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ISSERTATIONS | MINNA KOSONEN | CHANGES IN PROANTHOCYANIDIN PATHWAY AFFECT OTHER... | No 242
MINNA KOSONEN
CHANGES IN PROANTHOCYANIDIN PATHWAY AFFECT OTHER PHENOLICS AND GROWTH OF ASPEN AND BIRCH
Plants allocate limited resources between growth and various defense agents, including
phenolics. Understanding the changes occurring in phenolics is crucial to finding
out the roles of various environmental factors affecting not only phenolics but the plants’ growth or reproduction. This thesis provides further insights into our current
understanding of phenolic pathway and plant resource allocation under different environmental conditions through powerful
tools; genetically modified plants.
MINNA KOSONEN
Changes in
proanthocyanidin pathway affect other phenolics and growth of aspen and birch
Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences
No 242
Academic Dissertation
To be presented by permission of the Faculty of Science and Forestry for public examination in the Auditorium N100 in Natura Building at the University of Eastern
Finland, Joensuu, on December, 09, 2016, at 12 o’clock noon.
Department of Environmental and Biological Sciences
Grano Oy Jyväskylä, 2016
Editors: Research Dir. Pertti Pasanen,
Profs. Pekka Toivanen, Jukka Tuomela and Matti Vornanen
Distribution:
University of Eastern Finland Library / Sales of publications P.O.Box 107, FI-80101 Joensuu, Finland
tel. +358-50-3058396 www.uef.fi/kirjasto
ISBN: 978-952-61-2316-5 (nid.) ISSNL: 1798-5668
ISSN: 1798-5668 ISBN: 978-952-61-2317-2 (PDF)
ISSN: 1798-5676 (PDF)
FI-80101 Joensuu FINLAND
email: minna.kosonen@uef.fi
Supervisors: Professor Riitta Julkunen-Tiitto, Ph.D.
University of Eastern Finland
Department of Environmental and Biological Sciences P.O. Box 111
FI-80101 Joensuu FINLAND
email: riitta.julkunen-tiitto@uef.fi
Sari Kontunen-Soppela, Ph.D.
University of Eastern Finland
Department of Environmental and Biological Sciences P.O. Box 111
FI-80101 Joensuu FINLAND
email: sari.kontunen-soppela@uef.fi
Reviewers: Professor Ann Hagerman, Ph.D Miami University
Department of Chemistry and Biochemistry 160 Hughes Laboratories, 651 E. High St.
45056 Oxford, Ohio USA
email: hagermae@miamioh.edu
Professor Maike Petersen, Ph.D University of Phillipps
Institute of Pharmaceutical Biology and Biotechnology Deutschhausstraße 17a
35032 Marburg GERMANY
email: petersen@staff.uni-marburg.de
Opponent: Professor Hely Häggmann Ph.D Department of Biology
University of Oulu P.O.Box 3000
FI-90014 University of Oulu FINLAND
email: hely.haggmann@oulu.fi
Phenolics have major roles in defence against various biotic and abiotic threats in plants. Different environmental factors affect not only phenolics but also plants’ growth and reproduction.
Phenolics are formed via the phenolic pathway, and the end products of this pathway are e.g. proanthocyanidins (PAs). This thesis yields insights into changes in the responses of PA- pathway genes (ANR, ANS, DFR and LAR) and compounds of young deciduous plants of early-flowering birch (Betula pendula Roth) and hybrid aspen (Populus tremula × Populus tremuloides) to changing environmental factors (temperature, nitrogen fertilization) and over the early ontogeny phase (B. pendula). The essential research question was how the changes in the PA- pathway affect phenolics in other parts of the phenolic pathway.
The carbon trade-off between the different branches of the phenolic pathway was studied using genetically modified (GM) plants.
In the juvenile phase, resource allocation between growth and phenolic compounds is an important factor. Both quantitative and qualitative changes in the content of phenolics were found to be due to an ontogenetic shift. The maturation process decreased several phenolics in the leaves and stems of B. pendula. Increased PA concentration, caused by constitutive expression of the MYB134 gene, reduced concentrations of salicylates in P. tremula
× tremuloides, whereas inhibition of ANR gene expression (ANRi) in B. pendula resulted in decreased concentration of PAs and in growth reduction. Environmental factors also clearly affected phenolic accumulation. Especially low nitrogen level affected the content of individual phenolics in the stems of B. pendula. The concentrations of certain individual phenolics increased in the stems of wild-type (WT) birches, while the same phenolics decreased in ANRi birches. There was more nitrogen in the leaves of the ANRi birches than in the WT leaves. Elevated temperature reduced in particular salicylates, some individual flavanol glycosides and acids in the leaves of P. tremula × tremuloides.
and the regulation of trade-off in deciduous species, in particular in their juvenile phases. The genes that affect the formation of PAs have also various effects on the entire plant, for example on the regulation of growth. Important environmental factors, such as nitrogen and temperature, have a major impact on the concentrations of many phenolics in birch and aspen. A clear carbon trade-off was found between the different branches of the phenolic pathway. The results of this thesis indicate the importance of phenolics for various ecological interactions, and especially for the comprehensive undisturbed growth and reproduction of the plants studied.
Universal Decimal Classification: 547.973, 547.98, 577.218, 577.13, 582.622.2, 582.681.82
CAB Thesaurus: phenolic compounds, proanthocyanidins, anthocyanidins, tannins, salicylates, Betula pendula, Populus tremula, herbivory, environmental factors, temperature, nitrogen, growth, ontogeny, maturation, reproduction, gene expression, transgenic plants
Yleinen suomalainen asiasanasto: fenoliset yhdisteet, tanniinit, salisylaatit, koivu, haapa, hybridihaapa,
herbivoria, ympäristötekijät, lämpötila, typpi,
kasvu, lisääntyminen, geeniexpressio, muuntogeeniset eliöt
Acknowledgements
This study was carried out at the Department of Biology and Environmental Science, the University of Eastern Finland. I am sincerely grateful to my main supervisor, Professor Riitta Julkunen-Tiitto. This thesis would never have seen daylight without her encouragement and helpful advice. I would like to express my warmest gratitude to my other supervisors, Doctor Sari Kontunen-Soppela for the tireless help and the valuable discussions and golden questions, which opened my mind and gave me many new ideas throughout this long journey. I also wish to express my gratitude to co-authors, Doctor Virpi Virjamo, Doctor Mika Lännenpää, Docent Teija Ruuhola, Professor Peter Constabel and Ms Milla Ratilainen. I am especially grateful to Professor Peter Constabel and his group for sending the genetically modified aspen line clones.
I further wish to convey my sincere thanks to my collaques in Joensuu, Virpi, Anneli, Merja, Maarit, Line, Anu and Tendry, who gave me insightful thoughts, and valuable help with growing my plants, as well as many happy moments. I greatly appreciate the support of my colleagues in Mikkeli. Anni, Mervi and Anne, many thanks for your supportive words and your strong belief in my ability to complete this thesis. I extend my thanks to all unnamed people who have supported and encouraged me in many different ways.
I wish to thank Rosemary Mackenzie, M.A., who kindly revised the language of this summary. Professor Maike Petersen and Professor Ann Hagermann pre-examined this thesis.
The Financial supporters of this thesis, the Graduate School in Forest Sciences, the Academy of Finland (decision number 128652), grants of the University of Eastern Finland and the Finnish Cultural Foundation, South Savo Regional Fund, are acknowledged for providing the funding. This study would not
I also express my gratitude to my relatives and friends, and I am especially grateful to my mother Leena, for her help with child care and her great support to me during my life. I also thank my grandmother Eila who I know to be proud of my achievement.
And last but not by any means least, my utmost and sincere thanks go to my family, my husband Jarkko for his love, support and humour, which have helped me to complete this dissertation journey, and to Mertsi, Petrus and Tyyne, who have made it possible for me to think about other things than this work. You can light your own way, you have the capability to do whatever you want, you are the brightest little stars in my sky.
Don´t just grow your plants, let your plants nurture you.
ANR anthocyanidin reductase
ANRi birches birches in which the BpANR gene was inhibited using the RNA interference method.
ANS anthocyanidin synthase
BpANR Betula pendula ANR gene BpANS Betula pendula ANS gene BpDFR Betula pendula DFR gene BpLAR Betula pendula LAR gene
BPM5 early-flowering birch (Betula pendula) clone number 5
CNB carbon nutrient balance hypothesis
CaMV cauliflower mosaic virus
CoA coenzyme A derivative
DSDG dehydroshikimate dehydrosgenase
DFR dihydroflavonol 4-reductase
DHPPG 3,4′dihydroxypropiophenone 3-glucoside
DW dry weight
EST expressed sequence tag
GDB growth-differentiation balance hypothesis
GM genetically modified
GUS the Escherichia coli beta-glucuronidase gene
HT hydrolysable tannins
HPLC high pressure liquid chromatography LAR leucoanthocyanidin reductase
MYB myeloblastosis
mRNA messenger RNA
PA proanthocyanidin, condensed tannin
PAL phenylalanine ammonia-lyase
PAP1 and PAP2 MYB factor genes (1 and 2) that are involved in production of the anthocyaidin pigment
PCM protein competition model
Phe phenylalanine
PG phenolic glycosides
UV ultraviolet
UV-B ultraviolet B radiation (280–315 nm)
WT wild type
Juvenile (sapling): immature developmental stage in which plants are not yet sexually reproductive (flowering).
Mature stage: developmental stage characterized by the ability of plants for sexual reproduction.
Resource allocation: partitioning of resources to different plant structures or metabolic functions.
I Kosonen M, Virjamo V, Lännenpää M, Kontunen-Soppela S and Julkunen-Tiitto R. The pre-reproductive phases affect phenolics in early-flowering birches (Betula pendula Roth). Submitted manuscript
II Kosonen M, Lännenpää M, Ratilainen M, Kontunen- Soppela S and Julkunen-Tiitto R. Decreased anthocyanidin reductase (ANR) expression strongly decreases silver birch (Betula pendula) growth and alters phenolics accumulation. Physiologia Plantarum 155: 384- 399, 2015.
III Kosonen M, Keski-Saari S, Ruuhola T, Constabel P and Julkunen-Tiitto R. Effects of overproduction of condensed tannins and elevated temperature on chemical and ecological traits of genetically modified hybrid aspens (Populus tremula × P. tremuloides). Journal of Chemical Ecology 38: 1235-1246, 2012.
The above publications have been included at the end of this thesis. The pubilications are reprinted with kind permission from the publishers: John Wiley and Sons (II) and Springer (III)
In papers I, II and III Minna Kosonen (M.K.) planned the experiments together with her main supervisor. In all the papers (I, II and III) M.K. contributed to the founding and maintenance of the experiment, and M.K. had primary responsibility for measurements and samplings. In papers I and III M.K. was responsible for the phenolic analyses and, in papers II and III for the statistical data analysis. M.K. was the main author of all the papers. Genetically modified trees were provided by Mika Lännenpää for the birches and by the group of Prof. Peter Constabel for the aspens.
1 Introduction ... 15
1.1. Phenolic pathways ... 15
1.2. Biological roles of phenolics ... 17
1.3 Changing environment and juvenile phase affect phenolics .. ... 18
1.3.1 Direct and indirect effects of temperature on phenolics ... 19
1.3.2 Nitrogen effects on phenolics ... 20
1.4 Defensive structures and chemicals in Salicaceae and Betulaceae ... 21
1.5 Plant resource allocation theories ... 23
1.6 Genetic Modification techniques for regulation of the PA pathway ... 25
1.6.1 Regulation with transcription ... 25
1.6.2 Downregulation by the RNAi method ... 26
1.7 Aims of the thesis ... 27
2 Materials and methods ... 29
2.1 Study organisms ... 29
2.1.1 Birch (B. pendula Roth.) (I, II) ... 29
2.1.2 Aspen (P. tremula x tremuloides) (III) ... 30
2.1.3 Leaf beetle (Phratora vitellinae) (III) ... 30
2.2 Experimental procedures ... 31
2.2.1 Experiments and treatments ... 31
2.2.2 Chemical analyses (I, II, III) ... 32
2.2.3 Growing and sampling of the birches (I, II) ... 33
2.2.4 Gene expression analysis (I, II) ... 34
2.2.5 Cafeteria experiments: five-choice experiment and dual-choice experiment with aspen (III) ... 34
2.2.6 Statistical analysis ... 35
... 37
3.2 How do the downregulation and overexpression of the PA pathway genes affected phenolics? ... 39
3.2.1 Downregulation of ANR caused alterations of chemotype and phenotype ... 40
3.2.2 Overexpression of the MYB134 gene implied trade-offs ... 44
3.3 Abiotic factors affecting phenolics ... 44
3.3.1 Nitrogen levels ... 44
3.3.2 Elevated temperature ... 47
3.4 Effects of temperature and nitrogen to herbivore resistance ... 47
3.5 Can we increase the growth of trees by manipulation of the phenolic pathway ... 49
4 Conclusions ... 57
5 References ... 59
15
1 Introduction
1.1. PHENOLIC PATHWAYS
Carbon-based compounds, such as phenolics and terpenoids are numerous and widely distributed defense chemicals in the plant kingdom (Haslam et al. 1992, Cheynier et al. 2013). Simple phenolics consist of an aromatic 6-carbon ring bearing one or more hydroxyl groups. Phenolics range from simple, low molecular weight molecules, such as stilbenes or phenolic acids, to more complex polyphenols, such as lignans, coumarins, flavonoids, anthocyanidins and proanthocyanidins (PAs, condensed tannins).
Phenylalanine (Phe) is synthesized from chorismate produced by the shikimic acid pathway. Phe serves as a precursor for phenolics via the complex phenolic pathway (Herrmann & Weaver 1999, Bowsher et al. 2008, Maeda & Dudareva 2012). The phenylpropanoid pathway leads from Phe to coumaroyl-CoA and is initiated by phenylalanine ammonia-lyase (PAL). PAL catalyzes the deamination of Phe. It is the branch-point enzyme between primary metabolism (growth, development and reproduction) and the phenolic pathway. The trans-cinnamic acid is thus formed for further use in the biosynthesis of other phenolic compounds, such as flavonoids (Bowsher et al. 2008).
Phenolics are divided into several subgroups, such as flavonoids, tannins (including PAs and hydrolysable tannins), phenolic acids and salicylates. Flavonoids are mixed-origin compounds synthesized by the phenylpropanoid pathway and acetate-malonate pathway, providing the additional carbon units needed to form the phenyl ring for flavonoids and PAs (Bowsher et al. 2008). Phenolic glycosides (PGs) are formed from cinnamic acid derivatives via two possible routes, e.g. via the o-coumaric acid or benzyl alcohol routes (Zenk 1967, Babst et al. 2010). Higher PGs are salicyl alcohol-derived glucosylated compounds having 6-
hydroxy-2-cyclohexen-on-oyl-group (HCH-esters) (e.g. Boeckler et al. 2011). However, these phenolic formation pathways are not the only routes available for the variable phenolics in plants, and not all the metabolites of the phenolic pathway present in all plant species (Dixon et al. 2002). Although the hydroxycinnamic acids and several flavonoid classes are ubiquitous in vascular plants, many phenolic glycosides and stilbenes are present only in specific plant families, genera or even species (Bowsher et al. 2008, Gardner
& McGuffin 2013).
PA formation occurs via a biosynthetic pathway comprising four enzymes: dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS; also called leucoanthocyanidin dioxygenase, LDOX), leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR; in Arabidopsis thaliana the product of BANYULS gene) (Saito et al. 1999, Abrahams et al. 2003). DFR is a key enzyme that catalyzes and controls the biosynthesis of anthocyanins and PAs by converting dihydroflavonols into leucoanthocyanidins (Dixon & Paiva 1995, Xie et al. 2004). PAs and anthocyanins are synthesized via flavan-3,4-diols. LAR catalyzes the synthesis of (+)- catechins (Tanner et al. 2003), while ANR catalyzes the synthesis of epicatechins (Xie et al. 2003). Thus, (+)-catechin and (−)-epicatechin are the final building blocks of PAs (Xie et al. 2003, Dixon 2005, Pfeiffer et al. 2006). ANS produces colored anthocyanidins that are reduced by ANR to form colorless flavan-3-ols (Saito et al. 1999, Abrahams et al. 2003). Elaborated chart of the phenolic pathway can be found in figure 3.
The phenylpropanoid pathway is tightly controlled by several transcription factors (Weisshaar & Jenkins 1998, Nesi et al. 2001, Dixon et al. 2002, Winkel-Shirley 2002). One known gene family that regulates the phenylpropanoid pathway comprises myeloblastosis (MYB) transcription factors, which are common in several plant families (Martin & Paz-Arez 1997, Stracke et al. 2001).
The MYB transcription factor activates the PAL gene that encodes the first enzyme (PAL) of the phenylpropanoid metabolism (Nesi et al. 2001). MYB transcription factors have also been found to have a crucial role in plant hormone signal transduction, disease resistance and organ development (Du et al. 2009).
17
1.2. BIOLOGICAL ROLES OF PHENOLICS
Phenolics have been considered to play a key role as defense compounds when plants are exposed to different abiotic and biotic stresses (Dixon & Paiva 1995). In addition to defense, phenolics are known to have several biological functions in the ecosystem (e.g.
Castells 2008). Simple flavonoids are a large class encompassing over 10 000 compounds (Veitch & Grayer 2011, Agati et al. 2012).
Flavonoids are important for the regulation of essential physiological processes, such as growth and development in plants, by regulating plant hormone levels (Besseau et al. 2007, Pourcel et al. 2007, Bowsher et al. 2008, Feucht et al. 2014).
Interestingly, the transport of the plant hormone auxin has been thought to be inhibited by flavonoids, such as quercetin and kaempferol (Jacobs & Rubery 1988, Brown et al. 2001, Peer &
Murphy 2007, Brunetti et al. 2013). Auxin is also possibly guided into the nucleus by catechins (Talaat et al. 2014). In addition, the chromosomes can act as a target for flavanols (Polster et al. 2003).
Catechin and epicatechin may also interact with histone proteins, playing a key structural and regulatory role in packing DNA into chromosomes (Polach & Widom 1996, Mueller–Harvey et al. 2012).
Phenolics have a role in defending plants against biotic threats:
insects (e.g. Ossipov et al. 2001, Ruuhola et al. 2001), fungi and pathogens (Nicholson et al. 1987, Dixon & Paiva 1995, Harborne 1999). As well as phenolics have interactions with plant- mycorrhiza symbiosis and as chemical signals in symbiotic nitrogen fixation with legumes (Beyeler & Heyser 1997, Aoki et al.
2000, Ceccarelli et al. 2010). Many different factors, including plant species, tissue type and also the structural variations of phenolics, have a strong impact on ecological processes (e.g. Fierer et al. 2001, Nierop et al. 2006). Flavonoids provide protection against herbivores and microbes, also playing important roles in interactions with free radicals and reactive oxygen species and in avoidance of damaging oxidation of proteins, cell membranes and DNA (Pourcel et al. 2007, Bowsher et al. 2008, Agati & Tattini 2010, Agati et al. 2012, 2013, Cheynier et al. 2013).
Anthocyanins, which contribute to the colouring of leaves, stems, fruits and flowers, giving them red, blue, purple and brown pigmentation (e.g. Winkel-Shirley 2002, Bowsher et al. 2008), are induced by the influence of various stresses (Dixon & Paiva 1995, Winkel-Shirley 2002), including cold or limitation of macronutrients (such as phosphorous or nitrogen) (Dixon & Paiva 1995, Hilbert et al. 2003, Hernández & Munné-Bosch 2015).
Anthocyanins may also have a function in photoprotection as well as in resistance to herbivores and pathogens in leaves (Gould 2004).
In addition, spectral composition of light affect strongly to anthocyanin accumulation (Zoratti et al. 2014).
Polymeric PAs, one endproduct of the phenylpropanoid pathway are complex compounds that influence many biological processes in both terrestrial and aquatic ecosystems (Schweitzer et al. 2008). Like hydrolyzable tannins (HTs), PAs are protein-binding agents (Hagerman 1992). Although PAs and HTs are chemically distinc, they both act as feeding deterrents and are also toxic for many insects and mammals (e.g. Hagerman 1992, Bowsher et al.
2008, Quideau et al 2011).
1.3 CHANGING ENVIRONMENT AND JUVENILE PHASE AFFECT PHENOLICS
The production of phenolics in woody and herbaceous plants shows responses to changing environmental conditions and the ontogenic phase (e.g. Bryant et al. 1983, Muzika & Pregitser 1992, Laitinen et al. 2005a, Zvereva & Kozlov 2006, Veteli et al. 2007, Paajanen et al. 2011, Onyango et al. 2012, Lavola et al. 2013).
Nutrients and other environmental factors, such as temperature, elevated UV-B radiation, drought stress, heat, salinity, ozone stress, cold and CO2, affect the compound spectrum and content of phenolics as well the growth of plants (e.g. Dixon & Paiva 1995, Lavola et al. 1997, Rivero et al. 2001, Saleem et al. 2001, Tegelberg et al. 2001, Tattini et al. 2004, Hale et al. 2005, Bidart-Bouzat &
Imeh-Nathaniel 2008, Paajanen et al. 2011, Suzuki et al. 2014).
19 The juvenile phase is a critical time during a plant´s life history.
Plants go through processes of physiological aging from the juvenile to the mature phase (Hanley et al. 2004). The juvenile phase is defined as an immature developmental stage in which the plant does not flower, even though the environmental conditions are suitable for flowering (Boege & Marquis 2005).
1.3.1Direct and indirect effects of temperature on phenolics Temperature is a crucial factor that affects chemical reactions and plant production rate, both at the cellular and at the whole organism level (Berry & Björkman 1980, Luo 2011). Temperature influences, for example, vegetative growth, flowering, seasonal acclimation and accumulation of flavonoids (Veteli et al. 2002, Welling & Palva 2006, Miller-Rushing & Primack 2008). Plants have adapted to potential changes in temperature and can adjust to conditions around their optimum range by inducing numerous genetically regulated mechanisms (Berry & Björkman 1980, Hasdai et al. 2006). It is predicted that through global climate change, the average temperature will rise by 0.3-5°C above pre-industrial levels, and extreme weather conditions will become more frequent (IPCC 2014). Increasing global temperatures are challenging for plants’ biology and nutrient demands, because the distribution of plant species is incapable of keeping pace with global change (Lukac et al. 2010). Generally, phenolic levels decrease in response to increased temperature (Kuokkanen et al. 2001, Hansen et al. 2006, Veteli et al. 2007, Huttunen et al. 2008, Paajanen et al. 2011).
However, alterations in concentrations of phenolics are often species-specific (e.g. Hansen et al. 2006).
Temperature increases may considerably modify the dynamic interaction between plants and herbivores via changing defensive compounds. Lower concentrations of phenolics can directly enhance the performance of insect herbivores (Bale et al. 2002). On the other hand, the alterations may also modify the quality of plants as food for herbivores (Veteli et al. 2002, Lemoine et al. 2013).
Warming is also expected to accelerate leaf maturation, and as a consequence, further change the quality of plants as food for herbivores (Buse et al. 1998). As a result of global warming, insect
herbivores’ consumption, growth and survivorship may alter, suggesting that herbivory can impact plant communities with interaction between insects and plants (Lemoine et al. 2013, Heimonen et al. 2015). Rising temperatures accelerate insect metabolism and thus they demand more food, and moreover, the number of generations produced per season increases (Bale et al.
2002). Insects cause defoliation, which usually causes a decrease in concentrations of total phenolics under elevated temperature (Huttunen et al. 2008). Herbivore density should increase with rising temperatures (Zvereva & Kozlov 2006, Kozlov 2008, Garibaldi et al. 2011), although opposite results have also been found, especially among tundra species (Barrio et al. 2016).
1.3.2Nitrogen effects on phenolics
Nitrogen, the most necessary inorganic macronutrient, affects all levels of plant function, from growth and metabolism to resource allocation and developmental processes (Crawford 1995, Stitt &
Krapp 1999). Nitrogen is also an essential building block of amino acids. Scheible et al. (2004) have found that a nitrogen addition induces the genes for biosynthesis of amino acids, while repressing many of the genes involved in phenolics metabolism in Arabidopsis.
Generally, plants growing in conditions of poor nitrogen levels have more phenolics than do plants growing in nitrogen-rich conditions (Bryant et al. 1983, Herms & Mattson 1992, Jones &
Hartley 1999). Especially in birches a decrease in soluble PAs and flavonoids has been found as a result of increased nitrogen fertilization (Lavola & Julkunen-Tiitto 1994, Keski-Saari &
Julkunen-Tiitto 2003, Keski-Saari et al. 2005). On the other hand, foliar PA in developing leaves of Populus, impacts nitrogen and carbon utilization and distribution in the plant’s tissues (Harding et al. 2014).
1.3.3Ontogeny influences phenolics
The juvenile period before flowering is an important time for plant resource allocation and further growth (Hanley et al. 2004), and
21 during that time, plants are most vulnerable to external threats, such as herbivory (e.g. McCall & Fordyce 2010). The content of phenolic compounds often changes as plants grow and mature (e.g.
Bryant & Julkunen-Tiitto 1995, Laitinen 2005a, b, Boege & Marquis 2006). Young perennial plants are more likely than older plants to be restricted in their acquisition, storing and allocation of resources to multiple functions (e.g. growth and resistance). Differences in phenolic content at different developmental stages have been found in several species, such as Populus sp. (Rehill et al. 2006), Hypericum sp. (Ayan et al. 2006), Malus domestica (Treutter 2001) and Betula sp. (Laitinen et al. 2000, Riipi et al. 2002, Liimatainen et al. 2012). Concentrations of phenolics may diminish or increase during ontogeny, depending on the plant species (Basey et al. 1988, Laitinen et al. 2005a, b, Holeski et al. 2012).
Generally, juvenile individuals have been found to be better defended than older plants (Bryant & Julkunen-Tiitto 1995). A possible reason for that is the protection need during the highly vulnerable developmental stage (Reichardt et al. 1984, Bryant &
Julkunen-Tiitto 1995). Especially one reason for the ontogenic variation is thought to be involved in defense against herbivores (Rousi et al. 1989, Boege & Marquis 2006, Elger et al. 2009).
1.4 DEFENSIVE STRUCTURES AND CHEMICALS IN SALICACEAE AND BETULACEAE
Plants have different strategies for defending themselves against different threats, and these strategies may be used concomitantly.
Morphological structures, together with chemical defenses, may directly prevent the feeding of certain herbivores (Lucas et al. 2000, Martin & Glover 2007, Howe & Schaller 2008). Wax layers, trichomes or resin droplets are important structures for defense in trees. All these structures have been found in the Salicaceae and Betulaceae families (Curtis & Lersten 1978, Lapinjoki 1991, Vanhatalo et al. 2001, Langenheim 2003, Tomaszewski 2004).
Chemical defenses in both Betulaceae and Salicaceae families include low-molecular weight phenolics, such as hydroxycinnamic acids, flavonoids, PAs and phenolic glucosides (PGs). Many species belonging to the Salicaceae family, such as aspens and willows, form salicylates, in very large amounts (Julkunen-Tiitto 1986, Lindroth & Hwang 1996, Ruuhola et al. 2001). The Betulaceae family includes birches, alders and hornbeams. Salicylates have been found in only some of the Betulaceae species (Gardner &
McGuffin 2013). Especially the flavonols quercetin, myricetin and kaempferol function as antioxidants and UV screen in birches (Lavola et al. 1997, Tegelberg et al. 2001, Anttila et al. 2010).
Aspens are mainly characterized by their production of PAs and PGs, these compounds comprising 10 to 35% of aspen leaf dry mass (Randriamanana et al. 2014, Randriamana et al. 2015). PGs in particular are constitutively maintained in leaves and bark (e.g.
Julkunen-Tiitto 1985, 1986, 1989, Lindroth & Hwang 1996, Hakulinen et al. 1999, Osier & Lindroth 2001, Ruuhola et al. 2001).
The leaves of aspens also accumulate anthocyanins and numerous small phenolic acids (Lindroth & Hwang 1996, Randriamanana et al. 2015). The PGs in particular, such as salicortin and tremulacin, have a role in reducing the performance of generalist aspen insect herbivores (Hwang & Lindroth 1997, Osier & Lindroth 2001, Donaldson & Lindroth 2007, Philippe & Bohlmann 2007). High PG levels also decrease the consumption of willow or aspen leaves and/or stems by mammalian herbivores, such as hares and voles (Tahvanainen et al. 1985, Heiska et al. 2007, Boeckler et al. 2011).
In birch, the majority of leaf phenolics are tannins, especially PAs, which may constitute over 10% of the dry mass of leaves (Ossipov et al. 2001, Witzell & Martin 2008). Birches are also rich in PGs, flavonoids, and terpenoids, and to a lesser extent, hydrolyzable tannins (gallotannins) (e.g. Tahvanainen et al. 1991, Laitinen et al. 2005b, Salminen et al. 2001, Riipi et al. 2002).
Previously, phenolic compounds have been found in the pollen, buds, leaves, bark, cotyledons and roots of birches (Julkunen-Tiitto et al. 1996, Rozema et al. 2001, Keski-Saari & Julkunen-Tiitto 2003).
Phenolic compounds occur in the glandular trichomes (Strack et al.
1988, Tattini et al. 2007). The glandular trichomes (resin glands) of
23 birches contain mainly terpenes, which reduce the browsing of snowshoe hares (Lepus timidus) (Reichard et al. 1984, Tahvanainen et al. 1991, Rousi et al. 1991). The accumulation of phenolic compounds in birches is strongly affected by environmental factors, but also by genetic control and genotype (e.g. Rousi et al.
1989, Lavola & Julkunen-Tiitto 1994, Laitinen et al. 2000, Kuokkanen et al. 2001, Tegelberg et al. 2001, Veteli et al. 2002, Morales et al. 2010). In addition, seasonal variation, developmental stage and age also affect the phenolic concentrations and also composition of birches (Palo et al. 1985, Julkunen-Tiitto et al. 1996, Keinänen et al. 1999a,b, Riipi et al. 2004, Laitinen et al. 2005b).
Phenolics have been found to be a taxonomically significant tool for species recognition, especially among Betula species (Julkunen- Tiitto et al. 1996, Keinänen et al. 1999b, Valkama et al. 2003). Betula pubescens contained several flavonoids that are different compared with other Betula species (Keinänen et al 1999b). B.
pendula contained several myricetin derivatives compared to B.
pubenscens while B. pubenscens contains more kaempferols than B. pendula (Keinänen & Julkunen-Tiitto 1998)
Defensive phenolics have many important ecological functions in the Betulaceae and Salicaceae families. It is probable that new roles can be found for them in future.
1.5 PLANT RESOURCE ALLOCATION THEORIES
Allocation theory argues that plants have limited essential resources, which they have to allocate between different competing physiological functions, such as metabolic functions, reproduction and growth (Herms & Mattson 1992, Boege &
Marquis 2005, Caretto et al. 2015). Trade-offs between defense and growth may be transient (Orians et al. 2010), and processes of allocation and trade-offs between various activities and functions are thought to be improved by natural selection (Caretto et al. 2015).
Many theories/hypotheses have been formulated on carbon allocation between different physiological functions for plant defense.
The carbon-nutrient balance hypothesis (CNB) (Bryant et al.
1983) and growth-differentiation balance hypothesis (GDB) (Herms & Mattson 1992) are extensively used hypotheses on plant carbon and nutrient allocation for defense and growth. These hypotheses postulate that fertilization with growth-limiting nutrients will lead to decreased concentrations of phenolics (Bryant et al. 1983). Photosynthesis is not as sensitive as growth to limitation of water or nutrients, and thus moderately low resource availability has only a minor effect on photosynthesis, while it reduces growth and increases phenolic production (Herms &
Mattson 1992). In other words, these hypotheses predict that growth is limited by the absence of carbon or nitrogen, and the increased production of defensive compounds is connected with reduced growth (Massad et al. 2012).
The CNB hypothesis arises from attempts to explain differences in plant defenses against herbivory (Bryant et al. 1983). CNB suggests a trade-off between growth and the production of phenolics and other carbon-based defensive compounds.
According to CNB, low nitrogen levels limit the growth of plants, and plants allocate the extra assimilated carbon to the production of carbon-based metabolites (Bryant et al. 1983, Tuomi et al. 1990).
The CNB hypothesis has its limitations, for example problems with interpretation of the trade-off between growth and the production of phenolics (Hamilton et al. 2001). Owing to the limitations of CNB, another model, the protein competition model (PCM), has been developed (Jones & Hartley 1999). PCM is also a developmental system model, suggesting a trade-off between proteins and phenolic production. This hypothesis suggests that protein and phenol synthesis are in direct competition for the common branch-point enzyme L-phenylalanine. This enzyme is a building block for proteins that are needed both for carbon assimilation (e.g. photosynthesis) and growth but also serves as the precursor for the phenylpropanoid pathway (Haukioja et al. 1998, Jones & Hartley 1999). The PCM model predicts that trade-offs for carbon and also for nitrogen probably occur among and within metabolic pathways. The GDB hypothesis predicts resource allocation to growth or to differentiation processes, such as
25 production of phenolics, trichomes and thorns or production of reproductive organs (Herms & Mattson 1992). GDB is based on a trade-off between growth and differentiation, and it takes into account the interaction between photosynthetic rate and growth (Herms & Mattson 1992).
These hypotheses are conceptual models wherein impacts of the environment on plants’ internal demands for carbon, nitrogen and other nutrients have been taken into account. There is evidence that hypotheses can change within the species, depending on the phenolic groups under study (e.g. Randriamanana et al. 2014). In dioecious plants, differences have been found even between different sexes, since females of the aspen (P. tremula L.) invested more in phenolics, while males invested more in growth (Randriamanana et al. 2014).
1.6 GENETIC MODIFICATION TECHNIQUES FOR REGULATION OF THE PA PATHWAY
To gathering new information of biochemical pathways genetic modifications provides a modern technique. The regulation and synthesis of phenylpropanoids have been studied using genetic modification techniques (e.g. Dixon et al. 1996, Mellway et al. 2009).
Several genetic modification methods have been used to downregulate or upregulate the gene expression in plants.
Downregulation of endogenous genes is accomplished by silencing the target genes by means of antisense expression, post- transcriptional gene silencing, virus-induced gene silencing or RNA interference (RNAi) methods (Dixon 2005, Dixon et al. 2013).
1.6.1 Regulation with transcription factors
Transcription is regulated by proteins. These transcription factors can be expressed or silenced in order to control a known trait using the genetic modification technique. One member of the MYB transcription factor family is R2R3 MYB, which is involved in the regulation of the phenylpropanoid metabolism (Stracke et al. 2001).
The R2R3 MYBs constitute large gene families in plants, with 192
in poplar (Wilkins et al. 2009). R2R3 is subfamily for MYB protein MYB134 in Populus (Mellway et al. 2009, Li et al. 2015).
Transcription factor gene MYB134 is coinduced with PA biosynthetic genes. MYB134 upregulates the genes encoding the known enzymes (e.g. PAL, 4-coumarate-CoA, cinnamate 4- hydroxylase DFR, ANR, ANS, LAR) of general flavonoid and PA biosynthesis and activates the genes ANR, ANS, DFR and LAR in PA synthesis in Populus tremula x tremuloides and Arabidopsis thaliana (Nesi et al. 2001, Kao et. al 2002, Mellway et al. 2009). In an upregulation method, a constitutive and strong promoter such as cauliflower mosaic virus (CaMV) 35S is usually used (Benfey &
Chua 1990, Terada & Shimamoto 1990, Yang & Christou 1990). This promoter enhances transcriptional activation and causes overexpression of the genes. A CaMV 35S promoter with the coding sequence of MYB134 and the correct transcriptional enhancer sequence and terminator sequence has been used to create GM Populus lines (Mellway et al. 2009). Constitutive expression of MYB134 in Populus results in overexpression of the PA pathway genes and thereafter, increased production of PAs and reduced levels of salicylates (Mellway et al. 2009, Boeckler et al. 2014).
1.6.2 Downregulation by the RNAi method
The RNA interference (RNAi) method is a way of gene downregulation and it was reported for the first time in petunia (Napoli et al. 1990, Sen & Blau 2006). Napoli et al. (1990) determined that CHS is a key enzyme in flavonoid biosynthesis and they used the RNAi method to affect anthocyanin biosynthesis.
RNA silencing is thought to be based on the same mechanism in animals and plants (Voinnet 2001, Hannon 2002). RNA silencing inhibits translation or cleaves mRNA. The RNAi method is based on the ancient ability of cells to recognize and defend themselves against a virus (Sharp 2001). Restriction of virus growth in plants is normally associated with post-transcriptional gene silencing, which can be initiated by the production of double-stranded RNA (dsRNA) replicative intermediates. The silencing process is initiated using dsRNA which bears the known gene sequence. The
27 dsRNA is cleaved into short, interfering RNAs (siRNA), which leads further to cleavage of the target mRNA (Zamore et al. 2000).
This is followed by degradation of the target mRNA and silencing of the expression of the target gene. Downregulations of the gene IbDFR using an RNAi have been carried out in Sweet Potato (Ipomoea batatas Lam.), resulting in reduced anthocyanin accumulation (Wang et al. 2013a). In this thesis, the decreased PA concentration has been achieved by the RNAi method (II).
1.7 AIMS OF THE THESIS
The main focus of this doctoral work was to study changes in the phenolic pathway, especially PAs, and the growth of two common deciduous plant species in relation to pre-reproductive time (I), nitrogen availability (II) in early flowering birches (Betula pendula Roth.), and temperature (III) in aspens (Populus tremula × P.
tremuloides). Throughout the whole thesis I kept in mind a question based on plant defense theories (CNB, GDB and PCM): Would it be possible to increase the growth of the plants if the concentration of phenolics could be reduced? The tools I used in the thesis were the GM hybrid aspen (Populus tremula × P. tremuloides) (III) and the early flowering variety of silver birch (B. pendula Roth.) (I, II).
Concentrations of PAs were increased in hybrid aspen by overexpression of PtMYB134, which activates the genes ANR, ANS, DFR and LAR in the PA pathway (III). Concentrations of PAs were inhibited in the early-flowering silver birch using the RNAi method, by producing ANRi lines (II). The wild type (WT) of early- flowering birch was used also in the study (I). In addition, the palatability of hybrid aspen (III) to the leaf beetle (Phratora vitellinae) was investigated.
The following questions were studied:
1. How does the development in pre-reproductive time change the phenolic concentration and gene expression of genes in the PA pathway of the early-flowering birch (I)?
2. How does the decreased concentration of PAs affect concentrations of other phenolics in the phenolic pathway in birch (II)?
3. How does the altered accumulation of phenolics affect the growth of plants at different temperatures (III) or different nitrogen levels (II)?
4. Do the genetically modified plants react similarly to changes in different environmental conditions (temperature and nitrogen) as compared with WT trees (II, III)?
5. How do possible alterations in the phenolics of the leaves grown at different temperatures affect the food preferences of the specialist beetle herbivore (III)?
29
2 Materials and methods
2.1 STUDY ORGANISMS
Birch (Betula) and aspen (Populus) species are valuable deciduous trees in northern forests both ecologically, as a host and as a food source for herbivores and parasites, and economically, as sources for the papermaking industry (Ylitalo 2013). Silver birch (B.
pendula) has adapted to a range of different climates, and is found all across Europe and northern Asia, from China to southwest Asia. The hybrid aspen P. tremula x tremuloides is also a widely distributed and cultivated tree in the Northern Hemisphere.
2.1.1 Birch (B. pendula Roth.) (I, II)
Clonal, micropropagated plantlets of the early-flowering silver birch clone BPM5 (Stern 1961) were used in this study: the WT (I, II) and three ANRi-lines (1179, 1183 and 1201) (II). The genetic modification of birch was carried out by RNAi performed according to Keinonen-Mettälä et al. (1998). The silencing process is initiated using dsRNA which bears the known gene sequence.
The clone Q5FB34 that codes for ANR was selected from the birch expressed sequence tag (EST) library (Aalto & Palva 2006). The gene-specific region of 170 bp (nucleotides 679-849 from the translation start site) was selected from the BpANR sequence and amplified using PCR analysis. Detailed methods are found in article (II). The reporter gene in a binary vector was tranferred into birch BPM5 with Agrobacterium tumefaciens strain LBA4404 (Vancanneyt et al. 1990, Keinonen-Mettälä et al. 1998). After antibiotic selection of the transgenecity of each birch line was confirmed by PCR (Lemmetyinen et al. 1998). Ten kanamycin- resistant and PCR-positive lines were tested using quantitative real-time PCR (qRT-PCR) for the function of the transgene. Three
of these lines (1179, 1183, and 1201) with reduced level of ANR mRNA. The inhibition of the ANR gene is assumed to lead to the reduction of PA formation and to an increase in flavonol glycosides. Genetic modification has made doctor Mika Lännenpää.
2.1.2 Aspen (P. tremula x tremuloides) (III)
The plant material consisted of clonal, micropropagated plantlets from five lines of the INRA 353-38 clone of hybrid aspen: wild type (WT), GUS 41 (beta-glucuronidase gene, GUS), and three independently generated MYB 134 overexpressors (lines 46, 54 and 61). The genetically modified GUS line (pRD410), which serves as a control for the genetic modification, contains the GUS marker gene under the cauliflower mosaic virus promoter (CaMV 35S) (Datla et al 1992, Mellway et al. 2009). The lines (46, 54 and 61) contain the MYB134 gene under the 2X CaMV 35S promoter that was transferred using the Agrobacterium tumefaciens strain C58 (pMP90) (Mellway et al. 2009). Genetic modification in aspens were provided by the group of Professor Peter Constabel.
2.1.3 Leaf beetle (Phratora vitellinae) (III)
P. vitellinae is a chrysomelid herbivore, which is widely distributed in Europe (Pasteels et al. 1983, Pasteels & Gregoire 1984). Beetles are specialized in using salicylates for their own or their offsprings' defense (Pasteels et al. 1983, Pasteels & Gregoire 1984). P. vitellinae can produce a defensive secretion, salicylaldehyde to protect itself (Rowell-Rahier 1984a,b, Tahvanainen et al. 1985, Rowell-Rahier & Pasteels 1986, Kolehmainen et al. 1995, Rank et al. 1998).
P. vitellinae chews the leaves of various tree species which are rich in salicylates and poor in PAs (Tahvanainen et al. 1985, Veteli et al. 2002). Their entire life cycle is often spent on the same Salix or Populus species. P. vitellinae for cafeteria experiments were collected from a field population of Salix myrsinifolia in Eastern
31 Finland. The beetles were fed with leaves of S. myrsinifolia prior to the experiment.
2.2 EXPERIMENTAL PROCEDURES
2.2.1 Experiments and treatments
The thesis included the following four experiments: Two experiments with early-flowering silver birches (the effect of pre- reproductive time to phenolics (Experiment I) and the effect of inhibition of the anthocyanidin reductase gene (ANR) on growth and phenolics at two nitrogen levels, Experiment II). Two cafeteria experiments have been done with leaf beetle (Phratora vitellinae) in the hybrid aspen (five-choice experiment and dual- choice experiment). A list of the treatments, measured variables and studied plantlet tissues in each article included in this thesis is organized in Table 1. Detailed methodologies are provided in the articles in question.
Table 1. Outline of the plant material treatments and methods in each article.
Article I II III
Material
Early-flowering birch (Betula pendula Roth.)
Early- flowering birch (Betula pendula Roth.) ANRi lines 1179, 1183 and 1201
Hybrid aspen (Populus tremula x tremuloides), MYB134
overexpressing lines 46, 54, 61 and GUS line
Leaf beetle
(Phratora vitellinae) Plant tissues
used
Leaves, stems and
roots Leaves and stems Leaves
Treatment Growth stages Nitrogen level Elevated temperature
Treatment levels
Four experimental points before flowering
Low and optimum
nitrogen level Ambient and +2°C
Measurements
Phenolics, Growth,
Gene expression, Biomass of leaves and stems
Phenolics, Growth, Gene expression, Chlorophyll index, Biomass of leaves and stems, Foliar nitrogen concentration, Number of resin glands
Phenolics, Growth,
Leaf area consumed by beetles, Foliar nitrogen concentration, Biomass of leaves
2.2.2 Chemical analyses (I, II, III)
Phenolics from air dried leaves (II and III) and stems (II) and freeze-dried leaves, stems and roots (I) of the saplings were extracted using cold methanol. The biggest, fully developed leaves and a part of stem were taken from the middle part of birch stems (I, II) in order to obtain comparable sampling, since it is known that phenolic concentration may differ between younger and more mature leaves (Laitinen et al. 2005a). The phenolic concentrations of the samples were analyzed by high pressure liquid chromatography (HPLC) (Julkunen-Tiitto and Sorsa 2001).
The concentrations of each compound were calculated on the
33 basis of commercial or purified standards. The leaf and stem compounds were identified using ultraviolet spectra, retention times, and HPLC-MS (API-single quadrupole mass- spectrometry). The conditions for the MS analysis were according to Julkunen-Tiitto & Sorsa (2001). For root compounds (I) the UHPLC-quadrupole time-of-flight mass spectrometer (UHPLC Q-TOF/MS) was used. The conditions for the UHPLC Q-TOF/MS were as described in Randriamanana et al. (2014).
Soluble PAs were determined from the dissolved methanol extract, and insoluble PAs were determined from the extraction residue by acid-butanol assay, as described in Hagerman (2011).
The concentrations were calculated on the basis of the standard curve from purified tannin of Betula nana (I, II) and Populus tremuloides (III) leaves.
The total nitrogen content of the leaves was analysed using LECO FP-528 equipment (II, III). The chlorophyll index was measured from mature leaf blades using a CCM-200 chlorophyll meter (II).
2.2.3 Growing and sampling of the birches (I, II)
All the birch experiments were carried out at the same greenhouse in Joensuu, Eastern Finland. Micropropagated plantlets of the birch development experiment (I) (early- flowering birch clone BPM5) and the ANRi birch experiment (II) were planted in 0.08 dm3 pots (5 x 5.5 x 5 cm depth) containing vermiculite: fertilized Sphagnum peat (50:50) (Kekkilä, Finland, N:P:K=10:8:16) and they were acclimated for two weeks to 55%
relative humidity, at about 23°C. After acclimation, the plantlets were transferred to bigger pots. To avoid any site effects in the greenhouse, the positions of the pots were changed twice a week.
In the birch development experiment (I), fertilizer (Kekkilä, taimisuperX, N:P:K 19:40:20, Finland) was added twice a week in solutions approximating a total N input of 6 kg ha-1 year-1 (pH adjusted to 5.6–5.7). The roots, stems and leaves were sampled four times during the experiment.
Two levels of nitrogen for the ANRi birch experiment (II) were set according to Ingestad (1971): at the optimal level, 40.0 g N (12
ppm NH4NO3) dissolved in 10 l water, and at the low level 1.2 g N (400 ppm NH4NO3) dissolved in 10 l water. The birches were fertilized with 100 ml of the solution per pot seven times during the experiment. Other fertilizers were added as described in Ingestad (1971). The experiment continued for 10 weeks after the acclimation of the plants and was carried out in spring-time. The birches were fertilized every week, and the pH was adjusted to 5.6–5.7.
2.2.4 Gene expression analysis (I, II)
Gene expression levels were analyzed by qRT-PCR. For qRT-PCR, total RNA was extracted from birch leaves, stems and roots and reverse-transcribed to cDNA (II). Primers for quantification of the expression level of BpANR BpANS, BpDFR, BpLAR were selected outside the sequence used in the RNAi construct. Relative quantification was performed using birch 18S ribosomal RNA gene as an internal reference gene.
2.2.5 Cafeteria experiments: five-choice experiment and dual-choice experiment with aspen (III)
The micropropagated and acclimated plantlets of aspen were grown in sixteen chambers at the Mekrijärvi research station.
Eight growth chambers were kept at real-time ambient temperature and eight chambers at elevated temperature (ambient + 2 °C). The plantlets were fertilized as described in Häikiö et al. (2009).
Nine week-old plantlets were sampled for the chemical analyses and the cafeteria experiments for Phratora vitellinae food intake after 26 days of temperature treatment. In the five-choice experiment, the mature leaves of each plant were offered to the beetles in order to study the differences between the lines in terms of their palatability to the herbivore.
The food intake of five different aspen lines (WT, GUS, MYB:
46, 54 and 61) was tested as a five-choice experiment with aspens grown in ambient temperature. The experimental setup for
35 feeding is shown in Figure 1a. The beetles were allowed to feed for 24 hours at constant room temperature (22±2 oC).
The effect of the temperature at which the aspens were grown on the food intake of P. vitellinae was tested in a dual-choice experiment for three lines (WT, MYB lines 54 and 61). Two randomly selected aspens grown under both temperature conditions were used for each line (n = 8 = chambers). Hence, the experiment included 48 beetles (three lines, eight chambers, two aspens). The experimental setup for feeding can be seen in Figure 1b. The leaves were eaten through two holes by beetles. The beetles were allowed to feed for 26 hours.
Figure 1 Experimental setups for five-choice experiment (a) and dual- choice experiment (b).
2.2.6 Statistical analysis
All the statistical analyses were conducted with IBM® SPSS®
Statistics 19.0. The effect of the sampling point on the chemistry of the leaves stems and roots (I) was analyzed using Univariate ANOVA. The effect of reduced expression of BpANR (II) on the chemistry at two different N levels was analyzed, and the effects of temperature and plant line (III) on concentrations of phenolic compounds was analyzed using Multivariate ANOVA. Since the main results of MANOVA were significant (III), the individual compounds were tested using mixed model analysis.
Graphic vector analysis (GVA) was used to explain the association between mass accumulation caused by growth and phenolic chemistry (I) (Haase & Rose 1995, Koricheva 1999, Veteli
et al. 2007, Virjamo & Julkunen-Tiitto 2014). Interpretation of direction of GVA arrows is shown in Figure 1 in Article I.
37
3 Results and discussion
3.1 PHENOLICS BEFORE THE FLOWERING OF SILVER BIRCH (BETULA PENDULA)
The juvenile stage often differs from the mature stage with regard to physiological characteristics as well as phenolic composition.
In early-flowering birches, the lowest concentrations of all phenolics were found before the beginning of flowering in the leaves and stems (I), while the lowest concentration of phenolics in the roots was found already before planting in peat (I). Before flowering time, the growth of the birches was linear, while concentrations of phenolics decreased in the leaves and stems.
The dilution effect in phenolics was observed as a huge increase in biomass, mainly in the WT birches (I) (Figure 2a).
Figure 2.
Change in relative content to concentration ratio of total phenolics.
Open marks refer to leaves and closed marks to stems.
a) WT during maturation (first sampling as the reference point) (I).
Arrows represent time parameters from the first sampling to the last harvesting.
b) ANRi birches compared to WT (WT as the reference point). The results in Figure 2b are recorded at the optimum fertilization level.
The dilution effect of phenolics occurred earlier in the development of the leaves than in the development of stems of WT birches (Figure 2a). This is explained by the rapid leaf area increase, which is important, and even expected, for plant photosynthesis over the growth period.
However, some individual phenolics were accumulated, such as certain flavonol glycosides in the leaves, and salidroside and DHPPG in the stems (I). DHPPG is assumed to be associated with defense agents, especially in young leaves of birches (Keinänen et al. 1999a, Laitinen et al. 2002). By contrast, a concentration effect was observed in the ANRi birches, while concentrations of PAs were decreased (II), indicating that the small plants accumulated other phenolics and the concentration effect was considerable (Figure 2b). Previous studies have shown increased concentrations of PAs from seedling phase towards reproductive age in field-grown B. pendula (Tegelberg et al. 2001, Laitinen et al.
2005b). PA concentration is fairly sensitive to environmental conditions (Osier & Lindroth 2001, 2006), and this may be the reason for variations between studies.
The increased synthesis of total phenolics was found in the stems at the beginning of the experiment (Figure 2a) (I). Similarly, in roots the concentrations of phenolics increased (I). This may partly be due to the stress reaction induced by radical change in the growth environment. The change might have been induced when very juvenile birches were transferred from closed micropropagation dishes to the open pots containing Sphagnum peat and moved to the greenhouse. Several species have been found to increase the formation of phenolics when the growing environment has become more stressful (Bautista et al. 2016, Tattini et al. 2004). Another partial explanation may be a phase change of juvenile stems to a more mature stage accompanied by normal constitutive development of the phenolic content.
Gene expressions of the studied genes change slightly during the pre-reproductive time, but no clear connection can be found between the gene expressions of BpANR BpANS, BpDFR, BpLAR and PA or the precursors formation of PA (Figures 4 and 5 in article I). The reason for this may be that the regulation of the
39 phenolic pathway occurs post-translationally. Several studies have found that, between the gene expression and target enzyme, have not been correlation (Morse et al. 2009, Vogel & Macorette 2012, Agarwal et al. 2016). The lack of correlation may due to the possible role for co-regulators that affect via post-translational modification (Agarwal et al. 2016). There are found both negative and positive post-translationally regulation in phenolic formation caused by photosynthesis and amino acids in Arabidopsis (Lillo et al. 2008) and by UV-light in maize (Zea mays) (Casati et al. 2005). Interactions between multiple genes and transcription factors form a complicated network, and there is not necessarily a direct connection between gene expression and the end products (Dale & von-Schantz 2007).
3.2 HOW DO THE DOWNREGULATION AND OVEREXPRESSION OF THE PA PATHWAY GENES AFFECTED PHENOLICS?
On the basis of my results, it seems that genetic modifications in one gene of the PA pathway caused changes throughout the phenolic pathway. Previously, it has been found that GM plants may show unintended changes in other genes (e.g. Hjältén et al.
2007). These changes have been reported to induce non- predictable traits, such as growth changes (Hjältén et al. 2007, Axelsson et al. 2011). Genetic modification may also cause pleiotropic effects, if the locations and the numbers of the transgene are not completely controlled (Novak & Haslberger 2000).
Nevertheless, ANRi birches provided new and surprising changes in the concentration of phenolics and also in responses to nitrogen levels in the stems (II). Further, the ANRi birches showed a strong decrease in growth, and biosynthesis in the phenolic pathway turned towards phenolic acids and flavonols (Figure 3, II). Overexpression of the MYB134 gene resulted in the accumulation of PAs in hybrid aspen (Mellway et al. 2009, III) and in decreased content of PGs, which affected leaf consumption by leaf beetles (Figure 3, III).
The competition for biosynthetic precursors within the phenolic pathway may due to trade-off in P. tremuloides (Donaldson et al. 2006, Osier & Lindroth 2006). Especially environment with limited resources, such as low fertilization or weak light availability have caused trade off between PAs and PGs in several genotypes in P. tremuloides. (Donaldson et al. 2006, Osier & Lindroth 2006).
3.2.1 Downregulation of ANR caused alterations of chemotype and phenotype
Gene inhibition causes silencing or damage of a known gene. In other words, mRNA level is reduced, but not completely eliminated (Mocellin & Provenzano 2004). Inhibition of ANR gene caused dramatic changes in the phenolic pathway in B.
pendula. Concentrations of PAs decreased clearly in response to inhibition of the ANR gene. Similarly, antisense downregulation of the ANR gene in Populus trichocarpa has been found to cause a decrease of PA concentration and an increase of anthocyanidins in the leaves of Populus (Wang et al. 2013b). By contrast, concentration of flavonols accumulated in the studied tissues and the concentrations of several other compounds of the phenolic pathway changed (II) (Figure 3). As expected, most of the upstream phenolics of the phenolic pathway, such as flavonol glycosides, accumulated as much as ten times more in ANRi birches compared to WT due to PA inhibition (Figure 3 red and blue arrows). However, only kaempferols decreased due to ANR inhibition (Figure 3). There were also several phenolic compounds in ANRi birches that were not detected in WT birches (II). ANR inhibition caused alterations in the chemotype of the birches and several steps of the phenyl propanoid route changed.
Thus the available carbon was allocated differently in different parts of the pathway than normal (Figure 3). Generally, ANR genes play a crucial role in PA synthesis in all plant organs in different plant species (Kovinich et al. 2012, Wang et al. 2013b).
Leaves and stems responded differently to downregulation of ANR (II), both in chemistry (Figure 3 red and blue arrows, II) and
