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Studies on the Metabolism of Abscisic Acid Studies on the Metabolism of Abscisic Acid A thesis submitted for the degree of Doctor of Philosophy by Gary Thomas Vaughan School of Biochemistry University of New South Wales May, 1986 ii Declaration The work reported in this thesis was carried out between March 1981 and May 1986 on a part-time basis while I was employed as a full-time Professional Officer in the School of Biochemistry, University of New South Wales. This work represents original research which has not been submitted for examination for any other degree. All work was executed by the author unless otherwise acknowledged. Gary Vaughan iii Acknowledgements I wish to express sincere thanks to my supervisor, Professor B. V. Milborrow for providing me with the opportunity to undertake post-graduate studies while employed as a Professional Officer in his laboratory. I am grateful for his advice and guidance during the course of this work. I have benefited greatly from his considerable expertise and experience in the field and his infectious enthusiasm for research. I also acknowledge the contribution made by the following people: Associate Professor K.D. Barrow and Dr. A.G. Netting, for assistance and advice as co-supervisors while Professor Milborrow was on study leave. Dr. A.G. Netting, for his expertise and advice on gas chromatography and HPLC; and for constructive discussions and suggestions. Mr. D. Bourne, for his advice and assistance with the analysis of sugars as TMS-0-methyloximes. Professor Sir J.W. Cornforth, F.R.S., for his suggestions on the mechanism of the interconversion and oxidation of the 1' ,4'-diols of ABA. iv Dr. A.M. Duffield and Mr. R.O. Lidgard (Biomedical Mass Spectrometry Unit), for mass spectra. Dr. J. Saunders, Dr. K. Cross and Mr. G. Grossman, for 1H NMR spectrometry. Finally, I wish to acknowledge the patience and support of my wife, Sharon, especially during the preparation and typing of this manuscript; and the sacrifices unknowingly made by my two sons. V Summary The metabolism of the plant hormone, abscisic acid (ABA) has been investigated in higher plants. A procedure for the preparation of R- and S-ABA from racemic ABA was developed using high-performance liquid chromatography (HPLC) on optically-active columns. This method was used throughout this work to analyse the proportion of R- and S-enantiomers in samples and to prepare radiolabelled R- and S-ABA. A major, polar metabolite of ABA was isolated from shoots of Lycopersicon esculentum and was characterized as 4'-0-~-D-glucopyranosyl dihydrophaseic acid (DPAGS). This metabolite was produced from ~-ABA, phaseic acid and dihydrophaseic acid but not R-ABA or epi-DPA. The 1' ,4'-cis- and 1' ,4'-trans-diols of ABA were identified as metabolites of exogenous, racemic abscisic acid and were formed from R- and ~-[2-14c]ABA. In addition, the 1' ,4'-trans-diol was isolated as an endogenous constituent of pea (Pisum sativum) shoots and avocado (Persea americana). It was also released from extracts of the latter by basic hydrolysis. vi The 1' ,4'-cis- and trans-diols were found to interconvert and to be oxidized to ABA in solution on standing. Inversion was more rapid at low pH values and occurred by inversion at the C-4' and C-1' chiral centres. The inverted hydroxyl groups were labelled with 180 from H218o. The 4'-glucoside and the glucose ester of (+)-trans­ diol were isolated and characterized. The conjugates were also formed from (-)-trans-diol. The conjugates of the (+)- and (-)-trans-diol were separable by HPLC. When low concentrations of the (+)-trans-diol were fed most was converted into DPAGS. The absence of deuterium in DPAGS formed from [4'-2HJ-trans-diol indicated that an intermediate 4'-ketone was involved. The glucose ester of the 1' ,4'-cis-diol of ABA was also isolated and characterized as a metabolite of exogenous cis-diol. vii Abbreviations ABA abscisic acid Ac acetylated ABAGE abscisic acid-P-D-glucosyl ester ABAGS 1'-0-abscisic acid P-D-glucoside ax. axial BBOT 2,5-bis[5-t-butyl-benzoxazol-2-yl]thiophene BHT butylated hydroxytoluene CI chemical ionization COSY two-dimensional homonuclear correlated spectrum cpm counts per minute d doublet DPA dihydrophaseic acid DPAGS 4'-0-dihydrophaseic acid P-D-glucoside dpm disintegrations per minute ECD electron capture detection epi-DPA epi-dihydrophaseic acid eq. equatorial FID flame ionization detection GC gas chromatography GC-MS combined gas chromatography-mass spectrometry HMG-HOABA P-hydroxy-p-methylglutarylhydroxyabscisic acid HPLC high-performance liquid chromatography HPTLC high-performance thin layer chromatography IAA indole-3-acetic acid i.d. inner diameter IR infra-red m multiplet Me methyl viii MeAc methylated and acetylated MS mass spectrometry m.p. melting point m.w. molecular weight NMR nuclear magnetic resonance PA phaseic acid PPO 2,5-diphenyloxazole RF ratio of travel of compound to travel of solvent front s singlet t triplet TLC thin-layer chromatography TMS trimethylsilyl UV ultra-violet ix Publications Publications and abstracts arising from work presented in this thesis Publications Milborrow, B.V. and Vaughan, G.T. (1982) Characterization of dihydrophaseic acid 4'-O-~-D-glucopyranoside as a major metabolite of abscisic acid. Aust. J. Plant Physiol. 1, 361-72. Vaughan, G.T. and Milborrow, B.V. (1984) The resolution by HPLC of RS-[2-14c]Me l' ,4'-cis-diol of abscisic acid and the metabolism of (-)-R-- and (+)-S-abscisic- acid. J. Exp. Bot. 35, 110-120. Vaughan, G.T. and Milborrow, B.V. (1984) Resolution of RS-abscisic acid and the separation of abscisic acid metabolites from plant tissue by high-performance liquid chromatography. J. Chromatogr. 336, 221-228. Vaughan, G.T. and Milborrow, B.V. (1986) The chemistry and occurrence of the l' ,4'-diols of abscisic acid. In preparation. X Vaughan, G.T. and Milborrow, B.V. (1986) The metabolism of the 1' ,4'-diols of ABA. In preparation. Abstracts of papers presented Vaughan, G.T. and Milborrow, B.V. (1981) Conjugation of metabolites of abscisic acid. XIIIth International Botanical Congress.-Abstracts, 232. Vaughan, G.T. and Milborrow, B.V. (1983) Resolution by HPLC of RS-[2-14cJabscisic acid and metabolism of the enantiomers. Proc. Aust. Biochem. Soc. 15, 58. Milborrow, B.V. and Vaughan, G.T. (1984) The formation of the 1' ,4'-trans-diol from RS-[2-14cJabscisic acid. Proc. Aust. Biochem. Soc. 1:..&_, 66. Vaughan, G.T. and Milborrow, B.V. (1984) Resolution of RS-abscisic acid and the separation of abscisic acid metabolites from plant tissue by high-performance liquid chromatography. International Symposium on HPLC in the Biological Sciences, Melbourne, 1984-Abstracts, 143. xi Contents Declaration ii Acknowledgements iii Summary v Abbreviations vii Publications ix 1. GENERAL INTRODUCTION 1.1 DISCOVERY AND CHARACTERIZATION 2 1.2 NOMENCLATURE AND ISOMERIZATION 4 1.2.1 Nomenclature 4 1.2.2 Isomerization 6 1.3 OCCURRENCE 7 1.4 PHYSIOLOGICAL EFFECTS OF ABA 8 1.4.1 Seed dormancy 8 1 . 4 . 2 Bud dormancy 9 1. 4. 3 Abscission 10 1.4.4 Root geotropism 11 1.4.5 Response to stress 12 1.4.6 Stomatal closure 13 1.4.7 Effects on nucleic acid and protein 13 synthesis 1.4.8 Other effects of ABA 14 1.5 THE BIOSYNTHESIS OF ABA 14 1.5.1 Carotenoid pathway of biosynthesis 16 1.5.2 Biosynthesis of ABA in fungi 20 1.6 CATABOLISM OF ABA 22 1.6.1 Oxidative degradation 23 1.6.1.1 8'-Hydroxyabscisic acid 23 xii 1.6.1.2 Phaseic acid 27 1.6.1.3 Dihydrophaseic acid and 28 epi-dihydrophaseic acid 1.6.1.4 7'-Hydroxyabscisic acid 29 1.6.1.5 The 1' ,4'-trans-diol of ABA 29 1.6.1.6 Other metabolites 30 1.6.2 Conjugation 31 1.6.2.1 Abscisic acid glucose ester 31 1.6.2.2 1'-O-abscisic acid-P-D- 33 glucopyranoside 1.6.2.3 3-Hydroxy-3-methylglutaryl-8'- 33 hydroxy ABA 1.6.2.4 Dihydrophaseic acid glucoside 33 1.6.2.5 Other conjugates 34 1.6.3 Bacterial metabolism of ABA 35 1.6.4 Pathway of ABA metabolism 36 1.6.5 Further studies of ABA metabolism - 37 the present investigations 2. CHARACTERIZATION OF DIHYDROPHASEIC ACID 4'-~-D-GLUCOPYRANOSIDE 2.1 INTRODUCTION 39 2.2 MATERIALS AND METHODS 41 2.2.1 Chemicals 41 2.2.2 Plant material and feeding 41 2.2.3 Preparation of PA, DPA and epi-DPA 42 2.2.4 Extraction and partial purification 42 of metabolites 2.2.5 Preliminary chromatography on c18 43 Sep-Pak cartridges 2.2.6 High-performance liquid chromatography 43 xiii 2.2.7 Separation of metabolites by HPLC 44 2.2.8 Preparation of derivatives 44 2.2.8.1 Acetylation 44 2.2.8.2 Methylation 45 2.2.8.3 Oxidation with Jones' reagent 45 2.2.9 Thin-layer chromatography 46 2.2.10 Isolation of DPAGS 46 2.2.11 Melting point 47 2.2.12 Liquid scintillation counting 47 2.2.13 Autoradiography 47 2.2.14 Electrophoresis 48 2.2.15 Hydrolysis of DPAGS 48 2.2.17 Gas chromatography 49 2.2.16 a- and ~-Glucosidase assays 49 2.2.18 Chemical-ionization mass spectrometry 50 2.2.19 Nuclear magnetic resonance (NMR) 51 spectroscopy 2.3 RESULTS 51 2.3.1 Preliminary clean-up on Sep-Pak 51 cartridges 2.3.2 Separation of metabolites by HPLC 52 2.3.3 Extraction of DPAGS 53 2.3.4 Thin-layer chromatography 53 2.3.5 Isolation of DPAGS 56 2.3.6 Electrophoresis 57 2.3.7 Ultraviolet spectroscopy.
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