METABOLIC ENGINEERING for BETA-ALANINE OVERPRODUCTION and STRESS TOLERANCE in PLANTS: EXPRESSION of Escherichia Coli L

Home , Arogenate dehydratase, Methionine decarboxylase

METABOLIC ENGINEERING FOR BETA-ALANINE OVERPRODUCTION AND STRESS TOLERANCE IN PLANTS: EXPRESSION OF Escherichia coli L- ASPARTATE-ALPHA-DECARBOXYLASE IN TRANSGENIC TOBACCO

WALID MOHAMED MOUNIR FOUAD

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

2004

WALID MOHAMED MOUNIR FOUAD

This dissertation is dedicated to the memory of my beloved father, and to my mother, brothers, and sister. It is also dedicated to my wife Abeer for her love and support. It is also dedicated to my boy Youssef, wishing him a bright future.

ACKNOWLEDGMENTS

I would like to thank my supervisor Dr. Bala Rathinasabapathi for his guidance during my graduate studies. His advice and support were indispensable to the successful completion of this dissertation. I would also like to thank my doctoral committee Dr.

Karen Koch, Dr. Andrew Hanson, Dr. Curt Hannah and Dr. Nancy D. Denslow for serving on my committee and for their contributions to this work.

I would like to thank the previous and current members of my lab, Celia, Suresh,

Joel and Keri for making the lab a nice place to work. I would also like to thank Dr.

Charlie Guy and Dr. Daniel Cantliffe for making growth chamber facilities available to conduct very crucial experiments. In addition, thanks go to Dr. Harry Klee for providing the plant expression vector and giving me access to his lab and Michelle Auldridge for her help in using the HPLC.

I would like to express my deepest appreciation to the Egyptian government and the USAID for the financial support throughout my graduate education in the U.S. My appreciation is extended to the College of Agriculture and Life Sciences for providing me

an assistantship for two semesters that allowed me to finish this work.

I would also like to make special acknowledgments to my relatives in the US for

their great support since my first day in the States. I would like to acknowledge the

support of all my friends especially Gamal Riad and all the Egyptian students, families

and Islamic community in Gainesville.

iv Finally, I would like to thank my family members in Egypt; they were just a phonecall away in expressing their support. Special thanks and appreciation to my wife, who stood by and supported me, by all means, during the last four years, and my little boy who was a great motivation for me, thank you all, with love.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... iv

LIST OF TABLES...... ix

LIST OF FIGURES ...... x

ABSTRACT...... xii

CHAPTER

1 INTRODUCTION ...... 1

2 LITERATURE REVIEW ...... 4

Role of Beta-Alanine in Living Organisms ...... 4 B5 Vitamin Pantothenate...... 4 Beta-Alanine Betaine...... 7 Homoglutathione ...... 7 Beta-Alanine Biosynthesis...... 8 Uracil Degradation ...... 9 Polyamine Oxidation...... 10 Fatty Acid Metabolism as Source of Beta-Alanine...... 10 Prokaryotic L-Aspartate-α-Decarboxylation...... 11 Biosynthesis of Amino Acids in Plant and Its Metabolic Engineering ...... 12 Nitrogen Assimilation ...... 13 GS/GOGAT cycle ...... 13 Asparagine synthetase and its role in nitrogen assimilation ...... 14 Glutamate dehydrogenase assimilates ammonia...... 15 Aspartate-Derived Amino Acids Family...... 15 Sulfur-Containing Amino Acids...... 18 Cysteine...... 18 Methionine ...... 20 Metabolic Engineering of Proline Biosynthesis...... 22 Shikimic Acid Pathway and Biosynthesis of Tryptophan, Phenylalanine, and Tyrosine ...... 24

vi 3 OVEREXPRESSION AND PURIFICATION OF L-ASPARTATE-α- DECARBOXYLASE AND DEVELOPMENT OF POLYCLONAL ANTIBODIES ...... 30

Introduction...... 30 Material and Methods ...... 31 Materials...... 31 Construction of Expression Plasmid ...... 32 Expression of His-Aspartate Dcarboxylase in Yeast...... 33 Yeast Cell Lysate Preparation ...... 33 Purification of His-Aspartate Decarboxylase from Yeast ...... 33 Yeast RNA Etraction and RNA Bot...... 34 Cloning of panD into pET Blue-2 Epression Vctor ...... 34 Induction of the Recombinant ADC in E. coli ...... 35 Purification of ADC-His from E. coli ...... 35 SDS-PAGE Analysis...... 36 Immuonoblotting ...... 37 ADC Immunopreciptation...... 37 Peptide Mass Fingerprinting...... 38 Aspartate Decarboxylase Assay ...... 38 Results...... 39 Cloning the panD Gene from E. coli...... 39 ADC Expression in Yeast...... 40 Expression of Recombinant ADC in E. coli pET System...... 41 ADC Polyclonal Antibodies...... 43 Discussion...... 44

4 E. COLI L-ASPARTATE α-DECARBOXYLASE EXPRESSION IN TRANSGENIC TOBACCO...... 57

Introduction...... 57 Materials and Methods...... 58 Materials...... 58 Construction of the Expression Vector...... 59 Agrobacterium-Mediated Transformation of Tobacco ...... 59 DNA and RNA Blot Analyses...... 60 Genetic Analyses and Identification of Homozygous Lines ...... 61 SDS-PAGE and Immunoblot Analyses...... 61 ADC Activity Assays ...... 62 Germination Tests ...... 63 Growth Tests and High Temperature Stress...... 63 Salt Stress Experiment...... 63 Quantification of Free β-alanine and Aspartate Levels...... 64 Statistical Treatment of Data...... 65 Results...... 65 The E. coli panD Gene is Expressed in Transgenic Tobacco ...... 65

vii The E. coli ADC Protein Expressed in Transgenic Tobacco is Active...... 66 Free Amino Acid Analysis in Transgenic Lines ...... 67 Transgenic Expression of ADC Improves Vegetative Biomass...... 68 Thermotolerance Phenotype in ADC Transgenic Lines...... 69 Transgenic Expression of ADC Improves Tobacco Seed Germination at High Temperature ...... 70 Discussion...... 70

5 SUMMARIES AND CONCLUSIONS...... 91

LIST OF REFERENCES...... 95

BIOGRAPHICAL SKETCH...... 106

viii

LIST OF TABLES

Table page

3-1 Primers used for PCR cloning and screening...... 56

3-2 Peptide mass fingerprinting of the purified recombinant ADC-His...... 56

4-1 Retention times for PTC-derivatives of amino acids standards...... 89

4-2 Beta-alanine and L-aspartate Percentage of the total free amino acids in transgenic tobacco expressing the E. coli panD gene compared to vector control and wild-type...... 90

LIST OF FIGURES

Figure page

2-1 Beta-alanine metabolism in plants...... 26

2-2 Beta-alanine biosyntheses in plants...... 27

2-3 The prokaryotic route of β-alanine biosynthesis...... 28

2-4 Overview of amino acid biosynthesis in plants...... 29

3-1 PCR amplification of the panD gene...... 47

3-2 Restriction analysis of pUC-panD vector...... 47

3-3 Complementation test of the cloned panD open reading frame...... 48

3-4 Restriction analysis of pYES-panD vector...... 48

3-5 PCR amplification of the panD gene from yeast INVSc 1 recombinant strain...... 49

3-6 The amino acid residues of aspartate-α-decarboxylase fused with His-tag at N- terminus encoded by the pYES-panD yeast expression vector...... 49

3-7 SDS-PAGE for protein purified from yeast INVSc 1:pYES-panD yeast strains. ..50

3-8 RNA expression in recombinant yeast. RNA blot showing the panD expression in yeast INVSc 1:pYES-panD strains...... 51

3-9 PanD cloning into pET-Blue-2 vector...... 51

3-10 ADC induction with IPTG in E. coli DE3 cells...... 52

3-11 ADC purification with DEAC-Sepharose column...... 53

3-12 Peptide mass fingerprinting of the purified recombinant ADC-His...... 54

3-13 Recombinant ADC-His immunoprecipitation using anti-ADC polyclonal antibodies...... 55

3-14 Immunoblot analysis of recombinant ADC-His using ADC polyclonal antibodies...... 55

x 4-1 Restriction analysis of pMON-panD vector...... 76

4-2 PCR screening of primary transgenic tobacco...... 77

4-3 PanD integration and RNA expression in primary transgenic tobacco...... 78

4-4 Expression of L-aspartate-α-decarboxylase in two transgenic lines homozygous for the E. coli panD gene...... 79

4-5 PTC-amino acid chromatogram for total free amino acids extracted from tobacco leaves of plants stressed at 350C for one week...... 80

4-6 Transgenic tobacco expression of E. coli panD gene accumulates higher levels of β-alanine, aspartate and total free amino acids...... 82

4-7 Expression of E. coli panD gene in transgenic tobacco improves seedling growth...... 83

4-8 Expression of E. coli panD gene in transgenic tobacco improves seedling biomass...... 84

4-9 ADC-transgenics response’s to salt-stress...... 85

4-10 Expression of E. coli panD gene in transgenic tobacco improves growth and biomass under high temperature...... 86

4-11 Expression of E. coli panD gene in transgenic tobacco significantly improves recovery from heat-stress...... 87

4-12 Expression of E. coli panD gene in transgenic tobacco improves germination at high temperature...... 88

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

METABOLIC ENGINEERING FOR BETA-ALANINE OVERPRODUCTION AND STRESS TOLERANCE IN PLANTS: EXPRESSION OF Escherichia coli L- ASPARTATE-ALPHA-DECARBOXYLASE IN TRANSGENIC TOBACCO

Walid Mohamed Mounir Fouad

December 2004

Chair: Bala Rathinasabapathi Major Department: Horticultural Science

The Escherichia coli L-aspartate-α-decarboxylase, coded by the panD gene,

catalyzes the decarboxylation of L-aspartate to generate β-alanine and carbon dioxide.

This is a pyruvoyl-dependent enzyme that undergoes limited self-processing required for its activities and it is unique to prokaryotes. A hexahistidine-tagged E. coli L-aspartate-α- decarboxylase was overexpressed in E. coli using the inducible promoter in a pET expression vector. Purification of active recombinant protein was accomplished by using immobilized metal (Ni2+) affinity column chromatography and DEAE ion-exchange

chromatography. The identity of the purified protein was confirmed by peptide

fingerprinting using MALDI-TOF analysis and enzyme activities. Polyclonal antibodies,

raised against the native recombinant protein, recognized E. coli L-aspartate-α- decarboxylase in immunoprecipitation and immunoblotting experiments.

xii The E. coli panD gene was expressed under the control of a constitutive promoter

in transgenic tobacco. Transgene expression was verified by assays based on RNA and

immunoblots and enzyme activity in vitro. Analyses of leaf free amino acids showed that

the lines expressing the E. coli panD gene had 1.2-4 fold increase in their β-alanine

levels, and up to 3.7-fold increase in their aspartate and total free amino acid levels.

Homozygous lines expressing E. coli L-aspartate-α-decarboxylase gained an average of

17% and 19% more fresh weight and dry weight respectively than wild-type and vector

control lines when five week-old plants were grown at 300C for 30 days. Homozygous lines expressing E. coli L-aspartate-α-decarboxylase also showed significantly (P ≤ 0.05) improved growth when stressed for one week at 350C. When transferred from 350C to

300C for three weeks, the PanD transgenic lines recovered better than the control plants.

These lines had on average 13% and 11% increased fresh and dry weight respectively

compared to control plants. Homozygous lines expressing E. coli L-aspartate-α-

decarboxylase had significantly (P ≤ 0.05) greater thermotolerance during germination.

At 420C, 95% of two T3 PanD transgenic line seeds germinated after 12 days compared

to 73% for the wild type seeds. The results presented in this dissertation indicated that E.

coli L-aspartate-α-decarboxylase was active in the transgenic host and its expression

resulted in increased growth and thermotolerance. These two traits are valuable for crop

improvement in agriculture and forestry.

xiii CHAPTER 1 INTRODUCTION

β-Alanine, the non-protein amino acid, is found in all organisms. It was found to be

a weak osmoprotectant in bioassays. In plants and microbes, β-alanine is a precursor for

pantothenate, a coenzyme A precursor and an essential vitamin in human and animal

nutrition. In most members of the higher plant family Plumbaginaceae, β-alanine is also

methylated to β-alanine betaine, an osmoprotectant. In some legumes, β-alanine along

with γ-glutamate and cysteine forms the thiol tripeptide homoglutathione, an antioxidant

involved in heavy metal detoxification and protection against reactive oxygen species. β–

Alanine biosynthesis in plants is not well studied; however, there are three hypothesized

pathways: uracil and spermidine degradation, and propionate metabolism.

In Escherichia coli, β-alanine is synthesized via α-decarboxylation of L-aspartate catalyzed by the panD-encoded L-aspartate-α-decarboxylase. This route appears to be

unique to prokaryotes and absent in eukaryotes including yeast and plants. The L- aspartate-α-decarboxylase is an unusual enzyme because of its requirement for pyruvate as a covalently bound, catalytically active prosthetic group. The enzyme is initially translated as an inactive precursor protein (π-protein, 13.8 kDa). It undergoes self- processing at a specific Gly24-Ser25 bond to produce a β-subunit (2.8 kDa) with hydroxyl group at its C-terminus and an α-subunit (11.0 kDa) with a pyruvoyl group at its N-

terminus, derived from serine. The crystal structure of the E. coli L-aspartate-α- decarboxylase demonstrated that the active enzyme is a multimer containing three of each

1 2

α- and β-subunits plus an incompletely processed π-protein. However, there are no

available antibodies specific for L-aspartate-α-decarboxylase.

Metabolic engineering of plants for β-alanine overproduction is an important first

step toward generating transgenic plants capable of accumulating osmotically significant

quantities of β-alanine betaine. Elevated levels of β-alanine in plants could potentially be

useful in engineering plants for increased pantothenate, improving nutritional value and

for engineering plants for stress tolerance. However, plant genes involved in β-alanine

synthesis have not been characterized yet. Therefore, the use of prokaryotic pathway, α-

decarboxylation of L-aspartate, for engineering β-alanine level in plants could be a relatively easier and more efficient strategy as the substrate is available and abundant. An additional advantage of using this route is related to the enzyme properties: L-aspartate-α-

decarboxylase is not subject to feed-back inhibition by the metabolic end products β-

alanine, pantothenic acid, acetyl coenzyme A or coenzyme A. This pathway has not

evolved in plants, so it is unknown whether the L-aspartate-α-decarboxylase-proenzyme

would be properly self-processed and folded in plants, a mechanism required for its

activity. However, there are known examples of other pyruvoyl-dependent enzymes that

go through similar self-processing in plants (e.g. S-adenosyl-L-methionine decarboxylase

and phosphatidylserine decarboxylase).

The overall objective of this study was to examine the heterologous expression of

the E. coli panD gene in transgenic tobacco as a model plant and study the effect of the expressed gene on the β-alanine level in transgenic tobacco. The specific objectives were to (1) overexpress E. coli panD in an efficient expression system for L-aspartate-α-

decarboxylase protein purification, (2) develop polyclonal antibodies specific to L-

aspartate-α-decarboxylase, (3) generate transgenic tobacco expressing the E. coli panD

gene, (4) examine the activities of L-aspartate-α-decarboxylase in transgenic tobacco as

an eukaryotic system and (5) analyze the transgenic plants expressing active L-aspartate-

α-decarboxylase for β-alanine level and stress tolerance.

CHAPTER 2 LITERATURE REVIEW

Role of Beta-Alanine in Living Organisms

Beta-alanine is a non-protein amino acid found in all organisms. Bacteria and yeast mutants deficient in β–alanine biosynthesis are lethal as they are unable to synthesize pantothenate (Cronan, 1980; Merkel and Nichols, 1996; White et al., 2001). In plants, β- alanine is found to increase in response to environmental stress. In a cowpea cell culture,

β-alanine level was increased two-fold within four hours and more than five-fold after 24 hours of heat shock (Mayer et al., 1990). In arabidopsis, β-alanine level was significantly increased when plants were exposed to either heat stress and/or drought stress (Rizhsky et al., 2004; and Kaplan, et al., 2004). In mammals, addition of β-alanine to rat liver during transplantation increased liver resistance to osmotic stress (Vairetti et al., 2002). In preimplantation of mouse embryo, β-alanine was found to be accumulated to high level, protecting against increased osmolarity (Hammer and Baltz, 2003). Therefore, β-alanine itself has a very significant role in all living organisms and its increase could be beneficial for preventing damage from environmental stress. However, the major role for

β-alanine in plants is being a precursor of pantothenate (Figure 2-1). Some plant species use β-alanine as a precursor for two other important metabolites (Figure 2-1).

B5 Vitamin Pantothenate

Beta-alanine is a precursor of pantothenate (B5 vitamin; Figure 2-1). Pantothenate is synthesized by bacteria and plants but not mammals; therefore it is an essential vitamin

4 5

in human and animal nutrition (Smith and Song, 1996). Pantothenate itself is a precursor

of coenzyme A and acyl carrier protein involved in fatty acid metabolism. In bacteria, the

biosynthetic pathway of pantothenate is well characterized and all the genes involved

have been cloned and overexpressed (Cronan et al., 1982; Merkel and Nichols, 1996;

Dusch et al., 1999; Merkamm et al., 2003). There are four enzymes involved in the

pathway distributed in two branches. The first branch has three enzymes. The first is

ketopantoate hydroxymethyltransferase (KPHMT) encoded by the panB gene. It

generates ketopantoate from α-ketoisovalerate (Power and Snell, 1976). The second

enzyme, ketopantoate reductase (KPR) encoded by the panE gene, reduces ketopantoate

to pantoate in NADPH-dependent reaction (Frodyma and Downs, 1998). The last enzyme

in the pathway is pantothenate synthetase (PtS) encoded by the panC gene (Cronan et al.,

1982). PtS catalyzes an ATP-dependent condensation of pantoate and β-alanine to generate pantothenate. Beta-alanine is synthesized in the second branch from L-aspartate by L-aspartate-α-decarboxylase (Williamson and Brown, 1979 and Cronan, 1980)

The pantothenate biosynthetic pathway in plants is not well characterized.

However, there are several experiments indicating that the pathway in plants is similar to

the bacterial pathway. The first experiment was conducted by Savage et al. (1979) who

isolated a ketopantoate auxotroph cell line of Datura innoxia. This mutant was unable to

grow on α-ketoisovalerate whereas supplying either ketopantoate or pantoate promoted its growth. It was suggested that this mutant is deficient in KPHMT activity. However,

there were no detectable activities of KPHMT in wild-type D. innoxia (Sahi et al., 1988).

In conclusion, the pantothenate pathway is operating in plants as the two intermediates

ketopantoate and pantoate were converted to pantothenate (Savage et al., 1979). This

finding was further confirmed by Jones et al. (1994) who fed radioactive 14C-valine to

pea leaf disks and recovered radiolabeled intermediates of pantothenate. In the same year

a study demonstrated the first assay of a plant enzyme involved in pantothenate pathway

was reported (Julliard, 1994). A partially purified enzyme from spinach reduced ketopantoyl-lactone to pantoyl-lactone not ketopantoate. However the purified enzyme

had higher affinity for other substrates indicating that it may not be specific for

pantothenate synthesis. Also, there is no evidence of a plant PtS that uses pantoyl-lactone instead of ketopantoate.

The first report of identifying a plant gene involved in the pantothenate pathway was by Genschel et al. (1999). The panC gene, encoding PtS, was cloned from Lotus japonicus and Oryza sativa. Both clones encoded active enzymes and complemented an

Escherichia coli panC- mutant. Most recently, the arabidopsis gene was also cloned and

PtS enzyme was found to be localized in the cytosol (Ottenhof et al., 2004). In the same

study two arabidopsis genes coding for KPHMT were identified based on sequence

homology. The two genes were cloned and their gene products were active and localized

in mitochondria (Ottenhof et al., 2004). They also reported the identification of a putative

arabidopsis panE gene based on comparing the protein structure of KPR with arabidopsis

putative proteins (Ottenhof et al., 2004).

In conclusion, the pantoate biosynthetic branch in the pantothenate biosynthesis

pathway is operating in plants in the same manner as in bacteria. However, in the β-

alanine branch, L-aspartate-α-decarboxylase was not found in plants. Several attempts to

assay ADC in plants were not successful (Rathinasabapathi et al., 2000). Also, attempts

to identify active ADC using functional complementation of an E. coli panD- mutant with

arabidopsis cDNA expression library were not successful (Ottenhof et al., 2004).

Furthermore, there are no homologues of ADC in the arabidopsis genome based on

conventional sequence similarity search or when using ADC structural properties to find

matches with the predicted arabidopsis proteins (Ottenhof et al., 2004).

Beta-Alanine Betaine

Beta-alanine betaine is an efficient osmoprotectant found in members of the plant

family Plumbaginaceae and some marine algae (Hanson et al., 1994). It as been proposed

that β-alanine betaine is more a suitable osmoprotectant under saline hypoxic conditions

(Hanson et al., 1994). The substrate for β-alanine betaine is β-alanine (Rathinasabapathi

et al., 2000). In Limonium latifolium plants, S-adenosyl-L-methionine-dependent β- alanine-N-methyltransferase generates β-alanine betaine through three sequential N- methylations of β-alanine (Figure 2-1). The enzyme catalyzing this step was purified

(Rathinasabapathi et al., 2001) and its gene was isolated from L. latifolium (Raman and

Rathinasabapathi, 2003). This route is a target for metabolic engineering to improve stress tolerance in crops; therefore understanding and improving β-alanine biosynthesis in plants is beneficial.

Homoglutathione

Most plants synthesize the thiol tripeptide glutathione that performs several important functions such as scavenging of reactive oxygen species and heavy metal detoxification (May et al., 1998). The pathway for glutathione involves two steps: first γ- glutamylcysteine synthetase forms the dipeptide γGlu-Cys and in the second step L- glycine is added to the C-terminus of γGlu-Cys to generate the tripeptide γGlu-Cys-Gly.

However there are at least three other forms of this tripeptide found in some plant

species. These forms are distinguished by the second step’s substrate. In cereals, a serine

residue is used instead of glycine to form hydroxymethyl-glutathione (Klapheck et al.,

1992). In maize, glutamic acid is used in the second step to form the tripeptide γGlu-Cys-

Glu, which was detected in seedlings exposed to cadmium (Meuwly et al., 1995).

Some legumes use β-alanine in the second step to form the thiol tripeptide

homoglutathione (γ-Glu-Cys-β-Ala; Figure 2-1) instead of or in addition to glutathione

(Klapheck et al., 1988). In soybean, common bean, and mungbean homoglutathione is the

major thio-tripeptide in nodules (Matamoros et al., 2003). However it is also found in

roots and leaves of pea and alfalfa (Matamoros et al., 1999). The role of homoglutathione

in legume nodules is believed to be the protection of the nitrogen fixation machinery

against reactive oxygen species (Matamoros et al., 2003). Homoglutathione also acts as a

precursor for homo-phytochelatins that are involved in heavy metal detoxification (Grill

et al., 1986; Klapheck et al., 1995; Oven et al., 2001). A plant homoglutathione

synthetase has recently been characterized at the molecular level (Moran et al., 2000;

Iturbe-Ormaetxe et al., 2002).

Beta-Alanine Biosynthesis

Beta–alanine biosynthesis in plants is not well studied; however, there are three

predicted pathways for its biosynthesis in plants. The three pathways include uracil

degradation, polyamine oxidation and propionate catabolism (Figure 2-2). There are

several studies that provide evidence for the existence of the three pathways; however

there is only one detailed study on the last enzyme in the uracil degradation pathway

(Walsh et al., 2001). In prokaryotes, however, decarboxylation of L-aspartate is the major pathway for β-alanine biosynthesis.

Uracil Degradation

Degradation of uracil to β-alanine is known in animal, bacterial, and plant species.

There are three enzymatic steps in the pathway (Figure 2-2). These enzymes were

purified and their genes were cloned from various non-plant sources (Wasternack et al.,

1979; Xu and West, 1992; Matthews et al., 1992; Gojkovic et al., 2001, Walsh et al.,

2001).

In the first step in the pathway, uracil is reduced by uracil reductase to

dihydrouracil, in a reversible reaction (Figure 2-2). In tomato cell suspension cultures and

Euglena gracilis this step was found to be rate-limiting for uracil degradation (Tintemann et al., 1985). In the second step (Figure 2-2), dihydropyrimidinase catalyzes the ring-

opening hydrolysis of dihydrouracil generating β-ureidopropionate (N-carbomyl-β-

alanine). This enzyme was first characterized in pea (Mazus and Bochowicz, 1966). The

last step catalyzed by β-ureidopropionase (also called N-carbomyl-β-alanine

aminohydrolase or β-alanine synthase) hydrolyzes β-ureidopropionate to produce carbon

dioxide, ammonia and β-alanine (Figure 2-2). This enzyme was partially purified and

characterized from shoots of etiolated maize seedlings and a cDNA for β-

ureidopropionase was cloned from arabidopsis and overexpressed in E. coli (Walsh et al.,

2001). The two upstream enzymes in the plant pathway are not yet characterized.

However there is strong evidence for the operation of this pathway in plants, particularly

in tomato (Tintemann et al., 1985) and Limonium latifolium (Duhaze et al., 2003). In the

latter report the authors fed 3H-uracil to L. latifolium seedlings and recovered 3H β-

alanine. The authors also reported an increase in β-alanine level in roots and shoots of L.

latifolium seedling in response to supplying with N-carbomyl-β-alanine for 10 days.

Polyamine Oxidation

The direct relationship of polyamines to β-alanine was first established by Terano and Suzuki (1978). The authors reported the recovery of [3H]β-alanine in maize after

feeding [3H]spermidine, [3H]spermine or [3H]1,3-diaminopropane. However, in another feeding experiment using tomato fruit, the catabolism of [3H]spermidine but not

[3H]spermine resulted in β-alanine, while [3H]spermine was catalyzed into γ-

aminobutyric acid (Rastogi and Davies, 1989). In L. latifolium, supplying seedlings for

10 days with 1,3-diaminopropane, an intermediate in polyamine catabolism, resulted in

an increase in β-alanine level in the roots (Duhaze et al., 2003). In the polyamine

oxidation predicted pathway Spm and Spd get oxidized by polyamine oxidase (PAO)

generating 1,3-diaminopropane, which get oxidized by diamine oxidase (DAO)

generating 3-aminopropion-aldehyde (Figure 2-2). There are two predicted genes coding

for DAO in the arabidopsis genome. The last step in the pathway is catalyzed by 3-

aminopropion-aldehyde dehydrogenase generating β-alanine (Figure 2-2). There are three predicted genes coding for that enzyme in arabidopsis. However, there are no enzymes or genes linked to β-alanine biosynthesis from polyamines characterized in plants.

Fatty Acid Metabolism as Source of Beta-Alanine

Beta-alanine could also be synthesized directly from malonate semialdehyde, a

reversible reaction catalyzed by β-alanine-pyruvate transaminase (Figure 2-2). This

reaction is not yet confirmed in plants. However, genes encoding omega-amino

acid:pyruvate transaminase have been characterized from soil microorganisms (Yonaha

et al., 1992; Yun et al., 2004). The malonate semialdehyde itself could be derived from

propionate and malonyl-CoA. There are two reports confirming the biosynthesis of β-

alanine from radioactive propionate in wheat, sunflower and pea, (Hatch and Stumpf,

1962) and L. latifolium (Rathinasabapathi, 2002). Also, L. latifolium seedlings that were

supplied with 5 mM propionate for 10 days showed increase in β-alanine level in their roots and shoots (Duhaze et al., 2003). However, there are no studies examining the malonyl-CoA, malonate and acetyl-CoA contribution in β-alanine biosynthesis in plants.

Prokaryotic L-Aspartate-α-Decarboxylation

The prokaryotic route to β-alanine through the α-decarboxylation of L-aspartic acid

was identified (Figure 2-3) and the bacterial L-aspartate-α-decarboxylase (ADC) had been purified and characterized (Williamson and Brown, 1979; and Cronan, 1980). The panD locus coding for ADC was characterized in E. coli (Markel and Nichols, 1996).

Corynebacterium glutamicum and Mycobacterium tuberculosis panD genes were cloned

and overexpressed in microbial expression systems (Ramjee, et. al., 1997; Dusch, et. al.,

1999; Chopra et al., 2002). The crystal structure of ADC was also reported (Albert et al.,

1998).

Unlike other amino acid decarboxylases that require pyridoxal-5'-phosphate,

(Sandmeier et. al., 1994), the bacterial ADC is a pyruvoyl-dependent enzyme. It belongs

to a small group of enzymes that includes S-adenosyl-L-methionine decarboxylase and phosphatidylserine decarboxylase (Xiong et al., 1997; Rontein et al., 2003). Most, if not all, of the enzymes belonging to this group are known to be translated as a proenzyme that undergoes self-processing at a certain serine residue to generate the covalently bound

active group pyruvate (Ramjee et al., 1997; Albert et. al., 1998). In the E. coli ADC the

self-processing takes place between Gly24-Ser25 bond to generate a β-subunit (2.8 kDa)

and an α-subunit (11.0 kDa) with a pyruvoyl group at its N-terminus, derived from serine

(Ramjee et al., 1997). This processing is essential for generating a pyruvoyl as a

catalytically active prosthetic group and hence enzyme activity (Ramjee et al., 1997;

Albert et. al., 1998). Point mutations at Gly24 or Ser25 demolished the self-processing mechanism and hence, the enzyme activity (Schmitzberger et al., 2003; and Kennedy and

Kealey, 2004).

Biosynthesis of Amino Acids in Plant and Its Metabolic Engineering

The synthesis of amino acids is central to plant survival, growth, development, and

environmental adaptations. Some segments of amino acid synthetic pathways interact

with carbon metabolism and certain amino acids are precursors of other amino acids or

secondary products (Figure 2-4). Several carbon metabolites work as precursors for many

amino acids and their availability could control the flux toward amino acid biosynthesis

(Coruzzi and Last, 2000; Coruzzi and Zhol, 2001 and Stitt at al., 2002). At the very early

stage of nitrogen metabolism, two important TCA intermediate metabolites are essential

for nitrogen assimilation. The first one is α–ketoglutarate, the glutamate precursor, and

the second is oxaloacetate, the aspartate precursor (Figure 2-4). Pyruvate also is a

precursor for several amino acids (Figure 2-4). Pyruvate can be directly converted to

alanine or through sequential reactions can generate either valine or leucine. Two other

amino acid precursors are 3-phosphoglycerate and phosphoenolpyruvate (Figure 2-4).

The first is a serine precursor and the second is a shikimic acid pathway precursor,

tryptophan, phenylalanine, and tyrosine are products of this pathway.

Glutamate is the precursor of proline, arginine, aspartate and glutamine; the latter in part contributes in histidine, asparagine and glutamate biosynthesis. Aspartate also acts as a precursor for several amino acid including lysine, threonine, isoleucine and methionine.

Among the above mentioned amino acids, the best studied for metabolic engineering are

the sulfur-containing amino acids, cysteine and methionine, proline for improving stress

tolerance, and aspartate-derived amino acids lysine, threonine and isoleucine, to improve crop nutritional qualities.

Nitrogen Assimilation

Plants use four amino acids to assimilate inorganic nitrogen and transfer the nitrogen containing amino acids from the source organs to the sink. These N-transport amino acids are glutamate and glutamine, which play the major role in nitrogen assimilation, and aspartate and asparagine, which are suitable for organic nitrogen transport and storage. The nitrogen assimilated in these amino acids can be donated for the biosynthesis of other amino acids and nitrogen containing metabolites. Therefore, these four amino acids are the most abundant ones in the phloem (Coruzzi and Last,

2000; Coruzzi and Zhol, 2001).

GS/GOGAT cycle

The nitrogen assimilation pathway involves the combined action of two enzymes.

Glutamine synthetase (GS) catalyzes the incorporation of ammonia into glutamate resulting in glutamine. The second enzyme glutamate synthase (GOGAT; glutamine- 2- oxoglutarate amino transferase), catalyzes the transfer of the amide group from glutamine to α-ketoglutarate to form two molecules of glutamate. There are chloroplast and cytosolic GS isoforms. Their functions are not overlapping as revealed from mutant analyses (Coschigano at al., 1998). The GS-2 mutant deficient in the chloroplast isoform showed a normal level of cytosolic GS-1 isoform, and displayed a conditional lethality in air but grew well when CO2 was increased to 1%. The increased CO2 prevented photorespiration, indicating that the chloroplast isoform (GS2), but not the cytosolic

isoform, has a role in reassimilation of ammonia generated from photorespiration

(Coschigano at al., 1998). The two GS isoforms were overexpressed in transgenic plants and conferred significant improvement in plant growth rate and nitrogen utilization

(Migge et al., 2000; Fuentes et al., 2001; Oliveira et al., 2002). When the chloroplast GS-

2 isoform was overexpressed in tobacco chloroplast, the increased GS-2 activity was correlated with a decrease in the leaf ammonium pool and an increase in the levels of

glutamate by 2.5-fold and glutamine by 2.3-fold compared to the control (Migge at al,

2000). Overexpressing the cytosolic GS-1 in transgenic tobacco has resulted in a

significant improvement of plant growth and nitrogen assimilation under nitrogen-

limiting condition but not under optimum nitrogen fertilization conditions (Fuentes et al.,

2001; Oliveira et al., 2002).

On the other hand, when NADH-GOGAT was constitutively expressed in tobacco

and the transgenic plants grown under greenhouse conditions and supplied with either

nitrate or ammonium, they showed higher total carbon and nitrogen content in shoots and

increased shoot dry weight when plants were entering into the flowering stage, as

compared to control plants (Chichkova et al., 2001). The observed phenotype of the

transgenic plants was interpreted as reflecting a higher capacity to assimilate nitrogen due

to a higher NADH-GOGAT activity. Thus the two enzyme GS and GOGAT are very

important in utilizing nitrogen and their engineering could help increase nitrogen and

hence reduce nitrogen fertilization.

Asparagine synthetase and its role in nitrogen assimilation

The amino acid asparagine is synthesized by asparagine synthetase enzyme (AS)

which catalyzes the amidation of aspartate by glutamine or ammonia. Three routes were

proposed, of which the one utilizing the glutamine-dependent enzyme AS is now

15 considered the major route for asparagine biosynthesis in plants (Coruzzi and Last, 2000;

Coruzzi and Zhou, 2001). Biochemical studies on the partially purified plant enzyme have been hindered by the copurification of a heat-stable inhibitor, the instability of AS in vitro, and the presence of asparaginase activity. Glutamine is the preferred substrate for nearly all the AS enzymes although some evidence indicate that ammonium- dependent asparagine synthesis is possible (Coruzzi and Last; 2000). Overexpression studies indicate that constitutively expressed AS in transgenic arabidopsis led to elevations of soluble seed protein contents, and higher tolerance of young seedlings when grown on nitrogen-limiting media. These changes in transgenic plants were accompanied by higher levels of free asparagine in flowers and developing siliques when compared to wild type (Lam et al., 2003).

Glutamate dehydrogenase assimilates ammonia

The last enzyme involved in ammonium assimilation is glutamate dehydrogenase

(GDH) existing in two isoforms in the mitochondria and chloroplast. It was initially thought that GDH only catalyzes the deamination of glutamate. However, the arabidopsis gdh-1 mutant, which displayed an impaired growth phenotype when grown under conditions of high inorganic nitrogen, provided evidence that the GDH1 gene product functions in both glutamate synthesis and catabolism (Oliveira, at al 1996). It is believed that GDH plays a role in ammonia assimilation under conditions of excess inorganic nitrogen (Miflin and Habash, 2002).

Aspartate-Derived Amino Acids Family

The four aspartate-derived amino acids, lysine, threonine, isoleucine and methionine, are among the essential amino acids whose synthetic pathways have been extensively studied. Aspartate, which is derived from the citric acid cycle, serves as a

precursor for the lysine, threonine and isoleucine; it also provides the carbon backbone

for methionine (Azevedo at al, 1997; Coruzzi and Last, 2000). This pathway is branched

and has many enzymes, most of which are well characterized. The first enzyme in this

pathway is aspartate kinase (AK), which is common for all aspartate-derived amino acid

biosynthesis. AK converts aspartate into aspartate 4-phosphate, using ATP in the

presence of Mg++. This enzyme has multiple isoforms, with different regulatory properties, like differences in their sensitivities to inhibition by lysine and threonine

(Galili 1995). Aspartate 4-phosphate then is converted to aspartate 4-semialdehyde, catalyzed by aspartate semialdehyde dehydrogenase (ASDH), in the presence of NADH+.

The aspartate 4-semialdehyde is a common substrate for two enzymes, homoserine dehydrogenase (HSDH) and dihydrodipicolinate synthase (DHPS).

In the threonine biosynthetic branch, HSDH generates homoserine which is phosphorylated to generate O-phosphohomoserine (OPHS), a common substrate for

methionine in one branch, and threonine and isoleucine in the other branch (Coruzzi and

Last, 2000). There are many isoforms for HSDH with different sensitivities to threonine.

Threonine inhibits HSDS activity, while potassium stimulates its activity. One isoform of

HSDH, the T-form, is highly sensitive to threonine inhibition, a second one, K-form, is

insensitive to threonine inhibition and is highly abundant in the presence of potassium.

One more factor that controls the abundance of one isoform over the other is light. In the

light, chloroplasts have higher internal concentrations of potassium and lower

concentrations of threonine than chloroplasts in the dark. OPHS is converted into

threonine by the action of threonine synthase (TS). TS competes with cystathionine

gamma-synthase (CgS) for their common substrate OPHS. In plants, TS is strongly

17 activated by low concentration of SAM and is inhibited by cysteine. SAM is synthesized from methionine. As SAM increases, it stimulates TS activity, and therefore withdraws

OPHS towards threonine synthesis and away from methionine and SAM synthesis (Hesse and Hoefgen, 2003). Threonine deaminase (TD) is the only unique enzyme in the isoleucine biosynthetic pathway. This enzyme deaminates and dehydrates threonine to give α-ketobutyrate and ammonia. A set of four enzymes carry out sequential-parallel reactions in isoleucine, and valine biosynthetic pathway. Leucine and valine are both synthesized from pyruvate resulting from glycolysis.

In the lysine biosynthetic branch, dihydrodipicolinate synthase (DHPS) is the first enzyme committed to the synthesis of lysine. It catalyzes the condensation of aspartate semialdehyde and pyruvate to form 2,3-dihydrodipicolinate (Coruzzi and Last, 2000).

DHPS is a major control point in lysine synthesis; it is more sensitive to lysine than AK.

In the last step in lysine biosynthesis pathway, lysine is generated by the decarboxylation of meso-2,6-diaminopimelate. Except for the first and last steps, other enzymes in lysine biosynthetic pathway have not been characterized.

Engineering plants for overaccumulation of threonine and/or lysine was investigated early on by using bacterial threonine and lysine feedback-insensitive AK and/or DHPS for plant transformation. The cytoplasmic expression of these genes resulted in elevated level of the two amino acids in leaves; however, increasing lysine content in transgenic lines resulted in abnormal phenotypes (Shaul and Galili, 1993).

When the two genes were expressed in seed, transgenics expressing AK accumulated up to 6-fold higher threonine in their seeds than the wild type. Transgenics expressing DHPS showed an initial increase in free lysine, which declined upon seed maturity. When

investigated, it turned out that the activity of lysine α–ketoglutarate reductase was

significantly high in transgenic plants compared to the control and correlated to the free

lysine reduction (Karchi et al., 1994). When DHPS was expressed in barley, there was a

significant increase up to 14-fold in lysine compared to control, while the AK

overexpression did not alter the free amino acid composition (Brinch-Pederson at al,

1996). Thus, different plants respond differently to elevated levels of lysine and threonine

which reflect differences in their enzymes’ properties. The up-regulation of lysine

catabolism was also detected in Arabidopsis, where transgenics did not show any direct

correlation between the levels of free lysine and the activity of DHPS (Ben-Tzvi et al.,

1996). Free lysine level in arabidopsis is subject to control by two enzymes, lysine α– ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH) encoded by one gene (Tang et al., 1997). Interestingly, Zhu et al., (2001) isolated an arabidopsis knockout mutant in LKR/SDH gene; this mutant over accumulated lysine up to 5-fold in mature seed compared with the wild type. When the bacterial DHPS was overexpressed in this mutant a tremendous increase in seed free lysine level up to 80-fold compared to the wild type was detected (Zhu and Galili, 2003). The elevated lysine was also accompanied by an increase of up to 38-fold in free methionine and reductions in glutamate and aspartate pools. These results support the direct regulatory role of LKR and SDH in lysine catabolism and lysine interaction with the other amino acids in arabidopsis seed (Galili et al., 2001).

Sulfur-Containing Amino Acids

Cysteine

In plants, cysteine is synthesized from serine by two consecutive enzymes, serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL), also named cysteine

synthase (CSase). The last enzyme incorporates sulfur ions to O-acetylserine generating cysteine. Both enzymes have been reported to be localized in plastids, mitochondria and the cytosol of different plant species (Nikiforova et al., 2002; Hesse and Hoefgen, 2003).

Several SAT cDNAs were cloned from different plants and their enzymes showed a wide

range of feedback inhibition by cysteine (Noji and Saito, 2002; Wirtz and Hell, 2003).

Up-regulation for some arabidopsis SAT isoforms was confirmed in response to

cadmium (Howarth et al., 2003), where the increased cysteine production increases

glutathione and synthesis of phytochelatins, required for heavy-metal detoxification.

When bacterial and plant SATs cysteine-sensitive isoforms were overexpressed in

transgenic arabidopsis, tobacco and potato, there was an increase in cysteine and

glutathione in transgenic plants of 2-4 fold compared to wild type levels (Blaszczyk et al.,

1999; Harms et al., 2000; Liszewska et al., 2001; Noji and Saito, 2002). This increase was improved up to 10-fold when a feedback-insensitive arabidopsis SAT isoform was overexpressed in tobacco (Wirtz and Hell, 2003).

The second enzyme involved in cysteine biosynthesis, OAS-TL, was also overexpressed in transgenic plants. Overexpressing OAS-TL in chloroplast or cytosol of transgenic tobacco showed increased accumulation of cysteine and glutathione several- fold compared to the wild type plants (Noji at al, 2001). The tobacco hybrid overexpressing the two isoforms showed elevated accumulation of both cysteine and glutathione. When exposed to sulfur dioxide, the lines expressing the cytosolic form fixed

SO2 into cysteine and glutathione more efficiently than other lines (Noji and Saito, 2002).

Increased cysteine and glutathione in transgenic tobacco overexpressing OAS-TL

confirmed that resistance to oxidative stress in transgenic tobacco was through the

induction of Cu/Zn superoxide dismutase (Youssefian et al., 2001).

In conclusion, the above mentioned studies indicate that both enzymes, SAT and

OAS-TL contribute in controlling the flux toward cysteine biosynthesis. Yet, no study has been done where the two enzymes were overexpressed in transgenic plants, which is expected to improve sulfur assimilation several-fold compared to single gene overexpression.

Methionine

Increasing methionine level in crops is a major target in research because it is a nutritionally important essential amino acid. The first step in methionine synthesis is condensation of cystathionine from cysteine and O-phosphohomoserine (OPHS) by

cystathionine gamma-synthase enzyme (CgS). The OPHS is an intermediate metabolite

in the aspartate family pathway that could be converted directly to threonine by threonine

synthase (TS). It was initially expected that increasing the flux toward threonine via

expressing threonine-feedback-insensitive AK would provide enough OPHS for

methionine synthesis if it was the rate limiting substrate. However, when threonine-

feedback-insensitive AK was overexpressed in tobacco seed, there was no significant

increase in free methionine (Galili, 1995). This result directed attention to the other

substrate, cysteine, and the regulation of CgS as well as its downstream enzymes

cystathionine beta-lyase and methionine synthase, CbL and MS respectively (Galili and

Hoefgen, 2002). Transgenic potato expressing serine acetyltransferase (SAT) in

chloroplasts with a 2-fold increase in cysteine did not exhibit any increase in methionine

(Nikiforova et al., 2002). Thus there is apparently no substrate limitation for methionine

biosynthesis in plants.

At the enzyme level, transgenic potato expressing CbL antisense, exhibiting 50% of the enzyme activity below wild type level, showed a reduction in methionine content

(Maimann et al., 2000). It was also proved that higher reduction in the enzyme activity was lethal, which reflects the importance of this enzyme in methionine biosynthesis.

However, transgenic potato overexpressing CbL did not show significant increase in methionine in spite of increased enzyme activity (Maimann et al., 2001). When the last enzyme in methionine biosynthesis, MS, was overexpressed, transgenic plants did not show detectable increase in methionine (Nikiforova et al., 2002). Thus, the two enzymes

CbL and GS do not seem to influence the flow of metabolites to increase methionine biosynthesis.

On the other hand, arabidopsis mutant mto2 with 40% threonine synthase (TS) activity of that in wild type, showed an increase up to 22-fold in methionine level

(Bartlem et al., 2000). Consistent with this result, transgenic potato expressing TS antisense exhibited up to 239-fold increase in methionine level (Zeh et al., 2001). These results spotlight the interaction of CgS and TS and their competition for their shared substrate, OPHS. Once again, mutation studies on arabidopsis revealed the significant role of CgS for regulation in methionine synthesis. In the mto1 mutant the CgS mRNA was resistant to methionine-dependent degradation, and therefore accumulated methionine up to 40-fold (Chiba et al., 1999). When CgS was overexpressed in arabidopsis the methionine level increased several-fold but it was subject to tissue regulation (Gakiere et al., 2000; 2002; Kim et al., 2002). In contrast, when the same gene was overexpressed in potato, there was no change in the soluble methionine level (Kreft et al., 2003). The results for the transgenic studies demonstrated that not only methionine

biosynthesis is tightly controlled by TS and CgS enzymes but there are other regulations

at the tissue level and these regulations are different between species (Amir et al., 2002;

Galili and Hoefgen 2002; Hesse and Hoefgen, 2003). Hence, the only ways to increase methionine in plants are engineering CgS that is insensitive to methionine feedback or reducing the flux toward threonine synthesis by altering TS expression.

Metabolic Engineering of Proline Biosynthesis

Because of the significant role of proline in osmoregulation and plant tolerance to environmental stress, proline biosynthesis is well known and characterized in plants.

Proline is synthesized from two different substrates, glutamate and ornithine. Proline is synthesized from glutamate via ∆1-pyrroline-5-carboxylate (P5C) by two enzymes, P5C

synthetase (P5CS) and P5C reductase (P5CR). Expression studies in arabidopsis revealed

the important role of P5CS in controlling the flux toward proline biosynthesis. When

plants were exposed to various types of environmental stresses with monitoring of the

transcription level of both genes, P5CS and P5CR, it turned out that P5CS is induced by

dehydration, high salt treatments while P5CR is not (Yoshiba et al., 1995). The P5CS induction was correlated with proline accumulation in the stressed plants. When P5CS was overexpressed in transgenic tobacco, the overproduction of proline enhanced root biomass and flower development in transgenic plants under drought-stress conditions

(Kishor et al., 1995). However, it turned out that this enzyme is subject to proline- feedback inhibition (Zhang et al., 1995). Removal of the feedback inhibition was achieved using site-directed mutagenesis and the mutated gene was overexpressed in tobacco. The transgenic plants expressing the mutated P5CS accumulated 2-fold more free proline under normal conditions and showed improved growth rate under 200 mM

NaCl compared to transgenic plants expressing the wild type P5CS (Hong et al., 2000).

Studying P5CS expression and proline content in two rice cultivars differing in their salt

tolerance indicated that the P5CS expression increased strongly in the salt-tolerant

cultivar exposed to salt stress while the salt sensitive cultivar showed much less induction

(Igarashi at al, 1997). Further studies of transgenic arabidopsis expressing P5CS

antisense confirmed the significant role for P5CS and proline in plant growth and salt

stress. The transgenic plants exhibited morphological abnormalities of epidermal and

parenchymatous cells in their leaves. When exposed to osmotic stress the transgenic was

hypersensitive (Nanjo at al, 1999a).

For further understanding of proline metabolism, studies were conducted to

determine the free proline contents and the P5CS level under different osmotic stress and recovery conditions. It turned out that during recovery, the P5CS declines while another gene, proline dehydrogenase (PDH) is induced; this enzyme catalyzes the first step in proline degradation and conversion to glutamate (Peng et al., 1996). The PDH gene was also significantly induced by exogenously applied proline and inhibited by salt stress.

Transgenic arabidopsis plants expressing PDH antisense were obtained, and showed higher levels of proline than wild type plants (Nanjo et al., 1999b). Moreover it was tolerant to freezing and salinity. Although it is not yet characterized, transgenic plants expressing the feedback-insensitive P5CS with antisense suppression for PDH are expected to accumulate much more free proline which could contribute in conferring higher level of stress tolerance.

Shikimic Acid Pathway and Biosynthesis of Tryptophan, Phenylalanine, and Tyrosine

Shikimic acid is an intermediate in the common pathway of aromatic amino acid tryptophan, phenylalanine, and tyrosine biosynthesis. The shikimate pathway begins with the condensation of phosphoenolpyruvate and erythrose 4-phosphate catalyzed by 3- deoxy-d-arabino-heptulosonate-7-phosphate synthase (Coruzzi and Last, 2000). The final step in this pathway is the formation of chorismate, which is the branch point for biosynthesis of the three amino acid in plastids (Coruzzi and Last, 2000).

For tyrosine and phenylalanine branch, chorismate is catalyzed by chorismate mutase (CM) generating prephenate, which accepts an amine group from glutamate through a reaction catalyzed by prephenate aminotransferase, generating arogenate

(Coruzzi and Last, 2000). Arogenate is the branch point in the tyrosine and phenylalanine biosynthesis pathway. It can either be converted to phenylalanine by the action of arogenate dehydratase or to tyrosine by NADP-dependent arogenate dehydrogenase

(Coruzzi and Last, 2000). Both enzymes are regulated by their amino acid end product.

Additionally, the plastidic CM is activated by tryptophan and inhibited by phenylalanine and tyrosine; however, the cytosolic CM is insensitive (Eberhard et al., 1996).

Chorismate can also be utilized by anthranilate synthase (AS), the first enzyme in the tryptophan biosynthesis branch. It was confirmed by genetic studies that AS is the key enzyme in tryptophan synthesis. When the arabidopsis trp5 mutant, which accumulated 3-fold higher free tryptophan was compared to the wild type, it turned out that its AS was mutated in a region responsible for its feedback inhibition by tryptophan

(Li and Last, 1996). This hypothesis was later confirmed when analyzing transgenic tobacco and rice overexpressing their mutated feedback-insensitive AS. In the AS

transgenic plants, the tryptophan increased several-fold compared to the wild type and

transgenic expressing the wild type tryptophan-sensitive enzyme (Zhang et al., 2001;

Tozawa et al., 2001).

Beta-Alanine

γ-glutamyl- AdoMet Pantoate cysteine NMTase ATP AdoHcy ATP PtS hGSHS N-Methyl β-alanine AMP ADP AdoMet + + PPi Pi NMTase Homoglutathione Pantothenate AdoHcy

N,N-Dimethyl β-alanine

AdoMet NMTase

AdoHcy

Homo- phytochelatins β-Alanine betaine Coenzyme A

Figure 2-1. Beta-alanine metabolism in plants. Pantothenate biosynthesis is predicted to be present in all plants. Pantothenate synthetase (PtS) catalyzes the condensation reaction of β-alanine and pantoate. β-Alanine betaine is an osmoprotectant found in the members of the plant family Plumbaginaceae. It is synthesized through three sequential methylation of β-alanine catalyzed by one enzyme β-alanine-N-methyltransferase (NMTase). Legumes use β-alanine to synthesize the thiol tripeptide homoglutathione involved in heavy metal detoxification.

Propionate Uracil Spm Spd

H2O + O2 NAD PAO Dihydrouracil H2O2 dehydrogenase

NADH Aminopropyl- ∆1-Pyrroline pyrroline Malonyl-CoA Dihydrouracil 1,3-Diaminopropane

H O 2 H2O + O2 Dihydropyrimidinase DAO H2O2 +

NH3

Malonate N-Carbamyl-β-alanine 3-Aminopropion- semialdehyde (β-Ureidopropionate) aldehyde

H2O NAD L-Alanine β-Ureidopropionase + (β-ananine synthase) H2O CO + NH Transaminase 2 3 3-Aminopropion- aldehyde dehydrogenase Beta-alanine

Pyruvate NADH

Figure 2-2. Beta-alanine biosyntheses in plants. The polyamines spermine (Spm) and spermidine (Spd) oxidation is one of the possible routes for β-alanine biosynthesis. The pathway involve two sequential oxidation ractions catalyzed by polyamine oxidases (PAO) followed by diamine oxidases (DAO). Uracil degradation is the second route. Uracil degradation is catalyzed by three enzymes in three sequential steps, the first being reversible. The enzyme that catalyzes the last step is the only characterized enzyme in plants. The third predicted route is from malonate semialdehyde via transamination. Malonate semialdehyde could be generated from propionate or malonyl-CoA.

CO2

Aspartate β-Alanine ADC

Figure 2-3. The prokaryotic route of β-alanine biosynthesis. The α-decarboxylation of L- aspartic acid catalyzed by L-aspartate-α-decarboxylase (ADC) generating β- alanine and carbon dioxide is the major route for β-alanine biosynthesis in E. coli and other prokaryotes.

Cys, Ser, Gly 3-Phosphoglycerate

Phosphoenolpyruvate Trp, Tyr, Phe

Pyruvate Ala, Val, Leu

Asp, Asn, Lys Thr, Met, Ile

Acetyl CoA

Oxaloacetate Citrate

Malate Isocitrate

Fumarate Oxalosuccinate

Succinate α-Ketoglutarate

Glu, Pro, Gln, His, Arg

Figure 2-4. Overview of amino acid biosynthesis in plants. Metabolites generated from glycolysis, citric acid cycle and the calvin cycle provide carbon skeletons for amino acid biosynthesis.

CHAPTER 3 OVEREXPRESSION AND PURIFICATION OF L-ASPARTATE-α- DECARBOXYLASE AND DEVELOPMENT OF POLYCLONAL ANTIBODIES

Introduction

The non-protein amino acid β-alanine is an essential precursor of pantothenate

(vitamin B5) in plants and microorganisms (Cronan, 1980). It was also found to be a

weak osmoprotectant in bioassays (Yancey et al., 1982). In most members of the higher

plant family Plumbaginaceae, β-alanine is methylated to β-alanine betaine, an excellent

osmoprotectant (Rathinasabapathi et al., 2002). A cDNA for β-alanine N-

methyltransferase was recently isolated from a member of the Plumbaginaceae (Raman

and Rathinasabapathi, 2003). Engineering the accumulation of β-alanine betaine in crops

could potentially enhance their stress tolerance.

Despite the importance of β-alanine in the production of pantothenate and β-

alanine betaine, β-alanine synthesis routes in plants have not been elucidated well. In

bacteria, β-alanine is synthesized via α-decarboxylation of L-aspartic acid (Willianson and Brown 1979; and Cronan, 1980) catalyzed by L-aspartate-α-decarboxylase (ADC).

Both ADC and its gene, panD were isolated and characterized from E. coli and other prokaryotes (Merkel and Nichols, 1996; Ramjee et al., 1997; Dusch et al., 1999; Chopra et al., 2002). E. coli ADC is initially translated as an inactive precursor protein (π-protein,

13.8 kD) and undergoes self-processing between a specific Gly24Ser25 bond to produce a

β-subunit (2.8 kD) with hydroxyl group at its C-terminus and an α-subunit (11 kD) with a pyruvoyl group at its N-terminus, derived from serine (Ramjee et al., 1997 and Albert et

30 31

al., 1998). The active enzyme is a multimer with three each of α- and β-subunits and an

incompletely processed π-protein. Aspartate-α-decarboxylation appears to be unique to

prokaryotes and absent in eukaryotes including plants (Naylor et al., 1958;

Rathinasabapathi et al., 2000; White et al., 2001; Ottenhof et al., 2004)

The E. coli panD gene is currently used for metabolic engineering experiments that

aim to increase β-alanine levels in transgenic plants (Chapter 4). Toward this aim, a

highly purified E. coli L-aspartate-α-decarboxylase protein will allow production of

polyclonal antibodies. Such antibodies will be useful for the analysis of the protein level

and processing in transgenic plants. The antibodies would also be useful for screening a

wide range of organisms for the presence of ADC. The objective of the study presented in

this chapter was thus to overexpress E. coli ADC fused with a His-tag, purify the recombinant enzyme to near homogeneity, and develop and characterize polyclonal antibodies against the purified protein.

Material and Methods

Materials

Yeast expression vector pYES NT, Pro-Bond Ni-NTA resin, and pCR 2.1-TOPO cloning kit and secondary antibodies were from Invitrogen (Carlsbad, CA). Bacterial expression vector pET-Blue-2 and E. coli BL21-DE3 were from Novagen (Madison, WI).

L-[U-14C]aspartate (217 mCi/mmol) from ICN Biomedicals (Irvine, CA) was used

without further purification. Yeast and bacterial media, antibiotics, buffers, protein A-

agarose, DEAE-Sepharose fast flow (bead size 45-165 µm) and protease inhibitors were

from Sigma (St. Louis, MO). Gradient SDS-PAGE protein gels, protein stains and PVDF

membranes were from BioRad (Hercules, CA).

Construction of Expression Plasmid

Genomic DNA was isolated from E. coli DH5α (Sambrook et al., 1989) and used

as a template for a polymerase chain reaction (PCR) with forward primer PAND+ and

reverse primer PNAD- (Table 3-1) using the Expand High fidelity PCR system (Roche,

Indianapolis, IN). The reaction in a volume of 50 µL contained 1.5 mM magnesium

chloride, 200 µM dNTPs, and 1 unit of thermostable DNA polymerase. Each PCR cycle included denaturation 940C/30 seconds, 580C/1 min for annealing and 720C extension.

Following 40 cycles in a thermal cycler (GeneAmp 9600, Roche, Indianapolis, IN), the product was separated in an agarose gel and purified from the gel (Sambrook et al.,

1989). The amplified product of 426 bp containing the entire E. coli panD gene was subcloned into pCR2.1-TOPO (Invitrogen Inc., Carlsbad, CA) according to manufacturer’s instructions and sequenced. The sequence information indicated that the

PCR amplified panD gene had G to A mutation at the 78th basepair. This point mutation

resulted in changes in the amino acid residue 26 from Cys to Tyr. To test whether the

cloned gene is capable of encoding an active ADC, the panD gene was subcloned in

the right frame into the pUC-18 vector after the lac promoter. The pUC-panD

vector was used in a complementation test of an E. coli mutant (strain AB543)

defective in β-alanine biosynthesis. For the yeast vector cloning, the panD gene

from the pUC-panD was isolated as an EcoRI fragment and subcloned into EcoRI-

digested pYES2/NT B (Invitrogen, Carlsbad, CA) to get pYES-panD. The pYES vector

has the advantage of His-Tag on the N-terminus, and also the ability to cleave the

His-Tag using enterokinase. The gene insertion and its orientation were confirmed by

restriction digestion. The panD sequence was placed in frame with the polyhistidine

peptide adding 53 amino acid residues to the panD gene product at the N-terminus.

Expression of His-Aspartate Dcarboxylase in Yeast

The pYES-panD was transferred into yeast INVSc 1 strain using the lithium

acetate-polyethylene glycol method. The recombinant yeast was cultivated in SC-U

medium supplemented with 2% (w/v) glucose at 300C overnight. Then the cells were

harvested by centrifugation and resuspended in the induction medium (SC-U medium

0 supplemented with 2% (w/v) galactose) to obtain 0.4 OD600 and incubated at 30 C for 12 h. At the end of the induction period, the cells were harvested by centrifugation and resuspended in sterile distilled water. After removing the water by centrifugation, the cells were stored at –800C until lysate preparation.

Yeast Cell Lysate Preparation

The cell pellet was suspended in buffer A (50 mM sodium phosphate, pH 7.4) containing 1X protease inhibitor mixture as following, 10 µM leupeptin, 0.2 mM 4-(2- aminoethyl) benezenesulfonyl fluoride, 1 µM pepstatin A, 1 µM bestatin, 1 µM E-64 and

1 mM 1,10-phenanthroline. Cells were broken by vigorous mixing using a vortex with acid-washed glass beads and freezing in liquid nitrogen and thawing at 250C in a water

bath. The cell lysate was obtained as a supernatant following centrifugation for 30 min at

6000 g, at 40C.

Purification of His-Aspartate Decarboxylase from Yeast

The crude cell lysate was loaded onto a 2 mL column of Probond equilibrated in buffer A containing the protease inhibitor mixture. The column was washed in buffer A and eluted in buffer A containing 250 mM imidazole. The eluted protein solution was

concentrated in a 10-kD cutoff Centriprep (Millipore, Bedford, MA) centrifugal filter

device.

Yeast RNA Etraction and RNA Bot

Total RNA was extracted from INVSc 1 yeast strain using TRIzol reagent

according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA). Two INVSc 1

colonies harboring pYES-panD were induced with galactose and cells harvested after 0, 10 and 15 hours from the induction. A yeast INVSc 1 strain harboring pYES-lacZ was used as a negative control. Total RNA (5 µg per lane) was separated in formaldehyde 1.2 % (w/v) agarose gel. Equal loading of RNA was verified by ethidium bromide staining. RNA was blotted by capillary transfer onto nylon membranes with 20 x SSC. Hybridization with a 32P-labeled probe was conducted at 420C following the formamide procedure as described (Sambrook et al., 1989) and the membranes were washed at high stringency conditions prior to autoradiography.

Cloning of panD into pET Blue-2 Epression Vctor

The panD gene was amplified from E. coli DH5α genomic DNA template using panDBlue2+ forward primer and panDBlue2-Hi reverse primer (Table 3-1). A BspH I restriction site was introduced on the panD ATG start codon by the forward primer and a

Pvu II restriction site was introduced at the 3’end by the reverse primer. The Advantage-

HF 2 PCR Kit (Clontech; Palo Alto, CA) was used for the PCR reaction. The PCR product was digested with BspH I and Pvu II and ligated directly into Nco I, Pvu II

digested pET-Blue-2 vector. The ligated products were used to transform E. coli

NovaBlue competent cells (Novagen; Madison, WI). Transformants selected for

ampicillin resistance were analyzed for the recombinant plasmid by restriction digestion.

The insert was further verified by DNA sequencing. Thus the panD sequence was placed

between the bacterial ribosomal binding site, right before the panD ATG start codon

without any extra amino acid residues, and polyhistidine peptide fused at the panD 3’ end, resulting in pETB-panD. The vector was introduced into competent BL21-DE3 cells via transformation.

Induction of the Recombinant ADC in E. coli

Recombinant E. coli BL21-DE3 strains harboring pETB-panD or vector alone were grown in LB medium supplemented with 1% (w/v) glucose at 370C. When the culture

reached a cell density of 0.8 OD600, IPTG was added to 0.1 mM final concentration for induction. Cells were harvested after 3.5 h following induction by centrifugation at 5000

X g for 15 min at 40C. Cells were resuspended in BugBuster reagent (Novagen; Madison,

WI) 5 ml g-1 fresh weight of cells, at room temperature for total soluble protein

extraction. Benzonase (Novagen; Madison, WI) 1 µl ml-1 and β-mercaptoethanol to 5 mM final concentration were added. Protease inhibitor mix was added to a final concentration to achieve 10 µM leupeptin, 0.2 mM 4-(2-aminoethyl) benezenesulfonyl fluoride, 1 µM pepstatin A, 1 µM bestatin, 1 µM E-64 and 1 mM 1,10-phenanthroline. The cell suspension was incubated for 20 min at room temperature. Cell debris was removed by centrifugation at 14000 Χ g and protein concentration was determined in the supernatant as described by Peterson (1977) using a Lowry protein assay kit (Sigma; St. Louis, MO).

Purification of ADC-His from E. coli

ProBond purification system (Invitrogen; Carlsbad, CA) was used for ADC-His affinity purification from cell lysate. The column was equilibrated with native binding buffer C (50 mM Tris-HCl, 0.5 M NaCl, 5 mM β-mercaptoethanol, 0.2 X of the protease inhibitor mix and 20 mM imidazole, pH 8.0) then 200 mg crude protein was incubated

with the equilibrated ProBond resin overnight at 40C with slow shaking. After collecting the unbound fraction, the column was washed with 5 column volumes of buffer C. A second wash with 5 column volumes of Buffer C supplemented with 0.5 M NaCl was done. The bound proteins were eluted using 20 ml of buffer C supplemented with 250 mM imidazole. The imidazole was removed from the eluted fraction using a 10-kD cutoff

Centriprep (Millipore, Bedford, MA) centrifugal filter device and the buffer C was substituted with buffer D (50 mM Tris-HCl, pH 7.5, 5 mM β-mercaptoethanol and 1X of the protease inhibitors mix). The second and last step of ADC-His purification employed a DEAE-Sepharose fast flow ion-exchange column, bead size 45-165 µm (Cat # DFF100,

Sigma; St. Louis, MO). The ProBond eluted protein fraction was loaded into 5 mL

DEAE-Sepharose column, equilibrated with buffer D. The column was washed with 10 column volumes of buffer D, and the bound proteins were eluted with a 0 to 0.5 M linear

NaCl gradient in buffer D. Fifteen fractions, 4 ml each, were collected. The purified

ADC-His was detected using SDS-PAGE gels.

Three different rabbits were used for raising polyclonal antibodies against ADC according to a commercial protocol (Cocalico Biological, Reamstown, PA). Two rabbits were injected with SDS-PAGE-separated ADC and one rabbit was injected with purified native ADC. After the initial inoculation, the rabbits were given four boosters of the antigen at 14, 21, 42 and 56 days from the beginning of the protocol.

SDS-PAGE Analysis

SDS-PAGE was performed in 10% to 20% Tris-Tricine gradient PAGE gel (Bio

Rad; Hercules, CA) or 12% (w/v) Tris-glycine polyacrylamide gels. Protein samples were diluted with 2 X SDS-PAGE sample buffer containing 0.1 M Tris-HCl, pH 6.8, 4%

37 w/v SDS, 20% v/v glycerol, 5mM dithiotheritol, and 0.08% w/v bromophenol blue and denatured at 95oC for 10 min. The separated proteins were visualized with Coomassie

Brilliant Blue or silver stain.

Immuonoblotting

The SDS-PAGE-separated proteins were transferred by electroblotting onto a

PVDF membrane. The membranes were incubated with different dilutions of antibodies after blocking with blocking buffer (20 mM Tris-HCl, pH 7.5, 140 mM NaCl, and 5% w/v nonfat dry milk). For anti-His-COOH-alkaline phosphatase conjugated monoclonal antibody, 1:8000 dilution was used. For the primary anti-ADC polyclonal antibodies,

1:20000 dilution was used. The secondary anti-rabbit IgG antibody conjugated to alkaline phosphatase (Sigma; St. Louis, MO) was used at a 1:30000 dilution. After washing, binding of the antibody was recorded using a colorimetric substrate. The alkaline phosphatase activity was detected in 10 ml alkaline phosphatase buffer containing 0.1 M

Tris-HCl pH 9, 0.1 M NaCl, 5 mM MgCl2, 3.3 mg nitro blue tetrazolium and 1.7 mg bromochloroindolyl phosphate.

ADC Immunopreciptation

Recombinant ADC-His was immunoprecipitated using anti-ADC polyclonal antibodies. Purified ADC-His (100 µg) was incubated for 4 h with ADC-antiserum (1:50 dilution) in 100 µl binding buffer (50 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 10 % v/v glycerol, 1% v/v Triton X-100 and 1X of the protease inhibitor mix). Protein A-agarose was then added to the reactions and the mixture was incubated for 4 h. After washing the matrix four times with binding buffer, proteins were solubilized by heating in 50 µl SDS-

PAGE sample buffer and separated by 12% (w/v) SDS-PAGE and proteins transferred to

PVDF membrane and probed with anti-His-COOH monoclonal antibody (1:8000

dilution). Control treatments were conducted in which either no protein was added or

ADC antiserum or protein A-agarose was omitted. One control treatment involved the

use of the pre-bleed at 1:50 dilution.

Peptide Mass Fingerprinting

Purified ADC was separated in SDS-PAGE and stained with Coomassie Brilliant

Blue. Bands corresponding to the three ADC polypeptides were eluted from the gel, and

subjected to proteolytic cleavage using trypsin (Matsudaira, 1989). Peptide fragments

were analyzed for their mass to charge ratio using MALDI-TOF mass spectroscopy.

MALDI mass spectra were acquired on a Voyager DE-Pro MALDI-TOF mass

spectrometer (Applied BioSystems, Framingham, MA) operated in reflector mode. Ions

were accelerated by 22 kV after an extraction delay time of 200 ns. Mass spectra were a

signal average of 150 laser shots and mass-calibrated using a four-point external

calibration or internally calibrated to minor trypsin autolysis peaks at m/z 650 and 3200.

Peptide masses were compared to theoretical peptide masses using MS-Fit search program from the web site (Protein Prospector, http://prospector.ucsf.edu).

Aspartate Decarboxylase Assay

Aspartate decarboxylase was assayed using a radiometric procedure employing L-

[U-14C]aspartate. The assays contained in 50 mM potassium phosphate, pH 7.0, 1.2 mM

-1 of aspartate, 0.2 µCi of L-[U-14C]aspartate (217 mCi.mmol ), 5 mM DTT, and crude or

purified protein in a total volume of 50 µL. Following incubation at 37oC for 1 h, the

reaction was terminated by adding 5 µL of 72% (w/v) trichloroacetic acid and the

14 proteins were removed by centrifugation at 14000 X g for 10 min. Radiolabeled CO2 generated by ADC was trapped during the reaction with Whatman 3 paper saturated with

20% (w/v) KOH. At the end of the reaction period, the reaction products in the reaction

mixture were separated from the substrate using thin layer chromatography 20x20 cm

cellulose plates, 100 micron, (Selecto Scientific, Georgia, USA) developed with 1-

butanol: acetic acid: water (60:15:20, v/v/v), followed by autoradiography. The [14C]β- alanine formed was quantified by isolating the zone corresponding to β-alanine standard.

14 In a variation of the assay, CO2 trapped from the assay was quantified in 50% (v/v)

Ready Gel (Beckman Instruments, Fullerton, CA) in a Beckman liquid scintillation counter. The counting efficiency was 30%.

Results

Cloning the panD Gene from E. coli

A 429 bp fragment containing the aspartate decarboxylase open reading frame

(ORF) was amplified using panD-specific primers and E. coli DH5α genomic DNA as a template (Figure 3-1). The PCR product was cloned using a TOPO-TA cloning kit, and the panD DNA sequence was confirmed by sequencing. There was a single point mutation in the clone’s open reading frame converting amino acid residue 26 from Cys to

Tyr. An alignment of ADC protein sequences from 35 different organisms showed that the Cys26 amino acid residue is not conserved (http://pbil.univ-lyon1.fr/cgi-bin/acnuc-

link-ac2aln?db=Hobacprot&query=Q9EYU1). To examine the gene product activity,

the panD gene was sub-cloned in the right frame into the pUC-18 vector after the lac

promoter (Figure 3-2). The pUC-panD vector successfully complemented the E. coli

mutant (strain AB543) defective in β-alanine biosynthesis after gene induction with IPTG

(Figure 3-3). This confirmed that the cloned gene codes for an active aspartate

decarboxylase enzyme. This clone was used for further subclonings.

ADC Expression in Yeast

The panD sequence from pUC-panD vector was subcloned into pYES-B yeast

expression vector under the control of the Gal inducible promoter. The panD fusion with

the ATG start codon was verified by restriction digestion (Figure 3-4) and sequencing.

The generated pYES-panD vector was used for INVSc 1 yeast strain transformation. The recombinants were selected on SC medium lacking uracil and further confirmed by PCR using panD-specific primers (Figure 3-5) and sequenced (Figure 3-6).

The unprocessed fused His-ADC recombinant protein is expected to have a molecular weight of 19.956 kDa. When it is processed it will give two polypeptides, the β subunit with 8.897 kDa (with 53 extra amino acid residues), and the α subunit with

11.077 kDa. In the first purification, the protein extraction and Ni++ column binding were conducted at 40C while the column washes and protein elution were completed at room temperature. A partially purified fraction that had two intensive bands in the same molecular weight range as the expected processed His-ADC was obtained (Figure 3-7.

A). Further analysis indicated that this protein was degraded upon storage.

For the second purification, methods were modified to allow all extraction, column binding and elution to be conducted at 40C; a protease inhibitor mix was included in the

extraction buffer. SDS-PAGE gel analysis revealed an abundant protein band at the

expected molecular weight. This band was subjected to trypsin digestion and the resulted

peptides were analyzed by MALDI-TOF-MS. The resulting peptide masses (m/z) from

MALDI-TOF-MS were used to identify the purified protein-fingerprinting pattern using

MS-Fit program. The best hit was for a yeast hypothetical 95.1 kDa protein. When

investigated it turned out that this protein is rich in His amino acid residues, which

explains its binding to the Ni++ column.

A third purification was done from yeast using the same conditions as the first. A

partially purified fraction having a protein band within the expected molecular weight

was detected. Figure 3-7 B and C represent the purified fraction of the third purification on silver stained gel and Coomassie blue stained gel, respectively. The two bands

indicated by arrows in figure 3-7 C, named as upper and lower bands, were eluted and subjected to trypsin digestion and separated on MALDI-TOF-MS. The lower band gave

18 peptides while the upper band gave 25 peptides, of all these 15 peptides were identical based on their mass/charge, which confirm the relationship between these two bands.

However, when MS-Fit program was used to identify these peptides, it identified yeast

alcohol dehydrogenase (0.52 Score and 38.6% of sequence covered).

To examine the panD induction at the RNA level, the total RNA was extracted

from two yeast lines that were used for protein purification. The two lines were induced

with galactose and cells harvested after 0, 10 and 15 hours from the induction. A yeast

strain harboring pYES-lacz was used as a negative control. The RNA blot indicated that

the panD mRNA was detected after 10 h of galactose induction (Figure 3-8).

Expression of Recombinant ADC in E. coli pET System

The E. coli panD gene was PCR-amplified (Figure 3-9. A) and cloned in pET-

Blue-2 expression vector with no fusion at the N-terminus, but with a His-tag fusion at

the C-terminus to facilitate protein purification. The gene orientation was confirmed by

restriction digestion (Figure 3-9. B) and sequencing. Then the gene was introduced into

E. coli DE3 strain using CaCl2 transformation. Total soluble proteins, extracted from the

recombinant strain following induction with IPTG, were analyzed in a SDS-PAGE gel. A

42 protein band corresponding to 16.8 kD was visible (Figure 3-10), at low levels without induction and with higher intensities upon IPTG induction, suggesting the expression of the recombinant π protein. Whether some of the expressed protein was also self- processed was not discernible in these gels. Protein fraction from vector control (Figure

3-10. A) lacked this 16.8 kDa band. Radiometric assays for the recombinant ADC indicated that the expressed enzyme was active. Figure 3-10. B, showing the products of such assays separated on a TLC indicated that the endogenous ADC activity in E. coli was too low to detect by this method since protein extracts from vector control E. coli had no detectable activity.

The recombinant ADC was purified from E. coli DE3 overexpressing panD, using nickel affinity chromatography, followed by DEAE anion exchange chromatography.

Protein purified from nickel affinity chromatography had the expected protein bands corresponding to recombinant ADC (Figure 3-11. A, lane L) but further purification was needed. Following DEAE anion exchange chromatography, fractions were analyzed in a silver-stained gel. In some of the fractions, bands corresponding to 16.8 kDa, the π protein, 14 kDa, the α subunit and 2.8 kDa the β subunit were visible, with few other protein contaminants (Figure 3-11. A, lanes F4 and F5). Two bands corresponding to

16.8 kDa and 14 kDa were recognized in immunoblot analyses using monoclonal antibodies against the His tag (Figure 3-11. B, lanes F4 and F5). The recombinant ADC was purified by pooling fractions 4 and 5.

This two-step protocol provided purified recombinant ADC protein with an average specific activity of 256 nmol h-1 mg-1 protein. Together, these results suggest that the

recombinant ADC underwent correct processing and assembled into an active multimer,

despite the presence of the His-tag in its carboxyl terminus.

The purified ADC protein was further analyzed using MALDI-TOF mass spectrometry to confirm the identity of the enzyme. The m/z of tryptic fragments (Figure

3-12) identified E. coli ADC when analyzed using MS-FIT program. A total of 8 mass spectral peaks matched with mass/charge ratios for expected peptides covering 46.4% of the recombinant ADC protein sequence. Out of these, five peaks numbered in Figure 3-

12 as 1 to 5 are listed in table 3-2.

ADC Polyclonal Antibodies

Polyclonal antibodies against the native recombinant ADC or pre-immune serum were tested in an immunoprecipitation experiment. Following incubation of the protein with the antiserum, the protein-antibody complex was precipitated using protein A. The eluted proteins in these complexes were analyzed in an immunoblot probed with anti-His-

COOH monoclonal antibody. A 16.8 kDa protein was recognized only when anti-ADC antibodies were employed for immunoprecipitation (Figure 3-13, lanes 2 and 3). No protein was precipitated if no antiserum was added (Figure 3-13, lanes 1 and 5), or pre- immune serum was used (Figure 3-13, lane 4) or when no protein A was added (Figure 3-

13, lane 6). This shows that compared to the pre-immune serum, the polyclonal antibodies can recognize recombinant ADC in solution.

An immunoblot analysis of purified recombinant ADC is shown in Figure 3-14.

Anti-ADC antibodies recognized a 16.8 kDa band in total protein extracts from the recombinant E. coli (Figure 3-14, lane 2). Little cross-reacting protein was found in the region where native ADC 13.8 kDa would migrate, in cell extracts from the E. coli containing vector alone or AB354, a panD mutant (Figure 3-14, lane 3), suggesting that

the wild-type E. coli does not express ADC at high levels. There were some weak

background protein bands but these were common to both ADC antibodies and pre-

immune antibodies (Figure 3-14, lanes 2-4). The background bands were distinguishable from the over expressed recombinant-ADC protein based on their weaker signals and lack of staining with His-COOH specific antibodies.

Discussion

ADC, an enzyme unique to prokaryotes, undergoes an unusual self-processing. It is also a potential target for antimicrobial agents. Therefore, this enzyme has been purified by others from E. coli (Williamson and Brown, 1979; Ramjee et al., 1997),

Helicobacter pylori (Kwon et al., 2002), and Mycobacterium tuberculosis (Chopra et al.,

2002). The attempt to express the E. coli panD gene in yeast did not meet with success.

There are several reasons that could explain such failure: loss of the vector, inefficient

induction, and protein instability. The first was ruled out by screening the recombinant

yeast strains with PCR using panD specific primers where panD product was generated

from the yeast strains harboring the pYES-panD vector (Figure 3-5). To verify the panD

induction, the total RNA was extracted after 0, 10 and 15 hours from the galactose

induction, and assayed in RNA blot. The RNA blot indicated that the panD mRNA was

detected after 10 h of the galactose induction (Figure 3-8). Most probably, the long N-

terminus tag (53 amino acids) resulted from sequence carried on from the pUC-panD

vector and the fusion with the His-tag in pYES vector is the reason for the instability of the recombinant protein in yeast (Figure 3-6). Even though part of the protein fusion could be removed in vitro using enterokinase, the resulting β subunit will have 22 extra amino acids residues.

In conclusion, the yeast expression system failed to provide purified ADC protein.

This failure is however not due to lack of mRNA expression. Therefore either there could be a problem at the translational or post-translational levels. Such a problem at the post- translational level could be due to the long add-on at the N-terminus which affected the protein folding and stability, or this protein undergoes certain kind of modifications in yeast that altered its MS-fingerprinting. However, to overcome the yeast expression problem, a bacterial expression system was used for ADC induction and purification.

Using a bacterial expression system, the panD gene was cloned in E. coli pET-

Blue2 expression vector (Figure 3-9). The ADC was purified in an active form despite its fusion with a His-tag at its carboxyl terminus (Figure 3-10. B). The purification technique employed a combination of affinity and anion exchange chromatography. In a recent study Schmitzberger et al. (2003) also reported that they had used a similar system to overexpress and purify E. coli ADC coded from various mutant versions of the panD gene (Schmitzberger et al., 2003). Their constructs were designed to provide a His-tag at the N-terminus. While Schmitzberger et al. (2003) provided data on the role of specific mutations on protein processing (Schmitzberger et al., 2003), it was not reported whether

the expressed protein was active or not.

The current study shows that the endogenous expression of ADC in E. coli is

generally low (Figure 3-10). Therefore, recombinant E. coli overexpressing ADC with a

carboxyl terminal His-tag is an efficient way to purify this enzyme. The results shown in

this study indicate that a carboxyl terminal fusion with His-tag does not abolish the

activity of the enzyme. In the current study, polyclonal antibodies against purified native

ADC were raised in rabbits. Two other attempts to make polyclonal antibodies against

SDS-denatured ADC protein in rabbit failed (data not shown), perhaps because of poor antigenicity of the ADC subunits. Immunoprecipitation and immunoblotting experiments

(Figure 3-13 and 3-14) revealed that the antibodies against the native ADC, however, were specific enough for their general use in probing the expression and processing of

ADC in heterologous transgenic systems.

Developing ADC-polyclonal antibodies would be beneficial for analyzing transgenic tobacco expressing the E. coli panD gene for their recombinant ADC expression and activity.

M12 (-)

panD 429 bp (+)

Figure 3-1. PCR amplification of the panD gene. The panD open reading frame was amplified from E. coli chromosomal DNA. M, 100 bp ladder, 1 and 2, two independent PCR reactions.

172 M 3 4 5 6 (-)

(+)

Figure 3-2. Restriction analysis of pUC-panD vector. Lane 1, pUC-18 uncut. Lane 2, pUC-panD uncut. M, λ HinD III digested marker. Lane 3, pUC-18 digested with SacI. Lanes 4-7 pUC-panD digested with SacI. Clones 4 and 7 have the panD in the right orientation.

AB354

D A an B -p 35 C 4 U :p p U 4: C 35 1 B 8 A

Figure 3-3. Complementation test of the cloned panD open reading frame. The panD ORF in pUC-panD vector complemented the E. coli panD- mutant, AB354 growing in a minimal medium. AB354= mutant, AB354:pUC18= vector control and AB354:pUC-panD= panD construct.

172 3 4 5 6 8 9 10 11 12 13 14 M (-)

462 bp (+)

Figure 3-4. Restriction analysis of pYES-panD vector. M, λ HinD III digested marker. Lanes 2-14, pYES-panD; lane 1, pYES-B digested with SacI and XhoI. Clones 2, 3, 5, 7, 10, 12, 13 and 14 have the panD in the right orientation.

12345M

(-)

(+)

Figure 3-5. PCR amplification of the panD gene from yeast INVSc 1 recombinant strain. Lane 1 and 2, INVSc 1:pYES-panD strains. Lane 3, INVSc 1:pYES-lacZ strain, negative control. Lane 4, positive control, pYES-panD vector. Lane 5, negative control, no DNA template. M, 100 bp ladder marker.

MGGSHHHHHHGMASMTGGQQMGRDLYDDDDKVPKDPVWWNSPFRARQGRK VEV+MIRTMLQGKLHRVKVTHADLHYEG/SYAIDQDFLDAAGILENEAIDIWNVTNGK RFSTYAIAAERGSRIISVNGAAAHCASVGDIVIIASFVTMPDEEARTWRPNVAYFEGDNE MKRTAKAIPVQVA

Figure 3-6. The amino acid residues of aspartate-α-decarboxylase fused with His-tag at N-terminus encoded by the pYES-panD yeast expression vector. Underlined is the add-on of 53 amino acid residue that could have affected the protein activity. Italic, the aspartate-α-decarboxylase, when self processed at GS processing site, in bold, it gives β subunit of 77aa and α subunit of 102aa.

A) B) C) MM12 3 4 56M

Figure 3-7. SDS-PAGE for protein purified from yeast INVSc 1:pYES-panD yeast strains. M is polypeptide marker (26.6, 16.95, 14.43, 6.51, 3.94 KDa). A) Silver stained gel for the first purification lane 1 purified protein (2 µg), lane 2 crude extract (5 µg). B) Silver stained gel for the third purification lane 3 purified protein un-boiled (5 µg) lane 4 purified protein boiled for 10 min before loading. C) Coomassie blue stained gel for the third purification, the bands indicated by arrows are the ones recovered from the gel and used for MS analysis.

1234567

Figure 3-8. RNA expression in recombinant yeast. RNA blot showing the panD expression in yeast INVSc 1:pYES-panD strains. Lanes 1 to 6 INVSc 1:pYES-panD harvested after different time from galactose induction; lanes 1 and 4 at 0 h.; lanes 2 and 5 at 10 h.; lanes 3 and 6 at 15 h. Lane 7 INVSc 1:pYES-lacZ; negative control. The lower panel showing ethidium bromide stained total RNA.

A 12345M B 123456 78910M (-) (-)

panD 390 bp 364 bp (+) (+)

Figure 3-9. PanD cloning into pET-Blue-2 vector. A) PCR amplification of the panD. M, 100 bp ladder; lane 1, negative control, no DNA added; lanes 2-5, four independent panD PCR produces. B) Restriction digestion of pET-panD vector to confirm insertion and orientation and restriction digestion of pETB- panD vector. Lane 1 and 2 pET vector; lanes 3-10 pET-panD vector. Lane 1, uncut pET vector; lanes 2-10, vectors digested with PstI and PvuII. Arrows indicate the expected fragment with the right size.

2 2 pET-panD e- e- u u l l B B 0 25 100 500 µM IPTG pET pET A)

26.6 kDa

16.9 KDa π 14.4 KDa

6.5 KDa

Asp

β-Ala

Figure 3-10. ADC induction with IPTG in E. coli DE3 cells. A) SDS-PAGE (15% w/v Tris-glycine gel) of crude protein extracted from DE3 strains harboring pETBlue-2 or pET-panD vector. DE3:pET-panD bacteria was induced with different IPTG concentrations (0, 25, 100 or 500 µM). The arrow indicates the position of the recombinant pro-protein (π) of ADC-His. B) An autoradiogram of TLC containing recombinant ADC-His reaction products of the same protein samples shown in A. The positions of L-aspartate (Asp) and β-alanine (β-Ala) are shown by arrows.

L F3 F4 F5 F6 F3 F4 F5 F6 A) B)

36.4 KDa

26.6 KDa 26.6 KDa

16.9 KDa ππ 16.0 KDa 14.4 KDa α α

6.5 KDa

Figure 3-11. ADC purification with DEAC-Sepharose column. A) DEAE-Sepharose purification of recombinant ADC-His. Affinity purified proteins (L) were loaded into DEAE-Sepharose column. Proteins were eluted by NaCl gradient (0-0.5M) and 4 ml fractions (F3 to F6) were collected. Protein fractions were separated by 10-20 % (w/v) Tris-tricine gel and silver-stained (A). Positions of recombinant ADC-His π α and ß subunits are indicated by arrows. B) Western blot for the DEAE-Sepharose fractions using anti-His-COOH monoclonal antibody (1:8000 dilutions). Recombinant ADC-His π and α subunits identified by the antibody are shown by arrows.

4 5

Figure 3-12. Peptide mass fingerprinting of the purified recombinant ADC-His. Purified ADC-His was separated by 10-20 % (w/v) Tris-Tricine gradient SDS-PAGE and the 16.8 kDa band was excised and digested with trypsin. The extracted peptides were analyzed by MADLI-TOF. Representative mass spectra of the trypsin-digested recombinant ADC-His. Asterisks indicate internal standards and numbers indicate peaks matching recombinant ADC.

ADC + +

ADC-antiserum + I Pre-bleed I +

Protein-A + I + I + I I + I I ADC 6 5 4 3 2 1

26.6KD a

16.0KD a

Figure 3-13. Recombinant ADC-His immunoprecipitation using anti-ADC polyclonal antibodies, (lanes 1-6). Purified ADC (100 µg) was incubated for 4 h (lanes 1- 5) with no antiserum, (lane 1 and 5); or ADC-antiserum (1:50 dilutions; lanes 2, 3 and 6); or pre-bleed (1:50 dilutions; lane 4). Protein A was then added to reactions 2, 4, 5 and 6 and mixture incubated for 4 h. Immunoprecipitated proteins were separated by 12 % w/v SDS-PAGE and proteins transferred to PVDF membrane and probed with anti-His-COOH monoclonal antibody (1:8000 dilutions).

ADC antiserum Pre-bleed

1 2 3 4 4 3 2 1

Figure 3-14. Immunoblot analysis of recombinant ADC-His using ADC polyclonal antibodies. Purified recombinant ADC lane 1 and crude protein from: DE3:pET-panD (lane 2); DE3:pETBlue2 (lane 3) and AB354 strain (panD-; lane 4) were separated by 15 % (w/v) SDS-PAGE and used for Immunoblotting with ADC polyclonal antibodies. Pre-bleed and ADC antiserum were used with 1:5000 dilutions and the secondary antibodies 1:30000 dilutions respectively. The arrow shows the position of the recombinant ADC-His at 16.8 kD.

Table.3-1. Primers used for PCR cloning and screening. Restriction sites added to the PCR products from the primers are underlined.

Primer Sequence (5′-3′) PAND+ CCGAGCTCGACAGGGTAGAAAGGTAGA PAND- CCCCATGGGGATAACAATCAAGCAACC panDBlue2+ TCATGATTCGCACGATGCTGCCAGG panDBlue2-Hi CAGCTGAGCAACCTGTACCGGAATCGC

Table. 3-2. Peptide mass fingerprinting of the purified recombinant ADC-His. Matched peptide sequence and their starting and ending amino acid residues, modifications and missed cleavage.

Peak Matched peptide sequence Start End Modification Missed number cleavage 1 F S T Y A I A A E R 55 64 none 0 2 T W R P N V A Y F E G D N E M K 110 115 none 0 3 T W R P N V A Y F E G D N E M K 110 115 1 Met-ox 0 4 T W R P N V A Y F E G D N E M K R 110 116 1 Met-ox 1 5 A S Q P E L A P E D P E D L E H H H 131 151 none 0 H H H I I S V N A A A H C A S V G D I V I I 68 99 none 0 A S F V T M P D E E A R

CHAPTER 4 E. COLI L-ASPARTATE α-DECARBOXYLASE EXPRESSION IN TRANSGENIC TOBACCO

Introduction

The non-protein amino acid β-alanine is found in all living organisms. In plants and

microbes, β-alanine is a precursor for pantothenate, a coenzyme A precursor and an

essential vitamin in human and animal nutrition. In most members of the higher plant

family Plumbaginaceae, β-alanine is also methylated to β-alanine betaine, an

osmoprotectant (Raman and Rathinasabapathi, 2003). In some legumes, β-alanine along

with γ-glutamate and cysteine forms the thiol tripeptide homoglutathione, an antioxidant

involved in heavy metal detoxification and protection against reactive oxygen species

(Klapheck, 1988; Moran et al., 2000). In Escherichia coli β-alanine is synthesized via α- decarboxylation of L-aspartate catalyzed by the panD-encoded L-aspartate-α-

decarboxylase (Williamson and Brown, 1979; Merkel and Nichols, 1996). This route

appears to be unique to prokaryotes and absent in eukaryotes including yeast and plants

(Rathinasabapathi et al., 2000; White et al., 2001).

E. coli L-aspartate-α-decarboxylase is an unusual enzyme because of its

requirement for pyruvate as a covalently bound, catalytically active prosthetic group

(Williamson and Brown, 1979, Ramjee et al., 1997). The enzyme is initially translated as

an inactive precursor protein (π-protein, 13.8 kDa). It undergoes self-processing at a specific Gly24-Ser25 bond to produce a β-subunit (2.8 kDa) with a hydroxyl group at its

C-terminus and an α-subunit (11.0 kDa) with a pyruvoyl group at its N-terminus, derived 57

58 from serine (Ramjee et al., 1997; Albert et al., 1998). The crystal structure of the E. coli

L-aspartate-α-decarboxylase demonstrated that the active enzyme is a multimer containing three α- and β-subunits and an incompletely processed π-protein (Albert et al.,

1998).

Metabolic engineering of plants for β-alanine overproduction is an important first step toward generating transgenic plants capable of accumulating osmotically significant quantities of β-alanine betaine. Elevated levels of β-alanine in plants could potentially be useful in engineering plants for increased pantothenate, improving nutritional value and for engineering plants for heavy metal detoxification. However, plant genes involved in

β-alanine synthesis have not been characterized yet. The aim of this work is to use the E. coli panD gene encoding L-aspartate-α-decarboxylase for engineering tobacco. The bacterial route to β-alanine is a well-characterized, it is a single step decarboxylation of aspartate that is not subject to feedback inhibition by the metabolic end products β- alanine, pantothenic acid, acetyl coenzyme A, or coenzyme A (Williamson and Brown,

1979; Cronan, 1980). Since the E. coli L-aspartate-α-decarboxylase decarboxylates aspartate and releases CO2 that could improve plant growth, the vegetative growth of the transgenic plants was examined. Additionally, the transgenic plants were examined for thermotolerance since the L-aspartate-α-decarboxylase’s processing and activity in vitro are stimulated by high temperature (Ramjee et al., 1997).

Materials and Methods

Materials

Bacterial media, antibiotics, buffers, and protease inhibitors were from Sigma (St.

Louis, MO). Plasmid purification and gel extraction kits were from Qiagen (Valencia,

-1 CA). L-[U-14C]aspartate (217 mCi mmol ) from ICN Biomedicals (Irvine, CA) and dGTP (α-32P, 800 Ci mmol–1) from Amersham Bioscience (Piscataway, NJ) were used

without further purification. Gradient SDS-PAGE protein gels, protein molecular weight

markers, protein stains and PVDF membranes were from BioRad (Hercules, CA). DNA

Mr marker, Taq polymerase, dNTPs, restriction enzymes, pCR 2.1-TOPO cloning kit,

TRIzol reagent, pro-Bond Ni-NTA resin, and secondary antibodies were from Invitrogen

(Carlsbad, CA). Oligonucleotide primers were synthesized by the custom primer

synthesis unit of Invitrogen (Carlsbad, CA).

Construction of the Expression Vector

The plant expression vector pMON979 contains a multiple cloning site (MCS)

between an enhanced Cauliflower mosaic virus 35S promoter and NOS3’ terminator, a

kanamycin resistance selectable marker for plant selection and a spectinomycin resistance

gene for bacterial selection. A 34-bp synthetic dsDNA containing BamHI, SacI, HpaI,

ApaI, KpnI and EcoRI sites was ligated into the BglII and EcoRI sites of the MCS to

create pMON-R5. The E. coli panD ORF in pUC-panD vector (Chapter 3) was digested with EcoRI and sub-cloned into EcoRI-digested pMON-R5 to derive pMON-R5-panD.

Recombinants with the insert in the correct orientation were identified by restriction analyses (Figure 4-1) and confirmed by sequencing.

Agrobacterium-Mediated Transformation of Tobacco

The pMON-R5-panD and pMON-R5 vectors were transferred into Agrobacterium tumefaciens ABI strain via triparental mating (An et al., 1988). Transformation of

Nicotiana tabacum cv. Havana 38 (Wisconsin 38) was performed as described previously

(An et al., 1988; Rathinasabapathi et al., 1994). More than 200 tobacco leaf-disks were used for six treatments and two controls. In the two control treatments the

leaf-disks were either cultivated without Agrobacterium or co-cultivated with

Agrobacterium that does not have any vector. In two other treatments the disks were

incubated with Agrobacterium harboring empty pMON-R5 vector (vector control

treatment). The last four treatments employed Agrobacterium that was harboring four

pMON-R5-panD vectors derived from four independent transformations.

-1 The leaf-disks were maintained under kanamycin selection (50 mg L ) starting

from the third day after the co-cultivation with Agrobacterium and were sub-cultured

every 2-3 weeks. No kanamycin-resistant shoots were obtained with the two control

treatments. All these plants were maintained under kanamycin till the rooting stage and

then moved to soil. A total of 10 and 29 independent putative transgenic plants were

generated from the leaf disks infected with agrobacterium harboring pMON-R5 and

pMON-R5-panD vectors respectively.

DNA and RNA Blot Analyses

Genomic DNA was extracted from leaves using a CTAB method according to

Wong and Taylor (1993). DNA (20 µg) digested with restriction enzymes, was separated by 1.2 % (w/v) agarose gel, and transferred to nylon membranes by capillary transfer.

PanD sequence (446 bp) was labeled with [32P]dGTP (800 Ci mmol–1, Amersham

BioSciences) using a random primer method (Invitrogen) according to manufacturer's instructions. Following hybridization at 420C using the formamide procedure (Sambrook

et al., 1989), the membranes were washed at high stringency prior to autoradiography.

Total RNA was extracted from leaves using TRIzol reagent according to the

manufacturer’s protocol (Invitrogen, Carlsbad, CA). Total RNA (20 µg per lane) was

separated in formaldehyde 1.2 % (w/v) agarose gels. Equal loading of RNA was verified

by ethidium bromide staining. RNA was blotted by capillary transfer onto nylon

membranes with 20 x SSC. Hybridization with a 32P-labeled probe was conducted at

420C following the formamide procedure as described (Sambrook et al., 1989) and the

membranes were washed at high stringency conditions prior to autoradiography.

Genetic Analyses and Identification of Homozygous Lines

Primary transgenic lines and wild-type (WT) tobacco were grown in a greenhouse and selfed. Surface sterilized T2 seeds of each line were placed on Petri plates containing half-strength MS salts media supplemented with kanamycin (200 mg L-1). Seeds were

0 -2 -1 germinated at 24 C under continuous fluorescent light (40-50 µmol m s ) for 14 d.

Seedlings were scored for their resistance to kanamycin and segregation was analyzed

2 using a X test. Several T2 lines with single gene segregation for kanamycin resistance were grown in a greenhouse for flowering and their seeds were analyzed for the segregation of the marker. Those lines that did not segregate at the T3 generation were considered homozygous.

SDS-PAGE and Immunoblot Analyses

SDS-PAGE was performed in 10% to 20% Tris-Tricine gradient PAGE gel (Bio

Rad; Hercules, CA) or 12% (w/v) Tris-glycine polyacrylamide gels. Protein samples were diluted with 2 X SDS-PAGE sample buffer containing 0.1 M Tris-HCl, pH 6.8, 4% w/v SDS, 20% (v/v) glycerol, 5 mM dithiotheritol, and 0.08% (w/v) bromophenol blue and denatured at 950C for 10 min. The separated proteins were visualized with

Coomassie Brilliant Blue or silver stain. The SDS-PAGE-separated proteins were

transferred by electroblotting onto a PVDF membrane. The membranes were incubated

with different dilutions of antibodies after blocking with blocking buffer (20 mM Tris-

HCl, pH 7.5, 140 mM NaCl, and 5 % (w/v) nonfat dry milk). The primary anti-ADC

polyclonal antibodies were used at 1:5000 dilution. The secondary anti-rabbit IgG

antibody conjugated to alkaline phosphatase (Sigma; St. Louis, MO) was used at a

1:30000 dilution. After washing, binding of the antibody was recorded using a

colorimetric substrate. The alkaline phosphatase activity was detected in 10 ml alkaline

phosphatase buffer containing 0.1 M Tris-HCl pH 9, 0.1 M NaCl, 5 mM MgCl2, 3.3 mg nitro blue tetrazolium and 1.7 mg bromochloroindolyl phosphate.

ADC Activity Assays

Aspartate decarboxylase was assayed using a radiometric procedure employing L-

[U-14C]aspartate. The assays contained in 50 mM potassium phosphate, pH 7.0, 1.2 mM

-1 of aspartate, 0.2 µCi of L-[U-14C]aspartate (217 mCi mmol ), 5 mM DTT, and crude

protein or PEG (25% w/v)- precipitated fraction in a total volume of 50 µL. Following

incubation at 37oC for 1 h, the reaction was terminated by adding 5 µL of 72% (w/v)

trichloroacetic acid and the proteins were removed by centrifugation at 14000 X g for 10

14 min. Radiolabeled CO2 generated by ADC was trapped during the reaction with

Whatman 3 paper saturated with 20% (w/v) KOH. At the end of the reaction period the reaction products in the reaction mixture were separated from the substrate using thin layer chromatography 20x20 cm cellulose plates, 100 micron, (Selecto Scientific,

Georgia, USA) developed with 1-butanol: acetic acid: water (60:15:20, v/v/v), followed by autoradiography. The product formed was quantified by isolating the zone

14 corresponding to β-alanine. In a variation of the assay, CO2 trapped from the assay was quantified in 50% (v/v) Ready Gel (Beckman Instruments, Fullerton, CA) in a Beckman liquid scintillation counter. The counting efficiency was 30%.

Germination Tests

Seeds of panD homozygous transgenic lines, vector-alone transgenic and wild-type

control lines were surface sterilized. Around 100 seeds of each line were placed on 100 x

15 mm Petri plates containing half-strength MS salts. The plates were incubated in a

growth chamber set at four different temperatures 240C, 300C, 360C and 420C under

-2 -1 continuous fluorescent light (20 µmol m s ). Percent germination was scored over a

period of 12 d. Seeds were considered germinated when the white radicle emerged.

Growth Tests and High Temperature Stress

For growth analysis and seedling temperature stress, five-week-old seedlings of

two homozygous transgenic lines, vector control (R5) and WT control lines growing in

350-mL pots in Metromix 220 (Scotts-Sierra Horticultural Products Company,

Marysville, OH) at 240C were supplemented with 1 g slow release fertilizer Osmocote,

(N:P:K - 26:10:6). Seedlings were germinated and maintained in a culture room at 240C

-2 -1 under 50 µmol m s light intensity 16 h light/8 h dark period for five weeks. Then the

pots were transferred to growth chambers adjusted to 240C, 300C or 350C with same light period and intensity. After one week, or four weeks, aerial biomass, plant height, and number of leaves per plant were recorded.

Salt Stress Experiment

Seedlings of two homozygous transgenic lines, pD2 and pD7, vector control transgenic line (pR5) and WT control lines were geminated and maintained at 24oC.

Three-week-old seedlings were transferred to 350-mL pots containing sterilized vermiculite media and irrigated with 100 mL 1 X Hoagland solution once a day. After two weeks, plants were transferred to the greenhouse and maintained for one week prior to salt treatment. For the salt stress treatment, NaCl was added to the 1 X Hoagland

solution at 50 mM concentration. The NaCl concentration was increased by 50 mM every

three days. When the NaCl concentration reached 300 mM, plants were harvested for

their aerial biomass.

Quantification of Free β-alanine and Aspartate Levels.

Leaf tissue (0.5 to 1.0 g fresh wt.) was extracted using a

methanol:chloroform:water mixture as described in Hanson and Gage (1991). The

aqueous fraction was evaporated under a stream of nitrogen, redissolved in water and

purified using ion exchange resin as described previously (Rhodes et al., 1989). One mL

of the redissolved extract was loaded onto a 1-ml Dowex 50-H+ ion exchange column, the column was washed with 8 mL sterilized water and total free amino acids were eluted with 6 mL NH4OH. The eluted fraction was evaporated under a stream of nitrogen, and

redissolved in 0.4 ml double distilled-water. Amino acids were then incubated with

phenylthiocyanate (PITC) in the presence of trimethylamine and methanol at room

temperature for 30 min. Following pre-column derivatization, 20 – 50 µl of the PTC-

amino acid derivatives were separated and quantified by HPLC using a Waters 515

HPLC Pump, Waters 717 plus Autosampler, Waters 2410 Refractive Index Detector, and

YMC-Pack ODS-AM, S-5µm, 12 nm 250 x 4.6 mm I.D. column, as described by

Sherwood (2001). The mobile phase was composed of buffer A (10mM sodium acetate,

pH 6.4) and buffer B (60% acetonitrile, 10mM sodium acetate, pH 6.4). The column was

equilibrated with buffer A and eluted as following, 100 % buffer A for 20 min.; 87%

buffer A and 13% buffer B for 35min.; 80% buffer A and 20% buffer B for 10min.; 70%

buffer A and 30% buffer B for 10min.; 60% buffer A and 40% buffer B for 10min.; 45%

buffer A and 55% buffer B for 20min.; 100% buffer B for 5 min. and finally, 100%

buffer A for 20min.

Table 4-1 lists the retention time of PTC-derivatives of amino acids standards; nor-

leucine was used as an internal standard. Amino acid derivatives were detected at 254 nm

and identified based on retention times for pure standards run under identical conditions.

Statistical Treatment of Data

All experiments were done at least twice with 3 to 6 replicates per treatment. The data were processed using analysis of variance in a completely randomized design model using the SAS software package (SAS, 2002). The mean separations were done using

Duncan’s multiple range test at P ≤ 0.05.

Results

The E. coli panD Gene is Expressed in Transgenic Tobacco

Tobacco was transformed with Agrobacterium tumefaciens strain ABI carrying

(pMON-R5) vector or pMON-R5-panD vector, which contained the E. coli panD gene

under the control of the CaMV 35S promoter and NOS3’ terminator. A total of 10 and 29

independent putative transformants were obtained for pMON-R5 and pMON-R5-panD

constructs, respectively, based on their kanamycin resistance. In a PCR screen using

genomic DNA template and primers specific for the panD gene (Table 3-1), all the panD

putative transgenic plants amplified the expected 429 bp band which was not present in

vector controls (Figure 4-2).

Total RNA samples from 22 panD and 4 vector-control transformants were

analyzed in RNA blots, probed with 32P-labeled panD DNA. Fifteen panD transformants showed low, moderate or high levels of the expected ≅ 1 kb transcript which was absent in the vector alone controls (pR5; Figure 4-3. A). Some transformants with high levels of

panD transcript were analyzed by Southern blot to determine the panD copy number.

Genomic DNA, digested with Hind III, was separated on an agarose gel and blotted on

nitrocellulose and probed with a panD-specific DNA probe. The panD gene sequence

does not have a Hind III site but there is a Hind III site upstream of the CaMV 35S

promoter. Eight panD transformants showed a single band, consistent with a single panD

gene insertion (Figure 4-3. B).

Some of the transformants that had single panD gene in their genome based on

DNA blot analysis and positive for gene expression based on RNA blots were grown in a

greenhouse and selfed. The progeny from these plants segregated 3:1 for kanamycin

resistance: sensitivity in a seedling bioassay, consistent with a single gene insertion.

Further analyses were done on two lines homozygous for the panD transgene (pD2 and

pD7) and a vector control line (pR5) homozygous for the kanamycin resistance gene.

The E. coli ADC Protein Expressed in Transgenic Tobacco is Active

Protein extracts from the leaves of pD2 and pD7 showed detectable activities for

ADC (Figure 4-4. A). However, there was little activity present in extracts from the pR5

transgenic line (Figure 4-4. A). The ADC activities in the PEG-concentrated crude

protein extracted from transgenic lines pD2 and pD7 were 465 and 793 pmol of β-alanine

-1 -1 mg h respectively. The stoichiometry of β-alanine and CO2 in an ADC assay of transgenic tobacco was determined. For each molecule of β-alanine generated one molecule of CO2 was formed. The ADC activity in the E. coli BL21-DE3 crude protein

extract or purified ADC-His protein was used as control for the enzyme assay. In an

immunoblot analysis with ADC-specific polyclonal antibodies, a band corresponding to

13.9 kDa, the unprocessed protein, was revealed (Figure 4-4. B). Neither WT nor pR5

plants protein extracts showed reaction with the ADC-specific polyclonal antibodies at

13.9 kDa. However, in the two transgenic lines there was no signal for the 11.01 kDa and

2.83 kDa protein bands corresponding to α and β peptides, respectively.

Free Amino Acid Analysis in Transgenic Lines

Total free amino acids, β-alanine and L-aspartate levels in leaves were evaluated using HPLC separation of amino acid-PTC derivatives (Figure 4-5). Table 4-1 lists the

retention times of PTC-amino acid standards. Five week old plants were grown at 240C and at 350C for one week prior to analysis because of a thermotolerance phenotype of the

ADC lines (see below). There was no significant difference in the β-alanine for plants that were growing at 240C (Figure 4-6. A). However, when comparing β-alanine percentage relative to the total free amino acids, the two transgenic lines pD2 and pD7 had a significantly higher per cent compared to pR5 and WT (Table 4-2). Beta-alanine

was about 0.35% of the total free amino acids in control lines and about 0.56% of the

total amino acids in the pD2 and pD7 lines (Table 4-2). Additionally, both pD2 and pD7

lines had significantly (p < 0.05) higher levels of β-alanine up to 4 fold increase

compared to the pR5 and WT lines when the plants were stressed at 350C for one week

(Figure 4-5; Figure 4-6). Furthermore, the β-alanine percentage of the total free amino

acids was up about 0.6% in the pD2 and pD7 lines compared to 0.4% in the control

0 0 (Table 4-2). In both treatments, 24 C and 35 C, the free L-aspartate level was elevated by two-fold in the ADC transgenic lines compared to control (Figure 4-6. B). The aspartate percentage of the total free amino acid did not show significant changes for the stressed plant (Table 4-2). However, for the plants that were growing at 240C, the aspartate percentage of the total free amino acid was 15.3% for the ADC transgenic lines and 5.6%

for the control (Table 4-2). Along with the β-alanine and L-aspartate increases, the total free amino acid levels in the ADC transgenic lines was also significantly elevated in the stressed plants (Figure 4-6. C).

Transgenic Expression of ADC Improves Vegetative Biomass

In general, young seedlings of pD2 and pD7 lines were larger than the pR5 and WT plants from visual inspection. Figure 4-7. A shows one each of the smallest and the largest seedlings and two intermediate size seedlings for pD2, pD7, pR5 and WT lines, germinated and grown in a greenhouse for 4 weeks. Figure 4-7. B shows ten week old

seedlings for pD2, pD7, pR5 and WT lines, germinated and grown in a greenhouse. In a

controlled temperature experiment to evaluate growth of these lines, seeds were

germinated at 240C and the seedlings maintained for five weeks prior to transferring to

environmental growth chambers set at either 240C or 300C. The aerial biomass was

evaluated after one week and four weeks. While there were no significant differences

between the four genotypes at 240C (Figure 4-8. A), pD2 line had higher mean aerial biomass compared to pR5 and WT lines when grown at 300C for a week (Figure 4-8. B).

This increase ranged between 11.2 and 19.2 % compared to the control. Also these two lines had a higher mean number of leaves per plant at 300C. When plants were grown for

an additional three weeks at 300C, lines pD2 and pD7 had significantly (P < 0.05) higher

mean aerial biomass (Figure 4-8. C and D). The increase in the biomass ranged between

12.6 and 22.3 % compared to the control based on the fresh weight. The average dry

weight of pD2 and pD7 lines showed 17.7 % increase compared to the average dry

weight of pR5 and WT (Figure 4-8. D).

In salt stress experiments, six weeks old plants were challenged with NaCl up to

300 mM added to the nutrient solution. All the lines showed reduction in their growth in response to salt treatments compared to control (Figure 4-9). The pD2 and pD7 lines did not show improved response compared to WT and pR5 when comparing the aerial fresh and dry weight (Figure 4-9. A and B).

Thermotolerance Phenotype in ADC Transgenic Lines

To study the high temperature stress effect on transgenic lines, seeds were germinated and seedlings maintained at 240C for five weeks and then transferred to environmental growth chambers set at 350C. After one week of high temperature stress,

WT and pR5 plant’s height was reduced by 14.8 and 15.1 % respectively compared to

plants growing at 240C (Figure 4-10. A). On the other hand, pD2 and pD7 transgenic lines gained 8 and 12 % greater height at 350C compared to plants growing at 240C

(Figure 4-10. A). Additionally, the pR5 and WT plants showed significant reduction in their leaf number compared to plants growing at 240C while pD2 and pD7 did not (Figure

4-8. B). The pD2 and pD7 lines had also significantly higher aerial biomass compared to

pR5 line but not the WT plants when plants were evaluated after one week of growth at

350C (Figure 4-10. C). In another experiment, the plants were stressed for one week at

350C and then were allowed to recover for three weeks at 300C. The pD2 and pD7 lines gained significantly (P < 0.05) higher aerial biomass compared to WT and pR5 lines

(Figure 4-11. A). The increase in fresh weight was between 8.5 and 13% greater than that achieved by the pR5 and WT plants during recovery from high temperature stress. The increase in the aerial fresh weight was translated to the dry mass where the pD2 and pD7 lines average dry weight was 11.2 % greater than that of the average dry weight of pR5 and WT (Figure 4-11. B).

Transgenic Expression of ADC Improves Tobacco Seed Germination at high Temperature

When seeds of lines pD2 and pD7, vector control pR5 and wild-type seeds were

germinated at 240C, all the lines germinated equally well (Figure 4-12. A). However, when the imbibed seeds were incubated at 420C, pD2 and pD7 lines germinated better

than the vector control and wild-type tobacco (Figure 4-12. B). A time course experiment

at 420C showed that pD2 and pD7 lines had significantly (P < 0.05) higher germination rate than the vector control and wild-type (Figure 4-12. C). Vector control line had significantly (P < 0.05) lower germination rate than the wild-type at 420C (Figure 4-12.

C). Improved germination at high temperature was also found in two other independent

lines expressing the panD gene (Figure 4-12. D).

Discussion

This study documents evidences for the heterologous expression of the prokaryotic

pyruvoyl-dependent L-aspartate-α-decarboxylase (ADC) in eukaryotic cells. The

transcriptional level of panD gene varied within primary tobacco transformants (Figure

4-3. A). The gene was integrated in the tobacco genome (Figure 4-2 and Figure 4-3. B),

and stable for three generations.

At the protein level, the bacterial ADC was active in the transgenic tobacco (Figure

4-4. A). The ADC is known to be translated as a proenzyme that undergoes self-

processing at a Gly24-Ser25 bond to produce a β-subunit (2.8 kDa) and an α-subunit (11.0

kDa) with a pyruvoyl group at its N-terminus, derived from serine (Ramjee et al., 1997).

This processing is essential for generating a pyruvoyl as a catalytically active prosthetic

group and hence enzyme activity (Ramjee et al., 1997). Although ADC is unique to

prokaryotes and not found in eukaryotes (Rathinasabapathi et al., 2000; White et al.,

2001), it was expected that the proenzyme will be correctly processed in its plant host

since there are other pyruvoyl-dependent enzymes requiring similar post-translational

modifications in plants such as S-adenosyl-L-methionine decarboxylase and

phosphatidylserine decarboxylase (Xiong et al., 1997; Rontein et al., 2003). The protein

blot analysis for tobacco crude protein using ADC-specific antibodies labeled a band

corresponding to the unprocessed peptide (Figure 4-4. B). However, the blot analyses

were not sensitive enough to detect the processed α and β subunits. This could be due to

incomplete processing of the proenzyme, resulting in low abundance of α and β subunits.

The crude protein extracted from DE3:pET-panD E. coli showed similar reaction pattern

(Figure 3-14).

When leaf free amino acids were analyzed in plants grown at 350C for one week, there was a significant increase in β-alanine levels up to 4 fold in the ADC transgenic lines compared to control lines growing at the same conditions (Figure 4-6. A). While wild-type and vector control transgenic plants synthesize their β-alanine through the endogenous pathway, the ADC transgenic lines had elevated levels of β-alanine due to L- aspartate decarboxylation. ADC is not subject to feedback inhibition by β-alanine, pantothenate, coenzyme A or acetyl coenzyme A (Williamson and Brown, 1979; Cronan,

1980). This is consistent with the increase in β-alanine level in the ADC transgenics. In spite of L -aspartate being the substrate, its levels increased up to 660 nmoles per g f.wt.

in the ADC transgenics growing at 350C (Figure 4-6). It was expected that expression of

bacterial ADC in tobacco would not negatively affect the aspartate pool since other work

showed that the aspartate pool in plants is flexible for metabolic engineering (Galili and

Hoefgen, 2002). Although many studies have been done on free amino acid levels in

plants, only a few of them quantified non-protein amino acids. Here, the β-alanine and L- aspartate levels were quantified using HPLC separation of PTC-derivatized amino acids.

Future experiments are needed to evaluate whether the increase in β-alanine levels could be further improved by modifying the expression elements used in the vector.

Plants expressing the bacterial ADC showed increased growth under controlled growth conditions (Figure 4-8. C and D) especially when grown at 300C. Plants grown at lower temperature (240C) did not exhibit statistically significant differences in their biomass, suggesting that higher temperature had an influence in promoting the growth of

ADC transgenic lines. Accordingly, when the plants were exposed to high temperature stress (350C), transgenic lines expressing bacterial ADC, showed less inhibition by stress

(Figure 4-10). In a high temperature stress recovery experiment, the ADC transgenic lines

recovered significantly better than the wild-type and vector control lines (Figure 4-11).

Thermotolerance phenotype could also be found in germinating seeds. Seeds of ADC

lines had an increased germination rate and germination percent at 420C compared to the vector control and wild-type lines (Figure 4-12. B, C and D). This suggests that the transgene is expressed from the earliest stages of plant development and improves thermotolerance. However, it is not clear if β-alanine has any significant role in improving seed germination under high temperature. The thermotolerance phenotype seems to be consistent with the properties known for bacterial L-aspartate-α- decarboxylase. Self-processing and specific activity of bacterial ADC were promoted in vitro by a high temperature treatment (Ramjee et al., 1997; Chopra et al., 2002).

Therefore, it is conceivable that ADC was self-processed and has better activity at high

temperature and this therefore, may have resulted in the increased β-alanine under high

temperature.

Indeed, the ADC transgenic plants described in this work exhibit increased biomass

and thermotolerance. Both traits will be valuable for crop improvement. Although the

mechanisms by which L-aspartate α-decarboxylation interacts with plant metabolism are not clear, it could be speculated that the two traits could be mediated via the products of

ADC reaction, β-alanine, CO2 or both.

Firstly, β-alanine could have biological activities when accumulated in plant cells, such as interaction with chaperones, Hsp proteins, membranes and enzymes. This interaction could provide protection for plant cell metabolism under elevated high temperature. Consistent with this, the increased level of β-alanine in ADC transgenic lines provided thermotolerance at seed germination and whole plant stage. Other work using cultured plant cells or whole plants showed that endogenous β-alanine levels increased under heat stress (Mayer et al., 1990; Kaplan et al., 2004) or drought stress

(Rizhsky et al., 2004). However, the mechanism by which β-alanine promotes such function is yet to be addressed.

Secondly, β-alanine could work as a signaling molecule, turning on pathways that improve plant metabolism and growth, similar to γ-aminobutyric acid (GABA; Bouche and Fromm, 2004; Beuve et al., 2004), a four carbon analog of β-alanine. Supplying

GABA to Brassica nupus roots resulted in significant increase in the expression of

- BnNrt2, a component of a high-affinity NO 3 transport system that operates at low

external nitrate concentration (Beuve et al., 2004). Transgenic lines with elevated levels

of β–alanine could be useful for examining such interaction at the RNA level for key

genes in plant metabolism using RNA blots or microarrays.

Thirdly, the elevated level of β-alanine may increase the flux toward L-aspartate biosynthesis which in turn could have improved nitrogen utilization and reduced C/N ratio. Although other metabolic engineering efforts in amino acid metabolism such as over expressing glutamine synthetase and glutamate synthase resulted in improved total free amino acid and growth phenotypes in transgenic plants (Ameziane et al., 2000;

Migge et al., 2000; Fuentes et al., 2001; Chichkova et al., 2001; Oliveira et al., 2002; Fu et al., 2003), this is the first report of expressing the unusual bacterial L-aspartate-α-

decarboxylase in transgenic plants. Together, the data reported here suggested that free

amino acids, vegetative growth and thermotolerance can be improved in plants by simply

expressing a bacterial gene involved in nitrogen metabolism.

Fourthly, the elevated level of β-alanine could have affected the endogenous β-

alanine biosynthetic pathway(s) by feedback inhibition. This in return could have

provided extra β-alanine-precursors, such as uracil and/or polyamines, to be available for

other biologically significant use.

Fifthly, increased β-alanine and free amino acid levels could have led to an increase

in pantothenic acid and its derivatives. Consistent with this hypothesis, expressing

Corynebacterium glutamicum panD gene in E. coli resulted in elevated level of both β-

alanine and Pantothenate (Dusch, et. al., 1999).

Finally, ADC-generated CO2 can be transferred to the chloroplast and be available for fixation by Rubisco. Such increase in CO2 could alter the CO2/O2 ratio and increase

the carboxylation activity of the Rubisco reducing photorespiration and improve leaf-

water status (Long et al., 2004). Since the ADC activities found in the ADC transgenics

are relatively low (Figure 4-4. A), this increase can only be expected to be marginal.

However, such low increases in CO2 could have had an effect stimulating nitrogen or carbon assimilation. Elevated CO2 is known to increase growth (Stitt and Krapp, 1999),

improve stress adaptation (Wullschleger et al., 2002) and protect photosynthesis against

high temperature stress (Taub et al., 2000). The transgenic material generated in this

work should be valuable to test these hypotheses and understand the role of β-alanine in

thermotolerance.

M12 3

6119

1433 1144

693

Figure 4-1. Restriction analysis of pMON-panD vector. Cloning of E. coli panD ORF into the pMON-R5 plant expression vector. M, 100bp marker. Lane 1 and 2 pMON-panD digested with PstI. Lane 3 pMON-R5 vector control.

D D I n I an I a ont. p -p c -

3 nd 7 1 6 B i 1 2 5 4 2 g. TB 1 1 7 1 1 T H e E pD pD pD pD pR N p λ- pD pD pD pD pR pE 564 bp 429 bp

Figure 4-2. PCR screening of primary transgenic tobacco. panD sequence was PCR amplified from genomic DNA of primary transgenics pETB-panD is positive control. Negative control, no DNA was added. pR and pD lines are primary transgenic lines containing pMON-R5 and pMON-R5-panD-A9 respectively.

A PR1 PD1 PD2 PD3 PD4 PD5 PD6 PD7 PD8

panD

rRNA PD9 PD10 PR2 PD11 PD12 PD13 PD14 PD15 PD16 PD17

panD

rRNA

PD17 PD7 PD16 PD9 PD14 PD13 PD6 PD5 PD4 PD3 PD12 kb 9.4 6.5

4.5

Figure 4-3. PanD integration and RNA expression in primary transgenic tobacco. (A) RNA blot probed with panD DNA probe (top) and EtBr stained RNA gel showing equal load (lower panel). (B) DNA blot probed with panD DNA probe. pR and pD lines are primary transgenic lines containing pMON-R5 and pMON-R5-panD-A9 respectively.

A 6

5 ) -1

ies 4 .h it -1 iv 3 ADC act (nmol. mg 2

0 pR5 pD2 pD7 E. coli

B WT pR5 pD2 pD7 13.9 KDa

Figure 4-4. Expression of L-aspartate-α-decarboxylase in two transgenic lines homozygous for the E. coli panD gene. (A) Activities of L-aspartate-α- decarboxylase in protein extracts. (B) Western blot probed with E. coli L- aspartate-α-decarboxylase-specific polyclonal antibodies. WT=Wild-type, pR5=Vector control, pD2 and pD7 = panD transgenic homozygous lines, E. coli strain BL21-DE3.

0.024 pD7 β-Ala 0.4

0.3

0.000 AU 0.2 35.0 38.5 42.0

0.1 Asp

0.0 0.0 11 22 33 44 55 66 77 88 99 Minutes

0.024 pD2 0.4 β-Ala

0.3

0.000 AU 0.2 35.0 38.5 42.0

0.1 Asp

0.0 0.0 11 22 33 44 55 66 77 88 99 Minutes

Figure 4-5. PTC-amino acid chromatogram for total free amino acids extracted from tobacco leaves of plants stressed at 350C for one week. WT=Wild-type, pR5=Vector control, pD2 and pD7 = panD transgenic homozygous lines.

0.024 pR5 0.4

β-Ala

0.3

0.000 AU 35.0 38.5 42.0 0.2

0.1 Asp

0.0 0.0 11 22 33 44 55 66 77 88 99 Minutes 0.024 WT 0.4

β-Ala

0.3

0.000 AU

0.2 35.0 38.5 42.0

0.1 Asp

0.0 0.0 11 22 33 44 55 66 77 88 99 Minutes

Figure 4-5. Continue.

WT pR5 pD2 pD7

) 100 90 A fwt

-1 80 70 60 50 40 30 20

ta-alanine (nmol.g 10

Be 0 240C350C

0.8 t)

fw 0.7

-1 B 0.6 0.5 mol.g.

µ 0.4

e ( 0.3 at 0.2

part 0.1 s

A 0 240C350C

20 18 C 16 14 fwt) ino acids 12 -1 10 8 mol.g ee am

(µ 6 4 2 Total fr 0 240C350C

Figure 4-6. Transgenic tobacco expression of E. coli panD gene accumulates higher levels of β-alanine, aspartate and total free amino acids. Levels of β-alanine (A), aspartate (B), and total free amino acids (C), in transgenic tobacco expressing E. coli panD gene compared to controls. Fully expanded leaves were sampled from 6-week old seedlings either growing at 240C or after one week at 350C. Values are means and standard error for three independent analyses.

WT pR5 pD2 pD7

Figure 4-7. Expression of E. coli panD gene in transgenic tobacco improves seedling growth. (A) Representative of four-week-old seedlings from WT, pR5 pD2 and pD7 lines growing in the greenhouse. (B) Ten-week-old seedlings from pD2, pD7, pR5 and WT lines germinated and grown in a greenhouse.

20 25 A B a 18 a a a,b a b ) b 16 a 20

14 FW FW) g g 12 15

omass ( 8 10

bi 6 al

rial biomass ( 5 4 e Aeri A 2 0 0 WT pR5 pD2 pD7 WT pR5 pD2 pD7

C 70 6 a D a )

) a,b 60 a 5 b,c W b b c D FW 50 g g 4 40 3 30 2 20 rial biomass ( rial biomass ( e e 1 A A 10

0 0 WT pR5 pD2 pD7 WT pR5 pD2 pD7

Figure 4-8. Expression of E. coli panD gene in transgenic tobacco improves seedling biomass. (A) Aerial biomass of plants germinated and grown at 240C for six weeks. (B) Aerial biomass of plants grown at 300C for one week after five weeks of growth at 240C. Aerial biomass of plants grown at 300C for four weeks after five weeks of growth at 240C, Fresh weight (C), Dry weight (D). WT=Wild-type, pR5=Vector control, pD2 and pD7 = panD transgenic homozygous lines. Data are means (± S.E.) from six plants.

A 50 45

) 40 35 30 25 ass (g FW 20 15 10 al biom i 5

Aer 0 WT pR5 pD2 pD7

B 4 3.5

3 DW)

g 2.5

1.5 omass (

bi 1 al 0.5

Aeri 0 WT pR5 pD2 pD7

Figure 4-9. ADC-transgenics response’s to salt-stress. Plants were treated with gradual NaCl increase up to 300 mM over 18 days and harvested for their aerial fresh weight (A) and dry weight (B). WT=Wild-type, pR5=Vector control, pD2 and pD7 = panD transgenic homozygous lines. Open-bar = control and shaded-bar = salt-stress treatment.

A WT pR5 pD2 pD7 B WT pR5 pD2 pD7 12 9 a a a

8 a ant a a l a a a a a b 10 a P ) 7 b b b 6 8

5 ves per ght (cm 6 4 3 4 of Lea ant Hei l er

P 2 b 2 1 0 Num 0 0 0 24 C35C 240C350C

C 16 a a 14 a,b

12 b

10 ass (g FW) 8 om

Bi 6 al i 4 Aer 2

0 WT pR5 pD2 pD7

Figure 4-10. Expression of E. coli panD gene in transgenic tobacco improves growth and biomass under high temperature. Five-week-old seedlings from wild-type, pR5, pD2 and pD7 lines were grown at 240C and 350C for one week and harvested for plant height (A), number of leaves per plant (B), and aerial biomass (C). Aerial biomass data for plants at 240C are shown in Fig 4-8.A. WT=Wild-type, pR5=Vector control, pD2 and pD7 = panD transgenic homozygous lines. Data are means (± S.E.) from six plants.

A 60 a a 50 b b

ss (g FW) 30

10 Aerial bioma

0 WT pR5 pD2 pD7

5 B a a 4.5 b b ) 4 W

D 3.5 g 3 2.5 2 1.5 rial biomass ( e

A 1 0.5 0 WT pR5 pD2 pD7

Figure 4-11. Expression of E. coli panD gene in transgenic tobacco significantly improves recovery from heat-stress. Five-week-old seedlings from wild-type, pR5, pD2 and pD7 lines were stressed for one week at 350C then allowed to recover for three weeks at 300C and harvested for aerial biomass (A) fresh weight, (B) dry weight. WT=Wild-type, pR5=Vector control, pD2 and pD7 = panD transgenic homozygous lines. Data are means (± S.E.) from six plants.

87 88

B pR5

WT pD2

pD7

C 100 100 90 pD7 90 D ) ) 80 80 % % )

70 pD2 % 70 on ( on ( 60 i i 60 t t ion ( a a 50 t 50

WT a n n i i 40 40 m m r r 30 pR5 rmin e e 30 20 G G Ge 10 20 0 10 8 9 10 11 12 0 5 2 7 3 7 3 D2 1 Days after imbibition WT pR pR p pD D1 pD pD p

Figure 4-12. Expression of E. coli panD gene in transgenic tobacco improves germination at high temperature. (A) Seeds germinated at 240C photographed after 6 days of imitation (B) Seeds at 420C photographed 12 d after sowing. (C) Germination percent at 420C scored over 12 d after sowing. (D) Germination percent at 420C at day 12 after sowing. WT, pR vector control, pD = panD transgenic lines. Data are means (± S.E.) from six independent Petri dishes.

Table 4-1. Retention times for PTC-derivatives of amino acids standards. The numbers marked with asterisks represent mean and standard error for 5 determinations. Other retention times were derived from a single run of the standards.

Amino acid Retention time (min)

Asp 15.6 ± 0.08 *

Glu 20.32

Ser 34.759

Asn 35.53

Gly 36.05

Glu 36.87

β-Ala 39.28 ± 0.08 *

Thr 43.65

L-Ala 45.92 ± 0.11 *

Met 82.48

Ile 87.56

nor-Leu 89.86 ± 0.04 *

Lys 95.50

Table 4-2. Beta-alanine and L-aspartate Percentage of the total free amino acids in transgenic tobacco expressing the E. coli panD gene compared to vector control and wild-type. Fully expanded leaves were sampled from 6-week old seedlings either growing at 240C or after at 350C for one week right before sampling. Values are means and standard error for three independent analyses.

Temp Genotype Beta Ala Asp Total Free amino acid % of Total % of Total (nmol.g-1fwt) amino acids amino acids WT 0.33 ±0.05 7.3 ± 1.2 4687 ±965

pR5 0.37 ±0.07 4 ± 1 5023 ±1072 240C pD2 0.54 ±0.06 13 ± 3.4 3699 ±170

pD7 0.58 ±0.05 17.7 ± 3.6 3672 ±502

WT 0.41 ±0.03 6.3 ±0.03 4047 ± 345

pR5 0.39 ±0.03 5.0 ±1.01 6178 ± 1347 350C pD2 0.62 ±0.05 4.7 ±0.17 10855 ± 816

pD7 0.59 ±0.1 5.0 ±1.01 14891 ± 4127

CHAPTER 5 SUMMARIES AND CONCLUSIONS

Environmental stress is one of the major factors negatively affecting agricultural

production world wide. Traditional approaches to improve stress tolerance of agricultural

crops have led to some improved cultivars. However, in the biotechnology era, better

tools are available to investigate and understand plant responses to various environmental

stresses, and to breed crops tolerant to environmental stresses. The objective of this study

was to introduce a single-step prokaryotic pathway for β-alanine overproduction into the

model plant tobacco. The advantages of increasing the β-alanine pool in plants are: (1)

engineering plants for stress tolerance and (2) engineering plants with increased B5

vitamin (pantothenate) for improving nutritional value.

In this study, the bacterial gene panD, coding L-aspartate-α-decarboxylase (ADC),

was cloned from E. coli using PCR and gene specific primers. The cloned gene was then subcloned into yeast and bacterial expression systems for the purpose of protein purification. The yeast expression did not meet with success, likely due to technical problems. However, the objective of the study was achieved using the bacterial expression system. The ADC was purified to near homogeneity and the purified protein was used for developing ADC-polyclonal antibodies.

The E. coli panD gene was introduced into tobacco plants using agrobacterium transformation and several panD transgenics were recovered. The panD transgenics expressed an active ADC, and the ADC-polyclonal antibodies recognized the ADC protein in an immunoblot of fractionated crude protein extracted from panD transgenics.

91 92

The β-alanine levels were analyzed in the ADC transgenic plants. The ADC transgenic plants developed in this study accumulated up to 4-fold more β-alanine than controls under high temperature stress conditions. The ADC transgenic plants also showed improved growth at 300C accumulating 17% and 19% more fresh and dry weight, respectively, compared to the controls. Furthermore, the ADC transgenic plants maintained their growth under high temperature stress and recovered better from stress than controls. The seeds of the ADC transgenic plants exhibited thermotolerance during germination where 94% of their seed germinated at 420C compared to only 72% in the case of control plants.

Therefore, the results of this work can be summarized in the following points:

1. PanD expression in a bacterial system allowed ADC purification and

developing ADC-polyclonal antibodies.

2. The E. coli panD gene integration in transgenic tobacco was verified by assays

based on RNA, immunoblots and in vitro enzyme activity.

3. The ADC transgenic plants accumulated up to 4-fold more β-alanine and had

about 2-and 3.7-fold increases in their aspartate and total free amino acids,

respectively, compared to controls.

4. The ADC transgenic plants gained 17% and 19% more fresh and dry weight,

respectively, than control plants when grown at 300C for 30 days.

5. The ADC transgenic plants recovered better at 300C from high temperature

stress at 350C than control plants. Following an imposed high temperature

treatment, the ADC transgenic plants accumulated 13% and 11% more fresh

and dry weight over a three-week recovery period, respectively, compared to the

controls.

6. The seeds of the ADC transgenic plants had greater thermotolerance during

germination.

Both thermotolerance and increased growth components of the phenotype observed

in the ADC transgenic plants were accompanied by elevated levels of β-alanine

suggesting that β-alanine could have a significant role in both traits. More extensive

investigations to understand the exact role of β-alanine in stress tolerance are needed.

This could be achieved using the materials generated in this study and also through

engineering transgenic plants with high and low levels of β-alanine from various species

known to be stress sensitive.

β-Alanine also could have a regulatory role affecting nitrogen assimilation and metabolism in the plant. Such a hypothesis is supported by the fact that GABA, an analog of β-alanine, has such a role. The transcription pattern and activities of the enzymes involved in nitrogen assimilation and metabolism can be analyzed in plants accumulating high levels of β-alanine. Additionally, the CO2 generated from the decarboxylation of aspartate by ADC could have positive effect in plant water status and growth.

Accordingly, expression of ADC in chloroplasts might further improve plant growth and thermotolerance. The transgenic plants generated in this study would be very useful for testing these and other hypotheses.

Future work to follow up on this work:

1. Assay the ADC transgenic plants for levels of polyamines and uracil as a

potential precursor(s) of β-alanine, and pantothenate as end product.

2. Analyze the ADC transgenic plants for their carbohydrate, total nitrogen

contents, free and bound amino acids.

3. Analyze the ADC transgenic lines under high temperature stress for their

photosynthesis and oxidative stress.

4. Study the effect of high levels of β-alanine on transcriptional levels and

activities of enzymes involved in nitrogen and carbon assimilation and

metabolism.

5. Chloroplast expression of ADC either using chloroplast-transit peptide or

chloroplast transformation.

6. Expand the developed technology on economically important crops for

generating stress-tolerant varieties necessary for agricultural production.

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BIOGRAPHICAL SKETCH

Walid Mohamed Mounir Fouad was born few miles away from the great Egyptian pyramids in Giza, Egypt. He joined the college of Agriculture, Cairo University, after finishing his high school education in 1989. His father was the head of the Horticultural

Sciences Department in Cairo University at the time to whom he takes after this great love to research. Walid had a chance to get practical training in the Cairo University tissue culture labs during his undergraduate studies. He graduated in June 1993 with a

Bachelor of Sciences in horticultural sciences from Cairo University. Seeking further education, he joined the Agricultural Genetic Engineering Research Institute (AGERI) in

1994, and enjoyed his work there as a research assistant. In 1998, he earned his master’s degree in horticultural science. In 2000, he was awarded a scholarship by the Egyptian

Government/USAID to pursue his doctorate degree in the US; after which he joined Dr.

Rathinasabapathi’s lab conducting research in stress physiology.

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