Evolution of Homospermidine Synthase in Pyrrolizidine Alkaloid Biosynthesis of the Morning Glory (Convolvulaceae) Family

Evolution of homospermidine synthase in pyrrolizidine alkaloid biosynthesis of the morning glory (Convolvulaceae) family

by Arunraj Saranya Prakashrao

Dissertation in fulfillment of Requirements of the Doctoral Degree Doctor rerum naturalium of the Faculty of Mathematics and Natural Sciences at Christian-Albrechts-Universität zu Kiel

Kiel, October 2020

Chairperson: Prof. Dr. Axel J. Scheidig (Zoological Institute, Christian-Albrechts- Universität zu Kiel, Kiel, Germany)

First referee: Prof. Dr. Dietrich Ober (Botanical Institute, Christian-Albrechts- Universität zu Kiel, Kiel, Germany)

Second referee: Prof. Dr. Eva H Stukenbrock (Botanical Institute, Christian-Albrechts- Universität zu Kiel, Kiel, Germany)

Additional examiner: Prof. Dr. Birgit Classen (Pharmaceutical Institute, Christian- Albrechts-Universität zu Kiel, Kiel, Germany)

Date of thesis defense: 24.11.2020 Approved for printing: 24.11.2020

ABSTRACT

Pyrrolizidine alkaloids (PAs) are toxic compounds produced in many different species of flowering plants as a chemical defense against herbivores. The PAs are also economically important as they can cross-contaminate herbals medicines and food products and are harmful if ingested. The biosynthesis of PAs evolved several times independently in different plant lineages, such as Apocynaceae, Asteraceae, Boraginaceae, Convolvulaceae, Fabaceae,

Poaceae, and Orchidaceae. Homospermidine synthase (HSS) is the first pathway-specific enzyme of PA biosynthesis. Interestingly, HSS repeatedly evolved by gene duplication events from the primary metabolic gene of deoxyhypusine synthase (DHS) and was successfully recruited into PA metabolism in all the PA producing plant species. DHS is involved in activating the eukaryotic translation initiation factor 5a (eIF5a) by transferring an aminobutyl group from spermidine to a particular lysine residue in eIF5a. The HSS transfers the aminobutyl group from spermidine to putrescine to form homospermidine, which serves as PAs' backbone.

In the morning glory (Convolvulaceae) family, the PAs occur in two different clades called

Ipomoea (sweet potato clade) and Distimake (wood roses). However, only a single gene duplication event was reported in the morning glory family long before Ipomoea and Distimake diverged. Thus, it was hypothesized that the recruitment of the duplicated copy to PA biosynthesis occurred independently in these two clades, i.e., the functional shift from DHS activity to HSS activity occurred two times.

In this thesis, an analytical method was developed to directly measure both DHS and HSS enzymes' activities using reversed-phase high-pressure liquid chromatography (HPLC). The new method uses chromophore to derivatize substrates and products that can be monitored by

UV absorbance and fluorescence, thus replacing the old radio-active labeled tracer experiments. The HPLC method proved to be robust, fast, and reliable. This method can also

i detect various side reactions of both DHS and HSS in addition to their main activities, thus shedding light on the previously unexplored promiscuous activities.

Furthermore, an extensive search for hss-like genes in the morning glory family and reconstructing the phylogenetic tree revealed the history of gene loss, neo- and non- functionalization, and duplicate retention. Alkaloid analyses using gas chromatography and mass spectrometry revealed that not all the species that retained the hss-like genes produced

PAs. Molecular evolutionary analysis of selective pressure using coding sequences of hss and dhs combined with ancestral sequence reconstruction of ancient HSS enzymes revealed that in both Ipomoea and Distimake clades, the hss genes experienced a similar pattern of selection pressure. The resurrected last common ancestor of hss genes from Ipomoea and Distimake was under strong purifying selection and retained its original ancestral DHS specific activity.

However, this last common ancestor also showed high promiscuity in the usage of aminobutyl- acceptors and can produce homospermidine and canavalmine additional to the activation of the eIF5a. In the Ipomoea clade, HSS enzymes of PA producing species were refined towards HSS activity by positive Darwinian selection. In the Distimake clade, relaxed selection pressure resulted in refining the HSS activity in PA producing species. In summary, this work elucidated the independent functional optimization of HSS activity in PA biosynthesis that occurred twice in the morning glory family. This parallel evolution of HSS enzymes occurred predominantly via divergent mutations with few identical amino acid replacements in both genera.

KURZFASSUNG

Pyrrolizidin-Alkaloide (PA) sind giftige Verbindungen, die in vielen verschiedenen Arten der

Angiospermen als chemische Verteidigung gegen Herbivoren produziert werden. PAs sind auch von wirtschaftlicher Bedeutung, da sie pflanzliche Arzneimittel und Lebensmittel kontaminieren können und bei oraler Einnahme gesundheitsschädlich sind. Die Biosynthese der PAs entstand in diversen Pflanzenfamilien, wie den Apocynaceae, Asteraceae,

Boraginaceae, Convolvulaceae, Fabaceae, Poaceae und Orchidaceae, mehrmals unabhängig voneinander. Die Homospermidin-Synthase (HSS) ist das erste spezifische Enzym der PA-

Biosynthese und initiiert diese. Interessanterweise entwickelte sich die HSS wiederholt durch

Genduplikation aus dem primären Stoffwechselgen der Deoxyhypusin-Synthase (DHS) und wurde erfolgreich in den PA-Stoffwechsel aller PA-produzierenden Pflanzenarten rekrutiert.

Die DHS ist an der Aktivierung des eukaryotischen Translations-Initiationsfaktors 5a (eIF5a) durch Übertragung der Aminobutylgruppe von Spermidin auf den eIF5a beteiligt. Die HSS

überträgt die Aminobutylgruppe von Spermidin auf Putrescin. Dabei entsteht Homospermidin, welches wiederum als „Rückgrat“ des PA Grundkörpers dient. In der Familie der

Windengewächse (Convolvulaceae) kommen PAs in zwei verschiedenen, nicht näher verwandten Gattungen vor: Ipomoea (Prunkwinden) und Distimake (Waldrosen). Es wurde jedoch nur ein einziges Genduplikationsereignis in der Familie der Convolvulaceae gefunden, von dem die HSS-Gene in den rezenten Arten stammen. Darauf basierend vermutet man, dass das duplizierte Gen zweimal unabhängig in den beiden Gattungen für die PA-Biosynthese rekrutiert wurde und damit auch die funktionelle Veränderung der DHS-Aktivität zu HSS-

Aktivität zweimal unabhängig entstand.

In dieser Arbeit wurde eine analytische Methode entwickelt, um die Enzymaktivitäten sowohl der DHS- als auch der HSS-Enzyme mit Hilfe der Hochdruck-Flüssigkeitschromatographie

(HPLC) direkt zu messen. Die neue Methode ersetzte die alten radioaktiv markierten Tracer-

iii

Experimente und nutzt die Derivatisierung von Substraten und Produkten durch chromophore

Verbindungen, die anschließend durch UV-Absorption und Fluoreszenz quantifiziert werden können. Die HPLC-Methode erwies sich als robust, schnell und zuverlässig. Diese Methode war auch in der Lage, verschiedene Nebenreaktionen von DHS und HSS zusätzlich zu ihren

Hauptaktivitäten zu erkennen und somit Licht auf die promisken Aktivitäten zu werfen, die nie zuvor erforscht wurden.

Darüber hinaus ermöglichte eine umfangreiche und erfolgreiche Suche nach hss-ähnlichen

Genen in der Familie der Convolvulaceae die Rekonstruktion des Gen-Stammbaums, der eine

Geschichte des Genverlusts, des Funktionsverlusts, der Entwicklung einer neuen Funktion sowie der Beibehaltung von Duplikaten erzählt. Analysen des PA-Gehaltes mittels

Gaschromatographie und Massenspektrometrie zeigten, dass nicht alle Arten, die die hss-

ähnlichen Gene behielten, PAs produzieren. Untersuchungen zur molekularen Evolution basierend auf Selektionsanalysen der kodierenden Sequenzen von hss und dhs in Kombination mit der Rekonstruktion der Sequenz der Vorfahr-Enzyme der heutigen HSS ergaben, dass die hss-Gene sowohl in Ipomoea als auch in Distimake ein ähnliches Muster des Selektionsdrucks erfuhren. Der rekonstruierte, letzte gemeinsame Vorfahre der hss-Gene von Ipomoea und

Distimake war unter starker negativer Selektion (auch reinigender Selektion; englisch: purifying selection) und behielt seine ursprüngliche DHS-spezifische Aktivität bei. Allerdings zeigte dieser Vorfahr auch eine hohe Promiskuität bezüglicher der Verwendung verschiedener

Aminobutyl-Akzeptoren und war neben der Aktivierung des eIF5A auch in der Lage,

Homospermidin und Canavalmin zu produzieren. In der Gattung Ipomoea wurden die hochfunktionellen HSS-Enzyme der PA-produzierenden Arten durch positive darwinistische

Selektion bezüglich der HSS-Aktivität verfeinert. In Distimake führte ein reduzierter negativer

Selektionsdruck zu einer Verfeinerung der HSS-Aktivität bei PA-produzierenden Arten.

Zusammenfassend konnte diese Arbeit zeigen, dass in der Familie der Convolvulaceae die

iv funktionelle Optimierung der HSS-Aktivität für die PA-Biosynthese tatsächlich zweimal unabhängig entstand. Diese parallele Evolution der HSS-Enzyme erfolgte vorwiegend durch divergente Mutationen und mit nur wenigen identischen Mutationen, die in beiden Gattungen auftraten.

Table of Contents 1. INTRODUCTION ...... 1 1.1. Specialized metabolism ...... 1 1.2. Chemical Defenses ...... 2 1.3. Genetics and evolutionary significance ...... 3 1.4. Pyrrolizidine Alkaloids...... 6 1.5. Homospermidine synthase (HSS)...... 7 1.6. Deoxyhypusine synthase (DHS) ...... 8 1.7. PAs in the Morning Glory family ...... 8 2. OBJECTIVES AND OUTLINES ...... 11 3. Development of an activity assay for characterizing deoxyhypusine synthase and its diverse reaction products ...... 13 3.1. ABSTRACT ...... 14 3.2. INTRODUCTION ...... 15 3.3. RESULTS ...... 21 3.3.1. Development of the method – factors affecting the derivatization procedure ...... 21 3.3.2. Chromatographic separation and reproducibility ...... 23 3.3.3. Possible co-precipitation of polyamines ...... 26 3.3.4. DHS activity assays with recombinant DHS and HSS enzymes ...... 27 3.3.5. HSS activity assays with recombinant DHS and HSS enzymes ...... 30 3.3.6. Substrate competition studies of SvDHS ...... 30 3.4. DISCUSSION ...... 32 3.4.1. Effects of the assay buffer ...... 32 3.4.2. New insights into SvDHS and SvHSS activity ...... 33 3.5. CONCLUSION ...... 35 3.6. EXPERIMENTAL PROCEDURES ...... 36 3.6.1. Chemicals...... 36 3.6.2. Bioinformatic tools to calculate structure-based predictions of polyamines and proteins ...... 36 3.6.3. Heterologous expression in E. coli and purification of DHS, HSS, and eIF5A precursor protein from S. vernalis ...... 36 3.6.4. Activity assays of recombinant DHS and HSS ...... 37 3.6.5. Derivatization, HPLC system, and conditions ...... 38 3.6.6. LC-MS ...... 40 3.7. Acknowledgments ...... 40 3.8. Supplementary material ...... 41

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4. The recruitment of a duplicated gene to pyrrolizidine alkaloid biosynthesis in “morning glories” (Convolvulaceae, Solanales) ...... 43 4.1. ABSTRACT ...... 44 4.2. BACKGROUND ...... 45 4.3. RESULTS ...... 51 4.3.1. Identifying homologs of dhs and hss in Convolvulaceae ...... 51 4.3.2. Presence of PAs in the Convolvulaceae...... 55 4.3.3. The ability of HSS from D. quinquefolius and C. umbellata to produce homospermidine ...... 56 4.4. DISCUSSION ...... 59 4.4.1. Functional role of the hss gene copy ...... 61 4.4.2. Presence of the functionally characterized H/V/D amino acid motif ...... 63 4.5. CONCLUSIONS ...... 64 4.6. METHODS ...... 65 4.6.1. Plant material ...... 65 4.6.2. Total RNA and genomic DNA isolation ...... 65 4.6.3. cDNA synthesis ...... 65 4.6.4. Amplification of hss and dhs homologs ...... 66 4.6.5. Gene tree reconstruction ...... 67 4.6.6. Estimation of the amount of pairwise synonymous substitution (Ks) ...... 67 4.6.7. PA extraction from plant tissues ...... 67 4.6.8. PA analysis by GC and GC-MS...... 68 4.6.9. Heterologous expression, purification and activity assays of HSS encoding cDNAs from D. quinquefolius and C. umbellata ...... 69 4.6.10. HPLC system and conditions ...... 70 4.7. Availability of data and material ...... 70 4.8. Acknowledgments ...... 71 4.9. Supplementary material ...... 72 5. Evolution of homospermidine synthase by divergent mutations and parallel adaptation in pyrrolizidine alkaloid biosynthesis in morning glories ...... 81 5.1. ABSTRACT ...... 82 5.2. INTRODUCTION ...... 83 5.3. RESULTS ...... 86 5.3.1. Sequences encoding dhs and hss homologs ...... 86 5.3.2. Phylogenetic tree of dhs and hss homologs ...... 86 5.3.3. Evolutionary selection on HSS genes in PA producing species ...... 90

viii

5.3.4. Increased nucleotide substitution on the branches of Ipomoea and Distimake HSS clade in parallel ...... 90 5.3.5. Positive Darwinian selection on the HSS sequences of Ipomoea ...... 94 5.3.6. Parallel selection on Distimake HSS sites ...... 94 5.3.7. Structural model of HSS from I. neei ...... 95 5.3.8. Activities of extant HSS sequences ...... 95 5.3.9. Activities of reconstructed ancestral enzymes ...... 99 5.4. DISCUSSION ...... 101 5.4.1. Gene losses and evolutionary mechanisms for duplicate retention ...... 101 5.4.2. Parallel adaptation of HSS specific activity ...... 102 5.4.3. Nuanced evolution of HSS ...... 105 5.4.4. Parallel and divergent routes of HSS optimization in Convolvulaceae...... 106 5.5. MATERIALS AND METHODS ...... 108 5.5.1. Plant materials ...... 108 5.5.2. Genetic materials ...... 108 5.5.3. Isolation and database retrieval of DHS/HSS encoding genes ...... 109 5.5.4. Analyses of DHS/HSS encoding genes ...... 110 5.5.5. Reconstructing the gene tree of dhs/hss homologs ...... 110 5.5.6. Selection analysis in Convolvulaceae hss clade ...... 111 5.5.7. Ancestral sequences reconstruction ...... 112 5.5.8. Heterologus expression of DHS, HSS, eIF5A and reconstructed ancestors ...... 113 5.5.9. Biochemical characterization ...... 113 5.5.10. Structural homology modeling of HSS ...... 114 5.7. Acknowledgement ...... 115 5.8. Supplementary Material ...... 116 6. CONCLUSIONS AND GENERAL PERSPECTIVES...... 127 6.1. OUTLOOK ...... 129 6.1.1. Divergence of gene expression among duplicates ...... 129 6.1.2. Structural mechanistic aspects of enzyme promiscuity ...... 130 7. CONTRIBUTIONS ...... 133 8. FUNDING INFORMATION ...... 135 9. REFERENCES...... 137 ACKNOWLEDGEMENTS ...... 151 CURRICULUM VITAE ...... 153 DECLARATION ...... 155

1. INTRODUCTION

1.1. Specialized metabolism

Plants produce a wide variety of chemical compounds that can be grouped in two types: primary and specialized metabolites (Moghe and Kruse 2018). Primary metabolites are involved in intrinsic functions like normal growth, development, and reproduction. Examples are bases in DNA/RNA, proteinogenic amino acids, carbohydrates, and fatty acids. The specialized metabolites are low molecular weight compounds not immediately required for the survival of the plant but produced to specific needs of the plant local environments to help in reproductive success or fitness. Examples include alkaloids, glycosides, tannins, saponins, amines, terpenes, organic acids and others. (Fraenkel 1959; Wink 2003; Wink 2018). These specialized compounds, also referred as “secondary metabolites”, and were considered as plant waste products as long as their functions had not been elucidated even (Fraenkel 1959). Today, about 200000 compounds have been isolated and identified from different plant species, thus attesting a unique richness and chemical diversity (Dixon and Strack 2003; Pichersky and

Lewinsohn 2011; Wink 2018). Specialized metabolites such as monoterpenes in fragrances, anthocyanins and carotenoids in flower colors act as plant signal compounds to attract pollinators (Wink 2003). Another most significant function of specialized metabolites is their role as plant chemical defense against herbivores and pathogens (Levin 1976; Wink 1988;

Wink 2003).

1.2. Chemical Defenses

A majority of specialized metabolites are from the repository of plant defense compounds against predation. Plants are sessile organisms that cannot run away from predators and thus evolved, both, mechanical and chemical responses to protect themselves (Levin 1976; Wink

2003). The mechanical strategies include morphological modifications like barks, thorns

(modified branches), trichomes and spines (modified leaves) (Levin 1976; War et al. 2012;

Burns 2014). As part of the chemical responses specialized compounds are produced to deter predators with noxious odor, repulsive tastes, or even to harm with lethal toxic properties upon ingestion by herbivores (Levin 1976; Wink 2003). Some of the specialized metabolites are widely distributed in plant families like phenolic compounds. They can interact non- specifically with proteins (e.g. enzymes) resulting in a reduced nutritional value of the plant tissue consumed by the herbivores or infected by the pathogens (Wink 2003; War et al. 2012).

Some specialized metabolites have lineage specific distribution and have functions by specifically targeting enzymes or ion-channels of herbivores, for example cardiac glycosides blocking sodium-potassium ATPase pump in cardiac muscle cells or alkaloids intercalating with DNA molecules (Wink 2003). Plants often produce complex mixtures of specialized metabolites that synergistically interact with multiple targets improving the effectiveness of defense (Nelson and Kursar 1999; Liu et al. 2017; Wink 2018). However, the exact composition and concentration varies between organs, tissues, developmental stages, population and even between individuals of species (Stegemann et al. 2018; Wink 2018), still a single group of specialized metabolites and its derivatives dominates within a given taxon

(Wink 2003; Wink 2018). Above mentioned interesting yet complex characteristics represent the adaptive nature of plant specialized metabolites as defense compounds that are continuously exposed to selection pressures of herbivory and subsequent evolution.

1.3. Genetics and evolutionary significance

Major questions have been asked about how such exceptional diversity of specialized metabolism arose in evolution and, especially at the molecular level, how pathway specific enzymes evolved? Gene duplication was postulated to provide source material for the origin of new enzymes (Ohno 1970). Gene duplicates are the extra copies of existing genes that arise via various mechanisms such as tandem duplication resulting from ectopic recombination (unequal crossing-over) during meiosis, replication slippage, retrotransposition, chromosome duplication (aneuploidization), or whole genome duplication (WGD) (also called polyploidization) (Zhang 2003; Herron and Freeman 2015; Panchy et al. 2016). Although other mechanisms were proposed to contribute to gene novelties including gene co-option (True and

Carroll 2002) and de novo gene evolution (Tautz and Domazet-Lošo 2011; Schlötterer 2015), gene duplication remains a major contributor to plant novelties (Panchy et al. 2016). Gene duplicates are quite abundant in plant genomes; comparative genomic study of land plants by

Panchy et al. 2016 have shown that on average, 65% of annotated genes in land plant genomes have duplicate copies. Most of these duplicates were derived from WGDs, particularly angiosperms experienced multiple WGD events (paleopolyploidization) in the last 200 million years of their evolution (Panchy et al. 2016). Next significant contributor were tandem gene duplication events (Panchy et al. 2016). However, gene duplication is just an initial step in the evolution of a new gene or protein. Various evolutionary models were proposed to explain the preservation and evolution of new gene copies to new function after gene duplication (Innan and Kondrashov 2010). The most common fate is believed to be the loss of a duplicated gene copy by a process known as “non-functionalization” or “pseudogenization” (Ohno 1970;

Conant and Wolfe 2008; Innan and Kondrashov 2010). In this process, one copy of the duplicated gene is freed from selective constraints and accumulates mutations that destroy the function by introducing premature stop codons or negatively affecting the protein structure

(Hurles 2004). Plant genomes are cluttered with such pseudogenes (Panchy et al. 2016). In the second scenario the accumulated mutations in one of the gene copies evolves a new beneficial function and will be selected, while the other copy retains the ancestral function, a process called “neofunctionalization” (Ohno 1970; Conant and Wolfe 2008). The third type called

“sub-functionalization” refers to the partitioning of the activity of the ancestral gene between the two duplicates. This scenario is only possible, if the ancestor gene encoded a bifunctional or multifunctional enzyme. Each of the duplicates specializes into different subfunctions of the ancestor gene (Conant and Wolfe 2008). Further models have been proposedto explain the evolution of new gene functions after duplication such as the “gene dosage balance hypothesis”

(Birchler and Veitia 2012; Kondrashov 2012), “duplication-degeneration-and- complementation” (DDC) (Force et al. 1999), “escape from adaptive conflict” (EAC) (Des

Marais and Rausher 2008), and “paralog interference” (Baker et al. 2013; Kaltenegger and

Ober 2015). Despite the differences in the models a common idea is the postulate that, in order to maintain both the gene copies, there need to be differences in functions, expression patterns, or interaction partners in one or both of the copies on which natural selection can act (Panchy et al. 2016).

The majority of plant specialized metabolic enzymes characterized so far were the product of duplication followed by divergent evolution (Pichersky and Lewinsohn 2011; Panchy et al.

2016). In this process enzyme promiscuity also suggested to have played a significant role in the evolution of these enzymes (Tawfik and S 2010; J.-K. Weng and Noel 2012; Weng 2014).

Many of the specialized metabolic enzymes evolved from genes encoding metabolic enzymes of primary metabolism and have been successfully recruited to their new role (Pichersky and

Gang 2000; Ober 2005; Weng 2014). The substrates for the various pathways of specialized metabolites are also products of primary metabolism. Examples include isoprenoid biosynthesis, which uses isopentenyl-diphosphate and dimethylallyl-diphosphate of

4 mevalonate pathway as substrates, caffeine biosynthesis starts with purine of nucleotide biosynthesis, and alkaloids are derived from amino acids (Weng 2014). Another evidence for the recruitment of enzymes from primary metabolism are the protein folds of the specialized metabolic enzymes. Most of the protein folds and catalytic mechanisms are exploitations of pre-existing protein family folds rather than the emergence of new folds (Weng 2014).

However, often new functions can already be acquired by reassembling only a few catalytic residues in the active sites of a conserved fold (Jing-Ke Weng and Noel 2012; Weng 2014).

Although divergent evolution dominates specialized metabolism, identical metabolic or molecular traits appears independently through parallel and convergent evolution in plants

(Pichersky and Lewinsohn 2011). The convergence can present at various levels: i) Different chemicals are produced for same functions as it is the case for the red color pigments in different fruits like anthocyanin in strawberry (pelargonidin-3-O-glucoside), betain in dragon fruit (betaxanthin), carotenoid in tomato (lycopene) (Pichersky and Lewinsohn 2011). ii) Same chemicals are produced by different enzymes, for example the flavone apigenin produced in

Apiaceae family by flavone synthases (belonging to the oxoglutarate-dependent dioxygenase enzyme family) whereas in most other plant species flavone is produced by enzmyes belonging to the cytochrome P450 family (Pichersky and Lewinsohn 2011). iii) Enzymes of the same family evolved independently in different species to catalyze an identical conversion of a specific substrate to a specific product. An example is the formation of stilbenes in different taxa like Pinus, Arachis, and Vitis (Pichersky and Lewinsohn 2011). It was shown that the stilbenes synthase , the key enzyme in stilbene biosynthesis, evolved from ubiquitous chalcone synthase independently in the different lineages (Tropf et al. 1994; Pichersky and Lewinsohn

2011; Parage et al. 2012). Another well-studied example of such repeated independent evolution is the biosynthesis of pyrrolizidine alkaloids (PAs). The gene encoding homospermidine synthase (HSS), the first specific enzyme in PA-biosynthesis, evolved

5 repeatedly and independently from the gene encoding deoxyhypusine synthase (DHS), an enzyme involved in primary metabolism of eukaryotes (Ober and Hartmann 1999a; Reimann et al. 2004; Kaltenegger et al. 2013).

1.4. Pyrrolizidine Alkaloids

Alkaloids constitute one of the major groups of plant specialized metabolites and occur in about

20% of all land plant species (De Luca and Laflamme 2001). Many of these compounds are allelochemicals produced as a toxic chemical defense against herbivores and about 12000 different structures of alkaloids have been identified (De Luca and Laflamme 2001; Wink

2003). Due to the allelochemical nature, alkaloids are often evolutionarily shaped to mimic the compounds that binds to the receptors of the animal’s neural system and thus display a wide variety of pharmaceutical properties such as stimulants, narcotics, and poisons. Examples are caffeine, morphine, or nicotine etc. (De Luca and Laflamme 2001; Wink 2003). The majority of alkaloids are derived from the amino acid metabolisms. One major class of alkaloids are the

PAs. There are about 400 structures PAs and their occurrence has been reported in many unrelated species of plant families, such as Apocynaceae, Asteraceae, Boraginaceae,

Convolvulaceae, Fabaceae, and also in the monocot families of Poaceae and Orchidaceae

(Hartmann and Witte 1995; Hartmann 1999a; Anke et al. 2008; Eich 2008; Langel et al. 2011;

Gill et al. 2018a; Livshultz et al. 2018). PAs are toxic substances and act as a chemical deterrent against herbivores, mainly insects (Hartmann and Witte 1995). Due to their toxicity PAs can also cause irreversible damage to the livers of livestock, wild-animals, and humans when consumed (Wiedenfeld and Edgar 2011). Reports suggest that PAs can contaminate food products such as honey, herbal drugs, and tea most likely if PA-containing plants are co- harvested (Bodi et al. 2014). Despite their vast structural diversity, all PAs possess a common backbone structure, the N-containing double ring system, called necine base (Hartmann and

Witte 1995). One or more organic acids are esterified to this necine base to form different chemical varieties of PAs (Hartmann and Witte 1995). The first committed step in PA biosynthesis is the formation of the triamine homospermidine which is the precursor for the necine base moiety (Ober and Hartmann 1999a; Hartmann and Ober 2000).

Fig. 1. A) Skeleton structure of pyrrolizidine alkaloids. Necine base is the nitrogen containing bicyclic ring structure in red. Organic acids esterify to the positions at R1 and R2 B) Lycopsamine – a common type of pyrrolizidine alkaloid found in plants.

1.5. Homospermidine synthase (HSS)

The homospermidine is synthesized by the first pathway specific enzyme of PA biosynthesis, known as homospermidine synthase (HSS, EC 2.5.1.45; spermidine specific) (Ober and

Hartmann 1999a). HSS catalyzes the transfer of an amino-butyl group from spermidine to a putrescine molecule using NAD+ as a cofactor thus forming homospermidine and 1,3-diamino- propane as byproduct (Böttcher et al. 1994; Ober and Hartmann 1999a). HSS was first characterized in Senecio vernalis (Asteraceae family) (Ober and Hartmann 1999a; Langel et al. 2011). Since then, HSS-like genes were identified from many plant species of various lineages (Reimann et al. 2004; Kaltenegger et al. 2013; Livshultz et al. 2018). The expression of HSS has been studied in the species of Asteraceae and Boraginaceae and was found to exhibit different tissue specific and cell specific patterns. These observations along with the

7 phylogenetic analyses of cDNA sequences provided further support for a polyphyletic origin of PA biosynthesis (Moll et al. 2002; Anke et al. 2004; Reimann et al. 2004; Frölich et al. 2007;

Niemüller et al. 2012). Sequence based homology studies showed that HSS was originated via gene duplication events from Deoxyhypusine synthase (Ober and Hartmann 1999a; Ober,

Harms, et al. 2003a).

1.6. Deoxyhypusine synthase (DHS)

DHS is an enzyme ubiquitous in eukaryotes that is involved in the posttranslational activation of a regulatory protein, the eukaryotic initiation factor (eIF5A). EIF5A was shown to be essential for cell growth and viability (Ober and Hartmann 1999b; Cano et al. 2008). DHS functions by transferring the amino-butyl group from spermidine to a specific lysine residue in the eIF5A precursor proteinresulting in the formation of the unusual amino acid deoxyhypusine

(Ober and Hartmann 1999b; Umland et al. 2004; Wolff et al. 2007; Cano et al. 2008). Due to its intrinsic function, DHS has long been a potential therapeutic target in cancer and other diseases in biomedical research (Hauber et al. 2005; Nakanishi and Cleveland 2016).

1.7. PAs in the Morning Glory family

The Morning Glory family (Convolvulaceae, also called ‘sweet potato family’) are a large group of flowering plants, which includes 60 genera and about 1600 species (Stefanović et al.

2002). They occupy a wide variety of habitat including tropical to temperate regions around the world (Stefanović et al. 2002). They also exhibit a great diversity in their morphological characteristics, such as flowers (Fig. 2), leaves, and appear mostly as herbaceous twining vines, and rarely as trees or shrubs (Austin 1997). Convolvulaceae are thought to have originated around 65-85 Mya (million years ago) and the oldest fossils reported from the late Paleocene

(Thanetian; 58.7–55.8 Mya) of India, which was a part of East Gondwana during this time

(Srivastava et al. 2018). Convolvulaceae also have two major agricultural crops, sweet potato

(Ipomoea batatas) and water spinach (Ipomoea aquatica). Several other species used as ornamental plants and many of the species are known to have central nervous system efficacies and are used for medicinal purposes (Chen et al. 2018). The phylogenetic tree of

Convolvulaceae reconstructed using molecular sequences grouped the species into six monophyletic clades (Stefanović et al. 2003). Convolvuloideae is one of the six clades, which is further sub-divided into 4 tribes (Ipomoeeae, Merremieae, Convolvuleae, and Aniseieae)

(Stefanović et al. 2003). Of note, the Merremieae tribe was recently renamed to Distimake according to a prominent genus of this tribe (Simões and Staples 2017). The majority of PA occurrences have been reported within the Convolvuloideae and especially within the species of Ipomoeeae and Distimake (formerly Merremieae) tribes (Eich 2008).

Fig. 2: Flowers of selected species of Convolvulaceae. A) Ipomoea batatas (Sweet potato), B) Ipomoea purpurea, C) Distimake tuberosus, D) Jacquemontia paniculate, E) Distimake quinatus, F) Distimake cissoides, G) Ipomoea indica, H) Camonea umbellata, I) Ipomoea lobata, and J) Ipomoea nil (Japanese morning glory). Figure sources and license information were provided in Chapter 3.8 Suppl. Table 1.

Interestingly, the occurrence of PAs in Convolvulaceae is sporadic but follows a taxon-specific distribution (Eich 2008). PAs of the retronecine type are present in species of Distimake tribes

(Eich 2008) of Convolvulaceae. Retronecine type PAs contain retronecine as necine base with a 1,2-double-bond. They are the predominant type of PAs that occurs in many other plant families like Asteraceae, Boraginaceae, Apocynaceae etc. (Eich 2008). Platynecine-type PAs, a class of PAs that encompass derivatives like ipangulines and minalobines, are characterized by a saturated necine base moiety that is esterified to aliphatic and/or an aromatic necic acid moiety , occur only in some species of Ipomoeeae tribe (Eich 2008). From the phylogenetic tree of (Stefanović et al. 2003), it was hypothesized that the presence of different types of PAs in only a few and not closely related species indicates an independent origin with parallel or repeated adaptation (Reimann et al. 2004; Kaltenegger et al. 2013). However, the HSS or HSS- like sequences of all the Convolvulaceae species were shown to originate from a single duplication event (Kaltenegger et al. 2013). The study also reported that there a functional gene copy was lost after the duplication event, a fate attributable to gene loss or pseudogenization

(Kaltenegger et al. 2013). Furthermore, the study showed that after the duplication event the duplicates were subjected to various selection pressures in HSS-like clade of the Ipomoeeae, including purifying selection, relaxed functional constraints and finally positive Darwinian selection. In the Distimake clade such a scenario was not detectable due to the limited amount of species studied in this lineage (Kaltenegger et al. 2013).

2. OBJECTIVES AND OUTLINES

PA biosynthesis evolved multiple times independently in different plant lineages. Studies suggest that in all the PA producing species HSS initiates the PA biosynthesis. But little is known about how the functional shift from DHS to HSS happened after the gene duplication event. Which changes in the amino acid sequence of HSS were involved to change the substrate preference from the eIF5A precursor protein to putrescine as aminobutyl-acceptor? The main objective of this thesis is to understand the evolution of the PA-specific HSS function in the

Convolvulaceae. In the Morning Glories, this functional shift is postulated to have occurred after an ancient gene duplication of dhs gene two times independently in the lineages leading to the extant genera Ipomoea and Distimake. Therefore, the Morning Glory family is an excellent model system to study the functional recruitment of the HSS.

The data of this thesis is presented in three chapters:

First, in order to study the functional shift, it is essential to understand the comprehensive biochemical characteristics of both, HSS and DHS. Therefore, the Chapter 3 describes the development of new HPLC- based pre-column derivatization method for measuring the enzyme activities of HSS and DHS. We used well studied DHS and HSS enzymes from Senecio vernalis, a PA-producing species from Asteraceae, to quantify and compare the enzyme activities and to validate the new method in comparison with the previously used radioactively labeled tracer assays. In contrast to previous assays, this new method allows the direct detection and quantification of substrates and products involved in the enzyme reaction.

Chapter 4 reports the identification of additional HSS-like genes in various species from sister clades of PA producing Morning Glory species. These species were also tested for their ability to produce PAs using gas chromatography and mass spectrometric analysis. Also, this chapter discusses the possibilities and challenges in functional prediction of putative hss genes and their use to predict the presence of PAs in individual species of the Morning Glory family.

Chapter 5 describes comprehensive evolutionary analyses of HSS- and DHS-coding sequences in the Morning Glory family. An extensive phylogenetic analysis was done to recreate the history of duplicated genes and to identify the various selection pressures that might have acted on the sequences of the HSS-clade. To understand the functional divergence between the duplicates, various sequences encoding ancestral HSS were reconstructed and the enzymatic activities of the encoded proteins were compared with the DHS and HSS of extant species.