Acclimation of plants to combinations of abiotic factors Connecting the lab to the field

Zsófia Réka Stangl

Umeå Plant Science Centre Department of Plant Physiology Umeå University Umeå, Sweden, 2017

© Zsófia Réka Stangl, 2017 Umeå Plant Science Centre Department of Plant Physiology Umeå University, 901 87 Umeå ISBN: 978-91-7601-700-5 Cover: “Inner Glow” by Jolene Mackie Electronic version available at http://umu.diva-portal.org Printed by: KBC Service Centre, Umeå University Umeå, Sweden, 2017

“One’s destination is never a place, but rather a new way of looking at things” /Henry Miller/

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Table of Contents Table of Contents iAbstract iiiSammanfattning ivList of papers vAcknowledgements viiIntroduction 1

Stress perception and signalling 2Thermal stress 8Carbon dioxide 14Nitrogen 17Significance of the ectomycorrhizal symbiosis 18Stress physiology of plants under a combination of stressful environmental conditions 19

Aim and significance of the presented work 21Discussion 22

Carbon release under warming conditions: Modelling the thermal response and acclimation of respiration 22Modelling the thermal acclimation of respiration 24Shifts in the environmental carbon-nitrogen balance drives the dynamics of tree-mycorrhiza interaction 28The effect of nitrogen limitation on thermal acclimation 31The role of the alternative respiratory pathway under thermal stress and nitrogen limitation 35Tree carbon balance under warming growth conditions 35The transcriptional underpinning of thermal sensitivity in Norway spruce 40The significance of the C-N balance under constrains of growth temperature 45

Conclusions and future outlook 48References 50

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Abstract Increasing atmospheric CO2 and other greenhouse gasses coupled to the accelerated rate of global warming puts plants and ecosystems under the strain of a rapidly changing abiotic environment. Understanding the impacts of changing global climate is a strong focus of plant science and the establishment of more resilient crop variants is an important goal for breeding programs. Our understanding of plant responses and acclimation to abiotic conditions has improved substantially over the last decades but the combination of a complex abiotic environment and high biological diversity, both on molecular as well as on species level, leaves us still with a lot of uncertainties. The aim of this doctoral thesis was to establish a link between plant thermal responses and the carbon-nitrogen balance of plants. The work in this thesis focused on ecologically significant species of the boreal region: Picea abies, Pinus sylvestris and Betula pendula; and Betula utilis, which is one of the prominent tree species in the high altitudes of the Himalayas. The results presented demonstrate that sub-optimal temperatures combined with other abiotic factors can have additive effects that are not easily deducible from the effect of the two factors separately. Low nitrogen availability enhanced the negative effect of low temperature, while elevated CO2 enhanced plant growth under moderate increases in temperatures but under a more extreme temperature increase it exacerbated the negative effect of heat. I also show evidence that species, despite being grouped into the same functional group or inhabiting the same biome can have different thresholds to temperature and to shifts in the C/N balance of their environment and that these differences can, to some extent, be explained by their differential growth strategies. Furthermore, I demonstrate results supporting the hypothesis that the C-N fluxes between mycorrhizal fungi and tree are strongly dependent on the C and N in the environment, highlighting the significance of the tree-mycorrhiza associations in the C sequestration capacity of the boreal region. In this thesis I also present a generalised empirically based mathematical model that can describe the respiration-temperature response of plant functional types or biomes with high precision, giving a more accurate estimate of NPP when implemented in global climate models, and has the potential to incorporate the thermal acclimation of respiration, further increasing the precision of estimating carbon fluxes under future warming temperatures. My results provide novel insights into the interactive temperature-carbon-nitrogen responses of plants, taking a step towards better understanding the response of plants and forests to future climates.

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Sammanfattning Acklimatisering av växter till en kombination av abiotiska faktorer: Ett steg mot att länka laboratoriet till utemiljön Ökande atmosfäriskt CO2 och andra växthusgaser kopplade till den accelererande globala uppvärmningen utsätter växter och ekosystem för stressen av en snabbt förändrande abiotisk miljö. Att förstå påverkan av ett globalt klimat i förändring står i fokus inom växtforskning och utvecklandet av mer motståndskraftiga grödor är ett viktigt mål inom programmen för växtförädling. Vår förståelse av växters responser och acklimatisering till abiotiska förhållanden har förbättrats avsevärt under de senaste decennierna, men på grund av kombinationen av en komplex abiotisk miljö och stor biologisk mångfald, både på molekylär nivå såväl som på art-nivå, kvarstår en del frågetecken. Syftet med denna avhandling var att upprätta ett samband mellan växters responser på temperaturförändringar och kol-kvävebalansen hos växter. Arbetet i denna avhandling inriktades på ekologiskt betydande arter i den boreala regionen, Picea abies, Pinus sylvestris and Betula pendula; samt Betula utilis som är en av de framträdande trädarterna på höga höjder i Himalaya. Resultaten som presenteras visar att suboptimala temperaturer i kombination med andra abiotiska faktorer kan ha additiva effekter som inte enkelt kan härledas från effekten av de två faktorerna var för sig. Låg kvävetillgänglighet ökade den negativa effekten av låg temperatur, medan förhöjd CO2-halt förbättrade planttillväxt under måttliga temperaturökningar, men under en mer extrem temperaturökning förvärrades dock den negativa effekten av värme. Jag framför även bevis på att arter, trots att de grupperas i samma funktionella grupp eller finns inom samma biom, kan ha olika tröskelvärden beträffande temperatur och förskjutningar i C/N-balansen i sin miljö och att dessa skillnader, i viss utsträckning, kan förklaras av deras olika tillväxtstrategier. Vidare visar jag resultat som stöder hypotesen att C-N - flöden mellan mykorrhiza och träd är starkt beroende av C och N i miljön. Detta belyser i sin tur betydelsen av samarbetet mellan träd och mykorrhiza gällande kolbindningskapaciteten i den boreala regionen. I denna avhandling presenterar jag även en generaliserad empiriskt baserad matematisk modell som med hög precision kan beskriva respiration-temperatur svar av växtfunktionella typer eller biom, vilken ger en mer exakt uppskattning av NPP i globala klimatmodeller. Mina resultat åstadkommer nya insikter i de interaktiva temperatur-kol-kväve-responserna hos växter, och tar ett steg mot bättre förståelse för växters och skogars reaktion på framtida klimat.

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List of papers

I. Mary A. Heskel, Odhran S. O’Sullivan, Peter B. Reich, Mark G. Tjoelker, Lasantha K. Weerasinghea, Aurore Penillard, John J. G. Egerton, Danielle Creek, Keith J. Bloomfield, Jen Xiang, Felipe Sinca, Zsofia R. Stangl, Alberto Martinez-de la Torre, Kevin L. Griffin, Chris Huntingford, Vaughan Hurry, Patrick Meir, Matthew H. Turnbull, and Owen K. Atkin. Convergence in the temperature response of leaf respiration across biomes and plant functional types. Proceedings of the National Academy of Sciences of the United States of America. 113, 14 (2016), 3832–7.

II. Niles Hasselquist, Dan Metcalfe, Erich Inselsbacher, Zsofia R.

Stangl, Ram Oren, Torgny Näsholm and Peter Högberg. Greater carbon allocation to mycorrhizal fungi reduces tree nitrogen uptake in a boreal forest. Ecology. 97, 4 (2016), 1012–1022.

III. Leonid V. Kurepin*, Zsofia R. Stangl*, Alexander G. Ivanov, Vi

Bui, Marin Mema, Norman P.A. Hüner, Gunnar Öquist, Danielle Way, Vaughan Hurry. Contrasting acclimation abilities of two dominant northern conifers to elevated CO2 and temperature (Plant Cell & Environment, submitted April 6th)

IV. Kathryn M. Robinson*, Zsofia R. Stangl*, Alexander Vergara,

Leonid V. Kurepin, Alexander G. Ivanov, Danielle Way, Norman P.A. Hüner and Vaughan Hurry. Elevated CO2 does not mitigate the effect of increased temperature at the whole plant or transcriptome scale (manuscript)

V. Zsofia R. Stangl, Catherine D. Campbell, Torgny Näsholm and

Vaughan Hurry. Nitrogen limitation differentially affects acclimation by two contrasting Betula species to low and high growth temperatures and decreases the advantage of fast growth strategy under warming climates (manuscript)

* joint first authors Paper I and II are reprinted with kind permission of the publishers. In the following, the papers are referred to by their roman numbers.

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The contribution of Zsofia R. Stangl to the papers included in this thesis was as follows:

I. Planned and performed the sampling and gas-exchange measurements in Umeå, together with M. A. H., and contributed to writing and revising the manuscript.

II. Performed the photosynthetic gas-exchange measurements and the subsequent data analysis, and contributed to writing and revising the manuscript.

III. Contributed to the anatomical analysis of the wood, to the

growth measurements and to the measurements of needle traits, such as SLA and C/N content. Performed the gas-exchange measurements and the statistical analysis of all the data in the manuscript. Contributed with figures, tables and text to the submitted manuscript.

IV. Planning of the transcriptomic analysis of the plants in the

experiment for Paper III. Performed the sampling of the needle and wood tissues and the RNA extraction. Performed the statistical analysis of the plant physiology related data and contributed to the planning and discussion of the transcriptomic data analysis. Contributed with tables and text to the manuscript.

V. Planned and executed the experiment, performed all

measurements, sampling and biochemical analysis. Performed the statistical analysis of all data and prepared all text and figures in the manuscript. All this, with the support of V. H. and C. D. C.

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Acknowledgements First and foremost I would like to thank my supervisor, Prof. Vaughan Hurry for giving me the opportunity to work in his group in the last five years. I highly appreciate all the support and encouragement I got from you over these years and I would like to thank you for making it possible for me to participate in diverse experiments and work with many exceptional researchers. Special thanks to Catherine Campbell, my assistant supervisor, for all her help especially in the beginning of my PhD studies, and to Prof. Torgny Näsholm and Prof. Per Gardeström for their invaluable insight and contribution to my work and progress as members of my reference group. I would like to thank Prof. Norman Hüner for welcoming me in his lab at the University of Western Ontario and for his support during my stay. I would like to thank members of the Hüner lab, especially Marianna Krol and Alexander Ivanov for sharing their knowledge and experience with me in regards to various lab techniques and measurement techniques, and Leonid Kurepin for his help with technical and logistical arrangements during my stay at UWO. Alex, I especially thank you for welcoming me in your home during the months of my stay in Canada, and for your great hospitality. I would like to thank Danielle Way at UWO for her support and hospitality and for all the insightful discussions regarding our experiment. I would like to thank Kathryn Robinson and Alexander Vergara for their invaluable contribution to Paper IV. Especially, Kathryn I would like to thank you for all the discussions that made working on this manuscript really fun. I would like to thank Mary Heskel for introducing me to a new measurement technique and Niles Hasselquist for inviting me to participate in his experiment. Special thanks to the following people for their technical and practical help with various aspects of my work during these five years: Gunilla Malmberg, Margareta Zetherström, Totte Niittylä, Junko Takahashi Schmidt, Mathieu Castelain, Åsa Boily, Jonas Lundholm, Jan Karlsson, Olivier Keech, Nils Henriksson, Nicolas Delhomme, Nathaniel Street and Ioana Gaboreanu. Finally I would like to express my appreciation to all my colleagues at Umeå Plant Science Centre, researchers and fellow PhD students as well as technical staff, for creating such a friendly and supportive work environment.

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Introduction The Industrial Revolution has enabled humanity access and control over diverse forms of abundant energy and raw materials and marked the beginning of the most productive period of human history. These innovations had many positive effects on our ways of life and largely disconnected us from the constraints and challenges of our environment. This intensive human activity together with the unprecedented increase in population has changed our global environment enormously. Anthropogenic emissions have caused the CO2 concentration of the air to increase by approximately 40%, and increased the concentrations of other potent greenhouse gases such as methane (CH4) and nitrous oxide (N2O) (Ciais et al. 2013). As a consequence, the earth mean surface temperature has increased by close to 1°C compared to the pre-industrial level (Hartmann et al. 2013), with 2016 being the warmest year on record. This rapid change in the global temperature has caused imbalances in the global climate that on a regional scale is responsible for more frequent extreme weather events, prolonged drought periods, changes in precipitation patterns and patterns of seasonal temperatures, heat waves and unusual cold spells (Seneviratne et al. 2012). Although a large proportion of the over 7 billion humans is not dependent on their local environment for their food supply and the supply of raw material, on a global scale we cannot disconnect ourselves from global climatic conditions and the global ecosystem. Non-optimal weather conditions can negatively impact crop yields and potentially jeopardise secure food and raw material supply. Understanding the impacts of extreme weather events and changing global climate has therefore long been the focus of plant science and the establishment of more resilient crop variants has been an important goal for breeding programs. There is now a scientific consensus that the current rate of emissions and the course of rising global temperature is not sustainable and will have dangerous consequences in the long term. Scientific focus to improve predictions of climate change and its impacts, political efforts such as the Paris agreement (http://unfccc.int/paris_agreement) and technological innovation towards utilising renewable energy sources are all efforts towards the goal of stopping the increase of the CO2 concentration in the atmosphere. In this context, the CO2 sequestration capacity of forests is of utmost importance and has gained substantial focus. Tropical forests account for over 40% of annual carbon sequestration and boreal forests account for approximately 25% (Bonan 2008), while accounts for the temperate regions are less accurate. In addition to carbon sequestration, forests have an important role in sustaining the global hydrological cycle (Bonan 2008). It is

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not that straight forward to predict how vulnerable forest ecosystems are to long-term prolonged changes in regional climatic conditions. While tropical forests have a high albedo and strong cooling effect on their environment through evapotranspiration, boreal forests have comparably low albedo and weak evapotranspiratory capacity (Bonan 2008). This might cause these forests to be vulnerable to warming and decrease their carbon sequestration capacity under a warming climate, especially considering that the highest temperature increase of about 4°C in the next 50 years has been predicted to occur at these higher latitudes (Hartmann et al. 2013). Our understanding of plants response and acclimation to abiotic conditions has improved substantially over the last decades but the combination of a complex abiotic environment and high biological diversity, both on molecular as well as on species level, leaves us still with a lot of uncertainties. It will be very important to decipher these uncertainties if we are to improve our understanding of, and prediction capacity for, the impact of climate change on both crops and forest species. This understanding and these predictions will motivate political decisions and social efforts and form the basis for technological solutions. Stress perception and signalling Heat, cold, drought and high salinity have been identified as major abiotic factors that all have negative effects on survival and biomass production of plants. Success of efforts to breed for tolerance with traditional plant breeding techniques or genetic approaches have been limited (Limin and Fowler 1991, Cushman and Bohnert 2000, Mittler 2002), because stress tolerance is encoded in complex sets of genes; from perception to transcriptional activation and response to adaptation. Some of these pathways are shared between stresses; others are specific to one or the other environmental condition. Stressful conditions induce a complicated signalling cascade of stress signal perception, transmission and triggering stress response. Environmental cues are perceived by receptors that trigger the generation of signalling molecules, which translate the external signal into intracellular signals by inducing phosphoprotein cascades that activate various transcription factors to induce the expression of stress responsive genes. Abiotic stress signals are complex, comprising of a combination of physical and chemical signals. For example drought stress might be perceived as osmotic stress, ionic stress or mechanistic stress. Similarly, cold stress can induce osmotic stress, oxidative stress, as well as mechanistic stress. Due to the complexity of the

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environmental signals, plants have multiple cellular sensors that respond to the various signals or some aspects of it. The redundancy of signal perception makes it difficult to identify each sensor related to each specific stress factor because knocking out one receptor might not affect substantially the stress signalling as a whole. A few putative sensors for perceiving stress signals have been identified. For example the plasma membrane is widely accepted to have a key role in perceiving and transmitting external stress signals. The physical properties of the plasma membrane change under various stresses and this can trigger primary responses within the cells. For example the permeability of the plasma membrane changes under salinity stress, or the fluidity of the plasma membrane and membranes of cell organelles change under thermal stress (Murata and Los 1997, Sangwan et al. 2002, Falcone and Ogas 2004). Proteins embedded in the membranes can also function as sensors. For example two-component systems consisting of a histidine protein kinase, which senses the signal input and a response regulator that mediates the response output (Urao et al. 2000, Hwang et al. 2002). The signalling is initiated through the autophosphorylation of the histidine protein kinase in response to the environmental stimulus, and the phosphoryl group transferred to the response regulator, will alter its activity and induce the signalling cascade (Hwang et al. 2002). In Arabidopsis, three groups of histidine protein kinase homologs have been identified: ethylene receptors, phytochrome photoreceptors and the AHK family including an osmosensing receptor and a cytiokinin receptor (Hwang et al. 2002). For instance AHK1 was described to be a positive regulator of the ABA-dependent and ABA-independent signalling pathways in drought and slat stress, while AHK2 and AHK3 were found to be negative regulators of the ABA-dependent signalling pathway (Tran et al. 2007). Elements of the two-component system of histidine protein kinases and response regulators have also been described in genetic analysis of other plant species, such as rice and soybean (Mochida et al. 2010). Another protein family, the lecithin receptor-like kinases (LecRLKs) were suggested to play a role in plant development as well as signal perception (Vaid et al. 2013). Co-expression analysis has connected this protein family to genes of the brassinosteroid mediated signalling pathways, and the MAP kinase signalling pathway (Vaid et al. 2012), as well as the ABA-dependent signalling pathway, with putative functions in the response to drought, salt-, osmotic- and thermal stress (He et al. 2004, Deng et al. 2009, Vaid et al. 2012, Sun et al. 2013). A group of small signalling molecules, such as reactive oxygen species (ROS), reactive nitrogen species (RNS), lipid phosphatases, ions, such as Ca2+, and some plant hormones respond to the primary stress perception and function in signal transduction. Various environmental stresses have been described

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to induce transient Ca2+ influx into the cell cytoplasm (Tuteja 2009), serving so as one of the primary stress signals. Ca2+ influx occurs through ion channels, and Ca2+ signal is maintained through the interplay between influx and efflux. Cytosolic Ca2+ concentration can be increased through influx from the apoplast or through release from intracellular stores, like the endoplasmic reticulum, vacuoles, mitochondria, chloroplast or the nucleus, and the magnitude and kinetics of the signal is dependent on the stress (Reddy 2001). Mechanical signals and ABA signals, like signals to the guard cells under drought stress, primarily mobilise Ca2+ from intracellular stores, whereas low temperature signals induce Ca2+ influx from the extracellular space (Knight 1999, Wood et al. 2000). The ion channels regulating Ca2+ influx during cold stress were found to be mechanosensitive, so that their function depends on membrane rigidity (Sangwan et al. 2002). Hyperpolarization-activated channels and cyclic ADP-ribose mediated Ca2+ flux were found to respond to the ABA signal, and facilitate Ca2+ influx from intracellular stores (Wu et al. 1997, Sangwan et al. 2002). Targets of Ca2+ signalling can be Ca2+-dependent protein kinases (CDPKs), which have been suggested to play a role in the activation of the heat-shock-activated MAP kinase (HAMK) and the cold induced stress-activated MAP kinase (SAMK)(Sangwan et al. 2002). Reactive oxygen species is a collective term for oxygen radicals, like superoxide (O2-) and hydroxyl (-OH), and non-radicals, such as hydrogen peroxide (H2O2), singlet oxygen and ozone (O3). ROS are produced under stressful conditions. H2O2 is produced as a by-product of the catalytic reaction of glycolate oxidase and acetyl-CoA oxidase in the photorespiratory pathway and the fatty acid ß-oxidation pathway, respectively, and by the cell wall bound peroxidases (del Río 2015). The plasma membrane bound NADPH oxidase has been identified as one of the major sources of superoxide (for a review see: del Rio, 2015). Other sources of H2O2 and superoxide are various oxidative and electron transport processes in the chloroplast, mitochondria and peroxisomes. For example in the chloroplasts, ROS are produced in PSI as well as PSII under stress conditions, when the absorbed light exceeds the capacity of electron utilisation in downstream processes (Pospíšil et al. 2004, Asada 2006). In particular hydroxyl radicals and singlet oxygen are powerful oxidants that can react with lipids, proteins and nucleic acids and so can cause substantial damage within the cells, termed oxidative stress (for a review see Mittler, 2002). Plants have a range of enzymatic and non-enzymatic antioxidants that can scavenge the excess ROS and protect cells from oxidative damage. A few examples are catalase, superoxide dismutase, NAPD-dehydrogenase, gluthatione and ascorbic acid (for a review see: del Rio, 2015). Since genetic evidence suggested that the extent of purely oxidative damage in plant cells could be more limited than

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previously thought, the significance of ROS has been re-evaluated and their role as signal transductors is more and more acknowledged (Foyer and Noctor 2005). It has been shown that plant cells can induce and amplify ROS production for the purpose of signalling and regulate their function through the equilibrium between their production and scavenging (Torres and Dangl 2005, Suzuki et al. 2011, Marino et al. 2012, Mittler 2017). Highly reactive oxygen radicals, and radicals with relatively short half-life, like for example hydroxyl radicals or singlet oxygen, can have localised functions, whereas H2O2 has the capacity to diffuse through membranes and travel long distances within the plant (for a review see: Sewelam et al. 2016). ROS can react with target proteins, and induce protein modification. This can lead to activation of plant hormones or other secondary signalling molecules like MAPKs (mitogen activated protein kinase) and activate various stress response cascades leading to activation of stress specific transcription factors (TFs) (Asai et al. 2000, Sewelam et al. 2013, Sewelam et al. 2014, Sewelam et al. 2016). Furthermore ROS can have a signalling function by altering the cell redox homeostasis in response to imbalances induced by environmental factors, which in turn can induce the response of different pathways to restore homeostasis. Antioxidants, such as ascorbate, gluthatione and carotenoids for instance, have been described to influence processes of cell division and senescence and so play a role in plant growth and development in response to stress (for a review see: Sewelam et al. 2016). ROS can also indirectly induce signalling pathways through other molecules that respond to the products of ROS-inflicted damage (Evans et al. 2005, Møller and Sweetlove 2010). Reactive nitrogen species (RNS) is a collective term for radicals, like nitric oxide (NO-) and nitric dioixide (NO2-), and non-radicals, like nitrous acid (HNO2) and dinitrogen tetraoxide (N2O4), that are produced in metabolism. NO- can be produced by enzymatic reactions, by nitrate reductase during N assimilation, or by peroxidise and a non-enzymatic source of nitric oxide is nitrite. Furthermore a putative nitric oxide synthetase activity has been suggested as possible source. Similar to ROS, RNS can have beneficial and harmful effects, so that they can function as signalling molecules in low concentrations. NO binds to proteins in a process called nitration or S-nitrosylation, which is the binding of an NO group to an SH group in a cysteine residue and plays an important role in the NO-mediated signalling (Stamler et al. 2001). NO release and NO synthase (NOS)-like activity has been associated with a range of abiotic stresses. For example NO has been suggested to play a role in the ABA-induced stomatal closure under drought stress (Garcia-Mata and Lamattina 2001). NO can interact with ROS under various stress conditions and serve as an antioxidant (Neill et al. 2003). Cold acclimation and increased freezing tolerance have been associated with

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proline accumulation in Arabidopsis, which has been suggested to be modulated by NO production (Zhao et al. 2009). Signalling lipids are rapidly formed in response to various stimuli through lipid kinases and the activation of phospholipases. They can activate enzymes by connecting to the lipid-binding domain or they can recruit proteins to membranes, leading to induction of signalling pathways. Phosphatidic acid (PA) has been identified, as one of the most significant lipid secondary messengers in plants (Testerink and Munnik 2011), while it is also an intermediate in lipid biosynthesis. PA can be directly formed by phospholipase D (PLD), or the interaction of phospholipase C (PLC) and diaglycerol kinase (DGK) (Munnik and Testerink 2009). Osmotic stress was found to trigger the transient formation of PA and the expression of PLD and PLC associated genes. PA was found to play a role in the induction of ABA-dependent response to osmotic stress and in turn, promote drought and salt tolerance (for a review see: Testernik and Munnik, 2011). Furthermore, cold and frost induced the formation of PA, which was suggested to play a role in cold acclimation and freezing tolerance (Li et al. 2004). PA has also been associated with influencing growth and root development, by regulating the phosphorylation of proteins of the PIN family, that are auxin efflux transporters (Friml et al. 2003). Furthermore, PA was also connected to sensing of nitrogen availability and balancing plant and root growth with the nutrient status (Hong et al. 2009). Phytohormones, in general, regulate plant growth and development and play a role in the regulation of nitrogen allocation, and therefore they are involved in abiotic stress signalling and regulation of stress response, by induction of gene expression associated with transcription factors (TFs) involved in stress response. For example pre-treatment with ethylene or salicylic acid (SA) has been found to increase thermotolerance (Larkindale and Knight 2002). Brassinosteroids can induce stress tolerance to different abiotic stresses by triggering H2O2 generation (Cui et al. 2011). ABA plays a key role in regulating water and osmotic balance, and is involved in ABA-dependent stress response pathways that can be induced parallel to ABA-independent pathways during stress (Tuteja 2007). ABA is synthesized from ß‑carotene via the oxidative cleavage of neoxanthin and conversion of xanthoxin to ABA via ABA‑aldehyde. The biosynthesis of ABA is suggested to be mediated by ROS and/or by calcium-dependent phosphorylation pathways (Xiong et al. 2002, Chinnusamy and Schumaker 2004). ABA plays a vital role in draught and salt stress response and has also been connected to acclimation to cold and heat (Larkindale and Knight 2002, Cutler et al. 2010).

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Protein kinases control the phosphorylation of other proteins, having a significant role in regulating and activating many signal transduction pathways. For example ABA-activated SNF1-kinases activate ion channels by phosphorylation and have so been connected with drought stress in several plant species (Shen et al. 2001, Kobayashi et al. 2005, Huai et al. 2008). Calcium-dependent protein kinases (CDPKs) are one of the largest protein kinase families. They perceive intracellular Ca2+ concentration signals under various stresses and translate them into specific phosphorylation events, playing a significant role in the rapid stress signalling responses (Schulz et al. 2013). The mitogen-activated protein kinase cascade (MAPK), consisting of three components a MAPK kinase kinase (MAPKKK), a MAPK kinase (MAPKK) and a MAPK connected to each other by the event of phosphorylation, is a signal transduction module involved in transducing extracellular signals to the nucleus for appropriate cellular adjustment (Sinha et al. 2011). They play a role in the “cross-talk” between various signalling pathways and are induced by a wide range of abiotic and biotic stimuli. MAPK cascades have recently been found to be activated in response to ROS (Jalmi and Sinha 2015). Abiotic stresses induce large groups of genes, and in addition to stress-specific gene responses there are some large transcription factor (TF) families, the members of which are induced generally in response to a wide range of stress conditions. Examples for such TF families are the WRKY, NAC, MYB or the DREB/CBF family. Some of these TF families, like DREB/CBF, NAC and WRKY, were found to be specific for the plant kingdom (Riechmann et al. 2000) and play key roles in plant acclimation processes. For example, the DREB/CBF family play a central role in the response to cold, heat and drought. While these cascades of transcriptional responses lead rapidly to acclimation, they also play a role in inducing epigenetic changes such as chromatin remodelling, histone modifications and miRNA-mediated stress memory, which have been suggested to play a significant role in the long-term adaptation to stressful conditions (Kim et al. 2015, Ohama et al. 2017). The response of plants to any stress factor on a phenotypic scale involves decreasing exposure and mitigating impact in order to facilitate recovery of impaired or damaged systems. Generic stress-induced morphogenic responses (SIMRs) have been observed in a range of species and in response to a range of mild but chronic stress factors (Potters et al. 2009). SIMRs are characterised by inhibition of cell elongation, induction of local cell division and alteration of cell differentiation. On a whole plant level, this results in slower apical growth and induction of lateral growth, both in below- and aboveground organs, which is often associated with an overall increase of

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mass to area ratio together with early induction of flowering (Potters et al. 2009). Furthermore, plant growth phenology can also be influenced by stress-induced changes in assimilate partitioning, as for example temperature has significant effect on transport processes (Taiz 2015). For example in wheat it was found that assimilate movement out of the leaves had an optimal thermal range, while assimilate movement in the stem was not affected by temperature in the range of 1-50°C, indicating that the differential effect of temperature on the source and sink transport activities influenced the assimilate allocation (Yang et al. 2002, Wahid et al. 2007). Thermal stress When we talk about plant physiology under thermal stress, we need to differentiate between acclimation to mild stress and acclimation strategies to severe thermal conditions. In terms of cold acclimation, we can discuss chilling temperatures, typically 0°C < 20°C, and freezing temperatures. When it comes to heat stress, we differentiate between mild heat conditions and adaptation to extreme hot environments. Furthermore we differentiate between short-term thermal responses and acclimation to long-term mild changes in growth temperatures. The scope of this thesis is to discuss acclimation and growth under prolonged, mild non-optimal thermal conditions and therefore we will not discuss freezing tolerance and adaptation to extremely high temperatures. Cold acclimation is the term summarising biochemical and physiological processes by which low temperature tolerance and freezing tolerance develops in plants through prolonged exposure to chilling temperatures. Cold-hardy temperate and high latitude biennial and woody species have high capacities to acclimate to cold and grow under low temperatures. Cold is perceived through increased membrane rigidity, cold-induced increase in the cytosolic Ca2+ concentration and secondary signals, such as ROS and ABA (Chinnusamy et al. 2007). The calcium signal activates protein kinases that activate ICE1 through sumoylation and phosphorylation. ICE1 induces the expression of CBFs/DREB1s (C-repeat binding factors, also called dehydration-responsive element-binding protein1s), which bind to the cis-element in the promoters of COR (cold-responsive) genes and induce their expression. Simultaneously ICE1 is a negative regulator of MYB15, which supresses the expression of CBF/DREB1 (for review see: Chinnusamy et al. 2007). The CBF regulatory pathway is the best described cold response pathway, although a microarray analysis has demonstrated that it only accounts for about 10-15% of cold responsive genes in Arabidposis (Guy et al. 2008), highlighting the fact that our knowledge of the regulatory network of cold acclimation is still limited. About 50% of the COR genes have been

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assigned known or putative functions, coding for cryoprotective, biosynthetic or regulatory proteins. Cryoprotective proteins are members of the LEA (late embryogenesis abundant) and dehydrin protein families, and are typically hydrophyllic (Fowler and Thomashow 2002, Maruyama et al. 2004). COR15 is one of the polypeptides, which function has been experimentally investigated. It was found that COR15 is targeted to the chloroplast, where it helps to stabilise the membrane against freezing damage and increases its freezing tolerance (Artus et al. 1996). Some COR proteins have been suggested to protect other proteins from cold-induced inactivation and renature unfolded proteins (Bravo et al. 2003, Sanchez-Ballesta et al. 2004, Wise and Tunnacliffe 2004). Other proteins encoded by COR genes are biosynthetic proteins, enzymes involved in cold acclimation related metabolic processes, such as proline metabolism. Proline is a compatible solute with cryoprotective properties that can protect proteins and membranes under cold stress and was found to increase freezing tolerance (Fowler and Thomashow 2002, Hayat et al. 2012). Another group of enzymes expressed in response to cold are involved in lipid biosynthesis (Maruyama et al. 2004). Changes in membrane fatty acid composition is one of the key factors of thermal acclimation, and increased levels of unsaturated fatty acids, which lead to increased membrane fluidity, are associated with cold acclimation and increased freezing tolerance (Somerville 1995, Falcone and Ogas 2004). The third most significant group of biosynthetic proteins induced by cold are enzymes related to the Calvin cycle and sugar metabolism (Strand et al. 1999, Gilmour et al. 2000, Fowler and Thomashow 2002). Sugars can act as compatible solutes in cold and under freeze-thaw cycles, protect proteins and membranes from cold damage and were suggested to regulate the volume of cells and cell organelles during expansion and dehydration (Stitt and Hurry 2002). Heat stress is defined as increase in temperature beyond the optimal threshold, which can differ largely between species (Wahid et al. 2007). The severity of heat stress depends on intensity, duration and the rate of temperature increase. Plants under prolonged mild heat stress can acclimate to the higher temperature and increase their heat tolerance. The Heat shock TF A1 (HsfA1) has been identified as one of the key regulators of the heat stress response (HSR) in plants (Richter et al. 2010, Ohama et al. 2017). It has been suggested that the constitutively expressed heat shock proteins HSP70 and HSP90 negatively regulate HsfA1, by binding to Hsf1 monomers. According to the chaperon titration model, upon heat stress the increasing number of unfolded proteins will bind to HSP70 and HSP90 and release the Hsf1 monomers, which after a series of posttranslational modification, such as phosphorylation and sumoylation, will form the final active TF complex (for a review see: Richter et al. 2010). On the other hand, other studies have

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highlighted the importance of Ca2+ and ROS signalling in HSR, suggesting that the chaperon titration model alone cannot explain the activation of HsfA1 and HsfA1-independent HSR (for a review see: Ohama et al. 2017). It is well established that membrane fluidity is directly connected to temperature and increased membrane fluidity in response to heat, induces membrane depolarisation through the influx of Ca2+ into the cytosol from the extracellular space, through the opening of ion channels (Sangwan et al. 2002). Furthermore, inositol-1,4,5-triphosphate (IP3) generated in response to heat is also believed to increase Ca2+ concentration in the cytosol, presumably from intracellular stores, like the endoplasmatic reticulum (Zheng et al. 2012). In response to Ca2+, Calmodulin 3 (CaM3), a calcium sensor (Zhang et al. 2009), interacts with the calmodulin binding protein kinase 3 (CBK3), which plays a role in the post translational activation of HsfA1 (Ohama et al. 2017). The signal transduction pathway connected to ROS is not well described, but studies have highlighted their role in the early stages of HSR (Volkov et al. 2006, Ohama et al. 2017). Downstream, HsfA1 regulates the expression of DREB2A, which directly targets heat inducible genes (Yoshida et al. 2011). DREB2A is also involved in the ABA-dependent response to draught, although the connection between DREAB2A and ABA in HSR is not clear (Ohama et al. 2017). HsfA1 also regulates the expression of other TFs, for example HsfA2, which has been associated with histone modification and heat stress memory (Lamke et al. 2016). Heat stress inducible genes encode for proteins with a large variety in function, for example components of the proteolytic system, clearing misfolded and aggregated proteins from the cells. Other classes of heat induced proteins are involved in RNA and DNA repair, such as the heat-induced methylation of ribosomal RNAs (Bügl et al. 2000), in sustaining cellular structures, or are involved in transport, detoxification and membrane modulation (Ohama et al. 2017). A large group of heat induced proteins are involved in metabolic processes. A screening study of the Arabidopsis heat shock metabolome revealed coordinated increase in levels of free amino acids, such as asparagine, leucine, theorine and alanine, amine containing metabolites, such as ß-alanine, a precursor of Co-A and fatty acid biosynthesis, GABA and putrescine, and carbohydrates, such as maltose, sucrose, galactinol, raffinose and monosaccharide cell wall precursors (Kaplan et al. 2004). Perhaps the most studied group if heat induced proteins function as molecular chaperones, which are grouped by their size as HSP60, HSP70, HSP90, HSP100 and small HSP (sHSP). HSP 60 and HSP70 have a primary role in attaining the functional conformation of newly imported proteins to the chloroplast, mitochondria or endoplasmic reticulum (Al-Whaibi 2010). Besides these roles, HSP90s have been found to have a signalling function associated with pathogen resistance and heat response (for a review see: Al-Wahibi, 2010). HSP100s have a role in the reactivation and resolubilisation

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of aggregated proteins, and degrade irreversibly damaged polypeptides (Parsell and Lindquist 1993). Small HSPs can bind to partially folded or denatured proteins and prevent misfolding or aggregation. Some members of these molecular chaperone families are not specific for stress response and are constitutively expressed, while other members are typically expressed during stress. The substantial increase in HSP expression during heat stress is a well-known acclimatory response, although HSP expression can also be induced by other stress factors due to their general role as chaperones (Swindell et al. 2007). Both photosynthesis and respiration are thermosensitive processes and their acclimation is of vital importance for pant growth under sub-optimal temperatures. For the scope of this thesis, I will focus on the thermal response and acclimation of C3 photosynthesis. Photosynthesis (A) can acclimate to long-term moderate low and high temperatures, through different physiological changes and this acclimation typically results in a shift in the thermal optimum. The thylakoid membranes, like any other membrane structures, are temperature sensitive and change their physical properties due to temperature. Under warm conditions the membrane fluidity increases, causing a decrease in the charge density of the membrane surface and a destabilisation of the lipid-protein interactions in the membrane. It has been hypothesised that this can lead to disruption of the organisation and function of PSII and lead to reduced electron transport capacity (Sharkey 2005). On the other hand, at cold temperatures, thylakoid membrane fluidity decreases and they become more rigid. Therefore, thermal acclimation of the thylakoids involves changes in the ratio of unsaturated to saturated fatty acids. While this ratio decreases under warm conditions, to stabilise the membrane, under cold conditions the ratio increases in order to maintain membrane fluidity (Somerville 1995, Falcone and Ogas 2004). Cold- and heat-tolerance of photosynthesis depends on a wide range of physiological adjustments and cannot be directly linked to thylakoid membrane stability only. Nevertheless, in a study on mutant tobacco with reduced unsaturated fatty acid levels, it has been shown, that although the plants were not more susceptible to chilling, they were impaired in their ability to recover from cold induced photoinhibition. This suggested that due to the changed physical properties of the membrane, the rate at which D1 could be inserted into the PSII complex during repair, was reduced (Moon et al. 1995). The most important aspects of thermal acclimation of the photosystems is adjusting the electron flow according to the non-optimal temperature and protecting PSII and PSI from photooxidative damage. A study on winter wheat (Yamasaki et al. 2002), a crop that exhibits high thermal acclimation capacity, found that two regions of PSII electron transport have thermal acclimation potential; one is the

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electron transport on the water oxidation side of PSII, the other is the electron transport on the plastoquinone side, i.e. electron transport from PSII to PSI. Thermal stress leads to an imbalance between harvested light energy and the capacity to utilise this energy in carbon metabolism (Huner et al. 1998). The resulting increased excitation pressure can lead to build up of radicals that can cause photoxidative damage. Xanthophyll cycle pigments are involved in the dissipation of excess energy through heat, in a process we call non-photochemical quenching, which is the deactivation of the excited 1Chl* in the antenna of PSII (for a review see: Jahns and Holzwarth 2011) and thermal stress has been associated with increased concentration of xanthophyll cycle pigments and increase in the de-epoxidation state of the xanthophyll cycle. Some plants also have a lutein cycle, which has an effective photoprotective function and acts through the quenching of 3Chl (Jahns and Holzwarth 2011). Furthermore, the Mehler-ascorbate peroxidase pathway (water-water cycle) and direct reduction of oxygen through a PTOX-mediated electron transport pathway have been suggested as important photoprotective mechanisms under photoinhibiting conditions (Lovelock and Winter 1996, Ivanov et al. 2012). Cold reduces carbon assimilation and carbon utilisation through reduced Pi cycling, due to a cold induced reduction in the activity of enzymes associated with sucrose synthesis. Acclimation to cold has been connected to increased abundance and increased activation state of these cytosolic enzymes, together with Rubisco, which resulted in increased enzymatic activity, and was found to restore carbon assimilation rate (Hurry et al. 1994) within a range of moderate temperatures. Rubisco limitation occurs with increasing temperature, in the range of 15°C-35°C (Sharkey, 2005) One reason for this limitation is the decrease in solubility of CO2 combined with the decreasing affinity of Rubisco to CO2, which together result in increased photorespiration (Sharkey 2005). Another reason is the decline of Rubisco activity due to decreasing activity of Rubisco activase (Crafts-Brandner and Salvucci 2000), which can happen because of the temperature sensitivity of the enzyme or alternatively due to an active regulatory function that protects the plant from increasing photorespiration and the build-up of its potentially damaging products, such as H2O2 (Sharkey 2005). In some species, acclimation of A to heat stress has been connected to the accumulation of heat stable isoforms of Rubisco activase that consequently restored the activation state of Rubisco (Law et al. 2001, Law and Crafts-Brandner 2001). Respiration is regulated by a balance between substrate availability and energy demand. Under cold conditions, the activation state of the respiratory enzymes, especially NADH dehydrogenase (Svensson et al. 2002) and ATPase (Atkin et al. 2002) is limiting for respiration, affecting both reduction and oxidation of the ubiquinone pool. Under warm conditions,

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adenylate and substrate supply are more important limiting factors (Atkin and Tjoelker 2003). Adenylates are limiting, when the turnover rate of ATP/ADP is inadequate due to insufficient ATP use of growth and/or inefficient ATP/ADP transport, resulting in a build-up of protons in the inter-membrane space of the mitochondria (Atkin and Tjoelker 2003). Increasing evidence suggest that the alternative pathway is involved in the maintenance of metabolic and redox homeostasis under abiotic and biotic stress (Vanlerberghe, 2013). The alternative pathway couples the oxidation of the ubiquinol to the reduction of O2 to H2O, bypassing the proton pumping complexes III and IV. There is a reduced ATP production through the alternative pathway when electron flow to AOX occurs through the proton pumping complex I. However, if the electron flow to AOX occurs through an alternate dehydrogenase or complex II, which is not proton pumping, no ATP is produced and electron flow through AOX is completely decoupled from ATP turnover. Therefore it can maintain electron flow when the cytochrome pathway is adenylate limited. For example under limited N supply, the alternative pathway can maintain the capacity of the electron transport chain by dissipating excess reducing equivalents and maintaining cellular redox homeostasis (Wang et al., 2016). This function of the alternative pathway was suggested to play a direct role in modulating the growth rate under nutrient limitation to one that is appropriate to the nutrient supply in order to maintain cell ion homeostasis (Sieger et al., 2005, Noguchi and Terashima, 2006). Furthermore, the capacity of tolerance to photoinhibition under conditions of high steady-state chloroplast excitation pressure (EP), have been correlated to increased abundance of AOX. It has been hypothesised that AOX plays a role in keeping the primary quinone electron acceptor (QA) oxidised by facilitating maintenance of carbohydrate metabolism under such conditions (e.g. Dahal et a., 2015 and 2016, Gonzalez-Meler et al., 1999). R is enhanced by increasing temperature, with typically a higher thermal maximum than A (e.g.: O’Sullivan et al., 2013), which could theoretically mean increase in the respiratory CO2 flux under warmer future climates. However, R acclimation to elevated temperatures can eliminate up to 80% of the expected increase in CO2 released by non-acclimated plants (Reich et al., 2016). Atkin and Tjoelker (Atkin & Tjoelker, 2003) proposed two contrasting acclimation strategies for respiration. Type I acclimation is characterised by a change in Q10 (change in R rate over 10°C change in temperature) of the temperature response of respiration, resulting in relatively low adjustment of the respiration rate. This type of acclimation is presumably controlled by adenylate restriction, which is suggestive of it being a short-term thermal response (Atkin and Tjoelker 2003). Whereas Type II acclimation is characterised by a change in the intercept of the temperature response curve of respiration, resulting in a higher degree of respiratory adjustment. Type II acclimation is underpinned by temperature-

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induced changes in the respiratory capacity (Atkin and Tjoelker 2003), either through changes in per unit mitochondria in the tissue, or through changes in the capacity per mitochondria, for instance due to changes in enzyme abundance.

Root respiration (Rroot) is driven by three factors: Energy demand of root growth, energy demand of ion uptake and energy demand of maintenance. Growth uses 25-45% of the energy produced by R in roots, while ion uptake can use 50-70% of the respiratory energy, and both are highly dependent on the growth rate, together with environmental conditions, like nutrient availability and temperature. Maintenance energy is a relatively small proportion, only 5-10%, under optimal conditions. However, it might represent a much higher proportion under non-optimal conditions where growth rate and ion uptake decline (Atkin et al., 2000). Theoretically the thermal acclimation of leaf and root R should be similar, and Loveys et al. (Loveys et al. 2003) found no significant difference between the long-term acclimation capacity of leaf R and root R over a moderate range of temperature (18-28 °C). In contrast to leaves however, acclimation of Rroot to cold temperature was not correlated with root N concentration (Atkinson et al., 2007), and lateral root growth was found to be impaired under cold conditions (Marschner, 1995b). Both the temperature sensitivity of Rroot, reported as Q10, and Rroot thermal acclimation capacity was found to be highly variable in a meta-analysis of a wide range of studies (for a review see: (Atkin et al. 2000)) and it is unclear if these variations can be attributed to biological or methodological differences. Carbon dioxide The first step in CO2 assimilation is the reaction of CO2 with the 5-carbon sugar, ribulose 1,5-bisphosphate (RuBP), which is catalyse by the enzyme ribulose bisphosphate carboxylase/oxygenase (Rubisco). With such a key role, Rubisco is one of the most influential rate controlling enzymes of carbon assimilation, having a direct influence on plant growth and productivity (Taiz 2015). Phosphorylated 5C compounds, other than RuBP, can bind to the active sites of Rubisco and inhibit its activity, while another enzyme, Rubisco activase, is responsible for releasing the inhibitory sugar compounds and activate the binding sites (Taiz 2015). The activity of Rubisco is measured as the catalytic turnover rate of an active site, kcat, and it has been suggested to be an adaptive trait to thermal conditions. There is substantial variation in kcat among species, but an average of 3.55 ± 0.54 s-1 was found in C3 plants growing in cold habitats (Sage 2002), where high kcat is beneficial to maximise the carbon assimilation rate and the nitrogen use

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efficiency, because CO2 saturation point of Rubisco is lower and so it will most likely operate at a Ci close to that. In war habitats kcat is lower, around 2.46±0.52 s-1 in C3 plants (Sage 2002). In warm climates the CO2 saturation point is higher, furthermore Ci/Ca is typically lower due to the decreased solubility of CO2, therefore a lower kcat is preferred, in order to avoid high rates of oxygenation (Sage 2002). In a study with limiting nitrogen supply, a direct relationship between PSI content and Rubisco activity has been found (Eichelmann et al. 2009), in fact the activity of the enzyme was directly related to the PSI to Rubisco active site ratio. Based on the results in this study Eichelmann et al. proposed the model that Rubisco activase is re-activated, possibly by an ADP-ATP exchange on the thylakoid and then shuttled back to the stroma to activate Rubsico, connecting Rubisco activity to electron transport and PSI. If different parts of this activation chain are out of proportion compared to the others that will result in inhibition effects and decline of Rubisco activity, therefore they proposed that it is the balance between photosynthetic electron transport, ADP/ATP ratio, Rubisco activase and Rubisco content that will determine kcat and it is not one factor that regulates the others (Eichelmann et al. 2009). This implies that kcat has a stronger influence on carbon assimilation rate than the amount of Rubisco per se. Increasing atmospheric [CO2] could theoretically mitigate the negative effect of temperature on Ci/Ca so that an improved kcat might be beneficial for plant growth (Carmo‐Silva and Scales 2015). However, despite its prominent role in carbon fixation, it is not Rubisco and Rubisco activity on its own that regulates carbon assimilation capacity, therefore it has to be considered in connection with other processes, such as RuBP regeneration. Increasing [CO2] progressively shifts A from being Rubisco limited toward being RuBP regeneration limited, furthermore, increasing temperature increases the limitation of RuBP regeneration (Sage and Kubien 2007), highlighting the increasing significance of RuBP regeneration limitation with future climates. Hence it has been suggested that genetic improvements of RuBP regeneration could be a promising approach for improving crop yields in the future (Lefebvre et al. 2005, Ainsworth and Ort 2010). A meta-analysis of data collected from large scale free-air carbon dioxide experiments (FACE) reveal the general findings regarding the response of C3 plants to elevated CO2 (for a review see: (Medlyn et al. 1999, Ainsworth and Long 2005, Leakey et al. 2009)). High CO2 enhanced carbon assimilation (A) by 13-47%, despite the down regulation of photosynthetic capacity. Large variation between plant functional groups was observed, with higher enhancement in tree and grass species, and lower in shrubs and some crops. Seasonal variation was also observed in A, with A being more stimulated by elevated CO2 in the spring compared to autumn, in connected with changes in sink capacity over the season. The enhanced CO2 uptake also meant an

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increase in the photosynthetic nitrogen use efficiency, although this was not correlated with reduced leaf nitrogen per unit leaf area. Parallel with A, Rdark was also stimulated by CO2 in soybean, driven by increased substrate availability and energy demand. This stimulation of R was not observed in many other species, and it is not yet clear if this is due to methodological differences between measurements, or species-specific difference. The over-all growth and yield stimulating effect of CO2 was found to be much lower in the FACE experiments, than what was predicted based on previous chamber studies (Leakey et al. 2009). Already the stimulation of A was lower than expected, and it was found that a relatively small ratio of the extra carbon is allocated to grain production and realised as yield gain. This is probably due to environmental conditions that were not accounted for under chamber experiments, such as increased herbivory (Ainsworth and Long 2005). Increasing atmospheric [CO2] will occur in parallel with increasing average surface temperature (Hartmann et al. 2013) and increasing temperature increases the risk for draught, highlighting the importance of water use efficiency (WUE) of plants. Stomatal conductance decreases with elevated CO2, and the long-term FACE experiments provided evidence that the reduced conductance is maintained over time and it translates into decreased canopy evapotranspiration and higher water use efficiency (WUE) (Leakey et al. 2009), which could be beneficial for growth under low water availability. On the other hand forests with high evapotransipratory capacity have been predicted to be less vulnerable to warming (Bonan 2008), therefore it has yet to be established how the interactive effect of increasing [CO2] and temperature will affect plant physiology. Several studies have investigated wood quality and wood production of industrially significant tree species in response to elevated CO2. They found no effect of high CO2 on stem width growth, observed an increase of wood density and found no effect on the wood chemical composition and lignin content in P. abies (Hättenschwiler et al. 1996, Kostiainen et al. 2004). At the same time, they found that fertilisation increased radial increment, decreased wood density and cell wall thickness and increased lignin content and wood N content. Kostiainen et al. described that the observed effect of fertilisation overshadowed the effect of CO2, and the effect of CO2 in the non-fertilised trees was inconclusive over subsequent growing seasons. Similarly, increase in mean wood density and no change in wood composition was reported in P. sylvestris, although this species increased diameter growth, mostly through increased early wood ratio (Kilpeläinen et al. 2005). However, it is unclear whether the difference in radial growth response is a species specific or a site specific difference, maybe due to different soil nutrient status. In a study on coppiced Cottonwood (Popolus deltoides),

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Druart et al. found enhanced stem and branch biomass production together with increased wood density under elevated CO2 of 800 ppm, but no further enhancement with 1200 ppm, possibly due to enhanced allocation to leaf and root instead of stem. Furthermore, they reported no significant change in gene expression in leaves and stem under 800 ppm CO2, but found an induction of expression in growth related genes in leaves and lignin biosynthesis related genes in the stem at 1200 ppm CO2 (Druart et al. 2006). Nitrogen Nitrogen (N) is a primary constituent of essential molecules and as such it is directly correlated to growth and development of plants. N availability is a limiting factor in many regions and so the use of N-containing fertilisers has increased crop yields and food production substantially (Peñuelas et al. 2013). Nitrogen use efficiency (NUE) has therefore been the focus of much research and the target for genetic improvement for many years. NUE can be discussed in many aspects of plant physiology, but on a whole organism scale it is the combination of N utilisation and N assimilation efficiency (Xu et al. 2012). Most of N is taken up from soil solution by the roots in the form of inorganic N, mostly as nitrate and in case of wetlands and acidic soils, as ammonium (Buchanan 2015). The concentration of these N forms can have a wide range (100 µM – 10 mM) in the soil, and to cope with these variations plants have developed a combination of high- and low-affinity transport systems, regulated by N-form and concentration, together with diurnal and seasonal plant physiology (Buchanan 2015). Nitrate and ammonium can be assimilated directly in the roots or loaded into the xylem for transport and assimilated in the leaves. Nitrate is first reduced to nitrite by nitrate reductase (NR). The activity of this enzyme is induced by light and by the availability of reduced carbohydrates, and so it has a diurnal pattern, being most actively transcribed pre-dawn. Reduction of nitrite to ammonium is the next step in nitrate assimilation and is mediated by nitrite reductase activity (NiR). Since the accumulation of nitrite is toxic for the plant, activity of NR is tightly regulated by NiR activity as well, and signals of oxidative stress, such as Ca2+, will down regulate NR activity (Krapp 2015). Ammonium can be directly incorporated into amino acids, and the primary step of this is the metabolism of glutamine (Gln), by glutamine synthetase, which has so a central role in primary N assimilation. Another key enzyme is asparagine synthetase that forms asparagine and glutamate from glutamine and aspartate. Asparagine is the most significant storage amino acid in plants, and its synthesis is induced when further utilisation of N is limited, for example by light or by the availability of reduced carbohydrates (sucrose or glucose) (Buchanan 2015). In summary, it is clear that N assimilation is under strong regulation of photosynthetic activity and the general C status of

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the plant (Nunes-Nesi et al. 2010, Bussell et al. 2013). Whereas genetic approaches to increase NUE on the N assimilation side have not yielded the expected results (for a review see: Krapp, 2015), the over-expression of sugar transporters in Arabidopsis concomitantly increased NUE as well (Schofield et al. 2009). Significance of the ectomycorrhizal symbiosis The majority of land plants form symbiotic association with mycorrhizal fungi. In general, this symbiosis benefits the host plant by enhancing its capacity to acquire mineral nutrients and benefits the fungi by acquiring carbon from the host plant. Arbuscular, ericoid and ectomycorrhizal (EM) fungi form the three main types of mycorrhizal associations. For the scope of this thesis we will focus on the ectomycorrhizal association that is typical for a wide range of woody species, for example birch, pine and spruce, and is therefore a significant form of mycorrhizal symbiosis in the typically N limited boreal region. EM have saprotrophic capabilities, they are able to degrade and mineralise insoluble organic N compounds, increasing thereby the available N pool for utilisation by the host plants (Gobert and Plassard 2008). Besides inorganic N, organic compound, such as small peptides and amino acids can be absorbed as N source in fungi (Nehls et al. 1999) and taken up by the host plant, bypassing so the energy demanding mineralisation process (Buchanan 2015). Various studies have described the potential significance of these small organic compounds as N source in the boreal region (e.g.:(Näsholm et al. 1998, Persson et al. 2003)), and have identified several amino acid and peptide transporters (Näsholm et al. 2009) in plants. The general understanding is that the regulation of N and C cycle between host plant and fungi is interlinked. This has been demonstrated for example in a study that looked at the N supplied to two plants connected by the same mycelium, and they found that the plant supplying more carbon received higher amount of N (Ek et al. 1997). However, the mechanisms and regulation of exchange between fungi and host cells are not yet fully understood. Furthermore, there is no clear consensus about the contribution of fungi to the host plant N uptake. For example in an experiment with Pinus pinaster and Hebeloma cylindrosporum it has been demonstrated that non-colonised plants grew better than colonised plants when the N source was primarily inorganic, while mycorrhizal colonisation was beneficial for the plant when the N source was primarily organic (Plassard et al. 2000).

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Stress physiology of plants under a combination of stressful environmental conditions It is a long-discussed paradigm in plant stress research that a combination of stress factors is not equal to the sum of the individual stress factors and therefore should be considered separately. This is due to the fact that even though many stress response pathways are inter-connected, different stress factors can induce synergistic as well as antagonistic responses. In this respect, the combination of two environmental factors can have a relatively positive interaction, by inducing responses that are supporting each other, they can be neutral, or they can be in a negative relationship, by inducing responses that counteract each other. A common negative interaction is between heat and drought. While heat induces stomatal opening with the aim to increase transpiration and cool leaf surface, drought induces stomatal closing in order to retain humidity in the leaves. Therefore the combination of heat and drought has a more severe effect on the plant than the two stress factors would have individually (Mittler 2005). The rising air CO2 concentration is considered to have a fertilisation effect and possibly promote plant growth, rather than being a stress factor. However, it is not yet clear how much of this growth enhancing effect can be realised in combination with rising temperatures. We have discussed previously that increasing temperature increases the Rubisco limitation of carbon assimilation, due to decreasing Rubisco activity and decreasing CO2 solubility, i.e. decreasing Ci. Arguably, rising CO2 could counteract the problem of decreasing solubility but it is not clear whether it can be counteracted to an extent to realise a positive CO2 response. In fact it has been suggested that engineering Rubisco to be more thermotolerant, so that it will not be down regulated by increasing temperature, could cause increased rates of photorespiration even under elevated CO2 and make the plants more sensitive to heat (Sharkey 2005). Another aspect of warming to consider is VPD. VPD typically increases with increasing temperature, reducing thereby the stomatal conductance and increasing Ci/Ca (Sage 2002). This could possibly counteract the decreasing CO2 solubility especially under elevated CO2, and allow Rubisco to work closer to its saturation point. However, predictions are made complicated if we consider that rising temperatures often co-occur with extending drought periods that would induce stomatal closure instead. It is well known that thermal stress and low nutrient supply negatively affect plant growth and crop yield and eliminating nutrient constrains by fertilisation has been a long-time practice in agriculture. In studies across

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different plant species, it has been established that there is a linear relationship between N limitation and the suppression of growth rate (Ågren 1985). Furthermore, N deficiency results in increased root to shoot ratio (Ågren and Ingestad 1987), which means increased resource allocation to below ground growth and enhanced leaf senescence due to the remobilisation of N from the older leaves towards the new growth (Marschner 1995). However, it is not clear how N availability affects the thermal acclimation potential. As described earlier, thermal acclimation involves increase in the abundance of certain key enzymes and in leaf N concentration (Campbell et al. 2007), which leads to the hypothesis the N limitation in turn limits thermal acclimation. Since N limitation is one of the most prominent production limiting factors across a wide range of natural habitats, it is essential to evaluate thermal response under the interference of N supply. I have described earlier that N acquisition is tightly linked to the carbon balance in pants. While darkness and low levels of reduced carbohydrates can supress N acquisition it has also been found that the enhancing plant productivity with elevated CO2 can also be limited by low N supply. From various high CO2 studies it can be concluded that CO2 induces N uptake and growth given that there is sufficient N available for uptake, and elevated [CO2] increases the activity of NR (Medlyn et al. 1999, Stitt and Krapp 1999, Ainsworth and Long 2005, Leakey et al. 2009). Although it is beyond the scope of this thesis to examine the interaction between mineral nutrients and CO2, it is still worth it to make a general consideration. In low nitrogen regions, like the boreal forests, it has been found that elevated CO2 has no fertilising effect and does not increase growth (Kostiainen et al. 2004, Kostiainen et al. 2009) because nitrogen is limiting growth already under current conditions. Extrapolating this finding, it can be hypothesised that regions where plant productivity is currently not limited by mineral nutrients might progressively become nutrient limited by increasing demand due to increasing abundance of carbon, and so carbon fertilisation might actually enhance growth belowground rather than aboveground. Plants in this region are typically growing in symbiosis with ectomycorrhiza and we could hypothesise that an increased carbon flow to the fungi could increase its activity and enhance N degradation from insoluble compound and enhance N turnover in the soil. This could benefit the host plant by making new N source available for uptake and utilisation in growth. However, as describe earlier, there is no clear consensus regarding the mutual C-N cycle between host and fungi and it is not clear yet to what extent increased below ground carbon allocation would in turn benefit the host plant with increased N supply.

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Aim and significance of the presented work The over-all aim of this doctoral thesis was to establish a link between thermal response and the carbon-nitrogen balance of plants. The work in this thesis focused on ecologically significant species of the boreal region: Picea abies, Pinus sylvestris and Betula pendula; and Betual utilis, which is one of the prominent tree species in the high altitudes of the Himalayas. With my experiments I aimed to gain detailed insights into the following subjects:

1. Examining the carbon-nitrogen cycle between host and mycorrhiza with the aim of elucidating whether this symbiosis could be promoting or limiting the positive effect of carbon fertilisation under elevated CO2.

2 Evaluating the role of N availability in the thermal acclimation capacity of trees with contrasting growth strategies, and the consequences of N limitation under non-optimal growth temperatures.

3 Quantifying the carbon fertilisation effect of elevated air CO2 and the carbon sequestration potential under increasing growth temperature in the boreal region.

4 Elucidating the underlying transcriptional responses to warming growth temperatures and elevated CO2 in P. abies.

5 Establishing a better understanding of the carbon sequestration potential of large ecosystems through an improved model of the thermal response of respiration.

My experiments provide a novel insight into the interactive temperature-carbon-nitrogen responses of plants. My aim was to establish a link between knowledge, gained by controlled environment experiments, and the natural environment, with focus on gaining more predictive power when modelling plant and ecosystem response to future climates.

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Discussion

Carbon release under warming conditions: Modelling the thermal response and acclimation of respiration Photosynthesis and respiration are parameters with major uncertainties in terrestrial biosphere models (TBMs) and the associated land surface components of earth-system models (ESMs). These processes account for 120 Gt of yearly carbon exchange (Ciais et al. 2013) in the atmosphere, yet they are not well described in predictive models. Despite the widely accepted observation that the sensitivity of respiration to temperature is not linear over a wide range of temperatures (Tjoelker et al. 2001), in most models respiration is still represented with the assumption that that it follows an exponential curve with stable Q10 = 2, or is modelled using the Arrhenius model, which does not account for changes in thermal sensitivity and gives very similar results to the fixed Q10 approach. The study described in Paper I had three goals:

1. to quantify the T response of leaf R through use of a new and comprehensive set of thermally high-resolution field measurements of leaf R across large T ranges;

2. to assess the shape of T-response curves in leaves of species representing diverse environments and plant functional types (PFTs);

3. to assess the implications of altered T sensitivity of R for simulated carbon fluxes using the land-surface component of a leading ESM

High resolution instantaneous R-T response curves (O'Sullivan et al. 2013) were measured in 231 species, on 18 field sites globally, representing 7 biomes. Five alternative models were compared in order to establish the best fit for the data set: exponential model with fixed Q10; Arrhenius model; an Arrhenius model with modified Ea description to improve temperature sensitivity (Lloyd and Taylor 1994); a T-variable Q10 model (Tjoelker et al. 2001); and a second order polynomial model (O'Sullivan et al. 2013). Based on the mean residual produced from each model, it was established that the T-variable Q10 model and the second order polynomial model fit the data best (Supplementary information Paper I). The conceptual argument for using the second order polynomial model for down-stream analysis was that it is independent of the underlying concept of the Q10 formulation and does not rely on biological assumptions about activation energies: lnR = a + bT + cT2 Equation 1

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where R is respiration at T, a is lnR at 0°C, b is the slope of the R-T response and c is the curvature describing the T-dependent change of the thermal sensitivity of R. One of the key questions of this global study was whether R-T is spatially invariant or if it varies due to genotypic and environmental factors. Coefficient a, which corresponds to the absolute rate of R, was highly variable between biomes, with highest values in the cold biomes, i.e. with higher altitude and latitude (Paper I, Figure 1). This is in accordance with earlier findings that leaf R is higher in plants grown in cold environments (Atkin et al. 2000). Furthermore it was variable between PFTs, with highest values in C3 herbaceous species, needle-leaved and broad-leaved evergreen plants (Paper I, Figure 1). These variations are significant and therefore they must be accounted for when modelling global R or global R-T responses. A wide range of publications describe the non-exponential nature of the R-T response, still there is no clear consensus on the shape of the curve. In a recent analysis Reich et al. (Reich et al. 2016) have reported that about half of the R-T responses in their data set exhibited decreasing temperature sensitivity, while the other half was better characterised by increasing temperature sensitivity. This highlights the need for further studies in order to gain confidence in models and modelling approaches. In the data collected and analysed in this study, there was high degree of similarity between the R-T responses across all biomes and PFTs, with no significant variation in the b and c coefficients (Paper I, Table 1), suggesting an intrinsic T sensitivity of R. This implies that the enzymatic controls of the various pathways connected to R are highly conserved in C3 plants. These findings support the conclusion that it is possible to implement a generalized empirically based mathematical model (GPM) on the instantaneous R-T response, based on a second order polynomial fit: lnR = a + 0.1012T – 0.0005 T2 Equation 2 where a is lnR at 0 °C. This model has the capacity to represent the mean R-T response within biomes and within PFTs, across the whole range of measurement temperatures (10-45°C), and also when we fitted to only shorter segments of the temperature range (Paper I, Supplementary information). This model allows prediction of R at any given temperature, using a, or a reference rate RTref at a reference temperature, as

RT = RTref * e[0.01012(T-Tref) – 0.0005(T2-Tref2)] Equation 3

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where RTref = exp(a + 0.1012Tref – 0.0005Tref2) Equation 4

Using the GPM instead of fixed Q10 = 2 in the JULES model (Joint U.K. Land Environmental Simulator) had significant effects on the estimates of R in the temperate and cold sites, while estimates for tropical regions was similar with both models. For biomes characterised by freezing temperatures in the winter, i.e. winters with minimal metabolic activity, R estimates were lower with the GPM approach, on average across the whole year. In temperate biomes with evergreen woodland sites, the choice of model had the largest effect in the cool winter months, with an average annual decrease in R estimate by 10-20% (Paper I, Figure 3). These findings highlight that NPP of temperate and cold biomes is systematically underestimated in TBMs that use fixed Q10 or the Arrhenius model in their estimations of the R-T responses. Furthermore the GPM presented here can be easily integrated into TBMs and gives a more accurate formulation of the R-T response.

Modelling the thermal acclimation of respiration R-T differs both in magnitude and mechanism with time, meaning that short-term R-T is not equal to long-term R-T. In case of long-term R-T we have to account for acclimation processes that can alter not only the rate of respiration but also the sensitivity of its thermal response (Atkin and Tjoelker 2003). Since the 2nd order polynomial model described above accounts for all these aspects of respiration, theoretically it can be parameterised to account for long-term acclimation and so allow for a more accurate estimate of R under future climate conditions. In the following I show an example of how this model can be used to account for thermal acclimation of R, based on a small data set including two needle-leaved evergreen and two broad-leaved deciduous trees, acclimated to three different growth temperatures (from experiments described in Paper III and Paper V). I decided to only look at AC conditions in case of Paper III, and FN conditions for Paper V for the sake of this analysis, because the small size of the data set would not be sufficient to account for added variation due to elevated CO2 or limited N. I estimated the model parameters from individual polynomial fits to four replicate high-resolution instantaneous R-T response curves (Table 1). In order to evaluate acclimation, I took the median values of the three temperature treatments from Paper III, and the growth temperatures from Paper V.

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Table 1 Descriptive parameters of the models fitted to the R-T response curves of P. sylvestris, P. abies, B. pendula and B. utilis acclimated to three-three different growth temperatures The coefficients are estimated based on second order polynomial fits to four replicate high-resolution instantaneous R-T response curves. The table presents the mean and standard error of all coefficients and the R2 of the fit.

a b c R2

Pinus sylvestris ACT0 19.48 °C -3.200 ± 0.47 0.1290 ± 0.02 -0.0008 ± 0.00 0.92

ACT4 23.40 °C -3.725 ± 0.67 0.1450 ± 0.04 -0.0009 ± 0.00 0.94 ACT8 27.05 °C -3.875 ± 0.66 0.1140 ± 0.03 -0.0005 ± 0.00 0.81

Picea abies ACT0 19.48 °C -3.075 ± 0.37 0.0890 ± 0.02 -0.0004 ± 0.00 0.94

ACT4 23.40 °C -3.675 ± 0.69 0.1140 ± 0.02 -0.0007 ± 0.00 0.79

ACT8 27.05 °C -2.850 ± 0.40 0.0550 ± 0.02 0.0002 ± 0.00 0.93

Betula pendula FN10 10 °C -1.11 ± 0.14 0.1033 ± 0.0092 -0.00065 ± 0.00 0.99 FN20 20 °C -1.40 ± 0.10 0.1066 ± 0.0058 -0.00075 ± 0.00 0.99

FN30 30 °C -2.20 ± 0.26 0.0895 ± 0.0062 -0.00055 ± 0.00 0.99 Betula utilis FN10 10 °C -0.38 ± 0.056 0.0880 ± 0.0044 -0.00054 ± 0.00 0.99 FN20 20 °C -0.57 ± 0.045 0.0867 ± 0.0048 -0.00069 ± 0.00 0.99

FN30 30 °C -1.55 ± 0.035 0.0860 ± 0.0071 -0.00071 ± 0.00 0.97 In a recent meta-analysis it has been found that Type II acclimation, as a change in the intercept of the R-T response, is more common than Type I acclimation across a wide range of species and thermal conditions, occurring in about 65% and 75% of the observed cases in evergreen conifers and deciduous broad-leaved species respectively (Slot and Kitajima 2015). Furthermore, based on the global data in Paper I, parameter a was highly variable among PFTs, while b and c were highly conserved. Based on these arguments, I hypothesised that accounting for acclimation induced changes in a will be sufficient to model acclimated R. I excluded spruce under ACT8 from this analysis because it did not show any acclimation, which will be discussed in a later chapter. Statistical analysis showed no significant change in a of the two conifers (Paper III, Table 1, 2 and 3), due to the small number of replication. Statistical analysis of a of the two birches (Paper V) showed a significant

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response to growth temperature in both B. pendula (BP) and B. utilis (BU), with p = 0.009 and p > 0.001 respectively. I plotted values of a against the respective growth temperatures and found a high degree of overlap between the two conifer species, and so for further analysis I considered them as one group. I fitted a linear model to the a-Tg plots for the conifers and for BP and BU separately, and found that the slope describing the relationship was -0.1048 for conifers, and -0.0546 and -0.0587 for BP and BU respectively (Figure 1). Since the a-T slope was so similar in BP and BU I decided to group them and calculate a common acclimation parameter of -0.0567 for the broad-leaved group. These results point to a higher acclimation capacity of R in conifers compared to broad-leaved species, which is in accordance with other studies (Reich et al. 2016).

Figure 1 Regression of the change of parameter a in response to different growth temperatures in the two conifers (grey triangles), in B. pendula (black squares) and in B. utilis (black circles)

These data suggest that species within one PFT exhibit similar degrees of respiratory acclimation, and so they could be modelled using a common acclimation parameter (α) for each, as lnR = (a+(α𝛥T)) + bT + cT2 Equation 5 where R is respiration at T, α is the acclamation parameter and 𝛥T is the mean change in the annual growth temperature expressed in °C. Coefficients b and c showed a significant thermal response in spruce but not in pine (Paper III, Table 2 and 3), and they were significantly different between the 20°C and the 30°C grown seedlings of both BP and BU. Based

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on this it was not possible to rule out the importance of these parameters in modelling of R acclimation. Therefore I evaluated the difference between estimates with and without accounting for changes in b and c. I modelled the lnR-T responses using the coefficients of the reference experiments, i.e. ACT0 and FN20, and the respective α for conifers or broad-leaved trees and compared it to the measured lnR-T, essentially accounting for the acclimation of all three parameters (Table 1).

Figure 2 Comparison of modelled (M0, M4, M8, M10, M30) and measured (T4, T8, BP10, BP20, BP30, BU10, BU20, BU30) R-T response curves in the two conifers and the two broad-leaved species

Comparing the curves of measured and modelled lnR-T, it can be concluded that the models followed the trend of acclimation well in pine, BP and BU, and the error between modelled and measured data was between 1.5% and 25%, while the model failed to correlate with measured data in spruce. The thermal sensitivity of spruce will be discussed in a later chapter. This example did not intend to build a general model for the acclimation of R and the R-T response, because of its small sample size and small number of species. Nevertheless it highlighted some important aspects of R thermal acclimations, such as the high acclimation potential of R, which has been discussed in earlier studies (Atkin and Tjoelker 2003, Reich et al. 2016).

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Global climate change models predict 4°C to 8°C increase in mean annual surface temperature for the higher latitude regions (Hartmann et al. 2013) and therefore not accounting for R acclimation would result in substantial overestimation of leaf R and in turn underestimation of NPP under future climate conditions. Furthermore the presented example demonstrated that accounting for only the acclimation of parameter a, has the potential to account for 75% to 98.5% of the acclimation change in R. If this can be confirmed with a larger data set and in more species, then it can provide a simple way to incorporate thermal acclimation of R in TBMs, by correcting parameter a of the GPM (Paper I) with an acclimation parameter, as suggested in Equation 5.

Shifts in the environmental carbon-nitrogen balance drives the dynamics of tree-mycorrhiza interaction The key aspect of the plant-mycorrhiza association is the bi-directional exchange of resources across the mycorrhizal interface, most importantly carbon (C) assimilated via photosynthesis, and nutrients taken up by an extensive underground network of mycorrhizal hyphae. It is commonly assumed that mycorrhizal fungi transfer nutrients to their host in exchange for C at a rate favourable for plant growth (Cowden and Peterson 2009), while plants transfer 4% to 20% of their assimilated carbon to the fungi. However, there is increasing evidence that the benefits EM fungi convey to host plants vary from mutualistic to parasitic (Johnson et al. 1997, Franklin et al. 2014). Several studies (Alberton et al. 2007, Corrêa et al. 2008, Alberton and Kuyper 2009) have shown under greenhouse conditions that EM fungi represent a strong N sink under low-N conditions, irrespective of plant demand, and that increased C fluxes to the mycorrhiza resulted in reduced N fluxes to the host plant, having in turn a negative effect on seedling growth. Furthermore Näsholm et al. (2013) found in a field study that high rates of N addition reduce the proportion of N immobilized by EM fungi, thereby increasing the transfer of N to host plants. In addition to saturating the fungal demand for N, it is well documented that the addition of N at high rates, or over long-time periods, causes a severe reduction in below-ground tree C allocation to EM fungi (Wallenda and Kottke 1998, Treseder 2004, Högberg et al. 2010), which provides an explanation for increase in aboveground tree growth and foliar N concentrations in response to long-term N addition (Hogberg et al. 2006, Högberg et al. 2011), with a subsequent reduction in the plant-EM interaction. These studies suggest that in ecosystems where nitrogen (N) is the most limiting nutrient for plant growth, such as boreal forests, the C-N exchange between host and EM is strongly depend on the availability of C and N in the environment (Näsholm et al. 2013, Franklin et al. 2014). Both C and N is changing in abundance due

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to anthropogenic activity and it is not well understood what effects these shifts will have on the plant-mycorrhizal associations and N exchange. The central hypothesis of Paper II was that greater C allocation from trees to EM fungi under N-limiting conditions increases the fungal demand for N to build biomass, and as a consequence EM fungi may exacerbate rather than alleviate N limitation for tree growth in boreal forests. The aim of the experiment was to provide a first field-based assessment of the independent and interactive effects of the shifts in environmental C and N conditions on the plant-mycorrhiza C-N relations. Large-scale shading treatments designed to reduce plant C supply were installed in full factorial combination with three levels of N additions in a natural stand of young Pinus sylvestris trees in northern Sweden. Trace amounts of a highly enriched 15NO3–label was added to the EM-dominated mor-layer in the autumn in order to assess the dynamics of N transfer in EM associations, and the 15N label was followed in plant foliage, fungal chitin isolated from EM root tips, and EM sporocarps over a 1-month period. The shading resulted in reduction of photosynthetic carbon uptake by about 60%, in a 30% reduction of cumulative soil respiration and a ca. 40 % lower fungal biomass on EM root tips (Paper II, Figure 1). Foliar N content was over all higher in the shaded plots, and was increased by N fertilisation (Paper II, Figure 2). Shading, i.e. the reduction of belowground C flux, resulted in lower amount of cumulative 15N label in EM sporocarps (µg 15N excess m-2) compared to the unshaded plots (5.2 ± 2.3 µg 15N and 11.7 ± 5.5 excess m-2, respectively) (Paper II, Table D1). At the same time, the ratio of the amount of 15N label in the tree foliage relative to the amount in EM fungal biomass on root tips (i.e., an index of N transfer) was ca. twice as great in the shaded compared to unshaded plots (2.26 ± 0.9 and 1.09 ± 0.56, respectively; P = 0.02) (Paper II, Figure 4). These results indicated that decreased C flux to the fungi decreased the fungal demand for N, which in turn resulted in increased N flux to the tree. The addition of N also resulted in greater N transfer, with the ratio of 15N foliage:EM root tips being significantly higher in the low N plots (2.37 ± 0.79) compared to the unfertilized control plots (0.75 ± 0.24, p = 0.03), however the ratio in the high N plots was not different from the control plots. When comparing among all treatment combinations, there was a general trend of increasing N transfer to the trees with increasing N additions both in the unshaded plots and in the shaded plots, except for the addition of 150 kg N ha-1 in the shaded plots, which resulted in lower 15N foliage:EM root tip ratios compared to the low N plots (P = 0.07; Fig. 4). This can be explained by the relatively reduced recovery rate of the 15N tracer, which might indicate a saturation of tree and fungi N demand due to reduced C availability in the high N plots. In summary these results show in a natural stand of Scots pine,

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that both changes in C allocation from trees to EM fungi as well as changes in soil N supply are important drivers influencing the transfer of N in mycorrhizal associations. Short-term addition of N shifted the incorporation of 15N label from EM fungi to tree foliage, even with no changes in belowground tree C allocation to EM fungi. However, on average more N was transferred to the tree when C supply to mycorrhiza was limited. On the other hand under N-limited conditions greater C allocation to EM fungi resulted in reduced N transfer to the trees. These findings support the hypothesis that the C-N fluxes between fungi and tree are strongly dependent on the C and N in the environment (Näsholm et al. 2013, Franklin et al. 2014) and furthermore they show that the more limiting supply drives the direction of the fluxes, meaning that surplus of one element will rather reduce than increase the flux of the other element as long as its availability is limited. Based on these results, the dynamics of tree and mycorrhiza associations has a significant influence on forest productivity and C sequestration capacity, and therefore it has to be accounted for in prediction on forest C-N fluxes under future climate scenarios. N deposition is mostly concentrated to densely populated temperate latitudes, with high proportions of intensely managed agricultural lands, and is less pronounced in the high latitude boreal ecosystems (Peñuelas et al. 2013). Therefor it is not expected that these forests will become less N limited due to N deposition. At the same time, according to the predictions of global climate models, the high latitude regions will experience the highest rate of average temperature increase (Hartmann et al. 2013), which also means increase in soil temperature, especially in the active humus layer close to the surface. Increase in soil temperature induces soil microbial activity and in turn increases N availability in the soil. Based on the observations described in Paper II, increased soil N will increase the transfer of N to the trees and increase tree growth. Soil warming has indeed enhanced stem diameter growth, by about 115%, over a 6-year long soil warming experiment in a mature Norway spruce (Picea abies L.) stand in northern Sweden (Strömgren and Linder 2002). The authors connected the observed growth to increased nutrient availability for the trees, but also expressed concerns about the uncertainty of whether this was a transitional effect or if it would be sustained over long periods of time. Parallel with increasing temperature, atmospheric CO2 concentration will continue to increase as well, resulting in overall higher C transfer belowground to the mycorrhiza. According to the results described here, this would increase the mycorrhizal demand for N and limit N transfer, thereby progressively increasing the N limitation of the tree. In summary I can conclude with the hypothesis that as a consequence of increasing CO2 concentration, progressive N limitation, enhanced by the increasing N demand of the mycorrhiza, will in the future limit the C

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sequestration capacity of high latitude ecosystems where N deposition is negligible.

The effect of nitrogen limitation on thermal acclimation Thermal acclimation is a process of metabolic adjustment in order to maintain growth and metabolic and redox homeostasis under sub-optimal thermal conditions. Photosynthetic tissues are especially exposed to thermal fluctuations and are most likely to deal with redox imbalances and photosynthetic carbon assimilation together with leaf respiration are major drivers of whole plant metabolic balance. Therefore thermal acclimation in leaves, both to short-term and long-term exposure to thermal stress, have been the focus of many studies, while other plant organs, such as roots or stem have received less attention. Cold acclimation in leaves has been connected to increased relative nitrogen content (e.g.: Campbell et al. 2007) and this has been attributed to higher abundance of various photosynthetic and respiratory enzymes, enzymes related to the scavenging of reactive oxygen species, increased leaf chlorophyll content and increased concentration of compatible solutes. For example, the increased relative abundance of Rubisco, enzymes involved in RuBP regeneration, ATPase, NADH dehydrogenase and AOX have all been connected to cold acclimation (Hurry et al. 1995, Strand et al. 1999, Atkin et al. 2002, Svensson et al. 2002, Campbell et al. 2007). Proline and carotenoids also accumulate under cold stress (Hare and Cress 1997, Strzałka et al. 2003) and they have a role in maintaining the osmotic balance in the cells and reduce oxidative stress. Acclimation to warm growth conditions has not been directly connected to high nitrogen content in leaves (Tjoelker et al. 1999, Campbell et al. 2007), although proline level for instance tend to increase under heat stress as well (Hare and Cress, 1997) and the accumulation of heat stable isoforms of Rubisco activase has also been connected to acclimation to heat (Law et al. 2001, Law and Crafts-Brandner 2001). The activity of enzymes increases with increasing temperature, therefore the maintenance of linear electron transport from light harvesting to carbohydrate metabolism and the balance between carbon assimilation and utilisation gains importance under warm conditions, which is intrinsically connected to nitrogen assimilation and whole plant C:N balance (for a review see: Nunes-Nesi et al. 2010). In Paper V I discuss the hypothesis that nitrogen limitation negatively affects thermal acclimation capacity, based on a study with two Betula species. The seedlings were grown under optimal (20°C), cold (10°C) and warm (30°C) growth temperatures (Tg), combined with optimal (FN) and limited (LN) nitrogen availability. The fast growing Betula pendula (BP) and the slow growing Betula utilis (BU) were used in this experiment in order to compare the influence of growth strategy on thermal acclimation under nitrogen

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limitation. In a meta-analysis of 24 species it has been found that fast growing species allocate relatively more nitrogen to the leaves, have over-all higher photosynthetic nitrogen use efficiency and use a smaller portion of assimilated carbon for respiration (Poorter et al. 1990). Therefore, I hypothesised that the fast growing BP would be more sensitive to nitrogen limitation and would be more susceptible to an imbalance in the whole plant C:N homeostasis. Cold and N limitation induced similar responses in both BP and BU regardless of their growth strategy. The seedlings grown under 10°C with unlimited nitrogen supply (FN10) had a series of characteristic that resembled a similar response to plants grown under nitrogen limitation and optimal temperature (LN20). Their growth rate was significantly suppressed, they had higher root to shoot ratio (Paper V, Figure 1, 2), together with increased belowground N allocation, similar to plants grown under LN20. Even though Amax was upregulated in cold, the Anet:Rdark ratio did not fully acclimate and was lower than under FN20, suggesting a stronger upregulation of respiration compared to photosynthesis. The general trend of these results was very similar to that observed in seedlings under LN20 conditions. For example, N content as mg g-1 dry weight decreased in leaves, roots and stem of BP under FN10 as well as LN20 compared to FN20 (Figure 3a, c, e). I observed a similar response in BU roots, although not in leaves and stem (Figure 3d). In contrast, root ATPb and GlnA content was higher under FN10 compared to FN20, suggesting enhanced root metabolism and N assimilation. These results indicate that the high energy demand of N assimilation is one of the limiting factors to plant growth in cold temperatures, to a degree that it induces a series of responses that are similar to the responses to N limitation. N limitation under cold temperature further enhanced the effect of cold, and in addition it limited the acclimation capacity of photosynthesis, further disrupting the metabolic homeostasis of the seedlings, so that the combination of cold and N limitation had an additive negative effect on plant growth and acclimation capacity. Under LN10, significantly higher proportion of N was allocated to the roots and the stem, resulting in substantially lower relative N allocation to the foliage, over 50% lower compared to the relative allocation under FN10 (Paper V, Figure S1). The shift towards belowground N allocation, together with increased R:S ratio and relatively high GlnA abundance in the roots suggests increased root activity in an effort to increase N acquisition and N assimilation. Fv/Fm and Y(II) declined to a higher degree under LN10 than under FN10, with parallel increase in photosynthetic quenching, quantified as Y(NO) and Y(NPQ) (Paper V, Figure 2S).

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Figure 3 Nitrogen mg g-1 dry mass in the leaf, root and stem tissues of BP (a, c, e) and BU (b, d, f). Bars represent means and standard errors, white bars represent optimal N conditions and grey bars represent limited N conditions. The annotations represent the Tukey HSD pair-wise comparisons, where p ≤ 0.05 was considered a significant difference.

Acclimation of Amax, Rdark, leaf A:R homeostasis and Rroot was impaired under LN10, while seedlings under FN10 and LN20 displayed well-acclimated photosynthesis and respiration, and maintained C:N allocation homeostasis, despite suppressed growth rates. Decreased abundance of the photosynthetic proteins Lhcb1, PsbA and Rubisco correlated with the suppressed carbon assimilation (as Amax) and low abundance of AOX, both in leaves and roots, together with low abundance of COXII in leaves correlated with suppressed respiration. These results support the hypothesis that N limitation limits the acclimation capacity to cold growth temperature. However they did not support the hypothesis that fast growth strategy would result in higher

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vulnerability to N limitation, although the fast growing BP lost its advantage to BU at 10°C, so that seedlings of both species grew at a similar rate, suggesting a relatively higher thermal sensitivity of BP. 30°C supressed the growth rate of BP by about 30% even under optimal N conditions, whereas BU grew at a similar rate under both 20°C and 30°C, suggesting that 30°C exceeded the thermal optimum for growth of BP. Carbon assimilation in BP was suppressed under 30°C, whereas BU maintained a relatively high Amax, which can be part of the explanation for how the growth rate of BU was not different between FN20 and FN30. With increasing temperatures, a decline in Rubisco activity becomes increasingly limiting to A (Sage and Kubien 2007), and acclimation to warm Tg has been correlated with increased abundance of Rubisco activase, especially the expression of thermo stable isoforms of Rubisco activase (Sage et al. 2008). In accordance with this, in BU we observed similar Vcmax under FN30 and FN20 when measured at a common temperature, despite low Rubisco abundance, and correlating with high Amax. Nevertheless, acclimation of photosynthesis was similar under FN30 and LN30 in both species, so that N limitation had no additional effect on the acclimation capacity of either species. This is not surprising, since enzymatic activity typically increases with increasing temperature, up to a thermal maximum, therefore N requirement for enzyme metabolism is less pronounced under warmer Tg. Instead, the balance between carbon assimilation and carbon utilisation, i.e. electron transport and balanced ATP/ADP cycle, gain significance under warming Tg (e.g. Atkin et al., 2002, Sage and Kubien, 2007) and so N limitation can increase thermal sensitivity if it disturbs the whole plant C:N homeostasis due to insufficient metabolic adjustment. I found that N limitation exacerbates the thermal sensitivity of the fast growing BP under warm conditions. The growth rate of BP decreased below the growth rate of BU at FN30 and even more so under LN30. Increased R:S in BP under LN30, together with increased Rroot and high GlnA abundance in the roots suggested up-regulation of N assimilation, pointing to a growth rate maximising growth strategy, even under low N supply and non-optimal Tg. At the same time, in BU Rroot was similar under FN30 and LN30, and GlnA abundance in the roots decreased, pointing to a relative adjustment of N assimilation to the supressed growth. On the leaf level, Rdark decreased in BP under LN30, resulting in an increased A:R and leaf C:N ratio, consequently resulting in an imbalance in the whole plant C:N allocation homeostasis. In contrast, BU maintained Rdark, A:R and C:N homeostasis under LN30 at a similar level as under FN30. Overall the abundance of the analysed photosynthetic and respiratory proteins was not affected significantly by low N supply under 30°C, except for significantly high AOX content in BU leaves. Taken together, I concluded that BU maintained its metabolic homeostasis

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under warm Tg and limited N, which can be attributed to its conservative growth strategy. In contrast, while the Gr maximising strategy yielded high growth rate and gave advantage to BP under moderate temperatures, it was not beneficial under high temperature conditions, and N limitation exacerbated its sensitivity.

The role of the alternative respiratory pathway under thermal stress and nitrogen limitation Several studies suggested that the alternative pathway of respiration has a role in maintaining the metabolic and redox homeostasis in leaves by facilitating carbohydrate metabolism in a pathway uncoupled from the ADP/ATP cycle and so alleviating steady-state chloroplast excitation pressure under stressful conditions, such as cold, drought, high light or N limitation (Gonzàlez-Meler et al. 1999, Dahal et al. 2016). In accordance with this, in Paper V I observed high AOX abundance in both the root and the leaf tissues, in BP as well as BU, under cold conditions. Under LN30, high AOX abundance in the leaves of BU correlated with balanced Rdark, maintained A:R homeostasis and C:N ratio. In contrast, in BP, AOX abundance was similar under FN30 and LN30, Rdark decreased, leading to increased A:R ratio and leaf C:N. Although these results do not directly confirm or describe the functioning of the alternative pathway under N limitation, they add to an increasing body of evidence that it has an important role in in maintaining metabolic homeostasis under thermal stress and also in adjusting metabolism to growth under N limitation.

Tree carbon balance under warming growth conditions Photosynthesis (A) and respiration (R) are thermo-sensitive processes, regulating vegetation-atmosphere carbon exchange and directly influencing plant growth and performance. Acclimation to long-term moderate warm growth conditions typically increases photosynthetic thermal optimum (Topt) and can increase Anet at the new growth temperature compared to an unacclimated plant (Way and Yamori 2013, Yamori et al. 2014). However, many plants have reduced photosynthetic performance when grown at higher temperatures (Way and Yamori, 2014) and there is growing evidence that Anet at high leaf temperatures (>35 °C) is similar in acclimated and unacclimated plants in many species (Dusenge and Way 2017). The capacity for photosynthetic thermal acclimation to warming varies among species, and a recent meta-analysis indicated that evergreen trees had the least capacity for photosynthetic acclimation to warming of all measured plant functional types (Way and Yamori 2014). In contrast to low capacity for photosynthetic acclimation, conifers have been reported to possess a

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relatively high capacity for respiratory thermal acclimation compared to broad leaved species (Reich et al. 2016), so that thermal acclimation of R could reduce respiratory CO2 emissions by up to 80% in these species (Reich et al. 2016). Both photosynthesis and respiration can be affected by an increase in CO2 concentration. While a short-term exposure to elevated CO2 concentrations stimulates Anet by increasing the substrate availability for Rubisco (Ogren and Bowes 1971, Sage and Kubien 2007, Marshall and Linder 2013), growth at high CO2 often leads to a down-regulation of photosynthetic capacity (Curtis and Wang 1998, Ainsworth and Long 2005), although the strength of these two effects is not necessarily equal and trees grown at elevated CO2 tend to have higher Anet than ambient-grown trees in their respective growth CO2 concentrations (Medlyn et al. 1999, Ainsworth and Long 2005). Respiration, on the other hand, is not affected by short-term changes in CO2 (Amthor 2000) while longer-term exposure to a high CO2 environment either has no effect on Rdark or increases Rdark, with these two outcomes being about equally likely across the studies to date (Way et al. 2015). The relative acclimation capacity of Anet and Rdark the interactive effects of elevated temperature and CO2 is crucial to understand tree growth and performance under future climates and for more accurate predictions of NPP and carbon sequestration capacity on ecosystem level. In five North American boreal species, seedlings down-regulated photosynthesis at elevated CO2, thermally acclimated Rdark, and growth was most stimulated by high CO2 in the warmer treatments; species-specific responses to the treatments were largely explained by differences in leaf-level plasticity between broad-leaved seedlings and evergreen conifers (Tjoelker et al. 1998, 1998, Tjoelker et al. 1999). In Douglas fir seedlings (Psuedotsuga menziesii (Mirb.) Franco), elevated CO2 treatments also down-regulated photosynthesis, while increasing Topt, and warming increased Anet. A 50-day exposure to warmer temperatures increased growth in Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies L.) seedlings, a result that was enhanced at high CO2, but decreases in Rubisco, chlorophyll and soluble protein concentrations were also seen in warm-grown needles, which could reduce CO2 uptake (Sallas et al. 2003) on longer time scales. In a long-term experiment on mature Scots pine trees, elevated CO2 reduced Rdark and down-regulated photosynthesis but warming stimulated both photosynthetic capacity and Rdark and the combined treatments led to slight increases in Anet and decreases in Rdark (Wang et al. 1995, Kellomäki and Wang 1997). In contrast, in a similar experiment in Norway spruce there was little effect of either CO2 or temperature on tree carbon fluxes and growth, a result attributed to soil nutrient limitations (Kostiainen et al. 2004, Kostiainen et al. 2009, Sigurdsson et al. 2013, Wallin et al. 2013).

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Together with leaf carbon fluxes, changes in temperature and CO2 also alter plant growth. Evergreen tree species typically tend to show less enhancement of growth than deciduous species when exposed to warmer growth conditions and higher CO2 and there is considerable variation between species and studies in these patterns. For example, in black spruce, warmer growth temperatures reduced both seedling biomass and the root to shoot ratio by 58% (Way and Sage 2008). In Norway spruce, neither CO2, nor increased temperature had an effect on radial stem growth under natural (i.e. limited) N conditions, and a positive growth effect was only observed when N was added (Hättenschwiler et al. 1996, Kostiainen et al. 2009). In Scots pine both elevated CO2 and temperature enhanced stem radial growth, even on a low fertility site (Kilpeläinen et al. 2005). In both Norway spruce and Scots pine wood density increased with elevated CO2, whereas wood chemical composition was not significantly affected by CO2 or temperature (Kostiainen et al. 2004, Kilpeläinen et al. 2005). In Paper III, I evaluated the effects of future climate conditions on the performance of two dominant northern tree species, Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies L.), by exposing seedlings to ambient CO2 (400 ppm) (AC) or elevated CO2 (750 ppm) (EC) combined with ambient temperatures (T0), a +4 °C warming treatment (T4), or a +8 °C warming treatment (T8) for three growing seasons. Scots pine and Norway spruce are dominant species in the Eurasian temperate and boreal forest and are significant, high-value forestry species, widely cultivated in Europe and in North America. They have different niches in natural forests, pine is a pioneer species and spruce is a late successional species (Engelmark 1999). Nevertheless, they both inhabit the same biome and are grouped within the same plant functional type as evergreen conifers, and are often grouped together for global-scale modelling purposes that focus on changes in carbon fluxes. Therefore I hypothesised that in general they would respond similarly to the warming and CO2 conditions. Furthermore I hypothesised that photosynthesis would acclimate to warming and elevated CO2, by altering Aopt, Topt together with the light harvesting capacity, while Rdark would acclimate to warming, but would be unaffected by elevated CO2. Based on earlier findings about growth under elevated CO2 and temperature, I hypothesised, that moderate warming (+4°C) and high CO2 would enhance stem and canopy growth, given that in this experiment mineral nutrients were not limiting, however extreme warming (+8°C) would supress seedling growth due to potentially higher number of days with temperatures exceeding Topt during the growing season. Furthermore, that tracheid diameter would increase in response to both elevated CO2 and temperature, in order to accommodate increased water supply to a larger canopy.

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In Paper III I show that photosynthesis in pine was remarkably stable across all the treatment conditions, and Topt showed overall a small increase, while in spruce Aopt was reduced by ~35% by warming and Topt did not change. Interestingly Topt under control conditions was very similar in both species, around ~28°C, suggesting that Topt alone is not a direct indicator of the thermal sensitivity of photosynthesis. In spruce warming stimulated Y(II) and ETR (electron transport rate) while Y(NPQ) was significantly reduced. This suggests, that although light energy was sufficiently used in downstream processes, it was utilised in processes other than CO2 assimilation, possibly including photorespiration. This decoupling between light harvesting and C assimilation can lead to light-induced oxidative stress under warm conditions in spruce, already under T4. Similar to photosynthesis, Rdark also responded in a species-specific manner to the treatment conditions. Rdark, measured at 25°C was lower under T4 and T8 conditions in pine, compared to T0, resulting in a clear thermal acclimation pattern across the treatment temperature range. Parameters a, b and c, describing the intercept, the slope and the curvature of the temperature response curve of lnRdark did not show a significant response pattern, although a showed a weak negative correlation to increasing growth temperature. In spruce Rdark measured at 25°C was similar under all treatments, despite that parameters b and c responded significantly to warming, in that b declined and c increased, except under ACT4. Similar to pine, parameter a was lower under T4 compared to T0, but higher under T8. These subtle changes of a were not significant in our relatively low sample size, but considering the correlations with the responses of growth and photosynthetic parameters, it can be suggestive of a negative heat stress response in the respiratory processes due to extreme warming in spruce. Species-specific thermal acclimation capacity has been reported in other studies, for instance Tjoelker et al. (1999) noted that a 12 °C warming reduced shoot Rdark assessed at a common temperature by 55% in Picea mariana, but only 36% in Pinus banksiana. Despite the close relatedness to our experimental species, we observed the opposite, higher acclimation in Pinus compared to Picea. This might indicate that even family-level relatedness is not necessarily a good predictor of response to thermal conditions. Taken together the observation of A and R, pine displayed an increasing Anet:Rdark ratio over the range of the measurement temperatures, suggesting an overall more positive carbon balance compared to spruce. This is in accordance with its relatively high growth rate as a pioneer species, in contrast to spruce, which is a late succession species. Needle anatomy traits were for the most part unresponsive to the treatment conditions, suggesting that the observed acclimation in Rdark and Anet are underpinned by biochemical changes rather than changes in needle internal structure. This

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finding is in accordance with previous observations in field-grown Norway spruce (Hall et al. 2009). Some studies have suggested that elevated CO2 can ameliorate the negative effects of rising temperature (Drake and Leadley 1991, Long 1991). In the experiment described in Paper III, the effect of warming was not consistent across the two CO2 treatments, and a warming of +8 °C tended to suppress growth at EC more than at AC. Especially in spruce there were a number of non-linear responses to warming at EC: height growth, aboveground biomass, needle investment and the Topt of net photosynthesis all increased under a +4 °C warming but decreased again at +8 °C warming. One of the most unexpected findings was that the number of buds set was consistently reduced by warmer temperatures at EC in spruce, but increased at AC. In summary, T8 did not always supress growth and EC did not always support growth, suggesting that above a certain thermal threshold, heat is most detrimental when in combination with elevated CO2. In spruce the thermal response of stomatal conductance (gs), measured in dark was stable up to about 50°C and a VPD of 8 kPA. In pine, gs was stable until 35-40°C, when it sharply increased, offsetting the VPD in the cuvette, which then remained stable above 40°C. At this point I do not have a clear explanation to the mechanisms underlying this observation, but it is possible that the more dynamic temperature response of gs in pine is part of the explanation to why it is coping better with the high growth temperatures. The high gs at these extreme temperatures can promote latent heat loss and prevent heat damage in the needles (Teskey et al. 2014). At the same time it also suggests that this species can potentially become sensitive to draught under increasing future temperatures. At ECT8, CO2 induced reduction in gs might be causing the temperature increase of the needles inspruce to a damaging level and the lack of a dynamic temperature response of gs means that the needles will not be sufficiently cooled. This is a phenomenon that has been observed under the combined effect of heat and drought, in that drought induced stomatal closure, while under heat stress stomatal opening would be more beneficial and due to these contrasting preferences the combination of the two factors is a high risk for leaf damage (Mittler 2005). My observations in spruce suggest that under heat stress, the CO2 induced stomatal closure can result in a similarly damaging situation as drought in some species. In summary, the most significant finding in this experiment was the striking difference between pine and spruce in their response to temperature and CO2. Pine responded more positively to warming with increased height growth, annual ring width and lumen diameter. Overall these effects were not observed in spruce, although warming induced allocation to leaves and reduced allocation to stem was consistent in both species and in agreement

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with findings in previous studies (Way and Oren 2010). In contrast to pine, higher temperatures strongly negatively impacted the photosynthetic functioning of spruce. These results add to a growing body of literature indicating that photosynthetic processes in spruce species appear to have a limited ability to cope with warming (Picea mariana, (Way and Sage 2008, Way and Sage 2008); Picea likiangensis, Picea koraiensis and Picea meyeri,(Zhang et al. 2015); Picea abies, (Kroner and Way 2016), a limitation that has been linked to declines in growth at high temperatures (Way and Sage 2008, Way and Oren 2010). The results presented here also support the previous findings in field studies that found positive responses to warming and elevated CO2 in Scots pine (Wang et al. 1995, Kellomäki and Wang 1997, Peltola et al. 2002) but not in Norway spruce (Slaney et al. 2007, Sigurdsson et al. 2013, Wallin et al. 2013). Forest community composition under climate change will result from the physiological responses of component species, which are a product of their intraspecific variation (Reyer et al. 2015). Due to its thermal sensitivity, Norway spruce will presumably migrate towards cooler habitats, i.e. higher altitudes and latitudes, possibly altering the current species dynamics in the boreal ecosystem. Such migration events are not unprecedented in global history. However the human induced rate of warming of the earth surface is unprecedented and it is not clear if migration of species, especially in case of a slow growing late succession species like spruce, can keep up with the rate of warming. In a worst case scenario spruce could be lost from large portions of its current habitat.

The transcriptional underpinning of thermal sensitivity in Norway spruce In Paper III I identified Norway spruce as a thermal-sensitive species, with low acclimation capacity relative to Scots pine. Growth and carbon fluxes were negatively impacted by warming, Aopt decreased parallel with decreased use of electron transport for CO2 fixation and weak thermal acclimation of Rdark. Furthermore the responses of spruce to elevated CO2 were not consistent across all temperatures, while it had a positive effect on height under T0 and T4, under T8 CO2 seemed to exacerbate the negative effects of temperature, rather than ameliorating them. The aim in Paper IV was to elucidate the processes underpinning the thermal sensitivity observed on physiological level. I contrasted the responses to warming and CO2 of source tissues (needles) and sink tissues in the stem (xylem and phloem) on transcriptional level. This study was conducted as an ecological transcriptomics experiment with the aim to describe general processes in

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response to the experimental conditions and with the goal to connect these processes to the phenotypic observations. Needle, xylem and phloem tissues were collected in August 2014, and the total mRNA was extracted. The RNA was sequenced using paired-end high-throughput Illumina sequencing. Raw reads pre-processing and the alignment to a reference Picea abies genome (version 1.0) was performed by means of a pipeline previously described (Delhomme 2014) and implemented in the bioinformatics platform at Umeå Plant Science Centre (https://bioinformatics.upsc.se/). Subsequent quality control of the obtained count data and the differential gene expression analysis was performed using broadly accepted and utilised RNAseq analytical packages, available through the R interface (see details in Paper IV). Firstly, a model-testing approach was used to partition the variation attributed to CO2, temperature and CO2 x temperature interaction. Model reduction was used to examine the variation attributed to the independent variables and select the most suitable model. A model was selected where CO2 was used as a covariate, with temperature as the independent variable, because there were only few DE genes responding to CO2 (Paper IV, Table 3). The P. abies genome contains ~80,000 gene models, of which 13,314 are annotated (Nystedt et al. 2013). Differentially expressed genes (significant at P < 0.05) were tested for enrichment of Gene Ontology (GO) terms in the experimental background of annotated genes in each tissue, for up- and down- regulated genes separately. GO terms for the genome annotation were obtained from ConGenIE (Sundell et al. 2015) and tested for enrichment using the GOstats package (Falcon and Gentleman 2007) in R, using a significance threshold of P < 0.05. Redundancy of over-represented GO terms was reduced using the REViGO algorithm (Supek et al. 2011), based on the GO and adjusted P-values. Furthermore, the KAAS web server was used to assign KEGG (Kyoto Encyclopedia of Genes and Genomes) (Kanehisa et al. 2016) annotations to amino acid coding sequences and then used to perform molecular function enrichment tests (Moriya et al. 2007). The first important finding was that while the differential expression (DE) analysis showed clear remodelling of the transcriptome in response to warming in all three tissues, there was only negligible effect of CO2 (Paper IV, Table 3). Previous studies with focus mostly on leaf tissue in annual plants and broad-leaved trees grown under elevated CO2 have identified DE genes regulating sugar metabolism, cell wall loosening and cell expansion related to organ size in response to elevated CO2 (Cheng et al. 1998, Ainsworth and Long 2005, Druart et al. 2006, Leakey et al. 2009, Fukayama et al. 2011), and down-regulation of genes related to photosynthesis (Nie et al. 1995, Cheng et al. 1998, Kontunen-Soppela et al. 2010, Fukayama et al.

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2011). One study on Cottonwood (Populus deltoides) identified differentially up-regulated genes connected to lignin modification in the stems in response to high CO2 (Druart et al. 2006). However, in spruce our transcriptomic analyses identified only 3 DE genes responding to CO2, which indicates a clear insensitivity of spruce to high CO2 on a transcriptomic level. This is in accordance with the observations in the physiological assessment of spruce (Paper III), where we did not find any significant response to elevated CO2, except in height growth. The lack of the ameliorating effect of CO2 might be rooted in the complete lack of transcriptional response to CO2 in spruce. The warming sequentially induced the expression of genes with the highest number of DE genes found in the T8 treatment. The separation between the T0, T4 and T8 conditions was clearly visible in the 3rd component of the principle component analysis (Paper IV, Figure 1b). The proportion of warming induced DE genes was highest in the needles and lowest in the xylem, and within individual tissues the number of temperature-induced up- and down-regulated DE genes were approximately equal. Despite source (needles) and sink (phloem and xylem) tissues, with separate biological functions and requiring the expression of different genes, there was a significant number of common DEGs (Paper IV, Figure 5). The most striking response of up-regulated DEGs common to the three tissues was the enrichment of processes related to HSPs, molecular chaperones and protein folding and ubiquitination, identified in the GO enrichment analysis, as well as verified by the enrichment of the KEGG identities. The finding of major classes of HSPs, such as HSP70, HSP90, chaperone and DNJ domains in all tissues, indicate a universal heat response, rather than an organ specific expression. A previous study on boreal species of Picea and Pinus also reported increased levels of HSPs in warming treatments (Gifford and Taleisnik 1994). Furthermore, increased metabolism of small HSPs was reported among northern latitude tree species under 5 °C warming (Vahala et al. 1990, Jia et al. 2016), while higher molecular weight HSPs have been quantified in acute heat treatments in Picea spp. (Colombo et al. 1995, Valcu et al. 2008). Further to increased HSP activity, other important responses at T+8 were associated with oxidative stress, ROS and hydrogen peroxide that are symptomatic of stressed tissues (Larkindale et al. 2005) but also putatively involved in stress signalling (Suzuki et al. 2011). This correlates with the observed increase in the ETR:Jopt ratio that is suggestive of the decoupling between electron transport through light harvesting and electron utilisation for CO2 assimilation and implies the role of alternative electron sinks, such as photorespiration. Among the several components of the plant heat stress responses are changes in the composition and signalling of hormones (Larkindale et al.

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2005). Ethylene regulation genes were up-regulated in phloem samples at T+8 and there were significant responses to ABA in phloem at T+8 and in needles under both warming treatments. Brassinosteroid modifications have been reported in conifers undergoing warming (Escandón et al. 2016), and accordingly GO terms associated with detection or response to brassinosteroids were significantly enriched in all tissues at T+8. Enrichment of GO terms associated with lipid and steroid were detected universally at T+8, consistent with well-documented alterations in lipid metabolism caused by long-term heat stress (Qin et al. 2008, Zhang et al. 2013, Szymanski et al. 2014) and suggesting that stress in these seedlings may impact on membrane composition, permeability and stability. At the gene expression level, heat has been shown to affect transcripts encoding trehalose metabolism (Qin et al. 2008). I found GO terms related to trehalose metabolism were enriched in needles at both elevated temperature in agreement with Delorge et al. (Delorge et al. 2014). Furthermore, I note the up-regulation of sucrose-phosphate synthase, an enzyme in the molecular temperature stress response (Suzuki et al. 2011). Terpenoids and anthocyanins have an antioxidant capacity (Rodziewicz et al. 2014) and can increase in concentration under warming in P. abies (Sallas et al. 2003, Virjamo et al. 2014). In needles at T+4 and in phloem at T+8 there was significant enrichment of proanthocyanidin biosynthetic process. Warming stimulates the metabolism of terpenes in P. abies (Esposito et al. 2016) and as such the enrichment of terpene biosynthetic processes in needles at T+8 may reflect this. In the needle transcriptome at T+4, evidence of stress responses, heat acclimation and oxidative stress, suggest that warming by 4°C brings this species close to its thermal maximum. Together with altered tetrapyrrole metabolism, the down-regulation of genes for photosynthesis suggests the sensitivity of needle metabolism to the warming treatments. Analysis of the significantly intersecting genes involved in these processes at both temperatures confirmed that similar functions were enriched in the needles under both temperatures. GO enrichments shared by samples at T+4 and T+8 included small chaperone proteins and HSP90 proteins, GOs associated with photosynthetic processes, thylakoid lumen proteins and haem oxygenase (decyclizing) family proteins. However, the greater abundance of transcripts in the T+8 samples suggests a progressively increasing impact of temperature across the warming gradient. These results correlate with the suppressed Aopt and the lack of Topt acclimation discussed in Paper III. In all tissues, there was evidence from GO for the modification of growth and development by temperature, supported by the observed increase in biomass (Paper III, Figure 1) and a significant effect of e[CO2] on ring width,

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which was greatest under ECT4. However, as with needle and stem biomass, these effects were completely overridden at ECT8 where radial increment was the lowest among all treatment conditions. Fibre radial diameter was greatest in the samples at ECT8, suggesting an increased water transport capacity in these stems, which could ameliorate the effect of thermal stress. Indeed, in xylem at T+8 we observed GO enrichment of terms related to water and fluid transport. However, increased fibre diameter at high temperatures can increase the vulnerability to cavitation. These results, in conjunction with the results in Paper III, all point to the conclusion that in spruce, e[CO2] does not mitigate the effect of high temperature, but rather enhances it under ECT8. In summary, this transcriptomic analysis has revealed strong heat stress related response in both needle and stem tissues that was increasingly pronounced with increasing temperature. The high number of heat stress induced DE genes already under T4 in the needles was unexpected, since based on the physiological result, T4 enhanced overall growth, especially growth allocation to the canopy, with no apparent sign of oxidative stress in the fluorescence parameters. Nevertheless, the suppression of carbon assimilation at T4 is in clear accordance with the down-regulated photosynthetic processes observed in the GO enrichment analysis. The successive stress response was clear in all tissues under T8, underlying the stress related negative shifts in the carbon balance, and reduced proportion of electrons utilised in carbon assimilation. The results presented here are among the first studies of a conifer transcriptome under e[CO2] and warming treatments. The goal of this analysis was to perform an eco-physiology oriented transcriptomics analyses, and reveal the regulatory-level responses of spruce seedling to future climate conditions. The results revealed the differential regulation of some key processes in response to warming, like photosynthesis, that were in accordance with the findings based on physiological analysis. At the same time it also allowed for a deeper insight into the regulatory processes and revealed a substantial up-regulation of heat stress induced protective processes, the magnitude of which we would not necessarily deduce from the physiology. This can be an indication that some of the commonly accepted tools to detect plant stress, like for instance chlorophyll fluorescence, might not be indicative of subtle, latent states of constant stress, which in the long-term can put a strain on plant performance. Therefore I found that transcriptomic analysis was a useful tool to increase our knowledge on the response of spruce to warming. Furthermore these findings also indicated that it is possible that even pine, which was found to do much better than spruce under warming temperatures based on the physiological assessment,

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might be under latent heat stress, just not strong enough to manifest itself within a relatively short-term experiment.

The significance of the C-N balance under constrains of growth temperature The studies presented in this thesis explored different aspects of thermal acclimation; the significance of nitrogen availability for cold and warm acclimation and the interactive effects of elevated CO2 and elevated temperatures. Both studies in Paper III (and IV) and Paper V were conducted under controlled conditions, which made it possible to eliminate confounding environmental effects, such as precipitation, heat waves or cold spells and effects of soil nutrition (in case of Paper III and IV highlighting the effect of specific treatment conditions in a way that would be impossible under field conditions, and so these findings can serve as a basis for future filed studies. Furthermore the studies were conducted on young trees, or seedlings, and evaluating the treatments effects on young, developing trees can have implications on regeneration of forests under these combined stress scenarios. Small trees are also more vulnerable to C/N imbalances due to a smaller sink capacity in the stems relative to canopy size so that they tend to be more sensitive to environmental impacts and show stronger responses, while the same responses might be overlooked in mature trees due to overall higher buffer capacity of the source-sink system. In the described experiments the effects of nitrogen availability and elevated CO2 were investigated separately, however the C-N cycle in plants is tightly interconnected (Nunes-Nesi et al. 2010) and therefore one should not be discussed without considering the other. The strong relation between C and N has been described in earlier studies with high CO2. For example high CO2 induced photosynthetic down-regulation was found to be stronger when nutrient availability was low, and in the nitrogen-poor soils of the northern temperate and boreal forest, elevated CO2 did not always stimulate Anet (Marshall & Linder, 2013; Sigurdsson et al. 2013). Furthermore in their experiment on mature Norway spruce trees, Kostiainen et al. (Kostiainen et al. 2004) found no effect of elevated CO2 on stem growth, which they attributed to the low soil fertility at the site. Based on these findings, plants response to future conditions of elevated CO2 and elevated growth temperature has to be evaluated in the context of plant mineral nutritional status and the acclimation to thermal stress under nitrogen limitation has to be considered under elevated CO2 when discussing plant productivity in the future. In a review paper, Stitt and Krapp (Stitt and Krapp 1999) emphasise two reasons for the tight connection between carbon and nitrogen metabolism: First that sucrose utilisation for growth requires appropriate

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amounts of amino acids, and second that nitrate assimilation requires suitable amounts of reducing equivalents and ATP. They concluded that excess amounts of carbohydrates lead to metabolic imbalance, if not accompanied with increased nitrogen assimilation. In the following I will describe some considerations connecting the observations in the two experiments described in Papers III - V. I described the slow growing Betula utilis to be more resilient to stressful thermal conditions, especially heat, compared to the fast growing Betula pendula. I attributed this difference to the conservative growth strategy in BU, that ensured a more balanced C:N homeostasis across all treatments on whole plant level as well as in the leaves (Figure 4d). On the other hand BP followed a growth maximising strategy, i.e. high Anet even under low N conditions, which resulted in substantially increased leaf C:N ratio (Figure 4c) which was enhanced under sub-optimal growth temperatures due to suppressed growth and limited stem C sink capacity (Paper V Figure 9). Elevated CO2 has been described to increase leaf C:N ratio (Ainsworth and Long 2005, Leakey et al. 2009), therefore high CO2 might further increase the C:N ratio observed in BP when low N supply is combined with high CO2, especially under stressful thermal conditions of cold or warm. In BU leaves C:N balance was less responsive especially under moderate and warm temperatures. However it is not clear whether elevated [CO2] would push this metabolic balance to its limits, in which case, young seedlings with low C sink capacity might be especially vulnerable. Picea abies was sensitive to warming growth conditions and was especially negatively affected by the combination of elevated [CO2] and warm growth temperature, i.e. ECT8, and it is easily recognisable how low N conditions of natural soils would enhance this negative combined CO2-temperature effect, especially if the warming of 6°C to 8°C, predicted to high latitude regions, will be realised in the future. Pinus sylvestris on the other hand coped better with warming and the interactive effects of warming and elevated [CO2]. Nevertheless, comparing the response of Anet:Rdark (Figure 4e) ratio to the response in BP and BU, a similar pattern is recognisable, in that Anet:Rdark ratio increases with increasing temperature. While this positive carbon balance was beneficial for pine under high CO2, it was not as beneficial for BP under low N. It is of course not possible to compare conifers and broad-leaved species directly in this way, but these observed similarities raise the concern that pine might also become progressively more sensitive and less productive when growing under the three-fold constrains of low N, e[CO2] and warming climate. In summary, the results presented in this thesis support the formulation of the hypothesis that warming, combined with shifts in the environmental C:N balance can have detrimental effects on

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plants, and that positive, growth enhancing effects of warming and e[CO2] are limited, especially in regions of limited soil fertility.

Figure 4 Anet:Rdark and C:N ratio of BP (a, c), BU (b, d), PS (e, g) and PA (f, h) under their respective treatment temperatures. Bars represent means and standard errors, white bars represent optimal N conditions (a-d) or ambient [CO2] (e-h), grey bars represent limited N conditions (a-d) and black bars represent elevated [CO2]. The annotations represent the Tukey HSD pair-wise comparisons, where p ≤ 0.05 was considered a significant difference.

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Conclusions and future outlook There are wide ranging efforts to improve the predictive power of earth surface models by increasing the precision of the representation of the main carbon fluxes connected to vegetation, photosynthesis and respiration, because of their significance in the global carbon cycle, representing a flux of ~120 GT CO2 y-1. In this thesis I presented a generalised empirically based mathematical model (GPM) that can describe the R-T response of plant functional types (PFTs) or biomes with high precision and demonstrated that it can be easily implemented in global climate models, such as JULES, leading to the observation that the current practice of representing R-T with a fixed Q10 of 2 underestimates the NPP in cold climates and during winter month in evergreen woodland sites. Furthermore on a small dataset I demonstrated that this model also has the potential to incorporate the thermal acclimation of respiration, further increasing the precision of models estimating carbon fluxes under future climates. In order to precisely parameterise the GPM for this, a large data set with preferably high number of species would be necessary, which will probably be available in the future from current large-scale warming experiments, that aim at evaluating thermal responses, often combined with e[CO2] in naturally growing forest stands. I described the results of two experiments, both connected to thermal acclimation and one combined with low nitrogen, while the other was combined with elevated CO2. These results highlighted the importance of studies combining various abiotic factors under the controlled conditions of growth facilities, because in this setting both allowed for valuable insights into the combined effects of CO2 + temperature or temperature + N limitation without further confounding environmental factors. Both studies supported the previous assumption that the combination of these abiotic factors create stronger constraints on plant growth and performance than when they are evaluated separately. Furthermore the results emphasised that different species, despite being grouped into the same PFT or inhabiting the same biome, can have different sensitivity thresholds to temperature and to shifts in the C/N balance of their environment, and these differences could, to some extent, be explained by their differential growth strategies. The vulnerability of dominant species, especially in ecosystems with low tree species diversity such as the boreal forest, can have profound effects on the stability and composition of the ecosystem as a whole. I aimed to summarise the possible combined effects of elevated CO2, limited nitrogen availability and thermal stress in this thesis and also emphasise the

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importance of confounding biotic components, such as mycorrhizal associations. However in order to get a more complete picture of the abiotic constrains on forest production under current and future climates there are still many factors that need to be considered. I would like to highlight two factors that are, in my opinion, of high importance towards a more precise picture of plant-environment relations: water and phosphorous availability. The antagonistic responses to drought and heat, and the mechanisms in plants to cope with these effects has been the focus of many studies, especially concerning significant crop plants (for a review see: Mittler, 2005). In connection to N, it has been described that low N decreases water use efficiency (WUE) in plants, whereas elevated CO2 was found to increase WUE (Leakey et al. 2009). However, it is not well established how the combination of heat, drought, e[CO2] and low N might affect plant growth and performance. Phosphorous (P) is the second and slightly more neglected major macronutrient, after N, that can significantly limit plant growth. For example in densely populated temperate regions, where N limitation is decreasing due to anthropogenic N deposition, P limitation becomes progressively more significant (Peñuelas et al. 2013). Therefore in ecosystems in those regions, it is of high importance to initiate studies evaluating the combined effect of phosphorous-carbon-temperature-water relations. The findings summarised in this thesis emphasise the need for further studies that aim at a holistic evaluation of plant-environment interactions and examine plant physiology under combinations of multiple environmental factors.

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