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MOLECULAR GENETIC ANALYSIS OF JASMONATE SIGNALING
IN TOMATO (LYCOPERSICON ESCULENTUM)

presented by

Lei Li

has been accepted towards fulfillment
of the requirements for the

P. degree in Genetics

 

 

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8/01 CZICIRCJDBIODue.p65-p.15

 

MOLECULAR GENETIC ANALYSIS OF JASMONATE SIGNALING
IN TOMATO (L Y COPERSICON ESC ULEN TUM)

By

Lei Li

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Genetics Program

2003

ABSTRACT

MOLECULAR GENETIC ANALYSIS OF JASMONATE SIGNALING IN TOMATO
(L YCOPERSICON ESCULENTUM)

By

Lei Li

Synthesized from polyunsaturated fatty acids via the octadecanoid pathway,
jasmonic acid (J A) and its cyclic precursors and derivatives, collectively called
jasmonates, play critical roles in regulating many plant defensive and developmental
processes. Extensive studies of the wound signaling pathways in tomato have led to a
proposed model in which systemin, an 18-amino-acid polypeptide, acts as a mobile signal
to evoke de novo synthesis of J A, which in turn activates the expression of defense-
related genes. A major gap in our understanding of the function of j asmonates concerns
how jasmonate perception is coupled to transcriptional activation of j asmonate-inducible
genes in response to developmental and environmental cues. The focus of this
dissertation research was to dissect the role of j asmonates in tomato defense and
developmental processes by isolating and characterizing mutants with impaired responses
to exogenous J A. To this end, a fast neutron-mutagenized tomato population was
screened for plants that were deficient in methyl-JA-induced accumulation of polyphenol
oxidase and proteinase inhibitor-II, two jasmonate-regulated defensive proteins. One
recessive mutant (called .14 insensitive; -1 ) was isolated that was completely defective in
jasmonate signaling in roots, leaves, and flowers. Failure of jail-1 plants to express
jasmonate-regulated genes was correlated with increased susceptibility to herbivores.

Reciprocal grafting experiments using jail and sprZ, a tomato mutant defective in J A

biosynthesis, showed that spr2 plants are defective in the production, but not recognition,
of a graft-transmissible wound signal, whereas jai 1 plants are compromised in the
recognition but not the production of this signal. These results indicate that JA or a
related jasmonate species is an essential component of the long-distance wound signal.
Plants homozygous for the jai 1-1 mutation exhibited several novel development
phenotypes, including female sterility and impaired glandular trichome development.
These findings extend the role of the jasmonate signaling pathway to developmental
processes in tomato that have not been previously associated with jasmonates. In a
separate screen for ethyl methane sulfonate-induced mutations that suppress prosystemin—
mediated responses, 3 mutant (called jail-2) that was unresponsive to wounding and
methyl-J A was shown to be allelic to jai 1-1 . The jail mutants were determined to harbor
mutations in a gene that is homologous to the Arabidopsis CORONA T [NE

INSENSI T I VB 1 (C011), which encodes an F-box protein involved in ubiquitin-dependent
protein degradation. Stable transformation of jail-1 plants with the tomato JAII / C011
cDNA restored jasmonate-induced expression of defense genes, fertility, and trichome
development. We conclude that JAII/COII is a key regulator of the jasmonate signaling
pathway in tomato and anticipate that the jai 1 mutants will be useful for future
investigations aimed at elucidating in greater details the function of the jasmonate

signaling pathway in defense and development.

ACKNOWLEDGEMENTS

First and foremost, I would like to thank my advisor Dr. Gregg Howe, for taking
the chance on me as an inexperienced graduate student. Gregg has been a great mentor to
me. He has inspired and challenged me in my work yet has always shown a willingness

to help.

I wish to thank members of my guidance committee, Drs. Hans Kende, Christoph
Benning and Sheng-Yang He, for their constructive criticisms and insightful suggestions
to my work. Their expertise, encouragement, and support have helped make my graduate
study a very rewarding experience.

I would also like to thank all the members of the Howe lab, past and present, for
their companionship. I thank Drs. Chuanyou Li, Youfu Zhao, and Bonnie McCaig for
collaboration in several experiments presented in this thesis; Tony Schilmiller, Guanghui
Liu, and Gyu-In Lee for their entertainment and friendship. David Shaffer and Josh
Picotte are acknowledged for their help with isolating the jai I -1 mutant.

I owe many thanks to my family, for years of support, love across the miles, and
for having imperishable faith in me.

This dissertation research was supported by grants from the National Institute of
Health and the Department of Energy, and a Dissertation Completion Fellowship from

the Michigan State University Graduate School.

iv

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................. x

LIST OF FIGURES .......................................................................................................... XI
CHAPTER 1

INTRODUCTION: JASMONATE BIOSYNTHESIS, ACTION, AND FUNCTION ----- 1

I. Biosynthesis of j asmonates: The octadecanoid pathway -------------------------------------- 4

1.1. Release of a-linolenic acid from membrane lipids ------------------------------------ 9

1.2. Lipoxygenase ........................................................................................... 10

I. 3. Allene oxide synthase .............................................................................. 12

1.4_ Allen oxide cyclase .................................................................................. 13

I. 5. 12-Oxo-phytodienoic acid reductase ........................................................ 14

1.6. B—Oxidation .............................................................................................. 17

II. Regulation of J A synthesis ................................................................................. 19

11.1. Regulation of J A biosynthetic genes ...................................................... 19

11.2. Cellular compartmentation of J A biosynthesis -------------------------------------- 20

11.3. Up-regulation of JA synthesis by the prosystemin/systemin pathway 21
III. The jasmonate Signal transduction pathway ..................................................... 24
111.1. Perception ijasmonates ...................................................................... 24

111.2. The ubiquitin-mediated proteolysis pathway in jasmonate signal
transduction ................................................................................................... 26

111.3. Transcriptional regulation of j asmonate responses ------------------------------- 31

111.4. Integration ofjasmonate signaling with other defense signaling
pathways ........................................................................................................ 32

IV. Physiological roles ofjasmonates ..................................................................... 36

IV. 1. J asmonates play a direct role in resistance to herbivores and pathogens

........................................................................................................................ 36
IV.2. Role ofjasmonates in emission of plant volatiles and tritrophic
interactions ..................................................................................................... 39
IV .3. Role of jasmonates in plant growth and development -------------------------- 41
References ............................................................................................................... 44
CHAPTER 2
ALTERNATIVE SPLICING OF PROSYSTEMIN PRE-MRNA PRODUCES TWO
ISOFORMS THAT ARE ACTIVE AS SIGNALS IN THE WOUND RESPONSE
PATHWAY ...................................................................................................................... 60
Abstract ................................................................................................................... 61
Introduction ............................................................................................................. 62
Materials and Methods ............................................................................................ 65
Results ..................................................................................................................... 70
ProsysA and prosysB are generated by alternative splicing of intron 3 --------- 70
Expression pattern of prosys A and prosys B ................................................... 79
Overexpression ofprosysB confers constitutive wound signaling ---------------- 87
Discussion ............................................................................................................... 91
References ............................................................................................................... 95
CHAPTER 3
THE TOMATO MUTANT jail -I IS INSENSITIVE TO JASOMANATES AND
DEFECTIVE IN DEFENSE AGAINST HERBIVORES ............................................... 99
Abstract ................................................................................................................. 100
Introduction ........................................................................................................... 1 01

vi

Materials and Methods .......................................................................................... 104

Results ................................................................................................................... 109
Isolation of jasmonate-in sen sitive 1_ 1 .......................................................... 109
Jasmonate-induced phenotypes of seedlings ............................................... 1 12
Reproductive phenotypes of fat 1_ 1 plants ................................................... 112
Jasmonate-induced gene expression in jail -1 plants ----------------------------------- 118
Effects of jail-1 on the developmental expression pattern of PI genes ------- 124
Expression of wound response genes in jail -1 plants -------------------------------- 127
Responses of jail-I plants to PI-inducing compounds -------------------------------- 129
jai [-1 plants are compromised in resistance to herbivores ------------------------- 130

Discussion ............................................................................................................. 1 3 3
Forward genetic analysis of the jasmonate signaling pathway -------------------- 133
Jail is a positive regulator ofjasmonate responses ------------------------------------- 134

Jail -dependent and -independent wound-signaling pathways in tomato 136
References ............................................................................................................. l 39
CHAPTER 4

GENETIC ANALYSIS OF WOUND SIGNALING IN TOMATO: EVIDENCE FOR A
DUAL ROLE OF JASMONIC ACID IN DEFENSE AND FEMALE FERTILITY " 146

Abstract ................................................................................................................. 1 4‘7
Introduction ........................................................................................................... 1 4 8
Genetic analysis of wound signaling .................................................................... 150
A role for JA in female reproductive development? ............................................. 159
References ............................................................................................................. 1 61

vii

CHAPTER 5

DISTINCT ROLES FOR JASMONATE SYNTHESIS AND ACTION IN THE

SYSTEMIC WOUND RESPONSE OF TOMATO ...................................................... 164
AbStraCt ................................................................................................................. 1 65
Introduction ........................................................................................................... 166
Materials and MethOdS .......................................................................................... 169
Results ................................................................................................................... 1 73

Mutations affecting either JA biosynthesis or JA signaling abolish wound-
induced systemic expression of PI genes ..................................................... 173

Jasmonate signaling is required for functional recognition of a long-
distarlce wound Sigllal .................................................................................. 176

Jasmonate biosynthesis is required for generation of a long-distance wound

signal ............................................................................................................ 1 79
Roles for jasmonate biosynthesis and signaling in 35S: :prosys-mediated PI

expression .................................................................................................... 1 85

Discussion ............................................................................................................. 188

References ............................................................................................................. 194
CHAPTER 6

MOLECULAR CLONING OF J01] .............................................................................. 198

Abstract ................................................................................................................. 199

Introduction ........................................................................................................... 200

Materials and Methods .......................................................................................... 203

Results ................................................................................................................... 207

The tomato C011 gene ................................................................................. 207

jail plants harbor mutations in LQCOII ....................................................... 210

Molecular complementation of the jai 1 -I mutation by LeCOII ----------------- 218

viii

Discussion ............................................................................................................. 225

References ............................................................................................................. 228

CHAPTER 7

CLUSIONS AND FUTURE DIRECTIONS ................................................................. 230
References ............................................................................................................. 238

APPENDIX .................................................................................................................... 241
References ............................................................................................................. 247

ix

LIST OF TABLES

Table 1.1. J A biosynthetic mutants in Arabidopsis and tomato -------------------------------------- 16
Table 1H2 Jasmonate response mutants in Arabidopsis ................................................... 25

Table 3.1. List of genes that are differentially regulated in wild-type and jai 1 -1 plants by
exogenous MeJA ............................................................................................................ l 19

Table 3.2. Response of wild-type and jai [-1 plants to PI-inducing factors ------------------- 128
Table 4.1. Proteinase inhibitor H accumulation in response to wounding and MeJ A 154
Table 4.2. Genetic complementation tests between jail «J and 124A ---------------------------- 155

Table 5.1. Proteinase inhibitor II accumulation in wild-type and spr2 scion leaves in
response to wounding .................................................................................................... 186

Table 5.2. Proteinase inhibitor H accumulation in grafted tomato plants in response to a
long-distance signal generated in a 35S::prosys transgenic line ---------------------------------- 187

Table 6.1. Phenotypes of the primary (T1) jail -I transgenic plants transformed with the
35S...C011 transgene ...................................................................................................... 216

LIST OF FIGURES

Figure 1.1. Structure of representative jasmonates and coronatine ---------------------------------- 5
Figure 1.2. The octadecanoid pathway for jasmonate biosynthesis ---------------------------------- 7
Figure 1.3. Hypothetical model showing presumed function of C01] in the jasmonate
signaling pathway ............................................................................................................ 29
Figure 2.1. Mapping of the prosystemin gene by Southern blot analysis of tomato
introgression lines ............................................................................................................ 71
Figure 2.2. Proposed model for alternative splicing of prosystemin pre-mRNA ------------ 74

Figure 2.3. S] nuclease cleavage assay for detection of heteroduplex DNA formed by
prosyStemin CDN AS ......................................................................................................... 77

Figure 2.4. Quantitative PCR amplification of prosysA and prosysB cDNAs in a single
reaction ............................................................................................................................. 80

Figure 2.5. Relative abundance of prosysA and prosysB transcripts in different tissues of
tomato .............................................................................................................................. 83

Figure 2.6. Relative abundance of prosysA and prosysB transcripts in response to methyl
jasmonate treatment ......................................................................................................... 85

Figure 2.7. Overexpression of either prosysA or prosysB in transgenic tomato results in a
constitutive wound response phenotype .......................................................................... 89

Figure 3.1.jai1-1 plants exhibit normal vegetative growth and insensitivity to exogenous

MCJA .............................................................................................................................. 1 10
Figure 3.2. Reproductive development of wild-type and jail -1 plants ------------------------- 113
Figure 3.3. Accumulation of MeJA-responsive transcripts in wild-type and jail -1 leaves
......................................................................................................................................... 1 16
Figure 3.4. Developmental accumulation of transcripts encoding defense-related proteins
......................................................................................................................................... 1 22
Figure 3.5. Response of wild-type and jail —1 plants to mechanical wounding ------------- 125

Figure 3.6. Accumulation of defense-related transcripts in response to tobacco homworrn
attack .............................................................................................................................. 1 3 1

xi

Figure 4.1. Proposed action of mutations in the wound response pathway ------------------- 151

Figure 4.2. Proteinase inhibitor H levels in wild-type and jail-2 plants in response to J A
and systemin ................................................................................................................... 1 57

Figure 5.1. Induction of the proteinase inhibitor 11 gene in response to wounding and
MeJA .............................................................................................................................. 1 74

Figure 52 Photograph Of a typical grafted tomato plant .............................................. 177

Figure 5.3. Wound-inducible PI-II expression in grafts between WT plants and mutants
defective in jasmonate signaling (jail) or jasmonate biosynthesis (sprZ) -------------------- 180

Figure 5.4. Wound-induced systemic PI-II expression in grafis between jail and spr2

plants .............................................................................................................................. 182
Figure 5.5. Genetic model for the role of j asmonate synthesis and signaling in the

systemic activation of wound-responsive PI genes in tomato plants ---------------------------- 192
Figure 6H1 Sequence alignment Of AtCOIl and LCCOII .............................................. 208
Figure 6.2. jail-1 plants harbor a 6,243 bp deletion in the LeCOII locus --------------------- 211

Figure 6.3. RNA blot analysis of LeCOII transcript in tissues of wild-type and jai I -1
plants .............................................................................................................................. 21 4

Figure 6.4. DNA blot analysis of the primary (T1) jai 1 -1 transgenic plants expressing

35S3L€C011 .................................................................................................................. 221
Figure 6.5. RNA blot analysis of representative T2 transgenic plants -------------------------- 223
Figure A. 1. Scanning electron micrograph of wild-type and jail -1 plants ------------------- 245

xii

CHAPTER 1

Introduction: Jasmonate Biosynthesis, Action, and Function

Plants and insects have coexisted for over 350 million years, when the earliest
forms of land plants and insects existed (Gatehouse, 2002). Although some of the
relationships between the two phyla, such as pollination and seed dispersal, are mutually
beneficial, the most common interaction involves insect feeding on plants, and plant
defenses against herbivorous insects (Gatehouse, 2002; Pichersky and Gershenzon, 2002).
Paleobotanic studies indicated that predation of angiosperrn plants by insects can be dated
as far as 97 million years ago (Labandeira et al., 1994). According to the co-evolutionary
theory developed by Ehrlich and Raven (1964), insect predation on plants has been a
determining factor in increasing species diversity in both herbivores and their plant hosts

(Harbome, 1988; Gatehouse 2002).

In the face of this long-standing relationship, it is not surprising that plants have
developed diverse and sophisticated means to resist or evade their insect predators.
Sessile as they are, plants seek to minimize herbivore damage through rapid grth and
development, dispersion, or choice of habitat (Gatehouse, 2002). They also develop
morphological structures that physically repel or trap insect predators. But often times,
plants can accumulate high levels of pre-formed compounds which function as
biochemical defenses through their toxicity, or their physical properties (Wittstock and
Gershenzon, 2002). This defense mechanism can be described as constitutive, in contrast
to induced defenses in which the synthesis of defensive compounds is triggered by insect
attack (Harbome, 1988; Ryan, 2000; Walling, 2000). Since the latter mechanism cannot
come into play until plants are attacked, it does not involve the commitment of plant
resources to the synthesis of defensive compounds that must be accumulated and stored

(Gatehouse, 2002). Thus, the fitness cost of induced resistance is less than that involved

in constitutive defense (Sims and Fritz, 1990; Baldwin, 1998; Gatehouse, 2002; Heil

and Baldwin, 2002).

An important aspect of many induced defense responses is their occurrence not
only at the site of damage but in undamaged tissues located distal to the site of attack
(Karban and Baldwin, 1997). Wound-inducible proteinase inhibitors (HS) in tomato
(Lycopersicon esculentum), which are expressed within ~ 2 h after mechanical wounding
or herbivory, represent one of the best examples of this phenomenon (Karban and
Baldwin, 1997; Howe et al., 2000; Ryan 2000). In their landmark study of wound-
inducible PIs, Green and Ryan (1972) proposed that specific signals generated at the
wound site travel through the plant and activate PI expression in undamaged responding
leaves. Several chemical and physical signals have since been implicated in the systemic
wound response (reviewed by Ryan, 2000; Walling, 2000; Leon et al., 2001). One of
these signals is the fatty acid-derived hormone jasmonates. There is an ever-increasing
body of evidence indicating that jasmonates are essential signals for the control of
defense responses, and partitioning of metabolic resources between growth and defense
(Creelman and Mullet, 1997; Walling, 2000; Berger, 2002; Turner et al., 2002;

Wastemack and Hause, 2002; Weber, 2002).

I. Biosynthesis of Jasmonates: The Octadecanoid Path way

Jasmonic acid (JA) is representative of a family of plant signaling molecules
derived from fatty acids. These compounds are notably similar to the eicosanoid family
of animal hormones (Bergey et al., 1996). The methyl ester of IA (MeJA) was first
identified as a major fragrance in the essential oil of jasmine plants (Demole et al., 1962),
and J A was later obtained from a culture filtrate of the fungus Botryodiplodia
theobromae (Aldridge et al., 1971). It is now believed that J A and its cyclic precursors
and derivatives (Figure 1.1), collectively referred to as jasmonates, occur ubiquitously in
the plant kingdom (Sembdner and Parthier, 1993; Creelman and Mullet, 1997;

Wastemack and Hause, 2002).

The octadecanoid pathway for jasmonate biosynthesis was first proposed by Vick
and Zimmerman (1984) and has since been elucidated in detail in Arabidopsis and a few
other plants species (Schaller, 2001). The pathway starts with the release of a-linolenic
acid (a-LA; 18:3) from membrane lipids. Oxygenation of free a-LA by a 13-
lipoxygenase (LOX) generates (9Z,1 1E,1 5Z,l 3S)—13-hydroperoxy-9,1 1,15-
octadecatrienoic acid (13S-HPOT) that serves as a substrate for allene oxide synthase
(AOS). AOS then converts 13S-HPOT to 12,13(S)—epoxy-9(Z),11,15(Z)-octadecatrienoic
acid (12,13-EOT), which is cyclized by allene oxide cyclase (AOC) to the first cyclic and
biologically active compound of the pathway, 12-oxo-10,l 5(Z)-phytodienoic acid
(OPDA). Reduction of the 10,11-double bond in OPDA by OPDA reductase (OPR) then
yields 3-oxo-2(2'(Z)-pentenyl)-cyclopentane-l-octanoic acid (CFC-8:0), which

undergoes three rounds of B-oxidation to produce JA (Figure 1.2; Table 1.1).

Figure 1.1. Structure of representative jasmonates and coronatine. OPDA, 12-oxo-
10,15(Z)—phytodienoic acid; JA, jasmonic acid; MeJA, methyl jasmonate; coronatine, a
phytotoxin, produced by Pseudomonas syringae, that has biological activity similar to

jasmonates (F eys et al., 1994).

O

_ \ _
COOH COOH

OPDA JA

0

COOCH3 W
0%
MeJA H
HOOC

coronatine

Figure 1.1

Figure 1.2. The octadecanoid pathway for jasmonate biosynthesis. The pathway

originates with the release of a-LA from chloroplast membrane by DAD] , a

phospholipase A1. a-LA is then converted in the chloroplast to OPDA by the sequential

action of LOX, AOS, and AOC. Reduction of the cyclopentenone ring and subsequent
B-oxidations take place in the peroxisome. The spatial separation of OPDA and J A
formation implies that OPDA is transferred from the chloroplast to the peroxisome to
be further metabolized. JA can be methylated by JMT to the volatile MeJA in the
cytosol. JA can also be adenylated in the cytosol by JARl , a process that might lead to
the formation of IA conjugates. Enzymatic steps and fluxes of the intermediates are
shown. Question marks indicate the steps without explicit experimental evidence. See

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1.1. Release of a-linolenic acid from membrane lipids

The octadecanoid pathway originates from the release of a-LA from membrane
lipids (Vick and Zimmerman, 1984). The critical requirement of a-LA in J A biosynthesis
was explicitly demonstrated by the analysis of an Arabidopsis “triple” fatty acid
desaturase mutant (fad3-2/fad7-2/fad8). The mutant contained negligible levels of
trienoic fatty acids. Although photosynthesis and vegetative growth of the mutant were
unaffected, the triple mutant was male sterile. Application of a-LA and JA restored
fertility (McConn and Browse, 1996). When wounded, free a—LA levels in plants doubled
within 1 h after wounding, while JA levels increased lO-fold (Conconi et al., 1996).
Given that the level of free a-LA measured before wounding was many times higher than
the maximum JA accumulated after wounding, the wound-induced increase in J A levels
could have resulted from release of a-LA from lipids, or the utilization of a-LA present

before wounding (Conconi et al., 1996).

By analogy with mammalian eicosanoid biosynthesis, a phospholipase A may
catalyze the release of a-LA from membrane lipids in plants. This has recently been
confirmed for JA biosynthesis in flowers by characterizing the male-sterile Arabidopsis
mutant defective anther dehiscence] (dadl, Ishiguro etal., 2001). The dad] mutant was
isolated from a transposon-tagged population on the basis of its male sterility, which
could be rescued by a-LA or JA application. DADl was shown to be a lipase that
hydrolyses phosphatidylcholine at the sn-1 position, and targeted to the chloroplasts
(Ishiguro et al., 2001). The DAD] promoter was strongly activated in filaments of
stamens prior to the stage at which JA is required for development of the filament,

maturation of pollen grains, and dehiscence of the anther (Ishiguro et al., 2001). The

involvement of DAD] in wound-induced JA synthesis in leaves is less clear. The DAD]
transcript was wound-inducible but, significantly, dad] plants were competent for
wound-induced JA formation in leaves (Ishiguro etal., 2001). Therefore, DAD] is
required for developmentally regulated production of IA for stamen development, but not

for wound-induced JA accumulation in leaves.

On the other hand, there is evidence suggesting that a phospholipase D (PLD) is
required for wound-induced J A formation in both Arabidopsis and tomato leaves. For
example, wounding of wild-type plants promoted a substantial increase in a-LA levels,
whereas transgenic Arabidopsis plants in which PLDa was suppressed by the antisense
technique showed no significant increase in a-LA (Zien et al., 2001). Antisense
suppression of Arabidopsis PLDa also reduced wound-induced JA and the accumulation
of JA-inducible transcripts (Wang et al., 2000). These transgenic plants were male fertile
(Wang et al., 2000), suggesting that PLDa is required for wound-induced J A biosynthesis
but not for JA biosynthesis in stamen development. However, McConn and Browse
(1996) observed that the threshold level of a-LA for male fertility was less than 5% of the
a-LA levels in wild-type plants. Therefore, it is possible that the flower J A content was

reduced in PLDa antisense plants, but not to a level to cause male sterility.

1.2. Lipoxygenase

LOXs constitute a family of dioxygenases that catalyze the oxygenation of fatty

acids to their corresponding hydroperoxy derivatives. Plant LOXs oxygenate a-LA at the

10

9 or 13 position to give 9- or 13- hydroperoxy-octadecatrienoic acid (HPOT). Those
involved in J A biosynthesis produce 13-HPOT and are called l3-LOXs (F eussner and
Wastemack, 2002). The role of 9-hydroperoxides and their catabolites in plants is unclear,
though studies in potato suggested a role for these compounds in defense responses
against fungal pathogens (Gobel et al., 2001). Generally speaking, higher plants contain
multiple isoforms of LOXs (F eussner and Wastemack, 2002). Consequently, the specific
role of a given LOX in the regulation of IA synthesis has been difficult to analyze
because different LOX isoforms may have different enzymatic specificity and may be

located in different cell compartments (Feussner and Wastemack, 2002).

The involvement of 13-LOXs in IA biosynthesis was apparent from the
observations that plants treated with LOX inhibitors (Pena-Cortes et al., 1993), or
transgenic plants with suppressed LOX activity, exhibit reduced ability to synthesize J A
(Bell et al., 1995; Royo et al., 1999). The stroma—localized plastidial LOX2 appears to be
responsible for the wound-induced biosynthesis of JA in Arabidopsis. LOX2 mRNA
levels were high in leaves and inflorescences but low in seeds, roots, and stems.
Suppression of LOX2 expression by the antisense technique caused reduction in the
wound-induced accumulation of J A in leaves. However, no obvious changes in the
growth and fertility of the antisense plants were observed (Bell et al., 1995). Therefore,
these results suggested that LOX2 is required for wound-induced JA formation, but may

not be required for J A production during pollen and stamen development.

11

I.3. Allene oxide synthase

AOS catalyzes the dehydration of 13-HPOT to an unstable epoxide, which is
converted to OPDA by allene oxide cyclase (AOC). Tomato has at least two chloroplast-
targeted 13-AOSs (Howe et al., 2000; Sivasankar et al., 2000), whereas there is only a
single AOS gene in Arabidopsis (Kubigsteltig et al., 1999). Therefore, the Arabidopsis
AOS must function in JA formation both in leaves and flowers. Analysis of AOS
knockout mutants indeed indicated so. The mutant plants were completely abolished the
wound-induced J A accumulation and the induction of wound response genes (Park et al.,
2002; von Malek et al., 2002). Analysis of the mutant plants also confirmed the
requirement of AOS in male reproductive development. The mutants displayed defects in
filament elongation, anther dehiscence and pollen maturation that could be rescued by

exogenous JA (Park et al., 2002; von Malek et al., 2002).

The reaction catalyzed by AOS is the first committed step in JA synthesis.
Therefore, regulation of AOS expression and activity has been considered a major control
point for JA biosynthesis. The Arabidopsis AOS promoter was shown to be activated by a
variety of signals including JA, OPDA, wounding, and salicylic acid (SA; Laudert and
Weiler, 1998). However, overexpression of AOS in transgenic Arabidopsis and tobacco
did not alter the basal level of J A. Only when the transgenic plants were wounded did
they produce a higher level of J A than wild-type plants (Laudert et al., 2000; Park et al.,
2002). In Arabidopsis and in tobacco, therefore, it appears that AOS expression is
limiting JA levels in wounded plants. In unwounded plants, on the other hand,

availability of the AOS hydroperoxide substrate 13-HPOT might determine JA levels

12

(Park et al., 2002). By contrast, overexpression of the flax AOS in transgenic potato
plants delivered a chloroplast-localized AOS protein, and increased the endogenous J A
level. However, the JA-regulated Pin2 gene was not up-regulated in these transgenic
plants, indicating that the elevated J A in these transgenic plants was not biologically

active (Harms et al., 1995).

1.4. Allen oxide cyclase

AOC catalyzes the stereo-specific cyclization of allene oxide to OPDA, thus
establishing the stereochemistry of OPDA and JA. Because of the acute instability of the
epoxide, AOS and AOC are probably fimctionally and physically connected (Vick and
Zimmerman, 1984; Ziegler et al., 2000). Potato AOC combined with recombinant
Arabidopsis AOS can indeed produce OPDA from l3-HPOT in vitro (Laudert et al.,
1997). DNA gel blot analysis revealed a single AOC gene in tomato, whereas
Arabidopsis appears to have four AOC genes encoding proteins that contain a chloroplast

targeting transit peptide (He et al., 2002; Wastemack and Hause, 2002).

Immunohistochemical methods showed that the tomato AOC is localized to the
chloroplast by an N-terrninal transit peptide (Ziegler et al., 2000). AOC was found to
express at low levels in stems, young leaves, and young flowers, contrasting with a high
accumulation of the transcript in flower buds, flower stalks, and roots. AOC transcript
was transiently induced in tomato leaves when the plant was wounded or treated with J A

or systemin (an 18-amino-acid peptide signal molecule, see section 11.3), where its

13

expression was primarily confined to the vascular bundle tissues (Hause et al., 2000;
Stenzel et al., 2003). It should be noted that the vascular bundle—specific localization of
tomato AOC transcript and activity coincides with the spatial expression of the
prosystemin gene, suggesting that vascular bundle tissues are the site of active J A

biosynthesis in tomato (Jacinto et al., 1997, 1999; Ryan, 2000; Stenzel et al., 2003).

I. 5. 12-oxo-phytodienoic acid reductase

OPRs belong to the flavin-dependent oxidoreductase family and catalyze the
reduction of cyclopentenones in J A biosynthesis. Three OPR isoforms have been
characterized in both tomato and Arabidopsis. However, only the isoform OPR3 was
found to participate directly in the octadecanoid pathway for JA biosynthesis, as only
OPR3 reduces the 9S,13S-stereoisomer of OPDA, the biological precursor for J A
formation (Schaller and Weiler, 1997; Biesgen and Weiler, 1999; Miissig et al., 2000;
Strassner et al., 2002). The Arabidopsis ddeI (DELAYED DEHISCENCE 1) and opr3
(oxo-phytodienoic acid reductase 3) mutants, in which OPR3 is knocked out, are
deficient in J A but not OPDA accumulation in response to wounding (Sanders et al.,
2000; Stintzi and Browse, 2000). The opr3/dde1 mutants also display a male sterile
phenotype that can be restored by application of J A but not OPDA (Sanders et al., 2000;

Stintzi and Browse, 2000).

OPR3 transcripts can be induced by JA (Mfissig et al., 2000) and wounding

(Strassner et al., 2002). In tomato, the wound induction kinetics of OPR3 was found to

14

resemble that of the other forth-mentioned octadecanoid pathway genes (Strassner et al.,
2002). But in contrast to these octadecanoid pathway genes, which all encode enzymes
localized in the chloroplast, OPR3 does not contain a chloroplast targeting sequence.
Rather, OPR3 possesses a three-amino-acid carboxy-terminal extension (Ser-Arg-Leu)
that constitutes a putative peroxisome-targeting signal (Olsen, 1998) that is absent from
CPR] and OPR2 (Stintzi and Browse, 2000). Indeed, both the OPR3 protein and activity

were found exclusively in the peroxisome (Strassner et al., 2002).

Localization of OPR3 to the peroxisome provides strong support for the
hypothesis that the later phase of IA formation occurs in the peroxisome (see section 11.2).
This result also indicated that in both tomato and Arabidopsis the biosynthesis of
cyclopentanones (e. g. J A) and cyclopentenones (e. g. OPDA) is confined to the plastid
and the peroxisome, respectively. This apparent spatial separation of cyclopentenones
and cyclopentanones implies that transport processes might exist to shuttle OPDA from
the chloroplast to the peroxisome (Strassner et al., 2002). Interestingly, most OPDA was
found to be esterified to chloroplast galactolipids (Stelrnach et al., 2001), suggesting that
OPDA could be transferred between organellar membranes in lipid-bound form

(Strassner et al., 2002).

15

 

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I. 6. fl-Oxidation

Shortening of the CFC-8:0 side chain is achieved by three rounds of B-oxidation
(Vick and Zimmerman, 1984). These reactions probably occur in the peroxisome, where
enzymes for B-oxidation are known to be located in plants (Beevers, 1979; Gerhardt,
1983). There is little direct evidence for the subcellular localization of this part of the
pathway for JA biosynthesis until recently. The specific B-oxidation enzymes involved in
J A biosynthesis have yet to be identified. However, the demonstration of the peroxisomal
localization of OPR3 (Strassner et al., 2002), which catalyzes the formation of OPC-8:O
as a substrate for subsequent B-oxidation, provides a strong support to the hypothesis that
J A is finally formed in the peroxisome. Also noteworthy is that (Z)-jasmone, a common
component of plant volatiles, is thought to be formed from JA by a further round of [3-

oxidation (Birkett et al., 2000).

I. 7. Modification of Jasmonic Acid

JA can be metabolized to form its methyl ester MeJA and numerous conjugates
and catabolites, many of which have biological activity (Hamberg and Gardner, 1992;
Kramell et al., 1995). The methylation of IA to MeJA is catalyzed by an S-adenosyl-L-
methioninezjasmonic acid carboxyl methyltransferase (JMT, Seo et al., 2001). High levels
of JM T transcript were found in developing flowers and in wounded leaves. Transgenic
Arabidopsis plants overexpressing JMT accumulated MeJA without altering JA content,

stimulated the expression of j asmonate-responsive genes, and displayed enhanced

17

resistance to infection by Botrytis cinerea (Seo et al., 2001). However, it was not clear
whether these phenotypes of the transgenic plants were caused by a general increase in

j asmonate levels or by the specific accumulation of MeJA. Because Me] A is volatile, its
production by JMT could conceivably mediate intracellular and intercellular signaling in
planta, and could also function as an airborne signal mediating intra- and interplant
communications to orchestrate defenses against insects (Farmer and Ryan, 1990; Karban

et al., 2000; Seo et al., 2001).

The Arabidopsis jar] mutant exhibited reduced sensitivity to MeJA with regard to
root growth inhibition and enhanced susceptibility to the soil fungus Pythium irregulare
(Staswick et al., 1992; Staswick et al., 1998). However, even knockout alleles of jar]
exhibited no obvious defects in stamen development (Staswick et al., 2002). JAR] was
recently cloned and its predicted structure suggested that it belongs to the acyl adenylate-
forrning firefly luciferase superfamily of enzymes that activate the carboxyl groups of a
variety of substrates for their subsequent biochemical modification (Staswick et al., 2002).
The specificity of JAR] for JA adenylation was demonstrated by an ATP-PPi isotope
exchange assay (Staswick et al., 2002). These findings indicated that covalent
modifications of J A are important for many (e. g. root growth inhibition and defense
against soil pathogens) but not all (e. g. stamen development) jasmonate-mediated

processes in plant (Staswick et al., 2002).

18

11. Regulation of JA Biosynthesis

The octadecanoid pathway leading to the formation of J A involves the coincident
activation of at least five biosynthetic genes, the products of which are targeted to either
the chloroplast or the peroxisome. The similar expression profile of the biosynthetic
genes and the similar localization of the corresponding enzymes they encode suggest that
the octadecanoid pathway is highly regulated and coordinated. This view is reinforced by
the observations that transgenic overexpression of individual genes failed to increase J A
levels (Laudert et al., 2000; Stenzel et al., 2003) or led to elevated JA levels that were

functionally inactive (Harms et al., 1995).

II. 1. Regulation of JA biosynthetic genes

Most if not all of the octadecanoid pathway genes are transcriptionally activated
by treatment of plants with JA, wounding, or other stress conditions. Within 1 h of the
treatment, induction of J A biosynthetic genes was documented in several plant species.
These genes include DAD] (Ishiguro et al., 2001), LOX (Royo et al., 1996; Heitz et al.,
1997), AOS (Laudert and Weiler, 1998; Howe et al., 2000; Maucher et al., 2000;
Sivasankar etal., 2000), AOC (Stenzel et al., 2003); and OPR3 (Miissig et al., 2000;
Strassner et al., 2002). The promoter region of the Arabidopsis AOS was analyzed in
detail (Kubigsteltig et al., 1999). The AOS promoter contains cis—elements such as CAAT
and ACGT boxes similar to other known stress response elements (Goldsbrough et al.,
1993). When the promoter was fused with the GUS reporter gene, GUS activity was

found to be induced by wounding and JA treatment (Kubigsteltig et al., 1999). These data

19

were taken to be indicative of a positive feedback regulation in JA production (Laudert

and Weiler, 1998; Sivasankar et al., 2000).

II. 2. Cellular compartmentation of JA biosynthesis

The conversion of a-LA to OPDA occurs in the chloroplast, which contains an
abundance of a-LA esterified in glycerolipids. At present, at least one isoform for each of
the four octadecanoid enzymes (e. g. DAD], LOX2, AOS, and AOC) has been shown to be
localized in the chloroplast. These four enzymes contain a chloroplast transit peptide and
their localization to the chloroplast was demonstrated by immunocytochemical analysis
and by in vitro chloroplast import experiments. Furthermore, the corresponding
enzymatic activities were found in the chloroplast (Bell et al., 1995; Maucher et al., 2000;
Ziegler et al., 2000; Froehlich et al., 2001; Ishiguro et al., 2001). Co-localization of these
enzymes in the chloroplast has been suggested to facilitate the metabolism of lipophilic

or unstable intermediates of the JA branch of oxylipin metabolism (Froehlich et al., 2001).

The conversion of OPDA to J A, on the other hand, occurs in the peroxisome as
indicated by the peroxisome—specific compartmentation of OPR3 and the B-oxidation
enzymes (Beevers, 1979; Gerhardt, 1983; Strassner et al., 2002). It is interesting to note
that more than 90% of the OPDA in Arabidopsis leaves is present as a novel lipid, snl-O-
( l 2-oxophytodienoyl)-sn2-O-(hexadecatrienoyl)-monogalactosy1 diglyceride in
chloroplast membranes (Stelmach et al., 2001). OPDA could be released from chloroplast
membranes enzymatically by snl-specific lipases, and this could account for the rapid

transient increase in free OPDA and JA when leaves are wounded (Stelmach et al., 2001 ).

20

The spatial separation of OPDA and JA biosynthesis suggests that transfer of OPDA
from chloroplast to peroxisome might be an important regulatory step in JA biosynthesis.
The endogenous store of the lipid-bound OPDA also hints at its potential to rapidly
supply OPDA as a signal molecule. However, a precursor-production relationship

between lipid-bound OPDA and JA remains to be established.

II. 3. Up-regulation of JA synthesis by the prosystemin/systemin path way

A unique aspect of the wound signaling pathway of solanaceous plants is the
involvement of the peptide signal systemin (Ryan and Pearce, 1998; Ryan, 2000).
Systemin is an 18-amino-acid polypeptide originally isolated from leaves of tomato
plants using a bioassay in which accumulation of P18 is measured in young excised
tomato plants afier a compound is supplied through their transpiration stream. Systemin
was found to be active at a concentration of fmol/plant in the bioassay, which ranks it
among the most powerful plant gene-activating compounds known (Pearce et al., 1991).
Systemin is derived from the C—terminal end of a ZOO-amino-acid precursor called
prosystemin (McGurl et al., 1992), whose cDNA has been found in potato, nightshade
and bell pepper (Constabel et al., 1998). Most recently, two degenerate systemin-like
peptides that can activate PI synthesis were isolated from suspension-cultured tobacco
cells (Pearce et al., 2001). Molecular manipulation of the prosystemin cDNA has
provided convincing evidence that prosystemin plays a critical role in the transduction of
systemic wound signals. Antisense suppression of prosystemin expression in tomato
plants compromised the systemic wound response (McGurl et al., 1992), whereas

overexpression of the prosystemin cDNA driven by the CaMV 358 promoter (a genotype

21

designated as 35S::prosys) resulted in constitutive activation of wound response genes in

unwounded plants (McGurl et al., 1994).

The requirement of the octadecanoid pathway in mediating systemin signaling
was first suggested by Farmer and Ryan (1992) and has been confirmed by several lines
of evidence. When fed through the cut stem of tomato plants, systemin rapidly elevates
JA levels (Doares et al., 1995; Howe et al., 1996). Conversely, inhibitors of the
octadecanoid pathway, such as diethyldithiocarbamate (which converts 13-HPOT into a
hydroxyl derivative) and SA (which represses AOS activity), prevent systemin-mediated
activation of defense genes (Farmer et al., 1994; Doares et al., 1995). The most
convincing evidence has come from the analysis of a tomato mutant called def] (Table
1.1), which is impaired in IA accumulation upon wounding and systemin feeding, and is
deficient in the activation of defense genes (Howe et al., 1996). In keeping with this
finding, a genetic screen in the 35S::prosys background for mutants suppressed in the
constitutive activation of defense genes has identified genes required for both J A
synthesis (e. g. spr2, Table 1.1) and perception (Howe and Ryan, 1999; Li et al., 2001;

2002b)

Radioactively labeled systemin, when placed on fresh wounds of tomato leaves,
was shown to move from wound sites to the petiole phloem, supporting its possible role
as a mobile wound signal (Pearce et al., 1991; Narvéez-Vasquez et al., 1995). However,
the exact mode of action for systemin is still obscure. Recent models propose that
systemin is the mobile systemic wound signal that is released from prosystemin upon
wounding or herbivore attack and is transported throughout the plant where it interacts

with its specific receptor at the surface membrane of target cells. As a consequence of

22

this interaction, a-LA is released and converted along the octadecanoid pathway to J A
(Ryan and Pearce, 1998; Ryan, 2000). A systemin-binding activity was identified that
displayed characteristics of a functional systemin receptor (Meindl et al., 1998; Scheer
and Ryan, 1999). This putative receptor was purified to homogeneity from plasma
membrane fractions of suspension cultured cells of L. peruvianum. It was found to be a
l60-kD protein belonging to the leucine rich repeat (LRR) receptor kinase family that is

most similar to the brassinolide receptor kinase BRIl (Scheer and Ryan, 2002).

Interaction of systemin with its receptor is associated with several rapid
biochemical events including cytosolic calcium influx, membrane depolarization,
inhibition of a plasma membrane proton ATPase, and activation of a MAP kinase activity
(Felix and Boller, 1995; Moyen and Johannes, 1996; Stratmann and Ryan, 1997; Moyen
et al., 1998; Schaller and Oecking, 1999). These early signaling events in the systemin

and wound response pathways are believed to culminate in the activation of a

phospholipase A2 (PLA2) activity that presumably releases fatty acid from membrane
lipids (Narvaez-Vasquez et al., 1999; Ryan, 2000). The FLA; activity, as measured by

the accumulation of 14C-lysophosphatidylcholine, was rapidly induced by wounding and

systemin, but not by MeJA treatment (N arvaez-Vasquez et al., 1999). The

unresponsiveness of PLAZ to Me] A contrasts other J A biosynthetic enzymes, indicating
that this PLAZ activity is specific to the systemin pathway. However, a causal role for

this PLA2 activity in wound— and systemin-mediated J A biosynthesis remains to be

established.

23

III. The Jasmonate Signal Transduction Path way

In the past decade jasmonates have gone from little-regarded secondary
metabolites to the well-recognized central regulator of many plant defensive and
developmental processes. Like other plant hormones, jasmonates have a profound impact
on the biochemistry, metabolism and mRNA population of plant cells that can perceive
and transduce the hormonal signal (Creelman and Mullet, 1997). The following section

discusses our current understanding of the jasmonate signaling pathway.

III. I. Perception of jasmonates

By analogy to other plant hormones (Kende and Zeevaart, 1997), the jasmonate
signal is thought to be transduced by the activation of a receptor that binds the hormone
(Creehnan and Mullet, 1997). Lack of high-specific-activity JA and the lipophilic or
volatile nature of jasmonates made J A binding experiments, and thus the direct analysis
of JA receptors, difficult (Creelman and Mullet, 1997). Another approach to identify the
jasmonate receptor has been through analysis of j asmonate response mutants. In
Arabidopsis, exhaustive mutant screens for response mutants to Me] A or its structural
analog coronatine (Figure 1.1) have identified a number of loci important for jasmonate
signaling (Table 1.2). Cloning and biochemical analysis indicated that CO]! (Xie et al.,
1998), CE V1 (Ellis et al., 2002), and JAR] (Staswick et al., 2002) are not JA receptors.

Evidence regarding the molecular basis of other mutations is still lacking.

24

 

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J asmonate perception is firrther complicated by the fact that OPDA acts as a
signaling molecule in its own right. For instance, OPDA has been shown to be more
potent than JA in inducing tendril coiling in Bryom'a dioica (Weiler et al., 1993). The
assessment of the relative contributions of JA and OPDA to the various physiological
responses in Arabidopsis was made possible by the isolation of the ddeI/opr3 mutants
(Sanders et al., 2000; Stintzi and Browse, 2000). The male sterility defects of the mutants
(see section 1.5) could be alleviated by application of JA but not by OPDA, providing
clear evidence for a role of J A rather than OPDA in male developmental processes
(Sanders et al., 2000; Stintzi and Browse, 2000). Surprisingly, ddeI/opr3 plants were
found to be fully resistant to the dipteran insect Bradysia impatiens and the nectrotrophic
fungus Alternaria brassicicola, demonstrating that OPDA can substitute for J A in
mediating plant resistance (Stintzi et al., 2001). This suggests that, in Arabidopsis, OPDA
and JA have overlapping as well as distinct functions. Accordingly, there may exist at
least two pathways to transduce the OPDA and J A signals, one for recognition of either
OPDA or J A for defense responses, and one for recognition of JA, but not OPDA, for

stamen development.

III. 2. The ubiquitin-mediated proteolysis path way in jasmonate signal transduction

Selective proteolysis performed by the ubiquitin-proteasome pathway plays a key
regulatory role in numerous cellular processes in both animals and plants, in which
ubiquitin serves as a reusable tag to target proteins for degradation by the 26S proteasome

(Voges et al., 1999). Covalent attachment of ubiquitin to substrate proteins involves a

26

cascade of three protein complexes called the ubiquitin activating enzyme (E1), the
ubiquitin conjugating enzyme (E2) and the ubiquitin ligase (E3). Whereas eukaryotic
organisms contain only a few Els and dozens of E2s, the specificity and timing of
substrate ubiquitination are primarily controlled by a large number of E3 complexes

(Deshaies, 1998; Voges et al., 1999).

In plants, ubiquitin monomers are encoded by gene fusions that contain the
polyubiquitin and the ubiquitin extension protein (UbEP) genes. The polyubiquitin gene
consists of several tandem repeats of the ubiquitin coding unit whereas the UbEP gene
encodes a single ubiquitin unit fused in frame with either of two ribosomal proteins
(Finley et al., 1989). Both the polyubiquitin and the UbEP gene have been shown to be
up-regulated by wounding or MeJA (Garbarino et al., 1992; Garbarino et al., 1995).
Similarly, wound-induced accumulation of mRN A encoding the a proteasome subunit
was described in Arabidopsis (Genschik et al., 1992). These observations suggest the
involvement of the ubiquitin/proteasome pathway in the wound and the jasmonate

signaling pathways, presumably through selective degradation of regulatory proteins.

In keeping with these earlier results, analysis of the Arabidopsis cot] mutants
provided a clear link between ubiquitin-mediated proteolysis and jasmonate signaling.
The coil mutants were first isolated in a screen for plants insensitive to growth inhibition
by the bacterial toxin coronatine (Figure 1.1; Table 1.2; Feys et al., 1994). Subsequently,
it was found that cat] plants fail to express jasmonate-regulated genes, and also are
unresponsive to grth inhibition by Me] A, male sterile, and susceptible to insect

herbivores and to pathogens (Benedetti et al., 1995; McConn et al., 1997; Thomma et al.,

27

1998). The C011 gene encodes a 66-kD protein containing an F -box motif and an LRR
domain (Xie et al., 1998). The F -box motif is a hallmark of proteins that associate with
cullin and Skpl proteins to form an E3 ubiquitin ligase known as the SCF complex (Bai
et al., 1996; Deshaies, 1998). LRRs are short sequence motifs that mediate protein-
protein interaction (Kobe and Deisenhofer, 1994). Therefore, F-box proteins are believed
to function as receptors that recruit regulatory proteins as substrates for ubiquitin-

mediated degradation (Bai et al., 1996; Deshaies, 1998).

The involvement of an F —box protein in jasmonate signaling suggests that
jasmonate-inducible genes are transcriptionally activated by selective recruitment of a
repressor of j asmonate responses for proteolysis (Figure 1.3). It has been postulated that
upon binding with its receptor, a jasmonate signal activates a protein kinase cascade
(Rojo et al., 1998), which leads to the phosphorylation of the repressor of j asmonate
responses (Creelman, 1998). The phosphorylated repressor can then be recognized by the
C011-containing complex, polyubiquitinated and subsequently degraded by the 26S

proteasome (Deshaies, 1998). Removal of the repressor then allows jasmonate-responsive

. 2 .
genes to be transcribed (Creelman, 1998). The structure of SCFSkp (superscript denotes

the F -box protein) has been recently determined. The complex adopts an elongated shape,
with Cull forming the scaffold and bel and the Skpl/Skp2 complex segregated to
opposite ends (Zheng et al., 2001). Physical association of C011 with other SCF
components has been demonstrated by immunoprecipitation experiments (Devoto et al.,

2002; Xu et al., 2002), confirming that COIl participates in the formation a functional

E3-type ubiquitin ligase, SCFCOH, in plants.

28

Figure 1.3. Hypothetical model showing presumed function of C01] in the
jasmonate signaling pathway. In this model, an elevated level of j asmonate is
perceived as a signal resulting in the activation of a kinase catalyzing the

phosphorylation of a regulatory protein (R). The phosphorylated protein R binds to

€011 in the SCFCOIl complex via the LRR domain. Following transfer of ubiquitin

(Ub) from E1 to E2, the SCFC011 complex (composed of AtCULl, Atbel, either of

the Arabidopsis Skpl-like proteins ASKl or ASK2, and C011) acts as a ubiquitin
ligase complex to transfer ubiquitin to R. Following polyubiquitination, R is targeted to

the 26S proteasome for degradation. Removal of R then allows the transcription of

. . . . COIl
Jasmonate-responsrve genes. Composmon of the SCF complex was recently

determined by immunoprecipitation experiments (Devoto et al., 2002; Xu et al., 2002).

Images in this figure are presented in color.

29

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30

[11.3. Transcriptional regulation of jasmonate responses

Microarray analysis showed that exogenous MeJA affects the mRNA level of
numerous genes (Schenk et al., 2000). However, our knowledge of the cis acting
elements of j asmonate responsive genes and the transcriptional factors involved in
recognizing these elements is incomplete. The promoters of two jasmonate-inducible
genes, Pin2 and VspB, have been studied in some detail (Kim et al., 1992; Mason et al.,
1993). A SO-bp domain was identified in both promoters that conferred Me] A
responsiveness on truncated reporter gene constructs. Both domains contain a motif
(CACGTG) called the G-box, which in other promoters has been shown to bind bZIP
transcription factors (Williams et al., 1992). But the relevance of this G-box in conferring
jasmonate responsiveness is still unsettled because it is found in numerous promoters that
are not responsive to jasmonates. Further, mutation of the G-box in the Pin2 promoter did

not prevent Me] A induction of the gene (Lorbeth et al., 1992).

J asmonates induce the biosynthesis of many classes of secondary metabolites in
different plant species. Using the biosynthesis of a terpenoid indole alkaloid in
Catharanthus roseus as a biochemical marker, a gene called ORCA3 was identified.
ORCA3 mRNA was rapidly induced by MeJ A treatment and its overexpression in
untreated cells resulted in enhanced expression of several biosynthetic genes of the
alkaloid pathway (van der Fits and Memelink, 2000). ORCA3 was shown to be a MeJA-
responsive APETALAZ (AP2)-domain transcription factor that specifically activates
transcription via a JA- and elicitor-responsive element (JERE) in the promoters of
jasmonate-response genes, including the alkaloid biosynthetic gene strictosidine synthase

(van der Fits and Memelink, 2001). OCRA3 has similarity to the ethylene response

31

factors (ERFs), which were originally isolated as GCC box binding proteins from tobacco
(Ohmetakagi and Shinshi, 1995). Arabidopsis ERF genes (AtERFI to AtERF 5) are
differentially regulated by different stress conditions (Fujimoto et al., 2000). Particularly
relevant to jasmonate signaling, AtERFI was found to be JA-inducible and required for
induction of other jasmonate-responsive genes (Lorenzo etal., 2003). Cycloheximide,
which inhibits protein synthesis, induces marked accumulation of AtERF mRNAs,
suggesting that ERF transcription can be stimulated by protein turnover (Fujimoto et al.,

2000). Thus, it seems possible that jasmonate responses in Arabidopsis require ERF-like

. . . . . C011 . .
transcription factors, whose activation 18 dependent on SCF -med1ated proteolySIS

(Lorenzo et al., 2003).

111.4. Integration of jasmonate signaling with other defense signaling path ways

The jasmonate pathway is implicated in regulating responses to many abiotic
stresses, defenses against insect herbivores and necrotrophic as well as biotrophic
pathogens (Walling 2000; Ellis and Turner, 2001; Berger, 2002; Gatehouse 2002).

J asmonates also appear to regulate a broad spectrum of developmental processes
(Creelman and Mullet, 11997), many of which also involve other plant signaling pathways.
For instance, microarray analysis of gene expression in wild-type and coil Arabidopsis
plants that were subjected to wounding, insect attack, or water stress revealed a large
overlap of C011-dependent genes regulated by wounding and by water stress (Reymond
et al., 2000). This result suggests crosstalk between the jasmonate and the abscisic acid

(ABA) pathway, which is involved in water stress responses (Kende and Zeevaart, 1997).

32

In tomato, however, ABA was found not to be the primary signal for defense gene

activation (Birkenmeier and Ryan, 1998).

The jasmonate signal pathway also interacts with the ethylene signal pathway in
controlling some developmental and defensive responses. This is best exemplified in the
expression of the Arabidopsis defensin gene PDFI.2, which encodes a S-kD protein
possessing antifungal properties. PDFI .2 could be induced by challenge of pathogen,
Me] A or ethylene but not SA treatment (Penninckx et al., 1996). Further analysis
demonstrated the synergistic effect of J A and ethylene on the induction of PDFI.2 and
that activation of PDF 1.2 by pathogen depended on both the jasmonate and ethylene
signal transduction pathway (Penninckx et al., 1998). It has been shown that ERF 1, an
ethylene response factor that binds to ethylene response elements, could be induced by
both ethylene and J A treatments. Furthermore, overexpression of ERF] in transgenic
plants could rescue the defense defects of both coil and ethylene insensitiveZ plants
(Lorenzo et al., 2003). These results suggest that the ethylene and jasmonate pathways

converge at the transcriptional activation of ERF] .

The interaction between the SA and jasmonate pathways is often antagonistic
(Niki et al., 1998). The inhibitory effect of SA on JA-inducible genes has been previously
reported for the wound-induction of P13 in tomato (Doares et al., 1995). Conversely, JA-
responsive genes were hyper-induced in transgenic plants expressing a bacterial salicylate
hydroxylase gene (NahG) that fail to accumulate SA during pathogenesis (Reymond and
Farmer, 1998). Phenylalanine ammonia lyase (PAL) is a key enzyme in SA biosynthesis.

Silencing the expression of PAL in tobacco was found to reduce SA-mediated systemic

33

acquired resistance (SAR) to tobacco mosaic virus but enhanced herbivore—induced
systemic resistance to the insect Heliothis virescens. Overexpression of PAL, on the other
hand, enhanced SAR but reduced resistance to the insect (F elton et al., 1999). This
inverse relationship has been observed in other plants species as well. In Arabidopsis, the
coil mutant exhibited robust resistance to the bacterial pathogen Pseudomonas syringae
strain DC3000, which was correlated with elevated accumulation of SA and
hyperactivation of the pathogenesis related (PR) genes following infection. These
experiments suggest that the SA-mediated defense response pathway is sensitized in coil
plants (Kloek et al., 2001). Further, the enhanced disease susceptibility 4 (eds4) mutation
caused reduced accumulation of SA and enhanced susceptibility to infection by P.
syringae. Not surprisingly, the eds4 mutation also caused heightened j asmonate responses

(Gupta et al., 2000).

In apparent contradiction to the evidence above, other studies suggest that the
jasmonate and ethylene signaling pathways might be required for SA responses. The
Arabidopsis mutant nonexpression of PR1 (nprl) is insensitive to SA, fails to express
SA-induced PR genes, and has reduced SAR (Cao et al., 1994). A screen for suppressor
mutations of nprl yielded a dominant mutation named ssil for suppressor of SA
insensitivity]. The ssil mutant has elevated levels of SA, constitutive expression of PR
genes and restored resistance to P. syringae. A striking novel phenotype of ssil plants
was the constitutive expression of PDFI.2, which is normally induced by JA and
ethylene. When SA accumulation in ssil plants was prevented by expressing the NahG
gene, all of the ssil phenotypes were also suppressed, including the expression of

PDFI.2 (Shah et al., 1999). The results indicate that SS]! is a negative regulator of the

34

SA pathway and that in ssil plants, elevated SA does not antagonize but rather enhances

the expression of the J A and ethylene responsive PDFI.2.

Investigation of the Arabidopsis cevl mutant revealed interaction of the jasmonate
signaling pathway with cell wall metabolism. The cevl mutant displayed constitutive
expression of stress response genes and enhanced resistance to fungal pathogens (Ellis
and Turner, 2001). These phenotypes were in keeping with the finding that cevl plants
have increase production of JA and ethylene (Ellis et al., 2002). Cloning the CE V] gene
showed that it encodes the cellulose synthase CeSA3, which is expressed preferentially in
the roots. Consequently, cevl roots contained less cellulose than that in wild-type roots
(Ellis et al., 2002). Significantly, the cevl mutant can be phenocopied by treating wild—
type plants with inhibitors of cellulose synthesis. Other cellulose synthesis mutants, such
as rswl, also exhibited constitutive activation of J A-inducible genes (Ellis et al., 2002).
These experiments established a link between cell wall metabolism and jasmonate

signaling in plants.

35

IV. Physiological Roles of Jasmonates

J asmonates play a dual role in regulating plant development and responses to
numerous stresses. This conclusion was first evident by the development- and stress-
regulated accumulation of JA (Creelman and Mullet, 1997). Levels of endogenous J A are
highest in young growing tissue (Creelman and Mullet, 1995) and increase after
treatment of cell cultures with elicitors or after subjecting plants to wounding, UV light,
water deficit, pathogens and ozone (Conconi et al., 1996; Creelman and Mullet, 1997;
Rao et al., 2000). Application of j asmonates induces the expression of a large number of
genes that are also responsive to other stresses such as wounding and pathogen infection
(Reymond et al., 2000; Schenk et al., 2000). The identification and analysis of mutants
that are impaired in jasmonate biosynthesis, perception or signaling, and the analysis of
transgenic plants with altered expression of J A biosynthetic genes or jasmonate signaling

factors has begun to offer new insights into the function of j asmonates in plant.

IV. I. Jasmonates play a direct role in resistance to herbivores and pathogens

Several remarkable discoveries in the 1990s’ triggered an awakening to the
importance of jasmonates in plant defense. Farmer and Ryan (1990) observed that
volatile MeJ A produced by sagebrush could evoke defense gene expression in adjacent
tomato plants. Zenk and colleagues subsequently found that many plant species tested as
suspension cultured cells could accumulate defensive secondary metabolites in response
to MeJA (Gundlach et al., 1992; Mueller et al., 1993). In tomato, several complementary

pieces of evidence show that j asmonates play a crucial role in the defensive response to

36

herbivores. Plant defense against herbivore attacks was first reported by the wound-
induced accumulation of Pls that inhibit digestive proteases of herbivores and reduce the
nutritional quality of the ingested tissues (Green and Ryan, 1972). Further studies in
tomato revealed that wounding causes a systemic reprogramming of leaf cells that results
in the synthesis of over 20 defense-related proteins (Bergey et al., 1996; Ryan, 2000).
Treatment of plants with JA or MeJA induced the same set of genes (Farmer and Ryan
1992; Howe et al., 1996; Howe et al., 2000), suggesting that jasmonates are the main
regulator for the activation of defense genes. Finally, characterization of the tomato def]
and spr2 mutants (Table 1.1) showed that the mutant plants accumulate significantly less
J A afier wounding, herbivore predation and systemin treatment. The mutant plants also
produced negligible levels of P15 and several other defensive proteins in response to, and
were more susceptible to, lepidopteran insect attacks (Howe et al., 1996; Li et al., 2002a).
In Arabidopsis, mutants that are defective in jasmonate biosynthesis or perception also
exhibited impaired defense responses to insect herbivores (McConn et al., 1997; Stintiz et

aL,2001)

In conifer trees, the production of terpenoid-based resins has long been studied for
its role in defense against herbivores and pathogens (Phillips and Croteau, 1999; Trapp
and Croteau, 2001). For example, in the genus Picea, stem resin is stored in axial resin
canals in the cortex and in axial traumatic resin ducts (TDs). The resin appears after the
tree has been subjected to mechanical wounding, insect predation, or fungal elicitation
(Marin et al., 2002). Recent studies in the Norway spruce showed the induced formation
of TDs by insect and pathogen attack, wounding and MeJA treatment (Franceschi et al.,

2000; Martin et al., 2002). The complex defense responses induced by Me] A, including

37

de novo formation of TDs, terpenoid accumulation, and activation of enzymatic activities
for terpene synthesis (Martin et al., 2002) provided new avenues to evaluate the role of

jasmonates in plant defense.

Several observations in Arabidopsis implicated jasmonates in pathogen defense.
J A levels in plants increase after treatment with the necrotrophic fungus Alternaria
brassicicola (Penninckx et a1. 1996). It was also shown that Arabidopsis mutants affected
in jasmonate synthesis or signaling are more sensitive to attack by the necrotrophic
pathogens Pythium, A. brassicicola, and Erwinia carotovora (Staswick et al.; 1998;
Thomma et al.; 1998; Vijayan et al., 1998; Norman-Setterblad et al., 2000). In addition,
transgenic plants overexpressing JM T are more resistant to the necrotrophic pathogen
Botrytis cinerea (Seo et al., 2001). In contrast, coil plants show higher resistance to the
biotrophic bacterial pathogen Pseudomonas syringae (Kloek et al., 2001). Taking these
results together, the jasmonate pathway seems to positively regulate the resistance of
Arabidopsis plants to necrotrophic pathogens while negatively regulating resistance to
biotrophic pathogens (Thomma et al., 1998). However, in disagreement with this scenario,
the cevl mutant with elevated levels of JA is more resistant to the biotrophic fungal
pathogen powdery mildew (Ellis and Turner, 2001). The jasmonate signaling pathway is
also involved in induced systemic resistance (ISR) in which growth of Arabidopsis plants
in soil containing the rhizobacterium Pseudomonasfluorescens results in enhanced
resistance to subsequent pathogen infection (Pieterse and van Loon, 1998). Thus, the
mode of action of j asmonates in regulating pathogen responses in Arabidopsis is

determined by both the type of pathogen and the type of pathogenicity.

38

I V.2. Role of jasmonates in emission of plant volatiles and tritrophic interactions

The emission of specific volatile blends from plant tissue has long been
recognized as an important component in the interaction between plants and insects, both
in the attraction of pollinators and the deterrence of predators (Harbome, 1988;
Gatehouse, 2002; Pichersky and Gershenzon, 2002). Often times, the synthesis and
release of volatiles as part of the wounding response occurs both locally and systemically
in plants (Rose et al., 1996). Activation of volatile synthesis and release by jasmonates

has been reported (Thaler, 1999; Rodriguez-Saona et al., 2001).

Plant volatiles contribute directly to defense either through their toxicity or by
inducing defensive genes. One such an example is the C6 aldehydes, alcohols and esters
referred to as the “green leaf volatiles” (Pare and Tumlinson, 1999). These volatiles are
formed by a branch of the octadecanoid pathway involving the action of hydroperoxide
lyase (Hatanaka, 1993; Howe et al., 2000) and were found to be able to induce defense-
related genes (Bate and Rothstein, 1998). Antisense suppression of the hydroperoxide
lyase gene in transgenic potato plants led to lower levels of the volatile compounds and
improved performance of the aphid Myzus persicae (Vancanneyt et al., 2001),
demonstrating the importance of the green leaf volatiles in plant defense. As a further
example, the fragrant compound MeJA has been studied as a volatile for long distance
intraplant and plant-to-plant signaling. Me] A volatized from sagebrush was found to
evoke defense gene expression in adjacent tomato plants (Farmer and Ryan, 1990).
Genetic manipulation of Mel A production was achieved by overexpressing the

Arabidopsis JMT gene. The JMT transgenic plants contained three times more Me] A

39

compared with wild-type plants and exhibited enhanced resistance to fungal pathogens
(Seo, et al., 2001). In field experiments, tobacco plants grown adjacent to sagebrush were
found to experience reduced natural herbivore damage when the sagebrush was clipped to

release MeJA (Karban et al., 2000).

Plant volatiles also play a critical role in indirect defense strategies employed by
plants, such as the tritrophic interaction in which the plant-derived compounds signal
directly to the natural enemy of the herbivore infesting the host plant. Volatiles produced
by com, cotton, and Brassica plants during herbivory have been found to attract parasitic
wasps to lepidopteran larvae preying on the plants (Geervliet et al., 1994; Turlings et al.,
1995; Pare and Tumlinson, 1999). Volicitin, a conjugate of LA and L-glutamine purified
from beet arrnyworm oral secretion, was identified as a powerful elicitor of volatile
release (Albom et al., 1997). The LA moiety is apparently plant-derived, whereas the
glutamine is provided by the insect, which also performs the chemical reactions required
to produce volicitin (Pare et al., 1998). Volicitin has been shown to up-regulate the
expression of genes involved in biosynthesis of both indole (Frey et al., 2000) and
terpene (Shen et al., 2000) volatiles in maize. Similar fatty acid conjugates have been
isolated in oral secretions of other lepidopteran larvae (Halitschke et al., 2001).
Identification of volicitin linked insect feeding damage to plant recruitment of natural
enemies of the pest. Future experiments aimed at determining the requirement of the
jasmonate biosynthetic and signaling pathways in volicitin-mediated volatile production
should be helpful to elucidate the molecular control over the diverse responses elicited by

herbivores.

4o

I V. 3. Role of jasmonates in plant growth and development

Reports suggesting that J A might be involved in regulating plant growth, e. g. to
promote senescence, first appeared in 1980 (U eda and Kato, 1980). Later, a wide variety
of physiological responses to applied jasmonates were reported (Sembdner and Parthier,
1993; Creelman and Mullet, 1997). Besides their active role in plant defense, jasmonates
are also implicated in responses to abiotic stimuli including salt and drought stress
(Creelman and Mullet, 1995), UV irradiation (Conconi et al., 1996) and ozone exposure
(Rao et al., 2000;). In addition, jasmonates appear to play a role in regulating diverse
plant developmental processes including seedling growth (Staswick et al., 1992), tendril
coiling (Weiler et al., 1993), tuberization (Pelacho and Mingo-Castel, 1991), fruit
ripening (Czapski and Sarriewski, 1992), pollen maturation and anther dehiscence (Feys
et al., 1994; McConn and Browse, 1996; Sanders et al., 2000; Stintzi et al., 2000;

Ishiguro et al., 2001), and senescence (Ueda and Kato, 1980; He et al., 2002).

Implication of j asmonates in many developmental processes has largely been
based on observations of plant responses to applied jasmonates. While possibly
informative, these observations do not necessarily indicate the endogenous activity of the
hormone. In fact, evidence from analyzing jasmonate signaling and biosynthetic mutants
suggests that jasmonates may be disposable for many aspects of plant growth and
development, and that jasmonates may exert different effects on a particular plant process
in different species. For instance, the reported effects of exogenous JA on seed
germination are contradictory. Inhibition was noted in Brassica napus, flax (Wilen et al.,

1991), and sunflower (Corbineau et al., 1988), whereas stimulation was reported in apple

41

(Ranjan and Lewak, 1992) and no effect was evident in Arabidopsis (Staswick et al.,
1992). These findings suggest that a role for endogenous J A in seed germination, if any,
is at least variable among species. The possible link between JA and senescence is also
questionable because highest endogenous levels of J A generally occur in young growing
tissues and not in senescing tissues (Creelman and Mullet, 1997). Furthermore, tomato
and Arabidopsis mutants affecting J A synthesis or action all appear to senesce normally

(F eys et al., 1994; Howe et al., 1996; McConn and Browse, 1996).

The best example for the involvement of j asmonates in development is their role
in anther and pollen development in Arabidopsis. Initial evidence for this conclusion
came from the observation that the Arabidopsisfad3-2/fad7—2/fad8 mutant fails to
produce viable pollen. Subsequently, mutants defective in other JA biosynthesis genes
(Table 1.1) as well as the JA perception mutant coil were all found to be male sterile.
Significantly, all mutants exhibited identical characteristics in the male-sterile phenotype
(McConn and Browse, 1996; Xie, et al., 1998; Sanders et al., 2000; Stintzi et al., 2000;
Ishiguro et al., 2001). Floral organs of the mutants develop normally within closed buds,
but the anther filaments do not elongate sufficiently to position the locules above the
stigma at anthesis, and the anthers lack the proper dehiscence of the stomium at the time
of flower opening. Although the tapetum is correctly broken down, pollen cannot be
released from locules. The mutant plants produce mature tricellular pollens albeit in
much smaller amounts as compared with wild-type plants. However, the mutant pollen
does not germinate properly (McConn and Browse, 1996). The sterile phenotype of the
triple-fad mutant can be restored by application of o-LA, OPDA, J A, and MeJA whereas

the opr3/ddel mutants require JA/MeJ A for recovery. Therefore, three distinct steps in

42

Arabidopsis male reproductive development can be distinguished in which J A/MeJA may
function as a signal: elongation of anther filaments, maturation of pollen, and timing of
anther dehiscence. It has been postulated, based upon the expression pattern of the DAD]
gene, that temporal and spatial up-regulation of J A synthesis in filaments promotes water
uptake from the locules and subsequent transport to the filaments, resulting in the
elongation of filaments, maturation of pollen grains and dehiscence of anthers (Ishiguro

et al., 2001).

It is clear from the above discussion that our knowledge of the function of
jasmonates at the molecular and genetic levels is largely limited to studies conducted
with Arabidopsis. Therefore, a comprehensive understanding of the action and function
of jasmonates requires molecular genetic studies in diverse plant species. In the current
dissertation, efforts to utilize cultivated tomato (L. esculentem) as an experimental model
to dissect the role of j asmonates in the systemic wound response and the defense against
herbivores, and in several developmental processes will be described. The first part of
this dissertation research concerns the regulation of the tomato prosystemin gene. This
work is presented in Chapter 2. The remainder of the dissertation (Chapter 3-6) focuses
on the role of j asmonates in tomato defense and development using J A-insensitive
mutants developed in this thesis research. Chapter 7 summarizes the major results of this
dissertation research and suggests a few experiments to fiirther elucidate the jasmonate
pathway in tomato. The finding for a role of j asmonates in tomato glandular trichome

development is presented in the Appendix.

43

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59

CHAPTER 2

Alternative Splicing of Prosystemin Pre-mRNA Produces Two Isoforms That Are

Active as Signals in the Wound Response Pathway

The work presented in this chapter has been published:

Li L, Howe GA (2001) Plant Mol. Biol. 46, 409-419.

60

Abstract

Systemin and its precursor protein, prosystemin, play an essential role in the
systemic wound response pathway of tomato plants. We report here the isolation from
tomato of a novel prosystemin cDNA (prosysB) that differs from the reported cDNA
sequence (prosysA) by the addition of a CAG trinucleotide. Inspection of the prosystemin
genomic sequence, which was mapped to the central region of chromosome 5, indicated
that prosysA and prosysB transcripts are generated by an alternative splicing event that
utilizes different 3' splice sites within intron 3. Quantitative RT-PCR analysis showed
that prosysB transcripts accumulated to approximately twice the level of prosysA in all
tissues that express the prosystemin gene. The relative abundance of the two mRNAs was
unaffected by wounding or methyl jasmonate treatment, conditions that increase the level
of total prosys mRNA. These findings indicate that alternative splicing of prosys pre-

mRNA is a constitutive process. The amino acid sequence of prosysB is predicted to

differ from that of prosysA by replacement of Arg57 with Thr-Gly in the non-systemin
portion of the protein. Overexpression of the prosysB cDNA in transgenic tomato plants
conferred constitutive expression of defense genes that are regulated by wounding and

systemin. We conclude that prosysB is the major prosystemin-encoding transcript in

tomato, and that this isoform is active as a signal in the wound response pathway.

61

Introduction

Systemin and its precursor protein, prosystemin, play an essential role in the
regulation of systemic wound responses in tomato plants. An increasing body of evidence
indicates that systemin regulates a complex signaling pathway that culminates in the
systemic expression of proteinase inhibitor and other defense-related genes (reviewed by
Bowles, 1998; Ryan and Pearce, 1998; Schaller, 1999; Ryan, 2000). Systemin is
proposed to activate the signaling cascade upon binding to a 160-kD cell surface protein
that displays characteristics of a functional systemin receptor (Meindl et al., 1998; Scheer

and Ryan, 1999). Interaction of systemin with its receptor is associated with several rapid

. . . . . . 2+ . .
biochemrcal events 1nclud1ng increased cytosolic Ca levels, membrane depolarrzatron,

inhibition of a plasma membrane proton ATPase, and activation of a MAP kinase activity
(Felix and Boller, 1995; Moyen and Johannes, 1996; Stratmann and Ryan, 1997; Moyen

et al., 1998; Schaller and Oecking, 1999). These early signaling events in the systemin

and wound response pathways culminate in the activation of a phospholipase A2 activity

that releases fatty acid precursors of jasmonic acid (J A) from membrane lipids (Narvaez-
Vasquez et al., 1999). Current models indicate that J A, together with wound-induced
ethylene, is required as downstream signals for the activation of wound-responsive genes

(Farmer and Ryan, 1992; O’Donnell et al., 1996; Ryan, 2000).

Systemin is derived from the C-terminal end of a 200-amino-acid precursor called
prosystemin. Molecular cloning and analysis of a cDNA encoding the precursor has
provided evidence that prosystemin plays a critical role in the transduction of systemic

wound signals. First, anti-sense expression of the prosystemin cDNA in transgenic

62

tomato plants dramatically reduced the systemic wound response (McGurl et al., 1992).
Second, overexpression of prosystemin from the CaMV 35 S promoter in transgenic
plants resulted in constitutive expression of wound response genes in unwounded plants
(McGurl et al., 1994). Third, mutations that disrupt prosystemin-mediated signaling also
impair wound-induced expression of downstream target genes (Howe and Ryan, 1999).
Finally, recombinant prosystemin, like synthetic systemin, is a potent inducer of wound-
responsive genes when supplied to tomato seedlings through the transpiration stream.
Structure-function analysis of recombinant prosystemin further indicates that the
bioactivity of prosystemin resides exclusively in the systemin domain of the molecule
(Dombrowski et al., 1999). These results suggest that prosystemin itself might be active

as a signaling component for wound-induced gene expression.

Despite the evidence that (pro) systemin is a required component of the systemic
wound response pathway, the precise mechanism by which this polypeptide participates
in the transduction of systemic signals remains to be elucidated. At least two hypotheses
have been proposed. One model holds that systemin is transported through the plant
following its proteolytic release from the C-terrninal end of prosystemin (Pearce et al.,
1991; Ryan and Pearce, 1998; Ryan, 2000). Proteolytic activities that digest prosystemin
to smaller systemin-containing polypeptides have been identified in the apoplastic fluid
from tomato leaves (Dombrowski et al., 1999). Characterization of these proteases and
study of their role in systemic signaling is likely to provide insight into the proposed link
between prosystemin processing and the production of a mobile peptide signal. An
alternative hypothesis is that prosystemin functions locally to facilitate or amplify a very

rapid systemic signal, such as electrical signals that are produced in response to

63

wounding (Wildon et al., 1992; Bowles, 1998). Both hypotheses appear to be consistent
with the observation that prosystemin expression is restricted to vascular tissues, through
which systemic signals are likely to travel (J acinto et al., 1997). The observed increase in
vascular-specific expression of prosystemin in response to wounding and other elicitors
of the wound response, such as JA, may reflect a mechanism to amplify (pro) systemin-

mediated signals (McGurl et al., 1992; J acinto et al., 1997).

Here we describe a novel prosystemin cDNA that differs from the reported
sequence by including a CAG trinucleotide located at the boundary of intron 3 and exon
4. Our results indicate that two isoforms of prosystemin are produced by an alternative
splicing event that utilizes two 3’ splice sites within intron 3 of the prosystemin gene.
Analysis of transgenic tomato plants that overexpress either of the two cDNAs indicates
that both isoforms of prosystemin are active signaling components of the wound response

pathway.

64

Materials and Methods

Plant material and treatments

Lycopersicon esculentum cv Micro-Tom was used for all experiments except
where otherwise indicated. The 358::prosysA transgene present in cv Castlemart was
introduced into the Micro-Tom genetic background by repeated backcrossing (three times)
using Micro-Tom as the pistillate parent, with selection for the semi-dwarf and
constitutive wound response phenotypes in each generation. Seeds of L. pennellii
(LA0716), L. pimpinellifolium (LA2184), L. chmielewskii (LA1306), L. chilense
(LA1963), L. parviflorum (LA1326), L. hirsutum (LA1223), and the introgression lines
used for RFLP mapping were obtained from the Tomato Genetics Resource Center
(Davis, CA). Plants were grown in Jiffy peat pots (Hummert International) and
maintained in grth chambers as described previously (Howe et al., 2000). Methyl
jasmonate (MeJ A) treatments were accomplished by placing 4O three-week-old plants in
a closed Lucite box (3 1 x27 x 14 cm). Two pl of MeJA (Bedoukian Research Inc.) was
diluted into 50 ul ethanol and the resulting solution was applied to nine cotton wicks
evenly distributed within the box. For each time point after MeJA treatment, five plants
were removed from the box for extraction of RNA from leaf tissues. Plants were
challenged with tobacco homworrn larvae as previously described (Howe et al., 2000).
Proteinase inhibitor and polyphenol oxidase levels were measured as previously

described (Howe and Ryan, 1999).

65

Cloning of prosys cDNAs and intron sequences

Five pg of total RNA isolated from tomato leaf tissue was reverse transcribed
using the SuperScript Preamplification System (Gibco-BRL) and an oligo(dT)12-13

primer as recommended by the manufacturer. cDNA products of the reaction were used
as template for a PCR reaction that employed the primers P81 (5'- GCG AAT TCG ATG
AGT ATA TAA AGC TCA GC) and PS2 (5'- GCG GAT CCG AAG TTA CTT TTC
TAA CGG GAG AC). The resulting 0.76-kb PCR products were digested with BamHI
and EcoRI, gel purified, and ligated into the corresponding sites of pBluescript SK(-) to
generate plasmid pPS-B. The prosysB cDNA insert was sequenced in its entirety using
T7 and T3 primers. The prosysB cDNA contained the entire open reading frame for

prosysB as well as 57 bp of the 5' UTR and 100 bp of the 3' UTR.

For DNA sequencing of prosystemin intron 3 from wild tomato species, genomic
DNA prepared from these plants was PCR-amplified using primers PSDl (5' - GCG
GAT CCA TCT CTT CAT ATG T) and PSD2 (5' - GCG AAT TCC CTC CTC ATG
TTC CAT). The resulting PCR products were cloned into a pGEM-T Easy vector
(Promega, Madison, WI) and sequenced in using a T7 primer. Alignment of intron 3
sequences from various tomato species was performed using the Clustal method in the

Megalign program (DNAStar, Madison, WI).

66

Northern and Southern blot analyses

Total RNA was isolated from tomato tissue and analyzed by RNA blot

hybridization as previously described (Howe et al., 2000). 32P-labeled DNA probes were

prepared using a T7 Quickprime Kit (Pharmacia Biotech) according to the
manufacturer’s instructions. A 760-bp EcoRI-BamHI fragment containing the entire
prosysA cDNA was used as a probe for detection of prosystemin transcripts.
Hybridization signals were visualized by autoradiography using Kodak XAR-S film, or
were measured using a Phosphor-Irnager (Molecular Dynamics). Hybridization signals
were normalized to those obtained using a probe for translation initiation factor eIF 4A

mRNA as previously described (Howe et al., 2000).

Tomato genomic DNA was purified from young leaf tissue as previously

described (McCouch et al., 1988). Five pg aliquots of DNA were digested with HindIII,
. . . TM +
fractionated on a 0.8% agarose gel, and blotted by capillary action to Hybond -N

membrane (Amersham). Probes were prepared as described above. Pre-hybridization and
hybridization were performed in a solution containing 5xSSPE, 5xDenhardt’s reagent,
0.5 % SDS (w/v), and 50 pg/ml denatured sahnon testes DNA. Blots were washed at
65°C in a solution of 2xSSC and 0.1 % SDS (w/v), followed by repeated washing at

65°C in 1xSSC and 0.1% SDS. Probes were stripped from hybridized blots by boiling the

membrane in 0.5% SDS (w/v) for 2 min.

67

Quantitative PCR

Five pg of total RNA was reverse transcribed in a reaction volume of 20 pl using
the SuperScript Preamplification kit as described above. One p1 of this reaction served as
DNA template for a 25-pl PCR reaction containing 2.5 pl 10X reaction buffer supplied

by the manufacturer (Gibco-BRL), 5 pmol each of primers PSDl and PSD2, 5 nmol

dNTPs, and 1.25 U T aq DNA polymerase. Primer PSD2 was end-labeled with 32P using

T4 polynucleotide kinase (Gibco-BRL), as recommended by the manufacturer. PCR
reactions were denatured at 94°C for 3.5 min, and subjected to 20 cycles of amplification
(94°C, 1 min; 56°C, 1 min; 72°C, 35 sec). Two pl PCR products were mixed with 4 pl
formamide loading buffer (Sambrook et al., 1989), heated at 70°C for 5 min, and chilled
on ice. The samples were loaded on an 8% denaturing (7.6 M urea) polyacrylamide gel
and electrophoresed at 1,500 volts for 2 hrs. The gel was pre-electrophoresed at 1500
volts for 0.5 hr prior to running the samples. Gels were washed in 5% methanol and
acetic acid solution for 30 min and dried in gel-dryer (model 583, Bio-Rad). The signals

were visualized by autoradiography and quantified with a Phosphor-Imager.

S1 nuclease experiments

81 nuclease (Gibco-BRL) was used to probe PCR products for regions of
heteroduplex DNA as described by Eckhart et al. (1999). Reactions (30 pl) contained

approximately 100 ng template DNA, 33 mM sodium acetate, 50 mM NaCl, 0.03 mM

ZnSO4, pH 4.5. S] nuclease was added to a concentration of 20U/ pg template DNA, and

68

reactions were incubated at 37°C for 1 hr. Approximately one ng of reaction products
- TM +
was fractionated on a 2% agarose gel and blotted on Hybond -N membrane. Blots

were hybridized to a radiolabeled prosysA cDNA probe, as described above, for detection

of SI cleavage prbducts.

Transformation

The pPS-B plasmid harboring the prosysB cDNA was digested with EcoRI and
BamHI. The resulting 766-bp fragment containing the cDNA was gel-purified and blunt-
ended with Klenow fi'agment (Gibco-BRL). This fragment was cloned into SmaI and SstI
sites, blunt-ended with Klenow fragment, of the binary vector pBIlZl (Clontech). The
resulting construct was transformed into Agrobacterium tumefaciens strain LBA4404.

Transformation of cotyledon explants (cv Micro-Tom) was performed as previously

described (McCormick, 1991). Eleven independent primary transforrnants (T1) were
regenerated on kanamycin-containing medium. Introduction of the transgene was
confirmed by PCR using a primer set of PS3 (5’- GCG GAT CCG TGG AGA TGA CAA
AGA GAC TCC) and PS2 (see above). These primers were designed to amplify 1030-bp

and 290-bp products corresponding to the endogenous prosystemin gene and the

35S::prosysB transgene, respectively. Regenerated plants were transferred to the

greenhouse for collection of T2 seeds.

69

Results

ProsysA and prosysB are generated by alternative splicing of intron 3

During the sequencing of prosystemin cDNAs from L. esculentum, we isolated
several clones that contained a CAG insertion between nucleotides 273 and 274 of the
cDNA sequence (accession number M84801) reported by McGurl et a1. (1992). For
clarity, we have designated the novel CAG-containing cDNA as prosysB and the original
sequence reported by Mch1 et al. as prosysA. Of a total of 24 independent cDNAs that
were isolated, sequence analysis showed that 15 of them (62.5%) corresponded to
prosysB and the remainder corresponded to prosysA. To rule out the possibility that the
two transcripts are encoded by different genes, we used Southern blot and RF LP analysis
to confirm the single copy nature of the gene and to map its chromosomal position. In
agreement with previous results (McGurl et al., 1992), extensive Southern blot analysis
failed to provide evidence for more than one copy of the prosystemin gene (data not
shown). A set of introgression lines that harbor defined segments of L. pennellii DNA
(Eshed and Zamir, 1994) was screened for the presence of a Hind III-generated RFLP
that distinguishes the prosystemin gene in L. pennellii from that in L. esculentum. Only
one line (IL5-3; LA3497) displayed the L. pennellii RFLP pattern, indicating that the
prosystemin gene is located within the central region of chromosome 5 (Figure 2.1a).
Hybridization of blots to a RFLP marker (CT118A) known to map to the introgressed
DNA segment contained in LA3497 confirmed this conclusion (Figure 2.1b). These

results indicate that that prosysA and prosysB are not derived from two different genes.

70

Figure 2.1. Mapping of the prosystemin gene by Southern blot analysis of tomato

introgression lines. (a) Genomic DNA from L. esculentum (L.e.), L. pennellii (L.p.), an
F1 derived from a L. pennellii x L. esculentum cross (Le. x L.p.), and introgression
lines (IL5-2, IL5-3, IL5-4 and IL3-5) carrying different segmental substitutions of L.
pennellii DNA were digested with Hind III and analyzed by Southern blotting with a
prosysA cDNA probe. The position of migration of molecular weight standards (kb) is

shown. (b) Hybridization of the blot shown in (a) to CT118A, an RFLP marker known

to map to the introgressed DNA segment present in IL5-3.

71

(a)

_ _
o. o.
8 6

 

4.0 —

 

 

2.0 —

(b)

m-m.__
v.9:
m-m.:
N-m.__

.Q.I— x .m.l_

.Q.l—

.0i_ ...

. _
o o.
8 6

 

4.0 —

2.0 —

Figure 2.1

72

Comparison of the prosys cDNA and genomic sequences revealed that the CAG
insertion within prosysB coincided precisely with the location of intron 3 (Figure 2.2).
That the CAG polymorphism resulted from alternative splicing was suggested by the
location of a second 3' splice site (site B) three nucleotides upstream from the AG 3'
splice site (site A) used for generation of prosysA. The A and B splice sites are 71% and
57% identical to the UGYAGIGU consensus sequence for 3’ splice sites of plant introns
(Lorkovic' et al., 2000), with both sites containing the invariant AG dinucleotide. These
observations strongly suggest that prosysA and prosysB are the products of an alternative
splicing event that utilizes two juxtaposed 3' splice sites (A and B, respectively) in intron

3 (Figure 2.2).

To determine whether the two juxtaposed 3' splice sites are conserved in different
genetic backgrounds of tomato, we used primers PSDl and PDSZ (Figure 2.2a) to PCR-
amplify and clone prosystemin intron 3 from L. esculentum and six wild species of
tomato including L. pimpinellifolium, L. chmielewskii, L. chilense, L. pennellii, L.
parviflorum, and L. hirsutum. DNA sequencing showed that intron 3 of L. esculentum is
109 bp in length, whereas the same intron in the six wild species ranged between 97 bp (L.
pennellii) to 118 bp (L. chmielewskii) (data not shown). Sequence comparisons indicated
that L. esculentum intron 3 was most similar (95.3% identical) to that of L.
pimpinellifolium, and most dissimilar to that of L. hirsutum (62.6% identity). Despite the
various levels of polymorphism between L. esculentum and the wild species, the
juxtaposed 3’ splice site (CAGCAGIGA) was perfectly conserved in all wild species
examined. This result suggests that the alternative event splicing observed in L.

esculentum also occurs in distantly related tomato species.

73

Figure 2.2. Proposed model for alternative splicing of prosystemin pre-mRNA. (a)
Schematic diagram of the tomato prosystemin gene organized into 11 exons (vertical
bars) and 10 introns (horizontal lines). Systemin is encoded within exon 11 (black bar).
The vertical arrow indicates the location of the alternative splicing at the 3' end of
intron 3. Also shown are the annealing sites for the‘primers used in this study
(horizontal arrows). (b) Proposed scheme for alternative splicing of prosystemin intron
3. Exon-intron boundaries are indicated by “l”. The extreme 3' end of intron 3 contains
two “ag” dinucleotides (underlined) that compete as splice acceptor sites. Splicing at

the upstream ag (site B) results in production of prosysB, whereas splicing at the

downstream ag (site A) generates prosysA. At the protein level, the deduced effect of

alternative splicing is substitution of Arg57 in prosysA with a Thr-Gly sequence in

prosysB.

74

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75

An S1 nuclease assay was used to obtain additional evidence for the accumulation
of prosysA and B transcripts in samples of total RNA isolated from L. esculentum. This
assay, which takes advantage of the ability of SI nuclease to cleave heteroduplex DNA in
regions of nucleotide mispairing, has been used to detect small insertion/deletion DNA
polymorphisms (Shenk et al., 1975; Eckhart et al., 1999). Control experiments were
performed with plasmids pPS-A and pPS-B that harbor full-length cDNAs for prosysA
and prosysB, respectively. Primers (PS1 and PS2; Figure 2.2a) corresponding to the 5'-
and 3'-UTRs of the cDNAs were used to PCR-amplify the prosystemin open reading
frame. PCR products were treated with 81 nuclease and then analyzed by Southern
blotting (Figure 2.3). PCR products derived from a single plasmid template (pPS-A or
pPS-B alone) did not produce specific cleavage products when treated with 81 (Figure
2.3, lanes 1-4). However, Sl treatment of PCR products generated from a mixture of
pPS-A and pPS-B generated cleavage products of approximately 530 and 220 bp (Figure
2.3, lanes 5 and 6). The sizes of these fragments are consistent with that expected for
cleavage at the site of the CAG polymorphism. To test for the presence of prosysA and
prosysB transcripts in tomato leaf tissue, total RNA was reverse transcribed (RT) and
PCR-amplified using the same primer set. Analysis of the SI cleavage products revealed
the 535-bp band corresponding to the DNA located 3' to the polymorphism (Figure 2.3,
lane 8). Longer exposures of the autoradiograph also revealed the smaller 220-bp
cleavage product (not shown), which was ofien difficult to detect owing to its diffusion
from the gel. These results demonstrate the co-existence of prosysA and prosysB

transcripts in RNA isolated from tomato. The absence of additional Sl-generated

76

Figure 2.3. S] nuclease cleavage assay for detection of heteroduplex DNA formed
by prosystemin cDN As. PCR products were generated by amplification of various
prosys cDNA templates with primers PSI and PS2 (Figure 2.2a). For control reactions,
cloned prosys cDNAs contained in plasmids pPS-A and pPS-B were used as PCR
templates as follows: prosysA alone (A; lanes 1-2), prosysB alone (B; lanes 3-4), or an
equal mixture of prosysA and prosysB cDNAs (A+B; lanes 5-6). Alternatively, prosys
cDNAs were generated by reverse transcription of total leaf RNA (RT; lanes 7-8). PCR
products were incubated in the presence (+) or absence (-) of 81 nuclease and a portion
(approximately one ng) of the reaction was separated by gel electrophoresis, blotted to
Hybond membrane, and hybridized to a prosysA cDNA probe. Hybridization signals
were visualized by autoradiography. The band indicated by the asterisk (*) denotes the
larger of the 'two DNA fragments produced by 31 cleavage of the prosysA/prosysB

heteroduplex.

77

Template: A B A+B RT
S1: — + — + — + _ +

 

763 —

535 —

 

228 —

Figure 2.3

78

fragments (Figure 2.3, lane 8) further suggests that prosysA and B comprise the two most

abundant prosystemin transcripts in tomato leaves.

Expression pattern of prosysA and prosysB

Alternative splicing of pre-mRNA is often regulated by specific factors expressed
in response to developmental or environmental cues. To address this question in relation
to prosystemin pre-mRNA splicing, we developed a quantitative RT-PCR assay to
estimate the relative abundance of the prosysA and prosysB transcripts in total RNA
samples. Primers (PSDl and PSD2; Figure 2.2a) that span the site of the CAG
polymorphism were used to amplify both forms of the cDNA in a single PCR reaction.
The resulting isoform-specific fragments were then separated by PAGE. Radiolabeling of
one primer (PSD2) permitted visualization and quantification of the displayed products
after PAGE. Control experiments showed that PCR amplification of the cloned prosysA
and prosysB cDNAs yielded prosysA- and prosysB-specific products of 78 and 81
nucleotides, respectively (Figure 2.4). Mixing of the two templates in various pr0portions
prior to PCR yielded an excellent correlation (R value = 0.99) between the ratio of
template DNA in the reaction and the relative abundance of the two isoform-specific
products obtained (Figure 2.4). These results indicate that the PCR assay is quantitative

with respect to the relative abundance of the two templates in the reaction.

To examine the possibility that the abundance of prosysA and B is regulated

during development, total RNA prepared from different tomato tissues was reverse-

79

Figure 2.4. Quantitative PCR amplification of prosysA and prosysB cDNAs in a

. single reaction. (a) ProsysA and prosysB cDNA fragments contained in plasmids pPS—
A and pPS-B, respectively, were gel-purified and mixed in various ratios such that the
proportion of prosysA in the mixture was as follows: 100% (lane 1), 90% (lane 2), 80%
(lane 3), 70% (lane 4), 60% (lane 5), 50% (lane 6), 40% (lane 7), 30% (lane 8), 20%
(lane 9), 10% (lane 10), and 0% (lane 11). The total amount of prosysA + prosysB DNA

in each mixture was 30 fg. These samples served as templates for a PCR reaction using

primers PSDl and PSD2, where the latter primer was end-labeled with 32P. PCR

products obtained from 20 cycles of amplification were separated by denaturing PAGE
and visualized by autoradiography. The PCR products derived from prosysA and
prosysB were 78 and 81 bp in size, respectively. (b) The products shown in (a) were
quantified using a Phosphor-Imager. The relative proportion of the two products in each
reaction (expressed as % prosysA) was calculated and plotted against the proportion of

prosysA present in the template DNA.

80

(a) 1234567891011

 

(b)

 

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o
O

on
c?

or
c?

5

N
C?

 

°/o prosysA in product

 

 

O
O

o 23 4b 60 so 100
°/o prosysA in template

Figure 2.4

81

transcribed and subjected to the quantitative PCR analysis described above. Display of
the radiolabeled products on PAGE revealed the presence of prosysA- and prosysB-
specific bands in all tissues in which prosys mRNA is known to be expressed (Figure
2.5a). Furthermore, the absolute abundance of prosys mRNA (prosysA + B) observed in
different organs was consistent with that previously observed by RNA blot analysis
(McGurl et al., 1992). The ratio of prosysA to prosysB was approximately 1:2 in RNA
prepared from all tissues examined (Figure 2.5b). This finding indicates that prosysB is

the most abundant form of prosys mRN A.

Previous studies have shown that prosys mRNA accumulation is up-regulated by
wounding or by treatment of plants with methyl jasmonate (MeJ A), a potent inducer of
wound response genes (J acinto et al., 1997; McGurl etal., 1992). To determine whether
this effect reflects increased accumulation of one or both forms of the mRNA, plants
were exposed to MeJ A and total leaf RNA was prepared at different times thereafter for
analysis by RT-PCR. The effectiveness of the MeJ A treatment was evident by a several
hundred-fold increase in accumulation of proteinase inhibitor II (PI-II) mRNA during the
time course (Figure 2.6a). Although the effect of MeJ A on prosystemin mRNA
accumulation was considerably less, we did observe an increase of at least 5-fold.
Analysis of prosysA and prosysB transcripts in these RNA samples showed that the level
of both forms increased in parallel during the time course, such that the relative
abundance of the two transcripts remained constant (Figure 2.6b and c). Similar results
were obtained using RNA prepared from tomato leaves that were subjected to wounding
by tobacco homworrn (Manducta sexta) larvae (data not shown). Taken together, these

results indicate that alternative splicing of prosystemin pre-mRNA within intron 3 is

82

Figure 2.5. Relative abundance of prosysA and prosysB transcripts in different
tissues of tomato. (a) Total RNA isolated from cotyledon (lane 1), stem (lane 2),
petiole (lane 3), leaf (4), flower bud (5), or open flower (6) was reverse transcribed and
subjected to the quantitative PCR assay described in Figure 2.4. “A” and “B” denote
the isoform-specific products derived from prosysA and prosysB, respectively. (b)
Products in (a) were quantified by Phosphor-Irnage analysis. The gray and black
portions of each bar represent the relative abundance of prosysA and prosysB,

respectively.

83

(a)

 

 

ii

 

 

Hill

0 0 0 0 0
8 6 4 2

100

08353 o\o

Figure 2.5

84

Figure 2.6. Relative abundance of prosysA and prosysB transcripts in response to
methyl jasmonate treatment. (a) Three-week-old Micro-Tom plants were exposed to
MeJA vapor and leaf tissue was harvested for RNA isolated at various times (h)
thereafter. Five pg aliquots of total RNA were subjected to Northern blot analysis using
cDNA probes for prosysA (top panel) and proteinase inhibitor 11 (PI-II; middle panel).
elF 4A (bottom panel) was the loading control. (b) The RNA samples analyzed in (a)
were reverse transcribed and subjected to the quantitative PCR assay described in
Figure 2.4. (c) The relative proportion of the isoform-specific products shown in (b)
was quantified. The gray and black portions of each bar represent the relative

abundance of prosysA and prosysB, respectively, in each reaction.

85

(a) 00.512481224

P'OSys

Pl-ll

 

 

WW,

00.5124 81224

(c)

#0)“,
Coo

% abundance
N
O

     

Figure 2.6

86

constitutive, and is not regulated by developmental or environmental conditions that

regulate the expression of the prosystemin gene.

Overexpression of prosysB confers constitutive wound signaling

The above results indicate that prosysB is the predominant species of prosystemin

mRNA in all parts of the plant that express the prosystemin gene. Because the amino acid

sequence of prosysB is predicted to differ from prosysA by an Arg57—>Thr-Gly

substitution, it was of interest to determine whether this change affects the ability of
prosystemin to signal the expression of wound responsive genes. To address this question
we relied on the fact that expression of a 35S: :prosysA transgene in tomato activates the
constitutive expression of several wound-inducible genes, including those encoding PI-I,
PI-II and polyphenol oxidase (PPO) (McGurl et al., 1994; Constabel et al., 1995; Bergey
et al., 1996). Genetic analysis has shown that this phenotype results from constitutive
activation of the endogenous wound response pathway (Howe and Ryan, 1999). To
determine whether overexpression of prosysB confers such a constitutive signaling
phenotype, Agrobacterium-mediated transformation was used to generate transgenic
tomato plants (cv Micro-Tom) that harbor a 35S: :prosysB transgene. The presence of the
transgene in regenerated plants was confirmed by PCR and Southern blot analysis (data
not shown). As a control for these experiments, the 35S::prosysA transgene originally
introduced into the Better Boy cultivar (McGurl et al., 1994) was backcrossed into the
Micro-Tom genetic background (see Experimental procedures). Northern blot analysis
showed that leaf tissue from the 35S::prosysA and 35S::prosysB lines accumulated

significantly greater levels of prosys mRNA than were observed in untransformed control

87

plants (Figure 2.7a, upper panel). RT-PCR analysis of the same RNA samples showed
that the prosysB transcript accounted for 96% of the total prosys mRNA detected in
35S: :prosysB plants, whereas the prosysA transcript accounted for 90% of the total
prosys mRNA detected in 35S::prosysA plants (Figure 2.7a, lower panel). This result
established that the two transgenic lines have dramatically different isoform-specific
expression patterns. The RT-PCR assay did not distinguish the endogenous prosysA
transcript from 35S: :prosysA-derived transcripts, or endogenous prosysB transcripts fi'om
those derived from 35S: :prosysB. However, this experiment did indicate that
overexpression of one isoform does not increase the accumulation of the other isoform
derived from the endogenous prosystemin gene. This finding suggests that ectopic
expression of prosystemin from a 35S: :prosys transgene does not positively regulate the

expression of the endogenous prosystemin gene.

We next compared the different genotypes with respect to the level of PI-II and
PPO produced in leaf tissue from unwounded plants (Figure 2.7b). The level of PHI in
both 35S::prosysA and 35S::prosysB plants was extraordinarily high, and represented an
approximate 30-fold increase over the level observed in untransformed plants. Similarly,
the constitutive level of PPO activity in both transgenic lines was significantly elevated
(>10-fold) over that of control plants. The PI-II and PPO levels observed in 35S: :prosysB
plants were comparable to those in 35S::prosysA plants. These results demonstrate that
alteration of the ratio of prosysA to prosysB in planta does not affect the constitutive
signaling phenotype. We therefore conclude that prosysB, like prosysA, encodes a form

of prosystemin that activates the expression of wound-response genes.

88

Figure 2.7. Overexpression of either prosysA or prosysB in transgenic tomato
results in a constitutive wound response phenotype. (a) Five pg total RNA prepared
from leaf tissue of untransformed control plants (lane 1), 35S:.'prosysB plants (lane 2),
or 35S: :prosysA plants (lane 3) was subjected to Northern blot analysis for
determination of prosys mRNA levels (top panel). A second blot was hybridized to an
elF 4A probe as a loading control (middle panel). The relative proportions of prosysA
and prosysB in the same samples were determined using the RT-PCR assay described
in Figure 2.4 (lower panel). (b) Proteinase inhibitor 11 (PI-II; black bars) and
polyphenol oxidase (PPO; gray bars) levels in leaves of five-week—old unwounded
plants (1, untransformed; 2, 35S::prosysB; and 3, 35S::prosysA). Values represent the

mean i sd of six plants of each genotype.

89

(a)

S
w.
m
P

eIF4A

 

UUO moz<=<
3593.230 906.3

 

 

 

0
0
0 .
1004

$05.. .62 _E\c_ouoa m3
_o>o_ in

Figure 2.7

90

Discussion

The prosystemin gene of tomato consists of 10 introns and 11 exons that are
organized into five repeated regions encompassing the first ten intron-exon pairs (McGurl
and Ryan, 1992). Interestingly, these repeats exclude the eleventh exon in which the
systemin sequence is found (MchI and Ryan, 1992). The repeat structure of the gene is
also evident in the prosystemin protein, which contains several repeated motifs that also
exclude the C-terminal systemin domain. The repeated motifs are thought to have arisen
through successive gene duplication-elongation events (Mch1 and Ryan, 1992). This
observation, together with the highly conserved systemin region found in other
solanaceous plants (Constabel et al., 1998), suggests that the non-systemin portion of the
polypeptide plays only a minor role in the protein’s function. This view is consistent with
a recent study showing that the bioactivity of prosystemin as a signal for wound

responses resides exclusively in the systemin domain (Dombrowski et al., 1999).

In the present study, we show that different forms of prosystemin are produced by
alternative splicing. Initial insight into this phenomenon came from the identification of a
novel prosystemin cDNA (prosysB) that differs from the reported cDNA sequence
(prosysA) by the addition of a CAG within the non-systemin portion of the open reading
frame. An 81 nuclease assay was used to confirm the co-existence of prosysA and
prosysB transcripts in total RNA, and further indicated that prosysA and B comprise the
two major forms of prosystemin mRNA in tomato. The single copy nature of the
prosystemin gene, which we mapped to the central region of chromosome 5, excludes the

possibility that the two transcripts are derived from different genes. Rather, our results

91

indicate that prosysA and B are generated by an alternative splicing event that uses one of

two juxtaposed 3' splice sites in intron 3. At the protein level, the predicted outcome of

alternative splicing is an Arg57—>Thr-Gly substitution. Based on comparisons of deduced

prosystemins isolated from various solanaceous plants (Constabel et al., 1998), it is
apparent that this polymorphism is located in one of the most variable regions of
prosystemin. Constabel et a1. (1998) suggested that amino acid deletions found in bell
pepper prosystemin and one isoform of potato prosystemin may result from a shift in
intron-exon boundaries in this region. Our results suggest that alternative splicing may

also be responsible for generating sequence variability in this region.

ProsysA and prosysB transcripts accumulate at a relatively fixed ratio in all tissues
that express the prosystemin gene. The relative proportion of the two transcripts was not
significantly altered under conditions (e. g., wounding or MeJ A treatment) that increase
the accumulation of total prosys mRNA. These results indicate that alternative splicing of
prosystemin intron 3 is constitutive, and is not likely to be regulated by developmental or
environmental cues. Quantitative PCR experiments showed that the level of prosysB
transcripts was approximately twice that of prosysA transcripts. This value is consistent
with the higher proportion (62%) of prosysB cDNAs revealed by direct sequencing of
cloned prosystemin cDNAs. In further support of this, we found that all the prosystemin
cDNAs sequenced to date as part of the tomato EST project (http://www.tigr.org/tdb/lgi/)
correspond to the prosysB isoform. Taken together, we conclude that prosysB is the most
abundant prosystemin transcript in cells that express the gene. Assuming that both

transcripts are subject to similar post-transcriptional regulation, we predict that prosysB is

92

the major protein isoform in cell-types that express prosystemin. It may be possible to

test this hypothesis using chromatography techniques to separate the two protein isoforms.

The prevalence of prosysB over prosysA is consistent with the spliceosome
scanning model for recognition and selection of 3' splice sites (Smith et al., 1993). This
model proposes that 3' splice site selection is determined by spatial relationships between
the branch point, the polypyrimidine tract downstream of the branch point, and the 3'
splice site. The spliceosome scans in a 5’-to-3' direction from the splice branch point and
selects the first AG encountered. In the event that an additional AG lies downstream of
the proximal AG, the most competitive splice site is selected. Determinants of splice site
competition include not only the proximity of the AG to the branch point, but also the
sequence context surrounding the splice site. In the case of prosystemin, it is interesting
to note that the downstream splice site (site A) conforms to the 3' splice site consensus
better than does the upstream site (site B). However, our data indicate that the upstream
AG is the preferred splice site in vivo. This result lends direct support to the scanning
model, and in particular to the notion that proximity of the splice site to the branch point

is an important determinant in splice site competition.

The functional significance of alternative splicing of prosystemin pre-mRNA
remains to be determined. However, strict conservation of the two juxtaposed 3’ splice
sites in all wild tomato species examined suggests that alternative splicing of this intron
serves an important role. For example, it is possible that the efficiency of splicing is
enhanced by the close juxtaposition of two 3’ splice sites. In support of this idea is the

fact that several other genes have been shown to utilize an AGNAGI motif at the 3' end

93

of an intron to affect alternative splicing (Maurer et al., 1981; Shelness and Williams,
1984; Cook et al., 1985; Sun and Baltimore, 1991; Manrow and Berger, 1993; Vogan et
al., 1996). Given the close juxtaposition of the two AG splice sites, and their separation
by three nucleotides, such a mechanism to increase splicing efficiency is likely to
minimize alteration to the function of the protein. The constitutive signaling phenotypes
observed in both 35S: :prosysA and 35S: :prosysB plants support this view. The functional
equivalence of prosysA and prosysB in the transgenic assay is also consistent with the
recent finding that the systemin portion of prosystemin is necessary and sufficient for the
biological activity of prosysterrrin (Dombrowski et al., 1999). Of course, our results do
not exclude the possibility that prosystemin isoforms differ in functional attributes that
cannot be discerned from the overexpression phenotype. For example, prosystemin
isoforms could differ in their intracellular location within the specific cell types in which
prosystemin is normally expressed, interaction with other components of the wound
signaling pathway, or susceptibility to proteolysis. Additional experiments aimed at
determining the precise role of prosystemin in the transduction of systemic wound signals

should provide further insight into these possibilities.

94

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98

CHAPTER 3

The Tomato Mutant jail-l Is Insensitive to Jasmonates and Defective in Defense

against Herbivores

The work presented in Figure 3.2 and Table 3.1 was done in collaboration with Bonnie

McCaig and Youfir Zhao, respectively.

99

Abstract

J asmonates are endogenous signaling compounds that regulate a wide variety of
developmental and defense-related processes in plants. Here we report the
characterization of a fast neutron-induced recessive mutant (called jasmonic _a_cid-
insensitivel -1 ) of tomato that is deficient in jasmonate signaling in many, if not all,
tissues. jai 1-1 plants exhibited normal vegetative grth but produced fruit that lacked
viable seed. Although jai 1 pollen showed reduced germination and viability, sterility of
the mutant resulted from a defect in female reproductive development. Expression
profiling of ~ 500 tomato genes identified 40 transcripts whose levels were up-regulated
by exogenous methyl jasmonate in wild-type but not jail -1 leaves. Floral tissues of the
mutant were also deficient in the expression of j asmonate-regulated defensive proteinase
inhibitor (P1) genes that are constitutively expressed in wild-type flowers. Wound-
induced local and systemic expression of PI genes and other ‘late’ wound response genes
was abolished in jail-l leaves, as was PI expression in response to the PI-inducing
compounds systemin, 12-oxo-phytodienoic acid, bestatin, and hydrogen peroxide.
Expression of ‘early’ wound response genes in jail -1 plants was either reduced or not
affected, indicating the existence of multiple wound signaling pathways that differ in
their requirement for Jail. Failure of jai [-1 plants to express jasmonate-regulated genes
was correlated with increased susceptibility to herbivores. These findings demonstrate

that Jail is an essential component of the jasmonate signaling pathway in tomato.

100

Introduction

J asmonic acid (JA) and its cyclic precursors and derivatives, collectively called
jasmonates, comprise a family of oxylipins that are synthesized from linolenic acid via
the octadecanoid pathway (Schaller, 2001; Turner et al., 2002; Weber, 2002). J asmonates
act either alone or in combination with other phytohormones to promote a wide range of
biological activities (Creelman and Mullet, 1997; Wastemack and Hause, 2002).
Jasmonates are perhaps best known for their ability to regulate plant defense responses to
attack by herbivores and some microbial pathogens (Walling, 2000; Weber, 2002). For
instance, J A synthesized in response to herbivory activates the expression of genes
involved in the production of phytochemicals that have direct toxic or anti-nutritive
effects on the herbivore, or that act indirectly to attract natural predators to the herbivore
(Creelman and Mullet, 1997; Walling, 2000; Memelink et al., 2001; Kessler and Baldwin,
2002). J asmonates are also implicated in responses to abiotic stimuli including salt and
drought stress (Creelman and Mullet, 1995), UV irradiation (Conconi et al., 1996) and
ozone exposure (Rao et al., 2000). In addition, j asmonates play a role in regulating a
number of plant developmental processes including tendril coiling (Weiler et al., 1993),
pollen and anther development (Feys et al., 1994; McConn and Browse, 1996; Sanders et
al., 2000; Stintzi and Browse, 2000; Ishiguro et al., 2001), and senescence (He et al.,

2002)

Regulation of gene expression by endogenous jasmonates requires numerous
cellular activities to integrate the biosynthesis and perception of the hormone in specific
tissues at specific times. The octadecanoid pathway for JA biosynthesis from linolenic

acid is initiated by lipoxygenase in the chloroplast, with the terminal steps of

101

cyclopentenone reduction and oxidation taking place in peroxisomes (Schaller, 2001;
Strassner et al., 2002). Genes encoding all of the J A biosynthetic enzymes have been
identified and studied in detail (Schaller, 2001; Turner et al., 2002; Feussner and
Wastemack, 2002). In contrast to knowledge about JA synthesis, much less is known
about the process by which jasmonates regulate changes in gene expression. The isolation
and characterization of j asmonate response mutants of Arabidopsis has begun to yield
valuable insight into this question (Berger, 2002; Turner et al., 2002). For instance, the

J A-insensitive coil mutant defines a gene encoding an F-box protein that is essential for
many stress-induced and developmental responses (Feys etal., 1994; Xie et al., 1998).
The C011 protein is involved in the formation of an ubiquitin-E3-ligase complex that
presumably targets transcriptional repressors of j asmonate responsive genes for
degradation by the 26S proteasome (Devoto et al., 2002; Xu et al., 2002). Forward
genetic analysis also led to the identification of JARI, which encodes an enzyme involved
in the covalent modification (e. g., adenylation) of JA (Staswick et al., 2002). Despite
these significant advances, the mechanism by which J A and its derivatives are perceived

in the cell remains to be elucidated.

A complete understanding of the jasmonate signaling pathway and its role in the
plant life cycle may be facilitated by the identification of jasmonate response mutants in
plants other than Arabidopsis. In our efforts to characterize the systemic wound response
pathway in tomato (Lycopersicon esculentum), we conducted various genetic screens that
led to the identification of mutants that are deficient in j asmonate perception (Li et al.,
2001; Howe et al., 2002). Reciprocal grafting experiments performed with these mutants

showed that jasmonate perception is essential for the recognition of the transmissible

102

wound signal in undamaged responding leaves, whereas JA synthesis is essential for the
production of that signal in damaged leaves (Li et al., 2002b). In addition to providing a
powerful tool for studying long-distance wound signaling, j asmonate perception mutants
of tomato may prove useful for studying other processes for which tomato has been used
as a model system, including fruit development (Giovannoni, 2001), peptide signaling
(e.g., systemin; Ryan, 2000), and plant interactions with herbivores, pathogens, and
nematodes (Berger, 2002; Kessler and Baldwin, 2002; Li et al., 2002a; Kennedy, 2003).
Here, we report the identification and characterization of a fast-neutron-induced mutant,
called jai l -1 , that is defective in IA signaling in many, if not all, tissues of the plant. Our
results indicate that the signaling pathway defined by jail is essential for the expression
of a subset of wound responsive genes in leaves, as well as the constitutive expression of
several defense-related genes in flowers. Our results also provide strong evidence for a

role of jasmonate in female reproductive development.

103

Materials and Methods

Plant material and treatments

Tomato seedlings (Lycopersicon esculentum Mill cv Micro-Tom and cv

Castlemart) were grown under 17 h days at 27 °C with light at 200 pmol m-2 sec-1 and 7

h at 16 °C in darkness. The original mutant 406A was backcrossed twice using wild-type
plants (cv Micro—Tom and cv Castlemart) as the recurrent pistillate parent. All

experiments involving jai l —1 were performed with homozygous (jail -I/jai1-l) lines. For

selection of mutant plants in F2 populations, resistance to inhibition of root grth and

anthocyanin accumulation by MeJ A was assayed as follows. Seeds were placed on a
piece of water-saturated filter paper in a shallow Tupperware box and allowed to
germinate in the dark at ambient temperature for 4-5 days until the emerging radicals
were 1 cm in length. The filter paper was then re-saturated with a solution of 1 mM
MeJA, which was prepared by mixing 2 pl pure MeJA (Bedoukian Research, Danbury,
CT) with 75 pl ethanol. This mixture was then diluted into 10 ml distilled water.
Following growth in the dark for approximately 24 to 36 h, phenotypes of the seedlings
were scored for the presence of jai l -1/jail -1 homozygotes using two criteria: root growth
and anthocyanin accumulation. Me] A treatment of wild-type seedlings causes root
grth inhibition and anthocyanin accumulation in the hypocotyls. In contrast, jai l-
I/jail-l roots grow in the presence of MeJ A, and hypocotyls do not accumulate

anthocyanin (see Figure 3.10).

104

MeJ A treatment of adult plants was performed by incubating three-week-old plants
(cv Micro-Tom) in a sealed Lucite box (60 x 32 x 17 cm) in which 1 pl pure MeJA was
distributed to several evenly spaced cotton wicks. For each time point of sampling, five
plants were removed from the box for extraction of RNA from leaf tissues (Li and Howe,
2001). Tobacco homworrn (Manduca sexta) feeding trials were performed as described

previously (Howe et al., 1996).

Isolation of jail -1

Fast neutron irradiation of tomato (Lycopersicon esculentum cv Micro-Tom) seed

was performed at the International Atomic Energy Agency (Seibersdorf, Austria), using

calibrated doses in the range of 12.7 and 17.9 Gy. M2 seed was collected separately from

each M1 plant. The screen for MeJA-insensitive mutants was conducted as follows.

Approximately 120 eighteen-day-old M2 seedlings were enclosed in the Lucite box

containing 5 pl MeJA applied to cotton wicks. Plants were exposed to MeJA vapor for 24
h, followed by an additional 24 h of incubation in the absence of MeJA. A small piece of
leaf tissue was then sampled from each individual plant and assayed for PPO activity as
previously described (Howe and Ryan, 1999). Plants showing reduced PPO activity were
re-tested for PI-II accumulation using a radial immunodifusion assay (Howe and Ryan,

1999)

105

Pollen germination

Freshly collected pollen was incubated in germination medium (10% sucrose, 100
mg/L boric acid, 300 mg/L calcium nitrate, 200 mg/L magnesium sulfate, and 100 mg/L
potassium chloride) for 2 h at room temperature and then analyzed for pollen tube
formation. Pollen tube length was recorded with a digital video camera (Model MDSlOO,
Kodak). Pollen grains were considered germinated if the tube length was greater than the
diameter of the grain. The germination rate was calculated as the average percent

germination from 10 arbitrarily selected microscopic fields.

cDNA microarray and RNA gel blot analysis

Tomato EST (expressed sequence tags) clones were obtained from the Clemson
Genomics University Genomics Institute (Clemson, SC). cDNA inserts were amplified
by polymerase chain reaction (PCR) in a lOO-pl reaction volume using pBluescript KS(-)
primers T3 and T7, or amplified using gene specific primers. PCR products were
precipitated with ethanol and resuspended in 25 pl of 3 x SSC (1x SSC 0.15 M NaCl and
0.015 M sodium citrate). One p1 of PCR product was run on 1% agarose gel for quality
control before arraying. DNA was printed onto arnine-coated glass slides (Telechem,
Sunnydale, CA) using an Omnigridder robot (Gene Machines, San Carlos, CA) equipped
with ArrayIt chipmaker 4 pins (Telechem). Each DNA sample was printed in triplicate on

each slide. Slides were blocked according to the recommended protocol from Telechem.

Total RNA (100 pg) isolated fi'om tomato leaves was used to synthesize the

cDNA probes for microarray analysis. The RNA was purified according to the RNAeasy

106

kit cleanup protocol (Qiagen, Valencia, CA) and was labeled by direct incorporation of
Cy3- or Cy5-conjugated deoxy UTP (Amersham Pharmacia Biotech, Piscataway, NJ)
during reverse transcription. Briefly, RNA was mixed with 6 pg oligo-dT23V (Invitrogen)
in a total volume of 16.5 pl and incubated at 70 °C for 10 min. The mixture was then
chilled on ice for 5 min and 2 pl of FluoroLink Cy3- or CyS-dUTP, 3 pl of 0.1 M DTT, 6
pl of 5X first-strand buffer, 0.5 pl of 50X dNTPs mix (25 mM dATP, dCTP, dGTP, 9
mM dTTP), and 2 pl of Superscript II (Invitrogen) were added to the mixture. Reactions
were incubated at 42 °C for 120 min. RNA was hydrolyzed by adding 0.5 p1 of RNase A
(10 mg/ml) and 0.25 p1 of RNase H (Invitrogen) at 37 °C for 30 min. The labeled cDNA
probe was first purified on a Microcon YM-30 filter (Millipore, Bedford, MA) and then
by a PCR purification kit (Qiagen). The purified, Cy3- and Cy5-labeled probes were then
combined, concentrated, and resuspended in 4 pl 10 mM EDTA, pH8.0. The labeled
probes were denatured at 95 °C for 10 min and 30 pl SlideHyb buffer 1 (Ambion, Austin,
TX) was added to the denatured probes. The mixture was then hybridized to slides at 54
°C in a hybridization chamber for 16 to 20 h. After hybridization, slides were washed
twice in 2X SSC, 0.5% SDS for 5 min at 65 °C, in 0.1X SSC, 0.2% SDS for 5 min, and
in 0.1X SSC for 5 min at room temperature. After washing, slides were dried by
centrifugation and scanned by Affyrnetrix 428 Array Scanner (Affymetrix, MA). Spot
intensities were quantified using Axon GenePix Pro 3 image analysis software (Axon,

Foster City, CA). Ratio data were extracted and normalized as previously described

(Schaffer et a1. 2001).

107

For RNA gel blot analysis, total RNA isolation and gel blot hybridization were
carried out as previously described (Li and Howe, 2001). Gels were also stained with
ethidium bromide to check for equal loading. A cDNA for tomato translation initiation

factor eIF4A(cLED1D24) was used as the loading control.

108

Results

Isolation of jasmonate-insensitive] -I

Polyphenol oxidase (PPO) and proteinase inhibitor 11 (PI-H) are defense-related
proteins that accumulate in tomato leaves in response to wounding, systemin, and
exogenous JAs (Farmer and Ryan, 1992; Constabel et al., 1995). To identify tomato
mutants affected in the j asmonate signaling pathway, we used a rapid PPO activity assay
to screen a fast-neutron-mutagenized population of Micro-Tom plants for individuals that

fail to accumulate PPO in response to exogenous MeJA (Figure 1.1a; Howe and Ryan,

1999; Howe et al., 2002). Out of a total of 24,077 M2 plants tested, eighteen putative

mutants were identified that accumulated reduced levels of both PPO and PI-II in
response to MeJA. One plant (406A) showing normal vegetative growth (Figure 3.1b)

and no detectable expression of either PPO or PI-II in response to MeJA was chosen for

further analysis. F1 plants derived from a cross between 406A pollen and a wild-type

pistillate parent accumulated normal levels of leaf PPO and PI-II in response to MeJA,

and thus were sensitive to the hormone. Analysis of 687 F2 plants showed that the ratio

of MeJA-sensitive to -insensitive plants was 519: 168 (Figure 3.1a; x2: 0.089 for 3:1

hypothesis). These results indicate that the deficiency in MeJA-induced accumulation of
PPO and PI-II in line 406A is caused by a single recessive mutation, which we designated

jasmonic goal-insensitive l -I (fail - l ).

109

Figure 3.1. jail-1 plants exhibit normal vegetative growth and insensitivity to
exogenous MeJA. (a) MeJA-induced PPO activity in tomato leaves. Fourteen-day-old
plants were treated with MeJ A and assayed for PPO activity as described in the

Material and Methods. Wild-type (WT) plants were treated with Me] A (+) or ethanol (-)

as a control. All F2 plants, which were derived from a cross between jail -I and WT (cv

Castlemart), were treated with MeJA. (b) Four-week-old WT (left) and jail —I (right)

plants. (c) Response of germinating seedlings to Me] A. WT (cv Micro-Tom), jail -I,

and F1 seeds were germinated and exposed to Mel A (+) for 2 days. A WT seedling

grown in the absence of MeJA (-) is shown as a control. Images in this figure are

presented in color.

110

(3) WT 406A x WT F2

 

 

 

 

 

 

 

MeJA —+ +
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WT jai1-1
ééiséés‘é‘fiifi 3.) 3...,
(C)
Figure3.l

111

Jasmonate-induced phenotypes of seedlings

Exogenous jasmonates have several well-documented effects on seedling growth
and development (Corbineau et al., 1988; Staswick et al., 1992), including inhibition of
root growth in tomato (Tung et al., 1996). Treatment of four-day-old wild-type seedlings
with 1 mM MeJA resulted in inhibition of root and hypocotyl growth, and accumulation

of anthocyanin in the hypocotyl (Figure 3. 1 c). To determine whether these responses are

suppressed by jail-l , an F 2 population segregating for the mutation was exposed to

MeJ A. Approximately one-quarter of the seedlings appeared to be identical to untreated
wild-type seedlings (Figure 3.10), indicating that they were MeJA-insensitive. Re-testing
of these MeJA-insensitive seedlings, after growth for three weeks in soil, showed that
they also lacked MeJA-induced PPO/PI-II accumulation in leaves. These results
demonstrate that MeJA-induced responses in both seedlings (i.e., root growth, hypocotyl
elongation, and anthocyanin production) and mature plants (PPO and PI-II accumulation

in leaves) require Jail.

Reproductive phenotypes of jail -1 plants

The gross morphology and timing of development of jai l -1 flowers was similar to
that of wild-type flowers (Figure 3.2a, b). One exception to this was that stigma of mutant
flowers protruded from the anther cone during later stages of flower development (Figure
3.2a, b). Although stigma exertion can reduce the efficiency of self-pollination and fruit
set, the number of fruit produced by self-fertilized jai l -1 plants was comparable to that of

wild-type. The size of immature (green) jail -1 fruit was also similar to wild-type (Figure

112

Figure 3.2. Reproductive development of wild-type and jail-l plants. (a) Flowers of
wild-type (WT) plants. (b) jail —1 flowers. Arrowheads indicate the protruded stigma. (c)
Developing fruits of WT plants. ((1) Deve10ping fruits of jail-l plants. (e) Mature WT
(top) and jail -1 (bottom) fruits. (t) Enlargement of the jai I -1 fruit showing the small,
undeveloped seeds. (g and h) WT and jail -I pollen, respectively, after germination.

Images in this figure are presented in color.

113

 

Figure 3.2

114

3.20, d), as was the general timing of fruit ripening. The size of ripe jail -1 fruit, however,
was significantly less than ripe fruit from wild-type plants (Figure 3.2e). The average
weight of ripe fruit from wild-type and mutant plants was 5.4 i 1.1 g and 2.9 :l: 0.4 g,
respectively (mean :1: SD; n = 100 fruit/ genotype; P<0.0001). Wild-type fruit yielded
approximately 30 seed, each having a dry weight of approximately 2.5 mg. Ripenedjail—
1 fruit contained numerous undeveloped seeds having a dry weight < 0.7 mg (Figure 3.21).
Based on these observations, we estimated that the yield of mature seed from jail -1 fruit
was <0.01% of that from wild-type fruit. Examination of several hundred fruit from jail -
l/jail-l plants obtained from three successive backcrosses showed that the sterility

strictly co-segregated with the MeJA-insensitive phenotype of seedlings and leaves.

Reciprocal crosses between wild-type and jail -1 plants showed that sterility of
the mutant results from a defect in female reproductive development (Li et al., 2001).
Nevertheless, because Arabidopsis mutants that are impaired in J A synthesis or J A
perception are male sterile (Feys et al., 1994; McConn and Browse, 1996), we examined
jail -1 plants for possible defects in pollen development. Staining of pollen grains with
Alexander’s triple stain (Alexander, 1969; 1980) showed that the proportion of non-
aborted pollen grains (i.e., containing cytoplasm) fiom similarly-staged wild-type and
mutant anthers was approximately 94% and 67%, respectively. Fluorescien
diacetate/propidium iodine co-staining (Oparka and Read, 1994) fitrther revealed a
significant reduction in the viability of mutant pollen (28% viability) compared to that of
wild-type pollen (82% viability, P<0.0001). The general trend toward reduced vigor of
jail -I pollen was reflected in measurements of in vitro germination rates. In three

independent experiments, the germination rate of jail -I pollen ranged between 9 and

115

Figure 3.3. Accumulation of MeJA-responsive transcripts in wild-type and jail-l
leaves. Three-week-old plants (cv Micro-Tom) were exposed to MeJA vapor and leaf
tissues harvested for RNA extraction at various times (h) thereafter. RNA was also
prepared from untreated plants (0) for control. RNA gel blots were hybridized to cDNA

probes indicated on the right. eIF 4A was used as a loading control.

116

wild-type jai1-1

 

012481224 012481224

 

 

PI-I
PI-II
CDI
TD
....-.. “a..-“ .-.... LOXD
' ..... in...“ Aos2
.— ._ an?” t- : OPR3
“.m .Cuuuwnu Prosys
Vfi-~~~“-- u--,”“, eIF4A

Figure 3.3

117

10%, compared to >55% germination of wild-type pollen from flowers at a similar stage.
Despite this reduced germination, the overall morphology and tube length of germinated
jail -I pollen was comparable to that of germinated wild-type pollen (Figure 3.7g, h).
These observations indicate that the defects in jail-l pollen development, although

significant, are not sufficient to cause male sterility.

Jasmonate-induced gene expression in jail -1 plants

To investigate the effect of jail-1 on jasmonate-induced gene expression in leaves,
RNA blot analysis was used to determine the steady state level of several transcripts
whose expression is known to be wound- and JA-inducible in wild-type leaves (Figure
3.3). One set of genes, represented by Pl-I, PI-II, cathepsin D inhibitor (CD1), and
threonine deaminase (TD), was expressed at relatively low levels in untreated (0 h) wild-
type plants. Upon treatment with Me] A, these mRNAs began to accumulate within 2 to 4
h, and reached maximum levels 12 h after treatment. Pl-l, PI-Il, CD1, and TD mRNA
accumulation was undetectable in MeJA-treated jail -1 plants, with the exception that
very weak expression (< 2% of wild-type) was observed at the 24 h time point. Exposure
of wild-type plants to Me] A also stimulated moderate and transient accumulation of
transcripts encoding the octadecanoid pathway enzymes lipoxygenase D (LoxD; Heintz
et al., 1997), allene oxide synthase 2 (AOSZ; Howe et al., 2000), lZ-oxo-phytodienoic
acid reductase 3 (OPR3; Strassner et al., 2002), as well as prosystemin (Prosys; J acinto et
al., 1997). In contrast to P1 and TD genes, the steady state level of LoxD, AOS2, OPR3,

and Prosys mRNA was reduced but not abolished in untreated (0 h) jail -1 plants.

118

 

Table 3.1. List of genes that are differentially regulated in wild-type and jail-1

 

 

 

plants by exogenous MeJA.
GenBank Description Expression Ratio
accession no.

wild-type jail -I
A1485] 16 Threonine dearninase 41.88 ND
Q10712 Leucine arninopeptidase 32.98 1.33
AI485529 Unknown 25.89 0.66
A1486] 73 Similar to protein translation inhibitor 19.84 ND
AI897750 Miraculin;Kunitz-type protease inhibitor 14.83 0.76
AI490282 Unknown 14.32 0.91
AW037833 Metallocarboxypeptidase inhibitor 12.70 ND
AI487422 Pto-responsive gene 1 12.46 1.00
K0329] Proteinase Inhibitor II 12.4] 0.75
AI488657 Cathepsin D inhibitor 11.18 ND
AW649919 Putative GDSL-motif lipase/acylhydrolase 10.95 1.28
AW624058 Lipoxygenase A-9 10.79 1.37
U09026 Allene oxide cyclase 9.23 0.84
Z12838 Polyphenol oxidase F 8.38 1.28
AI4903 l 8 Unknown 8.24 1 .01
AW0925 79 Nucleoside diphosphate kinase 5.73 1.00
A1897184 Unknown 5.50 0.87
K03290 Proteinase Inhibitor I 5.46 0.84
AI486025 4-coumarate:coenzyme A ligase 5.17 1.1 1
AW040669 Thioredoxin M-type 3 chloroplast precursor 5.13 0.95
AI483889 NAC domain protein NAC2 4.77 0.87
AI486916 Proteinase inhibitor PID 4.53 1.08
AI486546 Wound-inducible carboxypeptidase 4.47 1.09
AI489221 WIZZ 4.32 1.03
AW032472 Unknown 4.20 0.65
Z21793 DAHP-synthase 2 4.18 1.17
AW034958 12-oxophytodienoate reductase 3 4.16 1.07
AI771886 RD2 protein 4.00 1.13
AI483527 Unknown 3.99 0.92
AW220064 Glutathione S-transferase 3.87 1.41
AF 230371 Allene oxide synthase 2 3.76 0.99
AW648549 Indole-3-glycerol phosphate synthase 3.46 1.15
U37840 Lipoxygenase D 3.38 1.01

 

119

 

Table 3.1. cont’d.

 

GeneBank Description Expression Ratio

 

accession no.
wild-type jail -I

 

AI895589 Allene oxide synthase 1 3.04 0.95
AI897620 Putative prephenate dehydratase 2.91 1.20
M84800 Prosystemin 2.91 1.18
AI483536 TMV response-related protein 2.67 0.86
BE459901 Caffeoyl-CoA O-methyltransferase 2.58 0.65
AW03 8929 Cathepsin B-like cysteine proteinase 2.48 1.12
AI485737 Glucosyl transferase 2.35 0.98
M488332 Unknown 2.19 0.94
AI489097 Putative embryo-abundant protein 1.4] 2.70
AI488782 Cold-inducible glucosyl transferase 1.30 6.47
AI484542 Eukaryotic initiation factor 4A-2 1.01 0.98

 

Three-week-old wild-type and jai l-l plants (cv Micro-Tom) were treated with MeJA
or ethanol (mock) for 8 h. Leaf tissue was harvested for RNA isolation after the
treatment. RNA from ethanol mock untreated plants was also prepared as controls. For
microarray analysis, customer-made slides were hybridized simultaneously to probes
derived from RNA isolated from mock and MeJA-treated plants. Numbers (average of
two independent biological replicates) represent the expression ratio of Mel A and
mock treated samples. Genes that were differentially regulated (up-regulated > 2-fold
in response to MeJA in either wild-type or jai [-1 plants) are listed and their GeneBank
Accession numbers and annotation given. The cDNA for eukaryotic initiation factor
4A-2 (AI484542) was included in the array as a spiking control. ND, not detectable.

 

120

Moreover, a low level of LoxD, AOSZ, OPR3, and Prosys mRNAs was maintained in

jai [-1 plants throughout the time course of MeJA treatment. These results suggest the
existence of two classes of MeJA-responsive genes in tomato leaves: those genes whose
induction by MeJ A is completely dependent on Jail (e. g., PI-l, PI-II, CD1, and TD), and
those whose expression is only partially dependent in Jail (e. g., LoxD, AOS2, OPR3, and

Prosys).

DNA microarray analysis was used to further examine the spectrum of genes
whose expression in response to MeJA is abrogated by jail-1 . For this purpose, we
constructed a microarray slide containing 607 tomato cDNAs corresponding to
approximately 500 unique genes involved in various aspects of herbivore and pathogen
defense, signal transduction, lipid metabolism, and hormone synthesis. To identify
jasmonate-responsive genes among this collection of sequences, the slide was hybridized
simultaneously to probes derived from mRNA isolated from mock— and MeJA-treated (8
h) wild-type leaves. This time point was chosen because RNA blot analysis indicated that
it maximizes the chance of detecting MeJA-responsive genes with different response
kinetics (Figure 3.3). This experiment identified 40 genes that were up-regulated > 2-fold
in response to MeJA (Table 3.1). Included among these were all eight of the genes
analyzed by RNA blot analysis (Figure 3.3), as well as other previously identified JA-
responsive genes including leucine amino peptidase (LAP; Chao et al., 1999), polyphenol
oxidase (PPO; Constabel et al., 1995), metallocarboxypeptidase inhibitor (MCPI;
Villanueva et al., 1998), allene oxide cyclase (A 0C; Hause et al., 2000), lipoxygenase A

(LoxA; Beaudoin and Rothstein, 1997), a wound-inducible serine carboxypeptidase (CP;

121

Figure 3.4. Developmental accumulation of transcripts encoding defense-related
proteins. Tissues from wild-type (WT, cv Micro-Tom) and jail -1 plants were
harvested for RNA extraction. R, roots; P, petioles; L, leaves; and F, young flower buds
(< 10 mm). cDNA of several defense-related genes were used as probes. A blot was

also probed with eIF 4A as a loading control.

122

 

 

PM

PM!

CDI

Pros ys

 

a .... o . «— «~- a .- eIF4A

Figure 3.4

123

Moura et al., 2001), and AOS] (Sivisankar et al., 2000). To determine whether MeJA-
induced expression of these genes involves Jail, slides were hybridized to probes derived
from mock— and MeJA-treated mutant plants. Of the 40 MeJA-responsive genes
identified in wild-type, none of them were up-regulated by MeJA in jail-1 (Table 3.1).
Interestingly, transcript levels for two genes encoding putative glucosyl transferases were

up-regulated by MeJA in jail -1 plants but not in wild-type plants (Table 3.1).

Effects of jail -I on the developmental expression pattern of PI genes

Several genes that are induced by wounding and J As in leaves have been shown
to be constitutively expressed in reproductive organs (Wingate and Ryan, 1991; Pef'ra-
Cortes et al., 1991; Benedetti et al., 1995; Chao et al., 1999). The accumulation of high
levels of JAs in flowers (Hause et al., 2000) and young fruit (Fan et al., 1998) of tomato
suggested to us that constitutive expression of wound-responsive genes in reproductive
organs is caused by constitutive activation of the JA response pathway in these tissues.
To test this hypothesis, we compared the organ-specific expression of PI genes in wild-
type and jail -1 plants (Figure 3.4). The results showed that Pl-I, PM], and CD1 mRNAs
accumulated to high levels in wild-type flowers, whilst little or no expression of these
genes was detected in jai l -1 flowers. Consistent with this, the level of PM] protein in
wild-type flowers was 209 A: 42 pg/ml crude extract, but undetectable in mutant flowers.
In contrast to P1 genes, constitutive accumulation of Prosys mRNA in flowers was not
significantly affected by jai l-l . This finding indicates that although Prosys expression in
leaves is partially dependent on JA signaling (Figure 3.3; J acinto et al., 1997), expression

of prosystemin in floral tissues is not.

124

Figure 3.5. Response of wild-type and jail-1 plants to mechanical wounding.
Tomato seedlings (cv Castlemart) at the two-leaf stage were wounded with a hemostat
on the lower leaves. Total RNA was isolated separately from the lower wounded (local)
and the upper unwounded (systemic) leaves at various times after wounding. RNA was
prepared from unwounded plants (0) as a control. cDNA probes representing different
classes of wound responsive genes (see text for details) were used for hybridization as

shown on the right. elF 4A was the loading control.

125

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126

Expression of wound response genes in jail -1 plants

Recent studies indicate that wound responsive genes in tomato can be divided into
at least two classes that differ with respect to their temporal and spatial pattern of
induction (Ryan, 2000). Transcripts of so-called ‘late’ response genes, including
defensive PIs, begin to accumulate both locally and systemically about 2 h after
wounding, and reach maximal levels 8 to 12 h after wounding. By contrast, mRNAs
transcribed from ‘early’ response genes accumulate rapidly (within 1 h) and transiently in
response to wounding, and include genes encoding J A biosynthetic enzymes as well as
other signaling components. To investigate the role of Jai l in wound-induced gene
expression, we determined the temporal and spatial (i.e., local and systemic) expression
pattern of representative early and late response genes in wild-type and jai [-1 plants.
Local and systemic expression of the late response genes (PI-I, PH] and CD1) was
detected in wild-type plants within 2 h of wounding, with transcript levels reaching
maximal levels about 10 h after wounding (Figure 3.5). Transcripts representing four
early response genes (LoxD, AOS], OPR3, and Prosys) accumulated in wild-type leaves
2 to 4 h after wounding, and were induced to relatively low levels in the unwounded
systemic leaves. Wounding of wild-type plants also resulted in very rapid (within 0.5 h)
and transient local and systemic accumulation of mRNA encoding a putative mitogen-
activated protein kinase (WIPK; Seo et al., 1995, 1999). Wound-induced expression of
late response genes (PI-I, PI-II and CD1) was undetectable in both local and systemic
leaves of jai l -1 plants. In contrast, local and systemic accumulation of WIPK mRNA was
not affected in the mutant, indicating that wound induction of this gene does not require

J A signaling. Wound- induced expression of other early response transcripts (LoxD,

127

 

Table 3.2. Response of wild-type and jail-1 plants to PI-inducing factors.

 

 

 

Elicitors PI-II (pg/ml juice)a

Wild-type jail -I

Bufferb 14 i 10 ND
Systemin 124 :1: 24 4 i ll

OPDA 166 d: 21 ND

Bestatin 126 i 14 ND

Glucose 74 j: 12 ND

Glucose + oxidase 145 :l: 35 ND

 

a Fifteen-day-old wild-type and jail -I seedlings were tested for the accumulation of
PHI in leaves using a radial immuno-diffusion assay. ND, not detectable. The
detection limit of the assay is ~ 5 pg/ml leaf juice.

b Seedlings were supplied through their cut stems with a buffer control (15 mM
sodium phosphate, pH 7.0), or with elicitors dissolved in the buffer: systemin (5 pmol
per plant), OPDA (10 nmol per plant), bestatin (50 nmol per plant), glucose (50 pM),
and glucose (50 pM) plus glucose oxidase (0.01 U per plant). Data represent the
mean i SD of six plants.

 

128

AOS], OPR3, and Prosys) was also evident in jail -1, although transcript levels for these
genes were reduced to approximately 20% of wild-type levels. These results indicate that
the J A signaling pathway defined by jai l is essential for wound-induced expression of
defensive PI and other late response genes. However, this pathway appears to function
together with a J A-independent wound signaling pathway to control the expression of

early wound response genes.

Responses of jail -1 plants to PI-inducing compounds

Two general classes of PI-inducing compounds that act at different points in the
jasmonate signaling pathway have been described. One class of elicitors functions either
to activate the octadecanoid pathway leading to J A biosynthesis (e. g., systemin) or as
metabolic precursors of JA (e. g., 12-OPDA). Because jail -1 plants are insensitive to JA,
we hypothesized that these compounds would not induce PI expression in the mutant.
Indeed, measurement of PHI protein accumulation in response to exogenous systemin
and 12-OPDA confirmed this prediction (Table 3.2). A second class of compounds

induces PI expression by acting downstream of J A. Included within this group are the

aminopeptidase inhibitor, bestatin, and the reactive oxygen species, H202 (Schaller et al.,

1995; Orozco-Cérdenas et al., 2001). We found that neither bestatin nor a

glucose/ glucose oxidase mixture capable of generating H202 induced PI-H accumulation

in jail -1 plants (Table 3.2). These results indicate PI expression in response to bestatin

and H202 requires the action of Jail .

129

jail -1 plants are compromised in resistance to herbivores

Previous studies have established that the octadecanoid pathway for J A
biosynthesis plays an important role in defense of cultivated tomato against a broad
spectrum of arthropod herbivores (Howe et al., 1996; Li et al., 2002a). To determine
whether JA signaling is required for anti-herbivore defense responses, wild-type and jai 1-
1 plants were challenged with Manducta sexta (tobacco homworm) larvae for five days.
Homworrn feeding on wild-type plants resulted in PI-II accumulation in damaged (176 :l:
17 pg/ml, n=9 leaves) and undamaged (205 :t 16 pg/ml, n=9 leaves) leaves. In contrast,
very low levels (< 20 pg/ml) of PHI were detected in damaged jail -1 leaves. RNA blot
analysis of the same tissues showed that transcript levels for several defense-related
genes were induced by homworm attack both locally and systemically in wild-type but
not in jai [-1 plants (Figure 3.6). In addition to these effects on host gene expression, we
also found that jai [-1 plants were defoliated much faster than wild-type plants. The
average weight of larvae fed on jail -1 plants (71.9 :t 25 mg, n=35 larvae) was
approximately twice that of larvae fed on wild-type plants (44.5 :1: 4 mg, n=30 larvae) for
the same period of time. These results indicate that foliage from jail -1 plants is a better

food source for homworm larvae.

130

Figure 3.6. Accumulation of defense-related transcripts in response to tobacco
hornworm attack. Newly hatched homworm larvae were allowed to feed on wild-type
(cv Micro-Tom) and jail -1 plants for 5 days. Total RNA was then isolated separately
from homworm damaged (L) and intact (S) leaves of the same plant. RNA from leaf
tissue of the same plants immediately before the trial was also prepared as a control (C).

cDNA probes used are indicated on the right. elF 4A was used as loading control.

131

wild-type jai1-1

 

PM

PM!

_ CDI

 

TD

drummed“ GIF4

Figure 3.6

132

Discussion

Forward genetic analysis of the jasmonate signaling path way

Forward genetic approaches have been instrumental in dissecting the jasmonate
signaling pathway in Arabidopsis. Various genetic screens for jasmonate responsive
mutants have revealed a number of loci that play important roles in the jasmonate signal
transduction pathway (Staswick et al., 1992; Feys et al., 1994; Berger et al., 1996; Ellis
and Turner, 2001; Hilpert et al., 2001; Xu et al., 2001). Cloning the corresponding genes
and subsequent biochemical analysis of the gene products have begun to yield valuable
insight into the mechanism of j asmonate signal transduction and the interaction of
jasmonate signaling with other cellular processes (Xie et al., 1998; Ellis et al., 2002;

Staswick et al., 2002).

Whereas additional genetic screens in Arabidopsis are likely to further our
understanding of the jasmonate response pathway, forward genetics in tomato constitutes
another powerful approach expand to our knowledge in this important field of research.
The rational lies in the fact that tomato, the most advanced model for species bearing a
fleshy berry type of fruit, has a well studied and distinctive wound repose pathway. The
best characterized wound response genes in tomato, including P15 and PPO, are absent in
Arabidopsis (van der Hoeven et al., 2002). The systemin pathway that is essential for the
systemic wound responses in tomato can not be detected in Arabidopsis. Therefore,
studies aimed at elucidating the jasmonate signaling pathway in tomato promise to
identify new components of the pathway and to complement our knowledge of the

functions of genes already identified.

133

Tomato is well suited for forward genetics thanks to recent technical and genomic
advances. Adoption of the determinate miniature L. esculentum cultivar Micro-Tom
provided further benefits for analysis of the wound responses. Meissner et a]. (1997)
reported that Micro-Tom has several attributes that make it ideal for genetic analysis.
Included among these are a short life cycle, growth of populations at high density, and
amenability to high frequency of transformation by Agrobacterium. We have found that
Micro-Tom is well suited for the study of wound— and herbivore- induced responses. For
example, brief (<10 min) feeding by a single tobacco homworm on a lower leaf triggered
a >50-fold increase in Pl-Il mRNA in upper undamaged leaves of the plant (Howe et al.,
2000). In the current research, we report the isolation and characterization of a tomato
loss of function mutant, jail -I, from a fast-neutron mutagenized population of Micro-

Tom plants.

Jail is a positive regulator of jasmonate responses

The jail-1 mutant was isolated in a screen for plants that fail to accumulate
MeJA-induced PPO and PI—II in leaves (Figure 3.1.a). Further characterization of jai l-l
plants indicated that the mutation defines a positive regulator of j asmonate signaling in
most if not all tomato tissues. This conclusion was first evident from expression profiling
of approximately 500 genes contained on a custom DNA microarray. In wild-type leaves,
MeJA treatment increased the mRNA level of 40 genes (Table 3.1). MeJA induction of
40 genes was abolished by the jai 1-1 mutation, suggesting that expression of most if not
all J A-responsive genes require Jail. Furthermore, wound- and herbivore-induced

defense gene activation was also abrogated in jail -1 leaves, which correlated with

134

enhanced susceptibility of jail -1 leaves to insect attack (Figure 3.5, 3.6). In germinating
seedlings, Jail is required for MeJA-mediated root growth and hypocotyl elongation

inhibition, and anthocyanin accumulation (Figure 3.10).

With respect to reproductive development, the timing of flower and fruit onset in
jail -1 plants appears normal. Nevertheless, jail -1 plants were sterile and only produced
nonviable seed with arrested embryos (Li et al., 2001; McCaig B and Howe GA,
unpublished data). We found that pollen grains from jail -1 plants have reduced viability
and in vitro germination. Nevertheless, a small portion of jail -I pollen (~2% of total
pollen) appears to germinate and grow normally (Figure 3.2g, h). This finding is
consistent with the observation that pollen collected from jail -1 plants is capable of
inducing normal fruit and seed set when manually applied to emasculated wild-type
pistils (Li et al., 2001). These results indicate that while reduced pollen fitness might
contribute to the sterility of jail-l plants, the jasmonate signaling pathway is not strictly

required for pollen development in tomato.

Reciprocal crosses between wild-type and jail-1 plants clearly showed that the
sterility of the mutant lies in female reproductive development (Li et al., 2001). A
proposed role for jasmonate signaling in female reproductive development in tomato
contrasts the well-documented studies in Arabidopsis where J A biosynthesis and
perception are essential for male, but not female, garnetophyte development (Feys et al.,
1994; McConn and Browse, 1996; Sanders et al., 2000; Stintzi and Browse, 2000;
Ishiguro et al., 2001). A role for jasmonate in female reproduction in tomato is consistent
with several observations. In a number of plant species including tomato, pistil and ovary

as well as developing embryo contain relatively high levels of j asmonates (Wilen et al.,

135

1991; Creelman and Mullet, 1997; Hause et al., 2000). Also, many JA biosynthetic genes
have been found to be highly expressed in female flower organs (Hause et al., 2000;
Sanders etal., 2000; Wastemack and Hause, 2002). In addition, a number of jasmonate-
inducible genes in leaves are highly expressed in female reproductive tissues (Pena-
Cortés et al., 1991; Kim et al., 1998; Chao et al., 1999). We found that the constitutive
expression of defense genes in reproductive organs was abolished in jail -1 flowers
(Figure 3.4). While it is conceivable that these proteins might play defensive roles, they
might also serve as storage proteins or be involved in supply of nutrients from maternal
tissues to the developing embryos. Further characterization of jai l mutants with regard to
pistil- or ovary-specific gene expression will likely provide clues for how jasmonate

signaling is coupled to female fertility in tomato.

Jail -dependent and -independent wound-signaling path ways in tomato

Based on their genetic requirement for Jail, the wound response genes in tomato
can be classified into at least three groups, i.e. the late wound response genes that encode
defensive proteins, the early response genes that include J A biosynthetic genes, and the
third group of genes that rapidly respond to wounding in a jasmonate-independent
manner (Figure 3.5). The late response genes were strongly induced by wounding both
locally and systemically in wild-type plants, with maximal induction at about 10 h after
wounding (Figure 3.5). The activation of the late response genes was completely
abolished in jail -1 plants, indicating a requirement for JA signaling in the activation of
these genes. Furthermore, the induction of late response genes by several chemical

elicitors was also completely abolished by the jail-l mutation (Table 3.2), indicating that

136

the signaling events mediated by these elicitors converge at Jail to orchestrate induction
of the late response genes. Expression of the early response genes was rapidly induced by
wounding in wounded leaves of wild-type plants though systemic induction of these
genes was rather weak (Figure 3.5). In jail -1 plants, wound induction of these genes was
much reduced compared to wild-type plants (Figure 3.5), suggesting that the jasmonate
pathway functions together with a J A-independent wound signaling pathway to control
the expression of early wound response genes. This finding is in keeping with the
previous observation that activation of early response genes was only partially dependent
on JA synthesis (Howe et al., 2000). The third group of genes, (e. g. WIPK), was very
rapidly (< 0.5 h) induced by mechanical wounding both locally and systemically in wild-
type as well as jail -1 plants. The apparent jasmonate perception-independent expression
of WIPK indicates the existence of yet another jasmonate-independent signaling pathway

in mediating the wound responses in tomato.

Although jasmonates play a central role in the wound response, jasmonate-
independent wound responses have previously been observed in plant species such as
tomato (O'Donnell et al., 1998; Howe et al., 2000) and Arabidopsis (Rojo et al., 1998;
Leén et al., 2001; LeBrasseur et al., 2002). Based on the differential induction kinetics of
the wound responsive genes, our results support a scenario in which jasmonate-
independent wound signaling pathways could be responsible for the induction of WIPK
Giigure 3.5) and other genes, which might lead to increased jasmonate levels (Seo, et al.,
1995; 1999). The jasmonate-independent pathways may function together with the Jail-
mediated jasmonate signaling pathway to activate the expression of early wound response

genes, which might contribute to further mobilization of the jasmonate pools (Ryan,

137

2000). These early signaling events might ultimately lead to the induction of the late
response genes whose expression requires jasmonate perception (Figure 3.5). A variety of
signal molecules have been implicated in wound signaling. Included among them are
physical signals such as electrical pulses (Wildon et al., 1992) and hydraulic waves
(Malone and Alarcon, 1995), and chemical signals such as systemin (Pearce et al., 1991),
ABA (Pefia-Cortés et al., 1991), and ethylene (O'Donnell et al., 1996). Although the
signaling pathways mediated by these signals are important for optimal wound responses,
none has been explicitly demonstrated as the primary wound signal. The interaction
between these and the jasmonate signaling pathways is also need to be further elucidated.
Isolation and characterization of the jail -l mutant, therefore, offers an opportunity to
gain greater insight into the genetic complexity of the wound response pathways and the
mechanism by which different signaling pathways interact to regulate wound responsive

genes in plants.

138

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145

CHAPTER 4

Genetic Analysis of Wound Signaling in Tomato: Evidence for a Dual Role of

Jasmonic Acid in Defense and Female F ertility

The work presented in this chapter has been published:

Li L, Li CY, Howe GA (2001) Plant Physiol. 127, 1414—1417.

The experiment described in Table 4.1 was done in collaboration with Chuanyou Li.

146

Abstract

Tomato provides an attractive system in which to study jasmonate biosynthesis
and signaling in regulating plant defense against herbivores and other processes. To
genetically dissect the jasmonate pathway, several screens have been conducted in tomato
for mutants that block wound-, prosystemin-, or MeJA-induced expression of
downstream target genes (Lightner et al., 1993; Howe and Ryan, 1999; Chapter 3).
Included among these mutants are jail -I and 124A (jail—2) that are defective in MeJA-
induced responses. In the current chapter, we demonstrated that the two jasmonate
signaling mutants are genetically allelic. Therefore, the jai 1 mutations define a single
locus required for jasmonate signaling in tomato. Furthermore, reciprocal crosses to wild-
type plants revealed that jail plants are female sterile, indicating a role for jasmonate

signaling in female reproductive development in tomato.

147

Introduction

Genetic analysis of the wound response pathway in tomato (Lycopersicon
esculentum) indicates that prosystemin and systemin are upstream components of a
defensive signaling cascade that involves complex regulation of jasmonic acid (JA)
biosynthesis and the ability of cells to perceive and respond to JA. Recent identification
of J A response mutants provides evidence for the hypothesis that the J A transduction

pathway also plays an important role in female reproductive development.

Many plants respond to insect attack and wounding by synthesizing an array of
phytochemicals that decrease the ability of herbivores to colonize, feed, or reproduce on
the plant (Green and Ryan, 1972; Karban and Baldwin, 1997). Wound-inducible
proteinase inhibitors (PIS) provide an attractive model system in which to study the signal
transduction pathways that regulate this form of defense. In tomato, damage to a single
leaflet by mechanical wounding or herbivory results in localized and systemic expression
of two Ser PI-encoding genes (PI-I and PH!) within about 2 h (Ryan, 2000; Howe et al.,
2000). These proteins can accumulate to high levels in leaves of the damaged plant,
where they play a defensive role by inhibiting digestive proteases of some lepidopteran
insects. In their pioneering study of wound-inducible P15 30 years ago, Green and Ryan
(1972) proposed that chemical signals generated at the site of wounding traverse the
vascular system to activate the systemic expression of P15. Although many of the signals
involved in this response have been identified, relatively little is known about the

mechanisms by which they are produced and transported between cells.

148

A unique component of the wound response pathway in tomato is the peptide
signal systemin and the precursor protein prosystemin, from which it is derived (Pearce et
al., 199]; Mchl et al., 1992). Tomato prosystemin is encoded by a single gene whose
primary transcript is alternatively spliced to generate two active forms of the protein (Li
and Howe, 2001). Several lines of genetic evidence indicate that prosystemin is essential
for wound-induced expression of PI and other defense-related genes. First, transgenic
plants expressing an antisense prosystemin cDNA are deficient in wound-induced
systemic expression of PI genes (McGurl et al., 1992). Second, overexpression of
prosystemin from a 35S: :prosys transgene constitutively activates PI expression in
unwounded plants (McGurl et al., 1994). Third, mutations that suppress 35S::prosys-
mediated signaling block wound induction of P13 (Howe and Ryan, 1999). It has been
proposed that systemin functions as a mobile wound signal following its proteolytic
release from prosystemin (McGurl et al., 1992). Expression of PI genes in tomato leaves
in response to wounding and systemin is mediated by J A, a terminal product of the
octadecanoid pathway (Farmer and Ryan, 1992; Creelman and Mullet, 1997). This model
has been refined to reflect the fact that wound- and systemin-induced expression of P15
involves synergism between J A and ethylene (O'Donnell etal., 1996). Recent studies
provide evidence that reactive oxygen species function downstream of J A to amplify
wound- and systemin-induced responses (Orozco-Cardenas et al., 2001). Due to space
limitations, the reader is referred to recent reviews for a detailed discussion of the wound-

signaling pathway (Bowles, 1998; Ryan, 2000; Walling, 2000; Leon et al., 2001).

149

Genetic Analysis of Wound Signaling

We are using tomato as a model system for genetic dissection of signaling
pathways that regulate wound responses and, more broadly, defense against herbivores.
To further define the function of prosystemin and systemin in the wound response, we
conducted a screen for mutations that suppress 35S::prosys-mediated expression of
downstream target genes (Howe and Ryan, 1999). We identified 13 independent mutants,
designated spr (suppressed in prosystemin-mediated responses). Eight mutants define
four genetic complementation groups called Sprl, 2, 3, and 4. Two mutants define new
alleles of def] , a JA-deficient mutant that is compromised in wound-inducible PI
expression and resistance to Manduca sexta larvae (Lightner et al., 1993; Howe et al.,
1996). The three remaining mutants were sterile and thus were not further characterized in
the initial study. Mutations in Defl, Sprl , and Spr2, in addition to suppressing the action
of 35S::prosys, impair wound- and systemin-induced PI expression. This finding provides
strong genetic evidence that prosystemin is an essential upstream component in the
wound response pathway. The ability of def] , sprl , and spr2 plants to respond to
exogenous J A suggests that these mutations affect processes required for JA biosynthesis
or accumulation (Figure 4.1). Support for this interpretation comes from the finding that
def] plants are deficient in J A accumulation in response to wounding and systemin

(Howe et al., 1996).

Identification of mutants that are impaired in JA perception would provide a

valuable tool to further elucidate the mechanism of wound signaling and its relationship

150

Figure 4.1. Proposed action of mutations in the wound response pathway. The
signaling pathway depicted is consistent with the model proposed by Farmer and Ryan
(1992). All mutants listed are deficient in wound-inducible systemic expression of P15
and also lack PI expression in response to systemin and 35S: :prosystemin. defl, sprl,
spr2, and spr5 plants are responsive to applied MeJA and JA, whereas jail plants are

insensitive to these signals. See text for details.

151

 

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152

to induced defense. Toward this goal, we screened a fast-neutron-mutagenized population
of tomato (0v Micro-Tom) for plants that fail to express polyphenol oxidase and PM]
upon exposure to gaseous methyl-IA (MeJA; Li et al., 2001). One mutant, designated
jasmonic acid-insensitive l-1 (jail-l), completely lacked these proteins upon treatment
with MeJA or wounding. jai [-1 plants displayed normal vegetative growth but produced
fruit that lacked mature seed. In over 1,000 jail -1 fruit examined, only two viable seeds
were recovered. Reciprocal crosses to wild-type showed that jail-l is female-sterile; it

failed to set seed following pollination with wild-type pollen but readily pollinated and

fertilized wild-type pistils. F 1 plants derived from this cross were fully responsive to

JA/MeJA and fertile. In a segregating population (108 F 2 plants), the JA-insensitive and

sterile phenotypes always co-segregated as if conditioned by a single recessive mutation.

The female-sterile phenotype of jai 1 -1 plants prompted us to investigate the
sterile spr lines that were previously generated by ethyl methane sulfonate-mutagenesis
(see above; Howe and Ryan, 1999). Attention was focused on two lines, 124A and 436G,
that developed flowers but produced either no fruit or fruit containing no viable seed.

Reciprocal backcrosses to wild-type indicated that both lines were female-sterile.

Analysis of F 2 populations derived from these crosses showed that one-quarter of the
progeny lacked wound-inducible PI-II expression both in the wounded leaf and the
undamaged systemic leaf (Table 4.1). Wound-insensitive 436G plants accumulated

normal levels of PI-II in response to exogenous MeJA (Table 4.1), similar to the

phenotype of def] , sprl, and spr2 plants (Figure 4.1). Complementation tests showed

153

 

Table 4.1. Proteinase inhibitor 11 accumulation in response to wounding and
MeJA.

Values indicate the mean i SD of PHI levels (pg/ml leaf juice) in leaf tissue of
wild-type and two mutant lines that are suppressed in 35S:.°prosys-mediated
signaling.

 

 

Genotypea Unwounded Local Systemic MeJAb

Wild-type 6 i 5 77 :1: 5 72 i 4 140 :1: 7
436G (spr5) 5 :t 5 3 i 5 ND 135 i 11
124A (jail-2) ND ND ND ND

 

a F 2 populations segregating for spr-5 (line 436G) or jail -2 (line 124A) were
wounded with a hemostat on the lower leaf, and PH] levels were measured 24 h later
in both the wounded leaf (Local) and the distal undamaged leaf (Systemic). The ratio
of woun -responsive to wound-insensitive plants was 59:21 for spr5 and 4022118 for
jail -2 (x = 0.067 and 1.477, respectively, for the 3:1 hypothesis). Data are shown
for wound-insensitive F2 plants and unwounded control plants grown in the same
flat. ND, not detectable.

b spr5 and jai [-2 plants were selected by screening the corresponding segregating F 2
population for seedlings (l l-d-old) that do not accumulate PI-II in wounded
cotyledons. Eighteen-day-old selected plants were treated with MeJ A as previously
reported (Li and Howe, 2001), and PI-II levels were measured in leaves 24 h later.

 

154

 

Table 4.2. Genetic complementation tests between jail-l and 124A.

 

 

Male Parent Female Parent JA sensitivea JA 12 b
insensitivea
124A Jail—l/jail-l 26 29 0.16 (1:1)
jail-l/jail-l WT x 124A (F1)c 48 59 1.13 (1:1)
Jail-l/jaiI-l WT " 124A<F1)C 93 27 0.40 (3:1)

 

a Progeny from the indicated crosses were tested for responsiveness to applied MeJA
as described in Table 4.1, using both PM] and polyphenol oxidase as JA-responsive
markers. The number of progeny displaying a J A-sensitive or JA-insensitive

phenotype is shown.

b x2 values were calculated for the expectation that jail -I and the mutation in 124A

are allelic, where the predicted segregation ratio is indicated in parenthesis (JA
sensitive:J A insensitive). If jai l -l and the mutation in 124A were non-allelic, all

progeny from each cross would be J A sensitive.

c A line heterozygous for the mutation in 124A was produced by crossing 124A as a

staminate parent to wild-type (WT).

 

155

that the recessive mutation harbored by 436G defines a novel locus, designated Spr5.
Backcrossed lines that are homozygous for spr5 produced viable seed, albeit at reduced
levels relative to wild-type. This finding indicates that the sterile phenotype of 436G can

be attributed in part to a mutation other than spr5.

In contrast to spr5, MeJA-treated 124A plants expressed no detectable PI-II
(Table 4.1) or polyphenol oxidase (data not shown). This indicates that 124A, like jai 1 -l ,
is blocked in J A perception or the ability to respond appropriately to the hormone.
Complementation tests between 124A and jai-l were performed using heterozygous
maternal parents, and the results showed that the two mutants define the same locus
(Table 4.2). We henceforth refer to the fast-neutron allele of jail as jail -I and the EMS
allele of 124A as jail -2. Plants homozygous for jail -2 were also unresponsive to
relatively high concentrations of J A (50 nmol/plant) and systemin (5 pmol/plant) supplied
through the cut stem (Figure 4.2). Recovery of a J A-insensitive mutant in a screen for
suppressors of 35S: :prosys is significant because it demonstrates that 35S::prosys-
mediated signaling requires a functional J A response pathway. The unresponsiveness of
jai [-1 plants to wounding and applied systemin likewise indicates that JA action is

essential for wound- and systemin-induced PI expression (Figure 4.1).

156

Figure 4.2. Proteinase inhibitor [1 levels in wild-type and jail-2 plants in response
to JA and systemin. Fifteen-day-old seedlings were supplied with buffer (B; 15 mM
sodium phosphate, pH 7.0), JA (J; 50 nmol/plant), or systemin (S; 5 pmol/plant)
through the cut stem. PI-II levels were measured in the leaves 24 h later. Plants
homozygous for jai 1-2 were selected as described in Table 4.]. Values represent the

mean i SD of at least six plants.

157

 

 

 

 

0
2
1

. q . 1 u q .
0 0 0 O O 0
0 8 6 4 2

1

A85. u=3. .503 __-_n_

S

jai1-2

J

 

 

 

wild-type

Figure 4.2

158

A Role for JA in Female Reproductive Development?

The JA insensitivity and female sterility of independent mutants (jail-1 and jail -2)
argues against the possibility that the two phenotypes result from mutations in different
genes. Rather, our results support the hypothesis that a single gene, Jail, is required both
for wound-induced expression of defensive genes and female reproductive development.
Conclusive proof will require cloning of Jail and fimctional complementation of the
mutant. The sterility of jail -1 plants could result from a defect in ovule development,
embryo genesis, or another maternal process required for seed production. A dysfunction
in embryogenesis would be consistent with previous studies implicating J A as an
endogenous regulator of embryo development in oilseeds (Wilen et al., 1991). A similar
situation could exist in tomato, where JA, its precursor 12-oxo-phytodienoic acid, and
various amino acid conjugates of J A are abundant in female organs of the flower (Hause

etaL,2000)

A proposed role for J A in female reproductive development in tomato stands in
contrast to well-documented studies in Arabidopsis where J A biosynthesis and perception
are essential for male, but not female, gametophyte development (McConn and Browse,
1996; F eys et al., 1994; Sanders et al., 2000; Stintzi and Browse, 2000). Although we
cannot exclude a role for J A in male gametophyte development in tomato, the ability of

jail -I pollen to induce norrnal seed set when crossed to a wild-type pistillate parent
indicates that JA perception and downstream signaling events are not essential for the
production of viable pollen. How might such species-specific differences in jasmonate

function be explained? One possibilityis that oxylipins such as J A first evolved as low-

159

abundance signaling molecules for the regulation of stress responses (0. g. defense) in
vegetative tissues and, subsequently, these compounds were recruited to perform other
physiological functions (e. g. reproduction) in specific plant lineages. This scenario was
previously suggested to account for species-specific differences in the requirement for
flavonoids in male fertility (Burbulis et al., 1996). The range of physiological processes
controlled by JA may ultimately reflect the function of specific genes whose expression is
regulated by the hormone in a tissue- or cell type-specific manner. The wound response
mutants described herein should provide useful tools to investigate the molecular

mechanisms by which jasmonates regulate diverse physiological processes.

160

References

Bowles D (1998) Signal transduction in the wound response of tomato plants. Phil. Trans.
R. Soc. Lond. B 353, 1495-1510.

Burbulis IE, Iacobucci M, Shirley BW (1996) A null mutation in the first enzyme of
flavonoid biosynthesis does not affect male fertility in Arabidopsis. Plant Cell 8,
1013-1025.

Creelman RA, Mullet JE (1997) Biosynthesis and action of jasmonates in plants. Annu.
Rev. Plant Physiol. Plant Mol. Biol. 48, 355-381.

Farmer BE, Ryan CA (1992) Octadecanoid precursors of j asmonic acid activate the
synthesis of wound-inducible proteinase inhibitors. Plant Cell 4, 129-134.

Feys BJF, Benedetti CE, Penfold CN, Turner JG (1994) Arabidopsis mutants selected for
resistance to the phytotoxin coronatine are male sterile, insensitive to methyl
jasmonate, and resistant to a bacterial pathogen. Plant Cell 6, 751-759.

Green TR, Ryan CA (1972) Wound-induced proteinase inhibitor in plant leaves: a
possible defense mechanism against insects. Science 175, 776-777.

Hause B, Stenzel I, Miersch O, Maucher H, Kramell R, Ziegler J, Wastemack C (2000)
Tissue-specific oxylipin signature of tomato flowers: allene oxide cyclase is highly
expressed in distinct flower organs and vascular bundles. Plant J. 24, 113-126.

Howe GA, Lee G], Itoh A, Li L, DeRocher A (2000) Cytochrome P450-dependent
metabolism of oxylipins in tomato: cloning and expression of allene oxide synthase
and fatty acid hydroperoxide lyase. Plant Physiol. 123, 711-724.

Howe GA, Lightner J, Browse J, Ryan CA (1996) An octadecanoid pathway mutant (J L5)
of tomato is compromised in signaling for defense against insect attack. Plant Cell 8,
2067-2077.

Howe GA, Ryan CA (1999) Suppressors of systemin signaling identify genes in the
tomato wound response pathway. Genetics 153, 1411-1421.

Karban R, Baldwin IT (1997) Induced responses to herbivory. Chicago University Press,
Chicago.

Leon J, Rojo E, Sénchez—Serrano JJ (2001) Wound signalling in plants. J. Exp. Bot. 52,
1-9.

161

Li L, Howe GA (2001) Alternative splicing of prosystemin pre-mRNA produces two
isoforms that are active as signals in the wound response pathway. Plant Mol. Biol.
46, 409-419.

Li L, Shaffer D, Howe GA (2001) Annual Meeting of the American Society of Plant
Physiologists, July 2001, Providence, Rhode Island. (Abstract no. 128).

Lightner J, Pearce G, Ryan CA, Browse J (1993) Isolation of signaling mutants of tomato
(Lycopersicon esculentum). Mol. Gen. Genet. 24], 595-601

McConn M, Browse J (1996) The critical requirement for linolenic acid is pollen
development, not photosynthesis, in an arabidopsis mutant. Plant Cell 8, 403-416.

McGurl B, Orozco-Cardenas M, Pearce G, Ryan CA (1994) Overexpression of the
prosystemin gene in transgenic tomato plants generates a systemic signal that

constitutively induces proteinase inhibitor synthesis. Proc. Natl. Acad. Sci. USA 91,
9799-9802.

McGurl B, Ryan CA (1992) The organization of the prosystemin gene. Plant Mol. Biol.
20, 405-409.

O’Donnell PJ, Calvert C, Atzom R, Wastemack C, Leyser HMO, Bowles DJ (1996)
Ethylene as a signal mediating the wound response of tomato plants. Science 274,
1914-1917.

Orozco-Cardenas M, Narvaez-Vasquez J, Ryan CA (2001) Hydrogen peroxide acts as a
second messenger for the induction of defense genes in tomato plants in response to
wounding, systemin, and methyl jasmonate. Plant Cell 13, 179-191.

Pearce G, Strydom D, Johnson S, Ryan CA (199]) A polypeptide from tomato leaves
induces wound-inducible proteinase inhibitor proteins. Science 253, 895-898.

Ryan CA (2000) The systemin signaling pathway: differential activation of plant
defensive genes. Biochim. Biophys. Acta 1477, 112-121.

Sanders PM, Lee PY, Biesgen C, Boone JD, Beals TP, Weiler EW, Goldberg RB (2000)
The arabidopsis DELA YED DEHISCENCE] gene encodes an enzyme in the j asmonic
acid synthesis pathway. Plant Cell 12, 1041-1062.

Stintzi A, Browse J (2000) The Arabidopsis male-sterile mutant, opr3, lacks the 12-

oxophytodienoic acid reductase required for jasmonate synthesis. Proc. Natl. Acad.
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162

Walling LL (2000) The myriad plant responses to herbivores. J. Plant Growth Regul. 19,
195-216

Wilen RW, van Rooijen GJH, Pearce DW, Pharis RP, Holbrook LA, Moloney MM (1991)
Effects of J asmonic Acid on embryo-specific processes in Brassica and Linum
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163

CHAPTER 5

Distinct Roles for Jasmonate Synthesis and Action in the Systemic Wound Response of

Tomato

The work presented in this chapter has been published:
Li L, Li C, Lee G], Howe GA (2002) Proc. Natl. Acad. Sci. USA 99, 6416—6421.
Quantification of j asmonates was performed by Gyu In Lee. Experiements presented in

Figure 5.2 were done with collaboration with Chuanyou Li.

164

Abstract

Plant defense responses to wounding and herbivore attack are regulated by signal
transduction pathways that operate both at the site of wounding and in undamaged distal
leaves. Genetic analysis in tomato indicates that systemin and its precursor protein,
prosystemin, are upstream components of a wound-induced, intercellular signaling
pathway that involves both the biosynthesis and action of jasmonic acid (J A). To examine
the role of J A in systemic signaling, reciprocal grafting experiments were used to analyze
wound-induced expression of the proteinase inhibitor II gene in a JA biosynthetic mutant
(spr2) and a JA response mutant (jail). The results showed that spr2 plants are defective
in the production, but not recognition, of a graft-transmissible wound signal. Conversely,
jail plants are compromised in the recognition of this signal, but not its production. It
was also determined that a graft-transmissible signal produced in response to 35S::prosys
expression in rootstocks was recognized by spr2 but not by jai 1 scions. Taken together,
the results indicate that (pro)systemin-mediated activation of the jasmonate biosynthetic
pathway in wounded leaves is required for the production of a long-distance signal whose
recognition in distal leaves requires jasmonate signaling. These findings provide strong
support for the hypothesis that jasmonate is an essential component of the transmissible

wound signal.

165

Introduction

Higher plants respond to insect attack and wounding by activating the expression
of genes involved in herbivore deterrence, wound healing, and other defense-related
processes (Karban and Baldwin, 1997). An important aspect of many induced defense
responses is their occurrence in undamaged leaves located distal to the site of attack
(Green and Ryan, 1972). Wound-inducible proteinase inhibitors (P15) in tomato
(Lycopersicon esculentum), which are expressed within about 2 h after mechanical
wounding or herbivory (Howe et al., 2000; Ryan, 2000), represent one of the best
examples of this phenomenon. In their landmark study of wound-inducible PIs, Green
and Ryan (1972) proposed that specific signals generated at the wound site travel through
the plant and activate PI expression in undamaged responding leaves. Although several
chemical and physical signals have since been implicated in the systemic wound response
(reviewed in Ryan, 1992;Bow1es, 1993; Malone, 1996; Ryan, 2000; Walling, 2000; Leon
et al., 2001), very little is known about how these signals interact with one another to

effect cell-to-cell communication over long distances.

Among the proposed intercellular signals for wound-induced PI gene expression
are systemin, an 18-amino-acid peptide derived from proteolytic cleavage of a larger
precursor protein called prosystemin (Pearce et al., 199]; Mchl et al., 1992), and
jasmonate signals such as jasmonic acid (J A) and its methyl ester, methyl-JA (MeJA;
Farmer and Ryan, 1990; Farmer et al., 1992). According to a recent model of wound
signaling in tomato (Ryan, 2000), systemin is transported through the plant as a mobile
signal following its proteolytic release fiom prosystemin. Interaction of systemin with a

plasma membrane-bound receptor (Meindl et al., 1998; Scheer and Ryan, 1998) then

166

triggers a signaling cascade leading to activation of a lipase that releases linolenic acid
from membrane lipids (N arvaez-Vasquez et al., 1999; Ishiguro et al., 2001). J asmonates
are synthesized from linolenic acid via the octadecanoid pathway, and are considered to
be key regulators for stress-induced gene expression in virtually all plants (Reymond et
al., 2000; Schaller, 2001). Recent studies have shown that 12-oxo-phytodienoic acid
(OPDA), a cyclopentenone precursor of JA/MeJ A, is a signal for defense gene expression
without its prior conversion to J A (Stintzi et al., 2001). Activation of PI expression in
response to wounding, systemin, and jasmonates involves the coordinate biosynthesis and
action of ethylene (Felix and Boller, 1995; O’Donnel etal., 1996), and is also associated
with the production of reactive oxygen species that act downstream of J A (Orozco-

Cardenas et al., 2001).

Of relevance to the mechanism of wound-induced intercellular signaling is the
observation that genes encoding prosystemin and some J A biosynthetic enzymes are
expressed in vascular bundle cells, whereas defensive PI genes are expressed in adjacent
palisade and spongy mesophyll cells (Shumway et al., 1970; J acinto et al., 1997;
Kubigsteltig et al., 1999; Hause et al., 2000). The cell-type-specific expression pattern of
these signaling components has led to the hypothesis that wound-induced release of
systemin into the vascular system activates J A biosynthesis in surrounding vascular
tissues in which J A biosynthetic enzymes are located (Ryan, 2000). Active transfer or
diffusion of a jasmonate signal from its site of synthesis could, in turn, induce PI
expression in neighboring mesophyll cells. A role for jasmonates in intercellular
signaling is supported by the fact that application of J A/MeJ A to one leaf induces PI

expression in distal untreated leaves (Farmer et al., 1992), and that exogenous J A is

167

readily transported in the phloem (Zhang and Baldwin, 1997). In addition, it has been
demonstrated that cultured plant cells secrete J A into the medium (Parchmann et al.,
1997). Recent studies suggest that the conversion of JA to MeJA by a specific JA
carboxyl methyltransferase is an important regulatory step in j asmonate-mediated

intercellular signaling (Seo et al., 2001).

We are using tomato as a model system for genetic analysis of systemic wound
signaling and its role in plant defense. Toward this goal, plant genotypes defective in
wound-induced systemic expression of PI and other defense-related genes have been
identified in various genetic screens (Lightner et al., 1993; Howe and Ryan, 1999; Li et
al., 2001). These mutants can be classified into two phenotypic groups: jasmonate
biosynthesis mutants that are insensitive to systemin but responsive to J A/MeJA, and
jasmonate response mutants that are insensitive to both systemin and JA/MeJ A (Li et al.,
2001). Here we report the use of grafting experiments to detem'rine whether these mutants
are defective in the production of a long-distance wound signal, or the recognition of that
signal in distal undamaged leaves. The results reveal distinct roles for jasmonate
biosynthesis and signaling in the generation and recognition, respectively, of a long-

distance wound signal for activation of defense gene expression.

168

Materials and Methods

Plant material and treatments

Lycopersicon esculentum cv Castlemart was used as the wild-type variety for all
experiments. Plants were grown and maintained as described previously (Howe et al.,
2000). The original spr2 and jail -2 mutants (Howe etal., 2000; Li et al., 2001) were

backcrossed to wild-type, and homozygous mutants lacking the 35S: :prosys transgene

were selected from the resulting F2 population. Seed for the spr2 mutant was collected

from a spr2/spr2 homozygote that had been back-crossed three times to wild-type.

Because of the reduced fertility of jail -2 plants (Li et al., 2001), plants homozygous for

jai 1-2 were obtained from a segregating F 2 population using the screening procedure

described by Li et al. (200]). Seed for the 35S: :prosys transgenic line was collected from
a 35S::prosys/35S::prosys homozygote that had been back-crossed five times to wild-
type. Wounding was performed as described in the figure legends. Treatment of plants
with MeJA (Bedoukian Research) was performed as described previously (Li and Howe,

2001).

Grafting experiments

Four-week-old plants were grafted using a modification of the procedure
described by McGurl et al. (1994). A longitudinal incision of approximately 1.5 cm was

made in the middle of the rootstock stem. The scion stem was trimmed to the shape of a

169

wedge, and then tightly fastened to the cortex flaps of the rootstock using water-soaked
raffia. All but one leaf immediately beneath the apical meristem were excised from the
scion. Scions were enclosed in a plastic bag that was fastened at the graft junction. One
week after grafting, the plastic bag and raffia were removed. Grafted plants contained
two or three leaves on the rootstock and two newly emerging leaves on the scion. Four
days after removal of the bag, plants were subjected to mechanical wounding as follows.
All leaflets (approximately 10) on leaves of the stock were crushed with a hemostat
across the midvein. This procedure was repeated 2 h later, such that the second wound
was parallel to the first wound and proximal to the petiole. Eight hours after the second
wound, wounded leaflets fi'om the stock and undamaged leaflets of the scion were
harvested separately for RNA isolation. Equal amounts of leaf tissue from three plants of
the same graft combination were pooled prior to RNA isolation. RNA blot analysis of PI-
Il mRNA levels was performed as previously described (Li and Howe, 2001), using an
eIF 4A cDNA probe as a loading control. PI-II protein levels in grafted plants were

measured by radial immunodiffusion assay (Ryan, 1967).

Quantification of jasmonic acid

Plants containing two fully expanded leaves and an emerging third leaf were
wounded with a hemostat on each leaflet of the two expanded leaves. Leaves (10 g FW)
were harvested for extraction and quantification of J A using a modification of the
procedure described by Weber et a1. (1997). Harvested leaves were frozen in liquid
nitrogen and ground to a fine powder using a chilled mortar and pestle. The tissue was

dissolved in 28 ml methanol containing 500 ng dihydrojasmonic acid (DHJA) as an

170

internal standard, and then homogenized with a Polytron for 1 min at 4°C. The
homogenate was incubated for 2 h at 4°C with shaking, diluted with 12 ml ice-cold water,

and then centrifiiged at 3500 x g. The resulting supernatant was recovered and the pH

adjusted to 8.0 with NH4OH. This solution was passed through a tClg-SepPak cartridge

(Waters) preconditioned with 70% (v/v) methanol and collected in a new tube. The
cartridge was washed with 7 ml 75% (v/v) methanol. Eluates from both the sample and

the wash steps were combined and adjusted to pH 4.0 with 10% (v/v) formic acid. This

solution was diluted with 160 ml ice-cold water and then loaded on a tClg-SepPak

column that was prewashed sequentially with methanol, diethylether, methanol, and
water. After washing the column with 7 ml 15% (v/v) ethanol and 7 ml water, the J A

fraction was eluted with 10 ml diethylether. The eluate was partially dried over

anhydrous MgSO4 and then dried completely under a stream of nitrogen gas. The dried

paste was dissolved in 0.5 ml methanol and subjected to methylation by the addition of

diazomethane in 0.5 ml diethylether. This mixture was dried under nitrogen gas and

resuspended in 20 pl hexane.

The amount of JA/MeJA in leaf extracts was quantified by GC-MS using a
Hewlett—Packard GC 5890 equipped with a Hewlett-Packard 5970 mass detector. The GC
was fitted with a DB-S column and run at 250 °C in the isothermal mode. GC-MS
analysis was performed in the SIM mode with monitoring of ions specific for MeJ A (m/z
= 224) and MeDHJA (m/z = 226). For quantification of JA/MeJ A, a standard curve was
generated from samples in which MeJA and MeDHJA were mixed in known ratios.

Because peaks corresponding to both the 3R,7S and 3R,7R isomers of endogenous

171

J A/MeJ A were detected, the areas of the two peaks were combined. JA levels reported in

the Results section represent the mean 3: standard error of at least three independent
experiments. DHJ A was prepared by PtOz-cataylzed hydrogenation of (2t)-J A (Sigma) as

previously described (Weber et al., 1997). The authenticity of the standard, as well as the

absence of endogenous DHJA/MeDHJ A in tomato leaf extracts, was verified by GC-MS.

172

Results

Mutations affecting either JA biosynthesis or JA signaling abolish wound-induced

systemic expression of PI genes

Wound response mutants that are defective either in J A biosynthesis or J A
responsiveness were used to study the role of JA in systemic wound signaling. The spr2
and jai 1-2 mutations were previously identified as suppressors of defense-related
responses that are constitutively activated in transgenic tomato plants that overexpress
prosystemin from the 35S::prosys transgene (Howe et al., 2000; Li et al., 2001). spr2
plants lack wound-induced systemic expression of the well-characterized Pl-II gene, but
nevertheless respond normally to applied MeJA (Figure 5.1). This phenotype is very
similar to that conditioned by defl , a non-allelic mutation that reduces wound-induced J A
accumulation to about 30% of wild-type levels (Howe et al., 1996). To determine
whether spr2 plants are defective in JA synthesis, JA was extracted from wounded and
control (unwounded) leaves of wild-type and spr2 plants, and quantified using gas
chromatography-mass spectrometry. The results showed that undamaged wild-type
leaves contained 12 i 1 pmol JA/g F W. In response to mechanical wounding, JA levels
increased to 262 i 41 and 151 i 26 pmol JA/ g FW 1 h and 3 h, respectively, after
wounding. The JA level in unwounded spr2 leaves was 3 i 1 pmol JA/ g FW, which rose
to 22 i: 9 and 7 i 1 pmol JA/g F W 1 h and 3 h, respectively, after wounding. This finding

indicates that the wound response phenotype of spr2 plants results from a defect in

jasmonate biosynthesis.

173

Figure 5.1. Induction of the proteinase inhibitor II gene in response to wounding
and MeJA. Two-leaf stage wild-type (WT) and mutant (spr2 and jail -2) tomato plants
were wounded once with a hemostat across the midvein of the lower leaf. Eight hours
later, leaf tissue was harvested separately from the wounded leaf (L; Local response)
and the upper undamaged leaf (S; systemic response) for RNA isolation. RNA was also
isolated from a set of plants treated for 12 h with MeJA (J), and a set of untreated
control plants (C). Five-pg aliquots of total RNA were analyzed by RNA blot analysis
for PI-II mRNA levels. As a loading control, a duplicate blot was probed with a cDNA

encoding elF 4A.

174

' O
11131333

10.11

WT

spr2

jai1-2

 

 

 

 

 

CLSJ CLSJ CLSJ

Q

 

 

 

Figure 5.1

175

PH]

eIF4A

Wounded jail -2 plants accumulate Pl-II mRNA to < 5% of the levels observed in wild-
type (Figure 5.1). The residual wound-induced expression of PM] in this mutant likely
results from partial activity of the jai [-2 allele, as we have observed that plants
homozygous for a deletion allele of jail (jail-l) accumulate no detectable PI-II
transcripts in response to wounding (Figure 3.5). In contrast to J A biosynthetic mutants,
jail -2 plants fail to accumulate PI-II transcripts in response to exogenous J A/MeJ A
(Figure 5.]; Li et al., 2001). This phenotype is indicative of a defect in jasmonate

perception or subsequent signaling events that are necessary for PI activation.

Jasmonate signaling is required for functional recognition of a long-distance wound

signal

To examine the role of jasmonate action in the systemic wound response, we
analyzed wound-induced PI-II expression in grafts between wild-type and jai [-2 plants.
Four-week-old plants were grafted such that both the rootstock (stock) and the scion
contained at least two healthy leaves (Figure 5.2). Following sufficient time for healing
of the graft junction, stock leaves were wounded and PI-II mRNA levels were measured
11 h later in both the damaged stock leaves (local response) and the undamaged scion
leaves (systemic response). Control experiments showed that the grafting procedure itself
induced some PI-II expression in wild-type stock and scion leaves (Figure 5.3a, lane 1;
data not shown). However, subsequent wounding of stock leaves induced local and
systemic Pl-II expression well above this background level (Figure 5.3a, lane 2). This

experiment demonstrates that wounding of wild-type stock leaves leads to the production

176

Figure 5.2. Photograph of a typical grafted tomato plant. The arrow indicates the
position of the graft junction between the stock and scion. Systemic PI-II expression
was measured in undamaged scion leaves ]1 hr after wounding of the stock leaves. The
distance between wounded leaflets on the stock and undamaged leaflets on the scion is

approximately 20 cm. Images in this figure are presented in color.

177

 

xi

Graft junction

\ K Kiln“

.1 {5r‘ A

 

Figure 5.2

178

of a graft-transmissible signal that is recognized in undamaged scion tissues. Consistent
with the wound response observed in jail -2 seedlings (Figure 5.1), wound-induced
expression of PI-Il in both stock and scion leaves of grafted jail plants was < 5% of that
in wild-type plants (Figure 5.3a, lanes 3-4). Analysis of jai-l /wild-type hybrid grafts
showed that wounding of jai 1 stock leaves resulted in full activation of PHI expression

in wild-type scion leaves (Figure 5.3a, lanes 5-6). In the reciprocal combination, however,
undamaged mutant scion leaves failed to express PM] in response to wounding of the
wild-type stock (Figure 5.3a, lanes 7-8). These results demonstrate that jail does not
affect the production of the graft-transmissible systemic signal at the site of wounding,

but rather disrupts the recognition or proper interpretation of that signal in distal

undamaged leaves.

Jasmonate biosynthesis is required for generation of a long-distance wound signal

Wound-induced PI-II expression in graft combinations between wild-type
and spr2 plants was examined to investigate the role of j asmonate biosynthesis in
the systemic wound response. In contrast to the results obtained with jai 1 plants,
wild-type scions responded very weakly to wounding of spr2 stock leaves (Figure
5.3b, lanes 5-6). Moreover, spr2 scions exhibited a strong response to a signal
emanating from wounded leaves of WT stock (Figure 5.3b, lanes 7-8). Analysis
of grafts between wild-type plants and the def] mutant, which is also deficient in
J A biosynthesis (Howe et al., 1996), gave results that were similar to those for

spr2/wild-type hybrid grafts (data not shown). These results indicate that JA, or a

179

Figure 5.3. Wound-inducible PI-II expression in grafts between wild-type plants
and mutants defective in jasmonate signaling (jail) or jasmonate biosynthesis
(spr2). Wild-type (WT) and jai [-2 plants (a) or WT and spr2 plants (b) were grafted in
the four combinations indicated. The genotypes listed above and below the horizontal
line correspond to the scion and stock, respectively. For each graft combination, plants
were divided into a control (C) and experimental (W) group consisting of three grafted
plants per group. For the experimental group, each leaflet on the stock was wounded as
described in the Experimental procedures. Eleven hours after wounding, leaf tissue was
harvested separately from wounded stock leaves (stock) and undamaged scion leaves
(scion) for RNA extraction. The control set of plants received no wounding, other than
that inflicted by the grafting procedure itself. Levels of PHI mRNA were analyzed by

RNA blot analysis, using an eIF 4A cDNA probe as a loading control.

180

 

(8) WT jai1 WT jai1
WT jai1 jai1 WT
C W C W C W C W

1 Q PI-Il

mum‘suflaI-n“ eIF4A

.' -_-- . a.”

“wflflflrwum eIF4A

 

 

     

scion

 

 

 

 

PI-II

   

 

 

stock

 

12345678

(b) WT spr2 WT spr2

WT spr2 spr2 WT
CWCWCWCW

1...... « . PHI

«4. am. PM!

wwm~m«m~~~elF4A

 

 

 

scion

 

 

 

stock

 

 

 

12345678

Figure 5.3

181

Figure 5.4. Wound-induced systemic PI-II expression in grafts between jail and
spr2 plants. Wound-induced systemic expression of PHI was assessed in the various
graft combinations indicated, as described in Figure 5.3. WT, wild-type. PH] and
eIF 4A (loading control) mRNA levels in the undamaged scion leaves of unwounded

control (C) plants, and plants wounded on the stock leaves (W), are shown.

182

 

scion

Lefl WT sgr2 sgr2 [fl
jai1 jai1 spr2 jai1 spr2

CWCWCWCW CW
“. . PI-Il

 

 

 

 

12345678910

Figure 5.4

183

related octadecanoid pathway-derived compound, is an essential component of the graft-
transmissible wound Signal. The results shown in Figure 5.3b (lanes 7—8) further
suggested that recognition of the long-distance signal and subsequent PI expression in
undamaged spr2 scions does not require J A biosynthesis in these tissues. However, an
alternative explanation was that grafting of spr2 scions to wild-type stock simply restored
the ability of the mutant scion to synthesize J A in response to a signal produced in
wounded wild-type stock leaves. To examine this possibility, spr2 scion leaves that had
been grafted to wild-type stock were wounded and then tested for PI-II protein
accumulation. Control experiments showed that wild-type scion leaves grafted to either
wild-type or spr2 stock were responsive to wounding (Table 5.1). spr2 scion leaves
grafted to spr2 stock failed to accumulate PI-II in response to wounding, and this
deficiency was not relieved by grafting to wild-type stock. This finding supports the
interpretation that PI-II activation in spr2 scions (Figure 5.3b, lanes 7-8) does not involve
de novo J A biosynthesis in these leaves, but rather requires a functional octadecanoid

biosynthetic pathway in the wild-type stock.

Despite the fact that both spr2 and jail abrogate systemic wound signaling, the
reciprocal nature of the grafting phenotypes conditioned by each mutation (Figure 5.3)
predicted that grafted plants lacking both jasmonate responsiveness in stock leaves and
jasmonate biosynthesis in scion leaves would be capable of systemic signaling. To test
this idea, we examined PI-II expression in spr2 scion leaves in response to wounding of
jail stock leaves (Figure 5.4). The results showed that wounded jail stock leaves produce
a graft-transmissible signal that activates PI—II expression in spr2 scions (Figure 5.4,

lanes 7-8), at a level comparable to that observed in wild-type scions (Figure 5.4, lanes 3-

184

4). As is also predicted from the grafting phenotypes of individual mutants, wound-
induced systemic expression of PI-Il was abolished in grafted plants that are deficient in
both J A biosynthesis in stock (i.e., spr2) leaves and JA responsiveness in scion (i.e., jai1)

leaves (Figure 5.4, lanes 9-10).

Roles for jasmonate biosynthesis and signaling in 35S::prosys-mediated PI expression

Previously it was shown that ectopic expression of prosystemin from a
35S: :prosys transgene leads to constitutive PI expression in the absence of wounding
(McGurl etal., 1994). Grafting experiments presented in the same study further
demonstrated that unwounded 35S::prosys stock tissue produces a graft-transmissible
signal that activates PI expression in wild-type scion leaves. To investigate the role of
jasmonate synthesis and perception in the 35S::prosys-mediated signaling pathway, PI-II
protein accumulation was measured in spr2 and jail scions that were grafted to either
wild-type or 35S::prosys stock. As previously reported by Mch1 et a1. (1994), wild-
type scion leaves accumulated high levels of PI-II in response to a signal emanating from
the 35S::prosys stock (Table 5.2). The responsiveness of spr2 scion leaves to the
35S::prosys-derived signal was comparable to that of wild-type scions. In contrast, jai1

scions were completely unresponsive to the 355::prosys-derived signal.

185

 

Table 5.1. Proteinase inhibitor 11 accumulation in wild-type and
spr2 scion leaves in response to wounding.

 

 

 

Graft combination PI-H in scion (pg/ml)
(Scion / Stock)

Unwounded Wounded
WT/WT 26:10 117i27
spr2/spr2 35:2 11i13
WT/spr2 6i] 113-:21
spr2/WT 14:7 17:7

 

Three plants of each of the indicated graft combinations were
constructed from four-week-old tomato plants. Three weeks after
grafting, PI-II levels were measured in two newly-developed leaves
of the scion, using one excised leaflet per leaf. At the same time, ten
of the remaining leaflets on the scion were wounded with a
hemostat. PI-II levels in four of the wounded leaflets were
determined 48 h later. Values represent the mean PI-H concentration
of three plants per combination i SD. WT, wild-type.

 

186

 

Table 5.2. Proteinase inhibitor 11 accumulation in grafted
tomato plants in response to a long-distance signal generated in
a 35S::prosys transgenic line.

 

Graft combination PI-II (pg/ml leaf juice)

 

 

(Scion / Stock)

Scion Stock
WT/WT 12i7 l8i14
35S::prosys / 35S::prosys 229 i 14 304 i 45
WT / 35S::prosys 140 i- 36 252 i 61
spr2/WT 14i7 l6i9
spr2 / 35S::prosys 150 i 12 263 i 10
jail / WT ND 30 i ll
jai1 / 35S::prosys ND 279 i ll

 

Five-week-old tomato plants were grafted in the indicated
combinations. Three weeks after grafting, PI-II accumulation was
measured in three plants of each graft combination. PI-II levels were
determined using three leaflets per leaf. Values represent the mean
PI-II concentration of three plants per combination i SD. WT, wild-
type; 35S::prosys, transgenic line that overexpresses prosystemin
from the 35S::prosys transgene. ND, not detectable.

 

187

Discussion

Systemic activation of defensive PI genes in tomato is orchestrated by signaling
events that operate both within and between cells. A wealth of biochemical and genetic
data support the original proposal (Farmer and Ryan, 1992) that (pro)systemin functions
in this pathway to regulate the synthesis of J A, which in turn activates the expression of a
subset of target genes including those encoding defensive PIs. However, very little is
known about the relationship between systemin-induced JA synthesis and the cell non-
autonomous processes by which mobile signals are produced at the wound site,
transported through the plant, and perceived by target cells distal to the wound site. To
address this question, we used classical grafting techniques to examine long-distance
wound signaling in mutants that are deficient either in JA biosynthesis or JA signaling. A
model consistent with our results and other available genetic data is shown in Figure 5.5.
This model accounts for the following observations. First, a graft-transmissible signal for
systemic PI expression is produced in response to either wounding or 355::prosys.
Second, in non-grafted plants, def] , spr2, and jail suppress both wound- and
35S::prosys-induced PI expression. Third, jail plants are insensitive to both systemin and
J A/MeJA, whereas the JA synthesis mutants spr2 and def] are insensitive to systemin but
responsive to J A/MeJ A. Fourth, jai1 scions do not respond to a graft-transmissible signal
generated either by wounding or 35S: :prosys, whereas spr2 scions do. Conversely, spr2
and defl stock leaves that are deficient in wound-induced JA accumulation are also
deficient in wound-induced generation of a graft-transmissible signal, whereas jai1 plants

are functional in this respect. Taken together, these findings indicate that activation of the

188

octadecanoid pathway in damaged leaves is required for the production of a long-distance

signal whose functional recognition in distal leaves requires jasmonate action.

It should be emphasized that other systemic wound responses may operate
independently of or in parallel to the systemin/j asmonate pathway that regulates the
synthesis of P15 and other defensive phytochemicals. For example, hydraulic signals may
be involved in rapid systemic wound responses such as the activation of a wound-
inducible protein kinase activity in tomato leaves (Malone, 1996; Stratmann and Ryan,
1997). There is also evidence that genes whose expression in tomato plants is rapidly and
systemically induced by wounding are regulated by signaling pathways that operate
independently of systemin and JA (O’Donnell et al., 1998; Howe et al., 2000; Walling
2000). Such a pathway may account for the residual signaling activity observed in wild-
type scions in response to wounding of srp2 (Figure 5.3b) or defl (data not shown) stock
leaves. Alternatively, this residual signaling may reflect incomplete loss of function of

Spr2/Defl .

Although systemic activation of PI genes clearly involves jasmonate-mediated
signaling events in undamaged (scion) responding leaves, grafting experiments conducted
with spr2 and defl plants indicate that J A biosynthesis is likely not required in these
leaves. This observation raises the question of whether PI expression in undamaged

leaves is mediated by JA or a related octadecanoid signal. Previous studies aimed at

addressing this question suggest that JA, rather than C13 precursors of JA, is the active

signal for PI expression in tomato leaves (Wastemack et al., 1998). If J A is a signal for

PI expression in undamaged leaves, our results, together with reports of wound-induced

189

systemic increases in JA levels in tomato (Herde et al., 1996), suggest that JA is
transported from its site of synthesis in stock tissues to undamaged responding leaves. An
alternative hypothesis is that the requirement for Jail -dependent signaling in undamaged

leaves is fulfilled by a jasmonate signal other than JA. Candidates for such a signal

include OPDA and its C16 analog, dinor-OPDA (Weber et al., 1997). A more precise

understanding of how defl and spr2 affect the octadecanoid pathway should provide
additional insight into this possibility, as will grafting experiments using transgenic plants

that are engineered for a deficiency in specific octadecanoid pathway enzymes.

The roles of j asmonate biosynthesis and perception in wound-induced systemic
signaling appear to be similar to their roles in 35S: :prosys-mediated PI expression
(Figure 5.5). This conclusion is based on the finding that spr2 and defl (L. Li and G.
Howe, unpublished data) scions respond to a graft-transmissible signal generated in
35S::prosys stock, whereas jai-l scions do not (Table 5.2). Because spr2 and defl plants
are insensitive to exogenous systemin (Howe et al., 1996; Howe and Ryan, 1999), it
seems unlikely that the graft-transmissible signal produced in 35S: :prosys plants is
systemin. Rather, our results suggest that this signal is a compound that acts downstream
of SprZ/Defl and through Jail. Possible candidates for this signal include JA/MeJA and
OPDA. Measurement of these compounds in 35S: :prosys plants may help to address this
question. It is interesting to note that while spr2 and defl scions respond to the
35S: :prosys-derived signal, these mutations effectively suppresses 35S::prosys-mediated
signaling when present in homozygous state in the 35S::prosys genetic background

(Howe et al., 1996; Howe and Ryan, 1999). This observation indicates that normal Spr2

190

and Def] activity, and thus a functional octadecanoid pathway, is required for production

of the 35S::prosys-derived graft-transmissible signal.

The grafting experiments reported herein demonstrate that jasmonate biosynthesis
and action, while both required for long-distance activation of PI genes, operate at
distinct spatial positions along the systemic signaling pathway. More specifically, we
propose that jasmonate biosynthesis is required for the generation of a long-distance
wound signal, whereas jasmonate action is involved in the recognition of this signal in
responding leaves. The most straightforward interpretation is that jasmonate is an
essential component of the transmissible wound signal. This is consistent with other
reports implicating J A/MeJA as intercellular signals for stress-induced gene expression
(Farmer et al., 1992; Parchmann et al., 1997; Zhang and Baldwin, 1997; L6bler and Lee,
1998; Ryan, 2000; Seo et al., 2001). Such a scenario raises the question of the role of
systemin in the systemic response. As discussed previously (Farmer et al., 1992; Hause et
al., 2000; Ryan, 2000), localized production of systemin at the site wounding may induce
the synthesis of J A/MeJ A, which in turn could promote gene expression in neighboring
cells. Although this model suggests that J A/MeJ A act in a paracrine fashion analogous to
eicosanoid signals in arrima] cells, it is conceivable that jasmonates exert their effects
over much longer distances. Alternatively, systemin-induced activation of the
octadecanoid pathway could further amplify the signaling cascade through positive
feedback on (pro)systemin production or action (J acinto et al., 1997; Scheer and Ryan,
1998). Identification of mutants that are defective in (pro)systemin perception may
provide additional insight into the role of this polypeptide in the systemic wound

response, and the mechanism by which it regulates the octadecanoid pathway.

19]

Figure 5.5. Genetic model for the role of jasmonate synthesis and signaling in the
systemic activation of wound-responsive PI genes in tomato plants. Wounding or
expression of 35S: :prosys leads to the production of a long-distance signal that

activates PI gene expression in distal leaves (i.e., scion). A functional octadecanoid
pathway for j asmonate biosynthesis (Hatched line), which is disrupted by defl and spr2,
is required for the production of the long-distance signal (X). J asmonate signaling,

which is blocked by jail , is required for the recognition of the long-distance signal.

192

 

Systemic Response

 

 

 

 

 

 

(scion)
F P, W
jai1 —|I
L l J
X
i
K JA > PI\
9 "l'
spr2 jai1
_| :

def1
Qound 35S::Prosys/

Local Response
(rootstock)

 

 

Figure 5.5

193

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197

CHAPTER 6

Molecular Cloning of Jail

198

Abstract

The jai [-1 mutant, isolated from a fast-neutron irradiated Micro-Tom population,
is impaired in jasmonate signaling. The mutation results in compromised wound
responses, increased susceptibility to herbivores, defects in glandular trichome
development, and female sterility. In a separate screen for ethyl methane sulfonate (EMS)
induced mutations that suppress prosystemin-mediated responses, a second jail allele
(jai1-2) was identified. Using a candidate gene approach, the jail mutations were found
to be lesions in the tomato homolog of the Arabidopsis CORONA T lNE INSENSIT I VE l
(AtCOII) gene that encodes an F-box protein involved in ubiquitin/proteasome-
dependent proteolysis. Stable transformation of jail -1 plants with the tomato COII
(LeCOII) cDNA restored MeJA-induced expression of defense genes, fertility, and
glandular trichome development. These findings demonstrate that LeCOIl is an essential
component of the jasmonate signaling pathway governing defense and developmental

processes in tomato.

199

Introduction

Tomato (L. esculentum) provides an excellent model system in which to study the
role of jasmonates in defense- and development-related processes. In our efforts to
characterize the systemic wound response pathway in tomato, we conducted various
genetic screens that led to the identification of j asmonate-insensitive (jai1) mutants that
are deficient in jasmonate signaling. The jail -1 mutant was isolated from a fast-neutron
mutagenized Micro-Tom population (Li et al., 2001; Howe et al., 2002). In a separate
genetic screen for mutations that suppress prosystemin-mediated responses, an EMS-
induced allele of jail called jail -2 was recovered (Howe and Ryan, 1999; Li et al., 2001).
Characterization of the jai1-1 mutant showed that Jail is required for MeJA-mediated
root grth inhibition, and anthocyanin accumulation in germinating seedlings (Chapter
3). In leaves, Jail was found to be essential for wound-, MeJA-, and herbivore-induced
defense gene activation and defense against hervibores (Chapter 3; Howe et al., 2002).
Furthermore, the critical importance of Jail for pollen viability, trichome development
on green fruits, female fertility, and constitutive expression of defense genes in flowers
was evident by the defects of jail -1 plants in these processes (Chapter 3, Li et al., 2001).
Taken together, these results indicated that Jail is a positive regulator of j asmonate

signaling in most if not all tomato organs.

In Arabidopsis, extensive genetic screens have revealed a number of loci required
for the function of the jasmonate signaling pathway (see Table 1.2). One of the genes
identified was AtCOII encoding a leucine rich repeat (LRR) containing F -box protein

that participates in the formation of an E3 ubiquitin 1i gase complex involved in ubiquitin-

200

dependent proteolysis (Xie et al., 1998; Devoto et al., 2002; Xu et al., 2002). Based on
the biochemical function of F-box proteins from yeast and Drosophila (Deshaies, 1999),
it was postulated that COI] serves as a receptor in the ubiquitin ligase complex to recruit
a phosphorylated substrate protein for ubiquitination (Creelman, 1998; Xie et al., 1998).
Subsequent degradation of the substrate, which was proposed to be a repressor of
transcription, by the 26S proteasome, would then allow transcriptional activation of

jasmonate-responsive genes (Creelman, 1998; Xie et al., 1998).

Mutation of AtCOIl has global effects on jasmonate signaling that are reminiscent
of the phenotypes of tomato jail plants. Arabidopsis coil mutants are unresponsive to
root grth inhibition by the bacterial toxin coronatine that is structurally related to J A
(Figure 1.1), fail to express jasmonate-regulated genes, are male sterile, and highly
susceptible to insect herbivores and fiingal pathogens (Feys et al., 1994; Benedetti et al.,
1995; McConn et al., 1997; Thomma et al., 1998). Further, exhaustive genetic screens in
Arabidopsis for mutants that affect jasmonate responses in both defense and development
have only yielded alleles of coil (Ellis and Turner, 2002). This finding suggests that
COIl plays a central role in jasmonate signaling and that there is genetic redundancy in
other components of the pathway. Given that the ubiquitin/proteasome pathway is well-
conserved among all eukaryotes, we hypothesized that Jail corresponds to the tomato

ortholog of AtCOIl .

In this chapter, a candidate gene approach was used to test whether jai 1 is a
mutation in the LeCOII gene. Analysis of the full-length LeCOII cDNA indicated that it
encodes a 603 amino acid protein that is 67% identical to AtCOIl. DNA sequencing of

LeCOIl from jail -2 plants revealed a single base-pair mutation that results in the

201

Gly261—»Cys amino acid substitution. The jail -I mutation was found to be a 6.2-kb

deletion in LeCOIl that completely abolished expression of the gene. Agrobacterium-
mediated transformation of jail -1 plants with the LeCOII cDNA driven by the CaMV
358 promoter restored MeJA-induced PI expression, fertility and glandular trichome
development. These findings demonstrate that jasmonate signaling and jasmonate-

regulated processes in tomato require LeCOIl.

202

Materials and Methods

Plant material and treatments

Tomato seedlings (Lycopersicon esculentum Mill cv Micro-Tom and cv

Castlemart) were grown under 17 h days at 27 °C with light at 200 pmol m.2 sec-1 and 7

h at 16 °C in darkness. Because the jai 1 mutants are sterile, they are maintained as
heterozygotes (crossed with wild-type). The jail -1 plants used in this study were derived
from the original mutant 406A (Li et al., 2001; Howe et al., 2002) backcrossed three

times using wild-type plants (cv Micro-Tom) as the recurrent pistillate parent. The jail-2
plants were derived from the original 124A mutant (Howe and Ryan, 1999; Li et al., 2001)
backcrossed twice using wild-type plants (0v Castlemart) as the recurrent pistillate parent.

All experiments involving jail mutants were performed with homozygous lines. For
selection of jai [-1 mutant plants in F2 populations, resistance to inhibition of root growth
and anthocyanin accumulation by MeJ A was assayed as described in the Materials and
Methods of Chapter 3. Homozygous jai1-2 plants were selected from the F2 populations

by assaying wound-induced PI-II accumulation in cotyledons of twelve-day-old plants.
MeJ A treatment of adult plants (three-week-old wild-type plants or transgenic plants after
transferring to soil for three weeks) was performed as described in the Materials and

Methods of Chapter 3.

203

Molecular biological techniques

A 1.1-kb tomato EST clone (AI48297 8) that shows significant sequence identity
with the 3' end of AtCOIl was used to screen a Bacterial Artificial Chromosome (BAC)
library (Budiman et al., 2000). A single BAC clone (BAC24909) was identified. A 9.5-
kb sequence was obtained from this BAC clone by sequencing several polymerase chain
reaction (PCR) products designed to cover the LeCOII locus. Two open reading frames
(ORFs) in this 9.5-kb region were revealed by the GENSCAN program
(http://genes.mit.edu/GENSCAN.html): one is LeCOIl and the other is annotated as a
putative Myb transcription factor (AI488165) that is transcribed in the opposite direction

relative to LeCOIl .

Two primers, C1 (5'- CGG GAT CCC TCT CCT CCA TCT TCT TCA A) and C2
(5'- CGA GCT CAT ACA TAT GGA CAA GAC ACC T), were designed according to
the LeCOII sequence to amplify the cDNA by reverse transcription-PCR (RT-PCR). Five
pg of total RNA isolated from tomato leaf tissue was reverse transcribed using the
Enhanced Avian HS RT-PCR-20 Kit (Sigma) and C2 primer as recommended by the
manufacturer. cDNA products of the reaction were used as template for a PCR reaction
that employed the primers C 1 and C2. The resulting 2,044-kb PCR products were ligated
into pGEM-T vector (Promega) to generate plasmid pGEM-COI]. The C011 cDNA
insert was sequenced in its entirety by primer walking. To obtain the CO]! cDNA from
jai1-2, the same set of primers, C1 and C2, was used for RT-PCR. The PCR products
were cloned into vector pGEM-T for sequencing. Three primers, CS (5'- GAG GCA

ATA TGT GGA TTT GAT GGA), C6 (5'- CCA CAC CGT GTT CTT TTG AAG TGG

204

A) and C7 (5'- GGA GAC GAT ATG TTG AGA CTA AGT) and the arbitrary primer
AD2 were used in a thermal asymmetric interlaced-PCR (TAIL-PCR) reaction to clone a
fragment of DNA from jail -1 plants. This fragment was cloned into pGEM-T and

sequenced.

Transformation

The pGEM-COI] plasmid harboring the full-length LeCOIl cDNA was digested
with BamHI and Sstl. The resulting 2,040-bp fragment containing the cDNA was cloned
into the BamHI and Sstl sites of the binary vector pBIlZ] (Clontech), replacing the GUS
reporter gene. The resulting construct was introduceded into Agrobacterium tumefaciens
strain AGLO (Lazo et al., 1991). Transformation of cotyledon explants (cv Micro-Tom)

was performed as previously described (Li and Howe, 2001). Twenty-eight independent

primary transformants (T1) were regenerated on kanamycin-containing medium and

transferred to soil. Introduction of the transgene was confirmed by a PCR assay using a
primer set of C3 (5'- CTG CAA GTT AGG GCT GAA GAT CTT) and C4 (5'- GGC
CAA GCA CTT CCA ATC CTC TAT). These primers were designed to amplify a 1116-

bp and a 433-bp product from the endogenous COII gene and the 35S::LeCOIl transgene,

respectively. T1 plants were then tested for MeJA-induced PI-II accumulation and were

subsequently transferred to the greenhouse for collection of T2 seeds.

205

RNA and DNA blot analyses

Total RNA was isolated from tomato tissue and analyzed by RNA blot
hybridization as described in the Materials and Methods of Chapter 2. The full-length
LeCOII cDNA was used as a probe for detection of the corresponding transcripts.
cDNAs for the Prosystemin and eIF 4 genes were used as loading controls. Genomic
DNA purification and blot analysis were also performed as described in the Materials
and Methods of Chapter 2. Five pg aliquots of DNA were digested with either BglII or
EcoRI and hybridized with either the full-length LeCOIl cDNA or different portions of

the LeCOII gene as indicated in the text.

206

Results

The tomato C011 gene

Identification of AtCOIl as an essential component of j asmonate signaling
implies that the well-conserved ubiquitin-mediated proteolysis pathway plays an
important role in transducing the jasmonate signal. Many phenotypes of the jail -1 plants,
such as altered expression of JA-responsive genes, alleviated root grth inhibition by
MeJ A, compromised resistance to herbivores, and reduced pollen viability in jail-I
plants, are similar to those of the Arabidopsis coil mutants. These observations prompted
us to hypothesize that Jail corresponds to the tomato ortholog (LeCOII) of C01] . As a
first step to test this, we isolated a full-length LeCOIl cDNA. The 2,050 bp cDNA
contained a 1,812 nucleotide open reading frame (ORF) that is predicted to encode a 603
amino acid protein. The deduced primary sequence of LeCOIl shares 67.2% sequence
identity with AtCOIl (Figure 6.1). The region of conservation is spread throughout the
protein. The secondary structure of LeCOIl is virtually identical to that of AtCOIl, as
both contain a degenerate F-box motif at the N-termini and 16 imperfect LRRs that

comprise almost the entire remainder of the proteins (Figure 6.1; Xie et al., 1998).

Extensive DNA blot analyses (Figure 6.2b and data not shown) and EST database
searches (www.tigr.org) suggest that LeCOII is a single copy gene in tomato. Screening
of a bacterial artificial chromosome (BAC) library of tomato genomic DNA (Budiman et
al., 2000) yielded a single positive clone (BAC24909). DNA sequence obtained from
BAC24909 indicated that LeCOII is composed of 3 exons and 2 introns (Figure 6.2a),

similar to the structure of AtCOIl. The size of LeCOI] exons (482, 508, and 822 bp for

207

Figure 6.]. Sequence alignment of AtCOll and LeCOIl. Identical amino acids are
shown in black boxes while similar residues are shaded. The F-box motif is underlined.

* indicates the position (residue 261) of the mis-sensejai1-2 mutation.

208

 

 

LeCOIl
AtCOIl

LeCOIl
AtCOIl

LeCOIl
AtCOIl

LeCOIl
AtCOIl

LeCOIl
AtCOIl

LeCOIl
AtCOIl

LeCOIl
AtCOIl

LeCOIl
AtCOIl

LeCOIl
AtCOIl

LeCOIl
AtCOIl

LeCOIl
AtCOIl

  

118
121

178
I81

238
241

298
292

358
352

418
412

478
472

538
532

596
589

Figure 6.1

209

1 IE

 
 

NNLREI: it

 

exon 1, 2, and 3, respectively) is similar to that of AtCOIl exons, but the LeCOIl introns
(3,078 and 683 bp for intron 1 and 2, respectively) are much larger than their AtCOIl
counterparts. Located approximately 3 kb downstream of the LeC 011 stop codon is a
small ORF annotated as a Myb transcriptional factor, whick is transcribed in the opposite

direction relative to LeCOIl as indicated by the GENESCAN program (Figure 6.2a).

jai1 plants harbor mutations in LeCOIl

As shown in Figure 6.2b, DNA blot analysis using full-length LeCOIl cDNA as a
probe detected three BglII-derived restriction fragments with an additive size of ~ 12 kb
from wild-type DNA. In jail -1 plants, a single 5.5 kb band was detected. This result
indicated a partial deletion of LeCOIl in jail -1 plants. Further DNA blot analyses using
different portions of the LeCOIl gene as a probe revealed the presence of the first exon
and the downstream intergenic region but not the remainder of the gene in jail -1 plants.
This observation prompted us to perform TAIL-PCR reactions from jai1-l DNA using
forward primers annealing to the middle region of intron 1. We expect the PCR product
to contain sequences flanking the deletion. Indeed, the 1.3-kb PCR product thus obtained
contained sequence identical to part of wild-type intron 1 fused with sequence located
near the putative Myb gene (Figure 6.2a). The sequence of the wild-type DNA in this
region indicated that jail -1 corresponds to 6,243 bp deletion, which removes 3' half of
intron 1, exon 2, intron 2 and exon 3, is deleted in jail -1 plants (Figure 6.23). Consistent
with with this scenario, RNA blot analysis showed that accumulation of LeCOIl

tmascript was completely abolished in jail —1 plants, which was readily detected in all

210

Figure 6.2.jai1-I plants harbor a 6,243 bp deletion in the LeCOIl locus. (a)
Schematic diagram of LeCOIl on chromosome 5. El, E2, and E3, LeCOIl exon],

exon2, and exon3. Myb, an open reading framing encoding a putative Myb gene located
adjacent to LeCOIl . (b) DNA blot analysis of wild-type (1), jail -I (2), and F1 hybrid (3)

plants. BglII—digested DNA was probed with the full-length COll cDNA (A), exon]
(B), exon3 (C), the intergenic space between exon3 and Myb (D), and the Myb ORF

(E). The position of migration of molecular weight standards (kb) is shown on the left.

211

 

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212

tissues examined in wild-type plants (Figure 6.3). The jail-l deletion is not likely to
affect the putative Myb gene, as the deletion occurs more than 500 bp away from the stop

codon of the Myb gene (Figure 6.2a).

The jail -2 mutant was identified from a genetic screen for suppressors of
35S::prosys-mediated responses (Howe and Ryan, 1999; Li et al., 2001). jai1-2 plants
were insensitive to jasmonates, deficient in wound responses, and female sterile (Howe
and Ryan, 1999; Li et al., 2001). To determine whether LeCOIl is affected in jail -2
plants, we obtained the LeCOIl cDNA from these plants by RT-PCR. Sequence

comparison between the jai [-2 C011 cDNA and that of wild-type identified a mis-sense

change (G—rT) that convertes Gly261 in the 12th LRR to a Cys (Figure 6.1). This

polymorphism was confirmed by sequencing PCR products amplified from wild-type and
jai1-2 genomic DNA (data not shown). The jail -2 mutation did not abolish the
expression of LeCOIl as indicated by the RT-PCR reaction (data not shown).

Nevertheless, jail -2 exhibited complete insensitivity to MeJA treatment regarding

defense gene activation (Figure 5.1), indicating that residue Gly261 is critical for the

function of LeCOIl. Taken together, these results demonstrate that the jai [-2 allele is a

mis-sense mutation in the LeCOIl gene.

213

Figure 6.3. RNA blot analysis of LeCOIl transcript in tissues of wild-type and jail-
1 plants. R, roots; P, petioles; L, leaves; F, flower buds, S, sepals; and G, green fruits.
Probes representing the prosystemin gene (Prosys) and eIF 4 were used as loading

controls.

214

 

wild-type jai1-1

 

 

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217

Molecular complementation of the jail -I mutation by LeC OI I

To confirm that LeCOII corresponds to Jail , the wild-type LeCOIl cDNA was
cloned into the plant transformation vector pB1121 under control of the CaMV 358
promoter. The resulting construct (35S: :LeCOII) was introduced into jai1-1 plants by

Agrobacterium-mediated transformation. A total of 28 independent primary transgenic

plants (T1) were obtained. The presence of the 35S::LeC011 transgene in these plants

was confirmed by a PCR assay that detects a transgene-specific PCR product (Table 6.1).

Upon treatment of T1 plants (four-week-old) with MeJA, seven of the 28 T1 plants

showed no accumulation of PHI in their leaves. PI-II accumulation in the other 21 T1

plants ranged from 30 to 220 ug/ml leaf juice (Table 6.1). As a control for this
experiment, jai1-1 plants grown under the same condition produced no PI-II in response
to MeJ A treatment, whereas wild-type plants accumulated around 160 pg PI-II/ml leaf
juice (data not shown). These resultes indicated that MeJA-induced PI-II accumulation
was restored in these 21 lines. All the 28 primary transgenic plants flowered and set fruit.
However, glandular trichomes were observed on developing fruits from only 10 of these
plants (Table 6.1; also see Figure A1). Fertility was restored in 12 of the primary
transgenic plants as determined by the recovery of mature seed from these plants (Table
6.1). Taken together, these experiments demonstrate that the jail mutant phenotypes are
caused by the loss-of-function of LeCOI. All seven plants that lacked MeJA-induced PI-
11 were sterile and defective in trichome development. Thus, it is likely that the transgene

is not expressed in these lines.

218

To determine the copy number of the transgene in representative primary

transgenic lines, four T1 plants in which all jail -I phenotypes were complemented were

subjected to DNA blot analysis. When genomic DNA from wild-type plants was digested
with EcoRI and probed with the full-length LeCOIl cDNA, two hybridizing bands were
detected (Figure 6.4). Since the 3' half of C011 is deleted in jail -1 plants, only the higher
molecular weight EcoRI band corresponding to the 5' half of LeCOIl was detected
(Figure 6.4). The four transgenic plants (in jai [-1 background) all contained hybridizing
bands in addition to the jail -1-specific band, indicating that they all contained the
transgene (Figure 6.4). The different hybridizing pattern of the four lines further indicated
that these transgenic plants were derived from independent transformation events. As
there is no internal EcoRI site within the LeCOIl cDNA (i.e. the transgene), the number

of these additional bands should correspond to the copy number of the transgene. As

shown in Figure 6.4, T1-08 and T1-13 appear to each have two copies of the transgene,

whereas T1-10 and T1-19 appeare to contain multiple copies of the transgene. Therefore,

T1-08 and T1-13 were chosen for further analysis.

The progeny (T2) from T1-08 and T1-13 each segregated for plants with and

without the transgene as revealed by the PCR assay (data not shown). RNA blot analysis

showed that T2 plants containing the 35S: :LeC 011 transgene also accumulated LeCOIl

transcript, whereas T2 plants without the transgene did not. Further inspection by RNA

blot analysis revealed that T2 plants containing the transgene accumulated high levels of

219

PI—II transcript in response to Me] A treatment whereas T2 plants that lacked the

transgene were deficient in MeJA-induced PI-II expression (Figure 6.5). These results

established a correlation between the presence of the transgene and the MeJA-induced

PI-II expression in the T2 generation, and thus confirmed that the jai1 phenotypes are

caused by the loss of function of LeCOIl .

220

Figure 6.4. DNA blot analysis of the primary jai1-I transgenic plants expressing

35S::LeC011. Genomic DNA from wild-type,jai1-1, and four representative T1 plants

was digested with EcoRI, and probed with the full-length LeC 011 cDNA. The position

of migration of molecular weight standards (kb) is shown on the left.

221

 

2-;
2-:
or;
8-:
0-20.0
8.0.0:;

_
0

11.

 

Figure 6.4

222

Figure 6.5. RNA blot analysis of representative T2 transgenic plants. Total RNA

was prepared from wild-type (WT), jai1 -I (fail), and T2-08 and T2-13 plants before (-)

and after (+) exposure to MeJ A vapor for 12 h. The T2 transgenic plants were scored

for the presence (+) or the absence (-) of the 35S::LeC011 transgene by the PCR assay
prior to MeJA treatment. cDNAs for PI—II and LeCOIl were used as probes. eIF 4A was

the loading control.

223

 

l Ru

barbs»

WT jai1 T1'08 T1-13

 

Transgene + + _ _ + + _ _

MeJA _+_+_+_+_+_+

' ' ' PI-II

LeCOI1

2‘.

Figure 6.5

224

Discussion

Selective proteolysis performed by the ubiquitin-proteasome pathway plays a key
regulatory role in numerous cellular processes in both animals and plants. In this system,
ubiquitin serves as a reusable tag to target proteins for degradation by the proteasome
(Voges et al., 1999). Covalent attachment of ubiquitin to substrate proteins involves a
cascade of three protein complexes called ubiquitin activating enzyme (E1), ubiquitin
conjugating enzyme (E2) and ubiquitin ligase (E3; Voges et al., 1999). SCF type of E3
enzymes represent one of the best characterized classes, which are composed of four core
subunits: a §kp1-like adaptor protein; the cullin subunit gull; an E-box containing

protein; and a RING-finger protein called be1 (Deshaies, 1999). The structure of

2 .
SCFSkp (superscript denotes the F-box protein) has been recently determmed. The

complex adopts an elongated shape, with Cull forming the scaffold and bel and the
Skpl/Skp2 complex positioned to opposite ends (Zheng et al., 2002). bel and Cull
constitute the heterodimeric core, which can interact with distinct substrate-recognizing
units (such as the Skpl/F-box protein complex) to form a large number of E3 complexes

(Deshaies, 1999; Zheng et al., 2002).

Isolation and characterization of the coil mutants in Arabidopsis established an

important role of C01] in jasmonate signaling. Identification of C011 as a LRR-

. . . . . . . . . . . COIl .
contalmng F-box protein implied its part1c1pation 1n the formation of SCF (Xie et al.,

1998). This hypothesis was later supported by biochemical experiments demonstrating

the direct physical association of AtCOIl with AtCULl , Atbel , and either of the

225

 

Arabidopsis Skpl-like proteins ASKl or ASK2 (Devoto, et al., 2002; Xu et al., 2002).

Recent demonstration of the physical association of SCFC011 with the COP9

signalosome and the genetic requirement of the signalosome in jasmonate signaling

. . COIl . . . . .
further sustamed the notion that SCF IS the central mediator of Jasmonate sngnalmg

(Feng et al., 2003).

In the current chapter, we reported that Jail corresponds to the tomato ortholog of
AtCOII . This conclusion was based on the fact that LeCOIl shares high sequence
identity with AtCOIl and that mutations in either gene affect jasmonate signaling.
Interestingly, mutations in the C 011 gene in both tomato and Arabidopsis affect
jasmonate signaling in both vegetative and floral tissues (Chapter 3; Feys et al., 1994; Xie
et al., 1998). These observations indicated that the core components of j asmonate

signaling pathway are conserved in leaves and floral organs. Based on the biochemical

function of SCFCOII, a model for jasmonate signaling has been proposed in which

transcriptional repressors of j asmonate-responsive genes would be targeted for

degradation by SCFC011 in the presence of jasmonates, allowing transcription of these
genes (Creelman, 1998; Xie et al., 1998). Were this model to hold true, it can be
proposed that in vegetative and floral tissues, COIl selectively removes different

transcriptional repressors to activate different transcriptomes that control diverse

physiological events.

A major gap in understanding the j asmonate signal transduction pathway regards

the identification of C011 -interacting proteins that are presumably targeted for

226

 

ubiquitination and subsequent degradation in the 26S proteasome. Efforts have been
made in Arabidopsis using the yeast two-hybrid technique to isolate C011-interacting
proteins (Devoto etal., 2002). This study identified the SKPl homologues and a histone
deacetylase, though the functional relevance of this histone deacetylase in the context of
jasmonate signaling has yet to be determined (Devoto et al., 2002). With the adoption of
protein-complex purification methods such as the Tandem Affinity Purification-tag
system (Rigaut et al., 1999) in plants, it can be foreseen that fiiture biochemical
approaches will help bridge the gap and provide new insights into the jamonate signal

transduction pathway.

227

References

Benedetti CE, Xie D, Turner JG (1995) C011-dependent expression of an Arabidopsis
vegetative storage protein in flowers and siliques and in response to methyl jasmonate.
Plant Physiol. 109, 567-572.

Budiman MA, Mao L, Wood TC, Wing RA (2000) A deep-coverage tomato BAC library
and prospects toward development of an STC framework for genome sequencing.
Genome Res. 10, 129-136.

Creelman RA (1998) J asmonate perception: characterization of coil mutants provides the
first clues. Trends Plant Sci. 3, 367-368.

Deshaies RJ (1999) SCF and cullin/RING H2-based ubiquitin ligases. Annu. Rev. Cell.
Dev. Biol. 15, 435-467.

Devoto A, Nieto-Rostro M, Xie D, Ellis C, Harmston R, Patrick E, Davis J, Sherratt L,
Coleman M, Turner JG (2002) C011 links j asmonate signaling and fertility to the
SCF ubiquitin-ligase complex in Arabidopsis. Plant J. 32, 457-466.

Ellis C, Turner JG (2002) A conditionally fertile coil allele indicates cross-talk between
plant hormone signalling pathways in Arabidopsis thaliana seeds and young
seedlings. Planta 215, 549-556.

Feng F, Ma L, Wang X, Xie D, Dinesh-Kumar SP, Wei N, Deng XW (2003) The COP9

signalosome interacts physically with SCFCOIl and modulates j asmonate responses.
Plant Cell 15, 1083-1094.

Feys BJF, Benedetti CE, Penfold CN, Turner JG (1994) Arabidopsis mutants selected for
resistance to the phytotoxin coronatine are male-sterile, insensitive to methyl
jasmonate, and resistant to a bacterial pathogen. Plant Cell 6, 751-759.

Howe GA, Lee GI, Itoh A, Li L, DeRocher AE (2000) Cytochrome P450-dependent
metabolism of oxylipins in tomato: cloning and expression of allene oxide synthase
and fatty acid hydroperoxide lyase. Plant Physiol. 123, 711-724.

Howe GA, Li L, Lee GI, Li C, Shaffer D (2002) Genetic dissection of induced resistance
in tomato. In “Induced resistance in plants against insects and diseases”. A. Schmitt &
B. Mauch-Mani, Eds. Vol. 26, pp. 47-52.

Howe GA, Lightner J, Browse J, Ryan CA (1996) An octadecanoid pathway mutant (J L5)
of tomato is compromised in signaling for defense against insect attack. Plant Cell 8,
2067-2077.

228

Howe GA, Ryan CA (1999) Suppressors of systemin signaling identify genes in the
tomato wound response pathway. Genetics 153, 1411-1421.

Li L, Howe GA (2001) Alternative splicing of prosystemin pre-mRNA produces two
isoforms that are active as signals in the wound response pathway. Plant Mol. Biol.
46, 409-419.

Li L, Li CY, Howe GA (2001) Genetic analysis of wound signaling in tomato: evidence
for a dual role of j asmonic acid in defense and female fertility. Plant Physiol. 127,
1414—1417.

McConn M, Creelman RA, Bell E, Mullet JE, Browse J (1997) J asmonate is essential for
insect defense in Arabidopsis. Proc. Natl. Acad. Sci. USA 94, 5473-5477.

Rigaut G, Shevchenko A, Rutz B, Wilrn M, Mann M, Séraphin B (1999) A generic
protein purification method for protein complex characterization and proteome
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Thomma B, Eggermont K, Penninckx I, MauchMani B, Vogelsang R, Cammue BPA,
Broekaert WF (1998) Separate jasmonate-dependent and salicylate-dependent
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microbial pathogens. Proc. Natl. Acad. Sci. USA 95, 15107-151 1 1.

Voges D, Zwickl P, Baumeister W (1999) The 26S proteasome: a molecular machine
designed for controlled proteolysis. Annu. Rev. Biochem. 68, 1015-1068.

Xie DX, Feys BF, James S, Nieto-Rostro M, Turner JG (1998) C011: an Arabidopsis
gene required for jasmonate-regulated defense and fertility. Science 280, 1091-1094.

Xu LH, Liu F Q, Lechner E, Genschik P, Crosby WL, Ma H, Peng W, Huang DF, Xie DX

(2002) The SCFC011 ubiquitin-ligase complexes are required for jasmonate response
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229

CHAPTER 7

Conclusions and Future Directions

230

The past decade saw the transformation of JA from a little regarded secondary
metabolite to a well-recognized phytohormone with a dual role in plant defense and
development. While the large volume of literature on J A has solidified our understanding
of the chemistry and biosynthetic pathway of J A (reviewed by Creelman and Mullet,
1997; Walling, 2000; Schaller, 2001; Berger, 2002; Turner et al., 2002; Wastemack and
Hause, 2002; Webber, 2002), additional research is needed to elucidate the mechanisms
that regulate the production and action of j asmonates in response to developmental and
environmental cues. The current dissertation, with the use of tomato (L. esculentem) as a
model system, is focused on the identification of components of the jasmonate signaling
pathway using a forward genetic approach, and on exploring J A perception mutants to

dissect roles of jasmonates in plant defensive and developmental processes.

Two allelic J A insensitive mutants, called jai1, are described (Chapter 3, 4, and 6).
Several lines of evidence indicated that Jail corresponds to the tomato ortholog (called
LeCOIl) of AtCOII . First, the deduced LeCOIl sequence shares high identity with that of
AtCOII (Figure 6.1). Second, both jai 1-1 and jai 1 -2 were found to be mutations in
LeCOIl (Figure 6.1). Third, stable transformation of jail -1 plants with the LeCOIl
cDNA restored all of the defective phenotypes of the mutant (Figure 6.5, A1, and Table

6.1). Identification of C011 as an F—box protein implies its participation of formation of

SCFCOIl (Xie et al., 1998). This hypothesis has been supported by biochemical

experiments that demonstrate direct physical association of AtCOIl with other SCF
subunits: AtCULl , Atbel, and either of the Arabidopsis Skpl-like proteins ASKl or

ASK2 (Devoto, et al., 2002; Xu et al., 2002). Molecular cloning of LeCOIl and

231

. Il . .
phenotypical analysrs of the jail mutants suggest that SCFCO rs also a key mediator of

jasmonate signaling in tomato.

Future experiments aimed at identifying C011-interacting proteins (CIPs) will
likely shed new light on the jasmonate signal transduction pathway. Previous experiments
using the yeast two-hybrid technique have implicated a histone deacetylase as a CIP,
though the functional relevance of this histone deacetylase in the context of j asmonate
signaling remains to be determined (Devoto etal., 2002). An attractive alternative
approach to identify CIPs is to use the Tandem Affinity Purification-tag system (TAP-tag;
Rigaut et al., 1999) to purify C011-containing protein complexes from transgenic tomato
plants that express a TAP-tagged derivative of C011. The major advantage of the TAP-
tag system is the ability to highly purify multiprotein complexes under mild conditions
(Rigaut et al., 1999). CPS purified in this manner can then be identified by mass
spectrometry-based methods. Expression and transgenic analysis of the CIP genes can be

sought to confirm a role for CIPs in jasmonate signaling.

Using the jai1-1 allele, a significant amount of new information regarding the
wound response pathway in tomato has been obtained. Based on studies of the jai1-1
mutant, the wound responsive genes in tomato can be classified into three groups (Figure
3.6). The first group represented by WIPK, is rapidly induced by wounding locally and
systemically in a Jail-independent manner. In contrast, wound induction of the so-called
“late response genes” (Ryan 2000) is completely abolished in jai 1 plants (Figure 3.5).
The third group, or the so-called “early response genes” (Ryan, 2000), requires Jail for

maximum wound induction. This classification is meaningful in that it fits well with the

232

assumed function of these genes. WIPK is likely involved in the initial phase of the
wound response during which J A production is triggered (Seo et al., 1995; 1999). The
early response genes that include J A biosynthetic genes and prosystemin may function to
modulate J A levels. The late response genes, wichi encode proteins with defensive
properties (Bergey et al., 1996; Ryan 2000), are then activated in response to elevated
levels of J A. Additional experiments comparing the expression of these different classes
of wound-inducible genes in response to various elicitors of the wound response may

help to elucidate the signal transduction events associated with the wound response.

We demonstrated that jai [-1 plants are compromised in resistance to both
chewing insects (e. g. tobacco homworm, Chapter 3) and cell-content-feeding arachnid
herbivores (e. g. two-spotted spider mite; Howe GA, unpublished data). These results
indicate that jasmonate signaling is necessary for cellular processes that are essential for
protection of tomato to a broad spectrum of arthropod herbivores. This adds to our
knowledge that the octadecanoid pathway for JA biosynthesis is important in defense of
cultivated tomato against herbivores (Howe et al., 1996; Li et al., 2002). Together, these
findings establish a central role of j asmoantes in mediating tomato resistance to
herbivores. Future experiments using the jail mutants to identify all the target genes that
require COIl for J A- or herbivore-induced expression might reveal novel aspects of the
defense process. For instance, microarray analysis can be conducted to compare the
expression profiles of wild-type and jail -1 plants subjected to wounding or MeJ A
treatment. Identification of novel genes that are differentially regulated in wild-type and
jai1-1 plants will likely provide clues to the cellular and metabolic processes that are

regulated in a C011-dependent manner.

233

The jail mutant was used to gain insight into the role of JA in systemic wound
signaling. In one set of experiments, wild-type, jail and spr2 (defective in JA synthesis;
Howe and Ryan, 1999) plants were reciprocally grafted. Analysis of wound-induced PI
expression in these chimeric plants showed that jai 1 plants lack the capacity to recognize
the graft-transmissible wound signal, but are not affected in the generation of the signal.
In contrast, spr2 displayed the reciprocal phenotype; they can perceive the long-distance
signal but are unable to generate it (Figure 5.3, 5.4). Thus, it appears that JA biosynthesis
in response to wounding is required for the production of a mobile signal whose
functional recognition is distal leaves depends on JA signaling (Figure 5.5). This finding
is consistent with the hypothesis that a member of the j asmonate family of oxylipins acts
as an essential component of the transmissible wound signal. Future grafting experiments
using other J A biosynthetic mutants as they become available (e. g. the tomato opr3
mutant) may help pinpoint which jasmonate species is involved in the transmission of the

the long-distance wound signal.

The finding that jasmonates could act as a mobile wound signal for defense gene
activation further demonstrated the genetic complexity of wound signaling in tomato.
Incorporating this new information, the most recent model of the systemic wound
response pathway proposes that both systemin and J A are released at wound sites, with
systemin being processed from prosystemin and JA initially produced from the
degradation of membranes (Ryan and Moura, 2002). Given that both systemin (McGurl
et al., 1992) and J A (Chapter 5) are important for systemic wound signaling, and that
systemic and JA could trigger the production of each other, it is proposed that systemin

and JA would both move away from the wound site and modulate the production of each

234

other thought positive feedback that results in the mutual amplification of systemin and

J A as a cascade along the stems and petioles (Ryan and Moura, 2002). The process would
eventually be limited by the presence of extracellular systemin-inactivating enzymes

(J anzik et al., 2000). Elevated levels of JA in the vascular bundle cells in the distal leaves
could then diffuse to nearby palisade and mesophyll cells where the defense proteins are

synthesized and compartmented (Ryan 2000; Ryan and Moura, 2002).

The role for jasmonates in regulating several developmental processes in tomato
was revealed by analyzing the jai 1 mutants. The surface of wild-type tomato leaf, stem,
sepal and developing fi'uit contains abundant glandular trichomes that provide an
important constitutive anti-herbivore defense (Figure A1; Kennedy, 2003). However,
jai1-1 immature fruit is completely devoid of trichomes whereas trichome density on the

jail -1 green tissues is significantly reduced (Figure 3.1). This phenotype likely
contributed to the compromised resistance of jail plants to herbivores. Agrobacterium-
mediated transformation of jail -I with the LeCOIl cDNA complemented the trichome
defects in the mutant plants (Figure A1 and Table 6.1). These results demonstrated, for
the first time, a role of the IN C011 pathway in trichome development. In this context,
analysis of the effects of the jail -I mutation in the wild species L. hirsutum, which has
different types of glandular trichomes, may provide further information regarding the role

of j asmonate in trichome development and in trichome-based defense mechanisms.

Another novel phenotype of jail plants is female sterility. Fruit production and
development on jai1-1 plants depended on pollination with either wild-type or mutant
pollen, though the fruit lacked viable seed. Plants homozygous for jai1 -2 also exhibited

female sterility, albeit with reduced severity (Chapter 4). Although jai l pollen showed

235

reduced germination and viability, reciprocal crosses between wild-type and jail plants
unambiguously demonstrated that sterility of the mutant resulted fi'om a defect in female
reproductive development (Chapter 3, 4). The fact that ripened jail fruit contained
numerous undeveloped seeds suggests that pollination and early embryonic development
of jaz' 1 plants are normal. Rather, defects in late embryonic development or seed filling
might contribute to the sterility phenotype. The sporophytic nature of jai 1 sterility
suggests that jasmonate-regulated processes in maternal tissue are required for embryo

development in tomato.

A proposed role for J A in female reproductive development in tomato contrasts
the well-documented studies in Arabidopsis where JA biosynthesis and perception are
essential for male, but not female, gametophyte development (McConn and Browse, 1996;
Feys et al., 1994; Sanders et al., 2000; Stintzi and Browse, 2000; Ishiguro et al., 2001;
Park et al., 2002; von Malek et al., 2002). Significantly, we found that jai1 flower buds
and ovaries were completely deficient in the expression of PI and other defense gene,
indicating that constitutive activation of defense-related genes in reproductive tissues
requires C011 (Figure 3.4). The expression pattern of PI is in keeping with the level of
endogenous J A, which is high in young apical sink tissues and reproductive structures but
inducible in older parts of the plant (Creelman and Mullet, 1997; Hause et al., 2000).
Thus, PIs provide a convenient biochemical marker for the activity of the JA signaling
pathway, and suggest a scenario in which defense gene activation is common to the
physiological processes controlled by C011 in both vegetative and reproductive organs.
Interestingly, this group of genes, including P15 and several other defense genes (Bergey

et al., 1996), is not found in Arabidopsis (van der Hoeven et al., 2002). One possibility is

236

that this cocktail of defense proteins could serve as seed storage proteins during embryo
development. Alternatively, some of the JA/COIl regulated hydrolytic enzymes might be

required for nutrient release during seed development.

The range of physiological processes controlled by jasmonates may ultimately
reflect the function of specific genes whose‘expression is regulated by the hormone in a
tissue- or cell type-specific manner. The well-characterized wound response pathways of
tomato, together with the availability of j asmonate signaling mutants, should provide
useful tools to investigate the molecular mechanisms by which jasmonates regulate
diverse physiological processes. Future experiments aimed at identifying all C011-
dependent jasmonate-responsive genes and C011—interacting proteins in tomato will no
doubt add to our understanding of the function of j asmonate signaling. Comparison of
this information with that obtained fi'om other plant systems would provide greater
insight into the evolution of plant defense signaling. This knowledge would have an
enormous potential to shape our understanding of the biological characteristics of plant
species found in various ecosystems. This knowledge may also be applied in ways that

will enhance agronomic traits that exploit natural defense strategies.

237

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240

APPENDIX

241

Glandular trichomes are specialized multi-cellular appendages occurring on many
vascular plants that produce and secrete secondary metabolites (Dell and McComb, 1978;
Kelsey et al., 1984; Wagner, 1991; Phillips and Croteau, 1999). These phytochemicals
have been implicated in attracting pollinators, reducing leaf temperature or water loss in
desert plants, repelling or intoxicating pests, and tritrophic interactions (Kelsey et al.,
1984; Kennedy, 2003). In wild tomato species (e. g. L. hirsutum and L. pennellii),
glandular trichomes have been extensively implicated in the resistance to various
anthopod insects (Williams et al., 1980; Carter and Snyder, 1985; Snyder and Carter,
1985; Weston et al., 1989; Kennedy 2003). In particular, type VI trichomes have been
found to physically entrap small insects such as aphids (McKinney, 1938). The tips of
type VI trichomes contain various compounds with anti-herbivore properties. For
instance, sesquiterpenes from type VI trichomes of L. hirsumtumf typicum were found to
contribute to resistance against beet arrnyworrn and Colorado potato beetle (Carter et al.,
1989; Eigenbrode et al., 1996). The phenolic compounds in trichomes, when released by
insect damage, can be toxic to the insect or can reduce the nutritive value of leaves
(Duffey and Isman, 1981; Felton and Duffey, 1991). The most potent trichome toxin
identified is the methyl ketones 2-tridecanone (Williams et al., 1980). Comprising up to
90% of the tip contents of type VI trichome of the accession P1134417 of L. hirsutum, 2-
tridecanone has been found to be acutely toxic to a wide range of herbivores (Williams et

al., 1980; Dimock and Kennedy, 1983).

Contrasting our knowledge of the physiological roles of glandular trichomes, the
molecular mechanism by which glandular trichome development and content are

regulated is obscure. Some studies have found that traits controlling 2-tridecanone levels

242

are linked to those controlling trichome density (Nienhuis et al., 1987; Kauffrnan and
Kennedy, 1989). Here we report that a tomato mutant (jai1-1) defective in the perception
of j asmonates (Li et al., 2001; Li et al., 2002) is defective in the production of glandular
trichomes. Molecular cloning of the gene defined by jai1-1 identified it as the tomato
ortholog(LeC011) of the Arabidopsis CORONA T [NE INSENSITIVE] gene that encodes
an F-box protein involved in ubiquitin-dependent proteolysis (Xie et al., 1998; Xu et al.,
2002). These results establish a role for the jasmonate signaling pathway in glandular
trichome development and suggest a strategy to genetically modify the content of gland-
derived compounds, which are widely used in products such as pesticides, medicines,
flavor and aroma substances, and cosmetic ingredients (Kelsey et al., 1984; Duke et al.,

1999; Mahmoud and Croteau, 2001).

Two types of glandular trichomes (type I and VI) are present on the aerial
surfaces of cultivated tomato (L. esculentum). The type I trichome has an elongated stalk
and a unicellular gland, whereas the type VI trichome has a relatively short stalk and a
four-celled glandular head (Figure A1). Type VI trichomes are the most conspicuous
glandular type on stems, leaves (Figure Al; Snyder and Carter, 1985) and sepals (Figure
A1). On the surface of immature green fruit, both type I and VI are abundant (Figure Al).
Strikingly, neither type of trichome is present on jail -I fruits, resulting in the glabrous
appearance of the fruits (Figure A1). The density of type VI on jail -I sepals and leaves is
reduced to approximately 30% that of wild-type tissues (Figure A1). In addition, the size
of type VI trichome on fat] -1 sepals and leaves of was smaller and their stalks shorter as
compared with those of the wild-type (Figure A1). To determine whether the jail -I

deletion is responsible for the trichome-related phenotypes of the mutant, Agrobacterium-

243

mediated transformation was used to introduce a 35S::LeC011 transgene into jail -1
plants. As shown in Table 6.1 and Figure Al, the LeCOIl cDNA restored trichome
development. These results provide conclusive evidence that COIl plays an essential role

in glandular trichome development in cultivated tomato.

244

 

Figure A1. Scanning electron micrograph of wild-type and jai1-1 plants. Scanning
electron micrography (SEM) was performed using a JEOL 6400V scanning electron
microscope (Tokyo, Japan) at an accelerating voltage of 15 kV. To examine the general
pattern of trichome distribution on leaves, sepals and green fruit, small pieces of
representative tissues (5x5 mm) were fixed in 4% glutaraldehyde in 20 mM sodium

phosphate buffer (pH 7.4), dehydrated through an ethanol series, critical point dried in

C02, and coated with gold using an EMSCOPE SC500 sputter coater (Ashford, UK). (a)

Developing wild-type fruit containing both type I and type VI glandular trichomes. (b)
Picture of green fruit from j ail-1 plants, which appears glabrous due to the lack of
trichomes. (c) Complementation of the glabrous phenotype in transgenic jail-I plants
expressing the 35S: : C011 transgene. (d, e) SEM picture of the surface of wild-type (d)
and jail -I (e) green fruit. (f, g) SEM showing wild-type (t) and jail -I (g) sepals. (h, i)

SEM picture of the surface of wild-type (h) and jail -I (i) young leaves.

245

 

 

Bar = 400 ,um

Figure A1

246

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248