Introduction - Responses of growth and defense in dioecious Populus tremula (L.) to simulated c

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

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

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

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).

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

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.

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

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

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

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.

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

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