AN ABSTRACT OF THE THESIS OF Paul Aikens Jones for the degree of Master of Science in Microbiology presented on November 29, 1984. Title: The Biosynthesis of Ethylene from Methionine in Saccharomyces cerevisiae. Redacted for Privacy Abstract approved: °Ferro Methionine metabolism, leading to the biosynthesis of ethylene in Saccharomyces cerevisiae does not appear to involveS-adenosyl- methionine (SAM) and aminocyclopropanecarboxylic acid(ACC), based on the lack of ethylene synthesiswhen both SAM and ACC were incubated in the presence and absence of cells. Thus, the mechanism leading to the formation of ethylene from methionine inSaccharomyces cerevisiae is different than that of higher plants. Incubation of cells without methionine included in the reactionmixture formed no appreciable amounts of ethylene, also, theoNceto(KMB) analogue of methionine produced more ethylene than methioninesupplementations in both the absence and presence of cells, andthe decarboxylated product of KMB (methional) accumulatedapproximately four times more than this. There was also no appreciable ethyleneaccumulation when thea-hydroxy (HMB) analogue of methionine wasutilized as a precursor. The determining factors in theaccumulation of ethylene from methionine and other substrates werethe concentration of substrates and glucose, along with the pH of reaction mixture. The presence or absence of light had a definate effect on ethylene production also, showing no signs of accumulation if reaction mixtures were incubated in the dark. In the light, the only medium component needed for ethylene production from substrates was riboflavin. Radiolabeled methionine in the 2,3-4, and methyl positions revealed the accumulation of ethylene products from carbon numbers 3 and 4 of methionine. The treatment of cell and medium extracts with dansyl hydrazine and 2,4-dinitrophenylhydrazine showed the build up of an aldehyde and/or ketone derivative, with the ketone showing the highest accumulations in the culture medium. Inhibition of ethylene synthesis occured when chloramphenicol, adenosine and azaserine were added to methionine supplemented cultures. Thus, the possibility that protein synthesis is occuring while ethylene synthesis is taking place is supported, along with the need for hydroxyl radicals, which are scavenged if adenosine is present. The inhibition of ethylene synthesis from methionine supple- mented cultures with the addition of azaserine imply that transami- nation is also involved in ethylene biosynthesis from methionine in Saccharomyces cerevisiae. Copyright by Paul Aikens Jones November 29, 1984 All Rights Reserved The Biosynthesis of Ethylene fro!Methionine in Saccharomyces cerevisiae by Paul Aikens Jones A Thesis submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science CompletedNovember 29, 1984 Commencement June 1985 APPROVED: Redacted for Privacy Associate P7fessfor of Microbiology in Charge of Major Redacted for Privacy Chaf4an of the D/ artment of Microbiology Redacted for Privacy Dean of Grade to School Date thesis is presented November 29, 1984 Typed by Peggy Hammer for Paul Aikens Jones ACKNOWLEDGEMENT I would like to extend special thanks to Dr. Al Ferro and all the personnel who have performed research in his laboratory. They have contributed much academic stimulation which aided me in my construction of a productive research program. Special thanks is also extended to Dr. Leong, Dr. Gamble, Dr. V. Brooks, Rick Parker, Manley Wong, members of Dr. Leong's laboratory, and all the members of the Microbiology Department. Finally, special thanks to my Mother, Father and Son, for without their love and support, this thesis would not have been possible. TABLE OF CONTENTS INTRODUCTION 1 MATERIALS AND METHODS 8 Strains and Culture conditions 8 Radioactive Tracer Experiments 8 Dansyl hydrazine Derivatives of Aldehydes and Ketones 10 2,4-Dinitrophenyl Hydrazone Quantification of Aldehydes and Ketones 10 Gas Chromatography 11 Anaerobic Investigations of Ethylene Synthesis 11 Chemicals 12 RESULTS 13 Ethylene Standard Curve 13 Effect of Methionine on Ethylene Production by S. cerevisiae 13 Dose Response Curve to Methionine 16 Time of Incubation vs Ethylene Production 16 Effect of Methionine on Cell Growth 21 Ethylene Synthesis at Various Stages of Growth 26 Effect of Various Compounds on Ethylene Production 26 Effect of Various Concentrations of Glucose on Ethylene Synthesis 29 Effects of HMB, KMB and Methional on Ethylene Production 29 Optimum Concentrations of HMB, KMB and Methional 33 Effect of pH on Ethylene Synthesis by KMB Supplementations 39 Effect of Light and Dark on Ethylene Synthesis 46 Ethylene Synthesis with Transfer of Cells from a Light to a Dark Environment 49 Role of Riboflavin in Ethylene Formation 52 Role of Riboflavin and Methionine in Ethylene Synthesis 56 Role of Methionine and Riboflavin in Ethylene Synthesis by 37018 in the Dark 56 Ethylene Synthesis in the Presence of Riboflavin and Dark 59 Accumulation of Ethylene in an Anaerobic Environment 59 Specificity of Mercuric Perchlorate for Absorption of Ethylene 62 Which Carbons of Methionine Give Rise to Ethylene Carbons 62 Inhibition of Ethylene Synthesis from L-D,414g_ Methionine by HMB, KMB and Methional 63 Radiolabeled Ethylene in the Presence of Boiled Cells 66 Accumulation of Products During Ethylene Synthesis 69 Aldehyde and Ketone Derivatives from Dansyl Hydrazine and 2,4-Dinitrophenyl Hydrazine 70 Elevated Induction of Ethylene Synthesis by Increasing Light Intensity 78 Inhibition of Transamination (Azaserine), Protein Synthesis (Chloramphenicol) and Electron Quenching (Adenosine) During Ethylene Formation 81 DISCUSSION 85 BIBLIOGRAPHY 102 LIST OF FIGURES Figure Page 1 Methionine metabolism and its relation to ethylene biosynthesis 2 2 The methionine transamination pathway 4 3 Standard curve of ethylene as determined by dilution volumes 14 4 Ethylene accumulation in three strains (3701B, R3720 and 7752) of S. cerevisiae after an eleven hour incubation period 17 5 Dose response curve to methionine 19 6 Time of incubation vs ethylene production in S. cerevisiae 22 7 Effect of methionine on cell growth 24 8 Ethylene synthesis at various stages of cell growth 27 9 Effect of various concentrations of glucose on ethylene synthesis 31 10 Optimum concentration of HMB yielding maximum ethylene production 35 11 Optimum concentrations of KMB yielding maximum ethylene production 37 12 Ethylene production from methional supplemented medium in the presence and absence of cells 40 13 Effect of pH on ethylene synthesis by KMB supplementation 42 14 Ethylene formation from methionine supplemented cultures as a function of pH 44 15 Effect of light and dark on ethylene formation 47 Figure Page 16 Ethylene synthesis when transfered from a light to a dark environment 50 14 17 Accumulation of medium products from methionine during ethylene synthesis 71 ] 18 Accumulation of medium products fromL13,4 14C- methionine during ethylene synthesis 73 19 Aldehyde and ketone derivatives from dansyl hydrazine and 2,4-dinitrophenyl hydrazine 76 20 Stimulation of ethylene synthesis by varying the intensity of light environment 79 21 Inhibition of ethylene synthesis by chloramphenicol, adenosine and azaserine 83 LIST OF TABLES Table Page 1 Effect of various compounds on ethylene production 30 2 Effects of HMB, KMB and methional on ethylene production 34 3 Role of riboflavin in ethylene formation 53 4 Role of riboflavin in ethylene formation 54 5 Role of riboflavin and methionine in ethylene synthesis by 3701B in light vs. dark 57 6 Ethylene production in the presence of riboflavin and dark 58 7 Ethylene accumulation under aerobic and anaerobic environmental conditions 60 8 Specificity of mercuric perchlorate for the absorption of ethylene 64 9 Which carbons of methionine give rise to ethylene 65 10 Inhibition of ethylene synthesis by non- labeled HMB, KMB and methional 67 11 Ethylene production from boiled cells 68 The Biosynthesis of Ethylene from Methionine in Saccharomyces cerevisiae INTRODUCTION Ethylene is a plant hormone (11,56,57), that is also produced by some bacteria (5,18,34,44), fungi (8,9,12,17), yeast (12,18) and mammalian cells (24,29,30,31,33). Precursors of ethylene, from these various biological systems, include methionine, alpha-keto-gamma- methylthiobutyric acid (KMB), alpha-hydroxy-gamma-methylthiobutyric acid (HMB), 3-methylthiopropionaldehyde (methional), S-adenosyl- methionine (SAM), and 1-aminocyclopropane-l-carboxylic acid (ACC) (4,24,30). In over 90% of the above cases examined, methionine was the most consistant precursor of ethylene synthesis. In plants, the mode of methionine metabolism resulting in the formation of ethylene is well known (2,11). However, in mammalian, yeast and bacterial cells, the mechanism for ethylene synthesis continues to be a source of controversy among many scientists in this area. In higher plants, ethylene formation from methionine, via SAM and ACC as intermediates, has been reported by Adams and Yang (62). Ammonia and carbon dioxide were coproducts along with the generation of ethylene. In their most recent investigations, Adams and Yang (65) utilizedLiMethylqmethionineandL-P5gmethionineto show that the methylthio group is recycled to methionine via methylthio- ribose (MTR). Kushad et al(75) found that in mature avacado fruits and tomato, MTR is phosphorylated to methylthioribose-l-phosphate (MTR -1- phosphate) prior to being metabolized to KMB. Figure 1 shows 2 CO2 O NH3 cH3-s-cH2-cH2-CH-000