STUDIES ON THE MODE OF ACTION OF ETHYLENE IN PIANT TISSUES AND ITS ROLE IN AUTOCATALYTIC ETHYLENE PRODUCTION Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY , EVANGELOS MICHAEL SFAKIOTAKIS 1972 ~ 5—5 LIBRARY ” III III IIIIIIIIIIIIIIIIIIIIIIII I III 3 1 10200 ichigan State ' University This is to certify that the thesis entitled STUDIES ON THE MODE OF ACTION OF ETHYLENE IN PLANT TISSUES AND ITS ROLE IN AUTOCATALYTIC ETHYLENE PRODUCTION I. ‘ presented by Evangelos Michael Sfakiotakis has been accepted towards fulfillment of the requirements for Ph.D. degree in Horticulture 4%; fl/fl/Zé, Major professor Date Jigvember 2 , 19 72 0-7639 E? ‘ V IINDING BY :— ‘ HOAB & SONS' BOOK BINDERY INC. ‘SL'IRB’RARY amozns {I H .1 u 'I ‘ LL3- crustutcmsgj ABSTRACT STUDIES ON THE MODE OF ACTION OF ETHYLENE IN PLANT TISSUES AND ITS ROLE IN AUTOCATALYTIC ETHYLENE PRODUCTION BY Evangelos Michael Sfakiotakis Investigations were conducted to determine the mechanism of ethylene action in causing diverse physiological and morpho- logical changes in plant tissues. Studies were also made to determine the processes involved in the regulation of ethylene synthesis during fruit maturation. Several investigative approaches were employed dealing with; pollen germination and tube growth, DNA polymerase activity in etiolated pea, DNA and RNA polymerase activity in potato tuber mitochondria, induction of autocatalytic ethylene production in apple fruits, and control of the internal ethylene concentration in apple fruits during maturation. The influence of ethylene and CO on pollen germination 2 and tube growth was investigated employing ventilated culture systems. Ethylene had no effect on pollen germinability or tube growth. Germinating pollen did not produce a detectable amount of ethylene (less than 0.1 nl/g/hr). Supplementing the cultures with CO2 caused a marked increase in germination Evangelos Michael Sfakiotakis and tube growth. The half—maximal response for germination was less than 0.5%. CO2 levels ranging from 1.08 to 2.22% were found in the internal cavity of lily styles. CO2 de- rived from stylar metabolism may, therefore, modulate pollen tube growth thus integrating the events leading to fertili- zation. An extract from the apical portion of etiolated seed- lings of Pisum sativum L. was used as a test system to examine the action of ethylene on DNA polymerase activity. Extracts from plants previously treated with ethylene showed less activity to synthesize DNA than extracts from untreated plants. Ethylene in_vitgg showed no activity. Inhibition of cell division by ethylene observed in this and other tissues may be the result of impaired synthesis of DNA polymerase. RNA and DNA polymerase activity was studied in mito- chondria isolated from potato tubers respiring at an accel- erated rate in response to treatment with ethylene (10 ul/l). Whole tuber respiration rate increased about 6 hrs after ethylene treatment began and reached a peak value 4°7-fold higher than the initial rate in 24 hrs. The rise in respira- tion preceded by an increase in DNA polymerase activity initiated within 6 hrs of ethylene application. RNA poly- merase activity began to increase after 6 hrs and reached a peak value 2.5-fold higher than the initial rate in 18 hrs. Cytochrome c oxidase activity, although increasing initially Evangelos Michael Sfakiotakis subsequently paralleled the respiration rate pattern of whole tubers. Activity of DNA and RNA polymerase of mitochondria isolated from tubers that did not receive ethylene treatment was not affected by ethylene treatment in_viE£g. Apparently the stimulation of respiration by ethylene is mediated in zizg_by enhancing mitochondrial DNA, RNA and protein syn- thesis. Ethylene biosynthesis becomes autocatalytic as fruit ripening proceeds. PrOpylene, which fruits do not produce, was employed to determine the stage of maturity apples must attain to autocatalytically produce ethylene and the effect of O2 tension on autocatalysis. The efficacy of propylene to stimulate ethylene production increased progressively with fruit maturation, but rate of production following treatment with 500 ppm propylene was constant. A shorter lag time to the onset of autocatalytic production was observed for the more mature fruit which reflects a natural increase in sensi- tivity. PrOpylene administered at 6.5% 02 or less did not induce ethylene production, but an anaerobic atmosphere was necessary to completely inhibit ethylene synthesis in fruits once autocatalysis began. Internal ethylene concentration was followed in apples while attached to or detached from the tree throughout the maturation period. Similar internal gas levels were found in fruits on the tree when compared to levels observed in fruits Evangelos Michael Sfakiotakis immediately after harvest. Harvesting fruits stimulated their ethylene production capacity more quickly as the fruits gained maturity. Isolating the fruit from leaves by girdling and defoliation stimulated an earlier increase in internal ethylene concentration. Leaves apparently provide the fruit with a substance that retards or prevents the onset of auto- catalytic ethylene production. STUDIES ON THE MODE OF ACTION OF ETHYLENE IN PLANT TISSUES AND ITS ROLE IN AUTOCATALYTIC ETHYLENE PRODUCTION BY Evangelos Michael Sfakiotakis A THESIS Submitted to. Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1972 CR 9?“ DEDICATION to my children, Despina and Michael ii ACKNOWLEDGEMENTS The author wishes to express his sincere appreciation to Dr. D. R. Dilley, for his counsel and assistance through- out the course of this work and during the development of the manuscripts contained herein. The author is also indebted to Drs., F. G. Dennis, M. J. Bukovac, R. S. Bandurski and P. Markakis who served as the guidance committee and for their review of this disser- tation. The advice and assistance offered by Drs. J. A. Boezi, N. E. Tolbert and D. H. Dewey are greatly appreciated. Appreciation is also extended to Drs. A. Apelbaum and D. H. Simons with whom the author in collaboration conducted part of this work. Thanks are extended to Dr. J. D. Downes for donating the potato tubers used in the mitochondrial study. The author gratefully acknowledges the support of the National Science Foundation for providing a portion of the financial assistance which made this study possible. Special thanks to my wife, Diamanta, for her patience and understanding throughout the course of this graduate study. iii Guidance Committee: The thesis is organized in the journal style with the various sections following the format of the respective journals to which they have been or will be submitted for publication. "Pollen Germination and Tube Growth: Dependent on Carbon Dioxide and Independent of Ethylene" conducted in c00peration with Dr. D. H. Simons has been published in Plant Physiology. "Inhibition by Ethylene of Soluble DNA Polymerase Activity in Pisum Sativum Seedlings" conducted in collaboration with Dr. A. Apelbaum will be submitted to Science; "DNA and RNA Polymerase Activity of Potato Tuber Mitochondria Enhanced by Ethylene" conducted also in collaboration with Dr. A. Apelbaum will be submitted to Biochemical and BiOphysical Research Communications; "Induction of Autocatalytic Ethylene Produc- tion in Apple Fruits by Propylene in Relation to Maturity and Oxygen Dependency" and "Internal Ethylene Concentrations in Apple Fruits Attached or Detached from the Tree" will be sub- mitted to Journal of the American Society for Horticultural Science. iv TABLE OF CONTENTS Page PREFACE O O I I O O O O O O O O O O O O O O O O O O O O 1 Introduction . . . . . . . . . . . . . . . . . . . l Mechanism of Ethylene Action . . . . . . . 2 Role of Ethylene in the Autocatalysis of Ethylene PrOduction O O O O O O O I O O I O O I I O O O O O 6 TheSiS Objectives 0 O O O O I O C O O O O O O O O O O 7 Literature Cited 0 O I O I O O O O O I O C O O O O 0 10 SECTION I: POLLEN GERMINATION AND TUBE GROWTH: DEPENDENT ON CARBON DIOXIDE AND INDEPENDENT OF ETHYLENE O C O O O O C O O O I O O O O O O O O O O O l 9 Abstract 0 O O O O O O O O O O O O I O O O O O O O O 19 Introduction . . . . . . . . . . . . . . . . . . . 20 Materials and Methods. . . . . . . . . . . . . . . . 21 Results and Discussion . . . . . . . . . . . . . . . 25 Literature Cited . . . . . . . . . . . . . . . . . . 38 SECTION II: INHIBITION BY ETHYLENE OF SOLUBLE DNA POLYMERASE ACTIVITY IN PISUM SATIVUM SEEDLINGS . . . 39 Abstract I I O O O I O O O O O O O O O O O O O O O O 39 Literature Cited . . . . . . . . . . . . . . . . . . 57 SECTION III: DNA AND RNA POLYMERASE ACTIVITY OF POTATO TUBER MITOCHONDRIA ENHANCED BY ETHYLENE. . . . . . . 59 Summary. . . . . . . . . . . . . . . . . . . . . . . 59 Introduction . . . . . . . . . . . . . . . . . . . 60 Materials and Methods. . . . . . . . . . . . . . . . 60 Results. . . . . . . . . . . . . . . . . . . . . . . 63 Discussion . . . . . . . . . . . . . . . . . . . . . 80 Literature Cited . . . . . . . . . . . . . . . . . . 82 TABLE OF CONTENTS-—Continued Page SECTION IV: INDUCTION OF AUTOCATALYTIC ETHYLENE PRO- DUCTION IN APPLE FRUITS BY PROPYLENE IN RELATION TO MATURITY AND OXYGEN DEPENDENCY. . . . . . . . . . . 83 Abstract. . . . . . . . . . . . . . . . . . . . . . 83 Introduction. . . . . . . . . . . . . . . . . . . . 84 Materials and Methods . . . . . . . . . . . . . . . 86 Results and Discussion. . . . . . . . . . . . . . . 87 Literature Cited. . . . . . . . . . . . . . . . . . 104 SECTION V: INTERNAL ETHYLENE CONCENTRATIONS IN APPLE FRUITS ATTACHED OR DETACHED FROM THE TREE. . . . . 107 Abstract. 0 I O O O O O O O O O O O O O O O O O O I 107 Introduction. . . . . . . . . . . . . . . . . . . . 107 Materials and Methods . . . . . . . . . . . . . . . 108 Results and Discussion. . . . . . . . . . . . . . . 112 Literature Cited. . . . . . . . . . . . . . . . . . 121 APPENDIX: APPARATUS TO TREAT PLANT MATERIAL WITH GASES AND MEASURE CO2 AND ETHYLENE PRODUCTION . . . 123 Abstract. I O O O O O O O O O O O O O O O O O O O O 123 Literature Cited. . . . . . . . . . . . . . . . . . 137 vi TABLE LIST OF TABLES Section I Concentrations of C02, 02 and ethylene in the stylar cavity of lily flowers at the time of anthesis. Samples (0.5 to 1 ml) were withdrawn from the cavity by a syringe fitted with a hypo- dermic needle. The flowers were submerged in water while the samples were taken to prevent entrance of outside air. . . . . . . . . . . . . Percent germination of lily pollen treated with 10 ppm ethylene and 5% CO in the ventilated hanging drop culture for 3 hr, The data are means of 17 observations . . . . . . . . . . . . The influence of 1 ppm ethylene on percent germination of peach and pear pollen cultured on agar and sealed in plastic bags for 5 hr . . . . Section II Dependency of DNA polymerase activity on compon- ents of the reaction mixture . . . . . . . . . . Utilization of different templates by pea DNA polymerase . . . . . . . . . . . . . . . . . . . Effect of in_vitro ethylene treatment, DNAase and perphosphate on TMP incorporation into acid-insoluble product . . . . . . . . . . . . . Section III Dependency of mitochondrial DNA polymerase activity on components of the reaction mixture . Dependency of mitochondrial RNA polymerase activity on components of the reaction mixture . vii Page 29 29 31 43 44 46 64 65 LIST OF FIGURES FIGURE Section I 1. Hanging drop culture chamber. . . . . . . . . . . 2. Effect of C02 concentration on percent germina- tion of lily pollen (bar indicates Tukey's w-test at p = 0.05). O O O O O O O O O O 0 O I O O C O 0 Effect of 10 ppm ethylene and 5% C02 on the dis- tribution of lily pollen tube length after 2 hr . Growth of a single pear pollen tube in a hanging drop culture continuously ventilated with 5% C02. Section II The relationship between incorporation of 3H-TMP and protein content in the assay. . . . . . . . . Time course of TMP incorporation into acid- insoluble product . . . . . . . . . . . . . . . . Effect of template concentration on TMP incorpora- tion 0 O O O O I I O I I I O O O I O O O O I O O 0 Section III Isolation of mitochondria from potato tubers. . . Time course of TMP incorporation into acid- insoluble product by mitochondria isolated from potato tubers . . . . . . . . . . . . . . . . . . Time course of UMP incorporation into acid- insoluble product by mitochondria isolated from potato tubers . . . . . . . . . . . . . . . . . . Concentration study for the incorporation of TMP into acid-insoluble product by mitochondria iso- lated from potato tubers. . . . . . . . . . . . . viii Page 23 27 48 52 54 62 68 7O 72 LIST OF FIGURES—-Continued FIGURE 5. Concentration study for the incorporation of UMP into acid-insoluble product by mitochondria iso— lated from potato tubers. . . . . . . . . . . . . The effect of ethylene treatment in vivo for various durations on the incorporEEion of UMP into acid-insoluble product by mitochondria iso- lated from potato tubers. . . . . . . . . . . . . The effect of ethylene treatment on respiration, cytochrome c oxidase, and incorporation of TMP- UMP into acid-insoluble product in mitochondria isolated from potato tubers . . . . . . . . . . . Section IV Effect of propylene treatment on ethylene pro— duction in Red Delicious apples at 52, 58, 65 and 72% maturity. . . . . . . . . . . . . . . . . Effect of 500 ppm propylene treatment on rate of ethylene production in Red Delicious apples treated at 52% (I), 58% (II), 65% (III), and 75% (IV) maturity . . . . . . . . . . . . . . . . . . Effect of prOpylene concentration on respiration in Red Delicious apples at 30% (A), 35% (B), 52% (C), 58% (D) and 65% (E) maturity, measured the 3rd-4th day after propylene treatment . . . . . . Effect of oxygen concentration in the stimulation of ethylene production by 100 ppm propylene in Red Delicious apples treated at 83% maturity. . . Effect of oxygen concentration at the time of exposure in the stimulation of ethylene produc- tion by 100 ppm propylene in Red Delicious apples treated at 90% maturity . . . . . . . . . . . . . Effect of oxygen concentration in the respiration of Red Delicious apples treated with 100 ppm propylene at 99% maturity . . . . . . . . . . . . ix Page 74 77 79 89 92 94 97 99 102 LIST OF FIGURES--Continued FIGURE Section V Method of sampling the internal ethylene concen- tration in Red Delicious apple attached to the tree. 0 O O I O O O O I O O O O c O O O O O O 0 Internal ethylene concentrations in Red Delicious apples followed A) by the method with needles attached to the fruit, and B) by the method of withdrawing a gas sample from harvested fruit sub— merged in water. Values represent means for 4 (attached) or 6 (detached) fruits per sampling date 0 O O O O O O O O O O O O O I O O I O O O 0 Internal ethylene concentrations in four Red Delicious apples left on the tree, or harvested 113 days after bloom. . . . . . . . . . . . . . Effect of defoliation and girdling on the intern— al ethylene concentration in Red Delicious apples Appendix Apparatus for measuring C02 and ethylene produc— tion of plant material under constant 02 concen— tration I O O O O O I O O I O O O O O C O O O 0 Standard curves obtained from the BaCl and the two indicator titration methods for C02 determin- ation. Known amounts of C02 were injected into closed containers with KOH. The data were cor- rected for STP conditions . . . . . . . . . . . Stimulation of ethylene production by propylene in Red Delicious apples treated 126 days after anthesis. The data indicate rate of ethylene production the 6th day after propylene treatment was Started 0 O O O O O O O O O O O O C I I O 0 C02 and ethylene production by carnation flowers. Three flowers were cut at the tight bud stage and placed in preservative solution (200 ppm 8—hy- droxyquinoline and 1.5% sucrose) in each chamber. The data represent the averages of measurements from four replications. . . . . . . . . . . . . X Page 111 114 116 119 126 130 132 134 LIST OF FIGURES--Continued FIGURE Page 5. Effect of ethylene on respiration of dormant cherry twigs collected from orchard on December 21, 1971. . . . . . . . . . . . . . . . 136 xi PREFACE Introduction: Ethylene has been implicated in many stages of growth and development from germination of seeds to senescence of plants. As a normal plant metabolite, the gaseous hormone controls hook formation in etiolation (36,37,54), ripening of fruits (16), sex expression in cucurbits (24) and the on— set of senescence of leaves (22,30), flowers (22) and other tissues (65). Application of ethylene produces a variety of responses characterized by suppression or stimulation of normal morpho- genetic events. The list of such responses includes: suppression of the shoot apex growth (17) with inhibition of leaf expansion in several dicots (22,37) or stimulation of growth in a few monocots (52,60,89), flower initiation (18), inhibition of flowering (3), inhibition of bud growth (22), breaking of dormancy in resting tubers (92), corms and bulbs (38,92), stimulation of germination of some seeds (5,34,38,57, 88,90,92) inhibition of root growth (25,26), induction of adventitious roots (59,96), epinasty of leaves (28,29,95) altering of sex expression (24,71), induction of abscission (4,81,95), enhancement of fruit growth (69) and promotion of ripening (10,14,16,4l,69,82). ’ I’ \L‘ I! Although knowledge of the physiological role of ethylene has become extensive in recent years little is known about the mechanism of action. Mechanism of Ethylene Action: Despite the diversity of phenomena regulated by ethylene nearly all responses have the same dose-response curve (8,67, 82). Another common feature of all ethylene reSponses is the inhibitory effect of CO (2,4,20,25,56,58,86,94). Lineweaver- 2 Burk plots (20,37) revealed that the CO2 inhibition of ethyl— ene action is competitive, similar to that observed for competitive inhibitors of enzyme reactions. This observation gave rise to the hypothesis by Burg and Burg (16,20) of a single receptor molecule in a metal containing enzyme requir- ing oxygen for activation. They suggested that interaction of ethylene with the receptor site caused a primary chemical change. This in turn produced a multiplicity of secondary changes resulting in a variety of physiological responses depending upon the kind and the age of tissue involved (23, 67). While little evidence has been presented to contradict the above proposal, there are a few physiological effects with different apparent kinetics (37,82). Pratt and Goeschl (82) suggest that multiple sites of action may exist. The mechanism by which ethylene initiates a variety of responses has not been determined. Four possibilities have been prOposed; 1) ethylene may act as an allosteric effector on enzyme activity, 2) it may enhance the permeability of membranes, 3) it may regulate auxin action, and 4) it may regulate the metabolism of nucleic acid and protein synthesis. In yitrg studies have shown no effect of ethylene on the activity of enzymes controlling rate-limiting reactions. The activity of carbonic anhydrase, which contains zinc and has the ability to combine with CO did not change in the 2, presence of ethylene (8, personal communication with H. Ku). Similar results were obtained in_yi££g studies with B-glucosidase (32), a-amylase (31), invertase (31,85), peroxi- dase (75) and adenosine triphOSphatase (80). On the contrary, activity of many enzymes increased when the tissue was pretreated in XiXE.With ethylene. These in- clude peroxidase (42,50,51,68,75,76,84), malic enzyme (48,49, 53,74), phenylalanine ammonia lyase (33,50.78), acid phospha- tase (42), chlorOphyllase (63), cytochrome c reductase (53), polygalacturonase (70), polyphenol oxidase (42,50,87), pro- tease (73), and cellulase (6,7,27,77). These changes in enzyme activity are probably secondary to the primary action of ethylene. The hypothesis that ethylene regulates physiological phenomena through an effect on the permeability of membranes has received considerable attention in connection with fruit ripening. The proposal that ethylene may exert its effects by disrupting the membranes and bringing changes of com- partmentalization (11) has been criticized because the changes in the characteristics of membranes may be the result of ripening rather than the cause (8,13,15,22,83). Lyons and Pratt (64) showed that ethylene caused mito- chondrial swelling, suggesting a change in permeability and Olson and Spencer (79,80) found an increase in adenosine tri- phosPhatase when mitochondria were exposed to ethylene. Recent studies have revealed that the effect on mitochondrial swelling was not typical of ethylene action. The concentra- tion of the gas needed for this reSponse was markedly higher than biologically active condentrations, and saturated hydro- carbons like propane and ethane had similar effects (61,72). The theory that ethylene affects auxin by altering auxin transport, increasing auxin destruction, or inhibiting the synthesis of auxin has received considerable support recently. Burg and Burg observed that ethylene inhibits polar (l9) and lateral (l7) IAA transport in pea epicotyl. Hall and Morgan (39) showed an increase in IAA oxidase activity in cotton after ethylene treatment. Valdovinos et 31. (93), reported that ethylene inhibited the conversion of tryptophan to auxin in Coleus. They concluded that ethylene regulates auxin levels by controlling auxin biosynthesis. Changes in nucleic acids and proteins are often associ- ated with responses to ethylene. Inhibition of cell division by ethylene was found in fig fruits during stage I of their growth (69). Holm and Abeles (44,45) treated soybean seed- lings with ethylene and found that DNA synthesis ceased in the apex where growth was inhibited, but was promoted in the subapical tissue where swelling took place. In more recent studies with pea Apelbaum and Burg (9) showed that low con- centrations of ethylene (50 ppb) prevented cell division in the apical meristem of the shoot and root within a few hours by inhibiting DNA synthesis and preventing the cell from entering prophase. The mechanism whereby ethylene inhibits DNA synthesis has not been elucidated. Promotion of RNA synthesis was associated with ethylene- induced epinasty in tomato leaves (91), and abscission (1). Ethylene also increased RNA in fig fruits (66). Hulme et 31. (49) found an ethylene stimulated synthesis of RNA in apple fruit slices associated with the climacteric phenomenon. Ethylene increased protein synthesis in ripening (12,35, 43,46,47,62) and abscission (l), and increased the activity of enzymes mentioned earlier. Application of inhibitors of nucleic acid and protein synthesis diminished the response to ethylene. Frenkel gt 31. (35) found that fruit ripening was not stimulated by ethylene unless protein synthesis proceeded. Inhibitors of protein and RNA synthesis abolished the suppression of hook opening caused by ethylene (55). It is not clear whether the above mentioned changes in nucleic acids and protein are due to the direct action of the gas, or whether they occur during the expression of phenomena regulated by ethylene. Thus the observed changes may be the result rather than the cause of the response induced by ethylene (8). Studies with inhibitors of nucleic acid and protein synthesis cannot resolve the problem because both processes are required in the response studied. Basic studies of the role of ethylene as well as of other hormones in the control of gene activity have suffered for lack of kinetic studies on changes in DNA, RNA, or protein. These would separate cause from effect. Role of Ethylene in the Autocatalysis of Ethylene Production: The action of ethylene in ripening of climacteric fruits and other senescing organs is amplified by the fact that the hormone stimulates its own production. Autocatalysis of ethylene production is common to senescing organs detached from the mother plant. However, the phenomenon does not occur in actively growing plant tissues. Fruits attached to the tree exhibit resistance to ripening by exogenous ethylene. This has been attributed to an inhibitor of ethylene action which is transported from the leaves to the fruit (16). Autocatalysis has been associated with ripening of cli- macteric fruits (16,58) and senescence of orchid flowers (21,40). The phenomenon may be very common in other tissues as well, but is not well understood. Lack of the appropriate tissue and the difficulties to separate the input from the output gas makes the study more complex. The mechanism of autocatalysis of ethylene production in detached organs, the nature of the resistance to autocatalysis and ripening in attached organs, and the factors controlling these processes have not been resolved. Thesis Objectives: It is quite apparent that ethylene causes many and diverse phenomena in plant growth and development. Plants, explants, and tissue organs respond to the gas they produce normally or are made to produce in response to environmental or chemical stimuli. They also respond to ethylene supplied exogenously. It is possible that ethylene affects a funda— mental process and depending upon the kind and age of tissue the response evoked varies accordingly. In general, ethylene activates a potential that exists within the tissue. The po- tential is governed by the intricate programming of plant growth and development. This suggests that ethylene may affect the process of transcription and the response is ob- served as the message is eventually translated into enzyme activities which cause the physiological and morphological changes. The complexity of tissue organs handicaps investigations on the mechanism of ethylene action. Several systems were investigated. A search made to find a simple tissue, reported to respond to ethylene, resulted in studies with pollen germination and tube growth. An hypothesis was formulated that ethylene would enhance germination and tube growth and that CO would competitively inhibit its action. The hypothe- 2 sis was not verified but a fundamental discovery on the role of CO2 in pollen germination and tube growth was made. Reports of inhibition of cell division by ethylene led to studies on DNA polymerase. An hypothesis was formulated that ethylene inhibits the synthesis or action of DNA poly- merase in pea stem. This was, in part, substantiated since ethylene applied in 2132 to etiolated peas reduced the activ- ity of DNA polymerase to the same extent (nearly 90%) that cell division was inhibited. However, ethylene did not affect DNA polymerase in yitrg, These studies led to investigations on DNA and RNA poly- merase in mitochondria from quiescent potato tubers since ethylene causes a marked stimulation of respiration in this tissue. An hypothesis was formulated that the respiratory increase in potato in response to ethylene was due to inhibi- tion of DNA synthesis and stimulation of RNA and protein synthesis. Increased activity of RNA polymerase and cyto- chrome c oxidase were found to precede the respiratory increase but DNA polymerase activity was increased rather than decreased as postulated. The importance of ethylene in causing fundamental changes in DNA and RNA metabolism which in turn could markedly alter subsequent changes in enzyme activities and resultant physio- logical responses led to studies of the control of autocatalytic ethylene production. An hypothesis was formu- lated that ethylene production in tissues was closely regu- lated perhaps via feed back control. And loss of this control would lead to autocatalytic production with irrevers- ible consequence such as fruit ripening and senescence. Induction of autocatalytic ethylene production was investi- gated by employing propylene, a biologically active homolog of ethylene, which allowed for the measurement of endogenous ethylene during maturation of apple fruit. 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Several growth substances which cause initiation of roots and other responses in plants. Contrib. Boyce Thompson Inst. 7:209-222. SECTION I 11(11‘ III-'11" {[1 [:1 I‘ll-3,1. POLLEN GERMINATION AND TUBE GROWTH: DEPENDENT ON CARBON DIOXIDE AND INDEPENDENT OF ETHYLENE Abstract. The influence of ethylene and CO on pollen germi- 2 nation and tube growth was investigated employing ventilated culture systems. Ethylene had no effect on pollen germin- ability or tube growth. Germinating pollen did not produce a detectable amount of ethylene (less than 0.1'n1/g/hr1. Supplementing the cultures with CO2 caused a marked increase in germination and tube growth. The half-maximal response for germination was less than 0.5%. CO2 levels ranging from 1.08 to 2.22% were found in the internal cavity of lily styles. CO2 derived from stylar metabolism may, therefore, modulate pollen tube growth thus integrating the events lead- ing to fertilization. 19 INTRODUCTION Recent reports that ethylene increases pollen germin- ation and tube growth (1,8) prompted us to examine pollen as a simple biological system for studies on ethylene action. Pollen is anatomically simple compared to other highly dif- ferentiated tissues and plant organs generally employed. Furthermore, appropriate germination media and culture sys- tems have been exhaustively investigated. Conventionally, hanging drOp or other closed environment culture systems are employed. Germinating pollen has a high respiration rate (4) which causes CO to accumulate in closed cultures, and fixa- 2 tion (9) would then be an uncontrolled variable. More importantly, CO is a competitive inhibitor of ethylene (2) 2 whose action was the point of investigation. Nakanishii _et 21. (6) have shown that CO increases the number of pollen 2 tubes penetrating the stigma papilla cells in self-incompat- ible Brassica while ethylene has no effect. Because of these interrelated effects of the production and action of CO2 and ethylene, the conventional closed or poorly ventilated pollen germination systems were inappropriate for this study and continuously ventilated systems were develOped. 20 MATERIALS AND METHODS Pollen used in these experiments was collected from freshly Opened flowers of greenhouse lily, Lilium longiflorum Thanh, cv Ace, except where otherwise specified (Table III, Fig. 4). Each morning freshly dehiscing anthers were har- vested and placed on weighing paper in front of a fan until fully dehisced. The pollen was then scraped from the anthers and mixed prior to storage or use. The medium used was that of Dickinson (4) modified by use of MES buffer as described below. All glassware was washed in dichromate cleaning solu- tion and thoroughly rinsed in distilled water prior to use. Hanging drop cultures were prepared by placing a 10 pk drop of medium on a microscope cover slip; with the use of a dissecting microscope and two sharpened toothpicks approxi- mately one hundred pollen grains were added. All inoculations and experiments were carried out at 27 C in a room where the relative humidity was maintained above 80%. Pollen was always equilibrated in the assay room before inoculation. A thin film of vaseline was used to seal cover slips to a brass cylinder fitted with gas inlet and exhaust ports as illus- trated in Figure 1. A11 gases were humidified by bubbling through water, and the filter paper in each brass cylinder was moistened with 0.2 ml of distilled water to ensure 21 22 Figure 1. Hanging drop culture chamber. 23 POltEN COVER SUP IN HANGING oaor ’/////’ MOISTENED FIIJEI PAPER BRASS CYLINDER Figure 1 24 saturation of the atmosphere surrounding the hanging drOp. With gas flows as high as 300 ml/min through these chambers for up to 24 hr no decrease in diameter of the hanging drOp could be detected. A metered flow of various measured, constant ethylene and CO mixtures (in cylinders) was admitted 2 to the cultures and the concentration of each gas was moni- tored at the exhaust from each chamber by gas chromatography. Ethylene was measured by a Varian Aerograph model 1700 equipped with an activated alumiua column and a flame ioniza- tion detector. CO2 and 02 were measured by gas chromatograph employing silica gel and molecular sieve in parallel and a katharometer. After the appropriate germination period, growth was stopped by rapid freezing at -10 C. Percent germi- nation was measured by examining and counting the grains in the drop under a dissecting microscope. Tube length measure- ments were made from projections of photographs of the drop. RESULTS AND DI SCUSS ION Buffering of the Medium. Because varying CO2 concentra- tions were used, it was necessary to buffer the medium to eliminate any possible effect of CO on pH. Several buffering 2 systems were tested for their effects on percent germination and maintenance of pH in submerged shake cultures with con- tinuous flow of air or 5% CO2 in air through the cultures. Tulip, Tulipa gesneriara, cv Madam Spoor and Gander, pollen was used for these experiments. High germination was observed in unbuffered cultures at pH 5.0 so this pH was selected. Cultures with 0.01 M MES (pK 6.15) maintained pH 5.0 and had the same germination as unbuffered controls. Buffering at higher pH decreased the germination slightly. High germin- ability of lily pollen was observed in unbuffered media at pH 5.4 so MES at this pH was used for the CO2 studies. Carbon Dioxide effect. The germination of lily pollen in hanging drop cultures increased rapidly when the CO2 con- centration was increased from 0.03% (in air) to 1.3%, with very little further effect at concentrations up to 5% (Fig. 2A). The half-maximal CO2 concentration was 0.25% to 0.5% as measured by double reciprocal plots (Fig. 2A and 2B). Similar results were obtained with submerged shake cultures where CO2 concentrations up to 8.4% were used (Fig. ZB). 25 Figure 2. 26 Effect of CO2 concentration on percent germina- tion of lily pollen (bar indicates Tukey's w-test at p = 0.05). A. Ventilated hanging drop culture for 2 hr. B. Submerged shake culture for 2.5 hr. % Gnumnnou % Gummmou m 27 "- n-- U 0 v U 0 l I I» GEIMINAHON U 0 \ III -2 .1 _ u 2 a o I/cnnou mono: coucmnmou o 1 1 1 r_ 0 I 2 3 4 5 I" ’ 1' B 90 - ——_f I -/ .0 " L 10 ' I II - 50 " a 23 u- “ ' E . .- E 5 30 + 3 .4. 1 2' ’ 1. . .. -; -; -: 1 a a a 1/cauou mono: couccunmou . t A l 1 ___ I ’1 0 I 2 3 a a canon mono: coucsmnnou (i) Figure 2 28 The internal CO2 concentration in the stylar cavity of the lily flower was found to be 1.59% (Table I) which is very close to the optimum concentration found for best germination Ethylene Production. The ethylene content in the air stream leaving the hanging drOp cultures was the same as the inlet concentration even when the number of pollen grains was increased and the flow rate decreased to a level that respiratory CO accumulated. The minimum ethylene concentra- 2 tion attributable to pollen was observed in closed shake cul- tures after 3 1/2 hrs, during which 79% germination occurred. These cultures contained 5 mg of pollen in one m1 of medium per 25 ml flask. The ethylene concentration in 10 flasks with and without pollen was 19 ppb 1,3 and 19 ppb i 2, respectively. Response to Applied Ethylene. When air or air containing 10 ppm ethylene was continuously supplied to lily pollen in hanging drOp cultures very poor and erratic germination was obtained with no difference between the treatments (Table II). Similarly ethylene had no effect on germination in 5% CO2 where high germination was obtained. These results contradict those of Search and Stanley (8) and Buchanan and Biggs (1). In an attempt to resolve this paradox we used peach (Prunus persica cv Elberta) and pear (Pyrus communis cv Packham), pollen obtained from a commercial source and stored dry at -10 C. The medium and culture system were those previously 29 Table I. Concentrations of CO and ethylene in the stylar cavity of 111% flgwers at the time of anthesis. Samples (0. 5 to 1 ml) were withdrawn from the cavity by a syringe fitted with a hypo- dermic needle. The flowers were submerged in water while the samples were taken to prevent entrance of outside air. Sample Carbon dioxide Oxygen Ethylene % % ppm 1 1.46 17.59 0.045 2 2.22 15 36 -- 3 1.08 18.60 -- Mean 1.59 17.18 Table II. Percent germination of lily pollen treated with 10 ppm ethylene and 5% CO in the ventilated hanging drop culture for 2 hr. The data are means of 17 observations. Treatment Percent Range Standard germination* Deviation Air 8.2a 0.6 - 28.2 i 8.2 Air + 10 ppm ethylene 10.6a 0.6 - 47.8 i11.5 Air + 5% CO2 72.2b 44.2 - 91.8 113.7 Air + ethylene + CO 70.9b 17.7 - 94.6 :19.2 2 *Means followed by different letters differ significantly at P = 0.01, by Tukey's w-test. 30 reported (1) except that our air controls were sealed in polyethylene bags. The bags containing the cultures had air inlets and exhausts and were aerated for 5 mins before clos- ing and injecting ethylene into some of them to yield a concentration of 1 ppm. Ethylene did not alter the percent germination (Table III). The increased germination observed by Buchanan and Biggs (1) may have occurred in response to CO2 produced by the pollen in the sealed plastic bags used for the ethylene treatments. Ethylene was reported (7) to increase pollen germination only in the absence of boron in the medium. Using peach pollen in hanging drops continuously ventilated with 5% CO2 containing 10 ppm ethylene, we found no effect of 10 ppm boron on percent germination. Evidence that ethylene did not affect the rate of growth of lily pollen tubes was obtained from tube length distribu- tions in hanging drop cultures (Fig. 3). Adding ethylene did not significantly alter the distribution pattern in air or in 5% CO . However, CO with or without ethylene increased 2 2 pollen tube length. The influence of ethylene on rate of pollen tube growth was checked by measuring single pollen tubes in hanging drop culture on a microscope stage. The in- cubation chamber was continuously flushed with the apprOpriate gas mixture which could be changed at will. Adding or re- moving ethylene did not alter the growth rate of pear pollen tubes (Fig. 4). The slight shifts in the growth curve are due to curvature which introduces an error into the length measurement. 31 Table III. The influence of 1 ppm ethylene on percent germination of peach and pear pollen cultured on agar and sealed in plastic bags for 5 hr. €229.21. 22a; Experiment Air C2H4 Air C2H4 1. 15.1 19.8 62.5 44.7 2. 21.4 27.4 48.7 65.1 3. 26.4 26.7 52.9 65.2 4. 27.1 27.2 30.2 60.6 5. 25.7 16.1 64.4 61.8 6. 24.8 21.1 52.0 56.7 7. 19.4 20.2 60.4 47.9 8- 12.-.4. as 29.1 .5210. Mean 21.5 22.0 54.0 57.6 S.D. 5.5 t 6.5 111.1 1 7.6 Figure 3. 32 Effect of 10 ppm ethylene and 5% CO2 on the distribution of lily pollen tube length after 2 hr. Gill! NA! [0 'Oltifl Gill “5 ‘0' 33 Air n ‘5' 9 C2“. co, jleAAAg :- CO! I ciut 5. . . y ,. A+kn4 Ll" ! 4 6 810120416182022240 2 4 6 0801214ibilio [INGIN 0! '0Lth "1.65 Figure 3 34 .mcmawspm usonuHB Nov wm ou cusumu mmumoapcfl + .mcmamnum Edd oon N mcflcflmucoo 00 mm mo wusuxae m on mmsmso m mwumoflpcfl + .NOU mm SDHz pmpmHHucm> hamsossflucoo musuaso doup mcflmcm: m CH obsu cmHHOQ Hood mamsflm 8 mo buzouu .v musmflm 35 L l l l l l J l 1 18 3 3 g: 9 an o v N (,ouww) 38n1 N3110d JO H19N31 80 IOO 120 140 160 I'IME AFIER INOCUtAHON (min) 60 40 Figure 4 Ill y I 1 1 II II" [.111 J Ill '1 u, . l‘.‘ 111 I I'll \ 36 The extreme variability of all pollen material used was a constant frustration throughout these studies. The range in percent germination within replicates is seen in Table II. Similarly there was extreme variation in pollen tube length both between and within individual replicates whether employ- ing hanging drop, submerged, or shake culture. Tube lengths ranged from just emerging to 20 or more times the grain diameters. Where the growth of two neighboring pollen tubes was measured under the microscope very different rates were obtained. The same variability was observed in hanging drop cultures of lily pollen when undiluted stigmatic fluid was used as the medium. It is doubtful that the artificial growth medium used is the source of the variation. Perhaps the method of collecting the pollen results in a sample of varying maturity. Further experimentation is needed to clarify this point. The apparent inability of pollen to synthesize ethylene and its insensitivity to exogenous ethylene are unique. Pollen is a rich source of auxin which induces ethylene syn- thesis in stylar tissue of orchids, and eventually the other floral parts, causing them to senesce (3). Auxin stimulation of ethylene synthesis in vegetative tissue and fruits is a general phenomenon resulting in many diverse morphological and physiological changes. The marked stimulatory effect of CO2 on pollen germination and tube growth has not been generally recognized. External application of CO2 apparently enhanced . ll".‘lllllull. III: till ,I ll 1| 1 1|! II]! I lull" llllll. Ill, ‘1 [I’ll 1|.- 11 I‘ I 37 pollen tube growth in Brassica (6). Participation of endo- genous CO in this process is likely because stimulatory 2 levels were found within the stylar cavity of lily (Table I). CO2 from stylar metabolism may in fact modulate pollen tube growth because the internal CO2 concentration would reflect respiration rate changes. In addition, a declining concen- tration of 02 observed (5) within the style implies a correr sponding increase in CO2 concentration. The biochemical role of CO2 in germination and tube growth has not been ascer- tained. CO2 fixation which is known to occur in pollen (9) may regulate the supply of oxaloacetic acid and hence affect the rate of metabolism. The marked influence of CO2 on pollen germination and tube growth may explain the well known "population effect" wherein germination increases as the pollen population in- creases due to CO2 accumulation from respiration. LITERATURE CITED Buchanan, D. W. and R. H. Biggs. 1969. Peach fruit abscission and pollen germination as influenced by ethylene and 2-chloroethane phosphonic acid. J. Amer. Soc. Hort. Sci. 94:327—329. Burg, S. P. and Ellen A. Burg. 1965. Ethylene action and the ripening of fruits. Science 148:1190-1196. Burg, S. P. and M. T. Dijkman. 1967. Ethylene and Auxin participation in pollen induced fading of Vanda blossoms. Plant Physiol. 42:415-420. Dickinson, D. B. 1965. Germination of lily pollen: Respiration and tube growth. Science 150:1818-1819. Linkskens, H. F. and J. Schrauwen. 1967. Measurement of oxygen tension changes in the style during pollen tube growth. Planta 71:98-105. Nakanishii, T., Y. Esashi and K. Hinata. 1969. Control of self-incompatability by CO gas in Brassica. plant Cell Physiol. 10:925—937. Sauls, J. W. and R. H. Biggs. 1970. Ethrel, boron, sucrose and cellulase effects on peach pollen germi- nation. HortScience 5:341. Search, R. W. and R. G. Stanley. 1968. The effect of ethylene on pollen tube elongation. Plant Physiol. 43:8-52. Stanley, R. G., L. C. T. Young and J. S. D. Graham. 1958. Carbon dioxide fixation in germinating pine pollen (Pinus ponderosa). Nature 182:1462-1463. 38 SECTION I I INHIBITION BY ETHYLENE OF SOLUBLE DNA POLYMERASE ACTIVITY IN PISUM SATIVUM SEEDLINGS Abstract. An extract from the apical portion of etiolated seedlings of Pisum sativum L. was used as a test system to examine the action of ethylene on DNA polymerase activity. The extract was capable of catalyzing the polymerization of labeled deoxyribonucleoside triphosphates into a trichloro- acetic acid-insoluble product. The system required Mg++, nicked DNA, and all four deoxyribonucleoside triphosphates for maximum activity. Inorganic pyrophosphate and DNAase inhibited the polymerase activity. Extracts from plants previously treated with ethylene showed less activity to synthesize DNA than extracts from untreated plants. Ethylene in 21332 showed no activity. Inhibition of cell division by ethylene observed in this and other tissues may be the result of impaired synthesis of DNA polymerase. Recent studies on the effects of ethylene on growth of etiolated pea seedlings have shown (1) that the gas suppresses growth of the root and shoot apices, lateral branching, and leaf expansion predominantly by inhibiting cell division. Furthermore, ethylene inhibits DNA synthesis in the plumular 39 4O hook and subapical region of etiolated pea seedlings (1). Growth inhibition was therefore attributed to inhibition of DNA synthesis by ethylene. DNA polymerase activity is re- quired for DNA synthesis and increases when cells approach the S phase and begin replication (5,6). Nucleic acid polymerases have been isolated from plant tissues (20,22,23) and some plant growth hormones affect their activity (2,11, 14,15). The purpose of this study was to determine whether ethylene controls DNA synthesis by altering DNA polymerase activity. Seeds of Pisum sativum (cv Alaska) were soaked for 5 hours in tap water, planted in moist vermiculite and grown in complete darkness at 23°C. Seven days after planting, seedlings were treated with 50 ul/R ethylene in air. Approxi- mately 500 plants were utilized in each experiment. Plants were placed in an air-tight chamber into which a gas mixture with the desired ethylene concentration was introduced at a flow rate of 470 ml/min. All manipulations were carried out under dim green light, otherwise the seedlings were kept in total darkness. At the end of 24-hour treatment, seedlings were transferred to 2°C, and the apical portions, including the plumule and plumular hook, were excised. The harvested tissue was weighed and kept at 2°C for preparation of the tissue extract. Five grams of chilled sections were sterilized with 5% sodium hypochlorite (diluted 1:100) and ground for 20 min in 41 a chilled mechanized mortar and pestle with 2 weights of grinding medium composed of 0.05M Trizma and 0.02M mercapto- ethanol at pH 8.0. The suspension was filtered through 4 layers of cheesecloth plus 2 layers of miracloth and centri- ‘fuged for 5 min at 400g to remove residual cell wall debris. The supernatant solution was used for DNA polymerase assay. Nicked DNA was prepared from calf thymus DNA (2.5 mg per ml in 0.01M tris buffer) incubated with pancreatic DNAase I (40 ng DNAase per mg DNA) and 5mM MgCl2 at 37°C for 25 min. The reaction was stopped by heating for 10 min at 60°C. Double stranded DNA treated in this manner is rendered 25% acid soluble (21). The incorporation of TMP (thymidine monophosphate) into acid insoluble material was used to measure DNA polymerase activity. Unless otherwise stated, 25 pi aliquots of tissue extract containing an appropriate amount of enzyme based on the protein content, were added to 225 ufl reaction mixture containing in umoles: Hepes buffer, pH 7.5, 20; KCl, 12.5; MgCl, 1; Cleland's reagent, 1; dATP, 0.05; dCTP, 0.05; dGTP, 0.05; TTP, 0.02; 6.2 uc 3H-TTP (specific activity 15.5 c/m mole) and 100 ug nicked calf thymus DNA. The incubation was carried out at 37°C. The reaction was stopped by adding 2 ml of 10% cold TCA containing 1% PPi. Fifty microliters of BSA (bovine serum albumin) were added to the mixture and centri- fuged for 10 min at 10,0009. The pellet was dissolved in 0.5 ml of 0.2M NaOH and 5 ml of 10% cold TCA + 1% PPi were added. 42 The acid insoluble pellet was collected on a Whatman FG/C glass filter paper and washed five times with 5% cold TCA and 1% PPi. The filters containing the acid insoluble precipitate were placed in scintillation Vials containing 10 ml Bray's solution (3) and 3H was determined with a Beckman scintilla- tion spectrometer with a counting efficiency of 35% for 3H. Incorporation of 3H—TMP into acid insoluble material was ob- tained by subtracting zero time counts from those of experi- mental samples. Protein content in the tissue extracts was determined by the method of Lowry et 31. (12) on cold TCA precipitated samples. Soluble DNA polymerase activity as measured by the incorporation of TMP into acid insoluble product was dependent upon the various components of the reaction mixture (Table 1). Highest enzyme activity was obtained in the presence of Mg++, nicked DNA and four deoxyribonucleoside triposphates (dNTP). Omission of Mg++ reduced the incorporation rate to 17% of that obtained with the complete mixture. Omission of dATP, dCTP, or dGTP reduced the incorporation to 20-23%, while omitting all three of them reduced the activity to only 10% of that of the complete reaction mixture. The incorporation of TMP into acid insoluble product was dependent upon the addition of DNA to the reaction mixture since only 3% in- corporation was observed without added DNA. Various templates were tested for their priming activity (Table 2). The most active for pea DNA polymerase was 43 Table l. Dependency of DNA polymerase activity on components of the reaction mixture. Reaction Mixture % Incorporation Complete 100 -Mg++ l7 -dATP 20 -dCTP 23 -dGTP 23 -dATP, dCTP, dGTP ll -Nicked DNA 3 v v Soluble DNA polymerase was extracted from the apical portion of etiolated pea seedlings. 100% incorporation represents incorporation of 140 pmoles TMP per mg protein. 44 Table 2. Utilization of different templates by pea DNA poly- merase. DNA Templatel % Incorporation3 Nicked 100 Nicked denaturated 47 Native 36 Native denaturated 16 poly dT:rA2 o 1Calf thymus DNA. 2Polydeoxythymidylate and polyriboadenylate. 3100% incorporation represent incorporation of 140 pmoles TMP per mg protein. 45 "nicked" DNA, i.e., DNA in which 3' hydroxyl termini have been introduced by the action of pancreatic enzymes. The priming activity of nicked calf thymus DNA was found to be 2.5 fold higher than that of native calf thymus DNA. Heat denaturation of native or nicked DNA reduced the priming activity by about 50%, indicating that the enzyme requires some double stranded structure for optimal priming activity. Utilizing the synthetic template poly dT:rA, which is used for detection of RNA—directed DNA polymerase enzyme (21), resulted in no incorporation of TMP into acid insoluble product. This may indicate that RNA cannot meet the template requirement of pea DNA polymerase. Adding l umole perphos- phate, one of the reaction products, to the reaction mixture inhibited the incorporation of TMP by 86% (Table 3). Pancreatic DNAase degraded the newly formed DNA and reduced the amount of TMP recovered by 94%. Gasing the incubation mixture with ethylene (1 ml/l in air) for 30 min prior to the incubation reduced the incorpor- ation rate by only 3-5% (Table 3). In contrast, ethylene treatment of intact pea plants lowered DNA polymerase activ- ity in the apical region to approximately 14% of the activity found in control plants (Fig. l) at low levels of protein in the assay and to 40% when compared at their respective peak values. The rate of incorporation increased at a decreasing rate with increasing amounts of soluble protein added to the assay mixture, and reached a maximum of 3400 cpm at 100 pg 46 Table 3. Effect of in vitro ethylene treatment, DNAase and pyrophosphate on TMP incorporation into acid- insoluble product. ;.___‘.___ AA- Treatment % Incorporationl Control 100 in vitro incubation w1th ethylene (l ml/l) 97 in vitro 30 min pre- lncubation with ethylene (1 ml/l) 95 pyrophosphate 1 umole l4 DNAase 20 mg/assay 6 1100% incorporation = 4630 cpm/assay 47 .Iol mmcflapmwm pmummup mamamnum H: am no no: Houucoo ampln Scum pmumaomfl mos mmemE>H0d ézo .mmmmw opp CH usmusoo samuoum Ucm mzenm m mo coHDMHOQHOOQA cmwzumn QHSmQOHumHmH one .H mnsmflm 48 H musmwm 335391 com 09 our om ov q u 4 q q i u u q - 009 mu d oi. \ .. 000w W I . 0’. 0‘ If. \ I.’ .s. l o’.’ 0‘ .b. \o\ I, 4x - ooom IIIIAW \\\\ b p n n n p p p b - 49 protein per assay in control tissue. Similar incremental additions of enzyme from ethylene treated plants yielded a maximum rate of only 1500 cpm at 140 ug protein in the assay mixture. Control tissue yielded a nonlinear rate of incorpor- ation whereas a linear rate was observed for ethylene treated tissue until the maximum rate was reached. In non-treated tissue, incorporation of TMP was reduced at high protein levels, suggesting degradation of the newly formed DNA by endogenous DNAase. The degradation of DNA appeared to be more rapid in the control than in the ethylene treated tissue, since at 180 ug protein per assay 50% of the newly formed DNA may have been hydrolyzed. Similarly, only 17% of the DNA was degraded by the extract from ethylene treated tissue at a level of 218 ug Protein per assay and perhaps less than that up to the maximum since the incorporation rate was linear. The amount of TMP recovered was apparently the net result of DNA polymerase and DNAase activity. Careful examination of Fig. 1 reveals that reduced DNA polymerase activity in ethylene-treated tissue may have been the result of reduced enzyme synthesis. Twenty ug extract protein from ethylene treated tissue was only 1/7 as active as that from the control tissue. At the 140 ug extradt protein level (7 times as much) the activity of the ethy1ene treated tissue was equal to that of 20 ug of control tissue. This proportionality suggests that the enzyme quantity was limiting the incorporation rate in the ethylene treated tissue. Aliquots containing 100 ug 50 protein per assay were used for subsequent experiments and the results are expressed per mg protein. The kinetics of the reaction is illustrated in Fig. 2. Incorporation increased with incubation time up to 15 min, reaching maxima of 140 and 90 pmole TMP incorporated for control and ethylene treated tissue, respectively. A 10 min. incubation period was employed for further experiments. Incorporation of TMP increased sharply with increase in template concentration (Fig. 3) up to 2.5 (ethylene-treated) or 5 ug (control) DNA. Higher concentrations increased in- corporation but at a much lower rate, reaching 109 and 70 pmoles TMP incorporated by extracts from control and ethylene treated tissue, respectively. The data demonstrate activity of soluble DNA polymerase in crude extracts of etiolated pea seedlings. By utilizing the soluble enzyme in the crude extract rather than the chromatin associated DNA polymerase complex, the effect of ethylene on the enzyme activity, per se, was elucidated. This eliminates possible effects of the endogenous template on the reaction, since its activity or availability might be affected by hormones as well (8,9,10). Table 1 shows that very little endogenous template was available in the crude' extract where only 3% incorporation of TMP was obtained with- out exogenously added template. The almost complete destruction of the acid insoluble product by pancreatic DNAase indicates that the product is a 51 .10: mmCHHpmmm pmpmouu MCmH>Cum CC «N no lo: HouuCoo CH nonpoud mHDCHOmCHIpHom ODCH COHumHOQHOOCH mze mo mmusoo mEHB .m mHCmHm 52 00 N wusmHm 3235:; NE: on mp q ON C O O C e w (D V (quJOJd Bw/senowd) Nouvuoauoom am 0 N P OV- 53 .uxmu 0CD CH meHuommp mm pmuwdmud mmB Czo msahzu Mano pmonz .Ion mmCHHpmmm pmumwuu mCmHmnum HC am no: HOHuCou COHHMHOQHOUCH mze Co CoHumuquoCoo mumHmEmu mo pomwmm .m mnsmHm m oupmHm 33 2 DOCUOHQ mHnsHOmCHIpHom OUCH COHuoHOQHOoCH ase mo mmusoo mEHB .N .OHh 68 I I I n O -l b d h . q - . -I - \. _ '0 o '1 (“Mom 'NOOIIW Bun/33mm!) uouvaouoam am nu! (minutes) 60 15 30 Figure 2 69 .mumnsu ounuom Scum meMHomH MHCUCOCOODHE ha Hosponm ansHOmCHlpHom ouCH COHDMHOQHOOCH CED mo mmusoo oEHB .m .mon 70 T I 7 T . d . Id cud . HI I l 0 V N (umom 'uoouw Owlse|owd) Houvaoaaoam awn 120 30 60 "ME (minutes) 15 0 7.5 Figure 3 71 .mnmnsu oumuOQ Eouw pmumHOmH mHHUCOCoouHE wn DUCUOHQ mHQCHOmCH IpHom OUCH CZB mo COHDCHOQHOUCH mCu Mom mpsum COHDMHHCQOCOU .w .mHm 72 Go" on. v musmHm 33 5205 ca. 56852.: c... + A 1 oov com d oom— coo— 73 .mumnsu cannon Scum pmuoHomH CHHpConoouHE wn nonpoud mHQCHomCH IpHom ODCH CED mo COHHMHOQHOUCH mnu How mpsum COHumeCmoCOU .m omflm 74 Com m stmHm 33 £205 .2552 0222 on. oo. - _ on com oov 000 Well? 75 A time course study showed maximal enhancement of the enzyme activity after an 18—24 hr ethylene treatment and a subse- quent decline. A three to four-fold increase in activity was obtained at the peak at all three concentrations of mito- chondria added per assay (Fig. 6). The lower incorporation rate observed at the highest level of mitochondria used is attributed to the action of ribonuclease. The effects of ethylene treatment on respiration and activity of cytochrome c oxidase, DNA polymerase, and RNA polymerase in a parallel experiment are shown in Fig. 7. The increase in RNA polymerase activity of mitochondria pre- ceded the increase in respiration rate of intact tubers. The peak in respiration rate occurred almost six hrs later than the peak in RNA polymerase activity (2 l/2-fold increase in the activity). DNA polymerase activity increased six hrs after the initiation of the ethylene treatment, declined at 18 hrs, coinciding with the period of maximum RNA polymerase activity, and subsequently rose. Ethylene treatment also enhanced the activity of cyto- chrome c oxidase. With the exception of the 6th hr measure- ment the increase in cytochrome c oxidase activity slightly preceded but otherwise paralleled the increase in respiration induced by ethylene. 76 .mmmmM\pmppm MHHUCOEO IouHE “1 mm >....> .H: om Ol.l.lo .Hn 00H IIIIIIIII .UmumoHpCH mEHu HOw mCmHECpm CpHs quEummHu Hmpmm muons» cumu0d EOHw pmuMHomH CHHUCOCUODHE En poopoum mHDCHOmCHIpHom ouCH mED mo COHumpomHOOCH me Co mCOHumHsp msoHHm> HOW o>H>.mm quEummHD mCmHEEDm mo nommmm one 77 l 1 I I- ...D .’. " 0.. 0’ 0'. 0’ .' .’ .' .I .0. .I .' I / .0 I If i - \ °. \, '-. \, ... \. r- 35. ‘ D d I- 1‘ J O a (um: 09/ ugelom um O O N — Owlseuowd) Mouvaouoqm awn 30' to Home 12 Figure 6 78 .ooopon o oEounoov>o«Inll.. .CoHuoHOQHooCH mED II.II .CoHuouomuooCH mE9.U.II|;U .HOHDCOU mo CoHuoHHdmoH o...o...o .moououom pouoouu oCoHEnuo mo COHuoHHQmoH o c .mnonsu onouom Scum popoHOmH oHupConoouHE CH nonpoum oHnsHomCHlpHoo ouCH mEDtmEB mo coHuoHomHOOCH pCo .omopon o oEonnooumo .COHuoHHmmou Co uCoEuooHu oConnuo mo poommo one .5 omaafirm 79 (ugw 09/ugalmd 'uw fiwlsanow d) NOIIVIOJUODNI awn—aw: 0’. O..0...o-.‘o..°...°~.o".o..O..o..°..O“ 00 o o _l 0"00.... Coo-.0..." l 33.. J l g :9 s (Ju-Bx/Zoo 6w) uouelgdseu l l 1 L L J a e s a 2 ° (|onuoo aAoqe [in/mos pads 09 efiueqo'x.) asepgxo o ewomooMo 0 M 30 18 12 Hou rs Figure 7 80 Discussion Isolated mitochondria possess many characteristics which enable in_yit£g examination of processes occurring in 3139. The technique has revealed increased enzyme activities in mitochondria isolated from both animal (14,15) and plant tissues (16) treated with hormones. Our data indicate that ethylene, a plant hormone, can induce changes in the activity of mitochondrial DNA and RNA polymerase. RNA polymerase activity in potato tubers rose during the first 18-24 hrs of treatment with ethylene, then declined during an additional 6 hrs. Respiratory activity was greatest when RNA polymerase activity began to decline. Ethylene induced a smaller increase in DNA polymerase activity, noticeable 6 hrs after the initiation of ethylene treatment. However, the relatively long intervals between measurements . did not allow determination of the time required for induction. RNA polymerase may allow the synthesis of mitochondrial proteins necessary for respiratory activity. The increase in specific activity of cytochrome c oxidase slightly before the rise in respiration supports this hypothesis. The changes in activity of RNA polymerase and cytochrome c oxidase paralleled, but preceded the change in respiration rate of ethylene-treated whole tubers. The decline in enzy- matic and metabolic activity suggests that this is of func- tional significance in mitochondrial metabolism. The Specific 81 mode of action by which ethylene affects nucleic acid metabo- lism is not known. The failure to induce in_yitrg, any changes in the enzyme activity of the isolated intact mito- chondria suggests the involvement of the nucleus or intact cell in the interaction of ethylene with the site of action. Potato tubers normally do not encounter ethylene at levels sufficient to cause this striking increase in respira- tory activity. Their respiration rate is sensitive to numerous environmental stresses and to circadian rhythm. The sequential rise and fall in DNA and RNA polymerase activ- ity, cytochrome c oxidase activity, and respiration rate in response to applied ethylene suggests that a mechanism exists to restore order and balance to metabolism. Perhaps other stress factors which induce less marked respiratory changes produce similar but more subtle metabolic changes. 10. 11. 12. 13. 14. 15. 16. LITERATURE CITED Reid, M. S. and Pratt, H. K., Nature, 226, 976 (1970). Reid, M. S. and Pratt, H. K., Plant Physiol., 42, 252 (1972). Wang, C. Y., Mellenthin, W. M. and Hansen, E., J. Amer. Soc. Hort. Sci., 21, 9 (1972). Nichols, R., J. Hort. Sci., 43, 335 (1968). Miller, L. A. and Romani, R. J., Plant Physiol., 41, 411 (1965). Switzer, C. M., Plant Physiol., 32, 42 (1957). Key, J. K., Hanson, J. B. and Bils, R. F., Plant Physiol., 35, 177 (1960). Jones, J. D., Hulme, A. C. and Wooltorton, L. S. C. New Phytol. 64, 158 (1965). Dilley, D. R., Dewey, D. H. and Dedolph, R. R., J. Amer. Soc. Hort. Sci., 24, 138 (1969). Huang, A. H. C. and Beevers, H., Plant Physiol., 48, 637 (1971). Simon, E. W., Biochem. J., 69, 67 (1957). Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., J. Biol. Chem., 193, 267 (1951). Bray, G. A., Anal. Biochem., l, 279 (1960). Tata, J. R., Ernster, L., Lindberg, 0., Arrhenius, E., Peterson, S. and Herdman, R., Biochem. J., 86, 408 (1963). Boodyn, D. B., Freeman, K. B. and Tata, J. R., Biochem. J., 94, 628 (1965). 'Baxter, R. and Hansen, J. B., Planta, 82, 246 (1968). 82 SECTION IV INDUCTION OF AUTOCATALYTIC ETHYLENE PRODUCTION IN APPLE FRUITS BY PROPYLENE IN RELATION TO MATURITY AND OXYGEN DEPENDENCY Abstract. Ethylene, the fruit ripening hormone, and other olefinic compounds cause apples and other climacteric fruits to ripen. Ethylene biosynthesis becomes autocatalytic as ripening proceeds. Propylene, which fruits do not produce, was employed to determine the stage of maturity apples must attain to autocatalytically produce ethylene and the effect of O2 tension on autocatalysis. Red Delicious apples har- vested at developmental stages representing 52, 58, 65 and 75% of maturity were gassed with propylene at conc. of 0, 10, 50, 100, 500 and 1000 ppm for one week at 20°C. The ability of propylene to stimulate ethylene production increased progres- sively with fruit maturation, but rate of production following treatment with 500 ppm propylene was constant. A shorter lag time to the onset of autocatalytic production was observed for the more mature fruit which reflects a natural increase in sensitivity. PrOpylene administered at 6.5% 02 or less did not induce ethylene production, but an anaerobic atmosphere was necessary to completely inhibit ethylene synthesis in fruits once autocatalysis began. 83 84 Introduction Autocatalysis of ethylene production (6,7,17) is a strik- ing feature of ripening in climacteric fruits, and is triggered by exposure to ethylene at concentrations above a threshold level (14,28). In most studies of the role of ethylene in ripening of climacteric fruits two processes, ethylene action and production, are interrelated because of the autocatalysis phenomenon. Experimentally it is difficult to study them separately. One way to assess the role of ethylene in fruit ripening consists of treating the fruit with ethylene at various concen- trations and measuring the ripening response (18,21,26,28). Numerous investigations have been undErtaken to determine the threshold concentration which triggers ripening (l,2,3,l4,25, 27,28). However, it is not clear whether this threshold con- centration itself causes ripening or whether it stimulates ethylene production to physiologically active levels which in turn cause the fruit to ripen. Hackett gt 213 (14) have sug- gested that the action of ethylene as a ripening hormone in fruits may be separated into two distinct processes; the first is the initiation of ethylene synthesis, the second the physio- logical response. Sensitivity to ethylene is not constant throughout the life of the fruit (7), for threshold level needed to cause ripening of climacteric fruits is dependent on the age of the tissue. Fruits attached to the tree are less sensitive to 85 ethylene than harvested fruits (23), suggesting that ethylene is rendered ineffective by a ripening inhibitor supplied by the parent plant (5). The threshold sensitivity of fruit to exogenous ethylene may depend upon the supply of oxygen (16,24). This was attributed to the decrease of ethylene production under the low oxygen tension (4,9,11,12,15,16). In this paper the sensitivity of the apple fruit to auto- stimulation was studied in relation to the age of the tissue and to 02 levels required. To avoid complications with the ripening process, premature apples were used starting from a very early stage (52% maturity). In order to measure only endogenous ethylene, prOpylene was used to trigger ethylene production. Thus, the potential for autocatalysis was de- termined by the fruit's response to propylene at different stages. Propylene, like ethylene, promotes fruit ripening, inhibits elongation of pea subapical sections and causes epinasty (10). Burg and Burg (8) determined the molecular requirements for ethylene action and found propylene to be the next most active compound to show ethylene-like action. The equivalent concentration of propylene to cause half-maximum ethylene response was found to be 130 times that of ethylene. The role of oxygen in autocatalysis was studied by applying prOpylene at different oxygen tensions. 86 Materials and Methods Plant material: Red Delicious apples were obtained from trees at the Horticultural Research Center at Michigan State Univer- sity. Fruits were harvested at 52, 58, 65 and 75% maturity, based on a l45-day post-bloom growth period. For experiments on the effect of O2 in the stimulation of ethylene production by propylene, apples were harvested at 83 and 90% maturity. The fruits used for each experiment were harvested from the same tree and were about equal in size. The experiments described were commenced approximately two hr after harvesting the fruits. Propylene treatments: Propylene at concn. of 0, 10, 50, 100, 500, and 1000 ppm was applied by injecting known amounts into chambers in the apparatus described in the Appendix. Concen- trations were checked by gas chromatography. When prOpylene was administered at 100 ppm at different 0 levels the gas was 2 injected into fruit chambers previously flushed with gas mix- tures prepared by cylinders. Measurements of ethylgne and CO2 production: Ethylene produc- tion was measured by gas chromatography of samples withdrawn at intervals from the chambers, using a Varian Aerograph 1700 gas chromatograph equipped with a flame ionization detector and a 1/8" x 4' column of activated alumina. The column temp- erature was 50°C with a N2 carrier gas flow of 40 ml/min. 87 The instrument was capable of measuring 10 ppb of ethylene in a 1 ml sample with a peak height : background noise level of 2. Measurements were made every one or two days to determine the rate of ethylene production. Fruit reSpiration was measured by CO absorption in 2N KOH as described in the 2 Appendix. The two-indicator method was used for the titri- metric determination of carbonate and bicarbonate. The fruit chambers were aerated every two or three days to avoid excessive accumulation of ethylene. Results and Discussion Sensitivity_of the tissue to propylene: Propylene triggered the fruits to produce ethylene. The efficacy of each concen- tration of propylene to stimulate the fruit to produce ethylene increased progressively during maturation (Fig. l). The mini- mum concentration of applied propylene and the time required to stimulate ethylene production decreased progressively dur- ing maturation (Fig. l, I, II, III and IV). Propylene at 500 or 1000 ppm applied at 52% maturity stimulated the fruit to produce ethylene by the 7th day at a rate of 50 ul/kg-24 hr (Fig. l, I) indicating that a rudimentary mechanism for ethyl- ene production existed at this early stage of development. At 75% maturity, treatments with 50, 100 and 500 ppm stimulated the fruit to produce ethylene at the 7th day at the rate of 400 ul/kg-24 hrs, which is very close to the highest rate of 88 Figure 1. Effect of propylene treatment on ethylene production in Red Delicious apples at 52, 58, 65 and 72% maturity. 600 500 400 300 200 100 300 200 pl C3015] Kg-24 hr: 300 200 100 100 89 0—0 CO H? I0 I. 0"'“". IO pun V-—v so .. Ann... '00 u D-‘--O 500 u I—I 1000 '0 75 x unuanv (IV) as a: MAIUIIIY (m) 53 x MAVUIHY (u) 1 2 a 4 5 o 7 ma (non) Figure 1 90 ethylene production reported for apples (14,24). Intermediate rates of ethylene production were found for fruit at 58 and 65% of full maturity. Following treatment with 500 ppm propylene, the rate of ethylene production rose exponentially with time, as illus- trated on log scale in Figure 2. This behavior is compatible with the concept of autocatalysis in ethylene production. Rate of production did not vary with stages of maturity as indicated by similar slope. However, more mature fruits re- quired less time to initiate production which may reflect an increase in the sensitivity of the fruit for autocatalysis with maturation. All fruits of different ages treated with propylene showed moderate increases in respiration rates as measured on the 3rd or 4th day (Fig. 3). The increase in respiration was more marked in the early stages of maturation and at propylene concentrations above 50 ppm. Propylene even at 1000 ppm had no effect on ripening. Although the gas induced the fruit to produce large amounts of ethylene the 7th day, flesh softening and content of soluble solids were little affected when measured 2-3 weeks after the prOpylene treatment. This suggests that although the mechan- ism for autocatalysis of ethylene production is functional in the early stages of maturation the fruit has not obtained the required potential for ripening. 91 .annsnms A>Hv wms tam .AHHHV wmm .AHHD wmm .AHV wmm no powwow» moHddo msoHoHHoD pom CH COHuospoud oConnuo mo ouou Co uCoEuooHu oCoHEdond Edd oom mo poommm .m ousde 92 N ousmHm ....a. w¢‘_ — J l ON O? OO O 0 OO— 00“ 00¢ 000 'I‘ ma vz-Gx/MZD 93 .uCoEuoouu oCondoud Condo woo nuvtpum onp nonsmooe .EuHuouoE Amv wmm pco ADV wmm .AUV mmm .Amv wmm .ACV mom no moHddo mCOHoHHoD pom CH CoHuoCHdmoH Co COHpoHuCoOCoo oCoHEdoud mo uoowmm .m oHCDHd 94 occ— can 1 OO— m mhfimwh :2... o: no On - O— l Goa oov con 95 Stimulation of ethylene production by propylene'in atmos— pheres deficient in oxygen: Autocatalysis of ethylene produc- tion was retarded by low 0 levels (Fig. 4). Fruits at 83% 2 of full maturity and treated with 100 ppm propylene required 6.5% or more 02 in the atmOSphere to trigger the mechanism for autocatalysis of ethylene production. Concentrations of 02 less than 6.5% gave very low rates of ethylene production which was not different from that obtained from fruit not treated with propylene. Above the threshold value of 6.5% a linear dependency upon 0 concentration was observed, with a satura- 2 tion level apparently above that of air. In order to further elucidate the role of 02, fruits were shifted to low or high 02 before or after the inductive treat- ment with prOpylene. The fruits held in 4.6% O and 100 ppm 2 propylene showed no sign of autocatalysis up to the fifth day (Fig. 5). When they were subsequently shifted to air plus propylene, ethylene production followed the same pattern as in fruit treated initially with prOpylene in air. Once the fruit gained capacity to produce ethylene, an anaerobic atmosphere was necessary to completely inhibit its synthesis. Transfer- ring the fruit to 4.6% 0 did not stop production, and even 2 at 1.2% 02 the fruit produced a considerable amount. At 10.5% 02 ethylene production was initially about 50% of that ob- served in air. However, there was little difference after 6 days. 96 .muHHCuoE wmm no powwow» moHddo mCoHoHHoD pom CH oCoHEdoud Edd OOH an CoHuonoud oCoHEnuo mo CoHuoHCEHum onu CH COHuouuCooCoo comwxo wo uoommm .v oHCDHd 97 ON 0. v oHCDHm 2w0>x0 i a. o ° 0 ' N "‘l n-Ox /',.ID .a O O on cop 98 .EanCnoE mom no oonoonn moHddo mCoHoHHoD pom CH oCondond Edd ooH En CoHnospond oConnno mo CoHnoHCEHnm onn CH onsmdeo mo oEHn onn no CoHnonnCooCoo Comwxo mo noomwm .m onsde 300 " 99 l O 8 MI vz-‘x flat: I“ I O O F I new.-- nu I. ‘5.+ C 3“ 0““ 4.6% +1: I’ 0.— I2 I 6 VIM! (Days) Figure 5 100 Thus, the stimulation of ethylene production by propylene is in some way linked to the utilization of atmospheric 02. Fruits kept at the threshold 0 concentration produced less 2 CO than fruits kept in air (Fig. 6). Our results are in 2 accord with those obtained by other investigators with apples (13,24) and bananas and tomatoes (20) where low 02 level prevented the onset of the climacteric by inhibiting ethylene production. They clearly indicate the importance of low 02 levels in retarding the autocatalysis of ethylene production in preclimacteric fruits, vs. their failure to retard ethyl- ene production in fruits which are already synthesizing ethylene at high rates. The results emphasize the importance of placing fruit in controlled atmosphere storage just before they have attained capacity for autbcatalysis, and the neces- sity for a reliable method of predicting the harvest date for fruit intended for long term storage. While this investigation was being conducted, McMurchie gt'al. (22), found that propylene induced ethylene production in bananas but not in citrus fruits. The authors suggested the existence of two ethylene producing systems in climacteric fruits, the one reSponsible for autocatalysis being missing from non-climacteric fruits. This suggestion receives support from the present study for the two ethylene producing systems in climacteric fruits, the non-autocatalytic system is under the control of limited supply of oxygen, while the autocataly- tic system is less dependent upon oxygen. This may indicate 101 .EnHHCnoE wmm no oCoHEdond Edd OOH nnHB oonoonn moHddo msoHoHHoD now no CoHnoanmon onn CH CoHnonnCooCoo Comwxo no noommm .o onCde 102 m ousde «gusty mi.» IO 0 4‘ q “5.-.-lo'o'o'o'o'o'o' 0'0..." 0300 no. 0000...... 00.000.00.00. 'o'W'Noo No 00.000 'o'o'o' 0.0.00.0. 0'. ‘0 8° ......” ‘1- l.ll 09 09 O O N M: n-‘x/‘OD Inl 0mm 103 differences in the affinity of enzymes with oxygen involved in the two ethylene producing systems. 10. LITERATURE CITED Biale, J. B., R. E. Young, and A. J. Olmstead. 1954. Fruit respiration and ethylene production. Plant Physiol. 29:168-174. Biale, J. B. 1960. The postharvest biochemistry of tropical and subtropical fruits. Advances in Food Res. 10:293-354. Brady, C. J., P. B. H. O'Connell, J. Smydzuk and N. L. Wade. 1970. Permeability, sugar accumulation, and respiration rate in ripening banana fruits. Austr. J. Biol. Sci. 23:1143-1152. Burg, S. P., and K. V. Thimann. 1959. The physiology of ethylene formation in apples. Proc. Nat. Acad. Sci. 45:335-344. Burg, S. P. 1963. Studies on the formation and func- tion of ethylene gas in plant tissues. Regulateurs Naturels de la France (Centre National de la Recherche Scientifique, Paris 1964), p. 719. Burg, S. P., and E. A. Burg. 1965. Relationship between ethylene production and ripening in bananas. Botan. Gaz. 126:200-204. Burg, S. P., and E. A. Burg. 1965. Ethylene action and the ripening of fruits. Science 148:1190-1196. Burg, S. P., and E. A. Burg. 1967. Molecular require- ments for the biological activity of ethylene. Plant Physiol. 42:144-152. Craft, C. C. 1960. Ethylene production by tomato tissue. Plant Physiol. 35 (Supp.) vii. Crocker, W., A. E. Hitchcock, and P. W. Zimmerman. 1935. Similarities in the effects of ethylene and the plant auxins. Contrib. Boyce Thompson Inst. 7:231-248. 104 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21- 105 Gane, R. 1934. Production of ethylene by some ripening fruits. Nature 134:1008. Gane, R. 1935. Identification of ethylene among volatile products of ripe apples. Ann. Rept. Food Invest. Board, Lond. 123:3. Hulme, A. C., M. J. C. Rhodes, and L. S. C. Wooltorton. 1971. The effect of ethylene on the respiration, ethylene production, RNA and protein synthesis for apples stored in low oxygen and in air. Phytochem- istry 10:1315-1323. Hackett, P. J., A. C. Hulme, M. J. C. Rhodes, and L. S. C. Wooltorton. 1971. The threshold value for physio- logical action of ethylene on apple fruits. J. Fd. Technol. 6:39-45. Hansen, E. 1942. Quantitative study of ethylene produc- tion in relation to respiration of pears. Botan. Gaz. 103:543-558. Kidd, F., and C. West. 1933. The influence of the compo- sition of the atmOSphere upon the incidence of the climacteric in apples. Ann. Rept. Food Invest. Board, London, pp. 51-57. Kidd, P., and C. West. 1945. Respiratory activity and duration of life of apples gathered at different stages of develOpment and subsequently maintained at a constant temperature. Plant Physiol. 20:467-504. Lyons, J. M., and H. K. Pratt. 1964. Effect of stage of maturity and ethylene treatment on respiration and ripening of tomato fruits. Proc. Amer. Soc. Hort. Sci. 84:491-500. Mapson, L. W., and J. E. Robinson. 1966. Relation between oxygen tension, biosynthesis of ethylene, respiration and ripening changes in banana fruit. J. Fd. Technol. 1:215-225. Mapson, L. W. 1970. Biosynthesis of ethylene and its control. Proc. Conf. Trop. Subtrop. Fruits, Tr0p. Prod. Inst., pp. 85-92. McGlasson, W. B., and H. K. Pratt. 1964. Effects of ethylene on cantaloupe fruits harvested at various ages. Plant Physiol. 39:120-127. 22. 23. 24. 25. 26. 27. 28. 106 McMurchie, E. J., W. B. McGlasson and I. L. Eaks. 1972. Treatment of fruit with propylene gives information about the biogenesis of ethylene. Nature 237:235- 236. Meigh, D. F., J. D. Jones, and A. C. Hulme. 1967. The respiration climacteric in the apple. Production of ethylene and fatty acids in fruit attached to and detached from the tree. Phytochemistry 6:1507-1515. Meigh, D. F., and J. Reynolds. 1969. Effect of low con- centrations of oxygen on the production of ethylene and on other ripening changes in apple fruit. J. Sci. Fd. Agric. 20:225-228. Palmer, J. K. 1970. The banana. In The Biochemistry of Fruits and Their Products. (A. C. Hulme Ed.), p. 75. (Academic Press, London and New York). Pratt, H. K., and M. Workman. 1962. Studies on the physiology of tomato fruits. III. The effect of ethylene on respiration and ripening behavior of fruits stored at 20°C after harvest. Proc. Amer. Soc. Hort. Sci. 81:467-478. Pratt, H. K., and J. D. Goeschl. 1968. The role of ethyl- ene in fruit ripening. In Biochemistry and Physiology of Plant Growth Substances (Wightman, F. and G. Setterfield, Eds.), pp. 1295-1302. (Runge Press, Ottawa). Wang, C. Y., W. M. Mellenthin, and E. Hansen. 1972. Maturation of 'Anjou' pears in relation to chemical composition and reaction to ethylene. J. Amer. Soc. Hort. Sci. 97:9-12. SECTION V INTERNAL ETHYLENE CONCENTRATIONS IN APPLE FRUITS ATTACHED OR DETACHED FROM THE TREE Abstract. A method is described for the measurement of intern- al ethylene concentration in fruits on the tree. Internal ethylene concentration was followed in Red Delicious apples attached to or detached from the tree throughout the matura- tion period. Levels observed in fruits immediately after harvest were similar to those in fruits on the tree. Harvest- ing fruits stimulated their ethylene production capacity more quickly as the fruits mature on the tree. Internal ethylene concentrations remained low in fruits left on the tree until ripening changes became evident. Isolating the fruit from leaves by girdling and defoliation hastened the increase in internal ethylene concentration. Leaves left above or below the fruit-bearing spur retarded the rise in ethylene, support- ing the concept of a ripening inhibitor originating in the leaf. Introduction Many fruits enter the climacteric soon after harvest, whereas they might not ripen for a few months if left on the tree (8,11,12,13). This is especially true for most avocado fruits which do not undergo a climacteric or ripen while attached to the tree. 107 108 It is commonly agreed that a substance entering the fruit from the tree inhibits the ripening process (5,6,7,10,l7). Burg (7) hypothesized that ethylene is rendered ineffective by a ripening inhibitor supplied by the parent plant, and that harvesting the fruit removes it from the source of this in- hibitor. Evidence derived from steam girdling experiments with avocados, mangos and other fruits suggested that the in- hibitor is transported in the phloem from leaf to fruit (5,6). Mapson and Hulme (17) suggested that the inhibitor either inhibits ethylene production or raises the threshold value at which ethylene becomes physiologically active in promoting ripening. The present work was undertaken to determine the effects of removal from the tree and branch girdling and or defoliation on the internal ethylene concentrations in apple fruits. A method was developed to monitor the internal ethylene con- centration of fruit attached to the tree. Material and Methods Various methods have been used for determining the intern- al ethylene concentration in tissues (2,4,14,15,18), the most common being the withdrawal of internal gas samples with a hypodermic syringe while the fruit is momentarily submerged in water (4,14,15). In other methods, vacuum is applied to the tissue and ethylene is measured in a sample of the extracted air (2,18). Such techniques (3,9,16) have been criticized on 109 the grounds that the application of vacuum might upset the equilibrium existing between dissolved gases and the gases in the intercellular spaces (1,3). Furthermore the same plant material cannot be used repeatedly with any of the above methods. In the present study a modification of the technique described by Williams and Patterson (19) was applied to measure the internal ethylene concentration (IEC) of the same fruit either attached to or detached from the tree. The sys- tem consisted of a 22 gauge 1 1/2" hypodermis needle fitted with two serum stOppers as shown in Figure 1. The needle was sterilized and inserted into the fruit from the calyx end to avoid excessive wounding and was sealed with silastic rubber (ARTV. Dow Corning) prepared with a nonphytotoxic catalyst (Herter T I, Wacker Chemie GmbH, Munish, Germany). The tip of the needle was bent to prevent clogging. Red Delicious apples were used from trees at the Horti- culture Research Center at Michigan State University. The needles were inserted into the fruit 80-100 days after bloom and remained for the duration of the season. Gas samples were taken every three to four days with hypodermic syringes and ethylene content was determined by gas chromatography with a Varian Aerograph model 1700 equipped with an activated alumina column and a flame ionization detector. The effect of harvest was examined for some of the fruits equipped with needles by leaving the fruits under the tree or bringing them to the laboratory at 23°C. 110 Figure 1. Method of sampling the internal ethylene con- centration in Red Delicious apple attached to the tree. 3 m f) (x n- 111 Figure 1 SILICONE IUIIEI SYRINGE NEEDLE IUIIEI STOPPEI 112 Results and Discussion Internal ethylene concentration in intact fruit attached to the tree. The technique proved valuable to follow the intern- al ethylene concentration in the same fruit during the period of maturation on the tree. Ethylene levels in attached fruits agree closely with initial levels in detached fruits between 85 and 150 days after anthesis (Fig. 2). Ethylene occurred at low but measurable concentrations until 130 days after anthesis, then increased about 1000-fold. Samples taken from 4 individual fruits at 4 hr intervals 111 days after anthesis (data not presented) revealed a slight fluctuation of IEC in the fruit with a minimum at night and a maximum during the day. This may have reflected either diurnal temperature changes or a natural rhythm. Effect of harvest. To determine if harvest stimulated the fruit to produce ethylene, IEC was followed in one set of 4 fruits left on the tree and in another set of 4 fruits har- vested 113 days after anthesis and placed in a well-aerated room at 23°C. Following harvest, the IEC remained relatively constant for 1 to 7 days and then increased almost 1000-fold in a 7 to 10 day period (Fig. 3). The difference in lag time may reflect natural differences in maturity or ripening re- sistance. In contrast, IEC in fruits left on the tree varied from a low of 27 ppb to a high of 776 ppb until 135 days after 113 .wumo mCHHmEmm mom muflsum Apmzomuwov o no Aomcomuumv v How mamma ucmmwummu mmoam> .¢IIIIIIIV nmums CH Ummuwanom uflzum omumm>umn Eoum mHmEmm mam m 0:43mupnuflz wo ponume msy an Am paw .A.I.I.|.I.V uflsum mnu ou pogomuum mmfipmmc Luw3 oosqu was >3 Ad UmBOHH0m mmammm mooaoflamo pom CH mCOHpmnpcmocoo mamamnpw HmchDCH .N musmwm 114 N ouamwm co .4 m-mu:.—Z< “whu( m><0 Omp 00— Gnu our 0:. SP 1 q - q u q u I .u / \soio / n \\ Jon 1 uleflWllli \ I, \ II \ IQ p6 'sz Illdd low 00' 115 Figure 3. Internal ethylene concentrations in four Red Delicious apples left on the tree (A), or harvested 113 days after bloom (B)._ 116 n- 9% l 90 IOO "0 ISO IOO I‘D I” DAYS AFYII AHflIISIE ‘2'“ 90 100 "0 I20 '30 ‘40 I90 DAYS AE'EI AN'HENS Figure'B 117 anthesis. Some fruits showed surprisingly high IEC (above 200 ppm) yet did not abscise. The results suggest that an inhibitor supplied by the tree delays autocatalytic ethylene production. Effect of girdling and defoliation. To determine if the postu- lated inhibitor originated from the leaves and was translocated through the xylem, 4 treatments were applied 100 days after bloom (Fig. 4). Stems were girdled by removing a section of bark 1.5 cm wide, and gas samples were taken from 4 fruits in each treatment. Complete isolation of the fruit from leaves via phloem transport (D in Fig. 4) hastened ethylene produc- tion by 3 weeks, while girdling alone (C) or defoliation alone (B) delayed ethylene production. Both treatments B and C showed high IEC and this discrepancy is not understood. The data support the concept that a substance originating in the leaves and transported into the fruit through the phloem, prevents autocatalytic ethylene production. The chemi- cal nature of this inhibitor is unknown. The variation be- tween individual fruits, harvested or left on the tree in lag time before accelerated ethylene production (Fig. 3), together with the results of girdling (Fig. 4), suggest that fruits receive differential amounts of the inhibiting sub- stance. The lag in the onset of ethylene production in fruits from defoliated but not girdled limbs (B) compared to fruits 118 Figure 4. Effect of defoliation and girdling on the internal ethylene concentration in Red Delicious apples. 119 DAYS AWE. ANNE“! Figure '4 120 from untreated limbs (A) suggests that availability of sub- strate (CHO) as well as an inhibitor may be involved in the regulation of ethylene synthesis. 10. ll. LITERATURE CITED Biale, J. B. 1960. Respiration of fruits. Encycl. Plant Physiol. 12(2):536-592. Blanpied, G. D. 1971. Apparatus for ethylene extraction from plant tissue. HortScience 6:132-134. Brooks, C. 1938. An apparatus for the extraction of internal atmOSpheres from fruits and vegetables. Proc. Amer. Soc. Hort. Sci. 35:202-203. Burg, S. P., and E. A. Burg. 1962. Role of ethylene in fruit ripening. Plant Physiol. 37:179-189. Burg, S. P., and E. A. Burg. 1964. Evidence for a natural occurring inhibitor of fruit ripening. Plant Physiol. 39 (supp.) x. ' Burg, S. P. 1964. Studies on the formation and function of ethylene gas in plant tissues. In Regulateurs Naturels de la Croissance Végétale (Centre National de la Recherche Scientifique, ed.), pp. 719-728 (Gif s/Yuette, Paris). Burg, S. P. and E. A. Burg. 1965. Ethylene action and the ripening of fruits. Science 48:1190-1196. Burroughs, A. M. 1923. Changes in the respiration rate of ripening apples. Proc. Amer. Soc. Hort. Sci. 19: Denny, F. E. 1946. Gas content of plant tissue and respiration measurements. Contrib. Boyce Thompson Inst. 14:257-276. Gazit, S. and A. Blumenfeld. 1970. Response of mature avocado fruits to ethylene treatments before and after harvest. J. Amer. Soc. Hort. Sci. 95:229-231. Gerhardt, F. 1947. A comparison of the ripening of attached and detached apple and pear fruits. Wash. State Hort. Assoc. Proc. 43:57-59. 121 12. l3. 14. 15. 16. 17. 18. 19. 122 Gore, H. C. 1911. Studies on fruit respiration. U. S. Department Agr. Bur. Chem. Bul. 142, 40 pp. Lewis, C. I., A. E. Murneek, and C. C. Cate. 1919. Pear harvesting and storage investigations in the Rogue River Valley. Oreg. Agr. Expt. Sta. Bul. 162, 39 pp. Lyons, J. M., W. B. McGlasson, and H. K. Pratt. 1962. Ethylene production, respiration, and internal gas concentrations in cantaloupe fruits at various stages of maturity. Plant Physiol. 37:31-36. Lyons, J. M., and H. K. Pratt. 1964. Effect of stage of maturity and ethylene treatment on respiration and ripening of tomato fruits. Proc. Am. Soc. Hort. Sci. 84:491-500. Magness, J. R. 1920. Composition of gases in intercellu- lar spaces of apple and potatoes. Botan. Gaz. 70:308-317. Mapson, W. and A. C. Hulme. 1970. The Biosynthesis, Physiological Effects, and Mode of Action of Ethylene. In Progress in Phytochemistry, Vol. 2 (Reinhold, L. and Y. Liwschitz, eds.), pp. 343-384 (Interscience Publishers, London). Staby, G. L. and A. A. De Hertogh. 1970. The detection of ethylene in the internal atmosphere of bulbs. HortScience 5:399-400. Williams, M. W. and M. E. Patterson. 1963. Internal atmospheres in Bartlett pears stored in controlled atmospheres. Proc. Amer. Soc. Hort. Sci. 81:129-136. APPENDIX APPARATUS TO TREAT PLANT MATERIAL WITH GASES AND MEASURE CO2 AND ETHYLENE PRODUCTION Abstract. A simple static system is described for treating plant material with olefinic gases and measuring respiration and or ethylene production at a constant 0 level. The pro- 2 cedure measures CO2 production by absorption and titration of an alkali scrubber and ethylene by sampling the gas phase of the chamber in which the plant material is enclosed. Several systems have been devised to treat plant material with gases and measure respiration and ethylene production. The tissue is placed either in sealed containers (2,4,6,7,16, 20) or in ventilated chambers (9,10,15). In the sealed con- tainers CO2 and ethylene production is measured by withdraw- ing samples from the gas phase. Monitoring the composition of O2 and CO2 in this system is difficult and the method has been used mainly for short term experiments. In ventilated chambers the treatments are applied either by using premixed gases in cylinders (3,18) or by mixing gases in prOper propor- tions in a continuous flowing system (21). CO and ethylene 2 production is measured by sampling the gas stream leaving the chambers. The method offers certain advantages over the sealed 123 124 containers. Monitoring the gases is accurate and is considered to be the most appropriate method for measuring rates of emana- tion of CO or ethylene. However, when CO or ethylene is 2 2 produced in small amounts the method has to be modified by passing the effluent through an alkali scrubber (1,5) to trap CD2 or through a mercuric perchlorate absorber (17,24) to re- move and measure ethylene. Although ventilated chambers have been the most widely used method for many years, an elaborate apparatus to control flow rates, and an appropriate method to scrub and release ethylene are required. A simple apparatus is described for treating plant material with gases and/or measuring CO and ethylene production. 2 The system was used by Magness and Diehl (11) and modi- fied by Haller and Rose (8) and by Platenious (14). It is essentially a combination of the apparatus used for determina- tion of CO2 and O2 and the Mariotte bottle system with minor modifications. The plant tissue is placed in a chamber (Fig. l A) connected to an oxygen bottle (B), which in turn is attached to the Mariotte bottle (C). The O2 inlets are fitted with small vials containing a saturated solution of ammonium sulfate to prevent backward diffusion of gases into the O2 supply bottle. As an extra safety for ethylene treatments or measurements and to avoid possible contamination from the oxygen supply, an ethylene absorbent is placed in the O2 inlet. A manometer is connected to the O2 container to indicate the 125 .coflpmnucwocoo ucmHm mo coHuosponm mcwamsum paw N NO ycmumcoo Hops: HMflumumE OU mcflpsmmmfi How msumummm< .H musmflm 126 H musmwm Inux q.~ Loewe t or: no: 9305 vommvzz. co::_ow “c3933 Emntomnm 39>ch 9.09:ch 550 07' ------__ + 127 pressure (hl) necessary to overcome the pressure (h3) in the respiratory chambers. This can be controlled by raising the water reservoir (C) and increasing the height (hz) of the water level at the ambient atmOSpheric pressure. One Mariotte bottle system can be used for a set of as many as 20 respira- tory chambers. As the plant material respires, CO is given off and 02 2 is absorbed. The CO2 is absorbed by the 2N KOH solution in the petri dish and the 0 used is replaced by 0 from the 2 2 oxygen supply bottle (B). The O2 withdrawn from the bottle is replaced by water from the water reservoir (A). Thus the sys- tem remains very close to atmospheric pressure and the concen- tration of O2 in the chamber remains practically unchanged. The system can also be used for 0 concentration studies since 2 the initial oxygen concentration is maintained during the course of the experiment. Samples can be withdrawn from the chamber and analyzed in a gas chromatograph for ethylene. The system may be Opened and aerated every one or two days to avoid excessive accumulation of ethylene in the chamber. The same system was used for respiration measurements. The CO2 trapped in the 2N KOH solution was determined by titra- tion with standard 0.5 N or 1 N HCl to the phenolphthalein end point and then to the methyl orange end point (22). In a second method, carbon dioxide was determined by adding excess 1 N BaCl2 and titrating the excess alkali against 0.5 N HCl to the phenolphthalein end point (23). Evaluation of the two 128 methods showed that the two indicator method was more reliable (Fig. 2). The amount of sample in the respiratory chamber should be sufficient to produce measurable amounts of ethylene with- out accumulation of physiologically active levels over a period of one or two days. Enough KOH should be present so that only a third of the alkali is neutralized by the CO2 evolved during a run. If more than half the KOH is neutralized, the phenolphthalein end point in the titration becomes indis- tinct. Changes in temperature should be avoided as the re- sulting pressure changes will affect the O2 supply. This procedure was used to determine ethylene production of preclimacteric apples (l9 and Fig. 3), and ethylene and CO2 production of carnation flowers (Fig. 4). Values obtained with carnations are close to those reported by Nichols (13). The same apparatus was used to treat dormant cherry twigs with ethylene to break bud dormancy and measure respiration rate (Fig. 5). Figure 2. 129 Standard curves obtained from the BaCl2 and the two indicator titration methods for CO2 determin- ation. Known amounts of CO2 were injected into closed containers With KOH. The data were cor- rected for STP conditions. 130 27;? :5! O E 00.90.... N00 90 40- p o M 120 '- 00 I20 160 :1: C02 absorbed 40 Figure 2 131 .omuumum maB ucmEummuu mcwawmonm kumm mop sum map coflposooum mamawnum mo mumn ODMOHUCH Bump one .mflmmnucw Hopwm mmmo mma pwummuu mmammm mSOHOHHmQ pom CH mcoammoum >3 COHOOSUOHQ mamamsum mo COHDMHSEHum .m Guzman 132 coo- 1 con q m 936.3 7...... «gnu 8. o... O— 0 cu 06— M rz-‘x/m ‘3 Id 133 .wCoflu ICOHHmmH HCOM Eoum mquEmHsmmmE mo mommum>m map pCmmmHmmu Chop 0C9 .HwnEMCo Comm CH Ammouosm wm.a UCm OCHHOCHDvmxouomCIm Ema oomv CoHp ICHOm m>Hum>Hmmmmm CH UmomHm UCm mmmum pan DCWHD mCu #m #50 OHOB mum3on meCB .mnm3on CoflumCHmo wn COHuodooum maHmCum UCm moo .v musmflm 134 cu. yz-unou/VHZD "I N— C— v muamwm auuaiv o at... v m CON w 3 O 7 z ./I x a 2: ..,. t W 000 135 .Hnma .HN HmQEwomo Co UHCCOHO Eonw pmuomaaoo mmflsu munmCo quEHOU mo Coflumsflmmmu Co waahCum mo pommmm .m mudmfim 136 coo— co— m cash at}; vzmu o. coo— coca OOOn “‘l n-‘x/‘oa Iw 10. LITERATURE CITED Biale, J. B. 1946. Effect of oxygen concentration on respiration of the fuerte avocado fruits. Amer. J. Bot. 33:363-373. Burg, S. P. and E. A. Burg. 1962. Role of ethylene in fruit ripening. Plant Physiol. 37:179-189. Byers, R. E., L. R. Baker, H. M. Sell, R. C. Herner, and D. R. Dilley. 1972. Ethylene: A natural regulator of sex expression of Cucumis melo L. Proc. Nat. Acad. Sci. 69:717-720. Chadwick, A. V. and S. P. Burg. 1970. Regulation of root growth by auxin-ethylene interaction. Plant Physiol. 45:192-200. Claypool, L. L., and R. M. Keefer. 1942. A colorimetric method for CO determination in respiration studies. Proc. Amer. S c. Hort. Sci. 40:177-186. Eaks, I. L. 1970. Respiratory responses, ethylene pro- duction, and response to ethylene of citrus fruit during ontogeny. Plant Physiol. 45:334-338. Fuchs, Y. and M. Lieberman. 1968. Effects of kinetin, IAA, and gibberellin on ethylene production, and their interactions in growth of seedlings. Plant Physiol. 43:2029-2036. Haller, M. H. and D. H. Rose. 1932. Apparatus for de- termination of C02 and 02 of respiration. Science 75:439-440. Looney, N. E. 1969. Control of apple ripening by succinic acid 2,2-dimethy1hydrazide, 2-chloroethyl trimethyl ammonium chloride and ethylene. Plant Physiol. 44:1127-1131. ‘ Lyons, J. M. and H. K. Pratt. 1964. Effect of stage of maturity and ethylene treatment on respiration and ripening of tomato fruits. Proc. Amer. Soc. Hort. Sci. 84:491-500. 137 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 138 Magness, J. R., and H. C. Diehl. 1924. Physiological studies on apples in storage. J. Agricult. Res. 27:1-38. Maxie, E. C. and J. C. Crane. 1967. 2,4,5-Trichloro- phenoxyacetic acid effect on ethylene production by fruits and leaves of fig tree. Science 155:1548- 1550. Nichols, R. 1968. The response of carnations (Dianthus caryophyllus) to ethylene. J. Hort. Sci. 43:335-349. Platenious, H. 1942. Effect of temperature on the respiration rate and the respiratory quotient of some vegetables. Plant Physiol. 17:179-197. Pratt, H. K., M. Workman, F. W. Martin, and J. M. Lyons. 1960. Simple method for continuous treatment of plant material with metered traces of ethylene or other gases. Plant Physiol. 35:609-611. Rasmussen, G. K., J. R. Furr and W. C. Cooper. 1969. Ethylene production by citrus leaves from trees grown in artificially salinized plots. J. Amer. Soc. Hort. Sci. 94:640-641. Sakai, S. and H. Maseki. 1970. Quantitative determina- tion of l4C-ethylene produced by plant tissues. Agr. Biol. Chem. 34:1584-1587. Sfakiotakis, E. M., D. H. Simons, and D. R. Dilley. 1972. Pollen germination and tube growth: Dependent on carbon dioxide and independent of ethylene. Plant Physiol. 49:963-967. Sfakiotakis, E. M. and D. R. Dilley. 1972. Stimulation of ethylene production in apple fruits by propylene. HortScience 7:325. Suge, H. 1971. Stimulation of cat and rice mesocotyl growth by ethylene. Plant and Cell Physiol. 12:831- 837. Wang, C. Y., W. M. Mellenthiu, and E. Hansen. 1972. Maturation of 'Anjou' pears in relation to chemical composition and reaction to ethylene. J. Amer. Soc. Hort. Sci. 97:9-12. Warder, R. B. 1881. Alkalimetry with phenolphthalein as indicator. Am. Chem. J. 3:55‘232. 139 23. Winkler, C. 1902. "Praktische Ubungen in der Massana- lyse," 3rd ed., Akademishe Verlagsgesellschaft, Leipzig. 24. Young, R. E., H. K. Pratt, and J. B. Biale. 1952. Manometric determination of low concentrations of ethylene. Anal. Chem. 24:551-555. "Illli'flllifllilllllr