‘‘‘‘‘‘‘‘‘ GIBBERELLIN METABOLISM IN HIGHER PLANT TISSUES Dissertation for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY BIBHUTI N. SINGH 1976 5‘“ C(fi-a .‘ ‘sni ‘ I " . 5mm“ fififiégzn Stat: " Ug.zi":'érrtiri7 an. aim-autumn"? magmas This is to certify that the thesis entitled GIBBERELLIN METABOLISM IN HIGHER PLANT TISSUES presented by Bibhuti N. Singh has been accepted towards fulfillment of the requirements for Ph.D. degree in Bot.iPl. Path. (kg/4v QflW/a/ut Major “lessor 0-7639 ABSTRACT GIBBERELLIN METABOLISM IN HIGHER PLANT TISSUES By Bibhuti N. Singh The objectives of the investigations were: (1) to determine whether gibberellic acid (6A3) and gibberellin A1 (6A1) are con- verted to other gibberellins or metabolites in aleurone layers; (2) to study the properties and, if possible, to determine the structures of any resulting compounds; (3) to ascertain the metabolic pathway by which 6A3 is inactivated or conjugated in aleurone lay- ers; and (4) to determine whether the metabolism of the phyto- hormones is related to their ability to evoke responses in the tissues. 6A3 was rapidly inactivated by barley aleurone layers when it was applied and it was the only biologically active compound detected at any time (5 minutes to 24 hours after application) in the tissue. Bioassays of sections of chromatograms of ethanol extracts revealed one area, corresponding to 6A3, which was active in the barley endosperm assay. In studies on 6A3 metabolism by barley (Hordeum vulgare cv. Betzes) and wheat (Triticum aestivum cv. Genesee) aleurone layers, tissues were incubated with radioactive phytohormone, removed from Bibhuti N. Singh the medium, thoroughly washed with water and subsequently boiled with methanol. The layers were ground with methanol, the extract was evaporated to dryness, and the residue dissolved in borax buf- fer, pH 8.4. An aqueous phase resulted after this solution was par- titioned successively against ethyl acetate at pH 8.4 and 2.4. The incubation and wash solutions were combined and extracted similarly. The radioactivity present in the acidic ethyl acetate phases of GAB treated tissue as well as in the combined medium and wash solutions was primarily unmetabolized 6A3. Two polar metabolites that were produced within 15 minutes and accumulated over a 24 hour period -were found in the aqueous phase of the layers. The major highly polar metabolite (M1) remained at the ori- gin of paper chromatograms developed with isopropanol-water (4:1, v/v) whereas the minor metabolite (M2) migrated behind the 6A3 area. Acid treatment of the minor metabolite, M2 of 6A3, and of 6A3 gave allogibberic and gibberic acids. M2 gave a positive test for hexoses and for pentoses. ‘4 l4 C-U-glucose was incorporated into the metabolite, C-U-galactose was not. A diasaccharide resulting from the partial acid hydrolysis of M2 migrated at the same rate as 3-0-B-D-glucopyranosyl-D-xylose. Complete acid hydrolysis of M2 resulted in the production of glucose and xylose, apparently in equal amounts. Radioactive xylitol was detected on paper chromato- grams after the sequential periodate oxidation, acid hydrolysis and reduction with NaBT4 of M2 of 6A3. The results, collectively, were indications that M2 is very likely O-B-D-glucose-(l+3)-0-B-D- xylopyranosyl-(l+3)-0-GA3. Bibhuti N. Singh Regarding the structure of M1 of 6A3: the basic 6A3 struc- ture appears to be modified in it since it does not give the same products as 6A3 on acid treatment. Alanine, glycine and serine were identified and their corresponding dinitrophenyl derivatives (DNP- alanine, DNP-glycine and DNP-serine) were prepared after hydrolysis of M1 of 6A3. The N-terminal amino acids of M1 of 6A3 are glycine and serine. 3H-GA1 was metabolized to 6A8 and to compounds tentatively identified as a GAl-glycoside (M2 of 6A1) and a GAB-glycoside (M3). These are thought to be congeners of M2 of 6A3. Hence, they are suspected to be 0-3-B-D-glucosyl-xylosyl-GA1 and the corresponding glycoside of 6A8, respectively, the sugars being attached to C-3 of the gntygibberellane ring. Additionally, 3H-GA1 was converted to a compound having pr0perties similar to the major highly polar metabolite, M1 of 6A3. Evidence was also obtained that GA8 is an intermediate in the forma- tion of M3 and M1 of GA.I since 3H-GA8 was metabolized by aleurone tissues to these compounds. A cell-free enzyme system from Phaseolus vulgaris L. used 3H-GA1 to 3H-GA8 also converted it to a less polar 14 to hydroxylate product. The enzyme system converted C-GA3 to two metabolites that are probably homologs of the GA1 products. Additionally, a compound more polar than the two metabolites, that is very likely more highly hydroxylated, was formed from gibberellic acid. In comparative studies on the conversion of gibberellins to polar metabolites by intact tissues from various plants, the Bibhuti N. Singh following results were obtained. Four-day-old seedlings of 14 Pharbitis nil Choisy that were devoid of roots converted C-GA3 to two polar metabolites that are unlike those formed by aleurone layers. ‘ 3H-GA1 was metabolized by four-day-old germinating Zga_mgx§ to two unidentified polar products. This gibberellin was converted to two radioactive substances less polar than M2 of GA1 or M3 by immature seeds of Scarlet Runner bean (Phaseolus coccineus L.). One of the metabolites appeared to be GAB-glucoside. The polar metabo- lites formed by P. coccineus seeds had identical properties as com— pounds formed by germinating Phaseolus vulgaris seeds. When mung beans (Phaseolus aureus) which were imbibing water 3 were incubated with H-GA], the hormone was converted to a major polar compound whose chromatographic and electrophoretic properties were similar to M3 of GA]. The highly polar metabolites, the Ml-like compounds, were only detected in aleurone layers. These studies indicate that reactions for the formation of the conjugated forms of the gibberellins are probably mechanisms for regulating the endogenous level of thephytohormones inasmuch as the metabolites are inactive in bioassays. In aleurone layers, the gib- berellins themselves rather than metabolites derived from them appear to be the biologically active hormonal species. GIBBERELLIN METABOLISM IN HIGHER PLANT TISSUES By an (Pi Bibhuti N‘.‘ Singh A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1976 To my parents, Raja Ram Singh and Shakuntala Devi Singh ii ACKNOWLEDGMENTS I would like to express my deepest gratitude and most sin- cere thanks and appreciation to Professor Clifford J. Pollard, my major professor. His qualities as an extraordinary teacher, coun- selor and human being will always be remembered. Appreciation is also extended to the members of my guidance Vcommittee, Professors E. S. Beneke, A. A. De Hertogh and N. E. Good, for their assistance and criticism of this dissertation. Very spe- cial thanks go to Professor Bruce E. Walker for providing employment for me and for encouragement and advice during this study and Mrs. Joann M. Bunwick for giving me an opportunity to learn about dif- ferent aspects of histotechniques. The assistance of Peter Felker in hydrolysing samples for identification of amino acids is grate- fully acknowledged. Thanks are also due to Dr. Axel Ehmann and Dr. Kofi Amuti for their help. I also wish to give thanks from the bottom of my heart to my parents, brothers and sister for their love and affection; to teachers and friends for all the advice, assistance and encouragement they have given me during the entire course of the study. This work was supported in part by the National Science Foundation Grant No. 68-38155 to Professor C. J. Pollard. TABLE OF CONTENTS LIST OF TABLES . LIST OF FIGURES INTRODUCTION . . . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . . I. II. III. IV. V. VI. VII. VIII. IX. XI. XII. Preparation and Incubation of Tissues Bioassays . Chromatography and Paper Electrophoresis Extraction of the Metabolites . . . Detection of Unlabeled Metabolites . . Acid and Alkali Treatment of the Gibberellins and Metabolites . . . Enzyme Treatment of the Metabolites . Purification and Characterization of the M2 Metabolite of 6A3. . . . . . Purification . Detection of Sugars in M2 of GA3 . Chemical Tests for Pentose in M2 of GA3 . Identification of Sugars in M2 of 6A3 by Thin Layer and Paper Chromatography and Borohydride Reduction of M2 of 6A3 . Incorporation of 4C U- Glucose into M2 by CA3 Treated Aleurone Layers . . Purification and Characterization of the M1 Metabolite of 6A3. . . . . . A. Purification B. Hydrolysis . . C. Identification of Amino Acids by Dinitro- phenylation . D Identification of the Terminal Amino Acids by Dansylation . l. Preparation of dansyl (DNS) derivatives . 2. Chromatographic separation of DNS- *1 m DOW) derivativei . . . . . . . . . . . Hydroxylation of .C-GA3 and 3H-GA1 by a Cell Free System . . . . . . . . . . . Chemicals Determination of Radioactivity iv Sequential Periodate Oxidation, Acid Hydrolysis. Page vii viii RESULTS I. II. III. IV. VI. VII. VIII. IX. XI. XII. XIII. Attempts to Find Biologically Active Metabolites of GA3 . . The Inactivation of Gibberellic Acid by Aleurone Layers . . . . The Metabolism of Gibberellic Acid . . A. Distribution of Radioactivity in Extracts of 14C- -GA3 Treated Layers . . . . B. Chromatography of Extracts The Metabolism of Gibberellin A1 . . . .gistribution of Radioactivity in Extracts of H -GA1 Treated Layers . . . . In the aqueous phase . . 2. In the acidic ethyl acetate phase . . Incubation of GA1 and GA3 with a Hydroxylating Enzyme System from Phaseolus vulgaris . . . A. Incubation of JH- -GA1 with the Enzyme . B. Incubation of 14C- -GA3 with the Enzyme . . . Metabolism of Radioactive GA by Aleurone Layers Time Course of Formation of he Metabolites Electrophoretic Pr0perties of the Metabolites Effect of Anaerobiosis, N2 and KCN on the Formation of Polar Metabolites from GA . . . . Abscisic Acid (ABA) and the Formation of Gibberellin Metabolites . . . . . . A. Influence on GA3 Metabolism B. Influence on GA] Metabolism . . The Effect of GA1 and GA7 on the Formation of Metabolites from l4c- -GA3 and 0 GA on the Formation of Metabolites from H- 1 . Studies on the Identification of the M2 Metabolites . . A. Effect of Enzymes on the M2 Metabolites from Both GA3 and GA1 and on M3 of GA1 . . The Effect of Acid Treatment on GA3 and M2 of GA3 . . . Detection of Sugars in M2 of GA3 . Incorporation of 14C- U- Glucose 1nto the Minor Metabolite, M2 of GA3 . Identification of the Sugars in M2 of GA3. l. Partial hydrolysis of M2 . . 2. Complete hydrolysis of M2 . F. Determination of the Linkages Involved in the Sugars of the M2 Metabolite of GA Studies on the Identification of the M1 Metabolites . . A. Effect of Enzymes on M1 Metabolites . B. Effect of Acid Treatment on M1 of GA3 . MUOW Page 27 Effect of NH4OH Treatment on the Metabolites of GA3 and GA1 . . Effect of Sodium Metaperiodate Treatment on M] Of GA1 . Identification of the Amino Acids Present in the Major Metabolite, M1 of GA3. . F. Terminal Amino Acids Present in M1 of GA3 MUD XIV. Comparative Study on Gibberellin Metabolism DISCUSSION . . . . . . . . . . . . . . . . I. The Metabolism of the Gibberellins and Hormonal Action . . II. On the Pathways of Gibberellin Metabolism . III. The Effect of ABA on Gibberellin Metabolism IV. The M2 Metabolites . A. M2 of GA3 . M2 of GA3 and M3 . V. Electrophoretic Properties of the M1 Metabolites VI. Effect of Acid, NH4OH and Periodate Treatment on the M1 Metabolites . . VII. Possible Structure of M1 of GA3 VIII. Hydroxylation of GA3. IX. Study of the Highly Polar Metabolite of GA1 from Rappaport' 5 Laboratory in Relation to the Findings Reported Here . . X. Gibberellin Metabolism in Tissues of Other Plants XI. Significance of the Research LITERATURE CITED . . . . . . . . . . . . . . vi Page 84 84 84 97 104 104 105 105 105 107 108 109 112 112 116 116 119 Table 10. 11. LIST OF TABLES Inactivation of gibberellic acid by barley aleurone layers . . . . . . . . . . . . Distribution of radioactivity in various extracts of 4C-GA3 treated aleurone layers RF values of GA3 and M2 of GA3 in various thin layer chromatograph1c systems . RF values for GA1, M3 of GA1, the metabolite from the acidic phase of the layers and authentic 6A8 Effect of prolonged incubation on the amount of radio- activity present in polar metabolites (M1 and M2) in the aqueous phase of 14C-GA3 treated aleurone layers . . . . Effects of anaerobiosis, KCN, and incubation in an atmosphere of nitrogen on the formation of the polar metabolites of 3H-GA1 by layers Effect of abscisic acid on 14C- ~GA3 metabolism in aleurone layers . . . . . Effect of abscisic acid on the amount of radioactivity from 3H-GA1 found in the aqueous and acidic phases of aleurone layers . . . . Effect of abscisic acid on the formation of the polar metabolites (M1, M3 and M2) from H -GA1 in the experiments given in Table 8 . Effect of GA and GA7 on the amount of radioactivity from 14C- 3 found in the methanol extract and polar metabolites of aleurone layers . Effect of GA3 on the formation of the polar metabo- lites (M1, M3 and M2) from 3H- -GA1, by aleurone layers . . vii Page 29 30 33 41 52 58 59 60 60 62 62 LIST OF FIGURES Figure 1. Structural formulas of some gibberellins discussed in the dissertation and the location of radio- activity (*) in the labeled compounds used . 2. Correspondence of the migration of 14C-GA3 with the biologically active area of extracts from barley aleurone layers treated with GA3 3. Pattern of distribution of radioactivity on paper chromatograms of the polar metabolites present in the aqueous phase of GA3 treated barley aleurone layers . . . . . . . . . . . . 4. Distribution of radioactivity of M2 of GA3 on thin layer chromatograms after development in various solvent systems . . 5. Migration patterns of radioactivity on a paper chro- matogram of M1 and M2 of GA3 in relation to GA3 6. Migration pattern of radioactivity on chromatograms of the major metabolite M1 of GA3 . 7. Distribution of radioactivity on paper chromatograms of the polar metabolites present in the aqueous phase of 3H-GA1 treated barley aleurone layers 8. Distribution of radioactivity on chromatograms of the acidic ethyl acetate phase of 3H-GA1 treated aleurone layers . . . . . . . . . . . 9. Migration pattern of radioactivity on thin layer chromatograms of the M3 metabolite of GA1, in relation to the products present in the acidic ethyl acetate phase of 3H-GA1 treated layers 10. Pattern of distribution of radioactivity on paper electrophoretograms of the acidic ethyl acetate phase resulting from the incubation of GA1 and GA3 with a hydroxylating enzyme system from Phaseolus vglgaris . . . . . viii Page 28 32 34 36 37 39 40 42 44 Figure 11. Distribution of radioactivity from the aqueous phase of 3H-GA3 treated aleurone layers on a paper chromatogram . . . . . . . . 12. Time course of the formation of polar metabolites (M1, M2 and M3) from H- -GA1 by aleurone layers 13. Time course of the formation of polar metabolites of14C-GA3 14. Distribution of radioactivity on a paper chromatogram of the aqueous phase of the combined medium and wash solution from aleurone layers incubated with 14 C- -GA3 for 72 hours l5. Distribution of radioactive M1 metabolites on paper electrophoretograms . . . . . . . . 16. Distribution of radioactivity in 3H- -GA1, 3H- -GA3 and M1 of GA1 on an electrophoretogram . . 17. Distribution of radioactivity on a paper chromatogram of the M2 metabolite treated with pectinase l8A. Distribution of radioactivity on a paper chromatogram of the M3 metabolite treated with pectinase 188. Distribution of radioactivity on a paper electro- phoretogram of the products obtained after pectinase hydrolysis of M3 19. Distribution of radioactivity on a thin layer chro- matogram of products from the treatment of M2 from GA1 with B-xylosidase . . . . . . . 20A. Distribution of radioactivity on a paper chromatogram of the aqueous phase of extracts from 3H- -GA1 treated Phaseolus coccineus seeds . 208. Distribution of radioactivity on a thin layer chro- matogram after treatment of the putative GA3- glucoside with B- -glucosidase . . 2l. Diagrammatic representation of the paper chromato- graphic separation of the products formed after acid treatment of M2 of GA3, and GA3 . 22A. Distribution of radioactivity on a paper chromatogram of the aqueous phase of aleurone layers which were incubated with GA3 and 14C-U-glucose . . ix Page 47 48 50 53 55 56 64 65 65 66 68 68 70 71 Figure 228. Pattern of distribution of radioactivity on a thin layer chromatogram of purified M2 of GA3 after incorporating 4C U- -glucose into it . 23. Diagrammatic representation of the paper chromato-. graphic separation of the sugars obtained after the minor metabolite, M2 of GA3, was partially hydrolyzed with dilute HCl . . 24. Thin layer chromatographic separation of the sugars obtained after the minor metabolite, M2 from GA3,. was partially hydrolyzed with dilute HCl . 25. Paper chromatographic separation of the sugars obtained after M2 from GA3 was partially hydrolyzed with dilute HCl . 26. Diagrammatic representation of the thin layer chro- matographic separation of the sugars obtained after the minor metabolite, M2 of GA3, was completely hydrolyzed with dilute HCl . 27. Linkages of glucosyl-xylose-GA3 that can give xylitol after sequential periodate oxidation, acid hydrolysis and reduction with Na8T4 of M2 of GA3 . 28A. Distribution of radioactivity on a paper chromatogram of the products resulting from sequential periodate oxidation, acid hydrolysis and reduction with radioactive Na8H4 of M2 of GA3 . 288. Distribution of radioactivity on a paper chromatogram of the product obtained in the xylitol area on the paper chromatogram of system E and rechromatographed in system F . . . . . . . . . . 28C. Distribution of radioactivity on a paper chromatogram of the product obtained in the xylitol area on the paper chromatogram of system F and rechromato- graphed in system G . . 29. Distribution of radioactivity on paper chromatograms of the products formed after acid treatment of 14C-GA3 and M1 from 14C-GA3 . 30A and 308. Distribution of radioactivity on paper chro- matograms of products resulting from the treatment of M1 of GA and M1 of GA1 with NH4OH for 30 minutes at 00°C . . Page 71 74 75 77 78 79 80 80 80 83 85 Figure 30C. Distribution of radioactivity on a paper electro- phoretogram of the product obtained after NH40H hydrolysis of M1 of GA3 . . 31. Distribution of radioactivity on a thin layer chro- matogram and a paper electrophoretogram of the product formed from M1 of GA1 after periodate treatment . . . . . 32. Diagrammatic representation of the thin layer chro- matographic separation of standards and the amino acids obtained after M1 of GA3 was hydrolyzed with HCl . 33. Chromatographic separation of the amino acids obtained after M1 of GA3 was hydrolyzed with 6 N HCl for 18 hours at 110° C . . 34. Diagrammatic representation of the thin layer chro- matographic separation of the amino acids obtained after the major metabolite, M1 of GA , was hydro- lyzed with 6 N HCl for 18 hours at 10°C 35. Diagrammatic representation of the thin layer chro- matographic separation of the amino acids obtained after the major metabolite, M11nyA3, was hydrolyzed with 6 N HCl for l8 hours at 110°C 36. Two-dimensional thin layer chromatographic separation of the DNP- -amino acids obtained after the dinitro- phenylation of the products of acid hydrolysis of M1 Of GA3. . . 37. Diagrammatic representation of a thin layer chromato- graphic separation of the DNP- -amino acids obtained after the dinitrOphenylation of the products of hydrolysis of M1 of GA3 38. Polyamide thin layer chromatographic separation of the DNS-amino acids obtained after the dansylation and subsequent acid hydrolysis of M] of GA3 . 39. Diagrammatic representation of a thin layer chromato- graphic separation of the DNS-amino acids obtained after the dansylation and subsequent acid hydroly- sis of M1 of GA3 . . . . . . . . 40. Distribution of radioactivity on paper chromatograms of the polar metabolites present in the aqueous phase of GA3 treated tissues xi Page 85 87 88 90 91 92 94 95 96 98 99 Figure Page 41. Distribution of radioactivity on paper chromatograms of the polar metabolites present in the aqueous phase of GA1 treated tissues . . . . . . . . 101 42. Acid hydrolysis of GA1/3H-ampho-GA1 mixture . . . . 114 43. Postulated pathways of gibberellin metabolism in aleurone layers . . . . . . . . . . . . . 117 xii INTRODUCTION The gibberellins have fascinated investigators for many years. Some have been concerned with the physiological effects of the gibberellins, others with the biochemical aspects of this group of hormones, and still others with the relationship of the structures of the various gibberellins with their function in the growth and the development of plants. Although the hormonal actions of gibberellic acid (GA3) in barley aleurone tissues have been studied extensively, the question still remains, how do these phytohormones really act? Studies on the metabolism of labeled gibberellins may provide significant information concerning their mode of action and the path- ways of their degradation and inactivation. They may also explain the specificity of plant responses to different gibberellins. The metabolic fate of labeled gibberellins has been investigated in relation to germination and maturation of seeds (Barendse gt_gl., 1968; Sembdner et_gl,, 1968; Barendse and de Klerk, 1975), dwarfism (Kende, 1967; Davies and Rappaport, 1975a), and flowering (Railton' and Hareing, 1973; Van Den Ende and Zeevaart, 1971; Durley et2gl,, 1975). In general, plants convert biologically active gibberellins to less active polar metabolites. The disappearance of endogenous gibberellin activity during certain stages of plant development that has been observed in several instances may be due to the inactivation of the hormones themselves or to their conversion to bound forms. The metabolic fate of gibberellic acid (GA3) in aleurone layers, a tissue that has been used extensively to study its hor- monal action, is unknown. The metabolism of 3H-91bb6V6111n A1 (GA1) the more readily available radioactive gibberellin, has been studied in several laboratories. The first report on gibberellin metabolism in cereal aleurone layers appeared from Kende's laboratory after he successfully prepared and purified tritium labeled GA1 by catalytic reduction of GA3 (Kende, 1967). Musgrave et_gl: (1972) treated barley aleurone layers with 3H-GA], 3H-GA5 and the biologically inactive 3H-GA5 methyl-ester. A major highly polar metabolite which remained at the origin of chromatograms was formed from each of the labeled gibberellins. Aleurone layers accumulated only 4% of the 3H-GA1 counts originally added, whereas layers treated with‘3H-GA5 and 3H-GA5-methyl-ester accumulated 18 and 22%, respectively. The aqueous phase of the layers contained most of the radioactivity which consisted of polar compounds. Unmetabolized GA1 was present in the acidic ethyl acetate phase. The accumulation of radio- activity by the layers was inhibited in the cold as well as by dinitrophenol and sodium fluoride. Nadeau et_§l, (1972) tentatively identified three polar metabolites resulting from the incubation of 3H-GA1 with barley aleurone layers as 3H-GA1-glucoside, 3H-GA8 and 3H-GAB-glucoside. A highly polar compound was produced in the greatest quantity. This compound was referred to as 3H-GA-X. The tentative identification of the metabolites was based on comparingtheir properties and that of their methyl ester-trimethyl silyl ethers with authentic standards on thin layers and in gas-liquid chromatographic systems, respec- tively. It is important to point out that in none of these studies was it shown that the sugar attached to the GAS is glucose. Treatment of the major highly polar metabolite with l N H2504 or 1 N NaOH for 4 hours at 90°C did not liberate a gibberellin- like compound. This metabolite migrated faster than GA1 or GAB- glucoside, each of which contains only one free carboxyl group, when electrophoresed in a buffer containing pyridine, acetic acid, and water (10:1:90) at pH 6.0. It was suggested, therefore, that the compound, 3H-GA-X, probably contains more than one carboxylic acid group. Another interesting point mentioned in the paper was that abscisic acid (ABA) enhanced the uptake of 3 H-GA1 and increased the amount of labeled metabolites formed. Later, Nadeau and Rappaport (1974) reported some of the properties of the major metabolite, 3H-GA-X, and renamed it 3 H-ampho- GA]. The formation of ampho-GA1 in aleurone layers was proportional to the logarithm of the ABA concentration. There was a lag period of 2.5 hours in the formation of ampho-GA]. They suggested that it may be necessary to induce an enzyme system responsible for the fbrmation of the conjugate. Ampho-GA1 was isoelectric at pH 2.2. It also formed a derivative with dinitrofluorobenzene. Ampho-3H-GA1 migrated faster than 3H-GA1 toward the anode upon electrOphoresis at pH 5.9, indicating that it contained more carboxyl groups than GA]. It was also said that the treatment of the major metabolite with l N HCJ athO°C for 12 hours resu1ted in the formation of compounds expected to be formed from the acid treatment of GA], indicating that the conjugate was formed directly from GA]. They suggested that the highly polar 3H-GA1-X formed from GA1 is amphoteric and is probably conjugated with a peptide. They concluded that the hydroxyl group at carbon-3 of the GA ring is in a "free" form andis not the site of conjugation. The treatment of ampho-GA] with strong alkali did not produce an ethyl acetate-soluble radioactive sub- stance, hence the possibility of having an amide or ester linkage was ruled out. Recently, Davies and Rappaport (1975a) have shown that a dwarf mutant of maize metabolized 3H-GA1 to 3H-GA8, 3H-GA8- glucoside and the unknown metabolite referred to as 3H-GA1-X. The 3H-GA1-X metabolite formed from the maize plant was similar to the ampho-3H-GA1 formed when aleurone layers were incubated with 3H-GA]. The biologically inactive pseudo GA1 (3a-0H-GA1), which differs from GA1 only in the orientation of the 3-OH group, was taken up by barley half seeds but was not metabolized (Stolp et_§l,, 1973). Abscisic acid enhanced the accumulation of CA1 and pseudo ' GA1 when incubated with barley half seeds, but it only affected the metabolism of the biologically active GA]. The important point men- tioned in this paper was the significance of the steriochemistry of the 3-OH position in relation to the uptake and metabolism of 3H-GA]. It is possible that the enzymes which are responsible for the hydroxylation or glycosylation of GA] at the 3-OH position are very specific and are unable to react with pseudo GA]. Barendse and de Klerk (1975) studied the metabolism of 14C-GA3 in Pharbitis nil Choisy. A single metabolite, tentatively 14 identified as GA3-glucoside, was observed when C-GA3 was applied to seedlings of Pharbitis. The identification was made by cochro- matographing the metabolite in different solvent systems with authentic 3—0-8-D-gluCopyranosyl-GA3. It gave GA3 and gibberic acid on mild acid hydrolysis as determined by thin layer chroma- tography. The GA3-glucoside was also hydrolyzed by enzyme prepa- rations of cellulase and B-glucuronidase whereas B-glucosidase had no effect on it. The formation of 2-0-8-glucosyl gibberellin A3, 2-0-8- glucosyl-isogibberellin A3, 2-0-8-glucosyl-gibberellinic acid and the B-glucoside of an unknown gibberellin-like substance has been reported by Asakava et_gl, (1974) in dwarf kidney bean plants treated with GA3. Yamane gt_gl, (1975) studied the pathways in which tritium labeled gibberellins (GA1, 8A4, GAS, GA8 and GAZO) are metabolized in maturing and germinating bean seeds (Phaseolus vulgaris cv. Kentucky Wonder). It was found that each of the 3H-GAs fed to the seeds was mainly converted to 3H-GAB-glucoside. 6A4 and GA20 were both converted to GA], whereas GA] and GA5 were converted to GA8' Furthermore, these gibberellins were converted to products tentatively identified as the corresponding glucosides and glucosyl- esters. The important point mentioned in this paper was that 6A4 and GAZO act as precursors of GAB-glucoside, and the intermediate products were GA1 and GA8; whereas GA5 was metabolized to CAS- glucoside, which involved the formation of GA8 as a free intermediate. The interconversion of gibberellins has also been studied in other plants. Usually these transformations take place through hydroxylation reactions at carbon 2, 3, 12 or 13 of the gibberellane ring. Thus, GA5 was converted to GA3 (hydroxylation on carbon 3) in seedlings of dwarf pea (Durley e§_gl,, 1973). GA4 was converted to GA] (hydroxylation on carbon 13) and GA34 (hydroxylation on carbon 2) by dwarf rice (Durley and Pharis, 1973). GA1 was con- verted to GA8 (hydroxylation on carbon 2) by dwarf rice seedlings and seedlings of Scarlet Runner bean (Railton e§_§l,, 1973; Reeve et_gl,, 1975). GA20 was converted to GA29 (hydroxylation on carbon 2) by etiolated shoots and germinating seeds of dwarf pea (Railton et al., 1974) and also by the plant Bryophyllum daigremontianum (Durley g§_§1,, 1975). GA9 was converted to GA20 (hydroxylation on carbon 13), GA10 (hydroxylation on carbon 16), and 2, 3 dihydro GA31 (hydroxylation on carbon 12) by dwarf pea (Railton gt_gl,, 1974). The objectives of the research reported here were: (a) to determine whether GA3 and GA1 are converted to other active gibber- ellins or metabolites in aleurone layers; (b) to study the properties and the structures of any resulting compounds; (c) to ascertain the metabolic pathway by which gibberellic acid is inactivated or conju- gated in aleurone layers,and (d) to relate, if possible, the metabo- lism of the hormone with its ability to evoke responses in the tissue. o 1 0 11 * / \ 12 2* / * co » co 3 5 8 1413 0H 0 H V / U H 0 H0 \\\v/,4 3 'COOH 16 2 CH3 -C|-| 2 *COOH GA] GA3 0 o / _—/\ no I /\ 50 /OH HO YV ”0 V . .___:CH2 CH - CH2 CH3 coon 3 0011 Figure 1. --Structural formulas of some gibberellins discussed in the dissertation and the location of radioactivity (*) in the labeled compounds used. MATERIALS AND METHODS 1. Preparation and Incubation of Tissues Barley seeds (Hordeum vulggre cv. Betzes) were obtained from the Michigan Crop Improvement Association, East Lansing, Michigan. The seeds were treated with 50% H2504 for 25-30 minutes by shaking them at medium speed on a mechanical shaker in order to remove the husk. They were then washed extensively with distilled water, sterilized with 1% hypochlorite for 2-3 minutes, rewashed and allowed to imbibe water by shaking on a mechanical shaker at room temperature for l6-20 hours. The seeds were then placed at room temperature for an additional 16-20 hours before use. The seeds were cut transversely with a razor blade to remove the anterior half containing the embryo. The extreme tip of the remaining posterior half was also cut off. The aleurone layers were made by making a superficial incision longitudinally passing through the median plane of the posterior halves of the seed in order to remove the starchy endosperm. Aleurone layers were prepared from deembryonated seeds of wheat (Triticum aestivum cv. Genesee, obtained from Dr. Albert Ellingboe) that had imbibed water for 16 hours. All tisSues were sterilized with hypochlorite immediately prior to use. The aleurone layers were shaken with the hormone on a nechanical shaker at room temperature in incubation media containing 3.4 x 10'4 M streptomycin sulfate and 0.02 M calcium chloride. As a control in some experiments, layers were boiled for 2-3 minutes in water before incubation with the hormone, and were subjected to the extraction procedure given experimental tissues. 11. Bioassays Several of the responses given by barley half seeds after application of gibberellic acid to them were the basis of bioassays in these studies, in addition to the commonly used amylase bioassay (Filner and Varner, 1967). Barley half seeds were prepared by merely 'removing the anterior embryo halves from imbibed seeds with a razor blade. To establish standard curves, 8 half seeds were shaken for 16-24 hours on a mechanical shaker in st0ppered test tubes with 1 ml of logarithmic concentrations of gibberellic acid ranging from 1 x 10‘3 to 10 pg per ml, each containing 250 pg streptomycin sulfate per ml and 0.02 M CaClz. At the end of the incubation, 5 ml of distilled water were added to the tubes, they were shaken thoroughly and aliquots assayed for phosphomonoesterase or phosphodiesterase activity. Typically, 0.5 ml of extracts from the half seeds, 0.5 m1 of 0.05 M sodium acetate buffer, pH 4.8, and 0.1 ml of either p-nitrophenyl phosphate or bis-p-nitrophenyl phosphate (0.11 mg/ml) were incubated at 37°C for 5-10 minutes. After the addition of 1 ml of 10% NaOH, the p-nitrophenol produced was read at 405 nm in a spectrophotometer. 10 III. Chromatography and Paper Electrophoresis Unless otherwise stated, paper chromatography was carried out on Hhatman 3 MM paper by the descending method and thin layer chromatography on precoated silica gel G (E. Merck or Macherey-Nagel Co., Germany) of 0.25 mm thickness by the ascending technique. The paper chromatographic solvents (by volume) were: (A) iSOpropanol- water (4:1), (8) methanol-ethanol-n-butanol-water-acetic acid (4:3:1:1.5:0.5), (C) propanol-methanol-34% NH40H (6:4:3), (D) ethyl acetate-acetic acid-water (9:2:2), (E) nitromethane-acetic acid- ethanol-water saturated boric acid (8:1:l:1), (F) n-butanol-pyridine- water (6:4:3) and (G) propanol-ethyl acetate-water (7:1:2). The thin layer solvents (by volume) were: (H) benzene-n-butanol-acetic acid (70:25:5), (I) acetone-acetic acid (97:3), (J) chloroform-methanol- acetic acid-water (40:15:3:2), (K) isopropanol-6 N NH40H (5:1), (L) propanol-ethyl acetate-water (7:2:2), (M) propanol-ethyl acetate- water (7:2:1), (N) n-butanol-acetic acid-water (4:1:1) and (0) propanol-methanol-34% NH40H (4:3:3). During the purification and identification of the metabolites and related compounds, the paper was initially washed with distilled water and dried, and_thin layer plates were washed by developing‘ them in a solvent system consisting of chloroform-methanol (1:1) before they were used for experimental purposes. All solvents used were redistilled. Thin layer solvents used for the identification of amino acids were: (P) phenol-water (3:1), (0) n-butanol-acetone- triethylamine-water (10:10:2:5) and (R) 2-butanol-methyl ethyl 11 ketone-dicyclohexylamine-water (10:10:2:5). The solvents used for the separation of DNP-amino acids were: (S) chloroform-tertiary amyl alcohol-acetic acid (70-30-3) and (T) toluene-Z-chloro-ethanol- pyridine-25% NH40H (100:70:30:l4). The composition of all solvent systems, by volume, will be given in parentheses hereafter in this dissertation. The electrolytes used for paper electrophoresis were: (A) buffer composed of 0.01 M tetrasodium EDTA containing 0.34 ml pyridine per liter and adjusted to pH 3.5 with glacial acetic acid; (8) 0.05 M ammonium borate buffer, pH 9.3 (Coombe and Tate, 1970); (C) buffer containing 2.5% formic acid and 8% acetic acid, in water, pH 2.2; and (D) 0.1 M ammonium carbonate buffer, pH 8.9; (E) buffer containing 0.5 ml pyridine plus 2.5 m1 acetic acid per liter of water and adjusted to pH 5.6. Electrophoresis (with a Durrum type unit and Hhatman 3 MM washed strips) was of 8 hours duration when buffer A was used, 4 hours with buffer 8 and 6 hours with buffers (C), (D), and (E), all at 400 volts potential. IV. Extraction of the Metabolites At the end of the incubation period, the medium was decanted and the layers were washed on a mechanical shaker for 40 minutes, two changes of water being made. The wash solution was combined with the medium. The layers were boiled for 1-2 minutes in methanol and the tissues were subsequently ground with methanol successively in a mortar and pestle or with a Haring blendor. The methanol extracts were decanted and filtered under suction pressure after 12 each extraction. Although the methanol insoluble residue was rou- tinely dried and assayed for radioactivity, very few counts were ever found. The methanol extract was evaporated to dryness under reduced pressure, the residue was taken up in borax buffer, pH 8.4, and the solution was extracted three times with ethyl acetate. The resulting top layer, the basic ethyl acetate phase, never contained significant amounts of radioactivity. The pH of the aqueous layer was adjusted to 2.4 with HCl and reextracted 4-5 times with ethyl acetate. The top layer was designated as the acidic ethyl acetate phase and the bottom layer as the aqueous phase. The aqueous phase was further extracted two times with petroleum ether to remove any remaining acidic, non-polar labeled compounds. The pH of the aqueous phase was adjusted to 7.0, evaporated to dryness and the residue taken up in-a known volume of water. The incubation media and com- bined wash solutions from the layers were similarly adjusted to the pH values given above and were partitioned 4-5 times against ethyl acetate. The ethyl acetate phases were dried over anhydrous Na2504 before being evaporated to dryness and the residues were dissolved in a known volume of 80% ethanol and chromatographed on paper in system A. A small amount of sample was taken from each resulting phase (basic ethyl acetate, acidic ethyl acetate and aqueous phases of both layers and medium) to determine the amount of radioactivity in the total sample. The aqueous phase of the layers was treated with charcoal. The adsorbent was washed 5-6 times with water by filtration with a Buchner funnel and the products were desorbed with a solution of 13 0.6% NH40H in 50% ethanol. The ethanolic solution was evaporated to dryness, the residue was taken up in a small portion of 50% ethanol and applied to paper for chromatography in solvent system A. Unlabeled metabolites were processed similarly by incubating a large number of layers with unlabeled hormone. In the time course experiments, layers were taken out at different intervals, washed and extracted as described above. V. Detection of Unlabeled Metabolites For detecting M2 metabolites and other gibberellins, the paper and thin layer chromatograms were sprayed with an ethanolic solution of H2504 (95 ml of ethanol containing 5 ml of conc. H2504) and the fluorescence developed by heating the chromatograms with a hot hair dryer was examined under a short wave length ultraviolet lamp. For detecting M1 metabolites, chromatograms were sprayed with ninhydrin reagent (0.3 g ninhydrin was dissolved in 100 ml n-butanol and mixed with 3 ml glacial acetic acid) and the color was developed by heating the chromatograms (l0-l5 minutes for thin layer, 8-10 minutes for paper) in an oven at 105°C. VI. Acid and Alkali Treatment of the Gibberellins and Metabolites The experimental sample was treated with conc. HCl (or NH40H) either in a round bottom evaporating flask with a stopper or in a small Pyrex glass tube with a Teflon cap. The reaction mixture was heated in a boiling water bath or in an oven at 100°C for the desired time. The sample was allowed to cool to room temperature l4 and then evaporated to dryness. The evaporation process was repeated 3-5 times after adding more water. The resulting residue was dissolved in a known volume of 80% ethanol and applied to chromatograms. VII. Enzyme Treatment of the Metabolites A cellulase preparation from Aspergillus niger (Sigma), B-glucosidase (Sigma) from almonds, emulsin (E. Merck, Darmstadt) from almonds, and pectinase (Nutritional Biochemicals Corporation) were used in attempts to hydrolyze M2 of both gibberellins and M3 from GA]. The metabolites and l-2 mg of the enzyme preparations in 0.4 ml of 0.05 M acetate buffer, pH 4.8, were incubated for 24 hours in a water bath at 37°C. The reaction was stopped by heating the sample in a boiling water bath for 6-10 minutes. The reaction mix- ture was centrifuged, the resulting supernatant solution was evapo- rated to dryness, and the residue was taken up in a known volume of water which was chromatographed on paper and thin layers along with standards. , VIII. Purification and Characterization of the_Mg Metabolite of GA3 A. Purification After M2 was located on the paper chromatogram developed in solvent system A, it was eluted with water and subjected to adsorp- tion on charcoal and desorption with NH40H in ethanol as described in section IV. The ethanol extract was evaporated to dryness, the l5 residue was dissolved in a known volume of 80% ethanol and was chromatographed further on thin layers in systems I, J, K and L; and on paper in system A. 8. Detection of Sugars in M2 of GA3 The diphenylamine-aniline-phosphoric acid reagent (Bailey and Bourne, 1960) and the ammoniacal silver nitrate reagent (Brown and Serrow, 1953) were used to locate all sugars on the paper and thin layer chromatograms. The diphenylamine reagent was made by dissolving 2 g diphenylamine, 2 ml aniline and 10 ml 80% ortho- phosphoric acid in 100 ml of acetone. The ammoniacal silver nitrate reagent was made as follows: 5 9 silver nitrate was dissolved in 50 ml of water and cone. NH40H was then added until the precipitate just redissolved. The dried chromatograms were sprayed with the diphenylamine reagent and heated to develop colors at 100°C for 1-2 minutes (Whatman No. 4 paper), 2-3 minutes (3 MM paper) or 10-25 minutes (thin layers). The silver nitrate dip technique (Robyt and French, 1963) was alSo used to detect sugar alcohols and sugars on paper chromato- grams. The dried chromatogram was dipped in solvent I (1 ml of saturated silver nitrate solution in 200 ml redistilled acetone); air dried, then dipped in solvent II (1 m1 of 40% NaOH in 100 ml methanol) to develop the color. C. Chemical Tests for Pentose in M2 Of GA3 The first test was performed as follows. To 0.5 ml ofasolu- tion containing metabolite of GA3 in a test tube 0.5 m1 of orcinol 16 _ ‘reagent (made by adding 10 m1 conc. HCl to 0.1 g orcinol and 0.05 g ‘ferric chloride) was added. The tube was heated in a boiling water bath for 20 minutes to develop the color. In the phloroglucinol test 0.2 m1 ofa solution containing the M2 metabolite of GA3 was added to 2.5 ml ofphloroglucinol reagent (Ashwell, 1966) which was made in the following manner: 110 nfl of glacial acetic acid, 2 m1 of concentrated HCl .andl m1 of 8% glucose were miXed with 5 m1 of 5% phloroglucinol in ethanol. The tube was heated for 15 minutes in a water bath to develop the color. 0. -Identification of Su ars in M2 Of GA3 by Thin Layer and aper Chromatography The purified M2 metabolite of GA3 from 4000 layers, isolated from experiments where 1000 layers/group were incubated in medium containing 500 ug GA3/ml in a'total volume of 30.0 m1,was hydrolyzed with 0.5 ml) of 2.5 N HCl‘ in a hot water bath at 95°C either for 40 minutes for partial hydrolysis or for 90 minutes for complete hydrolysis. The reaction was stopped by neutralizing the reaction mixture with potassium bicarbonate. The neutralized sample was treated first with Dowex SON-X8 cation exchange resin (H+ form), filtered and then treated with Dowex l-X8 anion exchange resin (Cl' form) to remove any positively and negatively charged materials. The resin was filtered, the filtrate evaporated to dryness and the residue dissolved in water. ‘ A known volume of the above sample and a standard mixture of glucose, galactose, xylose, arabinose and ribose were spotted on 17 a thin layer of silica gel G (E. Merck) or on Whatman No. 4 paper. The thin layer chromatogram was developed 3-4 times in solvent system M and the paper was developed in solvent system D for 24 hours. The dried chromatograms were sprayed with ammoniacal silver nitrate reagent or diphenylamine reagent to develop colors, as described earlier. E. Sequential Periodate Oxidation, Acid Hydrolysis and Borohydride Reduction of M2 of GA3 The metabolite M2 of GA3 from 1200 aleurone layers was treated with 2 mg of sodium metaperiodate in 0.2 ml water for 12 hours in the dark at room temperature. The reaction was stopped by adding a few drops of ethylene glycol and water was removed under reduced pressure. The mixture was hydrolyzed with 0.2 ml 2.5 N HC1 for 40 minutes at 105°C. It was then cooled and HC1 was removed by evaporating the sample to dryness. The residue was dissolved in 0.1 ml water, a few crystals (1-2 mg) of radioactive sodium boro- hydride (NaBT4) were added, and the mixture incubated for 90 minutes at room temperature. The reaction was stopped by the addition of acetone and the mixture was evaporated to dryness under reduced pressure. This process was repeated 7-8 times by adding successive portions of acetone and methanol. The residue was finally extracted 344 times with benzene and diethyl ether, these extracts being dis- carded. The residue was dissolved in a known vOlume of water and applied to the paper and chromatographed in system E with authentiC' xylitol along with a standard mixture of glucose, galactose, glucitol, 18 xylitol and xylose. The radioactive area corresponding to xylitol, which was located on paper by the silver nitrate dip technique, was eluted with water and filtered. The filtrate was treated suc- cessively with cation exchange (H+ form) and anion exchange (OH' form) resins to remove any inorganic material, as described earlier (section VIII-D). The filtrate was evaporated to dryness, the residue was taken up in water and further chromatographed on paper in solvent systems F and G. F. Incorporation of 14C-U-Glucose into Mg by GA3 Treated Aleurone Layers The aleurone layers (720) were incubated with 10 uc of glu- cose in the incubation medium containing GA3 (500 ug/ml) for 24 hours at room temperature on a mechanical shaker. The washing and extraction of the layers were carried out as described earlier. The resulting aqueous phase of the layers was chromatographed on paper in solvent system A alongside of authentic M2. The area correspond- ing to M2 on the paper chromatogram was eluted with 80% ethanol, evaporated to dryness and chromatographed further two times on a thin layer plate with system L. IX. Purification and Characterization of the M] Metabolite of GA3 A. Purification The major metabolite (M1) that remained at the origin of the paper chromatogram in solvent system A was eluted with water and the solution filtered. The filtrate was subjected to adsorption onto 19 charcoal and desorption with NH40H in ethanol. The ethanolic extract was evaporated to dryness and the residue dissolved in 50% ethanol. This was applied to paper and successively chromatographed on paper in systemsLBand C, and on thin layer in systems Nand 0. It was then electrophoresed successively in buffers C and D. The purity of the unlabeled compound was assessed by the presence of a single ninhydrin positive spot on the chromatogram which migrated along with radioactive M]. B. Hydrolysis The major metabolite, M] of GA3, was transferred to a thick Pyrex glass tube designed for evacuation and sealing. The sample was evaporated to dryness and 200 pl of 6 N HC1 were added to it. The liquid was then frozen by immersing the tube in'a dry ice- ethanol bath. After replacing the air with N2, the tube was evacu- ated to approximately'lmm of mercury and sealed subsequently. Hydrolysis was carried out for 16518 hours in a temperature regulated heated block (Lab Line Instrument, Melrose Park, 111.) at 110°C. HC1 was removed after hydrolysis by slow evaporation in a vacuum desiccator containing solid NaOH. The residue was dissolved in water and a portion of the hydrolyzed sample was chromatographed on thin layers of silica gel 6 (Macherey-Nagel) and cellulose plates (Brinkmann)of 250 u thickness in several solvent systems (N, P, Q, and R) along with amino acid standards. The dried chromatograms were sprayed with the ninhydrin reagent and heated to develop the color. Chromatograms developed in 20 systems 0 and R were dipped in petroleum ether for 20-30 seconds to remove excess amines, dried and then sprayed with the ninhydrin reagent. The purified M1 metabolite of GA3 from 4000 layers, isola— ted from experiments where1000 layers/group were incubated in medium containing 500 pg GA3/m1 in a total volume of 30 ml, was used hieach' of the following experiments on M1 identification. C. Identification of Amino Acids by Dinitrophenylation The dinitrophenylation technique was carried out in the fol- lowing manner. The reaction was performed in a small glass st0ppered test tube containing 0.2 ml sample (a known concentration of amino acids or acid hydrolyzed M1), 0.5 ml buffer (8.4 g NaHCO3 and 2.5 m1 of 1 N NaOH made up to 100 ml with distilled water, pH 8.8), 0.02 ml of 10% l-fluoro-2-4 dinitrobenzene (FDNB) solution in absolute ethanol and 0.4 m1 absolute ethanol. This was mixed thor- oughly and the reaction mixture was incubated in an oven in the dark for 1 hour at 65°C. After the reaction mixture was taken out of the oven and allowed to cool at room temperature, the pH was adjusted to approximately 12.0 with NaOH solution. Water (1 ml) was added to the mixture and it was extracted with 2 m1 portions of diethyl ether four times, the ether phase being discarded. The pH of the aqueous phase was adjusted to around 1.0 with dilute HC1 and it was reextracted 7-8 times with diethyl ether. The aqueous phase was discarded. The resulting ether extracts were combined and evaporated to dryness and the residue was taken up in a known volume of acetone. 21 These are the standard steps in the procedure for dinitrophenylation although they have been slightly modified (Pataki _e__t_a_1_., 1967). The UMP-derivatives of hydrolyzed products of MI were spotted on thin layer silica gel 6 (Macherey-Nagel) and a standard DNP-amino acid mixture of alanine, serine, and glycine was spotted alongside them on each chromatogram. *The chromatograms were then developed. Solvent system S was used for the development of the chromatogram in one dimension. For the two dimensional chromatography the thin layer plate was developed first in solvent system T, the chromatogram was dried with a hair dryer for 15-30 minutes and rechromatographed in the second dimension in solvent system S. All these operations (preparation and chromatography) were carried out under diffuse light conditions. 0. Identification of the Terminal Amino Acids by Dansylation 1. Preparation of dansyl (DNS) derivatives.--The technique for dansylation was based on the method of Parke (1975) and Heiner gt_gl. (1972), with some modifications. To a glass st0ppered test tube 0.1 m1 of a solution con- taining amino acids or M1 metabolite was added and the solution was subsequently evaporated to dryness. Then, 20 p1 of 0.2 M sodium carbonate buffer pH 9.8 was added to the sample and it was evapora- ted to dryness again. The residue was dissolved in 15 p1 of water. To the reaction mixture, 30 p1 of dansyl chloride stock solution was added and the test tube was sealed with Parafilm. Dansyl ,chloride was prepared by dissolving it in acetone (15 mg/ml) by 22 vortexing; the solution was centrifuged and an equal volume of water added to the supernatant solution. The reaction mixture was incubated at 37°C for an hour and then evaporated to dryness. After the completion of the dansylation of M], the product was transferred to a thick Pyrex glass tube designed for evacuation and sealing. The reaction mixture was evaporated to dryness and it was hydrolyzed with 0.2 m1 of 6 N HC1 at 110°C for 8 hours using a similar procedure as that described earlier (section IX-B) for the identification of amino acids in M]. Finally, the residue was dis- solved in a known volume of 50% aqueous pyridine (by volume) and stored in the freezer until it was ready to be used.' A similar pro- cedure was utilized for the dansylation of the M1 metabolite of GA3. 2. Chromatographic separation of DNS-derivatives.--The double-layer polyamide plate (Cheng-Chin Trading Co., Ltd.; dis- tributed by Gallard-Schlesinger) was cut with scissorsto 5 cm x 5 cm and 5 cm x 10 cm sizes from the full 15 cm square plate. The standard dansyl amino acids (DNS-alanine, DNS-serine and DNS-glycine; 1 p1) were spotted onto one side of the plate by touching the tip of a Hamilton microsyringe containing them to the polyamide sheet. The plate was dried with a hair dryer between applications. The experimental sample (3 p1 of hydrolyzed DNS-Ml) was spotted on the back of the plate, opposite the standards. The plate was placed vertically in a small beaker and dried with a medium hot air stream from a hair dryer for a few minutes. Then the polyamide plate was chromatographed at room temperature in three successive solvents in 23 an ascending manner according to the method given by Neiner gtgal, (1972) and Parke (1975). The chromatographic solvents were: (I) 1.5% formic acid in water; (11) acetic acid-benzene (1:9), and (III) acetic acid-methanol-ethyl acetate (1:1:20). The plate was first developed in solvent I for the first dimension and then successively in the other two solvents (II and III) in the second dimension. The chromatogram was dried after it was developed in each solvent under a stream of air from a hair dryer. The dried chromatogram was examined under a short wave length ultraviolet lamp and the results interpreted as described by Heiner §t_al, (1972) and Parke (1975). The solvents were freshly prepared each time 2-3 hours prior to chromatography. The chromatogram developing jars from Eastman Kodak were used for chromatography. All of the operations, including preparation and chromatography of DNS derivatives, were carried out under dim light conditions. X. Hydroxylation of 14C-GA3 and 3H-GA] Tby a CeTTTFree System A cell free system was prepared by a method described by Patterson §t_al, (1975), with a few modifications. The seeds used in this experiment were runner beans, Kentucky Wonder pole variety, purchased from a local store. Bean seeds (5 g) were scratched, surface sterilized and thoroughly washed with water before being . allowed to imbibe water for 20 hours at room temperature. After imbibition the seeds were decoated, sterilized, washed and ground in a mortar with a pestle for 5 minutes in the cold (3-4°C) with sand (200Q400 mg), polyvinylpyrrolidone (1.25 g) and 40 ml of a solution 24 of 0.05 M Tris-maleate buffer, pH 6.5, containing 0.2 M sucrose. The slurry was filtered through cheesecloth and the filtrate was centrifuged at 12,000 x g for 15 minutes. The supernatant fraction was decanted and recentrifuged at 95,000 x g in a Beckman model L ultracentrifuge (SH40 rotor) for 2 hours at 0°C. The resulting supernatant fraction was used. The unused portion of the fraction was stored in 10% glycerol in a freezer at -15°C for later use. 14 The radioactive hormones, C-GA3 (8.2 pg in 0.3 ml water) and 3H-GA1 (0.044 pg in 0.8 ml water), were incubated separately in st0ppered test tubes with 2 ml enzyme, 1.5 x 10'3 4 3 g ferric chloride, 8 x 10' g ascorbic acid, 1 x 10' g NADPH, 1 x 10'3 g streptomycin sulfate and enough 0.05 M Tris—maleate buffer, pH 6.5, to make a final volume of 3 ml. The reaction mixtures were incubated on a A mechanical shaker for 16-18 hours at room temperature. The reaction was stopped by heating the tubes in a boiling water bath for 7-10 minutes, cooled, and centringed at 1,000 x g for 5 minutes. The residue was discarded, the pH of the supernatant solution was adjusted to 2.5 with dilute HC1 and then extracted 4-5 times with ethyl acetate. The resulting ethyl acetate and the remaining aqueous phase (whose pH was adjusted to 7.0) were evaporated to dryness. The residue was dissolved in 80% ethanol and electrophoresed in the borate buffer system. XI. Chemicals 14 Gibberellic acid (8-methylene- C-GA3), which was synthesized by the method of Hanson and Hawker (1973), was obtained from 25 Amersham/Searle Corporation, Arlington Heights, Illinois. It had a specific activity of 4.45 pCi/mg. Gibberellic acid (1, 7, 12, 18- 14C-GA3), also obtained from Amersham/Searle, was used in some experiments and it had a specific activity of 4.9 pCi/mg. The radio- active gibberellin A1 [1,2-3H(N)] with a specific activity of 31 3 cpm/2.8 x 10'2 pg) was purchased from New Ci/m mole (5.2 x 10 England Nuclear Corporation, Boston, Massachusetts. ' The unlabeled gibberellic acid (GA3) was obtained from Eastman Kodak Co., Rochester, N.Y.; gibberellin A7 (6A7) from ICN Pharmaceuticals, Inc., Cleveland, Ohio; gibberellin A1 (GA1) was a gift from Professor Hans Kende and abscisic acid was kindly donated by Professor A. De Hertogh. The radioactive GAs were as active, biologically, as the unlabeled hormones in barley half seed bioassays. The purity of the compounds was assessed by thin layer and paper chromatography. The single peaks,1ocated on chromatograms by counting the area or located with a short wave length ultraviolet lamp after spraying with ethanolic sulfuric acid and heating, corresponded to areas that exhibited biological activity. Hence, they were very pure. The radioactive gibberellin A8 (3H-GA8) was prepared from 3H-GA1 by utilizing the cell free system reported by Patterson gt_al. (1975). D-glucose-U-14C was obtained from International Chemical and Nuclear Corporation, Irvine, California, and sodium borohydride [3H] (NaBH4) from New England Nuclear; whereas 2,4-dinitrof1uorobenzene (DNEB) and dansyl chloride (5-dimethylamino-napthalene-l-su1fonyl 26 chloride) were kindly donated by Professor Derek Lamport. All other chemicals were purchased commercially. XII. Determination of Radioactivity Segments of paper chromatograms and electrophoretograms were cut off, and zones from thin layer chromatograms were scraped off and counted directly in scintillation counting fluid which was made by dissolving 5 g of 2,5-diphenyloxazole (PPO) and 0.3 g of 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene (dimethyl POPOP) in a liter of toluene. Portions of liquid samples were counted directly in Bray's (1960) scintillation fluid. Radioactivity was determined with a Packard Liquid Scintillation Spectrometer (Model 3003). RESULTS I. Attempts to Find Biologically Active Metabolites of GA3_ The only biologically active form of the hormone present in ethanolic extracts at any time (5 minutes to 24 hours) after incuba- tion of gibberellic acid with barley aleurone layers was the com- pound itself. Thus, bioassays of sections of paper and thin layer chromatograms of extracts in systems A and H revealed only one area, corresponding to gibberellic acid, which was active in the barley endosperm assay at RF 0.51 after paper chromatography in system A and RF 0.54 after thin layer chromatography in system H (Figure 2). Radioactivity was also located there when 14 C-GA3 was utilized. Additionally, the material was shown to be gibberellic acid by thin layer chromatography in systems K (RF 0.41) and I (RF 0.81). Hence there was no evidence that gibberellic acid was converted to any other biologically active form of the hormone. II. The Inactivation of Gibberellic Acid by Aleurone Layers The inactivation of the hormone by barley aleurone layers progresses with time although inspection of the data in experiment 1 of Table 1 revealed an apparent lag in the destruction during the first 30 minutes of incubation. The rate of destruction approached linearity thereafter. A plot of the rate of loss of the hormonal activity between hours 14 and 22 of experiment 2 also gave a straight 27 28 . .umgamcme mm; cowpmeumm mmmpmsamogm .mmw mm: Emgmopmeoccu gmamp ems» mg“ .vwwpaam we: uumcuxm we» Lmu$< .—ocm;pm we; saw: nmuumtuxm cmgp use gmxm;m Pmu_:m;ums a co mczpmcmasmp Eco; pm mmuzcvs m Low .5 o.m we mE=Fo> Fmpou m cw pe\mwpum appmuw opowa mgu saw: M GA r_q 2 42> 3 400« 200. . ' '4' ' 8 ' 12 16r <:> M2 GA 600‘ <:> 3 600‘ 400. zooT 600. 400 1 200‘ 400- 200‘ 8 '12' Section Number ‘1 I“: 36 .Empmzm pcm>—om Am.onm._”_um"¢v uwum uwumucicmumzi—ocmuznic-Pocmnum-_ocm;um2 use cw umao_m>mu mm; Ememoumeoczu use .mmuomowume we mccwupma cowumcmwziu.m mczmwu cansaz comuomm om o_ N_ w a oov no mv mm: Ewemouwsocgm mg» .¢.~ ucm e.m :a pm mpwummm —»;pm pmcwwmm umcowuwpewa cmmn we; mmwga maomzcm mcp .mcao; m_ we mewsm mewcmcmms w :o mezuwgmaamu Eco: um —E m.o mo mas—o> prow :? Asam ooo.mm xpmumswxoeaamv wpmmowvwg mo cowpspwgpmwoii.~P mesmmu LmaEsz cowuuwm om mp NF w v 48 Figure 12. --Time course of the formation of polar metabolites (M1, M2 and M3) from 3H- -GA1 by aleurone layers. Aleurong layers (62/ group) were incubated in media containing 6. 3 x 10'3 H- -GA1/ml in a total volume of 3. 95 m1, on a mechanical shaker at room temperature. Some layers were removed at different intervals. They were washed, extracted with methanol and the extract evaporated to dryness. The residue was dissolved in a known volume of borax buffer, pH 8.4, which was partitioned against ethyl acetate at pH 8.4 and 2.4. The aqueous phase was treated with charcoal which was subsequently elu- ted with ammoniacal ethanol and the extract chromatographed on paper in the i50propanol-water (4: 1) system. CPM 7.000. 3,000. 2,000. 1,200‘ 1,000, 800. 600. 400. 2001 49 '2 Hours Incubation 50 .cowpzpom m.xmem cw uwucaom mm: mmwza mzomzcw mcwupamme mg» mo mE=Po> ::o:: < .¢.N new ¢.m :5 am mpmummm Phepm umcwmmm umcowuwuewa :o_pzpow men use .e.m :a .ememsn xmeon we mE=Po> ::ocx w c: um>_ommwu msuemwe ms» .mmmcxen on cmmmeoam>m mew: mummeuxm wee .Focmsmms saw: uwuumeuxm wcm emum: cmppeumwc new: cmsmm: .mpw>emp:w ucmemeewu um vm>oeme mew: memzmp msom .mezpmemaEmu Eooe pm emstm Pmmwcmgmws m co FE mo.¢ mo mssFo> Pwuou m cw Pe\mH Azm-o~ x FV owe mom mum zug m AFEV ms=Fo> Pmuoh moo.p Foe mom ownoemwc< m mic, x n.m Aps\mnv .ucom F eaeoe mea.~ eem m_¢ oeeoeoae< m_ m-oe x e.~ e_e\m:v .oeoo _mo cowu_vcou meme xmmpe mgu ea ucmucou .uaxm mswemouwsoecu emawe co cowpmazmcn mmw_Foawpmz empoe cw ucmmmem “Emmy xuw>wuumownwm .memamp An Pmauwowuwe mo comusnmepmmoi-.- mezmwe emnssz :owpmmm NdD toom 1114 1114 ooc < Ix: Origin <:3 (22> I 300. 2 £3 2001 100. I A ' 4' ' ' '87 '12' it Section Number GA3 AL GI " c:9 c:D 6:3 1 8001' L‘T 600. E B U 4001 '0" M a, ,4, , , '8' . . ('2. . . '16' Section Number Figure 29.--Distribution of radioactivity on paper ghro- matograms of the products formed after acid treatment of 4C-GA3 and M] from 14C-GA3. The labeled M] (A) and GA3 (B) were treated with strong HC1 for 30-40 minutes in a boiling water bath. The chromatogram was developed in the isopropanol-water (4:1) solvent system. The drawings at the top represent the fluorescent spots of unlabeled GA3 and its acid treatment products, allogibberic (AL) and gibberic (GI) acids located under ultraviolet light after the papers were sprayed with 5% H2504 in ethanol and heated. 84 C. Effect of NH4OH Treatment on the Metabolites of GA3 and 501 The hydrolysis of M1 with strong NH40H resulted in the forma- tion of products that were less polar than M1 and which migrated in front of M1 and behind the GA3 area on the paper chromatogram in solvent system 8 (Figure 30A). One of the compounds may represent the partially hydrolyzed product. The products of NH4OH treatment of MI of GA3 migrated in front of GA3 (Figure 30C). Treatment of M1 from GA1 with NH40H resulted in the formation of a similar less polar product which migrated in front of M1 and behind GA1 (Figure 308). 0. Effect of Sodium Metaperiodate Treatment on M] of GA] The product obtained after periodate treatment of M1 migrated in front of M1 on thin layer chromatograms himethanol-ethanol-water- acetic acid (3:3:3.5:0.5) and when electrophoresed in borate buffer, the rate of its migration was less than M]: it migrated with GA1 (Figure 31). This suggested that periodate treatment oxidized vicinal hydroxyl groups present in M]. E. Identification of the Amino Acids Present in the Major Metabolite, M] of GA3 Three distinctive areas that gave a pinkish purple color with ninhydrin spray resulted after thin layer chromatography of the acid hydrolyzed products of M1 in system N. These spots were located in the areas corresponding to glycine, serine and alanine/threonine (Figure 32). Thin layer chromatography in system P gave a better 85 Figures 30A and 308.--Distribution of radioactivity on paper chromatograms of products resulting from the treatment of M] of GA3 and M] of GA] with NH40H for 30 minutes at 100°C. Reaction mixtures were evaporated to dryness and the residues were taken up in a known volume of 80% ethanol. Chromatograms were developed for either 6 hours in the case of M] of GA3 (A) or 4.5 hours in the case of M] of GA] (8) in methanol-ethanol-n-butanol-water-acetic acid (4:3:l:l.5: 0.5). Figure 30C.--Distribution of radioactivity on a paper elec- trophoretogram of the product obtained after NH40H hydrolysis of M] of GA3. The products, I and II from the paper chromatogram of Figure 30A, were eluted with 80% ethanol and electrophoresed in ammonium borate buffer, pH 9.3, for 4 hours. CPM CPM CPM M1 GA3 400. l——’ t..._1 I ___F"‘ , II 200. -1_|_I— A '4" ' '8' ' '12' ' ' '16' ' 20 M 1 GA 800. L__L Product 400- B ' '4 ‘ 7 T8 I I l 1121 1 v '16' 1'1 600 ' .l_, 1’ 14C_GA Hydrolysis Products of M1 300- a: . . . '—.-"—."‘ . . . 2 4 6 8 10 12 14 87 M ._L 3000- E 2000 g Q A 1000. ___e4 4.2: F——" "TL=———————- h—F-—'—F w r , fi . T 1 . u i v v v v v v 4 8 . 12 16 Section Number GA1 M] - .___. 1_.__1_+J 200- 2 es 3 100- 2 4 6 8 30 '12 34 Section Number Figure 31.--Distribution of radioactivity on a thin layer chromatogram and a paper electrophoretogram of the product formed from M] of GA} after periodate treatment. The thin layer chro- matogram (A) was developed in methanol-ethanol-water-acetic acid (3:3:3.5:0.5). The resulting product from TLC was eluted with 80% ethanol, evaporated to dryness, the residue was dissolved in 80% ethanol and electrophoresed in ammonium borate buffer, pH 9.3, for '4 hours (B). . M' v 88 .mcwcwme<--e< new mnemucmpm mew FPw u-~_< .mcwpm>ii> .wcwcomezpiue .mcwcoweuwsuuz .mcwsmpsfimiipu .mceewmiim .mcwma_11>m .mcwmampomwiuH .wcwm:m_11m4 .mcwmxpmiiu .mcwcmpwui< .Pz um~xpoevxe cemmiiaxm .mmv mew: mamemoumEoegm wee .Uoopp um mesa; mp ewe xmopn mcwumm; umpmpsmme mezuwewasmu w cw Po: 2 o cue: umpmmmem mm: mwmxpoewxz .Fu: sue: vaAFoew»; mm: m nu . z nu AU AU H H mw nHv we mw mW. m4 89 separation of these compounds, revealing the presence of alanine, glycine and serine (Figure 33). The best separations of these amino acids were obtained when the hydrolyzed material from M1 was chro- matographed on thin layers of silica gel G and cellulose in systems R and 0 (Figures 34 and 35). The amino acids from M1 were identified in these systems solely as alanine, glycine and serine. A tentative identification of serine resulting from the hydrolysis of M1 was also made by using periodate/Nessler's reagent (Brenner et_pl,, 1965). It gave a positive yellowish orange color with the reagent. The same color was obtained with M]. The presence of these three amino acids in M1 was further confirmed by preparing their corresponding DNP-derivatives from the products of hydrolysis of M1 and separating them by chromatography. DNP-alanine, DNP-glycine and DNP-serine were observed on thin layer chromatograms (Figures 36 and 37). F. Terminal Amino Acids Present in M1 of GA3 Two yellowish green fluorescent spots were seen under ultra- violet light on polyamide plates when M1 was dansylated, subsequently hydrolyzed and chromatographed. One corresponded to DNS-serine and the other to DNS-glycine (Figure 38). On 5 cm x 5 cm polyamide plates, a yellowish blue fluorescent spot was present in front of standard DNS-alanine. This was identified as DNS-amine. A distinct separation was obtained when a 5 cm x 10 cm polyamide plate was used (Figure 388). It was concluded that Ml has two N-terminal amino acids: serine and glycine. Thin layer chromatography on silica gel G 90 o 0000 0 Origin Figure 33.-~Chromatographic separation of the amino acids obtained after M] of GA3 was hydrolyzed with 6 N HC1 for 18 hours at 110°C. The chromatogram was developed two times in the phenol-water (75:25) system, dried, and sprayed with ninhydrin reagent holocate the amino acids. G1--glutamine, A--alanine, T--threonine, G--glycine, S--serine. 91 BAQ] 011191n ————— Origin ———————-— Figure 34.--Diagrammatic representation of the thin layer chromatographic separation of the amino acids obtained after the major metabolite, M] of GA3, was hydrolyzed with 6 N HC1 for 18 hours at 110°C. (A) A cellulose (Brinkmann) plate developed two times in the 2-butanol-methyl ethyl ketone-dicyclohexyl amine-water (10:10:2:5) solvent system. (B) A silica gel G plate deve10ped in the 2-butanol- methyl ethyl ketone—dicyclohexyl amine—water (10:10:2:5) system. Amino acids were located with the ninhydrin reagent spray. A--alanine, BA--B-alanine, G--glycine, S--serine, T--thre- onine, Exp--acid hydrolyzed M]. 92 .P2 nmszoeuxc uwmmiiaxm .wcwcomeepiih .mcwemmiim .mcwmapmium .mcecmpwimiiPom men cw umaopm>mu mew: Am mesmwev o me wwwpwm new A< mesmwe .ccwExcwemv mmo:__mm we mwuw—a emzwp seep .uaoFF um mesa; mp eoe Fu: 2 m ;u_: um~aeoeuxc we: .m Figure 36.--Two-dimensional thin layer chromatographic sepa- ration of the DNP-amino acids obtained after the dinitrophenylation of the products of acid hydrolysis of M] of GA3. The chromatogram was developed by the ascending technique in direction I with the toluene-Z-chloroethanol-pyridine-25% NH40H (100:70:30:l4) solvent system; the dried chromatogram was developed in direction 11 with the solvent system containing chloroform-tert.-amyl alcohol-acetic acid (70:30:3). A--DNP-alanine, G--DNP-glycine, S--DNP-serine. .12' 95 —- — -— —- Origin Figure 37.--Diagrammatic representation of a thin layer chromatographic separation of the DNP-amino acids obtained after the dinitrophenylation of the products of hydrolysis of M] of GA3. The chromatogram was developed in the chloroform-tert.-amyl alcohol- acetic acid (70:30:3) system, under diffused light condition. A-—DNP-alanine, G--DNP-glycine, S--DNP-serine, Exp-~DNP derivatives of acid hydrolyzed M]. 96 .JIE, '225‘ A G (:2 (3% G S i 8 8 5 II Origin X X Origin II I <9- -€> I Back A Front Exp Standard 11 Originx x Origin 11 <-—- I -—) Front I Back Exp Standard 3 Figure 38.--Polyamide thin layer chromatographic separation of the DNS-amino acids obtained after the dansylation and subsequent acid hydrolysis of M] of GA3. The experimental sample was applied on the back of the plate, opposite the standards. For one experi- ment a 5 cm x 5 cm size polyamide plate (Figure A) was employed and for another experiment a 5 cm x lO cm size plate (Figure B) was used. The polyamide plate was developed by the ascending technique in dimension I with the solvent consisting of l.5% formic acid in water (w/v). The dried chromatogram was develOped in dimension II with acetic acid-benzene (l:9) first. It was dried, then deve10ped with acetic acid-methanol-ethyl acetate (l:l:20). The chro- matogram was examined under a short wave length ultraviolet lamp in order to locate the dansylated amino acids. Exp-~acid hydrolyzed dansylated M], A--DNS-alanine, G--DNS-glycine, S--DNS-serine. 97 of the DNS-M] hydrolyzed products, also showed only DNS-serine and DNS-glycine under ultraviolet light (Figure 39). Only a very small quantity (nanomoles) of the material was required for detec- tion on polyamide plate, but ten times this amount was required on thin layers. The polyamide plates were very sensitive to overloading of the material, smearing being produced. XIV. Comparative Studygon Gibberellin Metabolism A preliminary study of the nature of the polar products resulting from the metabolism of radioactive gibberellins by various plants (as assessed by the properties of labeled material from the aqueous phase on chromatograms) was made. Four-day-old morning glory seedlings, devoid of roots, converted 14C-GA3 to two compounds that are unlike those formed by barley andwheat aleurone layers (Figure 40C). The product which migrated immediately behind GA3 on paper in system A was further chromatographed on a thin layer in system H where it migrated like GA3-glucoside (RF 0.23). The iso- lation of a singlg_polar metabolite (tentatively identified as GA3- glucoside) from 14 C-GA3 treated seedlings of Pharbitis nil Choisy, has been reported by Barendse and de Klerk (1975). 3H-GA1 was metabolized by intact four-day-old germinating corn seeds to metabolites having the properties of 6A8 and M3 formed by barley and wheat. But, unlike what occurs in the other grasses, M1 of GA1 was not formed (Figure 418). 3H-GA1 was converted to two compounds, both less polar than M2 and M3 of GA], by immature seeds of Scarlet runner bean (Phaseolus 98 _—_E-X—[; m Origin Figure 39.--Diagrammatic representation of a thin layer chromatographic separation of the DNS-amino acids obtained after the dansylation and subsequent acid hydrolysis of M] of GA3. The chro— matogram was developed in the benzene-pyridine-acetic acid (16:4:l) system. The dried chromatogram was examined under a short wave length ultraviolet lamp in order to locate the dansylated amino acids. A--DNS-alanine, G--DNS-glycine, S--DNS-serine, Exp--acid hydrolyzed dansylated M1. 99 Figure 40.--Distribution of radioactivity on paper chro- matograms of the polar metabolites present in the aqueous phase of GA3 treated tissues. The aqueous phase had been partitioned against ethyl acetate at pH 8.4 and 2.4. The radioactive metabolites were adsorbed onto charcoal, desorbed with ammoniacal ethanol, the solutions concentrated, placed on paper and chro- matographed. Chromatograms were develOped in isopropanol-water 4:1 . A. Barley aleurone layers. B. Wheat aleurone layers. C. Morning glory, 4-day-old seedlings devoid of roots. 100 M 100 1 14 c-GA3 Z O. U 500* M2 A Jud-"WP... 4 8 12 I6 20 Section Number "1 mm 14 z c-GA3 O. Q 5004 B M2 7 ' 94' ' T '8 T12' ' ' ’16' ' ‘920‘ Section Number 800. ES ”c-GA3 U W 400. . c --L__r_—I——1"L__. ‘ "'4""8 "12""16' Section Number 1 103 coccineus) (Figure 41E). One compound was hydrolyzed by B-glucosi- dase to 6A8 and appears to be a GAB-glucoside. The other product migrated just behind GA1 on the paper chromatogram and was not identified. Kentucky Wonder beans which were imbibing water con- verted 3H-GA1 to metabolites whose properties were identical to those of the compounds formed by the Scarlet runner beans (Figure 410). When mung beans were incubated with 3H-GA], the phytohormone was converted to one major polar product (Figure 4lC), which migrated in a manner similar to M3 of GA1 on paper in system A as well as on thin layer chromatograms in system J (RF 0.15). Upon electrophoresis in ammonium borate buffer, pH 9.3, it migrated in a manner similar to M2 and M3 of GA . Like N3 of GA1 it was not hydrolyzed by l B-glucosidase. All of these properties indicate that it may not be a simple glucoside. Thus, it appears that the mung beans probably have a mechanism for metabolizing GA1 that differs from the other two beans studied. The highly polar metabolites (M1-like compounds) were not detected in any other plant tissues studied here; only in aleurone layers. DISCUSSION I. The Metabolism of the Gibberellins and Hormonal Action The results indicate that GA3 was not converted to any other fbrm that is more active than the applied gibberellin itself. The hormone was inactivated by aleurone layers and its destruction was almost linear with time. Extrapolation of the results of the time course (Figure l2) of the formation of metabolites demonstrated that the hormone was metabolized and inactivated during early stages of incubation. In preliminary results (data not presented) none of the gibberellin metabolites detected in aleurone layers (except 6A8) was biologically active in the barley half seed bioassay. It appears that when the metabolites are formed hormonal activity is lost. II. On the Pathways of Gibberellin Metabolism The reduction by GA1 and GA7 of the accumulation of radio- activity in barley aleurone layers incubated with ‘4 C-GA3 and the reduction by GA3 of the formation of polar metabolites in layers incubated with 3H-GA1 indicate that the pathways for the metabolism of these gibberellins are similar (Tables 10 and ll). GA7 con- tains only one hydroxyl group, at C-3. If the conjugation site in M2 is C-3 of the GA ring, GA7 will compete with the small amount of labeled giberellinu Therefore, the inclusion of it in the medium 104 105 should inhibit the formation of metabolites from GA3. The possi- bility exists, however, that GA7 might be converted to some unknown compound which in turn inhibits the formation of these metabolites. III. The Effect of ABA on Gibberellin Metabolism The effect of ABA on the production of the polar metabolites appeared to be due to increased uptake of the hormone by aleurone tissues rather than an effect on the formation of a specific metabolite (Tables 7, 8 and 9). Abscisic acid might affect the permeability of the aleurone layers to the hormone. It has been shown that it increases the permeability of carrot root cells and stem discs of Pelargonium to water (Glinka and Reinhold, 197l, l972). IV. The M2 Metabolites A. M2 of GA3 The metabolite M2 of GA3 migrated very close to GA3 upon electrophoresis, an indication that it contained one free carboxyl group.) It was not hydrolyzed by glucosidases from a variety of sources and its rate of migration in chromatographic systems was less than an authentic GA3-glucoside. M2 of GA3 was hydrolyzed by a preparation of pectinase (whose active component appeared to be B-xylosidase) to give gibberellic acid (Figure 17). Treatment of GA3 and M2 (from GA3) with hot mineral acid gave allogibberic and gibberic acid (Figure Zl). Therefore, M2 is a GA3 derivative. This metabolite gave a positive test for hexoses, and a positive test for pentoses with orcinol and phloroglucinol reagents. Radio- active glucose was incorporated into M2 of GA3 (Figure 22); 106 radioactive galactose was not. Partial acid hydrolysis of M2 resulted in the production of a product that migrated slower than hexoses and faster than hexose disaccharides on paper chromatograms; a characteristic feature expected of a disaccharide consisting of a hexose and a pentose. This product of partial acid hydrolysis of M2 migrated in the same manner as the disaccharide, 3—0-8- glucopyranosyl-D-glucose. Complete acid (2.5 N HCl) hydrolysis and subsequent chromatography of the products on paper as well as on thin layers showed that the minor metabolite contained glucose and xylose in apparently equal amounts (Figures 23-26). Xylitol was a major radioactive product obtained after the sequential reaction of Mzof GA3 with periodate, acid hydrolysis and NaBT4 reduction (Figure 28). It appears that glucose is the terminal sugar in M2 and xylose is in the pyranose form where C-1 of glucose is linked to xylose at C-3 and C-1 of xylose to GA3 (probably at C-3 of the entrgibberellane ring). Thus, it seems likely that the minor metabolite, M2 of GA3, is O-B-D-glucose-(l+3)-O-B-D- xylopyranosyl-(l+3)-O-GA3. To the best of my knowledge, this is the first report that GA3 is conjugated with xylose and glucose. The simple glucosides of gibberellins, such as 3-O-B-glucosyl-gibberel1in A3 (Tamura gt_a1., l968), 2-0-8-glucosyl-gibberellin A8 (Schreiber gt_g1,, 1967, 1970), ll-O-B-glucosyl-gibberellin 35 (Yamane gt;gl,, 1971) etc. and a partially characterized GA1-B-D-glucoside (Hemphill et al., 1973) have been reported as metabolic products of the corresponding gib- berellins in many dicotyledonous plants. 99 Figure 40.--Distribution of radioactivity on paper chro- matograms of the polar metabolites present in the aqueous phase of GA3 treated tissues. The aqueous phase had been partitioned against ethyl acetate at pH 8.4 and 2.4. The radioactive metabolites were adsorbed onto charcoal, desorbed with ammoniacal ethanol, the solutions concentrated, placed on paper and chro- matographed. Chromatograms were developed in isopropanol-water A. Barley aleurone layers. B. Wheat aleurone layers. C. Morning glory, 4-day-old seedlings devoid of roots. CPM CPM CPM 100 M 100 1 14 c-GA3 500‘ M2 A ' ' '4 ' '8 712' 15 '20' Section Number M1 14 c-GA3 500, M2 ‘7 ' 7 T8 12 15 20' Section Number 8004 14 c-GA3 W 400. . -1__4___} “‘1... 4 8 12 16 Section Number 101 Figure 41.--Distribution of radioactivity on paper chro- matograms of the polar metabolites present in the aqueous phase of GA] treated tissues. The aqueous phase had been partitioned against ethyl acetate at pH 8.4 and 2.4. The radioactive metabo- lites were adsorbed onto charcoal, desorbed with ammoniacal ethanol, the solutions concentrated, placed on paper and chro- matographed. Chromatograms were developed in i50propanol-water Barley aleurone layers. Four-day-old germinated corn. One-day-old germinated mung bean. One-day-old germinated Kentucky Nonder bean. Immature seeds of Phaseolus coccineus. MUOCD> CPM CPM CPM CPM CPM 2000. 3H-6A1 1'11“ 1000- v 161 W 300. 3H-GA 150 . H r # T a I v —T'—'_—-=_T-'—'T_"'—‘ 4 8 12 16 300. 3H-GA1 200. I“? 100« H 9* I 4 8j_—‘iT 12' 16 1000. 3H-6A1 mm 500- 4 8 12' 16 1 2000. 3 H-GA1 (”W 1000. I *r u v 4 u v v '8 v r v 1121 t I '16 Section Number 103 coccineus) (Figure 41E). One compound was hydrolyzed by B-glucosi- dase to 6A8 and appears to be a GAB-glucoside. The other product migrated just behind GA1 on the paper chromatogram and was not identified. Kentucky Wonder beans which were imbibing water con- verted 3H-GA1 to metabolites whose properties were identical to those of the compounds fbrmed by the Scarlet runner beans (Figure 410). When mung beans were incubated with 3 H-GA], the phytohormone was converted to one major polar product (Figure 41C), which migrated in a manner similar to M3 of GA1 on paper in system A as well as on thin layer chromatograms in system J (RF 0.15). Upon electrophoresis in ammonium borate buffer, pH 9.3, it migrated in a manner similar to M2 and M3 of GA]. Like M3 of GA] it was not hydrolyzed by B-glucosidase. All of these properties indicate that it may not be a simple glucoside. Thus, it appears that the mung beans probably have a mechanism for metabolizing GA1 that differs from the other two beans studied. The highly polar metabolites (M1-like compounds) were not detected in any other plant tissues studied here; only in aleurone layers. DISCUSSION 1. The Metabolism of the Gibberellins and Hormonal Action The results indicate that GA3 was not converted to any other fbrm that is more active than the applied gibberellin itself. The hormone was inactivated by aleurone layers and its destruction was almost linear with time. Extrapolation of the results of the time course (Figure 12) of the formation of metabolites demonstrated that the hormone was metabolized and inactivated during early stages of incubation. In preliminary results (data not presented) none of the gibberellin metabolites detected in aleurone layers (except GAB) was biologically active in the barley half seed bioassay. It appears that when the metabolites are formed hormonal activity is lost. II. On the Pathways of Gibberellin Metabolism The reduction by GA] and GA7 of the accumulation of radio- activity in barley aleurone layers incubated with ‘4 C-GA3 and the reduction by GA3 of the formation of polar metabolites in layers incubated with 3H-GA1 indicate that the pathways for the metabolism of these gibberellins are similar (Tables 10 and 11). GA7 con- tains only one hydroxyl group, at C-3. If the conjugation site in M2 is C-3 of the GA ring, GA7 will compete with the small amount of labeled giberellin.‘ Therefore, the inclusion of it in the medium 104 105 should inhibit the formation of metabolites from GA3. The possi- bility exists, however, that GA7 might be converted to some unknown compound which in turn inhibits the formation of these metabolites. III. The Effect of ABA on Gibberellin Metabolism The effect of ABA on the production of the polar metabolites appeared to be due to increased uptake of the hormone by aleurone tissues rather than an effect on the formation of a specific metabolite (Tables 7, 8 and 9). Abscisic acid might affect the permeability of the aleurone layers to the hormone. It has been shown that it increases the permeability of carrot root cells and stem discs of Pelargonium to water (Glinka and Reinhold, 1971, 1972). IV. 'The M2 Metabolites A. M2 of G53 The metabolite M2 of GA3 migrated very close to GA3 upon electrophoresis, an indication that it contained one free carboxyl group. It was not hydrolyzed by glucosidases from a variety of sources and its rate of migration in chromatographic systems was less than an authentic GA3-glucoside. M2 of GA3 was hydrolyzed by a preparation of pectinase (whose active component appeared to be B-xylosidase) to give gibberellic acid (Figure 17). Treatment of GA3 and M2 (from GA3) with hot mineral acid gave allogibberic and gibberic acid (Figure 21). Therefore, M2 is a GA3 derivative. This metabolite gave a positive test for hexoses, and a positive test for pentoses with orcinol and phloroglucinol reagents. Radio- active glucose was incorporated into M2 of GA3 (Figure 22); 106 radioactive galactose was not. Partial acid hydrolysis of M2 resulted in the production of a product that migrated slower than hexoses and faster than hexose disaccharides on paper chromatograms; a characteristic feature expected of a disaccharide consisting of a hexose and a pentose. This product of partial acid hydrolysis of M2 migrated in the same manner as the disaccharide, 3-0-8- glucopyranosyl-D-glucose. Complete acid (2.5 N HCl) hydrolysis and subsequent chromatography of the products on paper as well as on thin layers showed that the minor metabolite contained glucose and xylose in apparently equal amounts (Figures 23-26). Xylitol was a major radioactive product obtained after the sequential reaction of M2 of GA3 with periodate, acid hydrolysis and NaBT4 reduction.(Figure 28). It appears that glucose is the terminal sugar in M2 and xylose is in the pyranose form where C-1 of glucose is linked to xylose at C-3 and C-1 of xylose to GA3 (probably at C-3 of the entrgibberellane ring). Thus, it seems likely that the minor metabolite, M2 of GA3, is O-B-D-glucose-(l+3)-O-B-D- xylopyranosyl-(1+3)-O-GA3. To the best of my knowledge, this is the first report that GA3 is conjugated with xylose and glucose. The simple glucosides of gibberellins, such as 3-O-B-glucosyl-gibberellin A3 (Tamura gt_al., l968), 2-O-B-glucosyl-gibberellin A8 (Schreiber §t_gl,, 1967, 1970), ll-O-B-glucosyl-gibberellin 35 (Yamane gt_al,, 1971) etc. and a partially characterized GA1-B-D-glucoside (Hemphill et al., 1973) have been reported as metabolic products of the corresponding gib- berellins in many dicotyledonous plants. 107 B. M2 of GA] and M3' The metabolites M2 and M3 of GA], like M2 of GA3, were not affected by treatment with B-glucosidases although they were hydrolyzed by a preparation of B-xylosidase to GA1 and 6A8, respec- tively. Incubation of 3H-GA8 with aleurone layers gave M3, indi- cating that the M3 of GA1 is a 6A8 derivative (Figure 11). M2 from GA], and M3 (derived from GA1) are probably congeners, along with M2 of GA3. Since none of these metabolites was hydrolyzed by B-glucosidases from various sources and since acid hydrolysis of M2 of GA3 gave xylose and glucose, it appears that these are not simple glucosides. Very likely all of them are glycosides consisting of O-3-B-glucosyl-xylopyranose attached to C-3 of egg-gibberellane ring. The aglycone moieties are gibberellins GA], 6A8 and GA3, respectively. Nadeau gt_§1, (1972) have reported that the material referred to here as M3 (from GA1) is a GAB-glucoside. No evidence was pro- vided by these workers that the products studied by them are simple glucosides nor was the identity of the sugar conjugated with the gibberellins determined. The variations in the activity of B-glucosidases from a variety of sources against gibberellin glucosides have been discussed by Sembdnerial. (1973). The enzyme, B-glucosidase or emulsin isolated from almond and obtained from Darmstadt, Germany, was highly active in hydrolyzing GA1 and 6A8 glucosides; however, B-glucosidase obtained from L. Light & Co., Ltd., England, and cellulase from Aspergillus niger (obtained from Heidelberg) were less active. None 108 of these enzymes gave significant hydrolysis of GA3 glucoside. GA1 and GA8 glucosides were hydrolyzed completely and GA3-glucoside was hydrolyzed partly by the enzyme cellulase, a commercial product from Aspergillus niger. Whether any stereo-chemical differences that may exist between the glucopyranosides of GA1 and GA3 might be responsible for the different results obtained in enzymatic hydroly- sis could not be determined. V. Electrophoretic Properties of the M1 Metabolites The results obtained here indicate that the major highly polar metabolite (M1) is probably charged to the same extent as GA3, i.e., there is probably one free carboxyl group present. Since M] I migrates faster than GA3 on electrophoretic paper in ammonium borate buffer (pH 9.3), the presence of vicinal hydroxyl groups which are capable of forming a borate complex is suggested (Figure 15A). The formation of a borate complex has been reported (Coombe and Tate, 1970) by GA8’and the water-soluble apricot gibberellin A32, both of which contain hydroxyl groups adjacent to each other. The fact that M1 migrates faster than GA3 in electrophoretic buffer E, pH 5.6, towards the anode may be due to the presence of extra hydroxyl or carboxyl groups. It has been observed (Pridham, 1959) that phenolic hydroxyl groups are highly dissociated at higher pH values and have a profound effect on mobilities during electro- phoresis. These compounds, in most instances, migrated faster in alkaline than in acidic buffers because of the increased charge due to hydroxyl dissociation. Gibberellenic acid, which contains two 109 carboxyl groups migrated even faster than M1 in pyridine-acetic acid buffer, pH 5.6 (Figure 15C). M.I from GA1 migrates slightly faster than 6A8 when electro- phoresed in borate buffer, indicating that it is slightly more negatively charged (Figure 16). The migration of M1 towards the cathode at pH 2.2 is prob- ably attributable to the presence of the amino acids (Figure 158). A similar migration pattern for 3H-GA1-X was observed earlier (Nadeau and Rappaport, 1975). Since M1 contains three amino acids, it was assumed that it would migrate faster than serine, glycine and ala— nine upon electrophoresis in the low pH (2.2)Vbuffer. The only simple explanation for its failure to do so is that the slow mobility of M1 may be due to the presence of many hydroxyl groups. Hydroxy lysine migrated slower than lysine. The mobility of some amino acids in this buffer (< = slower than) can be represented in the following manner: dopa GA3-diol ——> —> -->M1 (GA -conjugated wi h amino acids) G"‘3 Glycosidation >:M2 (GA3-glycoside, Glucosyl-xylosyl-GAB) Hydroxylation ( > GA -diol -—> —-> —3‘ M GA1-conjugated ‘ 71 ”,”‘ ‘ with amino acids) I ,I’ GA Hydroxylation /‘,,” l ama’ mcosidation >M3 (GA -glycoside, Glu osyl-xylosyl-GAB) Glycosidation )1M2 (GA1-glycoside, Glucosyl-xylosyl-GA1) Figure 43.--Postulated pathways of gibberellin metabolism in aleurone layers. The significant aspects of this research are probably the following: 1. The revelation that the inactivation of the hormonal activity of the gibberellins in aleurone layers is probably effected by a series of reactions involving glycosidation or hydroxylation/ oxidation, with subsequent glycosidation or conjugation. 2. The finding that there is no apparent relationship between the metabolism of the hormones and their biological effects in layers other than the fact that the gibberellins are inactivated when they are metabolized by the tissue. 118 3. The revelation that, at least in the case of GA3, the gibberellins are glycosidated with glycosyl xylose and that glycine, alanine and serine are attached to a metabolite. This basic informa- tion should be invaluable in efforts to establish, unequivocally, the structure of the metabolites. 4. 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