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ISBN 978-952-62-0683-7 (Paperback)ISBN 978-952-62-0684-4 (PDF)ISSN 0355-3191 (Print)ISSN 1796-220X (Online)

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U N I V E R S I TAT I S O U L U E N S I SACTAA

SCIENTIAE RERUM NATURALIUM

OULU 2014

A 644

Suvi Sutela

GENETICALLY MODIFIED SILVER BIRCH AND HYBRID ASPEN – TARGET AND NON-TARGET EFFECTS OF INTRODUCED TRAITS

UNIVERSITY OF OULU GRADUATE SCHOOL;UNIVERSITY OF OULU, FACULTY OF SCIENCE, DEPARTMENT OF BIOLOGY

A 644

ACTA

Suvi Sutela

A C T A U N I V E R S I T A T I S O U L U E N S I SA S c i e n t i a e R e r u m N a t u r a l i u m 6 4 4

SUVI SUTELA

GENETICALLY MODIFIED SILVER BIRCH AND HYBRID ASPEN – TARGET AND NON-TARGET EFFECTS OF INTRODUCED TRAITS

Academic dissertation to be presented with the assent ofthe Doctora l Train ing Committee of Health andBiosciences of the University of Oulu for public defence inAuditorium IT116, Linnanmaa, on 12 December 2014, at12 noon

UNIVERSITY OF OULU, OULU 2014

Copyright © 2014Acta Univ. Oul. A 644, 2014

Supervised byProfessor Hely HäggmanProfessor Riitta Julkunen-TiittoDocent Karoliina Niemi

Reviewed byAssociate Professor Gerd BossingerProfessor Lise Lejus-Jouanin

ISBN 978-952-62-0683-7 (Paperback)ISBN 978-952-62-0684-4 (PDF)

ISSN 0355-3191 (Printed)ISSN 1796-220X (Online)

Cover DesignRaimo Ahonen

JUVENES PRINTTAMPERE 2014

OpponentDoctor Markku Keinänen

Sutela, Suvi, Genetically modified silver birch and hybrid aspen – target and non-target effects of introduced traits. University of Oulu Graduate School; University of Oulu, Faculty of Science, Department ofBiologyActa Univ. Oul. A 644, 2014University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland

Abstract

The efforts to improve forest trees could be accelerated by means of genetic engineering. Thus,the performance and effects of genetically modified (GM) trees have been investigated innumerous studies, which have generally concluded that GM trees have similar effects onenvironment and/or other organisms as do conventionally bred trees. In the present study, GMsilver birch (Betula pendula Roth) and hybrid aspen (Populus tremula L. × tremuloides Michx.)lines were utilized to study the influence of transgenes to the transcription of related endogenousgenes and to the production of soluble phenolic compounds in relation to ectomycorrhizalsymbiosis or herbivory.

The GM silver birch lines had altered lignin composition, whereas the hybrid aspen linesproduced the hemoglobin of Vitreoscilla sp. (VHb). The Pt4CL1a lines were generated usingbiolistic transformation and monitored under greenhouse conditions for three growing seasons.The Pt4CL1a and PtCOMT silver birch lines, with altered lignin syringyl/guaiacyl ratio, had alsoreduced transcript levels of endogenous genes, Bp4CL1 and BpCOMT, respectively. Thisindicates that these members of the 4CL and COMT multigene families are likely to contribute tothe monolignol biosynthesis pathway of silver birch. No unintended effects were detected in thePtCOMT or Pt4CL1a lines in relation to ECM symbiosis or performance of insect larvae.Moreover, in soluble phenolic compounds, alterations were found mainly in cinnamic acidderivatives, a group of compounds involved in the biosynthesis of monolignols. In addition, theresponses of the studied hybrid aspen lines that were exposed to herbivory for 24 hours were foundto be comparable. Furthermore, the proportional weight gain of lepidopteran larvae was alikewhen fed with leaves of the VHb and non-transgenic hybrid aspen lines. Taken together, nounintended changes were found in the GM silver birch lines with altered lignin composition or inthe VHb hybrid aspen lines. However, it is acknowledged that these short-term studies that wereconducted under controlled conditions have certain limitations.

Keywords: 4CL, Betula, COMT, Genetic engineering, GM, lignin, phenylpropanoidpathway, Populus

Sutela, Suvi, Geneettisesti muunnettujen rauduskoivujen ja hybridihaapojenominaisuudet ja vaikutusten arviointi. Oulun yliopiston tutkijakoulu; Oulun yliopisto, Luonnontieteellinen tiedekunta, Biologian laitosActa Univ. Oul. A 644, 2014Oulun yliopisto, PL 8000, 90014 Oulun yliopisto

Tiivistelmä

Puiden ominaisuuksia on mahdollista muuttaa geenitekniikkaa käyttämällä huomattavasti perin-teistä jalostusta nopeammin. Geneettisen muuntamisen vaikutuksia puiden ominaisuuksiin javuorovaikutussuhteisiin on selvitetty useissa tutkimuksissa geenitekniikkaan liitettyjen riskienarvioimiseksi. Muunnettuja kohdeominaisuuksiaan lukuun ottamatta geneettisesti muunnettujen(GM) puiden ei ole yleisesti ottaen tutkimuksissa havaittu eroavan ympäristövaikutuksiltaanperinteisellä jalostuksella tuotetuista puista. Tässä työssä tutkittiin siirrettyjen geenien vaikutuk-sia GM-rauduskoivun (Betula pendula Roth) sekä hybridihaavan (Populus tremula L. × tremu-loides Michx.) endogeenisten geenituotteiden ja liukoisten fenoliyhdisteiden määriin. Lisäksityössä tarkasteltiin ligniinirakenteeltaan muunnettujen rauduskoivulinjojen ektomykorritsasym-bioosia sekä ligniinimuunnettujen ja Vitreoscilla sp. -bakteerin hemoglobiinia (VHb) tuottavienhybridihaapalinjojen lehtien laatua perhostoukkien ravintona.

Biolistisella geeninsiirrolla tuotetuista Amerikan haavan 4-kumaraattikoentsyymi A-ligaasi -geeniä (Pt4CL1) ilmentävistä rauduskoivulinjoista yhdessä havaittiin ligniinin syringyyli- jaguaiasyyliyksikköjen suhteessa muutos. Havaittu muutos aiheutui todennäköisesti koivunBp4CL1-geenituotteiden määrän vähenemisestä. Myös kaffeaatti/5-hydroksylaatti O-metyyli-transferaasi -geeniä (PtCOMT) ilmentävissä, ligniinirakenteeltaan muunnetuissa rauduskoivulin-joissa havaittiin endogeenisen BpCOMT-geenin tuotteiden määrän väheneminen. Tulokset viit-taavat siihen, että Bp4CL1- ja BpCOMT-geenien tuottamat entsyymit toimivat rauduskoivunmonolignolien biosynteesissä. Ligniiniominaisuuksiltaan muunnettujen rauduskoivujen liukoi-sista fenoliyhdisteistä todettiin muutoksia ensisijaisesti kanelihappojohdannaisissa, jotka liittyvätläheisesti monolignolien biosynteesireittiin. Ektomykorritsasymbioosissa tai perhostoukkienkasvunopeudessa ei havaittu kasvien geneettisestä muuntamisesta johtuvia eroja. Merkitseviäeroja ei todettu myöskään hybridihaapalinjojen herbivoria-vasteissa. On kuitenkin otettava huo-mioon, että kaikki tutkimuksen kokeet suoritettiin kasvihuoneissa käyttäen vasta juveniilivai-heessa olevia kasveja. Jotta abioottisten ja bioottisten ympäristötekijöiden sekä GM-puiden vuo-rovaikutusta olisi mahdollista arvioida kokonaisvaltaisesti, puita pitäisi tutkia pitkäaikaisissakenttäkokeissa.

Asiasanat: 4CL, Betula, COMT, fenyylipropanoidireitti, geenitekniikka, GM, ligniini,Populus

7

Acknowledgements

This study was conducted at the Department of Biology at the University of Oulu.

For the financing of this thesis, I wish to acknowledge the Academy of Finland,

the Biological Interactions Graduate School, the Jenny and Antti Wihuri

Foundation, the Oulu University Scholarship Foundation, the Niemi Foundation,

and the University of Oulu Graduate School.

I wish to express my greatest gratitude to my supervisors, Prof. Hely

Häggman, Prof. Riitta Julkunen-Tiitto, and Dr. Karoliina Niemi. I am glad I have

had the opportunity to work with such ambitious, quick-witted and strong

researchers. I have always received guidance and support from you. I am

especially grateful to Hely, who spent her summer holidays reading, commenting

and correcting the manuscript of this thesis.

The members of my follow-up group, Prof. Anja Hohtola, Dr. Anna Maria

Pirttilä, and Dr. Kari Taulavuori, are acknowledged for their valuable comments. I

owe my gratitude to all the researchers who have contributed to this work

including Dr. Tuija Aronen, Jaanika Edesi, Kaisa Haapala, Terhi Hahl, Dr. Soile

Jokipii-Lukkari, Dr. Pauli T. Kallio, Tapio Laakso, Dr. Pekka Saranpää, Dr. Heidi

Tiimonen, and Dr. Tiina Ylioja. Dr. Mirka Rauniomaa is acknowledged for

revising the language of this thesis.

At the Biology Department, I have had the privilege to share an office with

great people: Emmi Alakärppä, Terttu Kämäräinen-Karppinen, Janne Koskimäki,

Dr. Mysore Tejesvi, Dr. Jaana Vuosku, and Laura Zoratti. I have really enjoyed

your company. I am also grateful for all the support that I have received from the

Plant Biology work community: Dr. Laura Jaakola, Dr. Katja Karppinen, Dr. Jana

Krajňáková, Riina Muilu-Mäkelä, Dr. Kaloian Nickolov, Johanna Pohjanen, Dr.

Marian Sarala, and Marko Suokas.

The personnel of the Biology Department at the University of Oulu are

acknowledged for their support during this process. Soile Alatalo, Marja

Nousiainen, Tuulikki Pakonen, Hannele Parkkinen, Niilo Rankka, Matti Rauman,

and Tarja Törmänen are thanked for their assistance, and especially Taina

Uusitalo, without whom the plant material would not have survived. I wish to

thank the staff of the Botanical Gardens of the University of Oulu for taking good

care of the plants at the greenhouse and Aino Hämäläinen, Tuomas Kauppila, and

Annikki Kestilä for their advices regarding the acclimation of plants and

experimental set-ups. The personnel of the Finnish Forest Research Institute

Punkaharju Research Unit are acknowledged for all their excellent work related to

8

silver birch and hybrid aspen that were utilized in the present study. Sinikka Sorsa

and the other members of the Natural Product Research Laboratory at the

University of Eastern Finland are thanked for their guidance regarding the

analyses of phenolic compounds. Irmeli Luovula and Tapio Laakso at the Vantaa

Research Unit of the Finnish Forest Research Institute are acknowledged for their

expertise on the lignin analyses.

Sincere thanks to my family and relatives for all their empathy and support.

Tytsit, former and current biology students of the University of Oulu, Otso, Mape,

and Ea, you are magnificent! Olli-Heikki has in many ways made this thesis

possible, not least by letting me to be immersed in my work. I do value your

attitude towards life, and I want to thank you for all your patience with me. Olli-

Heikki has also introduced a bunch of wonderful people to me, including his

parents and the families of his siblings. My late grandparents valued education

high, and the same is true for my parents, Säde and Jaakko, who have, however,

always encouraged me to be whatever I please. I cannot thank you enough. In

addition to being the best mam, Säde is acknowledged for language revisions and

surprise goodie post packages.

Oulu, August 2014 Suvi Sutela

9

Abbreviations

35S cauliflower mosaic virus 35S promoter

4CL 4-coumarate:CoA ligase

5-OH-G 5-hydroxy-quaiacyl

ACS 4CL-like acyl-CoA synthetase

AldOMT 5-hydroxyconiferaldehyde O-methyltransferase also COMT

ANR anthocyanidin reductase

ANS anthocyanidin synthase

Atub silver birch α-tubulin gene

BA 6-benzyladenine

BRDA breeding with rare defective alleles

C3H 4-coumarate 3-hydroxylase

C4H cinnamic acid 4-hydroxylase

CAD cinnamyl alcohol dehydrogenase

CAld5H coniferaldehyde 5-hydroxylase

CCoAOMT caffeoyl-CoA O-methyltransferase

CCR cinnamoyl-CoA reductase

cds coding sequence

CHI chalcone isomerase

CHS chalcone synthase

CoA coenzyme A

COMT caffeate/5-hydroxyferulate O-methyltransferase also AldOMT

CTs condensed tannins

DFR dihydroflavonol 4-reductase

ECM ectomycorrhizal

F3’H flavanoid 3’-hydroxylase

F3H flavanone 3-hydroxylase

F5H ferulic acid 5-hydroxylase

FLS flavonol synthase

FNR fumarate and nitrate reductase

FNS flavone synthase

G guaiacyl

GM genetically modified

GSNOR S-nitrosoglutathione reductase

H p-hydroxyphenyl

Hb hemoglobin

10

Hb1 class 1 non-symbiotic hemoglobin

Hb2 class 2 non-symbiotic hemoglobin

HCT p-hydroxycinnamoyl-CoA:shikimate p-

hydroxycinnamoyltransferase

HPLC high-performance liquid chromatography

IAA indole-3-acetic acid

LAR leucoanthocyanindin reductase

LPI leaf plastochron index

ML maximum-likelihood

NGS next-generation sequencing

NO nitric oxide

nptII neomycin phosphotransferase II gene

OxyR oxidative stress regulator

PAL phenylalanine ammonia-lyase

PKMF predictive kinetic metabolic-flux model (Wang et al. 2014a)

PP2A gene encoding protein phosphatase 2A regulatory subunit

PTGS post-transcriptional gene silencing

qPCR real-time RT-PCR

RGR the relative growth rate

RNAi RNA interference

RNA-seq RNA sequencing

S syringyl

SNP sodium nitroprusside

TrHb truncated hemoglobin

TUA hybrid aspen α-tubulin gene

UbB1 sunflower polyubiquitin promoter

UBC2 gene encoding ubiquitin-conjugating enzyme 2

VHb Vitreoscilla hemoglobin

WPM Woody Plant Medium

11

List of original publications

This thesis is based on the following publications, which are referred to in the text

by their Roman numerals:

I Sutela S, Hahl T, Tiimonen H, Aronen T, Ylioja T, Laakso T, Saranpää P, Chiang V, Julkunen-Tiitto R & Häggman H (2014) Phenolic compounds and expression of 4CL genes in silver birch clones and Pt4CL1a lines. Accepted (PLOS ONE).

II Sutela S, Niemi K , Edesi J, Laakso T, Saranpää P, Vuosku J, Mäkelä R, Tiimonen H, Chiang V, Koskimäki J, Suorsa M, Julkunen-Tiitto R & Häggman H (2009) Phenolic compounds in ectomycorrhizal interaction of lignin modified silver birches and Paxillus involutus. BMC Plant Biology 9: 124.

III Sutela S, Ylioja T, Jokipii-Lukkari S, Anttila A-K, Julkunen-Tiitto R, Niemi K, Mölläri T, Kallio PT & Häggman H (2013) The responses of Vitreoscilla hemoglobin-expressing hybrid aspen (Populus tremula × tremuloides) exposed to 24-h herbivory: expression of hemoglobin and stress-related genes in exposed and nonorthostichous leaves. Journal of Plant Research 126: 795–809.

IV Häggman H, Sutela S, Walter C & Fladung M (2014) Biosafety considerations in the context of deployment of GM trees. In: Fenning T (ed) Challenges and opportunities for the world’s forests in the 21st century. Dordrecht, Springer Verlag: 491–524.

Author contributions.

Paper Original

idea

Study design Data collection

(with help of others)

Data

analysis

Manuscript preparation

(with help of others)

I HH HH, TA, SS, TY SS, TH, HT, TA, TY SS SS*, HH, TY, RJT

II HH, KN KN, SS SS, JE SS SS*, KN, HH, RJT

III HH, TY HH, TY, SJL SS, AKA, TY SS SS*, HH, KN, SJL, TY, RJT

IV HH - - - HH, SS, CW, MF

Suvi Sutela (SS), Anna-Kaisa Anttila (AKA), Jaanika Edesi (JE), Mathias Fladung (MF), Terhi Hahl (TH),

Hely Häggman (HH), Soile Jokipii-Lukkari (SJL), Riitta Julkunen-Tiitto (RJT), Karoliina Niemi (KN),

Christian Walter (CW), Tiina Ylioja (TY), *drafted the first version of the manuscript and generated the

figures and tables

12

13

Contents

Abstract

Tiivistelmä

Acknowledgements 7 

Abbreviations 9 

List of original publications 11 

Contents 13 

1  Introduction 15 

1.1  Phenolic compounds ............................................................................... 16 

1.1.1  Phenylpropanoid pathway ............................................................ 17 

1.1.2  Monolignol biosynthetic pathway ................................................ 19 

1.1.3  4CLs function in branch point of flavonoid and

monolignol biosynthesis route ...................................................... 21 

1.1.4  COMT is required for synthesis of sinapyl alcohol ...................... 23 

1.2  Plant hemoglobins ................................................................................... 24 

1.3  Hemoglobin of Vitreoscilla, Vhb ............................................................ 25 

1.4  Tree improvement by means of genetic engineering .............................. 27 

1.4.1  Biosafety considerations of GM forest trees ................................ 28 

1.4.2  Interactions of GM forest trees ..................................................... 29 

1.5  Aims of the study .................................................................................... 30 

2  Materials and methods 31 

2.1  Plant material .......................................................................................... 31 

2.2  Regeneration of Pt4CL1a lines (I) .......................................................... 32 

2.3  Greenhouse experiments with Pt4CL1a lines (I) .................................... 32 

2.4  Wounding experiment (I) ........................................................................ 32 

2.5  Co-cultivation of PtCOMT lines and Paxillus involutus (II) .................. 33 

2.6  Herbivory exposure of VHb hybrid aspens (III) ..................................... 33 

2.7  Relative growth rate (RGR) experiments (I, III) ..................................... 34 

2.8  Southern and Northern blots (I) .............................................................. 34 

2.9  RNA isolation .......................................................................................... 36 

2.10 Isolation of silver birch 4CL and COMT genes (I, II) ............................. 36 

2.11 Relative gene expression analyses .......................................................... 36 

2.12 Phylogenetic analyses (I) ........................................................................ 37 

2.13 Microarray analyses (III) ........................................................................ 38 

2.14 Lignin analyses of silver birch roots and stems (I, II) ............................. 38 

2.15 Analyses of soluble phenolic compounds ............................................... 38 

14

2.16 Condensed tannin analyses ...................................................................... 39 

2.17 Histochemical stainings (II) .................................................................... 39 

2.18 Statistical methods .................................................................................. 39 

3  Results 41 

3.1  Characterization of Pt4CL1a silver birch lines (I) .................................. 41 

3.2  Isolation of silver birch putative COMT and 4CLs (I, II) ........................ 41 

3.3  Clonal differences in expression of Bp4CL1-4 (I) .................................. 43 

3.4  Effect of Pt4CL1 to transcript levels of Bp4CL1-4 (I) ............................ 44 

3.5  Relative expression of BpCOMT and lignin characteristics of

PtCOMT silver birch lines (II, unpublished results) ............................... 44 

3.6  Variation in phenolic compounds of silver birch clones (II) ................... 45 

3.7  Soluble compounds of silver birch lines (I, II) ........................................ 45 

3.8  Effect of wounding to 4CL genes and phenolics (I) ................................ 46 

3.9  Exposure of VHb hybrid aspen to herbivory (III, unpublished

results) ..................................................................................................... 46 

3.10 RGRs of insect larvae on Pt4CL1a and VHb leaves (I, III) .................... 49 

3.11 ECM symbiosis of PtCOMT lines and P. involutus (II).......................... 49 

4  Discussion 51 

4.1  Lignin, the target trait of PtCOMT and Pt4CL1a silver birches .............. 51 

4.1.1  Alterations in lignin-related soluble phenolic compounds ........... 53 

4.2  Positive effects of VHb limited to abiotic stress in higher plants? .......... 54 

4.3  Introduced traits had minor unintended effects ....................................... 55 

5  Conclusions and future prospects 59 

References 61 

Appendices 79 

Original publications 85 

15

1 Introduction

Conventional tree breeding programs are expensive long-term efforts that require

established mating designs, evaluation of parental trees, progeny testing and

monitoring, and assessment of economic traits (Mullin & Lee 2013, El-Kassaby

et al. 2014). Traits targeted for breeding include stem characteristics and

resistance to pathogens and pests, and these have been found to follow complex

inheritance patterns. Moreover, the breeding cycles for angiosperm and

gymnosperm species require more than seven and fifteen years, respectively

(White et al. 2007). Next-generation sequencing (NGS) technologies have made

the sequencing of large datasets more affordable and efficient and, thus, will

enable the expansion of sequence data and applications that are also utilizable for

tree-breeding practises (Harfouche et al. 2012, El-Kassaby et al. 2014, Isik 2014).

In addition to basic research where plants are frequently modified genetically in

order to uncover the functions of genes and the proteins that they encode, genetic

engineering could be used to improve and tailor trees to be more suitable for

specific purposes. The improvement of forest trees could be accelerated by

integrating modern sequence-data-dependent applications, clonal propagation and

metabolomics, to conventional tree-breeding practises, while target traits could be

introduced to trees by means of genetic engineering (Harfouche et al. 2012,

Häggman et al. 2013).

Silver birch (Betula pendula Roth) is one of the key species in boreal forest

ecosystems and, economically, the most important deciduous tree species in the

Nordic countries that is utilized by the pulp, paper, and plywood industries (e.g.

Lemmetyinen et al. 2008). The breeding efforts for silver birch started in Finland

in the late 1940s and have contributed substantially to the silver birch cultivars

that are exploited in forest plantations today (Haapanen & Mikola 2008). The

Betula genus is highly polymorphic and the species hybridizes frequently (e.g.

Järvinen et al. 2004, Lemmetyinen et al. 2008, Wang et al. 2013 and references

therein), and the genetic variation of the species is therefore wide.

The hybrid aspen (Populus tremula L. × tremuloides Michx.) is a fast

growing (Yu 2001) artificially produced F1 hybrid that is propagated clonally for

silviculture (Haapanen & Mikola 2008). The wood fibre properties of aspens are

suitable, for instance, for producing high-density paper sheets (Mansfield &

Weineisen 2007). However, in Finland the area of aspen cultivations is relatively

limited (Haapanen & Mikola 2008). The Populus species are considered the

woody model species in plant science, which has been ascertained by the first

16

whole-genome sequence of a woody perennial, Populus trichocarpa Torr. & Gray

(Tuskan et al. 2006).

1.1 Phenolic compounds

A benzene ring, with one or more hydroxyl groups, is a common structural

property of phenolic compounds. Otherwise the structures and chemical

properties of phenolics are numerous, and this enables them to have various roles

in different plant developmental and non-developmental processes. Phenolic

compounds function in defense against herbivores, pathogens, and other plants,

provide protection against physical stress, play a role in auxin transport and in

controlling pollen fertility, and attract pollinators and seed-dispersing animals

(Boeckler et al. 2011, Hichri et al. 2011, Cheynier et al. 2013). Furthermore,

lignin, the second most abundant plant polymer, has a vital role in land plants and

provides, among other things, mechanical strength and protection against

pathogens and herbivores (Xu et al. 2009, Weng & Chapple 2010, Albersheim et

al. 2011).

Phenolic compounds include phenolic glycosides, cinnamic acids, lignin,

lignans, chalcones, flavonoids (flavonols, flavones, flavanones, flavan-ols, and

isoflavonoids), anthocyanins and hydrolysable and condensed tannins (CTs or

proanthocyanidins) (Fig. 1) and are stored in different compartments of plant cells

(Wink 2010). In general, the vacuole contains many soluble phenolics, whereas

lipophilic flavonoids can also be found in the cuticle, resin ducts, and laticifers.

Lignin and insoluble conjugated tannins, in addition to some flavonoids, are

located in the cell walls (Wink 2010). However, there is great variation in the

distribution of different phenolic compounds within a plant species. For instance,

the flavonoid biosynthesis genes are absent in microalgae, whereas some genes

encoding anthocyanin-production-related enzymes are only present in dicots

(Tohge et al. 2013a, 2014). The production of phenolic compounds is,

furthermore, often restricted to a certain organ, tissue, or cell type and connected

to certain developmental stages and environmental conditions of plants (Wink

2010, Cheynier et al. 2013).

In higher plants, phenolic compounds are produced in the phenylpropanoid

route, while the A-ring of flavonoids is derived from the acetate-malonate

pathway (Wink 2010, Petersen et al. 2010, Cheynier et al. 2013). The

biosynthesis of phenolic compounds is under strict control, and phenylpropanoid

pathway regulators include R2R3-type MYB domain, basic WD repeat protein,

17

and basic helix-loop-helix transcription factors (Hichri et al. 2011, Zhao & Dixon

2011, Tohge et al. 2013a). In addition to the transcription factors, also subunits of

a co-regulatory complex, Mediator, have been identified and found essential for

the phenylpropanoid homeostasis in Arabidopsis thaliana (L.) Heynh. (Bonawitz

et al. 2012). The complex regulation of phenylpropanoid-route-associated genes

ensures the flexible production of phenolics that are required for specific plant

tissues and developmental phases and, moreover, for the modulation of phenolic

contents depending on abiotic and biotic factors.

1.1.1 Phenylpropanoid pathway

The phenylpropanoid pathway, which is the main source of phenolic compound

precursors, consists of families of enzymes located in cytosol or endoplasmic

reticulum (Wink 2010, Peterson et al. 2010, Wang et al. 2013). The carbon

backbone for phenylalanine is derived from the highly conserved shikimate route

that produces chorismate from phosphoenolpyruvate and d-erythrose 4-phosphate

(Maeda & Dudareva 2012, Tohge et al. 2013b). There are two alternative routes

for the production of phenylalanine from chorismate, of which the arogenate

pathway has been shown to function in A. thaliana, Petunia × hybrida cv

Mitchell, and Solanum lycopersicum cv M82 (Cho et al. 2007, Maeda et al. 2010,

Dal Cin et al. 2011). However, phenylalanine has been discovered to be formed in

Petunia × hybrida also from tyrosine via phenylpyruvate aminotransferase (Yoo

et al. 2013).

The phenylpropanoid pathway can be divided into a general phenylpropanoid

pathway that gives rise to cinnamoyl coenzyme A (CoA) esters, differing in the

degree of hydroxylation of the phenol ring, and a general flavonoid biosynthesis

route (Fig. 1). In addition, potential genes and enzymes responsible for the

synthesis of coumarin, flavonol, flavone, isoflavone and anthocyanin derivatives

as well as CTs have been identified, but these are not covered in this context. In

contrast to the conserved shikimate pathway, the general flavonoid biosynthesis

route shows great variation among plant lineages (Tohge et al. 2013a, 2013b,

2014). The flavonoid biosynthetic routes of A. thaliana and Zea mays L. are

among the best-defined (Nakabayashi et al. 2010, Falcone Ferreyra et al. 2012,

Saito et al. 2013, Wen et al. 2014). In woody species, the general

phenylpropanoid and flavonoid biosynthesis routes of Populus species have been

studied most extensively (Morreel et al. 2006, Tsai et al. 2006, Chen et al. 2009,

Polle et al. 2013).

18

Fig. 1. A schematic and simplified presentation of the general phenylpropanoid (with

light grey background) and general flavonoid routes giving rise to the phenolic

compound precursors in Populus according to Morreel et al. 2006 and Chen et al.

2009. PAL, phenylalanine ammonia-lyase; 4CL, a protein complex consisting of two 4-

coumarate:CoA ligase isoforms (Chen et al. 2014); C4H, of cinnamic acid 4-

hydroxylase; C4H-CH3, protein complex consisting of C4H and 4-coumarate 3-

hydroxylase (C3H) (Chen et al. 2011); HCT, p-hydroxycinnamoyl-CoA:shikimate p-

hydroxycinnamoyltransferase; CHS, chalcone synthase; CHI, chalcone isomerase;

F3'H, flavonoid 3'-hydroxylase; FNS, flavone synthase; F3H, flavanone 3-hydroxylase;

FLS, flavonol synthase; DFR, dihydroflavonol reductase; ANS, anthocyanidin

synthase; LAR, leucoanthocyanindin reductase; ANR, anthocyanidin reductase.

19

The phenylpropanoid route is initiated with the deamination of L-phenylalanine to

cinnamic acid in a reaction catalyzed by phenylalanine ammonia-lyase (PAL; EC

4.3.1.24) (Fig. 1). Cinnamic acid can subsequently be converted to cinnamoyl-

CoA by 4-coumarate:CoA ligase (4CL; 6.2.1.12) or to p-coumaric acid by

cinnamic acid 4-hydroxylase (C4H; EC 1.14.13.11). 4CL also catalyzes the

production of p-coumaroyl-CoA from p-coumaric acid. Caffeoyl-CoA is

generated from p-coumaroyl-CoA in steps catalyzed by p-hydroxycinnamoyl-

CoA shikimate p-hydroxycinnamoyltransferase (HCT, EC 2.3.1.133) and a

protein complex composed of C4H and 4-coumarate 3-hydroxylase (C3H) (C4H-

C3H, Chen et al. 2011). The cinnamic and hydroxycinnamic acids and esters

produced by the general phenylpropanoid pathway are utilized for the synthesis of

phenolic glycosides, lignin, and lignan. The chalcones, pinocembrin, naringenin,

and eriodictyol chalcone, arise from cinnamoyl-CoA, p-coumaroyl-CoA and

caffeoyl-CoA, respectively, in reactions catalyzed by chalcone synthase (CHS,

EC 2.3.1.74). In the subsequent steps of the general flavonoid biosynthetic route,

the chalcones are converted to flavanones by chalcone isomerase (CHI, EC

5.5.1.6). The flavanones are utilized as substrates for the production of flavones

and dihydroflavonols in reactions catalyzed by flavone synthase (FNS, EC

1.14.11.22) and flavanone 3-hydroxylase (F3H, EC 1.14.11.9), respectively, and

finally, the flavonols kaempferol and quercetin are produced from their

dihydroforms. In Populus, dihydroquercetin and possibly also dihydromyricetin

are utilized as precursors for synthesis of CTs and anthocyanins (Peters &

Constabel 2002).

1.1.2 Monolignol biosynthetic pathway

The monolignol biosynthetic route produces p-coumaryl, coniferyl, and sinapyl

alcohol, which give rise to p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S)

units, respectively, after conversion to phenoxy radicals and subsequent

incorporation to lignin polymer (Polle et al. 2013, Wang et al. 2013). In the

monolignol biosynthetic pathway, p-coumaric acid is converted to caffeic acid

and further to ferulic, 5-hydroxy-ferulic, and sinapic acid (Chen et al. 2013, Wang

et al. 2014a). These hydroxycinnamic acids can be utilized in the formation of

hydroxycinnamic esters and subsequently converted to aldehydes and finally to

alcohols in reactions catalyzed by 4CL, cinnamoyl-CoA reductase (CCR, EC

1.2.1.44), and cinnamyl alcohol dehydrogenases (CADs, EC 1.1.1.195),

respectively (Fig. 2).

20

Fig. 2. A schematic presentation of the principal monolignol biosynthesis route of

P. trichocarpa according to Chen et al. 2013, Wang et al. 2014a and Suzuki & Suzuki

2014. 4CL, a protein complex consisting of two 4-coumarate:CoA ligase isoforms

(Chen et al. 2014); HCT, p-hydroxycinnamoyl-CoA:shikimate p-hydroxycinnamoyl-

transferase; C4H-C3H, protein complex consisting of cinnamic acid 4-hydroxylase

(C4H) and 4-coumarate 3-hydroxylase (C3H); CCoAOMT, caffeoyl-CoA O-methyltrans-

ferase; CCR, cinnamoyl-CoA reductase; AldOMT, 5-hydroxyconiferaldehyde O-methyl-

transferase; CAld5H, coniferaldehyde 5-hydroxylase; CAD, cinnamyl alcohol

dehydrogenase.

The 5-hydroxyconiferaldehyde O-methyltransferase (AldOMT, EC 2.1.1.68) is

able to methylate the C-3 and C-5 positions of esters, aldehydes, and alcohols

derived from caffeic and 5-hydroxyferulic acids utilizing S-adenosyl methionine.

In the monolignol route, the C-3 position is methylated prior to the hydroxylation

of the C-5 position catalyzed by the coniferaldehyde 5-hydroxylase (CAld5H),

which can subsequently be methylated by AldOMT (Wang et al. 2014a).

However, the production of hydroxycinnamoyl alcohols, mostly coniferyl and

sinapyl alcohols in angiosperms, does not require all possible reactions, and in

21

this way, the metabolic grid provides flexibility to the monolignol biosynthesis

(Wang. et al. 2014).

In the monolignol biosynthetic route of P. trichocarpa (Fig. 2), p-coumaric

acid is converted to ester by a heterotetrameric protein complex composed of

subunits of two 4CLs (Chen et al. 2014). Two isoforms of HCT produce p-

coumaroyl shikimic acid followed by the hydroxylation of C-3 position by the

C4H-C3H complex (Chen et al. 2011). In the next step, caffeoyl shikimic acid is

converted to caffeoyl-CoA by two HCT isoforms. To a lesser extent, caffeoyl-

CoA is also produced from p-coumaric acid via caffeic acid in reactions catalyzed

by C4H-C3H and 4CL complexes (Fig. 2). Three isoforms of caffeoyl-CoA O-

methyltransferase (CCoAOMTs, EC 2.1.1.104) catalyze the incorporation of

methyl group to the C-3 position of the phenol ring producing feruloyl-CoA,

which is then reduced by CCR. The resulting coniferaldehyde is also produced

from caffeoyl-CoA in a reaction catalyzed by CCR and AldOMT. Coniferyl

alcohol is generated from coniferaldehyde by two CAD isoforms. Sinapyl alcohol

can also be produced from coniferaldehyde in three steps catalyzed by two

CAld5H isoforms, AldOMT, and two CAD isoforms. Alternatively, sinapyl

alcohol is produced from coniferyl alcohol by hydroxylation of the C-5 position

and the subsequent methylation catalyzed by two CAld5H isoforms and AldOMT,

respectively.

1.1.3 4CLs function in branch point of flavonoid and monolignol biosynthesis route

4CLs catalyze the last step of the general phenylpropanoid route and, in this way,

direct the metabolic flux to the flavonoid and/or lignin biosynthesis route. The

angiosperm 4CLs are categorized to classes I and II (Ehlting et al. 1999)

generally, the function of the former has been connected to lignin biosynthesis

and the function of the latter to both flavonoid and lignin biosynthesis. The small

4CL multigene families consist of two to nine members, depending on the plant

species, and are absent in microalgae (Tohge et al. 2014). Additionally, 4CL-like

genes and 4CLs converting sinapate (Schneider et al. 2003, Hamberger &

Hahlbrock 2004) are sometimes included into the 4CL families. 4CL isoforms

show distinct expression patterns according to plant parts and tissues, and the

isoforms have also been found to prefer different substrates (e.g. Lee et al. 1996,

Hu et al. 1998, Ehlting et al. 1999, Harding et al. 2002, Kumar & Ellis 2003,

Endler et al. 2008, Gui et al. 2011, Sun et al. 2013). Furthermore, the substrate

22

inhibition of 4CL shows isoform dependency (Harding et al. 2002, Chen et al.

2013, Wang et al. 2014a).

The P. trichocarpa genome contains seventeen 4CL and 4CL-like genes, of

which five have been suggested to belong to class I and II on the basis of

phylogenetic analyses (de Azevedo Souza et al. 2008). The expression of P.

trichocarpa 4CLs showed that three genes had xylem-specific expression patterns

(Shi et al. 2010), and recently Chen et al. (2013) confirmed the involvement of

two functionally distinct 4CL isoforms in the biosynthesis of monolignols.

Moreover, it was found that the two 4CLs, Ptr4CL3 and Ptr4CL5, catalyzed all

the necessary steps of the principal monolignol biosynthetic route and formed a

heterotetrameric protein complex (Chen et al. 2014). The A. thaliana genome

contains four genes encoding 4CLs (Ehlting et al. 1999, Raes et al. 2003,

Schneider et al. 2003, Vanholme et al. 2012) and more than ten 4CL-like genes

(Schneider et al. 2003, de Azevedo Souza et al. 2008). Of the four genes, one

seems to be specific to the monolignol route (Vanholme et al. 2012) and another

converts sinapate (Schneider et al. 2003, Hamberger & Hahlbrock 2004). In

Physcomitrella patens (Hedw.) B.S.G. (Silber et al. 2008), Oryza sativa L. (de

Azevedo Souza et al. 2008, Gui et al. 2011), Rubus idaeus L. cv. Meeker (Kumar

& Ellis 2003), and Selaginella moellendorffii (Tohge et al. 2014) the 4CL families

consist of four, five, three, and two genes, respectively.

The reduction of 4CL expression, with antisense and RNAi approaches, has

resulted in decreased lignin content and increased levels of cell-wall-bound or

soluble phenolic compounds, indicating suppressed supply of metabolites to the

monolignol route (Kajita et al. 1996, Lee et al. 1997, Hu et al. 1999, Jia et al.

2004, Wagner et al. 2009, Voelker et al. 2010, Gui et al. 2011, Tian et al. 2013;

Appendix 1). The effects of 4CL-downregulation on lignin composition vary:

reduced levels of G (Lee et al. 1997, Gui et al. 2011, Wagner et al. 2009, Tian et

al. 2013), H (Gui et al. 2011, Tian et al. 2013), and/or S units (Kajita et al. 1996,

Gui et al. 2011) have been reported, but so have increased levels of H (Kajita et

al. 1996) and S (Tian et al. 2013) units. The lignin content of Pinus radiata D.

Don and P. tremula × alba L. has been decreased up to 50% by supressing the

4CL expression. However, the lines with drastic changes in lignin quantity also

showed reduced growth (Wagner et al. 2009, Voelker et al. 2010) and

abnormalities in wood structure (Wagner et al. 2009, Kitin et al. 2010) as well as

stiffness (Voelker et al. 2011a) causing enhanced xylem drought susceptibility in

poplar lines (Voelker et al. 2011b).

23

1.1.4 COMT is required for synthesis of sinapyl alcohol

The function of AldCOMT (hereafter COMT) has puzzled researches for a few

decades. The kinetic preferences of COMTs (Parvathi et al. 2001, Zubieta et al.

2002, Wang et al. 2014a) and substrate inhibition (Li et al. 2000, Wang et al.

2014a) indicated that COMT catalyzes methylation at the C-5 position of the

phenol ring (Maury et al. 1999, Parvathi et al. 2001, Zubieta et al. 2002, Wang et

al. 2014a). In contrast to the 4CL gene family, the number of COMT orthologs is

low (Tohge et al. 2013a, 2014). This does not, however, suggest that the function

of COMTs would be more unambiguous as in addition to a range of

hydroxycinnamate derivatives (Li et al. 2000, Wang et al. 2014a), A. thaliana

COMT has been shown to methylate flavonol (Muzac et al. 2000, Do et al. 2007,

Tohge et al. 2007). Moreover, COMT-like sequences are abundant and have likely

risen from independent duplication events (Li et al. 2006; Tsai et al. 2006). In

mosses and monocotyledons, the number of COMTs ranges between one and two,

and of the dicotyledon species, only Malus domestica Borkh. and Glycine max

(L.) Merr. contain more than two COMT sequences (Tohge et al. 2013a).

Of the 23 COMT-like genes of P. trichocarpa, one (PtrCOMT2) was found to

be expressed extensively in the differentiating xylem and also, but to a lesser

extent, in the differentiating phloem and leaves (Shi et al. 2010). Most of the

studied COMTs were not expressed, and four COMTs, in addition to PtrCOMT2,

showed expression mostly in other tissues than xylem. Further studies on

PtrCOMT2 confirmed that the enzyme is efficiently able to methylate the C-3 and

C-5 positions of caffealdehyde, 5-hydroxyconiferaldehyde, caffeyl alcohol, and 5-

hydroxyconiferyl alcohol. Moreover, it was shown that substrate self-inhibition

regulated the activity of the enzyme so that aldehydes and alcohols were preferred

over hydroxycinnamic acids (Wang et al. 2014a). It is noteworthy that in A.

thaliana, too, only one COMT (At5g54160) was connected to the O-methylation

of C-5 positions (Humphreys et al. 1999, Goujun et al. 2003). However, A.

thaliana COMT functions also in the production of soluble phenolic compounds,

namely sinapoyl malate and isorhamnetin (Muzac et al. 2000, Do et al. 2007,

Tohge et al. 2007, Nakatsubo et al. 2008, Vanholme et al. 2012).

COMT-deficient plants have now been studied for more than 20 years

(Appendix 2). First studies with Nicotiana tabacum L. expressing heterologous

COMT in antisense orientation showed that COMT contributed to lignin

biosynthesis, although the effects detected in lignin content and composition

varied (Dwivedi et al. 1994, Ni et al. 1994). Subsequent studies on COMT-

24

deficient N. tabacum (Atanassova et al. 1995) and P. tremula × alba (Van

Doorsselaere et al. 1995) showed that the downregulation of COMT did not alter

the lignin content but increased the G and reduced the S monomer composition

and caused the incorporation of 5-hydroxy-quaiacyl (5-OH-G) units to lignin

polymer. The importance of COMT in the production of S unit monomers was

further demonstrated with COMT-deficient P. tremuloides (Boerjan et al. 1997,

Tsai et al. 1998), P. tremula × alba (Lapierre et al. 1999, Jouanin et al. 2000,

Pilate et al. 2002), and Z. mays (Piquemal et al. 2002). Comparable results were

later obtained with Betula pendula Roth (Aronen et al. 2003, Tiimonen et al.

2005, 2008) and Leucaena leucocephala (Lam.) de Wit (Rastogi & Dwivedi

2006), whereas in Medicago sativa L. and Z. mays the suppression of COMT

caused, in addition to reduced S units, a reduction in lignin content that was up to

20% and 45%, respectively (Marita et al. 2003, Guillaumie et al. 2008).

Moreover, 5-OH-G units were revealed to rise from 5-hydroxyconiferyl alcohol

through benzodioxane structures (Marita et al. 2001, Ralph et al. 2001a, 2001b,

Morreel et al. 2004) by cross coupling with O-4-positions of S and G units of

lignin and, subsequently, with coniferyl or sinapyl alcohol or with 5-OH-G (Lu et

al. 2010).

1.2 Plant hemoglobins

Plant 3-on-3 and 2-on-2 (truncated) hemoglobins (Hbs) have been suggested to be

of bacterial origin and to be transferred horizontally to an ancestor shared by

algae and land plants or to an ancestor of all eukaryotes (Vinogradov et al. 2007,

Vázquez-Limón et al. 2012). The symbiotic 3-on-3 Hbs were discovered first, and

they have been shown to function in controlling the oxygen level in legume

nodules (Appleby 1984), whereas the non-symbiotic 3-on-3 and truncated Hbs

probably play a role in nitric oxide (NO) metabolism (Hill 2012). Several studies

have shown that the expression of non-symbiotic 3-on-3 and truncated Hbs is

modulated in response to abiotic and biotic stress and also in developmental

processes connected to NO-mediated signalling pathways (Hill 2012, París et al.

2013).

The plant 3-on-3 Hbs are grouped into two classes which have presumably

derived from a common Hb gene that originates in a eukaryotic ancestor (Gupta

et al. 2011). However, the oxygen-binding properties of class 1 and 2 Hbs differ

(Smagghe et al. 2009). Class I Hbs may function in NO scavenging in plants

(Jokipii-Lukkari 2011, Hill et al. 2012), which was first suggested after exposing

25

plants to oxygen-limited conditions (Dordas et al. 2003, Seregélys et al. 2004,

Perazzolli et al. 2006). Their potential role in NO metabolism was further

supported by alteration in NO emissions when class 1 genes were downregulated

(Perazzolli et al. 2004, Hebelstrup et al. 2006, Shimoda et al. 2009, Hebelstrup et

al. 2012). Recently, Jokipii-Lukkari (2011) demonstrated that, with flavin-domain

containing reductase, class I Hb is able to rescue NO-sensitive yeast mutant in

vivo from the detrimental effects of NO. Class 2 Hbs, in turn, possibly play a role

in oxygen sensing or transport (Smagghe et al. 2009, Hill et al. 2012). The

distribution of class 2 Hbs in plant species is more restricted than that of class 1

Hbs. However, in A. thaliana, class 2 Hbs possess important functions as the

silencing of both class 1 and class 2 Hbs led to the death of A. thaliana seedlings

at an early vegetative stage (Hebelstrup et al. 2006). In A. thaliana, both Hbs

contribute to the emission rates of NO and ethylene (Hebelstrup et al. 2012) and

are found to reduce nitrite to NO in vitro (Tiso et al. 2012). Studies on A.

thaliana, O. sativa, and Z. mays have also shown that class 2 Hbs are induced by

cytokinins (Hunt et al. 2001), cold (Trevaskis et al. 1997), nitrate, and NO

(Ohwaki et al. 2005) and are expressed together with class 1 Hbs in the

embryonic tissues of O. sativa (Lira-Ruan et al. 2011).

Recently, it was shown that Chlamydomonas reinhardtii PA Dong. cells

require truncated hemoglobin (TrHb) in an NO-dependent signalling pathway in

order to survive in hypoxic conditions (Hemschemeier et al. 2013). Similarly,

experimental data obtained from higher plants indicated that TrHbs could

participate in NO metabolism by acting as oxygen transporters. The NO-donor,

sodium nitroprusside (SNP), can induce the expression of genes encoding TrHbs

in roots (Jokipii-Lukkari 2011, Kim et al. 2013) as well as the accumulation of

TrHb in stems (Dumont et al. 2014). TrHb has also been connected to plant

developmental processes (Jokipii et al. 2008, Hossain et al. 2010) and stress

tolerance (Hossain et al. 2011) together with NO.

1.3 Hemoglobin of Vitreoscilla, Vhb

The single-domain globin of Vitreoscilla bacterium, VHb, has been used in a

variety of microorganisms to enhance their growth under oxygen-limited

conditions and to improve the production of primary and secondary metabolites

(Jokipii-Lukkari et al. 2009, Frey et al. 2011). The advantageous effects of VHb

rise potentially from the two proposed functions, oxygen transport and protection

of cellular respiration from NO (Frey et al. 2011), which would be beneficial if an

26

aerobic organism inhabited an oxygen-poor environment with decaying material.

Indeed, the strictly aerobic Vitreoscilla sp. (Strohl 2005) was found in still ditches

and ponds as well as in dung that were rich in organic material (Pringsheim

1951), and it was shown to produce VHb under hypoxic conditions (Boerman and

Webster 1982, Wakabayashi et al. 1986, Dikshit et al. 1990).

The vhb promoter functions in several microorganisms, including Escherichia

coli (Migula) Castellani & Chalmers (Frey and Kallio 2003). Hence, studies

conducted on E. coli have significantly contributed to research on VHb because

there is only a limited number of methods applicable to Vitreoscilla. The vhb

promoter contains potential binding sites for putative homologs of E. coli

transcription regulators ArcA (Yang et al. 2005) and FNR (fumarate and nitrate

reductase, Tsai et al. 1995), controlling the transcriptome response to the switch

from aerobic to anaerobic conditions (Frey et al. 2011), catabolite repressor

protein (Khosla & Bailey 1989), and OxyR (oxidative stress regulator, Anand et

al. 2010) that is activated by superoxide. These recognition sites overlap partially,

suggesting possible interaction/competition of transcription factors that could

enable the multiple functions of VHb at different prevailing oxygen conditions, as

well as responses to oxidative and nitrosative stress (Frey et al. 2011).

By utilizing E. coli cells, it was discovered that heterologous VHb expression

is able to promote ATP production and respiratory activity in hypoxic conditions

(Kallio et al. 1994, Tsai et al. 1996), aided by oxygen delivery of VHb to the

terminal cytochrome (Kallio et al. 1994, Ramandeep et al. 2001, Park et al. 2002,

Chi et al. 2009). The E. coli cells expressing vhb and reductase-domain were also

found to have improved nitrosative stress tolerance (Frey et al. 2002, Kaur et al.

2002), which could be mediated via FNR as it has been shown to activate the

flavohemoglobin-encoding gene of E. coli, for instance (Frey et al. 2011). The

FNR was suggested also to play a role in improved oxidative-stress tolerance

because OxyR is unable to bind to the vhb promoter in the presence of FNR

(Anand et al. 2010). Moreover, the activation of antioxidant genes by OxyR has

been found to require oxidization, which could be facilitated by VHb. As OxyR is

believed to act as a repressor in vhb transcription, VHb might in this way also

regulate its own transcription (Anand et al. 2010). In general, the beneficial

effects of VHb in microorganisms are probably due to improvements in aerobic

respiration under oxygen-limited conditions since the expression of globins could

prevent NO production (Frey et al. 2011, Stark et al. 2011). The heterologous

expression of the VHb-encoding gene, vhb, has also enhanced growth (Jokipii-

27

Lukkari et al. 2009) as well as tolerance of nitrosative (Frey et al. 2004, Wang et

al. 2009) and oxidative (Wang et al. 2009) stress in certain plant species.

1.4 Tree improvement by means of genetic engineering

By using genetic engineering, recombinant and endogenous genes can be

expressed or downregulated/silenced at certain developmental stage, in different

tissues or by specific environmental cues (Hernandez-Garcia & Finer 2014).

Depending on the sequence similarities of introduced and endogenous genes, the

heterologous expression of a transgene may cause suppression of one or more

endogenous genes, regardless of transgene orientation (sense/antisense).

Suppression is mediated via RNA interference (RNAi), an antiviral defense

system that has been utilized successfully in the downregulation/silencing of

endogenous plant genes. Besides the antisense strategy, RNAi-mediated post-

transcriptional gene silencing (PTGS) can be achieved with hairpin RNAi vectors,

artificial micro RNAs, and virus-induced gene silencing, while small RNAs can

be exploited in transcriptional gene silencing (Busov et al. 2010).

The transformation of forest tree species has mostly been achieved using

biolistic transformation (also called particle or microprojectile bombardment) and

Agrobacterium tumefaciens (Smith & Townsend) Conn bacteria. The

disadvantage of using Agrobacterium and biolistic bombardment in gene transfer,

i.e. the randomness of transgene integration, could be overcome in the future with

new techniques. These techniques include artificial nucleases including zinc

finger, transcription activator-like or LAGLIDADG homing nucleases (Curtin et

al. 2012), site-specific recombination systems, such as Cre/lox and FLP/FRT, that

have already been shown to be applicable to P. tremula × tremuloides (Fladung et

al. 2010, Fladung & Becker 2010), and the combination of short palindromic

repeat (CRISPR) technology with the RNA-guided nucleases, called the CRISPR-

Cas9 system (Sander & Joung 2014).

The economically significant characteristics of trees, the properties of wood

and the vigour of plant growth, have successfully been modified with genetic

engineering (e.g. Vanholme et al. 2008, Dubouzet et al. 2013, Häggman et al.

2013, Séquin et al. 2014). Other traits introduced to forest tree species include

insect, disease, and herbicide resistance and abiotic stress tolerance (e.g. Hinchee

et al. 2009, Chen & Polle 2010, Osakabe et al. 2011, Häggman et al. 2013,

Séquin et al. 2014). Wood lignin, varying naturally from 15% to 40% among

species, has been studied intensively: it is considered the main factor limiting the

28

conversion of biomass to pulp, paper, and biofuel production (Vanholme et al.

2010, Häggman et al. 2013). Furthermore, lignin could potentially be utilized as a

resource for high-value products, such as carbon fiber, thermoplastic elastomers,

and polymeric membranes, if the processing technologies and lignin chemistry

were improved (Ragauskas et al. 2014).

1.4.1 Biosafety considerations of GM forest trees

The main concerns of genetically modified (GM) forest trees relate to the

dispersal of recombinant DNA to native or managed ecosystems and to any

unintended impact on non-target organisms and ecosystems. To tackle these

questions, more than 700 confined field trials, in addition to numerous

greenhouse experiments, have been carried out both by commercial companies

and the public sector on a variety of introduced traits (Walter et al. 2010,

Häggman et al. 2013). In general, the unintended effects detected in GM forest

tree lines or in monitored non-target organisms have not been significant or

unambiguously explained by the introduced trait/gene construct. There are only a

few exceptions, which will be discussed in section 1.4.2. Most of the pleiotropic

effects found in GM tree lines rise from the effects of the transgene integration

site and, thus, could usually be detected within the early phases of the

characterization process of GM tree lines that was taking place in in vitro and/or

greenhouse conditions. Moreover, transgene expression has been found to remain

stable in natural environments in trees that have passed preceding investigations

in in vitro and/or greenhouse conditions (Ahuja 2009, Walter et al. 2010). In

addition, no horizontal gene transfer has been detected in field trials (Zhang et al.

2005, Walter et al. 2010), and several strategies have been developed to avoid

vertical gene transfer (Vining et al. 2012, Sang et al. 2013). It should be noted,

however, that the duration of most studies conducted on GM trees has been

limited when taking into consideration the longevity of forest trees. Nevertheless,

the validity of the results should not be underestimated because the gene

transformation techniques per se have proved to be trustworthy. However, GM

trees should be followed under natural conditions in long-term field trials to

uncover the impacts and stability of introduced traits.

Concerns have risen about the possible impacts of the use of clonal tree lines

with narrow genetic background and about the potential local loss of natural

biodiversity if GM forest trees are grown commercially. The cultivation of GM

forest trees will most likely be first realized in plantation forests, which in many

29

respects resemble agricultural crop fields that are managed with well-established

practices of plantation forestry (Häggman et al. 2013). Hence, the benefits and

disadvantages of the utilization of GM forest trees should be debated in relation to

semi-natural or plantation forests.

1.4.2 Interactions of GM forest trees

As a key species in forests, trees interact with a myriad of organisms. Generally,

studies conducted to reveal the performance and possible unintended effects of

GM trees to their natural habitats and to non-target organisms are diverse and

reflect the various interactions of forest tree species. In field studies conducted on

GM forest tree species with altered disease resistance and wood properties as well

as with lines expressing marker genes, no significant changes have been found

that would be due to genetic modification (Halpin et al. 2007, Walter et al. 2010)

in relation to non-target organisms, including fungi (Stefani and Hamelin 2010,

Lamarche et al. 2011), bacteria (Pilate et al. 2002, Vauramo et al. 2006),

microfauna of rhizosphere (Vauramo et al. 2006), and arthropods (Pilate et al.

2002, Vihervuori et al. 2008, Axelsson et al. 2010, Schnitzler et al. 2010).

However, unintended changes have been detected in field trials in the richness of

species and structure of insect communities in GM Populus lines (Gao et al.

2003, 2006, Jiang et al. 2009, Axelsson et al. 2011, Zhang et al. 2011) and in the

rhizosphere microbial community of GM Picea glauca (Moench) Voss (LeBlanc

et al. 2007) producing δ-endotoxin. The lines were targeted to deter coleopteran-,

dipteran-, and lepidopteran-caused damage by expressing the δ-endotoxin of

Bacillus thuringiensis (Schnepf et al. 1998). The detected indirect effects of tree

lines producing δ-endotoxin were probably a consequence of the target effects: a

reduction in the target species caused alterations in the richness of non-target

species and, in this way, affected the dynamics of the insect populations that were

monitored.

30

1.5 Aims of the study

The aim of the present study was to determine the potential influence of A)

transgenes involved in the phenylpropanoid and monolignol biosynthetic

pathway, and B) vhb gene of Vitreoscilla to the transcription of related

endogenous genes and to the production of soluble phenolic compounds. These

topics were studied in relation to ectomycorrhizal (ECM) symbiosis or herbivory

in order to determine whether genetic engineering affects these interactions. The

specific aims of studies I–IV were

1. to characterize the silver birch (Betula pendula Roth) lines expressing

heterologous Pt4CL1 gene in antisense orientation (I),

2. to study the effects of PtCOMT1 and Pt4CL1 on the expression of

endogenous COMT and 4CL genes and lignin characteristics of silver birch

(I, II),

3. to study the soluble phenolic compounds of GM silver birch and hybrid aspen

(Populus tremula L. × tremuloides Michx.) lines (I, II, III),

4. to study the ectomycorrhizal symbiosis of silver birch PtCOMT lines and

Paxillus involutus in greenhouse conditions (II),

5. to examine the potential effects of heterologous Pt4CL1 and vhb on leaf

quality by assaying the relative growth of lepidopteran larvae (I, III),

6. to analyze the herbivory response of VHb hybrid aspen lines (III), and

7. to review the current state of knowledge on the environmental effects of GM

forest trees with a focus on field trials (IV).

31

2 Materials and methods

2.1 Plant material

The silver birch clones A, E5382, E5396, and R and hybrid aspen line V617 were

used in the present study. Clones A and R originated from crosses between trees

E1970 and E1980 and V5411 and V5402, respectively, whereas clones E5382 and

E5396 originated from elite trees cultivated in the clonal archive in Punkaharju

(61°49' N; 29°18' E). The hybrid aspen line originated from a plus tree grown in

eastern Finland (61°48' N; 29°22' E). The gene constructs pRT9/35S-PtCOMT

and pRT9/UbB1-PtCOMT containing the COMT gene of P. tremuloides

(PtCOMT, U13171) under the control of either cauliflower mosaic virus 35S

promoter (35S) or sunflower polyubiquitin (UbB1) promoter were utilized in the

generation of lines A23, A44, and A65 (Aronen et al. 2003, Table 2). In vitro stem

pieces of clones A, E5382, and E5396 were transformed with pRT99/35S-

Pt4CL1-a containing the 4CL gene of P. tremuloides (Pt4CL1, AF041049) in

antisense orientation under control of 35S promoter. Lines A1, A2, A5, E5382/3,

and E5396/4 were produced with biolistic transformation (I, Table 2). Line V617

was transformed using A. tumefaciens with pBVHB plasmid containing the vhb of

Vitreoscilla under control of 35S promoter (Farrés & Kallio 2002) which gave rise

to lines V617/45 and V617/60 (Häggman et al. 2003).

Table 2. Transgenic lines and non-transgenic clones studied.

Clone and species Line Construct Transgene

Orientation

Paper

A silver birch Pt4CL1a A1 pRT99/35S-Pt4CL1-a antisense I

Pt4CL1a A2 pRT99/35S-Pt4CL1-a antisense I

Pt4CL1a A5 pRT99/35S-Pt4CL1-a antisense I

PtCOMT A23 pRT9/35S-PtCOMT sense II

PtCOMT A44 pRT9/35S-PtCOMT sense II

PtCOMT A65 pRT9/UbB1-PtCOMT sense II

E5382 silver birch Pt4CL1a E5382/3 pRT99/35S-Pt4CL1-a antisense I

E5396 silver birch nptII

(Pt4CL1a)

E5396/4 pRT99/35S-Pt4CL1-a antisense I

V617 hybrid aspen VHb V617/45 pBVHb sense III

VHb V617/60 pBVHb sense III

32

2.2 Regeneration of Pt4CL1a lines (I)

After the transformation of the silver birch stem pieces with pRT99/35S-Pt4CL1-

a, the material was first cultivated on growth regulator free Woody Plant Medium

(WPM, Lloyd & McCown 1980) for a week, after which potentially transgenic

cells were selected by supplementing growth media with kanamycin. Lines A1,

A2, A5, E5382/3, and E5396/4 were regenerated as well as rooted on WPM as

described in Aronen et al. (2003).

2.3 Greenhouse experiments with Pt4CL1a lines (I)

The greenhouse experiments were conducted at a greenhouse of the Finnish

Forest Research Institute Punkaharju Unit (61°48' N; 29°24' E).

In the first greenhouse experiment, 51 plants of clones A and E5382 and lines

A1, A2, and A5 and 43 plants of line E5382/3 were placed in randomly assigned

design into four replicates and surrounded by additional birches that were not

used as experimental material (Fig. 3G). The experiment started in mid-June, and

growth and phenology were followed during the first and second growing

seasons, after which the samples were collected for analyses. During the third

growing season, nine individual silver birches per line/clone were cultivated in a

different experimental set-up.

The second greenhouse experiment started at the beginning of August and

consisted of clone E5396 and line E5396/4. The 60 potted plants of clone E5396

and line E5396/4 were organized into parallel, randomly assigned design

surrounded by additional birches. The growth parameters were determined at the

end of August and at the beginning and end of the following growing season, after

which the samples were collected for analyses.

2.4 Wounding experiment (I)

The silver birch clones A, R, E5382, and E5396 and lines A1, A5, E5382/3, and

E5396/4 were multiplied on WPM with 2.2 µM 6-benzyladenine (BA) and 2.8

µM indole-3-acetic acid (IAA) and supplemented with 200 mg/L kanamycin in

the case of transgenic lines. The rooting was conducted on growth regulator free

WPM, and plants were acclimatized for five weeks before transplanting and

placing to a randomly assigned experimental design in a greenhouse at the

Botanical Gardens of the University of Oulu in April. Plants of similar heights

33

were selected for the control and wounding treatments, and around 20% of the

leaf margins of two leaves representing the leaf plastochron index (LPI) of LPI 2

to LPI 4 (the first fully expanded leaf was LP1 0) were crushed with pliers.

Samples were collected immediately after wounding, and 1, 3, 12, and 24 h and 3,

7, and 21 d after treatment. Unwounded plants of all clones and lines were grown

in a different experimental set-up at the end of the growing season, and their

growth was determined at the beginning of August.

2.5 Co-cultivation of PtCOMT lines and Paxillus involutus (II)

Silver birches A, A23, A44, and A65 were multiplied on WPM supplemented with

2.2 μM BA and 2.8 μM IAA and rooted for six weeks on growth regulator free

WPM. The root systems were placed on Petri dishes, of 14 cm in diameter, filled

with sterile peat-vermiculite (1:10; v/v) mixture moistened with Melin-Norkrans

nutrient solution 2 (Marx 1969) without glucose. The inoculation was conducted

by placing three agar plugs containing mycelium of Paxillus involutus (Batsch)

Fr. (strain ATCC 200175), cultivated on Hagem media (Modess 1941) in darkness

at 21°C for two weeks, in proximity to roots. The inoculated and control cultures

supplemented with three plain agar plugs were cultivated for eight weeks in a

greenhouse at the Botanical Gardens of the University of Oulu (65°03'N, 25°27'E)

under a 16-h photoperiod at 20°C in a randomly assigned design with biological

replicates of 38 (Fig. 3A). At harvest, the growth characteristics and ECM status

of roots were monitored (Fig. 3B, C).

2.6 Herbivory exposure of VHb hybrid aspens (III)

The progeny of Conistra vaccinii L. originated from adult moths collected from

Salix caprea L. (61°48'N, 29°18'E) and reared on mixed diet of Betula species, P.

tremula, and hybrid aspen. Two leaves of six V617 and V617/45 plants (n = 3)

grown 1.5 months in a greenhouse at the Botanical Gardens of the University of

Oulu were positioned inside a cloth bag (Fig. 3D). One active last instar of C.

vaccinii was placed on a leaf inside a cloth bag for 24 hours, whereas in the

control treatment the bags were kept empty, and the leaves were subsequently

collected and their areas documented. In addition, the same experimental set-up

was conducted with plants of the same lines but only with one cloth bag and C.

vaccinii larvae (in herbivory treatment). Leaves positioned in the bags as well as

34

adjacent leaves, positioned above and below the bagged leaves, were collected for

analyses from both set-ups.

2.7 Relative growth rate (RGR) experiments (I, III)

Polyphagous larvae of moth species Aethalura punctulata Denis & Schiff.,

Cleora cinctaria Denis & Schiff., Epirrita autumnata Bork., and Orthosia gothica

L. were used to determine the quality of the Pt4CL1a lines whereas Ectropis

crepuscularia Denis & Schiff., O. gothica, and Orgyia antiqua L. were used in

assaying leaves of the VHb lines. The no-choice tests were conducted at

greenhouses of the Finnish Forest Research Institute Punkaharju Unit and the

Botanical Gardens of the University of Oulu (Fig. 3E, F). Adult moths of A.

punctulata, C. cinctaria, and O. gothica and larvae of E. autumnata were

collected from south-eastern Finland. The larval progeny of moths and E.

autumnata larvae reared on diet consisting of Betula, hybrid aspen, and P.

tremula. The full-grown leaves of clones and lines were offered to active larvae,

which were weighed at the start and end of the experiment, conducted as

described in papers (I, III).

2.8 Southern and Northern blots (I)

The Southern and Northern analyses were conducted in order to confirm the

integration of Pt4CL1 and nptII to the genome and their expression, respectively.

The genomic DNA was isolated from silver birch clones and lines transformed

with pRT99/35S-Pt4CL1-a construct using the procedure described in Valjakka et

al. (2000) and, subsequently, DNA samples were restricted with BamHI and XbaI.

All samples were probed as described in Aronen et al. (2003) for the presence of

35S-Pt4CL1 and nptII, expect for clone E5396 and line E5396/4, which were only

probed for nptII. The total RNA was isolated from leaves, phloem, and

developing xylem of clones A and E5382 and lines A1, A2, A5, and E5382/3

using the modified method of Chang et al. (1993) described in Aronen et al. 2003.

The Northern plots were carried out as described in Aronen et al. (2003) using

specific probes for Pt4CL1 and nptII. The expression of Pt4CL1 and nptII in line

E5396/4 was examined by real-time RT-PCR and standard RT-PCRs runs as

described in sections 2.10 and 2.11.

35

Fig. 3. (I) The start of the co-cultivation of PtCOMT lines and P. involutus in

greenhouse (A) and example of the silver birch at the end of 8-week experiment (B, C);

(II) hybrid aspen with two leaves positioned inside cloth bags (D); (I, III) the set-up of

the RGR experiment (E, F); (I) One block of greenhouse experiment 1 with Pt4CL1a

lines at the beginning of experiment (G) with A1, A2, A5, A, E5382/3, and E5382 plants

(from left to right, H); example of the growth difference between line E5396/4 (I) and

clone E5396 (J) grown for three months under normal greenhouse conditions in the

Botanical Gardens of the University of Oulu.

36

2.9 RNA isolation

The RNA was extracted from leaves and developing xylem using the protocol of

Jaakola et al. (2001), and the GeneJET™ Plant RNA Purification Mini Kit

(Thermo Scientific) and E.Z.N.A.® Plant RNA Kit (Omega Bio-Tek Inc) were

used in RNA isolation from stem and root material, respectively. The RNA quality

was determined with agarose gel electrophoresis and ND-1000 UV-Vis

spectrophotometer (NanoDrop Technologies). SuperScript II and III (Invitrogen)

and RevertAid™ Premium (Thermo Scientific) were used in the synthesis of

cDNA with anchored oligo-dT primers. The genomic DNA was either omitted by

gel purification of cDNAs (II, III, Jaakola et al. 2004) or by treating RNAs with

DNase I (Thermo Scientific) and checking the quality of RNAs again prior to

conversion to cDNA (I).

2.10 Isolation of silver birch 4CL and COMT genes (I, II)

The primers for silver birch 4CL genes (I) were designed based on the expressed

sequence tags obtained from Helariutta and Kauppinen (University of Helsinki,

Finland). Partially degenerated primers were designed for COMT on the basis of

the sequences in GenBank of NCBI database (II). The Blast tools available at

NCBI, ClustalW alignment tools (EMBL-EBI) and Primer3 (Koressaar & Remm

2007, Untergrasser et al. 2012) were used in the primer design process. The

amplification was conducted in standard PCR runs with DyNAzymeTM II or EXT

or Phusion® (Thermo Scientific), whereas the 3’- and 5’-ends were in some cases

obtained using the SMART™ RACE cDNA Amplification Kit (Clontech

Laboratories) performing the cDNA conversion in accordance with the

manufacturer’s instructions. The fragments were gel purified and subcloned using

the TOPA TA Cloning® Kit (Invitrogen). Three to five plasmids were sequenced

per putative fragment using the BigDye Terminator v3.1 Cycle Sequencing Kit

(Applied Biosystems) and the ABI PRISM 377 DNA sequencer (Perkin-Elmer).

2.11 Relative gene expression analyses

The RNA extraction and purification as well as the cDNA conversion were

conducted as described in the previous paragraph and publications (I–III), and the

relative gene expression was determined using real-time RT-PCR (from now on

qPCR) run with LightCycler® 480 (Roche Applied Science). The design of

37

primers for COMT, 4CLs, and protein phosphatase 2A regulatory subunit (PP2A)

was based on the sequence data obtained in this study, whereas the design of

primers for silver birch α-tubulin (Atub, AJ279695), PAL2 (AJ278116), PtCOMT,

Pt4CL1, and for hybrid aspen non-symbiotic class 1 Hb (PttHb1, EF180083),

vegetative bark storage protein (DN494285), copper chaperone (BU832694),

CuZn-superoxide dismutase (AJ643591), cytochrome P450 (CK112685), nitrate

reductase (BI071715), PAL1 (AF480619), plasma membrane intrinsic protein 1

(BU829500), pop3-/SP1-like protein (CF231524), and ubiquitin-conjugating

enzyme 2 (UBC2, XM_002325824) genes was based on the sequences in

GenBank. Primers presented in Jokipii et al. (2008) were used for the qPCR runs

of hybrid aspen truncated Hb (PttTrHb), α-tubulin (TUA), and vhb genes. The

runs contained LightCycler 480 SYBR green 1 Master mix (Roche, Meylan,

France), 0.5 µM each primer and cDNa template. The specificity of primers was

confirmed with melting curve analysis in LightCycler 480 software (Roche),

agarose gel electrophoresis and sequencing the purified qPCR products using

BigDye chemistry and the ABI PRISM 377 DNA. PP2A and Atub and UBC2 and

TUA were used as reference genes for silver birch and hybrid aspen, respectively.

2.12 Phylogenetic analyses (I)

Initial phylogenetic tree for COMT was generated using COMT-like sequences

presented in Appendix 3. The COMT-like unigenes (KA202529, KA202985,

KA213727, KA218668, KA232971, KA234110, KA242367, KA244536,

KA246429, KA261311, KA265181) produced of RNA sequencing (RNA-seq) of

Betula platyphylla Sukaczev developing xylem (Wang et al. 2014b) were

searched against the preliminary assembly of Betula nana L. genome (Wang et al.

2013, http://birchgenome.org). All phylogenetic analyses were conducted with

MEGA6 (Tamura et al. 2013). The COMT and 4CL cds were aligned using

MUSCLE (Edgar 2004), and the Model Selection feature was used to evaluate the

substitution models for maximum-likelihood (ML) algorithm (Felsenstein 1981).

The initial analysis for COMT was conducted by using predicted amino acid

sequences and the LG model (Le & Gascuel 2008) with gamma distributed with

invariant sites model. Based on an initial ML tree, sequences potentially involved

in the monolignol and phenylpropanoid routes were selected for generation of

second ML together with Selaginella moellendorffii COMT (GQ166949)

(Appendix 3). The ML trees were reconstructed using General time reversible

(Tavaré 1986) and Tamura 3-parameter substitution model (Tamura 1992) with

38

the gamma distributed with invariant sites model for COMT and 4CL,

respectively. All codons were included, and partial deletion was used to positions

containing missing data or gaps (Hall 2013). The confidence of ML trees was

evaluated with the bootstrap method using 500 replicates, and bootstrap values ≥

70% were considered reliable (Soltis and Soltis 2003).

2.13 Microarray analyses (III)

The SuperScript III (Invitrogen) was used to synthesize cDNA of total RNA in

accordance with instructions by the manufacturer of reverse transcriptase and the

3DNA Array 50 Expression Array Detection Kit (Genisphere Inc.) on which also

the conduction of hybridizations and post-washings was based. The cDNA

samples representing the control and herbivory treatment of either hybrid aspen

line V617 or V617/45 of bagged or adjacent leaves were hybridized to each

microarray containing salt stress-related oligos of Populus euphratica (Brosché et

al. 2005). The cDNA slides were produced by the Finnish DNA Microarray

Centre (University of Turku, Finland). The scanning of slides at 633 and 543 nm

was conducted using ScanArray® Gx PLUS (PerkinElmer) with 98% laser

power, resolution of 10 µm and varying PMT values. The fixed-circle method was

used in the quantification of spots, and the total-algorithm in the normalization

(ScanArray Express software, PerkinElmer). The statistical processing of the data

was conducted separately on the hybrid aspen lines and different leaf samples by

using identical procedures as described in paper III.

2.14 Lignin analyses of silver birch roots and stems (I, II)

The lignin contents were determined with the acetyl bromide method described in

Koutaniemi et al. (2007) and the Klason method as described in Tiimonen et al.

(2005). The S and G monomers of lignin were evaluated with the modified

method of thioacidolysis (Rolando et al. 1992) using the procedure and the

chromatographic conditions described in Tiimonen et al. (2005).

2.15 Analyses of soluble phenolic compounds

The samples were homogenized in methanol using either Ultra-Turrax T8 (IKA-

Labortechnik) or Precellys®24 (Bertin Technologies) homogenizers. The

extraction was conducted by repeating the homogenization followed by

39

incubation and centrifugation steps and combining the extracts and evaporating

methanol. The dried samples were dissolved in water:methanol (1:1, v/v) and

analysed with Agilent 1100 Series HPLC or HPLC Value System (Agilent

Technologies) using a diode array detector DAD. The hypersil ODS (4.6 mm × 60

mm, 3 μm particles, Hewlett-Packard) and Zorbax RRHD SB-C18 (2.1 mm × 50

mm, 1.8 μm, Agilent Technologies) columns with gradient elution were used as

described in Peltonen et al. (2005) and Häggman et al. (2003) in separation of

silver birch and hybrid aspen soluble compounds, respectively. A set of silver

birch extracts was also run with HP 1100 Series LC/MSD (Hewlett-Packard)

using conditions described in Peltonen et al. (2005). The identification and

quantification of compounds were based on their retention times, spectral

characteristics, HPLC-MS (API-ES, positive ions), and commercial standards

(Julkunen-Tiitto & Sorsa 2001).

2.16 Condensed tannin analyses

The extract run with HPLC was used in the determination of soluble CTs,

whereas the insoluble CTs were determined from the dried extract residues. The

quantification was conducted with modified acid butanol assay (Porter et al.

1986) using purified CT from B. nana as a standard.

2.17 Histochemical stainings (II)

The root tips were fixed with 4% paraformaldehyde in 0.1 M phosphate-buffered

saline, dehydrated in graded ethanol, treated with 2-methyl-2-propanol and

embedded into paraffin (Merck). The sections were stained with 0.05% toluidine

blue O solution. The hand-cut cross-sections were made of fresh roots and stems

and stained using phloroglucinol-HCl and Mäule protocols described in Guo et al.

(2001) with an additional potassium iodide treatment. The samples were

examined with a Nikon Optiphot 2 microscope and imaged using Nikon Coolpix

950 or Infinity1-3C camera (Lumenera Corporatiom) with the IMT iSolution Lite

imaging program (IMT i-Solution Inc.).

2.18 Statistical methods

R software (Ihaka and Gentleman 1996) with a graphical user interface, the R

Commander (Fox 2005), was utilized in statistical analyses except for the

40

microarray data for which the analyses were run with limma: linear models for

microarray data package (Smyth 2005) following the instructions for two-colour

data (III). In general, parametric tests (one-way, multi-way Anova, two-sample t

test, and Welch Two Sample t-test) were preferred if the data met the required

assumptions, and therefore some variables were also transformed. The Kruskal-

Wallis and Wilcoxon rank sum test were also utilized. The Bonferroni and the

false discovery rate (Benjamini & Hochberg 1995) correction were applied when

necessary and, when used, the Anova was complimented with Tukey Contrasts. In

addition, Fisher's exact test (II), Pearson's Chi-squared test (I), and Spearman’s

rank correlation rho (III) were used in the study.

41

3 Results

3.1 Characterization of Pt4CL1a silver birch lines (I)

Pt4CL1 was introduced by means of biolistic transformation to two silver birch

clones, A and E5382. Three lines were regenerated from clone A, of which two,

A1 and A5, were confirmed to contain and express nptII and Pt4CL1, based on

the Northern and Southern blots. Lines E5382/3 and E5396/4 were regenerated

from clones E5382 and E5396. However, only E5382/3 contained and expressed

both nptII and Pt4CL1. The growth characteristics and phenology of the lines

were examined under greenhouse conditions, and some differences were found

among non-transgenic clones and lines during the first growing seasons.

Concerning the Pt4CL1a lines, only the growth of line A1 differed significantly

from the control clone at the end of the third growing season. The phenotype of

line E5396/4 expressing only nptII showed suppressed elongation growth and

more frequent lateral shoots (Fig. 3I & J). The Klason lignin and lignin monomer

composition were determined from two-year-old stems. The lignin contents were

similar in the non-transgenic clones and Pt4CL1a lines, whereas the S/G ratio of

line A1 had increased significantly.

3.2 Isolation of silver birch putative COMT and 4CLs (I, II)

A putative full-length coding sequence (cds) of COMT (BpCOMT, FJ667539) and

four putative full-length cds of 4CL-like genes (Bp4CL1-4, KM099195-8) were

obtained from the stems of silver birch clone A. The BpCOMT and Bp4CL1 were

identical at the nucleotide sequence level with the COMT (KC292201) and 4CL1

(AY792353) mRNA sequences of B. platyphylla. The putative COMT and 4CL

genes were found in the preliminary assembly of B. nana genome (Wang et al.

2013, http://birchgenome.org). The full-length cds of COMT and Bp4CL3 were

99% similar to the nucleotide sequences and 98% similar to the predicted amino

acid sequences of B. nana (scaffold3434 and scaffold5102, respectively). Of the

other 4CL sequences, up to 440 bp (either from the 3’- or 5’-end) of

corresponding B. nana sequences was located to another scaffold. The partial B.

nana cds of 1412 bp was identical with Bp4CL1, whereas the B. nana sequences

of 1286 and 1509 bp in length showed 99% and 98% similarity with Bp4CL2 and

Bp4CL4 when compared to nucleotide and predicted amino acid sequences,

42

respectively. The P. tremuloides PtCOMT and Pt4CL1 showed similarity of 72%

and 73% at the nucleotide level with BpCOMT and Bp4CL1, respectively.

The preliminary assembly of B. nana genome (Wang et al. 2013,

http://birchgenome.org) and B. platyphylla unigenes produced from RNA-seq of

developing xylem (Wang et al. 2014b) were utilized to determine the putative

COMT-like sequences of Betula species. In addition to B. nana scaffold3434

showing high similarity to BpCOMT, scaffolds 17988, 7599, 1560, and 20949

contained homologs of B. platyphylla unigenes. The full-length cds were

generated based on homology searches, and the cds of scaffolds 1560 and 20949

were found to be identical. The phylogenetic analysis (Appendix 4, unpublished

data) conducted with predicted amino acid sequences suggested that only cds

retrieved from scaffold3434 of B. nana belonged to the same group as the

monolignol and phenylpropanoid biosynthesis pathway related COMTs including

P. trichocarpa PtrCOMT2, P. tremuloides PtCOMT, and A. thaliana At5g54160.

The phylogenetic analysis of these cds potentially involved in the monolignol and

phenylpropanoid route suggested the separation of monocot and dicot COMTs

into two main branches and grouping of Asterids, Rosales, Betulaceae,

Brassicaceae, Fabaceae, and Salicaceae COMT cds into separate clades (Fig. 4,

unpublished data).

Based on the phylogenetic analysis of 4CL and 4CL-like cds, two of the silver

birch 4CL sequences, Bp4CL1 and Bp4CL4, belonged to class I, whereas one 4CL

sequence (Bp4CL2) belonged to class II 4CLs (I). Bp4CL1, Bp4CL2, and Bp4CL4

showed closest relationship with the 4CL sequences of Rubus idaeus L.

(AF239685-7). The phylogenetic analysis suggested that Bp4CL3 could be

putative 4CL-like acyl-CoA synthetase (ACS) (de Azevedo Souza et al. 2008) by

grouping Bp4CL3 together with the putative ACS of P. trichocarpa, A. thaliana,

and O. sativa. The phylogenetic tree of 4CL produced using predicted amino acid

sequences did not differ substantially from the ones generated with cds (data not

shown).

43

Fig. 4. The phylogenetic ML tree, reconstructed using cds of COMT genes.

3.3 Clonal differences in expression of Bp4CL1-4 (I)

The expression of the putative 4CL genes and 4CL-like gene showed clone

dependency. Of the four examined clones, the expression patterns of clone A were

most distinct: the Bp4CL1 and Bp4CL2 expression was found to be higher,

whereas the transcript levels of the 4CL-like gene Bp4CL3 were low in both

leaves and stems. The transcript levels of the putative 4CL genes and 4CL-like

gene were most similar in clones E5382 and E5396.

44

3.4 Effect of Pt4CL1 to transcript levels of Bp4CL1-4 (I)

The expression of Pt4CL1 in antisense orientation generally reduced the transcript

levels of Bp4CL1 and Bp4CL2 genes in stems of Pt4CL1a lines A1 and A5.

However, with both reference genes that were used, significant reduction was

found only in the Bp4CL1 expression in line A1. In line E5382/3, the transcript

levels were similar or increased in comparison with clone E5382. The Pt4CL1

expression was found to be highest in line A1.

3.5 Relative expression of BpCOMT and lignin characteristics of PtCOMT silver birch lines (II, unpublished results)

The expression of putative BpCOMT and lignin characteristics were determined

from mycorrhizal and non-inoculated PtCOMT lines. The mycorrhizal status had

no effects on either the BpCOMT transcript levels of roots or the composition or

quantity of root or stem lignin. When the BpCOMT expression levels were

statistically examined by combining together the values of non-inoculated and

mycorrhizal roots, the BpCOMT levels showed significant reduction in the

PtCOMT lines A23 and A44 in comparison with clone A (Table 3, unpublished

results). Similarly, a significant reduction was detected in the S/G ratio of lignin

when the A23 and A44 root and stem samples were compared to clone A, without

taking the effect of treatment into account. In addition, the stem lignin percentage

that was determined using the acetyl bromide method was found to differ between

clone A and PtCOMT line A23 as well as A44 (Table 3, unpublished results).

Table 3. The expression of BpCOMT gene and lignin characteristics of roots and

stems derived from original publication (I) by combining the values of non-inoculated

and mycorrhizal roots of clone A and PtCOMT lines A23, A44 and A65 together.

Stem Root

Clone

/Line

Lignin (%) S/G ratio

Lignin (%) S/G ratio

BpCOMT

(Atub1)

BpCOMT

(PP2A1)

A 22.4 ± 1.49 2.16 ± 0.23 27.3 ± 3.0 1.52 ± 0.19 1.1 ± 0.52 2.91 ± 1.84

A23 20.4 ± 0.82* 1.48 ± 0.43* 25.7 ± 1.82 0.42 ± 0.39*** 0.32 ± 0.21** 0.66 ± 0.43*

A44 19.6 ± 1.08** 0.58 ± 0.43** 26.7 ± 1.78 0.48 ± 0.05** 0.41 ± 0.2** 1.1 ± 0.99*

A65 21.0 ± 0.94 2.55 ± 0.12* 27.1 ± 2.17 1.95 ± 0.19** 0.86 ± 0.46 2.9 ± 1.84

Values represent means ± SD (n = 6–9), *P < 0.05, **P < 0.005, ***P < 0.0005 according to one-way

Anova with Tukey Contrasts or by pairwise comparisons of clone A with PtCOMT line using two-sample t

test, Welch Two Sample t-test or Wilcoxon rank sum test, 1the housekeeping gene used as a reference.

45

3.6 Variation in phenolic compounds of silver birch clones (II)

The soluble phenolic compounds were more alike in stems than in leaves of silver

birch clones A, E5382, E5396, and R. In stems the concentration of p-OH-

cinnamic acid glucoside was higher in clones E5396 and A in comparison with

clones E5382 and R. The stems of clone A did not contain p-OH-cinnamic acid

derivative, which was found in all other clones. In addition, the leaves of clone A

differed from other clones in the contents of kaempferol, myricetin, and quercetin

3-acetyl-glucosides, as well as quercetin derivative and myricetin 3-arabinoside,

the levels of which, if found, were low in clones E5382, E5396, and R. The leaves

of clone A also contained more soluble CTs and flavonoids compared to other

clones, whereas in clone R the cinnamic and p-OH-cinnamic acid derivative

contents were found to be higher in comparison with other clones.

3.7 Soluble compounds of silver birch lines (I, II)

From Pt4CL1a silver birches, the contents of soluble phenolic compounds and

CTs were determined from stems and leaves (I). Concerning individual

compounds, differences were found in concentrations of flavonoids (e.g. catechin,

myricetin, and quercetin derivatives), phenolic glycosides, and cinnamic acid

derivatives between non-transgenic clones (A, E5382, and E5396) and individual

Pt4CL1a lines. However, the altered compounds varied among Pt4CL1a lines and

neolignan represented the only compound, the concentration of which differed in

two Pt4CL1a lines, in comparison with a non-transgenic clone/s. In line A1, with

increased S/G ratio, only the content of p-OH-cinnamic acid glucoside, from all

the cinnamic and p-OH-cinnamic acid derivatives analyzed, was found to be

altered.

Of the identified soluble phenolic compounds in leaves, roots, and stems, the

contents of flavonoids, phenolic glycosides, and CTs were similar in clone A and

PtCOMT lines (II). In general, the total levels of cinnamic and p-OH-cinnamic

acid derivatives were lower in both PtCOMT lines A23 and A44. However, the

differences were significant in comparison with clone A only in the case of

cinnamic acid derivatives of stems. In addition, line A23 had significantly

reduced contents of p-OH-cinnamic acid derivatives in leaves.

46

3.8 Effect of wounding to 4CL genes and phenolics (I)

The expression of 4CL genes and 4CL-like gene was examined in relation to the

mechanical wounding of leaves. The responses were similar among clones and

lines. The Bp4CL1 transcript levels increased 3 h and to some extent also 24 h

after treatment, whereas Bp4CL2 transcript levels showed induction mostly 24 h

after the wounding treatment. Bp4CL3 and Bp4CL4 showed no response to the

wounding.

In general, the mechanical wounding did not result in drastic alterations in the

soluble phenolic or CT content of leaves determined 21 d after treatment. As an

overall trend, the quercetin, kaempferol as well as total flavonoid concentrations

decreased in all silver birches, whereas the content of soluble CTs increased in all

lines and clones E5382, E5396, and R. Otherwise the responses of clones E5382

and E5396 and corresponding lines were more similar in comparison with clones

R and A as well as lines A1 and A5.

3.9 Exposure of VHb hybrid aspen to herbivory (III, unpublished

results)

A cDNA microarray containing approximately 6,340 stress-related oligos was

used in determining the transcriptome response of non-transgenic V617 and VHb

hybrid aspen line V617/45 after exposing leaves to larvae of C. vaccinii for 24 h.

In both lines, the transcriptome changes were similar, even though, according to

the statistical analysis, the non-transgenic line V617 had more significant up- and

downregulation of genes. In the adjacent leaves positioned above and below the

consumed leaves, the transcriptome changes were relatively mild, as based on the

hierarchical clustering the below and above positioned leaves grouped together

within line. In general, the C. vaccinii exposure caused transcriptome changes that

are typical of the Populus species. These are required for resource reallocation

and production of compounds, such as Kunitz-type trypsin protease inhibitors,

pop3 peptides, and CTs, which are potentially harmful/poisonous to insect

herbivores. Additionally, the insect herbivory increased the transcripts of genes

encoding vegetative bark storage proteins in the leaves of the VHb line, whereas

in the non-transgenic line, the transcript levels decreased as a result of herbivory.

The microarray analyses were verified with qPCR, which was also used in

the determination of PttHb1, PttTrHb, and vhb transcript levels. The relative

expression of PttHb1 decreased somewhat in the leaves exposed to C. vaccinii as

47

well as the leaves positioned below the consumed ones. However, the reduction

was significant only in the herbivory-exposed leaves of VHb line V617/45, which

also showed significant increase in the transcript levels of PttTrHb. A similar

trend was observed in the other herbivory exposed leaves of VHb and also in the

leaves of the non-transgenic line V617. The vhb expression was unresponsive to

the herbivory treatment.

Originally, the soluble phenolic compounds and CT were determined from

the same plants/leaves that were used in the transcriptome analyses and showed

no response to herbivory and, moreover, only few significant alterations were

found between lines. To reveal whether treatment and line or treatment, line, and

leaf position/ontogeny (LPI 1–3 or LPI 4–6) affected the concentration of

compounds, leaves from backup experiment were included in the statistical

analyses. In the herbivory exposed leaves, the concentration of flavonoids was

dependent on treatment × line (Table 4, Fig. 5, unpublished results), whereas in

intact adjacent leaves only line and leaf ontogeny had an effect on flavonoids

(Table 5). In addition, the concentration of phenolic glycosides was reduced as a

response to herbivory in all leaves (Table 4 & 5). The line and the leaf ontogeny

affected the concentrations of cinnamic and p-OH-cinnamic acid derivatives in

intact leaves (Table 5). The interaction of treatment, lines, and leaf position were

insignificant in adjacent leaves (data not shown).

Table 4. Anova test of differences in phenolic compounds of leaves positioned to

cloth bags between treatment (control, herbivory) and line (V617, V617/45).

Treatment Line Treatment/Line Residuals

Sum Sq Df F Sum Sq Df F Sum Sq Df F Sum Sq Df

Flavonoids1 0.47 1 6.36* 0.10 1 1.34 0.36 1 4.84* 1.18 16

Kaempferol der 0.02 1 0.04 0.002 1 0.005 1.97 1 3.98 7.93 16

Quercetin der1 0.68 1 6.08* 0.21 1 1.89 0.30 1 2.65 1.79 16

Cinnamic acid der2 0.01 1 0.08 0.50 1 3.54 0.08 1 0.55 2.25 16

p-OH-cinnamic acid der3 W = 71 0.123 W = 74 0.075

Phenolic glycosides3 W = 84 0.009 W = 28 0.105

Condensed Tannins 602.00 1 0.78 16.90 1 0.02 38.40 1 0.05 12297.40 16

1Log+1 transformed values; 2Sqr transformed values; 3Analyse conducted with Wilcoxon rank sum test, W

and P values given; der = derivatives; *P < 0.05, **P < 0.01, ***P < 0.001

48

Fig. 5. Soluble phenolic compounds and CTs in leaves (mean + SE, n = 5) of lines V617

and V617/45. Leaves above (LPI 1–3) and below (LPI 4–6) of leaves positioned to cloth

bags were empty (C) or with larvae of C. vaccinii. der = derivatives.

Table 5. Anova test of differences in phenolic compounds of intact leaves positioned

above or below the bagged leaves between treatment (control, herbivory), line (V617,

V617/45) and leaf position (above: LPI 1–3, below: LPI 4–6).

Treatment Line Leaf position Residuals

Sum Sq Df F Sum Sq Df F Sum Sq Df F Sum Sq Df

Flavonoids1 0.20 1 2.39 0.79 1 9.70** 0.55 1 6.73* 2.62 32

Kaempferol der 0.004 1 0.007 0.008 1 0.015 0.000 1 0.001 16.48 32

Quercetin der1 0.24 1 2.77 0.99 1 11.25** 0.99 1 11.29** 2.81 32

Cinnamic acid der 0.12 1 3.44 0.24 1 6.93* 0.31 1 8.72** 1.12 32

p-OH-cinnamic acid der1 0.04 1 1.86 0.19 1 6.58* 0.02 1 0.73 0.9 32

Phenolic glycosides1 0.48 1 6.4* 0.04 1 0.51 0.007 1 0.099 2.38 32

Condensed Tannins1 1.3 1 2.5 0.009 1 0.017 0.35 1 0.68 16.59 32

1Log+1 transformed values; der = derivatives;*P < 0.05, **P < 0.01, ***P < 0.001

49

3.10 RGRs of insect larvae on Pt4CL1a and VHb leaves (I, III)

The larvae of A. punctulata, C. cinctaria, O. antiqua, and E. autumnata fed on the

leaves of clones A and E5282 and Pt4CL1a lines A1, A2, A5, and E5382/4 were

found to have similar RGRs (I). Similarly, the larvae of O. gothica, O. antiqua,

and E. crepuscularia RGRs did not differ from the non-transgenic line V617 and

VHb lines V617/45 or V617/60 (III).

3.11 ECM symbiosis of PtCOMT lines and P. involutus (II)

The number of ECMs root tips per root system and the morphology of

mycorrhizal root tips were similar after eight-week co-cultivation of clone A and

PtCOMT silver birches with P. involutus under greenhouse conditions. The ECM

plants did not differ from the non-inoculated ones in lignin quantity, S/G ratio or

phenolic compound concentrations.

50

51

4 Discussion

4.1 Lignin, the target trait of PtCOMT and Pt4CL1a silver birches

In order to determine the effect of PtCOMT and Pt4CL1 on the expression of

silver birch endogenous COMT and 4CL genes, one BpCOMT and three putative

full-length Bp4CL cds were isolated. In most dicots, the number of COMTs

involved in the biosynthesis of lignin monomers is between one and two (Tohge

et al. 2013a), and it seems that one COMT may be sufficient for the C-5 O-

methylation in P. trichocarpa (Wang et al. 2014a) and A. thaliana (Humphreys et

al. 1999, Goujun et al. 2003, Vanholme et al. 2012). This might also be the case

in Betula sp., based on homology searches in the preliminary assembly of the B.

nana genome (Wang et al. 2013, http://birchgenome.org) and unigenes produced

of RNAseq of B. platyphylla developing xylem (Wang et al. 2014b) and,

moreover, on the phylogenetic analysis conducted in the present study. 4CLs

specific to the monolignol route have been identified from A. thaliana (Vanholme

et al. 2012), P. tremuloides (Hu et al. 1998, 1999), and P. trichocarpa (Chen et

al. 2013, 2014). Based on phylogenetic analyses, these particular 4CLs of P.

tremuloides and P. trichocarpa were suggested to belong to the same group as

Bp4CL1, together with other 4CLs of class I. Bp4CL4 could also belong to the

same class I, whereas Bp4CL2 was grouped together with 4CLs of class II with

possible functions in the biosynthesis of both lignin and flavonoids (Hu et al.

1998, Ehlting et al. 1999, Kumar and Ellis 2003, Soltani et al. 2006, Pan et al.

2013).

Lignin quantity and composition have previously been determined from

PtCOMT lines A23, A44, and A65 grown under in vitro and greenhouse

conditions (Aronen et al. 2003, Tiimonen et al. 2005, 2008, Tiimonen 2007). Line

A65, having PtCOMT under control of UbB1, has been found to be similar with

clone A regarding its lignin characteristics. This is probably because of unchanged

BpCOMT levels and low PtCOMT levels in comparison with lines A23 and A44,

as revealed in the present study. UbB1 is considered a strong constitutive

promoter (Hernandez-Garcia & Finer 2014), and the weak PtCOMT expression

may therefore be related to unfavourable integration sites of transgenes. The

lignin of PtCOMT lines A23 and A44 was found to contain 5-OH-G units

(Aronen et al. 2003) and a reduced rate of S/G monomers, which was also

demonstrated in this study, in addition to the modest reduction in lignin content.

52

Of the Pt4CL1a lines, only line A1 showed changed monomeric composition,

whereas the two other lines expressing Pt4CL1 showed unaltered lignin

composition and quantity. The S/G ratio of monomers was increased in line A1.

In addition, line A1 had the highest transcript levels of Pt4CL1. Furthermore, the

transcript levels of endogenous Bp4CL1 showed greatest reduction in line A1.

The altered lignin composition and transcript levels of Bp4CL1 indicate that the

Pt4CL1 levels in lines A5 and E5382/3 were insufficient to repress the

endogenous expression of Bp4CL1 and, moreover, that Bp4CL1 may function in

the monolignol biosynthesis pathway of silver birch.

The detected differences in the lignin of PtCOMT lines A23 and A44 are in

accordance with previous literature on COMT-deficient plants (Appendix 2),

whereas the decrease in lignin content, accomplished with several 4CL-deficient

plants (Appendix 1), was not achieved with Pt4CL1a silver birches. Based on the

predictive kinetic metabolic-flux (PKMF) model, the reduction of COMT and

4CL should exceed 90% of the abundance of the enzyme in the wild type in order

to cause changes to lignin (Wang et al. 2014a). In the present study, the relative

expression of BpCOMT in lines A23 and A44 was between 20% and 40% of the

expression of non-transgenic clone A. In Pt4CL1a line A1, the decrease in

Bp4CL1 expression was approximately half of the expression of clone A.

Therefore, it is possible that the homology between heterologous and endogenous

genes was insufficient to cause necessary reduction in the abundance of 4CL. It

should be noted, however, that a reduction in the lignin content of tobacco has

been achieved by using a 4CL sequence (in antisense orientation) originating

from Pinus massoniana Lamb. (Huan et al. 2012).

The monolignol biosynthesis route of P. trichocarpa utilizes 21 enzymes, of

which CCR and COMT have been suggested to be the only ones without isoforms

simultaneously functioning in the metabolic fluxes (Wang et al. 2014a). The

successful suppression of COMT should reduce the overall flux of metabolites in

contrast to suppression of 4CL, which based on the PKMF model, should activate

an alternative pathway, where ferulic acid is produced via 3-hydroxylation of p-

coumaric acid and 3-methylation of caffeic acid. Moreover, if the abundance of

4CL, among other early enzymes of the monolignol route, is drastically reduced,

the metabolic flux to coniferyl alcohol is affected to a greater extent, resulting in

an increased S/G ratio (Wang et al. 2014a). This was also observed in the

Pt4CL1a silver birch line A1. In P. trichocarpa, two 4CL isoforms operate in the

ligation of CoAs, which have been shown to differ in kinetics and regulation

(Chen et al. 2013). Hence, the modification of lignin by altering COMT

53

abundance may be more straightforward than that of 4CL. Moreover, it was

recently demonstrated that the two 4CL isoforms form a heterotetrameric protein

complex that affects the direction and rate of metabolite fluxes in the monolignol

biosynthesis route of P. trichocarpa (Chen et al. 2014).

4.1.1 Alterations in lignin-related soluble phenolic compounds

The suppression of 4CL has been found to increase the contents of cinnamic acid

derivatives (Kajita et al. 1996, Lee et al. 1997, Hu et al. 1999, Jia et al. 2004,

Wagner et al. 2009, Voelker et al. 2010, Gui et al. 2011, Tian et al. 2013). Wang

et al. (2014) explain this by their PKMF model by the utilization of the alternative

pathway leading to the production of ferulic acid, which would facilitate the

accumulation of both cell wall bound and soluble derivatives. Indeed, ferulic acid

and its derivatives may contribute to the brownish stem coloration observed in

lignin-modified plants (Piquemal et al. 1998, Leplé et al. 2007). By contrast,

COMT-deficient plants, characterized by the presence of 5-OH-G units, have not

been found to accumulate other cinnamic acid derivatives than the ones derived

from coniferaldehyde and/or coniferyl alcohols. Moreover, the reddish wood

phenotype observed in stems of some COMT-deficient plants can originate in

coniferaldehyde (Tsai et al. 1998). In Z. mays, the suppression of COMT led to

reduced levels of p-coumaric acids (Piquemal et al. 2002), whereas P. tremula ×

alba was found to produce benzodioxane moieties containing soluble phenolics

derived from 5-hydroxyconiferyl alcohol or 5-hydroxyconiferaldehyde (Morreel

et al. 2004). That is why, in the present study, the possible alterations in the

cinnamic acid derivatives of PtCOMT and Pt4CL1a silver birch lines were of

particular interest. In the PtCOMT and Pt4CL1a silver birch lines showing altered

lignin composition, the changes in cinnamic acid derivatives were relatively

moderate, and the suppression of BpCOMT and Bp4CLs mainly caused reductions

in the contents of cinnamic acids. Moreover, two Pt4CL1a lines were found to

have reduced content of neolignan, which is a derivative of coniferyl alcohol

(Satake et al. 2013). The detected reduction of neolignan is in accordance with

the increased S/G ratios that probably resulted from the higher flux of cinnamic

acids to sinapyl alcohol. In the present study, the analyses of phenolic compounds

were, however, limited to methanol-soluble compounds, and thus the possible

changes in the cell-wall-bound phenolics were not determined further than being

non-soluble CTs. In an A. thaliana COMT mutant, reduction was found in

isorhamnetin glycosides (Vanholme et al. 2012). In the PtCOMT lines

54

investigated in the present study, no changes were detected in the content of

quercetin derivatives or isorhamnetin 3-glucoside of stems, possibly indicating

that O-methylation of quercetin in silver birch is catalyzed by another OMT than

BpCOMT.

4.2 Positive effects of VHb limited to abiotic stress in higher plants?

Nitric oxide (NO) has been found to act as a signal molecule in diverse

physiological processes of plants, which suggests that Hbs also play a role in

signalling pathways that involve phytohormones (Hill 2012). Non-transgenic and

VHb hybrid aspens were studied in relation to herbivory as NO functions in the

herbivory defense pathways (Wünsche et al. 2011) and in the wound-healing

process together with H2O2 (París et al. 2007, Arasimowicz et al. 2009). The role

of NO was detected by studying the S-nitrosoglutathione reductase (GSNOR) that

is responsible for reducing S-nitrosoglutathione. S-nitrosoglutathione is a stable

storage form and major pool of plant cellular NO. The GSNOR is involved in the

accumulation of ethylene and jasmonic acid as well as in certain methyl-

jasmonate-triggered herbivory defenses (Wünsche et al. 2011).

The PttHb1 and PttTrHb of hybrid aspen potentially function in the

modulation of NO levels (Jokipii et al. 2008, Jokipii-Lukkari 2011, Dumont et al.

2014). Treating hybrid aspen with SNP increased the accumulation of PttTrHb in

stems (Dumont et al. 2014) and the transcript levels of PttHb1 and PttTrHb in

roots (Jokipii-Lukkari 2011). Similarly, in interaction with ECM fungi, both genes

were induced in roots, possibly relating to root development, i.e. a process

involving NO (Jokipii et al. 2008). However, Jokipii-Lukkari (2011) showed that

only PttHb1 with applicable reductase is able to rescue NO-sensitive yeast mutant

from the detrimental effects of NO. In the present study, a 24-hour exposure of

leaves to insect herbivore increased the transcript levels of PttTrHb and decreased

those of PttHb1. However, the change was significant only in the VHb line as

variation within treatments in the non-transgenic line was substantial. One

explanation to the different expressions of Hb genes could be that, in the

herbivory response, NO scavenging is not favored but that the signaling pathway

leading to transcriptome changes requires PttTrHb.

The VHb and non-transgenic hybrid aspens responded very similarly to the

herbivory, even though the responses were more drastic in the non-transgenic line

based on fold change values. In another study on the biotic interactions of VHb

hybrid aspen lines, no molecular or biological process could be detected that

55

would have been altered in all studied VHb lines when they were challenged with

pathogenic fungus (Häggman et al. 2011). It is therefore possible that the positive

effects of VHb are restricted to abiotic stress tolerance. In the present study, the

total contents of flavonoids, cinnamic acid derivatives and CTs were at a higher

level in almost all intact leaves of the VHb line in comparison with the non-

transgenic line. This may indicate that the energy status of the VHb lines had

improved, as suggested by Häggman et al. (2003). However, the 24-hour

herbivory treatment of the present study did not induce accumulation of soluble

phenolic compounds or CTs but reduced their concentrations in the VHb line in

particular. Yet, it is also possible that the timing of the sampling was not optimal,

as the induced defenses of Populus species have usually been detected, for

instance, in the accumulation of CTs (Peters & Constabel 2002). In general,

heterologous VHb expression has led to varying effects on growth, stress

tolerance, and metabolite production in plants (Jokipii-Lukkari et al. 2009, Stark

et al. 2011), which reflects the differences of bacterial and eukaryotic cells, the

complexity of higher plants, and, moreover, the absence of a native promoter of

vhb and/or the absence of effective reductase acquired for VHb-directed NO

scavenging.

4.3 Introduced traits had minor unintended effects

Of the five silver birch lines regenerated after biolistic bombardment with

pRT99/35S-Pt4CL1-a, three lines were confirmed to express Pt4CL1a in leaves

and stems. All regenerated lines were grown under greenhouse conditions, and the

lines not expressing Pt4CL1a were found to have the greatest alterations in

growth, morphology and phenology, which is typical to transgenic plants where

the transgene integration is disadvantageously located in the genome. The growth

of two Pt4CL1a lines was reduced, whereas the growth of one Pt4CL1a was

improved during the first growing seasons, but only in the A1 line the stem was

found shorter at the end of the third growing season. In general, it is considered

that lignin quantity and S/G content would correlate negatively with

growth/biomass (Novaes et al. 2010). In relation to 4CL-deficient tree lines,

however, the growth of lines has in most cases not increased (Li et al. 2003, Jia et

al. 2004, Hancock et al. 2007, Wagner et al. 2009, Voelker et al. 2009, Stout et al.

2014). Moreover, a drastic suppression of 4CLs and decrease in lignin content

have been shown to cause stunted growth and abnormal wood structure in P.

radiata (Wagner et al. 2009) and P. tremula × alba (Voelker et al. 2010) lines. In

56

the case of P. tremula × alba, naringenin and dihydrokaempferol as well as their

glucosides were found in the 4CL-deficient lines, but not in their non-transgenic

controls (Voelker et al. 2010). Similarly, the silencing of HCT led to increased

contents of kaempferol and quercetin derivatives and anthocyanins in A. thaliana

lines (Besseau et al. 2007).

The soluble phenolic compounds and CTs of silver birch lines showed mostly

line-specific alterations, apart from neolignan and cinnamic acid derivatives, and

can hardly be explained by the expression of transgenes. Interestingly, the total

content of flavonoids in two Pt4CL1a lines had almost doubled in comparison

with the non-transgenic line. The increased content of flavonoids resulted from

higher levels of (+)-catechin and catechin derivative, but the differences were not

significant. The composition of soluble phenolic compounds has been found to be

highly dependent on the silver birch genotype (Keinänen et al. 1999, Laitinen et

al. 2000, 2002, 2004) as well as ontogeny (Bryant and Julkunen-Tiitto 1995,

Julkunen-Tiitto et al. 1996, Laitinen et al. 2004, Laitinen et al. 2005). Often, the

constitutive chemical defenses increase through the juvenile stage of tree species

and tend to decrease in boreal species after transition to the mature stage, which

indicates stronger selection pressure in juvenile plants for defense against

herbivores (Barton and Koricheva 2010). In silver birch, the phenolic compound

contents have been higher in 3-year-old saplings in comparison with 1- and 4-

year-old plants (Laitinen et al. 2005). Moreover, the phenolic profiles of juvenile

birches are more alike than the profiles of older saplings (Bryant and Julkunen-

Tiitto 1995, Julkunen-Tiitto et al. 1996, Laitinen et al. 2005). In the present study,

both the hybrid aspens and silver birches under investigation were in a juvenile

state, and it is therefore possible that more profound alterations would have been

detected among lines and clones with older plant material. However, significant

alterations were found in the phenolic compounds among silver birch clones, and

it seems that soluble phenolic compounds and CTs were more alike in the silver

birch lines and corresponding clones than among the clones.

Previously, the quality of PtCOMT silver birch leaves has been examined in

food selection and RGR assays and no association has been found in the feeding

preferences or growth performance with lignin modification (Tiimonen et al.

2005). In the present study, larvae representing Noctuidae and Geometridae

families were used to determine if the leaf chemistry of Pt4CL1a silver birch lines

was comparable to their non-transgenic clones. The RGRs of the studied species

were found to be similar among the silver birch clones and Pt4CL1a lines. In the

present study, the lignin characteristics of Pt4CL1a lines were examined from

57

stem samples. Based on studies conducted, for instance, on PtCOMT lines, the

changes in lignin have been comparable across different plant parts (Tiimonen et

al. 2005, Tiimonen 2007). Therefore, it is presumed that in this study the change

in the S/G ratio of Pt4CL1a line A1 was also present in leaves. Leaf lignin

content, however, varies during ontogeny (e.g. Coleman 1986); it is connected to

the leaf venation type; and the composition of lignin, in general, is species-

dependent (Albersheim et al. 2011). The A. punctulata, C. cinctaria, O. antiqua,

and E. autumnata larvae are polyphagous and consume both common shrubs and

leaves of deciduous trees. Hence, it is unlikely that the change in the S/G ratio of

Pt4CL1a line A1 affected the RGRs of larvae. The same is true with the soluble

phenolics and CTs, which also differed more among silver birch clones than

among clones and Pt4CL1a lines.

Changes in the RGRs of lepidopteran larvae among non-transgenic and VHb

lines may arise from differences in the quality of leaves, starch or secondary

metabolites, which were found to be altered in an earlier study by Häggman et al.

(2003). The effect of starch on the performance of insect herbivores has been

addressed in relation to elevated CO2 (e.g. Johnson & Lincoln 1991, Will &

Ceulemans 1997, Oksanen et al. 2001, Hättenschwiler & Schafellner 2004). The

increase in carbohydrates/nitrogen ratio has species-specific effects (Lindroth

2010). It has been shown to enhance the consumption of leaves (Johnson &

Lincoln 1991, Goverde et al. 1999) by insect herbivores and to decrease

(Hättenschwiler and Schafellner 2004) or increase (Goverde et al. 1999) their

performance. After an eight-week period under normal greenhouse conditions, the

VHb lines were characterized by an increased volume of chloroplast starch

(Häggman et al. 2003). Because significant changes were not detected in the

RGRs of lepidopteran larvae in the present study, the leaf starch levels of the two-

year-old hybrid aspens were not analyzed. With the RGR experiment, however,

the possible effects of the composition of leaf metabolites were examined and

found to be similar, regardless of their transgenic status. This indicates that the

VHb did not cause deleterious alterations to the leaf chemistry of hybrid aspen.

The VHb hybrid aspen have previously been shown to form ECM symbiosis

comparable to the non-transgenic hybrid aspen line in in vitro conditions (Jokipii

et al. 2008). This is in accordance with studies on GM forest trees and

mycorrhizal fungi (Stefani & Hamelin 2010 and references therein, Danielsen et

al. 2012, 2013). The effect of COMT suppression to ECM symbiosis has been

studied on silver birch (Tiimonen et al. 2008) and poplar lines (Danielsen et al.

2013). In the latter, the ECM community composition of field-grown poplars was

58

similar among conventionally bred poplar clones and COMT, CCR, and CAD

lines (Danielsen et al. 2013). Previously, it has been found that the Hartig net of

ectomycorrhizas of two PtCOMT lines, A23 and A130, differed from that of clone

A after eight weeks of co-cultivation under in vitro conditions (Tiimonen et al.

2008). In the present study, the P. involutus and silver birches were co-cultivated

in a mixture of peat:vermiculate under greenhouse conditions for eight weeks, and

the ECM structure was found to be similar between the PtCOMT lines and clone

A. The alteration found in the penetration of the fungus between the epidermal

cells of line A23 (Tiimonen et al. 2008) was probably not caused by the

expression of PtCOMT but could be related to the in vitro conditions, thus

emphasizing the need to study GM trees in their natural conditions.

59

5 Conclusions and future prospects

The flexibility of the lignin biosynthesis route originates in enzymes utilizing a

variety of esters, aldehydes or alcohols as substrates; multiple enzyme isoforms

produced in excess; and the possibility to incorporate different alcohols to the

lignin polymer. Genome and transcriptome sequencing facilitates more precise

studies on enzymes that are coded by multigene families, as has been seen, for

instance, in studies on the monolignol pathway genes and enzymes of P.

trichocarpa (Tsai et al. 2006, Tuskan et al. 2006, Shi et al. 2010, Chen et al.

2013, 2014, Wang et al. 2014a) and A. thaliana (e.g. Costa et al. 2003, Raes et al.

2003, Vanholme et al. 2012). In addition to the genome of B. nana (Wang et al.

2013), the genome of silver birch, sequenced in cooperation between the

University of Helsinki and the Finnish Forest Research Institute will shortly be

available. This will enable more detailed studies and increase knowledge about

the regulation and function of the phenylpropanoid and monolignol routes.

In the present study, it was demonstrated that the heterologous expression of

sense PtCOMT and antisense Pt4CL1 caused reduction in the silver birch

endogenous BpCOMT and Bp4CL1 transcript levels, respectively. The silver birch

lines with altered transcript levels had changed lignin composition, indicating that

BpCOMT and Bp4CL1 are both likely to contribute to the biosynthesis of

monolignols. No unintended effects were detected in the PtCOMT or Pt4CL1a

lines in relation to ECM symbiosis or performance of insect larvae. Moreover, the

soluble phenolic compounds of lines and clones were similar, except in the case

of cinnamic acid derivatives, which are involved in the monolignol biosynthesis

route. Furthermore, the phenolic compound profiles were found to be less similar

among the non-transgenic silver birch clones than among the transgenic lines and

corresponding clones. By contrast, the VHb line was shown to have somewhat

increased levels of soluble phenolics and CTs under standard greenhouse

conditions, but no changes were observed in the proportional weight gain of

lepidopteran larvae and, thus, the leaves were found to be of similar food quality.

However, all studies conducted were performed on juvenile plants under

controlled conditions in the absence of abiotic and biotic stresses, which can

influence the stability of introduced traits, as recently reported by Stout et al.

(2014). In general, transgenes have been shown to be stable and GM trees have

not been found to cause unintended effects to non-target organisms when studied

under natural environmental conditions. Therefore, if long-term field trials will

reach similar results and if the dispersal of recombinant DNA can be avoided,

60

there seems to be no scientific reason for forbidding the exploitation of genetic

engineering among other tree-improvement means.

Tree domestication may in the near future be speeded up if genotyping

platforms become feasible and enable marker-assisted selection and genomic

selection in trees (Isik 2014). This would potentially reduce time and efforts

needed for progeny testing but requires reference genome sequences together with

high-density genetic maps. Another potential method to accelerate tree-

improvement efforts could be breeding with rare defective alleles (BRDA), a

method that utilizes knowledge of characterized genes affecting specific traits

(Vanholme et al. 2013). The single nucleotide polymorphism of selected

candidate genes would be identified by means of NGS in order to reveal rare

alleles (Vanholme et al. 2013). The trees with rare alleles might have higher

fitness compared to GM trees and could be exploited in controlled crosses and,

subsequently, in conventional breeding programs (Tsai 2013). Application of

BRDA could facilitate the identification of naturally stable alleles and overcome

the major obstacle for GM-tree-breeding efforts, the opinion of the general public.

61

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Appendices

Appendix 1. Effects of 4CL antisense constructs to lignin and phenolics

Appendix 2. Effects of COMT constructs to lignin and phenolics

Appendix 3. COMT-like sequences used in the generation of ML trees

Appendix 4. ML tree, reconstructed using predicted amino acids sequences of

COMTs

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6. T

he

eff

ects

of

4C

L a

nti

se

ns

e c

on

str

uc

ts t

o lig

nin

an

d p

he

no

lic

co

mp

ou

nd

s.

Pla

nt sp

eci

es

Gene

Lig

nin

conte

nt

Lig

nin

com

posi

tion

C

hanged p

henolic

s R

efe

rence

Ara

bid

opsi

s th

alia

na (

L.)

Heyn

h.

E

Decr

ease

d u

p to 5

0%

D

ecr

ease

d G

units

Lee e

t al.

1997

Populu

s tr

em

ulo

ides

Mic

hx.

E

D

ecr

ease

d u

p to 4

5%

N

o e

ffect

In

crease

d h

ydro

xyci

nnam

ic a

cids

Hu e

t al.

19

99

E

D

ecr

ea

sed

up

to

40

%

De

cre

ase

d S

/G r

atio

1

NA

Li e

t al.

2003

Populu

s to

mento

sa C

arr

. E

D

ecr

ea

sed

up

to

42

%

NA

A

ccu

mu

latio

n o

f re

d c

olo

ure

d

com

pounds

in w

oo

d

Jia e

t al.

20

04

E

D

ecr

ease

d u

p to 2

8%

D

ecr

ease

d G

& H

, in

crease

d S

units

In

crease

d h

ydro

xyci

nnam

ic a

cids

Tia

n e

t al.

2013

Po

pu

lus

tre

mu

la L

. ×

alb

a L

. H

D

ecr

ease

d u

p to 5

0%

In

crease

d S

/G r

atio

1

Acc

um

ula

tion o

f p

henolic

s1

Vo

elk

er

et al.

2010

Populu

s tr

ichoca

rpa

E

De

cre

ase

d u

p t

o 4

6%

In

cre

ase

d o

r d

ecr

ea

sed

S/G

ra

tio1

S

tou

t et al.

2014

Nic

otia

na tabacu

m L

. E

D

ecr

ease

d 1

3%

In

crease

d H

, decr

ease

d V

& S

units

In

crease

d h

ydro

xyci

nnam

ic a

cids

Kajit

a e

t al.

1996

Ory

za s

ativ

a (

L.)

japonic

a

E

Decr

ease

d 2

1%

D

ecr

ease

d H

, G

& S

units

In

crease

d 4

-coum

ara

te &

feru

late

Gui e

t al.

20

11

Betu

la p

endula

Ro

th

E

No c

hange

N

o c

hange

N

A

Seppänen e

t al.

2007

Pin

us

radia

ta D

. D

on

E

up to 5

0%

D

ecr

ease

d G

units

In

crease

d p

hen

olic

s W

agner

et al.

200

9

1V

ariatio

n a

mong li

nes;

E =

end

ogen

ous;

H =

hete

rolo

gous;

NA

= d

ata

not

ava

ilable

; *p

ote

ntia

lly fla

vonoid

s or

tannin

-lik

e c

om

pou

nds

81

Ap

pe

nd

ix 2

. E

ffe

cts

of

CO

MT

co

ns

tru

cts

to

to

lig

nin

an

d p

he

no

lic

s

Ta

ble

7. T

he

eff

ects

of

CO

MT

co

nstr

uc

ts t

o l

ign

in a

nd

ph

en

oli

c c

om

po

un

ds

.

Pla

nt sp

eci

es

Gene,

Orienta

tion

Lig

nin

conte

nt

Lig

nin

com

posi

tion

C

hanged p

henolic

s R

efe

rence

Zea m

ays

L.

E, as

Reduce

d u

p to 2

4%

R

educe

d S

, 5-O

H-G

U

Reduce

d p

-coum

ari

c aci

ds

Piq

uem

al e

t al.

20

02

E

, a

s R

ed

uce

d u

p t

o 4

5%

R

ed

uce

d S

U1

NA

G

uill

aum

ie e

t al.

2008

Medic

ago s

ativ

a L

. E

, as

Reduce

d 1

1%

R

educe

d G

& S

U

No c

hange

* G

uo e

t al.

20

01

, M

arita

et al.

2003

Po

pu

lus

tre

mu

la L

. ×

alb

a L

.

H, s

Reduce

d 1

7%

+

Re

du

ced

S,

5-O

H-G

U2

,+

Ste

ms

mo

re b

row

n

Jou

an

in e

t a

l. 200

0

H, as

No e

ffect

R

educe

d S

/G r

atio

3 , 5

-OH

-G U

P

ale

ro

se s

tem

s, a

ltere

d

meth

anol-so

luble

phenolic

s**

Van D

oors

sela

ere

et al.

1995,

La

pie

rre

et al.

199

9,

Pila

te e

t al.

200

2

Populu

s tr

em

ulo

ides

Mic

hx.

E, s

No e

ffect

R

educe

d S

/G r

atio

3 , 5

-OH

-G2 U

R

eddis

h-b

row

n w

ood

Tsa

i et al.

1998

E, as

No e

ffect

N

A

No c

hange in

the c

olo

ur*

**

Boerjan e

t al.

199

7

Nic

otia

na tabacu

m

L.

E, as

No e

ffect

R

educe

d S

/G r

atio

3 , 5-O

H-G

U

NA

A

tanoss

ova

et

al.

1995

E, s

No e

ffect

R

educe

d S

/G r

atio

3 , 5-O

H-G

U4

NA

A

tanoss

ova

et

al.

1995

H

, as

No e

ffect

R

educe

d S

U

NA

D

wiv

edi e

t al.

199

4

H

, as

Reduce

d

No c

hange

N

A

Ni e

t al.

19

94

H

, as

Reduce

d

Incr

ease

d S

/G r

atio

N

A

Sew

alt

et al.

19

97

Betu

la p

endula

Roth

.

H, s

No e

ffect

R

educe

d S

/G r

atio

, 5-O

H-G

U

NA

A

ronen e

t al.

2003

, T

iimonen e

t al.

2005

E =

endogenous;

H =

hete

rolo

gous;

as

= a

ntis

ense

; s

= s

ense

; U

= u

nits

; N

A =

data

not

ava

ilable

; 1S

clere

nch

yma-s

peci

fic;

2V

aria

tion a

mong li

nes;

3 Incr

ease

d G

an

d r

ed

uce

d S

un

its;

4In

most

of

the s

lin

es

CO

MT

act

ivity

and e

xpre

ssio

n i

ncr

ease

d,

how

eve

r, i

n o

ne l

ine (

A17)

act

ivity

and e

xpre

ssio

n w

ere

dra

stic

ally

reduce

d;

+In

most

of

the s

lin

es

CO

MT

was

ove

rexp

ress

ed a

nd n

o c

hang

es

in li

gnin

, but

in o

ne li

ne (

70S

OM

T-3

) th

e C

OM

T a

ctiv

ity w

as

reduce

d d

rast

ically

and

line s

ho

wed a

ltere

d li

gnin

pro

pert

ies;

* In w

all-

bound (

or

solu

ble

Kota

et

al.

2004)

hyd

roxy

cinn

am

ic a

cid

s; **

Mo

rre

el e

t al.

20

04

; **

*sta

ted

in T

sai e

t al.

1998

82

Appendix 3. COMT-like sequences used in the generation of ML tree

List of species and sequences used in the generation of ML tree presented in

Appendix 4. The species name is followed by utilized gene name and ID/gene

model/scaffold in parenthesis. The underlined sequencis were used in the

generation of ML tree presented in Fig. 4.

Ammi majus AmCOMT (AY443007); Arabidopsis thaliana AT1G21100,

AT1G21110, AT1G21120, AT1G21130, AT1G33030, AT1G51990, AT1G63140,

AT1G76790, AT1G77520, AT1G77530, AT3G53140, AT4G35160, AT5G37170,

AT5G53810, AT5G54160; Betula nana scaffold1560, scaffold7599,

scaffold17988, scaffold3434, Betula pendula BpCOMT (FJ667539);

Brachypodium distachyon BdCOMT (Bradi3g16530); Capsella rubella CrCOMT

(XM_006280619); Catharanthus roseus CrOMT (AY127568), CsCOMT

(AY028439); Chrysosplenium americanum CaCOMT (U16793); Citrus

aurantium CauCOMT (HM641694); Clarkia breweri CbCOMT1 (U86760),

CbCOMT2 (AF006009); Corylus avellana CavCOMT (KA429980), CavOMT

(KA430068); Eucalyptus camaldulensis EcCOMT (GU109375); Fragaria x

ananassa FaCOMT (AF220491); Glycine max GmCOMT (HQ651806), GmOMT

(NM_001254233); Hevea brasiliensis HbCOMT1 (JQ037840); Hordeum vulgare

HvCOMT1 (EF586876), HvCOMT2 (U54767), HvOMT (X77467); Liquidambar

styraciflua LsCOMT (AF139533); Lolium perenne LpCOMT (AF010291); Lotus

japonicus LjCOMT (AB091686), LjOMT (AB091686); Malus domestica

MdCOMT1 (DQ886018), MdCOMT2 (DQ886019), MdCOMT3 (DQ886020),

MdCOMT4 (DQ886021); Medicago sativa MsCOMT1 (GU066087), MsCOMT2

(M63853M), MsOMT (U97125); Medicago truncatula MtOMT

(XM_003622309); Nicotiana tabacum NtCOMT (X71430); Notholithocarpus

densiflorus NdCOMT (GAOS01025159); Oryza sativa Os01g54969,

Os02g57760, Os04g01470, Os04g09604, Os04g09654, Os04g11970,

Os05g43930, Os05g43940, Os06g13280, Os06g16960, Os07g27880,

Os07g27970, Os07g28040, Os08g06100, Os08g07260, Os08g19420,

Os08g35310, Os09g17560, Os10g02880, Os12g25820, OsCOMT

(NM_001067566), OsCOMT2 (DQ288259); Phyllostachys edulis PeOMT

(FP099862); Pisum sativum PsOMT (U69554); Populus tremuloides PtCOMT

(U13171); Populus trichocarpa PoptrOMT1 (Potri.015G003100.1,

83

estExt_fgenesh4_pg.C_LG_XV0035), PoptrOMT13 (Poptr_0013s13990),

PoptrOMT14 (Poptr_0014s10220), PoptrOMT15 (Poptr_0191s00210),

PoptrOMT16 (Poptr_0004s04980), PoptrOMT17 (Poptr_0019s12280),

PoptrOMT18 (Poptr_0002s18160), PoptrOMT19 (Poptr_0011s15360),

PoptrOMT2 (Potri.012G006400, estExt_fgenesh4_pm.C_LG_XII0129),

PoptrOMT20 (Poptr_0011s05850), PoptrOMT21 (Poptr_0015s00550),

PoptrOMT22 (Poptr_0019s13400), PoptrOMT23 (Poptr_0013s12580),

PoptrOMT24 (Poptr_0013s12510), PoptrOMT25 (Poptr_0012s00670),

PoptrOMT3 (Poptr_0014s10210), PoptrOMT4 (Poptr_0013s12620), PoptrOMT5

(Poptr_0013s12600), PoptrOMT6 (Poptr_0151s00220), PoptrOMT7

(Poptr_0002s18150), PoptrOMT8 (Poptr_0004s04990), PoptrOMT9

(Poptr_0013s14000); Prunus amygdalus PaCOMT (X83217); Punica granatum

PgCOMT (KJ713968); Pyrus x bretschneideri cultivar PbCOMT (KC905086);

Ricinus communis RcCOMT (XM_002525772); Ricinus communis RcCOMT2

(XM_002520245); Rosa chinensis RocCOMT (AJ439740), RocCOMT2

(AB121046); Rosa hybrida RhOMT (AF502433); Secale cereale ScOMT

(AY177404); Selaginella moellendorffii SmCOMT (GQ166949); Sorbus

aucuparia SaCOMT (KC903138), SaCOMT2 (ES789987); Theobroma cacao

TcCOMT (XM_007039153), TcCOMT2 (XM_007019028); Triticum aestivum

TaCOMT (DQ223971); Zea mays ZmCOMT1 (AAB03364), ZmCOMT2

(NM_001279714); Zinnia elegans ZeCOMT (U19911)

84

Appendix 4. ML tree, reconstructed using predicted amino acids sequences of COMTs

Fig. 6. A phylogenetic ML tree, reconstructed using predicted amino acids sequences

of COMT-like sequences (see Appendix 3 for detailed information on used sequences

and species). Closed squares, dicot COMTs possible involved in monolignol and

phenylpropanoid routes; open squares, monocot COMTs possible involved in

monolignol and phenylpropanoid routes, closed circles, dicot COMTs possible

involved in flavonoid biosynthesis; closed diamonds, full length putative COMTs

retrieved from preliminary assembly of B. nana (Wang et al. 2013,

http://birchgenome.org) based on BLAST searches with B. platyphylla unigenes

produced of RNA-seq of developing xylem (Wang et al. 2014b).

85

Original publications

I Sutela S, Hahl T, Tiimonen H, Aronen T, Ylioja T, Laakso T, Saranpää P, Chiang V, Julkunen-Tiitto R & Häggman H (2014) Phenolic compounds and expression of 4CL genes in silver birch clones and Pt4CL1a lines. Accepted (PLOS ONE).

II Sutela S, Niemi K , Edesi J, Laakso T, Saranpää P, Vuosku J, Mäkelä R, Tiimonen H, Chiang V, Koskimäki J, Suorsa M, Julkunen-Tiitto R & Häggman H (2009) Phenolic compounds in ectomycorrhizal interaction of lignin modified silver birches and Paxillus involutus. BMC Plant Biology 9: 124.

III Sutela S, Ylioja T, Jokipii-Lukkari S, Anttila A-K, Julkunen-Tiitto R, Niemi K, Mölläri T, Kallio PT & Häggman H (2013) The responses of Vitreoscilla hemoglobin-expressing hybrid aspen (Populus tremula × tremuloides) exposed to 24-h herbivory: expression of hemoglobin and stress-related genes in exposed and nonorthostichous leaves. Journal of Plant Research 126: 795–809.

IV Häggman H, Sutela S, Walter C & Fladung M (2014) Biosafety considerations in the context of deployment of GM trees. In: Fenning T (ed) Challenges and opportunities for the world’s forests in the 21st century. Dordrecht, Springer Verlag: 491–524.

Reprinted with permission from BioMed Central (II) and Springer (III, IV).

Original publications are not included in the electronic version of the dissertation.

86

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642. Kraus, Klemens (2014) Security management process in distributed, large scalehigh performance systems

643. Toivainen, Tuomas (2014) Genetic consequences of directional selection inArabidopsis lyrata

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