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dissertation entitled

Synthesis and Metabolism ofdfi6 Monounsaturated
Fatty Acids in Developing Seed of Umbelliferae Species
and Thunbergia alata

 

presented by

Edgar Benjamin Cahoon

has been accepted towards fulfillment
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Ph.D. degree in Botany and Plant
Pathology

 

 

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Date December 29, 1993

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SYNTHESIS AND METABOLISM OF AGMONOUNSATURATED FATTY ACIDS
IN DEVELOPING SEED OF

UMBELLIFERAE SPECIES AND THUNBERGIA ALATA

BY

Edgar Benjamin Cahoon

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

1994

 

ABSTRACT

SYNTHESIS AND METABOLISM or A‘NONOUNSATURAIED FATTY ACIDS IN DEVELOPING
SEED or UMBELLIFERAE SPECIES AND THUNBERGIA ALATA
By

Edgar Benjamin Cahoon

Studies were conducted to characterize the synthesis and metabolism
of the unusual fatty acids petroselinic acid and Aéhexadecenoic acid.
Petroselinic acid, the cisA6 isomer of octadecenoic acid (18:1) , composes
up to 85 wt% of the seed oil of Umbelliferae (Apiaceae), Araliaceae, and
Garryaceae species, and Aéhexadecenoic acid (16:1A5) accounts for more
than 80 wt% of the seed oil of Thunbergia alata. Both fatty acids have
potential economic value.

Results of 1"C-radiolabeling studies performed with seed endosperm
of coriander (Coriandrum sativum) , an Umbelliferae species, suggested that
petroselinic acid is formed through an acyl-acyl carrier protein (ACP)
desaturation pathway. Consistent with this, antibodies raised against the
avocado Agstearoyl (18:0)-ACP desaturase detected a 36 kDa peptide on
western blots that was specific to tissues that synthesize petroselinic
acid. A cDNA for this peptide was isolated from a coriander endosperm
cDNA library and introduced into tobacco. The resulting transgenic calli
produced nearly 5 wt% each of petroselinic acid and A‘hexadecenoic acid
(16:1A"), neither of which was detected in control calli. Further
metabolic studies with coriander endosperm and transgenic tobacco cell
cultures revealed that the 36 kDa peptide is functionally a Akpalmitoyl
(l6:0)-ACP desaturase. As such, the biosynthetic pathway of petroselinic
acid likely involves the A" desaturation of palmitoyl-ACP followed by two-
carbon elongation of the resulting l6:lA"—ACP. Extending these results
to the biosynthesis of AGhexadecenoic acid, the activity of a soluble
Aspalmitoyl-ACP desaturase was detected in extracts of T. alata endosperm.
A cDNA for the A6palmitoyl-ACP desaturase was isolated from a T. alata

endosperm cDNA library using PCR with oligonucleotides designed against

 

 

conserved regions of A918:O-ACP desaturases of various species. Amino
acid sequences deduced from cDNAs for the coriander A‘16:0-ACP desaturase
and the T. alata A616:0-ACP desaturase share significant identity with the
A918:O—ACP desaturase. These results indicate that variations in the
primary structures of acyl-ACP desaturases can result in enzymes with
altered substrate recognition and double bond positioning properties.
In addition, incubation of endosperm of the Umbelliferae carrot
(Daucus carota) and coriander in [1-1“C]acetate revealed that petroselinic
acid readily enters phosphatidylcholine (PC) prior to deposition in
triacylglycerol. This result indicates that PC can participate in the

metabolism of an unusual fatty acid in developing seeds.

 

Copyright by

 

Edgar Benjamin Cahoon

1994

ACKNOWLEDGMENTS

I thank my thesis advisor John Ohlrogge for taking me into his lab
on short notice and for being an all-around good boss. John gave me many
ideas and plenty of encouragement, yet he allowed me enough freedom to do
things my own way (for better or worse). I also thank Chris Somerville
for providing suggestions and resources that were invaluable to the
completion of work presented in this thesis. I am also grateful to Ray
Hammerschmidt and Jan Zeevaart for their input as members of my guidance
committee.

There are a number of other people who in some way contributed to
the work presented here. Among these people is John Shanklin who provided
antibodies, help, and enthusiasm that were essential for results reported
in Chapter 2. Dave Shintani patiently mentored me in molecular biology
and was a constant source of suggestions. Also, Peter Dormann provided
a large amount of technical advice as well as late-night lab company. In
addition, Dr. Z.-H. Huang from the Michigan State University mass
spectrometry facility introduced me to a variety of mass spec derivatives
for analysis of double positions of fatty acids. One of these
derivitization methods shows up throughout this thesis. Furthermore,
Grattan Roughan, during his stay in the Ohlrogge lab, provided me with a
perspective of lipid biochemistry that was important to the planning of
some of the experiments presented here. Finally, the radiolabeling study
using nonanoic acid that is reported in Chapter 3 was based on a
suggestion by Akira Shibahara (Kobe-Gakuin University).

Just as importantly, I thank everyone that I have known in the
Ohlrogge lab [including Adrian. Butt, Kathy Schmid, Alenka (I can't
remember how to spell her last names), Dave Shintani, Becky Peck, Dusty

Post-Beittenmiller, Linda Savage, Ray (Raimundo) Pacovsky, Paul and Keith

V

 

 

 

 

Roes(s)ler, Nicki Engeseth, Peter Dormann, Kuni Nakahira, Vicki Eccleston,
Basil Shorrosh, Todd Barkman, Di Do, James & Amy, and Sarah Hunter] for

putting up with me and the many "messes" I left around the lab.

vi

TABLE OF CONTENTS

Pages
LIST OF TABLES ...................................................... x
LIST OF FIGURES ................................................... xii
ABBREVIATIONS ...................................................... xv
FATTY ACID NOMENCLATURE .......................................... xvii
CHAPTER 1: Introduction ............................................ 1
Natural Occurrence of Petroselinic Acid
and AéHexadecenoic Acid ................................. 1
Physical, Industrial, and Nutritional Properties of
Petroselinic Acid and AGHexadecenoic Acid ............... 5
Biosynthesis of Petroselinic Acid and
A6Hexadecenoic Acid ..................................... 7
Metabolism of Petroselinic Acid and
AGHexadecenoic Acid .................................... l3
Rationale .................................................... 21
Literature Cited ............................................. 27
CHAPTER 2: Expression of a Novel Coriander Desaturase
Results in Petroselinic Acid Production
in Transgenic Tobacco ................................ 37
Abstract ..................................................... 37
Introduction ................................................. 38
Materials and Methods ........................................ 39
Results ...................................................... 43
Discussion ................................................... 52
Literature Cited ............................................. 58

 

CHAPTER 3: Metabolic Evidence for the Involvement of a
A“Palmitoyl-Acy1 Carrier Protein Desaturase

in Petroselinic Acid Synthesis in Coriander

Endosperm and Transgenic Tobacco Cells ............... 62
Abstract ..................................................... 62
Introduction ................................................. 64
Materials and Methods ........................................ 66
Results and Discussion ....................................... 73
Conclusions .................................................. 95
Literature Cited ............................................. 97

CHAPTER 4: Apparent Role of Phosphatidylcholine in the

Metabolism of Petroselinic Acid in Developing

Umbelliferae Endosperm ............................... 102
Abstract .................................................... 102
Introduction ................................................ 104
Materials and Methods ....................................... 105
Results ..................................................... 112
Discussion .................................................. 130
Literature Cited ............................................ 137

CHAPTER 5: A6Hexadecenoic Acid is Synthesized by the
Activity of a Soluble AGPalmitoyl-Acyl

Carrier Protein Desaturase in Thunbergia

alata Endosperm ..................................... 143
Abstract .................................................... 143
Introduction ................................................ 144
Materials and Methods ....................................... 145
Results ..................................................... 152
Discussion .................................................. 167
Literature Cited ............................................ 170

viii

CHAPTER 6: Conclusions and Future Research Perspectives ........... 175

Conclusions ................................................. 175
Future Research Perspectives ................................ 177
Literature Cited ............................................ 182

APPENDIX 1: GC-MS Identification of 016 and C18

Monounsaturated Fatty Acids in Coriander

and Thunbergia alata Endosperm ...................... 185
APPENDIX 2: cDNAs for Apparent Isoforms of the A918:0-

ACP Desaturase of Thunbergia alata Endosperm ........ 196

ix

Table

Table

Table

Table

Table

Table

Table

Table

Table

.i _._. as." -....,..._.

LIST OF TABLES

Pages

.lMetabolism of exogenous [lei‘C] medioum- and long-

chain fatty acids by slices of coriander endosperm ........ 75

..Oxygen- and ferredoxin-dependence of petroselinic acid

and oleic acid synthesis from [25”C]malonyl-C0A by

homogenates of coriander endosperm ........................ 79

..Glycerolipid and fatty acid products of [24“C]malonyl

-CoA metabolism by crude homogenates of coriander

endosperm ................................................. 82

..Products of the metabolism of [24“C]malony1-C0A by

homogenates of coriander endosperm ........................ 83

..Products of [UiuC]nonanoic acid labeling of coriander

endosperm slices ......................................... 91

..Glycerolipid content and fatty acid composition of

glycerolipids of developing coriander (A)

and.carrot (B) endosperm ................................. 113

...Changes in total fatty acid and petroselinic acid

content of phosphatidylcholine (PC) and
triacylglycerol (TAG) from early (16 DAF) to mid

(20 DAF)-development of coriander endosperm .............. 117

..Distribution of radiolabel incorporation into

glycerolipids of coriander endosperm following 3.5 h

incubation in [14”C]acetate ............................. 123

..Redistribution of radioactivity in phosphatidylcholine

(PC), triacylglycerol (TAG), and diacylglycerol (DAG)
following transfer of coriander endosperm slices from

[InJ‘C]acetate to media containing unlabeled acetate ..... 125

X

Table 4.5...Stereospecific fatty acid composition of

phosphatidylcholine of developing coriander endosperm....127
Table 4.6...Stereospecific fatty acid composition of

triacylglycerol of developing coriander endosperm

(22 to 26 DAF) ........................................... 128
Table 4.7...Stereospecific distribution of radioactivity in PC

following 15 min incubation of coriander

endosperm slices in.[1=4‘C]acetate ....................... 129
Table 5.1...In vitro substrate specificities of acyl-ACP or

-CoA desaturases of T. alata endosperm ................... 158
Table Appendix 1.1...Fatty acid compositions of triacylglycerol

(TAG), diacylglycerol (DAG), and phosphatidylcholine

(PC) of Thunbergia alata endosperm ....................... 195

xi

LIST OF FIGURES

ngm
Figure 1.1...Comparison of the chemical structures and melting
points of oleic acid (A), petroselinic acid (B), and
A6hexadeceno ic ac id ....................................... 2

Figure 1.2...Potential pathways for the biosynthesis of petroselinic

acid ...................................................... 9
Figure 1.3...Potentia1 pathways for the metabolism of petroselinic

acid ..................................................... 17
Figure 1.4...Examp1es of plants that accumulate petroselinic acid

(A) or A6 hexadecenoic acid (B) in their seed oil ........ 23
Figure 1.5...Mericarps (fruits) and dissected endosperm of carrot

(Daucus carota) and coriander (Coriandrum sativum)

(left to right) .......................................... 25
Figure 2.1...Western blot analyses of seed extracts of Brassicaceae

(Brass.) and Umbelliferae spp. (A) and leaf, root and

seed extracts of coriander (Coriandrum sativum L.) (B)...45
Figure 2.2...Nucleotide sequence of the coriander Type II acyl-ACP

desaturase cDNA (TII) and a comparison of the deduced

amino acid sequences of T11 and castor A918:0-ACP

desaturase cDNA (Cas) (Shanklin and Somerville, 1991)...48

Figure 2.3...Western blot analysis of protein extracts from callus

of transgenic tobacco .................................... 52
Figure 2.4...Gas chromatograms of fatty acid methyl esters prepared

from tobacco callus transformed with the pBI121

vector (A) and pBI121 containing the coriander Type 11

cDNA insert (B) .......................................... 53

xii

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

..Mass spectra of thiomethyl adducts of fatty acid methyl

esters detected exclusively in tobacco callus

transformed with the Type II coriander cDNA insert

in pBIlZl ............................................... 54
..Products of the metabolism of [24“C]malonyl-C0A by

crude homogenates of developing coriander endosperm ...... 80
..Analysis of long/medium chain acyl-ACPs of suspension

cultures of wild-type tobacco and transgenic tobacco

expressing a cDNA for the coriander 36 kDa desaturase....86
..Products of [lnl‘C]acetate time-course labeling of

developing carrot endosperm ............................. 118
..Autoradiograms of intact (A) or oxidized (B) products

of [1:‘“C]16:0-ACP desaturation assays conducted with

a 100,000xg supernatant of Thunbergia alata endosperm

homogenates ............................................. 153
...Time course of [lJMCJ16:O-ACP desaturation activity

in a 100,000xg supernatant of a T. alata endosperm

homogenate .............................................. 156
...Effect of catalase, ferredoxin (Fd), nitrogen (N2),

potassium cyanide (KCN), or hydrogen peroxide

(H502) on A616:0-ACP desaturation activity in T. alata

endosperm extracts ..................................... 159
..Nucleotide sequence of the cDNA insert of pTAD4 (A5),

and a comparison of the deduced amino acid sequences

of pTAD4 (A6) and cDNAs for the coriander A“16:0-ACP

desaturase (Cahoon et al., 1992) and the castor A91810-

ACP desaturase (A9) (Shanklin and Somerville, 1991) ..... 163
..Comparison of the A616:0-ACP desaturase activity of

extracts of E. coli BL21 pLysS containing only the

vector pET3d or pET3d with insert derived from the

T. alata cDNA of pTAD4 (with or without IPTG

induction) .............................................. 165

xiii

Figure

Figure
Figure

Figure

Figure

Figure

Figure

5.6...Mass spectrum of derivatives of [1:“C]16:1 formed by

16:0-ACP desaturation activity in extracts

of E. coli expressing the mature peptide-encoding

region of pTAD4 ......................................... 166
6.1...Proposed.pathway for petroselinic acid biosynthesis ..... 176
Appendix 1.1...Mass spectra of thiomethyl adducts of

C1,5 and C18 monounsaturated fatty acid standards

(A and B) and C16 and C18 monounsaturated fatty

acids of coriander (C-H) and Thunbergia alata (I-L)

endosperm glycerolipids ................................ 189
Appendix 2.1...Nuc1eotide and deduced amino acid sequences

of the cDNA insert of pTADl ............................. 198
Appendix 2.2...Nucleotide and deduced amino acid sequences

of the cDNA insert of pTAD2 ............................. 199
Appendix 2.3...Nucleotide and deduced amino acid sequences

of the cDNA insert of pTAD3 ............................. 200
Appendix 2.4...Comparison of the deduced amino acid

sequences of cDNA inserts of pTADl, 2, and 3 ............ 201

xiv

ABBREVIATIONS

ACP ........................ acyl carrier protein

ATP ........................ adenosine triphosphate

CaMV 358 ................... cauliflower mosaic virus 358 promoter
cDNA ....................... complimentary deoxyribonucleic acid
GDP ....... ' ................. cytidine diphosphate

CoA ........................ coenzyme A

CPT ........................ CDP-choline:DAG choline

phosphosphotransferase

DAF ........................ days after flowering

DAG ........................ diacylglycerol

DAGAT ...................... diacylglycerol acyltransferase

DGDG ....................... digalacatosyl diacylglycerol

dNTP ....................... dideoxy nucleotide triphosphate

DTT ........................ dithiothreitol

EDTA ....................... ethylenediamine tetraacetic acid

EGTA ....................... ethylene glycol-bis(B-aminoethyl ether)-

N,N,N',N'-tetraacetic acid

FA ......................... fatty acid

Fd ......................... ferredoxin

FFA ........................ free (or unesterified) fatty acid
FNR ........................ ferredoxinzNADPH reductase

G-3-P ...................... glycerol-3-phosphate

GC-MS ...................... gas chromatography-mass spectrometry
GPAT ....................... glycerol-3-phosphate acyltransferase
IPTG ....................... isopropyl-8—D-thiogalactopyranoside
LPA ........................ lysophosphatidic acid

LPAT ....................... lysophosphatidic acid acyltransferase
Mes ........................ 2-(N-morpholino) ethanesulfonic acid
MGDG ....................... monogalactosyl diacylglycerol

mRNA ....................... messenger ribonucleic acid

NADH ....................... B-nicotinamide dinucleotide (reduced)

NADPH ...................... B-nicotinamide dinucleotide phosphate
(reduced)

PA ......................... phosphatidic acid

PC ......................... phosphatidylcholine

PCR ........................ polymerase chain reaction

PE........ ................. phosphatidylethanolamine

PI ......................... phosphatidylinositol

Pipes ...................... piperazine-N,N-bis(2-etanesulfonic acid)

PMSF ..................... ..phenylmethyl sulfonyl fluoride

PVPP ....................... polyvinylpolypyrrolidone

Rf ......................... retention factor

RNA ........................ ribonucleic acid

SatFA ...................... saturated fatty acid

SDS/PAGE ................... sodium dodecyl sulfate/polyacrylamide gel
electrophoresis

TAG ........................ triacylglycerol

TCA ........................ trichloroacetic acid

TLC ........................ thin-layer chromatography

TIn ......................... gel-to-liquid cyrstalline phase transition
temperature

Tris ....................... tris(hydroxymethy1)amino methane

xvi

FATTY ACID NOMENCLATURE

xzy ........................ x is the number of carbon atoms in the fatty

acid or acyl chainzy is the number of double
'bonds in the fatty acid, e.g., 18:1 is a fatty
acid that contains eighteen carbon atoms and

one double bond.

A" ......................... Indicates that the double bond(s) is positioned

at the xth carbon atom relative to the
carboxyl end of an unsaturated fatty acid,
e.g., 18:1A6 is an eighteen carbon fatty acid
that contains one double bond at the sixth
carbon atom relative to the carboxyl end of

the acyl chain.

0" ......................... Indicates that the double bond(s) is positioned

at the xth carbon atom relative to the methyl
end of an unsaturated fatty acid, e.g.,
18:1m92 is an eighteen carbon fatty acid that
contains one double bond at the twelth carbon
atom relative to the methyl end of the acyl
chain. (Note: 18:1A6 and18zlml2 are the same

fatty acid.)

9:0 ........................ nonanoic acid

10:0 ....................... decanoic acid

12:0 ....................... lauric acid

14:0 ....................... myristic acid

16:0 ....................... palmitic acid

16 : 1A" ..................... A‘hexadecenoic acid
16:1A6 ..................... Ashexadecenoic acid
16:1A9 ..................... palmitoleic acid
17:0 ....................... heptadecanoic acid
18:0 ....................... stearic acid

xvii

 

18:1A6 ..................... petroselinic acid

18:1A8 ..................... Aaoctadecenoic acid

18:1A9 ..................... oleic acid

18:1A11 .................... cis-vaccenic acid

18:1A9,12-0H ............... ricinoleic acid (Note: 12-OH, hydroxyl residue
positioned at the twelth carbon atom from the
carboxyl end of the acyl chain.)

18:1A9, 12-epoxy ........... vernolic acid (Note: 12-epoxy, epoxy residue
positioned at the twelth carbon atom from the
carboxyl end of the acyl chain.)

18:2A9'12 ................... linoleic acid

18: 3A""""'12 .................. y-linolenic acid

18:3A9J2J5 ................. a-linolenic acid

20:1A5 ..................... Aseicosenoic acid

22:1A13 ..................... erucic acid

xviii

CHAPTER 1
Introduction

Seed oils of plants can contain a diversity of unusual fatty acids.
The term "unusual" indicates that a given fatty acid is found in only a
limited. number' of' families or species. In. addition, the chemical
structures of unusual fatty acids of seed oils often differ significantly
from those of the common C16 and 018 acyl moieties such as palmitic acid
(16:0) and oleic acid (18:1A9). In fact, more than 200 different unusual
fatty acid structures have been identified in plants (Badami and Patil,
1981; van de Loo et a1., 1993). Structural divergence in these acyl
moieties can include variations in carbon chain lengths as found in
medium-chain fatty acids (Ca-C“) such as lauric acid (12:0) and decanoic
acid (10:0) and in very long chain fatty acids (Zczo) such as erucic acid
(22:1Ad3). Unusual fatty acids can also contain functional groups such
as hydroxyl residues and epoxide rings as present, for example, in
ricinoleic acid (18:1A9,12-OH) and vernolic acid (18:1A9,12-epoxy),
respectively.

A more subtle variation observed in the chemical structures of
certain unusual fatty acids involves the positioning of double bonds in
monounsaturated acyl chains. Present in various amounts in nearly all
seed oils is oleic acid which has a double bond at the A9 carbon atom.
However, monounsaturated.fatty acids with double bonds at positions other
than the A9 carbon are known to exist as major components of the seed oil
of a few families or species of plants. This thesis examines two examples
of such acyl chains, the cisA6 monounsaturated fatty acids petroselinic
acid and.A6hexadecenoic acid (Figure 1.1). Oils containing these unusual
fatty acids are of potential economic value. Despite this, the synthesis
and.metabolism of petroselinic acid and.A6hexadecenoic acid.have received
little previous characterization. '

Natural Occurrence of Petroselinic Acid and AFHexadecenoic Acid

Petroselinic acid was first identified in the seed oil of parsley

(Petroselinum sp . ) , an Umbelliferae (or Apiaceae) species , by Vongerichten

Figure 1.1. Comparison of the chemical structures and melting points of

oleic acid (A), petroselinic acid (B), and A6hexadecenoic acid (C).

 

Oleic Acid (18:1A9)

0 mp. = 14°C
ll
HO \
B
Petroselinic Acid (18:1A6)
m.p. = 30°C
HO \
||
0
C
A6 Hexadecenoic Acid (16:1A6)
m.p. = ?
HO \

4

and Kahler (1909) and was later detected in the seed oil of English ivy
(Hedera helix), an Araliaceae species, by Palazzo and Tamburello (1914).
Since these early reports, petroselinic acid has been identified in a wide
range of Umbelliferae, Araliaceae, and Garryaceae species where it can
compose up to 85 wt% of the seed oil (Kleiman and Spencer, 1982) .
Petroselinic acid has also been found in the seed oil of a few genera of
other families including the Cornaceae (Kleiman and Spencer, 1982; Breuer
et'al., 1987) and Simarubaceae (Spencer et a1., 1970). Furthermore, small
amounts of petroselinic acid have been detected in human hair (Weitkamp
et a1., 1947) and other human tissues (Ohlrogge et a1., 1981).

Petroselinic acid is apparently a seed-specific fatty acid in plants
as only trace amounts have been found in leaves and roots of at least two
Umbelliferae species examined (Ellenbracht et‘ a1., 1980; Dutta and
Appelqvist, 1991). In.addition, cell cultures of'a number of Umbelliferae
species have been established, and the use of such systems for the study
of petroselinic acid synthesis has been proposed (Ellenbracht et a1.,
1980). However, in the case of callus cultures of parsley (Petroselinum
crispum) (Ellenbracht et a1., 1980) and embryogenic cultures of carrot
(Daucus carota) (Dutta and.Appe1qvist, 1989), petroselinic acid was found
to compose 51% of the total fatty acid. In contrast to these results,
levels of petroselinic acid are "strongly increased" in triacylglycerol
during somatic embryogenesis of the 'Umbelliferae anise (Pimpinella
anisum), as reported in.a recent meeting abstract (Theimer et a1., 1993).
Disregarding this preliminary report, the virtual lack of petroselinic
acid in carrot embryo cultures may suggest that synthesis of this fatty
acid in Umbelliferae seed is associated with endosperm, as this is the
predominant cell type, rather than embryo, in this tissue.

In contrast to petroselinic acid, Ashexadecenoic acid. is a
relatively obscure fatty acid. This is due in part to its more recent
discovery as a major component of a plant seed oil (Spencer et: a1. , 1971) .
'The most significant natural source of Afihexadecenoic acid is seed of

Thunbergia alata (or black-eyed susan vine) , which is generally classified

5

in the Acanthaceae family (Cronquist, 1988). The oil of this tissue
contains more than 80 wt% Ashexadecenoic acid (Spencer et a1., 1971).
Whether the occurrence of this fatty acid is limited to the seed of T.
alata has not been determined (though, given what is known of the typical
tissue distribution of unusual fatty acids, this is presumed to be the
case). Trace amounts of Ashexadecenoic acid have also been detected in
seed extracts of plants including English ivy (Grobois, 1971) and
Pricamnia selowii (Spencer et a1., 1970). In addition, Ashexadecenoic
acid has been identified in lipids of human skin (Shinohara, 1970) and
adipose tissue (Jacob and Grimmer, 1968).
Physical, Nutritional, and Industrial Properties of Petroselinic Acid

The presence of unsaturation at the A6 carbon atom instills
petroselinic acid with properties distinct from those of its A9 isomer
oleic acid. Perhaps the most dramatic divergence in the physical
properties of petroselinic acid and oleic acid is the difference in
melting points of these fatty acids. Depending on the reference, the
melting point of petroselinic acid is either 30°C (The Merck Index) or
33°C (The Lipid Handbook). As a result, petroselinic acid and
tripetroselinin (m.p. 28°C, The Lipid Handbook), a triacylglycerol
containing exclusively petroselinic acid, exist as solids at room
temperature. In contrast, the melting point of oleic acid is
approximately 14°C (The Lipid Handbook) or well below room temperature.
The high melting point of tripetroselinin has implications for nutritional
uses of this oil. For example, tripetroselinin and possibly other oils
rich in petroselinic acid could be used in the manufacture of unsaturated
margarines without requiring hydrogenation. This process is currently
used to convert conventional polyunsaturated plant oils to a solid form
as is necessary for margarine production. Hydrogenation of
polyunsaturated oils, however, results in extensive conversion of cis
double bonds to the trans configuration (Weidermann, 1978). Trans
unsaturated fatty acids fed to humans have been shown to result in

lipoprotein levels that favor the onset of cardiovascular disease (Zack

 

b-

 

and Katan, 1992; Siguel and Lermann, 1993). Therefore, a margarine

produced from petroselinic acid-rich oils may have more favorable health-
related properties than one produced from conventional plant oils.

Because of the location of its double bond at the A6 carbon atom,
petroselinic acid can also serve as a precursor of two valuable industrial
chemicals: lauric acid (12:0) and adipic acid (6:0 dicarboxylic). These
compounds are formed by the oxidative cleavage of petroselinic acid at its
double bond through methods such as ozonolysis. Lauric acid, the C12
product of this cleavage, is used extensively in the production of
detergents and surfactants and is derived commercially from palm kernel
and coconut. Neither palm (Elaeis guineensis) nor coconut (Cocos
nucifera) is grown for oil production in the U.S. or other nations of
temperate climate (Battey et a1. , 1989). An oilseed that produces large
amounts of petroselinic acid and that was adapted to climatic conditions
of the U.S. could therefore serve as a potential domestic source of lauric
acid. In addition, adipic acid, the C,5 product of petroselinic acid
oxidation, is a precursor of nylon 6,6. This polymer is the most
extensively manufactured nylon and is used primarily in the production of
carpet fibers (Putscher, 1984). Though adipic acid is derived
commercially from cyclohexane (Danly and Campbell, 1985) , a petroleum by-
product, plant oils rich in petroselinic acid could provide a renewable
source of this compound.

Furthermore, it has been reported that triacylglycerols containing
petroselinic acid as well as other 018 monounsaturated fatty acids with
double bonds between the A2 to A7 positions are resistant to hydrolysis
by porcine pancreatic lipase (Heimermann et: a1., 1973). In contrast,
triacylglycerols containing oleic acid are more readily digested by this
enzyme. A similar phenomenon has also been observed with lipases of
cotyledons of germinating Brassica napus seeds (Hills et a1. , 1990). In
this case, it was speculated that differences in rates of lipase
hydrolysis of oleic acid and petroselinic acid may relate to the different

syn-/anti- orientations of the double bonds of these fatty acids.

 

7

(Because its unsaturation is positioned at an even-numbered carbon, the
double bond of petroselinic acid exists in an anti- orientation relative
to the carboxyl group. Conversely, the A9 double bond oleic acid is
present in a syn- configuration.) If it is assumed that human pancreatic
lipase functions in the same manner as its porcine counterpart, then plant
oils rich in petroselinic acid would likely not be hydrolyzed as well as
conventional vegetable oils (i.e. , those containing oleic acid and its
polyunsaturated derivatives) in the small intestine. As a result,
petroselinic acid-rich oils could be potentially useful as low-caloric
edible oils.

A number of other physical properties and possible economic uses of
petroselinic acid have been reviewed previously by Placek (1963) .

In contrast to petroselinic acid, the melting point and other
physical properties of Ashexadecenoic acid have not been reported.
However, like petroselinic acid, A6hexadecenoic acid can be oxidatively
cleaved at its double bond to produce adipic acid for nylon synthesis.
The other compound formed in this process, decanoic acid (or capric acid)
(10:0), is used in the manufacture of artificial fruit flavorings and
perfumes (The Merck Index). It is also likely that oils rich in
Ashexadecenoic acid are resistant to hydrolysis by pancreatic lipase and,
as such, could serve as a component of low-caloric edible oils. In
addition, the use of Aéhexadecenoic acid in cosmetics and skin creams has
been patented (Steifel, 1977). These formulations apparently have
excellent skin-care properties, and as Aéhexadecenoic acid is a natural
component of skin lipids (Shinohara, 1970), the likelihood of allergic
response to such cosmetics is minimal (Steifel, 1977).

Biosynthesis of Petroselinic Acid and A‘Hexadecenoic Acid

Prior to the research presented in this thesis, no studies of
Ashexadecenoic acid biosynthesis had been reported. In addition, the only
previous work that directly addressed the metabolic origin of petroselinic
acid was a proceedings paper by Grobois and Mazliak (1978). In these

studies, 1"C-acetate was provided to intact English ivy seeds and membrane

 

fractions of this tissue. The radiolabel was incorporated into
petroselinic acid which was associated primarily with polar lipids. From
these results, it was speculated that petroselinic acid may arise from
reactions involving phospholipid precursors.

With such little background information available, attempts to
characterize the biosynthesis of petroselinic acid and AGhexadecenoic acid
must take into consideration all potential pathways (as summarized in
Figure 1.2) . Based on what is known of the synthesis of other unsaturated
fatty acids, one possibility is that the double bonds of petroselinic acid
and A6hexadecenoic acid result from aerobic desaturation of an acyl moiety
bound to either a glycerolipid (Browse and Somerville, 1991) or coenzyme
A (CoA) (Cook, 1985). Fatty acids formed via glycerolipid-linked
desaturation include, for example, linoleic acid, which can be derived
from the A12 (or n-6) desaturation of oleic acid esterified to
phosphatidylcholine in plant and algal cells (Gurr et a1. , 1969; Slack et
a1., 1978). In addition, acyl-CoA desaturation is the primary route of
monounsaturated fatty acid (particularly oleic acid) biosynthesis in
animals (Cook, 1985). Though such reactions have yet to be conclusively
demonstrated in plants, evidence has been presented for the involvement
of an acyl-CoA desaturase in the formation of Aseicosenoic acid (20:1A5)
in meadowfoam (Limanthes alba) seed (Pollard and Stumpf, 1980; Moreau et
a1. , 1981).

If produced through either acyl-CoA or glycerolipid-linked
desaturation, the likely precursors of petroselinic acid and
Aéhexadecenoic acid would be esters of stearic acid and palmitic acid,
respectively. Seed oils of species which synthesize petroselinic acid and
A6hexadecenoic acid typically contain 70 to 85 wt% of these fatty acids.
Therefore, such species would not only require a likely A6 desaturase but
also a mechanism for the release of large amounts of stearic acid and
palmitic acid from the de novo reactions of fatty acid synthesis, in order
to generate the appropriate acyl-CoA or glycerolipid substrate for the

desaturase. The stearic acid or palmitic acid needed as precursors in the

Figure 1.2. Potential pathways for the biosynthesis of petroselinic acid.

10

Potential Pathways of Petroselinic Acid (18:1A6°’S) Biosynthesis

A) Desaturation on a Glycerolipid

18:0 18:1A6
A6 Desat.

18:1A9 18:1A9

P-Choline P-Choline

B) Desaturation of an Acyl-CoA

A6 Desat.

18:0-GOA ——> 18:1A5-CoA

C) Desaturation/Elongation of AcyI-ACP

A6 Desat.

18:0-ACP ———> 18:1A6-ACP

A4 +20
16:0-ACP 6—7» 16:1A4-ACP ——> 18:1A6-ACP
esa.

A2 +20
14:0-Acp [TS—t» 14:1A2-ACP —-> 16:1A4-ACP -+-2—C-> 18:1A6-ACP
e a.

D) Isomerization of an Unsaturated Precursor

14:1A2m5.ACP -> 14:11:?“ -ACP -> 16:1A‘°"9-ACP -> 18:1A6c’S-ACP

18:1A9-X ——-> 18:1A6-X
(X: glycerolipid, CoA, ACP, etc.)

11

above biosynthetic scenario could result from an acyltransferase or
thioesterase specialized for the efficient removal of these moieties from
acyl carrier protein (ACP).

Routes of petroselinic acid and A5hexadecenoic acid synthesis
involving desaturation of an acyl moiety bound to a glycerolipid or CoA
would therefore seemingly require both a unique desaturase and a unique
acyl-ACP thioesterase or acyltransferase. The degree of metabolic
specialization necessary for such a pathway is not without precedent. For
example, oil of meadowfoam is composed of approximately 60 wt% of
Aseicosenoic acid (Pollard and Stumpf, 1980). It has been proposed that
this fatty acid arises from the activity of a Aseicosanoyl—COA desaturase,
as alluded to above (Pollard and Stumpf, 1980; Moreau et: a1. , 1981). The
eicosenoyl-CoA substrate is believed to originate from palmitoyl-COA or
stearoyl-CoA that is elongated by reactions separate from those of de novo
fatty acid synthesis. The pathway for Aseicosenoic acid formation would
therefore need an unprecedented desaturase, a non-ACP-track fatty acid
elongation system, and a means of releasing significant amounts of
palmitic acid or stearic acid from pools of acyl-ACPs.

The involvement of aerobic desaturation in the synthesis of
petroselinic acid and Ashexadecenoic acid could also entail reactions that
use acyl-ACPs as substrates. The only previously identified acyl-ACP
desaturase is the A9stearoyl (18:0)-ACP desaturase, which catalyses the
production oleoyl-ACP from 18:0-ACP. This enzyme was originally
identified in photoauxotrophic cultures of Euglena gracilis (Nagai and
Bloch, 1965), and its activity was subsequently detected in tissue and
chloroplast extracts of higher plants (Nagai and Bloch, 1968; Jacobson et:
a1., 1974; Jaworski and Stumpf, 1974; McKeon and Stumpf, 1982). In
contrast to all other known desaturases, the “18:0-ACP desaturase
displays soluble activity (Nagai and Bloch, 1968). In addition, this
enzyme uses reduced ferredoxin as its electron donor rather than
cytochrome b5, in contrast to the membrane-bound desaturases of animals

and the endoplasmic reticulum-localized desaturases of plants (Nagai and

 

 

12

Bloch, 1968). The soluble nature of the A918:0—ACP desaturase has
facilitated its purification from tissues of higher plants (McKeon and
Stumpf, 1982; Cheesbrough and Cho, 1990; Kinney et a1. , 1990; Shanklin and
Somerville, 1991; Thompson et a1. , 1991) and a number of cDNAs encoding
this enzyme have recently been isolated (Kinney et a1. , 1990; Shanklin and
Somerville, 1991; Shanklin et: a1. , 1991; Thompson et a1. , 1991; Nishida
et a1., 1992; Sato et a1, 1992; Slocombe et a1., 1992; Taylor et a1.
1992).

If petroselinic acid and AGhexadecenoic acid result from the
activity of an analogous enzyme, the most direct pathways would involve
the A6desaturation of 18:0-ACP and palmitoyl (16:0)-ACP, respectively.
Tissues that synthesize petroselinic acid, however, also produce oleic
acid. Therefore, in order to form the large amounts of petroselinic acid
typically found in seed oils, a “18:0-ACP desaturase would potentially
have to "outcompete" the A918:0-ACP desaturase for their common substrate
(assuming that these enzymes occur together in plastids and in comparable
amounts). This competition could be effectively avoided if the double
bond of petroselinic acid were inserted prior to the C18 stage of de novo
fatty acid synthesis. Alternatively, a A6 desaturase could have a lower
KIn for 18:0-ACP than the “18:0-ACP desaturase.

A further possibility is that petroselinic acid and Ashexadecenoic
acid are formed by isomerization of double bonds of pre-existing
monounsaturated fatty acids. For example, Shibahara et: a1. (1990) have
Presented data suggesting that the A9 double bond of oleic acid can be
e’I'IZYInatically shifted to the A11 position to form cis-vaccenic acid in
pulp of kaki (Diospyros kaki) . This reaction is apparently reversible as
the A11 double bond of cis-vaccenic acid can also be moved to the A9
Carbon to produce oleic acid. These results were obtained by monitoring
the metabolism of deuterated fatty acids using GC-MS. However, the
mechanism through which double bond migration occurs, including the exact
form of the substrate, has yet to be determined. If a similar reaction

13 associated with petroselinic acid synthesis, it could be envisioned

 

 

 

13

that the double bond of oleic acid is isomerized to the A6 position. In
the case of Aéhexadecenoic acid, such a biosynthetic pathway is more
difficult to propose. Seeds typically contain only trace amounts of Cl,5
monounsaturated fatty acids (Harwood, 1980). Therefore, a potential
precursor of Ashexadecenoic acid would likely be absent in developing seed
of Thunbergia alata.

Another form of double bond isomerization is observed in the
anaerobic reaction leading to the synthesis of palmitoleic acid (16:1A9)
and cis-vaccenic acid (18:1An) in Escherichia coli. The double bond of
these fatty acids has been shown to arise from an isomerization step that
occurs between the C8 and C10 stages of de novo fatty acid biosynthesis.
More specifically, the double bond of the transAzdecenoyl (10:1)-ACP
intermediate is shifted to the cisA3 configuration by the activity of the
3-hydroxydecanoy1-ACP dehydrase (Bloch, 1970) . The resulting cisAa-
decenoyl-ACP is subsequently elongated to yield palmitoleic acid and cis-
vaccenic acid. Because double bonds of petroselinic acid and
Aéhexadecenoic acid are located at even-numbered carbon positions, the E.
coli pathway of unsaturated fatty acid formation cannot be easily related
to the synthesis of these fatty acids. Instead, schemes involving, for
example, the isomerization of a transAzenoyl-ACP intermediate to a cisA2
moiety could be proposed. In the case of petroselinic acid, such a
pathway would likely entail the conversion of transAztetradecenoyl (14:1) -
ACP to cisAztetradecenoyl-ACP followed by four-carbon elongation.

Though petroselinic acid and A5hexadecenoic acid could arise through
other biosynthetic scenarios, those described above are the most likely
routes based on what is known for the synthesis of other unsaturated fatty
acids in various organisms.

Metabolism of Petroselinic Acid and A‘Hexadecenoic Acid

As with the synthesis of petroselinic acid and Ashexadecenoic acid,
the routes through which these fatty acids are incorporated into
glycerolipids of developing seeds have not been determined. With regard

to A6hexadecenoic acid, there have been no previous reports of the levels

14

of this fatty acid in specific glycerolipid classes of Thunbergia alata
seed. Previous studies, however, have indicated that amounts of
petroselinic acid are disproportionately distributed.between the storage
lipid triacylglycerol and polar or membrane-type glycerolipids in
Umbelliferae seed. In the report by Prasad et a1. (1987), petroselinic
acid.was found to compose approximately 70 wt% of the seed oils of carrot
and coriander but only 16 to 22 wt% of the fatty acids of the total
phospholipid.fractionu Similar results have been reported for developing
carrot seed (Dutta and.Appelqvist, 1991). In this case, triacylglycerols
contained >75 wt% petroselinic acid , whereas polar lipids contained <15
wt% of this fatty acid. This difference in amounts of petroselinic acid
in polar and storage lipids was observed despite the fact that polar
lipids synthesis occurs simultaneous to periods of rapid petroselinic acid
formation and deposition into triacylglycerols (Dutta and Appelqvist,
1991). It‘would.thus appear that Umbelliferae seed.possess some mechanism
for limiting the accumulation of petroselinic acid in membrane—type
glycerolipids while concentrating this fatty acid into a storage form in
triacylglycerols.

This phenomenon is similar to that which has been previously
described for the metabolism of other unusual fatty acids (as reviewed by
Browse and Somerville, 1991; Stymne et a1., 1990), particularly acyl
moieties whose chemical structures differ significantly from those of
common fatty acids such as oleic acid. For example, in seeds that
synthesize high levels of'medium-chain (Slabas et a1., 1982; Bafor et.al.,
1990) and very-long chain fatty acids (Gurr et a1., 1974; Pollard et a1.,
1980), these unusual acyl chains typically compose less than.a few weight
percent of the fatty acids of polar lipids but can account for 50 to 90
wt% of the fatty acids of triacylglycerols. In theory, the ability of
seeds to limit the accumulation of unusual fatty acids in polar lipids
insures that "proper" membrane function is not disrupted by the divergent
chemical structures of these acyl moieties (Stymne et a1., 1990). In this

regard, the melting point of petroselinic acid is approximately 30°C.

15

Therefore, Tms of polar lipids that contain large amounts of this fatty
acid would likely be quite elevated compared to those containing primarily
oleic acid and linoleic acid. As a result, bilayers containing
glycerolipids rich in petroselinic acid might exist in the non-
physiological gel phase at normal ambient temperatures. Furthermore,
because additional double bonds are not inserted into petroselinic acid,
the presence of large amounts of this fatty acid in polar lipids might
reduce the ability of cells to adjust levels of membrane unsaturation in
response to changes in their environment.

The mechanism through which developing seeds limit amounts of
unusual fatty acids in membrane-type glycerolipids while accumulating
these acyl moieties in triacylglycerols (TAG) has been the subject of
considerable speculation in the literature. One idea is that enzymes
involved in polar lipid and TAG metabolism have different substrate
specificities. In this regard, phosphatidylcholine (PC) is usually the
most abundant polar lipid in seeds (Harwood, 1980). In addition, PC and
TAG are both derived from diacylglycerol (DAG). The conversion of DAG to
PC and TAG is catalyzed by the enzymes GDP—cholinezDAG choline
phosphotransferase (choline phosphotransferase) and DAG acyltransferase,
respectively. As such, the incorporation of unusual fatty acids into PC
could be avoided if choline phosphotransferase discriminates against DAG
rich in unusual acyl moieties. Through the selective use of DAG species
containing common fatty acids for PC synthesis, a pool of DAG enriched in
unusual fatty acids would remain for incorporation into TAG. In contrast,
results reported by Bafor et a1. (1990) have indicated that the opposite
scenario is involved in the deposition of medium-chain fatty acids such
as decanoic acid (10:0) into TAG of" developing seeds of Cuphea
lanceolata. litthese studies, diacylglycerol acyltransferase rapidly and
preferentially converted DAG containing medium chain fatty acids into TAG.
By channeling DAG rich in medium chain fatty acids into the synthesis of

storage lipids, these molecules were made unavailable for conversion to

16

PC. Such a.nmmhanism thus effectively precludes the accumulation of
medium-chain fatty acids in membranes.

Based on the above discussion, the unequal distribution of
petroselinic acid in polar and storage lipids could be explained by the
fact that choline phosphotransferase uses DAG species containing common
fatty acids in preference to those containing petroselinic acid (see
Figure 1.3). Alternatively, DAG acyltransferase may rapidly shunt DAG
rich in petroselinic acid into TAG, leaving behind molecules containing
reduced amounts of this unusual fatty acid for subsequent incorporation
into PC. Either scenario could be used to explain results reported by
Dutta et a1. (1992) regarding the metabolism of petroselinic acid. In
these studies , 1"C-glycerol-3-phosphate provided to carrot seed microsomes
was converted primarily to phosphatidic acid and TAG in the presence of
petroselinoyl-COAH ILittle of the radiolabel, however, was detected.in.PC.
This finding suggests that, through some mechanism, movement of
petroselinic acid into polar lipids such as PC is partially prevented (as
shown in Figure 1.3, Pathway A).

Another possibility is that petroselinic acid is not excluded from
polar lipids (as shown in Figure 1.3, Pathway B). Instead, an "editing"
medhanism might exist which prevents high levels of petroselinic acid
accumulation in polar lipids. Such a scenario could arise, for example,
if choline phosphotransferase is incapable of distinguishing DAG moieties
that contain petroselinic acid from those containing oleic acid” Grobois
and Mazliak (1979), for example, reported that large amounts of
radiolabeled petroselinic acid appear in polar lipids following incubation
of English ivy seeds irll‘C-acetate. In addition, PC has been shown to
readily participate in the movement of fatty acids into TAG in seeds of
species (e.g., safflower, soybean, and linseed) that synthesize primarily
(L3 polyunsaturated fatty acids such as linoleic acid (18:2A9J2) and «-
linolenic acid (18:3A9A2A5)(Slack et a1., 1978). In such seeds, oleic

acid first enters PC where it can serve as a substrate for one or

17

Figure 1.3. Potential pathways for the metabolism of petroselinic acid.
Diacylglycerol (DAG) is formed through reactions of the "Kennedy" pathway
(Kennedy, 1961). Alternatives routes of DAG metabolism may explain why
petroselinic acid is typically found in much lower amounts in
phospholipids such as phosphatidylcholine (PG) than in triacylglycerols
(TAG): (1) DAG rich in petroselinic acid may be partially excluded from
incorporation into PC (Pathway A). This partial exclusion may result
from the substrate specificities of choline phosphotransferase (CPT) or
diacylglycerol acyltransferase (DAGAT) (as described in the text). (2)
Alternatively, DAG rich in petroselinic acid.may be incorporated into PG
and TAG at comparable rates (Pathway B). Petroselinic acid may then be
removed or "edited" from PC by the reverse reaction of CPT or by
phospholipases that cleave this fatty acid directly from the glycerol
backbone. In the latter case, petroselinic acid may be replaced by GoA
esters oficommon fatty acids (e.g., oleic acid, 18:1A9) from the acyl-GOA
p001. G 3-P, glycerol-3-phosphate; GPAT, G 3-P acyltransferase; LPA,
LYSOphosphatidic acid; LPAT, LPA acyltransferase; PA, phosphatidic acid;
IV\ P'tase, PA. 'phosphatase; DAG, Diacylglycerol; GPT, choline
'PIHJSphotransferase; PG, Phosphatidylcholine; DAGAT, DAG acyltransferase;
TEN}, Triacylglycerol.

 

18

Potential Pathways of Petroselinic Acid Metabolism

 

Pathway A

GDP-Choline

1am“ _/
18:1A°-[ "
P-Choline

PC
18:1A6-CoA

DAGAT

13m“
18:1A6-[
18:1A6

TAG

 

   

 

 

OH
Ho{
O-PO3

G 3-P

18:1A6-CoA
GPAT
13:11:6
0—P03

LPA

18:1A6-CoA
LPAT k

 

CDP-

 

Choline

gr,
DAGAT
a 123m6
1821A -CoA 18:1A6-[

Pathway B
. 9
18.1A-CoA 1821A6_00A

13:1A°
18:1A6-[
P-Choline

18:1A9
PC 9
18:1A
P—Choline

PC

GDP-
Choline

18:1A6
TAG

 

19

two desaturation steps. The resulting polyunsaturated acyl moieties may
then be incorporated into TAG. A similar route of metabolism has also
been described for the unusual fatty acids ricinoleic acid in castor
(Ricinus communis) endosperm (Bafor et: a1., 1991) and y-linolenic acid
(18:3A6'9'12) in borage (Borago officinalis) seed (Griffiths et: a1. , 1988) .
However, as with C18 polyunsaturated fatty acids, the synthesis of these
fatty acids occurs on PC from oleic acid or linoleic acid (Bafor et a1.,
1991; Stymne and Stobart, 1986).

The removal of fatty acids from PC for subsequent storage in TAG can
occur by release of the intact glycerol backbone from PC and subsequent
incorporation of the resulting DAG into TAG as has been shown by
experiments of Slack et a1. (1978). The conversion of PC to DAG can
potentially result from the reverse reaction of choline phosphotransferase
(Slack et a1., 1983) or by phospholipase C activity. Results of other
studies have also indicated that fatty acids synthesized on PC can be
directly removed from the glycerol backbone of this lipid and be exchanged
with other fatty acids in the acyl-GOA pool (Stymne and Stobart, 1987).
The released fatty acids are subsequently re-incorporated as GoA esters
onto glycerol backbones targeted for TAG synthesis. This direct removal
of fatty acids from PC may occur through activity of phospholipases (e.g. ,
phospholipases A1, A2, or B). In this regard, Banas et: a1. (1992) have
demonstrated the existence of phospholipases in plant extracts that cleave
unusual oxygenated fatty acids from PC. This activity likely prevents the
accumulation of unusual fatty acids such as ricinoleic acid and vernolic
acid in PC following their synthesis on this lipid (Stymne, 1993) . Based
on the above examples, if petroselinic acid does readily enter PC and
other polar lipids in developing Umbelliferae endosperm, accumulation of
this fatty acid could be restricted by conversion of phospholipids rich
in petroselinic acid to DAG or by the direct removal of petroselinic acid
from the glycerol backbone of phospholipids.

A final consideration regarding the metabolism of petroselinic acid

and A‘hexadecenoic acid in developing seeds is the substrate specificities

 

20

of acyltransferases that give rise to DAG. This molecule is formed
through reactions of the Kennedy pathway (Kennedy, 1961) involving the
successive incorporation of two acyl-GOA moieties onto glycerol-3-
phosphate and the subsequent removal of the phosphate residue from the sn-
3 carbon of the glycerol backbone. The first acylation step is catalyzed
by glycerol-3-phosphate acyltransferase (GPAT) . This enzyme has a rather
broad substrate specificity in seeds of species examined to date
(Ichihara, 1984; Griffiths et a1. , 1985; L5hden and Frentzen, 1992;
Eccleston and Harwood, 1990) and can incorporate CoA esters of unusual
fatty acids (Sun et a1., 1988). In contrast, the substrate specificity
of lysophosphatidic acid acyltransferase (LPAT) , the enzyme that catalyzes
the second acylation step of the Kennedy pathway, is typically more
defined. Studies reported by Ichihara et a1. (1987), for example, have
shown that LPAT of safflower seed microsomes displays a marked preference
for fatty acids such as oleic acid and linoleic acid that contain A9
unsaturation. Significantly less activity was detected in the presence
of saturated substrates or unsaturated substrates such as petroselinic
acid with double bonds at positions other than the A9 carbon.

The narrow substrate specificity of LPAT may explain that rapeseed
TAG, for example, contains significant amounts of erucic acid in only the
sn-1 and sn-3 positions of TAG. As a result, the oil of this plant can,
in theory, contain no more than 67 mol% erucic acid. However, TAG of
certain species are composed of significantly more than 67 mol% of a given
unusual fatty acid. Examples of this include the seed oil of many Cuphea
species and the kernel oil of palm which can contain 80 to 90% medium
chain fatty acids (The Lipid Handbook). This composition suggests that
LPAT of such tissues is specialized for the incorporation of these unusual
acyl chains. In the case of both palm kernel and Cuphea embryo extracts,
LPAT has been shown to catalyze the incorporation of medium chain acyl-
CoAs (00 and Huang, 1989; Bafor et: a1. , 1990) . This was particularly true
when the acceptor molecule for the acyltransferase was lysophosphatidic

acid that contained a medium chain fatty acid. Such results are of

 

21

relevance to genetic engineering studies of seed oils. In this regard,
transgenic Brassica species have recently been developed that synthesize
significant amounts (>25 mol% of the total seed oil) of lauric acid
(Voelker et al., 1992). Levels of lauric acid accumulation in excess of
67 mol% in oils of these plants, however, may also require the
introduction of an LPAT such as present in Cuphea embryo and palm kernel
that is specialized for the metabolism of this fatty acid.

TAG of species that produce petroselinic acid and Afhexadecenoic
acid can contain 70 to 85 mol% of these unusual fatty acids. In addition,
Gunstone (1991) has demonstrated using 13’C—NMRthat petroselinic acid can
compose nearly 50% of the sn-2 position of TAG in several Umbelliferae
species. The contributions of acyltransferases to levels of petroselinic
acid in Umbelliferae seed oils has been examined by Dutta et a1. (1992).
In these studies, the ability of acyltransferases of carrot seed
microsomes to incorporate oleoyl-GoA and petroselinoyl-GoA onto glycerol
backbones was compared. In the case of GPAT, the rate of oleoyl-GoA
incorporation was nearly twice that of petroselinoyl-CoA. LPAT also
incorporated oleoyl-GoA at rates significantly higher than that for
petroselinoyl-CoAH However, relative to those of rapeseed.and safflower,
microsomes of carrot seed were the only plant extract examined that could
introduce petroselinoyl-GoA onto a petroselinoyl-lysophosphatidic acid
acceptor. Still, the rate of this esterification was four-times lower
than that observed for oleoyl-CoA. These findings suggest that
acyltransferases may contribute somewhat to the total levels and
stereospecific distribution of petroselinic acid found in Umbelliferae
TAG. However, other factors may be of equal or greater importance. Such
factors may include, for example, the relative levels of petroselinoyl-COA
present in acyl-GOA pools that are available for incorporation on tn
glycerol backbones in vivo.

.Rationale
As described above plant oils containing petroselinic acid and

Afhexadecenoic acid have potential industrial and nutritional value, yet

 

22

there are currently no established oilseed crops that accumulate large
amounts of these fatty acids. Such crops, however, could be produced by
introducing genes encoding enzymes specialized for the synthesis and
metabolism of petroselinic acid and A6hexadecenoic acid into existing
oilseeds such as a rapeseed and sunflower. Limiting this endeavor is the
lack of understanding of pathways of biosynthesis and metabolism of these
unusual fatty acids. Studies presented in this thesis are an attempt to
provide such information.

In addition to the biotechnological implications of this work,
aspects of the synthesis and metabolism of petroselinic acid and
Ashexadecenoic acid are of general significance to plant lipid
biochemistry. For example, studies of the biosynthetic origin of the
double bonds of these fatty acids would likely provide new information
regarding desaturation mechanisms in plants. Also, characterization of
pathways through which petroselinic acid and Aéhexadecenoic acid are
incorporated into TAG of developing seeds may be useful for understanding
the metabolism of other unusual fatty acids.

Studies of petroselinic acid biosynthesis presented in this thesis
were conducted primarily with developing endosperm of the Umbelliferae
coriander (Coriandrum sativum) (Figures 1.4A, 1.5). This tissue
accumulates petroselinic acid in its seed oil to levels of nearly 75 wt%
of the total fatty acid (Kleiman and Spencer, 1982). In addition, unlike
many Umbelliferae, coriander is an annual rather than a biennial. As
such, seeds can be harvested year-round from this plant without requiring
vernalization of roots. Furthermore, the endosperm of coriander is larger
than that produced by many other annual Umbelliferae, which makes this
tissue particularly attractive for biochemical studies. Studies in
Chapter 4 were performed with endosperm of the Umbelliferae wild carrot
(Daucus carota) (Figure 1.5) as well as with endosperm of coriander.
Petroselinic acid composes at least 70 wt% of the seed oil of carrot
(Kleiman and Spencer, 1982). In addition, the campus of Michigan State

University has extensive natural stands of wild carrot that typically

23

Figure 1.4. Examples of plants that accumulate petroselinic acid (A) or
Afimmadecenoic acid (B) in their seed oil. Shown in A are flowers of
coriander (Coriandrum sativum), an Umbelliferae species. The seed oil
of this plant can contain petroselinic acid at levels of more than.75 wt%
of the total fatty acid. Shown in B are vines and flowers of Thunbergia

alata. AQHexadecenoic acid can compose more than 80 wt% of the seed oil

of this plant.

 

24

 

(A)

25

    

llllllllllllll'llil

Figure 1.5. Mericarps (fruits) and dissected endosperm of carrot (Daucus

MFTDII‘. 1

carota) and coriander (Coriandrum sativum) (left to right). Studies in
Chapters 2, 3, and 4 were conducted using endosperm (white tissue above)

of either of these plants.

 

 

26

bloom in August. The ready availability of developing seeds of this plant
was a major reason for its use in studies detailed in Chapter 4.
Experiments described in Chapter 5 were conducted with endosperm of
Thunbergia alata (Figure 1.4B) , which is the only plant identified to date
that accumulates large amounts of Aéhexadecenoic acid in its seed oil.
Advantageous qualities of this plant include its perennial growth habit
(under greenhouse conditions) and the fact that it flowers bountifully
throughout its life cycle.

Finally, the chapters that follow are presented as publication
units. However, studies in Chapters 2 and 3 are complementary as a
portion of the research presented in the latter chapter provided the basis

for experiments described in Chapter 2.

 

 

27

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CHAPTER 21

Expression of a Novel Coriander Desaturase Results in Petroselinic Acid

Production in Transgenic Tobacco

Abstract

Little is known about the metabolic origin of petroselinic acid
(18:1A6c—1"), the principal fatty acid of the seed oil of most Umbelliferae,
Araliaceae, and Garryaceae species. To examine the possibility that
petroselinic acid is the product of an acyl-acyl carrier protein (ACP)
desaturase, western blots of coriander (Coriandrum sativum L.) and other
Umbelliferae seed extracts were probed with antibodies against the
Agstearoyl-ACP desaturase of avocado. In these extracts, proteins of 39
and 36 kDa were detected. Of these, only the 36 kDa peptide was specific
to tissues that synthesize petroselinic acid. A cDNA encoding the 36 kDa
peptide was isolated from a coriander cDNA library, placed under control
of the cauliflower mosaic virus 35$ promoter, and introduced into tobacco
via Agrobacterium tumefaciens-mediated transformation. Expression of this
cDNA in transgenic tobacco callus was accompanied by the accumulation of
petroselinic acid and A‘hexadecenoic acid, both of which were absent in
control callus. These results demonstrate the involvement of a 36 kDa
putative acyl-ACP desaturase in the biosynthetic pathway of petroselinic
acid and the ability to produce fatty acids of unusual structure in

transgenic plants by the expression of a gene for a novel desaturase.

 

1Material in this chapter was published previously [Cahoon EB,
Shanklin J, Ohlrogge JB (1992) Proc Natl Acad Sci USA 89: 11184-11188].

37

38

Introduction

Petroselinic acid (18:1A693) is an unusual fatty acid that occurs
primarily in seeds of Umbelliferae (or Apiaceae), Araliaceae, and
Garryaceae species (Kleiman and Spencer, 1982). This fatty acid composes
as much as 85% of the total fatty acid of Umbelliferae seeds but is
virtually absent in leaves and other tissues of these plants (Ellenbracht
et a1. , 1980; Kleiman and Spencer, 1982; Dutta and Appelqvist, 1991) . The
structure of petroselinic acid differs from that of oleic acid (18:1A9fl) ,
a common plant fatty acid, by the position of its double bond. Because
of the presence of unsaturation at the A6 carbon atom, petroselinic acid
is of potential industrial significance. Through chemical cleavage at its
double bond, petroselinic acid can be used as a precursor of‘ lauric acid
(12:0), which is a component of detergents and surfactants, and adipic
acid (6:0 dicarboxylic), which is the monomeric component of nylon 6,6
(Murphy, 1992).

The pathway for petroselinic acid biosynthesis has not been
previously determined. Monounsaturated fatty acids of plants typically
derive from the desaturation of C1,5 and C18 saturated fatty acids bound to
acyl carrier protein (ACP) or to glycerolipids (Browse and Somerville,
1991; Jaworski, 1987). Our preliminary results from a variety of [“G]
labelling studies suggest that petroselinic acid is the product of an
acyl-ACP desaturase (see Chapter 3). The only such enzyme to have been
identified in plants is the Agstearoyl-ACP (18:0-ACP) desaturase (EC
1.14.99.6) which catalyzes the conversion of 18:0-ACP to 18:1A9-ACP (Nagai
and Bloch, 1968; Jaworski and Stumpf, 1974). This reaction is readily
assayable in tissue extracts of most plants using [1"G]18:0-ACP and
cofactors including ferredoxin, NADPH, and ferredoxin-NADPH reductase
(Jaworski and Stumpf, 1974; McKeon and Stumpf, 1981). However, the in
vitro synthesis of petroselinic acid from [“C]acyl-AGPs, including [1-
1l’C]18:0-ACP (or from [1-“C]18:0-CoA), has yet to be detected using seed
extracts of the Umbelliferae species coriander and carrot (see Chapter 3) .

Lack of a direct assay complicates any attempt to characterize the

 

39

biosynthetic pathway or to purify the acyl-ACP desaturase believed to be
involved in petroselinic acid synthesis.

As an alternative approach, the possibility that this proposed acyl-
ACP desaturase is antigenically related to “18:0-ACP desaturase was
examined. Zhn this study, western blots of extracts of tissues that
synthesize petroselinic acid were probed with antibodies raised against
the A918:0-AGP desaturase of avocado (Shanklin and Somerville, 1991) to
identify any related peptides. These antibodies were also used to isolate
cDNAs from a coriander endosperm expression library to directly explore
the possible involvement of an acyl-ACP desaturase in the synthesis of

petroselinic acid.

Materials and Methods
Western Blot Analysis
Plant tissues were homogenized in 50 mM potassium phosphate (pH
7.2), 2 mM phenylmethanesulfonyl fluoride (PMSF), 5 mM sodium
metabisulfite, 5 mM EDTA, 5 mM isoascorbate (5 ml/g fresh weight) with a
Polytron PT10/35 (Brinkman), passed through two layers of miracloth
(Calbiochem), and mixed with SDS/PAGE sample buffer.

Protein extracts of transgenic tobacco calli were obtained by
homogenization of tissue with an Elvehjem homogenizer in 2 ml of a
solution of 0.7 M sucrose, 0.5 M Tris, 50 mM EDTA, 0.1 M potassium
chloride, pH 9.4, containing 2% (v/v) B-mercaptoethanol and 2 mM PMSF
added just prior to use. The homogenate was mixed thoroughly with 2 ml
of phenol and centrifuged at 3000x g for 10 min. The upper phenol phase
was recovered, and proteins were precipitated with the addition of 10 m1
of 0.1 M ammonium acetate in methanol, and overnight incubation at -20°C.
The protein pellet obtained following centrifugation was washed
sequentially with methanolic ammonium acetate and acetone then air-dried
prior to addition of SDS/PAGE sample loading buffer.

Proteins of plant extracts were separated by SDS/PAGE (Laemmli,

1970) using 11% (w/v) acrylamide gels. Proteins were transferred from

40

gels and fixed to nitrocellulose as described (Post-Beittenmiller et a1.,
1989). Western blot analyses were performed using polyclonal,
immunoaffinity-purified antibodies raised against the 6918:0-ACP
desaturase of avocado (Shanklin and Somerville, 1991) and colorimetric
detection as described (Jabben et a1., 1989).
cDNA Library Construction

Total RNA was isolated from the endosperm and embedded embryo of
developing seed of coriander (Coriandrum sativum L.) cremocarps (fruits)
collected at stages ranging from early through mid-development. Tissue
samples were ground in liquid nitrogen, transferred to a Elvehjem tissue
grinder, and homogenized for an additional 2 min in a buffer (3.5 ml/g
tissue) preheated to 80°C consisting of 0.2 M sodium borate, 30 mM EGTA,
1% SDS, 1% deoxycholate, 2% polyvinylpyrollidone 40,000, 10 mM
dithiothreitol (freshly added), pH 8.5. The remainder of the total RNA
purification was performed as described by Hall et a1. (1978). Poly A+
RNA isolation from coriander total RNA and subsequent cDNA library
construction were performed by Stratagene, Inc. cDNA was synthesized
using an oligo-dT primer and inserted into the EcoRI site of the l-ZAP II
vector (Stratagene).
Isolation and Characterization of cDNA Clones

The coriander endosperm.cDNA library was subjected to immunological
screening as described (Sambrook et a1., 1989) using an immunoaffinity-
purified polyclonal antibody against A918:0-ACP desaturase of avocado
(Shanklin and Somerville, 1991). Immunopositive clones were purified to
homogeneity, and pBluescript SK(-) phagemid was excised as described
(Short et a1., 1988). Nucleotide sequence was obtained for both strands
of DNA by dideoxy chain-termination using Sequenase 2.0 (U.S.
Biochemical).

To obtain a full-length Type II clone, the coriander cDNA endosperm
library was re-screened using an [afinP]dCTP random-labelled DNA probe

derived from a 394 bp NcoI restriction fragment of a partial Type II cDNA

 

41
clone. Library screening was performed as described (Sambrook et: a1. ,
1989) .
Expression of cDNAs in E. coli

Polymerase chain reaction (PCR) primers were designed from the 5'
and 3' ends of Type I and II cDNA sequences encompassing the mature
peptide (native protein minus the plastid transit peptide). Type I cDNA
primers contained flanking XbaI and NdeI restriction site recognition
sequences (5' primer, 5'TGGTCTAGACATATGGCGTCTAGTCTTGGCATG3' and 3' primer,
5'ACCTCTAGACATATGTACAGACCACAATAAA3' ) . Type II cDNA primers were designed
with flanking EcoRI and NdeI restriction sites (5' primer,
5'TAGGAATTGATATGGCTTCAACTCTTGAT3' and 3' primer,
5'ACCGAATTCATATGATGATCTGAGG3') . PCRs consisted of 50-100 ng of Type I or
TypeII cDNAs contained in pBluescript SK(-), 0.25 M of primers, 0.16 mM
dNTPs, 1x reaction buffer, and 1 U Taq DNA polymerase (Boehringer
Mannheim) in a 25 pl reaction volume. Reactions were performed with a
Perkin-Elmer/Cetus thermal cycler with a program of 5 min at 94°C; 30
cycles of 1.1 min at 94°C, 2 min at 55°C, 3 min at 72°C; followed by 10
min at 72°C.

The Type I and Type II-derived PCR products were digested with XbaI
and EcoRI, respectively, and ligated into the corresponding sites of
pBluescript KS(+). Following amplification in E. coli strain DHSa,
plasmids were isolated, digested with NdeI, gel-purified, and inserted
into the NdeI site of the pET3a E. coli expression vector (Studier et a1. ,
1990) . Type I and II-containing plasmids were introduced into the E. coli
strain BL21 and grown under carbenicillin selection as described (Studier
et a1. , 1990) .

The Type II sequence contained an indigenous NdeI restriction site
within its translational stop codon. As a result, the protein expressed
from this cDNA included at its C-terminus three additional amino acids
encoded by the pET3a vector.

Cells containing the expression plasmids lacking insert or with Type

I or Type II mature peptide-encoding sequence were grown to [two-0.6,

42

induced with isopropyl B-D-thiogalactopyranoside (IPTG) (0.4 mM final
concentration), and grown for an additional 4 h. Protein extracts from
these cells were separated by SDS/PAGE and analyzed.by Coomassie staining
or by western blotting as described above. In the case of BL21 cells with
Type I-containing plasmids, washed pelleted cells were lysed by four
cycles of freezing in liquid nitrogen followed by thawing in a 25°C water
bath. Cell debris was pelleted and the soluble extract was used for
A918:0-ACP desaturase assays as described (McKeon and Stumpf, 1981).
Expression of Type II cDNA in Tobacco

PCR primers were designed from 5' and 3' terminal sequences of the
full-length Type 11 cDNA. These primers included a flanking BamHI
restriction site on the 5' primer and flanking BamHI and SacI restriction
sites on the 3' primer (5' primer, 5'TAGGATCCATGGCCATGAAACTGAAT3' and
3'primer, 5'ACGGATCCGAGGTCTCGACGACCACTCATATG3'). PCRs were performed as
described.above except1Vent.DNAupolymerase (New England.Biolabs) was used.
The PCR product was gel purified, digested with BamHI, and cloned into
pBluescript KS(+). The insert was excised by cleavage with BamHI and
SacI, gel-purified, and ligated into the BamHI and SacI sites of pBI121
(Jefferson et a1., 1987) behind the cauliflower mosaic virus (CaMV) 35$
promoter. The resulting plasmid pEG301 was subsequently introduced into
Agrobacterium tumefaciens strain LBA4404 by electroporation (Mersereau et
a1., 1990). The transformed Agrobacterium cells were grown at 27°C under
kanamycin selection and co-cultivated with leaf disks of tobacco
(Nicotiana tabacum L.) as described (Rogers et a1., 1986).
Fatty Acid Analysis of Transgenic Tobacco

Fatty acid methyl esters were prepared from transgenic tobacco calli
by heating of tissue at 90°C for 40 min in 10% (w/v) boron
trichloride/methanol (Alltech) supplemented. with 15% toluene (v/v).
Resulting methyl esters were analyzed by gas chromatography using a
Hewlett-Packard 5890 gas chromatograph with a 50 m x 0.25 mm i.d. CP-Sil88
column (Chrompack) and flame ionization detection. Oven temperature was

programmed from 155°C (60 min hold) to 175°C at 2.5°C/min with a column

43

head pressure of 7.5 psi He. Samples of monounsaturated fatty acid methyl
esters for gas chromatography-mass spectroscopy (CC-MS) analyses were
purified by argentation TLC (Morris et a1., 1967). TLC plates were
prepared by saturation of silica gel K6 (Whatman) TLC plates in 15% (w/v)
silver nitrate/acetonitrile. Fatty acid methyl esters were separated by
development of argentation TLC plates in toluene at -20°G. Double bonds
of purified monounsaturated fatty acids were converted to thiomethyl
adducts by reaction with dimethyl disulfide (Aldrich) in the presence of
I2 as described (Yamamoto et a1., 1991) except that derivitization was
performed for two hours. Derivatized samples were analyzed by GC-MS with
a Hewlett-Packard 5890 GC coupled to a Hewlett-Packard 5970A mass
selective detector (MSD) using a 15 m x 0.25 mm i.d. DB-17 column (J&W
Scientific) with the oven temperature programmed from 185°C to 230°C at
10°C/min. The MSD inlet temperature was 280°C, and the ionizing potential
of the MSD was 70 eV.
Results

Immunodetection of Two Putative Acyl-ACP Desaturases in Umbelliferae Seed
Extracts

A primary goal of our research was to determine the metabolic origin
of the cisA° double bond of petroselinic acid using seeds of the
Umbelliferae species coriander and carrot. In experiments described in
Chapter 3, [1-“C]stearic acid (18:0) or [1-1‘C]palmitic acid (16:0) fed
to endosperm slices of coriander and carrot was incorporated into
glycerolipids but not desaturated. However, crude homogenates of
coriander endosperm were capable of the de novo synthesis of petroselinic
acid from [2-1"C]malonyl-C0A. The majority of the resulting
[1‘C]petrose1inic acid was detected as free fatty acid. A smaller portion
of the recovered petroselinic acid was associated with protein, presumably
representing the acyl-ACP fraction. These results, therefore, suggested
that petroselinic acid derives from an acyl-ACP rather than a

glycerolipid-type desaturase. Despite this, we were unable to demonstrate

 

 

44

the in vitro synthesis of petroselinic acid from [l-“C]stearoy1- or [1-
1"C]palmitoyl-AGP as well as [l-“C]stearoy1-CoA (Chapter 3).

Using an alternate approach, we investigated the possibility that
the proposed acyl-ACP desaturase associated with petroselinic acid
synthesis is related to the A918:0-ACP desaturase. This enzyme was
recently purified to homogeneity from avocado (Shanklin and Somerville,
1991), safflower (Thompson et a1., 1991), and soybean (Cheesbrough and
Cho, 1990; Kinney et a1., 1990) and corresponding cDNAs isolated from
castor (Shanklin and Somerville, 1991) , cucumber (Shanklin et a1. , 1991) ,
safflower (Thompson et a1. , 1991), and soybean (Kinney et a1. , 1990). A
polyclonal antibody raised against the A918:0-AGP desaturase of avocado
(Shanklin and Somerville, 1991) was used to probe western blots of
extracts of developing seeds of coriander and several other Umbelliferae
species. In these extracts, two immunoreactive proteins of an apparent
mass on SDS/PAGE of 39 and 36 kDa were detected (Figure 2.1A, Lanes G-F) .
In contrast, tissues that do not synthesize petroselinic acid, e.g.,
vegetative tissues of coriander (Figure 2.1B) and seeds of species outside
of the Umbelliferae, Araliaceae, or Garryaceae families (Figure 2.1A, Lane
B), contained only a 39 kDa protein with antigenic recognition by anti-
A918:0-ACP desaturase antibodies. From these results, it was hypothesized
that the 39 kDa peptide is a A918:0-ACP desaturase, and the additional 36
kDa peptide, seen only in tissues which contain petroselinic acid, is an
acyl-ACP desaturase associated with the synthesis of this fatty acid.
Isolation of Putative Coriander Acyl-ACP Desaturase cDNA Clones

As an initial step in determining the function of the 39 and 36 kDa
peptides identified on western blots , clones encoding acyl-ACP desaturases
were isolated from a cDNA expression library prepared from coriander
endosperm. Screening of the library with antibodies against avocado
A918:0-ACP desaturase yielded two classes of clones, designated Type I and
Type II, as determined by partial nucleotide sequencing. Translation of
the 5' nucleotide sequence revealed that both Type I and Type II cDNAs

contained considerable amino acid sequence similarity with those of

45

Figure 2.1. Western blot analyses of seed extracts of Brassicaceae
(Brass.) and Umbelliferae spp. (A) and leaf, root, and seed extracts of
coriander (Coriandrum sativum L.) (B). Blot (A) lanes are: A, castor
recombinant A918:0-ACP desaturase (D5) (600 ng) (see Shanklin and
Somerville, 1991); B, crambe (Crambe abyssinica L.); C, coriander; D,
wild carrot (Daucus carota L.); E, sweet cicely (Myrrhis odorata L.); and
F, angelica (Angelica archangelica L.) seed extracts (50-60 pg). (Lane
B is a Cruciferae species. Lanes C-F are Umbelliferae species). Blot
(B) lanes are: leaf (110 pg), root (110 pg), and seed (60 pg) extracts

of coriander.

46

 

Ds Brass. Umbelliferae
kDa A B C D E F
97
66
43
~ _"‘ ‘ m ” <39 kDa
.-_.._. C. -—.... ---- -' <36 kDa
31
22

Coriander
Leaf Root Seed

«nu—— <39 kDa
—- <36 kDa

47

previously isolated A918:0-ACP desaturase cDNAs of castor (Shanklin and
Somerville, 1991), cucumber (Shanklin and Somerville, 1991), and safflower
(Thompson et a1., 1991). However, a region near the amino-terminus of
translated Type II clones exhibited marked divergence with a similar
region in translated Type I (data not shown) and A918:0-ACP desaturase
clones (Figure 2.2). This divergence included the absence of a sequence
encoding 15 amino acids in the Type II clone relative to the Type I and
to the previously reported A918:0-AGP desaturase cDNAs (Shanklin and
Somerville, 1991; Thompson et a1., 1991).

The A91820—ACP desaturase is found in the plastid of plant cells
(Jaworski, 1987). A similar localization would be expected for other
acyl-ACP desaturases. However, the longest Type I and II cDNA clones
obtained through antibody screening of the expression library lacked
nucleotide sequence encoding for an entire plastid transit peptide. A
full-length Type II clone (Figure 2.2) was isolated by screening the
coriander cDNA library with a nucleotide probe derived from a 394 bp NcoI
restriction fragment of a partial Type II cDNA. The full-length 1309 bp
cDNA clone contained a methionine codon at nucleotide 7 with surrounding
bases that differed by only one nucleotide from a plant consensus
translational start site (Lutcke et a1., 1987). The open-reading frame
of the Type 11 cDNA consisted of 1155 nucleotides encoding a 385-amino
acid peptide with a calculated.molecular weight of 43.8 kDa. By homology
with the deduced amino acid sequence of castor (Shanklin and Somerville,
1991), cucumber (Shanklin and Somerville, 1991), and safflower (Thompson
et a1., 1991) A918:0-ACP desaturase cDNAs, the encoding sequence of the
mature protein likely begins at nucleotide 115 of the Type II cDNA. The
108 bases preceding the start of the mature peptide encoded a 36 amino
acidsequence with properties similar to that of plastid transit peptides
(Keegstra et a1., 1989). The transit peptide encodedey the Type 11 cDNA
was 3 amino acids longer than those encoded by the A918:0-ACP desaturase
cDNAs reported to date (Shanklin and Somerville, 1991; Thompson et a1.,

1991) and.possessed.a markedly different amino acid sequence composition.

 

48

Figure 2.2. Nucleotide sequence of the coriander Type II acyl-ACP
desaturase cDNA. (TII) and. a <comparison. of the deduced. amino acid
sequences of T11 and castor A918:0-ACP desaturase cDNA (Gas) (Shanklin
and Somerville, 1991). Identical amino acids are indicated by colons.
Amino acids which are absent relative to either of the two sequences are
indicated by dashed lines. Alignment of the TII nucleotide sequence is
maintained with a dotted line. The underlined alanine (amino acid 37)
indicates the likely amino-terminus of the mature peptide encoded by the

Type II cDNA.

T11
T11
CAS

TI!
TI!
CAS

T11
T11
CAS

TII
TII
CA8

T11
T11
CAS

TII
TII
CAS

T11
T11
GAS

T11
T11
CAS

TII
TI!
CAS

TII
TII
CAS

TII
TII
CAS

TII
TIT
CAS

T11
T11
CA3

T11

T11

CAS
TII
TII
CAS
T11

T11

49

GCAAAAATGGCCATGAAACTGAATGCCCTCATGACTCTTCAGTCCCCAAAAAGGAACATGTTTACGAGAATTGCCCCTCCT
H A H K L N A L M T L Q C P K R N M F T R I A P P
H A L K : N P F L S O T 0 K L P S F A L : :

CAAGCAGGGAGAGTGAGATCAAAGGTGTCCATGGCTTCAACTCTTCATGCTAGCCCACTGGTGTTCGACAAGCTGAAGGCTGGG
O A G R V R S K V S H A S T L H A S P L V F D K L K A G
H : S T R S P : F Y : : : : : K S G S K E V E N : : K P

AGGCCT.........GAGGTG......... .............. .............GATGAATTGTTCAACTCTCTGGAGGGT
R P - - E V - - - - - - - - - - - D E L F N S L E G
F H P P R : : H V 0 V T H S H P P O K I : I : K : : D N

TGGGCCAGGGACAACATCCTTGTGCACCTGAAATCCGTAGAGAACTCATGGCAGCCGCAAGACTATCTGCCCGATCCCACATCC

A R D N I L V H L K S V E N S H O P O D Y L P D P T S
: E E : : : : : : ' P : : K C : : : : : F : : : : A :

C T TCAAGGAGATGAGAGAACGGGCCAAGGACATCCCTGATGAATACTTTGTTGTTCTTGTTGGAGAC
A F V K E N R E R A K D I P D E Y F V V L V G D
G : : R : L : : : : : E : : : D : : : : : : : :
ATGATCACTGAAGAGGCACTCCCAACTTACATGTCTATGCTTAACAGATGTGATGGCATTAAGGATGACACTGGCGCTCAACCT
N I T E E A L P T Y N S N L N R C D G I K D D T G A Q P
: : : : : : : : : : O T : : : T L : : V R : E ' : : S -
ACTTCTTGGGCCACTTGGACCAGGGCTTGGACTGCTGAGGAGAACCGCCATGGCGATCTTCTCAACAAGTATCTTTATCTCTCT
T S U A T N T R A H T A E E N R H G D L L N K Y L Y L S
: : : : I : : : : : : : : : : : : : : : : : : : : : :
GGCCGAGTTGATATGAGGA TGATTGAGAAGACTATTCAATATCTTATCGGCTCTGGAATGGATACAAAAACAGAGAACTGTCCC
G R V D N R N I E K T I O Y L I G S G H D T K T E N C P
: : : : : : Q : : : : : : : : : : : : : : P R : : : S :
TAC ATGG GGCTTCATCTACACATCTTTCCAGGAAAGAGCCACATTCATCTCCCATGCCAACACAGCCAAACTTGCTCAACACTAC
Y H G F I Y T S F 0 E R A T F I S H A N T A K L A 0 H Y
: L : : : : : : : : : : : : : : : : G : : : R Q : K E H

G TG CA GA CC AG TCAAG GTGTG CAACATTGCTTCTGACGAGAAACGCCATGCCACCGCCTACACCAAAATCGTGGAG
0 C N I A S D E K R H A T A Y T K I V E
: T : : A : : : : : E : : : : : : : :
AAATGCCAGCTCATGCA
0 H P A H A
S

GATTGACCCAGACACCACTGTTATCGCATTTTCTGACATGATGA GAAGAAAATAC
K K I
: : : : : : : L

G
I D P D T T V l A F S D M M R

: : : : G : : L : : A : : :

eel“)

GCCG TGCTCAGCAGATTGGAGTCTACTCTGCATGGGATTAC
A A Q 0 I G V Y S A U D Y
: : : R L : : : T : K : :

A CT CGATG CT CGATGATA
D D D

u wi-c

T WC
H T
: : N S
CATAATTGATTTTCTGGTGGATAAATGGAACGTTGCGAAGATGACAGGGCTGTCGGGTGAAGGGAGAAAGGCTCAAGAA
I I D F L V D K W N V A K H T G L S G E G R K A Q E
: : L E : : : G R : K : D : L : z : : A : : 0 : : z D

T TC

0)

TG TTGTAGCTTGGCTGCTAAGATCAGGAGAGTTGAGGAGAAGGTTCAAGGCAAGGAGAAGAAAGCTGTGTTGCCTGTGGCT
C S L A A K I R R V E E K V 0 G K E K K A V L P V A
: R : P P R : : : L : : R A : : R A : E : P T - M P

T CA AGCTGGATTTTCAACCGTCAGATCATCATATGAGTGGTCGTCGACATTCAATATTAGACTTTTCAATTATGCTTATGCTTT
S W I F N R Q I I I

: : : D : : V K L *

TCCTTTTGATGTTATTATGTTTATGCTTATGCTATCGGTCGGTGTTTGTTGTCAGATCTGGTTATGTAAAACTTATATTTAAAT

GAATGTTGGATTT

81
22

165
53
50

204

78
288
106

372
122
134

456
150
162

540
178
190

624
206
218

708
234
246

792
262
274
876
302
960
318
330
1044
346
358
1128
374
385
1212
385
1296

1309

 

 

 

50

In addition to the absence of 15 amino acids near its amino-terminus, the
Type II cDNA-encoded peptide differed from that of “18:0-ACP desaturase
(Shanklin and Somerville, 1991; Thompson et a1. , 1991) by the presence of
one additional amino acid near its carboxyl-terminus. Excluding missing
amino acids, the overall amino acid sequence identity of the mature Type
II-encoded peptide and the mature castor A918:O-ACP desaturase (Shanklin
and Somerville, 1991) was 70%.
Expression of Type I and Type II Coriander cDNAs in E. coli

In an attempt to establish the identity of the Type I and Type II
coriander clones , the mature peptide-encoding sequences of both cDNAs were
engineered into the pET3a vector and expressed in the E. coli strain BL21
(Studier et a1., 1990). Protein encoded by the Type I cDNA was readily
detectable in Coomassie-stained SDS-polyacrylamide gels of crude E. coli
protein extracts (data not shown). This protein displayed immunological
cross-reactivity with anti-A918:O-ACP desaturase antibodies and exhibited
identical mobility on SDS/PAGE as the 39 kDa protein detected in coriander
seed extracts (data not shown). In addition, “18:0-ACP desaturase
activity could be detected in soluble extracts of E. coli expressing this
protein (data not shown). The Type I cDNA was therefore identified as
encoding a A918:0-ACP desaturase. The Type II coriander cDNA was also
expressed to high levels in E. coli, but the resulting protein was
detected almost entirely as an inclusion body (data not shown). Western
blots of urea-solubilized extracts from E. coli expressing the Type II
clone revealed a 36 kDa immunoreactive peptide indistinguishable from the
lower band of coriander seed extracts (data not shown). Because the Type
II peptide was insoluble, we were unable to assay for activity directly,
and preliminary attempts to obtain in vitro activity of the Type II-
encoded protein after urea solubilization of the inclusion body were
unsuccessful .
Expression of the Coriander Type II cDNA in Tobacco

As a direct means to test the hypothesis that the 36 kDa polypeptide

is an acyl-ACP desaturase that results in petroselinic acid accumulation,

51

the Type II cDNA was cloned into the plant expression vector pBIlZl
(Jefferson et a1., 1987) and transformed into tobacco, a plant which
contains no detectable petroselinic acid. Western blot analysis of the
transgenic tobacco callus using anti-A918zo-ACP desaturase antibodies
revealed an additional peptide of 36 kDa in extracts of callus containing
the Type II cDNA that was absent in callus transformed with only the
pBIlZl vector (Figure 2.3). The lack of detectable higher molecular
weight precursors onnwestern blots suggests that tobacco calli are capable
of properly processing the transit peptide of the expressed product of the
coriander Type II cDNA.

To determine whether the peptide encoded by the Type II cDNA
possessed in vivo activity, the fatty acid composition of the transgenic
callus was examined by gas chromatography (Figure 2.4A,B). Callus
transformed with the Type II cDNA contained two fatty acids not present
in callus transformed with the pBIlZl vector alone (Figure 2.4B, Peaks 2
& 5). The retention times of methyl ester derivatives of these fatty
acids coincided with those of methyl hexadecenoic acid (16:1) and methyl
petroselinic acid. In ten callus samples analyzed, levels of each of
these fatty acids ranged from 1 to 4 wt% of the total. To determine the
double bond position of hexadecenoic acid and confirm the double bond
position of petroselinic acid, monounsaturated fatty acid methyl esters
of the transgenic calli were derivatized with dimethyl disulfide (Yamamoto
et a1., 1991), and analyzed by GC-MS (Figure 2.5A,B). The mass spectrum
of the derivatized petroselinic acid contained the same diagnostic ions
as an petroselinic acid standard. In addition, the mass spectrum of the
derivatized hexadecenoic acid of transgenic calli contained ions

diagnostic for a A“ isomer (Francis, 1981).

Discussion
The synthesis of petroselinic acid in Umbelliferae species is an

apparently tissue-specific event. Seeds of these plants are rich sources

52

<39kDa
<36kDa
(Do on O'T' 0-0-0
312 08- 0% 83
38' Zn) 2:» {Do-o-
”3 >0 >0 03
£2 a; as
m to
2‘ 3‘

Figure 2.3. Western blot analysis of protein extracts from callus of
transgenic tobacco. Proteins of crude extracts were separated by 11%
SDS/PAGE, transferred to nitrocellulose, and probed with polyclonal
.antibodies against the A918:0-ACP desaturase antibody of avocado. Lanes:
.A, coriander seed extract; B, C, tobacco callus transformed with pBIlZl
containing the coriander Type II cDNA; D, tobacco callus transformed with
the pBIlZl expression vector without insert. Lanes B and C represent
extracts from two independent samples of calli transformed with the Type

II cDNA.

S3

 

 

 

 

 

 

 

 

 

 

 

 

 

1O 5 8
A 1 B
10
1
$5
3
7
JL
I i
4 23 5
l 67’ 9 ‘1 i6 9
ALWMI A A ALLJCJJLML __1 I //7 LLJL...
15 30 45 60 75 15 30 45 60 75
Retention Time (min) Retention Time (min)

Figure 2.4. Gas chromatograms of fatty acid methyl esters prepared from
tobacco callus transformed with the pBIlZl vector (A) and pBIl2l
containing the coriander Type II cDNA insert (B). Arrows indicate fatty
acids present only in callus transformed with the Type II insert. The
double bond positions of “hexadecenoic acid (16:1A‘) (Peak 2) and
petroselinic acid (18:1A6) (Peak 5) were determined by GC-MS (see Figure
5) . Inset in B is an enlargement of peaks (Peaks 5, 6 & 7) corresponding
to the closely eluting isomers of 18:1. Peak identification: 1, 16:0; 2,
16:1A‘; 3, 17:0 (internal standard); 4, 18:0; 5, 18:1A6; 6, 18:1A9; 7,
18:1A11; 8, 18:2; 9, 20:0; 10, 18:3.

 

A
”me
o
c
:0
g %
3
.Q
a:
8
188
B
0 188
o
c
«s
'0
c %
3
.o
E
8
188
Figure 2.5.

 

54

 

 

Al
16ߣf $H3
§
8 / T
8-32 147 215 CH3!
//’ ‘ 3E3\
158 288 258 388 358
Mass/Charge
Al
ieflép §Fb
1?
S
3 B 15 éH
14 17s 3 +
8-32 / A I B 31238
11 :1 Ah 1 L 1.1 I: fl L
88 388
Mass/Charge

Mass spectra of thiomethyl adducts of fatty acid methyl

esters detected exclusively in tobacco callus transformed with the Type

II

coriander cDNA insert

in pBIlZl.

Shown are mass spectra of

A‘hexadecenoic acid (16:1A") (Peak 2. Figure 4B) (A) and petroselinic acid

(18:1A6) (Peak 5, Figure 43) (B) derivatives prepared from fatty acid

methyl esters of transgenic tobacco callus as described in the text.

55

of this fatty acid (Ellenbracht et a1. , 1980; Kleiman and Spencer, 1982;
Dutta and Appelqvist, 1991) , but other tissues including leaves and roots
contain little or no petroselinic acid (Ellenbracht et a1., 1980; Dutta
and Appelqvist, 1991). Results of preliminary metabolic studies have
suggested that petroselinic acid in Umbelliferae seed is derived from the
activity of an acyl-ACP desaturase (see Chapter 3) . In the current study,
using a polyclonal antibody against the A918:0-ACP desaturase of avocado,
proteins of 39 and 36 kDa were detected on western blots of seed extracts
of coriander and various other Umbelliferae species. Consistent with a
role in petroselinic acid synthesis, the 36 kDa peptide, in contrast to
the 39 kDa peptide, was identified only in tissues that produce this fatty
acid. A cDNA clone (Type II) encoding a mature peptide of similar
mobility on SDS/PAGE as the 36 kDa protein was isolated from a coriander
endosperm cDNA library using anti-A918:0-ACP desaturase antibodies
(Shanklin and Somerville, 1991). Agrobacterium-mediated transformation
of tobacco, a species which lacks petroselinic acid, with a full-length
Type II clone resulted in the expression of the 36 kDa peptide and the
appearance of petroselinic acid in the transgenic callus. These results
demonstrate that the biosynthetic pathway of petroselinic acid involves
a desaturation step catalyzed by a 36 kDa peptide with antigenic relation
to the A91820-ACP desaturase. The high degree of amino acid sequence
identity with the “18:0-ACP desaturase, the presence of a plastid transit
peptide in its native form, and results of previous metabolic studies (see
Chapter 3) strongly suggest that the 36 kDa desaturase uses an acyl-ACP
as its in vivo substrate. Previously, oleic acid was the only plant fatty
acid known to be synthesized via an acyl-ACP desaturase .(Nagai and Bloch,
1968; Jaworski and Stumpf, 1974; McKeon and Stumpf, 1982).

An unexpected finding from this work was the appearance of nearly
equal levels of “hexadecenoic acid and petroselinic acid in tobacco
callus expressing the 36 kDa desaturase. Several hexadecenoic acid (16:1)
isomers are detectable in lipid extracts of coriander seed. One of these

isomers has been identified by mass spectrometry as A‘hexadecenoic acid

56

(unpublished data). However, amounts of this fatty acid are typically
50.2 wt% of the total fatty acid of coriander seed extracts. In contrast,
petroselinic acid accounts for ,>_70 wt% of the total fatty acid of
coriander seed (Kleiman and Spencer, 1982).

Despite our demonstration that the synthesis of petroselinic acid
involves a unique 36 kDa putative acyl-ACP desaturase, the immediate
precursor .of this fatty acid cannot be definitively identified from these
experiments. The detection of A‘hexadecenoic acid in addition to
petroselinic acid in transgenic tobacco callus, however, does suggest
possible biosynthetic origins of the A6 double bond of petroselinic acid.
If the location of double bond placement is determined by the position of
carbon atoms from the methyl (or to) end of acyl chains, then it is
conceivable that the 36 kDa peptide is an 012 desaturase. As such, this
enzyme could catalyze the 012 desaturation of both palmitoyl (16:0)- and
stearoyl (l8:O)-ACP resulting in A‘hexadecenoyl- and petroselinoyl-ACP,
respectively. Alternatively, if double bond placement is dictated by the
position of carbon atoms from the carboxyl (or A) end of acyl chains, then
it is unlikely that the 36 kDa peptide catalyzes both the A" and A6
desaturation of palmitoyl- and stearoyl-ACP, respectively. Instead,
petroselinic acid might result from the desaturation of a shorter chain
acyl-ACP by the 36 kDa peptide followed by elongation to petroselinoyl-
ACP. For example, a possible biosynthetic pathway might consist of the
A" desaturation of palmitoyl-ACP to A“hexadecenoyl-ACP with subsequent
two-carbon elongation to petroselinoyl-ACP.

The synthesis of petroselinic acid and Al‘hexadecenoic acid in
transgenic tobacco demonstrates the ability to produce new unsaturated
fatty acids via gene transfer technology. This finding suggests the
potential for the development of a new plant oil (i.e. , a high
petroselinate oil) in an existing oilseed crop. However, levels of
petroselinic acid in the transgenic tobacco callus were 1 to 4 wt% of the
total fatty acid. This rather low amount of petroselinic acid may suggest

that other factors are required for high levels of accumulation. Such

57

factors may include, for example, an acyl-ACP thioesterase specific for
petroselinoyl-ACP. It is also possible that petroselinic acid may not be
a preferred substrate for membrane lipid metabolism. As such, higher
levels of this fatty acid might be expected to accumulate in the seed oil
of transgenic plants.

Finally, the A918:0-ACP desaturase (Shanklin and Somerville, 1991;
Thompson.et a1., 1991) and.the 36 kDa.desaturase of coriander share a high
degree of amino acid identity; 'The primary difference between.these amino
acid sequences occurs near their amino-termini. The divergence in this
region includes the absence of 15 amino acids in the 36 kDa desaturase
relative to the A918:0-ACP desaturase. It is intriguing to speculate that
the alteration in double bond placement relates to this difference in the
primary structures of these enzymes. Regardless, comparative biochemical
studies of" the A918:0-ACP desaturase and. the 36 ‘kDa. desaturase of
coriander may allow for a better understanding of the relationship between

the catalytic mechanism and the active sites of fatty acid desaturases.

58
Literature Cited
Browse J, Somerville C (1991) Glycerolipid synthesis: Biochemistry and
regulation. Annu Rev Plant Physiol Plant Mol Biol 42: 467-506

Cheesbrough TH, Cho SH (1990) Purification and characterisation of
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Lipid Biochemistry, Structure and'Utilization. Portland Press, London, p
129-130

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Francis GW (1981) Alkylthiolation for the determination of double-bond

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29-38

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1,”!

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CHAPTER 31

Metabolic Evidence for the Involvement of a A‘Palmitoyl-Acyl Carrier
Protein Desaturase in Petroselinic Acid Synthesis in Coriander

Endosperm and Transgenic Tobacco Cells

Abstract

We have previously demonstrated that the double bond of petroselinic
acid (18:1A591—’) in coriander seed results from the activity of a 36 kDa
desaturase that is structurally related to the Agstearoyl-acyl carrier
protein (ACP) desaturase (E.B. Cahoon, J. Shanklin, J.B. Ohlrogge [1992]
Proc Natl Acad Sci USA 89: 11184-11188). To further characterize the
biosynthetic pathway of this unusual fatty acid, 1"C-labeling experiments
were conducted using developing endosperm of coriander. Studies were also
performed using suspension cultures of transgenic tobacco that express the
coriander 36 kDa desaturase and as a result produce petroselinic acid and
A‘hexadecenoic acid. When supplied exogenously to coriander endosperm
slices, [1-1‘C]pa1mitic acid and stearic acid were incorporated into
glycerolipids but were not converted to petroselinic acid. This suggested
that petroselinic acid is not formed by the desaturation of a fatty acid
bound to a glycerolipid or by reactions involving acyl-CoAs. Evidence
instead was most consistent with an acyl-ACP route of petroselinic acid
synthesis. For example, the exogenous feeding of [1-1"C]lauric acid and
myristic acid to coriander endosperm slices resulted in the incorporation
of the radiolabels into long-chain fatty acids, including primarily
petroselinic acid, presumably through acyl-ACP-associated reactions. In
addition, using an in vitro fatty acid biosynthetic system, homogenates
of coriander endosperm incorporated [2-“C]malonyl-CoA into petroselinic
acid, of which a portion was detected in a putative acyl-ACP fraction.

Furthermore, analysis of transgenic tobacco suspension cultures expressing

 

1Material in this chapter has been accepted for publication [Cahoon
EB, Ohlrogge JB (1994) Plant Physiology (in press)]

62

 

63

the coriander 36 kDa desaturase revealed significant amounts of
petroselinic acid and A‘hexadecenoic acid in the acyl-ACP pool of these
cells. Also presented is evidence derived from [U-“C]nonanoic acid
labeling of coriander endosperm which demonstrates that the coriander 36
kDa desaturase positions double bond placement relative to the carboxyl
end of acyl-ACP substrates. Data obtained in these studies are
rationalized in terms of a biosynthetic pathway of petroselinic acid
involving the A" desaturation of palmitoyl-ACP by the 36 kDa desaturase

followed by two-carbon elongation of the resulting A‘hexadecenoyl-ACP.

 

 

64

Introduction

Seed oils of most species of the Umbelliferae (or Apiaceae),
Garryaceae, and Araliaceae are characterized by their high content of the
unusual monounsaturated fatty acid petroselinic acid (18:1A9L‘fi) (Kleiman
and Spencer, 1982) . Prior to a recent report of this lab (Cahoon et a1. ,
1992), little was known of the biosynthetic origin of the cisA‘5 double
bond of this fatty acid. Such information is of possible biochemical and
biotechnological significance. Because petroselinic acid is the likely
product of a previously uncharacterized desaturase (Cahoon et a1. , 1992) ,
data regarding its synthesis may contribute to an understanding of fatty
acid desaturation mechanisms in plants. In addition, petroselinic acid
can be used as a chemical precursor of the industrially valuable compounds
lauric acid (12:0) and adipic acid (6:0 dicarboxylic) and is therefore a
potential target for oilseed modification research (Murphy, 1992).

Double bonds of plant fatty acids may result from enzymatic activity
involving several different substrate types. The most common desaturation
mechanism in plants involves the insertion of double bonds into acyl
moieties esterified to glycerolipids. Examples of this include the
desaturation of oleic acid to linoleic acid on phosphatidylcholine (Gurr
et a1., 1969; Slack et a1., 1978; Stymne and Appelqvist, 1978) and the
desaturation of palmitic acid to transA3hexadecenoic acid on
phosphatidylglycerol (Bartels et a1., 1967). In addition, fatty acid
unsaturation may arise from reactions which use saturated acyl chains
bound to acyl carrier protein (ACP). The primary example of such a
pathway is the desaturation of stearoyl-ACP to oleoyl-ACP through the
activity of the “stearoyl-ACP desaturase (Nagai and Bloch, 1969; Jaworski
and Stumpf, 1974). Acyl-coenzyme A (CoA) esters may also serve as
substrates for fatty acid desaturases. Though such reactions have yet to
be conclusively demonstrated in higher plants, the desaturation of
eicosanoyl-CoA is the proposed biosynthetic route of Aseicosenoic acid in
seeds of meadowfoam (Limanthes alba) (Moreau et a1. , 1980; Pollard and

Stumpf, 1980) . Finally, Shibahara et a1. (1991) have proposed a mechanism

 

65

in pulp of kaki (Diospyros kaki) in which the double bonds of oleic acid
and cis-vaccenic acid can be enzymatically shifted between the A9 and All
positions.

With regard to the biosynthesis of petroselinic acid, we have
previously identified a 36 kDa peptide in Umbelliferae endosperm that
displays immunological cross-reactivity with antibodies against the
A’stearoyl-ACP desaturase of avocado (Cahoon et a1. , 1992) . This peptide
is absent in tissues that do not synthesize petroselinic acid, including
leaves and roots of coriander, an Umbelliferae species. In addition,
expression of a coriander endosperm cDNA for the 36 kDa peptide in tobacco
resulted in the production of petroselinic acid and A‘heXadecenoic acid
(16:1A‘) in transgenic callus. Furthermore, translation of the coriander
cDNA indicated that the 36 kDa peptide is synthesized with a plastid
transit peptide and, in its mature form, possesses 70% amino acid identity
with the Agstearoyl-ACP desaturase of castor. These results lead us to
propose that petroselinic acid is the product of an acyl-ACP desaturase
that is related to the Agstearoyl-ACP desaturase (Cahoon et a1., 1992).
However, expression of the 36 kDa desaturase in E. coli resulted in the
recovery of nearly all of the recombinant protein as an insoluble
aggregate. Upon urea solubilization, this protein possessed no detectable
in vitro desaturase activity with saturated C“, 016, and C18 acyl-ACPs.
Therefore, we were unable to prove directly that the 36 kDa peptide of
coriander endosperm is an acyl-ACP desaturase rather than, for example,
an acyl-CoA desaturase. The chain length of the fatty acid substrate of
the 36 kDa desaturase was also not determined. In this communication,
results are presented from a variety of 1"C- labeling studies using
endosperm of coriander and suspension cells of transgenic tobacco that
express the cDNA for the coriander 36 kDa desaturase. These results
demonstrate the involvement of an acyl-ACP desaturation pathway in the
synthesis of petroselinic acid. Also presented are details of the double
bond positioning properties of the coriander 36 kDa desaturase and a

proposed biosynthetic pathway for petroselinic acid.

 

66

Materials and Methods

Plant Material

Experiments were conducted using either endosperm or endosperm
homogenates obtained from developing seeds of coriander (Coriandrum
sativum L.). Plants were grown and mericarps harvested as described
(Chapter 4). Studies were also performed with suspension cells derived
from calli of tobacco (Nicotiana tabacum L.) transformed with a cDNA for
the 36 kDa (or Type II) desaturase of coriander endosperm as previously
described (Cahoon et a1. , 1992). The coriander cDNA was inserted behind
the cauliflower mosaic‘virus (CaMV) 358 promoter in the plant expression
vector pB1121 (Clontech, Palo Alto, CA). Transgenic and wild-type
suspension cells were maintained in liquid media (Linsmaier and Skoog,
1965) with constant shaking at 100 rpm at 28°C. Media of transgenic cells

also contained kanamycin (100 mg/L) and carbenicillin (250 mg/L) .

Labeling of Coriander Endosperm Slices with [1-1‘C]Fatty Acids
Transverse slices (approximately 1 to 2 mm thickness) of coriander
endosperm (40-100 mg fresh weight) were incubated in loosely-capped 13 x
100 mm tubes containing 3 pCi of the ammonium salts of either [1-
1"C]lauric acid (12:0) (57 mCi/mmol, Amersham, Arlington Heights, IL),
myristic acid (14:0) (55 mCi/mmol) (American Radiolabeled Chemicals, St.
Louis, MO), palmitic acid (16:0) (58 mCi/mmol, New England Nuclear,
Boston, MA), stearic acid (18:0) (55 mCi/mmol, American Radiolabeled
Chemicals, St. Louis, MO), or oleic acid (52 mCi/mmol, Research Products
International, Mt. Prospect, IL) dispersed in 250 uL of 0.1 M potassium
phosphate (pH 7.2) . Labeling studies were also performed using 1 pCi of
[U-“C]nonanoic acid (9:0) that was synthesized as described below.
Sample tubes were rotated at 100 rpm and maintained at room temperature.
Endosperm slices were incubated in lauric acid and myristic acid for 3.5
h, palmitic acid, oleic acid, and nonanoic acid for 6 h, and stearic acid

for 10 h. At each time point, label was removed, and endosperm was washed

 

 

 

67

twice with ice-cold water. Lipids were then extracted as described for
radiolabeling studies reported in Chapter 4. To assess the ability of
endosperm to synthesize petroselinic acid after extended labeling periods
such as that used in the stearic acid feeding experiment, coriander
endosperm slices were pre-incubated for 8.5 h in 50 mM Mes, pH 5.0. At
the end of this period, endosperm slices were supplied with 3 uCi of [1-
1"C]acetate and incubated for an additional 1.5 h at which time the tissue
was homogenized and lipids extracted as described above.

Elongation and/or desaturation of the labeled fatty acids was
determined by a combination of argentation and reverse-phase TLC of fatty
acid methyl esters prepared from recovered lipids. Extracted
glycerolipids were transesterified by incubation for 45 min at room
temperature in 1.5 mL of 0.5 M sodium methoxide in methanol supplemented
with 20% (v/v) toluene. At the end of this period, 1.5 mL of 125 mM
sulfuric acid was added, and fatty acid methyl esters were extracted three
times with 3 mL of hexane. Fatty acid methyl esters were subsequently
separated by 15% argentation TLC with development in toluene at -20°C as
described (Chapter 4; Morris et a1. , 1967) . TLC plates were prepared by
10 min incubation of silica gel K6 pre-poured TLC plates (250 [1 layer
thickness) (Whatman) in 15 wt% silver nitrate in acetonitrile followed by
air-drying. To further characterize the distribution of radioactivity in
fatty acid methyl esters, labeled bands on argentation TLC plates were
eluted from scrapings using hexanezethyl ether (2:1 v/v) . The recovered
fatty acid methyl esters were further resolved by reverse-phase TLC using
K618 (200 u layer) plates (Whatman) developed sequentially to 75% and 100%
of their length (20 cm) in acetonitrile:methanol:water (65:35:O.5 v/v).
In addition, to characterize the distribution of label in molecules,
monounsaturated products of [l-“C]lauric acid and myristic acid that co-
migrated with petroselinic acid were cleaved by permanganate-periodate
oxidation (Christie, 1982). Chain lengths of oxidation products were
determined using reverse phase TLC as described above. This method was

also used to determine double bond positions of monounsaturated products

68

of [U-“C]nonanoic acid. In this case, oxidation products were analyzed
by reverse phase TLC using a solvent system of acetonitrile:methanol:water
(75:25:0.5 v/v). Radiolabeled fatty acid methyl esters and oxidation
products were visualized by autoradiography of TLC plates, and
radioactivity was quantified by liquid scintillation counting of TLC

scrapings in a non-aqueous complete cocktail.

In vitro Synthesis of Petroselinic Acid from [2-1‘C]Malonyl-CoA

The synthesis of petroselinic acid from [2-“C]malonyl-COA was
studied using crude homogenates of endosperm dissected from the pericarp
and seed coat of coriander mericarps. In these experiments, the highest
activity was obtained from endosperm at latter stages of mid-development
(22-24 DAF under our growth conditions), and all assays were performed
using freshly collected plant material. Endosperm was ground with a glass
Elvehjem homogenizer in an ice-cold buffer composed of 100 mM Tris-HCl,
pH 7.5, 1 mM NazEDTA, 2.5 mM DTT, 1mM MgClz, 1mM KCl, 1 mM isoascorbate,
and 0.1% (w/v) bovine serum albumin (Fraction V, Sigma, St. Louis, MO).
The homogenate was passed through two layers of miracloth (Calbiochem, La
Jolla, CA). The approximate protein concentration of homogenates was 7.5
mg/mL as estimated using the method of Bradford (1976) and a bovine
albumin standard. [2-“C]Malonyl-CoA was either purchased (55 mCi/mmol,
Amersham, Arlington Heights, IL) or chemically synthesized from [2-
1"C]malonate (56 mCi/mmol, American Radiolabeled Chemicals, St. Louis, MO)
using the method of Rutkowski and Jaworski (1978) but without dilution of
specific activity.

Reactions were performed in 13 x 100 mm glass screw cap test tubes.
Typical reaction mixtures consisted of 5 uL of 100 mM ascorbate, 7.5 uL
of 25 mM NADPH, 5 ML of 50 mM NADH (NADPH and NADH were freshly prepared
in 100 mM Tricine, pH 8.0), 1 pL of catalase (800,000 U/mL), 7.5 uL of 1
mg/mL recombinant spinach ACP-I, 5 pL of 100 mM ATP, 80 uL of 100 mM Pipes
(pH 6.0) , 34 pL of water, and endosperm homogenate (200-300 pg protein).

The reaction mixture was brought to a total volume of 245 [IL with tissue

69

homogenization buffer. Reactions were started with the addition of 2.4
to 3.1 nmoles of [2-“C]malony1-CoA (275,000-350,000 dpm) in a 5 pL
volume. This assay was based in part on that described by McKeon and
Stumpf (1981) for measurement of “stearoyl-ACP desaturase activity. Upon
addition of the radiolabeled malonyl-CoA, the loosely-capped reaction
tubes were rotated at 100 rpm and maintained at 25°C. At time points of
7 or 20 min, 40 ML of glacial acetic acid and 4.5 mL of acetone were added
to each tube, and the contents were dried under N2. The reaction products
were subsequently methyl esterified by heating in 10% (w/v) boron
trichloride/methanol (Alltech) at 90°C for 35 minutes. Fatty acid methyl
esters were then analyzed by argentation TLC as described above. To
confirm the identity of 1“C-methyl petroselinic, this fatty acid was
recovered from argentation TLC plates and subsequently analyzed by reverse
phase TLC as either an intact moiety or as products of permanganate-
periodate oxidation using methods described above.

The oxygen dependence of [2-“C]malonyl-CoA incorporation into
petroselinic acid was assessed using the assay conditions described above
except that reaction tubes were purged with nitrogen prior to and after
addition of the radiolabeled substrate. In addition, tubes were
completely sealed for the duration of the incubation period. The
ferredoxin requirement of petroselinic acid synthesis from [2-1‘C]malonyl-
CoA was determined by supplementing assay components described above with
15 pL of 1 mM spinach ferredoxin (Sigma) and 3.1 uL of ferredoxin:NADPH
reductase (10 U/mL) (Sigma) in place of a corresponding amount of water.
In both experiments, reactions were carried out for 20 min. Assays were
stopped and products analyzed as described above.

As one method of characterizing the radiolabeled products of in
vi tro reactions, assay mixtures were extracted according to the method of
Bligh and Dyer (1959). At time points of 7 or 20 min following addition
of [2-1‘C]malonyl-CoA, reactions were stopped with 50 uL of glacial acetic
acid and 4 mL of chloroformzmethanol (1:1, v/v). Two phases were formed

with the addition of 1.5 mL of H20. Following centrifugation, the lower

70

chloroform layer was removed and the upper aqueous methanolic phase was
extracted twice with 2 mL of hexane:isopropanol (7:2, v/v) . The hexane
extract was pooled with the recovered chloroform layer, and the combined
extracts were dried under N2. Lipids obtained in the organic phase were
resolved by silica TLC using sequential developments in polar and non-
polar solvent systems as described (Chapter 4). In addition, the free
fatty acid band on TLC plates was scraped into 1.5 mL of boron
trichloride/methanol and heated at 90°C for 15 min. The resulting fatty
acid methyl esters were analyzed by argentation TLC as described above.
The aqueous-methanolic phase from the Bligh and Dyer extract was
saponified as described by Browse and Slack (1985) . The recovered fatty
acids were converted to methyl ester derivatives by reaction in 10% (w/v)
boron trichloride/methanol and were then analyzed by argentation TLC.
Saturated fatty acid methyl esters were purified from argentation TLC
plates and further resolved by reverse phase TLC as described above.

To analyze the acyl-ACP products of in vitro fatty acid biosynthetic
assays, a modified version of the method of Mancha et a1. (1975) was
employed. The procedure used was essentially the same as that described
by Roughan and Nishida (1990) except that stopped reactions were extracted
three times with 2 mL of 50% isopropanol saturated petroleum ether prior
to ammonium sulfate precipitation of acyl-ACPs , and the precipitated acyl-
ACPs were washed three times with 4 mL of chloroformzmethanol (1:2, v/v) .
For analysis of acyl-ACPs, reactions were typically carried out for 7 min.
Petroleum ether extracts were dried under N2 and converted to fatty acid
methyl esters by reaction in 10% (w/v) boron trichloride/methanol as
described above. Fatty acid methyl esters from petroleum ether extracts
and from the sodium methoxide transesterified acyl-ACPs were resolved by
argentation TLC as described above.

Radioactivity in TLC-separated products of these studies was

visualized by autoradiography and quantified as described above.

71
Metabolism [l-l‘C]Acy1-ACPs and -CoAs By Coriander Endosperm Homogenates

The ability of coriander endosperm homogenates to convert saturated
[l-“C]acy1-ACPs and -CoAs to petroselinic acid was determined using
essentially the same assay conditions and product analysis as described
above for studies of malonyl-CoA metabolism. In addition, some assays (as
described in "Results and Discussion" section) were supplemented with 10
ILL of 1 mM spinach ferredoxin (Sigma) and 8 [1L of ferredoxin: NADPH
reductase (Sigma) (10 U/mL) and/or 10 pL of 2 mM malonyl-CoA in place of
a corresponding volume of water. Reactions were carried out with 15,000
dpm (6.8 nCi or 117 to 124 pmoles) of [1-1‘C]acyl-ACP and -CoA as
described below. To assess the ability of homogenates to synthesize
petroselinic acid in vitro, reactions using [2-1‘C]malonyl-CoA (2.4 to 3.1
nmole) as the substrate were conducted in parallel.

Radiolabeled acyl-ACPs were prepared from E. coli ACP and [1-
1"C]myristic acid, palmitic acid, stearic acid according to the method of
Rock and Garwin (1979) . [l-“C]Stearoyl-CoA was synthesized enzymatically
as described by Taylor et a1. (1989). [l-“C]Palmitoyl-CoA (56 mCi/mmol)
was purchased from Amersham.

Analysis of the Long/Medium-Chain Acyl-ACP Pool of Transgenic Tobacco
Suspension Cultures

To characterize the composition of long/medium-chain acyl-ACP pools
of tobacco suspension cells transformed with the 36 kDa coriander
desaturase (Cahoon et a1., 1992), 2.5 mL of transgenic and wild-type
cultures were incubated with shaking in 25 pCi of [1-“C]acetate (52
mCi/mmole, New England Nuclear, Boston, MA) in 13 mL polypropylene screw
cap tubes. (Suspension cells had been subcultured in fresh media four
days prior to use in these experiments.) Following a 12 min labeling
period, 100% (w/v) TCA was added to suspension cultures to a final
concentration of 5% (w/v). Sample tubes were immediately vortexed and
frozen in liquid N2. After thawing on ice, labeled cells were homogenized
with a Polytron PTlO/35 (Brinkman) , and the homogenate was centrifuged at

10,000 x g for 20 min at 5°C. The resulting pellet was washed with 2.5

72

mL of 1% (w/v) TCA and centrifuged again at 10,000 x g for 20 min. The
supernatant was discarded, and the pellet was centrifuged at 10,000 x g
for an additional 5 min in order to remove traces of TCA. Proteins
including free and acylated ACPs in the TCA pellet were resuspended in
1.25 mL of 50 mM Hepes, pH 7.8 containing 10 mM N-ethylmaleimide. The pH
of the resuspended pellet was adjusted as necessary to approximately pH
6.5 with.l M Tris, pH 9.0 and debris was removed.by centrifugation of the
sample at 10,000xg for 10 min.

Proteins in the resuspended TCA pellet were separated on a.13%
native polyacrylamide gel containing 1 M urea, and blotted to
nitrocellulose as previously described (Post-Beitenmiller et a1., 1991).
Radiolabeled.long/medium-chain.acy1 ACPs were identified on western blots
by mobility equivalent to that of [lJHC]palmitic acid or myristic acid
esterified to a mixture of tobacco ACP isoforms. Radioactivity on western
blots was detected by phosphorimaging. Using this gel system,
long/medium-chain acyl-ACPs migrated as essentially one'bandu Regions of
the nitrocellulose containing 1"C-long/medium chain acyl-ACPs were cut
from western blots with scissors. Acyl-ACPs on the cut nitrocellulose
pieces were subsequently transesterified by reaction for 45 min in 0.5 M
sodium methoxide in methanol as described above. Approximately, 1.5 mL
of sodium methoxide were used per 25 to 30 mg of nitrocellulose. In
preliminary studies, approximateLy 80% of [an‘C]palmitoyl-ACP blotted
onto nitrocellulose was recovered as fatty acid methyl esters using this
procedure.

Fatty acid methyl esters derived from the long/medium-chain acyl-ACP
pool of wild type and transgenic tobacco cells were further analyzed by
argentation TLC. Samples were loaded onto 15% argentation TLC plates,
which were sequentially developed to heights of 10 cm, 14 cm, 18 cm, and
20 cm in toluene at -20°C. Radioactivity in separated fatty acid methyl
esters was detected by phosphorimaging of TLC plates and mobilities
compared to those of radiolabeled standards. To more completely

characterize the identity of 16:1A“ in the acyl-ACP pool of transgenic

73

tobacco calli, the methyl ester derivative of this radiolabeled fatty acid
was recovered from argentation TLC plates and analyzed by reverse-phase
TLC as an intact or permanganate-periodate oxidized moiety using methods
described above.
Synthesis of [U-“C]Nonanoic Acid

[U-“C]Nonanoic acid (9:0) was synthesized from the methyl ester of
[U-“C]oleic acid (900 mCi/mmol, New England Nuclear, Boston, MA) for use
in radiolabeling studies described above. [U-“C]Oleic acid (a gift from
Dr. Edward Emken, USDA Northern Regional Research Center, Peoria, IL) was
converted to a methyl ester derivative using 10% (w/v) boron trichloride
in methanol and purified from contaminating radiolabeled material using
argentation TLC. Radiolabeled methyl oleic acid was subsequently
converted to [U-“C]nonanoic acid and methyl azelaic acid using
permanganate-periodate oxidation as described by Christie (1982). The
products were separated by silica TLC using a solvent system of
hexanezethyl ether:acetic acid (60:40:l v/v) . Radiolabeled nonanoic acid,
which migrated above methyl azelaic acid, was recovered from silica gel
with three washings of scrapings with 2 mL of chloroform:ethyl
ether:methanol (1:1:1 v/v) (Kates, 1972). The resulting [U-“CJnonanoic

acid was found to be radiochemically pure by reverse-phase TLC analysis.

Results and Discussion

Metabolism of Exogenous 1‘C-Fatty Acids by Coriander Endosperm Slices

Results of a previous study of this lab demonstrated that the double
bond of petroselinic acid of Umbelliferae endosperm arises from the
activity of a 36 kDa peptide that is structurally related to the
Agstearoyl-ACP desaturase (Cahoon et a1., 1992). The identity of the
substrate of the 36 kDa desaturase, however, was not determined in this
report. Based on what is known of the synthesis of other unsaturated
fatty acids in plants, the double bond of petroselinic acid most likely
results from a reaction(s) involving a saturated acyl chain bound to

either (1) a glycerolipid, (2) coenzyme A (CoA), or (3) acyl carrier

 

 

74

protein (ACP). In this regard, kinetics of [1-“C]acetate radiolabeling
of carrot and coriander endosperm shown in the companion paper gave no
indication that petroselinic acid is synthesized via the desaturation of
saturated acyl chains esterified to glycerolipids, including
phosphatidylcholine (PC) (Chapter 4). As another method of determining
whether a saturated fatty acid bound to a glycerolipid (or CoA) is the
precursor of petroselinic acid, the metabolism of [1-1‘C]1ong chain fatty
acids by coriander endosperm was examined. Such studies using whole plant
tissues and green algae have been previously conducted to demonstrate the
in vivo activity of desaturases that use fatty acids bound to
glycerolipids or CoA as substrates (e.g. , Curr et a1., 1969; Pollard and
Stumpf, 1980; Stymne and Stobart, 1986).

In the present study, incubation of coriander endosperm slices in
a buffered solution containing [l-“C]stearic acid for 10 h resulted in
the incorporation of radioactivity into all major glycerolipid classes
including PC, which contained approximately 17% of the incorporated label
(data not shown). Despite this, no A6 desaturation of [l-“C]stearic acid
to petroselinic acid was detected (Table 3.1) . These results thus suggest
that the A6 desaturation of stearic acid bound to a glycerolipid is not
the biosynthetic route of petroselinic acid. Furthermore, it is generally
presumed that fatty acids are incorporated into glycerolipids as coenzyme
A esters (Roughan and Slack, 1982). As such, the inability to synthesize
petroselinic acid from exogenous stearic acid would suggest the absence
of a A‘stearoyl-CoA desaturation pathway in Umbelliferae endosperm.

As with [l-“C]stearic acid, radiolabeled palmitic acid was not
metabolized to petroselinic acid when provided exogenously to slices of
coriander endosperm (Table 3.1) . The lack of palmitic acid incorporation
into petroselinic acid would suggest, for example, that petroselinic acid
does not derive from a palmitoyl-CoA desaturation/elongation pathway.

In addition to radiolabeled C15 and C1,3 saturated fatty acids, the
metabolism of [l-“C]oleic acid by coriander endosperm slices was

examined. In this regard, Shibahara et a1. (1991) have provided evidence

 

'75

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76

suggesting that the A9 double bond of oleic acid can be shifted to the A11
position to form cis-vaccenic acid in pulp of kaki. However, when
supplied exogenously to coriander endosperm, [1-“C]oleic acid was not
converted to petroselinic acid (Table 3.1) . As such, it is unlikely that
metabolic shifting of the double bond of oleic acid is associated with
petroselinic acid formation in coriander endosperm.

To further characterize petroselinic acid biosynthesis, coriander
endosperm slices were incubated in buffered solutions containing [1-
1"C]lauric acid and myristic acid. Several previous studies have
demonstrated that medium-chain length fatty acids such as lauric acid and
myristic acid can be converted to long-chain derivatives including
palmitic acid and oleic acid by green algae and intact tissues of higher
plants (Kannangara et a1. , 1973; Norman et a1. , 1985; Norman and St. John,
1986). These modification reactions presumably occur via acyl-ACP-
associated reactions since this is the principal (if not only) route of
016 and C18 saturated fatty acid synthesis and A9 stearic acid desaturation
in most plants. In contrast to medium-chain length fatty acids,
exogenously supplied long-chain fatty acids (e.g. palmitic acid and
stearic acid) are not readily modified by acyl-ACP-associated reactions
by plant tissues (Roughan et a1. , 1987). Consistent with an acyl-ACP
route of metabolism, radiolabeled lauric acid and myristic acid were
converted to a number of long-chain derivatives including palmitic acid
and oleic acid by coriander endosperm (Table 3.1). However, the major
elongation product of both [l-“C]lauric acid and myristic acid was
petroselinic acid, which accounted for approximately 60% of the fatty
acids derived from both medium-chain length precursors. Analysis of 1"C-
petroselinic acid following permanganate-periodate oxidation indicated
that lauric acid and myristic acid were incorporated as intact moieties.
In this regard, radioactivity in petroselinic acid was found not to be
randomized as might be expected if the lauric acid and myristic acid had
been first fl-oxidized and the radioactivity re-incorporated into fatty

acids. Instead, radiolabel was detected only in the lauric acid product

 

77

of oxidized petroselinic acid derived from [l-“C]1auric acid and only in
the methyl adipic acid product of oxidized petroselinic acid formed from
[l-“C]myristic acid (data not shown). Significant amounts of [1-
1"Cllauric acid and myristic acid were also converted to a hexadecenoic
acid (16:1) moiety that was identified as a A5 isomer by argentation TLC
analysis and by permanganate-periodate oxidation. Interestingly, this
fatty acid. composed approximately 10 to 15% of the elongation products of
[l-“C]lauric acid and myristic acid but 52.5% of the [“C] fatty acids
resulting [l-“C]acetate labeling of coriander endosperm for a similar
time period (Chapter 4) The basis for this difference is not known.
However, because desaturases, including the stearoyl-GOA desaturase
(Bloomfield and Bloch, 1960; Holloway et: a1., 1963) and presumably the
stearoyl-ACP desaturase (Cheesebrough and Cho, 1990) , typically introduce
double bonds into only one specific position of fatty acid substrates
regardless of chain length, it is presumed that the A6 double bond of
petroselinic acid and Ashexadecenoic acid result from the same enzymatic
reaction.

In summary, [l-“C]1ong-chain fatty acids (e.g., stearic acid,
palmitic acid, oleic acid) were incorporated into glycerolipids but were
not converted to petroselinic acid in detectable levels by coriander
endosperm. This suggests that petroselinic acid is not formed via the
desaturation of an acyl moiety esterified to a glycerolipid or coenzyme
A. In contrast to the metabolic fate of long-chain fatty acids, [1-
1"C]medium-chain length fatty acids were converted in relatively high
levels to petroselinic acid. Because these fatty acids, unlike long-chain
fatty acids, presumably enter the acyl-ACP track of de novo fatty acid
synthesis, these results suggest the involvement of acyl-ACP-linked

reactions in petroselinic acid biosynthesis.

In vitro Synthesis of Petroselinic Acid from [2-1‘C]Malonyl-CoA
To further characterize the biosynthetic origin of petroselinic

acid, an in vitro system for the synthesis of this fatty acid was

78

developed using crude homogenates of coriander endosperm and [2-
1"C]malonyl-CoA. Included in this assay were a variety of potential fatty
acid biosynthetic and desaturation co-factors, including NADPH, NADH, ACP
and catalase. Depending upon the experiment, assays were carried out for
7 or 20 minutes. The major radiolabeled acyl products of both 7 and 20
minute reactions were palmitic acid and stearic acid, which typically
accounted for 55 to 70% of the radioactivity recovered in fatty acids
(Figure 3.1A). In addition, 20 to 35% of the label incorporated into
fatty acids was typically associated with petroselinic acid. The
remainder of the labeled fatty acid (5 to 10%) was present as oleic acid,
and no radioactivity was detected in dienoic fatty acids or in any
hexadecenoic acid (16:1) isomers (e.g. A" or Aéhexadecenoic acid) that
might potentially co-migrate with oleic acid or petroselinic acid on
argentation TLC plates. The identity of 1"C-petroselinic acid was
confirmed by the mobility of its methyl ester derivative on argentation
and reverse-phase TLC plates and by reverse-phase TLC analysis of its
permanganate-periodate oxidation products. Using the latter method, a
nearly proportional distribution of radiolabel was detected between the
lauric acid and methyl adipic acid oxidation products, indicating that the
1"C-petroselinic acid formed in the in vitro assay resulted from de novo
fatty acid biosynthesis rather than from the elongation of a pre-formed,
non-radioactive fatty acid (data not shown). Finally, the conversion of
[2-“C]malonyl-CoA to petroselinic acid appeared to be dependent upon the
developmental stage of coriander endosperm as well as upon maintaining
high protein concentrations during tissue homogenization and in the
subsequent assays (see "Materials and Methods").

The incorporation of [2-1‘C1malonyl-CoA into petroselinic acid was
reduced by more than three-fold when the assay was conducted under oxygen-
limiting conditions (Table 3.2) . The apparent dependence of petroselinic

acid synthesis on molecular oxygen is consistent with the known catalytic

79

Table 3.2. Oxygen- and ferredoxin-dependence of petroselinic acid and
oleic acid synthesis from [Z-HCJmalonyl-COA by homogenates of coriander
endosperm

Control (or complete) assays contained ferredoxin and
ferredoxin:NADPH reductase and.were performed as described in "Materials
and Methods" . Oxygen-dependence (+N2) of petroselinic acid and oleic acid
synthesis was examined by carrying out reactions in tightly-capped tubes
that had been purged with nitrogen prior to and after addition of
substrate ([25”C]malony1-CoA). Ferredoxin-dependence (-Fd/FNR) of
petroselinic acid and oleic acid synthesis from [2-1‘C]malonyl-CoA was
studied by omitting spinach ferredoxin (Pd) and ferredoxin:NADPH-reductase
(FNR) from assays. Shown is the distribution ofqu recovered in fatty
acid methyl esters obtained by transesterification and subsequent

argentation TLC analysis of the products of 20 min assays.

 

 

Fatty Acid %Distribution of 1"C
Treatment Products in Products
Complete SatFA‘ 56 (5,300)b
18:1A6 29 (2,740)
18:1A9 15 (1,460)
-Fd/FNR SatFA 65 (5,430)
18:1A6 28 (2.340)
18:1A9 7 (620)
+N2 SatFA 85 (9,790)
18:1A6 8 (960)
18:1A9 7 (830)

 

‘SatFA, saturated fatty acids. bNumbers in parenthesis indicate dpm of 1"C

recovered in each acyl moiety.

80

SATURATED SATURATED

FATTY Acros> ~ “ <FATTY ACIDS

181149) n 18'1A9> <1821A9

I 6 _‘
18.1A > . 18:11‘1 <18:1A6

" hat 51

Figure 3.1. Products of the metabolism of [2-“C]malonyl-C0A by crude
homogenates of developing coriander endosperm. Shown in (A) is an
autoradiogram of fatty acid methyl esters prepared from the total products
of a 20 min assay performed as described in "Materials and Methods".
Shown in (B) is an autoradiogram of products of a 7 min assay extracted
according to the method of Mancha et a1. (1978). In Lane 1 are fatty acid
methyl esters prepared from the isopropanol saturated—petroleum ether
extract consisting primarily of free fatty acids. In lane 2 are fatty
acid methyl esters derived by transesterification of precipitated acyl-
ACPs. Fatty acid methyl esters in (A) and (B) were separated by
argentation TLC (see "Materials and Methods"). Radiolabeled material near
the origin in lane 2 of panel (B) was not identified.

81
properties of plant fatty acid desaturases (Jaworski, 1987).
Unexpectedly, the addition of ferredoxin and ferredoxin:NADPH reductase
had only a small effect upon petroselinic acid synthesis from [2-
1‘C]malonyl-C0A. In contrast, the presence of these co-factors in assays
resulted in a two-fold stimulation of oleic acid production.

To determine the exact form (e.g., glycerolipid- or ACP-esters) of
fatty acids produced in Vitro from [2-“C]malonyl-CoA, reactions were
extracted according to the method of Bligh and Dyer (1959). Using this
procedure, the radiolabel in the total lipid extract (or chloroform layer)
of in vitro assays after 7 and 20 min was detected primarily as free or
unesterified fatty acids (Table 3.3). More than 70% of the radioactivity
in free fatty acids of a 7 minute assay was associated with petroselinic
acid. Saponification of the aqueous-methanolic upper phase of Bligh and
Dyer extracts revealed a much different distribution of radiolabeled fatty
acids. In this phase, 290% of the radioactivity recovered in acyl
moieties of 7 and 20 min reactions was present in saturated fatty acids,
most likely in the water soluble form of CoA- or ACP-esters. In contrast,
petroselinic acid accounted for less than 8% and 4% of the radiolabeled
fatty acids recovered in the aqueous-methanolic phase of extracts of 7 and
20 min reactions, respectively.

Products of in vitro assays were also analyzed using the method of
Mancha et a1. (1975) as modified by Roughan and Nishida (1990). Using
this method, acyl-ACPs are recovered exclusively in an ammonium sulfate
pellet obtained following methanol:chloroform precipitation. Analysis of
acyl-ACPs of a 7 min reaction in this manner, revealed a preponderance of
radioactivity associated with saturated fatty acids (Figure 3.13, Table
3.4). This result was consistent with the radiolabeled fatty acid
composition of the aqueous-methanolic phase of Bligh and Dyer extracts.
In addition, approximately 15 to 20% of the total [2-“C]malonyl-CoA
incorporated into petroselinic acid in a 7 min reaction was present in the
putative acyl-ACP fraction. The majority of 1"C—petroselinic acid,

however, was recovered in the initial isopropanol saturated-petroleum

 

_~—

 

82

Table 3.3. Glycerolipid and fatty acid products of [Z-HCJmalonyl-COA
metabolism by crude homogenates of coriander endosperm

Reactions products of seven and twenty minute assays were analyzed
following extraction according to the method of Bligh and Dyer (1959).
Aqueous-methanolic phase and organic phase refer to reaction products
recovered in the upper and lower phases, respectively, of the Bligh and

Dyer extract.

 

Reaction Time

 

Glycerolipid/Fatty Acid
Products 7 min 20 min

 

dpm
Aqueous-methanolic phase

16:0 2130 5570
13:0 700 4690
18:1A‘ 250 510
18:1A9 100 200

Organic phase

 

FFA‘ 760 1760
SatFAb-+ 18:1A9 <50 n.a.°
18:1A6 720 n.a.

PCd 140 210

DAGe 80 <50

TAGf 60 180

Other <50 <50

Total Lipid

SatFA n.a. 540

18:1A6 n.a. 1550

18:1A9 n.a. 330
a‘FFA, Free fatty acid. bSatFA, Saturated fatty acids . °n.a. , Not
analyzed. dPC, Phosphatidylcholine. °DAG, Diacylglycerol. fTAG,
Triacylglycerol.

83

Table 3.4. Products of the metabolism of [Z-QKUmmlonyl-COA by
homogenates of coriander endosperm

Reaction products of seven minute assays were analyzed using the
extraction method.of Mancha et a1. (1975) with.modifications as described
by Roughan and Nishida (1990). Fatty acids and glycerolipids recovered
from the initial 50% (v/v) isopropanol saturated petroleum ether extract
were converted to fatty acid methyl ester derivatives. Results shown were
Obtained following argentation TLC-separation of fatty acid.methyl esters

and represent data from three independent experiments.

 

(NH, ) 280,, Pellet/Acyl -ACP
Fatty Acid Petroleum Ether Extract Pool

 

dpm recovered dpm recovered

Experiment I

SatFA‘ 110 (3)b 3300 (97)
18:1A‘ 1360 (86)° 220 (14)
18:1A9 24o (59)d 170 (41)

Experiment II

SatFA 180 (4) 3870 (96)
18:1A6 700 (81) 160 (19)
18:1A9 150 (65) 80 (35)

Experiment III

SatFA 60 (1) 5840 (99)
18:1A6 600 (79) 160 (21)
18:1A9 100 (62) 60 (38)

aLSatFA, saturated fatty acids. b% of total dpm of 1"C recovered in
saturated fatty acids. °% of total dpm of l"C recovered in petroselinic

acid” d% of total dpm of 1"C recovered in oleic acid.

84

ether wash, most likely in the form of free fatty acid as indicated by
results of Bligh and Dyer extraction described above. The relatively low
recovery of petroselinic acid as an acyl-ACP ester is consistent with the
presence of high levels of petroselinoyl—ACP thioesterase activity in
coriander endosperm extracts as reported by Darmann et a1. (1994). This
is also consistent with the detection of the majority of radiolabeled
petroselinic acid as free fatty acid in these assays.
In vitro Metabolism of [1-1‘C]Acyl-ACPs and -CoAs by Coriander Homogenates
The metabolic evidence presented above and.previously reported.data
demonstrating that the double bond of petroselinic acid arises from the
activity of a Agstearoyl-ACP desaturase-like peptide (Cahoon et a1. , 1992)
are consistent with an acyl-ACP route of petroselinic acid biosynthesis.
To test this directly, the ability of crude coriander homogenates to
convert [l-“C]saturated acyl-ACPs to petroselinic acid was examined.
Because it was known from experiments above that coriander endosperm can
incorporate exogenous [1-1‘C]myristic acid into petroselinic acid,
saturated acyl-ACPs of 14 carbon atoms and longer were used as potential
substrates. However, under conditions sufficient for petroselinic acid
synthesis from [2-1"C]malony1-CoA and for oleic acid synthesis from [1-
1"C]stearoyl-ACP, no conversion of radiolabeled acyl-ACPs (or -CoAs) to
petroselinic acid was detected (results not shown). Also, in the case
of assays carried out with [1-“C]myristoyl- and palmitoyl-ACP, the
addition of unlabeled malonyl-CoA resulted in nearly complete elongation
of the acyl-ACP substrates to stearic acid; however, no detectable amount
of radiolabeled petroselinic acid was formed. Furthermore, the presence
of ferredoxin and ferredoxin:NADPH reductase in assays resulted in
significant enhancement of oleic acid synthesis but had no apparent
influence on petroselinic acid production from [1-1‘C]acyl-ACPs (data not
shown).
Analysis of the Fatty Acid Composition of the Long/Medium-Chain Acyl-ACP
Pool of Transgenic Tobacco

To provide further evidence for the involvement of acyl-ACP-

85

associated reactions in the synthesis of petroselinic acid, the
long/medium-chain acyl-ACP pools of cell suspension cultures of tobacco
transformed with a cDNA for the coriander 36 kDa desaturase (Cahoon et
a1. , 1992) were examined. These cells, which were derived from transgenic
callus, produce approximately 4 wt% petroselinic acid and 5 wt%
A‘hexadecenoic acid (16:1A") (neither fatty acid is detectable in control
callus). Because tobacco likely does not contain a petroselinoyl-ACP-
specific thioesterase such as that present in Umbelliferae endosperm
(D5rmann et a1., 1994), it was expected that petroselinic acid might be
more easily detectable in the acyl-ACP pool of these cells than in
homogenates of coriander endosperm as described above. The method used
in this experiment also provided a more direct means of analyzing acyl-
ACPs than that of Mancha et a1. (1975) used above in studies of [2-
1“C]mal-CoA metabolism by coriander endosperm homogenates. To enhance the
sensitivity of acyl-ACP analyses, wild-type and transgenic tobacco
suspension cells were first incubated in [1-“C]acetate. Proteins
recovered after radiolabeling of tobacco cells and subsequent TCA
precipitation were resolved on native polyacrylamide gels containing 1 M
urea and blotted to nitrocellulose. Phosphorimaging of the resulting
western blots indicated the presence of radiolabel associated primarily
with a protein band that co-migrated with a tobacco long/medium-chain
acyl-ACP standard (Figure 3.2A) . Examination of fatty acid methyl esters
derived from acyl-ACPs of the transgenic tobacco revealed the presence of
a radiolabeled band that co-migrated with a methyl petroselinic acid
standard on argentation TLC plates (Figure 3.2B). The major
monounsaturated moiety in these samples, however, was a fatty acid methyl
ester identified as methyl Al‘hexadecenoic acid based on its mobility on
argentation and reverse-phase TLC and the chain lengths of its
permangana'te-periodate oxidation products (data not shown). Neither
methyl petroselinic acid nor Ahhexadecenoic acid waspresent in fatty acid

methyl esters prepared from acyl-ACPs of wild-type tobacco cells.

86

Figure 3.2. Analysis of long/medium chain acyl-ACPs of suspension
cultures of wild-type tobacco and transgenic tobacco expressing a cDNA
for the coriander 36 kDa desaturase. Shown in (A) is a phosphorimage of
a nitrocellulose blot of TCA-precipitated proteins of wild-type and
transgenic tobacco cells resolved by native PAGE following [1-“C]acetate
labeling of suspension cultures. In the standard (Std.) lane of (A) is
a mixture of tobacco ACP isoforms esterified to [l-“C]myristic acid.
Regions of blots co-migrating with the acyl-ACP standard were
transesterified, and the resulting fatty acid methyl esters were
separated by argentation TLC as shown on the phosphorimage in (B). Lanes
2 and 3 are methyl esters derived from acyl-ACPs of wild-type and
transgenic suspension cells, respectively. Lanes 1 and 4 of (B) are
methyl esters of 1"C-fatty acid standards. Shown in lane 1 are [1-
ll'C]methyl palmitic acid and oleic acid. Standards in lane 4 were
Prepared by [1-1"C]acetate labeling of coriander endosperm and consist

0f 1"C-methyl petroselinic acid, oleic acid, and saturated fatty acids.

87

 

 

. . ..E a. ...

22035....

2.3- Es . a

 

 

 

 

 

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i. .
a ..
a a
u.» D
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1. u .
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f. l. . . .
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88

SATURATED
FATTY ACIDS>

18:1A9>

 

SATURATED
<FATTY ACIDS

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<16:1A

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89

Interestingly, at least 60% of the radioactivity found in the long/medium-
chain acyl-ACP pool of transgenic cells was in the form of A‘hexadecenoic
acid and petroselinic acid despite the fact that these acyl moieties
together compose 510 wt% of the total fatty acids of these cells.

In summary, the presence of petroselinic acid in the acyl-ACP pool
of transgenic tobacco cells provides direct evidence that the synthesis
of this fatty acid involves acyl-ACP associated reactions. In addition,
because tobacco cultures expressing the 36 kDa desaturase contain
A‘hexadecenoic acid in their long/medium-chain acyl-ACP pool, it is
unlikely that, for example, this fatty acid is a partial B-oxidation
product of petroselinic acid. As discussed below, a more likely
explanation is that A"hexadecenoy1-ACP is the direct precursor of
petroselinic acid.

Metabolism of [U-“ClNonanoic Acid by Coriander Endosperm Slices

We have previously reported that the expression of a cDNA for the
36 kDa desaturase of coriander endosperm in tobacco resulted in the
production of petroselinic acid as well as A‘ hexadecenoic acid in
transgenic callus. The occurrence of both of these fatty acids in
transgenic tobacco can be interpreted in two ways. First, the 36 kDa
desaturase may position the placement of double bonds with respect to the
methyl end of acyl-ACPs and is therefore functionally an (012 (or n-12)
desaturase. Such an enzyme would be capable of inserting a double bond
at the A‘5 (or 012) carbon of stearoyl-ACP to form petroselinic acid and at
the A" (or on) carbon of palmitoyl-ACP to form Ahhexadecenoi-c acid.
Alternatively, the position of carbon atoms from the carboxyl (or A) end
of acyl-ACP substrates may dictate where the 36 kDa desaturase introduces
double bonds. In this regard, it would be unlikely that such an enzyme
could possess the dual properties of a A“ and A6 desaturase. Therefore,
the co-occurrence of petroselinic acid and A‘hexadecenoic acid in
transgenic tobacco would suggest that petroselinic acid is an elongation

product of Al'hexadecenoic acid.

90

To determine whether the 36 kDa desaturase positions the placement
of double bonds with respect to the carboxyl (A) or methyl (GI) end of acyl
chains, the metabolism of [U-“C]nonanoic acid (9:0) supplied exogenously
to slices of coriander endosperm was examined. Based on results of
radiolabeling experiments described above using exogenous medium-chain
length fatty acids, it would be expected that nonanoic acid would be
readily elongated by slices of coriander endosperm. In addition, because
nonanoic acid contains an odd number of carbon atoms, the mode of double
bond positioning by the 36 kDa desaturase can be directly assessed. For

example, if this enzyme functions as an 012

(or n-12) desaturase,
monounsaturated products would possess double bonds at odd-numbered carbon
atoms (e.g. 17:1A5 or 19:1A7). Conversely, given that petroselinic acid
(18:1A6) is the major fatty acid of coriander endosperm, double bond
placement with respect to the carboxyl end of fatty acids would likely
result in monounsaturated acyl chains with unsaturation primarily at the
A6 position (e.g. , 17:1A6 or 19:1A6).

As detailed in Table 3.5, the major products of nonanoic acid
labeling were 17:1A6 and 19:1A6 which together accounted for nearly 60%
of the total elongation/desaturation products. The identities of these
radiolabeled molecules was determined by their mobility on argentation and
reverse-phase TLC following conversion to methyl esters. In addition, the
free fatty acid generated from the permanganate-periodate oxidation of the
double bonds of the 17:1 and 19:1 moieties displayed mobility on reverse-
phase TLC plates equivalent to that of C11 and 013 moieties, respectively
(data not shown). This result is consistent with the location of the
double bond at the A6 position of both the 17:1 and 19:1 products. Among
the other fatty acids formed from [U-“C]nonanoic acid were [1"C]17:0 and
15:1A6.

Overall, the detection of radiolabeled 17:1A6 and 19:1A6 (rather
than 17:1A5 or 19:1A7 monounsaturated fatty acids) conclusively

demonstrates that the 36 kDa desaturase does not function as an 012

91

Table 3.5. Products of [U-ERUnonanoic acid labeling of coriander
endosperm slices

Labeling was conducted over a 6 h period with 53 mg of tissue and
approximately 1 pCi of [U4”C]nonanoic acid. Fatty acid products were
analyzed by combination of argentation and reverse-phase TLC following
transesterification of the total lipid extract in 0.5 M sodium methoxide
in methanol. Double bond positions were determined by reverse-phase TLC

analyses of permanganate-periodate oxidation products.

 

 

Elongation/Desaturation % of Total Elongated/
Products of'[IL4‘C]Nonanoic Acid Desaturated Fatty Acid
15:0 4.5
15:1A6 4.9
17:0 18.3
17:1A6 46.4
17:1A"9‘ 10.6
19:11:6 10.4
otherb 4.8

Total 1"C Elongated/Desaturated 369,000 dpm

 

aConsists of mixture of A“ and A9 isomers. bIncludes 51.3% each of

11:0, 13:0, 15:11:”, 19:0, and 19:11:”.

92

desaturase but instead positions double bond insertion from the carboxyl
end of acyl chains. In this regard, the 36 kDa desaturase of coriander
endosperm is similar to the A9stearoyl-CoA desaturase of rat liver
(Holloway et a1., 1963) and yeast (Bloomfield and Bloch, 1960) and the
A’stearoyl-ACP desaturase of soybean (Cheesbrough and Cho, 1990) which can
introduce double bonds at the A9 carbon atom of both C18 and 016
substrates.
Synopsis of Metabolic Studies of Petroselinic Acid Synthesis

From the data presented above and from previously reported results
(Cahoon et a1. , 1992) , we propose that petroselinic acid is formed by the
A“ desaturation of palmitoyl-ACP followed by elongation of the resulting
A‘hexadecenoyl-ACP to petroselinoyl-ACP. The rationale for this
conclusion is as follows. Petroselinic acid can be synthesized from [1-
ll’C]myristic acid by slices of coriander endosperm (Table 3.1). This
would suggest that the A6 double bond of petroselinic acid can arise from
a A2, A“, or A6 desaturation step at a point between the C1,, and C18 stages
of de novo fatty acid synthesis by one of the following pathways:

(a) 14:0-ACP - 14:1A2-ACP - 16:1A‘-ACP - 18:1A5-ACP

(b) 16:0-ACP - 16:1A‘-ACP - 18:1A6-ACP

(c) 18:0-ACP - 18:1A6-ACP
However, [1-1‘C]myristic acid can also be converted to “hexadecenoic acid
by coriander endosperm (Table 3.1) . This product cannot result from a A2
desaturation step as in pathway (a) (i.e. , A2 desaturation of myristoyl-
ACP followed by two-carbon elongation would produce l‘Ahexadecenoyl-ACP
rather than A6hexadecenoyl-ACP) . Assuming that the same desaturase is
involved in the insertion of the double bond of both petroselinic acid and
A‘hexadecenoic acid, pathway (a) would therefore be an unlikely route of
petroselinic acid formation. In addition, transgenic tobacco calli
expressing the coriander 36 kDa desaturase produce not only petroselinic

acid but also A‘hexadecenoic acid (Cahoon et a1. , 1992) , both of which are

93

detectable in the acyl-ACP pool of these cells. A‘Hexadecenoic acid and
petroselinic acid, however, likely cannot arise from both pathways (b) and
(c), respectively. Results of nonanoic acid labeling, for example,
demonstrate that the 36 kDa desaturase does not function as an
mlzdesaturase. Such a catalytic property would be necessary for this
enzyme to synthesize both A‘hexadecenoic acid and petroselinic acid
directly. 1 Furthermore, desaturases typically introduce double bonds into
specific positions of fatty acid substrates regardless of chain length
(e.g., Bloomfield and Bloch, 1960; Cheesebrough and Cho, 1990; Halloway
et a1. , 1963; Higashi and Murata, 1993) . As such, it is unlikely that the
36 kDa desaturase acts as both a A‘palmitoyl-ACP desaturase (pathway b)
and as a A‘stearoyl-ACP desaturase (pathway c) . More consistent with the
double bond-positioning properties of the 36 kDa desaturase and the fatty
acid and acyl-ACP profile of transgenic tobacco is that a double bond is
first introduced at the A" position of palmitoyl-ACP via the activity of
the 36 kDa desaturase. Subsequent elongation of 16:1A1-ACP would result
in the synthesis of petroselinoyl-ACP. As such, we propose that pathway
(b) as shown above is the most likely route of petroselinic acid formation
in Umbelliferae endosperm. The fact that 1"C-short and medium chain-
length fatty acids (e.g. , nonanoic acid, lauric acid, and myristic acid)
can be converted to other A6 monounsaturated fatty acids in addition to
petroselinic when supplied exogenously to coriander endosperm (Tables 3.1
and 3.5) suggests that the 36 kDa desaturase does not have an absolute
specificity for palmitoyl-ACP.

If petroselinic acid is synthesized from A‘hexadecenoyl-ACP, the
efficiency of this elongation reaction would appear to be quite different
between coriander endosperm and transgenic tobacco calli. In coriander
endosperm, for example, the mass ratio of petroselinic acid to
A‘hexadecenoic acid in the total lipid extract is on the order of 500:1
(72 wt% petroselinic acid: 0.1 to 0.2 wt% A‘hexadecenoic acid) (Chapter
4). This ratio in transgenic tobacco callus, however, is approximately

0.8:1 (4 wt% petroselinic acid: 5 wt% A‘hexadecenoic acid) (Cahoon et a1. ,

94

1992). This substantial difference might indicate that some component(s)
of A‘hexadecenoyl-ACP elongation in coriander endosperm is absent in
transgenic tobacco callus. This component may, for example, be a B-keto-
acyl-ACP synthetase that is specialized for the metabolism of
AAhexadecenoyl-ACP. Whatever its nature, the enzyme(s) involved in
Afhexadecenoyl-ACP elongation in Umbelliferae endosperm is likely to be
an essential factor for achieving high levels of petroselinic acid
production in transgenic plants.

Finally, a puzzling aspect of this study has been the inability to
detect in vitro synthesis of petroselinic acid by crude homogenates of
coriander endosperm using radiolabeled acyl-ACP substrates. This result
is somewhat surprising given the high degree of structural similarity
between the 36 kDa desaturase and the Agstearoyl-ACP desaturase (Cahoon
et a1. , 1992). Activity of the latter enzyme can be readily detected in
extracts of most plant tissues, including coriander endosperm (as
described above), using radiolabeled stearoyl-ACP in the presence of added
NADPH, ferredoxin, and ferredoxin:NADPH reductase. It cannot be ruled out
that the acyl-ACP desaturase assay used in our studies lacked an essential
co-factor(s). For example, ferredoxin may not be the preferred electron
donor for the 36 kDa desaturase“ However, this is not consistent with the
plastid localization of this enzyme and its structural similarity to the
Agstearoyl-ACP desaturase [i.e., the in vitro activities of all plastid
desaturases characterized t1) date, including the A?stearoyl-ACP
desaturase, are stimulated by ferredoxin (Jaworski, 1987; Schmidt and
Heinz, 1990)]. It is also possible that the 36 kDa desaturase may be
inactive with fatty acids esterified to E. coli ACP. This enzyme instead
might only be capable of using fatty acid substrates bound to higher plant
ACP or, more specifically, to Umbelliferae endosperm ACPs. However, plant
and E. coli ACP typically have similar in vitro activity in studies of
plant fatty acid biosynthetic reactions (Guerra et a1., 1986), and the 36
kDa desaturase is functional in vivo in tobacco, a "non-Umbelliferae"

Species. Another possibility is that the 36 kDa desaturase operates in

 

95

close association with one or more fatty acid biosynthetic enzymes. In
such a scenario, growing acyl chains may be channelled through an
elongation/desaturation pathway that is inaccessible in vi tro to exogenous
acyl-ACPs. The existence of metabolic channels or "metabolons" have been
proposed for a number of biosynthetic pathways; however, such entities are

often elusive to biochemical characterization (Srere, 1987).

Conclusions

(1) [l-“C]Stearic acid, palmitic acid, and oleic acid supplied
exogenously to coriander endosperm slices were incorporated into
glycerolipids but not converted to petroselinic acid. This suggests that
petroselinic acid is not formed by desaturation reactions involving fatty
acids esterified to glycerolipids or coenzyme A or, in the case of oleic
acid, by reactions involving the shifting of A9 double bonds.

(2) [1-1"C]Lauric acid and myristic acid provided exogenously to coriander
endosperm slices were apparently modified by acyl-ACP-associated reactions
and incorporated into petroselinic acid in high levels. To a lesser
extent, both radiolabels were also converted to Ashexadecenoic acid.

(3) Crude homogenates of coriander endosperm were capable of incorporating
[2-“C]malonyl-COA into petroselinic acid in an oxygen-dependent manner.
The resulting radiolabeled petroselinic acid was detected primarily as
free fatty acid and in lower levels as putative acyl-ACP esters.

(4) Analysis of transgenic tobacco suspension cells expressing the
coriander 36 kDa desaturase following [l-“C]acetate labeling revealed
significant amounts of petroselinic acid and “hexadecenoic acid in the
long/medium chain acyl-ACP pool. Neither fatty acid was detected in the
t10t:a1 lipids or acyl-ACPs of wild-type tobacco cultures.

(5) [U-“C]Nonanoic acid (9:0) supplied exogenously to coriander endosperm
was incorporated into A‘5 isomers of 15:1, 17:1, and 19:1 demonstrating
that the 36 kDa desaturase positions double bond placement relative to the
carboxyl terminus of fatty acid substrates.

(6) Considered in total, the metabolic data reported in this communication

 

96

are most consistent with a biosynthetic pathway of petroselinic acid

involving the A‘ desaturation of palmitoyl-ACP followed by two-carbon

elongation of the resulting A‘hexadecenoyl-ACP. Relative to coriander

endosperm, elongation of A‘hexadecenoyl-ACP appears to be a limiting

reaction in the synthesis of petroselinic acid by transgenic tobacco.

97

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Anal Biochem 184: 311-316

 

CHAPTER 41

Apparent Role of Phosphatidylcholine in the Metabolism of Petroselinic
Acid in Developing Umbelliferae Endosperm

Abstract

Studies were conducted to characterize the metabolism of the unusual
fatty acid petroselinic acid (18:1cisA6) in developing endosperm of the
Umbelliferae species coriander and carrot. Analyses of fatty acid
compositions of glycerolipids of these tissues revealed a dissimilar
distribution.of petroselinic acid in triacylglycerols (TAG) and the major
polar lipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE).
Petroselinic acid composed 70 to 75 mol% of the fatty acids of TAG but
only 9 to 20 mol% of the fatty acids of PC and PE. Though such data
appeared to suggest that petroselinic acid is at least partially excluded
from polar lipids, results of [liuC]acetate radiolabeling experiments
gave a much different picture of the metabolism of this fatty acid. In
time-course labeling of carrot endosperm, [l-“C]acetate was rapidly
incorporated into PC in high levels. Through 30 min, radiolabel was most
(concentrated.in PC, and of this, 80 to 85% was in the form of petroselinic
Tacidu One explanation for the large disparity in amounts of petroselinic
acid in PC as determined by fatty acid mass analyses and 1"C radiolabeling
is that turnover of these lipids or the fatty acids of these lipids
:results in relatively lOW' accumulation. of 'petroselinic acid. mass.
(Zonsistent with this, the kinetics of KLJJC]acetate time-course labeling
<>f carrot endosperm and "pulse-chase" labeling of coriander endosperm
suggested a possible flux of fatty acids from PC into TAG. In time-course
‘experiments, radiolabel initially entered PC at the highest rates but
accumulated in TAG at later time points. Similarly, in pulse-chase

studies, losses in absolute amounts of radioactivity from PC were

 

1Material in this chapter has been accepted for publication [Cahoon
EB, Ohlrogge JB (1994) Plant Physiol (in press)].

102

103

accompanied by significant increases of radiolabel in TAG. In addition,
stereospecific analyses of unlabeled and [1-1"C]acetate labeled PC of
coriander endosperm indicated that petroselinic acid can be readily
incorporated into both the sn-1 and sn-2 positions of this lipid. Because
petroselinic acid is neither synthesized nor further modified on polar
lipids, the apparent metabolism of this fatty acid through PC (and
possibly other polar lipids) may define a function of PC in TAG assembly

apart from its involvement in fatty acid modification reactions.

104

Introduction

Petroselinic acid, the A693 isomer of octadecenoic acid (18:1), is
the major component of the seed oil of most Umbelliferae (or Apiaceae),
Araliaceae, and Garryaceae species where it may compose up to 85 wt% of
the total fatty acid (Kleiman and Spencer, 1982). Because of its limited
natural occurrence, petroselinic acid is considered to be an unusual fatty
acid. Many unusual fatty acids of seed oils possess chemical structures
that differ significantly from those of the common C16 and Cl,3 plant fatty
acids. Examples of such acyl moieties include erucic acid (22:1A1391—’) of
Brassicaceae species and the medium-chain length (Cs-C“) fatty acids of
coconut, palm, and Cuphea species. In contrast, the structure of
petroselinic acid differs from that of oleic acid (18:1A9c—1’), a common
plant fatty acid, by only the position of its double bond.

Because of its structural similarity to oleic acid, petroselinic
acid offers an interesting case study of the metabolism of an unusual
fatty acid in a developing oilseed. Unusual fatty acids of seeds,
especially acyl groups of atypical chemical structures, are often absent
or found in only small amounts in membrane (or polar) glycerolipids
including phosphatidylcholine (PC) , the primary membrane lipid of seeds.
These fatty acids are instead concentrated in storage form as
triacylglycerol (TAG) (as reviewed by Ohlrogge, 1988; Battey et a1.,
1989) . In theory, the partitioning of unusual fatty acids away from polar
glycerolipids insures that "proper" membrane function is not disrupted by
the aberrant structures of these acyl moieties (Stymne et a1. , 1990) . It
has been proposed that this selective partitioning of unusual fatty acids
may result from substrate specificities of enzymes such as diacylglycerol
acyltransferase and GDP-choline:diacylglycerol cholinephosphotransferase
(Bafor et a1., 1990; Browse and Somerville, 1991). Together these two
enzymes serve as primary determinants of fatty acid flux via
diacylglycerol into storage (TAG) and membrane glycerolipids. It has also
been suggested that specialized acyltransferases may contribute to the

channeling of unusual fatty acids into TAG (00 and Huang, 1989). Examples

105

(of such. enzymes are lysophosphosphatidic acid. acyltransferases
characterized in extracts of palm endospenm (00 and Huang, 1989) and
CUphea lanceolata embryo (Bafor et a1., 1990) which display a nmrked
preference for CoA esters of medium-chain length fatty acids. Recent
evidence has also suggested that phospholipases may contribute to the
exclusion of unusual fatty acids from membrane lipids (Banas et a1.,
1992).

In contrast, oleic acid and its polyunsaturated derivatives (e.g.,
linoleic acid.and.a-linolenic acid) are not excluded from.PC in developing
oilseeds such as those of soybean, safflower, and linseed (Slack et a1.,
1978). In these tissues, PC appears to readily participate in the flux
of'C218 polyunsaturated fatty acids into TAG. In addition, the unusual
fatty acids y-linolenic acid and ricinoleic acid may also be metabolized
through PC in seeds of borage (Stymne and.Stobart, 1986; Griffiths et a1.,
1988) and castor (Bafor et a1., 1991), respectively. In each of these
cases, however, the metabolic flow of fatty acids through PC can be
attributed to the role of this lipid as a substrate for fatty acid
'modification reactions including desaturation and hydroxylation.

In this communication, we describe the metabolism of the unusual
fatty acid petroselinic acid in the endosperm of developing seeds of the
Umbelliferae species coriander and carrot. Reported below are results
that are consistent with an apparent movement of petroselinic acid through
both stereospecific positions of PC (and perhaps other polar lipids).
This route of metabolism occurs despite the fact that petroselinic acid
is neither synthesized nor further modified on this glycerolipid in

coriander or carrot endosperm (Cahoon et a1., 1992; Chapter 3).

Materials and Methods
Plant Material
Developing mericarps (fruits) of coriander (Coriandrum sativum L.)
were obtained from plants grown under greenhouse conditions of at least

12 h illumination provided by natural or supplemental lighting.

106

Developing mericarps of wild carrot or Queen Anne's lace (Daucus carota
L.) were collected from natural stands on the Michigan State University
campus. All experiments and lipid analyzes were performed using endosperm
dissected from the seed coat and pericarp of coriander and carrot
mericarps. The endosperm contained small amounts of an embedded embryo
that was not readily separable from endosperm. Collected plant tissue was
placed on ice and used immediately after dissection (30 to 45 min after
collection). Endosperm was placed in isopropanol for use in lipid
analyses or in cold water or 50 mM Mes, pH 5.0 until use in radiolabeling
experiments described below.
Total Lipid Extraction and Analyses
Developing endosperm of coriander (approx. 150 mg fresh weight) and

carrot (approx. 75 mg fresh weight) was heated at 80°C in 2 mL of
isopropanol for 10 minutes. Upon cooling, total lipids were extracted by
homogenization of endosperm with a Polytron PT 10/35 (Brinkman, Westbury,
NY) in 10 mL of hexane:isopropanol (3:2 v/v) (Hara and Radin, 1978; Post-
Beittenmiller et a1. , 1989). Following 4-6 h of incubation, debris was
removed by centrifugation, and the total lipid extract in
hexane:isopropanol was dried under N2 and resuspended in 2 mL of
chloroform:acetic acid (100:1 v/v). Lipids were subsequently separated
into neutral lipid, glycolipid, and phospholipid fractions by column
chromatography using a silica Sep-Pak cartridge (Millipore, Milford, MA)
essentially as described by Lynch and Steponkus (1987) except that neutral
lipids were eluted with 10 mL of chloroform:acetic acid (100:1 v/v)
followed by 5 mL of chloroform:acetone (80:20 v/v). Fractionated lipids
were dried under N2 and stored at ~20°C in chloroformzmethanol (6:1 v/v)
until further use.

The fatty acid composition of glycerolipid classes was determined
by GLC following TLC separation and fatty acid transesterification.
Neutral lipids, primarily triacylglycerol (TAG) and diacylglycerol (DAG),
were separated on silica K6 (0.25 mm thickness) TLC plates (Whatman,

Maidstone, England) using a mobile phase of hexanezethyl ether:acetic acid

107

(60:40:l v/v). Glycolipid and phospholipid classes were separated by
silica TLC using a solvent system of chloroformzmethanol:acetic acid
(75:25:8 v/v). Following development, TLC plates were dried in a N2
atmosphere and separated lipid classes were lightly stained with I2 and
identified by co-chromatography with lipid standards. Lipid bands were
scraped from the TLC plate into 1-2 mL of boron trichloride/methanol (10%
w/v) (Alltech, Deerfield, IL) containing 17:0 (internal standard) and
transesterified at 90°C for 30 min. In the case of neutral lipids, the
boron trichloride/methanol was supplemented with 25% (v/v) toluene to
increase the solubility of these lipids, and transesterification was
carried out for 45 min. Following heating, fatty acid methyl esters were
extracted as described (Morrison and Smith, 1964).

The composition of fatty acid methyl esters of individual lipid
classes was determined by capillary GLC with a Hewlett Packard 5890 gas
chromatograph interfaced to a Spectra-Physics SP4290 integrator.
Separation of fatty acid methyl esters was achieved using a 50 m x 0.25
mm ID CP-Sil 88 capillary column (Chrompack, Middelburg, Netherlands) with
the oven temperature programmed from 155°C (70 min hold) to 170°C at
2.5°C/min. Injection port and FID detector temperatures were 215°C, and
the column head pressure was 7.5 psi He. Under these conditions, the 18:1
isomers methyl petroselinic acid (18:1A°-°-1-3), oleic acid (18:1A9°—1°-), and
cis-vaccenic acid (18:1A119-13) were sufficiently resolved for separate
integration of each. The CP-Sil 88 column, however, was not capable of
resolving 18:1 and 16:3 in the case of monogalactosyldiacylglycero1
(MGDG). Instead, this separation was achieved with a 30 m x 0.25 mm ID
DB23 (J&W Scientific, Folsom, CA) column using the same chromatographic
conditions as described above.

Several isomers of hexadecenoic acid (16:1) of unknown double bond
position were identified by GC-MS of dimethyl disulfide derivatives
prepared from monounsaturated fatty acid methyl esters of coriander

phospholipids as described (Cahoon et a1. , 1992; Yamamoto et a1. , 1991).

108

Lipid Stereospecific Analyses

Phosphatidylcholine (PC) of coriander endosperm was purified by TLC
from the total phospholipid fraction isolated as described above.
Phospholipids were separated by silica TLC with chloroform:methanol : acetic
acid (75:25:8 v/v). The PC band was visualized with light iodine
staining, scraped from the TLC plate, and eluted from the silica with 3
mL of methanolzchloroformzwater (100:50:40 v/v). Following
centrifugation, the supernatant was recovered, and the silica scrapings
were washed with an additional 2 mL of chloroform:methanol:water.
Supernatants from the two washes were pooled, and two phases were formed
with the addition of 1.8 mL of water and 1.5 mL of chloroform. After
centrifugation, PC was recovered in the resulting chloroform layer. The
stereospecific fatty acid composition of PC was determined using
phospholipase A2 from Naja naja venom (Sigma) dissolved in 100 mM Tris-HCl
pH 7.6 and 4 mM CaCl2 using the reaction conditions and extraction
procedure described by Griffiths et a1. (1985). This method produced
nearly complete hydrolysis of PC as judged by TLC analysis of the
products. The lysoPC and free fatty acid (FFA) products were separated
by silica TLC with development to a height of 10 cm with a mobile phase
of chloroform:methanol: water (65:25:4 v/v). After drying in a N2
atmosphere, TLC plates were developed to their full length (20 cm) in
hexanezethyl ether:acetic acid (60:40:l v/v). The separated lysoPC and
FFA were transesterified and analyzed by gas chromatography as described
above. The fatty acid composition of the sn-2 position of PC was
calculated by subtraction of the fatty acid composition of lysoPC from
that of total PC. This number agreed closely with the acyl composition
of FFA released upon hydrolysis.

The sn-2 composition of TAG was determined by Grignard hydrolysis
of coriander TAG using the method of Myher and Kuksis (1979) . This method
was used instead of pancreatic lipase treatment because TAGs rich in
petroselinic acid are resistant to lipase digestion (Heimermann et a1.,

1973). TAG of coriander endosperm was purified by TLC. TAG was

109

hydrolyzed with ethylmagnesium bromide (1.0 M in tert-butyl methyl ether,
Aldrich), and reaction products were recovered as described by Myher and
Kuksis (1979). Hydrolysates were separated using borate impregnated-
silica TLC plates with development in 50:50 hexane:ethyl ether (v/v)
(Christie, 1982) . TLC plates were prepared by incubation of silica K6 TLC
plates for 10 minutes in a solution of 5% (w/v) boric acid in
acetonitrile:methanol (60:40 v/v) followed by air-drying. The TLC-
purified l,2;2,3 DAG was transesterified and analyzed by gas
chromatography as described above. To minimize isomerization (acyl-
migration) of 1,2;2,3 and 1,3 DAG products, all steps were performed in
rapid succession. The sn-2 acyl composition of TAG was calculated using
the formula [(4 x fatty acid mol% of 1,2;2,3 DAG)-(3 x fatty acid mol% of
TAG)] for a given fatty acid as described by Christie (1982) and Lawson
and Hughes (1988). As a check of this procedure, the erucic acid-rich
TAG of Crambe abyssinica was analyzed as described above. This TAG, which
is composed of approximately 55 mol% erucic acid, is known to contain
virtually no erucic acid at its sn-2 position (Gurr et a1., 1972). In
close agreement, only 1.3 mol% of the sn-2 fatty acids were determined to
be erucic acid using the above calculation.
[l-“CJAcetate Time-Course Labeling of Carrot and Coriander Endosperm
Approximately 15-20 mg fresh weight of freshly harvested carrot
endosperm was incubated in 250 pL of 50 mM Mes-NaOH, pH 5.0, containing
0.54 mM sodium [l-“C]acetate (56 mCi/mmol, Amersham, Arlington Heights,
IL) in five loosely capped 13 mm x 100 mm test tubes (Slack et a1. , 1978) .
Incubations were performed at 25°C with shaking at 100 rpm. At time
points of 2, 5, 15, 30, and 60 min, the incubation buffer was quickly
removed from each tube, and endosperm was washed two times with 1 mL of
ice-cold water. The endosperm was then heated at 80°C in 1 mL of
isopropanol for 10 min. After cooling, 1.5 mL of hexane was added, and
the endosperm was homogenized in the original incubation test tube with

the pestle of a Elvehjem homogenizer. To enhance abrasion of the

110

endosperm, fine glass beads were added to the tube prior to grinding.
Following homogenization of the tissue, an additional 1.5 mL of hexane and
1 mL of isopropanol were added, and lipids were extracted for 4 h. Lipids
were recovered following a wash of the hexane:isopropanol extract with 2.5
mL of 6.7% (w/v) sodium sulfate (Hara and Radin, 1978) . The aqueous phase
was re-extracted three additional times with 2 mL of hexane:isopropanol
(7:2 v/v) .

A similar procedure was used for 1"C-acetate labeling of coriander
endosperm slices. In this experiment, transverse slices (approximately
1 mm thick) of coriander endosperm were incubated in 0.23 mM of [1-
1"C]acetate in 50 mM Mes, pH 5.0. After 3.5 h, label was removed, and the
endosperm slices were washed and lipids extracted as described above.

Lipid classes were separated by silica TLC with sequential
development of TLC plates to heights of 4 cm and 12 cm in chloroform:
methanol:acetic acid (75:25:8 v/v) with drying in a N2 atmosphere between
developments. TLC plates were subsequently developed to their full
lengths (20 cm) in hexanezethyl ether:acetic acid (60:40:l v/v). This
method allowed analysis of polar and neutral lipids on the same TLC plate
with full resolution of all major glycerolipid classes. Some cross-
contamination of DAG with radiolabeled free sterols was detected,
particularly in the 3.5 h labeling experiment with coriander endosperm
slices. However, free sterols represented only a small fraction of
labeled lipids and could be distinguished upon argentation TLC analysis
of fatty acid methyl esters as described below. In addition,
identification of radiolabeled PC was confirmed by two-dimensional TLC
analysis (Christie, 1982) . Radiation in lipid classes was determined by
autoradiography and quantified by liquid scintillation counting of lipid
bands of TLC plates scraped into 3a20 (Research Products International,
Mt. Prospect, IL) scintillation cocktail. Alternatively, the distribution
of radiation in fatty acids of specific lipid classes was determined
following TLC separation and transesterification of lipids as described

above. The resulting fatty acid methyl esters were separated by

lll

argentation TLC (Morris et a1., 1967). TLC plates were prepared by
immersion of silica plates in a solution of 15% (w/v) AgNO3 in
acetonitrile for 10 minutes followed by air-drying. Argentation TLC
plates (20 cm in length) were developed sequentially at -20°C to heights
of 10 cm, 15 cm, and 20 cm in toluene. TLC plates were air-dried between
developments. This procedure resulted in full resolution of saturated
fatty acid, di-unsaturated fatty acid, and, in particular, petroselinic
acid and oleic acid methyl esters. Separated fatty acid methyl esters
were visualized by autoradiography as well as by light spraying with 0.1
wt% 2,7 dichlorofluorescein in methanol (Kates, 1972). Radioactivity in
fatty acid methyl ester bands was determined as described above.
[l-“C]Acetate Pulse-Chase Labeling of Coriander Endosperm

Transverse slices (approx. 1 mm thickness) of freshly harvested
coriander endosperm (300 mg fresh weight) were incubated in 1 mL of 0.14
mM sodium [1-1"C]acetate in 50 mM Mes-NaOH, pH 5.0, for 15 min (Slack et
a1. , 1978) in a loosely-capped 13 mm x 100 mm test tube. The incubation
tube was shaken at 100 rpm and maintained at 25°C. After 15 min, the
labeling buffer was removed, and endosperm slices were washed three times
with 2 mL of ice-cold water and once with 2 mM unlabeled sodium acetate
in 50 mM Mes, pH 5.0. The endosperm slices were quickly partitioned among
five pre-weighed test tubes. One test tube contained 1 mL isopropanol
which was subsequently heated at 80°C for 10 minutes to stop further
metabolism of the label. The remaining four test tubes which constituted
the chase contained 1 mL of 2 mM unlabeled sodium acetate in 50 mM Mes pH
5.0. At times of 30, 90, 180, and 360 min following the 15 min pulse,
buffer was removed from one of the four chase tubes, and the endosperm was
washed with 1 mL of ice-cold water. One mL of isopropanol was added to
the tube which was subsequently heated at 80°C for 10 min. Lipids were
extracted from endosperm slices as described above for [l-“C]acetate
time-course labeling experiments. During the chase period, the incubation
buffer was removed from each of the four tubes and replaced with fresh

buffer containing 2 mM sodium acetate after 15 minutes and again at 30 min

112

intervals during the first two hours. This was done in order to minimize
incorporation of residual [1:1‘C]acetate.

The distribution of label in lipid classes and fatty acid methyl
esters of specific lipid classes was determined as described above. In
addition, stereospecific analysis of labeled fatty acids of PC following
the initial 15 min 'pulse was performed. as described above. The
distribution of label in fatty acid.methyl esters derived from lysoPC and
FFA was determined.by liquid scintillation counting following separation

of acyl derivatives by argentation TLC as described above.

Results
Distribution of Petroselinic Acid in Glycerolipids of Carrot and Coriander
Endosperm
Petroselinic acid has been previously identified in
phosphatidylcholine (Dutta et a1., 1992) and the total polar lipid
fraction of carrot seed (Dutta and Appelqvist, 1991) as well as the total
phospholipids of carrot and coriander seed (Prasad et a1., 1987). A more
detailed analysis indicated that petroselinic acid is a component of all
detectable glycerolipids of developing carrot and coriander endosperm
(Table 4.1). Relative amounts of this fatty acid in individual lipid
classes, however, were quite different. As is the case with most unusual
fatty acids, the highest levels of petroselinic acid.were detected in TAG.
In extracts of both carrot and coriander endosperm, petroselinic acid
accounted for about 70 to 75 mol% of the total fatty acid of TAG.
Petroselinic acid was also the major fatty acid of DAG of both coriander
and carrot endosperm, which is consistent with the primary role of this
lipid as a precursor of TAG in oil-accumulating seeds. In marked
contrast, the major phospholipids PC, phosphatidylethanolamine (PE), and
phosphatidic acid (PA), which together accounted for nearly 75 mol% of the
total polar lipids of carrot and.coriander endosperm, contained about one-
fourth to one-eighth as much petroselinic acid as TAG. PC of carrot and

coriander endosperm, for example, contained 15 and 20 mol% petroselinic

113

 

 

 

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115

acid, respectively. In addition, petroselinic acid composed <10 mol% of
PE of the endosperm of both species. In contrast to TAG, the major fatty
acids of these polar lipids were oleic acid and/or its derivative linoleic
acid. Minor glycerolipids, particularly phosphatidylinositol (PI) as well
as the plastid galactolipids monogalactosyldiacylglycerol (MGDG) and
digalactosyldiacylglycerol (DGDG) of'carrot.endosperm, contained.somewhat
higher levels of petroselinic acid than did PC and PE. Still, relative
amounts of petroselinic acid in these lipids were roughly one-half to one-
third as much as in TAG.

In addition to petroselinic acid” small amounts (51.5 mol%) of other
unusual fatty acids were detected.in glycerolipids of carrot and.coriander
endosperm. These included cis-vaccenic acid (18:1A11E) and three
hexadecenoic acid (16:1) isomers. Mass spectral analyses of thiomethyl
derivatives of hexadecenoic acid methyl esters indicated that these fatty
acids were A“, A°, and A9 isomers (Appendix 1).

It is notable that PA levels in developing carrot and coriander
endosperm were higher than might be expected for a plant tissue. Slack
et a1. (1978), for example, reported that PA composed between 4 to 13 mol%
of the phospholipids of developing cotyledons of safflower, soybean, and
linseed. In our analyses, PA accounted for approximately 16 and 24 mol%
of the polar lipids of the developing endosperm of coriander and carrot,
respectively. It is difficult to attribute this PA to the degradation of
PC and PE during or prior to lipid extraction. In order to inactivate
potential lipolytic enzymes, endosperm was incubated in hot isopropanol
before extraction of lipids (Kates and Eberhardt, 1957). Also, in
radioisotope labeling experiments described.below, PC was heavily labeled
with [lJMC]acetate, but no significant breakdown.of this lipid.to PA was

detected in either time-course or pulse-chase labeling experiments.

Lipid Synthesis During the Development of Coriander Endosperm
The nearly four-fold higher relative amounts of petroselinic acid

in TAG versus PC in coriander endosperm described above might reflect

116

temporal differences in TAG and PC synthesis. For example, the majority
of PC might be synthesized during early stages of endosperm development
prior to the rapid synthesis and accumulation of petroselinic acid in TAG.
To examine this possibility, changes in absolute amounts of total fatty
acid and petroselinic acid in PC and TAG were examined over a period of
coriander endosperm development ranging from early to mid-maturity.
Depending upon greenhouse conditions, a distinct endosperm (i.e. ,
endosperm readily separable from pericarp) could be detected by
approximately 12 to 16 DAF and mid-maturity was reached by 18 to 22 DAF.
In the example detailed in Table 4.2, the fresh weight of coriander
endosperm more than doubled between 16 and 20 DAF. During this period,
TAG and PC were both actively synthesized as the fatty acid content of
these lipids increased by more than six-fold and five-fold, respectively.
Despite this, relative levels of petroselinic acid deposition into TAG and
PC were significantly different. Of the increase in fatty acid content
of TAG, about 75% could be accounted for by increases in amounts of
petroselinic acid [(A nmol petroselinic acid/A nmol total fatty acid) x
100%] , whereas changes in petroselinic acid content constituted about 19%
of the increases in fatty acids of PC. These data therefore suggest that
differences in relative amounts of petroselinic acid in PC and TAG are not
the result of temporal differences in the synthesis of these lipids.
[1-“C]Acetate Time-Course Labeling of Carrot and Coriander Endosperm
Analyses of fatty acid compositions of lipids and developmental
changes in these compositions as described above give only an indication
of the net products of a myriad of metabolic reactions. To gain a better
understanding of how these fatty acid compositions arise, [l-“C]acetate
labeling studies of carrot and coriander endosperm were performed. In
time-course labeling of carrot endosperm (Figure 4.1A), at least 85% of
the radioactivity recovered in glycerolipids was detected in PC, TAG, DAG,
and PE at time points through 1 h. Of these lipids, PC and TAG were the

most heavily labeled.

117

Table 4.2. Changes in total fatty acid and petroselinic acid content of
phosphatidylcholine (PC) and triacylglycerol (TAG) from early (16 DAF) to
mid (20 DAF)-development of coriander endosperm

Fatty acids were analyzed by gas chromatography as methyl ester
derivatives and amounts determined relative to methyl heptadecanoic acid

(17:0) (internal standard).

 

 

TAG PC
DAFa Fresh Weight Total FAb/l8:lA6 Total FA/l8:1A6
mg/endosperm nmol fatty acid/endosperm
16 1.7 199/127 7.2/0.8
18 2.7 719/534 25.3/4.7
20 3.9 1,260/930 38.3/6.7

 

aDAF, Days after flowering. bFA, Fatty acids.

118

Figure 4.1. Products of [l-“C]acetate time-course labeling of
developing carrot endosperm. Shown is the distribution of radioactivity
recovered in the major glycerolipid classes (A) and fatty acids of
phosphatidylcholine (PC) (B), triacylglycerol (TAG) (C), diacylglycerol
(DAG) (D), and phosphatidylethanolamine (PE) (E) at time points through
1 h.

DPM/ 15 mg Carrot Endosperm
(Thousands)

119

 

150

120

30

 

 

 

 

Time (min)

120

 

 

 

 

DPM/15 mo Carrot Endosperm

 

 

 

 

 

 

 

C
E 130
§ 104 r
LU
‘5 7e -
8
g 52 -
‘2 26 r
E o 4

o 12 24 36 4a 60

Time (min) 18:2

DPM/ 15 mg Carrot Endosperm

DPM/15 mg Carrot Endosperm

7O

4O

2O

10

20
18
15
14
12
1O

ONJEOICD

121

 

 

 

 

Time (min)

 

 

 

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12 24 36 48 18260

Time (min)

 

122

The most striking result was the rapid incorporation of high levels
of [l-“C]acetate into PC as petroselinic acid. At time points through
30 min, PC was the most heavily labeled glycerolipid. Of the
radioactivity detected in PC during this period, nearly 85% was in the
form of petroselinic acid, and even after 1 h, 75% of the label in PC was
associated with this fatty acid (Figure 4.1B). Relative to PC, the
radiolabel appeared to initially enter TAG at a slower rate. However, by
1 h, the highest amounts of radioactivity were detected in TAG. At this
time point, TAG contained approximately 1.1 times as much 1“C as PC. In
addition, at least 90% of the radiolabel in TAG was present as
petroselinic acid throughout the 1 h labeling period (Figure 4.1C). The
incorporation of high amounts of [l-“C]acetate initially into PC followed
by accumulation into TAG at later time points was suggestive of the
metabolic movement of fatty acids from PC into TAG. Such precursor-
product labeling kinetics, in fact, were similar to those previously
described for the metabolism of oleic acid-derived C18 polyunsaturated
fatty acids in seeds of soybean and linseed (Slack et a1., 1978), in
which acyl chains were shown to move from PC into TAG.

In addition to PC and TAG, significant amounts of radioactivity were
detected in DAG as well as the polar lipid PE (Figure 4.1D and E).
Throughout the l h time-course, >80% of the radiolabel in these lipids was
detected as petroselinic acid. With regard to PE, however, the total
amount of label in this lipid was five- to seven-times less than that in
PC.

Similar to carrot endosperm, in pulse-chase labeling experiments of
coriander endosperm described below, [l-“C]acetate was also incorporated
into PC in high levels as petroselinic acid during an initial 15 min
"pulse" labeling period. In addition, in coriander endosperm that had
been incubated in [1-1"C]acetate for an extended period of time (3.5 h),
PC persisted as a major radiolabeled glycerolipid, accounting for nearly
30% of the total radioactivity recovered in the total lipid extract (Table

4.3). Of the label in PC, about 55% was detected as petroselinic acid.

 

123

Table 4.3.

of coriander endosperm following 3.5 h incubation in [1~¥%Uacetate

Distribution of radiolabel incorporation into glycerolipids

 

% Distribution of Radiolabel

Lipid Class % of Total in Fatty Acids

 

IncorporatediMC

 

 

SatFA“ 18:1A°b 18:1A9 18:2

Total 100 8.1 72.6 18.3 2.6

TAG 39.4 2.0 90.0 5.6 2.3

PC 28.7 6.4 56.4 31.6 5.5

DAG 18.9 4.4 78.3 15.0 2.3

PE 4.0 16.2 63.3 18.6 1.9
PI 3.1 ---ND°--
PA 2.8 ---ND---
Other 3.1 ---ND---

Total Incorporation 25,800 DPM/mg endosperm

 

aTotal saturated fatty acid. bMay include small amounts of 16:1A°.

acyl moiety composes 2.6% of the total radiolabeled fatty acid.

determined.

This

cNot

124

In comparison, 39% of the label in the total lipid extract was recovered
as TAG, of which 90% was present in the form of petroselinic acid. DAG
also contained significantly high levels of recovered label (19% of the
total label), and the relative proportion of radiolabeled petroselinic
acid in this lipid (78%) was roughly similar to that in the total extract.
Although the total amount of label in PE was more than seven-fold less
than that in PC, this polar lipid also contained.high relative amounts of
label in the form of petroselinic acid (63%).
[laj‘C]Acetate Pulse-Chase Labeling of Coriander Endosperm
[an‘C]Acetate labeling of carrot and.coriander endosperm described
above suggested a possible flux of fatty acids from PC into TAG. To
determine whether ll'C-petroselinic acid does move from PC to TAG, [l-
1"C]acetate pulse-chase labeling of tissue slices of developing coriander
endosperm was conducted. Following a 15 min incubation in labeled
acetate, endosperm slices were placed in unlabeled acetate for periods of
up to 6 h. After the initial 15 min labeling ("pulse"), more than 85%
of the total label was detected in PC, TAG, and DAG (Table 4.4).As with
time-course labeling of carrot endosperm (see above), PC was the most
heavily labeled lipid after 15 min of incubation of coriander endosperm
in. [lxl‘C]acetate. At the end of the 15 min. pulse, 42% of the
incorporated [14”C]acetate recovered in the lipid fraction was detected
in PC, and 85% of the label in PC was present as petroselinic acid. Some
difficulty was encountered in completely removing unincorporated [l-
ll'C]acetate from the endosperm slices at the end of the pulse period” As
a result, a 60% increase in levels of incorporated label was detected
during the subsequent chase period” Despite this, the absolute amount of
radioactivity in PC had declined by close to 2.5-fold at the end of the
6 h chase. This change in amounts of radiolabel in PC included
proportional losses ixtl‘C-petroselinic acid. Accompanying this change
was a marked increase in the amount of label in TAG. At the end of the
6 h chase, radioactivity in TAG had increased nearly 7-fold, and this

change included increases in amounts of radiolabeled petroselinic acid.

125

Table 4.4. Redistribution of radioactivity in phosphatidylcholine (PC),
triacylglycerol (TAG), and diacylglycerol (DAG) following transfer of
coriander endosperm slices from [l-MCJacetate to media containing
unlabeled acetate

Slices of coriander endosperm at mid-development were incubated in
0.14 mM [l-“C]acetate for 15 min, washed, and transferred to media
containing 2.0 mM unlabeled acetate (see Materials and Methods). Results
below indicate recovery of radioactivity in PC, TAG, and DAG at time

points following transfer to unlabeled media.

 

Time in Total 1“0 PC TAG DAG

unlabeled media Incorporated Tot . ‘ A‘S’b Tot . A6 Tot .

 

dpm (x103)/

min mg endosperm dpm (x103)/mg endosperm

0 10.4 4.3 3.7 2.7 2.5 2.2
30 11.0 3.6 2.9 4.3 3.8 2.1
90 12.9 3.4 2.9 5.3 4.8 2.2
180 15.1 2.9 2.5 7.5 6.6 3.2
360 16.7 1.7 1.4 9.1 8.1 3.8

 

‘Tot., Total radioactivity in fatty acids of given lipid class. bA‘s,

Radioactivity in petroselinic acid of given lipid class.

126

Because of the increase in total recovered label during the chase period,
it was not possible to definitively assign label lost from PC to that
gained by TAG. However, losses in absolute amounts of radioactivity in
PC accompanied by increases in radiolabel in TAG during the chase was
consistent with a precursor-product relationship between these lipids.
Stereospecific Patterns of Petroselinic Acid Metabolism in Glycerolipids
of Coriander Endosperm

In many seeds, the sn-2 position of glycerolipids contains almost
exclusivelyC218 fatty acids with A9 unsaturation, e.g., oleic acid and
linoleic acid (Stymne and Stobart, 1987). The biochemical basis for this
acylation pattern is believed to reside in the substrate specificity of
lysoPA acyltransferase, the enzyme which catalyzes the esterification of
acyl-CoA moieties to the sn-2 position of the glycerol backbone (Ichihara
et a1., 1987). However, in seeds which synthesize certain unusual fatty
acids, specialized lysoPA acyltransferases appear to exist with
substratespecificities that permit the accumulation of unusual acyl
moieties at the sn-2 position of TAG (00 and Huang, 1989; Bafor et a1.,
1990; Cao et a1., 1990).

With regard.to petroselinic acid, this fatty acid, in terms of mass,
was found to be most concentrated in the sn-1 position of PC isolated from
coriander endosperm (Table 4.5). However, significant amounts of
petroselinic acid were also present in the sn-2 position of this lipid.
In fact, approximately 40% of the total petroselinic acid in PC was
detected in this stereospecific position. Similarly, petroselinic acid
accounted for nearly 50 mol% of the fatty acid mass of the sn-2 position
of coriander TAG (Table 4.6). This value closely agreed with that
determined by Gunstone (1991) using 13C NMR. Furthermore, in PC from
coriander endosperm labeled with [lJHC]acetate for 15 min, nearly 85% of
the total radiolabeled.fatty'acids were composed.ofJAG-petroselinic acid.
Of the labeled fatty acids of the sn-2 position of PC, slightly more than
80% was detected as petroselinic acid (Table 4.7). Thus, fatty acid.mass

and.radiolabeling data demonstrate the ability of coriander endosperm to

 

 

127

Table 4.5. Stereospecific fatty acid composition of phosphatidylcholine

of developing coriander endosperm

PC was purified from phospholipid extracts of developing coriander

endosperm (22-26 DAF).

Fatty acid compositions are expressed as mol% of

the total fatty of PC or the total fatty acid of the sn-1 or sn-2

 

 

stereospecific positions of PC. Values are the average of two
determinations.
Fatty Acid Total FA‘ sn-l sn-2
mol%
16:0 15.3 30.0 0.5
16:1b 1.4 2.0 0.9
18:0 2.2 3.9 0.5
18:1A6 21.8 26.7 16.9
18:1A9 14.4 6.4 22.5
18:1A11 1.4 2.0 0.7
18:2 43.6 29.3 58.0

 

aFA, Fatty acids.

bSum of three detectable 16:1 isomers.

128

Table 4.6. Stereospecific fatty acid composition of triacylglycerol of
developing coriander endosperm (22 to 26 DAF)

Fatty acid compositions are expressed as mol% '2 SE (n-3) of the
total FA of TAG, l,2;2,3-DAG (Grignard hydrolysis of TAG), or the
calculated sn-2 position of TAG. The sn-2 position of TAG was calculated
using the formula [(4 x l,2;2,3-DAG fatty acid mol%)-(3xTAG fatty acid

m01%)] (see Materials and Methods).

 

 

 

Fatty Acid Total FAa l,2;2,3-DAG sn-2
mol%

16:0 3.9: 0.7 3.3: 0.7 1 5: 0 7
16:11) 0.4: 0.1 0.4: 0.2 0 8: 0 1
18:0 1.0: 0.2 0.7: 0.1 0
18:1A5 74.0: 0.9 67.6: 0.9 48.5: 0.7
18:1A9 7.2: 0.8 10.7: 1.0 21.2: 2.1
18:1A11 0.9: 0.1 0.9: 0.1 0 9: 0.1
18:2 12.7: 0.6 16.4: 1.0 27.4: 2.1

a‘FA, Fatty acids. bSum of three detectable 16:1 isomers.

129

Table 4.7. Stereospecific distribution of radioactivity in PC following

15 min incubation of coriander endosperm slices in [1--1—"C] acetate

 

 

Fatty Acid % of Total % of Total % of Total
1"C in PC ll‘C in sn-l 1"C in sn-2
Saturated FAa 2 3 2
18:1A6 85 90 81
18:1A9 13 7 18
Ratio of ll‘C sn-l/“C sn-2 42/58

‘

aFAA , Fatty acids .

 

130

readily use both the sn-1 and sn-2 positions of the glycerol backbone of

lipids in the metabolism of petroselinic acid.

Discussion

Like.many'other unusual fatty acids of seeds, petroselinic acid was
most concentrated in TAG of developing carrot and coriander endosperm.
In contrast, relative amounts of petroselinic acid in the major polar
lipids PC and PE were much reduced. In coriander and carrot endosperm,
for example, petroselinic acid composed 70 to 75 mol% of the fatty acids
of TAG but only 15 to 20 mol% and <10 mol% of the fatty acids of PC and
PE, respectively (Table 4.1). One explanation for such differences is
that metabolic mechanisms exist in cells of these tissues which maintain
active flux of petroselinic acid into TAG but limit or partially exclude
the accumulation of this fatty acid in polar lipids. In seeds of Cuphea
lanceolota, for example, it has been proposed that medium-chain length
fatty acids are excluded from PC by the high activity of DAG
acyltransferase for DAG molecules rich in these fatty acids (Bafor et a1. ,
1991). In such a scenario, unusual fatty acids are rapidly shunted into
TAG synthesis and effectively precluded from incorporation into PC and
other polar lipids. Our results from [14“0]acetate labeling studies of
carrot and coriander endosperm, however, give a much different view of
petroselinic acid metabolism. In this regard, incubation of carrot and
coriander endosperntiri[1214C]acetate for short periods (15 to 30 minutes)
was accompanied by the rapid incorporation of label into PC in high
levels, and >80% of the radioactivity in PC and PE was associated with
petroselinic acid. Relative to PE, however, incorporation of radiolabel
into PC was more significant as this lipid typically contained about seven
times more radioactivity than PE in these experiments. Thus, results of
[lsJ‘C]acetate radiolabeling studies indicated that there is virtually no
exclusion of petroselinic acid incorporation into polar lipids, especially
PC, despite the fact that this fatty acid does not accumulate to high

levels in these lipids in terms of mass.

131

If little or no exclusion of petroselinic acid from PC and perhaps
other polar glycerolipids occurs, what prevents high levels of
accumulation of petroselinic acid in these lipids? One possibility is
that turnover of polar lipids, particularly PC, results in reductions in
amounts of petroselinic acid” Such turnover could involve either removal
of fatty acids from the glycerol backbone or metabolic movement of the
complete DAG moiety of PC into TAG. Turnover of PC in coriander and
carrot endosperm is suggested.by at least three observations. First, in
time-course labeling of carrot endosperm with [124“C]acetate, radiolabel
most rapidly entered PC at early time points (15 to 30 minutes), and the
radioactivity detected in PC was primarily in the form of petroselinic
.acid. (Figure 4.1). IHowever, as the rate of incorporation. of [l-
1"C]acetate into PC declined at later time points, radioactivity
accumulated in TAG at an accelerating rate and after 1 h, amounts of
radiolabel in this lipid exceeded that in PC. Of the label accumulated
in TAG, >90% was detected in petroselinic acid. Such labeling kinetics
suggest that petroselinic acid first enters PC prior to its accumulation
in TAG.

A second indication of fatty acid turnover in PC was obtained from
pulse-chase labeling of coriander endospenm with [123“C]acetate (Table
4.4). In this study, significant amounts of label whichnwere incorporated
into PC during a 15 min incubation in [l-“C]acetate were lost in a
subsequent "chase" period. Accompanying this loss were significant
increases in radioactivity in TAG, primarily in the form of petroselinic
acid. The results of this experiment are thus consistent with a turnover
of fatty acids in PC and” more specifically, suggest the possible movement
of petroselinic acid, in route to TAG, through PC.

A third indication of fatty acid turnover in PC is the large
disparity in the rates of fatty acid accumulation in PC and TAG as
determined by radiolabeling studies and fatty acid mass analyses. In
time-course labeling studies with carrot endosperm, amounts of

radiolabeled fatty acids in TAG and PC were essentially equal after 1 h

132

incubation in [1-1“C]acetate (Figure 4.1). Similarly, after 3.5 h of
incubation of coriander endosperm in [l-“C]acetate, levels of
radioactivity in fatty acids of TAG only 1.6-fold greater than in PC
(Table 4.3). Based on these results, if no turnover of the fatty acids
of PC occurs, one would expect to find high, perhaps nearly equal levels
of fatty acid mass in PC and TAG. However, in developmental studies of
coriander endosperm, the rate of fatty acid mass accumulation into TAG was
at least 35 times greater than into PC (Table 4.2). One way to explain
this large difference is that fatty acids (primarily petroselinic acid)
are metabolized through PC such that they are readily detectable in this
lipid in radiolabeling experiments. However, movement of petroselinic
acid in the form of acyl or DAG moieties from PC results in relatively low
levels of fatty acid mass accumulation in this lipid.

The involvement of PC in TAG biosynthesis in developing oilseeds has
been previously documented for fatty acid flux in seeds which accumulate
oleic acid derived-C18 polyunsaturated fatty acids (e.g., linoleic acid
and a-linolenic acid) (Slack et a1., 1978). In seeds such as those of
safflower, linseed, and soybean, (he polyunsaturated fatty acids
synthesized from 1"C-acetate appear to move from PC into TAG in both time
course and pulse-chase labeling experiments (Slack et a1., 1978) in a
manner similar to that described here for petroselinic acid. However,
with regard to Cu,polyunsaturated fatty acids, PC acts as a substrate for
the A32 desaturation of oleic acid and the A35 desaturation of linoleic
acid in the endoplasmic reticulum (Browse and Somerville, 1991). It is
generally believed therefore that the movement of polyunsaturated fatty
acids through PC in developing oilseeds is related to the role of this
lipid in fatty acid desaturation. In other words, in order for the cell
to maintain. a ‘pool of ‘unsaturated fatty acids for membrane lipid
synthesis, oleic acid. must. be directed first through PC prior to
incorporation into TAG. The unusual fatty acids y-linolenic acid and
:ricinoleic acid.are also metabolized.through.PC in seeds of borage (Stymne

.and Stobart, 1986; Griffiths et a1., 1988) and castor (Bafor et a1.,

 

133

1991), respectively. Like the (he polyunsaturated fatty acids, though,
these unusual fatty acids are synthesized on PC via reactions involving
oleic acid or linoleic acid. The A6 double bond of petroselinic acid,
however, does not derive from fatty acid desaturation on PC (Cahoon et
a1., 1992; Chapter 3), and no further modification of petroselinic acid
occurs on this lipid. Therefore, the apparent flux of petroselinic acid
through PC suggests that the role of PC in TAG metabolism is not limited
to its involvement in fatty acid desaturation or other modification
reactions. PC, for example, could participate in the metabolic or
physical movement of fatty acids to intracellular or intramembrane sites
of TAG synthesis. In this regard, the radiolabeled PC detected in our
studies might correspond to a pool of PC dedicated to TAG assembly rather
than to membrane biogenesis. Alternatively, because of the similarity in
structures of oleic acid.and petroselinic acid, phospholipid biosynthetic
enzymes such as CDP—choline:DAG cholinephosphotransferase may not be able
to effectively distinguish DAG moieties rich in petroselinic acid. As
such, the movement of petroselinic acid out of PC and perhaps other polar
lipids might represent an editing or retailoring activity. Such activity
has recently been demonstrated in studies by Banas et al. (1992) in which
microsomal extracts of several oilseeds were able to remove unusual
oxygenated fatty acids from PC via endogenous phospholipase activity.
It is interesting to speculate why petroselinic acid does not
accumulate to high levels in polar lipids. It has been proposed that
certain unusual fatty acids of oil seeds may be disruptive to "proper"
membrane structure and function (Stymne et a1., 1990). In this regard,
the melting point of petroselinic acid is twice as high as that of oleic
acid (30°C vs. 14°C). In addition, plants cells apparently lack the
ability to further desaturate petroselinic acid (i.e., the occurrence of
petroselinic acid-derived 18:2 or 18:3 has yet to be detected in plants).
As a result, PC molecules rich in petroselinic acid would have
significantly' higher' Tgs than. those rich in. oleic acid, and. cells

containing large amounts of petroselinic acid in PC would.be less capable

 

134

of increasing levels of membrane unsaturation in response to environmental
changes. However, coriander and carrot endosperm do contain significant
amounts of petroselinic acid in polar lipids compared to seeds rich in
other unusual fatty acids including medium-chain length fatty acids,
erucic acid, and ricinoleic acid. Because the structure of petroselinic
acid does not differ greatly from that of the common fatty acid oleic
acid, it is likely that membranes of plant cells can tolerate somewhat
higher levels of petroselinic acid than other unusual fatty acids of more
divergent structures.

Another finding of this study was the ability of coriander endosperm
to incorporate high levels of petroselinic acid in the sn-2 position of
glycerolipids, particularly PC and TAG. In this regard, Dutta et al.
(1992) have demonstrated that microsomes of carrot endosperm are capable
of incorporating petroselinoyl-CoA at the sn-2 position of lysoPA.
However, oleoyl-CoA was a much preferred substrate for this reaction. It
was also reported that, in the presence of lysoPC, microsomes of carrot
endosperm incorporate petroselinoyl-CoA more readily than oleoyl-CoA at
the sn-2 carbon (Dutta et a1. , 1992) . Therefore, our results may indicate
the activity of a lysoPA and/or lysoPC acyltransferase specialized for the
metabolism of petroselinic acid. The detection of significant amounts of
1"C-petroselinic acid in the sn-2 position of PC may also reflect the
composition of the acyl-CoA pool available for esterification at the sn-2
carbon. That is, the acyl-CoA pool may be enriched in 1"C-petroselinoyl-
CoA relative to oleoyl-CoA following acetate radiolabeling of coriander
endosperm.

The results of labeling experiments presented here provide a
somewhat unique perspective of the metabolism of an unusual fatty acid in
a developing seed. In this regard, in a proceedings report by Grobois and
Mazliak (1979) , it was noted that high levels of radiolabeled petroselinic
acid were detected in the total phospholipid fraction of seed of English
ivy (an Araliaceae species) that had been incubated in ll‘C-acetate. This

finding provides at least a partial, independent confirmation of our

 

135

results and suggests that the metabolism of petroselinic acid may be
similar in seeds of families other than the Umbelliferae which accumulate
high levels of this fatty acid.

An unanswered questions of this study is the mechanism through which
the apparent flux of petroselinic acid from PC (and perhaps other polar
lipids) to TAG occurs in developing coriander and carrot endosperm.
[“C]Acetate labelling as used in our experiments gives only an indication
of the metabolism of fatty acids. To confirm the results presented above
and to determine whether the intact glycerol backbone moves from PC to
TAG, [l,3-“C]glycerol labelling of coriander endosperm was attempted
(results not shown). However, in these preliminary studies, only low
levels of radiolabeled glycerol were incorporated into lipids, and a clear
interpretation of the movement of petroselinic acid among lipid classes
was not possible. The majority of the glycerol incorporated in these
experiments was associated with DAG. Lesser amounts were detected in TAG,
and only a small portion of the label was present in PC (data not shown).
With time, levels of 1"C-glycerol in TAG increased. This distribution
of radioactivity in glycerolipids is similar to that recently reported for
3H-glycerol labeling of Brassica napus embryos (Perry and Harwood, 1993) .
The detection of low amounts of 1‘°C-glycerol in PC was also observed by
Dutta et al. (1992) in studies of ll‘C-glycerol-3-phosphate metabolism by
carrot seed microsomes.

Another unanswered question is the relevance of our results to the
metabolism of other unusual fatty acids, especially those of more atypical
chemical structures. In particular, are these fatty acids metabolized
through PC during periods of rapid TAG synthesis? Results of
radiolabeling studies with seeds that accumulate medium-chain fatty acids
have suggested a more limited involvement of PC in the flux of these fatty
acids into TAG (Slabas et a1., 1982; Bafor et a1. , 1990). In studies with
seeds of plants (e.g., Brassicaceae sp., Limanthes sp., and Tropaeolum
majus) that synthesize high levels of very long-chain fatty acids (Zczo) ,

radiolabel associated with these acyl moieties is often detected in PC and

 

136

other polar lipids (Gurr et a1., 1974; Pollard and Stumpf, l980a,b;
Fehling et a1., 1990; thden and Frentzen, 1992; Taylor et a1., 1992).
The relative amounts of radiolabeled very long-chain fatty acids in PC and
other 'polar lipids are typically' in. excess of that determined. by
measurement of the fatty acid mass composition of these lipids. Very
long-chain fatty acids, however, are generally believed to enter TAG
directly (through reactions of the Kennedy pathway) (Fehling et a1. , 1990;
L5hden. and. Frentzen, 1992; Taylor et a1., 1992). Still, it is
interesting to speculate that a small, rapidly metabolized pool of PC
might participate in.the movement of other unusual fatty acids, including

very long chain fatty acids, into TAG.

137

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CHAPTER 5

A‘Hexadecenoic Acid is Synthesized by the Activity of a Soluble
A°Palmitoyl-Acyl Carrier Protein Desaturase in Thmbergia alata

Endosperm

Abstract

The seed oil of Thunbergia alata Bojer ex Sims is composed of more
than 80 wt% of the unusual fatty acid.AFhexadecenoic acid. Studies were
conducted to determine the biosynthetic origin of the double bond of this
fatty acid. Assays of soluble fractions of developing T. alata seed
endosperm with [1-1"C]palmitoyl (16:O)-acyl carrier protein (ACP) revealed
the presence of significant amounts of A‘5 desaturase activity. This
activity was greatest when 16:0-ACP was provided as a substrate, whereas
no desaturation of the coenzyme A ester of this fatty acid.was detected.
In addition, A°l6:0-ACP desaturase activity in T. alata endosperm
extracts was dependent on.the presence of ferredoxin.and.molecular oxygen
and.was Stimulated.by catalase. Most of the desaturase activity was lost
by the addition.of 1 mM potassium cyanide or hydrogen.peroxide to assays.
Using PCR with degenerate oligonucleotides corresponding to conserved
regions of A9stearoyl (18:0)- and A‘16:0-ACP desaturases, a nucleotide
probe encoding a portion of a diverged acyl-ACP desaturase was amplified
from a T. alata endosperm cDNA library. A full-length cDNA corresponding
to this probe was isolated. The mature peptide encoded by this cDNA.
shared.66% amino acid identity to the mature castor A918:0-ACP desaturase
and.57% amino acid identity to the mature coriander'A‘l6:0-ACP desaturase.
Extracts of Escherichia coli that express the T. alata cDNA catalyzed the
A° desaturation of 16:0-ACP. These results demonstrate that
Afhexadecenoic acid in T. alata endosperm is formed by the activity of a
soluble A°16:O-ACP desaturase that is structurally related to the A918:O-
and A‘l6:0-ACP desaturases. Implications of this work to an understanding

of active site structures of acyl-ACP desaturases are discussed.

143

144

Introduction

Fatty acid desaturases of plants have received considerable
attention because of their contributions to the physiology and economic
value of plants. ‘The activity of fatty acid.desaturases, for example, may
be a component of the ability of certain species to adjust levels of
membrane unsaturation in response to stresses such as chilling
(Cheesbrough, 1990; Wada.et.al., 1990; Miquel et a1., 1993). In addition,
the degree of fatty acid unsaturation resulting from desaturase activity
is often a major determinant of the nutritional and industrial quality of
plant seed oils (Battey et a1., 1989).

Plants typically contain a variety of fatty acid desaturases. The
most common are membrane-associated desaturases that use fatty acids bound
to glycerolipids as substrates (Browse and. Somerville, 1991). In
addition, the synthesis of oleic acid (18:1A9) in plants and certain other
organisms such as Euglena is catalyzed by a desaturase that functions on
fatty acids esterified to acyl carrier protein (ACP) (Nagai and Bloch,
1965; Nagai and Bloch, 1968; Jaworski and Stumpf, 1974). In contrast to
all previously characterized desaturases, this enzyme, the Aflstearoyl
(l8:0)- ACP'desaturase, displays soluble activity'(Nagai anthloch, 1965).
In the presence of radiolabeled 18:0-ACP and cofactors including NADPH,
ferredoxin, and.ferredoxin-NADPH reductase, the activity of the A918:0-ACP
desaturase is readily detectable in extracts of most plant tissues (McKeon
and Stumpf, 1981). Due in part to its soluble nature and relative ease
of assay, the A918:0-ACP desaturase has been purified from several plant
sources (McKeon and Stumpf, 1982; Cheesbrough and Cho, 1990; Kinney et
a1., 1990; Shanklin and Somerville, 1991; Thompson et a1., 1991), and a
number of cDNAs encoding this enzyme have been isolated (Kinney et a1.,
1990; Shanklin and Somerville, 1991; Shanklin et a1., 1991; Thompson et
a1., 1991; Nishida.et a1., 1992; Sato et a1., 1992; Slocombe et a1., 1992;
Taylor et a1., 1992). In.addition to the A918:0-ACP desaturase, a.A‘16:0-
ACP desaturase has recently been identified in plants (Cahoon et a1.,

1992; Chapter 3). This enzyme is a component of the petroselinic acid

145

(18:1A°) biosynthetic pathway in endosperm of coriander (Coriandrum
sativum L.) and other Umbelliferae species. Translation of a cDNA for the
A“l6:0-ACP desaturase has revealed that this enzyme shares extensive amino
acid identity with the A918:0-ACP desaturase (Cahoon et a1. , 1992).

The existence of structurally-related acyl-ACP desaturases with
different substrate recognition and double bond-positioning properties
offers the opportunity to characterize the active site structure of this
family of enzymes using techniques such as site-directed mutagenesis.
These studies would be aided by the isolation of cDNAs for other
functionally variant acyl-ACP desaturases in addition to those for the
A918:0- and A“l6:0-ACP desaturases. A potential source of such a
desaturase is seed of Thunbergia alata (a member of the Acanthaceae
family). The oil of this tissue consists of nearly 85 wt% of the unusual
fatty acid A°hexadecenoic acid (16:1A°) (Spencer et a1. , 1971). In this
report, we have used biochemical and molecular biological approaches to
examine whether 16:1A6 in T. alata seed endosperm is synthesized via an
acyl-ACP desaturase that is related to the A918:0- and “16:0-ACP
desaturases.

Materials and Methods
Plant Material

Studies were conducted using developing endosperm dissected from
fruits of Thunbergia alata Boj er ex Sims (black-eyed susan vine) (Northrup
King, Minneapolis, MN). Fruits were collected from plants grown either
outdoors in pots during summers in East Lansing, Michigan or under
greenhouse conditions with natural illumination. In the latter case,
flowers required extensive hand-pollination for adequate fruit set.
Endosperm was frozen in liquid nitrogen following dissection and stored
at -70°C until use in enzyme assays or RNA extraction.

Acyl-ACP Desaturation Assays

Approximately 200 mg of developing T. alata endosperm was
homogenized in 3 mL of buffer consisting of 100 mM Tris-H01 (pH 7.5) , 2.5
mM DTT, 1 mM isoascorbate, 10% (v/v) glycerol; and 1.5% (w/w)

 

146

polyvinylpolypyrrolidone (PVPP) using an Elvehjem tissue grinder. The
extract was subsequently centrifuged for 5 min at 14,000xg in order to
remove debris and PVPP. The supernatant was then passed through two
layers of miracloth (Calbiochem) and spun for an additional 10 min at
30,000xg. The soluble phase was removed while attempting to avoid
recovery of the floating fat layer. A portion of contaminating fat was
extracted by passing the supernatant through glass wool loosely packed in
a Pasteur pipet. The 30,000xg supernatant was then centrifuged at
100,000xg for 60 min. All centrifugation steps were performed at 5°C.
The resulting supernatant was used immediately for desaturation assays
described below or frozen in aliquots in liquid N2 and stored at -70°C
until further use. Of note, extensive phenolic oxidation occurred in
extracts developed a brown color, presumably due to extensive phenolic
oxidation, when maintained at —20°C for longer than one to two weeks.
Acyl-ACP desaturation assays were based on those previously
described by Jaworski and Stumpf (1974) and McKeon and Stumpf (1981).
Assays were performed in a total volume of 150 pL in loosely-capped 13 x
100 mm tubes and consisted of 1.25 mM NADPH (from a freshly prepared stock
in 100 mM Tricine, pH 8.2), 3.3 mM ascorbate, 0.7 mM DTT, 8000 U bovine
liver catalase (Sigma), 5 pg bovine serum albumin (Fraction V) (Sigma),
20 pg spinach ferredoxin (Sigma), 80 mU spinach ferredoxin:NADPH
reductase (Sigma), 33 mM Pipes (pH 6.0) and 118 pmoles of [1-1‘C]acyl-ACP
or -CoA. Reactions were started with the addition of the 100,000xg
supernatant of homogenized T. alata endosperm (typically 20 to 25 pg total
protein) and were conducted at room temperature (approx. 22°C) with
shaking (100 rpm). Assays were terminated with the addition of 850 pL of
2.35 M NaOH and carrier fatty acids (30 pg of palmitic acid and
petroselinic acid). The stopped reactions were then heated at 85°C for
l h. Following acidification with 350 pL of 4 M HZSO,” the resulting free
fatty acids were recovered by three extractions with 2.5 mL of hexane.
Fatty acids were converted to methyl ester derivatives with 10% (w/v)

boron trichloride (Alltech) using the method of Morrison and Smith (1964) .

 

147

Reaction products were then analyzed on 15% AgNO3 TLC plates developed
sequentially to heights of 10 cm and 20 cm in toluene at -20°C.
Radioactivity was detected by autoradiography and quantified by liquid
scintillation counting of TLC scrapings in 3a20 complete cocktail
(Research Products International).

To confirm the identity of 16:1A° produced from palmitoyl-ACP,
assays were conducted as described above using [U-“C]palmitoyl-ACP as the
substrate. The methyl ester derivative of the monounsaturated product was
purified by argentation TLC as described above and eluted from TLC
scrapings with hexane:ethyl ether (2:1 v/v) . The monounsaturated methyl
ester was then cleaved at its double bond using permanganate-periodate
oxidation (Christie, 1982). Chain lengths of oxidation products were
determined relative to ll‘C-fatty acid standards by reverse-phase TLC using
a mobile phase of acetonitrile:methanol:water (75:25:0.5).

Inhibition of desaturase activity was examined by supplementing
assays with 1 mM KCN (neutralized) or 1 mM H202. In the latter case,
catalase was omitted from reactions. Oxygen-dependence of desaturase
activity was characterized by purging assay tubes completely with nitrogen
prior to and after addition of plant extract, and the reaction tube was
tightly capped for the duration of the assay.

Radiolabeled acyl-ACPs were synthesized enzymatically according to
the method of Rock and Garwin (1979) using E. coli ACP. The following
fatty acids were used in the synthesis of acyl-ACPs: [lrl‘C]myristic acid
(American Radiolabeled Chemicals, St. Louis, MO) (sp. act. 55 mCi/mmol),
[l-“C]palmitic acid (New England Nuclear, Boston, MA) (sp. act. 58
mCi/mmol), [U-“C]palmitic acid (New England Nuclear) (sp. act. 800
mCi/mmol), and [l-1°C]stearic acid (American Radiolabeled Chemicals) (sp.
act. 55 mCi/mmol). [l-“C]Palmitoyl-COA (sp. act. 52 mCi/mmol) was
purchased from Amersham.

Thunbergia alata Endosperm cDNA Library Construction
Total RNA was isolated from T. alata endosperm using the method of

Hall et al. (1978) with minor modifications. The ratio of buffer to

 

148

endosperm used in tissue homogenization was 3.5 mL/g, and the buffer was
heated to 80°C prior to adding to the ground endosperm. In addition, the
homogenization buffer contained freshly added 10 mM DTT rather than 5 mM
DTT as previously described (Hall et a1. , 1978) and precipitated RNA was
washed three times with ice-cold 2 mM LiCl. Furthermore, the LiCl-washed
RNA was dissolved in 10 mM Tris-HCl (pH 7.5) and centrifuged at 12,000xg
for 20 min to remove any insoluble material. RNA was subsequently
precipitated with the addition of potassium acetate (pH 5.5) to a final
concentration of 200 mM and 2.5 volumes of ethanol. '

RNA was subsequently passed through a column of Sigma Cell 50
(Sigma) in order to reduce amounts of polysaccharides potentially
recovered along with the RNA. The column consisted of 0.25 g of Sigma
Cell 50 placed in a Pasteur pipet. The Sigma Cell 50 was then
sequentially washed three times with 1 mL aliquots of water; three times
with 1 mL aliquots of 100 mM sodium hydroxide, 5 mM EDTA; three times with
water; and five times with 1 mL aliquots of 20 mM Tris (pH 7.5), 0.5 M
sodium chloride, 1 mM EDTA, 0.1% (w/v) SDS. RNA resuspended in 1 mL of
the final column wash buffer was loaded onto the column and eluted with
5 mL of the final column wash buffer. The purified RNA was recovered
following ethanol precipitation.

Poly A+ RNA was enriched by passing total RNA once through a column
of oligo-dT cellulose (Pharmacia) and subsequently used in the
construction of a Uni-ZAP XR (Stratagene) cDNA expression library
according to the instructions of the manufacturer. A portion of the total
amplified library packaged in phage was in vivo excised as described by
Hay and Short (1992) yielding pBluescript II SK (-) harboring cDNA
inserts. The recovered plasmid DNA was used for cDNA isolation by colony
hybridization and PCR amplification as described below.

PCR Amplification of Nucleotide Sequences Encoding Acyl-ACP Desaturases

Fully-degenerate sense and antisense oligonucleotides were prepared
which corresponded respectively to the conserved amino acid sequences

GlyAspMetIleThrGluGlu and GluLysThrIleGlnTereu present in A9stearoyl-

 

149

(Shanklin and Somerville, 1991; Shanklin et a1., 1991; Nishida et a1. ,
1992; Sato et a1., 1992; Slocombe et a1., 1992; Taylor et a1., 1992) and
A‘palmitoyl-ACP desaturases (Cahoon et a1., 1992). The sequence of the

resulting sense and antisense oligonucleotides were
5'GG(A/C/G/T)GA(C/T)ATGAT(A/C/T)AC(A/C/G/T)GA(A/C)GA3' and
5 'A(A/G) (A/G)TATTG (A/G/T)AT(A/C/G/T)GT(C/T)TT(C/T)TC3 ' , respectively .

Included on the 5' terminus of each oligonucleotide was sequence
(5'CAUCAUCAUCAU3' or S'CUACUACUACUA3') that allowed for insertion of PCR
products into the pAMPl vector (GibcoBRlJ. Template for PCR amplification
was generated by transformation of the SOLR strain (Stratagene) of E. coli
with an aliquot of the in vivo excised T. alata endosperm cDNA library.
Following growth of transformed E. coli to stationary phase in a 3 mL
liquid culture, plasmid DNA was purified for use as template in PCR
amplification. Reactions were performed in a 50 pL'volume and consisted
of 10 uM sense and antisense oligonucleotides, 150 to 300 ng plasmid DNA
derived from the T. alata cDNA library, 2 mM MgClz, 0.2 mM dNTPs, 1x Taq
reaction buffer (Gibco BRL), and 5 units Taq polymerase (Gibco BRL).

Temperature conditions for PCR amplification were 5 min at 95°C and 25
cycles of 1 min at 95°C, 1.5 min at 55°C, and 1.5 min at 72°C. This was
followed by an additional 10 min extension at 72°C. PCR fragments of
approximately 215 bp were gel-purified, ligated into the pAMPl vector
using the CloneAmp system (GibcoBRL) according to the manufacturer's
protocol, and introduced into E. coli DHSa. The resulting colonies were
screened using colony hybridization as described by Sambrook et a1.
(1989). A "negative" screening protocol was used to reduce the chances
of re-isolating cDNAs (pTADl, 2, and 3) encoding A918:0—ACP desaturases
that were previously obtained by antibody screening of the T. alata
endosperm cDNA library (see "Results"). DNA probes for library screening
were formed by PCR amplification of portions of pTAD 1, 2, and.3. IPrimers
and PCR reaction conditions were the same as those described above. An
equimolar mixture of the PCR products derived from pTADl, 2, and 3 was

used as template for the synthesis of [awaiP]dCTP random-primed labeled

 

150

probes. Hybridization of plasmids of lysed colonies with radiolabeled
probes was carried out in 6x SSC and 0.05x BLOTTO with shaking for 4 h at
53°C as described by Sambrook et a1. (1989). Filters were washed three
times in 1x SSC and 0.1% SDS at 60°C for 45 min and exposed to
autoradiography. Plasmid DNA was subsequently isolated from ten of these
colonies, and nucleotide sequence was obtained by dideoxy chain
termination using Sequenase 2.0 (US Biochemical) according to the
manufacturer's instructions. Two classes of plasmids were identified
(designated pEC6 and 7), both of which contained inserts encoding for
portions of apparent acyl-ACP desaturases (based on amino acid identity
with known Agstearoyl- and Al‘palmitoyl-ACP desaturases) .
Screening of the T.a1ata Endosperm cDNA Library

Aliquots of the in vivo excised-T. alata endosperm cDNA library were
used to transform E. coli SolR cells. Approximately 50,000 of the
resulting colonies were screened using colony hybridization as previously
described (Sambrook et a1. , 1989) . Nucleotide probes for screening were
generated by [a-32P]dCTP random-primed hexamer labeling of inserts of pEC6
and 7. Hybridization and washing conditions were the same as those
described above. Colonies containing plasmid DNA that strongly hybridized
to the probe derived from pECé were isolated, and nucleotide sequence was
obtained for the longest of these cDNAs (the corresponding plasmid was
designated pTAD4) using Sequenase 2.0. Because of a relative lack of
abundance, colonies containing plasmid hybridizing to pEC7 were not
further characterized.
E. coli Expression of a Putative cDNA for A‘Palmitoyl-ACP Desaturase

To determine the activity of the desaturase encoded by pTAD4, the
portion of the clone corresponding to the mature peptide (total protein
minus plastid transit peptide) was expressed in E. coli. This region of
the cDNA insert of pTAD4 was first amplified by PCR using Vent DNA
polymerase (New England Biolabs). The nucleotide sequence of the sense
primer was 5'GCTTCGACTATTACTCAC3'. M13 (-20) primer was used as the

antisense oligonucleotide. The PCR product was blunt-end ligated into the

 

 

151

Nco I site of the E. coli expression vector pET3d (Novagen) as described
(Sambrook et a1., 1989). The NcoI-digested vector had been previously
treated with Klenow to fill-in 5' protruding ends. The junction between
the vector and the 5' terminus of the insert was sequenced.to confirm that
the PCR product was ligated into pET3d in the proper reading frame. This
construct was subsequently introduced into the E. coli strain BL21 pLysS
and grown in LB media with carbenicillin (125 ug/mL) and.chloroamphenicol
(30 pg/mL) selection. At a cell density of 0.D.“m~0.8, cultures were
induced with the addition of IPTG to a final concentration of 0.5 mM and
grown for an additional 4_h. Cells were then washed in 50 mM.Tris-HCl (pH
7.5), lysed by two freeze-thaw cycles (using a liquid nitrogen bath for
freezing and a 22°C water bath for thawing) and incubated with bovine
pancreas DNase I (Boehringer Mannheim Biochemical) (20 ng/mL) for 15 min
at 22°C. The extract was subsequently centrifuged at 14,000x g for 5
min" The resulting supernatant was used for 16:0-ACP desaturation assays
as described above. Reactions were performed using 230 pg of protein from
the E. coli extract. Radiolabel in the TLC—analyzed reaction products was
detected using a Bioscan System 200 image scanner.

The double bond. position of the monounsaturated product was
determined. by GC-MS analysis of its dimethyl disulfide derivative
(Francis, 1981). In these studies, desaturation assays described above
were scaled up six-fold, and reactions were conducted with 2.6 nmole of
[lxl‘C]l6:0-ACP and 1 mg of E. coli protein. Assays were conducted for
4 h. High protein concentrations and long incubation periods were used
to insure the synthesis of sufficient amounts of monounsaturated fatty
acid for mass spectral analyses. Reaction products were converted to
fatty acid.methyl esters as described above and subsequently reacted with
100 pL of an iodine solution (60 mg/mL.ethyl ether) and 350 uL.of dimethyl
disulfide (Aldrich). After 2 to 3 h incubation with shaking (250 rpm at
37°C), dimethyl disulfide derivatives of unsaturated fatty acid methyl
esters were extracted as previously described (Yamamoto et a1., 1991).

Dimethyl disulfide derivatives dissolved in hexane were analyzed by GC-MS

152

using a Hewlett Packard HP58901I gas chromatograph interfaced to a HP5971
mass selective detector. Separation of analytes was achieved using a DB23
(30m x 0.25 mm I.D.) column (J&W Scientific) with the oven temperature

programmed from 185°C (3 min hold) to 230°C at rate of 2.5°C/min.

Results

Demonstration of a Soluble A‘Palmitoyl-ACP Desaturase in Thunbergia alata
Endosperm Extracts

The seed oil of Thunbergia alata is composed of nearly 85 wt% of the
unusual monounsaturated fatty acid A°hexadecenoic acid (16:1A6). To
examine whether this acyl moiety is the product of a A°palmitoyl (16:0)-
ACP desaturase, the 100,000xg supernatant of a homogenate of developing
T. alata seed endosperm was incubated with 1"C-16z0-ACP and potential co-
factors including ferredoxin, NADPH, and ferredoxin-NADPH reductase.
Significant amounts of 16:0-ACP desaturase activity were detected in the
soluble endosperm extract. The 16:1 moiety produced as a result of this
desaturase activity displayed mobility on argentation TLC plates similar
to that of the A6 monounsaturated fatty acid petroselinic acid, when
analyzed as methyl ester derivatives (Figure 5.1A). In addition, the
chain lengths of the permanganate-periodate oxidation products of this
16:1 moiety were consistent with the insertion of the double bond at the
A5 carbon (Figure 5.1B).
Substrate Properties of the A‘Acyl-ACP Desaturase

To confirm that the A°desaturase identified above is most active
with 16:0-ACP, assays were conducted using 1"C-saturated acyl-ACP
substrates containing 14, 16, and 18 carbons. Under the assay conditions
used, “16:0-ACP desaturase activity in the 100,000xg supernatant of T.
alata endosperm homogenate was essentially linear over ten minutes (Figure
5.2). When assays were conducted over this time period with either [1-

ll'C]14:0- or 18:0-ACP provided as a substrate, low but detectable levels

153

Figure 5.1. Autoradiograms of intact (A) or oxidized (B) products of
[1°C]l6:0-ACP desaturation assays conducted with a 100,000xg supernatant
of Thunbergia alata endosperm homogenates. Shown (A, Desat.) is the
methyl ester derivative of 16:1 produced by desaturation activity in T.
alata endosperm extracts. Also shown (A, Desat.) is the methyl ester of
16:0 derived from unreacted [l-“C]l6:0-ACP. Fatty acid methyl esters
were resolved by argentation TLC as shown. In the standard (Std.) lane
of panel A are methyl 16:0, 18:1A9 (A9), and l8zlA° (A6). Radiolabeled
18:1A6 was produced by the incubation of coriander endosperm in [l-
1"C]acetate (see Chapter 4). In panel B are the permanganate-periodate
oxidation products of the methyl ester derivative of 16:1 formed by acyl-
ACP desaturation activity in T. alata endosperm homogenates. Oxidation

products were separated by reverse-phase TLC as shown.

 

 

154

 

 

155

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70

 

 

 

60 - -——-/1
E 50 -
‘6 4O '
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2 30 - /
Q.
g 20 -
Q.

10 -

0‘ l l l l

o 12 24 36 48 60

Time (min)

Figure 5.2. Time course of [l-“C]16:0-ACP desaturation activity in a
100,000xg supernatant of a T. alata endosperm homogenate. Assays were

conducted with 118 pmoles of [l-“C]16:0-ACP and 23 pg protein.

157

of A6 desaturase activity were measured (Table 5.1) . Values obtained with
the latter substrate, however, were obscured because of the presence of
competing A918:0-ACP desaturase activity in the endosperm extract. In
contrast to results obtainedwwith 14:0- and.l8:0-ACP, approximately 7-fold
higher levels of activity were detected when the 100,000xg supernatant was
assayed with 16:0-ACP. Because double bonds were inserted at the A6
carbon of 14:0-, 16:0-, and 18:0-ACP, it can be concluded that the A°
desaturase of T. alata endosperm positions the placement of unsaturation
with regard to the carboxyl- rather than the methyl-terminus of acyl-ACP
substrates. This double bond positioning property has been previously
observed with the A918:0- and “16:0-ACP desaturases (Cheesbrough and Cho,
1990; Chapter 3). Finally, no desaturase activity was detected.when [l-
1"C]16:0-CoA was presented as a potential substrate. Overall, these
results indicated that A6 desaturase is most active in vitro with
palmitic acid (16:0) esterified to ACP.
Catalytic Properties of A‘Palmitoyl-ACP Desaturase Activity

Virtually no “16:0-ACP desaturase activity was detected in the
100,000xg supernatant of T. alata endosperm homogenates when assays were
conducted in the absence of ferredoxin and.molecu1ar oxygen (Figure 5.3).
A616:0-ACP desaturase activity was also reduced when catalase was omitted
from assays. Furthermore, the inclusion of 1 mM KCN or H202 in reactions
resulted in the loss of most of the desaturase activity. Such catalytic
properties of the T. alata “16:0-ACP desaturase are similar to those
previously described for the A918:0-ACP desaturase (Nagai and Bloch, 1968;
Jaworski and Stumpf, 1974; McKeon and Stumpf, 1982).
Isolation of a cDNA Encoding a Diverged Acyl-ACP Desaturase from T. alata
Endosperm

In the studies described above, the A616:0-ACP desaturase appeared
t0 possess catalytic properties similar to those previously reported for
the A918:0-ACP desaturase. These included the ability to catalyze the
Soluble desaturation of acyl-ACPs and the requirement of reduced

ferredoxin for detectable activity. Based on these functional

 

158

Table 5.1. In vitro substrate specificities of' acyl-ACP or -CoA
desaturases of T. alata endosperm

Desaturase assays were conducted for 10 min.using 118 pmoles of [l-
1"C]acyl-ACP or -CoA substrate and 23 pg total protein from a 100,000xg

supernatant of a T. alata endosperm homogenate.

 

Monounsaturated Products1

 

Substrate A6 A9
pmol
14:0-ACP 3.0 n.d.2
16:0-ACP 22.7 n.d.
16:0-00A n.d. n.d.
18:0-ACP 2.8 39.8

 

1A6 and A9 isomers were identified by their mobilities on
argentation TLC plates.

2Not detected.

 

159

 

pmole product (16:1)

 

 

 

Control -Catalase +H202 +KCN -Fd +N2

Figure 5.3. Effect of catalase, ferredoxin (Fd), nitrogen (N2) , potassium
Cyanide (KCN), or hydrogen peroxide (H202) on A616:0-ACP desaturation
aetivity in T. alata endosperm extracts. Assays were conducted for 10 min
using 118 pmoles of [l-“C]l6:0—ACP and 23 pg protein from a 100,000xg
Supernatant of endosperm homogenate. Assays with potassium cyanide and
hydrogen peroxide contained 1 mM of each compound. Catalase was omitted

from assays containing hydrogen peroxide. n.d., not detected.

160

similarities, we examined whether the A°l6:0-ACP desaturase is also
structurally related to the A918:0-ACP desaturase. To address this
question, attempts were made to isolate a cDNA for the “16:0-ACP
desaturase using A918:0-ACP desaturase-derived probes. As a first
approach, a cDNA expression library prepared from polyA(+) RNA of T. alata
endosperm was screened using antibodies against the “18:0-ACP desaturase
of avocado (Shanklin and Somerville, 1991). This method was previously
used to obtain a cDNA for the “16:0-ACP desaturase of coriander endosperm
(Cahoon et a1. , 1992) . In the present study, however, antibody screening
of the T. alata endosperm expression library yielded only cDNAs for three
apparent isoforms of the A918:0-ACP desaturase, which were designated
pTADl, 2, and 3 (see Appendix 2).

As an alternative approach, PCR amplification of a A616:0-ACP
desaturase-specific nucleotide probe was attempted using degenerate sense
and anti-sense oligonucleotides prepared against two conserved regions of
amino acid sequences of A918:0- and “16:0-ACP desaturases. One of the
regions GlyAspMetIleThrGluGlu is encoded by the cDNA for the “16:0-ACP
desaturase and all known cDNAs for the A918:0-ACP desaturase. The second
region IleGluGlnThrIleTereu is also encoded by the A‘16:0-ACP desaturase
cDNA and all known “18:0-ACP desaturase cDNAs except that of safflower
(Thompson et a1., 1991). Products of approximately 200 bp obtained
following one round of PCR amplification of the total T. alata cDNA
library (in plasmid form) were screened after subcloning into the pAMPl
vector. To delineate products of the previously isolated cDNAs pTADl, 2,
and 3, colonies containing PCR-derived clones were screened in a
"negative" manner with random-labeled probes for pTADl, 2, and 3 and
conditions of moderate to high stringency. One of the resulting clones
(pEC6) which displayed weak or no hybridization to the probes encoded an
amino acid sequence that was somewhat diverged from those of known A918:0-
ACP desaturases.

When the T. alata endosperm library was screened with a random-

labeled probe prepared from pEC6, >0.1% of the total cDNAs examined

 

161

strongly hybridized to this probe. The longest of a selected portion of
these cDNAs (designated pTAD4) contained 1279 bp and had an open-reading
frame encoding a 387-amino acid peptide with considerable identity to
known. A°l6:0- and A918:0-ACP desaturases (Figure 5.4). Based on
similarity of flanking bases to the consensus sequence proposed by Lfitcke
et a1. (1987), the translational start site of the cDNA insert of pTAD4
likely occurs at nucleotide 17. In addition, from homology with A‘l6:0-
and A918:0-ACP desaturases, the mature peptide encoded by pTAD4 likely
begins at amino acid 33. As such, the 32 amino acids preceding this
residue correspond to a putative plastid transit peptide as is present in
all acyl-ACP desaturases characterized to date. Interestingly, the cDNA
insert of pTAD4 lacks nucleotide sequence for six to seven amino acids
found near the amino-terminus of all previously characterized A918z0-ACP
desaturases. This region is also altered in the cDNA for the coriander
“16:0-ACP desaturase (Cahoon et a1., 1992) as compared to cDNAs for
A918:0-ACP desaturases. In this case, the encoding sequence for 15 amino
acids is absent in the “16:0-ACP desaturase cDNA relative to the castor
9A18:0-ACP desaturase cDNA (Shanklin and Somerville, 1991). The pTAD4-
encoded peptide also contains two less amino acids at its carboxyl-
terminus than.both the A“l6:0- and.A9l8:0-ACP desaturases. Despite these
differences, the interior regions of the putative desaturase encoded by
pTAD4 share significant identity with portions of the primary structures
of A“16:0- and A918:0-ACP desaturases, and the spacing between conserved
regions of amino acids is the same in all three desaturase types.
Overall, the mature peptide encoded by the cDNA insert of pTAD4 has 67%
identity to the castor A918:0-ACP desaturase and 57% identity to the
coriander A“l6:0-ACP desaturase, disregarding any missing amino acids.
Activity of an.E. coli-Expressed cDNA for a Diverged Acyl-ACP Desaturase
0f T. alata Endosperm

To determine the activity of the desaturase corresponding to the
cDNAinsert of pTAD4, the mature peptide-encoding region of this clone was

Placed behind the T7 polymerase promoter of the pET3d vector (Novagen) and

 

162

Figure 5.4. Nucleotide sequence of the cDNA insert of pTAD4 (A6), and
a.comparison of the deduced amino acid sequences of pTAD4 (A6) and cDNAs
for the coriander A“16:0-ACP desaturase (A‘) (Cahoon et a1., 1992) and
the castor A918z0-ACP desaturase (A9) (Shanklin and Somerville, 1991).
Identical amino acids are indicated by colons. Amino acids that are
absent relative to the castor A?18:0-ACP desaturase are indicated by
dashed lines. Alignment of the nucleotide sequence of the cDNA insert
of pTAD4 is maintained with a dotted line. The underlined alanine at

amino acid 33 is the likely start of the mature-peptide encoded by pTAD4.

 

178
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164

expressed in E. coli. When assayed with [1-1"C]l6:0-ACP, crude extracts
of IPTG-induced recombinant E. coli catalyzed the synthesis of 1"C-16zl
(Figure 5.5) . In addition, the methyl ester of the 16:1 product displayed
mobility on argentation TLC plates similar to that of a methyl
petroselinic acid standard, suggesting that this monounsaturated product
is a A°isomer. Furthermore, like the activity found in T. alata endosperm
extracts, the desaturase expressed in E. coli displayed a substrate
preference in in vitro assays for 16:0-ACP rather than 14:0- or 18:0—ACP
(data not shown). No acyl-ACP desaturase activity was detected in
extracts of E. coli harboring the pET3d vector without cDNA insert or in
uninduced recombinant E. coli (Figure 5.5).

The 1"C-16zl moiety produced in vitro from the E. coli-expressed
desaturase was conclusively identified as a A6 isomer through GC-MS
analysis of its dimethyl disulfide derivative (Figure 5.6). Significant
amounts of non-radiolabeled or 12C-16zlA° were also detected among the
desaturase assay products. This was indicated by the presence of the
additional ions 143, 175 and 362 m/z in the mass spectrum of 1"C-16:1A° as
well as by an enrichment in the abundance of ion 187 m/z in this spectrum.
The unlabeled 16:1A6 likely did not result from in vivo synthesis by
E.coli. In this regard, E. coli does not normally produce 16:1A6 (Cronan
and Rock, 1987) . Furthermore, gas chromatographic analysis of fatty acids
of E. coli expressing the pTAD4-encoded desaturase failed to detect any
16:1A° in the bacterial lipids (data not shown). However, given the
relatively high concentrations of E. coli protein used in these assays,
unlabeled 16:1A6 likely arose from the in vitro desaturation of endogenous
E. coli 16:0-ACP present in crude extracts.

Of note, expression levels of the T. alata cDNA in E. coli appeared
to be low relative to expression levels often obtained with cDNAs placed
behind the T7 polymerase promoter (Studier et a1. , 1990). The expressed
Protein, for example, could not be distinguished on Coomassie-stained SDS-
POlyacrylamide gels of either the total soluble or insoluble protein

fractions of the lysed E. coli (data not shown).

 

165

pET3d 160

M

 

g T. alata cDNA (uninduced)

8 16:0

0..

(D

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a: W
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W
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Figure 5.5. Comparison of the A516:0-ACP desaturase activity of extracts
of E. coli BL21 pLysS containing only the vector pET3d or pET3d with
insert derived from the T. alata cDNA of pTAD4 (with or without IPTG
induction). The methyl ester of 16:1 formed by the E. coli expressed
desaturase was separated from methyl 16:0 of the unreacted substrate by
argentation TLC as shown. Radiation on TLC plates was detected using a
Bioscan 200 image scanner. Assays were conducted for 60 min.using 230 pg
0f E. coli protein and 118 pmoles of [1-1l‘C]16:0-ACP. Shown in the
Standard chromatogram (Std.) are methyl l"C-16z0, -l8:lA9, and -18:lA°.

 

166

 

 

 

 

 

 

 

 

 

 

100; \

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‘1 1

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50 100 1 50 200 250 300 350
m/z

Figure 5.6. Mass spectrum of derivatives of [1-1"C]16:l formed by 16:0-
ACP desaturase activity in extracts of E. coli expressing the mature
peptide-encoding region of pTAD4. The [l-“C]16:l desaturation product

was converted to a methyl ester derivative and reacted with dimethyl

disulfide prior to mass spectral analysis.

 

 

167

Discussion

The results presented here demonstrate the involvement of a soluble
A61620-ACP desaturase in the synthesis of A°hexadecenoic acid in the
endosperm of Thunbergia alata. The activity of this enzyme has several
properties similar to those previously described for the A918:0-ACP
desaturase. These include the requirement of reduced ferredoxin for
detectable in vitro activity, the stimulation of activity by catalase, and
the inhibition of activity by potassium cyanide and hydrogen peroxide.
In addition, a cDNA encoding an acyl-ACP desaturase that shares
considerable amino acid identity with the A918z0-ACP desaturase was
isolated from a cDNA library prepared from polyA(+) RNA of T. alata
endosperm. Extracts of E. coli that express this cDNA catalyzed the A6
desaturation of 16:0-ACP. These data thus indicate that the “16:0-ACP
desaturase is a functional and structural analog of the A918:0-ACP
desaturase.

The major difference between the primary structures of the mature
4616:0- , A“16:0- , and 4918:0-ACP desaturases occurs in a region near their
amino termini. In this region, the T. alata “16:0-ACP desaturase
contains six less amino acids than the castor A918:0-ACP desaturase.
Similarly, this portion of the coriander “16:0-ACP desaturase lacks 15
less amino acids relative to the castor 4918:0-ACP desaturase. One
possibility is that differences in substrate recognition (16:0-ACP vs.
18:0-ACP) and double bond positioning properties of these desaturases are
associated with this divergence in their primary structures. For example,
as a result of amino acids that are missing relative to A918:0-ACP
desaturases, the binding pocket of the A" and “16:0-ACP desaturases may
be smaller and active site residues involved in double bond insertion may
be positioned closer to the carboxyl end of acyl chains. Alternatively,
this amino-terminal region of the A918:0-ACP desaturase may not contribute
significantly to the catalytic properties of this enzyme. As such, if

the A“ and A616:0-ACP desaturases evolved from the A918:0-ACP desaturase,

 

168

then there may have been little selective pressure to maintain this region
in the variant 16:0-ACP desaturases.

Ultimately, an understanding of how differences in the amino acid
sequences of A‘16:0-, A616:0-, and A91820-ACP desaturases contribute to
variations in their functional properties will require comparisons of the
three-dimensional structures of these enzymes. In this regard,
elucidation of the crystal structure of the castor “18:0-ACP desaturase
is currently in progress (Schneider et a1. , 1992) . With such information,
it may be possible to overlap amino acid sequences of the A“ and 4516:0-
ACP desaturases onto the three-dimensional structure of “18:0-ACP
desaturase to more precisely identify residues associated with the
different substrate recognition and double bond positioning properties of
these enzymes. This could eventually lead to the design of "tailor-made"
desaturases that are capable of inserting double bonds into a variety of
positions of acyl moieties of a range of carbon chain lengths.

An interesting observation from the studies described above was the
lack of detectable amounts of 16:1A° in lipids of E. coli expressing the
T. alata cDNA. Similarly, Thompson et a1. (1991) reported that expression
of the safflower A918:0-ACP desaturase cDNA did not lead to the in vivo
production of oleic acid in recombinant E. coli. The latter result can
be explained by the fact that E. coli contains little 18:0-ACP (Rock and
Jackowski, 1982; Ohlrogge, unpublished data). However, 16:0-ACP is a
major component of the acyl-ACP pool of E. coli (Ohlrogge, unpublished
data). Therefore, the lack of 16:1A° synthesis in E. coli expressing the
T. alata cDNA is not due to the presence of insufficient substrate for the
desaturase. In addition, E. coli has been reported to contain ferredoxin
(Knoell and Knappe, 1974) , the apparent electron donor for the “16:0-ACP
desaturase. However, as proposed by Thompson et a1. (1991), E. coli
ferredoxin may not be functional with plant acyl-ACP desaturases.
Alternatively, E. coli may not contain adequate amounts of ferredoxin in

a reduced form as required for “16:0-ACP desaturase activity.

 

169

In addition to 16:1A6, T. alata seed contains the unusual fatty acid
18:1A8, which composes about 2 wt% of the oil of this tissue (Spencer et
a1, 1971). We have previously shown that petroselinic acid (182146) is
formed by elongation of 16:1A"-ACP in Umbelliferae endosperm (Chapter 3) .
In an analogous manner, we would predict that 18:148 arises from the
elongation of 16:1A6-ACP rather than from the A8 desaturation of 18:0-ACP.
Unlike the synthesis of petroselinic acid, though, elongation of 16:1-ACP
in T. alata endosperm would likely not be a major pathway as the ratio of
amounts of 16:1A°:18:1A8 in this tissue is approximately 40:1. In
contrast, the ratio of amounts of 16:1A":18:1A6 in endosperm of the
Umbelliferae coriander is more than 1:500 (Chapter 3).

Finally, significant efforts have been directed toward the
development of transgenic crops that produce high-value specialty oils
(Murphy, 1992) . In this regard, the physical and industrial properties
of 16:1A6 have received little study. However, like petroselinic acid,
16:1A6 can be oxidatively cleaved at its double bond to yield adipic acid,
which is used in the synthesis of nylon 6,6. In addition, high palmitic
acid (16:0) mutants of crop plants including soybean (Bubeck and Fehr,
1989) and Brassica campesteris (Perrsson, 1985) exist that may serve as
appropriate backgrounds for the transgenic expression of the cDNA for the
T. alata “16:0-ACP desaturase. Still, the success of such research
would likely require additional studies to determine whether enzymes other
than “16:0-ACP desaturase are specialized for the synthesis and
metabolism of 16:1A° in T. alata endosperm. For example, a petroselinoyl-
ACP specific thioesterase has been identified in Umbelliferae endosperm
extracts that efficiently releases this fatty acid following its synthesis
on ACP (Dormann et a1. , 1994) . A related-enzyme may also be required for

high levels of A°hexadecenoic acid production in transgenic plants.

 

170

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Browse J, Somerville C (1991) Glycerolipid synthesis: Biochemistry and
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Bubeck DM, Fehr WR, Hammond EG (1989) Inheritance of palmitic and stearic

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Cahoon EB, Shanklin J, Ohlrogge JB (1992) Expression of a coriander
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Proc Natl Acad Sci USA 89: 11184-11188

Cheesbrough TM (1990) Decreased growth temperature increases soybean
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559

Cheesbrough TH, Cho SH (1990) Purification and characterisation of
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Cronan Jr JE, Rock CO (1987) Biosynthesis of membrane lipids. In FC
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DSrmann P, Frentzen M, Ohlrogge JB (1994) Specificities of acyl-acyl
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Jaworski JG (1987) Biosynthesis of monoenoic and polyenoic fatty acids.
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Kinney AJ, Hitz WD, Yadav NS (1990) Stearoyl-ACP desaturase and a B-
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Portland Press, London, p 126-128

 

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Perrson C (1985) High palmitic acid content in summer turnip rape
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CHAPTER 6

Conclusions and Future Research Perspectives

Conclusions

Studies presented in this thesis have resulted in the identification
of two "new" acyl-acyl carrier protein (ACP) desaturases in plants: (1)
a.A‘palmitoyl (l6:0)-ACP desaturase that is associated with the synthesis
of petroselinic acid in Umbelliferae endosperm and (2) a “16:0-ACP
desaturase that is involved in the synthesis of A°hexadecenoic acid (16:1)
in.Thunbergia alata endosperm” cDNAs encoding both desaturases were also
isolated. Translation of these cDNAs revealed that the primary structures
of the A‘ and A51620-ACP desaturases are related to that of the
Agstearoyl-ACP desaturase, the enzyme that catalyses the synthesis of
oleic acid in plants. This finding indicates that it may be possible to
manipulate the amino acid sequences of acyl-ACP desaturases to produce
functional enzymes with altered substrate recognition and double bond
positioning properties.

Work described in this thesis has also led to the proposal of a
biosynthetic pathway for petroselinic acid in Umbelliferae endosperm as
shown in Figure 6.1. This pathway involves the conversion of 16:0-ACP to
16:1A4-ACP via the activity of the A‘16:0-ACP desaturase followed by two
carbon elongation to form petroselinoyl-ACP. Essential to an
understanding of the biosynthesis of petroselinic acid were results
obtained from.the transformation of tobacco with a cDNA for the coriander
A‘16:0-ACP desaturase. Though the resulting transgenic callus expressed
the desaturase in an active form, these cells apparently lacked other
enzymes specialized for the synthesis of petroselinic acid” As a result,
the transgenic tobacco callus, in contrast to Umbelliferae endosperm,
accumulated significant amounts of 16:14‘ as well as petroselinic acid in
glycerolipids. These fatty acids were also found to be major components

of acyl-ACP pools of transgenic tobacco cells. This detection of 16:1A‘

175

 

176

Proposed Pathway for Petroselinic Acid Biosynthesis

16:0-ACP

A4 Palmitoyl-ACP
Desaturase

16:1A4-ACP

B-Keto-AcyI-ACP
Synthase

18nAQACP

PetroselinoyI-ACP
Thioesterase

1831A6 + ACP

Figure 6.1. Proposed pathway for petroselinic acid biosynthesis. Studies
described in Chapters 2 and 3 have provided substantial evidence for the
involvement of a “16:0-ACP desaturase in the synthesis of petroselinic
acid in Umbelliferae endosperm. Dormann et a1. (1994) have also
demonstrated that Umbelliferae endosperm contain a petroselinoyl-ACP-
specific thioesterase. Furthermore, results of preliminary research
indicate that the elongation of 16:1A"-ACP is a specialized process in the
Synthesis of petroselinic acid in Umbelliferae endosperm (Cahoon,

unpublished data) .

 

177

in transgenic tobacco provided. a perspective of petroselinic acid
synthesis that could not have been obtained from studies of Umbelliferae
endosperm. These results indicate that it is possible to produce fatty
acids of unusual structure in transgenic plant cells. Moreover, these
results demonstrate the potential usefulness of transgenic plant material
in the elucidation of biochemical pathways associated with unusual fatty
acid synthesis.

Finally, through the use of [14”C]acetate radiolabeling, it was
shown that petroselinic acid readily enters phosphatidylcholine (PC) of
carrot and coriander endosperm yet does not accumulate to high levels in
this lipid relative to triacylglycerol (TAG). This result indicates that
an unusual fatty acid can be metabolized through PC in a developing seed.
As petroselinic acid is neither synthesized nor further modified on PC,
this finding also suggests that flux of fatty acids into PC in seeds is
not necessarily related. to the role of this lipid in fatty acid
modification reactions such as desaturation.

Future Research Perspectives
Acyl-ACP Desaturase Structural Studies

One area in Which.the work.described.in.this thesis can be continued
is the characterization of active site structures of acyl-ACP desaturases.
Now available are cDNAs for three acyl-ACP desaturases with related
primary structures but with different substrate binding and double bond
positioning properties. Also available are amino acid sequences derived
from at least 10 cDNAs encoding A918:0-ACP desaturases of a wide range of
plant species (Kinney et a1., 1990; Shanklin and Somerville, 1991;
Shanklin et a1., 1991; Thompson et a1., 1991; Nishida et a1., 1992; Sato
et a1., 1992; Slocombe et a1., 1992; Taylor et a1., 1992; Appendix 2).
In addition, efforts to determine the three-dimensional crystal structure
of the castor A918:0-ACP desaturase are at an advanced stage (Schnieder
et a1., 1992). With this cumulative information, it is (or will be)
possible to make predictions about specific amino acids or regions of

amino acids that may contribute to the catalytic and substrate binding

 

178

properties of acyl-ACP desaturases. Based on such predictions, it may be
possible to change the primary structure of a given acyl-ACP desaturase
through techniques such as site—directed mutagenesis to produce a "new"
desaturase that inserts a double bond at a different position or
recognizes a substrate of a different chain length. A similar approach
has been successful in the modification of enzymes including trypsin
(Craik et a1., 1985), substilisin (Wells et a1., 1987), lactate
dehydrogenase (Wilks et a1., 1988), and lipoxygenase (Sloane et a1.,
1991) . Not only would protein engineering studies of acyl-ACP desaturases
provide new information regarding structure-function properties of these
enzymes but might also result in new renewable sources of industrially
valuable chemicals. For example, if an acyl-ACP desaturase were designed
that could introduce a double bond at a position beyond the A9 carbon, the
resulting fatty acid might be useful in long-chain nylon production. As
a result, the introduction of such a desaturase into an existing oilseed
crop could potentially lead to the development of a new high-value
industrial oil.

From the amino acid sequences of the A“ and “16:0-ACP desaturase
presented in this thesis, the most obvious starting point for mutagenesis
is the region near the N-termini of these enzymes. Both desaturases
contain deletions of amino acids in this region relative to the A918z0-ACP
desaturase. One possibility is that these deletions reduce the size of
the binding pocket of 16:0-ACP desaturases so that they more effectively
accommodate C16 rather than C18 acyl chains. These deletions might also
position catalytic residues involved in double bond insertion closer to
the carboxyl end of acyl chains. As a result, unsaturation may be
introduced at the A“ or A6 carbon rather than at the more distant A9
carbon.

Of the desaturases identified in this thesis, the Thunbergia alata
A61620-ACP desaturase represents a useful tool in mutagenesis studies.
Like the “18:0-ACP desaturase, this enzyme displays soluble activity and

can be expressed in E. coli in an active form. Therefore, the nucleotide

 

179

sequence of the cDNA for the A616:0-ACP desaturase could be altered and
then introduced into E. coli. Subsequent assay of extracts of the
recombinant E. coli could then be performed to determine whether
manipulations of the primary structure of the desaturase lead to
alterations in its activity. In addition, large amounts of the A°l6:0-ACP
desaturase could potentially be generated through expression in E. coli
or other systems and used for X-ray crystallographic determination of its
three-dimensional structure. Comparisons of the crystal structure of this
enzyme to that of the A918:0-ACP desaturase would allow a more direct
assessment of the basis for the different substrate recognition and double
bond positioning properties of these enzymes. In addition, the parameters
that have been devised for crystallization of the castor A918:0-ACP
desaturase (Schneider et a1., 1992) might also be useful for similar
studies with the A616:0-ACP desaturase.
Development of Transgenic Crops that Produce Petroselinic Acid and
A‘Hexadecenoic Acid

As described in Chapter 1, oils that contain petroselinic acid and
A°hexadecenoic acid are of economic value. In this regard, with cDNAs for
the A“ and A516:0-ACP desaturases, it should be possible, in theory, to
develop transgenic crops that accumulate petroselinic acid or
A°hexadecenoic acid in their seed oil. Results presented in Chapter 2
partially support such an assertion. In these studies, tobacco callus
expressing the coriander A‘l6:0-ACP desaturase behind the CaMV 358
promoter synthesized petroselinic acid as well as 16:1A" to levels of 4
wt% each
of the total fatty acid. However, not reported in this thesis, amounts
of petroselinic acid in tissues, including seeds, of tobacco plants
derived from this callus were generally 51 wt% of the total fatty acid.
Based on further biochemical studies, it is believed that other enzymes
in addition to the desaturase are specialized for the synthesis of
petroselinic acid in Umbelliferae endosperm. The absence of these enzymes

in tobacco likely limits the transgenic production of petroselinic acid.

 

180

For example, a petroselinoyl-ACP-specific thioesterase has been identified
in Umbelliferae endosperm that is required for the efficient release of
petroselinic acid from the reactions of de novo fatty acid synthesis
(DSrmann et a1. , 1994) . Furthermore, preliminary investigations indicate
that the elongation of l6:lA"-ACP is also a specialized process in
Umbelliferae endosperm (unpublished data). Therefore, it may be necessary
to express genes encoding such enzymes along with the gene for the A‘16:0-
ACP desaturase in transgenic crops to produce high levels of this fatty
acid. Furthermore, acyltransferases, acyl-CoA synthetases, and lipases
may also be specialized for the metabolism of petroselinic acid in
Umbelliferae endosperm.

No work has yet been initiated with regard to the expression of
Thunbergia alata A616:0-ACP desaturase cDNA in transgenic plants. As with
the synthesis of petroselinic acid, specialized enzymes other than the
desaturase may be needed for high levels of A°hexadecenoic acid production
in a transgenic oilseed. One such enzyme, for example, might be a
A°hexadecenoyl-ACP-specific thioesterase. However, high palmitic acid
mutants of crop plants and Arabidopsis exist that may provide good genetic
backgrounds for initial studies involving the transgenic expression of the
T. alata A616:0-ACP desaturase cDNA (Perrson, 1985; Bubeck, 1989; James
and Dooner, 1990).

An additional consideration is the possibility that naturally
occurring plants that produce unusual monounsaturated fatty acids,
particularly petroselinic acid, could be developed into oilseed crops.
Many Umbelliferae species are grown commercially as vegetable and spice
crops and for essential oils. In addition, significant efforts have been
initiated to establish coriander as an agronomic oilseed crop for the
production of petroselinic acid (Anonymous, 1991). The major limitation
is the relatively low oil content of its seed (18 wt%). It is possible
that Umbelliferae species such as coriander could be developed as
agronomic sources of petroselinic acid-rich oils through conventional

Plant breeding (Anonymous, 1991) . Such efforts represent an alternative

 

181

to duplicating entire pathways for petroselinic acid synthesis and
metabolism in transgenic plants. Regardless, one must also consider the
large amount of basic knowledge that could be gained by characterizing
enzymes and corresponding genes specialized for these pathways. Like the
acyl-ACP desaturases, variant acyl-ACP thioesterases, B-keto-acyl-ACP
synthases, and acyltransferases may also be present in Umbelliferae and
Thunbergia alata endosperm. These enzymes could serve as comparative
biochemical tools in understanding active-site structures of fatty acid
and lipid biosynthetic enzymes. Such information may be important for
precisely modifying various enzymes as attempts are made to produce fatty
acids of more divergent structure in transgenic plants.
Further Characterization of Petroselinic Acid Metabolism

Finally, radiolabeling studies presented in Chapter 4 indicated that
petroselinic acid may be metabolized through PC during periods of rapid
triacylglycerol (TAG) production in Umbelliferae endosperm. However, no
details of' the 'mechanism involved. in, this process were 'presented.
Therefore, additional radiolabeling studies are required to determine
whether, for instance, the intact glycerol backbone moves from PC into
triacylglycerol or whether PC is edited or retailored by the activity of
phospholipases such as those recently described by Banas et a1. (1992).
More general questions that need to be addressed are what role, if any,
does PC have in the metabolic or physical movement of fatty acids into TAG
in developing seeds , and are the results obtained regarding the metabolism

of petroselinic acid applicable to other unusual fatty acids?

 

182

Literature Cited

Anonymous (1991) European researchers develop oil sources. INFORM 2: 690-
691

Banas A, Johansson I, Stymne S (1992) Plant microsomal phospholipases
exhibit preference for phosphatidylcholine with oxygenated acyl groups.
Plant Sci 84: 137-144

Bubeck DM, Fehr WR, Hammond EG (1989) Inheritance of palmitic and stearic

acid mutants of soybean. Crop Sci 29: 652-656

Craik.CS, Largman C, Fletcher T, Roczniak S, Barr PJ, Fletterick:R4 Rutter

WJ (1985) Redesigning trypsin: Alteration of substrate specificity.
Science 228: 291-297

D3rmann P, Frentzen M, Ohlrogge JB (1994) Specificities of acyl-acyl
carrier protein (ACP) thioesterase and glycerol-3—phosphate
acyltransferase for octadecenoyl—ACP isomers: Identification. of a

petroselinoyl-ACP thioesterase in Umbelliferae. Plant Physiol (in press)

James Jr DW, Dooner MK (1990) Isolation of EMS-induced mutants in

Arabidopsis altered in seed fatty acid composition. Theor Appl Genet 80:
241-245

Kinney AJ, Hitz WD, Yadav NS (1990) Stearoyl—ACP desaturase and a 8-
ketoacyl-ACP synthetase from developing soybean seeds. In PJ Quinn, JL
Harwood, eds, Plant Lipid. Biochemistry, Structure and 'Utilization.

Portland Press, London, p 1267128

 

183

Nishida I, Beppu T, Matsuo T, Murata N (1992) Nucleotide sequence of a
cDNA encoding a precursor to stearoyl-(acyl-carrier-protein) desaturase

from spinach, Spinacia oleracea. Plant Mol Biol 19: 711-713

Perrson C (1985) High palmitic acid content in summer turnip rape
(Brassica campestris var. annua L.) . Cruciferae Newslett 10, Scottish Crop

Research Institute, Invergowerie, Dundee, p 137

Sato A, Becker CK, Knauf VC (1992) Nucleotide sequence of’a complimentary
DNA clone encoding stearoyl-acyl carrier protein desaturase from

Simmondsia chinensis. Plant Physiol 99: 362-363

Schneider G, lindqvist Y, Shanklin J, Somerville C (1992) Preliminary

crystallographic data for stearoyl-acyl carrier protein desaturase from

castor seed. J Mol Biol 225: 561-564

Shanklin J, Somerville C (1991) Stearoyl-acyl carrier protein desaturase
from higher plants is structurally unrelated to the animal and fungal

homologs. Proc Natl Acad Sci USA 88: 2510-2514

Shanklin J, Mullins C, Somerville C (1991) Sequence of a complementary DNA
from. Cucumis sativus L. encoding the stearoyl-acyl-carrier protein

desaturase. Plant Physiol 97: 467-468

Sloane DL, Leung R, Craik CS, Sigal E (1991) A primary determinant for

lipoxygenase positional specificity. Nature 354: 149-152

Slocombe SP, Cummins I, Jarvis RP, Murphy DJ (1992) Nucleotide sequence
and temporal regulation of a seed-specific Brassica napus cDNA encoding

a stearoyl-acyl carrier protein (ACP) desaturase. Plant.Mol Biol 20: 151-
155

 

 

184

Taylor MA, Smith SB, Davies HV, Burch LR (1992) The primary structure of
a cDNA clone of the stearoyl-acyl carrier protein desaturase gene from

potato (Solanum tuberosum L.). Plant Physiol 100: 533-534

Thompson GA, Scherer DE, Foxall-Van Aken 8, Kenny JW, Young HL, Shintani
DK, Kridl JC, Knauf V6 (1991) Primary structures of the precursor and
mature forms of stearoyl-acyl carrier protein desaturase from safflower

embryos and requirement of ferredoxin for enzyme activity. Proc Natl Acad

Sci USA 88: 2578-2582

Wells JA, Cunningham BC, Graycar TP, Estell DA (1987) Recruitment of
substrate-specificity properties from one enzyme into a related one by

protein engineering. Proc Natl Acad Sci 84: 5167-5171

Wilks HM, Hart KW, Feeney R, Dunn CR, Muirhead H, Chia WN, Barstow DA,
Atkinson T, Clarke AR, Holbrook JJ (1988) A specific, highly active malate

dehydrogenase by redesign of a lactate dehydrogenase framework. Science

242: 1541-1544

 

Appendix 1

GC-MS Identification of 015 and C18 Monounsaturated Fatty Acids of

Coriander and Thunbergia alata Endosperm

Seed oils of coriander and Thunbergia alata contain a number of
other monounsaturated fatty acids in addition to petroselinic acid or A6
hexadecenoic acid. As shown below, GC-MS analysis of derivatives of fatty
acid methyl esters was used to conclusively identify the C16 and Cu
monounsaturated fatty acids of endosperm of these plants.

Derivatives were prepared by reaction of fatty acid methyl esters
with dimethyl disulfide in the presence of iodine. This is a relatively
new technique that is superior to many of the older methods for the
preparation of monounsaturated fatty acids for GC-MS analysis (Francis,
1981). In this regard, the synthesis of dimethyl disulfide (or
thiomethyl) derivatives is not technically difficult and can be performed
in one step. In addition, dimethyl disulfide reacts directly with double
bonds. As a result, the mass spectra obtained from these derivatives
contain, in addition to a molecular ion, at least three abundant ions
diagnostic for the position of the double bond. These ions include those
of fragments corresponding to portions of the molecule on either side of
the double bond and an ion formed by the loss of methanol from the
fragment containing the carboxyl end of the fatty acid (this ion
corresponds to Y-32 in the spectra shown below). Many previous methods
of GC-MS analysis of double bond position have used derivatives prepared
by reaction of the carboxyl group of fatty acids with reagents such as
pyrrolidine (Andersson and Holman, 1974; Christie, 1982). In contrast to
mass spectra of dimethyl disulfide adducts, diagnostic ions of these
"indirect" derivatives are typically of lower abundance and, as a result,
assigning the exact location of a double bond of a fatty acid can be
somewhat ambiguous.

Of the monounsaturated fatty acids identified below, “hexadecenoic

185

 

186

acid (16:1A“) had been not previously detected in lipids of Umbelliferae
seeds. As described in Chapters 2 and 3, the presence of this fatty acid
in transgenic tobacco expressing the A“l6:0-ACP desaturase cDNA provided
an essential clue in determining the biosynthetic pathway of'petroselinic
acid. The detection of 16:1A“ in lipids of coriander endosperm provides
further support for the pathway proposed in Chapter 3. Amounts of this
fatty acid in the total lipid extract of coriander endosperm were 50.1
wt%, but somewhat higher levels (50.5 wt%) were detected in
phosphatidylcholine of this tissue. The other two 16:1 isomers (16:1A‘
and A6) identified in extracts of coriander endosperm were present in
levels of 0.1 to 0.2 wt% of the total fatty acid. cis-Vaccenic acid
(18:1A11) composed between 0.9 to 1.6 wt% of fatty acids of the detectable
glycerolipid classes of coriander endosperm. Amounts of petroselinic acid
(18:1A°) and oleic acid (18:1A9) in glycerolipids of coriander endosperm
are reported in Chapter 4.

Fatty acid compositions, including 016 and C18 monounsaturated
isomers, of glycerolipids of developing,T. alata endosperm is provided in
Table Appendix 1.1. The unusual monounsaturated isomers A°hexadecenoic
acid (16:1A6) and.Asoctadecenoic acid (18:1A8) were previously identified
ixllfi alata seed extracts by Spencer et a1. (1971) usinggmass spectrometry
of ozonolysis products.

Dimethyl disulfide derivatives were prepared from fatty acid methyl
esters of phospholipids or neutral lipids of coriander and T. alata
endosperm and analyzed. by GC-MS using, methods and instrumentation

described in Chapter 5.

 

187

Literature Cited

Andersson BA, Holman RT (1974) Pyrrolidides for mass spectrometric
determination of the position.of the double bond in monounsaturated fatty

acids. Lipids 9: 185-190

Christie WW (1982) Lipid Analysis, 2nd Edition. Pergammon Press, Oxford

Francis CW (1981) Alkylthiolation for the determination of double-bond

position in unsaturated fatty acid esters. Chem Phys Lipids 29: 369-374

Spencer CF, Kleiman R, Miller RW, Earle FR (1971) Occurrence of cis-6-
hexadecenoic acid as the major component of Thunbergia alata seed oil.

Lipids 6: 712-714

 

188

Figure Appendix 1.1. Mass spectra of thiomethyl adducts of 016 and 013

monounsaturated fatty acid standards (A and B) and C16 and C18

monounsaturated fatty acids of coriander (C-H) and Thunbergia alata (I-L)

endosperm glycerolipids.

189

(A) 16:1A9 Standard

 

“b398838% 2,,
. 217 1€F1Z§

. CH

100000- 1, 3 MW 362

l .
CHCH co CH
145 I ( 2)? 2 3
s
61 185

J 8797 362

 

 

 

 

 

 

I 119
4 J 235 283 331
..l_l J ‘1

M[Z -> 50 100 150 200 250 300 350

 

 

 

 

(B) 18:146 Standard

 

Abundance
3 143 215
450005

‘ 17$ .
40000 -‘ x I CH 18.1A6
3 MW 390

j I
35000; I T

3 cuamaz), ocu ——CH(CH2)4COZCH3
300001 175 § I

25000; Y

2w

zoooof

 

15000{ 61
10000{ 67

127

97 390

 

sooofi
. 199

I
' l
0‘ r1 irT U fif‘r I '1 I T f—lififi 1

I
M Z -> 50 100 150 200 250 300 350

 

 

 

263 311
I

 

 

 

 

190

Coriander Endosperm C1,5 and C1,3 Monounsaturated Fatty Acids

(C) l6:lA" (Phospholipid fraction)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

bundance
215
60000- x|147 16:12?
(EH3 MW 362
. | S
I
50000 1 CH 3(CH 2)1OCH —————CH(CH2)ZCOZCH 3
I
s l
' l
40000~ 147 CH3 ‘
‘ 215 Y
30000;
1 115
J
20000-
‘ 87
. 61%?
10000—
- 362
I I w 174 m 235 265 299314 1
0" [11 1.1 I I] ‘I’ 1 111 I k Y I Yjfi ‘I I I I
M/Z -> 50 100 150 200 250 300 350
(D) 1621A6 (Phospholipid fraction)
abundance
E 143
45001
I 187 6
4000-: X ”5 16:1A
: .CHS MW 362
J
35001 ,
3000{ §
. I
: CH
25004 175 3 y
3 107
2000i
15001 6
3 £7 95
l 127
1000 -
362
500?
1 I
o 1
I r I ' I T l 'a 1 I I I Ifi I U I I I I I U I D
[Z -> 50 100 150 200 250 300 350

 

 

 

 

191

(E) 16:1A9 (Phospholipid fraction)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

bundance
‘ 217
. m7 . 9
500001 x I CH 16.141
‘ . 3 Mw 362
s
I 145 I c
40000 _ CH3(CH2)5pM CH(CH2)7COZCH3
. s l
1 I
4 Y
30000- ‘45
20000~ 61 185
1
. 87
10000: 69 168 362
109
1 235 315331
0 “ I l I 1 I 1 I t l I 11 l
M/z -> 50 100 150 200 250 300 350
(F) 18:146 (Phospholipid fraction)
bundance
3 143
450001
I 175 , 6
3 g 3 Mw 390
35000: I a
. CH CH _____.
: 3( 240C311 CH(CH2)40020H3
30000§ § I
: 175 CH3
25000 1 215 I Y
200005
15000{
1 127
10000:
3 97
5000{ 390
3 199 245253 L
o J U i ii] Tl I I 1 l Ufi‘ Y I I I 7 fl |
M z ~9' 50 100 150 200 250 300 350

 

 

 

192

(G) 18:149 (Phospholipid fraction)

 

Abundance

70000 “7 181i?

MVV 390
60000

I
173 CH3(CH2)7?H-—-——CH(CH2)7COZCH3

50000 S I
40000 I
30000

20000
137

 

95
10000
149 310

1E7 Ifl 245263 293
o . I t L P fi 1 ll 1 I
T ‘Ifi T 1 j I U U T j Y

M/Z -> 50 100 150 200 250 300 350

 

 

 

 

 

 

 

 

(H) 18:1A11 (Phospholipid fraction)

 

bundance
14000- 245

120001 x 2“ 18an‘
T 145 I :“3 Mw 390
1
213 CH CH CH—————-
3( 2),.“ I CH(CH2)90020H3
s
‘ I
I CH8
3000- us Y

1

100005

60001

4

61

 

4ooo§
97
69

 

 

 

 

 

135 165 196 I

 

 

 

‘ I 1
Z -> 50 100 150 200 250 300 350

 

193

Thunbergia alata Endosperm C16 and C1,3 Monounsaturated Fatty Acids

(I) 16: 1A6 (Triacylglycerol)

 

Abundance
I 14

.I

1
120000-
1

I3

1000001

800005
- 175

600005

1

61 127

 

400005
83

200003 97

 

0..
->

 

 

50 100

 

[Z

187

 

 

16:1A6
MW 362

l
CH(CH2)4COZCH3

 

(J) 18:1A8 (Phosphatidylcholine)

 

%bundance

1

8000:

4

70005

1
60005

1

50005
. 171

40005

 

1
30005
: 55
20005

J

154

95 123

10005

 

 

 

 

0.1

2¢3

 

203
X

Iv

CH3(CH2)8CH
I
- s
I
CH3
137

C

13:148

”3 MW 390

I
CH(CH2)GCOZCH 3

 

—> 150

 

50 100

12

 

 

 

(K) 18:1A9 (Phosphatidylcholine)

194

 

Abundance

160005

140005
120005
100005

80005
i
6000-

1
1

40005

4

I
2000:

 

0.1
"’>

 

M/Z

69

61

50

98

100

137

173

 

 

217

 

18:1A9
MMISQO

I
CH(CH2)7COZCH3

 

(L) 18:1A11 (Phosphatidylcholine)

 

Abundance

.4

60005

50003

4

40001

q

30005

2000

 

 

55

50

74

96

100

123

145

 

173

213

 

24

I5

 

X

CH3(CH2)5CH
s
I
CH3

I
l
I

245

18:1A11
‘EHS Mw 390
s

I
——CH(CH2)90020H 3

Y

390

 

 

 

 

 

195

Euommopao\moaoa:4

4444 6:4 :244444 .onaa 46664664.

 

N.Hw m.~ « m.n~ o.H « m.oN m.o a m.¢ «.0 « m.H
H.ow ¢.o « n.~ m.H « n.oH m.o « m.m m.o « m.m

 

m.o « H.om «.0 « n.m Am.wmv om
H.m « «.mm m.H « «.ma Am.nav 64a

 

4.04 4.o 4 4.4 4.6 4 4.4 N.o 4 «.4 ~.o 4 4.0 4.4 4 «.64 4.o 4 4.4 nxoeav 649
wHoa
.46446 4444 444444 444444 o444 444444 o444 44440 64644
640< manna

 

.Aalav mm « wHoa mm couaommum Cum mfiowuwmomaoo Cwom %uumm .¢ Hoummno

CH Confluence mcoHUHpCoo owsamuwCUMEoufio mafia: teamwuamsv mtwom %uuwm Cam CCHMHHGQ one: mCHmHHoHoo%HU

 

Appendix 2

cDNAs for Apparent Isoforms of the APStearoyl-ACP Desaturase

in Thunbergia alata Endosperm

As described in Chapter 5, screening of a Thunbergia alata endosperm
cDNA library with antibodies against the avocado A918:0-ACP desaturase
(Shanklin and Somerville, 1991) lead to the isolation of three cDNAs
encoding A918:0-ACP desaturases (designated pTADl, pTAD2, and pTAD3).
pTADl and.2 were initially obtained by antibody screening of the library,
and pTAD3 was isolated by screening with a random labeled probe derived
from pTAD2. Shown below are the nucleotide sequences of these cDNAs as
well as the corresponding amino acid sequences (Figures Appendix 2.1, 2,
and 3). From comparisons with previously isolated.A918:0-ACP desaturases
cDNAs, pTAD2 and 3 appear to contain open reading frames for complete
transit and mature peptides. The cDNA insert of pTADl, however, lacks
coding sequence for the plastid transit peptide and at least two amino
acids of the mature peptide. The amino acid sequences encoded by the
inserts of pTAD2 and 3 share 87% identity, whereas amino acid sequences
corresponding to cDNAs of pTADl and 2 Share 78% identity (Figure Appendix
2.4).

The double bond positioning properties of desaturases encoded by
pTADl, 2, and.3 were determined by in vitro assay of recombinant protein.
cDNAs were expressed.in E. coli using the pET3a'vector (Novagen). Inserts
were prepared by PCR.amp1ification.of the mature peptide-encoding regions
of cDNAs. In the case of pTADl, the sense PCR primer contained additional
sequence for an alanine and serine residue. Extracts of E. coli
expressing each of the cDNAs catalyzed the A9 desaturation of [1-
1"C]stearoyl (l8:0)-ACP (data not shown).

Overall, the isolation of three A918z0-ACP desaturase cDNAs from a
library derived from polyA(+) RNA of T. alata endosperm suggests that
isoforms of this enzyme can exist in a given species and in a single

tissue.

196

 

197

Literature Cited
Shanklin J, Somerville C (1991) Stearoyl-acyl carrier protein desaturase
from higher plants is structurally unrelated to the animal and fungal

homologs. Proc Natl Acad Sci USA 88: 2510-2514

198

pTADI ACGAGAACTATTCTAAGGGAGGTGGAAACTGCCAATAAACATTTTACTCCCCCCAGCGAGGTTCATGTTCAAGTGACGCACTCA 84
PTAD1 T R T I L R E V E T A N K H F T P P S E V H V 0 V T H S 28

pTAD1 ATGCCCCCACAGAAAATTGAGATATTCAAATCCTTGGAAGACTGGGCTGCAGATAATATATTAGTTTACCTTAAGCCTGTTGAA 168
pTAD1 H P P 0 K I E I F K S L E D W A A D N I L V Y L K P V E 56

pTADI AAATGTTGGCAACCTCAGGATTTCCTTCCAGATCCAGCATCTGATGGTTTTCATGATCAGGTCAAGGAATTGAGAGAAAGAGCC 252
pTADI K C U Q P 0 D F L P D P A S D G F H D 0 V K E L R E R A 84

pTAD1 AGGGAGATACCAGATGATTATTTTGTTGTTCTAGTTGGTGATATGATCACAGAAGAAGCTCTTCCAACATATCAGACAATGTTG 336
pTAD1 R E I P D D Y F V V L V G D H I T E E A L P T Y 0 T N L 112

pTAD1 GATACCTTAGATGGTGTGCGGGATGAAACAGGAGCGAGCCCAACTCCTTGGGCAATTTGGACAAGGGCATGGACTGCTGAAGAA 420
pTAD1 D T L D G V R D E T G A S P T P W A I W T R A W T A E E 140

pTAD1 AATAGGCATGGAGACCTTCTTAATAAATATCTTTATCTTTCTGGGAGAGTGGATATGAGACAGATTGAGAAGACTATTCAGTAT 504
pTADI N R H G D L L N K Y L Y L S G R V D M R Q I E K T I 0 Y 168

pTAD1 CTAATCGGCTCTGGAATGGACCCAAGGACAGAAAACAGTCCATACCTTGGATTTATATACACATCCTTCCAAGAAAGGGCTACT 588
pTADI L I G S G H D P R T E N S P Y L G F I Y T S F 0 E R A T 196

 

pTAD1 TTCATCTCCCATGGAAACACTGCCCGGCTCGCCAGACAACATGGGGACAATAAGCTAGCCCAAATCTGTGGTACAATTGCCTCA 672
pTAD1 F I S H G N T A R L A R O H G D N K L A Q I C G T I A S 224

pTAD1 GATGAGAAGCGTCATGAAACTGCATACACCAAGATAGTGGAGAAGCTATTCGAGATTGACCCTGATGGAACAATGCTATCTTTG 756
pTADI D E K R H E T A Y T K I V E K L F E I D P D G T M L S L 252

pTADI GCCGACATGATGAAGAAGAAGGTCTCTATGCCAGCCCACTTGATGTATGATGGCCATGACGAAAACCTCTTTGAGAACTTCTCA 840
pTADI A D N M K K K V S M P A H L H Y D G H D E N L F E N F S 280

pTADI GCTGTTGCTCAGCGACTTGGTGTTTACACAGCCAAAGACTATGCTGACATCCTAGAACATTTGATTGCCAGATGGAAAGTGTCA 924
pTAD1 A V A Q R L G V Y T A K D Y A D I L E H L I A R H K V S 308

pTAD1 GATTTGACCGGGCTATCAGGGGAAGGTCAGAAAGCTCAGGACTATGTGTGTCGGCTGCCACCAAGAATCAGAAAACTGGAGGAG 1008
pTAD1 D L T G L S G E G 0 K A 0 D Y V C R L P P R I R K L E E 336

pTAD1 AGAGCTCAAGCTCGAATAAAAGAAGGACCAAGAATCCCATTTAGCTGGATATACAATAGAGAGTTGCTACTATAGATTTGATGT 1092
pTAD1 R A Q A R I K E G P R I P F S H I Y N R E L L L * 360

pTAD1 GTTCTCTCTGGGCAGCCGTTGCTCACCTGTAGGTGAAAAGCTCAAGGAAATTTGCATTGTAGGTTGTTTGCAGCTTAAAATTAT 1176
pTADI GTTGAGAATGAAGAATGCTGGTGCAAGTGTTACTGTGTCGTGTCATTGTGTTCTAATATTTTTTCGTAGCTCTCAGGACTGTAA 1260

pTAD1 AAACTGTCAGAACGTTGTAAAAGGATTTTGGTGATGATTGCTGTGTTTTGCACTTTGACCAACAAAAAAAAAAAAAAAAAAAA 1343

Figure Appendix 2.1. Nucleotide and deduced amino acid sequences of the

cDNA insert of pTAD1.

 

 

 

199

pTAD2 CACAAAACCTCATTTTTCTGAAGAAATATAGACGAAATGGCGCTGAAATGTAGCGTAACCCCCCACCAGGTGCCTTCTTTTCCT 84
PTAD2 M A L K C S V T P H 0 V P S F P 16

pTAD2 GTTAATCAGCTCAGATCTCACCGAGTTTACATGGCTTCAACTCTCGATTCGGCATCCGCTAATGTTGGGAAAGGTAAAAAGGCT 168
pTAD2 V N 0 L R S H R V Y M A S T L D S A S A N V G K G K K A 44

pTADZ TTCACCCCCCCTCGAGAAGTCAAGGTTCAACTGACGCATCCCATGGCTCCAGAAAAGCGCGAGATCTTCCATTCACTGCACGGT 252
pTAD2 F T P P R E V K V 0 L T H P H A P E K R E I F H S L H G 72

pTAD2 TGGGCGGAAGAAAACCTTCTGTCTCTCTTGAAGCCTGTTGAGAAGTGTTGGCAGCCCAACGACTTTCTTCCCGACCCTTCTTCA 336
pTAD2 H A E E N L L S L L K P V E K C N O P N D F L P D P S S 100

pTAD2 GAAGGCTTTGATGAACAGGTCAGGGAGCTTCGGCTAAGAACCAAGGAACTACCCGATGAATACTTTGTTGTGTTGGTTGGCGAC 420
pTAD2 E G F D E O V R E L R L R T K E L P D E Y F V V L V G D 128

pTADZ ATGATCACGGAAGAAGCTCTCCCTACTTATCAGACGATGATCAACACTTTAGATGCAGTCCGTGATGAGACCGGTGCAAGCCTT 504
pTAD2 M I T E E A L P T Y Q T H I N T L D A V R D E T G A S L 156

pTAD2 ACTCCTTGGGCTATTTGGACTAGAGCATGGACTGCGGAAGAGAATAGGCACGGTGATCTTCTCAACAAATATCTTTACCTTTCG 588
pTAD2 T P U A I U T R A N T A E E N R H G D L L N K Y L Y L S 184

pTAD2 GGACGCGTGGACATGAGGCAAATTGAGAAGACGATTCAATACCTCATCGGTTCAGGAATGGATCCTCGCACGGATAACAACCCG 672
pTADZ G R V D H R Q I E K T I 0 Y L I G S G N D P R T D N N P 212

pTAD2 TACCTCGGATTCATCTACACCTCGTTCCAGGAGAGGGCGACATTCATTTCTCATGGGAACACGGCTAGGCTTGCCAAGGAACAC 756
pTAD2 Y L G F I Y T S F G E R A T F I S H G N T A R L A K E H 240

pTAD2 GGGGACCTTAAACTGGCACAGATATGCGGCAGCATAGCTGCAGATGAGAAACGGCACGAAACTGCTTACACCAAAATTATCGAA 840
pTAD2 G D L K L A 0 I C G S I A A D E K R H E T A Y T K I I E 268

pTAD2 AAGCTGTTTGAGATCGACCCTGATGGGACAGTACTTGCTCTAGCCGATATGATGAGGAAGAAAGTGTCTATGCCGGCACATTTG 924
pTAD2 K L F E I D P D G T V L A L A D H M R K K V S M P A H L 296

pTAD2 ATGTATGATGGACAAGATGAAAATCTGTTTGAACACTTTGCAGCTGTGGCGCAACGCATTGGAGTGTACACTGCGAAAGACTAT 1008
pTADZ M Y D G 0 D E N L F E H F A A V A 0 R I G V Y T A K D Y 324

pTAD2 GCTGATATACTGGAATTTTTAGTTGGGAGATGGGAGGTGGAGAAACTAACAGGACTTTCAGGGGAGGGTCGTAACGCGCAGGAG 1092
pTAD2 A D I L E F L V G R H E V E K L T G L S G E G R N A O E 352

pTAD2 TACGTGTGCGGGTTGGCTCCTCGGATCAGAAGGTTGGAAGAGAGAGCACAGGCACGGGCAAAGCAGGGGGCACCTGTGCCCTTC 1176
pTAD2 Y V C G L A P R I R R L E E R A O A R A K O G A P V P F 380

pTADZ TCCTGGGTTTATGGTCGAGAAGTTAAAATTTGAACCGTCGGACGGATTCCGAGTTAGTTTGCTTCTGTGAACTCCTCTTTTCAG 1260
pTAD2 S U V Y G R E V K I * 390

pTAD2 CTTTGCTATTAGCAAGAAAATTTTTATCAAGTTTATGTGTTTTTAAAATATATAATATCAATTGTTTTTGGGTTTTAAAAAAAA 1344

pTADZ AAAAAAAAAAA 1355

Figure Appendix 2.2. Nucleotide and deduced amino acid sequences of the
cDNA inserts of pTAD2. The underlined amino acid corresponds to the

likely transit peptide cleavage site.

200

pTAD3 TTTTTCTGGTGTCGATTTGAAGAAATATAGAGGAAATGGCGCTTAAATTTAGCATTACGCCCCACAAGATGCCTTCCTTCCCT 83
PTAD3 M A L K F S I T P H K M P S F P 16

pTAD3 GATTTCCAGCTCAGATCTCACCGGGTTTGCATGACTTCAACTCTCTATTCTGCATCCGTTGAGGTCGGCAATGGTAAAAAGCCT 167
pTAD3 D F 0 L R S H R V C M I S T L Y S A S V E V G N G K K P 44

pTAD3 TTCAGTCCCCCTCGAGAAGTCAATATTCAAGTGACACATCCCATGCCTCCAGAAAAGCGCGAGATCTTCAACTCATTGCATGGA 251
pTAD3 F S P P R E V N I 0 V T H P H P P E K R E I F N S L H G 72

pTAD3 TGGGCGGAAACTAATCTTCTGTCTCTCTTGAAGCCTGTTGACAAGTGTTGGCAGCCCAGTGACTTTCTACCCGACCCTTCCGCA 335
pTAD3 U A E T N L L S L L L P V D K C U 0 P S D F L P D P S A 100

pTAD3 GATGGCTTTGACGAGCAGGTCAGAGAGTTACGGAAAAGAACCAAGGAACTACCCGATGAATATTTTATTGTGTTGATTGGTGAC 419
pTAD3 D G F D E 0 V R E L R K R T K E L P D E Y F I V L I G D 128

pTAD3 ATGATTACGGAGGAAGCTCTCCCAACTTATCAGACTATGATCAACACGCTTGATGCAGTCCGGGATGAGACTGGTGCAAGCCTT 503
pTAD3 M I T E E A L P T Y Q T M I N T L D A V R D E T G A S L 156

pTAD3 ACTCCTTGGGCTATTTGGAATAGAGCATGGACTGCTGAAGAGAATAGGCATGGTGATCTTCTCAACAAATATCTCTACCTTTCG 587
pTAD3 T P N A I U N R A U T A E E N R H G D L L N K Y L Y L S 184

pTAD3 GGACGTGTGGACATGAAGCAAGTCGAAAAGACAATTCAGTACCTGATTGGTTCAGGAATGGACCCTCATACTGATAACAACCCG 671
pTAD3 G R V D H K O V E K T I 0 Y L I G S G M D P H T D N N P 212

pTAD3 TACCTTGGATTCATCTACACATCGTTTCAGGAGAGAGCAACATTCATTTCTCATGGGAACACGGCTAGGCTTGCCAAGGAACAC 755
pTAD3 Y L G F I Y T S F O E R A T F I S H G N T A R L A K E H 240

pTAD3 GGTGATATGAAACTGGCACAGATTTGTGGTACCATCGCTGCAGATGAGAAACGTCATGAAACTGCCTACACAAAAATTATTGAA 839
pTAD3 G D N K L A Q I C G T I A A D E K R H E T A Y T K I I E 268

pTAD3 AAGCTGTTTCAACTGGACCCTGATGGGACAATACTCGCTCTAGCTGACATGATGCGGAAGAAAGTGTCGATGCCTGCCCACCTG 923
pTAD3 K L F 0 L D P D G T I L A L A D H M R K K V S M P A H L 296

pTAD3 ATGTTTGATGGGAAAGATCAAAATCTATTTGAACACTTCTCTGCTGTGGCGCAACGTATTGGAGTGTACACTGCCAAGGACTAT 1007
pTAD3 H F D G K D O N L F E H F S A V A 0 R I G V Y T A K D Y 324

pTAD3 GCTGATATATTGGAATATCTAGTGGCGAGATGGGAGGTGGAGAAGCTGACAGGGCTTACAGGAGAGGGGCGTAAAGCGCAAGAG 1091
pTAD3 A D I L E Y L V A R U E V E K L T G L T G E G R K A Q E 352

pTAD3 TATGTGTGTGGCTTGGCTCCGAGGATCAGAAGGTTGGATGAGAGAGCACAGGCACGTGCAAAGGAAGCAGCGCCTGTGCCCTTT 1175
pTAD3 Y V C G L A P R I .R R L D E R A Q A R A K E A A P V P F 380

pTAD3 ACATGGATTTTTGGCCGAGAAGTTCGTCTCTAGTAATACTATTGAATGGTCAAATAAAACATGCAGCAAGGATGTGGAAACGGG 1259
pTA03 T u I F c R E v R L * 39o

pTAD3 CTATTAGGTCGATTTTGCCATAAGCCTATTTACTTGAAGAAAACCTTAAATTATTAAAAGGATTGCTGTTGAGGCCTAAAAATT 1343
pTAD3 GCAATACATCAAAGACACATATTCACCAACATTATTAATGTATTGTGTTTTTGTGAGGTTATGAAGTACTCATTTATTAAAAAA 1427

pTAD3 AAAAAAAAAAAA 1439

Figure Appendix 2.3. Nucleotide and deduced amino acid sequences of the
cDNA insert of pTAD3. The underlined amino acid corresponds to the likely

transit peptide cleavage site.

201

pTAD2 MALKCSVTPHQVPSFPVNQLRSHRVYMASTLDSASANVGKGKKAF 45
pTAD1 TRTILRE:ETAN:H: 15
pTAD3 ::::F:I:::KM::::DF:::::::C:l:::Y:::VE::N:::P: 45
pTAD2 TPPREVKVQLTHPMAPEKREIFHSLHGWAEENLLSLLKPVEKCWQ 90
pTAD1 :::S::H::V::S:P:Q:I:::K::ED::AD:I:VY::::::::: 60
pTAD3 S::z::NI:V::::P:::::::N::::::: :::::::::D:::: 90
pTADZ PNDFLPDPSSEGFDEQVRELRLRTKELPDEYFVVLVGDMITEEAL 135
pTAD1 :Q::::::A:D::HD::K::: :AR:I::D::::::::::::::: 105
pTAD3 :S:::::::AD::z:::::::K::::::::::I::I::::::::: 135
pTAD2 PTYQTMINTLDAVRDETGASLTPWAIWTRAWTAEENRHGDLLNKY 180
pTADl ::::::LD:::G::::::::P:::::::::::::::::::::::: 150
pTAD3 :::::::::::::::::::::::::::N::::::::::::::::: 180
pTAD2 LYLSGRVDMRQIEKTIQYLIGSGMDPRTDNNPYLGFIYTSFQERA 225
pTAD1 ::::::::::::::::::::::::::::E:S:::::::::::::: 195
pTADB :::::::::K::::::::::::::::H:::::::::::::::::: 225
pTAD2 TFISHGNTARLAKEHGDLKLAQICGSIAADEKRHETAYTKIIEKL 270
pTADl :z::::::::::RQ:::N:::::::T::S::::::::::::V::: 240
pTAD3 :::::::::::::::::::::::::T::::::::::::::::::: 270
pTADZ FEIDPDGTVLALADMMRKKVSMPAHLMYDGQDENLFEHFAAVAQR 315
pTAD1 ::::::::M:S:::::K:::::::::::::H::::::N:S::::: 285
pTAD3 :QL:::::I::::::::::::::::::F::K:Q::::::S::::: 315
pTAD2 IGVYTAKDYADILEFLVGRWEVEKLTGLSGEGRNAQEYVCGLAPR 360
pTAD1 L:::::::::::::H:IA::K:SD::::::::QK::D:::R:P:: 330
pTAD3 z:::::::::::::Y::A::::::::::T::::K::::::::::: 360
pTADZ IRRLEERAQARAKQGAPVPFSWVYGREVKI* 390
pTAD1 ::K::::::::I:E:PRI::::I:N::LLL* 360
pTAD3 :::: ::::::::EA:::::T:IF::::RL* 390

 

Figure Appendix 2.4. Comparison of amino acid sequences encoded by cDNA

inserts of pTAD1, 2, and 3. As shown, the primary structures of
polypeptides corresponding to pTAD1 and 3 are compared to that of pTAD2.
Colons indicate identical amino acids, and the underlined amino acids in

the pTAD2 and 3 sequences represent likely transit peptide cleavage sites.

   

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