THE EXPORT OF POLYAMINES IN PLANTS IS MEDIATED BY A NOVEL CLADE OF BIDIRECTIONAL TRANSPORTERS
Lingxiao Ge
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 2015
Committee:
Paul Morris, Advisor
Nancy Ann Orel, Graduate Faculty Representative
Vipa Phuntumart
Ray Larsen
Scott Rogers ii ABSTRACT
Paul Morris, Advisor
Ubiquitously existent in nature, polyamines play an important role as signaling compounds in plant responses to both biotic and abiotic stresses. My research has shown that the clade of Arabidopsis thaliana and rice BIDIRECTIONAL AMINO ACID TRANSPORTERS
(BAT) function as antiporters of polyamines and amino acids. The method used in this study takes advantage of the genetic resources of the model organism Escherichia coli. A double knockout E. coli strain that is deficient in all polyamine antiporters was created and then used to heterologously express candidate plant transporters AtBAT1.1, AtBAT1.2, and OsBAT1. Inside- out membrane vesicles of these transgenic E. coli cells were generated by ultrasound sonication or French press. A radioisotope assay was then performed using these vesicles to confirm the specificity of the target proteins as polyamine antiporters.
To determine the role of BATs in polyamine homeostasis we tested their GFP fusions by transient expression in Nicotiana benthamiana. The GFP-tagged AtBAT1 and OsBAT1 displayed plastid localization in tobacco leaves by using confocal microscopy. Furthermore, the overexpression of OsBAT1 in A. thaliana wild-type results in novel phenotypes that could have potentially economical usage. This indicates that by altering the expression of a single gene of polyamine transporter, we can manipulate the plants phenotypes similar to natural variations.
Metabolic pathways can be localized to one organelle or distributed across several cellular compartments. Previous work has identified only a single cytosolic pathway for putrescine synthesis in Arabidopsis. Here we show that both A. thaliana and soybeans have a plastid-localized putrescine pathway consisting of an arginine decarboxylase and an agmatinase that combine to synthesize putrescine from arginine.
iii
I dedicate my dissertation work to my parents, Ling Li and Ge Jizhong.
iv ACKNOWLEDGMENTS
I would like to express my sincere gratitude to my mentor Dr. Paul Morris for his support, guidance, and advisory that benefited me much in the completion of this study. In addition to mentoring in school work and lab tasks, Paul offered me the best opportunities to attend national and international conferences and workshops. His intelligence and passion in research always inspired me on the way to become a successful scientist.
I have a distinct appreciation for Dr. Vipa Phuntumart on my Dissertation Committee.
Vipa always treated me like her own student, welcomed me to use her lab space and equipment, and provided valuable comments on my research. I would like to thank my other committee members, Dr. Ray Larsen for providing the P1 bacteria phage strain and permitting me to use the ultrasound sonicator, and Dr. Scott Rogers for training me to use the ultracentrifuge, in addition to their service on the committee. I also thank Dr. Nancy Orel for being the graduate faculty representative on my committee.
I would like to acknowledge Dr. Andrea Kalinoski of the University of Toledo for helping us take great pictures on the confocal laser microscope. In addition, many thanks to my labmates, Jigar Patel and Sheaza Ahmed, who often gave assistance and advice to my projects. A special thank you to Menaka Ariyaratne, who did all the HPLC analyses for this study. At last but not least, I thank my family and all my friends in the US and China for their support and encouragement.
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TABLE OF CONTENTS
Page
CHAPTER I GENERAL INTRODUCTION ...... 1
1.1 What Are Polyamines? ...... 1
1.2 Importance of Polyamines in Plants ...... 2
1.3 Polyamine Metabolism in Plants ...... 4
1.4 Polyamine Transporters ...... 7
1.5 Project Overview ...... 9
1.6 References ...... 10
CHAPTER II A NOVEL METHOD TO IDENTIFY PLANT ANTIPORTERS OF AMINO
ACIDS AND POLYAMINES ...... 16
2.1 Introduction ...... 16
2.2 Materials and Methods ...... 20
2.2.1 Bioinformatics ...... 20
2.2.2 DNA Cloning and Constructs ...... 20
2.2.3 E. coli DKO Mutant with P1 Transduction ...... 21
2.2.4 Expression of Target Genes in E. coli DKO Mutant ...... 22
2.2.5 Generation of Inside-Out Membrane Vesicles ...... 22
2.2.6 Isotope Assay for Polyamine Antiporters ...... 23
2.2.7 HPLC Analysis of Polyamine Levels in E. coli Cells ...... 24
2.3 Results ...... 25
2.3.1 Identification of Candidate Plant Polyamine/Amino Acids Antiporters
...... 25
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2.3.2 Functional Characterization of BAT Transporters in E. coli mutant ... 30
2.3.2.1 OsBAT1 is an antiporter of putrescine and arginine...... 30
2.3.2.2 AtBAT1.1 is an antiporter of spermidine and arginine...... 31
2.3.2.3 AtBAT1.2 is an antiporter of spermidine and arginine...... 33
2.3.3 HPLC Analysis of Polyamine Levels in E. coli Mutant Expressing BAT
Transporters ...... 34
2.4 Discussion ...... 35
2.4.1 The first plant antiporters of polyamines and amino acids ...... 35
2.4.2 Substrate specificity of BATs ...... 36
2.4.3 Codon optimization is a useful tool for characterizing heterologous proteins
in E. coli ...... 38
2.4.4 E. coli inside-out membrane vesicles as a model system for characterizing
plant antiproters ...... 38
2.5 References ...... 41
CHAPTER III OVEREXPRESSION OF BAT AFFECTS PLANT DEVELOPMENT ..... 46
3.1 Introduction ...... 46
3.2 Materials and Methods ...... 48
3.2.1 Organ/Tissue expression of OsBAT1 and AtBAT1 ...... 48
3.2.2 Generation of transgenic plants ...... 48
3.2.3 Characterization of transgenic plants ...... 49
3.2.4 Subcellular localization ...... 49
3.3 Results ...... 52
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3.3.1 The tissue/organ expression of AtBAT1 and OsBAT1 by microarray
analysis ...... 52
3.3.2 OsBAT1 overexpression results in delayed flowering and thicken stems due
to elevated polyamine levels ...... 53
3.3.3 OsBAT1, AtBAT1.1, and AtBAT1.2 are localized to plastids ...... 57
3.4 Discussion ...... 60
3.4.1 Subcellular localization of BATs ...... 60
3.4.2 Overexpression of OsBAT1 in Arabidopsis and results in phenotypic
changes ...... 61
3.5 References ...... 64
CHAPTER IV HIDING IN PLAIN SIGHT: A THIRD ROUTE FOR PUTRESCINE
BIOSYNTHESIS IN PLANTS ...... 70
4.1 Abstract ...... 71
4.2 References ...... 80
4.3 Supplementary Materials ...... 83
4.3.1 Methods ...... 83
4.3.1.1 DNA Sources and Constructs...... 83
4.3.1.2 Subcellular Localization Analysis...... 83
4.3.1.3 Yeast Complementation Assay...... 84
4.3.1.4 Agmatinase Activity Assay...... 84
4.3.1.5 Phylogenetic Analysis...... 85
4.3.1.6 Phyre2 Analysis...... 85
4.3.2 References ...... 92
viii
CHAPTER V SUMMARY ...... 94
5.1 Identifying plant polyamine antiporters that affect plant development ...... 94
5.2 Revealing routes for putrescine biosynthesis in plants ...... 96
5.3 References ...... 97
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LIST OF FIGURES
Figure Page
I-1 Polyamine biosynthesis and degradation pathways in plants...... 6
I-2 Polyamine transporter systems in E. coli and yeast...... 8
II-1 Cladogram of selected polyamine transporters sequences from Arabidopsis thaliana (At),
Leishmania major (Lm), Oryza sativa (Os or AK), Phytophthora sojae (Ps),
saccharomyces cerevisiae (Sc), and Trypanosoma cruzi (Tc)...... 18
II-2 The Gateway® Entry cloning procedures for generating recombinants of target genes.
...... 21
II-3 Alignment of BATs from Arabidopsis and rice and the known putrescine/GABA
transporter ScUGA4 from yeast...... 28
II-4 Predicted transmembrane protein structures of OsBAT1 and the splice variants of
AtBAT1...... 29
II-5 Uptake of putrescine by inside-out membrane vesicles of OsBAT1-DKO...... 31
II-6 Uptake of spermidine by inside-out membrane vesicles of AtBAT1.1-DKO...... 32
II-7 Uptake of spermidine by inside-out membrane vesicles of AtBAT1.2-DKO...... 33
II-8 Putrescine and Spermidine levels in E. coli DKO transformed with AtBAT1.1,
AtBAT1.2, and OsBAT1 and untransformed DKO...... 34
III-1 Schematic of the pGWB2 vector ...... 49
III-2 Schematic of the pGWB5 vector ...... 50
III-3 Agrobacterium-mediated plant transformation of BAT genes...... 51
III-4 Predicted Tissue/Organ Expression of AtBAT1 ...... 52
III-5 Predicted Tissue/Organ Expression of OsBAT1 ...... 53
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III-6 Phenotype of wild-type (WT, Col-0), OsBAT1 overexpressing (OX) lines, and AtBAT1
knockout (KO) A. thaliana plants...... 55
III-7 Soluble, soluble conjugated, and insoluble conjugated polyamine levels in A. thaliana
plants measured via HPLC...... 56
III-8 Subcellular localization of BAT-GFP fusion proteins...... 58
III-9 Subcellular localization of AtBAT1.1-GFP fusion proteins with shorter expression time
...... 59
IV-1 Localization analysis of AtADC2 and AtARGAH2 by transient expression in N.
benthamiana leaves...... 78
IV-2 Characterization of Arabidopsis agmatinase by complementation in yeast...... 78
IV-3 Predicted 3D structure of AtARGAH2 is highly similar to the crystal structure of
Deinococcus radiodurans agmatinase...... 79
IV-4 Subcellular localization of OsODC...... 79
IV-S1 Localization analysis of AtADC1 by transient expression in N. benthamiana leaves
...... 87
IV-S2 Phylogenetic analysis of plant arginine decarboxylases...... 88
IV-S3 Phylogenetic analyses of Plant arginase/agmatinases...... 89
IV-S4 Characterization of Soybean agmatinase by complementation in yeast...... 90
IV-S5 Comparison of active site residues of Dienococcus radiodurans agmatinase and arginases
from Arabidopsis thaliana, Glycine max, and Populus trichocarpa...... 91
xi
LIST OF TABLES
Table Page
II-1 Percentage of identity and similarity between BAT proteins ...... 27
III-1 Predicted localization of BAT transporters ...... 57
IV-S1 Predicted localization of arginine decarboxylase genes from sequenced plant genomes
...... 86
IV-S2 Predicted localization of arginase/agmatinase genes from sequenced plant genomes
...... 87
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CHAPTER I GENERAL INTRODUCTION
1.1 What Are Polyamines?
The compound polyamine may be little known to many biologists, but it has a much longer history than those biological compounds such as DNA that we are familiar with. Indeed, polyamines are one of the oldest groups of substances known in biochemistry. The earliest record about polyamines can be traced back to more than 300 years ago (Bachrach 2010). It was
Antonie van Leeuwenhoek (1632-1723), “the Father of Microbiology”, who recorded the discovery of polyamine for the first time. In 1678, Leeuwenhoek observed some crystalline substance in aging human semen samples with his primitive microscope (Wu 2013). According to the description in his notes, those crystals were spermine, one of the very important polyamines in nature. In 1924, a German chemist Otto Rosenheim (1871-1955) published his work on the isolation and structure of the spermine crystals from human and yeast samples
(Rosenheim 1924). This is the first time that polyamines were isolated and the structures were addressed.
It is known that polyamines are universally present in all living organisms (Cohen 1998).
The most common polyamines in nature are diamine putrescine, triamine spermine, and tetramine spermidine. While all of these polyamines are synthesized as derivatives starting from putrescine, cadaverine (originally isolated from dead bodies) is a polyamine that is synthesized from the degradation of lysine. Unlike putrescine and spermidine that can be found in both eukaryotic and prokaryotic cells, spermine is mostly found in eukaryotes (Cohen 1998). In plants, a fourth polyamine-thermospermine, a structural isomer of spermine, is a key signaling molecule controlling vascular development (Takano, Kakehi, Takahashi 2012). 2
Polyamines in cells either exist in free forms or conjugated to other molecular compounds.
At physiological pH, polyamines are positively charged. The protonated polyamines can bind to some important negatively-charged molecules such as DNA, proteins, and some membrane sites.
Therefore such interactions have been attributed to account for the various effects that polyamines have been shown to have on biological processes such as cell division, embryogenesis, and tissue development (Evans and Malmberg 1989; Paschalidis and
Roubelakis-Angelakis 2005).
1.2 Importance of Polyamines in Plants
Since polyamines are positively charged at cellular pH, they can conjugate different macromolecules that are negatively charged in the cell, such as DNA, RNA, phospholipid, ATP and proteins (Galston 1991; Igarashi and Kashiwagi 2000). An RNA-polyamine complex is considered as the most common format for interacellular polyamines in bacteria (Igarashi and
Kashiwagi 2010a). It has been reported that the interaction of RNA and polyamines changed the secondary structures of RNA, thus influenced specific protein synthesis (Igarashi and Kashiwagi
2000). Especially, as the structural isomer of spermine, thermospermine can modify the secondary structure of specific mRNAs at the upstream ORFs, thereby enhance the chance of ribosome reaching the main ORF to synthesize target proteins more efficiently (Takano, Kakehi,
Takahashi 2012). The plastid membrane-bound polyamines also play important roles in chloroplast photodevelopment. In cucumber, the amount of polyamines bound to etioplast membranes changes to stabilize thylakoid membranes (Sobieszczuk‐Nowicka et al. 2007). In fact, plastids are the organelles that have the most abundant polyamines rather than the nucleus, as demonstrated by the metabolomics analysis using non-aqueous fractionation and mass spectrometry profiling (Krueger et al. 2011). 3
Present evidence suggests that polyamines play regulatory roles in developmental processes. In planta, they often act as signaling molecules that interact with hormone molecules such as auxins, ethylene, and gibberellins (Tiburcio et al. 2014). For instance, when ADC2, one of ADC (arginine decarboxylase) -encoding genes in Arabidopsis, was overexpressed, it caused dwarfism and delayed flowering by affecting gibberellins metabolism (Alcazar et al. 2005). In that case, putrescine concentration was observed to increase in response to environmental stresses (Alcazar et al. 2005). The function of spermidine in cell division has been linked with its ability to activate eIF5A hypusination, an essential modification to ensure eIF5A’s action in translation elongation (Saini et al. 2009; Tiburcio et al. 2014). Additionally, spermidine and spermidine conjugates are involved in pollen development, such as pollen viability and pollen tube growth (Rodriguez‐Enriquez et al. 2013; Song, Nada, Tachibana 2002). In Arabidopsis, exogenous thermospermine can inhibit the expression of genes involved in auxin signaling pathway, thus affecting xylem differentiation (Tong et al. 2014).
More significantly, polyamines play essential roles in plant responses to abiotic and biotic stresses (Alcázar and Tiburcio 2014; Marco et al. 2011). Experimental evidence and transcriptomic data have revealed the contribution of plant polyamines in response to abiotic stresses including cold, heat, drought, salinity, and many more (Gill and Tuteja 2010; Marco et al. 2011; Minocha, Majumdar, Minocha 2014). For instance, increase of endogenous putrescine contents due to low temperature was reported in Arabidopsis to upregulated abscisic acid levels in order to acclimate to cold (Cuevas et al. 2008). Transgenic plants which overexpress certain polyamine biosynthetic genes could have higher tolerance to stress. The overexpression of ADC gene from Datura stramonium in rice induces drought tolerance, which results from the conversion of putrescine to spermidine and spermine (Capell, Bassie, Christou 2004). In contrast 4 an insertion mutant of one of Arabidopsis ADC2 gene showed more sensitivity to salt stress than control plants and the sensitivity was relieved by adding exogenous putrescine (Urano et al.
2004). The connection between polyamines and biotic stress is usually via polyamine conjugation or polyamine catabolism (Jiménez-Bremont et al. 2014; Tiburcio et al. 2014). The level of conjugated polyamines is usually higher in plant tissues infected by pathogens (Walters
2003). For example, overexpression of a human SAMDC gene in transgenic tobacco plants increased their resistance to infection by Verticillium dahliae and Fusarium oxysporum due to the accumulation of free and conjugated polyamines (Waie and Rajam 2003). One of the catabolic products, H2O2, is important for the hypersensitive response in plant-pathogen responses (Gonzalez et al. 2011; Walters 2003).
1.3 Polyamine Metabolism in Plants
All the three natural polyamines, putrescine, spermine, and spermidine, can be synthesized from amino acids in the cells. As the precursor for the synthesis of the other polyamines, putrescine can be synthesized via two pathways: arginine decarboxylase pathway (ADC) and ornithine decarboxylase (ODC) pathway (Figure I-1a). The ADC pathway starts from the decarboxylation of arginine to form agmatine, which is then converted to putrescine by successive canalizations by agmatine imonohydrolase (EC 3.5.3.12) and N-carbamoylputrescine amidohydrolase (EC 3.5.1.53) (Tabor and Tabor 1976; Tiburcio et al. 1997). Ornithine, as the important intermediate from arginine in the urea cycle, can be directly converted to putrescine in the reaction catalyzed by ornithine decarboxylase (EC 4.1.1.17) (Canellakis et al. 1979; Pegg and
Williams-Ashman 1968). While most of higher plants have both ADC and ODC pathways, it is well known that Arabidopsis lacks the ODC pathway (Hanfrey et al. 2001). The absence of the 5
ODC pathway has also been discovered in the protozoan eukaryote, Trypanosoma cruzi (Carrillo et al. 1999).
The synthesis of spermidine starts with the addition of an aminopropyl group donor to putrescine carried out by spermidine synthase. The aminopropyl group is provided by decarboxylated S-adenosyl-L-methionine (AdoMetDC) converted from L-methionine (Bowman,
Tabor, Tabor 1973). Another enzyme spermine synthase also recruits the aminopropyl group from AdoMetDC onto spermidine to produce spermine (Tabor and Tabor 2006).
Polyamines play such significant roles in cells as mentioned above, so their concentration must be delicately regulated at the cellular level. In addition to polyamine synthesis, polyamine degradation is also an important process to regulate the contents of polyamines. The main enzymes involved in polyamine catabolism are diamine oxidases (EC 1.4.3.6) and polyamine oxidases (EC 1.5.3.3) (Figure I-1b). Diamine oxidase, is responsible for the oxidization of diamines such as putrescine and cadaverine to produce 4-aminobutanal, which can be spontaneously converted to Δ1-pyrroline and then to γ-Aminobutyric acid (GABA) (Seiler 2004).
The other enzyme polyamine oxidase catalyzes the degradation of spermidine and spermine
(Moschou, Paschalidis, Roubelakis-Angelakis 2008). It has been proposed that polyamines could act through their degradation to produce molecules that may function as secondary messengers signaling developmental and stress adaptation processes (Moschou, Paschalidis, Roubelakis-
Angelakis 2008). For example, in both degradation pathways, polyamine oxidation produces
H2O2, which is considered to be a signal to activate defense pathways against pathogens (Kusano et al. 2007). 6
Figure I-1 Polyamine biosynthesis and degradation pathways in plants. a. Biosynthesis: ODC pathway is indicated by red arrows; green arrows show the ADC pathway. b. Degradation. All the key enzymes are highlighted in yellow. 7
1.4 Polyamine Transporters
Polyamine transporters have been most completely characterized in Escherichia coli and
Saccharomyces cerevisiae (Figure I-2). There are two polyamine uptake systems in E. coli:
PotABCD and PotFGHI, and both systems are composed of ABC transporters (Igarashi and
Kashiwagi 2010b). PotABCD transports spermidine while PotFGHI is responsible for putrescine uptake (Kashiwagi et al. 1993; Terui et al. 2014). PotE and CadB belong to another class of polyamine transporters. At acidic pH, PotE is an antiporter of putrescine and ornithine, while
CadB is cadaverine and lysine exchanger (Igarashi and Kashiwagi 2010b; Schiller et al. 2000). In yeast, there are four kinds of polyamine uptake proteins (SAM3, DUR3, GAP1, AGP2) and five kinds of polyamine excretion proteins (TPO1~TPO5) (Aouida et al. 2005; Sampathkumar and
Drouin 2014; Uemura, Kashiwagi, Igarashi 2005; Uemura, Kashiwagi, Igarashi 2007). In addition, a GABA transporter UGA4, localized on the vacuolar membrane, can take up putrescine (Uemura et al. 2004).
In contrast, not many polyamine transporters have been identified in plant cells. The first published record of plant uptake transporter was in 2012 by Mulangi et al.. A rice POLYAMINE
UPTAKE TRANSPORTER (OsPUT1) was characterized to be a spermidine-specific transporter through heterologous expression in yeast AGP2Δ mutant in parallel with complementation assay and isotope uptake assay (Mulangi et al. 2012b). Furthermore, three other rice proteins
(OsPUT2, OsPUT3.1, and OsPUT3.2) and three Arabidopsis proteins (AtPUT1, AtPUT2, and
AtPUT3) were all confirmed to have polyamine uptake ability (Mulangi et al. 2012a). It has been reported that multidrug transporters could be potential candidates for transporting polyamines
(Brill, Falk, Schuldiner 2012). As structural analog of polyamines, the herbicide paraquat is suggested to share cellular transport system with polyamines (Fujita et al. 2012; Hart et al. 1992). 8
Thus, the identification of paraquat transporters is an indirect strategy for identifying polyamine transporters (Fujita and Shinozaki 2014).
Figure I-2 Polyamine transporter systems in E. coli and yeast. a. Escherichia coli polyamine transporter systems. b. Saccharomyces cerevisiae polyamine transporters. (Adopted from Igarashi and Kashiwagi 2010b)
9
1.5 Project Overview
Understanding the importance of compartmentation is essential to study the function and mechanism of enzymes and transporters at subcellular level (Lunn 2007; Sweetlove and Fernie
2013). In the complex organization within the cell, active molecules are usually synthesized in one compartment and then relocated to another for their designated functions. Therefore, the proteins involved in translocation of polyamines in order to are an essential component of how these compounds are regulated in plant cells. As mentioned above, two pathways involve in polyamine synthesis in plants and some members of the PUT family of transporters have been characterized. PUTs are unidirectional importers, yet no polyamine exporters have been identified. To address this problem, I started with the phylogenetic analysis of amino acid transporters derived from rice and Arabidopsis genomes with known polyamine transporters from protozoan parasites, yeast, and the PUT family. As a result, I focused on a clade of transporters that contain UGA4, the yeast vacuolar transporter of GABA and putrescine, and
AtBAT1, an Arabidopsis Bidirectional Amino Acid Transporter. I hypothesized AtBAT1 and
OsBAT1 from this clade as antiporters of polyamines and amino acids and designed a series of experiments to test my hypothesis. The characterization of OsBAT1 and two splice variants of
AtBAT1 (AtBAT1.1 and AtBAT1.2) will be described in Chapter II. Their subcellular localization and effects on plant development can be found in Chapter III. In Chapter IV, I will talk about the subcellular localization of enzymes revealing a new biosynthetic pathway of polyamines in Arabidopsis and soybean discovered by our lab.
10
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Amino acids: Biochemistry and nutrition. CRC Press. 145 p. 16
CHAPTER II A NOVEL METHOD TO IDENTIFY PLANT ANTIPORTERS OF AMINO ACIDS AND POLYAMINES
2.1 Introduction
Polyamines are low-molecular-weight aliphatic compounds that are positively charged at physiological pH in the cells. They are present in almost all living organisms and involved in many cell mechanisms such as cell signaling, gene expression, and cell defense (Kusano et al.
2008).
As positively charged metabolites, they have associations with DNA and particularly
RNA. In E. coli, Igarashi and Kashiwagi suggested that most polyamines are associated with nucleic acids, for instance, nearly 50% of putrescine and 90% of spermidine bind to RNA
(Igarashi and Kashiwagi 2006). Since both the mitochondria and plastids have DNA and RNA and originated from bacteria, it’s likely that polyamines are present in those organelles and play structural and functional roles.
In contrast to the findings in bacteria, non-aqueous fractionation of Arabidopsis leaf metabolites followed by MS analysis suggests that the two common nature polyamines, spermidine and putrescine, are preferentially localized to the chloroplasts (Krueger et al. 2011).
Thus the export of these polyamines out of the chloroplasts is necessary to maintain an equilibrium and for the mechanisms in other compartments.
The eukaryotic polyamine transporters have been well studied in S. cerevisiae. The major polyamine uptake proteins in yeast are DUR3 and SAM3, with higher affinity for spermidine uptake while lower affinity of putrescine uptake (Uemura, Kashiwagi, Igarashi 2007). In addition to their polyamine uptake activity, DUR3 also carries out the uptake of urea and SAM3 can import glutamine, lysine, and S-denosylmethionine into yeast cells (Uemura, Kashiwagi, Igarashi 17
2007). Polyamine excretion proteins (TPO1 to TPO5) were also reported in yeast cells. TPO2 and TPO3 are spermidine-specific excretion transporters, whereas TPO1 and TPO4 have broader substrate preference with spermidine, spermine, and putrescine; TPO5 catalyzes the excretion of putrescine and spermidine. (Tachihara et al. 2005; Tomitori et al. 2001; Uemura et al. 2005). The plasma membrane-localized TPO1 and Golgi membrane-localized TPO5 are the major polyamine exporters. In addition, an uptake transporter UGA4 which is localized on the tonoplast is both a GABA and putrescine transporter (Uemura et al. 2004). AGP2, was first identified as high-affinity polyamine permease (Aouida et al. 2005) but now appears to be a sensor that responds to environmental changes and controls the expression of other polyamine transporters (Aouida et al. 2013).
Our lab identified the first known plant polyamines transporter family POLYAMINE
UPTAKE TRANSPORTERS (PUTs) (Figure II-1). Using the yeast mutant complementary assays and isotope uptake assays, members of the PUT family, including three Arabidopsis transporters (AtPUT1, AtPUT2, and AtPUT3) and four rice transporters (OsPUT1, OsPUT2,
OsPUT3.1, OsPUT3.2), were found to have higher affinity for spermidine uptake than putrescine
(Mulangi et al. 2012a; Mulangi et al. 2012b). According to the Hidden Markov model described by Mulangi et al. (2012), there are different clusters of polyamine uptake transporters, which were evolutionarily expanded independently in diatoms, oomycetes, plants, Leishmania and
Trypanosoma. The independent expansion of these genes within each species makes it impossible to use orthology to predict where and when these genes are expressed in each organism. 18
Figure II-1 Cladogram of selected polyamine transporters sequences from Arabidopsis thaliana (At), Leishmania major (Lm), Oryza sativa (Os or AK), Phytophthora sojae (Ps), saccharomyces cerevisiae (Sc), and Trypanosoma cruzi (Tc). Sequences were aligned using ClustalX and subjected to parsimony analysis using PAUP (Larkin et al. 2007; Swofford 2003). The tree was generated in Dendroscope (Huson and Scornavacca 2012). Boot strapped values are shown in nodes. The PUTs family is highlighted in blue and green. The target transporters of this project, AtBAT1 and OsBAT1 are highlighted in orange. (Modified from Mulangi et al. 2012b)
19
In addition to PUTs, the polyamine transport activity has been reported from other groups of transporters. Paraquat, the widely used herbicide, is a structural analogue of polyamines, and it has been known that polyamines inhibit paraquat uptake (Hart et al. 1992). The natural variation of paraquat resistance in Arabidopsis accessions can be used as a tool to target genes involved in the transport of paraquat as well as polyamines. One of Arabidopsis PUTs, AtPUT3-also named as RMV1, is also responsible for paraquat resistance (Fujita et al. 2012). Other candidates for polyamine transport could be organic cation transporters, such as human OCT1, OCT2, and
OCT3 that were confirmed with polyamine transport activity in oocytes (Sala-Rabanal et al.
2013).
In order to further illustrate the mechanisms of polyamine movement in plant cells, we adapted a phylogenetic approach to identify plant proteins that were structurally related to polyamine transporters in other organisms. We targeted on an amino acid bidirectional transporter from Arabidopsis, AtBAT1 (At2g01170), which has two alternative splice forms
(AtBAT1.1 and AtBAT1.2) and shares 40% similarity to ScUGA4. This protein was previously characterized to have export activity of lysine and glutamine and import activity of arginine and alanine in a yeast assay (Dündar and Bush 2009). Later research suggested that this protein also had GABA-permease activity and was localized to mitochondria in tobacco mesophyll protoplasts (Michaeli et al. 2011). There are six rice proteins clustered in this clade. OsBAT1
(AK105656) was chosen for further characterization since it has the closest homology to
AtBAT1. Based on the sequence similarity of these proteins to ScUGA4, we hypothesized that all the three proteins AtBAT1.1, AtBAT1.2, and OsBAT1 function as polyamine/amino acid antiporters. In order to prove our hypothesis, we designed a set of assays to characterize the function of these transporters by expressing these candidate genes in E. coli mutant deficient in 20 polyamine antiporters and testing their exchange capability by means of inside-out membrane vesicles.
2.2 Materials and Methods
2.2.1 Bioinformatics
Sequences of Arabidopsis, rice, yeast, Leishmania major and Trypanosoma cruzi polyamine transporters were taken from (Vaishali Mulangi 2011). Sequence alignments were made with ClustalX and ClustalW2 programs (Larkin et al. 2007) and edited with Jalview
(Waterhouse et al. 2009), phylogenetic tree was constructed using PAUP* 4.0 (Swofford 2003) and Dendroscope (Huson and Scornavacca 2012). The transmembrane helices in the target proteins were predicted using TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) (Tusnády and Simon 2001) and the transmembrane protein models were created with TMRPres2D
(Spyropoulos et al. 2004).
2.2.2 DNA Cloning and Constructs
In this study, the rice clone of OsBAT1 (AK105656) was ordered from the Rice Genome
Resource Center (Japan). The AtBAT1 (At2g01170) clone was obtained from the Arabidopsis
Biological Resource Center (ABRC, Ohio, USA) A synthetic gene of AtBAT1.2 with codons optimized for E. coli was synthesized by GenScript (Piscataway, NJ, USA). The full-length sequence of each gene was amplified by PCR and inserted into a Gateway entry vector pENTR/D-TOPO (by Life Technologies, Carlsbad, CA, USA), following protocols suggested by the manufacturer (Figure II-2). All PCRs were performed using Phusion® High-Fidelity DNA
Polymerase (New England Biolabs). The cDNAs of OsBAT1, AtBAT1.1, and AtBAT1.2 were introduced into the E. coli expression vector pBAD-DEST49 (from Life Technologies, Carlsbad,
CA, USA) using the Gateway LR reaction (Figure II-2) with LR Clonase II (from Life 21
Technologies, Carlsbad, CA, USA), respectively. Successful recombinants were selected by ampicillin and plasmid DNA was prepared and sequenced to confirm the correct insertion.
Figure II-2 The Gateway® Entry cloning procedures for generating recombinants of target genes. (Modified from Akbari et al. BMC Cell Biology 2009)
2.2.3 E. coli DKO Mutant with P1 Transduction
The E. coli single-knockout mutant strains ΔPotE and ΔCadB were obtained from the E. coli Genetic Stock Center (http://cgsc.biology.yale.edu). The ΔPotE strain [potE740(del)::kan] is a deletion of the PotE gene and kanamycin resistant (Baba et al. 2006); the ΔCadB mutation
(cadB2231::Tn10) is tetracycline resistant with the Tn10 insertion into the CabB gene (Nichols,
Shafiq, Meiners 1998). Construction of PotE/CadB double knockout (DKO) strain was done using the P1 transduction protocol adapted from Sauer (2011). The P1 bacteriophage culture was kindly provided by Dr. Ray Larsen (Bowling Green State University). The P1 phage was first grown on the ΔPotE strain as a donor. The subsequent phage lysate was collected to infect the ΔCadB strain as a recipient. Recombinant strains were selected on LB agar plates with both 22 kanamycin and tetracycline and then confirmed by PCR with primers designed according to
(Baba et al. 2006; Nichols, Shafiq, Meiners 1998). The PCR fragments were verified by sequencing.
2.2.4 Expression of Target Genes in E. coli DKO Mutant
The plasmids from 2.2.2 were transformed into the E. coli DKO mutant and selected on
LB media with kanamycin, tetracycline, and ampicillin. The transformed E. coli strains were named OsBAT1-DKO, AtBAT1.1-DKO, and AtBAT1.2-DKO, correspondingly. The target genes were expressed under the control of the araBAD promoter in LB media with the above antibiotics and L-arabinose. In order to select the optimal concentration of arabinose for induction, a pilot expression assay was done as suggested by the manufacturer (Invitrogen). Cell pellets were collected from the E. coli culture of the three genes with gradient concentrations of arabinose. The expression of OsBAT1, AtBAT1.1, and AtBAT1.2 proteins was assessed by electrophoresis using Novex® Tris-Glycine precast gels (by Life Technologies) and visualization of protein bands.
2.2.5 Generation of Inside-Out Membrane Vesicles
E. coli OsBAT1-DKO, AtBAT1.1-DKO, and AtBAT1.2-DKO were cultured in 1 liter of polyamine-free growth medium (Igarashi and Kashiwagi 2010b) with optimal concentration of arabinose as tested in 2.2.4 until cell density reached and OD600 of 0.6~0.8. Cell pellets were collected and washed in Buffer 1 (100 mM potassium phosphate buffer, pH 6.6, 10 mM EDTA) with 2.5 mM arginine unless otherwise stated. Inside-out membrane vesicles were prepared either by French press (Kashiwagi and Igarashi 2011) or ultrasound sonication (Benov 2002) from the intact cells. The unbroken cells and cell debris were removed by centrifugation at low speed (3000~5000 x g) and membrane vesicles were pelleted by ultracentrifugation at 170,000 x 23 g for one hour. The vesicles were washed with Buffer 2 (10 mM Tris–HCl, pH 8.0, 0.14 M KCl,
2 mM 2-mercaptoethanol, and 10% glycerol) and resuspended in the same buffer. The protein concentration of the membrane vesicles was determined using the Bio-Rad DC Protein Assay
(Bio-Rad Laboratories, Hercules, CA) and NanoDrop (Thermo Fisher Scientific, Waltham, MA), following the manufacturers’ instructions.
The inside-out membrane vesicles of OsBAT1-DKO, AtBAT1.1-DKO, and AtBAT1.2-
DKO without amino acids wrapped inside were produced in the same way. In addition, membrane vesicles from DKO cells with or without arginine were created as a negative control.
2.2.6 Isotope Assay for Polyamine Antiporters
The reaction mixture of 100 µL containing 50 µg of membrane vesicle proteins from 2.2.5 was activated by incubation at room temperature for 5 min. To start the reaction, 3H-putrescine or 3H-spermidine was added to get a final concentration of 50 μM (unless otherwise stated). A series of the same reactions incubated on ice instead of at room temperature were set up aside as an indication for nonspecific binding.
At designated time points, the reactions were stopped by adding ice-cold Buffer 3 (10 mM
Tris–HCl, pH 8.0, 10 mM potassium phosphate buffer, pH 8.0, and 0.14 M KCl) with 10-fold concentration of corresponding polyamines. The vesicles were trapped by filtering the solution through nitrocellulose membrane filters (Millipore, 0.45 μm) and the filters were washed three times with ice-cold Buffer 3 to reduce nonspecific binding of the labeled isotope to the vesicles.
The filters were transferred to scintillation vials with 10 mL of scintillation cocktail Ecoscint
(National Diagnostics, Atlanta, GA) and radioactivity was determined by using a Beckman LS-
6500 liquid scintillation counter. 24
In order to calculate kinetic constants of BATs transporters, Km and Vmax of their corresponding substrates (putrescine or spermidine), the corresponding inside-out membrane vesicles were incubated with increasing concentrations of labelled polyamine for 1 minute and the radioactivity was counted as mentioned above. The values of Km and Vmax were determined by creating a Michaelis-Menten curve using GraphPad Prism version 5.01 for Windows
(GraphPad Software, La Jolla California USA, www.graphpad.com).
2.2.7 HPLC Analysis of Polyamine Levels in E. coli Cells
The protocol for HPLC analysis of E. coli polyamines was modified from (Lee et al. 2009) and (Ariyaratne 2014). Cell cultures of DKO mutant and OsBAT1-DKO, AtBAT1.1-DKO,
AtBAT1.2-DKO were grown in LB medium or polyamine-free amino acid medium (Igarashi and
Kashiwagi 2010b) with ampicillin for transformants. When cultures reached an OD600 of 0.5, arabinose was added to the transformants to a final concentration of 0.0002%. After another two hours of growth, all cell cultures were harvested at an OD600 of 0.8~1.2; and then pelleted at
4000 x g at room temperature. The cell pellets were washes three times in saline phosphate buffer (pH 7.4; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) and resuspended in lysis buffer (20 mM MOPS, pH 8.0, 10 mM NaCl, 4 mMMgCl2) (Lee et al.
2009). The cells were repeatedly frozen in liquid nitrogen and thawed on ice for three cycles. 5% perchloric acid was then added into the cell samples to precipitate proteins and extract polyamines. After five-minute incubation on ice, cell samples were centrifuged at 13,000 x g for
3 minutes at room temperature. The supernatants were then separated from cell debris and stored at -20⁰C until dansylation and HPLC analysis. The following HPLC procedures were performed according to (Ariyaratne 2014).
25
2.3 Results
2.3.1 Identification of Candidate Plant Polyamine/Amino Acids Antiporters
A previous bioinformatics analysis of Arabidopsis, rice, yeast, Leishmania major and
Trypanosoma cruzi polyamine transporters (Figure II-1) identified AtBAT1 (At2G01170) and
OsBAT1 (AK105656) as potential antiporters of polyamine and amino acids, based on their similarity to a yeast putrescine exporter ScUGA4 (Uemura et al. 2004) and the known property of AtBAT1 as a bidirectional amino acids transporter (Dündar and Bush 2009). AtBAT1 functions as an alanine, lysine, glutamate, and arginine transporter, but it has not yet been tested as a polyamine transporter. However, according to Dündar’s GUS expression, AtBAT1 expression was enhanced when the plant was exposed to different stresses such as cold and wounding (Dündar 2009). This phenotype connects AtBAT1 with features related to polyamines as it is well known that polyamine levels are significantly increased in plant cells under environmental stresses (Gill and Tuteja 2010).
AtBAT1 and AK105656 proteins share 71% identity and 79% similarity, so we hypothesized that they share the same function and henceforth named the rice protein as
OsBAT1 (Figure II-3). The AtBAT1 gene has two splice variants: AtBAT1.1 and AtBAT1.2
(Figure II-4d). The cDNA of AtBAT1.1, with a slightly longer 3’-UTR, is 17 bp shorter than
AtBAT1.2 with a much longer 5’-UTR. However, AtBAT1.1 has a longer protein sequence with
516 amino acids and AtBAT1.2 with 437 amino acids. Their C-terminal protein sequences are identical while the N-terminal of AtBAT1.1 is 79 residues longer (Figure II-3). All confirmed to be transmembrane proteins by TMHMM, AtBAT1.1 is predicted to have 13 transmembrane domains (Figure II-4a), while AtBAT1.2 has 11 transmembrane domains (Figure II-4b), and
OsBAT1 has 12 (Figure II-4c). 26
In addition to the mentioned Arabidopsis and rice BATs, there are at least five more BATs proteins from rice that have not been characterized (Figure II-1). The ClustalW alignment shows high conservation of the BAT clade (Figure II-3). In contrast with the known polyamine uptake transporters PUTs family, which shares from 35% to 86% identity at the amino acid level
(Vaishali Mulangi 2011), the conservation within the BATs family is much higher. The identity of Arabidopsis and rice BAT proteins varies from 48% to 91% and their similarity ranges from
55% to 93% (Table II-1).
27
Table II-1 Percentage of identity and similarity between BAT proteins (Calculated on http://imed.med.ucm.es/Tools/sias.html) Identity (%)
AtBAT1.1 AtBAT1.2 OsBAT1 LOC_Os LOC_Os LOC_Os LOC_Os AK065371 ScUGA4 01g71740 01g71760 01g71700 01g71710 AtBAT1.1 - 86.44 74.44 65.69 62.26 64.66 48.02 66.89 25.9 AtBAT1.2 86.44 - 65.52 59 55.4 58.14 61.57 59.86 20.92 OsBAT1 81.47 71.69 - 68.26 64.49 67.23 48.88 68.26 25.72
(%) LOC_Os01g71740 78.55 70.15 78.38 - 91.42 83.19 60.2 79.93 24.87 LOC_Os01g71760 74.61 66.03 74.61 93.65 - 81.47 62.43 78.04 25.21 LOC_Os01g71700 75.98 68.09 75.98 89.02 86.62 - 66.03 79.41 24.87 imilarity imilarity S LOC_Os01g71710 56.94 70.49 55.23 64.83 66.72 68.26 - 58.14 17.66 AK065371 77.35 68.78 78.38 87.3 85.24 86.79 63.97 - 25.72 ScUGA4 40.65 34.3 39.62 42.02 41.16 41.33 28.98 40.65 -
28
Figure II-3 Alignment of BATs from Arabidopsis and rice and the known putrescine/GABA transporter ScUGA4 from yeast. The alignment was performed using ClustalW2 (Larkin et al. 2007) and the color was coded by conservation using Jalview (Waterhouse et al. 2009). 29
Figure II-4 Predicted transmembrane protein structures of OsBAT1 and the splice variants of AtBAT1. a. The transmembrane structure of AtBAT1.1. It has 13 predicted transmembrane domains. N-terminus of AtBAT1.1 is predicted to be at the extracellular side, while the C-terminus is at the cytoplasmic side. b. The transmembrane structure of AtBAT1.2. It is predicted with 11 transmembrane domains. Its N-terminus is at the extracellular side, while the C-terminus is at the cytoplasmic side. c. The transmembrane structure of OsBAT1. It is predicted to have 12 transmembrane domains. Both of its N-terminus and C-terminus are predicted at the cytoplasmic side. d. exon-intron structures (including 5’- and 3’- UTRs) of AtBAT1.1 and AtBAT1.2. AtBAT1.1 has eight exons while AtBAT1.2 has seven (dark blue bars). The transmembrane structures were predicted by TMHMM and the models were created in TMRPres2D. 30
2.3.2 Functional Characterization of BAT Transporters in E. coli mutant
In order to demonstrate the exchange of polyamines for other amino acids by BATs,
individual BAT genes (OsBAT1, AtBAT1.1, and AtBAT1.2) were expressed the E. coli
PotE/CadB-double knockout (DKO) mutant. Export of polyamines from transformed DKO cells
was determined by measuring its uptake into inside-out membrane vesicles of the transformed
cells. The membrane vesicles were created by ultrasound sonication or French press with amino
acids added and then fed with tritium labelled spermidine or putrescine for its uptake activity.
The radioactivity of labelled polyamine substrates collected by the vesicles was measured using
a liquid scintillation counter.
2.3.2.1. OsBAT1 is an antiporter of putrescine and arginine
The cDNA of OsBAT1 was cloned to DKO and the functional assay was conducted as
mentioned above. OsBAT1 was demonstrated to have the ability to exchange putrescine with
arginine as a counterion. As shown in Figure II-5a, putrescine uptake was clearly observed only
with the membrane vesicles prepared from DKO cells transformed with OsBAT1 but not with
the membrane vesicles of DKO cells. The activity of OsBAT1 exchanging putrescence for
arginine could be seen within 1 minute of incubation time, which indicates that the rice
transporter does compensate the deficient activity of polyamine exchange on the plasma
membrane of E. coli DKO at a significant rate. 31
90 80 70 60 50 with Arg 40 without Arg 30 [3H]PUT uptak [3H]PUT
(pmol/mg protein) (pmol/mg 20 10 0 0 1 2 3 4 5 Incubation Time (min)
Figure II-5 Uptake of putrescine by inside-out membrane vesicles of OsBAT1-DKO. Assays for uptake of [3H]putrescine were performed as descibed in 2.2.6. The slight increase of [3H]putrescine in membrane vesicles without arginine as counterions was due to non-specific binding of isotopes. The uptake of [3H]putrescine was significant in the membrane vesicles with arginine added, during the incubation time from 1 min to 5 min. One sample for each reaction was read.
2.3.2.2. AtBAT1.1 is an antiporter of spermidine and arginine.
The exchange activity of AtBAT1.1 was determined in the same way as OsBAT1. The
inside-out membrane vesicles generated from E. coli DKO mutant expressing AtBAT1.1 had
distinct uptake of spermidine if the vesicles were pre-incubated with arginine or glycine before
French press (Figure II-6a). The time-dependent assay showed a significant uptake of spermidine
after 1 minute of incubation at room temperature (Figure II-6b). This suggests that AtBAT1.1 is
able to exchange spermidine using arginine as a counterion. 32
b 30 with Arg 25 without Arg
20
15
H] SPD SPD uptake H] 10 3 [ (pmol/mg protein) (pmol/mg 5
0 0 0.5 1 1.5 2 Incubation time (min)
Figure II-6 Uptake of spermidine by inside-out membrane vesicles of AtBAT1.1-DKO. (a) Assays for uptake of [3H]spermidine were performed at 37⁰C. The orange bars stand for counts of [3H]spermidine in vesicles at time 0 and the blue bars stand for the data of vesicles collected after 15 minutes incubation. DKO = DKO with arginine added; AtBAT1= AtBAT1.1-DKO without amino acid added; Arg = AtBAT1.1-DKO with arginine added; Gly = AtBAT1.1-DKO with glycine added. (b) Assays for uptake of [3H]spermidine were performed as descibed in 2.2.6. The uptake of [3H]spermidine was significantly increased from 0.5 minutes to 1.5 muniutes and then dropped afterwards. The assays were repeated three time independently. Each reaction was repeated three times.
33
2.3.2.3. AtBAT1.2 is an antiporter of spermidine and arginine.
The cDNA of AtBAT1.2 was initially cloned into DKO and its expression was verified with
SDS-PAGE protein gel. However no transport activity was detected using the inside-out
membrane vesicle assay. Subsequently a codon-optimized AtBAT1.2 for E. coli was synthesized
for this assay. As shown in Figure II-7, AtBAT1.2 was shown to function as an antiporter of
spermidine in exchange for arginine in the inside-out membrane vesicle assay. The efficiency of
AtBAT1.2 is similar to that of AtBAT1.1.
30
25
20 with Arg 15 without Arg [3H]SPD [3H]SPD uptak 10 (pmol/mg protein) (pmol/mg
5
0 0 0.5 1 1.5 2 Incubation Time (min)
Figure II-7 Uptake of spermidine by inside-out membrane vesicles of AtBAT1.2-DKO. Assays for uptake of [3H]spermidine were performed as descibed in 2.2.6. The uptake of [3H]spermidine was only observed if arginine was added as counterions. The exchange activity started after 0.5 minutes and reached an equilibrium after 1.5 minutes. Each reaction was repeated three times.
34
2.3.3 HPLC Analysis of Polyamine Levels in E. coli Mutant Expressing BAT
Transporters
Since the function of the three BAT proteins were successfully characterized in the isotope uptake assay of E. coli inside-out membrane vesicles, we decided to test if E. coli cells growing on LB media and expressing BATs exhibited any changes in polyamine contents compared to
DKO cells. Spermine is absent in E. coli cells (Igarashi and Kashiwagi 2006), so only putrescine and spermidine levels were quantified in transformed DKO and DKO mutant cells via HPLC analysis. Our data showed that putrescine levels remained the same in transformed cells or DKO cells. However, there is a significant increase of spermidine contents in OsBAT1-DKO cells compared to others.
1.6 1.4 PUT SPD 1.2 1 0.8 0.6 0.4 0.2 Polyamine concentration (µM) concentration Polyamine 0 AtBAT1.2-DKO AtBAT1.1-DKO OsBAT1-DKO DKO
Figure II-8 Putrescine and Spermidine levels in E. coli DKO transformed with AtBAT1.1, AtBAT1.2, and OsBAT1 and untransformed DKO. The E. coli cells were grown in liquid LB media, with ampicillin added for transformants. At an OD600 of 0.5~0.6, 0.0002% arabinose was added to the culture of the transformants in order to induce the expression of BAT genes. Harvested E. coli cells were treated according to (Lee et al. 2009) and the HPLC analysis was performed by Menaka Ariyaratne (Ariyaratne 2014). 35
2.4 Discussion
In the present study, we successfully characterized the function of three plant transporters, by using heterologous expression of BAT proteins in E. coli double knockout deficient in polyamine antiporters, radioisotope uptake assay of inside-out membrane vesicles prepared from
E. coli, and HPLC analysis of polyamine levels in these transformed cells.
2.4.1 The first plant antiporters of polyamines and amino acids
From the functional assay of inside-out membrane vesicles expressing BAT proteins, we have shown that both AtBAT1.1 and AtBAT1.2 are spermidine-specific antiporters, while
OsBAT1 functions as an antiporter for putrescine and arginine. The transport activity of
OsBAT1 on membrane vesicles was about two to three times higher than the two AtBAT1 proteins (Figure II-5, Figure II-6, and Figure II-7). They are the first characterized plant antiporters of polyamines and amino acids to date.
The vesicle assays seem to only illustrate the bidirectional property of BATs but not clear enough to suggest what the natural orientation of the transporters are (if they pump out or pump in polyamines). In order to address this question, we measured the polyamine levels inside the intact cells and expected difference between transformed E. coli and DKO E. coli. No significant difference in putrescine or spermidine contents was seen between AtBAT1.1/AtBAT1.2-DKO and DKO (Figure II-8). Although OsBAT1 is a putrescine transporter, there was a remarkable accumulation of spermidine in OsBAT1-DKO (Figure II-8). Since we measured the net change of polyamines in the intact E. coli cells, this result was not surprising. The polyamine titers in E. coli are mainly regulated by the two major polyamine uptake systems (PotABCD and PotFGHI) and biosynthesis systems (Igarashi and Kashiwagi 2010a). The fact that OsBAT1-DKO had different concentration of spermidine indicated that the protein was working. Since it is an 36
antiporter, I can postulate that I should be able to spike the medium with specific amino acids and detect changes in polyamine levels within the cells. Hence, a specific experiment has been designed to examine this hypothesis. First, the transformed E. coli will be grown in LB medium until reaching a high cell density (e.g. OD600=2). Cells will be harvested and a few of them collected for polyamine content determination via HPLC. The rest of the cells will be resuspended with minimal medium (e.g. M9 minimal medium) with 1mM arginine added.
Afterwards, the cells will be spin down and both the cell pellets and supernatant will be tested for polyamine levels. If we can detect polyamines in the supernatant and the polyamine levels in the cells from the second harvest are lower than the first harvest, we can conclude that BATs indeed export polyamines and import arginine.
Unlike the divergent PUT family, the BAT family are highly conserved between rice and
A. thaliana genomes. The rice genome contains six proteins in this clade including two genes which represent a recent tandem duplication event (LOC_Os01g71740 and LOC_Os01g71760).
Due to the conservation of sequences we hypothesize that all other BATs are also antiporters for polyamines and amino acids. The same experiments can be performed with the other rice BATs.
We expect to have similar results of their exchange activity, but they do not necessarily have the same substrate specificity. It is very possible to find a spermidine-specific antiporter from the other five rice candidates.
2.4.2 Substrate specificity of BATs
The current data collected from the already finished experiments are still limited in terms of addressing the preferential substrates of BAT proteins. The amino acid substrates used in the membrane vesicle uptake assay were selected based on the experimental results of Dündar’s work. AtBAT1.1 was identified as bidirectional transporter exporting lysine and glutamate while 37
importing arginine and alanine but showed no activity in transporting GABA or proline (Dündar and Bush 2009). The results of my earlier experiments indicated arginine was the best substrate as the counterion for polyamine exchange. Surprisingly, AtBAT1.1 was later determined to be a
GABA permease in a yeast complementary assay (Michaeli et al. 2011). In this paper, the authors failed to mention the previous work done by Dündar on this exactly same gene and even gave a new name to this gene as AtGABP according to their findings. These observations intrigued me to test whether GABA could function as a counterion for putrescine or spermidine exchange by AtBAT1.1. However, inside-out membrane vesicles of AtBAT1.1-DKO with
GABA did not uptake significant amount of spermidine or putrescine compared to the same vesicles without GABA. Therefore, we concluded that GABA was not a counterion in exchange for polyamines by BATs, though GABA may be exchanged with something else, likely the other amino acids.
OsBAT1 as a polyamine antiporter, its preferred substrates have yet to be defined. The hypothesis is that it’s likely to be putrescine, compared with the lower activity of AtBAT1.1 and
AtBAT1.2 with spermidine. This established system enables me to measure the Km of OsBAT1 and the other AtBATs with different substrates and the experiments are still in process. The Km will tell us the specificity of the transporters for polyamines in the E. coli vesicles. However, given that the polyamine levels (in uM) in the cytosol may be lower than the amino acids such as glutamatic acid and glutamine (in mM), it may not tell what the transporters is doing in the intact cells or in plants. 38
2.4.3 Codon optimization is a useful tool for characterizing heterologous proteins in E.
coli
The difference at the N-terminus of AtBAT1.1 and AtBAT1.2 has no effect on their functions as the antiporter. However, the alternative splicing does affect the expression of
AtBAT1.2 in E. coli DKO. Initially, when the cDNA of AtBAT1.2 was transformed into DKO, the transporter failed to function in the inside-out membrane vesicle assay. It was postulated that this lack of activity was due to the different codon choice in Arabidopsis and E. coli. Codon bias at the N terminus of genes could inhibit protein expression in E. coli and codon optimization can impressively increase the amount the heterozygous proteins that E. coli cells can make
(Goodman, Church, Kosuri 2013). Therefore, we used the synthesized AtBAT1.2 gene which was codon-optimized for E. coli and had it successfully expressed in DKO. In the vesicle assay, the transporter translated from the synthesized AtBAT1.2 showed the similar exchange activity as
AtBAT1.1 in E.coli DKO cells. It demonstrated that codon optimization was helpful to enhance the expression and function of heterlogously expressed proteins in bacterial cells.
2.4.4 E. coli inside-out membrane vesicles as a model system for characterizing plant
antiporters
The significance of this study is not only the discovery of the first plant polyamine antiporter, but also the utilization of E. coli mutant system to identify other kinds of amino acid antiporters. Currently, plant membrane transporters have been studied by expression in bacteria
(especially E. coli), yeast, mammalian cells, insects, and plant cells. Amongst these methods, expression of plant transport proteins in Xenopus oocytes is one of the most popular. After the first plant membrane transporters KAT1 and STP1 were characterized in Xenopus oocytes in
1992 (Boorer et al. 1992; Schachtman et al. 1992), there have been many other plant membrane 39
proteins analyzed using this method, since the oocytes are easy to manipulate due to their large size and few endogenous transporters may interfere with the activity of the inserted transporters
(Haferkamp and Linka 2012; Sobczak et al. 2010). However, it is not easy to use Xenopus oocytes to study intercellular transporter system, nor to characterize exchange system. Moreover, professional knowledge and skills are required for preparing the oocytes; thus, it is not applicable in many plant labs. In contrast, the model organism E. coli becomes an ideal system in terms of characterization of plant antiporters. With a fully sequenced genome in 1997 (Blattner et al.
1997), the molecular and physiological characteristics of E. coli are well studied and genetic tools including transformation and gene knockouts are completely available. There is no difficulty to grow and maintain both wildtype and mutant strains of E. coli in a laboratory condition. Foreign DNAs can also be easily inserted and expressed in E. coli cells using basic molecular techniques. Some obstacles of expression can even be overcome by means of codon optimization.
Our methods and results have shown the possibility of using bacteria mutant system to study the function of plant antiporters. In particular, we showed that a double knockout E. coli can be used to characterize polyamine antiporters using inside-out membrane vesicles prepared by French press and ultrasound sonication from the transformed knockout cells. Membrane vesicles have been commonly used to characterize E. coli transporters for both passive and active transport since 1960s (Sze 1985). As the vesicles themselves lack the essential energy source such as ATP and enzymes in the cytoplasm, the interference from active transporters and other metabolic activity is minimal. Thus, this system is ideal for the detection of non-energy-required translocation such as metabolite exchanges. The everted membrane vesicles, in particular, can be applied to the characterization of exporters and antiporters since the topology of transporters is 40
reversed on such membrane. Using French press or ultrasound sonication is fairly efficient to make inside-out membrane vesicles from intact cells. 70% of E. coli cells could be transformed into vesicles by sonication (Meuller and Rydstrom 1999) and 95% of the vesicles generated by ultrasound sonication or French press have everted membrane (Futai 1974; SECKLER and
WRIGHT 1984). PotE, the E. coli antiporter of putrescine and ornithine, is the first polyamine antiporter that was characterized using inside-out membrane vesicles (Kashiwagi et al. 1992).
This method will have general utilization for the functional analysis of other kinds of metabolite antiporters in plants. In A. thaliana there are1628 membrane proteins with six or more transmembrane (TM) regions, the minimum number TM domains thought to be sufficient for a functional transporter. The majority of these membrane proteins have yet to be characterized. We envision that a similar approach to the one described here, can be developed to characterize other amino acid, sugar, nucleotide, or plant metabolite antiporters in A. thaliana. A potential application of this method in the future work of our lab is to characterize soybean transporters from the Multidrug and Toxic Compound Extrusion (MATE) family. The
MATE1 protein in human has been shown to have antiport activity of putrescine and/or agamatine (Winter, Elmquist, Fairbanks 2010). The MATE family has orthologs in soybean and some of the soybean MATE genes have been correlated to stress response. Thus, they can be considered as the potential candidates for polyamine antiporters in soybean and characterized in a similar way as we studied the BAT family.
41
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46
CHAPTER III OVEREXPRESSION OF BAT AFFECTS PLANT DEVELOPMENT
3.1 Introduction
Polyamines directly affect developmental processes or have indirect influence via their interface with other hormones, though the detailed mechanisms of these effects are still not very clear (Alcazar et al. 2010; Kusano et al. 2008).
Experimental evidence of double mutants deficient in putrescine or spermidine synthesis indicate that putrescine and spermidine are vital for plant embryogenesis and viability (Ge et al.
2006; Imai et al. 2004; Urano, Hobo, Shinozaki 2005). Spermidine is synthesized from putrescine and is a precursor of hypusine, an unusual amino acid required for the essential translational modification of eIF5A (Scuoppo et al. 2012; Wolff and Park 2015). A specific lysine residue of N-terminus eIF5A has to undergo hypusination by the aminobutyl group of spermidine, in order to activate eIF5A for the elongation step in translation (Wolff and Park
2015).
Changes in putrescine levels can also cause developmental changes in plants. The accumulation of putrescine in transgenic rice plants overexpressing of an oat arginine decarboxylase gene prevented the callus tissue from further differentiation (Capell et al. 1998).
Overexpression of ADC2 resulted in elevated levels of putrescine in leaves and Arabidopsis plants that were dwarfed and delayed in flowering (Alcazar et al. 2005). Surprisingly, the inhibition of ornithine decarboxylase in tobacco significantly reduced the amount of polyamines in plant cells and generated similar dwarfism phenotypes (Nölke et al. 2005).
In Arabidopsis, acl5 gene encodes thermospermine synthase that can synthesize thermospermine from spermidine (Knott, Romer, Sumper 2007). Arabidopsis homozygous mutants in this gene showed defects in stem elongation, higher expression level of a set of genes 47
involved in auxin signaling and xylem differentiation. Exogenously applied thermospermine to the seedlings of acl5 mutants lowered the expression auxin related genes (Kakehi et al. 2008;
Tong et al. 2014). This evidence suggest that thermospermine promotes stem elongation but negatively controls auxin signaling and xylem differentiation (Kakehi et al. 2008; Tong et al.
2014).
As important signaling molecules, cellular polyamine homeostasis is precisely regulated by biosynthesis, segregation, and transport between cellular compartments. Because transport of polyamines can create localized changes in the concentrations of these metabolites, identifying where metabolite transporters are localized is central to an understanding of plant metabolism
(Sweetlove and Fernie 2013). At present, various databases, software, and online tools are available to help us predict the localization of proteins in silico (Hooper et al. 2014; Wang et al.
2014). Data from cellular fractionation and proteomic analyses can also be used to indicate the localization of proteins and metabolites (Drissi, Dubois, Boisvert 2013). However experiments that provide direct verification of predicted or potential subcellular localizations are still indispensable. One of the most common tools for subcellular localization is to use fluorescent protein fusions (Collings 2013). Green fluorescent protein (GFP) was first isolated from jellyfish
Aequorea victoria in 1961 (Shimomura 2005) and its first application as a gene marker for protein expression was in 1994 (Chalfie et al. 1994). Since then, GFP and its derivatives YFP,
CFP, BFP, etc. have been widely applied to protein localization, genetic labeling, and immunolabeling (Giepmans et al. 2006). Recently, plant scientists have effectively taken advantage of this tool by combining transient expression of target proteins tagged with fluorescent proteins and confocal fluorescence microcopy to visualize the in vivo localization at the subcellular level (Collings 2013). The transient expression can be conveniently approached 48
via either particle bombardment or agrobacterium-mediated transformation (Rivera et al. 2012;
Sparkes et al. 2006).
In the work presented in this Chapter, I have determined the subcellular localization of the three BAT proteins, AtBAT1.1, AtBAT1.2, and OsBAT1, using GFP tagging and transient expression in tobacco leaves. I have also generated transgenic Arabidopsis plants overexpressing
OsBAT1, characterized as putrescine and arginine antiporter as mentioned in Chapter II. The transgenic plants accumulate higher levels of spermidine before flowering and show altered phenotypes such as delayed flowering, bigger rosette leaves, and thicker stems.
3.2 Materials and Methods
3.2.1 Organ/Tissue expression of OsBAT1 and AtBAT1
Gene expression of OsBAT1 and AtBAT1 at the tissue and organ level was analyzed using
Arabidopsis eFP Browser (http://bar.utoronto.ca/efp/) and Rice eFP Browser
(http://bar.utoronto.ca/efprice/) at the Bio-Analytic Resource for Plant Biology (BAR) (Jain et al.
2007; Winter et al. 2007).
3.2.2 Generation of transgenic plants
Arabidopsis thaliana ecotype Col-0 was used as wild-type plants for all experiments and as the genetic background for the generation of transgenic plants. Seeds of homozygous AtBAT1 mutant plants were ordered from Arabidopsis Biological Resource Center (ABRC, Ohio, USA).
All seeds were geminated on basic MS medium (1x MS salts, Bacto Agar, pH 5.7) with selective antibiotics if applied and transferred into pots at stage 1.03 (3 rosette leaves) (Boyes et al. 2001).
Plants were kept in a growth chamber at 22°C under long-days (16h light/8h dark) condition.
The entry clone of OsBAT1 was used to make the destination clone in pGWB2 vector
(Figure III-1) via LR Clonase reaction (Nakagawa et al. 2007b). Afterwards, p35S:OsBAT1 49
construct was confirmed by PCR and sequencing and then transformed into Agrobacterium strain
GV3101. Agrobacterium-mediated transformation of Arabidopsis plants was performed via floral dip method (Clough and Bent 1998). The seeds collected from the transformed plants were screened on MS medium containing kanamycin antibiotic selection and the insertion of OsBAT1 gene was further confirmed by PCR for every generation.
Figure III-1 Schematic of the pGWB2 vector (Nakagawa et al. 2007a).
3.2.3 Characterization of transgenic plants
The phenotypes of different cultivars and lines were examined by measuring the stem thickness, numbers of siliques, leaf sizes, and photosynthetic activity using FluorPen (FP 100,
Photon Systems Instruments, Czech Republic).
Rosette leaf samples from wild-type, AtBAT1 KO (knockout), and OsBAT1 OX (over- expression) plants were collected at two-week, four-week, and six-week stages, respectively.
Four to six individuals from each plant type were sampled for HPLC analysis of polyamines. The
HPLC analyses were performed by Menaka Ariyaratne as described (Ariyaratne 2014). The
HPLC data were analyzed using two-way ANOVA program in GraphPad Prism version 5.01 for
Windows (GraphPad Software, La Jolla California USA, www.graphpad.com).
3.2.4 Subcellular localization
Standard Gateway cloning was used for the transformation of genes of interest. From the entry vector, the cDNAs of OsBAT1, AtBAT1.1, and AtBAT1.2 were introduced into the destination vector pGWB5 (35S promoter, C-sGFP, Figure III-2) (Nakagawa et al. 2007b) via 50
LR Clonase reaction according to the manufacturer’s instructions (Life Technologies). E.coli containing recombinant plasmids was selected for on LB plates containing kanamycin and hygromycin, and the plasmids were then isolated and transformed into Agrobacterium (GV3101) using the freeze/thaw transformation method (Sparkes et al. 2006). The workflow of E. coli and
Agrobacterium cloning is shown in the left panel of Figure III-3.
Figure III-2 Schematic of the pGWB5 vector (Nakagawa et al. 2007a) The corresponding Agrobacterium cultures with target constructs were then infiltrated into tobacco mesophyll cells for transient expression as described by Sparkes et al. (2006). 1ml of overnight culture of each Agrobacterium construct was centrifuged at low speed to collect cell pellets. The cells were washed and resuspended with infiltration buffer made with MgCl2 and acetosyringone to make the transformation culture. A further dilution was needed to maintain an
OD600 of 0.1. Then a 1 mL syringe with no needle attached was used to inject the transformation culture against the underside of a selected leaf from the tobacco plants of four to five weeks old. The transformed plants were placed back under normal growth conditions and the expression of fluorescent fusion proteins were examined after 16-40 hours.
The infiltrated tobacco leaves were examined with a Leica laser confocal microscope under a 63X oil immersion objective. The GFP image was excited at 488nm and detected at 510nm-
554nm; the autofluorescence of chlorophyll was excited at 633nm and detected by emission bandwidth of 645nm – 726nm, indicating the localization of plastids. GFP signals were false- colored green and chlorophyll autofluorescence signals were false-colored red. ImageJ was used to overlay the fluorescent images with brightfield. 51
Figure III-3 Agrobacterium-mediated plant transformation of BAT genes. The cDNA of BATs was cloned through entry cloning and LR reaction into a binary vector pGWB2 or pGWB5 for generating overexpression line or subcellular localization, respectively. A. tumefaciens transformed with pGWB5-BATs were used to infect tobacco leaves via infiltration. (Modified from Tanaka et al. 2012)
52
3.3 Results
3.3.1 The tissue/organ expression of AtBAT1 and OsBAT1 by microarray analysis
The expression of AtBAT1 and OsBAT1 was analyzed using the microarray database by
The Bio-Analytic Resource for Plant Biology (BAR). The microarray results showed that
AtBAT1 has a uniform expression in almost all tissues and highest expression in the petals during the flowering stage 12 (Figure III-4). OsBAT1 mRNAs show a wider range of expression than AtBAT1 and it has the highest expression in seedling roots, seeds, and mature leaves
(Figure III-5).
Figure III-4 Predicted Tissue/Organ Expression of AtBAT1 (generated by Arabidopsis eFP Browser (Winter et al. 2007)) 53
Figure III-5 Predicted Tissue/Organ Expression of OsBAT1, generated by Rice eFP Browser (Patel et al. 2012)
3.3.2 OsBAT1 overexpression results in delayed flowering and thicken stems due to
elevated polyamine levels
It is known that changes of polyamine levels result in phenotypic alteration in Arabidopsis and other plants (Alcazar et al. 2010; Kusano et al. 2008). Most of these reported changes were caused by differentiated expression of genes involved in polyamine metabolism (Alcazar et al.
2005; Capell et al. 1998; Nölke et al. 2005). In our case, we examined if overexpression of a rice polyamine antiporters OsBAT1 in Arabidopsis produced any phenotypic changes. OsBAT1 gene was transformed into wild-type Arabidopsis plants via floral dip mediated by Agrobacterium.
The transgenic plant overexpressing OsBAT1 were planted together with the wild-type and homozygous AtBAT1 plants under long-day conditions. Increased expression of this OsBAT1 results in distinct phenotypic changes in Arabidopsis. At five weeks, wild-type plants had already flowered but transgenic plants were delayed for one to two weeks with bigger rosette 54
leaves (Figure III-6a). After bolting, the flowering stems of the transgenic plants are thicker, almost as twice large as those of the wild-type (Figure III-6 b and c). In contrast, the mutant plants grew normally without the AtBAT1 gene and the knockouts have similar stem thickness to that of wild-type plants (Figure III-6 b and c).
To determine whether these phenotypic changes were correlated with changes in polyamine levels we measured polyamine contents in these plants using HPLC. The leaves of wild-type, AtBAT1-KO, and OsBAT1-OX were collected at certain time points according to the growth stages of wild-type plants. At two weeks, all the plants were at vegetative growth stage; at four weeks, wild-type and AtBAT1-KO plants started to flower but OsBAT1-OX had not yet bolted; at six weeks, wild-type and AtBAT1-KO plants already developed siliques while
OsBAT1-OX plants were at the flowering stage.
HPLC results (Figure III-7) indicated that AtBAT1-KO had similar polyamine levels with wild-type plants at all the three stages expect for the putrescine contents at 6-week. After flowering, AtBAT1-KO plants seemed to have less soluble putrescine than wild-type. In contrast,
OsBAT1-OX plants had different polyamine titers through the three stages, which may have contributed to the delayed flowering and thicker stems. At two weeks before flowering stage,
OsBAT1-OX accumulated more soluble conjugated putrescine and spermidine. At four weeks, even though the plants were present at different development stages, the flowering wild-type and
AtBAT1-KO plants had similar putrescine levels with OsBAT1-OX without flowering. At the same stage, soluble conjugated spermidine and spermine in OsBAT1-OX were more than that in the other two plants. At six weeks, the flowering OsBAT1-OX had much less putrescine than the other two plants after flowering. There was no significant difference of the spermidine levels at this stage but OsBAT1-OX had more soluble and soluble conjugated spermidine. 55
Figure III-6 Phenotype of wild-type (WT, Col-0), OsBAT1 overexpressing (OX) lines, and AtBAT1 knockout (KO) A. thaliana plants. All the plants were grown under long-day (16h light) condition. a. Comparison of 5-week-old OsBAT1 OX with the wild-type plant. b. Comparison of 6-week-old OsBAT1 OX, wild-type, and homozygous AtBAT1 KO plants. The top inset shows an enlargement of the the box area of the bottom picture indicating the thicker stems of OsBAT1. c. Comparison of stem thickness between OsBAT1 OX, wild-type, and homozygous AtBAT1 KO plants. The diameters of stems were measured in 6-week-old lants. Bars show mean±SE (n=8). 56
Figure III-7 Soluble , soluble conjugated , and insoluble conjugated polyamine levels in A. thaliana plants measured via HPLC. All the plants were grown under long-day (16h light/8h dark) condition. Leaves were collected from the same plants through the three growth stages (2-week, 4-week, and 6-week). Bars show mean±SE. HPLC analysis performed and data collected by Menaka Ariyaratne. 57
3.3.3 OsBAT1, AtBAT1.1, and AtBAT1.2 are localized to plastids
Before the in vivo localization of BAT proteins, the amino acid sequences of each target proteins were retrieved and run through online predictors. The programs used for this project are
WolF PSORT (Horton et al. 2007) , TargetP (Emanuelsson et al. 2000), BaCelLo (Pierleoni et al.
2006), Plant-mPLoc (Chou and Shen 2010), and iPSORT (Bannai et al. 2002). The predicted results are shown in Table III-1. The predictions are not compatible from these different software, which suggests that the results are not reliable and in vivo localization is essential for the accurate localization of the BAT antiporters.
Table III-1 Predicted localization of BAT transporters
Gene WolF PSORT TargetP BaCelLo Plant-mPLoc iPSORT AtBAT1.1 vacuole no data plastid plasma membrane no data AtBAT1.2 vacuole no data plastid plasma membrane plastid OsBAT1 plasma membrane unknown unknown plasma membrane no data
The transient expression of cGFP-tagged OsBAT1, AtBAT1.1, and AtBAT1.2 was examined using confocal microscopy. We found that all of the BATs transporters in this study are localized to the chloroplasts of the tobacco leaf cells (Figure III-8). The images were taken at
1 micron intervals and this picture is a stack of 60 sections. The autofluorescence from the chlorophyll was used as a reference for the chloroplast localization. The GFP signals false- colored in green overlay well with the chlorophyll autofluorescence false-colored in red, indicating that all the proteins are localized to the chloroplast. In order to further confirm the chloroplast localization of AtBAT1.1, the same transient expression was done using the leaves of
Arabidopsis plants. The same pattern of chloroplast localization was observed in both
Arabidopsis and tobacco cells after 18-hour incubation following the infiltration (Figure III-9). 58
Figure III-8 Subcellular localization of BAT-GFP fusion proteins. 35S::BAT-GFP fusion proteins (middle) were expressed in tobacco leaf via Agrobacterium infiltration and viewed under confocal fluorescence microscope with lasers. Autoflorescence of chloroplasts and merged images with bright field are shown in the left and right, respectiviely. Fluorescent images were taken 48 hours after infiltration. 59
Figure III-9 Subcellular localization of AtBAT1.1-GFP fusion proteins with shorter expression time. a. AtBAT1.1-GFP was expressed in tobacco leaf. b. AtBAT1.1-GFP was expressed in Arabidopsis leaf. Both leaves were infiltrated with the same amount of transfomred Arogrobacterium and examined 18 hours after infiltration.
60
3.4 Discussion
In this chapter, I have shown that the overexpression of a rice polyamine and amino acid antiporter OsBAT1 in Arabidopsis resulted in phenotypic changes that include a delay in flowering and thicker stems. HPLC analysis revealed that polyamine levels in the transgenic plants were higher than wild-type plants before flowering. In addition, all the three characterized polyamine/amino acid antiporters, AtBAT1.1, AtBAT1.2, and OsBAT1 are localized to the chloroplast by transient expression in tobacco leaves. The microarray data indicated that
AtBAT1 has more even distribution in tissues/organs compared with OsBAT1 (Figure III-4 and
Figure III-5), which may be due to the fact that Arabidopsis has only one BAT protein while rice has multiple genes in this clade. This characterized OsBAT1 has critical roles in roots, mature leaves and developmental seedling according to its in vivo expression patterns.
3.4.1 Subcellular localization of BATs
In our GFP tagging experiments, all the BATs appear to localize to the chloroplasts (Figure
III-8). The confocal image of tobacco leaf cells expressing BAT-GFP fusion proteins clearly indicated the location of the proteins in the chloroplasts but not any other parts of the cell according to their overlays with autofluorescence by chlorophyll.
However in earlier work, Michaeli et al. characterized AtBAT1.1 as a GABA permease that was localized in the mitochondria of both tobacco protoplasts and tobacco leaves (Michaeli et al. 2011). There are a number of experimental discrepancies that could account for the differences in their finds from ours. First, the expression time of their GFP fusions in protoplasts could be too long. They viewed both the protoplasts 48 hours after transformation but it is recommended to check the GFP expression within 16 hours (Wu et al. 2009). Secondly, protoplasts may not reflect the in planta condition of the tissue that they were isolated from 61
(Faraco et al. 2011). It is possible that the product of one gene may be distributed to multiple cellular compartments (Yogev and Pines 2011). Some proteins have been reported to have dual localization to mitochondria and chloroplasts since the targeting sequences for the two organelles are similar (Berglund et al. 2009; Carrie, Giraud, Whelan 2009; Pujol, Maréchal-Drouard,
Duchêne 2007). In this case, we could expect AtBAT1.1 to potentially be targeted to both the mitochondria and the chloroplast. In our experiments GABA was not an effective competitor of polyamine exchange with amino acids (not shown). While their assay showed there was less uptake in mitochondria isolated from the knockout strain, this assay did not demonstrate that this protein was directly involved in the uptake of GABA. In addition, they did not address the other known transport activities of this protein (Dündar and Bush 2009).
3.4.2 Overexpression of OsBAT1 in Arabidopsis and results in phenotypic changes
The transgenic Arabidopsis plants overexpressing OsBAT1 driven by 35S promoter displayed different phenotypes including delayed flowering, bigger rosette leaves, and thicker stems, compared to wild-type and AtBAT1 knockout plants. According to the HPLC results, the transgenic plants contained higher levels of soluble conjugated putrescine and spermidine and soluble spermidine before flowering (Figure III-7). The accumulation of polyamines may contribute to the delayed flowering phenotypes. Since what we measured here was the total pool of polyamines in Arabidopsis leaves, it certainly demonstrates that transport is integral to the regulation of cellular polyamines. However, we are not yet clear with the mechanism of how exactly overexpression of OsBAT1, a polyamine antiporter, leads to these changes of polyamine levels and phenotypes. We have no data of the net change of export or import of polyamines because directionality of the transporter in vivo has not been established. 62
The HPLC analysis also indicated a significant reduction of spermidine in the leaves of all plants entering the flowering stage (Figure III-7), which suggests the involvement of spermidine in flowering development. The exogenously applied spermidine stimulates bolting and flowering of Arabidopsis under short-day condition but delayed the processes under long-day condition
(Applewhite, Kaur‐Sawhney, Galston 2000). Although the mechanism is not very clear, spermidine is known to affect flowering through its interaction with hormones such as gibberellin. Arabidopsis ADC2 overexpressor lines display an altered phenotype of dwarf and delayed-flowering. It was reported that genes involved in gibberellin synthesis were repressed in these transgenic plants and the late flowering was a result of deficiency in gibberellin (Alcazar et al. 2005). During the flowering stage, the spermidine levels dropped expressively and reached a very low level in both wild-type and AtBAT1 KO, but the spermidine contents in transgenic plants were still relatively higher (Figure III-7). This could be explained by the transfer of spermidine from leaves to flowers. Additionally, this was the flowering time for wild-type and
AtBAT1 KO lines, but transgenic plants overexpressing OsBAT1 was actually in an earlier developmental stage – just started bolting. Its usage of spermidine might not be as much as that in the other plants. Overall, endogenous spermidine levels are higher in flowers than the rest of the plant (Applewhite, Kaur‐Sawhney, Galston 2000). Further experiments could be conducted to measure spermidine accumulation in flower buds and compare the concentration in different lines.
Polyamines have been known to play important roles in plant developments (Alcázar and
Tiburcio 2014; Neily et al. 2010; Tiburcio et al. 2014). The most common ways of manipulating polyamine levels in cells are by regulating homologous or heterologous genes involved in polyamine metabolisms. The high level of putrescine induced by overexpression of the 63 homologous ADC2 gene in Arabidopsis delayed flower time and reduced plant height (Alcazar et al. 2005). In a spermidine synthase gene (SPDS) double-mutant Arabidopsis plant, putrescine was accumulated but spermidine level was reduced, which resulted in infertile seeds (Imai et al.
2004). Here we have shown that overexpression of a single polyamine transporter is also a tool that can be used to regulate cellular polyamine contents.
These experiments have proven that we can disrupt the equilibrium pf polyamine levels by altering the expression of transporters. Additionally, we have learned that by regulating the expression of a single transporter, we can drive certain phenotypes via the changes in basal levels of its substrates. In nature, the changing of phenotypes is mediated by transcription factors, which activate specific genes. We are not suggesting that the membrane transporters are acting as transcription factors but that an altered expression of transporters can be used to mimic such affection. Our experiments certainly suggest that cells must have a polyamine sensing mechanism that can then activate other regulatory pathways. 64
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CHAPTER IV
HIDING IN PLAIN SIGHT: A THIRD ROUTE FOR PUTRESCINE BIOSYNTHESIS IN
PLANTS
Jigar Patel1, Lingxiao Ge1, Sheaza Ahmed1, Menaka Ariyaratne1, Vipaporn Phuntumart1, Andrea
Kalinoski2, Paul F. Morris1*.
1Department of Biological Sciences, Bowling Green State University, Bowling Green OH 43403.
2Department of Surgery, University of Toledo, Toledo OH 43614
*Corresponding author.
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4.1 Abstract
Metabolic pathways can be localized to a single organelle or distributed across several cellular compartments. Previous work has identified only a single cytosolic pathway for putrescine synthesis in Arabidopsis. Here we show that both A. thaliana and soybeans have a plastid-localized putrescine pathway consisting of an arginine decarboxylase and an agmatinase that combine to synthesize putrescine from arginine. Since the sequences of plant arginases show conservation of key residues and the predicted 3D structure of plant agmatinases overlaps the crystal structure of the enzyme from Deinococcus radiodurans, we suggest that these enzymes can synthesize putrescene, whenever they have access to the substrate agmatine. Finally, we demonstrate that the synthesis of putrescine by ornithine decarboxylase is localized to the endoplasmic reticulum. Thus A. thaliana has two, and soybeans have three separate pathways for putrescine synthesis.
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A distinguishing feature of eukaryotic cells is the presence of membrane-bound organelles and vesicles. In plants, these cellular rooms have multiple functions that include the specialized compartmentation of metabolic processes such as carbon fixation, and respiration; thylakoid membranes to capture light energy, and the sequestering of waste products or anti-feedants that are normally released only upon cell lysis (Lunn 2007). In addition to its role in photosynthesis, the plastid plays a major role in the biosynthesis of amino acids, nucleotides, fatty acids, and vitamins. This compartmentation of metabolism requires the organized import and export of metabolites, but also prevents futile cycling of metabolites (Linka and Weber 2010). An additional feature of plant metabolism is the redundancy of certain metabolic pathways (Plaxton
1996, Krueger, Niehl et al. 2009, Ros, Munoz-Bertomeu et al. 2014). This is also the case for polyamine biosynthesis. Ornithine decarboxylase (ODC) converts ornithine directly to putrescine
(Fuell, Elliott et al. 2010) and then putrescine serves as one of the building blocks for spermidine, thermospermine, and spermine. A second route for putrescine biosynthesis, utilizes arginine decarboxylase (ADC) to convert arginine to agmatine, and two additional enzymes, agmatine deiminase (AtAI) and N-carbamoyl putrescine aminohydrolase (At-NLP1) are needed to complete the pathway. Polyamines are essential for cell viability, and levels of putrescine along with the other polyamines spermidine, thermospermine, and spermine are strongly correlated with cellular responses to development and various environmental stresses (Tiburcio,
Altabella et al. 2014). These two independent pathways for polyamines enable plants to differentially activate the pathways in response to an environmental stimulus. In the course of evolution, A. thaliana has lost ODC, but now has two ADC’s (Hanfrey, Sommer et al. 2001).
The two ADCs also show a strong divergence in expression, and up-regulation of ADC2 has been implicated in multiple stress responses (Winter, Vinegar et al. 2007).
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In E coli and mammals, putrescine can be synthesized in a two-step process from arginine by arginine decarboxylase, which makes agmatine; and agmatinase, which converts agmatine to putrescine with the release of urea (Heller, Rostomily et al. 1983, Satishchandran and Boyle
1986, Mistry, Burwell et al. 2002). Enzymes with agmatinase activity are members of the ureohydrolase superfamily that also include enzymes with arginase, forminoglutamase, and proclavaminate amidoinohydrolase activities (Ahn, Kim et al. 2004). Phylogenetic analyses suggest that plant arginases may be more similar to bacterial agmatinases than bacterial or mammalian arginases (Chen, McCaig et al. 2004). However protein characterization of both tomato arginase/agmatine enzymes indicated that they exhibited a clear preference for arginine over agmatine in in vitro assays. Since plant agmatinases are known to be strongly conserved, it has been assumed that this pathway did not exist in plants. Co-transformation of an oat ADC and either a human or an E. coli agmatinase gene has previously been used to reconstitute a putrescine biosynthetic pathway in yeast (Mistry, Burwell et al. 2002). Since A. thaliana contains two genes with predicted arginase/agmatinase activity (At4G08870 and At4G08890) we adopted a similar strategy to assess the potential agmatinase activity of these genes. AtADC1 was transformed into the yeast mutant Spe1 and this strain was co-transformed with either
AtARGAH1 or ATARGAH2. Transformants expressing single genes did not grow in the absence of exogenous putrescine (Fig. 1). However expression of AtADC2 and either AtARHAH1 or
AtARGAH2 fully complemented the growth defect of Spe1. Thus both of the A. thaliana agmatinases are capable of synthesizing putrescine when supplied with agmatine.
To address gaps in the localization of polyamine biosynthesis, we first sought to confirm the cytosolic location of ADC1 by transient expression of N-terminal and C-terminal GFP fusions in tobacco leaves. Both constructs were found to localize to the cytosol (Fig. S1). Since
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AtAIH and AtNLP have been previously localized to the cytosol in a proteomic study (Ito, Batth et al. 2011) all of the enzymes of this putrescine pathway are localized in the cytosol. AtADC2 contains a predicted chloroplast localization signal and transient expression of AtADC2 in N. benthamiana leaves confirmed that this protein was localized to the plastid. Since AtARGAH2 had been localized to the chloroplast in one proteomic experiment, we tested the localization of this gene by transient expression in N. benthamiana. AtARGAH2-GFP constructs were found to be localized in the chloroplast. Thus we concluded that the leaves of A.thaliana have a complete plastid biosynthesis pathway for putrescine.
To investigate whether this pathway might exist in other plants, we used the Phytozome database to identify species with multiple ADCs and arginase/agmatinase genes. We found that,
23 of 43 sequenced plant genomes had more than one ADC, and 12 of these genomes had at least one ADC with predicted localization to the plastid (Table S1). Within in this subset of plant genomes, 13 genomes included more than one gene with predicted ARGAH activity.
Phylogenetic analysis showed that all of the ADCs from seven Brassica genomes grouped together in two clades of nine and five sequences (Fig. S2). AtADC2 was in a clade separate for
AtADC1 and all the members of the clade with AtADC2 which included at least one gene from each species, were predicted to be localized to the plastid. Other dicot species with two ADC genes included cacao, strawberry, tomato, potato, soybeans and Mimulus guttatus. With the exception of soybeans, at least one of the ADCs in each of these species, had a predicted plastid localization. The monocots; corn, rice and Panicum virgatum also contained two ADC genes, but none of these genes were predicted to be targeted to the chloroplast. However in oats, immunological staining indicated that ADC was localized in the plastid, so other monocots may have chloroplast localized ADCs. We also noted that moss has three predicted ADC genes, and
75 one of them is predicted to be localized in the chloroplast. A smaller number of species were found to have two genes with predicted ARGAH activity (Fig. S3). Species that contained two members of both ADC and ARGAH included four members of the Brassicaceae, along with tomatoes, and soybeans. Dicots including cassava, flax, and poplar had duplications in only
ARGAH, but not ADC. No monocot species were identified with more than one ARGAH.
To determine whether a complete biosynthetic pathway for putrescine in the chloroplast might exist outside of the Brassicaceae, we used transient expression assays of both
Glyma03G028000 (ADC2) and Glyma03g03270 (ARGAH) in N. benthamiana leaves to show that these two genes are localized to the chloroplast (Fig. S4.). In the yeast complementation assay, the expression of AtADC2 and Glyma03g03270 in the yeast mutant strain Spe1 enabled the yeast strain to grow in the absence of exogenous putrescine. Thus soybeans, along with A. thaliana have a complete chloroplast-localized putrescine biosynthetic pathway. Sequence alignment of plant agmatinases retrieved from Phytozome showed that these proteins have a high level of sequence conservation.
Protein homology modeling of these three ARGAHs from A. thaliana and Glycine max using PHYRE2 (Kelley and Sternberg 2009) showed that these proteins are structurally similar to agmatinase from Deinococcus radiodurans (DR) (Fig 3). These plant genes along with the human mitochondrial agamatinase (Ahn, Kim et al. 2004) have conservation of the critical manganese (Mn)-binding sites and the predicted catalytic residues. PHYRE2 analysis also revealed that the Mn-binding sites and catalytic sites are highly conserved amongst the ARGAHs investigated and DR agmatinase (Fig S5). A similar pattern was observed for an ARGAH from
Populus trichocarpa. Based on our analyses, it is likely that all plant arginases also have agmatinase activity.
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The localization of ornithine decarboxylase has not yet been clearly established. However, since we noted spatial separation of the two putrescine biosynthesis pathways starting with ADC, we postulated that synthesis of ornithine decarboxylase might be spatially separated from the cytosolic ADC pathway. The subcellular localization of OsODC was determined by transient expression of GFP fusions in N. benthamiana leaves mediated by Agrobacterium. The GFP signals overlapped with the mCherry signals of the endoplasmic reticulum (ER) marker (Fig
3a,b). Bombardment assay of onion epidermal cells confirmed the localization of ODC to the
ER (Fig. S7). Localization of ornithine decarboxylase to the ER may enable simultaneous operation of the urea cycle, and polyamine synthesis, which share a common substrate. It may also enable putrescine that is synthesized in the ER to be directly exported from the cell by secretion to the apoplast.
In this study we have confirmed the localization of two enzymes that enable the synthesis of putrescine in the chloroplast of A. thaliana and G. max. Our study also identified many plant species with two ADCs, some of which have been predicted to be localized to the plastid.
However, plant genomes with two ADCs and two agmatinases are not common, and notably, none of the sequenced monocot genomes contain two agmatinases. Thus the retention of a complete plastid pathway is relatively uncommon in plant genomes, assuming that arginase/agmatinases in plants with only one gene hae that copy localized to the mitochondria.
Of the seven sequenced Brassica genomes with two ADCs, only four were found to have retained two ARGAH genes. In this scenario, agmatine would then have to be exported from the plastid to be metabolized in the cytoplasm by the cytosolic pathway, or imported into the mitochondria to be converted to putrescine by agmatinases located there. Given the importance of interspecies hybridization and whole genome duplication events in the evolutionary history of
77 plants, the absence of a separate plastid pathway hints that for most plants, this pathway would be disadvantageous. Perhaps this is a consequence of that putrescine may also act as a permeant buffer in the thylakoid spaces, and under certain conditions impair the normal activity of the
ETC (Ioannidis, Cruz et al. 2012, Ioannidis and Kotzabasis 2014). Some plant genomes such as flax and cassava have two agmatinases. In these plants, synthesis of putrescine may also occur, if there is a plastid-localized agmatine transporter or antiporter.
The persistence of two or more biosynthetic pathways for polyamines is a likely consequence of the multiplicity of roles that these compounds have been shown to have in developmental and stress responses of plants. The spatial organization of these pathways also highlight the importance of PA transporters in providing an additional means of regulating localized changes of these signaling compounds. We hope that the elucidation of this pathway may renew interest in manipulating PA levels to optimize crop responses to climate change.
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Fig IV-1. Localization analysis of AtADC2 and AtARGAH2 by transient expression in N. benthamiana leaves. N. benthamiana leaves were infiltrated by Agrobacterium tumefecians (GV3101) containing expression plasmids harboring the full-length AtADC2 or AtARGAH2 fused to GFP at its C terminus under the control of the CaMV 35S promoter. GFP fluorescence and chlorophyll autofluorescence of transformed leaves were observed by confocal microscopy.
Fig IV-2. Characterization of Arabidopsis agmatinase by complementation in yeast. WT, spe1, spe1+ AtADC2, spe1 +AtARGAH2 and spe1 + AtADC2 +AtARGAH2 were plated on SC minimal media in the presence and absence of exogenous putrescine
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Fig. IV-3 Predicted 3D structure of AtARGAH2 is highly similar to the crystal structure of Deinococcus radiodurans agmatinase. (A) DR agmatinase, (B) AT agmatinase (C) superpose of A and B. Structure of AT agmatinase was predicted using PHYRE2 and superposition of DR agmatinase and AT agmatinase was done using Chimera.
Fig IV-4 Subcellular localization of OsODC. (Top) Confocal images of Nicotiana benthamiana leaf cells expressing OsODC-GFP under control of a cauliflower mosaic virus (CaMV) 35S constitutive promoter and fused with a C-terminus GFP tag. Scale bars, 25µm. Pictures were taken 3 days after the infiltration of Agrobacterium into tobacco leaves. (Bottom) Confocal images of onion epidermis cells expressing OsODC-GFP under control of a cauliflower mosaic virus (CaMV) 35S constitutive promoter and fused with N-terminus GFP tag. Scale bars, 50µm. Pictures were taken 16 hours after the particle bombardment.
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Ahn, H. J., K. H. Kim, J. Lee, J. Y. Ha, H. H. Lee, D. Kim, H. J. Yoon, A. R. Kwon and S. W.
Suh (2004). "Crystal structure of agmatinase reveals structural conservation and
inhibition mechanism of the ureohydrolase superfamily." J Biol Chem 279(48): 50505-
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arginase by wounding, jasmonate, and the phytotoxin coronatine." J Biol Chem 279(44):
45998-46007.
Fuell, C., K. A. Elliott, C. C. Hanfrey, M. Franceschetti and A. J. Michael (2010). "Polyamine
biosynthetic diversity in plants and algae." Plant Physiol Biochem 48(7): 513-520.
Hanfrey, C., S. Sommer, M. J. Mayer, D. Burtin and A. J. Michael (2001). "Arabidopsis
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arginine decarboxylase activity. ." Plant J 27: 551-560.
Heller, J. S., R. Rostomily, D. A. Kyriakidis and E. S. Canellakis (1983). "Regulation of
polyamine biosynthesis in Escherichia coli by basic proteins." Proc Natl Acad Sci U S A
80(17): 5181-5184.
Ioannidis, N. E., J. A. Cruz, K. Kotzabasis and D. M. Kramer (2012). "Evidence that putrescine
modulates the higher plant photosynthetic proton circuit." PLoS One 7(1): e29864.
Ioannidis, N. E. and K. Kotzabasis (2014). "Polyamines in chemiosmosis in vivo: A cunning
mechanism for the regulation of ATP synthesis during growth and stress." Front Plant Sci
5: 71.
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Kelley, L. A. and M. J. Sternberg (2009). "Protein structure prediction on the Web: a case study
using the Phyre server." Nat Protoc 4(3): 363-371.
Krueger, S., A. Niehl, M. C. Lopez Martin, D. Steinhauser, A. Donath, T. Hildebrandt, L. C.
Romero, R. Hoefgen, C. Gotor and H. Hesse (2009). "Analysis of cytosolic and plastidic
serine acetyltransferase mutants and subcellular metabolite distributions suggests
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3(1): 21-53.
Lunn, J. E. (2007). "Compartmentation in plant metabolism." J Exp Bot 58(1): 35-47.
Mistry, S. K., T. J. Burwell, R. M. Chambers, L. Rudolph-Owen, F. Spaltmann, W. J. Cook and
S. M. Morris, Jr. (2002). "Cloning of human agmatinase. An alternate path for polyamine
synthesis induced in liver by hepatitis B virus." Am J Physiol Gastrointest Liver Physiol
282(2): G375-381.
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and functions." Trends Plant Sci 19(9): 564-569.
Satishchandran, C. and S. M. Boyle (1986). "Purification and properties of agmatine
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Tiburcio, A. F., T. Altabella, M. Bitrian and R. Alcazar (2014). "The roles of polyamines during
the lifespan of plants: from development to stress." Planta.
Winter, D., B. Vinegar, H. Nahal, R. Ammar, G. V. Wilson and N. J. Provart (2007). "An
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83
4.3 Supplementary Materials
4.3.1 Methods
4.3.1.1 DNA Sources and Constructs cDNA clones of Arabidopsis thaliana ADC2 (At4G34710), ARGAH 1 (At4G08900), and
ARGAH2 (At4G08870) were obtained from ABRC. Genes were amplified via PCR using gene- specific primers. AtADC1 was amplified from genomic DNA of A. thaliana Col-0. Rice genomic DNA was isolated from the rice cultivar Nipponbare using the CTAB method (Clarke
2009). The full length OsODC (LOC_Os02g28110) gene was amplified from the genomic DNA.
GmODC (Glyma04g020200.1) was amplified by PCR using genomic DNA from the cultivar
Williams. The sequence of Glyma.03g028000 (GmARGAH) was codon-optimized for expression in yeast and synthesized by GenScript, Pistcataway, NJ.
4.3.1.2 Subcellular Localization Analysis
AtADC2, AtARGAH2, AtADC1, OsODC, GmADC, GmARGAH, and GmODC were cloned into plant expression vectors pGWB5/pGWB6 to generate constructs with C- or N-terminal GFP, using GATEWAY recombination system (Nakagawa et al. 2007). Inserts were verified by PCR and/or sequencing. The resulting constructs were then transformed into Agrobacterium tumefaciens strain GV3101 and infiltrated into tobacco (Nicotiana benthamiana) leaves (Sparkes et al. 2006). Infiltrated leaves were subjected to confocal laser-scanning analysis after 12-48 hours of infiltration with a TCS SP5 multi-photon laser scanning confocal microscope.
Fluorescence of chlorophyll or mCherry tagged ER Marker (ER-rbCD3960) (Nelson et al. 2007) was used as organelle markers. Images were merged using ImageJ (Rasband, 2008).
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4.3.1.3 Yeast Complementation Assay
The SPE1 yeast knockout strain that lacks ornithine decarboxylase (YSC6273-201936543) was obtained from GE Darmacon, Lafayette, CO. BY4741 served as a wild-type control. Yeast strains were maintained on enriched medium (YEPD) or SC minimal medium supplemented with
1 mM putrescine. The two plasmid constructs pAG303-ADC2 and pYES-DEST52-ARGAH2 were introduced to yeast SPE1 mutant strain separately. Competent yeast spe1 mutant cells were grown on liquid YEPD medium and incubated overnight at 30°C. The pAG303-ADC2 plasmid construct was introduced to yeast spe1 mutant cells by electroporation. The resulting transformants were selected on SC minimal medium lacking histidine and containing putrescine
(1 mM). Selected colonies were transferred to liquid YEPD medium to obtain competent cells.
The pYES-DEST52-ARGAH2 plasmid construct was then introduced to the above competent cells by electroporation. The resulting transformants were again selected on SC minimal medium plates lacking uracil and containing putrescine (1mM). Finally, selected transformants were tested for growth in SC minimal media with 1% raffinose and 2% galactose lacking exogenous polyamines.
4.3.1.4 Agmatinase Activity Assay
Arabidopsis agmatinase was cloned into pBAD-DEST49 Gateway destination vector according to manufacturer’s instructions. The enzyme was purified using HIS-Select™ kit from Sigma-
Aldrich by following manufacturer’s instruction. Enzyme assay was performed at 25°C in a buffer modified from (Chen et al. 2004). Agmatinase activity was determined by measuring the product (putrescine) by HPLC.
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4.3.1.5 Phylogenetic Analysis
Protein sequences of ADC and agmatinase/arginase were obtained from assembled plant genomes (Goodstein et al. 2012). The amino acid sequence alignment was created using
MUSCLE (Edgar 2004). Phylogenetic trees were constructed by MEGA 6.06 (Tamura et al.
2013) using the maximum likelihood method based on the Jones-Taylor-Thornton (JTT) matrix- based model. The reliability of the trees was tested using a bootstrapping test with 1000 duplicates.
4.3.1.6 Phyre2 Analysis
Three-dimensional structures of arginases/agmatinases from Arabidopsis, soybeans, and poplar were predicted using Phyre2 (Kelley & Sternberg, 2009). The predicted structures were then compared to the three-dimensional structure of Dienococcus radiodurans (DR) agmatinase (PDB entry 1WOHA) using Chimera (http://www.cgl.ucsf.edu/chimera). The 3D structures were superimposed using matchmaker tool of Chimera. Active site regions of DR agmatinase and plant arginases were compared using match-align tool of Chimera.
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Table IV-S1. Predicted localization of arginine decarboxylase genes from sequenced plant genomes
Species Name Gene PLPred TargetP Predotar WolfPSort Arabidopsis lyrata 491183Id Plastid Plastid Arabidopsis lyrata 931833 Plastid Plastid Arabidopsis thaliana AT4G34710 Plastid Arabidcoplusismb tiahal iana AT2G16500 Plastid Plastid Plastid Brasscoluicamb naia pa BnaA03g53010D Plastid Plastid Plastid Brassica napa BnaC07g45200D Plastid Plastid Plastid Brassica rapa Brara.K00332 Plastid Plastid Plastid Brassica rapa Brara.A00352 Plastid Capsella grandiflora Cagra.2350s0020 Plastid Plastid Plastid Capsella grandiflora Cagra.21579s0001 Plastid Capsella rubella Carubv10004246m.g Plastid Plastid Plastid Capsella rubella Carubv10013090m.g Plastid Eutrema salsugineum Thhalv10024529m.g Plastid Plastid Plastid Eutrema salsugineum Thhalv10022578m.g Plastid Fragaria vesca gene00390-v1.0-hybrid Plastid Plastid Fragaria vesca gene01668-v1.0-hybrid Plastid Plastid Glycine max Glyma.06G007500 Plastid Glycine max Glyma.04G007700 Plastid Mimulus guttatus Migut.N01279 Plastid Plastid Mimulus guttatus Migut.B00171 Plastid Oryza sativa LOC_Os06g04070 Oryza sativa LOC_Os04g01690 Panicum virgatum Pavir.J13691 Plastid Panicum virgatum Pavir.J33899 Physcomitrella patens Phpat.006G071900 Physcomitrella patens Phpat.005G013600 Physcomitrella patens Phpat.016G006200 Plastid Solanum lycopersicum Solyc10g054440.1 Plastid Solanum lycopersicum Solyc01g110440.2 Plastid Solanum tuberosum PGSC0003DMG400026 Plastid Plastid Solanum tuberosum P67G1S C0003DMG400001 Plastid Plastid Theobroma cacao 662 Thecc1EG006773 Plastid Plastid Plastid Theobroma cacao Thecc1EG020310 Plastid Plastid Zea mays GRMZM2G396553 Zea mays GRMZM2G374302 Plastid
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Table IV-S2 Predicted localization of arginase/agmatinase genes from sequenced plant genomes
Species Name Gene PLPred TargetP Predotar WolfPSort Arabidopsis lyrata 489723Id Plastid Plastid Arabidopsis lyrata 489781 Plastid Arabidopsis thaliana AT4G08900 Plastid Arabidopsis thaliana AT4G08870 Plastid Brassica napa BnaC03g28300D Plastid Plastid Brassica napa BnaA03g23800D Plastid Plastid Capsella rubella Carubv10001349m.g Plastid Capsella rubella Carubv10001337m.g Plastid Glycine max Glyma.17G131300 Plastid Glycine max Glyma.01G140200 Plastid Glycine max Glyma.03G028000 Plastid Linum usitatissimum Lus10000795.g Plastid Linum usitatissimum Lus10030288.g Plastid Plastid Manihot esculenta cassava4.1_011300m.g Plastid Manihot esculenta cassava4.1_011323m.g Plastid Plastid Populus trichocarpa Potri.002G146200 Plastid Plastid Populus trichocarpa Potri.014G067700 Plastid Solanum lycopersicum Solyc01g091160.2 Plastid Plastid Solanum lycopersicum Solyc01g091170.2 Plastid
Fig S1. Localization analysis of AtADC1 by transient expression in N. benthamiana leaves. N. benthamiana leaves were infiltrated by Agrobacterium tumefecians (GV3101) containing expression plasmids harboring the full-length AtADC1 fused to GFP at its C terminus under the control of the CaMV 35S promoter. GFP fluorescence and chlorophyll autofluorescence of transformed leaves were observed by confocal microscopy. (Not yet available.)
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Fig IV-S2 Phylogenetic analysis of plant arginine decarboxylases.
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Fig IV-S3 Phylogenetic analyses of Plant arginase/agmatinases.
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Fig IV-S4 Characterization of Soybean agmatinase by complementation in yeast. WT, spe1, spe1+ AtADC2, spe1 +GmARGAH , and spe1 + AtADC2 + GmARGAH were plated on SC minimal media in the presence (left) and absence (right) of exogenous putrescine.
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Fig IV-S5. Comparison of active site residues of Dienococcus radiodurans agmatinase and arginases from Arabidopsis thaliana, Glycine max, and Populus trichocarpa. Red boxes show the active site residues.
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4.3.2 References
Chen H, McCaig BC, Melotto M, He SY, Howe GA (2004) Regulation of plant arginase by
wounding, jasmonate, and the phytotoxin coronatine. J Biol Chem 279 (44):45998-
46007. doi:10.1074/jbc.M407151200
Clarke JD (2009) Cetyltrimethyl ammonium bromide (CTAB) DNA miniprep for plant DNA
isolation. Cold Spring Harbor protocols 2009 (3):pdb prot5177.
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Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high
throughput. Nucleic Acids Res 32 (5):1792-1797. doi:10.1093/nar/gkh340
Goodstein DM, Shu S, Howson R, Neupane R, Hayes RD, Fazo J, Mitros T, Dirks W, Hellsten
U, Putnam N, Rokhsar DS (2012) Phytozome: a comparative platform for green plant
genomics. Nucleic Acids Res 40 (Database issue):D1178-1186. doi:10.1093/nar/gkr944
Nakagawa T, Kurose T, Hino T, Tanaka K, Kawamukai M, Niwa Y, Toyooka K, Matsuoka K,
Jinbo T, T. K (2007) Development of series of Gateway binary vectors, pGWBs, for
realizing efficient construction of fusion genes for plant transformation. J Bioscience
Bioeng 104:34-41
Nelson BK, Cai X, Nebenfuhr A (2007) A multicolored set of in vivo organelle markers for co-
localization studies in Arabidopsis and other plants. Plant J 4:1126-1136
Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image
analysis. Nature methods 9 (7):671-675
Sparkes IA, Runions J, Kearns A, Hawes C (2006) Rapid, transient expression of fluorescent
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Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary
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94
CHAPTER V SUMMARY
5.1 Identifying plant polyamine antiporters that affect plant development
In nature, metabolic and phenotypic changes of an organism can result from altered gene expression regulated by transcription factors (Mitsuda and Ohme-Takagi 2009). In this present study, we have shown that by altering the expression of a single polyamine antiporter (OsBAT1) we can mimic the natural phenotypic changes caused by transcriptional regulation. This evidence in turn reaffirms the important roles of polyamines as signaling molecules in plant developments as described in a number of reviews (Alcázar and Tiburcio 2014; Moschou et al. 2012; Tiburcio et al. 2014; Wimalasekera, Tebartz, Scherer 2011).
A radioisotope uptake assay of membrane vesicles generated from transgenic E. coli mutant cells was developed for the characterization of AtBAT1.1, AtBAT1.2, and OsBAT1 as antiporters for polyamines and amino acids (Chapter II). E. coli double-knockout mutants deficient in polyamine antiporters (DKO) were generated from available single-knockout strains by means of P1 phage transduction. The target proteins were transformed into DKO and inside- out membrane vesicles of these E. coli cells were formed using French press or ultrasound sonication. When arginine was used as a counterion, the vesicles containing the BAT proteins accumulated labeled spermidine or putrescine, thus establish their role as polyamine/amino acid antiporters.
There are different kinds of membrane transporters in plant cells: active transporter, symporters, antiporters/exchangers, ion channels, and aquaporins (Buchanan, Gruissem, Jones
2000). Among these transporters, antiporters are really hard to characterize since there is no targeted assay established for them due to their bidirectional nature. This mehods represents a
95 new tool for characterizing plant antiporters and sets up a successful example for examining other types of antiporters using the vesicles of E. coli as the expression platform. Our lab has a particular interest in characterizing all the cellular transporters of polyamines. This system will enable us to expand research on plant polyamine antiporters, such as the orthologs of BATs in soybeans as well as antiporters in other clades.
Compartmentalization is fundamental for the action of membrane transporters in cells. To study where the BATs proteins are localized in vivo, cGFP was used to tag the proteins of interest and the localization was detected using transient expression and confocal fluorescent microcopy (Chapter III). It was determined that all the three BATs are chloroplast transporters.
To study the biological impact of BAT transporters on plant development, the rice protein
OsBAT1 was overexpressed in the A. thaliana wild-type. The transgenic plants were compared with wild-type and AtBAT1 knockout plants and showed altered phenotypes such as delayed flowering and thicker stems (Chapter III). Polyamine contents were analyzed to unravel the biochemical factors that affected these phenotypes. We have found out that spermidine was accumulated in the leaves of the over-expressor lines but not putrescine or spermine. This high level of spermidine matches the HPLC results of E. coli strains expressing the plant BAT proteins (Chapter II). Although we are unclear with the mechanism, the evidence certainly links the expression of polyamine antiporters, polyamine concentration in cells, and plant development.
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5.2 Revealing routes for putrescine biosynthesis in plants
In planta, it is well known that putrescine is synthesized through the ornithine decarboxylase (ODC) pathway and the arginine decarboxylase (ADC) pathway. The model plant organism Arabidopsis, however, has lost the ODC pathway and has two ADC genes instead
(Tiburcio et al. 1997). Here we show that AtADC1 is localized to the cytosol while AtADC2 has chloroplast localization. We further illustrated that another chloroplast-targeted protein
(AtARGAH2) had both arginase and agmatinase activity and it functioned together with
AtADC2 to synthesize putrescine in the yeast mutant (Chapter IV). This particular pathway was unknown in plants but has been found in E. coli and mammals (Mistry et al. 2002; Szumanski and Boyle 1990). In this pathway, arginine is first converted to agmatine catalyzed by arginine decarboxylase and then agmatine is synthesized by agmatinase to form putrescine. We first confirmed the existence of the chloroplast ADC pathway in Arabidopsis and extended this observation to soybeans as a third route for putrescine synthesis. Indeed, these findings change our knowledge about polyamine synthesis in plants and also hint at the importance of compartmentation of polyamine synthesis in plant cells.
Our research also demonstrated that the plant ornithine decarboxylase (ODC) pathway is a cytosolic pathway for putrescine synthesis, by localizing OsODC to the ER in rice. It has been well known that most plants have both ADC and ODC pathways to synthesize putrescine, but the subcellular localization of ornithine decarboxylase has never been clearly addressed in plants.
This discovery will certainly help us elucidate the distribution of polyamines in cells and compartmentation of polyamine metabolism.
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5.3 References
Alcázar R and Tiburcio AF. 2014. Plant polyamines in stress and development: An emerging
area of research in plant sciences. Frontiers in Plant Science 5:319.
Buchanan BB, Gruissem W, Jones RL. 2000. Biochemistry & molecular biology of plants.
American Society of Plant Physiologists Rockville.
Mistry SK, Burwell TJ, Chambers RM, Rudolph-Owen L, Spaltmann F, Cook WJ, Morris
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Mitsuda N and Ohme-Takagi M. 2009. Functional analysis of transcription factors in
arabidopsis. Plant Cell Physiol 50(7):1232-48.
Moschou PN, Wu J, Cona A, Tavladoraki P, Angelini R, Roubelakis-Angelakis KA. 2012. The
polyamines and their catabolic products are significant players in the turnover of
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Tiburcio AF, Altabella T, Borrell A, Masgrau C. 1997. Polyamine metabolism and its regulation.
Physiol Plantarum 100(3):664-74.
Wimalasekera R, Tebartz F, Scherer GF. 2011. Polyamines, polyamine oxidases and nitric oxide
in development, abiotic and biotic stresses. Plant Science 181(5):593-603.