Viewed by Maurel, 1997)

CLONING AND CHARACTERIZATION OF GENES RELATED TO BETAINE SYNTHESIS, THE EFFECT OF SALT ON CELL DEATH, AND COMPETITION ON ATRIPLEX PROSTRATA

A dissertation presented to

the faculty of

the College of Arts and Sciences of Ohio University

In partial fulfillment

of the requirements for the degree

Doctor of Philosophy

Li-Wen Wang

August 2002

This dissertation entitled

CLONING AND CHARACTERIZATION OF GENES RELATED TO BETAINE SYNTHESIS, THE EFFECT OF SALT ON CELL DEATH, AND COMPETITION ON ATRIPLEX PROSTRATA

LI-WEN WANG

has been approved

for the Department of Environmental and Plant Biology

and the College of Arts and Sciences by

Allan M. Showalter Professor of Environmental and Plant Biology

Leslie A. Flemming Dean, College of Arts and Sciences

WANG, LI-WEN Ph.D. August 2002. Environmental and Plant Biology / Molecular and Cellular Biology

Cloning and Characterization of Genes Related to Betaine, the Effect of Salt on Cell

Death and Competition on Atriplex Prostrata (248 pp.)

Director of Dissertation: Allan M. Showalter

Soil salinity is a major concern to agriculture all over the world because excess salts in the soil inhibit crop growth. Halophytes such as Atriplex prostrata are able to grow and reproduce in saline environments. One of the reasons A. prostrata is salt- tolerant is that it accumulates osmoprotectants such as glycine betaine in the cytosol and sequesters Na+ and Cl- into the vacuoles. In higher plants, glycine betaine is synthesized via the two-step oxidation of choline catalyzed by choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH). The cDNAs encoding CMO and BADH are cloned from A. prostrata (ApCMO and ApBADH1, respectively) by RT-PCR and 3’-RACE. The composite cDNA, ApCMO, is 1669 bp long and encodes a full-length protein of 438 amino acids, with similar characteristics and a high degree of identity to other CMOs. The composite cDNA of

A. prostrata BADH, designated as ApBADH1, is 1755 bp long and encodes a full- length protein of 500 amino acids. As in amaranth and in mangrove, Avicennia marina, there are two BADHs in A. prostrata. The second BADH, designated as

ApBADH2, was discovered as a partial cDNA by RT-PCR. This ApBADH2 may be a peroxisomal BADH in A. prostrata. Both ApCMO and ApBADH1 expressions are salt-inducible and regulated in a developmental and organ-specific manner. However,

ApCMO expression is ABA-independent while ApBADH1 expression may be ABA- dependent.

The importance of glycine betaine was further demonstrated in a cell line of A. prostrata that does not express CMO. Approximately 70 % of these suspension- cultured cells die after being treated with 340 mM NaCl. NaCl also significantly reduces growth. Most of these cells are protected by exogenous addition of betaine indicated by reduced percentage of cell death and growth inhibition. In addition,

NaCl-induced cell death in A. prostrata exhibits characteristics of programmed cell death.

Since the growth of halophytes in a natural habitat is affected by both salinity and competition, the effects of intraspecific competition on growth and photosynthesis of A. prostrata are examined. The growth of A. prostrata from higher densities is inhibited as a consequence of reduced photosynthesis due to competition for light.

Approved: Allan M. Showalter

Professor of Environmental and Plant Biology

Acknowledgments

The author would like to express her deep appreciation to Dr. Allan M.

Showalter and Dr. Irwin A. Ungar for their enthusiastic guidance and encouragement throughout this research. To them and to Dr. Ivan K. Smith and Dr. Frank Horodyski

I give thanks for serving on my dissertation committee and reading and editing of this manuscript.

The author is indebted to Dr. Howard Dewald for advice and help on glycine betaine quantification with HPLC.

I would also like to thank Dr. Shuxia Li and Dr. Ayyappan Nair for their help at earlier stage of my study. To all the other graduate students in the lab, I thank them for their interactions. I am also grateful to other faculty, staff members and graduate students in the Department of Environmental and Plant Biology and in the

Molecular and Cellular Biology Program. The financial support from the department and the program is appreciated as well.

Finally, I would like to thank my family and friends for their patient support.

Table of Contents

Page Abstract...... 3

Acknowledgments ...... 5

Table of Contents ...... 6

List of Tables...... 9

List of Figures...... 11

List of Abbreviations...... 15

Chapter 1. Introduction ...... 18

1.1 Salinity: a world problem ...... 19

1.2 Halophytes: the solution ...... 20

1.3 Mechanisms of salt tolerance ...... 21

1.3.1 Morphological and developmental levels of salt tolerance .....21

1.3.2 Cellular and molecular levels of salt tolerance...... 23

1.4 Improvement of salt tolerance in crops ...... 33

1.5 In this research...... 36

Chapter 2. Cloning and Expression of Choline Monooxygenase in Atriplex

prostrata ...... 38

Abstract...... 39

Introduction ...... 40

Material and Methods...... 45

Page

Results ...... 53

Discussion...... 83

Chapter 3. Cloning and Expression of Betaine Aldehyde Dehydrogenase in

Atriplex prostrata ...... 91

Abstract...... 92

Introduction ...... 93

Material and Methods...... 95

Results ...... 101

Discussion...... 136

Chapter 4. Effects of Intraspecific Competition on Growth and Photosynthesis

of Atriplex prostrata...... 140

Abstract...... 141

Introduction ...... 142

Material and Methods...... 144

Results ...... 146

Discussion...... 157

Chapter 5. Cell Death and Growth Inhibition Induced by NaCl in Suspension-

Cultured Cells of Atriplex prostrata...... 169

Abstract...... 170

Introduction ...... 171

Page

Material and Methods...... 173

Results ...... 177

Discussion...... 212

Chapter 6. Summary and Conclusion...... 223

Bibliography ...... 232

Appendix I. Components in MS (M5524) and McCown’s (M6774) media ...... 248

List of Tables

Page

Table 1-1. Functions and synthesis of selected osmolytes...... 28

Table 1-2. Summary of metabolic engineering for salt tolerance...... 34

Table 1-3. Summary of genetic engineering with non-osmolytes for salt tolerance...... 35

Table 2-1. Primers used in cloning Atriplex prostrata CMO ...... 48

Table 2-2. A comparison of the sequences of the actual clone and the primers...... 60

Table 2-3. Nucleotide and amino acid sequence identity (left- and right- hand side of the diagonal matrix with similarity of amino acid sequence in parentheses) among available CMOs...... 72

Table 3-1. A list of primers used to clone BADH of Atriplex prostrata ...... 97

Table 3-2. A comparison of the sequences of the actual clone and the primers...... 111

Table 3-3. Identity and similarity of amino acid sequence (left- and right-hand side of the diagonal matrix) among ApBADH and other available BADHs...... 118

Table 3-4. Identity of nucleotide and amino acid sequence between reported two BADHs from the same species...... 121

Table 4-1. Effect of density on the length of internode (cm) in Atriplex prostrata ...... 149

Table 4-2. Effect of density on the fresh mass (gram/plant) of Atriplex prostrata ...... 150

Page

Table 4-3. Effect of density on the dry mass (gram/plant) of Atriplex prostrata ...... 152

Table 5–1. A two-way ANOVA for the effects of glycine betaine pretreatment and simultaneous addition of glycine betaine and NaCl on the death of Atriplex prostrata suspension cells ...... 197

Table 5–2. “Time to double” for cells treated with 170 and 340 mM NaCl...... 201

Table 5–3. Comparison of the growth inhibition and cell death induced by NaCl and KCl ...... 203

Table 5–4. A two-way ANOVA for the effects of salt type and salt concentration on the growth of Atriplex prostrata suspension cells at the 6th day ...... 209

Table 5–5. “Time to double” for 340 mM NaCl-treated cells with glycine betaine pretreatment or simultaneous addition of glycine betaine...... 215

List of Figures

Page

Figure 1-1. The schematic depiction of a plant cell includes the vacuole, chloroplast (cp), mitochondria (mt) after salt adaptation ...... 24

Figure 2-1. The conversion of glycine betaine from choline is catalyzed by two enzymes: choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) ...... 43

Figure 2-2. A schematic map of 1669 bp composite cDNA sequence encoding CMO in Atriplex prostrata ...... 49

Figure 2-3. A nucleotide sequence alignment of four available CMO cDNAs with ApCMO ...... 54

Figure 2-4. An alignment of four clones that are possible 3’ ends of ApCMO...... 61

Figure 2-5. The nucleotide and deduced amino acid sequence of ApCMO ...... 64

Figure 2-6. Alignments of CMOs from Atriplex prostrata, A. hortensis, spinach (Spinacia oleracea), sugarbeet (Beta vulgaris), and amaranth (Amaranthus tricolor)...... 67

Figure 2-7. Comparison of the deduced amino acid sequences of five reported CMOs ...... 70

Figure 2-8. CMO expression in Atriplex prostrata is induced by NaCl ...... 74

Figure 2-9. CMO expression in various organs of Atriplex prostrata treated with 2% (=340 mM) NaCl for 3 days...... 76

Figure 2-10. CMO expression in 1-, 3-, 5-, 10-, and 15-day old seedlings of Atriplex prostrata...... 79

Figure 2-11. Time course of NaCl-induced CMO expression in Atriplex prostrata ...... 81

Page

Figure 2-12. The role of ABA in CMO expression in Atriplex prostrata...... 84

Figure 3-1. A schematic map of 1755 bp composite cDNA sequence encoding BADH in Atriplex prostrata ...... 99

Figure 3-2. Nucleotide sequence alignment of BADHs from spinach, sugar beet, barley and Atriplex hortensis...... 102

Figure 3-3. An alignment of three 3’ ends of ApBADH ...... 108

Figure 3-4. Nucleotide and deduced amino acid sequence of ApBADH1 ...... 112

Figure 3-5. Multiple amino acid alignments of BADHs from available BADH clones revealed conserved regions ...... 115

Figure 3-6. Examples of amino acid substitutions between ApBADH1 and ApBADH2...... 122

Figure 3-7. BADH expression in Atriplex prostrata is induced by NaCl ...... 124

Figure 3-8. Time course of NaCl-induced BADH expression in Atriplex prostrata ...... 127

Figure 3-9. BADH expression in various organs of Atriplex prostrata treated with 340 mM NaCl for 3 days ...... 129

Figure 3-10. BADH expression in 1-, 3-, 5-, 10-, and 15-day old seedlings of Atriplex prostrata...... 131

Figure 3-11. The role of ABA in BADH expression of Atriplex prostrata ...... 134

Figure 4-1. Effect of density on height (cm) of Atriplex prostrata ...... 147

Page

Figure 4-2. Effect of density on water content of Atriplex prostrata stems, leaves and roots ...... 153

Figure 4-3. Effect of density on area of leaves from the 1st, 2nd, and 3rd nodes of Atriplex prostrata ...... 155

2 Figure 4-4. Effect of density on net photosynthetic rate (μmol CO2/m /s) of Atriplex prostrata...... 158

2 Figure 4-5. Effect of density on transpiration rate (mmol H2O/m /s) of Atriplex prostrata ...... 160

Figure 4-6. Effect of density on water use efficiency (μmol CO2/mmol H2O) of Atriplex prostrata...... 162

2 Figure 4-7. Effect of density on stomatal conductance (mol CO2/m /s) of Atriplex prostrata ...... 164

Figure 5-1. PI-determined cell death induced by 340 mM NaCl over time in suspension-cultured cells of A. prostrata...... 179

Figure 5-2. PI-determined cell death induced by 170 and 340 mM NaCl in suspension-cultured cells of A. prostrata ...... 181

Figure 5-3. Cell death induced by 340 mM NaCl in suspension-cultured cells of A. prostrata...... 183

Figure 5-4. SYTO®-11-stained Nuclei in cells treated with 340 mM NaCl...... 185

Figure 5-5. DNA fragmentation in cells treated with 340 mM NaCl...... 188

Figure 5-6. Expression of CMO and BADH in suspension-cultured cells of Atriplex prostrata...... 190

Page

Figure 5–7. PI-determined cell death induced by 340 mM NaCl in the presence of 50mM glycine betaine ...... 193

Figure 5–8. Effect of glycine betaine pretreatment on cell death induced by 340 mM NaCl ...... 195

Figure 5–9. Effect of NaCl on the growth of Atriplex prostrata suspension-cultured cells...... 199

Figure 5–10. Effect of KCl on cell death in suspension-cultured cells of Atriplex prostrata ...... 204

Figure 5–11. Effect of KCl on growth inhibition of Atriplex prostrata suspension-cultured cells...... 206

Figure 5–12. Effect of simultaneous addition of glycine betaine with 340 mM NaCl on the growth of Atriplex prostrata suspension- cultured cells...... 210

Figure 5–13. Effect of glycine betaine pretreatment on the growth of Atriplex prostrata suspension-cultured cells under salt stress ...... 213

List of Abbreviations

2,4-D 2,4-dichlorophenoxyacetic acid aa amino acid

ABA abscisic acid

ABRE ABA-responsive element

Ala alanine

ALDH aldehyde dehydrogenase

ANOVA analysis of variance

Arg arginine

Asp aspartic acid

ATP Adenosine 5’- triphosphate

ATPase Adenosine 5’- triphosphatase

BADH betaine aldehyde dehydrogenase

BLAST Best Local Alignment Search Tool bp base pair

CAM Crassulacean Acid Metabolism

CDH choline dehydrogenase cDNA complementary DNA

CMO choline monooxygenase

Cys cysteine

DNA deoxyribonucleic acid

DNase deoxyribonuclease

FDA fluorescein diacetate

FUE far upstream element

Glu glutamic acid

Gly glycine

His histidine

HPLC high performance liquid chromatography

LEA late embryogensis abundant

Lov Lovastatin

MAPK mitogen-activated protein kinase

M-MLV Moloney Murine Leukemia Virus

MOPS Morpholinepropanesulfonic acid mRNA messenger RNA

MS Murashige and Skoog

NAD+ nicotine adenine dinucleotide (oxidized form)

NADP+ nicotine adenine dinucleotide phosphate (oxidized form) nt nucleotide

NUE near upstream element

PBS phosphate buffer saline

PCD programmed cell death

PCR polymerase chain reaction

PCV packed cell volume

PEPC phosphoenolpyruvate carboxylase

PI propidium iodide

PPi pyrophosphate

PSORT Prediction of Protein Localization Sites

RACE rapid amplification of cDNA ends

RNA ribonucleic acid

RNase ribonuclease

ROS reactive oxygen species rRNA ribosomal RNA

RT reverse transcription

SDS sodium dodecyl sulfate

Ser serine

SSC sodium chloride- sodium citrate buffer

TBE tris-borate-EDTA buffer

Thr threonine

TUNEL Terminal deoxynucleotidyl transferase-mediated dUTP Nick-

End Labeling

WUE water use efficiency

CHAPTER 1. INTRODUCTION

1.1 Salinity: a world problem

Salinization of soil is widespread on a global scale and causes a reduction in available land for agriculture. It normally is characterized by excessive amounts of salts, mainly NaCl, present in the soil that are transported into plants. However, salinity is not simply a matter of high Na+ and Cl- concentrations. Other ions such as

2+ 2+ - - - Mg , Ca , SO2 , HCO3 , H3BO3 , may accumulate in soils and reduce plant growth

(Breckle, 1995). One major cause of salinization in arid areas besides natural salinization is improper irrigation practices. This has occurred in Northern Africa, the Middle East, Australia, South America, the Western USA, and in Central Asia

(Rozema, 1996). There are about 3.8 billion acres of salt-affected land in the world.

As for cultivated land, at least one quarter of it has become saline (Tanji, 1990;

Yensen, 1995). As a result, crop production is reduced due to excessive salts and annual losses are estimated to be in the billons of dollars.

The presence of these ions, when they exceed a certain threshold, may inhibit plant growth for the following reasons: 1) they cause a specific ion toxicity; 2) more negative soil water potential prevent plants from absorbing water; 3) reduce uptake of K+ due to the competition from excess Na+; 4) alters physical properties of soil due to a high exchangeable Na+ percentage, which dispense clay particles and causes a reduction in hydraulic conductivity and poor aeration of soils (Rozema, 1996); 5) a lower amount of nutrients such as phosphorus and nitrogen may be available in the soil (Gorham, 1996).

1.2 Halophytes: the solution

Regardless of the definition found in the literature, halophytes are generally considered to be salt-tolerant plants that can grow and reproduce in saline environments (Ungar, 1991). Some halophytes can survive salinities which exceed that of seawater (approximately 500 mM NaCl, or 3% NaCl), although their maximum growth occurs at lower salt concentrations. On the other hand most crops, are considered as glycophytes, and exhibit severe growth inhibition at concentrations as low as 50 mM NaCl (Gorham, 1996).

Consequently, there is an increased interest in investigating various aspects of the biology of halophytes to determine whether they can be used for agricultural purposes. One possibility is that halophytes may be used as crop plants, mostly fodder. For example, Avicennia marina is used for cattle forage and firewood in

Pakistan, whereas the seeds, after being processed, can be used for human consumption (Aronson and Le Floc’h, 1996). Distichlis palmeri, a halophyte grain, can be used to make bread that has balanced proteins, high fiber and low fat (Yensen et al., 1995). Some mangrove extracts can be used as medicine for their protective effect against mosquitoes and viruses (Kathiresan et al., 1995). Other potential uses of halophytes include reclamation of salt-affected soils, protection of coastlines, and biopurification of water for irrigation (Lieth, 1999). However, most human food still comes from glycophytes. Improving crop resistance to salt stress is therefore an important goal of agricultural biotechnology. Halophytes can offer insights to assist

21 us in discovering salt tolerance mechanisms that may be targets for genetic engineering of salt-tolerant crops.

1.3 Mechanisms of salt tolerance

Salt causes two general problems for glycophytes: water stress due to the negative osmotic potential, and ion toxicity to ions such as sodium and chloride.

These two problems often result in wilting and growth inhibition of plants.

Halophytes, on the other hand, have adaptations to tolerate and flourish under saline conditions. Some mechanisms of salt tolerance revealed by studying halophytes are described below. These mechanisms are categorized into two levels for the convenience of organization. One should note that the mechanisms discussed in different categories may be related to each other. An integration of cellular level tolerance to whole plant mechanisms of adjustment to salinity is essential to maximize salinity tolerance of crops in the future.

1.3.1 Morphological and developmental levels of Salt tolerance

Germination is a crucial stage for establishment of any given individual plant.

In order to germinate under saline conditions, Atriplex prostrata has developed seed dimorphism (Khan and Ungar, 1984). Some halophytes develop waxy cuticles to reduce water loss from leaves since water can be drawn out of the plant tissue due to the excess salt in the soil (Weber, 1995). Another way of reducing water loss is through the closing of stomata. Certain halophytes double the endodermis in the root in order to retard the passage of salt into xylem. Anderson (1974) found that some

22 halophytes such as Spartina and Salicornia develop an aerenchymous root system with lignified cell walls and extensive intercellular spaces to improve aeration.

In order to deal with ion toxicity, halophytes develop an integrated system to exclude, sequester or excrete salts. Roots can exclude Na+ and Cl- as long as some type of osmoregulating organic compounds is accumulated within the cells to adjust cell water potentials so that plants can adjust their water status. Halophytes also control the salt content in plant tissue by developing a number of the following structures. Atriplex has epidermal salt hairs on the surface of leaf blades to store excess salt that is absorbed (Karimi and Ungar, 1989). As a mechanism to excrete salts from leaves, mangroves such as Avicennia marina and members of Poaceae such as Spartina have developed salt glands (Gorham, 1996). Halophytes in the families Aizoaceae and Chenopodiaceae develop bladder cells that act to eliminate salts from leaves (Breckle, 1995). Halophytes such as Suaeda and Salicornia have succulent leaves or shoots that serve to dilute salt concentration. These succulent leaves are composed of enlarged cells in which salts are sequestered and/or compartmentalized in vacuoles (Flowers, 1985). In addition, halophytes can remove salts by shedding older leaves that are primary sites for salt accumulation (Weber et al., 1977).

Halophytes can switch their photosynthetic pathway to remain metabolically active under salt stress. For example, when the ice plant (Mesembryanthemium crystallinum) is treated with 340mM NaCl, it switches from a C3 photosynthetic pathway to Crassulacean Acid Metabolism (CAM) (Dai et al., 1994; Cushman and

Bohnert, 1997). Some other species such as Spartina possess an active C4 pathway to increase water use efficiency (Langdale and Nelson, 1991).

These morphological and developmental mechanisms are complex and require the function of many gene products. Unless all of the genes involved in one mechanism are functionally characterized, these mechanisms are less likely to be targets for genetic engineering of glycophytes in order to increase salt tolerance.

1.3.2 Cellular and molecular levels of Salt tolerance

In order to tolerate saline environments, plants have to keep water flow into cells by osmotic adjustment, and to avoid ion toxicity by maintaining low cytosolic

Na+ and Cl- concentrations through selective uptake and ion compartmentalization.

Osmotic adjustment is accomplished by accumulating osmolytes in the cytosol and the osmolyte accumulation also plays a role in osmotic balance for Na+ and Cl- compartmentalized in vacuoles. In addition, ion transporters and channels are also essential to maintain a low cytosolic salt concentration. Fig. 1-1 represents a salt- tolerant plant cell that compartmentalizes K+ and osmolytes, such as proline and betaine, in the cytosol and Na+ and Cl- in the vacuole. The details of these two major cellular mechanisms as well as the upstream signaling events will be discussed later.

In addition, aquaporins, water channel proteins that enhance membrane permeability to water in both directions depending upon osmotic pressure, are reported to respond to drought and hormone treatments (reviewed by Maurel, 1997).

Fig. 1-1. The schematic depiction of a plant cell includes the vacuole, chloroplast

(cp), mitochondria (mt) after salt adaptation. Under NaCl stress, Na+ and

Cl- are sequestered to the vacuole, and K+ and osmolytes such as betaine

and proline are present in the cytosol. Various transporters and channels are

identified by the corresponding numbers in the box: 1. Cl- channel; 2.

H+/Cl- symporter; 3. K+/Na+ non-selective channel; 4. K+/Na+ symporter; 5.

K+/H+ symporter; 6. Na+/H+ antiporter; 7. H+-ATPase; 8. pyrophosphatase;

9. H+-ATPase; 10. Na+/H+ antiporter; 11. H+/Cl- antiporter. Transmembrane

proton gradients established by proton-ATPases and pyrophosphatase are

indicated (+/-). For organelles (mt and cp) no transporters have been

characterized through molecular techniques. This figure is modified from

Bohnert et al. (1999) and Serrano and Rodriguez-Navarro (2001).

? + betaine proline K - - c p - 7 + + + ? 1 + - ATP + Na Cl 8 H mt + Cl- H Cl- Vacuole PPi Cl- 1 H+ + H+ 11 Na - + 10 - - ATP - + Cl + + H 9 2 + + + + + K Na K Na K+ H+ + H H

3 4 5 Na+ 6

Regulation of water channels by salt stress has been documented at the level of post- translational modification in animal systems (King and Agre, 1996).

Reactive oxygen species (ROS) such as hydrogen peroxide, superoxide, and hydroxyl radicals are produced in photosynthetic tissues as well as in mitochondria and the cytosol. Moreover, production of these ROS is increased during drought and low temperature stress (Price et al., 1991). ROS scavenging systems therefore provide protection under salt stress. It has been suggested that osmolytes may act as compounds that neutralize the activity of free radicals (Verma, 1999). In addition, there are other proteins induced by salt or osmotic stress that might be involved in detoxification by scavenging ROS or preventing the damage of plant cellular structures (Zhu, 2001). For example, overexpression of HVA1, a late embryogensis protein (LEA) originally identified as genes induced in seeds during maturation, conferred salt tolerance in transgenic rice but we have no understanding of the mechanism for this observation (Xu et al., 1996).

Accumulation of compatible solutes as osmolytes

Osmolytes accumulated for osmotic adjustment must be compatible, not inhibitory to normal metabolism. Generally, they are easily synthesized from compounds diverted from basic metabolism. These osmolytes include amino acid derivatives (e.g. glycine betaine, proline, β-alanine betaine, proline betaine), sulfonium compounds (e.g. choline-ο-sulfate, dimethylsulfoniopropionate), and polyols (e.g. glycerol, pinitol, mannitol, sorbitol, trehalose, fructans, and methylated

27 inositols). Some osmolytes, named osmoprotectants, can stabilize proteins and membranes under environmental stress (Yancey, 1994). These osmoprotectants might have other functions. For example, polyols such as mannitol can protect against reactive oxygen species (Shen et al., 1997; Smirnoff, 1998). Glycine betaine may promote cell division (Akula et al., 2000) and protect mitochondria complex II

(Hamilton and Heckathorn, 2001). Proline possibly plays roles in nitrogen storage,

NADP+ regeneration and free radical scavenging (Verma, 1999). Some of these osmolytes and/or osmoprotectants are listed in Table 1-1 with summaries of their known function sand synthesis.

Ion uptake and membrane transporters

To actively control the ion toxicity due to excess salts, halophytes evolved mechanisms for selective ion uptake (Breckle, 1995). It was essential to develop more protective mechanisms since potassium plays an important role in metabolism, growth and development, and Na+ inhibits growth and alters development in glycophytes by inhibiting K+ uptake under high external Na+ concentrations

(Schachtman and Liu, 1999). The capacity for transporting potassium into cells and restricting sodium influx by increased K+ discrimination over Na+ is therefore an essential element for salt tolerance acquisition in halophytes. In addition, compartmentalization of Na+ in the vacuoles instead of in the cytosol eliminates Na+ toxicity. Transmembrane proton gradients, transporters and channels for K+, Na+, and Cl- together mediate ion homeostasis (Bohnert et al., 1999; Fig. 1-1).

Table 1-1. Functions and synthesis of selected osmolytes

Compounds Precursors Enzymes* Suggested functions References Glycine betaine Choline CMO & BADH Osmoprotection Papageorgious & Murata, 1995 codA Promoting cell division Akula et al., 2000 Proline Glutamate P5CR and P5CS Osmoprotection Delauney & Verma, 1993 Reducing oxidative damage Verma, 1999 DMSP Methionine MMT & DDH Osmoprotectant Gröne & Kirst, 1991 Pathogen defense Gage & Rathinasabapathi, 1999 Mannitol Fructose-6-P PMI, MPR and MPP Osmotic adjustment Gorham, 1996 Protection against oxidative stress Shen et al., 1997 Pinitol myo-Inositol IMT & OEP Osmotic adjustment Gorham, 1996 Sorbitol Glucose-6-P S6PDH & SPP Osmotic adjustment Gorham, 1996 Trehalose Glucose-6-P TPS & TPP Osmoprotection Goddijn & van Dun, 1999

* Abbreviations of enzymes: CMO- choline monooxygenase BADH- betaine aldehyde dehydrogenase; codA- choline oxidase; P5CR- ∆1-pyrroline-5-carboxylate reductase; P5CS-∆1-pyrroline-5-carboxylate snythetase; MMT- methionine methyltransferase; DMSP- dimethylsulfoniopropionate DDH- dimethylsulfoniopropionaldehyde dehydrogenase; PMI: phospho-mannose isomerase; MPR- mannose-6-phosphate reductase; MPP- Mannitol-1-phosphate phosphatase; IMT- myo- inositol-6-о-methyltransferase; OEP- ononitol epimerase; S6PDH- sorbitol-6-phosphate dehydrogenase; SPP- Sorbitol-6- phosphate phosphatase; TPS- trehalose-6-phosphate synthase; TPP- trehalose-6-phosphate phosphatase.

28 29

A transmembrane proton gradient is maintained by three proteins: the plasma membrane H+-ATPase, the vacuolar H+-ATPase and a vacuolar pyrophosphatase. In

Salicornia, both plasma membrane and vacuolar H+-ATPases exhibit increased activity in response to NaCl (Ayala et al., 1996). As for pyrophosphatase, the response to NaCl is not clearly known (Rea and Poole, 1993; Zingarelli et al., 1994).

Since a transmembrane proton gradient drives the translocation of Na+ as well as other ions, it is not surprising that a functional yeast vacuolar H+-ATPase is needed for salt tolerance (Gaxiola et al., 1999).

There are two components involved in K+ uptake and transport. One is a low- affinity system active in the mM range of K+, and the other is a high-affinity system functioning at µM levels of K+. Functional complementation of yeast mutants deficient in K+ uptake helped to isolate K+ transporters and channels from plants

(Anderson et al., 1992; Schachtman and Schroeder, 1994). The low-affinity system is indeed an inward-rectifying K+ channel that is highly selective against Na+, and there is no evidence of specific regulation by salt for this channel (KAT-1 in

Arabidopsis; Bertl et al., 1995). The high-affinity system, HKT1 from wheat, is a

K+/Na+ symporter, and mutated HKT1 that increases K+ selectivity by reducing Na+ permeability conferred salt tolerance (Rubio et al., 1995; Rubio et al., 1999). The fact that high level expression of a rice homolog to wheat HKT1 occurs in a salt- sensitive variety but not in a salt-tolerant variety under salt stress supports the idea that this K+/Na+ symporter is involved in salt tolerance (Horie et al., 2001).

While Na+ transport across plasma membrane may be carried out by the high affinity K+ uptake system and plasma membrane Na+/H+ antiporter (Fig 1-1), Cl- influx is presumably through H+-Cl- symporter and anion channel (Hasegawa et al.,

2000). In terms of transport from cytosol to vacuole, vacuolar Na+/H+ and H+/Cl- antiporters (Fig. 1-1) are predicted to be responsible although the latter has not been identified. Recently, a plant vacuolar Na+/H+ antiporter, AtNHX1, was isolated via the Arabidopsis genome-sequencing project (Gaxiola et al., 1999). Overexpression of AtNHX1 in Arabidopsis caused an accumulation of Na+, presumably in the vacuole, and conferred salt tolerance (Apse et al., 1999). As for the Na+/H+ antiporter located on the plasma membrane (Fig. 1-1), overexpression of ApNhaP, a plasma membrane Na+/H+ antiporter isolated from a halotolerant cyanobacterium

Aphanothece halophytica, enabled Synechococcus sp. PCC 7942, a freshwater cyanobacterium, to exclude Na+ while living in seawater (Waditee et al., 2002). An

Arabdopsis counterpart to the Nha gene, SOS1, was identified as the first plasma membrane Na+/H+ antiporter in higher plants (Shi et al., 2000).

Sensing and signaling of salts

Due to the lack of a genetic model system for halophytes, a lot of the regulatory proteins involved in cellular signal pathways that lead to the expression of these salt- tolerant genes mentioned above were identified through genetic analysis of

Arabidopsis mutants. Studies in yeast involving regulation of ion homeostasis also led to some important discoveries (Hasegawa et al., 2000). Finally, salt stress

31 regulatory determinants discussed in this section were mostly discovered and identified based on functional complementation of yeast mutants.

As mentioned earlier, SOS1 from Arabidopsis is a plasma membrane Na+/H+ antiporter and exports Na+. Two other genes found at the same loci, SOS2 and SOS3, turned out to be the regulatory elements for proper function of SOS1 (Zhu, 2001).

SOS3 is a myristoylated protein with 3 EF hands for Ca2+ binding (Liu and Zhu,

1998; Ishitani et al., 2000). Physical interaction of SOS3 with SOS2 activates SOS2, which is a serine-threonine protein kinase, and this activation regulates SOS1 expression (Halfter et al., 2000; Liu et al., 2000). However, it remains to be determined whether or not the SOS3-SOS2 kinase complex directly regulates the activities of SOS2. It is also possible that other regulatory proteins may be involved.

In yeast, calcineurin, which is a Ca2+ and calmodulin-dependent protein phosphatase, regulates Na+, K+ and Ca2+ homeostasis and is required for salt tolerance (Nakamura et al., 1993). A family of proteins that are homologous to yeast calcineurin have been isolated from Arabidopsis (Kudla et al., 1999). SOS3 is indeed a member of this protein family. Another gene, AtGSK1, was identified to rescue a yeast calcineurin mutant (Piao et al., 1999). Overexpression of AtGSK1 in

Arabidopsis exhibited enhanced NaCl tolerance and accumulated more Na+ than wild-type (Piao et al., 2001). The fact that overexpression of AtGSK1 induced the expression of salt-responsive genes in the absence of salt stress indicated that it is an upstream component of the salt stress signaling pathway. Another protein, AtSLT1

(for sodium/lithium tolerance), also suppressed this yeast calcineurin mutant and partially suppressed yeast ATPase mutant (Matsumoto et al., 2001).

In yeast, Dbf2, which is a cell-cycle-regulated serine/threonine protein kinase, modulates expression of genes involved in salt tolerance as a component of a general transcriptional regulatory complex (Liu et al., 1997). The Arabidopsis homologue of yeast Dbf2 was identified by screening libraries for osmotolerance, and overexpression of AtDBF2 in tobacco BY-2 cells enhanced the tolerance of plant cells to salt and drought stress (Lee et al., 1999). Moreover, a general stress signaling cascade mitogen-activated kinase (MAPK) in plants may be essential for salt stress signaling as well, implicated by the fact that a pea MAPK, PsMAPK, suppressed the

NaCl-induced growth inhibition in salt-sensitive yeast mutants (Pöpping et al., 1996).

Although numerous signal or signal-like proteins have been identified, little is known in terms of how these proteins function in one or more signal pathways. For example, SOS genes and calcineurin-like genes and their related proteins in

Arabidopsis were identified but no definite pathway has been revealed. In addition, whether the SOS pathway and the calcineurin-like pathway, if they exist, cross-talk experience remains unclear and requires further investigation. Moreover, some of these proteins may be responsive to several environmental stresses (i.e., salt, drought and temperature), and it is unclear whether any of them function exclusively in salt stress.

1.4 Improvement of salt tolerance in crops

More and more experiments are designed to genetically engineer glycophytes for increased salt tolerance according to the cellular mechanisms discussed in 1.3.2.

Most targets of such transgenic research are either enzymes involved in osmolyte synthesis (Table 1-2) or proteins involved in detoxification of Na+ and ion homeostasis (Table 1-3). Accumulation of osmolytes/osmoprotectants has remained a target for plant genetic engineering for more than a decade (Nuccio et al., 1999).

As summarized in Table 1-2, introduction of corresponding enzyme(s) has led to modest accumulation of osmolytes and, consequently, an increase in salt tolerance.

However, there were cases where a growth defect was observed and attention was attracted to the fact that metabolic engineering creates an imbalance in primary metabolism (Sheveleva et al., 1998). A thorough analysis of metabolism is indeed needed in order to avoid such imbalances and side effects. In addition, a complete understanding of metabolism might benefit metabolic engineering by increasing the efficiency of biochemical processes. For example, in the case of engineering glycine betaine for increased salt tolerance in tobacco, it is not sufficient to introduce BADH and CMO into a betaine non-accumulator (Nuccio et al., 1998). After metabolic control analysis, it was determined that increasing the choline pool turned out to be essential (McNeil et al., 2000).

Table 1-2. Summary of metabolic engineering for salt tolerance

Osmolytes Source Gene* Product level† Species engineered References

Glycinebetaine Spinach cmo & badh 1 tobacco Nuccio et al., 1998

A. globifomis codA 20 Arabidopsis Hayashi et al., 1997

E. coli cdh 26 tobacco Lilius et al., 1996

Proline Mothbean P5CS 180 tobacco Kavi Kishor et al., 1995

D-Ononitol Ice plant imt1 10-70 tobacco Sheveleva et al., 1997

Mannitol E. coli mtldh 16 tobacco Tarczynski et al., 1992

E. coli mtldh 8-16 tobacco§ Karakas et al., 1997

Sorbitol Apple s6pdh 0.7-300 tobacco§ Sheveleva et al., 1998

Trehalose Yeast tps1 1.2 tobacco§ Romero et al., 1997

E. coli tps1 & tpp 0.8-28 tobacco § Goddijn et al., 1997

* More abbreviations: cdh- choline dehydrogenase; mtldh- mannitol dehydrogenase (see Table 1-1 for the others). † The level listed is the level of osmolyte synthesis in stressed transgenic plants as a percentage of the level found in a representative plant that naturally accumulates the same osmolyte under similar conditions. § A growth defect was reported. 34

Table 1-3. Summary of genetic engineering with non-osmolytes for salt tolerance

Product Source Gene Function* High tolerance? References

K+ transporter Arabidopsis HKT1 Enhanced K+ uptake Yes Rubio et al., 1995

Na+/H+ antiporter cyanobacterium ApNhaP Na+ exclusion Yes Waditee et al., 2002

Arabidopsis AtNHX1 Accumulation of Na+ in vacuole Yes Zhang et al., 2001

Ser/Thr Kinase Arabidopsis AtDBF2 Signaling Yes Lee et al., 1999

LEA protein Barley HVA1 Detoxification§ Yes Xu et al., 1996

GS† Rice GS2 Enhanced photorespiration Yes Hoshida et al., 2000

* Function listed here is solely based upon the evidence presented in the references with one exception (see “§” below).

§ This function is not clearly known yet, but proposed by Xu et al. (1996) as well as by Bohnert et al. (1999).

† chloroplastic glutamine synthetase

35 36

A few components involved in ion homeostasis were unveiled only in the past couple of years, which makes genetic engineering of these components possible in the near future for improving salt tolerance in crop plants. To date, there are only a few reports where salt tolerance was conferred to crops (Table 1-3). For example, overexpression of AtNHX1 encoding a vacuolar Na+/H+ antiporter in Arabidopsis

(Apse et al., 1999), tomato (Zhang and Blumwald, 2001), and Brassica (Zhang et al.,

2001) allow these transgenic glycophytes to grow, flower and produce seeds in 200 mM NaCl. This modification of a single trait significantly improves salt tolerance, suggesting that production of salt-tolerant crops with far less target traits than had been anticipated is possible (Zhang et al., 2001).

In addition, transgenic improvements in plant salt tolerance have been achieved by overexpression of enzymes involved in oxidative protection. By doubling the glutathione S-transferase/glutathione peroxidase activity in transgentic tobacco, seedlings and plants showed significantly faster growth than wild type under salt stress (Roxas et al., 1997).

1.5 In this research

Among all these mechanisms discussed above, I chose to study the accumulation of glycine betaine and its synthesis in Atriplex prostrata after studying salt-induced growth inhibition in the same plant. Atriplex, a large and widely distributed genus of the Chenopodiaceae, is highly salt tolerant and naturally occurs is on saline land (Ungar, 1991). It is known that Atriplex species tightly regulate the

37 uptake of Na+ and K+, and Na+ that is taken up is translocated to older leaves or stored in the vacuole and salt bladder cells of younger leaves to avoid damage of photosynthetically active young leaves (reviewed by Aslam, 1999). To date, the

Na+/H+ antiporter and H+-ATPase involved in regulating ion homeostasis have been cloned from Atriplex species (Hamada et al., 2001 and Niu et al., 1993, respectively).

Atriplex species also accumulate osmoprotectants such as glycine betaine. In fact, the

Chenopodeaceae is one of the few plant families that accumulate glycine betaine in response to salt (Weretilnyk et al., 1989). In this research, two genes encoding the enzymes catalyzing the synthesis of glycine betaine, choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), were cloned from Atriplex prostrata to investigate the expression of these two genes in response to salt. Moreover, suspension-cultures of A. prostrata were established and cell death induced by NaCl in this cell line that might not accumulate glycine betaine as a result of no expression of CMO was investigated. In addition, halophytes such as A. prostrata have relatively high light requirement, therefore their growth in saline habitats is affected by both salt stress and competition (Ungar, 1991). In this research, controlled laboratory conditions were used to mimic intraspecific competition (i.e., density) in the field in order to understand the effects of density on growth and photosynthesis of A. prostrata.

CHAPTER 2. CLONING AND EXPRESSION OF CHOLINE

MONOOXYGENASE IN ATRIPLEX PROSTRATA

Li-Wen Wang and Allan M. Showalter

Manuscript submitted to

Physiologia Plantarum

ABSTRACT

Plants accumulate osmoprotectants such as glycine betaine in response to salinity. In plants, glycine betaine is synthesized via the two-step oxidation of choline, and the first step is catalyzed by choline monooxygenase (CMO). Cloned by RT-PCR and

3’-RACE, the cDNA of Atriplex prostrata CMO (ApCMO) is 1669 bp in length and encodes a full-length protein of 438 amino acids. The deduced amino acid sequence of ApCMO revealed a Rieske-type [2Fe-2S] cluster motif and a mononuclear non- heme Fe binding motif, and shares 82.9% identity and 87.2% similarity with the deduced amino acid sequence of spinach CMO. Accumulation of CMO transcript and glycine betaine both increase in response to NaCl. Without salt treatment, CMO mRNA was detected in stems and 5-day-old seedlings and not in leaves, roots, and older seedlings. With salt treatments, CMO mRNA accumulated dramatically in stems, leaves and roots, and most abundantly in young stems where accumulation occurs within 24 hours. Although abscisic acid may initiate global physiological reactions in response to osmotic stress, it did not induce the expression of CMO in A. prostrata. In summary, salt-induction of CMO mRNA in A. prostrata is more substantial than that reported in spinach and sugar beet, and the plant may serve as a useful model to study regulation of glycine betaine synthesis by environmental stress.

INTRODUCTION

Plants have developed different mechanisms in order to survive in response to salt stress. Among the known mechanisms associated with salt tolerance, accumulation of osmoprotectants such as glycine betaine and proline is of particular interest because of the potential to genetically engineer their pathways into non-salt tolerant crops. When subjected to salt stress, all plants make an osmotic adjustment by accumulating inorganic ions and organic solutes. However, many metabolic pathways are inhibited at high concentration of ions such as Na+. Salt tolerant plants are known to accumulate organic solutes in the cytosol (reviewed by Wyn Jones and

Gorham, 1983). Among these organic solutes, osmoprotectants not only can raise the cytoplasmic osmotic potential, but also can stabilize metabolic enzymes (Rhodes and

Hanson, 1993).

Osmoprotectants, which are highly soluble compounds that carry no net charge at physiological pH, may be amino acids, polyols, sugars or quaternary ammonium compounds. They can maintain protein and membrane stability, and possibly reduce or reverse the inhibitory effects of ions such as Na+ (Bohnert et al., 1999). Two models are proposed to explain how these compounds stabilize protein. One is the

“preferential exclusion model” which assumes these soluble compounds are largely excluded from the hydration shell of proteins and thus leave a water shell around proteins (Arakawa and Timasheff, 1985). It is the water shell that stabilizes proteins and promotes or maintains the protein-protein interaction, without disturbing the

41 native hydration shell of proteins. The other is the “preferential interaction model” which, instead, emphasizes the physical interactions between solution and proteins.

For example, during dehydration, compatible solutes may interact directly with the hydrophobic domains of proteins and thus prevent their destabilization (Schobert,

1977). Nevertheless, osmoprotectants play important roles not only in salt tolerance, but also in the adaptation of cells to other environmental stresses such as cold and drought.

Glycine betaine, one of the quaternary ammonium compounds, is a particularly well-known osmoprotectant that effectively stabilizes enzymes critical to physiological functions (Papageorgiou and Murata, 1995). Under salt stress, the level of glycine betaine in salt-tolerant plants typically rises. For example, in Atriplex griffithii treated with 360 mM NaCl for 30 days, the level of glycine betaine reached

160 mM on a tissue water basis, while in Suaeda fruticosa treated with 360 mM

NaCl, the level of glycine betaine is about 120 mM (Khan et al., 1998).

In some bacteria such as Arthrobacter globiformis, glycine betaine is synthesized from choline by choline oxidase. The gene for this choline oxidase, codA, has been cloned and transformed into Arabidopsis thaliana. This transformation enabled Arabidopsis to accumulate glycine betaine and enhanced its salt tolerance (Hayashi et al., 1997). E. coli, on the other hand, synthesizes glycinebetiane via a two-step reaction that is mediated by a membrane-bound oxygen-dependent choline dehydrogenase (CDH), which oxidizes choline to betaine aldehyde and then oxidizes betaine aldehyde to glycine betaine. The second step can

42 also be catalyzed by a NAD+-dependent betaine aldehyde dehydrogenase (BADH)

(Landfold and Strøm, 1986). In higher plants, glycine betaine is synthesized by a two-step oxidation of choline with betaine aldehyde serving as an intermediate (Fig.

2-1). The first step is catalyzed by choline monooxygenase (CMO) in the presence of ferredoxin, while the second step is catalyzed by BADH, in a NAD+-dependent reaction. Both enzymes are located in the stroma of chloroplasts and increase in activity in response to salinity treatments (Weigel et al., 1986; Brouquisse et al.,

1989).

After being identified as a unique plant oxygenase containing a Rieske-Type

[2Fe-2S] center, CMO was first cloned from spinach by RT-PCR, using primers corresponding to amino acid sequences in this iron-sulfur center, followed by cDNA library screening (Rathinasabapathi et al., 1997). So far, this enzyme, which catalyzes the committed step of glycine betaine synthesis, was cloned o from spinach

(Spinacia oleracea; Rathinasabapathi et al., 1997), sugar beet (Beta vulgaris; Russell et al., 1998), Atriplex hortensis, and amaranth (Amaranthus tricolor; Meng et al.,

2001). Little is known about the expression of CMO except that it increases in response to salt in spinach leaves and in sugar beet leaves and roots (Russell et al.,

1998). Recently, in amaranth, CMO protein was detected after salt and drought stress

(Meng et al., 2001).

Fig. 2-1. The conversion of glycine betaine from choline is catalyzed by

two enzymes: choline monooxygenase (CMO) and betaine

aldehyde dehydrogenase (BADH).

CH 3

+ H 3 C - N - CH 2 - CH 2 - OH choline CH 3

choline monooxygenase (CMO)

CH 3 O - N+ H 3 C - CH 2 - C betaine aldehyde H 3 CH

betaine aldehyde dehydrogenase

(BADH)

3 CH + O glycine betaine H 3 C - N - CH 2 - C OH CH 3

The questions I would like to address in this investigation in addition to cloning of CMO cDNA from Atriplex prostrata are: 1) whether CMO expression is regulated by salt; 2) whether CMO expression is differentially regulated in different organs and at different developmental stages, and 3) whether ABA regulates CMO expression.

MATERIALS AND METHODS

Plant Material and Growing Conditions

Seeds of Atriplex prostrata were collected in the salt marsh near Morton Salt

Co. at Rittman, OH, and germinated in flats filled with sand in an environmental growth chamber under a 25 ºC /5 ºC, 12 h day/12 h night regime (400-700 nm, 400

µmol m-2 s-1). Ten days after germination, seedlings were transferred to four-inch pots filled with sand at a density of four seedlings per pot. Pots were scattered in plastic trays. These trays were filled with half-strength Hoagland and Arnon No.2 solution (nutrient solution) with or without 170 or 340 mM NaCl and maintained in an environmental growth chamber under a 25ºC/15ºC, 14 h day/10 h night regime.

Distilled water was added daily to each tray to replace water lost because of evaporation and transpiration. Solutions were replaced weekly.

For seedling experiments, seeds were germinated on moist filter paper in large petri dishes for easy tracking of germination time. For ABA and Lovastatin treatments, plants were grown hydroponically for a week before being transferred to nutrient solutions supplemented with or without 10 µM (+/-) ABA (Sigma, St. Louis,

MO) or 10 µM Lovastatin (Sigma). After two days, plants were transferred to nutrient solutions with or without 340 mM NaCl.

RNA/mRNA Isolation from Salt-Treated Atriplex prostrata

Tissues from Atriplex prostrata treated with 170 or 340 mM NaCl for various times were collected and frozen in liquid nitrogen, prior to grinding in liquid nitrogen. Total RNAs were isolated as described using RNeasyTM Plant Total RNA

Kit (Qiagen Inc., Chatsworth, CA). Messenger RNA was isolated from total RNA using a mRNA isolation kit (Qiagen).

RT-PCR

Total RNA from leaves treated with 340 mM NaCl for 3 days was reverse- transcribed into cDNAs in the presence of MMLV-Reverse Transcriptase (Promega,

Madison, WI). PCR reactions were then carried out using Taq polymerase (Promega,

Madison, WI) with the following cycle profile: initial denaturation of cDNAs at

95°C for 2 min, 30 cycles of amplification by denaturing at 95°C for 1 min, annealing at 50°C for 1 min and extending at 72°C for 1 min for each cycle, and a final extension (i.e., polishing) at 72°C for 7 min. Annealing temperatures may vary but were set to be 4 °C lower than the lowest Tm of the paired primers (synthesized by Integrated DNA technologies, Inc., Coralville, IA). The sequence and Tm of all primers (calculated by Integrated DNA technologies, Inc. according to the nearest-

47 neighbor thermodynamic parameter) used in RT-PCR and PCR including RACE are listed in Table 2-1. The relative positions of these primers were shown in Fig. 2-2.

3’ Ends by RACE (Rapid Amplification of cDNA Ends)

Two µg of mRNAs isolated from NaCl-treated leaves was used to carry out first and second strand cDNA synthesis. The resulting double stranded cDNAs were then ligated to MarathonTM cDNA adaptors (Clontech, Palo Alto, CA) prior to 3’-

RACE. CMO6, a GC-rich primer, was designed based on the sequence of previous

RT-PCR products. CMO6 and the adaptor primer AP1 (Table 2-1) amplified the 3’ end product of ApCMO with the touchdown PCR program according to the instruction of MarathonTM RACE (Clontech, Palo Alto, CA).

Subcloning of PCR Products

All RT-PCR and RACE products were separated by electrophoresis with 1.0% agarose, and DNA fragments of desired length were excised from the gel and purified using GENECLEAN® (Qbiogene Inc., Carlsbad, CA) prior to subcloning.

 The vector, pCR II-TOPO (3.9 kb), included in TOPO cloning kit by Invitrogen

(Carlsbad, CA), was used to subclone the PCR products.

Sequence Analysis

Subcloned cDNAs were labeled using BigDye Terminator Cycle Sequencing

Reactions kit (Perkin-Elmer, Foster City, CA) and then sequenced with an automated sequencer (ABI 310, Perkin-Elmer, Foster City, CA). Sequence comparison with

Table 2-1. Primers used in cloning Atriplex prostrata CMO

Primersa Sequence Tmb Position

CMO3 TGTTTCGTGTGCCCTTACCAT 54.45 539-559c

CMO4 TGGTGGATTCCTTTCTCAATAGG 54.00 1265-1287d

CMO5 CCTGCCTTCTATTCCCATGAAC 54.34 320-341c

CMO6 CACGATTCAAAGGGTGGCTGGGACTTC 66.96 943-969c

AP1 CCATCCTAATACGACTCACTATAGGGC 59.40 NA (Adaptor)

CMO5S AATCATGGCAGCAAGTGCAA 57.30 1-20c

CMO5AS GGTCAAAAGCATGGGCCTTA 56.78 760-779d

a Cross-reference to Fig. 2-2 b Tm was calculated based on the nearest-neighbor thermodynamic parameter

(Allawi and SantaLucia, 1997) by Oligo Analyzer 2.5 available at

www.idtdna.com. c Sense primer d Antisense primer

Fig. 2-2. A schematic map of 1669 bp composite cDNA sequence encoding CMO in Atriplex prostrata. “ “:

Primers used for RT-PCR (see Table 2-1 for the sequence of these primers). “ “: cleavage site of restriction

enzymes. “ “: RT-PCR fragment, subcloned and sequenced. “ “: Positions of either start codon

(ATG) or stop codon (TGA).

CMO5S CMO5 CMO7 CMO3 CMO5AS CMO6 CMO4 1669 AAAA

5’ ATG 3’ HindIII PstI SpeI EcoRI TGA

ApCMO-0.7 745 bp

ApCMO-1.0 967 bp

ApCMO-3’ 730 bp

ApCMO-5’ 769 bp

50 51 other databases was performed through the National Center for Biotechnology

Information (http://www.ncbi.nlm.nih.gov) via BLAST. Sequence alignments and analysis were performed using AlignX® of Vector NTI suite 6 (InforMax, Inc.,

Bethesda, MD).

The sequences of cloned CMO were also subjected to additional sequence analysis such as PSORT, Motif Finder and ChloroP. The addresses of these web sites that perform these analyses are:

PSORT http://psort.nibb.ac.jp

Motif Finder http://motif.genome.ad.jp

ChloroP (V 1.1) http://www.cbs.dtu.dk

Northern Blotting

Either 15 or 20 µg of total RNAs were subjected to electrophoresis in 1% agarose gels containing MOPS and 7% formaldehyde. Gels were then transferred to

Zeta-probe GT blotting membranes (Bio-Rad, Hercules, CA) in 10X SSC.

Membranes were fixed by a Stratalinker® UV crosslinker (Model 1800, Stratagene,

La Jolla, CA) at an energy of 120 mJ for approximately 2 min and then air-dried. A partial CMO cDNA, ApCMO-0.7, was labeled with α-32P-dCTP (30ng of cDNA with 50 µCuries of α-32P-dCTP) using Prime-a-Gene® Labeling System (Promega,

Madison, WI) and used as a probe. Prehybridization and hybridization reactions were carried out in 7% SDS, 250 mM sodium phosphate buffer (pH7.2) at 65°C. Washing

52 was performed in ¼ strength prehybridization solution at room temperature for 5 min and twice at 65°C for 10 min.

Autoradiographs of northern blots and ethidium bromide-stained gels which were photographed were scanned using a digital imaging system (Fluor-S™

MultiImager MAX, Bio-Rad, Hercules, CA) and analyzed with Quantity One® quantitation software (version 4.2.2, Bio-Rad) to quantify levels of rRNA and CMO transcript. Levels of CMO transcript were normalized with respect to the amount of rRNA in each lane, determined by ethidium-bromide staining of the original gel.

Measurement of Glycine Betaine

Glycine betaine was extracted with boiling water and measured with a HPLC

(HP 1050 modular 3D, Hewlett Packard, Boise, ID) as described in Khan et al.,

(1998). Glycine betaine (Sigma, St. Louis, MO) standards were run at 1, 10, and 100 mmol / L.

Relative RT-PCR

In order to compare the amount of CMO mRNA present in each treatment, a relative RT-PCR was designed by incorporating ProSTAR® HF Single-Tube RT-

PCR (Stratagene, La Jolla, CA) and QuantumRNATM 18S Internal Standards

(Ambion, Austin, TX). Total RNAs (100ng) from each treatment were reverse- transcribed into cDNAs in the presence of MMLV-Reverse Transcriptase

(Stratagene). One-fifth of the RT reaction (2 µl) was then used in the PCR reaction

53 carried out by TaqPlus® Precision DNA polymerase, which is a blend of Taq and

Pfu DNA polymerase, from ProSTAR® HF Single-Tube RT-PCR. PCRs were performed using GeneAmp® PCR system 2400 Thermal Cycler (Applied Biosystems,

Foster City, CA) with the following cycle profile: 25 cycles of amplification by denaturing at 95°C for 30 sec, annealing at 50°C for 30 sec and extending at 68°C for 1 min for each cycle, and a final extension at 68°C for 10 min. Preliminarily,

RT-PCR was performed with different number of cycles (in increments of five) to determine the linear range of amplification specific for the target and primer pairs,

CMO3 and CMO4. In addition, the optimal 18S Primer: CompetimerTM ratio for

CMO3 and CMO4 was determined to be 3:7 also by preliminary experiments.

RESULTS

Cloning of CMO by RT-PCR and RACE

An alternative approach to conventional cDNA library screening for cloning

CMO is to clone by RT-PCR and RACE given that there were known cDNA sequences of CMOs. For cloning CMO from Atriplex prostrata, sequences of spinach and sugarbeet CMOs were aligned and compared to identify consensus regions (Fig. 2-3). Three primers, CMO3, CMO4 and CMO5, were designed based on the consensus region to amplify a partial CMO from Atriplex prostrata (see Table

2-1 for the sequence and Tm of these primers and Figs. 2-2 and 2-3 for their

Fig. 2-3. A nucleotide sequence alignment of four available CMO cDNAs with ApCMO. The nucleotide sequences of

AhCMO (Atriplex hortensis, accession number: AF270651), SpCMO (Spinacia oleracea, accession number:

U85780), AmaCMO (Amaranthus tricolor, accession number: AF290974), and beetCMO (Beta vulgaris,

accession number: AF023132) were aligned with that of ApCMO (accession number: AY082068) by AlignX®

of Vector NTI. The nucleotides conserved among all five clones are shaded in yellow, and those conserved

among three or four of the five clones are shaded in blue. Primers CMO3, 4, and 5, which are designed based

upon the sequence of spinach and sugar beet CMOs, are labeled.

1 10 20 30 40 50 60 70 80 90 100 $S&02 ------AAT $K&02 ATTGTTGATCATTCATCTCATCACCTCCTTATATATTATATTAAAATAAATTAATAAATATTAACAAGGAAGTGTTTAAGTTTGTTGATCATACAACAAT 6S&02 ------TAGTAAGGGTGTAGCTAATTAGCAAAATAAACAAAAAGGAAGTGTTGAGTTGGTTAAT EHHW&02 ------CTCGTGCCGAATTCGGCACGAGAAAATCTAGTAAC $PD&02 ------CGGCACCAGAAAAAAACATAGAAGAGTTTTGAGTGGTATTATTAG &RQVHQVXV A A AA A A A GG T T TAAT

101 110 120 130 140 150 160 170 180 190 200 $S&02 CATGGCAGCAAGTGCAA------CAACAATGTTGCTAAAATACCCAACTACTGTTTGCGGAATACCAAATTC------ATCCTCAAACAATTCTACTGAT $K&02 CATGGCAGCAAGTGCAA------CAACAATGTTGCTAAAATACCCAACTACTGTTTGTGGAATACCAAATTC------ATCCGCAAACAATTCTACTGAT 6S&02 GATGGCAGCAAGCGCAAGCGCAACCACAATGTTGCTAAAATACCCAACTACAGTTTGTGGTATTCCAAATCC------TTCATCAAACAATAATAATGAT EHHW&02 AATGGCAGCAAGTGCTA------CAACCATGTTGCTCAAATACCCAACTC---TTTGTGCTATGCCAAATTCCTCTTCATCTTCAAACAA--CAAC-GAT $PD&02 TATTGT-GCGATGGCATCATCAGCTTCAATGTTGATAAATTATCCAACTACTTTTTGTGGAGTTAGAAATTCAT---CAAATCCAAATAATGATCAATTT &RQVHQVXV ATGGCAGCAAGTGCAA CAACAATGTTGCTAAAATACCCAACTACTGTTTGTGGAAT CCAAATTC ATC TCAAACAAT CTACTGAT

201 210 220 230 240 250 260 270 280 290 300 $S&02 CCTTCAAATAACAT-CGTCCAAATTCCACAAACTAATACT------ACAAAAAGCCCGTTACTTAAGTGCCGTA------CTCCTAATAAACCCG $K&02 CCTTCAAATAACAT-CGTCCAAATTCCACAAACTACTACT------ACTAATAGCCCGCTACTTAAGTTCCGTA------CTCCTAATAAACCCG 6S&02 CCTTCAAACAATAT-AGTTTCTATTCCACAAAATACTACT------AA-TCCAACACTTAAGTCCCGTA------CACCTAATAAAATCA EHHW&02 CTTCCTACTAGCATTCCTCTTAATAACAATAACAATTTATTATCAAACAAAAACAAAATTCTTCAAACACCAAATATTAATACTTCTACTAATAAAATCA $PD&02 TCTGATCAAATTAACATTCCTTCTT-CATTAAATAATAAT------ATTAATATTAGTAAAATTACAAGTA------AAACCAATAAAATAA &RQVHQVXV CCTTCAAATAACAT CGTCC AATTCCACAAACTA TACT AC AAAA CCATTACTTAAGT CCGTA CTCCTAATAAAATCA

301 310 320 330 340 350 360 370 380 390 400 $S&02 T---TAACGCCGTCGCTGCCCCGGCTTTTCCGTCCG---CAACCACCATCA---CAACCACCACTCCGCCGTCCATCCAATCACTTGTCAAGGATTTCGA $K&02 T---TAACGCCGTCGCTGCCCCGGCTTTTCCGTCCG---TAACCACCACTA---CAACCACCACTCCGTCGTCCATCCAATCACTTGTCAAGGATTTCGA 6S&02 CCACCAACGCCGTCGCGGCACCGTCCTTTCCTTCTT---TAACCACCA------CTACACCGTCGTCCATCCAATCACTTGTCCACGAATTCGA EHHW&02 TCACTAAAGCTGTTGCTTCCCCTGTTTTTCCAACTC---TAAAAACCA------CATCCAACACACCTTCTTCCATTCGATCACTTGTTCATGAATTCGA $PD&02 TCCCAAAAGCAGTAGCATCCCCTGTGATTCCATCTTCTATAAATAGTAATAATATAACAACAACAACACCAAATATCAAAAGAATAATTCATGAATTTGA &RQVHQVXV TC CTAACGCCGTCGCTGCCCCGGCTTTTCC TCT TAACCACCA A CAACCACCACACCGTCGTCCATCCAATCACTTGTCCA GAATTCGA

401 410 420 430 440 450 460 470 480 490 500 $S&02 TCCTTCTATTCCGGCCGAGGATGCTTTTACTCCTCCTAGCTCTTGGTATACCGAACCTGCCTTCTATGCTCATGAACTTGACCGTATCTTTTACAAAGGA $K&02 TCCTCTTGTTCCGGCCGAGGATGCTCTTACTCCTCCTAGCTCTTGGTATACCGAACCTGCCTTCTATGCTCATGAACTTGACCGTATCTTTTACAAAGGA 6S&02 CCCTCAAATTCCCCCTGAAGACGCTCATACACCTCCTAGCTCTTGGTATACCGAACCTGCCTTCTATTCCCATGAACTTGAGCGTATCTTTTATAAAGGA EHHW&02 CCCCGAAATTCCACCTGAAGATGCTCTTACACCTCCTAGTACTTGGTACACTGAGCCTGCCTTTTATTCCCATGAACTTGAACGTATCTTTTACAAAGGA $PD&02 TCCAAAAGTTCCAGCTGAAGATGGTTTCACTCCTCCTTCTACTTGGTACACTGACCCTTCCCTTTATTCTCATGAACTTGACCGTATCTTTTCCAAAGGA &RQVHQVXV TCCT AAATTCC GCTGAAGATGCTCTTACTCCTCCTAGCTCTTGGTATACCGAACCTGCCTTCTATTCTCATGAACTTGACCGTATCTTTTACAAAGGA CMO5 55

Fig. 2-3. continued 501 510 520 530 540 550 560 570 580 590 600 $S&02 TGGCAAGTCGCAGGGTACAGTGATCAAGTTAAGGAGGCTAACCAATATTTCACCGGAACGTTAGGAAATGTTGAATATTTGGTGAGTCGAGATGGTGAAG $K&02 TGGCAAGTCGCAGGGTACAGTGATCAAGTTAAGGAGGCTAACCAATATTTCACCGGAACGTTAGGAAATGTTGAATATTTGGTGTGTCGAGATGGAGAAG 6S&02 TGGCAAGTTGCAGGGATCAGCGATCAAATAAAAGAGCCTAACCAATATTTCACTGGCAGCTTAGGAAATGTTGAATATTTGGTGTCTCGAGATGGTGAAG EHHW&02 TGGCAAGTTGCAGGCTACTCTGAGCAAGTAAAGGAGAAAAATCAATATTTCACTGGCAGTTTAGGGAATGTTGAATATTTAGTATCTCGAGATGGTCAAG $PD&02 TGGCAAGTCGCAGGATATAGTGATCAAATAAAGGAGCCTAATCAATATTTCACCGGAAGTCTAGGAAATGTTGAATATTTGGTATGCCGAGATGGTCAGG &RQVHQVXV TGGCAAGTCGCAGGGTACAGTGATCAAGTAAAGGAG CTAACCAATATTTCACCGGAAG TTAGGAAATGTTGAATATTTGGTGTGTCGAGATGGTGAAG

601 610 620 630 640 650 660 670 680 690 700 $S&02 GAAAAGTTCATGCATTTCACAATGTTTGCACCCATCGTGCATCTATTCTTGCTTGTGGAAGTGGCAAAAAGTCGTGTTTCGTATGCCCTTATCATGGATG $K&02 GAAAAGTTCATGCATTTCACAATGTTTGCACCCATCGTGCATCTATCCTTGCTTGTGGAAGTGGCAAAAAGTCATGTTTCGTATGCCCTTATCATGGATG 6S&02 GGAAAGTTCATGCATTTCACAATGTTTGCACCCATCGTGCATCTATTCTTGCTTGCGGTAGTGGCAAAAAGTCGTGTTTCGTGTGCCCTTACCATGGATG EHHW&02 GCGAACTTCATGCATTTCACAATGTTTGTACACATCGTGCATCAATTCTTGCTTGTGGAAGTGGCAAAAAGTCATGTTTCGTATGCCCTTACCATGGATG $PD&02 GGAAAGTTCATGCATTCCACAATGTTTGTACTCATCGTGCATCAATTCTTGCATGTGGAACTGGCAAGAAGTCTTGTTTTGTCTGCCCTTATCATGGATG &RQVHQVXV G AAAGTTCATGCATTTCACAATGTTTGCACCCATCGTGCATCTATTCTTGCTTGTGGAAGTGGCAAAAAGTC TGTTTCGTATGCCCTTATCATGGATG CMO3 701 710 720 730 740 750 760 770 780 790 800 $S&02 GGTATATGGCATGAATGGAACGCTTACGAAAGCTTCGAAAGCAACAGCAGAACAATCACTTAATCCCGATGAACTTGGGCTTGTACCACTAAAGGTTGCA $K&02 GGTATATGGCATGAATGGATCGCTTACGAAAGCTTCAAAAGCAACACCAGAACAATCACTAAATCCCGATGAACTTGGGCTTGTACCACTAAAAGTTGCA 6S&02 GGTATATGGCATGGACGGATCACTTGCGAAAGCCTCCAAAGCAAAACCTGAACAAAACTTGGATCCTAAAGAACTTGGGCTTGTACCCCTAAAAGTTGCA EHHW&02 GGTGTATGGCTTAGATGGATCACTCGCCAAAGCCAGCAAAGCAACTGAAACACAAAATTTGGATCCTAAAGAACTTGGGCTTGCACCCCTAAAAGTTGCA $PD&02 GGTATTTGGCTTAGATGGATCACTCATGAAAGCCACTAA---AACTGAAAATCAAGTATTTGACCCTAAAGAACTTGGGCTAGTAACTCTAAAGGTAGCA &RQVHQVXV GGTATATGGCATGGATGGATCACTTACGAAAGCCTC AAAGCAACAGCAGAACAA ATT GATCCTAAAGAACTTGGGCTTGTACC CTAAAAGTTGCA 801 810 820 830 840 850 860 870 880 890 900 $S&02 GAATGGGGCCCATTTATACTCATCAGTTTAGACAGATCAAGCCGTGAAGTAGGTGA---CGTTGGATCTGAATGGCTTGGTAGTTGTGCTGAAGATGTTA $K&02 GTATGGGGCCCATTTATACTCATCAGTTTGGACAGATCAAGCCGTGAAGTAGGTGA---CGTTGGATCTGAATGGCTTGGTAGTTGTGCTGAAGATGTTA 6S&02 GTATGGGGGCCGTTCGTTCTTATCAGCTTGGACAGATCACTTGAAGAAGGTGGTGA---TGTTGGAACTGAGTGGCTTGGTACTTCTGCTGAAGATGTTA EHHW&02 GAATGGGGCCCATTCATTCTTATCAGCTTGGACCGATCTCTAGATGCTAATGCTGA---TGTTGGAACAGAGTGGATTGGTAAATCTGCAGAAGATGTTA $PD&02 ATATGGGGGCCATTTGTTCTGATAAGCTTAGATAGATCAGGCTCTGAAGGAACTGAAGATGTTGGAAAAGAGTGGATTGGTTCATGTGCTGAAGAAGTTA &RQVHQVXV GTATGGGGCCCATTTATTCT ATCAGCTTGGACAGATCA GC TGAAG AGGTGA TGTTGGAACTGAGTGGCTTGGTA TTGTGCTGAAGATGTTA 901 910 920 930 940 950 960 970 980 990 1000 $S&02 AGGCCCATGCTTTTGACCCGAATCTGCAGTTTATTAATAGGAGTGAATTTCCAATTGAATCTAATTGGAAGATTTTCAGTGACAACTATTTGGATAGCTC $K&02 AGGCCCATGCTTTTGACCCGAATCTTCAGTTCATTAATAGGAGTGAATTTCCAATTGAATCTAATTGGAAGATTTTCAGTGACAACTATTTGGATAGCTC 6S&02 AGGCCCATGCTTTTGATCCTTCACTTCAATTCATTCACAGAAGTGAATTCCCAATGGAATCTAATTGGAAGATTTTCAGTGACAACTACTTGGATAGCTC EHHW&02 AGGCCCATGCTTTTGATCCTAATCTAAAGTTCACCCATAGAAGTGAATTCCCAATGGAATGCAACTGGAAGGTTTTCTGTGATAACTATCTGGATAGCTC $PD&02 AAAAACATGCTTTTGATCCTTCTCTTCAATTCATTAATAGGAGTGAATTCCCCATGGAATCCAATTGGAAGGTATTTTGCGACAATTATTTGGACAGTGC &RQVHQVXV AGGCCCATGCTTTTGATCCTAATCTTCAGTTCATTAATAGGAGTGAATTCCCAATGGAATCTAATTGGAAGATTTTCAGTGACAACTATTTGGATAGCTC 56

Fig. 2-3. continued 1001 1010 1020 1030 1040 1050 1060 1070 1080 1090 1100 $S&02 GTACCATGTTCCTTATGCACACAAATACTATGCAACTGAACTCGACTTTGACACTTACCAAACCGATATGGTTG-GAAATGTCACGATTCAAAGGGTGGC $K&02 TTACCATGTTCCTTATGCACACAAATACTATGCAACTGAGCTCGACTTTGATACTTACCAAACCGATATGGTTG-GAAATGTCACGATTCAAAGGGTGGC 6S&02 ATATCATGTTCCTTATGCACACAAATACTATGCAACTGAACTCAACTTTGACACTTACGATACCCAAATGATCG-AAAACGTTACAATTCAAAGAGTGGA EHHW&02 TTACCATGTTCCTTATGCTCACAAATACTATGCAGCTGAACTCGACTTTGACACTTACAACACTGAAATGATCGAGAAATGTGT-GATTCAAAGAGTTGG $PD&02 ATATCATGTTCCTTATGCTCACAAATACTATGCTGCTGAACTCGACTTTGACACCTATAAAACTGATTTGTTGGAGAAAGTTGT-GATTCAAAGAGTAGC &RQVHQVXV TACCATGTTCCTTATGCACACAAATACTATGCAACTGAACTCGACTTTGACACTTAC AAACCGATATG T G GAAATGT ACGATTCAAAGAGTGGC 1101 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200 $S&02 TGGGACTTCAAACAA------TGGTTTTAGTAGAATTGGAACTCAAGCCTTCTATGCTTTTGCATACCCAAACTTTGCTGTGGAAAGGTATGGCCCTTGG $K&02 TGGGACTTCAAACAA------TGGTTTTAATAGACTTGGAACTCAAGCCTTCTATGCTTTTGCATACCCTAACTTTGCTGTGGAAAGGTATGGCCCTTGG 6S&02 AGGAAGTTCAAACAAGCCTGATGGTTTTGATAGAGTTGGAATTCAAGCATTCTATGCTTTCGCGTATCCAAATTTCGCTGTGGAAAGGTATGGCCCTTGG EHHW&02 TAGCAGTTCAAACAAGCCAGATGGATTTGATAGACTTGGAACTGAAGCATTCTATGCTTTTATTTACCCCAACTTTGCTGTGGAAAGGTATGGCACTTGG $PD&02 AAGCAGTTCAAACAAGCCAAATGGGTTTGATAGACTTGGATCAGAAGCATTCTATGCTTTTATTTATCCAAACTTTGCTGTGGAAAGGTATGGCCCTTGG &RQVHQVXV TGG AGTTCAAACAAGCC ATGGTTTTGATAGACTTGGAACTCAAGCATTCTATGCTTTTGC TACCCAAACTTTGCTGTGGAAAGGTATGGCCCTTGG

1201 1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 $S&02 ATGACTACTATGCATATTGTTCCATTAGGACCAAGGAAATGCAAACTAGTGGTGGACTACTATATTGAAAAATCAAAGCTGGACGACAAGGATTACATCG $K&02 ATGACTACAATGCATATTGTTCCATTAGGACCAAGGAAATGCAAACTAGTGGTGGACTACTATATTGAAAAATCAAAGCTGGACGACAAGGATTACATCG 6S&02 ATGACTACAATGCATATTCACCCATTAGGACCAAGGAAATGCAAACTAGTGGTGGACTATTATATTGAAAATTCTATGTTGGATGACAAGGATTACATCG EHHW&02 ATGACTACAATGCATGTCGTTCCTATGGGACAAAGGAAATGCAAACTAGTGGTGGACTATTATCTTGAGAAAGCCATGTTGGACGACAAGGCTTACATTG $PD&02 ATGACCACAATGCACATTGGTCCATTAGGACCCAGGAAGTGTAAACTTGTGGTGGACTATTATCTTGAAAATGCCATGATGAACGACAAACCTTACATTG &RQVHQVXV ATGACTACAATGCATATTGTTCCATTAGGACCAAGGAAATGCAAACTAGTGGTGGACTATTATATTGAAAAATC ATG TGGACGACAAGGATTACATCG

1301 1310 1320 1330 1340 1350 1360 1370 1380 1390 1400 $S&02 AAAAGGGCATAGCAATCAATGATAATGTGCAGAAAGAAGATGTGGTGTTGTGTGAAAGCGTCCAAAAAGGGTTGGAGACACCAGCATATCGTAGTGGAAG $K&02 AAAAGGGCATAGCAATCAATGATAATGTGCAGAAAGAAGATGTGGTGTTGTGTGAAAGCGTCCAAAAAGGGTTGGAGACACCAGCATATCGTAGTGGAAG 6S&02 AGAAGGGCATAGCAATCAATGATAACGTACAGAGGGAAGATGTGGTGCTGTGTGAAAGTGTACAAAGAGGTTTGGAGACACCAGCATATCGTAGTGGGAG EHHW&02 ACAAGGGCATAGCAATCAACGATAACGTGCAGAAGGAAGATAAGGTGTTGTGTGAAAGTGTCCAAAGGGGACTGGAGACACCAGCATACCGCAGTGGCAG $PD&02 AAAAAAGCATAATGATCAACGACAACGTCCAGAAAGAAGATGTAGTGTTATGTGAAAGTGTGCAGAGGGGTCTAGAAACACCAGCATATAGAAGTGGAAG &RQVHQVXV AAAAGGGCATAGCAATCAATGATAACGTGCAGAAAGAAGATGTGGTGTTGTGTGAAAGTGTCCAAAGAGG TTGGAGACACCAGCATATCGTAGTGGAAG

1401 1410 1420 1430 1440 1450 1460 1470 1480 1490 1500 $S&02 ATATGTGATGCCAATTGAGAAAGGAATTCACCATTTCCACTGCTGGTTACACCAAGTCTTGAAGTGATTGCACC-CAGATTATCTATAATCTTGGTTTCT $K&02 ATATGTGATGCCAATTGAGAAAGGAATTCACCATTTCCACTGCTGGTTACACCAAGTGTTGAAGTGATTGCACC-CAGATTATATGTAATCTTGGTTTCT 6S&02 ATATGTGATGCCTATTGAGAAAGGAATCCACCATTTCCACTGCTGGTTGCAACAAACTTTGAAGTAATTGTTG---ACTTGAC------EHHW&02 ATATGTGATGCCAATTGAGAAAGGAATCCACCACTTCCACTGTTGGTTGCATGAAACTTTGCAGTGATTTTCGG-GAGCTTAT------T $PD&02 ATATGTGATGCCAATTGAGAAAGGAATCCATCATTTCCATTGCTGGTTGCACCAAACTTTGAACTGATCCTTCTTCATCTTCC------T---- &RQVHQVXV ATATGTGATGCCAATTGAGAAAGGAATCCACCATTTCCACTGCTGGTTGCACCAAACTTTGAAGTGATTGT C CAG TTAT T T CMO4 57

Fig. 2-3. continued

1501 1510 1520 1530 1540 1550 1560 1570 1580 1590 1600 $S&02 TTCCTTCTATGATTGGATATTATAATAATAAGTAAAATTATAATGTCATCATTTAGTTGAGATTGTTATTAGAGTTGAGCGTATGCTCCTTAGTAACGTG $K&02 TTCCTTCTATGATTGGATATTATAATAA---GTAAAATTATAATGTCATCATTTAGTTGAGATTGTTGTTAGAGTTGAGCTTATGCTCATTAGTAACGTG 6S&02 ----TTCACCGGACG----TTTGCGTG----CTCCTCTTGGC------TCTTGCAGTTA-----GTTA------TGTGTGTATGCTCATGGCC------EHHW&02 -----TCTATGGTT-----TTACCAT-----GTCACATT--A-----ATAATATAATTGA---TGTT----GGGTTGAGCCTATGCTCCTCA------$PD&02 --CTTCCTCTAGTTGG---GTCCAATATTAATTAAGCTTATGC-----TTATGAGTTTCA---TGTTA------AAAATATGT--ATGAA------G &RQVHQVXV C TTCTATGGTTGG TTA AATA GTAA ATTATA ATCAT TAGTTGA TGTTA G GTTGAGC TATGCTCAT A G

1601 1610 1620 1630 1640 1650 1660 1670 1680 1690 1700 $S&02 TGTATGTGTGT-AAGTGTTTGGTTATGGGCGAAAATGTTTGTTATTGCTAGAATTTTATAATAATAGTATGGTGCTGATGTCCAATATAAATAAAGACCA $K&02 TGTATGTGTGT-ATGTGTTTGGTCATGGTAAAAAATGTTTGTTATTGCTAGAATTTTATAATAATAGTATGGTGCTGATGTCCAGTATAAATAAAGAACA 6S&02 TAAACTTATAA-CATTTTATACTCAATTT--ATAATAAACACCATAGTAAGTACCTCAGA-TA----TCCCGTGC--ATTTTCATTTTCAGGGGAAATAA EHHW&02 TGCAATTAAGT-TATTTTGTGGTCATGGG--AAAACCCTTCC-ATTTCTAGTATAGTAG--TAGT-GTCTGGTGCTAATGTCCCATATAAATAAAAGCCA $PD&02 GGCAAGTATGTCTACTTGAAGATTATGGT----A-TGGTGCTAAATGCTAAT---GCATGTTA------TTGTAAT------ATTTTTAATAAACACCA &RQVHQVXV TG A GTATGT A TTT TGGTCATGGT AAAATG TT T ATTGCTAGTAT TTATA TA T GT TGGTGCT ATGTCCA TATAAATAAA ACCA

1701 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800 $S&02 TAGCACCCCCTTTCCCTACTTAAGAAATAATATCCCATACATATTTTCAGGGGAACTATGAGATTGTCTATGAACATTAATTTTCGGACAAAAAAAAAAA $K&02 TAGCACCCC-TTTCCCTACTTAAGAAATTATATTCCATA--TATTTTCAGGGGAACTATGAGATTGTCTATGAACA------AAAAAAAAAAA 6S&02 TGACA------TTGTCT--ATCAATATCAATAT-C--AA--TATCAATATCAATATTACCAG--TAATTTTAAACA------AAAAAAAAAAAAA EHHW&02 TAGCACCTAGTTTCCC--CTTCA-AAGTTATATCCTAAA--TATTTATGGGGAACATATGAGATTGAGTATGAACATTTTATCTAGGCATATGTGTGATT $PD&02 TCA-ACCTTCCTTGGCCCTTGCATTAAACTTAG----AACTTCTAATAATTAATCTTATTAA--TGTTTATGATTTTCA--T--CCAAAAAAAAAAAAAA &RQVHQVXV TAGCACC TTTCCCT CTTCA AAAT ATAT C AAA TATTTT AGGGAAA TATGAGATTGT TATGAACAT T AAAAAAAAAAAAA

1801 1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 $S&02 AAAAAAAAAAAAAAAAA------$K&02 AA------6S&02 AAAAA------EHHW&02 TTTAATTTCTTTGAACAATGAGGGTAAGATTTTTGTGGATGTTCGTCAGATTTTATTTTACTATTTATAGTAGAAATTGCTCCAATTATAAAAAAAAAAA $PD&02 AAAAA------&RQVHQVXV AAAAA

58 59 positions). CMO3, contains the one of the two Rieske-type Fe-S cluster binding sites that are conserved among the sequences of known CMO. CMO4 is located in the consensus region near the end of 3’ coding sequence. These two primers amplified a fragment of 745 bp, ApCMO-0.7 (Fig. 2-2), and this fragment was homologous to both spinach and sugar beet CMOs based upon a BLAST search and subsequent sequence alignment. CMO5 is located 220 bp upstream of CMO3, and along with

CMO4 they amplified a 967 bp fragment, ApCMO-1.0 (Fig. 2-2). This fragment again was homologous to known CMOs according to a BLAST search. After

ApCMO-1.0 was sequenced, the sequence of Atriplex CMO around CMO3 was elucidated (Table 2-2). Two additional primers, CMO6 and CMO7 which are rich in

GC content, were then designed based on the sequence of ApCMO-1.0 to perform 3’- and 5’-RACE respectively (Table 2-1). Fig. 2-2 presents the position of each primer and the resulting PCR fragments in the cloned Atriplex CMO.

While CMO7 failed to amplify the 5’ end of Atriplex CMO, CMO6 amplified a poly-A-tail-containing 726 bp fragment that contains a 323 bp perfect match with the

3’ end of ApCMO-1.0. After subcloning, several clones were recovered and sequenced. ApCMO-3’-2 and -17 are very similar in length and sequence, whereas -

5 and -38 are either longer or shorter respectively due to alternative polyadenylation sites (Fig. 2-4). In addition, ApCMO-3’-2 seems to be the most complete among these four selected clones in terms of the sequence of adaptor added during RACE.

Therefore, ApCMO-3’ -2 was selected to be the 3’ end of Atriplex CMO. The

Table 2-2. A comparison of the sequences of the actual clone and the primers.

The different nucleotides between the actual clone and primer appear in

bold.

Primers Sequence Comparison Similarity (%)

TGTTTCGTGTGCCCTTACCAT (primer) 90.5 CMO3 TGTTTCGTATGCCCTTATCAT

TGGTGGATTCCTTTCTCAATAGG (primer) 91.3 CMO4 TGGTGAATTCCTTTCTCAATTGG

CCTGCCTTCTATTCCCATGAAC (primer) 90.9 CMO5 CCTGCCTTCTATGCTCATGAAC

Fig. 2-4. An alignment of four clones that are possible 3’ ends of ApCMO.

CMO-3’-2, -5, -17 and -38 are four of the clones obtained from PCR

cloning of 3’-RACE products. The nucleotides conserved among all

four clones are shaded in yellow, and those conserved among three of

the four clones are shaded in blue. Three possible polyadenylation

signals are underlined at the concensus region. The G/T-rich region

that functions as “far-upstream element” (Hunt, 1994) needed for 3’

end formation is boxed.

1 10 20 30 40 50 60 PR TCACGATTCAAAGGGTGGCTGGGACTTCAAACAATGGTTTTAGTAGAATTGGAACTCAAG PR TCACGATTCAAAGGGTGGCTGGGACTTCAAACAATGGTTTTAGTAGAATTGGAACTCAAG &02 TCACGATTCAAAGGGTGGCTGGGACTTCAAACAATGGTTTTAGTAGAATTGGAACTCAAG PR TCACGATTCAAAGGGTGGCTGGGACTTCAAACAATGGTTTTAGTAGAATTGGAACTCAAG &RQVHQVXV TCACGATTCAAAGGGTGGCTGGGACTTCAAACAATGGTTTTAGTAGAATTGGAACTCAAG 6170 80 90 100 110 120 PR CCTTCTATGCTTTTGCATACCCAAACTTTGCTGTGGAAAGGTATGGCCCTTGGATGACTA PR CCTTCTATGCTTTTGCATACCCAAACTTTGCTGTGGAAAGGTATGGCCCTTGGATGACTA &02 CCTTCTATGCTTTTGCATACCCAAACTTTGCTGTGGAAAGGTATGGCCCTTGGATGACTA PR CCTTCTATGCTTTTGCATACCCAAACTTTGCTGTGGAAAGGTATGGCCCTTGGATGACTA &RQVHQVXV CCTTCTATGCTTTTGCATACCCAAACTTTGCTGTGGAAAGGTATGGCCCTTGGATGACTA 121130 140 150 160 170 180 PR CTATGCATATTGTTCCATTAGGACCAAGGAAATGCAAACTAGTGGTGGACTACTATATTG PR CTATGCATATTGTTCCATTAGGTCCAAGGAAATGCAAACTAGTGGTGGACTACTATATTG &02 CTATGCATATTGTTCCATTAGGACCAAGGAAATGCAAACTAGTGGTGGACTACTATATTG PR CTATGCATATTGTTCCATTAGGACCAAGGAAATGCAAACTAGTGGTGGACTACTATATTG &RQVHQVXV CTATGCATATTGTTCCATTAGGACCAAGGAAATGCAAACTAGTGGTGGACTACTATATTG 481490 500 510 520 530 540 PR ATTA-GAGTTGAGCGTATGCTCCTTAGTAAC-GTGTGTATGTGTGTAAGTGTTTGGTTAT PR ATTAAGAGTTGAGCGTATGCTCCTTAGTAACCGTGTGTATGTGTGTAAGTGTTTGGTTAT &02 ATTA-GAGTTGAGCGTATGCTCCTTAGTAAC-GTGTGTATGTGTGTAAGTGTTTGGTTAT PR ATTA-GAGTTGAGCGTATGCTCCTTAGTAAC-GTGTGTATGTGTGTAAGTGTTTGGTTAT &RQVHQVXV ATTA GAGTTGAGCGTATGCTCCTTAGTAAC GTGTGTATGTGTGTAAGTGTTTGGTTAT

541550 560 570 580 590 600 PR GGGCGAAAATGTTTGTTATTGCTAGAATTTTATAATAATAGTATGGTGCTGATGTCCAAT PR GGGCGAAAATGTTTGTTATTGCTAGAATTTTATAATAATAGTATGGTGCTGATGTCCA-T &02 GGGCGAAAATGTTTGTTATTGCTAGAATTTTATAATAATAGTATGGTGCTGATGTCCAAA PR GGGCGAAAATGTTTGTTATTGCTAGAATTTTATAATAATAGTATGGTGCTGATGTCCAAT &RQVHQVXV GGGCGAAAATGTTTGTTATTGCTAGAATTTTATAATAATAGTATGGTGCTGATGTCCAAT

601610 620 630 640 650 660 PR ATAAATAAAGACCATAGCACCCCCTTTCCCTACTTAAGAAATTATATCCCATACATATTT PR TTAAATAAAGACCATAGCACCCCCTTTCCCTACTTAAGAAATTATATCCCATACATATTT &02 ATAAATAAAGACCATAGCACCCCCTTTCCCTACTTAAGAAATTATATCCCATACATATTT PR ATAAAAAAAAACCATAGCCCCCCCTTTCCCTACT------&RQVHQVXV ATAAATAAAGACCATAGCACCCCCTTTCCCTACTTAAGAAATTATATCCCATACATATTT

661670 680 690 700 710 720 PR TCAGGGGAACTATGAGATTGTCTATGAACATTAATTTTC------PR TCAGGGGAACTATGAGATTGTCTATGAACATTAATTTTC------&02 TCAGGGGAACTATGAGATTGTCTATGAACATTAATTTTCGATACATGTATGATTTATTCA PR ------C----A------&RQVHQVXV TCAGGGGAACTATGAGATTGTCTATGAACATTAATTTTC

721730 740 750 760 770 780 PR ------GGAAAAAAAAAAAAAAAAAAAAAA-AAAAAAA------PR ------GGAAAAAAAAAAAAAAAAAAAAAA-AAAAAAAAA------&02 TCCCTTATGTAGGATGAGGAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAAAAAA PR ------AAAAAAAAAAAAAAAAAAAAAA-AAAAAAAAA------&RQVHQVXV GGAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAA

63 sequence of Atriplex CMO around CMO4 was completely resolved, and there are two mispaired bases (Table 2-2).

After 5’-RACE failed to amplify the 5’ end of the CMO mRNA, CMO5S was designed based on the sequence of the spinach CMO cDNA corresponding to the region around the start codon. In order to successfully amplify the 5’ end, CMO5AS was also synthesized to pair with CMO5S. These two primers generated a 769 bp fragment (ApCMO-5’, Fig. 2-2) and, according to the later sequence result, it overlapped with the ApCMO-1.0 (by 460 bp) as well as ApCMO-0.7 (by 241 bp), and it was homologous to the 5’ end of spinach CMO cDNA (Rathinasabapathi et al.,

1997). Therefore, the sequence of ApCMO around CMO5 was elucidated (Table 2-2) and the cloning of Atriplex CMO was completed.

Based on these overlapping clones, a composite cDNA encoding Atriplex prostrata CMO (ApCMO) was assembled. ApCMO is 1669 bp in length and encodes a 438 amino acid polypeptide (Fig. 2-5). This cDNA has a 3’-untranslated region of

348 bp. With this PCR cloning method, the 5’-untranslated region remains unclear.

According to the prediction of ChloroP (Version 1.1), it contains an N-terminal chloroplast transit peptide (score: 0.567), and the predicted cleavage site is Ala68

(CS-score: 2.571). However, Ala59 with a CS score of 4.736 should also be considered as a possible cleavage site for the transit peptide for the following reapons. First, the CS score of 4.736 is even higher than that of Ala68. Secondly, the experimentally determined N-terminal residue of the purified spinach CMO protein

Fig. 2-5. The nucleotide and deduced amino acid sequence of ApCMO. The

binding site of Rieske type Fe-S cluster is underlined with the key Cys

and His marked with asterisks. The dotted underline represents the non-

heme Fe binding motif, in which the conserved residues in this reported

motif (Jiang et al., 1996) are marked with daggers. The transit peptide

is underlined twice. The start and stop codons are boxed. Three possible

polyadenylation signals appear in bold. The accession number of

ApCMO is: AY082068.

1 AATCATGGCAGCAAGTGCAACAACAATGTTGCTAAAATACCCAACTACTGTTTGCGGA 1 M A A S A T T M L L K Y P T T V C G 59 ATACCAAATTCATCCTCAAACAATTCTACTGATCCTTCAAATAACATCGTCCAAATTCCA 19 I P N S S S N N S T D P S N N I V Q I P 119 CAAACTAATACTACAAAAAGCCCGTTACTTAAGTGCCGTACTCCTAATAAACCCGTTAAC 39 Q T N T T K S P L L K C R T P N K P V N 179 GCCGTCGCTGCCCCGGCTTTTCCGTCCGCAACCACCATCACAACCACCACTCCGCCGTCC 59 A V A A P A F P S A T T I T T T T P P S 239 ATCCAATCACTTGTCAAGGATTTCGATCCTTCTATTCCGGCCGAGGATGCTTTTACTCCT 79 I Q S L V K D F D P S I P A E D A F T P 299 CCTAGCTCTTGGTATACCGAACCTGCCTTCTATGCTCATGAACTTGACCGTATCTTTTAC 99 P S S W Y T E P A F Y A H E L D R I F Y 359 AAAGGATGGCAAGTCGCAGGGTACAGTGATCAAGTTAAGGAGGCTAACCAATATTTCACC 119 K G W Q V A G Y S D Q V K E A N Q Y F T 419 GGAACGTTAGGAAATGTTGAATATTTGGTGAGTCGAGATGGTGAAGGAAAAGTTCATGCA 139 G T L G N V E Y L V S R D G E G K V H A 479 TTTCACAATGTTTGCACCCATCGTGCATCTATTCTTGCTTGTGGAAGTGGCAAAAAGTCG 159 F H N V C* T H* R A S I L A C G S G K K S 539 TGTTTCGTATGCCCTTATCATGGATGGGTATATGGCATGAATGGAACGCTTACGAAAGCT 179 C F V C* P Y H* G W V Y G M N G T L T K A 599 TCGAAAGCAACAGCAGAACAATCACTTAATCCCGATGAACTTGGGCTTGTACCACTAAAG 199 S K A T A E Q S L N P D E L G L V P L K 659 GTTGCAGAATGGGGCCCATTTATACTCATCAGTTTAGACAGATCAAGCCGTGAAGTAGGT 219 V A E W G P F I L I S L D R S S R E V G 719 GACGTTGGATCTGAATGGCTTGGTAGTTGTGCTGAAGATGTTAAGGCCCATGCTTTTGAC 239 D V G S E W L G S C A E D V K A H A F D 779 CCGAATCTGCAGTTTATTAATAGGAGTGAATTTCCAATTGAATCTAATTGGAAGATTTTC 259 P N L Q F I N R S E F P I E S N W K I F 839 GTGACAACTATTTGGATAGCTCGTACCATGTTCCTTATGCACACAAATACTATGCAACT 279 S D† N Y L D† S S Y H† V P Y A H† K Y Y A T 899 GAACTCGACTTTGACACTTACCAAACCGATATGGTTGGAAATGTCACGATTCAAAGGGTG 299 E L D F D T Y Q T D M V G N V T I Q R V 959 GCTGGGACTTCAAACAATGGTTTTAGTAGAATTGGAACTCAAGCCTTCTATGCTTTTGCA 319 A G T S N N G F S R I G T Q A F Y A F A 1019 TACCCAAACTTTGCTGTGGAAAGGTATGGCCCTTGGATGACTACTATGCATATTGTTCCA 339 Y P N F A V E R Y G P W M T T M H I V P 1079 TTAGGACCAAGGAAATGCAAACTAGTGGTGGACTACTATATTGAAAAATCAAAGCTGGAC 359 L G P R K C K L V V D Y Y I E K S K L D 1139 GACAAGGATTACATCGAAAAGGGCATAGCAATCAATGATAATGTGCAGAAAGAAGATGTG 379 D K D Y I E K G I A I N D N V Q K E D V 1199 GTGTTGTGTGAAAGCGTCCAAAAAGGGTTGGAGACACCAGCATATCGTAGTGGAAGATAT 399 V L C E S V Q K G L E T P A Y R S G R Y 1259 GTGATGCCAATTGAGAAAGGAATTCACCATTTCCACTGCTGGTTACACCAAGTCTTGAAG 419 V M P I E K G I H H F H C W L H Q V L K 1319 TGATTGCACCCAGATTATCTATAATCTTGGTTTCTTTCCTTCTATGATTGGATATTATAA 1379 TAATAAGTAAAATTATAATGTCATCATTTAGTTGAGATTGTTATTAGAGTTGAGCGTATG 1439 CTCCTTAGTAACGTGTGTATGTGTGTAAGTGTTTGGTTATGGGCGAAAATGTTTGTTATT 1499 GCTAGAATTTTATAATAATAGTATGGTGCTGATGTCCAATATAAATAAAGACCATAGCAC 1559 CCCCTTTCCCTACTTAAGAAATTATATCCCATACATATTTTCAGGGGAACTATGAGATTG 1619 TCTATGAACATTAATTTTCGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

(Rathinasabapathi et al., 1997) corresponds to Ala59 not Ala68 based upon the comparison of N-terminal regions of Atriplex prostrata, A. hortensis, spinach and sugar beet CMOs (Fig. 2-6 A). These together suggest Ala59 of ApCMO is the cleavage site for the transit peptide. In order to further confirm the localization of this cloned CMO according to its sequence, the deduced amino acid sequence was submitted to the web site of PSORT (Prediction of Protein Localization Sites).

PSORT predicted that this CMO is most likely localized in chloroplast stroma

(certainty = 0.625), with a possible localization to the peroxisome (certainty = 0.535) or chloroplast thylakoid membrane (certainty = 0.438). The reasons that PSORT predicted this ApCMO as well as the other known CMO clones to be localized in the stroma of chloroplast are: 1) an apolar signal for intrachloroplastic sorting was found immediately after the transit peptide (aa 59-65); and 2) it scores very high for the consensus sequence for intrachloroplastic sorting within the transit peptide (aa 1-58)

(Fig. 2-6 A). However, since it also contains an SKL motif at amino acid 375-377, the possibility that ApCMO is a peroxisomal protein can not be excluded, despite the fact that the SKL motif in peroxisomal proteins is usually localized at the C-terminus.

According to Motif Finder, ApCMO contains both subunits of the Fe-S cluster binding sites that are the characteristic of Rieske iron-sulfur proteins. In fact, the registered Rieske-type [2Fe-2S] cluster motif in PROSITE is Cys-X-His-X15-17-Cys-

X-X-His (X represents any amino acid) and ApCMO contains this registered motif

(underlined region in Fig. 2-5), which is common to all reported CMOs (Fig. 2-6 B).

Fig. 2-6. Alignments of CMOs from Atriplex prostrata, A. hortensis, spinach

(Spinacia oleracea), sugarbeet (Beta vulgaris), and amaranth

(Amaranthus tricolor). A. N-terminal region. The N-terminal residue

of the processed spinach CMO polypeptide is bolded

(Rathinasabapathi et al., 1997); B. The region contains the two

Rieske-type Fe-S clusters; C. the region contains the mononuclear

non-heme Fe binding site. Numbering above the alignments

corresponds to that of ApCMO appearing in Fig. 2-5. Numbering in

parentheses represent the numbering of the first amino acid in that

line for each individual species.

1 38 A. prostrata (1) -MA--ASATTMLLKYPTTVCGIPNSSSNNSTDPSNNIVQIP A. hortensis (1) -MA--ASATTMLLKYPTTVCGIPNSSANNSTDPSNNIVQIP spinach (1) MMAASASATTMLLKYPTTVCGIPNPSSNNNNDPSNNIVSIP sugarbeet (1) -MA--ASATTMLLKYPTLCAMPNSSSSSNNNDLPTSIPLNN amaranth (1) -MASSAS---MLINYPTTFCGVRN-SSNPNNDQFSDQINIP consensus MA ASATTMLLKYPTTVCGIPNSSSNNNNDPSNNIVQIP

39 69 A. prostrata (39) QTNTTKSPLLKCRTP------NKP-VNAVAAPAFPSAT A. hortensis (39) QTTTTNSPLLKFRTP------NKP-VNAVAAPAFPSVT spinach (42) QNTTN--PTLKSRTP------NKITTNAVAAPSFP--- sugarbeet (39) NNNLLSNKNKILQTPNINTSTNKIITKAVASPVFP-TL amaranth (37) SSLNNNINISKITSKT-----NKIIPKAVASPVIPSSI consensus QT T PLLK RTP NKI NAVAAPAFPS

B 157 187 A. prostrata (157) HAFHNVCTHRASILACGSGKKSCFVCPYHGW A. hortensis (157) HAFHNVCTHRASILACGSGKKSCFVCPYHGW spinach (156) HAFHNVCTHRASILACGSGKKSCFVCPYHGW sugarbeet (163) HAFHNVCTHRASILACGSGKKSCFVCPYHGW amaranth (159) HAFHNVCTHRASILACGTGKKSCFVCPYHGW consensus HAFHNVCTHRASILACGSGKKSCFVCPYHGW

C 280 297 A. prostrata (280) DNYLDSSYHVPYAHKYYA A. hortensis (280) DNYLDSSYHVPYAHKYYA spinach (279) DNYLDSSYHVPYAHKYYA sugarbeet (286) DNYLDSSYHVPYAHKYYA amaranth (282) DNYLDSAYHVPYAHKYYA consensus DNYLDSSYHVPYAHKYYA

In addition, ApCMO also contains another concensus sequence, a mononuclear nonheme Fe binding motif: Glu/Asp-X3-4-Asp-X2-His-X4-5-His (dotted underlined region in Fig. 2-5). This motif is also conserved among spinach, sugar beet and A. prostrata (Fig. 2-6 C). Within the two binding motif mentioned above, the amino acid sequences are identical among Atriplex, spinach and sugar beet CMOs.

When the nucleotide sequence of ApCMO was aligned to that of spinach and sugar beet CMOs, it shared 73.4% and 69.7% identity, respectively (Fig. 2-3). At the amino acid level, the identity between A. prostrata and spinach is 82.9% and 72.8% between A. prostrata and sugar beet (Fig. 2-7). The identity increased to 95.8% at nucleotide level and 96.3% at amino acid level when CMO of A. hortensis was compared with the one of A. prostrata. A summary of the identities among available

CMOs is shown in Table 2-3.

Expression of CMO in Atriplex prostrata

In order to understand how salinity affects CMO expression, A. prostrata was treated with 1% NaCl (= 170 mM) and 2% NaCl (= 340 mM) in addition to the control media for 3 days. Total RNA was then isolated from young stems (i.e., the first internode from the top of the plant) and young leaves (i.e., fully expanded leaves from the top of the plant) to perform northern blotting. When the membrane was probed with ApCMO-0.7, a 1.9 kb transcript was detected (Fig. 2-8 A). In young stems, this transcript increased two fold when plants were treated with 170 mM

Fig. 2-7. Comparison of the deduced amino acid sequences of five reported CMOs. A. Multiple sequence alignment.

The amino acid sequences of AhCMO (Atriplex hortensis, accession number: AAF76895), SpCMO (Spinacia

oleracea, accession number: AAB52509), AmaCMO (Amaranthus tricolor, accession number: AAK82768),

and beetCMO (Beta vulgaris, accession number: AAB80954) were aligned with that of ApCMO by AlignX®

of Vector NTI. The amino acids conserved among all five clones are shaded in yellow, and those conserved

among three or more of the five clones are shaded in blue, whereas the similar amino acids are shaded green.

The consensus region of Rieske type Fe-S cluster is underlined, and the transit peptide is underlined twice.

The dotted underline represents the non-heme Fe binding motif. B. Phylogenetic tree of the five CMOs.

A 1 10 20 30 40 50 60 70 80 90 100 $S&02 ---MAASATTMLLKYPTTVCGIPNSSSNNSTDPSNNIVQIPQTNTTKSPLLKCRTP------NKP-VNAVAAPAFPSAT--TITTTTPPSIQSLVKDFDP $K&02 ---MAASATTMLLKYPTTVCGIPNSSANNSTDPSNNIVQIPQTTTTNSPLLKFRTP------NKP-VNAVAAPAFPSVT--TTTTTTPSSIQSLVKDFDP 6S&02 MMAASASATTMLLKYPTTVCGIPNPSSNNNNDPSNNIVSIPQNTTN--PTLKSRTP------NKITTNAVAAPSFP-----SLTTTTPSSIQSLVHEFDP EHHW&02 ---MAASATTMLLKYPTLCAMPNSSSSSNNNDLPTSIPLNNNNNLLSNKNKILQTPNINTSTNKIITKAVASPVFP-TL--KTTSNTPSSIRSLVHEFDP $PD&02 ----MASSASMLINYPTTFCGVRN-SSNPNNDQFSDQINIPSSLNNNINISKITSKT-----NKIIPKAVASPVIPSSINSNNITTTTPNIKRIIHEFDP &RQVHQVXV MAASATTMLLKYPTTVCGIPNSSSNNNNDPSNNIVQIPQT T PLLK RTP NKI NAVAAPAFPS T TTTTPSSIQSLVHEFDP

101 110 120 130 140 150 160 170 180 190 200 $S&02 SIPAEDAFTPPSSWYTEPAFYAHELDRIFYKGWQVAGYSDQVKEANQYFTGTLGNVEYLVSRDGEGKVHAFHNVCTHRASILACGSGKKSCFVCPYHGWV $K&02 LVPAEDALTPPSSWYTEPAFYAHELDRIFYKGWQVAGYSDQVKEANQYFTGTLGNVEYLVCRDGEGKVHAFHNVCTHRASILACGSGKKSCFVCPYHGWV 6S&02 QIPPEDAHTPPSSWYTEPAFYSHELERIFYKGWQVAGISDQIKEPNQYFTGSLGNVEYLVSRDGEGKVHAFHNVCTHRASILACGSGKKSCFVCPYHGWV EHHW&02 EIPPEDALTPPSTWYTEPAFYSHELERIFYKGWQVAGYSEQVKEKNQYFTGSLGNVEYLVSRDGQGELHAFHNVCTHRASILACGSGKKSCFVCPYHGWV $PD&02 KVPAEDGFTPPSTWYTDPSLYSHELDRIFSKGWQVAGYSDQIKEPNQYFTGSLGNVEYLVCRDGQGKVHAFHNVCTHRASILACGTGKKSCFVCPYHGWV &RQVHQVXV IPAEDA TPPSSWYTEPAFYSHELDRIFYKGWQVAGYSDQVKE NQYFTGSLGNVEYLVSRDGEGKVHAFHNVCTHRASILACGSGKKSCFVCPYHGWV 201 210 220 230 240 250 260 270 280 290 300 $S&02 YGMNGTLTKASKATAEQSLNPDELGLVPLKVAEWGPFILISLDRS-SREVGDVGSEWLGSCAEDVKAHAFDPNLQFINRSEFPIESNWKIFSDNYLDSSY $K&02 YGMNGSLTKASKATPEQSLNPDELGLVPLKVAVWGPFILISLDRS-SREVGDVGSEWLGSCAEDVKAHAFDPNLQFINRSEFPIESNWKIFSDNYLDSSY 6S&02 YGMDGSLAKASKAKPEQNLDPKELGLVPLKVAVWGPFVLISLDRS-LEEGGDVGTEWLGTSAEDVKAHAFDPSLQFIHRSEFPMESNWKIFSDNYLDSSY EHHW&02 YGLDGSLAKASKATETQNLDPKELGLAPLKVAEWGPFILISLDRS-LDANADVGTEWIGKSAEDVKAHAFDPNLKFTHRSEFPMECNWKVFCDNYLDSSY $PD&02 FGLDGSLMKATKTEN-QVFDPKELGLVTLKVAIWGPFVLISLDRSGSEGTEDVGKEWIGSCAEEVKKHAFDPSLQFINRSEFPMESNWKVFCDNYLDSAY &RQVHQVXV YGMDGSL KASKAT EQ LDPKELGLVPLKVAVWGPFILISLDRS SEE GDVGSEWLGSCAEDVKAHAFDPNLQFINRSEFPMESNWKIFSDNYLDSSY

301 310 320 330 340 350 360 370 380 390 400 $S&02 HVPYAHKYYATELDFDTYQTDMVGNVTIQRVAGTSN--NGFSRIGTQAFYAFAYPNFAVERYGPWMTTMHIVPLGPRKCKLVVDYYIEKSKLDDKDYIEK $K&02 HVPYAHKYYATELDFDTYQTDMVGNVTIQRVAGTSN--NGFNRLGTQAFYAFAYPNFAVERYGPWMTTMHIVPLGPRKCKLVVDYYIEKSKLDDKDYIEK 6S&02 HVPYAHKYYATELNFDTYDTQMIENVTIQRVEGSSNKPDGFDRVGIQAFYAFAYPNFAVERYGPWMTTMHIHPLGPRKCKLVVDYYIENSMLDDKDYIEK EHHW&02 HVPYAHKYYAAELDFDTYNTEMIEKCVIQRVGSSSNKPDGFDRLGTEAFYAFIYPNFAVERYGTWMTTMHVVPMGQRKCKLVVDYYLEKAMLDDKAYIDK $PD&02 HVPYAHKYYAAELDFDTYKTDLLEKVVIQRVASSSNKPNGFDRLGSEAFYAFIYPNFAVERYGPWMTTMHIGPLGPRKCKLVVDYYLENAMMNDKPYIEK &RQVHQVXV HVPYAHKYYATELDFDTYQTDMIENVTIQRVAGSSNKPNGFDRLGTQAFYAFAYPNFAVERYGPWMTTMHIVPLGPRKCKLVVDYYIEKSMLDDKDYIEK

401410 420 430 440 453 $S&02 GIAINDNVQKEDVVLCESVQKGLETPAYRSGRYVMPIEKGIHHFHCWLHQVLK B $K&02 GIAINDNVQKEDVVLCESVQKGLETPAYRSGRYVMPIEKGIHHFHCWLHQVLK ApCMO 6S&02 GIAINDNVQREDVVLCESVQRGLETPAYRSGRYVMPIEKGIHHFHCWLQQTLK AhCM AhCMO EHHW&02 GIAINDNVQKEDKVLCESVQRGLETPAYRSGRYVMPIEKGIHHFHCWLHETLQ ApCM AmaCMO AmaCM $PD&02 SIMINDNVQKEDVVLCESVQRGLETPAYRSGRYVMPIEKGIHHFHCWLHQTLN beetCMObeetCM &RQVHQVXV GIAINDNVQKEDVVLCESVQRGLETPAYRSGRYVMPIEKGIHHFHCWLHQTLK SpCMOSpCM 71

Table 2-3. Nucleotide and amino acid sequence identity (left- and right-hand side of the diagonal matrix with

similarity of amino acid sequence in parentheses) among available CMOsa.

ApCMO AhCMO SpCMO beetCMO AmaCMO Arabidopsis

ApCMO -- 96.3 (97.3) 82.9 (87.2) 72.8 (80.8) 69.8 (79.1) 43.9 (56.9)

AhCMO 95.8 -- 83.6 (87.8) 73.0 (81.5) 70.1 (79.6) 44.6 (57.1)

SpCMO 73.4 73.2 -- 76.2 (82.4) 72.5 (80.1) 45.3 (55.7)

beetCMO 69.7 66.2 69.3 -- 71.1 (79.8) 43.4 (54.9)

AmaCMO 69.4 70.2 70.3 67.1 -- 44.6 (56.3) Arabidopsis 54.6 54.7 55.7 53.4 54.6 -- a The percentage of identity and similarity shown is determined by alignments of each two sequences performed by

AlignX®. The accession number of ApCMO is: AY082068. The accession number of AhCMO (A. hortensis) is:

AF270651. The accession number of SpCMO (Spinacia oleracea) is U85780. The accession number of beet CMO (Beta

vulgaris) is: AF023132. The accession number of AmaCMO (Amaranthus tricolor) is: AF290974. The accession number

of Arabidopsis CMO-like protein is: CAB43664 and NM_119135 (coding region of the corresponding mRNA).

72 73

NaCl and increased ~5 fold when plants were treated with 340 mM NaCl. In young leaves, there was almost no expression of CMO when the plants were not treated with salt. When treated with 170 mM NaCl, CMO expression was dramatically induced to 1.6 fold of the expression in control (0%) YS, whereas with 340 mM

NaCl, the induction was further enhanced to more than twice as much as observed with 170 mM NaCl, after the transcript levels were standardized with respect to rRNA stained by ethidium bromide (Fig. 2-8 A).

The accumulation of the CMO transcript paralleled accumulation of glycine betaine. In stems of A. prostrata treated with NaCl, there was a two to three fold increase in glycine betaine content. In 170 mM NaCl-treated leaves, a two-fold increase in glycine betaine was observed, while a three-fold increase was detected in

340 mM NaCl-treated leaves (Fig. 2-8 B).

Since CMO mRNA levels in young leaves and stems was different (Fig. 2-8 A), it was of interest to investigate whether various organs differentially respond to NaCl treatment with respect to CMO expression. Total RNAs were isolated from roots as well as old and young stems and leaves after the entire plants were treated with 340 mM NaCl for 3 days. Without salt treatment, CMO transcripts were detected only in the stems (Fig. 2-9). When plants were treated with 340 mM NaCl, CMO transcripts were present in roots, stems and leaves. In order to obtain a more accurate estimate of changes in ApCMO transcript levels, a densitometric analysis of northern blots was performed (Fig. 2-9). Compared to 0% YS, an approximately 5-fold increase

Fig. 2-8. CMO expression in Atriplex prostrata is induced by NaCl. A. In both

young stems (YS) and leaves (YL), the transcript of CMO accumulated in

response to 1% (= 170 mM) and 2% (= 340 mM) NaCl after 3 days. 20

µg of total RNA was loaded in each lane and the membrane was probed

with ApCMO-0.7. Relative amounts of CMO transcript were compared to

0% YS and standardized with respect to the amount of rRNA in each lane.

B. Accumulation of betaine in plants subjected to 1% (= 170 mM) and

2% (= 340mM) NaCl. Different letters above the bars indicates a

significant difference between the two treatment groups as determined by

the Bonferroni t-test.

B 200

180 b 0% 1% 160 b e 2% 140

120 d mol / gmol dry weight)

µ 100 ( 80 a

60 c

40 Betaine Content Betaine 20

0 Stems Leaves

Fig. 2-9. CMO expression in various organs of Atriplex prostrata treated with

2% (=340 mM) NaCl for 3 days. Here young stems (YS) are the first

internode and young leaves (YL) are fully expanded leaves from the top

of the plant. Old leaves (OL) are green leaves close to the base of the

plants, and old stems (OS) are the second node from the base of the

plants. Roots (R) are mostly lateral roots. 15 µg of total RNA was

loaded in each lane and the membrane was probed with ApCMO-0.7.

Relative amounts of CMO transcript were compared to 0% YS and

standardized with respect to the amount of rRNA (EtBr/rRNA) in each

lane.

78 after salt treatment was detected in young stems whereas in young leaves the increase was ~ 4 fold. In old stems, there was still a 3-fold increase relative to the level of CMO transcript in 0% YS. In old leaves, the salt-induction of CMO expression was only minimal. In roots, with both old and young roots included, the increase was moderate (about two fold more than that detected in control, young stems).

Not only was CMO differentially expressed in old and young tissues, it was also differentially expressed in seedlings of different ages. CMO transcript was first detected in three-day-old seedlings (three days after germination), and the accumulation of the transcript reached a maximum in 5-day-old seedlings (Fig. 2-10).

However, CMO transcript was barely detectable in older seedlings. Again, if one takes into consideration that the amount of RNA loaded in the lanes of 3-d and 5-d was much less than what was loaded in the lane of the positive control: 2%YL, the level of CMO transcript detected in 5-day-old seedlings is approximately 2.5 fold more than that of 2%YL.

In young stems, CMO was dramatically induced by NaCl within 24 h (Fig. 2-

11 A), and the maximum induction was observed at 48 and 72 h. After 120 h (6 d), the level of CMO transcript started to decline. There was a certain amount of CMO transcript accumulated at corresponding time points in control, but the induction by salt at different time points was still substantial. In young leaves, relative quantitative

RT-PCR was used instead of traditional northern blots in order to better distinguish any CMO transcript difference. While CMO mRNA was barely detected in non-

Fig. 2-10. CMO expression in 1-, 3-, 5-, 10-, and 15-day old seedlings of

Atriplex prostrata. 15 µg of total RNA isolated from seedlings 1, 3, 5,

10, and 15 days after germination was loaded in each lane and the

membrane was probed with ApCMO-0.7. Relative amounts of CMO

transcript were compared to 2% YL and standardized with respect to

the amount of rRNA (EtBr) in each lane.

Fig. 2-11. Time course of NaCl-induced CMO expression in Atriplex prostrata. A.

In young stems treated with either control (0%) or 2% (= 340 mM) NaCl

for various times, 15 µg of total RNA was loaded in each lane and the

membrane was probed with ApCMO-0.7. Relative amounts of CMO

transcript were compared to 0%-120h and standardized with respect to

the amount of rRNA (EtBr/rRNA) in each lane B. In young leaves treated

with either control (0%) or 2% (= 340 mM) NaCl for various time,

relative RT-PCR with CMO3 and CMO4 was used instead of a northern

blot. The lower band (315 bp) is part of 18S rRNA and serves as internal

standard to verify the equal amount of RNA used in the RT-PCR

reactions.

83 treated leaves over time by RT-PCR (except at 120 h), it was detected at 12 and 24 hours after salt treatments (Fig. 2-11 B). A pronounced increase in CMO mRNA (i.e., more RT-PCR product) was further observed at 72 and 120 h after salt treatments.

Effects of Exogenous ABA on CMO Expression

In addition to participating in various developmental and physiological processes including stomatal closure and root growth, abscisic acid (ABA) may initiate global physiological reactions in response to osmotic stress (reviewed by Zhu et al., 1997). Therefore, it was of interest to investigate whether ABA can transduce signals related to salt stress in Atriplex protrata and induce CMO expression. ABA alone did not induce CMO expression (Fig. 2-12), and the only induction was observed when plants were treated with 340 mM NaCl. Moreover, Lovastatin, a general isoprenoid biosynthesis inhibitor used to block ABA accumulation (Thomas et al., 1992), did not alter NaCl-induced CMO expression either.

DISCUSSION

Using RT-PCR and RACE, a composite cDNA encoding CMO from Atriplex prostrata was cloned and characterized. This composite ApCMO is derived from

ApCMO-5’, ApCMO-0.7, and ApCMO-3’ which substantially overlaps with each other Fig. 2-2). These overlapping regions, not including the primer sites, match almost perfectly (~ 99%) with a less than 5 mistakes which were fully resolved with

ApCMO-1.0 and repeated sequencing. The size of the transit peptide was predicted

Fig. 2-12. The role of ABA in CMO expression of Atriplex prostrata. Total

RNA (15 µg) was loaded in each lane and the membrane was probed

with ApCMO-0.7. EtBr RNA is the original RNA gel stained with

ethidium bromide to verify the amount of RNA loaded in each lane.

YL: RNA isolated from young leaves. YS: RNA isolated from young

stems. ABA: 10 µM abscisic acid added to the media. Lov: 10 µM

Lovastatin added to the media. X: no ABA nor Lovastatin added.

Plants were grown hydroponically in nutrient solutions containing

one of these treatments for 2 days before being transferred to either

control or 2% NaCl-containing media.

86 to be 67 residues, according to ChloroP. However, based on the N-terminus sequence of the purified, mature protein of spinach CMO determined by Rathinasabapathi et al.

(1997), as well as the alignment between the N-terminal regions of available CMOs and ApCMO (Fig. 2-6 A), the most probable size of the ApCMO transit peptide is 58 residues. According to ChloroP, the cleavage site for spinach CMO is Ala70, not

Ala61 although this is the N-terminus of the purified mature protein. In both cases, although the earlier Ala scored higher, it was excluded because it is one amino acid outside of the algorithm-predicted window of possible cleavage site, indicating that these computer programs are not perfect. In any event, one should be extremely cautious about reaching conclusion based solely on the predictions from computer programs.

In CMO of Atriplex prostrata, there are at least 3 AATAAA-like motifs that are possible polyadenylation signals (Fig. 2-4). One is ATTAAT (4 of 6 bases in

AATAAA conserved) located 6 nts upstream of poly A site in ApCMO. This may explain why one of the 3’-RACE products, CMO-3’-5, had an additional 38 nts before the poly A sites and was longer than the others. Second AATAAA-like motif is AAATTA located 55 nts upstream of poly A site. This may be the near-upstream element of CMO-3’-2 and -17. Third one is AATAAA located 85 nts upstream of poly A site. This AATAAA is believed to be the polyadenylation signal of CMO-3’-

38 that is 57 nts shorter than -2 and -17. In plants, polyadenylation signals require 2 cis-regulatory elements: far-stream element (FUE) and near-upstream element (NUE)

(for review, see Hunt, 1994; Rothnie, 1996). There is no consensus sequence for

87 either element, but each has key sequence characterisitics based on nucleotide composition. Located approximately 40 to 150 bases upstream of the cleavage site, the FUE is required for efficient 3’ end formation. The most common motif among known FUEs is the presence of multiple U/G rich regions (Hunt, 1994). The NUE, similar to AATAAA in animal systems, is an A/U-rich element typically found 10 to

30 bases upstream of the cleavage site.

ApCMO is 72.8 to 96.3% identical and 80.8 to 97.3% similar to CMOs of other members in the Chenopodeaceae (i.e., A. hortensis, spinach, and sugar beet) (Table

2-3). Among CMOs of these four species, A. prostrata is most related to A. hortensis and sugar beet is least related to others. As a betaine-accumulator from another family, Amaranth CMO is more closely related to CMOs of the chenopods than to

Arabidopsis. These observations are in agreement with known phylogenetic relationships. The deduced amino acid sequence of ApCMO contains two motifs, the Rieske-type [2Fe-2S] cluster and the mononuclear non-heme Fe binding site, which are also conserved in all reported CMOs (Figs. 2-1B, 2-2B and 2-2C;

Rathinasabapathi et al., 1997; Russell et al., 1998; Meng et al., 2001). Although the

Rieske cluster is also present in various dioxygenases and chlorophyll oxygenase, the presence of these two motifs together make CMO unique.

Although CMO was cloned from spinach, sugar beet and amaranth earlier, the expression of CMO at mRNA level was not studied in much detail. In A. prostrata,

CMO expression is induced by NaCl (Fig. 2-8), which is consistent with other research (Rathinasabapathi et al., 1997; Russell et al., 1998; Meng et al., 2001).

Indeed, with higher salt concentration, accumulation of CMO transcript was observed to increase. In parallel, glycine betaine accumulates as a function of different salt concentration. Taken together, these data support that CMO catalyzes the rate-limiting step of glycine betaine production. In addition, the expression of

CMO in response to salt treatment differs in an organ-specific manner (Fig. 2-9).

CMO transcript is most abundantly expressed in stems, and the induction by 340 mM NaCl is also most pronounced in stems. In young leaves and roots, NaCl- induced CMO expressions were dramatic and infinitely large as CMO expressions in corresponding unsalinized tissues were undetectable.

Up-regulation of CMO expression by NaCl is less pronounced in older leaves and stems (Fig. 2-9), indicating that glycine betaine biosynthesis may be more active in younger cells. One explanation is that young cells are generally more sensitive to salt stress than older cells. Glycine betaine is not degraded once accumulated in the cytosol (Nuccio et al., 1998), therefore older cells, once accumulate glycine betaine, do not need to activate the pathway. Another piece of evidence demonstrating that

CMO is differentially expressed at different developmental stages is that CMO transcripts accumulate in 3- and 5-day-old seedlings without salt induction.

However, CMO is not expressed later in 10- or 15-day-old seedlings. This may be because 3- and 5-d-old seedlings require glycine betaine to deal with the particular physiological conditions unique to these developmental times.

Many observations have indicated that osmotic stress due to salt, drought and water stress, leads to an increase in endogenous levels of ABA, which in turn

89 induces expression of osmotic stress responsive genes (Skriver and Mundy, 1990;

Ishitani et al., 1997). Moreover, these genes can be induced by exogenous ABA application. However, accumulation of the CMO message was not dependent upon exogenous ABA. Furthermore, chemically inhibited accumulation of ABA by

Lovastatin did not block the NaCl-induced accumulation of CMO transcript (Fig. 2-

12). These results indicate that the CMO gene, at least in A. prostrata, is not one of the ABA-responsive genes whose transcript levels increase many-fold within hours of ABA application. Such ABA-responsive genes share similarity within their respective 5’ upstream regions (Guiltinan et al., 1990). In particular, specific nucleotide sequences or ABA-responsive elements (ABREs) in the promoter region are necessary for the induction of these ABA-responsive genes (Marcotte et al., 1989;

Thomas et al., 1992). Without the sequence of CMO gene from Atriplex or any species, it is difficult to demonstrate that an ABRE is absent. Recently, however, a

CMO-like protein in Arabidopsis was documented (protein ID: CAB43664), and no and no ABRE (i.e., ACGTGGC in either orientation) can be found upstream of start codon. Therefore, it appears unlikely that an ABRE is present and ABA is unlikely to induce CMO expression.

There are other examples in which the expression of osmotic stress-responsive genes may be independent of ABA (Binzel and Dunlap, 1995; Gosti et al., 1995). In ice plants (Mesembryanthemum crystallinum), the transition from the C3 photosynthetic pathway to Crassulacean Acid Metabolism (CAM) is one mechanism for water conservation and/or salt tolerance. A CAM specific isozyme,

90 phospoenolpyruvate carboxylase (PEPC), is strongly induced by salt, but ABA is not necessary for the expression of PEPC (Yen et al., 1995). Therefore, different signal transduction pathways (i.e., ABA-dependent and ABA-independent) exist that regulate the expression of osmotic stress-induced genes in plants. Based on the data presented here, the expression of the Atriplex prostrata CMO gene is ABA independent.

It will be useful to discover genomic clones and regulatory elements of CMO from members of Chenopodeaceae and investigate how signals are transduced and activate CMO expression. Since CMO expression in response to salt stress in A. prostrata is more substantial than that reported for other members of the

Chenopodiaceae, such as spinach and sugar beet (Rathinasabapathi et al., 1997,

Russell et al., 1998), Atriplex may serve as a particularly useful model plant for studies of regulatory mechanisms related to the activation of the glycine betaine biosynthetic pathway in response to environmental stress.

CHAPTER 3. CLONING AND EXPRESSION OF BETAINE ALDEHYDE

DEHYDROGENASE IN ATRIPLEX PROSTRATA

ABSTRACT

In plants, the second step of glycine betaine synthesis is catalyzed by betaine aldehyde dehydrogenase (BADH). Cloned by RT-PCR and 3’-RACE, the composite cDNA of Atriplex prostrata BADH (ApBADH1) is 1755 bp in length and encodes a full-length protein of 500 amino acids. The deduced amino acid sequence of

ApBADH2 contained a NAD+ binding motif and active sites for aldehyde dehydrogenase. According to amino acid sequence alignment, ApBADH2 is 90.8% identical and 96.6% similar to spinach BADH. Northern blot analysis revealed that accumulation of BADH transcript increased in response to NaCl. In young stems,

NaCl-induced accumulation occurred and peaked within 24 hours. Without salt treatment, BADH mRNA was not detected in old leaves or roots but was detected in stems, leaves and seedlings of different ages. With salt treatment, BADH mRNA accumulated substantially in stems, leaves and roots, and most abundantly in young stems. Unlike CMO, BADH expression was induced by abscisic acid in A. prostrata.

As in mangrove Avicennia marina, there are two BADHs in Atriplex prostrata.

Another BADH from A. prostrata was partially cloned. This partial cDNA clone,

ApBADH2, is 84.9 % identical to the corresponding cDNA of ApBADH1.

INTRODUCTION

Accumulation of osmoprotectants is one of the most studied mechanisms of salt tolerance because they not only can adjust osmotic potential but also can stabilize membranes and enzymes (Rhodes and Hanson, 1993). Glycine betaine is a particularly well-known osmoprotectant that effectively stabilizes enzymes critical to physiological functions (Papageorgiou and Murata, 1995). All known pathways for the synthesis of glycine betaine start with choline and the reactions involve either one or two enzymes for the oxidation of choline to glycine betaine. The two-enzyme reaction in mammalian cells and in microorganisms such as E. coli is catalyzed by choline dehydrogenase (CDH) and betaine aldehyde dehydrogenase (BADH)

(Landfold and Strøm, 1986). In plants, the two-enzyme reaction is catalyzed by choline monooxygenase (CMO) and BADH (Gorham, 1995).

Unlike CMO which is unique to plants, BADH has been well studied in plants and animals. BADH shares similar structure to other aldehyde dehydrogenases and contains 3 domains: a coenzyme binding domain, a catalytic domain and an oligomerization domain (Johansson et al., 1998). In bacteria and mammals, BADH is found as homotetramer (Figueroa-Soto and Valenzuela-Soto, 2001). In plants, it is a dimer of identical subunits of Mr 60-63 kDa and is encoded by a single nuclear gene

(Weretilnyk and Hanson, 1988). Consistent with the salt-induced level of BADH activity, the mRNA for BADH is also found to be induced by salinity (Weretilnyk and Hanson, 1990). In non-accumulating species, neither the BADH isozyme nor

94 activity has ever been detected (Weigel et al., 1986; Weretilnyk et al., 1989). In contrast, putative BADH-like genes were found in non-accumulating species such as

Arabidopsis (T8E24.4, protein ID: AAG50992; F25A4.11, protein ID: AAD55284;

T24C20; protein ID: CAB51064) and rice (AB001348; protein ID: BAA21098). In fact, the existence of a BADH gene in rice was confirmed by southern blot analysis with a barley BADH cDNA as a probe under high-stringency conditions (Nakamura et al., 1995). This rice BADH was further cloned with low expression under salt stress (Nakamura et al., 1997).

BADH cDNA clones were isolated from spinach (Weretilnyk and Hanson,

1990), sugarbeet (McCue and Hanson, 1992), barley (Ishitani et al., 1995), sorghum

(Wood et al., 1996) and amaranth (Legaria et al., 1998). Recently, two BADHs were cloned from mangrove Avicennia marina and functional analysis revealed that both

BADHs were more efficient enzymes than spinach BADH for 2 reasons: 1) mangrove BADHs were more stable at high temperature than spinach; 2) mangrove

BADHs have unique substrate specificity whereas spinach BADH may catalyze the oxidation of not only glycine betaine aldehyde but also 3-aminopropionaldehyde and

4-aminobutyraldehyde (Hibino et al., 2001).

Although BADH was first reported to be localized in the stroma of chloroplast

(Weigel et al., 1986), its occurrence in compartments other than the chloroplast can not be exclued. In fact, in barley, BADH may be localized in peroxisomes due to the targeting sequence SKL found at its C-terminus (Ishitani et al., 1995). In Avicennia marina, one of the two BADH also contains SKL at its C-terminus (Hibino et al.,

2001). In rice, BADH containing SKL motif was detected in peroxisomes by immunolocalization (Nakamurat et al., 1997). The significance of alternative localization remains to be elucidated.

The questions addressed in this investigation in addition to cloning BADH cDNAs from Atriplex prostrata are: 1) whether BADH expression is regulated by salt; 2) whether BADH expression is differentially regulated in different organs and at different developmental stages, and 3) whether ABA regulates BADH expression.

MATERIALS AND METHODS

Plant Material and Growing Conditions

Seeds of Atriplex prostrata were germinated in flats filled with sand in an environmental growth chamber under a 25ºC/5ºC, 12 h day/12 h night regime (400-

700 nm, 400 µmol m-2 s-1). Ten days after germination, seedlings were transferred to four-inch pots filled with sand at a density of 4 seedlings per pot. Pots were scattered in plastic trays. These trays were filled with half-strength Hoagland’s solution

(nutrient solution) with or without 1% (w/v) or 2% (w/v) NaCl and maintained in an environmental growth chamber under a 25ºC/15ºC, 14 h day/10 h night regime.

Distilled water was added daily to each tray to replace water lost because of evaporation and transpiration. Solutions were replaced weekly.

96 nutrient solutions supplemented with or without 10 µM (+/-) ABA (Sigma, St. Louis,

MO) or 10 µM Lovastatin (Sigma, St. Louis, MO). After 2 days, plants were transferred to nutrient solutions with or without 2% (w/v) NaCl.

Cloning of ApBADH by RT-PCR and 3’ RACE

Total RNA from leaves treated with 340 mM NaCl for 3 days was reverse- transcribed into cDNAs in the presence of MMLV-Reverse Transcriptase (Promega,

Madison, WI). Polymerase chain reactions (PCR) were then carried out using Taq polymerase (Promega, Madison, WI) with the following cycle profile: initial denaturation of cDNAs at 95°C for 2 min, 30 cycles of amplification by denaturing at 95°C for 1 min, annealing at 46°C for 1 min and extending at 72°C for 1 min for each cycle, and a final extension (i.e., polishing) at 72°C for 7 min. Annealing temperatures were set to be 46°C or 4 °C lower than the lowest Tm of the paired primers (synthesized by Integrated DNA technologies, Inc., Coralville, IA). The sequence and Tm of all primers (calculated by Integrated DNA technologies, Inc. according to the nearest-neighbor thermodynamic parameter) used in RT-PCR and

3’- RACE (rapid amplification of cDNA ends) are listed in Table 3-1.

For 3’-RACE, 2 µg of mRNA isolated from NaCl-treated leaves using a mRNA isolation kit (Qiagen Inc., Chatsworth, CA) was used to carry out first and ligated to MarathonTM cDNA adaptors (Clontech, Palo Alto, CA) prior to 3’-RACE.

Table 3-1. A list of primers used to clone BADH of Atriplex prostrata

Primersa Sequence Tmb Position

GB1 AGAATGGCGTTCCCAATTCC 55.29 1-20c

GB3 CAAGGAGACTTGTACCATCCC 50.23 1485-1505d

GB4 TATTTGCGTGCTATTGCTGC 51.87 232-251c

GB5 ATTCTCGATACCCCATTCCC 51.91 1438-1458d

GB6 CCATGGGGAGGAGTGAAGCGTAGTGG 66.20 1395-1420c

GB7 CTGTCCATAGGCAGGGTGACTGGAGCC 67.24 402-428d

GB5AS TCAATGTCCCATACTGCTTC 53.29 318-338d

AP1 CCATCTAATACGACTCACTATAGGGC 56.87 NA (adaptor)

a Cross-reference to Fig. 3-1 b Tm was calculated based on the nearest-neighbor thermodynamic parameter

(Allawi and SantaLucia, 1997) by Oligo Analyzer 2.5 available at

www.idtdna.com. c Sense primer d Antisense primer

GB6, a GC-rich primer, was designed based on the sequence of previous RT-PCR product, ApBADH-1.2 (Fig. 3-1). GB6 and the adaptor primer AP1 (Table 3-1) amplified the 3’ end product of ApBADH with the touchdown PCR program according to the instruction of MarathonTM RACE (Clontech, Palo Alto, CA).

Subcloning and Sequence Analysis of PCR Products

All RT-PCR and RACE products were subcloned using the TOPO cloning kit

 by Invitrogen (Carlsbad, CA) with the vector, pCR II-TOPO (3.9 kb). Subcloned cDNAs were sequenced using the BigDye Terminator Cycle Sequencing Reactions kit and an automated DNA sequencer (ABI 310, Perkin-Elmer, Foster City, CA).

Sequence comparison with other databases was performed through the National

Center for Biotechnology Information via BLAST (http://www.ncbi.nlm.nih.gov).

Sequence alignments and analysis were performed using AlignX® of Vector NTI suite 6 (InforMax, Inc., Bethesda, MD).

RNA Isolation and Northern Blotting

Total RNAs were isolated from tissues of Atriplex prostrata from various treatments as described using RNeasyTM Plant Total RNA Kit (Qiagen Inc.,

Chatsworth, CA). Total RNAs were subjected to electrophoresis in 1% (w/v) agarose gels containing MOPS and 7% (w/v) formaldehyde. Gels were then transferred to

Zeta-probe GT blotting membranes (Bio-Rad, Hercules, CA) in 10X SSC. RNA was

Fig. 3-1. A schematic map of 1755 bp composite cDNA sequence encoding BADH in Atriplex prostrata. “ “: Primers

used for RT-PCR (see Table 3-1 for the sequence of these primers). “ “ : cleavage site of restriction enzymes.

“ “: RT-PCR fragment, subcloned and sequenced. “ “: Positions of either start codon (ATG) and stop

codon (TGA).

5’ 3’

GB1 GB4 GB5AS GB7 GB6 GB5 GB3 1755 AAAA

ATG TGA HindIII HindIII PstI EcoRI

ApBADH-1.2 1206 bp

ApBADH-1.3 1274 bp

ApBADH-3’ 381 bp ApBADH-5’ 338 bp

ApBADH-7

100 101 fixed to membranes by a Stratalinker® UV crosslinker (Model 1800, Stratagene, La

Jolla, CA) at an energy of 120 mJ for approximately 2 min and then air-dried. A partial length BADH cDNA, ApBADH-1.2, was labeled with [α-32P]dCTP using the

Prime-a-Gene® Labeling System (Promega, Madison, WI) and used as a probe.

Prehybridization and hybridization reactions were carried out in 7% (w/v) SDS, 250 mM sodium phosphate buffer (pH7.2) at 65°C. Washing was performed in ¼ strength prehybridization solution at room temperature for 5 min and twice at 65°C for 10 min.

Autoradiographs of northern blots and ethidium bromide-stained gels were scanned using a digital imaging system (Fluor-S™ MultiImager MAX, Bio-Rad,

Hercules, CA) and analyzed with Quantity One® quantitation software (version 4.2.2,

Bio-Rad, Hercules, CA) to quantify levels of rRNA and BADH transcript. Levels of

BADH transcript were normalized with respect to the amount of rRNA in each lane, as determined by ethidium-bromide staining of the original gel.

RESULTS

Cloning of BADH by RT-PCR and RACE

To clone BADH from Atriplex prostrata, sequences of spinach, sugar beet, barley and A. hortensis BADHs were aligned and compared to identify consensus regions (Fig. 3-2). Three primers, GB4, GB3 and GB5, were designed based on

Fig. 3-2. Nucleotide sequence alignment of BADHs from spinach, sugar beet, barley and Atriplex hortensis. Sequences

were downloaded from GeneBank (www.ncbi.nlm.nih.gov); accession numbers are M31480, X58462,

AB063178 and X69770, respectively. The nucleotides conserved among all four clones are shaded in yellow,

and those conserved among three of the four clones are shaded in blue. Primers GB3, GB4, and GB5, which

were designed according to this multiple sequence alignment, are labeled with arrows.

102

1 10 20 30 40 50 60 70 80 90 100 $K%$'+ ------AGAATGGCGTTC-CCAA--TTCCTGCTCGTCAA EDUOH\%%' ------GACCCCGTCAACAACCACCACGCAAGCTCACTCCCCGCCCGCCGCGATGGTCGCGCCGGCCAAGATCCCGCAGCGGCAG EHHW%$'+' ------CTTTTTTTTTTCATCTCTATTTT----ACTGAATTTCTTCTACTGTTTTTTTAAAAAATGTCGATG-CCAA--TTCCTTCTCGCCAG 6S%$'+ CGTTGCGTGCTCGCCTTACCCTCTCAACTCAATTTCTTCAACCCAATTTCTTCGCAT---TTAACCAAGAATGGCGTTC-CCAA--TTCCTGCTCGTCAG &RQVHQVXV C TT T TCA CTCAATTTC C AC CAATTTCTTC CCTG TT CAAGAATGGCGTTG CCAA TTCCTGCTCGTCAG

101 110 120 130 140 150 160 170 180 190 200 $K%$'+ CTCTTCATCGATGGAGAGTGGAGAGAACCCCTTTTAAAAAATCGCATACCCATCATCAACCCTTCTACTGAAGAAATCATCGGTGATATTCCTGCAGCAA EDUOH\%%' CTCTTCATCGACGGCGAGTGGCGCGCGCCCGCGCTCGGCCGCCGCCTCCCCGTCATCAACCCGACCACCGAGGTCTCCATCGGCGAGATCCCGGCGGGCA EHHW%$'+' TTATTCATTGATGGAGAGTGGAGAGAACCCATCAAGAAAAATCGCATCCCTATTATCAACCCATCTAATGAGGAGATCATCGGTGATATTCCGGCGGGCA 6S%$'+ CTATTCATCGACGGAGAGTGGAGAGAACCCATTAAAAAAAATCGCATACCCGTCATCAATCCGTCCACTGAAGAAATCATCGGTGATATTCCGGCAGCCA &RQVHQVXV CTCTTCATCGATGGAGAGTGGAGAGAACCCATTATAAAAAATCGCATCCCCGTCATCAACCCGTCTACTGAGGAAATCATCGGTGATATTCCGGCGGGCA

201 210 220 230 240 250 260 270 280 290 300 $K%$'+ CTGCAGAGGATGTGGAGGTTGCAGTGGTGGCAGCTAGAAAAGCCTTTAAGAGGAACAAAGGCAGAGATTGGGCTGCA--CTCTGGT-CTCATCGTGCTAA EDUOH\%%' CCTCGGAGGACGTGGACGCCGCGGTGGCGGCCGCGCGGGCCGCCCTCAAGAGGAACCGCGGCCGCGACTGGTCCCGCGCCCCCGGCGCCGTCCGGGCCAA EHHW%$'+' GTTCCGAGGATATAGAGGTTGCGGTGGCGGCCGCTCGAAGAGCCCTCAAGAGGAATAAGGGAAGAGAGTGGGCTGCTACATCTGGAGCTCATCGTGCCAG 6S%$'+ CGGCTGAAGATGTGGAGGTTGCGGTGGTGGCAGCTCGAAGAGCCTTTAGGAGGAACAAT------TGGTCAGCAACATCTGGGGCTCATCGTGCCAC &RQVHQVXV CTTC GAGGATGTGGAGGTTGCGGTGGTGGCCGCTCGAAGAGCCTTTAAGAGGAACAA GGCAGAGA TGGTCTGCAACCTCTGG GCTCATCGTGCCAA

301 310 320 330 340 350 360 370 380 390 400 $K%$'+ ATATTTGCGTGCTATTGCTGCTAAGATAACAGAGAAAAAAGATCATTTTGTTAAACTGGAAACCCTTGATTCTGGAAAACCACGGGATGAAGCAGTGTTA EDUOH\%%' GTACCTCCGCGCAATCGCCGCCAAGATGATTGAGCGGAAATCTGATCTGGCTAGGCTAGAGGCACTTGATTGCGGGAAGCCTCTTGATGAAGCGGCATGG EHHW%$'+' ATATTTGCGTGCTATTGCAGCTAAAGTGACAGAAAGAAAAGATCATTTTGTTAAACTTGAAACGATTGATTCTGGGAAACCGTTTGATGAAGCAGTGTTG 6S%$'+ ATACTTGCGTGCTATTGCTGCTAAGATAACAGAAAAAAAAGATCATTTCGTTAAACTGGAAACCATTGATTCTGGGAAACCTTTTGATGAAGCAGTGCTG &RQVHQVXV ATATTTGCGTGCTATTGCTGCTAAGATGACAGAGAGAAAAGATCATTTTGTTAAACTGGAAACCCTTGATTCTGGGAAACCTTTTGATGAAGCAGTGTTG GB4 401 410 420 430 440 450 460 470 480 490 500 $K%$'+ GATATTGATGATGTTGCTACATGCTTTGAATACTTTGAATACTTTGCCGGTCAAGCAGAAGCTCTGGATGCTAAACAAAAGGCTCCAGTCACCCTGCCTA EDUOH\%%' GACATGGACGATGTTGCCGGCTGCTTTGAGTTCTTCG------CAGGTCATGCTGAAGCCTTGGACAAAAGGCAAAATGCTGCAGTTGCTCTCCC-- EHHW%$'+' GATATTGATGATGTGGCCACATGTTTTGAATATTTTG------CCGGTCAAGCAGAAGCCATGGACGCTAAACAAAAAGCTCCCGTCACCCTTCCAA 6S%$'+ GACATTGATGACGTTGCTTCATGTTTTGAATATTTTG------CCGGACAAGCAGAAGCTCTTGATGGTAAACAAAAGGCTCCAGTCACCCTGCCTA &RQVHQVXV GATATTGATGATGTTGCTACATGTTTTGAATATTTTG CCGGTCAAGCAGAAGCTCTGGATGCTAAACAAAAGGCTCCAGTCACCCTGCCTA

103

Fig. 3-2. Continued

501 510 520 530 540 550 560 570 580 590 600 $K%$'+ TGGAAAGATTTAAAAGTCATGTTCTCAGGCAGCCCATTGGTGTTGTTGGATTAATATCCCCATGGAATTACCCACTTCTAATGGATACATGGAAAATTGC EDUOH\%%' -GGAGAATTTTAAATGCCATCTTAAGAAGGAACCTATTGGTGTAGTTGCTCTAATCACACCATGGAACTATCCTCTCCTGATGGCTGTATGGAAGGTAGC EHHW%$'+' TGGAGAGATTCAAAAGTCATGTTCTCAGGCAGCCCATTGGTGTTGTTGGATTGATTACCCCATGGAATTACCCACTTCTAATGGCTACATGGAAAATTGC 6S%$'+ TGGAAAGGTTCAAAAGTCATGTTCTCAGGCAGCCCCTTGGTGTTGTTGGATTAATATCCCCATGGAATTACCCACTTCTAATGGCTACATGGAAAATTGC &RQVHQVXV TGGAGAGATTTAAAAGTCATGTTCTCAGGCAGCCCATTGGTGTTGTTGGATTAATATCCCCATGGAATTACCCACTTCTAATGGCTACATGGAAAATTGC

601 610 620 630 640 650 660 670 680 690 700 $K%$'+ TCCCGCACTTGCTGCTGGATGCACGACTGTACTTAAACCATCAGAATTGGCATCTGTGACTTGTCTAGAATTCGGTGAAGTGTGTAATGAAGTGGGACTT EDUOH\%%' CCCTGCTCTGGCAGCTGGTTGTACAGCTGTACTGAAACCATCTGAATTAGCTTCCGTGACTTGCTTAGAGCTTGGTGATGTGTGTAAAGAGGTCGGTCTT EHHW%$'+' TCCAGCTCTTGCTGCTGGGTGTACAGCTGTACTGAAGCCATCAGAGTTGGCATCTATAACTTGCCTAGAATTTGGAGAAGTTTGCAATGAAGTGGGACTT 6S%$'+ TCCAGCACTTGCTGCTGGGTGTACAGCTGTACTTAAGCCATCCGAGTTGGCATCTGTGACTTGTCTAGAATTCGGTGAAGTTTGCAACGAAGTGGGACTT &RQVHQVXV TCCAGCTCTTGCTGCTGGGTGTACAGCTGTACTTAAGCCATCAGAGTTGGCATCTGTGACTTGTCTAGAATTTGGTGAAGTTTGTAATGAAGTGGGACTT

701 710 720 730 740 750 760 770 780 790 800 $K%$'+ CCTCCAGGTGTGTTAAATATTTTGACAGGATTAGGTCCTGATGCTGGTGCCCCAATAGTATCTCATCCTGATATTGACAAGGTAGCATTTACTGGGAGTA EDUOH\%%' CCATCAGGTGTCTTAAACATTGTGACTGGACTAGGTAATGAAGCTGGTGCTCCTTTGTCATCACACCCTGACGTCGACAAGGTTGCATTTACCGGGAGCT EHHW%$'+' CCTCCGGGGGTGTTGAATATTGTGACTGGATTGGGTCCAGATGCCGGTGCACCGCTAGCAGCTCATCCTGATGTTGACAAGGTTGCATTTACTGGAAGTA 6S%$'+ CCTCCAGGCGTGTTGAATATCTTGACAGGATTAGGTCCAGATGCTGGTGCACCATTAGTATCACACCCCGATGTTGACAAGATTGCCTTTACTGGGAGTA &RQVHQVXV CCTCCAGGTGTGTTGAATATTTTGACTGGATTAGGTCCTGATGCTGGTGCACCATTAGTATCTCATCCTGATGTTGACAAGGTTGCATTTACTGGGAGTA

801 810 820 830 840 850 860 870 880 890 900 $K%$'+ GTGCCACTGGAAGCAAGATTATGGCTTCTGCTGCCCAACTAGTTAAGCCTGTTACTTTGGAGCTTGGAGGTAAAAGTCCTGTTATCATGTTCGAAGATAT EDUOH\%%' ATGCAACCGGTCAAAAGATTATGGTTGCTGCAGCTCCTACAGTCAAGCCTGTTACATTGGAGCTTGGTGGCAAAAGTCCTATTGTAGTATTTGATGATGT EHHW%$'+' GTGCCACTGGCAGCAAAGTGATGGCTTCAGCTGCTCAATTGGTTAAGCCTGTTACATTGGAACTTGGAGGTAAAAGTCCTATTATCGTGTTCGAAGATGT 6S%$'+ GTGCCACTGGAAGCAAGGTTATGGCTTCTGCTGCCCAATTGGTTAAGCCTGTTACATTAGAACTTGGGGGTAAAAGTCCTATTGTAGTGTTTGAAGATGT &RQVHQVXV GTGCCACTGGAAGCAAGGTTATGGCTTCTGCTGCTCAATTGGTTAAGCCTGTTACATTGGAGCTTGGAGGTAAAAGTCCTATTGTCGTGTTTGAAGATGT

901 910 920 930 940 950 960 970 980 990 1000 $K%$'+ TGATATTGAAACAGCTGTTGAATGGACTCTTTTTGGCGTTTTCTGGACAAATGGTCAAATCTGTAGTGCAACATCTAGACTGCTTGTGCATGAAAGCATT EDUOH\%%' CGACATTGACAAAGCTGTTGAGTGGACTCTATTTGGGTGCTTTTGGACCAATGGTCAGATTTGTAGTGCGACATCTCGTCTTCTTATCCATAAAAATATC EHHW%$'+' TGATATTGATCAAGTTGTTGAATGGACCATGTTTGGCTGTTTCTGGACAAATGGTCAAATATGCAGTGCAACATCCAGACTGCTTGTGCATGAAAGTATT 6S%$'+ TGATATTGATAAAGTTGTGGAATGGACTATTTTTGGCTGTTTCTGGACAAATGGTCAAATATGTAGTGCAACGTCTAGACTGCTTGTGCATGAAAGTATT &RQVHQVXV TGATATTGATAAAGTTGTTGAATGGACTCTTTTTGGCTGTTTCTGGACAAATGGTCAAATATGTAGTGCAACATCTAGACTGCTTGTGCATGAAAGTATT

104

Fig. 3-2. Continued

1001 1010 1020 1030 1040 1050 1060 1070 1080 1090 1100 $K%$'+ GCAGCTGAATTTGTTGATAGGATGGTGAAGTGGACAAAAAACATAAAAATTTCTGATCCATTTGAAGAAGGATGCCGGCTTGGCCCCGTTATTAGTAAAG EDUOH\%%' GCTAAAGAATTCGTTGACAGGATGGTTGCATGGTCCAAAAATATCAAGGTGTCAGACCCGCTCGAGGAGGGTTGCAGGCTTGGGCCAGTTGTTAGTGAAG EHHW%$'+' GCAGCTGAATTTATTGATAGGCTTGTAAAGTGGACCAAAAACATTAAGATATCTGATCCATTTGAGGAAGGCTGTCGACTTGGCCCTGTTATTAGTAAGG 6S%$'+ GCAGCTGAGTTTGTTGATAAGCTTGTAAAATGGACGAAAAACATTAAAATTTCTGACCCATTTGAAGAAGGATGCCGGCTTGGCCCTGTTATTAGTAAAG &RQVHQVXV GCAGCTGAATTTGTTGATAGGCTTGTAAAGTGGACCAAAAACATTAAGATTTCTGATCCATTTGAGGAAGGATGCCGGCTTGGCCCTGTTATTAGTAAAG

1101 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200 $K%$'+ GACAGTACGACAAGATTATGAAGTTCATATCAACAGCGAAGAGTGAAGGGGCAACAATTTTGTGTGGAGGCTCCCGTCCTGAGCATTTGAAGAAAGGGTA EDUOH\%%' GACAGTATGAGAAGATCAAGAAGTTCGTAGCGAATGCTAAAACTGAAGGTGCGACCATTCTCACTGGGGGTGTCAGGCCCAAGCATCTGGAGAAAGGTTT EHHW%$'+' GGCAGTACGATAAAATTATGAAGTTCATATCAACAGCAAAGAGTGAGGGGGCAACTATCTTGTGTGGAGGTTCCCGTCCTGAGCATTTGAAGAAAGGGTA 6S%$'+ GACAGTACGACAAAATTATGAAGTTCATATCAACAGCAAAGAGTGAGGGGGCAACTATTTTGTATGGAGGTTCCCGTCCTGAGCATTTGAAGAAAGGTTA &RQVHQVXV GACAGTACGACAAGATTATGAAGTTCATATCAACAGCAAAGAGTGAGGGGGCAACTATTTTGTGTGGAGGTTCCCGTCCTGAGCATTTGAAGAAAGGTTA

1201 1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 $K%$'+ TTATATTGAACCCACCATTATAACTGATATTACCACATCCATGCAAATATGGAAAGAGGAAGTGTTTGGCCCTGTCATATGTGTTAAAACATTTAAAACT EDUOH\%%' CTTCATTGAACCCACAATCATCACTGACATCAACACATCAATGGAGATTTGGAGGGAGGAAGTCTTTGGTCCAGTCCTGTGTGTGAAGGAATTTTCTACC EHHW%$'+' TTTCATCGAACCAACTATTATAAGTGATATCTCCACATCCATGCAAATATGGAGGGAAGAAGTTTTTGGCCCTGTCTTATGTGTTAAAACATTTAGTTCT 6S%$'+ TTACATTGAACCCACCATTGTAACTGATATCTCCACATCCATGCAAATATGGAAAGAGGAAGTTTTTGGCCCTGTCTTGTGTGTTAAAACATTTAGTTCC &RQVHQVXV TTTCATTGAACCCACCATTATAACTGATATCTCCACATCCATGCAAATATGGAGGGAGGAAGTTTTTGGCCCTGTCTTGTGTGTTAAAACATTTAGTTCT

1301 1310 1320 1330 1340 1350 1360 1370 1380 1390 1400 $K%$'+ GAAGATGAAGCCATTGAATTGGCAAATGATACAGAGTATGGTTTAGCTGGTGCCGTGTTTTCTAAAGATCTTGAAAGATGTGAGAGGGTAACTAAGGCTC EDUOH\%%' GAAGAGGAAGCCATCGAACTGGCCAACGATACTCATTATGGTCTGGCTGGTGCCGTGATTTCCGGTGACCGTGAGCGATGCCAGAGATTGGCTGAGGAGA EHHW%$'+' GAAGATGAAGCTCTTGATTTGGCAAATGATACTGAGTATGGTTTAGCTTCTGCTGTGTTTTCAAAAGACCTTGAAAGGTGTGAAAGGGTATCGAAGCTTT 6S%$'+ GAAGATGAAGCCATTGCATTGGCAAATGATACAGAGTACGGTTTAGCTGCTGCTGTGTTTTCTAATGATCTTGAAAGATGTGAGAGGATAACGAAGGCTC &RQVHQVXV GAAGATGAAGCCATTGAATTGGCAAATGATACTGAGTATGGTTTAGCTGGTGCTGTGTTTTCTAATGATCTTGAAAGATGTGAGAGGGTAACTAAGGCTC

1401 1410 1420 1430 1440 1450 1460 1470 1480 1490 1500 $K%$'+ TAGAAGTTGGAGCTGTTTGGGTTAATTGCTCACAACCATGCTTTGTTCATGCTCCATGGGGAGGAGTCAAGCGTAGTGGATTTGGACGTGAACTTGGGGA EDUOH\%%' TCGAGGCGGGATGCATCTGGGTGAACTGCTCGCAGCCTTGCTTCTGCCAAGCTCCGTGGGGTGGGAACAAGCGCAGCGGCTTTGGGCGAGAGCTCGGAGA EHHW%$'+' TGGAATCTGGAGCCGTGTGGGTTAATTGCTCACAACCATGCTTTGTTCATGCTCCATGGGGAGGCATCAAGCGTAGTGGTTTTGGACGTGAGCTTGGGGA 6S%$'+ TAGAAGTTGGAGCTGTTTGGGTTAATTGCTCACAACCATGCTTTGTTCAAGCTCCTTGGGGAGGCATCAAGCGTAGTGGTTTTGGACGTGAACTTGGAGA &RQVHQVXV TAGAAGTTGGAGCTGTTTGGGTTAATTGCTCACAACCATGCTTTGTTCATGCTCCATGGGGAGGCATCAAGCGTAGTGGTTTTGGACGTGAGCTTGGGGA

105

Fig. 3-2. Continued 1501 1510 1520 1530 1540 1550 1560 1570 1580 1590 1600 $K%$'+ ATGGGGTATCGAGAATTACTTGAATATCAAGCAGGTGACGAGTGATATCTCTGATGAACCATGGGGATGGTACAAGTCTCCTTGAA------EDUOH\%%' AGGGGGCATTGATAACTACCTGAGCATCAAGCAGGTGACGGAGTACACATCTGATGCGCCGTGGGGATGGTACAAGGCTCCGGCTAACTAAGATGCAATT EHHW%$'+' ATGGGGTATCGAGAATTACTTGAACATCAAGCAGGTCACGTCGGATATTTCTAACGAACCATGGGGATGGTACAAGTCTCCTTAAAGCTACTCTCCTATA 6S%$'+ ATGGGGTATCCAGAATTACTTGAATATCAAGCAGGTGACTCAAGATATTTCTGATGAACCATGGGGATGGTACAAGTCTCCTTGAAGCTATGATCAAATT &RQVHQVXV ATGGGGTATCGAGAATTACTTGAATATCAAGCAGGTGACG AGGATATTTCTGATGAACCATGGGGATGGTACAAGTCTCCTTGAAGCTA GATCCAATT GB5 GB3

1601 1610 1620 1630 1640 1650 1660 1670 1680 1690 1700 $K%$'+ ------EDUOH\%%' GAGCTGTTTCGAGGACCTGTATCTGTTCCATCAGGATAAATCTGTGTGCCGGCCTTTGTAGTGGTGAGAATAAATGTAATTTGCCATGGACTACTACTGG EHHW%$'+' AGAAACTTTGCACGAA-TGCTGCCGTTGTAAAGTGAACAG----TGGA---TAAGTTAA---GGATC-CAGTTTAGTTCTTG------AATATATGTG- 6S%$'+ TGAA----TG-ACGG--TGTTGTTTTTGTTAAGTGAGCAGCGGTTGGACTGTACCTTGAAATGGTTCGCAGAGAAGGTCGAGTTTAC--AGTAAAAATGG &RQVHQVXV GAA TTTG ACGA TGTTGCTGTTGTAAAGTGA CAG TGGAC GTAC TTGAA TGGTTCGCAGA AAGTTCTTG A A TA AA TGG

1701 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800 $K%$'+ ------EDUOH\%%' ACCTTTCTTGCAAAATAAATGGCATG--GGTTTTGTGGACATAGGGTG-TGTGACAAGGATTTATTTTGCAAAATTAAGGTGTTTGGAATATCTCCAACA EHHW%$'+' A------TTGAATAAAGG-TGGTTTGATGTTGGAGTAGGATTAGCAAAATGATTCAGACAATTGAGTTTGACCATGAAATCCT-TGTTTTTCTTCTAAAA 6S%$'+ A------TTGAATAAAGGGTTGGTTGATGCAGAAGTCCAACAAGCATA--GCTT-----AATTTTGTTGTATCATGTAATAGTGTGTATTATTTCAGACA &RQVHQVXV A TTGAATAAAGG TGG TTGATG TG AGT GAA TAGCATA TG TTCA AATT TGTTG A CATGAAAT GT TGTATTATTTC AACA

1801 1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 $K%$'+ ------EDUOH\%%' ATTCCCCAGTCTATATCAATGCTGTCTATATCGATGATGTGAATGTTATCATCGGCTACGCAAATTGAGGATGTAAAGGAAGCTAGGTCTTACCTTAAAG EHHW%$'+' AAAAA--AA------6S%$'+ ATTGA--GTTGGATCAGAAATAAGATGATACAGAGTTTGACAATG------&RQVHQVXV ATT A A T AT AA G ATA GA TG AATG

19011910 1925 $K%$'+ ------EDUOH\%%' ACGAGTTTGAGATAAAAAAAAAAAA EHHW%$'+' ------6S%$'+ ------&RQVHQVXV

106 107 consensus regions to amplify a partial BADH from Atriplex prostrata (see Table 3-1 for the sequence and Tm of these primers and Figs. 3-1 and 3-2 for their positions).

GB4 and GB5 together amplified a fragment of 1206 bp (ApBADH-1.2), and this fragment, after sequencing, is homologous to reported BADHs based upon a BLAST search and subsequent sequence alignments. Two additional primers, GB6 and GB7 which are rich in GC content, were then designed based on the sequence of

ApBADH-1.2 to perform 3’- and 5’-RACE respectively (Table 3-1). Fig. 3-1 presents the position of each primer and the resulting PCR fragments in the cloned

Atriplex BADH.

3’-RACE, using BADH6 and AP1, amplified a poly-A-tail-containing 381 bp fragment that contains a 60 bp perfect match with the 3’ end of ApBADH-1.2. After subcloning, several clones, designated as ApBADH-3’-12, -16, and -18, were recovered as a result of multiple polyadenylation sites (Fig. 3-3). In fact, there were at least 2 AATAAA-like motifs that are possible polyadenylation signals. One is

AAATAA located 24 nts upstream of poly A site. 2 of the 3 3’-RACE products,

ApBADH-3’-12 and -18, may be the result of this polyadenylation signal. BADH-3’-

16 has a ~40 nts shorter 3’ end because of a further upstream polyadenylation signal

AATAAA (~70 nts upstream of its own poly A site; Fig. 3-3). As far as FUE is concerned, there might be two GT-rich regions that separately control these two

AATAAA-like motifs (Fig. 3-3). Given that ApBADH-3’-12 and -18 were longer

108

Fig. 3-3. An alignment of three 3’ ends of ApBADH. BADH-3’-12, -16 and -18

are three of the clones obtained from PCR cloning of 3’-RACE

products. Nucleotides conserved among all three clones are shaded in

yellow, and those conserved among two of the three clones are shaded

in blue. The putative polyadenylation signals are dotted underlined.

The G/T-rich regions that functions as “far-upstream element” (Hunt,

1994) needed for 3’ end formation are boxed.

109

1 10 20 30 40 50 64 %$'+ CCATGGGGAGGAAGGTGAAGGGTAAGTGAAATTTGGGACGTAAAATTGGGGAATGGGGTATCGG %$'+ CCATGGG-AGGA--GTGAAGCGTA-GTGGATTT--GGACGTGAACTTGGGGAATGGGGTATCG- %$'+ CCATGGGGAGGA--GTGAAGCGTA-GTGGATTT--GGACGTGAACTTGGGGAATGGGGTATCG- &RQVHQVXV CCATGGGGAGGA GTGAAGCGTA GTGGATTT GGACGTGAACTTGGGGAATGGGGTATCG

65 70 80 90 100 110 128 %$'+ AAAATTACTTGAATATCAAAGACAGGTGACTAGCGGATATTTCTTGATGAACCATGGGGCTGGT %$'+ AGAATTACTTGAATATCAA--GCAGGTGACTAGCG-ATATTTCT-GATGAACCATGGGGCTGGT %$'+ AGAATTACTTGAATATCAA--GCAGGTGACTAGCG-ATATTTCT-GATGAACCATGGGGCTGGT &RQVHQVXV AGAATTACTTGAATATCAA GCAGGTGACTAGCG ATATTTCT GATGAACCATGGGGCTGGT

129 140 150 160 170 180 192 %$'+ ACAAGTCTCCTTAAAGCTGTGAGGAATTTGAACACCAGCATTATTGTAAAGTGAAATATGGTGA %$'+ TCAAGTCTCCTTAGAGCTGTGAGGAATTTGAACACCAGCATTATTGTAAAGTGAAATATGGTGA %$'+ ACAAGTCTCCTTAGAGCTGTGAGGAATTTGAACACCAGCATTATTGTAAAGTGAAATATGGTGA &RQVHQVXV ACAAGTCTCCTTAGAGCTGTGAGGAATTTGAACACCAGCATTATTGTAAAGTGAAATATGGTGA

193 200 210 220 230 240 256 %$'+ GACTGGACCTTGAAATGATTCACAAAGGTTTAGTTTAAAGTATGAATGGATTGAATAAAGGTTG %$'+ GACTGGACCTTGAAATGATTCACAAAGGTCTAGTTTAAAGTATGAATGGATTGAATAAAGGTTG %$'+ GACTGGACCTTGAAATGATTCACAAAGGTCTAGTTTAAAGTATGAATGGATTGAATAAAGGTTG &RQVHQVXV GACTGGACCTTGAAATGATTCACAAAGGTCTAGTTTAAAGTATGAATGGATTGAATAAAGGTTG

257 270 280 290 300 310 320 %$'+ TTTGATGCTGGAATTCGCTTAGCAAAGCTTAATTTTGTTGTGTGTATTATTACTTCAGACAATT %$'+ TTTGATGCTGGAATTCGCTTAGCAAAGCTTAATTTTGTTGTGTGTATTATTACTTCAGACGAAA %$'+ TTTGATGCTGGAATTCGCTTAGCAAAGCTTAATTTTGTTGTGTGTATTATTACTTCAGACAATT &RQVHQVXV TTTGATGCTGGAATTCGCTTAGCAAAGCTTAATTTTGTTGTGTGTATTATTACTTCAGACAATT

321 330 340 350 360 370 384 %$'+ GAATCGGATTAGAAATAAAATGACTACGTAGTTTGTCTTTCCAAAAAAAAAAAAAAAAAAAAAA %$'+ AAAAAAAAAAAAAAAAAAAAAAAAAA------%$'+ GAATCGGATTAGAAATAAGATG-CTACGTAGTTTGTCTTTTCAAAAAAAAAAAAAAAAAAAAAA &RQVHQVXV GAATCGGATTAGAAATAAAATGACTACGTAGTTTGTCTTT CAAAAAAAAAAAAAAAAAAAAAA

110 than ApBADH-3’-16 and ApBADH-3’-18 and -16 are almost identical in sequence,

ApBADH-3’-18 was selected to be the 3’ end of Atriplex BADH. The sequence ofAtriplex BADH around GB3 was thus elucidated, and there are two mispaired bases (Table 3-2).

After 5’-RACE failed to amplify the 5’ end of the BADH mRNA, GB1 was designed based on the sequence of the A. hortensis and spinach BADH cDNA corresponding to the region around the start codon. In order to successfully amplify the 5’ end, GB5AS was also synthesized to pair with GB1. These two primers generated a 338 bp fragment (ApBADH-5’, Fig. 3-1) and, according to the later sequence result, it overlaps with the ApBADH-1.2, and it is homologous to the 5’ end of A. hortensis and spinach BADH cDNA. Therefore, the cloning of Atriplex

BADH was completed.

Based on the overlapping clones, ApBADH-5’, ApBADH-1.2 and ApBADH-

3’-18, a composite cDNA encoding Atriplex prostrata BADH was assembled and designated as ApBADH1 (abbreviated as Ap1 in alignments). ApBADH1 is 1755 bp in length and encodes a 500 amino acid polypeptide (Fig. 3-4). This cDNA has a 3’- untranslated region of 250 bp. With this PCR cloning method, the 5’-untranslated region remains unclear.

In order to examine the localization of this cloned BADH according to its sequence, the deduced amino acid sequence was submitted to the web site of PSORT

(Prediction of Protein Localization Sites, http://psort.nibb.ac.jp). PSORT predicted that this BADH is most likely localized in peroxisome (certainty = 0.540), with a

111

Table 3-2. A comparison of the sequences of the actual clone and the primers. The

different nucleotides between the actual clone and primer appear in bold.

Primers Sequence Comparison Similarity (%)

TATTTGCGTGCTATTGCTGC (primer) 100 GB4 TATTTGCGTGCTATTGCTGC

CAAGGAGACTTGTACCATCCC (primer) 90.5 GB3 TAAGGAGACTTGTACCAGCCC

ATTCTCGATACCCCATTCCC (primer) GB5 100 ATTCTCGATACCCCATTCCC

112

Fig. 3-4. Nucleotide and deduced amino acid sequence of ApBADH1. The active

sites of aldehyde dehydrogenase are underlined. The dotted underline

represents the NAD binding motif (Johansson et al., 1998), in which the

conserved residues are marked with daggers. The proposed substrate-

binding site is marked with an asterisk. The start and stop codons are

boxed. The polyadenylation site for the ApBADH-3’-18 is underlined

twice. The sequence is deposited in GeneBank (accession number:

AY083902).

113

1 AGAATGGCGTTCCCAATTCCTGTTCGTCAACTCTTCATCGATGGGGAGTGGAGAGAACCC 1 M A F P I P V R Q L F I D G E W R E P 61 CTTTTAAAAAATCGCATTCCCATCATCAACCCTTCTACTGAAGAAATCATCGGTGATATT 20 L L K N R I P I I N P S T E E I I G D I 121 CCTGCTGCAACTGCAGAGGATGTAGAGGTTGCAGTAGTAGCAGCTAGAAAAGCATTTAAG 40 P A A T A E D V E V A V V A A R K A F K 181 AGGAACAAAGGTAGAGATTGGGCTGCAACTTCTGGTGCTCATCGTGCTAAATATTTGCGT 60 R N K G R D W A A T S G A H R A K Y L R 241 GCTATTGCTGCTAAGATAACAGAAAGAAAAGATCATTTCGTTAAACTGGAAACCCTTGAT 80 A I A A K I T E R K D H F V K L E T L D 301 TCTGGAAAACCATTCGATGAAGCCGTGTTGGACATTGATGATGTTGCTTCATGCTTTGAA 100 S G K P F D E A V L D I D D V A S C F E 361 TACTTTGCTGGTCAAGCAGAAGCTCTGGATGCTAAACAAAAGGCTCCAGTCACCCTGCCT 120 Y F A G Q A E A L D A K Q K A P V T L P 421 ATGGACAGATTTAAAAGTCATGTTCTCAGGCAGCCCATTGGTGTTGTTGGATTAATATCC 140 M D R F* K S H V L R Q P I G V V G L I S 481 CCATGGAATTATCCACTTCTAATGGCTACATGGAAAATTGCTCCCGCACTTGCTGCTGGA 160 P W N Y P L L M A T W K I A P A L A A G 541 TGCACAGCTGTACTTAAACCATCAGAATTGGCATCTGTGACTTGTCTAGAATTTGGTGAA 180 C T A V L K P S E L A S V T C L E F G E 601 GTGTGTAACGAAGTGGGACTTCCTCCAGGTGTGTTGAATATTTTGACAGGATTAGGTCCT 200 V C N E V G L P P G V L N I L T G L G P 661 GATGCTGGTGCCCCAATAGTATCTCATCCTGATATTGATAAGATTGCATTTACTGGGAGT 220 D A G A P I V S H P D I D K I A F T G S† 721 AGTGCCACTGGAAGCAAGATTATGGCTTCTGCTGCCCAACTAGTTAAGCCTGTTACGTTA 240 S A T† G S K I M A S A A Q L V K P V T L 781 GAGCTTGGAGGTAAAAGTCCTGTTATCATGTTCGAAGATATTGATATTGAAACAGCTGTT 260 E L G G K S P V I M F E D I D I E T A V 841 GAATGGACTCTTTTTGGCGTTTTCTGGACAAATGGTCAAATCTGTAGTGCAACATCTAGA 280 E W T L F G V F W T N G Q I C S A T S R 901 CTGCTTTTGCATGAAAGTATTGCAGCTGAATTTGTTGATAGGATGGTAAAGTGGACAAAA 300 L L L H E S I A A E F V D R M V K W T K 961 AACATAAAAATTTCTGACCCGTTTGAAGAAGGATGCCGACTTGGTCCTGTTATCAGTAAA 320 N I K I S D P F E E G C R L G P V I S K 1021 GGACAGTATGACAAGATTATGAAGTTCATATCAACAGCAAAGAGTGAAGGGGCTACAATT 340 G Q Y D K I M K F I S T A K S E G A T I 1081 TTGTGTGGAGGCTCCCGCCCTGAGCATTTGAAGAAAGGGTATTATATTGAACCCACCATT 360 L C G G S R P E H L K K G Y Y I E P T I 1141 ATAACTGATATTACCACATCCATGCAAATATGGAAAGAGGAAGTGTTTGGCCCTGTCATA 380 I T D I T T S M Q I W K E E V F G P V I 1201 TGTGTTAAAACATTTAAAACTGAAGATGAAGCCATTGAATTGGCAAATGATACAGAGTAT 400 C V K T F K T E D E A I E L A N D T E Y 1261 GGTTTAGCTGGTGCCGTGTTTTCTAAAGATCTTGAAAGATGTGAGAGGGTAACTAAGGCT 420 G L A G A V F S K D L E R C E R V T K A 1321 CTAGAAGTTGGAGCTGTTTGGGTTAATTGCTCACAACCATGCTTTGTTCATGCTCCATGG 440 L E V G A V W V N C S Q P C F V H A P W 1381 GGAGGAGTGAAGCGTAGTGGATTTGGACGTGAACTTGGGGAATGGGGTATCGAGAATTAC 460 G G V K R S G F G R E L G E W G I E N Y 1441 TTGAATATCAAGCAGGTGACTAGCGATATTTCTGATGAACCATGGGGCTGGTACAAGTCT 480 L N I K Q V T S D I S D E P W G W Y K S 1501 CCTTAGAGCTGTGAGGAATTTGAACACCAGCATTATTGTAAAGTGAAATATGGTGAGACT 500 P 1561 GGACCTTGAAATGATTCACAAAGGTCTAGTTTAAAGTATGAATGGATTGAATAAAGGTTG 1621 TTTGATGCTGGAATTCGCTTAGCAAAGCTTAATTTTGTTGTGTGTATTATTACTTCAGAC 1681 AATTGAATCGGATTAGAAATAAGATGCTACGTAGTTTGTCTTTTCAAAAAAAAAAAAAAA 1741 AAAAAAAAAAAAAAA

114 possible localization to the chloroplast stroma (certainty = 0.200) or chloroplast thylakoid membrane (certainty = 0.200).

According to InterProScan (http://www.ebi.ac.uk/interpro/scan.html),

ApBADH1 contains both active sites of aldehye dehydrogenase (ALDH): Cys and

Glu active sites (ID# in PROSITE: PS00070 and PS00687, respectively) (Fig. 3-4).

In fact, the registered active sites in PROSITE are [FYLVA]-X(3)-G-[QE]-X-C-

[LIVMGSTANC]-[AGCN]-X-[GSTADNEKR] (where X represents any amino acid, and letters in parentheses represents possible amino acids in this position) and

[LIVMFGA]- E-[LIMSTAC] - [GS] – G -[KNLM]-[SADN]-[TAPFV], respectively.

These active sites (underlined region in Fig. 3-4) are highly conserved among all reported BADHs (Fig. 3-5 C). ApBADH1 also contains another concensus sequence, a binding motif for NAD+ (dotted underlined region in Fig. 3-4). This motif is also conserved among all known BADHs (Fig. 3-5 B). The proposed substrate binding site, Phe143 (marked with an asterisk in Fig. 3-4; Hibino et al., 2001), was likewise conserved among all BADHs (Fig. 3-5 A).

When the amino acid sequence of ApBADH1 was aligned to that of spinach and sugar beet, it shared 90.8% and 89.0% identity, respectively (Table 3-3). The identity increased to 96.8% when BADH of A. hortensis was compared with

ApBADH1. The similarity between ApBADH2 and any known BADHs ranged from

75.2% (with sorghum BADH clone 15) to 98.0% (with A. hortensis BADH). A summary of the identities and similarities among available BADHs is shown in

Table 3-3.

115

Fig. 3-5. Multiple amino acid alignments of BADHs from available BADH clones

revealed conserved regions. A. Region around the proposed substrate-

binding site (Johansson et al., 1998; Hibino et al., 2001). B. Region

containing NAD+ binding site. C. Active sites of aldehyde dehydrogenase

(documented ID in PROSITE: PS00087 and PS00070) are shaded in blue

with conserved residues in red. D. C-terminal of the polypeptides.

116

A. 117 153 ApBADH1 CFEY...FAG QAEALDAKQK APVTLPMDRF KSHVLRQPIG SpBADH CFEY...FAG QAEALDGKQK APVTLPMERF KSHVLRQPLG AhBADH CFEYFEYFAG QAEALDAKQK APVTLPMERF KSHVLRQPIG AvmBADH2 CFEY...FAG IAERLDSEQR TPVSLPMETF KCHLLKEPIG AvmBADH13 CFEY...FAD LAERLDSNQ. IPVSLPMDTF KCHVLKEPIG barleyBBD1 CFEY...YAA LAEALDGKQH APISLPMEEF KTYVLKEPIG barleyBBD2 CFEF...FAG HAEALDKRQN AAVALPEN.F KCHLKKEPIG beetBADHAC CFEY...FAG QAEAMDAKQK APVTLPMERF KSHVLRQPIG beetBADHD CFEY...FAG QAEAMDAKQK APVTLPMERF KSHVLRQPIG riceBADH CFEY...YAD LAEALDGKQR APISLPMENF ESYVLKEPIG sorghumBADH15 CFEY...YAD LAEALDGKQR SPISLPMENF KSYVLKEPLG suaedaBADH CFEY...FAD QAEALDNKQK YPVKLPMDRF KSHVLRQPIG AmaBADH CFEY...YAD QAEALDAKQK APIALPMDTF KCHVLKQPIG

B. 204 243 ApBADH1 VGLPPGVLNI LTGLGPDAGA PIVSHPDIDK IAFTGSSATG SpBADH VGLPPGVLNI LTGLGPDAGA PLVSHPDVDK IAFTGSSATG AhBADH VGLPPGVLNI LTGLGPDAGA PIVSHPDIDK VAFTGSSATG AvmBADH2 VGLPPGVLNI LTGLGPEAGA PLVTHPHVAK ISFTGSDTTG AvmBADH13 VGLPAGVLNI LTGLGPEAGA PLASHPHVDK ITFTGSGATG barleyBBD1 IGLPSGVLNI ITGLGPDAGA PIASHPHVDK IAFTGSTATG barleyBBD2 VGLPSGVLNI VTGLGNEAGA PLSSHPDVDK VAFTGSYATG beetBADHAC VGLPPGVLNI VTGLGPDAGA PLAAHPDVDK VAFTGSSATG beetBADHD VGLPPGVLNI VTGLGPDAGA PLAAHPDVDK VAFTGSSATG riceBADH IGLPPGVLNI ITGLGTEAGA PLASHPHVDK IAFTGSTETG sorghumBADH15 IGLPPGVFNV ITGLGLKLVL HYPHIPCGIR LLLLGSTETG suaedaBADH VGLPPGVLNI LTGLGPDAGA PLVSHPDVDK VAFTGSSATG AmaBADH VGLPPGVLNI LTGLGPEAGG PLACHPDVDK VAFTGSTATG

117

Fig. 3-5. Continued

C. 259 298 ApBADH1 LELGGKSPVIMFED.IDIETAVEWTLFGVFWTNGQICSATS SpBADH LELGGKSPIVVFED.VDIDKVVEWTIFGCFWTNGQICSATS AhBADH LELGGKSPVIMFED.IDIETAVEWTLFGVFWTNGQICSATS AvmBADH2 LELGGKSPIVVFED.VDLDTAAEWTLFGCFWTNGQICSATS AvmBADH13 LELGGKSPIVVFED.VDLDTAAEWTLFGCFWTNGQICSATS barleyBBD1 LELGGKSPLVIFDDVADIDKAVEWAMFGCFFNGGQVCSATS barleyBBD2 LELGGKSPIVVFDD.VDIDKAVEWTLFGCFWTNGQICSATS beetBADHAC LELGGKSPIIVFED.VDIDQVVEWTMFGCFWTNGQICSATS beetBADHD LELGGKSPIIVFED.VDIDQVVEWTMFGCFWTNGQICSATS riceBADH LELGGKSPLIVFDD.VDIDKAVEWAMFGCFANAGQVCSATS sorghumBADH15 LELGGKSPLIVFDDIRDIDKAVEWTMFGILPNAGQVCSATS SuaedaBADH LELGGKSPIIVFEDVVDLDVAAEWTIFGVFWTNGQICSATS AmaBADH LELGGKSPIVIFED.VDLDKAAEWTAFGCFWTNGQICSATS -PS00087- ---PS00070---

D. 463 500 ApBADH1 KRSGFGRELGEWGIENYLNIKQVTSDISDEPWGWYKSP... SpBADH KRSGFGRELGEWGIQNYLNIKQVTQDISDEPWGWYKSP... AhBADH KRSGFGRELGEWGIENYLNIKQVTSDISDEPWGWYKSP... AvmBADH2 KRSGFGRELGERGLDIYLNVKQVTQYVSSEPWDWYKSP... AvmBADH13 KRSGFGRDLGEWGLDNYLNVKQVTRYVSSEPWDWYKSPSKL barleyBBD1 KRSGFGRELGEWGLENYLSVKQVTRYCKDELYGWYQRPSKL barleyBBD2 KRSGFGRELGEGGIDNYLSIKQVTEYTSDAPWGWYKAPAN. beetBADHAC KRSGFGRELGEWGIENYLNIKQVTSDISNEPWGWYKSP... beetBADHD KRSGFGRELGEWGIENYLNIKQVTSDISNEPWGWYKSP... riceBADH KRSGFGRELGQWGLDNYLSVKQVTKYCSDEPYGWYRPPSKL sorghumBADH15 KR.MFGRELGEWGLDNYMTVKQVTKYCSDEPWGWYQPPSKL SuaedaBADH KRSGFGRELGEWGIENYLNIKQVTSDISDEPWGWYKSP... AmaBADH KRSGFGRELGEWGIENYLNIKQVTEYISDEPWGWYKSP...

Table 3-3. Identity and similarity of amino acid sequence (left- and right-hand side of the diagonal matrix) among ApBADH and

other available BADHsa.

Ap1 Ap2 Ah Sp BBD1 BBD2 BVd BVac Avm13 Avm2 Am Su So15 Rice Ap1 -- 94.6 98.0 96.6 82.4 81.5 97.0 97.0 84.1 86.3 91.8 96.4 75.2 83.0 Ap2 84.7 -- 93.2 93.9 84.3 83.6 94.6 94.6 86.7 87.5 95.5 94.1 78.6 85.9 Ah 96.8 83.4 -- 94.8 80.7 80.7 95.4 95.4 82.6 84.8 90.7 85.0 74.4 81.7 Sp 90.8 84.7 89.2 -- 81.8 80.6 96.0 96.0 83.1 84.9 91.0 94.4 75.3 82.4 BBD1 71.5 70.1 69.9 70.9 -- 81.8 81.4 81.4 80.2 78.5 81.6 80.0 79.3 90.1 BBD2 69.6 72.1 70.6 70.8 70.4 -- 81.9 81.9 80.4 80.8 94.3 92.2 74.6 84.0 BVd 89.0 84.7 87.7 89.6 69.2 71.0 -- 100.0 84.3 86.1 92.6 96.2 74.1 82.0 BVac 89.2 84.9 87.9 89.6 69.4 71.2 99.8 -- 84.3 86.1 92.6 96.2 74.1 82.0

Avm13 74.0 77.5 71.9 74.0 69.6 69.8 72.0 72.2 -- 89.5 88.3 84.7 72.7 81.6 Avm2 74.6 76.4 83.1 75.1 66.7 69.7 72.6 72.4 84.0 -- 87.3 86.3 72.2 80.4 Am 82.6 89.9 81.7 83.4 70.9 72.8 82.8 83.0 79.1 77.1 -- 92.2 76.0 83.6 Su 89.2 85.4 88.1 88.6 67.8 83.6 88.6 88.8 76.2 75.4 83.6 -- 74.5 81.8 So15 62.2 64.6 61.5 52.4 71.2 61.7 61.0 61.2 60.0 59.8 62.4 60.0 -- 82.7 Rice 70.1 71.2 68.9 70.5 85.2 72.3 68.7 68.9 70.7 68.6 70.7 68.6 76.2 --

118

a The percentage of identity shown is by alignments of each two sequences performed by AlignX®. The accession number of Ah

(A. hortensis) is: CAA49425. The accession number of Sp (Spinacia oleracea) is A35994. The accession number of BVac and

BVd (Beta vulgaris) are CAA41377 and CAA41376 respectively. The accession number of BBD1 and BBD2 (Hordeum

vulgare) are BAB62847 and BAB62846 respectively. The accession number of Am (Amaranthus tricolor) is: AAB70010. The

accession number of Avm13 and Avm2 (Avicennia marina) are BAB18544 and BAB18543 respectively. The accession

number of So15 (Sorghum bicolor) is: T14729. The accession number of Su (Suaeda liaotungensis) is: AAL33906. The

accession number of rice (Oryza sativa) is: BAA21098.

119 120

During cloning of BADH from A. prostrata, GB4 and GB3 amplified a 1274 bp fragment, ApBADH-1.3, which is similar but not identical to ApBADH-1.2 (Fig.

3-1). In fact, when ApBADH-1.3 was aligned to ApBADH-1.2, they shared 84.9% identity at nucleotide level and 84.7% identity at amino acid level (Table 3-4).

ApBADH-1.3 and ApBADH-1.2 were highly similar since the different amino acids were mostly substitutions from the group of amino acids sharing similar properties

(Fig. 3-6). Therefore, ApBADH-1.3 was designated as a partial cDNA clone of a second BADH clone, ApBADH2 (abbreviated as Ap2 in alignments).

BADH Expression in Atriplex prostrata

In order to understand how salinity affects BADH expression, A. prostrata was treated with 1% NaCl (= 170 mM) and 2% NaCl (= 340 mM) in addition to the control media for 3 days. Total RNA was then isolated from young stems (the first internode from the top of the plant) and young leaves (fully expanded leaves from the top of the plant) to perform northern blotting. When the membrane was probed with ApBADH-1.2, a 1.9 kb transcript was detected (Fig. 3-7). In young stems, this transcript increased 2-fold when plants were treated with 1% NaCl and increased 3- fold when plants were treated with 2% NaCl. In young leaves, there was little expression of BADH when the plants were not treated with salt. When treated with

1% NaCl, BADH expression was dramatically induced, whereas with 2% NaCl, the induction was further enhanced to 3 times more than what was observed in young

Table 3-4. Identity of nucleotide and amino acid sequence between reported two BADHs from the same species.

b Nucleotide Identity Amino Acid Identity Species Accession Numbers (%) (%)

84.9 84.7 (94.6) AY083902 & AY083903a Atriplex prostrata

94.7 99.8 (100) X58462 & X58463 Beta vulgaris

66.2 70.4 (81.8) AB063178 & AB063179 Hordeum vulgare

79.8 84.0 (89.5) AB043539 & AB043540 Avicennia marina

69.4 61.0 (69.2) U12195a & U12196 Sorghum bicolor

a Partial cDNA clones b Similarities are shown in parentheses.

121 122

Fig. 3-6. Examples of amino acid substitutions between ApBADH1 and ApBADH2.

The amino acid substitutions between the two clones of ApBADH appear in

bold. Among these substitutions, those corresponding to the substitutions

between Avicennia marina and spinach are underlinedn in all five.

123

107 143 ApBADH1 AVLDIDDVASCFEYFAGQAEALDAKQKAPVTLPMDRF ApBADH2 ALLDIDDVAGCFEYYADQAEALDAKQKAPIALPMDTF AvmBADH2 ASLDMDDVIGCFEYFAGIAERLDSEQRTPVSLPMETF AvmBADH13 AAWDMDDVAGCFEYFADLAERLDSNQ.IPVSLPMDTF spBADH AVLDIDDVASCFEYFAGQAEALDGKQKAPVTLPMERF

197 238 ApBADH1 FGEVCNEVGLPPGVLNILTGLGPDAGAPIVSHPDIDKIAFTG ApBADH2 LADVCKEVGLPPGVLNILSGYGPEAGGPLASHPDVDKVAFTG AvmBADH2 LAEVCMEVGLPPGVLNILTGLGPEAGAPLVTHPHVAKISFTG AvmBADH13 LAQVCKEVGLPAGVLNILTGLGPEAGAPLASHPHVDKITFTG SpBADH FGEVCNEVGLPPGVLNILTGLGPDAGAPLVSHPDVDKIAFTG

259 298 ApBADH1 LELGGKSPVIMFEDIDIETAVEWTLFGVFWTNGQICSATS ApBADH2 LELGGKSPIILFEDVDLDQAAEWAAFGCFWTNGQICSATS AvmBADH2 LELGGKSPIVVFEDVDLDTAAEWTLFGCFWTNGQICSATS AvmBADH13 LELGGKSPIVVFEDVDLDTAAEWTLFGCFWTNGQICSATS SpBADH LELGGKSPIVVFEDVDIDKVVEWTIFGCFWTNGQICSATS

303 342 ApBADH1 HESIAAEFVDRMVKWTKNIKISDPFEEGCRLGPVISKGQY ApBADH2 HESIAAEYLDKLVKWCKNIKISDPFEDGCRLGPVVSKGQY AvmBADH13 HESIATTFLEKLVKWCEKIKISDPLEEGCRLGPIISRGQY AvmBADH2 HESIAATFLEKLVKWCEKIKISDPLEEGCRLGPIVSRRQY SpBADH HESIAAEFVDKLVKWTKNIKISDPFEEGCRLGPVISKGQY

124

Fig. 3-7. BADH expression in Atriplex prostrata is induced by NaCl. In both

young stems (YS) and leaves (YL), BADH transcript accumulated in

response to 1% (= 170 mM) and 2% (= 340 mM) NaCl after 3 days. 20

µg of total RNA was loaded in each lane and the membrane was probed

with ApBADH-1.2. Relative amounts of BADH transcript were compared

to 0% YS and standardized with respect to the amount of rRNA in each

lane.

125

126 stems without NaCl treatment, after the transcript levels were standardized with respect to rRNA stained by ethidium bromide (Fig. 3-7).

NaCl-induced BADH expression was detected and peaked within 24 hours and gradually declined thereafter in young stems (Fig. 3-8). A certain amount of BADH transcript was detected at corresponding time points in the control. Yet the induction by salt at different time points was still substantial. In order to examine whether

BADH gene expression in various organs differentially responds to NaCl, total

RNAs were isolated from roots as well as old and young stems and leaves after entire plants were treated with 340 mM NaCl for 3 days. Without salt treatment, BADH transcripts were not detected in old leaves or in roots (Fig. 3-9). When plants were treated with 340 mM NaCl, BADH mRNA was detected in all organs examined except in old leaves. A densitometric analysis of northern blots was included to obtain a more accurate estimate of changes in ApBADH1 transcript levels. A 5-fold increase after salt treatment was detected in young stems whereas in young leaves the increase was approximately 3-fold. In roots, the increase was moderate with a level similar to that detected in control young stems. In old stems, salt-induction was increased to a limited extent.

Not only was BADH differentially expressed in various organs, it was also differentially expressed in seedlings of different ages. BADH transcript was detected in all seedlings examined, and the accumulation of the transcript gradually increased and peaked at 5 days after germination (Fig. 3-10). Ten days after germination,

127

Fig. 3-8. Time course of NaCl-induced BADH expression in Atriplex prostrata. In

young stems treated with either control (0%) or 2% (= 340 mM) NaCl for

various times, 15 µg of total RNA was loaded in each lane and the

membrane was probed with ApBADH-1.2. Relative amounts of BADH

transcript were compared to 0%-24h and standardized with respect to the

amount of rRNA (EtBr/rRNA) in each lane.

128

129

Fig. 3-9. BADH expression in various organs of Atriplex prostrata treated with

340 mM NaCl for 3 days. Here young stems (YS) are the first internode

and young leaves (YL) are fully expanded leaves from the top of the plant.

Old leaves (OL) are green leaves close to the base of the plants, and old

stems (OS) are the second internode from the base of the plants. Roots (R)

are mostly lateral roots. 15 µg of total RNA was loaded in each lane and

the membrane was probed with ApBADH-1.2. Relative amounts of

BADH transcript were compared to 0% YS and standardized with respect

to the amount of rRNA (EtBr/rRNA) in each lane.

130

131

Fig. 3-10. BADH expression in 1-, 3-, 5-, 10-, and 15-day old seedlings of

Atriplex prostrata. 15 µg of total RNA isolated from seedlings 1, 3, 5,

10, and 15 days after germination was loaded in each lane and the

membrane was probed with ApBADH-1.2. Relative amounts of

BADH transcript were compared to 2% YL and standardized with

respect to the amount of rRNA (EtBr) in each lane.

132

133

BADH transcript declined to and returned to a basal level (i.e., the level detected in

1-day-old seedlings). The maximum level of BADH transcript detected in 5-day-old seedlings is approximately 3.5 fold more than that of 2% YL.

Effects of Exogenous ABA on BADH Expression

Besides participating in various developmental and physiological processes including stomatal closure and root growth, abscisic acid (ABA) may initiate global physiological reactions in response to osmotic stress (reviewed by Zhu et al., 1997).

Therefore, it is of interest to investigate whether ABA is able to transduce signals related to salt in Atriplex protrata and to induce the expression of BADH. Unlike the expression of CMO, ABA alone mimicked the effect of salt and induced the expression of BADH (Fig. 3-11). In fact, the level of BADH mRNA with ABA treatment was about 60% (in Fig. 3-11A) or 100% (in Fig. 3-11B) of what observed in 2% NaCl-treated young stems. However, Lovastatin, a general isoprenoid biosynthesis inhibitor used to block ABA accumulation (Thomas et al., 1992), did not inhibit the NaCl-induced BADH expression (Fig. 3-11).

DISCUSSION

Using RT-PCR and RACE, cDNAs encoding BADH from Atriplex prostrata were cloned and characterized. A composite cDNA, ApBADH1, was assembled from

ApBADH-5’, ApBADH-1.2, and ApBADH-3’, three overlapping RT-PCR clones

134

Fig. 3-11. The role of ABA in BADH expression of Atriplex prostrata. A. Total

RNA (15 µg) isolated from young stems (YS) was loaded in each lane

and the membrane was probed with ApBADH-1.2. B. A northern blot

with total RNAs of young stems isolated from a second set of plants. This

membrane was probed with ApBADH-7 (Fig. 3-1) instead of ApBADH-

1.2. For both panels, EtBr RNA is the original RNA gel stained with

ethidium bromide to verify the amount of RNA loaded in each lane. ABA:

10 µM abscisic acid added to the media. Lov: 10 µM Lovastatin added to

the media. X: no ABA nor Lovastatin added. Plants were grown

hydroponically in nutrient solutions containing one of these treatments for

2 days before being transferred to either control or 2% NaCl-containing

media.

135

136

(Fig. 3-1). These overlapping regions match perfectly except at the primer region

(Table 3-2). Another partial cDNA clone of BADH, ApBADH-1.3, was amplified by

RT-PCR. ApBADH-1.3 shared 84.9% nucleotide identity and 84.7% amino acid identity with ApBADH-1.2 (Table 3-4). From sequences deposited in GeneBank, there are 4 other examples where more than one BADH clone was reported. The nucleotide identity between any two clones varies from 66.2 to 94.7%, whereas the deduced amino acid sequence identity varies from 61.0 to 99.8% (Table 3-4). Other than the two BADH clones from sugar beet, which are highly identical, the similarity between two ApBADH clones are highest, with the percentage of identity being similar to that of two BADH clones from Avicennia marina.

Although most amino acid substitutions between two ApBADH clones were similar, there were substitutions around the substrate and coenzyme binding sites

(Johansson et al., 1998), as well as active sites of aldehyde dehydrogenase, that might be critical for enzyme specificity and activity (underlined in Fig. 3-6). The exact consequence of these substitutions remains to be elucidated. In Avicennia marina, both BADHs exhibited unique substrate specificity (Hibino et al., 2001).

The amino acids around the proposed substrate binding site may be responsible for this specificity (Fig. 3-5 A and underlined in Fig. 3-6); however, this remains to be clarified by approaches such as site-directed mutagenesis. There are two amino acid substitutions between two ApBADHs, Arg/Thr142 and Ser/Gly116 that may provide clues for specific sites to study (Fig. 3-6).

137

In addition, BADHs from monocots contain a peroxisomal targeting sequence at their C-terminus (Fig. 3-5 D), indicating a possible alternative localization for glycine betaine biosynthesis. This reinforced the idea that the choline-oxidizing enzyme in these plants may not be CMO, as CMO could not function in peroxisomes where there is no reduced ferredoxin (McNeil et al., 1999). In fact, CMO has not been cloned from any of these plants.

In Atriplex prostrata, the localization of BADH is not completely clear.

PSORT predicted ApBADH2 to be either peroxisomal or chloroplastic, due to the fact that BADH apparently lacks typical transit peptide. In spinach, it was experimentally demonstrated that BADH is localized to the chloroplast and it contains an unusually short transit peptide (Weretilnyk and Hanson, 1990). Based on multiple sequence alignments, ApBADH1 is highly similar to spinach BADH not only at the N-terminus but also throughout the entire polypeptide. Therefore it is most likely localized to the chloroplast. On the other hand, the partial BADH clone,

ApBADH2, shares higher identity and similarity with other peroxisomal BADHs than ApBADH1 does (Table 3-3); moreover, among those amino acid substitutions between two ApBADHs, ApBADH2 shares more identical amino acids with peroxisomal BADH from Avicennia marina (i.e., amino acids 127, 209, 233 in Fig.

3-6). Based on the above reasons, ApBADH2 may be the peroxisomal counterpart of

ApBADH1. This could be further confirmed if the 3’ end of ApBADH2 is cloned by another 3’-RACE.

138

Many observations have indicated that osmotic stress due to salt, drought and water treatments, leads to an increase in endogenous levels of ABA, and thus induces expression of osmotic stress responsive genes (Ishitani et al., 1997). These genes can also be directly induced by exogenous ABA application. Accumulation of BADH message was induced by exogenous ABA without NaCl although the level of induction varied (Fig. 3-11). This agrees with the report that BADH mRNA was dramatically induced by exogenously applied ABA at mRNA level in barley (Ishitani et al., 1995). However, chemically inhibited accumulation of ABA by Lovastatin did not block the NaCl-induced accumulation of BADH transcript (Fig. 3-11). This may simply be due to the possibility that Lovastatin did not penetrate into cells. In amaranth, the induction of BADH by ABA at both mRNA and protein levels was not as dramatic as seen in barley (Legaria et al., 1998). In terms of ABA-responsive elements (ABRE), which are specific nucleotide sequences (i.e. ACGTGGC) in the promoter region that induce ABA-responsive gene expression (Marcotte et al., 1989;

Thomas et al., 1992), only one from Arabidopsis, among all reported BADH genes, contains the sequence of ACGTGGC in the region upstream of start codon. In addition, ABRE is necessary but may not be sufficient for ABA-induction and other cis-elements are apparently required to interact with ABRE (Shen and Ho, 1997).

None of the reported cis-elements were found in any reported BADH genes.

Therefore it is not clear that ABA is involved in signal transduction pathway that leads to the BADH expression.

139

Unlike CMO which did not express in some tissues examined (Fig. 2-11),

BADH transcript in Atriplex prostrata was generally detected at a minimum level regardless of the treatments and tissue examined (Figs. 3-7~3-11). In various plants,

BADH protein was present even if betaine did not accumulate in response to salt

(Hibino et al., 2001). Meng et al. (2001) further demonstrated that BADH proteins were always detected in Amaranth with various treatments and the presence of

BADH protein was not parallel to the accumulation of glycine betaine. All the above investigations and the results regarding ApBADH expression presented here confirmed that the oxidizing step catalyzed by CMO is the rate-limiting step in biosynthesis of glycine betaine (Rathinasabapathi et al., 1997).

In conclusion, one complete and one partial cDNAs encoding for A. prostrata

BADH were cloned through RT-PCR and 3’-RACE. The expression of ApBADH1, which is the complete cDNA clone, was induced by NaCl in an organ-specific manner and was regulated by development.

140

CHAPTER 4. EFFECTS OF INTRASPECIFIC COMPETITION ON

GROWTH AND PHOTOSYNTHESIS OF ATRIPLEX PROSTRATA

Li-Wen Wang, Allan M. Showalter, and Irwin A. Ungar

Manuscript submitted to

International Journal of Plant Science

141

ABSTRACT

Growth of halophytes occurring in saline habitats may be affected by both salinity and competition. Intraspecific competition could cause a reduction in biomass of halophytes, due to the limitation of resources such as light, water and nutrients. In this study, the effect of intraspecific competition on growth parameters and photosynthesis of Atripelx prostrata was investigated in order to understand the nature of density-induced growth inhibition. High density (i.e., 16 plants per pot) caused a reduction in height as well as dry mass of stems, leaves, and roots. In addition, high density caused a pronounced reduction in leaf area and length of mature internodes. It also caused a decline in net photosynthetic rate. Indeed, the alteration of net photosynthesis paralleled growth inhibition, indicating the observed growth inhibition by intraspecific competition is mainly due to a decline in net photosynthesis. Light is obviously a limiting resource due to the fact that taller individuals overshadow shorter ones. In addition, plants grown at high density also exhibited reduced stomatal conductance and transpiration. Along with a significant reduction in fresh and dry mass of roots, this indicates that water is also a limiting resource. Therefore, we concluded that competition for both light as an aboveground resource and water as a belowground resource contributes to growth inhibition and morphological changes for Atriplex prostrata plant grown at high density.

142

INTRODUCTION

Growth and development of plants occurring in saline habitats may be determined by a combination of abiotic and biotic factors (Pennings and Callaway,

1992). Halophytes have relatively high light requirements, which could be limited by intraspecific or interspecific competition. Indeed, the growth of halophytes in saline habitats is reported to be affected by both salt stress and competition (Adam, 1990;

Ungar, 1991). In terms of interspecific competition, halophytes are unable to compete successfully with glycophytes in less saline habitats but are more competitive at higher salinities (Kenkel et al., 1991). Intraspecific competition is also known to cause a reduction in biomass production in halophytes from both field and laboratory investigations (Ellison, 1987; Rahman and Ungar, 1994).

Plant species that are salt tolerant vary considerably in their response to competition. The response of halophyte populations to increases in plant density may lead to an asymmetric frequency distribution of plants in which there are a few large individuals and numerous small plants (Weiner, 1986; Drake and Ungar, 1989) or to a symmetrical competitive response in which all individuals have an equal decline in biomass production (Ellison, 1989). In the case of asymmetric distribution, size variation among plants generally increases when there is competition for light because larger individuals may reduce light available to smaller individuals and thus suppress their growth (Weiner, 1990). Smaller individuals might be lost from a population due to high mortality, because halophytes have relatively high light

143 requirements. In the field, self-thinning in response to plants growing at high densities has been observed (reviewed by Westoby, 1984).

Intraspecific competition is reported to cause a reduction in biomass production in halophytes in both field and laboratory investigations (Ellison, 1987; Penning and

Callaway, 1992; Bertness and Yeh, 1994; Rahman and Ungar, 1994; van Klunen et al., 2001). Specifically, the growth of Atriplex prostrata is negatively affected by increased density within a natural population (Riehl and Ungar, 1983). Drake and

Ungar (1989) determined that increases in plant density under controlled laboratory conditions caused a reduction in shoot dry mass and reproductive dry mass of

Atriplex prostrata. Although much is known about the whole plant response to competition, little is known about the mechanisms responsible for such density/competition-induced growth inhibition.

To understand competitive mechanisms, it is important to consider that resource levels are spatially and temporally heterogeneous and that numerous physical and biotic processes limit availability of resources such as light, water and nutrients (Tilman, 1988; Tilman, 1990; Vargads-Mendoza and Fowler, 1998). In turn, the relative availability of aboveground and belowground resources will influence the rate of plant growth and the intensity of competitive relationships (Dyer and Rice,

1999). In the field, although the availability of the belowground resource water is also a function of the aboveground plant community, it is mostly dependent on seasonal precipitation (Dyer and Rice, 1999). In the laboratory, by replenishing water and nutrients regularly, investigators may simplify experiments.

144

To better understand the dynamics and consequences of intraspecific competition of A. prostrata, we used controlled laboratory conditions to address the following questions: (1) Which growth parameters of A. prostrata are density- dependent? (2) Does self-thinning happen when A. prostrata is growing at high density? (3) How are measurements of photosynthesis, stomatal conductance, and transpiration affected by plant density?

MATERIALS AND METHODS

Plant Material

Seeds collected from Atriplex prostrata plants growing in the salt marsh at

Rittman, Ohio in October, 1994 were spread out in four-inch white-sand-filled pots in plastic trays filled with water and germinated in an incubator under a 25ºC/5ºC, 12 h day/12 h night regime. Saran Wrap was used to cover the tray to prevent evaporation. Two weeks after germination, seedlings were thinned to desired densities (2, 4, 8, or 16 seedlings per pot). A total of 40 pots (10 pots per density treatment) were scattered in 4 plastic trays. These trays were filled with half- strength Hoagland and Arnon No.2 solution and maintained in an environmental growth chamber under a 25ºC/15ºC, 14 h day/10 h night regime (400-700nm, 400

µmol m-2 s-1). Distilled water was added daily to each tray to replace water lost because of evaporation and transpiration. Solutions were replaced weekly.

145

Growth Measurements

Following growth at the desired densities for 4 weeks, ten plants from each treatment were randomly chosen to measure the height, fresh weight of leaves, stems and roots, the length of the first, second, third, and fourth internodes. Area of leaves from the 1st, 2nd, and 3rd nodes was measured by a Licor portable area meter (Model:

LI-3000; LI-COR, Lincoln, NE). These ten plants were dried in a forced draft oven at 60ºC for three days to obtain their dry weight.

Photosynthesis

Following growth at the desired densities for 3 weeks, five plants from each treatment were randomly chosen to measure the photosynthetic rate (P), transpiration rate (T), and stomatal conductance with an ADC Infrared Gas Analyzer (Model:

LCA-3), and Water Use Efficiency (WUE) was calculated as P/T. Leaves from the second node were used for these measurements. The same measurements were taken again before harvest and reported.

Statistics

The results for growth and photosynthesis were analyzed with a one-way

ANOVA using the NCSS (version 2000) and/or SigmaStat (Version 2.03) statistical package. A log10 transformation was used to normalize data that were not normally distributed. If a significant difference was determined among means, a Bonferroni t-

146 test was used to determine significant differences between pairwise comparisons among individual treatments.

RESULTS

Growth parameters such as height, fresh mass and dry mass, were measured on these plants grown at different densities to examine the effect of intraspecific competition on growth. A one-way ANOVA indicated that Atriplex prostrata grown at lower densities (2 and 4 plants per pot) were significantly taller than those grown at higher densities (8 and 16 plants per pot) (F =16.99, P <0.0001) (Fig. 4-1).

However, density did not have such a pronounced effect with respect to length of upper internodes. There is no significant difference on the first and second internodes from the top of the plants at the time of harvest (F =1.30 and 0.61, P

=0.2892 and 0.6138, respectively). As for the more mature third and fourth internodes, internodes of plants grown at 4 plants per pot were significantly longer than those of plants grown at higher densities such as 8 and 16 plants per pot (F

=7.68 and 10.35, P =0.0004 and 0.00005, respectively; Table 4-1).

In terms of fresh mass, Atriplex prostrata grown at lower densities produced more leaf (F =23.74, P <0.0001), stem (F =18.38, P <0.0001), and root (F =16.76, P

<0.0001) biomass than those grown at higher densities (Table 4-2). Similarly,

Atriplex prostrata grown at lower densities produced greater stem dry mass (F =

16.89, P = <0.0001) (Table 4-3). A similar trend was observed for leaf dry

147

Fig. 4-1. Effect of density on height (cm) of Atriplex prostrata. Numbers plotted

here represent the average and standard error of data obtained from 10

randomly selected plants grown at designated densities. Different letters

above bars indicate that the two groups are statistically different

determined by Bonferroni t-test, after an overall significant difference

among groups was determined.

148

45 a 40 a

30 b b 25

20 Height (cm) 15

0 24816 Density ( plants/pot)

149

Table 4-1. Effect of density on internode length (cm) in Atriplex prostrata

2 plants/pot 4 plants/pot 8 plants/pot 16 plants/pot

1st internode 1.4 ± 0.21 a 1.0 ± 0.13 a 1.6 ± 0.34 a 1.1 ± 0.27 a

2nd internode 5.3 ± 0.51 a 4.3 ± 0.53 a 5.3 ± 0.76 a 5.2 ± 0.72 a

3rd internode 8.1 ± 0.52 a,b* 9.2 ± 0.40 b 6.2 ± 0.66 a 6.6 ± 0.37 a

4th internode 6.6 ± 0.77 a,b 8.7 ± 0.39 b 5.1 ± 0.63 a 4.7 ± 0.36 a

* Different letters shared by cells within the same row indicate that they are

statistically different as determined by the Bonferroni t-test, after a significant

difference was found overall among groups as determined by one-way ANOVA. In

this case, for the third internode, F= 7.68 and P= 0.0004, and for the fourth

internode, F= 10.35 and P= 0.00005.

150

Table 4-2. Effect of density on the fresh mass (gram/plant) of Atriplex prostrata

2 plants/pot 4 plants/pot 8 plants/pot 16 plants/pot

Stem 2.92 ± 0.54 a* 1.94 ± 0.23 a 0.79 ± 0.17 b 0.45 ± 0.05 b

Leaf 2.80 ± 0.42 a 1.90 ± 0.25 a 0.82 ± 0.19 b 0.41 ± 0.05 b

Root 3.27 ± 0.62 a 1.65 ± 0.29 a,b 0.87 ± 0.24 b,c 0.43 ± 0.14 c

* Different letters in cells within the same row indicate that they are statistically

different as determined by the Bonferroni t-test, after a significant difference was

found overall among groups as determined by one-way ANOVA.

151

Table 4-3. Effect of density on the dry mass (gram/plant) of Atriplex prostrata

2 plants/pot 4 plants/pot 8 plants/pot 16 plants/pot

Stem 0.45 ± 0.07 a* 0.36 ± 0.05 a 0.15 ± 0.04 b 0.08 ± 0.01 b

Leaf 0.46 ± 0.07 a 0.43 ± 0.07 a 0.18 ± 0.04 b 0.09 ± 0.02 c

Root 0.74 ± 0.26 a 0.26 ± 0.05 a,b 0.14 ± 0.04 b,c 0.05 ± 0.01 c

* Different letters in cells within the same row indicate that they are statistically

different as determined by the Bonferroni t-test, after a significant difference was

found overall among groups as determined by one-way ANOVA.

152 mass with the exception that Atriplex prostrata grown at a density of 16 plants per pot produced the smallest leaf dry mass (F =26.42, P < 0.0001). Atriplex prostrata grown at a density of 2 plants per pot accumulated significantly greater root dry mass than those at densities of 8 and 16 plants per pot (F = 16.86, P = <0.0001) (Table 4-

3). Based on the fresh and dry mass measurements, one can calculate water content as percent of water per gram of fresh weight. Density did not change the water content of stems and roots (F = 1.292, P = 0.292 and F = 1.894, P = 0.148, respectively; Fig. 4-2). However, leaves from plants grown at densities of 4, 8 and 16 plants per pot had significantly lower water content than leaves of plants from density 2 plants per pot (F = 10.748, P <0.001; Fig. 4-2).

In terms of leaf area, when leaves from a particular node, either 1st, 2nd, or 3rd, were sampled, the area of leaves from low densities (i.e., 2 and 4 plants per pot) were significantly larger than that from higher densities (i.e., 8 and 16 plants per pot) (F =

16.044, P <0.001 for leaves from 1st node; F = 12.558, P <0.001 for leaves from 2nd node; F = 8.677, P <0.001 for leaves from 3rd node; Fig. 4-3).

It was observed that there were some dramatically taller individuals among the plants grown at the density of 16 plants per pot. Considering these individuals might shade others, photosynthetic measurements were taken from both the taller (16t) and shaded (16s) groups. However, although the taller group (16t) has higher net photosynthesis than the shaded group, the difference was not significant. Overall photosynthesis results indicated that there was a significant difference among different density groups (F =10.63, P <0.001), and plants from lower densities (2 and

153

Fig. 4-2. Effect of density on water content of Atriplex prostrata stems, leaves and

roots. Different letters above bars indicate that the means are statistically

different as determined by the Bonferroni t-test, after an overall

significant difference among different densities of the same organ was

determined.

154

100 2 90 a a aaa 4 a aa a b 8 80 b b 16 70

Water Content (%) 30

0 Stem Leaf Root

155

Fig. 4-3. Effect of density on the area of leaves from the 1st, 2nd, and 3rd node of

Atriplex prostrata. Different letters above bars indicate that the means are

statistically different as determined by the Bonferroni t-test, after an

overall significant difference among different densities of the same organ

was determined.

156

20 2 plants 4 plants 8 plants a 15 16 plants a ) 2 a a a a

b Leaf Area (cm b b 5 b b b

0 1st node 2nd node 3rd node

157

4 plants per pot) had higher net photosynthesis than plants from higher densities (8 and 16 plants per pot) (Fig. 4-4).

The transpiration rates of plants grown at various densities were significantly different (F = 9.713, P <0.001; Fig. 4-5). Specifically, plants grown at 2 and 4 plants per pot had significantly higher transpiration rates than shaded plants grown at a density of 16 plants per pot. In terms of the calculated water use efficiency, there is an overall significant difference among density treatments (F = 13.214, P <0.001;

Fig. 4-6). According to a Bonferroni t-test, plants grown at a density of 2 plants per pot had significantly higher water use efficiency than plants grown at other densities examined (P <0.05).

In terms of stomatal conductance, there were significant differences among plants grown at various densities (F = 7.511, P <0.001; Fig. 4-7). Specifically, plants grown at 2 and 4 plants per pot had significantly higher transpiration rates than shaded plants grown at a density of 16 plants per pot, based upon the pair-wise comparison Bonferroni t-test.

DISCUSSION

Data collected for height, dry mass and fresh mass indicated that Atriplex prostrata had greater growth at densities of 2 and 4 plants per pot, while growth at

16 plants per pot was most inhibited. Although self-thinning has been observed for other species in plant monoculture (Gorham, 1979), none of the plants grown at

158

2 Fig. 4-4. Effect of density on net photosynthetic rate (µmol CO2/m /s) of Atriplex

prostrata. Leaves from the second node were used for these

measurements. Taller individuals (16t) and shaded individuals (16s)

grown at 16 plants per pot were measured separately. Different letters

above bars indicate that the means are statistically different as determined

by the Bonferroni t-test, after an overall significant difference among

groups was determined.

159

/s) 8.0 2 /m 2 a

6.0 mol CO µ ( b b

4.0 b

2.0 Photosynthetic Rate

0.0 24816t16s Density ( plants/pot)

160

2 Fig. 4-5. Effect of density on transpiration rate (mmol H2O/m /s) of Atriplex

prostrata. Leaves from the second node were used for these

measurements. Taller individuals (16t) were measured separately from

shaded individuals (16s) grown at 16 plants per pot. Different letters

above bars denote significant difference between means as determined by

the Bonferroni t-test.

161

7.0 a

/s) ab 2 6.0 ac

O /m bc 2 5.0

c (mmol H 4.0

3.0

2.0

Transpiration Rate Rate Transpiration 1.0

0.0 24816t16s Density ( plants/pot)

162

Fig. 4-6. Effect of density on water use efficiency (µmol CO2/mmol H2O) of

Atriplex prostrata. Leaves from the second node were used for these

measurements. Taller individuals (16t) were measured separately from

shaded individuals (16s) grown at 16 plants per pot. Different letters

above bars denote significant difference between means as determined by

the Bonferroni t-test.

163

2.0 O) 2

a 1.5 /mmol H 2

mol CO b µ

( 1.0 b b b

0.5 Water Use Efficiency Water Use

0.0 2 4 8 16t 16s

Density ( plants/pot)

164

2 Fig. 4-7. Effect of density on stomatal conductance (mol CO2/m /s) of Atriplex

prostrata. Leaves from the second node were used for these

measurements. Taller individuals (16t) were measured separately from

shaded individuals (16s) grown at 16 plants per pot. Different letters

above bars denote significant difference between means as determined by

the Bonferroni t-test.

165

1.2 /s) 2 1.0 a /m 2

ab 0.8 (mol CO (mol ac 0.6 bc

0.4 c

0.2 Stomatal Conductance Conductance Stomatal

0.0 24816t16s Density ( plants/pot)

166 the highest densities died during this experiment. This may be because the density examined here is simply not high enough for self-thinning to occur, and/or because the belowground resource was replenished regularly. Similarly, Salicornia in a dense stand under field conditions did not self-thin (Ellison, 1987). In a plant monoculture that does not self-thin, many investigators have shown that biomass distribution consisting of a few large plants and many small plants may develop when individuals compete for resources (Weiner, 1986). Such biomass distribution may result from suppression of smaller plants by the larger ones (the “dominance and suppression hypothesis” by Harper, 1977) or by exponential growth of plants whose biomass distribution is initially normal (the “growth rate” hypothesis by Turner and

Rabinowitz, 1983). It was noted that only at high densities (i.e., 8 and 16 plants per pot) did Atriplex prostrata exhibit an asymmetric size distribution. Plants grown at 2 and 4 plants per pot were uniformly taller than those at higher densities, which indicates that “dominance and suppression” occurred so that the degree of size inequality is larger in high density populations.

The number of nodes per plant from various densities was similar, but the length of mature internodes (i.e., the 3rd and 4th from the top) was shorter in plants grown at high densities, especially at 16 plants per pot. This indicates that density did not alter internode development, but limited their elongation. Density also dramatically altered the area of leaves with A. prostrata grown at high densities having significantly smaller leaves (Fig. 4-3). None of the leaves exhibited symptoms of nutrient disorders, confirming that nutrient deficiency was not a

167 significant factor in this system. None of the plants were wilted at any point, indicating that none of these plants were severely water-stressed. Therefore, the observed growth inhibition at higher densities was primarily due to competition for available light, resulting from changes in photosynthetic rate. Indeed, the net photosynthetic rate of plants grown at low densities (i.e., 2 and 4 plants per pot) was significantly higher than that for plants grown at high density (i.e., 16 plants per pot)

(Fig. 4-2), which parallels the density effect observed on fresh and dry mass of stems and leaves (Tables 4-2 and 4-3).

Density effects observed on fresh and dry mass of roots were slightly different, with plants grown at 16, but not 8 plants per pot, having significantly lower root mass than plants grown at low densities (Tables 4-2 and 4-3). Moreover, relatively more root biomass, compared to stem and leaf biomass, was produced in plants grown at 2 plants per pot (45% of total dry mass) than for those grown at 16 plants per pot (23% of total dry mass) (Table 4-3). According to the model of Vargas-

Mendoza and Fowler (1998) as well as that of “size symmetrical competition”, which represents a proportional division of resources by size (Weiner, 1990), water use is proportional to the relative size of plants. It implies that relative water availability for individuals is limited for plants grown at high density. When water availability is low, plants tend to lower their transpiration rate to avoid water loss by closing stomata. In fact, the effect of density on the transpiration rate follows the same decreasing trend with density as for stomatal conductance (Figs. 4-5 and 4-7).

A reduction in stomatal conductance may also contribute to the reduction in net

168

photosynthesis by decreasing CO2 diffusion (Cornic et al., 1992). However, as long as leaves are well illuminated and supplied with CO2, the photosynthetic rate will remain the same (Cornic, 1994). In other words, minor water stress does not affect photosynthetic capacity but may reduce net photosynthesis by reducing CO2 uptake through stoma. Based on these results, the decrease in net photosynthesis observed in plants grown in high density can be attributed to a reduction in light and, because of reduced transpiration rate, may be affected by water availability. This corroborates the results of Dyer and Rice (1999), who indicated both aboveground competition for light and belowground competition for water appeared to be important in biomass production of the inland California grasslands.

In conclusion, intraspecific competition did not cause self-thinning in Atriplex prostrata under laboratory conditions. Instead, it caused an asymmetric size distribution of plants, with some individuals of the population being suppressed. In addition, high density caused growth inhibition as indicated by declines in height, leaf area, and biomass of stems, leaves, and roots. Root biomass was more greatly reduced than shoot biomass at higher plant densities. This growth inhibition is mainly due to a reduction in net photosynthesis. Competition for an aboveground resource such as light and for a belowground resource such as water both contribute to the reduction of net photosynthesis, resulting in the reduction of plant height, leaf area and biomass.

169

CHAPTER 5. CELL DEATH AND GROWTH INHIBITION INDUCED

BY NACL IN SUSPENSION-CULTURED

CELLS OF ATRIPLEX PROSTRATA

Li-Wen Wang and Allan M. Showalter

Manuscript submitted to

Physiologia Plantarum

170

ABSTRACT

The effects of NaCl on cell death and growth at the cellular level were investigated using suspension-cultured cells of Atriplex prostrata. When these cells were placed in media containing 340 mM NaCl, approximately 70% of cells died within 30 min as determined by propidium iodide staining. The NaCl-induced cell death exhibited apoptotic-like characteristics such as nuclear degradation and DNA internucleosomal fragmentation. Surviving cells remained active in replicating, but grew at a slower rate than untreated, control cells. In contrast, 340 mM KCl caused 57% of cell death, surviving cells replicated significantly faster than cells treated with 340 mM NaCl. In order to examine the protective effect of glycine betaine on NaCl- induced cell death and growth inhibition, 50 mM of glycine betaine was added to the media either simultaneously or as a pretreatment. While simultaneous addition of glycine betaine and NaCl lowered the percentage of cell death to 40%, pretreatment with glycine betaine for 5 d, reduced the extent of cell death further to 27%.

Moreover, cells pretreated with 50 mM glycine betaine replicated faster than cells without glycine betaine pretreatment when subjected to salt stress. In conclusion,

NaCl induced cell death, most likely programmed cell death, and caused growth inhibition in suspension-cultured cells; moreover, glycine betaine was able to ameliorate these effects and afford protection against salt stress.

171

INTRODUCTION

When glycophytes are exposed to salinity, generally there is a reduction in growth that is associated with a decrease in cell enlargement, which is inferred to be due to the inability of the glycophyte cells to maintain turgor pressure. These cells, even after adaptation to salt, exhibited a reduced cell expansion rate possibly because of the alternation of cells walls (Cushman et al., 1990). Halophytes differ from glycophytes in that high salinity results in little or no growth inhibition, depending on the nature of the halophyte. Atriplex prostrata is a halophyte commonly found along sea beaches, marshes, and in salinized inland areas. It exhibits a certain degree of growth inhibition yet reproduces regularly to complete its life cycle. In fact, its seeds can germinate in up to 5% (850 mM) NaCl, and plants can grow in salinities as high as 3.6% (612 mM) NaCl (Khan and Ungar, 1984; Ungar, 1991).

Growth inhibition could be a result of 1) limited cell enlargement or expansion,

2) reduced cell division, and/or 3) cell death. Casas et al (1991) reported that the growth reduction observed in Atriplex nummularia L. cells is not attributable to a reduction in turgor pressure or cell enlargement, but rather to a reduction in the rate of cell division. Only a few articles have addressed salt-induced cell death in plants

(i.e., barley and Arabidopsis). Katsuhara (1997) reported that apoptosis-like cell death occurs in barley (Hordeum vulgare) root cells under salt stress. Recently, Huh et al. (2002) reported 200 mM NaCl caused DNA fragmentation detected by TUNEL staining in Arabidopsis thaliana.

172

Apoptosis, or programmed cell death, first described in animals cells at morphological level, is distinguished from necrosis and considered to be an important developmental process. Today, programmed cell death (PCD) is being revealed at biochemical and genetic levels, not only in animal cells but also in plant cells. PCD in plants occurs during normal morphogenesis and development in a variety of tissues and organs. For example, PCD occurs during the differentiation of tracheary elements (Groover and Jones, 1999), endosperm, and aleurone layers of the kernel (Young et al., 1997; Bethke et al., 1999), as well as during flower development (Calderon-Urrea and Dellaporta, 1999; Wang et al., 1999). Cell death also occurs when plants encounter certain pathogens (Bucker et al., 2000). This is also known as hypersensitive response, which results in the death of a limited number of cells at the site of infection. By sacrificing these few cells, plants restrict pathogen infection and are able to survive. Senescence, another type of PCD found in plants, occurs in specific organs such as fruits and leaves under delicate regulation by hormones such as ethylene and cytokinins (Bleeker and Patterson, 1997; Yen and

Yang, 1998). In response to oxygen deficiency as commonly occurs during flooding, the root cortical cells of plants such as maize form aerenchyma as a result of programmed cell death to aid in the transfer of oxygen (Drew et al., 2000). Abiotic environmental stresses such as salt (Katsuhara, 1997) and cold (Koukalová et al.,

1997) are also capable of inducing cell death, having apoptotic features such as nuclear DNA fragmentation as detected by TUNEL and gel electrophoresis.

173

My earlier work has already shown that growth inhibition in Atriplex prostrata occurs at the whole plant level (Wang et al., 1997). In contrast, this study was designed to examine growth inhibition at the cellular level using suspension-cultured cells, and also to investigate the effect of NaCl on death of these cells. At the whole plant level, Katsuhara (1997) demonstrated that death of certain root cap cells is essential for survival of the whole plant under salt stress. The questions I am asking in this investigation are: 1) Does salinity induce cell death and does it inhibit the rate of cell replication? 2) Does glycine betaine alleviate the effect of NaCl on growth of

A. prostrata suspension-cultured cells?

MATERIAL AND METHODS

Generation of Suspension Cell Culture from Leaves of Atriplex prostrata

Seeds of Atriplex prostrata were collected in a salt marsh at Rittman, OH. For sterilization, seeds were first immersed in 80% ethanol for 15 min with shaking, washed 3 times in sterile water with vigorous shaking, and treated with commercial bleach solution, Chlorox, (0.8% active chlorine) with a drop of Tween 80 per 20ml of bleach solution for 5 min. Sterile water was then added to dilute the bleach and the seeds were soaked in this 50% bleach solution for 15 min. After rinsing off the bleach, the seeds were placed on solid MS media (full strength, see Appendix for the composition) with addition of 2% sucrose (w/v), and 0.8% agar (w/v), for germination in magenta boxes. The medium was adjusted to pH 5.8 using 1M KOH

174 prior to sterilization by autoclaving. Approximately 10-14 days later, leaf segments were excised from new, yet fully developed, leaves under sterile conditions.

For callus induction, leaf segments were placed, abaxial side down, on solid

MS media with addition of 4.4 µM of 2, 4-D (2,4-dichlorophenoxy acetic acid, from

Sigma, St. Louis, MO), and 4.6 µM of kinetin (Sigma) in 100 × 15 mm Petri dishes.

About 3 weeks later, visible callus generated on the edge of leaf segments were transferred to new MS media for further growth before being placed into liquid MS media supplemented with 2,4-D and kinetin in order to generate suspension cell culture. After one week of shaking, the original calli were removed, and subcultured every 10 days for at least 10 rounds until they reached a stable state and before they were used in any experiment.

Maintenance and Treatments of Suspension Cells of Atriplex prostrata

McCown’s media was used to replace MS media for all the experiments involving NaCl treatment given the reduced content of chloride in the basal salt (see

Appendix I for the details). Suspension-cultured cells of Atriplex prostrata were grown in McCown’s liquid media supplemented with 20g of sucrose (Sigma) per liter, 4.4 µM of 2, 4-D, and 4.6 µM of kinetin. Cell cultures were maintained on a horizontal platform shaker (115 rpm, INNOVA 2300, New Brunswick Scientific Co.

Inc.) in a tissue culture room under a 16 h light/ 8h dark regime at 24 °C, and were subcultured every 7 days. These cells were maintained in McCown’s media for at least 5 generations prior to the initiation of any salt treatment.

175

For NaCl and KCl treatments in cell death experiments, cells were used 4 days after subculture, and maintained for the desired time before staining for viability, in the McCown’s media containing 170 mM or 340 mM of NaCl or KCl, in addition to supplemental sucrose, 2,4-D and kinetin. For NaCl and KCl treatments in growth inhibition experiments, cells were subcultured in McCown’s media containing 170 mM or 340 mM of NaCl or KCl, in addition to the supplemental sucrose, 2,4-D and kinetin.

For glycine betaine pretreatment in cell death and growth inhibition experiments, cells were grown in McCown’s liquid media with 50 mM glycine betaine (Sigma), in addition to the supplements for 5 days before transfer to the

NaCl-containing media.

Viability Stain

Cells were sampled by removing a 1 mL aliquot from the appropriate flask, quickly washed in 1X PBS 3 times, and then placed in PI (propidium iodine, 2 mg/ml of stock in H2O from Sigma) and FDA (fluorescein diacetate, 5 mg/ml of stock in acetone from Sigma) staining solution at a final concentration of 5 µg/ml of each in 1X PBS solution for 15 min at room temperature in the dark. 1X PBS replaced the staining solution and was used to wash the cells 3 times (5 min per

 wash). For cells stained with SYTO -11 (Molecular Probes, Inc., Eugene, OR), the rinsed cells were placed in the 5 µM solution for 60 min followed by three washes with fresh media. Cells were then placed on microscopic slides and observed with

176 the fluorescence microscope, Labophot-2 (Nikon, Melville, NY), connected to a digital camera, SPOT RT color (Diagnostic Instrument Inc., Sterling Heights, MI).

Multiple images were captured by SPOT software (version 3.1, Diagnostic

Instrument Inc.) to examine cell viability. In order to calculate the percentage of dead cells, at least 1000 cells from each treatment group, were scored by observing 5 to 10 different microscopic fields of view.

Isolation and Analysis of Nuclear DNA

Approximately 100 mg fresh weight of calli were frozen with liquid N2.

Nuclear DNA was isolated using MasterPureTM Plant Leaf DNA Purification Kit

(Epicentre, Madison, WI). Isolated DNA was treated with RNase A to remove RNAs.

DNA samples (5 µg) and molecular markers were subjected to electrophoresis on

1.0% agarose gels and stained with ethidium bromide.

Measurement of Growth

Cells were grown in supplemented McCown’s media with or without the addition of NaCl and/or glycine betaine (see previous section for details) in replicates of 4. Cells were collected every 2 days from the date of subculture (0, 2, 4, and 6 days, respectively) by harvesting one of the 4 replicates to measure packed cell volume (PCV). Growth is represented as relative growth in percentage and calculated as follows:

177

measured PCV at particular time point Relative Growth (%) = × 100% original PCV at the time of subculture

Statistics

Data presented here were analyzed with a one-way ANOVA using the NCSS

(2000) and SigmaStat (Version 2.03) statistical package. A log10 transformation was used to normalize data that were not normally distributed or equally variant. If a significant difference was determined among different treatment groups, a

Bonferroni t-test was performed to determine significant differences between pairwise individual treatments.

RESULTS

NaCl Induced Cell Death in Atriplex prostrata

When cells of Atriplex prostrata were placed in 340 mM NaCl, about 70% of cells died within 30min, while less than 20% of the cells in McCown’s medium were dead (Fig. 5-1). As the time of cells incubated in NaCl-containing medium extended up to 144 hours (6 days), the percentage of cell death remained similar without any statistically significant difference (P=0.406).

When the cells were placed in 170 mM NaCl, only 20% of the cells died in 5 hours, and the percentage of cell death increased to and remained at 30 to 40% with longer incubation in 170 mM NaCl (Fig. 5-2). Cell death caused by NaCl is dose-

178 dependent given the greater percentage of cell death seen at all time intervals in the

340 mM NaCl treatment compared to 170 mM NaCl treatment (Fig. 5-2).

Characteristics of the Cell Death Induced by NaCl

Vital stains such as FDA and PI were used to determine the death of suspension-cultured cells treated with 340 mM NaCl. Within 30 min of 340 mM

NaCl treatment, few FDA-stained cells were observed (Fig. 5-3). Among these few

FDA-stained cells, there were less visible nuclei, and the content of these cells were uniformly stained (Fig. 5-3 D). PI-stained cells lost integrity of their plasma membrane, since PI is known to penetrate only cells with damaged plasma membrane (MaCabe and Leaver, 2000). The dead cells (i.e., PI-stained) appeared to have lost most of their cell contents, resulting in empty cells, based upon the images from the light microscope (Fig. 5-3 F). In addition, some cells, after incubation in

NaCl-containing media for 30 and 60 min showed cytoplasmic condensation (Fig. 5-

3).

In order to better observe the nuclei, a nucleic acid-specific fluorescent dye,

 SYTO -11, was used to stain nuclei. While most nuclei were visible and stained in control cells, nuclei of cells treated with 340 mM NaCl for 5 days were degraded,

 since SYTO -11 uniformly stained entire cells (Fig. 5-4, E-H). Earlier at 30 min, both control and 340 mM NaCl-treated cells had intact nuclei (Fig. 5-4, A-D). In

179

Fig. 5-1. PI-determined cell death induced by 340 mM NaCl over time in

suspension-cultured cells of Atriplex prostrata. One mL of cells were

sampled from 340 mM NaCl-containing media at desired time points and

stained with PI. Numbers plotted here represent the average and standard

error of the data obtained from 8 fields of view for each treatment. The

same letters above each bar indicate that the two groups are statistically

similar as determined by the Bonferroni t-test.

180

80 30min b 12h b b b b 24h b 48h 72h 60 144h

40 Cell Death (%) a a a 20 a a a

0 Control 340 mM NaCl

181

Fig. 5-2. PI-determined cell death induced by 170 and 340 mM NaCl in

suspension-cultured cells of Atriplex prostrata. One ml of cells

were sampled at desired time points and stained with PI. Numbers

plotted here represent the average and standard error of the data

obtained from 8 fields of view for each treatment. The same letters

above each bar indicate that the two groups are statistically similar

as determined by the Bonferroni t-test.

182

80 5h c c c 12h c 1d c c 2d 3d 60 6d

b 40 b b b

Cell Death (%) Death Cell ab

a a a 20 a a a a

0 Control 170 mM NaCl 340 mM NaCl

183

Fig. 5-3. Cell death induced by 340 mM NaCl in suspension-cultured cells of

Atriplex prostrata. A. Cells in control media stained by FDA. B. Cells

in control media stained by PI. C. Cells in control media under the

light microscope. D. Cells in 340 mM NaCl-containing media for 30

min prior to FDA staining. E. Cells in 340 mM NaCl-containing

media for 30 min prior to PI staining. F. Cells in 340 mM NaCl-

containing media for 30 min under the light microscope. G. Cells in

340 mM NaCl-containing media for 60 min prior to FDA staining. H.

Cells in 340 mM NaCl-containing media for 60 min prior to PI

staining. I. Cells in 340 mM NaCl-containing media for 60 min under

the light microscope. In all panels, the size bar represents 100 μm.

184

185

 Fig. 5-4. SYTO -11 stained nuclei in cells treated with 340 mM NaCl. A. Cells

in control media stained by SYTO®-11. B. Cells in control media

under the light microscope. C. Cells in 340 mM NaCl-containing

media for 30 min stained by SYTO®-11. D. Cells in 340 mM NaCl-

containing media for 30 min under the light microscope. E. Cells in

control media for 5 days stained by SYTO®-11. F. Cells in control

media for 5 days under the light microscope. G. Cells in 340 mM

NaCl-containing media for 5 days stained by SYTO®-11. H. Cells in

340 mM NaCl-containing media for 5 days under the light

microscope. In all panels, the size bar represents 100 μm.

186

187 addition, internucleosomal DNA degradation as indicated by DNA laddering was observed in cells treated with 340 mM NaCl for 8 days (Fig. 5-5 A). At the 8th day,

DNA ladders consisting of multimers of approximate 180 bp were observed in both

170 and 340 mM NaCl-treated cells. In fact, at the 6th day, DNA isolated from 340 mM NaCl- treated cells started to be degraded. Again, at earlier stages (the 2nd and

4th day), none of the DNAs isolated from control nor NaCl-treated cells were fragmented (Fig. 5-5 B).

Exogenous Glycine Betaine Protected Cells from NaCl-Induced Cell Death

Although Atriplex prostrata is known to accumulate glycine betaine in response to salt treatments as part of the salt tolerance mechanisms, the particular cell line used here did not express much, if any, choline monooxygenase (CMO) (Fig.

5-6), which is the key enzyme involved in the biosynthesis of glycine betaine. On the same northern blot, the transcript of BADH was detected. However, the mRNA level in salt-treated cells was not much different from the one in control cells. Thus, this line is unlikely to produce much or any glycine betaine. In order to examine the protective effect of glycine betaine on NaCl-induced cell death, glycine betaine was therefore added to the medium, either simultaneously with or prior to salt treatments.

When 50 mM glycine betaine was added simultaneously with 340 mM NaCl, only 33% of the cells were dead at 30 min while 72% of the cells in the media without glycine betaine were dead (Fig. 5-7). Three days later, the death rates were

188

Fig. 5-5. DNA fragmentation in cells treated with 340 mM NaCl. 5 µg of DNA

from each treatment were separated on 1% agarose gel stained by

ethidium bromide. A. At the 8th day, DNA laddering is observed in

cells treated with 340 mM NaCl. Lane 1: DNA isolated from cells in

control media after 8 days. Lane 2: DNA isolated from cells in 170

mM NaCl-containing media for 6 days. Lane 3: DNA isolated from

cells in 340 mM NaCl-containing media for 6 days. Lane 4: DNA

isolated from cells in 170 mM NaCl-containing media for 8 days.

Lane 5: DNA isolated from cells in 340 mM NaCl-containing media

for 8 days. Lane 6: 100 bp DNA ladder marker. B. At the 2nd and 4th

day, DNA laddering is not clear. Lane 1: DNA isolated from cells in

control media after 4 days. Lane 2: DNA isolated from cells in 170

mM NaCl-containing media for 2 days. Lane 3: DNA isolated from

cells in 340 mM NaCl-containing media for 2 days. Lane 4: DNA

isolated from cells in 170 mM NaCl-containing media for 2 days.

Lane 5: DNA isolated from cells in 340 mM NaCl-containing media

for 2 days. Lane 6: 100 bp DNA ladder marker.

189

190

Fig. 5-6. Expression of CMO and BADH in suspension-cultured cells of Atriplex

prostrata. 20 μg of total RNA was loaded into each lane. C: RNA

isolated from cells in control media. S: RNA isolated from cells in 340

mM NaCl-containing media. CMO: membrane probed with partial

Atriplex CMO cDNA, ApCMO-0.7 (Fig. 2-2). EB: RNA gel stained by

ethidium bromide. BADH: membrane probed with an Atriplex BADH

cDNA, ApBADH-1.3 (Fig. 3-1).

191

192

40% and 69% respectively. At both time points, the percentages of cell death in glycine betaine-containing media were significantly lower than the ones in the media without betaine (P<0.001 in both cases).

In order to examine whether the accumulation of glycine betaine is more effective in protecting Atriplex prostrata cells treated with 340 mM NaCl from dying, cells were grown in 50 mM glycine betaine supplemented media for 5 days prior to salt treatment. After being placed in the media containing 340 mM NaCl, these cells had a higher survival rate than untreated cells (i.e., without glycine betaine pretreatment) (Fig. 5-8). Only 28% of cells died, which is significantly lower than those without glycine betaine pretreatment (P<0.001). During this particular study,

50 mM glycine betaine was also added to the media simultaneously with 340 mM

NaCl just 10 min before the PI staining to elucidate whether the pre-accumulation of glycine betaine is necessary. In contrast to what was observed in the previous experiment, simultaneous addition of glycine betaine and NaCl for a time period as short as 10 minutes killed 61% of the cells. This is not significantly different from the death rate of cells treated with only 340 mM NaCl (70%, P=0.876). Ten min was not sufficient for glycine betaine to exert its full protective effect. A two-way

ANOVA further summarizes the results. There is a significant effect of glycine betaine pretreatment (F= 85.967, P<0.001), but not of the simultaneous addition of glycine betaine and NaCl (F= 2.527, P= 0.131) nor the interaction of both (F= 0.327,

P= 0.576) (Table 5-1).

193

Fig. 5-7. PI-determined cell death induced by 340 mM NaCl in the presence

of 50 mM glycine betaine. Cells were incubated with or without

340 mM NaCl and with or without 50 mM glycine betaine for 30

min or 3 days before being subjected to PI staining. Numbers

plotted here represent the average and standard error of the data

obtained from 8 fields of view for each treatment. Different letters

above each bar indicate that the two groups are statistically

different as determined by the Bonferroni t-test.

194

30 min 80 b b 3 d

d 40 c Cell Death (%)

a 20 a a a

0 340 mM NaCl Control 50 mM Betaine 340 mM NaCl + 50 mM Betaine

195

Fig. 5-8. Effect of glycine betaine pretreatment on cell death induced by 340 mM

NaCl. Glycine betaine-pretreated cells were grown in 50 mM glycine

betaine-containing media for 5 days before being subjected to salt

treatment. Cells with or without glycine betaine pretreatment were then

transferred to either control (McCown’s media only), 340 mM NaCl, or

340 mM NaCl plus 50 mM glycine betaine-containing media 10 min prior

to the PI staining. Different letters above each bar indicate that the two

groups are statistically different as determined by the Bonferroni t-test.

196

80 w/o pretreatment w/ pretreatment b b

40 c Cell Death (%) Cell c

20 a a

0 340 mM NaCl Control 340 mM NaCl + 50 mM Betaine

197

Table 5-1. A two-way ANOVA for the effects of glycine betaine pretreatment

and simultaneous addition of glycine betaine and NaCl on the death of

Atriplex prostrata suspension cells

Source df Mean Squares F-ratio P

Pretreatment (P) 1 7982.81 85.967 <0.001

Simultaneous 1 234.68 2.527 0.131 addition of betaine and NaCl (S) P × S 1 30.33 0.327 0.576

Residue 16 92.86

Total 19 512.29

198

NaCl Inhibited Cell Growth of Atriplex prostrata

In order to compare the effect of NaCl on growth of Atriplex prostrata at the cellular level versus the whole plant level, 170 and 340 mM NaCl were added to suspension cultured cells and growth was measured as packed cell volume every two days after subculture. Packed cell volume was then converted to relative growth, which is the growth relative to the original packed cell volume of each treatment.

The 170 mM NaCl treatment significantly inhibited cell growth by 38% after 4 days and by 51% after 6 days (Fig. 5-9). By comparison, 340 mM NaCl significantly inhibited growth by 38% after 2 days, 67% after 4 days, and 76% after 6 days (Fig.

5-9).

Based on the microscopic images, cells from different treatments were similar in size (Figs. 5-3 and 5-4), indicating that the observed growth inhibition as shown in

Fig. 5-9 is less likely due to reduced cell enlargement. Therefore, not only cell death, but also inhibition of cell replication may result in the observed growth inhibition. In order to examine the effect that NaCl has on replication, the “time to double the volume” was calculated based on the relative growth and percentage of cell death measured according to the formula described in Table 5-2. During the first two days after subculture, cells treated with NaCl at both concentrations replicated significantly slower than control cells (Table 5-2). Later, after 4 days, surviving cells treated with 170 mM NaCl replicated at a rate slower than untreated control cells,

199

Fig. 5-9. Effect of NaCl on the growth of suspension-cultured cells of Atriplex

prostrata. The packed cell volume of cells grown in McCown’s

media, McCown’s media with 170 mM NaCl, or with 340 mM NaCl

were measured to calculate relative growth every 2 days for 6 days.

Numbers presented are the average and standard errors of the three

replicates. The entire experiment was repeated twice and the results

were similar.

200

800

700 Control 170 mM NaCl 340 mM NaCl 600

500

400

300 Relative(%) Growth 200

100

0 2 46

Days after subculture

201

Table 5-2. “Time to double” for cells treated with 170 and 340 mM NaCl.

Treatments 2nd day 4th day 6th day

Control 1.73 ± 0.047a* 1.83 ± 0.012a 1.93 ± 0.013a

170 mM NaCl 3.10 ± 0.216b 2.38 ± 0.046b 2.33 ± 0.011b

340 mM NaCl 3.71 ± 0.490b 3.66 ± 0.261c 3.20 ± 0.080c

* Same letter within each column indicates that there is no significant

difference between the two groups of data according to the Bonferroni t-

test.

** The death rate used to calculate “time to double” is the mean of 2

independent experiments with all the time points as described in Fig. 5-1.

The formula used to calculate “time to double” is:

−  Relative Growth − Death Rate  1 Time to double =  Log   2 100 − Death Rate 

202

while surviving cells treated with 340 mM NaCl still replicated even more slowly

(P<0.001).

KCl is Less Toxic Even at 340 mM

To examine whether induced cell death and growth inhibition were a specific effect of Na+, KCl was used to replace NaCl in the above experiments. Since little difference in percentage of cell death at different time points with each of the two

NaCl concentrations, cells were treated with 170 and 340 mM KCl and sampled at

30 min and at 3 days after subculturing. At 30 min, 170 mM KCl induced 23% cell death, while 340 mM KCl killed 57% of cells (Fig. 5-10). Three days later, the percentage of cell death induced by 170 mM increased to 34% while the percentage of death caused by 340 mM KCl remained at 56% (Fig. 5-10). This percentage of cell death is significantly less than that caused by 340 mM NaCl (P = 0.010). But there was no significant difference between the percentage of cell death caused by

170 mM NaCl and KCl (Table 5-3).

Not only did KCl induce less cell death, but it also inhibited growth to a lesser extent than NaCl (Table 5-3). Suspension-cultured cells in 170 mM KCl-containing media only exhibited 22% growth inhibition after 4 days, and 31% inhibition after 6 days, while cells grown in 340 mM KCl-containing media exhibited 49% and 64% growth inhibition respectively (Fig. 5-11). In terms of replication rate (i.e., time to double), cells treated with 170 mM KCl replicated at a rate similar but significantly

203

Table 5-3. Comparison of the growth inhibition and cell death induced by

NaCl and KCl.

Rate of Cell Death Relative Growth Treatments Time to Double** (3rd day) (6th day)

Control 15.5 ± 0.92a* 706.7 ± 2.57a 1.98 ± 0.003a

170 mM NaCl 33.4 ± 1.97b 379.2 ± 2.89b 2.33 ± 0.011b

170 mM KCl 34.3 ± 2.80b 486.3 ±2.24c 2.24 ± 0.006b

340 mM NaCl 69.5 ± 1.48c 187.9 ± 4.01d 3.20 ± 0.080c

340 mM KCl 56.5 ± 3.74d 251.9 ± 0.90e 2.74 ± 0.045d

* Same letter within each column indicates that there is no significant

difference between the two groups of data according to the Bonferroni t-

test.

** The average of percentage of cell death for each corresponding

treatment at 30 min and 3 days is used to calculate “time to double”

here.

204

Fig. 5–10. Effect of KCl on cell death in suspension-cultured cells of Atriplex

prostrata. One mL of cells were sampled at 30 min or 3 days after

subculture and stained with PI. Numbers plotted represent the average

and standard error of the data obtained from 8 fields of view for each

treatement. Different letters above each bar indicate that the two groups

are statistically different as determined by the Bonferroni t-test after a

significant difference among all treatments was determined by one-way

ANOVA.

205

80 30 min 3 d

d d 60

40 c Cell Death (%) b

20 ab a

0 Control 170 mM KCl 340 mM KCl

206

Fig. 5–11. Effect of KCl on growth inhibition in Atriplex prostrata suspension-

cultured cells. Packed cell volume of cells grown in McCown’s

media, McCown’s media with 170 mM KCl, or with 340 mM KCl

were measured to calculate relative growth every 2 days for 6 days.

Numbers presented are the average and standard errors of the three

replicates.

207

800

Control 700 170mM KCl 340mM KCl 600

500

400

300

Relative Growth (%) Growth Relative 200

100

0 2 4 6

Days after Subculture

208 slower than that of untreated control cells (P<0.001), and 340 mM KCl-treated cells replicated at a much slower rate (Table 5-3). Growth inhibition caused by KCl was significantly less than for NaCl as indicated by a two-way ANOVA, which demonstrated a significant effect of salt type (F= 124.49, P<0.001), salt concentrations (F= 5343.31, P<0.001), and salt by concentration interaction (F=

113.62, P<0.001) (Table 5-4).

Exogenous Glycine Betaine Protected Cells from NaCl-Induced Growth

Inhibition

To further elucidate the role of glycine betaine in protecting cells from damage by NaCl, suspension cells were treated with or without 50 mM glycine betaine in addition to 340 mM NaCl and examined with respect to growth. While suspension- cultured cells in the media containing 340 mM NaCl exhibited 68% growth inhibition after 4 days, and 75% after 6 days, cells grown in the media containing 50 mM glycine betaine and 340 mM NaCl exhibited only 47% and 25% growth inhibition, respectively (Fig. 5-12). These data indicated that 50 mM glycine betaine significantly reduced NaCl-induced growth inhibition.

As for the effect of glycine betaine pretreatment, suspension cells were first treated with or without 50 mM glycine betaine in McCown’s media for 5 days and then subcultured into McCown’s media with or without 340 mM NaCl. Cells were collected every two days to compare their relative growth under these four different conditions. As indicated by one-way ANOVA and the Bonferroni t-test, glycine

209

Table 5-4. A two-way ANOVA for the effects of salt type and salt concentration

on the growth of Atriplex prostrata suspension cells at the 6th day

Source df Mean Squares F-ratio P

Salt Type (T) 1 8995.4 124.49 <0.001

Concentration (C) 2 386106.8 5343.31 <0.001

T × C 2 8209.9 113.62 <0.001

Residue 12 72.3

Total 17 46970.3

210

Fig. 5-12. Effect of simultaneous addition of glycine betaine with 340 mM

NaCl on the growth of Atriplex prostrata suspension-cultured cells.

Packed cell volume of cells grown in McCown’s media,

McCown’s media with 340 mM NaCl, or with 340 mM NaCl and

50 mM glycine betaine were measured to calculate relative growth

every 2 days for 6 days. Numbers presented are the average and

standard errors of the three replicates.

211

800

Control 700 340 mM NaCl 340 mM NaCl+ 600 50 mM betaine

500

400

300 Relative Growth (%) Growth Relative 200

100

0 24 6

Days after subculture

212 betaine pretreatment did not significantly alter cell growth in McCown’s media (P=

1.000), but significantly alleviated growth inhibition caused by the addition of 340 mM NaCl in McCown’s media (P<0.001) (Fig. 5-13).

In order to compare the effect of simultaneous addition of betaine and betaine pretreatment on growth, “time to double” was again calculated using the average percentage of cell death and the relative growth data. Cells treated with 340 mM

NaCl replicated at a much slower rate than cells grown in the media containing 340 mM NaCl and glycine betaine, with the latter replicating at a rate similar to that of control cells (Table 5-5). On the other hand, glycine betaine pretreatment did not have a similar pronounced effect on replication. Cells pretreated with 50 mM glycine betaine for 5 days did not replicate as fast as either control cells or cells grown in the media containing 340 mM NaCl and glycine betaine, although they did replicate faster than those cells without glycine betaine pretreatment subjected to 340 mM

NaCl (P<0.001).

DISCUSSION

NaCl induces cell death as determined by vital stains such as FDA and PI. In suspension-cultured cells of Atriplex prostrata, a halophyte, NaCl killed 70% of the cells at different time points ranging from 30 min to 6 days, while NaCl killed more than 95% of the cells in Arabidopsis, which is considered a glycophyte, under similar conditions (Chen and Showalter, unpublished data). The reason that NaCl kills cells

213

Fig. 5-13. Effect of glycine betaine pretreatment on the growth of Atriplex

prostrata suspension-cultured cells under salt stress. Cells were

grown in McCown’s medium with (+B) or without (-B) 50 mM

glycine betaine for 5 days before transferred to either McCown’s

media (control) or media with 340 mM NaCl (NaCl). Numbers

presented are the average and standard errors of the three replicates.

214

600

-B / control -B / 340 mM NaCl 500 + B / control + B / 340 mM NaCl

400

300

200 Relative Growth (%) Growth Relative

100

0 2 4 6

Days after subculture

215

Table 5-5. “Time to double” for 340 mM NaCl-treated cells with glycine betaine pretreatment or simultaneous addition of glycine betaine.

Treatments 4 days 6 days

Control 1.94 ± 0.039a 2.04 ± 0.037a

340 mM NaCl 4.98 ± 0.326b 3.35 ± 0.195c

340mM NaCl + betaine 3.17 ± 0.032c 2.15 ± 0.058a,b

Betaine pretreatment + 2.02 ± 0.008a 2.09 ± 0.089a control media

Betaine pretreatment + 2.81 ± 0.063c 2.48 ± 0.066b 340 mM NaCl

* Same letter within each column indicates that there is no significant

difference between the two groups of data according to the Bonferroni t-

test.

216 might be that Na+ perturbs or compromises membrane integrity and results in increased Na+ influx into the cytosol, which leads to disruption of cellular metabolism and eventually to cell death (Mansour, 1998). Excess Na+ specifically impairs mitochondrial (Lu and Vonshak, 1999) and chloroplastic function (Hamilton and Heckathron, 2001).

Halophytes, at the whole plant level, develop mechanisms to exclude salts in order to survive (Ungar, 1991). At the cellular level, halophytic cells usually accumulate osmoprotectants such as glycine betaine and proline to tolerate the saline environment. Atriplex prostrata is known to accumulate glycine betaine as one mechanism for salt tolerance (Egan and Ungar, 1998). Also, at the whole plant level,

CMO and BADH were both expressed in leaves and stems of Atriplex prostrata treated with NaCl (Figs. 2-8 and 3-7). However, the transcript of CMO in this particular cell line did not accumulate in response to salt. It is not surprising that cell cultures established from different parts of plants may differ in terms of the response to salt stress (Vera-Estrella et al., 1999). In fact, CMO mRNA was barely detected in the particular cell line used here (Fig. 5-6). Moreover, this cell line appears to be yellowish and therefore might contain low level of endogenous glycine betaine as suggested in the literature. Tanimoto et al., (1997) reported that yellowish callus of

Suaeda japonica, compared to green callus, have markedly less chlorophyll as well as endogenous glycine betaine, and demonstrate more NaCl-induced growth inhibition. On the other hand, BADH was detected in this cell line of Atriplex prostrata. BADH was localized in the stroma of chloroplast (Weigel et al., 1986) as

217 well as in the cytosol (Weretilnyk and Hanson, 1988) and peroxisome (Nakamura et al., 1997). This may explain why BADH mRNA was detected in this cell line. In fact, according to the sequence of ApBADH-1.3, it is possibly targeted to both peroxisomes and chloroplasts (PSORT, see Discussion of Chapter 3).

The cell death reported here most likely represents a type of PCD given the observed morphological changes (Fig. 5-3), nucleus degradation (Fig. 5-4) and

 internucleosomal DNA fragmentation (Fig. 5-5). SYTO -11 uniformly stained entire cells treated with 340 mM NaCl for 5 days, indicating that the nuclei were no longer intact and that nucleic acids was distributed throughout cells (Fig. 5-4). In addition, some cells in Fig. 5-3, after incubating in NaCl-containing media for 30 min showed condensed cytoplasm. Longer incubation resulted in more cells with cytoplasm condensation and shrinkage (data not shown). NaCl-treated cells even lost their cellular content and became empty (Fig. 5-3). These morphological changes are consistent with characteristics of PCDs (MaCabe and Leaver, 2000). Furthermore, internucleosomal DNA fragmentation, as indicated by DNA ladders of 180 bp multimers, was observed perhaps as early as 6 days; however, the clearest evidence for such DNA laddering was seen 8 days after treatment (Fig. 5-5). The DNA laddering observed here is a result of DNA cleavage by specific endonuclease(s) activated during PCD (Schwartzman and Cidlowski, 1993), not by non-specific nucleases which would result in DNA smear on agarose gels (McCabe et al., 1997).

In general, DNA cleavage is a late event in PCD (McCabe and Leaver, 2000). In addition, the detection of DNA laddering with ethidium bromide staining alone can

218 be difficult (i.e., a more sensitive DNA dye or a southern blot of the gel with a total genomic DNA probe will increase the sensitivity (McCabe and Leaver, 2000). This explains why laddering was not observed before 6 d.

NaCl is reported to cause cell death in other plants. Katsuhara (1997) reported apoptosis-like cell death occurs in barley root cells under salt stress with 300mM

NaCl. They further determined that these treated roots, where nuclear degradation and cell death occurs, never recovered, but the whole plant survived and recovered by developing new roots after the removal of salt stress (Katsuhara and Shibasaka,

2000). Recently, Huh et al. (2002) reported 200 mM NaCl caused DNA fragmentation detected by TUNEL staining in Arabidopsis, and this DNA fragmentation was even more severe in sos1 mutant, which is defective for ion homeostasis.

The relative growth of Atriplex prostrata in regular McCown’s media without the addition of NaCl increased exponentially (Fig. 5-9). Calculations indicate that the surviving cells (approximately 90% in most cases) duplicated approximately every two days during the 6 days period of observation. In contrast, when cells were treated with 340 mM NaCl, only 30% of cells survived and these surviving cells duplicated at a slower rate (approximately 3-4 days). Therefore, the growth reduction cannot be explained merely by the low survival rate although cell death may have a more pronounced effect on growth inhibition. It seems that a high concentration of

NaCl (340 mM in this case) also inhibits cell replication while 170 mM NaCl does not have a significant effect in this regard (Table 5-2). These data agree with the

219 report of Michea et al. (2000) who indicated high salinity delays the cell cycle and causes cell death in renal medullary cells. In rice (Oryza sativa L.), Samarajeewa et al (1999) reported that NaCl remarkably suppressed cell division but not cell elongation, in the root meristem. They further suggested that these dividing cells, which lack prominent vacuoles, can not compartment ions and therefore NaCl is more likely to directly influence cytoplasmic events in meristematic cells. The suppressed cell replication observed in A. prostrata cells might be also due to failure to compartmentalize ions. Glycine betaine, as an osmoticum, usually accumulates in cytoplasm so that the excess salt can be counter-balanced and confined to vacuoles.

The particular cell line used here did not express CMO in response to salinity.

Therefore it is unlikely that these cells produce glycine betaine and use it to balance ions such as Na+ and to protect cells.

Potassium is considered necessary for plant growth and cases of K+ toxicity are rare. Here, however, it was observed that the addition of KCl to McCown’s medium which contains no Na salt (see Appendix I) caused cell death and growth inhibition.

This is consistent with several studies that focused on K+ toxicity in halophytes.

+ Egan and Ungar (1998) reported that K salt (both K2SO4 and KCl) severely inhibited the growth of A. prostrata at the whole plant level. Kefu et al (1995) also found that K+ could inhibit the growth of halophytes such as Suaeda salsa (L.) Pall. and Atriplex centralasiatica Iljn. in absence of Na+. Potassium salts also inhibited enzyme activity almost to the same extent as sodium salt (Gorham, 1996). One explanation for the deleterious effects of K+ observed here with respect to cell death

220 and growth inhibition is that it is not mainly due to ion toxicity but to ion disequilibrium as reported for the yeast ena1-4∆ mutant with a defective plasma membrane ATPase (Huh et al., 2002).

The effect of exogenous glycine betaine was examined in two ways. One is to add glycine betaine simultaneously with NaCl. This significantly reduced the percentage of cell death (Fig. 5-7). The other way is to pretreat cells with glycine betaine prior to exposure to salt stress. The pretreatment of glycine betaine successfully protected cells when exposed to 340 mM NaCl-containing media (Fig.

5-8). In fact, the glycine betaine-pretreated cells were even partially protected when cells were later placed in 680 mM NaCl-containing media (data not shown).

However, the simultaneous addition of glycine betaine to NaCl-containing media for as short as 10 min did not further protect glycine betaine-pretreated cells which indicates that 10 min does not appear to be sufficient for glycine betaine to penetrate and exert its full protective effect (Fig. 5-8). Instead, at least 30 min of glycine betaine treatment was required to significantly protect cells from dying. Thus, a pretreatment time as long as 5 days probably is not necessary or could be reduced without compromising the protective effect.

In terms of growth inhibition, cells pretreated with glycine betaine obviously exhibited less growth inhibition than when they were subsequently placed in 340 mM NaCl-containing media (Fig. 5-13). These glycine betaine-pretreated cells, when placed in control media, grew normally without any significant difference from the cells without glycine betaine pretreatment (Figs. 5-8 and 5-13), but the calculated

221 replication rate was significantly lower than that of control cells (Table 5-5). This rules out the possibility that glycine betaine, when compartmentalized in the cytosol, can trigger cell division. On the other hand, simultaneous addition of glycine betaine and NaCl significantly reduced the growth inhibition caused by 340 mM NaCl (Fig.

5-12). These cells even replicated at a rate similar to those in control media (Table 5-

5). Akula et al. (2000) reported that glycine betaine, rather than other osmotica such as mannitol and polyethylene glycol, rapidly induces somatic embryogenesis in tea

(Camellia sinensis (L.) O. Kuntze). It is unclear how glycine betaine present in the media, not in the cytosol, triggers cell division. Presumably, glycine betaine exerts its protective effects on growth inhibition and cell death by accumulating in the cytosol and serving as an osmoticum involved in osmotic adjustment and cytoplasmic compartmentation (Gorham, 1996). Furthermore, glycine betaine also directly stabilizes membranes and enzymes (Gorham, 1995; Lee et al., 1997;

Mansour, 1998). The stabilizing effect on membranes may be associated with the protective effect against NaCl-induced cell death because permeability changes of the mitochondial membrane is known to trigger cell death (Pedersen, 1999).

Specifically in mitochondia, while Complex II electron transport is disrupted by Na+ toxicity, glycine betaine provides a 20% higher rate of electron transport when compared with NaCl stress (Hamilton and Heckathron, 2001). In chloroplast, the activity of photosystem II in transgenic rice was protected by glycine betaine against salt stress (Sakamoto et al., 1998). Along with the data presented here, it is possible that glycine betaine may promote cell division and growth in suspension culture

222 when present in the media. Overall, these data together demonstrate that glycine betaine plays a role in protecting cells from damage caused by NaCl. In addition, glycine betaine, when present in the media, might trigger cell division and thus promote cell growth.

223

CHAPTER 6. SUMMARY AND CONCLUSION

224

Soil salinity is currently a major concern to agriculture all over the world.

Natural salinization and improper irrigation management both contribute to the problem of soil salinity and affect at least one quarter of cultivated land. Salts in the soil influence their chemical and physical properties and eventually affect the fertility of the soil. Because of osmotic inhibition and specific ion toxicity, salts in the soil inhibit crop growth and are estimated to result in the annual loss of billions of dollars.

Halophytes, or salt-tolerant plants, that can grow and reproduce in saline environments are therefore of great interest for two obvious reasons. First, some halophytes themselves may be used as crop plants. Second, through the study of halophytes we may better understand mechanisms involved in salt tolerance and be able to increase salt tolerance of conventional crop plants. In Chapter 1 of this dissertation, salt tolerance mechanisms at the morphological/developmental and cellular/molecular levels were summarized and discussed. Unlike the morphological mechanisms that require the function of many gene products, the cellular and molecular mechanisms typically involve only a few gene products and therefore it is feasible to genetically engineer these mechanisms into conventional crops to increase their salt tolerance.

Among all cellular mechanisms discussed in Chapter 1, two major mechanisms that may be utilized in developing salt-tolerant crops are: 1) accumulation of compatible solutes, and 2) regulation of ion homeostasis. Accumulation of compatible solutes has been a focus of research for the past two decades, while key

225 components involved in ion homeostasis were unveiled more recently. Glycine betaine, one of the compatible solutes, is produced and accumulated in only a few families of higher plants but not in most common crops. Therefore, substantial effort was devoted to investigating the function and biosynthesis of glycine betaine.

Atriplex prostrata, the species I studied for my master’s research, is a member of the

Chenopodeaceae, which happens to be one of the few families of higher plants that accumulate glycine betaine in response to salt stress.

In chenopods, glycine betaine is synthesized via the two-step oxidation of choline (Fig. 2-1). These two steps are catalyzed by choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) respectively. In Chapter 2, a cDNA encoding CMO enzyme was cloned from Atriplex prostrata by RT-PCR and RACE.

This cDNA, designated as ApCMO, is 1669 bp long and encodes a full- length protein of 438 amino acids. Similar to CMOs cloned from spinach, sugar beet and amaranth, ApCMO contains a Rieske type [2Fe-2S] cluster and a non-heme Fe binding motif. The cDNA and deduced amino acid sequenced of ApCMO were aligned with other CMOs and revealed a high degree of identity. Specifically, a dendrogram showing the relationships among CMOs revealed that ApCMO is most closely related to another CMO from Atriplex, AhCMO, than to other CMOs, which is in agreement with their phylogenetic relationship.

The expression and NaCl-induction of ApCMO were also investigated using a

ApCMO cDNA as a probe in northern blotting experiments. In leaves and stems of A. prostrata in response to NaCl treatments, CMO transcript accumulated in parallel to

226 the accumulation glycine betaine. This observation is consistent with the metabolic analysis that the CMO-catalyzed step is the rate-limiting step of glycine betaine synthesis (McNeil et al., 2000). In order to understand the regulation of CMO expression, detailed studies on CMO expression were pursued. The NaCl-induced expression of CMO was most pronounced in young stems and leaves. Interestingly, a

CMO transcript was also detected in 3- and 5-day-old seedlings without salt treatments Along with the fact that accumulation of CMO transcript was less substantial in old tissues, CMO expression is obviously regulated in a developmental manner. Although ABA is known to play a role in general responses to salt and osmotic stresses, it did not induce CMO expression in A. prostrata.

As for BADH, the enzyme that catalyzes the second step of glycine betaine synthesis, its cloning and expression was elucidated in Chapter 3. Cloned by RT-

PCR and 3’-RACE, the composite cDNA of A. prostrata BADH, designated as

ApBADH1, is 1755 bp long and encodes a full-length protein of 500 amino acids. As in amaranth and in mangrove, Avicennia marina, there are two BADHs in A. prostrata. During RT-PCR, a second partial cDNA, designated as ApBADH2, was also discovered. ApBADH2 is 84.9% identical to the corresponding region of

ApBADH1. Multiple sequence alignments of ApBADH1 with other BADHs suggested that it is localized in chloroplasts, whereas ApBADH2 might be localized in the peroxisome, although this latter location remains to be confirmed by future work. Constitutive expression of ApBADH2 in A. prostrata suspension-cultured cells

(Fig. 5-6), similar to the expression of peroxisomal BADH from Avicennia marina

227

(AvmBADH13), along with the amino acid sequence similarity between ApBADH2 and AvmBADH13 around the substrate binding site (Fig. 3-6) supports the notion that ApBADH2 may be a peroxisomal BADH in A. prostrata.

Expression of ApBADH1, as revealed by northern blotting, was induced by

NaCl as well. Similar to ApCMO, expression of ApBADH1 was also regulated in a developmental and organ-specific manner. However, the difference is that BADH expression was higher in young leaves without salt treatment than CMO expression, but in older leaves BADH transcript was barely detected. In addition, the salt- induction of BADH is less dramatic in older stems than that of CMO. This indicates that CMO expression is more sensitive to salt in older tissue than BADH expression.

Furthermore, time course studies of CMO and BADH expression revealed that they both markedly expressed in response to salt treatment within 24 hours, but after 120 hours (5 days) BADH expression declined to basal level (i.e., the level observed in stems without salt treatment) whereas CMO expression remained pronounced with only a slight decline compared to that of earlier time points. This observation may indicate that BADH mRNA has a higher turn-over rate than CMO so that the accumulation of BADH transcript detected after 5 days was much lower. The most notable contrasting in the expression of CMO and BADH is that NaCl-induced

ApBADH1 expression is ABA-dependent, whereas ApCMO expression is not. With the CMO-catalyzed step being rate-limiting, glycine betaine production may remain independent of ABA. Furthermore, NaCl-induced expression of CMO and BADH in

A. prostrata was more substantial than that reported in spinach and sugar beet,

228 suggesting that A. prostrata may serve as a useful model to study regulation of glycine betaine accumulation by environmental stresses as well as how stress signals are perceived and translated into the accumulation of glycine betaine.

Although recent advances in identifying Na+/H+ antiporters and their essential role in ion homeostasis have overshadowed the significance of glycine betaine accumulation in salt tolerance by the fact that overexpression of this Na+/H+ antiporter made freshwater cyanobacteria more salt-tolerant than the same freshwater cyanobacteria accumulating glycine betaine (Waditee et al., 2002). Organic osmolytes may play a more significant role in eukaryotic organisms to balance the osmotic concentrations of vacuolar and cytosolic compartments of cells. The importance of glycine betaine accumulation was demonstrated in a cell line of A. prostrata that did not express CMO and thus might accumulate no or little glycine betaine. In Chapter 5, approximately 70% of these suspension-cultured cells were dead as determined by PI staining soon after being treated with 340 mM NaCl. With the exogenous addition of glycine betaine, most of these cells were protected against salt stress as the percentage of cell death was significantly lowered. KCl also induced cell death in suspension-cultured cells of A. prostrata but to a lesser extent. The above observation, along with the data from Huh et al., (2002), suggests that this cell death is due to ion toxicity, not osmotic stress. Moreover, NaCl-induced cell death in

A. prostrata exhibited characteristics of programmed cell death (PCD) such as DNA fragmentation as detected by DNA laddering. This is one of the few studies that revealed NaCl-induced PCD.

229

Growth of A. prostrata cells in media containing 170 or 340 mM NaCl was also investigated. The presence of 170 and 340 mM NaCl significantly reduced growth by 51 and 75%, respectively, after 6 days. Inhibition of growth was similar to that observed at the whole plant level by Wang et al. (1997). Mathematical analysis

(calculated “time to double”) revealed that the growth inhibition observed here was not only due to cell death but also due to slower replication. This study also suggested a role for glycine betaine in promoting growth and cell division, since the addition of glycine betaine to the medium alleviated the growth inhibition induced by NaCl by maintaining the rate of replication.

Since the growth of halophytes in a natural habitat is affected not only by salt but also by competition, the effects of intraspecific competition on growth and photosynthesis of A. prostrata were examined. This work is presented in Chapter 4.

Intraspecific competition, mimicked by different densities of A. prostrata grown at under laboratory conditions, caused significant growth inhibition similar to that observed in NaCl-treated A. prostrata (Wang et al., 1997). A. prostrata grown at higher densities were significantly shorter, had smaller leaves, and accumulated less biomass of leaves, stems and roots. This growth inhibition is a consequence of a reduced net photosynthesis due to competition for light most likely and maybe for water as well at higher plant densities.

In order to genetically engineer salt-tolerant crops, overexpression of Na+/H+ antiporters to exclude Na+ or to more efficiently compartmentalize Na+ in vacuoles seems to be a simpler solution (usually only one gene is sufficient to manipulate salt

230 tolerance). However, genetic engineering of osmoprotectants may still be beneficial, considering their additional functions that are not quite clear yet. In addition, the fact that there are more than one CMO-like and BADH-like genes found in Arabidopsis suggests that these genes may be present but not expressed for unknown reasons.

One possible constraint is the absence or inactivation of upstream components in the signal transduction pathway(s). Therefore, future work proposed as follows may expand our knowledge of the molecular biology of glycine betaine synthesis:

1. To further elucidate how CMO expression is regulated. In other words, it

will be useful to understand how a salt signal is perceived and translated

into the expression of CMO and to understand signal transduction of

glycine betaine synthesis in response to salt. Specifically, since CMO

expression is ABA-independent, the involvement of other components in

the general ABA-independent pathway in osmotic stress response such as

various kinases will need to be examined.

2. To isolate the 3’ end of ApBADH2 to confirm that ApBADH2 is the

peroxisomal version of ApBADH1, as in the case of Avicennia marina. In

addition, localization experiments to confirm the predicted localization of

BADHs as well as CMO by computation tools such as PSORT may be

pursued. Moreover, similar functional analysis (Hibino et al., 2001) can be

pursued to elucidate whether CMO and BADH from Atriplex prostrata are

more substrate-specific and stress-tolerant under salt as well as other

stresses than CMO and BADH from spinach. If so, genetic engineering of

231

these two enzymes using cDNAs from A. prostrata into conventional crops

might further improve salt tolerance.

3. To establish a glycine betaine-accumulating cell line of A. prostrata and to

compare NaCl-induced cell death in both suspension cultures. This will

further elucidate whether glycine betaine is critical in salt tolerance. This

cell line can also be used to assist in the exploration of salt-induced signal

transduction pathway(s) involving CMO and glycine betaine as mentioned

above.

232

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Appendix I. Components in MS (M5524) and McCown’s (M6774) media.

Source: product information from Sigma (www.sigma-aldrich.com)