ESCLAWON AND QHARACTERJZATEON OF A GERMENATEQN ENHIBETQR FROM SUfiAR BEET FRUIT “fizasés {er the Degree of Ph. D. MECHIGAN STATE UNIVERSETY Earl D. Mitcheil, Jr. 1966 Tfinblé This is to certify that the thesis entitled The Isolation and Characterization of a Germination Inhibitor from Sugar Beet Fruit. presented by Earl D. Mitchell, Jr. has been accepted towards fulfillment of the requirements for _P_h..ll.__degree in—Bioehemistry :0? E . /' waded“ Major professor N. E. Tolbert Date M 0-169 nu ABSTRACT ISOLATION AND CHARACTERIZATION OF A GERMINATION INHIBITOR FROM SUGAR BEET FRUIT by Earl D. Mitchell, Jr. A seed germination inhibitor, gig n-cyclohexene- 1,2-dicarboximide, has been isolated from sugar beet fruit. The isolation procedure involved ether extrac- tion, silicic acid chromatography, cellulose chromato- graphy and crystallization after elution from a sepha- dex G-lO gel filtration column. Physical data was ob- tained by infrared, ultraviolet, nuclear magnetic re- sonance and mass spectral analyses. Identity of the compound was confirmed by synthesis and comparison of physical and biological data. Other inhibitors were present in the sugar beet fruit, but they were not identified. The same procedures were used to investi- gate inhibitors from wheat chaff, which appears to con- tain similar compounds. Fifty percent inhibition of lettuce seed germination was effected by 5 x 10'4fl gig h-cyclohexene-l,2-dicarbox- imide. Inhibition was not reversed by gibberellins, but the inhibitor could be removed by washing and then the seeds would germinate. Stimulated respiration of seeds after imbibing water did not occur in the presence of the inhibitor. Earl D. Mitchell, Jr. -2 The structure of cis h-cyclohexene-l.Z-dicarboximide is unusual for naturally occurring compounds. However, a similar structure occurrs in the synthetic N-ethyl maleimides which are herbicides and in the agricultural fungicide cycloheximide, which is a protein synthesis inhibitor. ISOLATION AND CHARACTERIZATION OF A GERMINATION INHIBITOR FROM SUGAR BEET FRUIT By J5“ J Earl Df’Mitchell, Jr. A THESIS Submitted to MichiganSState University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1966 r l ' 7 ' if“: .éy'yfié/E’; » V .4 e,» x/‘ I ACKNOWLEDGEMENTS The author wishes to express his earnest appre- ciation to Professor N. E. Tolbert for his guidance, support and understanding throughout the course of this investigation and for counsel in the preparation of this manuscript. He is also grateful to Profes- sors: H. M. Sell for use of laboratory equipment, F. w. Snyder for sugar beet material and literature, J. E. Varner for crystalline Q -amylase enzyme and advice on.the cx-amylase assay and P. Kindel for his counsel. The author acknowledges the support of the Herman Frasch Foundation for this research program. 11 TABLE OF CONTENTS Page INTRODUCTION..................................... 1 Inhibition of Germination................... l Germination................................. 8 Dormancy Factors............................ 10 Previous Research on the Dormancy Factors at Michigan State University............. 12 MATERIALS AND METHODS............................ 15 General Equipment and Chemicals............. 15 Bioassay.................................... l7 Cis h-cyclohexene l,2-Dicarboximide...... l7 «.Amylase Barley Endosperm Assay......... 18 (X-Amy1ase Inhibition Assay............... 20 qumylase Whole Seed Assay............... 2O Lettuce Seed Respiration................. 21 Avena Straight Growth Assay.............° 21 RESULTS AND DISCUSSION........................... 23 Isolation Procedure from Sugar Beet Fruit... 23 Other Isolation Procedure Attempted......... 29 Thin Layer Chromatography (TLC).......... 29 Gas Chromatography (GC).................. 30 Proof of Structure.......................... 33 Isolation Procedure from Wheat Chaff........ 50 Formation of Inhibitor in Wheat Chaff....... 53 Properties of Inhibitor from Wheat Chaff.... 56 iii Page BIOLOGICAL STUDIES WITH Cis U-CYCLOHEXENE 1’2 DICARBOXIMIDE (CCD)OOOQOOOOOOOOOOOOOOOOOOO 58 ‘.Light Gibberellins and d-amylase SyntheSj-SOOOOOOOOOOOOOOOOOOOOOOOOOO00000 58 Respiration.0.0.0.0000...OOOOOOOOOOOOOOOOO 62 Avena Straight Growth..................... 68 STRUCTURE OF Cis n—CYCLOHEXENE 1.2 DICARBOXIMIDE AND ITS BIOLOGICAL] ACTIVITY...0.00.00.00.00... 7O SUMMABYOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. 76 BIBLIOGRAPHY...00......OOOOOOCOOOOOOOCO0.0.0.0... 77 iv LIST OF TABLES Table No. Page 1. Isolation procedure for 8.“ kg of Sugar Beet Fruit...................... M 2. Elution pattern for Silicic acid column.. 25 3. Chemical Test for Inhibitor.............. 36 4. Comparison of the activity of the Isolated and Synthesized Inhi- bitorsOO0.0000000000000000.0.0.0000... “9 5. Isolation Procedure from Wheat Chaff..... 51 6. a-amylase synthesis in the Barley Endosperm..0...OOOOOOOOOOOOOOOOOOOOOOO 6O 7. a-amylase synthesis in the Whole seedOOOOOOOOOOOOOOOOOOOCOOOOOOOOOOOOOO 61 8. Effect of COD on Avena Straight Growth... 69 LIST OF FIGURES Figure Title Page 1. Sephadex G-10 column elution pattern...... 28 2. Infrared Spectrum of the Unknown InhibitorOOOOOOOOOOOOO0.00.00....000.00 35 . mass SpectMOOOCCC.........OOOOOOOOOOOCOO 39 3 4. U.V. Spectrum of unknown.................. #1 5. N.m.r. Spectrum........................... #4 6 . I.r. Spectra of the synthetic and unknown InhibitorOOOO00.00.000.900.0... n8 7. Elution of Wheat Inhibitors from Sephadex COl‘lmnCOOOOOOOOOOODOO00....0.0.0.000... 55 8. Respiration of White aris lettuce seeds (oxygen UptakeS.................. 65 9. Respiration rate of white paris lettuce seedsto0.00.00.000.00...0.00.00.00.00.0 67 vi INTRODUCTION INHIBITION OF GERMINATION In recent years evidence has been accumulating that dormancy in plants is regulated by many endogenous na- tural occurring inhibitors and growth substances (1). A seed may be viewed as a resting bud in which biological activity is the result of a marvelous interplay of sev- eral hormones that trigger different enzymes. Dormancy is a phase of development. In seeds, one finds a ba- lance between inhibitors and promoters (2). This ba- lance can be broken by removal or alteration of the in- hibitors. Thus, dormancy of hard winter wheat can be broken after a heavy rain before harvesting. when the water washes out the inhibitor(s). A variety of substances can inhibit germination. Inhibitors of germination have been found in grain (3), fruit (U), tubers (5) and buds (6). The many compounds which are toxic to living systems will also, at the toxic concentrations, prevent germination simply by killing the seed. In the latter cases the metabolic processes are irreversibly inhibited. Inhibition by toxic compounds is often the result of inhibition of certain metabolic pathways. Thus all compounds which interfere with normal metabolic pathways are likely to inhibit germination. Various respiratory 2 inhibitors such as cyanide. dinitrophenol, azide, fluo- ride and others, all inhibit germination at concentra- tions similar to those inhibiting metabolic processes. These substances act on germination as a result of their effect on metabolism and not necessarily that of in- ducing dormancy. The most interesting substances are those which prevent germination only when they are present but with- out effecting the seed viability. Of this type, pheno- lic compounds of various kinds will inhibit germination. Mayer and Evenari (7) found 50% inhibition of lettuce seed by such compounds as lO‘ufl coumarin. 10‘3fl ferulic acid, 10'3fl salicyclic acid, 10‘3 resorcinol and 10‘2 ca— techol. Because of the widespread occurrence of phenolic compounds in plants, van Sumere (8) has suggested that these substances might act as natural occurring germina- tion inhibitors. It is also possible to prevent germination of seed by placing them in a solution of high osmotic pressure. When the seeds are removed from such an enviroment and placed in water they can germinate. Some of the com- pounds responsible for high osmotic pressures may be su- gars and inorganic salts such as sodium chloride. One of the more commonly used laboratory compounds is man- nitol which gives results somewhat different than so- 3 dium chloride (9). Uhvits attributes this difference to the ionic toxicity of the salt. In the literature there are a number of reports of germination inhibitors being present in seeds (3) and fruits (h). The use of chromatographic technique has resulted in detection of many of these unknown sub- stances but few have been thoroughly characterized. Cou- marin and hydroxycinnamic acid and their derivatives have been shown to occur in barley husks. These same types of compounds have been identified in clusters of sugar beet seeds, and Massart (h) suggested that these substances act as germination inhibitors. However, as in the case of most of the inhibiting substance in sugar beet (4), the evidence is very uncertain and no definite conclusion can yet be drawn as to the nature of many of these inhibitory compounds. Herbicides also inhibit germination to a greater or lesser extent. Many of the commonly used herbicides, such as 2,4-D, affect germination at comparatively low concentrations. Sometimes herbicides are applied in or- der to kill the weed seedling immediately after germina- tion and before the main crops germinate, and in this case they are not being used as germination inhibitors. Coumarin, as shown below, has been extensively stu- died as a germination inhibitor and it is fairly wide- spread in nature. It is characterized by an aromatic ring and an unsaturated lactone ring. There is no ef- fect of changes in structure of the coumarin molecule on Coumarin its germination inhibition activity. Therefore there is no single essential group on the molecule which is the cause of its inhibitory activity. The mechanism of coumarin's activity on the germination seed is not clear. Coumarin has been studied with fat metabolism, lipid me- tabolism, nitrogen metabolism and respiration (10). An additional interaction is that between coumarin and gib- berellic acid; the latter is able to reverse the inhibi- tory action of coumarin in germination. The effect of coumarin on metabolism of the germinating seed has been studied to great depths, because this substance has ra- ther specific effects in the germinating seed. Many of the reported investigations serve to indicate some of the problems and achievements in metabolic studies du- ring germination even though they do not solve them un- equivocally. One natural occuring inhibitor, far more active than coumarin, was first isolated by Addicott from young cotton fruits as a factor involved in abscission of the leaf petiole (11). This inhibitor was called Abscisin. It is identical with Dormin which was isolated about the same time from dormant sycamore buds by Wareing (l3). Dormin causes a vegetative bud to change to a winter bud. This inhibitor is a sesquiterpene which contains a hy- droxyl group. a carboxylic acid residue and a conjugated ketone of the following structure. Abscisin II was la- ter synthesized by Cornforth (l3). Dormin and Abscisin II may be involved in compli- cated interactions with auxins and gibbere111ns as re- \\ \\ OH coin Abscisin II ported by Wareing (14). It was suggested that 'dormin' appears to inhibit a-amylase production in the gibbe- relic acid treated barley endosperm. The inhibitor re- duces the growth of normal plants and is reversed by gibberellic acid. Dormin was also found to inhibit au- xin induced growth which was later restored when gibbe- 6 rellic acid was applied. Coleoptiles did not respond to gibberellic acid in the absence of dormin. Another Abscisin II like inhibitor is the "QScom- plex" (15) from the potato tuber which showed marked inhibition of wheat coleoptile elongation and of sprou- ting potato eyes. This inhibition was reversed by gib- berellic acid. However, this inhibitor 6 complex, when stored for 6 months at a -lO°, lost the ability to inhibit both coleoptiles and potato buds. Rappaport (15) has also shown that Abscisin II (dormin) when ap- plied at very low dosages almost completely inhibited sprouting of potato buds. Dormin or Abscisin II may be the inhibiting component of the Q complex, since Con- forth (16) has recently shown that Abscisin II (dormin) is very widespread in being present in buds, leaves, tuber, seeds and fruit. While the search for the chemical identity of he. tural occurring inhibitors continued, several synthetic retardants have discovered through serendiptic research. Among these were amo-l6l8 (l7) and cycocel (18, 19). These growth inhibitors seem to inhibit the biosynthesis of gibberellic acid (20) and not the activity of gibbe- rellic acid once it has been formed. Therefore, growth retardants, or inhibitors, are thought not to enter into the processes initiating germination but are 7 operative only when growth has been started by light or gibberellins. Whether or not any of the natural occurring inhibi- tors are hormones is a question of semantics. One might define a plant hormone as a chemical which evoked a re- sponse, possibly at the DNA and RNA level, which has physiological significance. of men The the fan by try GERMINATION: A cereal seed has two major components; the embryo (germ) and the endosperm (the food reserve). The cells of the embryo are alive and contain the necessary hor- mones to initiate cell division and cell enlargement. The most obvious change associated with germination is the rapid uptake of water and oxygen. During the first four days of maize germination most of the water is held by the embryo (21). This water uptake activates the em- bryo, out of which the entire plant will develop. The storage tissue of the cereal seed endosperm is considered to be nonviable but it contains starch for the growth and respiration of the embryo. In the course of normal germination, the starch of the endOSperm is hydrolysed by a-amylase. Simultaneously Yomo (22) and Paleg (23) discovered that gibberellic acid is the chemical or hormone, which is secreted by the em- bryo, in order to activate the aleurone layer to secrete the hydrolytic enzyme a-amylase. Recently Varner (26) has demonstrated that gibberellic acid induces the d3 22' lg synthesis of a-amylase in the aleurone cells. Thus gibberellic acid probably works at the RNA level by con- trolling the synthesis of specific RNAs, which in turn controls the synthesis of Specific enzymes. In addition to inducing the dg novo synthesis of a-amylase, gibbe- 9 rellic acid activates the enzymes that promote the de- gradation of the cell wall (25). The germination of lettuce seeds in response to light is associated with a pigment system. Red light promotes the germination of these seeds (26). However, the seeds are sensitive to the illumination only after they have absorbed enough moisture. After inhibition of water, a one minute exposure to 60 foot-candles or red light is sufficient to give 100% germination. The light absorbing pigment is called phytochrome, a bluish colored protein (27). This light absorption may set in motion a mechanism by which the hormones in the embryo are formed or activated. Since gibberellic acid triggers the syn- thesis of a-amylase, then it is conceivable that the light may trigger the formation of gibberellic acid. The hydro- lytic enzymes may weaken the seed coat and thus allow the young root to break through (26). On the other hand, gib- berellic acid stimulates the germination of Lactuca sativa in the dark as shown by Kahn et. a1 (28). In this case gibberellic acid and far-red light with high temperature as done by Evenari (29) have shown that it is not always possible to effectively reverse the gibberellic induced stimulation by the use of far-red light. These experi- ments have led to the conclusion that gibberellic acid and red light act only partially in the same way and that 10 their mode of action is not identical. DORMANCY FACTORS: This investigation has been involved with the iso- lation of inhibitors of seed germination from sugar beet fruit and wheat seed or chaff. At least 9 organic sub- stances which inhibit seed germination have been isolated from sugar beet fruit (30, u, 31, 32, 33, 3t) but none from wheat. The chemical structures of many of the men- tioned compounds have not been determined. Also, only very limited data on the quantity of inhibitors in sugar beet fruits are presently available. Duym et.al,,(30) have concluded that the aqueous extracts were inhibitory because of the osmotic effect provided by the high concentration of salts present. FrSschel (33) observed that demineralized extracts of su- gar beet fruits inhibited the germination of Lepidium and other seeds and that the extracts also emitted volatile substances which caused inhibition. He indicated the presence of specific organic substances as the cause of inhibition. Dekok et a1.,(32) indicated that the ether extracts of sugar beet fruits contained inhibitors of germination and that these inhibitors were a mixture of organic substances having a synergistic effect on inhibi- tion of germination. The sugar beet seed inhibitors have also been investigated by Massart (4) who chromatographed ll extracted material and described these inhibitors as hy- droxy organic acids. According to Mankind and Mayamoto (33, 34), water soluble oxalate, which exceed two per. cent in sugar beet fruits of some sugar beet varieties, may be casually related to germination performance. Sny- der stain . (35) showed a significant correlation be- tween water soluble oxalate in the fruit and spud on germination of sugar beet seeds. They also had evi- dence that at least other inhibitors besides oxalate were present and depressed the speed of germination of sugar beet Seeds. However, as stated earlier, the inhibitory substan- ces in sugar beet seeds have not been fully characte— rized and evidence for one or more inhibitors is very uncertain. No definite conclusion can be drawn, from the literature, as to the nature of these inhibitory com- pounds. It has been the purpose of this investigation to look for additional inhibitory substances in seeds which may be very Specific, active and unique in reSpect to other known germination inhibitors. Masheov (36) was the first to study and report ger- mination inhibitors in wheat kernels. He studied the in- fluence of water extracts of wheat seeds upon their ger- mination and growth. These inhibitors in wheat were found in certain varieties. Everson and Hart (37) re- 12 ported on the varietal variation of the presence of in- hibitors in mature wheat. All wheat varieties with white kernels readily germinated. Those wheat kernels of red color were more resistant to Sprouting. There is also a morphological difference between the red and white seed coats (38). The seed coat of the red wheats tightly cover the embryo whereas the seed coat of the white wheats are often separated from the embryo. The morphological differences between the two types of wheat seed coats suggest that water may enter the embryo of the white wheat more easily than that of the red wheat. Previous Research On the Dormangy Factors At Michigan State University: This thesis has been a part of a long term conti- nuum of research on dormancy at this University. The orgin of our interest in wheat seed dormancy and at- tempts to alleviate it through crop breeding had been carried out in Professor Everson's group in the Crop Science Department (37). In the Biochemistry Depart— ment, Professor Tolbert's group undertook the task of isolating and characterizing chemical compounds which might be responsible for dormancy. If these compounds were known, it is possible chat, in the future, plant breeders may be able to expedite their program by rapid assays for the compounds and by selecting plants with de- 13 sired amounts of inhibitory material. Miyamota et a1.. (39) obtained a small amount of crystalline inhibitor from wheat seed which, however, was not characterized. The latter steps of their isola- tion procedure differed from the ones reported in this thesis. Gibberellic acid could reverse the effect of this inhibitor on the wheat embryo, but gibberellic acid had no effect on lettuce seed germination in the presence of the inhibitor. Niyamoto et a1.. (39) decided that postharvest dormancy in wheat was caused by inhibitors found in the seed coat of red wheat. Dormancy was not caused by restricted water or oxygen uptake, a mechani- cally tough seed coat or an immature embryo. A. A. Khan, N. E. Tolbert, W. J. Bruin and E. Ever- SOn (in manuscript) investigated the biolOgical distri- bution of the wheat dormancy factors in field grown wheat crops during the 1963 and 196A season. They found that, although there was some dormancy factor in the seed coat, that a far greater amount was present in the chaff. For that reason wheat chaff and the fruit of sugar beet seed was used in the present investigations. Khan et a1.. further deliniated the seasonal variation in amounts of inhibitors in chaff and seeds. Units of inhibitors ac- cumulated during maturation until the seeds were nearly mature, after which the amount dropped preceptibly lb during a rain storm. The urgency of properly harvested material was emphasized. Khan et al.,found that inhibi- tory material was stable if the slightly green chaff were stored at -18°. In the period from 1962 to 1965, A. A. Khan, W. J. Bruin and E. D. Mitchell worked with N. E. Tolbert on the isolation procedures for the dor- mancy factors from wheat chaff and developed the proce- dure essentially as outlined in Table 5. Because of li- mited supply of wheat chaff, F. W. Snyder suggested the use of sugar beet fruit which could be obtained in near- ly unlimited amounts. The isolation procedure developed for wheat chaff was found to be applicable to the sugar beet fruit. It has been the purpose of the present in- vestigation to complete the purification and crystalli- zation of one of the dormancy factors so that its iden- tity could be determined. MATERIALS All chemicals used in this work were standard re- agent grade unless otherwise stated. Anhydrous puriw fied diethyl ether and petroleum ether (300 - 60°) were obtained from J. T. Baker Chemical Co. (Phillipsburg, New Jersey). The Silicic acid powder was purchased from Mallinekrodt (St. Louis, Missouri) and the cellulose powder from W. T. R. Balstor (England). Sephadex G-10 was purchased from Pharmacia (Uppsala, Sweden). Gibbe~ rellic acid was obtained as Gibrel from Merk and Company (Rahway, New Jersey). Cis 4-cyclohexene-l,2-dicarboxylic acid anhydride was obtained from Aldrich Chemicals. Infrared spectra was obtained from a Beckman IR - 5 Spectrophotometer. Ultraviolet spectra were obtained from a Beckman DB Spectrophotometer. Nuclear magnetic resonance Spectra were received from a varian HA~60 Nu- clear Magnetic Resonance Spectrophotometer using the CAT 1054 computer. Elemental analysis was determined by Spang Micro Analytical Lab. (Ann Arbor, Michigan). A Soxhlet extractor of 5000 ml. capacity with a 145 mm id., female joint 145/60 was borrowed from Dr. H. M. Sell of this department. Crystalline barleyor-amylase was a gift from Dr. J. E. Varner of this department. The sugar beet fruit was the corky material from lot number (SL 121 x 133) ms x 5822-0 variety of Beta l6 vulgaris L. seed was a gift from the Farmers and Manu- factures Beet Sugar Association (Saginaw, Michigan). It was obtained as a very fine powder formed during milling of the sugar beet seed to remove part of the seed coat. I am grateful to Dr. F. W. Snyder for his help in ob- taining this material. Upon receiving the material in large bags it was stored at ~18O until used which was generally 1 to 10 months. The White paris lettuce seeds were obtained yearly from the Ferry-Morse Seed Company (Fulton. Kentucky). Ferric chloride: 2,2'bipyridyl reagent was used to detect weak reducing compounds. 1 ml solution of 0.2% ferric chloride in ethanol and a 1 ml solution of .2% 2,2'bipyridyl (freshly prepared) in ethanol were added to 1.0 ml of the test solution. The ferric chloride so- lution was added first in order that any Fe++ formed would then immediately be trapped by the addition of the 2,2'bipyridyl. A red color appears immediately or after setting for 10 - 15 minutes which can subsequently be measured Spectrophotometrically at 520 mu after diluting to 25 ml. For detection of phenolic and other hydroxyl com- pounds a ceric nitrate reagent was used. A 200 g quan- tity of ceric ammonium nitrate was dissolved in 500 ml of 2 N nitric acid. Bromine - water and KMnOu were re- 17 agents used for detecting unsaturation. BIOASSAY In all studies the inhibitory materials were de- tected by inhibition of germination of White_paris let- tuce seeds. A quantity of 25 lettuce seeds were germi- nated on a 5 cm Whatman No. I filter paper discs mois- tened with 2 ml of test solution in a petri dish. They were held at room temperature and light and percent ger- mination was recorded after 20 hours in a 2.0 ml medium. A unit of activity was defined as the amount of material that would completely inhibit the germination of 25 let- tuce seeds. Specific activity was in units of activity per mg. of dry material. In all fractions to be assayed, it was necessary to remove organic solvent completely or else solvent inhibition of germination would occur. Ei- ther the residue after evaporation of the organic sol- vent was dissolved in water or else an aliquot of the fraction in organic solvent was placed directly on the filter paper, evaporated to dryness at room temperature and then 2 ml of water was added to moisten the paper during germination. CIS 4-CYCLOHEXENE-l,2-DICARBOXIMIDE: Twenty-five milliliters of absolute ethanol in a py- rex tube was saturated with ammonia gas at 00 and then 4.1g of cis 4-cyclohexene-l,2-dicarboxylic acid anhy- 18 dride was added to the cold solution. The tube was sealed and placed in an autoclave with 20 lbs. pressure at 125° for 90 minutes. After being allowed to cool the contents were dissolved in benzene and the insoluble ma- terial was obtained by decanting. The benzene solution was evaporated to dryness leaving a crystalline material, mp. 1340 - 136O . The imide was recrystallized from wa- ter and a total of 0.62 g (15% yield) of crystalline ma- terial was obtained. mp. 1380 - 1390 (lit, (40) 136,5 - 137.3). The imide was also prepared in the following man- her, A 500 ml round bottom flask equipped with an air condenser was charged with 44.4 ml (0.66 moles) of 28% aqueous ammonium hydroxide. A 51.6 g (0.34 moles) quan- tity of cis-4-cyclohexene-l,2-dicarboxylic acid anhydride was added to the aqueous solution. The reaction mixture was heated with a flame for 2 hours at a temperature of about 3000 C to remove water. An oily residue was ob- tained which solidfied after cooling. The solid was re- crystallized from benzene leaving 35.4 g of white needle like crystals (yield 70%). After recrystallizing from water the melting point was 1380 - 1390 C. WW= This assay, was developed by Varner (41), and was used with minor modifications. The barley seed Hardeum 19 vulgare. variety Himalaya, were cut in half and the em- bryos were discarded. The emeSperm halves were soaked in 1% sodium hypochlorite for 15 minutes, rinsed in sterile deionized distilled water until the odor of so- dium hypochlorite was no longer present and then asepti- cally transferred to sterile moist sand in a petri dish. After three days at room temperature (preincubation pe- riod). 10 half seeds were transferred to a sterile War- burg flask containing 1 m1 of a solution of 10'2M sodium acetate buffer (pH 4.8). with 10-211 CaClz and 0.5 ml of appropriate dilutions of inhibitory substances. Each flask was treated with 50 ug/ml chloramphenicol to pre- vent the growth of micro organisms. The flasks were shaken on a Gilson differential respirometer for 20 hours at 25° for respiration readings. The gibberellic acid concentration, when used, was 10‘5 M. After incubation the medium was poured off and saved for a-amylase assay. The half seeds were rinsed with 3.0 ml of 0.01 M acetate buffer (pH 4.8) and then ground for one minute in a mor- tar with sand and 2.0 m1 of 0.2 M NaCl. The mortar with sand was rinsed with 3.0 m1 of 0.01 M acetate buffer (pH 4.8) and the combined solutions were centrifuged with a clinical centrifuge and designated as extract. The me- dium and extract were assayed separately by a modifica- tion of the Schuster and Gifford method (42). To 1.0 m1 20 of enzyme solution a 1.0 quantity of soluble starch (1.20 mg/ml) was added for a one minute incubation. The enzyme reaction was killed by adding a 1.0 ml solution containing 3.5 mM 12 and 2.5 mM KI in 0.05 M RCL. The mixture was diluted to 8. 0 ml and a Spectrophotometric reading was obtained at 620 mu . Units of a-amylase ac- tivity were calculated according to Varner's method as follows: units = J§A620 x vol aliquot x minutes a-AMYLASE INHIBITION ASSAY A 100 g quantity of crystalline d-amylase was in- cubated with a 16.8 units of inhibitor for a period of one hour in an acetate buffer (pH 4.8). The volume of the incubation medium was 1 m1. A 0.1 ml quantity was removed and diluted to 1 ml. This quantity was assayed as above and units were calculated as previously shown. g-AMYLASE WHOLE SEED ASSAZ This assay was nearly identical to the half seed assay except that barley seeds were used from which only the tip of the endosperm had been removed to facilitate the imbibition. Ten seeds were placed in a petri dish and covered with 3 ml of deionized distilled water. The seeds were then allowed to stand at room temperature for 21 30 hours. The medium was saved for assay and the seeds were washed with 2 m1 of .01 M acetate buffer (pH 4.8) containing .01 M CaClz. Preparation of the seed extract by grinding was performed as described for the half seeds. C SEED RESPIRATION 50 lettuce seeds (White paris) were placed in a Warburg flask with suspended trough and incubated with a 1.5 ml solution containing 0.29 mg/ml of the inhibi- tor and 1 drop of a 0.5 mg/ml penicillin and mysteclin solution to prevent growth of micro-organisms and fungi. Duplicates of the controls and treated flask were set up and manometric readings were obtained each hour on the respirometer. G OWTH This procedure was obtained from K. Schlender and H. M. Sell of this department. In a buffer solution containing 10-2 M citrate, 10-2 M phosphate (pH 5), 2% sucrose and 0.1% Tween 80 was dissolved IAA or the inhi- bitor. The IAA concentration was 10‘5 M and inhibitor concentration was 10'“ M. A control of the buffer so- lution was used. Oat seeds (variety Torch) were soaked in the dark for two hours in tap water. The seeds were then planted under green light, on Vermiculite in germinating dishes 22 and germinated under dim red light. After 24 hours, the seeds were covered with Vermiculite and placed in the dark for the rest of the germination period. All sub- sequent operations were carried out under green light or in the dark. Three days after the seeds were planted, 5 mm sections of the coleoptiles were cut. two to three mm below the tip. These sections were floated for two hours on a distilled water solution containing 1 mg MnSOu-HZO per liter. Six inch test tubes containing 1 m1 of the assay medium and 10 coleoptiles were placed in a revolving drum, (one revolution per minute). The drum was placed in a dark incubator at 26° and the coleoptiles were al- lowed to elongate for 22 hours and then removed and placed in a photographic enlarger. The projected sha- dows were enlarged 5x and measured in millimeters. The eXperiments were repeated for each solution at least three times. The mean length values were expressed as percent of values obtained by controls with or without IAA. RESULTS AND DISCUSSION ISOLATION PROCEDURE FOR SUGAR BEET FRUIT: A summary of the isolation procedure is given in Table I. A 1.4 kg quantity of sugar beet fruit was ex- tracted in the soxhlet with 3.5 L of ether for 8 hours. After 6 subsequent extractions ( a total of 8.4 kg of material extracted) the ether was removed by evapora- tion and the extracted material weighed about 65 g. The total material was placed on top of a silicic acid packing in a column of 45 x 5 cm dimensions. The organic substances were eluted with petroleum ether: ether (9:1 to 1:9 v/v) and eventually with ether, as shown in Table II. The silicic acid column was eluted with solvent under 3-6 lbs. air pressure. One liter fractions were collected until a total volumn of 13,000 m1 of eluent was obtained. Aliquots of each fraction were bioassayed. The strongest inhibitory fractions (80 - 90% of the total) always appeared between fractions 8 - 11 and usually fraction numbers 9 and 10 were combined and evaporated to near dryness for futher isolation. The residual material in fractions 9 and 10 was then mixed with powdered cellulose and placed on top a dry cellulose packing in a column of 25 x 5 cm dimen- sions. Substances were eluted with water and the first 250 ml fraction contained 90% of the inhibitory ma- 23 Table I Isolation Procedure for 8.4 Kg of Sugar Beet Fruit Residue Total Specific activity it, in 5 Units unitsfimg Ether extraction 65.0 32,500 .5 Silicic acid column 3.36 4.000 1.19 Cellulose column 1.33 3.800 2.86 Sephadex G-10 column 0.096 319 3.33 24 Elution Pattern for Silicic Acid Columns Fraction \OCDVO‘KA4rw N F4 r4 H a: +4 <3 I" \10 Volume 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 Table 2 Eluent Pet.Et: Ether (9:1) It I! " Ether Ether Ether Ether I! I! 25 (8:2) (7:3) (6:4) (5:5) (4:6) (3:7) (2:8) (1:9) unitslml 0.0 0.0 0.0 1.0 8.0 2.0 1.0 0.25 0.0 26 terials. Eluting with water removed only water soluble material and eliminated much of the organic soluble ma- terial which was not inhibitory. The pH of the eluent was 4.5. This procedure also removed much acidic ma- terial. The first 250 m1 aqueous fraction containing the. inhibitory substances from the cellulose column was evaporated to a 3 to 5 m1 volume. It was placed on top a sephadex G-lO column of 95 x 2.5 cm dimensions which had been packed and equilibrated with water. Sephadex G-10 is a gel filtration dextran with a molecular weight exclusion of 0 to 700. The eluting solvent was water and flow rate was adjusted between 7 - 8 ml per hour.. Fractions were collected every thirty minutes. Inhibi- tory substances were detected in two bands by the let- tuce assay (Figure 1). The inhibitory material in the first fraction after evaporation was an oily residue and it has not been further investigated extensively. The substance in the second band, fractions 121 - 132, upon combination and evaporation, yielded 96 mg of white so- lid residue (mp 105 - 110). The germination inhibitor in this preparation was crystallized from cold water to constant melting point. The yield was 9.9 mg. The va- cumn dried (15 mm Hg at 25° for 12 hours) crystalline preparation melted at 138° - 139O . 27 Amuse : pgobaom weapSHm nn\He m - a open Sofia SBSHOU oauo Novenamm m ache Hmaaopms mAOpananSH pcom gemsm one mo camppmm gofipzam .H oasmam Om. 0.1 h F 56.x. .# ZOFOdiu ON. a »- 0mg O_m ON Ore Om Om OO. % NOILIEHHNI 28 29 C S ATTEMPTED: Acid—base extraction was repeatedly tried with e- luent from the silicic acid column. This aqueous ex- tract contained much material including most of the units of inhibition which could be extracted into ether at acidic pH and which was soluble in water at a basic pH above 8 or 9. However. the biological activity was partially lost upon treatment with dilute alkali. There- fore. the principle of organic and aqueous extractions at different pH values was not employed with sugar beet fruit as a method of isolation. Thin layer chromatography (TLC) with silica gel G and GF was used for qualitative studies and H and HF for preparative work. 2537 A0 and 3660 A0 U.V. light were used for detection of components. Silica gel HF was preferred, since it could be used to detect non- fluorescent materials also. Of the many solvent systems tested, CHC13: MeOH (9:1) was found to give the best se— paration without moving the inhibitory material to the solvent front. Areas of the TLC were scraped off the plate and collected on a sintered glass suction funnel. The silica gel fractions were extracted with ether and acetone repeatedly and the extract evaporated to yield the active material. In all this work it was necessary to run controls to ascertain that the absorbent material 30 and solvents did not contain traces of impurities which would be inhibitory in the biological assay. In a ty- pical experiment 480 mg (about 2500 units) in a ml of ether was Spotted in 10 TLC plates (200 mm x 200 mm) coated with silica gel HF (0.5 mm). After development 8 components ranging from Rf 0.05 to 0.85 could be ob- served under U.V. lights. Each component was collected separately. Upon bioassay, only fractions designated as number 3 and 4 were found to contain inhibitory ac- tivity against lettuce seed germination. Fraction 3, Rf of 0.6, was pale yellow in color, did not fluoresce in 3660 A0 U.V.. but absorbed 2537 A° U.V. In about 35 mg of material there was 625 units. Fraction 4, Rf of 0.5. was pale yellow in color, showed fluorescence in 3660 A0 U.V., and absorbed 2537 A0 U.V. In 27 mg of material there was 830 units. When the TLC procedure was repeated with either fraction 3 or 4, about 8 components were again observed on the chromatogram and over half of the activity was lost. From many such disasters we conclude that most of the inhibitors were unstable to the TLC procedure with break down into several inactive components. The most likely reason for this loss of activity was either de- struction of the compounds by U.V. or oxidative degene- ration on the TLC. 31 For gas chromatography fraction 3 and 4 from TLC was injected onto a SE-30 (3% silicon rubber on Diato- port WAW, 60-80 mesh) gas chromatography column of an Aerograph 661 Gas chromatograph, using a flow rate of 2, and a temperature 2.6, a pressure of 40 lbs. per in. of 225°. The three major peaks were detected corre- sponding to a retention time of 0.7, 1.0 and 8.3 minutes respectively. The last peak generally contained the bulk of the material. The column temperature was 225°. A collection tube was used to collect the peak at a re- tention time of 8.3 minutes. Inside the tube a crystal- line material along with liquid was obtained. The tube was washed with ether and the solution was assayed for units in the usual manner. The weight of the sample collected was obtained by weighing the collection tube before and after washing with ether. There were 480 u- nits obtained in which the weight was 18 mg. TLC of this material in chloroform: Methanol (9:1) on silica gel HF showed non-fluorescent material with an Rf 0.60. The infrared Spectra (KBr pellet) showed carbonyl absorb- tion at 1775 cm-1 and 1725 cm"1 along with carbon-hydro- gen absorbtion at 2950 cm‘l, 2925 cm‘1 and 2845 cm'l. Collecting the material from gas chromatography was very difficult to duplicate because the crude sample when in- jected on the column left a residue which eventually 32 eluted with the sample. So gas chromatography was a- bandoned for the sake of larger preparative methods. 33 PROOF OF STRUCTURE: The inhibitory material in the second peak from the G-lO column was recrystallized to a constant melting point of 1380 - 139°. From elemental analysis the em- pirical formula was calculated to be CBH9N02. The i.r. spectrum (KBr pellet), figure (2), showed N-H next to a strong electro-negative atom (3215 cm‘l): unsaturated CH stretch (3058 cm-1, 2976 cm-l): methylene C-H (2924 cm'l, 2865 cm-l): carbonyl of a cyclic imide (1767 cm-1 and 1704 cm'l): methylene bending frequencies (1425 cm’l) and carbon-carbon double band (1637 cm'l). An absence or absorption at 1600 cm-1 (1580) and 1500 cm-1 (1450) indicated that the compound did not have an aromatic phenyl nucleus. The strongest indication of a functional group was the double carbonyl absorption at 1767 cm‘1 and 1704 cm"1 in which the lower band was stronger. This is characteristic of a 5 member cyclic imide ring (43)o The conclusion from i.r. is that the compound is non~aro~ matic, unsaturated and a cyclic imide. The unknown inhibitor gave a positive test with both KMnO,+ reagent and with Br2/H20 reagent (Table 3). This indicated the presence of an olefinic double bond° With the ferric chloride 2,2”bipyridyl reagent a red colora- tion indicated a reducing group. The ceric nitrate re- agent is a test for phenolic and alcoholic hydroxyl groups. 34 ems co ma002\waa coaace ems HoudnangH szogxmb mo scavenge nonmamSH .N maswdm cues—00m ..‘.\I. it‘. ..\«. N J.AQJ - i [‘3‘.» ‘. 462 r1. J 83533 414L453 _ z... On. P C...... 89 ._.._:_.:_:_::_ _ . .1. 8.. 00: 00». ‘U C3<€32H>mapas .3 maswam 00m 03 0mm :5 .reozmsm>Ss CNN 0mm 0mm OVN 0mm .ON m O_N DON « J l I l 1 O. ON Om OW Om O® .ON 0m 00 OD. O/o l 41 40 m2 "0 Ismem u used gouanaccH mzocxgs one no adhpooam poaofi>mapab .3 madman 00m 0mm 0mm :5 .reozmnm>Ss CNN 0mm 0mm O¢N 0mm ON m 9m DON u l I 01 ON .O® .ON Om Om OO. O/c 1 41 42 and are characterized by high molar absorptivity. Since the molar absorptivity was 128 then the transition must be from a R-band (German radikalartig) whose orgin is in the n-Yr* transition of a single non-conjugated chromo- phoric group. From all of the information thus far pre- sented, it was concluded that the compound contained a cyclic 5-membered imide, a non conjugated carbonyl and a non conjugated double bond. There was no aromatic character (from u.v and i.r) and there was probable an alicyclic ring to satisfy the atomic structure. From the n.m.r. spectrum in figure 5 was obtained additional information as to the nature of the protons. With only a small sample, there was difficulty in get- ting a good n.m.r spectrum. The n.m.r spectrum showed no aromatic protons which agreed with u.v and i.r data. There was only one set of methylene on a ring which would stand out as a Sharp singlet peak. Two vinyl pro- tons (in the same environment) should Show some Splitting although only a broad peak was observed. From the n.m.r Spectrum in figure 5 it was very difficult to measure spin-Spin coupling constants (J values). The absorb- tion peak at 6.867’ appeared as a triplet (J = 3.0 cps). A J value close to 6.863' could be due to tertiary pro- tons next to a methylene group to give a triplet. How- ever, the n.m.r spectrum was not satisfactory for abso- 43 omze senescence ca RowapamgH :zocxcs one no asapoomm .a.a.: .m oasmfim anal cIoI'A i g Jerome-Mrs ‘qu" ad a . 513. .m: A acts! as}! «IS. I. I s «Tom... in! I. (unison: in)! you I IIODW In! -1- chic £55.59 1 7a.... 155.- l. - -ma- gal. ( . . l3!!! 1 l - -T 9,- Steel...- 9. - 1_ I la; (.9 .m 6.39 «Slim a. ~{Ja4llwu 3:!00 i .wiiew 233mg; a m 214......" ——_»= 2..— '-.——._ _ L M -- 8w -§-§—§—§—s - F4} 45 lute proof of structure because the concentration of the solution was less than 1%. The evidence so far presented pointed to a compound whose structure contains a double bond, a 5-membered cyclic imide (dicarboximide) and no aromatic ring. Two of the likely structures are as follows. I II The above structures would fit the physical data and the empirical formula, u.v., mass and i.r. spectra. Excluding the proton on the nitrogen, structure II shows 6 different protons where as structure I Shows 3 dif- ferent kinds of protons. Structure I and 11 both have gl§_and Egang dicarboximides. Structure II can be elie minated since it does not agree with the observed n.m.r. spectrum which shows 4 different kinds of protons. Structure I, cis 4-cyclohexene 1,2-dicarboximide, ape peared to be the unknown dormancy factor from sugar beet fruit. It has been synthesized by Snyder and P008 (40). They reported a melting point of 134°— 1360 after recry- 46 stallizing from benzene which did not agree with the ma- terial isolated. Starting with gig 4-cyclohexene 1,2—dicarboxylic acid anhydride, the dicarboximide was synthesized and re- crystallized from benzene (mp 134° - 136°). The melting point corresponded to the value reported in the litera- ture (45). The isolated germination inhibitor had a melting point of 138° - 139°. However, when the synthe- tic compound was recrystallized from water, the melting point was 138° - 139°. Perhaps the crystallization from benzene carried some solvent with the crystals and thus lowered the melting point even though the crystals were dried in a vacuum desiccator for 15 hours. The infrared spectrum (figure 6) of the synthetic cis 4-cyclohexene 1,2-dicarboximide is identical with the spectra obtained with the isolated germination inhibitor. A mixed melt- ing point with the synthesized compound and the isolated inhibitor Showed no depression. The biological activity of the synthetic compound was essentially identical to the isolated material (table 4). There. the structure of the inhibitor was gig 4-cyclohexene 1,2-dicarboximide (as Shown) which will be referred to as CCD. A7 moaacscsamv moaaaxonamoHuum.H ogmxmsoaozoud mao “Soupom noeaaassH eoecaoma uace gouanammH gaogxgs can caponpczm ho mapocam doamaan .0 oaswfim AIOIU.‘ :— x»284~>(3 .5 .‘ t» 2 2 2 a. : o. a o a a a v «x x 2 1.20:5 JiufllUlé. 8 .44.,4uufé. A1114 ”41141.1(; . c -- I .. ., .. .. m . . _ Juleco 8. _ , . i!1_|l)x_ IIIWNIdrlEut. - .. -p. __ .hL .p. :1»...— I...::_ :._._a;:_._._::__ _._ ... __ __. _::_Z:_::::_157.5. _ N . on. 85 a 8. g. 8: 02. can. 8.. 002 002 09,. on: .. V .Ivu‘lalgdl I (n.- ‘5 iv} ' it‘s... (111;. .u... .' .Ill, p‘c‘ 1" l 0" 1| _ glvzlzozgt’ lavage-a a .CIExOOm , a 1.... 03 80535313 (Illifll...) .196. E1; hip _ -a. FL; .5). FL 7., _. I»; a: 5:33. I: I... .::::_.:_.r.__ I. _ _. __. .ZLT: ::_;:_.. : It; a. 88 8. 80 g. 8: 8a. 8a. 81. 8a. 0005 09.. 9.5a , . .‘V .3310)(’ . ‘4‘ 55 fire...“ I fi-‘II..' [Cl-r. 2' n, I!» .Q.‘ 1!. I'- mace: om ca 0mm no Uopmzdaaom mecca oofiuuoa man .I. we no. N. ca mo. N. ea co. s. an ca. e. ooH ma. m. mm ma. m. ooa mm. o.a ooa mm. o.a ooa on. m.m ooa om. n.m .r mewma mode Ha\me moax soaeaaassH a acancao: soacaaassa a aeancaoz .omoo .ocoo oddaawonamodelm.HiochoSOHOho Suede 1 hopanancH wondHOmH shopapassH oaeosesam one ocecaooH no aoasacos co a canoe acmaamaaoo 49 50 ISOLATION PROCEDURE FROM WHEAT CHAFF: The summary of the isolation procedure is exhibited in Table 5. The bioassay was the complete inhibition of germination of Hhitg_pgzi§ lettuce seeds and serial di- lutions were run on all samples. Hulls of red coat wheat which were harvested in July, 1964, and stored at -100 to -18°were used in 1964 through 1966. An 8.0 Kg quantity of wheat hulls (Chaff) were extracted with 141 liters of purified anhydrous ethyl ether for 12 hours. The hulls were removed by filtration through cheese cloth and the ether was evaporated in a continous flask evaporator under vacuum. The concentrate was mixed with minimum amount of silicic acid powder to form a paste and air dried at room temperature. The mixture was fur- ther dried overnight in a vacuum dessicator. It was then placed on top a silicic acid column of 39 x 5 cm dimensions. Final column height was 44 cm. The column was eluted as follows and aliquots were collected and assayed: (a) Fraction number 1: 1000 ml petroleum ether (30°- 60°), no inhibitor was eluted. (b) Fraction number 2: 1000 m1 75:25 mixture of petroleum ether: ethyl ether; no inhibitor eluted. (0) Fraction number 3: 3000 ml of 50:50 mixture Table 5 Units of activity Recovered in Isolation Procedure from 8 Kg of Wheat Hulls Units Ether EXtraction 11,000 31lL§1£_A&lfl_222222£2822221 (a) 50:50 Pet. Et: Ether 3,000 (b) 25:75 Methanol:Ether 6,000 Celluloge Column 5,000 Ether-water Partition (pH 4.0) 4,000 Ether-water Partition (pH 8.5) 3,000 Ether-water Partition (pH 3.0) 2,500 51 52 of petroleum ether:ethy1 ether: The first 300 to 1000 m1 of elute contained consider- able inhibitory activity. This fraction was stored in the cold for further investigation. It was unstable at room temperature over prolonged periods of time. (d) Fraction number 4: 1000 ml of 25:75 methanol: ethyl ether. The addition of a polar sol- vent immediately eluted a major inhibitory fraction which was used in subsequent steps. The inhibitory fraction was evaporated to a small volume and the concentrate was mixed with cellulose pow- der and dried in a vacuum desiccator. This mixture was subsequently applied on top of a cellulose packing which had been packed dry. The material was eluted with water. All of the inhibiting activity was eluted with 100 ml of water and subsequent fractions were inactive. The pH of the aqueous solution was adjusted to 3.5 and extracted three times with equal volumes of et- her. The inhibitor was found in the ether layer, thus leaving some impurities in the aqueous layer. The ether extracts were then extracted with aqueous sodium bicar- bonate. The aqueous layer turned yellow in color and all of the inhibitory material was found in the aqueous layer. The aqueous layer was neutralized with Dow - 53 50—H+ and again extracted with ether in order to trans- fer the material to an organic solvent. A small sample has been run through a sephadex G-10 column as described for the isolation procedure for su- gar beet fruit (Figure 7). Inhibitory activity was e- luted from the columnist about the same position as for the cis 4—cyclohexene-l,2-dicarboximide compound from sugar beet. However the amount of material was too small for any analysis except biological activity. EQEMAIIQN QE INHIBITIORS IN WHEAT CHAFF: The results in this section were collected by A. A. Khan and N. E. Tolbert and have not been published. Since their results were pertinent to the isolation of inhibitors from wheat. their findings are summarized herein with their permission. During the summer of 1963 and 1964 field tests were made on the amount of inhibitor in ripening grain. From these studies it was acertained that wheat hulls were a rich source of the dormancy factors whereas the seeds contained much less inhibitor. The amount of inhibitor increased to maximum values about 26 days after anthesis when the grain was fully formed. The amount of inhibitor in the more dormant Red Coat wheat was greater than in the Genesee wheat which is often not dormant at harvest. 54 genes HSO£\HEOHIB menace oauo finances: anocaaassH coca: esp u ashram ouch Soam Nmummmcm m Scam mo gacppma SoapsHm .m maswam IOO '- 90- 0 G3 I I O o P to NOUMNHM 9b 55 I A o 0 3 l O m 20 IO HO IRO a: so FRACTION ‘IO 20 :FF. 56 The amount of inhibitor in the Genesee wheat hulls de- creased more rapidly after 26 days than that in the hulls of the Red Coat wheat. A two day rain occured 41 days after anthesis and only 50% of the inhibitory ma- terial remained, probably, because the rain had leached or washed out the inhibitor. It is known that germi- nation will occur in the head after such rain storms. Consequently. all of the research was done on the Red Coat Wheat. Furthermore. the hulls were a more de- sirable starting material because ether extractions re- moved less bulk from hulls than from the seeds from which ether extracted much fat and lipoidal material. It was necessary to harvest the the hulls 35 days after anthesis which is before normal harvest in order to obtain them with maximum amounts of inhibitory ma- terial per gram of weight. The heads were harvested with a stripper and a fan was used to separate the grain from the hulls. The hulls have been stored for over ten months at -10° C but with an appreciable loss in the inhibitory material which can be removed by ether ex- traction. F M WHEAT CHAFF: Some of the chemical properties are as follows: (a) Inhibitor is a small molecular weight (b) (e) (d) (e) (f) 57 organic compound. It is a weakly acid or neutral compound since it is not absorbed appreciably on an ion exchange resin. It is soluble in polar solvents such as, water. ethanol, methanol. ethyl acetate. ether: but insoluble in carbon tetra- chloride and petroleum ether (30° — 60°). On paper chromatography the inhibitory material has an Rf of 0.60 - 0.90 with a variety of polar solvent systems used. Using thin layer chromatography with the same solvent systems the Rf is from 0.50- 0.85. Using chloroformzmethanol (9:1) the Rf of the strongest inhibitory band is 0.55., The inhibitory material forms a brown coloration on paper chromatograms with 1% permanganate, a red coloration with Rho- damine 6 G, and a yellow color with bro- mo cresol purple. A solution of the inhibitory material de- colorized bromine-water, decolorized per- manganate, and did not react with ferric chloride. These results suggested oeli- 58 finic unsaturation. (8) Biological activity was destroyed by re- duction with sodium borohydride. (h) I.r and u.v characteristics can not be discussed in absolute certanity but the i.r always Showed a strong carbonyl ab- sorbtion along with carbon-hydrogen (possible aliphatic) absorption. How- ever, there were other components (in- purities) with the inhibitor. C L STUDIES ON CIS 4-CYCLOXENENE-1 Z-DICARBOX- M D CCD : LIGHT, GIBBERELLINS AND a-AMYLASE SYNTHESIS: It is known that dormant seeds at harvest do not germinate regardless of the light treatment. Thus there is no evidence at present on any relationship between dormancy and the phytochrome system. So far no interaction between CCD inhibition of seed germi- nation and light treatment has been observed by us in the laboratory. Rather CCD seems to supress the phy- tochrome effect on germination until the CCD is re- moved. However further research on the effect of CCD on various phytochrome responses Should be explored. Lettuce seed treated with CCD will not germinate if 1 x 10-5 M gibberellin A3 is also added. AS far as 59 these test have gone, gibberellins do not appear to reverse the inhibition of germination induced by CCD. Since gibberellins regulate a-amylase synthesis, another way to test this conclusion was to examine a- amylase production in germinating seeds. Gibberellins are known to induce the de-novo synthesis of c-amylase in barley endosperm (24), and this was confirmed by my data in table 6. The CCD germination inhibitor at 10-3 M did not alter a-amylase production or release by barley endosperms as brought about by 10-5 M gib- berellin A3. As a further test, the effect of CCD was studied on c-amylase synthesis in the whole seed. The purpose of this experiment was to measure the synthesis of c- amylase using endogenously synthesized gibberellins from the embryo. About 70% as much d—amylase enzyme was synthesized with the CCD treated whole seeds (Tab- le 7). Thus CCD did not inhibit severely c-amylase synthesis by the whole seed. There was a lower amount of a-amylase in the medium which suggests that less enzyme was synthesized in the aleurone layer but that no CCD effect exists upon release of the enzyme from the aleurone layers. In the previous experiments with the barley endosperm, there was no effect upon a- amylase synthesis in gibberellic acid treated half Table 6 a-amylase synthesis in the barley endosperm Treatment Medium Extract Total units units units +10-5M :13 29.3 12.6 41.9 10-3 con + 10‘5M GA, 28.5 12,2 40.7 6O Table 7 d-amylase Whole Seed Assay Treatment Medium Extract Total units unitsfif ugits None 15.3 16.9 34.2 +ccn lo-“M 6.3 17.1 23.4 61 62 seeds. The smaller amount of enzyme produced in the whole seeds could mean that the CCD inhibitor possibly inhibited part of gibberellin synthesis in the embryo. However, there were still some gibberellin produced or there was another factor produced by the embryo which is involved in the formation of a-amylase. The latter has been suggested by MacLeod (45). At present we conclude there appears to be no re- lationship between gibberellin linked a-amylase syn- thesis and CCD. Thus CCD is not an "anti-gibberellin" and has little effect on gibberellic induced synthesis of c-amylase. CCD activity as a germination inhibitor must reside elsewhere. In this respect. CCD is difs ferent from Abscisin II which prevents the formation of a-amylase without inhibiting respiration, phOSphory- lation. protein synthesis or RNA synthesis (46). The mechanism of action of Abscisin II seems to be rather specific and clear, whereas CCD has no effect in this system at the concentrations used. BESEIBATION: The 002 ( 1 02 hour -1) for 10 barley half seeds measured at 25° was 31 1. For the half seeds treated With gibberellic acid the value was 31 1.and for those treated with gibberellic and CCD inhibitor the value 63 was 32 . It appears that respiration of the barley endosperms is not effected by inhibitor (CCD). Germination of White paris lettuce seeds was in- hibited at 10-3 to 10-4 M concentration of the inhibi- tor (CCD). In figures 8 and 9 the expression of this phenonmenon is shown as inhibition of respiration of the lettuce seeds. The seeds will begin to germinate at 16 hours after inbibition. The physical evidence for this in the protrusion of the radical which can be visually observed after 16 hours. However. respira- tion increases after 10 to 12 hours and the rate of respiration (figure 9) reaches a new higher level af- ter 16 hours. The seeds treated with CCD did not ger- minate nor change their respiration rate after 12 hours of inbibition. Rather CCD treated seeds contin- ued to respire at approximately the same slow initial rate for more than 20 hours . It appears that the in- hibitor CCD prevents germination without effecting the initial respiration rate. Thus it does not inhibit germination by killing the seed. In fact after treat- ment of seeds with a 10‘1+ M CCD for 24 hours, the seeds may be washed and left for an additional 20 hours with fresh water when they will then germinate. This remarkable experiment Shows the reversibility of 64 HouanancH z enoa I mica moaoafio oomoao aouanamcH peonpaz moaoafio Soao mooom oozppoa mahmm opanz mo manna: Sewage Hence .w ogsmfim mmDCI m. 9 ¢; .9 O. m m .V N motmiz. It>> O $02912. FDCICB o ICON .TOOV IOCO 00w OOO_ CON. (H ENVLdfl NBOAXO 65 66 sooacassH : suoa . muoa noaoaao ocnoao Houanammd psonpaz moHoHHo :mao mooom ooappoa magma opasz mo oxmpa: cowhxo mo mpmm .m oasmflm ~mmoo: 0m m. m._ 10.. m. o._ m w m motmiz. It; 0 motmiz. 561:; o . :2./\ a e CN 0% CC om CC. SW‘V/ldfl NBC/{X0 31178 “vi/IT) 67 68 the effect of this inhibitor. W: The results of the.A1ena straight growth assay are summarized in table 8. The IAA + CCD treated coleoptiles showed quite a variation in lengths; how- ever, the mean value was somewhat less than IAA treated coleoptiles. This suggests that the CCD in- hibitor at 10‘3 M may act only as a weak inhibitor of auxin induced growth. The mean value length of COD treated coleoptiles were the same as the control (buf- fer only). These length measurements were very con- sistent and did not vary as those with IAA + CCD. That all the above physiological experiments were negative indicated the uniqueness of the CCD structure on inhibition seed germination. It suggest that this inhibitor (CCD) does not prevent germination by kil- ling the seed, affecting the seed viability or inter- acting with other known plant hormones such as IAA and GA. Further studies or mechanism of action should be done at the molecular level for inhibition of RNA syn- thesis. Table 8 Effect of CCD on Avena Straight Growth Test Treatment Length at Length at % control 0 hoggs 24 heaps growth Control 4.9mm 6.8mm 100 IAA 4.9mm 10.0mm 147 IAA + CCD 4.9mm 9.2mm 135 CCD 4.9mm 6.8mm 100 69 7O STRUCTQRE 0F CIS 4-CYCLOHEXENE-l.Z-DICARBOXIMIDE AND ITS BIOLOGICAL ACTIVITY: This structure is unusual for a naturally occuring compound. It is not an alkaloid nor does it belong to the family of terpenoid compounds which contain the iso- prenoid unit. The structure fits no known biosynthetic family of compounds. In the structure of CCD. the dicarboximide func- tional group seems to be most important by comparison with other similar compounds to be described below. However the single double bond in the hexene ring is un- doubtedly of importance. In aqueous solution, the di- carboximide ring must be in equilibrium with two other resonance forms which are identical: 0 OH 0 no «+0000 0 on (3 CCD is a weak acid and its PK is about 6.5. Conse- quently CCD is soluble in both polar and non polar sol- vents, and this solubility is effected by pH shifts of importance in biological tissue. It can be readily solubilized by water from seeds and other tissue, yet it could penetrate lipoidal layers and non-polar sol- vents. CCD is a stable compound. It is not destroyed by 71 autoclaving; in aqueous solutions. The loss of inhibi- tory units experienced in the isolation procedure dur- ing thin layer chromatography and acid-base treatments must be attributed to destruction of compounds other than CCD. The amount of CCD per weight of sugar beet fruit has not been determined. Its effective concentration for complete inhibition of lettuce seed germination was 1 x 10'“ M. CCD is not the only seed germination inhi- bitor in sugar beet fruit, for in fact there are many inhibitors in the sugar beet fruit such as coumarin, organic acids and phenolic compounds. The nature of many of these other compounds, which are inhibitors. re- mains unknown. The first peak off the Sephadex G-10 column in the last step of the isolation procedure would be the next most logical inhibitor to identify. This unknown must be relatively pure and may have some of CCD properties, since it has been isolated by the same procedure. This unknown is not Abscisin, since it showed no cotton effect at 247 mu (16). The basic structure of CCD is very similar to the agricultural fungicide, Captan, which is shown below. 72 Captan is used as a agricultural fungicide. It was dis- covered by Kettelson (49) who also made other deriva- tives of similar cyclic imide structures. Captan has more effect as an agricultural fungicide, and it is now commonly used as in agricultural practices. Its pos- sible use on the sugar beet fruit before we received the material raises the serious question as to whether CCD is naturally occurring or whether CCD arose as a breakdown product from some chemical treatment of the seed. In a private communication, the West Coast Sugar Beet Seed Company from whom the seeds were obtained, stated that the seeds had not been treated with Captan. Further similar inhibitory material is present in wheat hulls which were grown here by us and which we also know were not treated with Captan. Captan is a very stable compound and is not easily decomposed. All these points suggest that CCD is a naturally occurring ma- terial, but further careful investigation will be neces- sary to establish this point. If CCD were a decomposition product or metabolite of Captan, then CCD might be the active component of Captan. This is the universal question about active structures of inhibitors such as herbicides, fungicides, pesticides and insecticides. In this case with Captan, is Captan the active structure, or is a decomposition 73 produce or metabolite such as CCD the active structure. For the present, the assumption is made that Captan it- self is active and that CCD is a similar naturally oc- curring compound. Since Captan inhibits fungi growth by inhibiting the germination of spores, both compounds may have similar mechanism of action. Another interesting compound which has a similar functional group to CCD is Cycloheximide, (shown below), which is produced by Upjohn Company. Cycloheximide has a cyclic imide structure and has been used as an inhi- bitor of protein synthesis in mammalian systems (48). CH5 Hsc< 7:0 0 CHOH‘CHg‘u (D CYCLOHEXIMIDE Cycloheximide has no effect upon protein synthesis in extracts of Echerichia 0011. It is also used as an agricultural fungicide but the mechanism of action in systems other than mammalian has not been thoroughly in- vestigated. However, cycloheximide has been studied in higher plants (24, 51, 52) and at 10 mg liter gives in- hibition to germination of Phacelia tanacetifolia seed 74 seed (49). It inhibits germination completely in dark- ness and in the light with gibberellic acid. Thus the mechanism of action of CCD and cycloheximide may be i- dentical and may involve inhibition of protein synthe- sis. Varner (24) has shown that cycloheximide inhibits de novo synthesis ofcx-amylase by inhibiting protein synthesis. A class of biological inhibitors may be evident from the above structural observations of compounds similar to CCD. They all contain the cyclic imide ring. In this group might go other biological inhibiting compounds such as the maleimides. C) l -R R = H, alkyl or aryl C) Of the maleimides, the N-ethyl maleimides has been the most widely used as an enzyme inhibitor. N-ethyl male- imides can react with amino groups in proteins under the same conditions which are used to alkylate sulfhydryl groups. Recently, Sharpless and Flavin (50) have studied the reactivity of maleimides with protein amino groups and reported that the amino group adds to the double bond of the maleimide. Thus the significance of the double bond in CCD is emphasized. 75 Another similar compound is maleic hydrazide (shown below) which acts as a metabolic poison. O I): O Maleic Hydrazide The above mentioned compounds have differend effect upon biological systems, but they do have similar func- tional groups. Namely, the cyclic imide. Perhaps this functional group is necessary for biological activity though each has a different function in similar systems. The spectficity or selectivity may be a result of the other differences in structures. SUMMARY A compound has been isolated and characterized from sugar beet fruit which is a germination inhibitor. The use of Sephadex G-lO column chromatography was very beneficial in separating low molecular weight organic compounds. This compound'8 activity is not Just due to destroying the seed but is due to action at a site other than some isolated metabolic process. It seems to be very specific in only inhibiting germination. However, more eXperiments are needed to determine the site of ac- tion of this Compound. There was no effect upon respi- ration, gibberellic acid induced synthesis of a-amylase, the activity of the a-amylase enzyme and a questionable effect upon avena straight growth. The compound is characterized by a 5-membered cyc- lic imide which is somewhat unusual as a natural occur- ing biological compound. The closest similar structure is a derivative CAPTAN which is used as an agricultural fungicide. Its action is inhibition of germination of fungi spores. The cyclic imide perhaps may be a parti- cular class of biologically active compounds along with glutarimides and N- substituted maleimides. 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