T H E A C T I N O R H I Z A L S Y M B I O S I S O F T H E

E A R L I E S T D I V E R G E N T F R A N K I A C L U S T E R

N g u y e n T h i T h a n h V a n

The actinorhizal symbiosis of

the earliest divergent Frankia cluster

Nguyen Thi Thanh Van

©Nguyen Thi Thanh Van, Stockholm University 2017

ISBN print 978-91-7649-716-6

ISBN PDF 978-91-7649-717-3

Printed in Sweden by US-AB, Stockholm 2017

Distributor: Department of Ecology, Environment and Plant Sciences

Stockholm University, Stockholm, Sweden

Con kính tặng Ba

Sammanfattning

Under de senaste åren har allt större tonvikt lagts vid vikten av att utveckla en

världsomspännande utvecklingsmässigt ekologiskt hållbar jordbruksnäring.

Nödvändigheten av att minska beroendet av syntetiska kvävebaserade

gödningsmedel har lett till ett alltmer växande forskningsfält baserat på vikten

av biologisk kvävefixering, med emfas på rotknölsymbios. Mina studier har

fokuserat på mekanismerna i och med actinorhizalsymbios, i.e den

symbiotiska interaktionen mellan olika kvävefixerande actinobacteria -genus

Frankia. i förhållande till en mycket skiftande grupp av växter från åtta olika

växtfamiljer, som sammantaget kallas actinorhizalväxter. Frankia kluster II

har visats vara en systerklad i förhållande till alla andra kluster, vilket gör att

denna speciella gren kan ge insikt i hur Frankia kluster II kan tillföra värdefull

information om den evolutionära bakgrunden i och med actinorhizalsymbios.

Det första fullt sekvenserade genomet av en medlem från detta kluster,

Candidatus Frankia datiscae Dg1 med ursprung från Pakistan, visar att

genomet infattar nod generna nodABC som svarar för framställandet av

lipochitooligosaccharid Nod-faktorer, vilket särskiljer denna medlem från

andra grupper av Frankia som saknar dessa gener. Arbetet i och med denna

avhandling är grundat på gDNA isolerat från sex inokula, tre från

Nordamerika (USA; två från Kalifornien och ett från Alaska), ett från Europa

(Frankrike), ett från Asien (Japan) och ett från Oceanien (Papua Nya Guinea).

gDNA isolerades från rotknölar från Datisca glomerata (Datiscaceae),

Ceanothus thyrsiflorus (Rhamnaceae), Coriaria myrtifolia och Coriaria

arborea (Coriariaceae). Sammantaget sekvenserades tretton metagenom.

Denna avhandling visar att de olika medlemarna i Frankia kluster II agerar

som en grupp, vilket kan förklara förmågan att just kluster II kan nodulera en

ovanligt stor och divers grupp av värdväxter, vilka innefattar fyra familjer från

två ordningar. Det inokulum som isolerades från Papua Nya Guinea (den enda

art från södra halvklotet som hittills sekvenserats), visade sig härbärgera en

hittills okänd Frankia-art, som namngavs Candidatus Frankia meridionalis.

Alla kluster II-arter studerade i detta arbete bär nod-generna nodABC i sitt

genom, med undantag för den art som isolerats från Papua Nya Guinea vars

genom enbart innehar nodB’C. Alla tre metagenom isolerade från USA

innefattade dessutom en sulfotransferas-gen, nodH. Denna gen har visat sig

vara uttryckt värdväxtspecifikt, då proteinet kunde spåras i rotknölar från

värdväxten C. thyrsiflorus men inte i de knölar som isolerats från D.

glomerata. Fylogenetiska analyser sammantagna med transposasfrekvensdata

i våra analyserade genom ger starkt stöd till hypotesen att den utökade

värdkretsen från Coriaria till Datisca för Frankia-arter i kluster II skedde i

Euroasien samt att kluster II-arter kom till nordamerika via Berings sund. För

att erhålla mer information om och hur värdväxten påverkar

mikrosymbiontens molekylära reaktioner jämfördes metabolismen i rotknölar

från D. glomerata (Cucurbitales) och C. thyrsiflorus (Rosales) på

transkriptionsnivå. Det system som skyddar nitrogenas från de negativa

effekterna av syre verkar vara mer effektivt i rotknölar från Ceanothus i

jämförelse med hur systemet verkar i knölar från Datisca, enär bakteriernas

kvävemetabolism sannolikt är likställd i samröret med båda växtarterna.

Aminosyraextrakten från rotknölarna från D. glomerata visar att profilen

utifrån kvävehaltiga aminosyror domineras av glutamat and arginin, vilket

stöder hypotesen att Frankia utsöndrar kväve, troligen i form av arginin,

speciellt anpassat för symbiosen med D. glomerata. Således visar våra data att

arter från Frankia II särskiljer sig från andra Frankia-arter i hänseendet att

deras repertoar av nod-gener och deras kvävemetabolism i symbios med sin

värdväxt är specifikt annorlunda.

List of publication

This thesis is based on the following publications and manuscripts:

I. Nguyen TV, Wibberg D, Battenberg K, Blom J, Vanden

Heuvel B, Berry AM, Kalinowski J, Pawlowski K (2016) An

assemblage of Frankia Cluster II strains from California con-

tains the canonical nod genes and also the sulfotransferase

gene nodH. BMC Genomics 17: 796.

II. Nguyen TV, Wibberg D, Vigil-Stenman T, Battenberg K,

Demchenko KN, Blom J, Fernandez MP, Yamanaka T, Berry

AM, Kalinowski J, Brachmann A, Pawlowski K. Meta-

genomes from the earliest divergent Frankia cluster: They

come in teams. Manuscript

III. Nguyen TV, Hilker R, Wibberg D, Battenberg K, Berry AM,

Kalinowski J, Pawlowski K. Cucurbitales vs. Rosales: Anal-

ysis of bacterial metabolism based on candidate gene expres-

sion profiling in root nodules of Datisca glomerata and Cea-

nothus thyrsiflorus. Manuscript

IV. Persson T, Nguyen TV, Alloisio N, Pujic P, Berry AM, Nor-

mand P, Pawlowski K (2016) The N-metabolites of roots and

actinorhizal nodules from Alnus glutinosa and Datisca glom-

erata: can D. glomerata change N-transport forms when nod-

ulated? Symbiosis 70: 149-157

My contributions to these papers/manuscripts were as follows:

I: Obtained the inoculum, grew and infected the plants, isolated

the DNA, participated in the evaluation of the genome data,

conducted and evaluated the RT-qPCR experiments, partici-

pated in writing the manuscript

II: Grew the plants and maintained the inocula, isolated the DNA,

participated in the evaluation of the genome data, conducted

and evaluated the RT-qPCR experiments, participated in writ-

ing the manuscript

III: Grew the D. glomerata plants, performed all RNA isolations,

conducted and evaluated the RT-qPCR experiments, wrote the

manuscript

IV: Grew the plants and extracted the metabolites for D. glomer-

ata, participated in the evaluation of the data and the writing

of the manuscript

Also partially written by the author but not included in this thesis:

V. Nguyen TV, Pawlowski K (2017). Frankia and actinorhizal

plants: symbiotic nitrogen fixation. In: Mehnaz S (ed). Mi-

crobes for sustainability - Rhizotrophs, Springer, New York.

In press.

VI. Normand P, Nguyen TV, Battenberg K, Berry AM, Vanden

Heuvel B, Fernandez MP, Pawlowski K. Proposal of Candi-

datus Frankia californiensis, the uncultured nitrogen-fixing

symbiont associated with a phylogenetically broad group of

hosts endemic to California. Int J Syst Evol Microbiol, in

preparation.

Contents

1. Introduction ......................................................................................... 13

1.1 Nitrogen-fixing symbiosis in root nodules ......................................... 13

1.2 Actinorhizal symbiosis ....................................................................... 14

1.3 Actinorhizal nodules .......................................................................... 19

1.3.1 Nodule induction ........................................................................ 19

1.3.2 Nodule structure ......................................................................... 21

1.3.3 Nodule metabolism ..................................................................... 23

1.4 Signal exchange between microsymbiont and host plant ................... 24

1.4.1 Signal exchange in the legume-rhizobia symbiosis .................... 24

1.4.2 Signal exchange in actinorhizal symbiosis ................................. 26

1.5 Evolution of actinorhizal symbiosis ................................................... 27

2. Aim of this thesis ................................................................................. 29

3. Comments on methodology ................................................................. 30

3.1 Frankia gDNA isolation for genome sequencing ........................... 30

3.2 Candidate gene expression profiling of bacteria in nodules of Datisca

glomerata and Ceanothus thyrsiflorus ..................................................... 30

3.3 Amino acid profiling of roots and nodules from Datisca glomerata . 32

4. Results and Discussion ........................................................................ 33

4.1 Genome sequencing of Frankia cluster II strains............................... 33

4.1.1 Frankia cluster II inocula represent strain assemblages ............. 33

4.1.2 Eurasian cluster II Frankia (meta-)genomes contain the canonical

nod genes nodABC and North American strains also contain the

sulfotransferase gene nodH.................................................................. 37

4.1.3 Cluster II genome (in-)stability: transposase frequencies in the

different strains .................................................................................... 38

4.1.4 Evolution of the symbiosis between actinorhizal plants and

Frankia cluster II strains ...................................................................... 39

4.2 Candidate gene expression profiling of bacterial metabolism in root

nodules of D. glomerata (Cucurbitales) and C. thyrsiflorus (Rosales) .... 40

4.2.1 Frankia nodABC genes are expressed in both types of nodules, and

nodH genes are expressed in nodules of C. thyrsiflorus ...................... 41

4.2.2 The expression levels of genes encoding bacterial truncated

hemoglobin and catalases in C. thyrsiflorus are higher than in D.

glomerata ............................................................................................. 41

4.2.3 Nodules induced by members of Frankia cluster II seem to share

the same nitrogen metabolism ............................................................. 42

4.2.4 Which carbon source does the host plant provide for symbiotic

Frankia in both systems? ..................................................................... 43

4.3 The N-metabolites of roots and actinorhizal nodules from Datisca

glomerata ................................................................................................. 46

4.3.1 Amino acid profiles of roots and nodules ................................... 46

4.3.2 Do D. glomerata plants change nitrogen transport forms when

nodulated? ............................................................................................ 47

5. Conclusions ......................................................................................... 49

6. Future perspectives .............................................................................. 50

7. Acknowledgments ............................................................................... 51

8. References ........................................................................................... 53

Abbreviations

AMF Arbuscular Mycorrhizal Fungi

AMS Arbuscular Mycorrhizal Symbiosis

ANI Average Nucleotide Identity

ATP Adenosine triphosphate

BNF Biological Nitrogen Fixation

CcaMK Calcium- and Calmodulin-dependent serine/threonine

protein Kinase

CSSP Common Symbiotic Signalling Pathway

DMI Does not Make Infection

DNA Deoxyribonucleic acid

EDGAR Efficient Database framework for comparative Ge-

nome Analyses using BLAST score Ratios

GABA γ-aminobutyrate

gDNA Genomic Deoxyribonucleic acid

GOGAT Glutamine oxoglutarate aminotransferase

(Glutamate synthase)

GS Glutamine Synthetase

HPLC High-Performance Liquid Chromatography

IS Insertion Sequence

LCO Lipochitooligosaccharide

LysM Lysin motif

Mbp Mega base pairs

Mya Million years ago

NFR Nod Factor Receptor

NFP Nod Factor Perception

NIN Nodule Inception

Nod Nodulation

NORK Nodulation Receptor Kinase

NSP Nodulation Signalling Pathway

ORF Open Reading Frame

PHYLIP Phylogeny Inference Package

RNA Ribonucleic acid

RT-qPCR Quantitative Reverse Transcription Polymerase Chain

Reaction

SYMRK Symbiosis Receptor-like Kinase

TCA Tricarboxylic Acid

Vc Vesicle cluster

13

1. Introduction

1.1 Nitrogen-fixing symbiosis in root nodules

Nitrogen is most often a limiting element for plant productivity even

though it is abundant in the Earth’s atmosphere (Bohlool et al. 1992;

Graham and Vance 2000). Since the invention of the Haber-Bosch pro-

cess, which made the industrial manufacture of ammonia economically

feasible, the extensive use of synthetic nitrogen fertilizer has been

boosting crop yields worldwide (reviewed by Frink et al. 1999). How-

ever, the production of nitrogen fertilizer requires significant amount of

fossil fuel energy (Galloway et al. 1995). In addition, it has caused sev-

eral adverse environmental issues such as eutrophication and hypoxia

in aquatic ecosystems (Galloway et al. 1995; Tilman et al. 2001), in-

creasing greenhouse gas emission (Socolow 1999; Kim and Dale 2008)

and contribution to acid rain and photochemical smog (Vitousek et al.

1997).

The growing international concern for environmental issues and de-

pletion of natural resources has led to emphasis on alternative nitrogen

resources. In nature, plants acquire reduced forms of nitrogen from dif-

ferent sources, organic matter decomposition, conversion of atmos-

pheric nitrogen into forms that plants could utilize by lightning and bi-

ological nitrogen fixation (BNF). BNF is a process carried out by a

number of prokaryotes that could convert atmospheric nitrogen to am-

monia thanks to the nitrogenase enzyme complex (Postgate 1982). Ni-

trogenase consists of two proteins, the MoFe protein and the electron-

transfer Fe protein, that associate and dissociate in a catalytic cycle in-

volving adenosine triphosphate (ATP) hydrolysis (reviewed by Hoff-

man et al. 2014). The reaction that this enzyme catalyses can be given

as

N2 + 8e- + 16ATP + 8H+ 2NH3 + H2 + 16ADP + 16Pi

14

BNF not only enhances agricultural production but also contributes

to nitrogen input into natural ecosystems (Giller and Cadisch 1995;

O’Hara et al. 2002). The contribution of symbiotic/associative nitrogen

fixation to the total fixed nitrogen was estimated to 70%, as compared

to 30% by non-symbiotic systems (Paul 1988). Nitrogen-fixing symbi-

oses include the cyanobacterial partnerships, e.g., Azolla/Nostoc, and

root nodule symbioses, namely the legume-rhizobia symbiosis and the

actinorhizal symbiosis. The legume-rhizobia symbiosis is established

between members of the legume family, and one non-legume genus

Parasponia (Cannabaceae), with a polyphyletic group of Gram-nega-

tive soil proteobacteria called rhizobia. Actinorhizal symbioses are es-

tablished between members from 25 plant genera belonging to eight

families from three orders, collectively called actinorhizal plants, with

Gram-positive soil actinobacteria of the genus Frankia (Benson and Sil-

vester 1993).

1.2 Actinorhizal symbiosis

1.2.1 Frankia

The genus Frankia includes nitrogen-fixing soil actinobacteria that

can establish a symbiotic relationship with a widely diverse group of

perennial trees and shrubs, representing more than 200 species from

eight families (Lechevalier 1994). The genus name was first used by J.

Brunchorst to honour his mentor, the Swiss biologist A.B. Frank who

coined the word symbiosis. Becking (1970) affirmed the name and

added six more Frankia species based on host plant specificity to the

existing four in the family Frankiaceae: Frankia alni, F. elaeagni, F.

brunchorstii, F. discariae, F. casuarinae, F. ceanothi, F. coriariae, F.

dryadis, F. purshiae, and F. cercocarpi. Based on its morphology, this

family was placed in the order Actinomycetales (now: Actinobacteria)

(Becking 1970) which later was confirmed based on 16S rRNA se-

quences (Normand et al. 1996).

Phylogenetically, Frankia strains can be divided into four main clus-

ters (Figure 1; Normand et al. 1996; Sen et al. 2014; Persson et al. 2015;

15

Nguyen et al. 2016). Members of cluster IV are either unable to induce

root nodules, or induce ineffective, i.e., non-nitrogen-fixing nodules.

Strains from cluster I enter nitrogen-fixing symbioses with members of

the actinorhizal plant families Betulaceae, Casuarinaceae (with the ex-

ception of the genus Gymnostoma) and Myricaceae (except the genus

Morella). Strains from cluster II nodulate a wide range of host plants

which include four families from two different orders, the Rosaceae and

the rhamnaceous genus Ceanothus from the Rosales, and Datiscaceae

and Coriariaceae from the order Curcubitales. This cluster is sister to

all other Frankia clusters (Sen et al. 2014; Persson et al. 2015). Strains

from cluster III induce nodules on members of two families belonging

to the Rosales (Rhamnaceae [with the exception of the genus Ceano-

thus] and Elaeagnaceae), two genera from the order Fagales (Gymnos-

toma, Morella) and occasionally on the genus Alnus.

Figure 1. Comparison of core genomes of 14 sequenced Frankia strains. Outgroups were

two actinobacterial genomes, Nocardia farcinica and Mycobacterium gilvum Spyr1. The phy-

logenetic tree was deduced from concatenated core gene alignments using PHYLIP (Felsenstein

2005). The bar below the phylogenetic tree represents the scale of sequence divergence. The

phylogenetic tree was kindly provided by Daniel Wibberg (University of Bielefeld, Germany)

and Jochen Blom (Justus Liebig University, Gießen, Germany).

Table 1 summarizes the sequenced Frankia genomes available at the

onset of this thesis. The size of Frankia genomes varies significantly,

from the 5.0 mega base pairs (Mbp) genome of the strain CeD (cluster

I) which infects Casuarina equisetifolia to the 10.45 Mbp genome of

16

R43 (cluster III) which was isolated from nodules of C. cunninghami-

ana but only infects members of the Eleaegnaceae family. Strains of

cluster I have genome sizes between 7 and 8 Mbp (Betulaceae- and My-

ricaceae-infective strains) or between 5 and 6 Mbp (Casuarinaceae-in-

fective strains). Cluster II strains have genome sizes between 5 and 6

Mbp. Cluster III genomes range from 6.61 Mbp to 10.45 Mbp while

genome sizes of cluster IV range from 6.9 Mbp to 10 Mbp. With only

one exception, Frankia strain BMG5.1, members of cluster II could not

be cultured despite numerous attempts. In contrast to other known

Frankia strains which show optimal growth at pH values of ca. 7,

BMG5.1 is alkaliphilic. It achieves optimal growth at pH 9.5 and barely

shows any growth at pH 7 (Gtari et al. 2015).

Table 1. Sequenced Frankia genomes with their GenBank accession numbers and the host

plants from which they were originally isolated.

Cluster Strain Host Size

(Mbp)

Accession

number

I

ACN14a Alnus crispa 7.49 GCA_000058485.1

ACN1ag Alnus viridis 7.52 LJPA00000000

AvcI.1 Alnus viridis 7.74 LJFZ00000000

Allo2 Allocasuarina verticillata 5.35 JPHT00000000

BMG5.23 Casuarina glauca 5.27 JDWE00000000

BR Casuarina equisetifolia 5.22 LRTJ00000000

CcI3 Casuarina cunninghamiana 5.43 CP000249

CcI6 Casurina cunninghamiana 5.57 AYTZ00000000

CeD Casuarina equisetifolia 5.00 JPGU00000000.1

CpI1-S Comptonia peregrina 7.62 JYFN00000000

CpI1-P Comptonia peregrina 7.61 LJJX00000000

G2 Casuarina equisetifolia 9.53 FAOZ00000000

QA3 Alnus nitida 7.59 AJWA00000000

Thr Casuarina cunninghamiana 5.30 JENI00000000

II

BMG5.1 Coriaria myrtifolia 5.79 JWIO00000000

Dg1 Datisca glomerata 5.32 CP002801

Dg2 Datisca glomerata 5.92 FLUV01000001

III

BCU110501 Discaria trinevis 7.89 ARDT00000000

BMG5.12 Eleaegnus sp. 7.58 ARFH00000000

EAN1pec Elaeagnus angustifolia 8.98 CP000820.1

EI5c Elaeagnus angustifolia 6.61 LRTK00000000

17

R43 Casuarina cunninghamiana 10.45 LFCW00000000

IV

CN3 Coriaria nepalensis 9.97 AGJN00000000

DC12 Datisca cannabina 6.88 LANG00000000

EuI1cT Elaeagnus umbellata 8.81 CP002299

The nitrogenase enzyme complex is very sensitive to oxygen in vitro

and in vivo. This is because one of its components, the iron-molyb-

denum complex, rapidly denatures upon exposure to oxygen (Gallon

1981). This leads to the so-called oxygen dilemma of nitrogen fixation:

on one hand, nitrogenase has to be protected from oxygen, on the other

hand, the nitrogenase reaction requires high amounts of ATP which are

best supplied by aerobic respiration. Most rhizobial strains can only fix

nitrogen inside root nodules because they rely on their host plant to pro-

vide protection for their nitrogenase complex from oxygen. Beta-rhizo-

bia can fix nitrogen in the free-living state but only under microaerobic

conditions (Patriarca et al. 2002). In contrast with rhizobia, Frankia

strains have the capability to protect their nitrogenase enzyme by de-

veloping highly specialized structures called vesicles at the end of hy-

phae or short side branches, and therefore can fix nitrogen in the free-

living state under aerobic conditions (Benson and Silvester 1993). Thus,

in symbiosis, both partners can contribute to the protection of nitrogen-

ase from oxygen. Frankia strains form vesicles in nodules of most host

plants; the shape, septation and subcellular position of the vesicles de-

pends on the host. i.e., the same strain can form different types of vesi-

cles in nodules of different plant species (Huss-Danell 1997). The

multi-layered envelopes of vesicles contain a high amount of

hopanoids, bacterial lipids (Berry et al. 1993). Like in culture, the thick-

ness of the envelope depends on the concentration of oxygen (Parsons

et al. 1987; Huss-Danell 1997; Kleemann et al. 1994).

In nodules of Casuarina and Allocasuarina species, the microsym-

biont does not form vesicles. The infected and adjacent uninfected cor-

tical cells have lignified cell walls which impede the diffusion of oxy-

gen (Berg and McDowell 1988). In addition, a large amount of protein

hemoglobin has been found in the infected cells of Casuarina nodules

(Fleming et al. 1987; Jacobsen-Lyon et al. 1995). The purified protein

possess similar characteristics as legume leghemoglobin’s, suggesting

18

that it plays a role in the transport of oxygen to the respiratory chain

(Gibson et al. 1989).

1.2.2 Actinorhizal plants

Actinorhizal plants are widely

distributed on Earth, from the arc-

tic (Lawrence et al. 1967) to the

tropics (Burleigh and Dawson

1994), but primarily located in the

temperate zone. They belong to

eight families from three different

orders (Table 2). The actinorhizal

species of the order Fagales in-

clude members of the families Bet-

ulaceae, Casuarinaceae and Myri-

caceae. The actinorhizal Rosales

include members of the families

Elaeagnaceae, Rhamnaceae, and

Rosaceae. The actinorhizal Cucur-

bitales include members of the

families Coriariaceae and Datisca-

ceae (Swensen 1996). All actino-

rhizal plants are woody with the

exception of two species of Da-

tisca. Datisca glomerata (common

name: Durango root) has a genera-

tion time of ca. six months. This

wind-pollinated, self-compatible

species is native to Baja California

in Mexico and California, USA

(Davidson 1973). Another actino-

rhizal genus that has its centre of

distribution in California is Ceano-

thus, belonging to the family

Rhamnaceae (Mabberely 1988).

Oder Family Genus

Betulaceae Alnus

Comptonia

Morella

Myrica

Allocasuarina

Casuarina

Ceuthostoma

Gymnostoma

Ceanothus

Colletia

Discaria

Kentrothamnus

Retanilla

Talguenea

Trevoa

Elaeagnus

Hippophae

Shepherdia

Cercocarpus

Chamaebatia

Cowania

Dryas

Purshia

Coriariaceae Coriaria

Datiscaceae DatiscaCucurbitales

Myricaceae

Casuarinaceae

Rhamnaceae

Elaeagnaceae

Rosaceae

Fagales

Rosales

Table 2. Actinorhizal plants and their

host specificity. Actinorhizal plants con-

sist of 25 genera belonging to eight di-

verse families from three orders. Host

plants of cluster I strains are depicted in

blue. Hosts of cluster II strains are de-

picted in red, and hosts of cluster III

strains are given in green.

19

Actinorhizal plants are the major source of fixed nitrogen in diverse

ecosystems including forests, swamps riparian zones, prairies and de-

serts (Silvester 1976; Dawson 1986). Alnus rubra, the red alder, which

is native to North America and Alnus glutinosa, the common alder, col-

onize riparian zones as well as nitrogen-poor wetlands (Dawson 2008).

Casuarina and Allocasuarina species play an important role in sand

dune stabilization and prevention of soil erosion in tropical climates and

warm temperate regions from Africa (Diagne et al. 2013) to Australia

(Barritt and Facelli 2001). Members of the genera Alnus, Shepherdia,

Hippophae and Dryas probably played a crucial role in soil formation

in vast areas of Europe, Asia and North America (Dawson 2008). Sea

buckthorn (H. rhamnoides) is currently being domesticated. This is be-

cause its seed oil can be used in cosmetics and the fruits are very rich

in vitamin C, carotenes and unsaturated fat and can be used in food in-

dustry as well as for phytopharmaceutical purposes (Suryakumar and

Gupta 2011).

1.3 Actinorhizal nodules

1.3.1 Nodule induction

In the legume-rhizobia symbiosis, the best examined way of root in-

fection is via root hairs. Just a few cases have been analysed where this

infection occurs via crack entry (Capoen et al. 2010; Goormachtig et al.

2004). In actinorhizal symbiosis there are currently two known ways of

root infection, intracellular infection for host plants from the order Fa-

gales and intercellular infection for hosts from the order Rosales. The

infection pathway in Cucurbitales has not been characterized yet. As in

legumes, the host plant of the actinorhizal symbiosis determines the

mechanism of infection.

Intracellular infection begins with the deformation of root hairs in

the presence of their respective microsymbionts (Callaham et al. 1979;

Berry et al. 1986). When a Frankia hypha is trapped in a root hair curl,

infection can occur (Berry and Torrey 1983). The Frankia hypha enters

20

the root in an infection thread-like structure, embedded in a plant-de-

rived pectin-rich matrix. The invasion of cortical cells by infection

thread-like structures is always preceded by the formation of a prein-

fection thread structure (Berg 1999). Concurrently, cell division in the

cortex is triggered, similar to the process in legume nodule formation.

Some of the newly-divided cells become infected, i.e., infection threads

branch and fill the cells from the centre outward. Eventually they form

the so-called prenodule which is part of the infection process of Fagales.

However, the prenodule is not directly involved in nodule formation.

Unlike in legume where the development of a nodule primordium takes

place in the cortex, actinorhizal nodule primordia originate in the peri-

cycle like lateral root primordia. The number of the primordia formed

per prenodule varies from a few up to a dozen or more (Callaham et al.

1979). Hyphae in infection threads grow from the prenodule to the nod-

ule primordium and infect primordium cells by branching. In the intra-

cellular infection process, infection threads grow from cell to cell

(transcellular growth). When the cells have been filled with hyphae in

branching infection threads, the microsymbiont starts to differentiate

vesicles wherein nitrogenase is formed and nitrogen fixation takes place

(reviewed by Pawlowski and Demchenko 2012).

The most obvious differences between the intracellular and the inter-

cellular infection pathway is that in the latter, there is no root hair de-

formation and no prenodule formation. A Frankia hypha enters the

young root by penetrating the middle lamella between epidermal cells

(Racette and Torrey 1989; Berry and Sunnel 1990). The hyphae grow

through intercellular spaces and proceed to colonize the apoplast of the

root cortex. During these stages the intercellular spaces become filled

with extensive darkly-staining matrix which contains high levels of

pectic compounds and proteins (Liu and Berry 1991). Concomitantly, a

nodule primordium is formed in the root pericycle. Hyphae infect pri-

mordium cells from the apoplast, and infection threads are formed di-

rectly in the infected cells. No transcellular growth of infection threads

is observed.

Berg et al. (1999), in the description of infection threads in actino-

rhizal Fagales, made a division between those containing invasive hy-

phae and those containing vegetative hyphae. The former invade the

21

plant cells and perform transcellular growth, while the latter branch

within infected cells, and eventually forms vesicles. Both types of hy-

phae/infection threads are involved in intracellular infection while only

vegetative hyphae are found in the intercellular pathway.

The mechanism of infection in the order Curcubitales shares features

with the processes in both Fagales and Rosales. No prenodules are

formed, but the infection threads show transcellular growth (Berg et al.

1999). However, unlike in legumes and actinorhizal Fagales, the for-

mation of preinfection threads does not take place (Berg et al. 1999),

and the infected cells are filled with hyphae from the periphery inward.

Furthermore, there is no visual difference between infection threads

containing invasive or vegetative hyphae (reviewed by Pawlowski and

Demchenko 2012).

1.3.2 Nodule structure

Root nodules of legume and actinorhizal plants are quite different in

many aspects. In legume-rhizobia nodules, the infected cells are located

in the central tissue and the vascular bundles are peripheral. The nod-

ules can be determinate or indeterminate depending on the host plant.

In determinate nodules, meristemic activity stops in the early stages of

development, leading to a rather spherical shape (Newcomb 1981). In-

determinate nodules have a more cylindrical shape because they main-

tain an active apical meristem which keeps producing new cells over

the life time of the nodule. The infected cells of indeterminate nodules

are arranged in a spatial developmental gradient, while the developmen-

tal gradient of the infected cells of determinate nodules is temporal.

All non-legume root nodules, i.e., actinorhizal nodules and nodules

of Parasponia sp., are characterized by their perennial coralloid struc-

tures. They consist of multiple lobes with each lobe resembling a mod-

ified lateral root with infected cells located in the cortex and a central

vascular system (Pawlowski and Bisseling 1996). As in indeterminate

legume nodules, the infected cells are arranged in a developmental gra-

dient from the apex to the base, allowing the delineation of zones. The

apical meristem is considered as zone I. Below it is the infection zone

(II) where infection thread-like structures containing Frankia invade

22

and branch in infected cells. The fixation zone (III) is where nitrogen is

fixed by the nitrogenase enzyme complex. The last one is the senes-

cence zone (IV) where bacteria are inactive and degraded (reviewed by

Pawlowski and Demchenko 2012). In the species of the orders Fagales

and Rosales, the infected cells are intermixed with the uninfected cells.

However, infected cells in root nodules of the order Cucurbitales form

a distinct sector located on one side of the acentric vascular system, not

intermixed with uninfected cells (Figure 2) (Newcomb and Pankhurst

1982; Hafeez et al. 1984).

When the host plants are grown in wet or waterlogged land, the nod-

ules’ access to oxygen or nitrogen can be limited. To cope with gas

deprivation, members of Casuarina, Comptonia, Datisca, Gymnos-

toma, and Myrica can form nodule roots at the tips of their lobes due to

a change of meristem activity (Silvester et al. 1990). Nodule roots show

negative geotropic growth and contain large air spaces in their cortex in

order to allow gas diffusion. They are not infected by Frankia thus do

not contribute to nitrogen fixation.

Figure 2. Comparison of longitudinal sections of lateral root and different nodule types.

The vascular system is depicted in black. (A) shows a standard lateral root with central vascular

bundle given in black, root hairs (rh), apical meristem (m) and calyptra (c). (B) shows an inde-

terminate legume nodule with peripheral vascular system and infected cells in the inner tissue.

Due to the activity of the apical meristem (I) the cells of the inner tissue (shaded in red) are

arranged in a spatial developmental gradient (Vasse et al. 1990): The prefixation zone (II) is

followed by the interzone (II-III) where nitrogen fixation commences. Zone III is the nitrogen

fixation zone and Zone IV is the zone of senescence. (C) shows a lobe of an actinorhizal nodule

formed by a member of the Fagales or Rosales. Like a lateral root, the lobe has a central vascular

bundle. The infected cells (shaded in yellow) are located in the expanded cortex, intermixed

with uninfected cells. (D) shows a nodule formed by a member of the Cucurbitales. The infected

cells (shaded in green) form a continuous section in the cortex, kidney-shaped in cross section,

on one side of the acentric stele, and are not interspersed with uninfected cells.

A B C D

23

1.3.3 Nodule metabolism

Root nodules are specialized organs formed on the plant root system

after communication with the bacteria. For the host plants, nodules rep-

resent strong carbon sinks and nitrogen sources. Berry and Sunell

(1990) showed that uninfected prenodule cells contain large amounts of

starch granules. The function of this storage is not clear but it has been

shown that nodule starch is reduced during carbon-stressed situations

(Vikman et al. 1990).

The most common form of carbon transported in higher plants is su-

crose, a disaccharide in which fructose and glucose are linked via an O-

glycosidic bond (Giaquinta 1983). Sucrose can be degraded by cyto-

solic sucrose synthase (SucS) or by cytosolic, vacuolar or apoplastic

invertases. In legume nodules, SucS is mostly responsible for sucrose

degradation (Gordon et al. 1999). SucS transcripts have also been

shown to be present at high levels in infected cells of actinorhizal nod-

ules of A. glutinosa, C. glauca and D. glomerata (Van Ghelue et al.

1996; Schubert et al. 2011; Schubert et al. 2013).

In legume-rhizobia symbioses, dicarboxylates, especially malate and

succinate, are the main carbon source supplied to the microsymbiont by

the host plant (reviewed by Udvardi and Poole 2013). More research

needs to be done to determine the carbon source that Frankia utilizes in

the infected cells of actinorhizal nodules. In A. glutinosa, a nodule spe-

cific dicarboxylate transporter, AgDCAT1, has been identified and

characterized (Jeong et al. 2004). This transporter locates to the peri-

symbiont membrane and facilitates the flow of dicarboxylates from the

cytoplasm to the microsymbiont (Jeong et al. 2004). Alternatively,

amino acids, i.e. Gln or Asp have been proposed as sole carbon source

for Frankia strain CpI1, isolated from Comptonia peregrina root nod-

ules (Zhang et al. 1992).

In all types of root nodules, bacteria reduce atmospheric nitrogen to

ammonium. In the legume-rhizobia symbiosis, ammonium assimilation

does not take place inside the microsymbiont. The product of nitrogen-

ase activity is exported to the plant cytosol where it is assimilated in the

glutamine synthetase-glutamate synthase (GS-GOGAT) cycle (Temple

et al. 1998). In actinorhizal root nodules from plants of two different

24

orders, A. glutinosa in Fagales and Discaria trinervis in Rosales, gluta-

mine synthetase (GS) is highly expressed in infected cells (Guan et al.

1996; Valverde and Wall 1999), implying that the primary nitrogen as-

similation mechanism is similar to that in the legume-rhizobia symbio-

sis. In root nodules of A. incana, bacterial GS protein cannot be detected

(Lundquist and Huss-Danell 1990), indicating that ammonium is ex-

ported to the plant cytosol where assimilation takes place.

However, D. glomerata of the order Cucurbitales shows a nitrogen

metabolism significantly different from other types of actinorhizal nod-

ules. GS transcripts and proteins were found in uninfected cortical cells

that surrounded the infected tissue but were not detected in the infected

cells (Berry et al. 2004). Gln, Glu and Arg were present at high levels

in the root nodule extract (Berry et al. 2004). Detailed analyses implied

that in nodules of D. glomerata, Frankia carries out primary nitrogen

assimilation and exports arginine to the plant cytosol. This arginine is

transported to the uninfected cells where it is broken down, and the am-

monium is re-assimilated (Berry et al. 2011). Assimilated nitrogen is

further processed into amino acids (glutamate, glutamine, aspartate and

asparagine) or ureides (citrulline, arginine) for transport via the xylem

and/or temporary storage (Schubert 1986).

1.4 Signal exchange between microsymbiont and host plant

1.4.1 Signal exchange in the legume-rhizobia symbiosis

The symbiotic interaction between legumes and rhizobia has been

studied extensively. Chemical communication must take place between

the bacteria and the host plant root. The plant secrets flavonoids which

are inducers of rhizobial nodulation genes (Phillips 1991). These com-

pounds bind the NodD protein, a transcriptional regulator of the LysR

family. NodD activates the genes that are responsible for the synthesis

of rhizobial signal molecules, lipochitooligosaccharide (LCO) Nod

(Nodulation) factors (reviewed by Oldroyd 2013). These molecules

have a chitooligosaccharide backbone bearing an acyl chain on the non-

reducing end (Mylona et al. 1995). The backbone of these compounds

25

is synthesized by proteins encoded by the nod genes nodABC, which

are present in all rhizobial species. NodC represents a chitin synthase

that synthesizes the N-acetyl-glucosamine oligomer. NodB, a deacety-

lase, removes the acetyl group of the sugar at the non-reducing end,

while the acyl transferase NodA transfers the acyl chain to the position

where the acetyl group was removed. There are also several other nod

genes that are involved in chemical modifications of the Nod factors,

influencing host specificity (Dénarié et al. 1996). Nod factors are per-

ceived by the plant and stimulate transcriptional changes in the plant

roots that lead to nodule formation and are required for bacterial infec-

tion (reviewed by Oldroyd 2013).

Nod factors are perceived by lysin motif (LysM) domain receptor-

like kinases in the root hair plasma membrane. In Lotus japonicus these

receptor kinases are Nod Factor Receptor 1 (NFR1) and Nod Factor

Receptor 5 (NFR5) while in Medicago truncatula, LysM receptor ki-

nase 3 (LYK3) and Nod Factor Perception (NFP) fulfil the equivalent

function (Madsen et al. 2003; Limpens et al. 2003). They form a recep-

tor complex that interacts with Nod factors secreted by the bacteria.

Other receptor kinases that also participate in the recognition of Nod

factor are Symbiosis Receptor-like Kinase (SYMRK) in L. japonicus

and its ortholog Does not Make Infections 2 (DMI2 in M. truncatula)

and Nodulation Receptor Kinase (NORK in M. sativa; Endre et al.

2002; Stracke et al. 2002). SYMRK/ DMI2/NORK are presumed to fa-

cilitate the perception of Nod factors by LysM receptor complexes.

SYMRK/DMI2 and LysM receptor kinases are also required in the

signal exchange between arbuscular mycorrhizal fungi (AMF) and their

host plant. This is unsurprising in view of the fact that AMF signal fac-

tors, the so-called Myc factors, represent LCOs or chitooligosaccha-

rides (Genre et al. 2013; Maillet et al. 2011; Op den Camp et al. 2011).

Downstream of the plasma membrane receptors in the signal transduc-

tion pathway are the cation channels CASTOR and POLLUX in L. ja-

ponicus (Charpentier et al. 2008) and DMI1 in M. truncatula (Peiter et

al. 2007). They are located on the nuclear membrane and are essential

for the symbiotic calcium oscillations that are caused by Nod (and Myc)

factor signalling which play the central role in both the infection process

26

and root nodule organogenesis (Capoen et al. 2011). The calcium spik-

ing signatures are decoded by the Calcium- and Calmodulin-dependent

serine/threonine protein kinase (CCaMK/DMI3) which phosphorylates

LjCYCLOPS/MtIPD3 in L. japonicus and M. truncatula, respectively

(Horváth et al. 2011; Yano et al. 2008). CYCLOPS/IPD3 is a transcrip-

tion factor that together with CCaMK/DMI3 activates the expression of

a further transcription factor, Nodule Inception (NIN) (Marsh et al.

2007; Singh et al. 2014). Other nodulation-specific transcription factors

functioning downstream of the calcium oscillations include Nodulation

Signalling Pathway 1 (NSP1) and NSP2 (Singh et al. 2014).

Arbuscular mycorrhizal symbiosis (AMS) were already present in

the first terrestrial plants (Redecker et al. 2000). It has been concluded

that when root nodule symbioses evolved, they recruited part of the ar-

buscular mycorrhizal signal transduction pathway (Markmann and Par-

niske 2009). The signalling pathway from SYMRK/DMI2 to CY-

CLOPS/IPD3 is shared between AMS and legume-rhizobia symbiosis

and it is called the “common symbiotic signalling pathway” (CSSP).

1.4.2 Signal exchange in actinorhizal symbiosis

There are commonalities between the signalling pathways used by

rhizobia and Frankia strains. Homologs of legume SYRMK have been

found in C. glauca (CgSYMRK) and D. glomerata (DgSYMRK) and

could be shown to be essential for the induction of nodules by Frankia

(Gherbi et al. 2008; Markmann et al. 2008). CCaMK, a key component

in the CSSP, is also crucial for the formation of nodules on roots of C.

glauca (Svistoonoff et al. 2013). Furthermore, an ortholog of NIN,

which plays a central role in nodulation in rhizobial symbiosis has been

found in C. glauca (Clavijo et al. 2015). Homologs of NIN were also

found to be expressed in nodules of A. glutinosa (Hocher et al. 2011)

and D. glomerata (Demina et al. 2013). These results show that the

CSSP is shared between rhizobial and actinorhizal symbiosis.

Since LCOs are used by both rhizobia and AM fungi to signal via

the CSSP, one would expect Frankia strains to utilize the same type of

27

molecule to communicate with its host plants. However, all the se-

quenced genome of members from cluster I and cluster III do not con-

tain the common genes nodABC (Normand et al. 2007).

Cérémonie et al. (1999) used high-performance liquid chromatog-

raphy (HPLC) to purify the putative symbiotic signal of the Alnus-in-

fective Frankia strains ACoN24d. However, the root hair deforming

factor was chemically different from rhizobial Nod factors. Recent re-

search shows that the signal molecule produced by Frankia cluster I

strain CcI3 is chitinase resistant, suggesting that it does not represent a

LCO (Chabaud et al. 2016). This factor was shown to trigger calcium

spiking and activate NIN expression in its host plant C. glauca

(Chabaud et al. 2016). LysM receptor kinases possess the ability to rec-

ognize not only LCOs but also peptidoglycans and exopolysaccharides

(Willmann et al. 2011; Kawaharada et al. 2015). Therefore, it is possible

that members of Frankia cluster I and III produce signalling molecules

that are different from LCOs.

The only Frankia genome available so far that contains nodABC

genes, Candidatus Frankia datiscae Dg1, belongs to Frankia cluster II,

the earliest divergent cluster (Persson et al. 2011, 2015). These genes

were shown to be expressed in root nodules of D. glomerata (Persson

et al. 2015). It was also shown that the phylum Actinobacteria contains

a much more diverse series of nodA homologs than the ones in rhizobia

(alpha- and beta-Proteobacteria), which led to the hypothesis that nodA

originated and evolved in Actinobacteria and was laterally transferred

to rhizobia (Persson et al. 2015). However, the second genome of a

cluster II strain, BMG5.1, does not contain the canonical nod genes

(Gtari et al. 2015). So the question is whether Dg1 or BMG5.1 repre-

sents an exception.

1.5 Evolution of actinorhizal symbiosis

All plants able to evolve a root nodule symbiosis contain a common

predisposition which is supposed to have emerged in the late Creta-

ceous ca. 100 millions years ago (mya) in the common ancestor of the

Rosales, Cucurbitales, Fagales and Fabales (Doyle 2016). Actinorhizal

28

plant species belong to eight families from the orders Rosales, Cucur-

bitales and Fagales (Figure 2). The oldest eudicot families containing

actinorhizal species include the families Betulaceae, Casuarinaceae and

Myricaceae with fossils of related species from the Cretaceous (re-

viewed by Swensen and Benson, 2008). Pollens from the families

Elaeagnaceae, Rhamnaceae and Rosaceae were dated much later, 39-

22.5 mya (Muller 1981). Although no fossil record is known for the

family Datiscaceae, fossil wood of related species from India suggests

that this family may have arisen in the Eocene (55-39 mya; Lankhanpal

1970). The divergence time of the species of the genus Coriaria (Cori-

ariaceae) between the Northern and the Southern hemisphere was dated

to 63-59 mya using molecular clock data (Yokoyama et al. 2000). Since

all Coriaria species form root nodules, the evolution of the symbiosis

must have preceded this date.

The distribution of the bacterial symbionts has been shown to have

a correlation with the distribution of the host plants (reviewed by Ben-

son et al. 2004). Since Frankia cluster II is sister to all other Frankia

clusters, it is of particular interest to study the evolution of the symbiotic

relationships between Frankia strains and actinorhizal plants. Frankia

cluster II strains have been suggested to be closely related as cross-in-

fection by crushed nodules have been successful between Ceanothus,

Coriaria, Datisca and Dryas species (Kohls et al. 1994; Torrey 1990).

In addition, phylogenetic analysis of the glnA and 16S rDNA region

suggested that there was a low genetic diversity among Ceanothus of

the family Rhamnaceae and Datisca glomerata of the family Datisca-

ceae (Vanden Heuvel et al. 2004). Nevertheless, more studies of

Frankia cluster II are needed to attain further insights into the evolution

of actinorhizal symbiosis.

29

2. Aim of this thesis

My research topic is the nitrogen-fixing actinorhizal root nodule

symbiosis, particularly of the Frankia cluster II. This cluster is earliest

divergent among Frankia strains (Persson et al. 2015). Even though

several genomes of the other clusters have been sequenced (starting

with Normand et al. 2007), just two genomes of members of cluster II

have been sequenced so far, Candidatus Frankia datiscae Dg1 (Persson

et al. 2011) and BMG5.1 (Gtari et al. 2015). While Dg1 contains the

canonical nod genes nodABC, BMG5.1 and members of the other

Frankia clusters do not. Therefore, one objective of this thesis was to

acquire more genome sequences of members of this cluster to (i) find

out whether the presence of nod genes in Dg1 is an exception; (ii) gain

insights into the evolution of actinorhizal symbiosis based on compari-

son of these genomes.

Members of cluster II nodulate a broad range of host plants belong-

ing to four families from two orders Cucurbitales and Rosales. In order

to investigate the diversity of microsymbionts in nodules of host plants

from different orders, we compared the expression levels of genes en-

coding enzymes involved in bacterial metabolism in nodules of a mem-

ber of the order Cucurbitales (Datisca glomerata) to that in nodules of

a member of the order Rosales (Ceanothus thyrsiflorus). To further un-

derstand the bacterial nitrogen metabolism in actinorhizal Cucurbitales,

the amino acid profiles of roots and nodules of D. glomerata were ob-

tained and analysed.

30

3. Comments on methodology

With only one exception, Frankia cluster II strains cannot be cul-

tured so far. This poses several technical challenges in obtaining suffi-

cient amount of pure, high quality DNA and RNA for next-generation

sequencing. Part of this thesis work was dedicated to develop methods

to acquire genomic DNA (gDNA) and total RNA from nodules induced

by Frankia cluster II strains. The lysis methods that worked on cultures

of cluster I Frankia strain ACN14a and strain CcI3 failed on cluster II

vesicle clusters; it seems cluster II cell walls are particularly difficult to

break.

3.1 Frankia gDNA isolation for genome sequencing

Different techniques to break Gram-positive cell wall described in

the literature were applied including the traditional grinding with mor-

tar and pestle (Kucho et al. 2009; Alloisio et al. 2010), using a tissue-

lyzer (Qiagen, Hilden, Germany) with iron beads or a FastPrep BIO101

benchtop homogenizer (Qbiogene, Carslbad, CA, USA) with glass

beads (Bickhart and Benson, 2011). Ultimately, the best results were

obtained by grinding in a mortar with a pestle in liquid nitrogen, fol-

lowed by breaking the cell walls using ultrasonic homogenizer Sono-

plus HD 2070 (Bandelin Electronic, Berlin, Germany), in spite of the

fact that this method also fragmented the gDNA.

3.2 Candidate gene expression profiling of bacteria in

nodules of Datisca glomerata and Ceanothus thyrsiflorus

In our group, bacterial and plant symbiotic transcriptomes were ana-

lysed in parallel. Therefore, total plant and bacterial RNA was isolated

from nodules, sequenced and later separated by bioinformatics means.

31

Prior to transcriptome sequencing, rRNA had to be removed from the

total RNA to produce an mRNA-enriched sample. This meant to re-

move both plant and bacterial rRNA. In the first attempt to deplete

rRNA, I used the Ribo-Zero rRNA Removal Kit (Illumina, California,

USA). Based on the Technical Application Staff’s advice two kits were

combined, the Ribo-Zero bacterial rRNA removal kit and the Ribo-Zero

kit for Plants. The two rRNA removal reagents were mixed with a ratio

of 1:1 to be used in one standard reaction. However, there was very little

RNA left after the experiment. To achieve a sufficient rRNA-degraded

preparation without losing too much RNA, finally the Terminator™ 5´-

Phosphate-Dependent Exonuclease (Epicentre, Wisconsin, USA) was

utilized. This enzyme efficiently digest RNA that has a 5’-monophos-

phate end without affecting other groups of RNA. However, in most

cases, repeated Terminator treatments were required to remove the

rRNA, and in some cases, most of mRNA was lost during these re-

peated treatments.

After rRNA depletion, the bacterial transcriptomes were sequenced

at the National Genomic Infrastructure, SciLifeLab (Stockholm, Swe-

den). Altogether, five transcriptomes from Datisca glomerata nodules

and three transcriptomes from Ceanothus thyrsiflorus nodules were se-

quenced (Table 3). The amounts of mapped reads vary significantly be-

tween different data sets, between 0.9 and 10.3 million reads. This could

be due to the fact that the RNA samples had been isolated using a tis-

suelyzer since the ultrasonic method had not been established at the

time. Hence the transcriptomes did not provide enough reads for relia-

ble statistical analyses. We decided to analyse the metabolic pathways

of interest based on quantitative reverse transcription polymerase chain

reaction (RT-qPCR) based on RNA isolated using the ultrasonic

method to break bacterial structures.

32

Table 3. Bacterial transcriptomes from root nodules of D. glomerata and C. thyr-

siflorus.

Transcriptome Dg (Millions of reads) Ct (Millions of reads)

Total Mapped

reads

Total Mapped

reads

1 31.1 0.9 25.4 6.6

2 28.08 3.0 22.4 9.2

3 65.0 3.3 30.6 5.02

4 65.0 6.0 n.a. n.a.

5 67.6 10.3 n.a. n.a.

3.3 Amino acid profiling of roots and nodules from Datisca

glomerata

While the metabolism of nitrogen-fixing legume nodules has been

studied extensively (reviewed by Oldroyd et al. 2011), less information

is available for the situation in actinorhizal nodules. To further under-

stand actinorhizal nitrogen metabolism, the metabolomes of nitroge-

nous solutes in roots and nodules of D. glomerata were obtained (part

of paper IV). For nodulation, D. glomerata plants were infected with

Candidatus Frankia datiscae Dg1. Nodulated plants were watered with

¼ strength nitrogen free Hoagland’s solution while uninfected plants

were provided with ¼ Hoagland’s solution supplemented with 10 mM

KNO3.

Six weeks after inoculation, young roots (4 cm from the root tips)

from un-infected plants and young roots and nodules from nodulated

plants were collected. For statistical analysis, four replicates of each

sample were harvested. The free amino acids were extracted according

to the protocol in Hacham et al. (2002). The analysis was performed on

a Hitachi L-8900 Amino Acid Analyzer (Ibaraki, Japan) at the Molec-

ular Structure Facility, UC Davis Genome Center, California, USA.

33

4. Results and Discussion

4.1 Genome sequencing of Frankia cluster II strains

We sequenced 13 genomes based on seven different inocula includ-

ing Candidatus Frankia datiscae Dg1. These inocula were collected

from different places on four different continents: Japan (Asia), France

(Europe), Papua New Guinea (Oceania) and three members from the

USA (North America).

4.1.1 Frankia cluster II inocula represent strain assemblages

Dg1 originated from nodules of Coriaria nepalensis in Pakistan

(Persson et al. 2011) whereas BMG5.1 originated from nodules of C.

japonica growing in Japan (Gtari et al. 2015). Therefore, obtaining a

genome from North America in an area where the genus Coriaria is not

present was likely to provide more information about the evolution of

Frankia cluster II. A soil-and-nodule sample was obtained from under

a growing Datisca glomerata plant in Vacaville, California, USA, and

this inoculum was used to infect D. glomerata plants in a growth cham-

ber. Bacterial gDNA was isolated from vesicle clusters isolated from

root nodules of these plants. While Dg1 represented a single strain, the

first member of Frankia cluster II from North America, Dg2, was

shown to represent a metagenome consisting of two closely related ma-

jor strains and one minor strain. It was not feasible to separate the two

major strains Dg2a and Dg2b (ca. 98–99 %) due to their strong se-

quence similarity and similar abundance (ca. 55 % Dg2a, ca. 45 %

Dg2b). The third strain, on the other hand, was underrepresented with

ca. 1% (Nguyen et al. 2016).

The fact that Dg1 represents one single strain and Dg2 consists of an

assemblage of Frankia might be due to the origin and maintenance of

the inocula. Since cluster II strains cannot be cultured, actinorhizal

34

plants growing in the greenhouse have to be infected by crushed nod-

ules of other plants. Dg1 was propagated in D. glomerata for more than

ten years in the greenhouse whereas Dg2 was isolated from nodules of

the first series that was infected with an inoculum from the field. Hence,

obtaining more inocula from other regions and host plants will help an-

swering the questions (i) how many strains are present in the actinorhi-

zal nodules; (ii) whether all the bacterial strains fix nitrogen in the nod-

ules or only the dominant ones and (iii) whether the composition of the

assemblage depends on the host plants.

Five more inocula were collected and named according to their orig-

inal host plants and the name of the country if needed: Cj1 from Cori-

aria japonica in Japan, Cm1 from Coriaria myrtifolia in France,

Crpng1 from Coriaria ruscifolia in Papua New Guinea, Dd1 from

Dryas drummondii in Alaska, USA. In all of those cases the host plants

represented the only endemic host species of Frankia cluster II in the

region. We also added one more inoculum, Cv1 from Ceanothus veluti-

nus from California where many host plant species are present (D.

glomerata, Ceanothus sp., Purshia sp., Cercocarpus sp., Chamaebatia

sp.). Two approaches of acquiring the bacterial gDNA were followed,

either from whole root nodules (nod) or from vesicle clusters (vc). The

metagenomes were named according to the name of the inoculum, the

plant species that was used for bacterial propagation, and the method of

gDNA extraction. For example, the metagenome Cj1_Dg_nod was se-

quenced from gDNA of whole nodules from D. glomerata infected with

Cj1 while Cj1_Dg_vc was obtained from gDNA of vesicle clusters of

nodules from the same plant species.

The features of all sequenced genomes and metagenomes including

the previously published Dg1 (Persson et al. 2015) are presented in Ta-

ble 4.

35

Table 4. Features of genomes and metagenomes analysed in this thesis. *maintained in D.

glomerata over a period of 10 years before isolation of vesicle clusters for sequencing; Persson

et al. (2015); **Nguyen et al. (2016). Dg1_Dg_nod2 and Dg1_Cn_nod contain several very

similar strains of which the exact relatedness could not be quantified.

A core genome tree of all genomes in Table 4 was calculated using

EDGAR (Blom et al. 2009), together with several Frankia genomes

from other clusters and two Micromonospora sp. genomes as outgroups

(Figure 3). A clear separation could be seen between strains from North

America, Eurasia and the Southern Hemisphere. Crpng1_Ca_nod,

based on a cluster II inoculum from Papua New Guinea, was phyloge-

netically basal to all cluster II strains, and to all other Frankia strains.

This finding is in accordance with previous studies where Frankia

strains from New Zealand usually formed the sister branch to other

Frankia strains (Benson et al. 1996; Clawson et al. 2004; Nouioui et al.

2014; Nguyen et al. 2016). Figure 3 also shows two cases of meta-

genomes based on the same inoculum displaying strong phylogenetic

differences (Cm1_Dg_nod/Cm1_Dg_vc and

Cj1_Dg_nod/Cj1_Dg_vc).

36

Figure 3. Core genome tree of sequenced Frankia strains from clusters I, III and IV and

of all cluster II (meta-)genomes available thus far. The tree was calculated using EDGAR

(Blom et al. 2009). The host plant orders and geographic origins of the original cluster II inocula

are color-coded, red for Cucurbitales, blue for Rosales, green for Eurasia, pink for North Amer-

ica, purple for the Southern hemisphere. Two actinobacterial genomes were used as outgroups,

Micromonospora lupini Lupac 08 (Trujillo et al. 2014) and Micromonospora aurantiaca ATCC

27029 (GenBank accession nr. CP002162). The phylogenetic tree was deduced from concate-

nated core gene alignments using PHYLIP (Felsenstein 2005). Bootstrap values were 100 for

every branch. The bar denotes 0.01 substitutions.

Comparisons of percentages of Average Nucleotide Identity (ANI)

show that the Crpng1_Ca_nod genome has less than 86% ANI with ge-

nomes of all other members of Frankia cluster II. This means that

Crpng1_Ca_nod, the only sequenced cluster II from the Southern Hem-

isphere so far, represents a novel Frankia species. We propose this

strain as type strain of Candidatus Frankia meridionalis

(me.riː.di.oːˈnaː.lis. N.L. masc./fem. adj. meridionalis, southern).

37

4.1.2 Eurasian cluster II Frankia (meta-)genomes contain the canonical nod genes nodABC and North American strains also contain the sulfotransferase gene nodH

4.1.2.1 Nod gene presence in Frankia (meta-)genomes As shown in Table 4, all sequenced (meta-)genomes of Eurasian

Frankia cluster II in the course of this study contain the canonical nod

genes nodABC and the nltIJ operon, a homolog of the nodIJ operon

which, like nodIJ, is linked to nodC (Persson et al. 2015). They are usu-

ally present in two genomic regions, the nod1 region being represented

by nodA’B1A, the nod2 region by nodB2CnltIJ (Persson et al. 2015;

Nguyen et al. 2016). Crpng1_Ca_nod contains nodB2’C as well as nltIJ

and a carbamoyl transferase gene that shows homology to rhizobial

nodU genes.

In addition, all North American strains contain one or two copies of

nodH, a sulfotransferase gene which in rhizobia is required for produc-

ing sulfated Nod factors. To investigate the origin of Frankia nodH

genes, the amino acid sequences of the two putative NodH proteins of

Dg2 together with 25 sequences from ten different rhizobial genera

were used to build a dendrogram in GARLI 2.0 (Zwickl 2006) with Cy-

anothece sp. PCC7424 as outgroup (Figure 4; Nguyen et al. 2016). This

tree shows that the two Dg2NodH proteins are sister to each other and

all other NodH proteins from Proteobacteria. The tree suggests that

nodH was laterally transferred between Frankia and rhizobia. The di-

rection of this transfer, however, cannot be determined based on phy-

logeny.

38

Figure 4. Phylogenetic tree of NodH amino acid sequences in Dg2. The tree shows that the

NodH proteins of Dg2 are sister to the rhizobial proteins. The scale bar indicates the number of

amino acid substitutions per site (Nguyen et al. 2016).

4.1.3 Cluster II genome (in-)stability: transposase frequencies in the different strains

In order to adapt to different conditions of different environment,

bacterial genomes need a certain flexibility, which is achieved by mo-

bile DNA elements, horizontal gene transfer, genome rearrangements,

etc. (Juhas et al. 2009). The frequency and size of insertion sequence

(IS) elements of genomes of all cluster II (meta-)genomes are depicted

in Figure 5. Dg1 contains more transposases than all other genomes

since it is the only fully assembled genome and assembly normally

breaks down in repetitive elements. The result shows that strains from

North America contain higher numbers of transposases than the Eura-

sian strains. The amount of IS elements in Crpng1, the Southern hemi-

sphere representative, seems to be closer to that of North American

strains; however, this result is not statistically significant.

39

Figure 5. Transposase frequencies. Numbers of transposase ORFs per Mbp in the cluster II

Frankia (meta-)genomes are given. The genome of Candidatus Frankia Datiscae Dg1 se-

quenced from DNA isolated from vesicle clusters (Dg1_Dg_vc) contains at least four times

more transposases in its genome than all other strains; this discrepancy is explained by the fact

that this genome is the only one in the group which is not in draft stage, but fully assembled.

Among the draft genomes, the Eurasian strains contain significantly less transposase ORFs than

the North American strains (Wilcoxon's p=0.006).

4.1.4 Evolution of the symbiosis between actinorhizal plants and Frankia cluster II strains

Cluster II host genus Coriaria currently has a disjunct distribution in

four isolated geographical locations of the world. Since all known Co-

riaria species can form root nodules, the evolution of the symbiosis

must have predated this separation. Therefore, it can be postulated that

the symbiosis between Coriaria sp. and Frankia cluster II evolved in

the Southern hemisphere. Together with Coriaria species, cluster II

Frankia strains probably spread to Eurasia ca. 60 mya (Yokoyama et

al. 2000). Together with Datisca sp., Frankia cluster II seemingly

reached North America 55-25 mya via the Bering Strait (Nguyen et al.

2016).

In the Southern hemisphere, currently cluster II Frankia strains only

nodulate Coriaria species. Yet, two scenarios are possible. Cluster II

symbioses might have originated in the Southern hemisphere in Gond-

wana in the common ancestor of Coriaria sp. and Datisca sp. In that

case, cluster II strains would have reached Asia via two routes, once

40

with Coriaria sp. and once with Datisca sp. which evolved in India

(Zhang et al. 2006). India separated from Gondwana ca. 120 mya and

collided with Eurasia ca. 55 mya (summarized by Ali and

Aitchison, 2008). Alternatively, cluster II symbioses would have origi-

nated in the Southern hemisphere in the ancestor of Coriaria sp. and

extended their host range after India had collided with Eurasia and Da-

tisca sp. reached Eurasia. The low level of diversity between the Eura-

sian cluster II genomes sequenced in this study suggests a single origin,

i.e., points toward an extension of the host range from Coriaria to in-

clude Datisca sp. After Frankia cluster II came to North America to-

gether with Datisca sp. via the Bering strait, it extended its host range

to include plants from the order Rosales.

An analysis of genome stability shows that with the exception of

Crpng1_Ca_nod, the nod gene regions show numerous signs of earlier

transposition events. These regions are highly conserved in the Eurasian

strains but less in the North American strains. This is consistent with

the significantly higher transposase frequency in North American than

in the Eurasian metagenomes. However, the size distribution of trans-

posases indicates that the time of transposition is in the past. The distri-

bution in Eurasia was probably associated with a burst of transposition.

Later the distribution in North America required a new burst of trans-

position, possibly related to the extensions of the host range that re-

quired more genomic flexibility.

4.2 Candidate gene expression profiling of bacterial

metabolism in root nodules of D. glomerata (Cucurbitales)

and C. thyrsiflorus (Rosales)

In order to gain information on the bacterial metabolism in host

plants from different orders, we compared gene expression levels of en-

zymes involved in bacterial metabolism from root nodules of D. glom-

erata (Datiscaceae, Curcubitales) and C. thyrsiflorus (Rhamnaceae,

Rosales). Candidatus Frankia datiscae Dg1 was used to infect D. glom-

erata and Frankia strain Dg2 was used to induce nodule on C. thyrsi-

florus. Gene expression levels were normalised against infC, the gene

41

encoding the bacterial translation initiation factor IF3 (Alloisio et al.

2010; Persson et al. 2015).

4.2.1 Frankia nodABC genes are expressed in both types of nodules, and nodH genes are expressed in nodules of C. thyrsiflorus

With one exception, all cluster II Frankia strains sequenced so far

contain the canonical nod genes nodABC (Persson et al. 2015; Gtari et

al. 2015; Nguyen et al. 2016). The Californian strain Dg2 also contained

two copies of gene encoding sulfotransferase nodH (Nguyen et al.

2016). In previous study, nodABC were found to be expressed in nod-

ules of D. glomerata but nodH was not (Persson et al. 2015; Nguyen et

al. 2016). Therefore, in this study nod gene expression was examined

in nodules of C. thyrsiflorus. As shown in Table 5, in C. thyrsiflorus

nodules, not only nodABC are expressed, but also both copies of nodH.

This result might suggest that nodH is involved in host specificity in the

symbiosis between Frankia cluster II and actinorhizal plants.

Table 5. Relative expression levels of Frankia nod genes from root nodules of D. glomerata

(Dg) and C. thyrsiflorus (Ct). Expression levels were normalised against the initiation factor

gene infC. Three biological replicates were analysed for each type of nodule. Except for nodC,

the differences between the relative expression levels of nod genes in D. glomerata and C.

thyrsiflorus are not statistically significant (p-value >0.05). n.a., not applicable.

4.2.2 The expression levels of genes encoding bacterial

truncated hemoglobin and catalases in C. thyrsiflorus are

higher than in D. glomerata

In D. glomerata and C. thyrsiflorus, different oxygen protection

mechanisms for the oxygen-sensitive nitrogenase enzyme complex are

applied (Pawlowski et al. 2007; Strand and Laetsch 1977). In D. glom-

erata, the vesicles have thin envelopes but a thick blanket of mitochon-

dria around the zone of vesicles creates a microaerophilic space (Berg

et al. 1999; Silvester et al. 1999). In nodules of actinorhizal Rosales,

nodA nodB1 nodC nodH1 nodH2

Dg 0.0087 0.0130 0.0009 n.a. n.a.

Ct 0.0396 0.0240 0.0064 0.0037 0.0036

42

Frankia strains form non-septate vesicles with multilaminate envelopes

(Strand and Laetsch 1977). A bacterial truncated hemoglobin trHbO

gene was found to be expressed in symbiotic Frankia and could act as

an oxygen diffusion facilitator under hypoxic conditions (Pawlowski et

al. 2007). The question was which oxygen protection system was more

efficient, the physiological protection by positioning of vesicles and mi-

tochondria in D. glomerata or the multi-layered vesicle envelopes in C.

thyrsiflorus.

Examining of the expression levels of the bacterial hemoglobin gene

trHbO showed it was ca. 15-fold higher in nodules of D. glomerata than

in nodules of C. thyrsiflorus (Table 6). This is combined with signifi-

cantly higher expression levels of catalase genes katA and katG in nod-

ules of Datisca than in nodules of Ceanothus. The result suggests that

the protection system of nitrogenase from oxygen in C. thyrsiflorus

leads to less reactive oxygen species (ROS) stress for the symbiotic bac-

teria than in D. glomerata. This could be explained by the fact that

Frankia metabolism is better adapted to utilizing their endogenous pro-

tection system of multilayered vesicles than the plant-controlled system

of physiological protection.

Table 6. Relative expression of genes involved in oxygen protection of nitrogenase and in

ROS detoxification in root nodules of D. glomerata (Dg) and C. thyrsiflorus (Ct). trHbO,

truncated hemoglobin; katA, catalase; katG, catalase-peroxidase. Three biological replicates

were analysed for each type of nodule. The relative expression levels of these genes are signif-

icantly different between nodules of D. glomerata and C. thyrsiflorus (p-value<0.05).

trHbO katA katG

Dg 0.240 0.197 0.560

Ct 0.016 0.039 0.001

4.2.3 Nodules induced by members of Frankia cluster II seem to share the same nitrogen metabolism

With the exception of D. glomerata, in all actinorhizal nodules stud-

ied so far, the assimilation of ammonium by glutamine synthetase (GS)

in the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle

takes place in the cytosol of the infected cells. However, in D. glomer-

ata GS accumulates in the uninfected cells surrounding the infected

43

cells (Berry et al. 2004). This result suggested that, unlike in other type

of root nodules where the bacteria export ammonia directly to the plant,

Frankia in D. glomerata nodules exports an assimilated form of nitro-

gen, most likely arginine (Berry et al. 2004, 2011). This type of nitrogen

metabolism would enhance the need for carbon supply from the host

plant to the intracellular bacteria.

In order to find out whether the unusual nitrogen metabolism of D.

glomerata (Berry et al. 2004, 2011) nodules was shared by C. thyrsiflo-

rus nodules, expression levels of genes encoding enzymes involved in

the GS-GOGAT cycle and in arginine biosynthesis were compared. The

results showed that the genes encoding the bacterial enzymes of the GS-

GOGAT cycle are highly expressed in nodules of both D. glomerata

and C. thyrsiflorus. The expression levels of genes encoding the en-

zymes of the arginine biosynthetic pathway are much lower, and there

are no significant differences between the two types of nodules. How-

ever, to ascertain that Frankia in C. thyrsiflorus nodules also exports a

metabolite of the arginine cycle, analysis of the nodule N-metabolite

profile is needed for C. thyrsiflorus.

4.2.4 Which carbon source does the host plant provide for symbiotic Frankia in both systems?

In legume nodules and in nodules of the actinorhizal species Alnus

glutinosa (Betulaceae, Fagales) dicarboxylic acids, mainly malate and

succinate, are supposed to be the main form of carbon source supplied

to the microsymbionts by the host plant (Vance and Gantt 1992; Jeong

et al. 2004; Udvardi and Poole 2013). These metabolites would be fed

into the tricarboxylic acid (TCA) cycle, which would break down the

substrates for production of ATP and reduction equivalents and also

provide precursors for the primary metabolism of Frankia, e.g., to pro-

vide sugars for cell wall biosynthesis. However, in D. glomerata and

probably also in C. thyrsiflorus, the plant would also have to provide

carbon skeletons (ultimately, alpha-ketoglutarate, the precursor of glu-

tamate) for ammonium assimilation.

Therefore, oxaloacetate would be shuttled out of the TCA cycle to

be used in gluconeogenesis via phosphoenolpyruvate carboxykinase

44

(pepck; Figure 6, 7). Alpha-ketoglutarate would be used for the synthe-

sis of glutamate via the GS/GOGAT cycle and maybe, in case of high

ammonium concentrations, also by glutamate dehydrogenase (gdh; Fig-

ure 7). Since the symbiotic bacteria provide nitrogen sources for the

entire host plant, a large supply with alpha-ketoglutarate would be re-

quired. So its removal from the TCA cycle would be disruptive. How-

ever, Figure 7 shows that the expression levels of genes of the pathway

between alpha-ketoglutarate and malate are not much lower than in the

part between malate and alpha-ketoglutarate. More importantly, all

cluster II Frankia strains sequenced so far lack the gene for the first

enzyme of the glyoxylate shunt, isocitrate lyase. So it is unlikely that

the production of alpha-ketoglutarate could be bypassed.

Figure 6. Comparison

of the expression levels

of genes encoding en-

zymes of the glycolysis

pathway of Frankia

from root nodules of D.

glomerata (Dg) and C.

thyrsiflorus (Ct). Ex-

pression levels were nor-

malised against the infC

gene. Glk, glucokinase;

pgi, phosphoglucose iso-

merase; pfk1, phos-

phofructokinase-1; fba,

aldolase; gap, glycer-

aldehyde 3-phosphate

dehydrogenase; pgk,

phosphoglycerate kinase;

pgm, phosphoglycerate

mutase; eno, enolase;

pyk, pyruvate kinase;

pepck, phosphoenolpy-

ruvate carboxykinase.

Three biological repli-

cates were analysed for

each type of nodule. Rel-

ative expression levels of

genes are depicted in red

for Dg and in blue for Ct.

45

Figure 7. Comparison of expression levels of Frankia genes encoding TCA cycle enzymes

from root nodules of D. glomerata (Dg) and C. thyrsiflorus (Ct). Expression levels were

normalised against the infC gene. AcnA, aconitase; icd, isocitrate dehydrogenase; lsc1, succin-

ate-CoA ligase subunit alpha; lsc2, succinate-CoA ligase subunit beta; sdhA/B/C/D, succinate

dehydrogenase subunit A/B/C/D; mdh, malate dehydrogenase; gltA, citrate synthase; gdh, glu-

tamate dehydrogenase; gltB and gltD, glutamate synthase large subunit and small subunit, re-

spectively. Three biological replicates were analysed for each type of nodule. Relative expres-

sion levels of genes are given in red for Dg, in blue for Ct.

One explanation could be that in D. glomerata and C. thyrsiflorus,

the plant provides a combination of succinate and malate to the bacteria

as was suggested for legume nodules (Udvardi and Poole 2013). The

citrate cycle would then work as a linear pathway from succinate to

alpha-ketoglutarate. The additional input of malate from the host plant

would compensate the oxaloacetate that leaves the cycle for gluconeo-

genesis and acetyl-CoA production. This would also explain the ab-

sence of the glyoxylate shunt in cluster II strains.

The export of an assimilated form of nitrogen would require more

energy-intensive transport processes. The question is why this type of

nodule metabolism would evolve in two different plant/bacterial com-

binations. As suggested above, Frankia cluster II strains seem to have

come to North America from Asia with Datisca sp. via the Bering Strait

55-25 mya (Nguyen et al. 2016). It is possible that by the time cluster

II strains had reached North America and expanded their host range to

46

Ceanothus sp. and Rosaceae host plants, they had already lost the gly-

oxylate shunt and had adapted to the symbiotic metabolism in actino-

rhizal Cucurbitales.

4.3 The N-metabolites of roots and actinorhizal nodules from

Datisca glomerata

4.3.1 Amino acid profiles of roots and nodules

The nitrogenous solute profiles of roots of uninfected plants as well

as roots and nodules of nodulated plants are shown in Table 7. The re-

sults showed that the profiles of nitrogenous solutes in roots of unin-

fected plants were dominated by glutamine (51.2%), followed by glu-

tamate (16%), γ-aminobutyrate (GABA) (3.4%), aspartate (6.2%), ser-

ine (3.4%) and alanine (2.7%). All other amino acids contributed less

than 2% of the total amount.

In nodulated roots, glutamine (25.1%) and glutamate (24.9%) were

still dominant even though the contribution of glutamine was reduced

by half in comparison with uninfected roots. As in roots of plants sup-

plemented with nitrate, GABA (5.4%), serine (3.4%) and alanine

(4.9%) played minor roles in nodulated roots. While the amount of ar-

ginine was insignificant in uninfected roots (0.9%), its contribution was

10.5% in nodulated roots.

In the profiles of nodules, glutamate (27.9%) was still dominant

while glutamine was present at 9.5%. Aspartate (6.6 %) and alanine (4.8

%) played minor roles, followed by serine (3.6 %) and GABA (2.5 %).

In contrast to the profiles in roots, arginine (26.9%) was dominant

among the nitrogenous solutes in nodules.

47

Table 7. Percentage of total nitrogenous solutes in D. glomerata plants. GABA, γ-ami-

nobutyrate. (Taken from Supplementary material, Persson et al. 2016)

Amino acid

Percentage in

roots of unin-

fected plants

Percentage in

roots of nodu-

lated plants

Percentage in

nodules

ala 2.7 4.9 4.8

asn 1.8 5.4 2.8

asp 6.2 10.7 6.6

arg 0.9 10.5 26.9

citrulline 0.0 0.0 0.2

ornithine 0.0 0.0 0.1

ethanolamine 6.8 0.0 0.9

GABA 3.4 5.4 2.5

gln 51.2 25.1 9.5

glu 16.0 24.9 27.9

gly 1.2 1.2 0.3

his 0.6 1.2 1.5

ile 0.7 1.0 1.2

leu 0.4 0.9 1.9

val 1.2 1.7 3.3

thr 1.6 1.7 3.3

phe 0.5 0.6 0.4

pro 0.8 0.7 0.7

ser 3.4 3.4 3.6

tyr 0.0 0.0 0.7

lys 0.5 0.6 1.0

4.3.2 Do D. glomerata plants change nitrogen transport forms when nodulated?

Root nodule nitrogen metabolism is a combination of nitrogen ex-

port from the microsymbiont, nitrogen assimilation, and nitrogen stor-

age. In all root nodules, the host plant provides the bacteria with carbon

sources in exchange for nitrogen. In legume-rhizobia symbiosis, the

product of nitrogenase activity, ammonium, is exported to the plant cy-

tosol where it is assimilated (Temple et al. 1998). The primary nitrogen

assimilation in root nodules of actinorhizal plants of Fagales (Alnus glu-

tinosa and A. incana) and Rosales (Discaria trinervis) is similar to that

48

in legume nodules (Guan et al. 1996; Valverde and Wall 1999;

Lundquist and Huss-Danell 1990). However, the situation was different

in D. glomerata where the microsymbiont exported an assimilated form

of ammonium (Berry et al. 2004, 2011). In this study, arginine was

shown to be one of the major nitrogenous solutes in nodules, suggesting

that D. glomerata nodules can export not only amides and their acids

but also arginine. Apart from arginine, glutamine and glutamate were

the major amino acids in nodules, as reported in previous studies (Berry

et al. 2004).

The amino acid profiles of roots of uninfected plants were dominated

by glutamine and glutamate while aspartate played a minor role and

arginine was present in a very low amount (0.9%). The profiles of roots

from infected plants were also dominated by glutamine and glutamate

followed by aspartate. However, these roots contained a significant

higher amounts of arginine (10.5%) compared to uninfected roots, in-

dicating that nodulated plants seemed to add arginine as nitrogen

transport form. Hence, for D. glomerata growth conditions may lead to

changes in the xylem N transport forms.

49

5. Conclusions

The range of sequenced genomes of Frankia cluster II, the earliest

divergent Frankia cluster, has expanded from Asia to three more conti-

nents, North America, Europe and Oceania. The results suggest that

cluster II inocula represent groups of strains which might explain their

wide host range. Since all Eurasian cluster II strains analysed in this

study contain the canonical nod genes nodABC and the North American

strains also contain the sulfotransferase nodH, it could be concluded

that the presence of the canonical nod genes is the standard in Frankia

cluster II. The host plant-specific expression of nodH might suggest that

this gene is linked to host specificity. Based on the analysis of trans-

posase frequencies, it could be postulated that the distribution of

Frankia in Eurasia was associated with a burst of transpositions. The

distribution in North America possibly came later and required a new

burst of transpositions. These events may have coincided with the ex-

pansion of the host range which necessitated a higher level of genomic

flexibility.

Analysis of the expression levels of genes involved in metabolism of

symbiotic bacteria in nodules of Datisca glomerata and Ceanothus

thyrsiflorus showed that the nitrogenase protection in Ceanothus sp.

nodules seemed to be more efficient than that in Datisca sp. nodules.

On the other hand, there was a similarity in expression levels of genes

involved in nitrogen metabolism, implying that in both systems an as-

similated form of nitrogen is exported to the plant. The result was sup-

ported by analyses of amino acid profiles of D. glomerata roots and

nodules, which showed that the amounts of arginine in roots and nod-

ules of nodulated plants were significantly higher than in roots of unin-

fected plants.

50

6. Future perspectives

This thesis has paved the way to understand the evolution of the

Frankia cluster II, yet there are several questions left unanswered.

- Are the canonical nod genes expressed in other host plants beside

Datisca sp. and Ceanothus sp.? To answer this question, the expressions

of nod genes in nodules of Coriaria sp. should be investigated.

- Is the expression of nodH necessary for the infection of North

American host plants other than D. glomerata? It would be necessary

to infect North American host plants from the Rosales, e.g., Ceanothus

sp., with Eurasian inocula.

- How do the genome sequences of the South American strains fit

into the phylogeny of cluster II? It is therefore essential to obtain meta-

genomes from Frankia cluster II inocula from Central and South Amer-

ica, i.e. from strains that infect Coriaria ruscifolia which is endemic to

this area, presumably brought from New Zealand (Yokoyama et al.

2000; Nouioui et al. 2014).

- To obtain reliable evidence on the function of the canonical nod

genes in members of cluster II, it is crucial to purify the signalling mol-

ecule and conduct bioassays with the purified products. In parallel, it is

important to invest effort in determining the culture conditions for nod-

gene-containing cluster II strains, e.g., Dg1, in order to set up a trans-

formation protocol and to knock out the nod genes.

- To gain additional data of carbon source supplied to the microsym-

biont in symbiosis, the metabolome of symbiotic Frankia needs to be

analysed. This would require non-aqueous fractionation to isolate bac-

terial vesicles under conditions where no enzyme activity is possible.

- To test the hypothesis that the microsymbiont exports assimilated

forms of ammonium in Ceanothus sp. nodules, like in D. glomerata, it

is necessary to analyse the amino acid profiles of roots and nodules of

a candidate of the genus Ceanothus.

51

7. Acknowledgments

I would like to thank the Department of Ecology, Environment and

Plant Sciences (DEEP) at Stockholm University for giving me the op-

portunity to conduct my doctoral study in this beautiful city and this

nice country. For me, it has been a life-changing experience.

I would like to express my deep gratitude to my supervisor, Dr.

Katharina Pawlowski. “Despite numerous attempts”, I was not success-

ful in convincing her to name Dg2 as Frankia nguyeni. I am still really

grateful for her valuable guidance and kind support. Without her, my

encounter with the fascinating and challenging research topic of actino-

rhizal symbiosis would have not happened.

I would like to thank Dr. Alison Berry at the University of California,

Davis, for providing me the opportunity to work in her lab and her sci-

entific support. I would also like to thank Dr. Daniel Wibberg and other

co-authors for their essential collaboration.

I am very grateful to Dr. Lisbeth Jonsson and Dr. Edouard Pesquet for

their constructive criticism of my thesis. I would also like to thank my

opponent Dr. Peter Young and the committee members Dr. Roger Fin-

lay, Dr. Anita Sellstedt and Dr. Per-Olof Lundquist for their time and

the scientific input.

Special thanks go to the current and former members of the Paw-

lowski’s group: Marco, Irina, Pooja, Tomas, and Theo for their cooper-

ation and stimulating discussions. Their encouragement during my

stressful times is very much appreciated.

I would like to thank my co-supervisor Dr. Sophia Ekengren, Dr. Ulla

Rasmussen, Dr. Sara Rydberg, Denis, Sandra, Andreas, Sara, Aleksan-

dra, Lina, Paul, Andrea, and other colleagues for their help during my

thesis. I am grateful to Peter, Anna, and Ingela for taking care of my

experimental plants in the greenhouse. I would also like to thank all the

colleagues at DEEP for fostering a friendly and positive working envi-

ronment.

52

I am heartily thankful to my beloved ones, Nhật Quang, Lisa, Xiaoyun,

Mai Trang, Nguyệt Minh, and Rickard for sharing my joyful and tearful

moments. I really enjoy their companionship on my journeys around

Europe. Though we are not always together, memories of all of them

remain deep within me. I especially want to thank my dear friend Katha

who has always been there for me through thick and thin.

I would also like to use this opportunity to extend my gratitude to my

family and relatives.

Con xin chân thành cảm ơn mọi người đã thương yêu, dạy dỗ và chăm

sóc con từ lúc nhỏ cho đến lúc trưởng thành. Cảm ơn em Bích Tuyền

đã hết lòng chia sẻ và giúp đỡ chị.

This thesis would have not been accomplished without the contribution

of many of my colleagues, relatives, and friends. To all whose names

have not been mentioned in this acknowledgement: Thank You!

53

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C, Yamaura M, Kakoi K, Kucho K (2010) The Frankia alni symbiotic

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Barritt AR, Facelli JM (2001) Effects of Casuarina pauper litter and grove

soil on emergence and growth of understorey species in arid lands of South

Australia. J Arid Environ 49:569-579.

Becking JH (1970) Frankiaceae fam. nov. (Actinomycetales) with one new

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