Studies on the Metabolism
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. 57 2.3.8 Mass Spectrometry 58 2.3.9 NMR Spectroscopy 61 2.3.10 Hydrolysis of DPAGS 65 xiv
2.3.11 Identity of the sugar moiety 65
2.3.12 Metabolism of degradation products 67 of [ 14c] ABA
2.4 DISCUSSION 69
2.4.1 Occurrence 69
2.4.2 Identity of the aglycone 69
2.4.3 The sugar moiety 70
2.4.4 Site of attachment of the glucosyl 70 residue
2.4.5 Relationship between DPAGS and 73 metabolites isolated by others
3. RESOLUTION BY HPLC OF RS-Me-cis-DIOL OF ABA AND METABOLISM OF R.- AND S-ABSCISIC ACIDS
3.1 INTRODUCTION 76
3.2 MATERIALS AND METHODS 80
3.2.1 Isolation of (+)-1-ABA 80
3.2.2 Preparation of Me-1'-,4'-cis- and 80 Me-1' ,4'-trans-diol of ABX--
3.2.3 Resolution of RS-Me-1'-,4'-cis-diol 81 of ABA - --
3.2.4 Improved resolution procedure 83
3.2.5 An improved oxidation using pyridinium 83 chlorochromate
3.2.6 Plant feeding and extraction of 84 metabolites
3.2.7 Thin-layer chromatography 84
3.2.8 Determination of the proportion of 85 R-[ 14c]ABA to S-[ 14c]ABA in ABA - - and its conjugates xv
3.3 RESULTS 85 3.3.1 Resolution of the enantiomers of 85 Me-cis-diol of ABA 3.3.2 Improved resolution and oxidation 87 procedure
3.3.3 Metabolism of R- and S-[2-14c]ABA 89
3.3.4 Proportion of R- and S-ABA in 92 conjugates.
3.4 DISCUSSION 94
4. THE CHEMISTRY, NATURAL OCCURRENCE AND METABOLISM OF THE 1' ,4'-DIOLS OF ABA
4.1 INTRODUCTION 99 4.2 MATERIALS AND METHODS 105
4.1.1 Plant materials 105 4.2.2 High-performance liquid chromatography 106 4.2.3 1' ,4'-cis- and 1' ,4'-trans-diols of 107 ABA as products of [2-14c]ABA
4.2.4 Isolation of 1' ,4'-cis- and 1' ,4'- 108 trans-diol of ABA
4.2.5 Measurement of endogenous 110 concentrations of ABA and 1' ,4' trans-diol of ABA
4.2.6 Interconversion of 1' ,4'-cis-diol of 112 ABA and 1' ,4'-trans-diol of ABA
4.2.7 Preparation and isomerization of 113
1' ,4'- [l 1 - 18o]trans-diol of ABA xvi
4.2.8 Isomerization of the cis- and trans- 113 diols in [18o]water
4.2.9 Preparation of [4 1 - 18o]cis- 114
and [4 1 - 18o]trans-diol of ABA
4.2.10 Isomerization of (+)- and (-) 114 [2-14c]trans-diol
4.2.11 The metabolism of 1' ,4'-[2-14c] 114 trans-diol of ABA
4.2.12 Thin-layer chromatography of 1' ,4'- 115 trans-diol metabolites and their derivatives
4.2.13 Derivatization and purification of 116 the metabolites of 1' ,4'-trans-diol of ABA
4.2.14 NMR spectroscopy 117
4.2.15 Chemical-ionization mass spectrometry 118
4.2.16 Hydrolysis of the diol conjugates and 119 identification of the sugar residue as the TMS-O-methyloxime derivative
4.2.17 Reduction of ABAGE to form the glucose 120 esters of 1' ,4'-cis- and 1' ,4'-trans- diol of ABA --
4.2.18 Metabolism of (-)-1' ,4'-[2-14c]trans- 120 diol of ABA and (+)- 1' ,4'-[2-14c] trans-diol of ABA
4.2.19 Metabolism of (-)-[ 14c]- and (+) 121 [3H]-cis- and trans- diols of ABA
4.2.20 Metabolism of (+/-)-1' ,4'-[2-14c,4'-2H] 122 trans-diol of ABA and (+/-)-[G-3H]ABA
4.2.21 Metabolism of (+/-)-1' ,4'-[2-14c]cis- 122 diol of ABA xvii
4.3 RESULTS 123
4.3.1 Reduction of [2-14c]ABA by various 123 higher plant species
4.3.1.1 The proportion of the cis- and trans- 128 diol derived from R-[2-14c1 and~ [2-14c]ABA by three plant species
4.3.2 The natural occurrence of the 1' ,4'- 129 trans-diol of ABA
4.3.3 Stability of the 1' ,4'-diols of ABA 131
4.3.3.1 The site of inversion during 135 interconversion of the diols
4.3.3.2 Mass spectrometry of the diols 135 of ABA
4.3.3.3 The interconversion of [1'-1801- 139 labelled 1' ,4'-diols of ABA
4.3.3.4 The stability of the 4'-hydrogen 139 atom during diol interconversion
4.3.3.5 Interconversion of the diols in 141 [l80]H20
4.3.3.6 Inversion of the 1'-hydroxyl group 141 during diol interchange
4.3.4 Metabolism of the 1' ,4'-trans-diol 144 of ABA
4.3.4.1 Isolation of metabolites 148 4.3.4.2 Ultra-violet spectroscopy 149 xviii
4.3.4.3 Characterization of compounds 149 I and J.
( a) Mass spectrometry 153
(b) NMR spectroscopy 155
(C) The glucosyl residue 159 (d) The aglycone 161
( e) Hydrolysis of the 4'-glucoside 162 of trans-diol
4.3.4.4 Characterization of compounds G 162 and H
(a) Reduction of ABAGE to the 164 glucose esters of 1' ,4'-cis and 1' ,4'-trans-diol of ABA
(b) Mass Spectrometry 164
(c) NMR Spectroscopy 168
4.3.4.5 The Metabolism of (+)- and (-) 172 trans-diol of ABA
4.3.4.6 Conversion of the 1' ,4'-trans-diol 176 into the 4'-glucoside of dihydrophaseic acid
4.3.5 Metabolism of the 1' ,4'-cis-diol of ABA 178
4.3.5.1 The glucose ester of 1' ,4'-cis-diol 180 of ABA
4.4 DISCUSSION 184
4.4.1 The 1' ,4'-diols of ABA as metabolites 184 of exogenous ABA and as endogenous constituents of higher plants xix
4.4.2 The stability and interconversion of 187 the diols
4.4.3 Metabolism of the 1' ,4'-trans-diol 192 of ABA
4.4.4 Metabolism of the 1' ,4'-cis-diol of ABA 193
4.4.5 The conversion of the 4'-dihydro- 195 abscisates into the 4'- glucoside of dihydrophaseic acid
4.4.6 Biological activity of the 1' ,4'- 198 diols of ABA
5. REFERENCES 200 1
General lntrod uction 2
1.1 DISCOVERY AND CHARACTERIZATION
The ancient Greeks were aware of an effect of ABA, the inhibition of seed germination, at about 200 B.C.
Theophrastus (cited by Milborrow, 1969a) tells of the practice of the people of Philippi who propagated native roses collected on Mount Pangaeus from cuttings rather than by seed because germination was slow. We now know that rose fruits are a rich source of ABA.
Research on endogenous inhibitory compounds had to wait for attention to be given to the study of growth promoting properties of auxins and extracts that inter fered with auxin-induced responses. Bennet-Clark et al.
(1952) used paper chromatography combined with wheat coleoptile growth bioassay when they were looking for auxin in their plant extracts. One area of their chromatograms was found to slow or prevent growth of
coleoptiles - they called this fraction "inhibitor-~"
This fraction was found in a wide range of plants and
other workers noted that extracts of dormant tubers and
buds showed higher growth-inhibitory activity than did
extracts from equivalent non-dormant tissue. Milborrow
(1967) used wheat embryo growth inhibition and optical
rotatory dispersion to measure the concentrations of the 3
recently discovered (+)-abscisic acid (ABA) in inhibitor
~ fractions. He discovered that the inhibitory activity of these fractions could largely, if not entirely, be accounted for by the ABA present.
The discovery of abscisic acid resulted from concurrent work in independent laboratories concerned with different research projects. In California,
Addicott and coworkers isolated a growth inhibitory compound they named "abscisin II" which was associated with the abscission of young cotton fruit. It was iso lated as crystals (Ohkuma et al., 1963) and a structure was proposed (Ohkuma et al., 1965) and confirmed by synthesis shortly afterwards (Cornforth et al., 1965a).
Previously, Wareing's group at Aberystwyth had begun investigating the role of inhibitory substances and their relationship to the dormancy of woody plants. An inhibitor of coleoptile growth was present in extracts of dormant buds of sycamore whose titre was correlated with the depth of dormancy of the buds. This dormancy factor, named "dormin", was isolated from sycamore leaves at the
Milstead Laboratory of Shell Research Ltd. Cornforth et al. (1965b) isolated the active substance in crystalline
form and showed it was identical to abscisin II. About the same time, Rothwell and Wain (1964), following up preliminary studies by Van Steveninck, isolated a sub
stance from lupin fruit which was responsible for the
abscission of immature fruit and was found to be 4
identical to abscisin II (Porter and Van Steveninck,
1966; Koshimizu et al., 1966; Cornforth et al., 1966).
Some confusion began to develop over the terms
"dormin" and "abscisin II" so the investigators of
Addicott's, Wareing's and Cornforth's groups met at the
1967 Sixth International conference on Plant Growth
Substances in Ottawa and agreed upon the name abscisic acid (Addicott et al., 1968).
1.2 NOMENCLATURE AND ISOMERIZATION
9'
2 5'
COOH 0 7'
[1] (+)-,§-abscisic acid
1.2.1 Nomenclature
Natural (+)-f-abscisic acid is the trivial name for
the compound defined by the systematic name (+)-(l'f,2~,
4E)-5-(1'-hydroxy-2' ,6' ,6'-trimethyl-4'-oxo-cyclohex-2'
enyl)-3-methylpenta-2,4-dienoic acid. Throughout this
thesis the expanded numbering system for ABA proposed by
(Boyer et al., 1986) will be used. The numbering system
of ABA has been expanded by numbering the methyl groups.
This was done to facilitate the naming of hydroxylated 5
metabolites of ABA. The expanded numbering system is
shown in [1]. Thus, for example, "metabolite C", or what was referred to as 6'-hydroxymethyl ABA becomes 8'-hydroxy ABA.
According to the rules of chemical nomenclature the
atoms which comprise the rings of phaseic acid (PA) [19]
are numbered differently from those of ABA (Milborrow,
1969b) but it is often clearer to apply the ABA numbering
system to PA. Throughout this thesis, positions in the
phaseic acid molecule will be referred to using the ABA
numbering system.
...... 'OH COOH
[2] (+)-cis-diol of ABA (1'§,4'8-dihydroABA)
...... 'OH COOH HO
[3] (+)-trans-diol of ABA (1'$,4'$-dihydroABA)
The 1' ,4'-cis-diol of ABA [2] and the 1' ,4'-trans
diol of ABA [3] should be referred to as 4'-dihydro- 6
abscisic acid provided that the stereochemistry is also defined. Thus "1'1-,4'1-trans-diol" of natural (+)-1-ABA should be referred to as 1'1-,4'1-4'-dihydroabscisic acid because "trans" in this situation is superfluous.
However, using the name "4'-dihydroabscisic acid" without specifying the stereochemistry is ambiguous and could refer to either the cis- or trans-diol of ABA.
1.2.2 Isomerization
The carbon atom at the 1'-position of ABA is chiral and ABA has one of the most intense optical rotatory dispersion spectra recorded. Only the (+)-1-enantiomer occurs naturally and the synthetic material, of course, is racemic, containing equal amounts of (+)-S-ABA and ( - ) - R- ABA [ 4 ] .
COOH 0
[4] (-l-.!3-abscisic acid
COOH
0
[5] 2-trans-abscisic acid 7
Under the influence of light, especially UV, ABA is converted into a 1:1 mixture of 2-cis- (or 2E-) and
2-trans- (or 2-Z) [5] geometrical isomers. Because ABA is defined as 2~,4E, only the 2-trans isomer needs to be specified. The 2-trans isomer has been isolated from plant tissue and because it is biologically inactive it has been assumed to have been derived by light-catalysed isomerization in vivo.
OH
COOH HO
[ 6] lunularic acid
1.3 OCCURRENCE
Abscisic acid has been found in all vascular plants in which it has been sought. ABA occurs in dicotyledons, monocotyledons, gymnosperms, ferns and horsetails. It has been detected in every major part of higher plants including leaves, roots, stem, seeds, fruit and buds.
The reported concentrations vary from 3 ng/g in a water plant to 20 µg/g in dormant cocklebur buds. However, most tissues contain between 20 and 100 ng/g fresh weight. Abscisic acid appears to be absent from liverworts and algae where lunularic acid [6] has been proposed to have the same physiological role as ABA in 8
higher plants. The situation with mosses is uncertain
(Milborrow, 1984) and culture of mosses on defined media will be necessary to demonstrate that any ABA they may contain has not been derived by contamination from a higher plant source. ABA has not been found in bacteria or in fungi, except for some species, such as Cercospora rosicola, Cercospora cruenta and Botrytis cinerea in which it may be involved in the mechanism of parasitism.
1.4 PHYSIOLOGICAL EFFECTS OF ABA
One approach used to investigate the physiological role of plant hormones is to correlate changes in the concentration of the hormone with a physiological event.
Another approach is to study the physiological response to application of exogenous hormone. In general, abscisic acid counteracts the responses to auxins, gibberellins and cytokinins and augments the effect of ethylene. However, there are exceptions to all these broad generalisations.
1.4.1 Seed dormancy
The idea that dormancy in potato tubers and trees may involve growth inhibitory substances was proposed by
Hemberg (1949). This hypothesis was extended to various
types of seed dormancy (Wareing, 1965). Although
exogenous ABA will inhibit the germination of a wide
range of species, ABA appears to play a role in dormancy
of seeds only in those that require stratification.
Dormant, immature embryos of yew (Taxus baccata) can be 9
induced to germinate without stratification if they are placed in a nutrient solution which causes an ABA-like fraction to be leached from them (Le Page-Degivry and
Garrello, 1973). Dormancy can be reimposed by immersing the leached embryos in ABA solutions - analogous results have been obtained with apple seeds (Durand et al.,
197 5) .
Attempts to correlate endogenous ABA concentrations with the state of dormancy have given contradictory results. In some species, ABA concentrations are high in dormant seeds and decline with stratification, whereas in other cases no decline is observed. Evidence suggests that the induction of embryo dormancy may involve endogenous ABA whereas the breaking of dormancy during stratification may be more directly dependent upon increased production or release of gibberellins and/or cytokinins rather than a reduction in ABA levels.
1.4.2 Bud dormancy
Phillips and Wareing (1958) showed that seasonal
changes in the inhibitor content of sycamore were
correlated with the dormancy of shoot apices. Attempts
to determine the nature of the endogenous inhibitor led
to the identification of ABA by Cornforth et al.
(1965b).
Endogenously supplied ABA has been shown to be able
to prolong bud dormancy or induce dormancy of normally 10
growing plants (El Antably et al., 1967) although subsequent attempts to repeat these results have been unsuccessful (Hocking and Hillman, 1975).
When measured by bioassay, the amount of inhibitory material in leaves grown in different day lengths was correlated with the ability of leaf extracts to induce bud dormancy. However, GC analysis for ABA gave results which were not in agreement with those of the bioassay
(Lenton et al., 1972). Thus, other factors may be
involved in the control of bud dormancy. Trewavas (1981)
suggests that the dormancy of different species may
involve a changing pattern of gibberellins or cytokinins
and changes in the tissue sensitivity.
The role of ABA in the formation of "turions"
(winter resting buds) in duckweed (Lemna polyrrhiza) is
clearer. The formation of turions can be induced by the
application of ABA and the turions so formed can be
induced to germinate by chilling or treatment with
cytokinin (Perry and Byrne, 1969; Stewart, 1969).
1.4.3 Abscission
Although ABA was originally discovered in the search
for an abscission-promoting substance, ABA has little
effect in promoting leaf abscission when applied to an
intact plant, except at very high concentrations
(Milborrow, 1974). However, ABA may have a role in the
regulation of flower and fruit abscission. 11
Van Steveninck's (1959) studies of the abscission of
immature fruits of yellow lupin pointed to the
involvement of a specific substance which was later
identified as ABA (Cornforth et al., 1966). Application
of ABA accelerated the abscission of mature fruits of peach, olive, citrus, apple, and the flowers and young
fruits of grape (Milborrow, 1974). Davis and Addicott
(1972) measured the ABA concentration in developing
cotton fruits and found high concentrations were
correlated with abortion and abscission of young fruit
and the dehiscence of mature fruits.
1.4.4 Root geotropism
It was thought for a long time that the downward
growth of roots under the influence of gravity was the
result of an asymmetric redistribution of IAA in the
root, however, more recent work has implicated ABA. The
removal of the root cap destroys the geotropic response
and removal of half of the root cap results in curvature
towards the side with the remaining half. These experi
ments suggested that the root cap is the source of a
growth inhibitory substance whose asymmetric distribution
controls the geotropic response. The inhibitory substance
was thought to be ABA because it is present in the root
cap and is known to be synthesized there. Furthermore,
application of ABA to the root surface at concentrations of 10-8 to 10-4 M causes inhibition of growth and
asymmetric application of ABA to the root surface causes
curvature towards the site of application (Wilkins, 1977; 12
Pilet, 1975). Experiments with ABA-deficient mutants
(Moore and Smith, 1985) and Fluoridone-treated plants
(Moore and Smith, 1984) have shown that these plants have normal geotropic responses in the absence (or at low levels) of ABA. ABA, therefore, may be involved as part of an integrated system but the rest of the system can function without the ABA component.
1.4.5 Response to stress
The concentration of ABA increases markedly when a plant is subjected to water stress (Wright and Hiron,
1969). When excised wheat leaves were wilted, the ABA
concentration increased by as much as 40-fold within 4 hours. The extent of ABA synthesis in response to wilting is dependent on the tissue. While avocado leaves
increased ABA content 40-fold the ABA contents of stems
doubled, roots showed a slight response but ripening
mesocarp, although synthesizing ABA rapidly, showed no
response to wilting (Milborrow and Robinson, 1973).
Waterlogging, heat, chilling, salinity and certain
pathological conditions also cause an increase ABA
content but the increase is not as high as that which
occurs on wilting. It could be suggested that all the
treatments cause loss of turgor and operate via the
wilting response. However, this does not appear to be
correct because stress conditions can be adjusted so that
leaf water potentials remain below the critical point for
wilting and the ABA content still increases. 13
1.4.6 Stomatal closure
The most obvious role of ABA in a wilted plant is to close stomata thus preventing further water loss (Cummins et al., 1971). Mutants that are ABA deficient, such as the "wilty" mutants of tomato, wilt more readily because of excessive stomatal opening (Tal, 1966). When "wilty" mutants are treated with exogenous ABA the stomata close, water loss decreases, and the plants become turgid.
Low levels of exogenous ABA applied to leaves of normal plants cause stomatal closure within 3-9 minutes
and this is thought to be brought about by the effect of
ABA on the proton-potassium pump in the guard cells.
Stomata open up when the guard cells are under turgor
which is caused by the accumulation of K+ ions and
organic acid counter ions in the guard cells (Raschke,
1975). ABA causes the efflux of K+ ions and the stomata
close within minutes of application.
1.4.7 Effects on nucleic acid and protein synthesis
The effects of ABA on nucleic acid and protein
synthesis have been studied in a wide variety of systems,
involving growing and mature plant tissues. Evidence
suggests that ABA does not exert a general inhibition of
DNA transcription but may inhibit the synthesis of a
limited number of specific mRNA species such as the
a-amylase mRNA of the cells of the aleurone layer in
barley seeds (Higgins et al., 1977). However, ABA exerts 14
its main effect by blocking protein synthesis at a post-transcriptional level such as mRNA processing or translation. For example, ABA has been shown to inhibit the synthesis of protease and isocitrate lyase in the
embryos of cotton seeds (Ihle and Dure, 1970) although
the appropriate mRNAs are present. Some effects of ABA
are manifested too rapidly to be accounted for by its
inhibition of protein synthesis (Milborrow, 1980) so
these actions have been attributed to an effect on
preformed sites, such as uptake carrier molecules in
membranes.
1.4.8 Other effects of ABA
ABA has been shown to inhibit elongation growth in
many systems, but there is little evidence that it acts
as a slight brake on normal growth. Abscisic acid has
even been found to promote growth of particular organ
preparations, especially at low concentrations. Other
processes in which ABA has been implicated are in
senescence, the ripening and development of fruit,
flowering, frost hardiness and tuberization.
1.5 THE BIOSYNTHESIS OF ABA
The structure of abscisic acid [1] suggests that the
molecule is made up of three five-carbon isoprene units.
Each of these units is derived from the six-carbon
mevalonic acid [7]. The biosynthetic pathway between the
known terpenoid precursors and ABA is not clear and two
possibilities have been suggested: i) a c15 route 15
CH 3 OH I I HOOC OH
[7] (+)-3!3-mevalonic acid
OPP
[8] farnesyl pyrophosphate
HO
[9) violaxanthin
involving cyclization and modification of farnesyl pyrophosphate [8] (direct pathway); ii) a route involving cleavage of a c40 intermediate such as violaxanthin [9] (carotenoid or indirect pathway).
Noddle and Robinson (1969) demonstrated that mevalonic acid was incorporated into ABA and ripening fruits gave the highest percentage incorporation of any 16
tissue studied. The stereochemistry of ABA biosynthesis has been determined using 14c-labelled and stereospecif ically tritiated mevalonic acids (Milborrow (1978a).
Unfortunately, the results did not discriminate between the direct and carotenoid pathways but they did show that the ~-2 double bond of ABA is formed in the trans config uration and must be isomerized at a later stage to the
cis configuration seen for ABA.
1.5.1 Carotenoid pathway of biosynthesis
Simpson and Wain (1961) observed that the amounts of
growth-inhibitory material increased when dark-grown plants were illuminated and the action spectrum resembled
the absorption spectrum of carotene. This observation as well as the structural similarity between ABA and the
terminal rings of some carotenoids suggested that a
carotenoid could give rise to ABA by photolytic cleavage.
Taylor and Smith (1967) exposed a mixture of carotenoids
to sunlight and found a potent, neutral growth inhibitor
was formed as well as other products, including loliolide
[10] and "butenone" [11]. They showed that violaxanthin
[9] produced the inhibitor which they named xanthoxin
(Taylor and Burden, 1970). Xanthoxin was originally
defined as a mixture of the 2-cis [12] and 2-trans [13]
isomers. 2-cis-xanthoxin is rapidly metabolized to ABA
and has been isolated from shoots and leaves of a number
of plants (Firn et al., 1972). Although cleavage of some violaxanthin to ABA by light may occur, it is unlikely to
be the main mechanism of ABA biosynthesis because wheat 17
C==O I 0
[ 10] loliolide
[11] "butenone·
HO CHO
[121 2-cis-xanthoxin
CHO
HO
[13] 2-trans-xanthoxin 18
leaves wilted in darkness and avocado fruit kept in darkness and supplied with 14c-mevalonic acid were able to synthesize ABA. Firn and Friend (1972) found that a lipoxygenase enzyme from soybean leaves cleaves viola xanthin to give xanthoxin and a similar range of products to those formed by photolysis. However, Nonhebel and
Milborrow (1986) have found that xanthoxin is not label led in tomato shoots which were synthesizing [2H]ABA from
2H2o. Therefore, under the experimental conditions xanthoxin did not appear to be a precursor of ABA.
Further evidence which suggests involvement of a carotenoid in the ABA biosynthetic pathway comes from studies on mutants that were deficient in carotenoids.
Moore and Smith (1985) have shown that the ABA levels in the embryos of carotenoid deficient corn mutants were significantly lower than control embryos. In addition, when Zea mays seedlings are treated with Fluoridone, an inhibitor of carotenogenesis, no ABA was detected (Moore and Smith, 1984). However, Fluoridone could inhibit an
analogous enzyme in the direct pathway and the carotenoid mutant could also have suffered the loss of an ABA
synthesizing enzyme. In other work, Li and Walton (1985)
found that when the epoxide oxygen of violaxanthin was
labelled in situ with 180 the ABA that was produced
contained 180 in the 1'-hydroxyl although there was
considerable dilution of 180. However, the 1'-hydroxyl
oxygen of ABA could have come directly from o2 in the experiment and although the evidence is suggestive it 19
does not establish rigorously the involvement of a carotenoid.
Evidence against the carotenoid pathway comes from the work of Robinson (see Milborrow, 1974) who fed
[14c]phytoene (a precursor of carotenoids) to avocado fruit with [3H]mevalonic acid. The ABA extracted contained 3H but no 14c whereas the carotene contained
14c and 3H. If ABA had been formed via a carotenoid it would be expected to contain 14c. A criticism of the experiment is that it is possible that [14c]phytoene failed to reach the cellular compartment where 3tt ABA was formed because intact cells were used.
Some recent work has produced evidence for a large pool of a precursor of ABA. Creelman and Zeevaart (1984) analysed ABA produced by Xanthium strumarium wilted in an atmosphere containing 180 2 . Analysis of the ABA by gas chromatography-mass spectrometry revealed that the ABA was labelled only in one of the carboxyl-group oxygens with 180. The absence of 180 in the 1'-,4'- and one of the C-1 positions suggests that the oxygens at these positions may come from water, although in a similar experiment in Cercospora rosicola the oxygen in all four positions was derived from molecular oxygen (R. Horgan
and D.C. Walton, cited by Creelmann and Zeevaart, 1984).
Another possibility is that there was a large pool of
precursor, such as violaxanthin, with oxygen already in
the 1'- and 4'-positions and the other C-1 oxygen atom
coming from water. Studies of the incorporation of 20
deuterium from 2tt2o into ABA in tomato shoots (Nonhebel and Milborrow, 1986) give further information on the pool size of precursor(s). Tomato shoots incubated with
~-[2-14c]ABA and 2tt2o for 6 hours incorporated no deuterium into ABA although up to 93% of the ABA had been synthesized in the presence of 2tt2o as shown by the dilution of [14c]ABA. When tomato shoots were incubated in 80% 2tt2o for 6 days up to 32% of ABA molecules were labelled with 1 to 14 deuterium atoms. Thus, there is a large pool of precursor whose minimum size was calculated to be 35 times that of the pool size of ABA in turgid shoots. The ABA produced in wilted plants contained significantly less deuterium (22%) than that from turgid plants (32%). This difference may be explained by either i) a release of ABA from a bound or sequestered form, or ii) by the drawing in of an increased pool of a precursor in wilted plants.
1.5.2 Biosynthesis of ABA in fungi
The finding that the pathogenic fungus, Cercospora rosicola biosynthesized ABA in culture (Assante et al.,
1977) prompted other workers to use the fungus as a means to study the biosynthetic pathway of ABA. Neill et al.
(1982) found [3H]mevalonic acid was incorporated into ABA
and a less polar compound, identified as 1'-desoxy ABA
[14], which was found to be the immediate precursor of
ABA in the fungus. Other compounds converted into ABA by
the fungus are: 2-cis-a-ionylidene ethanol [15]; 2-cis-a
ionylidene acetic acid [16]; 4'R- and 4'~-hydroxy-2-cis- 21
0 COOH
[14] 1'-desoxyabscisic acid
[15] 2-cis-a-ionylidene ethanol
COOH
[16] 2-cis-a-ionylidene acetic acid
COOH
[17] 4'-§-hydroxy-2-cis-a-ionylidene acetic acid 22
a-ionylidene acetic acid [17]; 1' ,4'-cis-[2] and 1' ,4' trans-diol of ABA [3]. Another fungus, Botrytis cinerea, has been found to use the 1' ,4'-trans-diol of ABA as the immediate precursor of ABA (Hirai et al., 1985).
The biosynthetic pathway of ABA in fungi appears to be via the c15 (direct) route but there is little evidence that the immediate precursors of ABA in fungi are the same as those in higher plants. Only one plant,
(Vicia faba), was found to convert a-ionylidene acetic acid into 1'-desoxy ABA and into ABA although in a much lower yield than Cercospora rosicola. When [2H]a-ionyl idene acetic acid was fed to Vicia faba plants 1'-desoxy
ABA was 100% labelled with deuterium which implies that
1'-desoxy ABA does not occur endogenously in Vicia faba or that it is rapidly turning over.
Thus the pathway of ABA biosynthesis in fungi appears to be different from that in higher plants.
Whereas the pathway in the fungus appears to be by a direct pathway, the burden of the evidence favours the carotenoid pathway of ABA biosynthesis in higher plants.
1.6 CATABOLISM OF ABA
Abscisic acid concentrations can vary widely and rapidly in response to some environmental stimuli and changes in the concentrations of ABA affect a wide range of physiological responses. The concentration of ABA present in a tissue is a balance between synthesis and 23
breakdown, modified by transport of ABA in and out of the tissue. As described above, the biosynthesis of ABA is not fully understood and the immediate precursors of ABA in higher plants are yet to be discovered. By contrast, more is known of. the catabolic pathway of ABA which involves the processes of conjugation and oxidative degradation. These processes mask and modify features of the molecule that are essential for its activity or increase the polarity of the molecule which may affect its subcellular partitioning.
1.6.1 Oxidative degradation
1.6.1.1 8'-Hydroxyabscisic acid
The first feeding experiments carried out with
RS-[2-14c]ABA were with petiole segments of bean and sycamore (Milborrow, 1967). Analysis of the tissue after
12 h incubation showed the presence of three labelled compounds, which were referred to as metabolites A, Band
C. Similar compounds were formed from [2-14c]ABA by tomato shoots (Milborrow, 1970). Metabolite B was
identified as ~-D-glucopyranosyl abscisate (ABAGE), which had previously been isolated from Lupinus luteus fruit
(Koshimizu et al., 1968). Metabolite A was shown to be
ABA methyl ester and was an artefact of extraction
produced by the methanolysis of ABAGE in neutral or basic
methanol (Milborrow and Mallaby, 1975). 24
HO~ \ \
COOH 0
(18] 8'-hydroxyabscisic acid
' ' 'OH 0 COOH
[ 19 J phaseic acid
Metabolite C was isolated as a crystalline solid
(m.p. 190°c) which gave a strong ORD spectrum similar to that of (+)-ABA and was shown to be 8'-hydroxy ABA
(6'-hydroxymethyl ABA) [18]. Upon heating or treatment with diazomethane the compound rearranged to a compound
(m.p. 205°c) with a different, very weak ORD spectrum and was identified as a compound previously isolated from beans (phaseic acid [19]). All subsequent attempts to isolate 8'-hydroxy ABA have failed, phaseic acid being obtained instead. 25
Despite the failure to reisolate 8'-hydroxy ABA there is further evidence for its existence. Adesomoju et al. (1980) have obtained mass spectral evidence for the existence of the metabolite in Vigna unguiculata.
Furthermore, 8'-hydroxy ABA has been isolated as the ester of hydroxymethylglutaric acid [21] (Hirai et al.,
1978). The methyl ester of ABA is also hydroxylated in the 8'-position and the 8'-O-glucoside of 8'-hydroxy ABA methyl ester [22] has been isolated as a metabolite of
[2-14c]ABA methyl ester in sunflower shoots (unpublished, cited by Milborrow, 1985).
A cell-free system capable of hydroxylating ABA has been prepared from Echinocystis lobata liquid endosperm
(Gillard and Walton, 1976). The activity, which was present in the particulate fraction, required o2 and NADPH, typical of a mono-oxygenase, and had a high substrate specificity for (+)-ABA. Acetylation of short term incubation products gave, presumably, acetylated
8'-hydroxy ABA which rearranged on hydrolysis to give a compound tentatively identified as phaseic acid. Also present in the liquid endosperm was a soluble enzyme
capable of reducing phaseic acid to dihydrophaseic acid
[20]. Further evidence for the involvement of an oxygen
ase in the hydroxylation of ABA comes from the work of
Creelman and Zeevaart (1984) who found that phaseic acid,
formed from ABA in the presence of 180 2 , contained 180 in the 8'-hydroxyl group. 26
HO COOH
[20] dihydrophaseic acid
0 COOH
[ 21] 3-hydrox y- 3-met hy lg lutary 1-8' -hydrox yab scisic acid
(HMG-HOABA)
0 COOCH 3
[22] 8'-0-glucoside of 8'-hydroxy ABA methyl ester 27
0 COOH
(23]
COOH
[24] epi-dihydrophaseic acid
1.6.1.2 Phaseic acid
Phaseic acid (PA) was originally isolated as a contaminant in the gibberellin fraction of Phaseolus multiflorus (MacMillan et al., 1960) and the structure
[23] proposed by MacMillan and Pryce (1968), who noted its similarity to ABA. This structure was incompatible with the rearrangement of 8'-hydroxy ABA to phaseic acid and a new structure was proposed for phaseic acid [19]
(Milborrow, 1969b). The absolute configuration has been confirmed by comparison with the NMR and IR spectra of methyl phaseate and the two epimers of methyl 4'-dihydrophaseate (Milborrow, 1975). Independent
confirmation can be deduced from the structure of
drummondone (Powell et al., 1985) 28
1.6.1.3 Dihydrophaseic acid and epi-dihydrophaseic acid
Two major metabolites of RS-[ 14c]ABA supplied to excised bean axes were designated M-1 and M-2 (Walton and
Sondheimer, 1972a) and were later shown to be phaseic acid [19] and dihydrophaseic acid (DPA) [20] (Tinelli et al., 1973). Dihydrophaseic acid was thought to be a major deactivation product in bean seeds where it was present at a concentration 100 times that of ABA. When phaseic acid is reduced with sodium borohydride approximately equal amounts of dihydrophaseic acid and its epimer, epi-dihydrophaseic acid (epi-DPA) [24] are produced. epi-DPA was formed from [2-14c]ABA by bean shoots and was also found to occur naturally at 2-50% the concentration of DPA (Zeevaart and Milborrow, 1976). In addition, epi-DPA and DPA also appear to occur as base-hydrolysable conjugates. When RS-[2-14c,4 1 - 18o]ABA was fed to bean shoots, 180 was present in the
4'-hydroxyl group of both DPA (41%) and epi-DPA (43%)
(Zeevaart and Milborrow, 1976). These results demonstrated that epi-DPA and DPA were formed rapidly
from phaseic acid and neither epimer had lost 180 by a mechanism that inverted the stereochemistry at C-4'.
1.6.1.4 7'-Hydroxyabscisic acid An acidic metabolite was detected in cell-suspension
and tissue cultures of Nigella damascena fed RS-[ 14c]ABA
(Lehmann et al.,1983a) and was identified as 3'-hydroxy
methyl ABA (7'-hydroxy ABA) [25] (Lehmann et al., 1983b).
The compound was claimed to be naturally occurring in the 29
leaves of Vicia faba by the correspondence of chromatographic mobility (HPLC and TLC) of A substance in extracts of untreated plant material with the compound
[25] produced from racemic [14c]ABA in Nigella. However,
Boyer and Zeevaart (1985) found 15-20% conversion of
RS-ABA into 7'-hydroxy ABA in leaves of Xanthium strumarium but the metabolite was a product of R-ABA and not S-ABA. Thus 7'-hydroxy ABA does not appear to be a normal metabolite of endogenous ABA.
0 COOH
(25) 7'-hydroxyabscisic acid
' ' 'OH COOH
(26) 4'-desoxyabscisic acid
1.6.1.5 The l' ,4'-trans-diol of ABA
The l' ,4'-trans-diol of ABA [3] was identified as a product of RS-[2-14c]ABA fed to pea shoots (Milborrow,
1983). A compound identified as 4'-desoxy ABA [26]
(Tietz et al., 1979) appears to be an artefact produced 30
during mass spectrometry by dehydration of the
1' ,4'-trans-diol of ABA. This metabolite will be discussed in detail in Chapter 4.
1.6.1.6 Other metabolites
Martin et al. (1977) have shown the presence of hydroxyphaseic acid or oxodihydrophaseic acid as an endogenous constituent of pear seeds. Evidence for the
same compound in the phloem exudate of coconut was obtained using mass spectrometry (Hoad and Gaskin, 1980)
Tietz (1985) tentatively identified 8'-hydroxy-
4'-dihydroabscisic acid [27], a product formed when
RS-[l-14c]ABA was supplied to pea seedlings. This
compound was produced in a higher yield from [1-14c]-
1' ,4'-trans-diol of ABA and Tietz (1985) suggests that
this represents an alternative pathway to DPA.
COOH HO
(27) 8'-hydroxy-4'-dihydroabscisic acid 31
1.6.2 Conjugation
There has been some confusion in the literature between the terms "bound" and "conjugated". Sembdner et al. (1972) proposed that "conjugation" should be used to describe a covalent link formed between the hormone and another small molecule such as a monosaccharide or amino acid, although attachment to a macromolecule is not excluded. "Bound" refers to non-covalent attachment to a molecule in some unspecified manner such that it is not susceptible to unhindered exchange or partition into free solution. An implicit assumption of this definition is that it does not include the binding to the active site
(although strictly not excluded) and that the binding is a form of storage or temporary sequestration.
1.6.2.1 Abscisic acid glucose ester
Abscisic acid glucose ester (ABAGE) [28] has the
systematic name of ~-D-glucopyranosyl(l'~,2~,4E)-5- (1'-hydroxyl-2' ,6',6'-trimethyl-4-oxo-cyclohex-2'-enyl)-3
-methylpenta-2,4-dienoate. ABAGE was the first conjugate
of ABA to be discovered (Koshimizu et al., 1968). Its
isolation from yellow lupin pods was monitored by a rice
seedling bioassay in which ABAGE had half the activity of
ABA on a weight basis and very similar to ABA on a molar
basis. Abscisic acid glucose ester has also been identified in Xanthium leaves (Zeevaart, 1980) and pear
receptacles (Martin et al., 1982). In addition, ABAGE was characterized as a metabolite of RS-[ 14c]ABA in 32
tomato shoots (Milborrow, 1970) and the ABA in the
conjugate had a preponderance of the (-)- enantiomer.
Formation of the glucose ester appears to be a means by which ABA is removed from the active pool since ABAGE
has been shown not to be a source of stress-induced ABA
(Milborrow, 1978b; Zeevaart, 1980; Pierce and Raschke,
1981; Neill et al., 1983). Furthermore, the findings
that the levels of ABAGE rise with increasing tissue age
(Weiler, 1980) and that 80 to 90% of ABAGE is located
within the vacuoles (Bray and Zeevaart, 1985) suggests
that ABA is deactivated by conjugation to glucose and is
stored in the vacuole where it is not available as a
source of ABA.
0
[28] abscisic acid glucose ester
0
CH 2 OH __ 1/ HO~o HO~ "\_ ,0----~~ OH
COOH
[29] abscisic acid 1'-glucoside 33
1.6.2.2 1'-O-abscisic acid-(3---D-glucopyranoside
ABA 1'-O-glucoside (ABAGS) [29] has the systematic name: (1'~,2~,4E)-5-(1'-[P-d-glucopyranosyloxy]-2' ,6' ,6' trimethyl-4'-oxo-cyclohex-2-enyl)-3-methylpenta-2,4-dieno ic acid. The 1'-0-glucoside of ABA was characterized as a product of RS-[2-14c]ABA fed to tomato shoots and was also found to occur naturally (Loveys and Milborrow,
1981). Unlike most glucosides, ABAGS is highly susceptible to hydrolysis with base and also undergoes slow spontaneous rearrangement to form ABA glucose ester. Its lability may reflect the unusual electronic environment of the 1'-tertiary hydroxyl group which is extremely difficult to derivatize.
1.6.2.3 3-Hydroxy-3-methylglutaryl-8'-hydroxy ABA
A conjugate of 8'-hydroxy ABA was isolated as endogenous constituent, from seeds of Robina pseudacacia by Hirai et al (1978). Conjugation of 8'-hydroxy ABA to
3-hydroxy-3-methylglutaric acid prevents its rearrangement to phaseic acid. The conjugate [21] was hydrolysed by base and when treated with diazomethane,
3-hydroxy-3-methylglutarate dimethyl ester and phaseic acid methyl ester were identified as the products.
1.6.2.4 Dihydrophaseic acid glucoside
The 4'-O-glucoside of dihydrophaseic acid (DPAGS)
[30] has been characterized as a product of RS-[2-14c]ABA fed to tomato shoots (Milborrow and Vaughan, 1982) and as
an endogenous constituent of avocado fruit (Hirai and 34
Koshimizu, 1983). The isolation of a characterization of this conjugate is discussed in Chapter 2.
HO COOH OH
[30] dihydrophaseic acid 4'-glucoside
1.6.2.5 Other conjugates
The increase in free metabolites after alkaline hydrolysis has been used used by some authors as evidence for the existence of "conjugates" or even "glucose esters" of that metabolite. Thus release of ABA by alkaline hydrolysis has been used to measure concentrat ions of ABA glucose ester, though it is now known that
ABA is also released by ABA glucoside under these conditions. Furthermore, some glycosidic linkages are resistant to alkaline hydrolysis (Milborrow and Vaughan,
1982; Hirai and Koshimizu, 1983) and an underestimation of conjugated metabolites is made when the metabolites released by alkaline hydrolysis are measured. Other sources of error are the partitioning of conjugates into solvents used to extract the acid fraction. Thus ABA glucose ester (Zeevaart, 1980) and ABA glucoside both partition into ethyl acetate to some extent (Loveys and Milborrow, 1981). Conjugates which yield rearranged 35
products on hydrolysis, such as 3-hydroxy-3-methyl glutaryl-8'-hydroxy ABA to give phaseic acid, can also lead to misinterpretation of the nature of the conjugates. Nonetheless, there is evidence for the existence of uncharacterized conjugates of abscisic, phaseic, dihydrophaseic and epi-dihydrophaseic acids.
1.6.3 Bacterial metabolism of ABA
Destruction of RS-[2-14c]ABA has been found in
microbial cultures isolated from soil or decaying fruit
(Milborrow and Vaughan, 1979). Several labelled metab
olites were found and one was identified as 1' ,4'-trans
diol of ABA. The decarboxylation of RS-[1-14c]ABA by
developing apple seeds (Rudnicki and Czapski, 1974) was
attributed to the microbial destruction of ABA (Milborrow
and Vaughan, 1979) and Corynebacterium, a soil bacterium,
has been found to decarboxylate [14c]ABA to produce
dehydrovomifoliol [31] (Hasegawa et al., 1984).
0
[31] dehydrovomifoliol 36
1.6.4 Pathway of ABA metabolism
A general pattern in the sequence of metabolic reactions has emerged in which ABA is oxidized to 8' hydroxy ABA which rearranges to PA and the subsequent reduction of PA produces the epimeric dihydrophaseic acids. In addition, ABA and the free acid metabolites are conjugated to glucose or another small molecule. This sequence is depicted in Scheme 1. Support for this path way comes from the measurement of the specific activity of metabolites of [14c]ABA. The specific activities decrease along the sequence ABA, PA, DPA / epi-DPA and the specific activity of each free acid is higher than the specific activity of the conjugates (Milborrow,
1978a) .
ABAGE
1' ,4'-trans-diol ABA of ABA ABAGS
8'-Hydroxyl ABA HMG-HOABA
epi-DPA PAl conjugate
conjugatel DPAl DPAGS
Scheme 1: Pathway of the metabolism of ABA 37
1.6.5 Further studies of ABA metabolism - the present investigations
Various aspects of ABA metabolism were studied in this present work and a specialized introduction is given
at the beginning of each section. Briefly, the aspects
of ABA metabolism investigated were:
i) The further metabolism of acidic metabolites of ABA
and the isolation and characterization of a conjugate of
dihydrophaseic acid (Chapter 2).
ii) The differential metabolism of natural S-ABA and
unnatural R-ABA:- A method was developed to prepare the
separate enantiomers of ABA from labelled, racemic
material and metabolism of the separate enantiomers was
studied (Chapter 3).
iii) The metabolic reduction of ABA. The 1' ,4'-trans
diol of ABA appears to be a side reaction and does not
fit into the general pathway of ABA metabolism. An
attempt was made to examine the relationship between the
metabolic reduction of ABA to the 1' ,4'-diols and the
major pathway of metabolism by investigating the form
ation and further metabolism of the diols (Chapter 4). 2
Characterization of Dihydrophaseic Acid 4 ·-p-D-g lucopyranoside
38 39
2.1 INTRODUCTION
Before the widespread use of high-performance liquid chromatography the study of metabolites and conjugates of
ABA usually involved solvent partitioning of the products followed by basic hydrolysis of conjugated products in the aqueous residue and a second partitioning. The dis advantages of this procedure are that some conjugates are extracted into solvents such as ethyl acetate (Zeevaart,
1980; Loveys and Milborrow, 1981), the conjugates remain ing in the aqueous phase are destroyed, and conjugates resistant to hydrolysis or compounds released by hydro lysis but do not partition into organic solvents may be lost. Furthermore, highly polar compounds are poorly resolved by thin-layer chromatography and are difficult to isolate.
In experiments where [2-14cJABA was supplied to tomato shoots (Milborrow, 1978b) and apple seeds
(Milborrow and Vaughan, 1979) a significant proportion of the radioactivity remained in the aqueous residue after basic hydrolysis, especially after long-term incubations.
Several other workers have reported unidentified polar products of ABA (Sondheimer et al., 1974; Dewdney and
McWha, 1978; Dashek et al., 1979; King, 1979). 40
This chapter describes the development of a reversed-phase HPLC method with which metabolites and conjugates of ABA were separated in a single chromato graphic step. The isolation and characterization a polar metabolite, dihydrophaseic acid 4'-O-~-D-glucoside
(DPAGS) [30] and the further metabolism of some metabolites of ABA are also described.
COOR 2
Rl R2 [20] H H
[32] H CH 3
[33] Ac CH 3 [30] ~-D-glucosyl H
[34] ~-D-glucosyl CH 3
[35] tetraacetyl-~-D-glucosyl CH 3 41
2.2 MATERIALS AND METHODS
2.2.1 Chemicals
RS-[2-14cJabscisic acid (11.1 mCi/mmol) was purchased from the Radiochemical Centre, Amersham,
England. RS-[l 1 - 18oJABA (36 atoms% enrichment) was from the sample used by Gray et al. (1974). RS-ABA
was the sample synthesized by Cornforth et al. (1965a).
~-ABA glucose ester was from the sample isolated by Koshimizu (1968). ABA l'-glucoside was from that isolated by Loveys and Milborrow (1981). HPLC solvents were from Millipore-Waters, or were redistilled before use. Distilled water was passed through a Milli-Q reagent water system (Millipore, Bedford, MA, U.S.A.).
2.2.2 Plant material and feeding
Tomato plants (Lycopersicon esculentum cv. Grosse
Lisse) were germinated in a mixture of peat and sand
(1:1, v/v) and grown under natural light in a glasshouse for 6-8 weeks, by which time they were 200 mm to 300 mm
high. Tomato plants were cut just above ground level and
placed in aqueous solutions of labelled compounds. Once
the solution had been taken up, the stems were rinsed
with distilled water and this solution was also
absorbed. 42
2.2.3 Preparation of PA, DPA and epi-DPA
[2-14c]PA was isolated from tomato plants supplied with RS-[2-14c]ABA by the methods of Zeevaart and
Milborrow (1976). [2-14c]DPA and [2-14c]epi-DPA were prepared by the reduction of [2-14c]PA with sodium borohydride in methanol/water (2:1, v/v) at o0 c for 30 min. The products were purified by reversed-phase HPLC as described below.
2.2.4 Extraction and partial purification of
metabolites
Plants were homogenized in acetone/acetic acid
(99:1, v/v) to which BHT (2,6-tert-butyl-4-methylphenol)
100 mg/1 (Sigma Chemical Co., St. Louis, U.S.A.) had been added. The acetone extract was decanted, filtered and the residue extracted twice more with acetone. The combined acetone extracts were evaporated and the aqueous residue acidified to pH 3.5 with acetic acid and extract ed with an equal volume of ether (three times). The aqueous phase, which contained polar metabolites and conjugates of ABA, was put aside for HPLC. An equal volume of water was added to the ether extract and the pH adjusted to 7.0 with saturated sodium bicarbonate. This was repeated three times and the ether phase which contained unlabelled neutral material was discarded. The aqueous phase was acidified with 1 M sulphuric acid to pH
2.5. ABA and ether-soluble metabolites were extracted with diethyl ether (three times). The ether extract and the initial aqueous phase were combined, the diethyl 43
ether was evaporated, the sample was concentrated and chromatographed on Sep-Pak c18 cartridges.
2.2.5 Preliminary chromatography on c18 Sep-Pak cartridges
Four Sep-Pak c18 cartridges (Millipore-Waters) were connected in series, without gaps, by 20 mm lengths of
1 mm-i.d. glass tubing. An upper reservoir was filled with solvent and pressurised by a peristaltic pump to maintain a flow rate of 5.0 ml/min. The sample (about pH
4), which was in a volume of about 50 ml, was loaded into the reservoir, pumped onto the Sep-Pak cartridges and washed with 20 ml water/acetic acid (500:1, v/v). Only a small amount of radioactivity was present in the washings and comprises uncharacterized polar metabolites of ABA.
Ethanol/water/acetic acid (256:475:1, by vol.) (20ml) eluted abscisic acid and its metabolites from the
Sep-Paks. Washing the cartridges with ethanol removed
less polar compounds, which contained no radioactivity,
and regenerated the column.
2.2.6 High-performance liquid chromatography
A Waters' HPLC system consisted of a 6000A pump, a
model M-45 pump, a U6K injector and a model 660 solvent
programmer. Effluent was monitored with a model 440
absorbance detector fitted with a 254 nm filter. 44
2.2.7 Separation of metabolites by HPLC
The metabolite fraction eluted from the Sep-Pak cartridges was applied to a 7.8 (i.d.) x 300 mm µBondapak c 18 column (Waters Associates) in ethanol/water/acetic acid (84:504:1, by vol.) and chromatographed in the same solvent at 4.0 ml/min for 22 min. This was followed by a linear gradient to ethanol/water/acetic acid (158:508:1, by vol.) over 15 min. After a further 10 min the column was washed with ethanol/water (19:1, v/v). Fractions were collected at 1 minute intervals, subsampled, and radioactivity determined by liquid scintillation counting. DPAGS [30] was found at a retention time of
5 min; DPA [20], 11 min; phaseic acid [19], 16 min;
ABAGE [28], 19 min; epi-DPA [24], 21 min; ABAGS [29],
33 min; t-ABA [5], 41.7 min and ABA, 46 min - these retention times were verified by comparison with standards. Other labelled materials were detected which did not correspond with any known metabolites of ABA.
2.2.8 Preparation of derivatives
2.2.8.1 Acetylation
The compounds were dissolved in pyridine (100 µl)
and acetic anhydride (100 µl) was added. The mixture was
incubated at 40°c for 30 min. 45
2.2.8.2 Methylation
Diazomethane was prepared by a modified method of
Schlenk and Gellerman (1960). N-Nitroso-N-methyl-p toluenesulphonamide (4.6 g) was dissolved in ether
(20 ml) and poured slowly into 3M potassium hydroxide in ethanol/water (5:2, v/v, 7 ml) contained in a distillation apparatus which had no ground glass joints.
The mixture was warmed (~ 40°c) and stirred continuously. Diazomethane in ethereal solution was collected in a flask on ice. When the diazomethane was for use with samples which were unstable in basic solutions the diazomethane in ether was redistilled through a cotton wool plug to remove any traces of KOH.
Samples were dissolved in methanol (100 µl) and ethereal diazomethane at o0 c. was added dropwise until a yellow colour persisted or, in the case of coloured samples, until bubbling had ceased. The diazomethane and ether were evaporated under nitrogen.
2.2.8.3 Oxidation with Jones' reagent Jones' reagent (Fieser and Fieser, 1967) [267 mg chromic acid in 4.5 M sulphuric acid (1 ml)] was used to
oxidise compounds by dissolving them in acetone (0.5 ml)
and adding reagent until the mixture remained permanently brown. Excess reagent was destroyed by adding excess
isopropanol. 46
2.2.9 Thin-layer chromatography
Chromatography on silica gel plates (60 F254 , 0.5 mm) (E. Merck, Darmstadt) was used initially in attempts to purify the metabolite. DPAGS remained at the origin of plates developed with toluene/ethyl acetate/acetic acid (25:15:2, by vol.) and butanol/propanol/ammonia/ water (6:2:1:2, by vol.). The RF of DPAGS in chloroform
/methanol/water (67:30:3, by vol.) was 0.35.
2.2.10 Isolation of DPAGS
The DPAGS fraction from reversed-phase HPLC (Section
2.2.7) was chromatographed on a 7.8 (i.d.) x 300 mm
µBondapak c18 column using ethanol/water/acetic acid (24:475:1, by vol.) at 4.0 ml/min and a radioactive fraction at 24 min was collected. The sample was methylated with diazomethane and chromatographed on the same µBondapak c18 column in ethanol/water/acetic acid (71:428:1, by vol.), and the fraction with a retention time of 17 min was collected. This fraction was acetylated and the product chromatographed on a 7.8
(i.d.) x 300 mm µBondapak c18 column in ethanol/water/ acetic acid (333:665:2, by vol.) at 4.0 ml/min. Two peaks were obtained, a minor peak at 20 min and a major peak at 24 min. Finally, the major peak was purified by
HPLC on a 7.8 (i.d.) x 300 mm µPorasil column with isopropanol/hexane (1:9, v/v) as the mobile phase at 4.0
ml/min. The retention time of acetylated DPAGS methyl
ester [34] was 19 min. 47
2.2.11 Melting point
The melting point of O-acetylmethyl ester of DPAGS
[35] was determined on a Koffler block and is uncorrected.
2.2.12 Liquid scintillation counting
Radioactively-labelled samples were placed into glass scintillation vials to which was added 10 ml of scintillation fluid comprising 6 g BBOT (2, 5-bis-[5' tert-butylbenzoxazol-2-yl] thiophene) and 80 g napthalene per litre of toluene/methoxyethanol (2:1, v/v) - 10 ml of this scintillant was miscible with up to 450 µ1 water.
Radioactivity was measured in a Packard Tri-Carb series
2650 scintillation spectrometer which gave an efficiency of 81% for 14c and 20.5% for 3tt. Samples were spiked with 10 µl [u-14c] toluene (5.27 x 105 dpm/g, Packard
Instrument Co., Ill., U.S.A.) to measure the degree of quenching in the sample.
2.2.13 Autoradiography
The position of radioactive metabolites on thin-layer plates was detected by placing Fuji RX X-ray
film over the dried plates which had been marked with spots of radioactive ink. The film was exposed for at
least 7 days and then developed in Kodak X-ray Developer and fixed in Kodak X-ray Fixer. The detection of weakly exposed areas was enhanced by the method described by
Randerath (1970). Areas of silica gel which contained
labelled material were scraped off the plates into grade 48
4 sintered glass funnels and eluted with methanol.
2.2.14 Electrophoresis
14c-labelled samples of ABA, glucose, DPAGS [30],
DPAGS methyl ester [34], ABAGE [28] and methyl green dye were loaded as separate zones along the mid-line of 200 mm x 100 mm plastic-backed cellulose (0.1 mm layer) sheets (Polygram CEL 300 - Machery Nagel and Co., Duren).
The sheets were soaked with 0.1 M phosphate buffer, pH
6.0. Electrophoresis in 0.1 M phosphate buffer, pH 6.0, was run at c. 200 V for 20 min under Varsol (Pylon
Chemicals, Botany, N.S.W.). Zones of radioactivity were detected by autoradiography of the dried sheet.
2.2.15 Hydrolysis of DPAGS
A sample of DPAGS (50 µg) formed from [2-14c]ABA was dissolved in 1 M HCl (50 µl) and heated (100°c, 6 h) in a glass vial sealed under nitrogen. The hydrolysate was partitioned against an equal volume of ethyl acetate
(three times). A sample of [14c]DPA [20] was treated similarly. The extracts, together with DPA and ABA markers, were chromatographed on a silica gel thin-layer plate in toluene/ethyl acetate/acetic acid (25:15:2, by vol.) and radioactive zones detected by autoradiography.
The aqueous residue of the hydrolysate was dried and
a portion (60%) trimethylsilylated at 90°c with Tri-sil-Z (Pierce, Illinois). Mass spectra of the trimethylsilyl
ated derivatives were obtained. The remaining 40% of the 49
aqueous residue was dried, dissolved in 0.1 M phosphate buffer, pH 7.0 (10 µl), and tested for D-glucose with
Tes-Tape (Eli Lilly, Aust.) urine sugar analysis paper which is entirely specific for D-glucose because the first reaction is catalysed by glucose oxidase.
2.2.16 Gas chromatography
GC analysis was carried out in a Pye-Unicam GCV gas chromatograph with a 1.5 m x 4 mm (i.d.) glass column containing 1% XE-60 on Gas Chrom W-HP 100/120 mesh. The gas flow was split so that 25 parts went to a flame ionization detector (FID) and one part to an electron capture detector (ECD). The injector temperature was
200°c and the detector temperature was 240°c. The following retention times were obtained using an oven temperature of 198°c and a carrier (N 2 ) gas flow rate of 33 ml/min.
Compound min
Me-ABA 9
Me-2-t-ABA 14
Me-PA 13.2
Me-epi-DPA 10
Me-DPA 13.7
Me-2-t-DPA 17.5 50
2.2.17 a- and ~-Glucosidase assays ~-Glucosidase from almonds (5.1 units/mg) and a-glucosidase from yeast (43 units/mg in a 5 mg/ml solution) were obtained from Sigma Chemical Co. Both were made up in sodium citrate-phosphate buffer (0.5 M): the a-glucosidase at pH 6.8, the ~-glucosidase at pH 5.2.
Enough enzyme calculated to hydrolyse 100 times the amount of conjugate (based on conventional substrates) was made up to 60 µland added to both DPAGS (500 ng;
24,000 dpm) and ABAGE (500 ng) in 1 ml Reacti-vials
(Pierce) and incubated at 20°c. Samples (10 µl) were withdrawn after 2, 10 and 60 min, mixed with 0.1 M citric acid solution (0.4 ml) and partitioned against ether.
The ether fraction was methylated with diazomethane and analysed by GC. The DPAGS samples were also analysed by
TLC and autoradiography.
2.2.18 Chemical-ionization mass spectrometry
Mass spectra were obtained on a Finnigan model 3200 chemical-ionization mass spectrometer interfaced to a
Finnigan model 6115 data system. Methane was the
ionization gas (source pressure 105 - 120 Pa). Samples
of silylated glucose were injected into a 1.5% OV-1 on
Gas Chrom Q (100-120 mesh) gas chromatography column with methane as a carrier gas (20 ml/min) held at 150°c for 1 min and then programmed to rise 10°c per min to 300°c. 51
2.2.19 Nuclear magnetic resonance (NMR) spectroscopy
Proton NMR spectra were obtained with a JEOL 100 -
MHz Fourier-transform NMR spectrometer. Samples were dissolved in [2H5 ]pyridine or [2H] chloroform (30 µl) in a 1 mm-i.d. tube. 300 MHz spectra were obtained on a
Bruker CXP-300 in 5 mm tubes with [2H] chloroform as the solvent and tetra-methylsilane as an internal reference.
Further details of NMR methods are in Section 4.2.14.
2.3 RESULTS
2.3.1 Preliminary clean-up on Sep-Pak cartridges
Chromatography (HPLC or TLC) was improved by first removing interfering material from plant extracts by a procedure using c 18 Sep-Paks. Sep-Paks are small dis posable columns of stationary phases, such as silica or c 18-coated silica particles, encased in a polythene cylinder. By joining four or five of these columns with
capillary glass tubing the capacity and efficiency of
separations was markedly improved. After removing neutral material from an acetone extract of tomato shoots
fed [14c]ABA for 5 days the aqueous residue was dissolved in 0.2% acetic acid and pumped through the Sep-Paks. In
this initial step, polar compounds passed through the
column and only 1.3% of the total radioactivity did not bind to the column. This represents a small amount of
very polar metabolites. 52
35% Ethanol in 0.2% aqueous acetic acid eluted most metabolites (98.3% radioactivity). The remainder of the extract (0.4% radioactivity) was eluted with ethanol.
The recovery of radioactivity was high, being 97-99% of the counts applied to the cartridges. The salts and polar compounds were removed by the first washing. The removal of so much inactive material before the chromatographic separation commenced allowed larger amounts of extracts to be loaded onto the column than could be separated if the preliminary washing step were omitted. It also removed compounds that bind irreversibly to the stationary phase of the HPLC column.
2.3.2 Separation of metabolites by HPLC
Reversed-phase HPLC with an ethanol/water gradient, which contained acetic acid to suppress ionization of weak acids, separated the metabolites of ABA in a single chromatographic step. The sample was dissolved in 100 µl
15%(95% ethanol) in 0.2% aqueous acetic acid - or ethanol
/water/acetic acid (84:506:1, by vol.). The same solvent was used as the mobile phase at 4.0 ml/min in the separ ation of metabolites of ABA on a semi-preparative c18 column. This initial isocratic step separated DPAGS, DPA,
PA, ABAGE and epi-DPA within 22 min. The metabolites most difficult to resolve on the c18 column were PA and ABAGE and so the chromatographic conditions selected were
those which achieved clear separation of these compounds.
After 22 min of isocratic conditions the remainder of the
metabolites were eluted with a linear gradient rising to 53
25% (95% ethanol) in 0.2% aqueous acetic acid (ethanol
/water/acetic acid [158:508:1, by vol]) over 15 minutes and then a further 10 min of isocratic elution.
The separation of the metabolite fraction eluted from Sep-Paks is shown in Figure 2.1. The products were identified by comparison with the retention time of standards (Table 2.1). A mixture of marker compounds: p-aminobenzoic acid, p-hydroxybenzoic acid, benzoic acid, t-ABA and ABA were chromatographed under conditions identical to those used to separate the metabolites and provided an effective means to test the performance of the system before a metabolite fraction was injected.
2.3.3 Extraction of DPAGS
When RS-[2-14c]ABA was fed to tomato shoots and the products extracted after 5 days, about 35% of the radioactivity remained in the aqueous residue after saponification and extraction of metabolites with ethyl acetate. The radioactivity partitioned into n-butanol at pH 2.5 but only partially at pH 8.0.
2.3.4 Thin-layer chromatography Thin-layer chromatography (TLC) was used in initial
attempts to isolate the metabolite. A small amount of radioactive compound (>1000 dpm) was used to form
derivatives which were chromatographed on silica gel
HPTLC plates. Radioactive material on the plates was 54
2.0
DPAGS E a. "O
~ > -> 1.0 -CJ «s ABAGE 0 "O «s a: X CD I 0 -- ABA
0 20 40 60 Retention time, min
Fig. 2.1 HPLC separation of the products of RS-[2-14CJABA in tomato shoots
2.8 µCi RS-[2-14cJABA were fed to tomato shoots (30g) and extracted after 2 days. The extract was subjected to the preliminary clean up procedure with c 18 Sep-Pak cartridges (Page 42). The fraction that contained metabolites of ABA was chromatographed on a 7.8 mm (i.d.) x 300 mm µBondapak c 18 column with ethanol/water/acetic acid (84:504:1, by vol.) delivered at 4.0 ml/min for 22 min. This was followed by elution with a linear gradient to ethanol/water/acetic acid (158:508:1, by vol.) over 15 min and elution with the final solvent for 20 min. The column was then washed with ethanol /water (19:1, v/v). Compounds identified were abscisic acid (ABA), ABA l'-glucoside (ABAGS), ABA glucose ester (ABAGE), phaseic acid (PA), dihydrophaseic acid (DPA). 55
Table 2.1 HPLC retention times of standard and marker compounds
Compound Retention time* (min)
ABA 46.0 t-ABA 41.7 ABAGS 33.0 epi-DPA 22.7 ABAGE 21.0 PA 18.0 DPA 12.0 DPAGS 5.3 E-aminobenzoic acid 5.7 E-hydroxybenzoic acid 12.3 Benzoic acid 28.0
* HPLC on a 7.8 mm (i.d.) x 300 mm µBondapak c 18 column with ethanol/water/acetic acid (84:504:1, by vol.) for 22 min at 4.0 ml/min, followed by a linear gradient with ethanol/water/acetic acid (158:508:1, by vol.) over 15 min and elution with the final solvent for 20 min.
detected by autoradiography. Valuable structural information about the metabolite was obtained using this procedure at the cost of a minute fraction of the labelled material. This information was used to plan strategies for the isolation of the metabolite. The relative mobilities of the metabolite and its derivatives
(Table 2.2) indicated the presence of a methylatable group and a number of acetylatable groups 56
Table 2.2 Chromatography of derivatives of DPAGS on silica gel HPTLC plates
Solvent A: toluene/ethyl acetate/acetic acid (25:15:2, by vol.) Solvent B: chloroform/methanol/water (75:22:3, by vol.)
RF Compound Solvent A Solvent B
DPAGS 0 0.1
Me-DPAGS 0 0.71
Ac-DPAGS 0.18 0.84
MeAc-DPAGS 0.44 1.00
2.3.5 Isolation of DPAGS
Radioactivity in the DPAGS fraction of tomatoes fed
[2-14c]ABA increased with incubation time up to about 6 days and then began to fall. The following proportions of counts in DPAGS were found: 1 day - 16.3%; 2 days -
21.0%; 5 days - 38%; 12 days - 28.5%.
DPAGS was purified by a series of HPLC steps. The metabolite was chromatographed as a free acid, a methyl ester and then as an O-acetyl methyl ester on a semi preparative reversed-phase (C 18) column. Although the compound gave a single symmetrical peak when run as a methyl ester, it split into two peaks when run as the
acetylmethyl derivative. The major peak was DPAGS and
the other peak, which has not been characterized,
increased in proportion to DPAGS with time. The retention time for each compound remained unchanged after 57
reacetylation so it is not an artefact of incomplete acetylation of DPAGS. Finally O-acetyl Me-DPAGS [35] was chromatographed on a normal-phase HPLC column and after the evaporation of the solvent the material was deposited as colourless crystals (m.p. 81 - 84°c, uncorrected).
2.3.6 Electrophoresis
Electrophoresis of DPAGS on plastic-backed cellulose plates at pH 6 produced evidence of a free carboxyl in the molecule (Table 2.3). Acidic compounds and DPAGS showed net movement towards the anode. The methyl ester of DPAGS and neutral standards did move slightly towards the cathode although this movement is attributed to electroosmosis.
2.3.7 Ultraviolet spectroscopy.
The UV spectrum of DPAGS in 4.8% ethanol in water is altered by pH. The wavelength of the single absorption maximum (~ax> shifted reversibly with a change in pH
(adjusted by the addition of lM HCl or lM KOH), while the methyl ester was unaffected.
pH ~ax (nm) DPAGS O-acetyl Me-DPAGS
11.5 250 265
3.0 268 265 58
Table 2.3 Movement of DPAGS, Me-DPAGS and related compounds during electrophoresis
+ indicates cationic movement; - indicates anionic movement. The movement (23 mm) of the neutral compounds towards the cathode is attributed to electroosmosis.
Compound Movement * Direction Distance (mm)
Methylene green dye (cation) + 83
ABA 35
DPAGS 14
ABAGE + 23
Me-DPAGS + 23
Glucose + 23
#Measured from point of application.
2.3.8 Mass Spectrometry
The chemical ionization mass spectrum of 0-acetyl
Me-DPAGS [35] (Table 2.4; Fig. 2.2) shows a molecular ion at m/z 626 and peaks at m/z 627 (M + 1), 655 (M + 29) and
667 (M + 41). This pattern of higher mass peaks frequently occur when methane is used as an ionizing gas.
The first fragment ion in both chemical ionization and electron impact spectra of ABA and DPA is the loss of 18 mass units which has been shown to be derived from the
1'- tertiary hydroxyl group (Gray et al., 1974, Zeevaart and Milborrow, 1976). The occurrence of this ion in the spectrum of methylated, acetylated DPAGS gives a clear indication that the 1'-hydroxyl group is unsubstituted. 59
Table 2.4 Chemical ionization mass spectrum of O-acetyl Me-DPAGS ester and the same compound derived
from RS-[l 1 - 18o]ABA
Methane was used as the ionizing gas
m/z % Comment m/z Percentage of region r16o]sample r18o]sample
110 5 Molecular-ion region 125 3 Sidechain and Me ester 626 58.45 54.07 169 42 S-2(CH3COOH)-CH3CO 627 33.80 31.35 211 3 S-2(CH3COOH) 628 6.34 10.97 247 34 A-CH3OH 629 1. 41 3.14 261 4 A-H2o 630 0 0.47 271 19 S-CH3COOH 279 27 M-(S+H2O)=A First-fragment-ion region 289 6 S-CH3CO 609 70.39 70.48 307 3 610 22.65 22.86 331 100 s 611 5.66 5.71 332 16 612 1.05 0. 96 567 11 MH-H2O-CH 3CO 613 0.24 0 577 2 MH-CH 3OH-H2O 595 10 MH-CH 3OH 609 79 MH-H 2O 610 25 611 5 626 4 molecular ion 627 3 M+l 655 6 M+29 667 1 M+41
S glucose tetraacetate - H2o 60
CH,OAc M/',-...;: -...;: AcO~o, 'o__ 'OH AcO~O COOCHl OAc 100 169
50 61 109 247
219 >, 100 "iii- 331 200 C: 100 Q) -C: Q) > 50 279 ~- Q) 271 a:
350 10x 100 1 609
50
567 595 626 655
500 600
m/z
Fig. 2.2 Chemical-ionization (methane) mass spectrum of 0-acetyl Me-DPAGS
The molecular ion at m/z 626 is also evidence that the
4'-hydroxyl has the glucosyl residue attached because if
it were free it would be expected to carry an additional
acetyl group giving a molecular weight of 668. The mass
spectrum of a sample biosynthesized from [l 1 - 18o]ABA
gives further evidence that the 609 peak is tetraacetyl
methyl ester of DPAGS minus the l'-hydroxyl group. The
molecular ion was enriched some 4% with 180 as shown by
the ion at M + 2/z (628). The absence of an increase in the peak at m/z 611 established that the loss of 18 mass
units in the normal spectrum is caused by the loss of the 61
1'-hydroxyl group (with the enriched sample losing 20 mass units, H218o).
The fragment ion at 331 can be attributed to tetraacetyl glucose - OH and the ions at m/z 289, 271,
169 due to loss of 2 molecules of acetic acid and one of ketene from the tetraacetyl glucose residue (Hogg and
Nagabhushan, 1972). Loss of the tetraacetylglucosyl residue from DPAGS produces the fragment ion at m/z 279 which then loses methanol from the methyl ester to give m/z 247. The fragment at m/z 125 is identified as the
side chain and methyl ester and indicates that the
1-carboxyl was methylated. Chemical-ionization mass
spectrometry of Me-DPAGS [34] with isobutane, ammonia and
[2H]ammonia as reagent gases has been studied in detail by Takeda et al (1984). The fragmentation of Me-DPAGS with these reagent gases was similar to MeAc-DPAGS with methane except the relative abundance of the ions from the glucosyl residue was lower.
2.3.9 NMR Spectroscopy
When [2H5 ]pyridine was used as a solvent, proton signals due to the water in the pyridine spread from 84.5
to 85. Homogated decoupling of the water signal
increased the quality of the rest of the spectrum but
could not overcome the loss of the part that contained
the anomeric proton (Fig 2.3). The methylated and acetylated metabolite was sufficiently soluble in
[2H]chloroform for a complete spectrum to be obtained at 62
(I) C: -0... >, a.. I\
(I) C: -0... >, a.. >, ·;:- ::, c. E +
7 6 5 4 3 2 1
ppm 6
Fig. 2.3 1H NMR spectrum of the O-acetylmethyl ester of DPAGS in [2H5 ]pyridine
The 100 MHz 1H NMR spectrum was obtained with 200 scans at 20°c. The region near o5 is distorted by homogated decoupling of the water signal. The superimposed signal of the anomeric proton is taken from another spectrum obtained without decoupling.
100 MHz, although there was little resolution of the glucosyl protons. A sample run at 300 MHz showed resolution of the glucosyl protons and enabled the sugar residue to be identified as glucose by comparison of the coupling constants with a sample of pentaacetyl
~-D-glucose (Table 2.5, Fig. 2.4). The spectra show features similar to those of phaseic, dihydrophaseic and abscisic acids. The C-4 and C-5 protons occur as doublets (J=l6 Hz at 88.06 and 86.33 respectively). The singlet at 85.77 is the C-2 proton, the singlet at 83.73 is the ester methyl and the 1H3 singlet at 82.01 is the 63
Crl,OAc ~,'', '-:: '-:: Ai.;0 ----l.-o 'o _ 'OH Ac~Q ' , COOCH, OAc
9 8 7 6 5 4 3 2 0 ppm o
Fig. 2.4 300 MHz 1H NMR spectrum of the O-acetylmethyl ester of DPAGS in [2H]chloroform
512 scans, 20°c. See table 2.5 for the identification of signals.
C-3 Me. The methyl signals at 80.96 and 1.20 are in similar positions to the same groups of 4'-O-acetyl
Me-DPA (80.94, C-6'Me; 1.16 C-2'Me).
The coupling constants for the glucosyl protons of
O-acetyl Me-DPAGS are the same as those of ~-glucose pentaacetate. However, the chemical shifts are different
for C-lH and C-5H which are 84.56 and 3.69 in the metabolite and 85.73 and 3.85 respectively in glucose pentaacetate. The position of the other glucosyl protons
are similar. 64
Table 2.5 1H NMR spectra of 4'-0-acetyl Me-DPA, 0-acetyl Me-DPAGS and ~-glucose pentaacetate in c2H]chloroform
The 4'-0-acetyl Me-DPA spectra were those reported previously (Milborrow, 1975). 300 MHz spectra of derivatized DPAGS were obtained on a sample of 500 µgin a 5 mm tube, 1 ml solvent, 512 accumulations 20°c. P-Glucose pentaacetate (1 mg) - 256 accumulations. s = singlet; d = doublet m = multiplet
0-acetyl Me-DPAGS 4'-0-acetyl Me-DPA
C-1 OMe s 03.73 03.72 C-2 H s 5.77 5.76 C-3 Me s 2.01 2.06 C-4 H d 8.06 J=l6 8.07 J=l6 C-5 H d 6.33 J=16 6.39 J=16 C-2' Me s 1.20 1.16 C-3' 2H m 1.6-2.0 1.20-2.14 C-4' H m 4.1-4.3 3.91-4.5 C-5' 2H m 1.6-1.7 1.20-2.14 C-6' Me s 0. 96 0.94 C-6' CH2 m 3.83 J=2,8 3.85 3.76 J=8 0-acetyl (3) 2.02-2.04 (1) 2.10 2.01
Glucosyl Residue P-Glucose pentaacetate
C-1 H d 4.56 J=8 5.73 J=8 C-2 H m 4.95 J=8,10 5.14 J=8,10 C-3 H m 5.20 J=l0,9 5.26 J=l0,9 C-4 H m 5.07 J=9,10 5.15 J=9,10 C-5 H m 3.69 J=2.5,5,10 3.85 J=2.2,5,10 C-6 H m 4.27 J=12,5 4.29 J=l2,5 C-6' H m 4.13 J=12,2.5 4.12 J=l2,2.2 65
2.3.10 Hydrolysis of DPAGS
Attempted hydrolysis of the conjugate by ammonia or alcoholic KOH failed to affect the bulk of the compound.
A small amount was hydrolysed to DPA and to a compound which co-chromatographed (TLC and GC) with PA. Acid hydrolysis (lM HCl, 100°c, 6 h) of DPAGS biosynthesized from [2-14c]ABA gave glucose and [14c]DPA (Fig. 2.5) as major products, as well as other 14c-labelled products.
An identical pattern 14c-labelled products was seen by thin-layer chromatography when free [14c]DPA was subjected to the same conditions.
a- and P-Glucosidase did not cause any detectable hydrolysis of DPAGS within 10 min, whereas ABAGE was almost completely hydrolysed by p-glucosidase and only slightly affected by a-glucosidase. After one hour a small amount of DPA (1/20 DPAGS) was released almost equally by a- and p-glucosidase. Therefore, in comparison with ABAGE, DPAGS is not a substrate for a- or p-glucosidase.
2.3.11 Identity of the sugar moiety
In addition to the identification of the sugar residue by NMR, it was confirmed by the use of Tes-tape paper. This is a commercial glucose test paper, which
contains glucose oxidase, peroxidase and o-tolidene. The
oxidation of glucose produces hydrogen peroxide which is
used by peroxidase to produce a blue compound from o-tolidene (Comer, 1956; Seltzer and Loveall, 1958). 66
1.0 a
0.5
~ ~ -en C: (1) ) '"C (1) 0 > 0 0.5 1.0 -co Rf Q) a: 1.0 b
0.5
0 .__....______. ______.,..._, _ __. 0.5 1.0 Rf
Fig. 2.5 Acid hydrolysis products of DPAGS
Densitometer scans of the autoradiogram of acid hydrolysis products formed from [14cJDPAGS and [14CJDPA. The two mixtures were chromatographed on one plate. (a) DPAGS (b) DPA 67
This assay is specific for D-glucose. The characteristic blue colour was produced by a sample of the products of acid hydrolysis of DPAGS.
This result was also confirmed by GC-MS of trimethylsilylated product. Two peaks were obtained with identical cracking patterns and at the same retention times as those of the a- and ~-anomers of trimethylsilylated glucose.
2.3.12 Metabolism of degradation products of [14c]ABA
Samples of 14c-labelled PA, DPA, epi-DPA were fed to tomato shoots and analysed after 39 h. Shoots fed
[14c]DPA were analysed after 7 days. Analysis of the extracts by HPLC (Fig. 2.6) showed that DPAGS was formed slowly from [14c]phaseic acid and more readily from
[14c]dihydrophaseic acid. epi-DPA was also converted
into a more polar product but this was well separated
from DPAGS. When [14c]DPAGS was refed to tomato shoots
an even more polar metabolite was formed and accounted
for 85% of the radioactivity fed. 68
100 PA 50 PA
DPAGS DPA "'C gl~i .. ,,...... ;[• Q) 100 I.. Q) > DPAGS DPA 0 0 50 Q) I...... ,> DPA ·- :::~;:.~$-:f.,... ,., ... '..... ·- ..... ·-· ··········· .,.,,. -.-;, ...... ,> 100 0 m epi-DPA 0 "'C 50 m a: ~iDPA
~ •7-~ ·,·,·,···.'.·o"O""• :~.;:,: ... ,...... ·::···:,,·:"
50
DPAGS o----+-----~---==~---~~= ·-~t ...... ------..------J + 0 5 10 15 20 25 Aqueous residue Retention time, min
Fig. 2.6 Metabolism of degradation products formed from [14CJABA
80 000 dpm of phaseic acid, DPA and epi-DPA (all 2.3 µCi/mol) and 800 000 dpm of DPAGS (£. 1.0 µCi/mol) were fed to tomato shoots (70 g) which were analysed after 39 h; the shoots fed [14cJDPAGS were analysed after 7 days. The first, most polar fraction was eluted from the Sep-Pak cartridges by 0.2% aqueous acetic acid. The remaining compounds were analysed by HPLC and the percentages of the total 14c recovered at various retention times are shown. Each labelled fraction was identified by further chromatography. 69
2.4 DISCUSSION
2.4.1 Occurrence
In the initial isolations of DPAGS, the metabolite was derived from RS-[2-14c]ABA and its purification was monitored by the presence of radioactivity. Once the methods for isolation had been developed it was possible to isolate endogenous DPAGS from tomato plants. Further evidence for the metabolite's natural occurrence comes from the specific activity of the metabolite which was several times less than that of the [14c]ABA supplied.
Subsequent work has shown that DPAGS is a metabolite of
S-ABA (see Chapter 3).
2.4.2 Identity of the aglycone
The 1H NMR spectrum of 0-acetyl Me-DPAGS shows signals attributable to DPA superimposed on the glucose moiety. The signals from DPA in DPAGS are very close to those of 4'-0-acetyl Me-DPA. The presence of a 1tt3 signal at 6 3.73 attributable to a methyl ester. The C-1 must therefore have a free carboxyl group. The pH-dependent alteration of the UV spectrum of the underivatized metabolite and its electrophoretic properties provide
further support for a free carboxyl. The mass spectrum
shows fragment ions that are attributable to DPA and an
acetylated hexose. Furthermore, DPA as well as
14c-labelled rearrangement products, similar to those
produced by DPA under the same conditions, were detected
after acid hydrolysis of the metabolite (Fig. 2.5). 70
2.4.3 The sugar moiety
The sugar released by acid hydrolysis was identified as glucose by GC-MS of the TMS-derivative and by a method based on glucose oxidase. This conclusion was supported by the 1H NMR data of a sample of MeAc-DPAGS. Coupling constants in excess of 8 Hz were found for protons on adjacent carbon atoms in the pyranose ring (C-1 to C-2,
C-2 to C-3 and so on). Coupling constants in the range of 8.6 - 11 Hz are found for vicinal protons which are diaxial, whereas protons in equatorial - axial or equatorial-equatorial position exhibit coupling constants less than 5.8 Hz (Coxan, 1972). Therefore, the protons on the pyranose-ring carbon atoms of the sugar are all axial, which is the configuration of ~-glucose. The presence of one signal for the anomeric proton is evidence that glucose is attached to DPA by the anomeric carbon. If this were not the case signals for both a and ~-glucose would be expected.
2.4.4 Site of attachment of the glucosyl residue
There are three possible loci for the attachment of the glucosyl residue to DPA: the C-1 carboxyl group and
the C-1' or C-4' hydroxyl group. The first can be eliminated as the C-1 carboxyl has been shown to be free
by the pH dependence of its UV spectrum, its partition
characteristics from acidic and basic solutions and its reaction with diazomethane. Furthermore, its stability 71
to alkaline hydrolysis is characteristic of a glycosidic linkage and not a glucose ester.
The 1'-hydroxyl group of abscisic, phaseic and dihydrophaseic acids cannot be acetylated by the conventional method of acetic anhydride in pyridine whereas the 4'-hydroxyl group of DPA reacts readily
(Zeevaart and Milborrow, 1976). Acetylation of the metabolite forms a tetraacetate as shown by the integral of the methyl signals in the NMR spectrum and the molecular ion in the chemical ionization mass spectrum.
The four acetyl groups are considered to be attached to the glucosyl moiety because, if the glucosyl residue were attached to the 1'-hydroxyl group, then the glucose would carry four acetyl groups and the C-4' hydroxyl group would also carry one, giving a molecular weight of 668.
This ion was not present; the molecular weight was 626.
Furthermore, a fragment ion at m/z 331 in the mass
spectrum of the acetylated metabolite is absent from that
of unacetylated material. This fragment ion is
attributed to the tetraacetyl glucose moiety, indicating
that all four acetyl groups are on the glucose. Barrow
et al. (1971) reported that a secondary hydroxyl group of
fusicoccin could not be acetylated by acetic anhydride in
pyridine, presumably because it was shielded in some way
by a nearby glucose residue. This secondary hydroxyl
group of fusicoccin was readily oxidized to a ketone by
Jones reagent while 0-acetyl Me-DPAGS was unaffected by
this treatment and when rerun on the c18 reversed-phase 72
column showed an identical retention time. It appears, therefore, that the secondary 4'-hydroxyl group of DPA in acetylated Me-DPAGS was already substituted.
The 4'-hydroxyl group of DPA has been shown to be readily oxidized by Jones reagent (Zeevaart and
Milborrow, 1976) , so the failure of the reagent to oxidize the metabolite shows that the 4'-hydroxyl is blocked and is, therefore, the site of attachment of the glucosyl residue. This unreactivity to Jones reagent provides further support for the attachment of C-1 of glucose to the aglycone.
The 1'-hydroxyl group of ABA can be glucosylated in vivo but the ABA 1'-0-glucoside is quite unstable to mild basic hydrolysis whereas the DPA metabolite is not hydro lysed by 3M KOH in aqueous ethanol (2:1, v/v). The resistance of the DPA metabolite to hydrolysis again suggests that the 4'-hydroxyl group is the site of attachment of the glucosyl residue.
The first fragment ion in the mass spectrum of ABA
is the result of the loss of 18 mass units caused by the elimination of the 1'-hydroxyl group (Gray et al., 1974).
This fragmentation mode is particularly prominent in the
chemical ionization mass spectrum (Loveys and Milborrow,
1981). Methyl DPA and 0-acetyl Me-DPA also show this
loss of 18 (Zeevaart and Milborrow, 1976). A similar
loss of 18 mass units occurs with the acetylated methyl 73
ester of the metabolite, and this also suggests that the
1'-hydroxyl group is not glucosylated.
Thus the metabolite was identified as 4'-0-P-D glucosyl dihydrophaseic acid to conform with the nomenclature proposed for conjugates of ABA (Loveys and
Milborrow, 1981) and using the numbering system of the atoms in ABA. The abbreviation "DPAGS" has been proposed
(Milborrow and Vaughan, 1982).
2.4.5 Relationship between DPAGS and metabolites
isolated by others
Sondheimer et al. (1974) partially characterized a product of ~-[2-14c]ABA fed to ash (Fraxinus americana) seed. This metabolite, designated "M-3", was more polar than DPA, was not hydrolysed by base, reacted with diazomethane and was acetylated by acetic anhydride. The
TLC properties of the derivatives were similar to DPAGS.
Periodate oxidation resulted in the formation of a
compound which co-chromatographed with DPA. M-3 was not
effected by treatment with p-glucosidase, emulsin or
p-glucuronidase. These properties are similar to those
of DPAGS and it is probable that M-3 and DPAGS are the
same compound.
A compound partially identified as the 4'-aldopyran
oside of DPA, based on mass-spectral evidence, was
isolated from soybean leaves and pods supplied with
RS-[2-14c]ABA (Setter et al.,1981). Although the sugar 74
moiety was not identified this compound is almost certainly identical to our material.
Hirai and Koshimizu (1983) have independently identified DPA 4'-0-glucoside as an endogenous constituent of avocado (Persea americana) fruit and fruit of grape (Vitis vinifera). The compound was characterized by a combination of HPLC, GC, IR, MS and
NMR methods and the structure was confirmed by the synthesis of the acetyl methyl ester of DPAGS from a-acetobromoglucose and Me-DPA.
DPA 4'-0-glucoside appears to be a major metabolite
of ABA and has been found to occur endogenously in tomato, avocado and grape. It is a metabolite of
exogenous ABA supplied to tomato, pea (see Chapter 4),
soybean, ash and there is chromatographic evidence for
its occurrence in spinach, cassava, guava, eggplant,
macadamia, sunflower, broad bean and grape as a product
of exogenously applied RS-[2-14C]ABA (Loveys and
Milborrow, 1984). Experiments in which metabolites of
ABA have been refed to tomatoes indicate that although
DPAGS is a major product of metabolism, it is not the
final one. 3
Resolution by HPLC of RS-Me-cis-diol of ABA and Metabolism of .B.- and .§.
Abscisic Acids
75 76
3.1 INTRODUCTION
All crystalline isolates of abscisic acid that have been subjected to optical rotatory dispersion-circular dichroism analysis have shown the same specific rotation
(Milborrow, 1974). It is assumed, therefore, that naturally occurring ABA exists exclusively as the (+)-S enantiomer. However, most experiments in which exogenous
ABA, especially radiolabelled material, has been fed have used racemic, synthetic material, although differences in the physiological effects and metabolism of the natural and unnatural enantiomers have been reported.
The resolution of racemic ABA was first achieved by
fractional crystallization of the (-)-brucine salt of ABA
in methanol by Cornforth et al. (1967). The fractions were further enriched by selective solubilization and
microsublimation. The resolved R- and S-enantiomers were
found to be equally active when assayed on dissected
wheat embryos (Milborrow, 1968) and a similar result was
obtained by Sondheimer et al. (1971) who used acetyl
cellulose chromatography to prepare fractions slightly
enriched with one or other enantiomer and optically
active ABA was isolated by selective solubilization.
They reported that R- and S- ABA were almost equally
active in a barley half-seed a-amylase and a cotton
explant abscission assay. In a bean embryo and barley 77
embryo assay the S-enantiomer was up to three times more potent than the R-enantiomer. By contrast, stomatal closure exhibits strict steric requirements. S-ABA rapidly causes stomatal closure while R-ABA has little effect (Cummins and Sondheimer, 1973; Kriedemann et al.,
1972). A similar stereospecificity was found for the uptake carrier in root segments of Phaseolus coccineus in which only ~-ABA is taken up (Milborrow and Rubery,
1985). The action of ABA is thought to be of two kinds: i) slow reactions involving protein synthesis which respond to both R- and S- ABA ii) fast reactions which occur before protein synthesis begins and respond to S-ABA (Milborrow, 1980).
Milborrow (1970) found that there was differential metabolism of R- and S- enantiomers of ABA in that when racemic [2-14c]ABA was fed to tomato shoots ABAGE contained an excess of the (-)-R-enantiomer and the balance (~-ABA) had been oxidised to 8'-hydroxy ABA.
These results have been confirmed by Sondheimer et al.
(1971, 1974) who performed experiments in which R-[2-14c]
and ~-[2-14c]ABA were fed to ash seeds and bean axes. S-ABA was found to enter bean axes at a higher rate and was metabolized to phaseic and dihydrophaseic acids
whereas R-ABA was metabolized more slowly with only a
small amount going to PA and DPA.
Mertens et al. (1982) have prepared R- and~- ABA of high optical purity by immunoaffinity chromatography. 78
When R- and ~-[ 3H]ABA, prepared by this method, were supplied to leaf discs of Vicia faba phaseic and dihydro phaseic acid were formed rapidly from S-ABA as well as two minor acidic metabolites and two base labile conjugates of ABA. The R-enantiomer was metabolized slowly to the two conjugates and minor acidic metabolites but not to PA or DPA. As well as metabolites that are derived principally from ~-ABA, one metabolite,
7'-hydroxyabscisic acid [18], has been shown to be formed from R-ABA only by leaves of Xanthium strumarium (Boyer and Zeevaart, 1985; Zeevaart, 1985).
The excess R- and S- ABA in a sample can be measured by optical rotatory dispersion and circular dichroism spectrometry (Milborrow, 1978a). Unfortunately the apparatus is costly and relatively large quantities of sample are required at a high level of UV purity. As can be deduced from the forgoing, there is a considerable need for a supply of labelled R- and S- ABA so that the source of metabolites and the concentrations of either enantiomer can be followed. The costs and technical difficulties of the existing methods were such that an
alternative HPLC method was sought by which ABA was
resolved in a column with a chiral stationary phase.
This method, which has advantages in time and cost, was
used to prepare 14c-labelled R- and~- abscisic acids and
their metabolism in tomato shoots was studied. 79
,; ,; R 1 0,,. COOR 2
Rl R2 [2] H H [ 36) H CH3 [37) Ac CH3
COOR2
R12
[ 38] H
[39) H
[ 40] Ac
COOR2
Rl
[3] H
[ 41] H
[ 42] Ac 80
3.2 MATERIALS AND METHODS
3.2.1 Isolation of (+)-S-ABA
Red wine is a rich and convenient source of natural
(~)-ABA (Loveys and Downton, 1979) and a sample was obtained from it to use as a chromatographic standard.
Red wine (2 1) was evaporated to remove alcohol and then the pH was adjusted to 7.0 with sodium bicarbonate. The residue was extracted three times with ether to remove neutral compounds. The aqueous phase was acidified (pH
2.5-3.0) with 2 M sulphuric acid and the acid fraction containing the ABA was extracted with an equal volume of ether (3 times). The acid ether fraction was injected onto a 7.8 mm (i.d.) x 300 mm µBondpak c18 HPLC column (Waters Associates), eluted at 4.0 ml/min with ethanol/ water/acetic acid (158:508:1, by vol.) and the peak with a retention time of 12 min was collected. This fraction was methylated with ethereal diazomethane and chromato graphed on a 7.8 mm (i.d.) x 300 mm µPorasil column
(Waters Associates) with hexane/isopropanol (39:1, v/v)
as a mobile phase at 4.0 ml/min. Me-ABA had a retention time of 12.5 min and was used as a standard of S-Me-ABA.
3.2.2 Preparation of Me-1'-,4'-cis- and Me-1'-,4'-trans
diol of ABA RS-ABA (10 mg) was methylated with ethereal
diazomethane. Me-ABA was dissolved in methanol (0.8 ml) and water (0.4 ml) was added. The sample was placed in ice and sodium borohydride (3 mg) added at 0, 5 and 81
10 min. After 30 min the reaction was stopped by the dropwise addition of 1 M sulphuric acid until the solution was pH 2.5-3.
Methanol was evaporated under nitrogen and the products extracted with ether (3 times). The products were separated by HPLC on a 7.8 (i.d.) x 300 mm µBondapak c18 column with a mobile phase of ethanol/water/acetic acid (317:517:1, by vol.) delivered at 4.0 ml/min.
Me-1' ,4'-trans-diol of ABA [41], Me-ABA and Me-1' ,4'-cis diol of ABA [36] had retention times of 8.2, 10 and 16 min respectively.
3.2.3 Resolution of RS-Me-1'-,4'-cis-diol of ABA
RS-Me-1' ,4'-cis-diol of ABA was loaded onto a 4.6
(i.d.) x 250 mm Pirkle Type 1-A column (Regis, Morton
Grove, IL., U.S.A.) and eluted with hexane/isopropanol
(9:1, v/v) at 1.0 ml/min. Peaks eluted after 21 and 23 min were the 1'~-,4'R- [36] and l'R-,4'~-4'-dihydro
abscisic acid methyl ester [39] ((+)-Me-cis- and
(-)-Me-cis-diol of ABA) respectively. A sample of
Me-1' ,4'-cis-diol prepared from natural ~-ABA gave one
peak only (21 min). Improved resolution of RS-Me-cis
diol with baseline separation between the peaks was
achieved by connecting a Pirkle Type 1-A and a Pirkle
covalent R-phenylglycine column in series, eluting with
hexane/isopropanol (97:3, v/v) at 2.0 ml/min and
recycling the eluate up to four times. 82
RS-1' ,4'-[2-14c]cis-diol of ABA methyl ester (100 µg
[13.2 mCi/mmol]) was resolved as described above. An equal amount of unlabelled l'R-enantiomer was added to 14 the l'S,4'R--- [2- c]-4'-dihydroabscisic acid methyl ester and the mixture resolved again. The initially unlabelled l'R,4'S-dihydroabscisic acid methyl ester was discarded.
The complementary procedure was used for the l'R,4'f-[2-14c] enantiomer [39]. This step would remove any contaminating, 14c-labelled enantiomer imperfectly separated by the first chromatography and any contaminating enantiomer remaining would be of extremely low specific activity.
R- and f-methyl abscisates were obtained by oxidation of the Me-1' ,4'-cis-diol of ABA with a 10-fold excess of Mno 2 . The oxidation mixture in 2 ml dry chloroform was continuously stirred at 20°c. The duration of the oxidation depended on the reactivity of the batch of Mno2 used, reaction times required ranged from 30 min to 72 h. The reaction mixture was loaded
onto a 200 x 200 mm silica gel 60 F254 TLC plate (E. Merck, Darmstadt) and chromatographed in hexane/ethyl
acetate (2:1, v/v) to separate Me-ABA from unreacted
Me-cis-diol and Mno2 . ABA was released by alkaline hydrolysis in 2 M aqueous KOH/ethanol (1:2, v/v) at 20°c
for 30 min. 83
3.2.4 Improved resolution procedure
RS-1' ,4'-cis-diol of ABA methyl ester (1 mg) was prepared as described above, and resolved into its enantiomers by HPLC on a 4.6 (i.d.) x 250 mm Pirkle covalent R-phenyl- glycine column connected in series with 2 Pirkle Type 1-A columns with the mobile phase: isopropanol/hexane (2:23, v/v), at 2.0 ml/min. The
S-enantiomer had a retention time of 33.5 min and the R-enantiomer 36.5 min.
3.2.5 An improved oxidation using pyridinium
chlorochromate
The Me-1' ,4'-diols of ABA were oxidized to Me-ABA using pyridinium chlorochromate on alumina (Cheng et al.,
1980). Pyridinium chlorochromate was prepared by the method of Corey and Suggs (1975). Chromium trioxide
(6 g) was added rapidly with stirring to 6 M HCl (11 ml).
After 5 min the solution was cooled to 10°c and pyridine
(4.8 ml) was added over 10 min. When a yellow-orange solid formed, it was heated to 60°c and alumina (50 g)
added to the solution with stirring. After rotary evaporation the orange solid was dried in vacuo for 2 h
at 20°c. The reagent was stored in the dark.
The sample to be oxidized was dissolved in 1 ml
hexane (dried by the addition of molecular sieve) and a
three-fold excess of pyridinium chlorochromate was added.
After stirring for 2 h in darkness, the hexane was
evaporated. The Me-ABA was separated from the reagent 84
and unreacted compounds by chromatography on silica gel
HPTLC plates developed with hexane/ethyl acetate (2:1, v/v).
3.2.6 Plant feeding and extraction of metabolites
Tomato plants were described in section 2.2.2.
2.8 µci RS-[2-14c]ABA [25.6 mCi/mmol] was fed to 30 g
(4) tomato (Lycopersicon esculentum cv. Grosse Lisse) shoots. 0.28 µCi f-[2-14c]ABA [1.05 nCi/mmol] was fed to two (7.0 g) tomato shoots and 0.23 µCi R-[2-14c]ABA
[1.05 mCi/mmol] fed to two (10g) tomato shoots. After 2 d the plants were homogenised in acetone/acetic acid
(99:1, v/v) containing BHT (100 mg/1). The metabolites were extracted (Section 2.2.4/5) and separated by HPLC
(Section 2.2.7) as described earlier.
3.2.7 Thin-layer chromatography
One-fifth of the acetone extract of tomatoes fed with R- or f-ABA was further analysed by TLC. Acetone was evaporated from each extract, the residue acidified
(pH 2.5-3.0) with 2 M sulphuric acid and extracted three times with ethyl acetate. The ethyl acetate extract of
each sample was applied to half of the origin of a 200 mm
x 100 mm silica gel 60 F254 HPTLC plate (E. Merck, Darmstadt) and developed in toluene/ethyl acetate/acetic
acid (25:15:2, by vol.). Narrow strips were removed from
the plates and radioactivity measured by liquid
scintillation counting. 85
3.2.8 Determination of the proportion of R-[ 14c]ABA to
S-[14c]ABA in ABA and its conjugates
Conjugates of ABA were hydrolysed with aqueous ammonium hydroxide (sp. gr. 0.880) at 27°c for 30 min as described by Loveys and Milborrow (1981). 400 µg RS-ABA were added to the samples of [14c]ABA released by hydrolysis and the samples were methylated with diazomethane. The Me-ABA was purified by HPLC on a 7.8
(i.d.) x 300mm µBondapak c 18 column with ethanol/water/ acetic acid (256:475:1, by vol.) at 4.0 ml/min. Me-ABA was reduced, resolved as described above and the radioactivity in the resolved enantiomers determined by liquid scintillation counting.
3.3 RESULTS
3.3.1 Resolution of the enantiomers of Me-cis-diol of
ABA
Pirkle HPLC columns have been used to separate enantiomers of several compounds (Pirkle and House, 1979) but attempts to separate the enantiomers of RS-ABA and
some of its derivatives on a Pirkle column were unsuccessful. Racemic ABA, Me-ABA, 2-trans-ABA,
Me-2-trans-ABA, Me-1' ,4'-trans-diol of ABA [3] and
4'-O-acetyl Me-1'4'-trans-diol of ABA [42] all
chromatographed as single peaks. However, the RS-Me
l',4'-cis-diol of ABA [36]/[39] (Fig. 3.la) and its
4'-O-acetyl derivative [37]/[40] were both separated into 86
0.4 a b C
E C "
0 10 20 0 10 20 0 10 20
Retention time, min
Fig. 3.1 Resolution of the enantiomers of RS-Me-1',4'-cis diol of ABA by HPLC on a Pirkle Type 1-A column with hexane/ isopropanol (9:1, v/v) as the mobile phase at 1.0 ml/min.
a) 200 µg RS-Me-1' ,4'-cis-diol of ABA
b) 100 µg Me-l'S,4'R-4'dihydroabscisic acid ((+)-Me-cis-diol)
c) 50 µg Me-l'S,4'R-4'-dihydroabscisic acid+ 50 µg RS-Me- 1' ,4'-cis-diol of ABA
their enantiomers by HPLC on a Pirkle Type 1-A column with a mobile phase of hexane/isopropanol (9:1, v/v) delivered at 1.0 ml/min. A sample of Me-1'~,4'R-4'
-dihydroabscisic acid of ABA [36] formed by the reduction 87
of a sample of natural ~-ABA gave a single peak at 21 min under the same conditions (Fig. 3.lb) and when injected with a sample of the racemic compound caused the peak at
21 min to increase in height while the peak at 23 min remained unchanged (Fig. 3.lc). This confirmed that separation of the enantiomers had been achieved and identified the first peak as the ~-enantiomer. The separation of the enantiomers was increased by recycling the column effluent through a Pirkle Type 1-A column and a Pirkle covalent R-phenylglycine column in series (Fig.
3.2). Better than baseline separation was achieved by four cycles through the column (248 min).
The reduction of Me-ABA to the Me-1' ,4'-cis- and trans- diols, separation of the enantiomers by HPLC, oxidation of the diols to Me-ABA and saponification to release R- and S-ABA gave an overall yield of 20% from the original ABA (i.e. 10% in each enantiomer). However, half of the ABA is reduced to Me-1' ,4'-trans-diol [41], which is not resolved. The yield can be increased by
re-oxidizing Me-1' ,4'-trans-diol to Me-ABA and carrying
out a second reduction.
3.3.2 Improved resolution and oxidation procedure
Two improvements to the procedure described above
shortened the time required for HPLC and increased the
yield of R- and S-ABA. The HPLC step was shortened by
using an extra Pirkle Type 1-A column in series with the
Pirkle covalent R-phenylglycine and Type 1-A columns used 88
0.3
E C ' Q) 0 C a, ..0.... 0 \ \. \. 0 \_ l 0 100 200 300 Retention time, min Fig. 3.2 Resolution of 300 µg RS-Me-1' ,4'-cis-diol of ABA; 2 Pirkle HPLC columns connected in series and eluted with hexane/isopropanol (97:3, v/v) at 2.0 rnl/rnin. The column effluent was recycled. previously. The enantiomers of racemic Me-cis-diol were resolved to baseline in under 40 min when eluted from the three pirkle columns with 8% isopropanol in hexane at 2.0 ml/min (Fig. 3.3). Oxidation of the methyl ester of the cis- and trans-diols with pyridinium chlorochromate on alumina markedly increased the yield of Me-ABA. When a sample of Me-cis-diol of ABA was oxidized with pyridinium chlorochromate and separated from the reagent and unreacted material by chromatography on silica gel HPTLC 89 E ,.....C CD C\I Q) u C 0 10 20 30 40 Retention time, min. Fig. 3.3 HPLC separation of the reduction products of 1 mg RS-Me-ABA on 3 Pirkle columns eluted with hexane/isopropanol (185:15, v/v) at 2.0 ml/min. Me-trans-diol, 18.3 min; Me-ABA, 24.5 min; (+)-Me-cis-diol, 33.5 min; (-)-Me-cis-diol, 36.5 min. plates, the Me-ABA eluted from the silica contained 85% of the radioactivity of the Me-cis-diol that was oxidized. This was an improvement on oxidation with manganese dioxide, which showed batch to batch variation and yields of Me-ABA which varied between 20 and 65%. 3.3.3 Metabolism of R- and S-[2-14c]ABA R- and ~-[2-14c]ABA, prepared as described in Section 3.2.3, were fed to tomato shoots. The metabolites were extracted after 2 days and separated by 90 reversed-phase HPLC (Fig. 3.4). There were distinct differences between the products formed from S-[2-14cJABA and R-[2-14cJABA. The clearest difference perceived was the occurrence of DPA-4'-glucoside (DPAGS) [30] as the major metabolite formed from ~-[2-14c]ABA. This compound was not detected when the fraction derived from R-[2-14c]ABA was analysed. The second most abundant product of ~-[2-14cJABA was phaseic acid (PA) [19] followed by ABA glucose ester [28] and ABA-1'-glucoside [29]. By contrast, ABA glucose ester and ABA glucoside were the major products from R-[2-14c]ABA. Small quantities of labelled material co-chromatographed with PA and DPA [20]. An uncharacterized conjugate of ABA was found (retention time, 28 min) in the extracts of R- and ~-[2-14cJABA. Other unidentified, labelled materials were detected including a few in the ethanol wash of the column (from 55 min, Fig. 3.4). Analysis of the ethyl acetate extracts by TLC gave results in agreement with the HPLC results for the free acid metabolites (Fig. 3.5). Conjugates extracted into the ethyl acetate fraction remained at the origin of TLC plates developed in toluene/ethyl acetate/acetic acid (25:15:2, by vol.). The ethyl acetate extract of tomatoes supplied with R-[2-14CJABA contained labelled materials which chromatographed at RFs corresponding to DPA [20], epi-DPA [24], PA [19] and ABA. ABA was the major unconjugated compound, phaseic acid was the most abundant of the free acid products formed from 91 150 a DPAGS E c. 10 -0 > -> 0 -as 0 -0 as 5 a: ..,X I ,...0 0 20 40 60 Retention time, min 15 b E c. -o 100 > > 0 -as 0 -0 ABAGE ~ 50 ..,X I ,...0 Retention time, min Fig. 3.4 HPLC separation of the products of ~-[2-14cJABA (a) and ~-[2-14cJABA(b) in tomato shoots. The chromato graphic conditions were the same as those described in the legend to Fig. 2.1. 92 4 a E 4 E Q b Q ~ ~ ~ ~ ·;;:: ~ - ~ .2: 0 -0 ~ ~ .Q .Q ~ 2 ~ 2 ~ ~ ~ ~ ~ X I ~ I 0 0 ~ ~ 0.5 ,n 0.5 ,n RF RF Fig. 3.5 Thin-layer chromatography of ethyl acetate extracts of tomatoes fed ~-[2-14c]ABA(a) and ~-[2-14cJABA(b). The samples were applied to the origin of 200 mm x 100 mm silica gel HPTLC plates and developed with toluene/ethyl acetate/acetic acid (25:15:2, by vol.). Standards had the following RF values: ABA, 0.67; !-ABA, 0.73; PA, 0.49; epi-DPA, 0.30; DPA, 0.24. ~-[2-14c]ABA, others were present in much smaller amounts. 3.3.4 Proportion of R- and S-ABA in conjugates. One of the advantages of the HPLC method of resolution is that it allows the determination of the ratio of R-ABA to S-ABA in small samples and even allows the specific activity of each enantiomer to be calculated. This is done by hydrolysing the conjugates of ABA and then the ABA released is methylated and reduced to the diols. The Me-cis-diol is resolved by 93 Table 3.1 The proportion of R-[2-14cJ and S-[2-14cJABA in ABA and its conjugates in extracts of tomato shoots fed RS-[2-14CJABA Compound ~-[2-14cJABA:~-[2-14cJABA ABA 6.1:1 ABAGE 5.8:1 ABAGS 29.4:1 Uncharacterised conjugate 8.3:1 HPLC on Pirkle columns and the amount of radioactivity in each enantiomer determined. The conjugates of ABA, produced from RS-[2-14c]ABA supplied to tomato shoots, were analysed with this procedure and the proportion of R- and ~-[2-14c]ABA in each conjugate was determined. Conjugates of ABA formed from ~-[2-14c]ABA contain a preponderance of the R-enantiomer (Table 3.1) and the ratio of R-[ 14c]ABA to ~-[14c]ABA ranged from 5.8:1 for the glucose ester to 29:1 for ABA glucoside. The free ABA remaining also contains an excess of the R-[ 14c] enantiomer (6.1:1) .) 94 3.4 DISCUSSION ABA is intensely optically active so it is surprising, at first sight, that in experiments in which growth is assayed, the R- and S-enantiomers are equally or almost equally active. It has been proposed that both R- and S- forms can attach to some active site(s). This could occur if the 7'-methyl group of one enantiomer takes the place occupied by a 6'-methyl of the other (Milborrow, 1974). The finding of Boyer and Zeevaart (1985) that the 7'-methyl group of ABA is hydroxylated and is formed from only R-ABA suggests that the site of hydroxylation may accommodate the two enantiomers in a similar fashion. The molecule is not intrinsically highly asymmetric and an alternative projection of the structural formula emphasizes this [43]. ; 0 CH= CH-CCH3 = CH-C '-OH [43] Reduction of the 4'-ketone of ABA generates a second chiral centre in close proximity to the l' chiral centre and enables RS-Me-1' ,4'-cis-diol to be separated into its enantiomers by HPLC on a column having a chiral 95 stationary phase. The chemical reactions involved in the production of samples of R- and ~-ABA do not give quantitative yields when carried out on the very small scale reported by Mertens et al. (1982). However, the HPLC resolution of RS-Me-1' ,4'-cis-diol of ABA on a chiral- stationary phase column enables large amounts of R- and ~-ABA of high optical purity to be produced at much less cost than existing methods. An added advantage is that the method also allows the proportion of R- and ~-ABA in a sample to be measured. If the ABA is labelled the specific activity of each enantiomer can be determined, although this is limited to samples containing in excess of 1 µg of each enantiomer. However, the proportion of labelled R- to S-ABA in very small samples of ABA may be determined by adding unlabelled ABA before reducing the sample and is only limited by the method of detecting the label. When RS-ABA is fed to tomato shoots the amounts of several metabolites rise because material derived from the unnatural R-enantiomer accumulates. The amounts of the 4'-glucoside of DPA shows the most extreme difference between the products formed from R- and ~-ABA. It is derived almost exclusively from ~-ABA and so is a more important metabolite of endogenous ABA than was first realized. By contrast, the conjugates of ABA are the principal products of R-ABA, and their importance is over emphasised when the products of RS-[14C]ABA are examined. 96 The metabolites formed from R- and ~-ABA samples were identified by co-chromatography with authentic standards. The presence of labelled material from R- and ~-ABA adjacent to PA and DPA markers (or at the same HPLC retention time) was taken as evidence that optical or stereoisomers of these compounds had been formed (Scheme 1), however, a more rigorous identification of the products is required. The insertion of the same new chiral centre into R- and S-ABA will produce two different stereoisomers ([19] and [45]) which should be separable by conventional chromatographic procedures, although this may be slight in some systems. However, if ABA was hydroxylated at the C-9'-position instead of C-8' (6'-pro-~-methyl group) a mirror-image form of PA [44] would be produced which would not be separable by conventional chromatography. The chemical reactions involved in producing R- and S-ABA from resolved Me-cis-diols has subsequently been shown to cause racemization of the resolved diols and results in ABA contaminated some 1% with the opposite enantiomer (see Chapt. 4). However, this degree of contamination is insufficient to account for the amount "PA" or "DPA" produced from R-ABA. Racemase activity has not been ruled out, although its presence is considered unlikely because no labelled DPA-4'-glucoside was detected in the plants supplied with R-ABA. This was in spite of there being more radioactivity in the DPA fraction of tomatoes supplied with R-ABA. This suggests 97 that the enzyme which glucosylates DPA is specific for the material derived from S-ABA and that no racemization of ABA or subsequent metabolites had occurred, either physicochemically or by the action of a racemase. COOH COOH 0 0 [1) .S-ABA [19) 1'~,2'8,6'8-PA COOH [44) 1'B,2'S,6'S-PA COOH 0 [4) !!-ABA 0 COOH [45) 1'8,2'8,6'8-PA Scheme 3.1 The formation of and isomeric phaseic acids from R- and S-ABA. 4 The Chemistry, Natural Occurrence and Metabolism of the 1' ,4'-diols of ABA 98 99 4.1 INTRODUCTION The reduction of abscisic acid with sodium borohydride produces the 1' ,4'-cis- [2] and 1' ,4'-trans diol of ABA [3] in approximately equal amounts. The diols have been used in structural and biosynthetic studies and have been isolated from biological material. The biological activity of crude "dormin" extracts was slowly destroyed by treatment with sodium borohydride (Robinson, 1962). Similar treatment of a purified sample of dormin caused a loss of biological activity which was accompanied by a change in the molecule (Cornforth et al., 1965b). The products of reduction were separated chromatographically and identified by comparison with a sample of 1' ,4'-cis-diol of ABA which was synthesized by an unequivocable route. When the racemic epidioxide [46] was hydrogenated over Lindlar's catalyst the cis diol only is produced. The cis-diol produced by this method was used as a chromatographic marker to identify the reduction products of ABA separated by column chromatography. The diols were then used to determine the absolute stereochemistry of ABA (Cornforth et al., 1967) and have also been used in conformational studies by Milborrow (1984b). 100 COOH [46] Reduction of ABA to the diols with sodium borohydride has been widely used to determine the radiochemical purity of ABA produced in biosynthetic studies (Noddle and Robinson, 1969; Milborrow and Noddle, 1970; Milborrow and Garmston, 1973). The conversion of both enantiomers of racemic cis and trans-diol into ABA in wheat seedlings has been reported by Milborrow (1970). However, this apparent lack of stereospecificity was attributed to non-enzymic oxidation because of the observation that aqueous solutions of cis- and trans- diols autooxidize to ABA (Milborrow and Garmston, 1973). The cis- and trans diols were also converted into ABA and DPA by excised embryonic axes of Phaseolus vulgaris (Walton and Sondheimer, 1972b) and to ABA in pea seedlings (Milborrow , 1983). As well as the diols being oxidized to ABA, the 1' ,4'-trans-diol has been found as a metabolite of ABA in pea seedlings and avocado fruit (Milborrow, 1983). The abscisic acid metabolite 4'-desoxy ABA (Tietz et al., 101 1979) was shown to be the 1' ,4'-trans-diol of ABA and the desoxy compound was an artefact produced during mass spectrometry (Milborrow, 1983). Apart from being a metabolite of exogenously applied ABA, the 1' ,4'-trans-diol of ABA occurs naturally in Botrytis cinerea, a phytopathogenic fungus. Studies using deuterated ABA and deuterated trans-diol indicated that the trans-diol is not a metabolite, but it is a precursor of ABA in the fungus (Hirai et al., 1985). In addition, the 1' ,4'-cis-diol of ABA has been isolated from immature seeds of Vicia faba and characterized by Dathe and Sembdner (1982) but no metabolic role was proposed. Most of the biological activity of the diols has been attributed to their oxidation to ABA. Walton and Sondheimer (1972) found that 50% growth inhibition of embryonic axes of Phaseolus vulgaris required 7 µM ABA, 30 µM cis-diol and 80 µM trans-diol. The lower concentration of the cis-diol required, compared with the trans-diol, was thought to be due to its more rapid oxidation to ABA. Milborrow and Rubery (1985) have found that in short-term experiments (4-10 min) the cis-diol was a potent inhibitor of carrier-mediated uptake of ABA by root segments of Phaseolus coccineus - the trans-diol was less effective. 102 The present work seeks to determine the following: 1. Whether the trans-diol occurs naturally in plants and, if so, what are its endogenous concentrations? 2. Are the diols natural metabolites of S-ABA? 3. How stable are the diols and what factors cause their breakdown during extraction? 4. How are the diols metabolised? Are they precursors or metabolites of ABA? COOR2 Rl R2 [ 3] H H [41] H CH 3 [42] Ac CH 3 [47] ~-D-glucosyl H [48] ~-D-glucosyl CH 3 [ 4 9] tetraacetyl-~-D-glucosyl CH 3 [ 50] H ~-D-glucosyl [51] Ac tetraacetyl-~-D-glucosyl 103 Rl R2 [ 52] H H [53] H CH3 [54] Ac CH3 [55] ~-D-glucosyl H [56] ~-D-glucosyl CH3 [57) tetraacetyl-~-D-glucosyl CH3 [58] H P-D-glucosyl [59] Ac tetraacetyl-~-D-glucosyl ...... 'OH COOR 2 Rl R2 [2] H H [36] H CH3 [37] Ac CH 3 [60] P-D-glucosyl H [ 61] H ~-D-glucosyl [62] Ac tetraacetyl-~-D-glucosyl 104 COOR2 R1 R2 [ 38] H H [39) H CH 3 [40) Ac CH 3 [63) ~-D-glucosyl H [ 64] H ~-D-glucosyl [65) Ac tetraacetyl-~-D-glucosyl COOR 0 R [28) ~-D-glucosyl [66) tetraacetyl-~-D-glucosyl 105 4.2 MATERIALS AND METHODS 4.1 Plant materials Peas (Pisum sativum cv. Massey Gem) were washed under cold running water for 24 h, transferred to seed trays containing vermiculite, placed in a greenhouse and watered daily. Twelve-day-old seedlings were cut at ground level and the shoots placed in 100 ml beakers containing aqueous solutions (10 ml) of labelled compounds so that the 5 mm above the cut surfaces were immersed. When the solution had been taken up, the walls of the beaker were rinsed with 10 ml water which was also taken up by the pea shoots. Avocado (Persea americana cv. Haas) fruit were purchased locally and used when the fruit began to soften. Four fruit were cut in half and the stones removed. In one half of each fruit the endogenous concentrations of ABA and the 1',4'-trans-diol of ABA were measured. In the other half longitudinal and transverse cuts 5-10 mm apart were made in the mesocarp without piercing the skin. Labelled compounds in aqueous solutions (3 ml), which contained 0.2% (v/v) Triton X-100 as wetting agent, were applied and each half fruit was distorted a few times to spread the solution evenly over the cut surfaces. Tomato (Lycopersicon esculentum cv. Grosse Lisse) seeds (Arthur Yates and Co., Milperra, N.S.W.) were 106 germinated in a mixture of peat and sand (1:1, v/v) and grown in a glasshouse for 7 to 8 weeks. Alternatively, tomato seedlings (200-300 mm high) of the same variety were purchased from local suppliers. The seedlings were cut at ground level and the cut ends of the shoots placed in vials (50 x 25 mm diameter) which contained 1-2 ml aqueous solutions of labelled compounds. When this solution was taken up (30-60 min), the vials were rinsed with 2 ml water. The shoots were then stood upright in 250 ml beakers containing 200 ml water. Broad bean (Vicia faba cv. Early Long Pod) seeds (Arthur Yates and Co., Milperra, N.S.W.), were germinated in vermiculite. Two-week-old seedlings (250-300 mm) were cut at ground level and the shoots fed with labelled compounds using the same procedure as used with tomatoes. 4.2.2 High-performance liquid chromatography The HPLC system included the following Waters' equipment: a Model 510 pump, a Model M45 pump, a U6K injector and a Model 660 solvent programmer (Millipore Waters, Milford, MA). Effluent was monitored with an HP 1040A Diode Array Detector (Hewlett-Packard, Waldbronn, F.R.G.) which was connected by a HP-IB interface bus (IEE-488) to an HP-85B personal computer (Hewlett Packard, Corvallis, OR). Chromatograms were recorded at 215, 254, 265, 275 and 330 nm with a bandwidth of 4 nm. The reference for each signal was 550 nm with a bandwidth 107 of 100 nm. Spectra were recorded on the upslope, apex and downslope of each peak monitored at 265 nm. The chromatograms and spectra were plotted on an HP 7470A plotter using the software supplied by Hewlett-Packard. Radioactive compounds were detected with a continuous-flow HPLC radioactivity monitor. The column eluent was mixed with scintillant [PPO (9 g/1) in Triton X-100/toluene/methanol (30:67:15, by vol.)] at a ratio of 1:3 (eluent:scintillant) with a Precision Splitter Mixer (Reeve Analytical, Glasgow). The eluent-scintillant mixture was pumped to a Precision Radioactivity Monitor (Reeve Analytical) fitted with a homogeneous flow cell. Chromatograms of radiolabelled compounds were recorded on an HP-85B personal computer and were integrated and plotted using INT/143 software (Reeve Analytical). Alternatively, radioactive compounds were detected by collecting the effluent in 1-2 ml fractions and measuring radioactivity by liquid scintillation counting. 4.2.3 1' ,4'-cis- and 1',4'-trans-diols of ABA as products of [2-14c]ABA 1.36 µCi RS-[2-14c]ABA [25.6 mCi/mmol] were supplied to 25 pea seedlings (10 g) and to 8 tomato shoots (18 g). The plants were homogenized in acetone after 48 h. 4 µCi RS-[2-14c]ABA was fed to 4 broad bean shoots (19 g) and extracted after 72 h. One-third of the extract was set aside for total metabolite analysis. 4 halves of avocado fruit (338 g) were prepared as 108 described in section 4.2.1. Each half was supplied with 1 µci [2-14C]ABA dissolved in 4 ml 0.1 M potassium phosphate buffer (pH 6.8) with 0.02 ml Triton X-100. 100 µg each of 1' ,4'-cis- and 1',4'-trans-diol of ABA, in a similar buffer solution (2 ml), were added to one half fruit and to the other halves at 2, 4, and 20 h. The products were extracted after 24 h. One-fifth of the extract was analysed for total metabolites and four fifths specifically analysed for the 1' ,4'-cis- and 1',4'-trans-diol of ABA. 4.2.4 Isolation of 1' ,4'-cis- and 1' ,4'-trans-diol of ABA Plant material was homogenized in acetone containing BHT (100 mg/1) and 1 mg each of 1' ,4'-cis- and 1' ,4' trans-diol of ABA. The slurry was centrifuged and the pellet extracted twice with acetone plus BHT. Acetone was evaporated on a rotary evaporator, 20 ml saturated sodium bicarbonate added to the aqueous residue, and the neutral fraction was obtained with two extracts of an equal volume of ether. The aqueous residue was acidified to pH 3 with 1 M sulphuric acid, BHT (2 mg) was added and the acid fraction extracted with an equal volume of ether (four times). The acid ether fraction was evaporated to approximately 3 ml and the metabolites separated by thin layer chromatography on two 200 x 200 mm silica gel (60 F254 ) plates (Merck) developed in toluene/ethyl acetate/acetic acid (25:15:2, by vol.) containing BHT 109 (200 mg/ml) to give the following RF values: 1',4'-cis-diol of ABA, 0.43; 1',4'-trans-diol of ABA, 0.58; ABA, 0.65; t-ABA, 0.69. One-tenth of the diol zones were left on the plate and radiolabelled compounds detected by autoradiography. Autoradiograms were scanned with a Model 1650 Transmittance/Reflectance Scanning Densitometer (Bio-Rad, Richmond, CA) used in the transmittance mode and at the lowest scanning speed. The bands corresponding to the 1' ,4'-cis- and 1' ,4'-trans diol of ABA markers were scraped off the plates and eluted from the silica with methanol. The 1',4'-cis- and 1' ,4'-trans-diol of ABA were methylated with ethereal diazomethane and chromatographed on an 8 (i.d.) x 250 mm Techsil 10 silica HPLC column (HPLC Technology, West Macclesfield, Cheshire) with isopropanol/hexane (1:19, v/v) as the mobile phase delivered at 4.0 ml/min. Me-1' ,4'-cis-diol of ABA [36] had a retention time of 10.8 min and Me-1',4'-trans-diol of ABA [41], 7 min. The Me-1' ,4'- trans-diol of ABA was oxidized to with pyridinium chlorochromate on alumina (Cheng et al., 1980) and the resultant Me-ABA reduced with sodium borohydride. The enantiomers of Me-1' ,4'-cis-diol of ABA produced from 1' ,4'-trans-diol of ABA and the Me-1' ,4'-cis-diol of ABA, a product of [2-14c]ABA, were resolved by HPLC on Pirkle columns (Section 3.2.4, p. 83). The remainder of the avocado (1/5) and broad bean (1/3) extracts were subjected to the preliminary clean-up procedure described previously (Section 2.2.5, p. 43). 110 The metabolites of [2-14c]ABA were separated by HPLC on a 4.6 (i.d.) x 250 mm Brownlee RP-18 Spheri-5 column (Brownlee Labs, Santa Clara, CA) eluted with a linear gradient of methanol/0.2% aqueous acetic acid (1:3, v/v) into methanol/0.2% aqueous acetic acid (1:1, v/v) over 30 min at 1.0 ml/min. Radioactive compounds were detected with a continuous-flow HPLC radioactivity monitor. 4.2.5 Measurement of endogenous concentrations of ABA and 1' ,4'- trans-diol of ABA Pea shoots (275 g) and 4 halves of avocado fruit (356 g) were homogenized in acetone containing BHT (100 mg/1). 50 000 dpm (181 pg) [G-3H]-trans-diol of ABA [33.2 Ci/mmol] and 50 000 dpm (234 ng) [2-14c]ABA [25.6 mCi/mmol] were added to each extract. Acetone was evaporated and the pH of the aqueous residue adjusted to 9-10 with saturated sodium bicarbonate solution. The aqueous residue was partitioned against ether (3 times). After the removal of neutral material the aqueous residue was acidified with 1 M sulphuric acid to pH 2.5-3.0 and partitioned against ether (4 times). A fifth ether extract contained negligible amounts of radioactivity. 10 M KOH was added to the aqueous residue until it was alkaline (pH 12). After 12 hat 20°c, the aqueous residue was acidified with 1 M sulphuric acid to pH 2.5-3.0 and extracted four times with ether. The acidic ether extracts were evaporated to dryness under nitrogen, dissolved in 0.2% aqueous acetic acid, and loaded onto 4 111 Sep-Pak c18 cartridges connected in series. ABA and the trans-diol of ABA were eluted from the Sep-Paks with ethanol/0.2% aqueous acetic acid (2:3, v/v). This fraction was evaporated to dryness and fractionated by HPLC on an 8 (i.d.) x 250 mm Techsil 10 Cl8 column (HPLC Technology) with methanol/0.2% aqueous acetic acid (2:3, v/v), at 4.0 ml/min. ABA had a retention time of 15 min and the trans-diol of ABA, 10.4 min. The ABA and trans diol fractions were methylated with ethereal diazomethane and purified by the following sequence of steps: i) Reversed-phase HPLC on an 8 (i.d.) x 250 mm Techsil 10 Cl8 column, methanol/0.2% aqueous acetic acid (1:1, v/v), 4.0 ml/min:- Me-ABA, 14 min; Me-1' ,4'-trans-diol of ABA [ 41] , 11 min. ii) Normal-phase HPLC on an 8 (i.d.) x 250 mm Techsil 10 silica column with isopropanol/hexane (3:97, v/v) at 4.0 ml/min. Me-ABA, 8 min; Me-trans-diol of ABA [41], 12.2 min. iii) Acetylation of Me-trans-diol of ABA and HPLC on a 4.6 (i.d.) x 250 mm Brownlee RP-18 Spheri-5 column with methanol/0.2% aqueous acetic acid (775:225, v/v), 1.0 ml/min. O-acetyl-trans-diol of ABA methyl ester [42], 7.8 min; Me-trans-ABA, 5.6 min. The absorbance of the eluent was measured at 265 nm and the amount of the methylated and acetylated l',4'-trans-diol determined by comparison with a standard curve constructed with injections of 0.1 to 5 µg of a standard of MeAc-1',4'-trans-diol of ABA. Spectra were 112 recorded across the peaks on a diode array detector and were used to determine the purity of the peaks. The concentrations of both Me-ABA and MeAc-trans-diol of ABA [42] were measured by GC-electron-capture detection on ov-225 at 210°c (Me-ABA, 10.5 min) and at 205°c (MeAc-trans- diol, 6.9 min). The identity of the MeAc-trans-diol of ABA, prepared from material isolated from peas and avocados, was verified by GC-MS. 4.2.6 Interconversion of 1' ,4'-cis-diol of ABA and 1',4'-trans-diol of ABA 8 000 dpm [2-14c]-cis-diol of ABA [25.6 mCi/mmol] or 8 000 dpm [2-14c]-trans-diol of ABA [25.6 mCi/mmol] were added to 10 ml 0.1 M sodium citrate-phosphate buffer at pH values between 2.4 and 7.2 or 0.1 M phosphate-NaOH buffer (pH 7.2 to 12) in increments of 0.6 pH unit. The solutions were left at 20°c for 72 hand 1' ,4'-cis-diol of ABA, 1' ,4'-trans-diol of ABA and ABA (1 mg each) added to each sample, which was then partitioned against ether (twice). If necessary, the aqueous residue was acidified to pH 4 with oxalic acid and then reextracted with ether. The radioactivity remaining in the aqueous residue was less than 1% in all cases. The combined ether extracts were chromatographed on 200 x 200 mm silica gel (60 F254 ) thin-layer plates (Merck) developed in toluene/ethyl acetate/acetic acid (25:15:2, by vol.). The compounds were visualised by UV light (254 nm), the zones scraped off the plates and radioactivity measured by liquid scintillation counting. 113 4.2.7 Preparation and isomerization of l' ,4' [11-18o]trans-diol of ABA 500 µg [l1- 18o]ABA methyl ester was reduced with sodium borohydride at o0 c for 30 min and the diols separated by normal-phase HPLC on an 8 x 250 mm Techsil 10 silica eluted with isopropanol/hexane (1:19, v/v) at 4.0 ml/min. Retention times: Me-ABA, 6.5 min; Me-1' ,4'-trans-diol of ABA, 7.1 min; Me-1',4'-cis-diol of ABA, 11 min. The diols were left in 0.1 M sodium acetate buffer (pH 4) for 72 h. The products of isomerization were separated by normal phase HPLC with the same conditions used above. The proportion of 180 in the products was measured by mass spectrometry. 4.2.8 Isomerization of the cis- and trans-diols in [18o]water (+)-1' ,4'-cis- and (+)-1',4'-trans-diol of ABA methyl ester (200 µg) were dissolved in 30 µl [18OJH2o (97.2 atoms% 180; Novachem, South Yarra, Aust.) and acetic acid (1 µl) added to give a final pH of 3.5. After 72 hat 20°c the solution was evaporated under N2 and the products chromatographed on three 4.6 (i.d.) x 250 mm Pirkle columns (one covalent R-phenyl glycine connected in series with two type 1-A columns) with isopropanol/hexane (7:93, v/v) as the mobile phase at 2.0 ml/min. Retention times: Me-trans-diol [41], 28.5 min; Me-ABA, 32.5 min; Me-(+)-cis-diol [36], 46.2 min; Me-(-)-cis-diol [39], 48.3 min. The products 114 were acetylated and the incorporation of 180 measured by mass spectrometry. 4.2.9 Preparation of [4 1 - 18o]cis- and [4 1 - 18o]trans-diol of ABA ABA (1 mg) was dissolved in 50 µ1 r18oJH2o to which a small piece of sodium metal had been added. After 16 h, 50 µl methanol was added and the [4 1 - 18o]ABA reduced with sodium borohydride at o0 c for 30 min. The mixture was acidified, extracted with ether and the products in the ether phase were methylated and separated by normal-phase HPLC. The [4 1 - 18o]cis- and trans-diols were acetylated and studied by mass spectrometry. 4.2.10 Isomerization of (+)- and (-)-[2-14c]trans-diol (+)- and (-)-[2-14c]trans-diol of ABA were prepared by the reduction of (+)- and (-)-[2-14c]ABA [25mCi/mmol]. The diols (77 000 dpm of each) were dissolved in 35 mM acetic acid (100 µl) and after 72 h the products were separated on three Pirkle columns as described above and radioactivity in the effluent detected with a continuous-flow radioactivity monitor. 4.2.11 The metabolism of l',4'-[2-14c]-trans-diol of ABA 1.18 µci (308 µg) RS-1' ,4'-trans-diol of ABA [1.01 mCi/mmol] was dissolved in 2 ml water and fed to 35 pea shoots (13 g) and to 5 tomato shoots (11 g). The metabolites were extracted after 7 days. 115 The homogenization and chromatographic procedure was the same as that used for the analysis of the metabolites of ABA (sections 2.2.4-7, pp. 42-44). 2 µci (7.5 mg) (+/-)-1' ,4'-[2-14c]trans-diol of ABA [70 µCi/mmol] was supplied to 17 300 mm-high tomato shoots (56 g). The tomatoes were homogenized after 4 days. Extraction and separation of metabolites was the same as above. A subsample (1/40) was separated by HPLC to determine the position of radioactive compounds. Fractions were collected every 30 sec (2 ml). 10 ml Scintillant (0.5% PPO in Triton X-100-toluene (2:1, v/v) was added and radioactivity determined by liquid scintillation counting. The remainder of the extract was separated by HPLC using the same conditions and peaks corresponding to radioactive metabolites were collected. These were: DPAGS [30], 6 min; G [58], 12 min; H [50], 13.3 min; I [55], 16.3 min; J [47], 19 min; ABAGE [28], 20.3 min; 1' ,4'-trans-diol of ABA, 33.7 min; ABA, 37.8 min. 4.2.12 Thin-layer chromatography of 1' ,4'-trans-diol metabolites and their derivatives Thin-layer chromatography was used to analyse radioactive metabolites of (+/-)-1' ,4'-trans-diol of ABA and the products of methylation, acetylation, acetylation and methylation, and basic hydrolysis (ammonia [s.g. 0.880] 40°c, 30 min). Samples were applied in 10 mm strips on the origin of 100 mm x 100 mm HPTLC plates (60 F254 ). 116 ABA and 1' ,4'-trans-diol of ABA markers were applied to each plate. The plates were developed in toluene/ethyl acetate/acetic acid (25:15:2, by vol.) or chloroform/ methanol/water (75:22:3, by vol.). Radioactive compounds were detected by autoradiography (Section 2.2.13, p. 47). 4.2.13 Derivatization and purification of the metabolites of 1' ,4'- trans-diol of ABA The products of 1' ,4'-trans-diol of ABA in tomatoes were methylated with ice-cold, ethereal diazomethane and chromatographed on an 8 (i.d.) x 250 mm Techsil 10 Cl8 HPLC column with a mobile phase of ethanol/0.2% aqueous acetic acid (1:3, v/v) at 4.0 ml/min. Retention times: G [58], 6.5 min; H [50], 7.1 min; I [55], 8.4 min); J [47], 9.3 min; Me-I [56],17 min; Me-J [48], 20 min. The methyl esters of I and J were acetylated (200 µl pyridine/acetic anhydride (1:1, v/v, 40°c, 30 min). Excess reagent was evaporated under N2 . The acetylated methyl esters were chromatographed on an 8 (i.d.) x 250 mm Techsil 10 Cl8 with ethanol/0.2% aqueous acetic acid (9:11, v/v) as the mobile phase at 4.0 ml/min. Retention times: Me-ABA, 5.8 min; MeAc-I [57], 8.5 min; MeAc-J [49], 9.5 min. MeAc-I [57] and MeAc-J [49] were further purified by normal phase HPLC on an 8 (i.d.) x 250 mm Techsil 10 silica with isopropanol/hexane (1:24, v/v) as the mobile phase. 117 Retention times: Me-ABA, 6.7 min; MeAc-J [49], 13.5 min; MeAc-I [57], 15.7 min. G [58] and H [50] were acetylated, and HPLC on an 8 (i.d.) x 250 mm Techsil 5 Cl8 column with ethanol/0.2% aqueous acetic acid (3:1, v/v) at 2.0 ml/min gave the following retention times: Ac-G [59], 8.7 min; Ac-H [51], 8.7 min; Me-1' ,4'-trans-diol of ABA [41],3.7 min; Me-1' ,4'-cis-diol of ABA [36], 4.8 min. 4.2.14 NMR spectroscopy 1H NMR spectra were recorded at 300 MHz on a Bruker CXP-300 spectrometer operating in the pulsed Fourier transform mode with quadrature detection. 5 mm outside diameter spinning sample tubes (Wilmad Glass Co., 527-PP grade) were used, and the probe temperature was maintained with a Bruker B-VT 1000 variable temperature unit. Typical spectral accumulation parameters were: sweep width 2600 Hz, 8192 addresses, 90° radiofrequency pulses (6.5-7.5 µs duration), 3 s recycle time, 500-2000 accumulations, probe temperature 27°c. In some cases, resolution enhancement was effected by means of a Lorentzian-Gaussian transformation. Chemical shifts were measured digitally, using TMS as an internal standard. The two-dimensional homonuclear correlated spectrum (COSY) was recorded with the pulse sequence: [9o 0 -t1-90°-t2 ]n, where t 1 and t 2 are the evolution and 118 observation periods, respectively. The measurement was repeated for 256 equidistant values of t 1 , from 0.01 to 98.5 ms. The spectral width was 3000 Hz in each dimension, and in f 2 the data block size 2048 addresses. Quadrature detection was employed in both dimensions, with appropriate phase cycling to select P-type peaks. Before Fourier transformation, the time domain matrix was multiplied in both dimensions by a sine-bell function. The frequency domain spectrum is shown in the absolute value mode, without symmetrisation. 4.2.15 Chemical-ionization mass spectrometry Positive and negative ion chemical ionization mass spectra were obtained using a Finnigan 3200 Quadrupole mass spectrometer equipped with a pulsed positive ion negative ion chemical ionization (PPINICI) attachment and interfaced for data acquisition and scan control with a Finnigan Incos 2300 Data System. Electron energy was 90-100 eV, emission current, 100 mA, and electron multiplier voltage 1.2- 1.3 kV. Commercially pure methane was used as the CI energy moderator and collisional stabilization gas (ion source pressure 0.8-0.9 Torr) and GC carrier gas (20 ml/min). Samples analysed by GC-MS were injected onto a 1.5 m x 2 mm (i.d.) column of 3% OV-17 on Gas Chrom Q (100-120 mesh) held at 150°c for 1 min and programmed to rise to 30o0 c at 10°c/min. 119 4.2.16 Hydrolysis of the diol conjugates and identification of the sugar residue as the TMS-O-methyloxime derivative The sample to be hydrolysed was dissolved in 1 M HCl (50 µl) and incubated at 100°c for 6 h in a 1 ml Reacti vial (Pierce Chemical Co., Rockford, Ill.) sealed under nitrogen. The acid was evaporated under nitrogen and the O-methyloxime of the sugar formed by the method of Mawhinney et al., (1980). The O-methyloximation reagent was prepared by dissolving 300 mg O-methylhydroxylamine hydrochloride in dry methanol (1 ml) and pyridine (1.78 ml) followed by the addition of 220 µl l-dimethyl amino-2-propanol. 100 µl of this reagent was added to the hydrolysate and the mixture heated at 70°c for 10 min. The volume of the mixture was reduced to about 20 µl by evaporation under nitrogen. The O-methyloxime of the sugar was silylated by the method of Leblanc and Bali (1978). The following reagents were added, in sequence, to the O-methyloxime: 350 µ1 dry dimethylsulphoxide, 350 µl cyclohexane, 60 µ1 hexamethyldisilazaine (Pierce) and 30 µl trimethyl chlorosilane (Pierce). The sample was shaken until fully dissolved and the silylated compound was found in the upper phase. The TMS-O-methyloxime was analysed by capillary gas chromatography-mass spectrometry. The sample was injected onto a 0.33 mm (i.d.) x 25m BP-1 capillary column held at 100°c for 1 min and programmed 120 to rise at 6°/min to 30o0 c - the TMS-O-methyloxime of glucose had a retention time of 19.75 min. 4.2.17 Reduction of ABAGE to form the glucose esters of 1' ,4'-ct.§.- and 1',4'-trans-diol of ABA RS-ABAGE [28] was isolated from tomato shoots supplied with RS-ABA. ABAGE [28] was reduced with sodium borohydride in 400 µl methanol/0.1 M potassium phosphate buffer, pH 6.0 (2:1, v/v) at o0 c for 50 min. The pH of the mixture was readjusted by the dropwise addition of a saturated solution of sodium dihydrogen orthophosphate after each addition of sodium borohydride (3 mg at 0, 5, 10, 20 and 30 min). The products were separated by HPLC on an 8 (i.d.) x 250 mm Techsil 10 Cl8 column with a mobile phase of ethanol/0.2% aqueous acetic acid (3:17, v/v) and a linear gradient into ethanol/0.2% aqueous acetic acid (1:3,v/v) over 15 min. The resultant chromatogram had two peaks at 12 and 13 min ( (-)- [58] and (+)- [50] 1',4'-trans- diol of ABA glucose ester respectively); a peak at 19 min (ABAGE [28]) and a peak at 38 min (1',4'-cis-diol of ABA glucose ester [61]). 4.2.18 Metabolism of (-)-1' ,4'-[2-14c]-trans-diol of ABA and (+)- 1' ,4'-[2-14c]-trans-diol of ABA (-)-[2-14c]ABA and (+)-[2-14c]ABA were prepared by the resolution of(+/-)-1' ,4'-[2-14c]cis-diol of ABA methyl ester [0.52 mCi/mmol] on Pirkle columns (Section 3.2.4, p. 83). The enantiomers of [2-14c]ABA were reduced to the diols with sodium borohydride at o 0 c for 30 min and the 121 diols separated by TLC on 200 x 200 x 0.5 mm plates developed in toluene/ethyl acetate/acetic acid (25:15:2, by vol.). RF values: 1' ,4'-trans-diol of ABA [3], 0.57; 1' ,4'-cis- diol of ABA [2], 0.47; ABA, 0.62. 82 200 dpm (18.7 µg) (-)-1' ,4'-[2-14c]trans-diol of ABA [52] [0.52 mCi/mmol] was fed to two tomato shoots (6 g) and 95 250 dpm (21.6 µg) (+)-1' ,4'-[2-14c]trans-diol of ABA [3] [0.52 mCi/mmol] to two tomato shoots (5.6 g). The products were extracted after 5 days. The extraction procedure and separation of metabolites was that described in Sections 2.2.4-7, pp. 42-44. 4.2.19 Metabolism of (-)-[ 14c]- and (+)-[ 3H]cis- and trans- diols of ABA 665 000 dpm (-)-[2-14c]ABA [25.6 mCi/mmol], 1 587 000 dpm (+)-[G-3H]ABA [44 mCi/mmol] and 40 mg (+/-)-ABA were mixed and reduced with sodium borodeuteride (98 atom% 2H, Sigma) at o0 c for 30 min in methanol/water (2:1, v/v). The diols were separated by TLC on 200 mm x 200 mm silica gel plates developed in toluene/ethyl acetate/acetic acid (25:15:2, by vol.) and purified by reversed-phase HPLC on an 8 (i.d.) x 250 mm Techsil 10 Cl8 with ethanol/0.2% aqueous acetic acid (7:13, v/v) 153 000 dpm (4 mg) (-)-1',4'-[2-14c, 4'-2H]trans diol of ABA [4µCi/mmol] and 366 000 (4 mg) (+)-1',4' [G-3H, 4'-2H]trans-diol of ABA [9.52 µCi/mmol] were 122 supplied to 11 tomato shoots (20 g). 114 000 (3.5 mg) (-)-1' ,4'-[2-14c, 4'-2HJ-cis-diol of ABA [4µCi/mmol] and (+)-1' ,4'-[G-3H, 4'-2HJcis-diol of ABA were supplied to 12 tomato shoots (21 g). The tomatoes were homogenized in acetone containing BHT (100 mg/1) after 7 days and the metabolites separated as described in Sections 2.2.4-7 (pp. 42-44) 4.2.20 Metabolism of (+/-)-1' ,4'-[2-14c, 4'-2HJtrans diol of ABA and (+/-)-[G-3HJABA 4 µCi (31 ng) (+/-)-[G-3H]ABA [33.2 Ci/mmol] and 0.65 µCi (1 mg) (+/-)-[2-14c, 4'-2H]trans-diol of ABA [0.17 mCi/mmol] (prepared by the reduction of [2-14cJABA with sodium borodeuteride) were fed to 6 tomato shoots (13.5 g). The tomatoes were extracted after 7 days and prepared for HPLC as described above. The metabolites were separated by HPLC: 8 (i.d.) X 250 mm Techsil 10 Cl8 with ethanol/0.2% aqueous acetic acid ( 1: 9, v/v) at 4.0 ml/min. Fractions were collected every 4 ml and 3H and 14c-labelled compounds detected by liquid scintillation counting. DPAGS [30) had a retention time of 9 min. The DPAGS fraction was methylated, acetylated and purified as described in Chapter 2 (Section 2.2.10 p.46). 4.2.21 Metabolism of (+/-)-1' ,4'-[2-14c]cis-diol of ABA 2 µg (7.5 mg) (+/-)-1' ,4'-[2-14cJcis-diol of ABA [70 µCi/mmol] was fed to 15 tomato shoots (47 g). After 4 days the metabolites were extracted and prepared for HPLC 123 as described in Sections 2.2.4-5, pp. 42-43. The metabolites were separated by HPLC on a 4.6 (i.d.) x 250 mm Brownlee RP-18 Spheri-5 column, eluted with methanol/water (0.2% aqueous acetic acid) (2:3, v/v) at 1.0 ml/min and 14c-labelled compounds were detected with a radioactivity monitor. 4.3 RESULTS 4.3.1 Reduction of [2-14c]ABA by various higher plant species Exogenous [2-14c]ABA was supplied to shoots of tomato (Lycopersicon esculentum c.v. Grosse Lisse), pea (Pisum sativum c.v. Massey Gem), broad bean (Vicia faba c.v. Early Long Pod) and fruit of avocado (Persea americana c.v. Haas) In each experiment a milligram of unlabelled 1' ,4'-cis- and/or 1',4'-trans-diol of ABA was added to the homogenate to act as a "scavenger" and improve the recovery of the diols. It also acted as a marker during the chromatography of the products. Avocado fruit and broad bean shoots reduced RS-[2-14c]ABA to both the 1' ,4'-cis-diol of ABA and the 1' ,4'-trans-diol of ABA. The acid ether fractions of avocado and pea extracts were chromatographed on silica gel thin-layer plates. Nine-tenths of the cis- and trans-diol zones were eluted immediately from the silica gel and X-ray film was placed on the remaining one-tenth of the plate to detect radioactive material. 124 Densitometer scans of the autoradiograms are shown in figure 4.1. The zones of radioactivity, and the intervening zones were scraped from the plate and radioactivity measured by liquid scintillation counting. In the extract of avocado fruit the cis-diol contained 2.7% and the trans-diol zone, 2.3% of the radioactivity supplied. However, 85.1% of the [2-14c]ABA supplied to the avocado was not metabolized and of the metabolites of ABA in the acid ether extract the cis- and trans- diols accounted for 22.5 and 19% respectively. The ABA fed to broad bean shoots was metabolized to a greater extent (34.8% ABA unmetabolized) and the cis-diol (17.9%) and the trans-diol (6.6%) accounted for a greater proportion of the radioactivity supplied compared to the same metabolites in avocado fruit. Expressed as a proportion of the metabolites in the acid ether extract the cis-diol was 37.8% and the trans-diol 14.7%. When a portion of the avocado (1/5) and broad bean (1/3) extracts were treated in a similar manner to extracts in which conjugates and free acids were separated by reversed phase HPLC, the proportion of the radioactivity in the trans-, and especially the cis-diol, decreased. Figures 4.2 and 4.3 show the separation of metabolites of [2-14c]ABA in extracts of avocado fruit and broad bean shoots. The trans-diol is present in a reduced amount compared to separation by TLC and there 125 a (/l C: Q) -a Q) > -ea Q) er: 0 0.5 1.0 Rr b (/l C: Q) -a Q) > -ea Q) er: 0 0.5 1.0 Rr Fig. 4.1 TLC separation of the metabolites of [ 14cJABA in avocado and broad bean The traces represent densitometer scans of the autoradio grams of the products of [ 14cJABA separated on thin-layer silica plates developed in toluene/ethyl acetate/acetic acid (25:15:2, by vol.) - a) acid ether extract of avocado fruit supplied with (2-14cJABA b) corresponding fraction from broad bean extract. RF values of standards: ABA - 0.65; 1' ,4'-trans-diol of ABA - 0.58; 1',4'-cis-diol of ABA - 0.45, t-ABA - 0.69. 126 50 40 IJ) 0. 0 >, -> 30 0 -ro 0 -0 ro 20 a: 10 Retention time, min Fig. 4.2 HPLC separation of metabolites formed from [2-14cJABA in avocado fruit Metabolites of [2-14CJABA supplied to avocado fruit were separated by HPLC on a 4.6 (i.d.) x 250 mm Brownlee RP-18 Spheri-5 column eluted with a linear gradient from methanol /0.2% aqueous acetic acid (1:3, v/v) to methanol/0.2% aqueous acetic acid (1:1, v/v) over 30 min at 1.0 ml/min. Radiolabel led material was detected with an on-line radioactivity monitor. Retention times of standards: ABA - 35 min; t-ABA - 31.5 min; l' ,4'-trans-diol of ABA - 23.5 min, l' ,4'-cis-diol of ABA - 45.5 min, DPAGS - 8 min. are few if any counts in the cis-diol fraction. An explanation for this discrepancy is that these samples were assayed two days after the samples which were extracted in ether and separated by HPLC and the cis- and trans- diols may have been lost by oxidation. 127 80 60 Cl) 0. 0 >, -> 40 -0 Cll 0 "O Cll a: 20 0 10 20 30 40 Retention time, min Fig. 4.3 HPLC separation of the metabolites formed from [2-14c]ABA in broad bean shoots The extraction and HPLC conditions were the same as those described in the legend to Fig. 4.2. In pea and tomato shoots the presence of only the 1' ,4'-trans-diol of ABA was investigated. Pea shoots metabolized 9% of the [14cJABA supplied to the trans-diol. Tomato shoots contained no radioactivity in the trans-diol fraction, although most of the (cold) trans-diol scavenger was recovered. 128 Table 4.1 Proportion of R- and~- enantiomers in cis- and trans- diols produced from RS-ABA Samples of 1' ,4' [2-14cJcis-diol of ABA produced RS-[2-14cJ ABA were methylated, purified and the enantiomers resolved by HPLC on optically-active Pirkle columns. The 1' ,4' trans-diol samples were methylated, oxidized to Me-ABA and then reduced to the Me-cis- and Me-trans-diols. The Me-cis diol was resolved on Pirkle columns. 1' ,4'-trans-diol of ABA* 1' ,4'-cis-diol of ABA* Source s- R- s- R- Avocado 3636 6171 389 2293 (1:1.7)** (1:5.9) Broad Bean 837 3790 300 541 (1:4.5) (1:1.8) Pea 846 1055 (1:1.25) * figures are in dpm ** ratio in brackets is the proportion of S- to R enantiomer 4.3.1.1 The proportion of the cis- and trans-diol derived from R-[2-14c] and S-[2-14c]ABA by three plant species The proportion of R- and S- enantiomers of the [2-14c]l' ,4'-cis- and [2-14c]l' ,4'-trans-diol produced from RS-[2-14c]ABA was determined by resolving the methyl ester of the cis-diol of ABA as described in Chapter 3. Samples of cis- and trans-diol were methylated with diazomethane and purified by normal-phase HPLC. The Me-trans-diol samples were oxidized to Me-ABA and then 129 reduced to the Me-cis- and Me-trans-diols. Each sample of Me-cis-diol of ABA was resolved into the R- and S enantiomers and radioactivity in each enantiomer was measured. The results (Table 4.1) show that the 1' ,4'-cis- and 1' ,4'-trans-diol of ABA are produced from natural S-ABA and unnatural R-ABA. In every sample there was a higher proportion of the R-enantiomer although the ratio of S- to R- varied. 4.3.2 The natural occurrence of the 1' ,4'-trans-diol of ABA The endogenous concentrations of ABA and the 1' ,4'-trans-diol of ABA were measured in avocado fruit and pea shoots; the amount of these compounds released by basic hydrolysis was also measured. HPLC of the compounds as free acids, methyl esters and acetyl derivatives on reversed-phase and normal-phase columns resulted in a product of high purity, and the peaks occurred in all systems at the identical retention time as those of authentic standards. Furthermore, the identity of the final samples were verified by GC-MS and UV spectroscopy. The inclusion of [2-14C]ABA and 1' ,4' [G-3H]trans-diol of ABA (50 000 dpm of each - 234 ng and 18 pg respectively) did not add significantly to the endogenous levels, and enabled the efficiency of the recovery of the endogenous compounds to be calculated. The recovery of the labelled internal standards ranged from 32.5% for avocado ABA to 2.2% for the trans-diol in the saponified pea extract. 130 Table 4.2 Endogenous concentrations of ABA and l' ,4'-trans-diol of ABA and ABA was determined The concentration of the ---trans-diol by adding internal standards of [2-14cJABA and [G-3H]trans- diol. The compounds were then isolated from the extracts by normal- and reversed-phase HPLC as O-acetyl Me-trans-diol and Me-ABA. The concentrations were measured by gas chromatography with electron-capture detection and by HPLC-uv265 nm (MeAc-trans-diol only). The concentrations measured by HPLC-UV are shown in brackets. Concentration (ng/g)* Compound Avocado fruit Pea shoots ABA 8451 40 ABA released by basic 1337 22 hydrolysis l' ,4'-trans-diol of ABA 93(71) 5.2(4.36) l' ,4'-trans-diol of ABA 6. 7 (5 .1) not detected released by basic hydrolysis * Corrected for losses. Recovery of the trans-diol was between 11.7% and 2.2% for all fractions. The amounts of ABA, as the methyl ester, and the 1' ,4'-trans-diol of ABA, as the 0-acetyl methyl ester [42], (Table 4.2) were measured by gas chromatography with electron capture detection. The concentrations of 131 MeAc-1' ,4'-trans-diol were also measured by HPLC (UV - 265 nm). In avocado fruit the trans-diol occurs at a concentration of 93 ng/g or 1.1% of the concentration of free ABA. In pea shoots the concentration of the trans diol (5.2 ng/g) was much lower than in avocado but relat ive to the concentration of free ABA in peas the trans diol (13% of free ABA) was higher than in avocado. The l' ,4'-trans-diol of ABA (7.2% concentration of free trans-diol) was released by basic hydrolysis of the aque ous residue of avocado extract after removal of the free trans-diol by ether extraction. However, no trans-diol was detected in the corresponding fraction from peas. The identity of the l' ,4'-trans-diol of ABA isolated from avocado fruit was verified by its UV spectrum recorded on a diode array detector, and its HPLC retention time was identical to that of a sample of authentic 0-acetyl Me-1' ,4'-trans-diol of ABA [42). The 0-acetylated methyl ester of the diol isolated from avocado fruit had the same GC retention time and positive-ion mass spectrum as standard MeAc-trans-diol (Fig. 4.4a). There was insufficient l',4'-trans-diol from peas to obtain a reliable UV spectrum, however, there was enough sample for a negative-ion mass spectrum to be obtained (Fig. 4.4b). 4.3.3 Stability of the l' ,4'-diols of ABA The poor recovery of the cis- and trans-diols was found to be due to their oxidation to ABA and to the 132 a 100 , 20x 61 AcO COOCH, 231 >, ;t::: Cl) C Cl) 245 .!;- Cl) 50 .:::> 213 a, a:a3 125 137 291 ••itt 273 303 ' I 100 200 300 m/z 260 b 100 '.:::: COOCH AcO 3 >, "iii- C Cl) -_!; Cl) 50 .:::> a, a3 a: 243 146 230 163 83 141 111 187 276 ----,~~.1-~,-1-y.-->Ju...,.._...~ 100 200 m/z Fig. 4.4 Chemical ionization (methane) mass spectra of 0-acetyl l' ,4'-trans-diol of ABA methyl ester a. positive ion b. negative ion 133 100 -·-·--· from cis-diol .... C ~ 50 i... Q) a.. unchanged cis-diol 3 6 9 12 pH Fig. 4.5 Effect of pH on the stability of the 1' ,4'-~ diol of ABA The products of [2-14cJ-cis-diol of ABA after 72 h in solutions buffered at pH 2.4 to 12 were separated by TLC and radioactivity determined by liquid scintillation counting. interconversion of the diols. The cis-diol in particular was unstable in acidic solutions and mixture of ABA, cis-diol and trans-diol resulted. The effect of pH on the isomerization and oxidation of the diols was investigated by dissolving 14c-labelled diol in buffers from pH 2.5 to 12 and separating the products by TLC 134 cis-diol from trans-diol -a3 50 (.) lo.. (I) a.. unchanged trans-diol 3 6 9 12 pH Fig. 4.6 Effect of pH on the stability of the l' ,4'-trans diol of ABA [2-14c]trans-diol of ABA was incubated in solutions buffered at pH 2.4 to 12. The products were separated by TLC and radioactivity determined by liquid scintillation counting. after 3 days. The cis-diol (Fig. 4.5) was relatively stable at high pH but below pH 6 oxidation to ABA occurred readily and isomerization to the trans-diol was highest at pH 3.6. At pH 3.6 a mixture containing cis-diol (19%), trans-diol (23%) and ABA(58%) was produced from cis-diol after 72 hat pH 3.6. 135 The trans-diol (Fig. 4.6) was more stable than the cis-diol at low pH with little isomerization to the cis-diol above pH 4.8. Below pH 4.8 oxidation and isomerization increased and at pH 2.5 the trans-diol was converted into a mixture containing ABA (57%), cis-diol (16%) and trans-diol (27%). 4.3.3.1 The site of inversion during interconversion of the diols The interconversion of the 1' ,4'-cis- and 1' ,4'-trans- diols could occur by inversion at either the C-1' or C-4' chiral center. Isomerization at C-4' would produce the opposite geometric isomer but the chirality at C-1' would be unchanged. Thus (+)-trans-diol would be converted into (+)-cis-diol. However, if isomerization occurred at C-1', (+)-trans-diol would be converted into (-)-cis- diol. The possible mechanisms were studied by analyzing, by mass spectrometry, the interconversion of diols labelled in the 1'- or 4' positions with stable isotopes. This study required that the composition of the fragment ions in the diol was known. 4.3.3.2 Mass spectrometry of the diols of ABA A mass spectrum of the trans-diol of ABA methyl ester does not usually show a molecular ion (Tietz et al., 1979; Milborrow, 1983). Instead the molecule dehydrates readily and although this major fragmentation path was attributed to dehydration at C-4'-position this 136 Ac1so [ 6 7] AcO [68] D--- AcO [ 6 9] AcO [ 70] 137 Table 4.3 Chemical-ionization (methane) mass spectra of MeAc-trans-diol Positive-ion mass spectra of O-acetyl Me-trans-diol of ABA containing oxygen-: or deuterium at various positions 1 18 1 2 2 MeAc-trans- MeAc-(4 - O1 MeAc-(1 - 18O1 MeAc-[4'- HJ MeAc-[6- H3 : diol trans-diol trans-diol trans-diol trans-diol [42] [67] (68] [69] [70] M + 41 365 (0. 3) 353 (2) 353 (0 .2) M + 29 351 (1) 351 ( 0. 2) 352 ( 0. 7) 307 (5) MH-H 2O 305 (2. 5) 305 (2) 306 (1) 293 (0. 4) 291 ( 3) 291 (3) 291 ( 0. 6) 292 (1) 265 (3) MH-60 263 ( 4) 263 (22) 263 (5) 264 (6) 266 (10) MH-78 245 (27) 245 (20) 245 (4) 246 (6) 248 (21) 233 (2) 233 ( 11) 233 (4) 234 (61) 232 (6) 232 (1 7) 232 (3) 232 ( 36) MH-92 231 (40) 231 ( 100) 231 ( 18) 231 (3) MH-110 213 (22) 213 (10) 213 (2) 214 (3) 216 (18) 203 (3) 203 (2) 205 ( 0. 3) 204 ( 1) 206 (3) 153 (2) 153 (2) 153 ( 1) 153 (1) 156 (2) 137 (5) 137 (4) 139 (1) 138 ( 3) 137 (19) 133 ( 4) 133 (1) 133 (0. 4) 134 (0. 4) 133 (9) 127 (2) 127 (1) 127 ( 1) 127 (1) 128 (14) 125 (13) 125 (7) 125 (4) 125 (5) 125 (12) 111 (3) 111 (1) 111 (1) 111 (1) 114 (28) 95 (4) 95 (1) 95 (1) 95 (1) 98 (10) 89 ( 1) 89 ( 10) 89 (3) 89 (27) 71 (1) 71 (1) 71 (100) 69 (1) 69 (1) 69 (3) 69 (1) 63 ( 0. 4) 63 (39) 63 ( 0. 4) 63 (0. 3) 65 (10) 61 (100) 61 (17) 61 (100) 61 (100) 61 (93) had to be established by 180 labelling. Chemical- ionization mass spectrometry of the 4'-acetyl trans-diol showed that the 4' group was lost as acetic acid (m.w. = 60) giving ions at m/z 263 and m/z 61. 138 0-acetylated trans-diol of ABA methyl ester containing oxygen-18 at C-1' or C-4' or deuterium at C-4' or C-6 positions was used to identify the origin of the fragment ions. The spectra are listed in table 4.3 and the fragmentation pathway is described in scheme 4.1. The same fragmentation occurs with the cis-diol with minor differences in intensities. MeAc-trans-diol -18 MH = m/z 323 305 CH 3 COOH (4'-0Ac) -60 -18 263 245 (4'-desoxy ABA) -32 -18 231 213 139 The two most abundant ions m/z 61 and m/z 231 containg oxygen derived exclusively from the 4'- and 1'-hydroxyl group respectively. These fragment ions could therefore be used to determine the proportion of oxygen-18 in the 4'- and 1'-hydroxyl groups. 4.3.3.3 The interconversion of [1 1 - 180]-labelled 1' ,4'-diols of ABA The incubation of Me-[l 1 - 18o]cis- and Me-[l 1 - 18o]trans-diol in pH 3.5 buffer for 72 h has shown that the 1'-hydroxyl group is exchangeable with the medium (Table 4.4). The Me-[l 1 - 18o]trans-diol recovered from the buffered solution had lost 3.4% of its 180 whereas the cis-diol produced by isomerization of the trans-diol lost 26.3% of its 180. When [l 1 - 18o]cis-diol was incubated at pH 3.5 the recovered cis-diol had lost 8.3% of its 180. On conversion into the trans-diol 11.2% of the 1 1 - 180 was lost from the cis-diol. Thus, the 1'-hydroxyl group is exchanged with the medium during interconversion of the diols and when the diol is recovered unchanged from the medium. 4.3.3.4 The stability of the 4'-hydrogen atom during diol interconversion When the cis- and trans-diol of ABA were reduced with sodium borodeuteride (98 atom% 2H) no ions corresponding to undeuterated diol were observed in the mass spectra of the diols produced (Table 4.3). The diols recovered after 72 h in acidic solution had 140 Table 4.4 Isomerization of [l 1 - 18o]cis- and [l 1 - 18o]trans diol of ABA methyl ester at pH 3.5 The methyl ester of [l 1 - 18o]cis- or [l 1 - 18o]trans-diol of ABA was left in pH 3.5 buffer for 72 hand the products separated by normal phase HPLC. The retention of l 1 - 18oH was measured by mass spectrometry of the O-acetyl derivative. The ions at m/z 231 (18o 0 ) and 233 (18o 1 ) were used to measure the proportion of 180 in the samples. Starting material After incubation 1 - 18 cis-diol (l o]trans-diol ---trans-diol fragment 61 100 61 100 61 100 containing 63 0.35 63 0.37 63 0.30 4'-OH fragment 231 17.75 231 17.52 231 7.27 containing 233 12.34 233 11. 48 233 3.14 l'-OH % l'-180 41.0 39.6 30.2 % starting 100.0 96. 5 73.7 material loss of l'-180 0% 3.4% 26.3 [l 1 - 18oJcis-diol cis-diol trans-diol fragment 61 100 61 100 61 100 containing 63 0.22 63 0.23 63 0.19 4'-OH fragment 231 6.89 231 6.81 231 16.82 containing 233 4.63 233 4 .11 233 9.37 l'-OH % l'-180 40.3 37.6 35.8 % starting 100.0 91. 7 88.8 material loss of l'-180 0% 8.3 11.2 identical mass spectra to diols before isomerization. Therefore, there was no exchange of the 4'-hydrogen atom during the interconversion of the diols. 141 4.3.3.5 ______Interconversion of____;____;,:_____;_:__;:_ the dials __in _.__-=------2- [18oJH o The 1' ,4'-diols of ABA methyl ester in [18oJH2o (97.2 atoms% 180), pH 3.5, exchanged 180 into the 4' and 1'-hydroxyl groups (Table 4.5). 0xygen-18 from water was readily exchanged with the 4'-hydroxyl group of Me-cis-diol (84% 4 1 - 180) but no 180 was detected in the 1'-hydroxyl. The trans-diol produced from cis-diol in [18oJH2o had 76.3% 180 in the 4'-hydroxyl and 11.7% 180 in the l'-hydroxyl. Thus, both the 1'- and 4'-hydroxyl groups are involved when the cis-diol is isomerized to the trans-diol. ABA produced from cis-diol also had a high proportion (74.5%) of 180 in the 4'-position as shown by the relative abundance of the ions at m/z 261 and 263, which are produced by the loss of 1'-0H from the molecular ion. Unlabelled trans-diol exchanged 180 from the medium into the 4'-hydroxyl group (23.6%) although not to the same extent as the cis-diol. The ABA (19.5% 4 1 - 18oH) produced reflected the proportion of 180 in the trans-diol. 4.3.3.6 Inversion of the 1'-hydroxyl group during diol interchange Although the l'- and 4'-hydroxyl groups exchanged oxygen with water, it is possible that exchange could take place at either position without inversion occurring. The isomerization of (+)- and (-)-Me-[2-14cJ trans-diol was investigated to determine whether the 1' and/or 4'-hydroxyl groups were inverted during the 142 Table 4.5 Isomerization of the Me-cis- and Me-~-diol of ABA at pH 3.5 in [18oJH2o The Me-1',4'-cis- and Me-1',4'-trans-diol of ABA were dissolved in [ 18oJH2o made to pH 3.5 with acetic acid, and the products separated by HPLC. Mass spectrometry of the acetylated products was used to measure the proportion of oxygen-18 in the 4'-(18o0 - m/z 61; 18o1 - m/z 63) and 1 1 -(180 0 - m/z 231; 18o1 - m/z 233) positions. Starting material After incubation cis-diol cis-diol trans-diol ABA fragment 61 100 61 19.00 61 30.99 261 16.04 containing 63 0.20 63 100 63 100 263 46.96 4'-OH fragment 231 7.75 231 6.14 231 31. 34 containing 233 0.19 233 233 4.16 l'-OH % 4,_180 0.2% 84% 76.3% 74.5% % l'-180 2.4% 11. 7% Starting material After incubation trans-diol trans-diol ABA fragment 61 100 61 100 261 20.74 containing 63 0.4 63 29.96 263 8.93 4'-OH fragment 231 40 231 28.6 containing 233 0.8 233 0.44 l'-OH % 4,_180 0.4% 23.6% 19.5% % l'-180 2.0% 1.5% 143 30 a Cl) Cl. u 20 :,:; > u «I 0 -0 «I a: 10 Retention time, min b 40 Cl) Cl. u :,:; > u «I 0 -0 20 «I a: 0 10 20 30 40 50 Retention time, min Fig. 4.7 The products of isomerization and oxidation of (+)- and (-)-trans-diol separated by HPLC on Pirkle columns The (-)- (a) and (+)-Me-1' ,4'-[2-14cJtrans-diol of ABA (b) were left in solution at pH 3.5 for 72 hand the products separated by HPLC on one covalent - and 2 Type 1-A Pirkle columns eluted with hexane/isopropanol (93:7, v/v) at 2.0 ml/min. Retention times: Me-trans-diol, 28.5 min; Me-ABA, 32.5 min; Me-(+)-cis-diol, 46.2 min; Me-(-)-cis-diol, 48.3 min. 144 interconversion of the diols. The products of isomerization in acidic solution were separated by HPLC on optically-active Pirkle columns. Thus, the cis-diol produced from the (+)- or (-)-trans-diol could be resolved into its enantiomers to determine whether inversion at the C-1'- or C-4'-positions had occurred. (-)-Me-[2-14c]trans-diol (Fig. 4.7a) was converted into the (+)- and (-)-cis-diols with slightly more of the (+)-enantiomer being produced. The identity of the (+) and (-)-cis-diol was verified by their HPLC retention times and their UV spectra. The isomerization of (+)-[2-14c]trans-diol (Fig. 4.7b) also produced approximately equal amounts of (+)- and (-)-cis-diol. Therefore, the isomerization of the diols involves inversion at C-1' and C-4'. 4.3.4 Metabolism of the 1' ,4'-trans-diol of ABA Tomato and pea shoots supplied with RS-1' ,4'-[214c] trans-diol of ABA produced similar metabolites over 7 days (Fig. 4.8). The major products were shown to be the same by comparison of the relative mobilities of the compounds and their derivatives on thin-layer plates (Table 4.6). The amounts of the products were increased by feeding a relatively large amount (7.5 mg) of low specific activity RS-1' ,4'-[214c] trans-diol of ABA to tomato shoots. The metabolites were separated by reversed-phase HPLC (Fig. 4.9) and were found to be DPAGS, ABA, ABAGE and four unknown compounds which were designated "G", "H", "I" and "J". The known metabolites 145 pea E a. 10 "C ;::,:; ;t: .2= -t) CU 0 "C a:CU 5 X "'I 0... Retention time, min tomato 4 E a. "C 3 ;::,:; .-:: .2= -t) CU .2 2 "C CU a: X "' ....0' 1 0 10 20 30 40 Retention time, min Fig. 4.8 HPLC separation of the metabolites of [2-14c1 trans-diol by pea and tomato shoots 1.18 µCi (308 µg) (+/-)-1' ,4'-trans-diol of ABA was supplied to 35 pea shoots (13 g) and to 5 tomato shoots (11.2 g). After 7d the metabolites were analysed by reversed-phase HPLC. For chromatographic conditions see the legend to figure 4.9. a) pea b) tomato 146 Table 4.6 Thin-layer chromatography· of metabolites of RS-1' ,4'-[2-14cJ-t_nns-diol of ABA A: Solvent - chloroform/methanol/water (75:22:3, by vol.) Compound Underivatized Me Ac MeAc Hydrolysed ABA 0.69 0.96 0.69 0. 96 0.69 trans-diol 0.63 0.94 0.75 1.0 0.63 ABAGE 0.54 0.54 1.0 1.0 0.69 DPAGS 0.10 0. 71 0.84 1.0 0.10 G 0.39 0.39 1.0 1.0 0.63 H 0.40 0.39 1.0 1.0 0.63 I 0.26 0.56 0.75 0.91 0.26 J 0.28 0.55 0.77 0.97 0.28 B: Solvent - toluene/ethyl acetate/acetic acid (25:15:2, by vol.) Compound Underivatized Me Ac MeAc Hydrolysed ABA 0.49 0.61 0.49 0.61 0.49 trans-diol 0.41 0.53 0.58 0.68 0.41 ABAGE 0 0 0.53 0.53 0.49 DPAGS 0 0.1 0.23 0.44 0 G 0 0 0.66 0.66 0.41 H 0 0 0.65 0.65 0.41 I 0 0.3 0.50 0.59 0 J 0 0.2 0.51 0.60 0 Me - reacted with diazomethane Ac - attempted acetylation MeAc - acetylation of the product of methylation Hydrolysed - the major product after basic hydrolysis 147 10 J E a. 8 "O >, .'!:: > G -u 6 «l 0 "O «l a: X 4 (') I 0,... 2 Retention time, min Fig. 4.9 Metabolites of (+/-)-1' ,4'-[2-14cJtrans-diol of ABA supplied to tomato shoots 2 µCi (7.5 mg) (+/-)-1' ,4'-[2-14cJtrans-diol of ABA was supplied to 17 tomato shoots (56 g). Metabolites were separated by reversed-phase HPLC on an 8 (i.d.) x 250 mm Techsil 10 Cl8 column with a mobile phase of ethanol/ 0.2% aqueous acetic acid (3:17, v/v) for 22 min and a linear gradient to ethanol/0.2% aqueous acetic acid (1:3, v/v) over 15 min. After 7 min at the final concentration a linear gradient to ethanol/water (19:1, v/v) over 10 min removed the remainder of metabolites from the column. Retention time of standards: DPAGS - 6 min; ABAGE -20.3 min; trans-diol - 33.7 min; ABA - 37.8 min. were identified by a combination HPLC retention times and the UV spectra measured with a diode array detector (Fig. 4.10). Confirmation of the identity of the metabolites was obtained by comparing the relative mobilities of the 148 compounds and their derivatives with standards on thin-layer plates in two solvent systems. The same procedure was used to gain structural information of the unknown products (Table 4.6). The compounds G and H behaved almost identically. Upon acetylation both compounds displayed a marked change in mobility but were unaffected by methylation. The compounds were readily hydrolysed by base to yield the 1',4'-trans-diol of ABA. Apart from their polarity, compounds G and H had properties similar to ABAGE. The chromatographic behaviour of compound I was similar to that of compound J both were methylated and acetylated but were unaffected by basic hydrolysis. 4.3.4.1 Isolation of metabolites Derivatization of the metabolites G, H, I and J coupled with separation by HPLC enabled materials of high purity to be isolated. The increased volatility and solubility of the derivatives in non-polar solvents was useful in the subsequent mass spectrometry and NMR spectroscopy. Compounds I and J were chromatographed as methyl esters and then 0-acetylated methyl esters. When a sample of methylated, acetylated I and J were combined the derivatives were separable by HPLC. By contrast, O-acetyl derivatives of compounds G and H were not separable by the HPLC methods tried so care had to be taken to separate G and H completely as free acids. 149 4.3.4.2 Ultra-violet spectroscopy A diode-array detector was used to record the UV spectra between 200 and 350 nm at the apices of peaks eluted from HPLC columns. The spectra of compounds I and J were almost identical and the absorbance maxima (Amax) are very similar to those of the cis- and trans- diols and their methyl esters (Fig. 4.10). Compounds G and H also have similar spectra but their absorbance maxima, 273 nm (underivatized) and 275 nm (acetylated), are very similar to those of ABAGE except that the former lack the shoulder at 240 nm, caused by the a, ~-unsaturated ketone chromophore, which is a characteristic of ABA in aqueous solutions. 4.3.4.3 Characterization of compounds I and J. Compounds I and J behaved similarly in various chromatographic systems. The underivatized compounds failed to move from the origin of silica gel thin-layer plates developed in toluene/ethyl acetate/acetic acid (25:15:2, by vol.) (Table 4.6) the same was seen for other conjugates: ABAGE [28], ABAGS [29] and DPAGS [30]. When chloroform/methanol/water (75:22:3, by vol.) was the solvent I (RF 0.26) and J (RF 0.22) had mobilities between those of DPAGS (RF 0.1) and ABAGE (RF 0.54). The same intermediate polarity was observed on reversed-phase chromatography with I and J eluting after DPAGS and before the less polar ABAGE. Acetylation of the metabolites produced a greater change in mobility and retention time on thin-layer plates and reversed-phase 150 !-ABA Amu=263 nm ABA Amax = 261 nm 90 Cl) .,"C: -e 0 IJ) .0 <( 'ii \ !-ABA 0 -10 200 250 300 350 Wavelength, nm ABAGE Amax=273 nm 90 Cl) .,"C: .0 0 IJ) .0 <( 'ii 0 -10 200 250 300 350 Wavelength, nm H Amax= 273 nm 90 Cl) .,"C: .0 0 IJ) .0 <( 'ii 0 -10 200 250 300 350 Wavelength, nm Fig. 4.10 (a} UV spectra of metabolites of 1' ,4'-trans-diol of ABA in ethanol/water/acetic acid recorded on a diode-array detector 151 !-diol of ABA Ama.=267 nm 90...------~ ., 0 .,C: n 0 n <("' ~ 0 -10+-r---,----,--,---,-,--,~---,---.-,---,,--,----,--,-~~~-.-.....-~~----.--.-.....-~r-1 200 250 300 350 Wavelength, nm J Amax=265 nm 90 ., 0 .,C: n 0 n"' <( ~ 0 -10+-r---,----,--,---,-,--,~---,---,-,---,,--,----,--,-~~~--,-.---,,-r---,--,-,-,-,7 200 250 300 350 Wavelength, nm DPAGS Amax =265 nm 90 ., 0 .,C: n 0 nU) <( ~ 0 - 1 O-h.-r---r-,-.---r-r---.-...-,--r-.-,....-,,--,---r---r-,--,~30TO:,--,-.--.--r--.-r-r---i;:3j5 0 200 250 Wavelength, nm Fig. 4.10 (b) UV spectra of metabolites of l' ,4'-trans-diol of ABA in ethanol/water/acetic acid recorded on a diode-array detector 152 Table 4.7 Absorbance maxima of trans-diol metabolites and their methyl and 0-acetyl derivatives in aqueous ethanol solutions Me - product of methylation, MeAc - product of methylation and acetylation A.Illax Compound Me MeAc DPAGS 265 267 267 G 273 273 275 H 273 273 275 I 265 267 267 J 265 267 267 ABAGE 273 273 275 trans-diol 265 267 267 cis-diol 265 267 265 t-ABA 263 263 263 ABA 261 261 261 HPLC than did methylation. The large change in polarity after acetylation reflects the derivatization of four hydroxyl groups on the glucosyl residue, while reaction with diazomethane is evidence for a free carboxyl group. The UV spectra of I, J, and their derivatives (Table 4.7) were like those of l' ,4'-trans-diol and its derivatives. 153 (a) Mass spectrometry Mass spectra of methylated and acetylated I and J were obtained by direct probe-chemical ionization with methane as the reagent gas. The mass spectra of MeAc-I and MeAc-J (Fig. 4.11) are almost identical with only slight differences in the abundance of some fragment ions, therefore the discussion of the features of the mass spectrum of MeAc-J applies to that of MeAc-I. The positive ion spectrum of MeAc-J has ions corresponding to M+l, M+29 and M+41 which are characteristic of spectra obtained with methane as the ionizing gas. This gives a molecular weight of 610. The lower mass fragment ions are of two groups : i) those derived from the sugar residue at m/z 331, 289, 271, 169, 109 ii) those derived from the aglycone at m/z 263, 245, 232, 231, 137, 125. A sample of J biosynthesized from 1' ,4'-[4'-2H]trans-diol of ABA when methylated and acetylated had a molecular weight of 611 with the ions from the aglycone increased by one mass unit (except for m/z 125) but the ions from the sugar residue were unchanged. The fragmentation of D-glucose pentaacetate has been described by Hogg and Nagabhushan (1972) and involves the loss of four molecules of acetic acid (60 mass units each) and one of ketene (42 mass units). The sugar residue of Me-J shows similar fragmentation. The aglycone of MeAc-J shows fragment ions characteristic of 154 a 100 231 50 125 169 61 109 137 213 245 271 >, 100 200 300 1 50x ·;;:; 100 C Q) -~ so 331 431 551 400 500 2.0 593 1.0 639 579 611 628 651 600 700 800 m/z b CH,OAc ~\--°"' '-' AcO~ OH /4 COOCH, AcO 0 OAo 100 263 231 50 125 169 109 137 245 271 289 100 200 300 ·;;:;~ C Q) .S Q) > -~ qi ·:1'-~33,.._(...... ,r...... ,so~x_.,...... ,_,..,-..--,-_,..4J,1-.-----r----r--r---.--r--,-1 -,-s+-1~...... -.-s..,...... ,,;• J a: 400 500 :1~. T ---.--'t'----,'T. / ....,....-,---- 1 --r--r-r-1~----,---,---,-, .r ~~r600" . 700 800 m/z Fig. 4.11 Mass spectrometry of MeAc-I and MeAc-J (a) methylated and acetylated compound I (4'-glucoside of (-)-1' ,4'-trans-diol of ABA) (b) methylated and acetylated compound J (4'-glucoside of (+)-1' ,4'-trans-diol of ABA) Positive ion. Methane was the ionizing gas 155 O-acetyl 1' ,4'-trans-diol of ABA methyl ester (Fig.4.4a; Table 4.3). The peak at m/z 263 corresponds to 4'-desoxy ABA [26] which is often the highest mass ion observed for Me-trans-diol or its O-acetyl derivative. The other ions (at m/z 245, 232, 231, 137 and 125) and their relative abundance are similar to those obtained with MeAc-trans-diol and the pentadienoic acid side chain methyl ester, m/z 125, is evidence that the C-1 carboxyl is methylated. A molecular weight of 610 and the mass spectrum is consistent with the metabolite being the 1' ,4'-trans-diol of ABA conjugated through the 4'-hydroxyl group to a hexose residue. Conjugation through the tertiary C-1 hydroxyl group is unlikely because this would leave the secondary 4'-hydroxyl group free to be acetylated and a molecular weight of 652 would be expected. Under the conditions used to acetylate the glucose residue, the 4'-hydroxyl group of the trans-diol is readily acetylatable but the tertiary 1'-OH was not acetylated (even in the presence of catalysts, 4-pyrrolidinopyridine or 4-dimethylaminopyridine, with isopropenyl acetate). (b) NMR spectroscopy 1H-NMR spectra of the O-acetyl methyl derivatives of compounds I and J (Figs. 4.12 and 4.13 respectively; Table 4.8) were obtained on a 300 MHz instrument. Unlike spectra of glucosides run at 100 MHz (Loveys and Milborrow, 1981; Milborrow and Vaughan, 1982) the signals of the glucosyl protons were well resolved allowing the 156 ·- t:5i' ' ;p;;;;} = •,;i! - .1111 IC/ 0 "" ,g - .. 7 6 5 4 3 2 ppm & Fig. 4.12 1tt 2-D NMR (COSY) spectrum of (-)-~-diol 4' glucoside 300 MHz 1tt shift-correlated 2-D NMR (COSY) spectrum of methylated and acetylated compound I in [2H)chloroform 157 CJ . •D •':'-!~ :- lfi;B'B 0 ... :• w,o•, ~-"'°"" A..:O~O ~OH /4 -...._ COOCH, AcO 0 OAc - .lJ ·--·· 7 6 5 4 3 2 ppm o Fig. 1 4.13 H 2-D NMR (COSY) spectrum of (+)-t.£fillli-diol 4' glucoside 300 MHz 1 H shift-correlated 2-D NMR (COSY) spectrum of methylated and acetylated compound Jin [2H]chloroform 158 Table 4.8 1H NMR spectral data for o-acetyl trans-diol glucosides 1H NMR spectroscopy at 300 MHz in [ 2H]chloroform, tetramethylsilane as internal standard. MeAc-I = O-acetyl 4'-glucoside of l'~,4'~-4' dihydroabscisic acid methyl ester (57]. MeAc-J = O-acetyl 4'-glucoside of l'f,4'f-4'-dihydroabscisic acid methyl ester (49]. The data for O-acetyl trans-diol of ABA methyl ester (42] are those of Milborrow (1984). MeAc-I (57] MeAc-J (49] MeAc-trans-diol (42] C-lOMe s 3. 71 3.73 s 3. 71 C-2H s 5. 72 s 5.73 m 5. 71 C-4H dd 7.75 (J=16, 1) d 7.73 (J=l6) dd 7.76 (J=l6, 1) C-5H dd 6.13 (J=l6, 1) d 6.13 (J=l6) dd 6.15 (J=l6, 1) C-6Me s 2.01 s 2.02 d 2.02 C-71Me s 1.57 s 1.56 m 1. 69 C-3'H d 5. 62 d 5. 64 d 5.53 C-4'H m 4.3-4.4 m 4.3-4.4 m 5.37 C-5'2H m 1. 7-1. 9 m 1. 7-1. 9 m 1. 70 (ax.) 1. 87 (eq. l C-8'Me s 1.03 s 1.03. s 1.08 C-9'Me s 0. 92 s 0.91 s 0. 92 4'O-AcMe s 2.07 acetyl glucose residue pentaacetyl glucose C-lH d 4.75 (J=8) d 4.76 (J=8) d 5.73(J=8) C-2H dd 4. 98 (J=8, 10) dd 4.97- (J=8,10) t 5.14 (J=l0) C-3H t 5.32 (J=9) t 5.29 (J=9) t 5.26 (J=9) C-4H dd 5.05 (J=9,10) t 5.07 (J=l0) t 5.15 (J=l0) C-5H ddd 3. 71 (J=2, 5, 10) ddd 3.91 (J=2,5,10) ddd 3.85 (J=2,5,10) C-6HA dd 4.20 (J=5,12) dd 4.25 (J=S,12) dd 4.23 (J=5,12l C-6HB dd 4.12 (J=2,12) dd 4.14 (J=2,12) dd 4.12 (J=2,12) O-AcMe 2.06 2.09 2.01 (x2) 2.02 2.00 2.00 1. 99 configuration of the sugar residue to be determined. Two-dimensional homonuclear shift correlated spectroscopy (COSY) was used to assign the signals obtained to the position of the protons in the glucose molecule by 159 determining which signals were coupled. In a COSY spectrum diagonal multiplets occur at each chemical shift and symmetrical off-diagonal multiplets indicate connectives via J-coupling. For a given pair of coupled spins the diagonal and off diagonal multiplets form the corners of a rectangle. This relationship is illustrated in figure 4.14, which shows the glucosyl region of MeAc-J. The anomeric, C-1, proton occurs as a doublet at 84.75 and is coupled to the proton at 84.98 which, therefore, is then the C-2 proton. The C-2 proton is in turn coupled to the proton signal at 85.32: the C-3 proton. This procedure was used to assign the signals to protons and their position in the glucose molecule. (c) The glucosyl residue The signals attributed to glucose from the COSY spectrum and the calculated coupling constants for the protons of ~-D-glucose pentaacetate, MeAc-I and MeAc-J are included in Table 4.8. The C-1 (anomeric) proton (84.76) is coupled to the C-2 proton (84.97) with a coupling constant, J, of 8 Hz. The coupling constants of the other pyranose ring protons are: J 2 , 3 (10 Hz), J 314 (9 Hz), J 415 ((10 Hz). The coupling constants calculated for glucose pentaacetate are the same as those of MeAc-I and MeAc-J. The coupling constants are the same order as that measured by Coxon (1972) for diaxial, vicinal protons of carbohydrates (J = 8.6 - 11 Hz). This gives the configuration of the sugar residue as ~-glucose (C-1-~). The integral of the spectrum indicated that 160 0 g3 g2-3 3 4 2 6' l 5.0 4.0 ppm 6 Fig. 4.14 Glucosyl proton region of the COSY spectrum of MeAc-J The glucosyl proton region of the 300 MHz 1H COSY spectrum of MeAc-J (Fig. 4.13) in [2H]chloroform there was only one glucose residue in the molecule and this was confirmed by the presence of only one anomeric proton in the spectrum. The chemical shifts of the glucosyl protons of MeAc-I and MeAc-J have similar chemical shifts to those of pentaacetyl glucose. The major exception is the anorneric proton which is upfield 161 in MeAc-I and MeAc-J by approximately 1 o and reflects the difference in conjugation of C-1 to acetate or the 4'-hydroxyl group of the trans-diol of ABA. The chemical shifts of the glucosyl protons of MeAc-I are similar to those of MeAc-J except that the C-5 glucosyl proton of MeAc-I is shifted upfield by 0.2 o and could be due to the different environment of this proton in glucose conjugated to (-)-trans-diol from that of the corresponding proton for glucose conjugated to (+)-trans-diol. (d) The aglycone The proton signals of the trans-diol residue of MeAc-I and MeAc-J (Table 4.8) show a close correspondence to the signals of 0-acetylmethyl 1',4'-trans-diol of ABA [42] (Milborrow, 1984b). The methyl signals of MeAc-J at o0.91, 01.03, 01.57, 02.02 and 03.73 are in similar positions to the same groups of 4'-0-acetyl-trans-diol methyl ester [42] (C-9' Me- o0.92, C-8' Me- 01.08, C-7' Me- ol.69, C-6 Me- 02.02, C-10 Me- 03.71). The signals of the sidechain protons of MeAc-J correspond to those of acetyl trans-diol methyl ester (C-2- 05.71, C-4- 07.76 and C-5- 06.15). The ring proton signals correspond except that the 4'-proton of MeAc-J (4.3-4.4) is downfield 1 o from its position in the spectrum of 4'-0-acetyl trans-diol (05.37) but is close to the position of the 4'-proton of DPAGS (04.1-4.3). This provides evidence that the 4'-hydroxyl group of MeAc-J is conjugated to glucose and is further evidence that it is 162 not acetylated. The integrals of the spectrum indicate that four acetyl groups and one glucosyl residue are present . (e) Hydrolysis of the 4'-glucoside of trans-diol Under basic conditions (ethanol/2M KOH (2:1, v/v), 2 hat 40°c), which are sufficient to hydrolyse glucose esters, compounds I and J released a small amount (5-12%) of a product with the same UV spectrum and HPLC retention time as the 1' ,4'-trans-diol of ABA. The compounds were more susceptible to acid hydrolysis being completely hydrolysed by lM HCl at 100°c for 6 h. The sugar released by acid hydrolysis of I and J was identified as glucose by comparison of the capillary GC retention time and mass spectrum of the TMS-O-methyloxime and that of a glucose standard. 4.3.4.4 Characterization of compounds G and H The chromatographic behaviour of compounds G, H, and their acetyl derivatives paralleled that of ABAGE (Table 4.6 ABAGE, G and Hall remain at the origin of silica gel thin-layer plates developed in toluene/ethyl acetate/ acetic acid (25:15:2, by vol.) and all compounds show a marked increase in mobility after acetylation: Ac-ABAGE (RF 0.53), Ac-G (RF 0.65), Ac-H (RF 0.66). When the solvent system chloroform/methanol/water (77:22:3, by vol.) was used, the following RF values were obtained: ABAGE (RF 0.54), G (RF 0.39), H (RF 0.40), Ac-ABAGE (RF 1.0), Ac-G (RF 1.0), Ac-H (RF 1.0). Although G and H 163 have a lower RF than ABAGE, acetylated G and H have a higher RF than acetyl ABAGE indicating the presence of an additional acetylatable group. Metabolites G and H, like ABAGE, did not react with diazomethane. However, if the diazomethane had not been redistilled before its use with G and H, sufficient KOH may have been present from the preparation of the diazomethane to hydrolyse the glucose ester and give Me-trans-diol. This hydrolysis by diazomethane was observed in methylation of ABAGS (Loveys and Milborrow, 1981). Compounds G and H were also hydrolysed under acidic conditions (lM HCl, 100°c, 6 h). The TMS-0-methyloxime produced from the sugar released by both G and H had an identical capillary GLC retention time and mass spectrum to the TMS-0-methyloxime of glucose. The UV spectra of compounds G and H (Amax= 273 (ethanol/water)) and the acetyl derivatives (Amax= 275 (ethanol/water)) had the same Amax as ABAGE and its acetyl derivative. The similarity of the UV spectra and chromatographic properties of G, Hand ABAGE, as well as the hydrolysis to release trans-diol indicated that G and H might contain trans-diol conjugated to a sugar through C-1. The glucose esters of cis- and trans-diol of ABA were prepared to compare with compounds G and H. 164 (a) Reduction of ABAGE to the glucose esters of 1' ,4'-cis- and 1' ,4'-trans-diol of ABA A sample of ABAGE, formed from RS-ABA, was reduced with sodium borohydride to prepare glucose esters of the cis- and trans-diols. The reduction was performed in phosphate buffer (0.1 M, pH 6.0) to avoid basic hydrolysis of the glucose ester. Reversed- phase HPLC, with the same conditions as those used to separate the metabolites of trans-diol, was used to separate the reduction products of ABAGE (Fig. 4.15). Two peaks with identical retention times to G and H (12 and 13 min respectively) were separated as well as unreduced ABAGE (19 min) and another peak at 38 min. The compounds eluting at 12 and 13 min had the same UV spectrum as G and H, and were hydrolysed by base to release 1' ,4'-trans-diol of ABA. These compounds co-chromatographed with G and Hand their derivatives and G and H were therefore identified as the glucose esters of (+)- and (-)-trans-diol ([50] and [58] respectively). The peak with a retention time of 38 minutes produced 1' ,4'-cis-diol of ABA on basic hydrolysis and was identified as the glucose ester of the cis-diol of ABA [ 61] . (b) Mass Spectrometry The chemical-ionization (methane) mass spectra of the acetylated glucose esters of the trans-diol of ABA, produced by the reduction of ABAGE and the spectra of Ac-G and Ac-H, formed biosynthetically from [4'-2H]- 165 E C: LO c.o C\I Q) 0 C: ctS ..c '- 0 en ..c <( 10 20 30 40 50 Retention time, min Fig. 4.15 HPLC separation of ABAGE reduction products The products of reduction of ABAGE with sodium borohydride were separated by HPLC as described in the legend to figure 4.9. Peaks at 12 and 13 min are the (-)- and (+)-trans-diol glucose esters respectively. ABAGE is at 19 min and cis-diol glucose ester at 38 min. trans-diol (Figs. 4.16 and 4.17 respectively) have many features in common. The mass spectra are dominated by the loss of acetic acid ([CH3COOH + H]+ = m/z 61) from the acetylated 4'-hydroxyl group of the trans-diol residue. This loss of acetic acid also occurs with the acetylated methyl ester of trans-diol (Fig. 4.4) to give an apparent molecular ion of m/z 262 ,that is, 4'-desoxy ABA [26]. The presence of the glucose ester of 4'-desoxy 166 100 61 50 169 231 109 133 177 100 200 >, 331 .-t:: 100 rlOOx (/J C Q) .E- 50 369 Q) .::: 271 -ctl ai a: 300 1.0 515 579 0.5 471 487 647 621 631 500 600 m/z Fig. 4.16 Mass spectrum of (-)-Ac-trans-diol glucose ester The positive ion chemical-ionization (methane) mass spectrum was obtained by direct probe insertion of a sample of (-)-0-acetyl trans-diol glucose ester (Ac-G) (59] (a) and the same compound biosynthesized from [4'-2H]trans-diol (b) ABA (tetraacetate m.w. = 578) is indicated in the spectra of the diol glucose esters by the occurrence of ions at m/z 579 [M + H] and 580 for the glucose esters derived from [4'-2H]-trans-diol. The ions at the higher-mass end of the spectrum are of low abundance and the presence of impurities complicated the spectrum. The lower-mass fragment ions are those of glucose (m/z 331, 271, 169, 109) and trans-diol (m/z 232, 231). 167 ' AcO OAc OAc H coo~,OAc 100 61 AcO M 50 >, 169 331 -Cl) C Q) 109 231 73 271 -C 127 Q) 100 200 300 > 100 ,2oox -«l Q) a: 50 519 5 9 400 500 600 m/z Fig. 4 .17 Mass spectrum of (+)-Ac-trans-diol glucose ester Positive ion (methane) chemical-ionization mass spectrum of (a) (+) -O-acetyl-trans-diol glucose ester [51 l (Ac-H) and (b) the (+)-[4'-2H]trans-diol glucose ester direct probe. 168 (c) NMR Spectroscopy 300 MHz 1H NMR spectra of the glucose esters of (-) -trans-diol (Fig. 4 .18a) , ( +) -trans-diol (Fig. 4 .18b) , cis-diol (Fig. 4.19a) and ABAGE (Fig. 4.19b) were obtained in [2H5 Jpyridine and are listed in Table 4.9. The compounds were separated by a single chromatographic step and some impurities in the spectra are evident. However, the spectrum of ABAGE was similar to the 100 MHz spectrum of ABAGE in [2H5 Jpyridine reported by Loveys and Milborrow (1981). In [2H5 Jpyridine the glucosyl protons were poorly resolved in comparison with those of O-acetyl trans-diol glucosides in [2HJchloroform (Fig. 4.14). All four glucose esters had signals for the C-2 to C-6 glucosyl protons between 83.8 and 84.4. However, the signal for the anomeric, C-1 proton occurring as a well-resolved doublet at 86.2 with a coupling constant of 8 Hz showed that the glucosyl residue was attached via a ~-configuration (Coxon, 1972). The main difference between the spectra of the diol glucose esters and ABAGE is the presence of a broad signal, at 4.44 8 to 4.48 8 in the spectra of the diols, which is attributable to the C-4' proton. The signals for the C-5' protons also show differences due to the effects of differences in coupling to the C-4' proton. Overall, as expected, the NMR spectra of the (+)-[50] and (-)-trans-diol glucose ester [58] are very similar but apart from the C-4' and C-5' proton signals the remainder 169 a 9 8 7 6 5 4 3 2 0 ppm I> b HO OH ·oH ~coo-b-:l:,1,011 HO , A. .. J .J ""-'+--- ~~J_ I 9 8 7 6 5 4 3 2 1 0 ppm 6 Fig. 4.18 1H NMR spectroscopy of trans-diol glucose esters 1H NMR spectroscopy at 300 MHz in [2H5 Jpyridine. (a) (-)-trans-diol glucose ester (b) (+)-trans-diol glucose ester signals at 87.00, 7.37, 8.52 are pyridine H2o is a broad signal at 85.0. Identification of signals is given in table 4.9. Reference is pyridine 87.00. 170 a ~ Hor----J:}--oH 0 H0',,~ ~ 'loo__L-O...._£cH,OH .·-·-·· -. II __L __jJL_- 9 8 7 6 5 4 3 2 0 ppm 11 b I I I I I __r-,.l't,1___, ... ~-.1JvrJ\~,LJj J_--uJ1I ...... ~. __ 9 8 7 6 5 4 3 2 0 ppm 11 Fig. 4.19 1H NMR spectroscopy of ABAGE and cis-diol glucose ester 1H NMR spectroscopy at 300 MHz in [2H5 ] pyridine. (a) cis-diol glucose ester (b) ABAGE. Pyridine 87.00, 7.37, 8.52 H2o is a broad signal at 85.0. Reference pyridine 87.00. Identification of signals is given in table 4.9. 171 Table 4.9 1H NMR of glucose esters of ABA and its 1' ,4'-diols 1H NMR spectra at 300 MHz in (2H5 ]pyridine of ABA glucose ester (28], (-)-1'~,4'~-4'-dihydroabscisic acid glucose ester [58], (+)-1'~,4'~-4'-dihydroabscisic acid .glucose ester (50], and (+/-)-1' ,4'-cis-diol of ABA glucose ester [61]/(64]. Reference 7.00 o pyridine; s, singlet; m, multiplet; d, doublet glucose ester ABA (-) -trans- (+) -trans- cis-diol diol [58] diol[50] (61]/[64] C-4 H d 8.30 (J=l6) 8.31 (J=l6) 8.29 (J=l6) 8.39 (J=16) C-5 H d 6.40 (J=l6) 6.38 (J=l6) 6.38 (J=l6) 6.27 (J=l6) anomeric 6.18 (J=7 .8) 6.16 (J=7_. 7) 6.18 (J=7.7) 6.21 (J=7 .6) proton d C-3'H s 6.00 5. 96 5.96 6.02 C-2 H s 5.67 5.58 5.59 5.64 C-4'H m 4.44-4.58 4.46-4.54 4.43-4.54 glucose C-2 3.81-4.31 3.81-4.35 3.81-4.33 3.84-4.33 to C-6 5H C-5' 2H m 2.54 (eq.) 1.88-2.04 1.88-2.03 2.14 (eq.) 2.28 (ax.) 1. 86 (ax.) C-6 Me s 1.80 1. 75 1. 75 1. 79 C-7'Me s 1. 65 1. 61 1. 61 1. 67 C-8' Me s 1.09 1.13 1.13 1.16 C-9'Me s 0.94 0.93 0.93 0.90 of the spectrum is close to that of ABAGE [28]. The spectrum of the cis-diol glucose ester [61] shows the presence of the C-4' proton signal but the remainder of the signals, with the exception of the C-5' proton signals, show more deviation from those of ABAGE than the spectra of the trans-diol glucose esters. 172 4.3.4.5 The Metabolism of (+)- and (-)-trans-diol of ABA Racemic l' ,4'-trans-diol of ABA was metabolized to two pairs of conjugates. The compounds of one pair were both identified as the 4'-glucoside of l' ,4'-trans-diol of ABA and the other pair as the l' ,4'-trans-diol of ABA glucose ester. It is likely that each component of a pair was derived from either the (+)- or the (-) enantiomer of the racemic dial which was fed. To test this possibility separate (+)- and (-)-[2-14c]trans diols were fed to tomato shoots. The resultant chromatograms are shown in figure 4.20. The (+)-trans diol (l'~,4'~-4'-dihydroabscisic acid) [3] was metabol ized mainly to the 4'-glucoside of DPA [30] with small amounts of radioactivity corresponding to compounds H, J and as well as ABAGE. The major products of the (-)-trans-diol (l'R,4'R-4'-dihydroabscisic acid) [52] were the l'-glucoside [29] and glucose ester of ABA [28]. Smaller amounts of material were found co-chromatograph ing with compounds G [58], I [55] and DPAGS [30]. In these feeding experiments small amounts (approximately 20 µg) of high specific activity [2-14c]trans-diol were supplied to tomato shoots. The major products were metabolites of ABA. In previous experiments 15 to 350 times the amount of trans-diol was supplied to a similar weight of tomato shoots. 173 (-} E 2 c. -0 ~ -~- -(.) «I .Q -0 1 «I 0: ..,>< I 0.... 0 10 20 30 40 Retention time, min 10~------, (+} E c. -0 >, -> -(.) 5 «I 0 -0 «I a: ..,X I ....0 0 10 20 30 40 Retention time, min Fig. 4.20 Separation of the metabolites of (+)- and (-)- 1' ,4~trans-diol by HPLC 82 200 dpm (18.7 µg (-)-1' ,4'-[2-14c]trans-diol of ABA (0.52 mCi/mmol] was fed to two tomato shoots (6 g) and 95 250 dpm (21.6 µg) (+)-1' ,4'-[2-14C]trans-diol of ABA (0.52 mCi/mmol to two tomato shoots (5.6 g). The products were extracted after 5 days. The HPLC conditions were the same as those described in the legend to figure 4.9. Radioactivity in fractions (4 ml) was determined by liquid scintillation counting. 174 However, the conjugates predominated when a larger amount of the trans-diol was fed. Apart from the problem of a small amount of trans-diol resulting in relatively small amounts of radioactivity in the conjugates of trans-diol, separate feeding experiments of (+)- and (-)- enantiomers cause some difficulty. Two separate batches of tomato shoots can mean differences in uptake and metabolic conditions. The subsequent extraction and chromatography can also differ slightly between each experiment and comparison between metabolites with close retention times can lead to uncertainties. To overcome these problems relatively large amounts of (-)-[2-14c]- and (+)-[G-3H]trans-diol were mixed and supplied to one batch of tomato shoots. The labelled enantiomers were diluted with a large amount of cold racemic material so that the amount of each enantiomer was practically identical. In addition, the diols were prepared by the reduction of the radiolabelled abscisic acids with sodium borodeuteride so that the diols were labelled with deuterium in the 4'-position. The products of the mixture of (+)-[ 3H] and (-)-[ 14c]trans-diol fed to tomato shoots and analysed after 7 days show, as expected, a large proportion of trans-diol conjugates (Fig. 4.21). The chromatogram of 3H-labelled compounds indicated that compounds H (13 min) and J (19 min) were derived from (+)-[ 3H]trans-diol. Other compounds containing tritium were trans-diol, ABA, 175 E c. -0 >, -> () -ro 0 -0 ro er: 0 10 20 30 40 50 Retention time, min Fig. 4.21 HPLC separation of the metabolites of (+}-[G-3HJ and (-)-[2-14c]trans-diol of ABA 153 000 dpm (4 mg-)-1' ,4'-[2-14c, 4'-2HJtrans-diol of ABA [4 µCi/mmol] and 366 000 dpm (4 mg} (+)-1' ,4'-[G-3H, 4'-2H]trans-diol were fed to 11 tomato shoots (20 g} and extracted after 7 days. The products were separated by reversed-phase HPLC (see legend to Fig. 4.9). Fractions (2 ml) were collected and radioactivity determined by liquid scintillation counting. Full scale for 3H is 3 000 dpm and for 14c is 500 dpm. ABAGE and small amounts of DPAGS. The products of (-)-[ 14c]trans-diol were compounds G and I, ABA, ABAGE and trans-diol. The unmetabolized trans-diol contained higher amounts of the R-enantiomer indicating that the unnatural, (-)-enantiomer is metabolized more slowly than 176 the natural (+)-enantiomer as reported earlier for (+/-)-ABA (Chapter 3). The trans-diol glucose esters and 4'-glucosides were purified by high-performance liquid chromatography as the methyl, acetyl derivatives. The purified compounds G and I contained carbon-14 label; Hand J contained tritium. However, 3H-labelled compounds contained a small amount (approximately 3%) of carbon-14 and a similar, small proportion of tritium was found in the principally 14c-labelled compounds. This small enrichment of the originally opposite enantiomer in these metabolites was probably produced by racemization of the diols during the preparation of the (+)- and (-)-trans-diol, which involved two (acidic) oxidation steps, because the configuration at C-1' of the diols was shown to be partially inverted when the cis- and trans-diol interconverted in acidic solutions (Section 4.3.3). 4.3.4.6 Conversion of the 1' ,4'-trans-diol into the 4'-glucoside of dihydrophaseic acid Most of a small quantity of (+)-trans-diol supplied to tomato shoots was converted into DPAGS [30]. Possible routes for this conversion which were considered were: i) Oxidation of the trans-diol to ABA and metabolism via the normal pathway to DPAGS. ii) Metabolism of the trans-diol to DPAGS by a direct, unique pathway. 177 An attempt to differentiate between these two possible routes was made by labelling [2-14c]trans-diol in the 4'-position with deuterium. If DPAGS was produced via ABA then the deuterium would be lost. However, if deuterium were present in the 4'-position of DPAGS then oxidation to ABA could be ruled out. Racemic [2-14c,4'-2H]trans-diol was of low specific activity for carbon-14 but mass spectrometry revealed that the compound was almost completely deuterated in the 4' position. The deuterated and 14c-labelled trans-diol was mixed with high specific activity [3H]ABA, to monitor the isolation of DPAGS. The mixture was fed to tomato shoots and the metabolites separated by HPLC. The DPAGS was isolated by the procedures previously used (Section 2.2.10) and the deuterium content determined by mass spectrometry. The mass spectrum showed no deuterium was present in the 4'-position, although approximately 65% of the sample was derived from the deuterated trans-diol (calculated using the specific activity of deuterated, 14c-labelled trans-diol and the absobance of MeAc-DPAGS [£=17,200; 267nm in ethanol (Hirai and Koshimizu, 1983)]). This result is consistent with oxidation of the 4'-hydroxyl group to a ketone giving either ABA or phaseic acid (via 8'-hydroxy-4'-dihydroabscisic acid [27]). These two possibilities will be discussed later. 178 4.3.5 Metabolism of the 1' ,4'-cis-diol of ABA The metabolism of the cis-diol was studied in tomato shoots and although many products were found some of these could be attributed to artefacts occurring during the extraction of products. The (+)-[ 3HJ- and (-)-[14c]cis-diol produced in the preparation of the corresponding trans-diol was fed to tomato shoots and the products separated by reversed-phase HPLC (Fig. 4. 22) . The majority of the metabolites of the cis-diol had longer retention times than metabolites of ABA or trans-diol. This reflects the longer retention time of the cis-diol compared to ABA and trans-diol on reversed phase columns. Some radioactive peaks contained mainly 3tt or 14c, however, a major peak contained both isotopes and had the same retention time as the cis-diol glucose ester [61] (38 min). Products were also found which had the same retention times and isotopic labelling as the glucosides and glucose esters of trans-diol. When the chromatographic conditions were changed to allow for the apparent lower polarity of the cis-diol metabolites, a better separation of the products resulted (Fig. 4.23). The metabolites of low specific activity (+/-)-[2-14cJcis-diol were separated on an analytical reversed-phase HPLC column methanol/0.2% aqueous acetic acid (2:3, v/v) at 1.0 ml/min and monitored on diode-array and radioactivity detectors. Because a relatively large amount of the cis-diol was fed, most of the absorbance at 265 nm was due to metabolites of 179 a{+)- 3 H D{-)-14C E a. -0 >, -> () -co 0 -0 lU 0: 0 10 20 30 40 50 Retention time, min Fig. 4.22 Separation of the metabolites of (+)-[G-3HJ and (-)-[2-14cJcis-diol of ABA by HPLC 114 000 dpm (3.5 mg) (-)-1' ,4'-[2-14c, 4'-2HJcis-diol of ABA and 320 000 dpm (3.5 mg) (+)-1',4'-[G-3H, 4'-2HJcis-diol of ABA were supplied to 12 tomato shoots (21 g) and the metabolites extracted after ?days. The metabolites were separated by reversed-phase HPLC on a 7.8 (i.d.) x 300 mm µBondapak c 18 column eluted with ethanol/water/acetic acid (84:504:1, by vol.) for 22 min at 4 ml/min followed by a linear gradient to ethanol/water/acetic acid (158:508:1, by vol.) over 15 min. After 7 min at the final concentration a linear gradient to ethanol/water (19:1, v/v) over 10 min removed the remainder of the metabolites from the column. The radioactivity in fractions (2 ml) was determined by liquid scintillation counting. Full scale for 3H is 3000 dpm and for 14c is 500 dpm. 180 the cis-diol. A close correspondence between the chromatogram of absorbance at 265 nm and radioactivity was observed (Fig. 4.23). UV spectra recorded between 200 and 350 nm were used partially to identify the products and some were found with retention times and spectra identical to standards: DPAGS [30] (5 min; Amax= 267 nm), trans-diol glucose ester (7.8 min; Amax= 273 nm), trans-diol glucoside (9.5 min; Amax= 265 nm), ABAGE [28] (13.9 min; Amax= 273 nm), ABA (23.4 min; Amax= 261 nm) and cis-diol glucose ester [61] (16 min; Amax= 273 nm). In addition peaks, at 25 min (Amax= 265 nm) and 28.5 min (Amax= 265 nm), which reacted with diazomethane and acetic anhydride were probably glucosides of cis-diol. When a small amount of high specific-activity cis-diol was supplied to tomato shoots the major metabolite was DPAGS as occured when a small amount of trans-diol was fed to tomatoes. 4.3.5.1 The glucose ester of 1' ,4'-cis-diol of ABA A metabolite of the cis-diol of ABA had the same UV spectrum and retention time as the cis-diol glucose ester [61] produced by the reduction of ABA glucose ester. This metabolite, which was produced from both (+)- and (-)-cis-diol, co-chromatographed with acetylated cis-diol glucose ester on an 8 (i.d.) x 250 mm Techsil 5 c18 HPLC column eluted with methanol/0.2% aqueous acetic acid (3:1, v/v) at 2.0 ml/min (retention time = 8.8 min; Amax= 275 nm). 181 40 30 10 20 30 40 en Retention time, mln c. 0 >, > 2 0 -co 0 "O co a: 1 0 10 20 30 40 Retention time, min Fig. 4.23 HPLC separation of the metabolites of (+/-)- 1' ,4'-[2-14cJcis-diol of ABA The metabolites of 2 µCi (+/-)-1' ,4'-[2-14cJcis-diol of ABA (7.6 mg; 70 µCi/mmol) fed to 15 tomato shoots (47 g) and extracted after 4 days were separated by HPLC. A 4.6 (i.d.) x 250 mm Brownlee RP-18 Spheri 5 HPLC column was eluted with methanol/0.2% aqueous acetic acid (2:3, v/v) at 1 ml/min. The effluent was monitored with a diode array detector (see inset) and 14C-labelled compounds detected with a radioactivity monitor. Retention time of standards: ABA 23.5 min, trans-diol glucose ester 7-8 min, cis-diol glucose ester 16 min. 182 100 61 50 169 109 231 69 139 100 200 2oox ....>, 100 1 ·oo C ....Q) £ 50 Q) 331 ....> ro 271 369 441 0.2 579 519 559 603 645 500 600 rn/z Fig. 4.24 Chemical-ionization (methane) mass spectrum of ~-diol glucose ester The mass spectrum of 0-acetyl cis-diol glucose ester [62] (Fig. 4.24) is similar to that of the 0-acetyl trans-diol glucose esters ([51] and [59]) in that the spectrum is dominated by the ion at m/z 61 (acetic acid from the 4'-0-acetyl group). No molecular ion was evident but ions attributable to 4'- desoxy ABA glucose ester (m/z 579) and fragment ions of the cis-diol and tetraacetyl glucose residues are present. A sample of [4'-2H]cis-diol glucose ester biosynthesized from 183 [4'-2H]cis-diol retained the 4'-deuterium atom and in the mass spectrum of the O-acetylated and derivative ions at m/z 580 and 232 replaced the ions at m/z 579 and 231. The metabolite was hydrolysed in base to products having retention times and UV spectra similar to glucose and cis-diol on reversed phase HPLC-diode array detection. The sugar residue was identified as glucose by mass spectrometry of the products of acidic hydrolysis. The TMS derivative of the O-methyloxime of glucose had the same retention time on capillary gas chromatography and a similar mass spectrum to that of a standard of the TMS-methyloxime of glucose. The cis-diol glucose ester [61] in weakly acidic solutions isomerized to the (+}- [50] and (-}-trans-diol glucose ester [58] diastereomers which were separated by reversed-phase HPLC (Fig. 4.25}. This isomerization was observed for the synthesized compound as well as the metabolite isolated from tomato shoots. Therefore, the products of [14c]- and [3H]cis-diol which had the same UV spectra and retention times as trans-diol glucosides and glucose esters may be products of the isomerization of cis-diol glucosides and glucose esters. Thus, the analysis of the metabolic products of racemic cis-diol is complicated by isomerization of the conjugates of cis-diol, the oxidation of cis-diol to ABA and isomerization to trans-diol as well as the separation of diastereomers on HPLC. 184 E C LO (0 C\I Q) 0 C tU .0... 0 (/) .0 0 10 20 30 40 Retention time, min Fig. 4.25 Isomerization of cis-diol glucose ester A sample of cis-diol glucose ester was left in an acidic solution (c. pH 4) and the products of isomerization were separated by HPLC - on a semi-preparative Techsil 10 c1 a column with a mobile phase of ethanol/0.2% aqueous acetic acid (1:4, v/v) at 4.0 ml/min. 4.4 DISCUSSION 4.4.1 The 1' ,4'-diols of ABA as metabolites of exogenous ABA and as endogenous constituents of higher plants In many studies of the metabolism of ABA, acidic solvents have been used in the extraction and separation of metabolites. The 1' ,4'-diols of ABA, especially the cis-diol, were found to be unstable in acidic solutions, so it is not surprising that the diols have been detected amongst ABA metabolites by few workers. When extracts of 185 plants supplied with [2-14c]ABA were processed rapidly in the presence of "cold scavenger" cis- and trans-diols, and precautions taken to minimize oxidation, then the diols recovered contained a substantial proportion of the [2-14C]ABA metabolized. The cis-diol zones eluted from TLC plates contained up to 2.7 times as much radio activity than the trans-diol zones. However, when the diols were derivatized and rechromatographed the radioactivity in the trans-diol contained several times as much radioactivity as the cis-diol (Table 4.1). This is because the yield of purified cis-diol (1.3%) from the zone on TLC plates was much lower than that of the trans-diol (8.2%). It is possible that some of the loss of yield was due to other labelled compounds which co-chromatographed with the diols. Derivatization and further chromatography could have removed these contaminants. In addition, there were substantial losses at most steps even when the diols would be expected to have a high degree of purity. It was assumed that the loss of material from the diols was due to oxidation and interconversion of the diols because the recovery of an internal standard of 3H-trans-diol, used to monitor the recovery of endogenous trans-diol, showed similar losses (2.2% to 12.7% recovery). The effect the duration and type of analytical procedure had on the yield of the diols was demonstrated by analysing a portion of the extract by the usual methods used for the separation of metabolites. Only 186 small amounts of trans-diol and little or no cis-diol were detected in the extracts by these methods. The yield of the diols is so susceptible to the conditions used during the isolation that a system comprising non-acidic solvents and chromatographic systems, a rapid extraction and analysis with minimal exposure to air is required. Although the yields of the purified diols were low, there was sufficient radioactivity present to determine the proportion of (+)- and (-)- enantiomers in the samples. The [14c]cis- and [14c]trans-diols were formed from R- and S-ABA, although there was a greater abundance of the enantiomers derived from unnatural R-ABA. However, it must be pointed out that the RS-[ 14c]ABA was subject to metabolism by the tissues during the course of the experiment and this characteristically causes the R-ABA to predominate. The balance in the 1' ,4'-diols, therefore may merely reflect the relative abundance of the two enantiomers of [ 14c]-ABA. The 1',4'-cis-diol of ABA has been isolated from immature broad bean seeds and characterized by Dathe and Sembdner (1982). The trans-diol also occurs naturally; it has been isolated as an endogenous constituent of cultures of the fungus, Botrytis cinerea (Hirai et al., 1985). In the present work the trans-diol has been found in two species of higher plant - avocado and pea. There were species differences between the concentrations of the trans-diol and the amounts relative to the endogenous 187 ABA. The trans-diol of ABA (93µg/g fresh weight) was present in avocado at 1.1% of the concentration of free ABA whereas in pea shoots the trans-diol (5.2µg/g fresh weight) was 13% of the free ABA (Table 4.2) In avocado fruit the trans-diol was released from an extract by basic hydrolysis indicating that conjugates of the trans-diol might be present. 4.4.2 The stability and interconversion of the 1' ,4'-diols of ABA The biological activity of the cis- and trans-diols was attributed to their oxidation to ABA (Walton and Sondheimer, 1972b) and the susceptibility of the diols to oxidation in air has been pointed out by Milborrow and Garmston (1973). The present work has shown that as well as being oxidized, the diols are also interconverted. The interconversion and oxidation of the diols, although occurring to some extent at all pH values measured, was more rapid at low pH values. The means by which the diols interconvert was shown to be by inversion at both C-1' and C-4' positions. The inversion at C-1' was surprising because of the unreactivity of the tertiary hydroxyl group to derivatization such as acetylation, the absence of exchange of the 1'-hydroxyl of ABA with [18o]H2o and the apparent absence of R-ABA in plants. The inversion at C-1' and C-4' caused samples of optically pure trans-diol to be converted into a mixture of enantiomers of the cis-diol (Fig, 4.7). 188 HO HO HO Scheme 4.2 The interconversion of the diols in acid may be initiated by the acceptance of a proton by one of the hydroxyl groups (Scheme 4.2). This is followed by the elimination of water to yield a carbocation, which is 189 attacked by a molecule of water and after deprotonation a diol is produced. In the absence of any side reactions, either diol would eventually give the same mixture of isomers in a ratio determined by their free energies in solution. The centre which is attacked by a molecule of water would not necessarily be inverted by exchange with the medium. This was indeed the case when the cis- and trans-diol of ABA were incubated in [ 18oJH2o - the cis-diol exchanged 84% of its 4'-oxygen and the trans-diol 23.6% without interconversion (Table 4.5). Similarly when the [l 1 - 18o]cis-diol was incubated at pH 3.5, 8.3% of the 1 1 - 180 was lost without any inversion occurring (Table 4.4). Interconversion of the diols with inversion at C-1' provides a mechanism by which (-)-ABA may be produced within the plant. This could occur by a sequence such as (+)-ABA ~ (+)-cis-diol ~ (-)-trans-diol ~ (-)ABA. However, because the diols occur at concentrations of 1% to 10% of those of ABA and because only a few percent of the diols isomerized at cellular pH, only a very small proportion of the ABA in a plant would be expected and indetectable by even the most sensitive measurements. (-)-ABA, however, would be expected to accumulate as the glucose ester of ABA (Milborrow, 1970). Hydrolysis of the glucose ester followed by separation of the enantiomers may reveal the presence of endogenous (-)-ABA. 190 The inversion of the chirality at C-1' also raises the possibility that interconversion of the diols by isomerization at C-1' may cause some racemization of the Me-cis-diol during the preparation of (+)- and (-)-ABA. A sample of (+)-Me-[2-14c]cis-diol, separated from the (-)- enantiomer by HPLC on Pirkle columns, was rechromatographed under the same conditions and no (-)-Me-cis-diol was detected. The Me-cis-diol was oxidized with pyridinium chlorochromate (an acidic reagent) to Me-ABA, which was then reduced and the resulting Me-cis-diol again chromatographed on the Pirkle columns. The (-)-Me-cis-diol contained 0.9% of the radioactivity in the (+)-Me-cis-diol. Thus it appears that the ABA produced by this method is approximately 99% optically pure and that some racemization at C-1' occurs during the oxidation. In addition to interconversion, the diols were oxidized to ABA in acidic solution. The degree of oxidation depended on the experimental conditions. More than twice as much ABA as the cis-diol was formed from the trans-diol when the products were separated by TLC (Fig. 4.6). By contrast, the amount of Me-ABA formed from Me-trans-diol was less than half of the cis-diol formed by interconversion of the diol and analysis of the products by HPLC (Fig. 4.7). Whether the increased oxidation of the trans-diol free acid to ABA was due to oxidation on silica gel thin-layer plates or whether the free acid diols are more rapidly oxidized than the methyl esters is yet to be determined. 191 Two routes for the oxidation of the l' ,4'-diols to ABA are possible: 1. The oxidation of the diols could occur by hydride transfer. The carbocation derived from a diol could abstract a hydride from the 4'-position of another molecule diol converting it into ABA and itself yielding a desoxy diol (Scheme 4.3). + H~ m\---~~OH 0 /2- Scheme 4.3 2. The oxidation of the diols may involve a free radical reaction. Some influence, such as light, could generate a free radical at the 4'-position of the diol, either directly or by exchange with another free radical. This could then accept a molecule of oxygen and form a hydroperoxy radical which attacks another molecule of the diol to continue the chain reaction. The resulting hydroperoxide then loses hydrogen peroxide to form ABA (Scheme 4.4). HO Scheme 4.4 192 4.4.3 Metabolism of the 1' ,4'-trans-diol of ABA The amounts of the 1' ,4'-trans-diol of ABA which were fed to tomato shoots were calculated to be about 100-fold greater than the endogenous concentrations. The metabolites produced were similar to those produced when (+)- or (-)-ABA was fed. The major product of the (+)-trans-diol was DPAGS (62%) while the (-)-trans-diol was metabolized to ABAGE (19%) and ABAGS (11%). In addition, to these metabolites both (+)- (16%) and (-)-trans-diol (16%) were conjugated to glucose (Fig. 20). The amounts of trans-diol supplied in a number of experiments varied somewhat, and it was observed that as the concentrations increased so too did the proportion of the trans-diol conjugated. It appears that there is a saturable system for oxidizing the trans-diol to ABA. The oxidation was not stereospecific but, because the proportions of the products altered with changing concentration, involvement of an enzymic mechanism is required over and above the simple aerial oxidation proposed previously (Milborrow and Garmston, 1973). Two conjugates of the trans-diol were isolated and characterized as the glucose ester, and the 4'-glucoside of the 1' ,4'-trans-diol of ABA. In accordance with the guidelines for the naming of conjugates of ABA (Loveys and Milborrow, 1981) the conjugates of natural (+)-trans-diol are: (+)-l'S,4'~-4'-dihydroabscisic acid -4'-~-D-glucopyranoside [47] and ~-D-glucopyranosyl 1'~,4'~-4'-dihydroabscisate [50]. 193 COOH OH [47] ' ' 'OH HO [50] The (+) and (-)- enantiomers of the trans-diol formed a pair of epimers when conjugated to ~-D-glucose. Because racemic trans-diol was used in feeding experiments, two pairs of diastereomers were formed and the components of each pair were separable by reversed-phase HPLC. This type of separation has not been observed for the glucose ester or 1'-glucoside of ABA although diastereomers are formed when racemic ABA is supplied (Milborrow, 1984a). 4.4.4 Metabolism of the 1' ,4'-cis-diol of ABA Although the metabolism of the 1' ,4'-cis-diol of ABA 194 was not studied as thoroughly as the metabolism of the trans-diol the overall features seem to be similar: at low concentrations the cis-diol was metabolized principally to DPAGS and conjugates of ABA but as the concentration increased more conjugates of the cis-diol were produced (Fig. 4.22, Fig. 4.23). One of these conjugates was isolated and characterized (for (+)-cis-diol) as ~-D-glucopyranosyl l'R,4'R-4'-dihydro- abscisate [61]. Unlike the trans-diol glucose ester the diastereomers of the cis-diol glucose ester were not separable by reversed-phase HPLC. This was a surprising result in view of the facile resolution of the cis-diol by the Pirkle columns and their inability to resolve the trans-diol. ...... 'OH / / HO" [61] The study of the metabolism of the cis-diol was complicated by the instability of the cis-diol and its metabolites. In general, the investigation of the metabolism of the racemic 1' ,4'-diols of ABA was confused by the following processes: 195 * Oxidation of the diol to ABA and then the production of metabolites of ABA * The interconversion of the diols * The separation of diastereomers in the analysis of metabolites by HPLC which increased the number of peaks to be identified. * The interconversion of the diol metabolites. All of the above factors combine to produce a complex mixture of products, especially when the cis-diol is fed. It is preferable to use only the (+)-enantiomer, avoid acid solvents and exposure to air, and to shorten the time between extraction and analysis. Prior to feeding the diols, interconversion of the diols can be minimized by storing the diols in the presence of a small amount of sodium bicarbonate. 4.4.5 The conversion of the 4'-dihydroabscisates into the 4'- glucoside of dihydrophaseic acid 4'-Dihydroabscisic acid was postulated by Walton et al. (1973) as a possible intermediate in the metabolism of ABA to dihydrophaseic acid (DPA) [20]. Three pathways to DPA were considered: i) ABA ~ 8'-hydroxy ABA [18] ~ PA [ 19] ~ DPA ii) ABA ~ 8'-hydroxy ABA ~ 4'-dihydro-8'-hydroxy ABA [27] ~ DPA iii) ABA ~ 4'-dihydro ABA [3] ~ 4'-dihydro-8'- hydroxy ABA ~ DPA 196 Pathway i) was preferred because phaseic acid had been isolated and was converted into DPA in high yield whereas the conversion of 4'-dihydroabscisic acid into DPA was very slight. Further support for pathway i) was the finding that, in a cell-free system, that metabolized ABA to DPA, phaseic acid was a product of hydroxylation of ABA. The cis- and trans-diols, however, were not substrates for the enzyme(s) (Gillard and Walton, 1976). If DPA was formed via 4'-dihydro ABA then the trans-diol would produce DPA and the cis-diol, epi-DPA. However, the cis-diol was metabolized to DPAGS with no epi-DPA being detected. In the present work, the (+)-trans-diol was metabolized to DPAGS in high yield whereas the (-)- enantiomer was metabolized to conjugates of ABA suggesting that the diols were oxidized to ABA and metabolized along the usual ABA metabolic pathways of ABA catabolism (Fig. 4.20). When [2-14c, 4'-2H]trans-diol was fed to tomato shoots a high proportion of the trans-diol was metabolized to DPAGS and about 65% of the DPAGS was derived from the deuterated trans-diol but no deuterium was retained in the product. This indicates that at some stage in the metabolism of the trans-diol to DPAGS the 4'-hydroxyl group was oxidized to a ketone. The presence of the ketone at C-4' is probably required to activate the the C-2' double bond and allow cyclization to give 197 phaseic acid. It is likely, therefore, that the conversion of the diols into DPAGS involved their oxidation to ABA and metabolism via the usual pathway to DPA. However, the results are also consistent with another pathway: iv) ABA ~ 4'-dihydro ABA ~ 4'-dihydro-8'-hydroxy ABA ~ 8'-hydroxy ABA ~PA~ DPA. In this pathway the cis-diol would produce DPA and [4'-2H]trans-diol would lose deuterium in its conversion into DPA. A compound, isolated from pea shoots fed with [l 1 - 14c]ABA and tentatively identified as 4'-dihydro- 8'-hydroxy ABA (Tietz, 1985), was produced in higher yield from the 1' ,4'-trans-diol of ABA than from ABA. The metabolism of 1' ,4'-trans-diol produced approximately equal amounts of 4'-dihydro-8'-hydroxy ABA and DPA. In these experiments racemic [14c]ABA and the trans-diol formed from it were fed to pea shoots. It is possible that the (+)-trans- diol was metabolized to DPA and the 4'-dihydro-8'-hydroxy ABA was formed from the (-)-trans-diol. This could occur if the l'R,4'R-4'-dihydro-8'-hydroxy ABA was not accepted by the enzyme oxidizing the 4'-hydroxyl group to a ketone. In an analogous situation it has been demonstrated that the (-)-1' ,2'-epi-2-cis-xanthoxin acid [71] is not oxidized to ABA whereas the (+)-2-cis-xanthoxin acid [72] is (Milborrow and Garmston, 1973). Thus the configuration of the 1' ,2'-epoxy group controls whether the 4'~-hydroxyl can be oxidized. A similar specificity for the oxidation of 198 HO COOH (72) HO COOH [ 71) 4'-dihydro-8'-hydroxy ABA would explain why only the (+)-trans-diol was metabolized to DPAGS. Therefore, two possibilities exist for the metabolism of the trans-diol to DPAGS, firstly the pathway via ABA (i) and secondly a pathway (iv) not involving ABA as in intermediate. In tomato shoots, at least, the evidence would favour metabolism via ABA to DPAGS. 4.4.6 Biological activity of the 1' ,4'-diols of ABA As discussed previously (Section 4.1) the 1' ,4'-diols of ABA were found to inhibit the growth of embryonic axes of Phaseolus vulgaris (Walton and Sondheimer, 1972b). The 199 activity of the diols was attributed to their oxidation to ABA with the cis-diol being more active than the trans-diol because of its more rapid oxidation to ABA. In a short-term bioassay, in which oxidation to ABA was minimal, both of the diols were inactive. Water loss by transpiration from excised shoots of Cyparus was strongly inhibited with in 10 minutes by 100 mg/1 ABA which was fed through the transpiration stream. There was no observable effect of a similar concentration of either of the diols during 30 minutes and thereafter a very slight decrease in the rate of uptake became apparent and was attributed to autooxidation or biological oxidation (B.V. Milborrow, unpublished results). The lack of activity of the diols in an assay over a short duration shows that the reduction of the 4'-ketone of ABA is sufficient to render the hormone inactive. The metabolism of ABA to the diols may be used to remove the active hormone. However, the biological oxidation of diols could mean that ABA is regenerated from the diols, although this may vary from species to species and conjugation of the diols to glucose may form part of an alternative pathway of ABA metabolism. 5 References 200 201 Addicott, F.T., Carns, H.R., Cornforth, J.W., Lyon, J.L., Milborrow, B.V., Ohkuma, K., Ryback, G., Smith, O.E., Thiessen, W.E., Wareing, P.F. (1968) Abscisic acid: A proposal for the redesignation of abscisin II (dormin). In F. Wightman and G. Setterfield (eds.), Physiology and Biochemistry of Plant Growth Substances, pp. 1527-29. Runge Press, Ottawa. Adesomoju, A.A., Okogun, J.I., Ekong, D.E.U., Gaskin, P. (1980) GC-MS identification of abscisic acid and abscisic acid metabolites in seed of Vigna unguiculata. Phytochemistry 19, 223-25. Assante, G., Merlini, L., Nasiai, G. (1977) (+)-Abscisic acid, a metabolite of the fungus Cercospora rosicola. Experientia 33, 1556. Barrow, K.D., Barton, D.H.R., Chain, E.B., Ohnsorage, U.F.N., Thomas, R. (1971) Fusicoccin: II. The constitution of fusicoccin. J. Chem. Soc. (C), 1265-1274. Bennet-Clark, T.A., Tambiah, M.S., Kefford, N.P. (1952) Estimation of plant growth substances by partition chromatography. Nature 169, 452-453. Boyer, G.L., Zeevaart, J.A.D. (1985) 7'-Hydroxy abscisic acid - an artefact of feeding racemic ABA to Xanthium. Plant Physiol. 77 (Suppl.), 76. Boyer, G.L., Milborrow, B.V., Wareing, P.F., Zeevaart, J.A.D. (1986) Nomenclature of abscisic acid and its metabolites. In M. Bopp (ed.) Plant Growth Substances 1985, Springer-Verlag, Berlin 202 Bray, E.A., Zeevaart, J.A.D. (1985) The compartmentation of abscisic acid and P-D-glucopyranosyl abscisate in mesophyll cells. Plant Physiol. 79, 719-722. Cheng, Y-S, Liu, W-L., Cheng, S. (1980) Pyridinium chlorochromate adsorbed on alumina as a selective oxidant for primary and secondary alcohols. Synthesis, 223-224. Comer, J.P. (1956) Semiquantitative specific test paper for glucose in urine. Anal. Chem. 28, 1748-1750. Corey, E.J., Suggs, J.W. (1975) Pyridinium chlorochromate. An efficient reagent for oxidation of primary and secondary alcohols to carbonyl compounds Tetrahedron Lett. 31, 2647-2650. Cornforth, J.W., Milborrow, B.V., Ryback, G. (1965a) Synthesis of (+/-)-abscisin II. Nature 206, 715. Cornforth, J.W., Milborrow, B.V., Ryback, G., Wareing, P.F. (1965b) Chemistry and physiology of 'dormins' in sycamore. Identity of sycamore 'dormin' with abscisin II. Nature 205, 1269-70. Cornforth, J.W., Milborrow, B.V., Ryback, G., Rothwell, K., Wain, R.L. (1966) Identification of the yellow lupin growth inhibitor as (+)-abscisin II ((+)-dormin). Nature 211, 742-43. Cornforth, J.W., Draber, W., Milborrow, B.V., Ryback, G. (1967) Absolute stereochemistry of (+)-abscisin II. Chem. Commun. , 114-116. 203 Coxon, B. (1972) Conformational analysis via nuclear magnetic resonance spectrometry. Methods Carbohydr. Chem.~, 513-539. Creelman, R.A., Zeevaart, J.A.D. (1984) Incorporation of oxygen into abscisic acid and phaseic acid from molecular oxygen. Plant Physiol. 75, 166-169. Cummins, W.R., Sondheimer, (1973) Activity of the asymmetric isomers of abscisic acid in a rapid bioassay. Planta 111, 365-369. Cummins, W.R., Kende, H., Raschke, K. (1971) Specificity and reversibility of the rapid stomatal response to abscisic acid. Planta 99, 347-351. Dashek, W.V., Singh, B.N., Walton, D.C. (1979) Abscisic acid localisation and metabolism in barly aleurone layers. Plant Physiol. _§i, 43-48. Dathe, w., Sembdner, G. (1982) Isolation of 4'-dihydroasbscisic acid from immature seeds of Vicia faba. Phytochemistry 21, 1798-1799. Davis, L.A., Addicott, F.T. (1972) Abscisic acid - correlations with abscission and with development in cotton fruits. Plant Physiol. i2_, 644-648. Dewdney, S.J., McWha, J.A. (1978) The metabolism and transport of abscisic acid during grain fill in wheat. J. Exp. Bot.~, 1299-1308. Durand, M., Thevenot, C., Come, D. (1975) Role des cotyledons dans la germination et la levee de dormance de l'axe embryonnaire du Pommier, apres traitement par l'acide abscissique. Physiol. Veg. U, 603-610. 204 El-Antably, H.M.M., Wareing, P.F., Hillman, J.R. (1967) Some physiological responses to D,L-abscisin (dormin). Planta 73, 74-90. Fieser, L.F., Fieser, M. (1967) in Reagents for Organic Synthesis p. 142, Wiley, New York. Firn, R.D., Burden, R.S., Taylor, H.F. (1972) The detection and estimation of the growth inhibitor xanthoxin in plants. Planta 102, 115-126. Firn, R.D., Friend, J. (1972) Enzymatic production of the plant growth inhibitor, xanthoxin. Planta 103, 263-266. Gillard, D.F., Walton, D.C. (1976) Abscisic acid metabolism by a cell-free preparation from Echinocystis lobata liquid endosperm. Plant Physiol. ~, 790-795. Gray, R.T., Mallaby, R., Ryback, G., Williams, V.P. (1974) Mass spectra of methyl abscisate and isotopically labelled analogues. J. Chem. Soc. Perking, 919-924. Hasegawa, S., Poling, S.M., Maier, V.P., Bennett, R.D. (1984) Metabolism of abscisic acid: Bacterial conversion to dehydrovomifoliol and vomifoliol dehydrogenase activity. Phytochemistry 23, 2769-2771. Hemberg, T. (1949) Significance of growth-inhibiting substances and auxins fo the rest period of the potato tuber. Physiol. Plant.~, 393-399. 205 Higgins, T.J.V., Zwar, J.A., Jacobsen, J.V. (1977) Hormonal control of the level of translatable mRNA for a-amylase in barley aleurone cells. In J.H. Weil and L. Bogorad (eds.) Acides Nucleiques et Synthese des Proteines chez les Vegetaux. Collogues Int. C.N.R.S. No. 261 Paris Hirai, N., Koshimizu, K. (1983) A new conjugate of dihydrophaseic acid from avocado fruit. Agric. Biol. Chem. j]__, 365-371. Hirai, N., Fukui, H., Koshimizu, K. (1978) A novel abscisic acid metabolite from the seeds of Robinia pseudacacia. Phytochemistry 17, 1625-1627. Hirai, N., Okamoto, M., Koshimizu, K. ( 1985) l' ,4'-trans-DiolABA, a potent precursor of ABA in Botrytis cinerea. 12th International Conference on Plant Growth Substances Abstracts, 30. Hoad, G.V., Gaskin, P. (1980) Abscisic acid and related compounds in phloem exudate of Yucca flaccida Haw and coconut (Cocus nucifera L.). Planta 150, 347-348. Hocking, T.J., Hillman, J.T. (1975) Studies on the role of of abscisic acid in the initiation of bud dormancy in Alnus glutinosa and Betula pubescens. Planta 125, 235-242. Hogg, A.M., Nagabhushan, T.L. (1972) Chemical ionization mass spectra of sugars. Tetrahedron Lett. j]__, 4827-4830. Ihle, J.N., Dure, L. (1970) Hormonal regulation of translation inhibition requiring RNA synthesis. Biochim. Biophys. Res. Commun.~, 995-1001. 206 King, R.W. (1979) Abscisic acid synthesis and metabolism in wheat ears. Aust. J. Plant Physiol. l, 99-108. Koshimizu, K., Fukui, H., Mitsui, T., Ogawa, Y. (1966) Identity of lupin inhibitor with abscisin II and its biological activity on growth of rice seedlings. Agr. Biol. Chem. 30, 941-943. Koshimizu, K., Inui, M., Fukui, H., Mitsui, T. (1968) Isolation of (+)-abscisic-P-D-glucopyranoside from immature fruit of Lupinus luteus. Agric. Biol. Chem. 32, 789-791. Kreidemann, P.E., Loveys, B.R., Fuller, G.L., Leopold, A.C. (1972) Abscisic acid and stomatal regulation. Plant Physiol . .i2_, 842-847. Le Page-Degivry, M.T., Garello, G. (1973) La dormance embrionnaire chez Taxus Baccata: Influence de la composition du milieu liquide sur l'induction de la germination. Physiol. Pl. 29, 204-207. Leblanc, D.J., Bali, A.J.S. (1978) A fast one-step method for the silylation of sugars and sugar phosphates. Anal. Biochem. _!!!, 574-578. Lehmann, H., Bohm, H., Schutte, H-R. (1983a) The metabolism of abscisic acid in cell cultures of various plant species. z. Pflanzenphysiol. 109, 423-428. Lehmann, H., Preiss, A., Schmidt, J. (1983b) A novel abscisic acid metabolite from cell suspension cultures of Nigella damascena. Phytochemistry 22, 1277-1278. 207 Lenton, J.R., Perry, V.M., Saunders, P.F. (1972) Endogenous abscisic acid in relation to photoperiodically induced bud dormancy. Planta 106, 13-22. Li, Y., Walton, D.C. (1985) Evidence for a carotenoid origin for ABA in water-stressed bean leaves. Plant Physiol. 77 (Suppl.), 23. Loveys, B.R., Downton, J.S. (1979) Abscisic acid in crushed grape berries during fermentation and in wine during storage. Am. J. Enol. Viticult. l.Q_, 232-235. Loveys, B.R., Milborrow, B.V. (1981) Isolation and characterization of 1'-O-abscisic acid-~-D-glucopyranoside from vegetative tomato tissue. Aust. J. Plant Physiol. ~, 571-589. Loveys, B.R., Milborrow, B.V. (1984) Metabolism of abscisic acid. In A. Crozier and J.R. Hillman (eds.) The Biosynthesis and Metabolism of Plant Hormones, pp. 71-104, Cambridge University Press, Cambridge. MacMillan, J., Pryce, R.J. (1968) Phaseic acid, a putative relative of abscisic acid from seed of Phaseolus multifloris. Chem. Commun., 124-126. MacMillan, J., Seaton, J.C., Suter, P.J. (1960) Plant hormones- I. Isolation gibberellin Al and gibberellin AS from Phaseolus multiflorus. Tetrahedron Lett.]:_!_, 60-66. 208 Martin, G.C., Dennis, F.G., MacMillan, J., Gaskin, P. (1977) Hormones in pear seeds. I. Levels of gibberellins, abscisic acid, phaseic acid, dihydrophaseic acid, and two metabolites of dihydrophaseic acid in immature seeds of Pyrus communis L. J. Am. Soc. Hort. Sci. 102, 16-19. Martin, G.C., Scott, I.M., Neill, S.J., Horgan, R. (1982) Identification of abscisic acid glucose ester, indole-3-acetic acid, zeatin and zeatin riboside in recepticles of pear. Phytochemistry ~, 1079-1082. Mawhinney, T.P., Feather, M.S., Barbero, G.J., Martinez, J.R. (1980) The rapid, quantitative determination of neutral sugars (as aldonitrile acetates) and amino sugars (as O-methyloxime acetates) in glycoproteins by gas-liquid chromatography. Anal. Biochem. 101, 112-117. Mertens, R. Stuning, M., Weiler, E.W. (1982) Metabolism of tritiated enantiomers of abscisic acid prepared by immunoaffinity chromatography. Naturwissenshaften 69, 595-597. Milborrow, B.V. (1967) The identification of (+)-abscisin II [(+)-dormin] in plants and measurement of its concentration. Planta ]__£, 93-113. Milborrow, B.V. (1968) Identification and measurement of (+)-abscisic acid in plants. In F. Wightman and G. Setterfield (eds.) Biochemistry and Physiology of Plant Growth Substances, pp. 1531-45. Runge Press, Ottawa 209 Milborrow, B.V. (1969a) The occurrence and function of abscisic acid in plants. Sci. Prog. 57, 533-558. Milborrow, B.V. (1969b) Identification of 'Metabolite C' from abscisic acid and a new structure for phaseic acid. Chem. Commun., 966-967. Milborrow, B.V. (1970) The biosynthesis and degradation of abscisic acid. In D.J. Carr (ed.) Plant Growth Substances, 1970, pp. 281-290 Springer-Verlag, Berlin. Milborrow, B.V. (1974) The chemistry and physiology of abscisic acid. Ann. Rev. Plant Physiol. 25, 259-307. Milborrow, B.V. (1975) The absolute configuration of phaseic and dihydrophaseic acids. Phytochemistry ..!.i, 1045-1053. Milborrow, B.V. (1978a) Abscisic acid. In D.S.Letham, P.B. Goodwin and T.J.V. Higgins ( eds.), Phytohormones and Related Compounds - A Comprehensive Treatise, Volume 1, pp. 295-347. Elsivier/North Holland, Biomedical Press. Milborrow, B.V. (1978b) The stability of conjugated abscisic acid during wilting. J. Exp. Bot. 29, 1059-1066. Milborrow, B.V. (1980) A distinstion between the fast and slow responses to abscisic acid. Aust. J. Plant Physiol. 2, 749-754. 210 Milborrow, B.V. (1983) The reduction of (+/-)-[2-14c]abscisic acid to the 1',4'-trans-diol by pea seedlings and the formation of 4'-desoxy ABA as an artifact. J. Exp. Bot. 1_!, 303-308. Milborrow, B.V. (1984a) Inhibitors. In M.B. Wilkins (ed.) Advanced Plant Physiology, pp. 76-110, Pitman, London. Milborrow, B.V. (1984b) The conformation of abscisic acid by n.m.r. and a revision of the proposed mechanism for cyclization during its biosynthesis. Biochem J. 220, 325-332. Milborrow, B.V. (1985) The shapes of abscisic acid and the active site. In M. Bopp (ed.) Plant Growth Substances 1985, Springer-Verlag, Berlin. Milborrow, B.V., Garmston, M. (1973) Formation of (-)-1' ,2'-epi-2-cis-xanthoxin acid from a precursor of ABA. Phytochemistry 12, 1597-1608. Milborrow, B.V., Mallaby, R. (1975) The occurrence of methyl (+)-abscisate as an artefact of extraction. J. Exp. Bot. 26, 741-748. Milborrow, B.V., Noddle, R.C. (1970) Conversion of 5-(1,2-epoxy-2,6,6-trimethylcyclohexyl)-3-methy l-penta-cis-2-trans-4-dienoic acid into abscisic acid in plants. Biochem. J. 119, 727-734. Milborrow, B.V., Robinson, D.R. (1973) Factors affecting the biosynthesis of abscisic acid. J. Exp. Bot. 24, 537-548. 211 Milborrow, B.V., Rubery, P.H. (1985) The specificity of the carrier-mediated uptake of ABA by root segments of Phaseolus coccineus L. J. Exp. Bot. 36, 807-822. Milborrow, B.V., Vaughan, G.T. (1979) The long term metabolism of (+/-)-[2-14c]abscisic acid by apple seeds. J. Exp. Bot. 30, 983-995. Milborrow, B.V., Vaughan, G.T. (1982) Characterization of dihydrophaseic acid 4'-O-P-D-glucopyrano side as a major metabolite of abscisic acid. Aust. J. Plant Physiol. ~, 361-372. Moore, R., Smith, J.D. (1984) Growth, graviresponsive ness and abscisic-acid content of Zea mays seedlings treated with Fluoridone. Planta 162, 342-344. Moore, R., Smith, J.D. (1985) Graviresponsiveness and abscisic-acid content of carotenoid-deficient mutants of Zea mays L. Planta 164, 126-128. Neill, S.J., Horgan, R., Walton, D.C., Lee, T.S. (1982) The biosynthesis of abscisic acid in Cercospora rosicola. Phytochemistry ~, 61-65. Neill, S.J., Horgan, R., Heald, J.K. (1983) Determination of the levels of abscisic acid-glucose ester in plants. Planta 157, 371-375. Noddle, R.C., Robinson, D.R. (1969) Biosynthesis of abscisic acid: Incorporation of radioactivity from [2-14c]mevalonic acid by intact fruit. Biochem. J. 112, 177-178. 212 Nonhebel, H.M., Milborrow, B.V. (1986) Incorporation of 2tt from 2tt2o into ABA in tomato shoots. Evidence for a large pool of precursors. Submitted to J. Exp. Bot. Ohkuma, K, Lyon, J.L., Addicott, F.T., Smith, O.E. (1963) Abscisin II, an abscission-accelerating substance from young cotton fruit. Science 142, 1592-1593. Ohkuma, K, Addicott, F.T., Smith, O.E., Thiessen, W.E. (1965) The structure of abscisin II. Tetrahedron Lett. 29, 2529-2535. Perry, T.O., Byrne, O.R. (1969) Turion induction in Spirodela polyrrhiza. Plant Physiol. 44, 784-785. Phillips, I.D.J., Wareing, P.F. (1958) Studies in dormancy of sycamore. I. Seasonal changes in the growth-substance content of the shoot. J. Exp. Bot. 1, 350-364. Pierce, M., Rashke, K. (1981) Synthesis and metabolism of abscisic acid in detached leaves of Phaseolus vulgaris L. after loss and recovery of turgor. Planta 153, 156-165. Pilet, P.E. (1975) Abscisic acid as a root root growth inhibitor: physiological analyses. Planta 122, 299-302. Pirkle, W.H., House, D.W. (1979) Chiral high-pressure liquid chromatographic stationary phases. 1. Separation of the enantiomers of sulphoxides, amines, amino acids, alcohols, hydroxy acids, lactones and mercaptans. J. Org. Chem. _ii, 1957-1960. 213 Porter, N.G., Van Steveninck, R.F.M. (1966) An abscission-promoting factor in Lupinus luteus L. Life Sci. 5, 2301-2308. Powell, R.G., Weisleder, D., Smith, C.R. (1985) Abscisic acid catobolites from Sesbania drummondii. 12th Int. Conf. on Plant Growth Subst.-Abstracts, 28. Randerath, K. (1970) An evaluation of film detection methods for weak ~-emitters, particularly tritium. Anal. Biochem. 34, 188-205. Raschke, K. (1975) Simultaneous requirement of carbon dioxide and abscisic acid for stomatal closing in Xanthium strumarium L. Planta 125, 243-259. Robinson, D.R. (1962) The Nature and Physiological Action of Growth Inhibitors. PhD thesis. Univ. Wales. Rothwell, K., Wain, R.L. (1964) Studies on a growth inhibitor in yellow lupin (Lupinus luteus L.) In J.P. Nitsch (ed.), Regulateurs Naturels de la Croissance Vegetale pp. 363-75, C.N.R.S., Paris. Rudniki, R., Czapski, J. (1974) The uptake and degrad ation of [1-14c]abscisic acid by apple seeds during stratification. Ann. Bot. l!!_, 189-192. Schlenk, H., Gellerman, J.L. (1960) Esterification of fatty acids by diazomethane om a small scale. Anal. Chem. 32, 1412-1444. Seltzer, H.S., Loveall, M.J. (1958) Improved accuracy of Tes-Tape in estimating concentration of urinary glucose. J. Am. Med. Assoc. 167, 1826-1830. 214 Sembdner, G., Weiland, J., Schmeider, G., Schreiber, K., Focke, I. (1972) Recent advances in the metabolism of gibberellins. In D.J. Carr (ed.), Plant Growth Substances 1970 pp. 143-150, Springer-Verlag, Berlin. Setter, T.L., Brenner, M.L., Brun, W.A., Krick, P. (1981) Identification of a dihydrophaseic acid aldopyranoside from soybean tissue. Plant Physiol. 68, 93-95. Simpson, G.M. and Wain, R.L. (1961) A relationship between gibberellic acid and light in the control of internode extension in dwarf pea (Pisum sativum). J. Exp. Bot. 12, 207-216. Sondheimer, E., Galson, E.C., Chang, Y.P., Walton, D.C. (1971) Assymmetry, its importance to the action and metabolism of abscisic acid. Science 174, 829-831. Sondheimer, E., Galson, E.C., Tinelli, E., Walton, D.C. (1974) The metabolism of hormones during seed germination and dormancy. IV. The metabolism of (S)-2-14c-abscisic acid in ash seed. Plant Physiol. 54, 803-808. Stewart, G.R. (1969) Abscisic acid and morphogenesis in Lemna polyrrhiza. Nature 221, 61-62. Takeda, N., Harada, K-I., Suzuki, M., Tatematsu, A., Hirai, N., Koshimizu, K. (1984) Structural characterization of abscisic acid and related metabolites by chemical ionization mass spectrometry. Agric. Biol. Chem.~, 685-694. Tal, M. (1966) Abnormal stomatal behavior in wilty mutants of tomato. Plant Physiol. 41, 1387-1391. 215 Taylor, H.F., Burden, R.S. (1970) Identification of plant growth inhibitors produced by photolysis of violaxanthin. Phytochemistry i, 2217-2223. Taylor, H.F., Smith, T.A. (1967) Production of plant growth inhibitors from xanthophylls: A possible source of dormin. Nature 215, 1513-14. Tietz, D. (1985) Characterization of a novel (+/-)-abscisic acid metabolite. Physiol. Plant. 65, 171-176. Tietz, D., Dorffling, K., Wohrle, D., Erxleben, I., Liemann, F. (1979) Identification by combined gas chromatography-mass spectrometry of phaseic acid and dihydrophaseic acid and characterization of further abscisic acid metabolites in pea seedlings. Planta 147, 168-173. Tinelli, E.T., Sondheimer, E., Walton, D.C., Gaskin, P., MacMillan, J. (1973) Metabolites of 2-14c abscisic acid. Tetrahedron Lett., 139-140. Trewavas, A. (1981) How do plant growth substances work? Plant Cell Environ.!, 203-228. Van Steveninck, R.F.M. (1959) Abscission accelerators in lupins (Lupinus luteus L.). Nature 183, 1246-1248. Walton, D.C., Sondheimer, E. (1972a) Metabolism of 2-14c-(+/-)abscisic acid in excised bean axes. Plant Physiol . .i2_, 285-289. Walton, D.C., Sondheimer, E. (1972b) Activity and metabolism of 14c-(+/-)-abscisic acid derivatives. Plant Physiol. 49, 290-292. 216 Walton, D.C., Dorn, B., Fey, J. (1973) The isolation of an abscisic acid metabolite, 4'-dihydrophaseic acid, from non-imbibed Phaseolus vulgaris L. and other plants. Planta 112, 87-90. Wareing, P.F. (1965) Endogenous inhibitors in seed germination and dormancy. Encycl. Pl. Physiol. 15, 909-924. Weiler, E.W. (1980) Radioimmunoassays for the differential and direct analysis of free and conjugated abscisic acid in plant extracts. Planta 148, 262-272. Wilkins, M.B. (1977) Guidance systems in roots and shoots. Syrop. Soc. Exp. Biol.~, 275-335. Wright, S.T.C., Hiron, R.W.P. (1969) (+)-Abscisic acid, the growth inhibitor induced in detached leaves by a period of wilting. Nature 224, 719-720. Zeevaart, J.A.D. (1980) Changes in the levels of abscisic acid and its metabolites in excised leaf blades of Xanthium strumarium during and after water stress. Plant Physiol. _§_§_, 672-678. Zeevaart, J.A.D., Milborrow, B.V. (1976) Metabolism of abscisic acid and the occurrence of epi-dihydrophaseic acid in Phaseolus vulgaris. Phytochemistry ~, 493-500. Zeevaart, J.A.D. (1985) Biosynthesis and catabolism of abscisic acid. 12th International Conference on Plant Growth Substances - Abstracts, 27.