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III

 

LIBRARY
Michigan State
University

 

 

 

This is to certify that the
dissertation entitled
"Stress-Induced Abscisic Acid Bio synthesis

in Higher Plants"

presented by

R. A. Creelman

has been accepted towards fulfillment
of the requirements for

Ph.D. degreeinBotany and Plant Pathology

Major professor

3W
fl

Date June 11, 1986.

 

"Hum—An: .- . ~ '1 ”‘r ' ' ' J 0-12771

 

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STRESS-INDUCED ABSCISIC ACID BIOSYNTHESIS IN HIGHER PLANTS

By

Robert Arthur Creelman

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1986

SQQZC? 70)r

ABSTRACT
STRESS-INDUCED ABSCISIC ACID BIOSYNTHESIS IN HIGHER PLANTS
By

Robert Arthur Creelman

In water-stressed leaves, levels of abscisic acid (ABA)
can increase 10- to 40-fold over those in turgid ones. Turgor
loss is the crucial parameter of cell water relations
governing the accumulation of ABA. Spinach leaf slices
incubated in the presence of ethylene glycol, a penetrating
solute, did not accumulate ABA, while slices incubated in
non-penetrating solutes, such as mannitol or Aquacide III,
did.

Two hypotheses have evolved concerning ABA biosyn—
thesis: ABA is formed (i) directly from farnesyl pyrophos-
phate, or (ii) indirectly from a carotenoid, with xanthoxin
as an intermediate in the pathway.

The immediate precursor(s) to ABA must be a C15 com-
pound. Three C15 compounds with structures similar to ABA
were synthesized. The biological activity of -ionylidene
acetic acid was greater than that of ABA, indicating that it
was active per se and not a precursor, while 1',2'—epoxy-io—
nylidene acetic acid had less biological activity than
ABA. When the deuterated analogue of l',2'—epoxy-ionylidene
acetic acid was fed to Xanthium leaves, deuterium was
incorporated into ABA. However, this compound is not endogen-
ous in Xanthium. Radioactive 1',2'-epoxy—ionylidene acetal-

dehyde was metabolized by plant tissues (spinach leaves,

 

tomato shoots, and tomato fruits), but no incorporation

into ABA or xanthoxin was detected.

Inhibitors of carotenoid biosynthesis decrease ABA
accumulation. While levels of carbohydrates and organic
acids were also changed, this result supports the hypothesis
that ABA is derived from a carotenoid.

The biosynthesis of ABA requires molecular oxygen. When
stressed leaves of Xanthfum were incubated in the presence of
1802 for six hours, the majority of the 180 present in ABA
was found in the carboxyl group. With longer incubations (12
and 24 hours), increasing amounts of 180 were found in the
ring oxygen atoms of ABA. However, with stressed Xanthium
roots, 180 was already present in the ring oxygen atoms after
only six hours. Incorporation of 180 into phaseic acid, a
catabolite of ABA, was also detected. These data are consis-

tent with the indirect pathway for biosynthesis of ABA.

Knowledge is good (Emil Faber)
The struggles you survive make you stronger (F. Nietzsche)

It is by the solution of problems that the strength of the
investigator is hardened; he finds new methods and new
outlooks and gains a freer horizon (D. Hilbert)

The thing that hath been is that which shall be; and that
which is done is that which shall be done; and there is no
new thing under the sun (Ecclesiastes)

The more things change, the more they remain the same
(Montaigne)

I write my book, whether it be read by the present age or
posterity imports little...has not God waited six thousand
years for an observer of his works? (J. Kepler)

For we do not think that we know a thing until we are
acquainted with its primary conditions or first principles
and have carried our analysis as far as its simplest elements
(Aristotle, Physics, book 1)

I have yet to see any problem, however complicated, which
when you looked at it the right way did not become more
complicated (P. Anderson)

Some day you will tax it (M. Faraday, on being asked by
P.M. Gladstone what was the use of electricity)

Where there is a great deal of light, the shadows are
deeper (Goethe)

It is the task of science to turn the impossible into the
boring (R. Ornstein)

Wherever you go, there you are (Buckaroo Banzai)

Vegetables are organized bodies, which extract from the
earth jucies proper to their nature. Vegetable substances
are much more compound than mineral. Their analysis is
consequently more difficult; certain principles, of too
great tenuity or volatility, escape us entirely (Beaume)

 

ACKNOWLEDGMENTS

There are many people I would like to thank for their
help, but here I can only offer my gratitude to a few. Many
thanks go to Jan Zeevaart, my major professor, for letting me
do the experiments I wanted to do. I also wish to thank the
members of my guidance committee, Ray Hammerschmidt, Hans
Kende, and Frank Dennis, for their helpful comments and
critical reviews. Much of this thesis consists of mass
spectra; without the MSU-NIH Mass Spectroscopy Facility
(RR00480) and the people who work there (among others, Brian
Musselman, Betty Baltzer, and John Stults) I could not have
accomplished what I did. Without a doubt, my interactions
with other graduate students, post-docs, and faculty at MSU,
the PRL, and the Botany and Plant Pathology Department (in
particular Alice and Jennifer) have not been unrewarding or
unpleasurable. Special thanks go to the Spuds/Citrus Can-
kers/Dynamo Buffuphna (past, present, and future) for all the
fun, good times, and exercise. I would have never been able
to get through this place without going insane if I had not
known Ted John, Brian Parks, and Jim Smith. Thanks guys, this
one is for you. I would also like my parents and family for
all the support and encouragement through this period. And
last but not least, SAK, who made these last few years so
nice.

Without money, nothing can be done, so I would like to
state that the research described in this thesis was support-
ed by the United States Department of Energy under Contract
DE-AC02-76ERO—l338.

TABLE OF CONTENTS

List of Tables

List of Figures

List of Abbreviations

Chapter 1. INTRODUCTION

1.

1.

1

1.

1.

. . e .
01 h (A) N
o . . o

7.
.8.
9.

Isolation and Identification of Abscisic
Acid

Occurrence in Plants

Chemical Properties

Biosynthesis in Fungi

Biosynthesis in Higher Plants

1.5.1. The Direct Pathway

1.5.2. The Indirect Pathway

1.5.3. Abscisic Acid Biosynthetic Mutants
Metabolism of Abscisic Acid

Physiology

Statement of Purpose

Literature Cited

Chapter 2. INHIBITION OF ABSCISIC ACID ACCUMULA-

TION IN WATER-STRESSED CORN SEEDLINGS IN THE

PRESENCE OF CAROTENOID BIOSYNTHETIC INHIBITORS

2.
2.

1.
2.

Introduction

Materials and Methods

2.2.1. Plant Material

2.2.2. Abscisic Acid Analysis

iv

Page
xi
xiii

xvii

34
35
36
36
36

 

 

Page

2.2.3. Organic Acid Analysis . . . . . . . . . . . 36
2.2.4. Soluble and Insoluble Sugar Analysis . . . . 38
2.2.5. Carotenoid Analysis . . . . . . . . . . . . 38
2.3. Results . . . . . . . . . . . . . . . . . . . . . . 39

2.3.1. Carotenoid, Sugar, and Organic Acid
Levels in Inhibitor and Control Corn
Seedlings . . . . . . . . . . . . . . . . . 39
2.3.2. Effects of Carotenoid Biosynthetic In—
hibitors on Abscisic Acid Accumulation . . . 41
2.4. Discussion . . . . . . . . . . . . . . . . . . . . 41
2.4.1. Inhibition of Abscisic Acid Accumula-
tion by Norflurazon and Fluridone . . . . . 41
2.4.2. Non-specificity of Carotenoid Biosyn-
thetic Inhibitors . . . . . . . . . . . . . 44
2.5. Literature Cited . . . . . . . . . . . . . . . . . 46
Chapter 3. BIOLOGICAL ACTIVITY AND METABOLISM OF

COMPOUNDS STRUCTURALLY RELATED TO ABSCISIC ACID . . . . . 48
3.1. Introduction . . . . . . . . . . . . . . . . . . . 49
3.2. Materials and Methods . . . . . . . . . . . . . . . 51

3.2.1. Synthesis of 2H-a-Ionone . . . . . . . . . . 51
3.2.2. Synthesis of 2H- and 3H-B—Ionone . . . . . . 51
3.2.3. Synthesis of a-Ionylidene Acetic Acid . . . 53
3.2.4. Synthesis of 1',2'-Epoxy-Ionylidene

Acetic Acid . . . . . . . . . . . . . . . . 55
3.2.5. Synthesis of I',2'-Epoxy-Ionylidene
Acetaldehyde . . . . . . . . . . . . . . . . 61

 

3.
3.
Chapter 4.

4.
5.

3.2.6.
3.2.7.
3.2.8.
3.2.9.

Results

3.3.1.

Spectrometry
Plant Material
Extraction and Purification Procedures

Biological Activity Assay

Biological Activity of a-Ionylidene
Acetic Acid (X) and 1',2'-Epoxy-Ionyl—
idene Acetic Acid (XV)

Incorporation of 1',2'-Epoxy-Ionylidene
Acetic Acid (XV) into Abscisic Acid
Metabolism of 1',2'-Epoxy—Ionylidene
Acetaldehyde (XVII) by Higher Plant .

Tissue

Discussion

Literature Cited

INCORPORATION OF OXYGEN INTO ABSCISIC

ACID AND PHASEIC ACID FROM MOLECULAR OXYGEN

4.

4

1.
.2.

Abstract

Introduction

Materials and Methods

4.3.1.
4.3.2.
4.3.3.
4.3.4.

Plant Material
Chemicals
Extraction and Purification Procedures

Mass Spectrometry

Results and Discussion

Literature Cited

Page
68
70
70
72
73

73

73

73
80
83

86
87
87
87
87
88
88
88
88
9O

 

Chapter 5. ACCUMULATION OF ABSCISIC AND PHASEIC

ACID IN XANTHIUM STRUMARIUM LEAVES UNDER DIFFERENT

OXYGEN TENSIONS

5.1.
5.2.

5.3.
5.4.
5.5.

Introduction

Materials and Methods

5.2.1. Plant Material

5.2.2. Extraction and Purification Procedure
5.2.3. Oxygen measurements

Results

Discussion

Literature Cited

Chapter 6. INCORPORATION OF MOLECULAR OXYGEN INTO

ABSCISIC AND PHASEIC ACID IN LEAVES AND ROOTS OF XANTHIUM

STRUMARIUM DURING LONG TERM INCUBATIONS IN 1802

6.1.
6.2.

Introduction

Materials and Methods

6.2.1. Culture of Plant Material

6.2.2. Extraction and Purification of Abscisic
and Phaseic Acid

.2.3. Oxygen Measurements

2.4. Purification of Carotenoids

2.5. Mass Spectrometry

03010103

.2.6. Chemicals
Results
6.3.1. Carotenoid levels in leaves and roots

6.3.2. Incorporation of 180 into abscisic

 

Page

91
92
92
92
93
93
94
94
97

98
99
100
100

101
101
102
103
104
104
104

and phaseic acid during long term
incubations in 1802
6.4. Discussion
6.5. Literature Cited
Chapter 7. INCORPORATION OF DEUTERIUM INTO ABSCISIC
ACID, STEROLS, AND CAROTENOIDS FROM DEUTERIUM OXIDE
7.1 Introduction
7.2. Materials and Methods
7.2.1. Plant Material
7.2.2. Extraction and Purification of Abscisic
Acid
7.2.3. Purification of Sterols
7.2.4. Purification of Carotenoids
7.2.5. Mass Spectrometry
7.3. Results and Discussion
7.4. Literature Cited
Chapter 8. THE ROLE OF XANTHOXIN IN ABSCISIC ACID
BIOSYNTHESIS
8.1. Introduction
8.2. Materials and Methods
8.2.1. Plant Material
Chemicals
Extraction and Purification Procedures
Determination of Xanthoxin Stability

Gas Chromatography .

oooooooooo
NNNNN
OlU'l-fiWN

. Mass Spectrometry

viii

 

Page

107
115
122

124
125
127
127

127
128
129
130
131
136

137
138
140
140
140
140
143
143
143

 

8.3. Results
8.3.1. Stability of Xanthoxin
8.3.2. Incorporation of 1802 into Xanthoxin
and Abscisic Acid
8.3.3. Xanthoxin Levels in Tissues Extracted in
Air and Nitrogen
8.4. Discussion
8.5. Literature Cited
Chapter 9. ABSCISIC ACID ACCUMULATION IN SPINACH
LEAF SLICES IN THE PRESENCE OF PENETRATING AND
NON-PENETRATING SOLUTES
9.1. Abstract
9.2. Introduction
9.3. Materials and Methods
9.3.1. Plant Material
9.3.2. ABA Purification Scheme
9.3.3. [14C]Mannitol Uptake and Catabolism
9.3.4. Water Potential Measurement
9.3.5. Mass Spectrometry
9.4. Results and Discussion
9.5. Literature Cited
Chapter 10. STRESS-INDUCED ABSCISIC ACID BIOSYNTHESIS
IN HIGHER PLANTS -- A MODEL
10.1. Introduction
10.2. A Model Explaining Stress-Induced Abscisic

Acid Biosynthesis

 

Page

144
144
144
144

144
148
152

154
155
155
155
155
155
156
156
156
156
158

159
160

165

 

 

Page

10.3. Literature Cited . . . . . . . . . . . . . . . . 168
Appendix. QUANTITATION 0F ABSCISIC ACID USING INTERNAL
STANDARDS . . . . . . . . . . . . . . . . . . . . . . . 171

 

LIST OF TABLES

Page

Table
2.1. The effect of norflurazon and fluridone

on levels of phytoene and total carotenoids

in corn seedlings . . . . . . . . . . . . . . . . . . . 42
2.2. The effect of norflurazon and fluridone

on organic acid levels in corn seedlings . . . . . . . . 42
2.3. The effect of norflurazon and fluridone

on levels of soluble and insoluble sugars

in corn seedlings . . . . . . . . . . . . . . . . . . . 43
2.4. The effect of norflurazon and fluridone on

ABA levels in corn seedlings . . . . . . . . . . . . . . 43
3.1. Selected ion monitoring response of standard

ABA and ABA isolated from leaves fed 1',2'-epoxy-

ionylidene acetic acid . . . . . . . . . . . . . . . . . 75
4.1. Effect of anoxia on PA accumulation in

Xanthium leaves . . . . . . . . . . . . . . . . . . . . 88
4.2. Effect of anoxia on ABA accumulation in

Xanthium leaves . . . . . . . . . . . . . . . . . . . . 89
6.1. Absorption maxima for some carotenoids found in

roots of Xanthium strumarium . . . . . . . . . . . . . 106
6.2. Levels of carotenoids and ABA in roots and

leaves of Xanthium strumarium . . . . . . . . . . . . 108
6.3. Incorporation of 180 into abscisic acid in

stressed Xanthfum leaves . . . . . . . . . . . . . . . 109

6

.4. Predicted and actual values for 180 incorpor-

xi

-. a .44; , 5&9“).

 

 

Page

Table

ation into phaseic acid . . . . . . . . . . . . . . . 120
7.1. Incorporation of deuterium from deuterium oxide

into abscisic acid . . . . . . . . . . . . . . . . . . 134
8.1. Xanthoxin levels from spinach leaves extracted

in air or N2 . . . . . . . . . . . . . . . . . . . . . 150
A.1. Example of abscisic acid quantitation by gas-

liquid chromatography-electron capture detection

with internal standards . . . . . . . . . . . . . . . 175

LIST OF FIGURES

Figure

1.
1.

0—4

H

0—4

p—a

NH

(.000

w

3

1. Structure of abscisic acid
2. Biosynthesis of abscisic acid in

Cercospora rosicola

.3. Biosynthesis of abscisic acid in higher

plants

.4. Some xanthophylls with terminal ring

structures similar to abscisic acid

.5. Compounds produced when violaxanthin is

photo-oxidized

.6. Biosynthetic scheme for trisporic acid in

the fungal order Mucorales

.7. Catabolites of abscisic acid

.1. Corn seedlings watered with either

nutrient solution or 10'4 M norflurazon

.1. Synthesis of deuterated a—ionone

.2. Synthesis of deuterated a-ionylidene acetic

acid (X)

.3. Mass spectrum of deuterated methyl—a—io-

nylidene acetic acid (X)

.4. Synthesis of deuterated 1',2'-epoxy-io—

nylidene acetic acid (XV)

.5. Mass spectrum of deuterated ethyl-cis,

trans-B-ionylidene acetate (XII)

.6. Mass spectrum of deuterated methyl-cis,

xiii

Page

11

18

4O
52

54

56

57

58

Page

Figure

trans-epoxy-ionylidene acetate (XV) . . . . . . . . . . 60
3.7. Synthesis of 1',2'-epoxy-ionylidene acet-

aldehyde (XVII) . . . . . . . . . . . . . . . . . . . . 62
3.8. Mass spectrum of ethyl-cis,trans-B-io-

nylidene acetate (XII) . . . . . . . . . . . . . . . . . 63
3.9. Mass spectrum of ethyl-cis,trans-1',2’-

epoxy-ionylidene acetate (XIV) . . . . . . . . . . . . . 64
3.10. Mass spectrum of cis,trans-1',2'-epoxy-

ionylidene ethanol (XVI) . . . . . . . . . . . . . . . . 66
3.11. Mass spectrum of cis,trans-1',2'-epoxy-

ionylidene acetaldehyde (XVII) . . . . . . . . . . . . . 67
3.12. Mass spectrum of deuterated cis,trans-1',

2'-epoxy-ionylidene acetaldehyde (XVII) . . . . . . . . 69
3.13. Biological activity of a-ionylidene acetic .

acid (X) and 1',2'-epoxy-ionylidene acetic ,

acid (XV) . . . . . . . . . . . . . . . . . . . . . . . 74
3.14. Mass spectrum of a compound which co-

chromatographed with standard methyl-cis,

trans-1',2'-epoxy-ionylidene acetate (XV) . . . . . . . 76
3.15. Metabolism of 3H-cfs,trans-1',2'-epoxy-

ionylidene acetaldehyde (XVII) in spinach leaves . . . . 77
3.16. Metabolism of 3H-cis,trans-1',2'-epoxy-

ionylidene acetaldehyde (XVII) in tomato shoots . . . . 78
3.17. Metabolism of 3H-cis,trans—1',2'-epoxy-

ionylidene acetaldehyde (XVII) in immature

Page

Figure

(breaker stage) tomato fruits . . . . . . . . . . . . . 79
4.1. Mass spectra of PA isolated from stressed

and subsequently rehydrated Xanthium

leaves incubated in room air (A), or 1802 (B) . . . . . 89
4.2. Mass spectra of ABA isolated from stressed

Xanthium leaves incubated in room air (A),

or1802(B) 90
5.1. Accumulation of phaseic acid under different

oxygen tensions . . . . . . . . . . . . . . . . . . . . 95
5.2. Accumulation of abscisic acid under different

oxygen tensions . . . . . . . . . . . . . . . . . . . . 96
6.1. Purification of Xanthium root carotenoids

by HPLC . . . . . . . . . . . . . . . . . . . . . . . 105

6.2. Mass spectra of ABA obtained by DP-NCI . . . . . . 110
6.3. Mass spectrum of ABA isolated from stressed

Xanthium roots incubated in 1802 for 6 h . . . . . . . 112
6.4. Mass spectra of PA obtained by DP-NCI . . . . . . . 113

6.5. Mass spectrum of ABA isolated from stressed

Xanthium leaves incubated in 1302 for 24 h . . . . . . 116
7.1. Incorporation of deuterium from deuterium

oxide into sitosterol and stigmasterol . . . . . . . . 132
8.1. Stability of xanthoxin under different environ-

mental conditions . . . . . . . . . . . . . . . . . . 145
8.2. Mass spectrum of xanthoxin isolated from

spinach leaves incubated in 1802 for 8 h . . . . . . . 146

XV

Page

Figure
8.3. Mass spectrum of ABA isolated from spinach

leaves incubated in 1802 for 8 h . . . . . . . . . . . 147
8.4. Mass spectrum of the 0-(2,3,4,5,6-penta-

fluorobenzyl)hydroxylamine hydrochloride

derivative of xanthoxin . . . . . . . . . . . . . . . 149
9.1. Changes in ABA content of detached control

(turgid), and in stressed (wilted) leaves . . . . . . 156
9.2. ABA accumulation in detached control (C),

stressed (S) spinach leaves, and in spinach

leaf slices and media . . . . . . . . . . . . . . . . 156
9.3. ABA accumulation in detached spinach leaves

and in spinach leaf slices and media . . . . . . . . . 156
9.4. Levels of ABA in detached spinach leaves

and in spinach leaf slices and media . . . . . . . . . 157
10.1. Representative sesquiterpenoids . . . . . . . . . 161

10.2. Compounds with structures similar to abscisic

acid . . . . . . . . . . . . . . . . . . . . . . . . . 162
A.1. Electron capture detector response of

abscisic acid and internal standards . . . . . . . . . 174
A.2. Typical standard curve obtained by injecting

internal standards with increasing concentrations

of methyl abscisic acid . . . . . . . . . . . . . . . 176

xvi

 

 

 

LIST OF ABBREVIATIONS

ABA-GE B-D-glucopyranosyl abscisate

BHT 2,6-di-tert-butyl-p-cresol

B.R. boiling range

BSA N,0-bis-(trimethylsilyl)-acetamide
CD circular dichroism

Ci Curie(s)

cv cultivar

d day(s)

DPA 4'—dihydrophaseic acid

DP direct probe

EtABA ethyl ester of abscisic acid

FAB fast atom bombardment

GC-NCI gas chromatography-negative chemical

ionization

 

GC-MS gas chromatography-mass spectrometry

GC-SIM gas chromatography-selected ion
monitoring

GLC-ECD gas-liquid chromatography-electron
capture detection

GLOTID gas-liquid chromatography-flame
ionization detection

GLCJCD gas—liquid chromatography-total
conductivity detector

h hour(s)

xvii

 

 

HMDS
8'-0H-ABA
HPLC
EI
MeABA
min
MVA
MVL
m/z
NMR
0RD
PA
PA-GE

Rf

SIM
TLC
TMCS
TMS
TMSi
UV

hexamethyldisilazane

8'-hydroxy ABA

high performance liquid chromatography
electron impact

methyl ester of abscisic acid
minute(s)

mevalonic acid

mevalonic acid lactone
mass/charge

nuclear magnetic resonance
optical rotary dispersion
phaseic acid

B-D-glucopyranosyl phaseate
ratio of the distance traveled of a
solute relative to solvent front
second(s)

selected ion monitoring

thin layer chromatography
trimethylchlorosilane
tetramethylsilane

trimethylsilyl

ultraviolet

xviii

 

CHAPTER 1
INTRODUCTION

2
1.1. ISOLATION AND IDENTIFICATION OF ABSCISIC ACID

The discovery of abscisic acid (ABA, Figure 1.1) stemmed
from investigations performed during the 1950’s and early
1960’s by three groups working on unrelated problems. One
group, led by Addicott at the University of California at
Davis, purified an active compound (abscisin II) from cotton
bolls which accelerated petiole abscission in explants from
young cotton seedlings (Ohkuma et a7., 1963; Ohkuma et
a7., 1965).

At Aberystwyth, Wareing’s group was investigating
the cause of dormancy in trees. An active extract (termed
dormin) Iuas obtained from leaves of sycamore (Acer pseudo-
platanus) which induced dormancy in buds of sycamore seed-
lings (Robinson and Wareing, 1964; Wareing et a7., 1964).
Cornforth et a7. (1965) later showed that dormin was identi-
cal to abscisin II.

Rothwell and Wain (1964) isolated a compound which
appeared to accelerate fruit and flower drop in yellow
lupin, later identified as abscisin II (Cornforth et al.,
1966b; Koshimizu et al., 1966; Porter and Van Steveninck,
1966). As a compromise, abscisin II (dormin) was renamed
abscisic acid and is abbreviated ABA (Addicott et al.,
1968). Recently, the numbering system of ABA was extended to
include previously unnumbered methyl groups (Boyer et al.,

1986).

   

Figure 1.1. Structure of abscisic acid.

 

1.2. OCCURRENCE IN PLANTS

ABA has been identified in angiosperms (both monocots
and dicots), gymnosperms, two ferns, a horsetail, and a
moss (Milborrow, 1978; Bearder, 1980), and several genera
of fungi (Assante et al., 1977; Ichimura et al., 1983;
Dérffling and Peterson, 1984; Marumo et al., 1984). ABA has
been detected in every major (and minor) plant organ from
shoot to root apices (Milborrow, 1978). ABA tends to occur
in highest concentration in young leaves, buds, fruits, and

seeds (Milborrow, 1978).

1.3. CHEMICAL PROPERTIES

ABA (MW 264 g/mole) is a sesquiterpenoid and contains
a carboxyl, keto, and hydroxyl group (Figure 1.1). It is a
weak acid (pka 4.8) and partitions into organic solvents,
such as diethyl ether, dichloromethane, and ethyl acetate
(but not hexane), at low pH. The molecule has one chiral
center at C-1'. The naturally occurring enantiomer is dextro-
rotary and has a sinister (S) configuration. ABA has high
optical activity, with extrema at 289 nm (positive) and 246
nm (negative) (Cornforth et al., 1966a). ABA absorbs in the
UV, with its maximum varying with the pH of the solution

(Dorffling and Tietz, 1983). UV, infrared, NMR, 0RD, CD, and

5
mass spectra are presented in Dorffling and Tietz (1983).

The configuration of the side chain at C-2 can be either
cis or trans. By convention, 2-cis,4-trans-ABA is ABA, and
the 2-trans,4-trans isomer t-ABA (Addicott et al., 1968).
Strong ligflrt catalyzes the isomerization at the 2,3 double
bond to establish an approximate 1:1 ratio of ABAzt—ABA in
solution.

In several biological assays the unnatural (-) enantiomer
was as active as natural (+)-ABA (Sondheimer et al., 1971).
However, (-)-ABA was much less active than (+)-ABA in closing

stomata of barley leaves (Cummins and Sondheimer, 1973).

1.4. BIOSYNTHESIS IN FUNGI

The discovery that the fungus Cercospora rosicola
produced large amounts of ABA (Assante et al., 1977) initiat-
ed studies on the biosynthetic pathway in that organism. When
3H-MVA was applied to C. rosicola mycelia it was incorporated
into two major fractions, ABA and 1'-deoxy-ABA (IV, Figure
1.2; Neill et al., 1981; Neill et al., 1982). Later it was
shown that a-ionylidene ethanol (1), a-ionylidene acetic acid
(11), and the epimeric 4'-hydroxy-<x-ionylidene acetic acids
(111) were also incorporated into ABA (Neill and Horgan,
1983; Horgan et al., 1983). Of these compounds, only 111 and
IV are endogenous in C. rosicola (Horgan et al., 1983). Com-
pounds with a B-ionylidene type structure are not converted

‘to ABA (Neill and Horgan, 1983). Recently, Ichimura et

 

O
/
/

\x

\\ II
1 COOH
Ho COOH
'V
0% 1 COOH

\\ \\ IABAI
OH
0% COOH

Figure 1.2. Biosynthesis of abscisic acid in Cercospora

rosicola.

Only III, IV, and ABA are endogenous in C. rosicola. It is

not known which epimer of III is the natural one.

 

7

a7. (1983) reported similar results with C. cruenta.

Interestingly, only the 2-cis,4-trans isomers of I and II
were metabolized to ABA, while the 2—trans,4-trans isomers
were converted only as far as 2-trans,4-trans-IV (Neill and
Horgan, 1983). This implies that the early oxidizing enzymes
are rather nonspecific, but the final hydroxylating enzyme
has high specificity for the geometry of the side chain.

In summary, labeling studies with C. rosicola and
C. cruenta suggest that ABA' in these fungi is formed from
a-ionylidene type compounds. Several possible intermediates
are also converted to ABA, but only two of them (111 and IV)
appear to be endogenous. The final step in the biosynthetic
pathway appears to be the hydroxylation of IV at the 1'
carbon to give ABA. Nothing is known about the steps from MVA

to the first cyclized intermediate.

1.5. BIOSYNTHESIS IN HIGHER PLANTS

In order to understand the physiology of ABA it is essen-
tial to understand how the ABA level is regulated. This
requires information about how ABA is biosynthesized and
catabolized. Suprisingly for this youngster of the plant
growth substances, nothing is known about the biosynthetic
precursors to ABA. Reasons for this are the low levels of ABA
in plant tissues and lack of specific inhibitors of its
formation. As ABA contains 15 carbons and fits the isoprene

rule, it is considered to be a sesquiterpenoid. However, the

8

structure of ABA is radically different from any of the known
sesquiterpenoids (Loomis and Croteau, 1980). Research has
focused on two pathways: (a) the direct pathway involving a
C15 precursoriderived from farnesyl pyrophosphate, and (b)
the indirect pathway involving a precursor derived from a
carotenoid (Figure 1.3). The relative contribution of each
pathway is unknown, and the possibility exists that both may
be operating iri higher plants at the same time. In either
case, MVA would be the ultimate precursor.

1.5.1. The Direct Pathway. Little evidence exists for the
immediate biosynthetic precursors of ABA, yet there is
evidence that MVA is a precursor. Label from MVA (or MVL)
has been incorporated into ABA on several occasions (Noddle
and Robinson, 1969; Milborrow and Robinson, 1973). However,
even though large amounts of radioactivity were supplied,
the amount of label found in ABA was a small fraction of
the total fed. Possible reasons for low incorporation are
competition for MVA by other terpenoid pathways (such as
sterols), or that the applied radioactive MVA is extensive-
ly diluted by a (large) precursor pool to ABA.

Results from feeding experiments with labeled MVA
indicate that three residues of the natural 3-R enantiomer
are incorporated into ABA (Robinson and Ryback, 1969). In
addition, the pro-4-R hydrogen of MVA is retained at carbons
Z and 5' of ABA (Robinson and Ryback, 1969), the pro-S-S
hYdrogen at carbon 5 (Milborrow, 1972), and a preponderance

of the pro-Z-R hydrogen at carbons 4 and 3' (Milborrow,

 

_-..

lsopentenyl—P-P

GeranyI-P-P

Farnesyl-P—P

GeranylgeranyI-P—P

Carotenoids

Figure 1.3.

It is not known whether xanthoxin

ABA, or

  

 

 

  

   

C5
V
C10
ABA
V ?
015 ; é COOH
o
I)
. /
ll Xanthoxin
C20 ' :::o\ \
HO CHO
?
4 \. ‘\ \. ‘\I
C40

HO Violaxanthin

Biosynthesis of abscisic acid in higher plants.

is a natural precursor to

if endogenous xanthoxin arises from a C15 or C40

compound (Adapted from Burden and Taylor, 1976).

10
1974b).

Similar retention of hydrogens from MVA has been observed
with carotenoid biosynthesis. This similarity suggests that
the same enzymes involved in carotenoid biosynthesis are also
used in ABA biosynthesis, or that different enzymes (in
different compartments?) operate with the same mechanism.

An 1%) vitro plastid system from avocado fruit that
synthesized ABA from MVL has been described (Milborrmw,
1974a), although the amount of radioactivity recovered in ABA
was very low. Recently, Hartung et a7. (1981) presented data
indicating that no biosynthesis of ABA occurred when isolated
spinach chloroplasts were fed MVA. Incorporation into ABA was
detected, however, when protoplasts or cytoplasmic fractions
were incubated with MVA.

1.5.2. The Indirect Pathway. Once ABA had been identified,
similarities between its structure and many xanthophylls,
e.g. lutein, zurtheraxanthin, and violanxanthin, were noted
(Figure 1.4). Taylor and Smith (1967) discovered that a
mixture of carotenoids irradiated in vitro were degraded to
an inhibitor with physiological properties similar to
ABA. The greatest amount of inhibitor was obtained when
Violaxanthin was degraded. Later work showed that lolio-
lide, butenone, and an isomeric mixture of xanthoxins (Figure
1.5) were produced when Violaxanthin was photooxidized, and
that 2-cfs,4-trans-xanthoxin was the active ingredient
causing growth inhibition in this mixture (Taylor and Burden,

1970; Burden zuui Taylor, 1970). Soon after this, Firn and

11
1 I I
:10.
a b E“) c

Ho d HO 9

R1\ \ \ \ \ \'\ \ \R,

R1 R2

B-carotene b b
Violaxanthin c c
antheraxanthin e c
lutein d e

Figure 1.4. Some xanthophylls with terminal ring structures

similar to abscisic acid.

 

 

 

12

 

Loliolide Butenone
CH0
0 \ \ o \ \
° 0
cwo
H' HO
c,t-—Xanthoxin ILL-Xanthoxin

Figure 1.5. Compounds produced when Violaxanthin is photo-ox-
idized.

These four compounds are also produced when Violaxanthin is
treated with zinc permanganate or soybean lipoxygenase. The
trans,trans isomer is usually present in 2 X greater amount

than the cis,trans isomer.

 

13

Friend (1972) showed that treatment of Violaxanthin with
soybean lipoxygenase could produce butenone and xanthoxin in
yields similar to those when Violaxanthin was photo—oxidized,
indicating that light was not needed for xanthoxin pro-
duction. In all instances more 2-trans,4-trans-xanthoxin was
produced than the 2-cis,4-trans isomer. Interestingly, the
pheromone trisporic acid, which regulates sexual reproduction
in the Mucorales, has been shown to be a breakdown product of
B-carotene (Figure 1.6; Gooday, 1974) However, it should be
noted that endogenous xanthoxin has never been shown to be
derived from Violaxanthin in viva. Futhermore, xanthoxin
could also be an intermediate in the direct pathway.

Studies on xanthoxin indicate that 2-cfs,4-trans-xan-
thoxin is more active in bioassays than 2-trans,4-trans—
xanthoxin (Taylor and Burden, 1972). Only 2-cis,4-trans-
xanthoxin was converted to ABA and PA when fed to tomato
and dwarf bean plants (Taylor and Burden, 1973). When 2-
cfs,4-trans—xanthoxin was applied through the transpiration
stream to leaves of several plants, stomata closed as fast
as when ABA was used (Raschke et al., 1975). However, when
applied to isolated epidermal strips it was ineffective.
Raschke et a7. (1975) suggested that xanthoxin was convert—
ed to ABA during its passage to the epidermis when intact
leaves were used. However, if xanthoxin was converted to
ABA it should not act as fast as it did.

An attempt to differentiate between the indirect and

direct pathways was performed by D.R. Robinson (cited in

14

4 oHYOROXY -fl.-C..KETONE

HEVALONATE
I 1’
1’1 0

OH

W on . 0 1”

\h.
\
3
'0
>9
:1

 

 

/ mmonomseonm 3' TRSPORIN
CAROTENE l I
P 15.15’ axidntlvo [H
cleave e
9 n H n
0 CNN" , omen
/ / / I L-omvonomsem TRISPORJL
paint of
RETINAL ’_ .I {an insertion OH I" I.‘
of 'extm' ' I
n o
/ / "/ / 2 omgggi‘msrom
cn and C12 4- 1: -
"CnKETONE 0,, “3'8 I I l R
I “stain H coon
. n
1 . coocw,
METHYL L-DIHYDRO
TRSPORATE

 

Figure 1.6. Biosynthetic scheme for trisporic acid in the
fungal order Mucorales.

On the left are shown reactions common to both mating
types. The compounds shown on the right are formed by the
respective strains ([+] or [-]). Compounds marked with an

asterisk are hypothetical and have not been isolated (From

Gooday et al., 1978).

15

Milborrow, 1983). The first colorless Carotenoid, phytoene
(labeled with 14C), was fed to avocado fruit slices along
with 3H-MVA. When the ABA isolated from this tissue was
analyzed, only 3H was found in ABA, yet both 3H and 141C were
detected in carotenoids. This work is inconclusive because
phytoene would have to penetrate to chloroplasts and then be
further metabolized to a xanthophyll. It is not known whether
this occurred, because a detailed account of this work has
never been published.

1.5.3. Mutants Deficient in Abscisic Acid. Several mutants
of higher plants have been found which appear to have an
ABA minus phenotype. The recognition of an ABA deficient
phenotype is based on excessive wilting or vivipary (prema-
ture germination). These mutants can be divided into two
classes, those that are green and contain carotenoids,
and those that are albino. It should be noted that are no
known mutants which produce no ABA at all. Perhaps this
genotype is lethal.

Green ABA-deficient mutants are found in potato (Quarrie,
1982), pea (Wang et al., 1984), tomato (Tal and Nevo, 1973),
and Arabidopsfs (Koornneef et al., 1982). Of these, the only
well documented mutants are the wilty mutants of tomato,
flacca (f7c), sftiens (sit), and notabilis (not). The
tomato mutants are recessive and are located at three
separate loci (Stubbe, 1957; Stubbe, 1958; Stubbe, 1959). ABA
'levels in the three mutants range from 12-15% of wild type

for sit, 17-26% for flc, and 31-49% for not (Tal and Nevo,

 

16
1973; Neill and Horgan, 1985). Since the decreased ABA
contents are not due to increased catabolism (Nevo and Tal,
1973), the lesions must be in enzymes involved in ABA
biosynthesis.

Normal water stress induced accumulation of ABA does
not occur in droopy (potato, Quarrie, 1982), sit and flc
(tomato, Neill and Horgan, 1985) and wilty (pea, Wang et al.,
1984). It is possible that these mutants are already stressed
and have reached their maximum accumulation of ABA, or these
mutants may not be able to accumulate stress-induced ABA. If
the latter case is true, the implication is that in higher
plants two pathways exist, one operating in turgid and the
other operating in water-stressed leaves.

Examples (rf mutants it: the second class are the maize
viviparous mutants (viviparous 2, 5, and 9 [Vp2, vp5, vp9],
pink scutellum [ps=vp9], white seedling [W3], and yellow
[y9]). All maize viviparous mutants are characterized by
pale yellcnv endosperms and white or almost white seedlings
(Robertson, 1975). The primary lesion in these mutants is
defective carotenoid biosynthesis (Fang et al., 1983a). All
carotenoid deficient mutants of maize have reduced levels
of ABA in the embryos, from 7-70% of wild type (Brenner et
al., 1977; Smith et al., 1978). It is possible that some of
the ABA has a maternal origin. No ABA was detected in
seedlings and roots of w3, Vp5, and vp7 (Moore and Smith,
1985), although the method used to detect ABA was rather

insensitive.

 

 

17
Although not conclusive, the biochemistry of the vivip-
arous maize mutants suggests the involvement of the indirect
pathway. However, it is also possible that the decreased ABA
levels in these mutants could simply be a secondary effect of
carotenoid deficiency. If the indirect pathway is indeed

operating, then the green wilty mutants represent blocks in

 

the pathway after xanthophylls, since Nevo and Tal (1973)

showed that flc had slightly higher levels of carotenoids
than the wild type ‘Rheinlands Ruhm’.

1.6. CATABOLISH 0F ABSCISIC ACID

While nothing is known about the intermediates in the
ABA biosynthetic pathway in higher plants, much more is
known about how ABA is degraded. ABA is rapidly catabolized
in plants by either conjugation to water soluble catabol-
ites or by oxidation to more polar compounds (Figure 1.7).
This subject has recently been reviewed (Loveys and Mil-
borrow, 1984). This section will deal with catabolites
that have been fully characterized. See Loveys and Milbor-
row (1984) or Walton (1980) for a discussion of other less
rigorously identified catabolites.

A major pathway in higher plants involves the hydroxyla-
tion of ABA at the 8'-geminal methyl group to give the
unstable intermediate HM-ABA, which rearranges to form
PA, and the subsequent reduction to DPA. This pathway

has been shown to operate in several plants, including beans

18

    

co2

l
/ B—D—glucose

 

 

['3-D-aldopyranoside
Figure 1.7. Catabolites of abscisic acid.
Only well characterized compounds are shown. PA-GE has also
been demonstrated; its structure is similar to ABA-GE except

with PA as the aglycone.

19

(Har‘rison and Walton, 1975), ash seeds (Sondheimer et al.,
1974 ), and castor bean (Zeevaart, 1977). In spite of many
atternpts to isolate it, 8’—OH-ABA has only been reported once
(Mil borrow, 1969). Its presence as an intermediate in the
conversion of ABA to PA in a cell-free system was inferred
from a product which was acetylated (Gillard and Walton,
1976). The epimer of DPA has also been reported (Zeevaart
and Milborrow, 1976).

In addition to catabolism to PA and DPA, conjugation
of ABA also occurs. ABA—GE appears to be widely distributed
in plants, but has been unequivocally identified only a few
times (Hirai et al., 1978; Boyer and Zeevaart, 1982b).
Other conjugates of PA (PA-GE; Boyer and Zeevaart, 1982a)
and DPA (DPA aldopyranoside; Setter et al., 1981) have been
reported.

Many studies dealing with the catabolism of ABA have
involved the use of radioactive (i)-ABA. Determination of
whether the catabolites are natural or not is difficult
unless the stereochemistry of the catabolite is report-
ed. When 14C-(t)-ABA was fed to plants, only the (+) enantio-
mer was converted to PA and DPA, whereas hydrolysis of the
ABA-GE gave predominantly (-)—ABA (Milborrow, 1978; Zeevaart
and Milborrow, 1976). Another catabolite, 7'-hydroxy (-)-ABA
has recently been reported (Boyer and Zeevaart, 1986). Thus,
all studies dealing with ABA catabolism should state the

stereochemistry of ABA fed and of the catabolites isolated.

20
1.7. PHYSIOLOGY

Numerous theories on the involvement of ABA as a plant
growth regulator have been proposed, including, but not
limited to, root gravitropism (Wilkins, 1978, 1984), seed
and bud dormancy (Addicott and Lyon, 1969; Wareing, 1978),
and stomatal closure (Raschke, 1975). However, Wareing
(1978) and Walton (1980) have concluded that too little is
known about the events occurring during any physiological
process to state unequivocally that ABA is involved in its
control.

Root gravitropism is thought to involve the asymmetric
redistribution of a growth inhibiting substance. Although the
identity of the growth inhibiting substance is unknown, ABA
has been proposed to be the agent causing inhibition. How-
ever, recent evidence (Moore and Smith, 1984, 1985) argues
against a role for ABA in gravitropism. Using carotenoid
deficient mutants of maize (which have non-detectable levels
of ABA), Moore and Smith (1985) demonstrated that gravitropic
curvature still occurred. In addition, Moore and Smith (1984)
showed that curvature also was present when ABA biosynthesis
was inhibited using carotenoid biosynthetic inhibitors.

The best evidence, although still correlative, for the
involvement of ABA in a physiological process has been
obtained with experiments on water stress. Detached or
attached leaves will accumulate ABA (usually 10 to 40 times

that found in a turgid leaf) upon the imposition of a water

 

21

stress. When ABA is given to epidermal strips or fed to
intact leaves through the transpiration stream, stomata
close rapidly (Mittelheuser and Van Steveninck, 1969;
Tucker and Mansfield, 1971). The stomatal response is
specific for the naturally occurring (+) enantiomer and is
reversible (Cummins and Sondheimer, 1973; Cummins et a7”
1971).

Thus, a role for ABA in controlling water deficit through
regulating stomatal closure seems reasonable. ABA is clearly
made during water stress, it will then travel to the guard
cells via the transpiration stream and cause stomatal
closure. However, stomata have been shown to close before a
measurable increase in bulk leaf ABA occurs (Henson, 1981b).
The results of Henson (1981a, 1981b) could be explained if
ABA were compartmentalized as a large inactive pool and a
small active one. Evidence exists for the compartmentation of
ABA in leaves (Cummins and Sondheimer, 1973; Raschke et al.,
1976; Raschke and Zeevaart, 1976). In addition, following
loss of turgor a sufficient amount of ABA moves into the
apoplast to cause stomatal closure (Cornish and Zeevaart,
1985).

The effect of ABA on stomata is rather rapid compared
to its effect on protein synthesis, as in the control of
a-amylase production in barley aleurone layers (Higgins et
al., 1982; Mozer, 1980). In this tissue, ABA inhibits the
gibberellin induced synthesis of a-amylase, which is needed

for the hydrolysis of endosperm starch during germination

22

(Ho and Varner, 1976). This inhibition is not due to competi-
tion for a common site of action, because high concentrations
of gibberellin do not overcome the effect (Ho and Varner,
1976). The effect of ABA can be prevented by cordycepin,
indicating that continued protein or RNA synthesis is needed
for the response to occur (Ho et al., 1985). ABA applied
alone also appears to induce the synthesis of several
proteins in barley aleurone layers (Higgins et al., 1982;
Mozer, 1980). The function of these proteins is unknown.

ABA has been proposed to be an inhibitor of vivipary
(precocious germination; see section 1.5.3) and a promoter
of embryo maturation. Vivipary occurs normally in mangroves
(Rhizophora mangle) and it has been shown that embryos of
this species are insensitive to ABA (Sussex, 1975). The vp
mutants of corn (see section 1.5.3) also have decreased
levels of ABA. Addition of fluridone, a carotenoid biosyn-
thetic inhibitor, induces vivipary in developing maize
kernels (Fong et al., 1983a, Fong et al., 1983b).

In several other studies, it has been shown that exogen-
ous ABA application to excised dicot embryos will inhibit
both germination and the appearance of germination—specific
proteins, and promotes embryo-specific proteins and mRNAs
(Ihle and Dure, 1970; Dure et al., 1983; Crouch and Sussex,
1981; Crouch et a7. 1984). Bray and Beachy (1985) showed
that the B-subunit of B-conglycinin, a storage protein,
accumulated when excised soybean cotyledons where treated

with ABA. Addition of fluridone decreased B-subunit product-

23
ion. The accumulation of wheat germ agglutinin (a lectin
found in wheat embryos, shoots, and roots) is also induced
by ABA (Raikhel et al., 1986; Quatrano et al., 1983). Ap-
plication of fluridone to wheat leaves decreases the amount
of lectin present (Raikhel et al., 1986).

ABA appears to be necessary for other processes. An
example is the biochemical, physiological and morphological
changes that occur in the tomato mutants flc and sit (see
section 1.5.3). These mutants appear shorter, produce
adventitious roots on stems, and the leaves show epinasty
compared with wild type (Tal et al., 1979). In addition,
levels of auxinlike substances and ethylene are higher than
normal (Tal et al., 1979). Even though ABA is present
(although in lower amounts than wild type) application of ABA
will cause reversion to the wild phenotype (Imber and Tal,
1970; Bradford, 1983).

Thus, there appears to be several different roles for ABA
in higher plants. One is a rapid response associated with
stomatal closure. Here the site of action of ABA appears to
be the plasmalemma (Hartung, 1983) where it appears to
inhibit proton extrusion (Shimazaki, et al., 1986). Another
role is associated with the regulation of protein accumu-
lation, such as that found in barley aleurone layers with
a-amylase or in wheat with wheat germ agglutinin. However,
the effects of ABA on ion transport and gene expression
cannot explain the phenotypes seen in the ABA deficient

mutants flc and sit.

 

24

1.8. STATEMENT OF PURPOSE

Earlier it was mentioned that ABA levels increased upon
the imposition of water stress, and that ABA appears to
ameliorate the stress by closing stomata. The increase in ABA
is apparently regulated by loss of turgor (Pierce and
Raschke, 1980). Environmental conditions other than water
stress have also been reported to cause ABA accumulation,
although to a lesser degree. Examples of this are low
relative humidity (Wright, 1972), flooding of soil (Wright,
1972), salinity or osmotic stress of roots (Mizrahi et al.,
1970), mineral deprivation (Mizrahi and Richmond, 1972), and
chilling (Raschke et al., 1976). While the tissue did not
appear to be visibly stressed in any of these cases, each of
these conditions can be expected to change the water status
or reduce translocation. Thus, the accumulation of ABA
appears to be a situation where a physical phenomenon (loss
of turgor) controls a biochemical pathway. I was interested
in understanding how this occurs. Unfortunately, there is
very little known about the intermediates of the ABA biosyn-
thetic pathway. Therefore, I wanted to determine how ABA is
made in higher plants. Specifically, I wanted to:

1. Determine which pathway, the indirect or
direct one, operates during water stress;
2. Determine the role of xanthoxin in the pathway,

and the intermediates between it and ABA and;

 

25
3. Confirm that loss of turgor regulates the accum-

ulation of stress-induced ABA.
1.10. LITERATURE CITED

Addicott, F.T., Lyon, J.L. (1969) Physiology of abscisic
acid and related substances. Ann. Rev. Plant Physiol.
20:139—164.

Addicott, F.T., Lyon, J.L., Ohkuma, K., Thiessen, W.E.,
Carns, H.R., Smith, O.E., Cornforth, J.W., Milborrow,
B.V., Ryback, G., Wareing, P.F. (1968) Abscisic acid: a
new name for abscisin II (dormin). Science 159:1493.

N

Assante, G., Merlini, L., asini, G. (1977) (+)-Abscisic
acid, a metabolite of the fungus Cercospora rosicola. Ex-
perientia 33:1556.

Bearder, J.R. (1980) Plant hormones and other growth sub-
stances: ‘their background, structures, and occurrence.
In: Hormonal regulation of development 1. Molecular
aspects of plant hormones, pp. 9-112, MacMillan, J. ed.,
New York: Springer-Verlag.

Boyer, G.L., Milborrow, B.V., Wareing, P.F., Zeevaart,
J.A.D. (1986) The nomenclature of abscisic acid and
its metabolites. In: Plant growth substances 1985, pp,
Bopp, M.,ed., New York: Academic Press.

Boyer, G.L., Zeevaart, J.A.D. (1982a) Metabolism of abscisic
acid in Xanthium strumarium. Plant Physiol. Suppl. 69:77.

Boyer, G.L., Zeevaart, J.A.D. (1982b) Isolation and quanti-
tation of B-D-glucopyranosyl abscisate from leaves of
Xanthium and spinach. Plant Physiol. 70:227-231.

Boyer, G.L., Zeevaart, J.A.D. (1986) 7'-hydroxy (-)-R-ab-
scisic acid: a metabolite of feeding (-)-R-abscisic
acid to Xanthium strumarium. Phytochemistry 25:1103-1105.

Bradford, K.J. (1983) Water relatios and growth of the flacca
tomato mutant in relation to abscisic acid. Plant
Physiol. 72:251-255.

Bray, E.A., Beachy, R.N. (1985) Regulation by ABA of B-con-
glycinin expression in cultured developing soybean
cotyledons. Plant Physiol. 79:746-750.

 

26

Brenner, M.L., Burr, B., Burr, F. (1977) Correlation of
genetic vivipary in corn with abscisic acid concentra-
tion. Plant Physiol. Suppl. 63:36

Burden, R.S., Taylor, H.F. (1970) The structure and chemical
transformations of xanthoxin. Tetrahedron Lett. 47:4071-
4074.

Burden, R.S., Taylor, H.F. (1976) Xanthoxin and abscisic
acid. Pure Appl. Chem. 47:203-209.

Cornforth, J.W., Milborrow, B.V., Ryback, G. (1966a) Iden—
tification and estimation of (+)-abscisin II (‘dormin’)
in plant extracts by spectropolarimetry. Nature 210: 627-
628.

Cornforth, J.W., Milborrow, B.V., Ryback, G., Rothwell, K.,
Wain, R.L. (1966b) Identification of the yellow lupin
growth inhibitor as (+)-abscisin II ((+)-dormin).
Nature 211:742-743.

Cornforth, J. . Milborrow, B. V. Ryback, G., Wareing,
P.F. (1965)w Identity of sycamore ‘dormin’ with abscisin
II. Nature 205: 1269- 1270.

Cornish, K., Zeevaart, J.A.D. (1985) Movement of abscisic
acid into the apoplast in response to water stress in
Xanthium strumarium L.. Plant Physiol. 78:623-626.

Crouch, M.L. Sussex, I.M. (1981) Development and storage
protein synthesis in Brassica napus L. in vivo and in
vitro. Planta 153. 64—74.

Crouch, M.L., Tenbarge, K., Simon, A., Finkelstein, R.,
Scofield, S., Solberg, L. (1985) Storage protein mRNA
levels can be regulated by abscisic acid in Brassica
embryos. In: Molecular form and function of the plant
genome, pp. 555—566, van Vloten-Doting, L., Groot,
G.S.P., Hall, T.C., eds. New York: Plenum.

Cummins, W R., Kende, H., Raschke, K. (1971) Specificity
and reversibility of the rapid stomatal response to
abscisic acid. Planta 99:347—351.

Cummins, W.R., Sondheimer, E. (1973) Activity of the asym-
metric isomers of abscisic acid in a rapid bioassay.
Planta 111:365-369.

Ddrffling, K., Peterson, W. (1984) Abscisic acid in phyto-
pathogenic fungi of the genera Botrytis, Ceratocystis,
Fusarium, and Rhizoctonia. Z. Naturforsch. 39C:683—684.

DOrf fling, K., Tietz, D. (1983) Methods for the detection
and estimation of abscisic acid and related compounds.

27

In: Abscisic acid, pp. 23-78, Addicott, F.T., ed. New

York: Praeger Press.

Dure, L., Galu, G., Chlan, C., Pyle, J. (1983) Develop-
mentally regulated gene sets in cotton embryogenesis.
In: Plant molecular biology, pp. 331-342, Goldberg,
R., ed. New York: Liss.

Firn, R.D., Friend, J. (1972) Enzymatic production of the
plant growth inhibitor, xanthoxin. Planta 103:263-266.

Fong, F., Koehler, D. E. Smith, J D. (1983a) Fluridone
induction of vivipary during maize seed development.
In: Third international symposium on pre- harvest sprout-
ing in cereals, pp. 186—196, Krueger, J.E., La Berge,
D.E., eds. Boulder: Westview Press.

Fong, F., Smith, J.D., Koehler, D.E. (1983b) Early events
in maize seed development. 1- methyl- -3- phenyl-S-(3-[tri-
fluoromethyl]phenyl)- -4- (1H) pyridinone induction of
vivipary. Plant Physiol. 73. 899— 901.

Gillard, D.F., Walton, D.C. (1976) Abscisic acid metabolism
by a cell-free preparation from Echinocystis lobata
liquid endosperm. Plant Physiol. 58:790-795.

Gooday, G. W. (1974) Fungal sex hormones. Ann. Rev. BioJ
chem. 43: 35- 49 ‘

Gooday, G. W. Jones, B. E. Leith, W.H. (1978) Trisporic
acid and the differentiation in the Mucorales. In: Re—
gulation of secondary product and plant hormone metabol-
ism, pp. 221-229, Luckner, M., Schreiber, K., eds. New
York: Pergamon Press. ,

Harrison, M.A., Walton, D.C. (1975) Abscisic acid metabol-
ism in water-stressed bean leaves. Plant Physiol. 56:
250-254.

Hartung, W. (1983) The site of action of abscisic acid at the
guard cell plasmalemma of Valerianella locusta. Plant,
Cell, and Environ. 6:427-428.

Hartung, W., Heilmann, B., Gimmler, H. (1981) Do chloro-
plasts play a role in abscisic acid synthesis? Plant
Sci. Lett. 22:235-242.

Henson, I.E. (1981a) Abscisic acid and after-effects of
water stress in pearl millet (Pennisetum americanum
(L.) Leeke). Plant Sci. Lett. 21:129-135.

Henson, I.E. (1981b) Changes in abscisic acid content
during stomatal closure in pearl millet (Pennisetum
americanum (L.) Leeke). Plant Sci. Lett. 21:129-135.

28

Higgins, T.J.V., Jacobsen, J.V., Zwar, J.A. (1982) Gibber-
ellic acid and abscisic acid modulate protein synthesis
and mRNA levels in barley aleurone layers. Plant Mol.
Biol. 1:191-215.

Hirai, N., Fukui, H., Koshimizu, K. (1978) A novel abscisic
acid metabolite from seeds of Robina pseudacacia. Phyto—
chemistry 17:1625—1627

Ho, T.H.D., Varner, J.E. (1976) Response of barley aleurone
layers to abscisic acid. Plant Physiol. 57:175-178.

Ho, T.H.D., Nolan, R.C., Uknes, S.J. (1985) On the mode of
action of abscisic acid in barley aleurone layers. In:
Current topics in plant biochemistry and physiology
1985, pp. 118-125, vol. 4, Randall, D.D., Blevins,

D.C., Larson, R.L., eds. Columbia, MD: University of
Missouri.

Horgan, R., Neill, S.J., Walton, D.C., Griffin, D. (1983)
Biosynthesis of abscisic acid. Biochem. Soc. Trans.
11:553-557.

Ichimura, M., Oritani, T., Yamashita, K. (1983) The metabol-
ism of (22, 4E)-a-ionylideneacetic acid in Cercospora
cruenta, a fungus producing (+)-abscisic acid. Agr.
Biol. Chem. 47:1895-1900.

Ihle, J.N., Dure, L. (1970) Hormonal regulation of transla-
tion requiring RNA synthesis. Biochem. Biophys. Res.
Comm. 38:995—1001.

Imber, D., Tal, M. (1970) Phenotypic reversion of flacca, a
wilty mutant of tomato, by abscisic acid. Science:
169:592-593.

Koornneef, M., Jorna, M.L., Brinkhorst-van der Swan, D L.C.,
Karssen, C.M. (1982) The isolation of abscisic acid (ABA)
deficient mutants by selection of induced revertants in
non-germinating gibberellin sensitive lines of Arabidop-
sis thaliana (L.) Heynh. Theor. Appl. Genet. 61:385-393.

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.

Loomis, W.D., Croteau, R. (1980) Biochemistry of terpen—
oids. In: The biochemistry of plants, a comprehensive
treatise, pp. 363-418, vol 4, Stumpf, P.K., Conn, E.E.,
eds. New York: Academic Press.

Loveys, B.R., Milborrow, B.V. (1984) Metabolism of abscisic

29

acid. In: The biosynthesis and metabolism of plant

hormones, pp. 71-104, Crozier, A., Hillman, J.R.,

eds. London: Cambridge University Press.

Marumo, S., Katayama, M., Komori, E., Ozaki, Y., Natsume,
M., Kondo, S. (1982) Microbial production of abscisic
acid by Botrytis cinera. Agr. Biol. Chem. 46:1967-1968.

Milborrow, B.V. (1969) Identification of ‘metabolite C’
from abscisic acid and a new structure for phaseic
acid. Chem.Commun., pp. 966-967.

Milborrow, 135V. (1972) Stereochemical aspects of the form-
ation of double bonds in abscisic acid. Biochem. J.
128:1135-1146.

Milborrow, B.V. (1974a) Biosynthesis of abscisic acid by a
cell-free system. Phytochemistry. 13:131-136.

Milborrow, B.V. (1974b) The chemistry and physiology of
abscisic acid. Ann. Rev. Plant Physiol. 25:259-307.

Milborrow, B.V. (1978) Abscisic acid. In: Phytohormones and
related compounds-a comprehensive treatise, pp. 295-
397, vol 1, Letham D.S., Goodwin, P.B., Higgins,T.J.
V., eds. Amsterdam: Elsevier.

Milborrow, B.V. (1983) Pathways to and from abscisic acid.
In: Abscisic acid, pp.79-111, Addicott, F.T., ed. New
York: Praeger Press.

Milborrow, B.V., Robinson, D.R. (1973) Factors affecting
the biosynthesis of abscisic acid. J. Exp. Bot. 24:
537-548.

Mittelheuser, (ZHJ., Van Steveninck, R.F.M. (1969) Stomatal
closure and the inhibition of transpiration induced by
(RS)-abscisic acid. Nature 221:281-282.

Mizrahi, Y., Blumenfeld, A., Richmond, A.E. (1970) Abscisic
acid and transpiration in relation to osmotic root
stress. Plant Physiol. 46:169-171.

Mizrahi, Y., Richmond, A.E. (1972) Abscisic acid in relation
to mineral deprivation. Plant Physiol. 50:667-670.

Moore, l2., Smith, J.D. (1984) Growth, graviresponsiveness,
and abscisic acid content of Zea seedlings treated
with fluridone. Planta 162:342-344.

Moore, R., Smith, J.D. (1985) Graviresponsiveness and
abscisic acid content of roots of carotenoid-deficient
mutants of Zea mays L. Planta 164:126-128.

 

30

Mozer, T.J. (1980) Control of protein synthesis in barley
aleurone layers by the plant hormones gibberellic acid
and abscisic acid. Cell 20:447-455.

Neill, S.J., Horgan, R. (1983) Incorporation of a-ionylidene
ethanol and a-ionylidene acetic acid into abscisic acid
by Cercospora rosicola. Phytochemistry 22:2469—2472.

Neill, S.J., Horgan, R. (1985) Abscisic acid production and
water relations in wilty tomato mutants subjected to
water deficiency. J. Exp. Bot. 36:1222-1231.

Neill, S.J., Horgan, R.t Lee, T.S., Walton, D.C. (1981)
3-methyl-5-(4-oxo-2 ,6',6'-trimethylcyclohex-2 -en-
yl)-2,4-pentadienoic acid, a putative precursor of
abscisic acid from Cercospora rosicola. FEBS Lett.128:30-
32.

Neill, S.J., Horgan, R., Walton, D.C., Lee, T.S. (1982) The
biosynthesis of abscisic acid in Cercospora rosicola.
Phytochemistry 21:61-65.

Nevo, Y., Tal, M. (1973) The metabolism of abscisic acid in
flacca, a wilty mutant of tomato. Biochem. Genet. 10:79-
90.

Noddle, R.C., Robinson, D.R. (1969) Biosynthesis of abscisic
acid: incorporation of radioactivity from [2—1 C]mevalon-
ic acid by intact fruit. Biochem. J. 112:547-548.

Ohkuma, K., Addicott, F.T., Smith, O.E., Thiessen, W.E.
(1965) The structure of abscisin II. Tetrahedron Lett.
29:2529-2535.

Ohkuma, K., Lyon, J.L., Addicott, F.T., Smith, O.E. (1963)
Abscisin 11, an abscission-accelerating substance from
young cotton fruit. Science 142:1592-1593.

Oritani, T., Ichimura, M., Yamashita, K. (1982) The metabol-
ism of (2Z,4E)-a-ionylideneacetic acid in Cercospora
cruenta, a fungus producing (+) abscisic acid. Agr.
Biol. Chem. 46:1959-1962.

Pierce, M., Raschke, K. (1980) Correlation between loss of
turgor and accumulation of abscisic acid in detached
leaves. Planta 148:174-182.

Porter, N.G., Van Steveninck, R.F.M. (1966) An abscission-
promoting factor in Lupinus luteus L. Life Sci.
522301-2308.

Quarrie, S.A. (1982) Droopy: a wilty mutant of potato
deficient in abscisic acid. Plant Cell Environ. 5:23-26.

31

Raikhel, N.V., Palevitz, B.A., Haigler, C.H. (1986) Abscisic
acid control (if lectin accumulation in wheat seedlings
and callus cultures. Plant Physiol. 80:167-171.

Raschke, K. (1975) Stomatal action. Ann. Rev. Plant Physiol.
26:309-340.

Raschke, K., Zeevaart, J.A.D. (1976) Abscisic acid content,
transpiration, and stomatal conductance as related to

leaf age in plants of Xanthium strumarium L.. Plant
Physiol. 58:169-174.

Raschke, K., Firn, R.D., Pierce, M. (1975) Stomatal closure
in response to xanthoxin and abscisic acid. Planta
125:149-160.

Raschke, K., Pierce, M., Popiela, C.C. (1976) Abscisic acid
content and stomatal sensitivity to C02 in leaves of
Xanthium strumarium L. after pretreatments in warm and
cold growth chambers. Plant Physiol. 57:115-121.

Robinson, D.R., Ryback, G. (1969) Incorporation of tritium
from: [(4)-4-3H] mevalonate into abscisic acid. Biochem.
J. 113:895-897.

Robinson, P.M., Wareing, P.F. (1964). Chemical nature and
biological prOperties of the inhibitor varying with
photoperiod in sycamore (Acer pseudopIatanus). Physiol.
Plant. 17:314-323.

Robertson, 0.5. (1975) Survey of the albino and white-endo-
sperm mutants of maize. J. Heredity 66:67-74.

Rothwell, K., Wain, R.L. (1964) Studies on a growth inhibitor
irI yellow lupin (Lupinus luteus L.). In: Régulateurs de
la Croissance Végétale, pp. 363-375, Nitsch, J.P.,
ed. Paris: Cent. Nat. Rech. Sci..

Setter, T.L., Brenner, M.L., Brun, W.A., Krick, T.P. (1981)
Identification (If a dihydrophaseic acid aldopyranoside
from soybean tissue. Plant Physiol. 68:93-95.

Shimazaki, K., Iino, M., Zeiger, E. (1986) Blue light-depend-
ent extrusion by guard-cell protoplasts of Vicia faba.
Nature 319:324-326.

Smith, J.D., McDaniel, S., Lively, S. (1978) Regulation of
embryo growth by abscisic acid in vitro. Maize Genet.
Coop. News Lett. 52:107-108.

Sondheimer, E., Galson, E.C., Chang, P.P., Walton, D.C.
(1971) Asymmetry, its importance to the action and
metabolism of abscisic acid. Science 174:829-831.

 

32

Sondheimer, E., Galson, E.C., Tinelli, E., Walton, D.
(1974) The metabolism of hormones during dormancy. IV.
The metabolism of (S)-2-14C-abscisic acid in ash seed.
Plant Physiol. 54:803-808.

Stubbe, H. (1957) Mutanten der Kulturtomate, Lycopersicon
esculentum Miller I. Kulturpflanze 5:190-220.

Stubbe, H. (1958) Mutanten der Kulturtomate, Lycopersicon
escuIentum Miller 11. Kulturpflanze 6:89-115.

Stubbe, H. (1959) Mutanten der Kulturtomate, Lycopersican
esculentum Miller 111. Kulturpflanze 7:82-112.

Sussex, 1. (1975) Growth and metabolism of the embryo and
attached seedling of the viviparous mangrove Rhizophora
mangle. Am. J. Bot. 62:948-953.

Tal, M., Imber, D., Erez, A., Epstein, E. (1979) Abnormal
stomatal behavior and hormonal imbalance in flacca, a
wilty mutant of tomato. V. Effect of abscisic acid on
indoleacetic acid metabolism and ethylene evolution.
Plant Physiol. 63:1044-1048.

Tal, M., Nevo, Y. (1973) Abnormal stomatal behavior and root
resistance, and hormonal imbalance in three wilty mutants
of tomato. Biochem. Genet. 8:291-300.

Tamura, 53., Nagao, M. (1970) Synthesis and biological
activities <rf new plant growth inhibitors structurally
related to abscisic acid. Agr. Biol. Chem. 34:1393-1401.

Taylor, H.F., Burden, RHS. (1970) Identification of plant
growth inhibitors produced by photolysis of Violaxan-
thin. Phytochemistry 9:2217-2223.

Taylor, H.F., Burden, R.S. (1972) Xanthoxin, a recently
discovered plant growth inhibitor. Proc. Roy. Soc.
B 180:317-346.

Taylor, ‘Purden, R.S. (1973) Preparation and metabol-
isnIIrf F2-1 C cis,trans xanthoxin. J. Exp. Bot. 24:873-
880.

Taylor, H.F., and Smith, T.A. (1967) Production of plant
growth inhibitors from xanthophylls: A possible source
of dormin. Nature 215:1513-1514.

Tucker, I)HJ., Mansfield, T.A. (1971) A simple bioassay for
detecting "antitranspirant" activity of naturally
occurring compounds such as abscisic acid. Planta 98:157-
163.

Walton, D.C. (1980) Biochemistry and physiology of abscisic

33
acid. Ann. Rev. Plant Physiol. 31:453-489.

Wang, T.L., Donkin, M.E., Martin, E.S. (1984) The physio—
logy of wilty pea: abscisic acid production under water
stress. J. Exp. Bot. 35:1222-1232.

Wareing, P.F. (1978) Abscisic acid as a natural growth reg-
ulator. Phil. Trans. Roy. Soc. Lond. B 284:483-498.

Wareing, P.F., Eagles, C.F., Robinson, P.M. (1964) Natural
inhibitors as dormancy agents. In: Régulateurs de la
Croissance Végétale, pp. 377-386, Nitsch, J.P., ed
Paris: Cent. Nat. Rech. Sci..

Wilkins, M.B. (1978) Gravity sensing guidance mechanisms in
roots and shoots. Bot. Mag. Tokyo Spec. Issue 1:255-277.

Wilkins, M.B. (1984) Gravitropism. In: Advanced plant
physiology, pp.163-185, Wilkins, M.B., ed. London:
Pitman.

Wright, S.T.C. (1972) Physiological and biochemical responses
to wilting and other stress conditions. In: Crop process-
es in controlled environments, pp. 349-361, Rees, A.R.,
Cockshull, K.E., Hand, D.W., Hurd, R.G., eds. New
York: Academic Press.

Zeevaart, J.A.D. (1974) Levels of (+)-abscisic acid and
xanthoxin in spinach under different environmental
conditions. Plant Physiol. 53:644-648.

Zeevaart, J.A.D. (1977) Sites of abscisic acid synthesis
and metabolism in Ricinus communis L. Plant Physiol.
57:788-791.

Zeevaart, J.A.D. (1980) Changes in the level of abscisic
acid and its metabolites in excised leaf blades of
Xanthium strumarium during and after water stress.
Plant Physiol. 66:672-678.

Zeevaart, J.A.D., Milborrow, B.V. (1976) Metabolism of
abscisic acid and the occurrence of epi-dihydrophaseic
acid in Phaseolus vulgaris. Phytochemistry 15:493-500.

 

CHAPTER 2
INHIBITION OF ABSCISIC ACID ACCUMULATION IN WATER-
STRESSED CORN SEEDLINGS IN THE PRESENCE OF CAROTENOID
BIOSYNTHETIC INHIBITORS

34

35
2.1. INTRODUCTION

If ABA is synthesized via the indirect pathway, then
inhibition (if carotenogenesis with specific inhibitors
should also prevent the accumulation of ABA. Pyridinone
(sucd1.as fluridone, 1-methyl-3-phenyl-5-[3-(trifluoro-
methyl)phenyl]-4(1H)pyridonone) and pyridazinone (such as
norflurazon, also known as SAN-9789, 4-chloro-5-(methyl-
amino)-2(aggna-trifluoro-m-tolyl)-3-(2H)pyridazinone) herbi-
cides block the desaturation of phytoene to phytofluene, the
first two colorless carotenoids (Bartels and Watson, 1978).

These inhibitors also have a number of effects aside
from inhibiting carotenoid synthesis. When plant tissue
lacking carotenoids is grown irI light, chlorophyll, chloro-
plast proteins, and plastid ribosomal RNA ch) not accumulate
(Bartels and Watson, 1978; Quarrie and Lister, 1984). In
addition, alterations in the fatty acid composition of
galactolipids also occur (St. John, 1976). How these alter-
ations in plant metabolism might affect ABA accumulation is
unknown. These non-specific effects require the measurement
of primary metabolites to determine if they might be in-
directly affecting ABA accumulation.

'The effect of these inhibitors on ABA accumulation was
determined using seedlings of Zea mays. Sugar (soluble and
insoluble) and organic acid levels were also measured. This
was done to determine if the inhibitors had a non-specific

effect on primary cell metabolism.

36
2.2. MATERIALS AND METHODS

2.2.1. Plant Material. Seeds of corn (Zea mays, hybrid MS
WFg x Bear 38, Custom Farm Seed, Decatur, IL, gift of
Dr. K. Poff) were imbibed overnight in solutions of norflura-
zon, fluridone or distilled water. In most experiments the
inhibitor concentration was 10'4 M. They were then planted in
vermiculite and watered with the appropriate solutions. Flats
were kept in darkness, except for a daily 2 h period when red
light was given to inhibit mesocotyl growth. After 9 to 10 d,
seedlings were harvested by clipping just above soil level.
For experiments involving measurement of ABA, seedlings were
either stressed until they lost 13% of their fresh weight,
followed by storage in a plastic bag for 8 h, or placed
immediately in a plastic bag. For analysis of primary
cellular metabolites leaves were frozen immediately in liquid
nitrogen (organic acids or sugars), or extracted in methanol
(carotenoids). All experiments were repeated at least once,
with two replicates per treatment.

2.2.2. Abscisic Acid Analysis. After the experimental
period, the tissue was frozen and lyophilized. Extraction
and quantitation were performed exactly as described in
Zeevaart (1980).

2.2.3. Organic Acid Analysis. The procedure used was basic-
ally that of Stumpf and Burris (1979). Approximately 1 g of
tissue was frozen, lyophilized, and powdered. This powder was

placed in a 50 ml Erlenmyer flask to which 15 ml 95% ethanol

37
was added. The ethanolic solution was boiled for 10 min. The
extract was centrifuged and the supernatant was taken to
dryness. The residue was redissolved in one nfl 11f distilled
water.

The extract was then subjected to ion exchange chromato-
graphy. Dowex 1 and 50 (Dow Chemical Co.) were both washed
with acetone, methanol, and toluene to remove colored
contaminants. Dowex 1 was converted to the formate form by
washing with 1 N formic acid until the pH of the eluate was
less than 2. The resin was back-washed with distilled water
until the {Hi was greater than 5. Dowex 50 was used as
provided by the manufacturer.

An aliquot (0.3-0.5 ml) of the aqueous residue was
applied to a Dowex 50 column (H+, bed volume 0.3 ml) on top
of a Dowex 1 column (formate, bed volume 0.1 ml). The Dowex
50 column was washed with 2 ml of distilled water, removed,
and the Dowex 1 column similarly treated. The organic acids
were eluted from the Dowex 1 column with 4 ml 2 N HCl and
dried. They were then redissolved in 400 pl distilled
water, transferred to a small vial, and dried.

The samples were resuspended in 50 pl pyridine to which
25 pl BSA was added 10 min prior to analysis. GLC-FID
analysis was performed with a 3% SE-30 (2 m, 80-120 mesh,
gas-chrom Q, 30 ml/min He flow) temperature programmed from
80 C to 240 C at 5 C per min. Quantitation was performed by
comparison 111th known amounts of malonic, succinic, malic,

and citric acids.

38

2.2.4. Soluble and Insoluble Sugar Analysis. The procedure
used was basically that of McCready et a7. (1950). Lyophil-
ized leaves were finely powdered and extracted four times
with 25 ml of hot 80% ethanol to give the soluble sugar
fraction. Distilled water (5 ml) was added to the residues,
followed with, after cooling in an ice bath, 6.5 ml of 53%
perchloric acid. The mixture was stirred for 20 min after
which 20 ml of distilled water-was added. The mixture was
next centrifuged. This was repeated once and the supernatants
combined to give the insoluble carbohydrate fraction. Both
fractions were lyophilized to dryness and known amounts of
distilled water were added.

Aliquots were analyzed by the phenol-sulfuric acid method
(Ashwell, 1966). To 2 ml of the sugar solution was added 50
pl 80% phenol followed by 5 ml of sulfuric acid. After 15
min, the absorbance was read at 488 nm. Quantitation was
performed by construction of a standard curve with known
amounts (if sucrose. To take into account water lost during
the biosynthesis of starch, the value obtained from the
standard curve was multiplied by 0.9.

2.2.5. Carotenoid Analysis. The procedure used was that
described in Davies (1976). After harvesting, leaves were
immediately extracted in methanol. Following filtration,
the filtrate was added to an equal volume of diethyl ether
to whicfli three volumes of aqueous 20% NaCl were added. The
diethyl ether phase was washed three times with distilled

water and dried over Na2504. The ether was removed by

39

rotary evaporation and the residue dissolved in methanol to
which a solution of 60% KOH in methanol was added to give a
final concentration of 6% KOH in methanol. The solution was
placed under‘Iritrogen and stored in the dark at 4 C over-
night. The saponified extract was added to an equal amount of
diethyl ether to which three volumes of aqueous 20% NaCl was
added. The ether phase (containing carotenoids) was washed
four times vrtth distilled water and the aqueous phase
(containing degraded chlorophyll) was discarded.

If total carotenoids were to be determined, suitable
aliquots were analyzed according to Davies (1976). If
phytoene was to be measured, a small aliquot was subjected
to silica gel TLC using hexanezacetone 9:1 as solvent. The
zone containirm; phytoene (Rf 0.97), which fluoresces under
UV as a weak violet zone, was eluted with hexane and quanti-

fied according to Davies (1976).

2.3. RESULTS

2.3.1. Carotenoid, Organic Acid, and Sugar Levels in Control
and Inhibitor Treated Corn Seedlings. Seedlings grown in the
Presence of inhibitor concentrations greater than 10‘3 M were
Stunted and generally had an abnormal appearance. At concen-
trations less than 10'6 M seedlings had the same appearance
as control plants. At a concentration of 10'4 M seedlings
were slightly shorter than control plants (Figure 2.1), but

were suitable for experimental use. Both inhibitors caused a

 

4O

CONTROL 10" M

NORFLURAZON

 

Figure 2.1. Corn seedlings watered with either nutrient

solution or 10'4 M norflurazon.

 

41

decrease in total carotenoid content and increased phytoene
levels at a concentration of 10'4 M compared with control
seedlings (Table 2.1). Organic acid levels were increased in
the inhibitor treated plants (Table 2.2), indicating that the
tricarboxylic acid cycle was perturbed. Insoluble carbohy-
drates (starch) were slightly decreased, whereas soluble free
sugars were slightly increased (Table 2.3) in seedlings grown
in the presence of inhibitors.

2.3.2. Effects of Carotenoid Biosynthetic Inhibitors on
Abscisic Acid Accululation. ABA levels in turgid, inhibitor
treated tissue, were decreased compared to green tissue
(Table 2.4). In addition, the ability to accumulate water-
stress-induced ABA was reduced in inhibitor-grown seed-

lings (Table 2.4).

2.4. DISCUSSION

2.4.1. Inhibition of Abscisic Acid Accumulation by Norflur-
azon and Fluridone. The results presented above show that the
accumulation of ABA was drastically reduced when corn
seedlings were grown in the presence of inhibitors of
carotenoid biosynthesis. This result has been observed by
other workers (pearl millet, Henson, 1984; barley, Quarrie
and Lister, 1984; corn, Moore and Smith, 1984). Interest-
ingly, fluridone induces vivipary when applied to developing
maize seeds 11 d after pollination (Fong et al., 1983a; Fong
et al., 1983b).

42

Table 2.1. The effect of norflurazon and fluridone on
levels of phytoene and total carotenoids in corn seedlings.
The data presented are from different experiments, and
are the average of two replicates per treatment. The values

in parenthesis are the per cent of control.

Control Norflurazon Fluri one
(10'4 M) (10- M)

ug/g fresh wt.
Phytoene 25(100) 63(252) 68(672)

Total carotenoids 62(100) 4.1(7) 0.8(2)

Table 2.2. The effect of norflurazon and fluridone on
organic acid levels in corn seedlings.
The data presented are from different experiments, and

are the average of two replicates per treatment.

Organic acid Control Norflurazon Fluridone
(10-4 M) (10-4 M)

pg/g fresh wt.

Malonic 21 nd* 63
Succinic 75 92 360
Malic 790 1890 2430
Citric 1000 1230 1520

* not detected

43

Table 2.3. The effect of norflurazon and fluridone on
levels of soluble and insoluble sugars in corn seedlings.
The data presented are from different experiments, and

are the average of two replicates per treatment.

Sugar Control Norflurazon Fluri one
(10‘4 M) (10' M)
""""""""""""""""" igig'iéééi'iif""""""'
Soluble 26 38 54
Insoluble 5.7 3.3 2.8

Table 2.4. The effect of norflurazon and fluridone on ABA
levels in corn seedlings. J
The data presented are from different experiments, and
are the average of two replicates per treatment.

pg/g fresh wt.

Control
Turgid 15
Stressed 307
Norflxrazon
(10' M)
Turgid l
Stressed 15
Fluri one
(10' M)
Turgid 0.5

Stressed 13

44

When green leaves (which already have a large carotenoid
pool) were treated with norflurazon, stress induced ABA
accumulation was not inhibited (Henson, 1984). In green bean
leaves labeled with 14C02 and then water-stressed, the
specific activities of ABA and xanthophylls were similar on
a per carbon basis. However, when green leaves of bean were
treated with norflurazon prior to 14C02 labeling, the
specific activities of ABA and xanthophylls were reduced to
the same extent, but total ABA biosynthesis was not affected
(Walton et al., 1985). Thus, the effect of norflurazon on ABA
accumulation in green leaves must be on the biosynthesis of
new carotenoids. These results (Henson, 1984; Walton et al.,
1985) are consistent with the idea that ABA arises from a
pool of carotenoids.
2.4.2. Non-Specificity of Carotenoid Biosynthetic Inhibi-
tors. Clearly, the experiments described in section 2.4.1
are strong evidence for the indirect pathway of ABA biosynth-
esis. However, the fact that these inhibitors have strong
effects on primary metabolism imply that the conclusions
should be interpreted cautiously with regard to ABA accumula-
tion. In addition to being grown in the presence of in-
hibitors, the seedlings were also grown in darkness to avoid
photo-bleaching. Because of these factors, the primary
metabolism of corn seedlings grown under these conditions is
different from plants grown under normal conditions. These
facts lead to the conclusion, that while the inability to

accumulate ABA is associated with carotenoid deficiency, it

45
cannot be separated from an indirect effect of altered
primary metabolism on ABA biosynthesis.

Quarrie and Lister (1984) used a mutant of barley (albos-
trians) which has green, chimeric, and white leaves, and
norflurazon-treated barley plants in studies dealing with ABA
biosynthesis. These workers concluded that plastid ribosomes
were needed for stress ABA and that the enzymes for ABA
biosynthesis are encoded in nuclear DNA (Quarrie and Lister,
1984). They stated that the decreased ability to accumulate
stress-induced ABA in the mutant and norflurazon treated
plants was not due to decreased levels of NADPH, ATP, or
carbohydrate because dark—grown etiolated plants accumulated
ABA just as well as green plants. However, it should be noted
that functional mitochondria are present in etiolated plants,
and these organelles could substitute for chloroplasts with
regard to energy production.

Quarrie and Lister (1984) also state that small amounts
of ABA are produced during stress in the absence of plastid
ribosomes, indicating that the nuclear genome codes for
enzymes in ABA biosynthesis. However, the data they present
are barely above the detection limit and they mention that
there was a impurity which co-chromatographed with MeABA
which made quantitation difficult. Since the data were
obtained with the mutant albostrians the variability observed
could simply represent ABA that was transported into the leaf

before the stress period began.

46
2.5. LITERATURE CITED

Ashwell, G. (1966) New colorimetric methods of sugar anal-
ysis. Methods Enzymol 8:85-95.

Bartels, P.G., Watson, C.W. (1978) Inhibition of carotenoid
synthesis by fluridone and norflurazon. Weed Sci. 26:
198-203.

Davies, B.H. (1976) Carotenoids. In: Chemistry and biochem-
istry of plant pigments, pp. 38-165, vol. 2, Goodwin,
T.W., ed. New York: Academic Press.

Fong, F., Koehler, D.E., Smith, J.D. (1983a) Fluridone
induction of vivipary during maize seed development. In:
Third international symposium on pre-harvest sprouting in
cereals, pp. 186-196, Krueger, J.E., La Berge, D.Eq
eds. Boulder: Westview Press.

Fong, F., Smith, J.D., Koehler, D.E. (1983b) Early events
in maize seed development. 1-methyl-3—phenyl-5-(3[tri-
fluromethyl]phenyl)-4-(1H)-pyridinone induction of
vivipary. Plant Physiol. 73:899-901.

Henson, I.E. (1984) Inhibition of abscisic acid accumula-
tion in seedling shoots of pearl millet (Pennisetum
americanum [L.] Leeke) following induction of chloro-
sis by norflurazon. Z. Pflanzenphysiol. 114:35-43.

McCready, R.M., Guggolz, J., Silviera, V., Owens, H.S.
(1950) Determination of starch and amylose in vegeta-
bles. Anal. Chem. 22:1156-1158.

Moore, R., Smith, J.D. (1984) Growth, graviresponsiveness
and abscisic-acid content of Zea mays seedlings
treated with fluridone. Planta 162:342-344.

Quarrie, S.A., Lister, P.G. (1984) Evidence of plastid
control of abscisic acid accumulation in barley
(Hordeum vulgare L.). Z. Pflanzenphysiol. 114:295-308.

St. John, J.B. (1976) Manipulation of galactolipid fatty
acid composition with substituted pyridazones. Plant
Physiol. 57:38-40.

Stumpf, D.K., Burris, R.H. (1979) A micromethod for the
purification and quantification of organic acids of
the TCA cycle. Anal. Biochem. 95:311-315.

Walton, D.C., Li, Y., Neill, S.J., Horgan, R. (1985)
Biosynthesis of abscisic acid: a progress report.
In: Current topics in plant biochemistry and physio-
logy 1985, pp. 111-117, vol. 4, Randall, D.D.,

47

Blevins, D.G., Larson, R.L., eds. Columbia, MO: Uni
versity of Missouri.

Zeevaart, J.A.D. (1980) Changes in the level of abscisic
acid and its metabolites in excised leaf blades of
Xanthium strumarium during and after water stress.
Plant Physiol. 66:672-678.

CHAPTER 3
BIOLOGICAL ACTIVITY AND METABOLISM OF COMPOUNDS
STRUCTURALLY RELATED TO ABSCISIC ACID

48

 

49
3.1. INTRODUCTION

Regardless of whether ABA is synthesized via the direct
or indirect pathway, the immediate precursor(s) to ABA must
be a C15 compound. I decided to synthesize three compounds
structurally related to ABA, a-ionylidene acetic acid, 1’,2'-
epoxy-ionylidene acetic acid, and 1',2’-epoxy-ionylidene
acetaldehyde, to determine if any of them would serve as a
precursor to ABA.

One of these compounds, a-ionylidene acetic acid, has
been shown to be a precursor to ABA in fungi (Neill and
Horgan, 1983; Oritani et al., 1982; Ichimura et al., 1983).
In several bioassays, this compound has greater biological
activity than ABA (Walton, 1983). However, it has been shown
that many plants can catabolize a-ionylidene acetic acid to
1'-deoxy-ABA and conjugates, but not to ABA (Lehmann and
Schfitte, 1976). Only with Vicia faba has any conversion to
ABA been demonstrated (Walton et al., 1985). Lack of convers-
ion in many other plants could be due to a failure of the
compounds to reach the proper compartment for metabolism to
ABA. It could also be that the results with V. faba are
anomalous because an enzyme is present in this plant that is
lacking in others.

The only C15 compounds known to be converted to ABA
are 1',2'-epoxy ionylidene acetic acid (Milborrow and
Noddle, 1970) and xanthoxin (Taylor and Burden, 1973). How-

ever, in both cases only 14C incorporation into ABA was

 

50
demonstrated instead of an unequivocal proof showing heavy-
isotope incorporation by GC-MS.

In some bioassays (such as stomatal closure, rice
seedling growth, and lettuce hypocotyl growth) 1',2'-epoxy-
ionylidene acetic acid had less biological activity than
ABA" Inhereas 111 others (lettuce seed germination and Avena
coleoptile growth) it had the same or greater activity
(Walton, 1983), depending (Hi the concentration used. Xan-
thoxin was highly active in all biological assays, except
when stomatal closure in epidermal strips was tested (Walton,
1983). Higher plants can convert 1',2'-epoxy ionylidene
acetic acid to xanthoxin acid and ABA and other unknown
catabolites (Milborrow and Noddle, 1970; Milborrow and Garms-
ton, 1973). Similar results have been obtained with the
fungus C. cruenta (Oritani and Yamashita, 1985). I decided to
synthesize this compound and confirm that it is indeed
converted to ABA.

Since the chemical synthesis of xanthoxin is very
laborious and difficult (Kienzle et al., 1978), I decided to
synthesize 11 compound very similar to both 1',2'-epoxy-io-
nylidene acetic: acid and xanthoxin, namely 1',2'-epoxy-io-
nylidene acetaldehyde. This compound is identical to xanthox-
in except that there is no hydroxyl group at C4'. Since it is
known that higher plants can insert a hydroxyl group at this
position with a-ionylidene type structures (Lehmann and
Schfitte, 1976; Walton et al., 1985), it seemed reasonable to

assume that this would occur when 1',2'-epoxy-ionylidene

51
acetaldehyde was fed. If xanthoxin was made, further metabol-
ism to ABA would occur. Incorporation of tritium and deuter-
ium into this compound would allow for purification by HPLC

and subsequent identification by GC-MS.

3.2. MATERIALS AND METHODS

3.2.1. Synthesis of 2H Labelled a-Ionone (V, Figure 3.1).
Deuterium was exchanged into a-ionone by dissolving a-ionone
(8.8 g, 25 mmol) and 2H20 (1 ml, 99% atom excess) in 12 ml of
d5-acetone and adding about 1 mg of Na metal. After stirring
at room temperature for 24 h the acetone was removed with the
aid of a rotary evaporator. Chloroform (2 ml) was added and
the mixture was partitioned against 3 ml 2H20 twice and 3 ml
H20 twice. Analysis by NMR and GC-MS indicated that the
deuterium was located in the side chain methyl group of
a-ionone (V) and had an isotopic composition, [2H0]:[2H1]:
[2H2]:[2H3], of 2:3:21z75 (2.69 deuterium/molecule).
3.2.2. Synthesis of 2H and 3H Labelled B-Ionone. Tritium
was exchanged into B—ionone in a custom synthesis by Amersham
Corporation by a method similar to that used by Walton et
a]. (1977). The exchange was performed by mixing B-ionone,
alumina, anisole, and 3H20 (25 C1) for 25 min at 120 C.
Deuterium was exchanged into B-ionone in a manner
identical to that done with a-ionone. NMR and GC-MS analysis
indicated that the deuterium was located in the side chain

methyI group of B-ionone with an isotopic composition of

 

Figure 3.1.

52

0
Na
2H,o
CH,-
\ \o \
ne‘-
c211,
\ \0

Synthesis of deuterated a-ionone.

53

1:200:180028000 (2.77 deuterium/molecule).

3.2.3. Synthesis of a-Ionylidene Acetic Acid (X, Figure
3.2). A mixture of deuterated a-ionone (V, 2 g, 12.5 mmol)
and carbethoxymethylenetriphenylphosphorane (V1, 8.8 g, 25
mmol) was heated at 170 C for 90 min. After cooling, the
red viscous product was triturated with hexane and stored
at 20 C overnight. Any triphenylphosphine oxide (VII) which
precipitated was removed by filtration. The filtrate was
concentrated to an oil by rotary evaporation. GLC—FID
analysis (3% SP-2100, 140 C to 240 C at 5 C/min, 30 ml/min
He flow) of the crude mixture indicated a 1:2 ratio of
cis,trans- (VIII, retention time 4.96 min) and trans,trans-
ethyl a-ionylidene acetate (IX, retention time 5.61 min).

The isomers were separated by silica gel chromatography
yielding 110 mg pure cis,trans- (VIII) and 260 mg trans,trans
ethyl a-ionylidene acetate (IX); both compounds were light
yellow oils. The cis,trans isomer was saponified overnight in
1 ml 10% KOH/methanol. After acidification with 6 N HCl a
white flocculate resulted which was partitioned into diethyl
ether. The solvent was removed with a stream of nitrogen to
give 96 mg of pure cis,trans-a-ionylidene acetic acid (X,
3-methyl-5—(2',6',6'-trimethyl-2'-cyclohexen-1'-yl)—cis,
trans-2,4-pentadienoic acid).

Methylation of a small aliquot of X with ethereal diazo-
methane gave a single peak with GLC-FID. The fragmentation
pattern of X obtained by GC-MS was similar to that of Neill

and Horgan (1983), except that no molecular ion was present

 

 

 

\ \o - -
-F (Ph)3P=CHCOOC2H5
V VI
noc
\ \
(Ph)3P=0 + .-
- cooc H
VII . 2 5
. . VIII
+
IX
Wittig Reaction
i.KOH MeOH ' - \ \
VIII . /c _ > .
2°” ‘ . _ coow
X

Hydrolysis, Formation of Free Acid

Figure 3.2. Synthesis of deuterated a-ionylidene acetic

acid (X).

55

because in this study a higher ionization potential was
used. MS (GC-MS, Figure 3.3), methyl X, m/z (rel. int.):
252(M+,O), 220(M+-32,3), 219(4), 195(4) (M+-56), 194(4),
193(3), 163(15), 162(20), 161(12), 160(8), 136(38), 135(55),
134(32), 133(18), 128(97), 127(100), 126(38), 125(6),
115(28), 114(19), 113(9), 112(5), 108(14), 107(31), 106(14),
105(16). The isotopic composition in the side chain methyl
group was 2:16:41:40 (2.2. deuterium/molecule), indicating
that some exchange occurred during the Wittig reaction
[compare with 2.69 deuterium/molecule in a-ionone (section
3.2.1)].

3.2.4. Synthesis of 1',2'-Epoxy-Ionylidene Acetic Acid
(XV, Figure 3.4). A mixture of deuterated B-ionone (XI,
0.7 g, 3.6 mmol) and carbethoxymethylenetriphenylphos-
phorane (V1, 1.5 g, 4.3 mmol) was heated at 155 C for 2.5
h. The product, a red oil, was triturated with hexane as
described above in section 3.2.6. GLC-FID analysis indicat-
ed that 60% of the B-ionone present was used in the re-
action. The crude mixture of isomers was purified as describ-
ed above (section 3.2.4) to give pure ethyl cis,trans-B-io-
nylidene acetate (XII). UV XII A max nm: 309, 260; MS
(GC-MS, Figure 3.5), XII, m/z (rel. int ): 265(M+,57),
264(42), 263(14), 262(2), 250(M+-15,13), 249(12), 248(5),
247(5), 236(8), 235(6), 234(2), 233(1), 220(34), 219(33),
218(15), 217(34), 204(32), 203(25), 202(9), 201(5), 181(11),
180(12), 179(12), 178(13), 177(26), 176(59), 175(54),
174(20), 173(9), 165(18), 164(38), 163(47), 162(68), 161(57),

56

 

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57

\ \
0 4. (Ph)p=c1-Icooc,I-I,
XI VI
155C
\ \
(Pthzo + w
cooc H
VII XII 2 ‘

Wittig Reaction

c1
//° \ \
XII + coow—> o
cooczH,
XIII XIV

Insertion of Epgxide

1. KOH/MeOH \ \

XIV 2.1—1c1 7

 

COOH
XV

Hidrolvsis. Formation of Free Acid

Figure 3.4. Synthesis of deuterated I’,2'-epoxy-ionyl-
idene acetic acid (XV).
In both XI and XV the deuterium is located in the side

chain methyl group.

 

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59

160(31), 159(16), 125(6), 124(18), 123(31), 122(100),
121(95), 120(49), 119(46). The side chain methyl group had
an isotopic composition of 2:12:37:50 (2.3 deuterium/mol-
ecule), indicating that some exchange of deuterium present in
the precursor B-ionone occurred during the Wittig reaction
[compare lefli 2.77 deuterium/molecule in B-ionone (section
3.3.2)].

The epoxide was inserted by adding dropwise m-chloro-
perbenzoic acid (XIII, 322 mg in 1.5 ml CHZClZ, 1 M) to
ethyl cis,trans-B-ionylidene acetate (XII, 396 mg in 1.5 ml
CHZClZ, 1 M) cooled in an ice bath. After stirring at room
temperature for 45 min the reaction mixture was washed
sequentially with 10% sodium bisulfite, water, 10% sodium
bicarbonate, and finally water. The solvent was removed
with :1 stream of nitrogen. GLC-FID analysis indicated 100%
conversion of in”; starting material into ethyl cis,trans-
1',2'-epoxy-ionylidene acetate (XIV). The ester was sapon-
ified as described above in section 3.2.7 to give cis,
trans-1',2'-epoxy-ionylidene acetic acid (XV; 3-methyl-
5-(1',2'-epoxy-2',6',6'-trimethyl-1'-cyclohexyl)-cis,
trans-2,4-pentadienoic acid). UV XV A max nm: 266; methyl
XV: 266; «ethyl XV: 266; MS (GC-MS, Figure 3.6), methyl
XV, Im/z (rel. int.): 267(M+,6), 266(1), 252(M+-15,12),
251(5),. 250(2), 249(2), 235(M+-32,18), 234(8), 233(3),
232(1), 224(35), 223(12), 222(4), 221(6), 192(20), 191(12),
190(6), 189(5), 183(15), 182(55), 181(23), 180(12), 179(13),
178(22), 177(27), 176(19), 150(53), 149(38), 148(22),

 

 

 

60

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73.5 699.0332

 

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61

147(10), 126(100), 125(45), 124(29), 123(19), 122(26),
121(20), 120(14), 119(13). There was an isotopic composition
of 10:10:17:63 (2.3 deuterium/molecule) in the side chain
methyl group.

3.2.5. Synthesis of 1',2'-Epoxy-Ionylidene Acetaldehyde
(XVII, Figure 3.7). This compound was first synthesized
without deuterium to provide material for GC-MS and NMR
analysis. A mixture of B-ionone (XI) and carbethoxymethyl-
enetriphenylphosphorane (VI) was reacted and purified
as described in section 3.2.7 to give ethyl cis,trans-B-io-
nylidene acetate (XII). UV XII Amax'nm: 311, 257; MS (GC-MS,
Figure 3.8) XII, m/z (rel. int.): 262(M+,15), 247(M"’-15,6),
233(2), 217(10), 189(8), 173(27), 161(14), 159(29), 145(20),
133(59), 119(100), 107(20), 105(54), 91(59), 77(48), 69(46),
67(22), 55(43).

The epoxide was inserted at the 1',2' double bond as
described ir1 section 3.2.7 in) give ethyl cis,trans-1',2'-
epoxy-ionylidene acetate (XIV). UV XIV Amax nm: 266; MS
(GC-MS Figure 3.9) XIV, m/z (rel. int.): 278(M+,1), 263(M+-
15,4), 235(12), 217(6), 193(16), 174(13), 165(24), 161(31),
159(24), 147(32), 133(29), 123(100), 121(35), 119(33),
105(56), 95(27), 93(36), 91(77), 79(44), 77(65), 69(66),
67(35), 55(45), 55(59), 53(41); 1H NMR: 0.94(3H, s),
1.10(3H,s), 1.18(3H,s), 1.25(3H,s), 1.96(3H,s), 4.05(2H,q),
5.5(1H,s), 6.15(1H,d), 7.3(1H,d).

Ethyl cis,trans-1',2'-epoxy-ionylidene acetate (XIV)
was reduced with LiAlH4 in ether at 0 C for 30 min. After

 

62

\ \ LiAlH, \ \
0 —6c—"" 0
cooc,H, CHpH
XIV XVI

Reduction to Alcohol

XVI ——Z—>M"° o\ \
24 mm
cwo
XVII

Oxidation to Aldehyde

 

Figure 3.7. Synthesis of 1',2‘-epoxy-ionylidene acet-

aldehyde (XVII).

 

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65

ecomposition of the excess LiAlH4 with ethyl acetate and
aturated ammonium chloride, the alcohol (XVI) was par-
itioned into ether. The starting material was converted
00% into cis,trans-1',2'-epoxy-ionylidene alcohol (XVI).
V XVII A max nm: 239; MS (GC-MS, Figure 3.10) XVI, m/z
rel. int.): 218(M+-18,4), 203(M+-33,5), 193(2), 185(1),
75(5), 145(36), 133(23), 123(91), 121(32), 119(31), 109(44),
17(37), 105(57), 95(48), 93(37), 91(66), 85(22), 81(44),
3(58), 77(58), 71(38), 69(100), 67(43), 55(84), 53(51); 1H
4R: 0.95(3H, s), 1.10(3H,s), 1.17(3H,s), 1.90(3H,s),
30(2H,d), 5.55(1H,s), 6.47(1H,s).

A solution of cis,trans-1',2'-epoxy-ionylidene alcohol
IVI) in chloroform was vigorously stirred with active MnOz
repared by the method of Attenburrow et al., 1952) at room
mperature for 24 h. Filtration and removal of solvent gave
e expected product, cis,trans-1’,2'-epoxy-ionylidene
etaldehyde (XVII, 3-methyl-5-(1',2'-epoxy-2’,6',6'—tri-
thyl-1'-cyclohexyl)-cis,trans-2,4-pentadienal). XVII
acted positively with 2,4-dinitrophenyl hydrazine, giving a
llow spot indicative of an aldehyde or ketone function
aylor and Burden, 1970). UV XVII A max nm: 282; MS (GC-MS,
gure 3.11) XVI, m/z (rel. int.): 234(M+,15), 219(MT-15,12),
5(M+-29,6), 201(M+-33,5), 191(8), 161(31), 149(66),
1(45), 105(56), 95(100), 93(37), 91(64), 82(46), 79(46),
(49), 71(16), 69(57), 67(42), 65(29), 55(37), 53(24),
(12); 1H NMR: 1.00(3H,s), 1.15(3H,s), 1.25(3H,s), 2.05(3H,
, 4.00(2H,m), 5.70(1H,d), 6.30(1H,d), 7.20(1H,d), 9.90(1H,

 

 

 

66

 

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68

)euterium labeled XVII was prepared from deuterium
|ed B—ionone by identical methods as described above in

section. MS (GC—MS, Figure 3.12) deuterated XVII, m/z

int.): 237(M+,6), 236(7), 222(M+-15,4), 221(5), 207(4),
9), 193(4), 192(4), 164(16), 163(13), 162(11), 161(10),
‘49), 151(36), 150(14), 149(12), 126(41), 125(26),
33), 123(23), 122(14), 121(14). The isotopic composition
Lhe side chain methyl group was 1:11:38z50 (2.4 deuter-
molecule).
Tritium labeled XVII was prepared from tritium labeled
inone by identical methods as described above. The final
:ific activity of tritiated XVII was 1.6 mCi/mmol.
.6. Spectrometry. Mass spectra were obtained with a
lett-Packard 5985 quadrupole mass spectrometer connected
a Hewlett Packard 5840A gas chromatograph. GLC con-
ions were: 3% SP-2100 on 100-200 mesh Gas Chrom Q in a
anized glass column (2 m x 0.2 cm) temperature pro-
nmed from 120 C to 240 C at 5 C/min. The ionizing poten—
l was 70 eV. To detect incorporation of deuterated
thetic analogues the base peak of ABA (m/z 190) in
ition to m/z 191, 192, and 193 was monitored by GC-

The dwell time for each ion was 140 ms. GLC condi-
ns were as described above, except that the initial
perature was 190 C.

NMR spectra were obtained with a Varian EM-360 NMR (60

) spectrometer. Sample concentration was 5% in CDCl3

.AHH>xV mnazmoamumom

wcmnflfiacofiLflxommli N c , Huang H a m: uwuouousmu mo Esupommm mmm: . NH . m muswfim

Ao\=: omcmco\mmm§

 

 

OVN CNN V CON Gas 00? OVF ONF 00— cm 00 O?
___ _ .3 _. _ _=_ :=_ _ ~=Ei+ 11a :1: 1|. ufi _ .3 _r:fir+
m cm
.0?
.oo
,om
-oo.

 

 

 

(94,) “ml 'le‘a

70

finingl% TMS as internal standard.
JV umctra were obtained with a Perkin-Elmer Lambda 7
IS spectrophotometer. Compounds were dissolved in 95%
01 mm scanned from 340 nm to 200 nm.
'.Plant Material. The metabolism of radioactive com-
5 was investigated with either spinach leaves (Spinacfa
rcea L., cv Savoy Hybrid 612), young tomato shoots
oersicon esculentum L., cv Moneymaker, 20-25 cm tall),
Hts of tomato (breaker stage). Compounds (usually 50 to
g of the deuterated analogues plus 106 dpm of the
ctive compound) were administered to leaves through the
iration stream or injected directly into fruits. Only
‘ated analogues of 1’,2'-epoxy-ionylidene acetic acid
sed in feeds.

Extraction and Purification Procedures. After applica-
F radioactive compounds there was an incubation period
6 h. The tissue was extracted with methanol containing
'L BHT. The extract was reduced to an aqueous residue
ary evaporation, frozen, and lyophilized. A small

was subjected to semi-preparative C18 reverse phase
0% ‘to 80% ethanol in aqueous 1% acetic acid in 1 h,
te 2.5 ml/min). Radioactivity in the column effernt
itoveed with a RadioAnalytic HP Flo-One radioactive
:ector.
oa<:tive peaks from tomato fruit were analyzed further
[Si lica gel, 0.25 mm, hexanezethyl acetate 1:1 (1X

2d) , or hexanezethyl acetate (3X), or hexanezethyl

 

 

71

:etatezacetic acid 123:0.1, (1X)]. One cm zones were removed
1d radioactivity was measured by liquid scintillation count-
ig. To determine if the catabolites were conjugates, small
quuots were subjected to either basic hydrolysis with 2 N
i4OH (2 h, 60 C), or they were treated with pectinase AC
{ohm and Haas, 0.1 M potassium phosphate, pH 4.7). These
"eatments distinguish between ester or glycosidic conjuga-
as, respectively. To determine if a carboxylic acid group
[S present, the Rf before and after treatment with ethereal
azomethane was compared.

To confirm that XV was converted to ABA, ABA from leaves
rd XV was purified as described in chapter 4. In an attempt
1 determine if XV was endogenous in X. strumarfum, leaves
00 g fresh weight) were extracted in methanol. The methanol
5 removed with the aid of a rotary evaporator to an aqueous
sidue to which was added phosphate buffer (1 M, 50 ml, pH
5). The pH of the solution was lowered to 2.5 and parti-
oned four times against petroleum ether (B.R. 40—60 C). In
is system, standard XV was partitioned into the petroleum
her. After removal of petroleum ether, the residue was
ssolved in ethanol to which KOH was added to give a final
ncentration of 6% KOH. The saponified extract was kept at 4
overnight. Distilled water was added, and after removal of
e ethanol, the pH was lowered to 2.5. The aqueous residue
5 partitioned three times against petroleum ether (B.R. 40-

C). The petroleum ether was removed by rotary evaporation

d the residue applied to a silica gel column (hexanezethyl

 

 

72

acetatezacetic acid 7:3:0.1). Using this solvent system
standamwi XV eluted in the early fractions. Thus, after
application of the residue, the identical fractions were
pooled and dried. These combined fractions were dried and
subjected to semi-preparative reverse phase C13 HPLC (20% to
80% ethanol in 1% aqueous acetic acid, gradient time was 25
min). The fractjcni which eluted between 23 and 26 min
(determined using standard XV) was collected, dried, and
methylated with ethereal diazomethane. This fraction was then
analyzed by GC-MS.

3.2.9. Biological Activity Assay. The biological activity
of )( and XV was determined using the IAA-induced Avena
coleoptile elongation bioassay (Milborrow, 1978). In this
bioassay ABA, or compounds structurally related to it,
inhibit elongation (Milborrow, 1978). IAA must be added to
cause significant elongation.

Oats (Avena sativa L., cv Korwood) were soaked in water
for 2 h, exposed to red light for 4 h, and then sown, embryo
side up, (”1 moist Kimpack. After 3 d, coleoptiles were cut
three mm below the tip into 10 mm sections and floated on 1
mg/L MnSO4 for three h. Sections were then placed in one ml
of the solution to be tested [IAA alone, IAA plus ABA (0.4
pM), cu" IAA plus synthetic compound (0.4 pM) containing 2%
sucrose, 1.8 g/L KZHPO4 and 1.0 g/L citric acid, pH 5.0). Co-
leoptiles were measured to the nearest mm after an incubation

period of 24 h at room temperature.

 

 

73
3.3. RESULTS

3.3.1. Biological Activity of oz-Ionylidene Acetic Acid (X)
and 1',2'-Epoxy-Ionylidene Acetic Acid (XV).The biological
activity of X and XV was measured with the IAA Avena coleop-
tile elongation assay. In this assay, at the concentration
used, X had greater biological activity than ABA and XV had
less (Figure 3.13).
3.3.2. Incorporation of 1',2'-Epoxy-Ionylidene Acetic Acid
(XV) into Abscisic Acid. After 24 h incubation, analysis
of Xanthium leaves fed XV indicated that approximately 5%
of the starting material was still present. No apparent
degradation of XV occurred when standard material was
subjected to the purification procedure used with plant
tissue. The SIM response of ABA isolated from leaves fed XV
showed that an isotope shift of the molecular ion had
occurred (Table 3.1), confirming that higher plants can
convert XV into ABA (Milborrow and Noddle, 1970).

However, it appears that XV is not endogenous in higher
lants, at least not in X. strumarium. A compound which
7-chromatographed with standard XV is clearly not identical

XV as determined by GC-MS (compare Figures 3.14 and 3.6).
3.3. Metabolism of 1',2'-Epoxy-Ionylidene Acetaldehyde
VII) by Higher Plant Tissue. Extensive metabolism of
II was observed, with little or no starting material
Iaining after 24 h (Figures 3.15, 3.16, and 3.17). How-

tr, only with immature tomato fruit was the majority of

 

 

74

50

Ma
~ leum Elongallon

7‘.
g
0

 

I

20 ‘—/
4 s 5
—Log [IAA]

‘x

rgur‘e 3.13. Biological activity_of a-ionylidene a'cetic

X) a'nd 1 ’,2'—epoxy-ionylidene acetic acid (XV).
= biological response of X (A) and XV (it) are compared

/e to ABA (x) and IAA alone ([3). The standard deviation

:0 2%, 100% was 6 mm.

 

 

 

75

Table 3.1. Selected ion monitoring response of standard ABA
and ABA isolated from leaves fed 1’,2’—epoxy-ionylidene
acetic acid (XV).

The data shown are the area (relative units) under the
peak of the respective ion calculated by the GC-SIM. There
is 2.5 deuterium per ABA melecule after taking into account
endogenous ABA and natural abundance effects. This number
was calculated by the weighted average method using the
difference between the relative SIM response of ABA from

standard material or that derived from fed XV.

m/z SIM Response Relative SIM Response
"""" s tandard-"X-V- 'F‘e'd"m""3:255:51""iv'fiéé""""
190 3 49890 41879 1.000 1.000
191 3 9928 9498 0.199 0.227
192.3 1422 4128 0.028 0.099
193 3 695 8058 0.014 0 192

 

76

.A>xv mumuwoe mcocflfiscoflixxogm: m..Hum=eco.mcu:H»:ums

cumocmum cuflz omzamumoumeouzoioo :oflzs vasomeoo a mo Esuuoomm mom: .«H.n ousmam

AEEV 0920335.

ovm ONN CON oww oww ovw ONF

 

_. .2 _._._ ________ _. ____ .__._.__.._ eéji:
mm
on
mn

, 00F

 

 

(%)'1UIWGH

 

 

77

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Spinach leaves
Xanthoxin
>
:2
>
2:
O
(U
.2
'0
(U
(I ABA
I
i
l U XI”
(3 5 To 1:5 2:0 25 3'6 35

Time, minutes
metabolisni of 3H cis,trans-1',2’-epoxy-ionylidene acetalde-
hyde (XVII) in spinach leaves.
The arrows indicate the retention times of standards, and are
not meant to indicate the actual presence of these compounds

in the extract.

78

 

 

 

 

 

Xanthoxhi

Tomato shoots
>-
: V)
> -xvn
fl;
0
<0
0 ABA
'0
<0
[I

5 5 1'0 1’5 2'0 2'5 30 35

Time, minutes .

Figure 3.16. Radioactivity in HPLC effluent demonstrating
metabolism of 3H cis,trans-1',2'-epoxy-ionylidene acetalde-
hyde (XVII) in tomato shoots.

The arrows indicate the retention times of standards, and are
not meant to indicate the actual presence of these compounds

in the extract.

 

 

79

.AQ£:;X:j9hoxhi

Tomato fruit ' l

 

Ivity

 

Radioact

 

 

 

 

 

 

 

 

 

 

a

L L_Ll____

6 s 11) 1'5 2'0 25 so 35

 

 

 

 

Time, minutes
Figure 3.17. Radioactivity in HPLC effluent demonstrating
metabolisnltaf 3H cis,trans-1',2'-epoxyeionylidene acetalde-
hyde (XVII) in immature (breaker stage) tomato fruit.
The arrows indicate the retention times of standards, and are

not meant to indicate the actual presence of these compounds

in the extract.

80

XVII converted to compounds which co—chromatographed with
ABA and xanthoxin. When these two peaks (termed fraction 23
for the ABA-like and fraction 27 for the xanthoxin-like)
were analyzed by TLC, no radioactivity co-chromatographed
with either xanthoxin or ABA. On the contrary, all radio—
activity remained at the origin. This behavior is sug-
gestive of either a highly hydroxylated metabolite, or a
conjugate.

Basic hydrolysis of a small aliquot and subsequent TLC
analysis indicated that the radioactivity still remained
at the origin. Thus, the catabolites were not conjugates
containing an ester linkage, such as that found in ABA-GE.
Another aliquot was hydrolyzed with pectinase AC, which
cleaves glycosidic linkages. After this treatment,-radio-
activity no longer remained at the origin on TLC plates.
The major peak of radioactivity did not co-chromatograph
with either xanthoxin or ABA, although a small minor peak
did with xanthoxin. Both fractions had similar Rf values
with TLC after hydrolysis with pectinase. The mobility was
not changed when a small sample was methylated with ethereal

diazomethane, indicating that no carboxyl group was present.
3.4. DISCUSSION
As shown in section 3.3.1, X had greater biological

activity than ABA. Similar results have been obtained by

Kumamoto et a7.(1970). It is highly unlikely that a precursor

 

81

to ABA would have more biological activity than ABA. It
appears that the structural similarity of X rather than
biochemical conversion to ABA is responsible for the results
obtained with the Avena coleoptile elongation bioassay.

In addition, the results described here with XV (section
3.3 -2) and those with xanthoxin (Taylor and Burden, 1973)
indicate that compounds that contain an epoxide at the 1',2'
posi tion can be converted to ABA. Thus it was unexpected that
XVI I was not converted to ABA by immature tomato fruit
(section 3.3.3). 5

Preliminary structural analysis of the catabolites formed
in tomato fruit indicate that they may be diastereomers, 1'.e.
compounds which contain two or more optically active cen-
ters - XV was synthesized as a racemic mixture at the epoxide
group, Addition of a sugar via a glycosidic linkage would
Create another chiral center. Since diastereomers have
different physical properties (such as melting points,
5°1ubilities in different solvents, etc.) it is possible to
SEapatrate them on HPLC. Removal of the sugar linkage elimin-
ates a center of optical activity and results in one and the
Same compound, although it should differ in the orientation
of the epoxide group. When the fractions treated with
peCtinase were analyzed by TLC they had identical chromato-
gr‘aphic behavior. The catabolites also appeared to retain the

a~ldehyde, rather than it being converted to a carboxylic acid

g"‘Olnp, since treatment with diazomethane did not alter the
Rf.

 

 

82

The fact that a glycosidic linkage is present in the
tomato fruit catabolites implies that XVII was hydroxylated
and then had a sugar attached to the hydroxyl group. The
'location of the hydroxyl group to which the sugar is con-
jLJgated is unknown, but it is not at C-4'. If this were the
<:ase, then the compound would be xanthoxin. This cannot be,
si nce by TLC analysis the majority of radioactivity (after
yae ctinase AC ‘treatment) did not co—chromatograph with
st.andard xanthoxin. It is possible that the epoxide opened up
gi ving rise to a di-hydroxy compound. Another possibility is
ttiat IX was hydroxylated at positions in the ring other than
at C-4'. This latter case seems unlikely given the relative
noni-polar nature (after pectinase AC treatment) of the
catabolite.

It is apparent that C15 compounds such as X, XV, and
X\III are extensively metabolized (Lehmann and Schfitte,
19 76; Milborrow and Noddle, 1970; Milborrow and Garmston,
19 73). In cases where incorporation into ABA (and ABA
Czatabolites) was described, the percent incorporation was
Vfiary low. This low amount of incorporation could be explained
1 F the ABA biosynthetic pathway is highly compartmentalized
arid the fed compounds never reached the site of ABA biosyn-
thesis. It is also possible that conjugation or oxidation of
these C15 compounds occurred rapidly as they entered the
CJ’toplasm. Thus, the majority of the fed compounds would not
have a chance to enter the ABA biosynthetic pathway.

Another possible explanation is that the ABA biosyn-

 

83
theztic pathway is catalyzed by a multi—enzyme complex
(Stza fford, 1981). In this case, due to metabolic channeling,
tile efficiency of incorporation would be very low, unless
ttie correct intermediate in the pathway was fed. Pene-
tr‘a't ion of the fed compound to the active sites in the
conip>lex would be low due to competition with endogenous

mat a rial already bound.

3.5. LITERATURE CITED

Attenburrow, J., Cameron, A.F.B., Chapman, J.H., Evams,
R M., Hems, B.A., Jansen, A.B.A., Walker, T. (1952) A
synthesis of vitamin A from cyclohexanone. J. Chem.
Soc. pp. 1094-1111.

ICFI‘imura, M., Oritani, T., Yamashita, K. (1983) The metabol-
ism of (2Z, 4E)-a-ionylideneacetic acid in Cercospora
cruenta, a fungus producing (+)-abscisic acid. Agr.
Biol. Chem. 47:1895-1900.

Klearwzle, F., Mayer, H., Minder, R.E., and Thommen, H. (1978)
Synthese von optisch aktiven, natflrlichen Carotinoiden
und strukturell verwandten Verbindungen; III. Synthese
von (+)-Abscisinsaure, (-)-Xanthoxin, (-)-Loliolid,
(-)-Actinidiolid und (-)-Dihydroactinidiolid. Helv.
Chim. Acta 61:2616-2627.

Kllnléimoto, J., Smith, O.E., Asmundson, C.M., Ingersoll, R.B.,
Sadri, H.A. (1970) Cis,trans-a-ionylideneacetic acid: a
bioactive analog of abscisic acid. J. Agr. Food Chem.
18:531-533.

Lehrnann, H., Schutte, H.R. (1976) Biochemistry of phyto-
effectors. 9. The metabolism of a-ionylideneacetic acids
in Hordeum distichon. Biochem. Physiol. Pflanzen. 169:55-
61.

M‘ 1 t>orrow, B.V. (1978) Abscisic acid. In: Phytohormones and
related compounds-a comprehensive treatise, pp. 295-397,
vol 1, Letham D.S., Goodwin, P.B., Higgins, T.J.V.,
eds. Amsterdam: Elsevier.

M1 113 orrow, B.V. (1983) Pathways to and from abscisic acid.
In: Abscisic acid, pp.79-111, Addicott, F.T., ed. New
\’ork: Praeger Press.

 

 

 

 

84

Mi 1 Inqrrow, B.V., Garmston, M. (1973) Formation of (—)—
1 ,2 -epf-2-cis—xanthoxin acid from a precursor of
abscisic acid. Phytochemistry 12:1597-1608.

Mi 1 t>orrow, B.V., Noddle, R.C. (1970) Conversion of 5-(1,2-
epoxy-2,6,6-trimethylcyclohexyl)-3-methyl-penta-cis-2-
trans-4-dienoic acid into abscisic acid in plants.
Biochem. J. 119:727-734.

Ne'i 1 l, S.J., Horgan, R. (1983) Incorporation of a-ionyli-
dene ethanol and a-ionylidene acetic acid into abscisic
acid by Cercospora rosicola. Phytochemistry 22:2469-2472.

Nei 1 l, S.J., Horgan, R., Lee, T.S., Walton, D.C. (1981)
3-methyl-5-(4-oxo-2',6 ,6’-trimethylcyclohex—2 -enyl)—
2,4—pentadienoic acid, a putative precursor of abscisic
acid from Cercospora rosicola. FEBS Lett. 128:30-32.

0r i t.ani, T., Ichimura, M., Yamashita, K. (1982) The metab-
olism of (22,4E)-a-ionylideneacetic acid in Cercospora
cruenta, a fungus producing (+) abscisic acid. Agr.
Biol. Chem. 46:1959-1962.

0r i 13ani, T., Yamashita, X. (1985) Conversion of (2Z,4E)-5-
(1 ,2 -epoxy-2 ,6 ,6 -trimethylcyclohexyl)-3-methyl-
2,4-pentadienoic acid to xanthoxin acid by Cercospora
cruenta, fungus producing (+)—abscisic acid. Phyto-
chemistry 24:1957—1961.

stafford, H.A. (1981) Compartmentation in natural product
biosynthesis by multienzyme complexes. In: The biochem-
istry of plants, pp. 117-37, vol. 7, Stumpf, P.K., Conn,
E.E., eds. New York: Academic Press.

Tay1or, H.F., Burden, R.S. (1970) Identification of plant
growth inhibitors produced by photolysis of Violaxan-
thin. Phytochemistry 9:2217-2223.

Tiib'T or, H.F., furden, R.S. (1973) Preparation and metabol-
ism of 2- C-cis,trans xanthoxin. J. Exp. Bot. 24:873-
880.

”511 1:0n, D.C. (1983) Structure—activity relationships of
abscisic acid analogs and metabolites. In: Abscisic
acid, pp.113-146, Addicott, F.T., ed. New York: Prae-
ger Press.

Na] t(Nl, D.C., Li, Y., Neill, S.J., Horgan, R. (1985) Biosyn-
thesis of abscisic acid: a progress report. In: Current
topics in plant biochemistry and physiology 1985,
pp. 111—117, vol. 4, Randall, D.D , Blevins, D.C.,
Larson, R.L., eds. Columbia, MO: University of Missouri.

 

 

 

 

85

w;3lton, D., Wellner, R., Horgan, R. (1977) Synthesis of
tritiated abscisic acid

of high specific activity.
Phytochemistry 16:1059-1061.

 

CHAPTER 4
INCORPORATION OF OXYGEN INTO ABSCISIC ACID
AND PHASEIC ACID FROM MOLECULAR OXYGEN

86

 

 

 

i
ad“

 

Plant Physiol. (1984) 75. 166- l 69
0032-0889/84/75/0166/04/501.00/0

87

Incorporation of Oxygen into Abscisic Acid and Phaseic Acid

from Molecular Oxygenl

Received for publication August 4. l983 and in revised form December 5. 1983

ROBERT A. CREELMAN AND JAN A. D. ZEEVAART‘
NSC-DOE Plant Research Laboratory, .ilichigan State University. Eds: Lansing. .l-[r‘c/zr’gan 48824

ABSTRACT

Abscisic acid accumulates in detached. wilted leaves‘iil' Xanthium
strumrr'um. When these leaves are subsequently rehydrated. phaseic
acid. a catabolite of abscisic acid. accumulates. Analysis by gas chroma-
tography-mass spectrometry of phaseic acid isolated from stressed and
subsequently rehydrated leaves placed in an atmosphere containing 20%
“O: and 80% N: indicates that one atom ot‘ “0 is incorporated in. the
6'-hydroxymethyl group of phaseic acid. This suggests that the enzyme
that converts abscisic acid to phaseic acid is an oxygenase.

Analysis by gas chromatography-mass spectrometry of abscisic acid
isolated from stressed leaves kept in an atmosphere containing "01
indicates that one atom of l‘0 is present in the carboxyl group of abscisic
acid. Thus. when abscisic acid accumulates in water-stressed leaves. only
one of the {our oxygen: present in the abscisic acid molecule is derived
from molecular oxygen. This suggests that either (a) the oxygen present
in the l'-, 4’-, and one of the two oxygens at the l-position of abscisic
acid arise from water. or (b) there exists a stored precursor with oxygen
atoms already present in the l’- and 4'-positions of abscisic acid which
is converted to abscisic acid under conditions of water stress.

 

Little is known about the biosynthetic pathway of ABA. except
that as a sesquiterpenoid. ABA is ultimately derived from MVA.2
When radioacrive MVA was applied to higher plant tissues. the
percentage of incorporation into ABA was always very low, and
no intermediates have ever been isolated. Some controversy
exisrs as to whether ABA is synthesized from a C-15 precursor,
presumably farnesyl perphosphate (the direct pathway), or re-
sults from the degradation of a C40 precursor (the indirect
pathway). such as the xanthophyll Violaxanthin ( 6). It is known
that the stereochemisuy of pretons in ABA derived from MVA
is identical to that found in carotenoids and it should be noted
that the terminal ring Structure of certain xanthOphylls is similar
to ABA (6).

Oxygen incorporation into camtenes to form xanthophylls is
a late Step in caratenoid biosynthesis occurring after ring for-
mation. The oxygen atoms in the hydroxyl groups of lutein and
the epoxide groups of antheraxanthin and Violaxanthin are de-
rived from molecular oxygen (12. 13). The keto group of spher-
oidenone also comes from molecular oxygen (8). By analogy
with xanthophyll biosynthesis. if ABA is derived from farnesyl
pyrophosphate. incorporation of oxygen at the l’- and 4’-caro

'Supported by the U nitcd States Department of Energy under Con-
tract DE-AC02-76ERO-l338.

’ Abbreviations: M VA. mevalonic acid: PA. phaseic acid: ABA-GE.
d-D-glucopyranosyl abscisate: GC-SIM. gas chromatography-selected ion
monitoring: m/z. mass/charge.

bons of the ABA molecule should be a late step in the pathway,

. ,lthg oxygens being derived from molecular oxygen.

With respect to ABA‘eetabolism. Gillard and Walton (2) have
shown with a crude enzyme preparation from Echinocystr's (about
that hydroxylation at the 6’-methyl group to g‘ve PA via the
unstable intermediate 6’ohydroxymcthyl-ABA. is inhibited by
CO and anaerobic conditions. They concluded that the enzyme
involved is very similar to Cyt P450 monooxygenases found in
animals. but it did not meet all the criteria necessary for calling
ABA hydroxylating enzyme a Cyt P450 monooxygenase. These
criteria are: (a) inhibition of the reaction in the presence of CO.
(b) presence in the reduced enzyme preparation of a CO-binding
pigment with a maximum A at 450 nm in the CO difference
specrrum. (c) reversal of CO inhibition by light with a maximum
in the aetion speed-um at 450 nm. (d) demonsrration of the
expected reaction stoichiometry, and (c) incorporation of one.
oxygen atom from "02 into each molecule ot‘produCt (l 1).

We decided to study the origin of the oxygen atoms in ABA
and PA by the use of “Oz. The number of oxygen atoms present
as well as their positions in the molecules can then be determined
by MS of the purified compounds. It is essential that during
incubation with "0;, large amounts of ABA and PA are synthe-
sized by theexperimental system under study. For this reason
we chose detached leaves of Xanthium. since upon wilting their
ABA content increases dramatically over a period of a few hours.
If these leaves are subsequently rehydrated by immersing them
in water. their ABA content decreases and PA. a catabolite of
ABA. rapidly accumulates (15). Xanthium leaves are. therefore.
an ideal system for rapidly inducing the accumulation of bOth
ABA and PA. depending on how the leaves are manipulated.

MATERIALS AND METHODS

Plant Material. Xanthium smrman'um L.. Chicago strain. was
grown as before (15). The youngest. fully expanded leaf blade.
hereafter called leaf. was used in all experiments. For experiments
involving ABA. PA. and ABA-GE. leaves were wilted until they
had lOSt 13% of their fresh weight and then were stored in plastic
bags for 6 h. Wilted leaves were rehydrated by immersing them
in water for 5 min. and then were rescaled in plastic bags. or in
a 250-ml Erlenmeyer flask sealed with a serum stepper. Flasks
were immediately evacuated until a final pressure of 7 to 13 Pa
was reached. and then were backflushed with N z. This procedure
was repeated two more times. To test the erl‘ect of vacuum on
PA accumulation. some flasks were unsealed to allow room air
to enter and then were immediately rescaled. If leaves were to be
incubated in the presence of "‘03, 50 ml of "O; was added to
the flask after 2 evacuation cycles and then the flask was filled
with N. For experiments involving only ABA. a similar proce-
dure was used except that after the leaves were wilted. they were
immediately placed under N3, or under a mixture of “O; and
N: as described above.

The primary leaves of Phaseoltu' vrdgarr's L.. cv Redkloud were

 

 

88

"O INCORPORATION INTO ABA AND PA

used in some experiments involving ABA accumulation. Leaves
were harvested 10 d after planting and were treated as described
above for X. smrmarr'tmt.

Incubation of leaves in the presence of "O; was carried out
2x in the case of the PA experiment. The ABA experiment was
performed 3x with Xanthium leaves. and 1x with Plies-cola:
leaves. The experiments described in Tables I and II were per-
formed twice with two replicates each time. Similar results were
obtained in repetitions of all experiments.

Chemicals. "0: was purchased from Stohler Isorope Chemi-
cals Inc. (49 Jones Road. Waltham. MA). H;"O was purchased
from Kor. Inc. (56 Rogers Street. Cambridge. MA). One atom
of"O was exchanged into the «V-keto group ot'(=)-ABA (Sigma)
by placing (:l-ABA in Hf‘O with 1% (v/v) acetic acid for
2.5 d at room temperature.

Extraction. Purification. and Quantification Procedures. For
experiments dealing with ABA. PA. and ABA-GE. the samples
were purified and quantified according to Zeevaart (15. 16). For
experiments involving only ABA. samples were eittracted accord-
ing to Zeevaart (15). ABA was further purified by semi-prepara-
tive reverse phase HPLC on a uBondapak C a (10 iii-particle size).
30 x 0.78 cm column (Waters Associates. Milford. MA). The
sample was eluted by means of a convex gradient (curve 5 on
the Waters Associates Model 660 Solvent Programmer) from 0
to 50% Solvent B in Solvent A (Solvent A: water with 1% acetic
acid: Solvent 8: ethanol with 1% acetic acid) in 30 min at a flow
rate of 2.5 ml/ min. The fracuon containing ABA was dried and
further purified by analytieel straight phase HPLC on a uBon-
dapak NH: column (Waters Associates). ABA was eluted by
means of a convex gradient (curve 5) from 50 to 100% ethyl
acetate containing 1% acetic acid in hexane in 15 min at a flow
rate of 2 ml/min. After elution from the analytical column. the
fraction containing ABA was dried and methylated with ethereal
diazomethane. Quantification of the methyl ester of ABA was
performed with a Hewlett-Packard 5840A gas chromatograph
equipped with a ”Ni-elecrron capture detector (15). Samples
were dissolved in ethyl acetate and analysis was done on a
Dumbond 08-1 (.1 & W Scientific. Inc. Rancho Cordova. CA)
gas capillary column (30 m X 0.32 mm x 0.25 um). GLC
conditions were: oven temperature 165'C. Hz carrier flow 10 ml/
min. split ratio 5:1: argon-methane (95:5) was used as make-up
gas and had a flow at the detector of 80 ml/min.

To determine if exchange from the 4'-keto group of ABA
occurred during the extraCtion and purification process. a leaf
sample with added 4’-"O—ABA was extracred and purified as
described above.

Mass Spectrometry. Mass speCtra were obtained with a Hew-
lett-Packard 5985 quadrupole mass specrrometer connecred to a
5840A gas chromatograph. GLC conditions were: 3% SE-30 on
100 to 200 mesh Gas Chrom Q in a silanized glass column (180
x 0.2 cm) held isOthermaIly at 205'C for the methyl ester of PA.

Table 1. Effect ofAnuxiu on PA Accumulation in Xanthium Leaves

Leaves were stressed. placed in a plastic bag for 6 h. and then
rehydrated by immersrng the leaves in water for 5 min. The leaves were
then either frozen. or subrecred to a vacuum-(lush treatment to remove
any oxygen present. Leaves SUDJCCICd to a vacuum-flush treatment were
then placed in an atmosphere of N3. or room air was allowed to enter
the flask and the flask was then rescaled.

 

 

Treatment of Leaves ABA PA ABA-GE
ug-g" fresh n-r
Stressed 6 h .. rehydrated -- frozen 3.1 1.5 1.0
Stressed 6 h _. rehydrated - vacuum 0.9 4.5 1.2
-- room air 5 h
Stressed 6 h -- rehydrated _. vacuum 2.9 1.3 1.0
- I003: | 2 5 h

 

and temperature programmed from 185 to 240’C at S'C/ min for
the methyl ester of ABA with a He flow rate of 30 ml/min. The
ionizing porential was 70 ev.

The extent of exchange of the 4'-lteto group during the extrac-
tion and purification procedure was determined by monitoring
m/z 190 (base peak ofABA) and m/z 192 (base peak of-V-“Oo
ABA) by GC-SIM (dwell time for each ion was 400 ms). GLC
conditions were as described above for ABA.

Analysis of air samples present around leaves during incuba-
tion in Erlenmeyer flasks was performed with a Varian-Mat CD-
150 magnetic sector mass spectrometer.

RESULTS AND DISCUSSION

The ABA level in stressed and subsequently rehydrated leaves
incubated in room air declined and PA accumulated (Table I).
By contrast. when stressed and subsequently rehydrated leaves
were incubated in N:. the ABA level remained high. and PA did
nor accumulate. The accumulation of ABA-GE did act appear
to be greatly afi‘ected by anaerobic conditions. However. little
ABA-GE accumulates in Xanthium leaves that have been stressed
and subsequently rehydrated ( 16). The possibility remains that
anoxia did not affect ABA catabolism directly. but rather indi-
rectly via an effect on cell metabolism. GC-MS analysis of PA
isolated from rehydrated leaves incubated in an atmosphere of
20% "Oz and 80% l 2 showed a new molecular ion at m/z 296
compared with a molecular ion of m/z 294 for PA from rehy-
drated leaves incubated in room air. This indicates the incorpo-
ration of one atom of "0 into the PA molecules (Fig. 1). The
presence of m/z 294 in the mass spectrum of PA isolated from
leaves incubated with "0; (Fig. 18) is due to PA already present
in turgid leaves and to PA synthesized during the stress portion
of the experiment ( 15. 16).

From the fragmentation pattern derived from a high resolution
mass spectrum of methylated PA (G. l... Boyer. R. A. Creelman.
and .l. A. D. Zeevaart. unpublished results), we conclude that
the atom of “O in the PA molecule is located in the 6’-
hydroxymethyl group for the following reasons: (a) m/z 125 (side
chain containing carboxyl group) is not shifted. (b) m/z 276
(arising from the loss of the l’-hydroxyl group as water from
m/z 294) is shifted by 2 mass units. (c) m/z 139 (m/z 167 gives
rise to m/z 139 with loss of CO'. the CO coming from the 4’-
keto group) is shifted by 2 mass units. and (d) m/z 233 (m/z
263. Which is shifted by 2 mass units. gives rise to m/z 233 with
the loss of CHgO') is not shifted. These data support the conclu-
sion (2) that ABA hydroxylating enzyme is an oxygenase and are
consistent with Gillett's rule (3) that biological hydroxylation of
a methyl group involves the direct participation of molecular
oxygen.

ABA levels in stressed leaves of X. strumarr'um increased. and
the vacuum-(lush treatment had no effect on accumulation of
ABA (Table II). On the other hand. accumulation of ABA in
stressed leaves was inhibited by anoxia. However. as with the PA
results (see above). one cannor rule out that anoxia had only an
indireCt effect on ABA biosynthesis. GC-MS analysrs of ABA
from leaves incubated in an atmosphere of 16% ”‘03. 4% ”’03.
and 80% l 3 shows a new molecular ion at m/z 280 compared
with the molecular ion (m/z 278) found with ABA from leaves
incubated in room air (Fig. 2). This indicates that only one atom
of "O is incorporated into the ABA molecule. Similar results
have been obtained with P. vulgaris cv Redkloud (data net
shown). The atom of "O is located in the carboxyl group ofABA
for the following reasons: (211 m/z 125 (side chain) is shifted by 2
mass units (4). and (b) the fragment derived entirely from the
methylated carboxyl group. m/z 59 (COCO-if) is shifted by 2
mass units to m/z 61.

The fragments m/z 125 and m/z 262 contain both oxygens of
the l-eerboxyl group of ABA (4). and as expected. the ratios

——._———-

CREELMAN AN D ZEEVAART

l

5"-.. -

89

Plant Physiol. Vol. 75. 1984

 

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276

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244-246
276-273 34-296

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223 ass—ass

237-
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240 260

19469

11
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180 200 280 300

rue (m/z)

Frc. 1. Mass spectra of PA isolated from stressed and subsequently rehydrated Xanthium leaves incubated in room air (A). or "O; (B).

Table 11. Efl'ecr of Anoxia on ABA Accumulation in Xanthium Leaves
Leaves were stressed and then either placed in a plastic bag for 8 h. or
in a 250-ml flask and subjected to a vacuum-flush treatment. Leaves
subjected to a vacuum-flush treatment were then placed in an atmosphere
of N3. or room air was allowed to enter the flask and the tlaslt was then
resealed. Control indicates a turgid leaf placed in a plastic bag.

 

 

Treatment of Leaves ABA

pg 3“ dry wt
Control 8 h 5.3
Stressed 8 h 24.5
Stressed - vacuum .. room air 8 h 21.0
Stressed -~ vacuum _. 100% N, 8 h 3.3

 

m/z 125/127 and 262/264 are similar. On the Other hand. the
fragment m/z 190 contains only one of the two oxygens found
in the earboxyl group (4). From a chemical viewpoint the two
oxygens in the carboxyl group are equivalent. Thus. if there is
only one "0 present in the carboxyl group. as concluded above.
only half the m/z 190 fragments will contain “0. resulting in a
higher ratio of m/z 190/192 than found for m/z 125/127 and
262/264. This is indeed observed in Figure 2B.

Exchange of oxygen from the l’-hydroxyl group of ABA with
oxygen present in water during sample extraction is highly un-
likely. since it was necessary to use synthetic methods to intro-
duce “O in the l’-hydroxyl group (,4). It is also unlikely that the
oxygen from the 4’~keto group or l-carboxyl group exchanged
with the oxygen in water. since synthesis of ABA containing u'0
in the carboxyl group or the keto group required Strongly alkaline
conditions and heat (4). Conditions used in the present experi-
ment for the isolation of ABA were weakly acidic. and do not
appear to cause exchange (17). However. when ABA was placed
in l-lz"O with 1% (v/v) acetic acid for 2.5 d one atom of "O was
exchanged into the 4'-lteto group of ABA. as determined by
analysis of the fragmentation pattern (data not shown). Thus. it
is possible that the keto group exchanged out during the extraco
tion process. To determine if this was indeed the case. a small
amount of 4‘-"O-ABA was added to a Xanthium leaf sample
and the sample was extracted and purified as described above.

At the same time. an equal amount of 4’-"O-ABA was stored in
a refrigerator. termed hereafter Stored aliquot. After purification.
the tissue sample was brought up to a final volume of 20 1:1. as
was the stored aliquot. Equal amounts of the extract and the
stored aliquot were analyzed by GC-MS and m/z 192 was
monitored by GC-SIM. The SIM response of the tissue and the
stored aliquot were comparable. indicating that little or no
exchange had occurred during extraction and purifieation. We
conclude therefore that no l‘0 was incorporated into the 4’-
keto-group of ABA under our experimental conditions.

The fact that only one "0 atom appeared in the carboxyl
group of ABA that accumulates in water-streased leaves is unex-
pecred. Based on the strong similarities between ABA and carot-
enoids. we expected that the 4’-keto and l’-hydroxyl groups
would contain “0. Since they remain unlabeled. the oxygen
atoms in the 4’-keto and l’-hydroxyl groups muSt either (a)
come from water. or (b) muSt already be present in a precursor.
such as xanthoxin or certain xanthophylls (such as violaxanthin),
that is converted to ABA under conditions of water stress. If this
latter case is correct. it would be futile to search for intermediates
in the ABA biosynthetic pathway by feeding radioactive MVA
as a precursor.

There is no firm evidence to support either the direct or
indirect pathway of ABA biosynthesis. Milborrow (see 6) rules
out the indireCt pathway on the basis of an experiment with [“C]
phytoene and [’H]MVA fed to avocado fruit. "C and ’l-l were
bOth found in carorenoids. yet only ’11 was found in ABA. This
work is n0t conclusive because phytoene would have had to
penetrate to chloroplasts and then be converted to a xanthophyll.
It is am known whether or not this occurred. since a detailed
account of this work has never been. published.

We believe that the data presented here are in accord with the
indirect pathway. although the existence of two separate. inde-
pendent pathways. one operating in turgid and a different one
in water-stressed leaves. cannor be ruled out. Xanthoxin. a
degradation product of violaxanthin (9). is endogenous to higher
plants (1. 14). When labeled xanthoxin was fed to tomato and
been plants. it was converted to ABA and catabolites of ABA
(10). Since xanthoxin is endogenous in higher plants. and is

 

90

I'O INCORPORATION INTO ABA AND PA

 

 

 

 

 

 

 

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Mass / Chorqe (tn/z)

F16. 2. Mass spectra of ABA isolated from stressed Xanthium leaves incubated in room air (A). or “O: (B).

converted to ABA. it is a likely precursor of ABA in higher
plants.

Neill er a1. (7) have isolated l’-deoxyoABA from Cereospora
rosicoia and have shown that it is the immediate precursor of
ABA in this fungus. There is no evidence. however, that 1'-
deoxy-ABA is endogenous in higher plants. GC~SIM analysis of
l’cdeoxy-ABA exrracted from Vicia faba (the only plant tested
which appears to convert l’-deoxy-ABA to ABA) cuttings fed
lH-a-ionylidene acetic acid showed that l’-deoxy-ABA was
100% labeled. Le. all the extramed l’-deoxy-ABA was synthe~
sized from the applied «at-ionylidene acetic acid (7). This implies
that either l’deoxyoABA is not endogenous to V. fitba. or that
it is present in a small pool rapidly turning over. Furthermore.
Lehmann and Schiitte (5) observed that when a-ionylidene acetic
acid was fed to barley plants. it was converted to l'deoxy-ABA

and conjugates. but not to ABA. Our data also indicate that l’-'

deoxy-ABA is not the immediate precursor of ABA in higher
plants. unless the oxygen present in the l’-hydroxyl group is
derived from water rather than from molecular oxygen.

in conclusion. we have demonstrated that when Stressed and
subsequently rehydrated Xanthium leaves are incubated in an
atmosphere containing "0:. one atom of "O is found in the 6'-
hydroxymethyl group or. PA. continuing that ABA hydroxylating
enzyme is an oxygenase. When stressed Xanthium leaves are
incubated in an atmosphere containing ‘30:. one atom of "O is
found in the carboxyl group of ABA. This implies that either the
oxygens in the l'chydroxyl group. 4’-keto group. and one of the
two oxygens in the l-carboxyl group come from water. or a
stored precursor exists w1th oxygen atoms already present at the
l'- and 4'-positions. and possibly the l-position.

Note Added in Proof. Since this work was completed. we have
learned of similar "0; labeling experiments with the fungus
C ercaspara rosicola. In this case. ABA contained four "0 atoms
when the fungus was cultured over a 48-h period under an
atmosphere containing 20% "O: (R. Horgan and D. C. Walton.
personal communications).

Ackmm-Ivdemmts—We would like to thank I. Throclt Watson and Bnan

Musselman for valuable discussions regarding the fragmentation pattern of PA and
the Michigan State U niver'sits-Nattonal institutes of Health Mass Spectroscopy
Facility (RR00480) for use or the GC-MS equipment. We also thank T. M. Shimer
for MS analysis or as samples

LITERATURE CITED

1. Frau RD. RS buaoen. HF TAYLOR I972 The detection and estimation of the
growth inhibitor xanthoxin in plants. Planta 102.: 115-116

2. Guam DF. DC WALTON 1916 Abscisic acid metabolism by a cell-free
preparation from Erbium-sits (abate liquid endosperm. Plant Physiol 58:
790-795

3. GILLETI’ JR 1959 Side chain oxidation ot‘alkyl substituted ring compounds I.
Enzymatic oxidation of p-niuoroluene. J Biol Chem 234: 139-l4}

4. GRAY RT. R MALLMY. G Rnacx. VP WILLIAMS I974 Masspeetiaof methyl
abscisate and isotoprcally labelled analogues. J Chem Soc Perkins Trans ll:
919-924

5. LEHMANN H. HR SCHUTTE 1976 Biochemistry ofphytoetTeetors 9 The metab-
olism or a-ionylidenaceuc acid in Hordeum disriclton. Biochem Physiol
Pflanzen 169: 55-6l

6. Mtuortrtow BV 1983 Pathways to and from abscisic acid. In FT Addicou. ed.

Abscisic Acrd. Praeger. New York. pp 79-1 ll
. New. SJ. R HORGAN. DC WALTON. D GRIFFIN 1982 Biosynthesis ofabsctsic
acid. In PF Wareing. ed. Plant Growth Substances 1982. Academic Press.
New York. pp 315-323
. SHNEOUR EA I962 1113 source of oxygen in Rhodopseudomonas spirerotdes
carorenoid p1gment conversion. Bioehim Biopnys Acta 65: SIG-5| l
. TAYLOR HF. RS BURDEN I972 Xanthoxin. a reeenrly discovered plant growth
inhibitor. Proc R Soc Lond 3 ISO: 3l7-3-36
IO. TAYLOR HF. RS BURDEN 1973 Preparation and metabolism 01' 2-{ "CI-c1:
trans-xanthoxm. J Esp Bot 24: 873-880
ll. Wm CA 1930 Hydroxylases. monookygenases. and cytochrome P450. In PK
Stumot. EE Conn. cos. The Biocnemistry 01‘ Plants. Vol 2. Academic Press.
New York. pp 3 l 7-364
ll. Yauamoro HY. C0 CHICMBTER 1965 Dark incorporation of "0 into anther-
axanthin 'oy bean lear'. B1ocrttm Biophvs Acta 109: 303-305
IJ. Yammoro HY. CO CHICHFSTEI. TOM Namibian“ 1962 Biosvnlneuc origin
or'oxygen in the leaf xanthopnyus. Arch Biochem Bioonvs 96: 045-649
l4. Zisviuitr MO 1974 Levels 01‘ (+)-auntie acid and xanthoxin 1n spinach
under oilTerent enwronmental conditions. Plant Physiol 53: 644-648
13. ZEEVAAIT JAD I980 Changes in the levels 01' obscure and and its metabolites
in caused leaves or' .t'unrmum strumonum during and after water stress.
Plant Physiol 66: 672-678

l6. Z£evanr MD 1983 Metabolism of abscisic and and its regulation in Xan-

thium 1am dunng and aster water stress. Plant Physiol 7 I: $77—11“

l7. Zssviurr JAD. 8V MthonRow 1976 Metabohsm ot‘ abscisic acid and the

occurrencagt' epidihyoropnasetc acid in Manila: vuieans. Phytochemrstry
l5: 493-5

q

0“

 

CHAPTER 5
ACCUMULATION 0F ABSCISIC AND PHASEIC ACID IN
XANTHIUM STRUMARIUM LEAVES UNDER DIFFERENT
OXYGEN TENSIONS

91

92
5. 1 . INTRODUCTION

In chapter 4 it was shown that the formation of ABA
and PA requires molecular oxygen. Hence, an enzyme(s) in
the ABA biosynthetic pathway must at some step(s) require
oxygen, as do mono—oxygenases and oxidases. The enzyme
1' nvolved in the conversion of ABA to PA is presumed to be a
mono-oxygenase (Gillard and Walton, 1976).

Some oxidases, such as ascorbic acid oxidase, are not
saturated at a normal atmospheric oxygen tension of 20%
(Thimann et al., 1954). With polyphenol oxidase, the Km for
0X.Ygen was found to vary with substrate; with the exception
01: Pyrocatechol, the enzyme-substrate complexes were saturat-
Ed at room oxygen partial pressure (Butt, 1980).

We decided to determine the effect of different oxygen
tens-i ons on the accumulation of ABA and PA. Xanthium leaves
are ideal for this study because they have the ability to
accurnulate rapidly either ABA or PA, depending on how the

1eaves are manipulated (Zeevaart, 1980).
5.2. MATERIALS AND METHODS

5'2- 1. Plant laterial. X. strumarium was grown as previous-
15’ described (Zeevaart, 1980). The youngest, fully expanded
leaVes were used in all experiments. For experiments involv-
ing ABA accumulation, leaves were excised and stressed by

al 7 Owing them to lose 15% of their fresh weight. They were

 

93

then placed in 125 ml Erlenmyer flasks. For PA, the same
procedure was followed as for ABA, except that after imposing
stress they were placed in a plastic bag for six h. The
leaves were rehydrated by submerging them in distilled water
for five min and then placed in 125 ml Erlenmyer flasks. The
f1 asks (closed with serum stoppers) were then subjected to a
vacuum-flush treatment as described in chapter 4. A vacuum
was created in the flasks with a vacuum pump. The flasks were
then flushed with nitrogen. This was repeated three times.
Suitable amounts of oxygen and nitrogen were then added to
the flasks. For experiments dealing with ABA leaves were
incubated under different oxygen concentrations for six h,
and for PA five h.

All experiments were performed four times with two
leaves per treatment. Similar results were obtained in all
eXperiments.

5- 2.2. Extraction and purification procedure. ABA and PA
We re extracted and purified as described in chapters 4
and 9 for ABA. Relevant HPLC fractions (determined with
standard ABA and PA) were collected, dried, and methylated
WT th ethereal diazomethane. ABA was analyzed by GLC-ECD as
described in chapters 4 and 9. PA was analyzed in a similar
manner except that the oven temperature was held isothermally
at 187 c.

5-2.3. Oxygen leasurements. Oxygen content in the flasks
was measured at the beginning and end of the incubation

Period by GLC-TCD using a Varian 3700 gas chromatograph.

 

94
Analysis of the gas mixtures was performed with a molecular
sieve column (5A, 45-60 mesh, 2 m x 1/8 inch stainless
steel). Quantitation was performed using standards of 0, 2,
20 and 100% oxygen. The balance, if any, consisted of
ni trogen. GLC conditions were: oven temperature 65 C,
injector temperature 100 C, detector temperature 120 C,

detector current 108 mA, He carrier flow 26 ml/min.

5 . 3 . RESULTS

The oxygen tension in the flasks varied from 0 to 90%
oxygen. Some respiration was noted in all flasks (except
0%); the amount of oxygen consumed was usually 2—3%. All
data presented are related to the concentration of oxygen
Present at the beginning of the experiment.

The production of PA was not increased at tensions
greater than 20% oxygen (Figure 5.1). On the other hand,
ABA accumulation was not saturated until 60% oxygen was

present (Figure 5.2).

5.4. DISCUSSION

The net accumulation of ABA depends on both its formation
a"Id degradation. Since the conversion of ABA to PA is not
Elf:Fected (Figure 5.1), the data described here imply that the
ox.Ygen requiring step(s) must be prior to ABA.

In addition, the enzyme responsible for the conversion

 

 

95

60

 

pg/g dry weight

 

 

 

 

 

o 20 45 6'0 100
Oxygen Content (73)

Fl'gure 5.1. Accumulation of phaseic acid under different
oXygen tensions. I

The accumulation of PA in stressed (D) and stressed and

Subsequently rehydrated leaves (X) was saturated at 20%

oX.Ygen content.

 

 

96

 

ABA Accumulation (7.1)

 

 

 

O K I I I
40 so 80 100
Oxygen Content (Z)

 

Figure 5.2. Accumulation of abscisic acid under different
0Xygen tensions.

The accumulation of ABA in stressed Xanthium leaves was not
Saturated until about 50-60% oxygen. Shown are the results
16"‘0m 3 experiments. The maximum accumulation in experiment 1
(D), 2 (A), and 3 (x) was 49.0, 59.5, and 43.0 g/ug dry

Weight, respectively.

 

 

97
of ABA to PA (ABA hydroxylase) appears to be similar to
other mono-oxygenases in that ABA hydroxylase is saturated
at atmospheric oxygen tensions. However, the enzyme(s)
responsible for ABA biosynthesis appear to be different. The
mechanism of the introduction of oxygen into ABA might be
similar to the mechanism used by ascorbic acid or polyphenol
oxidase. This is based on the observation that increasing the
oxygen tension does not saturate these oxidases nor the

ability to accumulate ABA.

5.5. LITERATURE CITED

Butt, V.S. (1980) Direct oxidases and related enzymes. In:
The biochemistry of plants, pp 81-123, vol 2, Stumpf,
P.K., Conn, E.E., eds. New York: Academic Press.

Gillard, D.F., Walton, D.C. (1976) Abscisic acid metabolism
by a cell-free preparation from Echinocystis lobata
liquid endosperm. Plant Physiol. 58:790-795.

Thimann, K.V., Yocum, C.S., Hackett, D.P. (1954) Terminal
oxidases and growth in plant tissues. III. Terminal
oxidation in potato tuber tissue. Arch. Biochem. Bio-
phys. 53:239-257.

Zeevaart, J.A.D. (1980) Changes in the level of abscisic
acid and its metabolites in excised leaf blades of
Xanthium strumarium during and after water stress.
Plant Physiol. 66:672-678.

Chapter 6
INCORPORATION OF MOLECULAR OXYGEN INTO ABSCISIC
AND PHASEIC ACID IN LEAVES AND ROOTS OF XANTHIUM
STRUMARIUM DURING LONG TERM INCUBATIONS IN 1802

98

 

 

 

99
6.1. INTRODUCTION

Nothing is known about the biosynthetic pathway of ABA,
except that as a sesquiterpenoid, ABA is ultimately derived
from MVA. Research on ABA biosynthesis has focused primarily
on two pathways: (a) the direct pathway involving farnesyl
pyrophosphate, and (b) the indirect pathway involving a
carotenoid, with xanthoxin as an intermediate in the pathway.

In chapter 4 evidence was presented indicating that one
atom of 180 was incorporated into the carboxyl group of ABA
isolated from stressed Xanthium leaves incubated in the
presence of 1802. This indicated that ABA is derived from a
carotenoid precursor. In addition, in chapter 4 data were
presented showing that the conversion of ABA to PA required
molecular oxygen, with one atom of 180 incorporated into what
eventually forms the lactone oxygen of PA. Since these
experiments were done using relatively short incubations (6
h), stressed Xanthium leaves were incubated with 1802 for 12
and 24 h to see if the incorporation of 180 was changed under
these conditions.

Detached roots of Xanthium also accumulate ABA, although
the levels are lower than in stressed leaves. ABA levels
increased up to 100-fold in stressed roots, while detached,
stressed leaves accumulated only 15 times that found in
turgid leaves (Cornish and Zeevaart, 1985). Thus, the
Possibility exists that the biosynthetic pathway for stress-

induced ABA may be different in roots from the one operating

 

 

 

100
in leaves.

In order in) measure low levels of 180 incorporation, a
highly sensitive mass spectrometer must be used. The pro-
cedure used must also measure the molecular ion as major
ion. ABA has a small molecular ion, and analysis of the
amount of incorporation in fragment ions is complicated. NCI
has high sensitivity to electrophilic compounds (such as ABA)
and causes little fragmentation (Watson, 1985). Furthermore,
because of the low amount of energy given to the analyte, CI
mass spectra contain fewer ions than the corresponding EI
mass spectra, with the molecular ion (M') usually predominat-
ing.

Ne incubated stressed Xanthium leaves and roots in
atmospheres containing 20% 1802 for various times and
determined the amount of incorporation and location of 180 in
ABA and PA using MCI and EI. Levels of carotenoids were
also measured in turgid leaves and roots because carotenoids

may be important as precursors to ABA.
6.2. MATERIALS AND METHODS

6.2.1. Culture of Plant Material. xanthfum strumarium L.,
Chicago strain, was grown as before (Zeevaart, 1980). The
youngest, fully expanded leaf was used in all experiments.
Leaves were wilted until they had lost 13% of their fresh
weight and were then placed in atmospheres of 80% N2 and 1302

as described in chapter 4. Leaves were incubated for 6, 12,

 

 

 

 

 

101
and 24 h.

Roots of Xanthium strumarfum were grown in solution
culture as follows. Seeds were soaked in water for 2 d and
embryos were removed and placed on moist filter paper in
Petri dishes. After a further 2 d incubation, young seedlings
were placed on a bed of soil and vermiculite was layered over
them. After approximately 2 weeks of growth, the young plants
were removed from the soil/vermiculite and suspended from
perforated boards over trays containing half-strength
Hoagland nutrient solution. The plants were then grown in a
growth chamber for 2 weeks, with a change of the nutrient
solution after 1 week.

In all experiments, roots were analyzed that had little
secondary thickening. For stress experiments, the roots were
detached from the plants, cut into approximately 2 cm
lengths, and stressed by allowing them to lose 60-70% of
their fresh weight (Cornish and Zeevaart, 1985). They were
placed in an atmosphere of 80% N2 and 1802 as described above
for leaves. Roots were incubated in this mixture for 6 h.
6.2.2. Extraction and Purification of Abscisic and Phaseic
Acid. ABA and PA were purified as described in chapters 4, 9,
and Cornish and Zeevaart (1984).

6.2.3. Oxygen measurements. To insure that depletion of
oxygen did not occur during the incubations due to respir—

ation, the oxygen concentration was monitored in all flasks
every few h. Oxygen content in the flasks was measured by

GLC-TCD using a Varian 3700 gas chromatograph. Analysis of

 

102

the gas mixtures was performed with a molecular sieve column
(5A, 45-60 mesh, 2 m x 1/8 inch stainless steel). Quantita-
tion was performed using standards of 0, 2, 20 and 100%
oxygen. The balance, if any, consisted of nitrogen. GLC
conditions were: oven temperature 65 C, injector temperature
100 C, detector temperature 120 C, detector current 108 mA,
He carrier flow 26 ml/min.
6.2.4. Purification of Carotenoids. In separate experiments,
leaves of Xanthium were extracted in methanol until a
color-less residue was left. Equal portions of methanol and
diethyl ether were mixed together to which three volumes of
10% sodium chloride were added. This had to be done carefully
to prevent the formation of emulsions. The combined ether
fractions were backwashed with water and dried over anhydrous
sodium sulfate. The ether was removed and the residue
dissolved in 6% KOH in methanol. After incubation overnight
at 4 C, diethyl ether was added to the methanolic solution
followed by water. By this procedure, chlorophyll was
separated from carotenoids. The ether solution was backwashed
with water and dried over anhydrous sodium sulfate. The
ether was removed and the residue dissolved in methanol. Tot-
al carotenoids were determined according to Davies (1976).

The extraction of carotenoids from roots was performed in
a similar manner. Roots were homogenized in methanol contain-
ing 100 mg/L BHT and 10 g/L sodium bicarbonate. These were
added to prevent oxidation and neutralize organic acids. Af—

ter extraction in the methanolic solution for 4 to 6 h, the

 

103

root residue was extracted twice with diethyl ether. The
methanolic solution was mixed with the ether extract and
water was added as described above. The ether was dried over
sodium sulfate, removed with a rotary evaporator, and the
residue dissolved in hexane. This yellow solution was further
purified tu/ analytical normal phase HPLC using a Porasil
column. Samples were purified by using a gradient of 0 to 66%
ethyl acetate in hexane over 40 min. The solvent was deliver—
ed by two Waters Model 510 pumps, controlled by a Waters
Automated Gradient Controller. Possible carotenoids were
detected by a Waters Lambda Max Model 481 spectrophotometer
at 450 run and collected. After removal of HPLC solvent the
samples were dissolved in hexane and their visible and UV
spectra were recorded. Spectra were obtained with a Perkin-
Elmer Lambda 7 UV-VIS spectrophotometer. Solutions were
scanned from 550 to 210 nm.

6.2.5. Mass Spectrometry. GC-MS (E1) was performed with the
methyl ester of ABA and PA as described in chapter 4 using a
3% SP-2100 column.

GC-NCI was performed using a modified Hewlett-Packard
5985A GC-MS (Her and Watson, 1985). The conditions were as
follows: a capillary on-column injector (J & W, Inc.) and a
GMCC/90 open split interface (Scientific Instrument, Inc.)
were mounted on the 5985A GC-MS. A 55 m DB-l capillary column
(0.32 mm, 0 & W, Inc.) was used with temperature programming
from 50 to 300 C at 10 C/min using He as carrier gas. The

pressure of the reagent gas methane was 1.2 X 10'4 torr.

104

DP-NCI was performed using a JEOL JMSJDOJIOHF double
focusing mass spectrometer with a JMA-DA5000 data system
using methane as the reagent gas (pressure in source 5 x 10‘6
torr). The ion source temperature was 115 (liwith a filament
emission current of 300 A and 70 eV. Approximately 2 l of
each solution used was applied to a direct probe which was
then inserted into the source and heated from 50 to 250 C at
32 C/min. The mass spectrum was scanned from 50 to 1000
daltons at 5 s/scan with a resolution (m/ m) of 1000. Sensi-
tivity of this procedure was 1 to 5 ng of ABA or PA.
6.2.6. Chemicals. 1802 (99%) was purchased from Stohler
Isotope Chemicals, Inc. (Waltham, MA) or Cambridge Isotopes
(Woburn, MA).

6.3. RESULTS

6.3.1. Carotenoid Levels in Leaves and Roots. It was rela-
tively easy to purify carotenoids from leaves compared with
roots. In roots, a compound which had absorption in the
yellow porticni of the visible spectrum, but was not a
carotenoid, necessitated further purification of the extract
by HPLC (Figure 6.1). This compound eluted in the void
volume, and appeared to be present in levels about 100 times
that (rf the carotenoids. Absorptione maxima in the visible
region of the spectrum (in hexane) for the carotenoids
purified from roots are given in Table 6.1. None appeared to
match with any of the known carotenoids (Davies, 1976),

except for the compound which had maxima at 467, 441, and 419

 

 

 

 

 

105
0.06 I;
0.03
c:
:3 l
< e
22
1
D! :. -.. :3 -

Time (min)

Figure 6.1. Purification of Xanthium root carotenoids by
HPLC.

Eight compounds having absorbance at 450 nm were resolved
using the lHHJZ system described in the text. The compound
eluting iri the void volume did not have a carotenoid type
spectrum. This figure represents analysis of 1/200 of the

total sample.

106

Table 6.1. Absorption maxima for some carotenoids found
roots of Xanthium strumarium.
Spectra were recorded in hexane. Numbers refer to peaks

Figure 6.1.

in

in

 

 

 

107

nm. These maxima are similar to those found for Violaxanthin
or neoxanthin. The total amount of carotenoids present in
roots and leaves are shown in Table 6.2. For comparison, ABA
levels in turgid and stressed tissue are also shown.
6.3.2. Incorporation of 180 into Abscisic and Phaseic Acid
During Long Term Incubations in 1302. After 6 h, there was a
prominent incorporation of an atom of 180 into one of the
carboxyl oxygen atoms. In addition, small amounts of 180 were
found in both ring oxygens of ABA (Table 6.3 and Figure
6.2). With longer incubations a steady decrease in the
molecular icni (M‘ 278) with an increase in M' 284 and 286
(two and three 180 atoms incorporated, respectively) was
observed. The base peak (M’ 280) remained constant at all
time points. The decrease in the molecular ion (M’ 278) is
due to turnover of unlabeled ABA that was present at the
beginning of the experiment. There was no indication at any
time of four 180 atoms incorporated into ABA. However, with
Xanthium roots, there was a clear indication of incorporation
of three atoms of 180 incorporated after 6 h (Figure 6.3).

Incorporation of 180 into PA was also detected during
long term incubations (Figure 6.4). In the procedure used
here, PA accumulation was not induced by a stress-rehydration
cycle. Thus, any PA formed represents turnover of ABA during
water-stress. It is for this reason that there is not a large
M'+2.

The DP-NCI spectra of ABA and PA also show a large

M‘+32. Presumably, this is due to the formation of a methanol

 

 

 

 

 

108

Table 6.2. Levels of carotenoids and ABA in roots and

leaves of Xanthium strumarium.

The ABA data are taken from Cornish and Zeevaart (1985).

Total carotenoids ABA
turgid stress
pg 9 1 dry wt ng g'1 dry wt
Root 9.4 50 1400

109

Table 6.3. Incorporation of 180 into abscisic acid in

stressed Xanthium leaves.

The values given were obtained by DP-NCI. A value of 100%

iridicates that it was the most prominent ion. All other ions

ar‘e shown relative to the base peak.

Incubation time (h) Per cent of base peak
m/e
278 280 282 284
6 78.6 100 5.5 1.1
12 35.3 100 7.0 2.0

24 27.3 100 10.5 5.4

110

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115
adduct formed during ionization in the sample chamber. The
adduct decreases as the amount of sample is lowered. The
origin of the adduct is unknown; however, it is possible that
both the methyl ester of ABA and PA could lose methanol in
the source. This methanol could then form an adduct with

another analyte in the source.
6.4. DISCUSSION

The results described are in agreement with those
presented lfl chapter 4. However, the technique used to
measure 180 incorporation here is much more sensitive than
the one used to obtain GC-MS (EI) spectra described in
chapter 4. The present results indicate that, in addition to
one atom of 180 found in the carboxyl group, low amounts of
180 incorporation into the rfing oxygens occurred. The
localization of the 180 atoms in ABA is based on a GC-MS (E1)
spectrum (Figure 6.5) of ABA isolated from stressed Xanthium
leaves incubated in 1802 for 24 h. In this spectrum, the
molecular ion indicates that three 180 atoms were incor-
porated. The ion corresponding to the side chain which
contains the carboxyl group (m/e 125) is shifted by 2 mass
units, indicating one atom of 180 is incorporated. 0n the
other hand, m/e 190, which contains the two ring oxygens and
one (Hi the carboxyl oxygens, is shifted by up to six mass
units (there are new ions at m/e 192, 194, and 196). This

indicates that both ring oxygen atoms as well as one of

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the carboxyl group oxygen atoms contain 180. While dehydra-
tion of the molecular ion (m/e 278 to 260) involves primarily
the oxygen atom in the tertiary oxygen group of ABA, there is
some evidence that the ketone oxygen atom (C-4') also plays a
part in this elimination (Gray et al., 1974). Because of
this, it is not possible to determine which of the two
oxygens in the ring contains the larger fraction of label.

The 180 incorporation into ABA suggests that there is one
large, primary precursor pool that forms ABA. This precursor
gives rise to the large M‘+2. With incubation periods greater
than 6 h, more 180 starts to appear in the ring oxygen
atoms. This incorporation implies that there may be other
compounds feeding into this large precursor pool which,
during the conversion to this pool, incorporate 180 into
positions which ultimately form the ring oxygens of ABA. 0n
the other hand, there could be three separate pathways to
ABA. In this case there would be three different precursors
which incorporate one, two, or three atoms of 180 respective—
ly during their conversion to ABA. One compound (one atom of
180 incorporated) would be rapidly and the other two slowly
converted to ABA. However, in roots, this precursor appears
to be depleted faster than in leaves because substantial
amounts of 180 were found in the ring oxygen atoms after 6
h. One would predict, therefore, that in ABA isolated
from stressed roots more 180 should be incorporated after 12
and 24 h of incubation in 1802 than with ABA from stressed

leaves.

 

 

 

118

The oxygen incorporation data suggest that carotenoids
are precursors to ABA. It is known that the hydroxyl groups
of lutein and the epoxide groups of antheraxanthin and
Violaxanthin are derived from molecular oxygen (Yamamoto and
Chichester, 1965; Yamamoto et al., 1962). The turnover of
carotenoids in green leaves is rather low (Goodwin, 1980).
Hence, the low incorporation of 180 into sites which ulti-
mately form the keto and hydroxyl groups of ABA could
represent the biosynthesis of lutein and antheraxanthin
and subsequent conversion to Violaxanthin. Since the pool of
Violaxanthin is so large, and turnover so small, the amount
of incorporation of 180 into the ring oxygens of ABA would be
predicted to be small if ABA was derived from Violaxanthin.

The amount of carotenoids found in Xanthium roots (9.4 pg
9'1 dry wt) is similar to that found in corn root caps (30
pg 9‘1 dry wt; Maudinas and Lematre, 1979). Interestingly,
the major carotenoid in corn root caps is Violaxanthin. The
fact that carotenoids are found in Xanthium roots indicates
that, if ABA is derived from a carotenoid, then the level of
total carotenoids in roots is not rate limiting. It is not
unusual that roots contain carotenoids. There are several
reports of carotenoids in roots in the literature (Goodwin,
1980; Karrer and Jucker, 1950). In commercial carrots
B-carotene predominates while in wild carrots xanthophylls
represent the majority of the small amount of carotenoids
present (Goodwin, 1980). Since the majority of the root

carotenoids described here eluted with increasing ethyl

 

 

119
acetate concentration, they are most likely xanthophylls, or
are at the very least more polar than carotenes.

The incorporation of 180 into PA was also demonstrated
(Figure 6.4; see also chapter 4). If the pool of ABA for the
conversion to PA is homogeneous, then the incorporation of
180 into PA may be predicted by knowledge of the extent of
180 incorporation into ABA and the percentage of PA that
contains no 180. The calculation is based on the fact that
ABA which contains zero, one, two, or three atoms of 180 will
give PA containing one, two, three, or four atoms of 180,
respectively. Hence, one may calculate the distribution of
180 in PA (Table 6.4) by multiplying the fraction of ABA by
(1 minus fraction endogenous PA). While this calculation
assumes that there is no discrimination between ABA molecules
(i.e. homogeneous distribution of labeled ABA in the pool
which forms PA), one may see that the predicted values are
very close to those actually obtained.

ABA levels in stressed Xanthium leaves reach a new,
steady state level after 6 h. This implies that the rate of
formation of ABA is equal to its rate of degradation. One may
distinguish between old (present before stress) and newly
synthesized ABA (present after stress) because any ABA made
after stress will contain 180. Hence, by following the
disappearence of m/e 278 (ABA present before stress), one may
calculate a half-life for stress ABA, assuming that the
ABA pool for catabolism is homogenous. That this assumption

is correct, at least for the conversion of ABA to PA, is

 

120

Table 6.4. Predicted and actual values for 180 incorporation
into phaseic acid.

The data shown were obtained from the 12 h incubation in

 

1802 and represent the contribution of each fraction to the

total amount of ABA and PA injected into the mass spectromet-

 

er.

Ion Actual per cent Predicted per cent
ABA PA PA
M' 24.5 38.8 38.8
M'+2 69.3 21.7 15.0
M‘+4 4.9 35.9 42.4
M'+6 1.4 2.8 3.0
M'+8 ---- 0.8 0.9

121
shown by the data in the last paragraph. Assuming a steady
state level of 30 pg ABA/g dry weight (Zeevaart, 1980), the

calculated (see below) turnover rate is 16.0 h (Aronoff,

 

1956).
logM1 - logM2 = — -§£%fi—
where M1 = amount of ABA unlabeled at 24 h (5.7 pg/g dry wt)
M2 = amount of ABA unlabeled at 6 h (12.7 pg/g dry wt)
M = steady state level of ABA (30 pg/g dry wt)
t = 18 h
0.35
k=zT§—FT(2.3)(30 pg/g dry wt)
= 1.3 pg/g dry wt/h
Now t1 = 0.693M = 0.693(30 pg/g dry wt)
2 k 1.3 pg/g dry wt/h

= 16.0 h

This value may be compared with that obtained from data
found in Zeevaart (1980). Since during water stress ABA is
primarily catabolized to PA, then a half-life may be calcu;
lated based on the rate of accumulation of PA. If one assumes
that ABA has reached a new, steady state level and that the
conversion to ABAGE is minimal during water stress (Zeevaart,
1980), then one obtains a value of 14.7 h for the half-life
of ABA in stressed Xanthium leaves. The differences between
the two methods is that the 180 method contains data on the
disappearence of ABA, while the PA method measures just one
aspect of ABA catabolism and neglects further metabolism of
PA and conversion of ABA to ABAGE.

In conclusion, it has been shown that during long term

incubations (12 and 24 h) of stressed Xanthium leaves in 1802

 

122

incubations (12 and 24 h) of stressed Xanthium leaves in 15
up to three atoms of 18O are incorporated into ABA. One a1
of 180 is located in the carboxyl group of ABA and two at(
are found in the ring oxygen atoms and. ABA purified f1
stressed roots of Xanthium incubated in 1802 shows a simi'
pattern, but with more incorporation of 180 into the r
positions within 6 h. Both roots and leaves of Xanthi
contain sufficient total carotenoids to account for i
amounts of ABA biosynthesized during water stress. Howew
if in roots only one carotenoid was converted to ABA, tl
the levels could be too low to support the accumulation

ABA.

6.5. LITERATURE CITED

Aronoff, S. (1956) Techniques of Radiobiochemistry.l
York: Hafner.

Cornish, K., Zeevaart, J.A.D. (1984) Abscisic acid metabol
in relation to water stress and leaf age in Xanth
strumarfum. Plant Physiol. 76 1029-1035.

Cornish, K., Zeevaart, J.A.D. (1985) Abscisic acid accumu
tion by roots of Xanthium strumarium L. and Lycopersfl
esculentum Mill. in relation to water stress. Pl:
Physiol. 79:653-658.

Davies, B.H. (1976) Carotenoids. In: Chemistry and biocm
istry of Plant Pigments, pp. 38-165, vol. 2, Goodw
T.W., ed. New York: Academic Press.

Goodwin, T.W. (1980) The biochemistry of carotenoids, voL
New York: Chapman and Hall.

Goodwin, T.W. (1980) Carotenoids. In: Secondary pL
products, Encyclopedia of plant physiology NS, vol
Bell, E.A., Charlwood, B.V., eds., New York: Spring
Verlag.

Grab], R.T., Mallaby, R., Ryback, G., Williams, V.P. (19

123

Mass spectra of methyl abscisate and isotopically
labelled analogues. J.C.S. Perkins Trans. 919-924.

Her, G. R., Watson, J. T. (1985) Quantitative methodology
for corticosteroids based on chemical oxidation to
electrophilic products for electron capture-negative
chemical ionization using capillary gas chromatography-
mass spectrometry. 1. Assessment of feasibilty in the
analysis of horse urine for dexamethasone. Anal. Bio-
chem. 151:292-298.

Karrer, P., Jucker, E. (1950) Carotenoids. New York: Else-
vier.

Maudinas, B., Lematre, J. (1979) Violaxanthin, the major
carotenoid pigment in Zea mays root cap during seed
germination. Phytochemistry 18:1815-1817.

YamaTgto, H.Y., Chichester, C.0. (1965) Dark incorporation of
0 into antheraxanthin by bean leaf. Biochim. Biophys.
Acta 109:303-305.

Yamamoto, H.Y., Chichester, C.0, Nakayama, T.O.M. (1962)
Biosynthetic origin of oxygen in the leaf xanthophylls.
Arch. Biochem. Biophys. 96: 645-649.

 

Chapter 7.
INCORPORATION OF DEUTERIUM INTO
ABSCISIC ACID, STEROLS, AND CAROTENOIDS FROM DEUTERIUM OXIDE

 

124

 

125
7.1 INTRODUCTION

A major problem in investigating the biosynthesis of
ABA is the inability to incorporate precursors, such as MVA,
into ABA. A reason for this is the competition of compounds,
such as carotenoids and sterols, for MVA and other inter—
mediates in the terpenoid biosynthetic pathway. Another
explanation for low MVA incorporation is that ABA is derived
from a pool removed from the general terpenoid pathway (i.e.,
the indirect pathway).

A way of testing this is to incubate leaves in the
presence of deuterium oxide (2H20). Deuterium oxide has the
advantage as an in vivo label of quickly entering all
subcellular compartments. Deuterium from 2H20 will be
incorporated into intermediates in the terpenoid biosynthetic
pathway at three points: (a) by exchange into acetoacetyl
coenzyme A, (b) during the isomerization of isopentenyl
pyrophosphate into dimethylallyl pyrophosphate, and (c) into
the C-5 of MVA via NADPH+ (Lehninger, 1970). In addition, one
deuterium should be incorporated during the cyclization of
farnesyl pyrophosphate into what will be the 5’ position of
ABA (Britton, 1986). ABA synthesized from an acyclic precurs-
or should, therefore, contain one deuterium at C-5'. ABA
arising from a post-cyclized precursor (such as a carotenoid)
would not contain any deuterium with short-term incubations,
but rather would become slowly labeled with several deuterium

atoms during long-term incubations (as would carotenoids).

 

 

126

In studies of de novo protein synthesis utilizing 2H20 it
has been noted that if H20 is present (at amounts of 10 to
20%) protons rather than deuteriums will be used prefer-
entially because of isotope discrimination. Little or no
deuterium is incorporated if less than 40% 2H20 is used
(Chrispeels and Varner, 1973). Because of this, and the fact
that 2H20 might be toxic at high concentrations, it is
necessary to also measure deuterium incorporation into
another compound known to be synthesized during the incuba-
tion period. Therefore, the presence of deuterium in the
plant sterols stigmasterol and sitosterol was also determined
by GC-MS.

To measure deuterium incorporation it is necessary to
use a mass spectrometer. Because the levels of ABA will be
quite low in turgid leaves, a requirement is that the mass
spectrometer have high sensitivity. The procedure used must
also measure the molecular ion as the major ion. ABA has a
small molecular ion, and analysis of the amount of incorpora—
tion in fragment ions is complicated. A technique that meets
these criteria is GC-NCI (Watson, 1985). This procedure
affords high sensitivity towards electron withdrawing
compounds such as ABA. Futhermore, a characteristic of CI
mass spectra is that they contain fewer ions than correspond-
ing EI mass spectra, with the molecular ion (M') predominat-
ing. However, GC—NCI (and most other mass spectroscopy
techniques) cannot be used with structures of high molecular

weight, low volatility, or poor electron withdrawing capabil-

 

 

127
ity, such as carotenoids. For these compounds, FAB may be
used to introduce the analyte into the ionization chamber
(Watson, 1985). FAB utilizes an energetic beam of atoms to
cause desorption/ionization of nonvolatile substances
dissolved in a viscous, low vapor pressure matrix.

We decided to incubate half-expanded Xanthium leaves in
80% 2H20 for 6 h or 4d and determine if deuterium was
incorporated into ABA, sterols, and carotenoids. Xanthium
leaves at this age are ideal for this study because they have

a high level of ABA and are synthesizing sterols.

7.2. MATERIALS AND METHODS

7.2.1. Plant Material. Half-expanded leaves of Xanthium
strumarium were allowed to take up ZHZO (80% enriched) for 3
h Via the transpiration stream. This allowed for complete
replacement of tissue water with the mixture of 2H20 and H20.
Leaves were then placed in a plastic bag for either 6 h or 4
d. Each experiment was performed twice.

7.2.2. Extraction and Purification of Abscisic Acid. The
plant material was homogenized in methanol until a colorless
residue was left. Water was added to the extract and the
methanol removed to give an aqueous extract. This aqueous
fraction had its pH adjusted to 7.0 and was partitioned 3
times against diethyl ether, giving free sterols in the ether
fraction. The aqueous extract had its pH lowered to 2.5 and

was partitioned against diethyl ether. This gave conjugated

 

128

sterols in the aqueous fraction and ABA in the diethyl ether
fraction. The two sterol fractions were combined and further
purified as described below. The ABA fraction was reduced to
an aqueous residue which was frozen and lyophilized. It was
then purified as described in chapters 9 and 4. After methyl-
ation with ethereal diazomethane, it was analyzed by GC-NCI.
7.2.3. Purification of Sterols. The combined fractions were
reduced to an aqueous fraction, frozen, and lyophilized. The
dry residue was dissolved in 100 ml of 10% KOH in 85%
methanol and was refluxed for 1 h. After cooling, the
hydrolysate was concentrated to a small volume and 40 ml
water added. This aqueous solution was then extracted with 3
volumes of diethyl ether. The combined diethyl ether extracts
were washed with water and dried over anhydrous sodium
sulfate. The solvent was removed to give the crude non-sapon—
ifiable sterol fraction.

The free fraction was dissolved in 6 ml acetone:diethyl
ether (1:1). To this solution was added 3 ml of an ethanolic
digitonin solution (3 mg of digitonin in 1.4 ml water and 1.6
ml ethanol). The solution was shaken at room temperature for
6 h and stored at 4 C overnight. The solution was centrifuged
and supernatant removed by pipetting. The resulting precipi-
tate was washed 3X with cold diethyl ether.

The digitonides were dissolved in 3 ml pyridine and
heated at 70 C for 1 h. The resulting solution was extracted
with 3 10 ml portions of diethyl ether. The combined ether

fractions were washed with 30 ml saturated cupric sulfate,

 

 

129

followed by 30 ml water, and were dried over anhydrous sodium
sulfate.

After evaporation to dryness, the residue was fraction-
ated by column chromatography. The residue was dissolved in a
small amount of diethyl ether to which 1 g of celite was
added. The ether was evaporated depositing the sterols onto
the celite which was placed on top of a neutral aluminum
oxide (grade III, 60 9) column. The column was eluted with
increasing amounts of diethyl ether (0, 1, 2, 6, and 30%) in
hexane; 4-demethylsterols (sitosterol and stigmasterol)
eluted in the 30% ether fraction.

After reducing the column eluate to a small volume, the
sterols were transferred to small vials and dried over P205
for 12 h. The sterols were dissolved in 20 pl pyridinezHMDS:
TMCS (9:3:1) and were heated at 60 C for 15 min. The result—
ing TMSi sterol ethers were then analyzed by GC-MS.

7.2.4. Purification of Carotenoids. In separate experiments,
leaves of Xanthium were extracted in methanol until a
color-less residue was left. Equal portions of methanol and
diethyl ether were mixed together to which three volumes of
10% sodium chloride were added carefully to prevent emul-
sions. The combined ether fractions were backwashed with
water and dried over anhydrous sodium sulfate. The ether was
removed and the residue dissolved in 6% KOH in methanol. Af-
ter incubation overnight at 4 C, diethyl ether was added to
the methanolic solution followed by water. By this procedure,

chlorophyll was separated from carotenoids. The ether

 

 

 

130

solution was backwashed with water and dried over anhydrous
SOdTlJm sulfate. After removal of the ether. the carotenoid
residIJe was dissolved in a small amount of hexane and applied
to ttie top of an aluminum oxide column (neutral, grade III,
30 g) . Carotenoids were eluted from the column with increas-
ing axnounts of diethyl ether in hexane. Identification of the
catwatenoid in each fraction was by absorbance maxima and
Orwier of elution from the column.
7-2.5. Mass Spectroscopy. GC-MS was performed as described in
Cllapter 4 using a 3% SP-2100 column, except the column
temperature was 275 C.

GC-NCI was performed using a modified Hewlett-Packard
5985A GC-MS (Her and Watson, 1985). The conditions were as
fol‘lows: a capillary on-column injector (J & W, Inc.) and a
GMCZC/90 open split interface (Scientific Instrument, Inc.)
”VEPe mounted on the 5985A GC—MS. A 55 m DB—l capillary column
(0.32 mm, J & W, Inc.) was used with temperature programming
FFOnI 50 to 300 C at 10 C/min using He as carrier gas. The
pressure of the reagent gas methane was 1.2 X 10‘4 torr.

FAB was performed with a Varian MAT CH5-DF double
fOcusing mass spectrometer (Ackermann et al., 1984) using a
Sample matrix of thiodiglycol:dithiothreitol:dithioerythritol
(1:1:1). The fast atom gun was operated to give 6 keV Xe

Atoms with a pressure inside the ion source of 8 X 10'6 torr.

 

131
7.3. RESULTS AND DISCUSSION

In most vascular plants, sitosterol (stigmasta-S-en—3B-
ol) and stigmasterol (stigmasta-S,22—diene-3B-ol) are the two
major 4—demethylsterols. Both sterols have 29 carbon atoms
and differ only in that stigmasterol has a double bond
at C-22 of its C-17 side chain, whereas sitosterol has a
saturated side chain. Sitosterol and stigmasterol were
identified in purified Xanthium extracts by GC-MS both as
free sterols and as their TMSi-ether derivatives. The
derivatives gave better separation with GLC and were used for
further analysis of deuterium content. Since the molcular ion
is the best indicator of deuterium incorporation, samples
containing sterols were scanned during GC-MS from m/e 290 to
m/e 350. Analysis of sitosterol and stigmasterol from leaves
incubated in the presence of 2H20 indicated that deuterium
was indeed incorporated into both molecules (Figure 7.1).
Although the amount of deuterium incorporation is low, more
deuterium was found in both compounds after 4 d of incubation
than after 6 h. Because of the structural similarities
between the compounds, it has been postulated that a common
biosynthetic pathway, sitosterol-————————stigmasterol,
exists. However, the evidence for this pathway is not
unequivocal, and it has been proposed that two different
pathways exist in dicots and monocots (Grunwald, 1986). The
data here indicate that after a 6 h incubation in the

presence of 2H20 more deuterium was present in sitosterol

 

132

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

'Sitosterol Stigmasterol
1004 1oo-4
a b
.J
5
.5m 5m
0
a:
ll Y I r I I [ ‘ v v I l I If!
‘00 410 440 460 ‘00 ‘20 $40 460
1004 ~ 100
c d
x25
‘5 l—x13 . |—
'1 so- so-
3
cc
l I I l I I 1 x I I 1 I I l
400 420 440 ‘60 400 420 440 460
1001 100
e i
.J
E I—X7
.:so
0 50
I
1 U“ L I I I I " 1 1 i l l I
400 420 440 460 400 420 440 480
m/e

Figure 7.1. IncorSECZtion of deuterium from deuterium oxide
inyo sitosterol and stigmasterol.

Standard sitosterol (M+ 396) and stigmasterol (M+ 394) are
shown in the top panels (a and b, respectively). Deuterium
was incorporated into sitosterol (c and e) and stigamsterol

(d and f) after 6 h (c and d) and 4 d (e and f).

 

 

 

133

than in stigmasterol. This would be in accord with the
pathway described above. It is possible that the results
described by Grunwald (1986) could represent metabolic
channeling. The deuterium incorporation into sterols indi-
cates that incubation in 80% 2H20 was not toxic to Xanthium
1 ea ves because the terpenoid biosynthetic pathway was
operating.

Similar to the incorporation of deuterium into sterols,
l 1' ttle deuterium was found in ABA analyzed by GC-NCI (Table
7 - 1 ) . However, slightly more deuterium was incorporated into
ABA after 4 d of incubation in 2H20 than after 6 h. There
were two fractions, one (m/e 279) that had incorporated one
deuterium, and another (m/e 284-290) that had 6-12 deuterium.

No deuterium was detected in B-carotene [Xmax, hexane,
470 , 446, 422(shoulder) nm], a precursor to other caroten-
oids, such as lutein and Violaxanthin. Analysis by FAB of
0th er carotenoids was not successful, possibly because of
mat rix difficulties. Successful use of FAB depends on many
Parameters and is still in the development stage. Another
Problem associated with FAB is high background. However,
B-Carotene gave a clear m/e 535 (M+—H). Because of the high
baC kground, it could be argued that some deuterium was
inCOrporated; if this was true then the amount of incorpor-
atl'On was very low (at least less than 5%).

If one assumes that ABA is derived from carotenoids, then
the fact that some deuterium was incorporated into ABA

and none appeared to be in B-carotene can be explained in

 

 

134

T'zalale 7.1. Incorporation of deuterium from deuterium oxide
into abscisic acid.
The extent of deuterium incorporation after 6 h or 4 d
v:a.s determined using GC-NCI. For comparison, standard ABA is

al so shown.

ABA

m/e Standard 6 h 4 d

278 100.00 100.00 100 00
279 11.39 15.66 15.52
280 4.04 3.95 3.96
281 0.33 0.52 0.43
282 ---- 0.13 0.09
283 ---- 0.04 0.05
284 -—-- 0.18 0.29
285 ---— 0.14 0.29
286 ---- 0.08 0.17
287 —--- 0.03 0.12
288 ---- 0.13 0.17
289 —--- 0.07 0.06
290 ---- 0.09 ----
291 ---- —--- O 04
292 ---- 0.09 0 07
293 ---- 0.04 0 08
294 --—- 0.04 O 07
295 —--— —--- 0.05
296 ---— ---- ----
297 ——-- --—- --—-
298 -—-— -—-- ----

 

135

several ways. One explanation is that deuterium was incorpor-

ated into B-carotene (and not observed when analyzed by

FAB). Another is that only a small fraction of the total

B—carotene was labeled with deuterium (in amount,

th at found

similar to
in ABA) and that the bulk B-carotene (unlabeled)

was observed by FAB. Another possibility is that the initial

as sumption (ABA arises from carotenoids) is incorrect.

Deuterium was incorporated into sterols, and the

terpenoid pathway did not seem to be deleteriously affected

by 2H20. However, no information was obtained on how fast ABA

and sterols were being synthesized. Half—expanded leaves have
a high rate of sterol biosynthesis. On the other hand, these

1 eaves were not stressed, so the turnover of ABA was probably

1 Ow. It was expected that more deuterium should have been

i ncorporated into ABA.

and 4 d,

Since the time points were 6 h

the possibility exists that the leaves became

Senescent in the course of the experiment. With these

CaVeats, the data indicate that the majority of ABA does not

aY‘ise from the cyclization of farnesyl pyrophosphate. If this

had happened, then ABA would have a large M‘+1, and this was

hot observed. It appears that one ABA fraction is derived

1:l‘om precursors that are removed from the general terpenoid

Pathway (m/e 284 to m/e 290), and that the other fraction

ml'ght arise from farnesyl pyrophosphate (m/e 279).

 

 

136
7.4. LITERATURE CITED

A<: kLermann, B. L., Watson, J. T., Newton, J. F., Hook,
J. B.,Braselton, Jr., W. E. (1984) Application of fast
atom bombardment mass spectrometry to biological sam-
ples: analysis of urinary metabolites of acetaminophen.
Biomed. Mass Spectrom. 11:502-511.

E3!‘ 1 tton, G. (1976) Later reactions of carotenoid biosynthe-
sis. Pure and Appl. Chem. 47:223-236.

Clfi‘r-ispeels, M. J., Varner, J. E. (1973) A test for de novo
synthesis of enzymes in germinating seeds: density label-
ing with 020. In: Molecular techniques and approaches
in developmental biology, pp 79-92, Chrispeels, M. J.,
ed. New York: John Wiley and Sons.

Gl"L1nwald, C. (1986) In vivo synthesis of stigmasterol in
Nicotiana tabacum. Phytochemistry 24:2915-2918.

I“let‘r', G. R., Watson, J. T. (1985) Quantitative methodology
for corticosteroids based on chemical oxidation to
electrophilic products for electron capture-negative
chemical ionization using capillary gas chromatography-
mass spectrometry. I. Assessment of feasibilty in the
analysis of horse urine for dexamethasone. Anal. Bio-
chem. 151:292-298.

L-ehninger, A.L. (1970) Biochemistry. New York: Worth.

wafltson, J. T. (1985) Introduction to mass spectrometry. New
York: Raven.

 

 

Chapter 8
THE ROLE OF XANTHOXIN IN ABSCISIC ACID BIOSYNTHESIS

137

 

138
8.1. INTRODUCTION

ABA is a sesquiterpenoid and appears to be derived from
three MVA residues. However, the structure of ABA is quite
different from all other known sesquiterpenoids, and in fact
resembles the terminal ring portions of many carotenoids.

Taylor and Smith (1967) showed that exposure of caroten-
Oids to bright light on damp filter paper gave rise to an
neutral compound which inhibited cress seed germination. Of
the carotenoids tested, the greatest amount of inhibition
occurred when Violaxanthin and neoxanthin were photo-oxidized
(Taylor, 1968). Extensive purification of the photo-oxidized
Products of Violaxanthin indicated that the inhibitor was a
mixture of cis,trans- and trans,trans—xanthoxin (Taylor and
Burden, 1970; Burden and Taylor, 1970). In addition, butenone

and loliolide were also identified.

It was later shown that cis,trans- and trans,trans-xan-_

thOxin could be efficiently formed from Violaxanthin,
neOxanthin, antheraxanthin and lutein epoxide by zinc
pelf‘manganate (Taylor and Burden, 1972). The chemical conver-
SIon of xanthoxin to ABA was also reported (Taylor and
BuY‘den, 1972). The biological activity of cis,trans-xanthoxin
Was greater than that of the trans,trans isomer in several
asSays (Taylor and Burden, 1972). Both isomers of xanthoxin
have been detected in several plants, including ferns, a
1 1‘Verwort, and wheat (Firn et al., 1972). Firn and Friend
(1972) showed that the isomers of xanthoxin, butenone, and

101 iolide could be produced by soybean lipoxygenase in the

139
presence of linoleate, indicating that these compounds could
be produced without the action of oxidizing agents or light.

ABA levels showed a large increase after plants had been
treated with solutions of cis,trans—xanthoxin (Taylor and
Burden, 1972). To determine whether the increase was due to
conversion to ABA rather than a stimulation of synthesis,
14C—cis,trans-xanthoxin was prepared and fed to tomato shoots
(Taylor and Burden, 1973; Taylor and Burden, 1974). After
8 h, 14C-cis,trans—xanthoxin was converted to ABA (10.8%)
and PA (4%). In addition, a small amount of conjugated
ci$,trans-xanthoxin acid was also formed (Taylor and Burden,
1974). When applied to tomato fruits and pea seeds, "small
but significant" amounts of radioactivity were also found in
ABA (Taylor and Burden, 1974).

Thus, xanthoxin appears to be an endogenous compound in
many plants, and it is converted to ABA. Because of these
Considerations, xanthoxin is a likely precursor to ABA in
h‘igher plants and thus could be a key intermediate between
Calr'otenoids and ABA. However, the possibility exists that the
19Vels of xanthoxin may be overestimated due to the lability
of: xanthophylls. We decided to determine if the extraction
pr‘Ocedure (anaerobic vs. aerobic) has any effect on xanthoxin
meaSurement. In addition, we wanted to determine if xanthoxin
Was as labile as described by Shen-Miller et a7. (1982).
TheSe workers showed that xanthoxin was rapidly destroyed at
r00"! temperature or -2 C. Finally, if xanthoxin arises from

an Oxidative cleavage of a xanthophyll, the mechanism should

¥

 

140
be similar to that operating in vitamin A biosynthesis
(Britten, 1983). Leaves were therefore incubated in the
presence of 1802 and the amount and location of 18O incorpor-

ated into xanthoxin was determined.
8.2. MATERIALS AND METHODS

8.2 -1. Plant Material. Spinach plants (Spinacia oleracea cv
Sav<3y Hybrid 612) were grown under short day as described
(Zetevaart, 1971) and transferred to long days and used after
10 to 12 d.

For experiments involving incorporation of 18O, spinach
leiives were detached from plants and incubated in an atmo-
Sptiere containing 80% N2 and 20% 1802 for 8 h as described in
Chapter4.

8o2.2. Chemicals. Xanthoxin was initially purchased from
Fllea. When this supply was depleted, xanthoxin was prepared
according to Taylor and Burden (1970) from Violaxanthin
1.Solated from Xanthium strumarium L. leaves.

8..2‘.3. Extraction and Purification Procedures. In the 180
i nCorporation experiment, spinach leaves were homogenized and
eXliracted according to Zeevaart (1974). After partitioning
aglai nst ethyl acetate to give neutral (xanthoxin) and acidic
(A‘BIX) fractions, the acidic fraction was further purified as
de$<2ribed in chapters 4 and 9. The neutral fraction contain-
ir'g xanthoxin was further purified by semi-preparative

reVerse phase HPLC (20 to 60% ethanol in water in 30 min with

 

141
a 2 min hold at the beginning of the run, flow rate 2.5
ml/min). The fraction which eluted between 20.5 and 23.5 min
was collected, reduced in volume, and lyophilized. If further
Purification was necessary, TLC was performed on the extract
as described in Zeevaart (1974)

To determine if extractions done in atmospheres contain-
ing 02 gave elevated levels of xanthoxin, some extractions
were performed entirely under N2. In addition, unless
otherwise noted, all solvents were purged of 02 by vigorously
bubbling N2 through them. Leaf material was detached from
Plants and crushed in liquid NZ to which 250 ml of methanol
was added. After extraction overnight at 4 C in the dark,
further purification was performed in a plexiglas chamber in
an atmosphere of N2. The tissue was homogenized, filtered,
and the residue washed with 150 ml methanol. To this was
added sufficient water to make an 80% aqueous methanol
50] ution. This solution was then rapidly applied to a
pr'eparative Bondapak C18 column (1 x 25 cm, particle size
37‘75 p) using a Beckman Accu-Flo pump. With this procedure,
p1 ant pigments and waxes are bound to the column packing and
xanthoxin elutes from the column (Shen-Miller et al.,
1982). After each sample the column is washed with 100%
methanol and equilibrated with 80% methanol. The column
e‘lllate was reduced to a small volume by a rotary evaporator
equ'ipped such that the vacuum release was done with N2
instead of air. This aqueous residue was then placed in a

pjeXiglas chamber with a 100% N2 atmosphere and partitioned 3

L..

 

 

142

times against diethyl ether. Water was added to the ether
phase and the ether removed leaving an aqueous fraction which
was frozen and lyophilized. After lyophilization, all further
operations were performed in room air. The dry residue was
then subjected to reverse phase HPLC using a Waters Radial
Pak 411 C18 cartridge (10 to 70% methanol in water in 45 min
with a 2.5 minute hold at the beginning of the run, flow rate
2.0 ml/min). Two Waters Model 510 pumps were controlled by a
Waters Automated Gradient Controller with the absorbance at
282 nm monitored using a Waters Lambda Max Model 481 spectro-
Photometer. This column was able to resolve the isomers of
xanthoxin (retention t,t-xanthoxin 31.00 to 32.00 min,
C,t-xanthoxin 32.75 to 33.75 min). The isomers were collected
Separately, reduced in volume, frozen, and lyophilized. As a
COntrol, the same procedure was followed as described above
eXcept that all procedures were carried out in room air. This
experiment was performed three times with two replicates per
t‘F‘Eatment.

To increase the sensitivity of xanthoxin to GC—ECD, it
Was derivatized with 0-(2,3,4,5,6-pentafluorobenzyl)hydroxyl-
alrnl'ne hydrochloride (Koshy et al., 1975). This reagent
r‘eacts with the aldehyde group of xanthoxin to give an
oxime. To the dry residue was added 100 pl of the derivatiza—
ti°n reagent in pyridine (2 mg/ml). Vials were heated at 65 C
for 30 min and the pyridine removed with a stream of N2 at 45
C“ One ml of hexane was added which was washed with 500 pl

W . .
ater. The water was removed and the hexane dried over sodium

¥

 

143
sul f‘ate.
8. 2-4. Determination of Xanthoxin Stability. To determine the
stability of xanthoxin, identical aliquots were stored at
room temperature (24 C), cold room temperature(4 C), re-
frigerator temperature (4 C), -20 C, and -80 C. MeABA was
a1 so present in this solution as an internal standard to
correct for evaporation. Xanthoxin was not derivatized in
this procedure.
8-2.5. Gas Chromatography. Underivatized xanthoxin was
detected by GC-ECD using a Hewlett-Packard 5840A gas chro-
matograph equipped with a 10 m Hewlett-Packard methyl silicon
wi de bore column (inner diameter 0.53 mm) in the splitless
mode. Column temperature was 175 C, column pressure 9 kg/cmz,
Ar-methane (make up gas) flow 60 ml/min.

The oxime derivative of xanthoxin was also detected by
GC-ECD. However, due to the large, bulky nature of the
derivative, it had a much longer retention time, so that the
analysis was performed with a 1.5% OV-I column operated
isothermally at 225 C (2 m, 80-120 mesh, Gas chrom Q, 30
ml/min Ar-methane flow).

3-2.6. Mass Spectrometry. To detect 180 incorporation in ABA
(as the methylated derivative) and underivatized xanthoxin,
mass spectra were obtained by direct injection on a modified
Hewlett-Packard 5895A mass spectrometer (Her and Watson,
1985) in the positive EI mode. Samples were injected with the
column (55 m DB-l) initially at 50 C. Following injection,
the column was rapidly heated to 150 C at 30 C/min after

144
which it was slowly heated to 240 C at 10 C/min.
The EI mass spectra (GC-MS) of the oxime derivative of
xanthoxin were obtained on a Hewlett-Packard 5895A mass

spectrometer. Gas chromatography was performed as described

above in section 8.2.5.
8.3. RESULTS

8- 3.1. Stability of Xanthoxin. As seen in Figure 8.1, under
al 1 conditions tested, xanthoxin was stable. No isomerization
of: c,t-xanthoxin to t,t-xanthoxin occurred.

8- 3.2. Incorporation of 180 into Xanthoxin and Abscisic
Acid. GC-MS (EI) analysis of xanthoxin isolated from spinach
leaves incubated in an atmosphere containing 1802 indicated
that no incorporation of 180 had occurred (Figure 8.2). On
the other hand, analysis of ABA isolated from the same leaves
indicated that incorporation into this molecule had occurred
(Figure 8.3).

8-3.3. Xanthoxin Levels in Tissues Extracted in Air and
Nitrogen. As is seen when making the acylated derivative of
xanthoxin, derivatization with 0-(2,3,4,5,6-pentafluoro-
benZyl)hydroxylamine hydrochloride gave two isomers. However,
Since the two isomers were separated by HPLC before derivati-
zation, it is possible to quantitate the levels of the
isomers actually present in the extract, not those created
during derivatization. The cis,trans oxime derivative

of xanthoxin gave a mass spectrum with a molecular ion (M+

 

 

 

 

 

 

145
2
_ a " - ,4:
< -
CD 1.5-
<
0)
2
\
.E
X
o
E 1 -
c
o
x
V
.9.
6
CE .5 -
:- 3 \“3’l————a
O I l I l
O l 2 3 4- 5
Day
-—:—- —20 C
—x—— —80 C

-e.— Cold Room
+ Room Temp.
—e— 4 C

Figure 8.1. Stability of xanthoxin under different environ-
mental conditions.
Plotted are the ratio of cis,trans-xanthoxin (upper lines)

and trans,trans-xanthoxin (lower lines) to MeABA.

 

146

 

so: I

2: r" ‘ .‘ I] 4411' . III'I.I:I. III':1|"111111
20 4e

         

Rel.lnt.

 

 

 

 

F1'gure 8.2. Mass spectrum of xanthoxin isolated from spinach
leaves incubated in 1802 for 8 h.
Standard xanthoxin has a molecular ion of 250, and is

identical to the spectrum shown here. There is no incorpora-

tIOn of 18O.

 

 

3;:‘1'09— ”
z" I
ahsfid ‘

50..

 

4o 99

r>x10
' 190-

:17:ch ‘

50-

 

 

 

 

147

 

140

L .LWLI 1!- .

 

uuuuuuuuuu I' ' ”

190

L1... LI
1

__ -.—

Figure 8.3.
incubated 18oz for 8 h.

L l u, .1 ILLLLL LL.L..,...J.

I

(m/o) amu

Mass spectrum of ABA isolated from spinach leaves

ABA Wad: purified from the same extract from which xanthoxin

was

xanthox «i n,

Ptlrified in Figure 8.2. Contrary to that seen with

there is incorporation of 180.

 

 

 

 

148

445) and fragmentation pattern consistent with the presumed
structure of the derivative (Figure 8.4). Absorption in the
UV region gave a maximum at 280 nm (xanthoxin has a maximum
at 282 nm). A standard series was created using the extinc-
tion coefficient for xanthoxin at 282 nm. Since the addition
of: the derivative did not change the absorption maximum, it
probably would not interfere with the extinction coeffici-
ent. The detection limit on GC-ECD was 100 pg/pl.

Using this derivative, values were obtained for xanthoxin
from spinach leaves incubated in room air or N2 (Table
8. 1). As can be seen, the levels of xanthoxin in spinach
Ieaves extracted under N2 were about 13X lower than those

found in extracts from leaves prepared in air.
8.4. DISCUSSION

Contrary to the results of Shen-Miller et al. (1982),
both isomers of xanthoxin were highly stable under all
conditions tested. The only differences between the procedure
used here and that in Shen-Miller et a1. (1982) is that these
workers stored xanthoxin in methanol, whereas in the present
work it was stored in ethyl acetate. However, xanthoxin was
Stored in methanol at both 4 C and -20 C with no apparent
d"Z‘Sll‘éldation for several months.

If xanthoxin is a degradation product of violaxanthin, it
Should have one atom of 18O inserted into the aldehyde group,

similar to what happens in the formation of vitamin A from

 

 

149

 

 

I I I '
O 4° 50 60 7O 80 90 100 11° 150 130 140 150 13° 17°

 

 

 

 

 

 

IUU
. < 181
~o— _ 250
C -
_
50-
-—. 245
:53
Q: - 230
(I: IHhJLII 1nd; I l I I :L I I ‘ 1 I I [L I
I | I a
1.70 180 2.50 200 2:0 220 230 240 so 260 270 230 290 300 310
133
J
..
‘ x 1.4 443
50‘ 427
j 313 3“ sea 405
11 II 1 I LL I

 

I
| I I . I I I l I I I I I
310 320 330 340 350 360 370 330 390 400 410 420 430 440 ‘50

m/e

Figuy-
b e 8.4. Mass spectrum of the 0-(2,3,4,5,6-pentafluoro-
enz .

yl )Iiydroxylamine hydrochloride derivative of xanthoxin.

SIIOWn -
l S the mass spectrum for the cis,trans derivative.

 

150

T’a.ble 8.1. Xanthoxin levels from spinach leaves extracted
i n air or N2.
l'h is experiment was performed 3 times with 2 replicates per

tr‘eatment. Similar results were obtained each time.

ng g'1 fresh wt

cis,trans trans,trans

 

 

 

 

151

B—carotene (Britton, 1983). However, as evident from the
mass spectrum of xanthoxin isolated from leaves extracted in
ai r, this did not occur (Figure 8.2). As a control, the
incorporation of 180 into ABA was detected (Figure 8.3). In
other words, 180 incorporation was detected in ABA, but not
i n its putative precursor xanthoxin. There could be several
reasons for this. One is that, in spite of all the evidence
that xanthoxin is a precursor, in reality xanthoxin is not a
true endogenous precursor to ABA. This would seem unlikely
wi th all the data that implicate carotenoids as precursors to
ABA (i.e carotenoid-less mutants, inhibitors, and the 18O
incorporation patterns in ABA). Another possible explanation
is that xanthoxin containing 180 is diluted by xanthoxin
produced from Violaxanthin (or other xanthophylls) during
extraction and purification. Because the purification and
extraction was done in room air, any xanthoxin produced
by Violaxanthin degradation would contain 160 instead of 18O
in its aldehyde group. Due to the relative lack of sensi-
tivity and large fragmentation that occurs with GC-MS (EI),
low amounts of incorporation would not be detected. This
result suggests that much of the xanthoxin seen in plants
could be an artifact caused by the purification procedure.

This was tested by extracting xanthoxin in room air and
N2 (Table 8.1). As can been seen, the levels of xanthoxin are
IOWEY‘ in the leaves extracted under N2. The values obtained
for- air extraction are similar to those reported by Zeevaart

(1974) - This same situation is seen in bean leaves extracted

 

152

uticiear conditions which degrade violaxanthin, yet do not
degrade xanthoxin (Walton, pers. com.). When bean leaves
i n ClJbated in 1802 were extracted carefully to prevent
V'i olaxanthin breakdown, no 180 was found in xanthoxin
(Vlalton, pers. com.). However, their method of detection

rni ght not be sensitive to low levels of incorporation.
A better chance to detect 180 incorporation would be to
u s e GC-NCI (or DP-NCI). Xanthoxin is an electrophilic com—
pc>und, although not as good as ABA. With the higher sensitiv-
itgy and low fragmentation that occurs with NCI, a low amount
O‘F incorporation would still be detected because, assuming
ttlat the molecular ion was the base peak, incorporation of 1

‘to 2% would still be detected as the M'+2.
8.5. LITERATURE CITED

Br‘itten, G. (1983) The biochemistry of natural pigments. Cam-
bridge: Cambridge University Press.

Bllrden, R.S., Taylor, H.F. (1970) The structure and chemical

transformations of xanthoxin. Tetrahedron Lett. 47:4071-
4074.

FT rn, 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.

He)”, G. R., Watson, J. T. (1985) Quantitative methodology
for corticosteroids based on chemical oxidation to
electrophilic products for electron capture-negative
chemical ionization using capillary gas chromatography—
mass spectrometry. 1. Assessment of feasibility in the
analysis of horse urine for dexamethasone. Anal. Bio-
chem. 151:292-298.

 

 

153

Kc>shy, K.T, Kaiser, D.G., VanDerSlik, A.L. (1975) O—(2,3,4,5,
6-pentafluorobenzyl)hydroxylamine hydrochloride as a
sensitive derivatizing agent for the electron capture
gas liquid chromatographic analysis of keto steroids. J.
Chromatogr. Sci. 13:97-104.

Riaschke, K., Firn, R.D., Pierce, M. (1975) Stomatal closure
in response to xanthoxin and abscisic acid. Planta
125:149-160.

Stien-Miller, J., Knegt, E., Vermeer, E., Bruinsma, J. (1982)
Purification and lability of cis-xanthoxin and its
occurrence in phototropically stimulated hypocotyls of
Helianthus annuus L.. Z. Pflanzenphysiol. 108:289-294.

T'a ylor, H.F. (1968) Carotenoids as possible precursors of
abscisic acid in plants. In: Plant growth regulators,
S.C.I. Monograph Number 31, pp. 22—35, London: Society of
Chemical Industry.

Tiaylor, H.F., Burden, R.S. (1970) Identification of plant
growth inhibitors produced by photolysis of violaxan-
thin. Phytochemistry 9:2217-2223.

Tdaylor, H.F., Burden, R.S. (1970) Xanthoxin, a new naturally
occurring plant growth inhibitor. Nature 227:302-303.

I'aylor, H.F., Burden, R.S. (1972) Xanthoxin, a recently
discovered plant growth inhibitor. Proc. R. Soc. Lond. B
180:317-346.

szylor, H.f4, Burden, R.S. (1973) Preparation and metabolism
of 2—[ C]—cis,trans-xanthoxin. J. Exp. Bot. 24:873-880.

Taaylor, H.F., Burden, R.S. (1974) The biochemistry of
xanthoxin and its relationship to abscisic acid. In: Bio-
chemistry and chemistry of plant growth regulators,
pp. 187—196, Schreiber, K., Schutte, H.R., Sembdner, G.,
eds., Halle (Saale): Academy of Sciences of the German
Democratic Republic.

Tayior, H.F., Smith, T.A. (1967) Production of plant growth
inhibitors from xanthophylls: a possible source of dor-
min. Nature 215:1513-1514.

Zeevaart, J.A.D. (1971) Effects of photoperiod and endogenous
gibberellins in the long day rosette plant spinach. Plant
Physiol. 71:477-481.

Zeevaart, J.A.D. (1974) Levels of (+)-abscisic acid and
xanthoxin in spinach under different environmental condi-
tions. Plant Physiol 53:644-648.

 

 

Chapter 9.

ABSCISIC ACID ACCUMULATION IN SPINACH LEAF
SLICES IN THE PRESENCE OF PENETRATING AND NON-
PENETRATING SOLUTES

 

 

154

 

 

 

 

Plant Physiol. (1985) 77. 25-23
0032-0889/85/77/0025/04/50 I .00/0

155

Abscisic Acid Accumulation in Spinach Leaf Slices in the
Presence of Penetrating and Nonpenetrating Salutesl .

Received for publimu'on July 6. 1984 and in revised form September 14. 1984

ROBERT A. CREELMAN AND JAN A. D. ZEEVAART‘
.-11$U DOE Plant Research Laboratory. .1! zc/ugan State Unnersm. East Lansmg. .lfz'c/iigan 488.. 74

ABSTRACT

Abscisic acid (ABA) accumulated in detached. wilted leaves of spinach
(Spinacia airmen L. cv Savoy Hybrid 612) and reached a maximum
level within 3 rod hours. The increase in ABA over that found in detached
turgid leaves was approximately lO-l'old. The effects of water stress could
be mimicked by the use of thin slices of spinach leaves incubated in the
presence of 0.6 molar mannitoL a compound which muses plasmolysis
(loss of turgor). About equal amounts of ABA were found both in the leaf
slices and in detached leaves. whereas 2 to 4 times more ABA accumu-
lated in the medium than in the slices. When spinach leaf slices were
incubated with ethylene glycoL a compound which rapidly penetrates the
cell membrane causing a decrease in the osmotic potential of the tissue
and only transient loss of turgor. no ABA accumulated. Ethylene glycol
was not inhibitory with respect to ABA accumulation. Spinach leaf slices
incubated in both ethylene glycol and mannirol had ABA levels similar
to those found when slices were incubated with mannitol alone. Increases
similar to those found with mannitol also occurred when Aquacide III. a
highly purified form of polyethylene glycol. was used. Aquacide III muses
cytorrhysis. a situation similar to that found in wilted leaves. Thus. it
nppmrs that loss of turgor is essential for ABA accumulation.

When spinach leaf slices were incubated with solutes which are
supposed to disturb membrane integrity (KI-150,. ZopropanoL or KCI)
no increase in ABA was observed. These data indimle that. with respect
to the accumulation of ABA. mannitol mused a physical stress (loss of
turgor) rather than a chemical stress (membrane damage).

 

ABA levels in water-stressed tissues are usually IO to 40 times
greater than those found in turgid tissues (21). Several workers
(1.2. 8. 22) have hated that ABA accumulation is dependent on
lmf water potential declining below a certain ‘threshold’ level.
usually around —l.O to -!.2 MP3. Pierce and Raschke (16)
concluded that turgor is the critical component of plant cell
water relations that controls ABA levels. 1.2.. loss ofturgor is the
signal that causes ABA accumulation.

We decided to use leaf slices of spinach incubated in various
solutes as a way of studying ABA accumulation. Several workers
have triggered ABA accumulation by incubating plant tissues in
hyperosmouc solutions of mannitol or sorbitol (7. l2. 13. 1.5.
18. 2.0). These compounds will cause plasmolysis (loss ofturgor).
Greenway and Leahy (6) have shown that ethylene glycol. since
it rapidly penetrates the cell membrane. will decrease a cell's
osmotic potential and muse only a transient loss of turgor. With
the use of penetrating and nonpenelmu'ng solutes. one should be

' Supported by the United States Department of Energy under Can-
an DE-AC02-76ERO-1338 and the National Science Foundation un-
der Giant PCM78-O7653.

able to determine whether or not loss of turgor is important in
musing ABA accumulation.

For those mses where mannitol mused increased ABA levels
(7, 12. 13. 15. 18. 20), the possibility exists that mannitol may
have a perturbing effect on the cell membrane. Riov and Yang
(17) used 100 mM mannitol to Stimulate ethylene production in
Citrus lmf discs. They concluded that mannitol exerted a chem-
iml Stress on the membranes since the mannitol concentration
used was too low to induce water stress. Other workers (5) have
observed that when cucurbit leaf discs were incubated in the
presence of 2-propanol. 10150;, or KCI, the production of
ethane increased. Since ethane producuon is a result of mem-
brane damage. addition ofthese compounds to the medium with
spinach lmf slices should indicate if ABA producdon is mused
by a chemical stress to the membranes.

MATERIALS AND METHODS

Plant Material. Spinach plants (Spinacia oleracea cv Savoy
Hybrid 612) were grown under SD as described (23) and were
transferred to LD for 8 to 14 d. For experiments involving
detached leaves. just fully expanded (light green in color) leaves
were excised and either placed immediame in plasdc bags (‘con-
trol’ leaves). or were allowed to lose 15% of their fresh weight
and then placed in plasric bags (‘stressed' leaves).

Leaf slices were prepared in the following manner. Just fully
expanded leaves were detached. midribs removed. and lmf blades
were sliced 1 to 2 mm in width with a sharp razor blade. Slices
were placed in BM2 ( 10 mM Hopes. pH 6.5 With KOH) containing
2.5 g-l'L PVP-40 for 10 min. then rinsed and placed in BM for
10 min. Next. slices were blotted dry and placed in the appro-
priate incubation medium (10 ml tatal volume). Total incubation
time in mos: experiments was 4 h. Slices were incubated in 60-
ml test tube: under light from a General Electric H4OORDX33-
I mercury-vapor lamp (50 w/m‘) which was filtered through a
5-cm layer of disu‘lled H20. To insure that the slices did not
experience anaerobic conditions. air was bubbled into the solu-
tion throughout the rinse and incubation period at a rate of
approximately 25 ml/mln.

All experiments were performed at least twice with two repli-
cates per treatment.

ABA Purifimtion Scheme. After the incubation period. slices
and medium were separated. Slices were washed for 10 min with
glass-distilled H20. and the wash was then combined with the
incubation medium. Slices were frozen and lyophilized for dry
weight determinations. and then extracted as described (4) ABA
was purified by HPLC with a CI. pBondapak column (4) using
a convex gradient from O to 50% ethanol containing 1% acetic
acid in aqueous 1% scene acid. The incrmse in ethanol with

1 Abbreviations: BM. basal medium: GCSIM. gas chromatography-
sclected ion monitoring: m/Z. mass/charge: ECD. electron capture detec-
tor. MeABA. methyl ester of ABA.

 

 

 

156

CREELMAN AN D ZEEVAART

_’.‘—-‘
. .I—

respect to time (t. mins) may be described as: % ethanol - 50(1-
(l-l/ZO)“]. The fracuon containing ABA. which eluted between
18 and 21 min. was dried. methylated. and quantified on a
Hewlett-Packard 5840A gas chromatograph (4) equipped with a
Hewlett-Packard 7672A automatic sampler. The ethyl ester of
ABA was used as an internal standard.

The medium plus wash was acidified with glacial acetic acid
to give 1% acetic acid and applied to a Cu Sep-Palt cartridge
(Waters Associates) equilibrated with 1% acetic acid. A small
amount of (:H’HIABA (16.4 Ci-mmol") was added before
acidification of the medium to determine losses during the
purification procedure. The Sep—Pak was washed sequentially
with 2 ml of 1% aqueous acetic acid and 20% ethanol in 1%
acetic acid. ABA was eluted with 5 ml of 40% ethanol in 1%
acetic acid. The eluate was dried. methylated. and quantified as
described above for the extracts from leaf slices.

The percentage recovery of [’HIABA from tissue was 60% to
80%. While for medium it was 70% to 90%. All data were
corrected for losses.

["ClMannirol Uptake and Catabolism. Spinach leaf slices (2.1
g fresh weight) were incubated with 0.6 M mannitol plus 1 uCi
D-{lo"C]mannitol (45 mCi-mmol", New England Nuclear) for
4 h. The tissue was treated as described above and the sugars
exnacted three times with boiling 80% ethanol. After removal of
the ethanol with a stream of N1. an aliquot of the aqueous
solution was applied in a narrow band to a 22-cm Strip of
Whatman 3MM paper and subjeCted to descending chroma-
tography for 18 h with methyl ethyl ketonezacetic acid:water
saturated with boric acid (9:1:1) (10) as solvent. Standards (10
pg each) of sucrose. glucose. fructose. and mannitol were also
applied to the origin. Sugars were visualized by spraying With
CrzorKMnO. spray (25). Peaks of radioactivity were detected
with a Packard model 7220/ 21 radiochromatogram scanner.

Water P0tential Measurement. Leaf water and osmou'c paten-
tial were measured with a Wescor HR-33T dew point microvolt-
meter equipped with 6 052 sample chambers as described (24).
Turgor porential was determined by subtraction. The osmotic
porential of the incubation medium was measured by placing
one drop of medium on a cellulose disc in a 052 sample chamber
for 2 11.

Mass Spectrometry. The GC-SIM response at m/z 190 (dwell
time. 100 ms) was monitored as described (4).

RESULTS AND DISCUSSION

Maximum accumulation of ABA occurred when detached
spinach leaves were allowed to lose 13% or more of their fresh
weight and were then placed in a plastic bag for 4 h (data not

 

 

 

 

 

 

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6 " wince

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Hours '

Fro. 1. Changes in ABA content of detached control (turgid). and in
stressed (wilted) spinach leaves. The fresh weight or‘ wilted leaves was
reduced by 15%.

Plant Physiol. Vol. 77. 1985

 

 

 

 

 

 

 

 

 

 

 

 

 

2’5 '- CJM-eium T '
Elma— T
2.0 l— 4
5 L5 I- c S -
1 ~05 -03 ‘l' MPO
i -o.9-os it.
:3
3. L0 _ 0.4 0.0 *0 q
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0.5 F. as
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C C S 0.0 0.2 0.4 0.5 0.8 L0 M Mon]
0h 4"! 4h ‘0.| ‘05 ‘l.0 ‘l.4 ‘13 ‘ZQMPQ‘Y,

Frc. 2. ABA accumulation in detached control (C). stressed (S) spin-
ach leaves. and in spinach leaf slices and media. Leaf slices were incubated
in increasing concentrations of mannitol (Man). Parameters for cell water
relations of detached leaves (insert) and osmotic potentials of the incu-
bation media (ordinate) are indieated.

 

 

 

 

 

 

 

 

 

 

   

 

L5 —' cl
UMedium
BWoen F_ E
.Tlssue
Loi— d
'3
I
5
O
6:
Z o.s~ .
m
<
’ 2H /
F'l tea I" E 1 E ’r _ r1.—
CCSlh2h3h4h6hBM
011 4h 411 h—fiM Monmtol—-0 41’)

FIG. 3. ABA accumulation in detached spinach leaves. and in spinach
leaf slices and media. The leaf slices were incubated in 0.6 mannitol
(Man) for the times indicated. in this experiment. the wash (see "Mate-
rials and Methods") was analyzed separately from the medium.

shown). A maximum level of ABA (about a 10-fold increase)
was reached within 3 to 4 h when such leaves had lost 15% of
their fresh weight (Fig. 1). Therefore. in all experiments described
here. leaves to be stressed were detached and allowed to lose 15%
of their fresh weight. At this point. turgor was zero (Fig. 2).

in a similar manner. ABA accumulation in spinach leaf slices
began only when mannitol concentrations greater than or equal
to 0.4 M were used. and reached a maximum at 0.6 to 1.0 M (Fig.
2). Concentrations greater than 1.0 M could n0t be used due to
solubility problems. In all further experiments 0.6 M mannitol

 

 

 

157

 

LOSS OF TURGOR CAUSES ABA ACCUMULATION

 

 

 

 

 

 

 

 

 

 

 

1
3'0- Omaha-n -
.Tim .1
zo— " -
'3
>-
5 .. -
O
b
.5
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El _._ E
C 5 3M EG G . M 0
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-O.| 4.! ~23 '1.4 -.4MP¢‘77

FIG. 4. Levels of ABA in detached spinach leaves and in spinach leu‘

slices and media The leaf slices were incubated in ethylene glycol (EG).
EG plus 0.6 M mannitol (Mari). 0.6 M mannitol. or Aquacide lII (AQ
III).

was used. When slices were incubated in 0.6 M mannitol. maxi-
mum accumulation of ABA in the tissue occurred within 3 h
(Fg. 3). while ABA levels continued to increase somewhat in the
medium. 50 ome ABA asalsot'ou rid in the wash. A was
probably due to et‘quitw from the slices. In addition. if slices were
incubated in 0. 6 M mannitol for 4 h and were then placed in BM
for 4 h. ABA levels in the tissue declined to those found in
control leaves or slices incubated in BM for 3 h (data not shown).

us. with regard to ABA accumulation spinach leer slices
incubated in mannitol solutions act in a manner similar to a
stressed. detached Xanthium leat (2 4).

To insure that the measured ECD response of ABA from tissue
and medium was due solely to MeABA. equal amounts or ABA
(as determined by ECD) extracted from tissue and medium.
along Wllh standard (:)- ABA (Sigma) were analyzed by GC-
SIM. If the MeABA peak from tissue or medium contained a
contaminant. its SIM response would be less than that of the
standard. The SIM response at m/z 190 (base peak) of MeABA
from tissue. medium. and standard were identical within exper-
imental error (data not shown). indicating that no contaminating
peaks cochromatogi-aphed wrth MeABA during the meuure-
ment ot'A

Some variation in the ABA level in turgid leaves and the
accumulation or ABA in stressed leaves occurred between exper-
iments. Possrble sources or variation include leat age (leaves were
harvested 8 to l4 c1 arterL transter to L0) and the stress history oi
the pill“. T0 initiu "th Drill-0min:
from goons of plants placed in trays containing deionized H O
for l2 to [6 h betore the onset ofthe experiment.

en leaf slices were incubated in 0. 6 M ethylene glycol. no
ABA accumulated in either the medium or the tissue (Fig. 4).
Similar results have been obtained with thiourea (data not
shown). another solute which is able to rapidly penetrate the cell
membrane (9.) However when slices were incubated in 0. 6 M
mannitol or Aquacrde III. ABA levels rose in both the medium
and tissue. Aquacide III. a highly purified form of polyethylene

 

glycol. average mol wt 18.000. is unable to penetrate the cell wall‘

uses qtorrhysis ( 14). Thus. treatment with Aquacide llI
mimicks the situation found in water-stressed leaves (14). when
slices were incubated with ethylene glycol plus mannitol. ABA
accumulation was similar to that with mannitol alone. Thus.
with regard to ABA accumulation. ethylene glycol was not toxic
or injurious to the leaf tissue Since ABA accumulation occurred
only when solutes were used which caused plasmolysis or mor-
rhysis. we conclude that ABA accumulation is dependent on the
log of turgor

Since Riov and Yang ( 17) observed uptake and metabolism or‘
mannitol in Cr'trttr leatdisc. we were concerned that mannitol
could penetrate the cell membrane in our system and cause
osmotic adjustment. or mannitol could be metabolized. Based
on radioactivity found in the time. 0. 739’ a or the total mannitol
found Iijn the medium penetrated the cell membrane. The con-

nof mannitol in the tissuewas approximately 20 mM.
or contributed about —0. 05 MPa to the osmotic pressure (assum
ing a 1.0 M ideal solution' IS equivalent to -7 ..4 MPa). This is not
enough to cause osmotic adjustment. In addition. contrary to
the results that Riov and Yang obtained With Cirrus ( 17). man-
nitol was not metabolized to sucrose in spinach (data not shown).

No signcitieent accumulation of ABA occurred when three
nt cooncentratt o KHSO ,6, IS, and 25 mM). 2-
propanol (I 5 and 10%). or KCI (3. 50. and 100 mM) wereused
(data not shown). These compounds caused the production ot
ethane (a measure of membrane damage) When incubated With
cucurbit leat dises (5). We conclude. theretore. that the effect of
mannitol on leat slicesis tsa physi one involving water stress.
and not a chemical one involving membrane damag

Of considerable interest is the large accumulation eof ABA in
the rriiued m(Figs. 2-4: see also Rel. 12). In mosr experiments.
approximately 400 mg fresh weight of slices were used per 10 ml
incubation medium. presenting a relatively int'inite’ free space
to the tissuea A likely explanation is that ABA Acrosses the plasma
membrane mediu um Where it cannot be
catabolized. 3On the C“basis of the model for cellular compartmen-
tation or ABA (3) and appropriate values tor the relative volumes
of free space. tissue. chloroplasts. and vacuoles. one can calculate
that most ofthe ABA will be found in the medium.

Attempts to develop similar systems with slices or bath ( Phas-
eolus vulgarr'r cv Redkloud) and Xanthium strumariitm failed.
Accumulation of ABA in slics of these ues was erratic and
irreproducible. Interestingly, Loveys (ll) reported higher levels
of ABA in View faba mesophyll tissue infiltrated with 0.88 M
mannitol than with buffer. but the total ABA in the tissue plus
medium was lower than the value reported for nonstressed turgid
leaves. Xanthium mesophyll cells have high photosynthetic acnv-
ity (19). yet do not appear to accumulate ABA when stressed
wtth mannitol (ML Pierce. EA Bray, personal communications)
even though stressed. detached leaves or Xanthium accumulate
large amounts 0! ABA (2 4). When spinach leaf slices were
incubated III 0. 6 M mannitol along with .‘lunt/ir'um mesophyll
cells (l9). the amount or" ABA that accumulated was reduced
(EA Bray RA Creelman JAD Zee eevaart. unpublished results),
indicating the presence ot a dilTusi‘ole inhibitor. This inhibitor IS
probably nonspecrl' c in action and most likely a result or tissue
wounding and cellular leakage. It is also possrble that the Xan-
thium mesophyll cell system was not fully optimized for ABA
production.

Pierce and Raschke (l6) observed that ABA accumulation
occurred When turgor was zero using data obtained from pres-
sure- -volume curves Using spinach 1w slics incubated with
plasmolyzing or nonplasmolyzing solutes. we obtained rsults
that support the hypothesis or Pierce and Raschke (16). We
conclude. therefore. that turgor is the criticil parameter of cell
water relations governing stress-induced ABA accumulation.

Adan-II -—We would like to thank the Miemgn SIate Unmet-my-
anomlm [Multan of Helm Mu may Family IRROOIMOI Io: use oI' the
GC-MS

LITERATURE GTE'D

I. Salaam-MP. DCoum I975 Relauonslunbetween [arr-mums
51....an In

207-212

1 But“. WK Fenm. I977 The man ":3” soIl 3nd xylem water
potenuaI. Inal‘ruxstace. an nd arms: adorn IIn dmu gnted seedling of
Douglas-firthnmuuum "If: an). thtlol Phnt 39: 106-I O9

3. Covus IR. JA RAE w HA WCD Fnocmn I981 A potable role for
IDSCXSIC and In COUOIH‘! swmual conductance and DHOIOSYHUWUC
meuooIIsm In lemex AustJ Phnt thm 9. ~89—9 8

4. mean EA. IAD Ban“: I984 lneomonuo no onygen Into :bmsie
and and chase: 3cm Iron-Int ecular oxvgen. PIant Phyuol 75: 166-16 9

5. FIL‘IEI P.H Rn women“ Sean. RA Busy ~.LG Iuon. L uux.
T Sulua I 983 Biosyn mm and emmn uI NIIyIII-Imgen amid; by hiner
plans In MJ Konol. FR Whauev. ads. Gaseous AI: Polluun andtPIaI-I

N 3
3 Ham-nu DD In. H3 TUKEY In I975 EITect ol'intermment mm on m:
and Ieveis In Hnnmznm

9. szrI'rJ I983 PIasmolysIs shaoe In relauon to freae- autumn; of mbbag:
plants and [0! me eII'ect oI peneu-aung squu: Punt Cell Samoa 6: 465-

470
I0. LEWIS DH. DC Sum! 1967 Sugr alecnols eIponoIs) In I'unp and ween plants
IL Metnoas ol daemon md quanutauv Inauo n In plan: um New

158

p

_____________/

CREELMAN AND ZEEVAART Plant Physiol. Vol. 77. I985

Phylot 66: IlS-ZO-t

. Invm BR I977 The mImeIIuIar Ioauon oI‘ absent: dad in maul and
new a! 0

.Phxuol Man 40: 6-I
Lavm BR. CJ Bum. PE Kmmtnm: I975 Bimmu-Icm oI‘ aha-me and
I:l sue: nudu: (undo dual Iabeung Ieenntquc. PhyuoI
Plant 33: 166-

I70
. Mano mp: 51'. B COLIAN. WR Cumulus l9ll Amuemds and Mammals

med lax mannyll ceIL Phnl Phynol 07: 113-2

. 0::1'u JJ I976 The m1: of water In the clam. In 0L Lange. LKa open. E-

Schulz: eds Water and Ham UI’: Sonnet-Verlag. New Yon- pp l9-

. PInczMLI‘Ial' , “ ' L

 

PhD them. \IIenIun State UnIveI-II Il\' Ea.“ L-mtI

. PIERCE .‘-IL K Pusan: 1950 CorreIauon Den-m log mm turgor and accu-

muuuon oI‘ :bsns: 9cm ndxn deucned com Plant: “8:174-

8-
. Rxov J. SF Y AM; I981 Stimulauon oI CKI'IHEI'IE produetxon In «:1qu led! disc:
-I—'b

bv man I. PIAIII PhquI' I

Rmzu I. .II- F Leos VII-o. J P Cdmn I983 Ramd eIIecI oI' osmone men on
the cement and umflun'on oI absent and In Zea mays roots. Phat 5d
hen 3|: 133-I 37

. Sun‘s? TD.K RASCHKE I980 Ems-Is oI‘ plume and and dihvdmnm

on mm at: andpm: atmmzuoa :Iupnntua. PIanPhnyol 65:19l-_97
Wino JK? IM Sum 1980 Isolauon of ohms: :nd- mam vananu from

:I97- 02
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Chapter 10
STRESS-INDUCED ABSCISIC ACID BIOSYNTHESIS IN
HIGHER PLANTS -- A MODEL

159

 

 

—==::w___m -_ fl- ,m .

160
10.1. INTRODUCTION

As mentioned in chapter 1, nothing is known about
intermediates in the biosynthetic pathway of ABA, except that
as a sesquiterpenoid, ABA is ultimately derived from MVA. Re-
search on the ABA biosynthesis centers around two path-
ways: the direct pathway involving a C15 precursor derived
from farnesyl pyrophosphate and the indirect pathway invol-
ving a precursor derived from carotenoids.

Sesquiterpenoids are characterized as compounds that have
a basic skeleton of fifteen carbon atoms formed by a regular
repetition of isoprene units. Almost 200 different carbon
skeletons containing up to four carbocyclic rings are found
in this group (Loomis and Croteau, 1980). Representative
sesquiterpenoids are illustrated in Figure 10.1. The carbon
skeleton of ABA can easily be derived from farnesyl pyrophos-
Phate by cyclization on paper. However, the abscisane
Skeleton is very unique, with ABA (and its derivatives, such
as PA) and xanthoxin being the only members of this class.
There are many compounds, however, with a ring structure and
Side chain similar to ABA, such as "allenic sesquiterpenoid",
and a component of the aroma of tea, theaspirone (Ina et al.,
1968; Figure 10.2). "Allenic sesquiterpenoid" was found in
the flightless grasshopper Romalea microptera and is consid-
Ered to be a breakdown product of neoxanthin (Meinwald et
37., 1968). Other similar compounds are loliolide, butenone

(Figure 1.5), a-ionone, B-ionone, and dihydroactinidiolide

 

161
O“ OH 1
HO
trans .m- Fumesol leidal fl -Fom¢sene c - Bis-social
l H l l 3
9%: are CZ /
coCurcummc Lanceol Sunniihuieno Compnermonc
O
. \ \
c- aerqomotene Zerum'oono Cunarene ,8 - Vetwono
: H of I /
H 0
a - Munrolene Eremoohilono Sativene Langifoleno
O
on o
OH
OR
Cedrol Culmorm Coriomyrtin Trichomecin

Figure 10.1. Representative sesquiterpenoids.
Note the large diversity of structures. From Loomis and

Croteau (1980).

16

,c\
¢C/\
OH
HQ '

AllenIc éTheaspirone
sesquiterpenoid

CHZOH
\ \ \
Vitamin A
/ /c=o
O
Dihydroactinidiolide a-ionone
. \ \O /&AOH
_ (D/
d-Ionone Vomifoliol

Flgure 10.2. Compounds with structures similar to abscisic

acid.

These compounds have a ring skeleton similar to ABA, and are

though to be derived from carotenoids.

163

(Figure 10.2). All of these compounds are found in plants
(except "allenic sesquiterpenoid"), and are considered to be
derived iflwnn carotenoids. However, it is not known whether
these compounds are endogenous or are formed by oxidative
destruction of carotenoids during extraction. Both B-ionone
and dihydroactinidiolide can be formed by an oxidative attack
on B-carotene (Isoe et al., 1969). The only known cases where
compounds have been shown to be derived from a carotenoid are
vitamin A (in animal intestine; Figure 10.2; Britten, 1983),
and trisporic acid (in fungi; Figure 1.6).

The best evidence for a compound being derived from a
carotenoid and being further catabolized to a physiologically
active substance is the production of trisporic acids from
B-carotene (Bu’Lock et al., 1976; Gooday et al., 1979; Figure
1.6). It should be noted that some uncertainty exists as to
whether the first compound formed is indeed a C20 (retinal)
derivative. Trisporic acids are produced by, and act only on,
fungi of the order Mucorales. The observed effect of trispor-
ic acid is in) induce zygospores in both mating types of a
particular species. Enzymatic conversion of B-carotene to
trisporic acids is believed to occur for the following
reasons: (a) radioactive retinol, B-Clg-ketone, and 4-hy-
droxy-B-Clg-ketone are converted to trisporic acids (Bu’Lock
at al., 1974); (b) the distribution of radioactive label from
MVA is correlated in both B-carotene and trisporic acid
(Austin at al., 1970); (c) in mated Blakesiea trispora,

inhibition Irf B-carotene biosynthesis by diphenylamine

 

 

164

prevents trisporic acid production (Austin at al., 1969); (d)
Phycomyces blakesieeanus mutants blocked in B-carotene
synthesis are also blocked in trisporic acid production
(Bergman et al., 1969). As described in chapter I, a similar
situaticni exists with regard to ABA production (carotenoid
mutants and biosynthetic inhibitors, conversion of radioac—
tive xanthoxin to ABA).

Thus, there are many compounds which appear to be
carotenoid degradation products. With regard to caroten-
oid biosynthesis, much more is known about the anabolism of
carotenoich; than their catabolism. The pathway of carotene
biosynthesis from isopentenyl pyrophosphate through the
cyclic carotenes has been established (Spurgeon and Porter,
1980). An enzyme complex from tomato fruit plastids that
converts 'isopentenyl pyrophosphate to phytoene (Porter and
Spurgeon, 1979) has been described. This complex is apparent-
ly located in the chloroplast envelope, as is phytoene
dehydrogenase (Lfitke-Brinkhaus et al., 1982). In view of the
extensive knowledge concerning the enzymatic formation of
carotenoids, surprisingly little is known about the rate of
carotenoid catabolism, the nature of the catabolites, or
Which enzymes are involved. Several enzyme systems, such as
peroxidase (Matile and Martinoia, 1982), or lipoxygenase
(Gardner, 1980) are known which will convert carotenoids to
colorless products; however, it is not known if these enzymes
perform the same role in vivo.

In conclusion, there are many compounds which can be

 

165
derived from carotenoids by paper chemistry. However, in many
cases it is not known if these compounds are artifacts of
extraction, or are endogenous. Only two compounds have been
shown to result from carotenoid degradation in viva, vitamin
A and trisporic acid. Enzymes have been described which will

degrade carotenoids, however, their role in viva is unknown.

10.2. A MODEL EXPLAINING STRESS-INDUCED
ABSCISIC ACID BIOSYNTHESIS

Any model proposing to explain the water stress induced
biosynthetic pathway in higher plants must explain the
following observations: (a) the lag time of 20 to 50 min
before the accumulation of ABA (Guerrero and Mullet, 1986;
Henson, 1981; Henson and Quarrie, 1981; Zeevaart, 1980),
(b) the fact that much more ABA appears to be made during
water stress than is needed for stomatal closure (Raschke,
1975), and (c) the heavy oxygen incorporation data described
in chapters 4 and 6.

The lag iri the accumulation of ABA after imposition of
water stress is consistent with the notion that an enzyme is
induced. This enzyme appears to be encoded in the nucleus
because ABA accumulation is inhibited by cycloheximide, an
inhibitor Irf cytosolic protein synthesis, and not with
chloramphenicol, lincomycin, or spectinomycin, inhibitors of

plastid protein synthesis (Quarrie and Lister, 1984; Guerrero

 

 

166
and Mullet, 1986). Accumulation of ABA was also inhibited
with actinomycin D and cordycepin, nuclear transcription
inhibitors (Guerrero and Mullet, 1986). The signal created by
water stress causing the accumulation of ABA appears to be
the loss of turgor (chapter 9; Pierce and Raschke, 1980).

The induced enzyme (or another enzyme in the ABA biosyn-
thetic pathway) appears to be sensitive to the oxygen tension
surrounding the leaf. The accumulation of ABA is not saturat-
ed until approximately 60% oxygen. While it is difficult to
extrapolate from data generated from a whole leaf system to a
specific enzyme, this result suggests that an enzyme in the
biosynthetic pathway of ABA has a low affinity for oxygen,
and may be a mono-oxygenase (or oxygenase).

If ABA is derived from a carotenoid(s), then there is a
huge excess of potential precursor in a green leaf. This
could explairliwhy more ABA is made than is needed for
stomatal closure. Water stress could change the topology of
chloroplasts membranes, exposing carotenoids to degradative
enzymes. There are also enough total carotenoids present in
roots to account for the amount of ABA made.

However, there are certain lines of evidence that
indicate that if leaf ABA is derived from carotenoids, it is
from a small fraction of the total carotenoid pool. Walton et
a]. (1985) introduced 180 into Violaxanthin via the xantho-
Phyll cycle. 131 this cycle, Violaxanthin loses its epoxide
groups, ultimately forming zeaxanthin when leaves are

incubated in light under nitrogen. If leaves are then placed

 

 

 

167

irI the dark, in”; level of Violaxanthin increases with the
incorporation of oxygen from molecular oxygen into the
epoxide group. When this was done in an atmosphere containing
1802, between 40 and 45% of the total Violaxanthin contained
180; no 180 was found in lutein or neoxanthin. When these
leaves were stressed, the ABA which accumulated contained
10-15% 180 in the ring oxygens. It was not possible to
determine the position of incorporation in the ABA mole-
cule. This result suggests that a portion of the ABA which
was made during the stress period came from Violaxanthin. An
explanation for the low amount of incorporation into ABA is
that ABA is produced from other xanthophylls or from a
non-xanthophyll precursor. Another possibility is that ABA is
derived from the Violaxanthin pool which was not labeled with
180. There exist two Violaxanthin pools, with the majority of
Violaxanthin found in the chloroplast envelope and the rest
in thylakoids (Douce et a7, 1973). An enzyme has been
isolated from spinach chloroplasts which will convert
Violaxanthin to zeaxanthin (Hager and Perz, 1970). This
enzyme is apparently located in the grana and stroma thyla-
koids (Siefermann and Yamamoto, 1976). Thus, if in the
experiment of Walton et al. (1985), ABA came primarily from
Violaxanthin in the outer envelope, then it might not have
much 180 present in the ring positions. The 18O incorporation
data described iri chapters 4 and 6 support the hypothesis
that ABA is derived from a carotenoid.

Most of 'the data described in this thesis suggest that

 

 

168

carotenoids are precursors to ABA. What is needed, however,
is a definitive experiment to prove that a carotenoid does
break down to form ABA. Since stressed Xanthium roots contain
low amounts of carotenoids, it might be possible to deplete
the tissue of carotenoids. A time course study of changes in
carotenoid levels and ABA accumulation in roots needs to be
done. If ABA came from a particular carotenoid in roots, then
the amount of that carotenoid should decline with water
stress.

Another question that needs to be answered is the role of
xanthoxin. Labeled xanthoxin needs to be synthesized and fed
to the three tomato mutants that do not accumulate stress-in-
duced ABA (flc, sit, and not; see chapter 1). If ABA does
arise from xanthoxin, it is possible that intermediates might
accumulate in these mutants. Possible intermediates between
xanthoxin and ABA are xanthoxin acid and abscisic alde-
hyde. These compounds can also be synthesized from interme-
diates used in the biosynthesis of labeled xanthoxin and used

in feeding studies or as standards.

10.3. LITERATURE CITED

Austin, D.J., Bu’Lock, J.D., Drake, D. (1970) The biosynthe-
sis Irf trisporic acids from B—carotene via retinal and
trisporal. Experientia 26:348-349.

Austin, D.J., Bu’Lock, J.D., Winstanley, D.J. (1969) TriSpor-
ic acid biosynthesis and carotenogenesis in Biakesea tri-
spara. Biochem. J. 113:34P.

Bergman, K., Burke, P.V., Cerda-Olmedo, E., David, C.N.,
Delbrfick, li., Foster, K.W., Goodell, E.W., Heisenberg,

 

 

169

M., Meissner, G., Zalocar, M., Dennison, D.S., Shrop-
shire, W. (1969) Phycomyces. Bacterial. Rev. 33:100—157.

Britton, G. (1983) The biochemistry of natural pigments. Cam-
bridge: Cambridge University Press.

Bu’Lock, J.D., Jones, B.E., Taylor, D., Winskill, N.,
Quarrie, S.A. (1974) Sex hormones in Mucorales. The
incorporation of C 0 and C18 precursors into trisporic
acids. J. Gen. Micro iol. 80:304—306.

Bu’Lock, J.D., Jones, B.E., Winskill, N. (1976) The apocarot-
enoid system of sex hormones and prohormones in Mucor-
ales. Pure and Appl. Chem. 47:191-202.

Douce, R., Holtz, R.B., Benson, A.A. (1973) Isolation and
properties of the envelope of spinach chloroplasts. J.
Biol. Chem. 248:7215-7222.

Gardner, H.W. (1980) Lipid enzymes: lipases, lipoxygenases,
and "hydroperoxidases". In: Autoxidation in food and
biological systems, pp 447-504, Simic, N.G., Karel, M.,
eds. New York: Plenum.

Gooday, E.W., Jones, B.E., Leith, W.H. (1978) Trisporic
acid and the differentiation in the Mucorales. In: Re—
gulation of secondary product and plant hormone metabol-
ism, pp. 221-229, Luckner, M., Schreiber, K., eds. New
York: Pergamon Press.

Guerrero, F., Mullet, J.E. (1986) Increased abscisic acid
biosynthesis during plant dehydration requires transcrip-
tion. Plant Physiol. 80:588-591.

Hager, A., Perz, H. (1970) Veranderung der Lichtabsorption
eines Carotenoids im Enzym (De—epoxidase)-Substrat
(Violaxanthin)-Komplex. Planta 93:314-322.

Henson, I.E. (1981) Changes in abscisic acid content during
stomatal closure in pearl millet [Pennisetum americanum
(L.) Leeke]. Plant Sci.Lett. 21:121—127.

Ina, K., Sakato, Y., Fukami, H. (1968) Isolation and struc-
tural elucidation of theaspirone, a compound of tea
essential oil. Tetrahedron Lett. 2777-2780.

Isoe, S., Hyeon, S.B., Sakan, T. (1969) Photo-oxygenation of
carotenoids. I. The formation of dihydroactinidiolide and
B-ionone from B-carotene. Tetrahedron Lett. 279-281.

Loomis, W.D , Croteau, R. (1980) Biochemistry of terpen-
oids. In: The biochemistry of plants: a comprehensive
treatise, pp. 363-418, vol. 4, Stumpf, P.K., Conn, E.E.,
eds. New York: Academic Press.

 

170

Lfitke-Brinkhaus, F., Liedvogel, B., Kreuz, K., Klenig,
H. (1982) Phytoene synthase and phytoene dehydrogenase
associated with envelope membranes from spinach chloro-
plasts. Planta 156:176-180

Matile, P., Martinoia, E. (1982) Catabolism of caroten-
oids: involvement of peroxidase? Plant Cell Reports
1:244—246.

Meinwald, J., Erikson, K., Hartshorn, M., Meinwald, Y C.,
Eisner, T. (1968) Defensive mechanisms of arthropods.
XXIII. An allenic sesquiterpenoid from the grasshopper
Ramalea microptera. Tetrahedron Lett. 2959-2962

Pierce, M.L., Raschke, K. (1980) Correlation between loss of
turgor and accumulation of abscisic acid in detached
leaves. Planta 148:174-182. '

Porter, J.W., Spurgeon, S.L. (1979) Enzymatic synthesis of
carotenes. Pure and Appl. Chem. 51: 609- 622.

Spurgeon, S.L., Porter, J.W. (1980) Carotenoids. In: The
biochemistry of plants, pp 420-484, vol 4, Stumpf, P.K.,
Conn, E.E., eds. New York: Academic Press.

Quarrie, S.A, Lister, P.G. (1984) Effects of inhibitors of
protein synthesis on abscisic acid accumulation in
wheat. Z. Pflanzenphysiol. 114 309-314.

Walton, D.C., Li, Y., Neill, S.J., Horgan, R. (1985)
Biosynthesis of abscisic acid: a progress report.
In: Current topics in plant biochemistry and physio-
logy 1985, pp. 111-117, vol. 4, Randall, D.D.,
Blevins, D.G., Larson, R.L., eds. Columbia, MO: Uni-
versity of Missouri.

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. 66:672-678.

 

APPENDIX
QUANTITATION 0F ABSCISIC ACID USING INTERNAL STANDARDS

171

 

 

172

Quantitation of ABA in large numbers of samples requires
the use of automated equipment in order to give the experi-
menter time in) perform other tasks than injecting samples
into a gas chromatograph. In our laboratory, ABA samples are
routinely analyzed vfiini a Hewlett-Packard 7672A automatic
injector coupled to a Hewlett-Packard 5840 gas chromatograph.

The use of such equipment requires that an internal
standard be used to take into account two possible events
that may occur during analysis: (a) evaporation of solvent
from vials as they wait for their turn to be sampled,
and (b) injections are not exactly reproducible from one
sample in) the next. Evaporation occurs because the seal on
the sample vials is not airtight; this is unlikely to
occur given the time scale of analysis. The latter event
(b) is inherent in any machine-operated analysis and is due
to the fact that an exact syringe volume cannot be obtained
with each injection.

Certain criteria must be met for a compound to be used
as an internal standard: it must have (a) the ability to
capture electrons, and (b) a retention time different from
contaminating peaks and MeABA. We commonly use EtABA and/or
enderin, a chlorinated pesticide.

'The procedure is; as follows. Once the samples have
been purified by HPLC, they are dried, and methylated with
ethereai Iiiazomethane. The manner in which the samples are
further analyzed depends on the amount of ABA expected to

be present. For small amounts of ABA the sample may be

 

 

173
ciissolved 'hi a small volume of a stock solution containing
the internal standard(s) in ethyl acetate. The dissolved
sample is then transferred to an injection vial.

For samples containing large amounts of ABA dilution
‘is necessary. This is commonly done by dissolving the
:sample in ethyl acetate (for instance 10 to 25 ml). A small
al iquot is transferred IX) an injection vial and the solvent
is allowed to evaporate. The stock solution containing the
iraternal standard is added and the samples run on the
aLItomatic injector (see Figure A.1 for a standard run).

Standard amounts of ABA are also dissolved in the same
st:ock solution. Because MeABA, EtABA, and enderin are
stLable, standards can be used several times. For a given
seat of standards, the stock solution (containing the internal
standards) may be used for dissolving samples as long as
evaporation does not significantly increase the peak area of
the internal standards. If this occurs, then a new set of
standards must be made.

If solvent evaporation or an injection error occurs,
the peak areas of both MeABA and the internal standards
will increase to the same extent. This occurs because the
detecrtor response is proportional to the amount of compound
Preseent in the injected solution (sample). Thus, comparison
0f tiie ratio of the peak areas of MeABA and the internal
stanciard in samples and in standards (see Table A.1 for
typl<:al data and Figure A.2 for typical standard curves)

will give an estimation of the concentration of ABA in the

 

 

174

94.2

9.51

18.52

 

Detector Response

 

 

 

 

 

 

Fl'Qure A.1. Electron capture dectectar response of abscisic

acid and internal standards.

The retention times of MeABA, EtABA, and enderin are 9.51,

7U3.52, and 11.68 min, respectively. The peak corresponding to
MeABA respresents an injection of 200 pg in 1 pl.

 

175

 

 

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176

 

1.5

 

 

 

 

 

 

(a)
1 -I
.9.
6
o:
a
.5 -
o r f e
O 50 100 150 200
MeABA. Concentration Used (pg/pl)
.2
(b)
.15 -I
.2
I? 1‘
.05 -
O T r
200

 

so too 150
MeABA, Concentration Used (pg/pl)
Figure A.2. Typical standard curve obtained by injecting
internal standards with increasing concentrations of methyl.

abscisic acid.

In figure (a) MeABA/EtABA vs. the concentration of MeABA used

is plotted; ‘Hl (b) MeABA/enderin vs. the concentration of

MeABA used is plotted.

177
sample vial. The amount of ABA present in sample is then
calcnrlated knowing the volume injected (usually 1 pl), per
cent recovery, dilution, and amount of tissue, using the

formula below:

. _ (concentration)(dilution factor)
ABA/g dry weight ’ (recovery)(g dry weight)

Using values obtained from Table A.1 the amount of ABA

present in the tissue extract was:

(91 pg/ul)(75000) .
(O.75)(O.4 g dry weight)‘ 22.7 #9/9 dry weight.

 

 

 

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