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THESlS

This is to certify that the
thesis entitled

JASMONATE REGULATION OF DEFENSE RESPONSES
IN TOMATO (L YCOPERSICON ESCULENTUM)

presented by

GUANGHUI LIU

has been accepted towards fulfillment
of the requirements for the

MS. degree in Genetics PrQLram

 

 

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6/01 c-JCIRC/DateDue.p65-p. 15

JASMONATE REGULATION OF DEFENSE RESPONSES IN TOMATO
(L Y C OPERSIC 0N ESCULENTUM)

By

Guanghui Liu

A THESIS

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

MASTER OF SCIENCE
Genetics Program

2004

ABSTRACT

JASMONATE REGULATION OF DEFENSE RESPONSES IN TOMATO
(L Y C OPERSIC ON ESCULENTUM)

By

Guanghui Liu

Jasmonic acid (JA) and methyl-IA (MeJA) are fatty acid—derived cyclopentanone signals
that regulate a broad range of plant defense responses against herbivores and microbial
pathogens. In my thesis research, I found that the expression of a lanthionine synthetase
C-like (LANCL) gene in tomato is induced in response to wounding and treatment with
MeJA, indicating that LeLANC L expression is regulated by the IA signaling pathway.
LANCL proteins are homologous to bacterial LanC, which is involved in the synthesis of
lantibiotic peptides exhibiting antimicrobial properties. Thus, LeLANCL may have a role
in jasmonate-mediated plant protection against biotic stress. In a second project, a tomato
mutant (1'11) that is defective in wound-induced expression of proteinase inhibitor (P1)
gene was shown to be compromised in resistance to the tobacco homworrn (Manduca
sexta). Wounded le plants accumulate normal levels of OPDA, but are compromised in
their ability to produce IA. The gene defined by jl 1 encodes an acyl-CoA oxidase (named
LeACXl) that catalyzes the first and rate-limiting step of fatty acid B-oxidation in the
peroxisome. This finding indicates that the final step in JA biosynthesis involves [3-
oxidation of 3-oxo-2(2’[Z]-penteny1)-cyclopentane-l-octanoic acid (CFC-8:0) to J A.
LeA CXI transcripts constitutively accumulate in tomato leaves, and are further induced
by wounding in a JA-dependent manner. These results show that LeACXl plays a major

role in the B-oxidation step of IA biosynthesis and induced resistance to herbivores.

ACKNOWLEDGEMENTS

First and foremost, I would like to express my earnest gratitude to my advisor Dr.
Gregg Howe. Gregg has always been an admirable mentor to me, and has shown great
support for my work with his inspiration and encouragement. His persistent efforts and

patience have assisted the writing and revision of this dissertation.

I wish to show appreciation to members of my guidance committee, Drs. Rebecca
Grumet, Sheng Yang He, and Jonathan Walton, for their constructive comments and

insightful suggestions on my work.

I am also grateful to past and present members of the Howe lab, for their
companionship and collaboration. I thank Drs. Chuanyou Li, Gyu-In Lee, Youfu Zhao for
contributing preliminary results that were included in this thesis; Drs. Hui Chen and
Bonnie McCaig for their cooperation in certain experiments presented in this thesis; Drs. '
Sastry Jayanty, Abe K00 and Lei Li, Tony Schilmiller, Leron Katsir for their
entertainment, friendship, and support. Jim Klug is acknowledged for his assistance with

taking care of plants in the greenhouse.

I owe many thanks to my family for years of love and support. It is their

encouragement and support that has inspired me to finish this work.

This dissertation research was supported in part by grants from the National

Institute of Health, the Michigan Life Science Corridor, and the Department of Energy.

iii

TABLE OF CONTENTS

LIST OF FIGURES ............................................................................... vii
LIST OF TABLES ................................................................................. ix
CHAPTER 1 ....................................................................................................................... 1
Introduction: Jasmonate Biosynthesis, Action, and Function ............................................. l
I. Biosynthesis of jasmonates .......................................................................................... 3
1.1. Linolenic acid and lipases ...................................................................................... 6
1.2. Lipoxygenase (LOX) ............................................................................................. 7
1.3. Allene oxide synthase (AOS) ................................................................................ 7
1.4. Allene oxide cyclase (AOC) .................................................................................. 8
1.5. 12-Oxo-phytodienoic acid reductase (OPR) .......................................................... 9
1.6. B-Oxidation ............................................................................................................ 9
1.7. Cellular compartmentation of IA biosynthesis .................................................... 10
1.8. Regulation ofjasmonate synthesis ....................................................................... 11
1.9. Prosystemin and systemin .................................................................................... 11
TI. The jasmonate signal transduction pathway ............................................................. 12
IT. 1. Perception of jasmonate ..................................................................................... 12
i 11.2. Role of ubiquitin-mediated proteolysis in jasmonate signaling ......................... 13
113. OPDA pathway .................................................................................................. 15
III. Physiological function of jasmonates ...................................................................... 16
[IL]. Role of IA in resistance to herbivores and pathogens ...................................... 16

111.2. Role of IA in systemic signaling ....................................................................... 18

111.3. Role ofjasmonates in plant growth and development ...................................... 19
References ..................................................................................................................... 21
CHAPTER 2 ..................................................................................................................... 29

A Lanthionine Synthetase C-Like Gene (LeLANCL) Is Regulated by the Jasmonate

Signaling Pathway in Tomato ........................................................................................... 29
Material and Methods .................................................................................................... 35
Plant material and growth conditions ........................................................................ 35
Wound- and MeJA- response assay ........................................................................... 35
RNA isolation and gel blot analysis ........................................................................... 36
Identification of MeJA-induced LA NCL gene ........................................................... 37
Agrobacterium tumcf aciens—mediated transformation .............................................. 38
Results ........................................................................................................................... 39
Identification of a LANC L gene in tomato ................................................................. 39

LeLANCL expression is highly induced in response to MeJ A and mechanical
wounding .................................................................................................................... 46
Tissue-specific expression of LeLANCL .................................................................... 49

Construction and preliminary characterization of transgenic plants altered in

LeLANCL expression ................................................................................................. 52
Discussion ..................................................................................................................... 56
References ..................................................................................................................... 58

CHAPTER 3 ..................................................................................................................... 61

Characterization of the Tomato/I I Wound Response Mutant .......................................... 61

Material and Methods .................................................................................................... 65
Plant material and growth conditions ........................................................................ 65
Identification of LeACXl .......................................................................................... 66
Wound response assay ............................................................................................... 67
RNA isolation and gel blot analysis ........................................................................... 67
Elicitor feeding experiments ...................................................................................... 68
Tobacco homworm feeding trials .............................................................................. 68
Measurement ofjasmonic acid .................................................................................. 69

Results ........................................................................................................................... 70
The jll mutant of tomato has a defective A CXI gene ................................................ 70
Expression of wound response genes is reduced in j]! plants ................................... 70
The jl] mutant is defective in resistance to tobacco homworm ................................. 75
Response of jl 1 plants to exogenous signaling compounds ....................................... 77

Discussion ..................................................................................................................... 88
LeACXl is required for wound-induced .I A biosynthesis ......................................... 88

LeA CXI is expressed constitutively in tomato leaves and induced in response to

wounding .................................................................................................................... 88
Role of J A and OPDA in plant defense ...................................................................... 89
References ..................................................................................................................... 95

vi

LIST OF FIGURES

Figure 1.1. The octadecanoid pathway for jasmonate biosynthesis ........................... 4
Figure 2.1. Schematic representation of the posttranslational modification of Pep5 33
Figure 2.2. Gene structure of LeLANC L and its homolog in Arabidopsis .................. 40

Figure 2.3. Amino acid sequence of LeLANCL and multiple alignment with other
members of the LanC-like protein family and four prokaryotic LanC proteins ........... 41

Figure 2.4. Phylogenetic relationship of LeLANCL to other LanC and LanC-like
proteins ............................................................................................... 47

Figure 2.5. LeLANCL expression in response to exogenous MeJ A in wild-type andjail

plants ................................................................................................ 48
Figure 2.6. LeLANCL expression in response to wounding in wild-type and SprZ

plants ................................................................................................. 50
Figure 2.7. Expression pattern of LeLANCL in different tissues ............................. 50
Figure 2.8. PCR-based detection of transgenes in S-LANC L and AS-LANCL
transforrnants ........................................................................................ 53
Figure 2.9. PI-II levels in LeLANCL transgenic plants ........................................ 55
Figure 3.1. Octadecanoid pathway for J A biosysthesis ....................................... 64
Figure 3.2. Map-based cloning of the ACX gene .............................................. 70

Figure 3.3. Gene expression in wild-type andle plants in response to mechanical
wounding ............................................................................................ 73

Figure 3.4. Accumulation of wound-induced transcripts in response to tobacco homworm
attack ................................................................................................ 77

Figure 3.5. Expression of LeA CXI in wild-type and jail plants in response to tobacco
homworm attack .................................................................................... 78

Figure 3.6. Challenge of wild-type and fl] plants with tobacco homworm larvae ........ 79

Figure 3.7. Effect of OPDA feeding on the expression of various wound-responsive genes
......................................................................................................... 83

vii

Figure 3.8. Dose effect of OPDA and OPC-8:0 on induction of PHI in wild-type and/'11
plants ................................................................................................. 84

Figure 3.9. JA accumulation in wild-type andle plants in response to application of
exogenous OPDA ................................................................................... 88

Figure 3.10. Model for the role of J A and OPDA in the fine control of gene expression in
tomato leaves ....................................................................................... 94

viii

 

LIST OF TABLES

Table 1.1. Jasmonate response mutants of Arabidopsis ...................................... 14
Table 3.1. Tobacco homworm feeding assay with wild-type and jl] plants ............... 75

Table 3.2. PI-II levels in leaves of wild-type andle plants in response to different I A
precursors ........................................................................................... 82

CHAPTER 1

Introduction: Jasmonate Biosynthesis, Action, and Function

Throughout their lives, plants interact with a wide array of organisms such as insects
and pathogens. Plants have evolved complex traits that affect their interactions with these
organisms at all levels. Although some of these relationships are mutually beneficial,
many other interactions cause plants to deploy defensive strategies that protect them
against invaders. To combat invasion by herbivorous insects effectively, plants make use
of pre-existing physical barriers such as the cuticle, bark, and trichomes to repel or trap
insect predators (Leon et al., 2001). Plants also accumulate high levels of pre-formed
chemical compounds that are toxic to insect invaders (Wittstock and Halkier, 2002). This
protection strategy can be described as constitutive, in contrast to induced defenses in
which the synthesis of toxins and anti-feedants is triggered by insect attack (Harborne,
1988; Ryan, 2000; Gatehouse, 2002). Because the latter protection mechanism does not
become activated until plants are attacked, the fitness cost of induced resistance is less
than that involved in constitutive defense (Simms and Fritz. 1990; Gatehouse, 2002; Heil

and Baldwin, 2002; Kessler and Baldwin, 2002).

An important aspect of inducible defenses is their expression occurrence both at the
site of wounding and in undamaged tissues distant from the site of primary attack (Green
and Ryan, 1972; Karban and Baldwin, 1997). This systemic induced response protects
plants against subsequent invaders. Wound-induced defenses in tomato (Lycopersicon
esculentum), which are typically triggered by feeding insects or mechanical wounding
(Howe etal., 2000; Ryan, 2000). represent one of best examples of systemic-induced
resistance to herbivores (Kessler and Baldwin, 2002). In their landmark study of wound-
inducible proteinase inhibitors (PIs), which are expressed within z2 hrs afier mechanical

wounding or herbivore attack, Green and Ryan (1972) proposed that specific signals

generated at the wound site travel through the plant and activate PI expression in
undamaged responding leaves. The fatty acid-derived plant hormones jasmonic acid (J A)
and methyl JA (MeJA) are essential signals for the control of wound-induced defense
responses (Li et al., 2002b; Turner et al., 2002; Weber, 2002). .I A also plays an important
role in plant developmental processes such as root growth, tendril coiling, trichome

formation, seed maturation, and production of viable pollen.

I. Biosynthesis of jasmonates

JA and its volatile methyl ester, MeJA, are potent signaling molecules that are
derived from fatty acids. J A and its cyclic precursors and derivatives, collectively referred
to as jasmonates (JAs), occur ubiquitously in the plant kingdom (Wastemack and Hause,
2002). Following the first identification of MeJA as a major fragrance in the essential oil
of jasmine plants (Demole et al., 1962), the octadecanoid pathway for jasmonate
biosynthesis (Figure 1.1) was elucidated by Vick and Zimmerman (1984). The pathway
starts with the release of u-linolenic acid (or-LA) from membrane lipids in the chloroplast
(Narvaez-Vasquez et al., 1999; Ishiguro et al., 2001). A 13-lipoxygenase (LOX) adds
molecular oxygen to a-LA resulting in the production of 13S-hydroperoxylinolenic acid
(HpOTrE). This hydroperoxy fatty acid is converted to an unstable allene oxide by the
action of allene oxidase synthase (AOS). Allene oxidase cyclase (AOC) then transforms
the allene oxide intermediate to the first cyclic compound in the pathway, 12-oxo-
phytodienoic acid (OPDA). The terminal reactions of JA biosynthesis occur in the
peroxisome, which is the site of fatty acid B-oxidation in plants. First, the cyclopentenone
ring of OPDA is reduced by OPDA reductase (OPR3) to yield 3-oxo-2(2’[Z]-pentenyl)-

cyclopentane-l-octanoic acid (OPC-8:0). Three cycles of B-oxidation remove six carbons

 

Figure 1.1. The octadecanoid pathway for jasmonate biosynthesis.

The pathway originates with the release of u-LA from the chloroplast membrane by a
lipase. a-LA is then converted to OPDA in the chloroplast by the sequential action of 13-
lipoxygenase (LOX), allene oxidase synthase (A08), and allene oxidase cyclase (AOC).
OPDA is transferred to the peroxisome and reduced to OPC-8:0, which is converted to J A
by three cycles of B-oxidation. JA can be methylated by jasmonate methyl transferase
(JMT) to the volatile MeJ A in the cytosol. JA can also be metabolized to other I A

conjugates such J A-Ile.

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from the carboxyl side chain, completing the biosynthesis of J A. JA also occurs in a
variety of modified forms, including Me] A, glycosyl esters, and amide-linked conjugates

with various amino acids (Sembdner and Parthier, 1993; Staswick and Tiryaki, 2004).

1.1. Linolenic acid and lipases

The critical requirement of a-LA in J A biosynthesis was explicitly demonstrated by
the analysis of an Arabidopsis “triple” fatty acid desaturase (FAD) mutant
(fad3/fad7/fad8), which produces no trienoic fatty acid and is completely deficient in J A
(Wallis and Browse, 2002). Although photosynthesis and vegetative growth of the mutant
are unaffected, the triple mutant is male sterile. Exogenous a-LA and JA restore fertility
to the mutant (McConn and Browse, 1996), indicating the JA plays an essential role in
plant reproductive development. Additional evidence for the role of (D3-FADS in J A
biosynthesis comes from the characterization of the spr2 mutant of tomato (Li et al.,
2003). This mutant is defective in anti-herbivore defense in response to wounding and the
18-amino-acid peptide wound signal, systemin (Howe and Ryan, 1999). The spr2 gene
encodes a chloroplast fatty acid desaturase (LeFAD7) that is homologous to Arabidopsis
F AD7/8. spr2 plants are severely deficient in a-LA and JA accumulation, indicating that
chloroplast pools of a-LA are required for wound- and systemin-induced J A biosynthesis

in tomato (Li et al., 2003).

Recent evidence indicates that phospholipases (PLs) that release fatty acid precursor
from membrane lipids play a critical role in JA biosynthesis. The male sterile delayed in
anther dehiscence I (dadI ) mutant of Arabidopsis is deficient in J A accumulation in

floral tissues (Ishiguro et al., 2001). DA D1 encodes a PLAl that presumably liberates a-

LA from membrane lipids (Figure 1.1). Localization of BAD] to the cholroplast is
consistent with the notion that it generates fatty acid substrate for the plastid-localized
enzymes of the octadecanoid pathway. The capacity of dad] mutant plants to accumulate
normal levels of J A in wounded leaves (Ishiguro et al., 2001) indicates that other lipases
are invloved in IA biosynthesis in response to insect and pathogen attack. PLA2 and PLD
have also been implicated in wound-induced JA biosynthesis (Creelman and Rao, 2002;

Howe and Schilmiller, 2002; Turner et al., 2002).

1.2. Lipoxygenase (LOX)

LOXs catalyze the conversion of fatty acids to their corresponding hydroperoxy
derivatives. Plant LOXs oxygenate a-LA at the 9 or 13 positions to generate 9- or 13—
HpOTrE, respectively. LOXs involved in JA biosynthesis produce l3-HpOTrE, and are
called 13-LOX (Feussner and Wastemack, 2002). Evidence for the involvement of 13-
LOX in JA biosynthesis came from the observation that plants treated with LOX
inhibitors or transgenic plants with suppressed LOX activity exhibited reduced .1 A levels
in response to wounding (Pena-Cortes et al., 1993; Bell et, al., 1995; Royo et al., 1999;

Halitschke and Baldwin, 2003).

1.3. Allene oxide synthase (A OS)

Production of IA requires the metabolism of 13-HpOTrE to an unstable epoxide
intermediate by A08. A08 is a cytochrome P450 enzyme (CYP74A) that contains an N-
terminal plastid targeting sequence (Laudert et al., 1996). ADS activity and protein has
been localized to the plastid outer envelope (Bleé and Joyard, 1996; Froehlich et al.,

2001). Tomato has two chloroplast-targeted 13-AOSs (Howe et al., 2000; Sivasankar et

al., 2000). A08 knockout mutants of Arabidopsis, which has a single AOS gene, are
completely defective the wound-induced JA accumulation and the activation of wound
response genes (Park et al., 2002; von Malek et al., 2002). Wounding increases AOS
expression and A08 activity (Laudert et al., 1996; Laudert and Weiler, 1998) in both
wounded and systemic leaves. The reaction catalyzed by A08 is the first committed step
in JA biosynthesis. Therefore, regulation of AOS expression and activity is considered a
major control point for JA biosynthesis (Laudert and Weiler, 1998). Overexpression of
A05 in transgenic Arabidopsis plants did not alter the basal J A levels, but these
transgenic plants produced more J A than wild-type plants in response to wounding
(Laudert et al., 2000; Park et al., 2002). Therefore, it appears that AOS expression limits
J A levels in wounded plants. The production of J A in unwounded plants appears to be

limited by substrate availability (Laudert et al., 2000; Ziegler et al., 2001).

1.4. Allene oxide cyclase (A DC)

AOC catalyzes the stereospecific cyclization of the unstable allene oxide to OPDA.
A single AOC gene in tomato encodes a protein that is targeted to the chloroplast by an
N-terminal transit peptide (Ziegler et al., 2000). In tomato, AOC expression is highest in
roots, flower buds, flower stalks, with a lower expression in stems. young leaves. and
pistils (Hause et al., 2000). A DC transcript is transiently induced in tomato leaves in
response to wounding or treatment with J A or systemin. A 0C expression is primarily
confined to the vascular bundle tissues, specifically in companion cells and sieve
elements (Hause et al., 2000; 2003; Stenzel et al., 2003). Vascular bundle-specific
localization of A 0C transcript coincides with the spatial accumulation of J A in main

veins of tomato leaves (Stenzel et al., 2003).

1.5. IZ-Oxo-phytodienoic acid reductase (OPR)

OPR catalyzes the reduction of OPDA to UPC-8:0. Both Arabidopsis and tomato
contain three OPR isozymes. However, only OPR3 has the capacity to reduce the 9S,
IBS-stereoisomer of OPDA, which is the biologically relevant precursor of JA (Mtissig et
al., 2000; Strassner et al., 2002). This was confirmed in studies demonstrating that an
opr3 null mutant lacks JA and is male sterile (Wallis and Browse, 2002). OPR3 transcript
is induced by JA (Mfissig et al., 2000) and wounding (Strassner et al., 2002; Li et al.,
2004). Localization of OPR3 to the peroxisome (Strassner et al., 2002) provides strong
support for the hypothesis that the later phase of J A formation occurs in this organelle.
The spatial separation of the octadecanoid pathway into two distinct compartments
(chloroplast and peroxisome) implies that transport processes should be existed to shuttle

OPDA from the chloroplast to the peroxisome.

I. 6. fi-Oxidation

Shortening of the side chain of OPC-8:0 to form JA is achieved by three rounds of 0-
oxidation. These reactions are thought to occur in the peroxisome, which is the exclusive
site of fatty acid B-oxidation in plants (Gerhardt et al., 1983; Strassner et al., 2002).
However, until recently, there has been little direct evidence for the involvement of [3-
oxidation in the biosynthesis of JA. The enzymatic activities involved in fatty acid B-
oxidation are acyl-CoA oxidase (ACX), multifunctional enzyme showing enoyl-CoA
isomerase and hydroxyacyl-CoA dehydrogenase activity, and thiolase (Gerhardt, 1992).
Castillo et a1. (2004) identified specific B-oxidation enzyme encoding genes that were

expressed in wounded leaves of Arabidopsis. Wound-activated synthesis of J A was

reduced in acyl-CoA oxidase 1 (A CX 1) and 3-ketoacyl-C 0A thiolase 2 (KA T2) antisense
plants, suggesting that ACXl and KAT2 play a role in the biosynthesis of JA in

Arabidopsis (Castillo et al., 2004).

I. 7. Cellular compartmentation of JA biosynthesis

The conversion of a-LA to OPDA occurs in the chloroplast, which contains an
abundance of a-LA. Localization studies using biochemical fractionation and
immunocytochemical and in vitro chloroplast import assays have demonstrated a
chloroplast location for DADl, LOX, A08 and AOC, which together catalyze the core
chloroplast reactions in the octadecanoid pathway. In addition to protein localization, the
corresponding enzymatic activities are also found in the chloroplast (Bleé and J oyard,
1996; Ziegler et al., 2000; Froehlich et al., 2001; Ishiguro et al., 2001). Co-localization of
these enzymes in the chloroplast has been suggested to facilitate the metabolism of
lipophilic or unstable intermediates of the J A branch of oxylipin metabolism (Froehlich et

al., 2001).

The conversion of OPDA to J A occurs in the peroxisome as indicated by the
peroxisome-specific compartmentation of OPR3 and the B-oxidation enzymes (Strassner
et al., 2002). The spatial separation of IA biosynthesis suggests that the transfer of OPDA
from chloroplast to peroxisome might be an important regulatory mechanism in J A
biosynthesis. OPDA could be released from chloroplast membranes enzymatically, and
this could account for the rapid transient increase in free OPDA and J A when leaves are
wounded. The presence of a large pool of OPDA esterified to chloroplast galactolipids

suggests that these pools might function as precursors for J A (Stelmach et al., 2001 ).

10

I. 8. Regulation of jasmonate synthesis

Most genes encoding JA biosynthetic enzymes are coordinately activated in response
to wounding or JA treatment (Li et al., 2004). Within 1 hr of treatment, transcripts
encoding DAD] (Ishiguro et al., 2001), LOX (Heitz et al., 1997), AOS (Howe et al., 2000),
AOC (Stenzel et al., 2003), and OPR3 (Strassner et al., 2002) begin to accumulate. The
AOS promoter contains motifs that are similar to other known stress response elements.
GUS activity expressed from an AOS promoter-GUS fusion was induced by wounding
and JA treatment (Kubigsteltig et al., 1999). Although these data indicate a positive
feedback regulation in JA biosynthesis (Sivasankar et al., 2000), wound induced
expression of octadecanoid pathway genes is not required for J A synthesis. Rather,
substrate availability appears to play a major role in the regulation of J A biosynthesis

(Ziegler et al., 2001).

I. 9. Prosystemin and systemin

The systemin signaling pathway is a unique aspect of the wound response pathway in
solanaceous plants. Systemin is an lS-amino-acid polypeptide that is produced at the
wound sites of tomato leaves. Systemin has a role in the systemic regulation of defensive
genes such as PIS (Ryan et al., 2000). Systemin is derived from a 200-amino-acid
precursor called prosystemin (McGurl et al., 1992). Genetic manipulation of the
prosystemin cDNA has provided convincing evidence that prosystemin plays a critical
role in the transduction of systemic wound signals. Antisense suppression of prosystemin
in tomato plants abrogated the systemic wound response (McGurl et al., 1992), whereas

overexpression of the prosystemin cDNA resulted in constitutive activation of wound

response genes in unwounded plants (McGurl et al., 1994). Narvaez-Vasquez and Ryan
(2004) recently presented in situ hybridization and immunocytochemical evidence that
wound- and MeJA-induced prosystemin mRNA and protein are exclusively found in
vascular phloem parenchyma cells of minor veins and midribs of leaves, and in the
bicollateral phloem bundles of petioles and stems of tomato. Prosystemin protein was
also found in parenchyma cells of various floral organs, including sepals, petals and
anthers. At the subcellular level, prosystemin was localized in the cytosol and the nucleus
of vascular parenchyma cells. These data indicate that vascular phloem parenchyma cells
are the sites for the synthesis and, presumably, proteolytic processing of prosystemin
(Narvaez-Vasquez and Ryan, 2004). Enzymes involved in the processing of prosystemin

to systemin have not yet been identified.

The systemin receptor, SR160, is a member of the leucine-rich repeat (LRR) receptor
kinase family (Scheer and Ryan, 2002). The interaction of systemin with SR160 activates
an intracellular signaling cascade, including depolarization of the plasma membrane, the
opening of ion channels, an increase in intracellular Ca2+, activation of a MAP kinase
activity and a PLA2 activity. These rapid changes are thought to play important roles in
the intracellular release of linolenic acid from membranes and its subsequent conversion

to JA (Ryan, 2000).

11. T he jasmonate signal transduction path way
11.1. Perception of jasmonate
Similar to other plant hormones (Kende and Zeevaart, 1997), JA-mediated responses

are thought to be transduced upon binding of J A to a receptor (Creelman and Mullet.

12

1997). The lipophilic and the volatile nature of JA has made the direct analysis of the J A
receptor difficult (Creelman and Mullet, 1997). Isolation of JA response mutants has been
used to identify a number of loci important for JA signaling (Table 1.1). Several of the
genes defined by these mutants have been identified, including COll (a LRR-containing
F-box protein; Xie et al., 1998), J ARI (IA-amino synthetase; Staswick et al., 2004), and
JIN 1 (AtMYC2 transcription factor; Lorenzo et al., 2004). Neither of these appears to
function as a JA receptor. It is possible that functional redundancy within the J A
perception apparatus precludes the identification of the components by mutational

analysis.

[1.2. Role of ubiquitin-mediated proteolysis in jasmonate signaling

Selective proteolysis mediated by the ubiquitin-proteasome pathway plays a key
regulatory role in numerous cellular processes in both animals and plants. Ubiquitin is a
small polypeptide that is covalently attached to target proteins by three protein complexes
called the ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and
ubiquitin ligase (E3). Ubiquitinated proteins are degraded by the 26S proteasome. The
specificity and timing of substrate ubiquitination are primarily controlled by the E3

complexes (Voges et a1, 1999).

The analysis of the coronatine insensitive l (coil) mutant of Arabidopsis. which is
fully insensitive to JA. provides a link between J A signaling and the ubiquitin-
proteasome pathway. The C 01] gene encodes an F-box protein that is a component of an

FCOII

E3-type ubiquitin ligase complex referred to as SC (named for the three major

proteins of the complex: §kp1 , Cullin, and E-box protein). This was confirmed by the

 

Table 1.1. Jasmonate response mutants of Arabidopsis.

 

 

Mutant Phenotype References

jar] Reduced root growth inhibition by MeJA, enhanced Staswick et
susceptibility to Pythium irregulare; JARl belongs to the al., 1992;
acyl adenylate-forming firefly luciferase superfamily, and it 1998; 2002;
adenylates JA; J ARl is a JA-amino synthetase that is 2004
required to activate JA for optimal signaling; allelic to jin-l

jin] Reduced root growth inhibition by MeJA, increased Berger et
resistance to necrotrophic pathogens; JINI encodes al., 1996;
AtMYC2, a nuclear-localized basic helix-loop-helix-leucine Lorenzo et
zipper transcription factor, and differentially regulates the al., 2004
expression of two groups of JA-induced genes

jue1/2/3 Reduced LOXZ mRNA level; not yet cloned Jensen et

aL,2002

coil Reduced root growth inhibition by coronatine, enhanced Feys et al.,
susceptibility to Pythium irregulare and Alternaria 1994;
brassicicola, male sterility; C011 is and LRR-containing F- Xie et al.,
box protein 1998

cetl Constitutive expression of the JA inducible gene Thi2.1; not Hilpert et
yet cloned al., 2001

cexl Constitutive JA-responsive phenotypes including JA- Xu et al.,
inhibitory growth and constitutive expression of JA- 2001
regulated AtVSP, Thi2.1 and PDF1.2; not yet cloned

cevl Stunted roots with long root hairs, accumulated anthocyanin. Ellis et al.,
constitutive expression of the defense-related genes VSPI, 2001; 2002
VSP2, Thi2.1, PDF1.2, and CHI-B, enhanced resistance to
powdery mildew diseases; CEVl encodes a cellulose
synthase

joe1/2 Enhanced sensitivity to MeJ A; not yet cloned Jensen et

al., 2002

 

Abbreviations: jar, jasmonate resistant; jin, jasmonate i_nsensitive; jai, jasmonate
insensitive; jue, jasmonate undergxpressing; coi, gronatine insensitive; cet,
constitutive expression of the thionin gene; cex, gonstant gpression of J A-inducible
genes; cev, gonstitutive prression of ESP] ; joe, jasmonate gvergxpressing; LRR,
leucine rich repeat.

 

l4

demonstration that AtCOIl associates physically with AtCULl, Atbe 1 , and either of the
Arabidopsis Skpl-like proteins ASKl or ASK2 to assemble a functional SCF-type E3
ubiquitin ligase complex (Devoto et al., 2002; Xu et al., 2002). Moreover, plants deficient
in other components of SCF complexes also show impaired responsiveness to JA (Devoto
et al., 2002; Xu et al., 2002; Feng et al., 2003). The existence of a conserved COIl
homologue in other species has been demonstrated recently by the identification of the
tomato C011 gene (LeC 011 ) (Li et al., 2004). The presumed function of the SCFCO”
multiprotein complex is to attach ubiquitin to regulatory proteins that interact with the
leucine-rich repeat domain of C 01 l. A model for J A signaling has been proposed
(Creelman and Rao, 2002). In the absence of J A, J A-responsive genes are repressed by a
negative regulator. Increased J A levels initiate a signaling cascade that results in
modification (e. g. phosphorylation) of the negative regulator, such that SCFm”

recognizes and targets this protein for degradation.

11.3. OPDA path way

A few studies indicated that OPDA was also active as signal without prior
metabolism to JA (Howe, 2001). The Arabidopsis opr3 mutant, which is defective in the
conversion of OPDA to J A, exhibited full resistance to the dipteran Bradysia impatiens
and the fungus A lternaria brassicicola (Stintzi et al., 2001). Several wound-inducible
genes previously known to be J A-dependent were activated in opr3 plants. In addition,
exogenous OPDA powerfully upregulated several genes. These results indicate that the
resistance of opr3 plants is mediated by a signal other than J A, the most likely candidate
being OPDA. This research concluded that OPDA works in concert with JA to fine-tune

the expression of defense genes. Because resistance to insect and fungal attack can be

observed in the absence of J A, it was suggested that OPDA could fulfill some I A roles in

vivo (Stintzi et al., 2001; Farmer et al., 2003).

[(1. Physiological function of jasmonates

Jasmonates play a dual role in regulating plant development and responses to
numerous stresses (Creelman and Mullet, 1997; Turner et al., 2002; Rojo et al., 2003).
Levels of endogenous J A are highest in young growing tissue (Creelman and Mullet.
1995) and increase after treatment with elicitors, wounding, UV light, water deficit,
pathogen infection and ozone treatment (Rao et al., 2000; Rojo et al., 2003). Application
of IA induces the expression of a larger number of genes that are responsive to these
stress signals (Reymond et al., 2000; Cheong et al., 2002). The identification and analysis
of mutants that are impaired in IA biosynthesis, signaling, and the analysis of transgenic
plants with altered expression of J A biosynthetic genes or J A signaling factors have

offered new insights into the function of IA in plants.

111.1. Role of JA in resistance to herbivores and pathogens

JAs play a central role in regulating plant defense (Kessler and Baldwin, 2002;
Turner et al., 2002; Wastemack and Hause, 2002). So-called “direct” defenses are
mediated by JA-regulated phytochemicals that interact directly with plant invaders to
negatively affect their feeding, growth or reproduction. Classic examples are P15 and
polyphenol oxidase (PPO), which reduce the digestibility of damaged leaf tissue (Ryan,
2000). Several lines of evidence demonstrate that J A is the main regulator for the
activation of direct defenses. First, treatment of plants with exogenous JAs results in

major re-programming of gene expression, including defense-related genes that are

activated by mechanical wounding and herbivore attack (Farmer and Ryan, 1992; Li et al.,
2004). Second, endogenous levels of J A increase rapidly in response to wounding and
other biotic stress (Penninckx et al., 1996; Lee and Howe, 2003). Third, mutants that are
defective in either the IA biosynthesis or signaling are compromised in resistance to
herbivores (Howe et al., 1996; McConn et al., 1997; Li et al., 2003; 2004). By contrast.
constitutive activation of JA signaling results in enhanced resistance to herbivores (Li et
al., 2002a). In addition to anti-herbivore defense, genetics studies in Arabidopsis showed
that JA signaling promotes direct defense against fungal pathogens (Wallis and Browse,

2002).

JA also plays an important role in “indirect” defenses (Kessler and Baldwin, 2002).
One of the best examples of this fascinating type of self-protection is the production of
plant volatiles (e. g. terpenoids) in response to fatty acid amide elicitors found in the oral
secretions of foraging lepidopteran herbivores. These emitted volatile chemicals attract
natural enemies of the herbivore. Increasing evidence indicates that production and
emission of volatiles in response to herbivore attack involves the host plant’s J A signaling
pathway (Thaler, 1999; Kessler and Baldwin, 2002). The ability of IA to induce the
production of extrafloral nectar which will attract ants to fend off insect herbivores is

another remarkable example of a JA-mediated indirect defense (Heil et al., 2001).

There is also some evidence to suggest that JA mediates plant-to-plant signaling
(Farmer, 2001). MeJA released from sagebrush induced the expression of PIs in
neighboring tomato plants (Farmer and Ryan, 1990), supporting the hypothesis that MeJA
is a natural wound signal for interplant communication of defense responses (Karban et

al., 2000). This view is supported by the demonstration that MeJ A released from A.

17

tridentate inhibited the germination of nearby N. attenuata plants (Preston et al., 2002).

111.2. Role of IA in systemic signaling

Many induced plant responses occur both locally at the damaged site and
systemically in undamaged tissues (Green and Ryan, 1972; Ryan, 2000). This implies that
signals generated at the wound site travel through the plant and activate defense
responses in unwounded tissue. Several chemical signals such as systemin, OPDA, JA,
and MeJA, have been implicated in the systemic wound response (Ryan, 2000; Leon et
al., 2001; Farmer et al., 2003). Although all of them induce PI accumulation in distal
untreated leaves after application to one leaf (Farmer and Ryan, 1992; Ryan, 2000),
grafting experiments indicate that J A is likely a long-distance wound signal for activation
of defense gene expression (Li et al., 2002b; Lee and Howe, 2003). Tomato .1 A
biosynthesis mutants (e. g. spr2), J A perception mutants (e. g.jail), and systemin
perception (e. g. sprl) mutants are all impaired in wound-induced systemic PI expression
(Lightner et al., 1993; Lee and Howe, 2003; Li et al., 2003; 2004). Classical grafting
techniques were used to determine whether a particular mutant is defective in the
production of the systemic wound signal in wounded leaves or the recognition of that
signal in unwounded leaves. Analysis of systemic wound signaling in reciprocal grafts
between wild-type, sprZ, and jail plants revealed that jasmonate synthesis is needed to
produce the systemic signal in wounded leaves, but is not required in systemic
undamaged tissues: Conversely, J A perception is required for recognition, but not
production, of the transmissible wound signal (Li et al., 2002b). These results suggest
that a signaling compound derived from the octadecanoid pathway acts as a transmissible

wound signal.

18

What then is the role of systemin in systemic wound signaling? Grafts between wild-
type and sprl plants that are insensitive to systemin showed that systemin functions at or
near the wound site to amplify jasmonate synthesis, but is likely not a long-distance
signal by itself (Lee and Howe, 2003). This was confirmed by grafting experiment
showing that spr2 scions which are insensitive to systemin, can perceive the signal
generated in wild-type rootstock leaves (Li et al., 2002b). These results suggest that

jasmonate acts as the long-distance signal (Stratmann, 2003).

[11.3. Role of jasmonates in plant growth and development

Exogenous J As exert both inductive and inhibitory effects on a variety of plant
developmental processes (Creelman and Mullet, 1997; Wasternack and Hause, 2002).
Mutants that are defective in .I A biosynthesis or perception provide an opportunity to
assay the function of J A in specific development processes. One of the most pronounced
effects of exogenous JA is general inhibition of growth. JA-mediated inhibition of root
growth has been used as a phenotype to screen mutants having reduced sensitivity to J A
(Staswick et al., 1992; Lorenzo et al., 2004) or constitutive JA signaling (Ellis et al, 2001).
Another effect of JA is decreased expression of genes involved in photosynthesis and
reduction in chlorophyll content. JA-mediated chlorosis and increased abscission suggest
that JA may play a role in promoting senescence (Wasternack and Hause, 2002). This is
consistent with the demonstration that senescence of Arabidopsis leaves is correlated with
increased expression of .IA biosynthetic genes and increased JA levels (He et al., 2002).
However, J A biosynthesis or signaling mutants of Arabidopsis and tomato do not show
obvious delayed-senescence phenotypes, suggesting that J A is not strictly required for the

normal process of senescence, or that senescence-like effects induced by exogenous .IA

19

do not accurately reflect the normal senescence program. The abnormal development of
glandular trichomes in tomatojail plants suggest a role of .IA signaling in the promotion
of glandular trichome-based defense (Li et al., 2004). Trichome production in leaves of
Arabidopsis is stimulated by mechanical wounding and exogenous J A (Traw and

Bergelson, 2003), indicating that trichome development is affected by J A.

JA biosynthesis and signaling mutants of Arabidopsis are all male sterile, indicating
that JA is essential for male gametophyte development in this plant (Berger, 2002; Wallis
and Browse, 2002). J A appears control multiple aspects of male fertility including
development of viable pollen, timing of anther dehiscence, and elongation of the anther
filaments. Jasmonate application experiments further demonstrated that IA is necessary
and sufficient to promote these reproductive processes. However, comparable J A
biosynthesis mutants in tomato display normal fertility (Li et al., 2003), and a J A
signaling mutant is female sterile (Li et al., 2001; 2004). These phenotypes are consistent
with the fact that wild-type tomato flowers accumulate high levels of J A (Wasternack and
Hause, 2002), and that tomato J A biosynthetic mutants have significant levels of JA in
floral tissue. These observations indicate that male fertility in tomato is not strictly
dependent on J A. The apparent differences in the roles of .I A in reproductive development
in tomato and Arabidopsis indicate that JA signaling pathway regulates distinct

development processes in different plants (Li et al., 2004).

20

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27

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28

CHAPTER 2

A Lanthionine Synthetase C-Like Gene (LeLANCL) Is Regulated by the Jasmonate

Signaling Pathway in Tomato

The cDNA Microarray analysis was done by Dr. Youfu Zhao.

29

Abstract

Jasmonic acid (J A) and methyl-J A (MeJ A) are fatty acid-derived cyclopentanone
signals that regulate a broad range of plant defense responses against herbivores and
microbial pathogens. We used cDNA microarray analysis to identify and characterize
genes in Lycopersicon esculentum (tomato) that are regulated by the J A signaling
pathway. Here, we report the identification and preliminary characterization of a novel
gene (LeLANCL) that is predicted to encode a new member of the lanthionine synthetase
C-like (LANCL) family of proteins found in both plants and animals. These proteins
appear to be homologous to bacterial LanC, which is part of a membrane-bound complex
involved in the synthesis and transport of lantibiotic peptides that exhibit potent
antimicrobial properties. We found that LeLANCL transcripts accumulated to their highest
level in reproductive tissues of healthy tomato plants. In vegetative tissues, LeLANCL
expression was highly induced in response to mechanical wounding and treatment with
MeJ A. Wound-induced expression of the gene was blocked in tomato mutants that are
defective in JA biosynthesis or J A perception. These results indicate that LeLANC L
expression is regulated by the J A signaling pathway, and further suggest that the gene
could serve a role in defense against herbivores or pathogens. The physiological function
of LANCL in plant growth and development is being investigated by analysis of tomato

plants that are altered in LANCL expression.

30

Introduction

Unlike animals, plants are anchored to the ground and thus are unable to easily avoid
injuries caused by chewing insects or larger herbivores. Pre-existing physical barriers
such as the cuticle, bark, trichomes, and thorns provide one line of defense against
herbivores (Leon et al., 2001). However, plants rely mainly on chemical barriers for
protection against insect attack. Plants constitutively synthesize a broad range of
secondary metabolites, including alkaloids and terpenoids, which are toxic to herbivores
and pathogens (Wittstock and Gershenzon, 2002). Also, plant cells become competent for
the activation of chemical defenses in responses to initial damage by an herbivore. These
wound-activated responses play a role in healing of the damaged tissues and the
production of chemicals that negatively affect herbivore performance. Wound-inducible
genes encode proteins that have one of the following functions: (i) anti-feedant proteins
such as proteinase inhibitors (P13) and polyphenol oxidase (PPO); (ii) activation of
wound-induced signaling pathways for defense; and (iii) metabolic changes such as
alkaloid and terpenoid production (Kessler and Baldwin. 2002). Wounding of a single
leaf activates defense mechanisms both in the tissues directly damaged (local response)
and in the undamaged tissues (systemic response) (Green and Ryan, 1972). Wound-
activated gene expression requires the synthesis, accumulation, and perception of
jasmonic acid (JA) (Ryan et al., 1993; Li et al., 2002a). which is synthesized by the

octadecanoid pathway (Vick and Zimmerman, 1984).

Lantibiotics are lanthionine-containing antibiotic peptides (Sahl and Bierbaum,
1998). These antimicrobial compounds are characterized by the presence of the unusual

amino acids lanthionine and B-methyllanthionine. These amino acids form intramolecular

31

thioether rings that originate from posttranslational modification of serine, threonine, and
cysteine residues (Schnell et al., 1988; Sahl et al., 1995). Lantibiotics are synthesized
from ribosomally made prepeptides which are encoded by the structure gene LanA
(Meyer et al., 1995; Siegers et al., 1996). The LanA prepeptides have N-terminal leader
peptide and C-terminal prepeptide regions in which the Ser, Thr, and Cys residues are
posttranslationally modified to the rare amino acids by a two-step reaction (Figure 2.1)
(Ingram, 1970; Sahl and Bierbaum, 1998). First, the hydroxyl amino acids Ser and Thr
are dehydrated to yield didehydroalanine (Dha) and didehydrobutyrine (Dhb),
respectively (Weil et al., 1990). Second, the thioethers are formed by an intramolecular
Michael addition that involves the thiol groups of neighboring Cys residues and the
double bonds of the didehydroamino acids. The lantibiotic modification enzyme LanB is
involved in the dehydration reaction, whereas LanC catalyzes thioether formation (Figure
2.1) (Meyer et al., 1995; Koponen et al., 2002). The leader peptide is removed
proteolytically from the prepeptide after the modification reactions have been completed,

thus releasing the mature peptide.

Members of the lanthionine synthetase C-like (LANCL) protein family have been
identified in animals and plants (Bauer et al., 2000; Mayer et al., 2001a). These
peripheral membrane proteins are homologous to the bacterial LanC (Bauer et al., 2000).
Two LANCLs have been characterized in detail. LANCL] was originally isolated from
human erythrocyte membranes and is mainly expressed in the brain and testis (Mayer et
al., 1998). This peripheral membrane protein has also been identified in mouse and rat
(Mayer et al., 2001a). Sequence analysis revealed that LANCL is similar to the bacterial

LanC (Bauer et al., 2000). which is a part of membrane-associated complex involved in

32

TAGPAIRASVKQCQKTLKATRLFTVSCKGKNGCK

 

0
ll
Nxcgcx
I
c
R/ \OH
H o .
’ (i o Dehydration
I
N‘C’é“ 1.3118 0
:I ”

/
R l
/\ ,c
HS
Thioether formation k

LanC o o
11 II

R,rl__s_..4
l /S\

TAG PAIRAA<K3AQKTLKATRLFAVAAKGKNGAK

S 8

Figure 2.1. Schematic representation of the posttranslational modification of Pep5.
Top, the unmodified Pep5 prepeptide. Middle, the dehydration of a hydroxyl amino acid
(R=H for Ser or R=CH3 for Thr) and the formation of the thioether (R=H for Lan or

R=CH3 for MeLan). Bottom, mature Pep5 (Modified from Sahl and Bierbaum, 1998).

33

the modification of peptides (Siegers et al., 1996; Kiesau et al., 1997). LANCL2 is also
highly expressed in testis and brain (Mayer et al., 2001b). Its expression increases cellular

sensitive to adriamycin, which is used as an anticancer drug.

The function of LANCL proteins family is not known. Based on the limited
homology to bacterial LanC, most notably the seven GXXG repeats, these proteins are
thought to be peptide-modifying enzyme components in eukaryotic cells. In eukaryotes.
the GXXG is a signature motif within the KH module, which is a sequence motif found
in a number of proteins that are found in close association with RNA (Musco et al., 1996).
Structural studies suggest that the GXXG loop functions as a DNA/RNA-binding surface
(Musco et al., 1996). The presence of this conserved sequence in LANCL suggests that it
may function as a single-strand nucleic acid binding protein (Park and James, 2003). The
high expression of LANCLs in testis and brain, organs separated by blood-tissue barriers,
may hint at a role in the immune surveillance of these organs. LANCL2 is coamplified
and overexpressed with epidermal growth factor receptor (EGFR) in glioblastoma, which
is the most aggressive form of primary brain tumors (Wang et al., 1998; Eley et al., 2002).
Recently, Park and James (2003) reported that LANCL2 increased cellular sensitivity to

adriamycin by decreasing the expression of P—glycoprotein (P-gp).

Here, we describe the identification and preliminary characterization of tomato
LeLANCL, a new member of LANCL protein family. We found that LeLA NC L
expression in tomato leaves was highly induced in response to mechanical wounding and
treatment with MeJA. Wound-induced expression of the gene was blocked in mutants that
are defective in J A biosynthesis or J A perception. LeLA NC L transcripts accumulated to

their highest level in reproductive tissues of healthy tomato plants. These results indicate

34

that LeLANCL expression is regulated by the J A signaling pathway, and further suggest
that the gene could serve a role in defense against herbivores or pathogens. The
physiological function of LANC L in plant growth and development is being investigated

by analysis of tomato plants that are altered in LeLANCL expression.

Material and Methods
Plant material and growth conditions

Lycopersicon esculentum Mill cv C astlemart and cv Micro-Tom were used as wild-
type. Tomato seeds were placed on a piece of water-saturated filter paper in a shallow
Tupperware box and allowed to germinate in the dark at room temperature for 4-5 days.
At this time (emerging radicals were 1-1 .5 cm in length), the seedlings were transferred
to Jiffy peat pots (Hummert International, Earth City, MO). Seedlings were grown in a
growth chamber maintained under 17 hr days at 28 °C with light (200 umol m'zsec") and

7 hr at 16 °C in the darkness.

All experiments involving the jai 1 mutant were performed with homozygous
(fail/jail) lines. Homozygous jail seedlings were selected from F2 populations as

previously described (Li et al., 2004).

Wound- and MeJA- response assay

Two-leaf-stage plants (18-day-old for cv Castlemart and 20-day-old for cv Micro-
Tom) containing two fully-expanded leaves and a third emerging leaf were wounded with
a hemostat across the midrib of all leaflets (typically three) on the lower leaf. Three hrs

later, the same leaflets were wounded again, proximal to the petiole. Wounded plants

35

were incubated under standard growth conditions. At different time point after wounding,
the wounded leaf (local response) and the upper, unwounded leaf (systemic response)
were harvested separately for extraction of RNA. Quantification of local and systemic PI-
11 protein levels were measured by radial immunodiffusion assay (Ryan, 1967; Trautman
et al., 1971) 24 hrs after wounding. Briefly. a S-ul aliquot of expressed leaf juice was
placed into a well (0.5 mm diameter) of an agar plate (2% (w/v) Noble agar, 0.9% (w/v)
NaCl, 20 mm Tris, pH 8.5) containing 1% (v/v) polyclonal antiserum obtained from a
goat that was immunized with tomato PI-II. One day later, the diameter of the
immunoprecipitate ring that results from the antibody-antigen interaction was measured
and used to calculate the amount of PHI per milliliter of leaf juice. Based on a standard
curve obtained using purified PI-II. the detection limit of the assay was estimated to be

about 5 pg PI-II per ml ofleafjuice.

MeJA treatment of tomato plants was performed by incubating two-leaf-stage plants
in a sealed Lucite box in which 2 pl pure MeJA was dissolved in ethanol and distributed
to several evenly spaced cotton wicks. For each time point of sampling, six plants were
removed from the box for isolation of total RNA from leaf tissues. PI-II level in leaves

was measured by radial immunodiffusion assay 24 hrs after MeJA treatment.

RNA isolation and gel blot analysis

RNA was isolated from tomato leaves or other tissues and analyzed by gel blot
hybridization as described previously (Li et al., 2002a). RNA concentration was
determined by absorbance at 260 nm. Gels were run in duplicate, with one set stained

with ethidium bromide to verify RNA quality and to check for equal loading of samples.

36

A cDNA for tomato translation initiation factor elF 4a (cLEDlD24) was used as the
loading control. DNA probes were isolated and radiolabeled with [32P-a]dCTP as
described previously (Howe et al., 2000). The cDNA insert in EST clone cTOA14M17
was amplified by polymerase chain reaction (PCR) in a 100-pl reaction volume with
pBluescript SK(-) primers T3 and T7. PCR products were precipitated with ethanol and
re-suspended in 50 pl of elution buffer. 1 pl of this PCR product was used for probe
labeling. Hybridization results were visualized by autoradiographic exposure of hybrized

blots to Kodak XAR-S film.

Identification of MeJA-induced LAN CL gene

cDNA microarray experiments performed with the12K element TOM] slide (Cornell
University) identified 288 genes that are differently regulated (3-fold or greater) by the
JA signaling pathway (Zhao and Howe, unpublished data). One of the genes, represented
by two EST clones (cTOA14M17 and cLED6G16), was among those chosen for further
analysis. The Institute for Genomic Research (TIGR) and Solanaceae Genomics Network
(SGN) designation for this gene is TC101615 and SGN-Ul47059, respectively. EST
clone cTOA14Ml 7 was obtained from the Clemson University Genomics Institute
(Clemson, SC, USA). The identity of cTOA14M17 was verified by single pass DNA
sequencing at the MSU Genomics Technology Support Facility (MSU GTSF). The DNA
sequence of cTOA14M1 7 showed that it contained a full-length cDNA fragment. We
designated it as LeLANCL. Two genomic DNA fragments (l461-bp and 3370-bp)
containing part of the LeLANCL gene were amplified from wild-type genomic DNA.
Primers GCRF (5’- TTT CAG TTC CAT TTT CAG GAA-3’) and 14M17-496R (5’-CCA

TAA AGG AGG TCA TAT GAC AT) generate 3370-bp product. and primers 14M 1 7-524

37

(5’-GCC CTT CCT GTT GGA CCT GAA-3’) and GCRR (5’-CCT TCT GAT TGA CCT
TAT ATA-3’) generate 146l-bp product. After ligation into pGEM-T vector (promega),
these clones were sequenced. These two overlapping fragments encompassed the full-
length LeLANCL gene. The intron and exon organization of LeLANCL gene was deduced

by comparison of cDNA and genome sequences.

A grobacterium tumefaciens—mediated transformation

EST clone cTOA14M17 was digested with Xbal and Xhol to release the full-length
(1.7-kb) LeLANCL cDNA. This fragment was subsequently cloned in sense orientation
into the Xbal and Xhol sites of the binary vector pBITONY under the control of the
Cauliflower mosaic virus (C M V) 358 promoter. Similarly, a cDNA fragment was released
from EST cTOA14M17 by digestion of Xhol and Sac] and cloned in antisense orientation
into the Xhol and Sac] sites of pBITONY under control of the C MV 3SS promoter. These
two constructs were designed as S-LANCL and AS-LANCL, respectively. The inserts
and cloning junctions were sequenced to verify the construction. The S-LANCL and AS-
LANCL constructs were transformed into A grobacterium tumefaciens strain AGLO (Lazo
et al., 1991). A grobacterium-mediated transformation of tomato cotyledon explants was
performed as described previously (Li and Howe, 2001; Li et al., 2003). The presence of
the transgene in independently regenerated kanamycin-resistant transformants (T0) was
confirmed by PCR with primer set GRCF and GCRR or primer set l4M17-524 and
GCRR. The former primer set amplified 1.4-kb product corresponding to the transgene.
The later primer set amplified 1.48- and 0.9-kb products corresponding to the endogenous
LeLANCL gene and transgene, respectively. T0 lines were potted into standard soil mix

and grown in a growth chamber under standard conditions. Approximately 3 weeks after

38

transfer to soil, plants were assayed for wound-induced PI-II accumulation. T1 seed was

collected after T0 plants were transferred to greenhouse.

Results
Identification of a LANCL gene in tomato

The full-length LeLANCL cDNA contained a 1275—base pair (bp) open reading frame
(ORF) predicted to encode a 424-amino-acid protein. The genomic sequence of
LeLANCL was determined by analysis of genomic PCR products. Comparison of cDNA
and genomic sequences showed that the gene contained 5 exons and 4 introns (Figure
2.2). A BLAST search (Altschul et al., 1990) of the protein database at National Center
for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov) showed that
LeLANCL is similar to several lanthionine synthetase C-like (LANCL) proteins. Proteins
in this family are homologous to bacterial lanthionine synthetase (LanC) (Sahl and
Bierbaum, 1998), which is part of a membrane-bound complex involved in the synthesis
and transport of lantibiotic peptides exhibiting potent antimicrobial properties (Siegers et

al., 1996; Kiesau et al., 1997; Bauer et al., 2000; Mayer et al., 2001a).

The amino acid sequence of tomato LANCL shows significant similarity to LANCL]
and LANCL2 in human and mouse, which have been characterized in detail. A key
feature of all LANCL proteins is seven repetitive hydrophobic domains containing a
GXXG motif (Figure 2.3) (Mayer et al., 1998; Bauer et al., 2000), which is proposed to
be a single-strand nucleic acid binding surface (Musco et al., 1996). This general
repetitive structure of the seven hydrophobic domains is identical in all LanC-like protein

family members and most of LanC proteins in prokaryotes (Figure 2.3). Several

39

Am TAA

I77 203 112 234 576
NW - Uta “c1; D .' :54an t

94 305 158 82

 

 

 

 

 

 

 

 

 

 

A15g65280 (genome l94lbp/cDNA 1302 bp)

ATCI TIIA
159 I91 112 471 342

e'mumwk - -.'.-.i'.'a‘s '.x',t.‘o.«.-. . .‘1 , , '."~e .«veu n .vys-a. D gangrqga-avA-‘ucu. -"'b".‘lhwg','.,.~.q-;- - --.--:.I:z.-,:;‘-.- wag-had???

656 1247 992 580

LeLANCL (genome 4750 bp/cDNA 1275 bp)

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2.2. Gene structure of LeLANCL and its homolog in Arabidopsis.

Intron and exon sequences are indicated by horizontal lines and closed boxes.
respectively, and are drawn to same scale. Numbers above and below each gene denote
the number of nucleotides in each intron and exon, respectively. Only the translated
region of the first and last exon of each gene is shown, together with the start (ATG) and

stop (TAA or TGA) codons within the respective exon.

40

Figure 2.3. Amino acid sequence of LeLANCL and multiple alignment with other

members of the LanC-like protein family and four prokaryotic LanC proteins.

Amino acid sequences were aligned by C LUSTAL W method and shaded by DNASTAR
(MegAIign 5.06, DNASTAR Inc.). Identical residues are shown as white text on black
background. Similar residues are shown as black text on grey background. Conserved
residues are marked by black background. Two conserved glycine residues that are
essential for the function of the LanC protein EpiC from Staphylococcus epidermidis are
labeled ‘G’. Two cysteine residues suggested to form part of the active site of LanC
enzymes are labeled ‘C’. The name, species, and GenBank access number of each
proteins aligned are: LeLANCL (tomato), 7-TM-GPCR (potato, putative 7-
transmembrane G—protein-coupled receptor, AAF75794), At5g65280 (A. thaliana,
NP_201331), At1g52920 (LANCL2, A. thaliana, NP_175700), At2g20770 (LANCLI , A.
thaliana, NP_850003), OSJNBa0074L08 (OSJNBa0074L08.13, rice, CAD41202),
LANCL (rice, NP_922162), LANCLI (human LANCLI, CAA72205), LANCLI (mouse
LANCL], AAH58560), LANCLl (rat LANCL], XP_343585), LANCLl (zebrafish.
CAC39613), LANCL (frog, AAH51600), LANCL2 (human LANCL2, AAH70049),
LANCL2 (mouse, AAH16072), EpiC (LanC, Staphylococcus epidermidis, 823417), NisC
(LanC, Lactococcus lactis, Q03202), PepC (LanC, Staphylococcus epidermidis, 858361),

SpaC (LanC, Bacillus subtilis, P33115).

41

_—I————~u———u—I_u——|—n—~—

21
21
21
15
I7
20
46
15
15
15
17
15
38
37
28

47
47
B
42
n
57
87
33
33
33
35
32
74
74
47
23
w
23

----------------------- MSSSVVQFTASQQNNSDDGN--
----------------------- MYSSVVQLTASQKNNSDDGN--
------------------------ MSSSVDFVTEQGRCGDDGNG-
--------------------------- MPEFVPEDLSGEEE----
.......................... MAGRFFDNVMPDFVKE---
-------------------------- MADRFFPNNMPGYADEGAP
MGNGGGGNKKGRRDSPSPPLETPPRGELPSTSSSSSSRATKRHRV
-------------------------- MAQRAFPNPYADYN-----
-------------------------- MAQRAFPNPYADYN-----
-------------------------- MAQRAFPNPYADYN-----
------------------------- MSEQRALKNPYPDYTG----
-------------------------- MEQRCFQNPYPDYN-----
........ MGETMSKRLKLHLGGEAEMEERAFVNPFPDYEAAAGA
........ MGETMSKRLKFHLG-EAEMEERSFPNPFPDYEAAASA
----------------- LAVLYTCVVIEYSVLILKKKNLFYLFL-

---------------------------- MRIMMN-----------
................................ MN..---------
-------------------------- MERGTVSR-----------
------------- ERLDS------DQHLHPAVHSVPHTSETFLQA
------------- ERVDT------DQHHQPTAHSVSHTSETFLQA
----------- AGETVKNGEl--DHLLSEPSAPTISLPTESFLRA
-------------- TVTECK----DSLTKLLSLPYKSFSEKLHRY
PPPA----AAAAAAIPSTYS----SSLHHLLSLPYPDLADRFLHA
AGMADRYFPNDLPDFVAEAP----DGGRGLLSLPYSSLSERLLRA
------------ KSLAEGY---------------FDAAGRLTPEF
------------ KSLAENY---------------FDSTGRLTPEF
------------ KSLAENY---------------FDSTGRLTPEF
------------ LGCAQDL---------------FDMQGNLTQHF
------------- HQASTA---------------FNSTGQLTFEF
L-LA -------- SGAAEETGCVRPPATTDEPGLPFHQDGKIIHNF
AGLA -------- AGSAEETGRVCPLPTTEDPGLPFHPNGKIVPNF
---------- MKLQKLKN----------------IGMVVININNl
----------- KKNIKRN----------------VEKIIAQ“D--
------------- RFLNT----------------YIKQVTNIDNV
---------- IEVEIVKE----------------MARQISNYDKV
AISLKDQVVEMTWK ----- ENGRSASSVTDPTMYT AFTCL
AISLKDQVVEMTWK ----- ENGRSAGSVTDPTMYI AFTCL
ATLLKNQVVEATWKGGV-EALASGSGPVLDPTVYT AFTCL
ALSIKDKVVWETWE -------- RSGKRVRDYNLYI AYLLF
------ KEMIET“G--------FSGQTVEDFTLYS AFLLF
ALHLKQKVVHETWDKRR-RAAAAAGEAVGDFTLYT ALLLF
ALRIKDKVMEETWT -------- RARRQVTDYTLYI ALLLF
SQRLTNKIRELLQQ----MERGLKSADPRDGTGYT AVLYL
SHRLTNKIRELLQQ----MERGLKSADPRDGTGYT AVLYL
SHRLTNKIRELLQQ----MERGLKSADPQDGTGYT AVLYL
ATSISSKISELLAI----LENGLKNADPRDCTGY1 ALLYL
VHCLNNKIKELLQA----MEKGLKSADPGDCTVYI ALLYL
lRRIQTKlKDLLQQ----MEEGLKTADPHDCSAYT ALLYL
IKRIQTKIKDLLQQ----MEEGLKTADPHDCSAYT ALLYL
KKILENKITFLSDIEKATYIIENQSEYWDPYTLSH ILFLS
ERTRKNKENFD ---------------- FGELTLST ILMLA
DSYINNLYGPEP --------------- IYKASLII AISLF
LEIVNQKDNFRS-IGEVPLIP ------ “KSTALSH CMLYG

 

LeLANCL
7-TM-GPCR(potato)
At5g65280
Atlg52920
At2g20770
OSJNBa0074L08(Rice)
LANCL(Rice)
LANCL1(Human)
LANCL l(Mouse)
LANCL l (Rat)
LANCLl(zebrafish)
LANC L( Frog)
LANCL2(Human)
LANCL2(Mouse)
EpiC

NisC

PepC

SpaC

LeLANC L
7-TM-GPCR(potato)
At5g65280

At 1 g5 2920
At2g20770

OSJN Ba0074L08(Rice)
LANCL(Rice)
LANC L 1 (Human)
LANC L 1 (Mouse)
LANC L l (Rat)
LANC L l (zebrafish)
LANCL( Frog)
LANCL2(Human)
LANCL2(Mouse)
EpiC

N isC

PepC

SpaC

LeLANC L
7-TM-GPCR(potato)
At5 g65280

At 1 g5 2920
At2g20770

OSJN Ba0074L08(Rice)
LANCL(Rice)
LANCL 1 (Human)
LANC L 1 (Mouse)
LANC L l (Rat)
LANCL1(zebrafish)
LANCL(Frog)
LANCL2(Human)
LANCL2(Mouse)
EpiC

NisC

PepC

SpaC

Figure 2.3. Amino acid sequence of LeLANCL and multiple alignment with other

members of the LanC-like protein family and four prokaryotic LanC proteins.

42

87

RS YESTGDRKDLELC‘S El VDSCADLARTFTRH-

 

 

m RSYEATGDRKDLELCSEIVDACADLARTVTRH-

W KSYEVTRNHQDLLTCAEIIDTCANVARATTRH-

m KSYQVTRNEDDLKLCLENVEACDVASRDSE-R-

“ RAYQVTGNANDLSLCLEIVKACDTASASSG-D-

m1 RAYLVTGDRADLATCAEIVAACDAASMGAE-l-

D4 KSFQVTGNRADLALAGDIVKECDAASRGLP-F-

M HLYDVFGDPAYIQLAHGYVKQSLNCLTKRS---

M HLHNVFGDPAYLQMAHSYVKQSLNCLSRRS---

M HLHNVFGDPAYLQMAHSYVKHSLNCLSRRS---

% HLHSVFGDPTFLQRALDYVNRSLRSLTQRWL--

n HLSDVYGDSSLLQKAHEYICKSLRCLTRRD---

H5 QLYRVTCDQTYLLRSLDYVKRTLRNLNGRR---

H5 QLYRVTGDQTYLLRSLDYVKRTLRNLSGRR---VTFLCHDAUPLA
Q ASEKVFHKD-LEKVIHQYlRKLG-PYLESGIDGFSLFSMLSUIGF
fl ELKNKDNSKIYQKKIDNYIEYlVSKLSTYGLLTGSLYSn. '

49 AIYKETNNFEYYELCNKYLEKTIELINDTPMYSTSLFEM

w ELHAHFPEEGWDDIGHQYLSILVNElKEKGLHTPSMFSM

n1 LGAVAASYCGDQHKRDLYLNHFLEVAQ ----- ERALPVG----PE
Bl LGAVAANYCGDQHKRDLYLNHFLEVAQ ----- ERALPVG----PE
Ml LGAIVANYRGDQSKRDFFLGLFLELAE ----- ERELPAG----PE
n2 LGAVAAKCLGDDQLYDRYLARFRGIRL ----- PSDLPY -------
m LGAVAAKLSGEEDLLNYYLGQFRLlRL ----- SSDLPN .......
M4 LGAVVAKHAGDEAGVAHYLSAFKElKl ----- HSKSPD -------
m7 LGAVIAKHCNDQLLLTHYLSSFDEI[V ----- TEKVPN -------
H6 VAAVLYHKMNNEKQAEDCITRLIHLN----KIDPHAPN -------
H6 VAAVLYHKMNSEKQAEECITRLIHLN----KlDPHVPN -------
H6 VAAVLYHKMNSGKQAEDCITRLIHLN----KIDPHVPN -------
H8 IAAVVYHRLQKHQESDECLNRLLQLQPSVVQGKGRLPD -------
H5 VGAVVFQKLGLTKEAEDCVKSLLQLHPSVVRPDSGLPD -------
U7 VGAVIYHKLRSDCESQECVTKLLQLQRSVVCQESDLPD -------
U7 VGAVIYHKLKSECESQECITKLLQMHRTIVCQESELPD -------
us ALDlASDKQYSYQSlLEQlDNLLVQYV ----- FDFLNN ----- DA
w SILHLREDDEKYKNLLDSLNRYIEYFV ----- REKlEGF----NL
M SLLVCSDSGSNYSNIIKNLLFEYKKIS ----- KNEIDRLRTKLKN
HI AAICLSQRFTYYNGLISDINEYLAETV ----- PQLLTEF----DQ
m7 DGGFGMSYDLLYM 7 LumALFIRKYLGv---ESVPDDSLMPVV
m7 DGGFGMSYDLLYMRAU!LW%ALFIRKYLGV---ESVPDDYLMPVV
H7 EGGFGMSYDLLY‘” ‘FL“MALFLNRYLGQ---GTVPDHLLSPIV
U5 -------- ELLYURAMYL“%CLFLNKHIGQ---ESlSSERMRSVV
n4 -------- EILvuRvu LW%CLFINKYIGK---ETLSSDTIREVA
n7 -------- ELLYURAM LWfiCTFLNKHLGD---NT1PPTTTDTVM
m0 -------- FLth LW%CLFLNTHLGE---KTIPHEHITSVA
U0 -------- EMLYURI lYALlFVNKNFGV---EKIPQSHIQQIC
Ho -------- EMLYHRIH lFALLFVNKNFGE---EKIPQSHIQQIC
Ho -------- EMLYthh lFALLFVNKNFGE---EKIPQSHIQQlC
H6 -------- ELLYU LYSLIFVNQQFQQ---EKIPFQY|QQIC
U3 -------- ELLYh LYSLLFVNKQFGE---EKIPSSYlQQVC
ms -------- ELLYMRAM LYALLYLNTE]GP---GTVCESAIKEVV
ms -------- ELLY‘ LYALLYLNTEIGP---GTVGETAIKEVV
no LEVTPTNYDIIQM GRYLLNRISYNYN---AKKALKHILNYF
B3 ENITPPDYDVIEMLSM LSYLLLINDEQYD ----- DLKILIINFL
B4 NNIQFYEFDIISM LS-LLLLATDIFP ----- ELSELLVDEI
M7 RQVCMSDYDVIEM ANYLLLFQEDKAMGDLLIDlLKYLVRLT
Figure 2.3. (cont’d)

43

LeLANC L
7-TM-GPCR(potato)
At5g65280

At 1 g52920
At2g20770
OSJNBa0074L08(Rice)
LANC L( Rice)
LANC L 1 (Human)
LANCL 1 (Mouse)
LANC L l (Rat)
LANC L l (zebrafish)
LANCL( Frog)
LANCL2( Human)
LANC L2(Mouse)
EpiC

NisC

PepC

SpaC

LeLANC L
7-TM-GPCR(potato)
At5g65280

At 1 g52920
At2g20770
OSJNBaOO74L08(Rice)
LANCL(Rice)
LANCL 1 (Human)
LANCL 1 (Mouse)
LANCL l (Rat)
LANC L l (zebrafish)
LANCL(Frog)
LANCL2(Human)
LANCL2(Mouse)
EpiC

NisC

PepC

SpaC

LeLANC L
7-TM-GPCR(potato)
At5g65280

Atl g52920
At2g20770

OSJN Ba0074L08(Rice)
LANC L( Rice)
LANCL1(Human)
LANC L 1 (Mouse)
LANCLl(Rat)
LANCL l (zebrafish)
LANC L( Frog)
LANCL2(Human)
LANCL2(Mouse)
EpiC

NisC

PepC

SpaC

  
  
 
 
    
 
 

  

  
     

  
  

m9 EAlLAGGRAC----ASDNSA---CPLMYRWHGTRY 7
m9 EAlLAGURAG----ASDNPA---CPLMYRWHGTRY I
M9 AA!LAGURVG----AADHEA---CPLLYRFHGTRF
H9 EEIFRAGRQ ----- LGNKGT---CPLMYEWHGKRY 1
Ha QEllKEURS ----- MAKKGS---SPLMFE“NGKRY 7
MI RDIIRDGRT ----- LSTlG--~-CPLMYE“NGEKY 1
M4 KDllDEURK ----- LAKKGN---CPLMYEWHGKKY
H4 ETILTSGENL----ARKRNFTAKSPLMYFWWQEYYV
H4 EN!LTSUENL----SRKRNLAAKSPLMYEWWQEYYV
H4 ETILTSGEKL----SRKRNFTTKSPLMYEWNQEYYV l
H0 DAthSGQlL----SQRNKlQDQSPLMYEWWQEEYV l
H7 STVLESUERL----AGRRNLQSQSPLMHEWNREYYV 1
n9 NAIIESUKTL----SREFRKTERCPLLYQWMRKQYV l
n9 SAllESGKSL----SREERKSERCPLLYQWHRKQYV
M2 KTlHYSKD---N“lVSNEHQFLDIDKQNFPSGNINL P
n3 SNLTKENNGLISLYlKSENQMSQSESEMYPLGCLN v
n3 VQITSlLT---ELV1KFNNDDYLLDTILS ..... NL l
H2 EDllVDGEKVPGWHlPSQHQFTDIEKKAYPYGNFN P
G
M7 LHV HFPLSQED----IEDVKETLRYMMSNRPPHSGVYPVS---
M7 LHV HFPLSQED----lEDVKKTLRYMMSNRFPHSUNYPVS---
M7 LYV HFPLSEED----VKDVQGTLRYMMSNRFPNSG\YPCS---
n6 MNV TELEPDE--.-1KDVKGTLSYMIQNRPP-suvass--.
H5 MHV VQLKPDE----AFDVKGTLKYMIKNRFP-SUVYPAS---
M7 MHV DMDLTKDD----TECVKGTLRYMlQNRYP-SUNYPVT---
n1 MHV MHTELKLDE----KDDVKNTLLYM]RNRYP-TUNYPSS---
Ms YYY QPSLQVSQGK-LHSLVKPSVDYVCQLKPP-sn\YPPC---
n5 YYY QPSLQVNQGK-LHSLVKPSVDFVCRLKFP-SUNYPPC---
ns YYY MQPSLHVSQGK-LHSLVKPsvoncoLKrP-soNYPsc---
M1 YYY QPULVAGQDR-VFSLVKPSVNYVCQLKFP-SU\YAPC---
us YYF QPECNVSCEK-LQNLVRPSMEYVRCLKPP-TGVFPPC---
no YY QPAAKVDQET-LTEMVKPSIDYVRHKKrR-SGVYPSS---
NO YY QPEAKVDQET-LTEMVKPSIDYVRHKKFR-SUNYPSS---
M4 LSLTALSKMNGlElEGHEEFLQDFTSFLLKPEFKNNNEWFDR---
Ms GCl YAHIKGYSNEASLSALQKIlFIYEKFELERKKQFLWKDGL
M0 INT NSYKRGYGllKTKKlLEQSIFTLLQNLKLENGTIYIP---
M7 lCV SALlQGlKVKGQEAAIEKMANFLLEFSEKEQDSLFWKGII
M5 --EGNP ----- RDK---LVQ T TITMCKVSKVLSDDREF
H5 --EGNP ..... RDK---lVQ -T TITMCKVSEVLSDDREF
M5 --EGNP ----- RDK---LVQ ~T AlTIAKASQVFPKERDF
28 --EGSK ----- SDR---LVH AP ALTLVKAAQVYN-TKEF
M2 --EEDKK----KDl---LVH -P lALTLGKAAEVFG-EREF
M4 --EEDK ----- HDR---FVH -P SLTLAKASQVFP-EERF
ms --EGSE-----SDR---LVH P AlTlAKAYQVFH-DEHF
M5 --IGDN ----- RDL---LVH -P IYMLIQAYKVFR-EEKY
M5 --LDDT ----- RDL---LVH P lYMLlQAYKVFK-EERY
2“ --LDDT ----- RDL---LVH P IYMllQAYKVFK-EEHY
M1 --VGDA ----- RDL---LVH SP lYMLlQAFKVFG-VRQY
2H --lGDR ----- RDL—--lVH P IYMLIQAYKVFG-EPQY
no --LSNE ----- TDR---LVH -P lHMLMQAYKVFK-EEKY
no --LSNE ----- TDR---LVH -P lHVlLQAYQVFK-EFKY
M6 -YDlLENYlPNYSV---RNG YMIT MNTLLLSGKALN-NEGL
M3 VADELKKEKVIREASFIRDA P lSLLYLYGGLALD-NDYF
32 --NDlES---PNDY---RDA LPSVAYTIFNVSSTLK-NKSL
n2 SFEEYQYGSPPNAVNFSRDA 'PlVCLALVKAGKALQ-NTEL
Figure 2.3. (eont’d)

44

Lel .ANCL
7-TM-GPCR(potato)
At5g65280

At 1 g52920
A12g20770

OSJ NBa0074L08(Rjee)
LANCL(Rice)
LANCl,l(Human)
l.ANCLl(Mouse)
l.ANCl.l(Rat)
LANC I . l (zebrafish)
LANCL(Frog)
LANCL2(Human)
LANCL2(Mouse)
EpiC

NisC

PepC

SpaC

LeLANCL
7-TM-GPCR(potato)
At5g65280

At 1 g52920

At2

OSJN 830074 L08( Rice)
LANCL(Rice)
LANC L 1 (Human)
LANCL 1 (Mouse)
LANCL 1 (Rat)
LANCL l (zebrafish)
LANCL(Frog)
LANC L2(Human)
LANCL2(Mouse)
EpiC

NisC

PepC

SpaC

LeLANCL
7-TM-GPCR(potato)
At5g65280

Atl g5 2920
At2g20770
OSJNBaOO74L08(Rice)
LANC L(Rice)

LAN CL 1 (Human)
LANCL 1 (Mouse)
LANCL | (Rat)
LANCL l (zebrafish)
LANCL( Frog)
LANCL2(Human)
LANCL2(Mouse)
EpiC

NisC

PepC

SpaC

no RDAAIEGGEVVWRSGIVEK ..... VGLAD s AYAFLSLYRLT
no RGAAlEGGEVVWKShLVER ----- VULAD s AYAFLSLYRLT
Mo REAAIEAonvuKsoLVKK ----- VGLAD VA AYAFLSLYRLT
n7 VEAAMEAGuvvusRGILKR ----- VGICH IS NTYVFLSLYRLT
M7 LEASAAAAEVV“NRM1LKR ..... VGICH lS AYVFLALYRAT
M8 LEAIAEAAvauNRoLIKR ----- VGICH vs AYTFLALFRLT
M2 KQTAAEAAtVVWNRULLKR ----- VGICH s AYVFLSLYRLT
”9 LCDAYQCADVIuQYUILKK----GYGLCH ‘A AYAFLTLYNLT
M9 LCDAQQCADVI“QthLKK----UYULCH A AYAFLALYNLT
m9 LCDAQQCADVIquoLLKK----GYGLCH A AYAPLALYNLT
M5 LEDALQCGPVIuoRo:LKK----GYGLCH A AYGPLALYKIT
M2 LVDALQCAEVA“MYULLKK----GYGLCH A AYSFLALYNQT
M4 LKEAMECSDVIuokoLLkK----GYGICH A NGYSPLSLYRLT
M4 LKEAMECSDVIuoRoLLRK----ovolcu rs GYSFLSLYRLT
M6 lKMSKNlLlNllDKNNDD--L|S-PTFCH LASHLTIIHQANKFF
M7 VDKAEKILESAMQRKLG---lDS-YMICH SMLIEICSLFKRLL
Ms lELSESLLHOVFLRSDNATKLlS-PTLCH FShVVMlS----LLM
n6 lNlGVQNLRYTlSDlRG---lFS-PTICH SMIGQILLAVNLLT
M0 GESIYEERAK ------- AlASCIYQNARTIMNEREHNEA ----- D
M0 GESIYEERAK ------- AtASCLYQNARTlMNERHHNEA ----- D
no GDVVYEER\K ------- Al\SYLCRDAlELVN-MTSQET ----- E
M7 RNPKYLYRAK ------- AIASFLLDKSEKLISEGQMHGG ----- D
M7 GRSEYLYRAK ------- ArAsPLLDRGPKLLSKGEMHGG ----- D
M8 KKKEHLYRAK ------- APACPLLDRAKQLIADGIMHSG ----- D
M2 GNVEYLYRAK ------- APACFLLEKADQLIADGAMHGG ----- D
Mo QDMKYLYRAC ------- KIAEurLuYGEHGCR ----- TP ----- D
Mo QDLKYLYRAC ------- KrAuurLDYGEHGCR ----- TA ----- D
Mo QDAKYLYRAC ------- KrAnerDYGPHGCR ----- TP ----- D
M6 QDPKHLYRAC ------- MPADwrMNYGRHGCR ----- TP ----- D
M3 QDVKFLYRAC ------- KYAEWTMDYGTHGCR ----- TA ----- D
M5 QDKKYLYRAC ------- KTAEWTLDYGAHGCR ----- lP ----- D
M5 QDKKYLYRAC ------- KIAPWtLDYGAHGCR ----- lP ----- D
M8 NLSQVSTYIDTIVR---KlISHYSEESSFMFQDIEYSYG-QKIYK
M8 NTKKFDSYMEEFNVNSEQlLEEYGDESGTGF--LEGISG-ClLVL
n8 NNNELSSKYQ--K----KllQSYlDQlDGLYFDlNDPSN----FS
M7 GQEYFKEELQEIKQ---KlMSYYDKDYIFGFHNYESMEGEEAVPL
M3 ACPIPD-—.LLAPKNSRPPUPIL

M3 ACFLFD---LLAPKNSRFPGYEL

M2 VCLWFD---LVSPVDSKFVGYLI

M0 AYMLLD---MNDPTQALFPGYtL

Mo AYLFLD---MVDPSEARFPGYFL

M1 AYLPLD-.-MINPLDSRPPGYPL

M5 AYLLLD---MVSPsESKPPAYrL

M8 IYPIAD...LLVP7KARPPAPLL

M8 lYFlAD---LLVPTKAKFPAFLL

M8 lYFLAD---LLVPTKAKFPAFYL

M4 rIYPLAD---LLQPARAKPPCPPV

3n lYFISD---lLEPTKAKFPSF[M

M3 IHFLSD—..VLGPETSRPPAPPLDSSKRD

M3 VHrLsn---1LVPETARFPAPPLGPLQKD

M9 LLAlLDYIDTQNQSRKNWKNMFLIT

M0 SKFEYSlNFTYWRQALLLFDDFLKGGKRK

M3 KDIGLLNIEAHLLLTLLSYD-NNKLINlRWFDFMIMS

M9 QYVGLLD v GLGVLN---MELGSKTD“¢KALLI

Figure 2.3. (cont’d)

 

45

LeLANCL
7-'I‘M-GPCR(potato)
A15g65280

Al I g52920
At2g20770
()S.lNBaOO74L08(Rice)
LANCL(Rice)
LANCL 1 (Human)
LANCLHMouse)
LANCLHRal)
IPANC‘L l (zebrafish)
LANCHFrog)
llANCl.2(Human)
l,/\N(‘L2(Mouse)
1mm

NisC

PepC

SpaC

LeLANCL
7-’l‘M-GPCR(potato)
At5g65280

AI] g52920
At2g20770
OSJNBa0074L08(Rice)
LANCL(Rice)
LANCLl(Human)
LANCLI(Mouse)
LANCLl(Rat)
LANC L l (zebrafish)
LANCL(Frog)
LANCL2(Human)
LANCL2(Mouse)
EpiC

NisC

PepC

SpaC

LeLANC L
7-TM-GPCR(potato)
At5g65280

Atl g52920
At2g20770
OSJNBa0074L08(Rice)
LANCL(Rice)
LANCLl(Human)
LANCLI(Mouse)
LANCL|(Rat)
LANCLI(zebrafish)
LANC I4( Frog)
LANCL2(Human)
LANCL2(Mouse)
EpiC

NisC

PepC

SpaC

residues are conserved within the single repeats (Figure 2.3). It is likely that these
residues are important for the function of LanC-related proteins. The conserved Gly
residues in repeats 2 and 4 (marked by “G” in Figure 2.3) were shown by mutation
analysis to be essential for the function of the LanC protein EpiC (Kupke and Gotz, 1996).
The Cys residues in repeats 5 and 6 (marked by ‘C’ in Figure 2.2) were suggested to play
a role in the active site of LanC enzymes ( Kupke and Gotz, 1996; Okeley et al., 2003).
However, these two Cys residues are not conserved in LeLANCL (Figure 2.3). The high
degree of evolutionary conservation within the LanC-like protein family suggests that

these proteins play a fundamental role in animals and plants (Mayer et al., 2001a).

LeLANCL is most closely related to a gene from Solanum chacoense (potato; 94%
identity) that is annotated as a putative 7-transmembrane G-protein-coupled receptor
protein (7-TM-GPCR) (Figure 2.3 and 2.4). In Arabidopsis, three genes are similar to
LeLANCL (Figure 2.4). Two of these, At2g20770 and At1g52920, are known to be
related to LANCL] and LANCL2, respectively (Mayer et al., 2001a). The third one,
At5g65280, is more related to LeLANCL (63% identity) (Figure 2.4). Two cysteine
residues in repeats 5 and 6 are not conserved in LeLANCL, At5g65280, and the S.

chacoense protein (Figure 2.3).

LeLANCL expression is highly induced in response to MeJA and mechanical

wounding

RNA gel blot analysis was used to confirm the cDNA microarray analysis showing

that LeLANCL is positively regulated by JA. LeLANCL transcripts began to accumulate

46

LANCL1 (Mouse)

LANCL1 (Rat)
LANCL1 (Human)
LANCL1(zebrafish)
LANCL(Frog)
l: LANCL2(Human)
LANCL2(Mouse)
i: LeLANCL
7-TM-GPCR(potato)
At5965280
OSJNBa0074L08(Rice)
LANCL(Rice)
‘ “‘ At1g52920
At2920770
[ EpiC
SpaC
NisC
PepC

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2.4. Phylogenetic relationship of LeLANCL’ to other LanC and LanC-like

proteins.

A rooted phylogenetic tree was constructed with the MegAlign 5.06 (DNASTAR Inc.)

based on the alignment by CLUSTAL W method.

47

Wild-type iai1
00.5124812240481224hrs

 

 

‘1‘“

"Ia-r A ..‘. ~ -
; ‘ .uI-‘r 15.4.411. ant-mafia
‘4 . 3 ' .' 20:41...‘ q. ‘
I “ “m; 1' PI-I/

. f. _ _
{iv-Vei' “A. ..' "
8+. w. '81:“, . .

   

"""""‘ ‘""' """* LeLANCL

eIF4a

EtBr

 

Figure 2.5. LeLANCL expression in response to exogenous MeJA in wild-type and

jail plants.

Three-week-old wild-type (cv Micro-Tom) and jail (IA-insensitive mutant) plants were
exposed to MeJA vapor for various lengths of time (hrs) in an enclosed box. Leaves from
plants of the same genotype were pooled and harvested for RNA isolation at different
time points. RNA isolated from untreated plants (O-hr time point) also was analyzed as a
control. RNA gel blots were hybridized to the LeLANCL cDNA and the well-
characterized JA response gene, PI-II. Blots also were hybridized to an eIF4a probe as a

loading control. Ethidium bromide (EtBr) staining was used to verify the quality of RNA.

48

within 1 hr of Me] A treatment and remained elevated for at least 24 hrs (Figure 2.5).
LeLANCL expression was not detected in untreated leaf tissue. These results indicate that
the expression of LeLANCL in tomato leaf tissue is JA-inducible. To determine whether
induced LeLANCL expression in leaves is dependent on a functional J A signaling
pathway, we measured the expression of LeLANCL in a tomato mutant (jaiI) that lacks
C011 (Li et al., 2004). The results showed that jaiI plants are deficient in MeJA-induced
expression of LeLANCL (Figure 2.5). We conclude that the expression of LeLANCL is

induced by J A in tomato leaves in a C011-dependent manner.

LeLANCL expression also was highly induced by mechanical wounding (Figure 2.6).
LeLANCL was expressed 0.5 hr after wounding and transcripts reached a maximum level
1 hr after wounding. After 8 hr, expression declined to a low level. To determine whether
wound-induced expression of LeLANC L depends on J A, expression of the gene was
analyzed in the spr2 mutant that is defective JA biosynthesis (Li et al., 2003). The results
showed that LeLANCL expression was undetectable in spr2 plant after wounding.
Consistent with a previous study showing that spr2 plants are fully responsive to
exogenous JA and its metabolic precursors (Li et al., 2003), expression of LeLANCL was

induced by exogenous Me] A in spr2 plants (data not shown).

T issue-specific expression of LeLAN C L

RNA gel blot analysis was used to determine the expression pattern of LeLANCL in
various tissues of tomato. In wild-type plants, LeLANCL transcripts were relatively
abundant in flower, especially in mature unopened flowers and young flower buds

(Figure 2.7). Lower expression was observed in stems and fruits. This highest expression

49

Wild-type spr2

 

 

 

o .5 1 2 4 8 12 24 36 o 4 12 24 hrs
PI-Il
LeLANCL
«W W m in. 1% w m m "'4' ”fl" ‘ e, F 48
that u w x 4.
EtBr

Figure 2.6. LeLANCL expression in response to wounding in wild-type and spr2

plants.

Two-leaf-stage wild-type (cv Micro-Tom) and spr2 (J A biosynthesis mutant) plants were
mechanically wounded on both lower and upper leaves. Total RNA was extracted from
the wounded leaf tissue at different times after wounding. RNA isolated from unwounded
plants (O-hr time point) also was analyzed as a control. RNA gel blot was analyzed as

described in Figure 2.5.

50

Sd R S L B UF OF IF GF

...
t
I

. u . w M. « l LeLANCL

 

m eIF4a

5517.111...’

 

Figure 2.7. Expression pattern of LeLANCL in different tissues.

Blots containing total RNA from seedlings (Sd), roots (R), stems (S), leaves (L),

developing flower buds (B), mature unopened flowers (UF), mature opened flowers (OF),
small (<0.5cm) immature green fruits (IF), and mature green fruits (GF) from Micro-Tom
plants were hybridized to a LeLANCL cDNA probe. Blots also were hybridized to a probe

for eIF 4a as a loading control. EtBr staining was used to verify RNA quality.

51

level in reproductive tissues is consistent with the fact that almost all tomato ESTs
corresponding to LeLANCL were identified in cDNA libraries constructed from either

flower or ovary mRN A (TIGR, http://www.tigr.org).

Construction and preliminary characterization of transgenic plants altered in

LeLANCL expression

To begin to assess the function of LANCL in plants, we constructed transgenic
tomato plants that overexpress the LeL/lNC L cDNA in either the sense or antisense
orientation. The LeLANCL cDNA was ligated into pBITONY vector in either sense (S-
LANCL) or antisense (AS-LANCL) orientation under the control of the CMV 35S
promoter. A grobacterium-mediated transformation was used to transform the constructs
into Micro-Tom plants. Thirty-one regenerated primary (T0) lines of S-LANCL and 23
lines of AS-LANCL were obtained. PCR analysis indicated that most T0 plants contained
the transgene (Figure 2.8). In 31 overexpression lines, only 2 of them (# 4 and 22) had no
LeLANCL transgene. In 23 antisense lines, 5 of them (# 3, 10, 18, 19, and 23) had no
transgene. T1 lines homozygous for the transgene were identified and characterized. PCR
analysis was performed to determine the presence of transgenes in T1 plants (Figure 2.9),
which showed that transgenes were separated in some Tl plants from the same T0 line.
However, in other T1 plants (S-LANCL-6 and AS-LANCL-l) from same T0 line
transgenes were not separated. This maybe due to the multicopy of transgenes in these T0

lines.

To determine whether the transgenic plants are affected in the normal wound

response, wound-induced systemic PI-II expression was measured in transgenic plants.

52

Figure 2.8. PCR-based detection of transgenes in S-LANCL and AS-LANCL

transformants.

The presence of the transgene in regenerated primary transformants (T0) was verified by
PCR with primer sets GCRF and GCRR (Figure A), or 14Ml7-524 and GCRR (Figure B
and C) in S-LANCL lines (Figure A and B) and AS-LANCL line (Figure C). Micro-Tom
served as the wild-type (WT) control. S-LANCL (Figure A and B) and AS-LANCL
(Figure C) constructions in pBITONY served as positive control (C) for PCR reaction.
Primer set GCRF and GCRR amplified 1.4-kb products corresponding to S—LANCL
transgene. The endogenous genomic PCR product was not detectable, presumably due to
its large size (4.7 kb) (Figure A). Primer set 14Ml7-524 and GCRR amplified 1.48- and
0.9-kb products corresponding to the endogenous LeLANCL and transgenes (both S-

LANCL and AS-LANCL), respectively (Figure B and C).

53

(A) (B)

S-LANCL T0 S—1./\NCL T0

 

 

WTCI 2 3 J (a T 8 9101112111415161718 \\'1’C Z 410192021113314353617 382931131712

    

14 kb ------------_.
"" 148 kl)

—-—-— ——---—-—_u— I) 1) kl)

(C)

AS-LANCLTO
WTC12 3 4 5 6 7 8 91011121314151617181920212223

 

1.48 kb
0.9 kb

 

Figure 2.8. PCR—based detection of transgenes in S-LANCL and AS-LANCL

transformants.

54

A

Transgene: - - - + - - + + + + + + + + + + + + — +
120 PI-ll

100

Pl-Il (pg/ml leaf juice)

 

  

$533;

  

 

 

 

 

 

 

   

 

 

:2:
24 y y
W1" 1 2 3 4 5 9 1 2 3 4 5
B S-LANCL-Z S-LANCL—7
Transgene: + + + + + + + + - + - - + + + + + + — + + +
A 90 E {III-Ill 2,
3 80 fl,
3;: 70
'6': so
a) ' I?» '2" ’
Z 50 >. 8 ,,. p. 2 «21" 9.4:: ya .. 2:2. 2- .-
E In: , it 3:. i x 8?“ " 'b‘ 323 l .3
T» 4‘" 3" 1" ”i? it i 3% E i " 23? ,2 § § 22 1“ ‘3‘:- V
3 30 " '2' E. a... - ' '
-_- 20 :2 . g, 2;; g it - 13;; g; a ..
_. f .1 2: 4.9 f. i . 3:25 if
D- 102 "3 2% 2: E 4 g: § 1 E: g 2 q
. 2' V ,‘3 f; ' 3 J L2,;
0 ‘ : w. 2 .- -. ‘ ’;
WT 1 2 3 4 6 7 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14
AS—LANCL-1 AS-LANCL-2

Figure 2.9. PI-II levels in LeLANCL transgenic plants.

Three-week-old T1 plants harboring S-LANCL (A) or AS—LAN CL (B) transgenes were

wounded with a hemostat across the midrib of all leaflets (typically three) on the lower

leaf. Twenty-four hours later, PI-II levels were measured in upper unwounded leaves by

radial immunodiffusion assay. The TI transgenic plants were scored for the presence (+)

or the absence (-) of the LeLANCL transgene by the PCR assay described in Figure 2.8.

55

Upon wounding, all S-LANCL lines and AS-LANCL lines showed wound response
similar to that observed in wild-type plants (Figure 2.9). These results suggest that

LeLANCL is not involved in regulating Pl-II expression in wounded tomato plants.

Discussion

Here, we report a new member of LANCL protein family from tomato, LeLANC L.
Interestingly, the expression of LeLANC L was highly induced in tomato leaves in
response to mechanical wounding and treatment with applied Me] A (Figure 2.5 and 2.6).
Wound-induced expression of the gene was blocked in tomato mutants that are defective
in .1 A biosynthesis (spr2) or JA perception (jaiI). RNA gel blot analysis showed that
LeLANCL mRNA accumulated to high levels in flower tissue, which constitutively
accumulates high levels of JA (Hause et al., 2000). Taken together, these results indicate

that the expression of LeLANCL is regulated by the IA signaling pathway in tomato.

Although the function of LANC L proteins in eukaryote is unknown, the high degree
of conservation within the LanC-like protein family, notably within the seven
hydrophobic repeats, suggests that these proteins play a fundamental role in animals and
plants (Mayer et al., 2001a). The presence of the GXXG motif in LANCL proteins
suggests that those proteins may function as single-stranded nucleic acid-binding proteins
(Musco et al., 1996; Park and James, 2003). Based on its homology to the LanC protein,
and its wound- and JA-inducible expression in tomato leaves, LeLANCL may function as

a peptide-modifying enzyme in plant defense against herbivores or pathogens.

Two Cys residues in LanC (marked by ‘C’ in Figure 2.3) were suggested to play a

role in the active site of LanC enzymes (Okeley et al., 2003). In a working model, Okeley

56

et a1. (2003) suggested that the two conserved cysteines may provide two of the ligands to
zinc, which may function to activate the Cys thiol of the peptide substrate toward
intramolecular Michael addition of the dehydroalanine and dehydrobutyrine residues.
However, these two Cys residues are not found in LeLANCL (Figure 2.3). These results
indicate that LeLANCL maybe have a different function than LANCL proteins in

eukaryotes and LanC proteins in bacteria.

57

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60

CHAPTER 3

Characterization of the Tomato le Wound Response Mutant

Map-based cloning of ACX ] was done by Dr. Chuanyou Li.

Dr. Sastry Jayanty measured the level of J A.

Dr. Bonnie McCaig provided the RNA used for the Northern blot in Figure 3.5.

We thank Dr. Yuichi Kobayashi for kindly providing the OPC-8:0 used in this study.

61

Abstract

The activation of proteinase inhibitor (PI) expression in tomato plants in response to
mechanical wounding and herbivore attack is mediated by jasmonic acid (.IA). J A is
biosynthesized from linolenic acid (LA) by the octadecanoid pathway. Here, we report
the characterization of a tomato mutant ([1]) deficient in wound-induced expression of P15.
Insect feeding assays showed that jl 1 plants are compromised in defense against the
tobacco homworm (Manduca sexta). Using a map-based cloning approach, we
demonstrated that the gene defined by jl 1 encodes an acyl-CoA oxidase (ACX; named
LeACXl), which catalyzes the first and rate-limiting step of fatty acid B-oxidation in the
peroxisome. A function for ACX in JA biosynthesis is consistent with the widely held
assumption that the final step in JA biosynthesis involves B-oxidation of 3-oxo-2(2’[Z]-
pentenyl)-cyclopentane-l -octanoic acid (OPC-8z0) to JA. Consistent with this, wounded
le plants accumulated normal levels of OPDA, but are deficient in the production of J A.
LeA CXI transcripts constitutively accumulated in tomato leaves, and were further
induced by wounding in a J A-dependent manner. Taken together, these results show that
LeACXl plays a major role in the B-oxidation step of J A biosynthesis and resistance to

herbivores.

62

Introduction

Many plants respond to insect attack and wounding by activating the expression of
genes involved in herbivore deterrence, wound healing, and other defense-related
processes. The synthesis of wound-induced phytochemicals is regulated by signal
transduction pathways that act locally at the site of wounding and systemically in
unwounded leaves (Green and Ryan, 1972). Several lines of evidence demonstrate that
jasmonic acid (J A) plays a central role in the regulation of wound-induced defense
responses (Farmer and Ryan, 1992; Conconi et al., 1996; Howe et al., 1996; Li et al.,
2002b). The peptide signal systemin, which is a unique component of the wound
response pathway in solanaceous plants, regulates the biosynthesis of wound-inducible
defensive proteinase inhibitors (PIS) through the JA pathway (Ryan, 2000; Leon et al.,
2001). Systemin initiates the wound signaling by binding to a 160-kD plasma membrane-
bound receptor called SR160 (Scheer and Ryan, 2002). Binding of systemin to SR160 is
thought to trigger the release of linolenic acid (LA) from membrane lipids (Narvaez-
Vésquez et al., 1999). Wound-induced production of JA in tomato can also occur by a
systemin-independent pathway (Lee and Howe, 2003). The LA is metabolized to J A via
the octadecanoid pathway (Farmer and Ryan, 1992; Ryan, 2000; Li etal., 2001), which is
initiated in the chloroplast by addition of molecular oxygen to LA. The resulting 13S-
hydroperoxylinolenic acid (13(S)-HpOTrE) is converted to 12-oxo-phytodienoic acid
(12-OPDA) by the action of allene oxide synthase (AOS) and allene oxide cyclase (AOC).
Subsequent reduction of OPDA in peroxisomes by OPDA reductase (OPR3) yields 3-
oxo-2(2’[Z]-pentenyl)-cyclopentane-1-octanoic acid (UPC-8:0), which is shortened by

three cycles of B-oxidation to yield JA (Figure 3.1).

63

 

OCH
(1 -Linolenic acid (LA)

1 LOX
C hIaroplast OOH
\ _
1 COOH
138—hydroperoxylinolenic acid (13(S)-HpOTrE)
1
o AOC

COOH
12-oxo-phytodienoic acid (OPDA)

 

 

 

 

OPC-8:0

Peroxisome

1 1 3x B—oxidation
o

\\

COOH
Jasmonic acid (JA)

 

 

 

Figure 3.1. Octadecanoid pathway for JA biosysthesis.

64

Most enzymes in the J A biosynthesis pathway have been well studied, and the
corresponding genes have been cloned and characterized (Creeman and Mullet, 1997;
Ishiguro et al., 2001; Schaller, 2001; Feussner and Wasternack, 2002; Turner et al., 2002;
Li et al., 2003). The exception to this is the B-oxidation enzymes in the peroxisome. It has
long been proposed that OPC-8:O undergoes three cycles of [i-oxidation in the
peroxisome to produce JA (Vick and Zimmerman, 1984). However, direct evidence for a

role of peroxisomal fatty acid B-oxidation in IA biosynthesis is lacking.

The le mutant line of tomato was identified in a screen of EMS-mutagenized tomato
plants for individuals that are defective in P1 accumulation in response to mechanical
wounding (Lighter et al., 1993). Exogenous MeJA was shown to restore the production of
P15 in jl] plants, suggesting that jl l is defective in the biosynthesis of JA. Experiments
conducted in the Howe lab showed thatjll plants failed to elevate J A levels in response
to wounding; however, OPDA levels in wounded and unwoundedjll plants were
comparable to those in wild-type (Lee and Howe, unpublished data). These results
indicate that j]! plants synthesize OPDA but appear to be defective in its conversion to
JA. Map-based cloning showed that jl 1 plants have a defective ACX] gene, which
encodes an acyl-CoA oxidase involved in fatty acid B-oxidation in the peroxisome (Li
and Howe, unpublished data). Here, we report the phenotypic characterization of the

tomato le mutant.

Material and Methods

Plant material and growth conditions

Tomato (Lycapersican esculentum) Mill cv Castlemart was used as wild-type except

where otherwise indicated. Plants were grown and maintained as described in Chapter 2.
A j]! homozygous line was back-crossed three times to cv Castlemart as the recurrent

parent.

Identification of LeA CX 1

The LeACXI gene was identified by a map-based cloning approach described
previously (Li et al., 2003). Briefly, a BCl mapping population was constructed from a
cross between a homozygous jl 1 mutant (L. esculentum) and the wild tomato species L.
pennellii, followed by backcross of a resulting F 1 plant tojl] . Bulked segregant analysis
(Michelmore et al., 1991; Li et al., 2003) was performed to identify amplified fragment
length polymorphism (Vos et al., 1995) markers linked tojll . In a mapping population of
1200 BC] plants, the gene defined byjl] was mapped to a region flanked by makers
GP40 and cLEDl4K7 on the long arm of chromosome 8. TG510 marker, which co-
segregated with the target gene in all 1200 BCl plants, was then used to screen a tomato
bacterium artificial chromosome (BAC) library constructed from L. cheesmanii genomic
DNA. Two overlapping BAC clones (166B24 and 232Ll3) were identified.
Hybridization of BAC end sequences was used to determine the orientation and relative
position of the BACs. Fine mapping studies localized the target gene to BAC232L13,
which was shot-gun sequenced to five-fold coverage. Basic local alignment search tool
(BLAST) searches were performed to identify candidate genes in the BAC. The strongest

candidate identified was LeA CX 1.

A full-length LeA CXI cDNA was amplified by reverse transcription-PCR (RT-PCR)

of RNA isolated from wild-type andjl] plants. The primers were TCPl (5’-CTG AGA

66

GTA AGA GAG ATG GAG-3’) and TC P8 (5’-CTG GGA GGA AAA GAA GCC AAA-
3’), which were designed based on LeA CXI sequence information. The resulting RT-PCR

products were cloned into the pGEM-T easy vector (Promega) and sequenced.

Wound response assay

Two-leaf-stage plants (18-day-old seedlings containing two full expanded leaves and
a third emerging leaf) were wounded with a hemostat as described in Chapter 2. At
different time points after wounding, wounded leaves were harvested for isolation of
RNA. Pl-II levels were measured in wounded leaves (local response) and unwounded
leaves (systemic response) by radial immunodiffusion assay (Ryan, 1967) 24 hrs after

wounding, as described in Chapter 2.

RNA isolation and gel blot analysis

RNA was isolated from tomato leaves and analyzed by gel blot hybridization as
described in Chapter 2. Gels were also stained with ethidium bromide (EtBr) to verify
RNA quality. A cDNA for tomato translation initiation factor eIF 4a (cLEDlD24) was
used as the loading control. A DNA fragment was amplified by PCR from EST clone
cLESl4H13, which contains the full-length LeA CX] cDNA. The PCR primers were P1
(5’-GCT CTA GAG CGT AAG AGA GAT GGA GGG T-3’) and P2 (5’-CGA GCT CGA
ACA GTT TGC TGC AGC TCT CG-3’). The resulting ~2 kb PCR product was gel-
purified and labeled with [32P-a]dCTP. To directly compare transcript levels in wild-type
and fl] plants, blots containing RNA from both genotypes were hybridized in the same

container, washed under same condition, and exposed to film for the same length of time.

67

 

Elicitor feeding experiments

Systemin was provided by Dr. Ryan (Washington State University). 13(S)-HpOTrE
and 12-OPDA were purchased from Cayman Chemical (Ann Arbor, MI). UPC-8:0 was
provided by Dr. Kobayashi (Tokyo Institute of Technology, Japan; Ainai et al., 2003). (3:)
JA was purchased from Sigma (St. Louis, MO). These compounds were supplied to
tomato plants through their cut stems. Briefly, two-leaf-stage plants were excised at the
base of the stem with a double-edge razor blade (Ted Pella, Inc.), and immediately placed
into 0.5 m1 centrifuge tubes containing various amounts of the elicitor diluted in 300 pl of
15 mM sodium phosphate buffer (pH 6.5). Two excised plants were placed in one tube.
Because 13(S)-HpOTrE, OPDA, OPC-8:O and JA were originally dissolved in ethanol,
the mock control solution contained same amount of ethanol. When 90% of the elicitor
solution was imbibed (approximately 50 mins), the plants were transferred to glass vials
containing ~20 ml of distilled water. Plants were kept in a sealed Lucite box in a growth
chamber under normal conditions. Leaves were harvested at different time points after
elicitor treatment for isolation of total RNA. PI-II levels in leaves were measured 24 hrs

afler treatment.

Tobacco horn worm feeding trials

Tobacco homworm (Manduca sexta) eggs and Ready-To-Use Hornworrn Diet were
obtained from Carolina Biological Supply Company (Burlington, NC). Eggs were
hatched at 27°C under continuous light as recommended by the supplier. Hatched larvae

were reared on the artificial diet for 3 days before being transferred to tomato plants.

In experiment 1, ll-newly hatched larvae were placed on leaves of each of 5

68

separately potted 6-week-old wild-type and jl] plants. The average weight of larvae at the
beginning of the feeding trial was 18 mg. After 10 days of feeding, larvae were recovered
from wild-type and jl 1 plants, and the weight of each larva was measured. PI-II levels in

damaged and undamaged leaf tissue were measured at that time.

In experiment 2, 60-newly hatched larvae were placed randomly on leaves of 20 4-
week-old plants of each genotype. Each plant genotype was grown in a separate flat. The
average weight of larvae at the beginning of the feeding trial was 15 mg. Larvae were
allowed to move freely between plants of the same genotype. After 3 days of feeding,
larvae were recovered from each genotype. Pl-II levels and larvae weight were measured
as described in experiment 1. Damaged and undamaged leaf tissue was collected for RNA

isolation.

Measurement of jasmonic acid

Tomato leaf tissues were collected and immediately frozen in liquid nitrogen. J A was
extracted and as according to the method described by Schmelz et al. (2003) and
Engleberth et al. (2003). Dihydroj asmonic acid was added to samples as an internal
standard. Methylated carboxylic acids from plant samples were volatilized and collected
on volatile collection traps® (VCT) (Analytical Research Systems Gainesville, Florida).
Samples were eluted from the VCT resin by methylene chloride and subsequently
analyzed by gas chromatography-mass spectroscopy (GC-MS). GC-MS analysis was
performed by selected ion monitoring, with isobutane chemical ionization as described
(Schmelz et al., 2003). The GC-MS system consisted of a 6890 Network GC connected

to a 5973 inert Mass Selective Detector (Agilent, Palo Alto, CA, USA).Compounds were

69

 

separated on a HPSMS column (30m x 0.25mm x 0.25um). The temperature regime for
GC was 40°C for a minute after injection, followed by sequential temperature ramps of
25 °C/min to 150 °C, 5 °C/min to 200 °C, 10 °C ramp to 240 0C. The 240 °C temperature

was maintained for 10 minutes.

Results
T he jll mutant of tomato has a defective ACX] gene

In a map-based cloning experiment performed by Dr. Chuanyou Li, two genes
(designated as ACX] and ACXZ, respectively) were considered to be candidates for the
gene that is defective in le plants (Figure 3.2.A; B). cDNA sequences corresponding to
ACX] were obtained from wild-type L. esculentum and j] I by reverse transcription-PCR
(RT-PCR). Sequence comparison of cDNA clones revealed that jll plants harbor a single
base mutation in ACX] that changes the nucleotide at position 414 of the ORF from a C
to a T. This mutation is predicted to change Thr139 to an isoleucine (I). This mutation
was confirmed in genomic sequences from wild-type and fl] plants. Alignment of ACX
proteins from various plants and animals showed that Thr139 is conserved in all ACXs
(Figure 3.2.C), suggesting a functional importance of this residue (Nakajima et al., 2002).
Indeed, the crystal structure of rat ACX showed that this Thr is involved in binding the
FAD co-factor (Nakajima et al., 2002). Comparison between genomic and cDNA
sequences showed that the LeA CXI gene contains 14 exons and 13 introns (Figure 3.2.B).

The C-to-T substitution in the mutant occurs within the fourth exon (Figure 3.2.B).

Expression of wound response genes is reduced in le plants
Wound responsive genes in tomato can be divided into two classes based on their

70

Figure 3.2. Map-based cloning of the ACX gene.

(A) Genetic and physical map of LeA CXI . The mutation was mapped to a region between
RFLP markers GP40 and cLED14K7 on chromosome 8. The gene’s location was
narrowed down to a region encompassed by overlapping BAC clones (166B24 and
232L13). Numbers in parentheses indicate the number of recombinants identified

between markers and the target gene.

(B) Structure of three genes (designated TG510, LeA CX] and LcACXZ) identified on
BAC323L13. Filled boxes represent exons and lines between boxes represent introns or
intergenic regions. TG510 is a RFLP marker that co-segregates with LeACXl in 1200

BCl plants.

(C) Alignment of the FAD binding region of various ACXs from plants and animals. The

le mutation changes the threonine (T) to an isoleucine (I).

71

 

 

 

 

 

\ a
\ \ Q/ Q
G '\ Q \
A \‘l‘ '3) V\ \> ©Q—Q Qr-l’ ~13
N 9 <23" '2 '2 " o
Ix" Q“ (3° 09), (3° 627 5(1)] (0
9 N W ’\ 2.9 w 6’
1 II 1 l l l l J NJ
,, ,, Chr.8
—166824
232L13
Q
Ix
3 c3"
&
? I Jl* ! "- ’1'
+- . ..I-H-I—l-l—l-H—H—H—ri—iri-l-iwz-H—
———-> > e
1 kb LcACX1 LcACXZ
C Tomato mm 131 IIGCYAQ?ELGHGSNVQGLETTATFD 156
Arab ACXl 131 IIGCYAQ T ELGHGSNVQGLETTATFD 156
Pumpkin ACOX 177 YPGCE‘AM T ELHHGSNVQGLQTTATE‘D 202
Rat ACO 132 ITGTYAQ T EMGHGTHLRGLETTATYD 157
Human BACO 147 IIATYAQ T ELGHGTYLQGLETEATYD 172
Yeast ACO 183 IYGCFAM T ELGHGSNVAQLQTRAVYD 208
Human MCDH 154 LMCAYCV T EPGAGSDVAGIKTKAEKK 179
Human LCDH 166 CIGAIAM T EPGAGSDLQGIKTNAKKD 191
Human VLCDH 210 TVAAE‘CLlEPSSGSDAASIRTSAVPS 235

 

 

Figure 3.2. Map-based cloning of the ACX gene.

72

temporal and spatial pattern of induction (Ryan, 2000; Lee and Howe, 2003). Transcripts
of so-called “early” response genes accumulate rapidly (within 1 hr) and transiently in
response to wounding. These include genes encoding JA biosynthetic enzymes such as
lipoxygenase D (LoxD; Heitz etal., 1997), allene oxide synthase 2 (A082; Howe et al.,
2000), 12-OPDA reductase 3 (OPR3; Strassner et al., 2002), as well as other signaling
components such as prosystemin (PSYS; Jacinto et al., 1997). By contrast, mRNA
transcribed from “late” response genes begins to accumulate locally and systemically
about 2 hrs after wounding. These genes mostly encode defense related proteins,
including proteinase inhibitor I and II (PI-I, PI-II; Graham et al., 1986), and cathepsin D
inhibitor (CD1; Hildemann et al., 1992). To investigate the expression pattern of wound-
induced genes in fl 1 plants, we determined the temporal expression pattern of
representative “early” and “late” genes, as well as LeA CXI . Local expression of the late
response genes (PI-II and CD1) was detected in wild-type plants within 2 to 4 hrs of
wounding, with transcript levels reaching a maximum 12 hrs after wounding (Figure 3.3).
Transcripts representing two early response genes (LaxD and OPR3) accumulated in
wild-type plants 0.5 to 1 hr of wounding, and the expression level declined 2 hrs after
wounding. Similar to LaxD and OPR3, LeA CXI mRN A accumulated in wild-type leaves
1 hr after wounding, and reached maximal levels 2 hrs after wounding. LeA CXI
transcripts declined to basal level 12 hrs after wounding. These results indicated that
LeACXI behaves as an “early” wound response gene, consistent with its role in J A
biosynthesis. Wound-induced expression of late response genes was undetectable (CD1)
or was reduced to a very low level (PI-II) in jl 1 plants. Early response gene transcripts

(LaxD, OPR3, LeACXI) were induced by wounding inle plant, although at a level that

73

Wild-type jl1
0.5124812240.512481224Hrs

 

 

   

Pl-l/

 

..-... '1 Q C. CD/

... -. ........ ~ LaxD
g; m, .,,... _ OPR3

., ..'. g... 4...... . «=- m- 2. LeACX1

.ufiflwwt—n- --Qmmwmm eIF4a

EtBr

 

Figure 3.3. Gene expression in wild-type and j]! plants in response to mechanical

wounding.

Tomato seedling (cv Castlemart and jl 1 ) at the two-leaf-stage were wounded with a
hemostat on both lower and upper leaves. Total RNA was isolated from the wounded
leaves at various times after wounding. RNA was prepared from unwounded plants (0
time) as a control. cDNA probes representing different classes of wound responsive genes
and LeACXI were used for hybridization, as shown on the right of the figure. eIF 4a was

the loading control. EtBr staining was used to verify the quality of RNA.

74

 

was approximately 20% of wild-type. These results are consistent with the expression
pattern of wound response genes in the tomato/ail mutant that lacks J A perception (Li et
al., 2004), and indicate that the expression of early genes is controlled by both JA-

depended and JA-independed pathway.

T he le mutant is defective in resistance to tobacco horn worm

Previous studies have established that the octadecanoid pathway for J A biosynthesis
plays an important role in defense of tomato against a broad spectrum of herbivores
(Howe et al., 1996; Li et al., 2002a; 2003). The inability of jl 1 plants to express
significant levels of defensive P13 in response to mechanical wounding suggested that this
mutant might be compromised in resistance to herbivorous insects. To test this possibility,
6-week-old (experiment 1) or 4-week-old (experiment 2) wild-type and j]! plants were
challenged with tobacco homworm larvae. After termination of the feeding trial, we
assessed the weight of larvae, the amount of leaf damage, and the level of PHI in leaves
of both genotypes. Homworm feeding on wild-type plants resulted in accumulation of
high levels of PHI in both damaged and undamaged leaves (Table 3.1). In contrast, little
or no PI-II accumulation was detected in damaged and undamaged le leaves. RNA gel
blot analysis showed that the expression of defense-related genes (C DI and PM!) was
induced by homworm attack both locally (damaged) and systemically (undamaged) in
wild-type but not in jl] plants (Figure 3.4). Similar to mechanical wounding,le plants
did express early response genes (LaxD and OPR3) locally and systemically when
attacked by homworm larvae, although the level of expression was much lower than that

in wild-type.

75

 

 

Table 3.1. Tobacco hornworm feeding assay with wild-type and fl] plants.

 

Damaged leaf Undamaged leaf

.7 Y b
121-11 (pg/ml) PI-II (pg/ml) Larval W981" (1:)

Experimenta Genotype

 

1 Wild-type 225 :t 47 199 i 66 0.99 1: 0.41 (n=38)
fl] 0 0 3.45 .2 1.68 (n=42)
2 Wild-type 141 1 73 151 3: 26 0.10 3: 0.04 (n=38)
1'11 15 4; 24 0 0.19 i 0.05 (n=4l)

 

a In experiment 1, 11 newly-hatched larvae were placed on leaves of each of 5
separately potted 6-week-old wild-type and fl] plants. In experiment 2, 60 newly-
hatched larvae were placed randomly on leaves of 20 4-week-old plants of each
genotype, in separate flats. Larvae were allowed to move freely between plants of
the same genotype. Experiment 1 and 2 were terminated 10 and 3 days after the
start of feeding trial, respectively. At that time, larvae were recovered from plants
and their weight was measured. PI-II levels in damaged and undamaged leaf tissue

also were measured. Data represent mean i standard deviation.

b In both experiments, the weight of larvae grown on wild-type and mutant plants

was significantly different at P<0.001 (Student’s t test).

 

76

 

Interestingly, homworm feeding activated LeA CXI expression locally and
systemically in both wild-type and jl 1 plants (Figure 3.4). To determine whether the
induction of LeA CXI in response to homworm attack was dependent on J A signaling,
LeA CXI expression was assessed in the J A insensitive jai 1 mutant that was challenged
with homworms for 4 days. RNA gel blot analysis showed thatjail plants accumulated a
basal level of LeA CXI transcript, and this accumulation was not affected by homworm
attack (Figure 3.5). These results indicate that the basal expression of LeA CXI is
controlled independently of J A, whereas induced expression by mechanical wounding or

homworm attack requires a functional J A signaling pathway.

In addition to these effects on gene expression, we also found that jl 1 plants were
defoliated by tobacco homworms much faster than wild-type plants (Figure 3.6). The
average weight of larvae grown on jl 1 plants was about 3.8-fold (experiment 1) and 2-
fold (experiment 2) greater than that of larvae reared on wild-type plants for the same
period of time (Table 3.1). These results indicate that jl] compromises the tomato’s
defense against herbivorous insects. As a result, foliage from jl I plants is a better food

source for homworm larvae.

Response of jll plants to exogenous signaling compounds

Previous studies have shown that exogenous systemin and various intermediates in
the octadecanoid pathway activate the biosynthesis of J A leading to accumulation of
defensive PIs (Farmer and Ryan, 1992; Lee and Howe, 2003). The defect in ACX] injl]
plants suggested that elicitors acting upstream of .1A would be unable to induce the

accumulation of P15 in jl 1 plants. To test this prediction, we supplied plants with systemin

77

171 Wild-type
C U D C U D

 

PI-II

CDI

LaxD

 

m OPR3

-. ” LeACX1

eIF4a

EtBr

 

Figure 3.4. Accumulation of wound-induced transcripts in response to tobacco

homworm attack.

Newly hatched tobacco homworm larvae were allowed to feed on wild-type and jl 1
plants for 3 days (experiment 2 in Table 3.1). Total RNA was isolated separately from
homworm damaged (D) and undamaged (U) leaves of the same genotype. RNA from leaf
tissue of unattacked plants was prepared as control (C). RNA blots were hybridized to
cDNA probes indicated on the right. eIF4a was used as loading control, and EtBr staining

was used to verify the quality of RNA.

78

Wild-type jai1

 

 

C W
”'4 LeA CX1

  
 

EtBr

 

Figure 3.5. Expression of LeA CX1 in wild-type and jail plants in response to tobacco

homworm attack.

Newly-hatched tobacco homworm larvae were grown on 6-week-old wild-type (cv Micro
Tom) and fat] plants for 4 days. Total RNA was isolated from homworm damaged plants
(W, both damaged and undamaged leaves) of the same genotype. RNA from leaf tissue of
unwounded plants was prepared as control (C). eIF 4a was used as a loading control. and

EtBr staining was used to verify the quality of RNA.

79

Figure 3.6. Challenge of wild-type and 171 plants with tobacco hornworm larvae.

Eleven newly-hatched larvae (about 18 mg each) were placed on leaves of each of 5
separately potted wild-type andjl] plants (6-week-old). Larvae were allowed to feed for

10 days.
(A) Representative Wild-type andjl] plants at the end of the feeding trail.

(B) Homworm larvae recovered from wild-type andjl] plants at the end of feeding trail.

80

Wild-type le

..'?
F-
"r
\P
"In.-
w
J
9“
cl.
1"
C.
w
“(119’

 

Figure 3.6. Challenge of wild-type and fl] plants with tobacco hornworm larvae.

(Image in this thesis is presented in color)

81

or with other octadecanoid pathway compounds including l3-HpOTrE, OPDA, OPC-8:0
and J A. Surprisingly, all elicitors tested induced the accumulation of PHI in jl 1 plants
(Table 3.2). This was confirmed by RNA gel blot analysis, which showed that the Pl-II
transcript accumulated in le plants supplied with OPDA (Figure 3.7). We also found that
the early wound response gene LaxD was induced by OPDA treatment in both wild-type
and fl] plants, but the level in j! 1 was much lower than those in wild-type. mRNA
accumulation observed in buffer-treated plants reflects the effect of cutting (Lee and

Howe, 2003). Notably,le plants showed no expression of PHI in response to cutting.

To test the effect of different concentration of JA precursors on induction of PHI
expression in le plants, we supplied wild-type and jl] plants with different amounts of
OPDA and OPC-8:O. Five nmol of each elicitor per plant was sufficient to trigger the
accumulation of PHI in both wild-type and fl] plants (Figure 3.8). PI-II levels in wild-
type and jl 1 plants were generally correlated with the concentration of elicitors (Figure
3.8). These results indicate that exogenous OPDA and CFC-8:0 induce PI expression in

le plants, despite the fact that this mutant is defective in LeACXl.

To determine if OPDA induction of PHI in le plants is J A-dependent or J A-
independent, we measured the level of J A after OPDA feeding through the cut stern.
Wild-type plants accumulated low levels of JA in response to buffer, but produced 198.1
i 9.6 pmol JA/g FW within 1 hr after application of OPDA. This level declined to the
basal level 3 hrs after feeding. However, I A levels in OPDA treatedjll plants were <5%
of that in wild-type (Figure 3.9). These finding indicated that ACXl is strictly required

for JA accumulation in response to exogenous OPDA.

82

 

Table 3.2. PI-II levels in leaves of wild-type and 171 plants in response to different JA

precursors.

 

Genotype Buffer S ystemin HpOTrE OPDA OPC-8 :0 .IA

 

Wild-type 18i10 123160 88:1:32 96126 1183:22 187i25

le 3i4 66i29 67134 1223:25 83i43 177119

 

l8-day-old wild-type and le seedlings were supplied through their cut stems with a
buffer control (15 mM sodium phosphate, pH 6.5), or with elicitors dissolved in the
buffer. The elicitors concentration used were as follows: systemin (5 nmol/plant),
HpOTrE (25 nmol/plant), OPDA (10 nmol/plant), OPC-8:0 (25 nmol/plant), and IA (10
nmol/plant). Excised plants were incubated in the elicitor solution for about 50 min to
allow uptake of the elicitor, and then transferred to glass vials containing distilled water.
PI-II levels (pg/ml leaf juice) in leaves were measured 24 hrs after treatment. Values

represent the mean and standard deviation of 6 plants.

The difference in the response between wild-type and j] 1 plants was not statistically
significant (P value for buffer control is 0.03; P values range from 0.1 to 0.4 for elicitor

feeding.)

 

83

Buffer OPDA
Wild-type jI1 Wild-type jl1
00261224 C0261224 C0261224 C 0261224 Hrs

I ..‘ w ' . .. LaxD

M”““-~ --“~r“

 

 

 

 

”width“- ww-v-whelF4a

illlll II 111 111111 If

‘

   

Figure 3.7. Effect of OPDA feeding on the expression of various wound-responsive

genes.

Eighteen-day-old wild-type and j]! seedlings were supplied through their cut stems with a
buffer control (15 mM sodium phosphate, pH 6.5) or OPDA (25 nmol/plant, dissolved in
buffer) as described in Table 3.2. Leaf tissue of 8 plants was harvested and pooled at each
sampling time point after OPDA treatment. RNA was prepared from untreated plants (C)
as a control. LaxD and PI-II cDNA probes were used for hybridization. eIF4a was the

loading control, and EtBr staining was used to verify the quality of RNA.

84

Figure 3.8. Dose effect of OPDA and OPC-8:0 on induction of PM] in wild-type and

fl] plants.

Eighteen-day-old wild-type and jl 1 seedlings were supplied with phosphate buffer (15
mM sodium phosphate, pH 6.5) containing various amounts of OPDA (A) and OPC-8:0
(B) as described in Table 3.2. PI-II levels in leaves were measured 24 hrs after treatment.

Date represent the mean and standard deviation of 6 plants.

85

140

120 -

100

P14! (pg/ml leafjuice)
O)
O

0

160

120
100
80
60
4o
20

Pl-II (pg/m1 leaf juice)

1:1 VWld-type

I j/1
1 ..IM _ .=T=_L 1:.1—1-3 1i [i
0 0.2 1 5 25

nmol OPDA per plant

[:1 WIId-type .
.111 1

 

 

0 0.2 1 5 25
nmol CPO-8:0 per plant

Figure 3.8. Dose effect of OPDA and OPC-8:0 on induction of PI-ll in wild-type and

jII plants.

86

250

 

 

 

 

 

 

DWiId-type

2. 200 2 w
E ,,
O)
E 150
0
g 100
<
7 50 L

~ 1

Buffer-1 hr OPDA-1 hr Buffer-3hr OPDA-3hr

Figure 3.9. JA accumulation in wild-type and ['11 plants in response to application of

exogenous OPDA.

Eighteen-day-old wild-type and fl 1 seedlings were supplied with OPDA (20 nmol per
plant) through out stem as described in Table 3.2. Leaves were harvested for J A
extraction at 1 hr and 3 hr after OPDA application. The amounts of J A in plant extracts

were quantified by GC-MS. Data represent the mean and standard deviation of three

independent replicates.

87

Discussion
LeA CX1 is required for wound-induced JA biosynthesis

We demonstrated that the tomato jll mutant has a defective ACXl gene and, as a
consequence, is deficient in wound-induced JA production. The expression of both early
and late wound response genes was also highly reduced in thele mutant in response to
mechanical wounding and homworm attack (Figure 3.3; 3.4). The wound response of the
le mutant could be rescued by exogenous J A (Lightner et al., 1993), which is consistent
with the conclusion that LeA CX1 is required for the biosynthesis of J A. These data
indicate that B-oxidation is needed for wound-induced JA biosynthesis. As expected, a
deficiency in B-oxidation results in increased plant susceptibility to insect attack. Castillo
et al. (2004) recently showed that reduced expression of the ACX] gene in Arabidopsis
caused a defect in wound-activated synthesis of JA and reduced expression of J A-
responsive genes. Also, induced expression of JA-responsive genes by exogenous

application of J A was unaffected in those transgenic plants.

LeA CX1 is expressed constitutively in tomato leaves and induced in response to

wounding

It is generally agreed that JA biosynthetic enzymes accumulate constitutively in
unwounded tomato leaves (Stenzel et al., 2003a). Here, we show that LeA CX1 is also
expressed constitutively at a basal level in leaves of wild-type,jl] , andjai] plants (Figure
3.3; 3.4; 3.5). However, transcripts of LeA CX1 accumulate to a high level in response to
mechanical wounding and homworm attack, in a manner that depends on JA signaling. A

similar result was found in Arabidopsis (Castillo et al., 2004). These results lead us to

88

 

 

 

conclude that the expression of LeA CX1 is controlled by both JA -dependent and -
independent mechanisms. This conclusion is consistent with previous a cDNA microarray
study showing that ACX] is more highly induced by wounding in wild-type plants than in
cat] plants (Reymond et al., 2000), which are insensitive to J A. The JA -independent _
basal expression of JA biosynthetic genes suggests that the respective gene products serve
an important function in the absence of stress conditions that trigger J A signaling. For
example, the role of ACXl in fatty acid B-oxidation suggests that this enzyme is involved
in germination and early postgerminative development in higher plants. Uncoupling of
the basal expression level of IA biosynthetic enzymes from JA signaling might provide a
mechanism to ensure that the amplitude and timing of JA biosynthesis in response to
stress is sufficient to activate downstream target genes (Li et al., 2004). This hypothesis is
consistent with the observation that wound-induced activation of JA biosynthetic genes
such as LaxD, OPR3, and ACX] occurs later than the wound-induced accumulation of J A
(Figure 3.3) (Stenzel et al., 2003b; Castillo et al., 2004). Thus, wound-induced J A
synthesis does not depend on the induced expression of J A biosynthetic genes (Miersch

and Wasternack, 2000; Ziegler et al., 2001).

Role of JA and OPDA in plant defense

Unexpectedly, le plants expressed Pl-II in response to octadecanoid intermediates
that proceed the action of ACXI in the of J A biosynthesis pathway (Table 3.2; Figure 3.8).
This response of jl I contrasts with the near complete lack of PI expression in wounded
le plants. The fact that j! 1 plants still respond to JA precursors such as OPDA and OPC-
8:0 likely means that J A is synthesized when these precursors are supplied exogenously

to j]! through the transportation stream. We hypothesize that high concentrations of ACX

89

substrate (e. g. OPCz8-CoA) generated in response to exogenous intermediates are
converted to JA by the action of other ACXs. In the Arabidopsis genome, there are six
ACX genes that differ in their expression pattern, subunit composition, and substrate
specificity (Graham and Eastmond, 2002; Rylotteta1., 2003). AtACXl prefers medium-
to long-chain saturated acyl-CoAs, AtACX2 displays activity against long-chain
unsaturated acyl-CoAs (Hooks et al., 1999), AtACX3 is a medium-chain (C 10-C14) acyl-
CoA oxidase (Eastmond et al., 2000; F roman et al., 2000), and AtACX4 exhibits short-
chain substrate specificity (Hayashi et al., 1999). These ACX isozymes are highly similar
at the amino acid sequence level (Hayashi et al., 1999).AtACX1, AtACX2 and AtACX3
have similar Km values (around 5 11M), similar molecular weights (75 kDa), and function
as homodimers in viva (Graham and Eastmond, 2002). Biochemical evidence suggests
that acyl-CoA oxidase isozymes in plants have partially overlapping acyl-CoA substrate
chain-length specificities (Kirsch et al., 1986). Even though jll plants synthesize very
little JA in response to wounding (unpublished data), other ACXs in tomato may

metabolize OPCz8-CoA generated in response to exogenous elicitors.

Recently, Narvaez-Vasquez and Ryan (2004) presented in situ hybridization and
immunocytochemical evidence that wound-induced and MeJA-induced prosystemin
mRNA and protein are exclusively located in vascular phloem parenchyma cells of minor
veins and midribs of leaves, and in the bicollateral phloem bundles of petioles and stems
of tomato. Wound-activated AOC accumulation was restricted to companion cells and
sieve elements of vascular bundles (Stenzel et al., 2003a). Compartmentalization of
prosystemin, which is a positive regulator of J A, and JA biosynthetic enzymes in vascular

bundles suggests the JA biosynthesis is also vascular bundle-specific (Hause et al., 2003;

90

Stenzel et al., 2003a; Narvaez-Vasquez and Ryan, 2004). The basal levels of IA and
OPDA in untreated tomato leaves are low, indicating that J A biosynthetic enzymes are
not a limiting step in JA accumulation. The rapid and transient rise of J A upon wounding
suggests that JA generation is substrate dependent and occurs in specific cells within the
vascular bundles (Stenzel et al., 2003a). In wounded tomato plants, systemin is proposed
to be processed from prosystemin and translocated into the apoplast of the vascular
bundles where it initiates a positive amplification loop in which systemin and J A are self-
induced as a wave through the plant vasculature to activate local and systemic responses
(Stenzel et al., 2003a; Narvaez-Vasquez and Ryan, 2004). Due to the defect in ACX] , we
propose that j]! plants cannot metabolize wound-induced OPCz8-CoA to J A in vascular
bundles. In case of feeding exogenous elicitors, intermediates in the IA biosynthesis
pathway may be produced non-specifically in all cell types, leading to induction of PHI.

In le plants, other ACXs may fulfill the role of ACXl to produce J A in this manner.

However, J A did not accumulate in jl 1 plants in response to applied OPDA (Figure
3.9). These results indicate that ACXl is required for the biosynthesis of J A in tomato
plants, and that other ACXs are not involved in J A biosynthesis in response to applied
OPDA. It appears that the expression of PHI in OPDA-treatedjll plants is mediated by a

signal other than JA, most likely OPDA itself.

Although JA is a physiological signal in the regulation plant defense responses
against herbivorous insects and pathogen attack, the possibility that the J A precursor, 12-
OPDA, is active without metabolism to JA has been proposed (Weiler et al., 1993; Weiler
et al., 1994; Weber et al., 1997; Stintzi et al., 2001). The best evidence for this comes

from studies of the Arabidopsis opr3 mutant (Stintzi and Browse, 2000; Stintzi et al.,

91

2001), which is defective in OPDA reductase (OPR). Wounded apr3 leaves do not
accumulate detectable bioactive J A, and levels of OPDA in wounded opr3 leaves are
about 50% of that in wounded wild-type leaves. Thus, the apr3 mutant provides a useful
tool to separate the effect of 12-OPDA and J A in viva. In contrast to J A-insensitive coil
plants and the fad3/ fad7/ fad8 mutant lacking the fatty acid precursors of J A, apr3 plants
exhibited a wild-type level of resistance to the dipteran Bradysia impatiens and the
fungus Alternaria brassicicola. This striking result indicated that resistance of apr3
plants is mediated by a signal other than JA, the most likely candidate being OPDA
(Howe, 2001). cDNA microarray analysis in apr3 plants showed the wound induction of
genes previously known to be JA-dependent, suggesting that OPDA could fulfill some
roles of J A in viva. Treating apr3 plants with exogenous OPDA up-regulated several
genes, indicating that this J A precursor can activate gene expression in the absence of J A

(Stintzi et al., 2001).

Like the Arabidopsis opr3 mutant, the tomato jll mutant appears to be defective in
the metabolism of OPDA to J A (Lee and Howe, unpublished data). However, we found
that jll plants are deficient in wound-induced expression of defensive PIs (Figure 3.3),
and are much more susceptible to homworm attack than wild-type (Figure 3.6; Table 3.1).
This finding indicates that in tomato, OPDA is not a signal for defense against insects.

Furthermore, we conclude that J A, but not OPDA, is essential for expression of wound-

induced PIs.

The expression of PM] in the absence of J A in j]! plants when supplied with OPDA
leads us to a hypothesis that exogenous OPDA and other J A precursors trigger JA-

independent but OPDA-dependent gene expression injl] plants. A model summarizing

92

the roles of IA and OPDA in the control of gene expression in OPDA treated tomato
plants is given in Figure 3.10. We postulate that JA and OPDA play distinct, but maybe
complementary roles in the fine-tuning of PHI expression. In wounded plants, the
pathway obviously involves OPDA and JA, and acts through the C011 complex. J A is
required for this pathway. But it is possible that OPDA can activate the PH] expression
directly in the absence of J A when exogenous OPDA or other J A precursors are applied
to tomato plants through cut stem. OPDA-induced Pl-II expression is also COIl-
dependent (Li and Howe, unpublished data). In this process. OPDA may bind to the same

receptor as J A, based on their structural similarity.

The idea that J A and related octadecanoid compounds regulate different target
processes in different plants has been proposed (Li et al., 2003). Mutants of Arabidopsis
and tomato have been instrumental in establishing the roles of J A and related compounds
both in regulating developmental and defense processes. In this context, the le mutant
provides a valuable genetic resource to further investigate the roles of J A and OPDA in

regulating defense responses in tomato.

93

 

 

 

 

 

1 Wounding

 

 

OPDA

OPC 8:0 ——|—

 

 

 

 

 

 

 

 

 

 

 

 

  
   

Exogenous

 

 

 

jail/coil

 

 

 

PI-II

 

 

 

Defense

 

 

 

Figure 3.10. Model for the role of JA and OPDA in the fine control of gene

expression in tomato leaves.

In wounded plants, the conversion of OPDA to IA is required for the activation of PHI
expression. When exogenous OPDA or other I A precursors are supplied to tomato plants
through cut stems, OPDA or OPC 8:0 can regulate the expression of PHI directly in a

C011 -dependent pathway.

94

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