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Role of Members of the Tomato Ethylene Receptor Family in Determining the Timing of Ripening

Permanent Link: http://ufdc.ufl.edu/UFE0021537/00001

Material Information

Title: Role of Members of the Tomato Ethylene Receptor Family in Determining the Timing of Ripening
Physical Description: 1 online resource (98 p.)
Language: english
Creator: Kevany, Brian Michael
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: 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: Tomatoes are an economically important crop and a significant dietary source of important phytochemicals, such as carotenoids and flavonoids. While it has been known for many years that the plant hormone ethylene is essential for ripening of climacteric fruits, its role in fruit growth and maturation is much less understood. In an attempt to better understand tomato fruit ripening we utilized both biotechnology and traditional breeding strategies. The multigene ethylene receptor family has been shown to negatively regulate ethylene signal transduction and suppress ethylene responses. Here, we demonstrate that a reduction in the levels of either of two family members, LeETR4 or LeETR6, causes an early ripening phenotype. We provide evidence that the receptors are rapidly degraded in the presence of ethylene and that degradation likely occurs through the 26S proteasome-dependent pathway. Ethylene exposure of immature fruits causes a reduction in the amount of receptor protein and earlier ripening. Fruit-specific suppression of the ethylene receptor LeETR4 causes early ripening while fruit size, yield and flavor-related chemical composition are largely unchanged. These results demonstrate that ethylene receptors likely act as biological clocks regulating the onset of tomato fruit ripening. In order to better understand the mechanism controlling the timing of ripening we screened a Lycopersicon hirsutum introgression population for QTLs responsible for reduced time from anthesis to breaker and/or increased ripening-associated ethylene biosynthesis. The L. hirsutum population was chosen because of unusual ripening characteristics and significantly higher levels of ethylene biosynthesis at maturity of L. hirsutum. A number of lines were identified that showed statistically significant differences from the control for both phenotypes. These lines are currently being refined for possible map-based cloning of loci controlling these phenotypes. These results demonstrate the power of using both molecular biology and traditional breeding for gene isolation/characterization and crop improvement.
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 Brian Michael Kevany.
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: UFE0021537:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021537/00001

Material Information

Title: Role of Members of the Tomato Ethylene Receptor Family in Determining the Timing of Ripening
Physical Description: 1 online resource (98 p.)
Language: english
Creator: Kevany, Brian Michael
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: 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: Tomatoes are an economically important crop and a significant dietary source of important phytochemicals, such as carotenoids and flavonoids. While it has been known for many years that the plant hormone ethylene is essential for ripening of climacteric fruits, its role in fruit growth and maturation is much less understood. In an attempt to better understand tomato fruit ripening we utilized both biotechnology and traditional breeding strategies. The multigene ethylene receptor family has been shown to negatively regulate ethylene signal transduction and suppress ethylene responses. Here, we demonstrate that a reduction in the levels of either of two family members, LeETR4 or LeETR6, causes an early ripening phenotype. We provide evidence that the receptors are rapidly degraded in the presence of ethylene and that degradation likely occurs through the 26S proteasome-dependent pathway. Ethylene exposure of immature fruits causes a reduction in the amount of receptor protein and earlier ripening. Fruit-specific suppression of the ethylene receptor LeETR4 causes early ripening while fruit size, yield and flavor-related chemical composition are largely unchanged. These results demonstrate that ethylene receptors likely act as biological clocks regulating the onset of tomato fruit ripening. In order to better understand the mechanism controlling the timing of ripening we screened a Lycopersicon hirsutum introgression population for QTLs responsible for reduced time from anthesis to breaker and/or increased ripening-associated ethylene biosynthesis. The L. hirsutum population was chosen because of unusual ripening characteristics and significantly higher levels of ethylene biosynthesis at maturity of L. hirsutum. A number of lines were identified that showed statistically significant differences from the control for both phenotypes. These lines are currently being refined for possible map-based cloning of loci controlling these phenotypes. These results demonstrate the power of using both molecular biology and traditional breeding for gene isolation/characterization and crop improvement.
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 Brian Michael Kevany.
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: UFE0021537:00001


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5a9c25b58c0c6723aefebdd59639da6115c61766







ROLE OF MEMBERS OF THE TOMATO ETHYLENE RECEPTOR FAMILY IN
DETERMINING THE TIMING OF RIPENING























By

BRIAN MICHAEL KEVANY


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 Brian Michael Kevany



































To my parents, who have supported every decision I have ever made and given everything to
their children









ACKNOWLEDGMENTS

Thanks to my entire committee for their patience and constructive criticism throughout this

process. I would like to especially thank my advisor, Dr. Harry Klee, for guiding me through my

Ph.D. and not only teaching me how to be a scientist but how to present myself and my science. I

thank the entire Klee lab for all their help throughout the years. Thanks to my bench-mate

Michelle Zeigler whose attention to detail has helped me to become a better scientist. I thank

Denise Tieman for sharing her knowledge in the lab and Mark Taylor for generating all the

transgenic plants used in my experiments. Thanks to Peter Bliss for taking care of my plants in

the greenhouse and doing just about everything around the Klee lab. I thank Valeriano Dal Cin

for all of this help on the mapping proj ect.

Thanks to everyone in the lab of Dr. Andrew Hanson for all their help and great

friendship. I would especially like to thank Dr. Gilles Basset and Dr. Sebastian Klaus for

teaching me everything they know about protein expression. Additionally I thank Dr. Gale

Bozzo, Dr. Rocio Diaz de la Garza, Dr. Giuseppe Orsomando, Dr. Aymeric Goyer, and Tariq

Ahktar for being there when I needed a break and to have some fun. Thanks to the lab of Dr.

David Clark for allowing me to come over and do my RNA extractions in their hood and also to

bother them when I needed a break. I thank Carol Dabney-Smith for teaching me all she knows

about custom antibodies, without this help I would not have been able to finish all my work.

Most importantly, thank you to my family for always being there for me when I needed

them. Also for understanding that moving from Ohio to Florida was what was best for my career

even though it was so far. I also thank all of my friends back in Ohio and Michigan for staying in

touch and giving me plenty of fun times outside of Gainesville. Lastly, thanks to Stephanie Violi

from the bottom of my heart for being the person I have leaned on for the past three years. She









has made me laugh when I needed it and always put things in perspective. Even though we

haven't been together she has remained the driving force in my life and is the love of my life.











TABLE OF CONTENTS


page

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


LIST OF TABLES ................ ...............8............ ....

LI ST OF FIGURE S .............. ...............9.....


AB S TRAC T ........._. ............ ..............._ 1 1...

CHAPTER

1 LITERATURE REVIEW .............. ...............13....


Ethylene in Plant Biology .........._..._ ......... .. .... ......_. ...........1
The Ethylene Receptor Family Arabidopsis and Tomato............... ...............17.
Protein Degradation Through the 26S Proteasome .............. ...............24....

2 ETHYLENE RECEPTOR DEGRADATION CONTROLS THE TIMING OF
RIPENING INT TOMATO .............. ...............3 1....


Introduction............... .............3
R e sults.................... ... ........ ... ...... ..... ..... ........ .........3
A Subset of the Receptor Family Shows Ripening-associated Expression and Is
Ethylene-inducible in Fruit ................ .. ......... .. ........ .... ..................3
LeETR6 Antisense Lines Show Phenotypes Consistent with a Constitutive Ethylene
R response .............. .. .... ..... ..... ... .... ......................3
Receptor Protein Levels Are Distinctly Different From Transcript Levels During
Fruit Developm ent .............. .... ............... .. ... .......3
Treatment of Leaf and Fruit Tissue with Ethylene Causes a Rapid Degradation of
Receptor Proteins That Likely Occurs Through a Proteasome-dependent Pathway ...35
Receptor Levels in Developing Fruit Determine the Timing of Ripening ................... ...3 7
Discussion............... ...............3


3 FRUIT-SPECIFIC SUPPRESSION OF THE ETHYLENE RECEPTOR LEETR4
RESULTS INT EARLY RIPENING FRUIT .............. ...............50....


Introduction............... ..............5
R e sults................... .. ......... .......... ..... .. .. ..... .............5
LeETR4 RNAi Transgenic Plants Produce Early Ripening Fruit ................. ................51
Early Ripening Lines Show Altered Ripening Coordination .................. ....... ........... ....52
Transgenic Fruits are Indistinguishable from Wild Type Fruits in Horticultural
Traits .............. ...............53....
Discussion............... ...............5












4 IDENTIFICATION OF QTLS THAT MODIFY TIME TO RIPENING AND
RIPENING-AS SSOCIATED ETHYLENE PRODUCTION. ........._.. ...... ._._._...........61


Introduction............... ..............6
Re sults........._..... ...._... ...............63.....
Discussion............... ...............6


5 C ONCLU SION ........._..... ...._... ...............80...


6 MATERIALS AND METHODS............... ...............83


Plant Materials and Growth Conditions .............. ...............83....
Development of Transgenic Plants ........._.. ...._._..... ...............83...
Pharmacological Treatments ............................... ................8
Recombinant Protein Expression and Antibody Production.........._.._.._ ......_.._.. .....84
RNA Expression Analysis................. ...........................8
Microsomal Membrane Isolation and Protein Blot Analysis .............. .....................8
Acid and Soluble Solids Analysis .............. ...............86....
Vol atil e Analy si s .............. ...............87....


LIST OF REFERENCES ................. ...............89................


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










LIST OF TABLES


Table page

2-1 Days from anthesis to breaker of LeETR6 antisense lines............... ...............49.

2-2 Days from anthesis to breaker of ethylene treated Microtom fruit............. .. ........._._ ...49

3-1 Weight, yield, brix, citric acid and malic acid from field grown fruits ................ ................59

3 -2 Weight, yield, brix, citric acid and malic acid from greenhouse grown fruits ................... .....59

3-3 Volatile organic compounds from field grown fruits ................. ....._._ .............. ...5

3-4 Volatile organic compounds from greenhouse grown fruits .............. .....................6

6-1 Oligonucleotide primers and probes............... ...............88.










LIST OF FIGURES


Figure page

1-1 Schematic representation of tomato ethylene receptor family ................ ................ ...._30

2-1 Ethylene receptor family mRNA levels during fruit development............... ..............4

2-2 Ethylene-inducibility of each receptor mRNA in immature fruit tissue. ............. ................42

2-3 Constitutive ethylene response phenotypes of LeETR6 anti sense lines ................ ...............43

2-4 Receptor gene expression and protein levels show distinct differences during fruit
development ................. ...............44.................

2-5 Ethylene binding induces degradation of receptors in detached immature fruits .................. .45

2-6 Ethylene binding induces degradation of receptor proteins in vegetative tissue. .................. .46

2-7 Ethylene treatment induces turnover of receptor leading to early ripening fruit. .................. .47

2-8 Ethylene treatment induces expression of receptor mRNAs in attached fruit ................... .....48

3-1 Fruit-specific ETR4 RNAi transgenic lines produce early ripening fruit ............... .... ...........56

3 -2 Suppression of LeETR4 is Fruit-specific ................. ...............57........... ..

3-3 ETR4-RNAi transgenic plants have altered ripening coordination .............. ...................58

4-1 Ethylene emissions of fully ripe fruit from L. hirsutum IL 3945 ..........._..._ .........._._.......69

4-2 Days from anthesis to breaker of tagged fruits from L. hirsutum I~s ........._..... ........._.....70

4-3 Ethylene emissions of breaker fruit from L. hirsutum I~s .............. ...............71....

4-4 Ethylene emissions of fully ripe fruit from L. hirsutum I~s. .........__........ _.. ........._...72

4-5 Genomic map showing locations of introgressed regions that contain putative ripening-
associated QTLs................ ...............73.

4-6 Days from anthesis to breaker of tagged fruits from L. hirsutum I~s ........._..... ........._.....74

4-7 Ethylene emissions of fruits from field-grown L. hirsutum ILs ................ ............. .......75

4-8 Ethylene emissions of leaves from L. hirsutum ILs .............. ...............76....

4-9 Nucleotide alignment of ETR4 genomic sequence ........__............_ ........_._.........77

4-10 mRNA expression of LeETR4 in WT and the L. hirsutum IL 3945 .................. ...............78










4-11 mRNA expression of LeETR4 in seedlings of WT, L. hirsutum and IL 3945......................79









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 MEMBERS OF THE TOMATO ETHYLENE RECEPTOR FAMILY IN
DETERMINING THE TIMING OF RIPENING

By

Brian Michael Kevany

December 2007

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

Tomatoes are an economically important crop and a significant dietary source of important

phytochemicals, such as carotenoids and flavonoids. While it has been known for many years

that the plant hormone ethylene is essential for ripening of climacteric fruits, its role in fruit

growth and maturation is much less understood. In an attempt to better understand tomato fruit

ripening we utilized both biotechnology and traditional breeding strategies. The multigene

ethylene receptor family has been shown to negatively regulate ethylene signal transduction and

suppress ethylene responses. Here, we demonstrate that a reduction in the levels of either of two

family members, LeETR4 or LeETR6, causes an early ripening phenotype. We provide evidence

that the receptors are rapidly degraded in the presence of ethylene and that degradation likely

occurs through the 26S proteasome-dependent pathway. Ethylene exposure of immature fruits

causes a reduction in the amount of receptor protein and earlier ripening. Fruit-specific

suppression of the ethylene receptor LeETR4 causes early ripening while fruit size, yield and

flavor-related chemical composition are largely unchanged. These results demonstrate that

ethylene receptors likely act as biological clocks regulating the onset of tomato fruit ripening.

In order to better understand the mechanism controlling the timing of ripening we screened

a Lycopersicon hirsutum introgression population for QTLs responsible for reduced time from









anthesis to breaker and/or increased ripening-associated ethylene biosynthesis. The L. hirsutum

population was chosen because of unusual ripening characteristics and significantly higher levels

of ethylene biosynthesis at maturity of L. hirsutum. A number of lines were identified that

showed statistically significant differences from the control for both phenotypes. These lines are

currently being refined for possible map-based cloning of loci controlling these phenotypes.

These results demonstrate the power of using both molecular biology and traditional breeding for

gene isolation/characterization and crop improvement.









CHAPTER 1
LITERATURE REVIEW

Ethylene in Plant Biology

The phytohormone ethylene is an important signaling molecule that is involved in many

plant processes including but not limited to abscission, leaf and flower senescence, germination,

sex determination and fruit ripening (Abeles et al., 1992). Ethylene also functions in both biotic

and abiotic stress responses. Exposure to environmental stresses like flooding, wounding,

herbivory, chilling or pathogen attack can enhance ethylene production (Boller, T. 1991; Abeles

et al., 1992). This ethylene then slows growth until the stress is removed. Interest in ethylene' s

importance as a plant hormone has resulted in thousands of peer-reviewed publications in the last

100 years and has laid the foundation for a real understanding of ethylene' s involvement in plant

growth and development.

Ethylene is a small, gaseous, two-carbon molecule that has the ability to diffuse through

hydrophilic and hydrophobic environments. This property allows it to pass into any compartment

in the plant cell. The ability of ethylene to alter plant development has been known for centuries,

with farmers from many cultures using smoke and wounding to induce flowering and ripening

(Abeles et al., 1992). Damage to city and greenhouse plants in the late 19th and early 20th

centuries was found to be caused by leaking illuminating gas that was used at the time for

lighting. Work done by Dimitry Neljuboy in 1901 proved that ethylene was in fact the active

component in illuminating gas that resulted in the plant damage (Abeles et al., 1992). Subsequent

work showed that ethylene was clearly important for fruit ripening but many scientists at the

time believed that the other phenotypes of endogenously produced ethylene were a by-product of

the ripening process (Abeles et al., 1992). Work done in the 1960s by the Burgs provided

definitive proof that ethylene is important for plant development beyond its involvement in fruit










ripening (Burg and Burg, 1962; Burg 1962). Their work was instrumental in classifying ethylene

as a plant hormone.

Early feeding experiments suggested that the amino acid methionine is a precursor of

ethylene (Lieberman et al., 1966; Burg and Clagett, 1967). Later work provided evidence that

oxygen is necessary for the production of ethylene. It was then hypothesized that if fruit tissue

was held in an anaerobic environment the precursor should build up and provide enough

compound to allow identification. This work led to the subsequent isolation of 1-

aminocyclopropane-1 -carboxylic acid (ACC), the immediate precursor of endogenous ethylene

(Adams and Yang, 1979). ACC is synthesized from S-adenosyl methionine (SAM) by a

pyridoxal phosphate-requiring enzyme termed ACC synthase (ACS). The conversion of ACC to

ethylene is subsequently performed by the oxygen-requiring enzyme ACC oxidase (ACO). The

conversion of ACC by ACO results in the production of CO2 and HCN in addition to ethylene.

Most tissues synthesize low levels of ethylene. Synthesis can be stimulated by a number of

means, including wounding, submergence, chilling and pathogen attack. Synthesis of ACC is

considered to be the rate limiting step in ethylene production. Thus, increased ethylene

production requires modulation of ACS expression and/or activity.

While ethylene is often characterized as the ripening hormone, not all fruit require ethylene

to complete the ripening process. Species are often characterized by the presence or absence of a

large increase in ethylene production concomitant with increased respiration at the onset of

ripening. Species whose fruit exhibit these increases are termed climactericc" while those that do

not are referred to as "non-climacteric." Climacteric species include apple, avocado, banana,

peach and tomato while non-climacteric species include strawberry, grape, cherry and citrus. The

increase in ethylene production associated with climacteric ripening is essential for ripening.









Blockage of either ethylene biosynthesis or perception results in an inability of the fruit to

complete its ripening program.

Tomato is an excellent model for the study of ethylene' s involvement in fleshy fruit

development because of a relatively short life cycle, ease of genetic manipulation and a wealth of

genetic resources. In addition, the tomato genome is being sequenced, which will be a

tremendous resource to those working on this species.

Ethylene's involvement in ripening, fruit softening, volatile production and lycopene

accumulation has been well documented. Ethylene biosynthesis during tomato fruit development

generally goes through three distinct stages. There is a slight burst of synthesis after successful

pollination that then falls to low levels until the onset of climacteric ethylene production at the

onset of ripening. Ethylene production during immature fruit development has been termed

system I and is characterized as low level production which cannot be stimulated by treatment

with exogenous ethylene (Yang, 1987). Ethylene biosynthesis in mature fruit, referred to as

system II, is autocatalytic, meaning it can induce its own synthesis (Yang, 1987). The induction

of ethylene synthesis at the onset of ripening is believed to be due to developmental induction of

an ethylene-inducible ACS (Barry et al., 2000; Nakatsuka et al., 1998).

Although immature tomato fruit do not produce significant levels of ethylene they do

respond to ethylene, but in a different manner to that of ripening fruit. This response manifests as

a change in gene expression but to a smaller set of genes to that of ripening fruits (Alba et al.,

2005). This difference in response suggests that there is developmental control of gene

expression in addition to that of ethylene. The developmental control of ethylene regulated genes

has been best characterized by research done on the E4 and E8 genes found in tomato.

Expression ofE4 is ethylene inducible throughout fruit development while E8 is only ethylene









inducible in ripening fruit (Lincoln et al. 1987, Wilkinson et al., 1995). Treatment of immature

fruit with ethylene induces a set of genes, proving a response to the hormone, but it does not

induce immediate ripening. However, that ethylene exposure does hasten the onset of ripening as

compared to untreated fruit of similar age, suggesting that the fruit can measure cumulative

ethylene exposure (Burg and Burg, 1962; Lyons and Pratt, 1964; McGlasson et al., 1975; Yang,

1987). Treatment of immature green tomato fruit with ethylene, or its analog propylene, could

reduce the time from anthesis to breaker by half that seen in non-treated controls (Lyons and

Pratt, 1964; McGlasson et al., 1975). The way fruits measure this ethylene exposure is unknown.

Along with temporal control of fruit ripening there is also a spatial aspect of control. Fruit do not

ripen evenly across the entire fruit, they begin to ripen at the basal end of the fruit and proceed

towards the calyx. Since ethylene is diffusible throughout the fruit, and accumulates to high

levels within the fruit, there appears to be a developmental control within individual fruit that

controls the spatial ripening of the fruit.

In addition to ethylene's role in fruit development it also plays an important part in

seedling emergence (Clark et al., 1999). During germination seedlings must be able to force their

way through any soil between them and a light source. When a seedling encounters a barrier in

the soil it often becomes slightly wounded which can induce ethylene production. Dark grown

seedlings, like those found underground, are often tall and spindly in the presence of air alone.

Upon exposure to ethylene its growth habit changes and exhibits growth that is referred to as the

"triple response." This response manifests as a shortening of both the hypocotyl and root, radial

thickening of the hypocotyl and an exaggeration of the apical hook. These changes allow the

seedling to push through any barriers without damaging the meristem. While this mechanism has

evolutionary importance, the ability to exploit this response has revolutionized the ethylene









biology field by allowing researchers to screen for mutants in ethylene biosynthesis and

signaling.

The Ethylene Receptor Family Arabidopsis and Tomato

Much of the initial ethylene perception and signal transduction research was done in

Arabidopsis thaliana and thus we have exploited the Arabidopsis system to identify the

orthologous genes in tomato. The Arabidopsis ethylene receptor ETR1 was the first

phytohormone receptor cloned in plants and was isolated from a mutagenized population that

was screened for plants deficient in the triple response (Bleecker et al. 1988; Guzman and Ecker

1990). Ethylene insensitive mutants grow tall and spindly even in the presence of ethylene while

constitutive ethylene response mutants will show a triple response in the absence of ethylene.

etrl-1 was isolated as an ethylene insensitive mutant in one of these screens and was later cloned

and shown to encode an ethylene receptor with homology to bacterial two-component sensors

(Chang et al. 1993). In subsequent work a total of five receptors were cloned from Arabidopsis.

The ethylene signal transduction pathway in Arabidopsis is believed to be relatively linear

but we are unsure if all of the elements have been identified. Epistatic analysis has allowed

researchers to putatively order the components starting with the receptors. The next component is

the Raf-like Ser/Thr protein kinase, CTR1, which has been shown to physically interact with the

receptors (Clark et al., 1998; Gao et al., 2003; Huang et al., 2003). CTR1 has significant

homology to MAPKKKs and although no MAPKK or MAPK have been found to be involved in

ethylene signal transduction, their involvement in this pathway cannot be ruled out. EIN2, a

protein showing homology to Nramp metal transporters, is the next member of the pathway. The

role and activity of this protein in the pathway is unknown but it is absolutely necessary since

knockouts show complete ethylene insensitivity in every assay tested. The end of the ethylene

signal transduction pathway is composed of the transcription factors EIN3 and ERF l. EIN3 loss-









of-function (LOF) mutants show partial ethylene insensitivity, which is probably due to

redundancy within a gene family containing at least three members (Chao et al., 1997). In the

absence of ethylene the EIN3 protein is targeted for degradation in the 26S proteasome by a pair

of F-box proteins, EIN3-binding factors 1/2 (EBF l/2). Upon ethylene binding this repression is

released and EIN3 binds to the promoter of ERF 1 activating its transcription and ERF 1 is

involved in regulating the transcription of ethylene responsive genes. ERF 1 over-expressors

show a slight constitutive ethylene response suggesting that there are other important players in

the transcriptional control of ethylene responsive genes (Berrocal-Lobo et al. 2002).

ETR1 was not only the first phytohormone receptor to be cloned in plants but was also the

first eukaryotic protein with homology to a histidine kinase (Chang et al. 1993). These receptors

are endoplasmic reticulum-localized proteins that have copper-mediated ethylene binding and are

present in vivo as dimers (Chen et al., 2002; Ma et al., 2006; Schaller and Bleecker 1995;

Schaller et al. 1995). The Arabidopsis receptor proteins (ETR1, ETR2, ERS1, ERS2 and EIN4)

can be separated into three structurally different regions.

The sensor domain is composed of three putative transmembrane (TM) sequences in ETR1

and ERS1 and four domains in ETR2, ERS2 and EIN4, with the first TM sequence representing

a cleavable ER-targeting peptide. These transmembrane sequences are where the copper-

mediated ethylene binding takes place. This region also contains all of the known mutations that

cause ethylene insensitivity, likely due to an inability to bind ethylene or transmit the signal

through a conformational change. The amino acids necessary for dimerization are present in this

region and homodimerization has been proven in vivo but heterodimerization has not been

demonstrated (Schaller et al. 1995).









The next domain present in this family is a region that shows homology to histidine

kinases (Dutta et al., 1999). Histidine kinase domains contain five highly conserved sub-

domains, N, G1, F, G2 and the histidine (H) that is autophosphorylated. The ETR1 and ERS 1

proteins contain all five of these sub-domains while the other three members lack at least one

sub-domain. The ETR1 protein is the only member of the family that exhibits HK activity in

vitro, but the conserved histidine is not necessary for protein function based on the ability for a

mutant lacking this residue to rescue a receptor mutant (Gamble et al. 1998; Gamble et al. 2002,

Wang et al. 2003). The other family members all exhibit Ser/Thr kinase activity based on in

vitro kinase assays (Moussatche and Klee, 2005). This lack of histidine kinase activity in these

family members fits well with the finding that most of the other family members do not contain

all of the conserved regions in the histidine domain. All kinase assays completed so far have

been done in vitro and there has been no kinase activity directly linked to ethylene signal

transduction in vivo.

The third and final domain found in these proteins is the receiver, located at the C-terminus

of the protein. This region shows homology to the output domains from bacterial two-component

sensors and contains an aspartate that is active in phosphorelay in these bacterial pathways. The

ERS 1 and ERS2 proteins lack this domain while the other family members contain it, suggesting

that it may play a role in some family member-specific functions.

Using sequence and exon/intron organization comparisons, ETR1 and ERS 1 have been

classified as Subfamily 1 receptors while ETR2, ERS2 and EIN4 have been classified Subfamily

2 receptors. Considering the degree of divergence within the family, there may be specific

functions for each of the family members. The evidence suggests that the receptors may not be

completely redundant, although most genetic evidence suggests functional overlap.









Mutant analysis of the Arabidopsis ethylene receptor family has allowed a better

understanding of the receptor' s role in transducing the ethylene signal. All of the initial receptor

mutants cloned were semidominant, insensitive mutants. Single gene LOF mutants have no

obvious phenotypes which is most likely due to functional redundancy within the family. Based

on all of the genetic data available the receptors appear to function as negative regulators of the

ethylene response (Hua and Meyerowitz 1998). The default state of the receptor is one in which

the receptor actively suppresses ethylene responses in the absence of the hormone and ethylene

binding removes this suppression. The double mutant etrl ers1 and triple or quadruple mutants

show constitutive ethylene responses even in the absence of increased ethylene biosynthesis

(Wang et al., 2003), presumably because basal ethylene levels are able to inactivate the

remaining receptors. This model suggests that a decrease in receptor content will increase

ethylene responsiveness while an increase in receptor levels will decrease tissue responsiveness.

This simplified model does not appear to tell the entire story because it presumes that all of the

receptors contribute equally to the signal and recent work has suggested this may not be true.

Overexpression of a Subfamily 2 member was unable to rescue the constitutive ethylene

response phenotype of the double Subfamily 1 mutant, suggesting some family member-specific

functions (Wang et al., 2003). Work done in our lab has found that the system in tomato may be

quite different from that of Arabidopsis.

The tomato ethylene receptor family is composed of six members, LeETR1-6 with LeETR3

corresponding to the NR gene (Fig. 1, Zhou et al. 1996a; Zhou et al. 1996b; Lashbrook et al.

1998; Tieman and Klee 1999). All receptor family members have been shown to bind ethylene

with the exception of LeETR6 because it was not available at the time of analysis (O'Malley et

al., 2005). The first of the tomato ethylene receptor genes to be cloned was NR. This gene was









isolated from a mutant that shows semidominant ethylene insensitivity which prevents floral

wilting and abscission, alters leaf senescence and prevents fruit ripening (Wilkinson et al. 1995).

The basic structures of the receptors are similar to those of the Arabidopsis family but within the

tomato family the sequences are quite divergent with less than 50% identity at the extremes

(Figure 1). The transmembrane domains show the highest levels of sequence similarity owing to

the importance of this domain in the transmission of the signal. LeETR1, 2 and NR have three

putative transmembrane domains while LeETR4, 5 and 6 have four putative transmembrane

domains. The NR protein is the only member of this family that lacks the C-terminal receiver

domain (Figure 1). LeETR4, 5 and 6 resemble the Subfamily 2 receptors found in Arabidopsis in

that they are missing at least one of the conserved sub-domains in the HK domain and contain

the fourth transmembrane sequence (Figure 1-1). Each of the receptors has a distinct expression

pattern throughout fruit development, with NR, ETR4 and ETR6 being ethylene inducible

(current work). NR and ETR4 are both pathogen inducible, with the increase in expression being

a function of the increase in ethylene production found during a disease response (Ciardi et al.,

2000). An increase in receptor expression is likely an important factor in reducing the amount of

damage that occurs as a result of this increase in ethylene production.

The basic model for ethylene response states that the receptors act as negative regulators of

ethylene response and that higher receptor expression reduces sensitivity and lower expression

increases sensitivity. This model explains why multiple gene knockouts in Arabidopsis show a

constitutive response. While much of the available data fit this model it does not address the

importance of ethylene dissociation from the receptor or protein turnover. The Kd of ethylene

dissociation was measured in yeast-expressed AtETR1 and was found to be approximately 12

hours. This is likely to be an underestimate since it did not factor in protein turnover (Schaller










and Bleeker, 1995). There is no evidence to suggest that ethylene is able to dissociate from the

receptor, suggesting this association may be permanent. Isoform-specific antibodies have been

generated for a number of the Arabidopsis receptors and the tomato NR protein but no work has

been done to study in vivo turnover rates or ethylene's effect on receptor turnover. This type of

evidence will be necessary to draw any conclusions about the receptor' s importance in a plant' s

response to ethylene.

The current model suggests that the only way that a plant can reduce its response to

ethylene is by synthesis of new receptors. Less receptor leads to more sensitive tissue and more

receptor leads to less sensitive tissue. Previous work has shown that the current data do fit the

receptor model. Plants overexpressing NR have been found to be less sensitive to ethylene in

triple response assays and pathogen studies (Ciardi et al. 2000). LeETR4 antisense lines with

significantly reduced expression show phenotypes consistent with a constitutive ethylene

response. Phenotypes of these lines include epinastic growth, premature flower senescence and

abscission and for fruit, a reduction in the time from anthesis to breaker and from breaker to red

ripe (Tieman et al 2000). The effect on time from anthesis to breaker is quite significant with a

decrease of as much as 11 days compared to WT controls. LeETR4 antisense lines also have an

altered response to pathogen infection because an increase in ETR4 expression is one way in

which the plant reduces the amount of tissue damage. These lines display an accelerated

hypersensitive response in response to infection with an incompatible pathogen with greater

ethylene production and hastened expression of pathogenesis-related genes (Ciardi et al. 2000).

Antisense suppression of LeETR1, 2 and NR have no observable phenotype but this result is

likely due to redundancy within the system. The NR antisense lines show an unusual phenotype

in that with the reduction of NR expression levels there is a concomitant increase in ETR4










expression and this may explain why the NR antisense lines do not show any constitutive

ethylene response phenotypes. This phenomenon has been termed functional compensation and

appears to be a built-in system that allows the increase in expression of one family member when

another has been reduced (Tieman et al 2000).

The expression level of each of the tomato receptors has been monitored in response to

multiple ethylene-related phenomenons and at least one receptor is up-regulated in each of the

responses. On the other hand, a reduction in receptor expression has never been seen even

though it would increase the tissue's responsiveness to ethylene. So it seems that as soon as a

plant starts producing ethylene more receptor is produced thus attenuating the response. While

this may seem counterproductive it is not uncommon for a phytohormone in plants to be

attenuated as soon as it' s induced (Rashotte et al., 2003). Increased response to ethylene can be

very detrimental to plant tissues and since ethylene slows the growth of a plant, it could have

long term effects. The expression levels of all the receptors remain low throughout immature

fruit development and show a sharp increase at the onset of ripening, the time at which ethylene

production is at its highest. So it seems that at the point when ethylene is having its greatest

effect on plant development, receptor levels are at their very highest. It has been known for some

time that ethylene is intimately involved in the timing of fruit ripening and our research seeks a

better understanding of its role. Based on our previous research and that of others we believe that

if ethylene production rates during fruit development exceed the level of receptor synthesis then

there would be a de-repression of the system that would lead to an increase in sensitivity to the

hormone. At some point in development, when the fruit are ripening competent, sensitivity to

ethylene would rise past a threshold level where ripening could be initiated. Based on this model

the reduction of receptor levels in transgenic plants should reduce time to ripening and based on










our previous results this is true, ETR4 antisense plants ripen faster than controls (Tieman et al

2000).

Protein Degradation Through the 26S Proteasome

Protein degradation is an important regulatory mechanism that has been adopted by many

organisms. It has emerged as a mechanism of control as important as gene expression in

controlling cellular processes. Protein degradation has been implicated in the control of signaling

cascades, defense against viral infection, breakdown of cellular regulators and arguably its most

important role is the removal of abnormal proteins (Jabben et al. 1989, Glotzer et al. 1991,

Scheffner et al. 1993). The degradation of proteins generally falls into two classes: (1) relocation

of proteins to degradative organelles such as the lysosome or vacuole and (2) targeting the

proteins for degradation by the 26S proteasome. These two pathways are the principal modes of

degradation for both soluble and membrane bound proteins, albeit less is known about how

membrane-bound proteins are degraded. While relocation to degradative organelles is an

important type of protein degradation the focus of this review will be on the role of the 26S

proteasome in protein degradation.

The 26S proteasome is one of the most important proteolytic systems in plants and our

understanding of this system has grown considerably in the past decade. This system utilizes the

76-amino acid protein ubiquitin (Ub) as a reusable tag to target specific proteins to the multi-

subunit 26S proteasome for proteolysis. The attachment of Ub occurs at lysine residues on the

target protein and often occurs as a polyubiquitin chain of Ub monomers. Upon proteolysis in the

proteasome the Ub monomers are released to be used in another round of targeting.

Ubiquitination of target proteins occurs in a three-step conjugation cascade and can occur on

proteins in the cytoplasm, nucleus, integral membrane proteins and ER resident proteins that are

retro-translocated across the ER membrane.









The ubiquitin attachment cascade occurs in a three-step process designated El, E2 and E3.

The El component of the cascade is an ubiquitin-activating enzyme that binds ubiquitin at a

conserved cysteine. This enzyme is constitutively expressed and has little impact on target

specificity. The Arabidopsis genome encodes two El isoforms (Hatfield et al., 1997). The E2, or

ubiquitin-conjugating enzyme, is encoded by at least 37 family members in Arabidopsis

(Vierstra, 1996). This enzyme shuttles the ubiquitin moiety between the El and E3 proteins

(Pickart, 2001). The size of this family suggests that different E2s may be involved in regulating

specific pathways, although no specific functions have been assigned to any plant E2s. The

specificity of individual E2s likely occurs through their interaction with specific E3s. In addition,

the E2s are not all specific to ubiquitin but are also used for conjugating ubiquitin-like proteins

including NEDD, RUB and SUMO (Li et al., 2006). The E3, or ubiquitin-protein ligases, is the

component of the cascade that specifically recognizes proteins for ubiquitination. Because of the

specificity of this protein/complex it is encoded by several large families of genes, with more

than 1300 members in Arabidopsis (Vierstra, 2003). Four different types of E3 ligases have been

identified in plants: HECT, RING/U-box, SCF and APC (Smalle and Vierstra, 2004). HECT E3

ligases are composed of a large single polypeptide (often >100kDa), with seven family members

present in the Arabidopsis genome (Downes et al., 2003). Little is known of the functions of

plant HECTs, although one is known to be important for trichome development. Like the HECT

family, each RING/U-box family member is a single polypeptide that acts to bring together the

E2-Ub and target substrate. This group of proteins is each encoded by a large family of proteins

with 480 RING finger-containing and 64 U-box containing proteins, respectively, in Arabidopsis

(Azevedo et al., 2001; Kosarev et al., 2002). This type of E3 ligase has been implicated in a

diverse number of cellular processes in plants, including, auxin signaling, photomorphogenesis,









self incompatibility and removal of abnormal proteins (Smalle and Vierstra, 2004). The SCF

type of E3 ligases are composed of a complex of four different polypeptides. This type of E3

ligase acts in a similar manner to that of RING/U-box proteins in that they bring together the E2-

Ub and the target substrate. Plants have the ability to synthesize a vast number of SCF type E3

ligases. The Arabidopsis genome contains two RBX1 subunits, five cullin subunits, 21 SKP-like

proteins and almost 700 F-box proteins (Farras et al., 2001; Gagne et al., 2002; Shen et al., 2002)

.The F-box proteins provide the target specificity for this complex and constitute one the largest

gene superfamilies in the Arabidopsis genome. The APC type of E3 ligases is the most complex

type of E3, being composed of eleven subunits. Most of these subunits are encoded by single

genes in Arabidopsis and thus it is likely that they only form a small number of APC type E3s

(Capron et al., 2003). The APC was first identified as being important for the regulation of

mitosis through degradation of mitotic cyclins in yeast; it has been subsequently shown to have a

similar function in plant cells (Blilou et al., 2002).

The 26S proteasome is an ATP-dependent proteolytic complex that is composed of 31

subunits organized in two maj or subcomplexes. The 20S core protease (CP) is the portion of the

complex that houses the proteolytic activity, alone it is an ATP- and Ub-independent protease.

The CP has hydrolyzing, trypsin-like and chymotrypsin-like activity allowing it to degrade a

broad range of peptide bonds (Voges et al., 1999). The 19S regulatory particle (RP) can bind to

both ends of the CP and is the portion of the complex that recognizes the Ubs attached to

targeted proteins (Voges et al., 1999). The RP performs a number of additional functions

including unfolding the target protein, Ub removal, opening the gate to the CP core and directing

the target protein into the CP lumen (Smalle and Vierstra, 2004). Regulation of the activity and

specificity of the proteasome is thought to be affected by a number of factors including









association with additional proteins and substitutions or modifications to complex subunits. The

Arabidopsis genome encodes two isoforms of nearly all proteasome subunits. Transcriptional

control of the complex subunits in yeast is facilitated by a single transcription factor, Rpn4, that

is negatively regulated at the protein level by the 26S proteasome itself.

The role of the 26S proteasome in regulating many signal transduction pathways has been

confirmed in plants. The proteasome has been implicated in the action of all plant hormones. In

addition, it is important for a plant's response to both abiotic and biotic stimuli. Its role in auxin

and ethylene signaling are arguably the best characterized roles in hormone signaling. The auxin

signal transduction pathway is negatively regulated by a family of proteins (AUX/IAAs) that

bind and inhibit the functions of a family of transcription factors, the auxin response factors

(ARFs). Upon auxin binding the AUX/IAAs are targeted for degradation, thus releasing the

transcription factors to initiate expression of auxin responsive genes. The use of mutants and

proteasome inhibitors has confirmed this pathway and has facilitated the identification of the

auxin receptor as the F-Box protein, TIR1. TIR1, and TIR1-like proteins, specifically target the

AUX/IAAs for polyubiquitination and is an interesting example of the importance of the 26S

proteasome in regulating hormone pathways (Dharmasiri et al., 2005).

The ethylene signal transduction pathway is also regulated by the proteasome, which

modulates transcription factor activity/abundance. The F-Box proteins EBF l/2 target the EIN3,

and EIN3-like, transcription factors for degradation in the absence of ethylene. Upon ethylene

binding, this repression is removed and the transcription factor is able to activate transcription of

primary ethylene responsive genes. In the case of EBFl1/2, each has a different role in response to

ethylene, with EBF 1 being more important during early ethylene response and EBF2 more










important later during the response and in the resumption of growth after ethylene removal

(Binder et al., 2007).

The importance of the proteasome in response to abiotic stimuli is best characterized by its

role in regulating light signaling. PhyA, a red/far red absorbing photoreceptor, is rapidly

ubiquitinated and turned over following photoconversion to the Pfr form. In addition to the

regulation of photoreceptor protein levels, regulation of transcription factors is also performed by

the proteasome. In the absence of a light source the RING-E3 COP1 is present in the nucleus

where it targets a number of transcription factors for degradation. Upon illumination COP 1 is

removed from the nucleus and the transcription of light responsive genes occurs. These examples

represent an extremely small percentage of the pathways in which the proteasome has been

implicated and there are many more that have not been characterized.

While much is known about the degradation of soluble proteins by the proteasome,

relatively little is known about integral membrane protein degradation, especially in plants. What

is known about this pathway has been elucidated in yeast and to a lesser extent in humans. A

maj or regulatory and house keeping pathway that involves degradation of proteins in the

endoplasmic reticulum (ER) or integrated into the ER membrane and has been termed ER-

associated degradation (ERAD) has been uncovered. ERAD is responsible for targeting

misfolded ER proteins that are retro-translocated back across the ER membrane and also

targeting misfolded integral membrane proteins that are subsequently extracted from the

membrane and degraded by the proteasome (Meusser et al., 2005). It has been hypothesized that

different targeting complexes may be present in cells that target membrane proteins with

misfolded cytosolic domains, internal membrane domains or ER luminal domains (Carvalho et









al., 2006). These complexes contain a number of different subunits but each contains a

membrane-bound E3 ligase that attaches the ubiquitin monomers to the substrate.

A number of membrane-bound ERAD substrates have been identified but an interesting

example is that of the inositol 1,4,5-triphosphate (IP3) receptor. Activation of a G protein-

coupled receptor (GPCR) increases phospholipase C activity that generates diacylglycerol and

the second messenger IP3. IP3 mOves through the cytoplasm to IP3 receptors located in the ER

membrane, which activate channels that mobilize internal reserves of Ca2+. A persistent

activation of GPCRs leads to a down-regulation of IP3 receptors in order to prevent any

deleterious effects of continually elevated cytosolic Ca2+. This down regulation requires the 26S

proteasome and it has been shown that IP3 binding induces ubiquitination of the IP3 receptor,

leading to degradation (Zhu and Woj cikiewicz, 2000). In addition, a binding-defective mutant

receptor was shown to be resistant to ubiquitination and this resistance is not caused by the

removal of potential ubiquitination sites. It was hypothesized that ligand binding causes a

conformational change that exposes a signal leading to ubiquitination (Zhu and Woj cikiewicz,

2000).

The 26S proteasome has emerged as an essential part of a cell's repertoire for maintaining

cellular integrity and regulating a myriad of different pathways. Its involvement in both plant

development and responses to environmental stimuli implies an important evolutionary

advantage allowing these sessile organisms to flourish in many different environments.
















The Tomato ETR Ftamily
H N GI F G2 D

II~r~~r~-I~ II:::l~:::::::::


Response
regulator


GAF


LeETRI

LeETR2




L~eETR4

LeETR5


LeETR6 rl
Senior


Histidine
kinase


Figure 1-1.


Schematic representation of tomato ethylene receptor family. Black bars in sensor
domain represent putative transmembrane domains. Black boxes in histidine kinase
domain represent conserved sub-domains while black box in response regulator
represents conserved aspartate involved in phosphorelay. (Klee, 2004)









CHAPTER 2
ETHYLENE RECEPTOR DEGRADATION CONTROLS THE TIMING OF RIPENING IN
TOMATO

Introduction

The plant hormone ethylene is a gaseous molecule that regulates multiple processes

including germination, organ senescence, stress responses and fruit ripening (Abeles et al.,

1992). The role of ethylene in fruit ripening has been intensively studied in a number of species,

but most notably tomato, which has emerged as an important model for the study of fleshy fruit

development. Ethylene plays a critical role in determining the timing of ripening and thus

provides an attractive point to control fruit ripening through genetic modification.

Climacteric fruits such as tomato are characterized by an increase in respiration and a

concomitant increase in ethylene biosynthesis just prior to the initiation of ripening. Ethylene is

essential for normal fruit ripening in these species and blockage of either ethylene production or

perception leads to improper ripening. In tomato fruits, ethylene has profoundly different effects

depending on the stage of development. There is a distinct developmental switch that occurs

upon fruit maturation (Giovannoni, 2001). Although applied ethylene does not initiate ripening

in immature fruits, it does significantly hasten the onset of subsequent ripening (Yang, 1987); the

more ethylene to which an immature fruit is exposed, the earlier it ripens. Similar effects have

been observed in banana where Burg and Burg (1962) demonstrated that treatment of immature

green banana fruits shortened the time to ripening relative to untreated controls. The mechanism

by which fruits measure cumulative ethylene exposure is unknown.

Genetic analysis in tomato and Arabidopsis has shown that ethylene receptors act as

negative regulators of the ethylene response pathway (Hua and Meyerowitz, 1998; Tieman et al.,

2000). In the absence of the hormone, receptors actively suppress ethylene responses. Upon

ethylene binding, that suppression is removed and the response occurs. In tomato there are six









known ethylene receptors (LeETR1,2, 4-6 and NR) (Wilkinson et al, 1995; Zhou et al., 1996;

Lashbrook et al., 1998; Tieman and Klee, 1999). Functional analyses have indicated that some

Arabidopsis family members have a more important role in ethylene signaling than others.

Further, no single loss-of-function mutation has a maj or effect on ethylene responses, indicating

a degree of functional redundancy. However a completely different picture emerges in tomato

where loss of a single subfamily II receptor, LeETR4, results in increased ethylene sensitivity.

Antisense LeETR4 plants show phenotypes consistent with a constitutive ethylene response

including significantly earlier fruit ripening (Tieman et al., 2000). This mutant phenotype can be

restored to wild type by over-expression of the Subfamily I receptor, NR. No ethylene-associated

developmental effects have been observed in lines with reduced expression ofNR (Tieman et al.,

2000), LeETR1, LeETR2 or LeETR5 (Tieman and Klee, unpublished results).

The receptor signaling model states that the receptors are acting as negative regulators of

ethylene response. Experimentally it has been shown that reduction of receptor content increases

ethylene sensitivity (Hua and Meyerowitz, 1998; Tieman et al., 2000; Cancel and Larsen, 2002;

Hall and Bleecker, 2003) while increased receptor content has the opposite effect (Ciardi et al.,

2000). We have previously shown that NR and LeETR4 transcripts are up-regulated in ripening

fruits (Wilkinson et al., 1995; Tieman et al., 2000). Since fruit ripening is dependent upon

ethylene action, it seems illogical to increase receptor content and thus decrease ethylene

responses. To better understand the role of the tomato ethylene receptor family during fruit

development we have characterized the behavior of both the receptor RNAs and proteins during

fruit development. Contrary to the RNA data, protein blot analysis showed that receptor protein

levels are at their highest during immature fruit development and significantly decline at the

onset of ripening. This paradox is explained by observations that ethylene treatment induces a










rapid degradation of receptor proteins. Here, we present data indicating an important role for

LeETR4 and LeETR6 in modulating the timing of ripening. Reduced levels of these receptors

mediated by either antisense RNA or protein degradation results in earlier fruit ripening.

Results

A Subset of the Receptor Family Shows Ripening-associated Expression and Is Ethylene-
inducible in Fruit

Expression of all six ethylene receptor genes was assayed throughout fruit development

to assess stage-specific expression. Quantitative RT-PCR (qRT-PCR) analysis of each receptor

transcript showed low expression of all receptors throughout immature fruit development but

upon maturation there was a significant increase in NR, ETR4 and ETR6 transcripts (Fig. 2-1).

This ripening-associated increase in expression constituted a 10-fold increase in total receptor

mRNA content by the breaker stage. Since the receptors are negative regulators of ethylene

responses, the observed increases in mRNA levels during an ethylene-dependent process seems

counter-intuitive as an increase in receptors would make the fruit less sensitive to ethylene.

Ripening-associated gene expression can be the consequence of increased ethylene

production. Previous analysis has shown that ETR4 and NR are in fact ethylene-inducible in leaf

tissue (Ciardi et al., 2000). To determine if the receptor gene family is regulated by ethylene in

fruit tissue, individual fruits were treated with 50 ppm ethylene for 15 h. Expression analysis of

each receptor showed a 9-, 10- and 7-fold increase in NR, ETR4 and ETR6, respectively (Fig. 2-

2). Expression of ETR1, ETR2 and ETR5 changed little in response to the ethylene treatment.

Based on this analysis it appears that expression of NR, ETR4 and ETR6 is the consequence of

the climacteric increase in ethylene production at the onset of ripening.









LeETR6 Antisense Lines Show Phenotypes Consistent with a Constitutive Ethylene
Response

Single gene knockouts of ethylene receptors in Arabidopsis show no obvious phenotypes

and only the subfamily I double mutant (Hall and Bleecker, 2003; Qu et al., 2007) or triple and

quadruple mutants (Hua and Meyerowitz, 1998) show any ethylene-related phenotypes. As

previously shown by Tieman et al. (2000) this is not the situation in tomato as lines having

significantly reduced LeETR4 expression show ethylene hypersensitive phenotypes. When

LeETR6 antisense lines were generated, we found similar phenotypes to those seen in LeETR4

antisense lines, including a reduction of time to ripening by as much as seven days (Table 2-1).

Additional ethylene-related phenotypes include epinastic leaf growth and premature flower

senescence (Fig. 2-3). These results indicate gene-specific reductions in expression of either

LeETR4 or LeETR6 but not the other four receptors (data not shown) results in a hypersensitivity

to ethylene, including premature fruit maturation and ripening.

Receptor Protein Levels Are Distinctly Different From Transcript Levels During Fruit
Development

A wealth of recent work has demonstrated that post-translational control is an important

component of hormone pathway regulation. In order to uncover any potential post-translational

regulation of ethylene receptors, antibodies against NR, ETR4 and ETR6 were produced. Tissues

were collected for a comprehensive study of mRNA and protein expression during fruit

development. Measurement of receptor mRNA expression showed an increase in transcript

levels at the onset of ripening and these levels often remained high until fruits were completely

red (Fig. 2-4A). Microsomal membranes were isolated to enrich for the receptor proteins and

were used for protein quantification. Analysis of protein levels throughout fruit development

revealed an unexpected result; levels were highest during immature fruit development and

significantly declined at the onset of ripening (Fig. 2-4B). Data from cy. Flora-Dade are










presented, although identical results were obtained in the Pearson and Micro-Tom cultivars. This

reduction in protein occurred despite increased RNA content (Fig. 2-4C). The results indicate

that RNA levels are not predictive of receptor protein content nor the signaling state of the tissue.

Rather, there must be an additional level of control of ethylene perception. Because the drop in

receptor content coincided with the onset of autocatalytic ethylene synthesis, we subsequently

examined whether ethylene binding induces receptor turnover.

Treatment of Leaf and Fruit Tissue with Ethylene Causes a Rapid Degradation of Receptor
Proteins That Likely Occurs Through a Proteasome-dependent Pathway

To determine whether ethylene binding induces receptor degradation, immature fruits and

vegetative tissues were exposed to exogenous ethylene. Ethylene treatment of fruits resulted in 4,

5 and 8-fold increases in NR, ETR4 and ETR6 mRNA, respectively (Fig. 2-5A). Concomitant

with this increase in transcripts there were reductions of 60%, 60% and 40% in NR, ETR4 and

ETR6 proteins, respectively, within 2 h and this reduction was sustained throughout the

treatment (Fig. 2-5B). Removal of ethylene after 8 h of treatment lowered transcripts to pre-

treatment levels but receptor proteins remained lower even 24 h after treatment ceased (Fig. 2-

5A). Ethylene-mediated receptor degradation was also observed in vegetative tissues. Treatment

of seedlings with 50 ppm ethylene for 2 h resulted in 10-, 5- and 13-fold increases in NR, ETR4

and ETR6 mRNA, respectively. Similar to the data collected from immature fruit there was 60%,

40% and 50% reduction in NR, ETR4 and ETR6 protein levels, respectively (Fig. 2-6B). Taken

together, the results indicate that ethylene exposure in both vegetative and reproductive tissues

results in an immediate drop in receptor protein levels that is independent of transcript levels.

The 26S proteasome-dependent degradation pathway has emerged as a key point of

regulation in many phytohormone signaling pathways (Guo and Ecker, 2003; Dill et al., 2004;

Gagne et al., 2004; Dharmasiri et. al., 2005; Kepinski and Leyser, 2005). To determine if this pathway









is responsible for the turnover of ethylene receptors, seedlings were treated with the proteasome

inhibitor MGl32 prior to ethylene treatment. Following ethylene treatment, levels of each

protein actually increased, likely because of ethylene-induced increases in

transcription/translation (Fig. 2-6B). Very little is known about mechanisms of ER-associated

protein degradation in any system (Meusser et al., 2006). Presumably ubiquitinated proteins are

rapidly extracted from the membrane and degraded by the cytoplasmic 26S proteasome complex.

We did not observe larger ubiquitinated forms of immuno-reactive receptors in the microsomal

membrane fractions. Even after several-fold concentration, no receptors could be detected in the

soluble fraction (data not shown). Nonetheless, the MGl32 results are consistent with a

ubiquitin-mediated receptor degradation.

In order to demonstrate that ethylene binding is necessary for degradation, seedlings were

pre-treated with the ethylene action inhibitor 1 -methylcyclopropene (1-MCP) prior to ethylene

treatment. 1-MCP is a competitive inhibitor of ethylene and its attachment to the receptor is

essentially irreversible (Sisler, 2006). If ethylene binding is essential for the degradation of the

receptor, 1-MCP should stabilize the protein. Pretreatment of tomato seedlings with 1-MCP

prevented the ethylene-induced receptor degradation (Fig. 2-6B) as well as the ethylene-induced

increase in mRNA (Fig. 2-6A), indicating that ethylene binding is essential for receptor

degradation. To further confirm that ethylene binding is necessary for protein degradation we

utilized the semi-dominant Nr mutant that has a greatly reduced ethylene response. The mutant

Nr protein is unable to bind ethylene when heterologously expressed in yeast (Klee and

Bleecker, unpublished data). Treatment of Nr seedlings with 50 ppm ethylene for 2 h caused a

50% and 62% decrease in ETR4 and ETR6 proteins (Fig. 2-6B), respectively, but caused

significantly less change in the level ofNR protein. Taken together, the results are consistent









with enhanced receptor degradation following ethylene binding. However, we cannot completely

exclude the existence of an ethylene-induced receptor degradation machinery.

Receptor Levels in Developing Fruit Determine the Timing of Ripening

To determine whether ethylene-induced receptor depletion is the cause of the early

ripening phenotype seen in ethylene treated fruit, immature fruits were exposed to ethylene while

still attached to the plant and then allowed to ripen. Protein and mRNA samples were collected

throughout the duration of the experiment to correlate lower protein levels with reduced time to

ripening. Treated fruits ripened on average three days prior to untreated fruits (Table 2-2).

Receptor protein levels were lowered upon treatment with ethylene at 15 days post anthesis

(DPA) and remained lower than untreated controls throughout fruit development, indicating that

lower receptor levels correlate with earlier ripening (Fig. 2-7). Transcript data show that the

fruits responded to the ethylene treatment and upon removal of the ethylene, transcripts returned

to pre-treatment levels (Fig. 2-8).

Discussion

Upon maturation, tomato fruits undergo a developmental transition that is defined by their

response to ethylene (Lincoln et al., 1987). A number of system 1 and/or system 2-associated

genes have been identified in fruits. The E4 and E8 genes are excellent examples with E4 being

ethylene inducible throughout fruit development (both in response to system 1 and system 2

ethylene) and E8 only being ethylene-inducible in mature fruit (system 2 specific). While much

is known concerning the role of ethylene during ripening its function during the immature phase

of fruit development is less well understood. When mature fruits are exposed to ethylene, a

ripening program is initiated. While treatment of immature fruits does not initiate ripening it

does hasten the onset of ripening; the more the fruit is exposed to ethylene, the earlier it ripens

(Burg and Burg, 1962; Yang, 1987). How the fruit measures cumulative ethylene exposure is not









known. We have provided evidence indicating a specialized role for two receptors, ETR4 and

ETR6, in modulating ethylene responses, including fruit maturation. Reduced level of these

receptors mediated by either antisense RNA or ethylene-mediated protein degradation results in

earlier fruit ripening. Ethylene exposure also resulted in a parallel depletion of the other

ethylene-inducible receptor protein, NR. Our results are consistent with a model in which

ethylene receptor content is a maj or determinant of when fruits initiate the ripening program.

Since the receptors are negative regulators of ethylene signaling, depletion would lead to a

progressive increase in hormone sensitivity. When a particular threshold sensitivity is reached,

ripening would commence. Alternatively, receptors may act as a brake on ripening initiation. It

must be noted that there are other elements independent of ethylene that also must be in place for

ripening to initiate; most notably the RIN transcription factor (Vrebalov et al., 2002).

Receptor gene expression is low and constitutive throughout immature fruit development

with little difference between any of the family members (Fig. 1). At the onset of ripening there

is an increase in expression of NR, ETR4 and ETR6 that results in a 10-fold increase in total

receptor mRNA content. In contrast to mRNA expression, protein levels are at their highest in

immature fruits and show a significant decrease at the onset of ripening and remain low (Fig. 2-

4B) as a consequence of ethylene exposure. Ethylene binding likely causes a conformational

change in the receptors that makes them susceptible to degradation. In this context it is

interesting to note the model of Arabidopsis receptor signaling presented by Wang et al. (2006).

These authors provide genetic evidence supportive of a transitional state in which a receptor

continues to actively suppress downstream ethylene responses after ethylene is bound. This

intermediate state subsequently transitions to a receptor-inactive state. Our results suggest that

the "transmitter-off' state may actually be receptor degradation. It would be most interesting to









determine whether the mutations that define this transition state stabilize the protein. This

receptor degradation is dependent upon the action of the 26S proteasome. At least in some cases,

ubiquitination is associated with phosphorylation state (Hochstrasser, 1996). Although the

ethylene receptors are considered to be ancestral histidine kinases, many do not possess histidine

kinase activity (Moussatche and Klee, 2004). However, all of the receptors are functional

kinases; those that do not have histidine kinase activity are serine kinases. In light of the

degradation of receptors following ethylene binding, it is possible that the phosphorylation state

of the receptor may mediate ubiquitin binding. Although ligand-induced receptor degradation has

not been reported for plant hormones, it has been observed in animals where growth hormone

(GH) signaling is mediated by receptor levels (Flores-Morales et al. 2006). The GH receptor, like

ethylene receptors, is a membrane-associated protein in which hormone binding also increases

ubiquitin-mediated turnover (Govers et al. 1999).

The ethylene receptor family in tomato, like Arabidopsis, is split into two groups with

LeETR1, LeETR2 and NR belonging to subfamily I and LeETR4-6 belonging to subfamily II. The

Arabidopsis results indicate that there is a distinct difference between subfamily I and II

members. With the exception of a subfamily I double mutant (etrlersl), single and double gene

knockouts in Arabidopsis show no obvious phenotypes. This is likely due to functional

redundancy within the gene family. Over-expression of a subfamily II member in an etrlers1

double mutant cannot rescue the ethylene-hypersensitive phenotype (Wang et al. 2003). In a

reciprocal experiment over-expression of a subfamily I member in a subfamily II triple mutant

was sufficient to rescue the ethylene response phenotype. Together these data indicate that the

subfamily I receptors are more important than the subfamily II receptors in determining

competency to respond to ethylene. The Arabidopsis paradigm does not hold for tomato (Fig. 2-










3, Tieman et al. 2000). Plants with reduced expression of either LeETR4 or LeETR6, both

subfamily II members, show phenotypes that are consistent with an exaggerated ethylene

response, including epinastic growth, premature flower senescence and early fruit ripening.

Over-expression of NR in a LeETR4 antisense line is able to rescue the ethylene response

phenotype, indicating functional redundancy between subfamily I and II members (Tieman et al.

2000). Apparently there is a large degree of plasticity within the ethylene signaling pathway and

different plants have adapted the signaling components as appropriate for their situation.

Plant hormones are involved in most developmental processes and are critical for abiotic

and biotic stress responses. Plants can regulate hormone action through synthesis, catabolism or

perception. We have shown that a significant part of the regulation of ethylene responses

involves ligand-mediated receptor degradation. Frequently ethylene responses, particularly those

related to stresses, are transitory. In order to shut down an ethylene response, synthesis of new

receptors is essential. Our results with ethylene exposure to immature fruits indicate that receptor

degradation is apparently an important level of developmental control. Our results also indicate

that conclusions concerning receptor functions based on RNA levels must be interpreted

cautiously. Whether ethylene-mediated receptor turnover and replenishment are important for

other ethylene-mediated processes remains to be determined.





HETR1 o ET2 2 NR o ET4 o ETR5 5 ETR6


5.0

4C.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0

0.5


10 DPA 20 DPA 30 DPA MG Breaker Turning Re~d


Figure 2-1


Ethylene receptor family mRNA levels during fruit development. qRT-PCR analysis
of each receptor transcript in fruit tissue from different stages of fruit development.
DPA, days post anthesis; MG, mature green; Breaker, first external color change;
Turning, ~30% red color. Expression levels are presented as percentage of total
RNA.








O Control II 1ppm Ethyle~ne


2.5 -

a 2.0 -


ETR5 ETR6


ETR1


ETR2


ETR4


Figure 2-2


Ethylene-inducibility of each receptor mRNA in 20 days post anthesis, immature
fruit. qRT-PCR analysis of expression of each receptor in response to 10ppm
ethylene as a percentage of total RNA (+SE).


.I





















Figure 2-3


Constitutive ethylene response phenotypes of LeETR6 antisense lines. Epinastic leaf
growth (A) and early flower senescence (B) of LeETR6 antisense lines. Equivalent
aged wild type flowers are shown for comparison (C).










LeETRfi









1 0 1.04 0.18 0.27 0.41 0.17


1_ m


LeETR4


WWTT WT WT
IMG Broaker Tuming Red


4AS-1 4AS-2
IMG IMS


Figure 2-4


Receptor gene expression and protein levels show distinct differences during fruit
development. qRT-PCR analysis of gene expression expressed as percentage of
total RNA (a SE) (A) and protein blot analysis (B) throughout fruit development in
L. esculentum cy. Flora-Dade (WT). Levels of RNA and protein are also shown for
independent LeETR4 (4AS-1, 4AS-2) and LeETR6 (6AS-1, 6AS-2) antisense lines.
Values below each receptor protein blot represent the amount of protein in each
lane relative to the IMG stage. BiP antibody was used as a loading control and used
to normalize protein values. C. Ratio of protein to mRNA. IMG: Immature green
stage. Protein quantification was determined by densitometric analysis of Western
blots using the NCBI software ImageJ.


A 301
2NR


a NR ...E TH4
10 0.25 042 0 .47 1 0 0.17 0.11 0.05 0 1 0D.DH
a-BIP" -BIP


._.
WT WT WT WT SAS-1 6AS-2
IMG BrunKer Turnlug Red NO IMD


WT WT WT WT
IMG Breaker Turning Red










HOh Zh 0 8h B 32h


T


a-NR



a-ETR4




a-ETR6


1.00


0.76


Figure 2-5


Ethylene binding induces degradation of receptors in detached immature fruits.
Fruits were exposed to 10 ppm ethylene for 8 h. 32h time point represents fruit that
were treated for 8 h and left in air for a further 24 h. qRT-PCR analysis of gene
expression (A) and protein blot analysis (B) of ethylene-treated immature fruits.
Values below protein blots represent the amount of protein in each lane relative to
the 0 h time point. BiP antibody was used as a loading control and used to
normalize protein values. Data represent the results of two independent experiments
(+SE).











A o Control H Ethylene MG 132 + Ethylene a MCP + Ethylene O Nr Control 0 Nr + Ethylene


1.6


S1.2




" 0.6


LeETR4


LeETR6G
T


a
r ar
a E
er
w fi g
+ w fl
o w
o +
a u L L
Z a

----


a.-NR



u-ETR4




o.-ETR6


1.00


0.73 1.28 0.94


1.00 0.89


1.00


0.64 1.45 1.07


1.00 0.50


1.00 0.62 1.93


0.94


1.00 0.38


Figure 2-6


Ethylene binding induces degradation of receptor proteins in vegetative tissue. qRT-
PCR analysis of gene expression (A) and protein blot analysis (B) of L. esculentum
cy. Micro-Tom and Never-ripe (Nr) seedlings after treatment with 50 ppm ethylene
for 2 h. Data represent the results of two independent experiments (+SE). Values
below protein blots represent the amount of protein in each lane relative to the 0 h
time point. BiP antibody was used as a loading control and used to normalize
protein values.
















-*-ontol -0- 50ppm Ethylene


2.0
5

O
x15-
YI
.E
B

P10
g


a
0.5 -




2.5




20
e
o

r
x15
LU
.C
d

P 1.0
J
B

: as


C
[7-


LeETR4


9


'----d


LeETR6


C
O
~i
O
x 1,5
YI
c
B

P 1,0
g
~
1Y
rr



10






Figure 2-7


30

Days Post Anthesis


Ethylene treatment induces turnover of receptor leading to early ripening fruit. 15

days post anthesis (DPA) fruit were treated with 50 ppm ethylene while attached to

the plant. Relative protein expression of NR, ETR4 and ETR6 normalized to an

internal control, BiP. Values are plotted relative to the pretreatment protein level.












] Control 550ppmEthylene


] Control a 50ppm Ethyine


0 Control 150Dpm Ethiene


NR ETR4 ETR6
S20








15 DPA 25 DPA 35 DPA Breaker 15 DPA 25 DPA 35 DPA Breaker 15 DPA 25 DPA 35 DPA Breaker




Figure 2-8 Ethylene treatment induces expression of receptor mRNAs in attached fruit. qRT-
PCR analysis of expression of NR, ETR4 and ETR6 in response to 50ppm ethylene
as a percentage of total RNA (+SE).













Table 2-1 Days from anthesis to breaker of LeETR6 antisense lines
Line Days % Reduction LeETR6 mRNA
WT 43.33 f 0.71
LeETR6AS-1 38.42 f 0.90* 85.1 f 2.4
LeETR6AS-2 37.00 & 1.46* 75.6 f 6.4
LeETR6AS-3 35.83 f 0.78* 72.8 &5.8
Values represent mean of at least fifteen fruit for each line.
*p-value<0.001 based on Student's t-test.





Table 2-2 Days from anthesis to breaker of ethylene treated Microtom fruit
Treatment Days
-Ethylene 45.33 f1.41
+ Ethylene 41.20 f 0.80*
Values represent mean of at least ten fruit for each treatment. Experiment repeated with similar results.
*p-value<0.05 based on Student's t-test.









CHAPTER 3
FRUIT-SPECIFIC SUPPRESSION OF THE ETHYLENE RECEPTOR LEETR4 RESULTS IN
EARLY RIPENING FRUIT

Introduction

Tomato is the most economically important vegetable crop grown in the USA. Worldwide,

~70 million metric tons are produced each year. Short growing seasons in higher latitudes often

reduce the number of cultivars a grower can use in outdoor cultivation. One mechanism to

circumvent climate-related limitations is to grow early-maturing varieties. This offers a distinct

advantage to growers, because the first fruit to market in a season can garner a higher price. As

our knowledge of the molecular control of fruit ripening expands, biotechnology can provide

useful tools for generating early ripening cultivars. While much effort has focused on delayed

ripening, particularly as it relates to the ripening hormone ethylene, opportunities to hasten fruit

development have been relatively neglected. We have developed a tissue-specific approach to

enhance ethylene responses in tomato fruits by depletion of an ethylene receptor. Transgenic

fruits mature 5-7 days earlier than controls with no deleterious effects on yield, fruit size or

quality. This technology should be applicable to any fruit whose ripening is dependent on

ethylene.

Ethylene is a phytohormone that controls or influences many aspects of plant growth and

development (Abeles, 1992). Many of the developmental processes controlled by ethylene such

as senescence, organ abscission and fruit ripening are critically important to agriculture. For

example, climacteric fruits, such as tomato, banana and apple, require an increase in ethylene

biosynthesis at maturity in order to ripen. Transgenic plants that are reduced in either synthesis

or perception of ethylene exhibit delayed ripening (Oeller et al., 1991; Klee et al., 1991;

Wilkinson et al., 1995; Hamilton et al., 1990). Conversely, it should be possible to speed up fruit

maturation by increasing synthesis or perception of ethylene. Indeed, it has been known for many










years that ethylene application to immature tomato fruits does cause earlier onset of ripening

(Yang, 1987). Because of the pleiotropic negative effects of excessive ethylene exposure on

plant growth, simply increasing ethylene synthesis is not practical. Here, we describe an

approach involving tissue specific depletion of an ethylene receptor resulting in early ripening

fruit.

Receptors function as negative regulators of the ethylene response pathway (Hua and

Meyerowitz, 1998; Tieman et al., 2000). In the absence of the hormone the receptor actively

suppresses ethylene responses and ethylene binding removes this suppression. In practical terms,

this means that ethylene sensitivity is inversely correlated with receptor levels; depletion of

receptors effectively increases ethylene sensitivity because there are fewer receptors to

inactivate. Recent work on the tomato ethylene receptor family has demonstrated that receptor

levels during fruit development determine the timing of ripening (Kevany et al., 2007). Protein

levels are at their highest during immature fruit development and significantly drop at the onset

of ripening, facilitating ethylene-mediated ripening processes. Ethylene treatment of immature

fruits causes receptor degradation and earlier fruit ripening (Kevany et al., 2007).

Results

LeETR4 RNAi Transgenic Plants Produce Early Ripening Fruit

Antisense-mediated reduction in either of two tomato ethylene receptors, LeETR4 or

LeETR6, results in premature ripening (Tieman et al., 2000; Kevany et al., 2007). However,

these plants are severely affected in many aspects of growth and it is not clear that the early

ripening is a direct effect of transgene expression. We postulated that fruit-specific suppression

of the LeETR4 receptor would result in early ripening without undesirable ethylene-related

effects. In order to test the hypothesis a strategy was developed to specifically reduce LeETR4

expression throughout fruit development. To achieve this goal we generated a construct









consisting of an LeETR4 RNAi inverted repeat sequence fused to the promoter of Tfmn7, a gene

that is expressed specifically in immature fruits (Santino et al., 1997). Transgenic plants were

generated by Agrobacterium-mediated transformation into the tomato cultivar Flora-Dade, a

large fruited variety developed for Florida fresh tomato production. Transgenic lines that showed

no vegetative expression of the silencing construct were identified and assayed in a greenhouse

for time from anthesis to breaker stage (the first visible signs of ripening) in two successive

seasons. Three lines that exhibited both a reduction in time from anthesis to breaker and a

reduction of LeETR4 transcript throughout fruit development were chosen for further

characterization. Transgenic lines began ripening between 5 and 7 days earlier than controls

(Figure 3-1). No significant effects were observed on time from breaker to fully ripe nor were

there differences in color of ripe fruits (data not shown). As expected, LeETR4 transcript levels

were reduced by as much as 73% in immature fruit and 95% in ripening fruit (Figure 3-2A).

While Tfm~n7 expression has been reported to be immature fruit-specific the RNAi effect persisted

into ripening fruit (Figure 3-2A). This gene-specific reduction in expression was not seen in non-

target tissues such as leaves (Figure 3-2A). Expression analysis of the other family members

showed no decrease in transcript levels in transgenic plants (Figure 3-3). Protein blot analysis

confirmed that ETR4 protein levels were correspondingly reduced at all stages of fruit

development relative to non-transgenic control fruit (Figure 3-2B).

Early Ripening Lines Show Altered Ripening Coordination

Performance of the transgenic plants was also assessed in the field using standard

commercial practices. Harvests were conducted on a weekly basis in which all fruit that had

begun to show external color development were picked and staged for their degree of ripeness.

Transgenic plants had more ripening fruit in the first harvest than the control plants and









transgenic lines were stripped of between 77% and 86% of their fruit within the first two harvests

(Figure 3-4).

Transgenic Fruits are Indistinguishable from Wild Type Fruits in Horticultural Traits

Early maturing varieties of fruits frequently lack the quality of slower ripening varieties.

To achieve maximum value it would be advantageous if early ripening fruits maintain the size,

yield and flavor qualities of later ripening cultivars. Altering the time to maturation could

potentially impact synthesis of sugars, acids and volatile compounds associated with flavor. In

addition, fruit size and yield could potentially be negatively affected by earlier maturation and

harvest. To address these questions, tests were performed to assess quality and yield attributes.

Analyses of yield and fruit size were conducted in both greenhouse and field- grown

plants. To assess yield, fruits were harvested at the onset of ripening and individually weighed.

Average fruit size for two of the transgenic lines was slightly lower than control fruit but this

difference was not statistically significant (Table 3-1 and Table 3-2). Total yield and the number

of fruit per plant were not affected by the presence of the transgene (Table 3-1 and data not

shown).

Tomato flavor is the sum of a complex interaction between taste and olfaction. Sugars

and organic acids stimulate taste receptors while a set of volatile organic compounds (VOCs)

stimulate olfactory receptors (Buttery et al., 1993; Buttery and Ling, 1993). In order to assess

potential effects on flavor, total soluble solids, citric acid, malic acid and the 16 most important

VOCs were measured (Table 3-1, Table 3-3 and Table 3-4). Similar results were obtained on

both field-grown and greenhouse-grown materials. Although a very few statistically significant

differences in citric acid and some VOCs were observed, they were not repeatable from season to

season. All of these differences are well within the range of observed season-to-season










variations. Therefore, we concluded that the transgenic and control fruits are essentially

equivalent.

Discussion

While the essential role of ethylene in mediating climacteric fruit ripening has been known

for many years, its role during immature fruit development is only now being elucidated.

Previous work has shown that ethylene treatment of immature tomatoes or bananas quantitatively

reduces the time to the onset of ripening (Burg and Burg, 1962; Lyons and Pratt, 1964;

McGlasson et al., 1975; Yang, 1987) but the mechanism by which fruits measure cumulative

ethylene exposure has remained unknown until now. We have identified a potential mechanism

by which plants use ethylene receptor levels to measure cumulative ethylene exposure (Kevany

et al., 2007). Ethylene binding triggers a ubiquitin-dependent receptor protein degradation. If

receptors are not replaced after ethylene-mediated degradation, as occurs in immature fruit

(Kevany et al., 2007), the fruit will become more sensitive to subsequent ethylene exposure and

ripen earlier. The precise, fruit specific targeting ofLeETR4, described here, validates the model.

These results define a critical role for LeETR4 in mediating ethylene responses. The special

importance of this and another subfamily 2 receptor, LeETR6, to ethylene responses (Kevany et

al., 2007) contrasts markedly with what is known about ethylene perception in Arabidopsis. In

Arabidopsis, no single loss-of-function receptor mutant has an obvious effect on ethylene

responses and the subfamily 1 receptors seem to have a more important role in ethylene signal

transduction (Wang et al., 2003). These results taken together with results described in Kevany et

al. (2007) more broadly demonstrate that plants have the capacity to regulate hormone responses

by modulating receptor levels.

Tissue-specific modulation of ethylene sensitivity in transgenic plants has resulted in fruits

with altered ripening without an agronomic penalty. A similar approach to precisely separate an









advantageous trait from pleitropic negative effects was employed by Davuluri et al., (2005) who

used fruit-specific suppression ofDET1, a photomorphogenesis regulatory gene, to increase both

carotenoid and flavonoid content in transgenic tomatoes. Previous work on DET1 had reported

increases in these phytochemicals in loss-of-function mutants but global suppression of DET1

led to a number of serious developmental defects that would prevent these plants from being

used commercially.

We present here a crop improvement that should provide significant value to producers.

Early season harvests of tomatoes and many other horticultural crops usually constitute a

substantial percentage of a season's profits. The first fruit picked can be sold at a premium

because supply is generally low and demand is high. We have generated transgenic lines in an

elite background that ripen up to a week earlier than their control (Figure 3-1). These lines have

none of the developmental defects associated with global receptor suppression (Tieman et al.,

2000; Kevany et al., 2007) because of fruit-specific suppression of the gene (Figure 3-2A). This

approach for engineering early ripening should be applicable to any climacteric fruit species.











55 -


50 -





u,40 -


35 -


E T4-RNAi ETR4-RNA i-2 ETR4-RNAi-3


Cont ro-l


Figure 3-1


Fruit-specific ETR4 RNAi Transgenic Lines Produce Early Ripening Fruit. (A) Days
from anthesis to breaker were measured by tagging open flowers and recording the
number of days until the first signs of color development. (B) Fruit from transgenic
lines are similar in shape and color to control fruit.












A 1.6


1.4 -1 a INtl4-KNAl-1I
0 ET4-RNAi-2
m 1.2 IIIET4-RNAi-3




X 0.6


;j 0.4-

0.2-


Leaf IMG Breaker Tuming Red








ETR4-RNi-1~

E TR4-R NAi-2 ITlilllj

E TR4-RNAi-3 ~IIII



Figure 3-2 Suppression of LeETR4 is Fruit-specific. (A) qRT-PCR analysis of ETR4 transcript
levels in leaf tissue and throughout fruit development in control and RNAi
transgenic lines. (B) Protein blot analysis of ETR4 protein levels in control and
transgenic lines. IMG, immature green; Breaker, first external color change;
Turning, ~30% red color.













































ETR4-RNAi Transgenic Plants Have Altered Ripening Coordination. Fruits showing
visible color development were harvested on a weekly basis. Values represent the
percent of total fruit harvested each week +SE.


CoGantro~l --eET4-RNAi-1 TE4-RNAi-2 + TEW4-NAl-3


80%~'

70%



50%~

40%,'

30%

20%O~

10%~


Week 1 W~ee~k 2 We~ek 3 W~eek 4


Cumulative Harvest as % of Total
Line Wreek 1
Control 7.,8%
ETR4-RZNAi-1 18.1% o
ETR4-RNAi-2 18.3%
ETR4-RN~Ai-3 22.3%


Week 2
49. 1.%
78 9~'
77.0% '
86..4%


Week 3
77.4'%
95.2%
95.8%
99.0%


Wi5eek 4
100.0%
100.0%
100.0%
100.0%


Figure 3-3











Table 3-1 Weight, yield, brix, citric acid and malic acid from field grown fruits
Weight Yield/Plant Citric Acid Malic Acid
Line (g) (n) (kg) (n) oBrix (n) (mg/gfw) (n) (mg/gfw) (n)
Control 135.513.1 218 4.5110.4 9 4.110.2 10 2.7610.04 5 0.2210.02 5
RNAi-1 130.713.9 185 4.5310.6 8 3.910.1 10 2.6310.06 5 0.2310.03 5
RNAi-2 131.413.1 262 4.4910.4 12 3.810.1 10 2.6910.14 5 0.2310.02 5
RNAi-3 142.914.1 137 4.5610.3 10 4.010.1 10 2.5710.16 5 0.2110.03 5
Table displays mean iSE. n=number of fruit examined, or plants in case of yield study.



Table 3-2 Weight, yield, brix, citric acid and malic acid from greenhouse grown fruits
Weight Yield/Plant Citric Acid Malic Acid
Line (g) (n) (kg) (n) oBrix (n) (mg/gfw) (n) (mg/gfw) (n)
Control 115.414.4 98 3.6810.2 3 4.210.1 10 3.1010.03 15 0.5010.03 15
RNAi-1 103.713.3 64 3.5810.5 2 4.210.1 10 2.9210.10 15 0.5210.04 15
RNAi-2 106.913.2 70 4.1310.2 2 3.810.1* 10 3.0110.04 15 0.4810.03 15
RNAi-3 118.914.4 142 4.6910.4 3 4.010.0 10 3.2410.03* 15 0.4810.03 15
Table displays mean iSE. n=number of fruit examined, or plants in case of yield study.
*Statistically significant p-value<0.05 based on Student's t-test


Table 3-3 Volatile organic compounds from field grown fruits


Compound
cis-3 -Hexenal
B-lonone
Hexanal
B-Damascenone
1-Peneten-3 -one
3 -Methylbutanal
trans-2-Hexenal
2-Isobutylthiazole
1-Nitro-2-phenylethane
trans-2-Heptenal
Phenylacetaldehyde
5-Methyl-5-hepten-2-one
cis-3 -Hexenol
2-Phenylethanol
3 -Methylbutanol
Methyl salicylate


Control
37.4816.48
0.05+0.01
83.38122.22
0.0210.00
0.4610.09
4.25+0.47
1.0610.17
4.8211.16
1.6010.33
0.1510.03
0.4510.06
3.4010.87
50.6218.52
1.8610.39
20.1613.27
0.1310.01


RNAi-1
50.7017.99
0.07+0.01
116.06119.78
0.0310.01
0.4810.07
4.1910.42
1.2710.29
5.7410.99
1.2010.43
0.1710.03
0.5010.1
3.8110.80
64.7813.63
1.8910.55
18.16+4.05
0.1910.05


RNAi-2
66.6914.76*
0.0610.01
166.23118.54*
0.0210.00
0.4410.02
4.4910.44
1.6310.06*
5.6610.65
1.9210.37
0.2010.04
0.5110.15
4.5710.86
69.1918.37
2.6110.67
17.1914.05
0.1710.05


RNAi-3
34.7615.53
0.0410.01
59.5114.08
0.0210.00
0.4010.04
4.6210.63
0.8610.08
3.7910.42
1.1210.40
0.1310.02
0.3710.01
3.0210.29
41.6014.54
1.4910.04
16.5613.40
0.1410.04


Values are ng g FW h and table displays mean iSE with n=6.
* Statistically significant p-value<0.05 based on Student's t test.











Table 3-4 Volatile organic compounds from greenhouse grown fruits


Compound
cis-3 -Hexenal
B-lonone
Hexanal
B-Damascenone
1-Peneten-3 -one
3 -Metlwlbutanal
trans-2-Hexenal
2-Isobutvlthiazole
1-Nitro-2-phenylethane
trans-2-Heptenal
Phendlacetaldelwde
5-Metlwl-5-hepten-2-one
cis-3 -Hexenol
2-Phem lethanol
3 -Metlwlbutanol
Methyl salievlate


Control
105.01126.92
0.07+0.01
97.50115.17
0.0210.01
0.4710.02
7.5111.00
2.1510.53
2.5510.54
0.07+0.01
0.25+0.07
0.2710.02
3.6610.63
53.7016.73
1.0410.29
47.15+3.01
0.17+0.06


RNAi-1
105.41129.49
0.08+0.03
131.17130.44
0.0210.01
0.5310.22
7.721.11
2.4410.67
2.3810.65
0.10+0.00
0.3610.13
0.2310.06
5.071.64
57.12111.99
1.2010.03
48.20+11.74
0.2310.07


RNAi-2
115.46119.79
0.1010.02
118.18123.15
0.0310.01
0.5910.15
8.0110.81
2.2610.50
2.3310.40
0.07+0.00
0.3310.06
0.3010.03
3.5110.49
50.5316.44
1.3210.19
43.5518.62
0.1110.03


RNAi-3
122.32150.31
0.0810.00
183.49111.45*
0.0210.00
0.3710.05
5.8210.43
2.6310.76
2.7010.57
0.10+0.01
0.2910.06
0.4210.04*
4.3411.48
59.9911.47
1.6710.03*
46.35111.81
0.3410.14


Values are ng g' FW 10 and table displays mean iSE with n=4.
* Statistically significant p-value<0.05 based on Student's t test.









CHAPTER 4
IDENTIFICATION OF QTLS THAT MODIFY TIME TO RIPENING AND RIPENING-
ASSOCIATED ETHYLENE PRODUCTION

Introduction

The use of wild germplasm has become an important method for crop improvement by

today's plant breeders. Genetic diversity in today's domesticated varieties is narrow and land

races that could provide traits necessary for crop improvement are being lost every year. The

development of introgression lines (ILs) that each contain a single chromosome segment

introgressed into an otherwise uniform background has allowed for the identification of many

monogenic traits and quantitative trait loci (QTLs) (Frary et al 2003; Doganlar and Tanksley

2000; Fridman et al 2002). In tomato, a number of introgression lines have been developed from

crosses with wild relatives, including L. pennellii (Eshed and Zamir 1995), L. hirsutum;

Monforte and Tanksley 2000), and L. peruvianum. These libraries are useful in identifying QTLs

because any phenotypic variation can be associated with the introgressed segment. The entire

library can be screened for a particular phenotype and individual lines can be isolated. Once

these lines are identified the introgressed segment can be further reduced into sub-ILs by

subsequent back crossing. This permits further refinement of the QTL location and potentially,

map-based cloning. ILs have been used to identify QTLs responsible for changes in yield, quality

and stress responses (Fridman et al 2004; Zamir 2001).

Tomato is the most economically important vegetable crop grown worldwide, providing

significant incentive for crop improvement research. Short growing seasons in higher latitudes

often reduce the number of varieties a grower can use or can force them to use greenhouses that

require a significant investment. Identification of loci that control the time it takes a fruit to reach

maturity could offer a tremendous opportunity for breeders. Early ripening loci could be

selectively bred into elite varieties that would be otherwise impossible to grow at higher









latitudes. In addition to traditional breeding transgenic approaches are being developed to

provide options for growers but here I will focus on the traditional method.

Regulation of ethylene biosynthesis at the molecular level is a poorly understood process.

Work done in Arabidopsis led to the identification of proteins that regulate the activity of the key

biosynthetic enzyme ACC synthase (ACS). The ETO1 and ETO-like proteins posttranslationally

regulate the stability of ACS by targeting it to the 26S proteasome. Loss-of-function and

dominant gain-of-functions mutants were isolated by screening mutagenized populations for

plants exhibiting a triple response in the absence of exogenous ethylene. Where ctrl mutants

exhibit this phenotype because of loss of signaling capability in the absence of ethylene, eto

mutants produce significantly more ethylene than controls because of enhanced ACS stability.

An obvious difference between Arabidopsis and tomato is that tomato fruit go through a

developmental switch that results in a significant increase in ethylene production. While we

understand that a developmental switch occurs that triggers the expression of particular ACS and

ACO isoforms an understanding of the regulation of the expression and activity of these enzymes

is lacking in tomato.

While we will assess early ripening and increased ethylene biosynthesis separately, there is

a significant possibility that a locus that leads to increased ethylene production could also lead to

early ripening. While increased ethylene production leading to early ripening could prove to be

easier to understand it could prove less useful in terms of breeding early ripening lines because

excessive ethylene production could lead to undesirable effects.

In an effort to identify QTLs associated with ripening modification and ethylene

production, a screen was performed on a set of ILs derived from the L. hirsutum genome. L.

hirsutum was chosen to conduct this experiment because it is an unusual relative of the cultivated









tomato. L. hirsutum produces small green fruit that never show any signs of ripening such as

softening, carotenoid accumulation or volatile production. Maturity can only be assayed by the

measurement of ethylene production rates and fruit do not reach maturity until approximately 70

days post-anthesis (Grumet et al, 1981). Once fruit reach maturity, there is a sharp increase in

ethylene production that peaks at between 2000-4000 uL kg-l day- roughly ten times that of

cultivated tomato varieties. These unusual phenotypes suggest the presence of loci that may

influence ripening and ethylene synthesis in unusual ways.

Results

In a preliminary experiment ethylene emissions of fruit grown in the field were measured

and a line (LA 3945) that produces up to four times the amount of ethylene produced by the

control at the red stage was identified. This phenotype was confirmed with greenhouse-grown

fruit (Figure 4-1). In an effort to conduct a more comprehensive analysis, 35 different lines, each

containing a different segment of the L. hirsutum genome, along with both isogenic parents were

grown in triplicate in the greenhouse. A randomized complete block design was utilized as an

experimental design in order to control for variation within the greenhouse. Flowers of each line

were tagged at anthesis to determine the number of days from anthesis to breaker (Figure 4-2).

Statistical analysis using Dunnett' s test identified three lines (3935, 3958 and 3968) that had

reduced time to ripening with a p-value<0.05. Fruit from the same plants were collected at the

breaker and red ripe stages to measure ethylene emission rates (Figure 4-3 & 4-4). Statistical

analysis using Dunnett' s t test identified four (3922, 393 5, 3944 and 4005) and three lines (3922,

3934 and 3969) with increased ethylene emission in breaker and red fruit, respectively. In

addition to the lines identified by statistical analysis we included a few lines for each trait

assayed that were close to the p-value<0.05 cut-off. Figure 4-5 is a representation of the

approximate locations of each introgressed segment in the tomato genome. This map was used as









a guide to develop a library of markers from the sequence information available in the SOL

Genomics Network database. A postdoctoral researcher in our lab has taken over this project and

will use the markers to fine map the exact locations of these pieces. The map also contains the

locations of all known ethylene receptors and ACC-synthase (ACS) isoforms because they are

possible candidates for these QTLs.

In order to replicate the results of the first experiment we grew the identified lines in a

greenhouse to assess the ripening trait and in the field to assess ethylene emission. Again, each

line was grown in triplicate along with both isogenic parents. In addition to the selected lines

additional lines from the collection that overlap the introgressed L. hirsutum segments were

included in the analysis to better map the location of each QTL. Greenhouse data for the ripening

lines is presented in Figure 4-6. Of the original lines selected, only those that had previously

showed a statistically significant change in ripening (3935, 3958 and 3968) repeated a reduction

that was again statistically significant. The other lines (3921, 3955 and 3964) were assayed again

because they were close to making our cutoff of 0.05. The fact that these lines did not show a

significant reduction in the following season strongly supports our confidence in the statistical

analysis of the data from the first season. Interestingly line 3921 did not itself show a reduction

in the second season but three overlapping lines 3922, 3923 and 3924 were found to be

significantly lower than the control. Due to the labor intensive nature of measuring ripening time,

only lines 3935, 3958 and 3968 will be further characterized.

In order to gain a better understanding of the increase in ethylene emission, field grown

fluit were harvested at four stages and ethylene emissions were measured. Figure 4-7 shows the

complexity of the trait, with some lines being statistically higher at some stages and not at others.

IL 3922 and two overlapping IEs had a higher level of ethylene emission during early ripening










(i.e. breaker) but returned to WT levels by the red stage. IL 3935 and its overlapping lines were

low early in ripening but statistically higher at the pink and red stages. In accordance with early

studies, line 3945 had the highest ethylene emissions of any of the lines tested, with more than 2-

fold higher rates at the pink stage. Additional lines overlapping 3945 had higher ethylene

emission at each ripening stage tested. IL 3969 had higher emission rates at the breaker and pink

stages but returned to WT levels by the red stage. The complex nature of this phenotype has

made analysis more difficult. While we were principally interested in ethylene emissions in fruit

we were interested to determine if the increases were limited to the fruit. Ethylene emissions of

young leaves were assayed (Figure 4-8). No statistically significant differences were seen for any

of the lines suggesting that the increases were confined to ripening fruit tissue. Interestingly the

L. hirsutunt isogenic parent (1777) showed the lowest leaf ethylene emissions and this was

confirmed by repeating this experiment. Due to the fact that many of the introgressed pieces

were quite large, backcrosses were made to the isogenic L. esculentunt parent for all lines that

repeated in the second season. All of the data displayed from the second season (Figures 4-6 and

Figure 4-7) were collected and analyzed by Dr. Valeriano Dal Cin, a postdoctoral researcher in

our lab. He has also isolated homozygous recombinants from the backcrosses performed during

the second season and is currently analyzing the progeny of those recombinants.

Due to our interest in receptor function we were intrigued to see that the IL that

consistently emitted more ethylene (3945) contains a chromosomal segment that potentially

encodes the L. hirsutunt ortholog of LeETR4. In order to determine which allele is present in

3945, the intron of this gene from the 3945 line was cloned and compared to both the L.

esculentunt and L. hirsutunt sequences (Figure 4-9). The IL was confirmed to contain the L.

hirsutunt allele. In order to understand whether this allele showed any differential expression I










performed qRT-PCR on RNA collected from vegetative and reproductive tissues (Figure 4-10).

While there are some differences at particular stages the basic trend of expression is similar

between the control and IL. In addition to an analysis of developmental expression both parents

and 3945 seedlings were exposed to ethylene in order to determine if the ethylene inducibility of

the L. hirsutum allele was altered (Figure 4-11). No significant difference was observed between

any of the genotypes. An additional time to ripening experiment showed no statistical difference

between the L. esculentum parent and 3945. Subsequent marker analysis of the introgression

region at 3945 found that both 3944 and 4005 overlap 3945 and all three have increased ethylene

emission. This region of the introgressed segment is not the area that contains LhETR4. These

additional data suggest that the L. hirsutum allele of LeETR4 is likely not the cause of the

increased ethylene phenotype.

Discussion

Marker assisted breeding is an important technique used to address fundamental

problems in plant biology and crop improvement. It is particularly important with the current

public attitudes toward genetically modified organisms. Breeders are increasingly going to

require the identification of markers that are linked to agronomically important traits. A close

relationship between researchers and breeders will allow for efficient introduction of newly

identified traits into existing varieties.

While a number of different resources are available for mapping traits of interest in

tomato, the development of introgression populations using different wild relatives has greatly

enhanced this process. These populations facilitate sorting an entire genome down to a small,

known segment that can be assayed for a particular phenotype. In addition, once the tomato

genome sequencing proj ect is complete it will be relatively straightforward to screen a

population and then search a particular genomic location for candidate genes.









While a great deal of research has been done on the ethylene biosynthetic pathway the

only proteins that have been identified that act to modulate this pathway are the ETO proteins.

This modulation is accomplished by inhibiting activity of the ACS protein and targeting it for

degradation via the 26S proteasome. Identification of additional regulators of ethylene synthesis

will broaden our understanding of the factors affecting synthesis, whether they be positive or

negative. We present here a strategy to identify QTLs that control ripening-associated ethylene

production by screening L. hirsutum IEs. The choice of the L. hirsutum introgression population

was originally made because of the unusual ability of this species to produce high levels of

ethylene during ripening (Grumet et al., 1981). We have identified six introgression lines that

produce significantly more ethylene during at least one stage of fruit ripening (Figures 4-3, 4-4

and 4-7). This increased ethylene emission is restricted to ripening fruit as no difference was

seen in leaf ethylene biosynthesis rates in selected introgression lines (Figure 4-8).

A significant amount of research surrounding fruit ripening has been completed in the past

century but we still do not understand how fruits regulate ripening at the molecular level. In

climacteric fruits it is clear that there are two important levels of regulation, developmental cues

and ethylene synthesis. Work done on tomato in the past five years has begun to unravel this

phenomenon at both levels with the identification of the RIN and NOR genes as well as the

ethylene receptor family (Vrebalov et al., 2002; Kevany et al., 2007). While these findings are

important steps, fruit development is a complex and there are likely many additional regulators

of this process. Due to the unusual nature of ripening, or lack of ripening, in the wild tomato

species L. hirsutum, we hypothesized it may allow for the identification of some additional

factors regulating fruit development and specifically the onset of ripening. In addition to the ILs

with increased ethylene emission our screen identified three lines that showed significantly









reduced time from anthesis to breaker that was confirmed in two experiments (Figures 4-2 and 4-

6).

Future work will involve finer mapping of the L. hirsutum QTLs. Backcrosses have been

performed for each ripening and ethylene line and recombinants are being identified by a post-

doctoral researcher in our lab. A library of cleaved amplified polymorphic sequence (CAPS)

markers has been generated to precisely map each introgressed segment. Eventually these loci

will be cloned by a map-based approach or by use of the tomato genome sequence and will

increase our knowledge of both these processes.










H Control( B 3945


30 -

25 -

20 -

15-
15 -

5-


3rd Exp~eriment


1 st Experiment


2nd Experim~ent


Figure 4-1


Ethylene emissions of fully ripe fruit from L. hirsutum IL 3945. Red fruit were
sealed in 500 mL jars for ~1 h and ethylene emission was measured by gas
chromatography. Values represent mean +SE. *Statistically significant values p-
value<0.05 based on Dunnett' s t test.













55


50


S45

40


35




O


Figure 4-2


Days from anthesis to breaker of tagged fruits from L. hirsutum IEs. Open flowers
were tagged at anthesis and the number of days to breaker were recorded.
*Statistically significant values p-value<0.05 based on Student' s t test.





IIIII I I I I I I I I I II III I 1 111111 11 I IIII I
m(o N~~~ ~~~~~m~~~~Nm~~m N~~~~~~~~~
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~000
NN~~Nmmmmmmbbbbb~~~~~~~~~~~~~000
mmmmommmmmmmomommmmmmommmmmmmom~~~


30.0


S25.0

S20.0

E 15.0




5.0


13r


Figure 4-3


Ethylene emissions of breaker fruit from L. hirsutum I~s. Breaker fruit were sealed
in 500 mL jars for ~1 h and ethylene emission was measured by gas
chromatography. *Statistically significant values p-value<0.05 based on Dunnett's t
test.
























- .t~N~~m ~ ~m~~N~ ~ ~ ~
~N m m~t t t~~~~~~~0
-~~~~~~~~~~~~~~~~0
-mmm m m m o m m m m m m m m m~


40.0


E 35.0

r= 30.0

r=25.0

*u 20.0
E

o 15.0


Figure 4-4


Ethylene emissions of fully ripe fruit from L. hirsutum ILs. Red fruit were sealed in
500 mL jars for ~1 h and ethylene emission was measured by gas chromatography.
*Statistically significant values p-value<0.05 based on Dunnett' s t test.












1 2


3 4 5


- TG178
- TG590
- ETR4
STG153


- TG33
- ACS7A~B
- CT140




TG353


LA3022(E)



TG620


- TG564
- ACS8
- CT171


- TG379
- ACS4
- TG318
- ACS5
- CT118A


STG59

- ACS2

STGB3


LA3034(E)






LA3035(E+R)


STG176
- ACS6
C T187A








- CT265
- ACS1A~B

- TG480


- TG180
LA3688(R)
- CT211
SETR1
STG3BO

LA3000(E)


- TG296
- TG350


- TG252
SETR2
STG202


Figure 4-5


Genomic map showing locations of introgressed regions that contain putative
ripening-associated QTLs. Black regions represent locations of QTLs controlling
ripening phenotype. White regions represent locations of QTLs controlling
increased ethylene emission phenotype. Dark grey regions represent portions of
introgressed pieces that are ambiguous based on original mapping done with
population. Map also contains locations of all known ethylene receptors and ACC
synthase isoforms.


LA3045(E)
STG164
S- TG292

SLA4005(E)
LA3844(E)


-TG557
- ETR5

- TG651
LA3058(R)

- TG286


-TG390

SETR6
- CT198
LA3058(R)
- CT112


LA3845(E)

STG241
- TG233











45-


40-


a1 35-


30-


25-






Figure 4-6


CT 3921 3922 3923 3924 3934 3935 3936 3937 3955 3958 396 968


Days from anthesis to breaker of tagged fruits from L. hirsutum IEs. Open flowers
were tagged at anthesis and the number of days to breaker were recorded.
*Statistically significant values p-value<0.05 based on Student's t test. CT, control.















-a Control
-a- 3944
-a-3945
-- 3946
--4005









-* Control
-= 3969












Green Breaker Pink Red


30

S25

20

:15







30

S25

20



15


-*-Contro
-e 3934
- 3935
-c 3937


Green Breaker Pink Red


Figure 4-7


Ethylene emissions of fruits from field-grown L. hirsutum ILs. Fruit at indicated

stages were sealed in 500 mL for ~1 h and ethylene emission was measured by gas

chromatography. Breaker, first external signs of ripening; pink, ~70% color

development.













E~3.5-

S3.0-




2.0-






0.5-





Figure 4-8


CT 3921 3922 3923 3924 3934 3935 3936 3937 3944 3945 3946 3955 3958 3964 3968 4005 1777


Ethylene emissions of leaves from L. hirsutum IEs. Young leaves were harvested
and immediately placed in 5 mL plastic tubes, but were left uncapped for ~1/2 h to
permit release of wound-induced ethylene. After sealing, tubes were left for ~ 3 h
and then ethylene emission was analyzed by gas chromatography. CT, control.










L.esoulentum
IL3945
L.hirsutum

L.esoulentum
IL3945
L.hirsutum

L.esoulentum
IL3945
L.hirsutum



L.esoulentum
IL3945
L.hirsutum

L.esculentum
IL3945
L.hirsutum

L.esoulentum
IL3945
L.hirsutum

L.esculentum
IL3945
L.hirsutum

L.esoulentum
IL3945
L.hirsutum

L.esculentum
IL3945
L.hirsutum

L.esoulentum
IL3945
L.hirsutum

L.esoulentum
IL3945
L.hirsutum

L.esoulentum
IL3945
L.hirsutum

L.esculentum
IL3945
L.hirsutum

L.esoulentum
IL3945
L.hirsutum

L.esculentum
IL3945
L.hirsutum



Figure 4-9 ~


899 F.bJ.bib
899 e.Y.;iY

NTucleotide alignment of ETR4 genomic sequence. Sequence isolated from L.
esculentum, L. hirsutum and IL 3945. Alignment was done using ClustalW and
presented using Shade Box software.


~i~i~mf~


~i~E~m~s~






~l~f3~W"I~E~S~


~S~S~


~,~i~Siiii~ii~i~i~~


~I~Si~Tii~~


~S~iffS~i~SiiJi~i~


~5iZl~tS~S~S~


~j~i;il~Sj~j~~


~5i~.~ii~~


~S~l~i~Si~J~


Eii~IJi~i~


F~i~E~


I~iii~E~E~i~











SControl a LA3945


1.2




S0.8-




2 0.4-

E0.2-


Le


Figure 4-10


Red


MG Breaker Tuming


mRNA expression of LeETR4 in WT and the L. hirsutum IL 3945. qRT-PCR
analysis of transcript levels in leaf and reproductive tissues. Values represent
mean +SE and presented as % of total RNA.


af Bud Flowner 10 Day 20 Day 30 Day











Ig Control n 10ppm Ethylene


,c 1.8
O 1.6-
~1 .4-

1





<( 0.4-
ce 0.2-





Figure 4-11


I


L. eisculentum


3945


L. hAirsutum


mRNA expression of LeETR4 in seedlings of WT, L. hirsutum and IL 3945. qRT-
PCR analysis of transcript levels in leaf tissue treated with 10ppm ethylene.
Values represent mean +SE and presented as % of total RNA.









CHAPTER 5
CONCLUSION

The solanaceous species Lycopersicon esculentum, tomato, has emerged as the model for

studying fleshy fruit development. Because tomato is a climacteric species it is also the species

of choice for studying ethylene's involvement in fruit development. Ethylene is essential for

normal fruit ripening in these species and blockage of either ethylene production or perception

leads to improper ripening. In tomato fruits, ethylene has profoundly different effects depending

on the stage of development with a distinct developmental switch that occurs upon fruit

maturation. Treatment of mature fruits results in the initiation of a ripening program. While

treatment of immature fruits does not initiate ripening, it does significantly hasten the onset of

subsequent ripening (Yang, 1987). Our understanding of how fruits measure this ethylene

exposure has not been previously determined. The primary obj ective of this proj ect was to

identify the mechanism controlling this phenomenon and to gain a better understanding of the

factors that control ripening in general.

Previous work in our lab showed that reduced accumulation of a single receptor, LeETR4,

resulted in fruits that ripen significantly earlier than control fruits (Tieman et al., 2000). In this

study LeETR6 reduced expression lines exhibited similar effects to those of LeETR4 (Table 2-1;

Kevany et al., 2007). While these results are consistent with the model that the receptors act as

negative regulators of ethylene signaling, they do not address the question of how fruits measure

cumulative ethylene exposure. Analysis of receptor mRNA expression during fruit development

indicated a significant increase in receptor mRNAs at the onset of ripening coincident with the

increase in ethylene biosynthesis (Figure 2-1, Figure 2-2). Contrary to the mRNA expression

data, protein blot analysis of NR, ETR4 and ETR6 showed receptor protein levels highest in

immature fruit with a significant decrease at the onset of ripening (Figure 2-4). While these data









are contradictory to the mRNA expression data, they are consistent with a model in which

ethylene binding affects receptor protein stability. In an attempt to validate this hypothesis, we

exposed fruit and vegetative tissues to ethylene and observed that receptor proteins are rapidly

degraded in response to ethylene and that this likely occurs through the 26S proteasome-

dependent pathway (Figure 2-5, Figure 2-6). While ligand binding-induced degradation of

receptors has been described in mammalian and yeast systems, this work is the first example in

plants. These results led to a hypothesis that reduced levels of receptor proteins, due to ethylene

exposure, control the early ripening in ethylene treated immature fruit. To test this hypothesis,

we treated immature fruits, while still attached to the plant, with ethylene and measured protein

levels throughout fruit development. Treated fruits had reduced receptor protein levels after

ethylene treatment and these fruits ripened earlier than untreated controls (Table 2-2, Figure 2-7).

Together these data are consistent with our model that ethylene exposure leads to a degradation

of receptor proteins and that ethylene receptor levels modulate the timing of ripening.

While reduction of receptor levels results in early ripening fruit, systemic reduction also

causes severe developmental effects that would prevent the use of this method for crop

improvement (Figure 2-3). A technique to reduce the time from fruit set to the onset of ripening

could allow for an increase in the number of varieties available to farmers in higher latitudes. To

generate early ripening lines, we developed a fruit-specific RNAi construct to reduce LeETR4

levels only in the fruit. Fruit-specific suppression of LeETR4 resulted in fruits that ripened up to

7 days early (Figure 3-1). While early ripening fruit would be advantageous they must also retain

the same quality as traditional varieties. To test fruit quality I measured average fruit size, yield,

soluble solids, malic and citric acid content as well as the most important tomato flavor volatile

organic compounds. There was little or no difference between transgenic and control fruits. In









addition to providing a unique method of crop improvement these data also validate our model

that receptor levels in the fruit control the timing of ripening.

While biotechnology has provided us with many tools for gene discovery and crop

improvement, current public concerns have limited the marketing of transgenic foods. In an

effort to identify additional factors that regulate the timing of ripening we undertook a genetic

approach. A screen of a L. hirsutum introgression population was conducted because of the

unusual ripening characteristics and high ethylene biosynthesis levels of this species. Individual

lines were screened for reduced time from anthesis to breaker and for increased ripening-

associated ethylene synthesis. Three lines with a reduction of time to breaker were identified and

the results were repeatable across seasons. Seven lines that had increased ethylene emissions at

the breaker or red stages were identified. Due to the large segments of the L. hirsutum genome

that are found in these lines they had to be backcrossed to the L. esculentum parent to better map

the loci controlling ethylene emissions. Recombinants that may provide material for a map-based

cloning approach for gene discovery have been identified.

The work presented here has significantly increased our understanding of how ethylene

regulates ripening in climacteric fruits. While ethylene is not required for the ripening of non-

climacteric fruits, it can have significant effects on fruit development in these species. Ethylene

can cause damage to the fruits of many different species and our understanding of receptor

function could greatly enhance our ability to limit these losses. Ethylene-related losses in

underdeveloped countries often account for a significant proportion of the postharvest losses and

are an opportunity for our research to have a serious impact.









CHAPTER 6
MATERIALS AND METHODS

Plant Materials and Growth Conditions

L. hirsutunt cy. Flora-Dade, LeETR4-AS, LeETR6-AS and TFM7-ETR4-RNAi lines were

grown in a greenhouse set at approximately 27oC. Individual plants were grown in 3 gal pots that

were watered twice a day and supplemented with slow release fertilizer. Time to ripening data

was collected by tagging open flowers and recording the number of days from anthesis to

breaker. L. hirsutunt cy. Micro-Tom and Nr plants were grown in a growth chamber under

standard conditions (16 h day/8 h night). Field plants were grown in randomized, replicated plots

in Live Oak, FL. Plants were grown using standard commercial practices in raised plastic

mulched beds.

Development of Transgenic Plants

LeETR4-AS and LeETR6-AS lines were generated by cloning the full-length LeETR4 or

LeETR6 coding region into a vector in the antisense orientation under the control of the Figwort

Mosaic Virus 35S promoter (Richins et al., 1987) and followed by the Agrobacteriunt

tunrefaciens nopaline synthase (nos) 3' terminator. The transgene was introduced into cy. Flora-

Dade by the method of McCormick et al. (1986), with kanamycin resistance as a selectable

marker. Transgenic lines with a reduction of >70% ofLeETR6 transcript were identified (Table

1). The specificity of the transgene was determined by quantification of every receptor mRNA

from leaf tissue. In each case there was no effect on RNA levels of any other receptor.

LeETR4 fruit-specific RNAi lines were generated using method outlined by Dexter et al.

(2006). Briefly, two overlapping fragments of coding region were PCR amplified from tomato

fruit cDNAs, one 400 bp and 200 bp in length, primer sequences found in Table 6-1. The two

PCR products were ligated end to end and subsequently ligated into an EcoRI site in the









pMON999 vector that contained the TFM7 fruit specific promoter. The cassette containing the

promoter, RNAi fragment and nos terminator were excised from the vector and ligated into the

pHK plant expression vector. The transgene was introduced into cy. Flora-Dade by

Agrobacterium-mediated transformation according to McCormick et al. (1986), with kanamyacin

resistance as a selectable marker.

Pharmacological Treatments

Ethylene treatments of plant material were done in sealed 38 L tanks. Treatments were

performed using either 10 or 50 ppm, as indicated, concentrations in tanks was monitored by gas

chromatography. These levels are both within the linear response range for NR and LeETR4

ethylene inducibility (Ciardi et al. 2000). Proteasome inhibitor studies were performed by

spraying seedlings with an 80 CIM MGl32 solution (8% DMSO) 4 h prior to 2 h ethylene

treatment. Control seedlings were sprayed with an 8% DMSO solution. 1-MCP treatment of

seedlings was performed at 1 ppm in a sealed 38 L tank for 16 h prior to 2 h ethylene treatment.

Control seedlings were sealed in identical tanks for the same duration of time. All microsomal

membrane preparations were performed immediately after treatment ended.

Recombinant Protein Expression and Antibody Production

Coding regions ofLeETR4 (a.a. 532-684) and LeETR6 (a.a. 522-688) were amplified with

primer pairs ETR4-PF, ETR4-PR, ETR6-PF and ETR6-FR (Table 6-1) from fruit cDNAs

generated with the Clontech One-step cDNA Synthesis kit. PCR products were digested with

BamHI and BglII and cloned into the Invitrogen pTrcXHisA vector and subsequently

transformed into the BL21(DE3) (Invitrogen) E. coli strain for recombinant protein expression.

100 mL cultures were grown at 30oC and induced with 1 mM IPTG for 4 h. Cells were spun

down at 8,000 x g, resuspended in 10mL of lysis buffer (8 M urea) and pulse sonicated for 1 min.

Lysate was spun down at 8,000 x g and supernatant was purified with Ni-NTA affinity column









as directed. Recombinant protein was submitted to Cocalico Biologicals (Reamstown, PA) for

antibody production in rabbits using their standard protocol. Antiserum was received and used to

probe both antigens to determine antiserum specificity for its respective antigen.

RNA Expression Analysis

Total RNA extractions were performed using the Qiagen RNeasy Mini Kit with

subsequent DNase treatment to remove any contaminating DNA. RNA was quantified by

spectroscopy and visually analyzed on ethidium bromide-stained gels to assure equal

concentrations of all RNAs. Quantitative RT-PCR assays were performed using the Applied

Biosystems Taqman One-step RT-PCR kit in an Applied Biosystems GeneAmp 5700 Sequence

Detection System as described (Tieman et al. 2001). PCR conditions were as follows, Step 1:

48oC for 30 min, Step 2: 95oC for 10 min and Step 3: 95oC 15 sec and 60oC for 1 min (40X).

Primer and probe pairs for each gene assayed can be found in Table 6-1. Levels ofLeETR RNAs

were quantified using RNAs synthesized by in vitro transcription from plasmids containing the

coding region of each gene using a Maxiscript in vitro transcription kit (Ambion, Austin TX

USA). Total Clg of in vitro-transcribed RNA were determined and the in vitro transcription

product used for a standard curve in real-time RT-PCR analysis. Results are reported as %

LeETR RNA in total RNA.

Microsomal Membrane Isolation and Protein Blot Analysis

Microsomal membrane fractions were isolated from fruit or seedlings with a

homogenization buffer containing 30 mM Tris (pH 8.2), 150 mM NaC1, 10 mM EDTA, and 20%

(v/v) glycerol with protease inhibitors (1 mM PMSF, 10 Clg/mL aprotinin, 1 Clg/mL leupeptin,

and 1 Clg/mL chymostatin) as described (Schaller et al., 1995). Tissue was homogenized at 4oC

using a polytron and then centrifuged at 8,500 x g for 15 min at 4oC. The supernatant was









strained through cheesecloth then centrifuged at 100,000 x g for 30 min at 4oC and the

subsequent membrane pellet was resuspended in 10 mM Tris (pH 7.5), 5 mM EDTA, and 10%

(w/w) sucrose with protease inhibitors and stored at -80 oC. Protein concentrations were

determined using the Bio-Rad Protein Assay reagent with BSA used for a standard curve. 20 Clg

of total protein was run out for each sample on a 12% Tris-HCI gel and proteins were transferred

to a nitrocellulose membrane using the Bio-Rad Mini Trans-Blot cell. Membranes were blocked

overnight in 10% Carnation milk/Tris Buffered Saline-Tween (TBST) at 4oC. Membranes were

washed 2x5 min in TBST and then incubated with primary anti-ETR4 (1:2000) or anti-ETR6

(1:5000) antibody diluted in 5% Carnation milk/TBST for 1 h. Membranes were subsequently

washed 3x10 min in TBST and then incubated with peroxidase conjugated goat anti-rabbit

(1:5000) secondary antibody (Kirkegaard & Perry Laboratories, Gaithersburg, Maryland) diluted

in 5% Carnation milk/TB ST for 45 min. Membranes were finally washed 3x10 min in TB ST.

Visualization of signal was performed using the Amersham ECL Detection reagents before being

exposed to film. Quantification of bands was accomplished by using the NCBI imaging software

ImageJ (http://rsb .info.nih.gov/ij/). Values were normalized to an anti-BiP endoplasmicc

reticulum immunoglobulin binding protein) antibody (generously provided by Alan Bennett,

Univ. of California, Davis) which was used as an ER-localized loading control.

Acid and Soluble Solids Analysis

Individual tomato fruit were homogenized in a blender for 30 s and frozen at -80 OC until

acid analysis. Samples were thawed, centrifuged at 16 000 g for 5 min. The supernatant was

analyzed for citric and malic acid content using citric acid and malic acid analysis kits (R-

Biopharm, Marshall, MI) according to the manufacturer's instructions. Soluble solids are

expressed as oBrix which is a measurement of the mass ratio of dissolved sucrose to water in a









liquid. Individual fruit were homogenized in a blender for 30 s. 1 mL of the homogenate was

centrifuged at 16,000 x g for 2 min. ~75 uL of supernatant was applied to a handheld

refractometer.

Volatile Analysis

Ripe tomato fruit from each line and its corresponding control collected from the field

were harvested and volatiles from pooled fruits were collected on the day after harvest. Fruits

collected from plants grown in the greenhouse were analyzed for fruit volatiles immediately after

harvest. Tomato fruit volatiles were collected from chopped fruit with nonyl acetate as an internal

standard as described by Schmelz et al. (2003). Chopped fruit was enclosed in glass tubes, air

filtered through a hydrocarbon trap (Agilent, Palo Alto, CA) flowed through the tubes for 1 h

with collection of the volatile compounds on a Super Q column. Volatiles collected on the Super

Q column were eluted with methylene chloride after the addition of nonyl acetate as an internal

standard. Volatiles were separated on an Agilent (Palo Alto, CA) DB-5 column and analysed on

an Agilent 6890N gas chromatograph with retention times compared to known standards (Sigma

Aldrich, St Louis, MO). Volatile levels were calculated as ng gl FW hl collection. Identities of

volatile peaks were confirmed by GCMS as described by Schmelz et al. (2001).










Table 6-1 Oligonucleotide Primers and Probes


ETR4-PF
ETR4-PR
ETR6-PF
ETR6-PR
ETR4-RNAi-F 1
ETR4-RNAi-R1
ETR4-RNAi-F2
ETR4-RNAi-R2
ETR1-TaqF
ETR1-TaqR
ETR1-Probe

ETR2-TaqF
ETR2-TaqR
ETR2-Probe
NR-TaqF
NR-TaqR
NR-Probe
ETR4-TaqF
ETR4-TaqR
ETR4-Probe
ETR5-TaqF
ETR5-TaqR
ETR5-Probe
ETR6-TaqF
ETR6-TaqR
ETR6-Probe


CCGGATCCCGTGATAACGCCTATATCAGG
CCAGATCTGACGATTTGGAATGAGGATAC
CCGGATCCCCGAGATCAAACTCATCCAATG
CCAGATCTGCCATCTAAATCAGGCAGATG
GGAGATCTGGCATTCCTGAATATGGGG
CCGGCGCGCCGAGGATACAGCAGGGCTAAG
CCGGATCCGGCATTCCTGAATATGGGG
GGGGCGCGCCCATCATTCTACTTCCCCGTAGC
TTCAAGGATTAAAGGTHTTGGTGAT
ATCACATCCAAGGTGTGTAAGCA
FAM-ATGAGAATGGTGTTAGCAGGATGGTAACCAAA-
BHQ
GCCGTCAGTGTACATGAGAAATTT
AGTTTTCTTTTGTCACTTGGTCAGTGT
FAM-AGAGGC CACTTATTGTGGCACTAACTGGG-BHQ
AGGGAACCACTGTCACGTTTG
CTCTGGGAGGCATAGGTAGCA
FAM-AGTGAAACTCGGAATCTGTCACCATCCAA-BHQ
GGTAATCCCAAATCCAGAAGGTTT
CAATTGATGGCCGCAGTTG
FAM-AAAGCATGGCTGTCGTTCTTGGGCT-BHQ
AGTCATCTTHTAGGAAACGCATGTT
AGGAGTACATGAAGGCCTCTGAA
FAM-AATACAGAAATCCTTTGGAGCAACCG-BHQ
ATTCCAAAGGCAGCCGTTAA
GGATGTGGATATGTGGGATTAGAAG
FAM-CTCCACATAHTCGGACATGCCTAAGGGA-TAMRA


BamHI
BglII
BamHI
BglII
BglII
AscI
BamHI
AscI


* Nucleotides in bold face represent restriction sites










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BIOGRAPHICAL SKETCH

Brian Michael Kevany was born in Cleveland, Ohio on September 28, 1980. When he

was one he and his mother, father and older brother Thomas moved to North Olmsted, Ohio

where his younger brother Daniel was born and where Brian spent his entire childhood. As a

young boy Brian enjoyed discovering things in his backyard and playing golf, baseball and

hockey, with hockey being a sport he played year round. When he was in high school he got a

j ob at a local nursery and really enjoyed learning about plants. After high school Brian attended

Michigan State University where he maj ored in horticulture specializing in biotechnology. While

at MSU he worked as an undergraduate researcher in the Postharvest Physiology lab of Dr.

David Dilley under the tutelage of Dr. John Golding. Dr. Golding allowed Brian to become

intimately involved in the proj ects in the lab and fostered a great interest in plant research. After

graduation, Brian j oined the Plant Molecular and Cellular Biology Ph.D. program at the

University of Florida as a pre-doctoral Alumni Fellow. While at UF he worked in the lab of Dr.

Harry Klee studying the importance of the tomato ethylene receptor family during tomato fruit

development. Upon completion of his Ph.D. degree, Brian will enter the lab of Dr. Michael

Thomas in the Department of Bacteriology at the University of Wisconsin-Madison as a

postdoctoral researcher.





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ROLE OF MEMBERS OF THE TOMATO ETHYLENE RECEPTOR FAMILY IN DETERMINING THE TIMI NG OF RIPENING By BRIAN MICHAEL KEVANY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007 1

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2007 Brian Michael Kevany 2

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To my parents, who have supported every decisi on I have ever made and given everything to their children 3

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ACKNOWLEDGMENTS Thanks to my entire committee for their patie nce and constructive cr iticism throughout this process. I would like to especia lly thank my advisor, Dr. Harry Klee, for guiding me through my Ph.D. and not only teaching me how to be a scientis t but how to present myself and my science. I thank the entire Klee lab for all their help throughout the years. Thanks to my bench-mate Michelle Zeigler whose attention to detail has he lped me to become a better scientist. I thank Denise Tieman for sharing her knowledge in th e lab and Mark Taylor for generating all the transgenic plants used in my e xperiments. Thanks to Peter Bliss for taking care of my plants in the greenhouse and doing just about everythi ng around the Klee lab. I th ank Valeriano Dal Cin for all of this help on the mapping project. Thanks to everyone in the lab of Dr. A ndrew Hanson for all their help and great friendship. I would especially like to thank Dr. Gilles Basset and Dr. Sebastian Klaus for teaching me everything they know about protei n expression. Additionally I thank Dr. Gale Bozzo, Dr. Rocio Diaz de la Ga rza, Dr. Giuseppe Orsomando, Dr. Aymeric Goyer, and Tariq Ahktar for being there when I needed a break a nd to have some fun. Thanks to the lab of Dr. David Clark for allowing me to come over and do my RNA extractions in their hood and also to bother them when I needed a break. I thank Ca rol Dabney-Smith for te aching me all she knows about custom antibodies, without th is help I would not have been able to finish all my work. Most importantly, thank you to my family fo r always being there for me when I needed them. Also for understanding that moving from Ohio to Florida was what was best for my career even though it was so far. I also thank all of my friends back in Ohio and Michigan for staying in touch and giving me plenty of fun times outside of Gainesville. Lastly, thanks to Stephanie Violi from the bottom of my heart for being the person I have leaned on for the past three years. She 4

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5 has made me laugh when I needed it and always put things in perspe ctive. Even though we havent been together she has remained the drivi ng force in my life and is the love of my life.

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................8 LIST OF FIGURES .........................................................................................................................9 ABSTRACT ...................................................................................................................................11 CHAPTER 1 LITERATURE REVIEW .......................................................................................................13 Ethylene in Plant Biology .......................................................................................................13 The Ethylene Receptor Family Arabidopsis and Tomato ..................................................17 Protein Degradation Through the 26S Proteasome ................................................................24 2 ETHYLENE RECEPTOR DEGRADATION CONTROLS THE TIMING OF RIPENING IN TOMATO ......................................................................................................31 Introduction .............................................................................................................................31 Results .....................................................................................................................................33 A Subset of the Receptor Family Shows Ripening-associated Expression and Is Ethylene-inducible in Fruit ..........................................................................................33 LeETR6 Antisense Lines Show Phenotypes Consistent with a Constitutive Ethylene Response ......................................................................................................................34 Receptor Protein Levels Are Distinctly Di fferent From Transcript Levels During Fruit Development .......................................................................................................34 Treatment of Leaf and Fruit Tissue with Ethylene Causes a Rapid Degradation of Receptor Proteins That Likely Occurs Through a Proteasome-dependent Pathway ...35 Receptor Levels in Developing Fru it Determine the Timing of Ripening ......................37 Discussion ...............................................................................................................................37 3 FRUIT-SPECIFIC SUPPRESSION OF THE ETHYLENE RECEPTOR LEETR4 RESULTS IN EARLY RIPENING FRUIT ...........................................................................50 Introduction .............................................................................................................................50 Results .....................................................................................................................................51 LeETR4 RNAi Transgenic Plants Produce Early Ripening Fruit ....................................51 Early Ripening Lines Show A ltered Ripening Coordination ..........................................52 Transgenic Fruits are Indistinguishable fr om Wild Type Fruits in Horticultural Traits ............................................................................................................................53 Discussion ...............................................................................................................................54 6

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7 4 IDENTIFICATION OF QTLS THAT MODIFY TIME TO RIPENING AND RIPENING-ASSSOCIATED ETHYLENE PRODUCTION .................................................61 Introduction .............................................................................................................................61 Results .....................................................................................................................................63 Discussion ...............................................................................................................................66 5 CONCLUSION .......................................................................................................................80 6 MATERIALS AND METHODS ............................................................................................83 Plant Materials and Growth Conditions ..........................................................................83 Development of Transgenic Plants ..................................................................................83 Pharmacological Treatments ...........................................................................................84 Recombinant Protein Expr ession and Antibody Production ...........................................84 RNA Expression Analysis ...............................................................................................85 Microsomal Membrane Isolati on and Protein Blot Analysis ..........................................85 Acid and Soluble Solids Analysis ...................................................................................86 Volatile Analysis .............................................................................................................87 LIST OF REFERENCES ...............................................................................................................89 BIOGRAPHICAL SKETCH .........................................................................................................98

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LIST OF TABLES Table page 2-1 Days from anthesis to breaker of LeETR6 antisense lines .......................................................49 2-2 Days from anthesis to breaker of ethylene treated Microtom fruit ..........................................49 3-1 Weight, yield, brix, citric acid an d malic acid from field grown fruits ...................................59 3-2 Weight, yield, brix, citric acid an d malic acid from greenhouse grown fruits ........................59 3-3 Volatile organic compounds from field grown fruits ..............................................................59 3-4 Volatile organic compounds from greenhouse grown fruits ...................................................60 6-1 Oligonucleotide primers and probes ........................................................................................88 8

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LIST OF FIGURES Figure page 1-1 Schematic representation of tomato ethylene receptor family ...............................................30 2-1 Ethylene receptor family mRNA levels during fruit development. ........................................41 2-2 Ethylene-inducibility of each recep tor mRNA in immature fruit tissue. ...............................42 2-3 Constitutive ethylene response phenotypes of LeETR6 antisense lines .................................43 2-4 Receptor gene expression and protein leve ls show distinct differences during fruit development .......................................................................................................................44 2-5 Ethylene binding induces degradation of receptors in detached immature fruits ...................45 2-6 Ethylene binding induces de gradation of receptor prot eins in vegetative tissue ....................46 2-7 Ethylene treatment induces turnover of receptor leading to early ripening fruit ....................47 2-8 Ethylene treatment induces expression of receptor mRNAs in attached fruit ........................48 3-1 Fruit-specific ETR4 RNAi transgenic lines produce early ripening fruit ...............................56 3-2 Suppression of LeETR4 is Fruit-specific ................................................................................57 3-3 ETR4 -RNAi transgenic plants have altered ripening coordination ........................................58 4-1 Ethylene emissions of fully ripe fruit from L. hirsutum IL 3945 ...........................................69 4-2 Days from anthesis to breaker of tagged fruits from L. hirsutum ILs ....................................70 4-3 Ethylene emissions of breaker fruit from L. hirsutum ILs .....................................................71 4-4 Ethylene emissions of fully ripe fruit from L. hirsutum ILs ...................................................72 4-5 Genomic map showing locations of introgr essed regions that c ontain putative ripeningassociated QTLs .................................................................................................................73 4-6 Days from anthesis to breaker of tagged fruits from L. hirsutum ILs ....................................74 4-7 Ethylene emissions of fruits from field-grown L. hirsutum ILs .............................................75 4-8 Ethylene emissions of leaves from L. hirsutum ILs ...............................................................76 4-9 Nucleotide alignment of ETR4 genomic sequence ................................................................77 4-10 mRNA expression of LeETR4 in WT and the L. hirsutum IL 3945 .....................................78 9

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10 4-11 mRNA expression of LeETR4 in seedlings of WT, L. hirsutum and IL 3945......................79

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ROLE OF MEMBERS OF THE TOMATO ETHYLENE RECEPTOR FAMILY IN DETERMINING THE TIMING OF RIPENING By Brian Michael Kevany December 2007 Chair: Harry John Klee Major: Plant Molecular and Cellular Biology Tomatoes are an economically important crop and a significant dietary source of important phytochemicals, such as carote noids and flavonoids. While it ha s been known for many years that the plant hormone ethylene is essential for ri pening of climacteric fru its, its role in fruit growth and maturation is much less understood. In an attempt to better un derstand tomato fruit ripening we utilized both biotechnology and traditional breeding strategies. The multigene ethylene receptor family has been shown to nega tively regulate ethylene signal transduction and suppress ethylene responses. Here, we demonstrate th at a reduction in the levels of either of two family members, LeETR4 or LeETR6 causes an early ripening phe notype. We provide evidence that the receptors are rapidly degraded in the presence of ethylene and that degradation likely occurs through the 26S proteasome-dependent pa thway. Ethylene exposure of immature fruits causes a reduction in the amount of receptor protein and earlier ri pening. Fruit-specific suppression of the ethylene receptor LeETR4 causes early ripening while fruit size, yield and flavor-related chemical com position are largely unchanged. Th ese results demonstrate that ethylene receptors likely act as biological clocks regulating the onset of tomato fruit ripening. In order to better understand the mechanism cont rolling the timing of ripening we screened a Lycopersicon hirsutum introgression population for QTLs responsible fo r reduced time from 11

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12 anthesis to breaker and/or increased ripening-associated ethyle ne biosynthesis. The L. hirsutum population was chosen because of unusual ripening characteristics and significantly higher levels of ethylene biosynthesi s at maturity of L. hirsutum A number of lines were identified that showed statistically significant di fferences from the control for both phenotypes. These lines are currently being refined for possible map-based cloning of loci controlling these phenotypes. These results demonstrate the power of using both molecular biology and traditional breeding for gene isolation/characteri zation and crop improvement.

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CHAPTER 1 LITERATURE REVIEW Ethylene in Plant Biology The phytohormone ethylene is an important signaling molecule that is involved in many plant processes including but not limited to abscission, leaf and flower senescence, germination, sex determination and fruit ripening (Abeles et al., 1992). Ethylene also functions in both biotic and abiotic stress responses. Exposure to environmental stresses like flooding, wounding, herbivory, chilling or pathogen attack can enhance ethylene production (Boller, T. 1991; Abeles et al., 1992). This ethylene then slows growth until the stress is removed. Interest in ethylenes importance as a plant hormone has resulted in thousands of peer-r eviewed publications in the last 100 years and has laid the foundation for a real un derstanding of ethylene s involvement in plant growth and development. Ethylene is a small, gaseous, two-carbon molecu le that has the ability to diffuse through hydrophilic and hydrophobic environments. This propert y allows it to pass into any compartment in the plant cell. The ability of ethylene to alter plant development has been known for centuries, with farmers from many culture s using smoke and wounding to induce flowering and ripening (Abeles et al., 1992). Damage to city and greenhouse plants in the late 19th and early 20th centuries was found to be cause d by leaking illuminating gas th at was used at the time for lighting. Work done by Dimitry Neljubov in 1901 pr oved that ethylene was in fact the active component in illuminating gas that resulted in th e plant damage (Abeles et al., 1992). Subsequent work showed that ethylene was clearly important for fruit ripening but many scientists at the time believed that the other phenotypes of endogenous ly produced ethylene were a by-product of the ripening process (Abeles et al., 1992). Work done in the 1960s by the Burgs provided definitive proof that ethylene is important for plant development beyond its involvement in fruit 13

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ripening (Burg and Burg, 1962; Burg 1962). Their wo rk was instrumental in classifying ethylene as a plant hormone. Early feeding experiments suggested that th e amino acid methionine is a precursor of ethylene (Lieberman et al., 1966; Burg and Claget t, 1967). Later work provided evidence that oxygen is necessary for the production of ethylene. It was then hypothesized that if fruit tissue was held in an anaerobic environment the precursor should build up and provide enough compound to allow identification. This work led to the subsequent isolation of 1aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of endogenous ethylene (Adams and Yang, 1979). ACC is synthesized from S-adenosyl methionine (SAM) by a pyridoxal phosphate-requiring enzy me termed ACC synthase (ACS ). The conversion of ACC to ethylene is subsequently perf ormed by the oxygen-requiring enzyme ACC oxidase (ACO). The conversion of ACC by ACO results in the production of CO2 and HCN in addition to ethylene. Most tissues synthesize low levels of ethylene. Synthesis can be stimulated by a number of means, including wounding, submergence, chilling and pathogen attack. Synthesis of ACC is considered to be the rate limiting step in ethylene production. Thus, increased ethylene production requires modulation of AC S expression and/or activity. While ethylene is often charac terized as the ripening hormone, not all fruit require ethylene to complete the ripening process. Species are ofte n characterized by the presence or absence of a large increase in ethyle ne production concomitant with increas ed respiration at the onset of ripening. Species whose fruit exhi bit these increases are termed climacteric while those that do not are referred to as non-cli macteric. Climacteric species include apple, avocado, banana, peach and tomato while non-climacteric species incl ude strawberry, grape, cherry and citrus. The increase in ethylene production associated with climacteric ri pening is essential for ripening. 14

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Blockage of either ethylene bi osynthesis or perception results in an inability of the fruit to complete its ripening program. Tomato is an excellent model for the study of ethylenes involvement in fleshy fruit development because of a relatively short life cycl e, ease of genetic manipulation and a wealth of genetic resources. In addition, the tomato genome is being sequenced, which will be a tremendous resource to those working on this species. Ethylenes involvement in ripening, fruit softening, volatile production and lycopene accumulation has been well documented. Ethylene bi osynthesis during tomato fruit development generally goes through three distin ct stages. There is a slight bur st of synthesis after successful pollination that then falls to low levels until the onset of climacteric ethylene production at the onset of ripening. Ethylene production during i mmature fruit development has been termed system I and is characterized as low level pr oduction which cannot be stimulated by treatment with exogenous ethylene (Yang, 1987). Ethylene bios ynthesis in mature fruit, referred to as system II, is autocatalytic, meaning it can induce its own s ynthesis (Yang, 1987). The induction of ethylene synthesis at the onset of ripening is believed to be due to developmental induction of an ethylene-inducible ACS (Barry et al., 2000; Nakats uka et al., 1998). Although immature tomato fruit do not produ ce significant levels of ethylene they do respond to ethylene, but in a different manner to th at of ripening fruit. This response manifests as a change in gene expression but to a smaller set of genes to that of ripening fruits (Alba et al., 2005). This difference in respons e suggests that there is developmental control of gene expression in addition to that of ethylene. The developmental control of ethylene regulated genes has been best characterized by research done on the E4 and E8 genes found in tomato. Expression of E4 is ethylene inducible thr oughout fruit development while E8 is only ethylene 15

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inducible in ripening fruit (Lin coln et al. 1987, Wilkinson et al ., 1995). Treatment of immature fruit with ethylene induces a set of genes, proving a response to the hormone, but it does not induce immediate ripening. However, that ethylene exposure does hasten th e onset of ripening as compared to untreated fruit of similar age, s uggesting that the fruit can measure cumulative ethylene exposure (Burg and Burg, 1962; Lyons and Pratt, 1964; McGlasson et al., 1975; Yang, 1987). Treatment of immature green tomato fruit with ethylene, or its analog propylene, could reduce the time from anthesis to breaker by half that seen in non-treate d controls (Lyons and Pratt, 1964; McGlasson et al., 1975). The way fruits measure this ethylene exposure is unknown. Along with temporal control of fru it ripening there is also a spatial aspect of control. Fruit do not ripen evenly across the entire fruit, they begin to ripen at the basal end of the fruit and proceed towards the calyx. Since ethylene is diffusible throughout the fru it, and accumulates to high levels within the fruit, there appears to be a de velopmental control within individual fruit that controls the spatial ripening of the fruit. In addition to ethylenes role in fruit deve lopment it also plays an important part in seedling emergence (Clark et al., 1999). During germ ination seedlings must be able to force their way through any soil between them and a light so urce. When a seedling encounters a barrier in the soil it often becomes slightly wounded which can induce ethylene production. Dark grown seedlings, like those found underground, are often tall and spindly in the pr esence of air alone. Upon exposure to ethylene its growth habit changes a nd exhibits growth that is referred to as the triple response. This response manifests as a shortening of both the hypoc otyl and root, radial thickening of the hypocotyl and an exaggerati on of the apical hook. These changes allow the seedling to push through any barriers without da maging the meristem. While this mechanism has evolutionary importance, the ability to exploit this response has revolutionized the ethylene 16

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biology field by allowing researchers to scr een for mutants in ethylene biosynthesis and signaling. The Ethylene Receptor Family Arabidopsis and Tomato Much of the initial ethylene perception and signal transduction research was done in Arabidopsis thaliana and thus we have exploited the Ar abidopsis system to identify the orthologous genes in tomato. The Arabidopsis ethylene receptor ETR1 was the first phytohormone receptor cloned in plants and was isolated from a mutagenized population that was screened for plants deficient in the triple response (Bleecker et al. 1988; Guzman and Ecker 1990). Ethylene insensitive mutants grow tall and spindly even in the presence of ethylene while constitutive ethylene response mutants will show a triple response in the absence of ethylene. etr1-1 was isolated as an ethylene insensitive mutant in one of these screens and was later cloned and shown to encode an ethylene receptor with homology to bacterial two-component sensors (Chang et al. 1993). In subsequent work a total of five receptors were cloned from Arabidopsis. The ethylene signal transduction pathway in Arab idopsis is believed to be relatively linear but we are unsure if all of the elements have been identified. Epista tic analysis has allowed researchers to putatively order the components star ting with the receptors. The next component is the Raf-like Ser/Thr protein kinase, CTR1, which ha s been shown to physica lly interact with the receptors (Clark et al., 1998; Gao et al., 2003; Huang et al., 2003). CTR1 has significant homology to MAPKKKs and although no MAPKK or MAPK have been found to be involved in ethylene signal transduction, thei r involvement in this pathway cannot be ruled out. EIN2, a protein showing homology to Nramp metal transporters, is the next member of the pathway. The role and activity of this protein in the pathwa y is unknown but it is absolutely necessary since knockouts show complete ethylene insensitivity in every assay tested. The end of the ethylene signal transduction pa thway is composed of the transcript ion factors EIN3 and ERF1. EIN3 loss17

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of-function (LOF) mutants show partial ethylene insensitivit y, which is probably due to redundancy within a gene family containing at le ast three members (Chao et al., 1997). In the absence of ethylene the EIN3 prot ein is targeted for degradation in the 26S proteasome by a pair of F-box proteins, EIN3-binding factors 1/2 (EBF 1/2). Upon ethylene bindi ng this repression is released and EIN3 binds to the promoter of ERF1 activating its tran scription and ERF1 is involved in regulating the tran scription of ethylene responsiv e genes. ERF1 over-expressors show a slight constitutive ethylen e response suggesting that there are other important players in the transcriptional c ontrol of ethylene responsive ge nes (Berrocal-Lobo et al. 2002). ETR1 was not only the first phytohormone receptor to be cloned in plants but was also the first eukaryotic protein with homology to a histid ine kinase (Chang et al. 1993). These receptors are endoplasmic reticulum-localized proteins that have copper-me diated ethylene binding and are present in vivo as dimers (Chen et al., 2002; Ma et al., 2006; Schaller and Bleecker 1995; Schaller et al. 1995). The Arab idopsis receptor proteins (E TR1, ETR2, ERS1, ERS2 and EIN4) can be separated into three st ructurally different regions. The sensor domain is composed of three put ative transmembrane (TM) sequences in ETR1 and ERS1 and four domains in ETR2, ERS2 and EIN4, with the first TM sequence representing a cleavable ER-targeting peptide. These tr ansmembrane sequences are where the coppermediated ethylene binding takes place. This regi on also contains all of the known mutations that cause ethylene insensitivity, likely due to an inability to bind ethylene or transmit the signal through a conformational change. The amino acids necessary for dimerization are present in this region and homodimerization has been proven in vivo but heterodimerization has not been demonstrated (Schaller et al. 1995). 18

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The next domain present in this family is a region that shows homology to histidine kinases (Dutta et al., 1999). Histidine kinase domains cont ain five highly conserved subdomains, N, G1, F, G2 and the histidine (H) that is au tophosphorylated. The ETR1 and ERS1 proteins contain all five of th ese sub-domains while the other th ree members lack at least one sub-domain. The ETR1 protein is the only member of the family that exhibits HK activity in vitro, but the conserved histidine is not necessary for protein function based on the ability for a mutant lacking this residue to rescue a recepto r mutant (Gamble et al. 1998; Gamble et al. 2002, Wang et al. 2003). The other family members all exhibit Ser/Thr ki nase activity based on in vitro kinase assays (Moussatche a nd Klee, 2005). This lack of his tidine kinase activity in these family members fits well with the finding that most of the other family members do not contain all of the conserved regions in the histidine domain. All kinase assays completed so far have been done in vitro and there has been no ki nase activity directly linked to ethylene signal transduction in vivo. The third and final domain found in these proteins is the receiver, located at the C-terminus of the protein. This region shows homology to th e output domains from bacterial two-component sensors and contains an aspartat e that is active in phosphorelay in these bacterial pathways. The ERS1 and ERS2 proteins lack this domain while the other family members contain it, suggesting that it may play a role in some family member-specific functions. Using sequence and exon/intron organization comparisons, ETR1 and ERS1 have been classified as Subfamily 1 receptors while ETR2, ER S2 and EIN4 have been classified Subfamily 2 receptors. Considering the degr ee of divergence within the family, there may be specific functions for each of the family members. The ev idence suggests that the receptors may not be completely redundant, although most geneti c evidence suggests functional overlap. 19

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Mutant analysis of the Arabidopsis ethyl ene receptor family has allowed a better understanding of the receptors role in transducin g the ethylene signal. Al l of the initial receptor mutants cloned were semidominant, insensitiv e mutants. Single gene LOF mutants have no obvious phenotypes which is most likely due to functional redundancy within the family. Based on all of the genetic data available the receptors appear to function as ne gative regulators of the ethylene response (Hua and Meyerowitz 1998). The de fault state of the receptor is one in which the receptor actively supp resses ethylene responses in the ab sence of the hormone and ethylene binding removes this suppression. The double mutant etr1/ers1 and triple or quadruple mutants show constitutive ethylene responses even in th e absence of increased ethylene biosynthesis (Wang et al., 2003), presumably because basal et hylene levels are able to inactivate the remaining receptors. This model suggests that a decrease in receptor content will increase ethylene responsiveness while an increase in rece ptor levels will decrease tissue responsiveness. This simplified model does not appear to tell the entire story because it pr esumes that all of the receptors contribute equally to the signal and re cent work has suggested this may not be true. Overexpression of a Subfamily 2 member was unable to rescue the constitutive ethylene response phenotype of the double Subfamily 1 mutant, suggesting some family member-specific functions (Wang et al., 2003). Work done in our lab has found that the system in tomato may be quite different from that of Arabidopsis. The tomato ethylene receptor family is composed of six members, LeETR1-6 with LeETR3 corresponding to the NR gene (Fig. 1, Zhou et al. 1996a; Zhou et al. 1996b; Lashbrook et al. 1998;Tieman and Klee 1999). All receptor family members have been shown to bind ethylene with the exception of LeETR6 because it was not available at the time of analysis (OMalley et al ., 2005). The first of the tomato ethylene receptor genes to be cloned was NR This gene was 20

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isolated from a mutant that s hows semidominant ethylene insens itivity which prevents floral wilting and abscission, alte rs leaf senescence and prevents fruit ripening (Wilkinson et al. 1995). The basic structures of the receptors are similar to those of the Arabidopsis family but within the tomato family the sequences are quite divergent with less than 50% identity at the extremes (Figure 1). The transmembrane domai ns show the highest levels of sequence similarity owing to the importance of this domain in the transmi ssion of the signal. LeETR1, 2 and NR have three putative transmembrane domains while LeETR4, 5 and 6 have four putative transmembrane domains. The NR protein is the on ly member of this family that lacks the C-terminal receiver domain (Figure 1). LeETR4, 5 and 6 resemble the Subfamily 2 receptors found in Arabidopsis in that they are missing at least one of the cons erved sub-domains in the HK domain and contain the fourth transmembrane sequence (Figure 1-1). E ach of the receptors has a distinct expression pattern throughout fruit development, with NR ETR4 and ETR6 being ethylene inducible (current work). NR and ETR4 are both pathogen inducible, with the increase in expression being a function of the increase in ethylene producti on found during a disease re sponse (Ciardi et al., 2000). An increase in receptor expr ession is likely an important f actor in reducing the amount of damage that occurs as a result of th is increase in et hylene production. The basic model for ethylene response states th at the receptors act as negative regulators of ethylene response and that higher receptor expr ession reduces sensitivit y and lower expression increases sensitivity. This model explains why multiple gene knockouts in Arabidopsis show a constitutive response. While much of the availa ble data fit this model it does not address the importance of ethylene dissociation from th e receptor or protein turnover. The Kd of ethylene dissociation was measured in yeast-expressed AtETR1 and was found to be approximately 12 hours. This is likely to be an underestimate sin ce it did not factor in protein turnover (Schaller 21

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and Bleeker, 1995). There is no evidence to suggest that ethylene is able to dissociate from the receptor, suggesting this association may be pe rmanent. Isoform-specific antibodies have been generated for a number of the Arabidopsis receptors and the tomato NR protein but no work has been done to study in vivo turnover rates or ethyle nes effect on receptor turnover. This type of evidence will be necessary to draw any conclusi ons about the receptors importance in a plants response to ethylene. The current model suggests that the only wa y that a plant can re duce its response to ethylene is by synthesis of new receptors. Less receptor leads to more sensitive tissue and more receptor leads to less sensitive tissue. Previous work has shown that the current data do fit the receptor model. Plants overexpressing NR have been found to be less sensitive to ethylene in triple response assays and pat hogen studies (Ciardi et al. 2000). LeETR4 antisense lines with significantly reduced expression show phenotypes consistent with a constitutive ethylene response. Phenotypes of these lin es include epinastic growth, pr emature flower senescence and abscission and for fruit, a reduction in the time fr om anthesis to breaker and from breaker to red ripe (Tieman et al 2000). The effect on time from anthesis to breaker is quite significant with a decrease of as much as 11 days compared to WT controls. LeETR4 antisense lines also have an altered response to pathogen infection because an increase in ETR4 expression is one way in which the plant reduces the amount of tissue damage. These lines display an accelerated hypersensitive response in response to infection with an incompatible pathogen with greater ethylene production and hastened expression of pathogenesis-relate d genes (Ciardi et al. 2000). Antisense suppression of LeETR1 2 and NR have no observable phenotype but this result is likely due to redundancy within the system. The NR antisense lines show an unusual phenotype in that with the reduction of NR expression levels there is a concomitant increase in ETR4 22

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expression and this may explain why the NR antisense lines do not show any constitutive ethylene response phenotypes. This phenomenon has been termed functional compensation and appears to be a built-in system that allows the increase in expression of one family member when another has been reduced (Tieman et al 2000). The expression level of each of the tomato receptors has been monitored in response to multiple ethylene-related phenomenons and at least one receptor is up-regulated in each of the responses. On the other hand, a reduction in r eceptor expression has never been seen even though it would increase the tissues responsiveness to ethylene. So it seems that as soon as a plant starts producing ethylene more receptor is produced thus attenuating the response. While this may seem counterproductive it is not unco mmon for a phytohormone in plants to be attenuated as soon as its induced (Rashotte et al., 2003). Increased response to ethylene can be very detrimental to plant tissues and since ethyl ene slows the growth of a plant, it could have long term effects. The expressi on levels of all th e receptors remain low throughout immature fruit development and show a shar p increase at the onset of ri pening, the time at which ethylene production is at its highest. So it seems that at the point when ethylene is having its greatest effect on plant development, receptor levels are at their very highest. It has been known for some time that ethylene is intimately involved in the timing of fruit ripening and our research seeks a better understanding of its role. Based on our previous research and that of others we believe that if ethylene production rates during fruit developmen t exceed the level of receptor synthesis then there would be a de-repression of the system that would lead to an increas e in sensitivity to the hormone. At some point in development, when th e fruit are ripening competent, sensitivity to ethylene would rise past a thresh old level where ripening could be initiated. Based on this model the reduction of receptor levels in transgenic plants should reduce time to ripening and based on 23

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our previous results this is true, ETR4 antisense plants ripen faster than controls (Tieman et al 2000). Protein Degradation Through the 26S Proteasome Protein degradation is an important regulat ory mechanism that has been adopted by many organisms. It has emerged as a mechanism of control as important as gene expression in controlling cellular processes. Prot ein degradation has been implicat ed in the control of signaling cascades, defense against viral infection, breakdown of cellular regulators and arguably its most important role is the removal of abnormal pr oteins (Jabben et al. 1989, Glotzer et al. 1991, Scheffner et al. 1993). The degradation of proteins generally falls into two classes: (1) relocation of proteins to degradative orga nelles such as the lysosome or vacuole and (2) targeting the proteins for degradation by the 26S proteasome. These two pathways are the principal modes of degradation for both soluble and membrane bound proteins, albeit less is known about how membrane-bound proteins are degraded. While re location to degradative organelles is an important type of protein degradation the focus of this review will be on the role of the 26S proteasome in protein degradation. The 26S proteasome is one of the most importa nt proteolytic systems in plants and our understanding of this system has gr own considerably in the past de cade. This system utilizes the 76-amino acid protein ubiquitin (Ub) as a reusable tag to target specific proteins to the multisubunit 26S proteasome for proteolysi s. The attachment of Ub occu rs at lysine residues on the target protein and often occurs as a polyubiquitin chain of Ub m onomers. Upon proteolysis in the proteasome the Ub monomers are released to be used in another round of targeting. Ubiquitination of target proteins occurs in a three-step conjugation cascade and can occur on proteins in the cytoplasm, nucleus integral membrane proteins and ER resident proteins that are retro-translocated across the ER membrane. 24

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The ubiquitin attachment cascade occurs in a th ree-step process designated E1, E2 and E3. The E1 component of the cascade is an ubiquitin-activating enzyme that binds ubiquitin at a conserved cysteine. This enzyme is constitutivel y expressed and has little impact on target specificity. The Arabidopsis genome encodes two E1 isoforms (Hatfield et al., 1997). The E2, or ubiquitin-conjugating enzyme, is encoded by at least 37 family members in Arabidopsis (Vierstra, 1996). This enzyme shuttles the ubiquitin moiety between the E1 and E3 proteins (Pickart, 2001). The size of this family suggests that different E2s may be involved in regulating specific pathways, although no specific functions have been assigned to any plant E2s. The specificity of individual E2s likel y occurs through their interaction with specific E3s. In addition, the E2s are not all specific to ubiquitin but are also used for conjugating ubiquitin-like proteins including NEDD, RUB and SUMO (L i et al., 2006). The E3, or ubiquitin-protein ligases, is the component of the cascade that specifically recogn izes proteins for ubiquitination. Because of the specificity of this protein/complex it is encoded by several large families of genes, with more than 1300 members in Arabidopsis (Vierstra, 2003). Four different types of E3 ligases have been identified in plants: HECT, RING/U-box, SCF an d APC (Smalle and Vierstra, 2004). HECT E3 ligases are composed of a large single polypeptide (often >100kD a), with seven family members present in the Arabidopsis genom e (Downes et al., 2003). Little is known of the functions of plant HECTs, although one is known to be important for trichome development. Like the HECT family, each RING/U-box family member is a single polypeptide that acts to bring together the E2-Ub and target substrate. This group of protei ns is each encoded by a large family of proteins with 480 RING finger-containing and 64 U-box contai ning proteins, respectiv ely, in Arabidopsis (Azevedo et al., 2001; Kosarev et al ., 2002). This type of E3 liga se has been implicated in a diverse number of cellular proc esses in plants, including, auxi n signaling, photomorphogenesis, 25

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self incompatibility and rem oval of abnormal proteins (Sma lle and Vierstra, 2004). The SCF type of E3 ligases are composed of a complex of four different polypeptid es. This type of E3 ligase acts in a similar manner to that of RING/U-box proteins in that they bring together the E2Ub and the target substrate. Plants have the abil ity to synthesize a vast number of SCF type E3 ligases. The Arabidopsis genome contains two RBX 1 subunits, five cullin subunits, 21 SKP-like proteins and almost 700 F-box proteins (Farras et al., 2001; Gagne et al., 2002; Shen et al., 2002) The F-box proteins provide the target specificity for this complex and constitute one the largest gene superfamilies in the Arabidopsis genome. The APC type of E3 ligases is the most complex type of E3, being composed of eleven subunits Most of these subunits are encoded by single genes in Arabidopsis and thus it is likely that they only form a small number of APC type E3s (Capron et al., 2003). The APC wa s first identified as being im portant for the regulation of mitosis through degradation of mito tic cyclins in yeast; it has been subsequently shown to have a similar function in plant cel ls (Blilou et al., 2002). The 26S proteasome is an ATP-dependent prot eolytic complex that is composed of 31 subunits organized in two major subcomplexes. The 20S core protease (CP) is the portion of the complex that houses the proteoly tic activity, alone it is an ATPand Ub-independent protease. The CP has hydrolyzing, trypsin -like and chymotrypsin-like ac tivity allowing it to degrade a broad range of peptide bonds (V oges et al., 1999). The 19S regulatory particle (RP) can bind to both ends of the CP and is th e portion of the complex that recognizes the Ubs attached to targeted proteins (Voges et al., 1999). The RP performs a number of additional functions including unfolding the target protein, Ub remova l, opening the gate to th e CP core and directing the target protein into the CP lumen (Smalle a nd Vierstra, 2004). Regulation of the activity and specificity of the proteasome is thought to be affected by a number of factors including 26

PAGE 27

association with additional protei ns and substitutions or modifi cations to complex subunits. The Arabidopsis genome encodes two isoforms of ne arly all proteasome s ubunits. Transcriptional control of the complex subunits in yeast is facilitated by a single transcription factor, Rpn4, that is negatively regulated at the protei n level by the 26S proteasome itself. The role of the 26S proteasome in regulati ng many signal transduction pathways has been confirmed in plants. The proteasome has been imp licated in the action of all plant hormones. In addition, it is important for a plant s response to both abiotic and bi otic stimuli. Its role in auxin and ethylene signaling are arguably the best charact erized roles in hormone signaling. The auxin signal transduction pa thway is negatively regulated by a fa mily of proteins (AUX/IAAs) that bind and inhibit the functions of a family of tr anscription factors, the auxin response factors (ARFs). Upon auxin binding the AUX/IAAs are targeted for degradation, thus releasing the transcription factors to initiate expression of auxin responsive genes. The use of mutants and proteasome inhibitors has confirmed this pathwa y and has facilitated the identification of the auxin receptor as the F-Box protein, TIR1. TIR1, and TIR1-like proteins, specifically target the AUX/IAAs for polyubiquitination and is an intere sting example of the importance of the 26S proteasome in regulating hormone pathways (Dharmasiri et al., 2005). The ethylene signal transduction pathway is also regulated by the proteasome, which modulates transcription factor activity/abundance. The F-Box proteins EBF1/2 target the EIN3, and EIN3-like, transcription factors for degrad ation in the absence of ethylene. Upon ethylene binding, this repression is removed and the transcript ion factor is able to activate transcription of primary ethylene responsive genes. In the case of EBF1/2, each has a differe nt role in response to ethylene, with EBF1 being mo re important during early ethyl ene response and EBF2 more 27

PAGE 28

important later during the res ponse and in the resumption of growth after ethylene removal (Binder et al., 2007). The importance of the proteasome in response to abiotic stimuli is best characterized by its role in regulating light signa ling. PhyA, a red/far red absorb ing photoreceptor, is rapidly ubiquitinated and turned over following photoconve rsion to the Pfr form. In addition to the regulation of photoreceptor protein levels, regulation of transcription factors is also performed by the proteasome. In the absence of a light sour ce the RING-E3 COP1 is present in the nucleus where it targets a number of tran scription factors for degradat ion. Upon illumination COP1 is removed from the nucleus and the transcription of light responsive genes occurs. These examples represent an extremely small percentage of th e pathways in which the proteasome has been implicated and there are many more that have not been characterized. While much is known about the degradation of soluble proteins by the proteasome, relatively little is known about in tegral membrane protein degradat ion, especially in plants. What is known about this pathway has been elucidated in yeast and to a lesse r extent in humans. A major regulatory and house keeping pathway that involves degradation of proteins in the endoplasmic reticulum (ER) or integrated into the ER membrane and has been termed ERassociated degradation (ERAD) has been uncovered. ERAD is responsible for targeting misfolded ER proteins that ar e retro-translocated back across the ER membrane and also targeting misfolded integral membrane proteins that are subsequently extracted from the membrane and degraded by the proteasome (Meusse r et al., 2005). It has been hypothesized that different targeting complexes may be present in cells that target me mbrane proteins with misfolded cytosolic domains, internal membrane domains or ER luminal domains (Carvalho et 28

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al., 2006). These complexes contain a number of different subunits but each contains a membrane-bound E3 ligase that attaches the ubiquitin monomers to the substrate. A number of membrane-bound ERAD substrates have been identified but an interesting example is that of the inositol 1,4,5-triphosphate (IP3) receptor. Activation of a G proteincoupled receptor (GPCR) increases phospholipas e C activity that genera tes diacylglycerol and the second messenger IP3. IP3 moves through the cytoplasm to IP3 receptors located in the ER membrane, which activate channels that mobilize internal reserves of Ca2+. A persistent activation of GPCRs leads to a down-regulation of IP3 receptors in order to prevent any deleterious effects of continually elevated cytosolic Ca2+. This down regulation requires the 26S proteasome and it has been shown that IP3 binding induces ubi quitination of the IP3 receptor, leading to degradation (Zhu and Wojcikiewicz, 2000). In addition, a binding-defective mutant receptor was shown to be resistant to ubiquitina tion and this resistance is not caused by the removal of potential ubiquitination sites. It was hypothesized that ligand binding causes a conformational change that expos es a signal leading to ubiquitination (Zhu and Wojcikiewicz, 2000). The 26S proteasome has emerged as an essential part of a cells repertoire for maintaining cellular integrity and regulating a myriad of different pathways Its involvement in both plant development and responses to environmental stimuli implies an impor tant evolutionary advantage allowing these sessile organisms to flourish in many different environments. 29

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30 Figure 1-1. Schematic representati on of tomato ethylene receptor family. Black bars in sensor domain represent putative transmembrane domains. Black boxes in histidine kinase domain represent conserved sub-domain s while black box in response regulator represents conserved aspartate invo lved in phosphorelay. (Klee, 2004)

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CHAPTER 2 ETHYLENE RECEPTOR DEGRADATION CONT ROLS THE TIMING OF RIPENING IN TOMATO Introduction The plant hormone ethylene is a gaseous molecule that regulates multiple processes including germination, organ senescence, stress responses and fruit ripening (Abeles et al., 1992). The role of ethylene in fruit ripening has b een intensively studied in a number of species, but most notably tomato, which has emerged as an important model for the study of fleshy fruit development. Ethylene plays a critical role in determining the timing of ripening and thus provides an attractive point to control fruit ripening th rough genetic modification. Climacteric fruits such as tomato are charac terized by an increase in respiration and a concomitant increase in ethylene biosynthesis just prior to the in itiation of ripening. Ethylene is essential for normal fruit ripening in these specie s and blockage of either ethylene production or perception leads to improper ripening. In tomato fruits, ethylene has pr ofoundly different effects depending on the stage of development. There is a distinct developmen tal switch that occurs upon fruit maturation (Giovannoni, 2 001). Although applied ethylene does not initiate ripening in immature fruits, it does significantly hasten the onset of subsequent ripening (Yang, 1987); the more ethylene to which an immature fruit is expo sed, the earlier it ripens Similar effects have been observed in banana where Burg and Burg (1 962) demonstrated that treatment of immature green banana fruits shortened the time to ripeni ng relative to untreated controls. The mechanism by which fruits measure cumulativ e ethylene exposure is unknown. Genetic analysis in tomato and Arabidopsis has shown that ethylene receptors act as negative regulators of the ethyl ene response pathway (Hua and Meyerowitz, 1998; Tieman et al., 2000). In the absence of the hormone, receptors actively suppress ethylene responses. Upon ethylene binding, that suppression is removed and the response occurs. In tomato there are six 31

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known ethylene receptors ( LeETR1,2,4-6 and NR ) (Wilkinson et al, 1995; Zhou et al., 1996; Lashbrook et al., 1998; Tieman a nd Klee, 1999). Functional analyses have indicated that some Arabidopsis family members have a more impor tant role in ethylene signaling than others. Further, no single loss-of-function mutation has a major effect on ethylene responses, indicating a degree of functional redundancy. However a comple tely different picture emerges in tomato where loss of a single subfamily II receptor, LeETR4, results in increased ethylene sensitivity. Antisense LeETR4 plants show phenotypes consistent wi th a constitutive ethylene response including significantly earlier fruit ripening (Tie man et al., 2000). This mutant phenotype can be restored to wild type by over-expre ssion of the Subfamily I receptor, NR No ethylene-associated developmental effects have been observed in lines with reduced expression of NR (Tieman et al., 2000), LeETR1, LeETR2 or LeETR5 (Tieman and Klee, unpublished results). The receptor signaling model states that the r eceptors are acting as negative regulators of ethylene response. Experimentally it has been shown that reduction of receptor content increases ethylene sensitivity (Hua and Meyerowitz, 1998; Tieman et al., 2000; Cancel and Larsen, 2002; Hall and Bleecker, 2003) while increased receptor content has the opposite effect (Ciardi et al., 2000). We have previously shown that NR and LeETR4 transcripts are up-r egulated in ripening fruits (Wilkinson et al., 1995; Tieman et al ., 2000). Since fruit ripening is dependent upon ethylene action, it seems illogical to increase receptor content and thus decrease ethylene responses. To better understand the role of th e tomato ethylene receptor family during fruit development we have characterized the behavior of both the receptor RNAs and proteins during fruit development. Contrary to the RNA data, prot ein blot analysis showed that receptor protein levels are at their highest during immature fruit development and significantly decline at the onset of ripening. This paradox is explained by observations that ethylene treatment induces a 32

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rapid degradation of receptor prot eins. Here, we present data i ndicating an important role for LeETR4 and LeETR6 in modulating the timing of ripening. Reduced levels of these receptors mediated by either antisense RNA or protein de gradation results in ear lier fruit ripening. Results A Subset of the Receptor Family Shows Ripening-associated Expression and Is Ethyleneinducible in Fruit Expression of all six ethylene receptor ge nes was assayed throughout fruit development to assess stage-specific expression. Quantitati ve RT-PCR (qRT-PCR) anal ysis of each receptor transcript showed low expre ssion of all receptors throughout immature fruit development but upon maturation there was a significant increase in NR ETR4 and ETR6 transcripts (Fig. 2-1). This ripening-associated increase in expression constituted a 10-fold increase in total receptor mRNA content by the breaker stage. Since the re ceptors are negative regulators of ethylene responses, the observed increases in mRNA leve ls during an ethylene-dependent process seems counter-intuitive as an increase in receptors w ould make the fruit less sensitive to ethylene. Ripening-associated gene e xpression can be the conse quence of increased ethylene production. Previous analysis has shown that ETR4 and NR are in fact ethyle ne-inducible in leaf tissue (Ciardi et al ., 2000). To determine if th e receptor gene family is regulated by ethylene in fruit tissue, individual fruits were treated with 50 ppm ethylene for 15 h. Expression analysis of each receptor showed a 9-, 10and 7-fold increase in NR ETR4 and ETR6 respectively (Fig. 22). Expression of ETR1, ETR2 and ETR5 changed little in response to the ethylene treatment. Based on this analysis it appears that expression of NR ETR4 and ETR6 is the consequence of the climacteric increase in ethylene production at the ons et of ripening. 33

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LeETR6 Antisense Lines Show Phenotypes Consis tent with a Constitutive Ethylene Response Single gene knockouts of ethylene receptors in Arabidopsis show no obvious phenotypes and only the subfamily I double mutant (Hall and Bl eecker, 2003; Qu et al., 2007) or triple and quadruple mutants (Hua and Meyerowitz, 1998) show any ethylene-related phenotypes. As previously shown by Tieman et al (2000) this is not the situa tion in tomato as lines having significantly reduced LeETR4 expression show ethylene hyper sensitive phenotypes. When LeETR6 antisense lines were generated, we f ound similar phenotypes to those seen in LeETR4 antisense lines, including a reduc tion of time to ripening by as much as seven days (Table 2-1). Additional ethylene-related phenotypes include ep inastic leaf growth and premature flower senescence (Fig. 2-3). These resu lts indicate gene-specific reduc tions in expression of either LeETR4 or LeETR6 but not the other four receptors (data not shown) results in a hypersensitivity to ethylene, including premature fruit maturation and ripening. Receptor Protein Levels Are Distinctly Differ ent From Transcript Levels During Fruit Development A wealth of recent work has demonstrated that post-translational control is an important component of hormone pathway regulation. In or der to uncover any poten tial post-translational regulation of ethylene receptors, antibodies ag ainst NR, ETR4 and ETR6 were produced. Tissues were collected for a comprehensive study of mRNA and protein expression during fruit development. Measurement of receptor mRNA e xpression showed an increase in transcript levels at the onset of ripening and these levels often remained high until fruits were completely red (Fig. 2-4A). Microsomal memb ranes were isolated to enrich for the receptor proteins and were used for protein quantification. Analysis of protein levels thr oughout fruit development revealed an unexpected result; levels were highest during immature fruit development and significantly declined at the ons et of ripening (Fig. 2-4B). Da ta from cv. Flora-Dade are 34

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presented, although identical results were obtained in the Pearson and Micro-Tom cultivars. This reduction in protein occurred despite increased RNA content (Fig. 2-4C). The results indicate that RNA levels are not predictive of receptor protein content nor the signaling state of the tissue. Rather, there must be an additional level of c ontrol of ethylene perception. Because the drop in receptor content coincided with the onset of auto catalytic ethylene synthesis, we subsequently examined whether ethylene bindi ng induces receptor turnover. Treatment of Leaf and Fruit Tissue with Ethy lene Causes a Rapid Degradation of Receptor Proteins That Likely Occurs Thro ugh a Proteasome-dependent Pathway To determine whether ethylene binding induces receptor degradation, immature fruits and vegetative tissues were exposed to exogenous ethylene. Ethylene treatment of fruits resulted in 4, 5 and 8-fold increases in NR ETR4 and ETR6 mRNA, respectively (Fig. 2-5A). Concomitant with this increase in transcripts there were reductions of 60%, 60% and 40% in NR, ETR4 and ETR6 proteins, respectively, w ithin 2 h and this reduction was sustained throughout the treatment (Fig. 2-5B). Removal of ethylene afte r 8 h of treatment lowered transcripts to pretreatment levels but receptor proteins remained lower even 24 h after treatment ceased (Fig. 25A). Ethylene-mediated receptor degradation was also observed in vegetative tissues. Treatment of seedlings with 50 ppm ethylene for 2 h resu lted in 10-, 5and 13-fold increases in NR ETR4 and ETR6 mRNA, respectively. Similar to the data co llected from immature fruit there was 60%, 40% and 50% reduction in NR, ETR4 and ETR6 prot ein levels, respectively (Fig. 2-6B). Taken together, the results indicate that ethylene e xposure in both vegetative and reproductive tissues results in an immediate drop in re ceptor protein levels that is inde pendent of transcript levels. The 26S proteasome-dependent degradation pathway has emerged as a key point of regulation in many phytohorm one signaling pathways (Guo and Ecker, 2003; Dill et al ., 2004; Gagne et al ., 2004; Dharmasiri et. al ., 2005; Kepinski and Leyser, 2005). To determine if this pathway 35

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is responsible for the turnover of ethylene recep tors, seedlings were treated with the proteasome inhibitor MG132 prior to ethylene treatment. Fo llowing ethylene treatment, levels of each protein actually increased, likely because of ethylen e-induced increases in transcription/transl ation (Fig. 2-6B). Very little is known about mechan isms of ER-associated protein degradation in any system (Meusser et al., 2006). Presumably ubiquitinated proteins are rapidly extracted from the membrane and degr aded by the cytoplasmic 26S proteasome complex. We did not observe larger ubiquitinated forms of immuno-reactive receptors in the microsomal membrane fractions. Even after several-fold concen tration, no receptors could be detected in the soluble fraction (data not shown). Nonethele ss, the MG132 results are consistent with a ubiquitin-mediated receptor degradation. In order to demonstrate that ethylene binding is n ecessary for degradation, seedlings were pre-treated with the ethylene action inhibitor 1-methylcycloprope ne (1-MCP) prior to ethylene treatment. 1-MCP is a competitive inhibitor of ethylene and its attachment to the receptor is essentially irreversible (Sisler, 2006). If ethylene binding is essen tial for the degradation of the receptor, 1-MCP should stabilize the protein. Pr etreatment of tomato seedlings with 1-MCP prevented the ethylene-induced re ceptor degradation (Fig. 2-6B) as well as the ethylene-induced increase in mRNA (Fig. 2-6A), indicating that ethylene bind ing is essential for receptor degradation. To further confirm that ethylene binding is necessa ry for protein degradation we utilized the semi-dominant Nr mutant that has a greatly reduc ed ethylene response. The mutant Nr protein is unable to bind ethylene when heterologously ex pressed in yeast (Klee and Bleecker, unpublished data). Treatment of Nr seedlings with 50 ppm ethylene for 2 h caused a 50% and 62% decrease in ETR4 and ETR6 protei ns (Fig. 2-6B), respectively, but caused significantly less change in the le vel of NR protein. Taken togeth er, the results are consistent 36

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with enhanced receptor degrada tion following ethylene binding. However, we cannot completely exclude the existence of an ethylene-i nduced receptor degr adation machinery. Receptor Levels in Developing Fruit Determine the Timing of Ripening To determine whether ethylene-induced recept or depletion is the cause of the early ripening phenotype seen in ethylene treated fruit, immature fruits were exposed to ethylene while still attached to the plant and then allowed to ripen. Protein and mRNA samples were collected throughout the duration of the experi ment to correlate lower protei n levels with reduced time to ripening. Treated fruits ripened on average three days prior to untreated fruits (Table 2-2). Receptor protein levels were lowered upon treatm ent with ethylene at 15 days post anthesis (DPA) and remained lower than untreated controls throughout fruit development, indicating that lower receptor levels correlate with earlier ripe ning (Fig. 2-7). Transcript data show that the fruits responded to the ethylene treatment and upon removal of the ethylene, transcripts returned to pre-treatment levels (Fig. 2-8). Discussion Upon maturation, tomato fruits undergo a develo pmental transition that is defined by their response to ethylene (Lincoln et al ., 1987). A number of system 1 and/or system 2-associated genes have been identified in fruits. The E4 and E8 genes are excellent examples with E4 being ethylene inducible throughout fruit development (both in response to system 1 and system 2 ethylene) and E8 only being ethy lene-inducible in mature fruit (system 2 specific). While much is known concerning the role of ethylene during ripening its function during the immature phase of fruit development is less well understood. When mature fruits are exposed to ethylene, a ripening program is initiated. While treatment of immature fruits does not initiate ripening it does hasten the onset of ripening; the more the fruit is exposed to ethylene, the earlier it ripens (Burg and Burg, 1962; Yang, 1987). How the fruit m easures cumulative ethylene exposure is not 37

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known. We have provided evidence indicating a specialized role for two receptors, ETR4 and ETR6, in modulating ethylene responses, includi ng fruit maturation. Reduced level of these receptors mediated by either antisense RNA or et hylene-mediated protein degradation results in earlier fruit ripening. Ethylene exposure also resulted in a parallel de pletion of the other ethylene-inducible receptor protein, NR. Our resu lts are consistent with a model in which ethylene receptor content is a major determinant of when fruits initiate the ripening program. Since the receptors are negative regulators of ethylene signali ng, depletion would lead to a progressive increase in hormone sensitivity. When a particular threshold sensitivity is reached, ripening would commence. Alternatively, receptors may act as a brake on ripening initiation. It must be noted that there are othe r elements independent of ethylene that also must be in place for ripening to initiate; most notably the RIN transcription f actor (Vrebalov et al., 2002). Receptor gene expression is low and cons titutive throughout immature fruit development with little difference between any of the family members (Fig. 1). At the onset of ripening there is an increase in expression of NR ETR4 and ETR6 that results in a 10-fold increase in total receptor mRNA content. In contrast to mRNA expr ession, protein levels ar e at their highest in immature fruits and show a significant decrease at the onset of ripening and remain low (Fig. 24B) as a consequence of ethylene exposure. Ethylene binding likely causes a conformational change in the receptors that makes them suscep tible to degradation. In this context it is interesting to note the model of Arabidopsis receptor signaling presen ted by Wang et al. (2006). These authors provide genetic evidence supportive of a transitional state in which a receptor continues to actively suppress downstream ethylene responses after ethylene is bound. This intermediate state subsequently transitions to a receptor-inactive state. Our results suggest that the transmitter-off state may actually be receptor degradation. It would be most interesting to 38

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determine whether the mutations that define th is transition state stab ilize the protein. This receptor degradation is dependent upon the action of the 26S proteasome. At least in some cases, ubiquitination is associated with phosphorylation state ( Hochstrasser 1996). Although the ethylene receptors are considered to be ancestral histidine kinases, ma ny do not possess histidine kinase activity (Moussatche a nd Klee, 2004). However, all of the receptors are functional kinases; those that do not have histidine kinase activity are serine kinases. In light of the degradation of receptors following ethylene bindi ng, it is possible that the phosphorylation state of the receptor may mediate ubiquitin binding. Al though ligand-induced receptor degradation has not been reported for plant hormones, it has b een observed in animals where growth hormone (GH) signaling is mediated by receptor levels (Flores-Morales et al. 2006). The GH receptor, like ethylene receptors, is a membra ne-associated protein in which hormone binding also increases ubiquitin-mediated turnover (Govers et al. 1999). The ethylene receptor family in tomato, like Arabidopsis, is split into two groups with LeETR1 LeETR2 and NR belonging to subfamily I and LeETR4-6 belonging to subfamily II. The Arabidopsis results indicate that there is a distinct difference between subfamily I and II members. With the exception of a subfamily I double mutant ( etr1ers1 ), single and double gene knockouts in Arabidopsis show no obvious phenot ypes. This is likely due to functional redundancy within the gene family. Over-expression of a subfamily II member in an etr1ers1 double mutant cannot rescue the ethylene-hypersensitive phenotype (Wang et al. 2003). In a reciprocal experiment over-expression of a subfam ily I member in a subfamily II triple mutant was sufficient to rescue the ethylene response ph enotype. Together these da ta indicate that the subfamily I receptors are more important than the subfamily II receptors in determining competency to respond to ethylene. The Arabidop sis paradigm does not hold for tomato (Fig. 239

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3, Tieman et al 2000). Plants with reduced expression of either LeETR4 or LeETR6 both subfamily II members, show phenotypes that ar e consistent with an exaggerated ethylene response, including epin astic growth, premature flower se nescence and early fruit ripening. Over-expression of NR in a LeETR4 antisense line is able to rescue the ethylene response phenotype, indicating functional re dundancy between subfamily I a nd II members (Tieman et al. 2000). Apparently there is a large degree of plas ticity within the ethylene signaling pathway and different plants have adapted the signaling co mponents as appropriate for their situation. Plant hormones are involved in most developm ental processes and are critical for abiotic and biotic stress responses. Plants can regulate hormone action through synthesis, catabolism or perception. We have shown that a significant pa rt of the regulation of ethylene responses involves ligand-mediated receptor degradation. Fre quently ethylene responses, particularly those related to stresses, are transitory. In order to shut down an ethylene res ponse, synthesis of new receptors is essential. Ou r results with ethylene e xposure to immature fruits indicate that receptor degradation is apparently an important level of developmental control. Ou r results also indicate that conclusions concerning receptor functions based on RNA levels must be interpreted cautiously. Whether ethylene-med iated receptor turnover and repl enishment are important for other ethylene-mediated processes remains to be determined. 40

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Figure 2-1 Ethylene receptor fa mily mRNA levels during fruit development. qRT-PCR analysis of each receptor transcript in fruit tissue from different stages of fruit development. DPA, days post anthesis; MG, mature green; Breaker, first external color change; Turning, ~30% red color. Expression levels are presented as percentage of total RNA. 41

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Figure 2-2 Ethylene-inducibility of each recepto r mRNA in 20 days post anthesis, immature fruit. qRT-PCR analysis of expressi on of each receptor in response to 10ppm ethylene as a percentage of total RNA (SE). 42

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Figure 2-3 Constitutive ethylene response phenotypes of LeETR6 antisense lines. Epinastic leaf growth (A) and early fl ower senescence (B) of LeETR6 antisense lines. Equivalent aged wild type flowers are shown for comparison (C). 43

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Figure 2-4 Receptor gene expressi on and protein levels show distinct differences during fruit development. qRT-PCR analysis of gene expression expressed as percentage of total RNA (SE) (A) and protein blot analysis (B) throughout fruit development in L. esculentum cv. Flora-Dade (WT). Levels of RNA and protein are also shown for independent LeETR4 (4AS-1, 4AS-2) and LeETR6 (6AS-1, 6AS-2) antisense lines. Values below each receptor protein blot represent the amount of protein in each lane relative to the IMG stage. BiP antibody was used as a loading control and used to normalize protein values. C. Ratio of protein to mRNA. IMG: Immature green stage. Protein quantification was determin ed by densitometric analysis of Western blots using the NCBI software ImageJ. 44

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Figure 2-5 Ethylene binding indu ces degradation of receptors in detached immature fruits. Fruits were exposed to 10 ppm ethylene fo r 8 h. 32h time point represents fruit that were treated for 8 h and left in air for a further 24 h. qRT-PCR analysis of gene expression (A) and protein blot analysis (B) of ethylen e-treated immature fruits. Values below protein blots represent the am ount of protein in each lane relative to the 0 h time point. BiP antibody was used as a loading control and used to normalize protein values. Data represent th e results of two independent experiments (SE). 45

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Figure 2-6 Ethylene binding indu ces degradation of receptor prot eins in vegetative tissue. qRTPCR analysis of gene expression (A) and protein blot analysis (B) of L. esculentum cv. Micro-Tom and Never-ripe (Nr) seedlings after treatment with 50 ppm ethylene for 2 h. Data represent the results of tw o independent experiments (SE). Values below protein blots represent the amount of protein in each lane relative to the 0 h time point. BiP antibody was used as a loading control and used to normalize protein values. 46

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Figure 2-7 Ethylene treatment induces turnover of receptor leading to ear ly ripening fruit. 15 days post anthesis (DPA) fruit were treate d with 50 ppm ethylene while attached to the plant. Relative protein expression of NR, ETR4 and ETR6 normalized to an internal control, BiP. Values are plotted relative to the pretreatment protein level. 47

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Figure 2-8 Ethylene treatment induces expression of receptor mRNAs in attached fruit. qRTPCR analysis of expression of NR, ETR4 and ETR6 in response to 50ppm ethylene as a percentage of total RNA (SE). 48

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49 Table 2-1 Days from anthesis to breaker of LeETR6 antisense lines Line Days % Reduction LeETR6 mRNA WT 43.33 0.71 LeETR6AS-1 38.42 0.90* 85.1 2.4 LeETR6AS-2 37.00 1.46* 75.6 6.4 LeETR6AS-3 35.83 0.78* 72.8 5.8 Values represent mean of at least fifteen fruit for each line. *p-value<0.001 based on Students t-test. Table 2-2 Days from anthesis to brea ker of ethylene treated Microtom fruit Treatment Days Ethylene 45.33 1.41 + Ethylene 41.20 0.80* Values represent mean of at least ten fruit for each tr eatment. Experiment repeated with similar results. *p-value<0.05 based on Students t-test.

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CHAPTER 3 FRUIT-SPECIFIC SUPPRESSION OF THE ETHYLENE RECEPTOR LEETR4 RESULTS IN EARLY RIPENING FRUIT Introduction Tomato is the most economically important vegetable crop grown in the USA. Worldwide, ~70 million metric tons are produced each year. S hort growing seasons in higher latitudes often reduce the number of cultivars a grower can use in outdoor cultivation. One mechanism to circumvent climate-related limitations is to grow early-maturing varieties. This offers a distinct advantage to growers, becau se the first fruit to market in a se ason can garner a higher price. As our knowledge of the molecular control of fr uit ripening expands, biotechnology can provide useful tools for generating early ripening cultivars. While much effort has focused on delayed ripening, particularly as it relates to the ripening hormone ethylene, opportunities to hasten fruit development have been relatively neglected. We have developed a tissue-specific approach to enhance ethylene responses in tomato fruits by depletion of an ethylene receptor. Transgenic fruits mature 5-7 days earlier than controls with no deleterious effect s on yield, fruit size or quality. This technology should be applicable to any fruit w hose ripening is dependent on ethylene. Ethylene is a phytohormone that controls or influences many aspects of plant growth and development (Abeles, 1992). Many of the developm ental processes controlled by ethylene such as senescence, organ abscission a nd fruit ripening are critically important to agriculture. For example, climacteric fruits, such as tomato, ba nana and apple, require an increase in ethylene biosynthesis at maturity in order to ripen. Transgenic plants that are reduced in either synthesis or perception of ethylene exhibit delayed ripening (Oeller et al., 1991; Klee et al., 1991; Wilkinson et al., 1995; Hamilton et al., 1990). Conve rsely, it should be possible to speed up fruit maturation by increasing synthesi s or perception of ethylene. Indeed, it has been known for many 50

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years that ethylene application to immature toma to fruits does cause earlier onset of ripening (Yang, 1987). Because of the pleiotropic negativ e effects of excessive ethylene exposure on plant growth, simply increasing ethylene synthe sis is not practical. Here, we describe an approach involving tissue specific depletion of an ethylene receptor resulting in early ripening fruit. Receptors function as negative regulators of the ethylene response pathway (Hua and Meyerowitz, 1998; Tieman et al., 2000). In the absence of the hormone the receptor actively suppresses ethylene responses and ethylene binding removes this suppression. In practical terms, this means that ethylene sensitivity is inversely correlated with receptor levels; depletion of receptors effectively increases ethylene sensi tivity because there are fewer receptors to inactivate. Recent work on the tomato ethylene r eceptor family has demonstrated that receptor levels during fruit development determine the ti ming of ripening (Kevany et al., 2007). Protein levels are at their highest duri ng immature fruit development and significantly drop at the onset of ripening, facilitating ethylenemediated ripening processes. Ethylene treatment of immature fruits causes receptor degradation and earlier fruit ripening (Kevany et al., 2007). Results LeETR4 RNAi Transgenic Plants Pr oduce Early Ripening Fruit Antisense-mediated reduction in either of two tomato ethylene receptors, LeETR4 or LeETR6 results in premature ripening (Tieman et al., 2000; Kevany et al., 2007). However, these plants are severely affect ed in many aspects of growth a nd it is not clear that the early ripening is a direct effect of transgene expression. We postulate d that fruit-specific suppression of the LeETR4 receptor would result in early ripeni ng without undesirable ethylene-related effects. In order to test the hypothesis a strategy was developed to specifically reduce LeETR4 expression throughout fruit development. To achi eve this goal we generated a construct 51

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consisting of an LeETR4 RNAi inverted repeat sequen ce fused to the promoter of Tfm7 a gene that is expressed specif ically in immature fruits (Santino et al., 1997). Transgenic plants were generated by Agrobacterium-mediated transformation into the tomato cultivar Flora-Dade, a large fruited variety developed fo r Florida fresh tomato production. Transgenic lines that showed no vegetative expression of the silencing construct were identif ied and assayed in a greenhouse for time from anthesis to breaker stage (the firs t visible signs of ripeni ng) in two successive seasons. Three lines that exhibi ted both a reduction in time fr om anthesis to breaker and a reduction of LeETR4 transcript throughout fruit deve lopment were chosen for further characterization. Transgenic line s began ripening between 5 and 7 days earlier than controls (Figure 3-1). No significa nt effects were observed on time from breaker to fully ripe nor were there differences in color of ripe fruits (data not shown). As expected, LeETR4 transcript levels were reduced by as much as 73% in immature fruit and 95% in ripeni ng fruit (Figure 3-2A). While Tfm7 expression has been reported to be immature fruit-specific the RNAi effect persisted into ripening fruit (Figure 3-2A). This gene-specific reduction in expres sion was not seen in nontarget tissues such as leaves (Figure 3-2A). Expression analysis of the other family members showed no decrease in transcript levels in transg enic plants (Figure 3-3) Protein blot analysis confirmed that ETR4 protein levels were co rrespondingly reduced at all stages of fruit development relative to non-transgen ic control fruit (Figure 3-2B). Early Ripening Lines Show Altered Ripening Coordination Performance of the transgenic plants was also assessed in the field using standard commercial practices. Harvests were conducted on a weekly basis in which all fruit that had begun to show external color development were picked and staged for their degree of ripeness. Transgenic plants had more ripening fruit in the first harves t than the control plants and 52

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transgenic lines were st ripped of between 77% and 86% of their fruit within the first two harvests (Figure 3-4). Transgenic Fruits are Indistinguishable from Wild Type Fruits in Horticultural Traits Early maturing varieties of fruits frequently lack the quality of slower ripening varieties. To achieve maximum value it would be advantage ous if early ripening fruits maintain the size, yield and flavor qualities of later ripening cultivars. Altering the time to maturation could potentially impact synthesis of sugars, acids a nd volatile compounds associated with flavor. In addition, fruit size and yield could potentially be negatively affected by earlier maturation and harvest. To address these questi ons, tests were performed to asse ss quality and yield attributes. Analyses of yield and fruit size were conducted in both greenhouse and fieldgrown plants. To assess yield, fruits were harvested at the onset of ripening and individually weighed. Average fruit size for two of the transgenic lines was slightly lower than control fruit but this difference was not statistically significant (Table 3-1 and Table 3-2). To tal yield and the number of fruit per plant were not affected by the pres ence of the transgene (Table 3-1 and data not shown). Tomato flavor is the sum of a complex in teraction between taste and olfaction. Sugars and organic acids stimulate tast e receptors while a set of volatile organic compounds (VOCs) stimulate olfactory receptors (Buttery et al ., 1993; Buttery and Ling, 1993). In order to assess potential effects on flavor, total soluble solids, citric acid, malic acid and the 16 most important VOCs were measured (Table 3-1, Table 3-3 and Table 3-4). Similar results were obtained on both field-grown and greenhouse-gr own materials. Although a very few statistically significant differences in citric acid and some VOCs were observed, they were not repeatable from season to season. All of these differences are well w ithin the range of obs erved season-to-season 53

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variations. Therefore, we concluded that the transgenic and control fruits are essentially equivalent. Discussion While the essential role of ethylene in media ting climacteric fruit ri pening has been known for many years, its role during immature fruit development is only now being elucidated. Previous work has shown that ethylene treatment of immature tomatoes or bananas quantitatively reduces the time to the onset of ripening (Burg and Burg, 1962; Lyons and Pratt, 1964; McGlasson et al., 1975; Yang, 1987) but the mech anism by which fruits measure cumulative ethylene exposure has remained unknown until now. We have identified a potential mechanism by which plants use ethylene receptor levels to measure cumulative ethylene exposure (Kevany et al., 2007). Ethylene binding triggers a ubiqui tin-dependent receptor protein degradation. If receptors are not replaced after ethylene-mediate d degradation, as occurs in immature fruit (Kevany et al., 2007), the fruit will become more sensitive to subsequent ethylene exposure and ripen earlier. The precise, fr uit specific targeting of LeETR4 described here, validates the model. These results define a critical role for LeETR4 in mediating ethylene responses. The special importance of this and another subfamily 2 recep tor, LeETR6, to ethylene responses (Kevany et al., 2007) contrasts markedly with what is known about ethylene perception in Arabidopsis. In Arabidopsis, no single loss-of-function receptor mutant has an obvious effect on ethylene responses and the subfamily 1 receptors seem to have a more important role in ethylene signal transduction (Wang et al., 2003). These results taken together with results described in Kevany et al. (2007) more broadly demonstrat e that plants have the capacity to regulate hormone responses by modulating receptor levels. Tissue-specific modulation of ethylen e sensitivity in transgenic plants has resulted in fruits with altered ripening without an agronomic penalty. A similar appro ach to precisely separate an 54

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advantageous trait from pleitr opic negative effects was employed by Davuluri et al., (2005) who used fruit-specific suppression of DET1 a photomorphogenesis regulatory gene, to increase both carotenoid and flavonoid content in transgenic tomatoes. Previous work on DET1 had reported increases in these phytochemicals in lossof-function mutants but global suppression of DET1 led to a number of serious developmental defect s that would prevent these plants from being used commercially. We present here a crop improvement that shou ld provide significant value to producers. Early season harvests of tomatoes and many ot her horticultural crops usually constitute a substantial percentage of a seas ons profits. The first fruit pick ed can be sold at a premium because supply is generally low and demand is hi gh. We have generated transgenic lines in an elite background that ripen up to a week earlier than their control (Figure 3-1). These lines have none of the developmental defects associated wi th global receptor suppression (Tieman et al., 2000; Kevany et al., 2007) because of fruit-specific suppression of the gene (Figure 3-2A). This approach for engineering early ripening should be applicable to any climacteric fruit species. 55

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Figure 3-1 Fruit-specific ETR4 RNAi Transgenic Lines Pr oduce Early Ripening Fruit (A) Days from anthesis to breaker were measured by tagging open flowers and recording the number of days until the first signs of co lor development. (B) Fruit from transgenic lines are similar in shape and color to control fruit. 56

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Figure 3-2 Suppression of LeETR4 is Fruit-specific. (A) qRT-PCR analysis of ETR4 transcript levels in leaf tissue and throughout fr uit development in control and RNAi transgenic lines. (B) Protein blot analys is of ETR4 protein le vels in control and transgenic lines. IMG, immature green; Breaker, first external color change; Turning, ~30% red color. 57

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Figure 3-3 ETR4 -RNAi Transgenic Plants Have Altere d Ripening Coordination. Fruits showing visible color development were harvested on a weekly basis. Values represent the percent of total fruit harv ested each week SE. 58

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Table 3-1 Weight, yield, br ix, citric acid and malic aci d from field grown fruits Weight Yield/Plant Citric Acid Malic Acid Line (g) (n) (kg) (n) oBrix (n) (mg/gfw) (n) (mg/gfw) (n) Control 135.53.1 218 4.51.4 9 4.10.2 10 2.76.04 5 0.220.02 5 RNAi-1 130.73.9 185 4.53.6 8 3.90.1 10 2.63.06 5 0.230.03 5 RNAi-2 131.43.1 262 4.49.4 12 3.80.1 10 2.69.14 5 0.230.02 5 RNAi-3 142.94.1 137 4.56.3 10 4.00.1 10 2.57.16 5 0.210.03 5 Table displays mean SE. n=number of fruit examined, or plants in case of yield study. Table 3-2 Weight, yield, brix, citric acid and malic acid from greenhouse grown fruits Weight Yield/Plant Citric Acid Malic Acid Line (g) (n) (kg) (n) oBrix (n) (mg/gfw) (n) (mg/gfw) (n) Control 115.44.4 98 3.68.2 3 4.20.1 10 3.10.03 15 0.500.03 15 RNAi-1 103.73.3 64 3.58.5 2 4.2.1 10 2.92.10 15 0.520.04 15 RNAi-2 106.93.2 70 4.13.2 2 3.8.1* 10 3.01.04 15 0.480.03 15 RNAi-3 118.94.4 142 4.69.4 3 4.0.0 10 3.24.03 15 0.480.03 15 Table displays mean SE. n=number of fruit examined, or plants in case of yield study. *Statistically significant p-value<0.05 based on Students t-test Table 3-3 Volatile organic com pounds from field grown fruits Compound Control RNAi-1 RNAi-2 RNAi-3 cis -3-Hexenal -Ionone Hexanal -Damascenone 1-Peneten-3-one 3-Methylbutanal trans -2-Hexenal 2-Isobutylthiazole 1-Nitro-2-phenylethane trans -2-Heptenal Phenylacetaldehyde 5-Methyl-5-hepten-2-one cis -3-Hexenol 2-Phenylethanol 3-Methylbutanol Methyl salicylate 37.486.48 0.050.01 83.3822.22 0.020.00 0.460.09 4.250.47 1.060.17 4.821.16 1.600.33 0.150.03 0.450.06 3.400.87 50.628.52 1.860.39 20.163.27 0.130.01 50.707.99 0.070.01 116.0619.78 0.030.01 0.480.07 4.190.42 1.270.29 5.740.99 1.200.43 0.170.03 0.500.1 3.810.80 64.783.63 1.890.55 18.164.05 0.190.05 66.694.76* 0.060.01 166.2318.54* 0.020.00 0.440.02 4.490.44 1.630.06* 5.660.65 1.920.37 0.200.04 0.510.15 4.570.86 69.198.37 2.610.67 17.194.05 0.170.05 34.765.53 0.040.01 59.514.08 0.020.00 0.400.04 4.620.63 0.860.08 3.790.42 1.120.40 0.130.02 0.370.01 3.020.29 41.604.54 1.490.04 16.563.40 0.140.04 Values are ng g-1 FW h-1 and table displays mean SE with n=6. Statistically significant p-value<0.05 based on Students t test. 59

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60 Table 3-4 Volatile organic com pounds from greenhouse grown fruits Compound Control RNAi-1 RNAi-2 RNAi-3 cis -3-Hexenal -Ionone Hexanal -Damascenone 1-Peneten-3-one 3-Methylbutanal trans -2-Hexenal 2-Isobutylthiazole 1-Nitro-2-phenylethane trans -2-Heptenal Phenylacetaldehyde 5-Methyl-5-hepten-2-one cis -3-Hexenol 2-Phenylethanol 3-Methylbutanol Methyl salicylate 105.0126.92 0.070.01 97.5015.17 0.020.01 0.470.02 7.511.00 2.150.53 2.550.54 0.070.01 0.250.07 0.270.02 3.660.63 53.706.73 1.040.29 47.153.01 0.170.06 105.4129.49 0.080.03 131.1730.44 0.020.01 0.530.22 7.721.11 2.440.67 2.380.65 0.100.00 0.360.13 0.230.06 5.071.64 57.1211.99 1.200.03 48.2011.74 0.230.07 115.4619.79 0.100.02 118.1823.15 0.030.01 0.590.15 8.010.81 2.260.50 2.330.40 0.070.00 0.330.06 0.300.03 3.510.49 50.536.44 1.320.19 43.558.62 0.110.03 122.3250.31 0.080.00 183.4911.45* 0.020.00 0.370.05 5.820.43 2.630.76 2.700.57 0.100.01 0.290.06 0.420.04* 4.341.48 59.991.47 1.670.03* 46.3511.81 0.340.14 Values are ng g-1 FW h-1 and table displays mean SE with n=4. Statistically significant p-value<0.05 based on Students t test.

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CHAPTER 4 IDENTIFICATION OF QTLS THAT MODI FY TIME TO RIPENING AND RIPENINGASSOCIATED ETHYLENE PRODUCTION Introduction The use of wild germplasm has become an important method for crop improvement by todays plant breeders. Genetic diversity in todays domesticated varieties is narrow and land races that could provide traits necessary for crop improvement are being lost every year. The development of introgression lines (ILs) that each contain a single chromosome segment introgressed into an otherwise uniform backgr ound has allowed for the identification of many monogenic traits and quantitative trait loci (QTLs) (Frary et al 2003; Doganlar and Tanksley 2000; Fridman et al 2002). In tomato, a number of introgression lines have been developed from crosses with wild relatives, including L. pennellii (Eshed and Zamir 1995), L. hirsutum; Monforte and Tanksley 2000), and L. peruvianum These libraries are usef ul in identifying QTLs because any phenotypic variation can be associated with the introgressed segment. The entire library can be screened for a particular phenot ype and individual lines can be isolated. Once these lines are identified the introgressed segm ent can be further reduced into sub-ILs by subsequent back crossing. This permits further refinement of the QTL location and potentially, map-based cloning. ILs have been used to identify QTLs responsible for changes in yield, quality and stress responses (Fridman et al 2004; Zamir 2001). Tomato is the most economically important vegetable crop grown wo rldwide, providing significant incentive for crop improvement researc h. Short growing seasons in higher latitudes often reduce the number of varieties a grower can use or can force them to use greenhouses that require a significant investment. Id entification of loci that control the time it takes a fruit to reach maturity could offer a tremendous opportunity for breeders. Early ripe ning loci could be selectively bred into elite varieties that w ould be otherwise impossi ble to grow at higher 61

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latitudes. In addition to trad itional breeding transgenic approaches are being developed to provide options for growers but here I will focus on the traditional method. Regulation of ethylene biosynthe sis at the molecular level is a poorly understood process. Work done in Arabidopsis led to th e identification of prot eins that regulate th e activity of the key biosynthetic enzyme ACC synthase (ACS). The ETO1 and ETO-like proteins posttranslationally regulate the stability of ACS by targeting it to the 26S pr oteasome. Loss-of-function and dominant gain-of-functions mutants were isol ated by screening mutagenized populations for plants exhibiting a triple response in the absence of exogenous ethylene. Where ctr1 mutants exhibit this phenotype because of loss of signa ling capability in the absence of ethylene, eto mutants produce significantly more ethylene than controls because of enhanced ACS stability. An obvious difference between Arabidopsis and tomato is that tomato fruit go through a developmental switch that resu lts in a significant increase in ethylene production. While we understand that a developmental swit ch occurs that triggers the e xpression of particular ACS and ACO isoforms an understanding of the regulation of the expression and activity of these enzymes is lacking in tomato. While we will assess early ripening and increased ethylene biosynthesis separately, there is a significant possibility that a lo cus that leads to increased ethylene production could also lead to early ripening. While increased ethylene production leading to earl y ripening could prove to be easier to understand it could prove less useful in terms of breed ing early ripening lines because excessive ethylene production could lead to undesirable effects. In an effort to identify QTLs associat ed with ripening modification and ethylene production, a screen was performed on a set of ILs derived from the L. hirsutum genome. L. hirsutum was chosen to conduct this experiment because it is an unusual relativ e of the cultivated 62

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tomato. L. hirsutum produces small green fruit that never show any signs of ripening such as softening, carotenoid accumulati on or volatile production. Matur ity can only be assayed by the measurement of ethylene producti on rates and fruit do not reach maturity until approximately 70 days post-anthesis (Grumet et al, 1981). Once fruit reach maturity, there is a sharp increase in ethylene production that p eaks at between 2000-4000 uL kg-1 day-1, roughly ten times that of cultivated tomato varieties. These unusual pheno types suggest the presen ce of loci that may influence ripening and ethylene sy nthesis in unusual ways. Results In a preliminary experiment ethylene emissions of fruit grown in the field were measured and a line (LA 3945) that produces up to four times the amount of ethylene produced by the control at the red stag e was identified. This phenotype was confirmed with greenhouse-grown fruit (Figure 4-1). In an effort to conduct a mo re comprehensive analysis, 35 different lines, each containing a different segment of the L. hirsutum genome, along with both isogenic parents were grown in triplicate in the gree nhouse. A randomized complete block design was utilized as an experimental design in order to control for vari ation within the greenhouse. Flowers of each line were tagged at anthesis to determine the number of days from anthesis to breaker (Figure 4-2). Statistical analysis using D unnetts test identified three lines (3935, 3958 and 3968) that had reduced time to ripening with a p-value<0.05. Fruit from the same plants were collected at the breaker and red ripe stages to measure ethylen e emission rates (Figure 4-3 & 4-4). Statistical analysis using Dunnetts t test identified four (3922, 3935, 3944 and 4005) and three lines (3922, 3934 and 3969) with increased ethylene emission in breaker and red fruit, respectively. In addition to the lines identified by statistical an alysis we included a few lines for each trait assayed that were close to th e p-value<0.05 cut-off. Figure 45 is a representation of the approximate locations of each introgressed segment in the tomato genome. This map was used as 63

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a guide to develop a library of markers from the sequence information available in the SOL Genomics Network database. A post doctoral researcher in our lab has taken over this project and will use the markers to fine map the exact locatio ns of these pieces. The map also contains the locations of all known ethylene receptors and A CC-synthase (ACS) isoforms because they are possible candidates for these QTLs. In order to replicate the results of the first experiment we grew the identified lines in a greenhouse to assess the ripening trait and in th e field to assess ethyl ene emission. Again, each line was grown in triplicate along wi th both isogenic parents. In addition to the selected lines additional lines from the collecti on that overlap the introgressed L. hirsutum segments were included in the analysis to better map the loca tion of each QTL. Greenhouse data for the ripening lines is presented in Figure 4-6. Of the original lines selected only those that had previously showed a statistically signif icant change in ripening (3935, 3958 and 3968) repeated a reduction that was again statistically si gnificant. The other lines (3921, 3955 and 3964) were assayed again because they were close to making our cutoff of 0.05. The fact that these lines did not show a significant reduction in the following season stro ngly supports our confidence in the statistical analysis of the data from the first season. Inte restingly line 3921 did not itself show a reduction in the second season but three overla pping lines 3922, 3923 and 3924 were found to be significantly lower than the control. Due to the la bor intensive nature of measuring ripening time, only lines 3935, 3958 and 3968 will be further characterized. In order to gain a better understanding of the increase in ethylene emission, field grown fruit were harvested at four stages and ethylen e emissions were measured. Figure 4-7 shows the complexity of the trait, with some lines being statistically higher at some st ages and not at others. IL 3922 and two overlapping ILs had a higher leve l of ethylene emission during early ripening 64

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(i.e. breaker) but returned to WT levels by the red stage. IL 3935 and its overlapping lines were low early in ripening but statisti cally higher at the pink and red stages. In accordance with early studies, line 3945 had the highest ethy lene emissions of any of the lin es tested, with more than 2fold higher rates at the pink stage. Additional lines overlapping 3945 had higher ethylene emission at each ripening stage tested. IL 3969 ha d higher emission rates at the breaker and pink stages but returned to WT leve ls by the red stage. The complex nature of this phenotype has made analysis more difficult. While we were princi pally interested in ethylene emissions in fruit we were interested to determine if the increases were limited to the fruit. Ethylene emissions of young leaves were assayed (Figure 4-8). No statistic ally significant differences were seen for any of the lines suggesting th at the increases were confined to ri pening fruit tissue. Interestingly the L. hirsutum isogenic parent (1777) showed the lowest leaf ethylene emissions and this was confirmed by repeating this experiment. Due to the fact that many of the introgressed pieces were quite large, backcrosses were made to the isogenic L. esculentum parent for all lines that repeated in the second season. All of the data displayed from the second season (Figures 4-6 and Figure 4-7) were collected and analyzed by Dr. Valeriano Dal Cin, a post doctoral researcher in our lab. He has also isolated homozygous recombinants from the backcrosses performed during the second season and is currently analyzing the progeny of those recombinants. Due to our interest in receptor function we were intrigued to see that the IL that consistently emitted more ethylene (3945) cont ains a chromosomal segment that potentially encodes the L. hirsutum ortholog of LeETR4 In order to determine which allele is present in 3945, the intron of this gene from the 3945 line was cloned and compared to both the L. esculentum and L. hirsutum sequences (Figure 4-9). The IL was confirmed to contain the L. hirsutum allele. In order to understand whether this allele showed any diff erential expression I 65

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performed qRT-PCR on RNA collected from vegeta tive and reproductive tissues (Figure 4-10). While there are some differences at particular stages the basic trend of expression is similar between the control and IL. In addition to an an alysis of developmental expression both parents and 3945 seedlings were exposed to ethylene in order to determine if the ethylene inducibility of the L. hirsutum allele was altered (Figure 4-11). No si gnificant difference was observed between any of the genotypes. An additional time to ripe ning experiment showed no statistical difference between the L. esculentum parent and 3945. Subsequent marker analysis of the introgression region at 3945 found that both 3944 and 4005 overlap 3945 and all three have increased ethylene emission. This region of the introgressed segment is not the area that contains LhETR4 These additional data suggest that the L. hirsutum allele of LeETR4 is likely not the cause of the increased ethylene phenotype. Discussion Marker assisted breeding is an importa nt technique used to address fundamental problems in plant biology and crop improvement. It is particularly important with the current public attitudes toward genetically modified organisms. Breeders are increasingly going to require the identification of markers that are lin ked to agronomically important traits. A close relationship between researchers and breeders will allow for efficien t introduction of newly identified traits into existing varieties. While a number of different resources are av ailable for mapping traits of interest in tomato, the development of introgression populations using different wild relatives has greatly enhanced this process. These populations facilitate sorting an entire genome down to a small, known segment that can be assayed for a partic ular phenotype. In a ddition, once the tomato genome sequencing project is complete it will be relatively straightforward to screen a population and then search a particular genomic location for candidate genes. 66

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While a great deal of res earch has been done on the ethyl ene biosynthetic pathway the only proteins that have been identified that ac t to modulate this pathway are the ETO proteins. This modulation is accomplished by inhibiting ac tivity of the ACS protein and targeting it for degradation via the 26S proteasome. Identification of additional regulators of ethylene synthesis will broaden our understanding of the factors affec ting synthesis, whether they be positive or negative. We present here a strategy to identify QTLs that control ripening-associated ethylene production by screening L. hirsutum ILs. The choice of the L. hirsutum introgression population was originally made because of the unusual abil ity of this species to produce high levels of ethylene during ripening (Grumet et al., 1981). We have identified six in trogression lines that produce significantly more ethylene during at leas t one stage of fruit ripening (Figures 4-3, 4-4 and 4-7). This increased ethylen e emission is restricted to ri pening fruit as no difference was seen in leaf ethylene biosynthe sis rates in selected introg ression lines (Figure 4-8). A significant amount of research surrounding fruit ripening has been completed in the past century but we still do not unders tand how fruits regulate ripeni ng at the molecular level. In climacteric fruits it is clear that there are two important levels of regul ation, developmental cues and ethylene synthesis. Work done on tomato in the past five years has begun to unravel this phenomenon at both levels with the identification of the RIN and NOR genes as well as the ethylene receptor family (Vreba lov et al., 2002; Kevany et al., 2007). While these findings are important steps, fruit development is a comple x and there are likely many additional regulators of this process. Due to the unus ual nature of ripening, or lack of ripening, in the wild tomato species L. hirsutum, we hypothesized it may allow for the identification of some additional factors regulating fruit development and specifically the onset of ripening. In addition to the ILs with increased ethylene emission our screen iden tified three lines that showed significantly 67

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reduced time from anthesis to breaker that was confirmed in two experiments (Figures 4-2 and 46). Future work will involve finer mapping of the L. hirsutum QTLs. Backcrosses have been performed for each ripening and ethylene line and recombinants are being identified by a postdoctoral researcher in our lab. A library of cleaved amplified polymorphic sequence (CAPS) markers has been generated to precisely map each introgressed segment. Eventually these loci will be cloned by a map-based approach or by use of the tomato genome sequence and will increase our knowledge of both these processes. 68

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Figure 4-1 Ethylene emissions of fully ripe fruit from L. hirsutum IL 3945. Red fruit were sealed in 500 mL jars for ~1 h and ethylene emission was measured by gas chromatography. Values represent mean S E. *Statistically significant values pvalue<0.05 based on Dunnetts t test. 69

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Figure 4-2 Days from anthesis to breaker of tagged fruits from L. hirsutum ILs. Open flowers were tagged at anthesis and the number of days to breaker were recorded. *Statistically significant values p-va lue<0.05 based on Students t test. 70

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Figure 4-3 Ethylene emissi ons of breaker fruit from L. hirsutum ILs. Breaker fruit were sealed in 500 mL jars for ~1 h and ethylene emission was measured by gas chromatography. *Statistically significant values p-value<0.05 based on Dunnetts t test. 71

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Figure 4-4 Ethylene emissions of fully ripe fruit from L. hirsutum ILs. Red fruit were sealed in 500 mL jars for ~1 h and ethylene emi ssion was measured by gas chromatography. *Statistically significant values pvalue<0.05 based on Dunnetts t test. 72

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Figure 4-5 Genomic map showing locations of introgressed regions that contain putative ripening-associated QTLs. Black regions re present locations of QTLs controlling ripening phenotype. White re gions represent locations of QTLs controlling increased ethylene emission phenotype. Dark grey regions repres ent portions of introgressed pieces that are ambiguous based on original mapping done with population. Map also contains locations of all known ethylene receptors and ACC synthase isoforms. 73

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Figure 4-6 Days from anthesis to breaker of tagged fruits from L. hirsutum ILs. Open flowers were tagged at anthesis and the number of days to breaker were recorded. *Statistically significant values p-value<0.05 based on Students t test. CT, control. 74

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Figure 4-7 Ethylene emissions of fruits from field-grown L. hirsutum ILs. Fruit at indicated stages were sealed in 500 mL for ~1 h and ethylene emission was measured by gas chromatography. Breaker, first external signs of ripening; pink, ~70% color development. 75

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Figure 4-8 Ethylene emi ssions of leaves from L. hirsutum ILs. Young leaves were harvested and immediately placed in 5 mL plastic tube s, but were left uncapped for ~1/2 h to permit release of wound-induced ethylene. Af ter sealing, tubes were left for ~ 3 h and then ethylene emission was analy zed by gas chromatography. CT, control. 76

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Figure 4-9 Nucleotide ali gnment of ETR4 genomic sequence. Sequence isolated from L. esculentum L. hirsutum and IL 3945. Alignment was done using ClustalW and presented using Shade Box software. 77

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Figure 4-10 mRNA expression of LeETR4 in WT and the L. hirsutum IL 3945. qRT-PCR analysis of transcript levels in leaf and reproductive tissues Values represent mean SE and presented as % of total RNA. 78

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79 Figure 4-11 mRNA expression of LeETR4 in seedlings of WT, L. hirsutum and IL 3945. qRTPCR analysis of transcript levels in leaf tissue treated with 10ppm ethylene. Values represent mean SE and presented as % of total RNA.

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CHAPTER 5 CONCLUSION The solanaceous species Lycopersicon esculentum tomato, has emerged as the model for studying fleshy fruit development. Because tomato is a climacteric species it is also the species of choice for studying ethylenes involvement in fruit development. Ethyl ene is essential for normal fruit ripening in these species and blockage of either ethylene production or perception leads to improper ripening. In tomato fruits, et hylene has profoundly diff erent effects depending on the stage of development w ith a distinct developmental switch that occurs upon fruit maturation. Treatment of mature fruits results in the initiation of a ripening program. While treatment of immature fruits doe s not initiate ripening, it does si gnificantly hasten the onset of subsequent ripening (Yang, 1987). Our understanding of how fru its measure this ethylene exposure has not been previously determined. The primary objective of this project was to identify the mechanism controlling this phenome non and to gain a bette r understanding of the factors that control ri pening in general. Previous work in our lab showed that reduced accumulation of a single receptor, LeETR4, resulted in fruits that ripen signi ficantly earlier than control fruits (Tieman et al., 2000). In this study LeETR6 reduced expression lines exhibite d similar effects to those of LeETR4 (Table 2-1; Kevany et al., 2007). While these results are consiste nt with the model that the receptors act as negative regulators of ethylene signaling, they do not a ddress the question of how fruits measure cumulative ethylene exposure. Analysis of r eceptor mRNA expression during fruit development indicated a significant increase in receptor mRNAs at the onset of ripening coincident with the increase in ethylene biosynthesi s (Figure 2-1, Figure 2-2). Cont rary to the mRNA expression data, protein blot analysis of NR, ETR4 and ETR 6 showed receptor protein levels highest in immature fruit with a significant decrease at the onset of ripening (Figure 2-4). While these data 80

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are contradictory to the mRNA expression data, they are consis tent with a model in which ethylene binding affects receptor pr otein stability. In an attempt to validate this hypothesis, we exposed fruit and vegetative tissues to ethylene and observed that recepto r proteins are rapidly degraded in response to ethylene and that th is likely occurs thr ough the 26S proteasomedependent pathway (Figure 2-5, Figure 2-6). While ligand binding-induced degradation of receptors has been described in mammalian and yeast systems, this work is the first example in plants. These results led to a hypothesis that redu ced levels of receptor proteins, due to ethylene exposure, control the early ripeni ng in ethylene treated immature fr uit. To test this hypothesis, we treated immature fruits, while still attached to the plant, with ethyle ne and measured protein levels throughout fruit development. Treated fr uits had reduced receptor protein levels after ethylene treatment and these fruits ripened earlier than untreated controls (T able 2-2, Figure 2-7). Together these data are consiste nt with our model that ethylene exposure leads to a degradation of receptor proteins and that ethylene recep tor levels modulate the timing of ripening. While reduction of receptor levels results in early ripening fruit, systemic reduction also causes severe developmental effects that w ould prevent the use of this method for crop improvement (Figure 2-3). A technique to reduce the time from fruit set to the onset of ripening could allow for an increase in the number of varie ties available to farmers in higher latitudes. To generate early ripening lines, we developed a fruit-specif ic RNAi construct to reduce LeETR4 levels only in the fruit. Fruit-specific suppression of LeETR4 resulted in fruits that ripened up to 7 days early (Figure 3-1). While ear ly ripening fruit would be advant ageous they must also retain the same quality as traditional varieties. To test fruit quality I measured average fruit size, yield, soluble solids, malic and citric acid content as well as the most important tomato flavor volatile organic compounds. There was little or no difference between transgenic and control fruits. In 81

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82 addition to providing a unique me thod of crop improvement these data also validate our model that receptor levels in the fruit control the timing of ripening. While biotechnology has provided us with many tools for gene discovery and crop improvement, current public concerns have limited the marketing of transgenic foods. In an effort to identify additional factors that regu late the timing of ripe ning we undertook a genetic approach. A screen of a L. hirsutum introgression population wa s conducted because of the unusual ripening characteristic s and high ethylene biosynthesis levels of this species. Individual lines were screened for reduced time from an thesis to breaker and for increased ripeningassociated ethylene synthesis. Three lines with a reduction of time to breaker were identified and the results were repeatable across seasons. Seven lines that had increased ethylene emissions at the breaker or red stages were identif ied. Due to the large segments of the L. hirsutum genome that are found in these lines they had to be backcrossed to the L. esculentum parent to better map the loci controlling ethylene emissions. Recombin ants that may provide ma terial for a map-based cloning approach for gene disc overy have been identified. The work presented here has significantly increased our understa nding of how ethylene regulates ripening in climacteric fruits. While ethylene is not required for the ripening of nonclimacteric fruits, it can have significant effect s on fruit development in these species. Ethylene can cause damage to the fruits of many different species and our unde rstanding of receptor function could greatly enhance our ability to limit these losse s. Ethylene-related losses in underdeveloped countries often account for a significant proportion of the postharvest losses and are an opportunity for our research to have a serious impact.

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CHAPTER 6 MATERIALS AND METHODS Plant Materials and Growth Conditions L. hirsutum cv. Flora-Dade, LeETR4AS, LeETR6AS and TFM7ETR4 -RNAi lines were grown in a greenhouse set at approximately 27oC. Individual plants were grown in 3 gal pots that were watered twice a day and supplemented with sl ow release fertilizer. Time to ripening data was collected by tagging open flowers and record ing the number of days from anthesis to breaker. L. hirsutum cv. Micro-Tom and Nr plants were grown in a growth chamber under standard conditions (16 h day/8 h night). Field plants were gr own in randomized, replicated plots in Live Oak, FL. Plants were grown using standard commer cial practices in raised plastic mulched beds. Development of Transgenic Plants LeETR4-AS and LeETR6-AS lines were generated by cloning the full-length LeETR4 or LeETR6 coding region into a vector in the antisense orientation under the control of the Figwort Mosaic Virus 35S promoter (Richins et al ., 1987) and followed by the Agrobacterium tumefaciens nopaline synthase ( nos ) 3' terminator. The transgene was introduced into cv. FloraDade by the method of McCormick et al (1986), with kanamycin resistance as a selectable marker. Transgenic lines with a reduction of >70% of LeETR6 transcript were identified (Table 1). The specificity of the transgene was determ ined by quantification of every receptor mRNA from leaf tissue. In each case there was no effect on RNA levels of any other receptor. LeETR4 fruit-specific RNAi lines were genera ted using method outlined by Dexter et al. (2006). Briefly, two overlapping fr agments of coding region were PCR amplified from tomato fruit cDNAs, one 400 bp and 200 bp in length, pr imer sequences found in Table 6-1. The two PCR products were ligated end to end and subse quently ligated into an EcoRI site in the 83

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pMON999 vector that contained the TFM7 fruit specific promot er. The cassette containing the promoter, RNAi fragment and nos terminator were excised from the vector and ligated into the pHK plant expression vector. The transgen e was introduced into cv. Flora-Dade by Agrobacterium-mediated transformation according to McCormick et al. (1986), with kanamyacin resistance as a selectable marker. Pharmacological Treatments Ethylene treatments of plant material were done in sealed 38 L tanks. Treatments were performed using either 10 or 50 ppm, as indicated concentrations in tanks was monitored by gas chromatography. These levels are both w ithin the linear re sponse range for NR and LeETR4 ethylene inducibility (Ciardi et al. 2000). Prot easome inhibitor studies were performed by spraying seedlings with an 80 M MG132 solution (8% DMSO) 4 h prior to 2 h ethylene treatment. Control seedlings were sprayed w ith an 8% DMSO soluti on. 1-MCP treatment of seedlings was performed at 1 ppm in a sealed 38 L tank for 16 h prior to 2 h ethylene treatment. Control seedlings were sealed in identical tank s for the same duration of time. All microsomal membrane preparations were performe d immediately after treatment ended. Recombinant Protein Expression and Antibody Production Coding regions of LeETR4 (a.a. 532-684) and LeETR6 (a.a. 522-688) were amplified with primer pairs ETR4-PF, ETR4-PR, ETR6-PF and ETR6-FR (Table 6-1) from fruit cDNAs generated with the Clontech One-step cDNA Synt hesis kit. PCR products were digested with BamHI and BglII and cloned into the Invitr ogen pTrcXHisA vector and subsequently transformed into the BL21(DE3) (Invitrogen) E. coli strain for recombinant protein expression. 100 mL cultures were grown at 30oC and induced with 1 mM IP TG for 4 h. Cells were spun down at 8,000 x g, resuspended in 10mL of lysis buffer (8 M urea) and pulse sonicated for 1 min. Lysate was spun down at 8,000 x g and supernatant was purified with Ni-NTA affinity column 84

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as directed. Recombinant protein was submitted to Cocalico Biologicals (Reamstown, PA) for antibody production in rabbits using their standard protocol. Antiser um was received and used to probe both antigens to determine antiserum specificity for its respective antigen. RNA Expression Analysis Total RNA extractions were performed us ing the Qiagen RNeasy Mini Kit with subsequent DNase treatment to remove a ny contaminating DNA. RNA was quantified by spectroscopy and visually analyzed on ethidi um bromide-stained gels to assure equal concentrations of all RNAs. Quantitative RT-PCR assays were performed using the Applied Biosystems Taqman One-step RT-PCR kit in an Applied Biosystems GeneAmp 5700 Sequence Detection System as described (Tieman et al. 2001). PCR conditions were as follows, Step 1: 48oC for 30 min, Step 2: 95oC for 10 min and Step 3: 95oC 15 sec and 60oC for 1 min (40X). Primer and probe pairs for each gene assayed can be found in Table 6-1. Levels of LeETR RNAs were quantified using RNAs synthesized by in vitro transcription from plasmids containing the coding region of each gene using a Maxiscript in vitro transcription kit (Ambion, Austin TX USA). Total g of in vitro -transcribed RNA were determined and the in vitro transcription product used for a standard curve in real-tim e RT-PCR analysis. Results are reported as % LeETR RNA in total RNA. Microsomal Membrane Isolation and Protein Blot Analysis Microsomal membrane fractions were is olated from fruit or seedlings with a homogenization buffer containing 30 mM Tris (pH 8.2), 150 mM NaCl, 10 mM EDTA, and 20% (v/v) glycerol with protease inhibitors (1 mM PMSF, 10 g/mL aprotinin, 1 g/mL leupeptin, and 1 g/mL chymostatin) as described (Schaller et al ., 1995). Tissue was homogenized at 4oC using a polytron and then centrif uged at 8,500 x g for 15 min at 4oC. The supernatant was 85

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strained through cheeseclo th then centrifuged at 100,000 x g for 30 min at 4oC and the subsequent membrane pellet was resuspended in 10 mM Tris (pH 7.5), 5 mM EDTA, and 10% (w/w) sucrose with protease inhibitors and stored at -80 oC. Protein concentrations were determined using the Bio-Rad Protein Assay reagent with BSA used for a standard curve. 20 g of total protein was run out for each sample on a 12% Tris-HCl gel and proteins were transferred to a nitrocellulose membrane using the Bio-Rad Mini Trans-Blot cell. Membranes were blocked overnight in 10% Carnation milk/Tris Buffered Saline-Tween (TBST) at 4oC. Membranes were washed 2x5 min in TBST and then incubated with primary anti-ETR4 (1:2000) or anti-ETR6 (1:5000) antibody diluted in 5% Carnation milk /TBST for 1 h. Membranes were subsequently washed 3x10 min in TBST and then incubated with peroxidase conjugated goat anti-rabbit (1:5000) secondary antibody (Kir kegaard & Perry Laboratories, Gaithersburg, Maryland) diluted in 5% Carnation milk/TBST for 45 min. Membra nes were finally washed 3x10 min in TBST. Visualization of signal was perf ormed using the Amersham ECL De tection reagents before being exposed to film. Quantification of bands was acc omplished by using the NCBI imaging software ImageJ (http://rsb.info.nih.gov/ij/). Values were normalized to an anti-BiP (endoplasmic reticulum immunoglobulin bind ing protein) antibody (generously provided by Alan Bennett, Univ. of California, Davis) which was used as an ER-localized loading control. Acid and Soluble Solids Analysis Individual tomato fruit were homog enized in a blender for 30 s and frozen at C until acid analysis. Samples were thawed, centrifuged at 16 000 g for 5 min. The supernatant was analyzed for citric and malic acid content using citric acid and malic acid analysis kits (RBiopharm, Marshall, M I) according to the manufacturer's instructions. Soluble solids are expressed as oBrix which is a measurement of the mass ratio of dissolved sucrose to water in a 86

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liquid. Individual fruit were homoge nized in a blender for 30 s. 1 mL of the homogenate was centrifuged at 16,000 x g for 2 min. ~75 uL of supernatant was applied to a handheld refractometer. Volatile Analysis Ripe tomato fruit from each line and its corresponding control coll ected from the field were harvested and volatiles from pooled fruits were collected on the day after harvest. Fruits collected from plants grown in the greenhouse were analyzed for fruit volatiles immediately after harvest. Tomato fruit volatiles were collected from chopped fruit with nonyl acetate as an internal standard as described by Schmelz et al (2003). Chopped fruit was en closed in glass tubes, air filtered through a hydrocarbon trap (Agilent, Palo Alto, CA) flowed through the tubes for 1 h with collection of the volatile compounds on a Super Q column. Volatiles collected on the Super Q column were eluted with methylene chloride after the addition of nonyl acetate as an internal standard. Volatiles were separated on an Agilent (P alo Alto, CA) DB-5 column and analysed on an Agilent 6890N gas chromatograph with retention times compared to known standards (Sigma Aldrich, St Louis, MO). Volatile levels were calculated as ng g FW h collection. Identities of volatile peaks were confirmed by GCMS as described by Schmelz et al (2001). 87

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88 Table 6-1 Oligonucleotide Primers and Probes ETR4-PF ETR4-PR ETR6-PF ETR6-PR ETR4-RNAi-F1 ETR4-RNAi-R1 ETR4-RNAi-F2 ETR4-RNAi-R2 ETR1-TaqF ETR1-TaqR ETR1-Probe ETR2-TaqF ETR2-TaqR ETR2-Probe NR-TaqF NR-TaqR NR-Probe ETR4-TaqF ETR4-TaqR ETR4-Probe ETR5-TaqF ETR5-TaqR ETR5-Probe ETR6-TaqF ETR6-TaqR ETR6-Probe CCGGATCC CGTGATAACGCCTATATCAGG CCAGATCT GACGATTTGGAATGAGGATAC CCGGATCC CCGAGATCAAACTCATCCAATG CCAGATCT GCCATCTAAATCAGGCAGATG GGAGATCT GGCATTCCTGAATATGGGG CCGGCGCGCCGAGGATACAGCAGGGCTAAG CCGGATCC GGCATTCCTGAATATGGGG GGGGCGCGCCCATCATTCTACTTCCCCGTAGC TTCAAGGATTAAAGGTTTTGGTGAT ATCACATCCAAGGTGTGTAAGCA FAM-ATGAGAATGGTGTTAGCAGGATGGTAACCAAABHQ GCCGTCAGTGTACATGAGAAATTT AGTTTTCTTTTGTCACTTGGTCAGTGT FAM-AGAGGCCACTTATTGTGGCACTAACTGGG-BHQ AGGGAACCACTGTCACGTTTG CTCTGGGAGGCATAGGTAGCA FAM-AGTGAAACTCGGAATCTGTCACCATCCAA-BHQ GGTAATCCCAAATCCAGAAGGTTT CAATTGATGGCCGCAGTTG FAM-AAAGCATGGCTGTCGTTCTTGGGCT-BHQ AGTCATCTTTTAGGAAACGCATGTT AGGAGTACATGAAGGCCTCTGAA FAM-AATACAGAAATCCTTTGGAGCAACCG-BHQ ATTCCAAAGGCAGCCGTTAA GGATGTGGATATGTGGGATTAGAAG FAM-CTCCACATATTCGGACATGCCTAAGGGA-TAMRA BamHI BglII BamHI BglII BglII AscI BamHI AscI Nucleotides in bold face represent restriction sites

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Deveaux, Y., Peaucelle, A., Roberts, G.R., Coen, E., Simon, R., Mizukami, Y., Traas, J., Murray, J.A., Doonan, J.H. and Laufs, P. (2003) The ethanol switch: a tool for tissuespecific gene induction dur ing plant development. Plant J. 36 918-930. Dexter, R., Qualley, A., Kish, C.M., Ma, C.J., Koeduka, T., Nagegowda, D.A., Duderava, N., Pichersky, E. and Clark, D. (2007) Characterization of a petunia acetyltransferase involved in the biosynthesis of the floral volatile isoeugenol. Plant J. 49, 265-275. Dharmasiri, N., Dharmasiri, S. & Estelle, M. (2005) The F-box protein TIR1 is an auxin receptor. Nature 435, 441-445. Dill, A., Thomas, S.G., Hu, J., Steber, C.M. and Sun, T. (2004) The Arabidopsis F-Box protein SLEEPY1 targets gibberellin signa ling repressors for gibberellin-induced degradation. Plant Cell, 16, 1392-1405. Doganlar, S. Tanksley, S.D. and Mutschler, M.A. (2000) Identification and molecular mapping of loci controlling fruit ripening time in tomato. Theor. Appl. Genet. 100, 249-255. Downes, B.P., Stupar, R.M., Gingerich, D.J. and Vierstra, R.D. (2003) The HECT ubiquitinprotein ligase (UPL) family in Arabidopsis: UPL3 has a specific role in trichome development. Plant J. 35 729-742. Dutta, R., Qin, L. and Inouye, M. (1999) Histidine kinases: dive rsity of domain organization. Mol. Micro. 34, 633-640. Farras, R., Ferrando, A., Jasik, J., Kleinow, T., Okresz, L. et al. (2001) SKP1-SnRK protein kinase interactions mediate proteasome binding of a plant SCF ubiquitin ligase. EMBO J 20, 2742-2756. Flores-Morales, A., Greenhalgh, C., Norstedt, G. & Rico-Bautista, E. (2006) Negative regulation of growth hormone receptor signaling. Molec. Endocrinology, 20, 241-253. Frary, A., Doganlar, S., Frampton, A., Fulton T., Uhlig, J., Yates, H. and Tanksley, S. (2003) Fine mapping of quantitat ive trait loci for improved fruit characteristics from Lycopersicon chmielewski chromosome 1. Genome 46, 235-243. Fridman, E., Carrari, F., Liu, Y.S., Fernie, A.R. and Zamir, D. (2004) Zooming in on a quantitative trait for tomato yield using interspecific introgressions. Science 305, 1786-1789. Fridman, E., Liu, Y.S., Carmel-Goren, L., Gur, A., Shoresh, M., Pleban, T., Eshed, Y. and Zamir, D. (2002) Two tightly linked QTLs modify tomato sugar content via different physiological pathways. Mol. Genet. Genomics 266, 821-826. 91

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BIOGRAPHICAL SKETCH Brian Michael Kevany was born in Cleveland, Ohio on September 28, 1980. When he was one he and his mother, father and older brother Thomas moved to North Olmsted, Ohio where his younger brother Daniel was born and where Brian spent his entire childhood. As a young boy Brian enjoyed discovering things in hi s backyard and playing golf, baseball and hockey, with hockey being a sport he played y ear round. When he was in high school he got a job at a local nursery an d really enjoyed learning about plan ts. After high school Brian attended Michigan State University where he majored in horticulture specializi ng in biotechnology. While at MSU he worked as an undergraduate research er in the Postharvest Physiology lab of Dr. David Dilley under the tutelage of Dr. John Go lding. Dr. Golding allowed Brian to become intimately involved in the projects in the lab and fo stered a great interest in plant research. After graduation, Brian joined the Plant Molecula r and Cellular Biology Ph.D. program at the University of Florida as a pre-do ctoral Alumni Fellow. While at UF he worked in the lab of Dr. Harry Klee studying the importance of the tomato ethylene receptor family during tomato fruit development. Upon completion of his Ph.D. degr ee, Brian will enter the lab of Dr. Michael Thomas in the Department of Bacteriology at the University of Wisconsin-Madison as a postdoctoral researcher. 98