between their growth and vegetative growth was reduced to a minimum. Removal of the leaves from the cherry branches, rather than having the intended effect of enhancing the rate of abscission, may have reduced competition with the fruit. Crane (h) suggested that growth hormones present in the seed function through the mobilization of substrates to them. Several studies have shown that auxins, gibberellins, and cytokinins are capable of causing the movement of inorganic and organic compounds, as well as other growth hormones to the site of application (8, 9, 13). Moreover, the levels of these hormones are known to be high in the sour cherry seed during the early stages of develop- ment1 (12). 0n.the other hand, ethylene has been shown to have a possible role in auxin destruction (ll). Possibly, the ethylene production induced by injury of the seed prior to the completion of growth of the embryo may result in the destruction of the metabolic gradient established by the hormones of the seed. Consequently, the transport of hormones through the peduncle: pedicel zone toward the fruit would be cut off and abscission induced. Ethylene appears to be involved in the abscission of immature fruit. However, as was shown for mature fruit (21), abscission is apparently controlled by an interaction of factors rather than ethylene alone. The absence of abscission at the peduncle:pedicel zone at maturity (20) may be due to either the presence of juvenil- ily factors or the absence of ethylene at a sufficient level 1Hopping, M. E. Isolation, characterization and role of endogenous auxins and cytokinins in sour cherry (Prunus cerasus L., cv. Montmorency) fruit development. Ph.D. Thesis, Miéhigan State university; 207 P. 1972. 55 to induce separation. The latter explanation would appear more reasonable, since late in the season the quantity of juvenility hormones is generally at a low level. Finally, the mechanism of abscission at both zones appears to be similar. Treatments which markedly enhanced ethylene evolution and abscission at the peduncle:pedice1 zone, also caused a reduction in FRF at the pedicel:fruit zone (Figures 5 and 6). However, because the FRF was initially much higher at the pedicel:fruit zone the reduction in FRF in response to the treatments, although nearly as great, did not result in separation at this zone. 10. LITERATURE CITED Beyer, E. M., Jr. 1973. Abscission: support for a role of ethylene modification of auxin transport. Plant Physio . 52: 1-5. Buchanan, D. W., R. H. Biggs, J.A. Blake, and‘W. B. Sherman. 1970. Peach thinning with 3CPA and Ethrel during cytokinesis. J. Amer. Soc. Hert. Sci. 95: 78l-78h. Bukovac, M. J ., F. Zucconi, V. A. Wittenbach, J. A. Flore, and H. Inoue. 1971. Effects of (2-chloroethyl)phosphonic acid on development and abscission of maturing sweet cherry (Prunus avium L.) fruit. J. Amer. Soc. Hert. Sci. 96: 777-78I. Crane, J. C. 1969. The role of hormones in fruit set and development. HortScience A: 108-111. Crane, J. C., and M. M. Nelson. 1970. Apricot fruit growth and abscission as affected by maleic hydrazide-induced seed abortion. J. Amer. Soc. Hort. Sci. 95: 302-306. Daniell, J. W., and R. E. Wilkinson. 1972. Effect of ethephon- induced ethylene on abscission of leaves and fruits of peaChese J0 Amer. SOCe Hort. SCie 97: 682-6850 Dennis, F. G. 1970. Effects of gibberellins and naphthalene- acetic acid on fruit development in seedless apple clones. J. Amer. Soc. Hort. Sci. 95: 125-128. Hatch, A. H., and 32 E. Powell. 1971. Hormone-directed transport of P in Malus s lvestris seedlings. J. Amer. Soc. Hort. Sci. 96: 230-23 . , and . 1971. Hormone-directed transport of certain organic compounds in Malus sylvestris seedlings. J. Amer. Soc. Hert. Sci. 96£_399:h00. Luckwill, L. C. 1953. Hormone production by developing apple seed in relation to fruit drop. J. Hort. Sci. 28: lh-2h. Morgan, P. E., and.J. L. Fowler. 1972. Ethylene: modification of peroxidase activity and isozyme complement in cotton. (Gossypium hirsutum L.). Plant Cell Physiol. 13: 727-736. 56 12. 13. 11+. 15. 16. 17. 18. 19. 21. 57 Naito, R., H. Inoue, and M. J. Bukovac. 1972. Endogenous plant growth substances in developing fruit of Prunus cerasus L. Levels of extractable gibberellin-like substances in the seed. J. Amer. Soc. Hort. Sci. 97: 7118-753. Seth, A. K., and P. F. Wareing. 1967. Hormone-directed transport of metabolites and its possible role in plant senescence. J. Exp. Bot. 18: 65-77. Sfakiotakis, E. M. and D. R. Dilley. 1973. Induction of autocatalytic ethylene production in apple fruits by propylene in relation to maturity and oxygen. J. Amer. Soc. Hort. Sci. 98: 50h-508. Stembridge, G. E., and C. E. Gambrell, Jr. 1971. Thinning peaches with bloom and postbloom applications of 2-chloroethylphosph0nic acid. J. Amer. Soc. Hort. Sci. 96: 7-9. , and . 1972. Peach fruit abscission as influenced by applied gibberellin and seed development. J. Amer. Soc. Hort. Sci. 97: 708-711. Teubner, F. G., and A. E. Murneek. 1955. Embryo abortion as mechanism of "hormone" thinning of fruit. Mo. Agr. E522. Sta. Res. Bul. 590. Tukey, H. B. 1936. Development of cherry and peach fruits as affected by destruction of the embryo. Bot. Gas. 98: 1-2140 Tukey, H. B., and J. 0. Young. 1939. Histological study of the developing fruit of the sour cherry. Bot. Gaz. Wittenbach, V..A., and M. J. Bukovac. 1972. A morphological and histochemical study of (2-chloroethyl)phosphonic acid-enhanced abscission of sour and sweet cherry fruit. J. Amer. Soc. Hort. Sci. 97: 628-631. , and . 197A. Cherry fruit abscission: evidence for a two-phase process and the involvement of ethylene. Plant Physiol. (in press). SECTION III PEROXIDASE ACTIVITY IN THE ABSCISSION ZONE IN RELATION TO SEPARATION 58 INTRODUCTION Maturation of sour cherry fruit is accompanied by a decline in fruit removal froce (FRF) at the pedicel:fruit abscission zone (21). This decline in FRF has been associated with the loss of pectic substances and polysaccharides and a change in cellulose orientation in the walls of cells comprising the abscission layer (19). These observations suggest the action of cell-wall degrad- ing enzymes in sour cherry fruit abscission as has been demonstrated for other fruit species (5, 10, 1h, 16). Enzymes not involved in cell-wall degradation have also been found in the abscission zone of cherry fruit during separation (12). One of these, peroxidase, has been associated with the break down of 3-indoleacetic acid (IAA) (3), an auxin long implicated in the abscission process. Moreover, peroxidase activity has been linked with the abscission of cotton and bean leaves (9, 11) in which both peroxidase activity and leaf separation were enhanced by ethylene. We have recently demonstrated a role for ethylene in sour cherry fruit abscission (21). Also, IAA is known to be present in sour cherry fruits during the early stages of growthl. Thus, peroxidase may play a role in the abscission of cherry fruit by regulating the auxin level. This study was designed to establish if there was a relation- ship between peroxidase and cherry fruit abscission. Data are 1Hopping, M. E., Isolation, characterization and role of endogenous auxins and cytokinins in sour cherry (Prunus cerasus L., cv. Mont- morency) fruit development. Ph.D. Thesis, Michigan State University. 207 P. 1972. 59 60 presented on localization, activity, and changes in isoenzyme pat- terns of peroxidase in the abscission zone and adjacent tissues prior to and during separation. MATERIALS AND METHODS General methods. Sour cherry fruit (m cerasus L., cv. Montmorency) were collected at weekly intervals from late Stage I of fruit development through maturity. The samples were frozen immediately and held in dry ice until used. Fruit development was monitored by taking fresh weight measurements twice weekly utiliz- ing a representative sample of 20 fruit. Abscission at the pedicel: fruit zone was followed by measuring the FRF on a sample of 20 fruit at weekly intervalsz. Abscission bioassay. Fruit explants of the same physiological age were treated with ethephon (10"3 m), cycloheximide (104* m), ethylene (10,41/1), or reduced pressure (0.2 atm) to produce explants with different degrees of. abscission layer development. The preparation of explants and the procedures used in the bioassay have been previously described (20, 21). Explants were treated with ethephon and cycloheximide by placing the cut pedicels of the fruit into the test solutions. Etrqune was administered to ex- plants held in desiccators, and reduced pressure treatments were performed in desiccators as described earlier (21). Localization of peroxidase activity. Longitudinal sections (2h pm thick) were cut from the abscission zone with a cryostat. 2Vittenbach, V. A. , Morphological and physiological aspects of cherry fruit abscission with reference to 2-(chloroethyl)phosphonic acid. M.S. Thesis. Michigan State University. 11% P. 1970. 61 Peroxidase activity was determined with benzidine using the method of Veech (18). Benzidine in the presence of peroxide and peroxidase yields an unstable blue intermediate, which undergoes autoxidation to give a brown precipitate. However, in the Veech method the blue intermediate is stabilized with nickelovs ammonium sulfate. The sec- tions were photographed immediately after staining to record the localization. A second procedure using g-dianisidine (22) was employed to confirm the results obtained with benzidine. Controls consisted of heat-treated sections or substrate-deficient reaction media. Measurement of Eroxidase activity. Peroxidase activity was quantitated for the abscission zone and related tissue at various stages of fruit development. A h m cork borer was used to remove the abscission zone from the frozen fruit. The zone tissue was then excised from most of the receptacle and pericarp tissues. Because of the semispherical shape of the abscission layer, its small size (6-8 cells wide), and its location within the fruit, the excised zone contained in addition to the actual layer a relatively large amount (nearly two-thirds by volume) of fruit (mesocarp) tissue. Tissue from the abscission zone 60.1 g from 5 fruits), receptacle, pedicel, or mesocarp ("0.2 g) was mechanically ground in a glass homogenizer with h ml of extraction medium containing 0.1% Tween 80 (polyoxyethylene sorbitan monooleate) and 0.5 mM ethylenediaminetetraacetic acid in 0.1 M phosphate buffer, pH 6.0, with 0.25 g insoluble polyvinylpyrrolidine . The extract was centrifuged at 10,000 g for 15 min and the supernatant solution was 62 decanted. The precipitate was reextracted twice with 2.0 m1 of extraction medium as described above. The supernatant solutions were combined and centrifuged at 25,000 g for 30 min. The resulting supernatant solution was dialyzed overnight against deionized dis- tilled water. All of the above procedures were carried out at o to h° c. Peroxidase activity was determined spectrophotometricaJJy using g-dianisidine (17). Parallel determinations were made using guaiacol (2) to verify the results obtained with g-dianisidine. Enzyme activity was based on the oxidation of g-dianisidine at 3460 nm or guaiacol at #70 nm in the presence of H202 and expressed per mg protein, determined by the Lowry method (6). Controls included heat-inactivated enzyme extract or H202 deficient medium. Gel electrophoresis. The enzyme extract, prepared as described above, was Jyophilized, and the resulting powder was dissolved in 0.5 ml 0.02 M phosphate buffer, pH 7.0 An aliquot (0.12 ml) of the concentrated enzyme extract, after mixing with a 60% (w/v) sucrose solution (2:1), was applied to the gel Just prior to electrophoresis. The amount of protein applied per gel was main- tained between 80 and 100 ,ug. Separation of the acidic proteins was carried out according to the discontinuous gel electrophoretic procedure of Davis (1) using Cyanogum hl (7), while separation of the basic proteins was performed by the procedures of Reisfeld et al. (15). The gel columns (5 mm diam.) were composed of two sections, the upper 10 mm of a 5.0% («v/v) and the lower 65 mm of a 9.0% Cyanogum #1. The gels were run at ho C using 1.5 ma per tube for the first 30 min and 3 ma per tube for the remaining 63 time (approx 90 min for the acidic protein gels and 150 min for the basic protein gels). Peroxidase activity was detected in the acidic protein gels with benzidine (18) or guaiacol (2), while only guaiacol was used for the basic protein gels, due to an inter- action between the gel and benzidine. The gels were photographed to record the isoenzyme patterns. IAA oxidase assay. A relatively large quantity of enzyme extract (ISO/“g of protein) from the abscission zone was applied to the gels, which were run as described above. After electro- phoresis the gels were frozen on dry ice and sliced into 1 mm sections. The sections were then incubated for 2h hr in the dark at 250 C in 0.2 ml of a reaction mixture containing 2 mM IAA, 0.1 mM 2,14- dichlorophenol, and 0.1 mM Mn012 in 0.1 M phosphate buffer, pH 6.0 (h). At the end of the incubation period 0.2 m1 of 0.5%‘p-N,N—.dimethylaminocinnamaldehyde in l N HCl was added to each tube (h, 8). After 1 hr the solutions were removed from the tubes and the optical density of the IAA oxidation products (8) was determined at 562 nM. RESULTS Localization ofgperoxidase. Peroxidase activity, as denoted by the blue reaction product, was localized principally in those cells through which the abscission layer forms (Figure 1, upper). However, a brown reaction product, indicating autoxidation of the blue intermediate, was observed in the receptacle tissue and to a much lesser extent in the fruit tissue. Autoxidation occurred irrespective of how soon after treating with benzidine the nickelous 6h Figure l.--Localization of peroxidase in the abscission layer and associated tissues at mid-Stage III (upper) and at the end of Stage I (lower). The abscission layer is denoted by the blue reaction product. 65 66 ammonium sulfate solution was added. Furthermore, both reaction products, denoting peroxidase activity, were observed in the abscis- sion layer aid the adjacent tissues from late Stage I (Figure 1, lower) to maturity. The endocarp also gave a positive reaction for peroxidase in the early stages of development (Figure 1, lower). A similar differential staining for peroxidase between the abscis- sion layer and the receptacle and fruit tissue was observed using g-dianisidine. In this case a green reaction product was formed in the abscission layer and a black reaction product in the adjacent fruit and receptacle tissues. Peroxidase activity. Peroxidase activity in the fruit (mesocarp), abscission zone, receptacle, and pedicel tissues determined at var- ious stages of fruit development is recorded in Table 1. Enzyme activity was highest in the receptacle, intermediate in the abscis- sion zone and pedicel and lowest in the fruit at all times. The activity of peroxidase in the abscission zone increased with fruit development until early Stage III of fruit growth (Table 2). In the latter weeks of maturation the activity declined. The initial decline in FRF which signals the start of separation ‘was observed June 26, coinciding with the peak of peroxidase activ- ity. Hawever, the marked drop in FRF which followed was accompan- ied by a decline in peroxidase activity. Although ethephon treatment resulted in a 78% reduction in FRF after 80 hr compared to a 59% reduction for the control, it had no significant effect on peroxidase activity (Table 3). Cyclohex- imide caused a significant reduction in enzyme activity after 10 hr compared with the control, and this decreased enzyme activity was 67 TABLE l.--Peroxidase activity in the abscission zone and associated tissues as related to stage of fruit development. Activity per mggproteinz Tissue June 7 June 21 July 5 Fruit (mesocarp) h.6 ey 6.2 d 3.5 c Abscission zonex 22.1 b 27.2 b 25.8 b Receptacle 31.0 a 33.h a 3h.9 a Pedicel 19.6 b 18.0 c 2h.6 b 2 Units of activity, based on 4Ah60 using 11202 as substrate and ‘g-dianisidine as H+ donor, per mg protein determined by Lawry's assay. yMean separation (in columns) by Tukey's no test, P I 0.01. 68 TABLE 2.--Peroxidase activity in the abscission ”zone as related to fruit removal force (FRF) and stage of fruit development. Measurement Date of Activity2 FRF (g) Fruit development measurement May 30 18.1; cy 1283 Late I June 5 23.2 bc lh96 Early II June 12 25.0 b 1571 Mid II June 19 27.1 b 1599 Late II June 26 33.8 a lh80 Early III July 3 2h.h be 876 Mid III JUIy 10 22.0 bc 326 Late III zUnits of activity based on M1460 ‘g-dianisidine as assay. at yMean separation by Tukey‘s 60 test, P = 0.05. using 0 as substrate and donor, per mg protein determined by Lawry's 69 .mod I m .1533 {neg .3 785500 n3 soapeummou 502% I Seems a g an congruence 50.5.3 we use .aosom +m pa 0533536 was 3.0353 as mom: made: 8:44 no women gauges mo 3.25» .o emu es ease one as case successes we a m.mm e >.mm u m.:m A: m-oav nonsense ma p%3 n93 993 Azfigvfi mm a m.e~ e m.m~ as H.am e.gm Homeeoo Nev he om hopes mum on someoaeom an o: om 0H 0 possesses hots 5539a gummy M3259”: .unvnsanxu 39C mo anon soauuaomps on» 3 EEC 3.8% H9690.” 39.5 one 23:33 0338qu so someones use E8 seaflonoaoho mo poommmnufi as 70 associated with a significant inhibition of abscission as indexed by the FRF after 80 hr. Neither treatment of explants with ethylene nor*lowering the endogenous ethylene level with reduced pressure affected peroxidase activity (Table h). However, ethylene caused a 75% reduction in FRF after 72 hr, while lowering the endogenous ethylene to one- fifth resulted in only an 8% reduction in FRF. Peroxidase isoenzymes. There was a change in the isoenzyme pattern of peroxidase extracted from the abscission zone with fruit development (Figure 2). Four acidic isoenzymes were observed (A—D), A and B representing the major and C and D the minor isoenzyme bands. The two minor bands remained relatively constant during development, whereas isoenzymes A.and B increased until mid-Stage II (Figure 2, gel 3) and thereafter decreased slightly or remained constant. Band A did not appear to be present during Stage I (Figure 2, gel 1) and decreased slightly after mid-Stage II. Only 1 basic isoenzyme (E) was observed and it moved only a short distance from the origin. The intensity of band E changed with fruit development, following a pattern similar to the acidic iso- enzyme A. Ethephon and ethylene had little or no effect on the acidic isoenzyme pattern (Figures 3 and h), however, ethylene appeared to increase the basic isoenzyme (Figure h). Cycloheximide caused a decrease in isoenzyme A (Figure 3), while lowering the level of endogenous ethylene by reduced pressure had no effect on the acidic isoenzymes (Figure h). IAA oxidase activity. The acidic isoenzymes of peroxidase 71 .36 u m 3.333 mimosa—.5. .3. $55.30 n3 soapsnsmou 50:." l Seems a E he. 605533 5090.3 ma non .uoeov +m ms 0533536 use opsnpmnsm as Noun wean: 8:44 no cause. .huggom ho spans» .0 omm es ease on» 3 See 383me m s cam . mam . «.8 as one as .mo 2. a mom e cram e 9mm as owe. as he 3 memo a}: 2 i: s dam a «.mm as mam m.mm as 8e es £2 3 .5 om seems Em 3 someosoom B 3 om S o eseaeseaa note howoponm a non. Nflufifii .uupqsamxo 39C no snow sowaaaouns on». 5 Arab mono.“ H268?" 39C use hugged manganese so 0.3395 moose?" 9:. 30.7390 no poofimcui mama. 72 Figure 2.--Zymograms of acidic (upper) and basic (lower) peroxidase isoenzymes extracted from the pedicel: fruit abscission zone at various stages of fruit development. 1, late Stage I; 2, early Stage II;' 3, mid-Stage II; R, late Stage II; 5, early Stage III; 6, mid-Stage III; 7, late Stage III. Arrows denote direction of run and letters denote iso- enzymes bands. 73 7h Figure 3.--Zymograms of acidic peroxidase isoenzymes extracted from the pedicel:fruit abscission zone of fruit explants treated with 10'” M cycloheximide (l, h, 7), distilled water (2, 5, 8), or 10'3 M ethephon (3, 6, 9). Explants were assayed for peroxidase isoenzymes after 10 (1, 2, 3), 20 (h, s, 6), and hO (7, 8, 9) hr. 75 IIIIIIIII 76 Figure h.--Zymograms of acidic (upper) and basic (lower) peroxidase isoenzymes extracted from the pedicel: fruit abscission zone of fruit explants subjected to oxygen at 0.2 atm (l, h, 7), air at 1 atm (2, 5, 8), or 10 Hl/l ethylene at 1 atm (3, 6, 9) pressure. Explants were assayed for peroxidase activity after 10 (l, 2, 3), 20 (h, 5, 6), and 1+0 (7, 8, 9) hr. 77 78 from the abscission zone degraded IAA (Figure 5). There was a close correlation between the intensity of the isoenzyme bands and IAA oxidase activity. The basic isoenzyme could not be analyzed because of an interaction between the reagent and the gel. DISCUSSION Peroxidase activity was highest in the abscission zone and receptacle, whereas activity in the fruit (mesocarp), by comparison, was very low (Table 1). Although the activity in the receptacle tissue was slightly higher than that of the abscission zone, this does not take into account the fact that the actual separation layer (6-8 cells wide) comprised only a portion (approx 1/3) of the abscission zone as assayed. Therefore, it is possible that the absolute activity in the abscission layer pg; Egywas over 2-fold greater than that observed for the excised zone tissue, due to the dilution of activity by the mesocarp tissue (Table l). The reason for the difference in reaction products formed by the peroxidase in the layer tissue from that in the receptacle, pedicel, and fruit (mesocarp) tissue is not known. These histo- chemical differences in activity were observed from late Stage I to maturity. One additional acidic isoenzyme band was observed in the peroxidase extracted from the pedicel tissue, which ran one-third the distance between bands B and A (unpublished data). However, this isoenzyme was not observed in the fruit and was present only as a minor band in the receptacle tissue, which may have resulted from incomplete separation of the pedicel tissue from that of the receptacle. Thus, these histochemical differences 79 Figure 5.--Degradation of IAA by isoenzymes of peroxidase extracted from the pedicel:fruit abscission zone of sour cherry fruit. The sketch (top) illus- trates the isoenzyme pattern in the electro- phoretic gel (direction of run - left to right). The graph (bottom) presents optical density of solutions of IAA after 2h hr incubation with seg- ments of gel, followed by addition of p—N,N- dimethylaminocinnamaldehyde. High OD indicates high IAA oxidase activity. 250 r- ISO)- :00- l O O N (39%) AllSNBO 'IVOlldO 50- 60 5O 40 3O 20 IO FRACTION NUMBER 81 are probably not due to differences in isoenzymes. Nevertheless, they may signify a higher capacity for oxidation in the receptacle tissue (18) and/Or the result of differences in the location of the enzyme within the cells of the respective tissues (13). A significant increase in peroxidase activity occurred in the abscission zone with development of the fruit (Table 2), and the maximal activity corresponded with the initiation of the separation phase (Table 2). Furthermore, the enzyme activity did not appear to be assOciated with lignification, as only a small amount of lignification of vascular bundle cells occurs in the abscission zonee. The increase in activity was most likely related to an increase in 2 of the major isoenzymes (B and E) and the appearance of a third (A) major isoenzyme (Figure 2). Cycloheximide resulted in a significant reduction of'perox- idase activity (Table 3), which was reflected by a decline in intensity of isoenzyme A (Figure 3). Moreover, this reduction in peroxidase activity was associated with cycloheximide-induced inhibition of abscission. Ethylene, however, had no effect on total peroxidase activity or on the acidic isoenzyme pattern, even though it markedly enhanced abscission. However, ethylene appeared to enhance the activity of the basic isoenzyme. Lowering the endog- enous ethylene level had no effect on peroxidase activity or on the acidic isoenzyme.pattern, but but appeared to slightly reduce the basic peroxidase isoenzyme. These results suggest that ethylene does not enhance cherry fruit abscission through an effect on total peroxidase activity, although it may exert some influence on the activity of the basic peroxidase isoenzyme. 82 Furthermore, it is not clear whether cycloheximide delays abscis- sion through its influence on peroxidase or whether the effect on peroxidase was simply due to a general inhibition of protein synthesis. The peroxidase isoenzymes from the abscission zone had the capacity to degrade IAA, and therefore, may play a role in abscis- sion through the control of IAA levels. Since activity was local- ized in the layer early in fruit development, peroxidase may have a general role in the preparation of these cells for separation. Possibly it maintains IAA at a low level and thereby prepares these cells for early senescence and separation. The observed increase in activity to a maximum at the time of induction of the separation phase certainly indicates the potential for a decline in IAA in the abscission zone with the onset of separation. 10. LITERATURE CITED Davis, B. J. l96h. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121: hon-h27. Evans, J. J., and N. A. Aldridge. 1965. The distribution of peroxidase in extreme dwarf and normal tomato (Lycopersicon esculentum Mill.). Phytochemistry h:75§9-503o Fowler, J. L., and P. W. Morgan. 1972. The relationship of the peroxidative indoleacetic acid oxidase system to in vivo ethylene synthesis in cotton. Plant Physiol. 157555-559. Frenkel, C. 1972. Involvement of peroxidase and indole- 3-acetic acid oxidase isozymes from pear, tomato, and blueberry fruit in ripening. Plant Physiol. 1‘9: 757-763. Jackson, M. B., I. B. Morrow, and D. J. Osborne. 1972. Abscission and dehiscence in the squirting cucumber, Ecballium elaterium. Regulation by ethylene. Can. J. .ESE- 50: lh65-lh7l. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. Melachouris, N. P. 1969. Discontinuous gel electrophoresis of whey proteins, casein, and clotting enzymes. J. Dairy fig. 52: 1456-1459- Meudt, W. J., and T. P. Gaines. 1967. Studies on the oxida- tion of indole-B-acetic acid by peroxidase enzymes. I. Colorimetric determination of indole-3-acetic acid oxidation products. Plant Physiol. he: 1395-1399. Morgan, P.‘w., and J. L. Fowler. Ethylene: modification of peroxidase activity and isozyme complement in cotton (Gossypium hirsutum L.). Plant Cell Physiol. 13: 727-736. Pollard, J. E., and R. H. Biggs. 1970. Role of cellulase in abscission of citrus fruits. J. Amer. Soc. Hort. Sci. 95: 667-673. 83 12. 13. 1h. 15. 16. 17. 18. 19. 20. 21. 22. 8h Poovaiah, B. W., and H. P. Rasmussen. 1973. Peroxidase activity in the abscission of bean leaves during abscis- sion. Plant Physiol. 52: 263-267. , and M. J. Bukovac. 1973. Histochemical localization of enzymes in the abscission zones of maturing sour and sweet cherry fruit. J. Amer. Soc. Hort. Sci. 98: 16-18. Res, J. 1973. Cytochemical localization of peroxidase in plant cells. Physiol. Plant. 28: 132-133. Rasmussen, G. K. '1973. Changes in cellulase and pectinase activities in fruit tissues and separation zones of citrus treated with cycloheximide. Plant Physiol. 51: 626-628. Reisfeld, R. A., U. J. Lewis, and D. E.'Williams. 1962. Disk electrophoresis of basic proteins and peptides in polyacrylamide gels. Nature 195: 281-283. Rogers, B. J., and C. Hurley. 1971. Ethylene and the appear- ance of an albedo macerating factor in citrus. J. Amer. Soc. Hort. Sci. 96: 811-813. Shannon, L. M., E. Kay, and J. Y. Lew. 1966. Peroxidase isozymes from horseradish roots. I.Isolation and physical properties. J. Biol. Chem. 2&1: 2166-2172. Veech, J. A. 1969. Localization of peroxidase in infected tobaccos susceptible and resistant to black shank. Phytopatholog. 59: 566-571. Wittenbach, V. A., and M. J. Bukovac. 1972. A morphological and histochemical study of (2-chloroethyl)phoflphonic acid-enhanced abscission of sour and sweet cherry fruit. J. Amer. Soc. Hort. Sci. 97: 628-631. , and . 1973. Cherry fruit abscission: effect of growth substances, metabolic inhibitors and environ- mental factors. J. Amer. Soc. Hort. Sci. 98:3h8-351. , and . 197h. 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