Responses of growth and defense in dioecious Populus tremula (L.) to simulated climate change and tissue damage

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PANU PIIRAINEN

PHARMACOKINETICS AND EFFICACY OF EPIDURAL OXYCODONE

Dissertations in Health Sciences

PUBLICATIONS OF

THE UNIVERSITY OF EASTERN FINLAND

Dissertations in Forestry and Natural Sciences

NORUL SOBUJ

Responses of growth and defense in dioecious Populus tremula (L.) to simulated climate change and

tissue damage

PUBLICATIONS OF

THE UNIVERSITY OF EASTERN FINLAND

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RESPONSES OF GROWTH AND DEFENSE IN DIOECIOUS Populus tremula (L.) TO SIMULATED

CLIMATE CHANGE AND TISSUE DAMAGE

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Norul Sobuj

RESPONSES OF GROWTH AND DEFENSE IN DIOECIOUS Populus tremula (L.) TO SIMULATED

CLIMATE CHANGE AND TISSUE DAMAGE

Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences

No 439

University of Eastern Finland Joensuu

2021

Academic dissertation

To be presented by permission of the Faculty of Science and Forestry for public examination in the Auditorium N100 in the Natura Building at the University of Eastern Finland, Joensuu, on December, 02, 2021, at 12 o’clock

noon

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Punamusta Oy Joensuu, 2021 Editor: Raine Kortet

Myynti: Itä-Suomen yliopiston kirjasto ISBN: 978-952-61-4356-9 (nid.) ISBN: 978-952-61-4357-6 (PDF)

ISSNL: 1798-5668 ISSN: 1798-5668 ISSN: 1798-5676 (PDF)

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5 Author’s address: Norul Sobuj

University of Eastern Finland

Depart. of Environmental and Biological Sciences P.O. Box 111

80101 JOENSUU, FINLAND email: norul.sobuj@uef.fi

Supervisors: Professor (emerita) Riitta Julkunen-Tiitto, Ph.D.

University of Eastern Finland

Depart. of Environmental and Biological Sciences P.O. Box 111

80101 JOENSUU, FINLAND email: riitta.julkunen-tiitto@uef.fi

Professor Heli Peltola, Ph.D.

University of Eastern Finland School of Forest Sciences P.O. Box 111

80101 JOENSUU, FINLAND email: heli.peltola@uef.fi

Professor Line Nybakken, Ph.D.

Norwegian University of Life Sciences

Faculty of Environmental Sciences and Natural Resource Management

P.O. Box 5003 1432 ÅS, NORWAY

email: line.nybakken@nmbu.no

Senior Researcher Virpi Virjamo, Ph.D.

University of Eastern Finland School of Forest Sciences P.O. Box 111

80101 JOENSUU, FINLAND email: virpi.virjamo@uef.fi

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Research Professor Lauri Mehtätalo, Ph.D.

Natural Resources Institute Finland P.O. Box 111

80101 JOENSUU, FINLAND email: lauri.mehtatalo@luke.fi

Reviewers: Research Professor Leena Finér, Ph.D.

Natural Resources Institute Finland P.O. Box 111

80101 JOENSUU, FINLAND email: leena.finer@luke.fi

Professor (emeritus) Pekka Niemelä, Ph.D.

University of Turku Depart. of Biology 20014 TURKU, FINLAND email: pnieme@utu.fi

Opponent: Professor Kurt Fagerstedt

University of Helsinki Viikki Plant Science Centre

P.O. Box 65

00014 HELSINKI, FINLAND email: kurt.fagerstedt@helsinki.fi

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7 Sobuj, Norul

Responses of growth and defense in dioecious Populus tremula (L.) to simulated climate change and tissue damage

Joensuu: University of Eastern Finland, 2021 Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences; 439 ISBN: 978-952-61-4356-9 (print)

ISSNL: 1798-5668 ISSN: 1798-5668

ISBN: 978-952-61-4357-6 (PDF) ISSN: 1798-5676 (PDF)

ABSTRACT

The aim of this thesis was to study the effects of simulated climate change and tissue damage on growth and defense traits in dioecious Populus tremula (L.). In a greenhouse experiment, I investigated the effects of elevated CO2 concentration and temperature on the growth and defensive phenolic compounds in four female and four male genotypes of P. tremula.

Moreover, in a controlled field experiment, I studied the responses of growth and phenolics due to developmentally regulated variation and lateral bud removal in six female and six male genotypes of P. tremula growing under elevated temperature and ultraviolet-B (UVB) radiation. Our results indicated that elevated temperature had the strongest effect among the three studied climatic factors. Elevated temperature substantially increased the growth and decreased the concentration of phenolics in stem bark, whole stems and leaves of P. tremula. On the other hand, elevated CO2

concentration induced phenolics in stem bark, but elevated UVB radiation had no effect on growth and phenolics. The plants seemed to acclimate to elevated temperature over time in the controlled field experiment. As a result, the stimulating effect of elevated temperature on the diameter growth rate decreased while the concentration of condensed tannins

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increased in P. tremula through time. Moreover, the removal of lateral buds simultaneously induced growth and phenolics in P. tremula. Therefore, bud- removed individuals had greater leaf, stem, and total biomass, as well as greater concentration of flavonoids and condensed tannins in leaves, as compared to the intact ones. Our results indicated that warming induced sex-based discrepancies in growth performance in P. tremula. In response to elevated temperature, growth stimulation was considerably higher in females, especially in bud-removed females. However, the sex-specific discrepancies in phenolic concentration mostly depended on the plant organ, clone, and compound in question, and were less influenced by the treatments. Overall, the findings of this thesis suggest that climate warming would stimulate the growth and decrease the concentration of defensive phenolics in P. tremula in the future, but the magnitude of the warming effect may reduce to some extent due to possible long-term temperature acclimation. Predicted warming may also offset the CO2-induced phenolic accumulation in P. tremula, and ecologically relevant UVB radiation may have little to no impact on growth and phenolic accumulation. Low-to-moderate levels of tissue damage may benefit P. tremula by increasing growth and defense simultaneously.

Keywords: Aspen, climate change, CO2, elevated temperature, growth, secondary metabolites, sexual dimorphism, tissue loss, trade-off, UVB radiation

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Acknowledgements

I express my greatest gratitude to Professor (emerita) Riitta Julkunen-Tiitto for maintaining her trust in me and granting me this wonderful opportunity to carry out this project. I have had a great learning experience in the early stage of my research career under your guidance. My sincere thanks are given to Professor Heli Peltola for her cooperation and advice during the preparation of the second and third manuscripts. Thank you Heli for providing your valuable remarks on those studies as well. I am very grateful to Professor Line Nybakken for her inspiration and her thoughtful comments on the manuscripts. Undoubtedly, your corrections, comments, and guidelines have greatly enhanced my publications.

I would like to express my gratitude towards senior researcher Virpi Virjamo for her coordination, support, and guidance in this study. Many thanks also for making valuable comments on the manuscripts. I am grateful to Research Professor Lauri Mehtätalo for guiding me in statistical analyses concerning the second and third manuscripts. It would have been very difficult to accomplish those studies without your guidance. Research Director Jarkko Akkanen is highly appreciated for his administrative support and for following the progression of this thesis.

Many thanks go to my co-authors for their contribution to the manuscripts based on which this thesis has been prepared. I am also grateful to my colleagues Unni, Katri, Paula, Yaodan, Tendry, Anneli, and Anu for their wonderful cooperation during the experiments, and for providing peer support during the laboratory and office work. I sincerely thank Sinikka Sorsa for her assistance in the laboratory work. The technicians and staff members of the department and Mekrijärvi Research Station are appreciated for their support during the experiments. I acknowledge the funding support from the Finnish Cultural Foundation, the Faculty of Science and Forestry (University of Eastern Finland), and the Finnish Society of Forest Science to carry out my doctoral studies.

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I wish to thank Research Professor Leena Finér and Professor (emeritus) Pekka Niemelä for reviewing this thesis. I am grateful to Professor Kurt Fagerstedt for accepting his role as opponent for my thesis.

I am grateful to my family, friends, and relatives for their constant support during my stay in Finland. It would have been very difficult to come that far without your unconditional love and sacrifice.

Joensuu, 12 October 2021 Norul Sobuj

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11 LIST OF ABBREVIATIONS

ASL above sea level CO2 carbon dioxide

HPLC high-performance liquid chromatography Q-TOF MS quadrupole time-of-flight mass spectrometry PPM parts per million

UV ultraviolet

UVA ultraviolet-A (315–400 nm) UVB ultraviolet-B (280–315 nm)

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LIST OF ORIGINAL PUBLICATIONS

This thesis is based on data presented in the following articles, referred to by the Roman Numerals I-III.

I Sobuj N, Virjamo V, Zhang Y, Nybakken L, Julkunen-Tiitto R. (2018).

Impacts of elevated temperature and CO2 concentration on growth and phenolics in the sexually dimorphic Populus tremula (L.).

Environmental and Experimental Botany, 146: 34–44.

II Sobuj N, Nissinen K, Virjamo V, Salonen A, Sivadasan U, Randriamanana T, Ikonen V, Kilpeläinen A, Julkunen-Tiitto R, Nybakken L, Mehtätalo L, Peltola H. (2021). Accumulation of phenolics and growth of dioecious Populus tremula (L.) seedlings over three growing seasons under elevated temperature and UVB radiation. Plant Physiology and Biochemistry, 165: 114–122.

III Sobuj N, Virjamo V, Nissinen K, Sivadasan U, Mehtätalo L, Nybakken L, Peltola H, Julkunen-Tiitto R. (2020). Responses in growth and phenolics accumulation to lateral bud removal in male and female saplings of Populus tremula (L.) under simulated climate change. Science of the Total Environment, 704: 135462.

The above publications have been included at the end of this thesis in the printed version with their copyright holders’ permission.

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13 AUTHOR’S CONTRIBUTION

The author took part in height and diameter measurements (I–III), sampling of the plants (I and III), and weighing plant biomass (I and III). In studies I and III, the author was responsible for the sample preparation, extraction, and quantification of phenolic compounds. In study II, the author was responsible for the quantification of phenolic compounds of the samples included in the first and second growing seasons. The author was primarily responsible for conducting the statistical analyses and writing the manuscripts of all three studies (I–III).

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1 Introduction

1.1 Plant growth responses to climate change

Terrestrial ecosystems have been subject to the human-induced climate change over the last decades and are projected to go through it during the current century. It is predicted that the concentration of CO2 in the atmosphere will reach 540–970 parts per million (ppm) by the year 2100, while the global mean air temperature could rise by 1.5–4 ˚C over the same period (IPCC 2014; Gherlenda et al. 2015). Although the concentration of CO2

is increasing uniformly throughout the world, temperature is increasing at an even higher rate at high latitudes due to the changes in Arctic sea ice and snow cover (Caldwell et al. 2007; Buermann et al. 2013; Ruosteenoja et al.

2016). Increased levels of CO2 concentration and temperature may have a profound impact on functioning and productivity of forest ecosystems (Kellomäki 2017). Under the current level of atmospheric CO2 concentration, maximum photosynthesis is a constraint for plants (Kim et al. 2003;

Stinziano and Way 2014). The predicted increase in atmospheric CO2

concentration would make more carbon available for photosynthetic carbon assimilation, hence it may promote height, diameter, and biomass accumulation in plants. However, these physiological and growth responses due to elevated levels of CO2 concentrations in plants may also depend on other factors such as duration of exposure, plant species, and availability of soil nutrients and water (Peñuelas and Estiarte 1998; Tjoelker et al. 1998;

Usami et al. 2001; Stinziano and Way 2014; Kellomäki 2017). Many experimental studies with temperate and boreal tree species have demonstrated that the initial photosynthetic rate is maintained in CO2- enriched plants through time, but growth is constraint due to low nitrogen availability (Tjoelker et al. 1998; Oren et al. 2001; Norby et al. 2010).

On the other hand, temperature is a major determinant of growth and distribution of plant species across different biomes (Mittler 2006; Stinziano

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and Way 2014). In the boreal region, forest growth is mainly limited by low temperature during the growing season (Kellomäki 2017). It is predicted that elevated temperature would increase the physiological activity and prolong the growing season, and therefore a significant increase in plant growth is widely expected in this region (Way and Oren 2010; Stinziano and Way 2014).

Almost all previous greenhouse and field studies have found that elevated temperature increases photosynthesis, height, diameter, leaf area, and biomass growth in deciduous tree seedlings of Betula, Salix, and Populus species (Veteli et al. 2002; Huttunen et al. 2007; Randriamanana et al. 2015;

Nissinen et al. 2020). Since increasing temperature in the atmosphere is due to the increase in the atmospheric CO2 concentration, and plants respond to both factors, one factor can modify the effect of the other. Several earlier studies found that under elevated temperature, stimulation of photosynthetic carbon assimilation and growth in CO2-fertilized plants increased as temperature increased (Usami et al. 2001; Zhao et al. 2012;

Kellomäki 2017). Thus, the stimulating effect of elevated CO2 concentration on growth performance in plants might be affected by warming in the forthcoming years, but this needs further investigation.

1.2 Effect of climate change on plant secondary metabolites

Secondary metabolites constitute one of the most common and widespread groups of chemical substances in plants. Among the secondary metabolites, phenolics represent the largest group, comprising almost 20% of total carbon in the terrestrial biosphere (Yu and Jez 2008). In boreal deciduous trees and shrubs, the most abundant phenolic compounds are composed of salicylates, flavonoids, phenolic acids, and condensed tannins (Veteli et al.

2007; Nissinen et al. 2017; Nissinen et al. 2018). Because of climate change, the predicted increase in CO2 concentration and temperature may alter the composition and quantity of phenolics in boreal tree species in the coming decades. In response to elevated CO2 concentration, plants typically increase photosynthetic carbon assimilation and decrease the concentration of

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19 nitrogen, therefore they generally exhibit increased carbon-to-nitrogen ratios in their foliar tissues (Bryant et al. 1983; Handa et al. 2005; Lindroth 2010). It is suggested that plants would allocate excess carbon to produce carbon-based secondary metabolites if there is a nitrogen deficiency in plant tissues which is essential for utilising carbon for biomass accumulation (Bryant et al. 1983; Peñuelas and Estiarte 1998). Therefore, we may expect that increased levels of CO2 concentrations would increase the synthesis of carbon-based secondary chemical constituents in boreal tree species under a changing climate. Although many empirical studies support this hypothesis, no change or even a decrease in the concentration of different carbon-based secondary metabolites in plants exposed to elevated levels of CO2 concentrations have been reported (Kuokkanen et al. 2003; Veteli et al.

2007; Lindroth 2012).

Temperature is one of the key parameters regulating primary and secondary metabolism in plants. Although plants would acquire more carbon under elevated temperature as it is predicted due to the increased levels of photosynthesis in plants, however plants may not use it to synthesize more carbon-based secondary metabolites in warming growth conditions. According to the carbon-nutrient and growth-differentiation balance hypotheses, under favorable circumstances, elevated temperature would decrease the accumulation of carbon-based secondary metabolites in plants because of plant’s preference for growth over defense (Bryant et al. 1983; Herms and Mattson 1992). In agreement with these hypotheses, many experimental studies have revealed that elevated temperature reduces the concentration of secondary metabolites in woody perennials (Nybakken and Julkunen-Tiitto 2013; Randriamanana et al. 2015; Sivadasan et al. 2018). Based on the above, we could assume that elevated temperature would counteract the stimulating effect of elevated CO2

concentration on phenolics accumulation in boreal tree species. Veteli et al.

(2007) found that elevated temperature lowered the stimulating effect of elevated CO2 concentration on salicylates, flavonoids, and phenolic acids in B. pendula (Roth.), B. pubescens (Ehrh.), and Salix myrsinifolia (Salisb.), although the magnitude of the interaction varied across the species. The

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altered chemical composition and quantity of phenolics in boreal tree species due to increased levels of CO2 concentration and temperature will most likely affect their susceptibility to microbes, insects, and other herbivores. Although the individual effect of elevated CO2 concentration or elevated temperature on secondary metabolites in boreal tree species has long been studied, the interactive effects of these two factors need more detailed studies.

1.3 Plant development and interaction with environmental conditions

As plants grow and develop, they pass through the juvenile seedling stage before reaching reproductive (vegetative and sexual) maturity. In long-lived tree species, the juvenile stage is particularly important because it includes the processes of physiological ageing and is vulnerable to external threats, such as herbivores (Hanley et al. 2004; Goodger et al. 2006). During the juvenile growth period before flowering, many tree species show a shift in chemical defense due to genetically determined developmental changes and phenotypic adjustment to the prevailing environmental conditions (Barton and Koricheva 2010; Cope et al. 2019). Boege and Marquis (2005) suggested that, as plants get older, during the juvenile growth period they should increase the concentration of defensive secondary metabolites due to a decreasing root-to-shoot ratio and a reduced growth rate over time.

Although many empirical studies support this trend, it appears that development-related changes in plant defense may also vary among plant species and classes of secondary metabolites (Goralka and Langenheim 1996; Ochoa-Lopez et al. 2015; Nissinen et al. 2018; Cope et al. 2019). For example, in many tree species belonging to the genera Salix and Populus, the concentrations of salicylates decrease whereas the concentrations of flavonoids and condensed tannins increase in leaves and twigs as plants aged (Julkunen-Tiitto 1989; Cole et al. 2016; Nissinen et al. 2018).

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21 If changes in the abiotic environments occur during plant development, the developmentally regulated changes in plant traits are also likely to change. However, long-term responses of perennial plants to elevated temperature is poorly understood. Many past studies have revealed that long-term exposure of plants to warmer conditions commonly leads to thermal acclimation (Leuzinger et al. 2011; Smith et al. 2016). Therefore, growth responses of perennial plants under elevated temperature may exhibit temporal variability due to the changes of growth rate as a result of possible long-term acclimation to a warming growth environment.

Moreover, changes in physiological demands and defence strategies as trees get older may lead to a variation in defensive secondary metabolites in plant tissues in response to elevated temperature over time. In a two year- long study, Nybakken et al. (2012) found that the magnitude of elevated temperature effects on phenolic concentration in the leaves of S. myrsinifolia were lower in the second year. Therefore, in the case of perennial plants, understanding their development-by-environment interactions on a long- term basis is particularly important, especially any that may be induced by climate change.

Ultraviolet-B (UVB) radiation is another important abiotic factor that may affect growth and other physiological processes of plants during their development. It has recently been reported that elevated levels of UVB radiations have no effect on photosynthesis of terrestrial plants when studies are conducted in open fields, but there may have subtle inhibitory effects on biomass accumulation (Ballaré et al. 2011; Suchar and Robberecht 2018). Yet, the effect of UVB radiation on growth performance in perennial plants may accumulate through time and may have a stronger long-term effect. As an example, elevated UVB radiation retarded diameter growth in B. pendula in the third growing season possibly due to the cumulative effect (Tegelberg et al. 2001). On the other hand, accumulation of phenolics, especially flavonoids and phenolic acids in response to elevated UVB radiation in terrestrial plants is well known (Newsham and Robinson 2009;

Suchar and Robberecht 2015). Since perennial plants very often show developmentally regulated variation in phenolics as they get older, it is

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expected that the cumulative effect of UVB radiation would either amplify or compensate for the concentration of phenolics during plant development.

Therefore, it is important to consider the long-term effect of UVB radiation on different perennial plants.

1.4 Responses of plants to tissue damage under climate change

Insect herbivores play important roles in terrestrial ecosystems. They consume a negligible amount of plant biomass unless an outbreak occurs, however this can alter vegetation structure and functions and can affect plant productivity substantially (Schowalter 1989; Tiffin 2000). Across all biomes, foliage, sap, and root feeding herbivores cause a loss of more than 20% of annual net primary productivity (Agrawal 2011). Although the consumption of plant tissues by insects and other herbivores is viewed traditionally as loss of production, the effects can be much more complex because there is no consistency in responses in plants after biomass loss.

Many experimental studies have demonstrated that plant growth is overcompensated after simulated tissue damage, while there are studies where undercompensation and no responses in plants due to tissue damage have also been reported (Reich et al. 1993; Fineblum and Rausher 1995; Nykänen and Koricheva 2004; Huttunen et al. 2013).

Overcompensation is typically seen in plants following low-to-moderate levels of tissue damage (Lehtilä 2000; Stowe et al. 2000; Jacquet et al. 2013).

Besides, it is suggested that the type of tissue damaged by insects and herbivores may also influence compensatory responses in plants (Honkanen et al. 1994). Lateral vegetative buds, containing meristematic tissue, are highly susceptible to damage by insect herbivores because they contain a high amount of protein and often also low concentrations of phenolics (Johnson and Lyon 1988; Maschinski and Whitham 1989; Herms and Mattson 1992). Damage to vegetative buds reduces the non- photosynthetic organs in plants, but this change increases the supply of photosynthates to the remaining buds (Honkanen et al. 1994; Ozaki et al.

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23 2004). Hence, insect herbivore feeding on vegetative buds commonly benefits plants by enhancing growth (Honkanen et al. 1994; Rasmussen et al. 2003). Of note, Schowalter (1989) reported a decrease in the production of shoots and new foliage in Pseudotsuga menziesii (Mirbel) Franco due to bud damage.

Provided the presence of allocational costs associated with both growth and defense, greater allocation of resources by plants to one trait may constrain allocation to other traits as it is hypothesized by Bryant et al. (1983) and Herms and Mattson (1992). Therefore, plants that overcompensate due to tissue damage may decrease the allocation of resources to secondary chemical constituents, such as salicylates, flavonoids, phenolic acids, and condensed tannins, in different plant organs. Accordingly, many plant–

herbivore interaction studies have reported that consumption of plant parts by their consumers results in greater growth and reduced defense in plants (Pilson 2000; Erwin et al. 2001; Prittinen et al. 2003; Hikosaka et al. 2005).

However, some recent evolutionary ecology studies have reported that growth and defense are complementary to each other, and that plants may employ both traits to compensate for tissue damage (de Jong and van der Meijden 2000; Tuller et al. 2018). It is suggested that if plants can compensate for the entire tissue damage, their growth and defense should be independent processes (Muola et al. 2010; Carmona et al. 2011). As an example, there were no trade-offs between growth and defense in Vachellia tortilis (Forssk.) Galasso & Banfi, which recovered completely after simulated browsing (Gowda 1997).

On the other hand, prevailing environmental conditions may affect a plant’s quality and its capacity to compensate following tissue damage. In some studies, it has been found that plants grown under elevated temperature increase their water content and carbohydrates and decrease the concentration of secondary metabolites in different organs (Nybakken et al. 2012; Lemoine et al. 2013; Randriamanana et al. 2015). Based on this, anticipated elevated temperature may substantially lower plant quality and make forest ecosystems more susceptible to herbivore damage. However, little is known about how plants would respond following tissue damage in

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the predicted climate warming in the future. Huttunen et al. (2007) found that a 25% defoliation of B. pendula resulted an increase of leaf area by 50%

in plants grown under elevated temperature than the ones grown in ambient conditions. This indicates that plants may modify their ability to tissue damage under changing climatic conditions.

The intensity of UVB radiation may also alter the quality of host plants and hence can affect the interaction between plants and their insect consumers. Although secondary compounds can act as attractants for some specialist herbivores (Lavola et al. 1998), the presence of these compounds in a greater quantity in plant tissues generally lower the forage quality (Julkunen-Tiitto et al. 2005; Ruuhola et al. 2007). Several empirical studies have revealed that exposure to elevated UVB radiation substantially reduces the consumption of plant tissues compared with the plants not exposed to UVB radiation (McCloud and Berenbaum 1994; Warren et al. 2002;

Rousseaux et al. 2004). On the other hand, UVB-induced secondary metabolites in plants may also be accumulated in response to herbivore damage. It was found that two groups of plants exposed to elevated UVB radiation and herbivory separately have accumulated similar secondary compounds in their tissues (Izaguirre et al. 2007). Therefore, if the UVB radiation exposed plants are challenged by herbivores simultaneously, the induction can be more pronounced than the single effects of either UVB radiation or herbivore attack.

1.5 Sex-dependent responses in growth and defense in dioecious plants

Dioecious plants represent ~5–6% of angiosperm species and are found in 43% of flowering plant families (Renner and Ricklefs 1995; Renner 2014). In many dioecious plants, males and females exhibit dimorphism in secondary plant traits, including physiology, morphology, and herbivory (Delph 1999;

Cornelissen and Stiling 2005). It is suggested that discrimination in allocation

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25 of resources by males and females to plant functions, namely growth, defense, and reproduction, results in dimorphism in secondary traits (Obeso 2002). Males often allocate a greater amount of resources to growth to become vigorous, while females prefer to invest more in defense to combat herbivores and pathogens, and in reproduction to produce fruits and seeds (Ågren et al. 1999; Cepeda-Cornejo and Dirzo 2010). This sex-based allocation of resources often leads to trade-offs between growth and defense in males and females, and such trade-offs are likely to be modified in interaction with abiotic and biotic factors, and as a result of plant development (Juvany and Munné-Bosch 2015; Jiang et al. 2016).

Sex-dependent responses of dioecious plants to abiotic and biotic factors can lead to changes in productivity of male and female individuals, and consequently may affect their distribution. Many earlier studies have reported that male populations of dioecious plants are more responsive to elevated levels of CO2 concentration and temperature than female individuals. For example, in response to elevated CO2 concentration, male individuals of P. tremuloides (Michx.) benefited more from CO2 fertilization by increasing photosynthetic carbon assimilation and total biomass accumulation than did females (Wang and Curtis 2001). In another study, when the two sexes of S. arctica (Pall.) were grown together under combined treatments of elevated CO2 concentration and temperature, males outperformed females in photosynthetic carbon assimilation (Jones et al.

1999). Therefore, if a predicted increase in CO2 concentration and temperature favors one sex over another, there could be changes in the sex ratio and, hence, an altered population structure of males and females.

On the other hand, because of climate change male individuals are predicted to face greater herbivore damage in the coming years due to their relatively higher growth rate and lower concentration of defensive secondary metabolites present in their tissues compared with females (Cornelissen and Stiling 2005). However, very few studies have investigated how male and female populations of dioecious species would respond following tissue damage (Wandera et al. 1992; Gronemeyer et al. 1997).

Because males and females differ from one another in their growth rate, we

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may expect sex-based differences in their compensatory ability. In fact, females should compensate for tissue consumed by herbivores slower than males, presumably as a consequence of their greater allocation of resources to defense in order to reduce further palatability, while the opposite should be true for males. Possible alterations of abiotic and biotic factors in the future can have differential effects on male and female populations of dioecious plants, which may have strong impacts on ecological processes of forest ecosystems.

1.6 Importance of studying P. tremula in the context of global climate change

Among trees, P. tremula is the second most widely distributed species in the world (Caudullo and Rigo 2016). It is native to cool temperate and boreal regions and occurs almost all over Europe and western Asia (Myking et al.

2011). It is regarded as a keystone species in the boreal forest ecosystem because of its fundamental ecological importance for other species. It hosts hundreds of herbivorous and saprophytic invertebrates, fungi, and epiphytic lichens, and is also a highly preferred forage species for large herbivores during winter (Lindroth et al. 2007; Boeckler et al. 2011; Myking et al. 2011).

Despite the broad spatial distribution and high ecological importance of P.

tremula, little is known about the impact of predicted climate change and associated factors on its growth and defense in boreal forests. The characteristic feature of P. tremula is of rapid growth (Myking et al. 2011).

This species also contains large amounts of carbon-based secondary metabolites in its leaves and twigs, which are plastic to abiotic and biotic stresses (Boeckler et al. 2011; Randriamanana et al. 2014). Therefore, predicted changes in climate and tissue damage due to increased levels of herbivory in the forthcoming years may have strong impacts on its growth and defense traits, and consequently may affect its productivity, reproductive success, and distribution.

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1.7 Aim of the thesis

The main aim of this thesis was to study the effects of simulated climate change and tissue loss on growth and phenolic accumulation in female and male individuals of P. tremula. A greenhouse experiment was conducted in which eight genotypes (four female and four male) of P. tremula were exposed to elevated levels of CO2 concentration and temperature for a single growing season. On the other hand, in a controlled field experiment, twelve genotypes (six female and six male) of P. tremula were grown for four consecutive growing seasons. For the first three growing seasons, the plants were grown under elevated temperature and UV radiation, and in the fourth growing season they were grown in ambient conditions. The Bud removal was performed in the third growing season and the responses in the plants due to the removal of buds were measured at the end of the fourth growing season. The specific research questions in different sub-studies (papers I–III) were as follows:

1) How does elevated CO2 concentration and/or temperature affect the growth traits and phenolic concentration in female and male P. tremula seedlings during the first growing season in a greenhouse experiment (I)?

2) How does the development phase affect the phenolic defense and growth of female and male P. tremula seedlings under elevated temperature and/or UVB radiation over three growing seasons in a controlled experimental field (II)?

3) How does the removal of lateral buds affect the growth and phenolic concentration in the leaves of female and male P. tremula seedlings grown under elevated temperature and/or UVB radiation in a controlled experimental field (III)?

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2 Materials and methods

2.1 Plant material

Plantlets of the same clonal material of P. tremula were used in all the studies (I–III). They were micropropagated from the axillary buds of 30–40-year-old mother trees that originated from different locations in eastern and southern Finland (Randriamanana et al. 2015). The plantlets were conventionally regenerated in glass jars, then acclimated in plastic trays and raised in greenhouses (Joensuu, Eastern Finland) until transfer to the experimental sites.

2.2 Outline of the experiments

The experiments were carried out in greenhouses (I) and a controlled experimental field (II, III). A summary of the experiments is presented in Table 1.

Table 1. Summary of the experiments included in the thesis

Article I II III

Experiment

type Greenhouse Field

No. of

genotypes

Eight (four females and

four males) 12 (six females and six males) Treatments Elevated temperature Elevated temperature

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Elevated CO2

concentration

Elevated UVB radiation

Lateral bud removal

Duration First growing season (10 weeks)

Growing seasons 1−3 (three years)

Growing seasons 3−4 (two years)

Measured parameters

Height Height Height

Basal diameter Basal diameter Basal diameter

Above-ground biomass

Above-ground biomass Stem bark phenolics Stem phenolics Leaf phenolics

2.2.1 Greenhouse experiment (I)

The study was conducted at the Mekrijärvi Research Station (62°47′N, 30°58′E, 145 m above sea level [asl], University of Eastern Finland) from 20 May to 5 August 2015. There were 16 greenhouse rooms which were randomly and evenly distributed to four different treatments including control (C), elevated CO2 concentration (CO2), elevated temperature (T), and CO2+T (Figure 1a). In each greenhouse room, one individual of each genotype (total of eight – four females and four males) were randomly placed. By employing a modulated system, the elevated temperature was set to 2 °C above the ambient level. Temperature sensors (PT1000, accuracy

± 0.3%, Czech Republic) were used to monitor the temperature within the measurement range of -40 to +60 °C. On the other hand, ambient and elevated CO2 concentrations were set at 400 and 720 ppm, respectively. In all the greenhouse rooms, an ambient level of UVB radiation was maintained by means of UV lamps (1.2 m long, UVB-313, Q-panel Co, Cleveland, OH), and the relative humidity was maintained at 60% (TRH-302A, accuracy ± 3%;

Nokeval Ltd, Finland).

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Figure 1. a) Photo of the greenhouses at the Mekrijärvi Research Station in 2015; b) photos of the P. tremula seedlings grown under different treatments in the greenhouses

2.2.2 Field experiment (II, III)

The experimental field was located at the botanical garden of Joensuu, eastern Finland (62°35′N, 29°46′E) (Figure 2). The plants were exposed to six different treatments including control (C), elevated ultraviolet-A (UVA) radiation (UVA), elevated UVB radiation (UVB), elevated temperature (T), UVA+T, and UVB+T, with six replicates of each treatment (total of 36 plots).

The UVA treatment was included in the experiment to serve as an additional

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control for UVB, because UV lamps also emit a small amount of UVA radiation. In each plot, six female and six male genotypes with five individuals of each genotype (total of 60 plantlets) were planted randomly in five rows on 11 June 2012. The elevated temperature and elevated UVB radiation were set to 2 °C and 30% above the ambient level, respectively, through a modulated system. The elevated temperature and UV radiation treatments were applied during the first three growing seasons (2012–2014).

During the fourth growing season (2015), the plants were left to grow under ambient climatic conditions.

For bud removal treatment, one individual of each genotype in each plot was randomly selected. Buds were removed during summer and autumn of 2014. On 2 July, 12 axillary buds were removed from four lateral branches (three from each) next to the leading shoot. Three more buds were removed from the same individuals again on 20 October. The number of buds removed was estimated to be ~5% of the total axillary buds.

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Figure 2. Photos of the experimental field during the summer time in (a) 2014 (photograph by Riitta Julkunen-Tiitto) and (b) 2015

2.3 Measurements and sampling

Height and basal diameter of all the plants were measured at about two- week intervals in the greenhouse (I) and three-week intervals in the field (II, III) experiments. The height was measured from the root collar to the tip of the longest shoot while the diameter was measured 1 cm above the root collar. All the plants were harvested at the end of greenhouse (4–5 August 2015) and field (17–18 August 2015) experiments. The plants were dried, and then leaf and stem biomass were weighted separately.

For the analysis of phenolics, a 10-cm-long fresh stem section from the part where the first mature leaves appeared (current season growth) (I, II) and the youngest and completely expanded leaves (III) were collected at the end of the growing seasons. The stem samples were cut into two longitudinal halves for proper drying. The samples were collected in small paper bags containing drying media (about 2 g of silica gel). They were dry-

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air dried at 22 °C (10% relative humidity) and kept in the freezer (-20 °C) until extractions.

2.4 Growth rate calculation

In the field experiment, the duration of the growth and treatment periods varied. Therefore, a similar growth period (in terms of the start date and overall length) from each year was selected to calculate the growth rates to compare growth increments between three growing seasons (II). The height and diameter of the plants measured on 13 June and 24 July in 2012, 10 June and 23 July in 2013, and 17 June and 29 July in 2014 were used to calculate the growth rates. The calculation was done by dividing the increment between the selected measurement dates of each year by the number of intervening days (2012: 41 days; 2013: 43 days; 2014: 42 days).

2.5 Phenolic analyses

Low-molecular-weight phenolics were extracted from stem (I, II) and leaf (III) samples by using cold methanol according to Nybakken et al. (2012). The individual compounds in the extracts were separated by high-performance liquid chromatography (HPLC). Most of the compounds were identified by using a mass spectrometer, quadrupole time-of-flight liquid chromatograph (QTOF LC/MS, 6540 series, Agilent, USA). The compounds that could not be confirmed by Q-TOF MS were identified separately based on their retention times. The available commercial standards were used to quantify the concentration of the compounds. The concentrations of condensed tannins were quantified by performing an acid-butanol assay (Hagerman 2011). The concentration of methanol-soluble condensed tannins was determined from dried sample extracts redissolved in methanol-water; the concentration of methanol-insoluble condensed tannins was determined

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35 from the dried sample residues of methanol extractions. Purified tannin extracts from P. tremula stems were used as a reference (Randriamanana et al. 2014).

2.6 Data analyses

The effects of fixed factors on growth and phenolic concentration were assessed by using a linear mixed effects model as described in the articles (I–III). All the statistical analyses were performed by using SPSS (version 21.0;

IBM Corp., Armonk, NY, USA) (I) or R (version 3.3.2; R Core Team 2016) (II, III).

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3 Results and discussion

3.1 Growth performance of P. tremula

3.1.1 Growth performance of seedlings under (simulated) climate change

In this thesis, I studied the height, diameter, and biomass growth in response to elevated CO2 concentration and temperature in P. tremula for a single growing season in greenhouses (I), and the variation in height and diameter growth rates in response to elevated temperature and UVB radiation during a three-year growth period in a field experiment (II). In the greenhouse experiment, the height growth of P. tremula decreased significantly under elevated levels of CO2 concentration (I). Although numerous studies have reported increased growth in plants exposed to elevated CO2 concentrations (McDonald et al. 1999; Lavola et al. 2013), some studies have shown a decrease or no response (Coley et al. 2002; Yazaki et al. 2004). Here, the CO2- fertilized P. tremula seedlings had a shorter apical portion of the main stem and more branches compared with the plants grown in the ambient CO2

condition, and this weak apical dominance is likely because of the effects of the higher CO2 concentration on hormonal production or transportation in the shoot apex (Conroy et al. 1990). On the other hand, elevated CO2

concentration did not affect diameter and biomass growth; however, diameter growth was considerably higher in the CO2-fertilised plants grown under elevated temperature (I). Elevated temperature might have increased the light-saturated rate of CO2 uptake in the plants, and this might have resulted in greater diameter growth under the combined treatments of elevated CO2 concentration and temperature (Farrar and Williams 1991;

Morison and Lawlor 1999; Dieleman et al. 2012).

Elevated temperature stimulated the height, diameter, leaf, and stem biomass of P. tremula seedlings when compared with the control plants (I).

These results are consistent with earlier studies. Many previous studies

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conducted in greenhouses and open fields for a single growing season have reported that elevated temperature increased growth in broad-leaf tree species in the northern hemisphere (Veteli et al. 2002; Huttunen et al. 2007;

Randriamanana et al. 2015), since tree growth is mainly limited by the below- optimal temperature in this region (Way and Oren 2010; Stinziano and Way 2014; Kellomäki 2017). In the field experiment, elevated temperature increased the height and diameter growth rates of P. tremula during the three-year growth period, however the stimulating effect of elevated temperature on the diameter growth rate became weaker as the plants aged (II). This result is in accordance with Nybakken et al. (2012), who also revealed a decrease of proportional increment in growth in response to elevated temperature in the second growing season when compared to the first one in S. myrsinifolia. The P. tremula seedlings might have acclimated in this study to the higher temperatures over the duration of the experiment, as long-term exposure of plants to a warmer growth environment commonly leads to thermal acclimation, which results in a decline in the proportional increment in carbon gain through photosynthesis in plants over time (Way and Yamori 2014; Way et al. 2015; Smith et al. 2016).

In response to elevated UVB radiation, the height and diameter growth rates of P. tremula did not vary over the entire three-year growing period or in any specific year (II). Although some experimental studies have revealed a cumulative effect of elevated UVB radiation on different growth traits in perennial plants (Sullivan et al. 1994; Tegelberg et al. 2001), there are also studies consistent with the findings of this thesis. For example, after seven years of exposure, no deleterious effect of elevated UVB radiation was detected on growth performance including dry weight, leaf thickness, and leaf area in the slow-growing S. polaris (Wahlenb.) plants (Rozema et al.

2006). In line with other contemporary studies (Sullivan 2005; Ballaré et al.

2011), it can be suggested that elevated UVB radiation may have a weak or no effect on growth increment in P. tremula young plants or seedlings and saplings in the case of ecologically relevant UVB radiation.

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39 3.1.2 Effect of bud removal on growth performance under elevated

temperature and UV radiation

The growth responses to the lateral bud removal of P. tremula seedlings grown under elevated temperature and UV radiation (III) were studied by removing artificially a small number of lateral buds from seedlings. This stimulated the height growth (except in temperature-treated male individuals), leaf, stem, and total aboveground biomass in P. tremula saplings when compared to the intact plants in all climatic treatments (III). Moreover, diameter growth also tended to be greater under all climatic treatments in bud-removed plants compared with intact individuals (III). Stowe et al. (2000) and Jacquet et al. (2013) suggested that damaged plants usually respond by overcompensation in growth after a low to moderate level of tissue loss. In this study, the removal of ~5% of the lateral buds was interestingly sufficient to alter the growth in P. tremula saplings. Overall, the removal of lateral buds increases the source-to-sink ratio by reducing the non-photosynthetic organs and increases the carbon supply to the remaining sinks (Honkanen et al. 1994; Ozaki et al. 2004). Therefore, in the bud-removed individuals, an increase in the source-to-sink ratio might have facilitated higher assimilation and induced greater growth.

There was no interaction between bud removal and UVB treatment on growth increment, however the magnitude of the increment in the leaf and total aboveground biomass due to bud removal was significantly higher in the temperature-treated plants (III). Moreover, after bud removal, UVA+T- treated plants had significantly greater stem biomass due to the additive effects of bud removal and elevated temperature (III). Due to bud removal, the plants might have improved their physiological competency and might have a greater ability to acquire resources under elevated temperature. It was found in the previous studies that tissue-damaged plants under warming conditions had higher concentrations of nutrients, greater water- use efficiency, and an increased photosynthetic rate (Huttunen et al. 2007;

Lemoine et al. 2013). Therefore, based on those and our findings, we may assume that if plants are subjected to low-to-moderate levels of tissue damage as a consequence of increased levels of insects and other

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herbivores due to climate warming, this may result in overcompensation in plant growth (Lehtilä 2000; Stowe et al. 2000).

3.1.3 Sex-dependent variation in growth performance

The sex-dependent variation in growth traits in P. tremula was studied both in greenhouse (I) and field (II, III) conditions. In the greenhouse experiment, there was a sex-dependent variation in males and females of P. tremula, since male individuals had significantly greater height growth than their female counterparts (I). In addition, leaf, stem, and total aboveground biomass tended to be higher in males when compared with females (I).

These results agree with the earlier studies that reported the differences between females and males in growth traits in dioecious tree species in greenhouse conditions (Nybakken and Julkunen-Tiitto 2013;

Randriamanana et al. 2014). In the field experiment, however, both female and male individuals of P. tremula maintained a similar level of growth (II, III).

Some earlier studies also detected the absence or only a few sex-specific differences in growth traits of S. myrsinifolia and P. tremuloides grown in field conditions (Nybakken et al. 2012; Nissinen et al. 2018; Cope et al. 2019).

Nissinen et al. (2018) suggested that plants grown in field conditions often face different stresses – for example, herbivory, plant diseases, competition by other plants, heat, heavy rain, and strong wind – all of which could play a part in maintaining a balance between males and females. However, in greenhouse experiments, plants are grown in a controlled growth environment. Therefore, the inherent secondary sexual dimorphism become very distinctive in such a controlled environment.

Under elevated temperature in the greenhouse experiment, height and diameter growth were considerably greater in females than in males (I). In the field experiment, on the other hand, height growth tended to be greater in males for temperature-treated and bud-removed individuals, but diameter growth was significantly higher in females (III). Moreover, leaf and stem biomass tended to be higher in temperature-treated and bud- removed females (III). These results indicate that under favorable conditions females also preferred to grow more, although plant ecological theory

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41 suggests that males are growth biased (Ågren et al. 1999; Obeso 2002).

These additive effects of elevated temperature and bud removal may also make dioecious females more susceptible to herbivores, although male- biased herbivory is frequently reported (Hjältén 1992; Ågren et al. 1999).

3.2 Phenolic concentration in stem bark, whole stems, and leaves of P. tremula

Among the identified low-molecular-weight phenolics in stem bark (I), stems (II), and leaves (III) of P. tremula, most of the individual compounds belonged to three different groups, namely salicylates, flavonoids, and phenolic acids.

Salicylates were the most concentrated compounds in stem bark and stems (I, II), while flavonoids were the most concentrated compounds in leaves (III).

As a Salicaceae species, P. tremula usually contains higher concentrations of salicylates than other phenolic compounds during the juvenile growth period (Randriamanana et al. 2015; Cope et al. 2019). The plants were about four years old at leaf sampling, which may explain their greater concentration of flavonoids than salicylates in leaves. It has been found that in Populus species, salicylates are gradually replaced by flavonoids and condensed tannins as plants age (Donaldson et al. 2006; Cole et al. 2016;

Cope et al. 2019).

3.2.1 Phenolic concentration under (simulated) climate change

In the greenhouse experiment, elevated CO2 concentration increased the concentrations of salicylates and phenolic acids in stem bark, and as a result the plants exposed to elevated CO2 concentration had a higher concentration of total low-molecular-weight phenolics compared with plants grown in ambient conditions (I). Many earlier studies with deciduous tree species have also reported that elevated CO2 concentration increased the synthesis of secondary metabolites, although there are reports that an elevated CO2 concentration decreased secondary metabolites or had no

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effect (Peltonen et al. 2005; Lindroth 2012; Lavola et al. 2013; Nissinen et al.

2016). Peñuelas and Estiarte (1998) suggested that elevated CO2

concentration increases the carbon-to-nitrogen ratio in plants, and consequently the utilisation of carbon for growth is diminished, therefore the surplus carbon is allocated to the synthesis of carbon-based secondary metabolites. In this study, the CO2-treated plants might have nitrogen deficiency and as a result the surplus carbon might have been allocated to synthesize carbon-based secondary metabolites.

On the other hand, elevated temperature decreased most of the individual compounds of salicylates, flavonoids, and phenolic acids, with a consequent decrease in the concentration of total low-molecular-weight phenolics in stem bark of P. tremula (I). In response to elevated temperature, the plants had greater growth. During a period of higher growth, L- phenylalanine, a precursor of proteins and phenolic compounds (Matsuki 1996), is apparently diverted away from phenolic synthesis, and thus downregulates synthesis of secondary metabolites relative to protein synthesis (Tuomi et al. 1991; McDonald et al. 1999; Fritz et al. 2006). In consistence with the greenhouse experiment, elevated temperature also decreased the concentration of total low-molecular-weight phenolics in P.

tremula stems in the field experiment, however it did not show the temporal variation during the three-year growth period, which contrasts with our expectation (II). Yet, the concentration of condensed tannins varied over time in response to elevated temperature, and as a result the concentration of condensed tannins increased in stems in the second growing season when compared to the first season (II). These results are in accordance with the growth differentiation balance hypothesis, which states that when growth increment in plants slows down, it makes the carbon substrates available for phenolic synthesis (Herms and Mattson 1992). The decline in proportional increment in the diameter growth rate in temperature-treated plants through time might have facilitated resources that could be allocated to condensed tannins.

There was no effect of elevated UVB radiation on salicylates, flavonoids, phenolic acids, and condensed tannins in stems of P. tremula in any

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43 particular year or over the entire three-year growth period. Although salicylates do not show very strong responses to elevated UVB radiation, flavonoids, such as quercetins and phenolic acids, are often reported to increase in plants exposed to elevated levels of UVB radiation (Julkunen- Tiitto et al. 2005; Nybakken et al. 2012; Nissinen et al. 2017). The induction of flavonoids and phenolic acids in plants in response to elevated UVB radiation, however, can differ depending on the plant part. Tissue thickness and anatomical surface features of different plant organs affect the attenuation of UVB radiation in plants (Day et al. 1992; Schreiner et al. 2009).

The relatively thick outer cell layer and exposed surface area of P. tremula stems may reduce the penetration of UVB radiation and thus minimize the synthesis of UVB-radiation-induced phenolics.

3.2.2 Effect of bud removal on phenolic concentration under elevated temperature and UV radiation

Alongside growth, I investigated the effect of mild (~5%) bud removal on phenolic concentration in the leaves of P. tremula which were grown under elevated temperature and UV radiation (III). The concentrations of salicylates and phenolic acids did not change due to the removal of buds as both intact and bud-remove individuals had a similar level of concentration in the leaves (III). However, bud-removed individuals had greater concentrations of flavonoids and condensed tannins as compared to the intact plants.

Although salicylates are very abundant in Populus species, they are highly genetically determined and are less responsive to environmental stresses such as lack of resources and tissue damage than other phenolic compounds (Roth et al. 1998; Hemming et al. 1999; Osier et al. 2000). Roth et al. (1998) found that salicylates are minimally affected, although levels of condensed tannins showed strong responses due to tissue damage in P.

tremuloides and Acer saccharum (Marshall.), even when the damage was severe.

In this study, greater accumulation of flavonoids and condensed tannins in leaves due to bud removal was not expected, because bud-removed individuals also had greater biomass growth. More growth in plants should

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cost them via a reduction in the synthesis of defensive chemical constituents (Bryant et al. 1983; Herms and Mattson 1992). Removal of buds might have activated induced defense in the plants, and as a result the concentration of flavonoids and condensed tannins increased to mitigate future damage. It has been suggested that induced defense is activated in plants when tissue is damaged or removed or in response to abiotic stresses (Kessler and Baldwin 2002; Young et al. 2010; Jiang et al. 2018). Although induced defense incurs costs, it may be relatively cheap because response to damage enhances a plant’s competitiveness, and induced secondary chemical constituents are only synthesized when needed (Miranda et al. 2007; Young et al. 2010; Hood and Sala 2015). Moreover, higher concentrations of flavonoids and condensed tannins in association with higher biomass growth in bud-removed individuals can be partly a result of the fact that P.

tremula is an inherently fast-growing species. Earlier studies found that fast- growing trees are generally better capable of compensating for the tissue damage than slow-growing ones owing to their greater soil nutrient acquisition capability, which increases their utilization of carbon for growth (Lavigne et al. 2001; Hikosaka et al. 2005; Endara and Coley 2011; O’Reilly- Wapstra et al. 2014). Accordingly, these bud-removed plants, under favorable conditions, might have prioritized both growth and defense instead of allocating resources to only one of these processes.

3.2.3 Sex-dependent variation in phenolic concentration

There were sex-dependent phenolic concentration variations in P. tremula stem bark in the greenhouse experiment (I), and in stems (II) and leaves (III) in the field experiment. In the greenhouse experiment, female individuals contained a greater concentration of phenolic acids, and the concentration of total low-molecular-weight phenolics also tended to be greater in females as compared to the males in the stem bark (I). Moreover, female individuals had higher concentrations of salicylates and total low-molecular-weight phenolics in stems in the field experiment (II). These results are expected because females are suggested to be inherently more defensive (Ågren et al. 1999; Cornelissen and Stiling 2005), and many empirical studies have

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45 reported greater concentrations of defensive chemical constituents in female individuals of Populus and other tree species (Jing and Coley 1990;

Nybakken and Julkunen-Tiitto 2013; Ruuhola et al. 2018).

In leaves, though the concentration of phenolic acids was higher in females, the concentrations of salicylates and condensed tannins were greater in males in both bud-removed and intact individuals (III). The higher concentration of salicylates in male individuals was due to the variation in clones over sex, as one of the male clones had an exceptionally higher concentration of salicylates in its leaves. However, male individuals containing higher concentrations of condensed tannins was unexpected because both female and male P. tremula had a similar level of growth.

Although an earlier study reported that male Spondias purpurea (L.) had greater concentrations of gallotannins, ellagitannins, and hydrolysable tannins in leaves compared with female individuals, but that was in cost of reduced growth (Maldonado-Lopez et al. 2014). Our results indicate that the high variation in defense between sexes occurs regardless of growth variation, although the trade-off between growth and defense in females and males of dioecious plants has often been reported (Cornelissen and Stiling 2005; Randriamanana et al. 2014; Cole et al. 2016). Moreover, higher concentrations of condensed tannins in males can be considered a fitness advantage because these compounds are one of the principal defensive compounds in Populus which are active against herbivores and pathogens (Erwin et al. 2001; Cole et al. 2016; Cope et al. 2019).

In the greenhouse experiment, both female and male P. tremula responded to elevated temperature almost equivalently in phenolic accumulation in stem bark (I). By contrast, the concentration of flavonoids increased in males compared with their female counterparts exposed to elevated CO2 concentration (I). Male individuals also accumulated greater biomass compared with females under elevated CO2 concentration. This indicates that when grown under elevated CO2 concentration, male individuals had surplus carbon after meeting the growth demand and that this surplus was then allocated for the synthesis of carbon-based phenolics, flavonoids. On the other hand, in the field experiment, sex-dependent

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variations in phenolic concentrations in stems and leaves were not influenced by the treatments, except for some individual compounds (II, III).

This discrepancy in individual compounds is expected because the plant responses to different individual compounds of phenolics vary due to various factors (Stamp 2004; Nybakken et al. 2012; Nissinen et al. 2017). The findings of this study provide further evidence on the fact that the sex- dependent variation in phenolic concentration depends on plant organ, clone, and the compound in question, and is less influenced by the treatments.

Responses of growth and defense in dioecious Populus tremula (L.) to simulated climate change and tissue damage

PANU PIIRAINEN

PHARMACOKINETICS AND EFFICACY OF EPIDURAL OXYCODONE

Dissertations in Health Sciences

Dissertations in Forestry and Natural Sciences

NORUL SOBUJ

Responses of growth and defense in dioecious Populus tremula (L.) to simulated climate change and

tissue damage

RESPONSES OF GROWTH AND DEFENSE IN DIOECIOUS Populus tremula (L.) TO SIMULATED

CLIMATE CHANGE AND TISSUE DAMAGE

RESPONSES OF GROWTH AND DEFENSE IN DIOECIOUS Populus tremula (L.) TO SIMULATED

CLIMATE CHANGE AND TISSUE DAMAGE

Acknowledgements

Table of contents

1 Introduction

2 Materials and methods

3 Results and discussion