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Role of Methylsalicylate in Tomato Flavor and the Response to Bacterial Pathogen Infection

Permanent Link: http://ufdc.ufl.edu/UFE0021392/00001

Material Information

Title: Role of Methylsalicylate in Tomato Flavor and the Response to Bacterial Pathogen Infection
Physical Description: 1 online resource (98 p.)
Language: english
Creator: Zeigler, Michelle L
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: acid, carboxyl, compound, defense, glucoside, methyl, methyltransferase, organic, plant, salicylate, salicylic, voc, volatile, wintergreen, xanthomonas
Plant Molecular and Cellular Biology -- Dissertations, Academic -- UF
Genre: Plant Molecular and Cellular Biology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Volatiles are aroma compounds with low molecular weight and high vapor pressure that evaporate at room temperature. Plants have evolved the use of volatiles for the attraction of pollinators, seed dispersing organisms, and to aid in the response to herbivory and pathogen attack. These plant volatiles are components of floral scent and the flavors of foods, and may even possess medicinal properties. Tomato (Solanum lycopersicum) flavor is due to a complex interaction between sugars, acids, and volatile compounds. Methylsalicylate (MeSA), or oil of wintergreen, is an important volatile component of tomato flavor. The purpose of this study was to characterize the contribution of MeSA to tomato flavor as well as any other biological functions in tomato. S-adenosyl-L-methionine: salicylic acid carboxyl methyltransferase (LeSAMT1) is the gene responsible for MeSA synthesis in tomato, and LeSAMT1 specifically converts SA to MeSA. Plants overexpressing LeSAMT1 were analyzed with respect to MeSA emissions and flavor. Tomato fruits overexpressing LeSAMT1 tasted different than the controls but were preferred equally to the controls by an untrained consumer panel. In addition, one of these transgenic lines overexpressing LeSAMT1 was used as a tool to examine the contribution of MeSA overproduction to infection by the bacterial pathogen Xanthomonas campestris pv. vesicatoria 93-1. The results indicated that MeSA is a key metabolite that affects the accumulation of SA during bacterial pathogen stress as well as the progression of disease symptoms.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Michelle L Zeigler.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Klee, Harry J.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021392:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021392/00001

Material Information

Title: Role of Methylsalicylate in Tomato Flavor and the Response to Bacterial Pathogen Infection
Physical Description: 1 online resource (98 p.)
Language: english
Creator: Zeigler, Michelle L
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: acid, carboxyl, compound, defense, glucoside, methyl, methyltransferase, organic, plant, salicylate, salicylic, voc, volatile, wintergreen, xanthomonas
Plant Molecular and Cellular Biology -- Dissertations, Academic -- UF
Genre: Plant Molecular and Cellular Biology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Volatiles are aroma compounds with low molecular weight and high vapor pressure that evaporate at room temperature. Plants have evolved the use of volatiles for the attraction of pollinators, seed dispersing organisms, and to aid in the response to herbivory and pathogen attack. These plant volatiles are components of floral scent and the flavors of foods, and may even possess medicinal properties. Tomato (Solanum lycopersicum) flavor is due to a complex interaction between sugars, acids, and volatile compounds. Methylsalicylate (MeSA), or oil of wintergreen, is an important volatile component of tomato flavor. The purpose of this study was to characterize the contribution of MeSA to tomato flavor as well as any other biological functions in tomato. S-adenosyl-L-methionine: salicylic acid carboxyl methyltransferase (LeSAMT1) is the gene responsible for MeSA synthesis in tomato, and LeSAMT1 specifically converts SA to MeSA. Plants overexpressing LeSAMT1 were analyzed with respect to MeSA emissions and flavor. Tomato fruits overexpressing LeSAMT1 tasted different than the controls but were preferred equally to the controls by an untrained consumer panel. In addition, one of these transgenic lines overexpressing LeSAMT1 was used as a tool to examine the contribution of MeSA overproduction to infection by the bacterial pathogen Xanthomonas campestris pv. vesicatoria 93-1. The results indicated that MeSA is a key metabolite that affects the accumulation of SA during bacterial pathogen stress as well as the progression of disease symptoms.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Michelle L Zeigler.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Klee, Harry J.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021392:00001


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ROLE OF METHYLSALICYLATE IN TOMATO FLAVOR AND RESPONSE TO
BACTERIAL PATHOGEN INFECTION




















By

MICHELLE LYNN ZEIGLER


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007
































O 2007 Michelle Lynn Zeigler
































This thesis is dedicated to my Mom, who has always supported and encouraged me.









ACKNOWLEDGMENTS

First I would like to thank Dr. Harry Klee for giving me this opportunity to work on

tomato flavor. I have learned a great deal throughout this process and I appreciate his patience

and understanding. I would like to thank Dr. Don Huber for his advice and helpful discussions. I

would also like to thank Dr. Bala Rathinasabapathi "Saba" for his advice and helpful discussions.

I wish to thank Dr. Denise Tieman for initiating this proj ect, all her help during the tomato

harvests, and especially her tomato-chopping expertise for the preference test. I appreciate all her

advice, patience, and encouragement throughout my time in the lab.

I would also like to thank those who helped me with the pathogen experiments. Thanks to

Dr. Jeffrey Jones for his encouragement and guidance with my pathogen experiments. I

appreciate him letting me use his greenhouse space and for taking care of my plants. I would also

like to thank Dr. Robert Stall and Jerry Minsavage for answering all my questions and for

maintaining the plants in the greenhouse. I also thank Dr. Eric Schmelz at the USDA for his

helpful discussions and for developing a new protocol to simultaneously measure plant hormones

and methyl esters. I appreciate his patience while my plant hormone extraction samples wreaked

havoc on his lab equipment.

I thank Dr. Amarat Simonne for giving me advice for the triangle taste test. I also thank

Dr. Charles Sims and the Sensory Testing Facility in the Food Science and Human Nutrition

Department at the University of Florida for coordinating the preference and likeability taste tests.

I especially thank all the volunteer panelists who participated in the taste tests.

I thank Dr. Ken Cline and Dr. Curt Hannah for allowing me to do rotations in their labs. I

thank Dr. Carole Dabney-Smith for teaching me the basics of protein expression. I also thank Dr.

Carla Lyerly-Linebarger for teaching me the basics of enzyme assays.










I want to thank all the members of the Klee Lab for their support and encouragement.

Thanks to Dr. Mark Taylor for making the transgenic plants and his fun attitude. Thanks to Peter

Bliss for all his help in the lab and for keeping a good sense of humor while chopping endless

amounts of tomatoes. Thanks to Brian Kevany, my bench-mate the past four years, for his

encouragement and no-nonsense advice. Thanks to Dr. Valeriano Dal Cin, Dr. Jonathan Vogel,

Dr. Sandrine Mathieu, and Greg Maloney for their help with the taste tests and their

encouragement. I appreciate everyone's willingness to keep tasting tomatoes and for coming to

my preference test in the rain! I would also like to acknowledge some past members of the Klee

Lab. Thanks to Dr. Joseph Ciardi for teaching me basic molecular biology. Thanks to Gina

Fonfara for doing the initial screening for this proj ect. Thanks to Dr. Anna Block for answering

my questions regarding pathogen experiments. I really enjoyed working with everyone and will

miss all the friends I have made.

Finally, I especially want to thank my Mom, my Dad, my sister, and the rest of my family

for all their patience, their loving encouragement, and for believing in me.












TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. ...............4.....


LI ST OF T ABLE S ................. ...............8................


LIST OF FIGURES .............. ...............9.....


AB S TRAC T ............._. .......... ..............._ 13...


CHAPTER


1 INTRODUCTION ................. ...............15......... .....


2 LITERATURE REVIEW ..........._..._ ...............17.......__......


Flavor Perception ................. ...............17......... ......
Volatiles and Tomato Flavor .............. ... ... ...._.._ .. .... ......... ... ..........1
Methylsalicylate Is a Ubiquitous Compound Involved in Tomato Flavor .............................21
SABATH Family of Methyltransferases ................... .. .....___ .. ......_.__ ......._._ .......22
Salicylic Acid--the Bridge between Methylsalicylate and Plant Defense ................... ..........23
Volatiles Are Involved in Plant Defense .............. ...............25....
Obj ectives of This Study ................. ...............27.......... ....

3 CHARACTERIZATION OF A TOMATO S-ADENOSYL-L-METHIONINE
CARBOXYL METHYLTRANSFERASE (LESAM~T1) IN METHYLSALICYLATE
SYNTHESIS AND TOMATO FLAVOR .....__.....___ ..........._ ............3


Introducti on ............ ..... ._ ...............33....
Results .............. .................... ...............35
Identification ofLeSA M~T 1.............. ...... .. .......................3
Substrate Specificity and Kinetic Properties ofLeSAMT1 .............. .....................3
Tissue-Specific Expression ofLeSAM~T1.............. ...............37....
Production of Transgenic Lines................... ... .. ..................3
Methylsalicylate Overproducers Taste Different and Are Preferred Equally to
Controls ................. ...............40........... ....
D iscussion................ .... .. .. .... .. ........ .................4
LeSAMT1 Is a Functional Salicylic Acid Carboxyl Methyltransferase .........................41
Flavor Panels .............. ...............42....


4 ROLE OF METHYLSALICYLATE IN RESPONSE TO BACTERIAL PATHOGEN
INFECTION IN TOMATO ................. ...............65........... ....


Introducti on ................. ...............65.................
Results ................. ...............67.................












Mature Leaves of LeSAM~T1 Overexpressors Produce More Methyl salicyl ate............... 67
LeSAM~T1 Overexpressing Lines Show a Delayed Disease Response after Xcy 93-1
Inoculation ........... ......... .. .. .. .. ................... ................6
LeSAM~T1 Overexpression Affects the Free Salicylic Acid and Methylsalicylate
Pools during Xcy Infection................... ........ .... .................6
LeSAM~T1 Overexpression Affects the Conjugated Salicylic Acid and
Methylsalicylate Pools during Xcy Infection .............. ...............69....
Discussion............... ...............7


5 GENERAL DI SCU SSION ............ ......_.._ ...............8_ 0....


6 EXPERIMENTAL PROCEDURES............... ...............8


Cloning ofLeSAM~T1............... ...............83....
Production of Transgenic Plants .........._.......... .... ...............83...
Expression and Purification of GST-LeSAMT 1 ................. ...............84...............
Kinetic As says .............. ...............85....
Volatile Collection.................. .... ...........8
LeSAM~T1 Expression Quantification .............. ...............86....
Pathogen Inoculations ........._.___..... .___ ...............87.....
Ion Leakage .............. ...............87....
Bacterial Growth Curves ............... .............. ... ..........8
Free Salicylic Acid and Methylsalicylate Extractions ................. ............... ......... ...87
Conjugated Salicylic Acid and Methylsalicylate Extractions .............. .....................8
Triangle Taste Test .............. .. ...............89...
Likeability and Preference Tests .............. ...............90....

LIST OF REFERENCES ................. ...............91................


BIOGRAPHICAL SKETCH ............. ..............98.....










LIST OF TABLES


Table page

3-1 Percent similarities between amino acid sequences of LeSAMT 1, LeMTs, and
known methyltransferases............... ...........4

3-2 Percent identities between amino acid sequences of LeSAMT 1, LeMTs, and known
methyltransferases ................. ...............46.................

3-4 MeSA emission from pST10E-6841-1 and Pearson ripe fruits. ........._ ..... .............. .58

3-5 MeSA emission from pST10E-5220-2a and M82 ripe fruits ................. ............... .....58

3-7 LeSAM~T1 expression from pST10E-5220-2a and M82 ripe fruits. ................ ...............59

3-9 MeSA emission from fruits used in the preference and likeability tests ...........................63

3-10 Hedonic scale parameters for the likeability taste test. ....._.__._ ... .....___ ........._.....63

3-11 Results for preference test between Pearson and pST10E-6841-1 fruits ................... .......64

3-12 Preference test results for Pearson and pST10E-6841-1 fruits by age range. .................64

3-13 Cross-tabulated scores for likeability test comparing flavor attributes between
Pearson and pST10E-6841-1. ............. ...............64.....










LIST OF FIGURES


Fiare page

2-1 Ripening patterns of tomato volatiles in the commercial processing tomato M82......_.....28

2-2 Important volatiles in tomato flavor .............. ...............31....

2-3 Routes of salicylic acid and methylsalicylate synthesis in plants ................. ................. 32

3-1 Phylogenetic tree of amino acid sequences of SABATH methyltransferases ...................45

3-2 Alignment of the LeSAMT 1 amino acid sequence with other known SABATH
m ethyltran sferas e s........._._._.. ...___.. ...............47...

3-3 Alignment of LeSAMT 1 and related tomato methyltransferase sequences ................... ...49

3-4 Purified GST-LeSAMT detected with co-GST antibodies. ................ ..................5

3-5 Substrate specificities of LeSAMT 1 ................ ...............52...............

3-6 Lineweaver-Burke plot for the Km of SA .............. ...............53....

3-7 Lineweaver-Burke plot for the Km of SAM ......___ ............. ...._._.. .........5

3-8 LeSAM~T1 tissue-specific expression in M82 .....___.....__.___ .......____ ...........5

3-9 LeSAM~T1 expression during M82 fruit ripening .............. ...............55....

3-10 Internal pools of MeSA during M82 fruit ripening. ................ ......_._ ........._.__...56

3-11 Internal pools of free SA during M82 fruit ripening............... ...............56

3-12 Comparison between cultivars M82 and Pearson ................. ...............57........... ..

3-13 Chromatograms comparing volatile emissions from Pearson and pST10E-6841-1
ripe fruits ................ ...............58........ ......

3-14 RNA gel of MeSA overproducing ripe fruits and control ripe fruits ........._..... ...............59

3-15 MeSA emission from pSTIAS-6831-1 and Pearson ripe fruits ................ ........._.......60

3-16 Me SA emi ssi on from LeSAM~T1 anti sense fruits in the M82 b background ................... ......6 1

3-17 LeSAM~T1 expression from pSTIAS-6831-1 and Pearson flower buds .........._................62

3-18 LeSAM~T1 expression from flower buds in M82 antisense lines .........._..... ........_.......62

4-1 MeSA emission from mature leaves of M82 and pST10E-5220-2a............. ............_...73











4-2 Secondary symptom development during Xcy 93-1 infection in tomato..............._..__........74

4-3 Bacterial growth during Xcy infection in tomato .....__ ................. ........._.._....7

4-4 Ion leakage during Xcy infection in tomato .............. ...............76....

4-5 Internal pools of free SA and MeSA during Xcy infection in tomato............... ................77

4-6 MeSA emissions during Xcy infection in tomato .............. ...............78....

4-7 Internal pools of conjugated SA and MeSA during Xcy infection in tomato ....................79









LIST OF ABBREVIATIONS

3, 7-Dimethylxanthine N-methyltransferase

Alcohol dehydrogenase

Flower bud

Benzoic acid

S-Adenosyl-L-methionine: benzoic acid carboxyl methyltransferase

Basic Local Alignment Search Tool

Breaker stage

S-Adenosyl-L-methionine: benzoic acid/salicylic acid carboxyl
methyltransferase

Colony forming units

Days after pollination

Days post inoculation

Ethanol

Farnesoic acid carboxyl methyltransferase

Flower

Figwort mosaic virus

Gibberellin carboxyl methyltransferase

Gram fresh weight

Glutathi one-S-tran sferas e

Indole-acetic acid carboxyl methyltransferase

Isochorismate synthase

Jasmonic acid

Jasmonic acid carboxyl methyltransferase

Methylbenzoate


3, 7-DMXMT

ADH



BA

BAMT

BLAST

Br

B SMT


efu

dap

dpi

EtOH

FAMT

FI

FMV

GAMT

gfw

GST

IAMT

ICS

JA

JMT

MeBA









MeSA Methyl sali cyl ate

MG Mature green

ML Mature leaf

MXMT 7-Methylxanthine N-methyltransferase

PAL Phenylalanine ammonia lyase

PBS Phosphate buffered saline

PL Pyruvate lyase

PR Pathogenesis related

RT-PCR Real-time polymerase chain reaction

SA Salicylic acid

SABATH Salicylic acid (SA), benzoic acid (BA), and theobromine (TH)

SABP2 Salicylic acid binding protein 2

SAH S-Adeno syl -hom ocy stein e

SAM S-Adeno syl -L-m ethi oni ne

SAMT S-Adenosyl-L-methionine: salicylic acid carboxyl methyltransferase

SAR Systemic acquired resistance

SE Standard error

SGN Solanaceae Genomics Network

STDEV Standard deviation

TCS Caffeine synthase

TIGR The Institute for Genomic Research

TMV Tobacco mosaic virus

Tu Turning stage

Xcy Xanthomona~s campestris py. vesicatoria

YL Young leaf









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ROLE OF METHYLSALICYLATE IN TOMATO FLAVOR AND RESPONSE TO
BACTERIAL PATHOGEN INFECTION

By

Michelle Lynn Zeigler

December 2007

Chair: Harry Klee
Maj or: Plant Molecular and Cellular Biology

Volatiles are aroma compounds with low molecular weight and high vapor pressure that

evaporate at room temperature. Plants have evolved the use of volatiles for the attraction of

pollinators, seed dispersing organisms, and to aid in the response to herbivory and pathogen

attack. These plant volatiles are components of floral scent and the flavors of foods, and may

even possess medicinal properties. Tomato (Solan2um lycopersicum) flavor is due to a complex

interaction between sugars, acids, and volatile compounds. Methylsalicylate (MeSA), or oil of

wintergreen, is an important volatile component of tomato flavor. The purpose of this study was

to characterize the contribution of MeSA to tomato flavor as well as any other biological

functions in tomato. S-adenosyl-L-methionine: salicylic acid carboxyl methyltransferase

(LeSAM~T1) is the gene responsible for MeSA synthesis in tomato, and LeSAMT1 specifically

converts SA to MeSA. Plants overexpressing LeSAM~T1 were analyzed with respect to MeSA

emissions and flavor. Tomato fruits overexpressing LeSAM~T1 tasted different than the controls

but were preferred equally to the controls by an untrained consumer panel. In addition, one of

these transgenic lines overexpressing LeSAM~T1 was used as a tool to examine the contribution of

MeSA overproduction to infection by the bacterial pathogen Xanthomona~s campestris py.

vesicatoria 93-1. The results indicated that MeSA is a key metabolite that affects the










accumulation of SA during bacterial pathogen stress as well as the progression of disease

symptoms.









CHAPTER 1
INTRODUCTION

Tomato (Solan2um lycopersicum) is a member of the Solanaceae, or nightshade family. Due

to their role as food sources, the Solanaceae are the most valuable family of vegetable crops and

are economically ranked third among plant taxa (Mueller et al., 2005). Consuming fruits and

vegetables such as tomato is highly correlated with disease prevention. However, consumers are

often dissatisfied with the flavor of tomatoes due to the quality of available genetic material as

well as postharvest handling and storage practices (Baldwin et al., 2000). Breeders and growers

have focused on traits such as yield, fruit size, color, and disease resistance while flavor has

largely been ignored. Since tomatoes contain a variety of vitamins and health-promoting

phytochemicals, including Vitamin A, Vitamin C, p-carotene, lycopene, and fiber, improving the

flavor of tomatoes would encourage more consumers to buy tomatoes and improve their overall

health. An ongoing proj ect in this lab is to identify the biosynthetic and regulatory genes

responsible for the synthesis of compounds linked to tomato flavor and to determine any

additional biological functions of these flavor compounds. Once these genes have been

identified, their sequences can be used to develop molecular markers for breeders to aid in flavor

selection.

The focus of this study was on methylsalicylate (MeSA), or oil of wintergreen, which is a

maj or component of tomato flavor and is also used commercially as a flavoring agent and as an

ingredient in topical ointments for muscle pain. MeSA is also known to be involved in pollinator

attraction and the gene responsible for MeSA synthesis was first identified in the California

annual plant Clarkia breweri (Ross et al., 1999). Thi s gene, S-adenosyl -L-methionine: salicylic

acid carboxyl methyltransferase (SAM~T) catalyzes the reaction of salicylic acid and the methyl










donor S-adenosyl-L-methionine (SAM) to MeSA. The tomato homolog, LeSAM~T1, was cloned

and overexpressed in tomato plants to determine if overproducing MeSA affected tomato flavor.

In addition, MeSA is emitted in response to biotic stress. For example, MeSA is emitted

from the leaves of tobacco mosaic virus-infected tobacco (Schulaev et al., 1997), spider mite-

infested tomatoes (Ament et al., 2004), and bacterial-inoculated pepper (Cardoza and Tumlinson,

2006). The current study examined the disease progression of the bacterial pathogen

Xanthomona~s campestris py. vesicatoria (Xcy) 93-1 in transgenic tomato plants overexpressing

LeSAM~T1. The purpose of this study was to characterize the gene responsible for the

biosynthesis of MeSA in tomato, S-adenosyl-L-methionine: salicylic acid carboxyl

methyltransferase (LeSAM~T1), and to identify any other biological roles of MeSA in response to

Xcy infection in tomato.









CHAPTER 2
LITERATURE REVIEW

Flavor Perception

Humans perceive flavor as a combination of taste and smell. Taste receptors in the taste

buds of the mouth contain microvilli that bind to food dissolved in the saliva. There are five

classes of taste receptors--sour, salty, sweet, bitter, and umami, which in Japanese means

"delicious." There is no unique flavor identified with umami--instead it is a flavor enhancer and

is associated with the amino acid glutamate and food additives such as monosodium glutamate

(MSG). Each taste receptor can respond to the presence of specific chemicals, but receptors in

different regions in the mouth are more sensitive to specific tastes (Germann and Stanfield,

2005). For example, sweetness is perceived by the binding of organic molecules to the receptor

and is most sensitive at the anterior tip of the tongue. In tomato these organic molecules include

fructose and glucose. Nitrogenous compounds are responsible for bitter taste, which is perceived

at the back of the tongue. Bitterness is often associated with an avoidance response. Saltiness and

sourness are perceived on the sides of the tongue, and the salty-sensitive receptors are located

closer to the anterior portion of the tongue. Sodium ions are responsible for salty taste and

hydrogen ions are responsible for sourness. In tomato, sourness is primarily due to citric acid and

malic acid (Mahakun et al., 1979). There are several mechanisms for the taste receptors to

transmit signals to the brain (Germann and Stanfield, 2005). Salty and sour receptors open

voltage-gated channels, sweet receptors utilize a G-protein-signaling cascade, and bitter

receptors can utilize either voltage-gated channels or G-protein signaling cascades. However,

flavor perception is complex, and the sense of smell must also be considered.

Molecules known as odorants or volatiles are responsible for the aroma component of

flavor, which gives each food its unique flavor. A volatile compound is a low molecular weight










molecule with high vapor pressure, so it evaporates at room temperature. Once inhaled, volatiles

become dissolved in mucus and are carried by olfactory binding proteins to the olfactory receptor

cells located in the nasal cavity. The volatiles bind to olfactory receptors, proteins with seven

transmembrane domains, which then activate a G-protein signaling cascade (Mombaerts, 1999).

These olfactory receptor cells are actually neurons and connect to the olfactory bulb in the brain.

Two regions of the brain eventually receive the signals transmitted by the olfactory neurons via

second-order neurons--the olfactory cortex and the limbic system. The olfactory cortex

perceives and discriminates smells, while the limbic system is responsible for emotions

associated with smells. In contrast to taste receptors, in which individual receptors can respond to

all classes of taste, each class of olfactory receptor responds to a unique set of volatile

compounds (Hallem et al., 2004). To add to the complexity, one volatile compound can activate

more than one type of receptor. This flexibility allows an organism to detect a vast range of

aromatic cues from the environment, including food sources and mates. For example, the

olfactory neuron responsible for MeSA recognition has been identified in the fruit fly Drosophila

melan2oga~ster (Hallem et al., 2004). This receptor, Orl0a, responds strongly to MeSA as well as

acetophenone, isoamyl acetate, and benzaldehyde. The complexity of aroma perception is due in

part to the large number of genes encoding olfactory receptors. It is estimated that 500 to 750

genes encode for the olfactory receptors in humans, while 1000 genes are estimated in mice,

which is a larger class than immunoglobulin and T-cell receptor genes (Moembarts, 1999). This

variability allows organisms to detect a broad range of sensory cues and is beneficial for animals

and insects, who rely heavily on their sense of smell for survival.

Volatiles and Tomato Flavor

Based on their chemical structures, tomato fruit volatiles are believed to be derived from

amino acids, carotenoids, and lipids. It has been suggested that volatile compounds originating









from these essential nutrients can serve as a cue linking the food to its nutritional components

(Goff and Klee, 2006). Over 400 volatile compounds have been identified in tomato, and no

single compound has been directly linked to a unique "tomato" flavor (Buttery and Ling, 1993).

Instead, it is a unique balance and blend of specific volatiles that contribute to the flavor of

tomato. These volatile compounds are mostly concentrated in the pulp and locular gel of

tomatoes rather than the skin or seeds (Buttery et al., 1988). This localization makes sense since

the locular gel houses the seeds, and concentrating the volatiles in this area will facilitate seed

dispersal. In addition, the majority of volatiles are not produced until the fruits reach the ripe

stage (Figure 2-1). This is either due to substrate availability or the localization of volatile-

producing enzymes in different compartments that cannot act on their substrates until the tissue

is disrupted (Buttery and Ling, 1993). The carotenoid-derived volatiles, such as p-ionone,

geranylacetone, and 6-methyl-5 -heptene-2-one, show this correlation because the pigmented

substrates, such as lycopene, would not be available until the fruits ripen and the seeds are

mature. The change in color along with the increase in aroma volatiles at the ripe stage would

attract seed-dispersing organisms to eat the fruit and disperse the mature seeds. Tomatoes also

produce higher levels of sugars when they are allowed to fully ripen on the vine as opposed to

being picked at an earlier stage of ripeness (Kader et al., 1977). Allowing tomatoes to ripen off

the vine resulted in "off" flavor and decreased sweetness, considered negative attributes to

flavor. Therefore, plants have evolved survival mechanisms by orchestrating the necessary

events for seed maturity with an increase in flavor compounds.

The contribution of individual volatile compounds to a particular flavor can be ranked

according to their log odor units. A log odor unit is a ratio measurement comparing the

concentration of a particular volatile to its detection threshold. Generally speaking, a compound










with a lower detection threshold is more easily recognized by smell. However, detection limits

can vary depending on the solution used during the evaluation (Tandon et al., 2000). For

example, when tomato volatiles were evaluated in water, a methanol/ethanol/water solution, and

a deactivated tomato homogenate, the detection threshold increased from water to the alcohol

solution to the homogenate, except for the branched-chain volatile 3-methylbutanal. In other

words, the volatiles became more difficult to detect as the viscosity and polarity of the solutions

changed and the solution medium had higher affinity for the volatile compounds. If the log of the

ratio between the concentration of a compound and its detection limit is greater than one, that

volatile is said to have a positive log odor unit and positively contributes to flavor.

Seventeen volatile compounds, shown in Figure 2-2, have been identified as being

important for tomato flavor (Buttery and Ling, 1993). These volatiles are ranked by log odor

units and the structures of the volatiles along with their precursors are listed. In addition, taste

descriptors are included. In general, lipid-derived volatiles are generally described as "green" or

"grassy," ketones are denoted as "sweet, floral and fruity," and the volatiles derived from the

branched amino acids leucine and isoleucine are "earthy, stale, or musty." Aromatic compounds

are often "flowery." The focus of this study is on methylsalicylate (MeSA), a phenolic

compound that is a major component of oil of wintergreen and has a "fragrant, sweet, and root

beer-like" aroma (Heath, 1981). The also lists representative values of these compounds from

two cultivars of tomato used in this study-M82, a commercial processing tomato and Pearson, a

larger variety with more locules. Both varieties are open-pollinated. The maj ority of the volatile

compounds are higher in the Pearson variety, including MeSA. However, it is common to see

varying volatile profiles between varieties (Baldwin et al., 1991).









Since tomatoes have not generally been bred for flavor, identifying the genetic components

of flavor has been of interest to improve the quality of tomatoes for consumers. A comparison

between volatile profiles of a wild species of tomato and a commercial cultivar has shown that

the wild species contains higher levels of most of the important tomato volatiles, indicating that

selection by breeding has generally led to reduced flavor (Goff and Klee, 2006). An ongoing

focus of this lab is to identify the genetic components of volatile synthesis since this is the flavor

constituent that gives tomato its unique flavor. The availability of public genomics databases and

germplasm has made it possible to begin to link quantitative traits, such as flavor, to positions of

candidate genes. For example, using an introgression line population between the wild species

Lycopersicon pennellii and the commercial processing tomato M82, Tieman et al. (2006)

mapped important tomato volatiles to specific regions of the tomato genome. The focus of this

study is on the volatile MeSA.

Methylsalicylate Is a Ubiquitous Compound Involved in Tomato Flavor

Methylsalicylate (MeSA), or oil of wintergreen, has been identified as an important

volatile in tomato flavor (Buttery and Ling, 1993). In a metabolomics study of 94 tomato

cultivars, Tikunov et al. (2005) found that MeSA and other phenolic-derived volatiles such as

guaiacol, eugenol, ethylsalicylate, and salicylaldehyde, were some of the most variable volatiles

in tomato flavor and were largely responsible for differences in volatile profiles between

cultivars. Figure 2-1 shows that MeSA emission gradually increases during ripening in M82.

MeSA is also used commercially in a variety of flavor and cosmetic products and is Generally

Recognized as Safe (GRAS) by the Expert Panel of the Flavor and Extract Manufacturers

Association (FEMA) (Adams et al., 2005). In addition to tomato flavor, MeSA is a flavor

constituent of strawberry, currant, root beer and various fruit juices--apple, cherry, and

raspberry (Heath, 1981; Burdock, 1995). MeSA is also present in black tea, and a study by









Abraham et al. (1976) showed that varying the concentrations of MeSA added to tea leaves

changed the flavor quality. MeSA levels up to 20 ppm imparted a desirable fragrant and flowery

flavor. However, when MeSA was present above 25 ppm, the wintergreen flavor was more

pronounced and the tea was described as bitter. In the pharmaceutical industry, MeSA is often

used to mask unpleasant odors and flavors and is a constituent of toothpaste and chewing gum. It

is also found as an active ingredient in topical ointments to relieve muscle pain and symptoms of

osteoarthritis (Hansen and Elliot, 2005). MeSA i s abundant in Gaultheria procumbens, or the

wintergreen plant. In the mid 1800s, it was shown that MeSA could be hydrolyzed to salicylic

acid, a compound present in the extract of willow tree (Salix sp) used for centuries as a pain

reliever (Mahdi et al., 2005). Therefore, plants that synthesize MeSA have been used by humans

due to this volatile's medicinal and desirable fragrant properties and its presence in tomato

suggests it is an important component for the balance of tomato flavor.

SABATH Family of Methyltransferases

MeSA is known to be involved in pollinator attraction and the gene responsible for MeSA

synthesis was first identified in the California annual plant Clarkia breweri (Ross et al., 1999).

Thi s gene, S-adenosyl -L-methionine: salicylic acid carboxyl methyltransferase (SAM~T) catalyzes

the reaction of salicylic acid and the methyl donor S-adenosyl-L-methionine (SAM) to MeSA.

The discovery of this methyltransferase led to the identification of a new class of O-

methyltransferases and N-methyltransferases called the SABATH family, named for the

substrates salic lic acid, benzoic acid, and theobromine (D'Auria et al., 2003 The O-

methyltransferases in the SABATH Family of methyltransferases can utilize substrates such as

salicylic acid (SAMT), benzoic acid (BAMT), jasmonic acid (JMT), indole-acetic acid (IAMT),

and gibberellic acid (GAMT). Floral SAMTs have been characterized from Stephanotis

floribunda-SfSAMT (Pott et al., 2004) and Atropa belladonna-AbSAMT~~ll~ll~~ll~ (Fukami et al.,









2002). Methyltransferases that recognize both benzoic acid (BA) and salicylic acid (SA), or

B SMTs, have been identified in Petunia x hybrid'a PhB SMT (Negre et al., 2003; Underwood et

al., 2005) and Arabidopsis thaliana AtBSMT (Chen et al., 2003). So far only the Arabidopsis

AtJMT (Seo et al., 2001), AtlAMT (Zubieta et al., 2003; Qin et al., 2005) and AtGAMT

(Varbanova et al., 2007) have been identified. Other family members include N-

methyltransferases that can act on substrates such as 7-methylxanthine (CaMXMT1) and

theobromine (TCS1) to produce the methylated products theobromine and caffeine, respectively.

CaMXMT1 is theobromine synthase from Coffea arabica (Ogawa et al., 2001) and TCS1 is

caffeine synthase from Camellia sinensis (Kato et al., 2000). Both the O- and N-

methyltransferases use the methyl donor SAM to methylate the substrates into methyl esters.

Interestingly, the substrates of the O-methyltransferases include several plant hormones, and

methylation may serve as a means for plants to regulate hormone levels. When AtGAM~T1 and

AtGAM~T2 were overexpressed in Arabidopsis plants, the transgenic plants assumed a dwarf GA-

deficient phenotype and the predicted GA substrates were depleted (Varbanova et al., 2007).

This finding suggests that methylation may serve as an additional mode of hormone regulation.

Salicylic Acid--the Bridge between Methylsalicylate and Plant Defense

Salicylic acid (SA), the precursor to MeSA, is a plant hormone known to be involved in

defense responses by inducing the transcription of pathogenesis-related genes (PR genes) and is

involved in establishing local resistance and systemic acquired resistance (SAR) (Dempsey et al.,

1999; Durrant and Dong, 2004). SA biosynthesis remains unclear, and studies have shown that

SA can be synthesized from phenylalanine via the phenylpropanoid pathway or isochorismate

via the shikimate pathway (Yalpani et al., 1993; Wildermuth et al., 2001; Strawn et al., 2007).

The shikimate pathway precedes the phenylpropanoid pathway, and chorismate is the last

common intermediate. The branch-point chorismate can be converted to isochorismate or can be









converted to phenylalanine via several intermediates to initiate the phenylpropanoid pathway

(Figure 2-3). Isochorismate synthase (AtlCS1) was identified in Arabidopsis as an enzyme

necessary for SA biosynthesis in response to the pathogen Erysiphe orontii (Wildermuth et al.,

2001). A follow-up study showed that AtlCS 1 is located in the stroma of the chloroplast, where

the precursor chorismate is localized (Strawn et al., 2007). On the other hand, SA biosynthesis

via phenylalanine has also been studied. Phenylalanine ammonia lyase (PAL) is the first

committed step of this pathway and it catalyzes the conversion of phenylalanine to trans-

cinnamic acid. Transcriptional activation of key enzymes as well as subcelluar

compartmentalization contribute to the regulation of this pathway (Samanani and Facchini,

2006). In Arabidopsis, PAL expression is rapidly induced after pathogen inoculation (Dong et

al., 1991). Yalpani et al. (1993) showed that in tobacco culture cells, 14C-trans-cinnamic acid

was converted to radiolabeled BA and SA. In addition, tobacco leaves pre-inoculated with the

precursors phenylalanine, trans-cinnamic acid, BA, or SA had smaller lesions after inoculation

with tobacco mosaic virus (TMV), indicating that the increased levels of SA increased the

resistance. In a complementary study, Leon et al. (1993) showed that a plant extract could

convert BA to SA. A follow-up study suggested that this conversion of BA to SA was due to an

oxygenase called benzoic acid 2-hydroxylase (BA2H) (Leon et al., 1995). However, few genes

involved in the route from phenylalanine to SA have been identified despite the evidence for its

existence (Wildermuth et al., 2006). Plants most likely have several routes to synthesize SA,

given its importance in plant defense, but conclusive studies have not been done to identify all

the enzymes involved.

In addition to de novo synthesis, it has been shown that SA can also be produced by the

action of an esterase that demethylates MeSA to SA. A MeSA esterase from tobacco, salicylic









acid-binding protein 2 (SABP2), has been identified, crystallized, and silenced in transgenic

plants (Kumar and Klessig, 2003; Forouhar et al., 2005). This esterase converts MeSA to SA and

is inhibited by its product, SA (Forouhar et al., 2005). SABP2-silenced tobacco plants infected

with TMV had larger lesions in the local infection and were impaired in SAR and their

responsiveness to SA (Kumar and Klessig, 2003). This result suggests that MeSA may function

as an important pool for conversion back to SA following pathogen infection. It also suggests

that SABP2 is involved in plant innate immunity. In addition, a methylj asmonate esterase has

been identified from tomato (Stuhlfelder et al., 2004), so this mode of regulation is not unique to

SA. It has been suggested that methylation via methyltransferases may allow the hormones to

move through cell membranes as a more nonpolar molecule and demethylation via esterases

allows the bioactive molecule to act in the appropriate tissue (Yang et al., 2006a). From work in

tobacco infected with TMV, it has been suggested that MeSA also participates in plant-to-plant

signaling or as a signal within the plant after pathogen attack (Schulaev et al., 1997). However,

the exact role of these methylated conjugates is not fully understood and has not yet been proven

experimentally.

Volatiles Are Involved in Plant Defense

Plant volatiles also function as chemical defenses for protection against feeding insects and

herbivores. These volatiles can either directly deter the feeding herbivore or attract predatory

insects to dispose of the feeding insect, also known as indirect defense. In addition, plants

respond differently to insect-feeding and general wounding, and the volatile profiles between

these two types of damage differ (Pare and Tumlinson, 1999). In contrast, insects possess

olfactory receptors that can recognize host plants that have been attacked. For example, the

strawberry weevil Anthonomus rubi has five neural receptors that recognize five classes of

induced volatile compounds, including MeSA (Bichao et al., 2005). This sensitivity to the









volatile profies of infested plants may help the insects find their food source, find mates, or it

may deter the insects from laying eggs on occupied plants. MeSA is often identified in the

volatile profies of plant defense studies, which is most likely a consequence of the role of its

precursor, SA, in plant defense. There have been numerous studies examining the volatile

profiles of damaged plants, but relatively few have linked volatile emissions to the responsible

plant genes. As mentioned before, volatiles can have a role in the indirect defense response, and

MeSA has been implicated in the attraction of the predatory mite Phytoseiubts persimilis to lima

bean in response to feeding by the herbivorous mite Tetranychus urticae (De Boer et al., 2004).

In a dual fungal infection/insect feeding study, Cardoza et al. (2002) found that peanut plants

infected with the white mold Sclerotium rolfsii emitted MeSA in addition to other volatile

compounds, but insect feeding alone did not induce MeSA emission. In a separate dual bacterial

infection/insect-feeding study, Cardoza and Tumlinson (2006) found that biochemical changes in

the plant during bacterial infection affect the plant' s response against insect feeding. Insects had

a lower survival rate on plants emitting volatiles in response to bacterial infection. Each plant-

pathogen interaction is unique, but MeSA emission is repeatedly induced from different hosts

and pathogens. This suggests that MeSA may also be important in tomato defense against

bacterial pathogens, which will be another focus of the current study.

Directly related to the current proj ect, the SAM~Tfrom tomato (LeSAM~T1) has been

implicated in cross-talk with j asmonic acid (JA) in defense against spider mite feeding (Ament et

al., 2004). The JA-synthesis mutant def-1 did not accumulate MeSA upon spider mite feeding in

tomato, and the LeSAM~T1 transcript did not accumulate in the def-1 mutant unless exogenous JA

was applied. This suggests that JA signaling is upstream of SA signaling in this indirect defense

response. AtBSM~T has been implicated in defense against a fungal elicitor and herbivory (Chen










et al., 2003). A SAM~T from rice (Oryza sativa), OsBSM~T, has also been implicated in wounding

and defense (Xu et al., 2005, Koo et al., 2007). To date, the role of MeSA and LeSAM~T1 in

response to a bacterial infection in tomato has not been studied, but this role will be examined in

Chapter 4.

Objectives of This Study

Plants have evolved to use volatiles for protection and reproduction. Humans have taken

advantage of these plant survival mechanisms for commercial and medicinal means--to sell

fragrances, improve flavors, and develop medicines. The development of public genomic and

expression databases has greatly increased the accessibility of information to identify the genes

involved in secondary metabolism. The purpose of this study was to identify and characterize the

tomato gene responsible for MeSA synthesis, S-adenosyl-L-methionine: salicylic acid carboxyl

methyltransferase (LeSAM~T1) and to determine its biochemical properties. An additional goal of

this proj ect was to use transgenic tomato plants overexpressing LeSAM~T1 to determine the role

of MeSA in tomato flavor and defense against the bacterial pathogen, Xanthomona~s campestris

py. vesicatoria.













160
cis-2-penten-1-ol
-trans-2-hexenal
140 ...... trans-2-pentenal
-+ isovaleronitrile

120 11 benzaldehyde
-0- cis-3-hexenal

Sgeranylacetone
S100 .)
a ~- B-ionone
-8- 6-methyl-5-hepten-2-one







40


20




Immature Green Mature Green Breaker Tumning Stage 5


Figure 2-1. Ripening patterns of tomato volatiles in the commercial processing tomato M82. A)
Maximum emission at Stage 5, B) Increase at breaker, C) Maximum emission at
turning, D) Decrease during ripening, E) No specific ripening pattern.
Methylsalicylate appears in B) and shows a slight increase during ripening. Immature
green, mature green, and breaker stage fruits were staged by cutting the fruits in half
lengthwise. The locular gel of immature green fruits was not fully developed and the
immature seeds could be cut with a knife. Mature green fruits had a developed locular
gel with a jelly-like consistency and seeds were not cut with a knife. Breaker fruits
showed the first signs of pink color on the blossom end of the fruit and showed signs
of red color in the locular gel. Turning and Stage 5 fruits were staged by the exterior
color. Turning fruits contained approximately 30% red color and Stage 5 fruits were
95% red. Data are presented as a percentage of volatile levels at Stage 5 from field-
grown fruits. Error bars represent + SE. n = 3.














160
- -- :
140 -= -

120 --

S100-

a80-

60 -0


401 -

20-



Immature G





300


250-


S200-


a 150-





501 -




Immature (


Figure 2-1. Continued


2-methylbutanal
2-phenylethanol
2-methyl-1-butanol
r-ans-2-heptenal
methylsalicylate


;reen Mature Green Breaker Turning Stage 5


Green MatureGreen Breaker Turning Stage 5














obutylthiazole


reen MatureGreen Breaker Turning Stage 5





isobutyl acetate
phenylacetaldehyde


Green MatureGreen Breaker Turning Stage 5


200
-* 2-isl
180-

160-



S120-


vl 80-

60-

40-

20

Immature G




180

160 -

140-

~i120-

100-

80-

60-

40-


20

Immature



Figure 2-1. Continued












Log
Structure Precursor odor
unit


Odor
Threshold
(ppb)


1982 Pearson
(ng/gfw/hr) (ng/gfw/hr)


Flavor
Descriptors


Compound


16 94


0 003


25 09


0 001


19 13


0 02


37 24


0 001


0 25


0 007 Floral, sweet


5 Green, grass


0 002 Fruity, floral

Fresh, sweet,
fruity taste
1~grassy e


ecl-3-He:Cenal 0~` Lipid


p-lonone Carotenold


He:Canal oLipid



P-Damascenoner Carotenold


1-Penten-3-one Lipid



2-Methylbutanal 0 soecie


3-Methylbutanal Leucine


trar-2-He:Cenal o Lipid


Isobutylthi azole Leucine .,,


1-Hitro-2-phenylethane o Ihenyl alanine


trairs-2-Heptenal o- Lipid


Phenyl acetaldehyde Phenyl alanine


6-Mlethyl-5-hepten-2-one =2~8_ arotenold


etc-3He:Ceol HOLipid


2-Phenylethanol Phenlalaine


3-Methylbutanol on\~ Leucne


Methylsalicyl ate 4, Ihenyl alanine


2 7 0 10


1 9:? 1 Pungent


4 32 0 2 Pungent


0 93 17 Green, grassy

Pungent,
1 72 3 5 medicinal,
tomato leaf

0 3:? 2 Musty, earthy


O 34 13 13reen


0 02 4 Floral/alcohol


2 20 2000 Sweet, floral,


44 75 70 13reen, leafy


0 06 750 Floral, roses


23 6:E 120 Earthy, musty


0 33 40 Wintergreen


1= 8


Figure 2-2. Important volatiles in tomato flavor. Compounds are arranged by decreasing log
odor unit. Levels of volatile emissions from field-grown ripe Pearson and M82 fruits
are also shown.
































Chorismic Acid


ICI 's


PAL



trans-Cinnamic Acid



P-oxidative or
/non P-oxidative
Sside-chain shortening



O OH



Benzoic Acid


Isochorismic Acid


A2H

L~eSAMTI


SABP2


Salicylic Acid


11sh.Ialk..la,


Figure 2-3. Routes of salicylic acid and methylsalicylate synthesis in plants. Abbreviations are
as follows: PAL, phenylalanine ammonia lyase; ICS1, isochorismate synthase;
BA2H, benzoic acid 2-hydroxylase; LeSAMT1, S-adenosyl-L-methionine: salicylic
acid carboxyl methyltransferase; SABP2, salicylic acid binding protein; PL, pyruvate
lyase, which has not yet been shown in plants but was identified in Pseudomona~s
aeruginosa (Serino et al., 1995).


OH
Shikimic Acid


OOH


NH2




Phenylalanine









CHAPTER 3
CHARACTERIZATION OF A TOMATO S-ADENOSYL-L-METHIONINE CARBOXYL
METHYLTRANSFERASE (LESAM~T1) IN METHYLSALICYLATE SYNTHESIS AND
TOMATO FLAVOR

Introduction

Plants contain a variety of scent compounds in their floral, fruit, and vegetative tissues.

These aroma compounds, also called volatiles, are organic compounds of low molecular weight

and high vapor pressure that evaporate at room temperature. Plant volatiles serve a variety of

functions, including protection, pollination, and seed dispersal. Methylsalicylate (MeSA), or oil

of wintergreen, is one such volatile compound found in a variety of flowers, fruits, and leaves.

For example, MeSA is a component of floral scent in approximately 80 species of plants

represented by approximately 25 families (Effmert et al., 2005). In addition, it is a flavor

component of fruits such as apple, strawberry, raspberry, and tomato (Burdock, 1995; Buttery

and Ling, 1993). MeSA is also emitted in response to biotic stress. For example, MeSA is

emitted from the leaves of tobacco mosaic virus-infected tobacco (Schulaev et al., 1997), spider

mite-infested tomatoes (Ament et al., 2004), and bacterial-inoculated pepper (Cardoza and

Tumlinson, 2006). MeSA has also been shown to have antioxidant activity in mouthwash

(Battino et al., 2002). Therefore, MeSA serves a variety of functions relating to reproduction and

protection in plants and in commercial products.

MeSA is synthesized from the plant hormone salicylic acid (SA) via S-adenosyl-L-

methionine: salicylic acid carboxyl methyltransferase (SAMT). The methyl donor S-adenosyl-L-

methione (SAM) transfers a methyl group to the carboxylic acid of SA to form the methyl ester

MeSA. SAMT was first identified in the Clarkia breweri and was classified into a new family of

plant methyltransferases, the SABATH family (Ross et al., 1999). SABATH stands for the

substrates utilized by the enzymes--salicylic acid (SA), benzoic acid (BA), and theobromine










(TH) (D'Auria et al., 2003). The SABATH family has been extensively studied with respect to

floral scent, since volatile emission is closely correlated with pollinator attraction (Effmert et al.,

2005). MeSA and methylbenzoate (MeBA) are structurally related compounds synthesized by

homologous enzymes. In some cases, both MeSA and MeBA can be synthesized by the same

enzyme. Methyltransferases with dual substrate action are called benzoic acid/salicylic acid

methyltransferases (BSMTs), while those acting only on benzoic acid are called benzoic acid

methyltransferases (BAMTs). BSM~Ts have been identified in Arabidopsis thaliana (Chen et al.,

2003), Petunia x hybrid'a (Negre et al., 2003; Underwood et al., 2005), and Oryza sativa (Koo et

al., 2007). A BAMT has been identified in Antirrhinum majus (Murfitt et al., 2000), while an

SA-specific SAMT has been identified in Hoya carnosa (Effmert et al., 2005). Other

Arabidopsis thaliana family members can utilize substrates such as jasmonic acid (AtJMT) (Seo

et al., 2001), indole-acetic acid (AtlAMT) (Zubieta et al., 2003; Qin et al., 2005), gibberellins

(AtGAMT) (Varbanova et al., 2007), and a sesquiterpene, farnesoic acid (AtFAMT) (Yang et

al., 2006b). Interestingly, the maj ority of these substrates are plant hormones, and it has been

suggested that methylation of these hormones may aid in their transportation across membranes

(Yang et al., 2006a). Based on studies of floral scent, methyltransferase activity is most likely

involved in the synthesis of the flavor compound MeSA in tomato as well.

Consumers are often dissatisfied with the flavor of tomatoes (Solan2um lycopersicum),

since breeders have focused on traits such as fluit size, color, firmness, yield, and disease

resistance, and flavor has generally been ignored. Tomato flavor is due to the interaction of the

non-volatile components, sugars and acids, in addition to volatile components. Sugars and acids

are responsible for the sweet and sour taste of tomatoes, while the volatile components contribute

to the unique flavor of tomato. Over 400 volatile compounds have been identified as constituents









of tomato flavor, but only about 30 of these volatiles are believed to positively contribute to

tomato flavor (Buttery and Ling, 1993). Based on their chemical structures, these compounds are

believed to be derived from carotenoids, amino acids, and lipids (Buttery and Ling, 1993). It has

been suggested that the volatile components of flavor serve as indicators of the nutritional

composition of foods (Goff and Klee, 2006). In general, most of the important volatiles of

tomato flavor are not synthesized until the fruits are ripe, most likely to facilitate seed dispersal

(Tieman et al., 2006). MeSA is an important volatile compound found in tomato (Buttery and

Ling, 1993), and has been identified as a key compound differentiating the volatile profiles of

tomato cultivars (Tikunov et al., 2005). To date, MeSA synthesis has not been characterized in

tomato or any other fruits. Since MeSA is an important component of tomato flavor, the purpose

of this study was to identify and characterize the tomato SAM~T gene (LeSAM~T1) responsible for

MeSA synthesis. In addition, the contribution of MeSA to tomato flavor was determined using

transgenic lines that produced elevated levels of MeSA.

Results

Identification ofLeSAMT1

Using BLAST analysis against the TIGR database, eight full-length tomato SAMT-related

ESTs were identified by sequence similarity to the amino acid sequence of CbSAMT. The

predicted amino acid sequence of LeSAMT1 was the closest homolog with 66% similarity and

54% identity to CbSAMT. The predicted protein is 362 amino acids with a molecular weight of

41.3 kDa. LeSAMT1 is even more closely related to solanaceous SAMTs as seen in the

phylogenetic tree (Figure 3-1). Other SABATH family members are also represented on the tree,

including AtlAMT (Zubieta et al., 2003; Qin et al., 2005) and AtGAMT (Varbanova et al.,

2007), which recognize the substrates indole-acetic acid and gibberellins, respectively. The

amino acid sequence alignment (Figure 3-2) shows that LeSAMT1 contains all the previously









identified SA-binding and SAM-binding residues of the SABATH family of methyltransferases

(Zubieta et al., 2003). Especially noteworthy are the substrate binding residues that are identical

to CbSAMT and PhBSMT1 at amino acid residues 153, 156, 232, 233, and 357 (Figure 3-2). The

seven other tomato homologs were 41% to 60% similar to CbSAMT based on amino acid

sequence (Table 3-1). These seven homologs were named LeM~T1-7 (Solan2um lycopersicum

methyltransferase) based on their amino acid sequence similarity to LeSAMT1 (Table 3-1). The

amino acid sequence identity of these homologs is also shown (Table 3-2). The amino acid

alignment of LeSAMT1 with the other LeMTS and two singleton sequences identified from the

SGN database shows that these LeSAMT1 homologs potentially contain SA-binding residues

(Figure 3-3). Both CbSAMT and PhBSMT 1 have preferred activity on SA (Zubieta et al., 2003;

Negre et al., 2003). However, when these residues were mutated by site-directed mutagenesis in

a study by Zubieta et al. (2003), the resulting CbSAMT protein had a broader substrate

specificity and could recognize substrates such as jasmonic acid and vanillic acid. Phylogenetic

analysis of the SABATH family has shown that SAMTs specific for SA activity have a Met at

position 153 of C. breweri, while BSMTs have a His residue at position 153 (Barkman et al.,

2007). This study went on to show that the wild-type SAMT of Datura wrightii with a Met- 153

only produces MeSA, while a M153H substitution of a recombinant SAMT results in the

production of MeSA and MeBA. Therefore, it appears that this active site Met is necessary for

SA specificity and a His broadens the specificity to BA. LeSAMT1 contains a Met at this

position, suggesting that it may preferentially have activity with SA.

Substrate Specificity and Kinetic Properties of LeSAMT1

To determine if LeSAMT 1 could convert SA to MeSA in vitro, a recombinant GST-tagged

LeSAMT1 was expressed in Escherichia coli. The N-terminal GST-LeSAMT1 was purified

using the GST-affinity purification method (Figure 3-4) and assayed for activity with SA and









structurally related compounds. Compounds on which other known methyltransferases were

active in this family were also tested, including j asmonic acid and indole-3 -acetic acid. GST-

LeSAMT1 had the highest activity with SA, which was normalized to 100% (Figure 3-5).

LeSAMT1 had the next highest activity on BA, which was only four percent of the activity seen

with SA. The kinetic properties of purified GST-LeSAMT1 were also determined. At 250C, SA

had a Km of 52 C1M (Figure 3 -6) and SAM had a Km of 15 C1M (Figure 3-7). VmaxSA WaS 138

pmol/mg/min and VmaxSAM WaS 85 pmol/mg/min. The kcatSA WaS 0.055 sec^' and kcatSAM WaS

0.028 sec^l. The Km values for SA and SAM are within the range of reported values in other

characterized methyltransferases, including CbSAMT, PhBSMT1, PhBSMT2, and SfSAMT

(Effmert et al., 2005). Since the in vitro data showed that LeSAMT 1 specifically converted SA

to MeSA, LeSAM~T1 was chosen for further analysis in plant.

Tissue-Specific Expression ofLeSAMT1

After determining the substrate specificities of LeSAMT1, the expression of LeSAM~T1 was

determined in various tissues. LeSAM~T1 transcript abundance and SA availability in ripening

fruits were determined. Studies in Petunia x hybrid'a have shown that volatile emissions can be

determined by both substrate availability and substrate preference of the methyltransferase

(Negre et. al., 2003; Underwood et. al., 2005). These studies have shown that PhBSMT1 has a

substrate preference for SA in vitro, but MeSA is not consistently detectable in plant because

the substrate BA is more abundant. Therefore, PhBSMT 1 catalyzes the synthesis of MeBA for

floral scent instead of MeSA. LeSAM~T1 expression was first quantified by real-time per in

flower buds, open flowers, young unexpanded leaves, and mature expanded leaves (Figure 3-8).

For transcript abundance during fruit ripening, five stages of M82 fruits were examined-1i5

days after pollination (dap), mature green, breaker, turning, and ripe (Figure 3-9). LeSAM~T1 is









most highly expressed in immature and mature green fruits, followed by flower bud tissue, open

flowers and young unexpanded leaves. LeSAM~T1 expression decreased as the fruits ripened, and

expression levels in immature fruits were comparable to levels seen in flowers. The internal

metabolite pools of MeSA (Figure 3-10) and SA (Figure 3-10) were also determined in ripening

fruits. Since immature green fruits generally release low levels of volatiles from fresh tissue, the

internal levels of MeSA were determined from frozen tissue of all fruit stages. Internal MeSA

pools were highest in 15 dap fruits (Figure 3-10). No obvious changes in free SA occurred

throughout fruit ripening in M82 (Figure 3-1 1). Internal pools of MeSA did not appear to

correlate with either transcript abundance or SA availability, except at the 15 dap stage.

However, MeSA is still present in ripe fruits and is considered important for tomato flavor.

Production of Transgenic Lines

To further elucidate the role of MeSA in tomato flavor and its other biological functions,

overexpression and antisense transgenic lines in the M82 and Pearson backgrounds were made.

The full-length cDNA of LeSAM~T1 was cloned under the control of the constitutive 3 5S figwort

mosaic virus (FMV) promoter in the sense and antisense orientations. M82 is a commercial

processing tomato, while Pearson is a variety with more locular compartments (Figure 3-12).

Pearson has higher levels of endogenous MeSA than M82 in any season (Tables 3-4, 3-5; see y-

axes in Figures 3-15 and 3-16). Transgenic fruits from plants grown in Live Oak, FL were

analyzed in the Spring and Fall of 2006. One transgenic line in the Pearson background,

pST10E-6841-1, had over a 100-fold increase in MeSA emission with a 3000-fold increase in

transgene abundance in the Spring of 2006 determined by quantitative real-time per (Tables 3-4

and 3-6). RNA was run on a gel as a loading control for each reaction (Figure 3-14). This

transgenic line also had a 100-fold increase in MeSA emission in the Fall of 2006 (Table 3-4).

The intensity of the MeSA peak was noticeably increased on the chromatogram from the









transgenic line (Figure 3-13). Another transgenic line in the M82 background, pST10E-5220-2a,

had an approximately 50-fold increase in MeSA emission in the Spring with over 3000-fold

increase in LeSAM~T1 expression determined by quantitative real-time per (Tables 3-5 and 3-7).

RNA was run on a gel as a loading control for each reaction (Figure 3-14). In the Fall this

transgenic line had over 100 times the MeSA emission of M82 (Table 3 -5). The absolute values

of MeSA emissions varied between the Spring 2006 and Fall 2006 seasons, with the MeSA

emissions being higher in the Fall. In addition, both cultivars varied in their endogenous levels of

MeSA, indicated by the scales of the y-axes. MeSA emissions from Pearson were ten-fold higher

than M82 in the Spring and six-fold higher than M82 in the Fall. Interestingly, LeSAM\~T1

overexpression in both cultivars significantly increased MeSA emission in the transgenic lines

over two seasons.

The antisense lines in Pearson (Figure 3-15) and M82 (Figure 3-16) showed approximately

50% reduction in MeSA emission that correlated with transcript abundance. Expression of

LeSAM~T1 in the Pearson antisense line, pSTIAS-6831-1, was reduced by about 50% in flower

buds (Figure 3-17), while the M82 antisense lines showed a similar reduction (Figure 3-18). The

flower bud tissue was chosen for expression analysis in the antisense transgenic lines because

LeSAM~T1 in this tissue was more easily quantified than in the fruits. There is some variability in

fruit MeSA emissions between the Fall and Spring 2006 seasons. For example, MeSA emissions

from M82 were higher in the Fall than in the Spring. However, the antisense lines in the Fall had

a 50% to 70% reduction in MeSA levels, while the Spring 2006 lines had a 40% to 50%

reduction. The MeSA emission from the Pearson antisense line was reduced by 60% in the

Spring and by 40% in the Fall.










Methylsalicylate Overproducers Taste Different and Are Preferred Equally to Controls

Ripe fruits from the LeSAM~T1 overexpressing line in the Pearson background, pSTIOE-

6841-1, were used in a triangle taste test to determine if panelists could taste the difference

between the transgenic and control tomatoes. The pST10E-6841-1 line was chosen for the taste

panel because the Pearson variety has a more pleasant taste than the commercial processing

tomato, M82. Even though pST10E-6841-1 fruits produce over 100-fold higher MeSA than

Pearson, these levels are well below the maximum daily MeSA intake of 740 Clg/kg body weight

determined by the Expert Panel of Flavor and Extract Manufacturing Association (FEMA)

(Adams et al., 2005). Therefore, these transgenic tomatoes have acceptable levels of MeSA for

human consumption.

The triangle taste test was done using an untrained consumer panel. Seeds were removed

from the control and transgenic tomatoes before giving the samples to the panelists. Tomatoes

were sliced into small wedges and placed in plastic cups marked with a random three-digit code.

Panelists were randomly given two transgenic pST10E-6841-1 samples and one control, or two

control samples and one transgenic, and asked to pick the odd sample. Results indicated that

there was a significant difference between the control and the transgenic fruits (p < 0.01), with

50% of the panelists choosing the correct sample (Table 3-8).

Since the triangle test determined that there was a difference between the taste of the

transgenic line and the control, a subsequent preference and likeability test was done on the same

lines. MeSA emission was increased by approximately 80-fold in the transgenic fruits used for

the preference test (Table 3-9). The likeability and preference tests were done at the Sensory

Testing Facility in the Food Science and Human Nutrition Department at the University of

Florida. The panel was an untrained consumer panel. Panelists were give two samples, Pearson










and pST10E-6841-1, and asked to rate the aroma, sweetness, sourness, tomato flavor, and

overall acceptability of the samples on a nine-point hedonic scale (Table 3-10). Panelists were

then asked to pick which sample they preferred. Out of 67 panelists, 36 preferred the control and

31 preferred the transgenic (Table 3-11). The difference was not statistically significant.

Therefore, the samples were preferred equally. Since this was a panel conducted on the

University of Florida campus, the maj ority of the panelists were in 18 to 24 year age range (36

out of 67 panelists). Interestingly, a significant number of panelists in the 25 to 34 year old range

preferred the transgenic sample (Table 3-12), but this was a small sample size (n = 11). The

results from the likeability scores (Table 3-13) also showed that the samples were equally

accepted and liked by the panelists, even though Pearson consistently scored higher than the

transgenic line. The panelists that preferred the control tomato believed that it tasted more

"tomato-like." These same panelists described the transgenic tomato as having a bland taste. In

contrast, the panelists who preferred the transgenic tomato believed it had more flavor overall, so

individual perception was a most likely a factor in choosing and describing the preferred sample.

Discussion

LeSAMT1 Is a Functional Salicylic Acid Carboxyl Methyltransferase

LeSAM~T1, a tomato homolog to a known SAM~T from Clarkia breweri (Ross et al., 1999),

was cloned, and recombinant GST-LeSAMT1 was shown to have specific in vitro activity to SA.

This is expected from the amino acid sequence that shows a Met at position 156, which is present

in SAMTs that specifically convert SA to MeSA (Barkman et al., 2007). The transcript

abundance and SA availability do not completely correlate with internal MeSA pools during fruit

development. However, we do not currently have an antibody to LeSAMT1, so we do not know

the protein levels during fluit ripening. When LeSAM~T1 was overexpressed, transgenic fruits had

up to 100-fold higher MeSA emissions than the control finits. Interestingly, overexpression of










LeSAM~T1 significantly increased the MeSA from two cultivars of tomato with different

endogenous levels of MeSA. MeSA production in the antisense lines from M82 and Pearson

were reduced by about 50% over two seasons. This incomplete reduction in the antisense lines

could be due to the presence of another methyltransferase present in fruits (Figure 3-3), since

both cultivars showed a similar trend. So far seven full-length and two singleton SAMT-related

methyltransferases have been identified in the tomato genome, so one of these may also be

responsible for MeSA synthesis in fruits.

Flavor Panels

It was determined that transgenic tomatoes overproducing MeSA tasted different and were

preferred equally to control tomatoes. It appears that the overproduction of MeSA in tomatoes is

not necessarily an unfavorable trait, but it is not an overwhelmingly preferred trait, either.

Personal preference and recognition of the trait may also influence which tomato is preferred. It

was not known how often the panelists in the preference test consumed tomatoes or if they liked

tomatoes. The transgenic and control tomatoes were preferred equally in a preference test, even

though the controls tended to score higher in the likeability scores. Since the maj ority of

panelists were under age 25, it is most likely that their familiarity with tomatoes is due to

tomatoes consumed from the food service industry. These tomatoes are not picked at a fully ripe

stage due to the transportation limitations of ripe fruits. Tomatoes picked before they are fully

ripe have been reported to have a negative "off-flavor" affecting sweetness, sourness, and overall

flavor (Kader et al., 1977). If this panel was repeated, it would be beneficial to know the how

frequently the panelists consume tomatoes and if they like tomatoes, which may produce

different results.

Other studies have examined the effects of overexpression of flavor genes in tomato. The

tomato alcohol dehydrogenase 2 gene (ADH2) was overexpressed in tomato and the resulting









transgenic fruits showed a disruption in the balance of lipid-derived 6-carbon volatiles (Speirs et

al., 1998), which are often described as "green" volatiles. The 6-carbon alcohols hexanol and cis-

3-hexenol increased, which lowered the ratios of these alcohols to their precursor aldehydes. An

increase in these alcohols correlated with an increase in ripe flavor according to a taste panel.

Davidovich-Rikanati et al. (2007) found that overexpressing the geraniol synthase gene from

lemon basil resulted in tomatoes that produced more monoterpenoid volatiles that were preferred

by panelists in a preference test. The transgenic tomatoes were altered in a variety of

monoterpenes not normally present in tomato and failed to develop a red color, but were still

preferred by 60% of the panel and described as "tomato-like," "perfume," "rose," "lemongrass,"

and "geranium." Furthermore, it has been shown that different classes of tomato volatiles can

decrease the effect of the taste of other classes of volatiles (Baldwin et al., 2004). For example,

increasing levels of "green" volatiles, such as the lipid-derived volatiles, can decrease the

perception of "floral" volatiles. It has also been shown that varying the levels of MeSA can

affect the desirable flavor of foods (Abraham et al., 1976). MeSA was added to black tea and the

flavor was rated. MeSA levels up to 20 ppm imparted a desirable fragrant and flowery flavor, but

above 25 ppm, the wintergreen flavor was more pronounced and the tea was described as bitter.

In the current study, the overproduction of MeSA was preferred equally, but those

panelists that preferred the controls commented that the transgenic tomatoes tasted bland.

Perhaps the overproduction of MeSA masked the other tomato volatiles which made the

tomatoes "lose" their tomato-like taste. On the other hand, panelists that preferred the MeSA

overproducing tomatoes believed that these tomatoes had more flavor. This particular volatile

was perceived differently by the panelists, and some may be more sensitive to it than others. The









results of this study showed that increasing MeSA in tomato was neither highly desirable nor

undesirable, and the flavor balance may have been perceived differently by panelists.

This is the first report of altering MeSA synthesis in fruits. Overexpression of LeSAM\~T1

resulted in tomatoes with significantly higher levels of MeSA, which affected the taste of

tomatoes. However, tomatoes with this "different" taste were preferred equally to control

tomatoes by an untrained consumer panel, even though the controls scored consistently higher in

likeability ratings. Since flavor is a highly complex trait involving primary and secondary

metabolism, genetic engineering is a valuable tool for targeting specific biosynthetic genes

involved in flavor. By altering the synthesis of single compounds in tomato fruits, the

contribution of these volatiles to tomato flavor can be studied in a common background. The

overexpression of LeSAM~T1 is an example of targeting a specific biosynthetic gene involved in

the synthesis of MeSA and evaluating its contribution to tomato flavor.











PhBSMT2 AbSAMT
PhBSMT14 I / LeSAMT1


AmBAMT


LeMT4 -
LeMT3


LeMT7


Figure 3-1. Phylogenetic tree of amino acid sequences of SABATH methyltransferases.
LeSAMT1 is bolded. LeSAMT1 and the seven full-length tomato homologs related to
LeSAMT1 are underlined. These tomato homologs are named LeMT1, LeMT2,
LeMT3, LeMT4, LeMT5, LeMT6, and LeMT7. See Experimental Procedures for
LeMT (tomato methyltransferase) sequence information. AbSAMT is Atropa
belladollll~~~~~~llllllnn (BAB39396), AmBAMT is Antirrhinus majus (BAB39396), AtGAMT 1,
AtlAMT, and AtJMT are Arabidopsis thaliana (NP_1943 72, NP_2003 36,
AAG23343), CaMXMT is Coffea arabica 7-MXMT theobromine synthase
(BAB3 9216), Cb SAMT is Clarkia breweri (AAF00 108), HcSAMT is Hoya carnosa
(CAI05934), PhBSMT 1 and PhBSMT2 are Petunia x hybrid'a (AAO45012 and
AAO45013), SfSAMT is Stephanotis floribunda (CAC33768), and TCS1 is Camellia
sinensis caffeine synthase (BABl2278). The unrooted dendogram was generated
using the Phylip format.











Table 3-1. Percent similarities between amino acid sequences of LeSAMT1, LeMTs, and known methyltransferases.
aa LeSAMT 1 CbSAMT~1 PhB SMT 1 PhBSMT2 LeMT1 LeMT2 LeMT3 LeMT4 LeMT5 LeMT6 LeMT7
LExSAlvf l 362 100 66 85 85 65 63 58 52 50 43 40
(13SAMTrf 359 100 67 67 60 56 57 51 52 41 42
PhB SMT 1 357 100 100 68 66 57 50 51 44 41
PhB SMT2 357 100 68 66 58 50 51 44 41
Le4T 1 347 100 71 58 49 52 40 40
Leh4T2 357 100 54 47 49 40 40
Leh4T3 353 100 78 49 42 39
Leh4T4 390 100 44 41 44
LEMTvf 322 100 42 44
Leh4T6 369 100 38
LEMTv7 305 100



Table 3-2. Percent identities between amino acid sequences of LeSAMT1, LeMTs, and known methyltransferases.
aa LeSAMT 1 CbSAMT~1 PhB SMT 1 PhBSMT2 LeMT1 LeMT2 LeMT3 LeMT4 LeMT5 LeMT6 LeMT7
LExSAlvf l 362 100 54 79 79 56 54 43 39 37 31 27
(13SAMTrf 359 100 56 56 46 46 43 38 37 31 30
PhB SMT 1 357 100 99 59 56 43 38 36 33 28
PhB SMT2 357 100 59 57 43 38 36 33 28
Le4T 1 347 100 63 43 37 37 30 27
LEMTv2 357 100 40 35 37 30 27
Leh4T3 353 100 75 33 30 28
LeMT4 390 100 32 28 32
LEMTvf 322 100 29 26
Leh4T6 369 100 27
LEMTv7 305 100




















































Figure 3-2. Alignment of the LeSAMT1 amino acid sequence with other known SABATH
methyltransferases. Amino acid sequences were aligned using ClustalW. SAM/SAH
binding residues are highlighted green, substrate-binding residues are blue, and other
active site residues are yellow according to Zubieta et al. (2003). See Figure 3-1 for
accession numbers and abbreviations.








47

























SAM/SAH Binding Residues

Substrate Binding Residues

Additional Active Site Residues

Figure 3-2. Continued









































Figure 3-3. Alignment of LeSAMT1 and related tomato methyltransferase sequences. Full-
length sequences of LeSAMT1-related homologs and two singleton sequences from
the SGN database were aligned using ClustalW. See Experimental Procedures for
LeMT (tomato methyltransferase) sequence information. The Clarkia breweri SAMT
(AAF00 108) and Petunia x hybrid'a BSMT1 (AAO45012) were also included in the
alignment since they have known activity with SA (Ross et al., 1999; Negre et al.,
2003). The two singleton sequences from the SGN database are SGN-U336017 (clone
cTSB-1-J1, from a seed library) and SGN-U343182 (clone FA21BAO2, from a mixed
fruit library containing immature green, mature green, breaker, turning, and red ripe
fruits). SAM/SAH binding residues are highlighted green, substrate-binding residues
are blue, and other active site residues are yellow according to Zubieta et al. (2003).




















































SAM/SAH Binding Residues

Substrate Binding Residues

Additional Active Site Residues

Figure 3-3. Continued




50











1 2 3 4 5


6 7 L 8 9


101 kDa
97 kDa

54 kDa
#11#
S38 kDa

29 kDa




Figure 3-4. Purified GST-LeSAMT detected with co-GST antibodies. Lane 1, uninduced culture;
Lane 2, induced culture; Lane 3, crude extract; Lane 4, flow through; Lanes 5,6,7,
washes; L, ladder (prestained SDS-PAGE low range standards, BioRad); Lane 8,
elution 1; Lane 9, elution 2. The GST-LeSAMT1 fusion protein is expected to be 67.6
kDa.













Stru cture Sub strat e Relative Activity


O

~~on


100 + 2.5


Salicylic acid


- 0.2



3 + 0.3


Benzoic acid



p-Aminosalicylic acid


0.4



< 1 + 0.2


Anthranilic acid



Nicotini c acid


<1 + 0.7



<1 + 0.3


Jasmoni c acid



Indole-3-acetic acid


Figure 3-5. Substrate specificities ofLeSAMT1. Purified GST-LeSAMT1 was assayed with 1
mM of each substrate and 30 C1M SAM. 2.85 Clg protein was assayed at 250C, pH 7.5
for one hour. Samples were performed in triplicate as well as a no enzyme control.
100% activity equaled 8.2 nmol/hr/mg protein.


OCO





NHZ


~Ton











y= 321.59x + 7.2568
40-
:sR2 =0.9384
30-

S20-
10 Km 52 CtM
10

0 0.02 0.04 0.06 0.08 0.1 0.12

OM-T SA



Figure 3-6. Lineweaver-Burke plot for the Km of SA. 14C-SAM was held constant at 75 CtM.
Assays were done at 250C for two hours. Samples were performed in triplicate as well
as a no enzyme control.







200
y = 166.92x + 11.706
73` 150 R2 = 0.9939

10 -o

S50 -Km, =15 ktM
0 Vmax= 85 pmol/mg/min


0 0.2 0.4


0.6 0.8


1 1.2


]M1 SAM


Figure 3-7. Lineweaver-Burke plot for the Km of SAM. SA was held constant at 1 mM. Assays
were done at 250C for two hours. Samples were performed in triplicate as well as a no
enzyme control.













0.0018
0.0016-
0.0014-
S0.0012-
0.0010-
5 0.0008
8 0.0006-
0.0004-
0.0002-
0.0000
B FI YL ML


Figure 3-8. LeSAM~T1 tissue-specific expression in M82. A) B = bud, FI = flower, YL = young
leaf, ML = mature leaf. Error bars = SE. n = 4. B) 300 ng of each RNA sample was
run on a 1% TBE gel and stained with ethidium bromide as a loading control for each
reaction. Lanes 1-4, B; Lanes 5-8, Fl; Lanes 9-12, YL, Lanes 13-16, ML. RNA was
collected from four biological replicates of vegetative and floral tissue from the field
at Live Oak, FL and analyzed by quantitative real-time per (RT-PCR).













0.035

0.03 -

0.025 -

0.02 -

0.015 -

0.01 -

0.005 -

0-


15 DAP MG


Tu Ripe


Figure 3-9. LeSAM~T1 expression during M82 fruit ripening. A) 15 DAP = 15 days after
pollination, MG = mature green, Br = breaker, Tu = turning. Error bars = SE. n = 4.
n.d. is not detectable. B) 200 ng of RNA from each sample was run on a 1% TBE gel
and stained with ethidium bromide as a loading control for each reaction. Lanes 1-4,
15DAP; Lanes 5-8, MG; lanes 9-12, Br; Lanes 13-16, Tu; Lanes 17-20, Ripe. RNA
was collected from 4 individual fruits from the greenhouse.












80

70-

60-

50-

40-


30-

20-


70

60 -IT


15 DAP


I


I


MG Br


16pe


Figure 3-10. Internal pools of MeSA during M82 fruit ripening.
individual greenhouse fruits of each stage. 15 DAP =
mature green, Br = breaker, Tu = turning. Error bars


Internal levels of MeSA from 4
15 days after pollination, MG =
= SE. n = 4.


50-
50 -

30-
40 -

30 -

0-


I


I


15 DAP MG


Tu 16pe


Figure 3-11. Internal pools of free SA during M82 fruit ripening. Internal levels of SA from 4
individual greenhouse fruits of each stage. 15 DAP = 15 days after pollination, MG
mature green, Br = breaker, Tu = turning. Error bars = SE. n = 4.



























B














Figure 3-12. Comparison between cultivars M82 and Pearson. A) Whole fruit of M82 (left) and
Pearson (right), B) Interior view of M82 (left) and Pearson (right).










Table 3-4. MeSA emission from pST10E-6841-1 and Pearson ripe fruits.
Sample Spring 2006 Fall 2006
pS T 10E-6 84 1 -1 48.97 78.08 MeSA ng/gfw/hr
5.96 33.10 SE


Pearson


0.40
0.08


0.32 MeSA ng/gfw/hr
0.10 SE


pST10E-6841-1 is an LeSAM~T1 overexpressing line in the Pearson background. Plants were
grown in Live Oak, FL. Error bars = SE. For Spring 2006, n = 17, 16. For Fall 2006, n = 4, 8.




Ani I II I Pearson





80 |n
70- S10-84-
60-.










20-g206 Fll20



M810 15 0 20 1 25 S 30 35 40
so- ~001 o 0 pS 10 -84 -
8S1E-20-2 a eAT oeepesn i i h 8 akrud lt we
70-w i i Ok L ro a S. FOH pig20,n=1,1.FrFl 06 ,8










Table 3-6. LeSAM~T1 expression from pST10E-6841-1 and Pearson ripe fruits.
% total mRNA STDEV
pST10E-6841-1 8.14E-03 3.14E-03
Pearson 3.23E-06 3.84E-07
Several fruits were pooled in Spring 2006 and assayed by quantitative real-time polymerase
chain reaction (RT-PCR) in duplicate. n = 2.



Table 3-7. LeSAM~T1 expression from pST10E-5220-2a and M82 ripe fruits.
% total mRNA STDEV
pST10E-5220-2a 1.12E-02 2.74E-03
M82 3.22E-06 3.72E-07
Several fruits were pooled in Spring 2006 and assayed by quantitative real-time polymerase
chain reaction (RT-PCR) in duplicate. n = 2.










Figure 3-14. RNA gel of MeSA overproducing ripe fruits and control ripe fruits. Lanes 1-2,
pST10E-6841-1; Lanes 3-4, Pearson; Lanes 5-6, pST10E-5220-2a; Lanes 7-8,
M82. 200 ng of RNA was run on a 1% TBE gel and stained with ethidium bromide as
a loading control for each reaction.













O -
0.45
0 -
0.45 -
0 -
0.5 -
0 -
0.35 -
0.-
0.S -
0-


pST1AS-6831-1


Pearson


0.4 ~

0.35 -

0.3 -

0.25 -

0.2 -

0.15 -

0.1 I

0.05 -


01 I

pST1AS-6831-1


Pears on


Figure 3-15. MeSA emission from pSTIAS-6831-1 and Pearson ripe fruits. A) Spring 2006 and
B) Fall 2006. pSTIAS-6831-1 is an LeSAM~T1 antisense line in the Pearson
background. Plants were grown in Live Oak, FL. Error bars = SE. For A), n = 3, 16.
For B), n = 4, 8.













0.05

0.04-


4 0.02
S0.0 -



pST1AS- pST1AS- pST1AS- M82
7001 6917 6918






0.14
S0.12-



0.04-

0.02-


pST1AS- pSTIAS- pST1AS- M8 2
7001 6917 6918




Figure 3-16. MeSA emission from LeSAM~T1 antisense fruits in the M82 background. A) Spring
2006 and B) Fall 2006. pSTIAS-7001, 6917, and 6918 are LeSAM~T1 antisense lines
in the M82 background. For A), n = 5, 3, 9, 13. For B), n = 6, 4, 5, 18.











*


0.008 -

0.007
0.0-
0.006 -
0.0-
90.005 -

0.004


0.001


0.000 I

pSTIAS-6831-1


Pearson


Figure 3-17. LeSAM~T1 expression from pSTIAS-6831-1 and Pearson flower buds. pSTIAS-
6831-1 is an LeSAM~T1 antisense line in the Pearson background. Several flower buds
were pooled and assayed by quantitative real-time polymerase chain reaction (RT-
PCR) in duplicate from Spring 2006. Error bars = STDEV. n = 2. The quality of RNA
was checked on a 1% TBE gel stained with ethidium bromide (not shown).


0.005 -

0.004 -

0.003 -

0.002 -

0.001

0.000 -


pSTIAS-
7001


pSTIAS-
6917


pSTIAS-
6918


M82


Figure 3-18. LeSAM~T1 expression from flower buds in M82 antisense lines. pSTIAS-7001,
6917, and 6918 are LeSAM~T1 antisense lines in the M82 background. Several buds
were pooled and assayed by quantitative RT-PCR from Spring 2006 in duplicate.
Error bars = STDEV. n = 2. RNA was run on a 1% TBE gel stained with ethidium
bromide as a loading control for each reaction (not shown).









Table 3-8. Triangle taste test results between Pearson and pST10E-6841-1 fruits.
Total Number Correct 30
Number incorrect 30
Total 60
30 correct for p-value < 0.01. A chi-s uare test was used to determine the si nificance of the
number of correct responses. The probability of random guessing was assumed to be 33.33%.



Table 3-9. MeSA emission from fruits used in the preference and likeability tests.
ng/gfw/hr SE
pS T 10E-6 84 1 -1 71.94 25.66
Pearson 0.87 0.13
pST10E-6841-1 is a line overexpressing LeSAM~T1 in the Pearson background. Ripe fruits were
collected in Spring 2007. Error bars = SE. n = 3.



Table 3-10. Hedonic scale parameters for the likeability taste test.
Value Descriptor
1 dislike extremely
2 dislike very much
3 dislike moderately
4 dislike slightly
5 neither like nor dislike
6 like slightly
7 like moderately
8 like very much
9 like extremely




















by age range.


preferred Age Range preferred Age Range Total in age range
22 underl18-24 15 underl18-24 37
1 25-34 *10 25-34 11
7 35-44 2 35-44 9
5 45-54 3 45-54 8
1 55-65 1 55-65 2
36 Total 31 Total 67
* indicates < 0.05 according to a two-sided directional difference test.



Table 3-13. Cross-tabulated scores for likeability test comparing flavor attributes between
Pearson and pST10E-6841-1.
Tomato Overall
Aroma Sweetness Sourness Flavor Acceptability
Pearson 429.00 414.00 387.00 435.00 435.00 Total Score
pS T 10E-6 84 1 -1 427.00 399.00 374.00 426.00 429.00

Pearson 7.00 6.00 5.00 7.00 7.00 Median
pS T 10E-6 84 1 -1 7.00 6.00 5.00 6.00 7.00

Pearson 6.40 6.18 5.78 6.49 6.49 Mean
pS T 10E-6 84 1 -1 6.37 5.96 5.58 6.36 6.40

Pearson 1.415 1.632 1.485 1.407 1.319 STDEV
pS T 10E-6 84 1 -1 1.774 1.637 1.539 1 .453 1.558
Values for total scores were calculated by totaling the hedonic scores for each attribute. Means
were calculated by dividing the total score by the total number of panelists (67). Differences
were not statistically significant according to a one-way ANOVA.


Table 3-11. Results for preference test between Pearson and pST10E-6841-1 fruits.
Pearson preferred 36
pST10E-6841-1 preferred 31
Total 67
The difference was not statistically significant according to a two-sided directional difference
test. The samples were preferred equally.


Table 3-12. Preference test results for Pearson and pST10E-6841-1 fruits
Pearson pST10E-6841-1









CHAPTER 4
ROLE OF METHYLSALICYLATE IN RESPONSE TO BACTERIAL PATHOGEN
INFECTION IN TOMATO

Introduction

Plants must respond to a variety of environmental stresses, including pathogen attack.

Plant-pathogen interactions can be classified as compatible or incompatible. In a compatible

interaction, the pathogen successfully infects the plant host and is able to multiply, therefore

causing expansive disease symptoms. In this case the pathogen is virulent. On the other hand, an

incompatible interaction occurs when the plant host is able to mount a resistance response to the

pathogen, which results in limited growth of the pathogen. The plant host usually accomplishes

this by localizing the cell death response to the area of infection, and the pathogen is considered

to be avirulent. In this case, the defense response is localized at the site of infection, which is

also called local resistance. In addition, plants can mount a systemic resistance to protect

themselves from subsequent attack by a pathogen. This systemic acquired resistance response is

designated SAR. In SAR, distal tissues not infected by a pathogen accumulate salicylic acid (SA)

and upregulate pathogenesis-related genes (PR genes) that provide protection against subsequent

infection. After the secondary infection, bacterial growth is restricted in the distal tissue.

SA has been implicated in both local resistance and SAR (Durrant and Dong, 2004). For

example, the sid2 (SA induction-deficient) mutant ofArabidopsis thaliana is more susceptible to

local infection by Pseudomona~s syringe and Peronospora pa~ra;sitica and is impaired in SAR

(Nawrtath and Metraux, 1999). The sid2 mutant fails to accumulate SA in response to biotic and

abiotic stress. The sid2 mutant was later defined as ICS1 (isochorismate synthase), a step in the

SA biosynthesis pathway, showing that normal levels of SA are required for local and systemic

responses (Wildermuth et al., 2001). Previous work in our lab has shown that action of the

phytohormones jasmonic acid, ethylene, and SA are required for a successful infection by the









virulent pathogen Xanthonzona~s canspestris py. vesicatoria (Xcy) 93-1 in tomato (Solan2un

lycopersicunt) (O'Donnell et al., 2001; O'Donnell et al., 2003). Xcy is the bacterial pathogen

responsible for bacterial spot disease in tomato. Disease progression during Xcy infection can be

divided into two phases. Primary symptoms include lesion formation on the abaxial surface of

the leaf around 4 days post inoculation (dpi), while secondary symptom development begins

around 8 dpi and includes the appearance of chlorotic patches on the blade of the leaf. Around 10

dpi, necrotic lesions appear within these chlorotic patches that eventually spread throughout the

blade of the leaf by 16 dpi (O'Donnell et al., 2001). Transgenic plants overexpressing a bacterial

SA hydroxylase (nahg) are deficient in SA and have been used as a tool to study the role of SA

in the response to Xcy (Gaffney et al., 1993). Previous work in our lab has shown SA is required

for symptom development during the course of Xcy infection in tomato (O'Donnell et al., 200 1).

SA can also be converted to the volatile compound methylsalicylate (MeSA), which has

also been implicated in defense in different plant-pathogen interactions (Schulaev et al., 1997;

Chen et al., 2003; Koo et al., 2007). MeSA is synthesized from SA via S-adenosyl-L-methionine:

salicylic acid carboxyl methyltransferase, or SAMT (Ross et al., 1999). The Arabidopsis S-

adenosyl -L-methionine: benzoic acid/salicylic acid carboxyl methyltransferase (AtBSM~T) was

upregulated in response to the fungal elicitor alamethicin and thrip (Phttella xylostella) feeding

(Chen et al., 2003). It has been shown that MeSA can be converted to SA via an esterase from

tobacco, salicylic acid-binding protein 2 (SABP2) (Kumar and Klessig, 2003; Forouhar et al.,

2005). This esterase is inhibited by its product, SA (Forouhar et al., 2005). SABP2-silenced

tobacco plants infected with tobacco mosaic virus (TMV) had larger lesions in the local infection

and were impaired in SAR and their responsiveness to SA (Kumar and Klessig, 2003),

suggesting a role for the MeSA pool in conversion back to SA. Here, we have examined the role









of MeSA in the tomato response to Xcy. We have used transgenic tomato lines overexpressing

LeSAM~T1 that have significantly more MeSA to examine the effects upon disease symptom

development.

Results

Mature Leaves of LeSAMT1 Overexpressors Produce More Methylsalicylate

In order to establish a baseline for understanding the role of MeSA in pathogen infection of

tomato plants, MeSA emissions from the control and MeSA overproducing line were examined.

Volatile emi ssions were collected from mature leaves of M82 and the transgenic line pSTIOE-

5220-2a. Mature leaves from the transgenic line had a three-fold increase in MeSA emission

over M82-a significant increase (p < 0.01) (Figure 4-1). Therefore, the constitutively expressed

LeSAM~T1 is functional in mature leaf tissue, the stage used for bacterial inoculations.

LeSAMT1 Overexpressing Lines Show a Delayed Disease Response after Xcy 93-1
Inoculation

The purpose of this study was to examine the effect of MeSA on the local disease response

to a virulent strain of Xcy (93- 1). Leaves 3 and 4 of the M82 and pST 10E-5220-2a plants were

inoculated with Xcy 93-1. The visible symptoms of disease progression in the M82 plants were

similar to previously described symptom development in other cultivars of tomato (O'Donnell et

al., 2001). Briefly, pinpoint lesions appeared on the abaxial side of the inoculated leaves 5-6

days post inoculation (dpi). Mild chlorosis appeared on the tips of the leaflets 7-8 dpi, and by 9-

10 dpi the chlorosis became even more severe and spread beyond the tip of the leaflet. The

increasing severity of the chlorosis was accompanied by the appearance of necrotic spots and

lesions. By 14 dpi, the necrotic lesions had grown larger and the tips of the leaflets became

necrotic, also described as expansive necrosis (Figure 4-2). In an initial experiment, the bacterial

growth was assayed in both the M82 and transgenic line during the course of disease. No









difference was observed between the two lines (Figure 4-3), indicating that overproduction of

MeSA did not affect bacterial growth.

Interestingly, the MeSA overexpressing line was delayed in the development of necrotic

lesions at 10 dpi. Ion leakage, a quantitative measure of cell death, showed that the transgenic

line had a 30% reduction in cell death, which correlated with the later appearance of necrotic

lesions (Figure 4-4). Eventually, the transgenic line succumbed to the disease and the infected

leaves became necrotic. However, the onset of necrotic lesions was delayed by several days.

Therefore, the symptom development was delayed in the transgenic line, prompting further

investigation.

LeSAMT1 Overexpression Affects the Free Salicylic Acid and Methylsalicylate Pools
during Xcy Infection

Since it is known that an increase in SA is essential for and precedes the appearance of

necrotic lesions (O'Donnell et al., 2001), internal levels of MeSA and SA were measured in the

control and transgenic lines during the course of the disease. Internal metabolite levels are

typically determined in frozen tissue and represent the pool of metabolites contained in the

leaves, while the volatile emissions are collected from fresh tissue. Current methods for

quantitating hormone levels in plant tissue rely on the derivatization of hormones to methyl

esters so that the volatile derivatives can be collected by vapor phase extraction (Schmelz et al.,

2004). For example, SA is converted to its methyl ester, MeSA. However, the purpose of this

experiment was to simultaneously measure SA and MeSA in the same tissue. Therefore, a new

protocol was developed to extract endogenous SA and MeSA from the same sample. First,

endogenous MeSA was extracted from leaves and the residual SA was derivatized to propyl-SA

with a strong acid catalyst (described in Experimental Procedures). At 10 dpi, the time point with

the greatest difference in necrotic symptoms between the samples, the internal MeSA pool of the









transgenic line was seven-fold higher than M82 (Figure 4-5). In addition, the free SA pool was

four-fold higher in the transgenic line at 10 dpi (Figure 4-5). Interestingly, an increase in MeSA

correlated with an increase in free SA. At 12 dpi, the free SA levels in the wild-type reached a

maximum, which correlated with the spread of necrosis. However, the MeSA levels were six-

fold higher in the transgenic line and kept increasing at 14 dpi. Apparently the overexpression of

LeSAM~T1 in the transgenic line resulted in an increase in MeSA as well as SA in response to the

pathogen. Both MeSA and SA pools reached a maximum after 10 dpi, and the secondary

chlorotic and necrotic symptoms developed until 14 dpi.

The rate of MeSA emissions from fresh leaves were also collected during the course of

disease (Figure 4-6). By 12 dpi, the MeSA emitted from the leaves in the transgenic line reached

a maximum. SA accumulation reached a maximum at 10 dpi in the transgenic line (Figure 4-5),

so the timing of MeSA emission from the transgenic line lagged behind the substrate availability.

At 14 dpi, the MeSA emission from the transgenic line remained higher than the control. During

the course of Xcy 93- 1 infection, the internal MeSA/SA pools and the MeSA emissions of the

transgenic line were significantly increased.

LeSAMT1 Overexpression Affects the Conjugated Salicylic Acid and Methylsalicylate Pools
during Xcy Infection

The conjugated pools of SA and MeSA were also examined. Glucoside conjugation is a

general mechanism for hormone inactivation, and both SA and MeSA glucoside conjugates have

been described (Dean et al., 2005). The conjugated SA and conjugated MeSA pools started to

increase only in the transgenic line after the levels of free metabolites had reached a maximum.

Both conjugated SA and conjugated MeSA followed the same trend. The conjugated pools

started to increase at 10 dpi and continued to increase as the disease progressed to 14 dpi (Figure

4-7). Interestingly, this conjugation began at the same time as the delay in disease symptom










development occurred, 10 dpi. Therefore, overexpression ofLeSAM~T1 caused the leaves of

transgenic plants to accumulate a significantly higher level of all forms of SA during pathogen

infection free MeSA, conjugated MeSA, free SA, and conjugated SA.

Discussion

The purpose of this study was to examine the effect of LeSAM~T1 overexpression on the

local plant-pathogen interaction between tomato and the virulent bacterial strain Xcy 93-1. The

transgenic line overexpressing LeSAM~T1 showed a delay in the appearance of secondary

chlorotic and necrotic symptoms, relative to the parental control, M82, but eventually succumbed

to the pathogen. SA is required for the development of these secondary symptoms (O'Donnell et

al., 2001). Surprisingly, once SA accumulation was initiated in the transgenic line, the internal

pools of free SA, free MeSA, conjugated SA, and conjugated MeSA increased significantly over

those of M82.

Since the transgenic line is constitutively expressing LeSAM~T1, data on SA and MeSA

levels were consistent with the availability of SA to be the cause of the significant increase in

MeSA accumulation and emission from the leaves after Xcy 93-1 inoculation. The results from

this work show that in LeSAM~T1 overexpressing plants, SA levels are higher than the control

after bacterial infection. Based on previous work in our lab (O' Donnell et al., 2001), the

increase in SA would suggest that the leaves should have more severe secondary symptoms.

However, this was not the case; the transgenic plants showed a delay in the onset of secondary

symptoms. In TMV-infected tobacco, an increase in MeSA and SA was correlated with a

decrease in lesion size (Schulaev et al., 1997). It may be that MeSA provides some protection to

the tissue to prevent the spread of lesions. Further work will be needed to determine the actual

mechanism involved in the reduced rate of symptom development.









SA accumulation in the LeSAM~T1 overexpressing plants resulted in an increase in the

entire SA/MeSA pool size. At 14 dpi the total SA pool of the transgenic line, including free and

conjugated SA, was approximately three-fold higher than the controls (10 nmol and 3 nmol,

respectively). The total MeSA pool of the transgenic line was approximately six-fold higher in

the transgenic line relative to the control (12 nmol and 2 nmol, respectively). In addition, the

pool of SA and all its derivatives in the transgenic line is approximately five-fold higher than the

control (22 nmol and 5 nmol, respectively) at 14 dpi. Excess MeSA may be converted back to

SA, or MeSA accumulation may be activating a feedback loop to SA synthesis. A MeSA

esterase from tobacco, salicylic acid-binding protein 2 (SABP2), has been identified and silenced

in transgenic tobacco plants (Kumar and Klessig, 2003; Forouhar et al., 2005). This esterase

converts MeSA to SA and is inhibited by its product, SA (Forouhar et al., 2005). SABP2-

silenced tobacco plants infected with TMV had larger lesions in the local infection and were

impaired in SAR and their responsiveness to SA (Kumar and Klessig, 2003). Therefore, it is

believed that SABP2 is involved in plant innate immunity in tobacco. The work in tobacco

further suggests that MeSA may function as an important pool for conversion back to SA

following pathogen infection. A more likely possibility for the increase in SA accumulation in

the transgenic tomato plants is induction of SA biosynthesis. However, SA biosynthesis has not

been fully characterized. Studies have shown that SA can be synthesized from phenylalanine via

the phenylpropanoid pathway or isochorismate via the shikimate pathway (Yalpani et al., 1993;

Wildermuth et al., 2001; Strawn et al., 2007). The induction of SA biosynthesis genes was not

examined in this study, but this system may be a useful tool to study the different branches of SA

biosynthesis in the future.









MeSA emission has also been studied in other plant-pathogen and plant-herbivore

interactions. A study by Huang et al. (2003) compared the volatile emissions, including MeSA,

from tobacco plants infected with different strains ofPseudomona~s syringe. They observed that

MeSA was most highly induced by an avirulent strain, induced to a lesser extent by a virulent

strain, and only induced in trace amounts by a nonpathogenic strain. In a dual fungal

infection/insect feeding study, Cardoza et al. (2002) observed that peanut plants infected with the

white mold Sclerotium rolfsii emitted MeSA in addition to other volatile compounds, but insect

feeding alone did not induce MeSA emission. In a separate dual bacterial infection/insect-

feeding study, Cardoza and Tumlinson (2006) observed that avirulent and virulent strains ofXcy

induced different volatile emission profiles in pepper, and the different bacterial strains affected

the timing of when insects preferred to feed on the plants. Notably, MeSA was induced during all

treatments except insect feeding, but was the highest during the combined virulent Xcy infection

and insect feeding treatment. In addition, the insect survival rate was increased by 25% on the

infected plants, which indicates that biochemical changes in the plant during bacterial infection

affect the plant's response to insect feeding. Therefore, MeSA emission is a common theme

found during different plant-pathogen interactions, but its exact function is still unknown. The

results of the current study suggest that MeSA may serve as a key metabolite regulating SA

biosynthesis in response to SA accumulation after bacterial infection.












9

8-

7-


6-
















M82 pST10E-5220-2a



Figure 4-1. MeSA emission from mature leaves of M82 and pST10E-5220-2a. Error bars = SE.
n = 10. < 0.01 by Student's t-test.



































H I













Figure 4-2. Secondary symptom development during Xcy 93-1 infection in tomato. A) Mock
infected M82; B) M82 10 days post inoculation (dpi); C) pST10E-5220-2a 10 dpi; D)
and E), M82 12 dpi; F) and G), pST10E-5220-2a 12 dpi; H) M82 14 dpi; I) and J),
pST10E-5220-2a 14 dpi.












I


-*- M82

- -=--pST10E-5220-2a


I


8.5


7-
7.5 -

4-
7.

5-


41

0 4 8

dpi



Figure 4-3. Bacterial growth during Xcy infection in tomato. dpi
colony forming units. Error bars = SE. n = 6.


12


days post inoculation. cfu





















-i ,,


1,


-


X 0

70


-*- M82
- ---pST10E-5220-2a


60 -

SO-
50 -


30 -

20 -


~.i


10O -

0 4 810
dpi


Figure 4-4. Ion leakage during Xcy infection in tomato. dpi
SE. n = 6.


12 14


days post inoculation. Error bars





















































Figure 4-5. Internal pools of free SA and MeSA during Xcy infection in tomato. A) SA internal
pool and B) MeSA internal pool. dpi = days post inoculation. Error bars = SE. n = 4.


I
0 4 8 10 12 14
dpi


1600 -

1400 -

1200 -

1000 -

X -
600 -

400 -

200 -


0


-*-- M82
- -=- -pST10E-5220-2a


,'




'_ ___ ,/
Fr~


1800


-*- M82
- ---pifflOE-5220-2a





.f *



7


1600 -
1400 -


~n1000 -

s -

600 -
400 -

200 -
0-


12 14










1


I


-*- M82
- -A- -pST10E-5220-2al


20 -


,...---I


Figure 4-6. MeSA emissions during Xcy infection in tomato.
bars = SE. n = 3.


dpi = days post inoculation. Error










800
-*- M82
700 ---pST10E-5220-2a
600 -

500-

400-

u 0300 -.


100-


0 4 8 10 12 14
dpi


600
-*- MS 2
S00 I--- pST10E-5220-2a .

400 -




200 --

S100-



0 4 8 dp 012 14


Figure 4-7. Internal pools of conjugated SA and MeSA during Xcy infection in tomato. A)
Conjugated SA pool and B) Conjugated MeSA pool. dpi = days post inoculation.
Error bars = SE. n = 4.









CHAPTER 5
GENERAL DISCUSSION

The purpose of this study was to characterize the contribution of methylsalicylate (MeSA)

to tomato flavor and as well as any function in response to bacterial infection in tomato (Solan2um

lycopersicum). The substrate specifieity as well as the enzyme kinetics of LeSAMT1 were

determined in vitro. Plants constitutively expressing the gene responsible for MeSA synthesis in

tomato, S-adenosyl-L-methionine: salicylic acid carboxyl methyltransferase (LeSAM~T1) were

analyzed with respect to MeSA emission and flavor. In addition, one of these transgenic lines

overexpressing LeSAM~T1 was used as a tool to examine potential roles of MeSA in bacterial

pathogen responses.

LeSAMT1 was the closest tomato homolog to a known SAMT from Clarkia breweri

(Ross et al., 1999), and the in vitro enzyme activity was specific for converting the plant

hormone salicylic acid (SA) to MeSA. Overexpression of LeSAM~T1 in transgenic tomatoes

resulted in a significant increase of MeSA in fruits and leaves. An untrained consumer panel

determined that MeSA overproducing fruits tasted significantly different than the control fruits.

Subsequently, a preference taste test from an untrained consumer panel determined that the

transgenic and control fruits were preferred equally. In addition, the transgenic and control fruits

were rated according to their aroma, tomato-like flavor, sweetness, sourness, and overall

acceptability. Even though the control fruits scored slightly higher in likeability and preference

choice, the overall means did not indicate a significant preference over the transgenic fruits.

Therefore, increasing the MeSA levels in the transgenic fruits did change the flavor of the

tomatoes, but overall preference was determined by how the panelists perceived this change and

both samples were liked by panelists.









Since the increased emission of MeSA was also seen in leaves, one of the transgenic lines

was used to determine the role of MeSA during bacterial pathogen stress. After inoculation with

Xanthomona~s campestris py. vesicatoria (Xcy) 93-1, the transgenic line showed a delay in the

onset of disease symptoms. Although delayed, the transgenic plants eventually reached the same

endpoint with necrosis of the tips of the leaves. Bacterial growth was not affected in the

transgenic line. However, the transgenic line accumulated significantly higher levels of SA,

MeSA, conjugated SA, and conjugated MeSA following infection. Therefore, overexpression of

LeSAM~T1 significantly altered the free and conjugated SA and MeSA pools in the transgenic

line, which could be due to a feedback loop to SA biosynthesis. These MeSA overproducing

lines may be useful tools for elucidating which branch of SA biosynthesis is affected in response

to Xcy 93-1 infection. In the future, it would be useful to examine the free SA levels in the MeSA

overproducing fruits to see if SA accumulates as it did in response to pathogen infection. In

addition, numerous studies have shown that MeSA is a common volatile seen in the emissions of

plants inflicted with herbivore damage. It would be interesting to see if increased levels of MeSA

affect tomato-herbivore interactions with regard to attracting or repelling feeding insects.

Plants have evolved to use volatiles to attract pollinators, seed dispersing organisms, and

to adapt to stress. These plant volatiles are components of floral scent and the flavors of foods,

and may even possess medicinal properties. Humans have devised ways to take advantage of

these volatile compounds for commercial use to improve upon fragrances, flavors, and

pharmaceuticals. Understanding the molecular mechanisms of plant volatile production may aid

in the development of improved crops by molecular breeding and may even help identify novel

compounds useful for industrial purposes. However, it is difficult to target specific traits using

traditional breeding practices. Genetic engineering has made it possible to study the effect of one










gene on specific biochemical pathways involved in a variety of plant processes. Using such a

tool is advantageous when studying a complex trait such as flavor, so only one target compound

will be altered. Using transgenic lines overexpressing LeSAM~T1, the gene responsible for MeSA

synthesis in tomato, this work demonstrated the effect of one volatile, MeSA, on tomato flavor

and plant hormone pools in response to pathogen stress.









CHAPTER 6
EXPERIMENTAL PROCEDURES

Cloning of LeSAMT1

The full-length EST ofLeSAM~T1 was identified in the TIGR database by homology to the

amino acid sequence of Clarkia breweri CbSAMT (Ross et al., 1999) and was amplified with

primers (Fwd) CACCATGAAGGTTGTTGAAGTTCTTCACATGAATGGAGG and (Rev)

TTATTTTTTCTTGGTCAAGGAGACAGTAACATTTATAAACTCGAC from Flora-

Dade (Solan2un lycopersicum) bud cDNA. For LeM~T(tomato methyltransferase) sequences, full-

length clones from the TIGR database were ordered and sequenced. LeM~Ts were named

according to their percent similarity to the predicted amino acid sequence of LeSAMT1 (Table 2-

1). The following full-length clones from the TIGR database were assigned as LeM~Ts:

LeSAM~T1, cTOA4C17; LeM~T1, cLEM709; LeM~T2, cTOAl4Pl; LeM~T3, cLEll3014; LeM~T4,

cTOD6Bl16; LeM~T5, cLEW1K6; LeM~T6, cTOA28E18; LeM~T7, cTOF25N7.

Production of Transgenic Plants

The full-length open reading frame of LeSAM~T1 was cloned into a vector containing the

constitutive FMV 35S promoter (Richins et al., 1987) in the sense or antisense orientation.

Solan2un lycopersicunt (M82 and Pearson) were transformed by Agrobacteriunt-mediated

transformation (McCormick et al., 1986) with the kanamycin selectable marker. Plants were

grown in the greenhouse for initial screening and planted in the field at the University of Florida

North Florida Research and Education Center--Suwannee Valley in Live Oak, FL for additional

analysis. In Spring 2006, the MeSA overproducing Pearson line pST10E-6841-1 and the M82

antisense lines pSTIAS-7001, 6917, and 6918 were a mixture of homozygous and heterozygous

per-positive lines. The antisense Pearson line pSTIAS-6831-1 and the M82 MeSA









overproducing line pST10E-5220-2a were homozygous in Spring 2006. In Fall 2006, all lines

were homozygous (pST10E-6841-1-4, pSTIAS-7001-1, pSTIAS-6917-2, and pSTIAS-6918-1).

Expression and Purification of GST-LeSAMT1

For protein expression and purification, LeSAM~T1 was cloned into the pENTR/D-TOPO

Gateway vector (Invitrogen). The open reading frame was recombined into the N-terminal-GST-

tag Gateway vector pDEST15 (Invitrogen) and transformed into E. coli strain BL21-AI

(Invitrogen) for arabinose-inducible expression. Bacterial cultures were grown with 100 Clg/mL

carbenicillin to an OD600 of 0.4 and were induced with a 20% L-arabinose solution for a final

concentration of 0.2%. Cells were induced overnight (16 hrs) at 15oC and harvested the next day.

To harvest cells, cultures were centrifuged at 5000 g for 15 minutes and resuspended in lysis

buffer--1X PB S, lysozyme, 10% v/v glycerol, and Bacterial Protease Inhibitor Cocktail (Sigma)

at 4oC. Cells were sonicated with a Fisher Sonic Desmembrator, Model 100 (Fisher) on level 1

for 10 cycles of 5 seconds on, 30 seconds off. Cells were centrifuged at 10000 g for 15 minutes

and the GST-tagged protein was purified on a Glutathione Uniflow Resin (BD Biosciences

Clontech) at 4oC. Columns with 1.5 bed volumes of resin were equilibrated with 1XPBS. The

extract was mixed with resin on a rotating wheel for 1 hour. The flow-through was collected and

run through the column a second time. The column was washed with 16 bed volumes of lX PBS

and the GST-LeSAMT1 was eluted with Elution Buffer (10 mM glutathione, 50 mM Tris-HCl--

pH 8.0, 20% v/v glycerol). Protein levels were quantified using Bradford Reagent (BioRad) and

purification was checked with protein blotting using co-GST antibodies and visualized with ECL

reagents (Amersham). The enzyme was stored on ice at 4oC.









Kinetic Assays

Assay conditions for substrate specificity and Km determination for GST-LeSAMT1

followed Zubieta et al. (2003) with some modifications. For substrate specificity assays, 2.85 Clg

of GST-LeSAMT 1 was assayed in a 100 CIL reaction containing 50 mM Tris-HCl--pH 7.5, 100

mM KC1, 2.8 mM BME, 1 mM substrate, and a 30 C1M solution of 4:1 unlabeled SAM: 14C

SAM, specific activity 11.04 mCi/mmol (Amersham). Substrates were diluted in EtOH with the

exception of nicotinic acid, which was diluted in water. Assays were done in triplicate, including

no enzyme controls. After one hour at 250C the reactions were stopped by adding an equal

volume of hexanes. 14C-MeSA was extracted from the organic layer by vortexing samples for 15

seconds and centrifuging at 13200 g for 2 minutes. Fifty C1L of the hexane layer was counted for

5 min in 3 mL Ready Gel Scintillation Fluid (Beckman Coulter). Counts for the no enzyme

controls were subtracted from the sample counts, and activity for SA was normalized to 100%.

For the Km of SA, 2.85 Clg of purified GST-LeSAMT1 was used. 14C-SAM was held constant at

75 C1M. Two 14C-SAM stock solutions were used with varying specific activity to minimize the

use of radioactivity and to make sure the product counts were detectable. For the lower [SA]

range, 200 C1M of a 2:1 dilution (unlabeled SAM: 14C-SAM) with a specific activity of 18.4

mCi/mmol was used. For the higher [SA] range, a 200 C1M of a 4: 1 dilution with a specific

activity of 1 1.04 mCi/mmol of SAM was used. For the Km of SAM, 3.42 Clg of GST-LeSAMT 1

was used and [SA] was held constant at 1mM. [SAM] was varied using two stock solutions with

different specific activities. An undiluted specific activity of 55.2 mCi/mmol (10 C1M) was used

as the stock solution for the lower [SAM] range. A 200 C1M stock of a 2: 1 dilution with a specific

activity of 18.4 mCi/mmol was used for the higher [SAM] range. The reactions for the Km









determinations were stopped after two hours as described above. A preliminary experiment

showed that the assay was linear after two hours.

Volatile Collection

Volatiles were collected from tomato fruits according to Tieman et al. (2006). For leaf

volatile collections, two whole leaves, approximately 4-5 g of fresh tissue, were carefully loaded

into glass collection tubes to avoid unnecessary damage. Briefly, air was passed over the samples

and volatiles were collected on a SuperQ Resin for one hour. Volatiles were eluted off the

column with methylene chloride and run on a GC for analysis as described in Tieman et al.

(2006).

LeSAMT1 Expression Quantification

Total RNA was extracted using the Qiagen RNeasy Plant Mini Kit and levels of LeSAM\~T1

mRNA levels were quantified by real-time polymerase chain reaction (RT-PCR) using Taqman

one-step RT-PCR reagents (Applied Biosystems). The pericarp and locular gel from several

fruits were pooled for each RNA extraction for the analysis of transgenic plants, and each

extraction was run in duplicate. For the tissue-specific expression and pathogen experiments,

four biological replicates were analyzed per time point. LeSAM~T1 expression was determined

using the following primer/probe set-Fwd: TCCCAGAAACATTATACATTGCTGAT, Rev:

AATGACCTTAACAAGTTCTGATACCACTAA, Probe: (56-FAM)

TGGGTTGTTCTTCTGGAGCGAACACTTT (3BHQ_1).

Samples were run on the BioRad iCylcer per detection system and quantified with the

MyiQ software. The following per conditions were used: 480C, 30 min; 950C 10 min; 40 cycles

of 950C, 15 sec; 600C 1 min. A sense strand was in vitro transcribed from plasmid DNA with 3H-

UTP (MAXIscript, Ambion) and was used to determine the absolute values of RNA in the

sample.









Pathogen Inoculations

Xanthomona~s campestris py. vesicatoria (Xcy) 93-1 inoculations were done on M82

control plants and the MeSA overexpressing line pST10E-5220-2a. Bacterial inoculations were

performed on leaves 3 and 4 of 5 to 6 week old plants. Virulent Xcy 93-1 cultures were grown

overnight in 100 mL of 0.7% Nutrient Broth (Difco Laboratories, Detroit, MI) at 280C. Cells

were centrifuged at 5000 g for 10 minutes and resuspended in mock buffer (10 mM MgCl2,

0.025% (v/v) Silwet L-77 in ultrapure H20). Cells were diluted to 1 x 106 ofu in mock buffer. For

inoculations, leaves 3 and 4 were dipped in the bacterial suspension for 15 seconds. Leaves of

mock-treated plants at 0 dpi were dipped in mock buffer only. Two to three plants were assayed

per time pomnt per measurement.

Ion Leakage

For ion leakage measurements, three plants were assayed, and each infected leaf was

assayed separately (n = 6). Measurements in microohms" per cm2 per hr (designated as

Clmho/cm2/hr) are described in Lund et al. (1998).

Bacterial Growth Curves

Bacterial colony counts were performed on two leaves of three plants for each time point.

Two V/2 cm2 discs were excised with a number 5 cork borer from representative leaflets for each

time point. Discs were ground in 10 mM MgCl2 and serial dilutions were plated on 0.7%

Nutrient Broth, 1.5% Bacto-agar (Difco Laboratories, Detroit, MI). Plates were incubated at

300C for 2 days and colonies were counted for each time point.

Free Salicylic Acid and Methylsalicylate Extractions

Vapor phase extraction of free metabolites and conjugated metabolites was performed

according to Engelberth et al. (2003) and Schmelz et al. (2004) with some modifications. In

previously reported vapor phase extraction protocols, free SA was derivatized to its methyl ester









MeSA to quantify the amount of SA in the leaves. However, the goal of this experiment was to

quantify the amounts of free SA and free MeSA in the same sample, so a different method of

derivatization was needed to analyze both metabolites without interference. Briefly, individual

leaves were frozen in liquid N2 and ground to a fine powder. Approximately 100 mg of frozen

tissue was weighed into a Fastprep tube containing 1 g ceramic beads (1.1 mm Zirmil beads;

SEPR Ceramic Beads and Powders, Mountainside, NJ, USA) and an internal standard mix

containing 100 ng 2H6-SA (CDN Isotopes, Pointe-Claire, Quebec, Canada) in EtOH and 100 ng

of a lab-prepared 2H4-MeSA standard in methylene chloride. The samples were extracted with

300 CIL Extraction Buffer (2: 1:0.005 1-propanol: H20: HC1) and shaken in a Fastprep FP 120

homogenizer (Qbiogene) for 30 sec. Then 1 mL methylene chloride was added and the samples

were shaken an additional 10 seconds. Samples were centrifuged at 11300 g for one minute and

the bottom methylene chloride layer was transferred to a 4 mL glass vial and sealed. The top

aqueous layer was later used for glucoside extractions. First, free MeSA was collected from the

methylene chloride phase. The glass vial was sealed with a cap containing a high-temperature

septa and a column containing SuperQ resin was inserted into the septa, followed by a needle

carrying a stream of N2. The glass vial with the methylene chloride phase was placed on a 700C

heating block and the vapor phase was collected just until the liquid evaporated. The column

containing the MeSA was saved for recollection after the SA derivatization. The free SA

remained in the dried vial and was derivatized to propyl-SA with 30 CIL of a 2: 1 mixture of 1-

propanol: HC1. The samples were vortexed and placed in a 700C oven for 45 minutes. Samples

were cooled to room temperature and 75 CIL of a 1 M citric acid solution was added to stop the

reaction. Samples were vortexed and the vapor phase was collected on the same column as

described above. After the liquid evaporated, the sample was left on the heat block for an









additional 2 minutes. Columns were rinsed with 200 CIL ultrapure water and dried. Then the

columns were eluted with 125 C1L methylene chloride for CI-GC/MS. Free SA and MeSA levels

were quantitated using the internal standard values as described in Schmelz et al. (2004). The

propyl-SA derivatized from the endogenous SA ran at a retention time of 10. 12 min and m:z of

181 and the 2H6-SA-propylated standard ran at a retention time of 10. 11 min and m/zof 185.

Conjugated Salicylic Acid and Methylsalicylate Extractions

The aqueous layer from the vapor phase extractions was transferred to a 4 mL glass vial

and dried in a speed-vac overnight. After drying completely, the 2H6-SA standard (100 ng) was

added and dried with a stream of N2. Then the 2H4-MeSA standard was added and the vial was

immediately sealed. The glucosides for MeSA and SA were acid hydrolyzed and derivatized in

the same step by adding 30 CIL of a 2: 1 mixture of 1-propanol: HCI as described above. Samples

were incubated for 45 minutes at 700C, followed by neutralization with 75 CIL of 1 M citric acid.

Hydrolyzed MeSA and SA were collected by vapor phase extraction in one step at 700C and kept

on the heat block 2 minutes after drying. Columns were rinsed and eluted as described above.

Samples were analyzed as described above.

Triangle Taste Test

Sixty untrained volunteer panelists participated in a triangle test to determine if the MeSA

overproducing line pST10E-6841-1 tasted different than the Pearson controls. Fruits were

collected from the field and the seeds and locular gel were discarded. Panelists were given three

samples of tomato slices: either two controls and one transgenic, or two transgenics and one

control, in random order. Each sample was given a random three-digit code. Panelists were asked

to smell the samples, taste the samples, and indicate which sample was different. Thirty panelists









were correct, and a p-value of 5 0.01 was assigned after a chi-square test assuming chance

probability was 33.33% (Meilgaard et al., 2007).

Likeability and Preference Tests

The likeability and preference tests were completed at the Sensory Testing Facility in the

Food Science and Human Nutrition Department at the University of Florida according to

Meilgaard et al. (2007). Seeds from the fruits of the MeSA-overproducing line and the Pearson

control were removed and the fruits were cut into wedges. Each sample was given a random

three-digit code and presented to the panelists in random order. Sixty-seven untrained panelists

were asked to rate the aroma, sweetness, sourness, and overall acceptability of the two samples

on a nine-point hedonic scale. Panelists were also asked to choose the tomato sample they

preferred overall. The statistical significance of the preference test was analyzed by a two-sided

directional paired-comparison test. The statistical significance of the likeability test was

determined using a one-way ANOVA.










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Yang, Y., Varbanova, M., Ross, J., Wang, G., Cortes, D., Fridman, E., Shulaev, V., Noel, J.
P. and Pichersky, E. (2006a) Methylation and demethylation of plant signaling molecules.
In Recent Advances in Phytochemistry, Vol 40. (Romeo, J., ed). NY: Elsevier Science, pp.
253-270.

Yang, Y., Yuan, J.S., Ross, J., Noel, J.P., Pichersky, E. and Chen, F. (2006b) An
Arabidopsis thaliana methyltransferase capable of methylating farnesoic acid. Arch.
Biochem. Biophys. 448, 123-132.

Zubieta, C., Ross, J.R., Koscheski, P., Yang, Y., Pichersky, E. and Noel, J.P. (2003)
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family. Plant Cell, 15, 1704-1716.









BIOGRAPHICAL SKETCH

Michelle Lynn Zeigler was born July 16, 1980 and was raised in Clearwater, Florida. As

an undergraduate student at the University of Florida, she participated in the University Scholars

Program, where she worked under the direction of Dr. Joseph Ciardi in Dr. Harry Klee' s lab and

became interested in plant molecular biology. She graduated from the University of Florida in

2002 with a bachelor' s degree in microbiology and was awarded an Alumni Fellowship in the

Plant Molecular and Cellular Biology Graduate Program at the University of Florida. In Dr.

Harry Klee' s lab, she studied the role of methylsalicylate, or oil of wintergreen, in tomato flavor

and its role in the response to the virulent bacterial pathogen Xanthomona~s campestris py.

vesicatoria.





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1 ROLE OF METHYLSALICYLATE IN TOMATO FLAVOR AND RESPONSE TO BACTERIAL PATHOGEN INFECTION By MICHELLE LYNN ZEIGLER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQ UIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Michelle Lynn Zeigler

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3 This thesis is dedicated to my Mom, who has always supported and encouraged me.

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4 ACKNOWLEDGMENTS First I would like t o thank Dr. Harry Klee for giving me this opportunity to work on tomato flavor. I have learned a great deal throughout this process and I appreciate his patience and understanding. I would like to thank Dr. Don Huber for his advice and helpful discussions. I would also like to thank Dr. Bala Rathinasabapathi I wish to thank Dr. Denise Tieman for initiating this project, all her help during the tomato harvests, and especially her tomato chopping expertise for th e preference test. I appreciate all her advice, patience, and encouragement throughout my time in the lab. I would also like to thank those who helped me with the pathogen experiments. Thanks to Dr. Jeffrey Jones for his encouragement and guidance with m y pathogen experiments. I appreciate him letting me use his greenhouse space and for taking care of my plants. I would also like to thank Dr. Robert Stall and Jerry Minsavage for answering all my questions and for maintaining the plants in the greenhouse. I also thank Dr. Eric Schmelz at the USDA for his helpful discussions and for developing a new protocol to simultaneously measure plant hormones and methyl esters. I appreciate his patience while my plant hormone extraction samples wreaked havoc on his lab equipment. I thank Dr. Amarat Simonne for giving me advice for the triangle taste test. I also thank Dr. Charles Sims and the Sensory Testing Facility in the Food Science and Human Nutrition Department at the University of Florida for coordinating the p reference and likeability taste tests. I especially thank all the volunteer panelists who participated in the taste tests. I thank Dr. Ken Cline and Dr. Curt Hannah for allowing me to do rotations in their labs. I thank Dr. Carole Dabney Smith for teachin g me the basics of protein expression. I also thank Dr. Carla Lyerly Linebarger for teaching me the basics of enzyme assays.

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5 I want to thank all the members of the Klee Lab for their support and encouragement. Thanks to Dr. Mark Taylor for making the tran sgenic plants and his fun attitude. Thanks to Peter Bliss for all his help in the lab and for keeping a good sense of humor while chopping endless amounts of tomatoes. Thanks to Brian Kevany, my bench mate the past four years, for his encouragement and no nonsense advice. Thanks to Dr. Valeriano Dal Cin, Dr. Jonathan Vogel, Dr. Sandrine Mathieu, and Greg Maloney for their help with the taste tests and their my pref erence test in the rain! I would also like to acknowledge some past members of the Klee Lab. Thanks to Dr. Joseph Ciardi for teaching me basic molecular biology. Thanks to Gina Fonfara for doing the initial screening for this project. Thanks to Dr. Anna Bl ock for answering my questions regarding pathogen experiments. I really enjoyed working with everyone and will miss all the friends I have made. Finally, I especially want to thank my Mom, my Dad, my sister, and the rest of my family for all their patien ce, their loving encouragement, and for believing in me.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF TABLES ................................ ................................ ................................ ........................... 8 LIST OF FIGURES ................................ ................................ ................................ ......................... 9 ABSTRACT ................................ ................................ ................................ ................................ ... 13 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .................. 15 2 LITERATURE REVIEW ................................ ................................ ................................ ....... 17 Flavor Perception ................................ ................................ ................................ .................... 17 Volatiles and Tomato Flavor ................................ ................................ ................................ .. 18 Methylsalicylate Is a Ubiquitous Compound Involved in Tomato Flavor ............................. 21 SABATH Family of Methyltransferases ................................ ................................ ................ 22 Sa licylic Acid the Bridge between Methylsalicylate and Plant Defense ............................. 23 Volatiles Are Involved in Plant Defense ................................ ................................ ................ 25 Objectives of T his Study ................................ ................................ ................................ ........ 27 3 CHARACTERIZATION OF A TOMATO S ADENOSYL L METHIONINE CARBOXYL METHYLTRANSFERASE ( LESAMT1 ) IN METHYLSALICYLATE SYNTHESIS AND TOMATO FLAVOR ................................ ................................ .............. 33 Introduction ................................ ................................ ................................ ............................. 33 Results ................................ ................................ ................................ ................................ ..... 35 Identification of LeSAMT1 ................................ ................................ .............................. 35 Substrate Specificity and Kinetic Properties of LeSAMT1 ................................ ............ 36 Tissue Specific Expression of LeSAMT1 ................................ ................................ ........ 37 Production of Transgenic Lines ................................ ................................ ....................... 38 Methylsalicylate Overproducers Taste Different and Are Preferred Equally to Controls ................................ ................................ ................................ ........................ 40 Discussion ................................ ................................ ................................ ............................... 41 LeSAMT1 Is a Functional Salicylic Acid Carboxyl Methyltransferase ......................... 41 Flavor Panels ................................ ................................ ................................ ................... 42 4 ROLE OF METHYLSALICYLATE IN RESPONSE TO BACTERIAL PATHOGEN INFECTION IN TOMATO ................................ ................................ ................................ .... 65 Introduction ................................ ................................ ................................ ............................. 65 Results ................................ ................................ ................................ ................................ ..... 67

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7 Mature Leaves of LeSAMT1 Overexpressors Produce More Methylsalicylate ............... 67 LeSAMT1 Overexpressing Lines Show a Delayed Disease Re sponse after Xcv 93 1 Inoculation ................................ ................................ ................................ ................... 67 LeSAMT1 Overexpression Affects the Free Salicylic Acid and Methylsalicylate Pools during Xcv Infection ................................ ................................ ........................... 68 LeSAMT1 Overexpression Affects the Conjugated Salicylic Acid and Methylsalicylate Pools during Xcv Infection ................................ ............................... 69 Discussion ................................ ................................ ................................ ............................... 70 5 GENERAL DISCUSSION ................................ ................................ ................................ ..... 80 6 EXPERIMENTAL PROCEDURES ................................ ................................ ....................... 83 Cloning of LeSAMT1 ................................ ................................ ................................ .............. 83 Production of Transgenic Plants ................................ ................................ ............................. 83 Expression and Purification of GST LeSAMT1 ................................ ................................ .... 84 Kinetic Assays ................................ ................................ ................................ ........................ 85 Volatile Collection ................................ ................................ ................................ .................. 86 LeSAMT1 Expression Quantification ................................ ................................ ..................... 86 Pathogen Inoc ulations ................................ ................................ ................................ ............. 87 Ion Leakage ................................ ................................ ................................ ............................ 87 Bacterial Growth Curves ................................ ................................ ................................ ........ 87 Free Salic ylic Acid and Methylsalicylate Extractions ................................ ............................ 87 Conjugated Salicylic Acid and Methylsalicylate Extractions ................................ ................ 89 Triangle Taste Test ................................ ................................ ................................ ................. 89 Likeability and Preference Tests ................................ ................................ ............................ 90 LIST OF REFERENCES ................................ ................................ ................................ ............... 91 BIO GRAPHICAL SKETCH ................................ ................................ ................................ ......... 98

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8 LIST OF TABLES Table page 3 1 Percent similarities between amino acid sequences of LeSAMT1, LeMTs, and known methyltransferases ................................ ................................ ................................ 46 3 2 Percent identities between amino acid sequences of LeSAMT1, LeMTs, and known methyltransferases. ................................ ................................ ................................ ............. 46 3 4 MeSA emission f rom pST1OE 6841 1 and Pearson ripe fruits. ................................ ....... 58 3 5 MeSA emission from pST1OE 5220 2a and M82 ripe fruits. ................................ ........... 58 3 7 LeSAMT1 expressi on from pST1OE 5220 2a and M82 ripe fruits. ................................ .. 59 3 9 MeSA emission from fruits used in the preference and likeability tests ........................... 63 3 1 0 Hedonic scale parameters for the likeability taste test. ................................ ...................... 63 3 11 Results for preference test between Pearson and pST1OE 6841 1 fruits. ......................... 64 3 12 Preference test results for Pearson and pST1OE 6841 1 fruits by age range. ................... 64 3 13 Cross tabulated scores for likeability test comparing flavor attributes between Pearson and pST1OE 6841 1. ................................ ................................ ........................... 64

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9 LIST OF FIGURES Figure page 2 1 Ripening patterns of tomato volatiles in the commercial processing tomato M82 ............ 28 2 2 Important volatiles in tomato flavor ................................ ................................ .................. 31 2 3 Routes of salicylic acid and methylsalicylate synthesis in plants ................................ ...... 32 3 1 Phylogenetic tree of amino acid sequences of SABATH methyltransferases ................... 45 3 2 Alignment of the LeSAMT1 amino acid sequence with other known SABATH met hyltransferases ................................ ................................ ................................ .............. 47 3 3 Alignment of LeSAMT1 and related tomato methyltransferase sequences ...................... 49 3 4 Purified GST LeSAMT detected w ith GST antibodies. ................................ ................ 51 3 5 Substrate specificities of LeSAMT1 ................................ ................................ .................. 52 3 6 Lineweaver Burke plot for the K m of SA ................................ ................................ .......... 53 3 7 Lineweaver Burke plot for the K m of SAM ................................ ................................ ....... 53 3 8 LeSAMT1 tissue specific expression in M82 ................................ ................................ ..... 54 3 9 LeSAMT1 expression during M82 fruit ripening ................................ ............................... 55 3 10 Internal pools of MeSA during M82 fruit ripening. ................................ .......................... 56 3 11 Internal pools of free SA during M82 fruit ripening ................................ .......................... 56 3 12 Comparison between cultivars M82 and Pearson ................................ .............................. 57 3 13 Chromatograms c omparing volatile emissions from Pearson and pST1OE 6841 1 ripe fruits ................................ ................................ ................................ ............................ 58 3 14 RNA gel of MeSA overproducing ripe fruits and control ripe fruits ................................ 59 3 15 MeSA emission from pST1AS 6831 1 and Pearson ripe fruits ................................ ........ 60 3 16 MeSA emission from LeSAMT1 antisense fruits in the M82 background. ........................ 61 3 17 LeSAMT1 expression from pST1AS 6831 1 and Pearson flower buds ............................. 62 3 18 LeSAMT1 expression from flower buds in M82 antisen se lines ................................ ........ 62 4 1 MeSA emission from mature leaves of M82 and pST1OE 5220 2a ................................ 73

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10 4 2 Secondary symptom development during Xcv 93 1 infection in tomato ........................... 74 4 3 Bacterial growth during Xcv infection in tomato ................................ ............................... 75 4 4 Ion leakage during Xcv infection in tomato ................................ ................................ ....... 76 4 5 Internal pools of free SA and MeSA during Xcv infection in tomato ................................ 77 4 6 MeSA emissions during Xcv infection in tomato ................................ .............................. 78 4 7 Internal pools of conjugated SA and MeSA during Xcv infection in tomato .................... 79

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11 LIST OF ABBREVIATIONS 3, 7 DMXMT 3, 7 Dimethylxanthine N methyltransferase ADH Alcohol dehydrogenase B Flower bud BA Benzoic acid BAMT S A denosyl L methionine: benzoic acid carboxyl methyltransferase BLAST Basic Local Alignment Search Tool Br Breaker stage BSMT S A denosyl L methionine: benzoic acid/salicylic acid carboxyl methyltransferase cfu Colony forming units dap Days after pollination dpi Days post inoculation EtOH Ethanol FAMT Farnesoic acid carboxyl methyltransferase Fl Flower FMV Figwort mosaic virus GAMT Gibberellin carboxyl methyltransferase gfw Gram fresh weight GST Glutathione S transfer ase IAMT Indole acetic acid carboxyl methyltransferase ICS Isochorismate synthase JA Jasmonic acid JMT Jasmonic acid carboxyl methyltransferase MeBA Methylbenzoate

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12 MeSA Methylsalicylate MG Mature green ML Mature leaf MXMT 7 Methylxanthine N methy ltransferase PAL Phenylalanine ammonia lyase PBS Phosphate buffered saline PL Pyruvate lyase PR Pathogenesis related RT PCR Real time polymerase chain reaction SA Salicylic acid SABATH Salicylic acid (SA), benzoic acid (BA), and theobromine (TH) SAB P2 Salicylic acid binding protein 2 SAH S A denosyl homocysteine SAM S A denosyl L methionine SAMT S A denosyl L methionine: salicylic acid carboxyl methyltransferase SAR Systemic acquired resistance SE Standard error SGN Solanaceae Genomics Network STD EV Standard deviation TCS Caffeine synthase TIGR The Institute for Genomic Research TMV Tobacco mosaic virus Tu Turning stage Xcv Xanthomonas campestris pv. vesicatoria YL Young leaf

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13 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ROLE OF METHYLSALICYLATE IN TOMATO FLAVOR AND RESPONSE TO BACTERIAL PATHOGEN INFECTION By Michelle Lynn Zeigler December 2007 Chair: H arry Klee Major: Plant Molecular and Cellular Biology Volatiles are aroma compounds with low molecular weight and high vapor pressure that evaporate at room temperature. Plants have evolved the use of volatiles for the attraction of pollinators, seed disp ersing organisms, and to aid in the response to herbivory and pathogen attack. These plant volatiles are components of floral scent and the flavors of foods, and may even possess medicinal properties. Tomato ( Solanum lycopersicum ) flavor is due to a comple x interaction between sugars, acids, and volatile compounds. Methylsalicylate (MeSA), or oil of wintergreen, is an important volatile component of tomato flavor. The purpose of this study was to characterize the contribution of MeSA to tomato flavor as wel l as any other biological functions in tomato. S adenosyl L methionine: salicylic acid carboxyl methyltransferase ( LeSAMT1 ) is the gene responsible for MeSA synthesis in tomato, and LeSAMT1 specifically converts SA to MeSA. Plants overexpressing LeSAMT1 we re analyzed with respect to MeSA emissions and flavor. Tomato fruit s overexpressing LeSAMT1 tasted different than the controls but were preferred equally to the controls by an untrained consumer panel. In addition, one of these transgenic lines overexpress ing LeSAMT1 was used as a tool to examine the contribution of MeSA overproduction to infection by the bacterial pathogen Xanthomonas campestris pv. vesicatoria 93 1. The results indicated that MeSA is a key metabolite that affects the

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14 accumulation of SA du ring bacterial pathogen stress as well as the progression of disease symptoms.

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15 CHAPTER 1 INTRODUCTION Tomato ( Solanum lycopersicum ) is a member of the Solanaceae, or nightshade family. Due to their role as food sources, the Solanaceae are the most valua ble family of vegetable crops and are economically ranked third among plant taxa (Mueller et al., 2005). Consuming fruits and vegetables such as tomato is highly correlated with disease prevention. However, c onsumers are often dissatisfied with the flavor of tomatoes due to the quality of available genetic material as well as postharvest handling and storage practices (Baldwin et al. 2000) B reeders and growers have focused on traits such as yield, fruit size, color, and disease resistance while flavor has largely been ignored. Since tomatoes contain a variety of vitamins and health promoting phytochemicals, including Vitamin A, Vitamin C, carotene, lycopene, and fiber, improving the flavor of tomatoes would encourage more consumers to buy tomatoes and im prove their overall health. An ongoing project in this lab is to identify the biosynthetic and regulatory genes responsible for the synthesis of compounds linked to tomato flavor and to determine any additional biological functions of these flavor compound s. Once these genes have been identified, their sequences can be used to develop molecular markers for breeders to aid in flavor selection. The focus of this study was on methylsalicylate (MeSA), or oil of wintergreen, which is a major component of tomato flavor and is also used commercially as a flavoring agent and as an ingredient in topical ointments for muscle pain. MeSA is also known to be involved in pollinator attraction and the gene responsible for MeSA synthesis was first identified in the Califor nia annual plant Clarkia breweri (Ross et al. 1999). This gene, S adenosyl L methionine: salicylic acid carboxyl methyltransferase ( SAMT ) catalyzes the reaction of salicylic acid and the methyl

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16 donor S adenosyl L methionine (SAM) to MeSA. The tomato homol og, LeSAMT1 was cloned and overexpressed in tomato plants to determine if overproducing MeSA affected tomato flavor. In addition, MeSA is emitted in response to biotic stress. For example, MeSA is emitted from the leaves of tobacco mosaic virus infecte d tobacco (Schulaev et al. 1997), spider mite infested tomatoes (Ament et al. 2004), and bacterial inoculated pepper (Cardoza and Tumlinson, 2006). The current study examined the disease progression of the bacterial pathogen Xanthomonas campestris pv. ve sicatoria ( Xcv ) 93 1 in transgenic tomato plants overexpressing LeSAMT1 The purpose of this study was to characterize the gene responsible for the biosynthesis of MeSA in tomato, S adenosyl L methionine : salicylic acid carboxyl methyltransferase ( LeSAMT1 ) and to identify any other biological roles of MeSA in response to Xcv infection in tomato.

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17 C HAPTER 2 LITERATURE REVIEW Flavor Perception Humans perceive flavor as a combination of taste and smell. Taste receptors in the taste buds of the mouth contain microvilli that bind to food dissolved in the saliva. There are five classes of taste receptors sour, salty, sweet, bitter, and umami, which in Japanese means instead it is a flavor enhancer and is associated with the amino acid glutamate and food additives such as monosodium glutamate (MSG). Each taste receptor can respond to the presence of specific chemicals, but receptors in different regions in the mouth are more sensitive to specific tastes (Germann and Stanfield, 2005). For example, sweetness is perceived by the binding of organic molecules to the receptor and is most sensitive at the anterior tip of the tongue. In tomato these organic molecules include fructose and glucose. Nitrogenous com pounds are responsible for bitter taste, which is perceived at the back of the tongue. Bitterness is often associated with an avoidance response. Saltiness and sourness are perceived on the sides of the tongue, and the salty sensitive receptors are located closer to the anterior portion of the tongue. Sodium ions are responsible for salty taste and hydrogen ions are responsible for sourness. In tomato, sourness is primarily due to citric acid and malic acid (Mah a kun et al. 1979). There are several mechanis ms for the taste receptors to transmit signals to the brain (Germann and Stanfield, 2005). Salty and sour receptors open voltage gated channels, sweet receptors utilize a G protein signaling cascade, and bitter receptors can utilize either voltage gated ch annels or G protein signaling cascades. However, flavor perception is complex, and the sense of smell must also be considered. Molecules known as odorants or volatiles are responsible for the aroma component of flavor, which gives each food its unique flav or. A volatile compound is a low molecular weight

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18 molecule with high vapor pressure, so it evaporates at room temperature. Once inhaled, volatiles become dissolved in mucus and are carried by olfactory binding proteins to the olfactory receptor cells locat ed in the nasal cavity. The volatiles bind to olfactory receptors, proteins with seven transmembrane domains, which then activate a G protein signaling cascade (Mombaerts, 1999). These olfactory receptor cells are actually neurons and connect to the olfact ory bulb in the brain. Two regions of the brain eventually receive the signals transmitted by the olfactory neurons via second order neurons the olfactory cortex and the limbic system. The olfactory cortex perceives and discriminates smells, while the limb ic system is responsible for emotions associated with smells. In contrast to taste receptors, in which individual receptors can respond to all classes of taste, each class of olfactory receptor responds to a unique set of volatile compounds (Hallem et al. 2004). To add to the complexity, one volatile compound can activate more than one type of receptor. This flexibility allows an organism to detect a vast range of aromatic cues from the environment, including food sources and mates. For example, the olfact ory neuron responsible for MeSA recognition has been identified in the fruit fly Drosophila melanogaster ( Hallem et al. 2004). This receptor, Or10a, responds strongly to MeSA as well as acetophenone, isoamyl acetate, and benzaldehyde. The complexity of ar oma perception is due in part to the large number of genes encoding olfactory receptors. It is estimated that 500 to 750 genes encode for the olfactory receptors in humans, while 1000 genes are estimated in mice, which is a larger class than immunoglobulin and T cell receptor genes (Moembarts, 1999). This variability allows organisms to detect a broad range of sensory cues and is beneficial for animals and insects, who rely heavily on their sense of smell for survival. Volatiles and Tomato Flavor Based on their chemical structures, tomato fruit volatiles are believed to be derived from amino acids, carotenoids, and lipids. It has been suggested that volatile compounds originating

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19 from these essential nutrients can serve as a cue linking the food to its nutr itional components (Goff and Klee, 2006). Over 400 volatile compounds have been identified in tomato, and no Instead, it is a unique balance and blend of specifi c volatiles that contribute to the flavor of tomato. These volatile compounds are mostly concentrated in the pulp and locular gel of tomatoes rather than the skin or seeds (Buttery et al. 1988). This localization makes sense since the locular gel houses t he seeds, and concentrating the volatiles in this area will facilitate seed dispersal. In addition, the majority of volatiles are not produced until the fruit s reach the ripe stage (Figure 2 1). This is either due to substrate availability or the localizat ion of volatile producing enzymes in different compartments that cannot act on their substrates until the tissue is disrupted (Buttery and Ling, 1993). The carotenoid derived volatiles, such as ionone, geranylacetone, and 6 methyl 5 heptene 2 one, show this correlation because the pigmented substrates, such as lycopene, would not be available until the fruit s ripen and the seeds are mature. The change in color along with the increase in aroma v olatiles at the ripe stage would attract seed dispersing organisms to eat the fruit and disperse the mature seeds. Tomatoes also produce higher levels of sugars when they are allowed to fully ripen on the vine as opposed to being picked at an earlier stage of ripeness (Kader et al. 1977). Allowing tomatoes to ripen off flavor. Therefore, plants have evolved survival mechanisms by orchestrating the necessary events for seed maturity with an increase in flavor compounds. The contribution of individual volatile compounds to a particular flavor can be ranked according to their log odor units. A log odor unit is a ratio measurement comparing the concentration of a parti cular volatile to its detection threshold. Generally speaking, a compound

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20 with a lower detection threshold is more easily recognized by smell. However, detection limits can vary depending on the solution used during the evaluation (Tandon et al. 2000). Fo r example, when tomato volatiles were evaluated in water, a methanol/ethanol/water solution, and a deactivated tomato homogenate, the detection threshold increased from water to the alcohol solution to the homogenate, except for the branched chain volatile 3 methylbutanal. In other words, the volatiles became more difficult to detect as the viscosity and polarity of the solutions changed and the solution medium had higher affinity for the volatile compounds. If the log of the ratio between the concentration of a compound and its detection limit is greater than one, that volatile is said to have a positive log odor unit and positively contributes to flavor. Seventeen volatile compounds, shown in Figure 2 2, have been identified as being important for tomat o flavor (Buttery and Ling, 1993). These volatiles are ranked by log odor units and the structures of the volatiles along with their precursors are listed. In addition, taste descriptors are included. In general, lipid derived volatiles are generally descr is on methylsalicylate (MeSA), a phenolic beer two cultivars of tomato use d in this study M82, a commercial processing tomato and Pearson, a larger variety with more locules. Both varieties are open pollinated. The majority of the volatile compounds are higher in the Pearson variety, including MeSA. However, it is common to see varying volatile profiles between varieties (Baldwin et al. 1991).

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21 Since tomatoes have not generally been bred for flavor, identifying the genetic components of flavor has been of interest to improve the quality of tomatoes for consumers. A comparison be tween volatile profiles of a wild species of tomato and a commercial cultivar has shown that the wild species contains higher levels of most of the important tomato volatiles, indicating that selection by breeding has generally led to reduced flavor (Goff and Klee, 2006). An ongoing focus of this lab is to identify the genetic components of volatile synthesis since this is the flavor constituent that gives tomato its unique flavor. The availability of public genomics databases and germplasm has made it poss ible to begin to link quantitative traits, such as flavor, to positions of candidate genes. For example, using an introgression line population between the wild species Lycopersicon pen n ellii and the commercial processing tomato M82, Tieman et al (2006) m apped important tomato volatiles to specific regions of the tomato genome. The focus of this study is on the volatile MeSA. Methylsalicylate Is a Ubiquitous Compound Involved in Tomato Flavor Methylsalicylate (MeSA) or oil of wintergreen, has been identif ied as an important volatile in tomato flavor (Buttery and Ling, 1993) In a metabolomics study of 94 tomato cultivars, Tikunov et al. (2005) found that MeSA and other phenolic derived volatiles such as guaiacol, eugenol, ethylsalicylate, and salicyla l dehy de, were some of the most variable volatiles in tomato flavor and were largely responsible for differences in volatile profiles between cultivars Figure 2 1 shows that MeSA emission gradually increases during ripening in M82. MeSA is also used commerciall y in a variety of flavor and cosmetic products and is Generally Recognized as Safe (GRAS) by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA ) (Adams et al. 2005) In addition to tomato flavor, MeSA is a flavor constituent of str awberry, currant, root beer and various fruit juices apple, cherry, and raspberry (Heath, 1981; Burdock, 1995). MeSA is also present in black tea, and a study by

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22 Abraham et al. (1976) showed that varying the concentrations of MeSA added to tea leaves chang ed the flavor quality. MeSA levels up to 20 ppm imparted a desirable fragrant and flowery flavor. However, when MeSA was present above 25 ppm, the wintergreen flavor was more pronounced and the tea was described as bitter. In the pharmaceutical industry, M eSA is often used to mask unpleasant odors and flavors and is a constituent of toothpaste and chewing gum. It is also found as an active ingredient in topical ointments to relieve muscle pain and symptoms of osteoarthritis (Hansen and Elliot, 2005). MeSA i s abundant in Gaultheria procumbens or the wintergreen plant. In the mid 1800s, it was shown that MeSA could be hydrolyzed to salicylic acid, a compound present in the extract of willow tree ( Salix sp) used for centuries as a pain reliever (Mahdi et al. 2005). Therefore, plants that synthesize MeSA have been used by humans suggests it is an important component for the balance of tomato flavor. SABATH Family of Me thyltransferases MeSA is known to be involved in pollinator attraction and the gene responsible for MeSA synthesis was first identified in the California annual plant Clarkia breweri (Ross et al. 1999). This gene, S adenosyl L methionine: salicylic acid c arboxyl methyltransferase ( SAMT ) catalyzes the reaction of salicylic acid and the methyl donor S adenosyl L methionine (SAM) to MeSA. The discovery of this methyltransferase led to the identification of a new class of O methyltransferases and N methyltrans ferases called the SABATH family, named for the substrates s a licylic a cid, b enzoic a cid, and th et al. 2003). The O methyltransferases in the SABATH Family of methyltransferases can utilize substrates such as salicylic acid (SAMT), benzo ic acid (BAMT), jasmonic acid (JMT), indole acetic acid (IAMT), and gibberellic acid (GAMT). Floral SAMTs have been characterized from St ephanotis floribunda SfSAMT (Pott et al. 2004) and Atropa belladonna AbSAMT (Fukami et al.

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23 2002). Methyltransferases that recognize both benzoic acid (BA) and salicylic acid (SA) or BSMTs, have been identified in Petunia x hybrida PhBSMT (Negre et al. 2003; Underwood et al. 2005) and Arabidopsis thaliana AtBSMT (Chen et al. 2003). So far only the Arabidopsis AtJM T (Seo et al. 2001), AtIAMT (Zubieta et al. 2003; Qin et al. 2005) and AtGAMT (Varbanova et al. 2007) have been identified. Other family members include N methyltransferases that can act on substrates such as 7 methylxanthine (CaMXMT1) and theobromine (TCS1) to produce the methylated products theobromine and caffeine, respectively. CaMXMT1 is theobromine synthase from C offea arabica (Ogawa et al. 2001) and TCS1 is caffeine synthase from Camellia sinensis (Kato et al. 2000). Both the O and N methyltra nsferases use the methyl donor SAM to methylate the substrates into methyl esters. Interestingly, the substrates of the O methyltransferases include several plant hormones, and methylation may serve as a means for plants to regulate hormone levels. When At GAMT1 and AtGAMT2 were overexpressed in Arabidopsis plants, the transgenic plants assumed a dwarf GA deficient phenotype and the predicted GA substrates were depleted (Varbanova et al. 2007). This finding suggests that methylation may serve as an addition al mode of hormone regulation. Salicylic Acid the Bridge between Methylsalicylate and Plant Defense Salicylic acid (SA), the precursor to MeSA, is a plant hormone known to be involved in defense responses by inducing the transcription of pathogenesis relat ed genes ( PR genes) and is involved in establishing local resistance and systemic acquired resistance (SAR) (Dempsey et al ., 1999; Durrant and Dong, 2004). SA biosynthesis remains unclear, and studies have shown that SA can be synthesized from phenylalanin e via the phenylpropanoid pathway or isochorismate via the shikimate pathway ( Yalpani et al. 1993; Wildermuth et al. 2001; Strawn et al. 2007). The shikimate pathway precedes the phenylpropanoid pathway, and chorismate is the last common intermediate. T he branch point chorismate can be converted to isochorismate or can be

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24 converted to phenylalanine via several intermediates to initiate the phenylpropanoid pathway (Figure 2 3). Isochorismate synthase (AtICS1) was identified in Arabidopsis as an enzyme nec essary for SA biosynthesis in response to the pathogen Erysiphe orontii (Wildermuth et al. 2001) A follow up study showed that AtICS1 is located in the stroma of the chloroplast, where the precursor chorismate is localized (Strawn et al. 2007). On the o ther hand, SA biosynthesis via phenylalanine has also been studied. Phenylalanine ammonia lyase (PAL) is the first committed step of this pathway and it catalyzes the conversion of phenylalanine to trans cinnamic acid. Transcriptional activation of key enz ymes as well as subcelluar compartmentalization contribute to the regulation of this pathway (Samanani and Facchini, 2006). In Arabidopsis, PAL expression is rapidly induced after pathogen inoculation (Dong et al. 1991). Yalpani et al. (1993) showed that in tobacco culture cells, 14 C trans cinnamic acid was converted to radiolabeled BA and SA. In addition, tobacco leaves pre inoculated with the precursors phenylalanine, trans cinnamic acid, BA, or SA had smaller lesions after inoculation with tobacco mosai c virus (TMV), indicating that the increased levels of SA increased the resistance. In a complementary study, Leon et al. (1993) showed that a plant extract could convert BA to SA. A follow up study suggested that this conversion of BA to SA was due to an oxygenase called benzoic acid 2 hydroxylase (BA2H) (Leon et al. 1995 ). However, few genes involved in the route from phenylalanine to SA have been identified d espite the evidence for its existence (Wildermuth et al. 2006). Plants most likely have several routes to synthesize SA, given its importance in plant defense, but conclusive studies have not been done to identify all the enzymes involved. In addition to de novo synthesis, it has been shown that SA can also be produced by the action of an esterase t hat demethylates MeSA to SA. A MeSA esterase from tobacco, salicylic

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25 acid binding protein 2 (SABP2), has been identified, crystallized, and silenced in transgenic plants (Kumar and Klessig, 2003; Forouhar et al. 2005). This esterase converts MeSA to SA an d is inhibited by its product, SA (Forouhar et al. 2005). SABP2 silenced tobacco plants infected with TMV had larger lesions in the local infection and were impaired in SAR and their responsiveness to SA (Kumar and Klessig, 2003). This result suggests tha t MeSA may function as an important pool for conversion back to SA following pathogen infection. It also suggests that SABP2 is involved in plant innate immunity. In addition, a methyljasmonate esterase has been identified from tomato (Stuhlfelder et al. 2004), so this mode of regulation is not unique to SA. It has been suggested that methylation via methyltransferases may allow the hormones to move through cell membranes as a more nonpolar molecule and demethylation via esterases allows the bioactive mole cule to act in the appropriate tissue (Yang et al. 2006a). From work in tobacco infected with TMV, it has been suggested that MeSA also participates in plant to plant signaling or as a signal within the plant after pathogen attack (Schulaev et al. 1997). However, the exact role of these methylated conjugates is not fully understood and has not yet been proven experimentally. Volatiles Are Involved in Plant Defense Plant volatiles also function as chemical defenses for protection against feeding insects an d herbivores. These volatiles can either directly deter the feeding herbivore or attract predatory insects to dispose of the feeding insect, also known as indirect defense. In addition, plants respond differently to insect feeding and general wounding, and the volatile profiles between these two types of damage differ (Pare and Tumlinson, 1999). In contrast, insects possess olfactory receptors that can recognize host plants that have been attacked. For example, the strawberry weevil Anthonomus rubi has five neural receptors that recognize five classes of induced volatile compounds, including MeSA (Bichao et al. 2005). This sensitivity to the

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26 volatile profiles of infested plants may help the insects find their food source, find mates or it may deter the ins ects from laying eggs on occupied plants. MeSA is often identified in the volatile profiles of plant defense studies, which is most likely a consequence of the role of its precursor, SA, in plant defense. There have been numerous studies examining the vola tile profiles of damaged plants, but relatively few have linked volatile emissions to the responsible plant genes. As mentioned before, volatiles can have a role in the indirect defense response, and MeSA has been implicated in the attraction of the predat ory mite Phytoseiulus persimilis to lima bean in response to feeding by the herbivorous mite Tetranychus urticae (De Boer et al. 2004). In a dual fungal infection/insect feeding study, Cardoza et al. (2002) found that peanut plants infected with the white mold Sclerotium rolfsii emitted MeSA in addition to other volatile compounds, but insect feeding alone did not induce MeSA emission. In a separate dual bacterial infection/insect feeding study, Cardoza and Tumlinson (2006) found that biochemical changes i n a lower survival rate on plants emitting volatiles in response to bacterial infection. Each plant pathogen interaction is unique, but MeSA emission is re peatedly induced from different hosts and pathogens. This suggests that MeSA may also be important in tomato defense against bacterial pathogens, which will be another focus of the current study. Directly related to the current project, the SAMT from tomat o ( LeSAMT1 ) has been implicated in cross talk with jasmonic acid (JA) in defense against spider mite feeding (Ament et al. 2004). The JA synthesis mutant def 1 did not accumulate MeSA upon spider mite feeding in tomato and the LeSAMT1 transcript did not accumulate in the def 1 mutant unless exogenous JA was applied. This suggests that JA signaling is upstream of SA signaling in this indirect defense response. At BS MT has been implicated in defense against a fungal elicitor and herbivory (Chen

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27 et al. 2003) A SAMT from rice ( Oryza sativa ), OsBSMT has also been implicated in wounding and defense (Xu et al. 2005, Koo et al. 2007). To date, the role of MeSA and LeSAMT1 in response to a bacterial infection in tomato has not been studied, but this role will b e examined in Chapter 4. Objectives of T his Study Plants have evolved to use volatiles for protection and reproduction. Humans have taken advantage of these plant survival mechanisms for commercial and medicinal means to sell fragrances, improve flavors, and develop medicines. The development of public genomic and expression databases has greatly increased the accessibility of information to identify the genes involved in secondary metabolism. The purpose of this study was to identify and characterize the tomato gene responsible for MeSA synthesis, S adenosyl L methionine: salicylic acid carboxyl methyltransferase ( LeSAMT1 ) and to determine its biochemical properties. An additional goal of this project was to use transgenic tomato plants overexpressing LeSA MT1 to determine the role of MeSA in tomato flavor and defense against the bacterial pathogen, Xanthomonas campestris pv. vesicatoria

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28 A Figure 2 1 Ripening patterns of tomato volatiles in the commercial processing tomato M8 2 A) Maximum emission at Stage 5, B) Increase at breaker, C) Maximum emission at turning, D) Decrease during ripening, E) No specific ripening pattern. Methylsalicylate appears in B) and shows a slight increase during ripening. Immature green, mature gree n, and breaker stage fruit s were staged by cutting the fruit s in half lengthwise The locular gel of immature green fruit s was not fully developed and the immature seeds could be c ut with a knife. Mature green fruit s had a develope d locular gel with a jell y like consistency and seeds were not cut with a knife Breaker fruit s showed the first signs of pink color on the blossom end of the fruit and showed signs of red color in the locular gel Turning and Stage 5 fruit s were staged by the exterior color Turn ing fruit s contained approximately 30% red color and Stage 5 fruit s were 95% red Data are presented as a percentage of volatile levels at Stage 5 from field grown fruit s Error bars represent + SE. n = 3.

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29 B C Figure 2 1. Continued

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30 D E F igure 2 1. Continued

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31 Figure 2 2. Important volatiles in t omato flavor. Compounds are arranged by decreasing log odor unit. Levels of volatile emissions from field grown ripe Pearson and M82 fruit s are also shown.

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32 Figure 2 3. Routes of s alicylic a cid and m ethylsalicylate s ynthesis in p lants Abbreviations are as follows: PAL, phenylalanine ammonia lyase; ICS1, isochorismate synthase; BA2H, benzoic acid 2 hydroxylase; LeSAMT1, S adenosyl L methi onine: salicylic acid carboxyl methyltransferase; SABP2, salicylic acid binding protein; PL, pyruvate lyase, which has not yet been shown in plants but was identified in Pseudomonas aeruginosa (Serino et al. 1995).

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33 CHAPTER 3 CHARACTERIZATION OF A TOMATO S ADENOSYL L METHIONINE CARBOXYL METHYLTRANSFERASE ( LESAMT1 ) IN METHYLSALICYLAT E SYNTHESIS AND TOMATO FLAVOR Introduction Plants contain a variety of scent compounds in their floral, fruit, and vegetative tissues. These aroma compounds, also called volatil es, are organic compounds of low molecular weight and high vapor pressure that evaporate at room temperature. Plant volatiles serve a variety of functions, including protection, pollination, and seed dispersal. Methylsalicylate (MeSA), or oil of wintergree n, is one such volatile compound found in a variety of flowers, fruits, and leaves. For example, MeSA is a component of floral scent in approximately 80 species of plants represented by approximately 25 families (Effmert et al. 2005). In addition, it is a flavor component of fruits such as apple, strawberry, raspberry, and tomato (Burdock, 1995; Buttery and Ling, 1993). MeSA is also emitted in response to biotic stress. For example, MeSA is emitted from the leaves of tobacco mosaic virus infected tobacco ( Schulaev et al. 1997), spider mite infested tomatoes (Ament et al. 2004), and bacterial inoculated pepper (Cardoza and Tumlinson, 2006). MeSA has also been shown to have antioxidant activity in mouthwash (Battino et al. 2002). Therefore, MeSA serves a v ariety of functions relating to reproduction and protection in plants and in commercial products. MeSA is synthesized from the plant hormone salicylic acid (SA) via S adenosyl L methionine: salicylic acid carboxyl methyltransferase (SAMT). The methyl dono r S adenosyl L methione (SAM) transfers a methyl group to the carboxylic acid of SA to form the methyl ester MeSA. SAMT was first identified in the C larkia breweri and was classified into a new family of plant methyltransferases, the SABATH family (Ross et al. 1999). SABATH stands for the subst rates utilized by the enzymes salicylic acid (SA), benzoic acid (BA), and theobromine

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34 et al. 2003). The SABATH family has been extensively studied with respect to floral scent, since volatile emission is closely correlated with pollinator attraction (Effmert et al. 2005). MeSA and methylbenzoate (MeBA) are structurally related compounds synthesized by homologous enzymes. In some cases, both MeSA and MeBA can be synthesized by the same enzyme. Methyltra nsferases with dual substrate action are called benzoic acid/salicylic acid methyltransferases (BSMTs), while those acting only on benzoic acid are called benzoic acid methyltransferases (BAMTs). BSMTs have been identified in Arabidopsis thaliana (Chen et al. 2003), Petunia x hybrida (Negre et al. 2003; Underwood et al. 2005), and Oryza sativa (Koo et al. 2007). A BAMT has been identified in Ant i rrhinum majus (Murfitt et al. 2000), while an SA specific SAMT has been identified in Hoya carnosa ( Effmert et al. 2005). Other Arabidopsis thaliana family members can utilize substrates such as jasmonic acid (AtJMT) (Seo et al. 2001), indole acetic acid (AtIAMT) (Zubieta et al. 2003; Qin et al. 2005), gibberellins (AtGAMT) (Varbanova et al. 2007), and a se squiterpene, farnesoic acid (AtFAMT) (Yang et al. 2006b). Interestingly, the majority of these substrates are plant hormones, and it has been suggested that methylation of these hormones may aid in their transportation across membranes ( Yang et al. 2006a ). Based on studies of floral scent, methyltransferase activity is most likely involved in the synthesis of the flavor compound MeSA in tomato as well. Consumers are often dissatisfied with the flavor of tomatoes ( Solanum lycopersicum ), since breeders have focused on traits such as fruit size, color, firmness, yield, and disease resistance, and flavor has generally been ignored. Tomato flavor is due to the interaction of the non volatile components, sugars and acids, in addition to volatile components. Sugars and acids are responsible for the sweet and sour taste of tomatoes, while the volatile components contribute to the unique flavor of tomato. Over 400 volatile compounds have been identified as constituents

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35 of tomato flavor, but only about 30 of thes e volatiles are believed to positively contribute to tomato flavor (Buttery and Ling, 1993). Based on their chemical structures, these compounds are believed to be derived from carotenoids, amino acids, and lipids (Buttery and Ling, 1993). It has been sugg ested that the volatile components of flavor serve as indicators of the nutritional composition of foods (Goff and Klee, 2006). In general, most of the important volatiles of tomato flavor are not synthesized until the fruits are ripe, most likely to facil itate seed dispersal (Tieman et al. 2006). MeSA is an important volatile compound found in tomato (Buttery and Ling, 1993), and has been identified as a key compound differentiating the volatile profiles of tomato cultivars (Tikunov et al. 2005). To date MeSA synthesis has not been characterized in tomato or any other fruit s Since MeSA is an important component of tomato flavor, the purpose of this study was to identify and characterize the tomato SAMT gene ( LeSAMT1 ) responsible for MeSA synthesis. In a ddition, the contribution of MeSA to tomato flavor was determined using transgenic lines that produced elevated levels of MeSA. Results Identification of LeSAMT1 Using BLAST analysis against the TIGR database, eight full length tomato SAMT related ESTs we re identified by sequence similarity to the amino acid sequence of CbSAMT. The predicted amino acid sequence of LeSAMT1 was the closest homolog with 66% similarity and 54% identity to CbSAMT. The predicted protein is 362 amino acids with a molecular weight of 41.3 kDa. LeSAMT1 is even more closely related to solanaceous SAMTs as seen in the phylogenetic tree (Figure 3 1). Other SABATH family members are also represented on the tree, including AtIAMT (Zubieta et al. 2003; Qin et al. 2005) and AtGAMT (Varba nova et al. 2007), which recognize the substrates indole acetic acid and gibberellins, respectively. The amino acid sequence alignment (Figure 3 2) shows that LeSAMT1 contains all the previously

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36 identified SA binding and SAM binding residues of the SABATH family of methyltransferases (Zubieta et al. 2003). Especially noteworthy are the substrate binding residues that are identical to CbSAMT and PhBSMT1 at amino acid residues 153, 156, 232, 233, and 357 (Figure 3 2). The seven other tomato homologs were 41 % to 60% similar to CbSAMT based on amino acid sequence (Table 3 1). These seven homologs were named LeMT1 7 ( Solanum lycopersicum methyltransferase) based on their amino acid sequence similarity to LeSAMT1 (Table 3 1). The amino acid sequence identity of these homologs is also shown (Table 3 2). The amino acid alignment of LeSAMT1 with the other LeMTS and two singleton sequences identified from the SGN database shows that these LeSAMT1 homologs potentially contain SA binding residues (Figure 3 3). Both CbS AMT and PhBSMT1 have preferred activity on SA (Zubieta et al. 2003; Negre et al. 2003). However, when these residues were mutated by site directed mutagenesis in a study by Zubieta e t al (2003), the resulting CbSAMT protein had a broader substrate speci ficity and could recognize substrates such as jasmonic acid and vanillic acid. Phylogenetic analysis of the SABATH family has shown that SAMTs specific for SA activity have a Met at position 153 of C. breweri while BSMTs have a His residue at position 153 (Barkman et al. 2007). This study went on to show that the wild type SAMT of Datura wrightii with a Met 153 only produces MeSA, while a M 153 H substitution of a recombinant SAMT results in the production of MeSA and MeBA. Therefore, it appears that this a ctive site Met is necessary for SA specificity and a His broadens the specificity to BA. LeSAMT1 contains a Met at this position, suggesting that it may preferentially have activity with SA Substrate S pecificity and Kinetic Properties of LeSAMT1 To deter mine if LeSAMT1 could convert SA to MeSA in vitro a recombinant GST tagged LeSAMT1 was expressed in E scherichia coli The N terminal GST LeSAMT1 was purified using the GST affinity purification method (Figure 3 4) and assayed for activity with SA and

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37 stru cturally related compounds. Compounds on which other known methyltransferases were active in this family were also tested, including jasmonic acid and indole 3 acetic acid. GST LeSAMT1 had the highest activity with SA, which was normalized to 100% (Figure 3 5). LeSAMT1 had the next highest activity on BA which was only four percent of the activity seen with SA. The kinetic properties of purified GST LeSAMT1 were also determined. At 25 o C, SA had a K m of 52 M (Figure 3 6) and SAM had a K m of 15 M (Figure 3 7). V maxSA was 138 pmol/mg/min and V maxSAM was 85 pmol/mg/min. The kcat SA was 0.055 sec 1 and kcat SAM was 0.028 sec 1 The K m values for SA and SAM are within the range of reported values in other characterized methyltransferases, including CbSAMT, PhBSMT 1, PhBSMT2, and SfSAMT (Effmert et al. 2005). Since the in vitro data showed that LeSAMT1 specifically converted SA to MeSA, LeSAMT1 was chosen for further analysis in planta Tissue Specific Expression of LeSAMT1 After determining the substrate specific ities of LeSAMT1, the expression of LeSAMT1 was determined in various tissues. LeSAMT1 transcript abundance and SA availability in ripening fruits were determined. Studies in Petunia x hybrida have shown that volatile emissions can be determined by both su bstrate availability and substrate preference of the methyltransferase (Negre et. al. 2003; Underwood et. al. 2005). These studies have shown that PhBSMT1 has a substrate preference for SA in vitro but MeSA is not consistently detectable in planta becau se the substrate BA is more abundant. Therefore, PhBSMT1 catalyzes the synthesis of MeBA for floral scent instead of MeSA. LeSAMT1 expression was first quantified by real time pcr in flower buds, open flowers, young unexpanded leaves, and mature expanded l eaves (Figure 3 8). For transcript abundance during fruit ripening, f ive stages of M82 fruit s were examined 15 days after pollination (dap), mature green, breaker, turning, and ripe (Figure 3 9) LeSAMT1 is

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38 most highly expressed in immature and mature gree n fruit s followed by flower bud tissue, open flow ers and young unexpanded leaves. LeSAMT1 expression decreased as the fruit s ripened, and expression levels in immature fruits were compa rable to levels seen in flowers. The internal metabolite pools of Me SA (Figure 3 10) and SA (Figure 3 10) were also determined in ripening fruits. Since immature green fruit s generally release low levels of volatiles from fresh tissue, the internal levels of MeSA were determined from frozen tissue of all fruit stages. Intern al MeSA pools were highest in 15 dap fruit s (Figure 3 10). No obvious changes in free SA occurred throughout fruit ripening in M82 (Figure 3 11). Internal pools of MeSA did not appear to correlate with either transcript abundance or SA availability, except at the 15 dap stage. However, MeSA is still present in ripe fruit s and is considered important for tomato flavor. Production of Transgenic L ines To further elucidate the role of MeSA in tomato flavor and its other biological functions, overexpression a nd antisense transgenic lines in the M82 and Pearson backgrounds were made. The full length cDNA of LeSAMT1 was cloned under the control of the constitutive 35S figwort mosaic virus (FMV) promoter in the sense and antisense orientations. M82 is a commercia l processing tomato, while Pearson is a variety with more locular compartments (Figure 3 12). Pearson has higher levels of endogenous MeSA than M82 in any season (Tables 3 4, 3 5; see y axes in Figures 3 15 and 3 16). Transgenic fruits from plants grown in Live Oak, FL were analyzed in the Spring and Fall of 2006. One transgenic line in the Pearson background, pST1OE 6841 1, had over a 100 fold increase in MeSA emission with a 3000 fold increase in transgene abundance in the Spring of 2006 determined by qua ntitative real time pcr (Tables 3 4 and 3 6). RNA was run on a gel as a loading control for each reaction (Figure 3 14). This transgenic line also had a 100 fold increase in MeSA emission in the Fall of 2006 (Table 3 4). The intensity of the MeSA peak was noticeably increased on the chromatogram from the

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39 transgenic line (Figure 3 1 3). Another transgenic line in the M82 background, pST1OE 5220 2a, had an approximately 50 fold increase in MeSA emission in the Spring with over 3000 fold increase in LeSAMT1 exp ression determined by quantitative real time pcr (Table s 3 5 and 3 7). RNA was run on a gel as a loading control for each reaction (Figure 3 14). In the Fall this transgenic line had over 100 times the MeSA emission of M82 (Table 3 5). The absolute values of MeSA emissions varied between the Spring 2006 and Fall 2006 seasons, with the MeSA emissions being higher in the Fall. In addition, both cultivars varied in their endogenous levels of MeSA, indicated by the scales of the y axes. MeSA emissions from Pear son were ten fold higher than M82 in the Spring and six fold higher than M82 in the Fall. Interestingly, LeSAMT1 overexpression in both cultivars significantly increased MeSA emission in the transgenic lines over two seasons. The antisense lines in Pearso n (Figure 3 15) and M82 (Figure 3 16) showed approximately 50% reduction in MeSA emission that correlated with transcript abundance. Expression of LeSAMT1 in the Pearson antisense line, pST1AS 6831 1, was reduced by about 50% in flower buds (Figure 3 17), while the M82 antisense lines showed a similar reduction (Figure 3 18). The flower bud tissue was chosen for expression analysis in the antisense transgenic lines because LeSAMT1 in this tissue was more easily quantified than in the fruit s There is some v ariability in fruit MeSA emissions between the Fall and Spring 2006 seasons. For example, MeSA emissions from M82 were higher in the Fall than in the Spring. However, the antisense lines in the Fall had a 50% to 70% reduction in MeSA levels, while the Spri ng 2006 lines had a 40% to 50% reduction. The MeSA emission from the Pearson antisense line was reduced by 60% in the Spring and by 40% in the Fall.

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40 Methylsalicylate O verproducers T aste D ifferent and Are Preferred Equally to Controls Ripe fruit s from the LeSAMT1 overexpressing line in the Pearson background, pST1OE 6841 1, w ere used in a triangle taste test to determine if panelists could taste the difference between the transgenic and control tomatoes. The pST1OE 6841 1 line was chosen for the taste panel because the Pearson variety has a more pleasant taste than the commercial processing tomato, M82. Even though pST1OE 6841 1 fruit s produce over 100 fold higher MeSA than Pearson, these levels are well below the maximum daily MeSA intake of 740 g/kg body weight determined by the Expert Panel of Flavor and Extract Manufacturing Association (FEMA) (Adams et al. 2005). Therefore, these transgenic tomatoes have acceptable levels of MeSA for human consumption. The triangle taste test was done usi ng an untrained consumer panel. Seeds were removed from the control and transgenic tomatoes before giving the samples to the panelists. Tomatoes were sliced into small wedges and placed in plastic cups marked with a random three digit code. Panelists were randomly given two transgenic pST1OE 6841 1 samples and one control, or two control samples and one transgenic, and asked to pick the odd sample. Results indicated that there was a significant difference between the control and the transgenic fruit s (p < 0 .01 ), with 50% of the panelists choosing the correct sample (Table 3 8). Since the triangle test determined that there was a difference between the taste of the transgenic line and the control, a subsequent preference and likeability test was done on th e same lines. MeSA emission was increased by approximately 80 fold in the transgenic fruit s used for the preference test (Table 3 9). The likeability and preference tests were done at the Sensory Testing Facility in the Food Science and Human Nutrition Dep artment at the University of Florida. The panel was an untrained consumer panel. Panelists were give two samples, Pearson

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41 and pST1OE 6841 1, and asked to rate the aroma, sweetness, sourness, tomato flavor, and overall acceptability of the samples on a nine point hedonic scale (Table 3 10). Panelists were then asked to pick which sample they preferred. Out of 67 panelists, 36 preferred the control and 31 preferred the transgenic (Table 3 11). The difference was not statistically significant. Therefore, the s amples were preferred equally. Since this was a panel conducted on the University of Florida campus, the majorit y of the panelists were in 18 to 24 year age range (36 out of 67 panelists). Interestingly, a significant number of panelists in the 25 to 34 ye ar old range preferred the transgenic sample (Table 3 12), but this was a small sample size (n = 11). The results from the likeability scores (Table 3 13) also showed that the samples were equally accepted and liked by the panelists, even though Pearson co nsistently scored higher than the transgenic line. The panelists that preferred the control tomato believed that it tasted more contrast, the panelists who pref erred the transgenic tomato believed it had more flavor overall, so individual perception was a most likely a factor in choosing and describing the preferred sample. Discussion LeSAMT1 I s a Functional Salicylic Acid Carboxyl Methyltransferase LeSAMT1 a t omato homolog to a known SAMT from Clarkia breweri (Ross et al. 1999), was cloned, and recombinant GST LeSAMT1 was shown to have specific in vitro activity to SA. This is expected from the amino acid sequence that shows a Met at position 156, which is pre sent in SAMTs that specifically convert SA to MeSA (Barkman et al. 2007). The transcript abundance and SA availability do not completely correlate with internal MeSA pools during fruit development However, we do not currently have an antibody to LeSAMT1, so we do not know the protein levels during fruit ripening. When LeSAMT1 was overexpressed, transgenic fruit s had up to 100 fold higher MeSA emissions than the control fruit s Interestingly, overexpression of

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42 LeSAMT1 significantly increased the MeSA from two cultivars of tomato with different endogenous levels of MeSA. MeSA production in the a ntisense lines from M82 and Pearson were reduced by about 50% over two seasons. This incomplete reduction in the antisense lines could be due to the presence of anoth er methyltransferase present in fruit s (Figure 3 3), since both cultivars showed a similar trend. So far seven full length and two singleton SAMT related methyltransferases have been identified in the tomato genome, so one of these may also be responsible for MeSA synthesis in fruit s Flavor Panels It was determined that transgenic tomatoes overproducing MeSA tasted different and were preferred equally to control tomatoes. It appears that the overproduction of MeSA in tomatoes is not necessarily an unfavora ble trait, but it is not an overwhelmingly preferred trait, either. Personal preference and recognition of the trait may also influence which tomato is preferred. It was not known how often the panelists in the preference test consumed tomatoes or if they liked tomatoes. The transgenic and control tomatoes were preferred equally in a preference test, even though the controls tended to score higher in the likeability scores. Since the majority of panelists were under age 25, it is most likely that their fami liarity with tomatoes is due to tomatoes consumed from the food service industry. These tomatoes are not picked at a fully ripe stage due to the transportation limitations of ripe fruit s Tomatoes picked before they are fully ripe have been reported to hav flavor (Kader et al. 1977). If this panel was repeated, it would be beneficial to know the how frequently the panelists consume tomatoes and if they like tomatoes, which may produce diff erent results. Other studies have examined the effects of overexpression of flavor genes in tomato. The tomato alcohol dehydrogenase 2 gene ( ADH 2 ) was overexpressed in tomato and the resulting

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43 transgenic fruit s showed a disruption in the balance of lipid d erived 6 carbon volatiles (Speirs et al. carbon alcohols hexanol and cis 3 hexenol increased, which lowered the ratios of these alcohols to their precursor aldehydes. An increase in these alcoho ls correlated with an increase in ripe flavor according to a taste panel. Davidovich Rikanati et al (2007) found that overexpressing the geraniol synthase gene from lemon basil resulted in tomatoes that produced more monoterpenoid volatiles that were pref erred by panelists in a preference test. The transgenic tomatoes were altered in a variety of monoterpenes not normally present in tomato and failed to develop a red color, but were still decrease the effect of the taste of other classes of volatiles (Baldwin et al. 2004). For example, iles, such as the lipid derived volatiles, can decrease the affect the desirable flavor of foods (Abraham et al ., 1976). MeSA was added to black tea and the flavor was rated. MeSA levels up to 20 ppm imparted a desirable fragrant and flowery flavor, but above 25 ppm, the wintergreen flavor was more pronounced and the tea was described as bitter. In the current study, the overproduction of MeSA was preferred equally but those panelists that preferred the controls commented that the transgenic tomatoes tasted bland. Perhaps the overproduction of MeSA masked the other tomato volatiles which made the like taste. On the other hand, panelists that preferred the MeSA overproducing tomatoes believed that these tomatoes had more flavor. This particular volatile was perceived differently by the panelists, and some may be more sensitive to it than others. The

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44 results of this study showed that incre asing MeSA in tomato was neither highly desirable nor undesirable, and the flavor balance may have been perceived differently by panelists. This is the first report of altering MeSA synthesis in fruit s Overexpression of LeSAMT1 resulted in tomatoes with s ignificantly higher levels of MeSA, which affected the taste of tomatoes by an untrained consumer panel, even though the controls scored consistently higher in likeab ility ratings. Since flavor is a highly complex trait involving primary and secondary metabolism, genetic engineering is a valuable tool for targeting specific biosynthetic genes involved in flavor. By altering the synthesis of single compounds in tomato f ruit s the contribution of these volatiles to tomato flavor can be studied in a common background. The overexpression of LeSAMT1 is an example of targeting a specific biosynthetic gene involved in the synthesis of MeSA and evaluating its contribution to to mato flavor.

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45 Figure 3 1. Phylogenetic tree of amino acid sequences of SABATH methyltransferases LeSAMT1 is bolded. LeSAMT1 and the seven full length tomato homologs related to LeSAMT1 are underlined. These tomato homologs are named LeMT1, LeMT2, LeMT3, LeMT4, LeMT5, LeMT6, and LeMT7. See Experimental Procedures for LeMT (tomato methyltransferase) sequence information. AbSAMT is A tropa belladonna (BAB39396), AmBAMT is A ntirrhinus majus (BAB39396), AtGAMT1, AtIAMT, and AtJMT are Arabidopsis thaliana (NP_194372, NP_200336, AAG23343), CaMXMT is C offea arabica 7 MXMT theobromine synthase (BAB39216), CbSAMT is C larkia breweri (AAF00108), HcSAMT is H oya carnosa (CAI05934), PhBSMT1 and PhBSMT2 are Petunia x hybrida (AAO45012 and AAO 45013), SfSAMT is S tephanotis floribunda (CAC33768), and TCS1 is C amellia sinensis caffeine synthase (BAB12278). The unrooted dendogram was generated using the Phylip format. LeMT7 PhBSMT1 PhBSMT2 AbSAMT LeSAMT1 LeMT 1 LeMT 2 CbSAMT AtJMT LeMT 6 AtIAMT AtGAMT1 AtGAMT2 TCS1 CaMXMT AtBSMT LeMT 5 LeMT 3 LeMT 4 AmBAMT HcSAMT SfSAMT

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46 Table 3 1. Percent similarities between amino acid sequences of LeSAMT1, LeMTs, and known methyltransferases. aa LeSAMT1 CbSAMT1 PhBSMT1 PhBSMT2 LeMT 1 LeMT 2 LeMT 3 LeMT 4 LeMT 5 LeMT 6 LeMT 7 LeSAMT1 362 100 66 85 85 65 63 58 52 50 43 40 CbSAMT1 359 100 67 67 60 56 57 51 52 41 42 PhBSMT1 357 1 00 100 68 66 57 50 51 44 41 PhBSMT2 357 100 68 66 58 50 51 44 41 LeMT 1 347 100 71 58 49 52 40 40 LeMT 2 357 100 54 47 49 40 40 LeMT 3 353 100 78 49 42 39 LeMT 4 390 100 44 41 44 LeMT 5 322 100 42 44 LeMT 6 369 100 38 LeMT 7 305 100 Table 3 2. Percent identities between amino acid sequences of LeSAMT1, LeMTs, and known methyltransferases. aa LeSAMT1 CbSAMT1 PhBSMT1 PhBSMT 2 LeMT 1 LeMT 2 LeMT 3 LeMT 4 LeMT 5 LeMT 6 LeMT 7 LeSAMT1 362 100 54 79 79 56 54 43 39 37 31 27 CbSAMT1 359 100 56 56 46 46 43 38 37 31 30 PhBSMT1 357 100 99 59 56 43 38 36 33 28 PhBSMT2 357 100 5 9 57 43 38 36 33 28 LeMT 1 347 100 63 43 37 37 30 27 LeMT 2 357 100 40 35 37 30 27 LeMT 3 353 100 75 33 30 28 LeMT 4 390 100 32 28 32 LeMT 5 322 100 29 26 LeMT 6 369 100 27 LeMT 7 305 100

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47 LeSAMT1 ---------------------MKVVEVLHM N GGNGDISYANN S LV Q KKVILMTKPIRD 37 CbSAMT ---------------------MDVRQVLHM K GGAGENSYAMN S FI Q RQVISITKPITE 37 PhBSMT1 --------------------MEVVEVLHM N GGNGDSSYANN S LV Q QKVILMTKPITE 37 AmBAMT --------------------MKVMKKLLCM N IAGDGETSYANN S GL Q KVMMSKSLHVLD 39 AtJMT ---------------------MEVMRVLHM N KGNGETSYAKN S TA Q SNIISLGRRVMD 37 AtBSMT MDPRFIN TIPSLRYDDDKCDDEYAFVKALCM S GGDGANSYSAN S RL Q KKVLSMAKPVLV 59 AtIAMT ---------MGSKGDNVAVCNMKLERLLSM K GGKGQDSYANN S QA Q AMHARSMLHLLE 49 AtGAMT1 ---------MES -------SRSLEHVLSM Q GGEDDASYVKN C YG P AARLALSKPMLT 41 ** : LeSAMT1 QAISDLYC -NLFPETLYIA D LGCSSGANTFLVVSELVKVIEK -ERKKHDLQ SPEFYF 92 CbSAMT AAITALYSG DTVTTRLAIA D LGCSSGPNALFAVTELIKTVEE -LRKKMGRENSPEYQI 94 PhBSMT1 QAMID LYS -SLFPETLCIA D LGCSLGANTFLVVSQLVKIVEK -ERKKHGFK SPEFYF 92 AmBAMT ETLKDIIGDHVGFPKCFKMM D MGCSSGPNALLVMSGIINTIED -LYTEKNINELPEFEV 97 AtJMT EALKKLMMS NSEISSIGIA D LGCSSGPNSLLSISNIVDTIHN -LCPDLDRP VPELRV 93 AtBSMT RNTEEMMM N LDFPTYIKVA E LGCSSGQNSFLAIFEIINTINV -LCQHVNKNS PEIDC 115 AtIAMT ETLENVHLNSSASPPPFTAV D LGCSSGANTVHIIDFIVKHISKRFDAAG -IDPP EFTA 106 AtGAMT1 TAINSIKLTEGCSS HLKIA D LGCAIGDNTFSTVETVVEVLGKKLAVIDGGTEPEMEFEV 100 : : ::**: *: : ::. : LeSAMT1 HFN DL PGNDFNAIFRSLGEFEQNLKKQI -----GEELGPCFFSGVAG SF YSRLFPSKSL 146 CbSAMT FLN DL PGNDFNAIFRSL PIENDVD ------------GVCFINGVPG SF YGRLFPRNTL 140 PhBSMT1 HFN DL PGNDFNTLFQSLGAFQEDLRKHI -----GESFGPCFFSGVPG SF YTRLFPSKSL 146 AmBAMT FLN DL PDNDFNNLFKLLSHEN ----------------GNCFVYGLPG SF YGRLLPKKSL 140 AtJMT SLN DL PSNDFNYICASLPEFYDRVNNNKEGLGFGRGGGESCFVSAVPG SF YGRLFPRRSL 153 AtBSMT CLN DL PENDFNTTFKFVPFFNKELMITN --------KSSCFVYGAPG SF YSRLFSRNSL 166 AtIAMT FFS DL PSNDFNTLFQLLPPLVSNTCMEE --CLAADGNRSYFVAGVPG SF YRRLFPARTI 163 AtGAMT1 FFS DL SSNDFNALFRSLDEKVN ------------GSSRKYFAAGVPG SF YKRLFPKGEL 147 :.**. **** : .**** **: : LeSAMT1 HFVH SSYSLMW LSQVPNLIEK ----------NKGNIYMASTSPPSVIKAYYKQYEKDFS 195 CbSAMT HFIH SSYSLMW LSQVPIGIES ----------NKGNIYMANTCPQSVLNAYYKQFQEDHA 189 PhBSM T1 HFVY SSYSLMW LSQVPNGIEN ----------NKGNIYMARTSPLSVIKAYYKQYEIDFS 195 AmBAMT HFAY SSYSIHW LSQVPEGLED ---------NNRQNIYMATESPPEVYKAYAKQYERDFS 190 AtJMT HFVH SSSSLHW LSQVPCREAEKEDRTITADLENMGKIYISKTSPKSAHKAYALQFQTDFL 213 AtBSMT HLIH SSYALHW LSKVPEKLE ----------NNKGNLYITSSSPQSAYKAYLNQFQKDFT 215 AtIAMT DFFH SAFSLHW LSQVPESVTDRRSAA ----YNRGRVFIHG AGEKTTTAYKRQFQADLA 217 AtGAMT1 HVVV TMSALQW LSQVPEKVMEKGSKS ----WNKGGVWIEG AEKEVVEAYAEQADKDLV 201 : :: ***:** ::: ** : LeSAMT1 IFLKYRSEELMKGGKMVLTF L GK ----RKXEDPFSKECCY IW ELLSMALNELVLEGLIE 250 CbSAMT LFLRCRAQEVVPGGRMVLTI L G -----RRSEDRASTECCL IW QLLAMALNQMVSEGLIE 243 PhB SMT1 NFLKYRSEELMKGGKMVLTL L G -----RESEDPTSKECCY IW ELLAMALNKLVEEGLIK 249 AmBAMT TFLKLRGEEIVPGGRMVLTF N GR -----SVEDPSSKDDLA IF TLLAKTLVDMVAEGLVK 244 AtJMT VFLRSRSEELVPGGRMVLSF L G -----RRSLDPTTEESCY QW ELLAQALMSMAKEGIIE 267 AtBSMT MFLRLRSEEIVSNGRMVLTF I GR ----NTLNDPLYRDCCH FW TLLSNSLRDLVFEGLVS 270 AtIAMT EFLRARAAEVKRGGAMFLVC L GRTSVDPTDQGGAG ---LL FG THFQDAWDDLVREGLVA 273 AtGAMT1 EFLKCRKEEIVVGGVLFMLM G GRPSGSVNQIGDPDSSLKHP FT TLMDQAWQDLVDEGLIE 261 **: *: .* :.: : : .:. **:: LeSAMT1 EEKVDSFNIPQ Y TPSQGEVKYVVDKEGSFTINKLETTRVHWNNASNNIEN ---------300 CbSAMT EEKMDKFNIPQ Y TPSPTEVEAEILKEGSFLIDHIEASEIYWSSCTKDGDGG ------GS 296 Ph BSMT1 EEKVDAFNIPQ Y TPSPAEVKYIVEKEGSFTINRLETSRVHWN ASNNEK ----------297 AmBAMT MDDLYSFNIPI Y SPCTREVEAAILSEGSFTLDRLEVFRVCWDASDYTDDDDQQD PSIFG 303 AtJMT EEKIDAFNAPY Y AASSEELKMVIEKEGSFSIDRLEISPIDWEGGSISEESYDLAIRSKPE 327 AtBSMT ESKLDAFNMPF Y DPNVQELKEVIQKEGSFEINELESH -GFDLGHYYEEDD --------319 AtIAMT AEKRDGFNIPV Y APSLQDFKEVVDANGSFAIDK --LVVYKGGSPLVVNEPD ------D 323 AtGAMT1 EEKRDGFNIPV Y FRTTEEIAAAIDRCGGFKIEKTENLIIADHMNGKQEELMK ------D 314 ** : : *.* :: Figure 3 2. Alignment of the LeSAMT 1 amino acid sequence with other known SABATH methyltransferases Amino acid sequences were aligned using ClustalW. SAM/SAH binding residues are highli ghted green, substrate binding residues are blue, and other active site residues are yellow according to Zubieta et al. (2003). See Figure 3 1 for accession numbers and abbreviations.

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48 LeSAMT1 INNDGYNVSKC MRAV AEPLLLSQFDPKLIDLVFQKYEEIVS -KC MAKEDTE F I N VTVSL 358 CbSAMT VEEEGYNVARC MRAV AEPLLLDHFGEAIIEDVFHRYKLLII -ERMSKEKTK F I N VIVSL 354 PhBSMT1 -NGGYNVSRC MRAV AEPLLVSHFDKELMDLVFHKYEEIVS -DCMSKENTE F I N VIISL 353 AmBAMT KQRSGKFVADC VRAI TEPMLASHFGSTIMDLLFGKYA KKIV -EHLSVENSS Y F S IVVSL 361 AtJMT ALASGRRVSNT IRAV VEPMLEPTFGENVMDELFERYAKIVG -EYFYVSSPR Y A I VILSL 385 AtBSMT FEAGRNEANG IRAV SEPMLIAHFGEEIIDTLFDKYAYHVT -QHANCRNKT T V S LVVSL 376 AtIAMT ASEVGRAFASS CRSV AGVLVEAHIGEE LSNKLFSRVESRATSHAKDVLVNLQ F F H IVASL 383 AtGAMT1 PDSYGRDRANY AQAG LKPIVQAYLGPDLTHKLFKRYAVRAA -ADKEILNNC F Y H MIAVS 372 : :: :: : : :* : : LeSAMT1 TKKK 362 CbSAMT IRKSD 359 PhBSMT1 TKIN 357 AmBAMT SRR -364 AtJMT VRTG 389 AtBSMT TKK -379 AtIAMT SFT -386 AtGAMT1 AVRV 376 SAM/SAH Binding Residues Substrate Binding Residues Additional Active Site Residues Figure 3 2. Continued

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49 LeSAMT1 ---------------MKVVEVLHM N GGNGDISYANN S LV Q KKVILMTKPIRDQAISDLY 44 CbSAMT ---------------MDVRQVLHM K GGAGENSYAMN S FI Q RQVISITKPITEAAITALY 44 PhBSMT1 ---------------MEVVEVLHM N GGNGDSSYANN S LV Q QKVILMTKPITEQAMIDLY 44 LeMT1 -----------------------M N GGMGDASYAKN S LL Q QKVILMTKSITDEAISSLY 36 LeMT2 -----------------------M T EGIGDSSYAKN S LF Q QKVILATKSITCEAISALY 36 LeMT3 ----------------------M N AGNGECSYASS S TL Q RKVIEVAKPVLEDAIKKMF 36 LeMT4 -----------------------------------------------MPVLEDAIKK -10 LeMT5 ---------------MDVEKVFHM T GGVGETSYSRN S SL Q KKASEMVKHITLETVEEVY 44 LeMT6 MAPLGDNNNNNVVVSNLK LERMLSM K GGKGEASYVNN S QA Q GQHARSMLHLLKDTLDGVQ 60 LeMT7 ---------------------------------------------MVRDAIIEKFDIKT 14 SGN U336017 -----------------------------------------------------------SGN U343182 ----------------------------------------------------------LeSAMT1 C -NLFP ETLYIA D LGCSSGANTFLVVSELVKVIEK ---ERKKHD LQSPEFYFHFN D 96 CbSAMT S -GDTVTTRLAIA D LGCSSGPNALFAVTELIKTVEE ---LRKKMGRENSP EYQIFLN D 98 PhBSMT1 S -SLFP ETLCIA D LGCSLGANTFLVVSQLVKIVEK ---ERKKHG FKSPEFYFHFN D 96 LeMT1 N -NLSSRETICIA D LGCSSGPNTFLSVSQFIQTIDK ---ERKKKGRHKAPEFHVFLN D 90 LeMT2 H -SLSTWETIRIA E LGCSSGPNTYLPVLQLIHTIRE ---KCTENG QKLPEFHVFFN D 89 L eMT3 SIIGEFPKSCLNMA D LGCSSGPNTLFTLSNIINIVQV ---LCGEKS CKMPEFQAYLN D 91 LeMT4 -IGE -KSCLNMA D LGCSSGPNTLFTISNIIKIVQI ---LCDEKR CKMPEFQVYLN D 61 LeMT5 V --ATKPKSIGIA D LGCSSGPNTLSNIKDILDKIEG ---ISHNKLKQSAPEFRVFLN D 97 LeMT6 L --NSPEIPFVIA D LGCSCGGNTIFIIDVIVEHMSKRYEATGQE ----PPEFSAFFS D 112 LeMT7 M --LSSSNTLCIV E FGCSVGPNTLIAMQHVVEALKDKYLSQIITNSTNDNLEIQIFFN D 71 SGN U336017 -----------------------------------------------------------SGN U343182 ---------------------------------------------------------D 1 LeSAMT1 L PGNDFNAIFRSLGEFEQNLKKQIGEELG -------PCFFSGVAG SF YSRLFPSKSLHF 148 CbSAMT L PGNDFNAIFRSLP IENDVDG -------------VCFINGVPG SF YGRLFPRNTLHF 142 PhBSMT1 L PGNDFNTLFQSLGAFQEDLRKHIGESFG -------PCFFSGVPG SF YTRLFPSKSLHF 148 LeMT 1 L PSNDFNTIFRLLPTFHQSLRKQNMGEDGS -LFDPSNCFVTGVAG SF YTRLFPSNSLHF 148 LeMT2 L PGNDFNTIFRLLTTFYEDLKKQNMRSEDG -LFDPP NCFVAAVAG SF YTRLFPSKKLHF 147 LeMT3 L PDNDFNTIFKSIPSFYQNHTN ---------------CFVSGVPG TF YERLFPSKSLHL 135 LeMT4 L PDNDFNNIFKSIPSFYQNHTN ---------------CFVSGVPG SF YERLFPSNSLHL 105 LeMT5 L PTNDFNAIFQALPEFHQWLKQKDGSDDDENRVTNSSNIYVAAYPG S F YGRLFPDHCLHF 157 LeMT6 L PSNDFNTLFQLLPPLANNGCGSMEECLTS --NSHRSYFAAGVPG SF YRRLFPARSIDV 169 LeMT7 H VNNDFNTLFRSLP ------------------IDR SYYACGVPG SF HGRLFPSRSIHF 111 SGN U336017 --------------------------------------------------LYYIKS FSN 9 SGN U343182 L VQNDFNTLFNYIH ------------------GNKPNYFTTGIPG SF YGRIFPKAFLHF 42 :: : Figure 3 3. Alignment of LeSAMT1 and related tomato methyltransferase sequences. Full len gth sequences of LeSAMT1 related homologs and two singleton sequences from the SGN database were aligned using ClustalW. See Experimental Procedures for LeMT (tomato methyltransferase) sequence information. The Clarkia breweri SAMT (AAF00108) and Petunia x hybrida BSMT1 (AAO45012) were also included in the alignment since they hav e known activity with SA (Ross et al. 1999; Negre et al. 2003). The two singleton sequences from the SGN database are SGN U336017 (clone cTSB 1 J1, from a seed library) and SGN U 343182 (clone FA21BA02, from a mixed fruit library containing immature green, mature green, breaker, turning, and red ripe fruit s ). SAM/SAH binding residues are highlighted green, substrate binding residues are blue, and other active site residues are yell ow according to Zubieta et al. (2003).

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50 LeSAMT1 VH SS YSLMW LSQVPNLIEKNKGN -----IYMAST SPPSVIKAYYKQYEKDFSIFLKYR 201 CbSAMT IH S SYSLMW LSQVPIGIESNKGN -----IYMANT CPQSVLNAYYKQFQEDHALFLRCR 195 PhBSMT1 VY S SYSLMW LSQVPNGIENNKGN -----IYMA RT SPLSVIKAYYKQYEIDFSNFLKYR 201 LeMT1 VH S SYSLHW LSQVPDGIKNNKGN -----IYLTST SPASVHKAYYEQFERDFVTFLKYR 201 LeMT2 VH SS YSLHW LSQVPDGIENNKGT -----IYASST SPSSVLKAYSKQYKRDFATFLKYR 200 LeMT3 VH S SYSLHW LSQAPEK IENNNN -----IYITRT SPPQVFE AYMKQFDNDFSRFLQVR 187 LeMT4 VH S SYSLHW LSQAPENYMENNNN -----IYITRT SPPHVVEAYMKQFDKDFSRFLQLR 158 LeMT5 IY SSY SLHW LSKVPRGLYDEQGNSLNKKSIYISEN SPCEVSKVYFDQFKEDFSLFLQSR 216 LeMT6 FY S AFSLHW LSQVPEIVLDKRSPAYNKGKIYIHGA -NESTANAYRKQFQTDL AYFLGCR 227 LeMT7 AH S SCSIHW LSKIPKELIGEKSPSWNKGLIHYIGT SNVEVVNAYFDQFEKDMEMFLNAR 170 SGN U336017 II C LY -LS IYQVPNFIEKNKGN -----IYITST SPQSYIKAYYKQFENDFSNFLKYR 60 SGN U343182 AH SS MSLLY LSRIPEEVLDRNSAAWNKGRIHYSSSGAAKEVEAAYADQFRKDIQAFLDAR 102 : : .. *: .* .*: ** LeSAMT1 SEELMKGGKMV LT F L GKRKXEDPFSKE CCY IW ELLSMALNELVLEGLIEEEKVDSFNIP 260 CbSAMT AQEVVPGGRMV LT I L G RRSEDRASTE CCL IW QLLAMALNQMVSEGLIEEEKMDKFNIP 253 PhBSMT1 SEELMKGGKMV LT L L G RESEDPTSKE CCY IW ELLAMALNKLVEEGLIKEEKVDAFNIP 259 LeMT1 SEELMKNGRMV LT M L G RKNEDRFSQG CSY EW ELLATTLKLLIAQESIDAEKVDSFNVP 259 LeMT 2 SEELVKGGRMV LA M P G KENEHHLSNV CRF ML EPLAIALKDLVTEGSIEEEKMDSFNVP 258 LeMT3 SEEIVTGG YM VL TF I G RGIPDPYGNH -SV HL DLLSKSFVDLIHEGLIEQAKLDSFNYP 244 LeMT4 SEEIVSGGRMV L TF M G STIPDPYGSH -YA LL ELLSNSLIDLIHEGLVEQAKLDSFSLP 215 LeMT5 SDELVSRGKM VL IL L G REGFNHVDRG NAF FW KILYQALTNLISKGEVEKEKLDSYEVH 274 LeMT6 SKEMKRGGS MFL AC L GRT SVDPTDQGGAGLL FG THFQDAWDDLVQEGLITSEKRDKFNIP 287 LeMT7 AEEIVHGGMM VL IT --------PFSTSYIR LV KFFGSSLTDLVNEGKLDESLVDSFNLP 221 SGN U336017 SEELMKGGKM VL TF L G RESEDLSIKEYCCY IW ELLAMVLNELVFEGLIEEDKVDSFDVP 119 SGN U343182 AQELVPGGLMT II T L TILDGVLPSDSP MAI NF TILGSCLQDMANMGIMSEEKLDSFNLP 161 :.*: : : : : : LeSAMT1 Q Y TPSQGEVKYVVDKEGSFTINKLETTRVHWNNASNNIEN ---INNDGYNVSKC MRAV A 316 CbSAMT Q Y TPSPTEVEAEILKEGSFLIDHIEASEIYWSSCTKDGDGG G SVEEEGYNVARC MRAV A 312 PhBSMT1 Q Y TPSPAEVKYIVEKEGSFTINRLETSRVHWN ASNNEK ------NGGYNVSRC MRAV A 311 LeMT1 A Y NPSPSEVMHIVEKERSFTIDILKTSEIQRNSCD ----------DEKYDMAKS FRSV A 308 LeMT2 T Y SPSPAEIQYVVEKEGSFTIDLLRTLEHQMDSS -----------CEGYN EAQS VRAF A 306 LeMT3 F Y TPYKDEVEKIVQMEGSFDVDTIKFFKVNWDERDNDDD ---DAYSSGKHIART MRAV S 300 LeMT4 F Y APNKDEVEKIVEMEGSFVVDTINFFKVKWDERDNDDDHICFDAYSSGKHIARN TRAV F 275 LeMT5 F Y APCKEEIEKVARENGCFEVERLEMFEIEKTIGKG --------MSYGTMVAMT VRSI Q 325 LeMT6 V Y APSIQDFKEVVEANDSFKINNLQVFRGGSPLVVSHPDD ---AAEIGRALANS CRSV S 343 LeMT7 M Y FPSVEDMTKVVEKNGCFSIERIELTYPKSKLVDEADAK ---TLIIN -----LRAV L 271 SGN U336017 N Y TPSPREVKYIVEKEGSFTINRLETTRVNWNNSSYESN ------NGGYKMTRC MRAV A 172 SGN U3 43182 Y Y YTSPMEFETLIKTHGCFDIIRFEK LPTPLRQVVIDVQ ---TAVLS -----VRVV T 210 : .* : : LeSAMT1 EPLLLSQFDP -KLIDLVFQKYEEIVSK CMAKEDTE F I N VTVSLTKKK --------362 CbSAMT EPLLLDH FGE -AIIEDVFHRYKLLIIE RMSKEKTK F I N VIVSLIRKSD -------359 PhBSMT1 EPLLVSHFDK -ELMDLVFHKYEEIISD CMSKEKTE F I N VIVSLTKIN --------357 LeMT1 EPLLVSHFGHDELNMDQVFHKYNQVIANDRKAMEKIM F V N VTISLTKIN --------357 LeMT2 QPLLVSHFGDDNKLMDVVFNKCR EIYAN TMAKEKNI F T N VIVSLIKS ---------353 Le MT3 EQMLVSHFQFGDHIVDYLFERYAYHLAC HLLVQKGK F S N IVISLRKK ---------347 LeMT4 EQMLVSHFQFGDSVVDYLFERYAYHLTC NLLVQKGN Y F N IVISLTKK ---------322 LeMT5 ESMLAHHFGE -AFIEGLFKEYGRLVDE EMEKEEIR P I T FLLVFRK ----------369 LeMT6 GVLVDAHIGE -QLSDELFTRVEER ---ATCHAKEL L Q N LQFFHIVASLSLV ----390 LeMT7 EGVLINHFGK -EIAKEACER -------TILKSDEI S -----AWMKAN -------305 SGN U336017 EPLLINQFGP -KLMDLVFQKYEKIISD CMAKEKAE F V N VTVSLTKENN ------219 SGN U343182 EQLFQQHFGN -DITEELFQRYTEKLDSHPLLKDDKY R T D ASYFVFLKRXAETSERES 266 : :: SAM/SAH Binding Residues Substrate Binding Residues Additional Active Site Residues Figure 3 3. Continued

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51 Figure 3 4 Purified GST LeSAMT detected with GST antibodies. Lane 1, uninduced culture; Lane 2, induced culture; Lane 3, crude extract; Lane 4, flow through; Lanes 5,6,7, washes; L, ladder (prestained SDS PAGE low range standards, BioRad) ; Lane 8, elution 1; Lane 9, elution 2. The GST LeSAMT1 fusio n protein is expected to be 67.6 kDa. 1 2 3 4 5 6 7 8 9 L 101 kDa 97 kDa 54 kDa 38 kDa 29 kDa

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52 Figure 3 5 Substrate specificities of LeSAMT1 Purified GST LeSAMT1 was assayed with 1 mM of each substrate and 30 M SAM. 2.85 g protein was assayed at 25 o C pH 7.5 for one hour. S ampl es were performed in triplicate as well as a no enzyme control. 100% activity equaled 8.2 nmol/hr/mg protein.

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53 Figure 3 6. Lineweaver Burke plot for the K m of SA. 14 C SAM was held constant at 75 M. Assays were done at 25 o C for two hours. Samples were performed in triplicate as well as a no enzyme control. Figure 3 7. Lineweaver Burke plot for the K m of SAM. SA was held constant at 1 mM. Assays were done at 25 o C for two hours. Samples were performed in triplicate as well as a no enzyme control. K m = 15 M V max = 85 pmol /mg/min K m = 52 M V max = 138 pmol/mg/min

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54 B Figure 3 8. LeSAMT1 tissue specific expression in M82. A) B = bud, Fl = flower, YL = young leaf, ML = mature leaf. Error bars = SE. n = 4. B) 300 ng of eac h RNA sample was run on a 1% TBE gel and stained with ethidium bromide as a loading control for each reaction. Lanes 1 4, B; Lanes 5 8, Fl; Lanes 9 12, YL, Lanes 13 16, ML. RNA was collected from four biological replicates of vegetative and floral tissue f rom the field at Live Oak, FL and analyzed by quantitative real time pcr (RT PCR). A

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55 B Figure 3 9. LeSAMT1 expression during M82 fruit ripening. A) 15 DAP = 15 days after pollination, MG = mature green, Br = breaker, Tu = turning. Error bars = SE. n = 4. n.d. is not detectable. B) 200 ng of RNA from each sample was run on a 1% TBE gel and stained with ethidium bromide as a loading control for each reaction. Lanes 1 4, 15DAP; Lanes 5 8, MG; lanes 9 12, Br; Lanes 13 16, Tu; Lanes 17 20, Ripe. RNA was collected from 4 individual fruit s from the greenhouse. A n.d.

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56 Figure 3 10. Internal pools of MeSA during M82 fruit ripening Internal levels of MeSA from 4 individual greenhouse fruit s of each stage. 15 DAP = 15 days after pollina tion, MG = mature green, Br = breaker, Tu = turning. Error bars = SE. n = 4. Figure 3 11. Internal pools of free SA during M82 fruit ripening Internal levels of SA from 4 individual greenhouse fruit s of each stage. 15 DAP = 15 days after pollinati on, MG = mature green, Br = breaker, Tu = turning. Error bars = SE. n = 4.

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57 Figure 3 12. Comparison between cultivars M82 and Pearson A) Whole fruit of M82 (left) and Pearson (right), B) Interior view of M82 (left) and P earson (rig ht). A B

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58 Table 3 4. MeSA emission from pST1OE 6841 1 and Pearson ripe fruit s Sample Spring 2006 Fall 2006 pST1OE 6841 1 48.97 78.08 MeSA ng/gfw/hr 5.96 33.10 SE Pearson 0.40 0.32 MeSA ng/gfw/hr 0.08 0.10 SE pST1OE 6841 1 is an LeSA MT1 overexpressing line in the Pearson background. Plants were grown in Live Oak, FL. Error bars = SE For Spring 2006, n = 17, 16. For Fall 2006, n = 4, 8. Figure 3 13. Chromatogram s comparing volatile emissions from Pearson and pST1OE 6841 1 ripe f ruit s A) Pearson chromatogram and B ) pST1OE 6841 1 chromatogram The MeSA peak ran at 26.13 min. Table 3 5 MeSA emission from pST1OE 5220 2a and M82 ripe fruit s Spring 2006 Fall 2006 pST1OE 5220 2a 1.77 12.69 MeSA ng/gfw/hr 0.76 3.33 SE M 82 0.04 0.10 MeSA ng/gfw/hr 0.01 0.02 SE pST1OE 5220 2a is an LeSAMT1 overexpressing line in the M82 background. Plants were grown in Live Oak, FL. Error bars = SE For Spring 2006, n = 17, 13. For Fall 2006, n = 7, 8.

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59 Table 3 6. LeSAMT1 expression f rom pST1OE 6841 1 and Pearson ripe fruit s % total mRNA STDEV pST1OE 6841 1 8.14E 03 3.14E 03 Pearson 3.23E 06 3.84E 07 Several fruit s were pooled in Spring 2006 and assayed by quantitative real time polymerase chain reaction (RT PCR) in duplicate. n = 2. Table 3 7. LeSAMT1 expression from pST1OE 5220 2a and M82 ripe fruits. % total mRNA STDEV pST1OE 5220 2a 1.12E 02 2.74E 03 M82 3.22E 06 3.72E 07 Several fruits were pooled in Spring 2006 and assayed by quantitative real time polymerase chain reaction (RT PCR) in duplicate. n = 2. Figure 3 14. RNA gel of MeSA overproducing ripe fruit s and control ripe fruit s Lanes 1 2, pST1OE 6841 1; Lanes 3 4, Pearson; Lanes 5 6, pST1OE 5220 2a; Lanes 7 8, M82. 200 ng of RNA was run on a 1% TBE gel and stained with ethidium bromide as a loading control for each reaction.

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60 A B Figure 3 15. MeSA emission from pST1AS 6831 1 and Pearson ripe fruit s A) Spring 2006 and B) Fall 2006. pST1AS 6831 1 is an LeSAMT1 antisense lin e in the Pearson background. Plants were grown in Live Oak, FL. Error bars = SE For A), n = 3, 16. For B), n = 4, 8.

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61 A B Figure 3 16. MeSA emission from LeSAMT1 antisense fruits in the M82 background. A) Spring 2006 and B) Fall 2006. pST1AS 7001 6917, and 6918 are LeSAMT1 antisense lines in the M82 background. For A), n = 5, 3, 9, 13. For B), n = 6, 4, 5, 18.

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62 Figure 3 17. LeSAMT1 expression from pST1AS 6831 1 and Pearson flower buds. pST1AS 6831 1 is an LeSAMT1 antisense line in the Pearso n background. Several flower buds were pooled and assayed by quantitative real time polymerase chain reaction (RT PCR) in duplicate from Spring 2006. Error bars = S TDEV. n = 2. The quality of RNA was checked on a 1% TBE gel stained with ethidium bromide ( not shown). Figure 3 18. LeSAMT1 expression from flower buds in M82 antisense lines. pST1AS 7001, 6917, and 6918 are LeSAMT1 antisense lines in the M82 background. Several buds were pooled and assayed by quantitative RT PCR from Spring 2006 in dupl icate. Error bars = STDEV. n = 2. RNA was run on a 1% TBE gel stained with ethidium bromide as a loading control for each reaction (not shown).

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63 Table 3 8. Triangle taste t est r esults between Pearson and pST1OE 6841 1 fruit s. Total Number Correct 30 Nu mber incorrect 30 Total 60 30 correct for p value < 0.01. A chi square test was used to determine the significance of the number of correct responses T he probab ility of random guessing was assumed to be 33.33%. Table 3 9. MeSA emission from fruit s u sed in the preference and likeability tests ng/gfw/hr SE pST1OE 6841 1 71.94 25.66 Pearson 0.87 0.13 pST1OE 6841 1 is a line overexpressing LeSAMT1 in the Pearson background. Ripe fruits were collected in Spring 2007. Error bars = SE. n = 3. Table 3 10. Hedonic scale parameters for the l ikeability taste test. Value Descriptor 1 dislike extremely 2 dislike very much 3 dislike moderately 4 dislike slightly 5 neither like nor dislike 6 like slightly 7 like moderately 8 like very much 9 like extremely

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64 Table 3 11. Results for preference test between Pearson and pST1OE 6841 1 fruit s Pearson preferred 36 pST1OE 6841 1 preferred 31 Total 67 The difference was not statistically significant according to a two sided directional difference te st. The samples were preferred equally. Table 3 12. Preference test results for Pearson and pST1OE 6841 1 fruit s by age range. Pearson preferred Age Range pST1OE 6841 1 preferred Age Range Total in age range 22 under 18 24 15 under 18 24 37 1 25 3 4 10 25 34 11 7 35 44 2 35 44 9 5 45 54 3 45 54 8 1 55 65 1 55 65 2 36 Total 31 Total 67 indicates p < 0.05 according to a two sided directional difference test. Table 3 13. Cross tabulated scores for likeability test comparing flavor attributes between Pearson and pST1OE 6841 1. Aroma Sweetness Sourness Tomato Flavor Overall Acceptability Pearson 429.00 414.00 387.00 435.00 435.00 Total Score pST1OE 6841 1 427.00 399.00 374.00 426.00 429.00 Pearson 7.00 6.00 5.00 7 .00 7.00 Median pST1OE 6841 1 7.00 6.00 5.00 6.00 7.00 Pearson 6.40 6.18 5.78 6.49 6.49 Mean pST1OE 6841 1 6.37 5.96 5.58 6.36 6.40 Pearson 1.415 1.632 1.485 1.407 1.319 STDEV pST1OE 6841 1 1.774 1.637 1.539 1.453 1.558 V alues for total scores were calculated by totaling the hedonic scores for each attribute Means were calculated by dividing the total score by the total number of panelists (67) Differences were not statistically significant according to a one way ANOVA.

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65 CHAPTER 4 ROLE OF METHYLSALICY LATE IN RESPONSE TO BACTERIAL PATHOGEN INFECTION IN TOMATO Introduction Plants must respond to a variety of environmental stresses, including pathogen attack. Plant pathogen interactions can be classified as compatible or incompatible. In a compatible interaction, the pathogen successfully infects the plant host and is able to multiply, therefore causing expansive disease symptoms. In this case the pathogen is virulent. On the other hand, an incompatible interaction occurs when the plant host is able to mount a resistance response to the pathogen, which results in limited growth of the pathogen. The plant host usually accomplishes this by localizing the cell death response to the area of infection and the pathogen is consi dered to be avirulent. In this case, the defense response is localized at the site of infection, which is also called local resistance. In addition, plants can mount a systemic resistance to protect themselves from subsequent attack by a pathogen. This sys tem ic acquired resistance response is designated SAR. In SAR, distal tissues not infected by a pathogen accumulate salicylic acid (SA) and upregulate pathogenesis related genes ( PR genes) that provide protection against subsequent infection. After the seco ndary infection, bacterial growth is restricted in the distal tissue. SA has been implicated in both local resistance and SAR (Durrant and Dong, 2004). For example, the sid2 (SA induction deficient) mutant of Arabidopsis thaliana is more susceptible to local infection by Pseudomonas syringae and Peronospora parasitica and is impaired in SAR (Nawrtath and Metraux, 1999). The sid2 mutant fails to accumulate SA in response to biotic and abiotic stress. The sid2 mutant was later defined as ICS1 (isochorismat e synthase), a step in the SA biosynthesis pathway, showing that normal levels of SA are required for local and systemic responses (Wildermuth et al. 2001). Previous work in our lab has shown that action of the phytohormones jasmonic acid, ethylene, and S A are required for a successful infection by the

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66 virulent pathogen Xanthomonas campestris pv. v esicatoria (Xcv) 93 1 in tomato ( Solanum lycopersicum ) (O'Donnell et al. 2001; O'Donnell et al. 2003). Xcv is the bacterial pathogen responsible for bacterial spot disease in tomato. Disease progression during Xcv infection can be divided into two phases. Primary symptoms include lesion formation on the abaxial surface of the leaf around 4 days post inoculation (dpi), while secondary symptom development begins a round 8 dpi and includes the appearance of chlorotic patches on the blade of the leaf. Around 10 dpi, necrotic lesions appear within these chlorotic patches that eventually spread throughout the et al. 2001). Transge nic plants overexpressing a bacterial SA hydroxylase ( nahg ) are deficient in SA and have been used as a tool to study the role of SA in the response to Xcv (Gaffney et al. 1993). Previous work in our lab has shown SA is required for symptom development du ring the course of Xcv et al ., 2001). SA can also be converted to the volatile compound methylsalicylate (MeSA), which has also been implicated in defense in different plant pathogen interactions (Schulaev et al. 1997; Chen et al. 2003; Koo et al. 2007). MeSA is synthesized from SA via S adenosyl L methionine: salicylic acid carboxyl methyltransferase, or SAMT (Ross et al. 1999). The Arabidopsis S adenosyl L methionine: benzoic acid/salicylic acid carboxyl methyltransfera se ( AtBSMT ) was upregulated in response to the fungal elicitor alamethicin and thrip ( Plutella xylostella ) feeding (Chen et al. 2003 ) I t has been shown that MeSA can be converted to SA via an esterase from tobacco, salicylic acid binding protein 2 (SABP2 ) (Kumar and Klessig, 2003; Forouhar et al. 2005) This esterase is inhibited by its product, SA (Forouhar et al. 2005) SABP2 silenced tobacco plants infected with tobacco mosaic virus ( TMV ) had larger lesions in the local infection and were impaired in SAR and their responsiveness to SA (Kumar and Klessig, 2003) suggesting a role for the MeSA pool in conversion back to SA. Here, we have examined the role

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67 of MeSA in the tomato response to Xcv We have used transgenic tomato lines overexpressing LeSAMT1 that have significantly more MeSA to examine the effects upon disease symptom development. Results Mature Leaves of LeSAMT1 Overexpressors Produce More Methylsalicylate In order to establish a baseline for understanding the role of MeSA in pathogen infecti on of tomato plants, MeSA emissions from the control and MeSA overproducing line were examined. Volatile emissions were collected from mature leaves of M82 and the transgenic line pST1OE 5220 2a. Mature leaves from the transgenic line had a three fold incr ease in MeSA emission over M82 a significant increase (p < 0.01) (Figure 4 1). Therefore, the constitutively expressed LeSAMT1 is functional in mature leaf tissue, the stage used for bacterial inoculations. LeSAMT1 O verexpressing L ines S how a D elayed D is ease R esponse after Xcv 93 1 I noculation The purpose of this study was to examine the effect of MeSA on the local disease response to a virulent strain of Xcv (93 1). Leaves 3 and 4 of the M82 and pST1OE 5220 2a plants were inoculated with Xcv 93 1. The vi sible symptoms of disease progression in the M82 plants were similar to previously described symptom development in other cultivars of tomato ( et al. 2001 ) Briefly, pinpoint lesions appear ed on the abaxial side of the inoculated leaves 5 6 days post inoculation (dpi). Mild chlorosis appeared on the tips of the leaflets 7 8 dpi, and by 9 10 dpi the chlorosis bec a me even more severe and spread beyond the tip of the leaflet The increasing severity of the chlorosis was accompanied by the appearance of necrotic spots and lesions By 14 dpi, the necrotic lesions ha d grown larger and the tips of the leaflets bec a me necrotic, also described as expansive necrosis (Figure 4 2). In an initial experiment, the bacterial growth was assayed in both the M82 and transgenic line during the course of disease. No

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68 difference was observed between the two lines (Figure 4 3), indicating that overproduction of MeSA did not affect bacterial growth. Interestingly, the MeSA overexpressing line was delayed in the development of necrotic lesions at 10 dpi I on leakage a quantitative measure of cell death, show ed that the transgenic line ha d a 30% reduction in cell death, which correlate d with the later appearance of necrotic lesions ( Figure 4 4). Eventually, the transgenic li ne succumb ed to the disease and the infected leaves became necrotic. However, the onset of necrotic lesions was delayed by several days Therefore, the symptom development was delayed in the transgenic line, prompting further investigation. LeSAMT1 O verex pression Affects the Free Salicylic Acid and Methylsalicylate Pools durin g Xcv I nfection Since it is known that an increase in SA is essential for and precedes the appearance of necrotic lesions ( et al. 2001 ), internal levels of MeSA and SA were measured in the control and transgenic lines during the course of the disease Internal metabolite levels are typically determined in frozen tissue and represent the pool of metabolites contained in the leaves, while the volatile emissions are collected f rom fresh tissue. Current methods for quantitating hormone levels in plant tissue rely on the derivatization of hormones to methyl esters so that the volatile derivatives can be collected by vapor phase extraction (Schmelz et al. 2004). For example, SA is converted to its methyl ester, MeSA. However, the purpose of this experiment was to simultaneously measure SA and MeSA in the same tissue. Therefore, a new protocol was developed to extract endogenous SA and MeSA from the same sample. First, endogenous Me SA was extracted from leaves and the residual SA was derivatized to propyl SA with a strong acid catalyst (described in Experimental Procedures). At 10 dpi, the time point with the greatest difference in necrotic symptoms between the samples, the internal MeSA pool of the

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69 transgenic line was seven fold higher than M82 ( Figure 4 5 ) In addition, the free SA pool was four fold higher in the transgenic line at 10 dpi ( Figure 4 5 ) Interestingly, an increase in MeSA correlate d with an increase in free SA At 12 dpi, the free SA levels in the wild type reach ed a maximum, which correlate d with the spread of necrosis However, the MeSA levels were six fold higher in the transgenic line and kept increasing at 14 dpi Apparently the overexpression of LeSAMT1 in the t ransgenic line result ed in an increase in MeSA as well as SA in response to the pathogen Both MeSA and SA pools reached a maximum after 10 dpi, and the secondary chlorotic and necrotic symptoms developed until 14 dpi. The rate of MeSA emissions from fres h leaves were also collected during the course of disease (Figure 4 6). By 12 dpi, the MeSA emitted from the leaves in the transgenic line reached a maximum. SA accumulation reached a maximum at 10 dpi in the transgenic line (Figure 4 5), so the timing of MeSA emission from the transgenic line lagged behind the substrate availability. At 14 dpi, the MeSA emission from the transgenic line remained higher than the control. During the course of Xcv 93 1 infection, the internal MeSA/SA pools and the MeSA emissi ons of the transgenic line were significantly increased. LeSAMT1 O verexpression Affects the Conjugated Salicylic Acid and Methylsalicylate Pools durin g Xcv I nfection The conjugated pools of SA and MeSA were also examined. Glucoside conjugation is a genera l mechanism for hormone inactivation, and both SA and MeSA glucoside co njugates have been described ( Dean et al. 2005 ) The conjugated SA and conjugated MeSA pools start ed to increase only in the transgenic line after the levels of free metabolites had re ached a maximum Both conjugated SA and conjugated MeSA follow ed the same trend The conjugated pools started to increase at 10 dpi and continu ed to increase as the disease progresse d to 14 dpi ( Figure 4 7 ) Interestingly, this conjugation began at the sam e time as the delay in disease symptom

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70 development occurred, 10 dpi. Therefore, o verexpression of LeSAMT1 caused the leaves of transgenic plants to accumulate a significantly higher level of all forms of SA during pathogen infection free MeSA, conjugated MeSA free SA, and conjugated SA. Discussion The purpose of this study was to examine the effect of LeSAMT1 overexpression on the local plant pathogen interaction between tomato and the virulent bacterial strain Xcv 93 1. The transgenic line overexpressi ng LeSAMT1 showed a delay in the appearance of secondary chlorotic and necrotic symptoms, relative to the parental control, M82, but eventually succumbed et al. 200 1). Surprisingly, once SA accumulation was initiated in the transgenic line, the internal pools of free SA, free MeSA, conjugated SA, and conjugated MeSA increased significantly over those of M82. Since the transgenic line is constitutively expressing LeS AMT1 data on SA and MeSA levels were consistent with the availability of SA to be the cause of the significant increase in MeSA accumulation and emission from the leaves after Xcv 93 1 inoculation. The results from this work show that in LeSAMT1 overexpre ssing plants, SA levels are higher than the control et al ., 2001), the increase in SA would suggest that the leaves should have more severe secondary symptoms. However, this was not t he case; the transgenic plants showed a delay in the onset of secondary symptoms. In TMV infected tobacco, an increase in MeSA and SA was correlated with a decrease in lesion size (Schulaev et al. 1997). It may be that MeSA provides some protection to the tissue to prevent the spread of lesions. Further work will be needed to determine the actual mechanism involved in the reduced rate of symptom development.

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71 SA accumulation in the LeSAMT1 overexpressing plants resulted in an increase in the entire SA/MeSA pool size. At 14 dpi the total SA pool of the transgenic line, including free and conjugated SA, was approximately three fold higher than the controls ( 10 nmol and 3 nmol, respectively). The total MeSA pool of the transgenic line was approximately six fol d higher in the transgenic line relative to the control (12 nmol and 2 nmol, respectively). In a ddition, the pool of SA and all its derivatives in the transgenic line is approximately five fold higher than the control ( 22 nmol and 5 nmol, respectively) at 14 dpi. Excess MeSA may be converted back to SA, or MeSA accumulation may be activating a feedback loop to SA synthesis. A MeSA esterase from tobacco, salicylic acid binding protein 2 (SABP2), has been identified and silenced in transgenic tobacco plants ( Kumar and Klessig, 2003; Forouhar et al. 2005) This esterase converts MeSA to SA and is inhibited by its product, SA (Forouhar et al. 2005) SABP2 silenced tobacco plants infected with TMV had larger lesions in the local infection and were impaired in S AR and their responsiveness to SA (Kumar and Klessig, 2003) Therefore, it is believed that SABP2 is involved in plant innate immunity in tobacco. The work in tobacco further suggests that MeSA may function as an important pool for conversion back to SA fo llowing pathogen infection A more likely possibility for the increase in SA accumulation in the transgenic tomato plants is induction of SA biosynthesis. However, SA biosynthesis has not been fully characterized. S tudies have shown that SA can be synthesi zed from phenylalanine via the phenylpropanoid pathway or isochorismate via the shikimate pathway ( Yalpani et al. 1993; Wildermuth et al. 2001; Strawn et al. 2007) The induction of SA biosynthesis genes was not examined in this study, but this system m ay be a useful tool to study the different branches of SA biosynthesis in the future.

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72 MeSA emission has also been studied in other plant pathogen and plant herbivore interactions. A study by Huang et al. (2003) compared the volatile emissions, including MeSA, from tobacco plants infected with different strains of Pseudomonas syringae They observed that MeSA was most highly induced by an avirulent strain, induced to a lesser extent by a virulent strain, and only induced in trace amounts by a nonpathogenic strain In a dual fungal infection/insect feeding study, Cardoza et al. (2002) observed that peanut plants infected with the white mold Sclerotium rolfsii emitted MeSA in addition to other volatile compounds, but insect feeding alone did not induce MeSA e mission In a separate dual bacterial infection/insect feeding study, Cardoza and Tumlinson (2006) observed that avirulent and virulent strains of Xcv induced different volatile emission profiles in pepper, and the different bacterial strains affected the timing of when insects preferred to feed on the plants Notably, MeSA was induced during all treatments except insect feeding, but was the highest during the combined virulent Xcv infection and insect feeding treatment In addition, the insect survival rat e was increased by 25% on the infected plants, which indicates that biochemical changes in the plant during bacterial infection to insect feeding Therefore, MeSA emission is a common theme found during different plant pathogen interactions, but its exact function is still unknown. The results of the current study suggest that MeSA may serve as a key metabolite regulating SA biosynthesis in response to SA accumulation after bacterial infection.

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73 Figure 4 1 MeSA emission from mature leaves of M82 and pST1OE 5220 2a. Error bars = SE. n = 10. p < test.

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74 Figure 4 2 Secondary symptom development during Xcv 93 1 infection in tomato A) Mock infected M82 ; B) M82 10 days post inocul ation (dpi); C) pST1OE 5220 2a 10 dpi ; D) and E), M82 12 dpi; F) and G), pST1OE 5220 2a 12 dpi; H) M82 14 dpi ; I) and J), pST1OE 5220 2a 14 dpi H I J D E F G A B C

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75 Figure 4 3 Bacterial growth during Xcv infect ion in tomato dpi = days post inoculation. cfu = colony for ming units. Error bars = SE. n = 6.

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76 Figure 4 4 Ion l eakage during Xcv infection in tomato. dpi = days post inoculation. Error bars = SE. n = 6.

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77 A B Figure 4 5 I nternal pools of free SA and MeSA during Xcv infection in tom ato. A) SA i nternal p ool and B) MeSA i nternal p ool dpi = days post inoculation. Error bars = SE n = 4

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78 Figure 4 6 MeSA emissions during Xcv infection in tomato. dpi = days post inoculation. Error bars = SE. n = 3.

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79 A B Figure 4 7 Internal p ools of c onjugated SA and MeSA during Xcv infection in tomato. A) Conjugated SA p ool and B) Conjugated MeSA p ool dpi = days post inoculation. Error bars = SE. n = 4.

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80 CHAPTER 5 GENERAL DISCUSSION The purpose of this study was to characterize the contrib ution of methylsalicylate (MeSA) to tomato flavor and as well as any function in response to bacterial infection in tomato ( Solanum lycopersicum ). The substrate specificity as well as the enzyme kinetics of LeSAMT1 were determined in vitro Plants constitu tively expressing the gene responsible for MeSA synthesis in tomato, S adenosyl L methionine: salicylic acid carboxyl methyltransferase ( LeSAMT1 ) were analyzed with respect to MeSA emission and flavor. In addition, one of these transgenic lines overexpress ing LeSAMT1 was used as a tool to examine potential roles of MeSA in bacterial pathogen responses. LeSAMT1 was the closest tomato homolog to a known SAMT from Clarkia breweri (Ross et al. 1999), and the in vitro enzyme activity was specific for converti ng the plant hormone salicylic acid (SA) to MeSA. Overexpression of LeSAMT1 in transgenic tomatoes resulted in a significant increase of MeSA in fruits and leaves. An untrained consumer panel determined that MeSA overproducing fruit s tasted significantly d ifferent than the control fruit s Subsequently, a preference taste test from an untrained consumer panel determined that the transgenic and control fruit s were preferred equally. In addition, the transgenic and control fruits were rated according to their aroma, tomato like flavor, sweetness, sourness, and overall acceptability. Even though the control fruit s scored slightly higher in likeability and preference choice, the overall means did not indicate a significant preference over the transgenic fruit s T herefore, increasing the MeSA levels in the transgenic fruit s did change the flavor of the tomatoes, but overall preference was determined by how the panelists perceived this change and both samples were liked by panelists.

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81 Since the increased emission of MeSA was also seen in leaves, one of the transgenic lines was used to determine the role of MeSA during bacterial pathogen stress. After inoculation with Xanthomonas campest r is pv. vesicatoria ( Xcv ) 93 1, the transgenic line showed a delay in the onset of disease symptoms. Although delayed, the transgenic plants eventually reached the same endpoint with necrosis of the tips of the leaves. Bacterial growth was not affected in the transgenic line. However, the transgenic line accumulated significantly higher levels of SA, MeSA, conjugated SA, and conjugated MeSA following infection. Therefore, overexpression of LeSAMT1 significantly altered the free and conjugated SA and MeSA pools in the transgenic line, which could be due to a feedback loop to SA biosynthes is. These MeSA overproducing lines may be useful tools for elucidating which branch of SA biosynthesis is affected in response to Xcv 93 1 i nfection. In the future, it would be useful to examine the free SA levels in the MeSA overproducing fruit s to see if SA accumulates as it did in response to pathogen infection. In addition, numerous studies have shown that MeSA is a common volatile seen in the emissions of plants inflicted with herbivore damage. It would be interesting to see if increased levels of MeSA affect tomato herbivore interactions with regard to attracting or repelling feeding insects. Plants have evolved to use volatiles to attract pollinators, seed dispersing organisms, and to adapt to stress. These plant volatiles are components of floral sc ent and the flavors of foods, and may even possess medicinal properties. Humans have devised ways to take advantage of these volatile compounds for commercial use to improve upon fragrances, flavors, and pharmaceuticals. Understanding the molecular mechani sms of plant volatile production may aid in the development of improved crops by molecular breeding and may even help identify novel compounds useful for industrial purposes. However, it is difficult to target specific traits using traditional breeding pra ctices. Genetic engineering has made it possible to study the effect of one

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82 gene on specific biochemical pathways involved in a variety of plant processes. Using such a tool is advantageous when studying a complex trait such as flavor, so only one target c ompound will be altered. Using transgenic lines overexpressing LeSAMT1 the gene responsible for MeSA synthesis in tomato, this work demonstrated the effect of one volatile, MeSA, on tomato flavor and plant hormone pools in response to pathogen stress.

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83 CHAPTER 6 EXPERIMENTAL PROCEDU RES Cloning of L eSAMT1 The full length EST of LeSAMT1 was identified in the TIGR database by homology to the amino acid sequence of Clar k ia breweri CbSAMT (Ross et al. 1999) and was amplified with primers (Fwd) CACCATG AAGGTTGTTGAAGTTCTTCACATGAATGGAGG and (Rev) TTATTTTTTCTTGGTCAAGGAGACAGTAACATTTATAAACTCAGTATCC from Flora Dade ( Solanum lycopersicum ) bud cDNA. For LeMT (tomato methyltransferase) sequences, full length clones from the TIGR database were ordered and sequence d. LeMT s were named according to their percent similarity to the predicted amino acid sequence of LeSAMT1 (Table 2 1). The following full length clones from the TIGR database were assigned as LeMT s: LeSAMT1 cTOA4C17; LeMT 1 cLEM7O9; LeMT 2 cTOA14P1; LeMT 3 cLEI13O14; LeMT 4 cTOD6B16; LeMT 5 cLEW1K6; LeMT 6 cTOA28E18; LeMT 7 cTOF25N7. Production of Transgenic Plants The full length open reading frame of LeSAMT1 was cloned into a vector containing the constitutive FMV 35S promoter (Richins et al. 1987) in the sense or antisense orientation. Solanum lycopersicum (M82 and Pearson) were transformed by Agrobacterium mediated transformation (McCormick et al. 1986) with the kanamycin selectable marker. Plants were grown in the greenhouse for initial screening an d planted in the field at the University of Florida North Florida Research and Education Center Suwannee Valley in Live Oak, FL for additional analysis. In Spring 2006, the MeSA overproducing Pearson line pST1OE 6841 1 and the M82 antisense lines pST1AS 70 01, 6917, and 6918 were a mixture of homozygous and heterozygous pcr positive lines The antisense Pearson line pST1AS 6831 1 and the M82 MeSA

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84 overproducing line pST1OE 5220 2a were homozygous in Spring 2006. In Fall 2006, all lines were homozygous (pST1OE 6841 1 4, pST1AS 7001 1, pST1AS 6917 2, and pST1AS 6918 1). Expression and Purification of GST LeSAMT1 For protein expression and purification, LeSAMT1 was cloned into the pENTR/D TOPO Gateway vector (Invitrogen). The open reading frame was recombined int o the N terminal GST tag Gateway vector pDEST15 (Invitrogen) and transformed into E. coli strain BL21 AI (Invitrogen) for arabinose inducible expression. Bacterial cultures were grown with 100 g/mL carbenicillin to an OD 600 of 0.4 and were induced with a 20% L arabinose solution for a final concentration of 0.2%. Cells were induced overnight (16 hrs) at 15 o C and harvested the next day. To harvest cells, cultures were centrifuged at 5000 g for 15 minutes and resuspended in lysis buffer 1X PBS, lysozyme, 10% v/v glycerol, and Bacterial Protease Inhibitor Cocktail (Sigma) at 4 o C. Cells were sonicated with a Fisher Sonic Desmembrator, Model 100 (Fisher) on level 1 for 10 cycles of 5 seconds on, 30 seconds off. Cells were centrifuged at 10000 g for 15 minutes an d the GST tagged protein was purified on a Glutathione Uniflow Resin (BD Biosciences Clontech) at 4 o C. Columns with 1.5 bed volumes of resin were eq uilibrated with 1 XPBS. The extract was mixed with resin on a rotating wheel for 1 hour. The flow through was collected and run through the column a second time. The column was washed with 16 bed volumes of 1X PBS and the GST LeSAMT1 was eluted with Elution Buffer (10 mM glutathione, 50 mM Tris HCl pH 8.0, 20% v/v glycerol). Protein levels were quantified using B radford Reagent (BioRad) and purification was checked with protein blotting using GST antibodies and visualized with ECL reagents (Amersham). The enzyme was stored on ice at 4 o C.

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85 Kinetic Assays Assay conditions for substrate specificity and K m determinat ion for GST LeSAMT1 followed Zubieta et al. (2003) with some modifications. For substrate specificity assays, 2.85 g of GST LeSAMT1 was assayed in a 100 L rea ction containing 50 mM Tris HCl pH 7.5, 100 mM KCl, 2.8 mM BME, 1 mM substrate, and a 30 M solu tion of 4:1 unlabeled SAM: 14 C SAM, specific activity 11.04 mCi/mmol (Amersham). Substrates were diluted in EtOH with the exception of nicotinic acid, which was diluted in water. Assays were done in triplicate, including no enzyme controls. After one hour at 2 5 o C the reactions were stopped by adding an equal volume of hexanes. 14 C MeSA was extracted from the organic layer by vortexing samples for 15 seconds and centrifuging at 13200 g for 2 minutes. Fifty L of the hexane layer was counted for 5 min in 3 mL Ready Gel Scintillation Fluid (Beckman Coulter). Counts for the no enzyme controls were subtracted from the sample counts, and activity for SA was normalized to 100%. For the K m of SA, 2.85 g of purifie d GST LeSAMT1 was used. 14 C SAM was held constant at 75 M. Two 14 C SAM stock solutions were used with varying specific activity to minimize the use of radioactivity and to make sure the product counts were detectable. For the lower [SA] range, 200 M of a 2:1 dilution (unlabeled SAM: 14 C SAM) with a specific activity of 18.4 mCi/mmol was used. For the higher [SA] range, a 200 M of a 4:1 dilution with a specific activity of 11.04 mCi/mmol of SAM was used. For the K m of SAM, 3.42 g of GST LeSAMT1 was used and [SA] was held constant at 1mM. [SAM] was varied using two stock solutions with different specific activities. An undiluted specific activity of 55.2 mCi/mmol (10 M) was used as the stock solution for the lower [SAM] range. A 200 M stock of a 2:1 dil ution with a specific activity of 18.4 mCi/mmol was used for the higher [SAM] range. The reactions for the K m

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86 determinations were stopped after two hours as described above. A preliminary experiment showed that the assay was linear after two hours. Volatil e Collection Volatiles were collected from tomato fruit s according to Tieman et al. ( 2006 ) For leaf volatile collections, two whole leaves, approximately 4 5 g of fresh tissue, were carefully loaded into glass collection tubes to avoid unnecessary damage. Briefly, air was passed over the samples and volatiles were collected on a SuperQ Resin for one hour. Volatiles were eluted off the column with methylene chloride and run on a GC for analysis as described in Tieman et al (2006). LeSAMT1 Expression Quanti fication Total RNA was extracted using the Qiagen RNeasy Plant Mini Kit and levels of LeSAMT 1 mRNA levels were quantified by real time polymerase chain reaction (RT PCR) using Taqman one step RT PCR reagents (Applied Biosystems) The pericarp and locular g el from several fruits were pooled for e ach RNA e xtraction for the analysis of transgenic plants, and each extraction was run in duplicate. For the tissue specific expression and pathogen experiments, four biological replicates were analyzed per time point LeSAMT1 expression was determined using the following primer/probe set Fwd : TCCCAGAAACATTATACATTGCTGAT Rev : AATGACCTTAACAAGTTCTGATACCACTAA Probe: (56 FAM) TGGGTTGTTCTTCTGGAGCGAACACTTT (3BHQ_1) Samples were run on the BioRad iCylcer pcr detection syste m and quantified with the MyiQ software The followi ng pcr conditions were used: 48 o C, 30 min; 95 o C 10 min; 40 cycles of 95 o C, 15 sec; 60 o C 1 min. A sense strand was in vitro transcribed from plasmid DNA with 3 H UTP (MAXIscript, Ambion) and was used to det ermine the absolute values of RNA in the sample.

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87 Pathogen Inoculations Xanthomonas campestris pv. vesicatoria ( Xcv ) 93 1 inoculations were done on M82 control plants and the MeSA overexpressing line pST1OE 5220 2a. Bacterial inoculations were performed o n leaves 3 and 4 of 5 to 6 week old plants. Virulent Xcv 93 1 cultures were grown overnight in 100 mL of 0.7% Nutrient Broth (Difco Laboratories, Detroit, MI) at 28 o C. Cells were centrifuged at 5000 g for 10 minutes and resuspended in mock buffer (10 mM Mg Cl 2 0.025% (v/v) Silwet L 77 in ultrapure H 2 O). Cells were diluted to 1 x 10 6 cfu in mock buffer. For inoculations, leaves 3 and 4 were dipped in the bacterial suspension for 15 seconds. Leaves of mock treated plants at 0 dpi were dipped in mock buffer on ly. Two to three plants were assayed per time point per measurement. Ion Leakage For ion leakage measurements, three plants were assayed, and each infected leaf was assayed separately (n = 6). Measurements in microohms 1 per cm 2 per hr (designated as mh o/ cm 2 / hr) are described in Lund et al. (1998). Bacterial Growth Curves Bacterial colony counts were performed on two leaves of three plants for each time point. Two cm 2 discs were excised with a number 5 cork borer from representative leaflets for each t ime point. Discs were ground in 10 mM MgCl 2 and serial dilutions were plated on 0.7% Nutrient Broth, 1.5% Bacto agar (Difco Laboratories, Detroit, MI). Plates were incubated at 30 o C for 2 days and colonies were counted for each time point. Free S alicylic A cid and Methylsalicylate E xtractions Vapor phase extraction of free metabolites and conjugated metabolites was performed according to Engelberth et al. (2003 ) and Schmelz et al. (2004) with some modifications. In previously reported vapor phase extraction protocols, free SA was derivatized to its methyl ester

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88 MeSA to quantify the amount of SA in the leaves. However, the goal of this experiment was to quantify the amounts of free SA and free MeSA in the same sample, so a different method of derivatization w as needed to analyze both metabolites without interference. Briefly, individual leaves were frozen in liquid N 2 and ground to a fine powder. Approximately 100 mg of frozen tissue was weighed into a Fastprep tube containing 1 g ceramic beads (1.1 mm Zirmil beads ; SEPR Ceramic Beads and Powders, Mountainside, NJ, USA) and an internal standard mix containing 100 ng 2 H 6 SA (CDN Isotopes, Pointe Claire, Quebec, Canada) in EtOH and 100 ng of a lab prepared 2 H 4 M e SA standard in methylene chloride. The samples were extracted with 300 L Extraction Buffer (2:1:0.005 1 propanol: H 2 O: HCl) and shaken in a Fastprep FP 120 homogenizer (Qbiogene) for 30 sec. Then 1 mL methylene chloride was added and the samples were shaken an additional 10 seconds. Samples were centrifuged at 11300 g for on e minute and the bottom methylene chloride layer was transferred to a 4 mL glass vial and sealed. The top aqueous layer was later used for glucoside extractions. First, free MeSA was collected from the methylene chloride phase. The glass vial was sealed wi th a cap containing a high temperature septa and a column containing SuperQ resin was inserted into the septa, followed by a needle carrying a stream of N 2 The glass vial with the methylene chloride phase was placed on a 70 o C heating block and the vapor p hase was collected just until the liquid evaporated. The column containing the MeSA was saved for recollection after the SA derivatization. The free SA remained in the dried vial and was derivatized to propyl SA with 30 L of a 2:1 mixture of 1 propanol: H Cl. The samples were vortexed and placed in a 70 o C oven for 45 minutes. Samples were cooled to room temperature and 75 L of a 1 M citric acid solution was added to stop the reaction. Samples were vortexed and the vapor phase was collected on the same colu mn as described above. After the liquid evaporated, the sample was left on the heat block for an

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89 additional 2 minutes. Columns were rinsed with 200 L ultrapure water and dried. Then the columns were eluted with 125 L methylene chloride for CI GC/MS. Free SA and MeSA levels were quantitated using the internal standard values as described in Schmelz et al. (2004). The propyl SA derivatized from the endogenous SA ran at a retention time of 10.12 min and m/z of 181 and the 2 H 6 SA propylated standard ran at a retention time of 10.11 min and m/z of 185. Conjugated Salicylic Acid and Methylsalicylate Extractions The aqueous layer from the vapor phase extractions was transferred to a 4 mL glass vial and dried in a speed vac o vernight. After drying completely, the 2 H 6 SA standard (100 ng) was added and dried with a stream of N 2 Then the 2 H 4 M e SA standard was added and the vial was immediately sealed. The glucosides for MeSA and SA were acid hydrolyzed and derivatized in the same step by adding 30 L of a 2:1 mixtur e of 1 propanol: HCl as described above. Samples were incubated for 45 minutes at 70 o C, followed by neutralization with 75 L of 1 M citric acid. Hydrolyzed MeSA and SA were collected by vapor phase extraction in one step at 70 o C and kept on the heat block 2 minutes after drying. Columns were rinsed and eluted as described above. Samples were analyzed as described above. Triangle Taste Test Sixty untrained volunteer panelists participated in a triangle test to determine if the MeSA overproducing line pST1O E 6841 1 tasted different than the Pearson controls. Fruits were collected from the field and the seeds and locular gel were discarded. Panelists were given three samples of tomato slices: either two controls and one transgenic, or two transgenics and one control, in random order. Each sample was given a random three digit code. Panelists were asked to smell the samples, taste the samples, and indicate which sample was different. Thirty panelists

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90 were correct, and a p value of < 0.01 was assigned after a ch i square test assumi ng chance probability was 33.33% (Meilgaard et al. 2007). Likeability and Preference Test s The likeability and preference tests were completed at the Sensory Testing Facility in the Food Science and Human Nutrition Department at the Un iversity of Florida according to Meilgaard et al. (2007). Seeds from the fruit s of the MeSA overproducing line and the Pearson control were removed and the fruit s were cut into wedges. Each sample was given a random three digit code and presented to the pa nelists in random order. Sixty seven untrained panelists were asked to rate the aroma, sweetness, sourness, and overall acceptability of the two samples on a nine point hedonic scale. Panelists were also asked to choose the tomato sample they preferred ove rall. The statistical significance of the preference test was analyzed by a two sided directional paired comparison test. The statistical significance of the likeability test was determined using a one way ANOVA.

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91 LIST OF REFERENCES Abraham, K O., Shankar anarayana, M .L., Raghaven, B. and Natarajan, C P. (1976) Determination of methyl salicylate in black tea. Mikrochimica Acta 65 11 15. Adams, T B., Cohen, S M., Doull, J., Feron, V J., Goodman, J I., Marnett, L J., Munro, I C., Portoghese, P S., Smith, R L., Waddell, W .J. and Wagner, B M (2005) The FEMA GRAS assessment of hydroxyl and alkoxy substituted benzyl derivatives used as flavor ingredients Food Chem. Tox 43 1241 1271. Ament, K., Kant, M.R., Sabelis, M.W., Haring, M.A. and Schuurink, R.C (2004) Jasmonic acid is a key regulator of spider mite induced volatile terpenoid and methyl salicylate emission in tomato. Plant Physiol. 135 2025 2037. Baldwin, E A., Nisperos Carriedo, M .O., Baker, R. and Scott, J W (1991) Quantitative analysis o f flavor parameters in six Florida tomato cultivars. J. Agric. Food Chem. 39 1135 1140. Baldwin, E.A., Scott, J A., Shewmaker, C K. and Shuch, W (2000) Flavor trivia and tomato aroma: biochemistry and possible mechanisms for control of important aroma components. Hort Sci. 35 1013 1022. Baldwin, E A., Goodner, K., Plotto, A., Pritchett, K. and Einstein, M (2004) Effect of volatiles and their concentration on perception of tomato descriptors. J. Food. Sci. 69 S310 S318. Barkman, T.J., Martins, T .R., Sutton, E. and Stout, J.T. (2007) Positive selection for single amino acid change promotes substrate discrimination of a plant volatile producing enzyme. Mol. Biol. Evol. 24 1320 1329. Battino, M., Ferreiro, M.S., Fattorino, D. and Bullon, P. (2002 ) In vitro antioxidant activities of mouthrinses and their components. J. Clin. Periodontol. 29 462 467. Bichao, H ., Borg Karlson, A., Araujo, J. and Mustaparta, H (2005) Five types of olfactory receptor neurons in the strawberry blossom weevil Antho nomus rubi : selective responses to inducible host plant volatiles. Chem. Senses 30 153 170. Burdock, G.A (1995) 3rd edn. Boca Raton, FL: CRC Press. Buttery, R G., T e ranishi, R., Ling, L C., Flath, R.C and Stern, D J (1988) Quantitative studies on origins of fresh tomato aroma volatiles. J. Agric. Food Chem 36 1247 1250. Buttery, R G and Ling, L C. (1993) Volatiles of tomato fruit and plant parts: relationship and biogenesis. In Bioactive v olatile compounds from plants (Teranishi, R., Buttery, R. and Sugisawa, H., eds). Washington, D.C.: ACS Books, pp. 23 34.

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98 BIOGRAPHICAL SKETCH Michelle Lynn Zeigler was born July 16, 1980 and was raised in Clearwater, Florida. As an undergraduate student at the University of Florida, she participat ed in the University Scholars became interested in plant molecular biology. She graduated from the University of Florida in obiology and was awarded an Alumni Fellowship in the Plant Molecular and Cellular Biology Graduate Program at the University of Florida. In Dr. and its role in the response to the virulent bacterial pathogen Xanthomonas campestris pv. vesicatoria