Volatile-Mediated Plant-To-Plant Communication in Hybrid Aspens in Ozone-Enriched Atmosphere

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Volatile-mediated plant-to-plant communication in hybrid aspens in ozone-enriched atmosphere

Mari Vesala Master’s thesis Program of biosciences, Plant biotechnology University of Eastern Finland, Department of Biosciences February 2013 ABSTRACT UNIVERSITY OF EASTERN FINLAND, Kuopio campus, Faculty of Science and Forestry, Program of biosciences, Plant biotechnology VESALA MARI HANNELI: Volatile-mediated plant-to-plant communication in hybrid aspens in ozone-enriched atmosphere Master’s thesis 65 pages Supervisors: Ph.D Arja Tervahauta, Ph.D Tao Li and Ph.D James Blande February 2013 ______Key words: VOC, plant defense, ozone exposure, hybrid aspen, Phyllodecta laticollis

Plants emit a wide array of volatile organic compounds (VOCs) when exposed to herbivores. These compounds protect the plants from damage caused by this stress. They also act as signaling molecules warning intact neighboring plants of the presence of herbivores. Receiving VOCs causes changes in the expression of defense genes and leaves the plants better equipped against an incoming herbivore attack.

Tropospheric ozone is the major air pollutant causing significant damage to plants. In addition, it interferes with VOC mediated ecological interactions such as plant-to-plant communication through degrading important signaling compounds. The concentrations of ozone continue increasing along with human activities producing ozone precursors. VOC communication is a considerable mechanism which protects plants from damage caused by herbivores. Hence, it is important to know how great an impact higher ozone concentration poses on the well-being of plants.

In this thesis, a chamber experiment was conducted in order to study the ability of herbivore- induced VOCs to prime the defense mechanisms of receiver plants in ozone exposure. Hybrid aspens (Populus tremula x P.tremuloides) were used as experimental plants. Changes in the expression of three defense genes were measured with quantitative reverse-transcribed polymerase chain reaction (qRT-PCR). The genes studied were 1-deoxy-D-xylulose-5- phosphate reductoisomerase (DXR), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and lipoxygenase 1 (LOX1). DXR and HMGR are key enzymes in two different branches of terpenoid biosynthesis pathways, DXR in MEP pathway and HMGR in MVA pathway. LOX1 is a key enzyme in octadecanoid pathway producing jasmonic acid and green leaf volatiles. In addition, the gene expression of the whole genome was analyzed using NGS.

Direct defense elicitation or defense priming was not seen in the gene expression analysis. However, these phenomena were observed in VOC emission measurements, 22 hours after herbivore infestation in ambient air but not in the ozone-enriched air, suggesting that ozone could disrupt plant-to-plant communication. It might also be interesting that MEP and MVA pathways which produce the same terpenoid precursors seemed to respond differently to herbivore damage. Ozone seemed to reduce the expression of all the defense genes. Changes in the transcription of DXR and LOX1 seemed to indicate the impairing effect of ozone on VOC communication at least in some time points.

FOREWORD

This master’s thesis was done in the cooperation of the Department of Biosciences and the Department of Environmental Sciences, in the Faculty of Science and Forestry, at the University of Eastern Finland, Kuopio campus during the academic year 2012-2013.

I wish to thank my supervisors Ph.D Arja Tervahauta, Ph.D Tao Li and Ph.D James Blande for the opportunity to work in their project. Their inspiring guidance and ability to answer my questions during the process were irreplaceable. I also want to thank Daniel Blande for his contribution in the statistical analysis and the staff of the Department of Biosciences for their help with the laboratory work and the data analyses.

Thanks are due to the Kuopio Naturalists' Society for awarding a scholarship which enabled me to concentrate on the thesis.

Special thanks to my parents and to my boyfriend Teemu for the support and the conversations which eventually helped me go the distance.

ABBREVIATIONS

ABA Abscisic acid

ACC 1-aminocyclopropane-1-carboxylic acid amb ctrl Ambient control treatment where intact emitter plants and receiver plants are infested with Phyllodecta laticollis larvae are in ambient air in an experimental chamber amb dam Ambient damaged treatment where emitter plants and receiver plants are infested with P. laticollis larvae in ambient air in an experimental chamber bp base pair

BVOC Biogenic Volatile Organic Compounds cDNA Complementary DNA

Co-A Coenzyme A

CTAB Hexadecyltrimetylammonium bromide

Ct value Cycle threshold value

DMAPP Dimethylallyl diphosphate

DMNT 4,8-dimethylnona-l,3,7-triene

DNA Deoxyribonucleic acid

DNase Deoxyribonuclease dNTP Deoxyribonucleotides: deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxyuridine triphosphate dUTP

DXR 1-deoxy-D-xylulose-5-phosphate reductoisomerase, an enzyme involved in the biosynthesis of terpenoids, in methylerythritol 4- phosphate (MEP) pathway

EFN Extrafloral nectar

ET Ethylene FPKM Fragments per kilobase million

FPP Farnesyl diphosphate

GGPP Geranylgeranyl diphosphate

GLVs Green leaf volatiles

GPP Geranyl diphosphate

HIPVs Herbivore-induced plant volatiles

HMGR 3-hydroxy-3-methylglutaryl-CoA reductase, an enzyme involved in biosynthesis of terpenoids, in mevalonate (MVA) pathway

HR Hypersensitive reactions

IPP Isopentenyl diphosphate

JA Jasmonic acid

LOX1 Lipoxygenase 1, an enzyme involved in biosynthesis of jasmonic acid and green leaf volatiles, in octadecanoid pathway

MAP kinase Mitogen-activated protein kinase

MeJA Methyl jasmonate

MeSA Methyl salicylate

MEP pathway Methylerythritol 4-phosphate pathway

MVA pathway Mevalonate pathway

NGS Next Generation Sequencing

NMOCs Non-methane volatile organic compounds

O3 ctrl Ozone control treatment where intact emitter plants and receiver plants infested with P. laticollis larvae are in elevated ozone concentration (100 ppb) in an experimental chamber

O3 dam Ozone damaged treatment where emitter plants and receiver plants are infested with P. laticollis larvae in elevated ozone concentration (100 ppb) in an experimental chamber

PAL Phenylalanine ammonia-lyase PCD Programmed cell death ppb Parts per billion

PR proteins Pathogenesis related proteins qRT-PCR Quantitative Reverse-Transcribed Polymerase Chain Reaction

RIN RNA integrity number

RNA Ribonucleic acid

RNase Ribonuclease

ROS Reactive oxygen species

Rubisco Ribulose-1,5-biphosphate carboxylase oxygenase

SA Salicylic acid

SOD Superoxide dismutase

Tbr DNA polymerase modified Thermus brockianus DNA polymerase

Tm Annealing temperature

TMTT 4,8,12-trimethyltrideca-1,3,7,11-tetraene

TPS Terpene synthase

VOC Volatile organic compounds

13-AOSs 13-allene oxide synthase

13HPL 13-hydroperoxide lyase

13HPOT Linolenic acid 13-hydroperoxide

TABLE OF CONTENTS

ABSTRACT ...... 2

FOREWORD ...... 3

ABBREVIATIONS ...... 4

TABLE OF CONTENTS ...... 7

1. INTRODUCTION ...... 9

2. LITERARY REVIEW ...... 12

2.1 Herbivore-induced plant volatiles (HIPVs) ...... 12

2.1.2 HIPVs and plant-to-plant signaling ...... 15

2.1.3 Structures and biosynthesis of HIPVs ...... 19

2.1.3.1 Terpenes (DXR and HMGR products) ...... 19

2.1.3.2 GLVs and jasmonates (LOX products) ...... 22

2.1.3.3 Other phytohormones ...... 23

2.2 Tropospheric ozone...... 24

2.2.1 Synthesis of tropospheric ozone ...... 25

2.2.2 Ozone effects on plant cells and tissues ...... 26

2.2.3 Ozone effects on photosynthesis ...... 28

2.2.4 The effects of ozone on VOCs and plant-to-plant signaling ...... 29

3. MATERIALS & METHODS ...... 31

3.1 Aim of study ...... 31

3.2 Experiment schedule ...... 31

3.3 RNA isolation and DNase treatment ...... 35

3.4 cDNA synthesis ...... 36

3.5 Quantitative reverse-transcribed polymerase chain reaction ...... 36 3.5.1 Optimization of qRT-PCR ...... 38

3.5.2 Primer design ...... 39

3.5.3 Statistical analysis...... 39

4 RESULTS ...... 40

4.1 RNA purity and integrity ...... 40

4.2 Amplification efficiencies ...... 41

4.3 VOC emissions of the emitter plants ...... 43

4.4 Quantitative reverse-transcribed polymerase chain reaction ...... 44

4.4.1 3-hydroxy-3-methylglutaryl-CoA reductase, HMGR ...... 44

4.4.2 1-deoxy-D-xylulose-5-phosphate reductoisomerase, DXR ...... 47

4.4.3 Lipoxygenase 1, LOX1...... 50

4.5 Next Generation Sequencing ...... 52

5 DISCUSSION ...... 52

6 CONCLUSIONS ...... 57

7 REFERENCES ...... 58

1. INTRODUCTION

Plants emit a set of compounds in response to biotic and abiotic stresses (Pinto et al. 2010). These compounds are called biogenic volatile organic compounds (BVOCs). They are released in the air where they take part in many biological and ecological functions on several levels. At leaf tissue and surface levels VOCs act as a part of signaling (Holopainen 2004; Heil & Silva Bueno 2007) and plant defense protecting cells and tissues from harm caused by abiotic factors like oxidative stress (Loreto et al. 2004) and biotic factors like microbes, herbivores and fungi (Dudareva et al. 2006; Dudareva & Pichersky 2008).

At ecosystem level VOCs participate in plant-insect interactions (Dicke & van Loon 2000; Kessler & Baldwin 2001), within- and between plant communications (Heil & Silva Bueno 2007). They can repel generalist herbivores (Dudareva et al. 2006; Laothawornkitkul et al., 2008), but on the other hand, attract specialist herbivores that use VOCs to locate their host plants (Kessler & Baldwin 2001; Pinto et al 2010). VOCs cause damage to herbivores directly for example by intoxication (Vancanneyt et al. 2001; Engelberth et al. 2004) or indirectly by attracting the natural enemies of the herbivores to consume them (Kessler & Baldwin 2001).

What is more, VOCs can help protecting intact plants nearby the infested ones by inducing their defense mechanisms (Heil & Silva Bueno 2007). In addition to defense VOCs act as odorous attractants for pollinators and animals dispersing seeds (Pichersky & Gershenzon 2002; Dudareva & Pichersky, 2008).

In the atmosphere many VOCs react readily and participate in photochemical reactions (Atkinson & Arey 2003; Pinto et al. 2010). Some of these reactions lead to formation of air pollutants like smog and tropospheric phytotoxic compounds like ozone. VOCs take part also in the formation of secondary organic aerosols (SOAs) which affect the radiation balance and have a cooling effect on the surface of the Earth (Laothawornkitkul et al. 2009, Pinto et al. 2010).

Approximately 1700 different plant volatiles have been identified so far (Dudareva et al. 2006; Knudsen & Gershenzon 2006). This comprises about 1 % of all plant secondary metabolites (Dudareva et al., 2004, 2006). The majority of VOC emissions entering the atmosphere are from biogenic producers, mostly the terrestrial vegetation (Guenther et al. 1995). VOCs compose more than one third (36 %) of all the carbon emitted annually (400- 800 Tg C/year) in the atmosphere by plants (Kesselmeier 2001; Kesselmeier et al. 2002; Maffei 2010).

Plant-to-plant communication mediated by herbivore-induced plant volatiles (HIPVs) is of special interest in this thesis. For a few decades there was a debate about whether this communication existed in nature or not but it was convincingly proven later on (Engelberth et al. 2004; Ton et al. 2006). When a plant leaf is damaged by a feeding herbivore VOCs called herbivore-induced plant volatiles (HIPVs) are released (Blande et al 2010, Engelberth et al. 2004; Heil & Silva Bueno 2007). Volatile compounds are carried in the air and can be received by a surrounding plant nearby. They can serve as signaling molecules inducing changes in the expression of defense genes of the receiving plant before it is exposed to actual herbivores. This way the plant will gain resistance against the herbivore beforehand. It is less vulnerable to herbivore damage than the plants unaffected by this signaling.

Tropospheric ozone is the major air pollutant causing significant damage to vegetation (Atkinson & Arey 2003; Ashmore 2005, Blande et al. 2010). Its harmful effects include interfering with plant-to-plant signaling as well. Neighboring plants can be “warned” by HIPVs only from rather short distances since these compounds react easily in the air. Ozone is known to degrade many herbivore-induced volatiles (Pinto et al. 2007; Blande et al. 2010). This is proposed to cause the observed reduction in the distance in which an effective defense signaling between two plants can occur. Blande et al. observed that a successful signaling occurred normally in 70 cm distance but because of ozone this was reduced to 20 cm between Phaseolus lunatus plants (Blande ei al. 2010).

The concentration of ozone began to increase after industrialization and has been increasing ever since (Sitch et al. 2007). It is expected to keep increasing further (0.5-2 % per year) along with human activities producing ozone precursors (Vingarzan 2004). Even though emission rates have declined in Europe and America, the importance of tropospheric ozone as a pollutant is expected to increase globally due to increasing emissions in Asia (Vingarzan 2004; IPCC 2007).

It is essential to know how severe impacts higher ozone concentrations can pose on plant-to- plant signaling and the wellbeing of crops and wild plants in the future. Studying the effects of excess ozone on plants and understanding the biochemical mechanisms behind them are hence important.

In this master’s thesis the effects of elevated ozone concentration on the VOC-mediated plant- to-plant signaling was studied in hybrid aspen. The hybrid aspens were infested with herbivores to induce the emissions of herbivore-induced volatiles in climatized chambers. VOCs were expected to be captured by a set of receiver hybrid aspens. During the experiment the plants were exposed either to ozone-enriched or ambient air. After three days of exposure the relative gene expressions of three defense genes were measured by quantitative reverse- transcribed polymerase chain reaction from the receivers. The effect of ozone on VOC signaling was expected to be shown in the differences in the expression of these genes in different treatments.

2. LITERARY REVIEW

2.1 Herbivore-induced plant volatiles (HIPVs)

Plants respond to biotic and abiotic stresses by emitting volatile organic compounds (VOCs) (Loreto et al. 2004, Dudareva & Pichersky 2008). Stressed plants have significantly different VOC profiles (Dicke & van Loon 2000) and their total VOC emissions can increase almost 2.5 fold compared to unstressed ones (Vuorinen et al. 2004a). Herbivore damage on leaves causes the release of a mix of VOCs consisting of 20 to 200 different compounds (Dicke & van Loon 2000; Arimura et al. 2009). These compounds are called herbivore-induced plant volatiles (HIPVs).

HIPVs provide protection against herbivores directly or indirectly (Kessler & Baldwin 2001). Direct defenses consist of compounds like cyanogenic glucosides, glucosinolates, alkaloids, phenolics and proteinase inhibitors that work against herbivores by hampering their life cycle or by causing direct damage (Bennett & Wallsgrove 1994). Some compounds repel herbivores from target plants (Kessler & Baldwin 2001). Some disturb the feeding (Vancanneyt et al 2001; Engelberth et al. 2004), reproduction, oviposition (Dicke & van Loon 2000; Kessler & Baldwin 2001) or growth of the herbivores (Maffei 2010). Indirectly HIPVs affect herbivores by attracting carnivorous arthropods, parasitoids and other natural enemies that feed on their eggs and larvae (Kessler & Baldwin 2001). For example, two-spotted spider mites (Tetranychus urticae) feeding on lima bean leaves elicit the emissions of HIPVs that attract carnivorous mites (Phytoseiulus persimilis) (Sabelis et al. 2007).

In addition to inducible defense plants also have constitutive defense mechanisms that confer continuous protection against stresses (Mithöfer et al. 2005). These include physical barriers like cuticle, trichomes and thorns and chemical defense by preformed toxic secondary metabolites. Some inducible VOCs are emitted also constitutively but in smaller quantities (Vuorinen et al. 2005). Also, extrafloral nectar can be secreted constitutively or after induction (Maffei 2010).

As immobile organisms plants are unable to defend themselves by changing location (Dudareva et al. 2006). Thus, they benefit of the large array of HIPVs and the ability to vary their composition to best survive the currently pressing threat. For example, different sets of HIPVs can be emitted depending on the attacking herbivore species or even its developmental stage (Sabelis et al. 2007). Also, egg deposition of herbivores can cause emissions of certain HIPVs attracting suitable carnivorous insects that feed on the eggs (Fatouros et al. 2008). Different sets of HIPVs are emitted also depending on the type of damage (Leitner et al. 2005). For example, chewing damage induces higher jasmonic acid (JA) levels but sucking damage induces both JA and salicylic acid (SA) in plants. Also, whether the damage is caused by a single or continuous wounding affects the emissions (Mithöfer et al. 2005).

The composition of emitted HIPV mixture varies depending on the characteristics of the plant, the herbivore and the environment. In addition to the genotype of the plant, the growth conditions such as availability of nitrogen and phosphorous (Schmelz et al. 2003b), soil salinity, air humidity (Vallat et al. 2005), temperature and day length have their effect on the emission rates (Ibrahim et al. 2010).

Biosynthesis of HIPVs requires considerable amounts of energy and resources making it too costly to keep up production continuously (Strauss et al. 2002). Thus, HIPVs are produced or activated and emitted only after the recognition of the herbivore by complex signaling (Wu & Baldwin 2009). Elicitors isolated from oral secretion of feeding herbivores, such as peptides and fatty acid-amino acid conjugates (Schmelz et al. 2003a,b), induce JA, SA and ethylene signaling pathways (Schmelz et al. 2007). These phytohormones act as signaling molecules that induce synthesis and emissions of other HIPVs in the vegetative parts of plants (Schmelz et al. 2003a,b; Arimura et al. 2008). It is the coordination of these JA, SA and ethylene pathways that is believed to control the composition of HIPV blend emitted in different situations (Engelberth et al. 2001; Arimura et al. 2009).

HIPVs are chemically a diverse group. Many of the volatiles belong to terpenoids or green leaf volatiles (GLV) (Pichersky & Gershenzon 2002; Holopainen 2004; Arimura et al. 2009; Wu & Baldwin 2009) (Table 1). Typical HIPV terpenoids include isoprene, homoterpenes, monoterpenes, sesquiterpenes and diterpenes. Isoprene is shown to deter herbivores from feeding (Laothawornkitkul et al. 2008) and protect plants from high temperature and ozone stress (Vickers et al. 2009; Ibrahim et al. 2010). Also, monoterpenes can quench ozone in tissues and in the leaf boundary layer (Holopainen 2004). Two important homoterpenes are 4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) and 4,8-dimethylnona-l,3,7-triene (DMNT) which are known to attract carnivorous insects and parasitoids (Kappers et al. 2005) and to induce defense reactions also in intact neighboring plants (Arimura et al. 2000).

Many monoterpenes such as (E)-β-ocimene and β-myrcene take part in plant-to-plant signaling as well (Arimura et al. 2000; Godard et al. 2008). Sesquiterpenes, such as (E)-β- farnesene and (E)-β-caryophyllene are important VOCs attracting pollinators to flowers and seed dispensers to fruits (Knudsen et al. 2006). (E)-β-caryophyllene has also been shown to take part in indirect defense as it attracts predatory nematodes that feed on insect larvae in maize roots (Rasmann et al. 2005). It usually takes hours after the attack before HIPVs like terpenoids and methyl jasmonate (MeJA) are released (Dudareva et al. 2006).

Green leaf volatiles are mostly isomers of C6 alcohols, aldehydes and esters (hexenols, hexenals and hexenyl acetates) (Matsui 2006). Plants also produce many alkanes, alkenes, and ketones. GLVs, the most important being (Z)-3-hexenyl acetate, (Z)-3-hexen-1-ol and (Z)-3- hexenal, induce defense reactions in intact neighboring plants (Engelberth et al. 2004; Ton et al. 2006). GLVs are released in the atmosphere immediately after a leaf is ruptured by wounding or herbivores or within a few minutes (Hatanaka 1993; Matsui 2006). GLVs can be distinguished by their scent of cut leaves.

2.1.2 HIPVs and plant-to-plant signaling

In addition to protecting an individual plant from herbivores emitted HIPVs can be beneficial to surrounding plants as well (Heil & Silva Bueno 2007). As volatiles they are drifted in the air and may reach the receiving structures of other plants of the same species in short distances. If the composition and concentration of the HIPV blend is a good indicator of the impending danger and has reached the physiological level for defense response it elicits or primes the defense mechanisms of this receiver (Engelberth et al. 2004; Heil & Silva Bueno 2007; Blande et al. 2010). The plant benefits by getting a head start to prepare for a very likely herbivore attack and is significantly better-off compared to a neighboring plant which could not receive the warning signal (Arimura et al. 2000; Engelberth et al. 2004; Ton et al. 2006; Heil & Silva Bueno 2007; Wu & Baldwin 2009). For instance, these volatiles include C6 GLVs such as (Z)-3-hexenyl acetate, (Z)-3-hexen-1-ol and (Z)-3-hexenal, terpenoids such as TMTT, DMNT, (E)-β-ocimene, linalool and β-myrcene and phytohormones such as MeJA and methyl salicylate (MeSA) (Table 1).

HIPVs can either elicit the expression of defense genes directly or prime the defense reactions of the receivers (Arimura et al. 2000; Engelberth et al. 2004; Ton et al. 2006). Primed intact plant is prepared for the herbivore attack but reacts only after being exposed to the herbivore, with faster and/or more intense defense reactions compared to unprimed plants. For example, HIPVs emitted by Spodoptera littoralis caterpillar-infested maize primed their neighbors which reacted with earlier and/or stronger responses to subsequent herbivore attack (Ton et al. 2006). In this case, the HIPVs activated JA-pathway regulating certain defense genes which led to induction of both direct and indirect defense mechanisms in the primed plants. They emitted terpenoids and other VOCs and increased their attractiveness to parasitic Cotesia marginiventris wasps. As a result, they suffered less from the feeding damage caused by the caterpillars.

In general, primed plants contain higher concentrations of proteinase inhibitors and other compounds interfering with the digestion of the herbivore, emit more VOCs (Engelberth et al. 2004), secrete more extrafloral nectar (Heil & Kost 2006) and attract natural enemies of the herbivore (Engelberth et al. 2004; Ton et al. 2006; Blande et al. 2010). In addition, primed plants benefit from reduced resource and fitness costs compared to directly induced defense in case the herbivore attack is altogether avoided (van Hulten et al. 2006). The molecular mechanisms of priming are still poorly understood (Pinto et al. 2010).

Table 1: Herbivore-induced plant volatiles (HIPVs) Compound VOC group Synthesis Structure Function Reference MeSA, SA Phytohormone Cinnamate and Indirect defense. Schmelz et al., isochorismate Attracts egg 2003a,b; Arimura et pathways, parasitoids, the al. 2008; Moraes et synthesized in natural enemies of al., 2009; Maffei wounded tissues major bug. 2010. Signaling for biosynthesis of HIPVs. MeJA, JA Phytohormone Oxylipin Signaling for Schmelz et al. pathway, biosynthesis of 2003a,b; Arimura et synthesized in HIPVs. al. 2008; Maffei 2010. secretory and herbivore- wounded tissues

Ethylene Phytohormone Signaling for Schmelz et al. H2C=CH2 biosynthesis of HIPVs 2003a,b; Arimura et al. 2008; Maffei 2010. Isoprene, Hemiterpene MEP pathway, Deters herbivores Laothawornkitkul et (2-methyl-1,3- synthesized in from feeding on al. 2008; Vickers et butadiene) chloroplasts of plants. al. 2009; Ibrahim et mesophyll Protects from high al. 2010, Maffei 2010. cells temperatures and ozone stress. 4,8- Homoterpene MVA and Indirect defense and Arimura et al. 2000; dimethylnona-l, MEP pathways, plant-to-plant Kappers et al. 2005; 3,7-triene synthesized in signaling. Attracts Maffei 2010. (DMNT) herbivore and predatory mites, the microbe- natural enemies of wounded spider mites. tissues Primes neighboring plants. 4,8,12- Homoterpene MEP pathway, Indirect defense and Arimura et al. 2000; trimethyltrideca- synthesized in plant-to-plant Moraes et al. 2009; 1,3,7,11-tetraene herbivore and signaling. Maffei 2010. (TMTT) microbe- Attracts egg wounded parasitoids, the tissues. natural enemies of major bug. Primes neighboring plants. (E)-β-ocimene Monoterpene MEP pathway, Indirect defense and Arimura et al. 2000; synthesized in plant-to-plant Godard et al. 2008; glandular signaling. Moraes et al. 2009; trichomes and Attracts egg Maffei 2010. wounded tissues parasitoids, the (mesophyll). natural enemies of major bug. Primes neighboring plants. (E)-β- Sesquiterpene MVA pathway, Indirect defense. Rasmann et al. caryophyllene synthesized in Attracts predatory 2005;.Knudsen et al. trichomes, nematodes, the natural 2006; Maffei 2010. secretory cells, enemies of insect mesophyll and larvae. wounded root Attracts pollinators tissues and seed dispensers. cis-3-hexenyl GLVs Oxylipin Involved in plant-to- Engelberth et al. acetate, (E)-2- pathway, plant signaling. 2004; Ton et al..2006; hexenal, (Z)-3- synthesized in Primes neighboring Maffei 2010. hexen-1-ol, herbivore and plants. alkanes, alkenes, microbe- ketones wounded tissues

Figure 1: Summary of the functions of herbivore-induced plant volatiles (HIPVs). Oral secretion of feeding herbivores contains elicitors that induce salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) production in plants. These phytohormones induce the synthesis of HIPVs. HIPVs protect the plant directly by deterring the life cycle of the herbivores (feeding, growth, oviposition) or by repelling them. Indirectly HIPVs provide protection by attracting natural enemies of the herbivores, insects and other predators. HIPVs mediate plant- to-plant communication by priming or elicitation of the defense reactions in uninfested neighboring plants. Ozone impairs this communication by degrading HIPVs. TMTT = 4,8,12- trimethyltrideca-1,3,7,11-tetraene, DMNT = 4,8-dimethylnona-l,3,7-triene, GLVs = green leaf volatiles, MeSA = methyl salicylate, MeJA = methyl jasmonate.

2.1.3 Structures and biosynthesis of HIPVs

In most vascular plants HIPVs, especially monoterpenes and sesquiterpenes, are synthesized and stored in specialized secretory tissues (Maffei 2010). HIPVs can be stored also in glandular trichomes, secreting ducts and cavities (Fahn 1988) from where they are rapidly released in large quantities after a leaf is ruptured by herbivores (Fahn 1988; Maffei 2010). Some HIPVs (C6 volatiles) might be toxic to plants in high quantities so in order to manage a rapid release of large amounts of HIPVs it is appropriate to store them in separate compartments, for example, trichomes or as harmless precursors like glucosides (Jerkovic & Mastelic 2001; Maffei 2010). The regulation of the biosynthesis of HIPVs is still largely unknown since 90 % of the regulating genes are unidentified (Laothawornkitkul et al. 2009).

2.1.3.1 Terpenes (DXR and HMGR products)

Terpenes, a group of terpenoids (=isoprenoids) are carbohydrates consisting of isoprene units (Dudareva et al. 2006). Isoprene (2-methyl-1,3-butadiene), a five carbon compound with two double bonds is the simplest terpenoid. Other terpenoids can be classified by the number of their isoprene units (Ruzicka et al. 1953). Monoterpenes are 10 carbon (C10) compounds which consist of two isoprene units (Dudareva et al. 2006). Sesquiterpenes (C15) have three, and diterpenes (20C) have four isoprene units. There are also homoterpenes which have 11 or 16 carbons in their structure.

All terpenoids are synthesized from either dimethylallyl diphosphate (DMAPP) or isopentenyl diphosphate (IPP) (Kesselmeier & Staudt 1999; Dudareva et al. 2006). These compounds are processed into other substrates for enzymes which eventually form all classes of terpenoids in different biosynthesis branches. There are two pathways leading to synthesis of DMAPP and IPP: the methylerythritol 4-phosphate (MEP) pathway, taking place in chloroplasts and the mevalonate (MVA) pathway, taking place in cytoplasm (Figure 2). Glycolysis leads to formation of D-glyceraldehyde-3-phosphate and acetyl-CoA which start the MEP pathway and the mevalonate pathway, respectively.

A diverse group of terpene synthases (TPSs) form a large proportion of terpenoids in many plants (Arimura et al. 2008, 2009; Wu & Baldwin 2009). DMAPP and IPP are converted into geranyl diphosphate (GPP), farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) which TPSs use in the biosynthesis of monoterpenes, sesquiterpenes and diterpenes, respectively (Dudareva et al. 2006; Arimura et al. 2009). Important HIPV homoterpene, TMTT, is proposed to be synthesized from geranyl linalool (diterpene) and another homoterpene, DMNT, from (E)-nerolidol (sesquiterpene), both by P450 enzymes (Degenhardt & Gershenzon 2000; Dudareva et al. 2006). Isoprene is synthesized from MEP pathway generated DMAPP by isoprene synthase in plastids (Sharkey et al. 2008). Monoterpenes and sesquiterpenes are synthesized in glandular trichomes and emitted after herbivore attack (Dudareva et al. 2006; Arimura et al. 2009).

Monoterpene and diterpene precursors are proposed to be formed predominantly in MEP pathway and precursors of sesquiterpenes in MVA pathway (Dudareva et al. 2005, 2006). However, some cross-talk is shown to occur between the two pathways by a metabolite IPP which is capable of shifting between plastids and cytoplasm. Especially MEP pathway originated IPP takes part also in MVA pathway forming sesquiterpenes after herbivore infestation (Dudareva et al. 2005).

Two genes encoding enzymes regulating terpenoid biosynthesis are of interest in this thesis: 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) and 3-hydroxy-3-methylglutaryl- CoA reductase (HMGR). DXR is a key enzyme in the MEP pathway and HMGR in the mevalonate pathway (Ibrahim et al. 2010). MEP pathway starts with D-glyceraldehyde-3- phoshate which is converted into 1-deoxy-D-xylulose 5-phosphate by 1-deoxy-D-xylulose-5- phosphate synthase (DXS). Then DXR catalyzes a reaction in which 1-deoxy-D-xylulose 5- phosphate is converted into methylerythritol 4-phosphate (MEP). DXR products include monoterpenes and diterpenes and also cytosolic sesquiterpenes produced from IPP originated from the MEP pathway (KEGG database). MVA pathway starts with acetyl-CoA which is converted into 3-hydroxy-3-methylglutaryl-CoA, which is converted into mevalonate by HMGR (KEGG database). HMGR products include cytosolic sesquiterpenes and homoterpenes.

Figure 2: Terpenoid biosynthesis in mevalonate (MVA) pathway and methylerythritol 4- phosphate (MEP) pathway. HMGR = 3-hydroxy-3-methylglutaryl-CoA reductase, DMAPP = dimethylallyl diphosphate, IPP = isopentenyl diphosphate, FPP = farnesyl diphosphate, DXR = 1-deoxy-D-xylulose-5-phosphate reductoisomerase, MEP = methylerythritol 4-phosphate, GPP = geranyl diphosphate, GGPP = geranylgeranyl diphosphate.

2.1.3.2 GLVs and jasmonates (LOX products)

LOX/hydroperoxide lyase pathway

Green leaf volatiles and jasmonates both derive from oxylipin metabolism by functions of lipoxygenase (LOX) enzymes in plastids (Figure 3) (Matsui 2006). They are produced when leaves suffer from biotic or abiotic stresses. LOX modifies C18 polyunsaturated fatty acids such as linolenic acid and linoleic acid by adding two oxygen molecules to form corresponding 13S and 9S hydroperoxides. For example the modification of linolenic acid leads to formation of linolenic acid 13-hydroperoxide (13HPOT) and 12-Oxo-dodecanoic acid (Howe & Schilmiller 2002). Fatty acid 13-hydroperoxide lyase (13HPL) metabolizes 13HPOT to (Z)-3-hexenal, a C6 aldehyde. (Z)-3-hexenal acts as a substrate for alcohol dehydrogenase to form corresponding C6 alcohol, (Z)-3-hexen-1-ol. (Z)-3-hexen-1-ol can be further metabolized into (Z)-3-hexen-1-yl acetate, a C6 ester, by acyltransferases.

Octadecanoid pathway

13S hydroperoxides like 13HPOT produced by LOX activity are substrates also to enzymes such as 13-allene oxide synthase (13-AOSs) forming jasmonates (MeJA, JA, other JA metabolites) in octadecanoid pathway (Figure 3) (Howe & Schilmiller 2002). Thus, LOX products include GLVs and signaling jasmonates. The biosynthesis pathways of GLVs and jasmonates might be competitive since they share the same substrates but this not confirmed. The pathways are distributed differently in thylakoid membranes so the competition is not considered likely (Farmaki et al. 2007).

Figure 3: Jasmonate and green leaf volatile biosynthesis pathways LOX/hydroperoxide lyase pathway and octadecanoid pathway. LOX = lipoxygenase, 13HPOT = linolenic acid 13- hydroperoxide, 13HPL = 13-hydroperoxide lyase, 13-AOS = 13-allene oxide synthase.

2.1.3.3 Other phytohormones

Other phytohormones, SA, MeSA and ethylene, are produced in different pathways. SA is formed in shikimate pathway either from benzoate via cinnamate, phenyalanine and chorismate or from isochorismate via chorismate (Chen et al. 2009). MeSA is synthesized in a reaction where a methyl group is transferred into SA by methyltransferase. The biosynthesis of ethylene starts with methionine (Wang et al. 2002). Methionine is converted first into S- AdoMet and then 1-aminocyclopropane-1-carboxylic acid (ACC). Ethylene is then formed from ACC.

2.2 Tropospheric ozone

Ozone is an essential gas in the upper atmosphere (stratosphere) where it forms the ozone layer and protects the Earth from UV light (Rao et al. 2000). However, in the lower atmosphere (troposphere) it is a toxic pollutant causing health problems to people and damage to vegetation (Ashmore 2005). Visible leaf injury and reduction in crop yield can be caused by ozone threshold concentration of >40 ppb. At present, the mean ozone concentrations are approximately 20-45 ppb in the Northern Hemisphere (Vingarzan 2004). It is estimated that globally the mean ozone concentration will increase to above 40 ppb and above 70 ppb in some areas by 2100 (Sitch et al. 2007). In troposphere, ozone is formed in photochemical reactions from precursors of anthropogenic and biogenic origin.

Ozone is a phytotoxic substance and its effects on plants are well documented but the molecular mechanisms behind are still somewhat unknown (Rao et al. 2000; Baier et al. 2005; Kangasjärvi et al. 2005). After entering tissues ozone causes oxidative stress which may damage some cell structures and lead to acute or chronic injuries. Ozone reduces photosynthesis and biomass production and causes changes in carbon flow which affects the synthesis of secondary metabolites (Karnosky et al. 2003). Ozone also enhances global warming by reducing the potency of plants to remove CO2 from the atmosphere by photosynthesis (Fiendlingstein et al. 2006). Furthermore, ozone could potentially interfere with VOC- mediated ecological interactions between plants and other community members, such as plant-to-plant communication by degrading bioactive volatile compounds (Blande et al. 2010).

Ozone and VOCs interact with each other in a positive feedback system (Llusià et al. 2002). Ozone formation is enhanced by increased VOC emissions and, on the other hand, the VOC emissions of plants may increase when exposed to ozone (Llusià et al. 2002, Atkinson & Arey 2003). The feedback mechanism can also be enhanced by high temperatures and thus increase VOC emissions, especially of GLVs (Hartikainen et al. 2009). Also the concurrent stresses of biotic and abiotic factors can significantly increase VOC emissions of plants (Blande et al. 2007).

2.2.1 Synthesis of tropospheric ozone

VOCs participate in photochemical reactions that form tropospheric ozone (Llusià et al.

2002). They react with nitrogen oxides (NOx) to ultimately form ozone (reaction (1))

(Atkinson & Arey 2003). The major NOx precursor of ozone is NO2. NO2 degrades in light and forms NO and a single oxygen molecule O (reaction (2)). O reacts with O2 and forms ozone (reaction (3)).

NOx + VOCs  O3 (1)

NO2 + hv (photon of UV radiation)  NO + O (2)

O + O2 + air  O3 + air (3)

Normally some NO is oxidized back to NO2 (Fuentes et al. 2000; Atkinson & Arey 2003). In polluted air some VOCs enhance ozone formation by increasing NO2 emissions. The oxidation of these non-methane volatile organic compounds (NMOCs) is triggered by hydroxyl radicals (OH*). NMOCs are oxidized by light and form organic alkyl peroxy (RO2*) and hydroperoxy (HO2*) radicals. RO2* reacts with NO to form NO2 which is ultimately photolyzed to form ozone. This process feeds itself by producing more OH* which is available to other NMOCs.

In rural areas the major VOCs contributing in ozone formation are plant alkenes, especially isoprene and monoterpenes (Fuentes et al. 2000, Atkinson & Arey 2003). In urban areas, the most important VOCs are alkenes such as propene and butene. Other important compounds include alkanes, aromatic hydrocarbons, alcohols, aldehydes, ketones, organic peroxides and halogenated organic compounds (Pinto et al. 2010).

VOC emissions are produced naturally by plants but also by human activities (Atkinson & Arey 2003). For example gasoline and diesel-fueled cars, trucks and other vehicles, fossil- fueled power plants, manufacturing, petroleum and chemistry industries release VOCs

(Atkinson & Arey 2003, Wang et al. 2008). Nitrogen oxides (NO and NO2) are produced mainly by traffic, generators and industrial processes. In low NOx concentrations VOCs stimulate the net formation of ozone but in high NOx concentrations the amount of ozone is reduced as it reacts with NO to form O2 and NO2 (Pinto et al. 2010).

2.2.2 Ozone effects on plant cells and tissues

Ozone is a phytotoxic substance with potential to cause damage to plant cells and tissues (Baier et al. 2005). Normally, the antioxidant mechanisms of plants make them tolerant to certain amount of oxidative stress. However, the oxidative stress caused by ozone is often too much and severe injury occurs. First ozone must enter the plant. Ozone is highly soluble in water and it is unable to diffuse through the waxy cuticle (Kerstiens & Lendzian 1989). The only possible entry is through open stomata (Overmyer et al. 2008).

Ozone ends up in the apoplasm where it is converted into reactive oxygen species (ROS)

(Kangasjärvi et al. 1994; Overmyer et al. 2008). These molecules for example H2O2, O2*, O and OH contain unpaired electrons which make them aggressively reactive (Kangasjärvi et al. 1994; Baier et al. 2005). If the defense and detoxification mechanisms of the plant fail to neutralize ROS, they will cause oxidative stress (Baier et al. 2005). ROS can attack other molecules, such as lipids, proteins and nucleic acids damaging and destroying them. Ozone itself can cause direct damage to sensitive molecules like unsaturated lipids, apoplastic proteins on the outer surface of cell membrane (Kangasjärvi et al. 1994). Ozone might cause some changes in the wax composition on cuticle as well (Kerstiens & Lendzian 1989). However, it is fairly well protected by antioxidative flavonoids.

Plants react to ozone and the oxidative stress by several mechanisms (Baier et al. 2005). Tolerant varieties react to ozone by rapidly closing their stomata, thus minimizing the accumulation of ozone in the cells (Kangasjärvi et al. 2005; Overmyer et al. 2008) Abscisic acid (ABA) is responsible of regulating stomatal movements (Kangasjärvi et al. 2005). The first line of defense in apoplasm or cell membranes includes antioxidant mechanisms: glutathione-S-transferase, pathogenesis related proteins (PR-proteins), phenylalanine ammonia-lyase (PAL) and L-ascorbate peroxidase (Heath 2008). Another common response to acute ozone exposure is the increase in capacity for scavencing ROS by superoxide dismutase (SOD) and related enzymes in Halliwell-Asada pathway (Conklin & Barth 2004).

Signaling by SA, JA, ABA, ethylene and Ca2+ is important in balancing the complex defense reactions and severity of damage (Kangasjärvi et al. 2005; Overmyer et al. 2008). Also, MAP kinase signaling cascade has been proposed to induce gene expression in ozone exposure (Kangasjärvi et al. 2005). The severity of injury depends on the concentration of ozone, duration of exposure and the tolerance of the plant. Acute effects occur when a plant is exposed to high concentrations (120-500 ppb) of ozone for a short time period (hours) (Kangasjärvi et al. 2001). Chronic effects occur slowly over longer time (months) in lower ozone concentrations (40-120 ppb) (Rao et al. 2000, Long & Naidu 2002). Both types of damage may lead to significant reductions in biomass production, agronomic traits and crop yield (Ashmore 2005).

Acute exposure to ozone causes reactions similar to hypersensitive reactions (HR) when exposed to pathogens (Rao et al. 2000). This leads to development of chlorosis (Baier et al. 2005) and to programmed cell death (PCD) which causes visible necrotic lesions on the leaves (Rao et al. 2000). PCD is controlled by phytohormones: it is initiated by salicylic acid, developed by ethylene and limited by jasmonic acid (Baier et al. 2005, Kangasjärvi et al. 2005). The emissions of lipoxygenase (LOX) products have been linked with the development of PCD and visible symptoms as well (Heiden et al. 2003). Visible leaf injury was observed to occur if LOX products were emitted after ozone stress. Ozone sensitive varieties tend to synthesize higher amounts of ozone inducible signaling molecules, ROS, ethylene, SA and jasmonates faster and longer than more tolerant varieties (Overmyer et al. 2008).

Chronic damage may occur with or without visible symptoms if plants have been exposed to relatively low concentrations of ozone for a long time (Long & Naidu 2002). Sometimes the only symptom of chronic exposure is decreased photosynthesis. It may also cause early leaf senescence (Rao et al. 2000), increased sensitivity to pathogens (Kangasjärvi et al. 2005) and changes in the plant physiology. For example the growth of the leaves is reduced in all layers, making them thinner than normally (Freiwald et al. 2008). Chronic exposure to ozone affects especially perennial plants like trees. Fast growing species, like hybrid aspen, are the most sensitive to ozone (Bortier et al. 2000; Häikiö et al. 2008). Almost one third of current forests are exposed to damaging ozone concentrations and the proportion is expected to increase close to 50 % by 2100 (Fowler et al. 2000).

2.2.3 Ozone effects on photosynthesis

The concentrations of ozone and CO2 are currently increasing due to increasingly active human activities (Vigarzan 2004; IPCC 2007). In general, plants benefit from high CO2 concentrations as they have more carbon to use for photosynthesis and biomass production, when water, light and nutrients are provided (Woodward 2002; Karnosky et al. 2003). However, plants tend to protect themselves from negative impact of ozone by stomatal closure (Vahisalu et al. 2010). By closing their stomata the plants also block the entry of CO2 (Vahisalu et al. 2010, Karnosky et al. 2003). Thus, the increase in photosynthetic potency can be significantly weakened by tropospheric ozone (Karnosky et al. 2003). This way ozone also accelerates climate change by reducing the ability of plants to accumulate carbon from the atmosphere (Fiendlingstein et al. 2006).

It has been considered that in simultaneous exposure to elevated CO2 and ozone, CO2 compensates for the decreased photosynthesis caused by ozone (Rao et al.1995). However, it has been suggested that CO2 may exacerbate the negative effects of ozone rather than compensate for them (Wustman et al. 2001; Karnosky et al. 2003). Oxidative stress causes the activation of defense mechanisms which means that less carbon is available to be used in photosynthesis and primary and secondary productions (Karnosky et al. 2003). This may mean a decrease also in the production of precursors for biosynthesis of herbivore-induced volatiles (Arimura et al. 2008). Biomass is decreased and onset of leaf yellowing and senescence is accelerated (Karnosky et al. 2003).

Ozone can reduce photosynthesis also directly by inhibiting different steps of the process (Long & Naidu 2002). The quantity of rubisco (ribulose-1,5-biphosphate carboxylase oxygenase) enzyme is reduced and photosynthetic pigments, chlorophyll a, b and carotenoids are reduced by chronic ozone exposure (Wustman et al. 2001). Almost all the steps from the light capture in photosystem II, starch accumulation and phloem loading are affected (Long & Naidu 2002; Pinto et al. 2010).

2.2.4 The effects of ozone on VOCs and plant-to-plant signaling

Induction or increase in VOC emissions is a common response to acute and sometimes chronic exposure of ozone (Rao et al. 2000). For example, ozone has been observed to induce the synthesis of MeSA and LOX products in tobacco plants (Nicotiana tabacum L.) and increased the emissions of some sesquiterpenes (Beauchamp et al. 2005). In some other experiments, increases has also been observed in other terpenoid and monoterpene emissions involved in antioxidant responses in Quercus ilex L. plant (Loreto et al. 2004), and homoterpenes DMNT and TMTT in lima bean plants commonly emitted in herbivore exposures (Vuorinen et al. 2004b). Also, isoprene contributes to protecting the plant from ozone stress and high temperatures (Vickers et al. 2009). The emissions of some VOCs may also decrease.

VOCs are emitted through the cuticle by diffusion (Holopainen 2004). Ozone induces changes in the synthesis and composition of epicuticular waxes changing its physiochemical properties (Kerstiens & Lendzian 1989). Also stomatal closure caused by ozone might affect the emissions of newly synthesized compounds though it does not have an effect on emissions through cuticle or from external glands and hairs (Kesselmeier & Staudt 1999).

Ozone-induced changes in emitted VOC mixtures may affect all processes involving VOC participation such as protection from micro-organisms, attraction of pollinators and the natural enemies of herbivores and plant-to-plant signaling (Holopainen 2004). However, the effects of ozone on these processes are still largely unknown (Pinto et al. 2010).

Once VOCs enter the atmosphere, ozone may degrade them by ozonolysis (Pinto et al. 2010). Ozone has been observed to degrade at least the following herbivore-induced volatiles: 2- butanone, β-myrcene, (Z)-3-hexenyl acetate, (E)-β-ocimene, (Z)-DMNT, (E)-DMNT, linalool, (E)-β-caryophyllene and TMTT (Blande et al. 2010). However, the degradation is significant only with (E)-β-ocimene, (E)-DMNT and TMTT. The degradation of these volatiles causes interference in their functions as signaling molecules between plants. When the concentration of signaling volatiles is reduced by ozone, the signal is weaker. Thus, the induction of effective defense reactions in the receiver can occur when the distance between an emitter and a receiver plant is reduced. This indirect effect of ozone on plant volatile mediated plant-to-plant communication is of interest in this thesis.

3. MATERIALS & METHODS

3.1 Aim of study

The aim of this master’s thesis is to study how herbivore-induced plant volatiles mediate elicitation and priming of the defense reactions of near-by plants in plant-to-plant communication and how this signaling is affected by ozone. The differences in defense reactions were detected by measuring the relative gene expression of defense genes determined by quantitative reverse-transcribed polymerase chain reaction (qRT-PCR). Also, the VOC emissions of the receiver plants were measured. The main focus is on the gene expression analysis in this thesis.

3.2 Experiment schedule

The following experimental set-up was conducted in order to study the impact of ozone on plant-to-plant communication by volatiles in hybrid aspens (Populus tremula x P.tremuloides) (figure 9.). Experiment conditions were put up in four separate chambers. In each chamber, eight hybrid aspens were placed in two parallel rows 35 cm apart from each other: the row of four plants emitting VOCs and the row of four plants receiving them.

Ambient air with low ozone concentration (15 ppb) was conveyed in two of the chambers and ozone-enriched air (100 ppb) in the other two chambers. The emitter plants in one ambient air and in one ozone-enriched chamber were infested with leaf beetle larvae (Phyllodecta laticollis), 5 larvae on each five leaves (figure 10.A) while the emitter plants in the other two chambers remained uninfested. Different conditions were labeled as ambient control (amb ctrl), ambient damaged (amb dam), ozone control (O3 ctrl) and ozone damaged (O3 dam). “Damaged” refers to the presence of larvae-infested emitters and “control” the presence of uninfested emitters. “Ambient” refers to 15 ppb and “ozone” to 100 ppb ozone concentrations in the chamber. Air flow was directed from the emitters to the receivers in all chambers.

Experiment schedule is presented in figure 11. P. laticollis larvae were added on the “damaged” emitters on day -3. The receivers were exposed to VOCs released from the emitters for three days in all chambers (days -3 to -1). One leaf from each receiver plant was harvested for RNA isolation and after that extrafloral nectar (EFN) and volatiles released from the receivers were collected on day 0. In addition, P. laticollis larvae were added also on the receivers, 5 larvae on each six leaves, in all chambers. Volatiles were collected again after the infestation.

One leaf from each receiver plant was harvested for RNA isolation and volatiles released by the receivers were collected in the morning and again in the afternoon on day 1. Yet, one leaf from each receiver plant was harvested for RNA isolation on day 2. Extrafloral nectar from the receivers was collected once more on day 2 and from the emitters on day 3. The leaves harvested for RNA extraction were stored in – 70 ºC for further use.

amb ctrl amb dam

O3 ctrl O3 dam

Figure 9. Experiment set-up on days -3 to -1. Experiment chambers with two sets of hybrid aspens exposed to different ozone and herbivore conditions. The emitters (four biological replicates) release volatile organic compounds that are expected to be captured by the receivers (four biological replicates) in each chamber. Air flow is indicated by red arrows. Amb ctrl: Intact control receivers in ambient air (ozone 15 ppb), that is, receivers that were exposed to volatiles emitted by undamaged emitters upwind. Amb dam: receives exposed to volatiles emitted by leaf beetle-damaged emitters under ambient condition. O3 ctrl: receivers that were exposed to volatiles emitted by undamaged emitters upwind under 100 ppb ozone condition. O3 dam: receives exposed to volatiles emitted by leaf beetle-damaged emitters under 100 ppb ozone condition.

A B C

Figure 10. A: Leaf beetle larvae (P. laticollis) on a hybrid aspen leaf. B: Condition amb ctrl, a receiver (left) and an emitter (right). C: Condition amb dam, a receiver (left) and an emitter (right).

Figure 11. Experiment schedule. The experiment was put up in four chambers with different conditions, amb ctrl, amb dam, O3 ctrl and O3 dam. There were four emitter plants and four receiver plants in parallel rows in each chamber. The distance between the rows was 35 cm. Day -4: Plants were placed in the experiment chambers and left to adapt for a day. Day -3: The four emitter plants in amb dam and O3 dam conditions were infested with P. laticollis larvae: larvae were added on leaves # 7-11, 5 larvae on each leaf. Days -3 to -1: The receivers were exposed to volatiles emitted by the emitters in each chamber. Day 0: Leaf #10 from each receiver replicate was harvested for RNA isolation. Volatiles emitted by the receivers and extrafloral nectar (EFN) from leaves #7-12 were subsequently collected. After that P. laticollis larvae were added on leaves #7-12 on all the receivers, 5 larvae on each leaf. Volatiles emitted by the receivers were collected again. Day 1: Leaf # 9 from each receiver replicate was harvested for RNA isolation. Volatiles emitted by the receivers were collected in the morning and again in the afternoon. Day 2: Leaves #8 and #11 from each receiver replicate were harvested for RNA isolation. Extrafloral nectar was collected from the receivers, from leaves #7-12. All the receivers were harvested. Day 3: Extrafloral nectar was collected from the emitters. The leaves were numbered starting from an apical leaf (#1) and then numbering sequential leaves.

3.3 RNA isolation and DNase treatment

Hybrid aspen leaves from the four receivers in each condition (amb ctrl, amb dam, O3 ctrl and

O3 dam) were harvested in three different time points, on day 0, day 1 and day 2. These 48 leaves were identified by numbers (1-4) and labeled by condition and time point. The receivers had been exposed to emitter VOCs for three days until day 0.

Total RNA was isolated from 12 leaves harvested on day 1, three biological replicates from each condition (isolation 1) and was sent to deep sequencing analysis. The Next Generation Sequencing (NGS) Illumina analysis was conducted at Cornell University, New York, USA. Total RNA was isolated again from these 12 leaves in order to complete day 1 samples for qRT-PCR (isolation 2).

At first, all leaves were separately ground with mortar and pestle in liquid nitrogen with sea sand. The sea sand mass was estimated to be approximately one third of the measured leaf mass. Leaf powder was stored in -70º C. Total RNA was isolated from the leaf powder using hexadecyltrimetylammonium bromide (CTAB) extraction and LiCl precipitation (Chang et al. 1993). The isolations were performed in 2 ml eppendorf tubes with maximum of 100 mg of leaf powder. About 150 mg of leaf/sand powder was used per isolation.

DNA contamination was removed from the RNA samples by deoxyribonuclease (DNase) digestions. The RNA samples of day 0 and day 2 were treated according to the instructions of Protocol for Preparation of DNA-free RNA Prior to RT-PCR (with DNase Ӏ, RNase-free) just before cDNA synthesis (Wiame et al. 2000). Ribolock™ RNase Inhibitor (40 u/µl) was added to prevent RNA degradation. The amount of RNA was adapted to 2 µg. The RNA samples of day 1 were treated either by the same protocol as day 0 and day 2 (isolation 2) or by QIAGEN RNeasy Mini Kit RNA Cleanup-protocol (RNeasy Mini Handbook 2010) right after the RNA isolation (isolation 1).

RNA concentrations and purity were determined by a spectrophotometer (NanoDrop ND- 1000 Spectrophotometer). Also, high integrity of the RNA was important for downstream applications. The integrity of the 12 RNA samples analyzed in NGS was determined by Agilent’s 2100 Bioanalyzer before sending. Eukaryote Total RNA Nano chip of Agilent RNA 6000 Nano Kit was used.

3.4 cDNA synthesis

Three biological replicates with the highest RNA concentrations were selected from each condition to be analyzed in qRT-PCR. Total RNA was first transcribed into complementary DNA (cDNA) by following the instructions of Thermo Scientific DyNAmo SYBR Green 2- step qRT-PCR Kit-protocol. PTC-100™ Programmable Thermal Controller with a heat lid was used. M-MuLV RNase H+ reverse transcriptase transcribes the RNA into cDNA and degrades the RNA afterwards. Random hexamer primer set was used (300 ng/µl). Reaction volume was 20 µl and the amount of template RNA was adapted to maximum of 1 µg in 7 µl of PCR water. The cDNA synthesis step at 37 °C was extended to 60 minutes. The cDNA samples were stored in -20 °C for further use.

3.5 Quantitative reverse-transcribed polymerase chain reaction

Quantitative reverse-transcribed polymerase chain reaction (qRT-PCR) was used to determine the relative expression of three selected defense genes in all the receiver plants in three different time points. These genes were lipoxygenase 1 (LOX1), 1-deoxy-D-xylulose-5- phosphate reductoisomerase (DXR) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). qRT-PCR is a common method to analyze relative gene expression (Wong & Medrano 2005). The gene expression is measured from the quantity of RNA in the sample. The RNA is first reverse-transcribed into complementary DNA (cDNA). This specific short fragment of double-stranded DNA is exponentially amplified by polymerase chain reactions and detected by fluorescent dye. The increase in the amplification of the cDNA is followed real-time cycle by cycle.

The qRT-PCR analysis was done by following the instructions of Thermo Scientific DyNAmo SYBR Green 2-step qRT-PCR Kit-protocol. cDNA was amplified by a heat stable, modified Thermus brockianus (Tbr) DNA polymerase. SYBR® Green Ӏ was used as a fluorescent dye to detect the progression of the amplification. The 2x Master mix contained all required reagents: the DNA polymerase, MgCl2 (5 mM), dNTP mix (2 mM each of dATP, dCTP, dGTP, and 4 mM dUTP), the dye and optimized PCR buffer. The reactions were performed in BioRad ICycler®. The optimal amount of cDNA was determined to be 1 ng per reaction. Primer concentration was 1 μM and reaction volume was 20 µl in each reaction. Reactions were set up in 96-well plates. Ordinal numbers were given to the samples and their places in the 96-well plate were randomized by a random number generator.

The expression of the gene of interest is normalized to the expression of an endogenous reference gene and related to the expression of a calibrator (Livak & Schmittgen 2001). In this study, α-tubulin was used as an endogenous reference gene and the condition with the lowest gene expression on day 0 as a calibrator. The reference gene ideally has constant expression in different tissues and conditions (housekeeping gene). In order to make the normalization more reliable more than one reference gene could have been used since the expression of housekeeping genes varies to some extent. The variation should be checked before the analysis. The variation in the gene expression of tubulin in different conditions was not checked in this study. However, tubulin is a commonly used reference gene (Ibrahim et al. 2010).

The quantity of cDNA eventually reached a fixed threshold level and a Ct-value was given for each sample. The Ct-value represents the number of PCR cycles required to reach the threshold level of amplification. Thus, the higher amount of cDNA in the sample, the smaller the Ct-value.

Gene expressions were quantified by exploiting the Ct-values in an equation 2^-ΔΔCt (Ct (2^- ΔΔCt)-method) (Livak & Schmittgen 2001).

ΔΔCt = (Ct (target gene) - Ct (inner reference gene)) sample x – (Ct (target gene) – Ct (inner reference gene)) calibrator

Ct-values were given for three technical replicates per each three biological replicate in all conditions and time points. These Ct-values (mean values of technical and biological replicates) were used in the equation 2^(-ΔΔCt) to calculate gene expressions of HMGR, DXR and LOX1.

3.5.1 Optimization of qRT-PCR

The optimal annealing temperatures and template (cDNA) concentrations were determined for primers designed for the three target genes, HMGR, DXR and LOX1, and the reference gene, tubulin. The annealing temperatures were optimized by using temperature gradients from 56 °C to 59 ºC. The optimal annealing temperature was 57-58 °C for LOX1 and DXR and 57 °C for HMGR.

A blank control including all reagents except the template cDNA was used. Additionally, a minus RT control was included in all the temperature optimizations. The minus RT control had RNA as a template. Thus, amplification should not occur unless there is contaminating DNA in the reaction.

The optimal template concentration was determined by using several dilution series. The concentration of cDNA in these series varied from 0,025-10 ng/µl. The amplification of cDNA of HMGR, DXR, LOX1 and tubulin was optimal with both herbivore damaged and undamaged samples in all time points when the template concentration was 0,5 ng/μl (1 ng per reaction) and the annealing temperature was 57 °C.

3.5.2 Primer design

Primers for HMGR, DXR, LOX1 and tubulin were designed with the primer designing tool of NCBI/ Primer-BLAST. HMGR and DXR primers were obtained from a paper studying the mRNA expression of HMGR and DXR (GenBank: DN495990) in European aspen (Populus tremula) (Ibrahim et al. 2010). LOX1 primers were designed from LOX1 cDNA sequence (GenBank: DQ131178.1) sequence of Populus deltoides (Cheng et al. 2006). Tubulin primers were designed from α-tubulin (GenBank: EF583814.1) sequence of Populus tremuloides (Oakley et al. 2007).

These primers produce amplicons sized 150-300 bp. The primers were designed to anneal in exon-exon junctions. Hence, the amplification of unwanted contaminating DNA would be highly inefficient. The annealing temperatures of all the primers were preferably as close to each other as possible. The formation of primer-dimers and secondary structures like hairpin loops was avoided.

Table 3. Primer sequences and annealing temperatures (Tm) of tubulin, HMGR, DXR and LOX1. Primers Sequences Tm ºC Reference

HaapaTubq_F 5’-GATCCTCGTCACGGAAAGTACA-3’ 60,3 HaapaTubq_R 5’-CAGCAACACTGGTAGAGTTGGA-3’ 60,3 PtHMGR_F 5’-GCTGGTGCTCTAGGTGGATT-3’ 59,4 Ibrahim et al. 2010 PtHMGR_R 5’-CGGATTGCGATGCAAGTTGA-3’ 57,3 Ibrahim et al. 2010 PtDXR_F 5’-GCCTGATATGCGCTTGCCTAT-3’ 59,8 Ibrahim et al. 2010 PtDXR_R 5’-CAGCACTGAGGACTCCTGTCA-3’ 61,8 Ibrahim et al. 2010 PtLOX1_F 5’-GCCACAGCTTCACCAGTCACA-3’ 61,8 PtLOX1_R 5’-CTGTGGACTGCTGGGTGACA-3’ 61,4

3.5.3 Statistical analysis

The statistical analysis of the effects of herbivore feeding and ozone on VOC emissions and gene expression were conducted with the software SPSS 19. statistics and R 2.14. statistics, respectively. Pairwise comparisons were made with the one-way ANOVA, post hoc Tukey test, Mann-Whitney and Kruskal-Wallis tests with the VOC emissions and with ANOVA t- test with the gene expression.

4 RESULTS

The impact of ozone on VOC-mediated defense elicitation and priming was studied in hybrid aspen in a climatized chamber experiment. A condition where receivers were exposed to VOCs released from larvae-infested emitters in ambient air (amb dam) and in elevated ozone

(O3 dam) was established in separate chambers. Control conditions where receivers were exposed to VOCs released from uninfested emitters in ambient air (amb ctrl) and in elevated ozone (O3 ctrl) were established in other two chambers.

Leaves from the receivers in each condition were harvested in three different time points, day 0, day 1 and day 2. Total RNA was extracted from the leaves, reverse transcribed into cDNA and used as a template in qRT-PCR. The impact of plant volatiles and ozone on the induction of defense reactions in the receivers was observed by measuring the relative gene expression of HMGR, DXR and LOX1. Also, the VOC emissions of the emitters and the receivers were measured. NGS analysis was conducted from the RNA samples obtained on day 1.

4.1 RNA purity and integrity

Good quality RNA was needed for the qRT-PCR reactions and the NGS analysis. The concentration and purity of total RNA was estimated by using a spectrophotometer. Adequate amount of RNA for cDNA synthesis was obtained from all the samples. The ratio of the absorbance in wavelengths 260 nm and 280 nm (A260/A280) between 1,8-2 indicates pure RNA. A260/A280 of all the RNA samples of days 0, 1 and 2 were between 1,89-2,28. Thus, the RNA in all samples was pure enough for both analyses.

The integrity of the RNA samples of day 1 was determined by Agilent’s 2100 Bioanalyzer before sending them to be sequenced by Illumina NGS sequencing. Integrity of RNA is indicated by RIN values. In animal material, the RIN value of 7-10 indicates high integrity. However, in plant material, especially in leaf tissues, the adequate RIN value is 6,6. The reduction is caused by additional peaks from chloroplast rRNA (Assessing integrity of plant RNA with the Agilent 2100 Bioanalyzer, Application note, 2011). All the RIN values determined varied from 6,8-8,1, hence, qualified for the NGS.

4.2 Amplification efficiencies

The Ct (2^-ΔΔCt)-method can be used in the relative quantification of gene expression only if the amplification efficiencies of the target gene and the reference gene are very similar and nearly 100 % (Wong & Medrano 2005). When the amplification efficiency is 100 % the amount of cDNA is doubled in every cycle and the equation (2^-ΔΔCt) holds true. If the amplification efficiency is less than 100% a quantification method considering reduced efficiency should be used.

In order to determine the amplification efficiencies a dilution series was made for each template (Livak & Schmittgen 2001). A regression curve was drawn showing the relation between Ct-values obtained from the dilution series and the logarithm of the amount of the template. The amplification efficiency is practically close to 100 % when the slope of the regression curve is between -3,1 and -3,4. Also, correlation coefficient should be more than 0,98 (Taverniers et al. 2005).

In this study, the slopes of the regression curves of the target genes and tubulin (tub) were as follows: HMGR: -3,2; tub: -3,3, DXR: -2,3; tub: -3,3 and LOX1: -3,2; tub: -3,3 (figure 12.a, b, c). The amplification efficiencies were close to 100 % for HMGR and LOX1. As for DXR, the slope was too gentle and the amplification efficiency too low to meet the requirements of using Ct (2^-ΔΔCt) -method for quantification. The correlation coefficients were high enough in all the curves: HMGR: 0,99; tub: 0,98, DXR: 0,99; tub: 0,98, LOX1: 0,99; tub: 0,98.

In order to determine how similar the amplification efficiencies of the target gene and the reference gene were another curve, a 2^(-ΔΔCt)- verification curve, was drawn (Livak & Schmittgen 2001). This curve shows the relation between the difference in the Ct-values of the target gene and the reference gene (ΔCt) and the logarithm of the amount of the template. The slope is near zero when the amplification efficiencies are similar. In this study, the amplification efficiencies of the target gene and the reference gene are close to each other with HMGR (slope: 0,14) and LOX1 (slope: 0,15) (figure 12.d, e, f). The amplification efficiencies of DXR and tubulin (slope: 0,72) could have been closer to each other.

Regression curves Verification curves a d 40 5 y (Tub) = -3,3497x + 29,186 R² = 0,9883 30 3 y (HMGR) = 0,1435x - 2,6184

R² = 0,2454

Ct 20 1 Ct 10 Δ -1 y (HMGR) = -3,2062x + 26,567 -1,5 -1 -0,5 0 0,5 1 R² = 0,9987 0 -3 -1,5 -1 -0,5 0 0,5 1 -5 Log (template) [ng] Log (template) [ng]

b e 40 0 y (Tub) = -3,3497x + 29,186 R² = 0,9883 -0,5 0 0,5 1

30 -2

Ct 20 -4

y (DXR) = 0,7161x - 7,2383 Ct 10 Δ -6 R² = 0,6677 y (DXR) = -2,3228x + 21,858 R² = 0,9998 0 -8 -1,5 -1 -0,5 0 0,5 1 -10 Log (template) [ng] Log (template) [ng]

c f

40 0 y (Tub) = -3,3497x + 29,186 R² = 0,9883 -1,5 -1 -0,5 0 0,5 1

30 -2

Ct 20 -4

Ct Δ 10 -6 y (LOX) = -3,2024x + 27,9 R² = 0,9938 -8 0 y (LOX) = 0,1473x - 1,2853 -1,5 -1 -0,5 0 0,5 1 R² = 0,5542 -10 Log (template) [ng] Log (template) [ng]

Figure 12. Regression curves of a: HMGR, b: DXR, c: LOX1 and (2^-ΔΔCt)-verification curves of d: HMGR, e: DXR, f: LOX1.

4.3 VOC emissions of the emitter plants

VOCs were collected from the emitter plants in each condition on day 2 (figure 16). The emitter plants infested with P. laticollis larvae emitted many VOCs in higher quantities than the control emitters both in ambient and ozone-enriched air. These VOCs were monoterpenes sabinene, limonene, β-ocimene and linalool and GLV cis-3-hexenyl isovalerate. Homoterpene (E)-DMNT and sesquiterpene caryophyllene were emitted in higher quantities in ambient air and GLV cis-3-hexenyl butyrate in ozone-enriched air. These differences were statistically significant. Especially, the emission of β-ocimene was considerably higher in the damaged conditions, approximately 150 ng g-1 DW h-1 in ambient air and 120 ng g-1 DW h-1 in ozone enriched air, compared to the control conditions, approximately 20 ng g-1 DW h-1.

Only methyl salicylate, a phytohormone, was more abundant in the control conditions compared to the damaged conditions. The difference was statistically significant in ozone- enriched air. Monoterpenes α-pinene and 3-carene, sesquiterpenes α-humulene and farnesene and GLV cis-3-hexenyl acetate were emitted in equal amounts in all conditions.

In general, the emissions of the VOCs were similar or somewhat lower in ozone damaged condition compared to the ambient damaged condition. Ozone seemed to reduce the emissions of α-pinene, limonene, β-ocimene, (E)-DMNT, caryophyllene, farnesene and cis-3-hexenyl acetate. However, none of these differences were statistically significant. There were no differences in the emissions of other VOCs between ambient and ozone-enriched air in control conditions.

Figure 16. The emission of VOCs released by the emitters in all conditions. amb ctrl: the VOCs emitted by uninfested emitter plants in ambient air (15 ppb). amb dam: the VOCs emitted by P. laticollis infested emitter plants in ambient air. O3 ctrl: the VOCs emitted by uninfested emitters in ozone-enriched air (100 ppb). O3 dam: the VOCs emitted by infested emitters in ozone-enriched air.

4.4 Quantitative reverse-transcribed polymerase chain reaction

4.4.1 3-hydroxy-3-methylglutaryl-CoA reductase, HMGR

The gene expression of HMGR was normalized to the expression of tubulin and related to the expression in ozone control condition on day 0 (calibrator). The fold change in the gene expression of HMGR compared to the expression of the calibrator in each condition and time point is shown in figure 13A. Receiver plants had been exposed to VOCs released from the emitters for three days by day 0. The RNA level of HMGR was at the level of the calibrator in ozone damaged condition and was approximately 2-fold in ambient control and ambient damaged conditions.

The receivers were exposed to herbivores in all conditions on day 1. The transcription of HMGR strongly increased in all conditions compared to day 0. The mRNA level was 8-9-fold higher than that of the calibrator in ambient control and ozone control conditions and 4-5-fold higher in ambient damaged and ozone damaged conditions. Due to high variation, especially in ambient control condition, the increase in the expression of HMGR from day 0 to day 1 was statistically significant only in the two ozone conditions.

The receivers had been exposed to the herbivores for two days in all conditions on day 2. The mRNA levels in ambient control and ozone control conditions decreased from day 1 to day 2 but were still 7 fold and 5 fold higher than the expression of the calibrator, respectively. In contrast, the mRNA level of HMGR kept increasing from day 1 to day 2 in ambient damaged condition. It might have increased slightly also in ozone damaged condition but due to high variation this is unclear. The differences in the expression were statistically significant between day 0 and day 2 in ambient control and ambient damaged conditions.

In general, the RNA expression of HMGR seemed to be lower in ozone conditions compared to ambient conditions separately and combined in all time points (figure 13B). The difference in mRNA levels in ozone and ambient conditions was the most noticeable on day 2. In ambient conditions the RNA expression was 7,5-fold and in ozone conditions less than 5-fold higher compared to the calibrator. Between day 0 and day 1 the difference in expressions was modest. The RNA level in ozone conditions was barely higher than that of the calibrator and less than 2-fold in ambient conditions on day 0. On day 1, the expression in ambient conditions and ozone conditions were 7-fold and 6-fold compared to the calibrator, respectively. None of these differences were statistically significant.

Figure 13. The relative gene expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) in hybrid aspen receivers calculated using the comparative 2^(-ΔΔCt)-method. A: the receivers were exposed to the following conditions: amb ctrl = Intact control receivers in ambient air (ozone 15 ppb), that is, receivers that were exposed to volatiles emitted by undamaged emitters upwind. Leaf beetle larvae (P. laticollis) were added on the receivers after leaf harvest on day 0; amb dam = receives exposed to volatiles emitted by leaf beetle-damaged emitters under ambient condition. Larvae were added on the receivers after leaf harvest on the day 0; O3 ctrl = receivers that were exposed to volatiles emitted by undamaged emitters upwind under 100 ppb ozone condition. Larvae were added on the receivers after leaf harvest on the day 0; O3 dam = receives exposed to volatiles emitted by leaf beetle- damaged emitters under 100 ppb ozone condition. Larvae were added on the receivers after leaf harvest on the day 0. Gene expression was measured on three time points, day 0, 1 and 2. On day 0 the receivers had been exposed to VOCs for three days and in addition to larvae the following two days (day 1 and 2). ). The gene expression between different time points (a and b) and conditions were compared in statistical analysis (P<0,05). B: combined results of ambient = amb ctrl and amb dam conditions and of ozone = O3 ctrl and O3 dam conditions.

4.4.2 1-deoxy-D-xylulose-5-phosphate reductoisomerase, DXR

The gene expression of DXR was normalized to the expression of tubulin and related to the expression in ozone control condition on day 0 (calibrator). The fold change in the gene expression of DXR compared to the calibrator in each condition and time point is shown in figure 14A. Receiver plants had been exposed to VOCs released from the emitters for three days on day 0. The mRNA level of DXR was higher in ambient control, ambient damaged, and ozone damaged conditions compared to the calibrator, 2,8-fold, 2,6-fold and 1,7-fold, respectively. The difference in the RNA expression was statistically significant between ambient control and the calibrator.

The receivers were exposed to herbivores in all conditions on day 1. The expression of DXR strongly decreased in all conditions from day 0 to day 1, except in ozone control condition where it was at the same level on both days. The mRNA level was approximately at the same level in all conditions compared to the calibrator on day 1. The decreases in the expressions were statistically significant in ambient control and ozone damaged conditions and might have been significant also in ambient damaged condition if not for high variation on day 2.

The receivers had been exposed to the herbivores for two days in all conditions on day 2. The RNA expression was slightly higher on day 2 than on day 1 in all conditions except in ozone damaged condition where it was at the same level on both days. The mRNA level was still lower compared to the mRNA level on day 0 in ambient control, ambient damaged and ozone damaged conditions, 1,5-fold, 1,4-fold and 0,8-fold compared to the calibrator, respectively. The expression had increased to 1,3-fold in ozone control condition. The differences in the mRNA levels were statistically significant between day 0 and day 2 in ambient damaged and ozone damaged conditions and between ozone control and ozone damaged conditions on day 2.

The gene expression of DXR was lower in ozone conditions than in ambient conditions on day 0, day 2 and in all time points combined (figure 14B). The differences in these time points were also statistically significant. The most noticeable differences between ambient and ozone conditions were on day 0, where the mRNA level of ambient conditions was approximately twice higher than the mRNA level in ozone conditions. The differences in the expressions on day 1 and day 2 were more modest. The RNA expression was at the same level as the expression of the calibrator in ambient and ozone conditions on day 1 and in ozone conditions on day 2. The expression was slightly, 1,5-fold higher in ambient conditions compared to the calibrator on day 2.

Also, figure 14C shows that the expression of DXR was lower in the ozone conditions. There was a statistically significant difference in the expressions between ambient control and ozone control conditions.

Figure 14. The relative gene expression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) in hybrid aspen receivers calculated using the comparative 2^(-ΔΔCt)-method A: the receivers were exposed to the following conditions: amb ctrl = Intact control receivers in ambient air (ozone 15 ppb), that is, receivers that were exposed to volatiles emitted by undamaged emitters upwind. Leaf beetle larvae (P. laticollis) were added on the receivers after leaf harvest on day 0; amb dam = receives exposed to volatiles emitted by leaf beetle-damaged emitters under ambient condition. Larvae were added on the receivers after leaf harvest on the day 0; O3 ctrl = receivers that were exposed to volatiles emitted by undamaged emitters upwind under 100 ppb ozone condition. Larvae were added on the receivers after leaf harvest on the day 0; O3 dam = receives exposed to volatiles emitted by leaf beetle- damaged emitters under 100 ppb ozone condition. Larvae were added on the receivers after leaf harvest on the day 0. Gene expression was measured on three time points, day 0, 1 and 2. On day 0 the receivers had been exposed to VOCs for three days and in addition to larvae the following two days (day 1 and 2). The gene expression between different time points (a, b and c) and conditions were compared in statistical analysis. Statistically significant differences were found between conditions amb ctrl and O3 ctrl on day 0 (*) and between O3 ctrl and O3 dam on day 2 (**) (P<0,05). B: combined results of ambient = amb ctrl and amb dam conditions and of ozone = O3 ctrl and O3 dam conditions. Statistically significant differences were found between amb and ozone conditions on day 0 (*), day 2 (**) and all time points combined (***) (P<0,05) C: gene expression of amb ctrl, amb dam, ozone ctrl and ozone dam conditions in combined time points. Statistically significant difference was found between amb ctrl and ozone ctrl conditions in all time points combined (*)(P<0,05). 49

4.4.3 Lipoxygenase 1, LOX1

The gene expression of LOX1 was normalized to the expression of tubulin and related to the expression in ozone control condition day 0 (calibrator). The fold change in the expression of LOX1 compared to the expression of the calibrator in each condition and time point is shown in figure 15A. Receiver plants had been exposed to VOCs released from the emitters for three days on day 0. The mRNA level of LOX1 was at the level of the calibrator in ambient control and ozone damaged conditions and 2-fold in ambient damaged condition. The difference in the expression of ambient damaged and ozone damaged was statistically significant.

The receivers were exposed to herbivores in all conditions on day 1. The RNA expression of LOX1 strongly increased in all conditions from day 0 to day 1. The mRNA level was 9-fold in ambient control, 13-fold in ambient damaged, 14-fold in ozone control and 9-fold in ozone damaged conditions compared to the calibrator on day 1. The differences between day 0 and day 1 were statistically significant in all conditions, except in ambient damaged condition where standard deviation was very high on day 1.

The receivers had been exposed to the herbivores for two days in all conditions on day 2. The RNA expression of LOX1 decreased in all conditions from day 1 to day 2. Now the mRNA levels were 7-fold in ambient control, 11-fold in ambient damaged, 8-fold in ozone control and 3-fold in ozone damaged conditions compared to the calibrator. The expression in ozone damaged condition strongly decreased from day 1 to day 2 but the difference in the expressions between day 0 and day 2 was still statistically significant.

The RNA expression of LOX1 was lower in ozone conditions than in ambient conditions on day 0 and day 2 and in all time points combined (figure 15B). The expression was at the same level with the calibrator in ozone conditions and almost 2-fold in ambient conditions on day 0. This difference was statistically significant. On day 2, the mRNA level was 9-fold and less than 6-fold in ambient and ozone conditions compared to the calibrator, respectively. LOX1 was expressed at the same level in ambient and ozone conditions, approximately 11-fold compared to the calibrator, on day 1. However, there were no statistically significant differences in the expression between ambient and ozone conditions on day 1, day 2 or all the time points combined.

Figure 15. The relative gene expression of lipoxygenase 1 (LOX1) in hybrid aspen receivers calculated using the comparative 2^(-ΔΔCt)-method. A: the receivers were exposed to the following conditions: amb ctrl = Intact control receivers in ambient air (ozone 15 ppb), that is, receivers that were exposed to volatiles emitted by undamaged emitters upwind. Leaf beetle larvae (P. laticollis) were added on the receivers after leaf harvest on day 0; amb dam = receives exposed to volatiles emitted by leaf beetle- damaged emitters under ambient condition. Larvae were added on the receivers after leaf harvest on the day 0; O3 ctrl = receivers that were exposed to volatiles emitted by undamaged emitters upwind under 100 ppb ozone condition. Larvae were added on the receivers after leaf harvest on the day 0; O3 dam = receives exposed to volatiles emitted by leaf beetle-damaged emitters under 100 ppb ozone condition. Larvae were added on the receivers after leaf harvest on the day 0. Gene expression was measured on three time points, day 0, 1 and 2. On day 0 the receivers had been exposed to VOCs for three days and in addition to larvae the following two days (day 1 and 2). ). The gene expression between different time points (a, b and c) and conditions were compared in statistical analysis. Statistically significant differences were found between conditions amb dam and O3 dam on day 0 (*) (P<0,05). B: combined results of ambient = amb ctrl and amb dam conditions and of ozone = O3 ctrl and O3 dam conditions. Statistically significant difference was found between amb and ozone conditions on day 0 (*) (P<0,05).

4.5 Next Generation Sequencing

Next Generation Sequencing (NGS) was used to analyze the gene expression of the whole genome in each condition. qRT-PCR was used to validate the NGS results. However, validation could not been made since the results in these analyses were not comparable. The reason for not comparable results between qPCR and NGS could be due to 1) we have not selected the correct sequences for enzyme genes to primer design; 2) we have hybrid aspen derived from two various species and probably several different alleles per gene not equally expressed in samples 3) after NGS we would have right gene sequences (of the differentially expressed genes) the selection of primer sequences would have been much easier.

5 DISCUSSION

Herbivore damage on plant leaves causes a release of a blend of herbivore-induced plant volatiles. These compounds might reach intact neighboring plants and elicit or prime their defense reactions. These important mechanisms help plants to survive herbivore attack with less damage. Tropospheric ozone impairs plant-to-plant communication by degrading many VOCs. In this study, plant-to-plant communication between hybrid aspens and the effect of ozone on this communication was studied in climatized chambers.

The objective of the experiment was to study VOC-mediated defense elicitation and priming in ambient air and in elevated ozone conditions. VOCs released from the emitter plants were collected and measured on day 2. The measurement showed that P. laticollis larvae feeding on the emitters induced higher emissions of herbivore-induced volatiles, especially that of β- ocimene, other monoterpenes (sabinene, limonene and linalool) and cis-3-hexenyl isovalerate both in lower and higher ozone concentrations. Also, (E)-DMNT, caryophyllene and cis-3- hexenyl butyrate emissions were increased either in ambient or in elevated ozone condition. Whether or not these HIPVs induced defense elicitation or priming in the receiver plants in terms of the mRNA expression of defense genes is discussed next.

Leaf samples for gene expression analysis were collected from the receivers before adding larvae on them on day 0. If HIPVs released from the damaged emitters were enough to elicit defense reactions in the receivers, the expression of the defense genes would be higher in damaged condition compared to controls on day 0.

HIPVs were not enough to directly elicit defense reactions, in this case by increasing the gene expression of HMGR, DXR or LOX1 compared to the VOCs emitted from the intact emitters on day 0. The expression of HMGR was at the same level in the two damaged and control conditions in this time point. The expression of LOX1 was higher, 2-fold, in the receivers exposed to HIPVs compared to the control condition in ambient air which might have indicated that HIPVs had elicited direct defense. However, this difference was not statistically significant so this conclusion cannot be made. The expression of LOX1 was similar in the receivers exposed to HIPVs and intact emitter VOCs alike in ozone conditions on day 0. In fact, ozone caused statistically lower expression of LOX1 in damaged condition compared to ambient air, which indicates the interference of ozone on VOC signaling on day 0.

The expression of DXR was also clearly higher in the receivers exposed to HIPVs compared to the receivers exposed to intact emitter VOCs but this time in ozone conditions. The direct defense elicitation by HIPVs was not seen here but this time the difference was very close to being statistically significant. Similar expression level of DXR in the receivers in damaged and control conditions in ambient air shows, though, that HIPVs were not enough to elicit defense reactions.

Larvae were added on the receivers in all conditions later on day 0. Thus, hybrid aspens in ambient damaged and ozone damaged conditions had been pre-exposed to emitter HIPVs and subsequently to herbivore attack. Also, hybrid aspens in ambient control and ozone control conditions had been exposed to herbivores but they had not been pre-exposed to emitter HIPVs by day 1. If HIPVs cause defense priming in the receivers, that is elicit more rapid or intense defense reactions after subsequent herbivore exposure, the expression of the defense genes would be significantly higher in the damaged condition compared to the control on day 1 and day 2.

The addition of herbivores caused considerable increases in the expression of HMGR and LOX1 in all conditions. However, pre-exposure to HIPVs did not elicit faster expression of HMGR in the receivers. In contrast, the mRNA levels were more intense in the two control conditions compared to the damaged conditions on day 1. On the other hand, on day 2, the mRNA level of HMGR in ambient damaged condition reached the same level of expression than the control conditions on day 1 while their expression had already started to decrease. Could this mean that pre-exposure to HIPVs elicits not faster but longer-lasting expression of the gene? This does not seem likely since the expression was slower and the delay did not make it more intense. The plant would probably benefit more of a rapid reaction than of a slower one, even if it was more intense. It is to be noted that there were no statistically significant differences in the expression between any conditions. Only the addition of the larvae elicited statistically significant increases in the gene expression of HMGR. This was seen in all conditions either on day 1 or day 2. Thus, the defense priming was not observed.

The increase in the expression of LOX1 after adding the larvae on the receivers was statistically significant in all other conditions, except in ambient damaged condition on day 1. The mRNA level was apparently higher in the damaged condition compared to the control condition in ambient air which might have indicated that defense priming had occurred, but very high deviation in ambient damaged condition on day 1 makes this unreliable. In contrast, the mRNA expression was higher in the control than in the damaged condition in elevated ozone. Thus, defense priming was not observed. The expression of LOX1 had already started to decrease in all conditions by day 2.

The addition of the larvae on the receivers caused statistically significant reductions in the expression of DXR in all conditions except in ozone control condition on day 1. This is interesting because DXR functions produce defense compounds, terpenoids, and the expression could be expected to increase rather than decrease when the plant is under herbivore attack. Anyhow, pre-exposure to HIPVs did not cause defense priming in DXR, either.

Why were the key enzymes, HMGR and DXR, producing the same defense compounds in two pathways so differently expressed after herbivore damage? Could it be that these MEP and MVA pathways producing the same terpenoid precursors are competitive and in the case of herbivore damage, the faster pathway, MVA pathway, first takes over to maximize terpenoid production? The activity of MVA pathway then decreases giving the MEP pathway the opportunity to reactivate? This would be consistent with the DXR results since the expression is slightly increased in all conditions, except in ozone damaged condition where the expression remained similar by day 2. There was nothing in the literature that would support this conclusion. MEP pathway originated terpenoid precursor IPP is known to shift from chloroplasts to cytoplasm and take part in the MVA pathway but otherwise these pathways should be separate. The DXR expression levels were quantified using the Ct (2^-ΔΔCt) method, though all the requirements were not met. It would be interesting to see how different the results would have been if a correct quantification method had been used.

Ozone seems to have affected the expression of DXR on day 0. The mRNA level of DXR is noticeably lower in the receivers in the two ozone conditions than in the two ambient conditions. The expression in ozone control receivers was less than half of the expression in ambient control receivers and this difference was statistically significant. The difference between ozone-enriched and ambient air in damaged conditions was also noticeable but not statistically significant. Why was the expression of DXR in ozone control condition so much lower than in the ambient control condition? These receivers had not been exposed to HIPVs so the difference does not tell about the interference of ozone on VOC signaling between plants. Could it indicate that ozone affects the MEP pathway directly in tissues? In that case, DXR seems to be sensitive to ozone. This conclusion is possible. Moderately elevated ozone has been observed to reduce the mRNA expression of DXR in Norway spruce and to indicate that ozone affects the regulation of terpenoid biosynthesis (Kivimäenpää et al. 2012).

There was a trend of ozone reducing the expression of HMGR and DXR in the receivers in all time points and of LOX1 on day 0 and day 2. This effect was fairly weak in HMGR, especially on day 1. The difference in the expression of HMGR was the most noticeable between the damaged conditions on day 2 where the expression was almost 2-fold in ambient air compared to ozone-enriched air. The difference in the control conditions was more modest but still clear. However it cannot be said that ozone interfered with the VOC signaling by affecting the expression of HMGR since the differences in the expression were not statistically significant in any time point.

Ozone caused noticeable reductions in the expression of LOX1 in damaged conditions on day 0 and day 2 and there were no differences in the expression between the control conditions in these time points. This difference was statistically significant in the damaged conditions and in combined results between ozone and ambient conditions on day 0, which indicates that ozone interfered with VOC signaling, possibly by degrading the volatile compounds. The results on day 2 also point to that direction, though this is not statistically proven. Thus, the impairing effect of ozone on VOC signaling was observed in the expression of LOX1 on day 0.

Ozone interference in VOC signaling is observed relatively clear in the expression of DXR. The expression is statistically lower in ozone conditions compared to the ambient conditions on day 0. Also, ozone seems to have reduced the expression of DXR in the damage condition on day 2 while the receivers in the control conditions were unaffected. This resulted in statistically significant difference between ozone conditions combined compared to ambient condition on day 2. Ozone seems to have reduced the expression of DXR statistically also in all time points combined. However, if ozone was interfering with VOC signaling by reducing the expression of DXR has to be considered carefully, since the significance shown in all time points combined mainly results of the relatively large difference in the expression on day 0.

Ozone seemed to reduce the emissions of many HIPVs, α-pinene, limonene, β-ocimene, (E)- DMNT, caryophyllene, farnesene and cis-3-hexenyl acetate, to some extent but this was not statistically proven. Thus, the impairing effect of ozone on VOC signaling was not seen in the VOC emissions.

6 CONCLUSIONS

The aim of this master’s thesis was to study plant-to-plant communication by herbivore- induced plant volatiles and how ozone affects this communication. The relative gene expression of HMGR, DXR and LOX1 was measured in ambient or ozone-enriched conditions.

Herbivore feeding increased the HIPV emissions of the emitter hybrid aspens. However, direct defense elicitation or defense priming by HIPVs was not observed at the gene expression level in the receivers. Changes in the expression of DXR and LOX1 seemed to indicate the impairing effect of ozone on VOC signaling at least in some time points. The impairing effect of ozone on VOC signaling was not seen in volatile emissions. Ozone seemed to reduce the expression of all the defense genes. In addition, there were some indications that MEP pathway might be more sensitive to ozone than the other branch of terpenoid biosynthesis, mevalonate pathway.

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