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This is to certify that the
dissertation entitled

BIOCHEMICAL AND PHYSIOLOGICAL STUDIES ON PLANT
OXYLIPINS

presented by
Anthony Louis Schilmiller

has been accepted towards fulfillment
of the requirements for the

Doctoral degree in Biochemistry and Molecular
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BIOCHEMICAL AND PHYSIOLOGICAL STUDIES ON PLANT OXYLIPINS
By

Anthony Louis Schilmiller

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Biochemistry and Molecular Biology

2005

ABSTRACT
BIOCHEMICAL AND PHYSIOLOGICAL STUDIES ON PLANT OXYLIPINS
By

Anthony Louis Schilmiller

Oxylipins comprise a diverse group of biologically active molecules that arise
from oxidative metabolism of polyunsaturated fatty acids, and have been implicated in
regulating diverse aspects of plant growth and development. Most studies of plant
oxylipins have focused on the hormone jasmonic acid (JA), which is a key regulator of
plant responses to insect attack, and recently has been implicated as a mobile signal for
activation of long distance defense responses.

The synthesis of oxylipins begins with lipoxygenase (LOX) catalyzed production
of hydroperoxy fatty acids, which are subsequently metabolized by the CYP74 family of
cytochromes P450. Different CYP74 enzymes direct the hydroperoxides into different
oxylipin biosynthetic routes. Part of this dissertation focuses on the characterization of a
novel C YP74 gene that was identified by searching the tomato expressed sequence tag
database. Biochemical analysis of the recombinant enzyme showed that it is an allene
oxide synthase (LeAOS3) that metabolizes both 9- and 13-hydroperoxides of linoleic and
linolenic acid. However, the increased specificity of LeAOS3 for 9-hydroperoxides
indicated that this enzyme functions in the 9-LOX pathway in tomato. LeAOS3
expression in roots was shown to be highly inducible by JA-treatment and wounding.
cDNA microarray experiments were performed to identify genes that are co-regulated

with LeAOSB in JA-treated roots. In contrast to roots, expression of LeAOS3 in

hypocotyls of germinating seedlings was constitutive and independent of JA.
Immunolocalization of LeAOS3 in roots and hypocotyl showed expression in cortex cell
layers. The dual modes of LeAOS3 regulation are discussed in the context of the function
of the 9-LOX pathway.

Pioneering studies in the 19805 demonstrated the involvement of B-oxidation in
JA biosynthesis. Until now, however, no genes encoding B-oxidative enzymes that are
required for the production of JA have been identified. The second half of this
dissertation focuses on the role in JA biosynthesis of acyl-COA oxidases (ACX), which
catalyze the first step of B-oxidation. Forward genetic analysis in tomato indicated that a
specific ACX isoform (ACXIA) was required for JA biosynthesis. Biochemical analysis
of recombinant ACXlA showed that this isoform has the catalytic capacity to oxidize
CoA-esters of JA biosynthetic intermediates. Arabidopsis thaliana mutants that are
defective in the orthologs of ACX 1A were isolated and used to study the role of ACX in
herbivore and pathogen defense. These results showed that JA is required for defense
against chewing insects, whereas cyclic intermediates are sufficient for defense against a
necrotrophic pathogen. The characterized Arabidopsis ACX mutants will be useful for
future studies aimed at further dissection of the signaling properties of JA and its cyclic

intermediates.

ACKNOWLEDGEMENTS

When I visited MSU to interview for grad school, my flight was delayed and got
into Lansing sometime around 2:00 AM in the morning. But there was Dr. Gregg Howe
at the Lansing airport ready to give me a ride to the hotel. From the very beginning and
over the years as a graduate student, Gregg has been a great advisor and mentor, and I
would like to thank him for all his guidance and support. I truly feel that I made the best
decision in joining his lab, and I look forward to continue collaborating with Gregg
during my time as a post-doc in Dr. Robert Last’s lab here at MSU.

I would also like to thank my guidance committee members: Dr. Christoph
Benning, Dr. Dean DellaPenna, Dr. Michael Garavito, and Dr. John Ohlrogge. They have
been an excellent source of help, especially for my projects involving fatty acid
metabolism. I would also like to acknowledge the other members of the Howe lab for
helpful discussions along the way, especially Aya Itoh for her tremendous help in getting
me started when I was a rotation student. There are many others throughout the PRL who
have helped in some way as well. I believe that the PRL is one of the best places for
graduate students to learn plant science, and I hope it stays this way for many years to
come.

Lastly I must thank my family who has supported me in my many (at least
according to them) years as a student. I only wish MSU was a little closer to home so I

could go plow a field or split some wood to relieve the stress sometimes.

iv

TABLE OF CONTENTS

LIST OF TABLES ................................................................................. ix
LIST OF FIGURES ................................................................................ x
CHAPTER 1
Introduction: Oxylipin Biosynthesis and Function ......................................... 1
I. Oxylipin biosynthesis and function .......................................................... 2
Lipoxygenase ................................................................................. 4
Other hydroperoxide-forming activities .................................................. 5
Hydroperoxide-metabolizing enzymes ................................................... 8
Lipoxygenase ........................................................................... 8
Epoxy alcohol synthase ............................................................... 9
Peroxygenase ........................................................................ 10
Reductase ............................................................................. 10
C YP74 cytochromes P450 ......................................................... 11
Hydroperoxide lyase ............................................................ 1 1
Divinyl ether synthase .......................................................... 13
Allene oxide synthase ........................................................... 15
II. Jasmonate biosynthesis .................................................................. 17
Chloroplast-localized reactions ........................................................ 21
Peroxisome-localized reactions ........................................................ 23
Metabolism of JA ........................................................................ 24
III. Physiological roles of jasmonates ..................................................... 25

Role of JAs in defense .................................................................. 26

Role of JAs in development ............................................................. 29
References .................................................................................... 30
CHAPTER 2
Identification and Biochemical Characterization of a New Tomato Allene
Oxide Synthase ............................................................................... 42
Abstract ................................................................................... 43
Introduction ............................................................................... 44
Materials and Methods .................................................................. 48
Results .................................................................................... 51
Identification of a new member of the C YP74 family of
cytochromes P450 ................................................................... 51
Biochemical properties of LeA 0S3 ............................................... 56
Tissue specific expression of LeA 0S3 in roots ................................. 66
Discussion ................................................................................. 68
References ................................................................................ 72
CHAPTER 3
Regulation of Allene Oxide Synthase3 Expression in Tomato ........................ 76
Abstract ................................................................................... 77
Introduction ............................................................................... 78
Materials and Methods .................................................................. 82
Results .................................................................................... 87

Wound-induced expression of LeAOS3 in roots requires the
JA signaling pathway ................................................................ 87

vi

Expression of LeA 0S3 in hypocotyls of germinating seedlings
is independent of C011 .............................................................. 92

LeA 053 protein accumulates in cortex cells of root tips and hypocotyls. . ..96

MeJA-induced gene expression in tomato roots ................................. 98
Discussion ............................................................................... 1 13
References ............................................................................... l 16

CHAPTER 4
Biochemical Analysis of a Tomato Acyl-CoA Oxidase Required for Wound-
Induced J asmonic Acid Biosynthesis ..................................................... 119
Abstract ................................................................................... 120
Introduction .............................................................................. 121
Materials and Methods .................................................................. 124
Results ..................................................................................... 129

The wound response phenotype of acx] results from a defect in ACX . . .....129

ACX 1A is required for JA biosynthesis ........................................... 138
Developmental expression of A CX 1A in tomato ................................ 138
Involvement of A CX 1A in ,B-oxidation of other substrates .................... 142
Discussion ................................................................................ 145
References ................................................................................ 1 5 1
CHAPTER 5

Acyl-COA Oxidases that Function in the B-Oxidation Stage of J asmonic Acid
Biosynthesis are Essential for Insect Resistance and Pollen Development

in Arabidopsis ............................................................................... 156
Abstract ................................................................................. 1 5 7
Introduction ............................................................................. 1 5 8

vii

Materials and Methods ............................................................... 163

Results .................................................................................. 170
ACX] and ACX5 catalyze wound-induced JA biosynthesis in
Arabidopsis leaves ............................................................... 170
acx1/5 plants are more susceptible to T richoplusia ni feeding
but maintain resistance to infection by Alternaria brassicicola ............ 176
acxl/5 plants are impaired in male fertility ................................... 179-
Discussion .............................................................................. 185
References .............................................................................. 1 90
CHAPTER 6
Conclusions and Future Directions ...................................................... 195

viii

LIST OF TABLES

Table 2.1. Analysis of LeAOS3 reaction products by GC-MS ........................... 62
Table 2.2. Substrate specificity of recombinant LeAOS3 ................................. 65
Table 3.1. MeJA-regulated genes in tomato roots ......................................... 103
Table 5.1. Reduction of seed-containing siliques in acx1/5 .............................. 181

ix

LIST OF FIGURES

Figure 1.1. Biosynthesis of oxylipins in response to environmental and
developmental cues ................................................................................... 3

Figure 1.2. Lipoxygenase-catalyzed production of hydroperoxy fatty acids

from linoleic and linolenic acids ................................................................... 4
Figure 1.3. ot-Dioxygenase pathway ............................................................... 6
Figure 1.4. Type I and Type II phytoprostanes synthesized from linolenic acid . . . . ....7
Figure 1.5. LOX-catalyzed production of ketodienes .......................................... 8
Figure 1.6. Epoxy alcohol synthase-catalyzed oxylipins ....................................... 9

Figure 1.7. Hydroperoxide lyase-catalyzed cleavage of fatty acid hydroperoxides ...... 12

Figure 1.8. Divinyl ether synthase ............................................................... 14

Figure 1.9. Allene oxide synthase-catalyzed reactions ........................................ 16

Figure 1.10. The pathway for J A biosynthesis ................................................. 20
Figure 1.11. Production of J A in vascular bundles of tomato leaves ........................ 28
Figure 2.1. Oxylipins derived from allene oxide synthase .................................... 46
Figure 2.2. Comparison of cDNA-deduced protein sequences of CYP74 P4505

in tomato ............................................................................................. 53
Figure 2.3. Phylogenetic analysis of plant CYP74 sequences ................................ 55
Figure 2.4. Affinity purification of LeA083 expressed in E. coli ........................... 57
Figure 2.5. Metabolism of fatty acid hydroperoxides by LeAOS3 ........................... 58
Figure 2.6. Mass spectra of products formed by incubation of LeAOS3

with 9-HPOD ....................................................................................... 61

Figure 2.7. Carbon monoxide difference spectrum of recombinant LeAOS3 .............. 64
Figure 2.8. Tissue specific expression of LeA 0S3 ............................................. 67

Figure 3.1. CYP74-catalyzed oxylipin biosynthesis .......................................... 80

Figure 3.2. Accumulation of LeA 0S3 transcript and protein in response to Me] A ....... 88

Figure 3.3. Induction of LeAOS3 protein and activity after wounding ..................... 91
Figure 3.4. LeA 0S3 expression in germinating seedlings .................................... 93
Figure 3.5. LeAOS3 protein levels in root and germinating seedlings ...................... 94
Figure 3.6. LeAOS3 activity in cell-free extracts of wild-type and jail .................... 95
Figure 3.7. LeAOS3 is expressed in non-root tissues of germinating seedlings . . . . .97
Figure 3.8. Irnmunolocalization of LeAOS3 protein ......................................... 100
Figure 4.1. Octadecanoid pathway for J A biosynthesis .................................... 123
Figure 4.2. Alignment of tomato ACXlA and ACXlB sequences ........................ 130
Figure 4.3. Plant ACX phylogeny ............................................................. 131
Figure 4.4. Genetic complementation of the wound-response phenotype of acxI ....... 133
Figure 4.5. UV/vis spectra of purified recombinant ACXlA and ACXlATlBs]

and substrate specificity of ACXIA ........................................................... 136
Figure 4.6. ACX activity assay with ACXlAT1381 .......................................... 137
Figure 4.7. ACXlA activity against OPDA and OPC-8 in a coupled assay with

acyl-COA synthetase ............................................................................. 139
Figure 4.8. ACX 1A expression in various tissues ............................................ 140
Figure 4.9. Analysis of ACXl protein in various WT and acxI tissues ................... 141
Figure 4.10. 2,4-DB treatment of wild-type and acxI germinating seedlings ............144
Figure 5.1. The octadecanoid pathway for J A biosynthesis ................................. 160
Figure 5.2. Identification of SALK lines harboring T-DNA insertions in

ACX] and ACX5 ................................................................................... 166
Figure 5.3. The Arabidopsis ACXl and tomato ACXlA isoforrns are

immunologically related ......................................................................... 172

xi

Figure 5.4. Effect of acx mutations on wound-induced JA accumulation and

gene expression .................................................................................. 175
Figure 5.5. acx1/5 plants are susceptible to attack by T richoplusia ni ................... 177
Figure 5.6. acx1/5 plants maintain resistance to Alternaria brassicicola ................ 180
Figure 5.7. acxI/5 plants are defective in JA-mediated pollen development ............184

xii

Chapter 1

Introduction: Oxylipin Biosynthesis and Function

Plants are constantly subjected to a variety of environmental insults including
drought, disease—causing pathogens, UV radiation, temperature stress, herbivory, and
mechanical wounding. Nevertheless, plants have evolved sophisticated ways to protect
themselves against these environmental hostilities. Mechanisms for protection involve
both constitutive and induced defenses. Constitutive defenses include morphological (e. g.
thorns) and physical (e. g. cuticle layer) barriers that prevent damage from such stresses as
herbivore attack or water loss during drought. Other constitutive defenses involve
preformed chemicals or proteins (“phytoanticipins”) that function to deter pest attack.
Alternatively, induced defense strategies involve recognition of stress or damage
followed by production of signals that activate the synthesis of defense compounds.

One of the first responses of plants to stress is altered lipid and fatty acid
metabolism. A wealth of evidence indicates that oxidized lipids and fatty acids,
collectively called oxylipins, play an important role in plant development and in plant
responses to environmental cues (Howe and Schilmiller, 2002). In addition to their role
as signals, oxylipins also function as defensive chemicals. These findings have fueled

interest in understanding how and why oxylipins are synthesized in plants.

I. Oxylipin biosynthesis and function

Oxylipin biosynthesis begins with the addition of oxygen to polyunsaturated fatty
acids. This step can be catalyzed by lipoxygenases or dioxygenases or can occur non-
enzymatically by auto-oxidation (Hamberg et al., 1999; Feussner and Wastemack, 2002;

Mueller, 2004). The resulting hydroperoxy fatty acids undergo a

Environment pevelgment

 

 

Herbivory/wounding Anther dehiscense
Pathogen attack Pollen development
Touch Response Tuberization

Osmotic shock Embryo development
Drought Trichome development
\ UV light Senescence J
V

RE/\=/\R.

/ 02 \l \f’x

Phytoprostanes 2—hydroperoxy FAs

 

 

 

 

 

 

 

 

OOH
Hydroperoxy FA
LOX / R HPL
Keto FAs ‘ Aldehydes
DES EAS

 

 

 

 

 

 

 

 

/ \

reductase POX & EH

/ \

Hydroxy FAs l Polyhydroxy FAs
Octadecanoids

 

 

 

 

 

Divinyl ether FAs Epoxyhydroxy FAs

 

 

 

 

 

 

Figure 1.1 Biosynthesis of oxylipins in response to environmental and
developmental cues. Many environmental and developmental signals can trigger LOX-
catalyzed production of hydroperoxy fatty acids that are further acted upon by various
enzymes to generate a wide range of oxylipins. LOX, lipoxygenase; a-DOX, ct-
dioxygenase; DES, divinyl ether synthase; AOS, allene oxide synthase; POX,
peroxygenase; EH, epoxide hydrolase; EAS, epoxyalcohol synthase; HPL,

hydroperoxide lyase (figure adapted from Howe and Schilmiller, 2002).

variety of cleavage and rearrangement reactions that give rise to the full suite of oxylipins

(Figure 1.1). Oxylipins are formed in all higher plants and are also found in mosses

(Dembitsky, 1993) and some species of marine alga (Gerwick, 1993). The similarity of

plant oxylipins to eicosanoids in animals, which can function in immune responses and

cell differentiation, demonstrates the importance of oxidative metabolism of lipids across

kingdoms.

Lipoxygenase

Lipoxygenases (LOX) are
non-heme, iron-containing di-
oxygenases that add molecular
oxygen to polyunsaturated fatty
acids (PUFAs, i.e. linoleic and
linolenic acids) to generate
hydroperoxy fatty acids (Figure
1.2). LOXs are classified
according to their positional
specificity of linoleic acid
oxygenation. Those isoforrns that
add a hydroperoxy group at the 9-
position are named 9-LOXs,
whereas those that add to the 13-

position are called 13-LOXs. In

linolenic acid (LnA)

9-L0y \f-Lox
w/ _ c3011 m \ coon
OOH OOH

9-hydroperoxy-LnA 1 3 -hydroperoxy-LnA

linoleic acids.

COOH

(13-HPOT)

COOH
OOH OOH

9-hydroperoxy-LA 1 3 -hydroperoxy-LA

(l3-HPOD)

9-LOX\\ /(13-Lox

COOH

linoleic acid (LA)

Figure 1.2 Lipoxygenase-catalyzed production

of hydroperoxy fatty acids from linolenic and

some plants, 16-carbon fatty acids can also serve as LOX substrates. A major focus of
oxylipin research is to determine the physiological roles of the 9- and 13-LOX pathways.
A useful approach has been to genetically alter expression of one or more LOX genes,
and then study the effects on development and defense. Silencing of the LOX-H1 gene of
potato showed for the first time that a LOX could supply substrate specifically to the
hydroperoxide lyase (HPL) branch of oxylipin biosynthesis (Leon et al., 2002). Antisense
suppression of a 13-LOX in tobacco caused reduction in wound-induced J A accumulation
and resulted in increased herbivore performance on the LOX-deficient plants (Halitschke
and Baldwin, 2003). Similar approaches have been used to study the role of the 9-LOX
pathway. Expression of some 9-LOX genes is induced upon treatment with fungal
pathogens (Veronesi et al., 1996). In one case, suppression of a 9-LOX gene in tobacco
resulted in enhanced susceptibility to Phytophthora parasitica (Rance et al., 1998).
Hydroperoxides derived from either the 9- and l3-LOX pathway have been shown to
accumulate during the hypersensitive response of plants to pathogen infection. However,
the specific profile of hydroperoxy fatty acids depends on the plant used in the study
(Montillet et al., 2002). A role for 9-LOX in plant development has also been suggested
based on the suppression of 9-LOX activity in potato, which caused fewer and deformed

tubers (Kolomiets et al., 2001).

Other hydroperoxide-forming activites
In addition to oxylipins derived from hydroperoxides generated by LOXs, there
are other routes for the synthesis of hydroperoxy fatty acids. In animals, cyclooxygenases

(COX) catalyze the formation of endoperoxides that undergo further metabolism to give

rise to prostaglandins, which function in diverse aspects of development and stress
responses (Smith et al., 2000). In plants, an enzymatic activity unrelated to LOX was
found to catalyze the dioxygenation of fatty acids at the 02 (a-) position. This enzyme
was named a-dioxygenase (a-DOX; Figure 1.3) (Hamberg et al., 1999). Sequence

analysis and comparison of the
COOH

predicted structure of ct-DOX
linolenic acid

revealed homology with the a-DOX¢
OOH

crystal structure of ovine COX

COOH
(Sanz et a1” 1998)' 2" Lhydroperoxylinolenic acid
- reduction 1) decarboxylation
hydroperoxy fatty acrds can be on /\ 2) oxidation
reduced to 2-hydroxy fatty acids, COOH fixCOOH

01' undergo succesSIve 2-hydroxylinolenic acid 8,11,14-heptadecatrienoic acid

decarboxylation and oxidation
Figure 1.3 or-Dioxygenase pathway

reactions to shorten the fatty acid
by one carbon. a-DOX was originally isolated by differential mRNA display in a search
for genes induced by a pathogen elicitor (Sanz et al., 1998). Later studies showed that
induction of a-DOX and increased production of 2-hydroxy fatty acids exert a tissue-
protective effect in pathogen infected leaves (Hamberg et al., 2003).

Auto-oxidation of fatty acids catalyzed by free-radicals generates hydroperoxy

fatty acids that are converted to prostaglandin—like compounds named phytoprostanes

(Figure 1.4) (Krischke et al., 2003). The production of phytoprostanes was shown to

Type I series Type 11 series

0 O
Gl-phytoprostanes | / COOH | /
O 0 COOH

OCH OCH
0 O
El-phytoprostanes < I x: : " COOH WCOOH
HO OH HO OH
O O
A -phytoprostanes COOH
l I I COOH
OH OH
O O
Brphytoprostanes WCOOH /
COOH
OH OH
HO HO
F l-phytoprostanes < | ,: : " COOH WCOOH
HO OH HO OH

Figure 1.4 Type I and Type II phytoprostanes synthesized from linolenic acid.
Non-enzymatic oxygenation of linolenic acid, which is catalyzed by free-radicals,

results in the production of two series of racemic phytoprostanes.

increase in tobacco cell cultures under peroxide stress and tomato leaves infected by the
necrotrophic fungus Botrytis cinerea. Application of in vitro synthesized phytoprostanes
to tobacco cell cultures resulted in the induction of phytoalexin synthesis, suggesting that
phytoprostanes act as signals to elicit responses during oxidative stress (Thoma et al.,

2003)

Hydroperoxide-metabolizing enzymes

Lipoxygenase
Whereas the primary reaction COOH
catalyzed by LOX is the linolenic acid (LnA)
. . . 9-L x -
peroxrdatlon of fatty acids, LOXs O / \ l3 LOX

also convert hydroperoxy fatty acids < /: _: CEOH < :\: :COOH
OOH OOH

to fatty acid ketodienes (KODE) and
9-hydroperoxy-LnA 13-hydroperoxy-LnA

ketotrienes (KOTE) from dienoic 9-LOX‘ *13-LOX

and trienoic fatty acids, respectively g >/~\/:COQH <_ XFOOH

(Figure 1.5) (Kuhn et al.,1991).
9-KOTE 13-KOTE
Until recently, relatively little was

Figure 1.5 LOX-catalyzed production
known about the function of KODEs 0f ketodienes
and KOTEs in plants. Wounded and pathogen infected leaves were found to accumulate
high levels of KODEs and KOTEs compared to untreated controls (Vollenweider et al.,
2000). Moreover, infiltration of Arabidopsis leaves with KODE or KOTE induced the

expression of the glutathione—S—transferase gene and caused cellular damage. These

effects of KODE and KOTE on plant physiology were attributed to the electrophilic
properties of the anti-unsaturated carbonyl feature of the molecules (Almeras et al.,
2003). A recently identified LOX from the moss Physcomitrella patens was found to
produce not only hydroperoxy and keto fatty acids, but was also found to possess a fatty-

acid lyase activity (Senger et al., 2005).

Epoxy alcohol synthase

Epoxy alcohol synthase (EAS) converts fatty acid hydroperoxides into epoxy
alcohol fatty acids (Figure 1.6) (Hamberg, 1999). The EAS products are then metabolized
by epoxide hydrolases to give trihydroxy fatty acids. Currently, genes encoding EAS
have not been identified. Oxylipin profiling experiments have shown that 9-HPOD-

COOH

linolenic acid

I 9-LOX

COOH
/ _ .—

OOH
EASA/ 9-HPOT \EAS

<1 ; : COOH < : 0; COOH
.... _ / _
HO O OH

epoxy alcohol
EH+ fatty acids +5}!
€1.10: _: :COOH Hs/ /: ; :COOH
HO OH HO OH
trihydroxy fatty acids

Figure 1.6 Epoxy alcohol synthase-catalyzed oxylipins.

(EAS, epoxy alcohol synthase; EH, epoxide hydrolase)

derived trihydroxy fatty acids from the EAS pathway are induced in potato leaves upon
pathogen infection (Gobel et al., 2002). These results, together with the observation that
trihydroxy fatty acids inhibit the grth of pathogenic fungi (Masui et al., 1989), suggest

a role for the EAS pathway in defense against plant pathogens.

Peroxygenase

The peroxygenase (POX) pathway catalyzes the production of trihydroxy fatty
acids that are regiochemically identical to products of the EAS pathway. However, POX-
derived trihydroxy fatty acids differ from those of the EAS pathway with respect to their
stereochemistry. POX is a membrane-bound, heme-containing oxidase distinct fiom
cytochrome P450 enzymes (Blee, 1998). Like EAS, no gene has been identified that
encodes POX. The production of trihydroxy fatty acids by the POX pathway suggests a
role in pathogen defense. More recently POX was shown to catalyze the epoxidation step

for the synthesis of C18-monomers for cutin biosynthesis (Lequeu et al., 2003).

Reductase

In the reductase pathway, hydroperoxy fatty acids are reduced to hydroxy fatty
acids by an unknown mechanism. The synthesis of l3-hydroxy fatty acids via the
reductase pathway was shown to be induced upon treatment with salicylic acid (SA)
(Weichert et al., 1999). This induction, and the ability of l3-hydroxy—octadecatrienoic
acid to induce expression of the PR] b gene, suggests a role for the reductase pathway in

establishment of SA-mediated defense. Another study implicated hydroxy fatty acids as

10

intermediates in the degradation of storage lipids in germinating seedlings (Feussner et

al., 1997).

C YP74 cytochromes P45 0
Of the enzymes that metabolize hydroperoxy fatty acids, the CYP74 family of
cytochromes P450 is the best characterized. CYP74 enzymes make up a divergent family

of P450s with catalytic properties distinct from typical P450 monooxygenases (Howe and
Schilmiller, 2002). Whereas P450 monooxygenases require 02 and a NADPH-dependent

P450 reductase for activity, CYP74 family members do not. Rather, the hydroperoxide
substrate of CYP74s serves as the oxygen donor and a source of reducing equivalents.
CYP74 P4503 also exhibit a reduced affinity for carbon monoxide. Interestingly, these
catalytic features of plant CYP74 P4508 are shared by prostacyclin synthase and
thromboxane synthase, two P4505 involved in the synthesis of eicosanoids (Hecker and
Ullrich, 1989). In plants, there are three types of CYP74 enzymes: hydroperoxide lyase
(HPL), divinyl ether synthase (DES), and allene oxide synthase (AOS). Based on amino
acid sequence similarity, CYP74s are divided into four subfamilies. CYP74A consists of
AOSS, whereas CYP74B consists of HPLs that metabolize 13-hydroperoxy fatty acids.
CYP74C includes AOSs and HPLs that have the capacity to metabolize both 9- and 13-
hydroperoxy fatty acids. CYP74D is made up of DESs that metabolize 9-hydroperoxy

fatty acids.

Hydroperoxide lyase

11

HPL catalyzes the cleavage of hydroperoxy fatty acids to aliphatic aldehydes and
(n-keto-fatty acids (Figure 1.7). The volatile aldehydes and their corresponding alcohols
contribute to the so-called “fresh green” odor of fruits and vegetables, and have generated
interest due to their use as food additives to restore the freshness of foods after
sterilization processes (Noordermeer et al., 2001). HPL was first described in 1973 in a
study of banana volatiles (Tressl and Drawert, 1973). The first gene encoding HPL was
later cloned from bell pepper, and demonstrated that the enzyme is a CYP74 cytochrome
P450 (Matsui et al., 1996). To date, HPL genes have been isolated and characterized from
a variety of plants including Arabidopsis, guava, alfalfa, cucumber, and tomato (Bate et

COOH

linolenic acid (LnA)

9-L0f/ \1‘3-LOX

< : : :COOH < I : :
/ _ _ — \ COOH
OOH OOH

9-hydroperoxy-LnA l3-hydroperoxy-LnA
‘9-HPL ¢ 13-HPL
O O
WCOOH 'K/WCOOH
9-oxo—nonanoic acid 12-oxo-(9Z)-dodecenoic acid
+ +
MCHO v_—_\,CHO

(32, 6Z)'n0nadienal (3Z)-hexenal

Figure 1.7 Hydroperoxide lyase-catalyzed cleavage

of fatty acid hydroperoxides.

12

al., 1998; Howe et al., 2000; Matsui et al., 2000; Noordermeer et al., 2000; Tijeta et al.,
2000). While many HPLs show specificity for l3-hydroperoxy fatty acids, some were
found to cleave both 9— and 13-hydroperoxides (Matsui et al., 2000; Tijet et al., 2001).
Localization experiments showed that 13-HPL, which lacks a transit peptide, is targeted
to the outer membrane of the chloroplast envelope (Froehlich et al., 2001).

Products of the HPL pathway not only contribute to the aroma of plant tissues, but
possess other physiological functions. The C12 product arising from cleavage of 13-
hydroperoxy linolenic acid is the precursor of the wound hormone traumatin that has
been implicated in wound healing (Zimmerman and Coudron, 1979). C6-aldehydes fi'om
the 13-HPL pathway induce a subset of wound-responsive genes, suggesting that HPL
products act as signals in plant defense (Bate and Rothstein, 1998). The HPL-derived
aldehydes have also been shown to have antimicrobial effects and reduce aphid fecundity
in vitro (Deng et al., 1993). More recently, antisense-meditated suppression of 13-HPL in
potato was shown to cause an increase in aphid performance. This finding indicated a
role for the HPL pathway in defense against sucking insects that feed on phloem contents
(Vancanneyt et al., 2001). More work is needed to identify physiological functions of the

9-HPL pathway.

Di vinyl ether synthase

Divinyl ether fatty acids are synthesized from polyunsaturated fatty acids via the
sequential action of LOX and DES (Figure 1.8). In 1972, Galliard and Phillips
demonstrated the synthesis of divinyl ethers in cell-free extracts from potato tubers, and

suggested 9-hydroperoxy fatty acids as an intermediate (Galliard and Phillips, 1972).

13

Later work demonstrated the same activity in
tomato roots (Caldelari and Farmer, 1998).

Homogenates of meadow buttercup leaves

/_/=W\/\,COOH
0 2

VA

etherolenic acid

13-DES
also possess a DES activity that acts on 13- 13-LOX
hydroperoxides of linoleic acid (Hamberg, _ _ CEOH
1998). Some species of marine algae can also lumen“: ac1d(LnA)
. . . . 9-LOX
synthesrze dIVInyl ether fatty acrds from 18-
9-DES
and 20-carbon fatty acids (Proteau and MCOOH
O __
W

Gerwick, 1993; Jaing and Gerwick, 1997).

colnelenic acid
The first DES gene was isolated from tomato,

and shown to encode. a CYP74D cytochrome Figure 1'8 Divinyl ether synthase
P450 (Itoh and Howe, 2001).

The physiological function of the DES pathway in plants is not known. However,
a role for divinyl ether fatty acids in defense against plant pathogens has been suggested
by a number of studies. Oxylipin profiling experiments showed that elicitors and
pathogen treatments led to an increase in the accumulation of the divinyl ethers colneleic
(CA) and colnelenic acid (CnA), which are produced from the 9-hydroperoxides of
linoleic and linolenic acid, respectively (Weber et al., 1999; Gobel et al., 2001; Gobel et
al., 2002). Treatment of Phytophthora infestans with purified CA and CnA resulted in
decreased mycelial growth and inhibition of cytospore germination, suggesting that
divinyl ether fatty acids could function in limiting the growth of this pathogen (Weber et

al., 1999). Expression of the potato DES gene was also shown to be induced by pathogen

treatment (Stumpe et al., 2001).

14

Allene oxide synthase

Over thirty years ago, it was shown that an enzyme activity in flaxseed extracts
converts 13—hydroperoxy linoleic acid (13-HPOD) to 13-hydroxy-12-oxo-9(Z)-
octadecenoic acid (or-ketol). This enzyme 'was named “hydroperoxide isomerase”
(Zimmerman, 1966; Zimmerman and Vick, 1970). Later experiments demonstrated the
conversion involved formation of an unstable allene oxide that undergoes spontaneous
hydrolysis to form 0t- and y-ketols or a cyclopentenone compound (Figure 1.9). This
finding prompted re-naming of the enzyme to allene oxide synthase (AOS) (Brash et al.,
1987; Hamberg, 1987). Purification of AOS from flaxseed showed the enzyme to be a
cytochrome P450 (Song and Brash, 1991). The AOS gene cloned from flaxseed became
the founding member of the CYP74 family of P4508 (Song et al., 1993). A OS genes have
since been isolated from several plants including Arabidopsis, tomato, tobacco, and
barley (Laudert et al., 1996; Howe et al., 2000; Maucher et al., 2000; Sivasankar et al.,
2000; Ziegler et al., 2001). Similar to 13-HPL, 13-AOS is located in the chloroplast
envelope. Unlike 13-HPL, however, 13-AOS contains an N-terminal chloroplast targeting
sequence and is located on the inner leaflet of the inner envelope membrane (Froehlich et
al., 2001). Interestingly, Plexaura homomalla (Caribbean sea whip coral) AOS occurs as
a fusion protein with LOX (Koljak et al., 1997). Although the AOS domain of the fusion
protein catalyzes the production of allene oxide fatty acids, sequence and structural data
indicate that this AOS is more similar to catalase, and is not a cytochrome P450 (Oldham

et al., 2005).

15

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Figure 1.9 Allene oxide synthase-catalyzed reactions. 9,10-EOT, 9,10-

epoxy-10,12,15-octadecatrienoic acid; 12,13-EOT, 12,13-epoxy-9,11,15-

octadecatrienoic acid; OPDA, oxophytodienoic acid

16

More research on the AOS pathway in plants has been focused on 13-AOS
because of its involvement in biosynthesis of jasmonic acid (J A). A knockout mutation in
the Arabidopsis AOS gene impairs the synthesis of JA and causes male sterility (Park et
al., 2002; von Malek et al., 2002). A more detailed discussion of the physiological roles
of the 13-AOS pathway and J A is presented later in this chapter.

9-AOS activity has been reported in several plant species (Gardner, 1970;
Graveland, 1973; Grechkin et al., 2000). However, only recently were cDNAs that
encode 9-AOSs isolated (Maucher et al., 2000; Itoh et al., 2002). The tomato 9-AOS
(LeAOS3), which is the subject of chapters 2 and 3 of this dissertation, represents the
first AOS shown to function in the 9-LOX pathway. In contrast to the clear understanding
of the 13-AOS pathway and its involvement in JA biosynthesis, virtually nothing is
known about the physiological function of the 9-AOS pathway. Part of the focus of this
dissertation was to study the regulation of LeA 0S3 in an effort to elucidate the role of the

9-AOS pathway in plants.

11. Jasmonate biosynthesis

Since the discoveries of MeJA in oils of jasmine (Demole et al., 1962) and JA in
fungal culture filtrates (Aldridge et al., 1971), JA and related members of the jasmonate
family (collectively referred to as I As) have been the most extensively studied of all the
plant oxylipins. The biosynthetic pathway for JA was elucidated by Vick and
Zimmerman in the early 19803 (Vick and Zimmerman, 1983). They demonstrated that
fatty acid hydroperoxides of linoleic acid undergo enzymatic cyclization to form 12-

oxophytodienoic acid (12-OPDA). It is now recognized that the cyclization to 12-OPDA

17

is catalyzed by the sequential action of AOS and allene oxide cyclase (AOC). Vick and
Zimmerman went on to show that 12-OPDA is further metabolized to JA through
reduction of the cyclopentenone ring followed by three successive rounds of B-oxidation
(Vick and Zimmerman, 1984). Work in the years following Vick and Zimmerman’s
contribution has identified most of the genes involved in IA biosynthesis (Schaller et al.,
2004). This work has also demonstrated the importance of JAs in plant growth and
development, and in regulating defense responses against herbivores and pathogens
(Wastemack and Hause, 2002).

The synthesis of IA is initiated in the chloroplast by LOX-catalyzed addition of
molecular oxygen to linolenic acid (Figure 1.10). The resulting 13-hydroperoxy-linolenic
acid (13-HPOT) is acted upon by AOS to form the allene oxide 12,13-epoxy-9,11,15-
octadecatrienoic acid. Allene oxide cyclase (AOC) converts the allene oxide to the first
cyclic intermediate in the pathway, 12-OPDA. At this point, 12-OPDA is transported to
the peroxisome where the cyclopentenone ring is reduced by OPDA reductase (OPR3) to
give 3-oxo-2(2’-pentenyl)-cyclopentane-l-octanoic acid (OPC-8z0). OPC-8:0 is esterified
to coenzyme A (COA) and undergoes three successive rounds of B—oxidation followed by

removal of CoA, presumably by a thioesterase, to yield J A.

18

Figure 1.10 The pathway for JA biosynthesis. JA synthesis begins in the
chloroplast with conversion of linolenic acid to 12-OPDA by the sequential action
of LOX, AOS, and AOC. 12-OPDA moves to peroxisome where it undergoes a
reduction step followed by three rounds of B-oxidatiOn to yield JA. JA
modifications include methylation by JMT or conjugation to amino acids by JARl.
The perception of JAs resulting in changes in gene expression requires COIl, an F -
box protein that is part of a E3-ubiquitin ligase complex. (JMT, JA
methyltransferase; JME, JA methyl esterase; JAR], jasmonate responsel; COIl,

coronatine insensitive 1 )

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Figure 1.10 The pathway for JA biosynthesis
20

Chloroplast-localized reactions

It is generally assumed that free linolenic acid (18:3), released from plastid
membrane lipids by a lipase, serves as the LOX substrate for the start of IA synthesis.
However, there also exists the possibility that a membrane-associated LOX acts on
esterified 18:3 prior to release from the membrane. Similarly, AOS and AOC could
convert lipid-bound 13-HPOT to 12-OPDA before cleavage from the membrane. Support
for this hypothesis came from the finding that OPDA and dinor OPDA (from 16:3) are
found esterified to galactolipids, primarily monogalactosyl diacylglycerides (Stehnach et
al., 2001; Hisamatsu et al., 2003). Evidence for LOX acting on free fatty acids comes
from the observation that wounding causes an increase in free fatty acids (including 18:3)
that correlates with the timing of IA accumulation (Conconi et al., 1996). The
chloroplast-localized DADl protein Of Arabidopsis is the only lipase known to be
involved in IA biosynthesis. However, the dad] (delayed gnther dehiscencel) mutant is
only affected in floral JA levels suggesting that other lipases must be involved in IA
synthesis in other tissues (Ishiguro et al., 2001). In vitro analysis of DADl activity
demonstrated that the enzyme hydrolyzes fatty acids from the sn-1 position of
galactolipids, triacylglycerol, and phospholipids. Finding the precise location of DADl
within the chloroplast would help to understand its physiological substrate(s). The lipase
responsible for the bulk of wound-induced JA biosynthesis in leaves has not yet been
identified.

The three plastidic enzymes (LOX, AOS, and AOC) in JA synthesis have been
studied extensively in a number of plants. Genomic sequencing and expressed sequence

tag (EST) projects have shown that plants typically cOntain multiple LOXs. For example,

21

there are six genes in Arabidopsis and at least eight in soybean with certain isoforms
involved in IA biosynthesis (F eussner and Wastemack, 2002). There is one 13—AOS gene
in Arabidopsis, whereas other plants contain multiple copies (Maucher et al., 2000; Howe
and Schilmiller, 2002). Both 13-LOXs and 13-AOSS, which are involved in IA
biosynthesis, contain chloroplast targeting sequences and are localized to the plastid
(Vick and Zimmerman, 1987 ; Bell et al., 1995; Froehlich etal., 2001). Overexpression of
AOS in different plants gave varying results with respect to basal JA levels. In potato,
overexpression of AOS caused a 6 to 12-fold increase whereas in Arabidopsis and
tobacco, no changes in basal JA levels were observed (Harms et al., 1995; Laudert et al.,
2000; Park et al., 2002). Upon wounding, however, higher levels of IA accumulated in
AOS overexpressing plants. This suggested that substrate is limiting for AOS in
unwounded plants and only after wounding does AOS activity become limiting.

AOC activity was first characterized in corn (Hamberg and Fahlstadius, 1990),
and the first AOC gene was cloned from tomato (Ziegler et al., 2000).
Imrnunocytochemical studies showed AOC protein is located in the plastid (Ziegler et al.,
2000). Although allene oxide products of 13-AOS can spontaneously cyclize to 12-
OPDA, AOC is required for synthesis of the correct stereoisomer of 12-OPDA. There is a
single AOC gene in tomato and barley, whereas four AOC genes are present in
Arabidopsis (Ziegler et al., 2000; Stenzel et al., 2003; Maucher et al., 2004). When AOC
was overexpressed in tomato, basal JA levels were not elevated in leaves, but higher
levels of JAs (including OPDA, JA, and MeJA) accumulated in various floral organs.
This indicated different points of regulation for the IA pathway in different tissues

(Miersch et al., 2004).

22

Peroxisome-localized reactions

The final steps of the J A biosynthetic pathway take place in the peroxisome where
12-OPDA is converted to J A. For this to occur, 12-OPDA has to move from the plastid to
the peroxisome. It is not known how 12-OPDA is transported from the plastid, however a
recent study has implicated the PXAl peroxisomal ABC-transporter in movement of 12-
OPDA into the peroxisome (Theodoulou et al., 2005). JA biosynthesis afler wounding in
a pm] null mutant was reduced approximately 50% compared to wild type. Therefore,
other routes of 12-OPDA entry into the peroxisome seem likely.

Once in the peroxisome, OPR3 catalyzes the reduction of the cyclopentenone ring
of 12-OPDA to yield OPC-8:0. Although plants contain a small family of 0PR genes,
only one isoform in Arabidopsis and tomato is capable of producing the specific isomer
of 12-OPDA that is a precursor for JA (Schaller et al., 2000; Strassner et al., 2002). In
vitro enzyme assays indicate that OPR3 is active with free 12-OPDA. This result,
together with the occurrence of detectable free OPC-8:0 in planta, suggests that OPC-8:O
is converted to OPC-8:0-COA for entry into the B-oxidation cycle. A recent in vitro study
demonstrated that certain peroxisome-localized acyl activating enzymes similar to 4-
coumaratezCoA ligase (4CL) can synthesize CoA esters of 12-OPDA and OPC-8:0
(Schneider et al., 2005). However, there is currently no genetic evidence that these 4CL-

like enzymes function in J A biosynthesis.

Vick and Zimmerman’s studies on the metabolism of [180]-12-OPDA suggested a

role for B-oxidation in conversion of lZ-OPDA to JA (Vick and Zimmerman, 1984).

Only recently, however, have specific B-oxidation enzymes been implicated in this stage

23

of the IA biosynthetic pathway. B-oxidation in plants takes place in the peroxisome and
involves the action of three enzymes: acyl-CoA oxidase (ACX), the multi-functional
protein (MFP, possessing 2-trans-enoyl-COA hydratase and L—3-hydroxyacyl-COA
dehydrogenase activities), and 3-keto-acyl-COA thiolase (KAT) (Graham and Eastmond,
2002). ACXS are encoded by a small gene family in plants, with six genes in Arabidopsis
and at least five in tomato (Adham et al., 2005; Li et al., 2005). In Arabidopsis, two MFP
and four KAT genes are present in the genome (Graham and Eastmond, 2002). Recent
work in Arabidopsis implicates ACXl and KAT2 in J A production. However, incomplete
suppression of JA levels in mutants or transgenic antisense plants altered in ACX] or
KAT 2 expression suggests that multiple isoforms contribute to JA synthesis (Castillo et
al., 2004; Pinfield-Wells et al., 2005). The identification and characterization of tomato
and Arabidopsis ACXS required for JA biosynthesis is the focus of chapters 4 and 5,
respectively, of this dissertation.

JA synthesis in the peroxisome presumably requires a thioesterase to cleave JA-
COA. Characterization of peroxisomal acyl—CoA thioesterases to date has shown activity
with medium- to long-chain acyl-CoAs or with B-hydroxyisobutryl-CoAs that function in
valine catabolism (Zolman et al., 2001; Tilton et al., 2004). It is not yet known if these

enzymes function in cleavage of JA-COA as well.

Metabolism of JA

J A is subject to a number of metabolic transformations that may play a role in fine

tuning the activity of the hormone. The routes of modification include: methylation at C1

to give MeJA; hydroxylation at C11 or C12 to form 11-hydroxyjasmonic acid (1 1-OHJA)

24

or 12-hydroxyjasmonic acid (lZ-OHJA, tuberonic acid); sulfation of 11-OHJA or 12-
OHJ A to produce hydroxy-J A sulfate; reduction of the keto group at C6 to form cucurbic

acid; O-glycosylation of the above mentioned hydroxylated jasmonates; conjugation of
the carboxyl group of IA to amino acids; and degradation of the carboxyl group to form
cis-jasmone (Schaller et al., 2004).

Several enzymes catalyzing JA modifications have been identified. JA-methyl
transferase (JMT) identified from Arabidopsis catalyzes the methylation of JA. When
JMT is overexpressed, MeJA levels are increased resulting in constitutive activation of
defense-related gene expression (Seo et al., 2001). The opposite reaction, cleavage of the
methyl group from MeJA, is catalyzed by a JA methyl esterase (JME) (Swiatek et al.,
2004). A gene encoding a putative JME was recently cloned from tomato (Stuhlfelder et
al., 2004). The Arabidopsis JARl protein is responsible for conjugation of J A with amino
acids, most likely with isoleucine in vivo (Staswick and Tiryaki, 2004). A jar] mutant
was isolated based on the reduction in JA-mediated root growth inhibition (Staswick et
al., 1992). The reduced sensitivity of jar] mutants to LA, and the increase in susceptibility
of jar] to Pythium irregulare, suggests that JA-Ile is an active signal for some jasmonate-
mediated responses (Staswick et al., 1998). A sulfotransferase with activity for sulfation
of 11-OHJA and 12-OHJA was identified from Arabidopsis, and it was suggested that

this enzyme may serve to inactivate JA (Gidda et al., 2003).

III. Physiological roles of jasmonates

Work by Green and Ryan demonstrated that wounding induces local and systemic

expression of proteinase inhibitor (PI) proteins that inhibit the activity of digestive

25

enzymes in the gut of herbivores (Green and Ryan, 1972). The Ryan group went on to
show that application of JAs causes the induction of PI gene expression (Farmer and
Ryan, 1990; Farmer et al., 1992). These properties of P13 led to their extensive use as a
marker to study systemic wound signaling in tomato (Howe, 2004). Grafiing experiments
with JA biosynthetic and JA perception mutants of tomato indicated that JA acts an
essential component of the long distance signal for activation of systemic responses (Li et
al., 2002). In some Solanaceous species, small peptides (named systemin in tomato) are
involved in the signaling pathway for the wound response. The current model in tomato
suggests that systemin works locally at the wound site to activate the synthesis of JA,
which acts as a phloem-mobile signal to promote systemic responses (Figure 1.11)
(Schilmiller and Howe, 2005). Other factors such as cell wall-derived oligosaccharides
and fatty acid-amino acid conjugates (from insect regurgitant) have also been found to

induce the synthesis of J A in plants.

Role of JAs in defense

JA-mediated plant defense responses have been classified as being direct or
indirect. Direct defenses include induced phytochemicals and proteins that directly inhibit
feeding, growth, or reproduction of herbivores (Halitschke and Baldwin, 2004). Indirect
JA-mediated defenses include volatiles that attract predators that parasitize insect
herbivores. A major portion of our knowledge concerning the role of I As in defense has
come from work with mutants affected in JA synthesis and perception. Tomato and
Arabidopsis mutants affected in IA signaling show increased susceptibility to various

herbivores (Howe et al., 1996; McConn et al., 1997; Li et al., 2002; Li et al., 2003).

26

Figure 1.1] Production of JA in vascular bundles of tomato leaves. The location
of IA biosynthetic enzymes in companion cells (cc) and sieve elements (se) along
with localization of prosystemin (ps) to phloem parenchyma (pp) cells supports the

hypothesis that J A is produced and transported in the phloem.

27

 

Figure 1.11 Production of JA in vascular bundles of tomato leaves

28

Similarly, JA-deficient tobacco plants with suppressed 13-LOX expression exhibit
reduced resistance to tobacco homwonn feeding (Halitschke and Baldwin, 2003).
Ecological studies with the suppressed 13-LOX tobacco lines showed the plants became
susceptible to two herbivores that normally do not feed on tobacco. This suggests that J A
is required for basal resistance as well as induced resistance (Kessler et al., 2004). This
phenomenon was also found for JA mutants exhibiting susceptibility to certain pathogens
(Vijayan et al., 1998; Thomma et al., 2001). While it is clear JAs are required for defense
against herbivores and pathogens, exactly which I As promote specific defense responses
is not fully understood. Arabidopsis plants defective in 0PR3 fail to synthesize JA but
accumulate the cyclopentenone precursor 12-OPDA (Stintzi and Browse, 2000; Stintzi et
al., 2001). The opr3 mutant retains resistance to the necrotrophic pathogen Alternaria
brassicicola and to the insect Bradysia impatiens (fungal gnat), whereas plants lacking all
JAs were susceptible to these pests. This suggested that precursors of IA (such as 12-
OPDA) have signaling properties as well. Work presented in chapter 5 of this dissertation
is focused on understanding the role of B-oxidative conversion of OPC—8:0 to J A in plant

defense.

Role of JAs in plant development

Early interest in JAs was bolstered by observations of plant growth-regulating
properties of JAs (Ueda and Kato, 1980; Dathe et al., 1981). To date, several aspects of
plant growth and development have been associated with JAs including root grth
inhibition, senescence, floral development, tendril coiling, and tuberization (Wastemack

and Hause, 2002). One of the best characterized processes in which JAs are involved is

29

floral development. An Arabidopsis mutant (fad3/fad7/fad8) deficient in trienoic fatty
acid synthesis was found to be male sterile due to pollen inviability and delayed
dehiscence of pollen (McConn and Browse, 1996). This defect was rescued by
application of JA, indicating that a defect in IA biosynthesis was the cause of male
sterility. Subsequent work with the Arabidopsis opr3 mutant showed that the requirement
for JA in male fertility cannot be compensated with 12-OPDA (Sanders et al., 2000;

Stintzi and Browse, 2000).

This dissertation is focused on the characterization of two enzymes involved in
oxylipin biosynthesis. Chapter 2 presents the characterization of a tomato CYP74
cytochrome P450 with 9-AOS activity. LeAOS3 represents the first cloned gene that
encodes an AOS that is active in the 9-LOX pathway. Chapter 3 describes the regulation
of LeAOS3 in roots and hypocotyls of germinating seedlings. Also included in this
chapter are the results from an expression profiling study to identify genes that are co-
regulated with LeA 053 in roots. Chapters 4 and 5 describe the characterization of tomato
and Arabidopsis mutants affected in ACX. Chapter 4 is focused on the tomato ACXlA,
whereas chapter 5 presents the isolation and characterization of Arabidopsis ACX

mutants impaired in J A biosynthesis.

30

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41

Chapter 2

Identification and Biochemical Characterization of a New Tomato Allene Oxide

Synthase

The work presented in this chapter has been published:

Itoh A, Schilmiller AL, McCaig BC, Howe GA (2002) J Biol Chem 277(48): 46051-

46058.

42

Abstract

Allene oxide synthase (AOS) is a cytochrome P-450 (CYP74A) that catalyzes the
first step in the conversion of l3-hydroperoxy linolenic acid to jasmonic acid and related
signaling molecules in plants. Here, we report the molecular cloning and characterization
of a novel AOS-encoding cDNA (LeAOS3) from Lycopersicon esculentum whose
predicted amino acid sequence classifies it as a member of the CYP74C subfamily of
enzymes that was hitherto not known to include AOSs. Recombinant LeAOS3 expressed
in Escherichia coli showed spectral characteristics of a P-450. The enzyme transformed
9- and 13-hydroperoxides of linoleic and linolenic acid to ct-ketol, y-ketol, and
cyclopentenone compounds that arise from spontaneous hydrolysis of unstable allene
oxides, indicating that the enzyme is an AOS. Kinetic assays demonstrated that LeAOS3
was ~10-fold more active against 9-hydroperoxides than the corresponding l3-isomers.
These findings suggest that LeAOS3 plays a role in the metabolism of 9-lipoxygenase-

derived hydroperoxides.

43

Introduction

Oxylipins comprise a group of biologically active compounds that are produced
by oxidative metabolism of polyunsaturated fatty acids. Members of the eicosanoid
family of lipid mediators have been studied extensively with respect to their biosynthesis
from arachidonic acid and their function in diverse physiological processes in animal
cells (Smith et al., 2000). In plants, which lack arachidonic acid, oxygenated derivatives
of C18 fatty acids participate in the regulation of many defense-related and
developmental processes. The biosynthesis of most phytooxylipins is initiated by
lipoxygenase (LOX), which adds molecular oxygen to either the C-9 or C-13 position of
linolenic or linoleic acid (Feussner and Wasternack, 2002). The resulting hydroperoxides
are further metabolized by several enzymes including three closely related members of
the CYP74 family of cytochromes P-450: allene oxide synthase (AOS), hydroperoxide
lyase (HPL), and divinyl ether synthase (DES). Indeed, much of the structural and
functional diversity in oxylipin metabolism in plants can be accounted for by the activity
of CYP74s that metabolize 9- and 13-hydroperoxides to a wide range of products (Howe
and Schilmiller, 2002). In contrast to typical P-450 monooxygenases, CYP74 P-4505 do
not require 02 and a NADPH-dependent P-450 reductase for activity. Rather, they use a
hydroperoxide group both as the oxygen donor and as a source of reducing equivalents
(Song and Brash, 1991; Song et al., 1993). This unique catalytic feature is shared by
thromboxane synthase and prostacyclin synthase, two P-450 enzymes involved in the
synthesis of eicosanoids (Hecker and Ullrich, 1989). A greater understanding of the

biochemical and physiological fimction of this atypical class of P-450 enzymes promises

44

to provide new insight into the evolution of fatty acid—based signaling pathways in
diverse biological systems.

Interest in plant oxylipins has focused mainly on the AOS branch of the 13-LOX
pathway that gives rise to the jasmonate family of compounds (collectively referred to as
JAs) including JA, methyl-IA (MeJA), and their metabolic precursor 12-oxo-10,15-
phytodienoic acid (12-OPDA) (Fig. 2.1). AOS catalyzes the transformation of 13-
hydroperoxy linolenic acid (13-HPOT) to a short-lived allene oxide intermediate, 12,13-
epoxyoctadecatrienoic acid (12,13-EOT) (Brash et al., 1988; Hamberg, 1989). In the
biosynthetic route to JA, allene oxide cyclase (AOC) converts 12,13-EOT to 12-OPDA
(Hamberg, 1988). Reduction of 12-OPDA by OPDA reductase (OPR) and three cycles of
B-oxidation yields JA (Vick and Zimmerman, 1983, 1984). During in vitro reactions
can'ied out in the absence of AOC, 12,13-EOT spontaneously hydrolyzes to a- and y-
ketols, and can also undergo non-enzymatic cyclization to produce racemic 12-OPDA
(Brash et al., 1988; Song and Brash, 1991). The physiological significance of or- and y-
ketols, and the extent to which they are produced in vivo, is unclear. Genes encoding
AOSs that metabolize 13-HPOT have been cloned from several plant species. Based on
comparisons of predicted amino acid sequence, these proteins are classified within the
CYP74A subfamily of P-4505 (Feussner and Wasternack, 2002; Howe and Schilmiller,
2002). Several AOSs have been shown to have a strong (~36:1) preference for the 13-
hydroperoxide relative to the corresponding 9-isomer (Feng and Zimmerman, 1979;
Howe et al., 2000). One exception to this is barley AOS, which despite its classification
as a CYP74A and proposed role in IA biosynthesis can metabolize both 9- and 13-

isomers in vitro (Maucher et al., 2000).

45

(3:;- _ 090:1 COOH

linolenic acid linolenic acid
I 9—LOX I 13-LOX
I _ COOH _ \ CBOH
OOH OOH
9-HPOT 13-HPOT
I A03 1 ADS
non-enzymatifi/ O ’ non-enzymatic
W 9,10-EOT 12, O‘l3-EOT COOH
+ non-enzymatic * AOC ' -
H00 a-ketol OOH

+ WCOOH mega... a-ketol
+
WEI-9'1 10- OPDA 12-0F’DA W

O OH : OHO

y-ketol ; :COOH y-ketol

O
jasmonic acid

Figure 2.1 Oxylipins derived from allene oxide synthase. The pathways
shown are for the metabolism of linolenic acid by the AOS branch of the 9-

LOX and l3-LOX pathways.

46

Plants also utilize a second type of AOS for the metabolism of 9-hydroperoxides
in non-photosynthetic tissues (Fig. 2.1). An AOS activity responsible for production of or-
and y-ketol fatty acids from 9-hydroperoxy linoleic acid (9-HPOD) was first
demonstrated in extracts from corn seed (Gardner, 1970; Gardner et al., 1975).
Subsequent studies showed that these compounds are formed by non-enzymatic
hydrolysis of an unstable allene oxide (9,10-epoxyoctadecanoic acid; 9,10-EOD) that is
generated by the action of AOS on 9-hydroperoxides (Hamberg and Hughes, 1988;
Hamberg, 2000). More recently, the production of ketols by the sequential action of 9-
LOX and AOS was demonstrated in cell-free extracts from tomato root (Caldelari and
Farmer, 1998), tulip bulb (Grechkin et al., 2000), and potato stolon (Hamberg, 2000). The
latter study (Hamberg, 2000) also showed that metabolism of linoleic and linolenic acids
via the 9-LOX/AOS pathway gives rise to lO-oxo-l l-phytoenoic acid (IO-OPEA) and
10-oxo-11,15-phytodienoic acid (lo-OPDA), respectively, novel cyclopentenones that
are structural isomers of 12-OPDA (Fig. 2.1).

The presence of 9—AOS in plants raises the question of the physiological function
of this branch of oxylipin metabolism and its relationship to AOSs that act in the l3-LOX
pathway for JA biosynthesis. Here we describe the identification and functional
characterization of a cDNA encoding a novel member of the CYP74 P-450 gene family
in tomato. We demonstrate that the recombinant protein (designated LeAOS3) is an AOS
by virtue of its ability to convert hydroperoxy fatty acids to ketol and cyclopentenone
oxylipins. The phylogenetic classification of LeAOS3 within the CYP74 family and the
substrate preference of the enzyme lead us to propose a role for this P-450 in the

production of 9-hydroperoxide-derived oxylipins.

47

Materials and Methods

Expression and purification of recombinant LeA 0S3

A construct for expression of LeAOS3 in E. coli was made using a PCR-based
method with a full-length EST (cLEIl612) used as template. Forward and reverse primers
were designed to contain NdeI and XhoI restriction sites, respectively to facilitate cloning
into the pET23b (Novagen, Madison, WI) expression vector. The sequence of the
forward primer was 5’-GGA-ATT-CCA-TAT-GGC-TAA-TAC-CAA-AGA-3’ and that
of the reverse primer, 5’-CCG-CTC-GAG-TGA-TGT-TGC-TTT-AG-3’. Following PCR
amplification, the 1.45-kb product was cut with NdeI and 20201 and subsequently ligated
into pET23b digested with the same enzymes. The resulting construct, which adds eight
amino acids (LEHHHHHH) to the C-terrninus of LeAOS3, was transformed into the E.
coli host BL21(DE3).

His-tagged recombinant LeAOS3 was expressed in BL21(DE3) host cells as
follows. An overnight culture (2 ml) of bacteria was inoculated into 200 ml of Terrific

Broth (TB) medium supplemented with 100 ug/ml ampicillin. Bacteria were grown at
37°C in a shaker at 250 rpm to an OD600 of 0.5. Cultures were cooled to 25°C, and

isopropyl-thio-B-D-galactopyranoside was added to a final concentration of 0.1 mM.
Induced cultures were incubated for 24 h at 25°C with gentle shaking (120 rpm). Cells
were collected by centrifugation and stored at -20°C until further use. Purification of
recombinant LeAOS3 was performed as described previously (Itoh and Howe, 2001)
with minor modification. Following centrifugation (100,000 x g for 60 min) of the

cleared E. coli lysate, the recombinant protein was solubilized from the particulate

48

fraction using 1.5% Triton X-100R. Solubilized protein was further purified using
TALON metal affinity column (cobalt-based IMAC, Clontech) and subsequent elution
with imidazole. Irnidazole was removed from the protein sample using a 2.5-ml spin
column prepared with Sephadex G-25 (Amersham Pharmacia, Piscataway, NJ)
equilibrated with 50 mM sodium phosphate (pH 7.0), 5% glycerol, and 0.02% Triton X-
100R. Protein measurements were performed using a BCA assay (Pierce, Rockford, IL)
and bovine serum albumin as a standard. The relative purity of recombinant LeAOS3 was
estimated by SDS-polyacrylamide gel electrophoresis (10% polyacrylamide) and staining

of gels with Coomassie Brilliant Blue R-250.

Product analysis

Affinity purified LeAOS3 (30 pg protein) was incubated with 250 pg fatty acid
hydroperoxide substrate (Cayman Chemical, Ann Arbor, MI) in 1 ml 0.1 M sodium
phosphate buffer (pH 7.0) containing 5% (v/v) glycerol. Reactions proceeded for 5 min at

25°C and then were stopped by acidification to pH 4.0 with l M citrate. Products were
extracted twice with chloroform, dried under N2 gas, and resuspended in 0.25 ml of
methanol. Compounds were methylated by treatment with ethereal diazomethane at 25°C
for 10 s and dried under N2 gas. Samples subjected to silylation were treated with 30 pl

of the following mixture: pyridine/hexamethyldisilazane/trimethylchlorosilane at a ratio
of 2/1/2 (v/v/v) at 25°C for 20 min (Gemzell-Danielsson and Hamberg, 1994). Following
removal of excess reagent under vacuum, the remaining residue was dissolved in hexane
and analyzed by gas chromatography-mass spectrometry (GC-MS) as previously

described (Howe et al., 2000), with the following modifications. The temperature

49

program was initiated at 50°C, ramped to 320°C at 10°C min], and maintained at 320°C

for 2 min. Stereochemical analysis of products was not attempted. However, it was noted
that incubation of LeAOS3 with hydroperoxides yielded two cyclopentenone products,
presumably diastereomers, which were separable by GC and gave identical mass spectra.
The peak area of these two compounds was added together to compute the total
cyclopentenone composition. The mass spectrum of 12-OPDA produced from LeAOS3-
catalyzed metabolism of 13-HPOT was identical to that of an authentic 12-OPDA

standard (Cayman Chemical).

Biochemical analysis of LeA 0S3

The hydroperoxide-metabolizing activity of recombinant LeAOS3 was measured
spectrophotometn'cally by monitoring the rate of decrease in absorbance at 234 nm
resulting from disruption of the conjugated diene bond of the substrate (Zimmerman and
Vick, 1970). Kinetic assays were performed at 30°C in 1 m1 of 100 mM sodium
phosphate (pH 7.0) containing 30 ng or 60 ng of purified LeAOS3 and varying
concentrations of hydroperoxide substrate (Cayman Chemical). Activity slopes obtained
during the first 0.5 min of the reaction were used for calculation of kinetic parameters.
Absorbance spectra were obtained using purified recombinant protein in 1 ml of 100 mM
sodium phosphate buffer (pH 7.0). Carbon monoxide treatments were performed by
bubbling CO gas through the sample for l min. The protein was reduced by the addition

of a few grains of sodium dithionite.

50

RNA blot analysis

RNA blot analysis was performed as previously described (Howe et al., 2000).
Total RNA was extracted from various tissues of soil-grown plants. The full-length
cDNA clone (cLEIl6112) was used to generate a probe for LeAOS3. RNA quality and

equal loading was assessed by staining a duplicate gel with ethidium bromide (EtBr).

Results

Identification of a new member of the C YP74 family of cytochrome P450s

A search of the tomato EST database (www.tigr.org/tdb/lgi/) was conducted to
identify potential new members of the CYP74 gene family in tomato. A tentative
consensus sequence (Quackenbush et al., 2000), constructed from multiple overlapping
EST clones, was identified that was similar to but clearly distinct from previously
characterized CYP74 sequences in tomato and other plant species. DNA sequence
analysis of one EST clone (cLEIl6112) revealed a l772-bp cDNA insert that contained
an open reading frame predicted to encode a 491-amino acid protein, having a calculated
molecular weight of 55,513. In keeping with the previous nomenclature of tomato
CYP74s (Howe and Schilmiller, 2002) and the biochemical characteristics of the protein
(see below), we henceforth refer to the cDNA as LeAOS3 (L. esculentum A053). The
deduced amino acid sequence of LeAOS3 showed several features that distinguish
CYP74S from P-450 monooxygenases (Fig. 2.2). For example, the I-helix region of P-450
monooxygenases contains an invariant threonine residue that serves an important role in

the binding and activation of oxygen (Chapple, 1998; Paquette et al., 2000). All reported

51

Figure 2.2 Comparison of cDNA-deduced protein sequences of CYP74 P4503 in
tomato. Sequences were aligned using the ClustalW1.8 program available at
SearchLauncher.bcm.tmc.edu. Sequences shown are LeAOSl (AJ 271093; CYP74A),
LeAOSZ (AF230371; CYP74A), LeAOS3 (AF454634; CYP74C), LeHPL
(AF230372; CYP74B), and LeDES (AF317515; CYP74D). Black and shaded boxes
in the alignment indicate positions that contain an identical and conserved amino
acid, respectively. The A symbol denotes the position of the conserved threonine
found in the I-helix of P-450 monooxygenases. The CYP74 consensus sequence

surrounding the cysteinyl heme ligand (*) is underlined.

52

LeAOSl
LeAOSZ
LeAOSB
LeDES
LeHPL

LeAOSl
LeAOSZ
LeAOS3
LeDES
LeHPL

LOADSI
LeAOSZ
LeAOSB
LeDES
LeHPL

LOADSI
LeAOSZ
LeAOS3
LeDES
LeHPL

LeAOSl
LeAOSZ
LeAOS3
LeDES
LeHPL

LeAOSl
LeAOSZ
LeAOS3
LeDES
LeHPL

LeAOSl
LeAOSZ
LaAOSS
LeDES
LeHPL

LeAOSl
LeAOSZ
LeAOSB
LeDES
LeHPL

68
46
23
10
14

136
114
91
78
84

   
   

---MASTSLSLPSLKLQE SSSRKNSSSYRVSIRPIQASVSEIPPYISSPSQSPSSSSSPPVKQAK
MALTLSFSLPLPSLHQKE ------------ TFRPIIVSLSDK ------------- STIEITQPIK
----- MENTKDSYHIITMDA? -------------------------------------------SIPS
------------------------------------------------------------- BLSN
--------------------------------------------------------- APVT
LP R: PGDYGLP GPLKDRLDYPYNQet' FEBS-{extoSTVPRTNMPPCP--PISINPN

L‘ IPCDYGLP CPHKDRLDYFYNQG! “FFES'f KYKSTSPBTNMPPCP-- Ijs PKVEVLLDF
LP EIPGDYSEPF GHIKD IEYNQ {3 FFES';1KYJSTVFRTNIPPGP--B Jaws -
LP LIPGDY P s IKD YFYNQGI gift? :N. {AB YKs'rvaGPu ‘

SIP SYGLP_ CPIHDRLDY * QI’-; FF <- B FKSTVFRT iPP PP

 
 

SEPNHAHLK: I
SEPNHISLKI!

   

206 It”

184
161
148
154

271 .
249 f,

230
216
220

340
318
299

285 »

290

409
387
367
354
360

479
457
436
423
430

 
  
   
 

g Lg- iKMP SVVYBSL-w PP ‘SQY
} Is KMPLfiiSVVYBHL-T PP -SQY
; ,3 3 KMPLVKSVVYBTL-j PPVP Q
T s SISLVKSVVYETL'P'PPVP _QY

 
 

GRAKgDymIISHDAsPBIKIGBLL GYQP ‘TKDPKIFDVSEE IR L GEKLLKHVLWSN SETE
CRAKSDv<IISHDAuPBfiKKCBiL GYQP ‘TKDPKIFDP PEBFVHDR ; GBKLLKHVLWSN B TE
r H'srsflIKKi- GYQP TKDQKID BB PIIR .4 CBKLLKEVEWSNOBI;
AP ‘OSHDAS <I§K or. GYQPA EI-PKI PQB BIR iNP-GEKELKHVLWSNG TE
-c-FKLSSHD TYBIKKGBLL-CYQP KDPKDPDIPBI ”RP «Bnec LL! L SN P-T

 

Figure 2.2 Comparison of cDNA-deduced protein sequences of CYP74 P450s in

tomato.

53

CYP74s, including LeAOS3, contain a small hydrophobic residue (I/AfV) at this
position. The CYP74 consensus sequence for residues surrounding the cysteinyl heme
ligand near the C terminus is NKQC(A/P)(G/A)K(D/N)XV. With the exception of a V —)
I substitution in the last position, this sequence is conserved in LeAOSB. Analysis of the
deduced amino acid sequence of LeAOS3 using the ChloroP program
(www.cbs.dtu.dk/services/ChloroP/) suggested that the protein does not contain a
predicted N-terminal targeting sequence that directs many, but not all (Maucher et al.,
2000; Froehlich et al., 2001), CYP74 P-4SOs to the chloroplast.

Phylogenetic analysis was used to gain insight into the functional relationship
between LeAOS3 and extant members of the CYP74 family, which is comprised of four
subfamilies (CYP74A through CYP74D). An unrooted neighbor-joining phylogeny
indicated that CYP74 members fall into distinct groups that reflect, to a large extent, the
known enzymatic identity and substrate specificity of individual members (Figure 2.3).
This phylogeny was in perfect agreement with that obtained using a maximum parsimony
algorithm (not shown). The CYP74A subfamily, which forms a monophyletic cluster
with monocots positioned basally to the dicots, consists mainly of AOSs that have
specificity for 13-hydroperoxides (i.e. 13-AOSS). The CYP74B subfamily also forms a
well-defined group comprised of HPLs that have specificity for 13-hydroperoxides (i.e.
13-HPLs). CYP74C P4505 from cucumber (CsHPL) and melon (CmHPL) are HPLs that
have a preference, but not absolute specificity, for 9-hydroperoxides (Matsui et al., 2000;
Tijet et al., 2001). Members of the CYP74D subfamily are DESs that have high
specificity for 9-hydroperoxides (Itoh and Howe, 2001; Stumpe et al., 2001). LeAOS3

was most similar to the mixed specificity HPLs (CYP74Cs) from melon (58% identity)

54

 
  
  
   
 
  

 

 

I CYP74A I

HvAOSZ
HvAOS1

OsAOS
AtAOS

 

 

NaAOS

 

StAOSZ
LeAOSZ

LeAOS1
StAOS1

LuAOS
PaAOS

     
 

NaHPL MsHPL1

AtHPL CYP74B

 

 

  

Figure 2.3 Phylogenetic analysis of plant CY P74 sequences. An unrooted
neighbor-joining phylogeny was created in PAUP4.0 from the deduced amino
acid sequences of representative CYP74 P-4SOs. Enclosed groups indicate the

four different CYP74 subfamilies (CYP74A through CYP74D).

55

and cucumber (57% identity). In accordance with cytochromes P-450 nomenclature,

LeAOS3 was classified as a CYP74C and was assigned the name CYP74C3.

Biochemical properties ofLeA 0S3

The full-length LeA 0S3 cDNA was subcloned into an expression vector (pET23b)
that adds a Hi56 tag to the C-terminus of the protein. Following introduction of this

construct into E. coli, recombinant LeAOS3 was expressed and purified using metal
affinity chromatography. The majority of LeAOSB was found in inclusion bodies in the
3,000 x g pellet after centrifugation of sonicated cells. Approximately 80% of enzyme
activity found in the 3,000 x g supernatant was subsequently recovered in the 100,000 x g
pellet, indicating that the active protein is associated with bacterial membranes.
Solubilization of membranes with Triton X-100R followed by cobalt-affinity

chromatography allowed purification of LeAOS3 to ~95% homogeneity (Fig. 2.4). The
apparent molecular weight of His6-tagged LeAOS3, as determined by SDS-

polyacrylamide gel electrophoresis, was in good agreement with the calculated molecular
weight of 56,578. The typical yield of affinity-purified LeAOS3 was 0.5 mg/liter of E.
coli culture.

A spectrophotometric assay (Zimmerman and Vick, 1970) was used to determine
whether purified LeAOS3 could metabolize fatty acid hydroperoxides (Fig. 2.5A). The
results showed that LeAOS3 was highly active against 9-HPOD. The enzyme also
metabolized 13-hydroperoxy linoleic acid (13-HPOD), albeit at a rate ~12-fold lower
than that observed for 9-HPOD. A similar preference for 9-hydroperoxy linolenic acid (9-

HPOT) compared with l3-HPOT was also observed (not shown; see below). These

56

 

U)
Q)
r:
E!
A
8 "a z: c:
s: E
<3 o :s g
36 E '3 "o'
"‘ E B o o
g .. N >. A
E z: 3:: t:
5 v a s a
E 212 '5 I: ‘4:
Q 'a')‘ m cu m
a a. o E E
oo o o o
3? >< '7‘ ¢ 9:
o o X r: t:
o o g: .9 2
o q o H *‘
r o 3:: 8 8
8 O t— i... u...
_ ._ l- LL. LL.
205 “i“
120 u" I” M W m
H
84 ’ . ...“--V‘ i. ‘- " .-
wm“? ‘
522 fire—mu“
“
36.3 M * ~
0‘“ M m
30-2 .._.._.. T “. “a

Figure 2.4 Affinity purification of LeAOS3 expressed in E. coli. Protein
fractions obtained during the purification of His-tagged LeAOSB were
analyzed by SDS-polyacrylamide gel electrophoresis. A coomassie stained
gel is shown with molecular weight marker sizes indicated at left. Crude cell
extract was centrifuged at 100,000 x g to give a soluble fraction and
membrane enriched fraction. Membranes were solublized using Triton X-
100R and spun again at 100,000 x g and the supernatant containing
solubilized membrane protein was loaded onto a Co-affinity column. After

washing, fraction were eluted from the column with imidazole.

57

 

0.40
A 0.35 r
0.30 -
0.25 .
0.20 -
0.15 4
0.10 ~
0.05

A234 nm

 

 

 

 

 

0.0 0.2 0.4 0.6 0.8 1.0

Time (min)

 

B 0.15 -
0.10 ~

0.05 ~

Absorbance

 

0.00 ~

 

 

 

I i I T

200 220 240 260 280 300

wavelength (nm)

Figure 2.5 Metabolism of fatty acid hydroperoxides by LeAOS3. (A)
utilization of 9-HPOD (solid line) and 13-HPOD (dotted line) by LeAOS3 in
a spectrophotometric assay that monitors the loss of absorbance at 234 nm of
the substrate. 9-HPOD was not metabolized by crude extract from E. coli
harboring the empty expression vector (dashed line). (B) UV spectra were
taken before (solid line) and 5 minutes afier (dotted line) addition of LeAOS3

to a reaction using 9-HPOD.

58

results indicate that LeAOS3 is active against both 13- and 9-hydr0peroxy fatty acids, but
with a marked preference for the latter substrates.

The UV spectra of the reaction product generated from 9-HPOD (Fig. 2.58) and
other hydroperoxides (data not shown) were featureless, indicating that the substrate

(Kmax = 234 nm) was largely consumed during the reaction, and that divinyl ethers

250—253 nm) were not among the major products. LeAOS3 also showed no

(kmax
detectable HPL activity using an NADH-coupled assay (Vick, 1991) that readily detected
products formed by the action of recombinant tomato HPL on 13-HPOT (data not shown
and (Howe et al., 2000)). These results suggested that LeAOS3 possess neither DES nor
HPL activity. To determine the enzymatic identity of LeAOS3, products generated from
various substrates were converted to the corresponding methyl ester/T MS derivatives and
subjected to GC-MS analysis (Fig. 2.6 and Table 2.1). The major product (77.2%)
obtained from reaction with 9-HPOD gave a mass spectrum that was identical to the
reported spectrum (Grechkin et al., 2000) for the a-ketol, 9-hydroxy—lO-oxo-12-
octadecenoic acid (Fig. 2.6A). The second most abundant (8.7%) product gave a mass
spectrum identical to that reported (Hamberg, 2000) for the cyclopentenone IO-OPEA
(Fig. 2.68). A third product (Fig. 2.6C), present in minor amounts (1.9%), was identified
as the y-ketol (lO-oxo-lB-hydroxy-ll-octadecenoic acid) by comparison to published
spectra of this compound (Grechkin et al., 2000). Because each of these products is
known to arise from spontaneous hydrolysis of 9,10-EOD that is generated by the action
of AOS on 9-HPOD (Grechkin et al., 2000; Hamberg, 2000), we conclude that LeAOS3
is an AOS. Analysis of products derived from other hydroperoxy fatty acids gave results

that were consistent with the identification of LeA083 as an AOS (Table 2.1). For

59

Figure 2.6 Mass spectra of products formed by incubation of LeA083 with 9-
HPOD. Purified LeAOS3 was incubated with 9-HPOD at 25°C for 5 min at pH
7.0. The resulting products were analyzed as the methyl ester TMS derivatives by

GC-MS. Shown are spectra for the a-ketol (9-hydroxy-10-oxo-lZ-octadecenoic

acid) (A), the cyclopentenone 10-oxo-l l-phytoenoic acid (IO-OPEA) (B), and the

y-ketol (l 0-oxo-13-hydroxy-l l-octadecenoic acid) (C).

60

 

259

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A 100 ~
,3 j COOCH3 383
2‘, 801 "‘
8 ( TMSO 0
g .
'2 60 367
B
if, 40 i 73 155 if i 398
30% i LLJi A r L - 7 i L
6‘2 201 109 *10.0
‘ 55 95 129
0 i ALL il .u....i'lh. -Lilr in . 7 AtzL27n +- 1 4 J]. v f fi g f
100 200 300 400
B
100 g 152 277
A j 95 0
°\° . coocu;
o .
c .
8 60 «
c .
3 .
g .
g 40 f
:3 j 82
Q} a
l 57 L123
Di lllL,LL. AAJAfl-u- , rial; .‘qufigk nun-file. - TL. .........
50 100 150 200 250 300 350 400
C
100: 327
1; j coocu,
E: 80 -
8 .
g 603 73 0 OTMS
g 4 199
‘3 l
<
.3 40; 184
g 1
o: 20 - 223
. 55 129
. l 319JJ5 i 277295 36., 383 398
0 Yr] .Ll IJ'. Jim} J'JalLLlhliL 11.1.
50 100 150 200 250 300 350 400

Figure 2.6 Mass spectra of products formed by incubation of LeAOS3 with 9-HPOD.

61

Table 2.1 Analysis of LeAOS3 reaction products by GC-MS. For analysis of
products formed by A083, purified recombinant protein was incubated with each of
the four substrates at 25 °C for 5 minutes. Following extraction and derivatization,
product identity was determined by GC-MS analysis. For each identified product, its

percentage of the total peak area as well as the prominent MS ions and their relative

abundance are given.

 

 

 

 

 

Substrate Product Rel. m/z of ion fragments (relative abundance)
abundance
9-HPOD a-ketol 77.2 % 398(1), 383(6), 259(100), 155(34),
129(10), 109(18), 73(37)
y-ketol 1.9 % 398(9), 383( 10), 327(100), 295(14),
227(8), 223(17), 199(53), 184(36),
129(14), 95(12), 73(65)
lO-oxo-l 1- 8.7 % 308(3), 277(9), 233(2), 192(4), 152(100),
phytoenoic acid 123(6), 109(14), 95(75), 82(23)
9 HP OT a-ketol 61.1 % 396(2), 381(4), 365(2), 259(100), 227(5),
' 155(39), 129(11), 109(23), 95(9), 73(44)
y-ketol 1.8 % 381(4), 365(4), 327(100), 295(11),
223(26), 197(8), 129(8), 73(75)
10-oxo-11,15- 3.5 % 306(1), 275(7), 238(7), 150( 19), 121(18),
phytodienoic acid 82( 100)
_ a-ketol 39.9 % 383(5), 367(2), 270(19), 173(100), 129(4),
13 HPOD 103(13), 73(43)
12-oxo-10- 8.1 % 308(7), 277(22), 238(45), 206(30),
phytoenoic acid 165(15), 151(23), 109(56), 96(100),
82(52), 67(21), 55(34)
_ a-ketol 37.4 % 381(4), 365(2), 270(21), 171(100),
13 HPOT 129(14), 103(9), 73(42)
12-ox0-10,15- 12.5 % 306(33), 275(32), 238(54), 206(22),
phytodienoic acid 163(54), 149(36), 121(36), 107(65),

96(86), 95(100), 82(51), 67(44), 55(53)

 

62

instance, when LeAOS3 was incubated with 9-HPOT, the reaction product contained the
corresponding a-ketol (9-hydroxy-10-oxo-12—l5-octadecadienoic acid, 61.1%), y-ketol
(10-oxo-l3-hydroxy-11—15-octadecadienoic acid, 1.8%), and the cyclopentenone 10-
OPDA (3.5%). Upon incubation of LeAOS3 with 13-HPOD or 13-HPOT, the major
products of catalysis were the corresponding a-ketol and cyclopentenone oxylipins. The
UV-visible spectrum of purified LeAOS3 was typical of the low spin ferric state, and
showed a main Soret band at 418 nm (Fig. 2.7). Reduction of the protein with sodium
dithionite and treatment with CO resulted in the appearance of a spectral peak (448 nm)
that is a hallmark of cytochromes P-450. Similar spectra have been observed for other
CYP74 P-4505 including flaxseed AOS, melon HPL, and tomato DES (Song et al., 1993;
Itoh and Howe, 2001; Tijet et al., 2001). The persistence of the 420-nm peak in the
LeAOS3 difference spectrum (Fig. 2.7, inset) was also observed in difference spectra
recorded on membranes from LeAOS3-expressing E. coli cells (data not shown). These
results suggest that the P-420 species does not result from inactivation of the enzyme by a
step (e. g. elution with imidazole) during affinity purification. Rather, the P-420 form may
reflect improperly folded protein or the weak interaction of CO with the active site of
CYP74 P-4508 (Song and Brash, 1991; Lau et al., 1993).

Purified LeAOS3 was used to determine the kinetic parameters of reactions

conducted with 9-HPOD, 9-HPOT, 13-HPOD, and l3-HPOT (Table 2.2). The apparent
Km of all four substrates ranged between 4 uM (13-HPOT) and 21 pM (9-HPOD). Km

values for 9-hydroperoxides were 2—4-fold higher than those for the corresponding 13-

hydroperoxides. LeAOS3 was most active against 9-HPOD, as determined both by the

estimated turnover rate (km) and catalytic efficiency (kw/Km). The estimated kw, value

63

 

0.2 -

Absorbance

10.02A

 

 

 

0-11 400 450 500

Absorbance

 

 

0.0

 

380 400 420 440 480 480 500
wavelength (nm)

Figure 2.7 Carbon monoxide difference spectrum of recombinant

LeAOS3. All spectra were recorded using 100 pg of purified LeAOS3 in 1

ml of 0.1 M sodium phosphate buffer (pH 7.0). The spectra shown are from

the native protein (solid line), protein afier reduction with sodium dithionite

(dotted line), and reduced protein after bubbling with CO for 1 minute

(dashed line). The inset shows the difference spectrum obtained by

subtracting the reduced protein spectrum from the CO-treated protein

spectrum.

64

Table 2.2 Substrate specificity of recombinant LeAOS3

 

 

Substrate (11:2) 2?; 3231:";
9-HPOD 21 3: 5 820 3. 93 39 x 106
9-HPOT 163:5 236i27 15x 106
13-HPOD 11 i 2 99 a: 3 9.0 x 106
13-HPOT 432 21 $2 5.3 x106

 

Assays were performed at 30 °C in 1 ml 0.05 M sodium phosphate
buffer, pH 7.0. Assays using the 9-hydroperoxy fatty acids as
substrate contained 30 ng of purified protein. Assays using the
l3-hydroperoxy fatty acids as substrate contained 60 ng of
purified protein. Values represent the average iSD of two

experiments using independent protein preparations.

65

(820 $4) for 9-HPOD was comparable to turnover rates reported for other recombinant

CYP74 enzymes (Song and Brash, 1991; Matsui et al., 2000; Itoh and Howe, 2001; Tijet

et al., 2001).

Tissue speafic expression of LeA 0S3 in roots

EST sequencing data (ww.tigr.ormtdb/lgifl indicated that all ESTs
corresponding to LeAOS3 were identified in cDNA libraries constructed from either
germinating seedlings (9 of 10 ESTs) or roots (1 of 10 ESTs). To further investigate the
developmental expression of LeAOS3, RNA blot analysis was used to determine the
abundance of LeAOS3 transcript in various tomato organs. The results showed that
LeAOS3 mRNA accumulated in roots of mature plants with no expression detected in
aerial tissues including cotyledons, leaves, stems, and flower buds (Figure 2.8). We also
confirmed that LeAOS3 was expressed early afier seed germination (4 days after seed
imbibition), when the radical had just emerged from the seed coat (data not shown).
These findings indicate that LeAOS3 transcript accumulation is tightly regulated by
developmental cues and further suggest that expression of the gene is restricted to soil-

exposed tissues.

66

BRSPCL

LeA 053 raw->1

 

Figure 2.8 Tissue specific expression of LeAOS3. Total RNA was extracted
from unopened flower buds (B) from six-week-old tomato plants and from
roots (R), stems (S), petioles (P), cotyledons (C), and leaves (L) from 18-day—
old plants. RNA blot was hybridized to a LeAOS3 cDNA probe. An ethidium

bromide (EtBr) stained agarose gel of the same samples is also shown.

67

Discussion

In the present study we report the functional characterization of a novel tomato
cDNA (LeAOS3) encoding an AOS that catalyzes the production of ketol and
cyc10pentenone oxylipins from both 9- and 13-hydroperoxy fatty acids. Several features
of the enzyme are distinct from previously characterized AOSs involved in JA
biosynthesis. First, the deduced amino acid sequence of LeAOS3 is more similar to
cucumber and melon HPLs than it is to l3-AOSs from tomato (i.e. LeAOSl and
LeAOS2) or other plants. In this context, LeAOS3 represents the first example of an AOS
that is classified as a CYP74C; all other AOSs are classified as CYP74As. Second, in
contrast to the specificity of most AOSs for 13-hydroperoxides, LeAOS3 exhibits a
marked preference for 9-hydroperoxides. A similar substrate preference was reported for
the two cucurbit HPLs that are the closest known relatives of LeAOS3 (Matsui et al.,
2000; Tij et et al., 2001). This observation suggests that the sequence relatedness between
various CYP74 P-4503 is an indicator of substrate specificity. Consistent with this notion,
CYP74Cs are more closely related to the 9-hydroperoxide-specific CYP74Ds than they
are to members of the CYP74A and B subfamilies, which have relative specificity for 13-
hydroperoxides. A third unique feature of LeA 0S3 is its tissue-specific expression pattern
in germinating seeds and roots of mature plants. This finding supports previous studies
showing that cell-free extracts from tomato roots catalyze the formation of a-ketols via
the 9-LOX/AOS pathway (Caldelari and Farmer, 1998). Taken together, our results
strongly suggest that LeAOS3 defines a class of A085 that is distinct from those involved

in JA biosynthesis in photosynthetic tissues. This idea is consistent with the notion that

68

oxylipin metabolism is organized into 9-LOX and 13-LOX pathways, each of which use
a specialized type of AOS for production of distinct oxylipins in specific cell and tissue
types (Howe and Schilmiller, 2002).

The overall characteristics of LeAOS3 suggest a biochemical function in the
production of one or more 9-hydroperoxide derived oxylipins. Given the expression of
LeAOS3 in roots, it is conceivable that the enzyme plays a defensive role against soil-
bome pests that affect roots or juvenile tissues (e.g. radical) as they emerge from the
germinating seed. This hypothesis is in keeping with increasing evidence for a role of the
9-LOX pathway in plant defense against pathogens (Masui et al., 1989; Rance et al.,
1998; Rusterucci et al., 1999; Weber et al., 1999; Gobel et al., 2001; Stumpe et al., 2001).
However, whereas pathogen-induced stimulation of the 9-LOX pathway has been shown
to activate the DES, epoxy alcohol synthase, and reductase branches of the 9-LOX
pathway (Gobel et al., 2001), there is little evidence that biotic stress activates the 9-AOS
pathway. A second possibility is that LeAOS3 catalyzes the production of oxylipins that
play a role in plant development. Support for this hypothesis comes from two recent
studies. One study provided evidence that 9-hydroxy-10-oxo-12,15-octadecadienoic acid,
the a-ketol produced by the action of LeAOS3 on 9-HPOT, functions as a signal for
flower development in Lemna paucicostata (Yokoyama et al., 2000). Another study
showed that transgenic potato plants depleted in the expression of a 9-LOX gene
exhibited abnormal tuber development (Kolomiets et al., 2001). Although specific
oxylipins that account for the tuber phenotype were not identified, the presence of 9-AOS
activity in potato stolons (Hamberg, 2000) is consistent with a role for 9-hydroperoxide-

derived compounds in tuber development.

69

One of the more interesting features of LeAOS3 is its involvement n the
formation of lO-OPEA and 10-OPDA from 9-hydroperoxides of linoleic and linolenic
acids, respectively. These novel cyclopentenones were recently identified as products of
the 9-LOX/AOS pathway in potato (Hamberg, 2000). Indeed, several similarities
between LeAOS3 and the potato stolon 9—AOS activity strongly suggest that the enzymes
are functionally equivalent. First, the relative abundance of lO-OPEA, a-ketol, and y-
ketol products formed by the action of LeAOS3 on 9-HPOD was comparable to that
reported for the potato enzyme. Second, the tissue-specific expression of LeAOS3 is
similar to that of the cyclopentenone-forming activity in potato, which was highest in
roots and not detectable in leaves. A third similarity between LeAOS3 and the potato 9-
AOS was the relatively high proportion of cyclopentenone product (i.e. lO-OPEA)
formed from 9-HPOD. This feature of 9-AOS-catalyzed metabolism of hydroperoxy
dienoic fatty acids contrasts other studies showing that non-enzymatic cyclization of
allene oxides requires the presence of a double bond in the [Sq-position relative to the
epoxide group (Vick and Zimmerman, 1979; Hamberg and Fahlstadius, 1990; Grechkin,
1994)

The formation of cyclopentenones by LeAOS3 is particularly interesting in light
of the signaling activity exhibited by cyclopentenones (e.g. 12-OPDA) produced from the
l3-LOX/AOS pathway. Recent studies (Stintzi et al., 2001) in Arabidopsis have shown
that 12-OPDA can activate the expression of defense-related genes via a signal
transduction pathway involving C011, an F-box protein that is required for J A-mediated
signaling (Turner et al., 2002). There is also evidence to suggest that the oc,B-unsaturated

carbonyl group located in the cyclopentenone ring 12-OPDA is involved in defense gene

70

activation via a C011-independent signaling pathway (Stintzi et al., 2001). Such a
signaling mechanism has been documented for cyclopentenone prostaglandins in which
the reactive 01,13-unsaturated carbonyl mediates conjugate addition to various intracellular
targets (Straus and Glass, 2001). Given the structural similarity of 9—AOS-derived
cyclopentenones to 12-OPDA (Figure 2.1), including the presence of an 01,13-unsaturated
carbonyl group in the cyclopentenone ring, a role for these compounds in signaling can
be hypothesized. Additional work is needed to determine whether 9-AOS catalyzes the

formation of 10-OPEA and lO-OPDA in plant tissues.

71

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74

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75

Chapter 3

Regulation of Allene Oxide Synthase3 Expression in Tomato

76

Abstract

Allene oxide synthase3 (LeAOS3) is a CYP74 cytochrome P450 from tomato that
metabolizes 9-hydroperoxides of linolenic and linoleic acid to unstable allene oxides that
spontaneously convert in the presence of water to 01- and y-ketols or racemic
cyclopentenones. This study focuses on understanding the regulation of LeAOS3
expression and activity as a means to gain greater insight into the physiological function
of the 9-AOS pathway. The expression of LeAOS3 was highly induced in roots upon
methyl-jasmonate (MeJA) treatment or wounding. Expression of LeAOS3 in roots was
abolished in the jail mutant that is defective in jasmonic acid (JA) perception. Whereas
expression of LeAOS3 in roots requires a functional JA signaling pathway, expression in
hypocotyls of germinating seedlings occurred constitutively and independently of JA,
indicating that there are different tissue-specific modes of regulation for the 9-AOS
pathway. Irnmunocytochemical studies showed that LeAOS3 protein is localized
specifically in cortex cell layers of both hypocotyls and growing root tips. cDNA
microarray analysis was used to identify other genes that are co-regulated with LeAOS3
in tomato roots. These findings suggest a possible role for the 9-AOS pathway in defense

against soil-home invaders.

77

Introduction

Oxidative metabolism of polyunsaturated fatty acids gives rise to a diverse set of
compounds collectively called oxylipins. Biosynthesis of oxylipins in plants is initiated
by lipoxygenase (LOX), which adds molecular oxygen to the 9- or 13-position of C16
and C18 polyunsaturated fatty acids (F eussner and Wasternack, 2002). The
hydroperoxide-containing fatty acids that are formed serve as substrates for the CYP74
family of cytochromes P450 that are responsible for generating different types of
oxylipins (Figure 3.1) Several recent studies provide evidence that oxylipins play a role
in a variety of developmental and stress-related processes (Howe and Schilmiller, 2002).
The best example of this is the jasmonate family of oxylipins (collectively referred to as
JAs) that includes jasmonic acid (JA) and its derivatives (Howe, 2005; Schaller et al.,
2005). Whereas JAs are known to regulate many processes in plant development and
defense, relatively little is known about the physiological function of other oxylipins.

LeAOS3 is a CYP74 cytochrome P450 that metabolizes 9-hydroperoxides of
linolenic and linoleic acid to 9,10-epoxyoctadecatrienoic and 9,10-epoxyoctadecadienoic
acids, respectively (Itoh et al., 2002). These unstable allene oxides spontaneously convert
in vitro to 01- and y-ketols or racemic cyclopentenones (Figure 3.1). Several lines of
evidence link the 9-LOX pathway to defense against pathogens. For example, oxylipin
profiling studies have shown that several 9-LOX pathway products accumulate upon
treatment with elicitors or pathogen infection (Gobel et al., 2001; Gobel et al., 2002).
Consistent with these findings, antisense-mediated depletion of a 9-LOX in tobacco

resulted in susceptibility to Phytophthora parasitica (Rance et al., 1998). Others have

78

Figure 3.1 CYP74-catalyzed oxylipin biosynthesis. Lipoxygenase (LOX) adds a
hydroperoxy group at the 9- or 13-position of polyunsaturated fatty acids. The
hydroperoxy fatty acids then serve as substrates for the CYP74 family of cytochromes
P450 (in bold) that target the hydroperoxides to various branches of oxylipin
biosynthesis. (DES, divinyl ether synthase; HPL, hydroperoxide lyase; AOS, allene
oxide synthase; AOC, allene oxide cyclase; OPDA, 12-oxophytodienoic acid; OPR,

OPDA-reductase)

79

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80

suggested a role for the 9-LOX pathway in the hypersensitive response to pathogen attack
(Rusterucci et al., 1999; Jalloul et al., 2002). However, there is no evidence to date to
indicate that the AOS branch of the 9-LOX pathway is involved in defense. An
alternative hypothesis is that 9-AOS functions in plant development. Support for this idea
comes from the observation that antisense suppression of a 9-LOX in potato (StLOXl)
results in reduced tuber yield, and defective tuber development (Kolomiets et al., 2001).
The presence of 9-AOS activity in potato roots, stolons, and tubers (Hamberg, 2000)
indicates the possibility that 9-AOS could function in tuber development.

To gain additional insight into the function of the 9-LOX pathway, we studied the
regulation and tissue—specific expression of LeAOS3 in tomato. Analysis of a tomato
mutant (jail) disrupted in the ortholog of Arabidopsis C011 showed LeAOS3 expression
in roots requires JA perception. However, LeAOS3 expression in hypocotyls of
germinating seedlings was independent of COD. Microarray analysis showed that the J A-
and wound-induced expression of LeAOS3 in roots was very similar to that of other
known defense genes. These advances in our understanding of the regulation of LeAOS3
lay the foundation for testing specific hypotheses concerning the physiological role of the

9-LOX pathway in plants.

81

Materials and Methods

Plant materials and growth conditions

Tomato (Lycopersicon esculentum) cv Micro-Tom was used as the wild type for
all experiments. Seeds were germinated on moist filter paper for experiments with
germinating seedlings. Older plants were grown in Jiffy peat pots (Hummert
International, Earth City, M0) for up to three weeks, and then transferred to a Baccto soil

and sand mix (1:1; Michigan Peat Company, Houston, TX). Plants were maintained in a

growth chamber under 17 h of light (200 pE/mZ/s) at 28°C and 7 h of dark at 18°C.

Homozygous jail -I mutants were obtained from a segregating F2 population as described

previously (Li et al., 2001). For methyl-1A (MeJA) treatment of tomato roots, plants
growing in peat pots or pots with a soil and sand mix were watered with a 0.1 mM
solution of MeJ A. 500 ml of solution was added to a tray containing ~ 25 peat pots or 12

four-inch pots.

Gene expression analysis

RNA extraction and analysis was performed as previously described (Howe et al.,
2000). A cDNA probe for LeAOS3 was made from a full-length EST clone (cLEIl6112)
obtained from Clemson University. As a loading control, identical RNA blots were
hybridized with a cDNA probe for translation initiation factor eIF 4A mRNA (EST clone
cLED1D24). The quality of RNA was assessed by staining of agarose gels with ethidium

bromide.

82

Antibody production and protein expression analysis

Recombinant His-tagged LeAOSB was purified from E. coli as described
previously (Itoh et al., 2002) and further purified by preparative SDS-PAGE. Acrylamide
gel slices containing LeAOS3 were sent for polyclonal antibody production (Cocalico
Biologicals, Reamstown, PA) following the company’s standard protocol for
immunization in rabbits. Total protein was extracted by grinding frozen plant tissue in
liquid nitrogen and adding extraction buffer (50 mM sodium phosphate pH 7.0, 150 mM
NaCl, 10% glycerol, and Roche mini-complete protease inhibitors) at a buffer to tissue
ratio of approximately 2:1 (v/w). Plant debris was removed by centrifugation at 13,000 x
g and the protein concentration of the supernatant was measured with the BCA assay
(Pierce, Rockford, IL), with BSA as a standard. Protein was separated on 10% SDS-
polyacrylamide gels and transferred to Irnmobilon-P membranes (Millipore, Bedford,
MA) by standard procedures (Harlowe and Lane, 1988). Membranes were probed with
anti-LeAOS3 serum (diluted 1:1000) in Tris-buffered saline (TBS) containing 1% (w/v)
nonfat dry milk and 0.1% (v/v) Tween 20. Antigen-antibody complexes were detected
with an alkaline phosphatase-conjugated secondary antibody (diluted 1:5000; Kirkegaard
& Perry Laboratories, Gaithersburg, MD) in TBS containing 1% (w/v) nonfat dry milk
and 0.1% (v/v) Tween 20, followed by detection with NBT/BCIP substrate (Roche,

Indianapolis, IN).
LeA 0S3 activity assay

LeAOS3 activity was measured as previously described with the following minor

modifications (Caldelari and Farmer, 1998). Cell-free extracts were collected by grinding

83

tissue in a chilled mortar, transferring the tissue to a microcentrifuge tube and pelleting
cell debris. The resulting supernatant was used immediately in the following activity
assay. The reaction (total volume 0.4 ml) was initiated by addition of 15 111 of cell-free

extract (~ 5 pg protein/111) to 40 mM sodium phosphate buffer (pH 7.0) containing 10%

glycerol and 0.56 11g of [1-14C]-linoleic acid (50 pCi/pmol; Perkin Elmer, Boston, MA).
After a 10 min incubation at 25°C, products were extracted with CHCl3-MeOH (2:1) and
the CHC13 phase was dried under a stream of nitrogen gas. Products were resuspended in

10 111 of CHC13 and separated by thin layer chromatography (TLC) using silica plates and

petroleum ether/diethyl ether/formic acid (70:30:1) as the solvent system. Following
separation, plates were analyzed by autoradiography. To generate oxylipin standards
(hydroperoxy fatty acids, (at-ketols, divinyl ethers), 9-LOX was partially purified from
ripe tomato fruit pericarp as previously described (Smith et al., 1997). This enzyme was
used alone or with purified recombinant LeAOS3 or LeDES (Itoh and Howe, 2001; Itoh

et al., 2002) in the assay described above.

Immunocytochemistry

Root tissue (from four-week old plants grown in sand/soil mix) or hypocotyl
tissue (from five-day old germinating seedlings) was fixed in FAA (3% w/v
formaldehyde, 5% v/v acetic acid, 50% v/v ethanol) at room temperature for 3 h and
embedded in polyester wax (Steedman, 1957) at 37°C overnight as previously described
(Vitha et al., 2000). Tissue sections (9-11m-thick) were made with a Leica RM2155
microtome (Leica, Germany) and attached to poly-L-lysine coated glass slides (Sigma,

St. Louis, MO). Sections were dewaxed and rehydrated as described (Vitha et al., 2000),

84

followed by an antigen retrieval procedure in which slides were submerged in 100 mM
Tris-HCl (pH 10.0) in plastic Coplin jars and heated in a microwave to near boiling. The

jars were allowed to cool at room temperature for 30 min and then washed once in PBS
(0.14 M NaCl, 2.7 mM KCl, 6.5 mM NazHPO4, 1.5 mM KH2P04, 3 mM NaN3, pH 7.3)

for 10 min. Slides were then transferred to blocking buffer (PBS containing 0.05% v/v
Tween 20 and 2% w/v nonfat dry milk) for 2 h followed by incubation with purified
LeAOS3 antibody (diluted 1:500) in blocking buffer overnight at room temperature.
Control slides were incubated with an equivalent amount of pre-immune serum. After
five 10 min wash steps in PBS containing 0.05% (v/v) Tween 20, slides were incubated
for 30 min in blocking buffer followed by a 4 h incubation in blocking buffer with goat
anti-rabbit secondary antibody conjugated with Alexa 488 fluorophore (diluted 1:300;
Sigma). Slides were then washed four times (10 min each) in PBS containing 0.05% (v/v)
Tween 20, then once for 10 min in PBS alone. After washing, 30 111 of mounting medium
(1 mg/ml p-phenylendiamine in 0.01 M potassium phosphate (pH 7.4), 0.15 M NaCl,
90% glycerol; adjust pH to 8.0 with Tris-HCl (pH 8.0) and add DAPI to 1 pg/ml) was
added to each slide. Slides were covered with a glass cover slip and sealed with nail
polish. Sections were analyzed with an epifluorescence Zeiss Axio Imager M1
microscope (Zeiss, Germany) equipped with the appropriate filter set (excitation 472 nm,
bandwidth 30 nm; emission 520 nm, bandwidth 35 nm).

To purify LeAOS3 antibodies, 225 11g of total protein from jaiI roots, which do
not express LeAOS3, was separated on a 10% polyacrylamide gel and transferred to an
Irnmobilon-P membrane (see above). The membrane was blocked with 2% non-fat milk

in PBS containing 0.05% (v/v) Tween 20 for l h, and then incubated with LeAOS3

85

antibodies (diluted 1:500) in blocking buffer for l h. LeAOS3 antibodies that remained in
the blocking buffer was used directly for incubation with sectioned tissue on slides, or

was stored at -80°C until needed.

Microarray analysis

Three-week-old wild-type and jail —I tomato plants grown in peat pots were
watered with a solution of 0.1 mM MeJA. At various times after treatment (1, 6, 12, and
24 h), roots from 10 plants were harvested, pooled, and frozen in liquid nitrogen. Roots
from untreated plants were harvested as the “0” h time point. Three biological replicates
were collected for each time point. For RNA extraction, tissue was ground under liquid
nitrogen and transferred to a 13 ml polypropylene tube containing 4 ml phenol and 4 ml
100 mM Tris-HCl (pH 8.0) and then mixed by vortexing. After centrifugation, the
aqueous phase was transferred to a new tube and extracted twice with 5 ml of chloroform.
RNA was precipitated with LiCl (2M final concentration) overnight at 4°C, and then
pelleted by centrifugation. The resulting RNA pellet was washed with cold 70% ethanol,
air dried, and then resuspended in 300 pl of diethyl pyrocarbonate (DEPC)-treated water.
RNA was then treated with RNase free DNaseI (Ambion, Austin, TX) according to the
manufacturer’s instructions. Following DNaseI treatment, RNA was extracted once with
phenol/chlorofonn/isoamyl alcohol (25/24/1), and then precipitated overnight with 2.5
volumes of cold ethanol and 1/ 10 volume of 2.5 M sodium acetate (pH 4.8) at -20°C.
RNA was collected by centrifugation and washed once with cold 70% ethanol, air dried,
and then resuspended in DEPC-water. The quality of RNA was analyzed by gel-

electrophoresis and quantified by UV absorbance.

86

All of the steps for microarray analysis (including cDNA production and labeling,
hybridization, and data normalization with LOWESS) were performed by the TIGR
Solanaceae Gene Expression profiling service according to their standard operating
procedures (http://www.tigr.org/tdb/potato/microarrav SOPs.shtml). Version 3 of the
TIGR potato cDNA microarray, which contains 15,264 potato cDNA clones with each
clone spotted twice on the array (http://wwwtigr.org/tdb/potato/microarrav_comp.shtml),
was used for all experiments. For our analysis, genes showing an expression ratio of 2 3
or S 0.33 in at least two of the three biological replicates were counted as being
differentially regulated. The final expression ratio was calculated as the average ratio

from the three biological replicates.

Results

Wound-induced expression of LeAOS3 in roots requires the JA-signaling pathway
Wound-induced expression of AOS genes in leaves of several plants has been
shown to involve the JA-signaling pathway (Laudert and Weiler, 1998; Howe et al.,
2000; Maucher et al., 2000; Sivasankar et al., 2000; Ziegler et al., 2001). To determine
whether JA signaling is involved in the regulation of LeAOS3 expression, we treated
wild-type and C011-deficient jaiI plants with a 0.1 mM solution of MeJA, and then
collected root tissue at various times after treatment for RNA and western blot analysis.
LeAOS3 expression increased after 1 h of treatment and peaked at the 12 h time point
(Figure 3.2A). Relative to mRNA levels, a delayed increase in LeAOS3 protein levels

was observed 8 h roots after MeJA treatment (Figure 3.2B). In contrast to the wild-type

87

A wild-type jail

 

 

Hrs:012481224 012481224

LeAOS3 M Q ”I.

 

 

 

 

eIF4Aflflflufluu1-a .«wfiflum

 

 

 

B
LeAOS3 “M ""

 

 

 

 

 

 

 

 

Figure 3.2 Accumulation of LeAOS3 transcript and protein in response to MeJA.

Four-week-old wild-type and jail plants were treated with a solution of 0.1 mM Me] A
and roots were collected at the indicated times after treatment for RNA or protein
extraction. Identical RNA blots (A) were probed with either LeAOS3 or eIF 4A
(loading control) cDNA probes. Western blots (B) were probed with antiserum raised

against recombinant LeAOS3.

88

expression of LeAOS3 was not detected in control or MeJA-treated jail roots (Figure
3.2A). Likewise, LeAOS3 protein was undetectable in jail roots (Figure 3.28).

It is well established that wounding causes a rapid and transient increase in the
accumulation of endogenous JA, which triggers the induction of JA-regulated genes
(Schilmiller and Howe, 2005). Experiments were performed to determine whether
wounding induces the expression of LeAOS3 in roots. Roots of three-week old wild-type
and jail plants were wounded by squeezing with a hemostat, which resulted in crushing
and breaking of root tissue. Western blot analysis showed that, similar to MeJA-
treatment, wounding induced the accumulation of LeAOS3 protein in wild-type but not
jail plants (Figure 3.3A). To determine whether increased LeAOS3 protein levels were
associated with increased LeAOS3 activity, we utilized a previously described assay to
measure oxylipin production in cell-free extracts from the unwounded and wounded roots
(Caldelari and Farmer, 1998). Incubation of unwounded wild-type root extract with [1-
l4C]-linoleic acid (MC-18:2) resulted in the production of several labeled compounds.
One of these compounds co-chromatographed with the product formed after incubation
of the same substrate with tomato 9-LOX (prepared from tomato fruit) and recombinant
LeAOS3 (Figure 3.3B). Based on our previous finding that 9-hydroxy-10-oxo-(12,l5)-
octadecadienoic acid (on-ketol) is the major 9-hydroperoxy linoleic acid (9-HPOD)
derived product formed by LeAOS3 in vitro (Itoh et al., 2002), this compound is likely
the or-ketol (marked with arrow, Figure 3.3B). Cell-free extracts prepared from wounded
wild-type roots catalyzed the formation of higher levels of the a-ketol compared to

extracts from unwounded roots. This is consistent with the wound-induced accumulation

of LeAOS3 protein. Extracts from jail roots showed little or no capacity to synthesize the

89

Figure 3.3 Induction of LeAOS3 protein and activity after wounding.

Roots of four-week-old wild-type and jail plants growing in peat pots were
wounding by crushing with a hemostat. Roots were collected at various times for
either protein extraction and western blotting with LeAOS3 antiserum (A) or
LeAOS3 activity assays (B). For activity assays, cell-free extracts from root tissue
was incubated with l"'C-linoleic acid followed by extraction of oxylipins and
analysis by thin layer chromatography. As controls, 9-LOX activity partially
purified from tomato fruit was used to generate l4C-9-hydroperoxy linolenic acid
standard (9-LOX). Co-incubation of 9-LOX with recombinant LeAOS3 (9-LOX +
AOS3) produced 9-hydroxy,10-oxo-octadecadienoic acid standard (or-ketol, marked

with arrow).

90

wild-type jail

 

 

 

A
Hrs: o 612 24 0 6 12 24
LeAOS3
M M
8 8
+ +
X X . X X .
wld- e '1
311 'W 311 M
._. o Ox 0 6 12 24 —- 0* 0* O 6 12 24
~ I ~ ‘4'
i u . ““ ...
b 505‘- anr J
O .1 ..QF - -
i .. i in ii a; - :4 -- a"- a- as

Figure 3.3 Induction of LeAOS3 protein and activity after wounding.

91

or-ketol from l4C-18:2 (Figure 3.3B). The low level of co-migrating product formed by

jail extracts is likely to be 13-hydroxy-12-oxo-(9,15)-octadecadienoic acid, the a-ketol
from the l3-AOS pathway, which migrates at the same position with this solvent system.
These findings indicate that the expression of LeAOS3 in roots is dependent on the JA-

signaling pathway.

Expression of LeA 0S3 in hypocotyls of germinating seedlings is independent of C 011
Analysis of LeAOS3 EST data (www.tigr.org/tdb/lgi/plant.shtml) showed that all
ESTs corresponding to LeAOS3 were from cDNA libraries constructed from either root
(1 of 12 ESTs) or germinating seedlings (11 of 12 ESTs). The relatively high abundance
of ESTs in the germinating seedling library prompted us to investigate LeAOS3
expression at this developmental stage. RNA blot analysis showed that LeA 0S3 in wild-
type germinating seedlings was expressed to its highest levels four days after seed
imbibition, and decreased substantially over the period of eleven days (Figure 3.4).
Unlike in root tissue, LeAOS3 expression in germinating jail seedlings was only slightly
decreased in comparison to the wild type (Figure 3.4). We also analyzed LeAOS3 protein
levels in roots and germinating seedlings of wild-type and jail plants. Consistent with
RNA-blot analysis, LeAOS3 protein was detected in roots and germinating seedlings of
both genotypes, while no LeAOS3 protein accumulated in jail roots (Figure 3.5).

LeAOS3 activity in extracts of wild-type and jail germinating seedlings was measured as

well. The major oxylipin formed from l4C-18:2 extracts from wild-type seedlings co-

chromatographed with the or-ketol produced by recombinant LeAOS3 (Figure 3.6). The

amount of or-ketol produced with extracts from jai I was comparable to wild-type levels

92

wild-type jail

 

 

days post-imbibition: 4 6 8 11 4 6 8 11
LeAOS3 . fl .

eIF4A ..fl..gum

Figure 3.4 LeAOS3 expression in germinating seedlings. Total RNA was
extracted from wild-type and jail seedlings germinated on wet filter paper for
the indicated times. Identical blots were hybridized to either LeAOS3 or

elF 4A (loading control) cDNA probes.

93

germ. germ. germ.
root seedling root seedling root seedling
M wt jail wt jail M wt jail wt jail M wt jail wt jail

 

 

 

 

 

 

 

 

 

 

 

 

207 m

119 "‘"“

98.5 ~ In:

56.6 ..... .. «.1 a

37.5 11111.: 1

29-5 ., L" __ .. =- -
Pre-immune a-AOS3 Coomassie blue-stained

Figure 3.5 LeAOS3 protein levels in root and germinating seedlings.

Total protein was extracted from roots of soil grown six-week-old wild-type (wt)
and jail plants and from four-day-old seedlings germinated on wet filter paper.
Proteins were separated by SDS-polyacrylamide gel electrophoresis (10% gels)
and either stained with Coomassie blue or transferred to PVDF membranes and
probed with antiserum raised against LeAOS3 or pre-immune serum. Numbers at

left indicate sizes of molecular weight standards (M).

94

 

. 18:2

wt GS
wt roots
jar] roots

;9—Lox
-1 9-LOX + DES

1 8 :2
Colneleic acid

a-ketol

 

Figure 3.6 LeAOS3 activity in cell-free extracts of wild-type and jail.

LeAOS3 activity was measured in extracts of 5-day old germinating
seedlings (GS) and roots from 3-week old wild-type and jail plants. In
addition to controls described in the legend to Fig 3.3, a control for colneleic
acid was made using partially purified 9-LOX with recombinant LeDES (9-

LOX + DES).

95

(Figure 3.6). These results show that LeA 0S3 expression in germinating seedlings is not
dependent on the J A-si gnaling pathway.

The C011-independent expression of LeAOS3 in germinating seedlings prompted
us to determine the spatial pattern of LeAOS3 expression within the seedling. Root or
non-root (i.e. hypocotyl, seed coat, cotyledons) tissues from 5-day-old seedlings was
collected and used for RNA-blot analysis. In contrast to older, soil-grown plants, LeA 0S3
expression was not detected in root tissue of germinating seedlings. At this stage of
development, expression was localized to non-root tissue of the tomato germinating
seedling (Figure 3.7A). Similar results were obtained for the analysis of LeAOS3 protein
levels (Figure 3.7B). These results show that the C011-independent constitutive
expression of LeAOS3 in germinating seedlings occurs in non-root tissues whereas in
older plants, LeAOS3 is expressed specifically in roots and is controlled by the IA

signaling pathway.

LeA 0S3 protein accumulates in cortex cells of root tips and hypocotyls

We used immunofluorescence microscopy to investigate the cell type-specific
expression pattern of LeAOS3 in roots and hypocotyls of germinating seedlings. As a
control for these experiments, we took advantage of the jail mutant that lacks expression
of LeAOS3 in root tissues. In hypocotyl cross-sections from wild-type germinating
seedling, LeAOS3 protein was detected in the first layer of cortex cells, adjacent to the
epidermal cell layer (Figure 3.8A’ and A”). LeAOS3 protein was detected at lower levels
in other cortex cell layers, but not in epidermal cells or central cell layers the comprise

the vascular tissues. A similar pattern of expression was observed in cross-sections of

96

 

 

 

 

 

 

 

 

 

A E» B E g
8 8 a ._.
8: *5 ‘g’; *5 >~ 8
E: e .1: e 7.1:. g
E E E E .2 a
LeAOS3 . “'AOS3
Pre-immune
eIF4A ..-

 

 

 

Figure 3.7 LeAOS3 is expressed in non-root tissues of germinating seedlings.

(A) Five-day-old wild-type germinating seedlings were dissected into root segments
and the remaining non-root tissues (labeled as hypocotyl) for RNA-blot analysis of
LeAOS3 expression.

(B) Wild-type and jail (5-day-old) germinating seedlings were dissected into root
and hypocotyl segments and total protein was extracted from the individual tissues.

Shown are western blots probed with LeAOS3 antibodies or pre-immune serum.

97

MeJA-treated wild-type root tips (Figure 3.83’ and B”). Longitudinal sections of this
tissue showed that LeAOS3 protein expression is highest in the cortex layer near the tip,
and decreases as cells elongate and mature (Figure 3.8C’ and C”). This expression pattern
closely resembles that of a subset of J A-responsive genes expressed in Arabidopsis roots
(Bimbaum et al., 2003). In sections incubated with pre-immune serum (Figure 3.8D’) or
in sections of jail roots incubated with LeAOS3 antibodies (Figure 3.8E’), no signal was
detected. In older root tissues, weak expression was seen in the cortex cells. Signal in the
epidermal cells was also seen in sections incubated with pre-immune serum, and thus was
non-specific labeling. High expression was observed in sections through a lateral root,

which showed LeAOS3 protein accumulating in the root tip (Figure 3.8F’ and F”).

MeJA-induced gene expression in tomato roots

To gain additional insight into the function of LeAOS3, we used microarray
analysis to identify genes that are co-regulated with LeAOS3. The microarray used in
these experiments was a Solanum tuberosum (potato) cDNA array constructed at The
Institute for Genome Research (TIGR). Hybridizations performed with RNA from
various Solanaceous species have previously established a high degree of genomic
similarity throughout the family and validated the use of the potato array to study gene
expression in tomato (see hybridization data available on-line at
http://www.tigr.org/tdb/potato/SGED index2.shtml). Our experimental set-up was to
compare expression in wild-type and jail roots at various times (1, 6, 12, and 24 h) after
treatment with MeJ A, as well as untreated controls. Of 11,512 sequenced verified cDNAs

on the array, 272 were identified as being regulated by MeJA in at least one time-point

98

Figure 3.8 Immunolocalization of LeAOS3 protein. Hypocotyls from S-day-old
germinating seedlings, or roots from 3-week-old plants, were used for
immunolabeling of LeAOS3. A-G, bright-field images; A’-G’, immunofluorescence
of same section shown in bright-field image. A-C, E, F were incubated with
antibodies raised against LeAOS3, D & G were incubated with pre-immune serum. A
cross section of untreated wild-type hypocotyl. B, D cross section of MeJA-treated
wild-type root tip. C, longitudinal section of MeJA-treated wild-type root tip. E, cross
section of MeJA-treated jail root tip. F, G cross section of MeJA-treated wild-type

root near root-shoot junction.

99

    

 

Bright-field Immunofluorescence
. , 1. ‘ i‘
'3; 1 0 .0,
*5 . _
o . 0“ o
0 j 1 9’ - .
0‘ . . .
>. 1' « ,
,1: .

‘Avf I‘VvV‘ 5.]-

 

jail root tip + MeJA WT root tip + MeJA. WT root tip + MeJA WT root tip + MeJA

Figure 3.8 Immunolocalization of LeAOS3 protein

100

WT root + MeJA

WT root + MeJA

Immunofluorescence

 

Figure 3.8 (cont’d)

101

(expression ratio 2 3 or S 0.33, wt vs. jail; Table 3.1). As seen for J A-regulated genes in
leaves (Schmidt et al., 2005), most of the identified genes in roots were up-regulated
(235) rather than down-regulated by the JA-signaling pathway (37). Several known JA-
regulated defense genes were found to be induced, including those encoding proteinase
inhibitors I and II, polyphenol oxidase, and cystatin (Zhao et al., 2003). Several genes
involved in oxylipin biosynthesis were also highly induced in a manner similar to
LeAOS3. Divinyl ether synthase (DES), another CYP74 enzyme that uses 9-hydroperoxy
fatty acids as its substrate, was not induced by MeJA treatment, indicating that only
certain branches of the 9-LOX pathway are regulated by JA-signaling. The most highly
expressed genes alter MeJA-treatment encoded patatin isoforms. Patatins are storage
proteins that have non-specific lipolytic activity and have been implicated as having
defensive roles as well (Dhondt et al., 2000; Sharma et al., 2004). It is possible that
patatins release fatty acids from membrane lipids for oxylipin biosynthesis during times

of stress.

102

Table 3.1 MeJA-regulated genes in tomato roots. Three-week-old wild-type and
jail plants were watered with 0.1 mM MeJA and roots were harvested for RNA
extraction at the indicated times. Roots from untreated plants were collected as the
“0” h time point. Microarray analysis was performed using the TIGR potato array (see
text for details). Genes that were up-regulated 2 3-fold or S 0.33-fold in wild-type at
any time point are listed, with the potato clone number and annotations given as well
as the expression ratios at each time point (sorted on the 24 h time point). A ratio of

“0.00” indicates flagged spot for which data was not reliable.

103

 

 

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Discussion

Our approach for determining the function of LeA 053 in tomato was focused on
understanding the regulation of LeAOS3 expression. Since other plant AOSS, particularly
those involved in JA production, are known to be regulated by wounding and JA
(Schaller et al., 2005), we decided to test the hypothesis that LeAOS3 is also regulated by
the JA signaling pathway. Analysis of mRNA, protein, and activity levels showed that
LeAOS3 is highly induced in roots in response to wounding and J A treatment (Figure 3.2
& 3.3). In the jail mutant that lacks a functional JA-signaling pathway, the expression of
LeAOS3 in roots was completely abolished. To our knowledge, LeAOS3 is the first
example of a gene whose expression in roots is dependent on JA-signaling.

Because most of the ESTs for LeAOS3 come from a germinating seedling cDNA
library, we also analyzed expression at this stage of development. To our surprise, the
level of LeAOS3 expression in jail seedlings was almost identical to wild-type (Figure
3.4). Further analysis showed that the C011-independent expression of LeAOS3 in
germinating seedlings occurs in hypocotyl tissue rather than in root (Figure 3.7). There
are likely different regulatory elements in the promoter of LeAOS3 that are responsible
for differential expression of the gene in root and hypocotyl tissue. This type of
regulation has been shown for wound-induction of the FAD7 gene in Arabidopsis, where
different promoter elements are active in different tissues (Nishiuchi et al., 1999). If
LeAOS3 is involved in defense against soil-borne pests, constitutive expression of
LeAOS3 in hypocotyls during germination may play a role in survival prior to emergence

from the soil. In contrast, the JA-inducible expression of LeAOS3 in roots would allow

113

for expression of this defense under stress conditions. It is not known whether other J A-
regulated root-expressed genes are constitutively expressed in hypocotyls, but genes
identified in our microarray analysis are candidates to test this hypothesis. A similar
expression pattern for a rice PR-protein was reported where expression in roots was
inducible by J A, while expression in shoots was unaffected by JA-treatment (Hashimoto
etaL,2004)

Previous studies of the 9-LOX pathway have been focused mainly on 9—LOX
rather than CYP74 P4508 that metabolize 9-hydroperoxy fatty acids. Several studies have
shown that 9-hydroperoxides accumulate to high levels upon induction of the
hypersensitive response (HR) by pathogens or elicitors (Rusterucci et al., 1999; J alloul et
al., 2002). However these studies were conducted with leaf tissue where LeAOS3
expression has not been detected. Another study showed that in Arabidopsis, induction of
HR coincides with the preferential accumulation of 13-hydroperoxides over 9-
hydroperoxides (Montillet et al., 2002). The most compelling argument for the 9-AOS
pathway not having a role in HR is the fact that Arabidopsis does not even have a 9-AOS,
yet can exhibit a HR. Therefore it seems that the involvement of the oxylipin pathway in
HR is at the level of hydroperoxides or possibly a branch other than the 9-AOS pathway.
Another study in tobacco showed that suppression of a 9-LOX gene resulted in
conversion of an incompatible interaction with Phytophthora parasitica to a compatible
interaction (Rance et al., 1998). This strongly indicated a role for the 9-LOX pathway in
defense against this fungal pathogen. Based on a search of the EST database at TIGR, a
sequence with high similarity to LeAOS3 is present in Nicotiana tabacum suggesting a 9-

AOS pathway could be involved in the suppression of the incompatible phenotype in

114

antisense 9-LOX tobacco plants. Consistent with a possible role of LeAOS3 in defense,
microarray analysis showed several genes co-regulated with LeAOS3 that have known
roles in defense.

A role for 9-AOS in plant development cannot be ruled out. Suppression of 9-
LOX genes in potato resulted in fewer and deformed tubers, suggesting a role for 9-LOX
pathway metabolites in tuber development (Kolomiets et al., 2001). The presence of 9-
AOS activity in potato stolons (Hamberg, 2000) is consistent with this, but it is unclear
whether the 9-AOS pathway is involved in tuber development. Also, no obvious
developmental phenotypes have been observed for tomato jail roots that lack LeAOS3
expression. Another hypothesis is that LeAOS3 functions as a scavenger under stress
conditions to prevent build-up of highly reactive hydroperoxy fatty acids. Measurements
of fatty acid hydroperoxide levels in plants lacking LeAOS3 are needed to test this.

Based on the work described in this chapter, future work will be aimed at testing
hypotheses for roles of the 9-AOS pathway in development and defense Using the jail
mutant that lacks LeA OS3 expression in roots as a starting point is possible, but with all
J A-regulated pathways affected in this mutant, it will be difficult to connect a particular
phenotype to lack of 9-AOS activity. Genetic manipulation of LeAOS3 expression in
transgenic plants and testing of individual pathogens or root-feeding insects is the next
step to determine what role, if any, LeAOS3 has in defense against soil-bome invaders

such as nematodes or fungal pathogens.

115

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J alloul A, Montillet JL, Assigbetse K, Agnel JP, Delannoy E, Triantaphylides C,
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Ziegler J, Keinanen M, Baldwin IT (2001) Herbivore-Induced Allene Oxide Synthase
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118

Chapter 4

Biochemical Analysis of a Tomato Acyl-CoA Oxidase Required for Wound-Induced

J asmonic Acid Biosynthesis

Part of the work presented in this chapter has been published:

Li C, Schilmiller AL, Liu G, Lee GI, J ayanty S, Sageman C, Vrebalov J, Giovannoni JJ,

Yagi K, Kobayashi Y, Howe GA (2005) Plant Cell 17: 971-986.

119

Abstract

Jasmonic acid (JA) is a lipid-derived signal that regulates plant defense responses to
biotic stress. Previous work identified a mutant of tomato (Lycopersicon esculentum),
JLl (since renamed acxl), that lacks local and systemic expression of defensive
proteinase inhibitors (P15) in response to wounding (Lightner et al., 1993). Map-based
cloning studies demonstrated that this phenotype results from loss of function of an acyl-
CoA oxidase (ACXIA) that catalyzes the first step in the peroxisomal B-oxidation stage
of J A biosynthesis (Li et al., 2005). Recombinant ACXlA exhibited a preference for C12

and C14 straight-chain acyl-CoAs and also was active in the metabolism of C18

T1381

cyclopentanoid-CoA precursors of JA. Recombinant ACXlA , harboring the

mutation found in acxl, failed to bind the FAD cofactor required for catalysis, and thus
showed no activity with C14 acyl-CoAs. While the overall growth, development, and
reproduction of acxl plants were similar to wild-type plants, acxl plants were slightly
more resistant to 2,4-DB treatment compared to wild-type. The expression of ACXlA
was detected throughout the plant with the highest levels in germinating seedlings and
flowers. Based on the ability of ACXlA to utilize cyclopentanoid-CoAs as substrate and
the finding that acxl is compromised in JA biosynthesis (Li et al., 2005), we conclude

that ACXlA is essential for the B-oxidation stage of J A biosynthesis.

120

Introduction

J asmonic acid (JA) and its cyclic precursors and derivatives, collectively referred
to as jasmonates (JAs), constitute a family of bioactive oxylipins that regulate plant
responses to environmental and developmental cues. JAs are perhaps best known for their
role in orchestrating plant defense responses to herbivores and certain microbial
pathogens (Liechti and Farmer, 2002; Turner et al., 2002). Activation of jasmonate-
mediated defenses is typically preceded by accumulation of JAs in response to biotic
stress (Wasternack and Hause, 2002). JAs are synthesized by the so-called octadecanoid
pathway that involves enzymes located in two different subcellular compartments (Vick
and Zimmerman, 1984; Schaller, 2001; Wasternack and Hause, 2002). The first part of
the pathway directs the conversion of linolenic acid to lZ-oxo-phytodienoic acid (OPDA)
by the sequential action of the plastid enzymes lipoxygenase (LOX), allene oxide
synthase (AOS), and allene oxide cyclase (AOC). The second part of the pathway takes
place in peroxisomes, where OPDA is reduced by OPDA reductase (OPR3) to give 3-
oxo-2(2’[Z]-penteny1)-cyclopentane-1-octanoic acid (OPC8). Removal of six carbons
from the octanoate side chain of OPC8 yields JA.

Studies on B-oxidation in plants have mainly focused on its role in the breakdown
of storage lipids in germinating seeds (Graham and Eastmond, 2002). Increasing
evidence, however, indicates that nonfatty plant tissues also depend on B-oxidation for
several other processes, including the synthesis of indole-acetic acid and J A (Graham and

Eastmond, 2002; Zolman and Bartel, 2004). Metabolic labeling experiments with 18O-

OPDA provided the first evidence that the conversion of OPC8 to JA involves three

121

cycles of B-oxidation (Vick and Zimmerman, 1984). These early studies are supported by
recent work showing that OPR3 is located in peroxisomes (Stintzi and Browse, 2000;
Strassner et al., 2002), which are thought to be the exclusive site of fatty acid B-oxidation
in higher plants (Graham and Eastmond, 2002; Tilton et al., 2004). Thus, it is generally
assumed that the final steps of JA synthesis are catalyzed by the three core enzymes of
the B-oxidation cycle, namely acyl-CoA oxidase (ACX), the multifunctional protein
(MFP; containing 2-trans-enoyl-CoA hydratase and L-3-hydroxyacyl-COA
dehydrogenase activities), and 3-ketoacy1-COA thiolase (KAT). An additional
thioesterase activity is also presumably involved in the release of JA from JA-CoA, the
product of the final round of B-oxidation (Figure 4.1). Identification of specific gene
products that participate in the conversion of OPC8 to J A has been hampered by the fact
that ACX, multifunctional protein, KAT, and thioesterases are encoded by small gene
families (Graham and Eastmond, 2002; Shockey et al., 2003; Reumann et al., 2004;
Tilton et al., 2004). Furthermore, isoforms within a particular enzyme family often
exhibit overlapping substrate specificity (Graham and Eastmond, 2002). Recent antisense
experiments conducted in Arabidopsis thaliana have provided evidence that specific
isoforms of ACX (ACXl) and KAT (KAT2) play a role in wound-induced JA synthesis
(Castillo et al., 2004). However, the precise contribution of these family members to J A

production in healthy and damaged tissues remains to be determined.

122

— o AOC AOS LOX
<— ‘— ‘— 18:3
OH

OPDA
10PR3
O
m
OH
OPC8
o 1 ACS 16:3
W 1“”
SCoA les
OPCB-CoA
02 ACX lec
H202 l
O
O __
- o m
\ SCoA dn-OPDA OH
2-trans-enoyl-CoA i OPR3
MFP
o l o
SCoA OH
3-hydroxyacyl-CoA OPC6
l MFP lAcs
0 o
W K“ m
SCOA SCoA
3-ketoacyl-COA OPC6-GOA
1 additional round ACX
of B-oxidation MFP
KAT
0
Figure 4.1 Octadecanoid pathway for {SSA
O
JA biosynthesis. Pathway for JA SCoA
JA—CoA
biosynthesis, including detailed steps of lThioesterase

O

B-oxidation in peroxisomes. See text for —
COOH
JA

further information.

123

Here, we describe the characterization of a wound-response mutant of tomato that
is deficient in JA biosynthesis. Positional cloning studies demonstrated that this defect
results from loss of function of a member (ACXIA) of the ACX family of enzymes that
participate in peroxisomal [i-oxidation (Li et al., 2005). Results obtained from the
biochemical characterization of ACXlA show its specificity profile for straight chain
acyl-CoA substrates is similar to other so-called medium-long chain ACXs. We also
show that ACXlA uses OPDA-CoA and OPC8-CoA as substrates. Taken together with
the finding that the acxl mutant is compromised in IA production (Li et al., 2005), we
propose that ACXlA is the major isoform essential for the B-oxidation stage of JA

biosynthesis.

Materials and Methods

Plant materials and growth conditions

Tomato (Lycopersicon esculentum) cv Castlemart was used as the wild-type
parent for all experiments. Seed for the acxl mutant was collected from an acxl /acx1
homozygous line derived from successive backcrosses of the original mutant (previously
called J L1; (Lightner et al., 1993) to the wild type. Seedlings were grown in Jiffy peat
pots (Hummert International, Earth City, MO) in a growth chamber maintained under 17
h oflight (200 uE/mz/s) at 28°C and 7 h of dark at 18°C. Adult acxl plants were
morphologically indistinguishable from wild-type plants and exhibited normal fruit and

seed production. The germination rate of acxl /acx1 seeds ranged between 10 and 92%,

124

depending on the seed batch. We reproducibly observed that the germination rate of
acxl /acx1 seed collected from field-grown plants was much higher than that of seed
obtained from potted plants grown in the greenhouse (A.L. Schilmiller, C. Li, and GA.

Howe, unpublished results).

Biochemical characterization of ACXlA
A full-length ACXIA EST clone (cLES14H13) obtained from Clemson University

Genomics Institute was used as the template for a PCR-based approach to construct a
vector for expression of ACXlA with an N-terminal Hi56 tag. The forward primer (5’-

GAGCTCGTAAGAGAGATGGAGGGTGTA-3’) was designed with a Sacl site and the
reverse primer (5’—CCGCTCGAGCGGAACAGTTTGCTGCAGCTC-3’) spanned a PstI
site located in the 3’—untranslated region of ACX 1A. PCR amplification yielded a 2047-bp
product that was subcloned into pGEM-Teasy (Promega). After digestion with SacI and
PstI, the ACXlA cDNA insert in this construct was cloned into the same sites of the
expression vector pQE30 (Qiagen, Valencia, CA). The resulting construct, which added
19 amino acids (MRGSHHHHHHGSACELVRE) to the N-terminus of ACXlA, was

transformed into the Rosetta strain of E. coli (Novagen, Madison, WI). The expression

T1381

construct for ACX was prepared by the same procedure, except the template for PCR

was the ACXIA cDNA derived fi'om the acxl mutant. Expression of recombinant

T1381

ACXlA proteins (ACXlWt and ACXl ) was initiated by inoculating 1 mL of an

overnight culture into 200 mL of Terrific Broth medium supplemented with 100 ug/mL
of ampicillin and 12.5 ug/mL of chloramphenicol. Bacteria were grown at 37°C in a

shaker (250 rpm) to an OD600 of 0.5. Cultures were then cooled to 30°C, and isopropyl-

125

thio-B-D-galactopyranoside (Roche, Indianapolis, IN) was added to a final concentration
of 0.1 mM. The induced cultures were incubated at 30°C for 18 h with gentle shaking
(120 rpm). Cells were harvested by centrifugation and either stored at -20°C or used
immediately for ACXl purification. The cell paste was resuspended in 50 mM potassium
phosphate, pH 7.6, 150 mM NaCl, and 10% (v/v) glycerol and then lysed by sonication.
His-tagged ACXl proteins were purified from the cleared lysate by nickel affinity
chromatography (nickel-nitrilotriacetic acid agarose resin; Qiagen) according to
manufacturer’s directions. During the purification procedure, FAD (Sigma-Aldrich, St.
Louis, MO) was added to all buffers at a final concentration of 10 uM. Protein
measurements were performed using the BCA assay (Pierce, Rockford, IL), with BSA as
a standard.

ACX activity with commercially available acyl-CoA substrates (Sigma-Aldrich)

was measured with an HzOz-coupled spectrophotometric assay as described previously

(Hyrb and Hogg, 1979). Typical enzyme assays contained 0.5 ug of affinity-purified
ACXl and 50 uM of acyl-CoA substrate. ACXlA-catalyzed metabolism of OPDA-CoA
and OPC8-CoA was measured with an enzymatic colorimetric assay (Roche) that is
typically used to measure free fatty acid levels in serum or plasma (Shimizu et al., 1980).
The method is based on the activation of nonesterified fatty acids by ACS to the
corresponding CoA ester, followed by detection of ACX catalyzed H202 production
(Shimizu et al., 1980). The test kit provided by the manufacturer uses a yeast ACS and an

unspecified ACX for the coupled reaction. For our studies, the ACX provided by the

manufacturer was replaced with affinity-purified ACXlA. Fatty acid substrate (dissolved

in 0.25% Triton X-100) was added to 100 uL of the manufacturer’s reaction mix A

126

(containing ATP, CoA, ACS, and sodium phosphate, pH 7.8). Concentrated stocks of
OPDA and OPC8 in ethanol were diluted directly in 0.25% Triton X-100 (v/v). The
sodium salt of myristic acid (14:0) was prepared according to Shimizu et a1. (1980).

Reactions were allowed to proceed for 30 min at 25°C, at which time excess CoA was

trapped by alkylation via the addition of 5 uL of N-ethyl-maleinimide. The ACX reaction
was then initiated by addition of 1 pg of purified ACXlA. H202 produced in this reaction

was used by a peroxidase to convert 2,4,6-tribromo-3-hydroxy-benzoic acid and 4-
aminoantipyrine to a red dye that was measured at 546 nm with a Beckman DU530
spectrophotometer (Fullerton, CA). OPC8 was chemically synthesized according to the
procedure of Ainai et a1. (2003). NMR spectroscopy showed that the OPC8 was >97%
pure and that the ratio of the cis and trans isomer was >20:1. OPDA was obtained from

Larodan Fine Chemicals (Malmo, Sweden).

Gene expression analysis

RNA extraction was performed as previously described (Howe et al., 2000).
cDNA clones for ACXIA (cLES14H13) and eIF4A (cLED1D24) were used to prepare
probes for hybridization. Duplicate gels were stained with ethidium bromide to assess the

quality of the RNA.

Antibody preparation and protein blot analysis
Recombinant His-tagged ACXIA was purified from E. coli as described above
and sent for polyclonal antibody production (Cocalico Biologicals, Reamstown, PA) in

rabbits following their standard protocol for inoculation. Total protein was extracted from

127

various tissues by grinding in liquid nitrogen and adding extraction buffer (50 mM
sodium phosphate pH 7.0, 150 mM NaCl, 10% glycerol, and Roche mini-complete
protease inhibitors) at a buffer to tissue ratio of approximately 2:1. Plant debris was
removed by centrifugation at 13,000 x g and protein concentration was measured in the
supernatant using the BCA assay (Pierce, Rockford, IL) with BSA as a standard. Protein
was separated on 10% SDS-polyacrylamide gels and transferred to Imobilon-P

membranes (Millipore, Bedford, MA) using standard procedures (Harlowe and Lane,
1988). Membranes were probed with anti-ACXIA antibodies at a 1:1000 dilution in Tris-

buffered saline (TBS) containing 1% nonfat milk (Krogers) and 0.1% Tween 20 (Sigma).
Antigen-antibody complexes were probed using a horseradish peroxidase conjugated
secondary antibody (Sigma) at 1:25,000 dilution in TBS + 1% nonfat milk and 0.1%
Tween 20 followed by detection using the Supersignal West Pico Chemiluminescent

substrate (Pierce, Rockford, IL).

2, 4-DB treatment of germinating seedlings

Seeds of wild-type (Castlemart) and acxl were surface sterilized with 40% beach
and rinsed several times with sterile water, then germinated for two days on plates
containing MS media (Plant Media, Dublin, OH). After three days, plants were
transferred to fresh MS plates containing various concentrations of 2,4-dichlorophenoxy

butyric acid (Sigma). Root lengths were measured five days after transfer.

128

Results

The wound-response phenotype of acxl results from a defect in ACX

Genetic mapping of acxl narrowed the location to a single Lycopersicon
cheesmanii BAC clone, which was sequenced (Li et al., 2005). Two candidate genes
located adjacent to each other were identified as putative ACX-encoding genes, which we
designated LcACXlA and LcACXlB (Figure 4.2). Given the likely role of ACX in JA
biosynthesis and wound signaling (Vick and Zimmerman, 1984; Castillo et al., 2004),
subsequent experiments were focused on testing the hypothesis that the wound-response
phenotype of acxl results from a defect in ACXIA or ACXIB.

Plant ACXs comprise a family of flavoenzymes that catalyze the initial step of
peroxisomal B-oxidation of a variety of acyl-CoA substrates (Graham and Eastmond,
2002). As a first step toward characterizing the tomato ACXIA and ACXIB genes, we
used RT-PCR to obtain full-length cDNAs (designated LeACXlA and LeACXlB) from
wild-type (cv Castlemart) L. esculentum. The predicted amino acid sequence of the two
tomato ACXs are 81.7% identical. ACXIA and ACXIB are most similar to ACXS
characterized from Arabidopsis (80.1 and 73.3% identical to AtACXl, respectively) and
soybean (81.8 and 75.7% identical to GmACXl-l, respectively) that have broad
specificity for medium- to long-chain acyl-CoAs (Figure 4.3)(Hooks et al., 1999;
Agarwal et al., 2001). Both LeACXlA and LeACXlB possess a C-terminal peroxisomal
targeting signal type 1 (PTSl) motif (Figure 4.2) (Reumann et al., 2004). The C-terrninal
region of ACXIB lacks a 15—amino acid sequence that is highly conserved in other

medium- to long-chain plant ACXs (Figure 4.2). Comparison of the ACXIB genomic

129

LeACXlA
LeACXlB

LeACXlA
LeACXlB

LeACXlA
LeACXlB

LeACXlA
LeACXlB

LeACXlA
LeACXlB

LeACXlA
LeACXlB

LeACXlA
LeACXlB

LeACXlA
LeACXlB

LeACXlA
LeACXlB

LeACXlA
LeACXlB

LeACXlA
LeACXlB

LeACXlA
LeACXlB

 

 
 

G I
I E E
E TMLRRY f‘?.T L'
S S H E EKLGY .- I S
+

 

 

  
 

VSLVDAFNYTDHY SILGRYDGNMF*FUV'fPHUILW
************LI FVICVCCS ..

'21

Figure 4.2 Sequence alignment of LeACXlA and LeACXlB. Predicted amino acid

sequences for ACXIA and ACXIB were aligned using the Multalin sequence analysis

package (http://prodes.toulouse.inra.fr/multalin/multalin.html)(Corpet, 1988). The "+"

denotes the Thr residue (Thr138) in LeACXlA that is affected by the acxl missense

mutation. Asterisks (*) denote residues that are missing in the deduced sequence of

LeACXl B.

130

 
     

Medium chain

  

0220503 AtACX3

 

 

AtACX6

 
 
 

AtACX5
AtACX‘l LeACX1 B

QA—Cflé .
J/é GmACX1-2 Med

GmACX1-1 “"9

Pumpkin ACX
AtACXZ

   
     
 

  

 

Long

 

chain .
PcACX HvACX Chem
ZmACX1 -1
Phalaenopsis ACX OsACX ZmACX1 .2

 
 
  

 

/7\ U223228

AtACX4
U216994
Short chain

     
   

Figure 4.3 Plant ACX phylogeny. An unrooted phylogenetic tree is shown,
constructed from deduced amino acid sequences from various plant ACXS.
Gray boxes indicate the substrate preference of members of each circled
group for acyl—CoA substrates of various chain lengths. Tomato sequences
are underlined. U220503, U216994, and U223228 are predicted tomato ACX

sequences compiled from EST sequence data at the Solanaceous Genomics

Network. (http://www.sgn.cornell.edu)

131

and cDNA sequences suggested that this polymorphism results from alternative splicing
at cryptic donor and acceptor splice sites.

Comparison of the sequence of wild-type- and acxl-derived ACX cDNAs
revealed a single C-to-T nucleotide substitution in LeACXlA. No sequence differences
were found in LeACXlB. The single-base change in LeA CXIA, which was confirmed by
sequencing of PCR-amplified genomic DNA from acxl, is predicted to replace a highly
conserved (i.e., invariant among all plant and animal ACXs) Thr residue at position 138
with an Ile. The three-dimensional structure of mammalian ACX (Nakajima et al., 2002)
and its conserved mitochondrial counterpart, acyl-CoA dehydrogenase (Kim and Miura,
2004), has shown that this Thr binds the flavin ring of FAD that defines the active site of
the enzyme.

Genetic complementation experiments were performed to determine whether the
missense mutation in LeA CX 1A was responsible for the deficiency in wound-induced PI-
II expression in acxl plants. The wild-type LeA CX IA cDNA was placed under the control
of the 35S promoter of Cauliflower mosaic virus in T-DNA vector pBIlZl. This
construct was introduced into the acxl genetic background by Agrobacterium

tumefaciens—mediated transformation. From 33 independent 35S-ACX1A transgenic
plants confirmed by genomic PCR, 20 primary lines (To) showed normal levels of

wound-induced PI-II expression both in the wounded leaf and in the systemic unwounded

leaves (Figure 4.4). Further characterization of two of these lines showed that the
complemented phenotype was inherited in the T1 generation (data not shown). These

results demonstrate that the wound-response phenotype of acxl results from loss of

function of ACXlA.

132

 

 

 

 

 

 

120-
81004
.2.
‘8 so-
2
_.|
I; 60-
U)
5': 40- T
EL' 20- I
0 J

 

 

 

 

 

 

WT JL1 35S-ACX1

Figure 4.4 Genetic complementation of the wound-response phenotype of
acxl. Wild-type, acxl, and 35S-ACX1A-expressing mutant plants (at the five-
to six-leaf stage) were wounded once across the midvein of each leaflet on the
two lower leaves. The same leaflets were wounded again 3 hrs later. Twenty-
four hours after the initial wounding, PI-II levels in the lower wounded leaves
(open bars) and the upper unwounded leaves (closed bars) were measured.
Data show the mean :h SD (n = 7 for the wild type and acxl; n = 20
independent transgenic lines confirmed by PCR to contain the 35S-ACX1A
transgene). PI-II levels in unwounded control plants were below the detection

limit of the assay (~5 pg PI-II/ml leaf juice).

133

To determine whether this mutation (T1381) affects the biochemical activity of

ACXlA, we expressed His-tagged derivatives of the wild-type (ACXIM) and mutant

T1381

(ACXl ) proteins in Escherichia coli. Affinity-purified ACX]Wt displayed 381- and

452-nm absorption peaks that are indicative of flavoenzymes (Figure 4.5A). An

T1381

equivalent amount of affinity-purified ACXl lacked this spectral signature. In vitro

activity assays with straight-chain acyl-CoAs showed that recombinant ACX]Wt has a

preference for C12 and C14 substrates (Figure 4.5B). The enzyme was less active with

C18 and C20 substrates and showed little or no activity with short-chain (3C6) acyl-

T1381

CoAs. ACXl showed no detectable activity against 14:0-CoA, which was a

preferred substrate of the wild-type enzyme (Figure 4.6). These results indicate that the
T1381 mutation in acxl renders ACXIA inactive, most likely by disrupting FAD binding

to the apoprotein.

134

Figure 4.5 UV/vis spectra of purified recombinant ACXIA and ACXIAT1381 and

substrate specificity of ACXlA.

(A) UV-visible spectra of affinity-purified recombinant ACXIA (solid line) and
ACXlAT‘38' (dotted line) were recorded at a protein concentration of 2 mg/ml.

Absorption peaks at 381 nm and 452 nm are indicative of FAD binding.

(B) Substrate specificity of ACXlA. The activity of recombinant ACXIA against
various fatty acyl-CoA esters was measured using a spectrophotometric assay linked
to H202 production. Data show the mean i SD from three replicate assays with the
same extract, and are representative of experiments performed with independent

enzyme preparations.

135

 

1.0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A
P, -—WT
0.8‘ ...... T1381
g :
(B 0.6 «
.0
h
0
g 0.41
<
0.2 1
0.0 .
300 400 500 600
W velen th
B a 9
C
E 8
e r“ 5
m 6‘
E " 5
.E
E 4« __ - -. _
N -.- .fl ' - FL

7

6‘

<9
7
7
77-
7
7c?
90.

Figure 4.5 UV/vis spectra of purified recombinant ACXIA and ACXIAT1381

and substrate specificity of ACXlA.

136

 

 

 

Absorbance 500 nm

 

 

 

Minutes

Figure 4.6 ACX activity assay with ACXIAT'33'. ACX activity was
measured for ACXIA (solid line) and ACXlAT‘38' (dotted line) against 14:0-
CoA. The change in absorbance at 500 nm results from production of a
colored product from a peroxidase catalyzed reaction utilizing the H202

released from the ACX reaction.

137

ACXIA is required for JA biosynthesis

Current knowledge of the J A biosynthetic pathway predicts that OPC8-CoA is the
substrate for entry into the fi-oxidation cycle via the action of ACX (Figure 4.1). To
determine whether ACXIA is capable of metabolizing OPC8-CoA, we used an in vitro
assay that couples ACXIA to a yeast acyl—CoA synthetase (ACS) that activates the fatty
acid substrate to the corresponding CoA ester. Addition of 50 uM OPC8 to the assay
resulted in ACXIA activity that was significantly greater than that obtained with the
same concentration of 14:0 (Figure 4.7). Control experiments showed that ACXlA
activity in these experiments was dependent on ACS and CoA and was proportional to
the amount of substrate added (data not shown). Interestingly, OPDA was comparable to
OPC8 in its ability to promote ACXIA activity. These results provide evidence that
ACXlA can metabolize OPC8-CoA and that reduction of the double bond in the

cyclopentenone ring of OPDA is not required for the enzyme’s activity.

Developmental expression of A CXIA in tomato

RNA- and westem-blot analyses were used to investigate the abundance of
ACXIA mRNA and its corresponding protein in various tissues of wild-type and acxl
tomato plants. ACXIA transcripts were detected in all tissues examined with highest
expression seen in germinating seedlings and flowers (Figure 4.8). Though acxl contains
a point mutation in ACXIA, expression in the mutant is unchanged compared to wild-
type. Using polyclonal antibodies raised against recombinant ACXlA, we also examined
the levels of ACXIA protein in various tissues (Figure 4.9). The antibody cross-reacted

with a protein at the expected size of 74 kD. In wild-type, ACXIA protein was

138

 

Absorbance 546 nm

0.2 '

 

 

0.0 ......... '...u......q.1 ......... . ‘

Time after addition of ACX1A (min)

Figure 4.7 ACX1A activity against OPDA and OPC-8 in a coupled assay with
acyl-CoA synthetase. ACX1A activity against OPDA-CoA and OPC8-CoA was
tested using the corresponding free acids in a coupled reaction using an ACS with
recombinant ACX1A. The indicated substrates (5 uL in 0.25% Triton X-100, 50 uM
final concentration) or mock control were added to a reaction mixture containing a
yeast ACS, CoA, and ATP. After 0.5 hrs at 25°C, the reaction was terminated by
addition of N-ethylmaleinimide and the ACX reaction was initiated by addition of 1
ug of purified ACX1A. ACX1A activity was monitored spectrophotometrically via
the production of a colored product from a peroxidase catalyzed reaction utilizing the

H202 released from the ACX reaction.

139

wild-type acx1

 

 

RGSCStLF RGSCStLF

 

 

/
ACX1A... ... . m,“ “I! n

 

 

elF4A ‘.,, . w .

 

 

 

 

 

 

 

Figure 4.8 ACX1A expression in various tissues. RNA was extracted from
roots (R), germinating seedlings (GS), cotyledons (C), stems (St), leaves (L),
and unopened flower buds (F) of wild-type and acxl plants. Identical RNA
blots were probed with either ACXIA or eIF4A (loading control) cDNA

probes.

140

g
E
3.7
s

5

 

 

 

 

8 8 E '3 g 8
3’: 8 8 .92 5 3’: 8 8 2 8
35385855 85388-35:
M 9. a, 8 .d 8 8 2 u: M 8 g 8 .d a 8. .2 a:
ii‘s1 194 --‘-~*‘--b"’""""
115 a
53 w “u— ----- ‘
53
ll 4"! 4
37 37 w \-
29 29 ,,

 

 

 

 

 

 

Figure 4.9 Analysis of ACXl protein in various WT and acxl tissues.
Twenty-microgram samples of protein from various tissues of wild type (left) or
acxl (right) was separated by SDS-PAGE, transferred to PVDF membranes, and

probed with antiserum raised against recombinant ACX1A. M, sizes of molecular

weight markers.

141

detected in each of the tissues examined in a pattern similar to that of its transcript. When
ACX1A protein was analyzed in extracts from acxl tissues, protein of the expected size
was reduced compared to that of wild-type. However, a much larger cross-reacting band
appeared in acxl extracts not seen in wild-type, and also not seen in the corresponding

pre-immune blots (data not shown).

Involvement of A CX 1A in fl-oxidation of other substrates

It has been shown for all ACXS studied to date that they tend to exhibit highly
variable substrate specificities, in terms of the chain length of fatty acyl-CoAs (Graham
and Eastmond, 2002). This was also seen for ACX1A as shown above (Figure 4.5B).
Since ACX1A was more active with OPDA-CoA and OPC8-CoA (both containing a
five—carbon ring), we also tested whether ACX1A could participate in B-oxidation of
another cyclic substrate, 2,4-dichlorophenoxybutryic acid (2,4-DB; containing a six-
carbon ring). 2,4—DB can undergo one round of B-oxidation to be converted to the
synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4D), which has been shown to inhibit
root grth of germinating seedlings (Hayashi et al., 1998). Root lengths were measured
five days after two-day old wild-type and acxl germinating seedlings were transferred to
plates containing media supplemented with 2,4-DB. Compared to seedlings that were
untreated or grown on 0.5 uM 2,4—DB, wild-type seedlings treated with 1 uM 2,4-DB
showed significant reduction in root growth (p=0.026, Mann-Whitney Rank Sum
test)(Figure 4.10). 10 uM was approximately the saturating concentration of 2,4-DB
needed to give maximum root grth inhibition for wild type. The root length for acxl

germinating seedlings grown on 1 uM 2,4—DB was not significantly different than either

142

untreated wild-type, untreated acxl, or acxl grown on 0.5 uM 2,4-DB, indicating the loss
of ACX1A impairs B-oxidation of 2,4-DB. This suggests that ACX1A can participate in
the B-oxidation of other cyclic substrates. At 10 uM however, root grth inhibition was

similar to wild type, likely indicating that another ACX isoform with a lower affinity for

2,4-DB can substitute for the loss of ACX1A at higher concentrations.

143

 

80
70 4
601
50l
40-
30 ~
20«
10-

Root Length (mm)

 

 

 

 

0.1 ‘1 1'0 100
2.4-DB (uM)

Figure 4.10 2,4-DB treatment of wild-type and acxl germinating
seedlings. Seed of wild type and acxl were germinated on MS plates, and
transferred after two days to plates containing various concentrations of 2,4-
dichlorophenoxy butyric acid (2,4-DB). Root lengths were measured five

days after transfer (wild-type, solid line; acxl, dashed line).

144

Discussion

Here we provide several lines of evidence demonstrating that the wound-signaling
defect in the JL1 mutant of tomato (renamed here acxl) results from a block in the [3-
oxidation stage of the octadecanoid pathway. First, acxl plants harbor a defective
member (ACX1A) of the ACX family of enzymes that catalyze the first step in the [3-
oxidation cycle. Second, recombinant ACX1A metabolizes OPC8-CoA, which is the
presumptive substrate for the first B-oxidation cycle in the JA biosynthetic pathway.
Finally, it has been shown that acxl leaves accumulate very little (~5% wild-type levels)
JA in response to wounding (Li et al., 2005). We thus conclude that ACX1A is
responsible for the majority of wound-induced J A in tomato leaves.

The deduced amino acid sequence and substrate specificity of LeACXlA is most
similar to that of ACXl and ACX5 of Arabidopsis and two ACXs from soybean (Hooks
et al., 1999; Eastmond et al., 2000; Agarwal et al., 2001). These ACXs have relatively
broad substrate specificity and, as yet, no definitive physiological function. Several
observations lead us to propose that LeACXlA and other members of the so-called
medium- to long-chain subfamily of ACXs (Figure 4.3) play a prominent role in JA
biosynthesis. First and foremost is the JA-deficient phenotype of acxl plants. Second,
results from the coupled ACS-ACX assay indicate that ACX1A exhibits a preference for
C 18 cyclopentanoid-CoAs over 14:0-CoA (Figure 4.7). This observation suggests that
ACX1A and related enzymes possess structural features that facilitate the metabolism of
C18 cyclopentanoid fatty acids. Third, recent studies have implicated Arabidopsis ACXl

in wound-induced J A synthesis and expression of wound responsive genes (Castillo et al.,

145

2004). Unlike the severe J A deficiency in tomato acxl plants, Arabidopsis acxl antisense
lines retained 50 to 60% of wild-type levels of wound-induced JA. This relatively weak
phenotype may reflect incomplete suppression of AtACXl or the capacity of other ACXs
(e. g., ACX5) to participate in J A biosynthesis in wounded Arabidopsis leaves. Hooks et
al. (1999) reported that anti-ACX] lines of Arabidopsis exhibit a modest reduction in root
growth, but J A levels were not assessed in that study.

Identification of LeACXlA as an essential component of the octadecanoid
pathway supports the original proposal by Vick and Zimmerman (1984) that JA
biosynthesis involves B-oxidation enzymes that remove six carbons from the octanoate
side chain of OPC8. The presence of a PTSI sequence at the C terminus of LeACXlA,
together with the fact that fatty acid B-oxidation in plants occurs in peroxisomes, are
consistent with the notion that ACX1A is a peroxisomal protein. Subcellular localization
experiments are needed to confirm this. Based on the ability of ACX1A to use OPC8-
CoA and the location of OPR3 in peroxisomes (Strassner et al., 2002), our results
indicate that the likely in vivo substrate for ACX1A is a peroxisomal pool of OPC8-CoA
(Figure 4.1). At present, the temporal sequence of metabolic events involved in the
conversion of plastid-localized OPDA to OPC8-CoA remains to be established. One
possibility is that OPDA is transported from the plastid as a CoA ester and is then
converted by OPR3 to OPC8-CoA in the peroxisome. Because OPR3 readily accepts free
OPDA as a substrate (Schaller,_2001; Strassner et al., 2002), an alternative scheme is that
OPR3 first reduces OPDA to OPC8, which is then converted to OPC8-CoA.
Identification of enzymes involved in the synthesis and transport of C18 cyclopentanoid-

CoAs may help to distinguish these possibilities.

146

Our results do not exclude the possibility that conversion of OPC8-CoA to JA-
CoA involves multiple ACXS that specifically act on OPC8-CoA, OPC6-CoA, or OPC4-
CoA. However, the ability of LeACXlA to metabolize a broad range of fatty acid chain
lengths (Figure 4.5B) argues against this hypothesis. Determination of the relative
specificity of ACX1A for different OPC-CoA derivatives would be helpful to further
address this issue, as would the identification of OPC-CoA intermediates that accumulate
in acxl tissues. In considering the role of ACX1A in JA biosynthesis, it is noteworthy
that the enzyme accepts both OPC8- and OPDA—CoAs as substrates in the ACS-ACX
coupled assay (Figure 4.7). Because we did not determine the extent to which each
substrate was activated to the CoA ester after the ACS reaction, it is not possible to draw
firm conclusions about the relative preference of ACX1A for different substrates.
However, assuming that OPC8 and OPDA were equally converted to the CoA ester, it
would appear that ACX1A does not discriminate between the cyclopentenone (i.e.,
OPDA) and the cyclopentenone (i.e., OPC8) derivatives. This observation raises the
possibility that OPR3 activity is not strictly required for entry of OPDA into the B-
oxidation cycle. Complete B-oxidation of OPDA is expected to yield 4,5-didehydro-JA.
Such a pathway may explain the production of this JA derivative in fungi (Miersch et al.,
1989) and plants (Dathe et al., 1989).

The acxl mutation affects a Thr residue (Thrl38) that plays a critical role in
positioning the FAD cofactor at the active site of the enzyme (Kim and Miura, 2004).
However, the point mutation in acxl does not seem to cause a change in the steady state

levels of the acxl transcript (Figure 4.8). Analysis of ACX1A protein levels in acxl

T1381

suggest that ACXl is mostly present as a multimer that is not separated even by

147

SDS-PAGE (Figure 4.9). It is not clear why such a high molecular weight form
accumulates, but one possible cause of the aggregation is improper folding due to lack of

FAD binding (Figure 4.5A). This result together with the observation that recombinant

T1381

ACXl lacked detectable activity against 14:0-CoA indicates that acxl is most likely

a null mutation. If this is the case, the residual JA production in acxl plants can be
attributed to one or more other ACX family members. The sequence of BAC232L13
revealed that LcACXlA is located adjacent to a closely related family member,
designated LeACXlB. Although RT-PCR experiments confirmed that ACXIB is
expressed in L. esculentum leaves, the presence of a lS-amino acid deletion in the
deduced sequence of ACXIB (Figure 4.2) raises the question of whether this gene
encodes an active enzyme. Attempts to express ACXIB in E. coli resulted in failure to
detect accumulation of the fusion protein (L. Katsir, A.L. Schilmiller, and GA. Howe,
unpublished results). Inspection of the tomato EST database (http://www.sgn.comell.edu)
provided evidence for additional ACX genes that are homologous to the short-chain
(AtACX4) and medium-chain (AtACX3) ACXs of Arabidopsis (Figure 4.3). An
additional partial-length sequence (SGN-U229242) appears to be homologous to the
Arabidopsis long-chain ACX (AtACX2). Given the distinct but overlapping substrate
specificity of different ACX family members (Eastmond et al., 2000; Graham and
Eastmond, 2002), it is reasonable to propose that one or more of these tomato ACXS is
responsible for the residual J A production in acxl leaves.

In higher plants, B-oxidation has been extensively studied for its role in
converting storage lipids to acetyl-CoA during early stages of seedling development. The

importance of this process in the plant life cycle is highlighted by the fact that mutants

148

defective in peroxisomal B-oxidation ofien exhibit developmental arrest at the seedling
stage (Hayashi et al., 1998; Zolman et al., 2001; Footitt et al., 2002). It is possible that
ACX1A, in addition to its role in JA biosynthesis, also is involved in fatty acid
catabolism during seed germination and seedling growth. This idea is supported by the
ability of ACX1A to use straight-chain fatty acyl-CoAs (Figure 4.5B) as well as the
accumulation of ACX1A transcripts and its encoded protein in germinating tomato
seedlings (Figure 4.8, 4.9). On the other hand, the ability of acxl seedlings to establish
photoautotrophic grth in the absence of sucrose (data not shown) indicates that if
ACX1A does participate in seed lipid mobilization, this role is not required for seedling
establishment. A nonessential role for ACX1A in seed lipid mobilization is consistent
with detailed biochemical analysis of the ACX family in Arabidopsis (Hooks et al., 1999;
Eastmond et al., 2000; Eastmond et al., 2000; Froman et al., 2000). These collective
studies indicate that the substrate specificities of AtACX2 and AtACX3 are likely
sufficient to metabolize, in the absence of AtACXl, the full range of medium- and long-
chain acyl-CoAs that exist in vivo.

B-Oxidation in nonfatty plant tissues has been implicated in diverse physiological
processes, including floral development, synthesis of indole-acetic acid and JA, and
production of acetyl-CoA substrate for primary and secondary metabolism (Graham and
Eastmond, 2002; Zohnan and Bartel, 2004). Aside from defects in JA biosynthesis and
JA-mediated defense responses, adult acxl plants did not exhibit overt morphological or
reproductive phenotypes. Based on these observations, we suggest that a primary
function of ACX1A in tomato leaves is catalysis of JA synthesis. However, with the

ability of ACX1A to also use 2,4-DB, demonstrated by the reduced sensitivity of acxl for

149

this proherbicide, we cannot rule out the possibility that ACX1A firnctions in B-oxidation
of other substrates.

We previously showed that the F-box protein CORONATINE INSENSITIVEI,
which is an essential component of the JA signaling pathway, is required for female
fertility in tomato (Li et al., 2004). This finding implies that normal female reproductive
development in tomato requires the action of endogenous J As, which are known to
accumulate to high levels in reproductive organs (Hause et al., 2000). Although acxl
homozygotes produce normal amounts of seed, we have observed that the rate of acxl
seed germination varies considerably (from <15 to >90%) between different seed
batches. This variability may be related to environmental growth conditions (see
Methods). The ability of acxl plants to produce viable seed is similar to that of the spr2
tomato mutant that is defective in the production of linolenic acid, the major precursor of
JAs (Li et al., 2003). We thus suggest that reproductive tissues of acxl plants, like those
of spr2, are capable of producing JAs at a level that is sufficient to promote seed
production. Assuming that acxl is a null mutation, this explanation implies that other
ACX isoforms can participate in J A biosynthesis in reproductive tissues.

In conclusion, we show that ACX1A in tomato is required for the B-oxidation
stage of JA biosynthesis. The involvement of B-oxidation in JA biosynthesis implies an
important role for peroxisomes in orchestrating plant responses to this stress hormone. A
remarkable feature of plant peroxisomes is their capacity to alter their enzymatic content
in response to environmental or developmental cues (Olsen, 1998; Corpas et al., 2001).
The induced expression of genes involved in B-oxidation and peroxisome biogenesis in

response to wounding and pathogen infection further suggests that biotic stress may be a

150

trigger for peroxisome proliferation in certain plant tissues (Lopez-Huertas et al., 2000;
Mysore et al., 2002; Schenk et al., 2003; Castillo et al., 2004). As noted by Strassner et
a1. (2002), this degree of metabolic plasticity raises the possibility that the terminal steps
in J A biosynthesis occur in a specialized type of peroxisome. Other results from studies
with acxl are consistent with this idea and further suggest that J A-producing peroxisomes
play an important role in the generation of signals that mediate systemic defense
responses to herbivore attack (Li et al., 2005). Recent studies have shown that plastid
enzymes of the octadecanoid pathway are spatially restricted to the companion cell-sieve
tube element complex of the vascular bundle (Ryan, 2000; Hause et al., 2003; Howe,
2005). That wound-induced JA accumulation is also enriched in vascular tissues (Stenzel
et al., 2003) would suggest that ACXI and other peroxisomal enzymes of the pathway are
located in these cell types as well. Support for this idea comes from studies showing that
peroxisomes exist in sieve tube elements of root phloem (J edd and Chua, 2002) and that
soybean ACXl is localized in the phloem cells of hypocotyl tissue (Agarwal et al., 2001).
Future studies aimed at determining the location of JA-producing peroxisomes may

provide additional insight into the role of oxylipins in long-distance wound signaling.

151

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155

Chapter 5

Acyl-CoA Oxidases that Function in the B—Oxidation Stage of J asmonic Acid

Biosynthesis are Essential for Insect Resistance and Pollen Development in

Arabidopsis

The work presented in this chapter has been submitted for publication: Schilmiller AL,

Koo AJK, Li C, Lee GI, Howe GA (2005) Plant J. Submitted for Review.

156

Abstract

The plant hormone jasmonic acid (J A) and its bioactive C18 precursors regulate a variety
of developmental and defense-related processes. The final steps in the production of JA
(a C12 cyclopentanone) require the action of peroxisomal B-oxidation enzymes that
remove six carbon atoms from a C18 precursor. Among the enzymes involved in this
metabolic transformation is acyl-CoA oxidase (ACX), which catalyzes the first step of
the B-oxidation cycle. The purpose of this study was to identify acx mutants of
Arabidopsis that are deficient in J A synthesis, and to use these mutants to investigate the
role of the B-oxidation in jasmonate-signaled processes. We show that JA biosynthesis in
Arabidopsis involves the so-called medium-to-long chain subfamily of ACXs that
includes ACX] and ACX5. acxl/5 double mutants were deficient in wound-induced JA
accumulation and gene expression, and were compromised in resistance to the
lepidopteran insect T richoplusia ni. Unlike mutants that are blocked in JA perception or
the production of C18 cyclopentenoids, acxl/5 plants maintained strong resistance to the
fungus Alternaria brassicicola. These results indicate that JA and its C 18 precursors
regulate defense responses against different types of biotic stress. The JA deficiency in
acxl/5 plants also resulted in reduced pollen viability and fecundity. Consistent with this
finding, analysis of transgenic lines expressing ACX] promoter-reporter fusions showed
that ACX] is highly expressed in pollen, as well as other reproductive and vegetative
tissues. We conclude that jasmonate signals derived from peroxisomal B-oxidation in

A rabidopsis are essential for both male fertility and defense against leaf-eating insects.

157

Introduction

Plant responses to biotic stress are coordinated by a network of signal transduction
pathways that control a wide range of physiological processes. Jasmonic acid (JA) and
related members of the jasmonate family signaling compounds (collectively called JAs)
play a central role in orchestrating these responses (Glazebrook, 2005; Halitschke and
Baldwin, 2005; Howe, 2005). Although JAs are often regarded as stress signals, it is now
clear that they also regulate a variety of developmental processes. Included among these
are carbon/nitrogen partitioning (Creelman and Mullet, 1997), tendril coiling (Weiler et
al., 1993), glandular trichome development (Li et al., 2004), root growth (Staswick et al.,
1992), and various aspects of male and female reproductive fimction (Feys et al., 1994;
McConn and Browse, 1996; Li et al., 2004). A current challenge in the field of jasmonate
signaling is to understand the molecular mechanisms by which individual bioactive JAs
regulate specific target processes.

The octadecanoid pathway for JA biosynthesis is initiated in the chloroplast and
terminated in peroxisomes (Figure 5.1). Many of the enzymes and corresponding genes
involved in the pathway have been identified (Schaller et al., 2005). A chloroplastic
lipoxygenase initiates JA synthesis by adding molecular oxygen to linolenic acid (18:3).
The resulting 13-hydroperoxy fatty acid is converted to 12-oxo-phytodienoic acid
(OPDA) by the sequential action of allene oxide synthase (AOS) and allene oxide cyclase
(AOC). Although little is known about the mechanism of plastid-to-peroxisome transport
of octadecanoids, a peroxisomal ATP-binding cassette transporter was recently

implicated in this process (Theodoulou et al., 2005). Within the peroxisome, OPDA

158

Figure 5.1 The octadecanoid pathway for JA biosynthesis.

Trienoic fatty acids (18:3 and 16:3) are converted within the chloroplast to 12-oxo-
phytodienoic acid (OPDA) and dinor-OPDA, respectively. These cyclopentenone
intermediates are transported to the peroxisome via a pathway that involves an
ABC-transporter (CTS/PED3/PXA1), and then reduced by OPDA reductase
(OPR3). The resulting cyclopentanone compounds (OPC8 and OPC6) are ligated to
CoA by an acyl-CoA synthetase (ACS). Successive rounds of B-oxidation yield J A.
See text for details. The three core enzymes in the B-oxidation cascade are acyl-
CoA oxidase (ACX), multifunctional protein (MFP), and 3-ketoacyl-CoA thiolase
(KAT). LOX, lipoxygenase; AOS, allene oxide synthase; AOC, allene oxide
cyclase; OPC8, 3-oxo-2-(2’-pentenyl)-cyclopentane-l-octanoic acid; OPC6, 3-oxo-
2-(2’-pentenyl)-cyclopentane-1-hexanoic acid; OPC4, 3-oxo-2-(2’-penetenyl)-

Cyc10pentane-1-butyric acid; JA, (+)-7-iso-jasmonic acid.

159

 

 

\\
((18:3 16:3

8

g 1 LOX, AOS, AOC I

5

 

 

 

 

OPDA dinor-OPDA
/ J)

k \. T

      

 

OPR3
OPC8 OPCG
lACS 1 ACS
OPC8-00A (a OPCG-CoA
l ACX l ACX
Az-OPCB-COA AZ-OPCG-COA
l MFP l MFP
E KAT l KAT
g OPC4-CoA
E l ACX
o .1 MFP
O.
l KAT

 

 

C

thioesterase
A .—— JA-Coy

Figure 5.1 The octadecanoid pathway for JA biosynthesis.

160

reductase (OPR3) converts OPDA to cyclopentanone 3-oxo-2(2’[Z]-pentenyl)-
cyclopentane-l-octanoic acid (OPC8). An acyl-CoA synthetase (ACS) is presumably
required to produce OPC8-CoA, which subsequently enters the B-oxidation cycle that
yields JA (Li et al., 2005). Peroxisomal enzymes related to 4-coumarate-CoA ligase were
recently shown to catalyze the formation of OPDA-CoA and OPC8-CoA in vitro
(Schneider et al., 2005). A parallel JA biosynthetic pathway starting from chloroplastic
pools of hexadecatrienoic acid (16:3) has also been described (Weber et al., 1997)
(Figure 5.1).

B-oxidative breakdown of fatty acids in plant cells occurs in peroxisomes. The
three “core” enzymes involved in this pathway are acyl-CoA oxidase (ACX), a multi-
functional protein (MF P) possessing 2-trans-enoyl-COA hydratase and L-3-hydroxyacyl-
CoA dehydrogenase activities and 3-keto-acyl-CoA thiolase (KAT) (Graham and
Eastmond, 2002) (Figure 5.1). Seminal work by Vick and Zimmerman (Vick and
Zimmerman, 1984) provided the first evidence that J A biosynthesis involves B-oxidation.
Only recently, however, has specific enzymes been implicated in this stage of the
pathway. The ACX IA gene product in tomato was shown to metabolize OPC8—CoA and
to contribute to the vast majority of JA production in wounded leaves (Li et al., 2005).
Genetic evidence also indicates that the ACX] and KAT 2 genes in Arabid0psis have a role
in wound-induced JA production (Cruz Castillo et al., 2004; Pinfield-Wells et al., 2005).
The persistence of significant levels of JA in acxl and kat2 mutants, however, suggests
that additional members of these gene families contribute to JA production in

A rabidopsis.

161

Increasing evidence indicates that OPDA, JA, and certain J A derivatives promote
different physiological responses (Mithofer et al., 2005; Schaller et al., 2005; Schilmiller
and Howe, 2005). For example, JA is strictly required for male fertility in Arabidopsis
(McConn and Browse, 1996; Stintzi and Browse, 2000). Conversely, OPDA rather than
J A is thought to be the active signal for the tendril coiling response of Bryonia (Weiler et
al., 1993). Studies of the Arabidopsis opr3 mutant have provided strong evidence that
OPDA promotes jasmonate-based resistance to Bradysia impatiens and Alternaria
brassicicola in the absence of J A (Stintzi et al., 2001). On the other hand, J A biosynthesis
is needed for production of the systemic wound signal in tomato, as well as for induced
resistance of this species to lepidopteran attackers (Li et al., 2005). These results raise the
possibility that plastid-derived OPDA and peroxisome-derived JA activate host defense
responses to different biotic threats, and that the transformation of OPDA to JA is
necessary for a subset of j asmonate-signaled defense responses.

A better understanding of the role of B-oxidation in jasmonate signaling would be
facilitated by the identification of Arabidopsis mutants that fail to convert OPC8-CoA to
J A. Although Arabidopsis mutants affected in the B-oxidation stage of JA synthesis have
been reported (Cruz Castillo et al., 2004; Pinfield—Wells et al., 2005), none have been
shown to exhibit physiological hallmarks of JA deficiency (e. g., male sterility). Here we
report that simultaneous disruption of two ACX genes (ACXI and ACX5) effectively
abolishes wound-induced JA accumulation in Arabidopsis leaves. We demonstrate that
this defect compromises defense against the lepidopteran insect T richoplusia ni, but does
not impair jasmonate-mediated resistance to the fungus Alternaria brassicicola. These

results support the hypothesis that JA and its C18 cyclopentenoid precursors promote

162

host resistance to different types of biotic stress. We also show that simultaneous loss of
function of ACXl and ACX5 depletes JA accumulation in flowers to a level below that
required for normal pollen development and fecundity. Taken together, our findings show
that the B-oxidation stage of IA biosynthesis is essential for both developmental and

defense-related processes in Arabidopsis.

Materials and Methods

Plant material and growth conditions

Plants (Arabidopsis ecotype Columbia) were grown in soil in a growth chamber
maintained under 16 h of light (100 pH In2 S") at 21°C and 8 h of dark at 21°C. T-DNA

tagged lines from the SALK collection (Alonso et al., 2003) were obtained from the
Arabidopsis Biological Resource Center (ABRC, Ohio State University). PCR assays
were used to screen for plants that are homozygous for T-DNA insertions in ACX]
(SALK_041464) and ACX5 (SALK_009998) (Figure 5.2). These assays employed the
LBbl T-DNA oligonucleotide amid/signal.salk.edu/tdn_a_FAOs.html) and appropriate
gene-specific primers (acxl, 64L 5’-GCAGACAGGAAGAATTTGTGAGAGTT TGG-
3’ and 64R 5’-GTGGTGGACATGGATACTTGTGGTG-3’; acx5, 98L 5’-CCGA
GTCATTGAGTGGATCCT-3’ and 98R 5’-CTGGAAAGGCTCCTTCTGGGA-3’). The
T-DNA insertion locations were confirmed by DNA sequencing of the amplified PCR
products. The acxl/5 double mutant was generated by crossing the single mutants, and

screening the resulting F 2 progeny for plants exhibiting reduced seed set. The identity of

163

Figure 5.2 Identification of SALK lines harboring T-DNA insertions in ACX] and
ACX5.

(A) Schematic diagram of the genomic organization of ACX] and ACX5. Introns and
exons are depicted as block boxes and horizontal lines, respectively. The location of
the T-DNA insertion (inverted triangle) within exon 10 of each gene, as well as the
location of primers (arrows) that were used for PCR-based genotyping, is shown.

(B) PCR assays were used to confirm the homozygosity of T-DNA insertions in ACX]
and ACX5. Genomic DNA isolated from WT, acxl, acx5, and acxl/5 plants was used
as a template for PCR reactions employing primer sets (see above) that flank the T-
DNA insertion site, or primer sets that included a gene-specific primer and a T-DNA
left-border primer. Lanes labeled “1”: primer pair (64L and 64R) that flanks the T-
DNA insertion in ACX]; lanes labeled “5”: primer (98L and 98R) that flanks the T-
DNA insertion in ACX5; lanes labeled “Tl”: ACX] gene-specific primer (64L) and a
T-DNA left-border primer (LBbl); lanes labeled “T5”: ACX5 gene-specific primer
(98R) and T-DNA left border primer (LBbl). The sizes (in kilobase pairs) of DNA

markers (M) are indicated on far left.

164

LBb1
64R " 64L
AtACX1 —> I‘-
SALK_041464
LBb1
+—

AtACX5 98"» l 38L
SALK_009998

B WT acx1 acx5 acx1/5

 

M15 T1T515T1T515T1T515T1T5M

 

Figure 5.2 Identification of SALK lines harboring T-DNA insertions in

ACX] and ACX5.

165

the double knockout was confirmed by PCR (Figure 5.2B). Prolonged exposures of
A CXl -probed northern blots showed that low levels of ACXl-related transcripts
accumulate in acxl leaves. RT-PCR analysis with primers that hybridize to the 5’
untranslated region (5'-CACACTCGAGAATCTGAGACAATAG-3') of ACX] and the 3’
end of the ACX] open reading frame (5'-
GGGTCGACTCAGAGCCTAGCGGTACGAAG-3') detected full length ACX]
transcripts in RNA isolated from WT but not acxl leaves (data not shown). DNA
sequencing of RT-PCR products from acxl revealed that the low abundance transcripts
are derived from transcriptional read-through of ACX] through the T-DNA insertion
followed by splicing out the exon containing the T-DNA (data not shown). The
corresponding ACXl proteins are predicted to lack amino acids encoded by at least one
ACX] exon, and thus can be assumed to be non-functional. PCR screening was also used
to identify a homozygous aos T-DNA insertion mutant (SALK_017756). Primers used
for these reactions were 5’-TTCTCTCCTTCTTCTCCGACG-3’ and 5’—

GATCCATCGGAGCCTAAACAC-3’ (data not shown).

Western blot analysis of A CX protein levels

Recombinant His-tagged LeACXlA was affinity purified as previously described
(Li et al., 2005). Rabbit polyclonal antibodies against this antigen were produced by a
commercial vendor (Cocalico Biologicals, Reamstown, PA) according to their standard
protocol. Western blot analysis was performed with 30 ug of total protein extracted from
three-week old rosette leaves. Proteins were separated on 10% SDS-polyacrylamide gels

and transferred to Irnmobilon-P membranes (Millipore, Bedford, MA) according to

166

standard procedures (Harlowe and Lane, 1988). Membranes were incubated at 24°C for 1
h with anti-LeACXlA antibodies that were diluted 1:1000 in TTBS (Tris-buffered saline
with 0.1% Tween 20) containing 1% nonfat milk. As a control, duplicate protein blots
were incubated with pre-immune serum obtained from the same rabbit that was
immunized with LeACXlA. Blots were washed 3 times with TTBS and then incubated
with a peroxidase-conjugated anti-rabbit secondary antibody (125,000 dilution; Sigma,
St. Louis, MO). ACX protein-antibody complexes were visualized with the SuperSignal
West Pico Chemiluminescent substrate (Pierce, Rockford, IL) according the

manufacturers instructions.

JA measurements

F our-week old plants were wounded twice with a hemostat across the mid-vein of
each rosette leaf. At various times after wounding, leaf tissue (200-300 mg) from at least
three different plants of the same genotype was pooled and fiozen in liquid nitrogen.
Tissue was also collected from unwounded control plants grown in the same flat. J A was
extracted according to the vapor-phase extraction procedure (Schmelz et al., 2004), and
quantified by GC-MS as previously described (Li et al., 2005). For JA measurements in
flowers, unopened buds and the first two opened flowers within the flower cluster were
pooled from at least five inflorescences of each genotype. A total of 50 to 60 mg of

flower tissue was used for each J A extraction.

167

RNA blot analysis

Wounding and harvesting of leaf tissue was done as described above. RNA
extraction and gel blot analysis was performed as previously described (Li et al., 2002).
Probes prepared from full-length cDNA clones for VSP-l (stock# 114D3), OPR3
(U13428), and ACX] (U14146) were obtained from the ABRC. RNA quality and equal
loading was confirmed by staining duplicate gels with ethidium bromide, as well as by
hybridization of blots to a cDNA probe for Actin-8. This cDNA was obtained by RT-PCR
with the following primers: 5’-GARAARATGACNCARATNATGTTYGARACNTT-3’

and 5 ’-TCYTTNCTNATRTCNACRTCRCAYTTCATDAT-3 ’.

Insect feeding trials and fungal pathogenicity assays
T richoplusia ni (cabbage looper) eggs were obtained from Benzon Research
(Carlisle, PA) and hatched at 30°C. Within 8 h of hatching, a single larva was transferred

to a four-week-old host plant. Feeding trials were conducted over a period of 10 days in a

grth chamber maintained under 12 h of light (100 uE/mZ/s) at 21°C and 12 h of dark at

21°C. At the end of the trial, individual live larvae were weighed and the plants were
photographed. In trials involving JA-treated plants, 20 ul of a solution containing 1 mM
JA (Sigma) and 0.1% Tween 20 was applied to each rosette leaf immediately before
insect challenge.

Alternaria brassicicola (strain MUCL20297) was grown on potato dextrose agar
at 25°C for 10 days, at which time conidia/conidiospores were collected in water. Four-

week-old soil-grown Arabidopsis plants were inoculated on the leaf surface by applying a

5 ul drop of a suspension containing 5x105 spores ml']. Flats containing the inoculated

168

plants were covered with a plastic transparent cover to maintain high humidity. Plants
were grown under standard conditions for 5 days prior to assessing the disease

phenotype.

Pollen viability measurements

Pollen viability was measured by double staining of pollen grains with fluorescein
diacetate (FDA) and propidium iodide (PI). The procedure was essentially as described
by McConn and Browse (McConn and Browse, 1996), with minor modifications. Two
mg ml'1 FDA in acetone was added dr0p wise to a 20% (w/v) sucrose solution. Pollen
from newly-dehisced anthers was transferred to a glass slide. Following the addition of
equal volumes of FDA and PI solution (1 pg ml'l) to the pollen, the sample was covered
with a cover slip and incubated in the dark for ~10 min. Pollen was visualized under UV
illumination with an epifluorescence microscope (Zeiss Axiophot, Germany) equipped
with a DAPI filter set (excitation at 365 nm; emission longpass at 450 nm). FDA is de-
esterified within living cells to fluorescein, which emits a green fluorescence signal under
UV excitation. Only non-viable cells incorporate PI, which fluoresces red-orange under

UV light. In vitro pollen germination was done as previously described (Thorsness et al.,

1993)

169

Results

ACX] and ACX5 catalyze wound-induced JA biosynthesis in Arabidopsis leaves

The persistence of significant levels of JA in acxl mutants of Arabidopsis
suggested that additional ACX isoforms likely contribute to production of the hormone
(Cruz Castillo et al., 2004; Pinfield-Wells et al., 2005). A good candidate for such an
enzyme is ACX5, which is highly related to both ACXl and the ACX1A isoform that
catalyzes the vast majority J A biosynthesis in tomato leaves (Figure 5.3A). Our approach
for testing this hypothesis was to isolate and characterize T-DNA knockout mutants that
are defective in ACXl (acx1 plants), ACX5 (acx5 plants), or both ACXl and ACX5
(acx1/5 plants) (see Methods). RT-PCR experiments showed that the acxl and acx5
mutants used in this study fail to express functional ACX] and ACX5 transcripts,
respectively, indicating that we were assessing the null phenotypes (data not shown).

To determine whether the various acx mutants are affected in the expression of
ACX isoforms that have a putative role in JA biosynthesis, we performed immunoblot
assays with a polyclonal antibody raised against LeACXlA. The antiserum reacted
strongly and specifically with a protein in wild-type (WT) leaves (Figure 5.3B). The
apparent molecular weight of this protein was in good agreement with the calculated
molecular weight of ACX1/5 (~74,300). That this polypeptide was not detected in acxl
leaves established its identity as ACXl. These results do not exclude the possibility that
the antibody cross-reacts with other ACX isoforms whose accumulation is below the
level of detection. This interpretation is consistent with the finding that acx5 leaves

accumulate WT levels of the LeACXlA-related protein, whereas acx1/5 leaves do not

170

Figure 5.3 The Arabidopsis ACX] and tomato ACX1A isoforms are
immunologically related.

(A) Phylogenetic analysis of the Arabidopsis ACX family. A neighbor-joining
phylogeny was constructed in PAUP4.0* from the deduced amino acid sequences of
the Arabidopsis ACX family members (AtACX1-AtACX6). Also included in the
phylogeny is the tomato ACX1A isoform that has an established role in JA
biosynthesis. Numbers indicate percent bootstrap support for each branch of the
phylogeny.

(B) Western blot analysis of ACX protein levels in WT and acx knockout plants.
Protein from three-week old rosettes of the indicated genotype was blotted and
probed with a polyclonal antibody against tomato ACX1A (LeACXlA; left panel) or
an equivalent amount of pre-immune serum (right panel). The arrow indicates an
Arabidopsis ACX protein that specifically cross-reacts with the immune serum.

Molecular weight standards (kDa) are indicated on the left side of each blot.

171

100 "— AtACX1 (At4g16760)
100 ll— AtACX5(At2935690)
-———- LeACX1A

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

T 100 r— AtACX3(At1906290)
eel :— AtACX6(At1906310)
AtACX2 (At59651 10)
AtACX4(At3951840)
B to
to \
\
h to F h '0 F
X x x X x ><
E 8 :3 g E 8 8 8
1945 194-
115~ 115-r
974 971
53'
37- 37-
29-1 ,fl_ 291i
a-LeACX1A pre-immune

Figure 5.3 The Arabidopsis ACXl and tomato ACX1A isoforms
are immunologically related.

172

(Figure 5.3B). These results demonstrate that AtACX1 and LeACXlA are
immunologically related, and that ACXl does not accumulate in either acx1 or acx1/5
leaves.

To determine the relative contribution of ACXl and ACX5 to JA synthesis, we
used gas chromatography-mass spectrometry to measure the endogenous levels of IA in
unwounded (control) and wounded leaves (Figure 5.4A). The basal level of JA in
unwounded acxl and acxl/5 plants (39 i 11 and 50 :t 37 pmol/gfw, respectively) was
reduced approximately two-fold in comparison to JA levels in WT and acx5 plants (95 i
31 and 83 i 23 pmol/gfw, respectively). Mechanical wounding of WT leaves caused a
sharp increase in JA accumulation. Wound-induced JA levels in acxl leaves were
reduced to 20.6% of WT levels, which is consistent with the results of previous studies
(Cruz Castillo et al., 2004; Pinfield-Wells et al., 2005). JA accumulation in wounded
acx5 plants slightly exceeded (by approximately 1.5-fold) that in WT plants. In contrast
to the relatively large (>25-fold) wound-induced increase in JA levels in WT, acxl, and
acx5 plants, JA levels in the acxl/5 mutant did not increase significantly in response to
wounding. We estimated that the total amount of J A in wounded acxl/5 leaves was ~1%
of WT levels.

RNA blot analysis was used to determine the effect of the acx mutations on the
expression of two wound-responsive genes, 0PR3 and VSP-l (Berger et al., 1995;
Mussig et al., 2000). Mechanical wounding activated the expression of both genes in WT,
acx1, and acx5 leaves (Figure 5.4B). The most notable difference between these three
genotypes was that, at the 12 h time point, 0PR3 expression in acx5 leaves was

considerably higher than that in WT and acx1 plants. Wound-induced expression of both

173

Figure 5.4 Effect of acx mutations on wound-induced JA accumulation and gene
expression.

(A) JA levels in acx knockouts in response to mechanical wounding. Leaves on four-
week old plants of the indicated genotype were wounded twice across the mid-vein.
At various times after wounding (1.5 or 3 h), tissue was collected for JA extraction
and quantification by GC-MS. Unwounded (UW) leaf tissue was collected as a
control. Values represent the mean and SD for three independent JA extractions per
genotype.

(B) Time-course expression of wound-induced genes. Leaves were wounded as
described above. At the indicated times after wounding, leaf tissue was harvested for
RNA extraction. Leaf tissue from unwounded control plants was used for the “0” h
time point. RNA blots were hybridized to 32P-labelled cDNA probes for 0PR3, VSP-
l, and ACX]. Blots were also hybridized to a probe for Actin-8 (AC T -8) as a loading

control.

174

 

A 10000

JA (pmollgfw)
I—-t

2000-

0 *nl -. ‘7—

WT acx1 acx5 acx1/5

 

 

 

 

 

 

 

WT acx1 acx5 acx1/5
hrs:02612 026120261202612

 

OPR3 a. .. u «I.

VSP1 ! Q Q *

 

 

 

 

 

 

 

ACT-8 '- me ”GI-«.mvnupo

 

 

 

Figure 5.4 Effect of acx mutations on wound-induced JA accumulation
and gene expression.

175

VSP-l and 0PR3 was severely diminished in acx1/5 leaves; expression of these genes in
the acxl/5 background was only detected upon prolonged exposure of autoradiographs.
As expected, expression of ACX] was detected in wounded WT and acx5 plants, but not
in wounded acxl and acx1/5 mutants. These results indicate that ACXl/S-mediated J A

production is required for the expression of wound-responsive genes.

acx1/5 plants are more susceptible to T richoplusia ni feeding but maintain resistance to
infection by Alternaria brassicicola

To better define the role of the B-oxidation stage of J A biosynthesis in jasmonate-
based plant defense against insect attack, WT and acx mutant plants were challenged with
T richoplusia ni larvae (cabbage looper worm). In three independent feeding trials, acxl/5
plants reproducibly suffered more damage than WT, acxl, or acx5 plants (Figure 5.5A).
Consistent with this observation, the average weight of larvae reared on acx1/5 plants
was significantly greater than that of larvae grown on the other host genotypes (Figure
5.5B). Treatment of acx1/5 plants with JA immediately before the start of the feeding
trail was sufficient to restore resistance of the mutant to T. ni attack (data not shown).
Moreover, larval weight measurements showed the T. ni performance on JA-treated
acx1/5 plants was not significantly different (P = 0.25) from that on untreated WT plants
(Figure 5.5B). These results demonstrate that ACX1/5 function is essential for resistance
of Arabidopsis to attack by T. ni.

Pathogenicity assays were performed to determine whether acxl/5 plants are
altered in their resistance to Alternaria brassicicola infection. This necrotrophic fungal

pathogen was previously shown to activate jasmonate-dependent defense responses in

176

Figure 5.5 acx1/5 plants are susceptible to attack by T richoplusia ni.

(A) Four-week-old WT and acx mutant plants were challenged with newly-hatched
T richoplusia ni larvae (one 15t-instar larva per plant). Plants were photographed 10 d
after the start of the feeding trial.

(B) The average weight of larvae reared on each host genotype was determined at the
end of the feeding trial. In this experiment, larvae were allowed to move freely
between acx1 and acx5 plants. Thus, larval weight data for caterpillars recovered from
these two genotypes were combined. Additional experiments showed that larval
performance on acx1 and acx5 plants was not significantly different (data not shown).
Also shown is larval weight data for acxl/5 plants that were treated with JA prior to
initiation of the feeding trial (acx1/5 + JA). Data show the mean and SD of the
following number of larvae: WT, 40; acxl + acx5, 42; acxl/5, 39; acx1/5 + JA, 8. The
asterisk indicates that the weight of larvae grown on acxl/5 plants was significantly
greater (P < 0.001; Mann-Whitney Rank Sum Test) than that of larvae reared on WT

plants.

177

 

   

30‘

20j

T. ni weight (mg)
8

 

 

 

Figure 5.5 acxl/5 plants are susceptible to attack by T richoplusia ni.

178

Arabidopsis (Penninckx et al., 1996; Thomma et al., 1998; Stintzi et al., 2001). WT and
acx1/5 rosette leaves were inoculated with a suspension of A. brassicicola spores. The
coil-l mutant that is susceptible to A. brassicicola (Thomma et al., 1998) was also
inoculated as an additional control. At 3 days post-inoculation (dpi), both WT and acx1/5
plants developed a typical resistance response, manifested by the formation of brown
necrotic lesions at the site of spore inoculation (Figure 5.6A and B). In contrast, coil
leaves were heavily colonized by the pathogen (Figure 5.6C and E). This susceptibility
phenotype was also observed in a mutant that harbors a T-DNA insertion in the AOS gene
that encodes a plastidic enzyme of the octadecanoid pathway (Figure 5.6D and F). The
most straightforward interpretation of these results in that resistance of Arabidopsis to A.
brassicicola does not depend on the B-oxidation stage of JA biosynthesis, but rather
requires the synthesis of an AOS-derived C18 signal that works through COIl, as

previously proposed (Stintzi et al., 2001).

acx1/5 plants are impaired in male fertility

Severe deficiencies in JA biosynthesis or perception in Arabidopsis result in male
sterility (Devoto and Turner, 2005). During the grth of F 2 plants derived from a cross
between acxl and acx5, we observed that the acx1/5 double mutant produced very few
seed-containing siliques and very few viable seeds. Both the acxl and acx5 single
knockout lines produced normal amounts of seed (data not shown). The acxl/5-mediated
decrease in fecundity was quantified in a controlled experiment in which WT and acx1/5

plants were grown side-by-side under identical conditions (Table 5.1). Treatment of

179

 

Figure 5.6 acx1/5 plants maintain resistance to Alternaria brassicicola.

Four-week-old plants of the indicated genotype were inoculated with
Alternaria brassicicola by applying a 5 m1 drop of a spore suspension (5 x
105 spores ml'l) on the leaf surface. Plants were photographed 5 days after
inoculation. Panels E and F show a close-up view of leaves depicted in panels

C and D, respectively.

180

Table 5.1. Reduction of seed-containing siliques in acxl/5

 

 

WT acx1/5
siliques / plant 115.1 d: 25.8 105 d: 34.3 p = 0.399
S‘l‘queSS/e‘e’ilam “h 83.8 :t 26.1 14.6 :t 9.3 p < 0.001

‘V '1' ’th d
o 31 rques wr sec 719 3:10.1 131$ 6.5 p < 0.001

 

The total number of siliques per plant as well as seed-containing siliques per
plant were quantified for 14 wild-type and 32 acxl/5 plants that were fully
senesced and the percentage of siliques per plant containing seed was
calculated. Values for wild-type (WT) and acx1/5 represent the mean i SD

and statistical significance was determined with the Student’s t test.

181

acx1/5 flowers with MeJA restored silique development and viable seed production
(Figure 5.7A). Seed collected from MeJA-treated acx1/5 plants did not require sucrose
for germination or seedling establishment (data not shown).

Male sterility in JA-deficient Arabidopsis mutants is caused by a combination of
defects in pollen viability, anther elongation, and anther dehiscence (McConn and
Browse, 1996; Stintzi and Browse, 2000). However, acx1/5 flowers did not exhibit
obvious abnormalities in the timing of anther dehiscence or anther elongation (data not
shown). We thus employed fluorescein diacetate-propidium iodide staining to compare
the viability of WT and acx pollen. In four independent experiments, pollen collected
from WT, acx1, and acx5 flowers exhibited approximately 80% viability (Figure 5.7B).
In contrast, only about 30% of the pollen collected from acx1/5 plants was viable.
Decreased pollen viability in acx1/5 plants was correlated with a steep decline in JA
levels in flower buds (P<0.001, Student’s t test; Figure 5.7C). These results indicate that
the JA deficiency caused by acxl/5 severely reduces plant fecundity, mainly as a result of

a defect in pollen development.

182

Figure 5.7 acx1/5 plants are defective in JA-mediated pollen development.

(A) Photograph of an acx1/5 inflorescence. In the absence of JA treatment, the
majority of siliques fail to develop and produced no viable seed. Treatment of stage-
12 flowers with J A restored silique development (arrows) and viable seed production.
(B) Pollen from newly-dehisced flowers was collected and double-stained with
fluorescein diacetate and propidium iodide to determine percent viability. All
measurements were performed in quadruplicate, with between 100 and 500
pollen/genotype. The asterisk indicates that pollen viability in acx1/5 plants was
significantly less than that in WT plants (P = 0.002, Mann-Whitney Rank Sum Test).
(C) JA levels are reduced in acx1/5 flowers. Data show the mean and SD of three
independent experiments. Experiment involved at least three JA extractions from

independent pools of similarly-staged flowers.

183

 

% Pollen viability

 

 

O

 

500

400
300 <

JA (pmollgfw)

100‘

 

 

200 — I
1

1‘1

WT acx1/5

 

 

Figure 5.7 acxl/5 plants are defective in JA-mediated pollen development.

184

Discussion

Arabidopsis provides an excellent model system in which to study the role of
jasmonates in the plant life cycle (Gfeller and Farmer, 2004; Devoto and Turner, 2005).
In this study, we took advantage of powerful genetic tools in Arabidopsis (Alonso et al.,
2003) to identify ACX isoforms that contribute to JA-dependent developmental and
defense-related processes. Our results indicate that the vast majority (>98%) of wound-
induced JA biosynthesis in leaves requires the activity of two members of the
Arabidopsis ACX family, namely ACX] and ACX5. Recent work has shown that a
single ACX isoform (ACX1A) fulfills this fimction in wounded tomato leaves (Li et al.,
2005). The high degree of sequence similarity between Arabidopsis ACXl/S and tomato
ACX1A indicates that the so-called medium-to-long chain subfamily of plant ACXS
plays a prominent role in JA biosynthesis. This information should facilitate the
discovery of ACXs that participate in JA biosynthesis in other plant species. As
determined by the three-dimensional structure of ACXl (Pedersen and Henriksen, 2005),
the wide fatty acyl-binding pocket of these enzymes is consistent with their ability to
metabolize a broad range of straight-chain acyl-CoAs, and cyclopentenoid-CoA
precursors of J A as well (Li et al., 2005).

Based on the analysis of acx1 and acx5 single mutants, it is evident that the
contribution of ACXI to wound-induced JA production is significantly greater than that
of ACX5. Our finding that wounded acx1 leaves accumulate ~20% of WT levels of IA is
in agreement with other studies on ACXl (Cruz Castillo et al., 2004; Pinfield-Wells et

al., 2005). Despite the major contribution of this isoform to J A production, loss of ACXl

185

function did not significantly reduce the expression of wound-responsive genes that are
regulated by the JA signaling pathway (Figure 5.4B). We propose that the amount of JA
produced in acx1 plants is sufficient to activate subsequent signal transduction events that
lead to gene expression, and that other ACXs contribute to JA-mediated defense
responses in Arabidopsis.

Introduction of an acx5 null mutation into the acx1 background nearly abolished
wound-induced J A accumulation and the expression of defense-related genes, indicating
that ACX5 plays a role in wound-induced expression of JA-responsive genes.
Paradoxically, however, JA accumulation in wounded acx5 leaves was not impaired, but
rather was slightly greater than that in WT. This result is in keeping with the idea that
ACX5 makes a relatively minor contribution to JA production, and that ACXl activity in
WT leaves is not limiting for JA biosynthesis. Although ACX5 is expressed in leaves,
northern blot analysis and quantitative RT-PCR experiments showed that its expression
level is low in comparison to ACX] (ALS and GAH, unpublished data)(Kamada et al.,
2003). That wound-induced JA levels in acx5 plants were slightly greater than in WT
plants raises the possibility that ACX5 may actually impede JA biosynthesis in the
presence of ACXl. For example, it is possible that ACX5 (or ACXl/S heterodimers) is
less efficient than ACXl in the metabolism of OPC8-CoA. In acx5 leaves, ACXl would
not compete with the less effective enzyme for substrate, thus allowing a higher flux
through the B-oxidation pathway. Comparison of the in vitro kinetic parameters of ACXl
and ACX5 will be useful to test this idea.

The reduced fecundity of acx1/5 plants demonstrates that B-oxidation plays a

critical role in male reproductive development in Arabidopsis. The Arabidopsis aim]

186

mutant that is defective in the MFP-catalyzed step of B-oxidation also exhibits severely
reduced fertility (Richmond and Bleecker, 1999). However, it has not yet been
determined whether aiml-mediated sterility is caused by decreased JA production. The
ability of exogenous JA to restore fertility to acxl/5 plants, as well as the reduced levels
of IA in acx1/5 flowers, demonstrates that the sterile phenotype of this mutant results
fi'om a block in the B-oxidative stage of JA biosynthesis. Thus, our results confirm and
extend previous studies showing that JA is strictly required for male fertility in
Arabidopsis.

It is noteworthy that acx1/5 flowers do not exhibit obvious defects in anther
elongation or pollen dehiscence that occur in many other JA-deficient mutants (e.g.,
opr3). Rather, the acx1/5 metabolic block appears to impair male fertility mainly by
decreasing pollen viability. Support for this hypothesis comes from the finding that ACXI
expression in the stamen is much more prominent in the pollen than it is in the anther or
filament (AJKK and GAH, unpublished results). Additional ACX family members that
are expressed in stamens may produce enough JA to satisfy these aspects of JA-
dependent anther development. The absence of anther elongation and dehiscence
phenotypes in acxl/5 flowers may explain the ability of the mutant to produce limited
amounts of viable seed under some growth conditions. We thus suggest that the amount
of J A in acxl/5 flowers (~25% of WT levels) is close to the threshold level that is needed
for normal reproductive vigor.

Peroxisomal B-oxidation plays a central role in the catabolism of storage lipids
during seedling establishment. Several mutations that disrupt B-oxidation have been

shown to arrest seedling development in the absence of exogenous sucrose (Hayashi et

187

al., 1998; Zolman et al., 2001). The demonstration that acx1 and acx5 single mutants, as
well as the acx1/5 double mutant, germinate and grow in the absence of sucrose indicates
that these isoforms are not absolutely required for normal seedling establishment (Adham
et al., 2005; ALS and GAH, unpublished results). Increasing evidence, however,
indicates that ACXl does in fact participate in the mobilization of seed storage lipids.
First, ACXI acts on a broad range of medium- and long-chain fatty acyl-CoAs in vitro
(Hooks et al., 1999). Second, enzyme extracts from acxl mutant seedlings show a
striking deficiency in the metabolism of long-chain acyl-CoAs (Adham et al., 2005).
Third, introduction of the acx1 mutation into a genetic background that lacks ACX2,
which also metabolizes long chain acyl-CoAs (Hooks et al., 1999), results in a sucrose-
dependent growth phenotype (Adham et al., 2005; Pinfield-Wells et al., 2005). These
biochemical and genetic data are further supported by our finding that ACX] is highly
expressed in germinating seedlings (AJKK and GAH, unpublished results). Thus, it can
be proposed that ACXl serves a dual function in bulk fatty acid catabolism during
seedling development and in JA biosynthesis. Direct support for this idea comes from the
finding that the orthologous tomato enzyme (ACX1A) metabolizes both straight-chain
acyl-CoAs and cyclopentenoid-CoA precursors of J A (Li et al., 2005).

A striking phenotype of acxl/5 plants is their susceptibility to herbivore attack.
The severe JA deficiency in wounded acx1/5 leaves, together with the ability of
exogenous J A to restore protection to herbivory (Figure 5.5B), indicates that J A performs
an essential role in defense of Arabidopsis to T. ni attack. Because acxl/5 plants fail to
convert OPC8-CoA to JA, this finding excludes the possibility that resistance to this

herbivore is mediated by a C18 jasmonate signal. We also have observed that acxl/5

188

 

plants have significantly reduced resistance to western flower thrips (Frankliniella
occidentalis) (ALS and GAH, unpublished results), a cell content-feeding herbivore that
activates the JA signaling pathway (Li et al., 2002; De Vos et al., 2005). The increased
susceptibility of acx1/5 plants to multiple leaf-eating insects is consistent with studies
showing that JA is required for defense responses of tomato to attack by Manduca sexta
larvae (Li et al., 2005). We thus conclude that the B-oxidation stage of JA biosynthesis is
an essential component of the plant immune system, and that J A (or its derivatives) is the
active signal for induced resistance to at least some types of phytophagous insects.
Analysis of C011-dependent defense responses in different JA biosynthetic mutants of
Arabidopsis provides a powerful approach to determine the contribution of specific
jasmonate signals to host resistance. In applying this approach to the Arabidopsis opr3
mutant, Stintzi and associates proposed that OPDA acts as a signal for defense responses
against A. brassicicola and the saprophagous insect B. impatiens (Stintzi et al., 2001).
Our finding that acxl/5 plants maintain resistance to A. brassicicola, whereas coil and
aos mutants do not, provides strong support for this idea. However, the increased
susceptibility of acxl/5 plants to T. ni feeding allows us to further define the role of
OPDA in defense signaling. Specifically, our work with Arabidopsis and tomato (Li et
al., 2005) acx mutants indicates that OPDA (or other C18 signals) is not sufficient to
promote the full range of C011-dependent defense responses. Rather, oxylipin-mediated
resistance to many herbivores depends on the ability of the host plant to convert OPDA to
JA via the B-oxidation pathway. This discovery distinguishes plants from their animal
counterparts in which B-oxidation plays a primary role in the inactivation of oxylipin

signals derived from arachidonic acid. Future studies aimed at understanding how plastid-

189

derived OPDA and peroxisome-derived JA regulate resistance to specific plant attackers

are clearly warranted.

190

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Chapter 6

Conclusions and Future Directions

195

 

At the beginning of this dissertation research, the identification of genes encoding
CYP74 cytochromes P450 was picking up pace. The goals of chapters 2 and 3 involved
studying the CYP74 family in tomato to better understand oxylipin biosynthesis in this
plant. Comparatively, tomato is a rich source of CYP74 genes, whereas the model plant
Arabidopsis thaliana contains only two CYP74 sequences in its genome. Identification
and characterization of the first AOS that functions in the 9-LOX pathway has laid the
groundwork towards understanding the function of the 9-AOS pathway in plants. There
still remains the question of why some plants, such as Arabidopsis, lack a 9-AOS gene
but contain multiple copies of 9-LOX genes. In all plants for which there is sequence
information, 9-LOXs can be identified. Likewise, all plants are believed to synthesize J A,
which requires the presence of a l3-A 0S gene. It seems as if the 9-AOS pathway evolved
from the 13-AOSs with some plants recruiting AOS genes for transformation of 9-LOX
products, while others have not. Alternatively, plants lacking the 9-AOS pathway may
have lost their 9-AOS genes. Regardless of how the 9-AOS pathway has evolved, we still
don’t understand the function or how plants benefit from having this pathway.

Future work for studying the 9-AOS pathway in tomato includes taking advantage
of reverse genetics approaches (i.e. antisense or RNAi suppression, overexpression) to
manipulate 9-AOS expression levels. Some of this work has been started with transgenic
plants suppressing or overexpressing LeAOS3 already generated. While no obvious
phenotypes were identified in primary transformants, generating homozygous lines for
further testing is the next step. With these plants, we can determine whether suppression
of LeAOS3 expression leads to any developmental phenotypes, particularly in growing

root tips where LeAOS3 is primarily expressed. Antisense plants will also be useful for

196

 

testing for susceptibility to tomato root pathogens such as Pythium and F usarium or to
root knot nematodes. Additionally, plants ectopically expressing LeAOS3 may exhibit
phenotypic changes, which could give clues for the function of LeAOS3. One possibility
we plan to test is whether the 9-LOX (and 9-AOS) pathway has a role in regulating JA
biosynthesis. The induction of 9-LOX and 9-AOS activities by J A may function as a way
to shift free linolenic acid away from J A production. If this is true we would expect to see
higher levels and/or prolonged production of IA in wounded roots of antisense LeAOS3
plants. Generation and testing of hypotheses like this will hopefully shed light on the
physiological function of the 9-AOS pathway in plants.

While the preliminary data from microarray analysis of MeJA-regulated gene
expression in tomato roots was presented in chapter 3, much more analysis is yet to be
done. There is currently little known about JA signaled responses in non-aerial tissues.
The genes identified in this experiment represent a valuable data set for looking at
pathways and processes in roots that are controlled by J A.

The other half of this dissertation focuses on the role of B-oxidation in JA
biosynthesis. At the time of cloning LeA CX 1A, there were no genes encoding B-oxidation
enzymes associated specifically with JA biosynthesis. Work in tomato and Arabidopsis
has shown conclusively that medium-long chain ACXs are required for JA production.
Using acx mutants demonstrated the requirement of the B-oxidation stage of J A
biosynthesis in insect defense. A recent disocvery in JA signaling is that cyclic precursors
of J A (namely OPDA) have signaling properties of their own, some of which are distinct
from J A. The finding that Arabidopsis acxl/5 mutants are susceptible to a chewing insect

but resistant to a necrotrophic pathogen supports this idea. For future studies, the tomato

197

and Arabidopsis acx mutants will be useful tools for further dissecting the roles of

individual cyclic J A precursors in signaling.

198