Characterization of Polyamine Transporters from Rice And

CHARACTERIZATION OF POLYAMINE TRANSPORTERS FROM RICE AND

ARABIDOPSIS

Gopala R Vaishali Mulangi

A Dissertation

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

August 2011

Committee:

Dr. Paul F. Morris, Advisor

Dr. Emilio Duran Graduate Faculty Representative

Dr. George S. Bullerjahn

Dr. Vipaporn Phuntumart

Dr. Scott O. Rogers

Gopala Reddy Vaishali Mulangi

ABSTRACT

Dr. Paul F. Morris, Advisor

Uptake and compartmentation of polyamines in plants is crucial for many developmental processes and resistance against biotic and abiotic stresses. Transport of polyamines is a poorly understood component of polyamine homeostasis in plants. In this study, a comparative genomics approach was used to identify and characterize putative polyamine uptake transporters (PUTs) in rice and Arabidopsis thaliana genomes which translocate polyamines. We identified three and four candidate transporter genes in

A. thaliana and rice, respectively. These genes are predicted to be membrane proteins with 9-12 transmembrane domains. Functional analysis of PUTs, using yeast complementation assay, demonstrated that all the PUTs tested transported polyamines and paraquat into the cells. Kinetic analysis and competition assays with alternate substrates revealed that all the genes except OsPUT3.2 are high affinity spermidine transporters with Kms ranging from 0.9 μM to 15 μM. Semiquantitative RT-PCR of

PUTs showed differential expression at the tissue level suggesting their specialized roles in plant growth and development. Expression analysis using promoter-GUS transgenic lines showed that AtPUT2 is expressed in the vascular tissues of leaves, roots, flower and flower buds indicating that this transporter may be involved in long distance transport of polyamines to the developing plant organs. Strong expression of AtPUT3 in root vascular tissue and lack of expression in the root hairs and root tips suggests that this gene may facilitate the long distance polyamine transport and redistribution within the plant. The iv subcellular localization of PUTs was determined by fusions with GFP and expression in onion epidermal cells. No convincing results could be obtained for all the PUTs except for OsPUT3.1 which localized to the mitochondria. Further localization studies of rice

PUTs in rice protoplasts showed that OsPUT1, OsPUT2 and OsPUT3.2 were localized to the plasma membrane and cytoplasm while OsPUT3.1 localized to the chloroplast. In conclusion, the overlapping substrate specificities along with individual expression patterns of PUTs suggest specific function of each PUT in plant growth and development. v

Dedicated to my husband, Sridhar and daughter, Nikhita

for their unconditional love, encouragement and support.

Thank you both for all that you have given and I love you so much.

ACKNOWLEDGMENTS

I would like to thank Dr. Paul Morris for giving me the opportunity to carry out this work. Thank you, Paul, for your advisory, starting from day one till today. I learnt a lot from you, mostly about the molecular biology techniques and bioinformatics. Your passion for science and optimism are infectious. Thank you for giving me an opportunity to participate in the International meetings and conference and meet new people and representing the department. I am grateful to Dr. Vipaporn Phuntumart who is very closely and most directly involved in my research. Vipa, many thanks for your help, both academically and personally. I also would like to thank my other committee members,

Dr. George Bullerjahn and Dr. Scott Rogers for their guidance and help. Thank you for your critical comments and suggestions. My special thanks to Dr. Emilio Duran, a

Graduate Faculty Representative, for accepting to be in the committee.

I would like to acknowledge the people who have helped me in carrying out my research: Dr. Dindial Ramotar, Maisonneuve-Rosemont Hospital, Montreal, for providing yeast mutant for my work; Dr. John Gray, University of Toledo for providing gene gun system; Chan Ho Park and Dr. G. L. Wang, Ohio State University for helping me in the isolation of rice protoplasts; Dr. Marilyn Cayer and Dr. Fengyu Li, Bowling Green State

University, for their help in obtaining the fluorescence images.

I would also like to thank all the people from the stock room and the office, especially Linda, Chris, Steve and Lorraine. Special thanks to Lingxiao, for her lovely friendship and wonderful time I spent with her. vii

This work in this dissertation was supported by Bowling Green State University,

Department of Biological Sciences; Bowling Green State University, Ohio Plant

Biotechnology Consortium fund to Paul Morris and Vipaporn Phuntumart.

This is the right opportunity to thank my husband, Sridhar, without whose inspiration

I would have never completed this work. My love to my beautiful daughter, Nikhita, who never complained about my absence during some of her activities.

viii

TABLE OF CONTENTS

Page

1. CHAPTER I. INTRODUCTION ...... 1

1.1. Polyamine metabolism ...... 1

1.1.1. Polyamine biosynthesis ...... 2

1.1.2. Polyamine conjugation ...... 3

1.1.3. Polyamine degradation ...... 4

1.2. Biological role of polyamines in plants ...... 4

1.2.1. Polyamines and stress ...... 4

1.2.2. Role of polyamine in disease response ...... 6

1.2.3. Role of polyamine in plant growth and development ...... 6

1.3. Prokaryotic polyamine transport systems ...... 7

1.3.1. Transport systems in Escherichia coli ...... 7

1.4. Eukaryotic polyamine transport systems ...... 9

1.4.1. Transport system in yeast ...... 9

1.4.2. Transport system in other eukaryotic systems ...... 10

1.4.3. Transport system in mammals ...... 11

1.4.4. Polyamine transport in plants ...... 11

1.5. Major goals of the study ...... 13

2. CHAPTER II. MATERIALS AND METHODS ...... 14

2.1. Sequence homology with other organisms ...... 14

2.2. Functional characterization of polyamine transporters in yeast ...... 14

2.2.1. Strains, media and reagents ...... 14 ix

2.2.2. DNA cloning ...... 15

2.2.3. Yeast transformation and selection ...... 15

2.2.4. Growth assays ...... 15

2.2.5. Polyamine transport assays ...... 16

2.2.6. Saturation kinetic experiments ...... 16

2.2.7. Competitive inhibition experiments ...... 17

2.3. Tissue specific expression of plant polyamine transporters by RT-PCR ..... 17

2.3.1. Plant Growth and sample collection ...... 17

2.3.2. RNA isolation ...... 18

2.3.3. Reverse transcription and cDNA Synthesis ...... 18

2.4. GUS-Promoter assays ...... 19

2.4.1. Plant Growth and transformation ...... 19

2.4.2. Promoter-Reporter Constructs and Plant Transformations ...... 19

2.4.3. Histochemical Staining and GUS Activity Assay ...... 20

2.5. Subcellular localization of PUT proteins ...... 20

2.5.1. Transient expression of PUTs in onion epidermal cells ...... 20

2.5.1.1.Construction of GFP –PUT fusion ...... 20

2.5.1.2.Particle Bombardment and GFP Fluorescence Detection ...... 21

2.5.2. Transient expression of OsPUTs in rice protoplasts ...... 21

2.5.2.1.Isolation of rice protoplasts ...... 21

2.5.2.2.DNA transfection ...... 22

2.5.2.3.Fluorescence microscopy ...... 22

3. CHAPTER III. RESULTS ...... 23 x

3.1. Identification and cloning of plant polyamine transporter genes ...... 23

3.2. Functional characterization of PUTs in yeast ...... 29

3.2.1. Growth assays ...... 29

3.2.2. Transport of polyamines by PUTs ...... 30

3.2.3. Transport capabilities of PUTs ...... 33

3.2.4. Substrate specificity of PUTs ...... 37

3.3. Tissue specific expression of PUTs ...... 42

3.3.1. Expression of PUT genes by RT-PCR ...... 42

3.3.2. AtPUT2 and AtPUT3 gene expression in plant organs by GUS reporter

assays ...... 43

3.4. Subcellular localization of PUT genes ...... 46

3.4.1. In Silico Analysis of Subcellular Localization of plant polyamine

transporters ...... 46

3.4.2. Subcellular localization of plant polyamine transporters in vivo ...... 46

3.4.2.1.Transient expression of PUTs in onion epidermal cells ...... 46

3.4.2.2. Transient expression of OsPUTs in rice protoplasts ...... 50

3.4.3. Localization of OsPUT3.1 to mitochondria and chloroplast ...... 52

3.5. OsPUT3.1 and OsPUT3.2 are alternatively spliced genes ...... 53

4. CHAPTER IV. DISCUSSION ...... 54

4.1. Phylogenetic analysis ...... 54

4.2. Polyamine transport by PUTs ...... 55

4.2.1. PUTs mediate the transport of polyamines and paraquat ...... 55 xi

4.2.2. PUTs (except OsPUT3.2) are high affinity spermidine and low affinity

putrescine transporters ...... 56

4.2.3. PUTs are spermidine specific transporters ...... 56

4.3. Tissue specific expression of PUTs ...... 57

4.3.1. Expression of PUT genes by RT-PCR ...... 57

4.3.2. AtPUT2 and AtPUT3 promoter - GUS assays ...... 58

4.4. Subcellular localization of PUT genes ...... 59

4.4.1. In silico analysis of subcellular localization of PUTs ...... 60

4.4.2. Subcellular localization of plant polyamine transporters in vivo ...... 60

4.5. Do isoforms OsPUT3.1 and OsPUT3.2 have different substrate specificities and

subcellular localizations? ...... 61

4.6. Conclusions ...... 62

5. REFERENCES ...... 64

6. APPENDIX ...... 74

xii

LIST OF FIGURES

Figure Page

Figure 1.1 Biosynthetic pathways of polyamines in plants ...... 3

Figure 1.2 Polyamine transport systems in Escherichia coli ...... 8

Figure 1.3 Polyamine transport in Saccharomyces cerevisiae ...... 10

Figure 1.4 Polyamine transport in mammalian cells ...... 11

Figure 3.1 Phylogenetic analysis of polyamine transporters ...... 25

Figure 3.2 Sequence alignment of PUTs ...... 27

Figure 3.3 Functional complementation of OsPUT1-4 and AtPUT1-3 in yeast

mutant agp2 ...... 31

Figure 3.4 Time course uptake of [14C]spermidine and [14C]putrescine ...... 32

Figure 3.5 Determination of Km values of PUTs for [14C]spermidine uptake

in yeast ...... 34

Figure 3.6 Determination of Km values of PUTs for [14C]putrescine uptake

in yeast ...... 36

Figure 3.7 Uptake of [14C]spermidine by PUTs in the presence of excess

substrates ...... 38

Figure 3.8 Uptake of [14C]spermidine by agp2-PUTs in the presence of amino

acids as substrates ...... 39

Figure 3.9 Expression of PUT genes in rice (A) and Arabidopsis (B) plants .... 42

Figure 3.10 GUS-expression driven by AtPUT2 promoter in Arabidopsis ...... 44

Figure 3.11 GUS-expression driven by AtPUT3 promoter in Arabidopsis ...... 45 xiii

Figure 3.12 Transient expression of GFP-OsPUT fusion proteins in onion

Epidermal cells ...... 48

Figure 3.13 Transient expression of GFP-AtPUT fusion proteins in onion

epidermal cells ...... 50

Figure 3.14 Subcellular localization of OsPUT – GFP proteins in rice

protoplasts ...... 51

Figure 3.15 Transient expression of OsPUT3.1 in onion epidermal cells and rice

protoplasts ...... 52

Figure 3.16 Intron-exon structure of OsPUT3.1and OsPUT3.2 ...... 53

xiv

LIST OF TABLES

Table Page

Table 3.1 GenBank accession numbers and predicted protein characteristics of

the seven plant polyamine transporters ...... 29

Table 3.2 Percentages of identity and similarity between PUT proteins ...... 29

Table 3.3 Affinity constants of PUTs for spermidine and putrescine ...... 33

Table 3.4 Effect of various amino acids and polyamine precursors on

spermidine uptake by PUTs ...... 41

Table 3.5 Predicted localization of plant transporters ...... 46

CHAPTER I: INTRODUCTION

1.1 Polyamine metabolism

Polyamines, putrescine, spermidine and spermine are small organic cations present in all living organisms (Cohen, 1998). They are essential for growth and development of prokaryotes and eukaryotes (Tabor and Tabor, 1984; Tiburcio et al.,

1997). Being positively charged at physiological pH, they interact with negatively charged macromolecules like DNA and RNA, proteins, phospholipids and regulate the physical and chemical properties of membranes and nucleic acids (Galston and Kaur-

Sawhney, 1990; Igarashi and Kashiwagi, 2000; Seiler and Raul, 2005; Alcazar et al.,

2006; Kusano et al., 2008). They accumulate in high concentrations in living cells and play a role in a variety of cellular processes including cell division and differentiation,

DNA replication transcription, RNA modification, protein synthesis (Tabor and Tabor,

1999). In addition to the standard polyamines, cadaverine, norspemidine, sym- homospermidine and thermine (norspermine) are found as less widely distributed cellular polyamines of many organisms. In plants, polyamines are known to play key roles in developmental processes such as cell division, embryogenesis, root formation; scenescence, ripening, floral initiation etc. (Kumar et al., 1997; Walden et al., 1997;

Cohen, 1998; Malmberg et al., 1998; Kakkar et al., 2000; Kakkar and Sawhney, 2002).

Polyamines are present in all plant cell compartments including nucleus (Slocum, 1991;

Bouchereau et al., 1999 and Kaur-Sawhney et al., 2003) and are suggested as hormonal secondary-messengers in plants, as they regulate the action of plant hormones. It is also established that polyamines modulate the defense response of plants to various abiotic 2 stresses including metal oxidative stress (Rider et al., 2007), drought (Yamaguchi et al.,

2007), chilling stress (Cuevas et al., 2008; Groppa and Benavides, 2008), metal stress

(Groppa et al., 2003) and salt stress (Duan et al., 2008; Urano et al., 2004). Intracellular polyamine pools in cells appear to be regulated by their synthesis, degradation, conjugation and transport across the cell membrane (Wallace et al., 2003).

1.1.1 Polyamine biosynthesis

Polyamine biosynthetic pathway has been well studied (Evans and Malmberg,

1989; Tiburcio et al., 1997; Slocum, 1991; Martin-Tanguy, 2001). In plants, putrescine is synthesized directly from ornithine (Fig. 1.1) by ornithine decarboxylase (ODC) or indirectly from arginine by arginine decarboxylase (ADC) via agmatine. Overexpression of ADC in Arabidopsis stimulated putrescine accumulation indicating that ADC is the rate limiting step for putrescine biosynthesis in plants (Alcazar et al., 2006). Spermidine and spermine are synthesized by successive attachment of aminopropyle first to putrescine and then to spermidine. These reactions are carried out by aminopropyltransferases, spermidine synthase (SPDS) and spermine synthase (SPMS).

Aminopropyl is formed due to decarboxylation of S-adenosylmethionine (SAM) by SAM decarboxylase (SAMDC) which is a rate limiting enzyme of spermidine and spermine biosynthesis (Franceschetti et al., 2004).

Figure 1.1: Biosynthetic pathways of polyamines in plants.

Putrescine and spermidine biosynthetic pathways and predicted polyamine transporters in plant cells. The predicted locations of polyamine transporters is based on localization of homologues in yeast or experimental evidence assessed from the literature. Specialized transporters may be need at each location for each polyamine. Transporters that export polyamines from cytoplasm ; transporters that import polyamines into the cytoplasm. ADC, arginine decarboxylase, AI, agmatine iminohydrolase; CAH, N-carbamoylputrescine amidohydrolase; MAT, methionine adenosyltransferase; ODC, ornithine decarboxylase; PAO, polyamine oxidase, SAMDC, S-adenosyl methionine decarboxylase; SMO, spermine oxidase; SSAT, spermine/spermidine acetyl transferase.

1.1.2 Polyamine conjugation

In plants polyamines are also present not only as free bases but also conjugated to various molecules, especially hydroxycinnamic acids and proteins (Martin-Tanguy,

2001). Formation of hydroxycinnamic acid conjugates is suggested as a mechanism to 4 buffer against excess polyamines (Antognoni and Bagni, 2008). When polyamines are conjugated with proteins, they add positive charges to the protein causing them to bind with non- covalent linkages to other molecules (Folk et al., 1980). This may represent a significant portion of the total pool of cellular polyamines.

1.1.3 Polyamine degradation (catabolism)

Polyamine degradation is one way of regulating the levels of polyamine in plant cells. PA degradation in plants is catalyzed by two oxidative enzymes: copper-containing diamine oxidase (DAO) and flavoprotein-dependent polyamine oxidase (PAO). Both of these enzymes are localized in the peroxisomes and cell walls. The production of H2O2 through polyamine oxidation has been correlated with the oxidative burst, cell death, lignification, and suberization processes occurring during development and defense responses (Allan and Fluhr, 1997; Angelini et al., 2008; Moller and McPherson, 1998;

Rea et al., 1998, 2002; Cona et al., 2003; Walters, 2003). Polyamines reduce the levels of superoxide radicals generated during scenescence (Drolet et al., 1986) and polyamine catabolism produces H2O2 as a signaling molecule for stress signal transduction (Mitsuya et al., 2009; Yoda et al., 2006). As a result of H2O2 production, spermine in the cells is converted back to spermidine and spermidine back to putrescine by PAO and DAO, thereby increasing the levels of putrescine in cells under stress (Cona et al., 2006;

Nobusada-Kamada et al., 2008; Moschou et al., 2008; Tavladoraki et al., 2006).

1.2 Biological role of polyamines in plants

1.2.1 Polyamines and stress 5

In the recent years there is an extensive study on role of polyamines in several environmental stresses (Kumar et al., 1997, Bouchereau et al., 1999, Kasukabe et al.,

2004), such as drought, metal toxicity, salinity and chilling stress. Polyamines, both in the free and soluble conjugated forms, are suggested to have important roles in stress- related conditions (Alcázar et al., 2006; Bouchereau et al., 1999; Groppa and Benavides,

2008; van Buuren et al., 2002; Martin-Tanguy, 2001). H2O2 produced as a result of polyamine catabolism by DAOs and PAOs plays a significant role in biotic and abiotic stress signaling and ABA-induced stomatal closure (Cona et al., 2006; An et al., 2008). In stressed plants, the increased levels of putrescine (Soyka and Heyer, 1999; Capell et al.,

2004) account for 1.2% of the dry matter, representing at least 20% of the nitrogen

(Galston, 1991; Alcazar et al., 2010).

Many genes involved in polyamine metabolism and their expression under different abiotic stresses have been studied in detail. In Arabidopsis, two different genes for ADC (ADC1 and ADC2) have been identified (Watson and Malmberg, 1996; Watson et al., 1997). The levels of putrescine increase during drought as a result of ADC acitivity

(Bouchereau et al., 1999). ADC2 is induced by osmotic stress (Soyka and Heyer, 1999), wounding, jasmonates and ABA (Perez-Amador et al., 2002). ADC2 mutants were more sensitive to salt stress (Urano et al., 2004; Urano et al., 2003), and had reduced levels of put and salt tolerance was restored by exogenous putrescine. The injurious effects of salt stress in rice seedlings are overcome by the exogenous application of putrescine and spermidine (Chattopadhayay et al., 2002; Roy et al., 2005; Prakash and Prathapasenan

1988). Depending on the duration of the stress the levels, polyamines increase or decrease in the cells. Bagni et al (2006) have shown that Arabidopsis plants under salt 6 stress conditions had upregulated polyamine metabolism with increased levels of free putrescine and spermine. Krishnamurthy and Bhagwat (1989) were able to demonstrate that the salt tolerant rice cultivars had increased levels of spermidine and spermine, and low levels of putrescine; while salt sensitive cultivars had high levels of putrescine.

1.2.2 Role of polyamines in disease response

Spermine plays a key role in defence signaling against plant pathogens

(Yamakawa et al., 1998; Takahashi et al., 2003; Takahashi and Kakehi, 2010). By accumulating in the apoplast, spermine triggers a series of defence-related genes like

PAOs leading to the production of H2O2 (Cona et al., 2006; Kusano et al., 2008;

Moschou et al., 2008; Yoda et al., 2006). Polyamine derived H2O2 plays a significant role in cell wall maturation and lignification during wound healing and cell-wall reinforcement during pathogen invasion (Cona et al., 2006).

1.2.3 Role of polyamine in plant growth and development

Polyamines are known to be involved in many plant developmental processes, including cell division, somatic embryogenesis, reproductive organ development, floral initiation and development, fruit ripening leaf scenescence etc. (Evans and Malmberg,

1989). It has been observed that increased ODC activity is correlated with cell division frequency in tobacco suspension cultures and tomato ovaries (Heimer et al., 1979). The exogenous application of polyamines to oat protoplasts stimulates DNA replication and mitosis (Galston et al., 1978). Polyamines are implicated in root initiation and root growth in Vigna and Phaseolus (Jarvis et al., 1985). Inhibition of Put biosynthesis is 7 found to prevent root initiation while exogenous Put supply reverses this effect (Tiburcio et al., 1989). Costa and Bagni (1983) have shown that in apples, spraying polyamines at millimolar concentrations on flowers nine days after full bloom increases both fruit set and yield, apparently by increasing fruit growth rate during the stage of rapid cell division.

1.3 Prokaryotic polyamine transport systems

1.3.1 Transport systems in Escherichia coli

In addition to putrescine and spermidine, cadaverine and aminopropyl cadaverine contribute to the normal growth of E. coli (Igarashi et al., 1986). Approximately 0.6% of the total E. coli genes are related to polyamine transport mechanisms (Igarashi and

Kashiwagi, 1999). There are two polyamine uptake systems in E. coli. Both systems belong to the group of ABC transporters. One system is a spermidine – preferential and another one is putrescine – specific (Fig 1.2A).

Spermidine – preferential system is made of a complex of four proteins: PotA

(membrane associated ATPase), PotB and PotC (channel forming proteins) and PotD

(substrate binding protein). Similarly, putrescine – specific uptake system consists of

PotG (membrane associated ATPase), PotH and PotI (channel forming proteins) and PotF

(substrate binding protein). All the four proteins of the uptake system are indispensable for polyamine uptake. 8

Figure 1.2 : Polyamine transport systems in Escherichia coli. A. spermidine- preferential and putrescine – specific uptake systems B. The polar and hydrophobic residues are drawn as ellipses and rectangles, respectively. The most crucial residues from the structural and mutational analyses are colored yellow, and the other residues, which affect spermidine binding, are colored pink. Spermidine is shown as red zig-zag lines. The polar interactions between PotD and spermidine are shown as broken black lines. (From Igarashi and Kashiwagi, 2010).

The structure of the substrate binding protein revealed that it has four acidic residues which recognize the three positively charged nitrogen atoms of spermidine and five aromatic side chains anchor the methylene backbone by van der Waals interactions

(Fig 1.2B) (Kashiwagi et al., 1996; Sugiyama et al., 1996). The Km values of spermidine and putrescine in the spermidine-preferential uptake system were 0.1 μM and 1.5 μM, respectively. The Km value for putrescine in the putrescine-specific uptake system was 9

0.5 μM. Other polyamine transport systems in E. coli are PotE and CadB (Kashiwagi et al., 1997; Kashiwagi et al., 2000; Soksawatmaekhin et al., 2004). These two systems catalyze both the uptake of putrescine and cadaverine at neutral pH and excretion at acidic pH. These proteins consist of 12 transmembrane segments with both N and C terminals located in the cytoplasm (Watson et al., 1992).

1.4 Eukaryotic polyamine transport systems

1.4.1 Transport system in yeast

In yeast, four plasma membrane localized polyamine transporters have been identified. DUR3 mediates the transport of both urea and polyamines; while SAM3 transports polyamines as well as S-adenosylmethionine, glutamate and lysine (Uemura et al., 2007) (Fig 1.3). Disruption of SAM3 and DUR3 reduced the cell growth of a polyamine requiring mutant suggesting that they are major polyamine importers in yeast.

Polyamines are also imported by GAP1 and AGP2 (Aouida et al., 2005; Uemura et al.,

2005a). UGA4, a GABA (4-aminobutyric acid) transporter in yeast was later established as a polyamine transporter localized on the vacuolar membrane (Uemura et al., 2004).

Four more (TPO1-4) genes have been identified which encode polyamine transport proteins in yeast (Uemura et al., 2005b). Among the four polyamine transporters, those encoded by TPO2 and TPO3 are specific for spermine, whereas those encoded by TPO1 and TPO4 recognize putresine, spermidine, and spermine (Timotori et al., 2001).

Polyamine transport by TPO1 was dependent on pH with uptake at pH 8.0 and export at pH 5.0 suggesting that TPO1-4 function similar to PotE and CadB proteins of E. coli.

Tachihara et al. (2005) identified, TPO5, a Golgi localized PA preferential excretion 10 protein. Polyamine uptake in yeast is energy-dependent and regulated by phosphorylation and dephosphorylation (Kaouass et al., 1997).

Figure 1.3: Polyamine transport in Saccharomyces cerevisiae. SAM3 and DUR3 are major polyamine importers. UGA4 is a vacuolar transporter. Excretion of polyamines is mediated by TPO1-5. SPD-spermidine, SPM-spermine, PUT-putrescine (From Igarashi and Kashiwagi, 2010).

1.4.2 Transport system in other eukaryotic systems

A plasma membrane localized polyamine transporter LmPOT1 was identified and characterized in protozoan parasite, Leishmania major (Hasne and Ullman, 2005).

LmPOT1 belongs to APC superfamily and consists of nine to 12 transmembrane segments like PotE. LmPOT1 is a high affinity polyamine transporter with Km values of

6.7 and 14.3 μM for spermidine and putrescine, respectively. A high affinity spermidine transporter, TcPAT12, was identified in another parasitic organism, Trypanosoma cruzi

(Carrillo et al., 2006). In Trypanosoma cruzi polyamine transport is critical as it lacks the ability to synthesize putrescine. The Km value for spermidine uptake by TcPAT12 is 14

μM.

1.4.3 Transport system in mammals

In mammalian cells, polyamine transporter proteins have not yet been identified.

However, polyamine uptake has been studied in several mammalian cells (Kakinuma et al., 1988; Mandel and Flintoff, 1978). An anitzyme is known to negatively regulate the polyamine transport by degrading enzyme ODC (Murakami et al., 1992). The antizyme also increased the export of polyamines suggesting antizyme plays an important role regulation of polyamine transport (Sakata et al., 2000). In mammalian cells, it has been suggested that an endocytic pathway of polyamine uptake may exist (Belting et al.,

2003).

Figure 1.4: Polyamine transport in mammalian cells. (Adopted from Igarashi and

Kashiwagi, 2010).

1.4.4 Polyamine transport in plants:

It is Friedman and his coworkers (1986) who first detected large amounts of polyamines in xylem sap and phloem exudates of four plant species (Sunflower, mung 12 bean, grapevine and orange). They also identified higher concentrations of putrescine and spermidine in the exudates of salt-stressed sunflower plants. In plants, long distance transport of polyamines has been studied extensively by Bagni’s group. In their studies, the uptake of putrescine by Malus domestica leaves and translocation to the fruitlet were observed (Bagni et al., 1984). The presence of polyamine transport system was reported in protoplasts and vacuoles of carrot cell cultures and sunflower mitochondria (Pistocchi et al., 1987, 1988 and 1990). They also reported that in carrot cells, the uptake of putrescine and spermidine occured at a very rapid rate reaching maximum within a minute (Pistocchi et al., 1987). It was also observed that cadaverine and putrescine were translocated acropetally via xylem and basipetally via phloem (Shevyakova et al., 2001;

Kuznetsov et al., 2002). It was also observed that cadaverine accumulated in the leaves as a response to salt stress or exogenous ethylene application. It was concluded that ethylene and other stress hormones like ABA, stimulated the long distance translocation of polyamines.

Very few studies have been done to identify the polyamine transporters at the cellular level. In an experiment with exogenous application of putrescine to the intact maize seedling roots, it was observed that roots absorbed 0.05 and 1 mM putrescine and transported into the root apoplast and across the plasma membrane (di Tamaso et al.,

1992). It was concluded that putrescine was transported across the plasma membrane by a carrier-mediated process. It was also shown that putrescine accumulated in the root- cell vacuoles, where it is stored.

1.5 Major goals of the study

Polyamines are most abundant organic cations present in all living organisms and absolutely essential for cell viability. Cellular levels of polyamines in plants are regulated by their synthesis, degradation, conjugation and transport. Polyamines are implicated in a variety of growth and developmental processes and response to different environmental stresses. While key enzymes of polyamine metabolism have been identified, knowledge about transmembrane proteins mediating the transport of polyamines and fluxes between different cellular compartments in plants is fragmentary.

Therefore, it is expected that knowledge gained about polyamine homeostasis can be used in genetic manipulation of crop plants to develop better stress-tolerant varieties.

The first step in this process is to implement a comparative genomics approach to identify putative plant polyamine transporters. The next step is to determine the substrate specificity of rice and Arabidopsis polyamine transporters by their heterologous expression in a yeast mutant, agp2. Yeast is chosen as a model system for the characterization because mutants of several transporters are available and their phenotypes are well characterized. The third objective is to express recombinant GFP fusion proteins and to identify the subcellular localization of candidate polyamine transporters by fluorescence microscopy. The final objective is to determine the role of alternate splicing in determining the subcellular localization of a polyamine transporter in rice.

CHAPTER II: MATERIALS AND METHODS

2.1 Sequence homology with other organisms

To identify candidate polyamine transporters, Blastp searches using the characterized yeast, L. major, and T. cruzi polyamine transporters were made against the

Arabidopsis and rice genomes. Additionally, sequences of distantly related clades of amino acid transporters from Arabidopsis were included. All the sequences were aligned using ClustalX (Thompson et al., 1997). Phylogenetic analysis was done using PAUP version 4.0 using both parsimony and distance analysis (Thompson et al., 1997)

(neighbor joining; NJ) with 100 bootstrap replicates. Phylogenetic trees were drawn using

Dendroscope (Huson et al., 2007).

2.2 Functional characterization of polyamine transporters in yeast

2.2.1 Strains, media and reagents

In this study, wild type, BY4741 (MATa his3 leu2 met15 ura3) and a yeast strain deficient in spermidine uptake, agp2 (Auioda et al., 2005) were used to characterize the candidate polyamine transporters. The cells were cultured in YPD or synthetic complete (SC) media at 30° C. For polyamine transport studies, cells were cultured in SC medium with 2% galactose. [1,4-14C]spermidine trihydrochloride 4.14

Gbq/mmol and [1,4-14C]putrescine dihydrochloride 3.96 Gbq/mmol were obtained from

Amersham Biosciences (Piscataway, NJ). Polyamine and amino acids were obtained from Sigma-Aldrich, St Louis, MO.

2.2.2 DNA cloning

Rice clones for OsPUT1, OsPUT2, OsPUT3.1 and OsPUT3.2 were obtained from the Rice Genome Resource Center (http://www.rgrc.dna.affrc.go.jp/index.html) and

Arabidopsis clones, AtPUT1, AtPUT2 and AtPUT3 were obtained from Arabidopsis

Carlsbad, USA) to generate constructs containing the cDNA flanked with the GAL1 promoter. Plasmid DNA was prepared and constructs were sequenced to ensure that the inserts were in the correct orientation.

2.2.3 Yeast transformation and selection

The resultant plasmids and empty vector were introduced into agp2 yeast mutant cells by lithium acetate method (Gietz et al., 1992) to obtain transformants. Transformed cells were selected on SC-ura plates.

2.2.4 Growth assays

Wild type, agp2-empty vector and agp2-candidate transporter gene transformants were grown in yeast extract-peptone-galactose (YPG) media. The density of exponentially growing cell cultures was normalized to an OD600 of 0.5. Cell suspensions were serially diluted and 3 l aliquots were spotted onto YPG plates containing 25 mM spermidine, 200 mM putrescine and 1.5 mM paraquat. Plates were 16 photographed after 3-4 d of incubation at 30ºC. All experiments were repeated 2-3 times each with three replications.

2.2.5 Polyamine transport assays

For time course transport studies, wild type, agp2-empty vector and agp2- candidate gene transformants’ cells were grown overnight in SC media with 2% galactose. Mid-logarithmic phase cells were harvested, washed three times with the uptake buffer (50 mM sodium citrate, pH 5.5, 2% galactose) and suspended in the same buffer at a concentration of 108 cells/ml. Aliquots of 100 μl cells (107 cells) were transferred to the eppendorf tubes and uptake of polyamines was initiated by the addition of [14C]spermidine or [14C]putrescine at 15 μM concentration. At selected times, the uptake was stopped by the addition of 1 ml ice-cold uptake buffer containing 10 – fold concentrations of polyamine, and filtered through a 0.45μm, Millipore cellulose acetate filter. The filters were washed three times with 2 ml ice-cold uptake buffer to remove exogenous labeled polyamines and transferred to scintillation vials containing 10 ml of scintillation cocktail Ecoscint (National Diagnostics, Atlanta, GA) . The radioactivity on the filters was counted in a liquid scintillation counter LS-7000, Beckman Coulter

(Fullerton, CA) after quenching.

2.2.6 Saturation kinetic experiments

Kinetic constants, Km and Vmax of [14C]spermidine and [14C]putrescine uptake by polyamine transporter genes were determined by incubating the yeast cells expressing the polyamine transporter genes with increasing concentrations of polyamine for 5 min. and measuring the intracellular radioactivity. Endogenous uptake by yeast transformed 17 with empty vector, pYES-DEST52 was subtracted as background activity. A Michaelis-

Menten curve was generated by Graphpad Prism software http://www.graphpad.com/prism/Prism.htm.

2.2.7 Competitive inhibition experiments

The uptake of [14C]spermidine was measured in the presence of 15 μM or 100 μM unlabeled putrescine and spermidine either singly or in combination. The results were presented as percentages of the total label taken up by yeast cells expressing the polyamine transporter genes. The uptake of [14C]spermidine by agp2-POTs was also assessed in the presence and absence of 500 μM levels of a variety of amino acids, polyamines and polyamine precursors.

2.3 Tissue specific expression of plant polyamine transporters by RT-PCR

2.3.1 Plant Growth and sample collection

Rice plants (Oryza sativa L. cv. Nipponbare) were grown in the greenhouse in plastic pots filled with soil at Bowling Green State University (OH, USA). Young stem and young roots were collected at 14 days after sowing. Leaf, stem, root, flower and panicle were collected around 12 days after flowering. Seeds were collected following maturity. Arabidopsis thaliana [ecotype Columbia (Col-0)] plants were grown on soil at

22°C under a 16-h-light/8-h-dark photoperiod. Roots, stems, rosette leaves, cauline leaves, flowers, siliques and seeds were collected from 30-day old plants. Seedlings were grown in Petri dishes on 0.5x MS media and harvested after 8 days.

2.3.2 RNA isolation

Total RNA from three biological replicates of different rice and Arabidopsis tissues was isolated using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. RNA concentrations were assessed using a ND-1000 NanoDrop Full spectrum spectrophotometer (Wilmington, DE, USA). The structural integrity of the

RNAs was checked with non-denaturing agarose gel and ethidium bromide staining. To remove contaminating genomic DNA, RNAs were treated with the TURBO DNA-free kit from Ambion Inc. (Austin, TX) according to the manufacturer’s instructions.

2.3.3 Reverse transcription and cDNA Synthesis

The total RNA (10 g) was mixed with 1 l of oligodT(18) primer (0.5 g/l,

MWG) and 1 l dNTP mix (10 mM each) in a 0.5 ml RNase-free tube (Eppendorf).

RNase-free water was then added to bring the final volume to 12 l. The tube was incubated at 65°C for 5 min and this was followed by adding 4 l 5x RT buffer, 1 l

RNaseOUT (40 U/ l (Invitrogen Carlsbad, USA), 2 l DTT, 1 l Superscript III (200

U/l, Invitrogen). The tube was briefly mixed by vortexing and spun down before incubating at 50°C for 1 h. The reaction was terminated by incubating the tube at 70°C for 5 min. RNase H (1 l, Invitrogen) was added to the RT reaction mixture to digest the remaining RNA. Polymerase chain reactions were performed with 1 L of the RT reaction using gene specific primers. Additional reaction components were 10 mM polymerase buffer, 1 mM dNTPs, 0.1 units Taq polymerase (Clontech, Palo Alto, CA), and 1 M specific primers. RT-PCR of rice and Arabidopsis actin genes served as internal controls. Primer sequences and fragment sizes along with number of PCR cycles are listed in the appendix. Products from 20 L of the PCR amplification were visualized 19 on a 1.2% TAE agarose gel containing ethidium bromide. Bands were photographed using the AlphaIamager Mini (San Leandro, CA).

2.4 GUS-Promoter assays

2.4.1 Plant Growth and transformation

Arabidopsis thaliana plants [ecotype Columbia (Col-0)] were grown in soil under a 16 h light/8 h dark photoperiod in an environment growth chamber at 22C under white fluorescent light 125 μmol/ m2 /s1. Tissues were harvested at required developmental stages.

2.4.2 Promoter-Reporter Constructs and Plant Transformations

Using Arabidopsis genomic DNA as a template, 5’ upstream regions of the

AtPUT2 and AtPUT3 genes, of 2.9 kb and 821 bp in length, respectively, were amplified by PCR (see Appendix). The amplified products were cloned into the pENTR/D-TOPO vector (Invitrogen, Carlsbad, CA) then moved into the Gateway plant transformation destination vector pBGWFS7 (Karimi et al., 2002) by LR recombination reactions. The constructs were electroporated into Agrobacterium tumefaciens strain GV3101 and used to transform Arabidopsis plants Col-0 by floral dip method ((Clough and Bent, 1998).

The seeds of floral dipped plants were collected and T1 generation was used for screening of transformants. BASTA (Sigma-Aldrich, St. Louis, MO) resistant seedlings were selected, transferred to soil and grown at 22 C in a growth chamber with a 12-h-light/-dark cycle.

2.4.3 Histochemical Staining and GUS Activity Assay

Histochemical staining of GUS expression was performed by staining (Jefferson et al., 1987) with X-Gluc buffer (2 mMX-Gluc, 50 mM NaPO4 pH 7.0, Triton-X (0.5%),

0.5 mM K-ferricyanide, 0.5 mM K-ferrocyanide) for 16 h at 37C. After staining, chlorophyll was extracted from photosynthetic tissues with 70% ethanol. For each promoter-reporter construct, 5 transgenic lines were analyzed and one representative line was selected for further experiments.

2.5 Subcellular localization of PUT proteins

2.5.1 Transient expression of PUTs in onion epidermal cells

2.5.1.1 Construction of GFP –PUT fusion

Rice clones for OsPUT1, OsPUT2, OsPUT3.1 and OsPUT3.2 were obtained from the Rice Genome Resource Center (http://www.rgrc.dna.affrc.go.jp/index.html) and

Arabidopsis clones, AtPUT1, AtPUT2 and AtPUT3 were obtained from Arabidopsis

Biological Resource Center (ABRC, Ohio, USA) (Alonso et al., 2003). Full-length cDNA of these candidate transporter genes were amplified (see Appendix for primer sequences) and cloned into pENTR/D-TOPO cloning vector (Invitrogen, Carlsbad, USA) according to the supplier’s protocol. Subsequently, the target genes were transferred to destination vector, destination vectors pGWB5 / pGWB6 (Nakagawa et al., 2007) by LR recombination reaction (Invitrogen, Carlsbad, USA) to generate constructs containing the cDNA flanked with C or N terminal GFP and under the control of the CaMV 35S promoter. Plasmid DNA was prepared and constructs were sequenced to ensure that the inserts were in the correct orientation. 21

2.5.1.2 Particle Bombardment and GFP Fluorescence Detection.

Inner epidermal peels of the onion were placed on MS medium with the inner side oriented upward. To coat gold particles, 10 g of purified DNA were precipitated onto

3 mg of gold particles. The DNA-coated particles were placed on a delivery disk. Onion cells were bombarded at 1300 psi using the PDS-1000 He Biolistic Particle Delivery

System (Bio-Rad, Hercules, CA, USA). Following bombardment, the cells were incubated in the same Petri dishes for 24 h at 22 °C before observation. After 24 h, GFP fluorescence was observed using a Zeiss Axiophot microscope equipped with a

Micromax cooled CCD digital camera, and electronic shutter control. Images were recorded with Metamorph software http://www.spectraservices.com/METAMRPH.html.

To stain mitochondria, MitoTracker Red CMXRos (Molecular Probes, Leiden) was used.

The dye was added to a final concentration of 10 nM and incubated for 45 min in darkness at room temperature.

2.5.2 Transient expression of OsPUTs in rice protoplasts

2.5.2.1 Isolation of rice protoplasts

Rice protoplasts were isolated according Chen et al. (2006). For isolating protoplasts from young seedling tissues, rice seeds were germinated on half-strength MS medium under light for 3 days. Seedlings were then cultured on half-strength MS medium in the dark at 26 °C for 10–12 days. Tissues of etiolated young seedlings were cut into approximately 0.5-mm strips and placed in a dish containing K3 medium (Kao and Michayluk, 1975) supplemented with 0.4 M sucrose, 1.5% cellulase R-10 (Yakult

Honsa) and 0.3% macerozyme R-10 (Yakult Honsha). The chopped tissue was vacuum- infiltrated for 1 h at 20 mmHg and digested at 25 °C with gentle shaking at 40 r.p.m. 22

After incubation, the K3 enzyme medium was replaced by the same volume of W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl and 2 mM MES, adjusted to pH 5.8 with KOH). Protoplasts were released by shaking at 80 r.p.m. for 1 h, followed by filtering through a 35-m nylon mesh. Protoplasts were collected by centrifuging at 300 g for 4 min at 4 °C. Pellets were resuspended in W5 solution.

2.5.2.2 DNA transfection

The collected protoplasts were resuspended in an appropriate volume of suspension medium (0.4 M mannitol, 20 mM CaCl2 and 5 mM MES, adjusted to pH 5.7 with KOH). Plasmid DNAs of OsPUT1, 2, 3.1 and 3.2 GFP constructs(see section

2.5.1.1) (about 10 g DNA of each construct) were mixed with 200 L of suspended protoplasts (usually 1.5–2.5 × 106 cells/mL).The DNA and protoplasts mixture was added to 40% PEG solution [40% PEG 4000, 0.4 M mannitol and 100 mM Ca(NO3)2,adjusted to pH 7.0 with 1 M KOH] and mixed immediately by gently shaking, and then incubated for 20 min at room temperature. After incubation, 1.0 mL K3 medium was added to the tube to dilute PEG.

2.5.2.3 Fluorescence microscopy

After 24 h, GFP fluorescence was observed using a Zeiss Axiophot microscope equipped with a Micromax cooled CCD digital camera, and electronic shutter control.

Images were recorded with Metamorph software http://www.spectraservices.com/METAMRPH.html.

CHAPTER III: RESULTS

3.1 Identification and cloning of plant polyamine transporter genes

A blastp search for the sequences with homology to characterized yeast,

Leishmania major and Trypanosoma cruzi polyamine transporter genes was conducted to identify homologous genes in Arabidopsis (Arabidopsis Genome Initiative, 2000) and rice genomes (Rice Genome Research Program). In addition, phylogenetic analysis also included the sequences of distantly related clades of amino acid transporters from

Arabidopsis. All the sequences were aligned using the ClustalX (Thompson et al., 1997) program. The best alignment was used in PAUP version 4.0 to derive a phylogenetic unrooted tree using parsimony and “neighbor joining”. Bootstrap analysis with 1000 replicates was used to asses the statistical reliability of the tree topology.

The plant sequences clearly cluster on a branch with LmPOT1 and TcPAT12 differing from the yeast proteins (Fig 3.1). The polyamine transporters LmPOT1 and

TcPAT12 clustered in a clade with 11 plant and three transporters from Phytophthora sojae. None of the genes in this clade are characterized for polyamine transport except for LmPOT1 and TcPAT12. No polyamine transporters from yeast clustered in this clade.

For this study, four genes from rice (OsPUT1, OsPUT2, OsPUT3.1 and OsPUT3.2) and three genes from Arabidopsis (AtPUT1, AtPUT2 and AtPUT3) were chosen as candidate polyamine transporters (PUTs). These genes shared from 35% up to 86% identity at the amino acid level (Table 3.2). OsPUT3.1 has an additional 75 amino acids at N-terminal end and OsPUT3.1 and OsPUT3.2 share 100% identity of 550 amino acids while AtPUT1 and AtPUT2 are 74% identical. 24

Using Transmembrane prediction program (TMHMM, http://www.cbs.dtu.dk/services/

TMHMM/), OsPUT1 and AtPUT3 are predicted to have 12 TM domains (Table 3.1) with both termini on the cytosolic side. OsPUT3.1, OsPUT3.2 and AtPUT2 have 10 TM domains with both termini inside. OsPUT2 and AtPUT1 have 9 domains with OsPUT2 having N-terminus in the cytoplasm and C-terminus outside while AtPUT1 has N- terminus outside and C-terminus inside (see Appendix for membrane topology of the

PUT proteins).

Figure 3.1: Phylogenetic analysis of polyamine transporters. Bootstrap values after analysis by parsimony are shown for an unrooted tree. Scale bar indicates the relative length of each branch. Gene IDs are preceded by a species prefix: Arabidopsis thaliana, At; Oryza sativa, Os; Phytophthora sojae, Ps: Saccharomyces cereviseae, Sc; Leishmania major, Lm; Trypanosoma cruzi: Tc. Genes in blue were used as candidates in this research study. 26

Figure 3.2: Sequence alignment of PUTs. OsPUT1, 2, 3.1 and 3.2 from Rice and AtPUT1-3 from Arabidopsis were used to create a sequence alignment by ClustalX algorithm. Black backgrounds show identical amino acids, gray-shaded areas represent similar amino acids and dashes indicate gaps in the sequence to allow maximal alignment.

OsPUT1 ------MADTGGRPEVSLATVRSPGHPAASTTAAAAADLGHADTGQEKPTVESAQPANGAAPMGECGTEYRGLPDGDAGGPMPSSAR OsPUT2 ------MTGACEAAPARRR OsPUT3.2 ------MEDCVGIKYSSVNEGEERKGGHGVP OsPUT3.1 MTNAWISPSVVALCPSPLPSSRLPGSVLSCWPDSRGIRRGAGEGTAGQTLRPARGFTVEKLRNTAITRANSACLPMEDCVGIKYSSVNEGEERKGGHGVP AtPUT1 ------MGDYNMNEFAYGNLYDDDDGDVGGSSKE-GNNSIQ AtPUT2 ------MQKRRIITVNPSASIEMSQYENNEVPYSSVGAD---EVPSSPPK-ATDKIR AtPUT3 ------MTELSSPNLDSASQKPRISTENPPPPPPHISIGVTTGDPATSPAR-TVNQIK

OsPUT1 TVSMIPLIFLIFYEVSGGPFGIEDSVGAAG-PLLAIIGFLVLPVIWSIPEALITAELGAMFPENGGYVVWVASALGPYWGFQQGWMKWLSGVIDNALYPV OsPUT2 GLTVLPLVALIFYDVSGGPFGIEDSVRAGGGALLPILGFLVLPVLWSLPEALVTAELASAFPTNAGYVAWVSAAFGPAAAFLVGFSKWASGTLDKALYPV OsPUT3.2 KVSIIPLIFLIFYEVSGGPFGIEDSVKAAG-PLLAIAGFLLFALIWSVPEALITAEMGTMFPENGGYVVWVSSALGPFWGFQQGWAKWLSGVIDNALYPV OsPUT3.1 KVSIIPLIFLIFYEVSGGPFGIEDSVKAAG-PLLAIAGFLLFALIWSVPEALITAEMGTMFPENGGYVVWVSSALGPFWGFQQGWAKWLSGVIDNALYPV AtPUT1 KVSMLPLVFLIFYEVSGGPFGAEGSVNAAG-PLLALLGFVIFPFIWCIPEALITAEMSTMFPINGGFVVWVSSALGTFWGFQVGWMKWLCGVIDNALYPV AtPUT2 KVSMLPLVFLIFYEVSGGPFGVEDSVNAAG-PLLALLGFVIFPFIWSIPEALITAEMGTMYPENGGYVVWVSSALGPFWGFQQGWMKWLSGVIDNALYPV AtPUT3 KITVLPLVFLIFYEVSGGPFGIEDSVKAAG-PLLAIVGFIVFPFIWSIPEALITAEMGTMFPENGGYVVWVTLAMGPYWGFQQGWVKWLSGVIDNALYPI

OsPUT1 LFLDYLKSGVPALGGGAPRAFAVVGLTAVLTLLNYRGLTVVGWVAICLGVFSLLPFFVMGLIALPKLRPARWLVID--LHNVDWNLYLNTLFWNLNYWDS OsPUT2 LFLDYLRSGGGLVLSPPARSLAVLALTAALTYLNFRGLHLVGLSALALTAFSLSPFVALAVLAAPKIRPSRWLAVN--VAAVEPRAYFNSMFWNLNYWDK OsPUT3.2 LFLDYVKSSIPALGGGLPRTLAVLILTVALTYMNYRGLTIVGWVAVFLGVFSLLPFFVMGLIAIPRIEPSRWLEMD--LGNVNWGLYLNTLFWNLNYWDS OsPUT3.1 LFLDYVKSSIPALGGGLPRTLAVLILTVALTYMNYRGLTIVGWVAVFLGVFSLLPFFVMGLIAIPRIEPSRWLEMD--LGNVNWGLYLNTLFWNLNYWDS AtPUT1 LFLDYLKSAVPALATGLPRVASILILTLLLTYLNYRGLTIVGWTAVFMGVFSMLPFAVMSLVSIPQLEPSRWLVMD--LGNVNWNLYLNTLLWNLNYWDS AtPUT2 LFLDYLKSGVPALGSGLPRVASILVLTILLTYLNYRGLTIVGWVAVLMGVFSILPFAVMGLISIPQLEPSRWLVMD--LGNVNWNLYLNTLFWNLNYWDS AtPUT3 LFLDYLKSGIPILGSGIPRVAAILVLTVALTYLNYRGLSIVGVAAVLLGVFSILPFVVMSFMSIPKLKPSRWLVVSKKMKGVNWSLYLNTLFWNLNYWDS

OsPUT1 ISTLAGEVKNPGKTLPKALFYAVIFVVVAYLYPLLAGTGAVPLDRG-QWTDGYFADIAKLLGGAWLMWWVQSAAALSNMGMFVAEMSSDSYQLLGMAERG OsPUT2 ASTLAGEVEEPRKTFPKAVFGAVGLVVGAYLIPLLAGTGALPSETAGEWTDGFFSVVGDRIGGPWLRVWIQAAAAMSNMGLFEAEMSGDSFQLLGMAEMG OsPUT3.2 ISTLAGEVENPKRTLPRALSYALVLVVGGYLYPLITCTAAVPVVRE-FWTDGYFSDVARILGGFWLHSWLQAAAALSNMGNFVTEMSSDSYQLLGMAERG OsPUT3.1 ISTLAGEVENPKRTLPRALSYALVLVVGGYLYPLITCTAAVPVVRE-FWTDGYFSDVARILGGFWLHSWLQAAAALSNMGNFVTEMSSDSYQLLGMAERG AtPUT1 VSTLAGEVANPKKTLPKALCYGVIFVALSNFLPLLSGTGAIPLDRE-LWTDGYLAEVAKAIGGGWLQLWVQAAAATSNMGMFLAEMSSDSFQLLGMAELG AtPUT2 ISTLAGEVENPNHTLPKALFYGVILVACSYIFPLLAGIGAIPLERE-KWTDGYFSDVAKALGGAWLRWWVQAAAATSNMGMFIAEMSSDSFQLLGMAERG AtPUT3 VSTLTGEVENPSKTLPRALFYALLLVVFSYIFPVLTGTGAIALDQK-LWTDGYFADIGKVIGGVWLGWWIQAAAATSNMGMFLAEMSSDSFQLLGMAERG

OsPUT1 MLPSFFAARSRYGTPLAGILFSASGVLLLSMMSFQEIVAAENFLYCFGMLLEFVAFILHRVRRPDAARPYRVPLGTAGCVAMLVPPTALIAVVLALSTLK OsPUT2 MIPAIFARRSRHGTPTYSILCSATGVVILSFMSFQEIVEFLNFLYGLGMLAVFAAFVKLRVKDPDLPRPYRIPVGAAGAAAMCVPPVVLITTVMCLASAR OsPUT3.2 MLPEFFAKRSRYGTPLIGIMFSAFGVVLLSWMSFQEIIAAENYLYCFGMILEFIAFIKLRVVHPNASRPYKIPLGTIGAVLMIIPPTILIVVVMMLASFK OsPUT3.1 MLPEFFAKRSRYGTPLIGIMFSAFGVVLLSWMSFQEIIAAENYLYCFGMILEFIAFIKLRVVHPNASRPYKIPLGTIGAVLMIIPPTILIVVVMMLASFK AtPUT1 ILPEIFAQRSRYGTPLLGILFSASGVLLLSGLSFQEIIAAENLLYCGGMILEFIAFVRLRKKHPAASRPYKIPVGTVGSILICVPPIVLICLVIVLSTIK AtPUT2 MLPEFFAKRSRYGTPLLGILFSASGVVLLSWLSFQEIVAAENLLYCVGMILEFIAFVRMRMKHPAASRPYKIPIGTTGSILMCIPPTILICAVVALSSLK AtPUT3 MLPEVFAKRSRYRTPWVGILFSASGVIILSWLSFQEIVAAENLLYCFGMVLEFITFVRLRMKYPAASRPFKIPVGVLGSVLMCIPPTVLIGVIMAFTNLK 28

OsPUT1 VAVVSLGAVAMGLVLQPALRFVEKKRWLRFSVNPDLPEIGVIRPPAAPDEPLVP------OsPUT2 TLVVSAAVAVAGVAMYYGVEHMKATGCVEFLTPVPPDSLRGSSSSSSSSAASDNGGDDDVEDVCALLLAAGEHAGEGVSVSKENY OsPUT3.2 VMVVSIMAMLVGFVLQPALVYVEKRRWLKFSISAELPDLPYSNVEEDSTIPLVC------OsPUT3.1 VMVVSIMAMLVGFVLQPALVYVEKRRWLKFSISAELPDLPYSNVEEDSTIPLVC------AtPUT1 VALVSFVMVVIGFLMKPCLNHMDGKKWVKFSVCSDLAEFQKENLDCEESLLR------AtPUT2 VAAVSIVMMIIGFLIHPLLNHMDRKRWVKFSISSDLPDLQQQTREYEETLIR------AtPUT3 VALVSLAAIVIGLVLQPCLKQVEKKGWLKFSTSSHLPNLM------

Table 3.1 GenBank accession numbers and predicted protein characteristics of the seven plant polyamine transporters

Protein GenBank no. No. of amino pI MW TM domains acids OsPUT1 AK068055 531 5.5 54.6 12 OsPUT2 AK099986 496 5.4 51.8 9 OsPUT3.1 AK071314 550 6.8 60.3 10 OsPUT3.2 AK062252 475 5.3 52.5 10 AtPUT1 At1G31820 482 4.9 52.5 9 AtPUT2 At1G31830 495 5.9 54.8 10 AtPUT3 At5G05630 489 9.2 53.5 12 pI, isoelectric point; MW, molecular weight; TM domains, number of transmembrane domains

Table 3.2: Percentages of identity and similarity between PUT proteins

Identity (%) OsPUT1 OsPUT2 OsPUT3.1 OsPUT3.2 AtPUT1 AtPUT2 AtPUT3 OsPUT1 - 42.1 60.4 61.2 55.6 60.7 56.5 OsPUT2 54.6 - 39.6 45.4 42.7 43.5 44.7 OsPUT3.1 72.7 52.4 - 86.3 53.8 60.3 55.6 OsPUT3.2 73.1 60.2 86.3 - 60.8 65.4 60.3 AtPUT1 68.0 58.4 69.2 78.1 - 74.4 61.6 Similarity (%) AtPUT2 73.8 58.1 73.2 78.0 83.3 - 66.0 AtPUT3 70.2 58.9 69.7 75.1 74.8 80.6 -

3.2.1 Functional characterization of PUTs in yeast

3.2.1 Growth assays

To investigate the potential role of these proteins in polyamine transport, cDNA fragment containing the ORF were amplified and inserted into the yeast expression vector, pYES-DEST52 allowing the expression under the control of the GAL1 promoter.

The plasmid constructs harboring the full-length cDNAs of the candidate polyamine transporter genes and the empty vector, pYES-DEST52 were used to transform the yeast mutant, agp2 in which the gene for high affinity spermidine permease is disrupted

(Aouida et al., 2005). Transformants were able to grow on SC-ura medium indicating 30 the presence of the expression vector. agp2 mutant strain is resistant to high concentrations of exogenous polyamines in the synthetic medium whereas wildtype

(BY4741) is unable to grow at very high polyamine concentrations. Complementation studies with wildtype, agp2-vector and candidate gene transformants showed that the expression of polyamine transporters under the control of GAL1 promoter in agp2, partially complemented the agp2 phenotype and conferred sensitivity to high concentrations of polyamines (Fig. 3.3). This clearly suggests the competence for polyamine transport. Transformants also showed sensitivity to paraquat indicating the transport of a polyamine structural analogue. The rate of growth of transformants was slower than the mutant indicating that the candidate transporters may not be expressed at a high level or be fully functional in yeast.

3.2.2 Transport of polyamines by PUTs

Further characterization of these genes in terms of their ability to uptake polyamines was done by transport experiments using radiolabeled spermidine and putrescine at a concentration of 15 μM. Yeast transformants of all the candidate genes tested showed increased uptake of spermidine than the agp2 mutant with the empty vector (Fig 3.4A). The uptake was linear up to 15 min. Among the PUTs tested, OsPUT2 and OsPUT3 showed significantly higher spermidine uptake compared to the other PUTs and OsPUT1 showed minimum enhancement in spermidine uptake. All the PUTs except

OsPUT3.2, showed enhanced putrescine uptake than the agp2 mutant with the empty vector (Fig 3.4B). OsPUT2 showed highest putrescine uptake among all the PUTs tested.

The uptake of putrescine by OsPUT3.2 was not significant than the vector control. 31

Figure 3.3: Functional complementation of OsPUT1-3.2 and AtPUT1-3 in yeast mutant agp2. WT (BY4741), agp2- PUTs and agp2-empty vector strains were grown overnight on SC medium supplemented with 2% galactose. The density of exponentially growing cell cultures was normalized to an OD600 of 0.5. Cell suspensions were serially diluted as indicated and 3 μl of each were spotted onto YP-galactose plates containing 25 mM spermidine, 200 mM putrescine or 1.5 mM paraquat. Plates were photographed after 3 -4 d of incubation at 30°C. The data is representative of three independent experiments.

Figure 3.4: Time course uptake of [14C]spermidine and [14C]putrescine. Log phase cells were incubated with [14C]spermidine (A) and [14C]putrescine (B) for 0, 5, 10 and 15 min to determine the intracellular amount of radiolabeled spermidine or radiolabeled putrescine. agp2 transformed with PUTs showed a significantly higher flux of spermidine and putrescine compared to the empty vector control.

3.2.3 Transport capabilities of PUTs

For the correct characterization of biochemical properties, Km and Vmax values were determined in the linear range of uptake (5 min). All the candidate transporters showed a clear Michealis-Menten kinetics with most of them having high affinity to spermidine and much lower affinity for putrescine (Fig 5 and Fig 6). OsPUT3.2 showed low affinity for spermidine (57.4μM). AtPUT2 showed very high affinity for spermidine

(0.94 μM) and a low affinity for putrescine (43.2 μM). OsPUT1 exhibited a moderate affinity for spermidine (15.2 μM). Putrescine was also taken up by all the PUTs except

OsPUT1 and OsPUT3.2, but with a much lower affinity. Table 2 summarizes the affinity constants of the transporters.

Table 3.3: Affinity constants of PUTs for spermidine and putrescine

Km values were obtained by incubating yeast cells, expressing the respective proteins in [14C]spermidine and [14C]putrescine for 5 min. Results are means ± SD for three independent experiments. ND, not determined.

Spermidine Putrescine Specific inhibitor for Transporter Vmax (pM/107 Vmax (pM/107 spermidine uptake (<70% Km (μM) Km (μM) cells/5 min) cells/5 min) residual uptake) methionine, asparagine, OsPUT1 15.2 ± 1 7.9 ± 0.4 ND ND glutamine, spermine OsPUT2 2.1 ± 0.3 8 ± 0.4 26.7 ± 4 8.7 ± 0.8 leucine, tyrosine OsPUT3.1 4.9 ± 0.8 10.3 ± 0.6 29.3 ± 7 32.9 ± 5 ornithine, spermine OsPUT3.2 57.4 ±2 24.64 ± 2 ND ND ND AtPUT1 3.3 ± 0.9 8.1 ± 0.7 28.7 ± 5 6.5 ± 0.7 leucine AtPUT2 0.94 ± 0.2 7.1 ± 0.4 43.2 ± 5 11.7 ± 0.8 None AtPUT3 1.2 ± 0.4 5 ± 0.4 32.6 ± 4 8.2 ± 0.6 leucine

Figure 3.5: Determination of Km values of PUTs for [14C]spermidine uptake in yeast. Michealis-Menten curves for [14C]spermidine uptake were obtained by subtracting the uptake in vector control expressing cells from that in PUT transformant cells. Cells were incubated with increasing concentrations of spermidine for 5 min, before determination of intracellular radioactivity.

Figure 3.6: Determination of Km values of PUTs for [14C]putrescine uptake in yeast. Michealis-Menten curves for [14C]putrescine uptake were obtained by subtracting the uptake in vector control expressing cells from that in PUT transformant cells. Cells were incubated with increasing concentrations of spermidine for 5 min, before determination of intracellular radioactivity. 37

3.2.4 Substrate specificity of PUTs

To test whether putrescine competes with spermidine for uptake by candidate polyamine transporters, the uptake of [14C]spermidine was measured in the presence of spermidine and putrescine added, either singly or in combination, at concentrations of 15

μM or 100 μM. For all the PUTs tested, the uptake of [14C]spermidine was not significantly inhibited by 15 μM or 100 μM unlabeled putrescine (Fig. 3.7). However, 15

μM unlabeled spermidine greatly inhibited the [14C]spermidine and same was the observation when both 15 μM unlabeled spermidine and 15 μM unlabeled putrescine were added. While the PUTs analyzed, also function as a putrescine transporters, uptake rates were not sufficiently higher than background levels in agp2 mutant to determine the competitive effects of other substrates (data not shown).

To analyze whether structurally related compounds affect the polyamine transport, competition experiments were conducted. For this the uptake of

[14C]spermidine at a concentration of 15 μM (OsPUT1), 2 μM (OsPUT2 and OsPUT3.1),

3 μM (AtPUT1) and 1 μM (AtPUT2, AtPUT3) was studied in the absence or presence of

10 fold excess of amino acids, polyamines and other polyamine precursors. Strongest competitors for [14C]spermidine uptake were leucine (OsPUT2, AtPUT1 and AtPUT3) and spermine (OsPUT1, OsPUT3.1) (Fig. 3.8, Table 3.4). All other substrates had little or no effect on spermidine uptake.

Figure 3.7: Uptake of [14C]spermidine by PUTs in the presence of excess substrates. Spermidine was provided at a concentration of 15 μM (OsPUT1), 2 μM (OsPUT2 and OsPUT3.1), 3 μM (AtPUT1) and 1 μM (AtPUT2, AtPUT3) and yeast cells were incubated for 2 min. Spermidine (SPD) and putrescine (PUT) were added, either singly or in combination, at concentrations of 15 μM or 100 μM, along with [14C]spermidine. The results were expressed as percent label uptake. Three independent experiments were performed (data points represent means ±SD). 39 40

Figure 3.8. Uptake of [14C]spermidine by agp2-PUTs in the presence of amino acids as substrates.

Spermidine was provided at a concentration of 15 μM (OsPUT1), 2 μM (OsPUT2 and OsPUT3.1), 3 μM (AtPUT1) and 1 μM (AtPUT2, AtPUT3) and yeast cells were incubated for 2 min. 500 μM concentrations of several amino acids and other alternate substrates were added, along with [14C]spermidine. The results were expressed as percent label uptake. Two to three independent experiments were performed (data points represent means ±SD).

Table 3.4: Effect of various amino acids and polyamine precursors on spermidine uptake by PUTs. Spermidine was provided at a concentration of 15μM (OsPUT1) , 2 μM (OsPUT2, OsPUT3.1), 3 μM (AtPUT1) and 1 μM (AtPUT2, AtPUT3) and yeast cells were incubated for 5 mins with the inhibitors (500μM) . The numbers represent percent reduction in uptake of [14C]spermidine. Results are means ±SD for three independent assays.

Amino acid / OsPUT1 OsPUT2 OsPUT3.1 AtPUT1 AtPUT2 AtPUT3 Polyamine precursor None 100 ± 2 100 ± 1 100 ± 10 100 ± 4 100 ± 8 100 ± 1 Arginine 6.2 ± 16 18.2 ± 15 5.4 ± 12 9.0 ± 5 9.3 ± 5 12.4 ± 16 Aspartate 5.9 ± 6 19.3 ± 12 3.6 ± 4 13.5 ± 0 6.5 ± 5 14.1 ± 4 Glutamate 7.2 ± 6 25.8 ± 8 8.7 ± 2 3.7 ± 10 2.5 ± 10 12.7 ± 10 Histidine 24.4 ± 1 25.5 ±2 16.2 ± 6 1 4.0 ± 6 1.4 ± 8 22.9 ± 7 Lysine 25.4 ± 12 17.5 ± 9 23.8 ± 2 4.5 ± 7 11.5 ± 7 11.4 ± 7 Glycine 2.0 ± 4 29.5 ± 3 8.3 ± 10 14.5 ± 7 3.2 ± 1 9.5 ± 6 Methionine 32.9 ± 1 26.5 ± 7 3.2 ± 14 9.8 ± 8 15.4 ± 7 34.0 ± 7 Leucine 13.7 ± 1 34.5 ± 4 7.9 ± 2 26.4 ± 8 19.4 ± 1 41.2 ± 4 Asparagine 44.3 ± 6 14.2 ± 3 10.8 ± 1 14.8 ± 8 1.8 ± 3 18.0 ± 1 Tyrosine 19.9 ± 4 34.2 ± 8 20.9 ± 1 3.7 ± 6 1.4 ± 2 8.8 ± 8 Tryptophan 12.4 ± 1 22.9 ± 0 4.0 ± 1 10.3 ± 7 7.2 ± 8 8.5 ± 7 Glutamine 35.5 ± 6 4.7 ± 17 4.7 ± 13 10.3 ± 8 3.9 ± 8 13.1 ± 8 Cadaverine 24.4 ± 13 23.6 ± 10 6.1 ± 0 16.9 ± 5 12.9 ± 2 8.8 ± 5 Agmatine 4.6 ± 5 26.5 ± 3 19.9 ± 1 14.0 ± 7 5.4 ± 7 12.4 ± 7 Ornithine 26.7 ± 13 5.5 ± 13 38.3 ± 1 15.0 ± 4 4.7 ± 4 15.0 ± 4 Spermine 33.6 ± 3 2.5 ± 6 48.4 ± 4 25.1 ± 4 18.3 ± 3 12.4 ± 6 42

3.2.2 Tissue specific expression of PUTs

3.3.1 Expression of PUT genes by RT-PCR

The expression of PUT genes was examined in various plant tissues by RT-PCR. Since

PUTs share partial homology, gene specific primers were designed in regions with higher variability. Rice and Arabidopsis actin mRNA was amplified as an internal control. Among the rice genes, OsPUT1 was not expressed in roots and seeds and expression of OsPUT3.2 could not be detected in roots (Fig 3.9A). OsPUT2 and OsPUT3.1 was expressed constitutively. AtPUT2 mRNA was detected in all tissues analyzed while AtPUT1 and AtPUT3 showed a tissue specific expression (Fig 3.9B). A high level of expression of OsPUT3.1 was observed in flowers and panicles.

A B

Figure 3.9: Expression of PUT genes in rice (A) and Arabidopsis (B) plants. Rice plants were grown in the greenhouse conditions for the collection of all the tissues except roots. Root tissue was collected from petridish grown rice plants. All the tissues except seedlings of Arabidopsis were collected from growth chamber grown plants. Arabidopsis seedlings were grown on 0.5x MS medium and harvested after 8 days of germination. Rice actin and Arabidopsis actin genes were used as internal controls.

3.3.2 AtPUT2 and AtPUT3 gene expression in plant organs by GUS reporter assays

To localize the expression of AtPUT2 and AtPUT3 genes at the cellular level, the promoters of these genes were cloned and used them to drive the expression of GUS as reporters

(Fig 3.10 and Fig 3.11). Arabidopsis plants expressing AtPUT2:GUS and AtPUT3:GUS constructs were produced and used for histochemical analysis of GUS activity in different plant tissues.

2.9 kb of the AtPUT2 promoter region was used to drive the expression of GUS reporter.

In AtPUT2:GUS transgenic plants, the GUS acitivity was detected in the vascular tissues of both cauline and rosette leaves (3.10A, C and F). GUS expression was also observed in the roots, flower and flower bud (Fig 3.10). Dark staining was observed in the tip of pedicel of the young silique (Fig 3.10D) and no staining was seen along the rest of the pedicel. 821 bp of the AtPUT3 promoter driven GUS was expressed very weakly in the vascular tissue of the mature rosette leaves. Weak stain was also observed in the flower. Strong staining was detected along the vascular bundles in the center of the roots (Fig 3.11A). The expression of AtPUT2 was slightly higher than AtPUT3 in vascular tissues of mature leaves. None of these genes were expressed in root hairs.

Figure 3.10: GUS-expression driven by AtPUT2 promoter in Arabidopsis. A-cauline leaf. Srong staining observed in the veins; B- Axillary bud; C- close-up of a rosette leaf. First true; D-inflorescence, Note the dark staining of the tip of the pedicel of the silique ; E- 3 week old plant; F-Close-up of a cauline leaf. Note a relatively strong GUS staining in the veins 45

Figure 3.11: GUS-expression driven by AtPUT3 promoter in Arabidopsis. A-Strong staining in the Root. Note that the root hairs lack GUS staining; B- root staining in a 3 week old plant; C- vascular tissues of the flower; D-vascular tissues of leaves of a seedling; E- close-up of a leaf of 3 week old plant

3.4 Subcellular localization of PUT genes

3.4.1 In Silico Analysis of Subcellular Localization of plant polyamine transporters

Heterologous expression of plant polyamine transporters in yeast indicated that these transporters are membrane localized, most likely to the plasma membrane. In order to test the reliability of this hypothesis, the subcellular localization prediction of PUTs was carried out using web-based programs WoLF PSORT (plant specific) (Horton et al, 2007), Noble

Foundation prediction (http://bioinfo3.noble.org/AtSubP/submit.html), TargetP (Emanuelsson et al., 2000) and Predator (Small et al., 2004). The results of in silico localization prediction performed are summarized in Table 3.5.

Table 3.5: Predicted localization of plant transporters Noble Foundation Gene WolFPSORT TargetP Predator prediction

OsPUT1 Cytoplasm Cytoplasm None None OsPUT2 Plasma membrane Chloroplast Mitochondria None OsPUT3.1 Mitochondria Mitochondria Chloroplast Chloroplast OsPUT3.2 Plasma membrane Unknown None None AtPUT1 Plasma membrane Plasma membrane None None AtPUT2 Plasma membrane Unknown None None AtPUT3 Chloroplast Chloroplast Chloroplast None

3.4.2 Subcellular localization of plant polyamine transporters in vivo

3.4.2.1 Transient expression of PUTs in onion epidermal cells

In order to study the localization in vivo, transient expression of PUTs was carried out in onion epidermal cells. Full-length coding sequences were amplified from and the amplified fragments were introduced in to pENTR/D-TOPO cloning vector (Invitrogen). After 47 confirmation of the sequences, they were cloned in frame with GFP in Gateway destination vectors pGWB6 (N-terminal GFP construct driven by CaMV 35S promoter) and pGWB5 (C- terminal GFP construct driven by CaMV 35S promoter) (Nakagawa et al., 2007). The fusion constructs were transiently expressed in onion epidermal cells. For all the PUTs except

OsPUT3.1, GFP fluorescence was observed all over the cells, including the nuclei (Fig 3.12 and

3.13). Fluorescence of cytoplasmic strands was clearly visible, suggesting that both GFP-PUT proteins must be distributed in the cytosol. Only OsPUT3.1with C-terminal GFP was found to be localized to the mitochondria in consistent with WolFPSORT and Noble foundation prediction

(Table 3.5). However, no localization was observed for AtPUT3.

Figure 3.12: Transient expression of GFP-OsPUT fusion proteins in onion epidermal cells

The constitutive 35S CaMV promoter was used to express these fusion proteins in onion epidermal cells. OsPUT1, OsPUT2 and OsPUT3.2 localized to the cytoplasm and OsPUT3.1 was localized to the mitochondria. OsPUT3.1- first panel is mitotracker staining, second panel is GFP florescence and third panel is superimposed picture.

Figure 3.13: Transient expression of GFP-AtPUT fusion proteins in onion epidermal cells

The constitutive 35S CaMV promoter was used to express these fusion proteins in onion epidermal cells. AtPUT1, AtPUT2 localized to the cytoplasm.

3.4.2.2 Transient expression of OsPUTs in rice protoplasts

Since the transient expression of PUTs in onion epidermal cells showed no clear localization except for OsPUT3.1, OsPUT gene – GFP fusion proteins were transiently expressed in rice protoplasts. GFP fluorescence for OsPUT1, OsPUT2 and OsPUT3.2 was detected along the plasma membrane and also in the cytoplasm (Fig 3.14). OsPUT3.1with C-terminal GFP was found to be localized to the chloroplast of rice protoplasts (Fig 3.14). 51

Figure 3.14: Subcellular localization of OsPUT – GFP proteins in rice protoplasts.

OsPUT1, OsPUT2 and OsPUT3.2 are localized to the plasma membrane. OsPUT3.1 is localized to the chloroplasts.

3.4.3 Localization of OsPUT3.1 to the mitochondria and the chloroplast

Online prediction programs predicted OsPUT3.1 to either the mitochondria or chloroplast

(Table 3.5). Transient expression of OsPUT3.1:GFP in onion epidermal cells showed that the protein is exclusively localized to the mitochondria. However, transient expression of the same protein in rice protoplasts was carried out; interestingly OsPUT3.1:GFP was found to be localized to the chloroplasts (Fig 3.15).

Figure 3.15: Transient expression of OsPUT3.1 in onion epidermal cells (A-C) and rice protoplasts (D-F)

A – Mitotracker stained onion epidermal cell B – GFP:OsPUT3.1 florescence in onion epidermal cell C - Superimposed picture of A and B D – Chlorophyll autofluorescence in rice protoplasts E – GFP:OsPUT3.1 expression in rice protoplasts F – Superimposed image of D and E. Observed green fluorescence corresponds to the red autofluorescence of chlorophyll. In onion epidermal cells, OsPUT3.1 is localized to the mitochondria while in the rice protoplasts, OsPUT3.1 is localized to the chloroplast

3.5 OsPUT3.1 and OsPUT3.2 are alternatively spliced genes

In the rice genome, the transcripts OsPUT3.1 and OsPUT3.2 are from the same locus (Fig.

3.15).

A B

Figure 3.16: Intron-exon structure of: A– (OsPUT3.1) AK071314, B–(OsPUT3.2) AK062252. Exons are represented by blue bars.

OsPUT3.1 is longer than OsPUT3.2 by 75 amino acids and OsPUT3.2 share 100% identity of 550 amino acids. Both the proteins have 10 predicted transmembrane domains. To test the substrate specificity of the two isoforms, these two genes were heterologously expressed in agp2 yeast mutant. The radiolabled transport experiment revealed that OsPUT3.1 is a high affinity spermidine transporter (4.9 μM) and low affinity putrescine transporter (29.3 μM).

Interestingly, OsPUT3.2 appeared to be a low affinity spermidine transporter (57.4 μM).

Putrescine uptake by OsPUT3.2 was not significantly higher than the vector control.

The expression of OsPUT3.1 and OsPUT3.2 genes was examined in various plant tissues by

RT-PCR (Fig 3.9A). Both isoforms were expressed in all the tissues except roots. Computer predictions suggest that OsPUT3.2 is localized to the plasma membrane, and OsPUT3.1 is localized to the chloroplast/mitochondria (Table 3.5). To confirm the subcellular localization of these two genes, transient expression in onion epidermal cells was performed. OsPUT3.1:GFP was localized to the mitochondria (Fig 3.15D-F) and OsPUT3.2:GFP fluorescence was observed all over the cells including the nucleus (Fig 3.12) indicating no proper localization. When the GFP fusions of

OsPUT3.1 and OsPUT3.2 were transiently expressed in rice protoplasts, OsPUT3.1 localized to the chloroplast and OsPOOT3.2 was localized to the plasma membrane and cytoplasm.

CHAPTER IV: DISCUSSION

Polyamines are shown to be involved in many physiological processes and are implicated in various abiotic stress resistances like chilling, salt, drought and pathogen attack. Biosynthesis, degradation and conjugation of polyamines have been extensively studied. However, not much information is available on the transport of polyamines in and out of the cell in plants. Transport and compartmentation of polyamines are important to further refine our knowledge about their metabolism and their role in stress resistance.

In the present work, the sequence information and the yeast mutant available was used identify and characterize putative polyamine transporters in rice and Arabidopsis.

4.1 Phylogenetic analysis

Phylogenetic analysis indicated that 10 plant genes clustered in clade with LmPOT1 and

TcPAT12, the two characterized protozoan polyamine transporters (Fig. 3.1). Therefore these plant genes appear to be related orthologs. None of the yeast polyamine transporters was identified in that cluster. Amino acid sequences from Arabidopsis are clustered in a different clade. For this study, four genes from rice (OsPUT1, OsPUT2, OsPUT3.1 and OsPUT3.2) and three genes from Arabidopsis (AtPUT1, AtPUT2 and AtPUT3) were chosen as candidate polyamine transporters (PUTs). The size of PUT proteins ranges from 475 – 550 amino acids

(Table 3.1). The predicted molecular weights of PUTs (Table 3.1) are approximately the same

(about 52-54 kDa), except OsPUT3.1, which is a little larger due to additional stretches (60 kDa) and is predicted to be targeted to mitochondria / chloroplast. In zucchini hypocotyls, spermidine was associated with 66 and 44 kDa proteins (Tassoni et al., 1998). All the PUTs chosen for the study have been predicted to be membrane proteins with 9-12 transmembrane (TM) domains 55 which is in agreement with two yeast polyamine preferential uptake proteins which have 12 TM domains (Uemura et al., 2007). Table 3.2 demonstrates the percent homology between the PUTs based on the alignment.

4.2.1 Polyamine transport by PUTs

Yeast has already proved to be a useful system for functional analysis of some plant amino acid transporters (Wittstock et al., 2000; Chen et al., 2001; Hsu et al., 1993). There are already a number of simple growth assays that can be used to prescreen polyamine transporters.

A particular advantage for using yeast as a model system to study polyamine transport is that in addition to transporter knockouts, there are a number of simple growth assays that can be used to prescreen heterologously expressed polyamine transporters.

4.2.1 PUTs mediate the transport of polyamines and paraquat

To shed light on the transport capabilities of PUTs, the full-length cDNAs of these genes were introduced into the yeast mutant, agp2 which is defective in high affinity spermidine uptake. High concentrations of polyamines or structural analogues such as paraquat in the media are toxic to wildtype yeast cells but the transport defective agp2 mutant cells are less sensitive to spermidine, putrescine and paraquat (Aouida et al., 2005). Hence, the initial growth analysis of agp2 cells expressing PUTs indicated that these genes transport polyamines by showing increased sensitivity to exogenous spermidine, putrescine and the herbicide paraquat (Fig 3.3). In maize seedlings, polyamine feeding experiments have shown that exogenously supplied polyamines can significantly inhibit the uptake of paraquat from the media (Hart et al., 1992).

These observations have provided the basis for the assumption that polyamine transporters 56 facilitate the movement of this herbicide throughout the plant (Hart et al., 1992). In the present study it is confirmed that PUTs can complement the paraquat transporting activity of yeast agp2 cells. Direct measurement of [14C]spermidine and [14C]putrescine by PUTs with agp2 and wildtype as controls, also demonstrated their ability to take up polyamines (Fig. 3.4).

4.2.2 PUTs (except OsPUT3.2) are high affinity spermidine and low affinity putrescine transporters

All the PUTs except OsPUT3.2 exhibited high affinity spermidine transport with Kms ranging from 0.9 μM (AtPUT3) to 15 μM (OsPUT1). These results are consistent with other reported spermidine transporters from yeast (Uemura et al., 2007) and human erythrocytes

(Fukumoto and Byus, 1996). OsPUT3.2 appeared to be a low affinity spermidine transporter with a Km of 57 μM. All the PUTs except OsPUT1 and OsPUT3.2 were found to be low affinity putrescine transporters. Uptake studies in carrot vacuoles showed a saturable spermidine transporter system with a Km of 61 μM (Pistocchi et al., 1988). Similarly, in sunflower mitochondria, a spermidine uptake system with a Km of 89 μM was identified (Pistocchi et al.,

1990).

4.2.3 PUTs are spermidine specific transporters

The uptake of radiolabeled spermidine by PUTs was also assessed in competition with 25 fold higher levels of amino acids, polyamines and polyamine precursors (Fig. 3.8). For

OsPUT1, the amino acids asparagine, glutamine and methionine inhibited uptake by 30% or more. Cadaverine, agmatine and ornithine had minimal effects, while high levels of spermine inhibited uptake by only 30%. Leucine inhibited spermidine uptake by OsPUT2 (34%), AtPUT1 57

(26%) and AtPUT3 (41%). In these experiments, inhibition of spermidine uptake can be due to either direct competition with spermidine for the transporter, or transporter competition for free energy. By comparison, uptake of arginine by AtCAT5 was reduced to less than 20% of control rates in the presence of 10 fold higher levels of the basic amino acids lysine and histidine. Lower levels of inhibition of AtCAT5 by other competing substrates (>55% of control rates) were attributed to some combination of direct competition for the binding site and competition for energy by other yeast transporters.

4.2.2 Tissue specific expression of PUTs

4.3.1 Expression of PUT genes by RT-PCR

Phylogenetic analysis identified 10 potential candidates as plant polyamine transporters.

Preliminary characterization of seven (4 in rice and 3 in Arabidopsis) of those genes by heterologous expression in yeast system revealed that six of them are high affinity spermidine transporters. These results give raise an important question – why there are multiple transporters with similar substrate specificities in a species? Table 3.2 shows that PUTs have undergone diversification in their amino acid sequences. AtPUT1 and AtPUT2 are two Arabidopsis genes which show a high similarity percentage at the amino acid level and are located in tandem on chromosome 1 indicating that they are result of a duplication event. Similarly, rice genes

OsPUT3.1 and OsPUT3.2 are of the same locus. These observations support the hypothesis of evolutionary gene duplication. Sequence divergence is often associated with functional divergence and thus contributes to the evolution of metabolic diversity in plants (Richmond and

Somerville, 2000). 58

Semiquantitative RT-PCR of PUTs showed variable expression at the tissue level (Fig

3.9). This is in consistent with publicly available microarray data

(http://www.ricearray.org/expression/ expression.php; https://www.genevestigator.ethz.ch/;

Zimmermann et al. (2004)). OsPUT1 expression was very low (Fig 3.9A) as 40 cycles of PCR were necessary to get expression to be observed on the gel. This is in consistent with the rice microarray data where the expression of OsPUT1 is low (http://www.ricearray.org/expression/ expression.php). Overlapping expression of PUT genes was also observed. For instance, all rice

PUTs tested are expressed in leaves, stems, flowers and panicles. OsPUT2 and OsPUT3.1 are ubiquitously expressed in all the tissues analyzed. RNA gel blot also showed that AtPUT1 is not expressed in the roots and AtPUT3 is not detected in leaves. Polyamines are implicated to the variety of plant growth and development processes. Diversity of the expression of PUTs confirms their diverse roles.

4.3.2 AtPUT2 and AtPUT3 promoter - GUS assays

Expression analysis of AtPUT2 (Fig 3.10) and AtPUT3 (3.11) was carried out using

AtPUT promoter – GUS constructs to observe the role of AtPUTs during plant growth and development. The expression analysis conducted using promoter – GUS constructs also in congruence with the RT-PCR expression profile of the AtPUTs. Expression of AtPUT2 and AtPUT3 in the vascular tissue of rosette leaves (Fig 3.10C) and cauline leaves (Fig 3.10A) suggests that they are involved in the long distance transport of polyamines from source tissues to sink tissues, such as developing floral organs. It is known that polyamines are transported over long distances between plant organs (Antognoni et al., 1998; Ohe et al., 2005). Large amounts of polyamines were detected in xylem and phloem vessels of different plant species (Friedman et al., 1986; 59

Shevyakova et al., 2001; Kuznestov et al., 2002). Strong staining at the tip of the pedicel (Fig

3.10D) just below the silique suggests that AtPUT2 plays a role in polyamine supply for the developing silique. Thus ATPUT2 gene seems to function in almost all the tissues. AtPUT3 expressed strongly in root vascular tissue (Fig 3.11A) and root of a 3 week old plant (Fig 3.11B).

Lack of expression of AtPUT3 in the root hairs and root tips suggests that this gene is not involved in the polyamine uptake from soil but facilitates long distance polyamine transport and redistribution within the plant. It appears to be involved in polyamine transport from the root cortex to the stele, where it can be loaded into the xylem and transported to other parts of the plant. Similar results were obtained in expression of Arabidopsis peptide transporters (AtOPTs)

(Stacey et al., 2006). Stronger GUS staining of AtPUT2 was observed also in the young axillary bud (Fig 3.10B) which is in the developmental stage than older flowers (Fig 3.10D). The reason may be that developing bud requires more polyamines. Lighter staining was also observed in the floral stigma.

4.2.3 Subcellular localization of PUT genes

Since, biosynthesis, degradation and conjugation of polyamines in the plant cells is highly compartmentalized, information on subcellular localization of the polyamine transporters would facilitate in understanding the physiological roles of these plant PUTs. N- or C –terminal

GFP –PUT GFP fusion constructs were analyzed by the transient expression in onion epidermal cells or rice / Arabidopsis protoplast cells.

4.2.4 In silico analysis of subcellular localization of PUTs

The in silico analysis of the localization of PUTs using several web-based programs

(Table 3.5) predicted different cellular locations. WoLF PSORT predicted most of the PUTs to be localized to the plasma membrane except OsPUT3.1 and AtPUT3.

4.4.2 Subcellular localization of plant polyamine transporters in vivo

CaMV 35S promoter driven GFP-PUT gene constructs were transiently expressed in onion epidermal cells. Twenty four hours after bombardment, fluorescence was recorded to identify the subcellular localization of different PUT genes. For all the PUTs except OsPUT3.1,

GFP fluorescence was observed all over the cells, including the nuclei (Fig 3.12 and 3.13). The results were in contradiction to the online predictions. Since no clear compartment could be assigned to the PUT proteins except OsPUT3.1, the GFP-OsPUT gene constructs were transiently expressed in rice protoplasts (Fig 3.14). In this experiment, OsPUT1, OsPUT2 and

OsPUT3.2 were localized to the plasma membrane and fluorescence was also observed throughout the cytoplasm. The cytoplasmic fluorescence could be due to the high expression of these constructs due to the strong 35S CaMV promoter or protein failed localize to a specific cellular compartment. In rice protoplasts, OsPUT3.1 was localized to the chloroplasts.

Sequencing of the gene-GFP constructs indicated that no sequence aberrations were present.

Vector pGWB6, utilized for transient expression, has been successfully used by other research groups. One of the plausible reasons for the failure to localize to a particular organelle or membrane could be the prevention of the proper folding of GFP by the interference of transmembrane domains of the native protein. To resolve the problem of mislocalization several alternative approaches that can be used. Instead of the full-length protein, only the N or C 61 terminal half of the gene fused with the GFP can be used to localize the protein. Another approach could be immunolocalization using immunogold labeled antibodies. To successfully observe the localization via this approach, expertise and availability of an electron microscope is necessary. However, this approach will be difficult to use if the proteins have reasonably high similarity (like AtPUT1 and 2) as finding a peptide motif specific to a PUT protein is difficult.

Also, generation of antibodies specific to different PUT proteins is expensive. Another way one could try to localize PUT genes to a cellular compartment is by fusing the protein to an epitope

(HA or myc) tag and detecting the localization using specific antibodies.

Online prediction programs predicted OsPUT3.1 to either mitochondria (WoLFPSORT,

Noble Foundation Program) or chloroplast (TargetP and Predator). Transient expression of

OsPUT3.1 in onion epidermal cells showed that the protein is exclusively localized to the mitochondria. However, when transient expression of the same protein in rice protoplasts was carried out, interestingly OsPUT3.1 was found to be localized to the chloroplasts (Fig 3.15).

There seems to be a discrepancy in the expression of the same fusion protein in onion and rice. If this protein is not targeted to both organelles, further study is required to find out the correct localization in the plant. In case, the correct localization is the chloroplast, then it is probably located on the inner envelope.

4.5 Do isoforms OsPUT3.1 and OsPUT3.2 have different substrate specificities and subcellular localizations?

The rice genes OsPUT3.1 and OsPUT3.2 are from the same locus (Fig. 3.16) with

OsPUT3.1 75 amino acids longer than OsPUT3.2. OsPUT3.1 gene has one intron and OsPUT3.2 has none. The radiolabled transport experiment showed that OsPUT3.1 is a high affinity 62 spermidine transporter (4.9 μM) and low affinity putrescine transporter (29.3 μM). On the other hand, OsPUT3.2 appeared to be a low affinity spermidine transporter (57.4 μM). The Km for putrescine uptake by OsPUT3.2 was not determined as the uptake was barely above background.

Semiquantitative RT-PCR revealed similar expression patterns for both OsPUT3.1 and

OsPUT3.2 (Fig 3.9A). Online prediction programs predicted OsPUT3.1 to either mitochondria or to the chloroplast and OsPUT3.2 to the plasma membrane (Table 3.5). Transient expression of both genes in onion epidermal cells localized OsPUT3.1 to the mitochondria and OsPUT3.2 to all over the cell. In a study with a divalent metal transporter 1 (DMT1) isoforms in human larynx carcinoma cells, distinct localization of the isoforms observed (Tabuchi et al., 2002). Yeast complementation of DMT1 isoforms did not show any difference in the phenotype. One isoform was localized to lysosomes and late endosomes while the other isoform was localized in early endosomes. This suggests that alternative translational start sites may be a common mechanism in eukaryotes to direct the same transporter to different subcellular compartments.

4.5.2 Conclusions

Polyamine transport in plants has still not been characterized at the molecular level. The results presented in this study describe the transport capabilities of a clade of previously uncharacterized membrane transporters from rice and Arabidopsis. As a preliminary characterization, four candidate genes from rice and three candidate genes from Arabidopsis were heterologously expressed in a yeast mutant system to identify the substrate specificity of these genes. Furthermore, promoter-GUS fusions assays and semiquantitative RT-PCR were carried out to understand the tissue specificity of PUTs. The characterization of PUTs also 63 included their subcellular localization by transient expression assays in onion epidermal cells and protoplasts.

The results of the study can be summarized as follows:

Computational analysis resulted in a distinct group of ten candidate polyamine

transporter genes from rice and Arabidopsis

Heterologous expression of PUTs in the agp2 yeast mutant showed that all seven PUTs

analyzed were polyamine transporters. Except for OsPUT3.2, all the PUTs are high

affinity spermidine importers and low affinity putrescine transporters.

Analysis of PUTs by RT-PCR revealed that they have diverse tissue expression.

GUS – reporter analysis showed that AtPUT2 and AtPUT3 are expressed mainly in the

vascular bundles indicating that they have a role in long distance transport of polyamines.

Transient expression of PUTs in onion epidermal cells showed no clear organellar

localization among the PUTs except OsPUT3.1. OsPUT3.1 appears to be localized to the

mitochondria.

Transient expression in rice protoplasts revealed that OsPUT3.1 might be localized to

chloroplast.

OsPUT3.1 and OsPUT3.2 are alternatively spliced forms of the same rice gene and

appear to have different affinities for spermidine uptake. Transient expression showed

OsPUT3.1 localized to the mitochondria (in onion) and chloroplast (in rice protoplast).

This work has shown that use of GFP-gene fusions might not be a good strategy to

resolve the subcellular localizations of PUTs.

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APPENDIX

Abbreviations

ABC transporter ATP Binding Cassette transporter

A .thaliana Arabidopsis thaliana

A. tumefaciens Agrobacterium tumefaciens

BLAST Basic Local Alignment Search Tool bp base pairs cDNA complementary DNA

CAT Cationic Amino acid Transporter

DNA Deoxyribonucleic acid

GABA Gamma Amino Butyric Acid

GFP Green Fluorescent Protein

GUS -glucurodinase kb kilo base kDa kilo Dalton mRNA messenger RNA nd not determined

ORF open reading frame

PCR polymerase chain reaction

PEG polyethylene glycol

PUT Polyamine Uptake Transporter

RT-PCR Reverse Transcriptase Polyamerase Chain Reaction

SC Synthetic Complete complements or medium 75

Tm melting temperature

TM Transmembrane domain

X-Gluc 5-bromo-4-chloro-3-indolyl--galactopyranoside

YPD Yeast extract Peptone Dextrose medium

YPG Yeast extract Peptone Galactose medium

Vector maps used in the study

Figure S1: Yeast Destination vector – pYES-DEST52 (Invitrogen, Carlsbad, USA) 77

Figure S2: GATEWAY GUS expression vector – pBGWFS7 (Karimi et al., 2002)

Figure S3: GATEWAY plant GFP expression vector – pGWB5/B6 (Nakagawa et al., 2007)

Table S1: Primer sequences used in this study

Primer Sequence 5’ to 3’ Primers used for GATEWAY cloning to create en try clone OsPUT1F CACCATGGCGGACACCGGCGGACG OsPUT1R CTACGGAACCAACGGCTCGTCCG OsPUT2F CACCATGACCGGAGCCTGCGAGGC OsPUT2R CTAATAATTCTCCTTGCTGACAC OsPUT3.1F CACCATGACGAACGCATGGATCTC OsPUT3.1R TCAGCACACAAGTGGGATTGTGC OsPUT3.2F CACCATGGAGGATTGTGTTGGTATC OsPUT3.2R GCACACAAGTGGGATTGTGCTGTCTT AtPUT1F CACCATGGGAGACTATAACATGAATG AtPUT1R ACGTAACAAAGACTCTTCACAATCC AtPUT2F CACCATGCAGAAGCGGAGAATCAT AtPUT2R ACGTATTAGAGTTTCTTCGTATTCCCGAG AtPUT3F CACCATGACTGAGCTTAGCTCTCCGA AtPUT3R CTCCATCAGGTTTGGTAGGTGAGAAC Primers used for Promoter-GUS cloning GUS-AtPUT1F CACCATCTATCCTACATACTTGTTCTCA GUS-AtPUT1R AACGTCATCCAGATTTTAAACGG GUS-AtPUT3F CACCACAAAAAATAAAAACTCAAGAGTAAGTCATATGATG GUS-AtPUT3R CGAGAATTGGAACTTAAAATAATTGAG Primers used for RT-PCR Primer Sequence Number of cycles RTOsPUT1F GATTGGAATCTGTACCTGAACAC 40 RTOsPUT1R GCAGTAGAGGAAGTTCTCGG RTOsPUT2F TTCAACTCCATGTTCTGGAACC 20 RTOsPUT2R AGAGGAAGTTGAGGAACTCGA RTOsPUT3.1F ACGAACGCATGGATCTCGCCGTCGG 20 RTOsPUT3.1R GCCACTTTGCCCAGCCTTGCTGAA RTOsPUT3.2F CTTCCTCGACTATGTTAAGTCCA 20 RTOsPUT3.2R TGAGAAATATCCATCCGTCCAG OsACT1F AGGAATGGAAGCTGCGGGTAT 25 OsACT1R GCAGGAGGACGGCGATAACA RTAtPUT1F CCTCATATTCTACGAAGTCTCAG 25 RTAtPUT1F AAATCCATCACAAGCCATCTC RTAtPUT2F GAATGTTTATAGCCGAGATGAG 25 RTAtPUT2R AGAACTGATGGAGAATTTGACC RTAtPUT3F ATCCGATTCTCTTCCTTGAC 25 RTAtPUT3F CCAAACTCCTCCTATAACTTTACC AtACT1F TATGTGGCTATTCAGGCTGT 25 AtACT1R TGGCGGTGCTTCTTCTCTG

Figure S4: Membrane topology of OsPUT1 as predicted by computer program HMMTM

Figure S5: Membrane topology of OsPUT2 as predicted by computer program HMMTM

Figure S6: Membrane topology of OsPUT3.1 as predicted by computer program HMMTM

Figure S7: Membrane topology of OsPUT3.2 as predicted by computer program HMMTM

Figure S8: Membrane topology of AtPUT1 as predicted by computer program HMMTM

Figure S9: Membrane topology of AtPUT2 as predicted by computer program HMMTM

Figure S10: Membrane topology of AtPUT3 as predicted by computer program HMMTM