NON-TARGET EFFECTS OF GENETICALLY MODIFIED

Patrik Blomberg

2007

Department of Ecology and Environmental Science Umeå University SE-901 87 Umeå Sweden

AKADEMISK AVHANDLING som med vederbörligt tillstånd av rektorsämbetet vid Umeå universitet för erhållande av filosofie doktorsexamen i ekologi kommer att offentligen försvaras lördagen den 6 oktober 2007, kl. 10.00 i Lilla hörsalen, KBC.

Examinator: Professor Lars Ericson, Umeå universitet

Opponent: Professor Jan Stenlid, Institutionen för skoglig mykologi och patologi, Sveriges lantbruksuniversitet, SLU, Uppsala

ISBN: 978-91-7264-391-8 © PATRIK BLOMBERG 2007 Printed by VMC, KBC, Umeå University, Umeå 2007

ORGANISATION DOCUMENT NAME Dept. of Ecology and Environmental Science Doctoral Dissertation Umeå University DATE OF ISSUE SE-901 87 Umeå, Sweden October 2007

AUTHOR Patrik Blomberg

TITLE Non-target Effects of Genetically Modified Trees

ABSTRACT To date, few studies have focused on the effects of genetically modified trees (GM trees) on the environment. One concern with GM trees is that they may have unanticipated effects on non-target organisms, i.e. effects on organisms that are not direct targets of the genetically modified trait. The main objective of this thesis was to study potential non-target effects from the interaction between GM trees and natural enemies, including phytopathogens and herbivorous . To study this I used a system consisting of GM trees featuring changes in growth-related characteristics, and naturally occurring enemies. The GM trees used were the aspen hybrids Populus tremula x tremuloides: one unmodified wild type clone T89 (control) and transgenic lines with altered expression of gibberellin (GA 20-oxidase), sucrose (SPS) or pectin (PME); and Populus tremula x alba: one unmodified wild type clone INRA 717-1-B4 (control) and lines modified to suppress the activity of the enzymes in the lignin biosynthetic pathway, i.e. CAD, COMT, CCR or CCoAOMT. The natural enemies used were the parasitic phytopathogens Melampsora pinitorqua, M. populnea and Venturia tremulae, and the herbivorous leaf- vitellinae. To address this question inoculation experiments, feeding preference experiments, analyses of secondary chemistry and field inventories were performed. The results of the studies showed that the GM trees significantly affected the interaction with the natural enemies, both in the laboratory as well as in the field. For instance, both M. pinitorqua and V. tremulae showed an altered disease incidence on the GM trees of P. tremula x tremuloides compared to the unmodified wild type T89, where all tested transgenic lines exhibited altered susceptibility to the pathogens. However, there were also differences in aggressiveness to the aspens depending on pathogen population. The results from the field inventory showed that lines within all tested transgenic construct, COMT, CAD, CCoAOMT and CCR of P. tremula x alba differed significantly from the wild type INRA 717-1-B4 in susceptibility to M. populnea. In addition, the susceptibility to the rust also differed significantly between lines carrying the same transgenic constructs. Furthermore, we found that overexpression of SPS in P. tremula x tremuloides, unintentionally induced changes in plant secondary chemistry, where the GM-line SPS33A exhibited the largest deviation from the wild type T89 in contents of plant phenolics and nitrogen, and that these changes coincide with a concurrent decrease in herbivory by P. vitellinae on this line. I argue that the altered interactions are the result of physiological changes in the trees. They can originate from direct effects i.e. altered expression of the modified trait, indirect effects of the genetic modification process e.g. pleiotropy, or effects from the transformation process e.g. position effects, to which the tested natural enemies respond. The result stresses the importance of further research on the causes and mechanisms responsible for the altered interaction between GM trees and non-target organisms, as well as evaluating the potential environmental effects of cultivation of GM trees in the field. Such research will require collaboration between researchers from different disciplines, such as plant ecology and physiology, functional genomics, proteomics and metabolomics.

KEY WORDS: Genetically modified, GM trees, Populus, transgenic, non-target effects, natural enemies, plant-pathogen/herbivore interaction, environmental effects, parasitic fungi, herbivorous , secondary metabolism, phytochemistry

LANGUAGE: English ISBN: 978-91-7264-391-8 NUMBER OF PAGES: 27 + 4 papers

SIGNATURE: DATE: 2007-09-03

NON-TARGET EFFECTS OF GENETICALLY MODIFIED TREES

Patrik Blomberg

2007

Department of Ecology and Environmental Science Umeå University SE-901 87 Umeå Sweden

Dept. of Ecology and Environmental Science Umeå University SE-901 87 Umeå Sweden

ISBN: 978-91-7264-391-8 © PATRIK BLOMBERG 2007 Printed by VMC, KBC, Umeå University, Umeå, Sweden, 2007

LIST OF PAPERS

This thesis is based on the following papers, which will be referred to in the text by the corresponding Roman numerals:

I Blomberg, P., Wennström, A., Hjältén, J., Lindau, A. & Ericson, L. Testing the effect of genetically modified hybrid aspen Populus tremula x tremuloides on the interaction with the non-target pathogen Melampsora pinitorqua. (Submitted manuscript)

II Blomberg, P., Wennström, A., Hjältén, J., Lindau, A. & Ericson, L. Genetically engineered aspen (Populus tremula x tremuloides) alters its interactions with the non-target pathogen Venturia tremulae. (Accepted manuscript)

III Hjältén, J., Lindau, A., Wennström, A., Blomberg, P., Witzell, J., Hurry, V. & Ericson, L. (2007). Unintentional changes of defence traits in GM trees can influence plant-herbivore interactions. Basic and Applied Ecology 8:434-443.

IV Blomberg, P., Wennström, A., Hjältén, J., Lindau, A., Ericson, L. & Pilate, G. Field survey of genetically modified trees (Populus tremula x alba) with altered lignification unveils altered susceptibility to the non-target pathogen Melampsora populnea. (Manuscript)

Paper III is reproduced with kind permission from the publisher.

TABLE OF CONTENTS

INTRODUCTION 7

OBJECTIVES OF THE THESIS 9

MATERIAL AND METHODS 9 Populus: a model 9 Study systems 10 Description of the studies 12

MAJOR RESULTS 14

DISCUSSION 15 Mechanisms responsible to altered interactions 15 Potential environmental effects 18 Future perspectives 19

ACKNOWLEDGEMENTS 20

REFERENCES 20

APPENDICES: PAPER I – IV

INTRODUCTION

Forestry and products are of great importance to the world economy, but are also important for , creating potential conflicts between production and environmental goals. Forest trees have a wide range of commercial uses, providing for heating and cooking, timber for construction, raw materials for paper and pulp production and biofuel. Furthermore, due to increases in the world’s human population, economic development, and pressures to reduce the use of non-renewable resources, global demands for forest products are increasing (FAO, 2001). Thus, there is an increasing need to increase forest productivity and to create novel forest products while finding ways to protect biodiversity in forest . Since improving trees by conventional breeding is heavily constrained by e.g. long reproductive cycles genetic engineering offers an attractive alternative, offering the potential to transfer specific traits into selected genotypes without affecting desirable aspects of their genetic background (Pena & Séguin, 2001). combined with forest biotechnology and genetic engineering of trees is likely to become a major source for wood products in the future (Tzfira et al., 1998, Pullman et al., 1998, Sedjo, 2001, Fenning & Gershenzon, 2002, Campbell et al., 2003).

Currently a number of ongoing research projects are exploring the possibilities to genetically modify forest trees for use in forestry applications. The major goals of such projects include: improving the productivity of trees by increasing their growth rates; altering wood quality and chemical parameters in desired ways for specific uses, e.g. to improve the cost efficiency of paper and pulp production; increasing their resistance to pests and ; enhancing their tolerance of various kinds of abiotic stresses; controlling flowering and maturation; and optimizing their suitability for bio/phytoremediation of polluted land and water (Tzfira et al., 1998, Pena & Séguin, 2001, Bradshaw & Strauss, 2001, Campbell et al., 2003, Brunner & Nilsson, 2004).

- 7 - Although genetically modified (GM) trees have great potential utility in forestry, a number of possible environmental risks associated with their release have been identified, as discussed in a number of reviews in the last decade (e.g. James et al., 1998, Mathews & Campbell, 2000, Wennström, 2004, Van Frankenhuyzen & Beardmore, 2004, et al., 2005, Hoenicka & Fladung, 2006). However, little empirical information is available for assessing the probability and scale of the possible risks. In contrast to crops, on which most research concerning risks associated with GM organisms has focused, trees are essentially undomesticated, having long lifespans and defining the structure of many communities (Raffa, 2001). These features increase not only the likelihood of transgene escape and hybridization with wild relatives, but also the trees’ exposure to various abiotic and biotic stresses (Raffa, 2001, Van Frankenhuyzen & Beardmore, 2004, Wennström, 2004).

One major concern is that GM trees could have unanticipated effects on non-target organisms (Mathews & Campbell, 2000, Raffa, 2001, Van Frankenhuyzen & Beardmore, 2004, Wennström, 2004, Hoenicka & Fladung, 2006), i.e. organisms that are not direct targets of the genetically modified trait. It is well known that various aspects of the transformation process in plant genetic engineering frequently have unintended consequences, such as: position effects; insertions of multiple intact or rearranged gene copies; random insertional mutagenesis, due to the randomness in which transgenes are inserted in host genomes; epistatic interactions between transgene constructs and native genes; pleiotropic effects, i.e. effects of transgenes on multiple traits; and somaclonal variation, i.e. stable genetic changes in somatic cells during the in vitro propagation step (Käppeli & Auberson, 1998, Conner & Jacobs, 1999, Van Frankenhuyzen & Beardmore, 2004, Snow et al., 2005, Hoenicka & Fladung, 2006). Unexpected consequences may include wide variability in gene expression, gene silencing and/or secondary effects on plant physiology and fitness (Maury et al., 1999, Eriksson et al., 2000, Casler et al., 2002, Kaldorf et al., 2002). For instance, Casler et al. (2002) have shown that genetically engineering lignin biosynthesis affects fitness in herbaceous perennials, and Maury et al. (1999) that reducing lignin contents can reduce resistance to viral disease in transgenic tobacco. Such changes may have profound effects on non-target organisms, such as plant pathogens and herbivores, for example by altering host plant susceptibility and palatability (Van Frankenhuyzen & Beardmore, 2004, Wennström, 2004, Hoenicka & Fladung, 2006, Brodeur-Campbell et al., 2006). For example, Brodeur-Campbell et al. (2006) found that survival rates of larvae of the gypsy moth (Lymantria dispar L.) were significantly reduced on transgenic aspen (Populus tremuloides) with reduced lignin contents. It is therefore of outmost importance to develop and apply methods to determine and evaluate such effects as they could influence many ecological and evolutionary processes (Gilbert & Hubbell, 1996, Van der Putten & Peters 1997, Clay & Holah 1999, Wennström, 1999). Further, natural enemies are major limiters of tree productivity, and the conditions under which trees are grown commercially, usually in evenly-aged, evenly-spaced monocultures, tend to increase their vulnerability to pests (Burdon & Chilvers, 1982, Raffa, 2001). Therefore, rigorous empirical data on the potential environmental effects of introducing GM trees into the environment are urgently needed to improve our ability to assess systematically the risks associated with their use in commercial applications.

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OBJECTIVES OF THE THESIS

The main objectives of the work underlying this thesis were to identify and evaluate potential ecological effects of genetically modified trees, focusing on non-target effects on the interactions between the trees and their natural enemies. A key issue addressed was whether the genetic modification of trees’ traits may unintentionally affect their interactions with natural enemies. To assess this possibility we performed experiments to test the effects of:

• Genetically modified Populus tremula x tremuloides on the non-target phytopathogens Melampsora pinitorqua and Venturia tremulae (Papers I, II).

• Genetically modifying non-defensive traits on secondary chemistry in P. tremula x tremuloides (Paper III).

• Genetically modified P. tremula x tremuloides on the feeding preferences of non-target Phratora vitellinae (Paper III).

• Field-grown lignin-modified Populus tremula x alba on the non-target rust Melampsora populnea under field conditions (Paper IV).

In accordance with these objectives, all of the GM lines used had modifications that were not intended to influence natural enemies, and clones with the same unmodified isogenic background as the modified trees were used as controls.

MATERIAL AND METHODS

Populus: a model tree Members of the genus Populus were the first trees to be genetically transformed and regenerated (Fillatti et al., 1987) and they now serve as the leading model systems in forest biotechnology and tree transgenesis (Bradshaw et al., 2000, Peña & Séguin, 2001, Bhalero et al., 2003, Brunner et al., 2004, Boerjan, 2005). Furthermore, following the recent sequencing of the first tree genome, that of black cottonwood (Populus trichocarpa), a new era in tree functional genomics, proteomics and metabolomics has started, offering great opportunities to acquire a deeper understanding of gene function and the development of woody perennial plants (Brunner et al., 2004, Busov et al., 2005). Poplars (members of the genus Populus and their hybrids) have several advantages as model systems for forest biotechnology, including rapid growth, clonability, small genomes and the availability of extensive genome resources (Bradshaw et al., 2000, Bhalero et al., 2003, Brunner et al., 2004). In addition, few other trees can currently be transformed, regenerated, vegetatively propagated and grown to tree size in a sufficiently short time to enable their use in functional genomic studies (Brunner et al., 2004).

- 9 - Poplars should also be well suited as model systems for studying the environmental impact of GM trees and their interactions with non-target organisms, since they are often keystone species in their natural habitats, harboring a vast number of organisms, including natural enemies such as parasitic fungi and herbivorous insects (Whitham et al., 1996 and ref. therein).

Study systems Our study systems consisted of four natural enemies of native aspens, the fungal pathogens Melampsora pinitorqua, M. populnea and Venturia tremulae, and the herbivorous leaf-beetle Phratora vitellinae; and lines of the aspen hybrids Populus tremula x P. tremuloides and P. tremula x P. alba carrying genetic modifications affecting various aspects of their metabolism, including their sucrose, pectin, gibberellin, and lignin contents or composition. As controls we used the corresponding, unmodified, isogenic, wild type aspen hybrids: clone T89 of P. tremula x tremuloides and clone INRA 717-1-B4 of P. tremula x alba.

PLANT MATERIAL Populus tremula x tremuloides is a hybrid between the native European P. tremula and the North American species P. tremuloides. The genetically modified plants of this hybrid used in the studies were produced in the transformation facility at Umeå Plant Science Centre (UPSC), Umeå University, Sweden, with the wild type clone T89 as background material. The transgenic constructs for all lines were generated by Agrobacterium tumefaciens-mediated transformation following the protocols described by Nilssson (1992). The following transgenic lines were used: line GA (Gibberellin), overexpressing a gene encoding a GA 20-oxidase (Eriksson et al., 2000); lines SPS33A and SPS26, overexpressing sucrose-phosphate synthase (SPS) 1.5 and 4 fold, respectively (Mouillon & Hurry, 2001); and lines PME2BS and PME5S, overexpressing and underexpressing pectin methyl esterases (PME), respectively (Mellerowicz et al., 2004).

GA 20-oxidase is a key regulatory enzyme for the biosynthesis of gibberellins, a class of compounds including important growth regulators that influence diverse growth and development processes in plants, including shoot elongation, the expansion and shape of leaves, flowering and germination (Kende & Zeevaart, 1997). In transgenic P. tremula x tremuloides increasing levels of GA biosynthesis has been found to promote growth, production and xylem fibre length, but also to have antagonistic effects on root initiation (Eriksson et al., 2000). SPS is a key regulatory enzyme in sucrose biosynthesis that affects cold acclimation in Populus trees (Shrader & Sauter, 2002). Overexpression of SPS in the lines used in our research results in increased mesophyll sucrose contents and biomass production (Mouillon & Hurry, 2001). PMEs catalyse de-methylesterfication of pectins and are involved in important developmental processes including pollen growth, seed germination, cellular adhesion and stem elongation (Micheli, 2001), but may also play roles in plant defences against pathogens (Lionetti et al., 2007). PMEs also affect major wood components such as cellulose and lignin (Mellerowicz, 2005). Overexpression of PME in line PME2BS results in shorter fibres and slower growth than in the wild type T89, while suppression of PME in line PME5S leads to longer fibres and faster growth (Siedlecka et al., 2003).

- 10 -

In addition, we used genetically engineered hybrids between P. tremula and P. alba, both of which are from section Populus and native to Europe, in which the activities of various enzymes involved in lignin (phenylpropanoid and monolignol) biosynthesis were suppressed. All of these transgenic lines were Agrobacterium- transformed according to Leplé (1992), and derived from the unmodified isogenic hybrid Populus tremula x Populus alba female clone, INRA 717-1-B4, which was used as the wild type control. The following lines were used: two (ASCADT21 and ASCADT52) transformed with an antisense cinnamyl alcohol dehydrogenase (CAD) construct; one (SCAD1) transformed with a sense CAD construct (Baucher et al., 1996, Lappierre et al., 1999); two (ASB2B and ASB10B) transformed with an antisense caffeate/5-hydroxyferulate O-methyltransferase (COMT) construct (Van Doorsselaere et al., 1995, Lappierre et al., 1999); two (FAS13 and FAS18) transformed with a full antisense cinnamoyl-CoA reductase (CCR) construct; two (FS3 and FS30) transformed with the full sense CCR construct (J.-C. Leplé, C. Lapierre & W. Boerjan, unpublished results); and four (101, 416, 429 and 823) carrying the caffeoyl-CoA O-methyltransferase (CCoAOMT) construct (Meyermans et al., 2000).

Lignin is synthesized by the phenylpropanoid and monolignol biosynthetic pathways, which regulate many functions in plant development and defense (Dixon et al., 2002, Boerjan et al., 2003, Rogers & Campbell, 2004). Lignin is a major component of wood that provides rigidity and resistance to mechanical stress, and enables xylem transport (Higuchi, 1985, Lewis & Yamamoto, 1990). However, it is also an undesirable compound in the . Therefore, lignin has been a major target for genetic engineering of trees. The lignin-modifying constructs used in the studies underlying this thesis have reportedly had the following effects: down- regulation of CCoAOMT and CCR has resulted in 12% - 20% reductions in lignin contents and increased syringyl/guaiacyl (S/G) ratios (Meyermans et al., 2000, J.-C. Leplé, C. Lapierre & W. Boerjan, unpublished results); suppressed CAD-synthesis has resulted in minor reductions in lignin contents, with no changes in the S/G ratio (Baucher et al., 1996, Lapierre, et al., 1999, Pilate et al., 2002); and COMT- suppressed aspens lines have shown both reduced (Jouanin, et al., 2000) and unchanged lignin contents (Van doorsselaere, et al., 1995, Lapierre, et al., 1999, Pilate, et al., 2002.), with in both cases dramatic reductions in the S/G ratio.

NATURAL ENEMIES The poplar leaf and shoot blight-causing Venturia tremulae is an ascomycete that is a common and destructive pathogen on P. tremula and their hybrids in Europe, causing wilting and death of terminal shoots in young stands, including forest and nurseries (Dance, 1961, Kasanen et al., 2001). In aspens, V. tremulae infections primarily occur on terminal shoots of trees less than 3 m tall (Dance, 1961). Primary infections in spring are caused by ascospores released from perithecia that survive the winter on dead tissues on the ground. Initial visible symptoms are lesions on leaves that turn brownish/black, indicating the development of conidia. After the symptoms have fully developed on the leaves the annual shoots are attacked, turn black and wilt. Killed shoots droop, forming a characteristic shepherd's crook shape (Weisgerber, 1968, Kasanen et al., 2001). Secondary infections are caused by conidia that are produced throughout the summer. The evolutionary potential of V. tremulae is considered to be high (Kasanen et al., 2004), suggesting it has the potential to overcome resistance in aspen tress, and studies of

- 11 - other Venturia species have revealed considerable differences in virulence on Populus hosts between Venturia races (Blenis & show, 2001).

Melampsora rusts are basidomycetes and foliar obligate parasites, having a complex life cycle involving several types of spores and a host shift. Melampsora pinitorqua Rostr. (I) is native to large areas of Eurasia and is host-alternating, having both Populus spp. and Pinus spp. as hosts (Kurkela, 1973). Melampsora populnea (Pers.) Karst. (IV) is a host-alternating aggregate species including four taxa with Populus spp. as the primary hosts and a specialized secondary host including: M. larici- tremulae Kleb. that alternates with Larix spp., M. magnusiana Wagn. that alternates with Chelidonium majus and Corydalis spp., M. pinitorqua Rostr. that alternates with Pinus spp. and M. rostrupii Wagn. that alternates with Mercurialis perennis (Wilson and Henderson, 1966). As the alternate host(s) have not been clarified in the area underlying the studies of Paper IV, we use the aggregate name M. populnea.

The primary hosts of Melampsora rusts, Populus spp., are infected by aeciospores during the summer, and the first visible symptoms are small, yellow abaxial pustules, in which uredospores are formed that reinfect the plants throughout the growing season. In the autumn overwintering telia with teliospores (the dormant stage of the rust) differentiate on senescent fallen leaves. In the spring basidia are formed from the teliospores. Basidiospores are then spread to the secondary host were fecundation takes place in structures called spermogonia, a process that gives rise to aeciospores, which are able to reinfect the primary host again. Although Melampsora rusts need both hosts to complete their life cycles, some species can perpetuate on the Populus host in the uredinal stage (Longo, 1975, Newcombe, 1996). Melampsora rusts are also known for their ability to create new races/pathotypes and overcome plant resistance (Newcombe, 1996, Pinon et al., 1998, Ramstedt, 1999, Newcombe et al., 2001, Pei et al., 2003). Melampsora rusts can cause defoliation, reduced growth and biomass production, in particular in young aspen stands, plantations and nurseries.

Phratora vitellinae (Chrysomelidae) is a herbivorous leaf-beetle belonging to a guild of Chrysomelids that is well adapted to aspen and . This species is an important herbivore in plantations of Populus hybrids in Europe (Gruppe et al., 1999), and is known to respond to inter and intra-specific variations in plant secondary chemistry (Tahvavainen et al. 1985, Hallgren et al. 2003). Eggs are deposited in batches, and preferentially at the lower level of aspen and shoots. When the larvae emerge they start to feed close to the eggs they hatched from. As the larvae grow they feed higher and higher up on the shoots. In the last instar the larvae crawl down to the litter on the ground and transform into a pupae. After a few weeks new adults emerge and to survive the winter they need to feed on the host plants for a couple of weeks before they overwinter.

Description of the studies To study the interactions between genetically modified trees and non-target organisms we performed inoculation and preference experiments in the laboratory, field inventories, and phytochemical analyses.

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Laboratory inoculation experiments to test the effects of genetically modified Populus tremula x tremuloides on the non-target phytopathogens Melampsora pinitorqua and Venturia tremulae (Papers I & II).

To evaluate the effects (if any) of genetically modified aspen trees on the non-target phytopathogens M. pinitorqua and V. tremulae we conducted inoculation experiments under controlled conditions in the laboratory. We used a leaf-disc assay for inoculation with M. pinitorqua (I) and a cut-shoot assay for inoculation with V. tremulae (II), both of which are standard methods that are frequently used in infection experiments to study resistance to these pathogens in Populus species (Shain & Cornelius, 1979, Dowkiw et al., 2003, Pei et al., 2003, Yanase & Takeda, 1987, Quencez et al., 2001). Several studies have shown that there are good correlations between resistance patterns in results obtained by these methods and from field inventories (Shain, 1976, Hamelin et al., 1994, Pei et al., 1996, Newcombe & van Oosten, 1997), indicating that they can provide accurate, reliable evaluations of the relative susceptibility of aspen trees to M. pinitorqua and V. tremulae. In addition, since pathogens are generally variable species that show considerable variation in pathogenicity to their hosts (Burdon, 1987), we used several field populations of each of the pathogens to assess the consistency and generality of the effects of the genetic modifications on them.

Testing the effect of genetic modification of non-defensive traits on secondary chemistry in P. tremula x tremuloides (Paper III).

Secondary metabolites play important roles in plants’ interactions with their environment, including their defenses against pests, such as herbivores and pathogens (Edwards, 1992). Therefore, one of the goals of the studies was to test the effects of genetically modifying non-defensive traits of aspens on the secondary chemistry of their leaves. Liquid chromatography (HPLC), acid butanol assays and an Elemental Analyzer (Perkins Elmer 2400) were used to determine the concentrations of individual phenolics, tannins and nitrogen, respectively. (For further description of the analyses see Julkunen-Tiitto & Sorsa, 2001, Witzell, Gref & Näsholm, 2003, and Paper III).

Feeding preference of non-target Phratora vitellinae beetles on genetically modified P. tremula x tremuloides (Paper III).

Herbivorous insects optimize their food selection by searching for sources with high quality, and both the nutrient value and secondary metabolite contents of available options are important variables in the decision-making process (Edwards, 1992). Therefore, a further goal was to examine whether phytochemical changes in transgenic P. tremula x tremuloides affect the feeding preferences of the phytophagus leaf-beetle Phratora vitellinae. For this purpose, the preference of adult P. vitellinae beetles, collected from P. tremula in the field, for transgenic and wild type P. tremula x tremuloides leaves was tested in a multiple choice cafeteria trial in the laboratory, by estimating the areas consumed of offered leaves.

- 13 -

Inventory of fungal attack by M. populnea on field-grown lignin-modified Populus tremula x alba (Paper IV).

Controlled laboratory and greenhouse studies can provide valuable information for evaluating various parameters of plant- interactions, e.g. the relative resistance of different varieties of hosts, or the preferences of various herbivores under standardized conditions, and thus the potential outcome of the interactions. However, field studies of natural infections may provide better indications regarding their interactions in natural environments (Alexander, 1990), because the outcomes of plant-pest interactions may be significantly affected by diverse abiotic and biotic factors, including environmental stresses, competition, and interactions with multiple organisms, ranging from parasites to obligate symbionts, that can vary markedly in both space and time (Burdon, 1987). Therefore, we monitored the incidence of rust attacks on control and lignin-modified P. tremula x alba in two field-plots; one with aspen tress planted in 1995, and one with aspen trees planted in 1999 at the INRA Research Centre in Orleans, France, by counting the number of pustules of the rust on sampled aspen leaves.

MAJOR RESULTS

The overall aim of the work underlying this thesis was to investigate the effects of genetically modified trees on their interactions with non-target organisms. The results of all our experiments in both the laboratory and the field clearly show that the modifications of the examined trees significantly affected their interactions with the tested phytopathogens and herbivorous insect.

• Effects of genetically modified P. tremula x tremuloides on the non-target phytopathogens Melampsora pinitorqua and Venturia tremulae in inoculation experiments (Papers I, II).

There were significant differences in the incidence of disease caused by both M. pinitorqua and V. tremulae between the GM trees and the corresponding unmodified wild types, were all tested transgenic lines exhibited altered susceptibility to the pathogens. In addition, there were also differences in aggressiveness to the aspens between the tested pathogen populations. However, the disease pattern varied between the two pathogens. Both of two tested lines, GA and PME5S, were more susceptible overall to the Venturia pathogen than the wild type, while the patterns of disease caused by the Melampsora rust were less consistent in terms of both the relative susceptibility of the GM and wild type lines, and between pathogen populations.

• Effects of genetic modification of non-defensive traits on secondary chemistry in P. tremula x tremuloides (Paper III).

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We found that overexpression of SPS, which is known to increase mesophyll sucrose contents and biomass production in GM aspens, also unintentionally induced changes in their concentrations of phenolics and nitrogen. For instance, one of the GM lines, SPS33A, had significantly higher concentrations of salicin, tremuloidin, condensed tannins and nitrogen, and lower concentrations of coumaric acid and four flavonoids than the unmodified wild type.

• Feeding preferences of the non-target Phratora vitellinae beetles towards genetically modified and control P. tremula x tremuloides (Paper III).

The herbivorous leaf-beetle P. vitellinae consumed less biomass from line SPS33A than from the unmodified wild type, indicating that it responded negatively to the phytochemical changes in this line (described above).

• Effects of field-grown lignin-modified P. tremula x alba on the non-target rust Melampsora populnea under field conditions (Paper IV).

We found that lines carrying all tested transgenic constructs, i.e. COMT, CAD, CCoAOMT and CCR, differed significantly in susceptibility to the rust compared to their respective, unmodified wild types, and that the susceptibility to the rust differed between lines carrying the same transgenic construct. Interestingly, all of the transgenic lines that exhibited significantly altered susceptibility to M. populnea were less susceptible to the rust than the corresponding wild types.

DISCUSSION

Mechanisms responsible for altered interactions The analyses of the trees’ secondary chemistry (III) and feeding preferences of the herbivorous P. vitellinae beetle (III) indicate that the observed effects of the modifications in the GM trees on their interactions with non-target organisms were due to changes in phytochemical variables that affect the responses of pathogens and herbivores. For example, genetic modification of SPS in P. tremula x tremuloides unintentionally induced changes in the trees’ secondary chemistry, most notably in the deviations from wild-type phenolic and nitrogen contents in the GM line SPS33A, and that these changes correlated with reductions in herbivory by P. vitellinae on this line. The line SPS33A contained higher than wild type concentrations of condensed tannins – compounds that have been shown to negatively affect the feeding performance and growth of P. vitellinae (Tahvanainen et al., 1985, Rank et al., 1998, Hallgren, 2003) – which may at least partly explain the reduced consumption of this line. The response of the leaf beetles to the changes in plant phenolics may have been partly counteracted by the higher concentrations of nitrogen in SPS33A, since nitrogen usually has a positive effect on herbivores’ food preferences. However, the proximate cause of the lower utilization of line SPS33A cannot be determined from this study; it could be due to a combination of effects or

- 15 - potentially even to changes in plant traits that were not measured in this study. Such factors may explain why the induced changes in plant secondary compounds described in Paper III were not associated with significant observed effects on the phytopathogens M. pinitorqua (I) and V. tremulae (II).

The mechanisms responsible for the altered phytochemistry and responses of the phytopathogens and herbivorous insect to the GM trees demonstrated in these studies remain to be elucidated. The changes in the responses could be directly related to the expression of the transformed traits. For example, increases in plant growth and biomass production, as seen in the GA, SPS and PME5S lines (Eriksson et al., 2000, Mouillon & Hurry, 2001, Siedlecka et al., 2003), could make the plants more susceptible to the pests and pathogens, by increasing the concentrations of photosynthetic products in their leaves (Edwards, 1992). In addition, it has been hypothesized that fast-growing genotypes are likely to be less well defended than more slowly growing genotypes, due to a trade-off in the allocation of resources to growth and defense (e.g. van der Meijden et al., 1988, Herms & Mattson, 1992). Thus, the increased growth and biomass production of line GA, which overexpresses GA 20-oxidase (Eriksson et al., 2000), may explain its increased susceptibility to V. tremulae (II). Similarly, the generally high susceptibility of line PME5S to the pathogen could also be associated with its increased growth rates relative to the control (Siedlecka et al., 2003). In contrast, overexpression of SPS, which Mouillon and Hurry (2001) found to be correlated with increased rates of growth and biomass production, was not accompanied by increased susceptibility to the pathogen (II), indicating that other factors are involved. Moreover, the lack of consistency in the susceptibility of the transgenic lines to the rust M. pinitorqua relative to the wild type described in Paper I shows that the increased growth rate of these lines cannot be the sole factor responsible for the observed changes in plant-pathogen interactions.

Similarly, the degree of downregulation of genes and changes in lignin content or composition cannot, in isolation, provide any simple rational explanation for the results described in Paper IV, since the resistance to M. populnea of the transgenic lines modified with the same construct varied, although their levels of downregulation and lignin parameters were similar. However, the chemical analyses of these lignin-modified lines were mainly done on wood tissues, whereas we monitored the incidence of fungal attacks on leaf tissue. Lignin content and composition vary between different tissues within the same plant and between different cell types of a single tissue. Furthermore, its deposition also varies under different kinds of biotic and , including attacks by pests and pathogens (Boudet, 2000, Dixon et al., 2002, Baucher et al., 2003, Hawkins & Boudet, 2003), indicating that data from wood analyses may not provide good indications of the lignin content and composition of the leaves. Further, lignins or “lignin-like materials” can play important roles in plant defense and protection (Vance, 1980, Nicholson & Hammerschmidt, 1982, Strange, 2001) and lignin deposited in response to wound/infection, i.e. “defence lignin” is chemically different from lignin involved in plant development (Southerton & Deverall, 1990, Messner & Boll, 1993, Lange et al., 1995, Hawkins & Boudet, 2003). Consequently, we cannot yet exclude the possibility that alterations in lignin content or composition per se are responsible for the results presented in Paper IV, and these considerations highlight the need for data on the lignin contents of the leaves and leaf tissues influenced by attacking

- 16 -

enemies in order to draw more meaningful conclusions regarding the effects of changes in lignin synthesis on non-target organisms.

Indirect effects of genetic modification may also be responsible for the changes in plant-pathogen interactions observed in the studies underlying this thesis. Studies have frequently shown that regulators of central biosynthetic pathways, such as GAs, SPS, PMEs and phenylpropanoids, could have secondary pleiotropic effects, affecting other traits in the trees (e.g. Rottman et al., 2000; Eriksson et al., 2000). For instance, phenylpropanoids regulate not only lignin biosynthesis, but also other developmental processes such as cell division (Teutonico et al., 1991) and auxin transport (Jacobs & Rubery, 1988), as well as the synthesis of various compounds involved in plant defense, such as salicylic acid (SA), flavonoids and condensed tannins (Dixon et al., 2002). These findings indicate that genetically modifying lignin biosynthesis by targeting genes in the phenylpropanoid pathway may also affect the synthesis of other compounds produced by this pathway, which in turn may affect secondary metabolism and hence plant fitness and susceptibility to pathogens. Similarly, Eriksson et al. (2000) found that overexpression of GA 20-oxidase not only promoted growth and biomass production, but also affected root initiation, indicating that GA 20-oxidase is involved in additional biosynthetic processes. Such pleiotropic effects could have caused changes in plant physiology that account for the high susceptibility of line GA to V. tremulae (II). Moreover, it has been proposed that PMEs play a role in resistance to fungal and bacterial pathogens (Wiethölter et al., 2003). For example, Lionetti et al. (2007) found that overexpression of PME inhibitors decreased the PME activity in genetically modified Arabidopsis thaliana, and increased its resistance to the necrotrophic fungus Botrytis cinerea. However, this pattern was opposite to our finding, reported in Paper II, that the lower PME activity in line PME5S was accompanied by decreased resistance to V. tremulae, suggesting that other factors are also involved.

As discussed above, there are good grounds for believing the altered interactions between GM trees and non-target organisms observed in these studies were probably due to phytochemical changes resulting from either direct effects of the genetic modifications, or indirectly due to secondary, pleiotropic effects to which the non- target organisms respond. However, we should also consider the possibility that other effects of the transformation process, including position effects and insertions of multiple intact or rearranged gene copies due to the randomness with which transgenes are inserted in the host genome, may at least partly account for some of the observed changes in responses to the rust between lines modified with the same transgene construct (Käppeli & Auberson, 1998, Conner & Jacobs, 1999, Snow et al., 2005). Such events frequently occur, they may cause variations in gene expression or gene silencing, and they may have secondary, unintended effects on plant physiology that subsequently affect plant fitness and interactions with other organisms. Thus, regardless of the underlying mechanisms, unintended effects of genetic engineering may potentially affect the fitness of plants and their interactions with non-target organisms (Maury et al., 1999, Casler et al., 2002, Tiimonen et al., 2005, Brodeur- Campbell et al., 2006). Consequently, there are no straightforward answers to questions concerning the underlying mechanisms responsible for the changes in GM trees’ interactions with non-target organisms observed in the studies underlying this

- 17 - thesis, and we should also consider the possibility that different mechanisms may have interactive effects.

Plant pathogens of the genera Melampsora and Venturia are known to show intraspecific variation in pathogenicity to their hosts (Morelet, 1986, Burdon, 1987, Ramstedt, 1999, Newcombe et al., 2001, Blenis & Chow, 2001, Samils et al., 2001, Pei et al., 2003), explaining the observed differences in aggressiveness to the plants between the pathogen populations (I & II). These findings also highlight the importance of using several pathogen strains or populations in tests, since experiments with single pathogen populations may lead to erroneous conclusions regarding plants’ resistance to a specific pathogenic species. Differences in disease patterns between M. pinitorqua and V. tremulae probably reflect differences in the properties of their pathosystems, since different pathogens do not necessarily respond in a similar fashion to phytochemical changes induced by genetic modification. The variability in disease incidence both between and within pathogen species is an important issue to consider in studies of interactions between GM trees and their natural enemies, and in assessments of the potential environmental effects of genetic modifications, since trees must frequently cope with multiple enemies, as well pathotypes with differing virulence.

The results from the field inventory (IV) clearly demonstrate that genetic modification of lignin synthesis in P.tremula x alba significantly affects the plant’s interactions with the rust M. populnea under field conditions. This is interesting as it has been hypothesized that as assay systems move closer to natural condition the effects of genetic modification on such interactions are likely to be weakened or lost, due to the greater complexity and heterogeneity of the biotic and abiotic interactions in more natural conditions (Halpin et al., 2007). Therefore we have to consider this result as a real and most probable effect that has to be seriously considered in future assessments of the benefits and environmental risk associated with the field release and cultivation of GM trees.

Potential environmental effects The results of the investigations described in this thesis clearly show that it will be difficult to draw general conclusions regarding the effects of genetic modifications of trees on their interactions with pathogens and insect herbivores, and thus difficult to predict the potential environmental consequences of their presence in the environment. However, changes in plant-pathogen or plant-herbivore interactions may have several ecological and evolutionary implications. For example, changes in the selection pressure on natural enemies may also affect the wild aspen hosts and have adverse effects on the natural host or its interactions with other organisms. In cases where Populus species are involved, this may potentially have major ecological effects since Populus trees are considered to be keystone species in many forest , hosting a high number of organisms including natural enemies, such as parasitic fungi and herbivorous insects (Whitham et al., 1996 and refs. therein). Consequently, for further research on the potential impact of GM trees on ecological interactions and ecosystem processes, the effects of GM trees on non-target organisms should not only be addressed in systems consisting of the GM trees and

- 18 -

their non-GM counterparts, i.e. the background hybrid clones, but also take in to account the variability in native host populations.

In addition, and no less importantly, adverse fitness effects may also undermine the commercial potential of GM trees. A possible negative consequence of changes in interactions between tree species and their pathogens and/or herbivores is that the spread of diseases is promoted by large, dense host populations, such as monocultural forest plantations (Burdon & Chilvers, 1982). Thus, if GM trees are highly susceptible to pathogens or herbivores that are present in the planting areas they may cause serious damage. Under such conditions, a pest outbreak may cause severe losses of biomass production, death of plants, and consequently greatly reduce profits. The considerations are applicable to the potential effects of both V. tremulae and Melampsora rusts especially in young aspen plantations and nurseries (Kasanen, et al., 2004, Van Oosten, 2004), and the herbivorous P. vitellinae, which can have significant effects on plant fitness in aspen plantations (Gruppe et al., 1999). Such scenarios are not exclusively problems for plantations with GM trees, since they have been frequently witnessed in unmodified Populus and Salix plantations (Royle and Ostry 1995, Ramstedt, 1999, Van Oosten, 2004). However, the issues raised emphasize the importance of considering potential non-target effects of the use of GM trees, and their potential consequences for plantation forestry.

Another important aspect to consider is that the effects of changes in interactions between perennial plants, such as trees, and natural enemies are not necessarily detectable during a single year of investigation (Gange, 1990). Thus, long-term field studies should be undertaken to evaluate non-target effects of transgenic trees more thoroughly (Van Frankenhuyzen & Beardmore, 2004, Snow et al., 2005). This is of particular relevance for their interactions with enemies that have short generation times, such as parasitic fungi and insects, since they have the potential to respond quickly to changes in selection pressure, overcome plant resistance and, consequently, have severe effects on the plants.

Future perspectives The findings presented in this thesis highlight the importance of further research on the impact of GM trees on non-target organisms. I believe that research should focus on two major topics: the mechanisms whereby the transformation procedures and/or genetic modifications affect plant responses to specific natural enemies; and the potential ecological consequences of the resulting changes in plant-pathogen interactions. A much better understanding is needed of the relationships between the expression of targeted genes and associated alterations in the metabolism of plants, their responses to pathogens and insect attacks, and more sophisticated methods for inserting transgenes into predetermined locations in plants’ genomes, in order to accurately predict the effects of genetic modifications on non-target organisms and ecosystem processes. Thus, to elucidate the causes and mechanisms involved in changes in interactions between plants and pathogens or insects associated with genetic modification, and the potential impact such changes may have on ecosystems, we need to combine approaches of various disciplines, including plant ecology and physiology, molecular biology and genetics. Rapid recent advances in functional genomics, proteomics and metabolomics, and the extensive genome

- 19 - resources that are available for the genus Populus, have provided a toolbox that enables such multi-disciplinary investigations to be undertaken. For instance, it will be interesting to combine gene expression array analyses and metabolic profiling to explore the relationships between changes in specific transcripts, alterations in plants’ metabolism and their responses to infection (Dixon et al., 2002). In addition, the complexity of our results suggests that ecological analyses of the non-target effects of GM trees should include assessments of GM trees’ interactions not only with numerous types of natural enemies, but also with numerous populations or pathotypes of the enemies. We also suggest that studies should include several base lines, i.e. not only the non-GM counterparts but also wild relatives with which the natural enemies may interact, and finally the interactions between GM trees and their surroundings should also be evaluated across a range of spatial and temporal scales.

Although this thesis focuses on the ecological effects of genetic modification of hybrid aspens, the data generated in the underlying studies are potentially useful for further research and development of GM trees, and assessments of the environmental risks associated with them. The results clearly show that it is difficult to draw general conclusions regarding the environmental effects of GM trees, especially their effects on interactions with non-target organisms. Our findings therefore support the conceptual framework of case-by-case and step-by-step environmental risk assessments applied in the EU, US and other OECD member states (e.g. EU, 2002; EFSA, 2006).

ACKNOWLEDGEMENTS

I would like to tank my supervisor Anders Wennsröm and my associate supervisor Joakim Hjältén for a good cooperation and support, valuable scientific input and for providing constructive comments and discussions on the work underlying this thesis. I am also thankful to John Blackwell for helping me correcting and improving the English of this thesis. The research underlying this thesis was primarily funded by grants from CMF (Centre for Environmental Research). Additional financial support was provided by FORMAS (the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning), the Gunnar and Ruth Björkman Foundation, and by the J.C. Kempes Minnes Akademiska Fond.

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