STUDIES ON THE VARIETAL SUSCEPTIBILITY IN WINTER WHEAT TO SUBSTITUTED PHENYLUREA HERBICIDES A Thesis submitted by REZA EMAMI-SARAVI a candidate for the degree of Doctor of Philosophy in BIOCHEMISTRY Department of Biochemistry Royal Holloway College University of London Egham Hill Egham January 1979 Surrey ProQuest Number: 10097467 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10097467 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 (i) ACKN OWLEDGEMENTS My sincere thanks and gratitude must go to my supervisor. Dr. W.J. Owen, for introducing me to such an interesting topic, for his help, encouragement, and patience throughout my research, and for his guidance in the preparation of this thesis. Secondly, I wish to thank Professor J.B. Pridham and Dr. W.A. Stevens for their interest, advice and helpful discussions throughout the duration of this work. My gratitude is also extended to all other members of staff and to students of this College who have helped me either practically or theoretically during my postgraduate research and in the preparation of this thesis. I am extremely grateful to Mrs. Valerie Owen for her care and enthusiasm throughout the typing of the manuscript, and to Mr. David Ward for the preparation of the photographic plates. Finally, I wish to give special thanks to my parents, without whose financial support and encouragement this thesis would not have been possible. (il) ABSTRACT The studies reported in the present thesis have been concerned with the biochemistry of the varietal susceptibility of winter wheat (Triticum aestivum L .) cultivars to the substituted phenylurea herbicide, metoxuron(N '-(3-chloro-4-methoxyphenyl)-N ,N-dimethylurea). The introduction includes an historical account of the substituted phenylureas and reviews of the relationships between structure and phytotoxicity and mechanism of action. In addition selective properties of phenylureas are discussed together with their metabolic fate not only in plants but also in microorganisms and mammalian systems. .Present concepts of the photosynthetic process are also outlined with special emphasis on those aspects directly pertinent to the mode of action of the substituted phenylureas. Studies of Hill activity of isolated chloroplasts incubated with metoxuron showed a similar inhibition of DCPIP and potassium ferricyanide photoreductions in both resistant (Capelle Desprez, Gama) and susceptible (Maris Nimrod, Maris Huntsman) plants. Examination of the Hill activity of chloroplasts isolated from herbicide-treated plants showed a greater inhibition of DCPIP and ferricyanide photoreductions in susceptible compared with resistant varieties. Whereas a severe inhibition of Hill activity in susceptible varieties was followed by scorching and eventual death of the plant, tolerant cultivars recovered from an initial slight visible injury in parallel with a return of Hill activity to normal levels. (iii) A reduction in the proportion of ethanol soluble material of leaves was also observed 24 h following treatment of wheat plants with metoxuron, the susceptible varieties showing the greater decrease. The distinction between resistant and susceptible varieties was more apparent when plants that had contacted the herbicide for longer periods were investigated. 14 These results were also reflected in a greater inhibition of QO^ fixation by metoxuron-treated leaves of susceptible plants compared to similarly treated resistant strains. Studies of absorption and translocation of (methoxy-^^C)-metoxuron in resistant and susceptible wheat varieties, indicated that a differential rate of herbicide uptake may represent a factor which contributes to the observed difference in varietal response. A subcellular organelle localisation study of absorbed (methoxy-^^G)-metoxuron indicated that on the basis of protein content the greatest amounts of radioactivity were associated with the chloroplast fraction. In addition, chloroplasts from susceptible varieties contained a quantity of metoxuron several fold greater than those of resistant plants. Metabolism studies made using (methoxy-^^C)-metoxuron indicated that a major degradative pathway in wheat involves a two-step N-dealkylation followed by hydrolysis of the ureido group to give the corresponding aniline derivative. The various metabolites were separated by thin- layer chromatography and their identity was revealed by a comparison of their values with those of known synthesised standards. Time course studies indicated that metoxuron metabolites represented a greater proportion of the radioactivity of the total methanol extract from (iv) resistant plants than from susceptible types. The metabolic route outlined was shown to result in an effective detoxication of metoxuron in in vitro assays of Hill reaction activity where the N-monodesmethylated derivative was found to be some 80% as inhibitory as metoxuron and the N-bisdesmethyl and aniline derivatives were ineffective as photosynthetic inhibitors. Experiments made using leaf discs contained in Warburg flasks in the presence of a COg trap demonstrated a loss of from (methoxy-^^C)- metoxuron indicating that cleavage of the methoxy substituent of the aromatic ring represents a significant additional step in the metabolism of the herbicide. Cleavage of the methoxy group occurred at an appreciably greater rate in leaf discs of resistant compared to susceptible plants. Preliminary experiments would indicate that metabolism of metoxuron by wheat leaves is achieved by the microsomal fraction of the cells which contains an N-demethylase activity requiring molecular oxygen and reduced pyridine nucleotide as co-factors. This activity was most apparent in the microsomal fraction prepared from leaves of resistant varieties. The various experimental results are discussed in terms of their significance in accounting for the differences in response to metoxuron of the wheat varieties studied. (v) ABBREVIATIONS The abbreviations used in this thesis are those listed in the Biochemical Journal (1976) 153, 1-21 (revised, 1978, 169, 1-27) with the following additions : CCP carbonylcyanide phenylhydrazone CMÜ N *-(3-chloro-4-phenyl)-N ,N-dimethylurea DAD diaminodurene(2,3,4,5-tetramethyl-£-phenylenediamine) DBMIB 2,5-dibromo-3-methyl-6-isopropylbenzoquinone DCMU N *-(3,4-dichlorophenyl)-N ,N-dimethylurea DCPIP 2,6-dichlorophenolindophenol DPC diphenylcarbazide Fd ferredoxin Fe-S bound iron-sulphur protein Fp ferredoxin-NADP reductase (flavoprotein) FRS ferredoxin reducing substance HEPES N -2-hydroxyethyl piperazine-N-2-ethanesulphonic acid MES 2-(N-morpholino)-ethane sulphonic acid PC plastocyanin PD £-phenylene diamine PMS phenazine methosulphate PQ plastoquinone PPG 2,5-diphenyloxazole PSl photosystem 1 PS2 photosystem 2 TMPD N,N,N',N'-tetramethyl-p-phenylenediamine Tricine N-tris(hydroxymethyl)methyl glycine CONTENTS Page Acknowledgements i Abstract ii Abbreviations v Chapter 1: INTRODUCTION A. The Substituted Urea Herbicides The advent of the herbicide era 1 The substituted phenylureas 4 Relationship between structure and herbicidal activity 6 Mode of action 10 Selective action of substituted phenylurea herbicides on plants 31 Molecular fate of the substituted phenylurea herbicides 42 B. Photosynthesis in Higher Plants The Light Reaction of Photosynthesis 57 The electron acceptor of photosystem 2 60 Artificial electron acceptors 62 The electron donor to photosystem 2 64 Artificial electron donors 66 Function of cytochrome b^^g in photosystem 2 68 The intermediate electron transport chain - Electron transport from photosystem 2 to photosystem 1 72 The electron acceptor of photosystem 1 76 Cyclic electron flow mediated by photosystem 1 79 Carbon Dioxide Assimilation in Photosynthesis 80 Page Chapter 2; MATERIALS AND METHODS Growth of wheat varieties 87 Application of herbicides to intact plants 89 Isolation of chloroplasts 89 Application of substituted phenylurea herbicides and their metabolites to isolated chloroplasts 94 Determination of chlorophyll 95 Isolation of mitochondria 96 Protein determination 97 Experimental methods for study of the light reaction in photosynthesis Spectrophotometric assays of electron transport in chloroplasts 98 Assay of potassium ferricyanide photoreduction 101 Assays involving the use of the oxygen electrode 102 Principles involved in the polarographic measurement of O 2 concentration 102 Calibration of the instrument 103 Calculation of change in oxygen concentration 104 Measurement of photosynthetic electron transport by oxygen evolution 105 Assay of cyclic photophosphorylation in chloroplasts 106 Determination of inorganic phosphate 108 Studies on the respiratory properties of isolated wheat mitochondria 109 Carbon Dioxide Assimilation Studies ^^COg fixation by whole wheat plants 110 ^^COg fixation by a single leaf 114 Isolation and estimation of ethanol-soluble carbohydrates from wheat 115 Paper chromatography of the ethanol-soluble carbohydrate fraction from wheat leaves 117 Experimental methods