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BIOCHEMICAL AND GENETIC ANALYSIS OF SYSTEMIC WOUND
SIGNALING IN TOMATO (LYCOPERSICON ESCULENTUM)

presented by

Gyu In Lee

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3 6/01 C'JCIRC/DateDquSS-p. 1 5

BIOCHEMICAL AND GENETIC ANALYSIS OF
SYSTEMIC WOUND SIGNALING IN TOMATO
(L YCOPERSICON ESCULENTUM)

By

Gyu In Lee

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Plant Biology

2003

ABSTRACT
BIOCHEMICAL AND GENETIC ANALYSIS OF SYSTEMIC WOUND
SIGNALING IN TOMATO (L YCOPERSICON ESCULENTUM)
By

Gyu In Lee

Tomato plants activate the synthesis of defense proteins such as proteinase
inhibitors (PIS) in response to insect attack. Systemic accumulation of PIS in wounded
plants is mediated by a long-distance signaling pathway that transmits a mobile signal
from the wound site to distal undamaged leaves. Previous studies have established that
the polypeptide signal systemin, which is derived from a precursor protein called
prosystemin, regulates systemic expression of PI genes via the jasmonic acid (J A)

Si gnaling pathway. However, the precise role of J A and systemin in systemic wound
signaling remains unclear. To address this question, two tomato mutants that are
compromised in the systemic wound response were characterized.

The suppressor of prosystemin-mediated responses] (sprI ) mutant was blocked
in J A biosynthesis and subsequent PI gene expression in response to systemin. The
systemin-insensitive phenotype of sprI plants indicates that the SprI gene product plays
a role in linking the perception of systemin to the activation of J A biosynthesis.
Reciprocal grafting experiments between wild-type and sprI plants demonstrated that
this mutant is deficient in the production of the long-distance wound signal in damaged

leaves, rather than the perception of that signal in neighboring undamaged leaves. This

result indicates that systemin acts at or near the site of wounding to increase J A
synthesis to a level that is required for the systemic response.

The role of JA in systemic wound signaling was investigated using the J L-l
mutant that is defective in both J A biosynthesis and wound-inducible expression of PI
genes. Gas chromatography-mass spectrometry analysis Showed that J L-l plants are
unable to produce J A from its precursor, 12-oxo-phytodienoic acid (OPDA),
presumably because of a defect in fatty acid B-oxidation. It was found that OPDA is not
an active signal for PI expression in tomato. Analysis of reciprocal grafis between wild-
type and J L—l plants showed that J A biosynthesis is required for the generation of the
systemic signal in wounded leaves. Taken together with the finding that J A perception
is essential for systemic expression of P1 in undamaged leaves, these results indicate
that JA or a derivative of J A is the long-distance Signal for systemic PI expression.

J A biosynthesis is regulated by substrate availability. Two related cytochrome
P4505, allene oxide synthase (A08) and hydroperoxide lyase (HPL), metabolize a
common hydroperoxy fatty acid substrate to JA and volatile C6 aldehyde, respectively.
To study the regulation of J A biosynthesis, genes encoding A08 and HPL were cloned
from tomato and expressed in E. coli. Wounding induced the expression of the ADS
gene in both wild-type and J A-deficient mutants, indicating that a JA-independent
signaling pathway may contribute to systemic AOS expression. These results, together
with those obtained from gene expression profiling in sprl plants, point to the existence
of both systemin/JA-dependent and ~independent signaling pathways that regulate

distinct sets of wound-inducible target genes in tomato plants.

Acknowledgements

My thesis could not have been completed without many people’s help and
contributions. First of all, I would like to thank my adviser, Dr. Gregg Howe, for his
support and guidance. I also thank my committee members, Drs. Hans Kende, Sheng
Yang He, and Christoph Benning, for advice, encouragement and critical reviews. It
was my honor to have Dr. Jan Zeevaart as an outside observer for my defense exam.

The Howe lab members, Amy DeRocher, Aya Itoh, Lei Li, Ying-Tsu Loh, Sara
Spencer, Dave Shaffer, Chuanyou Li, Mark Williams, Sara Birkeland, Liyan Liu, Sarah
Norris, Tony Schihniller, Youfu Zhao, Hui Chen, Guanghui Liu, and Bonnie McCaig,
have directly and indirectly contributed to this work. I appreciate their help and
friendship. As the first year graduate students, Julie Zwiesler-Vollick, Carl Andre, and
Wayne Riekhof, also participated in my research project during their lab rotations.

Many results in this study were obtained from GC-MS analysis. I learned how to
use GC-MS for JA measurement from Mr. Nate Riddle, Ms. Beverly Chamberlin, Dr.
Doug Gage and Dr. Hans Weber. Dr. Benjamin Travis helped me to prepare internal
standards. I appreciate their help.

Thanks to all members working in Plant biology building, especially for Drs.
Joong-Hoon Ahn, Kook-Ho Cho, Hyung Taeg Cho, Dongsu Choi, J eong-Hoe Kim, Yi
Lee, Dong Ju Lee, Seung-Hwan Yang, and Mi Jung Suh, for support, advice, and help.

Finally, I would like to thank our PRL editor, MS. Karen Bird, for critical

reading of my dissertation draft.

iv

Table of Contents

List of Tables .................................................................................................................... x
List of Figures .................................................................................................................. xi
Chapter 1
Systemic wound signaling in tomato ................................................................................ 1
Introduction ................................................................................................................ 2
Wound-induced gene expression ................................................................................ 3
Wound signals ............................................................................................................ 5
Oligogalacturonic acid ....................................................................................... 5
Systemin ............................................................................................................ 8
J asmonic acid ................................................................................................... 10
Physical Signals ............................................................................................... 1 5

Involvement of other plant hormones and signaling molecules in wound signaling. 15

Systemic wound signaling and the long-distance signal .......................................... 17

Systemic wound signaling in Arabidopsis ............................................................... 18

Wound response mutants of tomato ......................................................................... 19

Overview of thesis .................................................................................................... 21

References ................................................................................................................ 23
Chapter 2

The tomato mutant spr! is defective in systemin perception and the production of a
systemic wound signal for defense gene expression ...................................................... 34

Introduction .............................................................................................................. 35

Results ...................................................................................................................... 37

sprl preferentially affects wound-induced systemic PI expression ................ 37
sprl plants are impaired in systemin-mediated Signaling ............................... 42
Effect of sprl on wound- and systemin-induced J A accumulation ................. 50

sprl plants are defective in the generation of a systemic wound signal for PI

expression ........................................................................................................ 52
Discussion ................................................................................................................. 53
sprl defines a novel class of wound-response mutant .................................... 53
SprI-independent wound signaling ................................................................. 54
Role of systemin in wound signaling .............................................................. 56
Materials and Methods ............................................................................................. 57
References ................................................................................................................ 61
Appendix of Chapter 2
SprI does not encode SR160, the systemin receptor ...................................................... 65
Introduction .............................................................................................................. 66
Results ...................................................................................................................... 67
Cloning of SR160 from sprl plants ................................................................. 67
Analysis of linkage between Spr] and SR160 loci .......................................... 67
Expression of SR160 in sprl plants ................................................................. 75
Discussion ................................................................................................................. 75
Materials and Methods ............................................................................................. 78
References ................................................................................................................ 80
Chapter 3

vi

The wound response mutant J L1 is defective in the conversion of 12-oxo-phytodienoic

acid to jasmonic acid ...................................................................................................... 82
Introduction .............................................................................................................. 83
Results ...................................................................................................................... 85

J L1 plants are deficient in JA accumulation .................................................... 85
Exogenous OPDA does not activate the expression of PM] in J L1 plants ..... 85
J L1 plants fail to generate a systemic wound signal for PI—II expression ....... 86
Growth of J L1 seedlings is retarded ................................................................ 91
Discussion ................................................................................................................. 93
J L1 plants are unable to convert OPDA to JA ................................................ 93
OPDA is not a wound Signal for PI expression in tomato .............................. 97
Materials and Methods ............................................................................................. 99
References .............................................................................................................. 101
Chapter 4

Quantification of jasmonic acid and 12-oxo-phytodienoic acid in tomato plants by gas

chromatography-mass spectrometry ............................................................................. 105
Introduction ............................................................................................................ 106
Results .................................................................................................................... 1 10

Preparation of internal standards and determination of GC-MS parameters 110
Extraction and quantification of endogenous J A and OPDA ........................ 113
Quantification of IA and OPDA from wound response mutants of tomato.. 118
Discussion ............................................................................................................... 121

Regulation of IA biosynthesis in tomato ....................................................... 122

vii

Materials and Methods ........................................................................................... 123

References .............................................................................................................. 127
Chapter 5
Analysis of wound-induced root-to-leaf signaling in tomato plants ............................ 132
Introduction ............................................................................................................ 133
Results and Discussion ........................................................................................... 135
Materials and Methods ........................................................................................... 140
References .............................................................................................................. 142
Chapter 6

Cytochrome P450-dependent metabolism of oxylipins in tomato. Cloning and

expression of allene oxide synthase and fatty acid hydroperoxide lyase ..................... 144
Introduction ............................................................................................................ 145
Results .................................................................................................................... 149

cDNA isolation and sequence analysis .......................................................... 149
Functional expression of LeA 0S and LeHPL in E. coli ................................ 156
Developmental expression of LeA OS and LeHPL ......................................... 159

Wound-inducible expression of LeA OS is mediated by a Def] -independent

signaling pathway .......................................................................................... 162
Discussion ............................................................................................................... 167
Characterization of LeA 0S ............................................................................ 168
Characterization of LeHPL ............................................................................ 169

Wound-inducible expression of LeA OS is mediated by a Def] -independent

signaling pathway .......................................................................................... 171

viii

Materials and Methods ........................................................................................... 173

References .............................................................................................................. 1 83
Chapter 7
Conclusions and future perspectives ............................................................................ 189
References .............................................................................................................. 1 97

ix

List of Tables

Table 3-1. Wound response of wild-type and JLl plants ................................... 88
Table 4-1. Summary of IA levels in wound response mutants of tomato. . . . . . . . .......1 16
Table 4-2. Wound-inducible J A and OPDA levels in wound response mutants ........ 117
Table 6-1. Percent amino acid and nucleotide identity between different AOSS and

HPLS ................................................................................ 153

List of Figures

Figure 1-1. The current model of systemic wound signaling ................................. 7
Figure 1-2. The octadecanoid pathway ......................................................... 12
Figure 2-1. Spatial pattern of PH and PH! mRNA accumulation relative to the wound
Site ...................................................................................... 38
Figure 2-2. Time course of wound-induced gene expression in wild-type (WT) and
sprl plants .............................................................................. 41
Figure 2-3. PI-II accumulation in wild-type (WT) and sprl plants in response to
exogenous signaling compounds .................................................. 43
Figure 2-4. Response of sprl plants to exogenous systemin, oligogalacturonides
(OGA), and chitosan .................................................................. 44
Figure 2-5. Effect of exogenous systemin on gene expression in wild-type (WT) and
sprl plants ............................................................................. 46
Figure 2-6. J asmonic acid (J A) accumulation in response to systemin and mechanical
wounding .............................................................................. 49
Figure 2-7. Wound-inducible PI-II expression in grafts between wild-type (WT) and
Figure 2-8. Genomic DNA sequence of SR1 60 isolated from L. esculentum ............ 68
Figure 2-9. Comparison of deduced amino acid sequences of SR1 60 and BRII . . .. . ....70
Figure 2-10. Linkage analysis of SR] 60 and SprI ........................................... 73
Figure 2-11. Expression of SR] 60 in wounded wild-type and sprl plants. . . . . . . . . ..76

Figure 3-1. Conversion of OPDA to JA ........................................................ 87

xi

Figure 3-2. Accumulation of J A and OPDA in wild-type and JL1 plants in response to
wounding ............................................................................. 89

Figure 3-3. Effect of exogenous J A and OPDA on the accumulation of PI in wild-type
and JL1 plants .......................................................................... 90

Figure 3-4. Wound-inducible PI-II expression in grafts between J L1 and wild-type

plants .................................................................................... 92
Figure 3-5. Comparison of root length between wild-type and JL1 seedlings .......... 94
Figure 4-1. Structure ofJA, OPDA and their internal standards. . . . ...109
Figure 4-2. Chromatogram and mass spectra ofMeJA and MeDHJA. . ..1 11
Figure 4—3. Chromatogram and mass spectra of MeOPDA and MeH4OPDA. . . ..1 12
Figure 4-4. Standard curves for the quantification of IA and OPDA.........114

Figure 4-5. Accumulation of J A and OPDA in wild-type and def] plants challenged
with Spider mites .................................................................... 119
Figure 5-1. Systemic induction of PHI in leaves in response to wounding of roots..136
Figure 5-2. Systemic expression of wound-inducible genes in response to root
damage ................................................................................. 138
Figure 6-1. The octadecanoid pathway ....................................................... 146

Figure 6-2. Comparison of cDNA-deduced protein sequences of plant AOS and HPL

genes .................................................................................. 150
Figure 6-3. Southern-blot analysis of LeA OS and LeHPL ........................................... 155
Figure 64. Activity ofLeAOS and LeI-LPL expressed in E. colt .. 157

Figure 6-5. Expression of LeA 0S and LeHPL genes in different organs of tomato. . . 160

Figure 6-6. Accumulation of LeAOS protein in different organs of tomato. . . . . . . l 63

xii

Figure 6-7. Accumulation of LeA 0S, LeHPL, and Inh-II mRNAs in tomato plants in
response to herbivory ............................................................... 164

Figure 6-8. Analysis of wound-induced gene expression in wild-type and def] mutant
plants ................................................................................ 165

Figure 7-1. Revised model of systemic wound signaling in tomato. . . . . . . . . . . ..192

xiii

Chapter 1

Introduction:

Systemic wound signaling in tomato

Introduction

Herbivore attack causes plants to induce several defense responses (Kessler and
Baldwin, 2002; Walling, 2000). One of the most extensively studied defense responses
is the wound-induced synthesis of defensive proteinase inhibitors (PIS) in tomato
(Gatehouse, 2002; Ryan, 2000). Accumulation of PIS in response to herbivory and
mechanical wounding is regulated at the level of gene expression (Nelson et al., 1983).
Accumulated PI proteins in tomato adversely affect the grth of lepidopteran
caterpillars by interfering with digestive enzymes in the gut of the attacking insect
(Ryan, 1990).

Wound-induced synthesis of PIS occurs not only in the vicinity of the wound
site, but also in undamaged leaves throughout the plant (Green and Ryan, 1972). This
systemic response implies the existence of a long-distance signal transduction pathway
that connects local injury to the activation of gene expression in distal undamaged
tissue. Detachment of the damaged leaf immediately after wounding was Shown to
abolish systemic PI accumulation, indicating that the mobile Signal is synthesized in the
damaged leaf and transported to distal undamaged leaves (Green and Ryan, 1972).

Extensive research effort has been focused on the identification of the systemic
wound signal. Chemical elicitors of PI expression have been isolated from tomato
plants and characterized as candidates for the mobile signal (Bishop et al., 1981; Pearce
et al., 1991). These studies have Shown that a polypeptide signal called systemin, and
the plant hormone jasmonic acid (J A), play crucial roles in the systemic wound
response (Farmer and Ryan, 1992; McGurl et al., 1992; Pearce et al., 1991). Our current

understanding of systemic wound signaling is summarized in this chapter.

Wound-induced gene expression

Over 20 genes have been shown to be expressed systemically in response to wounding
of tomato plants. Based on their expression pattern, these genes have been classified
into two groups (Ryan, 2000). The first group is the ‘early genes’ that are induced
rapidly and transiently upon wounding. Expression of early genes is detected within 30
min of wounding. Maximal expression occurs about 2 hr after wounding, after which
mRNA levels quickly decrease. The group of early genes includes those encoding
calcium calmodulin (Bergey and Ryan, 1999), transcription factors (Stankovic et al.,
2000; Strassner et al., 2002), and enzymes involved in the biosynthesis of IA (Heitz et
al., 1997; Sivasankar et al., 2000; Strassner et al., 2002). Because the levels of cytosolic
calcium and endogenous J A rapidly increase in response to wounding and elicitor
treatment (Moyen et al., 1998; Parchmann et al., 1997), the induction of genes encoding
calcium calmodulin and JA biosynthetic enzymes may represent a positive feedback
mechanism to amplify the wound response.

The second group of wound response genes in tomato is referred to as ‘late
genes’. Transcript levels of these genes increase slowly and steadily in response to
wounding. The expression of late genes is detected about 2 hr after wounding, with the
highest expression level occun'ing 8 to 12 hr after wounding. Examples from this group
include genes encoding PIS, polyphenol oxidases, leucine aminopeptidase and LE
RNase (Chao et al., 1999; Constabel et al., 1995; Lers et al., 1998). Similar to P15,
polyphenol oxidases mediate plant defense against attacking insects by inhibiting insect
digestive processes (Constable et al., 1995). LE RNase and proteolytic enzymes such as

leucine aminopetidase (Chao et al., 1999; Schaller and Ryan 1996) may play a role in

reprogramming plant metabolism to allow for massive synthesis of PIS and other
defensive phytochemicals.

Advances in cDNA microarray technology have enabled investigators to
monitor global changes in gene expression in response to wounding. A microarray
experiment using 230 tomato cDNAs supports the idea that wound—inducible genes in
tomato can be classified according to their temporal expression pattern (Strassner et al.,
2002). The sequential expression of these genes indicates that transcription factors
encoded by early genes may activate the expression of late genes. Alternatively,
proteins encoded by the early genes may amplify the signaling pathway leading to the
expression of late genes, which results in maximal production of defensive chemicals.
Larger-scale microarray experiments have been performed in Arabidopsis. Mechanical
wounding significantly changed the expression level of 657 genes (Cheong et al., 2002).
About 20% of these wound—inducible genes encode signal transduction-related proteins
such as protein kinases, protein phosphatases, GTP-binding proteins, calcium binding
proteins, phosphatidylinositol-related enzymes, JA biosynthetic enzymes and
transcription factors. Although it is likely that these gene products are involved in
wound signaling, the precise contribution of each of gene remains to be determined.
Other wound-inducible genes encode enzymes involved in the phenylpropanoid
pathway. Activation of these genes leads to the synthesis of defensive chemicals and the
reinforcement of the cell wall (Constabel, 1999; F ranke et al., 2002; Peters and

Constabel, 2002), which may enhance resistance to pest attack.

Wound signals

A Simple bioassay has been used extensively to identify systemic wound signals in
tomato plants (Ryan, 1992). This assay involves supplementation of young plants
through the cut stem with compounds purified fiom tomato leaf extract, followed by
measurement of PI accumulation using an immunodiffirsion assay (Ryan, 1967). Two
structurally distinct molecules were found using this bioassay: oligogalacturonic acid
(OGA) derived from the cell wall (Bishop et al., 1981), and a polypeptide, systemin
(Pearce et al., 1991), In addition, the plant hormone J A and its methyl ester (MeJ A)
were identified as inducers of PI expression (Farmer and Ryan, 1990; Farmer and Ryan,
1992). Studies of these compounds have established a signaling cascade that regulates
wound-induced expression of PI and other defense-related genes (Figure 1-1; Ryan

2000).

Oligogalacturonic acid

Previously it was reported that a pectin-enriched fraction purified from tomato leaf
hydrolysate induced PI synthesis (Bishop et al., 1981). Further purification of the
fraction showed that the active component was the oligosaccharide OGA, which is a
hydrolysis product of pectin. Therefore, it was speculated that wounding results in the
release of OGA from the cell wall. A cDNA encoding a polygalacturonase that degrades
pectin to OGA was recently cloned from tomato (Bergey et al., 1999). The activity of

this enzyme is up-regulated locally and systemically upon wounding. This finding

supports the idea that OGA is an authentic signal for the activation of wound-inducible
genes in tomato. However, genetic evidence to support this hypothesis is lacking.

Although OGA is a potent elicitor of PI expression, the mobility of OGA in
plant tissue is very limited. Radioactively labeled OGA applied to wound sites was not
transported to unwounded tissue (Baydoun and Fry, 1985). Thus, it is unlikely that
OGA is the long-distance signal of the systemic wound response.

In addition to its effect on P1 expression, OGA treatment results in
phosphorylation of a plasma membrane protein (Farmer et al., 1989), depolarization of
the plasma membrane (Moyen and Johannes, 1996; Thain et al., 1995), a reactive
oxygen burst (Stennis et al., 1998), and an increase in endogenous JA levels (Doares et
al., 1995b). Among these rapid cellular responses, it was shown that activation of J A
biosynthesis is essential for OGA-mediated expression of PI genes. OGA failed to
induce PI accumulation in plants treated with inhibitors of J A biosynthesis (Doares et
al., 1995a), or in plants that are compromised in IA biosynthesis (Howe et al., 1996).
These results indicate that OGA activates PI expression through the J A signaling
pathway.

The oligosaccharide, chitosan, which is derived from fungal cell walls, also
induces J A synthesis and PI accumulation in tomato plants (Walker-Simmons and
Ryan, 1984). This observation is consistent with the fact that JA regulates defense
response against some fungal pathogens of tomato (Diaz et al., 2002). These results

indicate that plants use chitosan as a signal to trigger defense response.

systemin wounding

FEE \ ” I
prosystemin * \ Local

+\‘ / OGA response
t

jasmonic acid
++ ethylene

\ .1. /

SR160 \
\ I Systemic

jasmonic acid response
§ + ethylene

t

\ PIS j

 

 

 

 

 

 

 

Figure 1-1. The current model of systemic wound signaling. This schematic model is
adapted from Ryan (2000). Wounding activates J A biosynthesis via systemin- and
OGA-mediated signaling pathways in wounded tissue (local response). Systemin
functions as a mobile signal to activate PI expression in distal unwounded tissue
(systemic response). A recent study indicates that J A, not systemin, is the systemic
wound signal (Li et al., 2002). SR160, the systemin receptor.

Systemin

The 18-amino-acid polypeptide systemin was purified from tomato leaf extracts on the
basis of its ability to induce PI accumulation when supplied to young tomato plants
through the cut stem (Pearce et al., 1991). Synthetic systemin is active in the frnol
range, indicating that it is the most potent elicitor of PI accumulation ever found.
Systemin is derived fiom the C-terminal region of a 200 amino acid precursor protein
named prosystemin (McGurl et al., 1992). Tomato has a single copy of the prosystemin
gene, which is expressed at low levels in aerial parts of unwounded plants. Expression
of the prosystemin gene is elevated locally and systemically within cells of the vascular
bundles in response to wounding (J acinto et al., 1997; Mch1 et al., 1992).

Several lines of evidence indicate that systemin plays a critical role in systemic
wound signaling. First, systemic PI expression was severely reduced in transgenic
plants that express the prosystemin cDNA in antisense orientation (McGurl et al., 1992).
Moreover, these transgenic plants were more susceptible to insect attack than wild-type
plants (Orozco-Cardenas et al., 1993). Second, PI and other wound-inducible genes
were constitutively expressed in transgenic plants (called 35S::prosys) that overexpress
prosystemin from the cauliflower mosaic virus (CaMV) 35S promoter (McGurl et al.,
1994). In a grafting experiment in which wild-type scions were grafted onto
35S: :prosys transgenic rootstocks, the wild-type scions accumulated significant
amounts of Pls in the absence of wounding. This observation indicates that 35S:.°prosys
transgenic rootstocks produce a systemic wound signal that is transmitted to the wild-
type scions. Unlike OGA, systemin may be a mobile signal in vivo because

radioactively-labeled systemin applied to wound sites moved via the phloem to

unwounded tissue (Narvaez-Vasquez et al., 1995; Pearce et al., 1991). Therefore,
systemin was regarded as a strong candidate for the systemic wound signal.

Previous studies demonstrated that systemin requires J A biosynthesis for
induction of wound-inducible genes. Endogenous levels of J A rapidly increase in
response to systemin (Conconi et al., 1996; Doares et al., 1995b), followed by
expression of PI genes. Systemin treatment failed to induce PI expression in tomato
mutant plants that are impaired in J A biosynthesis (Howe et al., 1996). Also, inhibitors
of J A biosynthesis abolished systemin-induced PI accumulation (Doares et al., 19953).
These results indicate that systemin, like OGA, acts upstream of J A in the wound
signaling pathway (Figure 1-1).

In addition to up-regulating J A synthesis, exogenous systemin activates several
rapid cellular events. These events include increased cytosolic calcium levels,
inactivation of a proton-ATPase located on plasma membrane, activation of a wound-
inducible mitogen-activated protein kinase (MAPK), and membrane depolarization
(Felix and Boller, 1995; Moyen and Johannes, 1996; Moyen et al., 1998; Schaller and
Oecking, 1999; Stratrnann and Ryan, 1997). Pharmacological studies showed that
inhibition of the proton-ATPase and changes in calcium ion flux induced PI expression
(Schaller and Frasson, 2001; Schaller and Oecking, 1999). Thus, calcium mobilization
and modulation of proton-ATPase may be early causal events of the wound signaling
pathway.

Systemin specifically binds a plasma membrane protein called SR160 (Meindl et
al., 1998; Scheer and Ryan, 1999). This 160 kDa protein is a leucine-rich repeat

receptor kinase that shows high similarity to the brassinolide receptor, BRII , of

Arabidopsis (Scheer and Ryan, 2002). Interestingly, the tomato mutant curl3 is
insensitive to brassinolide as a result of a nonsense mutation in the SR160 gene
(Montoya et al., 2002). This finding indicates that SR160 is a dual receptor for systemin
and brassinolide. It is currently unclear how the same receptor regulates two distinct
signaling pathways.

Orthologues of the prosystemin gene have been found in members of the
Solanaceae family, such as potato, black nightshade, and bell pepper (Constabel et al.,
1998). In tobacco, two polypeptides were found to strongly induce PI expression. These
polypeptides are processed from the same precursor protein, and do not show sequence
similarity to tomato systemin (Pearce et al., 2001). Therefore, it is possible that other

plant species use polypeptide signals to regulate the wound response.

Jasmonic acid

The cyclopentanone compound J A is synthesized from linolenic acid by the
octadecanoid pathway (Figure 1-2; Wastemack and Hause, 2002). J A biosynthesis is
initiated in chloroplasts, where phospholipases release linolenic acid from membrane
lipids (Ishiguro et al., 2001). Lipoxygenase (LOX) converts linolenic acid to 13-
hydroperoxylinolenic acid (13-HPOT), which is metabolized by various enzymes
belonging to the CYP74 family of cytochrome P4508 (Howe and Schilmiller, 2002;
Howe et al., 2000). Among these, allene oxide synthase (AOS) commits 13-HPOT to
the biosynthesis of J A. AOS transforms l3-HPOT to an unstable epoxide intermediate

(12, 13-epoxy1inolenic acid), which is subsequently transformed by allene oxide cyclase

10

to 12-oxo-phytodienoic acid (OPDA). The remaining reactions of the octadecanoid
pathway take place in peroxisomes (Strassner et al., 2002). The double bond of OPDA
is reduced by OPDA reductase (OPR3) to yield 3-oxo-2-(22-pentenyl)-cyclopentane-1-
octanoic acid (OPC-8). Finally, three rounds of B-oxidation shorten the carboxylate side
chain of OPC-8, yielding J A. It remains unknown how OPDA is transported from
chloroplasts to peroxisomes.

J A is subject to several metabolic transformations such as methylation,
conjugation to amino acid or glucose, adenylation, hydroxylation and sulfonation
(Gidda et al., 2003; Hause et al., 2000; Kramell et al., 1997; Seo et al., 2001; Staswick
et al., 2002). Me] A, amino acid-conjugated J A, and glucose-conjugated J A have been
shown to induce expression of PI (Farmer and Ryan, 1990; Kramell et al., 1997 ;
Wastemack et al., 1998). These results indicate that some JA derivatives act as signals
for gene expression. In consistent, defective adedylation of J A abolishes JA-inducible
gene expression (Staswick et al., 2002). Alteration of the expression of J A-
metabolizing enzymes in transgenic Arabidopsis has provided insight into the role of J A
metabolism. For example, a Specific methyl transferase converts JA to volatile Me] A
(See et al., 2001). Overexpression of this enzyme in Arabidopsis results in elevated
endogenous Me] A levels and enhanced resistance to fungal pathogens. Thus, MeJ A
seems to be an active Signal for defense gene expression.

A wealth of evidence indicates that J A is an essential component of the wound
signaling pathway. First, tomato plants produce high levels of PIS in response to JA and
MeJA (Farmer and Ryan, 1990; Farmer and Ryan 1992). Second, wounding results in a

rapid increase in J A biosynthesis (Parchmann et al., 1997), which is followed by

11

Chloroplast

Peroxisome

 

Membrane Lipid

I Lipase

I - _ . . .
(m OOH a linolenic acnd

OOH ‘ Lipoxygenase
\
l 13-hydroperoxylinolenic acid
COOH
I Allene oxide synthase
0

I 12, 13-epoxylinolenic acid
0 Allene oxide cyclase

 

 

12-oxo-phytodienoic acid

COOH

I
I
V \
I OPDA reductase
- UPC-8
COOH
*

* ,B-oxidation

0 v

— Jasmonic acid
kNI:COOH

 

A /

 

 

 

J

Figure 1-2. The octadecanoid pathway. Linolenic acid is released from
chloroplast membranes and converted to OPDA by the sequential action of
three enzymes. OPDA is processed to J A in peroxisomes.

PI expression. Finally, wound-induced PI accumulation is blocked in JA-deficient
tomato mutants (Howe et al., 1996; Li et al., 2002). It has also been established that
wounding rapidly induces systemic expression of several J A biosynthetic genes
including LOX, AOS, ADC, and OPR3 (Heitz etal., 1997; Howe et al., 2000;
Sivasankar et al., 2000; Stenzel et al., 2003; Strassner etal., 2002). JA biosynthetic
enzymes such as ADC and OPR3 are highly enriched in vascular bundles, indicating
that IA is mainly synthesized in these tissues (Stenzel et al., 2003; Strassner et al.,
2002). Overexpression of A US and AOC did not result in increased J A levels or
constitutive expression of wound-inducible genes (Laudert et al., 2000; Stenzel et al.,
2003). These observations indicate that J A biosynthesis is not regulated by
transcriptional control of the genes. Because application of linolenic acid to tomato
results in P1 expression (Farmer and Ryan, 1992; Howe et al., 1996), JA biosynthesis is
probably regulated by substrate availability.

Despite extensive knowledge of the effects of J A on gene expression and
defense-related processes, our understanding of the signaling events that couple the
production of J A to the activation of target genes is still in its infancy. Our current
understanding of this problem has been significantly enhanced by the identification and
characterization of mutants that are insensitive to JA. For example, an Arabidopsis
mutant coronatine insensitive] (coil ) is unable to express wound-inducible genes in
response to J A and MeJA (Feys et al., 1994). The C011 gene encodes a protein
containing an F-box motif and leucine-rich repeats that functions in ubiquitin-mediated
protein degradation (Xie et al., 1998). As a component of ubiquitin ligase complex, F -

box proteins act as receptors that recruit regulatory proteins as substrates for ubiquitin-

13

mediated degradation (Bai et al., 1996). Previous studies demonstrated that the C011
protein participates in the assembly of a ubiquitin ligase complex that presumably
recruits regulatory proteins to the 26S proteosome for degradation (Feng et al., 2003;
Xu et al., 2002). A recent study indicates that COIl mediates degradation of a histone
deacetylase that modulates chromosome remodeling (Devoto et al., 2002). These results
indicate that C011 is involved in the removal of negative regulators acting downstream
of J A

There is also evidence indicating that a MAPK cascade operates downstream of
J A to regulate gene expression. JA failed to activate wound-inducible genes in
Arabidopsis plants deficient in MAPK4 activity (Petersen et al., 2000). In addition, a
pharmacological study indicated that protein dephosphorylation catalyzed by protein
phosphatase 2C is required for the induction of J A-responsive genes in Arabidopsis
(Rojo et al., 1998).

Previous studies indicated that OPDA, in addition to serving as an intermediate
in J A biosynthesis, is a signal for several plant developmental processes (Koch et al.,
1999; Weiler et al., 1993; Weiler et al., 1994). The Arabidopsis opr3 mutant is unable to
convert OPDA to J A due to the loss of OPDA reductase activity (Stintzi et al., 2001 ).
Whereas J A-deficient and J A-insensitive mutants are susceptible to insect attack, opr3
plants were shown to be as resistant as wild-type plants. Moreover, wild-type and opr3
plants showed very Similar gene expression patterns in response to wounding, and
OPDA treatment induced a subset of wound-inducible genes in opr3 plants. These

results demonstrate that OPDA is an active signal for defense responses in Arabidopsis.

14

Physical signals

Mechanical wounding generates an electrical pulse (Wildon et al., 1992) and changes in
xylem tension capable of producing a hydraulic signal (Malone and Alarcon, 1995;
Malone et al., 1994). Because these physical signals are rapidly propagated long
distance through the whole plant, they were proposed as candidates for the systemic
signal for PI expression (Malone, 1996; Wilden et al., 1992). To date, however, there is
no genetic evidence that an electrical signal is the causative agent of PI expression. In
the case of hydraulic Signals, it was proposed that mass flow of xylem sap might deliver
signaling molecules produced at the wound Site to undamaged leaves (Rhodes et al.,
1999). However, this idea is not consistent with the observation that steam girdling of
the petiole, which prevents phloem transport, but not xylem transport abolished
systemic P1 expression in tomato (N elson et al., 1983). Thus, a causal role of physical

signals in wound-induced systemic PI expression has not been established.

Involvement of other plant hormones and signaling molecules in wound
signaling

Ethylene is required for wound-inducible gene expression in tomato. Wounding and
systemin treatment activated ethylene biosynthesis. Inhibitors of ethylene perception,
such as silver thiosulphate, were Shown to prevent the induction of J A biosynthesis and
P1 expression. Also, wounding failed to stimulate PI synthesis in transgenic plants that
are deficient in ethylene biosynthesis (O'Donnell et al., 1996). These results
demonstrate that ethylene is a positive regulator of wound signaling in tomato.

However, ethylene is not an independent signal for wound-inducible gene expression

15

because ethylene treatment alone does not induce P1 expression. It was proposed that
ethylene and J A work together in a positive feedback loop to amplify wound-induced
expression of PIS (O'Donnell et al., 1996).

Abscisic acid (ABA) was proposed to be a positive regulator of wound signaling
in tomato because ABA-deficient mutants fail to express P1 in response to wounding
(Herde et al., 1996). It is not clear whether ABA alone is a signal for P1 expression
because studies involving ABA treatment gave contradictory results (Birkenmeier and
Ryan, 1998; Pena-Cortes et al., 1995). It is unlikely that ABA is the systemic wound
Si gnal because ABA-inducible genes were not expressed systemically upon wounding
(Birkenmeier and Ryan, 1998).

Hydrogen peroxide accumulates systemically in response to wounding (Orozco-
Cardenas and Ryan, 1999). Wounding, systemin, OGA, and JA did not induce PI
accumulation when hydrogen peroxide synthesis was suppressed by inhibitors (Orozco-
Cardenas et al., 2001). Interestingly, such inhibition did not affect expression of early
wound response genes. Consistent with this, application of the hydrogen peroxide-
generating enzyme glucose oxidase plus glucose to tomato plants resulted in the
induction of late wound response genes such as PI, but not early wound response genes.
Therefore, hydrogen peroxide appears to act downstream of J A to activate expression of
late wound response genes.

Other signals have been shown to inhibit wound signaling in tomato. For
example, salicylic acid (SA) treatment suppressed J A biosynthesis and PI expression
(Doares et al., 1995a). Likewise, nitric oxide (NO) was shown to decrease the level of

hydrogen peroxide and PI accumulation (Orozco-Cardenas and Ryan, 2002). Because

16

SA and NO regulate defense responses to pathogenic bacteria (Dumer and Klessi g,
1999; McDowell and Dangl, 2000), these results indicate that defense responses against
herbivores and bacterial pathogens are mediated by signaling pathways that antagonize

one another (Kunkel and Brooks, 2003).

Systemic wound signaling and the long-distance signal

Based on previous studies, Ryan and coworkers proposed a model for systemic wound
signaling in tomato (Orcozco-Cardenas et al., 2001; Ryan, 2000). According to this
model, wounding activates the processing of prosystemin to systemin by unknown
mechanism. Subsequently, systemin is loaded to the phloem and transported to distal
undamaged leaves. Upon binding to its receptor, SR160, systemin activates JA
biosynthesis in vascular bundle cells where prosystemin and JA-biosynthetic enzymes
are located. J A then activates expression of early wound response genes in vascular
bundles. In addition, J A induces release of OGA from cell wall by the activation of
wound-inducible polygalacturonase, which triggers an oxidative burst. Hydrogen
peroxide produced during the oxidative burst diffuses from vascular bundles to
mesophyll cells, where P1 expression is activated. In this model, systemin acts as the
long-distance signal for systemic gene expression.

Recent studies using reciprocal grafting techniques have challenged the idea that
systemin is the systemic signal for PI expression (Li et al., 2002). Analysis of wound-
induced systemic P1 expression in grafts using J A—deficient and JA-insensitive mutants
demonstrated that systemic P1 expression requires J A biosynthesis in wounded leaves

but not in distal undamaged leaves. Conversely, J A perception is essential for

17

recognition of the systemic wound signal in undamaged leaves, but not for the
generation of that signal in wounded leaves. The simplest interpretation of these results
is that J A or a derivative of J A is the systemic wound signal. Because systemin induces
PI expression by activation of JA biosynthesis (Doares et al., 1995a; Doares et al.,
1995b; Howe et al., 1996), these experiments indicate that systemin is unlikely to act in
unwounded tissue as the systemic Si gnal. Since prosystemin and J A biosynthetic
enzymes are enriched in vascular bundle cells, systemin and JA might interact
synergistically to amplify the production of the systemic wound signal along the
vascular bundles (Ryan and Moura, 2002).

Emerging evidence indicates that some systemic wound responses are mediated
by a signaling pathway that operates independently of J A and systemin. From example,
neither systemin nor J A activates expression of the wound-inducible glucosyl
transferase gene in tomato (O'Donnell et al., 1998). It was also Shown that rapid
systemic induction of a wound-inducible MAPK activity is unlikely to involve J A or
systemin (Stratmann and Ryan, 1997). Moreover, destruction of phloem transport by
steam girdling did not block the systemic induction of the kinase activity. This finding
clearly demonstrates that a systemin- and J A-independent signal is involved in this

particular systemic wound response.

Systemic wound signaling in Arabidopsis

Arabidopsis has been used as an alternative model system to study the wound responses
(Berger, 2002; Leon et al., 2001). In this system, it is clear that JA plays a crucial role in

wound-induced systemic expression of various defense-related genes. Wounding

18

induces JA biosynthesis in damaged leaves, which is followed by local and systemic
gene expression 0(ubigsteltig et al., 1999; Laudert and Weiler, 1998; Reymond et al.,
2000; Titarenko et al., 1997). Because these responses are impaired in J A-deficient and
JA-insensitive mutants (Berger et al., 1996; McConn et al., 1997; Park et al., 2002;
Reymond et al., 2000; Rojo et al., 1999), JA seems to be an essential component of the
systemic wound signaling pathway. Interestingly, the JA-mediated signaling pathway
appears to work independently of OGA in Arabidopsis, whereas J A is required for
OGA-induced gene expression in tomato (Doares et al., 1995b; Rojo etal., 1999;
Titarenko et al., 1997). Furthermore, expression of JA-responsive genes is inhibited in
wounded Arabidopsis leaves by OGA and ethylene (Rojo et al., 1999). Therefore,
although J A is required for systemic wound responses in both tomato and Arabidopsis,
the regulatory mechanism of J A-mediated signaling pathway may depend on the plant
species. This idea is consistent with the observation that systemin-mediated signaling is

absent in Arabidopsis.

Wound response mutants of tomato

Mutants impaired in the wound response provide valuable tools to elucidate the
systemic wound signaling pathway. J L1 and defenseless] (defI) were identified by
screening an ethyl methane sulfonate (EMS)-mutagenized population for plants unable
to produce PIS in response to wounding (Lightner et al., 1993). Genetic analysis Showed
that these mutations are recessive. Since Me] A treatment induced PI accumulation in
JL1 and def] plants, these mutants likely contain a defect in J A biosynthesis or an

upstream step in the wound signaling pathway. Additional characterization of def]

19

plants showed that this mutant is deficient in wound-inducible J A accumulation, and
has reduced AOC activity (Howe et al., 1996; Stenzel et al., 2003).

Transgenic plants (35S::prosys) that overproduce prosystemin constitutively
express defensive proteins such as PIS and polyphenol oxidase (PPO) in the absence of
wounding (McGurl et al., 1994). To investigate the systemin-mediated signaling
pathway, 35S::prosys plants were subject to EMS-mutagenesis, and mutant plants that
fail to accumulate PPO and PIS were isolated (Howe and Ryan, 1999). Genetic analysis
of these mutants showed four complementation groups, named suppressor of
prosystemin-mediated responses (spr) 1-4. Interestingly, sprI plants showed a severe
reduction in systemic but not local PI expression. Thus, Sprl appears to function
specifically in the systemic response. In contrast, wounded spr2 plants did not
accumulate PIS in either damaged or undamaged leaves. Recently, it was shown that
Spr2 encodes an omega-3 fatty acid desaturase that is required for the synthesis of
linolenic acid, which is the precursor of J A (Li et al., 2003).

Tomato mutants that are insensitive to J A and Me] A were isolated by screening
of EMS-mutagenized and fast-neutron mutagenized populations for plants that fail to
express P1 in response to volatile MeJ A (Li et al., 2001). Genetic analysis showed that
these lines define a Single gene named Jasmonic acid-insensitive! (Jail). As is
predicted from the current model of wound signaling (Figure 1-1), wounding, systemin,

and JA treatment are unable to induce PI expression in jail plants (Li et al., 2002).

20

Overview of thesis

Systemic wound responses play important roles in plant defense against insect pests.
Since the identification of wound-inducible PIS by Green and Ryan (1972), tomato has
been used as a model system for the study of systemic wound signaling. A wealth of
evidence indicates that systemin and J A are two essential signals required for the
systemic wound response. Recent studies using wound response mutants indicate that
JA or a derivative of J A acts as a long-distance Signal for the systemic P1 expression. In
addition, other systemic signals appear to be involved in systemic responses that are
induced rapidly upon wounding.

The current thesis is focused on understanding the role of systemin and J A in the
systemic wound response. Systemin-insensitive sprl plants were characterized to
investigate how systemin and J A regulate systemic PI expression. Chapter 2 describes
the characterization of sprl and discusses the role of systemin in the systemic wound
response. In Chapter 3, the role of J A and its precursor OPDA in systemic wound
signaling was examined by characterizing the J L1 wound response mutant. To study the
regulation of J A biosynthesis during the wound response, endogenous levels of J A and
OPDA were measured in wild-type and mutant plants by gas chromatography-mass
spectrometry (GC-MS). These results are presented in Chapter 4, along with a
discussion of the regulation of J A biosynthesis. Chapter 5 reports on JA-independent
and J A-dependent signaling events that occur during wound-induced root-to-leaf
Signaling. Chapter 6 presents a study aimed at characterizing two related cytochrome

P450 enzymes, AOS and hydr0peroxide lyase, that metabolize hydroperoxy fatty acids

21

to J A and volatile C6 aldehyde, respectively. In the final chapter, a model of systemic

wound signaling is proposed based on the results from thesis study.

22

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28

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31

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32

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33

Chapter 2

The tomato mutant spr1 is defective in systemin
perception and the production of a systemic wound

signal for defense gene expression

A version of this chapter has been published.

Gyu In Lee and Gregg A. Howe (2003) Plant J. 33: 567-576

34

Introduction

Many plants respond to insect attack and wounding by modulating the expression of
genes involved in various defense-related processes. The synthesis and deployment of
wound-induced phytochemicals is regulated by signal transduction pathways that
operate both locally at the site of wounding and systemically in undamaged leaves
throughout the plant (Green and Ryan, 1972; Karban and Baldwin, 1997). Wound-
inducible defensive proteinase inhibitors (PIS) in Solanaceous plant species provide an
attractive model system in which to study the mechanism of long-distance wound
signaling, and several ideas have been proposed regarding the identity of the systemic
signal transmitted from wound sites (reviewed by Bowles, 1998; Leén et al., 2001;
Malone, 1996; Ryan, 2000; Walling, 2000). Among the proposed intercellular signals
for wound-induced PI gene expression are systemin, an 18-amino acid peptide that is
produced from cleavage of a larger precursor protein called prosystemin, and the
octadecanoid pathway-derived hormone jasmonic acid (J A) (Farmer and Ryan, 1992; Li
et al., 2002a; McGurl et al., 1992; Pearce et al., 1991). A wealth of biochemical and
genetic evidence indicates that systemin and J A work together in the same signaling
pathway to activate expression of P1 and other defense-related genes (Li et al., 2001;
Ryan, 2000).

The systemin/J A signaling pathway is activated upon binding of systemin to a
l60-kDa plasma membrane-bound receptor called SR160 (Meindl et al., 1998; Scheer
and Ryan, 1999). This receptor was recently identified as a member of the leucine-rich
repeat (LRR) receptor kinase family (Scheer and Ryan, 2002). Binding of systemin to

the cell surface is associated with several rapid signaling events including increased

35

cytosolic Ca2+ levels, membrane depolarization, inhibition of a plasma membrane
proton ATPase, and activation of a MAP kinase activity (Felix and Boller, 1995; Moyen
and Johannes, 1996; Moyen et al., 1998; Schaller and Oecking, 1999; Stratmann and
Ryan, 1997). The systemin signaling pathway leading to P1 expression is thought to
cuhninate in activation of a phospholipase that releases linolenic acid, the metabolic
precursor of J A, from membrane lipids (F arrner and Ryan, 1992; Narvaez-Vasquez et
al., 1999). Chitosan oligomers and Oligogalacturonides (OGAS) derived fi‘om fungal and
plant cell walls, respectively, also activate PI expression via the octadecanoid pathway
(Bishop et al., 1981; Doares et al., 1995; Walker-Simmons and Ryan, 1984). The
presence of wound-inducible polygalacturonase activity in tomato leaves (Bergey et al.,
1999), together with the relative immobility of OGAS in the plant vascular system
(Aldington and Fry, 1996), indicates that these compounds induce P1 expression at or
near the site of wounding. J A synthesized in response to wounding, systemin, and
OGAS acts in concert with ethylene (O’Donnell et al., 1996) and hydrogen peroxide
(Orozco-Cardenas et al., 2001) to coordinate the induction of PI gene expression.
Recent studies in Arabidopsis indicate that J A signaling depends upon assembly of
ubiquitin-ligase complexes that presumably target transcriptional repressors of J A-
responsive genes for proteolytic degradation (Xie et al., 1998; Xu et al., 2002).

Mutants affected in the synthesis or perception of prosystemin and JA provide
useful tools to understand the mechanism of systemic wound signaling. For example,
antisense-mediated depletion of prosystemin expression in tomato plants abrogated
wound-induced systemic accumulation of P13, indicating that this gene is essential for a

normal systemic wound response (McGurl et al., 1992). To identify genes involved in

36

the systemin/J A signaling pathway using a forward genetic approach, we took
advantage of a transgenic plant (called 355::prosys) that overexpresses prosystemin
from the CaMV 358 promoter and, as a consequence, constitutively expresses PI and
other defense-related genes in the absence of wounding (Constabel et al., 1995; Mch1
et al., 1994). An ethyl methane sulfonate (EMS)-mutagenized population derived from
this line was screened for mutants that are impaired in 35S::prosys-mediated signaling
(Howe and Ryan, 1999). Among several 'spr' suppressed in prosystemin-mediated
responses mutations identified, five independent alleles were shown to define one locus
called SprI . Evidence presented herein indicates that SprI is involved in a Signaling
step that couples systemin perception to activation of the octadecanoid pathway. The
results of grafting experiments further indicate that SprI function is required at or near
the Site of wounding to amplify J A accumulation to a level sufficient to promote long-
distance signaling. The existence of a wound response pathway that operates
independently of SprI is also described. These results are discussed in the context of the

role of systemin in the wound response of tomato plants.

Results

spr1 preferentially affects wound-induced systemic Pl expression

Recessive mutations in Sprl were previously shown to suppress 35S::prosys-mediated
expression of the well-characterized serine PI genes, PH and PM] (Howe and Ryan,
1999). Further characterization of this mutant was conducted using spr1/spr1

homozygous lines in which the 35S::prosys transgene was removed by outcrossing (see

37

Figure 2-1. Spatial pattern of PM and PM] mRNA accumulation relative to
the wound site. A single wound was inflicted at the distal end of the terminal
leaflet of the lower leaf of two-leaf-stage plants. Eight hours thereafter, various
sections of the leaf blade and petiole were dissected for RNA isolation and
analysis. Leaf sections from six plants were pooled for RNA isolation. (A)
Schematic drawing illustrating the leaf and petiole sections that were harvested
for RNA extraction. Panels in (B) and (C) Show the results obtained for
analysis of wild-type (WT) and spr1-I plants, respectively. Lanes 1 through 6
rep resent analysis of RNA isolated from the corresponding tissue sections
shown in panel in (A). Lanes 1', 2', and 3' represent analysis of RNA isolated
from various tissue sections of unwounded plants: 1', pooled tissue from
sections 1 and 6; 2', pooled tissue from sections 2 and 5; 3', pooled tissue from
sections 3 and 4. RNA gel blots containing 5 pg total RNA were hybridized to
probes for PM and PI—II. To facilitate the comparison between WT and spr1,
RNA blots shown in panels in (B) and (C) were hybridized together in the same
containers and exposed to autoradiographic film for the same time. Blots were
hybridized to a probe for eIF 4A as a loading control. Note, however, that e1F4A
mRNA abundance is greater in petiole tissue relative to leaf lamina. As an
additional loading control, a picture of an ethidium bromide-stained gel of the
total RNA (rRNA) is shown. The results shown are representative from three
independent experiments.

38

(A)

 

|1|2I3|4I5>I¢6>l

(B) wild-type
control wounded

1'2'3'12 34 5 6
n“ ..

eIF4A w»vw~~~--um

 

 

PI-l

 

Pl-II

 

rRNA yapunu-Eu

rum-o

sat-‘— fill.

 

(C) an"
control wounded

 

 

1’2’3’1 23456

PM K“ ...--.
PM! ..

eIF4A ~ua-..-

  

I'RNA wgwwsswngr'

39

Materials and Methods). To determine the effect of spr1 on wound-induced local and
systemic gene expression, RNA gel blot analysis was used to measure PH and PH]
transcript levels in leaf tissue located at defined distances from a single wound inflicted
at the distal end of the lower leaf (Figure 2-1A). As previously observed in wild-type
(WT) tomato plants (Howe et al., 1996), P1 mRNA accumulation in the undamaged
section (section 2) of the wounded leaf was significantly greater than that in the
damaged section (section 1) of the same leaf (Figure 2-1B). A relatively strong systemic
response was observed in the undamaged leaf (sections 5 and 6), whereas WT petioles
(sections 3 and 4) showed little or no PI expression. In the case of spr1, PI mRNA
accumulation in the damaged section of the wounded leaf was comparable to that in WT
(Figure 2-1C). Mutant plants also showed PI expression in adjacent unwounded tissue
(section 2), albeit at a level lower than in WT. More significantly, however, the steady—
state level of P1 mRNA in the unwounded leaf (sections 5 and 6) of wounded spr1
plants was <10% of that observed in WT. This result indicates that spr1 impairs a
signaling pathway that mediates or amplifies wound-induced systemic P1 expression,
but contributes less to P1 expression near the wound site. This interpretation was
supported by measurements of PI—II protein levels in damaged (local response) and
undamaged (systemic response) leaves of wounded plants (data not shown). In six
independent experiments involving at least six plants per genotype, the local response
of spr1 plants ranged between 50 and 75% of the WT response. Systemic PI-II
accumulation in spr1 plants ranged between 0 and 35% of that in WT plants, with the
average response in the mutant being approximately 15% of WT levels. Analysis of

plants homozygous for an independent allele of spr1 (spr1-2) gave very similar results;

40

Local Systemic

 

 

wild-type spr1 wild-type spr1
012 48224012 481224 0124 812240124 81224

PH! .. fl 1“

 

 

   

an.“ ”a an».
0".” «mama-wuoou

fl. I

CD, ..... an. m.

LoxD g”..- ”a--- ._,.,,,_ e _.......,
3a :2; a; =.. .

A081 " 4... g. Q

WIPK

 

Ms. M w wu,u~~~~~o fi!‘uuhwfi'fi~whu
-- . o. a m..- \ .-- * 7 3L. v.. "
. .. vaim'..‘5

eIF4Awwumuzwnmufiwun.a «wfiflflfifififl‘bmwu

Figure 2-2. Time course of wound-induced gene expression in wild-type (WT)
and spr1 plants. WT and spr1 plants (15-day-old) were wounded once on the
lower leaf with a hemostat. Lower damaged (local response) and upper
undamaged (systemic response) leaves were harvested at various times (hours)
after wounding for RNA isolation and analysis as described in the legend for
Figure 2-1. For each time point, Six plants were harvested and pooled for RNA
extraction.

41

wound-induced local and systemic PI-II accumulation in spr1-2 plants was 51 and 13%,
respectively, of WT levels.

RNA gel blot analysis was used to determine the time course of local and
systemic expression of various wound-responsive genes in spr1 plants. Two classes of
genes that differ with respect to their timing of wound-induced expression have been
described in tomato (Ryan, 2000). Transcripts of so called 'late'-response genes,
including PI-I, PI—II, and cathepsin D inhibitor (CD1), accumulate to maximum levels
8-12 hr after wounding of WT plants (Figure 2-2). Consistent with the results shown in
Figure 1, sprl plants were deficient in the magnitude but not the timing of induction of
these genes. Genes whose expression is induced rapidly and transiently in response to
wounding comprise a second class of 'early'-response genes. Included among this group
are genes encoding signaling-related proteins such as lipoxygenase (LoxD), allene
oxide synthasel (A081), and a putative mitogen-activated protein kinase (WIPK).
Interestingly, wound-induced local and systemic expression of these early genes was
not affected in spr1 plants (Figure 2). These results indicate that spr1 specifically

affects the expression of late-response genes (i.e. PI genes).

spr1 plants are impaired in systemin-mediated signaling

To gain additional insight into the wound response phenotype of spr1 , the capacity of
the mutant to respond to various PI-inducing compounds was determined (Figure 2-3).
Consistent with the ability of spr1 to suppress 35S.'.°prosys-mediated P1 expression, spr1
plants did not accumulate PI-II in response to exogenous systemin or its bioactive

precursor, prosystemin (Dombrowski et al., 1999). However, the mutant was responsive

42

 

Pl-ll (pg/ml leaf juice)
CD
0

 

 

 

 

 

 

Con Sys PS Chit OGA LA JA

Figure 2-3. PM] accumulation in wild-type (WT) and spr1 plants in
response to exogenous signaling compounds. WT (filled bar) and spr1 (open
bar) seedlings (lS-day-old) were excised at the base of the stem and
supplied with 15 mM sodium phosphate buffer (Con), systemin (Sys, 15 nM),
recombinant prosystemin (PS, 0.1 pg ml"), chitosan (Chit,250 pg ml 'I),
oligogalacturonide (OGA, 250 pg ml '1), linolenic acid (LA, 5 mM), or
jasmonic acid (JA, 100 nM). PI-II levels were measured 24 hr after
treatment. Data points represent the mean and SD (n = 6).

43

160

 

(A)

_L ..s
N 1%
o o
1 1

..s
o
o

801

404

it; , i

0 0.01 0.1 1 10 100

Pl-ll (pg/ml leaf juice)
M O)
O O

 

 

O

systemin (pmollplant)

 

(B) 160

140 4
1204
100 -

80 4
60 -
4o 4
20 -
o - a r , .
o 15 40 100

061A (ngml)

   

PI-II (pg/ml leaf juice)

 

 

 

 

 

 

 

 

*1

1 80 250

160
140 .
120 «
100 4 I

 

(C)

   

 

 

PI-II (pg/ml leaf juice)

 

 

 

 

O 15 40 100 180 250
chitosan (pg/ml)

Figure 2-4. Response of spr1 plants to exogenous systemin,
oligogalacturonides (OGA), and chitosan. Two-leaf-stage wild-type (WT)
(filled bars), defl (gray bars), and spr1 (open bars) plants were excised at the
base of the stem and supplied with either phosphate buffer ('0') or buffered
solution containing various concentrations of systemin (A), OGA (B), or
chitosan (C). PI-II accumulation in leaves was measured 24 hr after
treatment. Values indicate the mean and SD (n = 6).

to octadecanoid signaling compounds (linolenic acid and J A) and to the polysaccharide
elicitors OGA and chitosan. Because exogenous chitosan, OGA, and systemin activate
P1 expression via the octadecanoid pathway (Doares et al., 1995), these findings
indicated that spr1 affects systemin-mediated signaling at a point upstream of the
octadecanoid pathway. To further test this hypothesis, the responsiveness of spr1 plants
to a range of concentrations of systemin, OGA, and chitosan was compared to that of
WT (Figure 2-4). Parallel analysis of the J A-deficient defenseless] (def?) that is
impaired in P1 expression in response to systemin, OGA, and chitosan (Howe et al.,
1996) was included as a control. WT plants showed a strong response to systemin
concentrations of 1 pmol per plant and greater, whereas def] accumulated low levels of
PI—II in response to high concentrations of systemin, as previously reported (Howe et
al., 1996). spr1 plants failed to accumulate significant levels of PHI (<5% WT levels)
in response to all concentrations of systemin tested. As expected, the spr1 mutant
responded normally to a range of concentrations of OGA and chitosan, whereas def]
plants were unresponsive to these elicitors (Figures 2-4B, C). These results support the
idea that spr1 specifically affects the systemin branch of the wound-response pathway.
RNA gel blot analysis was used to determine the effect of spr1 on systemin-
mediated expression of early and late wound-response genes. Excision of seedlings at
the base of the stem resulted in a gradual, low-level increase in PM] mRNA
accumulation in leaves of both WT and spr1 plants (Figure 2-5A). WT plants
accumulated high levels of PM] mRNA in response to exogenous systemin, whereas
spr1 showed no response above that observed in the buffer control. This finding is

consistent with the inability of spr1 plants to accumulate PI—II protein in response to

45

Figure 2-5. Effect of exogenous systemin on gene expression in wild-type
(WT) and spr1 plants. (A) Excised seedlings (2-week-old) were incubated
for 45 min in a solution containing 5 pmol systemin, and then transferred to
water. At various times (hours) after the beginning of systemin treatment,
leaf tissue was harvested for RNA isolation. Leaf tissue from six plants was
pooled for each RNA isolation. RNA gel blots containing 5 pg total RNA
were hybridized to probes for PI—II, LoxD, AOS], WIPK and, as a loading
control, eIF4A. (B) Excised seedlings were pre-incubated in water for 4 h
and then transferred either to 300 pl phosphate buffer (buffer) or the same
volume phosphate buffer containing 5 pmol of systemin ('0'). Following
uptake of this solution (approximately 45 min), seedlings were transferred
to water. At various times (hours) thereafter, leaves were harvested for
RNA isolation.

46

 

 

(A) buffer systemin
wild-type spr1 wild-type spr1
051248051248{0512480511248

"m

Pl'” :1 he. 4, au W “a . ‘1, 111:: .~,.... M, a”

 

 

 

 

LoxD 'w. 3.. "'e- .- 'e-
m [‘3' "2'
r?- "- m ..m
J 123. I!!!
('41 Inn 1531:. ‘ Linn ,
WIPK gm“ N r .101 M in!” . ‘~ .5”. .g....vdofi
ALL. ""3“... .. . mglum-Mm... “..1. ..1-{.1

eIF4A ”up- uflflfiflnfl QCQCCQOOGCOU

(8) buffer (after pretreatment) systemin (after pretreatment)

 

wild-type spr1 wild-type spr1
051248051248 0.51248051248

PI-II fififl- ..fiu U- a». u

 

 

LoxD .- ‘" “' "'
A031 . .....

. '1‘ . ’ p ‘ . . J.
WIPK " " '1 “ "f' "' j _"'".‘ 1;“. W

-\o-

eIF4A -----~flfl---~ uflflaauogubutui

47

systemin (Figure 2-4). Mock treatment (excision and incubation in buffer) of both WT
and spr1 seedlings resulted in rapid and transient expression of three early wound-
response genes: LoxD, AOS], and WIPK. In WT plants, systemin clearly enhanced the
accumulation ofLoxD and AOS] mRNA, as previouSly reported (Heitz et al., 1997;
Sivasankar et al., 2000). However, the expression of these genes in spr1 was not
enhanced by systemin. In contrast to LoxD and AOS], the steady-state level of WIPK
mRN A in both WT and spr1 plants was not affected by systemin treatment. The
analysis of systemin-induced gene expression in WT and mutant plants was
complicated by the fact that excision of seedlings at the base of the stem induced
significant expression of early-response genes (Figure 2-5A). To determine the effect of
systemin in the absence of this excision-induced effect, excised plants were pre-
incubated in water for 4 hr (to allow mRNA levels to return to basal level), and then
transferred to tubes containing either buffer or systemin (Figure 2-5B). A very low level
of LoxD and AOS] expression was detected in buffer-treated WT and spr1 plants,
presumably as a result of handling (i.e. touching) of plants during the transfer
procedure. Transfer of pre-incubated WT plants to a systemin-containing solution
resulted in a rapid and transient increase in the steady-state level of LoxD and AOS]
mRNAs, and a more gradual, massive accumulation of PHI transcripts. The level of
WIPK mRNA in WT plants was unaffected by systemin treatment, indicating that
exogenous systemin stimulates expression of some (e. g. LoxD, AOS], and PM!) but not
all (e. g. WIPK) wound-response genes. Treatment of pre-incubated spr1 plants with
systemin did not increase the accumulation of LoxD, AOS] , PM], or WIPK mRNAs

above the level observed in buffer-treated plants. In summary, these results show that

48

(A)

300

 

JA(pmolIgFW)
o 8 8 8 8 8

 

 

 

 

 

 

 

0 1 3
Incubation time (hour)

(3)

 

ears“

4'0“
1L1

JA (pmol/g FW)
0 8 8 8

 

fi—r—I

buffer systemin
Treatment

 

 

 

 

 

Figure 2-6. Jasmonic acid (JA) accumulation in response to systemin and
mechanical wounding. (A) Leaves of 2-week-old wild-type (WT) (filled
bar) or spr1 (open bar) plants were mechanically wounded with a
hemostat. At various times after wounding (1 or 3 hr), wounded leaf tissue
was harvested for J A extraction. J A was also extracted from leaves of
unwounded plants ('0').(B) Two-week-old seedlings were excised at the
base of the stem, pre-incubated in water for 4 hr, and then transferred to
either buffer unwanted plant ('0') or a buffered solution containing
systemin (5 pmol per plant). Leaves were harvested for JA extraction 2 hr
45 min after systemin application. The amounts of J A in plant extracts
were quantified by GC-MS. Data represent the mean and SD of three
independent experiments.

49

spr1 impairs systemin-mediated activation of both early- and late-response genes.
However, the mutation does not affect the rapid and transient activation of early genes
in response to excision or wounding, indicating the existence of an Spr]-independent

wound signaling pathway.

Effect of spr1 on wound— and systemin-induced JA accumulation

Because wound- and systemin-induced PI gene expression is dependent upon the
synthesis and subsequent action of J A, it was of interest to determine the capacity of
spr1 plants to accumulate J A in response to wounding and systemin. J A levels in
unwounded leaves of WT and mutant plants were 15.1 :t 1.5 pmol JA/g Fresh Weight
(FW) and 14.5 i 4.6 pmol J A/ g FW, respectively (Figure 2-6A). In wounded WT
plants, J A levels increased 15- and 7.5-fold, 1 and 3 hr after wounding, respectively.
Wounding also increased J A accumulation in spr1 plants, albeit to levels that were
significantly lower (P < 0.05) than WT levels. The amount of J A in wounded spr1
leaves throughout the time course was estimated to be approximately 57% of that in WT
leaves. Exogenous systemin induced high levels of J A accumulation in WT, but had no
effect in spr1 (Figure 2-6B). These findings indicate that Spr] is necessary for maximal
levels of JA accumulation in response to wounding, but is strictly required for systemin-

induced J A accumulation.

50

WT spr1 WT spr1

 

 

WT spr1 spr1 WT
- + - + - + - +
PHI
to
Q.
o
3
eIF4A
PHI 2)
o
9.
(I)
8
eIF4A S’-

 

 

Figure 2-7. Wound-inducible PI-II expression in grafts between wild-type
(WT) and spr1 plants. WT and spr1 plants were grafted in the four
combinations indicated. The genotypes listed above and below the
horizontal line correspond to the scion and rootstock, respectively. For each
graft combination, plants were divided into a control (-) and experimental
(+) group consisting of four grafted plants per group. For the experimental
group, each leaflet on the rootstock was mechanically wounded with a
hemostat. Eleven hours after wounding, leaf tissue was harvested separately
from wounded rootstock leaves 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 eIF4A cDNA probe as a loading
control. The results shown are representative of three independent
experiments.

51

spr1 plants are defective in the generation of a systemic wound signal for

PI expression

The deficiency of wound-induced systemic PI expression in spr1 plants could result
from a defect in production of a long-distance wound signal or a defect in the
perception of this signal in distal undamaged leaves. To address this question, wound-
induced PI-II expression was analyzed in reciprocal grafts between WT and spr1 plants.
F our-week-old plants were grafted such that both the rootstock (stock) and the scion
contained at least two healthy leaves. After the graft junction healed, stock leaves were
wounded and PH] mRN A levels were measured 11 hr after in both the damaged stock
leaves (local response) and the undamaged scion leaves (systemic response). Wounding
of WT stock leaves resulted in local and systemic accumulation of PHI transcripts to
levels well above that observed in unwounded control plants that had also been grafted
(Figure 2-7, lanes 1 and 2). This result demonstrates that wounding of WT stock leaves
leads to the production of a graft-transmissible signal that is recognized in undamaged
scion leaves. Consistent with the pattern of PHI expression in two-leaf-stage spr1
plants (Figure 2-1), wounded spr1 stock leaves showed a relatively strong local
response and a weak (10% WT) systemic response (Figure 2-7, lanes 3 and 4). Analysis
of spr1/WT hybrid grafts Showed that upon wounding of spr1 stock leaves, WT scions
failed to activate PI-II expression to levels greater than that in spr1 scions that had been
grafted to spr1 stock (Figure 2-7, lanes 5 and 6). In the reciprocal combination,
however, spr1 scions were responsive to a signal emanating from wounded leaves of
WT stock (Figure 2—7, lanes 7 and 8). Taken together, these findings indicate that spr1

impairs wound-induced systemic PI expression mainly by blocking the production of

52

the long-distance wound signal in damaged leaves, rather than the recognition of that

signal in systemic, undamaged leaves.

Discussion

spr1 defines a novel class of wound-response mutant

Forward genetic screens have identified two general classes of mutants that are
defective in wound-induced systemic PI expression (Howe and Ryan, 1999; Li et al.,
2001; Lightner et al., 1993). One group includes JA biosynthetic mutants (e.g. defl and
spr2) that are unresponsive to upstream signals (e.g. systemin, OGA, and chitosan) that
activate the octadecanoid pathway, but are responsive to exogenous J A. The second
group includes JA-insensitive mutants (e.g. jail) that are responsive neither to upstream
signals nor to J A. Here, we show that spr1 differs from existing wound-response
mutants in that it is responsive to OGA and chitosan but muesponsive to systemin and
its precursor, prosystemin. Consistent with the fact that OGA and chitosan activate PI
expression through J A (Doares et al., 1995), spr1 plants were responsive to exogenous
J A and its metabolic precursor, linolenic acid (Figure 2-3). These findings indicate that
the octadecanoid pathway and downstream signaling steps leading to P1 expression are
intact in sprl plants. The capacity of the mutant to accumulate significant levels of J A
(approximately 57% WT levels) in response to wounding supports this idea. Considered
collectively, the most straightforward interpretation of the results is that Spr] is
involved in the perception of systemin or a subsequent systemin-specific signaling

event necessary for activation of the octadecanoid pathway. Because systemin

53

perception occurs at the level of the plasma membrane (Meindl et al., 1998; Scheer and
Ryan, 1999) and the initial steps of the octadecanoid pathway occur in the chloroplasts,
it is possible that Spr] is involved in relaying a signal from the plasma membrane to the
chloroplast. Included among the early signaling events induced by systemin are
increased cytosolic Ca2+ levels, membrane depolarization, inhibition of a plasma
membrane proton ATPase, activation of a MAP kinase activity, and activation of a
phospholipase A2 activity (Felix and Boller, 1995; Moyen and Johannes, 1996; Moyen
et al., 1998; Narvaez-Vasquez et al., 1999; Schaller and Oecking, 1999; Stratmann and
Ryan, 1997). The insensitivity of sprl plants to both prosystemin and systemin indicates
that the mutant is not defective in the synthesis or proteolytic processing of
prosystemin. However, the data leave open the possibility that spr1 impairs the
interaction of systemin with SR160, or signaling output of the activated receptor

complex. Additional work is needed to distinguish these possibilities.

Spr1-independent wound signaling

It is noteworthy that sprl appears to impair wound-induced systemic PI expression
much more than it affects local P1 expression. This finding indicates that the signaling
pathway for systemic PI expression can be uncoupled from the signaling pathway that
operates in tissue adjacent to the wound site. This aspect of spr1 is reminiscent of the
wound-response phenotype of prosystemin antisense plants that are compromised in
prosystemin production but nevertheless have the capacity to respond to exogenous
prosystemin (Dombrowski et al., 1999; Mch1 et al., 1992). The robust local wound

response of sprl and prosystemin antisense plants supports the hypothesis that multiple

54

signals generated at the wound site activate the octadecanoid pathway in wounded
leaves (Doares et al., 1995; Farmer and Ryan, 1992; Ryan, 2000). This interpretation is
consistent with the observation that spr1 plants respond normally to OGAS, and
accumulate significant levels of J A in response to wounding (Figures 2-4 and 2-6).
Whether OGAS are responsible for the entire pool of wound-induced J A in spr1 leaves
or whether other mechanisms are involved in initiating the octadecanoid pathway
remains to be determined.

Genetic analysis of the wound response in tomato plants indicates that the bulk
of wound-induced systemic PI expression requires the systemin/J A signaling pathway.
However, we did observe that sprl plants exhibit a low but significant level of wound-
induced systemic Pl expression. This residual signaling activity could reflect
incomplete loss of Spr] function or, alternatively, a Spr] -independent pathway for
systemic PI expression. Given the complete lack of systemin-induced gene expression
in spr1 plants, however, the latter possibility seems more likely. The existence of a
systemin-independent wound-response pathway is clearly supported by the observation
that spr1 plants are not affected in wound- or cut-induced expression of early wound-
response genes such as LoxD and AOS] . The fact that exogenous systemin enhances
expression of LoxD and AOS] in WT plants (Figure 2-5) indicates that wounding and
exogenous systemin may regulate the expression of these genes in somewhat different
ways. For instance, it is possible that responses to systemin, when supplied through the
transpiration stream, do not accurately reflect the wound-induced activity of systemin
produced in vascular bundle cells of intact plants (Jacinto et al., 1997; Ryan, 2000). The

expression pattern of WIPK, which was wound and cut inducible in both WT and sprl

55

plants, provides fiirther evidence for systemin-independent wound signaling. Unlike the
LoxD and AOS] genes, exogenous systemin did not stimulate WIPK mRNA
accumulation in WT (or sprl) plants. Several other wound-induced rapid systemic
responses have been described in plants (e. g. O'Donnell et al., 1998; Seo et al., 1995;
Stratmann and Ryan, 1997), and may involve physical (e. g. hydraulic) Si gnals

propagated through the xylem (Malone, 1996) or the phloem (Rhodes et al., 1999).

Role of systemin in wound signaling

The signaling-related phenotypes of spr1 plants are fiilly consistent with a role for
prosystemin in regulating wound-induced systemic PI expression through the
octadecanoid pathway (Farmer and Ryan, 1992; Li et al., 2001; Mch1 et al., 1992).
What is less clear, however, is how systemin and J A (and other signals) interact to
effect wound signaling over long distances. Insight into this question was recently
provided by means of grafting experiments showing that J A biosynthesis in rootstock
leaves is essential for production of a long-distance Signal for P1 expression, whereas
J A biosynthesis in undamaged leaves is not required for PI expression (Li et al., 2002a).
Given that prosystemin works through J A, the most straightforward interpretation of
these data is that systemin, acting in the rootstock portion of the graft, amplifies the
synthesis of J A to levels that are required for long-distance signaling. Support for this
model comes from the analysis of systemic P] expression in reciprocal grafts between
WT and spr1 (Figure 2-7). These experiments Show that Spr] function is involved
primarily in the production of the long-distance signal, rather than the recognition or

processing of that signal in systemic undamaged leaves. The reduced level of wound-

56

induced J A in sprl plants further indicates that systemin may activate the synthesis of a
specific pool of J A that is necessary for the systemic response, which is consistent with
the lack of systemin-induced JA accumulation in spr1 (Figure 2-6). Along these lines,
two hypotheses have recently been proposed to explain the role of systemin in long-
distance P1 expression (Ryan and Moura, 2002). First, systemin may induce localized
production of J A that subsequently exits the wounded leaflet and activates PI
expression in distal leaves. Alternatively, systemin produced at the wound site may be
translocated in the phloem where it activates JA synthesis in vascular tissues of leaves,
petioles, and stems of the rootstock. The latter scenario indicates that a positive
feedback loop between systemin and J A may amplify and propagate the systemic signal
along the vascular system (Ryan, 2000; Ryan and Moura, 2002). Additional insight into
the function of Spr] and other genes involved in systemic wound signaling may help to

distinguish these hypotheses.

Materials and Methods

Plant material and treatments

Tomato (Lycopersicon esculentum Mill cv Castlemart) seedlings were grown in Jiffy
peat pots (Hummert International, Earth City, MO) in a grth chamber maintained
under 17 hr of light (200 pE m 2 sec '1) at 28°C and 7 hr of dark at 18°C. Seed for def]
was collected from a defl/defl homozygous line that was back-crossed four times using
Castlemart as the recurrent parent. To simplify and standardize the genetic
nomenclature, herein we refer to the previously described spr-l 593p and spr—l 9615

alleles (Howe and Ryan, 1999) as spr1-1 and sprl -2, respectively. Lines homozygous

57

for either of these two alleles were generated as described below. Unless otherwise
indicated, sprl-1 was used for all experiments.

Wounding and chemical elicitor experiments were performed with two-leaf-
stage plants (14 to 16-day-old) as previously described (Howe etal., 1996). To assay
the responsiveness of mutants to PI-inducing compounds, plants were excised at the
base of the stem and placed in 0.5 ml microfuge tubes containing 300 pl of the inducing
compound. When >75% of the elicitor solution had been imbibed (approximately 45
min), plants were transferred to glass vials containing 20 ml of water, and incubated in a
Lucite box for 24 hr under continuous light. PI-II levels in leaves were measured by
radial irnmunodiffusion assay (Ryan, 1967). Systemin, oligogalacturonic acid (OGA),
and recombinant prosystemin (Dombrowski et al., 1999) were obtained from Dr. C .A.
Ryan (Washington State University). Chitosan, J A, and linolenic acid were obtained
from Sigma. All inducers except linolenic acid were diluted from stock solutions into
sodium phosphate buffer (15 mm sodium phosphate, pH 6.5) prior to use. Linolenic
acid was diluted into 15 mm sodium phosphate, pH 6.5, containing 0.05% (v/v) ethanol.
Control experiments Showed that 0.05% ethanol in 15 mm soditun phosphate buffer did
not affect the background level of PI expression (data not shown). Grafting experiments

were performed as described by Li et al. (2002a).

Genetic analysis
sprl-l and spr1 -2 were originally isolated as recessive suppressors of 35S: :prosys-
mediated responses O-Iowe and Ryan, 1999). Segregation of spr1-1 and spr1-2 from the

35S::prosys transgene, for the purpose of isolating homozygous spr1 alleles in an

58

otherwise WT genetic background, was achieved by crossing mutant lines (homozygous
for the 35S::prosys transgene and either spr1-1 or spr1-2) to WT (cv Castlemart). Plants
in the resulting F2 populations were tested for wound-induced PI-II accumulation with
the assumption that spr1 homozygotes would display a Significantly reduced systemic
response, as is the case for prosystemin antisense plants (McGurl etal., 1992). F2 plants
showing a deficiency (<10% of WT levels) in the systemic response comprised
approximately one-quarter of the population (data not shown) and were selected as
putative spr1 homozygotes. A polymerase chain reaction (PCR)-based assay (Li and
Howe, 2001) was used to test these plants for the presence of the 35S::prosys transgene.
Individuals lacking the transgene were brought to the greenhouse for collection of F3
seed. To verify homozygosity of the spr1 allele, the deficiency in wound-induced
systemic PI-II expression and insensitivity to exogenous systemin were confirmed in F3
seedlings. Southern blot analysis confirmed the absence of 35S::prosys. Selected spr1
homozygotes (either spr1-l 0r spr1-2) were back-crossed again to WT (cv Castlemart).
Seedlings in the resulting F2 populations were scored for wound-induced systemic
accumulation of PI-H, as well as for systemin-induced PI-II accumulation. The results
were consistent with the expectation that spr1-l and sprl -2 impair both responses and

behave as single recessive mutations.

RNA gel blot analysis
RNA was isolated fiom tomato leaves and analyzed by gel blot hybridization as
described by Li et al. (2002b). Gels were run in duplicate, with one set stained with

ethidium bromide to check for equal loading of the samples and intactness of the RNA.

59

DNA probes were isolated and radiolabeled with [32P-a] dCTP as described by Howe et
al. (2000). The tomato EST clones, cLEC9C14 and cLET1D13, were used as probes for
detection of AOS] (Sivasankar et al., 2000) and WIPK transcripts, respectively.
Hybridization results were visualized by autoradiography using Kodak XAR-S film and,
when appropriate, quantified using a Phosphorimager (Molecular Dynamics).
Hybridization signals were normalized to the signal obtained using a cDNA probe (EST
clone cLED1D24) for translation initiation factor eIF 4A. To directly compare transcript
levels in WT and spr1 plants, blots containing RNA from both plant types were
hybridized in the same container, washed under the same conditions, and exposed to

film for the same length of time.

Measurement of jasmonic acid

J asmonic acid was extracted from leaves (10 g FW) of two-leaf-stage plants as
previously described (Li et al., 2002b). Dihydrojasmonic acid (DHJA) was used as an
internal standard for quantification of J A levels by gas chromatography-mass

spectrometry (Li et al., 2002a).

60

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Ryan, C.A., and Moura, BS. (2002). Systemic wound signaling in plants: a new
perception. Proc. Natl. Acad. Sci. USA. 99: 6519-6520.

Schaller, A., and Oecking, C. (1999). Modulation of plasma membrane HI-ATPase
activity differentially activates wound and pathogen defense responses in
tomato plants. Plant Cell, 11, 263-272.

Scheer, J .M., and Ryan, CA. (1999). A 160-kD systemin receptor on the surface of
Lycopersicon peruvianum suspension-cultured cells. Plant Cell 11: 1525-1536.

Scheer, J .M., and Ryan, CA. (2002). The systemin receptor SR160 from Lycopersicon
peruvianum is a member of the LRR receptor kinase family. Proc. Natl. Acad.
Sci. USA. 99: 9585-9590.

Sec, 8., Okamoto, M., Seto, H., Ishizuka, K., Sano, H., and Ohashi, Y. (1995). Tobacco
MAP kinase: a possible mediator in wound signal transduction pathways.
Science 270: 1988-1992.

Sivasankar, S., Sheldrick, B., and Rothstein, SJ. (2000). Expression of allene oxide

synthase determines defense gene activation in tomato. Plant Physiol. 122:
1 335-1342.

Stratmann, J .W., and Ryan, CA. (1997). Myelin basic protein kinase activity in tomato
leaves is induced systemically by wounding and increases in response to
systemin and oligosaccharide elicitors. Proc. Natl. Acad. Sci. USA. 94: 11085-
11089.

Walker-Simmons, M.K., and Ryan, CA. (1984). Proteinase inhibitor synthesis in
tomato leaves. Induction by chitosan oligomers and chemically modified
chitosan and chitin. Plant Physiol. 76: 787-790.

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

Xie, D.-X., Feys, B.F., James, S., Nieto-Rostro, M., and Turner, J .G. (1998). C011: an
Arabidopsis gene required for jasmonate-regulated defense and fertility. Science
280: 1091-1094.

Xu, L., Liu, F ., Lechner, E., Genschik, P., Crosby, W.L., Ma, H., Peng, W., Huang, D.

and Xie, D. (2002). The SCFCO" ubiquitin-ligase complexes are required for
jasmonate response in Arabidopsis. Plant Cell 14: 1919-1935.

64

Appendix of Chapter 2

Spr1 does not encode SR160, the systemin receptor

The cloning of SR160 from spr1 plants was performed by Mr. Carl Andre as part
of his lab rotation project.
The BC] mapping population used in this study was generated by Dr. Chuanyou

Li.

65

introduction

The previous chapter describes the putative role of the Suppressor of prosystemin-
mediated responses] (Spr]) gene in systemin-mediated signaling. Systemin failed to
induce synthesis of proteinase inhibitors (PIS) in spr1 plants, whereas other elicitors
such as oligogalacturonic acid (OGA) and jasmonic acid (J A) activate normal PI
accumulation in the mutant (Figure 2-3 and 24). Because both systemin and OGA
induce expression of P1s by stimulation of J A biosynthesis (Doares et al., 1995a;
Doares et al., 1995b; Howe et al., 1996), these results indicate that spr1 plants are
compromised in the perception of systemin or a subsequent systemin-specific Signaling
event necessary for activation of J A biosynthesis.

Systemin perception is mediated by the systemin receptor, SR160, which is
bound to the plasma membrane (Meindl et al., 1998; Scheer and Ryan, 1999). Using a
photoaffinify labeling approaCh, SR160 was purified from cultured cells of the wild
tomato species Lycopersicon peruvianum. This work led to the isolation of the
corresponding cDNA encoding SR160. DNA sequence analysis showed that SR160
encodes a leucine-rich repeat (LRR) receptor kinase that shows high similarity to the
BRII gene that encodes the brassinolide receptor of Arabidopsis (Scheer and Ryan,
2002).

The systemin-insensitivity of spr1 plants indicates the possibility that Spr]
encodes SR160. Experiments described here were designed to test this hypothesis. The
results demonstrate that the sprl mutation does not affect SR160. Thus, it is likely that
the Spr] gene product acts downstream of SR160 in the wound signaling pathway to

stimulate J A biosynthesis.

66

Results

Cloning of SR160 from spr1 plants

To determine whether spr1 plants carry a mutation in the SR160 gene, the sequence of
SR160 cloned from spr1 and its corresponding wild-type L. esculentum was compared.
Because the SR160 cDNA was previously isolated from L. peruvianum (Scheer and
Ryan, 2002), this sequence information was used to design two oligonucleotide primers
to clone the SR160 from spr1 and wild-type L. esculentum. Genomic DNA from these
plants was used as a template for PCR to clone SR160. Amplified products from these
reactions were cloned and sequenced. The results demonstrated that SR160 genes from
both sprl plants and wild-type (L. esculentum) contain an intronless 3624 bp open
reading frame, 62 bp of 5’ untranslated region (UT R), and 122 bp 3’ UTR.
Comparisons between SR160 from wild-type and spr1 plants Showed no sequence
differences (Figure 2-8). Comparison of the deduced amino acid sequence of SR160
from L. esculentum to L. peruvianum showed sequence variation at 10 amino acid

residues (Figure 2-9).

Analysis of linkage between Spr1 and SR160 loci

To further investigate whether Spr] and SR160 correspond to the same gene, linkage of
the two loci was examined in a BC 1 mapping population that segregates for both the
spr1 trait (i.e., systemin insensitivity) and a restriction fragment length polymorphism

(RF LP) at the SR160 locus. The mapping population was generated fiom a cross

67

Figure 2-8. Genomic DNA sequence of SR160 isolated from L. esculentum. A PCR-
strategy was used to clone SR160 from spr1 and wild-type L. esculentum. DNA
sequence analysis Showed no sequence differences in SR160 between sprl plants and L.
esculentum. Both genes contain an intronless ORF (3624 bp). The initiation and
termination codons are boxed. Nucleotide sequences corresponding to the PCR primers
are underlined.

1 AGAACTCAAGCTATAGATTCAAGAAAATCACCATTTAAGCTATAAAGTTTCAATCTTTGA

61 AQBIQAAAGCTCACAAAACTGTGTTTAACCAACATCCTTTGAGCTTAAACAAGCTTTTCT
121 TTGTTCTTCTTCTTATCTTTTTTCTTCCACCAGCTTCACCAGCAGCTTCTGTTAATGGTC
181 TTTATAAAGACTCCCAACAGCTTCTTTCCTTTAAAGCTGCACTCCCACCAACCCCAACTC
241 TGCTTCAGAACTGGTTGTCATCTACTGACCCTTGTAGTTTCACTGGTGTTTCATGCAAGA
301 ATTCTAGAGTTTCTTCTATAGATCTCAGTAACACTTTTTTAAGTGTGGATTTCAGTTTGG
361 TCACTTCTTATTTGCTTCCTCTTTCTAATTTGGAGTCTTTGGTGTTAAAGAATGCTAATC
421 TTAGTGGTTCTTTAACTTCTGCTGCAAAATCCCAATGTGGGGTTACTTTAGACTCCGTAG
481 ATCTAGCTGAGAACACAATTTCTGGACCTATTTCTGATATCTCTAGCTTTGGTGTTTGTT
S41 CAAACCTTAAGTCTCTTAATCTTTCTAAGAATTTCTTGGACCCTCCTGGTAAAGAAATGC
601 TTAACGCTGCAACCTTTAGCCTCCAAGTTCTTGATCTTTCTTACAATAATATCTCAGGGT
661 TTAACTTGTTTCCATGGGTTTCATCTATGGGGTTTGTTGAACTTGAGTTCTTTTCTCTCA
721 AGGGTAACAAGCTAGCTGGAAGTATTCCTGAATTAGACTTCAAGAATTTGTCATATTTGG
781 ATCTTTCTGCAAATAATTTCTCAACTGTTTTTCCTTCATTCAAAGATTGCTCCAATTTGC
841 AGCACTTGGATTTGTCATCCAACAAGTTTTATGGTGATATTGGTTCTTCACTTTCTTCAT
901 GTGGGAAGCTCAGTTTTCTCAACCTTACCAATAACCAGTTTGTAGGTTTGGTCCCTAAGC
961 TACCAAGTGAAAGTCTACAGTATTTGTACTTAAGAGGGAATGATTTTCAGGGTGTGTACC
1021 CAAACCAACTTGCTGATTTGTGCAAAACTGTGGTGGAATTGGACTTGTCATACAATAATT
1081 TCTCAGGCATGGTTCCTGAGAGCCTTGGTGAATGTTCAAGTTTGGAACTTGTTGATATTT
1141 CCTACAATAATTTCTCTGGTAAGTTGCCTGTTGATACTCTCTCCAAGTTGAGTAATATTA
1201 AGACTATGGTCTTATCATTCAACAAATTTGTTGGTGGTTTGCCTGATTCTTTCTCTAATT
1261 TACTGAAATTGGAGACTTTGGATATGAGTTCTAATAATCTCACAGGGGTTATTCCATCTG
1321 GGATTTGCAAAGATCCTATGAATAACTTGAAAGTGCTGTACCTTCAGAATAACTTGTTTA
1381 AAGGCCCTATACCTGACAGTCTAAGCAACTGTTCACAGCTGGTGTCACTTGATCTTAGCT
1441 TTAATTACTTGACTGGGAGTATACCATCTAGTTTGGGGTCATTGTCAAAGCTAAAGGATC
1501 TCATCCTTTGGTTAAATCAGCTTTCAGGGGAAATCCCACAGGAGTTGATGTACTTGCAGG
1561 CTTTGGAGAATTTGATTCTTGATTTTAATGACTTAACTGGACCAATACCTGCAAGTCTTA
1621 GCAACTGTACCAAGTTGAATTGGATTTCATTGTCAAATAACCAATTGAGTGGTGAGATAC
1681 CGGCTTCTCTTGGGCGTTTGTCAAATCTAGCTATTCTTAAGCTTGGAAACAACTCAATCT
1741 CAGGGAATATACCTGCTGAATTGGGTAATTGCCAGAGCTTGATATGGTTGGATCTCAATA
1801 CTAATTTCCTGAATGGATCCATTCCGCCACCTTTGTTCAAGCAATCTGGCAATATTGCAG
1861 TGGCATTACTGACCGGGAAGCGATACGTGTATATCAAGAATGATGGGAGTAAGGAGTGCC
1921 ATGGAGCAGGGAATCTGCTGGAGTTTGGAGGGATTAGACAGGAACAGCTGGATAGAATCT
1981 CAACAAGGCATCCTTGCAATTTCACAAGAGTTTATAGAGGTATCACTCAGCCAACATTTA
2041 ACCACAATGGCTCTATGATATTTCTTGATTTATCTTATAATAAGTTGGAAGGTAGTATCC
2101 CAAAGGAATTAGGGGCAATGTACTATCTGTCTATATTGAATTTGGGGCATAATGATCTGT
2161 CTGGTATGATTCCTCAACAACTTGGAGGCTTGAAGAATGTTGCAATTCTTGATTTGTCAT
2221 ATAATAGGTTCAATGGCACGATCCCGAATTCCCTCACCAGTCTTACATTGCTTGGAGAGA
2281 TTGACCTGTCAAACAATAATCTCAGTGGAATGATTCCTGAATCTGCACCATTTGACACAT
2341 TCCCTGATTATAGGTTTGCGAATAATTCCCTCTGTGGGTATCCTCTCCCCATACCTTGTA
2401 GCTCGGGGCCGAAATCGGATGCAAATCAGCATCAGAAGTCTCACCGCAGACAAGCATCGT
2461 TGGCAGGGAGTGTGGCCATGGGTTTGTTATTTTCCCTCTTTTGTATCTTTGGTTTGATTA
2521 TTGTTGCCATAGAGACGAAGAAGAGGAGGAGGAAGAAGGAGGCTGCTCTTGAAGCTTATA
2581 TGGATGGTCATTCACATTCTGCAACTGCCAACAGTGCCTGGAAGTTTACGAGTGCTCGTG
2641 AGGCGTTAAGCATCAACCTTGCAGCATTTGAGAAGCCTCTCAGGAAGCTCACATTTGCTG
2701 ATCTTCTCGAAGCCACCAATGGTTTCCACAACGACAGTCTTGTAGGCTCTGGTGGTTTTG

 

68

2761
2821
2881
2941
3001
3061
3121
3181
3241
3301
3361
3421
3481
3541
3601
3661
3721
3781

GTGATGTCTACAAAGCTCAGTTGAAGGATGGGAGTGTTGTAGCTATTAAGAAATTGATAC
ACGTCAGTGGACAGGGTGATCGAGAATTCACTGCTGAAATGGAAACCATAGGGAAGATCA
AGCACCGCAACCTTGTCCCTCTTTTGGGCTACTGCAAAGTAGGGGAAGAAAGACTACTGG
TTTATGAATACATGAAGTATGGAAGTCTTGAAGATGTCCTGCATGATCGGAAGAAAATTG
GGATCAAGCTGAATTGGCCTGCAAGAAGGAAAATTGCCATTGGAGCTGCGAGAGGTTTGG
CTTTCCTACACCATAACTGCATTCCACACATCATTCACCGGGACATGAAATCAAGTAATG
TCTTGCTTGATGAAAATTTGGAAGCCAGAGTATCTGATTTCGGAATGGCAAGGTTAATGA
GTGCTATGGACACTCATTTGAGTGTCAGCACTCTTGCCGGCACTCCAGGATACGTACCTC
CTGAATATTACCAAAGCTTTAGATGTTCTACAAAAGGAGACGTTTATAGTTATGGTGTCG
TATTACTTGAGCTTCTAACCGGCAAACAGCCAACAGATTCAGCTGATTTTGGTGACAACA
ATCTTGTCGGATGGGTAAAGCTGCACGCTAAGGGAAAAATAACAGATGTCTTTGACCGGG
AGCTATTGAAAGAGGATGCAAGCATTGAGATTGAACTTCTACAACACTTAAAGGTAGCTT
GTGCTTGCTTAGATGATCGACATTGGAAACGTCCCACAATGATACAAGTTATGGCTATGT
TTAAGGAGATTCAAGCAGGGTCAGGCATGGATTCGACATCGACAATCGGAGCTGATGATG
TTAATTTTAGTGGAGTTGAAGGAGGGATAGAAATGGGGATAAATGGAAGTATAAAAGAAG
GCAATGAGCTGAGCAAACACCrnfijzprcCACTAAATGAAGAGTTTATTGAAAGCTCACA
AATTTTCCAAAATCATCATATGCAAAGTGTAATTTTTAGCCCCCAATTATTGTATGTACC
ACTAGTTCCCATCCATAAAATCTTGTGT

 

69

Figure 2-9. Comparison of deduced amino acid sequences of SR160 and BRII.
SR160 genes were isolated from sprl plants, cultivated tomato (L. esculentum), and the
wild tomato species, L. peruvianum. Deduced amino acid sequences of these genes
were aligned with that of Arabidopsis brassinolide receptor BRII, using the Clustal W
program available at http://www.ch.embnet.org. Black boxes indicate conserved amino
acid residues among all aligned sequences.

  
 

 

 

 

 

 

   

 
     

 

 

spr1 VKAHKTVPNQHPLSLNKLFFVLLLlFFLPPASPAASVNGLYKDSQQLLEF
esculentum Hi AHKTVFNQHPLSLNKLFFVLLLIFFLPPASPAASVNGLYKDSQQLLSi
peruvianum Yfi-HRTVFNOHPLSLNRLFFVLLLTFFLPPASPAASVNGLIVDSOOLLQL
Arabidopsis “ ‘ '
spr1 V-PLPPTPTLLQNHLSSTDPCSFTG‘CCKNSRVSSIDLSNTFLSVDF L;
esculentum .UaLPPTPTLLQNWLSSTDPCSFTGVSCKNSRVSSIDLSNTFLSVDFQL-
peruvianum H-HLPPTPTLLONHLSSTDPCSFTGVSCKNSPVSSIDLSNTFLSVDFQI
Arabidopsis ‘ ‘

spr1 TSYLLPLSNLESLVLKNANLSGSLTSAAKSQCGVTLDSVDLAENTISGPI
esculentum TSYLLPLSNLESLVLKNANLSGSLTSAAKSQCGVTLDSVDLAENTISGPi
peruvianum

Arabidopsis
spr1lSSFGVCSNLKSLNLSKNFLDPPGKLNAATFSLQ\LDLSYNNISGE
esculentum DISSFGVCSNLKSLNLSKNFLDPPGREMLNAATFSLQVLDLSYNNISGb
peruvianum

Arabidopsis

spr1

esculentum

peruvianum THFPHVSSMGFVELFPFCIPGNKLAGSIPELDFFNLSYLDLSANNFSTVP
Arabidopsis “ 7

spr1 V3FKDCSNLQHLDLSSNKFYGDIGSSLSSCGKLSFLNLTNNQFVGLVPLL
esculentum IHFKDCSNLQHLDLSSNKFYGDIGSSLSSCGKLSFLNLTNNQFVGLVPl
peruvianum S3PKDCSNLOHLDLSSNKF{GDIGSSLSSCGRLSFLNLTNNQFVGLVPlL
Arabidopsis v ‘ «ISSNQFVGPIP'L
spr1 SLOYLYLRGNDFQGV PNQLADLCKTVVELDLSYNNFSGMVPBS
esculentum H)ESLOYLYLRGNDFOGVYPNOLADLCKTVVELDLS{NNFSGMVPESLGT
peruvianum ESLQ{LYLPGNDFOGVYPNOLADLCKTVVELDLSYNNFSGMVPECLEU
Arabidopsis ‘ .
spr1

esculentum

peruvianum

Arabidopsis

spr1 lPSGICKDPMNNLKVLYLONNLFKGPIPDSl
esculentum lPSGICKDPMNNLKVLYLONNLFKGPIPD”L”
peruvianum

Arabidopsis

 

70

spr1
esculentum
peruvianum
Arabidopsis

spr1
esculentum
peruvianum
Arabidopsis

spr1
esculentum
peruvianum
Arabidopsis

spr1
esculentum
peruvianum
Arabidopsis

spr1
esculentum
peruvianum
Arabidopsis

spr1
esculentum
peruvianum
Arabidopsis

spr1
esculentum
peruvianum
Arabidopsis

spr1
esculentum
peruvianum
Arabidopsis

spr1
esculentum
peruvianum
Arabidopsis

spr1
esculentum
peruvianum
Arabidopsis

spr1
esculentum
peruvianum
Arabidopsis

VNLI LDFIJDLTC'
HLI LEFHL‘LT
HLllLlHD

LSHCTI‘LI'

SHCTITLI

71

l

 

 

spr1 s4 ' Qfi'fiPLLM-KFLJHHI JCIPHl lHPDllKSSN \ LLDENLEHRVSDFG HPLl

esculentum JGAARGLAFLHHNCI PHI IHRDMKSSNVLLDENLEIARVSDFGMARLMSE
peruvianum J GAARGLAFLHHNC I PH I IHRDMKSSNVLLDENLEARVSDFGMARLMSR
Arabidopsis ian ARG LAFLHHNC PH I IHRDlVlKSSNVLLDENLE'ARVSDFGMARLl‘v‘lE‘ A

 

       
    
  

 
    
  

      

    

   
 
 

    
   
 

spr1 E3THLSVSTLAGTPGYVPPEYYQSFRCSTKGDVYSYGVVLLELLTGKQP?
esculentum HDTHLSVSTLAGTPGYVPPEYYQSFRCSTKGDVYSYGVVLLELLTGKQPT
peruvianum MDTHLSVSTLAGTPGYVPPEYYQSFRCSTKGDVYSYGVVLLELLTGKQPT
Arabidopsis DTHLSVSTLAGTPGYVPPEYYQSFRCSTKGDVYSYGVVLLELLTGKfiPJ
spr1 USADFGDNNLVGWVKLHAKGKITDVFDRELLKBDASIEIELLQHLKVAC;
esculentum D8ADFGDNNLVGWVKLHAKGKITDVFDRELLKEDASIEIELLQHLKVAC¥
peruvianum D‘DDFGDNNLVGWVKLHAKGKITDVFDRELLKEDASIEIELLQHLKVAC~
Arabidopsis US'DFGDNNLVGWV’OHAKL'ISDVFDPELMKEDP‘ EIELLQHLKVA Ts
spr1 ULDDRHWKRPTMlQVMAMFKEIQAGSGMDSTSTJG—ADDVNFSGVEGGJE
esculentum ”LDDRHWKRPTMIQVMAMFKEIQAGSGMDSTSTIG—’DDVNFSGVEGGIE
peruvianum -

Arabidopsis 81:.

spr1 HGINGSIKEGNELSKHJ

esculentum EGINGSIKEGNELSKHL

peruvianum JUJHGSTFEGNELC

Arabidopsis 7

 

72

systemin-insensitive systemin-responsive
(spr1/spr1) (Spr1/spr1)

       

 

Figure 2-10. Linkage analysis of SR160 and Spr]. To test linkage between
SR160 and Spr], a BCl mapping population was generated as described in
the text. RFLP analysis was used to test for linkage between SR160 and SprI
in this BCl population. XbaI digestion of genomic DNA generated a species-
specific RFLP pattern at the SR160 locus (L. esculentum, 2 kb band; L.
pennelliz', 12 kb band). To test whether the spr1 allele and the L. esculentum-
specific RF LP cosegregate in the BCI population, genomic DNA was
prepared from 26 BCl plants for RFLP analysis. The blot was probed with
a 2 kb EcoRI fragment from the SR160 DNA. Asterisks show the
cosegregation of the spr1 allele and L. esculentum-specific RFLP pattern.
The cosegreation of the Spr] allele and L. pennellii-specific RFLP is noted
with #. E, L. esculentum; S, the parental spr1 plant originated from L.
esculentum; P, L. pennellii; F, the F1 plant.

73

between a homozygous spr1 plant (L. esculentum) and the wild tomato species L.
pennellii (Spr]/Spr]), followed by a backcross of the resulting F 1 plant to the parental
spr1 line (L. esculentum).

To determine the genotype of individual plants within BCl population, each
BCl plant was assayed for systemin-induced PI accumulation. Systemin-insensitive
BCl plants do not accumulate P15 in response to systemin, and thus are homozygous for
spr1. In contrast, systemin-responsive BCl plants must be heterozygous; they contain
an spr1 allele inherited from L. esculentum and an Spr] allele transmitted fiom L.
pennellii.

If Spr] and SR160 correspond to the same gene or are tightly linked, all
systemin-insensitive BC 1 plants are expected to be homozygous for the SR160 allele
transmitted from L. esculentum (SR160m /SR160‘”‘). Conversely, systemin-responsive
BCl plants should have one allele of SR160 inherited from L. pennellii (SR160M’) and
another allele transmitted from L. esculentum (SR160“‘). To test this, the genotype
(SR16Oe‘C /SR160‘”C or SR160” 312160“) of the SR160 locus for each BCl plant was
determined using RFLP analysis that distinguishes the SR160” and SR160” alleles
(Figure 2-10). Among 26 BC] plants examined, only 9 plants showed a match between
the systemin-sensitivity phenotype and the expected RFLP pattern of SR160 (Figure 2-
10; lanes indicated with * or #). These results indicate that the Spr] and SR160 loci are
assorting independently of one another. Therefore, Spr] does not encode the systemin

receptor, SR160.

74

Expression of SR160 in spr1 plants

Linkage analysis demonstrated that Spr] and SR 1 60 are different genes. However, this
result does not rule out the possibility that the spr1 mutation may affect the expression
of SR160. To test this possibility, RNA gel blot analysis was used to determine the
steady state level of SR] 60 mRNA in spr1 and wild-type plants (Figure 2-11). Both
wild-type and spr1 plants accumulated comparable levels of SR160 mRNA in leaves.
Wounding did not significantly change the basal expression levels. Taken together with
DNA sequence analysis ofSR160, these results indicate that spr1 plants are not

compromised in SR160 or its expression.

Discussion

1 tested the hypothesis that the systemin-insensitive phenotype of spr1 plants results
from a defect in systemin receptor the SR160. Several lines of evidence disproved this
hypothesis. First, linkage analysis demonstrated that Spr] does not encode SR160.
Second, DNA sequence analysis showed that the SR160 gene in spr1 plants does not
harbor a mutation in the coding region. Furthermore, spr1 plants showed normal
expression of SR160. These results demonstrate that the spr1 mutation is not related to
SR160. The Spr] gene product may, therefore, act downstream of SR160 to activate J A
biosynthesis in systemin-mediated signaling. Alternatively, the Spr] product may assist
in the binding of systemin to SR160. Precedence for this hypothesis comes from the
observation that the Arabidopsis BRII associated receptor kinase 1 (BAKl) mediates

brassinolide binding by forming a heterodimer with BRII (Nam and Li, 2002).

75

wild-type spr1
Hr 012 4 812240124 81224

. - ' q
SR160 mu. m,“ «is .03». turn” “all“ 1.” fl

      
 

eIF4A wwnmwwufiflummu”

 

Figure 2-11. Expression of SR160 in wounded wild-type and spr1 plants. Two-leaf-
stage spr1 and wild-type plants were wounded once on each leaf with a hemostat.
Wounded leaves were harvested at indicated time (hour) after wounding for preparation
of RNA. For each time point, six plants were harvested and pooled for RNA extraction.
RNA gel-blot analysis was performed using a 2 kb EcoRI fragment of SR160 gene as a
probe. A duplicated blot was probed with cDNA encoded eIF4A as a RNA loading
control.

76

The tomato genome contains a single copy of SR1 60, which shows high
similarity to Arabidopsis BRII gene that encodes the brassinolide receptor (Scheer and
Ryan, 2002). Interestingly, a recent study indicates that SR160 acts as the brassinolide
receptor in tomato (Montoya et al., 2002). It was found that brassinolide-insensitive
curl3 plants have a nonsense mutation in SR160. This finding indicates that SR160
could be a dual ligand receptor of systemin and brassinolide. Additional insight into the
dual role of SR160 could be obtained by investigating systemin binding and the
systemic wound response in curl3 plants that lack a fimctional SR160. Because
brassinolide regulates plant growth, curl3 plants display a dwarf phenotype (Montoya et
al., 2002). If brassinolide and systemin compete for binding to SR160, it is expected
that increased levels of systemin may interfere with brassinolide action. In mammals,
the peptide hormone oxytocin and steroid hormone progesterone compete for the same
receptor (Grazzini et al., 1998). Recent in vitro experiments indicate that brassinolide
does not inhibit the binding of systemin to SR160 (Scheer and Ryan, 2002). Therefore,
the two ligands seem not to compete for binding to SR160 in tomato plants. It remains
to be determined how SR160 regulates two distinct signaling pathways.

Systemin binding to the receptor activates several rapid responses in tomato
cells, including increased cytosolic Ca2+ levels, depolarization of the plasma membrane,
inactivation of a proton-ATPase associated with the plasma membrane, and activation
of a mitogen-activated protein kinase (MAPK) activity (Felix and Boller, 1995; Moyen
and Johannes, 1996; Moyen et al., 1998; Schaller and Oecking, 1999; Stratmann and

Ryan, 1997). It is currently unclear how the activated SR160 promotes these effects and

77

how these changes are related to increased J A synthesis. Molecular cloning of Spr]

could provide a clue to address these questions.

Materials and Methods

Plant material
Plant growth conditions were the same as described in Chapter 2. The mapping
population for linkage analysis was generated from a cross between L. esculentum
carrying the homozygous spr1 mutation and wild-type L. pennellii. One of the resulting
Fl plants was backcrossed to the parental homozygous spr1 plant to produce the BCI
population. Individual BCl plants were tested for systemin-induced PI-II accumulation
as described in Chapter 2. Briefly, two-leaf-stage plants were excised at the base of the
stem and supplied with systemin dissolved in phosphate buffer (5 pmol systemin/plant).
Accumulation of PI-II in leaves was measured 24 hr later using a radioimmunodiffusion
assay. Each plant was transferred to a glass vial containing distilled water and incubated
in the grth chamber to promote rooting. Plants were transferred to soil afier roots
were established (approximately 7 days). For genotyping, DNA was isolated by leaf
tissue of 6-week-old plants.

To examine the expression of SR160, two-leaf-stage plants were wounded once
on the lower leaf with a hemostat. Leaves were harvested at various times after

wounding for extraction of total RNA.

78

Cloning of SR160

A PCR technique was used to clone SR160 from spr1 and wild-type L. esculentum. Two
oligonucleotide primers were designed using the sequence of SR1 60 cloned from L.
peruvianum: 5’-AGAACTCAAGCTATAGA-3’ and 5’-
ACACAAGATTTTATGGATGGGA—3’. These primers correspond to the 5’ and 3’
UTRs, respectively. Genomic DNA was prepared from spr1 and wild-type L.

esculentum to prepare template in PCR, following the method described in Chapter 6.
PCR was performed with Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA)
following the manufacturer’s instruction. The amplified products were subcloned into
EcoRV site of pBluescript SK (-) plasmid (Stratagene, La Jolla, CA) and sequenced

(Genomics Technology Support Facility, Michigan State University, East Lansing, MI).

Nucleic acid hybridization
To find a RFLP for SR160, genomic DNA was prepared from L. esculentum and L.
pennellii, and digested with several restriction enzymes. The reaction products were run
on 0.8% agarose gel and transferred to I-beond membrane (Amersham, Piscataway,
NJ). To prepare the radiolabeled probe, a 2 kb EcoRI fragment of SR160 was labeled
with [32P-a] dCTP. The DNA gel-blot analysis was performed following the method
describe in Chapter 6. The result showed that XbaI digestion generated an RFLP for
SR] 60.

Total RNA was extracted from leaves of spr1 and wild-type plants following the
method described in Chapter 6. The RNA gel-blot was hybridized to the 2 kb EcoRI

fragment of SR160, following the method described in Chapter 2.

79

References

Doares, S.H., Narvaez-Vasquez, J., Conconi, A., and Ryan, C.A. (1995a). Salicylic acid
inhibits synthesis of proteinase inhibitors in tomato leaves induced by systemin
and jasmonic acid. Plant Physiol. 108:1741-1746.

Doares, S.H., Syrovets, T., Weiler, E.M., and Ryan, C.A. (1995b). Oligogalacturonides
and chitosan activate plant defensive genes through the octadecanoid pathway.
Proc. Natl. Acad. Sci. USA. 92: 4095-4098.

Felix, G., and Boller, T. (1995). Systemin induces rapid ion fluxes and ethylene
biosynthesis in Lycopersicon peruvianum cells. Plant J. 7: 381-389.

Grazzini, E., Guillon, G., Mouillac, B., and Zingg, H. (1998). Inhibition of oxytocin
receptor function by direct binding of progesterone. Nature 392: 509-512.

Howe, G.A., Lightner, L., Browse, J ., and Ryan, CA. (1996). An octadecanoid pathway
mutant (JL5) of tomato is compromised in signaling for defense against insect
attack. Plant Cell 8: 2067-2077.

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

Meindl, T., Boller, T., and Felix, G. (1998). The plant wound hormone systemin binds
with the N-terminal part to its receptor but needs the C-terminal part to activate
it. Plant Cell 10: 1561-1570.

Montoya, T., Nomura, T., Farrar, K., Kaneta, T ., Yokota, T., and Bishop, GJ. (2002).
Cloning the tomato curl3 gene highlights the putative dual role of the leucine-
rich repeat receptor kinase tBRIl/SR16O in plant steroid hormone and peptide
hormone signaling. Plant Cell 14: 3163-3176.

Moyen, C., Hammond-Kosack, K.E., Jones, J ., Knight, M.R., and Johannes, E. (1998).
Systemin triggers an increase of cytoplasmic calcium in tomato mesophyll cells:

Ca2+ mobilization from intra- and extracellular compartments. Plant Cell
Environ. 21: 1101-1111.

Moyen, C., and Johannes, E. (1996). Systemin transiently depolarises the tomato
mesophyll cell membrane and antagonises fusicoccin-induced extracellular

acidification of mesophyll tissue. Plant Cell Environ. 19: 464-470.

Nam, K.H., and Li, J. (2002). BRIl/BAKl , a Receptor Kinase Pair Mediating
Brassinosteroid Signaling. Cell 110: 203-212.

80

Schaller, A., and Oecking, C. (1999). Modulation of plasma membrane H+-ATPase
activity differentially activates wound and pathogen defense responses in
tomato plants. Plant Cell 11: 263-272.

Scheer, J .M. and Ryan, CA. (1999). A l60-kD systemin receptor on the surface of
Lycopersicon peruvianum suspension-cultured cells. Plant Cell 11: 1525-1536.

Scheer, J .M., and Ryan, CA. (2002). The systemin receptor SR160 from Lycopersicon
peruvianum is a member of the LRR receptor kinase family. Proc. Natl. Acad.
Sci. USA. 99: 9585-9590.

Stratmann, J.W., and Ryan, CA. (1997). Myelin basic protein kinase activity in tomato
leaves is induced systemically by wounding and increases in response to
systemin and oligosaccharide elicitors. Proc. Natl. Acad. Sci. USA. 94: 11085-
11089.

81

Chapter 3

The wound response mutant JL1 is defective in the
conversion of 12-oxo-phytodienoic acid to jasmonic

acid

82

Introduction

The plant hormone j asmonic acid (J A) is synthesized from linolenic acid via the
octadecanoid pathway. The pathway is divided into two parts by the subcellular location
of the enzymes involved. The first part of the octadecanoid pathway occurs in
chloroplasts where the release of linolenic acid from plastid membranes leads to the
synthesis of 12-oxo-phytodienoic acid (OPDA). The second part takes place in
peroxisomes where OPDA, presumably transported from chloroplasts, is processed to

J A (Figure 3-1; Turner et al., 2002; Wastemack and Hause, 2002).

Several lines of evidence demonstrate that J A regulates the wound response of
tomato plants. First, application of J A or its methyl ester Me] A) triggers the
accumulation of wound-inducible proteins such as proteinase inhibitors (PIs; Farmer
and Ryan, 1992; Farmer et al., 1992). Second, the endogenous level of J A increases
rapidly upon wounding, which is followed by expression of PIs (Conconi et al., 1996).
Third, the expression of PIs is abolished by both chemical inhibitors and mutations that
block the octadecanoid pathway (Doares et al., 1995; Howe et al., 1996). Recent studies
show that J A is a component of the long-distance signaling pathway that relays signals
produced in the wound site to systemic expression of PIS in undamaged leaves (Li et al.,
2002)

Analysis of wound response mutants has expanded our knowledge of the role of
J A in the wound response. The J L1 and J L5 (renamed defenseless] ; defI) mutant lines
of tomato were isolated from an ethyl methanesulfonate (EMS)-mutagenized population
by screening for plants that are unable to accumulate P15 in response to wounding

(Li ghtner et al., 1993). Exogenous Me] A restored the production of P13 in both mutants,

83

indicating that they are defective in the biosynthesis of J A. Genetic analysis
demonstrated that the mutations defined by J L1 and def] are non-allelic and recessive.
Further study on def] plants showed that this mutant is deficient in the accumulation of
J A upon wounding (Howe et al., 1996). The Defl gene appears to be involved in the
regulation of allene oxide cyclase (AOC) that catalyzes the conversion of an unstable
allene oxide to OPDA (Stenzel et al., 2003).

The biochemical defect responsible for the wound response phenotype of J L1
remains to be established. Because JL1 plants synthesize P18 in response to exogenous
Me] A, it is likely that the mutant is compromised either in the octadecanoid pathway or
the activation of the pathway in response to wounding. To address this issue, the J L1
mutant was further characterized. Measurement of JA and OPDA levels in J L1 plants
indicate that the mutant is compromised in the conversion of OPDA to J A. This
hypothesis was supported by analysis of the response of J L1 plants to exogenous OPDA
and J A. The retarded growth phenotype of JL1 seedlings further indicated that JL1
plants are affected in a B-oxidation step that is required for the conversion of OPDA to
J A. The results of reciprocal grafting experiments indicate that J L1 plants are deficient
in the production of the systemic wound signal, but are able to recognize the signal in
unwounded leaves. Because J L1 plants accumulate OPDA but not J A, these results
indicate that J A, not OPDA, is an essential component of the transmissible wound

signal for PI expression.

84

Results

JL1 plants are deficient in JA accumulation

JL1 plants do not accumulate detectable levels of PHI protein in response to wounding
(Table 3-1). To test whether the absence of PHI accumulation is because of a defect in
J A synthesis, gas chromatography-mass spectrometry (GC-MS) was used to measure

J A and OPDA levels in wild-type and J L1 plants. Wild-type plants accumulated 14 d: 1
pmol JA/g FW (fresh weight) in unwounded leaves (Figure 3-2). This basal level
increased to 274 i 18 pmol J A/ g FW 1 hr after wounding. In contrast, unwounded J L1
plants produced significantly lower levels of JA (2 :1: 3 pmol/g FW) than wild-type
(student’s t-test, P < 0.05). Furthermore, wounding did not elevate the level of J A (4 i 5
pmol J A/ g FW) in the mutant. These results indicated that J L1 plants are impaired in J A
biosynthesis.

Quantification of OPDA showed that JL1 plants accumulated OPDA to levels
comparable to those in wild-type plants. Wild-type plants accumulated 597 i 41 pmol
OPDA/ g FW in unwounded leaves, which increased to 752 :1: 19 pmol OPDA/ g F W 1 hr
afier wounding. OPDA levels in JL1 plants were 413 i 205 pmol/g FW in unwounded
leaves and 578 :t 134 pmol/g FW in wounded leaves. Therefore, J L1 plants synthesize

OPDA but appear to be defective in its conversion to J A.

Exogenous OPDA does not activate the expression of PM! In JL1 plants

To further test the hypothesis that J L1 plants are unable to convert OPDA to J A, various
amounts of OPDA and JA were applied to JL1 and wild-type plants through the cut

stem, and the accumulation of PHI was measured 24 hr afier treatment. J L1 and wild-

85

type plants accumulated comparable amounts of PHI in response to J A (Figure 3-3).
This finding is consistent with the previous observation that volatile MeJ A induced the
synthesis of P15 in JL1 plants (Lightner et al., 1993). In wild-type plants, OPDA acted
as a potent elicitor of PHI accumulation (Figure 3-3). In contrast, exogenous OPDA
failed to induce PI- 11 production in J L1 plants. These results indicate that conversion of
OPDA to J A is necessary for expression of PI—II in wild-type plants, and that the JL1

mutant is impaired in this metabolic process.

JL1 plants fail to generate a systemic wound signal for PM! expression

The inability of J L1 plants to synthesize HS in response to wounding (Table 3-1;

Li ghtner et al., 1993) could result from a failure to produce or perceive a systemic
wound signal. To address this question, reciprocal grafting experiments were performed
with JL1 and wild-type plants. Mechanical wounds were inflicted to all leaves of the
rootstock, and the expression of PM! was monitored in both the wounded rootstock and
the undamaged scion leaves (Figure 3-4). Control experiments showed that the grafting
procedure alone resulted in a moderate expression of PHI in both rootstock and scion
leaves of grafted wild-type plants (Figure 3-4, lane 1). However, wounding of rootstock
leaves resulted in a significant increase of PHI expression in the scion, indicating that
the wounded rootstock produced a long-distance signal that was transmitted to the
undamaged scion (Figure 3-4, lane 2). Grafted JL1 control plants did not show
detectable local or systemic expression of PHI (Figure 3-4, lane 3), consistent with the

absence of PHI in wounded J L1 seedlings (Table 3-1). In response to wounding, weak

86

OPDA

O o
|

COOH

L OPDA reductase

OPC-8

3-oxo-2-(2Z-pentenyl)-
cyclopentane-1-octanoic acid

.0
3

0 * B-oxidation
OPC'6 . OOOH
3-oxo-2-(2Z-pentenyl)-
cyclopentane-1-hexanoic acid _ ,
l B—oxndatlon
O
OPC—4 .
OOOH

3-oxo—2-(22-pentenyl)-
cyclopentane-1-butyric acid
0 ‘ B-oxidation

.. e “
COOH

Figure 3-1. Conversion of OPDA to JA. OPDA is synthesized in chloroplasts and
converted to J A in peroxisomes by consecutive reactions of OPDA reductase and three
rounds of B-oxidation.

87

 

PI-ll levels (pg/ml Ieafjuice)

 

 

genotype
unwounded control wounded
wild-type ND. 68.5 1: 14.1
JL1 ND. ND.

 

Table 3-1. Wound response of wild-type and JL1 plants. Leaves of two-week-old
wild-type and JL1 plants were wounded with a hemostat. The accumulation of PHI was
measured in leaves harvested 24 hr after wounding. As a negative control, PI-II levels
were also measured in leaves of unwounded plants. The data present mean and standard
deviation from the measurement of three plants for each treatment. N.D. indicates that
the accumulation of PHI was not detected.

88

(A)
350

 

300 a

 

JA (pmol/g FW)
_.. _. N N
0 0| O 01
O O O O

O"
O
1

 

 

[—‘—*—1 i .-

 

 

 

O

unwounded wounded

(B)

 

900
800 1

OPDA (pmol/g FW)
# 01 O) \l
O O O O
O O O O

-‘ N (O
o O o
o o o
m 1 4

 

 

 

 

   

O

 

 

unwounded wounded

Figure 3-2. Accumulation of JA and OPDA in wild-type and JL1 plants in
response to wounding. Leaves of 2-week-old wild-type (open bar) and J Ll
(filled bar) plants were wounded with a hemostat. Leaves were harvested
for extraction of J A/OPDA 1 hr after wounding. JA/OPDA was also
extracted from leaves of unwounded control plants. J A (A) and OPDA (B)
levels were measured by GC-MS analysis. Data represent mean and SD of
three independent experiments.

89

(A)

(3)

Figure 3-3. Effect of exogenous JA and OPDA on the accumulation of P1
in wild-type and JL-l plants. Two-week—old wild-type (open bar) and J L-
1 (filled bar) plants were supplied through the cut stem with phosphate
buffer (pH 6.5) containing various amounts of J A (A) and OPDA (B).
Plants were assayed for PI-II accumulation in leaves 24 hr after
treatment. Data represent the mean and standard deviation of 6 plants.

Pl-ll ( pglml Ieafjuice)

Pl-ll (pg/ml leaf juice)

.3.3
#05
CC

.3

O

O
I‘LL

 

 

 

 

 

 

10
nmol JAlplant

 

.3 .3 .3 .3
N h 0) oo 0 N «F O)
O O O O O O O O
1 1 1 1 1 1 J

 

A

 

 

 

l—L

 

O

0

9O

10
nmol OPDA/plant

l

 

 

 

 

 

 

 

expression of PHI was observed in the damaged J L1 rootstock but not in undamaged
scion leaves (Figure 3-4, lane 4). In hybrid grafts between wild-type and J L1, wounding
of JL1 rootstock leaves did not significantly increase the expression of PM] in the wild-
type scion (Figure 3-4, lane 5 and 6). Thus, the wounded J L1 rootstock is defective in
the production of the long-distance signal for systemic PI-II expression. In the
reciprocal combination (Figure 3—4, lane 7 and 8), wounding of wild-type rootstock
leaves resulted in strong induction of PHI expression in the JL1 scion. This result
indicates that J L1 plants are able to respond to the long-distance signal that is produced
and transmitted from the damaged wild-type rootstock. Taken together with the
deficiency of J A in J L1 plants, these results indicate that the conversion of OPDA to J A

is required for the generation of a systemic wound signal in damaged tissue.

Growth of JL1 seedlings is retarded

Defects of J L1 plants were further investigated. In addition to JA deficiency,
J L1 plants displayed retarded growth at the seedling stage. To compare the grth rate
between J L1 and wild-type plants, the root growth of seedlings was measured. To
synchronize the seed germination, both J L1 and wild-type seeds were germinated on
filter paper saturated with distilled water. Gerrninating seeds having a protruding radicle
of the same size (about 2 mm) were then transferred to new filter paper saturated with
distilled water. These seedlings were incubated for four days in darkness, and then root

length was measured. The result shows that root length of J L1 seedlings was 42% of

91

scion

rootstock

wounding

scion

rootstock

 

scion “-.fl-“--
eIF4A

'°°t3t°°k C on up n on at b «I

Figure 3-4. Wound-inducible PI-II expression in grafts between JL1 and
wild-type plants. J L1(J L) and wild-type (WT) plants were grafted in the
four combinations indicated. The genotypes listed above and below the
horizontal line correspond to the scion and rootstock, respectively. For each
graft combination, plants were divided into a negative unwounded control
(-) and wounded (+) group consisting of four grafted plants per group. For
wound treatment, each leaflet on the rootstock was mechanically injured
with a hemostat. Eleven hours after wounding, leaf tissue was harvested
separately from wounded rootstock leaves and undamaged scion leaves
(scion) for RNA extraction. Levels of PHI mRNA were analyzed by RNA
gel blot analysis, using an eIF4A cDNA probe as a loading control.

92

that of wild-type (Figure 3-5). This finding indicates that root growth of J L1 seedlings
is significantly retarded relative to wild-type. However, growth retardation was not

observed in adult J L1 plants that developed fully expanded leaves (data not shown).

Discussion

JL1 plants are unable to convert OPDA to JA

The results of the present study indicate that the defective wound response of JL1 plants
results from a deficiency in J A accumulation. Three lines of evidence indicate that JL1
plants have a defect in the conversion of OPDA to J A. First, J L1 leaves contained less
than 2% of the level of J A observed in wild-type leaves (Figure 3-2A). This is the most
severe J A deficiency among known wound response mutants of tomato (Howe et al.,
1996; Lee and Howe, 2003; Li et al., 2002; Table 4-1, Chapter 4). Second, the OPDA
level of J L1 plants was comparable to that of wild-type (Figure 3-2B). In contrast, other
wound response mutants such as spr2 and def] are deficient in both of OPDA and J A
(Table 4-2, Chapter 4; Stenzel et al., 2003). This result indicates that the mutation in

J L1 affects a step downstream of OPDA in the octadecanoid pathway. Finally,
exogenous J A, but not OPDA, induced the accumulation of PHI in J L1 plants (Figure
3-3). Because OPDA is a precursor of J A, these results indicate that J L1 plants are

unable to metabolize OPDA to J A.

93

(D
O

 

 

 

 

 

 

 

 

 

 

 

 

root length (mm)
—8 N 00 -h 01 O) \1
C) O O O O O O O

wild-type JL1

Figure 3-5. Comparison of root length between wild-type and JL1 seedlings.
Germinated seeds at the same stage of development (2 mm radicle) were transferred to
3-MM filter paper saturated with distilled water, and incubated at room temperature in
darkness. Root length was measured after 4-day incubation. Data represent the mean
and standard deviation of 10 plants.

94

The conversion of OPDA to IA is accomplished by consecutive reactions
involving the transport of OPDA from chloroplasts to peroxisomes, reduction of the
cyclopentenone ring of OPDA by OPDA reductase, and the removal of 6 carbon units
from the carboxylate side chain by three rounds of B-oxidation (Figure 3-1; Wastemack
and Hause, 2002). Virtually nothing is known about the mechanism involved in
transport of OPDA to peroxisomes. Previous studies in Arabidopsis indicated that
substrates for B-oxidation are imported into the peroxisome by an ATP-binding cassette
transporter called anl/Ped3/COMATOSE, which is located on the peroxisomal
membrane (Footitt et al., 2002; Hayashi et al., 2002; Zolman et al., 2001b). It is unlikely
that the J L1 mutant is impaired in OPDA transport because exogenous OPDA failed to
induce the accumulation of PM]. Rather, it is more likely that JL1 plants are defective
in the conversion of OPDA to J A in peroxisomes.

OPDA reductase genes (OPRs) comprise a small gene family in Arabidopsis
and tomato. However, only OPR3 is involved in the biosynthesis of J A (Sanders et al.,
2001; Schaller and Weiler 1997; Schaller et al., 2000; Strassner et al., 2002). The
product of OPR3 is located in peroxisomes (Strassner et al., 2002), and the absence of
OPR3 in Arabidopsis results in J A deficiency and male sterility (Sanders et al., 2001;
Stintzi et al., 2001). Mapping experiments indicate that the mutation responsible for the
wound response phenotype of J L1 does not correspond to the OPR3 locus of tomato (C.
Li and GA. Howe, unpublished result)

JL1 shows growth retardation at the seedling stage (Figure 3-5). A similar
phenotype has not observed in the opr3 mutant of Arabidopsis or other J A-deficient

mutants of tomato (Howe and Ryan, 1999; Howe et al., 1996; Sanders et al., 2001). The

95

defect in J A biosynthesis and retarded seedling growth of J L1 indicates that this mutant
is compromised in B-oxidation. In addition to its role in J A biosynthesis, B-oxidation
also mediates degradation of fatty acids by the sequential removal of two carbon units in
the form of acetyl—coenzyme A (CoA; Graham and Eastrnond, 2002). In germinating
seeds, storage lipids are metabolized by B—oxidation with subsequent synthesis of
glucose. Thus, impeded seedling growth is often observed in mutants defective in [3-
oxidation (Hayashi et al., 1998; Zolman et al., 2000). In such mutants, the defect in
vegetative growth is typically alleviated after the transition to photoautotrophic growth
(Hayashi et al., 1998; Hayashi et al., 2002; Zolman et al., 2001b). This phenotype was
observed in JL1 plants; grth retardation was not observed in JL1 plants that had
developed more than four leaves (data not shown). Therefore, it is less likely that the
defective grth of JL1 seedlings results from a secondary mutation that adversely
affects general aspects of plant development. Instead, the JL1 mutant appears to be
compromised in B—oxidation, which inhibits both JA biosynthesis and seedling growth.
fi-oxidation in plants occurs in peroxisomes and involves the sequential action of
three enzymes: first, acyl—CoA oxidase; second, a multifunctional protein possessing L-
3-hydroxyacyl-COA hydrolyase, L-3-hydroxyacyl-dehydrogenase, D-3-hydroxyacyl-
CoA epimerase, and 3, 2-enoyl-C0A isomerase activities; third, 3-ketoacyl-CoA
thiolase (Eastmond et al., 2000; Germain et al., 2001; Richmond and Bleecker, 1999).
Substrates for B-oxidation are first activated by acyl-CoA synthetase (F ulda et al.,
2002). Enzymes involved in the B-oxidation cycle are encoded by small gene families in
Arabidopsis, which results in the partial overlap of the substrate specificity among

isozymes (Eastrnond and Graham, 2002; Eastmond et al., 2000; Germain et al., 2001;

96

Hayashi et al., 1998; Hayashi et al., 1999; Hooks et al., 1999; Richmond and Bleecker,
1999; Shockey et al., 2002). Additional work will be necessary to provide direct

evidence for a defect in fi—oxidation in J L1 plants.

OPDA is not a wound signal for PI expression in tomato

Previous studies have shown that OPDA, in the absence of the conversion to J A, is a
signaling molecule that regulates several aspects of plant development. For example,
OPDA was more effective than J A in inducing tendril coiling of Cucurbitaceae plants
(Weiler et al., 1993; Weiler et al., 1994). Furthermore, exogenous OPDA and J A
treatments resulted in distinct patterns of volatile emission in lima bean (Koch et al.,
1999). The study of the opr3 mutant demonstrated that OPDA is a signal for defense
responses in Arabidopsis (Stintzi et al., 2001). In spite of being unable to convert
OPDA to J A, opr3 plants were as resistant to insect and fungal pathogens as were wild-
type plants. The resistant phenotype of opr3 plants contrasts the susceptible phenotype
of other J A biosynthetic mutants of Arabidopsis that do not synthesize OPDA, or
mutants that are insensitive to J A. cDNA microarray experiments firrther showed that
wild-type and opr3 plants expressed defense-related genes in a similar manner upon
wounding. These results indicate that OPDA functions as a defense signal in
Arabidopsis.

In contrast to the situation in Arabidopsis, the results presented in this chapter
indicate that OPDA is not an active signal for the expression of wound-inducible PIs in
tomato. J L1 plants failed to express PIs upon wounding or application of OPDA,

whereas J A treatment restored PI-II accumulation in JL1 plants (Figure 3-3). Therefore,

97

the conversion of OPDA to J A is essential for PI expression in tomato. This finding is
consistent with a previous study demonstrating that fl-oxidation is required for
expression of P13 in tomato (Miersch and Wastemack, 2000). In this study, I examined
PI-II expression in response to J A analogues containing a carboxylate side chain of
differing length. PI-II expression was induced by analogues carrying an even-number of
carbons in the side chain (i. e., OPC-8, OPC-6, and OPC-4; Figure 3-1). In contrast, J A
analogues containing an odd-number of carbons in the side chain, which cannot be
converted to J A by B-oxidation, were inactive. These results indicated that B-oxidation
of OPC-8 is necessary for PM] expression in tomato.

Increasing evidence indicates that J A biosynthesis is essential for the systemic
wound response of tomato. Reciprocal grafting experiments using J A-deficient spr2
plants showed that J A biosynthesis is required for the production of the transmissible
wound signal (Li et al., 2002). Because spr2 plants lack both OPDA and J A because of
the loss of an co-3 fatty acid desaturase (Li et al., 2003; Table 4-2, Chapter 4), these
results do not rule out the possibility that OPDA is a mobile signal for the systemic
wound response. Grafting experiments performed with JL1 plants, which accumulate
OPDA but not J A, showed that this mutant failed to generate a systemic wound signal
in damaged leaves, but nevertheless was able to perceive that signal in unwounded
leaves. These results indicate that J A rather than OPDA is the a mobile signal for

systemic PI expression

98

Materials and Methods

Plant material

Seeds of J L1 were collected from a J L1 homozygous line that was back-crossed two
times using Lycopersicon esculentum Mill cv Castlemart as the recurrent parent

(Li ghtner et al., 1993). Wounding was performed on two-week-old plants as described
in Chapter 2. Grafting experiments were performed following the method described by

Li et al. (2002)

Treatment of JA and OPDA

(i)-J A and OPDA were purchased from Sigma (St. Louis, MO) and Cayman Chemical
(Ann Arbor, MI), respectively. Two-week-old plants were excised at the base of the
stem with a razor blade and immediately placed into 0.5 m1 plastic tubes containing
various amounts of elicitors diluted in 300 11.1 of 15 mM sodium phosphate solution (pH
6.5). Because J A and OPDA were originally dissolved in ethanol, the mock control
solution contained 0.1 % (vol/vol) ethanol. Excised plants were allowed to imbibe the
solution during a 50 min incubation period, and then transferred to glass vials
containing distilled water. PI-II levels in leaves are measured 24 hr after treatment as

described in Chapter 2.

RNA gel blot analysis

RNA extraction from leaves and RNA hybridization experiments were performed as

described in Chapter 2.

99

Root length measurement

Seeds were surface sterilized as described (Zolman et al., 2000). To synchronize the
seed germination, both J L1 and wild-type seeds were germinated on filter paper
saturated with distilled water. Germinating seeds with a radicle of about 2 mm length
were transferred to two layers of 3-MM filter paper soaked with distilled water. The
transferred seedlings were incubated for four days in darkness at room temperature.
Root growth was measured with a ruler using overhead projector to enlarge the image

of seedlings.

Analysis of JA and OPDA

Quantification of J A and OPDA was performed by GC-MS analysis as described in

Chapter 4.

100

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and Nishimura, M. (2002). Ped3p is a peroxisomal ATP-binding cassette
transporter that might supply substrates for fatty acid beta-oxidation. Plant Cell
Physiol. 43: 1-11.

Hayashi, M., Toriyama, K., Kondo, M., and Nishimura, M. (1998). 2, 4-
dichlorophenoxybutyric acid-resistant mutants of Arabidopsis have defects in
glyoxysomal fatty acid beta-oxidation. Plant Cell 10: 183-195.

Hooks, M., Kellas, F., and Graham, I. (1999). Long-chain acyl-CoA oxidases of
Arabidopsis. Plant J. 20: 1-13.

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

Howe, G.A., Lightner, J ., Browse, J ., and Ryan, CA. (1996). An octadecanoid pathway
mutant (J L5) of tomato is compromised in signaling for defense against insect
attack. Plant Cell 822067-2077.

Koch, T., Krumm, T., Jung, V., Engelberth, J ., and Boland, W (1999). Differential
induction of plant volatile biosynthesis in the lima bean by early and late
intermediates of the octadecanoid-signaling pathway. Plant Physiol. 121: 153-
162.

Li, C., Liu, G, Xu, C., Lee, G. 1., Bauer, P., Ling, H., Ganal, M., and Howe, GA.
(2003). The tomato suppressor of prosystemin-mediated responses 2 (spr2) gene
encodes a fatty acid desaturase required for the biosynthesis of jasmonic acid
and the production of a systemic wound signal for defense gene expression.
Plant Cell 15: 1646-1661.

Li, L., Li, C., Lee, G.I., and Howe, G (2002). Distinct roles for jasmonate synthesis and
action in the systemic wound response of tomato. Proc Natl Acad Sci USA. 99:
6416-6421.

Lightner, J ., Pearce, G., Ryan, C.A., and Browse, J. (1993). Isolation of signaling
mutants of tomato (Lycopersicon esculentum). Mol. Gen. Genet. 241: 595-601.

102

Miersch, 0., and Wastemack, C. (2000). Octadecanoid and jasmonate signaling in
tomato (Lycopersicon esculentum Mill.) leaves: endogenous jasmonates do not
induce jasmonate biosynthesis. Biol. Chem. 381: 715-722.

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diffiision in agar gels containing antibodies. Anal. Biochem. 19, 434-440.

Richmond, T., and Bleecker, A. (1999). A defect in beta-oxidation causes abnormal
inflorescence development in Arabidopsis. Plant Cell 11: 1911-24.

Sanders, P. M., Lee, P. Y., Biesgen, C., Boone, J. D., Beals, T. P., Weiler, E.W., and
Goldberg, R. B. (2000). The Arabidopsis DELAYED DEHISCENCEI gene

encodes an enzyme in the jasmonic acid synthesis pathway. Plant Cell 12:1041-
1061.

Schaller, F ., and Weiler, E. (1997). Molecular cloning and characterization of 12-
oxophytodienoate reductase, an enzyme of the octadecanoid signaling pathway
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Schaller, F ., Biesgen, C., Mfissig, C., Altrnann, T., and Weiler, E. (2000). 12-
Oxophytodienoate reductase 3 (OPR3) is the isoenzyme involved in jasmonate
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Shockey, J ., Fulda, M., and Browse, J. (2002). Arabidopsis contains nine long-chain
acyl-coenzyme a synthetase genes that participate in fatty acid and glycerolipid
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Stintzi, A., Weber, H., Reymond, P., Browse, B., and Farmer, E. (2001) Plant defense in
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octadecanoid biosynthesis in the local versus the systemic wound response.
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14S: 153-164.

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221.

103

Weiler, E., Kutchan, T., Gorba, T., Brodschelm, W., Niesel, U., and Bublitz, F. (1994).
The Pseudomonas phytotoxin coronatine mimics octadecanoid signalling
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Weiler, E., Albrecht, T., Groth, B., Xia, Z.., Luxem, M., Liss, H., Andert, L., and
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Phytochemistry. 32: 591-600.

Zolman, B., Monroe-Augustus, M., Thompson, B., Hawes, J ., Krukenberg, K.,
Matsuda, S., and Bartel, B. ( 2001a). chyI, an Arabidopsis mutant with impaired
beta-oxidation, is defective in a peroxisomal beta-hydroxyisobutyryl-COA
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Zohnan, B., Silva, 1., and Bartel, B. (2001b). The Arabidopsis pxaI mutant is defective
in an ATP-binding cassette transporter-like protein required for peroxisomal
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Zolman, B., Yoder, A., and Bartel, B. (2000) Genetic analysis of indole-3-butyric acid

responses in Arabidopsis thaliana reveals four mutant classes. Genetics
156:1323-1337.

104

Chapter 4

Quantification ofjasmonic acid and 12-oxo-
phytodienoic acid in tomato plants by gas

chromatography-mass spectrometry

105

Introduction

J asmonic acid (J A) and its methyl ester (MeJ A) regulate plant defense responses against
insects and pathogens (Cohen et al., 1993; Farmer and Ryan, 1992; Howe et al., 1996;
McConn et al., 1997; Penninckx etal., 1996; Seo etal., 2001), and also play an
important role in reproductive organ development in plants (McConn and Browse,
1996; Li et al., 2001; Sanders et al., 2000; Stintzi and Browse, 2000). JA is synthesized
from linolenic acid by the octadecanoid pathway (Turner et al., 2002; Wastemack and
Hause, 2002). The synthesis of J A is initiated in chloroplasts where linolenic acid is
released from plastid membranes and converted to 12-oxo-phytodienoic acid (OPDA)
by the consecutive action of lipoxygenase (LOX), allene oxide synthase (AOS), and
allene oxide cyclase (AOC). The remaining biosynthetic steps occur in peroxisomes,
where OPDA is processed to J A by OPDA reductase (OPR3) and three rounds of 5-
oxidation. Interestingly, OPDA functions as a regulatory signal for defense and
development, in the absence of its metabolic conversion to J A (Stelrnach et al., 1998;
Stintzi et al., 2001; Weiler et al., 1993). These derivatives of linolenic acid, OPDA, J A
and Me] A, are well-known examples of oxylipins that are linear or cyclic oxidation
products derived from the catabolism of fatty acids (Howe and Schilmiller, 2002).
Precise quantitative analysis of J A and OPDA is crucial for the study of the
biosynthesis and function of these molecules (Creelrnan and Mullet, 1995).
Quantification of J A and OPDA requires the use of appropriate internal standards and
an analytical method for the detection. Because internal standards are used to correct for
the loss of each oxylipin during extraction, it is important that the internal standard has

the same or very similar physiochemical properties of the endogenous compound being

106

analyzed. Two types of internal standards have been used for quantification of JA and
OPDA. The first type is isotope-labeled standards such as deuterium- and/or 13 C-labeled
J A (Baldwin et al., 1994; Creehnan and Mullet, 1995; Creehnan et al., 1992; Kramell et
al., 2000; Laudert and Weiler, 1998) and OPDA (Kramell et al., 2000; Parchmann et al.,
1997; Stelrnach et al., 1998). Alternatively, 9, lO-dihydrojasmonic acid (DHJ A;
Gundlach et al., 1992; Parchmann et al., 1997; Weber et al., 1997; Wilbert et al., 1998)
and tetrahydro-OPDA (H4OPDA; Weber et al., 1997) have been used as internal
standards for quantification of J A and OPDA, respectively (Figure 4-1).

Two detection methods have been used to quantify J A and OPDA. The first
method is an enzyme-linked immunosorbent assay (ELISA) that relies on a monoclonal
antibody raised against MeJ A (Albrecht et al., 1993). To avoid possible cross-reaction
of the antibody with J A-related molecules, the J A fraction was purified from plant
extracts using hi gh-performance liquid chromatography (HPLC) and methylated prior
to ELISA testing. Although this method has been used to measure endogenous J A levels
in several plants including tomato (Albrecht et al., 1993; Conconi et al., 1996; Doares et
al., 1995; Howe et al., 1996; Weiler et al., 1993), quantification of OPDA is net
possible with this method. The second method, gas chromatography (GC)-mass
spectrometry (MS), provides highly reliable identification and quantification of J A and
related oxylipins. GC-MS analysis has been successfully used for the monitoring of J A
levels in several plant species including soybean, broad bean, Arabidopsis, apple,
tobacco, potato, and barley (Baldwin et al., 1994; Creelman et al., 1992; Fan et al.,
1997; Kramell et al., 1997; McConn et al., 1997; Mueller and Brodschelm, 1994; Wang

et al., 1999; Weber et al., 1997). In contrast to the ELISA method, GC-MS analysis

107

enables simultaneous quantification of J A, OPDA, and other oxylipins (Kramell et al.,

2000; Vollenweider et al., 2000; Weber et al., 1997).

Wound-induction of proteinase inhibitors (P13) in tomato has been widely used
as a model system to study wound signaling in plants (Green and Ryan, 1972; Ryan,
2000). Several wound response mutants of tomato have been isolated with genetic
screens for plants that lack accumulation of P18 in response to wounding (Howe and
Ryan, 1999; Li et al., 2001; Lightner et al., 1993). Quantification of JA and OPDA in
these mutants is an essential step in investigating the molecular and biochemical basis
of these mutations.

Previously, GC-MS analysis was used to measure the amount of J A and OPDA
in tomato leaves (Parchmann et al., 1997). This published protocol relied on extensive
purification of JA and OPDA prior to GC-MS analysis, which reduced the recovery of
these compounds. The purpose of the experiments described in this chapter was to
optimize and simplify this procedure using a solid-phase extraction method that was
originally developed for oxylipin analysis in Arabidopsis (Weber et al., 1997). The
results indicated that this modified method can be used to efficiently quantify J A and
OPDA in tomato tissues. The method was used to measure the levels of J A and OPDA
in various wound response mutants. The results obtained are discussed in the context of

the regulation of JA synthesis.

108

O

 

7 _
C
3

(3R, 78)-JA (3R, 7R)-JA
0

(3R, 7S)-DHJA

98, 138-OPDA 98, 13R-OPDA

O
m...

98, 138-H4OPDA

 

Figure 4-1. Structure of JA, OPDA and their internal standards. The
octadecanoid pathway yields only the 3R, 7S-isomer of J A, which is
derived from its precursor 9S, 138-OPDA (Laudert et al., 1997). During
extraction and analysis by GC, 98, 13S-OPDA and 3R, 7S-JA epimerize
to 98, l3R-OPDA, and 3R, 7R-J A, respectively (Mueller and
Brodschelm, 1994). J A and OPDA were converted to DHJ A and
H4OPDA, respectively, for use as internal standards.

109

Results

Preparation of internal standards and determination of GC-MS parameters

Because I A and OPDA are commercially available, these molecules were used to
prepare the corresponding internal standards. J A has two chiral centers at the C-3 and
C-7 positions (Figure 4-1), which yield four possible stereoisomers (Creelman and
Mullet, 1997): 3R, 7S—JA; 3S, 7R-JA; 3R, 7R-JA; and 3S, 7S- JA [also called (+)-7-iso-
J A, (-)-7-iso-JA, (-)-J A, and (+)-JA, respectively]. Chemically synthesized JA [(i)-J A]
is a racemic mixture of these stereoisomers. OPDA also has two chiral centers at the C-
9 and C-13 positions. Commercially available OPDA [(i)-OPDA] consists of a mixture
of 9S, 13R-OPDA and 9S, 13S-OPDA (Figure 4-1). The internal standards DHJA and
H4OPDA were prepared from (i)-J A and (:t)-OPDA, respectively, by the reduction of
double bonds (Weber et al., 1997 ; Wilbert et al., 1998). Endogenous DHJA and
H4OPDA were not detected in the GC-MS analysis of tomato leaf extracts (data not
shown), indicating that these compounds would be suitable as internal standards.

(i)-J A, (i)-OPDA, and the corresponding internal standards were used to
establish GC-MS conditions. To increase the sensitivity of detection (Fans et al., 1997),
these molecules were methylated prior to injection into the GC. These methyl esters
were readily separable by GC, and more determined by MS (positive-ion detection
mode). Molecules were ionized in MS by the electron impact (EI) method to enhance
the fragmentation of the molecules. Two temperature programs were used for the GC-

MS analysis. The first program was optimized for quantification of J A (Figure 4-2). The

110

 

(A)
84055.:
g 3.0 551 .83

22055 I“._
31.0551 “
“ o ............. - .............. I.

 

 

 

310E5-
g 8.0 E4 ~
'g 6.0 E41
3 4.0 E4 - (3R. 7R) (3R. 78)

~20E4-
0M

 

' j

135' h 1610' ' ‘ 1615'T71'7To' ' '

Time (min)

 

 

 

 

 

 

(B)
3 1.5 E51 83
5 J COOCH
.51.OE5. 3
1: ; ‘ 153 153
a 0: ............ 1 I .............. 1:1
100 120 140 160 180 200 220
mlz
41
c
53054.
§20 E4
3R, 7R 3R,7S
N 1.0 E4 ( ) 1 )
0 . .A'_.‘:\j+‘_vaA_'/AI '__.-I-\____‘.
15.5 16.0 16.5 17.0
Time (min)

 

 

Figure 4-2. Mass spectrum and chromatogram of MeJA and MeDHJA. JA
was extracted from wounded leaves of wild-type plants and methylated prior to
GC-MS anaylsis. For quantification of endogenous JA levels, ions of m/z = 224
and 226 were monitored for MeJ A (A) and MeDHJA (B), respectively. Ions of
m/z = 83, 151, 153 were used for the identification of each peak in the
chromatogram. The configuration of each stereoisomer is indicated in the
chromatogram. GC-MS was run with the temperature program optimized for J A
quantification.

111

(A)

(B)

 

 

 

 

 

 

 

 

 

 

 

238
3 3.0 1551 -
1.: 95 coocrlsl
g 2.0 E5«
1: 1 0 E5 ’163
3 - x
In ' 153 , \\
N -:-| MD
100 120 140 160 180 200 220
mlz
01.055
=80E4
§ 6.0 E4 ‘
§ 4.0 E4
.2 .
I“ 2.0 E4. (98’ 13R)
4.22.: r k W f/YM‘ML’:
27.0 28. 0
Time (min)
240
g 3.0 E5 1 ~83
g 2.0 155 cooc113,
C 24
3 1.0 E5- 153., 1,163 0
“ .............. L ................ I
100 120 140 160 180 200 220 240
mlz
3 1.0 55*
GB 8.0 E4 (93’ 138)
§6.0E4l
.n
N 4.0 E41
04

 

27.0

28.0

 

Time (min)

 

 

Figure 4-3. Mass spectrum and chromatogram of MeOPDA and

MeDHJA. OPDA and J A were extracted from wounded leaves of wild-type

plants. For quantification of endogenous OPDA, ions of m/z = 238 and 240
were monitored for MeOPDA (A) and MeH4OPDA (B), respectively. The

configuration of each stereoisomer is indicated in the chromatogram. Ions of

m/z = 83, 95, 96, 153, 163 were used for identification of each peak.

112

second program has a longer running time, which is necessary to quantify J A and
OPDA simultaneously in the same plant extract. The latter program permits the
separation and detection of all peaks corresponding to MeJ A, MeOPDA (OPDA-methyl
ester), MeDHJ A (DHJA-methyl ester), and MeH4OPDA (H4OPDA-methyl ester;
Figure 4-3). For quantification of J A and OPDA, only a representative ion of each
molecule was counted in the single-ion monitoring (SI) mode. Molecular ions were
monitored for Me] A [mass-to-charge ratio (m/z) = 224] and MeDHJA (m/z = 226).
Because the molecular ions of MeOPDA (m/z = 306) and MeH4OPDA (m/z = 310)
were not sufficiently abundant for detection in SI mode, ions with m/z = 238 and 240

were monitored (Figure 4-3).

Extraction and quantification of endogenous JA and OPDA

To minimize unintended loss or modification of JA and OPDA during sample
preparation, it was desirable to simplify the extensive purification procedure described
by Parchmann et al. (1997). This was accomplished by a modification of the solid-phase
extraction method originally developed for oxylipin analysis in Arabidopsis (Weber et
al., 1997). Because this method does not require tedious purification steps such as
preparatory HPLC, multiple samples could be prepared at the same time. Both J A and
OPDA were extracted from tomato tissue with methanol and then partially purified on a
C13 column. Known amounts of internal standards were added to the methanol extracts
prior to the C13 column step. J A and OPDA recovered from the column were
methylated with diazomethane, which converts endogenous J A and OPDA to their

corresponding methyl esters. Therefore, the Me] A peak in the chromatogram represents

113

(A)

 

 

 

 

 

 

1o
9 _ y = 4.3392x _
3 9 A R2 = 0.9926
n
2
I!
3
N
N
II
a
E
H-
O
N
2
CB
0 T I I I
o 0.5 1 1.5 2 2.5
[JA]I[DHJA]
(B)
0.7
0.6 y = 0.0531x .
R2 = 0.9532

area of mlz=238l area of
mlz=240

 

 

 

o r I I T I
0 2 4 6 8 10 12

[OPDA]I[H40PDA]

 

Figure 4-4. Standard curves for quantification of JA and OPDA. The
standard curve of J A (A) was constructed using known amounts of J A and
DHJA as described (Wilbert et al., 1998). Similarly, the standard curve of
OPDA (B) was obtained using mixtures of OPDA and H4OPDA. The slope
of each standard curve was used to calculate the endogenous level of J A and
OPDA.

114

the sum total of endogenous J A and Me] A. For simplicity, the amount of endogenous

J A was calculated from the Me] A peak in the chromatogram without further distinction
between J A and Me] A. Endogenous MeOPDA was not detected in tomato leaves (data
not shown).

Two stereoisomers of JA (3R, 7R-J A and BK, 7S-JA) and OPDA (9S, 13R—
OPDA and 9S, 138-OPDA) were detected in extracts from tomato leaves (Figure 4-2;
Figure 4-3). The peak area of the two stereoisomers was added to calculate the level of
J A or OPDA. The amount of endogenous J A and OPDA was calculated from the
integrated peak areas using a standard curve constructed as follows. Various amounts of
J A and DHJ A were mixed, methylated, and analyzed by GC-MS to obtain the standard
curve for the quantification of endogenous JA in tomato (Figure 4-4). The area under
each representative ion peak was measured fiom the chromatogram, and the ratios of
these areas were plotted against the concentration ratios of J A and DHJ A, as described
previously (Wilbert et al., 1998). Similarly, the standard curve for OPDA was obtained
using mixtures of OPDA and H4OPDA (Figure 4-4).

A new standard curve was constructed for each independent experiment. The
resulting slope of the curve gave very consistent values among different experiments.
The standard curve for J A was linear in the range of 0.5 to 25 nmol, with a correlation
coefficient of 0.99. The standard curve for OPDA was linear in the range of 5 to 200
nmol with a correlation coefficient of 0.95. The recovery rate of internal standards was
in the range of 60 to 90% in most measurements. Results obtained from experiments in
which the recovery rate was less than 50% were discarded to avoid possible errors in

the quantification.

115

 

pmol JA/g FW tissue

 

 

 

 

 

 

genotype leaf (hour after wounding)
flower
0 hr 1 hr 3 hr
WT (cm) 12 1 1 (n=3) 262 1 41 (n=3) 151 :1: 26 (n=3) 1485 i: 290 (n=3)
spr2 3 t 1 (n=3) 22 :l: 9 (n=3) 7 :t 1 (n=3) 265 :t 36 (n=3)

WT (mt) 9 (n=2) 675 (n=2) ND. 555 :I: 58 (n=3)

jai1-1 15 (n=3) 493 (n=2) ND. 85 :t 34 (n=3)
WT (cm) 16 (n=1) 245 (n=1) 136 (n=1) N.D.

jai1-3 15 (n=1) 130 (n=1) 53 (n=1) N.D.

 

 

 

 

Table 4-1. Summary of JA levels in wound response mutants of tomato. J A
levels were compared between wild-type (WT) and wound response mutants.
Whereas spr2 and jail -3 plants originated in L. esculentum cv Castlemart (cm),
jail-1 plants were originated in a semi-dwarf cultivar, L. esculentum cv Micro-
Tom (mt). Therefore, J A levels of each mutant were compared to the
appropriate wild-type cultivar. Wound-induced levels of J A were compared
between WT and wound response mutants. Leaves of 2-week-old plants were
wounded with a hemostat and harvested at the indicated time after wounding
for J A extraction. As a negative control, leaves were harvested from
unwounded plants (0 hr). J A levels in flowers were compared between wild-
type and wound response mutants. Flowers were harvested from one month-
old plants grown in the greenhouse. The number of independent measurements
is indicated in parentheses. The mean of two independent measurements or the
mean i standard deviation from three independent experiments is presented.
N.D. indicates that the measurement was not performed.

116

unit: pmol/g FW

 

 

 

 

 

oxylipin genotype 0 hr 1 hr 3 hr
WT 15 (n=2) 241 (n=2) 120 (n=2)
JA spr1 13 (n=2) 162 (n=2) 37 (n=2)
spr2 5 (n=1) 11 (n=1) N.D.
WT 644 (n=2) 741 (n=2) 1307 (n=2)
OPDA spr1 301 (n=2) 499 (n=2) 556 (n=2)
spr2 133 (n=1) 179 (n=1) ND.

 

Table 4-2. Wound-inducible JA and OPDA levels in wound response
mutants. Leaves of wild-type (WT), spr1, and spr2 plants were wounded and
harvested for the extraction of oxylipins as described in the legend of Table 4-
1. Levels of J A and OPDA were simultaneously measured to examine the
effect of these mutations on the biosynthesis of J A. Except for spr2, each
value represents the mean of two independent measurements. ND, not

determined.

117

Quantification of JA and OPDA from wound response mutants of tomato

In wild-type plants (L. esculentum cv Castlemart), the JA level rapidly increased within
one hour after wounding (Table 4-1). In contrast, wound response mutants showed
reduced levels of J A. For example, the suppressor of prosystemin-mediated responsesZ
(spr2; Howe and Ryan, 1999) mutant accumulated extremely low levels of J A over all
time points. The OPDA level was also reduced in spr2 plants (Table 4-2).

The semi-dwarf tomato cultivar, Micro-Tom, has been shown to be a usefirl
model system for analysis of the wound response (Howe et al., 2000; Meissner et al.,
1997). The basal level of J A in unwounded Micro-Tom leaves was comparable to that
in Castlemart plants. However, Micro-Tom plants accumulated greater amounts of J A
in response to wounding. These results indicate that the genetic background of the
cultivar can affect the capacity for J A biosynthesis.

Both wounding and exogenous Me] A failed to induce the accumulation of HS in
jasmonic acid-insensitive] (jail) plants (Li et al., 2001). A deletion null allele, jail -I,
was isolated in a genetic screen of fast-neutron mutagenized Micro-Tom plants. Within
1 hr after wounding, the J A level in jail -1 plants increased to 73% of the level observed
in wild-type plants. A similar reduction of J A biosynthesis was found for the EMS-
induced jai 1-3 allele (originally named spr5) that was isolated in the Castlemart
background. Upon wounding, J A levels injai1-3 plants were approximately 50% of that
observed in wild-type plants. These results indicate that insensitivity to J A prevents the

maximal synthesis of J A in response to wounding.

118

 

 

 

   

 

 

 

 

 

 

 

 

 

 

 

   

 

 

(A) 60
50 -
E 40
U)
3 30 ~
E
3
< 20 .
_,
10 «
o T
control infested
(B) 1400
1200 — I
E 1000 ~ 1
0!
=0 800 ~
E
e 600 q
3
n_ 400 1
O
200 .
o .

 

control infested

Figure 4-5. Accumulation of JA and OPDA in wild-type and defl plants
challenged with spider mites. Sixteen-day-old wild-type (open bar) and def] (filled
bar) plants were infested by spider mites as described in Materials and Methods.
Two days after the challenge, leaflets showing visible symptom of damage
(infested) were harvested for the quantification of J A (A) and OPDA (B). J A and
OPDA were also quantified from leaves of untreated control plants (control).
Values indicate the mean and standard deviation of three independent

experiments.

119

The amount of JA in tomato flowers was higher than that in leaves (Table 4—1).
Micro-Tom flowers appeared to contain less than half the amount of J A found in
Castlemart flowers. Both spr2 and jail -I showed reduced JA levels in flowers. The J A
level in spr2 flowers was only 18% of the level observed in Castlemart flowers,
whereas jail -1 flowers accumulated less than 15 % of the level of JA observed in
Micro-Tom flowers.

Simultaneous monitoring of J A and OPDA levels showed that these oxylipins
displayed different kinetics of accumulation (Table 4-2). The J A level increased
transiently within 1 hr of wounding whereas the level of OPDA steadily increased up to
3 hr after wounding. This finding indicated that an increase in OPDA accumulation
does not necessarily result in a proportional increase in J A.

Consistent with a previous report (Lee and Howe, 2003), the basal level of J A in
the suppressor of prosystemin—mediated responses] (spr1) mutant was normal. One hr
afier wounding, this level increased to 65% of the level in wild-type. However, the
basal level of OPDA was significantly reduced in spr1 plants compared to wild-type
(Table 4-2). These results also indicate that the synthesis of OPDA is not proportional
to that of J A. The wound-induced accumulation of OPDA was impaired in spr1 plants
as well.

Changes in the levels of J A and OPDA were also observed in tomato plants
challenged with spider mites (Figure 4-5). This arachnid herbivore pierces epidermal
cells and sucks out the cellular contents of the underlying mesophyll cells (Lange and
Bronson, 1981). In infested leaves of wild-type plants, a moderate increase in JA was

observed (Figure 4-5). Although the OPDA level increased upon challenge of wild-type

120

plants (student’s t-test, P < 0.05), the induction of J A and OPDA was not equivalent.
The basal level of J A was comparable between wild-type and defenseless] (def?) plants,
but the basal level of OPDA was significantly reduced in def] plants compared to wild-
type. Neither J A nor OPDA levels increased in response to herbivore attack of defl
plants (P < 0.05). This result is consistent with the previous observation that def] plants
are defective in the induction of IA synthesis in response to wounding (Howe et al.,

1996).

Discussion

Efficient quantification of J A and OPDA is important for the study of wound signaling
because the induction of J A synthesis is one of the earliest events in the signaling
cascade that results in expression of wound-inducible genes (Doares etal., 1995;
Farmer and Ryan, 1992). Accordingly, this study was intended to establish a reliable
and simple procedure to measure endogenous J A and OPDA in tomato tissues. Recent
studies have shown the use of GC-MS to measure levels of J A and OPDA in flowers
(Hause et al., 2000) and leaves (Stenzel et al., 2003; Strassner et al., 2002) of tomato
plants. Despite minor differences in the methods used, the results are comparable to
those presented here.

A disadvantage of the method used in this study is that simultaneous
quantification of endogenous J A and MeJ A is not possible owing to methylation of the
plant extract prior to GC-MS analysis. Alternative derivatization methods (for example,
silylation of the hydroxy group in the carboxylate side chains of J A and OPDA with

trimethylsilyl reagents) may be helpful to circumvent this problem.

121

Regulation of JA biosynthesis in tomato

The results summarized in Table 4-1 show that endogenous J A levels change in
response to wounding, the particular tissue type under study, and the genetic
background of the cultivar. These factors have been reported to regulate the activity of
enzymes involved in IA biosynthesis (Hause et al., 2000; Lauder and Weiler, 1998;
Strassner et al., 2002).

In leaf tissue, J A levels seem to be regulated by substrate availability. Previous
studies have shown that application of linolenic acid to leaves activates the expression
of J A-inducible genes in tomato (Farmer and Ryan, 1992). Furthermore, linolenic acid
and J A showed a similar accumulation pattern in tomato leaves in response to wounding
(Conconi et al., 1996). Transgenic Arabidopsis plants that overexpress AOS did not
have altered basal levels of J A in leaves, but showed stronger induction of J A synthesis
in response to wounding (Laudert et al., 2000). These results indicate that the
biosynthesis of J A in leaves is limited by the release of linolenic acid from membranes.

The results of this study indicate that the conversion of OPDA to J A is another
rate-limiting step in J A biosynthesis (Table 4-2; Figure 4-5). The level of OPDA was
significantly higher than that of J A in unwounded control plants. Three hr after
wounding, the level of OPDA continued to increase, whereas the J A content almost
returned to the basal level. These results indicate that increased OPDA levels do not
necessarily lead to a proportional increase in J A levels. The differential accumulation of
OPDA and J A was also observed in Arabidopsis (Laudert and Weiler, 1998; Weber et

al., 1997) and potato (Weber et al., 1997).

122

Wound response mutants showed reduced levels of J A and OPDA compared to
wild-type plants (Table 4-1; Table 4-2; Figure 4-5). These observations provide clues to
understanding the signaling defect in these mutants. For example, the product of Spr2 is
expected to be involved in OPDA synthesis, because both J A and OPDA were severely
reduced in spr2 plants (Table 4-2). This speculation was confirmed by the recent
discovery that Spr2 encodes a fatty acid desaturase catalyzing the synthesis of linolenic
acid (Li et al., 2003). Therefore, the deficiency in linolenic acid blocks the production
of both OPDA and J A in spr2 plants.

Both spr1 and def] plants accumulated less OPDA than did wild-type plants in
response to wounding. These observations are consistent with recent findings that Spr]
is a positive regulator of IA biosynthesis (Lee and Howe, 2003), and that the activity of
AOC is reduced in def] plants (Stenzel et al., 2003). JA-insensitive jail -I and jail -3
plants showed reduced levels of J A in wounded leaves. These results indicate that J A

biosynthesis may be regulated by a positive feedback mechanism.

Materials and Methods
Preparation of internal standards
DHJ A and H4OPDA were prepared by PtOz-catalyzed hydrogenation of (:t)-J A (Sigma,
St. Louis, MO) and OPDA (Cayman Chemical, Ann Arbor, MI) as described (Weber et

al., 1997). The authenticity of the standards, as well as the absence of endogenous

DHJA/H4OPDA in tomato leaf extracts, was verified by GC-MS.

123

Extraction of JA and OPDA from tomato tissues

Plants containing two fully—expanded leaves and an emerging third leaf were wounded
with a hemostat on each leaflet. Three to five g FW of leaves were used for
quantification of J A. JA extraction was performed following the procedure described by
Weber et al. (1997) with modifications. Harvested leaves were frozen in liquid nitrogen
and ground to a fine powder with a chilled mortar and pestle. The tissue was dissolved
in 28 ml methanol containing 500 ng DHJ A and 500 ng H4OPDA as internal standards
and then homogenized with a Polytron for 1 min at 4°C. The homogenate was incubated
for 2 hr at 4°C with shaking, diluted with 12 ml ice-cold water, and then centrifuged at
3,500 X g. The resulting supernatant was recovered and the pH adjusted to 8.0 with
NH40H. This solution was centrifirged again at 3,500 x g, and the supernatant was
passed through a tClg-SepPak cartridge (Waters Corporation, Milford, MA) that was
preconditioned with 70% (vol/vol) methanol and collected in a new glass vials. The
cartridge was washed with 7 ml of 75% (vol/vol) methanol. Eluates from both the
sample and the wash steps were combined and adjusted to pH 4.0 with 10% (vol/vol)
formic acid. This solution was diluted with 160 ml ice-cold water and then loaded on
the same thg-SepPak column that was washed sequentially with methanol, diethylether,
methanol, and water immediately after the elution with 75% (vol/vol) methanol. After
washing the column with 7 ml of 15% (vol/vol) ethanol and 7 ml water, the J A fraction
was eluted with 10 ml diethylether. The eluate was partially dried over anhydrous
NaZSO4 and then dried completely under a stream of nitrogen gas at 35°C. The dried
paste was dissolved in 0.5 ml methanol and subjected to methylation by the addition of
diazomethane dissolved in 0.5 ml diethylether. This mixture was dried under nitrogen

gas, resuspended in 20 pl hexane, and injected into the GC. Diazomethane was prepared

124

prior to use: 0.2 g N-nitroso-methylurea (Sigma, St. Louis, MO) was dissolved in a

mixture of 2 ml ice-cold diethylether and 600 11.1 of 40 % (vol/vol) KOH solution.

GC-MS analysis
The amount of J A/MeJ A in leaf extracts was quantified by GC-MS by using a Hewlett-

Packard GC 5890 equipped with a Hewlett-Packard 5970 mass detector. The GC was
fitted with a DB-5 column (30 m X 0.25 mm id, J&W Scientific, Folsom, CA) and run
with a temperature gradient of 100°C for l min, 100°C to 170°C at 5°C/min, 170°C for
2.5 min, and 170°C to 250°C at 20°C/min. GC-MS analysis was performed in the SI
mode with monitoring of ions specific for Me] A (m/z = 224) and MeDHJ A (m/z = 226).
Molecules were ionized by electron impact in MS. For quantification of JA/MeJ A, a
standard curve was generated from samples in which MeJ A and MeDHJ A were mixed
in known ratios. Because peaks corresponding to the 3R, 7S and 3R, 7R isomers of

endogenous J A/MeJ A were detected, the areas of the two peaks were combined.

For simultaneous analysis of J A and OPDA, a modified temperature program
was used for GC: 100°C for 1 min, 100°C to 160°C at 20°C/min, 160°C to 238°C at
3°C/min, 238°C to 250°C at 30 °C/min. For the quantification, GC-MS was run in SI
mode while monitoring ions for MeJ A (m/z = 224), MeDHJA (m/z = 226), MeOPDA
(m/z = 23 8) and MeH4OPDA (m/z = 240). Retention times of these molecules were:
9.57 min for 3S, 7S—MeJA; 9.88 min for 3R, 7R-MeJA; 9.97 min for 3R, 7R-
MeDHJA;10.51 min for 3R, 7S-MeJA; 10.54 min for 3R, 7S-MeDHJA; 26.93 min for
98, 13R-MeH4OPDA (also called cis-MeH4OPDA); 27.30 min for 9S, l3R-MeOPDA;

27.70 min for 9S, 138-MeH4OPDA (also called trans-MeH4OPDA); and 28.60 min for

125

9S, 13S-MeOPDA. Standard curves for OPDA were constructed using a mixture
containing known amounts of OPDA and H4OPDA. The detection limit of Me] A (0.4
nmol) and MeOPDA (0.2 nmol) was determined by injection of known amounts of

these molecules.

Plant material

Seeds of spr1-1, spr2, def] and jail -I were collected from each homozygous line afier
at least two backcrosses using Castlemart cv (for spr1-1, spr2, and def!) or Micro-Tom
cv (for jai 1-1) as the recurrent parent. Leaves were harvested from two-week-old plants
for extraction of oxylipins. For the analysis of jail -3 plants, the second and the third
youngest leaves were harvested from 5-leaf-stage plants. Wounding was inflicted on
each leaflet with a hemostat. Flowers were harvested in both open and closed stages

from plants grown in the greenhouse. Growth conditions are described in Chapter 2.

Spider mite treatment

Two-spotted spider mite (T etranychus urticae Koch) treatment was performed as
described (Li et al., 2002). Spider mite eggs were hatched on leaves of lima bean
(Phaseolus lunatus cv Fordhook) plants and allowed to grow to a population density of
approximately 50 adult spider mites per fully expanded leaf. Infested leaves were cut
and placed onto the upper surface of leaves of l6-day-old wild-type and def] plants.
Two days later, tomato leaflets showing the visible symptom of damage were harvested

for J A/OPDA extraction.

126

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131

Chapter 5

Analysis of wound-induced root-to-leaf

signaling in tomato plants

132

Introduction

Tomato plants express many genes systemically in response to wounding. Previous
studies have shown that the polypeptide systemin and its precursor protein prosystemin
play crucial roles in the systemic wound response in tomato (Ryan, 2000). Exogenous
systemin induces the accumulation of defense proteins such as Proteinase Inhibitors
(PIs; Pearce et al., 1991). Systemic accumulation of PIs in response to wounding is
severely reduced in transgenic plants that constitutively express a prosystemin cDNA in
the antisense orientation (McGurl et al., 1992). Systemin activates the biosynthesis of
jasmonic acid (J A), which results in the expression of PIs in tomato (Doares et al.,
1995a; Howe et al., 1996). These observations, together with the mobility of systemin
applied at wound sites in the phloem (Pearce et al., 1991), led to the hypothesis that
systemin is a mobile signal that couples local injury to systemic gene expression (Ryan,
2000).

Results from recent studies do not support the proposed role of systemin as the
long-distance signal for systemic gene expression. Grafting experiments performed with
various wound response mutants of tomato indicate that J A, not systemin, is the long-
distance signal for systemic PI expression (Chapter 2; Li et al., 2002). Moreover, it was
shown that the systemin-insensitive suppressor of prosystemin-mediated responses]
(spr1) mutant is compromised in the generation but not the perception of the systemic
signal for PI expression (Chapter 2). This finding indicates that systemin is not the
long-distance signal. J A-deficient tomato mutants such as J L1 (Chapter 3), defenseless]
(def! ; Howe et al., 1996; Li et al., 2002), and spr2 (Li et al., 2002) are also defective in

the generation of the systemic wound signal. However, these plants are able to perceive

133

the systemic wound signal transmitted from wild-type rootstock leaves. In contrast, the
perception of the systemic wound signal was blocked in jasmonic acid-insensitive]
(jail) plants that fail to express P1 in response to JA (Li et al., 2002). Therefore, JA or a
derivative of J A appears to be synthesized in damaged leaves and transported to
undamaged tissue to induce expression of P13.

Increasing evidence indicates that not all systemic wound responses in tomato
are regulated by J A and systemin. For example, wound-induced activation of a mitogen-
activated protein kinase (MAPK) is mediated by a rapid, J A-independent signal
(Stratmann and Ryan, 1997). It was also reported that wound-induced systemic
expression of a glucosyl transferase gene occurs independently of J A and systemin
(O’Donnell et al., 1998). Finally, the observation that a subset of wound-inducible
genes is systemically expressed in spr1 plants (Figure 2-2 and 2-5, Chapter 2) points to
the existence of a systemin-independent signaling pathway.

Similar to systemic wound signaling in leaves, a long-distance signaling
pathway appears to transmit a wound signal between roots and leaves. For example,
mechanical wounding to roots induced PI expression in leaves of potato plants
(Dammann et al., 1997). Conversely, leaf damage activates expression of a fatty acid
desaturase gene in roots of Arabidopsis (N ishiuchi et al., 1997 ; 1999). At present, very
little is known about the signaling pathway that mediates this root-to-shoot systemic
wound response. The purpose of this study was to investigate the genetic basis of
wound-induced root-to-leaf signaling in tomato. This question was addressed by
analyzing the systemic expression of various wound-inducible genes in wild-type and

mutant plants that are defective in the systemin/JA-dependent signaling pathway. The

134

results show that a JA-dependent signaling regulates the systemic expression of a subset
of wound-inducible genes during root-to-leaf signaling, whereas a JA-independent
pathway is involved in the controlling the systemic expression of a different set of

genes.

Results and Discussion

In potato plants, damage to roots can induce gene expression in leaves (Dammann et al.,
1997). To determine whether this is also the case in tomato plants, PI-H levels in leaves
were measured in two-leaf-stage tomato plants afier wounding of roots. Mechanical
crushing of roots with a hemostat resulted in accumulation of PHI in undamaged leaves
(Figure 5-1). Although the level of PI accumulation was less than that observed in
wounded leaves, this finding indicates that a systemic signal travels between roots and
aerial parts of the plant.

To investigate whether root-to-shoot signaling depends on systemin and J A,
systemic accumulation of PHI was examined in systemin-insensitive spr1 and J A-
deficient def] plants. The results showed that root-damaged spr1 plants accumulated
normal levels of PHI in leaves. Thus, systemin action appears not to be required for
this systemic response. This observation contrasts the role of systemin in a leaf-to-leaf
systemic signaling (Chapter 2). A role for systemin in leaf-to-leaf but not root-to-leaf
systemic signaling is consistent with the previous report that prosystemin is expressed
in leaves but not roots (McGurl et al., 1992).

In contrast to spr1 plants, root damage failed to induce systemic PI-II

accumulation in def]. The lack of response in def] plants could result from a defect in

135

 

180

 

 

 

 

 

 

 

 

 

 

 

 

 

g 160 _
.5 140 -
i: 120 1
19
2 100 1 J
g 80 -
g 60 j
; 4o
5' 2o 1 j
0 __.__L..L__ , 1L
unwounded damaged leaf undamgged leaf systemic
leaf-to-leaf root-to-leaf

Figure 5-1. Systemic induction of PHI in leaves in response to wounding
of roots. The accumulation of PHI in leaves was compared among wild-
type (open bar), spr1 (gray bar), and def] (defI leaves did not accumulate
detectable amounts of PHI) plants. Mechanical wounding was inflicted to
roots of two-leaf-stage plants, and leaves were harvested 24 hr later for
measurement of PHI (systemic). As a negative control, PI-II was
measured in leaves harvested from unwounded plants (unwounded). For
comparison to leaf-to-leaf systemic signaling, a single wound was inflicted
on a lower leaf of two-leaf-stage plants, and PI-II levels were measured
separately in damaged lower leaves (damaged leaf) and undamaged upper
leaves (undamaged leaf) 24 hr later. Data represent the mean and
standard deviation of six plants.

136

the production of the systemic wound signal in wounded roots, or from a defect in the
recognition of that signal in unwounded leaves. Previous grafiing experiments showed
that leaf-to-leaf wound signaling requires J A biosynthesis in wounded leaves, not in
undamaged leaves (Chapter 3; Li et al., 2002). Because PI synthesis in leaves is
mediated by the action of J A (Doares et al., 1995a; Farmer and Ryan, 1992), these
results indicate that JA or a derivative of J A is synthesized in wounded leaves and
transported to undamaged leaves (Li et al., 2002). Therefore, the lack of PI production
in def] leaves in response to root damage indicates that def] roots are defective in
wound-inducible J A biosynthesis and the generation of a systemic wound signal.

RNA gel-blot analysis was used to determine whether root damage results in
activation of gene expression in unwounded leaves. In these experiment, cDNA probes
for two groups of wound-inducible genes were employed (Figure 5-2). The first group
of these genes is the so-called ‘early’ wound response genes, which shows rapid and
transient induction in response to wounding of leaves (Ryan, 2000; Figure 2-2). Early
genes include lipoxygenase D (Lox D) involved in J A biosynthesis and a wound-
inducible MAPK (WIPK) homologue (Seo et al., 1999). The second group is ‘late’
wound response genes, which are expressed slowly but steadily upon wounding (Ryan,
2002; Figure 2-2, Chapter 2). P13 are representative of this group.

Root damage activated strong expression of PHI in wild-type leaves, similar to
the pattern observed in response to leaf wounding or stem excision (Figure 2-5, Chapter
2). Wounding of wild-type roots also resulted in rapid and transient systemic expression
of LoxD and WIPK. The general pattern of systemic expression of early and late wound

response genes in spr1 plants was similar to wild-type plants, although differences in

137

WT spr1 iai1 spr2

hr 012481224012481224 012481224012481224

 

 

 

 

 

 

 

 

 

 

 

 

 

 

PI'” v: t 5. Q ’
LoxD "' 2*
WIPK ‘ -' L‘ - ; , 1.4L;
1 - ‘ .
eIF4A ..CQ. racmgytfioc

 

 

 

 

 

Figure 5-2. Systemic expression of wound-inducible genes in response to
root damage. Mechanical wounding was applied to roots of two-week-old
plants with a hemostat. Leaves were harvested from six plants at each time
point and pooled for RNA extraction. To facilitate direct comparison of
transcript levels in wild-type (WT) and mutant plants, blots containing
RNA from the four genotypes were hybridized in the same container and
washed under the same conditions. Then X-ray film was exposed to the
blots for the same length of time.

138

 

the magnitude of the response were noted. For example, the expression of PM] in spr1
leaves was significantly lower than in wild-type plants. Because the level of PHI
protein in leaves of root-damaged spr1 plants was similar to that of wild-type plants
(Figure 5-1), it appears that mRNA levels do not accurately reflect PI protein levels.
These phenomena have previously been observed in wounded tomato leaves (Howe et
al,1996)

Systemic PI-II expression was abolished in leaves of J A-insensitive jail plants,
indicating that a functional J A-dependent signaling pathway is required for this
systemic wound response. Consistent with the absence of PHI in def] plants (Figure 5-
1), J A-deficient spr2 plants also failed to express PM] in leaves upon root damage. This
result demonstrates that J A biosynthesis is required for systemic P] expression.
Interestingly, a wound-inducible MAPK homologue (WIPK) was uniformly expressed
in all genotypes in response to root damage. Therefore, expression of the WIPK appears
to be mediated by a J A-independent signaling pathway. J A-independent wound
signaling was previously proposed to account for the expression of an LE RNase gene, a
glucosyl transferase gene, and activation of a MAPK activity in tomato leaves (Figure
6-8, Chapter 6; O’Donnell et al., 1998; Stratmann and Ryan, 1997).

Unlike WIPK, LoxD expression in jail plants was less abundant than in spr1,
spr2, and wild-type plants. This result indicates that the J A-dependent signaling
pathway is required for maximal induction of LoxD. The residual expression of LoxD is
unlikely due to ‘leakiness’ of the jai 1 mutation because PI-II expression was not
detected in jail plants. Furthermore, this allele of jail (jail-1) corresponds to a fast-

neutron-induced deletion in the Jail gene (Li et al., 2002). The expression of LoxD in

139

wild-type plants therefore appears to be regulated by both J A-independent and J A-
dependent signaling pathways. This conclusion is consistent with the previous finding
that LoxD expression is regulated by a systemin-independent pathway (Figure 2-5,
Chapter 2).

In Arabidopsis, a J A-independent pathway regulates local wound response
through the action of oligogalacturonic acid (OGA) released from cell walls (Rojo et al.,
1999). It is unlikely that JA-independent signaling in tomato is mediated by OGA
because I A biosynthesis is required for OGA-induced gene expression (Doares et al.,
1995b). Previous studies indicated that the JA-independent signal could be rapidly
transmitted through the xylem (Malone et al., 1995; Rhodes et al., 1999; Stratmann and
Ryan, 1997). It may be informative to examine the expression of WIPK using steam-
girdled plants in which the xylem but not the phloem is intact. This experiment would
provide insight into the question of whether the JA-independent signal for WIPK

expression is phloem- or xylem-bome.

Materials and Methods

Plant material

Seeds of spr1-1, spr2, def], and jai 1-1 were collected from each homozygous line after
at least two backcrosses, using Castlemart cv as the recurrent parent. As a result, jai I -1
plants used in this study displayed normal growth compared to Castlemart cv. Plants
were grown in peat pots as described in Chapter 2. Root damage was applied on two-
leaf—stage plants by pinching the peat pot 7 times with a hemostat. The PI-II level was

measured as described in Chapter 2.

140

RNA gel-blot analysis
Tomato EST clones cLET1D13 and cLED1D24 were used as probes to detect WIPK
and eIF 4A mRNA, respectively. Total RNA was harvested and hybridized following the

methods described in Chapter 2.

141

References

Dammann, C., Rojo, E., and Sanchez-Serrano, J. (1997). Abscisic acid and jasmonic
acid activate wound-inducible genes in potato through separate, organ-specific
signal transduction pathways. Plant J. 11: 773-782.

Doares, S., Narvaez-Vasquez, J ., Conconi, A., and Ryan, C. (1995a). Salicylic acid
inhibits synthesis of Proteinase Inhibitors in tomato leaves induced by systemin
and jasmonic acid. Plant Physiol. 108: 1741-1746.

Doares, S., Syrovets, T., Weiler, E.M., and Ryan, C. (1995b). Oligogalacturonides and
chitosan activate plant defensive genes through the octadecanoid pathway. Proc.
Natl. Acad. Sci. USA. 92: 4095-4098.

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

Hause, B., Stenzel, I., Miersch, O., Maucher, H., Kramell, R., Ziegler, J ., and
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.

Li, L., Li, C., Lee, G.I., and Howe, G (2002). Distinct roles for jasmonate synthesis and
action in the systemic wound response of tomato. Proc Natl Acad Sci USA. 99:
6416-6421.

Malone, M. and Alarcon, J. (1995). Only xylem-bome factors can account for systemic
wound signaling in the tomato plant. Planta 196: 740-746.

McGurl, B., Pearce, G., Orozco-Cardenas, M. and Ryan, CA. (1992). Structure,
expression, and antisense inhibition of the systemin precursor gene. Science
255: 1570-1573.

Nishiuchi, T., Kodama, H., Yanagisawa, S., and Iba, K. (1999). Wound-induced
expression of the FAD7 gene is mediated by different regulatory domains of its
promoter in leaves/stems and roots. Plant Physiol. 121: 1239-1246.

Nishiuchi, T., Hamada, T., Kodama, H., and Iba, K. (1997). Wounding changes the
spatial expression pattern of the Arabidopsis plastid omega-3 fatty acid

desaturase gene (FAD7) through different signal transduction pathways. Plant
Cell 9: 1701-1712.

O'Donnell, P., Turesdale, M., Calver, C., Dorans, A., Roberts, M., and Bowles, D.

(1998). A novel tomato gene that rapidly responds to wound- and pathogen-
related signals. Plant J .14: 137-142.

142

Pearce, G., Strydom, D., Johnson, S., and Ryan, CA. (1991). A polypeptide from
tomato leaves induces wound-inducible proteinase inhibitor proteins. Science
253: 895-898.

Rhodes, J ., Thain, J ., and Wildon, D. (1999). Evidence for physically distinct systemic
signalling pathways in the wounded tomato plant. Annals Botany 84: 109-116.

Rojo, E., Leon, J, and Sanchez-Serrano, J. (1999). Cross-talk between wound signalling
pathways determines local versus systemic gene expression in Arabidopsis
thaliana. Plant J. 20:135-142.

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

Seo, S., Sano, H., and Ohashi, Y. (1999). Jasmonate-based wound signal transduction
requires activation of WIPK, a tobacco mitogen-activated protein kinase. Plant
Cell 11: 289-298.

Strassner, J ., Schaller, F ., Frick, U., Howe, G, Weiler, E., Amrhein, N., Macheroux, P.,
and Schaller, A. (2002). Characterization and cDNA-microarray expression
analysis of 12- oxo-phytodienoate reductases reveals differential roles for

octadecanoid biosynthesis in the local versus the systemic wound response.
Plant J. 32: 585-601.

Stratmann, J .W., and Ryan, GA. (1997) Myelin basic protein kinase activity in tomato
leaves is induced systemically by wounding and increases in response to
systemin and oligosaccharide elicitors. Proc. Natl. Acad. Sci. USA. 94: 11085-
11089.

143

Chapter 6

Cytochrome P450-dependent metabolism of oxylipins
in tomato. Cloning and expression of allene oxide

synthase and fatty acid hydroperoxide lyase

A version of this chapter has been published.
Gregg A. Howe, Gyu In Lee, Aya Itoh, Lei Li, and Amy E. DeRocher

(2000). Plant Physiol. 123:711-724.

Data presented in Figure 6-4 were obtained by Dr. Aya Itoh.
Data presented in Figure 6-6 were obtained by Dr. Amy DeRocher.
Data presented in Figure 6-7 were obtained by Dr. Lei Li.

Data presented in Figure 6-8 were obtained by Dr. Gregg Howe.

144

Introduction

Fatty acid hydroperoxides produced by lB-lipoxygenases are important intermediates in
the oxylipin pathway of fatty acid oxygenation in plants. In one branch of oxylipin
metabolism often referred to as the octadecanoid pathway, allene oxide synthase (AOS)
commits l3S-hydroperoxy—9(Z),11(E),15(Z)-octadecatrienoic acid (13-HPOT) to the
formation of j asmonic acid (J A) and related cyclopenta(e)nones (Creehnan and Mullet,
1997; Figure 6-1). Products of the AOS pathway are essential signals for plant defense
against pest attack (Staswick and Lehman, 1999), mechanical responses (Weiler et al.,
1993), and some developmental processes (McConn and Browse, 1996). An alternative
pathway for l3-HPOT metabolism is initiated by fatty acid hydroperoxide lyase (HPL;
Figure 6-1). Short chain aldehyde products of HPL, together with their corresponding
reduced alcohols, are important volatile constituents of the characteristic odor of fi'uits,
vegetables, and green leaves (Gardner, 1991; Hatanaka, 1993). C6 aldehydes produced
by HPL are also reported to act as phytoalexins against protozoa, bacteria, and fungi
(for review, see Blée, 1998), and may be signals for gene regulation (Bate and
Rothstein, 1998). The C12 oxo-acid product of HPL is the precursor of the previously
identified ‘wound signal’ known as traumatin (Zimmerman and Coudron, 1979). 13—
HPOT is metabolized by other plant enzymes including lipoxygenase (Salch et al.,
1995), peroxygenase (Blée et al., 1993), and divinyl ether synthase (Grechkin et al.,
1995; Hamberg, 1998), and may be subject to degradation by non-specific alkyl
hydroperoxide reductases (Baier and Dietz, 1999).

AOS and HPL comprise an unusual class of cytochrome (Cyt) P4503 that is

specialized for the rearrangement of fatty acid hydroperoxides. Unlike typical P450

145

COOH

Linolenic acid

1 Lipoxygenase

coon
_ \
Allene Oxide Synthase 13(9)::Por Hydroporoxide Lyase
(CYP74A) (CYP74B)

coon
<_X\ :\\_/\ Cfv‘coon + or-Ic’\=/\
o - CI-IO

12-oxo-(Z)-9 dodecenoic acid (3Z)-hexenal

Allene oxide
1 l 1
COOH COOH
O 12-oxo-(E)-9 dodecenoic acid (2E)-hexenal
12-oxo-phytodienoic acid (Traumatin)
l

Jasmonic acid

Figure 6-1. The octadecanoid pathway. Cyt P450-dependent metabolism of
13-HPOT. AOS (CYP74A) commits 13-HPOT to the production of J A and
related cyclopenta(e) nones. In the absence of allene oxide cyclase (AOC), the
epoxide product of AOS undergoes spontaneous hydrolysis to a- and 'y-ketols
and racemic l2-OPDA. HPL (CYP74B) cleaves l3-HPOT to produce C6 and
C12 products that are further metabolized as shown.

146

monoxygenases, AOS and HPL demonstrate low affinity for carbon monoxide and do
not require 02 or NADPH-dependent Cyt P450 reductase for their activity (Song and
Brash, 1991; Shibata et al., 1995a, 1995b). Identification of cDNA sequences encoding
AOS and HPL has provided additional insight into the relationship between these two
enzymes, and their divergence from classical P4508 (Song et al., 1993; Pan et al., 1995;
Laudert et al., 1996; Matsui et al., 1996; Bate et al., 1998). Based on the amino acid
sequence identity between AOS and HPL (approximately 38%), the two enzymes are
classified as subfamilies CYP74A and CYP74B, respectively, within the CYP74 family
of P4508 (Nelson, 1999).

The importance of oxylipins as signals for plant stress responses has prompted
interest in understanding the mechanisms by which their synthesis is regulated
(Creelman and Mullet, 1997; Farmer et al., 1998). JA accumulation, for example, is
stimulated by mechanical wounding and herbivory(Cree1man et al., 1992; Blechert et
al., 1995; Conconi et al., 1996), pathogen attack (Penninckx et al., 1996), treatment with
elicitors (Gundlach et al., 1992; Doares et al., 1995), and water or nutrient deprivation
(Creehnan and Mullet, 1995; Lehmann et al., 1995). Similarly, mechanical injury and
some plant-pathogen interactions lead to the production of HPL products (Hatanaka et
al., 1987; Gardner, 1991; Croft et al., 1993). Formation of AOS- and HPL-derived
oxylipins is controlled in large part by the availability of hydroperoxide substrates that
are generated from lipase/acyl hydrolase-mediated release of fatty acids from membrane
lipids, followed by lipoxygenase-catalyzed conversion to 9- and 13-hydroperoxides
(Galliard et al., 1977; Hatanaka, 1993; Mueller et al., 1993; Narvaez-Vasquez et al.,

1999). Nonenzymatic lipid peroxidation, such as that associated with the initial stages

147

of plant-pest interactions, may also contribute to the pool of hydroperoxides available to
AOS and HPL (Gardner, 1989; Harnmond-Kosack and Jones, 1996).

In addition to substrate availability, fatty acid hydroperoxide metabolism may
also be influenced by the spatial and temporal expression of enzymes that utilize these
substrates. For example, the localization of both AOS and HPL to the chloroplast (Vick
and Zimmerman, 1987; Song et al., 1993; Blée and Joyard, 1996; Laudert et al., 1996;
Bate et al., 1998; Froehlich et al., 1999) indicates that these enzymes utilize a common
pool of hydroperoxide substrates. Recent studies indicate that AOS expression is
positively regulated by wounding, as well as by terminal products of the AOS pathway
(Laudert and Weiler, 1998). These results, together with transgenic studies showing that
AOS is a rate-limiting step in J A biosynthesis (Harms et al., 1995), indicate that up-
regulation of AOS activity during the wound response may provide a mechanism to
amplify the octadecanoid signaling pathway. On the other hand, others have shown that
exogenous methyl J A stimulates oxylipin metabolism through the HPL pathway, and
thus may shift oxylipin metabolism away from J A biosynthesis (Avdiushko et al., 1995;
Kohlmann et al., 1999).

The aim of the present work was to gain an understanding of the molecular basis
of Cyt P450-dependent metabolism of fatty acid hydroperoxides in tomato
(Lycopersicon esculentum). Owing to the wealth of knowledge of plant-pest interactions
in tomato, this system is likely to provide a good model for assessing the role of
oxylipins in plant defense. The importance of HPL-derived volatiles in determining the
flavor and aroma of fruits and vegetables provides additional incentive for investigating

oxylipin metabolism in tomato (Kazeniac and Hall, 1970; Buttery and Ling, 1993).

148

Toward this goal, we report here the isolation of cDNAs that encode functional
members of the CYP74A (AOS) and CYP74B (HPL) subfamilies of P450 enzymes in
tomato. The results of expression studies in Escherichia coli indicate a role for AOS
and HPL in the commitment of l3-HPOT to the J A and C6 aldehyde/traumatin
pathways, respectively. We also report findings relevant to the developmental and
defense-related expression of these two genes in planta. The significance of these
results for understanding the regulation of fatty acid hydroperoxide metabolism is

discussed.

Results

cDNA isolation and sequence analysis

An AOS-encoding cDNA from Arabidopsis (Laudert et al., 1996) was used to screen
for related sequences in a tomato cDNA library. The longest clone obtained (designated
as LeA 05') contained a 1,533-bp open reading frame, a 57-bp 5'-untranslated region
(UTR), and a 11 l-bp 3'-UTR excluding the poly-(A) tail. The open reading frame was
predicted to encode a 510-amino acid protein having a calculated molecular mass of
57,202 D. The presence of an in-frame stop codon (UAA) 30 nucleotides upstream of
the putative AUG start codon indicated that LeA 0S contained the full-length coding
sequence. The deduced amino acid sequence of LeAOS was approximately 61%
identical to AOS from flax (Song et al., 1993), guayule (Pan et al., 1995), and
Arabidopsis (Laudert et al., 1996) (Table 6-1). Thus, LeAOS is classified as a new

member of the CYP74A subfamily of Cyt P450s. The N-terminal region of LeA US

149

Figure 6-2. Comparison of cDNA-deduced protein sequences of
plant AOS and HPL genes. LeAOS and LeHPL sequences were
aligned, using the ClustalW 1.7 program available at
http://mbcr.bcm.tmc.edu/searchlauncher. AOS sequences were
from flax (LuAOS; Song et al., 1993; accession no. U00428),
guayule (PaAOS; Pan et al., 1995; accession no. X78166), and

Arabidopsis (AtAOS; Laudert et al., 1996; accession no. Y12636).

HPL sequences were from bell pepper (CaHPL; Matsui et al.,
1996; accession no. U51674) and Arabidopsis (AtHPL; Bate et
al., 1998; accession no. AF087932). Black boxes indicate amino
acid residues that are conserved between all seven CYP74
members. Subfamily—specific substitutions are indicated with an
asterisk. The three subfamily-specific motifs discussed in the text
are underlined by the black bars. The " symbol denotes the T

-* (I/V) change within the I helix that is a hallmark of CYP74
enzymes. The conserved Cys within the heme-binding domain is
marked by a # symbol. The boxed residue (Pro—43) at the N
terminus of LeAOS denotes the site where the His-tag was added
in the pQE-AOS expression construct.

150

 

 

 

LuAOS
AtAOS
PaAOS
LeAOS
LeHPL
CaHPL
AtHPL

LuAOS
AtAOS
PaAOS
LeAOS
LeHPL
CaHPL
AtHPL

LuAOS
AtAOS
PaAOS
LeAOS
LeHPL
CaHPL
AtHPL

LuAOS
AtAOS
PaAOS
LeAOS
LeHPL
CaHPL
AtHPL

LuAOS
AtAOS
PaAOS
LeAOS
LeHPL
CaHPL
AtHPL

LuAOS
AtAOS
PaAOS
LeAOS
LeHPL
CaHPL
AtHPL

LuAOS
AtAOS
PaAOS
LeAOS
LeHPL
CaHPL
AtHPL

LuAOS
AtAOS
PaAOS
LeAOS
LeHPL
CaHPL
AtHPL

MASSALNNLVAVNPNTLSPSPKSTPLPNTFSNLRRVSAFRPIKASLFGDSPIKIPGITSQPPPSSDETTL
MA ------- SISTPFPISLHPKT--VRSKPLKFRVL--TRPIKASGSETP----DLTVATRTGSKD---L
------------------------------------------------------------ MDPSSK----
MAL----TLSFSLPLP-SLHQK—--IPSKYSTF ------ RPIIVSLS ------- DKSTIEITQEIK---L
------------------------------------------ MNSAP--—----——-LSTPAPVT----L
----------------------------------- M---IPIMSSAP-—--—----—LSTATPIS-—--L
----------------------- MLLRTMAATSPRP---PPSTSLTS----------QQPPSPPSQ---L

~||TFWFQG-PDK

 

138
120
75
114
84
88
101

208
190
145
184
154
158
171

277
259
214
253
224
228
241

345
327
282
321
293
297
311

414
395
351
389
362
366
378

483
465
420
459
432
436
448

LLFNLIKNRRDYVIPEFSSS
LFFLLKSSRNRIFPEFQAT
LLFFMLKNSSNRVIPQFETT
PFLLSSRRDHVIPEFHET
FSQDILKRGSKTWVPTLLKE
FSLDILKRSSKTWVPTLVKE
FAMETLKRSSKVWLQELRSN

* t i Q Q

 
  
 
  
  
  
 

  
  
   
 
 
   
  
   
  
 
 
  
  
  
 

FTDLCEVVEYDLATKGKAAFNDPAEQ"
YSELFDSLEKELSLKGKADFGGSSDGT‘E
YTELFEGLEAELAKNGKAAFNDVGEQ"

YTELFETLDKEMEEKGTVGFNSGSDQ"r
LDTMFTTFEADLSKSNTASLLPALQKF *
LDTLFGTFESDLSKSKSASLLPALQKF L
LNIFWGTIESEISKNGAASYIFPLQRCI*

SRAFFGVKP-IDTPLGKDAPSLIS
‘FYGTNP-ADTKLKADAPGLIT
RAYFNSNP-EETKLGTSAPTLIS
‘RSLFGVNP-VETKLGTDGPALIG

 

    
  
   
 

LP-KEVEEAT
LP-RVIEEPLI
LP-WFLQEPL
LP-KFLDDVL

 
   
  

YFQSVATPVMEQAE-KLGVPCl.
FFYTNSANLFIEAE-KLGISCDHX
QFVKNEAKEVLSRAQTEFQLTEQ‘L
IKFVKSEAKEVLTRAQTDFQLTE!

LKWIGVA-GENLHTQ
LKSIAKA—GVEIHTR

;_‘_ a e a a e

 

.,;ge5: ‘ -- ; ~ ' SSLGASITLTSLKRSTF
fifyfiégfih L 1 ' ' t “ SPLGSSVNFSSLRKASF
NHQCJQKU ' SPLGAAVTLTFLKRASI
YTI‘A KL “ SALGSSITITSLKKA--
-------- SLTSVKKAS-
-------- SLTSVKKAC-
-------- SIKAVVKAK-
-‘-“-- .

“WTITGDS

151

displayed features of a typical chloroplast targeting peptide including an enrichment of
hydroxylated amino acids. Conclusive evidence that LeA OS is localized to the
chloroplast was recently obtained (F roehlich et al., 1999). These findings indicate that
LeAOS is more similar to AOS from flax and Arabidopsis, which also reside in the
chloroplast (Song et al., 1993; Harms et al., 1995; Laudert et al., 1996), than it is to the
cytosolic AOS from guayule (Pan et al., 1995).

Values on the upper right diagonal of the matrix indicate the percentage of
amino acid identity between different family members. Values on the lower left of the
matrix indicate the percentage of nucleotide identity. Percentage identity within the
open reading frame of each pair of sequences was calculated using DNA Star software
(Clustal method). AOS sequences were from flax (Linum usitatissimum), guayule
(Parthenium argentatum), Arabidopsis, and tomato. HPL sequences were from pepper,
Arabidopsis, and tomato. GenBank accession nos. for the sequences are given in Figure
6-2.

A cDNA encoding HPL from bell pepper (Capsicum annum) was used to screen
a tomato cDNA library for related sequences. Among the 15 positive clones identified,
the longest cDNA (designated LeHPL) contained a 1,431-bp open reading frame that
was predicted to specify a 476-amino acid protein with a molecular mass of 53,542 D.
LeHPL contained a 169-bp 5'-UTR, and a 210-bp 3'-UTR excluding poly-(A) residues.
The presence of an in-frame stop codon (U GA) 93 nucleotides upstream of the putative
initiator AUG codon indicated that LeHPL encoded the entire protein. This was
confirmed by DNA sequence analysis of RACE products derived from the 5' end of

LeHPL transcripts (‘Materials and Methods’). The deduced amino acid sequence of

152

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

LuA OS AtAOS PaA OS LeA OS LeHPL CaHPL AtHPL

LuAOS 59.9 65.0 61.1 36.1 36.0 34.9
AtAOS 55.7 60.5 62.2 38.2 37.2 36.3
PaAOS 56.2 55.6 60.1 36.5 36.1 34.8
LeAOS 53.1 57.5 55.9 35.6 35.3 35.9
LeHPL 36.3 38.7 38.0 36.0 87.6 55.3
CaHPL 36.3 38.5 38.7 36.5 85.5 53.2
AtHPL 37.8 38.9 36.6 36.3 51.2 49.1

 

Table 6—1. Percent amino acid and nucleotide identity between different AOSs

and HPLs. Values on the upper right diagonal of the matrix indicate the

percentage of amino acid identity between different family members. Values
on the lower left of the matrix indicate the percentage of nucleotide identity.
Percentage identity within the open reading frame of each pair of sequences
was calculated using DNA Star software (Clustal method). AOS sequences

were from flax (Linum usitatissimum), guayule (Parthenium argentatum),

Arabidopsis, and tomato. HPL sequences were from pepper, Arabidopsis,
and tomato. GenBank accession nos. for the sequences are given in Figure 6-

2.

153

 

LeHPL was 88% and 55% identical to the published sequence of HPL from bell pepper
(Matsui et al., 1996) and Arabidopsis (Bate et al., 1998), respectively (Table 6-1). This
establishes LeHPL as a new member of the CYP74B subfamily of Cyt P4508. Unlike
HPL from Arabidopsis (Bate et al., 1998), LeHPL does not appear to contain a typical
chloroplast targeting sequence at the N terminus of the protein (Figure 6-2).
Comparisons between the primary structures of the seven known CYP74
members (four AOSs and three HPLs) showed 182 positions (38%) that were conserved
in all members of both subfamilies (Figure 6-2). Many conserved residues were
clustered at the N- and C-termini, and may be important for functions common to HPL
and AOS (e. g. heme or substrate binding). We also noted subfamily-specific amino acid
differences that might play a role in distinguishing AOS function fiom that of HPL.
Specifically, there were 39 positions at which an amino acid was invariant among all
AOSs, and was substituted to a different residue in all HPLS (Figure 6-2). One HPL-
specific motif was PPxF P, which represents a variation of the N-terminal PPGP
tetrapeptide that is important for stability and catalysis in many P4508 (Szczesna-
Skorpa et al., 1993). A hallmark of all CYP74 enzymes, including LeAOS and LeHPL,
is a T—)(V/I) substitution within the I helix that, in most P4508, participates in 0;
binding (Song et al., 1993). Sequences surrounding this site show a subfamily-specific
character, with the AOS and HPL consensus sequences being KI(L/F)F and (S/T)IFL,
respectively. Several other subfamily-specific signatures were located near the C-
terrninal heme-binding domain. The most striking of these was an eight-amino acid
insertion in AOS sequences relative to HPL sequences, at the extreme C-terrninal end of

the protein (Figure 6-2).

154

BEXBg BEXBg BEXBg

‘u h\ll" ' ‘

 

12.2 - X 11 . ‘.
1 M1 .
2‘ ‘ w“ “(in
7.1 - l
5.1 — G
”I“ ,
3.1 _ * v“
a " ‘
LeAOS LeHPL LeHPL 5’UTR

Figure 6-3. Southern-blot analysis of LeAOS and LeHPL. Genomic
DNA from tomato was digested with restriction enzymes BamHI (B),
EcoRV (E), XbaI (X), or BglII (Bg). DNA blots were hybridized to
labeled probes derived from the open reading frame of LeAOS (left),
LeHPL (middle), or the LeHPL 5'-UTR (right). Blots were hybridized
in 5x SSPE at 65°C and washed in 0.5)( SSPE at the same
temperature, as described in "Materials and Methods." Molecular
mass standards (in kb) are indicated on the left.

155

Genomic DNA-blot analysis using the LeA 0S cDNA as a probe showed a
simple hybridization pattern for each of the restriction enzymes tested (Figure 6-3). This
result indicates that LeA OS is derived from a single copy gene. However, detection of
additional hybridizing bands (data not shown) under conditions of reduced stringency
leaves open the possibility of related sequences in the genome. The results of
hybridization analysis using a LeHPL cDNA probe showed a more complex pattern of
weakly and strongly hybridizing bands (Figure 6-3). Use of a probe derived from the 5'-
UTR of LeHPL reduced the complexity of the hybridization pattern as expected, but
nevertheless still detected two or more bands for each restriction digest tested (Figure 6-
3, right panel). These results indicate that LeHPL is one member of a family of highly

related genes that may be clustered as tandem repeats in one region of the genome.

Functional expression of LeAOS and LeHPL in E. coli

To confirm that LeA 0S and LeHPL encode the expected P450 enzymes, the cDNAs
were subcloned into the pQE-30 expression vector to yield pQE-AOS and pQE-HPL,
respectively, and transformed into an appropriate E. coli host. Bacterial cultures induced
to express the constructs accumulated high levels of the recombinant proteins as
determined by SDS-PAGE of bacterial lysates (data not shown). Crude lysates from
cells expressing either pQE-AOS or pQE-HPL efficiently degraded l3-HPOT (Figure
6-4A) but did not metabolize 9-hydroperoxide derivatives of linolenic or linoleic acid
(data not shown). Recombinant LeAOS and LeHPL metabolized the C20 hydroperoxide
15S-hydroperoxy-11(Z),l3(E),l7(Z)-eicosatrienoic acid at a rate comparable to that

observed with 13-HPOT (Figure 6-4A), indicating that both enzymes can accommodate

156

>
m

 

 

— 13-HPOD — 134-WOO
5 15 « m 13-HP0T .5 15 . n— 1341901
3 _ 15”,“ {g- — 15-HPET
o
a 12‘ '5. 12‘
0’ a:
g 9 . g 9 .
g .5
S 6 . g 6 .
3 3 . g 3 .

0 0 _

 

 

 

 

 

 

 

Figure 6-4. Activity of LeAOS and LeHPL expressed in E. coli. Total lysates
of E. coli cells expressing pQE-AOS, pQE-HPL, or the empty vector (pQE-
30) were tested for their ability to metabolize C13 (l3-HPOD and l3-HPOT)
and C20 (15-HPET) fatty acid hydroperoxides. Activity was measured either
directly as a decrease in absorbance of the substrate at A 234 (A) or indirectly
as the production of aldehydes using a NADH-coupled assay (B). Error bars
represent the mean and SD of activity determined from three enzyme
preparations of each culture.

157

a range of fatty acid hydroperoxide substrates. However, the two enzymes differed in
their ability to metabolize 13S-hydroperoxy-9(Z), ll(E)-octadecadienoic acid (13-
HPOD), a common C13 hydroperoxide derived from linoleic acid. Whereas LeAOS
utilized l3-HPOD at about one-half the rate observed with 13-HPOT, the rate of
breakdown of 13-HPOD by LeHPL was less than 5% of that observed for 13-HPOT.
Similar results were obtained using a coupled enzyme assay (Vick, 1991) to measure
aldehyde production in the in vitro reactions. As expected from the known products of
AOS and HPL (Figure 6-1), aldehyde production was associated with reactions
catalyzed by LeHPL but not by LeAOS (Figure 6-4B). This assay also confirmed the
strong preference of LeHPL for l3-HPOT over 13-HPOD.

Gas chromatography-mass spectometry (GC-MS) was used to identify the
trimethylsilyl (TMS) derivatives of metabolites produced upon incubation of 13-HPOT
with lysates from bacteria that expressed either pQE-AOS, pQE-HPL, or the pQE-30
mock control. In the case of pQE-AOS, three prominent peaks (A, B, and C) that were
not present among the products of the mock reaction were observed. The relative
abundance of these compounds, as estimated by integration of the GC peak areas, was
22% (peak A), 100% (peak B), and 13% (peak C) (values normalized to peak B). The
retention time (11 min 9 8), molecular ion ([M]+' at m/z 364), and fragmentation pattern
of peak A were identical to that of an authentic 12-oxo-phytodienoic (12-OPDA)
standard (Cayman Chemical, Ann Arbor, MI). Peak B eluted at 12 min 37 8 and gave
the following mass spectrum as m/z (% relative intensity, ion structure): 526 (18%,
[M]+'), 511 (13%, [M - CH3]+'), 457 (100%, [M - C5H9]"), 367 (8%), 221 (5%), 179

(4%), 147 (12%), and 73 (28%, TMS+). This fragmentation pattern was consistent with

158

identification of the compound as the tri-TMS derivative of the a-ketol compound 12-
oxo-l3-hydroxy-9(Z), 15(Z)-octadecadienoic acid (enolization of the 12-oxo group
provided an additional hydroxyl for derivatization). The major fragment at m/z = 457
([M - C5H9]+') indicated that this compound represented the a-ketol rather than the y-
ketol (12-oxo-9-hydroxy-10[E],15(Z)-octadecadienoic acid). Peak C eluted with a
retention time of 13 min 10 s and produced a mass spectrum identical to that of peak B,
indicating probable double bond isomerization during derivatization or GC analysis. a-
Ketol and 12-OPDA, together with minor amounts of 'y-ketol, are known to arise by
spontaneous hydrolysis of the unstable epoxide product of AOS (Song and Brash, 1991)
(Figure 6-1). The products of the pQE-HPL-catalyzed reaction were analyzed by GC-
MS as the oxime, TMS derivatives. The major product eluted with a retention time of 8
min 59 s. A molecular ion, m/z 371 [M‘], was observed for the di-TMS derivative of
this product, and the fragmentation pattern was identical to that of an authentic 12-oxo-
trans-lO-dodecenoic acid standard, the expected product of HPL (Figure 6-1). These
results confirmed the identity of LeAOS and LeHPL as fimctional members of the

CYP74A and CYP74B subfamilies of P450 enzymes, respectively.

Developmental expression of LeAOS and LeHPL

RNA-blot analysis was used to investigate the distribution of LeHPL and LeA 0S
mRNA in different organs of tomato (Figure 6-5). LeHPL transcripts accumulated to
high levels in developing flowers, and decreased during flower maturation. LeHPL
mRNA levels were also relatively high in leaf tissue, with greater expression detected in

younger leaves compared to older leaves from the same plant (see Figure 6-7). Very

159

RSLBUFOFIFGFRF

 

LeA OS ”4 u ' MP6 119-In u. . ..

LeHPL 1 . .9 W

LeHPL

 

 

 

 

 

 

eIF4A _ " .

 

 

 

 

Figure 6-5. Expression of LeAOS and LeHPL genes in different
organs of tomato. Total RNA was extracted from roots (R), stems
(S), leaves (L), developing flower buds (B), mature unopened flowers
(UF), mature opened flowers (OF), small (<0.5 cm) immature green
fruit (IF), mature green fruit (GF), or mature red fruit (RF). Ten-
microgram samples of RNA were subjected to RNA-blot analysis.
Specific transcripts were detected by hybridization of blots to probes
corresponding to full-length LeAOS, full-length LeHPL, the 5'-UTR
of LeHPL, or an eIF4A probe used as a loading control. Also shown is
a photograph of an ethidium bromide-stained gel of the RNA used
for the experiment (EtBr).

160

low levels of LeHPL mRNA were detected in stems and immature green fi'uit, whereas
roots and mature green and red fruit lacked detectable transcripts. Hybridization probes
derived from either the full-length LeHPL cDNA or the LeHPL 5'-UTR showed a
similar organ-specific expression pattern (Figure 6-5). This result showed that LeHPL
transcripts detected by RNA-blot analysis are derived from a single LeHPL gene, or
highly related LeHPL genes that have a similar developmental expression pattern.

LeA 0S mRNA was broadly distributed among all organs examined (Figure 6-5).
LeA 0S transcript levels were relatively low in fruit, and appeared to decrease during
fruit development. Polyclonal antibodies raised against recombinant LeAOS, but not
preimmune serum fi'om the same rabbit, reacted with a polypeptide in the membrane
fraction of extracts prepared from different organs (Figure 6-6). The estimated mass of
the cross-reacting polypeptide as judged by SDS-PAGE was 55 kD, which is consistent
with that expected for the LeA 0S gene product. Furthermore, the distribution of this
polypeptide in different organs correlated with the distribution of LeA 0S mRN A. Taken
together, these results indicate that LeA OS is expressed in all tomato organs with the
possible exception of ripe fruit. A second cross-reacting polypeptide of slightly lower
Mr was often observed in immunoblot experiments, particularly in extracts derived from
flowers (Figure 6-6). This band could represent a polypeptide that shares common

epitopes with LeA OS, or a post-translationally modified form of LeA OS.

161

Wound-inducible expression of LeAOS is mediated by a Defl-independent

signaling pathway

The importance of oxylipin metabolism for wound-inducible defense gene expression in
tomato prompted us to examine the effect of wounding on LeAOS and LeHPL gene
expression. Damage inflicted to tomato leaves by Manduca sexta larvae resulted in a
modest (approximately 2-fold) increase in LeHPL mRNA accumulation (Figure 6-7).
Wound-induced accumulation of LeHPL mRN A was more apparent in the lower
damaged leaf than it was in the younger undamaged leaf. This is likely to reflect the
higher constitutive expression of LeHPL in younger leaves. Wound-induced
accumulation of LeA 0S transcripts was much more apparent, and thus became the focus
of additional experiments. The time course and amplitude of LeA 0S expression differed
in several ways from that of the well-characterized proteinase inhibitor II (Inh-II) gene
(Figure 6-7). Whereas the maximum level of induction of LeA OS in local and systemic
leaves was approximately 9- and 5-fold, respectively, Inh-II mRNA levels in these
tissues increased by at least 60-fold. Wound-inducible accumulation of LeA 0S mRNA
was also more transient than that of Inh-II. These results indicated that the mechanism
controlling wound-inducible expression of LeA 0S might be different from that

regulating Inh-II gene expression.

162

BRSPCL BRSPCL

 

81.0 -

51.2 -

 

 

 

 

33.6 -

 

LeAOS Immune pre-lmmune

Figure 6-6. Accumulation of LeAOS protein in different organs of
tomato. Fifteen-microgram samples of membrane protein prepared
from young flower buds (B), roots (R), stems (S), petioles (P),
cotyledons (C), and leaves (L) were separated by SDS-PAGE. Protein
was transferred to Immobilon-P membranes and probed with either
antiserum raised against LeAOS (left) or an equivalent amount of
preimmune serum (right). The numbers on the left of the figure
indicate the position of Mr standards.

163

Local Systemic
000.5124 81224 C00.5124 81224

Inh-II '0!!!

LeAOS o g - ..-

 

 

 

 

 

 

LeHPL .. f a etc-C...-

 

 

 

 

 

eIF4A --D--.--- vac-D----

 

Figure 6-7. Accumulation of LeAOS, LeHPL, and Inh-II mRNAs in
tomato plants in response to herbivory. Tobacco hornworm larvae
(third instar) were placed onto the lower leaf of 3-week-old cv
Micro-Tom plants and allowed to feed for 5 to 10 min. During this
period, approximately 5% to 10% of the area of the attacked leaf
was consumed by the larvae. Leaf tissue was harvested for
extraction of total RNA immediately after removal of the larvae

(0 point) or at the times indicated (in hr). RNA was prepared
separately from the lower damaged leaf (Local response) and from
the third leaf (counted from the base of the plant) (Systemic
response). RNA was also prepared from a set of control plants that
received no damage (C). Duplicate RNA blots containing 5 pg of
RNA per sample were hybridized to cDNA probes for proteinase
inhibitor 11 (Inh-II), LeAOS, LeHPL, and eIF4A as a loading control.

164

wild-type def1
Hours: 012 4 81224 012 4 81224

Inh-Il a Q Q. .

 

 

 

 

 

 

 

 

 

30’ n a...
AOS "fl““fi‘ «wise-911,11

v ‘ " r .
LE .. s?" “‘6'?“ fl “ 11.. ' 1 .. 1‘ '1 m ”a. ..W

 

 

 

 

eIF4A fi'fiflflfifiwflflflfl Ii i.
M" ..-

 

Figure 6-8. Analysis of wound-induced gene expression in wild-
type and def] mutant plants. F ifteen-day-old wild-type (cv
Castlemart) and def? mutant seedlings were mechanically wounded at
the distal end of the terminal leaflet of the lower leaf. Undamaged
tissue on the same leaflet was harvested for RNA extraction at the
indicated times after wounding. RNA blots were hybridized to cDNA
probes for proteinase inhibitor [1 (Inh-Il), cathepsin D inhibitor (CD1),
T ornLoxD (LoxD), LeA OS (A US), LE RNase (LE), and eIF 4A as a
loading control.

165

To further test this idea, we examined LeA OS expression in the tomato
defenseless] (defI) mutant that is deficient in the octadecanoid-based signaling pathway
that mediates the expression of Inh-II and other defense-related genes (Howe et al.,
1996). Previous characterization of def] showed that it is deficient in J A accumulation
in response to wounding and other elicitors (Howe et al., 1996). Genetic mapping
studies have shown that the def] phenotype does not result fiom a defect in the LeA OS
gene (A. Itoh and G.A. Howe, unpublished data). Moreover, direct measurements of 12-
OPDA levels in def] and wild-type plants indicated that the mutant has both AOS and
allene oxide cyclase activity (Stelmach, E. Weiler, G.A. Howe, unpublished data).
Taken together, the available evidence indicates that the Def] gene product plays a role
in the regulation of a late step in the biosynthesis of JA, or in the further metabolism of
J A (e. g. transport or stability). A dramatic aspect of the def] phenotype is the lack of
wound-induced accumulation of defensive proteinase inhibitor genes such as Inh-II and
cathepsin D inhibitor (CD1) (Figure 6-8). In contrast to this, the pattern of wound-
inducible LeA OS mRNA accumulation in defl plants was comparable to that in wild-
type plants. This effect was not specific for LeAOS, as other transcripts, including those
for lipoxygenase (LoxD) (Heitz et al., 1997) and a senescence-induced RNase (LE)
(Lers et al., 1998), were also induced by wounding in both mutant and wild-type plants.
These results demonstrate that wound-induced expression of LeA OS, LoxD, and LE

mRNA is Def] independent.

166

Discussion

Fatty acid hydroperoxides derived from lipoxygenase are precursors for an array of
oxylipins that function in diverse aspects of plant growth and development. In this paper
we report the isolation and characterization of tomato cDNAs encoding AOS and HPL,
two similar P450 enzymes that commit 13-HPOT to different branches of oxylipin
metabolism. The LeA OS and LeHPL proteins are 36% identical at the amino acid level,
and are classified as members of the CYP74A and CYP74B subfamilies of Cyt P4508,
respectively. Identification of AOS and HPL genes in tomato brings the total number of
reported AOS and HPL sequences to seven. In comparing the primary structures of
these, we noted 39 positions at which all AOS members contained one common amino
acid and all HPLS contained a different residue. The significance of these subfamily-
specific substitutions will become more or less apparent as additional CYP74 genes are
identified. Subfamily-specific substitutions might reflect differences in the catalytic
properties or substrate specificity of the two classes of enzymes. The facile expression
of recombinant forms of AOS and HPL in E. coli should facilitate studies aimed at
understanding the structure-function relationship that defines the catalytic identity of
these unusual P4508.

A major difference between the predicted amino acid sequence of LeA OS and
LeHPL was the presence of a typical chloroplast targeting sequence at the N-terrninus
of LeAOS (Figure 6-2). Previous studies indicate that chloroplast targeting peptides are
present on AOS from flax and Arabidopsis (Song et al., 1993; Harms et al., 1995;
Laudert et al., 1996), as well as HPL from Arabidopsis (Bate et al., 1998). A plastid

location for AOS and HPL is consistent with biochemical studies demonstrating that

167

AOS and HPL activity is associated with chloroplasts (Vick and Zimmerman, 1987;
Gardner et al., 1991; Blée and J oyard, 1996; Zhuang et al., 1996). Recently, we have
shown that LeAOS is imported into chloroplasts where it specifically targets to the
inner membrane of the chloroplast envelope (Froehlich et al., 1999). This finding
indicates that LeAOS obtains its hydroperoxide substrates from one or both of the
plastid-localized lipoxygenases (TomLoxC and TomLoxD) that have been described in
tomato (Heitz et al., 1997). In contrast to LeAOS, the deduced N-terminus of LeHPL
lacked a typical transit peptide. That the N—terminal sequence of LeHPL is very similar
to that of bell pepper HPL (Figure 6-2) indicates that these proteins share a similar
subcellular location. Given the preponderance of evidence indicating that HPL activity
is associated with the chloroplast, additional experiments aimed at determining the

subcellular location of LeHPL are clearly warranted.

Characterization of LeAOS

Expression of LeA OS in E. coli showed that the open reading frame encodes an
authentic CYP74A enzyme (LeAOS) that metabolizes 13- but not 9-hydroperoxides of
linoleic and linolenic acids. LeAOS expression was detected in all organs of the plant
except mature red fruit. Similar expression patterns were observed for AOS in
Arabidopsis and flax (Harms et al., 1998; Laudert and Weiler, 1998). Accumulation of
LeAOS mRNA and protein in flowers is consistent with previous studies in Arabidopsis
showing that AOS promoter activity is high in flowers, particularly in pollen sacs and
pollen grains (Kubigsteltig et al., 1999). It is presently not known whether AOS-derived

products are required for pollen development in tomato, as they are in Arabidopsis

168

(McConn and Browse, 1996). Detection of LeA OS mRNA and protein in leaves
supports previous reports of AOS activity (Caldelari and Farmer, 1997) and inducible

J A synthesis in tomato leaves (Pena-Cortes et al., 1993; Doares et al., 1995; Conconi et
al., 1996). The relatively high accumulation of LeAOS mRNA in stems (Figure 6-5)
compared to that in leaves indicates that LeAOS is expressed in vascular bundles, as was
reported to be the case in wounded leaves of Arabidopsis (Kubigsteltig et al., 1999).
LeA OS mRN A and protein were also detected in tomato roots. Given that root
development appears normal in J A-deficient mutants of Arabidopsis (McConn and
Browse, 1996), this result indicates that AOS-derived oxylipins serve a non-
developmental role in roots. LeA OS mRNA expression in green fi'uit, while being
relatively low, is consistent with previous studies showing increased levels of cis-JA
during the early stages of tomato fruit ripening (Fan et al., 1998). However, our results
do not exclude the possibility that J A synthesis in tomato fruit involves a different

AOS-encoding gene that is undetectable by high stringency nucleic acid hybridization.

Characterization of LeHPL

Expression of LeHPL in E. coli confirmed that this cDNA encodes a functional member
of the CYP74B subfamily of enzymes. LeHPL was similar to LeAOS in its ability to
use 13- but not 9-hydroperoxides of C18 fatty acids. However, LeHPL was clearly
distinguishable from LeAOS in its strong preference for l3-HPOT over 13-HPOD. This
feature is shared by HPL isolated from other sources, including bell pepper (Shibata et
al., 1995b), tea leaves (Matsui et al., 1991), and Arabidopsis (Bate et al., 1998).

F auconnier et al. (1997) reported the purification from tomato leaves of an HPL that,

169

like recombinant LeHPL, did not utilize 9-hydroperoxides and showed a strong
preference for l3-HPOT over l3-HPOD. However, the purified enzyme displayed a
molecular mass (73 kD) much greater than that predicted for LeHPL (53.5 kD).
Additional experiments are needed to clarify the relationship between LeHPL and this
purified form of tomato leaf HPL.

LeHPL mRNA was most abundant in developing flowers. This finding indicates
that HPL-derived products might have a role in the production of floral scent. Relatively
high levels of LeHPL mRNA were also detected in leaves. The overlapping expression
pattern of LeA OS and LeHPL in leaves is consistent with the idea that these two
enzymes compete for the same pool of substrate (Avdiushko et al., 1995; Blée and
Joyard, 1996; Blée, 1998). However, additional studies aimed at determining the
subcellular and tissue-specific location of both enzymes are needed to substantiate this
hypothesis. The paucity of LeHPL mRNA accumulation in mature green and red fruit
was surprising since cis-3-hexenal, derived from the action of HPL on l3-HPOT, is a
prominent volatile component of the aroma and flavor of tomato fruit (Buttery and
Ling, 1993). A possible explanation for these results is that LeHPL plays only a minor,
if any, role in the production of C6 volatiles during tomato fruit ripening. Additional
insight into the contribution of LeHPL to fruit aroma and flavor might be gained by

altering LeHPL expression in transgenic plants.

170

Wound-inducible expression of LeAOS is mediated by a Defl-independent
signaling pathway
Damage inflicted to tomato leaves by hornworm larvae triggered the accumulation of
LeA OS mRNA both in the damaged leaf and in the upper undamaged leaves of the plant
(Figure 6-7). Similar changes in LeA OS expression occurred in plants subjected to
mechanical wounding (Figure 6-8; data not shown). The transient accumulation of
LeA OS transcripts in these experiments was a consequence of the limited damage
inflicted to the plant (e. g. 5-10 min of feeding by the insect). It is likely that sustained
feeding by herbivores, such as that occurring in natural and agricultural ecosystems,
would result in much greater increases in LeAOS expression. Increased expression of
AOS, and possibly other octadecanoid pathway enzymes, could serve to amplify the J A
signaling cascade a8 a means of enhancing the induced resistance response. Wound-
inducible increases in AOS mRNA, protein, and activity have been documented in
Arabidopsis (Laudert and Weiler, 1998; Kubigsteltig et al., 1999) and flax (Harms et al.,
1998). LeHPL transcript levels also appeared to increase in response to insect attack
(Figure 6-7) and mechanical wounding (data not shown), but only by about 2- fold
relative to unwounded controls. Additional studies are needed to determine whether the
expression of LeHPL and LeA OS in tomato leaves is affected by other defense signals,
or by interactions with pathogens.

The wound-inducible expression pattern of LeA OS differed in several respects
from that of proteinase inhibitor genes. First, the time course of LeA OS mRNA
accumulation was more transient than that of the inhibitor genes. Second, the amplitude

of LeA OS mRNA accumulation in both damaged and systemic leaves was 10- to 20-

171

fold lower than that of Inh-II mRNAs. Finally, wound-inducible expression of LeAOS
was observed in the def] mutant, while that of the Inh-II and CD] genes was not. Two
additional genes, LoxD and LE, were also wound inducible in the def] background.
These results demonstrate the existence of two classes of genes whose requirements for
wound induction in tomato can be defined as being either Def] -dependent or Def] -
independent. Given the involvement of Def] in wound-inducible J A accumulation
(Howe et al., 1996), we indicate that endogenous J A is a signal for Def] -dependent, but
not Def] -independent, wound responses. It is noteworthy that some genes exhibiting
Def] -independent expression, such as LeA OS and LoxD, are inducible by exogenous J A
(Heitz et al., 1997; G.I. Lee and G.A. Howe, unpublished data). This raises the
possibility that genes whose expression is altered by exogenous J A might not be under
the control of the J A signaling pathway as it operates in planta. Alternatively, LeAOS
and LoxD may be controlled by both J A-dependent and -independent wound-response
pathways. This interpretation is consistent with the observation that wound-induced
accumulation of LeA OS and LoxD mRNA in def] plants was slightly less than that in
wild-type plants (Figure 6-8). Regulation of AOS and LOX activities by both J A-
dependent and -independent signaling pathways might allow amplification or increased
sensitization of wound responsiveness under different conditions. This notion is
consistent with other studies showing that wound and defense responses in tomato
involve multiple signaling pathways (Chao et al., 1999; Ryan, 2000). In Arabidopsis,

J A-dependent and -independent wound responses have been shown to be differentially
regulated by Ca2+/calmodulin, as well as by reversible protein phosphorylation events

(Titarenko et al., 1997; Leon et al., 1998; Rojo et al., 1998). Thorough analysis of

172

mutants such as def] or those that are suppressed in the action of systemin (Howe and
Ryan, 1999) may provide further insight into the role of oxylipins in wound and defense

signaling pathways in tomato.

Materials and Methods

Plant Material and Growth Conditions

cv Micro-Tom seed (Lycopersicon esculentum cv Micro-Tom) was obtained from Dr.
Avraham Levy (Weizmann Institute, Rehovot, Israel). Seed for the tomato def] mutant
was collected from a defl/def] homozygous line that had been back-crossed four times
to L. esculentum cv Castlemart, the wild-type parent of def]. Seedlings were grown in
Jiffy peat pots (Hummert International, St. Louis) in a growth chamber maintained
under 17 hr of light (300 .115 m2 s") at 28°C and 7 hr ofdark at 18°C. Flowers and fruit

were collected from plants maintained in a greenhouse.

cDNA Cloning and Sequencing

A 1.1-kb A7101 fragment derived from the coding region of an Arabidopsis AOS cDNA
(Laudert et al., 1996) was labeled with [or-32P]dCTP and used to screen a tomato cDNA
library constructed from tomato plants that overexpress the prosystemin gene, as
described by Heitz et al. (1997). Duplicate filters were hybridized at 42°C in a solution
containing 5X SSPE, 50% (v/v) forrnamide, 5x Denhardt's reagent, 0.5% (w/v) SDS,
and 50 11 g/mL denatured salmon sperm DNA. Filters were washed at 42°C in a solution

containing 5X SSPE and 0.5% (w/v) SDS, followed by an additional wash at 65°C. Four

173

positive clones were obtained among approximately 4 x 105 plaque-forming units
screened. Following excision of the cDNA from the phagrnid, DNA sequence analysis
showed that all clones were identical with the exception of minor differences in the
length of the 5' end. The longest cDNA insert, designated LeAOS, was sequenced
completely on both strands using a primer walking approach. The sequence of the
LeAOS cDNA was deposited to GenBank (accession no. AF 23037 1). A bell pepper
(Capsicum annum) cDNA encoding HPL was isolated using a reverse-transcription
PCR (RT-PCR) kit (Life Technologies/Gibco-BRL, Cleveland) and total RNA isolated
from bell pepper as a template. Two gene-specific primers were designed from the
cDNA sequence reported by Matsui et al. (1996) (GenBank accession no. U51674). The
sequence of the forward and reverse primers used for RT-PCR was 5'-(GTG-GAT-
CCA-TTC-ATA-AAA-CAA-CAA-CTA-C)-3' and 5'-(GTG-AAT-TCA-GCA-ACC-
TTT-AGT-ACC-TAC-C)-3', respectively. An amplified 1,477-bp product was
subcloned into pBluescript SK(-) (Stratagene, La J olla, CA) and sequenced to confirm
its identity to the published sequence (Matsui et al., 1996). This clone was used to
screen a tomato leaf cDNA library as described above but with the following
modifications. Filters were washed at 65°C in a solution containing 5X SSPE and 0.5%
(w/v) SDS. Fifteen positive plaques were identified among 3 X 105 plaque-forming units
screened. DNA sequence analysis of eight cDNA inserts showed that all cDNAs were
identical except for minor differences in the length of the 5' end and the number of poly-
(A) residues at the 3' end. The longest clone, designated LeHPL, was subcloned into
smaller fragments and sequenced in its entirety on both strands. The sequence of the

LeHPL cDNA was deposited to GenBank (accession no. AF 230372). A RACE

174

procedure (Life Technologies/Gibco-BRL, Cleveland) was used to obtain additional
sequence information at the 5' end of LeHPL. First strand cDNA was synthesized from
total RNA prepared from either tomato leaves or flowers as a template. The sequence of
the gene-specific primer used for this reaction was 5'-(ACT-TCC-TTG-GCT-TCA-
TTT-T)-3'. PCR amplification of the dC-tailed cDNA was performed using the
manufacturer's abridged anchor primer and a gene-specific primer having the sequence
5'-(AGC-GCC-GAG-GAT-AGT-GAG-GGA-GAA)-3'. PCR products were re-
amplified using the manufacturer's abridged universal amplification primer and a nested
gene-specific primer having the sequence: 5'-(TGG-AGT-GCA-GGA-AGA-AGA-
GAA-G)-3'. Amplified PCR products were subcloned into pBluescript SK(-). DNA
sequencing of 5' RACE products derived from both leaf and flower mRNA confirmed
the structure of the 5'-UTR of LeHPL, including the presence of the in-frame stop codon

upstream of the initiator Met.

Expression of LeA 0S and LeHPL in Escherichia coli

A PCR strategy was employed to subclone the LeA OS cDNA into the E. coli expression
vector pQE-30 (Qiagen USA, Valencia, CA). Forward and reverse primers were
designed to contain BglII and Pstl restriction sites, respectively. The sequence of the
forward primer was 5'-(GCT-AGA-TCT-CCT-ATA-AAA-TTA-TCT-ACC-AGG)-3'
and that of the reverse primer 5'-(GTT-CTG—CAG-CCG-ATA-GTG-ACA-GTG-TAG-
ACC)-3'. Using the LeA OS cDNA as a template, the PCR-amplified product was cut
with Bng and Pstl and cloned into BamHI and Pstl sites of pQE-30. The resulting

expression vector was called pQE-AOS. This strategy removed the first 42 amino acids

175

from the N terminus of LeA OS, and added the sequence MRGSHHHHHHGS to Pro 43
of LeA OS (Figure 6-2). A similar strategy was used to construct a vector for expression
of LeHPL. Forward and reverse primers were designed to contain BamHI and SstI sites,
respectively. The sequence of the forward primer was 5'-(CGG-GAT-CCC-CGA-TAA-
TGA-ATT-CTG-CTC)-3' and that of the reverse primer 5'-(GCG-AGC-TCT-CAT-
AAG-TCA-GAA-CAG)-3'. PCR products obtained using the LeHPL cDNA as a
template were digested with BamHI and SstI, and cloned into BamHI and SacI sites of
pQE-30 to give pQE-HPL. This strategy added the sequence MRGSHHI-II-II-IHGSPI to
the deduced initiator Met of LeHPL.

Expression constructs pQE-AOS and pQE-HPL were transformed into E. coli
strain M15. Bacteria grown under standard conditions (37°C in Luria-Bertani medium)
and induced with isopropylthio-fl-galactoside produced recombinant protein that was
associated with inclusion bodies (data not shown). Induction of cultures using the
following procedure significantly enhanced the recovery of active enzyme in the soluble
fi'action of lysed cells. Bacterial cultures (50 mL) were grown in Terrific Broth medium
at 37°C to logarithmic phase (A600 of 0.6), at which time the culture was induced by the
addition of isopropylthio-B-galactoside to a final concentration of 0.5 mM. Cultures
were incubated for an additional 8 hr at 26°C with gentle shaking (150 rpm). Bacteria
were harvested by centrifugation and resuspended in 5 mL of a solution containing 50
mM potassium phosphate (pH 7.5) and 5% (v/v) glycerol. Following one freeze-thaw
cycle, cells were broken by sonication and centrifuged at 10,000g for 20 min. SDS-

PAGE analysis of supernatant protein from induced culture extracts showed the

176

presence of the recombinant protein, migrating with the expected molecular mass (data

not shown).

Enzyme Assays and Preparation of Fatty Acid Hydroperoxides

The hydroperoxide-degrading activity of recombinant LeAOS and LeHPL was
measured spectrophotometrically using two methods described by Vick (1991). One
assay, which does not distinguish between AOS and HPL activity, involved monitoring
the decrease in A 2 34 that results from disruption of the conjugated diene bond in the
substrate. The second method was specific for HPL and involved an NADH-coupled
assay for detection of aldehyde reaction products. The protein content of cell extracts
was determined by the Bradford assay. F atty acid hydroperoxide substrates (9- and 13-
substituted) were prepared using soybean lipoxygenase (Sigma-Aldrich, St. Louis) or
com seed lipoxygenase as described (Vick, 1991). Fatty acids for these reactions were
obtained from Nu-Chek-Prep, Inc (Elysian, MN). Substrate specificity results were
confirmed using purified hydroperoxides (9-HPOD, 9-HPOT, 13-HPOD, 13-HPOT)

purchased fiom Cayman Chemical.

Identification of Metabolites

Two micromoles of 13-HPOT, dissolved in 30 mL of 50 mM potassium phosphate (pH
7.0), was mixed with 1 mg of soluble protein (enzyme source) obtained from E. coli
cells expressing either pQE-AOS, pQE-HPL, or the pQE-30 vector control. The
reaction was allowed to proceed for 10 min at room temperature and then stopped by

acidification to pH 4.0 with l M citrate. Products were extracted twice with diethyl

177

ether and dried under N2 gas. TMS derivatives of pQE-AOS reaction products were
prepared by treatment of the extract with 30 1.1L of BSTFA (bis[TMS]
trifluoroacetamide/trimethylchlorosilane) (99:1, v/v) (Supelco, Bellefonte, PA) and 10
11L of pyridine for 1 hr at 80°C. Oxirne TMS derivatives of pQE-HPL products were
prepared by first reacting enzyme products with hydroxylarnine hydrochloride at 80°C
for 1 hr, followed by treatment with BSTF A and pyridine as described above. One to 2
11L of the derivatized compounds was used for GC-MS analyses, which were carried out
on an AX 505H double focusing mass spectrometer (J EOL, Peabody, MA) equipped
with a 5890 gas chromatograph (Hewlett-Packard, Palo Alto, CA). GC separations
employed a DB-l methyl silicone capillary column (30 m X 0.25 mm id.) (J &W
Scientific, Folsom, CA) interfaced directly to the ion source via a heated transfer block.
The temperature program was initiated at 50°C and ramped to 225°C at 20°C min". The
temperature was then increased to 270°C at 2°C min". The ion source was operated at

70 eV with the scan rate of the instrument set to approximately 1 spectrum 8".

Antibody Production and Western-Blot Analysis

A 0.5-L culture of E. coli was induced for the expression of pQE-AOS as described
above, except that induced cells were grown at 37 °C for 4 hr in Luria-Bertani medium.
Cells were harvested by centrifugation and resuspended in 1/ 10 volume of lysis buffer
(50 mM sodium-phosphate, pH 8.0, 10 mM imidazole, 300 mM NaCl, and 0.25% [v/v]
Emulgen 911 [Kayo Corporation, Tokyo]). Following one freeze-thaw cycle, cells were
broken by two passes through a French press calibrated at 17,000 pounds per square

inch. Insoluble inclusion bodies containing LeAOS were recovered by centrifugation

178

for 20 min at 8,000g. The pellet was washed twice with 40 mL of lysis buffer and
recovered by centrifugation. Washed pellets were solubilized at 4°C in 35 mL of a
solution containing 6 M guanidine HCl, 100 mM sodium-phosphate, pH 8.0, and 10
mM Tris (tris [hydroxymethyl]aminomethane) HCl, pH 8.0. The mixture was sonicated
for 10 min at 4°C to facilitate solubilization, and then centrifuged at 12,000g for 20 min.
Recombinant LeAOS in the supernatant was purified by nickel affinity chromatography
as described by the manufacturer (Qiagen). His-tagged LeAOS, which eluted from the
nickel column at pH 4.5, was dialyzed twice for 4 hr against 100 volumes of 50 mM
Tris, and 0.2% (v/v) Emulgen 911. Approximately 0.5 mg of protein was solubilized in
Laemmli sample buffer and further purified by preparative SDS-PAGE (12% [w/v] gel).
Acrylarnide gel slices containing His-tagged LeAOS were stained with Coomassie
Brilliant Blue and macerated through a syringe as described (Harlowe and Lane, 1988).
For the initial immunization, 100 11g of antigen in the mashed gel slice was mixed with
F reund's complete adjuvant and injected at multiple subcutaneous and intramuscular
sites of a New Zealand white rabbit. Four boosts, each consisting of 50 ug of antigen
mixed with Freund's incomplete adjuvant, were administered over the course of a 90-d
immunization schedule.

Protein extracts for westem—blot analysis were prepared from fresh plant tissue
that was extracted with a mortar and pestle at 4°C in a buffer containing 50 mM
sodium-phosphate, pH 7.0. The buffer to tissue ratio (w/w) was about 2: 1. Crude
cellular debris was removed by centrifugation at 2,000g for 10 min. The membrane
fraction of the resulting supernatant was recovered by centrifugation at 100,000g for 15

min at 2°C. Pelleted membranes were washed with l M NaCl, and recovered by

179

centrifugation as described above. Membrane material equivalent to 15 ug of total
protein was solubilized in Laemmli sample buffer, boiled for 5 min, and separated by
SDS-PAGE (10% [w/v] gels). Separated proteins were electrophoretically transferred to
Immobilon-P membranes (Millipore, Bedford, MA) in a solution consisting of 25 mM
Tris, 192 mM Gly, and 20% (v/v) methanol, using standard procedures (Harlowe and
Lane, 1988). Membranes were probed with anti-LeAOS antibodies used at a 1:2,000
dilution in a Tris-buffered saline solution containing 1% (w/v) bovine serum albumin as
a blocking agent and 0.05% (w/v) Tween 20 to reduce non-specific binding. Anti gen-
antibody complexes were detected with the use of an alkaline phosphatase-conjugated
second antibody as described by the manufacturer (Kirkegaard and Perry, Gaithersburg,

MD).

Southern-Blot Analysis

Genomic DNA from young leaves of cv Micro-Tom plants was purified as described by
Rogers and Bendich (1985). Ten-micrograrn aliquots of DNA were digested with
restriction enzymes, electrophoresed on a 0.8% (w/v) agarose gel, and blotted to
Duralon-UV membranes (Stratagene) as indicated by the manufacturer. Blots were pre-
hybridized at 65°C in a solution containing 5)( SSPE, 5x Denhardt's solution, 100
ug/mL denatured salmon sperm DNA, and 0.5% (w/v) SDS. Blots were hybridized at
65°C and washed at the same temperature in a solution containing 0.5x SSPE and 0.5%
(w/v) SDS. DNA probes were prepared using a T7 Quickprime Kit (Pharmacia Biotech,
Piscataway, NJ). The following cDNA fragments were labeled for use as probes: a 1.7-

kb EcoRI-Xhol fragment containing full-length LeA OS; a 1.4-kb EcoRI-HindIH

180

fragment containing the coding region of LeHPL; and a 0.2-kb EcoRI-EcoRI fragment

containing the LeHPL 5'-UTR.

Wounding Experiments

Manducta sexta larvae were reared on artificial diet as described by the vendor
(Carolina Biological Supply, Burlington, NC) fiom which the eggs were purchased.
One larva (third instar) was placed on the terminal leaflet of the oldest leaf of a 3-week-
old cv Micro-Tom plant that contained three fully expanded leaves. Larvae were
allowed to feed on the leaf for 5 to 10 min, during which time 5% to 10% of the area of
the leaf was consumed. Plants were sampled for RNA analysis at different times after
the challenge. Leaf tissue from six to eight plants per time point was pooled prior to
RNA extraction. Mechanical wounding of plants was performed using a hemostat as

described previously (Howe et al., 1996).

RNA Gel Blot Analysis

Total RNA was isolated from tomato tissue and analyzed by RNA-blot hybridization as
previously described (Howe et al., 1996), except that Duralon-UV membranes were
used in place of nitrocellulose. All gels were run in duplicate, with one set stained with
ethidium bromide to ensure equal loading of the samples and intactness of the RNA.
Hybridization signals were visualized by autoradiography using Kodak XAR-S fihn, or
were measured using a Phosphorimager (Molecular Dynamics). These signals were
normalized to signals obtained using a probe for translation initiation factor eIF4A

mRN A (Taylor et al., 1993). Hybridization and subsequent washing of eIF4A-probed

181

blots was performed at 60°C in 2X SSPE. DNA probes were prepared as described

above.

182

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188

Chapter 7

Conclusions and future perspectives

189

The systemic wound response of tomato is an important defense mechanism against
insect herbivores. The discovery of systemin provided a breakthrough in our
understanding of how local injury induces systemic expression of defense genes such as
Proteinase Inhibitors (PIs; McGurl et al., 1992: Pearce et al., 1991). Manipulation of
prosystemin expression in transgenic plants demonstrated that this polypeptide signal is
an essential regulator of systemic PI expression (McGurl et al., 1992; Mch1 et al.,
1994). Subsequent studies showed that systemin induces PI expression by activating the
octadecanoid pathway for jasmonic acid (J A) biosynthesis (Doares et al., 1995; Howe et

al., 1996).

At the outset of this thesis research, a wealth of circumstantial evidence
indicated that systemin might function as a long-distance signal for PI expression (Ryan
and Pearce, 1998). To investigate this hypothesis, I characterized tomato mutants that
are defective in the systemic wound response. Systemin-insensitive spr1 plants were
previously isolated as suppressor of prosystemin-mediated responses (spr), and were
shown to be deficient in wound-induced systemic PI expression (Howe and Ryan,
1999). As described in Chapter 2, systemin failed to induce J A biosynthesis and
subsequent expression of PIs in spr1 plants (Figure 2-5; Figure 2-6). In contrast,
application of oligogalacturonic acid (OGA), which is regarded as a local wound signal,
resulted in normal PI expression in spr1 plants (Figure 2-3; Figure 2-4). These results
indicate that the spr1 mutation defines the systemin-dependent pathway of systemic
wound signaling, and further indicate that the Spr] gene product plays a role in
coupling systemin perception at the plasma membrane to the initiation of J A

biosynthesis in the chloroplast (Figure 7-1).

190

Reciprocal grafting experiments between wild-type and spr1 plants were used to
investigate how systemin regulates systemic PI expression (Figure 2-7). Whereas spr1
plants were impaired in the generation of the systemic wound signal in wounded leaves,
the mutant was able to perceive that signal in undamaged leaves. Because Spr] is
required for the recognition of systemin, these results indicate that systemin is likely not
the long-distance signal for PI expression. Systemin appears to act at or near the site of
wounding (i.e. in rootstock tissues) to increase J A synthesis to a level that is required
for the systemic response. Consistent with this interpretation, grafting experiments
using J A-insensitive and J A-deficient mutants indicated that J A or a derivative of J A is
the systemic wound signal (Li et al., 2002b). These experiments demonstrated that J A
biosynthesis is required for the generation of the systemic wound signal, and that J A
perception is essential for recognition of this signal in unwounded leaves. This finding
supports the idea that systemin amplifies the systemic wound signal in damaged tissue
by stimulating J A biosynthesis.

The role of J A in systemic wound signaling was investigated further by
characterization of J A-deficient J L1 plants. Gas chromatography-mass spectrometry
(GC-MS) analysis showed that J L1 plants are compromised in the conversion of 12-
oxo-phytodienoic acid (OPDA) to J A (Figure 3-2). OPDA application experiments
showed that this octadecanoid pathway intermediate is not an active signal for PI
expression in tomato (Figure 3-3). Reciprocal grafting experiments with wild-type and
JL1 plants confirmed that JA biosynthesis is required for the generation of the long-

distance signal for PI expression (Figure 3-4).

191

 

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\ ,’ response

 

 

 

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PI<----JA

A
I

iai1 {I‘— JL1 Local
1 response

 

+ OPDA

def1 —-n
l

spr2—-| : spr1
I 4

OGA/V

 

systemin

 

 

 

 

Figure 7-1. Revised model of systemic wound signaling in tomato. Wounding
activates systemin/JA-dependent (broken arrows) and J A-independent signaling (solid
arrows) pathways that regulate distinct sets of wound response genes in undamaged
leaves (systemic response). See text for details.

192

It was indicated that two related cytochrome P4508, allene oxide synthase
(AOS) and hydroperoxide lyase (HPL), regulate oxylipin levels in plants by competing
for a common substrate, 13-hydroperoxy linolenic acid (l3-HPOT; Bate et al., 1998;
Blee and J oyard, 1996; Lauder et al., 1996). AOS and HPL metabolize l3-HPOT to
J A and volatile C6 aldehyde, respectively. To investigate the regulation of J A
biosynthesis, genes encoding these two enzymes were cloned flom tomato. Expression
of these genes in E. coli confirmed that AOS and HPL efficiently metabolize the same
substrate, 13-HPOT, but do not metabolize other hydroperoxy fatty acid substrates
(Figure 6-4). Both genes are expressed in leaves, which supports the idea that AOS and
HPL compete for the same substrate in wounded tissue (Figure 6-5).

Previous studies indicated that a J A-independent signaling pathway mediates
some systemic wound responses in tomato (O'Donnell et al., 1998; Stratmann and Ryan,
1997). This idea is consistent with the observation that expression of AOS was up-
regulated by wounding in both wild-type and J A-deficient defenseless] (defI) plants
(Figure 6-7). In addition, early wound response genes such as lipoxygenaseD (LoxD)
were systemically expressed upon wounding of spr1 plants, which indicates that a
systemin-independent signaling pathway is involved in this systemic response (Figure
2-2; Figure 2-5). Wounding activated the systemic expression of a mitogen-activated
protein kinase (MAPK) gene homologue (WIPK) in J A-deficient and J A-insensitive
mutants. This observation indicates the existence of a J A-independent signaling.
Expression of LoxD was determined to be under regulation by both J A-independent and

-dependent signaling pathways.

193

Based on the findings flom this thesis study, a model of systemic wound
signaling in tomato is proposed (Figure 7-1). In this model, wounding activates two
distinct signaling pathways for systemic gene expression. One is the JA-dependent
signaling pathway, which requires the action of Spr], Spr2, Def], and Jail. fi-oxidation
is involved in this pathway, which is compromised in JL1 plants. This pathway
regulates systemic expression of late genes such as PIs. J A or a derivative of J A that is
transported by the phloem may be the long-distance signal in this pathway. Systemin
promotes the synthesis of J A at or near the site of wounding. The other systemic wound
signaling pathway operates independently flom J A and systemin, and is poorly
understood. A subset of early wound response genes is regulated by this signaling
pathway. The J A-independent pathway also regulates the systemic wound induction of a
MAPK. In this case, the long-distance signal appears to be xylem-home (Stratmann and
Ryan, 1997).

Further biochemical studies are needed to determine whether J A or one of its
derivatives is the long-distance signal of systemic PI expression. Because the systemic
wound signal for PI expression moves though the phloem (Nelson et al., 1983), the
relative level of the endogenous signal in phloem is expected to rapidly increase in
response to wounding. Measurement of JA and related oxylipins in phloem sap will be
useful to address this question. In addition, application of radiolabeled J A to the wound
site may be helpful to monitor the movement of IA in vivo.

As an initial step to identifying the Spr] gene by map-based cloning, a mapping
population was generated by crossing the spr1 mutant to the wild tomato species

Lycopersicon pennellii (Appendix of Chapter 2). Bulk segregant analysis and amplified

194

fragment length polymorphism (AF LP) analysis would be useful to find molecular
markers closely linked to the Spr] locus. A similar approach was used to map the
chromosomal location of Def] and Spr2 (Li et al., 2003; Li et al., 2002a). Molecular
cloning of the Spr] gene will provide insight into how the binding of systemin to the
SR160 receptor on the plasma membrane activates J A biosynthesis in chloroplasts
(Scheer and Ryan, 2002).

Map-based cloning of the gene defined by the J L1 mutant line is in progress in
the Howe lab (C. Li and G. Howe, unpublished results). This gene product is
presumably involved in B-oxidation, which is required for both degradation of fatty
acids and conversion of OPDA to JA. The cloning of this gene will extend our
understanding of how the conversion of OPDA to J A regulates endogenous J A levels.
OPDA itself is not an active signal for wound-induction of PHI, which is contrary to
the finding that OPDA regulates expression of defense genes in Arabidopsis (Stintzi et
al., 2001). Microarray experiments with JL1 plants will be helpful to investigate the
putative role of OPDA as a signal for gene expression in tomato plants.

In vitro import assays have established that AOS is targeted to the inner
envelope membrane of chloroplasts, whereas HPL is targeted to the outer envelope
membrane (Froehlich et al., 2001). This finding fiirther supports the possibility that the
two enzymes compete for the same substrate located in the envelope membrane. To
further address this hypothesis, it will be useful to determine whether the two enzymes
co-localize in the same cell and the same chloroplast.

Microarray experiments using J A-deficient and J A-insensitive mutants would be

helpful to identify wound-inducible genes that are regulated by the J A-independent

195

 

signaling pathway. It may also be informative to examine the expression of WIPK using
stearn-girdled tomato plants in which the xylem but not the phloem is intact. This
experiment would determine whether the JA-independent signal for WIPK expression is

phloem-bome or xylem-bome.

196

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