Genetically Modified Silver Birch and Hybrid Aspen – Target and Non-Target Effects of Introduced Traits

A 644 OULU 2014 A 644

UNIVERSITY OF OULU P.O.BR[ 00 FI-90014 UNIVERSITY OF OULU FINLAND ACTA UNIVERSITATISUNIVERSITATIS OULUENSISOULUENSIS ACTA UNIVERSITATIS OULUENSIS ACTAACTA

SERIES EDITORS SCIENTIAESCIENTIAEA A RERUMRERUM Suvi Sutela NATURALIUMNATURALIUM

ASCIENTIAE RERUM NATURALIUM Suvi Sutela Professor Esa Hohtola GENETICALLY MODIFIED BHUMANIORA SILVER BIRCH AND HYBRID University Lecturer Santeri Palviainen CTECHNICA ASPEN – TARGET AND NON- Postdoctoral research fellow Sanna Taskila TARGET EFFECTS OF DMEDICA Professor Olli Vuolteenaho INTRODUCED TRAITS ESCIENTIAE RERUM SOCIALIUM University Lecturer Veli-Matti Ulvinen FSCRIPTA ACADEMICA Director Sinikka Eskelinen GOECONOMICA Professor Jari Juga

EDITOR IN CHIEF Professor Olli Vuolteenaho PUBLICATIONS EDITOR

Publications Editor Kirsti Nurkkala UNIVERSITY OF OULU GRADUATE SCHOOL; UNIVERSITY OF OULU, FACULTY OF SCIENCE, ISBN 978-952-62-0683-7 (Paperback) DEPARTMENT OF BIOLOGY ISBN 978-952-62-0684-4 (PDF) ISSN 0355-3191 (Print) ISSN 1796-220X (Online)

ACTA UNIVERSITATIS OULUENSIS A Scientiae Rerum Naturalium 644

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 of the Doctoral Training Committee of Health and Biosciences of the University of Oulu for public defence in Auditorium IT116, Linnanmaa, on 12 December 2014, at 12 noon

Supervised by Professor Hely Häggman Professor Riitta Julkunen-Tiitto Docent Karoliina Niemi

Reviewed by Associate Professor Gerd Bossinger Professor Lise Lejus-Jouanin

Opponent Doctor Markku Keinänen

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

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

Cover Design Raimo Ahonen

JUVENES PRINT TAMPERE 2014 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 of Biology Acta Univ. Oul. A 644, 2014 University 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 in numerous studies, which have generally concluded that GM trees have similar effects on environment and/or other organisms as do conventionally bred trees. In the present study, GM silver 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 endogenous genes and to the production of soluble phenolic compounds in relation to ectomycorrhizal symbiosis or herbivory. The GM silver birch lines had altered lignin composition, whereas the hybrid aspen lines produced the hemoglobin of Vitreoscilla sp. (VHb). The Pt4CL1a lines were generated using biolistic transformation and monitored under greenhouse conditions for three growing seasons. The Pt4CL1a and PtCOMT silver birch lines, with altered lignin syringyl/guaiacyl ratio, had also reduced transcript levels of endogenous genes, Bp4CL1 and BpCOMT, respectively. This indicates that these members of the 4CL and COMT multigene families are likely to contribute to the monolignol biosynthesis pathway of silver birch. No unintended effects were detected in the PtCOMT or Pt4CL1a lines in relation to ECM symbiosis or performance of insect larvae. Moreover, in soluble phenolic compounds, alterations were found mainly in cinnamic acid derivatives, a group of compounds involved in the biosynthesis of monolignols. In addition, the responses of the studied hybrid aspen lines that were exposed to herbivory for 24 hours were found to be comparable. Furthermore, the proportional weight gain of lepidopteran larvae was alike when fed with leaves of the VHb and non-transgenic hybrid aspen lines. Taken together, no unintended changes were found in the GM silver birch lines with altered lignin composition or in the VHb hybrid aspen lines. However, it is acknowledged that these short-term studies that were conducted under controlled conditions have certain limitations.

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

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

Tiivistelmä Puiden ominaisuuksia on mahdollista muuttaa geenitekniikkaa käyttämällä huomattavasti perin- teistä jalostusta nopeammin. Geneettisen muuntamisen vaikutuksia puiden ominaisuuksiin ja vuorovaikutussuhteisiin on selvitetty useissa tutkimuksissa geenitekniikkaan liitettyjen riskien arvioimiseksi. Muunnettuja kohdeominaisuuksiaan lukuun ottamatta geneettisesti muunnettujen (GM) puiden ei ole yleisesti ottaen tutkimuksissa havaittu eroavan ympäristövaikutuksiltaan perinteisellä jalostuksella tuotetuista puista. Tässä työssä tutkittiin siirrettyjen geenien vaikutuksia GM-rauduskoivun (Betula pendula Roth) sekä hybridihaavan (Populus tremula L. × tremuloides Michx.) endogeenisten geenituotteiden ja liukoisten fenoliyhdisteiden määriin. Lisäksi työssä tarkasteltiin ligniinirakenteeltaan muunnettujen rauduskoivulinjojen ektomykorritsasym- bioosia sekä ligniinimuunnettujen ja Vitreoscilla sp. -bakteerin hemoglobiinia (VHb) tuottavien hybridihaapalinjojen lehtien laatua perhostoukkien ravintona. Biolistisella geeninsiirrolla tuotetuista Amerikan haavan 4-kumaraattikoentsyymi A-ligaasi - geeniä (Pt4CL1) ilmentävistä rauduskoivulinjoista yhdessä havaittiin ligniinin syringyyli- ja guaiasyyliyksikköjen suhteessa muutos. Havaittu muutos aiheutui todennäköisesti koivun Bp4CL1-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 rauduskoivun monolignolien biosynteesissä. Ligniiniominaisuuksiltaan muunnettujen rauduskoivujen liukoi- sista fenoliyhdisteistä todettiin muutoksia ensisijaisesti kanelihappojohdannaisissa, jotka liittyvät läheisesti monolignolien biosynteesireittiin. Ektomykorritsasymbioosissa tai perhostoukkien kasvunopeudessa ei havaittu kasvien geneettisestä muuntamisesta johtuvia eroja. Merkitseviä eroja 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 pitkäaikaisissa kenttäkokeissa.

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

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

7 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

8 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

9 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

10 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 Study design Data collection Data Manuscript preparation idea (with help of others) analysis (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 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

13 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

14 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

15 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,

16 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).

17 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.

18 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).

19 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-hydroxycinnamoyltransferase; C4H-C3H, protein complex consisting of cinnamic acid 4-hydroxylase (C4H) and 4-coumarate 3-hydroxylase (C3H); CCoAOMT, caffeoyl-CoA O-methyltransferase; CCR, cinnamoyl-CoA reductase; AldOMT, 5-hydroxyconiferaldehyde O-methyltransferase; 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

20 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

21 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).

22 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-

23 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

24 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

25 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-

26 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

27 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

28 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.

29 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).

30 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 Paper Orientation

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 E5396/4 pRT99/35S-Pt4CL1-a antisense I (Pt4CL1a) V617 hybrid aspen VHb V617/45 pBVHb sense III VHb V617/60 pBVHb sense III

31 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

32 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

33 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.

34 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.

35 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

36 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), PA L2 (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

37 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

38 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

39 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.

40 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,

41 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).

42 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.

43 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 Lignin (%) S/G ratio Lignin (%) S/G ratio BpCOMT BpCOMT /Line (Atub1) (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.

44 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.

45 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

46 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

47 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

48 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 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.

51 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

52 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

53 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

54 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

55 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

56 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

57 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.

58 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,

59 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.

60 References

Ahuja MR (2009) Transgene stability and dispersal in forest trees. Trees-Struct Funct 23: 1125–1135. Albersheim P, Darvill A, Roberts K, Sederoff R & Staehelin A (2011) Plant cell walls. New York NY, Garland Science, Taylor & Francis Group, LLC. Anand A, Duk BT, Singh S, Akbas MY, Webster DA, Stark BC & Dikshit KL (2010) Redox-mediated interactions of VHb (Vitreoscilla haemoglobin) with OxyR: novel regulation of VHb biosynthesis under oxidative stress. Biochem J 426: 271–280. Appleby C (1984) Leghemoglobin and Rhizobium respiration. Annu Rev Plant Physiol Plant Mol Biol 35: 443–478. Arasimowicz M, Floryszak-Wieczorek J, Milczarek G & Jelonek T (2009) Nitric oxide, induced by wounding, mediates redox regulation in pelargonium leaves. Plant Biol 11: 650–663. Aronen T, Tiimonen H, Tsai CJ, Jokipii S, Chen X, Chiang V & Häggman H (2003) Altered lignin in transgenic silver birch (Betula pendula) expressing PtCOMT gene. In: Espinel S, Barredo Y & Ritter E (eds) Sustainable forestry, wood products & biotechnology. Vitoria-Gasteiz, DFA-AFA Press: 149–161. Atanassova R, Favet N, Martz F, Chabbert B, Tollier MT, Monties B, Fritig B & Legrand M (1995) Altered lignin composition in transgenic tobacco expressing O- methyltransferase sequences in sense and antisense orientation. Plant J 8: 465–477. Axelsson EP, Hjältén J, LeRoy CJ, Julkunen-Tiitto R, Wennström A & Pilate G (2010) Can leaf litter from genetically modified trees affect aquatic ecosystems? Ecosystems 13: 1049–1059. Barton KE & Koricheva J (2010) The ontogeny of plant defense and herbivory: characterizing general patterns using meta-analysis. Am Nat 175: 481–493. Benjamini Y & Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B-Methodol 57: 289–300. Boeckler GA, Gershenzon J & Unsicker SB (2011) Phenolic glycosides of the Salicaceae and their role as anti-herbivore defenses. Phytochemistry 72: 1497–1509. Boerjan W, Baucher M, Chabbert B, Petit-Conil M, Leplé J, Pilate G et al. (1997) Genetic modification of lignin biosynthesis in quaking aspen and poplar. In: Klopfenstein NB, Chun YW, Kim M & Ahuja MR (eds) Micropropagation, genetic engineering, and molecular biology of Populus. Gen. Tech. Rep. RM-GTR–297. Forest Service, Rocky Mountain Forest and Range Experiment Station, Fort Collins CO, Department of Agriculture: 193–205. Boerman SJ & Webster DA (1982) Control of heme content in Vitreoscilla by oxygen. J Gen Appl Microbiol 28: 35–43. Bonawitz ND, Soltau WL, Blatchley MR, Powers BL, Hurlock AK, Seals LA, Weng J, Stout J & Chapple C (2012) REF4 and RFR1, subunits of the transcriptional coregulatory complex Mediator, are required for phenylpropanoid homeostasis in Arabidopsis. J Biol Chem 287: 5434–5445.

61 Brosché M, Vinocur B, Alatalo ER, Lamminmäki A, Teichmann T, Ottow EA, Djilianov D, Afif D, Bogeat-Triboulot MB, Altman A, Polle A, Dreyer E, Rudd S, Lars P, Auvinen P & Kangasjärvi J (2005) Gene expression and metabolite profiling of Populus euphratica growing in the Negev desert. Genome Biol 6: R101. Bryant JP & Julkunen-Tiitto R (1995) Ontogenic development of chemical defense by seedling resin birch - energy-cost of defense production. J Chem Ecol 21: 883–896. Busov VB, Strauss SH & Pilate G (2010) Transformation as a tool for genetic analysis in Populus. In: Stefan J, Rishikesh B & Andrew G (eds) Genetics and Genomics of Populus. New York NY, Springer: 113–133. Chang S, Puryear J & Cairney J (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep 11: 113–116. Chen F, Liu C, Tschaplinski TJ & Zhao N (2009) Genomics of secondary metabolism in Populus: Interactions with biotic and abiotic environments. Crit Rev Plant Sci 28: 375–392. Chen H, Li Q, Shuford CM, Liu J, Muddiman DC, Sederoff RR & Chiang VL (2011) Membrane protein complexes catalyze both 4- and 3-hydroxylation of cinnamic acid derivatives in monolignol biosynthesis. Proc Natl Acad Sci U S A 108: 21253–21258. Chen H, Song J, Wang JP, Lin Y, Ducoste J, Shuford CM et al. (2014) Systems biology of lignin biosynthesis in Populus trichocarpa: heteromeric 4-coumaric acid: coenzyme A ligase protein complex formation, regulation, and numerical modeling. Plant Cell 26: 876–893. Chen H, Song J, Williams CM, Shuford CM, Liu J, Wang JP et al. (2013) Monolignol pathway 4-coumaric acid:coenzyme A ligases in Populus trichocarpa: novel specificity, metabolic regulation, and simulation of coenzyme A ligation fluxes. Plant Physiol 161: 1501–1516. Chen S & Polle A (2010) Salinity tolerance of Populus. Plant Biol 12: 317–333. Cheynier V, Comte G, Davies KM, Lattanzio V & Martens S (2013) Plant phenolics: recent advances on their biosynthesis, genetics, and ecophysiology. Plant Physiol Biochem 72: 1–20. Chi P, Webster DA & Stark BC (2009) Vitreoscilla hemoglobin aids respiration under hypoxic conditions in its native host. Microbiol Res 164: 267–275. Cho M, Corea ORA, Yang H, Bedgar DL, Laskar DD, Anterola AM et al. (2007) Phenylalanine biosynthesis in Arabidopsis thaliana - Identification and characterization of arogenate dehydratases. J Biol Chem 282: 30827–30835. Costa MA, Collins RE, Anterola AM, Cochrane FC, Davin LB & Lewis NG (2003) An in silico assessment of gene function and organization of the phenylpropanoid pathway metabolic networks in Arabidopsis thaliana and limitations thereof. Phytochemistry 64: 1097–1112. Curtin SJ, Voytas DF & Stupar RM (2012) Genome engineering of crops with designer nucleases. Plant Genome 5: 42–50. Dal Cin V, Tieman DM, Tohge T, McQuinn R, de Vos RCH, Osorio S et al. (2011) Identification of genes in the phenylalanine metabolic pathway by ectopic expression of a MYB transcription factor in tomato fruit. Plant Cell 23: 2738–2753.

62 Danielsen L, Lohaus G, Sirrenberg A, Karlovsky P, Bastien C, Pilate G & Polle A (2013) Ectomycorrhizal colonization and diversity in relation to tree biomass and nutrition in a plantation of transgenic poplars with modified lignin biosynthesis. PLOS ONE 8: e59207. Danielsen L, Thürmer A, Meinicke P, Buée M, Morin E, Martin F, Pilate G, Daniel R, Polle A & Reich M (2012) Fungal soil communities in a young transgenic poplar plantation form a rich reservoir for fungal root communities. Ecol Evol 2: 1935–1948. de Azevedo Souza C, Barbazuk B, Ralph SG, Bohlmann J, Hamberger B & Douglas CJ (2008) Genome-wide analysis of a land plant-specific acyl:coenzymeA synthetase (ACS) gene family in Arabidopsis, poplar, rice and Physcomitrella. New Phytol 179: 987–1003. Dikshit KL, Dikshit RP & Webster DA (1990) Study of Vitreoscilla globin (vgb) gene expression and promoter activity in E. coli through transcriptional fusion. Nucleic Acids Res 18: 4149–4155. Do CT, Pollet B, Thévenin J, Sibout R, Denoue D, Barrière Y, Lapierre C & Jouanin L (2007) Both caffeoyl Coenzyme A 3-O-methyltransferase 1 and caffeic acid O- methyltransferase 1 are involved in redundant functions for lignin, flavonoids and sinapoyl malate biosynthesis in Arabidopsis. Planta 226: 1117–1129. Dordas C, Hasinoff BB, Igamberdiev AU, Manac'h N, Rivoal J & Hill RD (2003) Expression of a stress-induced hemoglobin affects NO levels produced by alfalfa root cultures under hypoxic stress. Plant J 35: 763–770. Dubouzet JG, Strabala TJ & Wagner A (2013) Potential transgenic routes to increase tree biomass. Plant Sci 212: 72–101. Dumont E, Jokipii-Lukkari S, Parkash V, Vuosku J, Sundström R, Nymalm Y, Sutela S, Taskinen K, Kallio PT, Salminen TA & Häggman H (2014) Evolution, three- dimensional model and localization of truncated hemoglobin PttTrHb of hybrid aspen. PLOS ONE 9: e88573. Dwivedi UN, Campbell WH, Yu J, Datla RSS, Bugos RC, Chiang VL & Podila GK (1994) Modification of lignin biosynthesis in transgenic Nicotiana through expression of an antisense O-methyltransferase gene from Populus. Plant Mol Biol 26: 61–71. Edgar R (2004) MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5: 1–19. Ehlting J, Büttner D, Wang Q, Douglas C, Somssich I & Kombrink E (1999) Three 4- coumarate:coenzyme A ligases in Arabidopsis thaliana represent two evolutionarily divergent classes in angiosperms. Plant J 19: 9–20. El-Kassaby YA, Isik F & Whetten RW (2014) Modern advances in tree breeding. In: Fenning T (ed) Challenges and opportunities for the world’s forests in the 21st century. Dordrecht, Springer Verlag: 441–459. Endler A, Martens S, Wellmann F & Matern U (2008) Unusually divergent 4- coumarate:CoA-ligases from Ruta graveolens L. Plant Mol Biol 67: 335–346. Falcone Ferreyra ML, Rius SP & Casati P (2012) Flavonoids: biosynthesis, biological functions, and biotechnological applications. Front Plant Sci 3: 222.

63 Farrés J & Kallio PT (2002) Improved cell growth in tobacco suspension cultures expressing Vitreoscilla hemoglobin. Biotechnol Prog 18: 229–233. Felsenstein J (1981) Evolutionary trees from DNA sequences - a maximum likelihood approach. J Mol Evol 17: 368–376. Fladung M & Becker D (2010) Targeted integration and removal of transgenes in hybrid aspen (Populus tremula L. x P. tremuloides Michx.) using site-specific recombination systems. Plant Biology 12: 334–340. Fladung M, Schenk TMH, Polak O & Becker D (2010) Elimination of marker genes and targeted integration via FLP/FRT recombination system from yeast in hybrid aspen (Populus tremula L. x P. tremuloides Michx.). Tree Genet Genomes 6: 205–217. Fox J (2005) The R commander: A basic-statistics graphical user interface to R. J Stat Softw 14: 1–42. Frey AD, Farrés J, Bollinger CJT & Kallio PT (2002) Bacterial hemoglobins and flavohemoglobins for alleviation of nitrosative stress in Escherichia coli. Appl Environ Microbiol 68: 4835–4840. Frey AD & Kallio PT (2003) Bacterial hemoglobins and flavohemoglobins: versatile proteins and their impact on microbiology and biotechnology. FEMS Microbiol Rev 27: 525–545. Frey AD, Koskenkorva T & Kallio PT (2003) Vitreoscilla hemoglobin promoter is not responsive to nitrosative and oxidative stress in Escherichia coli. FEMS Microbiol Lett 224: 127–132. Frey AD, Oberle BT, Farrés J & Kallio PT (2004) Expression of Vitreoscilla haemoglobin in tobacco cell cultures relieves nitrosative stress in vivo and protects from NO in vitro. Plant Biotech J 2: 221–231. Frey AD, Shepherd M, Jokipii-Lukkari S, Häggman H & Kallio PT (2011) The single- domain globin of Vitreoscilla: augmentation of aerobic metabolism for biotechnological applications. Adv Microb Physiol 58: 81–139. Gao B, Gao S, Liu J & Jian W (2006) Variation of nutritional structure and ecological niche of arthropod community in plantation of transgenic insect-resistance hybrid poplar 741. J Beijing For Univ 25: 3507–3507. Gao B, Zhang F, Hou D, Wu B, Zhang S & Zhao X (2003) Structure of arthropod community in stands of transgenic hybrid poplar 741. J Beijing For Univ 25: 62–64. Goujon T, Sibout R, Pollet B, Maba B, Nussaume L, Bechtold N, Lu FC, Ralph J, Mila I, Barrière Y, Lapierre C & Jouanin L (2003) A new Arabidopsis thaliana mutant deficient in the expression of O-methyltransferase impacts lignins and sinapoyl esters. Plant Mol Biol 51: 973–989. Goverde M, Bazin A, Shykoff J & Erhardt A (1999) Influence of leaf chemistry of Lotus corniculatus (Fabaceae) on larval development of Polyommatus icarus (Lepidoptera,

Lycaenidae): effects of elevated CO2 and plant genotype. Funct Ecol 13: 801–810. Gui J, Shen J & Li L (2011) Functional characterization of evolutionarily divergent 4- coumarate:coenzyme A ligases in rice. Plant Physiol 157: 574–586.

64 Guillaumie S, Goffner D, Barbier O, Martinant J, Pichon M & Barrière Y (2008) Expression of cell wall related genes in basal and ear internodes of silking brown- midrib-3, caffeic acid O-methyltransferase (COMT) down-regulated, and normal maize plants. BMC Plant Biol 8: 71. Guo D, Chen F, Inoue K, Blount JW & Dixon RA (2001) Downregulation of caffeic acid 3-O-methyltransferase and caffeoyl CoA 3-O-methyltransferase in transgenic alfalfa: Impacts on lignin structure and implications for the biosynthesis of G and S lignin. Plant Cell 13: 73–88. Gupta KJ, Hebelstrup KH, Mur LAJ & Igamberdiev AU (2011) Plant hemoglobins: Important players at the crossroads between oxygen and nitric oxide. FEBS Lett 585: 3843–3849. Haapanen M & Mikola J (2008) Metsänjalostus 2050 — pitkän aikavälin metsänjalostus- ohjelma. Working Papers of the Finnish Forest Research Institute. Helsinki, Finnish Forest Research Institute. Häggman H, Frey AD, Ryynänen L, Aronen T, Julkunen-Tiitto R, Tiimonen H, Pihakaski- Maunsbach K, Jokipii S, Chen XW & Kallio PT (2003) Expression of Vitreoscilla haemoglobin in hybrid aspen (Populus tremula x tremuloides). Plant Biotech J 1: 287–300. Häggman H, Sutela S, Jokipii-Lukkari S, Ylioja T, Kasanen R, Anttila AK, Suokas M, Dumont E & Kallio PT (2011) The effects of heterologous VHb expression to the functioning of stress-related genes in hybrid aspen lines exposed to biotic stress (abstract). BMC Proceedings 5(Supp 7): P88. Häggman H, Raybould A, Borem A, Fox T, Handley L, Hertzberg M et al. (2013) Genetically engineered trees for plantation forests: key considerations for environmental risk assessment. Plant Biotech J 11: 785–798. Hall BG (2013) Building phylogenetic trees from molecular data with MEGA. Mol Biol Evol 30: 1229–1235. Halpin C, Thain SC, Tilston EL, Guiney E, Lapierre C & Hopkins DW (2007) Ecological impacts of trees with modified lignin. Tree Genet Genomes 3: 101–110. Hamberger B & Hahlbrock K (2004) The 4-coumarate:CoA ligase gene family in Arabidopsis thaliana comprises one rare, sinapate-activating and three commonly occurring isoenzymes. Proc Natl Acad Sci U S A 101: 2209–2214. Hamberger B, Ellis M, Friedmann M, Souza CDA, Barbazuk B & Douglas CJ (2007) Genome-wide analyses of phenylpropanoid-related genes in Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa: the Populus lignin toolbox and conservation and diversification of angiosperm gene families. Can J Bot -Rev Can Bot 85: 1182– 1201. Hancock JE, Loya WM, Giardina CP, Li L, Chiang VL & Pregitzer KS (2007) Plant growth, biomass partitioning and soil carbon formation in response to altered lignin biosynthesis in Populus tremuloides. New Phytol 173: 732–742. Harding SA, Leshkevich J, Chiang VL & Tsai CJ (2002) Differential substrate inhibition couples kinetically distinct 4-coumarate:coenzyme A ligases with spatially distinct metabolic roles in quaking aspen. Plant Physiol 128: 428–438.

65 Harfouche A, Meilan R, Kirst M, Morgante M, Boerjan W, Sabatti M & Mugnozza GS (2012) Accelerating the domestication of forest trees in a changing world. Trends Plant Sci 17: 64–72.

Hättenschwiler S & Schafellner C (2004) Gypsy moth feeding in the canopy of a CO2- enriched mature forest. Global Change Biol 10: 1899–1908. Hebelstrup KH, Hunt P, Dennis E, Bjerregaard Jensen S & Østergaard Jensen E (2006) Hemoglobin is essential for normal growth of Arabidopsis organs. Physiol Plantarum 127: 157–166. Hebelstrup KH, van Zanten M, Mandon J, Voesenek LACJ, Harren FJM, Cristescu SM, Moller IM & Mur LAJ (2012) Haemoglobin modulates NO emission and hyponasty under hypoxia-related stress in Arabidopsis thaliana. J Exp Bot 63: 5581–5591. Hemschemeier A, Duener M, Casero D, Merchant SS, Winkler M & Happe T (2013) Hypoxic survival requires a 2-on–2 hemoglobin in a process involving nitric oxide. Proc Natl Acad Sci U S A 110: 10854–10859. Hernandez-Garcia CM & Finer JJ (2014) Identification and validation of promoters and cis-acting regulatory elements. Plant Sci 217: 109–119. Hichri I, Barrieu F, Bogs J, Kappel C, Delrot S & Lauvergeat V (2011) Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway. J Exp Bot 62: 2465–2483. Hill RD (2012) Non-symbiotic haemoglobins—What's happening beyond nitric oxide scavenging? Aob Plants pls004. Hinchee M, Rottmann W, Mullinax L, Zhang C, Chang S, Cunningham M, Pearson L & Nehra N (2009) Short-rotation woody crops for bioenergy and biofuels applications. In Vitro Cell Dev Biol -Plant 45: 619–629. Hossain KK, Itoh RD, Yoshimura G, Tokuda G, Oku H, Cohen MF & Yamasaki H (2010) Effects of nitric oxide scavengers on thermoinhibition of seed germination in Arabidopsis thaliana. Russ J Plant Physl 57: 222–232. Hossain KK, Nakamura T & Yamasaki H (2011) Effects of nitric oxide leaf non- photochemical quenching of fluorescence under heat-stress conditions. Russ J Plant Physl 58: 629–633. Hu WJ, Kawaoka A, Tsai CJ, Lung JH, Osakabe K, Ebinuma H & Chiang VL (1998) Compartmentalized expression of two structurally and functionally distinct 4- coumarate:CoA ligase genes in aspen (Populus tremuloides). Proc Natl Acad Sci U S A 95: 5407–5412. Hu W, Harding S, Lung J, Popko J, Ralph J, Stokke D, Tsai C & Chiang V (1999) Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees. Nat Biotechnol 17: 808–812. Huan HV, Giangv HV, Thanh NV, Zhang S & Wang Y (2012) Identification and functional analysis of the Pm4CL1 gene in transgenic tobacco plant as the basis for regulating lignin biosynthesis in forest trees. Mol Breed 29: 173–180.

66 Humphreys JM, Hemm MR & Chapple C (1999) New routes for lignin biosynthesis defined by biochemical characterization of recombinant ferulate 5-hydroxylase, a multifunctional cytochrome P450-dependent monooxygenase. Proc Natl Acad Sci U S A 96: 10045–10050. Hunt PW, Watts RA, Trevaskis B, Llewelyn DJ, Burnell J, Dennis ES & Peacock WJ (2001) Expression and evolution of functionally distinct haemoglobin genes in plants. Plant Mol Biol 47: 677–692. Ihaka R & Gentleman R (1996) R: A language for data analysis and graphics. J Comput Graph Stat 5: 299–314. Isik F (2014) Genomic selection in forest tree breeding: the concept and an outlook to the future. New Forests 45: 379–401. Jaakola L, Pirttilä AM, Halonen M & Hohtola A (2001) Isolation of high quality RNA from bilberry (Vaccinium myrtillus L.) fruit. Mol Biotechnol 19: 201–203. Jaakola L, Pirttilä AM, Vuosku J & Hohtola A (2004) Method based on electrophoresis and gel extraction for obtaining genomic DNA-free cDNA without DNase treatment. BioTechniques 37: 744–748. Järvinen P, Palmé A, Morales LO, Lännenpää M, Keinänen M, Sopanen T & Lascoux M (2004) Phylogenetic relationships of Betula species (Betulaceae) based on nuclear ADH and chloroplast matK sequences. Am J Bot 91: 1834–1845. Jia C, Zhao H, Wang H, Xing Z, Du K, Song Y & Wei J (2004) Obtaining the transgenic poplars with low lignin content through down-regulation of 4CL. Chin Sci Bull 49: 905–909. Jiang WH, Liu JX, Zhang F & Gai B.J. (2009) Population dynamic of target pest, non- target pests and major natural enemy in transgenic hybrid poplar 741. J Shandong Agric Univ (Nat Sci) 40: 195–199. Johnson RH & Lincoln DE (1991) Sagebrush carbon allocation patterns and grasshopper

nutrition - the influence of CO2 enrichment and soil mineral limitation. Oecologia 87: 127–134. Jokipii S, Häggman H, Brader G, Kallio PT & Niemi K (2008) Endogenous PttHb1 and PttTrHb, and heterologous Vitreoscilla vhb haemoglobin gene expression in hybrid aspen roots with ectomycorrhizal interaction. J Exp Bot 59: 2449–2459. Jokipii-Lukkari S (2011) Endogenous haemoglobins and heterologous Vitreoscilla haemoglobin in hybrid aspen. Acta Univ Oul A 572 (PhD thesis). Jokipii-Lukkari S, Frey AD, Kallio PT & Häggman H (2009) Intrinsic non-symbiotic and truncated haemoglobins and heterologous Vitreoscilla haemoglobin expression in plants. J Exp Bot 60: 409–422. Jouanin L, Goujon T, de Nadaï V, Martin M, Mila I, Vallet C, Pollet B, Yoshinaga A, Chabbert B, Petit-Conil M & Lapierre C (2000) Lignification in transgenic poplars with extremely reduced caffeic acid O-methyltransferase activity. Plant Physiol 123: 1363–1373. Julkunen-Tiitto R, Rousi M, Bryant J, Sorsa S, Keinänen M & Sikanen H (1996) Chemical diversity of several Betulaceae species: Comparison of phenolics and terpenoids in northern birch stems. Trees-Struct Funct 11: 16–22.

67 Julkunen-Tiitto R & Sorsa S (2001) Testing the effects of drying methods on willow flavonoids, tannins, and salicylates. J Chem Ecol 27: 779–789. Kajita S, Katayama Y & Omori S (1996) Alterations in the biosynthesis of lignin in transgenic plants with chimeric genes for 4-coumarate:coenzyme A ligase. Plant Cell Physiol 37: 957–965. Kallio PT, Kim DJ, Tsai PS & Bailey JE (1994) Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions. Eur J Biochem 219: 201–208. Kaur R, Pathania R, Sharma V, Mande SC & Dikshit KL (2002) Chimeric Vitreoscilla hemoglobin (VHb) carrying a flavoreductase domain relieves nitrosative stress in Escherichia coli: New insight into the functional role of VHb. Appl Environ Microbiol 68: 152–160. Keinänen M, Julkunen-Tiitto R, Mutikainen P, Walls M, Ovaska J & Vapaavuori E (1999) Trade-offs in phenolic metabolism of silver birch: effects of fertilization, defoliation, and genotype. Ecology 80: 1970–1986. Khosla C & Bailey JE (1989) Characterization of the oxygen-dependent promoter of the Vitreoscilla hemoglobin gene in Escherichia coli. J Bacteriol 171: 5995–6004. Kim DY, Hong MJ, Lee YJ, Lee MB & Seo YW (2013) Wheat truncated hemoglobin interacts with photosystem I PSK-I subunit and photosystem II subunit PsbS1. Biol Plant 57: 281–290. Kitin P, Voelker SL, Meinzer FC, Beeckman H, Strauss SH & Lachenbruch B (2010) Tyloses and phenolic deposits in xylem vessels impede water transport in low-lignin transgenic poplars: a study by cryo-fluorescence microscopy. Plant Physiol 154: 887– 898. Koressaar T & Remm M (2007) Enhancements and modifications of primer design program Primer3. Bioinformatics 23: 1289–1291. Kota P, Guo DJ, Zubieta C, Noel J & Dixon RA (2004) O-methylation of benzaldehyde derivatives by "lignin specific" caffeic acid 3-O-methyltransferase. Phytochemistry 65: 837–846. Koutaniemi S, Warinowski T, Kärkönen A, Alatalo E, Fossdal CG, Saranpää P, Laakso T, Fagerstedt KV, Simola LK, Paulin L, Rudd S & Teeri TH (2007) Expression profiling of the lignin biosynthetic pathway in Norway spruce using EST sequencing and real- time RT-PCR. Plant Mol Biol 65: 311–328. Kumar A & Ellis BE (2003) 4-Coumarate:CoA ligase gene family in Rubus idaeus: cDNA structures, evolution, and expression. Plant Mol Biol 51: 327–340. Laitinen J, Julkunen-Tiitto R, Rousi M, Heinonen J & Tahvanainen J (2005) Ontogeny and environment as determinants of the secondary chemistry of three species of white birch. J Chem Ecol 31: 2243-2262. Laitinen ML, Julkunen-Tiitto R & Rousi M (2002) Foliar phenolic composition of European white birch during bud unfolding and leaf development. Physiol Plantarum 114: 450–460.

68 Laitinen ML, Julkunen-Tiitto R, Tahvanainen J, Heinonen J & Rousi M (2005) Variation in birch (Betula pendula) shoot secondary chemistry due to genotype, environment, and ontogeny. J Chem Ecol 31: 697–717. Laitinen ML, Julkunen-Tiitto R, Yamaji K, Heinonen J & Rousi M (2004) Variation in birch bark secondary chemistry between and within clones: implications for herbivory by hares. Oikos 104: 316–326. Laitinen ML, Julkunen-Tiitto R & Rousi M (2000) Variation in phenolic compounds within a birch (Betula pendula) population. J Chem Ecol 26: 1609–1622. Lamarche J, Stefani FOP, Séguin A & Hamelin RC (2011) Impact of endochitinase- transformed white spruce on soil fungal communities under greenhouse conditions. FEMS Microbiol Ecol 76: 199–208. Lapierre C, Pollet B, Petit-Conil M, Toval G, Romero J, Pilate G, Leplé J, Boerjan W, Ferret V, De Nadaï V & Jouanin L (1999) Structural alterations of lignins in transgenic poplars with depressed cinnamyl alcohol dehydrogenase or caffeic acid O- methyltransferase activity have an opposite impact on the efficiency of industrial kraft pulping. Plant Physiol 119: 153–163. Le SQ & Gascuel O (2008) An improved general amino acid replacement matrix. Mol Biol Evol 25: 1307–1320. LeBlanc PM, Hamelin RC & Filion M (2007) Alteration of soil rhizosphere communities following genetic transformation of white spruce. Appl Environ Microbiol 73: 4128– 4134. Lee D & Douglas CJ (1996) Two divergent members of a tobacco 4-coumarate:coenzyme A ligase (4CL) gene family - cDNA structure, gene inheritance and expression, and properties of recombinant proteins. Plant Physiol 112: 193–205. Lee D, Meyer K, Chapple C & Douglas C (1997) Antisense suppression of 4- coumarate:coenzyme A ligase activity in Arabidopsis leads to altered lignin subunit composition. Plant Cell 9: 1985–1998. Lemmetyinen J, Järvinen P, Pasonen H, Keinonen K, Lännenpää M & Keinänen M (2008) Birches. In: Kole C & Hall TC (eds) Compendium of transgenic crop plants: transgenic forest tree species. Oxford, Blackwell Publishing Ltd.: 193–218. Leplé J, Dauwe R, Morreel K, Storme V, Lapierre C, Pollet B et al. (2007) Downregula- tion of cinnamoyl-coenzyme a reductase in poplar: Multiple-level phenotyping reveals effects on cell wall polymer metabolism and structure. Plant Cell 19: 3669–3691. Li L, Zhou Y, Cheng X, Sun J, Marita J, Ralph J & Chiang V (2003) Combinatorial modification of multiple lignin traits in trees through multigene cotransformation. Proc Natl Acad Sci U S A 100: 4939–4944. Li LG, Lu SF & Chiang V (2006) A genomic and molecular view of wood formation. Crit Rev Plant Sci 25: 215–233. Li LG, Popko JL, Umezawa T & Chiang VL (2000) 5-Hydroxyconiferyl aldehyde modulates enzymatic methylation for syringyl monolignol formation, a new view of monolignol biosynthesis in angiosperms. J Biol Chem 275: 6537–6545.

Lindroth RL (2010) Impacts of elevated atmospheric CO2 and O3 on forests: phytochemistry, trophic interactions, and ecosystem dynamics. J Chem Ecol 36: 2–21.

69 Lira-Ruan V, Ruiz-Kubli M & Arredondo-Peter R (2011) Expression of non-symbiotic hemoglobin 1 and 2 genes in rice (Oryza sativa) embryonic organs. Commun Integr Biol 4: 457–458. Lloyd G & McCown B (1980) Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture. Proc Int Plant Propagators’ Soc 30: 421–427. Lu F, Marita JM, Lapierre C, Jouanin L, Morreel K, Boerjan W & Ralph J (2010) Sequencing around 5-hydroxyconiferyl alcohol-derived units in caffeic acid O- methyltransferase-deficient poplar lignins. Plant Physiol 153: 569–579. Maeda H & Dudareva N (2012) The shikimate pathway and aromatic amino acid biosynthesis in plants. Annu Rev Plant Biol 63: 73–105. Maeda H, Shasany AK, Schnepp J, Orlova I, Taguchi G, Cooper BR, Rhodes D, Pichersky E & Dudareva N (2010) RNAi suppression of arogenate dehydratase1 reveals that phenylalanine is synthesized predominantly via the arogenate pathway in petunia petals. Plant Cell 22: 832–849. Marita J, Ralph J, Hatfield R, Guo D, Chen F & Dixon R (2003) Structural and compositional modifications in lignin of transgenic alfalfa down-regulated in caffeic acid 3-O-methyltransferase and caffeoyl coenzyme A 3-O-methyltransferase. Phytochemistry 62: 53–65. Marita JM, Ralph J, Lapierre C, Jouanin L & Boerjan W (2001) NMR characterization of lignins from transgenic poplars with suppressed caffeic acid O-methyltransferase activity. J Chem Soc Perkin Trans 1: 2939–2945. Marx D (1969) The influence of ectotrophic fungi on the resistance of pine roots to pathogenic infections. I. Antagonism of mycorrhizal fungi to root pathogenic fungi and soil bacteria. Phytopathology 59: 153–163. Maury S, Geoffroy P & Legrand M (1999) Tobacco O-methyltransferases involved in phenylpropanoid metabolism. The different caffeoyl-coenzyme A/5-hydroxyferuloyl- coenzyme A 3/5-O-methyltransferase and caffeic acid/5-hydroxyferulic acid 3/5-O- methyltransferase classes have distinct substrate specificities and expression patterns. Plant Physiol 121: 215–223. Modess O (1941) Zur kenntnis der mykorrhizabildner von kiefer und fichte. Symb Bot Upsal 5: 1–147. Morreel K, Ralph J, Lu FC, Goeminne G, Busson R, Herdewijn P, Goeman JL, Van der Eycken J, Boerjan W & Messens E (2004) Phenolic profiling of caffeic acid O- methyltransferase-deficient poplar reveals novel benzodioxane oligolignols. Plant Physiol 136: 4023–4036. Morreel K, Goeminne G, Storme V, Sterck L, Ralph J, Coppieters W, Breyne P, Steenackers M, Georges M, Messens E & Boerjan W (2006) Genetical metabolomics of flavonoid biosynthesis in Populus: a case study. Plant J 47: 224–237. Mullin TJ & Lee SJ (2013) Best practise for tree breeding in Europe. Stockholm, Skogforsk.

70 Muzac I, Wang J, Auzellotti D, Zhang H & Ibrahim R (2000) Functional expression of an Arabidopsis cDNA clone encoding a flavonol 3'-O-methyltransferase and characterization of the gene product. Arch Biochem Biophys 375: 385–388. Nakabayashi R, Yamazaki M & Saito K (2010) A polyhedral approach for understanding flavonoid biosynthesis in Arabidopsis. New Biotech 27: 829–836. Nakatsubo T, Kitamura Y, Sakakibara N, Mizutani M, Hattori T, Sakurai N, Shibata D, Suzuki S & Umezawa T (2008) At5g54160 gene encodes Arabidopsis thaliana 5- hydroxyconiferaldehyde O-methyltransferase. J Wood Sci 54: 312–317. Ni WT, Paiva NL & Dixon RA (1994) Reduced lignin in transgenic plants containing a caffeic acid O-methyltransferase antisense gene. Transgenic Res 3: 120–126. Novaes E, Kirst M, Chiang V, Winter-Sederoff H & Sederoff R (2010) Lignin and biomass: a negative correlation for wood formation and lignin content in trees. Plant Physiol 154: 555–561. Ohwaki Y, Kawagishi-Kobayashi M, Wakasa K, Fujihara S & Yoneyama T (2005) Induction of class-1 non-symbiotic hemoglobin genes by nitrate, nitrite and nitric oxide in cultured rice cells. Plant Cell Physiol 46: 324–331. Osakabe Y, Kajita S & Osakabe K (2011) Genetic engineering of woody plants: current and future targets in a stressful environment. Physiol Plantarum 142: 105–117.

Oksanen E, Sober J & Karnosky D (2001) Impacts of elevated CO2 and/or O3 on leaf ultrastructure of aspen (Populus tremuloides) and birch (Betula papyrifera) in the Aspen FACE experiment. Environ Pollut 115: 437–446. Pan X, Li H, Wei H, Su W, Jiang X & Lu H (2013) Analysis of the spatial and temporal expression pattern directed by the Populus tomentosa 4-coumarate:CoA ligase Pto4CL2 promoter in transgenic tobacco. Mol Biol Rep 40: 2309–2317. París R, Iglesias MJ, Terrile MC & Casalongué CA (2013) Functions of S-nitrosylation in plant hormone networks. Front Plant Sci 4: 294. París R, Lamattina L & Casalongué CA (2007) Nitric oxide promotes the wound-healing response of potato leaflets. Plant Physiol Biochem 45: 80–86. Park K, Kim K, Howard AJ, Stark BC & Webster DA (2002) Vitreoscilla hemoglobin binds to subunit I of cytochrome bo ubiquinol oxidases. J Biol Chem 277: 33334– 33337. Parvathi K, Chen F, Guo DJ, Blount JW & Dixon RA (2001) Substrate preferences of O- methyltransferases in alfalfa suggest new pathways for 3-O-methylation of monolignols. Plant J 25: 193–202. Peltonen PA, Vapaavuori E & Julkunen-Tiitto R (2005) Accumulation of phenolic compounds in birch leaves is changed by elevated carbon dioxide and ozone. Global Change Biol 11: 1305–1324. Perazzolli M, Dominici P, Romero-Puertas M, Zago E, Zeier J, Sonoda M, Lamb C & Delledonne M (2004) Arabidopsis nonsymbiotic hemoglobin AHb1 modulates nitric oxide bioactivity. Plant Cell 16: 2785–2794. Perazzolli M, Romero-Puertas MC & Delledonne M (2006) Modulation of nitric oxide bioactivity by plant haemoglobins. J Exp Bot 57: 479–488.

71 Peters DJ & Constabel CP (2002) Molecular analysis of herbivore-induced condensed tannin synthesis: cloning and expression of dihydroflavonol reductase from trembling aspen (Populus tremuloides). Plant J 32: 701–712. Peterson M, hams U & Matern U (2010) Biosynthesis of phenylpropanoids and related compounds. In: Wink E (ed) Biochemistry of plant secondary metabolism. Annual plant reviews volume 40, Singapore, Wiley-Blackwell: 182–257. Pilate G, Guiney E, Holt K, Petit-Conil M, Lapierre C, Leplé J-, Pollet B, Mila I, Webster EA, Marstorp HG, Hopkins DW, Jouanin L, Boerjan W, Schuch W, Cornu D & Halpin C (2002) Field and pulping performances of transgenic trees with altered lignification. Nat Biotechnol 20: 607–612. Piquemal J, Chamayou S, Nadaud I, Beckert M, Barrière Y, Mila I, Lapierre C, Rigau J, Puigdomenech P, Jauneau A, Digonnet C, Boudet A-, Goffner D & Pichon M (2002) Down-regulation of caffeic acid O-methyltransferase in maize revisited using a transgenic approach. Plant Physiol 130: 1675–1685. Piquemal J, Lapierre C, Myton K, O'Connell A, Schuch W, Grima-Pettenati J & Boudet AM (1998) Down-regulation of cinnamoyl-CoA reductase induces significant changes of lignin profiles in transgenic tobacco plants. Plant J 13: 71–83. Polle A, Janz D, Teichmann T & Lipka V (2013) Poplar genetic engineering: promoting desirable wood characteristics and pest resistance. Appl Microbiol Biotechnol 97: 5669–5679. Porter LJ, Hrstich LN & Chan BG (1986) The conversion of procyanidins and prodelphinidins to cyanidin and delphinidin. Phytochemistry 25: 223–230. Pringsheim EG (1951) The Vitreoscillaceae; a family of colourless, gliding, filamentous organisms. J Gen Microbiol 5: 124–49. Raes J, Rohde A, Christensen J, Van de Peer Y & Boerjan W (2003) Genome-wide characterization of the lignification toolbox in Arabidopsis. Plant Physiol 133: 1051– 1071. Ragauskas AJ, Beckham GT, Biddy MJ, Chandra R, Chen F, Davis MF, Davison BH, Dixon RA, Gilna P, Keller M, Langan P, Naskar AK, Saddler JN, Tschaplinski TJ, Tuskan GA & Wyman CE (2014) Lignin valorization: improving lignin processing in the biorefinery. Science 344: 1246843. Ralph J, Lapierre C, Lu F, Marita J, Pilate G, Van Doorsselaere J, Boerjan W & Jouanin L (2001a) NMR evidence for benzodioxane structures resulting from incorporation of 5- hydroxyconiferyl alcohol into lignins of O-methyltransferase-deficient poplars. J Agric Food Chem 49: 86–91. Ralph J, Lapierre C, Marita J, Kim H, Lu F, Hatfield R, Ralph S, Chapple C, Franke R, Hemm M, Van Doorsselaere J, Sederoff R, O'Malley D, Scott J, MacKay J, Yahiaoui N, Boudet A, Pean M, Pilate G, Jouanin L & Boerjan W (2001b) Elucidation of new structures in lignins of CAD- and COMT-deficient plants by NMR. Phytochemistry 57: 993–1003. Ramandeep, Hwang KW, Raje M, Kim K, Stark BC, Dikshit KL & Webster DA (2001) Vitreoscilla hemoglobin - Intracellular localization and binding to membranes. J Biol Chem 276: 24781–24789.

72 Rastogi S & Dwivedi UN (2008) Manipulation of lignin in plants with special reference to O-methyltransferase. Plant Sci 174: 264–277. Rolando C, Monties B & Lapierre C (1992) Thioacidolysis. In: Lin SY & Dence CW (eds) Methods in lignin chemistry. Berlin, Springer-Verlag: 334–349. Saito K, Yonekura-Sakakibara K, Nakabayashi R, Higashi Y, Yamazaki M, Tohge T & Fernie AR (2013) The flavonoid biosynthetic pathway in Arabidopsis: structural and genetic diversity. Plant Physiol Biochem 72: 21–34. Sander JD & Joung JK (2014) CRIRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32: 347–355. Sang Y, Millwood RJ & Stewart CN,Jr. (2013) Gene use restriction technologies for transgenic plant bioconfinement. Plant Biotechnology Journal 11: 649–658. Satake H, Ono E & Murata J (2013) Recent advances in the metabolic engineering of lignan biosynthesis pathways for the production of transgenic plant-based foods and supplements. J Agric Food Chem 61: 11721–11729. Schneider K, Hövel K, Witzel K, Hamberger B, Schomburg D, Kombrink E & Stuible H (2003) The substrate specificity-determining amino acid code of 4-coumarate:CoA ligase. Proc Natl Acad Sci U S A 100: 8601–8606. Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler D & Dean D (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62: 775–806. Schnitzler F, Burgess EPJ, Kean AM, Philip BA, Barraclough EI, Malone LA & Walter C (2010) No unintended impacts of transgenic pine (Pinus radiata) trees on above ground invertebrate communities. Environ Entomol 39: 1359–1368. Séguin A, Lachance D, Déjardin A, Leplé J & Pilate G (2014) Scientific research related to genetically modified trees. In: Fenning T (ed) Challenges and opportunities for the world’s forests in the 21st century. Dordrecht, Springer Verlag: 525–548. Seppänen S, Pasonen H, Vauramo S, Vahala J, Toikka M, Kilpeläinen I, Setälä H, Teeri TH, Timonen S & Pappinen A (2007) Decomposition of the leaf litter and mycorrhiza forming ability of silver birch with a genetically modified lignin biosynthesis pathway. Appl Soil Ecol 36: 100–106. Seregélyes C, Igamberdiev A, Maassen A, Hennig J, Dudits D & Hill R (2004) NO- degradation by alfalfa class 1 hemoglobin (Mhb 1): a possible link to PR-1a gene expression in Mhb1-overproducing tobacco plants. FEBS Lett 571: 61–66. Sewalt VJH, Ni WT, Jung HG & Dixon RA (1997) Lignin impact on fiber degradation: Increased enzymatic digestibility of genetically engineered tobacco (Nicotiana tabacum) stems reduced in lignin content. J Agric Food Chem 45: 1977–1983. Shi R, Sun Y, Li Q, Heber S, Sederoff R & Chiang VL (2010) Towards a systems approach for lignin biosynthesis in Populus trichocarpa: transcript abundance and specificity of the monolignol biosynthetic genes. Plant Cell Physiol 51: 144–163. Shimoda Y, Shimoda-Sasakura F, Kucho K, Kanamori N, Nagata M, Suzuki A, Abe M, Higashi S & Uchiumi T (2009) Overexpression of class 1 plant hemoglobin genes enhances symbiotic nitrogen fixation activity between Mesorhizobium loti and Lotus japonicus. Plant J 57: 254–263.

73 Silber MV, Meimberg H & Ebel J (2008) Identification of a 4-coumarate:CoA ligase gene family in the moss, Physcomitrella patens. Phytochemistry 69: 2449–2456. Smagghe BJ, Hoy JA, Percifield R, Kundu S, Hargrove MS, Sarath G, Hilbert J, Watts RA, Dennis ES, Peacock WJ, Dewilde S, Moens L, Blouin GC, Olson JS & Appleby CA (2009) Correlations between oxygen affinity and sequence classifications of plant hemoglobins. Biopolymers 91: 1083–1096. Smyth GK (2005) Limma: linear models for microarray data. In: Gentleman R, Carey V, Dudoit S, Irizarry R & Huber W (eds) Bioinformatics and Computational Biology Solutions using R and Bioconductor. New York, Springer: 397–420. Soltani BM, Ehlting J, Hamberger B & Douglas CJ (2006) Multiple cis-regulatory elements regulate distinct and complex patterns of developmental and wound-induced expression of Arabidopsis thaliana 4CL gene family members. Planta 224: 1226–1238. Soltis D & Soltis P (2003) The role of phylogenetics in comparative genetics. Plant Physiol 132: 1790–1800. Stark BC, Dikshit KL & Pagilla KR (2011) Recent advances in understanding the structure, function, and biotechnological usefulness of the hemoglobin from the bacterium Vitreoscilla. Biotechnol Lett 33: 1705–1714. Stefani FOP & Hamelin RC (2010) Current state of genetically modified plant impact on target and non-target fungi. Env Rev 18: 441–475. Stout A, Davis AA, Domec JC, Yang C, Shi R & King JS (2014) Growth under field conditions affects lignin content and productivity in transgenic Populus trichocarpa with altered lignin biosynthesis. Biomass Bioenergy 68: 228–239. Strohl WR (2005) Genus XII. Vitreoscilla Pringsheim 1949c, 70AL. In: Garrity GM (ed) Bergey's manual of systematic bacteriology. New York NY, Springer: 851–858. Sun H, Li Y, Feng S, Zou W, Guo K, Fan C, Si S & Peng L (2013) Analysis of five rice 4- coumarate:coenzyme A ligase enzyme activity and stress response for potential roles in lignin and flavonoid biosynthesis in rice. Biochem Biophys Res Commun 430: 1151–1156. Suzuki S & Suzuki H (2014) Recent advances in forest tree biotechnology. Plant Biotechnol 31: 1–9. Tamura K (1992) Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G+C-content biases. Mol Biol Evol 9: 678–687. Tamura K, Stecher G, Peterson D, Filipski A & Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30: 2725–2729. Tavaré S (1986) Some probabilistic and statistical problems in the analysis of DNA sequences. In: Miura RM (ed) Some mathematical questions in biology: DNA sequence analysis. Providence RI, Am Math Soc: 57–86. Tian X, Xie J, Zhao Y, Lu H, Liu S, Qu L, Li J, Gai Y & Jiang X (2013) Sense-, antisense- and RNAi-4CL1 regulate soluble phenolic acids, cell wall components and growth in transgenic Populus tomentosa Carr. Plant Physiol Biochem 65: 111–119. Tiimonen H, Aronen T, Laakso T, Saranpää P, Chiang V, Häggman H & Niemi K (2008) Paxillus involutus forms an ectomycorrhizal symbiosis and enhances survival of PtCOMT-modified Betula pendula in vitro. Silvae Genet 57: 235–242.

74 Tiimonen H, Aronen T, Laakso T, Saranpää P, Chiang V, Ylioja T, Roininen H & Häggman H (2005) Does lignin modification affect feeding preference or growth performance of insect herbivores in transgenic silver birch (Betula pendula Roth)? Planta 222: 699–708. Tiimonen H, Häggman H, Tsai CJ, Chiang V & Aronen T (2007) The seasonal activity and the effect of mechanical bending and wounding on the PtCOMT promoter in Betula pendula Roth. Plant Cell Rep 26: 1205–1214. Tiso M, Tejero J, Kenney C, Frizzell S & Gladwin MT (2012) Nitrite reductase activity of nonsymbiotic hemoglobins from Arabidopsis thaliana. Biochemistry 51: 5285–5292. Tohge T, de Souza LP & Fernie AR (2014) Genome-enabled plant metabolomics. J Chromatogr B 966: 7–20. Tohge T, Watanabe M, Hoefgen R & Fernie AR (2013a) The evolution of phenylpropanoid metabolism in the green lineage. Crit Rev Biochem Mol Biol 48: 123–152. Tohge T, Watanabe M, Hoefgen R & Fernie AR (2013b) Shikimate and phenylalanine biosynthesis in the green lineage. Front Plant Sci 4: 62. Tohge T, Yonekura-Sakakibara K, Niida R, Watanabe-Takahashi A & Saito K (2007) Phytochemical genomics in Arabidopsis thaliana: a case study for functional identification of flavonoid biosynthesis genes. Pure Appl Chem 79: 811–823. Tsai CJ, Popko JL, Mielke MR, Hu W-, Podila GK & Chiang VL (1998) Suppression of O-methyltransferase gene by homologous sense transgene in quaking aspen causes red-brown wood phenotypes. Plant Physiol 117: 101–112. Tsai C, Harding SA, Tschaplinski TJ, Lindroth RL & Yuan Y (2006) Genome-wide analysis of the structural genes regulating defense phenylpropanoid metabolism in Populus. New Phytol 172: 47–62. Tsai PS, Kallio PT & Bailey JE (1995) Fnr, a global transcriptional regulator of Escherichia coli, activates the Vitreoscilla hemoglobin (VHb) promoter and intracellular VHb expression increases cytochrome d promoter activity. Biotechnol Prog 11: 288–293. Tuskan GA, DiFazio S, Jansson S, Bohlmann J, Grigoriev I, Hellsten U et al. (2006) The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 313: 1596–1604. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M & Rozen SG (2012) Primer3 - new capabilities and interfaces. Nucleic Acids Res 40: e115. Valjakka M, Aronen T, Kangasjärvi J, Vapaavuori E & Häggman H (2000) Genetic transformation of silver birch (Betula pendula) by particle bombardment. Tree Physiol 20: 607–613. Van Doorsselaere J, Baucher M, Chognot E, Chabbert B, Tollier M-, Petit-Conil M, Leple J-, Pilate G, Cornu D, Monties B, Van Montagu M, Inzé D, Boerjan W & Jouanin L (1995) A novel lignin in poplar trees with a reduced caffeic acid/5-hydroxyferulic acid O-methyltransferase activity. Plant J 8: 855–864.

75 Vanholme B, Cesarino I, Goeminne G, Kim H, Marroni F, Van Acker R, Vanholme R, Morreel K, Ivens B, Pinosio S, Morgante M, Ralph J, Bastien C & Boerjan W (2013) Breeding with rare defective alleles (BRDA): a natural Populus nigra HCT mutant with modified lignin as a case study. New Phytol 198: 765–776. Vanholme R, Cesarino I, Rataj K, Xiao Y, Sundin L, Goeminne G et al. (2013) Caffeoyl shikimate esterase (CSE) is an enzyme in the lignin biosynthetic pathway in Arabidopsis. Science 341: 1103–1106. Vanholme R, Morreel K, Ralph J & Boerjan W (2008) Lignin engineering. Curr Opin Plant Biol 11: 278–285. Vanholme R, Storme V, Vanholme B, Sundin L, Christensen JH, Goeminne G, Halpin C, Rohde A, Morreel K & Boerjan W (2012) A systems biology view of responses to lignin biosynthesis perturbations in Arabidopsis. Plant Cell 24: 3506–3529. Vauramo S, Pasonen H, Pappinen A & Setälä H (2006) Decomposition of leaf litter from chitinase transgenic silver birch (Betula pendula) and effects on decomposer populations in a field trial. Appl Soil Ecol 32: 338–349. Vázquez-Limón C, Hoogewijs D, Vinogradov SN & Arredondo-Peter R (2012) The evolution of land plant hemoglobins. Plant Sci 191: 71–81. Vihervuori L, Pasonen H & Lyytikäinen-Saarenmaa P (2008) Density and composition of an insect population in a field trial of chitinase transgenic and wild-type silver birch (Betula pendula) clones. Environ Entomol 37: 1582–1591. Vining KJ, Contreras RN, Ranik M & Strauss SH (2012) Genetic methods for mitigating invasiveness of woody ornamental plants: research needs and opportunities. HortScience 47: 1210–1216. Vinogradov SN, Hoogewijs D, Bailly X, Mizuguchi K, Dewilde S, Moens L & Vanfleteren JR (2007) A model of globin evolution. Gene 398: 132–142. Voelker SL, Lachenbruch B, Meinzer FC, Jourdes M, Ki C, Patten AM et al. (2010) Antisense down-regulation of 4CL expression alters lignification, tree growth, and saccharification potential of field-grown poplar. Plant Physiol 154: 874–886. Voelker SL, Lachenbruch B, Meinzer FC, Kitin P & Strauss SH (2011a) Transgenic poplars with reduced lignin show impaired xylem conductivity, growth efficiency and survival. Plant Cell Environ 34: 655–668. Voelker SL, Lachenbruch B, Meinzer FC & Strauss SH (2011b) Reduced wood stiffness and strength, and altered stem form, in young antisense 4CL transgenic poplars with reduced lignin contents. New Phytol 189: 1096–1109. Wagner A, Donaldson L, Kim H, Phillips L, Flint H, Steward D, Torr K, Koch G, Schmitt U & Ralph J (2009) Suppression of 4-coumarate-CoA ligase in the coniferous gymnosperm Pinus radiata. Plant Physiol 149: 370–383. Wakabayashi S, Matsubara H & Webster DA (1986) Primary sequence of a dimeric bacterial hemoglobin from Vitreoscilla. Nature 322: 481–483. Walter C, Fladung M & Boerjan W (2010) The 20-year environmental safety record of GM trees. Nat Biotechnol 28: 656–658.

76 Wang C, Zhang N, Gao C, Cui Z, Sun D, Yang C & Wang Y (2014b) Comprehensive transcriptome analysis of developing xylem responding to artificial bending and gravitational stimuli in Betula platyphylla. PLOS ONE 9: e87566. Wang JP, Naik PP, Chen H, Shi R, Lin C, Liu J et al. (2014a) Complete proteomic-based enzyme reaction and inhibition kinetics reveal how monolignol biosynthetic enzyme families affect metabolic flux and lignin in Populus trichocarpa. Plant Cell 26: 894– 914. Wang N, Thomson M, Bodles WJA, Crawford RMM, Hunt HV, Featherstone AW, Pellicer J & Buggs RJA (2013) Genome sequence of dwarf birch (Betula nana) and cross-species RAD markers. Mol Ecol 22: 3098–3111. Wang Z, Xiao Y, Chen W, Tang K & Zhang L (2009) Functional expression of Vitreoscilla hemoglobin (VHb) in Arabidopsis relieves submergence, nitrosative, photo-oxidative stress and enhances antioxidants metabolism. Plant Sci 176: 66–77. Wen W, Li D, Li X, Gao Y, Li W, Li H, Liu J, Liu H, Chen W, Luo J & Yan J (2014) Metabolome-based genome-wide association study of maize kernel leads to novel biochemical insights. Nat Commun 5: 3438. Weng J & Chapple C (2010) The origin and evolution of lignin biosynthesis. New Phytol 187: 273–285. White TL, Adams WT & Neale DB (2007) Forest genetics. Cambridge MA, CAB International.

Will R & Ceulemans R (1997) Effects of elevated CO2 concentration on photosynthesis, respiration and carbohydrate status of coppice Populus hybrids. Physiol Plantarum 100: 933–939. Wink E (2010) Introduction: biochemistry, physiology and ecological functions of secondary metabolites. In: Wink E (ed) Biochemistry of plant secondary metabolism. Annual plant reviews volume 40, Singapore, Wiley-Blackwell: 1–19. Wünsche H, Baldwin IT & Wu J (2011) S-Nitrosoglutathione reductase (GSNOR) mediates the biosynthesis of jasmonic acid and ethylene induced by feeding of the insect herbivore Manduca sexta and is important for jasmonate-elicited responses in Nicotiana attenuata. J Exp Bot 62: 4605–4616. Xu Z, Zhang D, Hu J, Zhou X, Ye X, Reichel KL et al. (2009) Comparative genome analysis of lignin biosynthesis gene families across the plant kingdom. BMC Bioinformatics 10: S3. Yang JG, Webster DA & Stark BC (2005) ArcA works with Fnr as a positive regulator of Vitreoscilla (bacterial) hemoglobin gene expression in Escherichia coli. Microbiol Res 160: 405–415. Yoo H, Widhalm JR, Qian Y, Maeda H, Cooper BR, Jannasch AS, Gonda I, Lewinsohn E, Rhodes D & Dudareva N (2013) An alternative pathway contributes to phenylalanine biosynthesis in plants via a cytosolic tyrosine:phenylpyruvate aminotransferase. Nat Commun 4: 2833. Yu QB (2001) Can physiological and anatomical characters be used for selecting high yielding hybrid aspen clones? Silva Fenn 35: 137–146.

77 Zhang B, Chen M, Zhang X, Luan H, Tian Y & Su X (2011) Expression of Bt-Cry3A in transgenic Populus alba x P. glandulosa and its effects on target and non-target pests and the arthropod community. Transgenic Res 20: 523–532. Zhang J, Nieminen K, Serra JAA & Helariutta Y (2014) The formation of wood and its control. Curr Opin Plant Biol 17: 56–63. Zhang Q, Zhang Z, Lin S, Lin Y & Yang L (2005) Assessment of rhizispheric microorganisms of transgenic Populus tomentosa with cowpea trypsin inhibito (CpTI) gene. For Stud China 7: 28–34. Zhao Q & Dixon RA (2011) Transcriptional networks for lignin biosynthesis: more complex than we thought? Trends Plant Sci 16: 227–233. Zubieta C, Kota P, Ferrer JL, Dixon RA & Noel JP (2002) Structural basis for the modulation of lignin monomer methylation by caffeic acid/5-hydroxyferulic acid 3/5- O-methyltransferase. Plant Cell 14: 1265–1277.

78 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

80 80 Appendix 1. Effects of 4CL antisense constructs to lignin and phenolics

Table 6. The effects of 4CL antisense constructs to lignin and phenolic compounds.

Plant species Gene Lignin content Lignin composition Changed phenolics Reference Arabidopsis thaliana (L.) E Decreased up to 50% Decreased G units Lee et al. 1997 Heynh. Populus tremuloides Michx. E Decreased up to 45% No effect Increased hydroxycinnamic acids Hu et al. 1999 E Decreased up to 40% Decreased S/G ratio 1 NA Li et al. 2003 Populus tomentosa Carr. E Decreased up to 42% NA Accumulation of red coloured Jia et al. 2004 compounds in wood E Decreased up to 28% Decreased G & H, increased S units Increased hydroxycinnamic acids Tian et al. 2013 Populus tremula L. × alba L. H Decreased up to 50% Increased S/G ratio 1 Accumulation of phenolics 1 Voelker et al. 2010 Populus trichocarpa E Decreased up to 46% Increased or decreased S/G ratio 1 Stout et al. 2014 Nicotiana tabacum L. E Decreased 13% Increased H, decreased V & S units Increased hydroxycinnamic acids Kajita et al. 1996 Oryza sativa (L.) japonica E Decreased 21% Decreased H, G & S units Increased 4-coumarate & ferulate Gui et al. 2011 Betula pendula Roth E No change No change NA Seppänen et al. 2007 Pinus radiata D. Don E up to 50% Decreased G units Increased phenolics Wagner et al. 2009 1Variation among lines; E = endogenous; H = heterologous; NA = data not available; *potentially flavonoids or tannin-like compou nds

Appendix 2. Effects of COMT cons tructs to to lig nin and phenolics

Table 7. The effects of COMT constructs to lignin and phenolic compounds.

Plant species Gene, Lignin content Lignin composition Changed phenolics Reference Orientation Zea mays L. E, as Reduced up to 24% Reduced S, 5-OH-G U Reduced p-coumaric acids Piquemal et al. 2002 E, as Reduced up to 45% Reduced S U 1 NA Guillaumie et al. 2008 Medicago sativa L. E, as Reduced 11% Reduced G & S U No change * Guo et al. 2001, Marita et al. 2003 Populus tremula L. × H, s Reduced 17% + Reduced S, 5-OH-G U 2,+ Stems more brown Jouanin et al. 2000 alba L. H, as No effect Reduced S/G ratio 3, 5-OH-G U Pale rose stems, altered Van Doorsselaere et al. 1995, methanol-soluble phenolics** Lapierre et al. 1999, Pilate et al. 2002 Populus tremuloides E, s No effect Reduced S/G ratio 3, 5-OH-G 2 U Reddish-brown wood Tsai et al. 1998 Michx. E, as No effect NA No change in the colour*** Boerjan et al. 1997 Nicotiana tabacum E, as No effect Reduced S/G ratio 3, 5-OH-G U NA Atanossova et al. 1995 L. E, s No effect Reduced S/G ratio 3, 5-OH-G U 4 NA Atanossova et al. 1995 H, as No effect Reduced S U NA Dwivedi et al. 1994 H, as Reduced No change NA Ni et al. 1994 H, as Reduced Increased S/G ratio NA Sewalt et al. 1997 Betula pendula H, s No effect Reduced S/G ratio, 5-OH-G U NA Aronen et al. 2003, Tiimonen et al. Roth. 2005 E = endogenous; H = heterologous; as = antisense; s = sense; U = units; NA = data not available; 1Sclerenchyma-specific; 2Variation among lines; 3Increased G and reduced S units; 4In most of the s lines COMT activity and expression increased, however, in one line (A17) activity and expression were drastica lly reduced; +In most of the s lines COMT was overexpressed and no changes in lignin, but in one line (70SOMT-3) the COMT activity was reduced drastically and line showed altered lignin properties; *In wall-bound (or soluble Kota et al. 2004) hydroxycinnamic acids; **Morreel et al. 2004; ***stated in Tsai et al. 1998 81

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,

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)

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).

Original publications

Original publications are not included in the electronic version of the dissertation.

86 ACTA UNIVERSITATIS OULUENSIS SERIES A SCIENTIAE RERUM NATURALIUM

628. Päätalo, Heli (2014) Stakeholder interactions in cross-functional productization : the case of mobile software development 629. Koskela, Timo (2014) Interaction in asset-based value creation within innovation networks : the case of software industry 630. Stibe, Agnis (2014) Socially influencing systems : persuading people to engage with publicly displayed Twitter-based systems 631. Sutor, Stephan R. (2014) Large-scale high-performance video surveillance 632. Niskanen, Alina (2014) Selection and genetic diversity in the major histocompatibility complex genes of wolves and dogs 633. Tuomikoski, Sari (2014) Utilisation of gasification carbon residues : activation, characterisation and use as an adsorbent 634. Hyysalo, Jarkko (2014) Supporting collaborative development : cognitive challenges and solutions of developing embedded systems 635. Immonen, Ninna (2014) Glaciations and climate in the Cenozoic Arctic : evidence from microtextures of ice-rafted quartz grains 636. Kekkonen, Päivi (2014) Characterization of thermally modified wood by NMR spectroscopy : microstructure and moisture components 637. Pietilä, Heidi (2014) Development of analytical methods for ultra-trace determination of total mercury and methyl mercury in natural water and peat soil samples for environmental monitoring 638. Kortelainen, Tuomas (2014) On iteration-based security flaws in modern hash functions 639. Holma-Suutari, Anniina (2014) Harmful agents (PCDD/Fs, PCBs, and PBDEs) in Finnish reindeer (Rangifer tarandus tarandus) and moose (Alces alces) 640. Lankila, Tiina (2014) Residential area and health : a study of the Northern Finland Birth Cohort 1966 641. Zhou, Yongfeng (2014) Demographic history and climatic adaptation in ecological divergence between two closely related parapatric pine species 642. Kraus, Klemens (2014) Security management process in distributed, large scale high performance systems 643. Toivainen, Tuomas (2014) Genetic consequences of directional selection in Arabidopsis lyrata

Book orders: Granum: Virtual book store http://granum.uta.fi/granum/ A 644 OULU 2014 A 644

UNIVERSITY OF OULU P.O.BR[ 00 FI-90014 UNIVERSITY OF OULU FINLAND ACTA UNIVERSITATISUNIVERSITATIS OULUENSISOULUENSIS ACTA UNIVERSITATIS OULUENSIS ACTAACTA

SERIES EDITORS SCIENTIAESCIENTIAEA A RERUMRERUM Suvi Sutela NATURALIUMNATURALIUM

EDITOR IN CHIEF Professor Olli Vuolteenaho PUBLICATIONS EDITOR