Dissertations in Forestry and Natural Sciences
DISSERTATIONS | UNNIKRISHNAN SIVADASAN | CLIMATE CHANGE EFFECTS ON GROWTH A
PUBLICATIONS OF
THE UNIVERSITY OF EASTERN FINLAND
Phenological events of plant buds are highly sensitive to environmental fluctuations, and are useful for climate change investigations.
Climate change may also cause variation in the secondary metabolites concentration in plants.
This thesis provides new information regarding the phenology, growth and secondary meta- bolites of Populus tremula and Salix myrsini-
folia under predicted climatic conditions and it increases our knowledge about the possible
impacts of climate change on boreal forests.
UNNIKRISHNAN SIVADASAN
CLIMATE CHANGE EFFECTS ON GROWTH AND METABOLISM OF THE DIOECIOUS SALIX MYRSINIFOLIA
AND POPULUS TREMULA
Unnikrishnan Sivadasan
CLIMATE CHANGE EFFECTS ON GROWTH AND METABOLISM OF THE DIOECIOUS SALIX MYRSINIFOLIA AND POPULUS
TREMULA
Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences
No 303
University of Eastern Finland Joensuu
2018
Academic Dissertation
Grano Oy
Author’s address: Unnikrishnan Sivadasan University of Eastern Finland
Department of Environmental and Biological Sciences P.O. Box 111
80101 JOENSUU, FINLAND email: unnikrs@uef.fi
Supervisors: Professor Riitta Julkunen-Tiitto, Ph.D.
University of Eastern Finland
Department of Environmental and Biological Sciences P.O. Box 111
80101 JOENSUU, FINLAND email: riitta.julkunen-tiitto@uef.fi
Professor Line Nybakken, Ph.D.
Norwegian University of Life Sciences Faculty of Environmental Sciences and Natural Resource Management P.O. Box 5003
1432 ÅS, NORWAY
email: line.nybakken@nmbu.no
Tendry Randriamanana, Ph.D.
University of Eastern Finland
Department of Environmental and Biological Sciences P.O. Box 111
80101 JOENSUU, FINLAND email: tendry.randriamanana@uef.fi
Associate Professor Lauri Mehtätalo, Ph.D.
University of Eastern Finland School of Computing P.O. Box 111
80101 JOENSUU, FINLAND email: lauri.mehtatalo@uef.fi
Reviewers: Docent Annamari Markkola, Ph.D.
University of Oulu
Department of Ecology and Genetics P.O. Box 3000
email: maarit.karonen@utu.fi Opponent: Docent Anna Maria Pirttilä, Ph.D.
University of Oulu
Department of Ecology and Genetics P.O. Box 3000
90014 UNIVERSITY OF OULU, FINLAND email: am.pirttila@oulu.fi
Sivadasan, Unnikrishnan
Climate change effects on growth and metabolism of the dioecious Salix myrsinifolia and Populus tremula.
Joensuu: University of Eastern Finland, 2018 Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences 2018; 303 ISBN: 978-952-61-2733-0 (print)
ISSNL: 1798-5668 ISSN: 1798-5668
ISBN: 978-952-61-2734-7 (PDF) ISSN: 1798-5676 (PDF)
ABSTRACT
In this thesis, I studied how two Salicaceae species; Populus tremula and Salix myrsinifolia responded to predicted changes in climatic factors. The plant characteristics taken into account were bud phenology (bud break and bud set), growth, secondary metabolites (S. myrsinifolia buds and P.
tremula stems) and leaf herbivory (P. tremula). The climatic factors applied were both single and combined treatments of elevated temperature and UV radiation. P. tremula and S. myrsinifolia are closely related species, but they differ in growth forms; the former is a tree and the latter a shrub.
All the three studies were conducted in an open experimental field, where the levels of temperature and UV-B radiation were elevated by 2°C and 30%, respectively. The P. tremula bud phenology was scored for three years, including the year after the treatments had been discontinued, in order to study the acclimation responses. The influence of resource restriction on bud break and bud set was investigated by studying plants from which axillary buds had been removed.
Elevated temperature forced the spring bud break and delayed the autumn bud set in P. tremula.
In the first year, UV-B forced both bud set and bud break. These persistent phenological responses to UV-B declined over the years in P. tremula, showing an acclimation pattern. In bud-removed individuals, bud break was delayed under elevated temperature and under UV-B radiation.
Although elevated temperature alone delayed bud set in bud-removed individuals, it forced the bud set when combined with elevated UV radiation. P. tremula males were more responsive to the treatments than females. Growth of P. tremula individuals increased under elevated temperature, while the height growth decreased under combined UV-B and temperature treatment.
Salicylates were the most abundant secondary compounds in P. tremula stems, and their concentration declined under elevated temperature. Leaf herbivory increased under the elevated temperature treatment. Salicylates and condensed tannins were the major phenolic compounds in the buds of S. myrsinifolia. The temperature effect on the phenolic compounds in S. myrsinifolia buds was minor, while UV-B increased the amount of flavonoid hyperin. In both P. tremula and S.
myrsinifolia bud sizes were larger under temperature treatments. Male bud size was the largest for P. tremula, while female buds were the largest for S. myrsinifolia.
In conclusion, elevated temperature plays an important role in promoting growth, but can actively alter the amounts of some phenolic compounds. Elevated temperature also influenced the length of the active growing season by modifying bud break and bud set. Combined stresses may,
CAB Thesaurus: climate change; Populus tremula; Salicaceae; Salix nigricans; growth; metabolism; buds;
phenology; secondary metabolites; salicylates; tannins; phenolic compounds; flavonoids; herbivory;
air temperature; ultraviolet radiation; acclimatization
Yleinen suomalainen asiasanasto: ilmastonmuutokset; haapa; mustuvapaju; kasvu; aineenvaihdunta;
silmut; fenologia; aineenvaihduntatuotteet; lämpötila; ultraviolettisäteily; akklimatisaatio; salisylaatit;
tanniinit; fenoliset yhdisteet; flavonoidit
ACKNOWLEDGEMENTS
My four-year long journey is at its completion, and without the help of all these great people, it would have not been possible.
I must start with my supervisor, Professor Riitta Julkunen-Tiitto, to whom I owe my deepest gratitude for her continuous support of my work on my doctoral thesis. Her guidance, encouragement, and sharing her immense knowledge has made it possible to complete the thesis.
Working with her has motivated me daily, always to work harder and better.
I am also deeply grateful to my other supervisors, Line Nybakken, Tendry Randriamanana and Lauri Mehtätalo for their insightful comments, valuable guidance and support for completing this thesis.
It was always a pleasure coming to work with such lovely and engaging people. I am indebted to Norul, Katri, Virpi, Paula, Anu and Anneli for their collaboration and extraordinary support in this thesis process. I am also thankful to Chenhao and Apu for their support and assistance in the lab and in the field during their master theses.
I also wish to thank all the staff of the Environmental and Biological Sciences Department for the great working atmosphere. It gives me a special pleasure to acknowledge Sinikka Sorsa, Hannele Hakulinen, Mervi Kupari, Matti Savinainen and Kari Raitilainen for their generous help in the lab and in the experimental field. I also thank Rosemary Mackenzie for editing the English of this thesis.
I would like to express my appreciation to spearhead projects of the University of Eastern Finland and the Academy of Finland for their financial support and the facilities provided for the fulfillment of the thesis.
I would like to offer my special thanks to Docent Annamari Markkola and Docent Maarit Karonen for their reviews and Docent Anna Maria Pirttilä for accepting her role as an opponent.
I thank my homeland Kerala for its physical, spiritual and social nourishment. Therefore, the deepest gratitude and love goes to all my family: my parents Suseela and Sivadasan, my brothers Jayan and Hari, my in-laws Prasi and Vineetha. Also, my family in Finland, father-in-law Kalle, mother-in-law Terttu, my in-laws Jaakko, Olli, Mikko, Aino, Lauri, Hannu, Linda and Henna - thank you for following the progress of this thesis and for all your encouragement and support in my life. I also want to thank the little ones Väinö, Innu, Austin, Lila, Nikhilan and Ryan for sharing their playtime with me.
Sincerest thanks go to my dear friends Unmesh, Vivy, Anish & Deepa, Senthil & Gomathy, Parvathy & Jari, Maya & Deepak, Renjith & Remya, Bhabishya, Yaodan, Toivo, Iqbal, Nazmul, and Afrin as well everyone else who has touched my life and supported me until reaching the finishing line.
Most importantly I thank my loving and supportive wife Maija, for her unending inspiration and encouragement throughout my doctoral studies. I owe you everything.
Joensuu, 2nd March 2018 Unnikrishnan Sivadasan
There is a pleasure in the pathless woods, There is a rapture on the lonely shore,
There is society where none intrudes, By the deep Sea, and music in its roar:
I love not Man the less, but Nature more.
George Gordon Byron
LIST OF ABBREVATIONS
ABA Abscisic acid
ATP Adenosine triphosphate clmm Cumulative link mixed model CNB Carbon nutrient balance GDB Growth-differentiation balance HCH 6-hydroxy-2-cyclohexen-on-oyl
HPLC High-performance liquid chromatography IPCC Intergovernmental Panel on Climate Change lme Linear mixed-effects model
PCM Protein competition model
R Programming language
T Temperature
UV Ultraviolet Radiation UV-A Ultraviolet radiation-A UV-B Ultraviolet radiation-B UVR8 UV resistance locus 8
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the data presented in the following articles, referred to by the Roman Numerals I, II and III.
I Sivadasan, U., Randriamanana, T. R., Julkunen-Tiitto, R., & Nybakken, L. (2015). The vegetative buds of Salix myrsinifolia are responsive to elevated UV-B and temperature.
Plant Physiology and Biochemistry, 93: 66–73. doi: 10.1016/j.plaphy.2015.02.017.
II Sivadasan, U., Randriamanana, T., Chenhao, C., Virjamo, V., Nybakken, L., & Julkunen- Tiitto, R. (2017). Effect of climate change on bud phenology of young aspen plants (Populus tremula. L). Ecology and Evolution, 7: 7998–8007. doi:10.1002/ece3.3352.
III Sivadasan, U., Chenhao, C., Nissinen, K, Randriamanana, T., Nybakken, L., & Julkunen- Tiitto, R. Growth and defence of aspen (Populus tremula L.) after three seasons under elevated temperature and UV-B. Canadian Journal of Forest Research (accepted).
The above publications have been included at the end of this thesis with their copyright holders’ permission.
AUTHOR’S CONTRIBUTION
I) The author conducted the processing of the chemistry and measurement data, statistical analyses and wrote paper I.
II) The author planned the experiment with the main supervisor and was also responsible for the phenology scoring measurements and data processing, analyses and writing paper II.
III) The author was responsible for growth, photosynthesis, biomass measurements, phenolic compound analysis, statistical analyses, and writing of paper III.
CONTENTS
ABSTRACT ... 7
ACKNOWLEDGEMENTS ... 9
1 INTRODUCTION ... 17
1.1 Climate change ... 17
1.2 Salicaceae species ... 17
1.3 Seasonal phenology and climate change ... 18
1.4 Growth and allocation in relation to phenology under climate change... 19
1.5 Defence in relation to phenology under climate change ... 19
1.6 Objectives of the thesis ... 22
2 MATERIALS AND METHODS ... 23
2.1 Overview of experiments ... 23
2.2 Plant materials ... 24
2.3 Treatments and set-up of the experiments ... 24
2.4 Removal of vegetative axillary buds... 25
2.5 Measured variables ... 25
2.5.1 Measurement of growth, photosynthesis and leaf damage ... 26
2.5.2 Scoring the bud break and bud set stages ... 26
2.5.3 Analysis of phenolic compounds ... 26
2.5.4 Statistical analysis ... 27
3 RESULTS AND DISCUSSION ... 29
3.1 Spring phenology and seasonal growth of P. tremula under climate change ... 29
3.2 Autumn phenology of P. tremula under elevated temperature and UV radiation ... 30
3.3 Secondary metabolites and development of vegetative buds of S. myrsinifolia and P. tremula ... 30
3.4 Defensive chemistry of P. tremula under elevated temperature and UV radiation ... 31
3.5 Evolutionary implications for Populus and Salix ... 32
4 MAIN FINDINGS AND CONCLUDING REMARKS ... 33
5 BIBLIOGRAPHY ... 35
1 INTRODUCTION
1.1 CLIMATE CHANGE
The observed evidence shows that the effects of climate change on natural systems are both intensive and extensive (IPCC 2014). Multifaceted climate change involves changes in precipitation, glacier and ice melting, sea level rise, ecosystem imbalance resulting in heat waves, droughts, hurricanes, floods and air pollution. Fluctuations in seasonal activities, geographical ranges, migration patterns, species interaction, and the abundance of both animal and plant life have already been recorded (Menzel 2006, Parmesan 2006). Numerous climatic factors, such as ozone, temperature, UV-B, carbon dioxide and methane influence the change in climate (Manning
& Tiedemann 1995). Most of the climatic changes are results of increasing carbon dioxide and temperature. Anthropogenic greenhouse gas emissions continue and may increase the global mean temperature from 0.3 to 4.8°C by the end of 21st century (IPCC 2014).
Elevated temperature affects growth, development, physiological processes and reproduction, as well as the phenophases of woody plants. An increase in photosynthesis and growing season extension can lead to increased plant growth, especially at middle to high latitudes (Norby & Luo 2004). In cold environments, the temperature stimulates growth until the thermal optimum is reached, while in warmer environments exceeding the thermal optimum can reduce growth by lowering carbon dioxide assimilation. Warming effects could be either positive or negative, depending on both species and the interactions with other climatic factors (Kirschbaum 2000).
UV-B is an intrinsic part of the solar spectrum, which constitute about 0.5% of the total sunlight energy. UV-B also has the highest energy in the daylight spectrum, causing considerable effects on the biosphere (Caldwell et al. 2003, Jenkins 2009). Depletion of the stratospheric ozone layer allows more UV-B to pass into the earth’s atmosphere. Due to the successful implementation of the Montreal Protocol, the amount of ozone-depleting substances is decreasing, but the ozone layer’s recovery to its initial stage may still take several decades (McKenzie et al. 2011). Initially, UV-B was considered to be a generic stressor on plants, as plant physiology, morphology and metabolism were all affected by UV-B (Tevini et al. 1991, Allen et al. 1997, Boccalandro et al. 2001, Kakani et al.
2003). Recently, it has been shown that besides being a stress or damaging agent, UV-B is a specific regulator of plant growth and development (Jansen & Bornman 2012, Williamson et al. 2014, Parihar et al. 2015). Plants protect themselves against UV-B radiation by the synthesis of UV- absorbing phenolic compounds (Jordan 1996, Bornman et al. 1997, Rozema et al. 1997, Jenkins 2009). Lately, it has also been reported that the perception of UV-B by plants is mediated by a photoreceptor, UVR8 (e.g. Rizzini et al. 2011). UVR8 signalling results in the photomorphogenic responses and the accumulation of flavonoids (Tilbrook et al. 2013).
1.2 SALICACEAE SPECIES
The Salicaceae family consists of 800 species belonging to 60 genera. In Finland, they are mainly
environmental rehabilitation, phytoremediation and the alleviation of major environmental pollutants (e.g. Kuzovkina et al. 2008). Some Salix species are considered to be efficient bioenergy crops, due to their rapid growth and biomass yield. S. myrsinifolia (dark-leaved willow) is one of the best adapted willow species, e.g. as regards winter tolerance in northern Finland (Lumme &
Törmälä 1988). It is an important food source for several insects and mammals (Newsholme 1992).
S. myrsinifolia is also regarded as an appropriate species for herbal drug production, thanks to its high salicylate content (Julkunen-Tiitto et al. 2005, Paunonen et al. 2009 ). Due to its phenotypic and genetic diversity, it is also used as an excellent model system in numerous studies (e.g. Berlin et al. 2011).
1.3 SEASONAL PHENOLOGY AND CLIMATE CHANGE
Seasonal phenology plays an important role in carbon balance, agriculture, pest control and pollen forecasts (Penfound et al. 1945, Keeling et al. 1996, Fischer et al. 2002, Traidl-Hoffmann et al. 2003).
Plant phenology can be divided into two parts: bud phenology and growth phenology. Bud phenology includes the timing of bud break and bud set, while growth phenology includes the pattern and the duration of growth phases (Ceulemans 1997). The primary growth and reproduction ability of a plant is determined by bud development (Nitta & Ohsawa 1998). For growth, reproduction and other basic functions in plants, the assimilation of carbon is a primary factor. Plants have to assimilate the maximum amount of carbon for their functions during the growing season. A general trend globally is that the climatic warming will lead to earlier growth initiation and later leaf senescence in autumn, allowing longer active plant growth and thereby more carbon assimilation (Myneni et al. 1997, Root et al. 2003). However, these positive benefits will be challenged by the other climatic factors (e.g. UV), which could strengthen, counteract or reverse this phenomenon (Xia et al. 2012). Warming can accelerate the development of early active species, while late active species may have delayed phenology or may even remain unresponsive.
This phenological divergence may result in new distribution patterns for existing species (Sherry et al. 2007, Kelly & Goulden 2008). As the vegetative phenological processes also involve certain adaptive traits and fundamental trade-offs for a particular species, varying climate within the species range may result in divergent selection (Howe et al. 2003, Savolainen et al. 2007, Hereford 2009, Savolainen et al. 2013). Thus, the phenological shift patterns induced by climatic warming will severely affect the global carbon cycle, water cycle, plant-animal interactions and finally, the ecology of plants (White et al. 1999, Visser & Holleman 2001, Leinonen & Kramer 2002, Piao et al.
2008, Morin et al. 2008). In a two-year plant-herbivore phenology study under elevated temperature, the spring phenology of the host trees P. tremuloides and B. papyrifera, advanced more than that of the herbivore Malacosoma disstria, resulting in a phenological asynchrony (Schwartzberg et al. 2014). This may also be a result of the variable temperature sensitivity in both the herbivore and the plant (Visser & Holleman 2001, Memmott et al. 2007).
et al. 2017), while in hybrid poplar, certain day and night temperature combinations lead to early growth cessation (Kalcsits et al. 2009). The light quality perceived by the photoreceptors plays an important role in controlling growth, growth cessation and bud set (Cooke et al. 2012).
1.4 GROWTH AND ALLOCATION IN RELATION TO PHENOLOGY UNDER CLIMATE CHANGE
The timing of phenological events is crucial for plant growth and success. Perennial woody plants need a long-term allocation strategy for assigning resources to multiple functions. The phenological changes that are caused during the release and induction of dormancy by the perception of environmental stimuli are coupled with the substantial responses of the carbohydrates present (Chao et al. 2007). Carbon allocation studies show that during the spring bud break, both stored and recently fixed carbohydrates are translocated to the new growth (Webb 1977, Smith & Paul 1988). For instance, in P. tremuloides, bud break and early leaf area development are an autonomous process, as it relates to stored carbon reserves (Landhäusser 2011). In a genus such as Salix, developing buds and new leaves contain substantial amounts of nutrients (e.g.
nitrogen, phosphorus, potassium) transported from the plant's stored reserves (Chapin et al. 1980).
After bud set, deciduous trees reallocate their current resources from the senescing leaves to stems, branches and roots (Dickson 1989, Davis & Haissig 1994, Isebrands & Nelson 1983, Pregitzer et al.
1990). The carbohydrate reserves are lowest during bud break and highest during the dormancy (Johansson 1993, Bollmark et al. 1999, Landhäusser & Lieffers 2003). The levels will vary according to the usage and accumulation in woody plants and according to different seasons and age (Newell et al. 2002, Hoch et al. 2003, Richardson et al. 2013). Unique resource allocation patterns are seen in all phenological phases of growth (Friend et al. 1994), and this may become even more complex during the successive stages of plant life (Kozlowski 1992).
The phenological shifts that can be caused by climate change will alter the growing seasons and may induce negative impacts on these allocation patterns (Ericsson et al. 1996, Cleland et al. 2007, Xia et al. 2017). Reduced resource availability due to elevated temperatures may reduce growth and allocate more resources to defence, but at the same time temperature contributes to earlier and warmer growing seasons and may advance photosynthesis, favouring resource allocation to growth (Slack et al. 2017). This dual role of temperature will in the future result in aberrant plant responses. The growth differentiation balance hypothesis (GDB) proposes that limitation of the carbon pool by environmental factors will result in a trade-off between secondary compound synthesis and growth (Herms & Mattson 1992). According to the carbon-nutrient balance hypothesis (CNB), plant growth will decline more than photosynthesis under low nutrient availability, thereby resulting in an accumulation of carbon-based secondary compounds and carbohydrates (Bryant et al. 1983). According to the protein competition model (PCM), compared to carbon, nitrogen availability is more limited. Nitrogen availability has a direct effect on phenylalanine, a precursor, for which both protein and phenolic production compete, thus resulting in a trade-off between growth and secondary compound synthesis (Jones & Hartley 1999).
1.5 DEFENCE IN RELATION TO PHENOLOGY UNDER CLIMATE CHANGE
1). Phenolic glycosides, such as salicin, salicortin, tremulacin and tremuloidin are the most common defensive compounds against generalist herbivores in Salicaceae species (e.g. Palo et al.
1984, Tahvanainen et al. 1985, Julkunen-Tiitto et al. 1989, Rehill et al. 2005, Donaldson & Lindroth 2007). Salicortin and tremulacin in particular have been shown to have detrimental effects on herbivore growth and development (Osier & Lindroth 2006). Flavonoids may protect the plants from UV-B radiation, as well as being co-pigmentation compounds and pollinator attractants (Winkel-Shirley 2001b). The amounts of different flavonoids and phenolic acids are found to increase under the UV-B treatments of S. myrsinifolia and P. tremula (Tegelberg & Julkunen-Tiitto 2001, Randriamanana et al. 2015a).
Plant defensive traits are costly, as the biosynthesis of defensive compounds involves numerous biosynthetic pathways, and their production, maintenance, storage and transport require substantial amounts of resources (Herms & Mattson 1992, Heil & Baldwin 2002, Strauss et al. 2002).
During stress, resources are allocated to defence, often implying that plant growth is reduced.
Indirectly, it may also result in lower photosynthesis (Siemens et al. 2002, Preisser et al. 2007, Portman et al. 2015). Herbivore attacks may also decrease the photosynthesis levels by altering resource allocation and source-sink interactions (Kocal et al. 2008, Schwachtje & Baldwin 2008), thus enabling root nutrient uptake and use of resources from the existing pools for defence. From the evolutionary point of view, spring and autumn phenology are also important traits for herbivore and pathogen resistance (Dodd et al. 2008, Ghelardini & Santini 2009, McKinney et al.
2011, Sinkkonen et al. 2012). Low levels of quantitative/qualitative (tannins) and structural defences may leave the buds and expanding tissues particularly vulnerable to herbivores (e.g.
Herms & Mattson 1992).
Both plant resistance and tolerance to insect damage, as well as phenological synchrony will be influenced by climate warming (Bale et al. 2002, Parmesan & Yohe 2003, Jamieson et al. 2012).
Warming can directly influence the respiration and photosynthesis of trees, which can in turn indirectly reduce their secondary metabolism, plant resistance and carbon storage (Kirschbaum 2000, Hanson et al. 2005, Adams et al. 2009). In a meta-analysis, it was found that, under elevated temperature, the sugar and starch levels of woody plants declined, while the nitrogen concentrations remained unaffected (Zvereva & Kozlov 2006). Elevated temperature increased the concentrations of salicylates while reducing the concentrations of flavonoids, phenolic acids and condensed tannins in two-year-old P. tremula (Nissinen et al. 2017). In Salix repens, the concentrations of flavonoids were reduced under elevated temperature (Nissinen et al. 2016). In a two-year period, in an experiment with S. myrsinifolia, Nybakken et al. (2012) found that most of the phenolic compounds decreased under elevated temperature during the first year, while certain salicylates increased during the second year. In S. myrsinifolia, elevated temperature also increased the concentration of chlorogenic acid (Randriamanana et al. 2015b). UV-B exposure increased the concentration of quercetin, hyperin and some quercetin glycosides in S. myrsinifolia (Tegelberg et al. 2003, Nybakken et al. 2012). Elevated UV-B also increased the amount of flavonoids and phenolic acids in P. tremula (Randriamanana et al. 2015a, Nissinen et al. 2017). In Betula and Picea
1.6 OBJECTIVES OF THE THESIS
The main focus of this doctoral thesis was to study the influence of predicted climatic factors (T, UV) on phenological events, the growth and chemistry of two Salicaceae species in relation to the bud phenology: bud break and bud set (II) and axillary bud removal of P. tremula, bud chemistry and development (I) in S. myrsinifolia, and stem secondary metabolites and growth (III) in P.
tremula. In addition, the sex-related differences of the studied dioecious species were investigated in order to increase understanding of their responses to future climate change. Many of these traits have earlier been investigated mostly in growth chambers, while long-term outdoor studies are comparatively rare. In indoor experiments, the growth conditions are generally less variable than in field experiments. As the phenological events are generally influenced by natural light and temperature conditions, outdoor field experiments with modulated environmental factors will help us to gain a better understanding of how these environmental factors can influence plant activities under climate change. I used the same experimental set-up as Strømme et al. (2015) to investigate the effects of the treatments on P. tremula bud phenology, growth and secondary chemistry in the third year. The S. myrsinifolia studies were performed in the same experimental set up as Nybakken et al. (2012) and Randriamanana et al. (2015b). The following questions were addressed:
1) How do elevated temperature and UV influence the spring and autumn bud phenology of P.
tremula?
2) What are the effects of elevated temperature and UV on the seasonal growth of P. tremula?
3) Will bud development and the concentration of secondary metabolites in vegetative buds of S.
myrsinifolia be influenced by the elevated temperature and UV?
4) Will elevated temperature and UV affect the sex-related responses of S. myrsinifolia and P. tremula?
2 MATERIALS AND METHODS
2.1 OVERVIEW OF EXPERIMENTS
The experiments were conducted at a field site in the Botanical Garden in Joensuu, Finland (62°60’N, 29°75’E) (Fig. 2). The experimental set-up consisted of 36 plots with individual and combined treatments (Table 1).
Table 1. Outline of the conducted experiments
Article I II III
Species S. myrsinifolia P. tremula P. tremula
Treatment Elevated UV-B and temperature
Elevated UV-B and temperature
Elevated UV-B and temperature
Treatment duration 2 months 3 years One season
Total number of individuals
501 608 (2013)
552, 400 (2014) 818 (2015)
844
Number of replicates 6 6 6
Number of genotypes (clones)
8 (4 males and 4 females) 12 (6 males and 6 females) 12 (6 males and 6 females)
Plant traits analysed Bud length Bud weight Bud phenolics
Bud break Bud set
Basal diameter Height growth Shoot biomass Leaf biomass Leaf area Stem phenolics Gas exchange parameters Chlorophyll Content Index Rust intensity
Leaf herbivory damage Bud weight
2.2 PLANT MATERIALS
The P. tremula stands originated from six male and six female trees from: Loppi {Uotila (61°07’N, 21°34’E), Riihisalo (60°44’N, 24°10’E), Hirvijärvi (60°40’N, 24°36’E), Haapastensyrjä (60°36’N, 24°25’E), Hirvijärvi (60°43’N, 24°42’E)} in Southern Finland and Pieksämäki {Pirttimäentie (63°15’N, 28°04’E), Hiekkapuro (63°21’N, 27°14’E)}, Liperi {Polvijärventie (62°40’N, 29°34’E), Tornivaara (62°36’N, 29°32’E)}, Kaavi {Luikonlahti (62°55’N, 28°40’E)}, Kontiolahti {Onttola (62°38’N, 29°41’E)} and Polvijärvi {Saarivaara (62°16’N, 30°47’E)} in Eastern Finland (Sobuj et al.
2017). These P. tremula clones were micropropagated in a woody plant medium using 8.5 g L -1Agar
used to attain the supplemental levels of UV-B and temperature in the plots. The tubes in the UV- B plots were wrapped in cellulose diacetate (0.115 mm, Kotelorauma, Finland) filters in order to reduce the radiation to below 290 nm. In the UV-A plots, the lamps were wrapped in polyester film (0.175 mm, Kariplast, Finland) to exclude radiation below 315 nm. To maintain equal levels of shading, both unenergized UV lamps and wooden boards were used. The S. myrsinifolia plants studied were grown under these treatments for the years 2010 and 2011, while the P. tremula were under the same treatments for 2012, 2013, 2014 and 2015 (the last year without treatments).
FOREST
ROAD
Figure 3. Plot design executed in the experimental field with different treatments of ambient temperature and ambient UV radiation (C), elevated temperature (T), elevated ultraviolet radiation-B (UV-B), ultraviolet radiation-A (UV-A), combination treatment of elevated UV-B radiation and elevated temperature (UVB+T) and combination treatment of UV-A radiation and elevated temperature (UVA+T).
2.4 REMOVAL OF VEGETATIVE AXILLARY BUDS
The patterns of resource allocation change during the dormancy and growth induction stages of a plant’s growing season. In order to know whether the resource restrictions will play any role in
UVB+T
T
UVA+T T
UVA
UVA+T UVA+T UVA UVB
UVB UVB+T T
UVB+T C
UVB
C
UVB C
UVA
UVB+T UVB
UVA+T T
UVB+T UVA+T UVB T C UVB+T
UVA T
UVA C
UVB+T C
UVA
2.5.1 Measurement of growth, photosynthesis and leaf damage
The height and diameter of P. tremula were measured every third week (III) during the third growing season. The herbivory and rust damage to leaves of P. tremula were also calculated (III).
The lateral bud weight (I, III) of both S. myrsinifolia and P. tremula and the bud length of S.
myrsinifolia were also measured. In addition, photosynthesis and chlorophyll content of the P.
tremula plants were also recorded during the middle of the growing season (III).
2.5.2 Scoring the bud break and bud set stages
The bud break stages of P. tremula were scored according to Fu et al. (2012), and defined as follows:
(0)- a closed bud, (1)- a closed bud with protruding green leaf tips, (2)- a green leaf emerged from the bud with leaf bases hidden, (3)- a broken bud with at least one visible petiole, and (4)- an unfolded leaf with visible leaf blade and stalk (Fig. 4). The bud set stages were defined on the basis of Rohde et al. (2011) and were constituted of three stages: (1)- apices between full, active growth to apices with an open bud, (0.5)- a closed green bud, (0)- a brown closed bud (Fig. 4).
0
1
2
1 3 0.5
0
Bud break Growth Initiation Populus tremula Bud set
Growth
Cessation
were detected at 220 nm (salicyl alcohol, disalicortin, HCH-salicortin, salireposide, cinnamoyl salicortin), 270 nm (salicortin, salicin, tremulacin, diglucoside of salicyl alcohol, cinnamoyl salicortin derivative-2, dicoumaryl derivative-1, quercetin 3-galactoside, chlorogenic acid, chlorogenic acid derivatives 1-5, p-OH-cinnamic acid derivative, (+)-catechin, epigallocatechin, triandrin) and 320 nm (p-OH-cinnamoyl salicortin derivative-1, cinnamoyl salicortin derivatives, other flavonoids and phenolic acids), using corresponding commercial standards as a reference (III). The compounds were identified on the basis of their retention times and UV spectra (I, III).
The concentrations of the condensed tannins in the bud samples were determined as described by Hagerman (2002) (I) and quantified using purified Betula nana leaf tannins.
2.5.4 Statistical analysis
The effects of treatments on P. tremula bud break (2014, 2015) and bud set (2013, 2014) (II) were analysed using the cumulative link mixed model (clmm) in R (R Core Team 2016) by applying the clmm function in Ordinal package (Christensen 2015, II).
In this summary, the effects on bud break (2014, 2015) and bud set (2013, 2014) were also analysed using the linear and generalized linear mixed-effects models to compare them with the cumulative link mixed model. In both cases, the response variable was the Julian day of the bud stage shift and time lapse between bud stages. Whenever the bud was observed to have changed on a certain day of measurement, it was assumed that the shift had taken place at the mid-point of the current date and the previous day of measurement. If two shifts had taken place with a certain interval, they were assumed to have happened at relative times of 1/4 and 3/4 between the two dates. The fixed part includes the two levels of temperature (ambient and elevated), three levels of UV treatment (ambient, UV-A & UV-B) and two levels of sex (male and female). The model had random intercept for the crossed levels of clone and plot identity. The interactions between sex, temperature and UV were also included. In the case of bud set (2013, 2014), as many plants had already reached the second stage on the first scoring day, the bud stage on the first scoring day was analysed using binary logistic regression, with a random-effect structure similar to that of the linear model above.
The analyses were carried out using the lmer and glmer functions in the lme4-package (Bates et al.
2015, R core team 2016).
A linear mixed effects model was also used to check the effects of treatments on bud phenolics, bud development (I) and growth, stem phenolics, leaf herbivory, leaf rust and bud weight (III) and were analysed using IBM SPSS Statistics (Version 21, 24, Armonk, NY, USA).
3 RESULTS AND DISCUSSION
3.1 SPRING PHENOLOGY AND SEASONAL GROWTH OF P. TREMULA UNDER CLIMATE CHANGE
The first objective of the thesis was to identify the impacts of elevated levels of temperature and UV radiation on the vegetative bud break and shoot growth of P. tremula. I found that elevated temperature played a key role in forcing bud break. Even when the temperature treatment was terminated after three years, the aspen plants showed a slight forcing effect of the earlier increased temperature on bud break (II) the following spring. In the first year (Strømme et al. 2015), there was also a forcing effect of UV-B on bud break, but this was not seen in the following two seasons.
This suggested that the aspen plants acclimated to the elevated UV-B treatment over time.
However, when combined with elevated temperature, UV-B (UVB+T) reduced height growth (III) compared to the effect of the elevated temperature treatment alone. Generally, perennial plants adjust their dormancy and growth initiation according to the existing climatic conditions in order to allocate maximum resources during the growing season and to avoid frost damage (Körner 2012, Vitasse et al. 2014). The timing of the developmental processes of a tree normally changes during the growth cycle, depending either on internal or external factors (Sultan 2000, Gratani 2014). The earlier bud break in the elevated temperature plots might be a reason for the increased height and diameter growth of these plants compared to the control plot plants (III). In Picea abies warming accelerated bud burst (Hänninen et al. 2007), and warm spells enhanced the growth of primordial shoots (Sutinen et al. 2012).
Females showed earlier bud break compared to the males of the species (II), suggesting an extended growing season for females. This implies that females need to allocate more resources during the adult vegetative stage before they enter the adult reproductive stage. On the other hand, when resources were restricted by bud removal, only the males were responsive (II) to phenological shifts, delaying bud break under elevated temperature and UV-B treatments and advancing it under combined treatments (UVA+T). The higher responsiveness of males may be a result of their being more growth-oriented (Lloyd & Webb 1977). The lower response of the females may be due to their accumulating sufficient resources for the buds to break, despite the influence of any treatments.
In order to make a comparison with the cumulative linked mixed model (clmm) that was used for the published article (II), I also tested the phenology data using the linear mixed-effects model (lme) for the thesis summary. In clmm the response variable used was the bud stages and it is categorical, so that the model assumes all stages on all possible Julian days. In the case of lme, both the Julian day and the time lapse between bud stages were the response variables, so that the model assumes only particular stages for a particular interval period. This was done in order to show how well these two models would be suited for the analysis of phenological data and to see whether there is any significant difference between the two models. The results from the linear mixed- effects model also showed that elevated temperature was influential in forcing the bud break in
3.2 AUTUMN PHENOLOGY OF P. TREMULA UNDER ELEVATED TEMPERATURE AND UV RADIATION
The autumnal bud development of both males and females was delayed by elevated temperature (II). A delaying effect caused by elevated temperature was also detected in the first growing season of the same plants (Strømme et al. 2015), as well as in two-year-old P. deltoides × P. nigra (Rohde et al. 2011). With regard to UV, UV-B treatment alone had no effect on bud set. UV-A alone delayed bud set, while when combined with elevated temperature both UV-A and UV-B forced bud set, in males (II). This indicates that P. tremula was not able to acclimate to combined UV and temperature in successive years, in contrast to the case of bud break in spring. It is suggested that the hormone abscisic acid (ABA) and ethylene play important roles in the bud set process (Ruonala et al. 2006, Ruttink et al. 2007). In the scoring system for bud set proposed by Rohde et al. (2011), the last leaves of the apical shoot roll up and fold before the bud scale formation. In my case, this stage may have been prolonged due to the exposure to UV-A, as it has been shown that UV-A can induce leaf elongation (Cooley et al. 2001), resulting in a delayed bud set. Abscisic acid accumulation is responsible for the inhibition of growth during dormancy (Horvath et al. 2003). It is also found that under high ambient levels of UV radiation and high temperatures, ABA biosynthesis increases (Toh et al. 2008, Rakitin et al. 2008, Berli et al. 2010, 2011). Although not measured in my study, the higher accumulation of ABA under the combined treatments might have forced bud set. Bud removal, a form of resource depletion, also affected the bud set of males, and bud set was delayed under the temperature treatment while it was forced under the combined treatments. The axillary bud outgrowth depends on the ratio of auxin to cytokinin (Shimizu-Sato & Mori 2001), and by removing the buds the ratio may have been modified, resulting in apical dominance, thus supporting continued growth rather than bud set. Moreover, under high temperatures (20-30°C), auxin plays a predominant role (de Wit et al. 2014, Zheng et al. 2016), which may have led to the delayed bud set in my case.
I also compared the effects of treatments on bud set using clmm and lme. The results of lme showed that elevated temperature delayed the bud set of intact individuals in 2013 and of bud removed individuals in 2014. The interactive effects of UV and temperature that was seen with clmm was not found with the lme model. This disparity may have resulted from the consideration of time to start and time lapse for bud set as the response variable, and also from the usage of binary logistic regression for analysing the bud stage on the first scoring, as most of the plants were in the second stage.
3.3 SECONDARY METABOLITES AND DEVELOPMENT OF VEGETATIVE
BUDS OF S. MYRSINIFOLIA AND P. TREMULA
Randriamanana et al. 2015b, Sivadasan et al. 2015). Chlorogenic acid is a ubiquitous phenolic acid that exhibits multiple modes of protection roles (e.g. Isman & Duffey 1982, Matsuda & Senbo 1986, Ikonen et al. 2001, Hammerschmidt 2014). The presence of chlorogenic acid in buds and leaves of S. myrsinifolia may protect vulnerable tissues against herbivores or other pests during dormancy and the growth period. There were very few differences between genders in the phenolic chemistry of dormant buds (I) when compared to the leaves of S. myrsinifolia (Nybakken et al. 2012, Randriamanana et al. 2015b, Sivadasan et al. 2015).
Vegetative bud development was influenced by elevated temperature in both P. tremula and S.
myrsinifolia (I, III). The bud size of both species increased under elevated temperature, which could be expected as a result of increased growth in the preceding year (Little 1970, Kozlowski 1973, Nybakken et al. 2012, Nissinen et al. 2017). Male buds were larger than those of females in P.
tremula, while in the case of S. myrsinifolia, the female buds were larger (I, III), irrespective of treatments. This may be due to the fact that both S. myrsinifolia males and females were already flowering during the year 2011 (Randriamanana et al. 2015b), and at the reproductive stage, females also allocate more resources to photosynthetic organs, such as buds (Ueno et al. 2007). The P. tremula males had an early bud set during this season, which may have resulted in earlier initiation of carbon flow to the roots, as seen in Populus tristis x balsamifera (Nguyen et al. 1990).
This depletion of carbohydrate reserves increases the turgor pressure and results in the swelling of the buds (Kozlowski 1992), which may be the reason for the larger bud size in P. tremula males.
Female S. myrsinifolia buds were longer under ambient conditions, while under elevated temperature, the buds of males were longer (I). The bud length correlates positively with the previous season’s shoot growth (Hejnowicz & Obarska 1995), and in my case, the S. myrsinifolia male plants were taller under the elevated temperature treatment during the previous year (Nybakken et al. 2012), resulting in longer buds in males compared to those of females. During the pre-flowering period, the developmental stages of male plants are more sensitive to high temperature while the developmental stages of females are less sensitive to the temperature (Sage et al. 2015).
3.4 DEFENSIVE CHEMISTRY OF P. TREMULA UNDER ELEVATED TEMPERATURE AND UV RADIATION
The effect of elevated temperature on stem chemistry differed from that of leaf and bud chemistry.
Salicylates were the most abundant low molecular weight phenolics in P. tremula stems (III). In P.
tremula leaves the concentration of total salicylates was not affected by elevated temperature (Randriamanana et al. 2015a, Nissinen et al. 2017), while the concentration in stems was reduced (III). This result is in agreement with earlier studies on the effect of increased temperature on P.
tremula bark and S. myrsinifolia twigs (Nybakken & Julkunen-Tiitto 2013, Sobuj et al. 2017). The phenolic compounds present in Populus stems are suggested to serve as anti-browsing defences during the winter (Lindroth et al. 2007, Boeckler et al. 2013, Lindroth & St Clair 2013), and this could be the reason why, compared to other phenolic compounds, salicylates were found in higher concentrations in stems. The relatively lower amounts of flavonoids in stems compared to those found in leaves may also be a result of a lower need for UV protection in stems. Leaf herbivory on
3.5 EVOLUTIONARY IMPLICATIONS FOR POPULUS AND SALIX
Populus and Salix both belong to the family Salicaceae. Populus is classified into 29 species (Eckenwalder 1996) and Salix into 450-520 species (Argus 1997, Skvortsov 1999). The first appearance of Populus in Europe was during the early Eocene Epoch (56-34 million years ago) and that of Salix was during the Middle Oligocene Epoch (34-23 million years ago) (Collinson 1992).
Dorn (1976) and Skvortsov (1999) stated that Populus is more primitive than Salix, and the Salix species may derive from subtropical Populus-like forms (Hegi 1958, Jalas 1965). Fossil records also support this development (Collinson 1992). Even though P. tremula and S. myrsinifolia are assumed to have a similar ancestor, they have many dissimilar morphological features. In particular, the subgenus Salix has features typical of the Populus species, while the subgenus Caprisalix (S.
myrsinifolia belongs to this group) is considered younger, and also phylogenetically more developed and still developing when compared to the subgenus Salix or Populus (Skvortsov 1999).
The Populus genome sequencing by Tuskan et al. (2006) also points to the fact that both Populus and Salix share some similar genomic history. However, recently, some molecular phylogenetic studies of Salicaceae have shown that Populus and Salix are quite different groups (Leskinen & Alström- Rapaport 1999, Azuma et al. 2000, Cervera et al. 2005, Hamzeh et al. 2006), while Cervera et al.
(2005) found that the ancient species, P. mexicana has most similarities with Salix. Although the studied species have different evolutional histories, the growth, chemistry and phenology of both species are similarly affected by elevated temperature and UV-B (II, Strømme et al. 2015): elevated temperature improved all the growth parameters (height, diameter, leaf area, biomass etc.) in both species (III, Nybakken et al. 2012, Randriamanana et al. 2015a, Randriamanana et al. 2015b, Nissinen et al. 2017). Even though, to my knowledge there are no studies of S. myrsinifolia phenology under predicted climatic change, the bud break of Salix species (e.g. S. viminalis, S.
dasyclados) is largely dependent on the temperature sum (Lennartsson & Ögren 2004). Numerous other environmental and genetic factors influence the phenology of Salix species, also affecting their growth (Rönnberg-Wästljung 2001, Tsarouhas et al. 2003, Weih 2009). As regards secondary metabolites of the species, inter-species differences are found throughout their ontogeny: S.
myrsinifolia seems to keep a high level of defensive salicylates over seven years (Nissinen et al. 2018, submitted), while salicylates are high and are major compounds in P. tremula during the first two years of the plant’s life (Randriamanana et al.2015a, Nissinen et al. 2017). Secondary metabolites of the species whereas influenced, either increased or decreased by elevated temperature and UV, and the responses were also age-dependent (Nybakken et al. 2012, Randriamanana et al. 2015a, Randriamanana et al. 2015b, Nissinen et al. 2017). Even though these two species vary in numerous features, their responses to elevated temperature and UV radiation share a similar pattern.
Therefore, beneficial or detrimental, the impacts of climate change may be quite similar in the two species.
4 MAIN FINDINGS AND CONCLUDING REMARKS
The expected future increase in temperatures will lengthen the growing season of P. tremula due to a forcing effect on the plant’s spring phenology. This predictable situation may put P. tremula at a risk of frost damage. After three years’ treatment, UV-B in combination with elevated temperature was still functioning as a signal for the bud set of bud removed individuals. P. tremula and S. myrsinifolia generally show a pattern of acclimation after a certain period of exposure to stress factors.
The secondary metabolites of buds and stems are also responsive to climatic factors. The acclimation of S. myrsinifolia and P. tremula to UV-B strengthens the idea that UV-B has a role as a morphogenetic signal for plants. Regarding the sex-related treatment responses, in P. tremula the bud phenology of males was more responsive, while females were more responsive to the treatments concerning secondary metabolites.
Autumn phenology has been studied less compared to spring phenology. The interaction between temperature and UV-B on different plant development processes is still unclear, and studying the consecutive molecular pathways could resolve these mechanisms better. Future research taking into account the facts that UV-A is absorbed by UVR8 and that phytochromes function as thermosensors will give a better understanding regarding the interactive effects of temperature and UV radiation. Moreover, long-term natural field experiments focusing on molecular/hormonal pathways in combination with plant-stress mediated gene expression studies, will serve to elucidate the precise mechanism of environmental factors and their influence on plant growth cycle events.
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