F-_-_----I-----.II PHYSIOLOG¥CAL STUDlES 0F BRACT KEEPING QUALH’Y IN POENSETTIA Thesis for the Degree of Ph. D. M!CHIGAN STATE UNIVERSITY DAVID ALLAN GILBART 1969 may; ‘ LIBRARY Michigan Stew University ' This is to certifg that the thesis entitled Physiological Studies of Bract Keeping Quality in Poinsettia presented by David Allan Gilbart has been accepted towards fulfillment of the requirements for Ph.D. degree in Horticulture Major professor 0-169 I -.-‘u‘.'. u.‘..... ll“ ABSTRACT PHYSIOLOGICAL STUDIES OF BRACT KEEPING QUALITY IN POINSETTIA BY David Allan Gilbart A study was made of the factors governing differ- ential bract abscission rates and hence keeping quality among cultivars of poinsettia. Experiments were designed to measure changes in respiration rate, nitrogen pools, carbohydrate pools and ethylene evolution with aging. The effect of exogenous growth regulators such as indole- acetic acid (IAA), gibberellic acid (GA), abscisic acid (ABA), and ethylene on abscission rates of intact plants and eXplants was determined. A study was made of the extractable and diffusible endogenous growth regulators, especially IAA, together with changes in activity and isoenzymic forms of IAA-oxidase with aging. The respiration rate declined faster in the bracts of poor keeping cultivars while nitrogenous com- pounds were mobilized coincident with senescence. No significant correlation was found between any parameter and abscission rate. No ethylene evolution was detected with the method employed. David Allan Gilbart Exogenous IAA delayed the abscission of debladed bracts. Other growth regulators were not effective in promoting or delaying abscission. No interaction was found between any growth regulator and cultivar with re- spect to bract abscission. Both extractable and diffusible endogenous auxin levels in bracts decreased with time. The rate of de- crease was higher in the poor keeping cultivar. The de- crease in auxin could be related to an increase in the activity of IAA-oxidase and hydrogen peroxide content with time. The appearance of new isoenzymes was associated with increased enzyme activity. Differences in keeping quality among cultivars of poinsettia is the result of differential destruction of endogenous auxin through the appearance of new forms of peroxidase and increased levels of hydrogen peroxide together with differential synthesis of auxin through decreased rates of respiration leading to a decrease in the effective concentration of auxin. A threshold level of auxin which is reached earlier in poor keeping culti- vars triggers senescence and abscission resulting in mobilization of nitrogenous compounds which possibly further accelerates abscission. This hypothesis is compatible with inheritance studies of keeping quality in poinsettia. PHYSIOLOGICAL STUDIES OF BRACT KEEPING QUALITY IN POINSETTIA BY David Allan Gilbart A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1969 NOTE TO THE COMMITTEE This thesis has been prepared so that the body of the dissertation is in the form of two journal arti- cles designed for publication in the Journal of the American Society for Horticultural Science. Additional information, by way of a general introduction to the problem, additional data and a general interpretation of the results appear in the appendix. ii ACKNOWLEDGMENTS The author wishes to express his sincere appre- ciation to Dr. Kenneth C. Sink for guidance during the course of this investigation and preparation of the manuscript. Acknowledgment is also extended to the members of the committee; Dr. A. Kenworthy, Dr. A. De Hertogh, Dr. D. Dilley, Dr. F. Elliott and Dr. C. Pollard. Special appreciation is extended to Dr. Dilley for extensive use of laboratory facilities and to my wife Gill for her encouragement and assistance. iii TABLE OF CONTENTS I 0 LIST OF TABLES O O O O 0 O O O O O O O O O 0 II. LIST OF FIGURES . . . . . . . . . . . . . . III. ARTICLE I - The Effect of Exogenous Growth Regulators on Keeping Quality and Abscission in Poinsettia . . . . . . . . . A. IntrOdUCtion O O O O O O O O O O O O O B. Materials and Methods . . . . . . . . . C. ReSUlts O I I O I O O O O I O I O O O O l. Abscission of untreated and ethylene treated intact and explant poinsettia leaves and bracts . . . . . . . . . . . . 2. The effect of indoleacetate acid, abscisic acid and gibberellic acid on bract abscission of explants . . . . . . . . . . . . 3. The effect of concentration and point of application of indole- acetic acid and gibberellic acid on leaf and bract abscis- sion of explants . . . . . . . . 4. The effect of indoleacetic acid, gibberellic acid and abscisic acid on leaf and bract abscis- sion from intact plants . . . . 5. The effect of time of application of indoleacetic acid, chloram- phenicol and 2,4-dichlorophenol on bract abscission of explants. D. Discussion . . . . . . . . . . . . . . E. Literature Cited . . . . . . . . . . . IV. ARTICLE II - Regulation of Endogenous In- doleacetic Acid and Keeping Quality of POinsettia I O O O O O O O O I O O O O O O A. Introduction . . . . . . . . . . . . . iv Page vi vii \IWH H 12 14 15 20 28 30 3O B. Materials and Methods C. D. E. Results . . . . ~___ .._~. —u—--—-- 777 l. Extractable growth regulators 2. Diffusible growth regulators in the course of aging 3. IAA-oxidase activity 4. Gel electrOphoresis of enzyme . Discussion . . Literature Cited V. APPENDIX . . . . . . A. General Introduction peroxidase B. Metabolic Changes in the Poinsettia Relation to Aging, Senescence D. General Discussion Abscission . 1. Introduction . . . 2. Materials and Methods 3. Results . 4. Discussion . C. Additional Data on the Activity IAA-oxidase . VI. LITERATURE CITED . . 0 and Page 32 36 36 39 44 49 54 61 63 63 68 68 72 76 98 102 104 119 LIST OF TABLES ARTICLE I Page 1. Effect of ethylene on abscission of debladed or intact leaves and bracts of poinsettias . 8 2. Effect of growth regulator concentration and point of application on abscission of de- bladed leaf and bract petioles . . . . . . . 13 3. Effect of IAA, GA and ABA on leaf and bract abscission of intact poinsettias . . . . . . 16 ARTICLE II 1. Diffusible IAA with time from poinsettia bracts . . . . . . . . . . . . . . . . . . . 43 2. IAA-oxidase activity in healthy and senescent poinsettia bracts . . . . . . . . 45 APPENDIX 1. Time to 50% leaf and bract abscission and keeping time for 5 cultivars of poinsettia . . . . . . . . . . . . . . . . . 77 2. Metabolic activity and substrate levels of healthy and senescent leaves and bracts O O O O O O O O I O O O O O O O O O O 93 3. Coefficients of correlation between parameters of metabolism and the rate of bract abscission . . . . . . . . . . . . . . . . . 96 4. Optical density of active and inactive ex- tracts of poinsettia bracts . . . . . . . . 102 5. Optical density of active and inactive ex- tracts of poinsettia bracts before and after passing through Sephadex . . . . . . . 103 vi LIST OF FIGURES ARTICLE I Page 1. Sketch of a poinsettia plant showing the in- tact plant, an explant and the points of abscission in the flowering head . . . . . . 4 2. The interaction of IAA applied distally and GA or ABA applied proximally to debladed bracts on the rate of abscission . . . . . . 10 3. The interaction of IAA applied distally and 2,4-dichlorophenol or chloramphenicol applied proximally on the abscission of debladed bract petioles . . . . . . . . . . 17 ARTICLE II 1. Extractable growth regulators from leaves and bracts of 2 cultivars of poinsettia at anthesis and the time of abscission . . . . 37 2. Diffusible growth regulators from flowering heads of poinsettia at weekly intervals after antheSj-S O O O O O O O O O O O O O O O 40 3. Activity of IAA-oxidase and level of hydrogen peroxide in explants and intact plants of poinsettia at various times after anthesis . 47 4. Zymogram of the peroxidase isoenzymes in ex- plant and intact poinsettias at various times after anthesis . . . . . . . . . . . . 50 APPENDIX 1. Abscission rates and metabolic changes with time for leaves and bracts of Paul Mikkel-sen O I O O O O O O O O O O O O O O O 79 2. Abscission rates and metabolic changes with time for leaves and bracts of White Ecke . . 81 vii Page Abscission rates and metabolic changes with time for leaves and bracts of New Ecke Pink 0 I O O O O C O O O I O O O C O C O O O 83 Abscission rates and metabolic changes with time for leaves and bracts of Barbara ECke supreme O I O O O O O O O O O O O O O D 85 Abscission rates and metabolic changes with time for leaves and bracts of MSU 64—5 . . . 87 The theoretical interrelationships among factors regulating endogenous auxin, senescence and abscission in poinsettia bracts . . . . . . . . . . . . . . . . . . . 113 viii ARTICLE I THE EFFECT OF EXOGENOUS GROWTH REGULATORS ON KEEPING QUALITY AND ABSCISSION IN POINSETTIA The Effect of Exogenous Growth Regulators on Keeping Quality and Abscission in Poinsettia Abstract. A study was made of the response of several cultivars of intact and explant poinsettia cultivars to exogenously applied growth regulators. No treatment accelerated the abscission of explants over nontreated controls. Indoleacetic acid (IAA) delayed the abscis- sion of explants relative to its concentration and time of application. Gibberellic acid or abscisic acid alone had no effect but modified the IAA effect in interaction. Ethylene had no effect. There was no interaction be- tween any treatment and cultivar. INTRODUCTION Several naturally occurring metabolites and plant growth regulators may influence the rate of foliar and floral abscission when applied exogenously to an explant or intact plant. These include auxins, gibberellic acid, abscisic acid, ethylene, kinin, certain amino acids and protein synthesis inhibitors (11). Varying the experi- mental conditions may alter the response to any one regu- lator and 2 or more applied chemicals may interact to produce synergism or compensation. Indoleacetic acid as l an abscission retardant and ethylene as an accelerator have the most universal and the greatest quantitative effect. Most research on abscission has been done using explants of Phaseolus, Gossypium or Coleus. This has been particularly true in the testing of compounds for activity as abscission regulators. The poinsettia has been used in a few abscission studies. Gawadi and Avery (10) showed that applying ethylene chlorohydrin to in- tact poinsettias induced abscission of young leaves as fast as deblading while a 1% napthaleneacetic acid solu- tion applied to debladed leaf stumps delayed abscission from the normal 5 to 6 days to upward of 20 days. Car- penter (7) found that up to 6 ppm of 2,4,5—trichloro- phenoxyacetamide delayed abscission of both leaves and bracts on poinsettia while 2,4,5-trichlorophenoxy- propionamide slightly hastened it. However the results were variable, dependent on environmental conditions, and the treatments induced an epinastic response. MATERIALS AND METHODS Plants of the poinsettia cultivars Paul Mikkelsen, White Ecke, New Ecke Pink and Barbara Ecke Supreme were obtained as rooted cuttings in the fall or winter and grown as single stem plants to anthesis in 5 inch clay pots using standard greenhouse practices. At anthesis the plants were given treatments and transferred either to a laboratory simulating a home environment for intact plants, 21° and 50 ft-c of light for 12 hrs, or a dark room maintained at 21° for explants. A poinsettia explant is illustrated in Figure 1. All cyathia, growing points and primary bracts were re- moved. The lower 3 to 4 leaves were also removed as they were formed while the plant was under propagation stress and it was found in a preliminary study that they abscised abnormally early. Remaining leaves and bracts were removed to leave only 3 bracts on each of the 3 flowering stems arising from the first floral division and 6 leaves. These were then debladed by cutting directly below the leaf or bract blade. The growth regulators employed were indoleacetic acid (IAA), gibberellic acid (GA), abscisic acid (ABA), Figure 1. Sketch of a poinsettia plant showing an intact plant, an explant and the points of abscission in the flowering head. EXPLANT Poflolo End «flue! Slam End -pmimol Brod ”Hf I W "MP—N) & \ \ rows 0; assassaon .’ ‘ V . /) .l ‘ MATURE PLANT 4M , /"\ fllé": é‘téfi ‘ ‘ \\\=“. Primary Brae! \\\r V“‘—— Secondary and: on! fi'rmgoflon Loci ethylene, chloramphenicol or 2,4-dichlorophenol either alone or in combination. Ethylene treatment was applied by holding plants for 24 hrs in a sealed plastic bucket containing 10 ppm ethylene in air. Other treatments were applied in lanolin paste to the petiole or stem stumps of explants or as an aqueous solution with 100 ppm Tween 20 wetting agent applied as a spray to the drip off point on intact plants. Abscission was recorded on a daily basis and in— cluded lightly tapping each petiole to ensure complete separation. The abscission rate was based on the average number of days to abscission for all bract or leaf petioles on a plant. Petioles which had not abscised by the termi- nation date were given the maximum value. Explants held in the dark were measured for 12 days and intact plants held in the simulated home environment were maintained either for 18 days or until obviously senescent. None of the studies were replicated with time. Routinely 3 to 5 individual plant replicates were included for each treatment in each study and added confidence was achieved through the use of nontreated and IAA treated controls added to each of the explant studies. RESULTS Abscission of untreated and ethylene treated intact and explant poinsettia leaves and bracts. The cultivars studied exhibited a wide range of keeping times when held in a simulated home environment. In general, the rate of bract abscission determined keeping time. Paul Mikkelsen and Ecke White held their bracts the longest while New Ecke Pink was intermediate and Barbara Ecke Supreme lost its bracts the earliest. Leaf abscission appeared to be independent of bract abscission with little difference among cultivars. Abscission rates of bract petioles from explants differed among cultivars (Table l). The relative order of abscission among cultivars was the same as for intact plants although the rate was accelerated. The abscission rates of explant leaf petioles did not vary among culti- vars. In all cultivars prepared as explants, leaf petioles abscised sooner than bract petioles. Chronologically, bracts are the more juvenile tissue; however, both bracts and leaves were mature when debladed. Moreover, there was no apparent relationship between position on the stem and the time to abscission for either leaves or bracts. The effect of 10 ppm ethylene treatment on days Table l. to 50% abscission of debladed or intact leaves and bracts of 4 poinsettia cultivars. Cultivar Intact Debladed Ethylene Control Ethylene Control White Ecke bracts 11.6 12.0 6.6 6.5 leaves 12.0 12.0 4.6 4.8 Paul Mikkelsen bracts 12.0 12.0 7.3 7.2 leaves 12.0 12.0 5.4 5.0 New Ecke Pink bracts 11.6 11.2 5.5 5.6 leaves 9.8 10.0 4.5 4.5 Barbara Ecke bracts 10.8 11.2 5.7 5.8 Supreme leaves 8.6 9.8 4.8 4.9 The results of the study on exogenous treatment with ethylene are in Table l. Ethylene had no effect on abscission of leaves or bracts, whether intact or debladed in any of the cultivars. Over the length of the experi- ment few leaves or bracts abscised from intact plants, thus possibly masking a belated effect. A repetition of the experiment using the same design but with aged plants of 2 cultivars, Paul Mikkel- sen and Barbara Ecke Supreme, also gave negative results for an ethylene effect although there was considerable abscission among both control and treated intact and de- bladed plants. Epinasty was observed in many leaves and bracts treated with ethylene. This was more pronounced in explants and occurred least in the aged intact plants. The effect of indoleacetate acid, abscisic acid and gibberellic acid on bract abscission of eXplants. The bract petioles of pionsettia explants of the cultivars Paul Mikkelsen and Barbara Ecke Supreme were treated immediately after deblading with IAA applied distally and either ABA or GA proximally. Two concentrations, lO-SM and 10‘3 M of each chemical were used and all controls, single chemical treatments and interactions between IAA and the other 2 were included. The results of this study are presented in Figure 2. Although there were significant differences between Figure 2. 10 The interaction of 2 concentrations of IAA applied distally with 2 concentrations of GA or ABA applied proximally to de— bladed poinsettia bract petioles on the rate of bract petiole abscission. ll IIIIIIII s" o s o 10' v 0’. 6e, , 6 ’9' a. '9 10" 10's 53’ 0/ o’- llé V (‘6 99o ‘0 or \ 0° ’0'. 6° 0 0.0%, O ‘6‘ Molar concentration of growth regulator Ission A a-4 I Mean days to dbsc' t - .V I (d 12 the cultivars there was no interaction between cultivar and chemical treatment so treatment effects are presented as the average of both cultivars. Both main order and interaction effects between chemicals may be seen. Indoleacetic acid retarded the rate of abscission, more so at the higher concentration but also at the lower. Neither ABA nor GA at either concentration had any effect when used alone or with the lower concentration of IAA. However when interacted with the higher concentration of IAA, both ABA and GA at either concentration reduced the effectiveness of IAA in delaying abscission to approximately that of the lower concentration of IAA alone. Epinasty was observed in all treatments that included IAA. The effect of concentration and pgint of application of indoleacetic acid and*gibberellic acid on leaf and bract abscission of explants. Paul Mikkelsen and Barbara Ecke Supreme were treated immediately after deblading with 3 or lO-SMapplied either to the leaf IAA or GA at 10- petiole stump or the bract petiole stump (distal) or to the flowering stem terminal stump (proximal). Lanolin alone was applied to stumps not treated with chemical and to control plants. The results of this study are presented in Table 2. There was no interaction between cultivars and 13 Table 2. The effect of growth regulator concentration and point of application on days to 50% abscis- sion of debladed bract and leaf petioles. Indoleacetic Acid Gibberellic Acid Point of _3 _5 _3 _5 Application 10 M 10 M 10 M 10 M Bract Abscission Control 6.4 Leaf Petioles 6.8 6.0 6.6 6.3 Bract Petioles 11.6 9.3 5.6 5.8 Stem Terminals 8.8 7.3 6.8 6.4 Leaf Abscission Control 5.1 Leaf Petioles 9.2 6.7 4.7 4.6 Bract Petioles 4.7 4.6 4.8 5.2 Stem Terminals 5.3 5.0 5.0 4.7 - leaves .7 14 chemical treatment so the values were averaged across both cultivars. There were significant differences be- tween chemicals, points of application and their interaction. Indoleacetic acid delayed the rate of bract abscission of applied to the bract petiole stump or the stem terminal. Proximal application was effective in delaying abscission only at the higher concentration. Application to the leaf petioles did not affect the rate of bract petiole abscission. Indoleacetic acid delayed leaf petiole ab- scission only if applied at the higher rate directly to the leaf petiole stump. Gibberellic acid did not sig- nificantly affect abscission in any of the treatments studied although there appeared to be a consistent tend- ency for it to enhance bract abscission when applied distally to the bracts. It did not affect the leaf petiole abscission rate. In no case did IAA.acce1erate or GA slow the rate of abscission. The effect of indoleacetic acid, gibberellic acid and abscisic acid on leaf and bract abscission from intact plants. Paul Mikkelsen and Barbara Ecke Supreme plants were prepared as explants except that the remaining 6 leaves and 9 bracts were not debladed. The plants were then sprayed with either IAA, GA or ABA at lO—3M or water and held in a simulated home environment for 18 days. The first apparent result was pronounced epinasty of IAA treated plants which continued for the duration of 15 the experiment. The effect of these treatments on abscis- sion is summarized in Table 3. Because of the relatively short time span under observation, plants in several treat- ments had little or no abscission. This may have masked differences due to treatment as, for example, bract abscis- sion of Paul Mikkelsen. There were however, certain striking differences in abscission rates as a result of treatment. Abscisic acid tended to accelerate the rate of leaf abscission while having little or no effect on the bracts. Indole- acetic acid had the apparent effect of delaying leaf abscission in the cultivar Paul Mikkelsen while acceler- ating it in Barbara Ecke Supreme. Both IAA and GA tended to delay bract abscission in Barbara Ecke Supreme. The effect of time of application of indoleacetic acid, chloramphenicol and 2,4-dichlorophenol on bract abscis- sion of explants. Explants of the cultivar White Ecke were treated either immediately after preparation or 24.hrslater with IAA applied distally to the bract petiole Stumps and either 2,4-dichlorophenol or chloramphenicol proximally to the stem terminal. All chemical treatments were at 10-3M. Each alone and all possible combinations between IAA and the others were tested. As shown in Figure 3, IAA delayed abscission ir- respective of time of application. However, the later Table 3. The effect of 10' 16 3 bracts of 2 cultivars of poinsettia. M IAA, GA, and ABA on mean number of days to abscission of leaves and Control IAA GA ABA Paul Mikkelsen Bracts 18.0 18.0 18.0 17.6 Leaves 15.4 18.0 17.1 10.2 Barbara Ecke Supreme Bracts 14.7 18.0 18.0 16.9 Leaves 12.6 7.6 11.8 4.1 Figure 3. 17 The interaction of IAA applied distally immediately after deblading or 24 hours later with 2,4-dichlor0phenol or chlor- amphenicol applied proximally immediately after deblading or 24 hours later to de- bladed poinsettia bract petioles on the rate of bract petiole abscission. 18 \\ nono Time of application in hrs. after deblading Mean days to abscission 19 application had a lessened effect, approximately equal 5M IAA treatment applied immediately after de- to a 10- blading. Neither 2,4-dichlorophenol nor chloramphenicol alone had any effect on the abscission rate. Neither did they modify the effect of IAA applied at the time of de- blading. However, if 2,4-dichlorophenol or chloramphen- icol were applied immediately after deblading the effect of IAA added 24 hours later was nullified. In no case was the rate of abscission accelerated over the control. DISCUSSION The 4 cultivars studied exhibited a wide range in keeping time as evidenced by their differential rates of bract abscission. Paul Mikkelsen and Barbara Ecke Supreme which were chosen for more intensive study represent the extremes of commercial cultivars. The rate of bract petiole abscission from explants differed among cultivars but leaf abscission did not. This would indicate that leaves and bracts are under dif- ferent control systems or a system that is differentially active in the 2 tissues. If it is one system then it is active in both the blade and the petiole since remov— ing the blade hastened the rate of abscission yet left a residual varietal difference. While both leaves and bracts were fully mature the leaves were older. This relative difference in age in a mature flowering poin- settia may account for a portion of the different re- sponses of leaf and bract petioles after deblading. The effect of 10-3 M IAA applied immediately after deblading in delaying abscission of petioles confirms an accepted fact based on observations of the responses of many plants (11). However, the delay of abscission by proximal application, treatment after 24 hours aging with 20 21 -3 10 M IAA or immediately with 10—5 M IAA has not been so universally recognized. Gaur and Leopold (9) showed that dilute concentrations of IAA (lo-SM) stimulate abscission while 10'3 M IAA that will delay abscission if applied dis- tally to fresh explants may accelerate it if applied proximally (3) or if applied distally to aged explants (16). Later verification (1) has shown that it is pri- marily a question of auxin transport to the site of ab- scission as influenced by point of application, petiole length and environmental conditions. There was no apparent relationship between peti- ole length or distance from proximal treatment site and petiole response. This agrees with the findings of Lewis and Bakhshi (12) who used seedling orange explants analo- gous to those in this study. No attempt was made to monitor hormone transport rates in the poinsettia but it may be hypothesized that there is a sufficiently free flow of auxin basipetally to and across the abscission zones. The delay of abscission by proximal application of a high concentration of IAA would also indicate a functional acropetal transport. However, in the study on debladed leaf petioles application of auxin proximally either to the stem terminal or the bract petiole termi- nals had no effect on the rate of abscission. Either adequate transport does not continue down the stem or there is an active auxin destruction mechanism. 22 Neither GA nor ABA had any effect when applied alone to poinsettia explants. The effect of GA as an abscission accelerator has been debatable. According to Jacobs (11) the effect is slight and confined to young tissue. In these studies bracts and leaves were fully mature which may explain the lack of response. The ineffectiveness of ABA in promoting abscission is surprising in View of its effect on other plants (8). In part this may have been the result of proximal appli— cation only which is less effective than distal treatment (11). Although neither GA nor ABA was active as an abscission regulator either alone or in combination with -5 10 M IAA they both interacted in decreasing the effec- 3M IAA to approximately that of lO-SM IAA. tiveness of 10- Thus it would appear that both GA and ABA are effective in altering the abscission rate of debladed poinsettia bracts but only under conditions favoring maintained growth and maximum longevity. The results of spray application of IAA, GA, and ABA to intact plants are anomolous with those from the explant studies and with those from other studies. El- Antably, Wareing and Hillman (8) found very little activity when intact plants from a number of species were treated with ABA. Insufficient work has been done with ABA to allow an interpretation of these data although it again 23 illustrates the inherent difference between bracts and leaves. The effect of IAA in stimulating abscission in one instance is difficult to explain in View of a con- sistent inhibition of abscission on explants. The fact that the same treatment stimulated leaf abscission and delayed bract abscission on the same plants would argue against this being an artifact; however, the study was not repeated to confirm the observation. More likely, variable penetration and transport combined with endoge- nous growth regulators and uncontrolled variables in the environment resulted in this apparent discrepancy. It does support the observations made by other investigators that under some conditions auxin may stimulate abscission (3). Protein synthesis inhibitors have been reported to promote abscission (18) but were not effective on poinsettia bracts. Proximal application at a distance from the abscission zone could account for the lack of effect for chloramphenicol alone. However, the negation of the delaying effect of IAA added 24 hours later would indicate that chloramphenicol had some activity. Some possible pathways could be: interference with transport, action or destruction of auxin or the inhibition of pro- tein formation either necessary for auxin activity or normally formed as an integral part of the auxin effect. The lack of effect from simultaneous application may be 24 the result of differential tranSport times to the ab- scission zone. The IAA-oxidase co-factor 2,4-dichlor0phenol was similar to chloramphenicol in action. Schwertner and Morgan (17) found that IAA-oxidase co-factors as exempli- fied by 2,4-dichlorophenol could accelerate abscission of explants. The most feasible explanation to account for the effect of 2,4-dichlorophenol would be that prior application of the enzyme co-factor resulted in increased IAA-oxidase activity at the abscission zone sufficient to decrease the level of auxin from the later treatment to below an effective concentration. Several current theories regarding abscission control have attempted to explain several diverse obser— vations through a common channel; that of ethylene bio- synthesis (1,4). In vegetative poinsettias Gawadi and Avery (10) found that 2.8 ppm ethylene chlorohydrin caused intact poinsettia leaves to abscise as rapidly as deblading. Among studies of plants in flower Phan (14) working on iris and tulip and Nichols (13) on carnations found considerable ethylene evolution and a surge asso— ciated with wilting and petal drop. In View of the many positive results for an ethy- lene effect the lack of stimulation of poinsettia leaf or bract abscission is surprising. Similar experiments were conducted on 3 separate occasions; twice using plants 25 at anthesis and once with senescing plants and in no case was a positive result found. Bract and leaf petioles on treated explants had an epinastic response typical of ethylene treatment. The results of this study apparently are in sharp contrast to those of Gawadi and Avery (10). The 2 methodologies differed in several respects and may be re- sponsible for the anomalous results. The earlier work applied ethylene chlorohydrin to actively growing vege- tative plants while the present study used flowering plants with mature leaves and bracts and applied gaseous ethylene. It is possible that ethylene chlorohydrin and ethylene do not act the same particularly as they found that ethylene chlorohydrin could overcome the abscission delay resulting from a previous treatment with 1% NAA and also that it induced abscission of younger leaves first. These effects are not typical for ethylene as found in other studies (2,16,15). One possible reason for the lack of stimulation by ethylene may be that ethylene was always added imme- diately after deblading and discontinued after 24 hours. Rubinstein and Abeles (15) found that ethylene became ef- fective in stimulating abscission of bean petioles only after explants had aged for 12 hours. This agrees with the 2-stage process of abscission (16) in which ethylene is effective only during the second stage. It is 26 conceivable that after 24 hours the poinsettia was still in stage 1. The diminished delay of abscission resulting from the application of auxin 24 hours after deblading would support this. The only effect common to poinset- tias, both bracts and leaves, and other explants was a delay of abscission with auxin. Belated application of IAA may act in slowing the rate of abscission development already initiated. The ineffectiveness of abscission accelerators when applied alone or with lower rates of IAA could be a result of transport time to the site of activity being sufficiently long that the auxin level has dropped below the threshold value. One of the aims of this study was to elucidate, by analogy, a possible endogenous abscission control sys- tem which could explain the differential rates of abscis- sion found among poinsettia cultivars. Secondary division, prerequisite for separation, has been completed in the mature leaf or bract (10). When an explant is made by removing the leaf blade there is insufficient auxin in the petiole to delay the onset of the abscission process for a significant length of time. In the bract petiole there is sufficient endogenous auxin easily transported to the abscission zone to delay abscission in proportion to the limited amount of auxin. This could account for the increased longevity of bracts over leaves and also the residual varietal difference in bract petiole abscission. 27 In every explant study each cultivar responded the same relative to its own control. This would argue for one common control system. Therefore, a system with auxin as the central control and differences in the amount of diffusible auxin among cultivars would appear to be the most feasible. Above a certain threshold con- centration auxin delays the onset of abscission of a bract. When the effective concentration of diffusible auxin falls below the threshold at any of the well defined abscission zones then abscission results. This could come about through decreased synthesis, impaired transport or increased enzymatic destruction. LITERATURE CITED 1. Abeles, F. B. 1967. Mechanism of action of abscis- sion accelerators. Physiol. Plantarum 20:442-454. 2. Abeles, F. B. and B. Rubinstein. 1964. Regulation of ethylene evolution and leaf abscission by auxin. Plant Physiol. 39:963-969. 3. Addicott, F. T. and R. S. Lynch. 1951. Acceleration and retardation of abscission by indoleacetic acid. Science 114:688-689. 4. Burg, S. P. 1962. The physiology of ethylene forma- tion. Ann. Rev. Plant Physiol. 13:265-302. 5. Burg, S. P. 1968. Ethylene, plant senescence and abscission. Plant Physiol. 43:1503-1511. 6. Carns, H. R. 1966. Abscission and its control. Ann. Rev. Plant Physiol. 17:295-314. 7. Carpenter, W. J. 1956. The influence of plant hor— mones on the abscission of poinsettia leaves and bracts. Proc. Amer. Soc. Hort. Sci. 67:539-544. 8. El-Antably, H. M., P. F. Wareing and J. Hillman. 1967. Some physiological responses to D,L, abscisin (dormin). Planta 73:74—90. 9. Gaur, B. K. and A. C. Leopold. 1955. The promotion of abscission by auxin. Plant Physiol. 30:487-490. 10. Gawadi, A. G. and G. S. Avery, Jr. 1950. Leaf abscis- sion and the so-called abscission layer. Amer. J. Bot. 37:172-180. 11. Jacobs W. P. 1968. Hormonal regulation of leaf ab- scission. Plant Physiol. 43:1480-1495. 12. Lewis, L. N. and J. C. Bakhshi. 1968. Interactions of indoleacetic acid and gibberellic acid in leaf abscission control. Plant Physiol. 43: 351-358. 28 29 13. Nichols, R. 1966. Ethylene production during senescence of flowers. J. Hort.Sci. 41:279-290. 14. Phan, C. T. 1963. Production d'ethylene par les fleurs. C. R. Acad. Agric. 49:53-59. 15. Rubinstein, B. and F. B. Abeles. 1965. Ethylene evolution and abscission. Bot. Gazette 126: 255—259. 16. Rubinstein, B. and A. C. Leopold. 1963. Analysis of the auxin control of bean leaf abscission. Plant Physiol. 38:262—267. 17. Schwertner, H. A. and P. W. Morgan. 1966. Role of IAA-oxidase in abscission control in cotton. Plant Physiol. 41:1513-1519. 18. Valdovinos, J. G. and L. C. Ernest. 1967. Effect of protein synthesis inhibitors, auxin, and gib- berellic acid on abscission. Physiol. Plantarum 20:1027-1038. ARTICLE II REGULATION OF ENDOGENOUS INDOLEACETIC ACID AND KEEPING QUALITY OF POINSETTIA Regulation of Endogenous Indoleactice Acid and Keeping Quality of Poinsettia Abstract. A study was made of the changes with aging in the level of endogenous auxin and the activity of IAA- oxidase in the bracts of 2 poinSettia cultivars. The auxin level decreased with time in both cul- tivars but faster in the poor keeper. The activity of the IAA-oxidase system and the level of hydrogen peroxide increased with aging. The auxin level could be related to the activity of IAA-oxidase. The IAA-oxidase activity could be related to the appearance of new forms of perox- idase. A hypothesis is developed to explain differences in keeping quality among poinsettia cultivars based on changes in the level of endogenous auxin. INTRODUCTION Keeping quality of poinsettias is dependent on the rate of bract abscission and wide differences exist with respect to this factor among cultivars. Indoleace— tic acid (IAA) at concentrations above lO-SM applied to the petiole stump has been found to delay abscission of eXplants (10). This is also true in poinsettias for leaves and bracts. Studies with intact plants have 30 31 indicated a close relationship between the level of en- dogenous diffusible IAA and the propensity to abscise (19). A decrease in the level of auxin induces abscis— sion. Several hypotheses have been advanced to explain this auxin effect in_ziyg subscribing to a direct auxin effect (5), auxin interacting with aging and senescence (2), or auxin interacting with ethylene (l). The level of diffusible auxin may be regulated through snythesis, transport, or destruction. Control of abscission in cot- ton has been related to the activity of the IAA-oxidase system (18). This study was conducted to determine endogenous auxin relationships in the poinsettia. Experiments were designed to measure changes in the level of growth regu- lators and especially IAA with normal aging and after experimental treatment. A study of the IAA-oxidase sys- tem was undertaken to ascertain its role in the regulation of the level of IAA. MATERIALS AND METHODS The poinsettia cultivars Paul Mikkelsen, White Ecke, New Ecke Pink and Barbara Ecke Supreme were used in these studies. Paul Mikkelsen, a good keeping culti- var, and Barbara Ecke Supreme, a poor keeping type, were used in more intensive studies. Cultural methods, experi- mental methods, and preparation of explants were as described in Article 1. Both extractable and diffusible growth regulators were assayed by the A3233 coleoptile straight growth bio— assay. Five gram samples of both healthy and senescent freeze dried leaf and bract tissue from Paul Mikkelsen and Barbara Ecke Supreme were extracted 24 hrs at 0° with 2x 75 m1 aliquots of methanol and rinsed with a third. The methanol extracts were combined, dried in a flash evaporator and redissolved in 75 ml water. The pH was adjusted to 3.0 and extracted with 5x 50 ml aliquots of peroxide-free ether. The ether extract was taken just to the point of dryness in a flash evaporator and the final solution made to 1 ml with ethanol. Diffusible growth regulators were determined on either individual entire bracts or flowering stems out directly above the primary branch point. They were em- bedded in 1% agar at 21° and 100% RH in the dark and left 32 _ _ ”2.2:; 33 to diffuse for 24 hrs. The agar was extracted 3 times with 50 ml aliquots of methanol and subsequent concen- tration was the same as for the extractable growth regulators. Duplicate 100 pl samples of each extract were spotted on Whatman No. 1 paper and developed in descend- ing chromatography with isopropanolzammonium hydroxide: water, 10:1elw7v for 20 cm. Each chromatogram was cut into 10 equal sections and eluted in the bioassay tube with 1 ml 0.1 M phosphate-citrate buffer pH 5.0 with 2% sucrose added. The coleoptile straight growth bioassay using Avena sativa cv. Brighton was done according to Nitsch and Nitsch (15). All steps were carried out at 21°. Peroxidase or IAA-oxidase enzyme studies were carried out using fresh or freeze dried tissue. Freeze dried material was extracted for 2 hrs at 0° with suf- ficient 0.1 M phosphate-citrate buffer pH 6.0 to give a final protein concentration of approximately 1 mg/ml (usually 25 ml/gm tissue). For analyses using fresh tis- sue only the petioles were used. They were coarsely chopped then homogenized in a glass homogenizer at 0° with 5 volumes of phosphate-citrate buffer. In either case the extracts were given minimal further treatment as recommended by Steward and Barber (21). This usually involved a 3,000 x g centrifugation for 5 min followed 34 by a second centrifugation at 100,000 x g, for 30 min all at 0°. The supernatant was used directly for enzyme activity or gel electrOphoresis studies. For studies on the kinetics of IAA-oxidase the method of Meudt and Gaines (14) was followed. The best results were found when 0.5 m1 of the enzyme extract was incubated for 30 min in the dark at 21° with 1.5 ml of the incubation mixture which contained 0.5 mM IAA, 0.2 mM 2,4-dichlorophenol and 0.1 mM H in 0.01 M phOSphate- 202 citrate buffer pH 6.1. The reaction was stopped after 30 min with the addition of 1 ml of 1% p-dimethylamino- cinnamaldehyde in 2N HCl and the color read at 562 mu after a further 30 min. The method for vertical disc gel electrOphoresis was based on that of Davis (3). Routinely 0.2 ml enzyme extract was run in 7% polyacrylamide gel at pH 8.3 with 2.5 m amps/gel. After running, peroxidase activity was developed by incubating the gels in a mixture of 5 mM guaiacol and 1 mM H in 0.1 M phosphate buffer pH 6.1. 202 Color developed rapidly and the reaction was stopped by flooding with 10% acetic acid. Perioxide assay was based on the method of MacNevin and Urone (13). Fresh petioles were homogenized in excess cold ethanol. Peroxide was complexed by the addition of 0.5 m1 of 12.5% TiCl and precipitated with excess NH OH. 4 4 After centrifugation for 5 min at 2,000 x g the supernatent 35 was discarded and the precipitate solubilized in 10 ml of 3.0 N HCl. The yellow color was read at 405 mu. RESULTS Extractable growth regulators. The results of this study are shown in Figure 1. The extracts contained a complex of substances affecting coleoptile growth. There were differences between cultivars, between leaves and bracts, and between healthy and senescent tissue. Pure IAA Spotted on the chromatogram for standardization was found to have an RF of 0.3 to 0.4 with a tail in the 0.2 to 0.3 region. The equivalent region of the extract chromato- grams caused stimulated coleoptile elongation and showed variation among samples. In general there was greater activity in extracts from healthy tissue as compared to senescent tissue. There was also more growth stimulation from extracts of the healthy tissue of Barbara Ecke Su- preme than of Paul Mikkelsen. Healthy bracts showed greater activity than correSponding leaves. Differences between cultivars and tissues were not as apparent in the extract from senescing material. Other zones of promotion also showed differential activity. In the zone RF 0 to 0.1 the extracts of Paul Mikkelsen showed greater activity while in the zone 0.7 to 0.8 Barbara Ecke Supreme had the greater activity. 36 Figure l. 37 Extractable growth regulators from leaves and bracts of 2 cultivars of poinsettia at anthesis and the time of abscission measured as the effect on the growth of Avena coleoptile sections. Coleoptilo Growth 38 EXIRACTABLE GROWIH REGULATORS BARBARA ECKE SUPREME PAUL MIKKELSEN Leaves non senescent Rt leaves senescent Br ac ts non senescent Bracts senescent 39 There were zones of active inhibition and these too showed differences among samples. The zone from 0.6 to 0.7 showed less activity in the leaf samples of Bar- bara Ecke Supreme while the zone 0.8 to 0.9 showed greater inhibitory activity in Paul Mikkelsen. There were more inhibitory substances in Paul Mikkelsen than in Barbara Ecke Supreme. In comparing mature healthy and senescent abscised tissue the complex of stimulatory substances diminished while the inhibitory substances remained rela- tively static. Diffusible growth regulators in the course of aging. The growth regulators diffusing from the flowering head were measured at weekly intervals on 2 cultivars of poinset- tia, Paul Mikkelsen and Barbara Ecke Supreme, which were held in a simulated home environment. The results of this study are presented in Figure 2. There are definite zones of stimulation and inhibition on the chromatograms. Relative to the study on extractable growth regulators there are fewer, more clearly defined zones of activity. The zone corresponding to IAA, RF 0.3 to 0.4, stimulated coleoptile growth. The pattern for both cultivars was the same. After 1 week there was a small increase after which there was a steady decline. Extracts from Barbara Ecke Supreme declined in activity more rapidly. A second major zone of stimulation 0 to 0.1 also changed with time. 40 Figure 2. Diffusible growth regulators from flower- ing heads of poinsettia sampled at weekly intervals after anthesis measured as the effect on the growth of Avena coleoptile sections. .co:.co:>co 0E0; c. 3.0.»; n a .3 a: 05.0.9.5 .01 on £930.11: 30a mepM®H mm mm as me m.s m.oH o.m m.mH oaoa mood o o o I NMNH mpomufl op am as an m m m an H m N NH sea muse mm: mm mm me as e.m m.m~ 5.x «.ma «Hm mmmw memww on me so Hm H m o an m m a HH mma msmumsm mxom mumnumm NOH he no ma e.m «.ma w.m m.o~ can mama mm>mma mm om mm mm h.n ~.ma m.v m.wa cam mmaa muomun xcflm mxom 3oz as es en mm ~.HH o.~H m.o H.¢H mmm omen mm>mma pm mm mm mm ¢.m o.oa «.s ¢.m «mm mmma muomnn mxom mung; mm mm mm mm m.oa s.ma a.m o.na pom ones mm>mmH em as am mm m.v o.m v.v ¢.m mmm mmma muomun cmmamxxfiz Homm m m m m m m m m m m um>fluaso nonmwm Hmmsm z mHQDHom z cflmuoum coaumuflmmmm .u3 Map Em\wE ca mumuphnon IHmo cam :mmouuHG paw H£\u3 amp Em\mo H: CH pmHSmme mH coaumuflmmmm .mnm>fluaso mwuummsflom m «0 muomnn can mm>mma Amv unmommcmm Ummflomnm paw Amv musume hauammz mo mam>ma mumuquSm paw hufl>wuom Ofiaonmumz .N magma 94 The level of protein N decreased markedly in each case. This was not typical of the aging trends where protein either increased or maintained an overall con- stancy. Soluble non-protein N did not respond the same in different tissues or cultivars. In all cases it de— clined but most in the bracts of the poor keeping culti- vars and least in the leaves of the good keepers. The terminal values are again not in preportion and in some cases not in the direction of the trends during aging. Sugar accumulated in all leaves but only in bracts of the good keepers while it declined in the others. Starch, on the other hand, accumulated in every instance. In all cases where carbohydrates accumulated it was proportionately less than the trends during aging would have indicated. Correlation regression analysis was performed to establish possible relationships between 1 or more of the indices of metabolic activity and differences in keeping time. Values for each parameter at each sampling date were compared with the rate of bract abscission for that date. Comparisons were made over all cultivars and for each individual cultivar except for Barbara Ecke Supreme and MSU 64-5 which were pooled since they re- sponded similarly and each alone provided an insufficient sample. Correlations were also run between the same parameters and the rate of abscission 5 days later. 95 Preliminary tests had shown that a debladed bract abscises approximately 5 days later and it was felt that the condi- tion of the bract 5 days prior to abscission would provide a more realistic comparison. Results of this analysis, expressed as coefficients of correlation are summarized in Table 3. The results indicated a very poor degree of cor- relation and an inconsistency in the direction of the re- gression. Correlation over all samples gave a value intermediate to the individual cultivars and in no case was it a better fit than the best individual cultivar. In general the best fit was with respiration where in every case negative regression was found. Starch was also found to be negatively correlated. Sugars were gen- erally positively correlated with the exception of Paul Mikkelsen. Soluble non-protein N appeared to be posi- tively correlated with abscission in the good keepers and negatively correlated in the poor keepers. Protein N was negatively correlated in the poor keeping cultivars and gave the worst fit to abscission among all parameters. Very few of the correlations were significant and those which were did not indicate any particular pattern but were scattered among all parameters and cultivars. Cor- relation between the parameters and abscission 5 days later did not appear to describe as close a fit as with abscis- sion on the day of sampling. 96 Table 3. Coefficients of correlation between parameters of metabolism and the rate of bract abscission at the time of sampling and 5 days later. Protein Soluble Cultivar Respiration N N Sugar Starch Abscission Paul Mikkelsen -.673 -.051 +.770* -.615 -.021 White Ecke -.887* -.086 +.634 +.813* -.874 New Ecke Pink -.666 -.204 -.623 +.448 -.491 Barbara Ecke -.262 +.305 -.507 +.125 —.828 Supreme and MSU 64-5 Total -.677* -.012 +.l65 +.352 -.575* Abscission 5 Days Later Paul Mikkelsen -.416 -.433 +.509 -.744* -.674 White Ecke -.8l4* -.392 +.802* +.849* -.889 New Ecke Pink -.644 —.452 +.053 +.311 -.631 Barbara Ecke -.123 +.514 -.347 +.4l8 -.596 Supreme and MSU 64-5 Total -.481 -.019 +.182 -.275 -.555* *Significant at the 5% level. 97 Exhaustive attempts to monitor the level of en- dogenous ethylene in the poinsettia at different stages of aging and senescence gave only negative results indi— cating no detectable endOgenous ethylene. Extraction of the internal atmosphere from poinsettia bracts or leaves and control extraction of air from a water sample all indicated a trace of ethylene not greater than 0.01 ppm of the gas sample. Moreover the level of ethylene bore no apparent relationship to the size of sample nor to the time of sampling but was constant and indicative of the ambient level in the external environment. Long term collection of ethylene from air passed over poinsettia material in a closed system indicated ethylene levels not different from blank controls. Vari- ability was found between replications. Part of this variability could have been due to faulty apparatus allow- ing gas leaks or variable pressure altering the flow rate. However, the system was not so insensitive as to mask any large difference in ethylene concentration particularly between plant sample and blank. Therefore, the conclusion drawn from both methods is that the poinsettia does not have an endogenous ethylene concentration greater than that of the ambient atmOSphere. DISCUSSION Changes in metabolism with time could be associated with differences among poinsettia cultivars with respect to keeping quality. There were several differences be- tween good and poor keepers which may bear a relation to their behavior in a home environment. The assumption on which the study was based was that there were differential rates of bract or leaf aging among different cultivars of poinsettia. The respiration rate as a function of time most nearly describes the theoretical situation when compari- sons are made among cultivars and between samples from plants held in a greenhouse and a home environment. The reason for the differences in bract respiration is not completely clear. Conceivably it could be the result of limiting substrate level, limiting enzyme catalysts, or the buildup of rate limiting anti-metabolites. The first possibility may be eliminated on the basis of the data which shows a maintenance in the levels of both sugars and starch. Protein N maintained a steady level in the bracts of all cultivars though quantitative uniformity does not imply strict continuity of the same enzymes and as no qualitative protein studies were undertaken this 98 99 aspect must remain in doubt. Similarly the accumulation of anti-metabolites was not measured directly except in the case of soluble non-protein N. Valdovinos and Muir (56) showed that accumulation of amino acids and es- pecially those of the D—form could stimulate senescence and abscission through an inhibition of protein synthesis. There was an increase in the soluble non—protein N frac- tion in the bracts of all cultivars which was more pro- nounced in the poor keepers. This increase could con— ceivably indicate an increase in amino acids which, on being mobilized, could induce abscission. The possible importance of mobilization in bract abscission is apparent in the comparison of healthy and abscised bracts. Considerable mobilization of soluble nitrogenous compounds, both protein and non-protein N, occurred as the bracts senesced. It did not occur during the aging phase when there was accumulation with no ap- parent movement out of the bract. Mobilization was not apparent for either sugars or starch. Scott and Leopold (49) suggested that the mobilization of nutrients prior to abscission could accelerate senescence and abscission. More recent work by Abeles (2) on ethylene stimulated abscission has shown that mobilization is not necessarily an integral part of abscission. The present study does not elucidate the possible role of mobilization. It was shown to be a factor in senescence in poinsettia bracts 100 but whether it is a result of senescence, corollary with abscission, or whether it plays a role in enhancing the abscission process is speculative. The absence of measurable ethylene evolution at any sampling time and for any cultivar leaves open the question of the role of ethylene in abscission of intact plants. The methods employed were designed to measure gross ethylene. It is possible that ethylene evolved in or near the abscission zone at the time of senescence would have been masked by the bulk of non-ethylene evolv- ing tissue measured in the same sample. Most of the studies on ethylene mediated abscission have employed explants as their plant material and it has been shown (11) that the preparation of an explant induces a surge of ethylene which could possibly result in a shift in the metabolic pathways to one which is more receptive to additional ethylene. One problem in approaching a study on aging by the methods employed in this series of experiments is the in- troduction of a sampling bias which must be considered in any interpretation. Poinsettia plants in the home con- tinue to develop more bracts, albeit at a reduced rate, and senescence and abscission occurs randomly over an extended period of time. The sampling method measured the overall state of the leaves or flowering head. It did not take into account any differences among cultivars 101 with respect to continued growth nor did it account for the portion of the original sample already abscised. This latter aspect would be critical if it could be shown that differences in abscission among cultivars were not the result of differential aging rates but rather the ef- fect of a second factor inducing abscission at different relative degrees of aging. The results showing decreas- ing reSpiration rates and faster aging in the bracts of the poor keeping cultivars would argue that abscission is related to a certain degree of aging. A second aSpect of the sampling dilemma is the interaction with nutrients mobilized out of senescing tissue and accumulating in the remaining bracts or leaves. Where mobilization was shown to occur, as for nitrogenous compounds, it could alter the pattern with aging and make comparisons with studies using explants where there is no mobilization into distal tissue more difficult. 102 ADDITIONAL RELEVANT DATA ON IAA-OXIDASE ACTIVITY Table 4. Optical density at 562 am after incubation of active and inactivated by boiling extracts of the bracts of 2 poinsettia cultivars with and without the addition of H202. Incubation was for 0 and 30 min and color development was read 30 min later. j Paul Mikkelsen Barbara Ecke Supreme Inactive Active Inactive Active Time 0 without H202 .061 .041 .046 .046 with H202 .131 .137 .155 .149 Time 30 without H202 .066 .208 .066 .229 with H O .208 .357 .222 .432 2 2 103 Table 5. Optical density at 562 mu after incubation for 30 min of active and inactive extracts of the bracts of 2 cultivars of poinsettia before and after passing through a column of Sephadex G-25. Complete incubation mix contained 0.5 mM IAA, 0.2 mM 2,4-dichlorophenol, 0.2 mM MnClz in 0.01 M phosphate-citrate buffer pH 6.1 with or without 0.1 mM H202 added. Paul Mikkelsen Barbara Ecke Supreme Inactive Active Inactive Active Before Spehadex Without H202 complete .041 .208 .066 .237 less Mn .046 .215 .071 .244 less 2,4—DCP .032 .187 .046 .276 With H202 complete .168 .357 .174 .432 less Mn .187 .377 .194 .398 less 2,4-DCP .161 .387 .155 .432 After Sephadex Without H202 complete .000 .071 .000 .092 less Mn .000 .086 .000 .086 less 2,4-DCP .000 .000 .000 .000 With H202 complete .081 .155 .092 .174 less Mn .102 .143 .092 .161 less 2,4—DCP .076 .071 .081 .086 GENERAL DISCUSSION Results of the experiments conducted in this study indicated differences between good and poor keeping cultivars of poinsettia which could be related to observed differences in abscission rates. There are many recent historic and current hypotheses dealing with the physio- logical basis for abscission control and involving the aging process or the action of growth regulators such as auxin or ethylene. It is necessary to examine the data in the light of these hypotheses to establish a comprehensive hypothesis to explain abscission control in poinsettia and cultivar differences with respect to this character. Gaur and Leopold (22) proposed that the concentra- tion of auxin at the abscission zone determines whether a leaf will remain or abscise. A high concentration of auxin inhibits abscission while a low concentration promotes it. The application of exogenous IAA to poinsettia explants confirmed that a high concentration did inhibit abscission but no stimulation was found at lower concentrations. In the normal aging of intact leaves or bracts it is diffi- cult to say whether the normally reduced auxin level pro- motes the abscission process or merely allows it to proceed. 104 105 Jacobs (32) has proposed that auxin delays ab- scission indirectly by maintaining growth. Elongation measurements were not made of intact or debladed poin- settia bracts but there was no observable growth in ex- plants except where some treatments induced epinasty and bracts were retained on good keeping cultivars which had long ceased growth in the home environment. Perhaps it is more a matter of growth maintaining a high auxin level in vivo than the other way. Jacobs (30) and Addicott gt_al. (8) proposed sim- ilar hypotheses explaining abscission control as the re- sult of auxin-auxin interactions at the zone of abscission. Called respectively the auxin-auxin balance hypothesis and the auxin gradient hypothesis they both theorize that the auxin ratio across the abscission zone is the controlling factor. If the balance favors the distal side or the gradient is from the distall to the proximal then the leaf remains attached. However if the balance or gradient is reversed, irrespective of quantity, then abscission occurs. Attempts to establish reverse gradients or an unfavorable balance by proximal application of IAA to poinsettia explants resulted in a slight inhibition of abscission rather than the theoretically expected promotion. Rubinstein and Leopold (47) further elaborated on the control of abscission by auxin in proposing the 106 2-stage theory for auxin action. In explants there is a first stage or induction period where abscission is inhibited by auxin followed by a second stage which is promoted by the same concentration of auxin. Attempts to illustrate the 2-stage auxin action on poinsettia ex- plants were not successful. Several hypotheses have as their basis for ab- scission control the interaction between auxin and ethylene. Gawadi and Avery (23) hypothesized that abscis- sion was dependent on the balance of auxin and ethylene. They further suggested that ethylene could be active in hastening leaf aging at low auxin levels. Hall (26) con- sidered that when auxin synthesis is reduced or ethylene evolution increased abscission results. Attempts to reproduce the findings of Gawadi and Avery (23), using basically similar methods though differing in several details, failed to show any promotion of abscission with ethylene. Moreover, ethylene was not found to be evolved from normally aging intact poinsettia plants. Abeles and his group have continually stressed the importance of ethylene as the primary mechanism of abscission control while other factors may interact with the ethylene response or cause stimulation or inhibition of ethylene evolution. Abeles and Rubinstein (5) found a 2 stage ethylene action similar to that for auxin and also a positive correlation between auxin and ethylene 107 levels. They hypothesized that the auxin effect in ab- scission is ethylene mediated. Similarly (1) a wide range of abscission stimulators were found to have one common basis: that of stimulation of ethylene evolution. According to Abeles and Holm (3) ethylene has its effect in regulating protein synthesis through the stimulation of specific m-RNA's and r-RNA's. More recently Abeles (4) has expanded this hypothesis to include an interaction between aging and ethylene which could serve as a basis for abscission control in intact plants and explants. The aging-ethylene hypothesis states that the role of ethylene is to accelerate the formation of enzymes responsible for abscission but ethylene cannot act while there is a con- tinued supply of a juvenility factor such as auxin. A decrease in IAA concommitant with aging would result in a shift to the ethylene sensitive stage. Again the prob- lem of interpreting poinsettia bract abscission in terms of Abeles hypotheses has been the lack of response by intact or debladed poinsettia bracts to ethylene. Burg (11) has suggested a hypothesis dealing with auxin and ethylene effects on abscission in which the primary effect of ethylene in promoting abscission is in limiting the synthesis, transport and/or destruction of auxin. The decrease in effective auxin causes the onset of senescence and abscission. Ethylene may have a second effect in accelerating abscission through a number of 108 possible systems. This hypothesis is compatible with the findings for poinsettia if it is assumed that ethylene is not essential for the observed decrease in auxin. Ethylene could conceivably have different catalytic prop- erties depending on the plant species and tissue involved. Other hypotheses have based abscission control on other growth regulators in interaction with auxin. Os- borne (41) found a diffusible substance, capable of ac- celerating abscission, to increase in senescing leaves. She hypothesized that the increase in this senescence factor combined with the decrease in auxin in aging leaves is responsible for abscission. No experiments were de- vised to measure senescence factors directly in poinsettia; however, no diffusible substance inhibitory in the 53233 coleoptile straight growth test was found to increase markedly in aging bracts. Carns (12) suggested that gibberellic acid plays a role in abscission. He proposed that auxin, gibberellin and the senescence factor interact in one common mechanism which regulates abscission. The fault with this sort of hypothesis is that it is necessarily vague and much easier to formulate than substantiate. Exogenous gibberellin did not exert any pronounced effect on poinsettia bract abscission. Addicott et al. (6) described the senescence fac- tor as abscisic acid and linked this hormone to abscission regulation. Abscisic acid did not influence the rate of 109 abscission of intact poinsettia bracts though it did stimu- late leaf abscission. Several hypotheses have based abscission on mobil- ization phenomena with depletion or accumulation of meta- bolites or anti-metabolites. Osborne and Moss (42) showed that artificially induced mobilization could affect abscission and hypothesized that alterations in the bal- ance of growth regulators ifl.!l!2.°°uld induce mobilization resulting in localized senescence. Valdovinos and Muir (56) hypothesized that certain amino acids which may be mobilized at senescence could induce abscission through an anti-metabolite action interfering with normal protein synthesis. Mobilization, particularly of the amino acid fraction, was observed in senescent intact poinsettia bracts. No experiments were undertaken to test the pos- sible implications of this observation but, based on the hypothesis of Valdovinos and Muir (56) and supported by observations by Rubinstein and LeOpold (47), this aspect could conceivably play a role in stimulating abscission of poinsettia bracts. Scott and LeOpold (49) showed that stage 1 of bean explant abscission could be associated with the es— tablishment of a nutrient imbalance across the abscission zone and supported the hypothesis that mobilization and subsequent cellular senescence provide a component in abscission. The absence of a demonstrable 2-stage 110 relationship in poinsettia explants may possibly be ex- plained in terms of this hypothesis. One possible assump- tion is that mobilization is slower in aged, debladed poinsettia bract petioles. However, a more attractive assumption.h3that the debladed petiole has few mobilizable reserves relative to the vast proximal sink that is the main stem. In this view there is not a depletion-accumu- lation phenomenon but rather the gradual depletion of nutrients in the petiole which would accelerate the senes- cence process initiated by a primary fundamental component. Horton and Osborne (29) have hypothesized that senescence distal to the abscission zone gives rise to diffusion or mobilization products which initiate an in- crease in cellulase activity in the separation zone. The action of cellulase is to effect cell wall dissolution leading directly to abscission while the action of growth, anti-metabolites or growth regulators such as auxin or ethylene is to hasten or retard senescence. A hypothesis of this type in which growth regulator action is indirect and the only critical aSpect is senescence would appear to best fit the poinsettia data. Additional circumstan- tial evidence comes from the study of Gawadi and Avery (23) who showed that poinsettia leaves undergo secondary division forming a complete separation layer before the leaves have achieved full size and require only dissolu— tion of the middle lamella for abscission. 111 The foregoing hypotheses have attempted, by and large, to explain the primary cause and mechanism of ab- scission. This study was designed to test for differences among poinsettia cultivars that could serve as a basis to explain differential abscission rates. There was no elaboration as to what mechanism may be involved in poinsettia bract abscission but the results did not indi- cate the presence of more than one system. The general lack of an ethylene effect, the absence of any interac- tion between cultivar and exogenous growth regulator and differences in rate of change of endogenous factors rather than qualitative differences all point to one common mechanism but do not add to the elucidation of that mechanism. What the results do indicate is that differences in abscission rate among cultivars is the result of the differential appearance with time of the primary cause that initiates abscission and further that the primary cause of abscission in poinsettia is a decrease in dif- fusible auxin below a certain level at which point senes- cence is initiated in the abscission zone. The most characteristic internal changes in the bracts were a decrease in the level of diffusible auxin, a decrease in the rate of respiration, an increase in the capacity of the IAA-oxidase enzyme system, an increase in the IAA-oxidase co-factor, peroxide and mobilization 112 of soluble nitrogenous compounds. From these observations it is possible to formulate the hypothesis that senescence of poinsettia bracts is the result of a decrease in diffu- sible auxin below a certain critical level and that ab- scission is triggered by senescence and further hastened by senescence dependent mobilization. Within the framework of this hypothesis alterations in the respiration rate, the activity of IAA-oxidase, or the level of peroxide may all be interpreted as indirectly affecting abscission through a direct effect on the level of endogenous auxin. It is an assumption that the respiration rate is a valid estimate of the rate of auxin synthesis which was not measured directly. The hypothesis does not extend to the mechanism by which senescence and mobilization act in effecting abscission. A sketch of the ways in which these factors may interact in governing abscission is presented in Figure 6. It illustrates the hypothesis that senescence is based on the level of endogenous auxin which in turn is a combined function of auxin synthesis and destruction. The method chosen to illustrate this may be termed cumu- lative vector analysis. The sketch is designed to rep- resent the principle rather than any particular case. The 4 independent variables; respiration, growth, IAA- oxidase, and peroxide have each been assigned the same maximum and are assumed to run from zero to unity. Figure 6. 113 The theoretical interrelationships among factors regulating endogenous auxin, senescence and abscission in poinsettia bracts. 114 COMPONENTS OF ABSCISSION Endogenous auxin RETENTION J, \ ’: ABSCISSION TIME —> Mobilization IAA oxidase J, a: ANTHESIS T T SENESCENCE ABSCISSION 115 Assumed parameters and unmeasured values are presented as dotted lines while observed values are in the solid lines. Although the effect of growth was not measured and is completely speculative, conceivably it could play an important role during the early development stages of the bract. The dependent variable, endogenous IAA, is portrayed as the cumulative mean of the independent variables. The derived shape of the auxin curve fits closely to that observed for diffusible auxin. Finally, the point of senescence initiation is at an arbitrarily selected point on the auxin curve. The sketch has been interpreted as representing the mean of the entire flowering head. It could also be interpreted as repre- senting a single bract. The hypothesis may be visualized as one of devel- Opments among the quantitative relationships rather than qualitative differences or abrupt changes. In this way it is possible to account for observable differences among cultivars with respect to abscission rate. A change in the level of any factor controlling the level of auxin would influence the approach to senescence and a change in more than one factor would have either a cumulative or compensating effect. What was observed were relatively small increments in all factors between good and poor keeping cultivars. However, in every case the increment of the poor keepers was in the direction favoring lower 116 auxin levels thus leading to a magnified cumulative ef- fect and a disproportionate increment in diffusible auxin. The time scale, which is the measure of keeping quality, is directly dependent on the increment, inflec- tion point and slope of the auxin curve and the differences among cultivars may be a reflection of any or all of these. The data would indicate that the slope of decrease after inflection showed the greatest variation. Thus, the slower rate of decrease in diffusible auxin for the good keeping cultivar Paul Mikkelsen when compared to the poor keeper Barbara Ecke Supreme was the factor primarily re- sponsible for the characteristic of improved bract holding ability. The hypothesis explaining the physiological basis for abscission control may be related to the inheritance pattern for keeping quality. According to Stewart (53) and observations made by the author keeping quality is inherited quantitatively and is not linked to any other known character in poinsettia. The quantitative in- heritance pattern would be expected in view of the role of 3 independent factors in governing the auxin level. The large number of peroxidase isoenzymes alone would predicate quantitative inheritance or at least quantita- tive modification. Bempong and Sink (9) showed that the poinsettia may contain multiple loci making genetic analysis more complex. 117 Crosses between cultivars with different keeping times usually give progeny intermediate in nature or somewhat favoring either parent. One notable exception to this is MSU 64-5 a triploid seedling from a cross be- tween the diploid, White Ecke and the tetraploid, Barbara Ecke Supreme. In keeping time and metabolic characteris- tics it was very similar to Barbara Ecke Supreme, probably as a result of a gene dosage effect. Stewart (53) has found independent inheritance for keeping quality between leaves and bracts. The pre- sent study which showed large differences in bract abscis- sion and internal factors but no difference in leaf abscission or internal relationships among the cultivars studied would support this finding. Clearly then, the hypothesis explaining keeping quality differences must be limited in application to the bracts. Further investi- gation will be necessary to discover the underlying control of poinsettia leaf abscission. One of the anticipated benefits of this study was the development of an objective method to assess potential keeping quality in a breeding program. Analysis of the diffusible auxin level at anthesis and again after a pre- determined time of holding in a home environment would serve this end. More elaborate analysis of respiration rate, IAA- oxidase activity and peroxide level would give an even 118 clearer picture of the situation and allow better parental matching for mutual compensation. The disadvantage of this type of assay is that it entails sophisticated equip- ment and a great deal of time in performing the analysis and the plants must be kept nearly as long as for subjec- tive analysis. Preliminary investigation of the behavior of de— bladed bract petioles suggests that this may serve as a method of assaying for keeping quality. 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