Climate Change and Genetically Modified Insecticidal Plants

Transcription

1 KUOPION YLIOPISTON JULKAISUJA C. LUONNONTIETEET JA YMPÄRISTÖTIETEET 248 KUOPIO UNIVERSITY PUBLICATIONS C. NATURAL AND ENVIRONMENTAL SCIENCES 248 SARI J. HIMANEN Climate Change and Genetically Modified Insecticidal Plants Plant-Herbivore Interactions and Secondary Chemistry of Bt Cry1Ac-Toxin Producing Oilseed Rape (Brassica napus L.) Under Elevated CO 2 or O 3 Doctoral dissertation To be presented by permission of the Faculty of Natural and Environmental Sciences of the University of Kuopio for public examination in Auditorium ML1, Medistudia building, University of Kuopio, on Friday 5 th December 2008, at 12 noon Department of Environmental Science University of Kuopio JOKA KUOPIO 2008

2 Distributor: Kuopio University Library P.O. Box 1627 FI KUOPIO FINLAND Tel Fax Series Editors: Professor Pertti Pasanen, Ph.D. Department of Environmental Science Professor Jari Kaipio, Ph.D. Department of Physics Author s address: Department of Environmental Science University of Kuopio P.O. Box 1627 FI KUOPIO FINLAND Tel Fax [email protected] Supervisors: Professor Jarmo K. Holopainen, Ph.D. Department of Environmental Science University of Kuopio Docent Anne-Marja Nerg, Ph.D. Department of Environmental Science University of Kuopio Reviewers: Professor Jaakko Kangasjärvi, Ph.D. Department of Biological and Environmental Sciences University of Helsinki Nicole M. van Dam, Ph.D. Netherlands Institute of Ecology (NIOO-KNAW) Heteren, The Netherlands Opponent: Markku Keinänen, Ph.D. Faculty of Biosciences University of Joensuu ISBN ISBN (PDF) ISSN Kopijyvä Kuopio 2008 Finland

3 Himanen, Sari J. Climate change and genetically modified insecticidal plants: Plant-herbivore interactions and secondary chemistry of Bt Cry1Ac-toxin producing oilseed rape (Brassica napus L.) under elevated CO 2 or O 3. Kuopio University Publications C. Natural and Environmental Sciences p. ISBN ISBN (PDF) ISSN ABSTRACT Transgenic insect-resistant plants producing Bacillus thuringiensis (Bt) crystalline endotoxins are the first commercial applications of genetically modified crops and their use has steadily expanded over the last ten years. Together with the expanding agricultural use of transgenic crops, climate change is predicted to be among the major factors affecting agriculture in the coming years. Plants, herbivores and insects of higher trophic levels are all predicted to be affected by the current atmospheric climate change. However, only very few studies to date have addressed the sustained use and herbivore interactions of Bt-producing plants under the influence of these abiotic factors. The main objective of this study was to comparatively assess the performance of a Bt Cry1Ac toxin-producing oilseed rape line and its non-transgenic parent line in terms of vegetative growth and allocation to secondary defence compounds (glucosinolates and volatile terpenoids), and the performance of Bt-target and nontarget insect herbivores as well as tritrophic interaction functioning on these lines. For this, several growth chamber experiments with vegetative stage non-bt and Bt plants facing exposures to doubled atmospheric CO 2 level alone or together with increased temperature and different regimes of elevated O 3 were conducted. The main hypothesis of this work was that Bt-transgenic plants have reduced performance or allocation to secondary compounds due to the cost of producing Bt toxin under changed abiotic environments. The Bt-transgenic oilseed rape line exhibited slightly delayed vegetative growth and had increased nitrogen and reduced carbon content compared to the non-transgenic parent line, but the physiological responses (i.e. biomass gain and photosynthesis) of the plant lines to CO 2 and O 3 enhancements were equal. Two aphid species, non-susceptible to Bt Cry1Ac, showed equal performance and reproduction on both plant lines under elevated CO 2 and temperature despite differences in carbon and nitrogen concentrations between the non-bt and Bt line. Feeding of Bt-susceptible diamondback moth (Plutella xylostella) larvae was equally inhibited on the Bt line in control conditions, under elevated CO 2 and under O 3 exposure. Constitutive glucosinolate and volatile terpenoid profiles of the non-bt and Bt line plants were equal. Despite reduced feeding by Bt-susceptible P. xylostella on the Bt line, herbivore-induced increases in volatile terpenoids and glucosinolates were mostly comparable to those on the non-bt line, with the exception of lowered increases in foliar concentration of 4-methoxy-3- indolylmethylglucosinolate and emissions of (E)-4,8-dimethyl-1,3,7-nonatriene, (E,E)- -farnesene and - thujene in the Bt-transgenic line. The effectiveness of the transgenic trait, measured as Bt toxin concentration of leaves, was maintained under elevated CO 2 and temperature, separately and in combination, and enhanced under elevated O 3. As an effect of elevated CO 2, increased glucosinolate and volatile terpenoid concentrations were detected in both non-bt and Bt plant lines, whereas O 3 exposure changed concentrations of specific glucosinolates and decreased total terpenoid emissions in both plant types. In tritrophic bioassays, an endoparasitoid of P. xylostella, Cotesia vestalis, was attracted to host-damaged non-bt and Bt plants under elevated CO 2 compared to control plants, whereas with elevated O 3 the host-damaged Bt plants, which had reduced feeding damage, did not attract the parasitoids. This is in contrast to non-bt plants, which also remained attractive under O 3 exposure. To conclude, Bt-producing oilseed rape showed equal responses as the non-bt parent line to the studied abiotic variations in terms of vegetative growth, allocation to constitutive glucosinolates and volatile terpenoids and nontarget herbivore susceptibility. This suggests that these plants would respond similarly to the studied climate change factors in their vegetative growth stage. The herbivore resistance of the Bt line restricted certain specific herbivore-damage induced defences, which contributed to the disturbance of the tritrophic interaction studied under elevated O 3. However, even slight feeding damage was sufficient to induce major volatile terpenoid emissions and glucosinolates under control conditions indicating that endogenous defences were not excessively reduced by the transgenic modification rendering insect resistance. Elevated CO 2 and O 3 had clear effects on growth and secondary chemistry of oilseed rape, which promotes the significant role of climate change factors in affecting plant-herbivore interactions in this plant species in the future. Universal Decimal Classification: , , , 604.6, , , CAB Thesaurus: climatic factors; climatic change; insecticidal plants; transgenic plants; Brassica napus; herbivores; interactions; secondary metabolites; glucosinolates; terpenoids; Bacillus thuringiensis; endotoxins; carbon dioxide; ozone; temperature; growth; nitrogen; carbon; biomass; photosynthesis; Aphidoidea; Plutella xylostella; Cotesia

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5 ACKNOWLEDGEMENTS This work was conducted at the Department of Environmental Science, University of Kuopio. It was enabled by a project "Ecological and environmental constraints of direct and indirect defence in transgenic Bt plants - non-target effects on multitrophic interaction", funded by the Academy of Finland, the ESGEMO programme (Research Programme on Environmental, Societal and Health Effects of Genetically Modified Organisms, ). The thesis was also financed by The Finnish Cultural Foundation. This work would not have been possible without the premier effort of my main supervisor Professor Jarmo K. Holopainen, who provided me the opportunity to study plant-herbivore interactions and climate change from a highly novel aspect: this turned out to be an inspiring, yet a challenging topic. He had a major role in providing ideas for this study and always had confidence in this work, which I wish to sincerely thank him for. I am also most grateful to my other supervisor Docent Anne-Marja Nerg, Ph.D., whose expertise, help and friendship encouraged me along both the experimental and the writing phases. I sincerely thank Anne Nissinen, Ph.D., for collaboration throughout most of the experimental part of this work. Her help and good scientific expertise were invaluable for completion of this work. I also thank the Staff at the Department of Environmental Science and the Research Garden. Especially I wish to express my gratitude to Virpi Tiihonen for her invaluable help and excellent precise in laboratory analytics, enabling in large part my partial working from a distance. I wish to thank my coauthors: Professor C. Neal Stewart Jr. for his valuable and timely comments on my manuscripts and for sharing his excellent expertise in this research topic, Professor Guy M. Poppy for his ideas enabling this work in the first place and Delia M. Pinto, Ph.D., for her help and encouragement. I also thank James Blande, Ph.D., for revision of the language and express my gratitude to the two reviewers of this thesis: Professor Jaakko Kangasjärvi and Senior Research Scientist Nicole M. van Dam, Ph.D. Finally, I am most grateful to my family: thank you Petri and Heini just for being there to take my mind off work. I also wish to express my gratitude to my mother and to my father for their encouragement throughout these 30 years of my life and to my friends and relatives for their support. Your help with practical things was the greatest gift I could get when my work required absence and time for myself. Without you this would not have been possible, and for that I owe my deepest thanks to all of you. Mikkeli, November 2008 Sari Himanen

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7 ABBREVIATIONS Bt Bacillus thuringiensis C Carbon Chl Chlorophyll CO 2 Carbon dioxide DBM Diamondback moth DMNT 4,8-dimethyl-1,3,7-nonatriene DNA Deoxyribonucleic acid EFSA European Food Safety Authority EPA Environmental Protection Acengy EU European Union FAO Food and Agriculture Organization of the United Nations GFP Green fluorescent protein GC Gas chromatograph GLV Green leaf volatile GM Genetically modified GMO Genetically modified organism GS Glucosinolate GT Genotype HPLC High performance liquid chromatography 3-IM-GS 3-indolylmethyl-glucosinolate JA Jasmonic acid LD 50 Lethal dose to 50% of population MRGR Mean relative growth rate MS Mass spectrometer N Nitrogen O 3 Ozone OECD Organisation for Economic Co-operation and Development PAR Photosynthetically active radiation 2-PE-GS 2-phenylethyl-glucosinolate ppm Parts per million ppb Parts per billion PR-1 Pathogenesis-related protein 1 SA Salicylic acid SEM Standard error of mean TNC Total non-structural carbohydrates VOC Volatile organic compound WHO World Health Organization

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9 LIST OF ORIGINAL PUBLICATIONS This thesis is based on the following publications, which are referred to in the text by their chapter numbers: Chapter 2 Himanen SJ, Nissinen A, Auriola S, Poppy GM, Stewart CN Jr, Holopainen JK, Nerg A-M (2008) Constitutive and herbivore-inducible glucosinolate concentrations in oilseed rape (Brassica napus) leaves are not affected by Bt Cry1Ac insertion but change under elevated atmospheric CO 2 and O 3. Planta 227: Chapter 3 Himanen SJ, Nerg A-M, Nissinen A, Pinto DM, Stewart CN Jr, Poppy GM, Holopainen JK. Effects of elevated carbon dioxide and ozone on volatile terpenoid emissions and multitrophic communication of transgenic insecticidal oilseed rape (Brassica napus). New Phytologist (in press). Chapter 4 Himanen SJ, Nissinen A, Dong W-X, Nerg A-M, Stewart CN Jr, Poppy GM, Holopainen JK (2008) Interactions of elevated CO 2 and temperature with aphid feeding on transgenic oilseed rape: Are Bacillus thuringiensis (Bt) plants more susceptible to nontarget herbivores in future climate? Global Change Biology 14: Chapter 5 Himanen SJ, Nerg A-M, Nissinen A, Stewart CN Jr, Poppy GM, Holopainen JK. Elevated atmospheric ozone increases concentration of insecticidal Bacillus thuringiensis (Bt) Cry1Ac protein in Bt Brassica napus and reduces feeding of a Bt target herbivore on the non-transgenic parent. Environmental Pollution (in press).

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11 CONTENTS CHAPTER 1: General introduction Genetically modified crop plants Global meaning of plant biotechnology for agriculture today Transgenic insect resistant Bacillus thuringiensis (Bt) plants Regulation of GM plants - avoiding the possible environmental risks Climate change and crop plants Current climate change and predictions for the future Effects of elevated CO 2, temperature and O 3 on plant performance Effects of elevated CO 2, temperature and O 3 on insect herbivores Transgenic plants and climate change Plant secondary compounds and herbivory Glucosinolates and herbivory Volatile organic compounds (VOCs) - induction by herbivory and the role of VOCs in indirect defence Plant secondary compounds and climate change Effects of elevated CO 2 and O 3 on glucosinolates Effects of elevated CO 2 and O 3 on VOC emissions and indirect defence Aims of the study and description of experimental plants used Bt oilseed rape (Brassica napus L.) as a model plant Research objectives and outline of the experiments...31 CHAPTER 2: Constitutive and herbivore-inducible glucosinolate concentrations in oilseed rape (Brassica napus) leaves are not affected by Bt Cry1Ac insertion but change under elevated atmospheric CO 2 and O CHAPTER 3: Effects of elevated carbon dioxide and ozone on volatile terpenoid emissions and multitrophic communication of transgenic insecticidal oilseed rape (Brassica napus)...59 CHAPTER 4: Interactions of elevated CO 2 and temperature with aphid feeding on transgenic oilseed rape: Are Bacillus thuringiensis (Bt) plants more susceptible to nontarget herbivores in future climate?...75 CHAPTER 5: Elevated atmospheric ozone increases concentration of insecticidal Bacillus thuringiensis (Bt) Cry1Ac protein in Bt Brassica napus and reduces feeding of a Bt target herbivore on the non-transgenic parent...95 CHAPTER 6: General discussion Comparison of non-transgenic and Bt-transgenic oilseed rape in terms of growth and elevated CO 2, temperature and O 3 responses Effects of climate change factors and elevated O 3 on primary and secondary compounds - is the allocation pattern different in non-transgenic and Bt-transgenic oilseed rape? Stability of the transgenic trait and target herbivore susceptibility under elevated CO 2 or O 3 concentrations Nontarget herbivore performance on non-bt and Bt oilseed rape under elevated CO 2 and temperature - aphid responses

12 6.5 Induction of direct and indirect defence by herbivory in non-transgenic and Bttransgenic oilseed rape - the relation of feeding damage and defence induction with abiotic variation The tritrophic system oilseed rape -diamondback moth - Cotesia vestalis and its vulnerability against CO 2 or O 3 elevations Methodological considerations Conclusions Future prospects References

13 CHAPTER 1 GENERAL INTRODUCTION

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15 1. GENERAL INTRODUCTION 1.1 Genetically modified crop plants Global meaning of plant biotechnology for agriculture today The development of modern biotechnology, i.e. genetic engineering, has enabled the use of recombinant DNA techniques in plant breeding and thereby, modifying of the plant genome. Genome sequencing, discovering functions for specific genes and factors connected with their regulation, as well as targeted mutations in plants, are useful tools for studying many processes in plants and represent one of the main methods of advancement in plant science in recent decades and probably for many decades to come (Moose & Mumm 2008). Genetic engineering also provides the possibility to develop genetically modified (GM) plant varieties that are transformed to possess a specific trait that enhances the agronomic performance of the plant species. To date, this has been restricted to pest- and herbicide-resistant varieties in commercial use (James 2007). Biotechnological methods enable connecting particular gene modifications with phenotypic changes, which can significantly speed up the plant breeding process. There are substantial and accumulating arguments for the higher accuracy of genetic engineering over traditional breeding methods, and the need for GM technologies to improve future crops (Ainsworth et al. 2008, Moose & Mumm 2008). Transgenic crop varieties have also led to an approximately 224 million kg reduction in the usage of pesticides, to an estimated 14 % decrease in negative environmental impacts of pesticides and to significantly reduced production costs in certain agroecosystems (Romeis et al. 2006, Brookes & Barfoot 2007). However, this novel methodology has been raised as the most serious problem regarding public acceptance of transgenic crop plants, with concerns associated with environmental safety, human health and ethics (Stewart 2004). The overall cultivating area of transgenic crops has increased since their first wide commercial release in 1996, reaching 114 million hectares in 2007 with a yearly growth of 12% from 2006 to 2007 (James 2007). Soybean, maize, cotton and canola were the main GM crop species in 2007 (James 2007). Two traits have dominated in commercialised transgenic crop plants to date: insect resistance (Bt maize and cotton) and herbicide resistance (soybean and canola). At present, many varieties, e.g. 37 % of all GM crops used in the USA, have stacked properties, i.e. a combination of at least two different traits. The pace of adopting transgenic cultivars around the world has made plant biotechnology "the fastest adopted crop technology in recent history" (James 2007). In 2007, the top transgenic crop growing countries in terms of hectares were the USA (with 50 % of the global GM crop area), Argentina, Brazil, Canada Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008) 15

16 Sari J Himanen: Bt oilseed rape and climate change and India (James 2007). Recently, China has also become one of the top countries adopting GM crops. In Europe, several insect resistant Bt maize lines have been accepted ( for cultivation as agricultural GM crops. In 2007 developing countries had a 43% global share of transgenic crops. In these countries there are regions where GM varieties have clear dominance over traditional varieties (James 2007). The past decade has proven that GM crops are needed and wanted (Brookes & Barfoot 2007, James 2007), but they have to be adopted sensibly and substantial controlling efforts are required (EFSA 2004) Transgenic insect resistant Bacillus thuringiensis (Bt) plants In 1996, insect resistance based on insecticidal Bacillus thuringiensis (Bt) crystalline (Cry) endotoxins (Perlak et al. 1990) was the first trait to be commercially introduced into transgenic plants (James 2007). B. thuringiensis is a common soil bacterium, which was discovered in 1901 (Glare & O'Callaghan 2000). It produces Cry toxins as crystalline inclusion bodies upon sporulation. Over 200 different types of Bt toxin have been identified, each with specific toxicity against certain target herbivore groups, e.g. Bt subsp. kurstaki originating Cry1Ac is effective against Lepidopteran species and Cry1Ab targets Coleopteran species (OECD 2007). Truncated modes of Bt toxin genes that result in the production of active toxin instead of the original procaryotic protoxin gene, have been used to achieve sufficient expression in plants (Perlak et al. 1991). Bt toxins bind to gut membranes of susceptible herbivores (i.e. non-susceptible species lack these membranes) and cause gut disruption resulting ultimately in membrane leakage and finally the death of the herbivore (Glare & O'Callaghan 2000, OECD 2007). Bt maize and cotton are the main commercialised Bt applications in use today (James 2007). Various other plant species have been successfully transformed to express Bt genes for scientific purposes. Among these are a number of Brassica species including B. napus ssp. oleifera (canola, Stewart et al. 1996), B. oleracea (broccoli: Metz et al. 1995, Cao et al. 1999, Zhao et al. 2005, cabbage: Metz et al. 1995, Jin et al. 2000, collards: Cao et al. 2005) and B. juncea L. (Indian mustard, Cao et al. 2008). In recent trials Bt-transgenic Brassicas were used as trap crops located next to crop plants to successfully control Lepidoptera larvae (Shelton et al. 2008). Bt toxin production has been successfully controlled by constitutive promoters, i.e. Bt toxin is continuously produced in all plant parts. However, inducible promoters might help the plant to target defence to a time when it is needed (Christou et al. 2006). Bt-transgenic broccoli under PR-1 gene induction resulted in high expression when induced, but there were also background concentrations in control plants (Cao et al. 2006). In addition to traditional cry toxins, new types of 16 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008)

17 General introduction proteins called vip proteins, expressed in the vegetative stage of B. thuringiensis bacterial growth, are a recent target of Bt based stacked gene crop protection. Other pest resistance traits investigated in plants include protease inhibitors, amylase inhibitors and lectins (Christou et al. 2006). Bt based bacterial insecticide sprays have been used for pest control since the 1960s. Their extensive usage has already led to the development of resistance by the global Brassica herbivore Plutella xylostella L, the diamondback moth, in the field (Tabashnik et al. 1990, 1997, Shelton et al. 1993). Therefore, attention has focussed on effective dosing of Bt toxin in transgenic plants (i.e. first instructed by Environmental Protection Agency to be at least 25 times the LD 50 concentration of Bt toxin for the susceptible species), and the application of non-transgenic refuges for maintaining susceptible herbivore individuals in order to limit resistance development (Shelton et al. 2000, Bates et al. 2005, Tabashnik et al. 2003, 2008). A fixed percentage (circa 20 %) of the cultivation area has to be free of GM material to reduce the selection pressure on target herbivores to develop Bt toxin resistance (Cerda et al. 2006). This increases the probability that heterozygously resistant individuals will mate with homozygous non-resistant pests, and reduce the development of homozygous resistance. Pyramided plants with multiple Bt toxins (Cao et al. 2008) provide mechanism for delaying resistance development, but their concurrent use with plants that produce a single toxin could lead to even faster resistance development (Zhao et al. 2005). The current strategies appear to have substantially controlled resistance development over the last 10 years (Tabashnik et al. 2008) Regulation of GM plants - avoiding the possible environmental risks In the European Union (EU), the release of transgenic plants into the environment for commercial purposes is under the legislation of the European Food Safety Authority (EFSA, In the United States it is regulated by the Animal and Plant Health Inspection Service (APHIS), the Environmental Protection Agency (EPA) and the Food and Drug Administration (FDA). OECD currently has two active working groups (WG on harmonisation of regulatory insight in biotechnology and Task Force for the safety of novel foods and feeds) and it collects consensus documents on crops and technologies as guidance. In the case of Bt toxin expressing plants, a recent consensus document was released in November 2007 (OECD 2007). Although there are still substantial differences in the principles and methodology used between the different regulatory authorities, all these actions have a common goal: to ensure the safety of GMOs to both human health and the environment. The Comparative Safety assessment (Kok & Kuiper 2003) refers to the principle of Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008) 17

18 Sari J Himanen: Bt oilseed rape and climate change substantial equivalence and familiarity as proposed by OECD (1993) and later by WHO/FAO (2000), for the assessment of food and environmental safety of GMOs. This was the basis for regulation of the first GM foods in the United States. Since there is a history of growing non-gm agricultural plants "safely", they form the baseline on which to compare novel GM crop plants (EFSA 2004). However, it has to be recognised that gene insertion will not produce identical plants to the non-gm parents, yet both plant types can be safe. Therefore, the safety assessment includes an initial comparative analysis of GM and non-gm lines to identify possible differences, which is followed by a further assessment of the environmental and food safety effects of the identified intended and unintended differences (EFSA 2004). In the EU, prior to submitting a GMObased plant application, the applicant has to conduct extensive laboratory work and multi-year field studies to demonstrate that the new trait does not cause unintentional harm to humans, animals or the environment. Specifically, this will include a characterisation of the recipient and the parental plants, the genetic modification process, a detailed description of the GM plant partly in comparison to its non-gm counterpart, as well as tests on food safety aspects (including composition, toxicity, allergenicity and nutritional value) (EFSA 2004). After an environmental risk assessment report is made, the need for an environmental or a post-market monitoring plan is determined (EFSA 2004). In future, untargeted profiling techniques may also complement the existing protocols to recognize any possible unintended effects (Kuiper et al. 2003). The environmental fitness of GM plants is important with respect to possible risks for the receiving biotic and abiotic environment. Potential changes in interactions of GM plants with the biotic environment are assessed for persistence and invasiveness, selective advantage or disadvantage, potential for gene transfer, interactions between the GM plant and target or nontarget organisms (e.g. insects), effects on human and animal health and biogeochemical processes, and for specific cultivation, management and harvesting techniques (EFSA 2004). Potential interactions with the abiotic environment are assessed, including alteration of climatic conditions or mineralisation and sensitivity to, or tolerance of climatic conditions. An example of this is the assessment of the lignin content of transgenic plants, which might lead to reduced decomposition of the plant material (Flores et al. 2005, Stotzky 2005). Persistance in the soil (i.e root exudated Bt toxins) is also an important consideration with respect to soil quality (Saxena et al. 1999, 2004, reviewed by Icoz & Stotzky 2008) and soil microbial community structure (Griffiths et al. 2007). Impacts on population dynamics of competitors, herbivores, symbionts, predators, pollinators, parasites and pathogens are considered in the environmental risk assessment (EFSA 2004, Romeis et al. 2008). The occurrence 18 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008)

19 General introduction of a hazard and of exposure is combined to assess the general magnitude of the possible environmental risk. In the assessment, possible food chain effects, e.g. effects of prey reduction on parasitoid abundance, have to be distinguished from direct effects of the modification, e.g. toxin accumulation in the food chain. Furthermore, a crop ecosystem is not the same as a natural ecosystem, which is why effects of GM crop plants are compared to effects caused by comparable non-gm agricultural practices in current use (such as pesticide usage) (EFSA 2004). One major environmental issue concerning GM plants is the possibility of transgene flow via interspecific hybridization and backcrossing into natural plants (crop-towild) such as weeds. This could potentially enhance the fitness of the weeds and lead to the fixing of the transgene in nature (Ellstrand & Hoffmann 1990, reviewed by e.g. Chamberlain & Stewart 1999, Stewart et al. 2003, Chandler & Dunwell 2008). Crop-to-crop (interpecific hybridisation) gene flow could also lead to mixing of non-gm and GM material. Hybridization frequency and the possibility of transgene flow are affected by the preferences of the plant species for self-pollination or crossing, pollen dispersal methods and seed dispersal, the spatial and temporal suitability for the hybridisation to occur, and the vigour of the resulting hybrids (Stewart et al. 2003). Cruciferous plants have many wild relative species, which occur as common weeds in agricultural areas and are able to hybridize with Brassica crop plants (Wilkinson et al. 2003a, Warwick et al. 2003). If the transgene hybridizing or eventually even introgressing into a weed species has ecologically important characteristics (fitness enhancing properties such as insect resistance), the potential environmental impacts are predicted to be most problematic (Vacher et al. 2004, Sutherland & Poppy 2005, Poppy & Wilkinson 2005). However, no study so far can thoroughly predict the potential severity of an insect-resistant Bt plant of wild species origin in nature, including the actual probability of this transgene becoming fixed in weed populations by selection, or the potential for decreased genetic diversity as a result of this kind of introgression (Stewart 2004, Sutherland & Poppy 2005). After the acceptance and release of a GM variety case-specific monitoring and general surveillance procedures are typically used (EFSA 2004). Case specific monitoring practices are dependant on the ecological risk assessment of the variety approved. General surveillance is not experimental or hypothesis driven but is targeted at developing the current practice and identifying possible unanticipated adverse effects. This is done, for example, by routine observations and operator questionnaires. In spite of this relatively large scientific effort to ensure the safest possible use of GM crops, especially compared to that used for studying traditionally bred varieties with novel traits (Kok et al. 2008), there still seems to be worries about Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008) 19

20 Sari J Himanen: Bt oilseed rape and climate change the most efficient methodology for assessing GM crop safety. This is especially so with regard to possible pleiotropic effects, long-term nontarget effects of GM plants on different organisms of ecosystems, as well as the traceability, labelling and coexistence regulations between conventional, organic and GM crops. It is not easy to balance between comprehensive enough but not unreasonably wide environmental risk assessments that should be used for transgenic crop cultivars. The use of ecological assessment to cover whole complex ecosystem effects might not assist decision makers as well as specific hypothesis-based testings (Raybould 2007). A compromising practice has been to use a tiered case-by-case assessment, which is very timely but thorough (Wilkinson et al. 2003b). 1.2 Climate change and crop plants Current climate change and predictions for the future The current stage of climate change is that there are continuous rises in mean global atmospheric CO 2 level and mean global yearly air temperature, with the magnitude of these increases in the future highly dependent on controlling anthropogenic greenhouse gas emissions (IPCC 2007). The current atmospheric CO 2 level of ca. 380 ppm is the highest in nature for the last years. With current emissions, a doubling of this level is likely by the year The main reasons for CO 2 rises in the atmosphere are fossil fuel use and land-use changes (deforestation, decay of biomass, peat) (IPCC 2007). Together with the rising atmospheric CO 2 levels, the mean global temperature is predicted to increase by ca. 0.2 C per decade for the next two decades (IPCC 2007). Beyond this period the trend will depend on emission scenarios, with mean temperatures predicted to increase by C by the end of this century. In addition, changes on a regional-scale include wind pattern alterations, contraction of snow covered areas, increases in the frequency of hot extremes, heavy precipitation and tropical cyclone intensity (IPCC 2007). Biodiversity is likely to be affected, partly irreversibly. Sea level rise and melting of glaciers, ice caps and polar ice sheets are closely connected with temperature increases. Warming also results in lower uptake of CO 2 by the oceans, which increases CO 2 levels remaining in the atmosphere causing a feedback effect (IPCC 2007). Tropospheric background ozone (O 3 ) levels have significantly increased in recent decades due to anthropogenic pollution, i.e. fossil fuel use and biomass burning, even though extreme peak episodes have been generally reduced (IPCC 2007). O 3 is formed by phytochemical reactions involving nitrogen oxides (NO x ), carbon monoxide and volatile organic compounds (VOCs) in the atmosphere. Diurnal background levels of tropospheric O 3 above 40 ppb are already encountered in many parts of the world with a growing incidence of daily 20 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008)

21 General introduction and monthly peak fluctuations (Sitch et al. 2007). The radiative forcing by elevated O 3 has been estimated at 0.35 W m -2 (IPCC 2007), which corresponds to a 30% increase in tropospheric O 3 concentration since Additional indirect radiative forcing by O 3 effects on plants leading to suppression of the land-carbon sink has been suggested to be even higher (Sitch et al. 2007). Elevated temperature and CO 2 levels are predicted to add to the frequency of tropospheric O 3 episodes by the end of this century, but proposed increases in humidity will probably lead to reductions in tropospheric O 3 concentrations (Zeng et al. 2008). Other interactions between different climate change factors and O 3 are complex and hard to predict Effects of elevated CO 2, temperature and O 3 on plant performance Atmospheric carbon dioxide (CO 2 ) level is the main determinant of carbon availability for plants. It is the main fuel for photosynthesis, which provides plants with the carbohydrates needed for their growth and differentiation. Typically elevated atmospheric CO 2 stimulates photosynthetic rates, reduces stomatal conductance, leads to higher carbon concentration through accumulation of foliar carbohydrates and lowered nitrogen concentration, and in general results in higher biomass and increased leaf area in plants (Poorter et al. 1997, Urban 2003, Long et al. 2004, Gutschick 2007, Stiling & Cornelissen 2007). It may also lead to phenological differences in plant growth, i.e. decreased development times or changes in crop quality (Urban 2003). However, acclimation effects, such as lowered photosynthetic levels as a feed-back to inhibition by increased carbohydrates, reduced stomatal density and stomatal opening time have also been reported (Ainsworth & Rogers 2007), which may limit the beneficial effects of long-term increased CO 2 (Gutschick 2007). Carbonbased secondary compounds are thought to be increased under elevated CO 2, with excess carbon allocated to their production (Peñuelas & Estiarte 1998, Hartley et al. 2000). The opposite is proposed for nitrogen-based secondary metabolites, due to reduced nitrogen availability through increased competition for nitrogen allocated to enhanced biomass growth. The carbon-nutrient balance hypothesis of Bryant et al. (1983) serves as a primary basis for this assumption. Elevated temperature increases the optimal CO 2 level of photosynthesis, hastens plant metabolism and development and leads to decreased duration of the reproduction cycle (Batts et al. 1997, reviewed by Morison & Lawlor 1999). Ontogenetical changes such as earlier maturation and start of reproduction are responsible for many compositional changes observed in plants with increased temperatures (Morison & Lawlor 1999, Peñuelas & Filella 2001). Future predicted increased temperature combined with high light level and drought can cause sudden wilting of plants in warm and dry areas (IPCC 2007). Elevated CO 2 and temperature will occur together in future atmospheres, where Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008) 21

22 Sari J Himanen: Bt oilseed rape and climate change elevated temperature is expected to primarily promote the CO 2 effects or in some instances to counteract them (Batts et al. 1997, review by Morison & Lawlor 1999, Zvereva & Kozlov 2006). Increased levels of tropospheric O 3 (typically exceeding ppb) are phytotoxic and can reduce plant performance (Fuhrer et al. 1997). O 3 enters plant cells through stomata and O 3 flux to leaves is a significant contributor to the overall effects of O 3 on plants (reviewed by Ashmore 2005). Stomatal closure is one mechanism to prevent O 3 uptake and protect the photosynthetic apparatus (reviewed by Fiscus et al. 2005). However, this simultaneously leads to decreased photosynthesis due to reduced CO 2 inflow. O 3 induces various defence reactions such as formation of reactive oxygen species in the leaf apoplast and the synthesis of pathogenesis related-proteins and signalling molecules (stress hormones such as salicylic acid, jasmonic acid, abscisic acid and ethylene in plants) (reviewed by Kangasjärvi et al. 2005). Oxidative stress and reactive oxygen species can lead to programmed cell death, and the hypersensitive response, highly resembling the incompatible pathogen infection response in plants (Overmyer et al. 2005). Symptoms during the vegetative stage include lesion formation, the size of which is determined by the resistance of the species and interactions of the signaling hormones (Kangasjärvi et al. 2005). O 3 can damage sensitive proteins, cause carbon allocation changes and reduce root:shoot ratios (Grantz & Farrar 1999). High O 3 is often detrimental to flowers and interferes with seed production (Black et al. 2000) even if growth reductions are compensated (Ollerenshaw et al. 1999). Phenologically, O 3 can induce earlier senescence (Miller et al. 1999). Large differences in O 3 sensitivity exist between natural plants and cultivated crops and between different cultivars (Biswas et al. 2008a, b). Sensitivity is determined by the ability of plants to restrict O 3 inflow, the effectiveness of processes for detoxifying reactive oxygen species and differential activation of defence signalling pathways (reviewed by Fiscus et al and Kangasjärvi et al. 2005) Effects of elevated CO 2, temperature and O 3 on insect herbivores Insect herbivores are affected by changing abiotic conditions indirectly through compositional and biochemical changes in the metabolism of plants (Roth & Lindroth 1995, Coviella & Trumble 1999, Holopainen 2002, Jondrup et al. 2002, Percy et al. 2002, Holton et al. 2003, Reddy et al. 2004, Stiling & Cornelissen 2007, Zavala et al. 2008) and by direct effects on insect physiology or behaviour (Bale et al. 2002, Awmack et al. 2004). Indirect plant-mediated effects are often predominant. Effects of elevated CO 2 on plants, such as increased carbon and reduced nitrogen, can cause compensatory feeding in certain chewing herbivores (Docherty et al. 1996, Coviella & Trumble 22 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008)

23 General introduction 1999, Stiling & Cornelissen 2007) and lead to reductions in their relative growth rates (Reddy et al. 2004, Stiling & Cornelissen 2007). Piercing herbivores such as aphids have shown positive, negative and neutral responses to elevated CO 2 (Bezemer & Jones 1998, Holopainen 2002, Stacey and Fellowes 2002). CO 2 effects can also be mediated by changes in leaf longevity and phenology, as indicated by a reduced life span leading to a higher susceptibility of B. rapa to Trichoplusia ni (Marshall et al. 2008). An example of a specific interaction between elevated CO 2 - herbivory was recently revealed: downregulation of the defence genes lipoxygenase 7, lipoxygenase 8, and 1- aminocyclopropane-1-carboxylate synthase under elevated CO 2 led to reduced cysteine proteinase inhibitor production and enhanced performance of two pest herbivores on soybean (Glycine max) (Zavala et al. 2008). Similar findings are likely to appear in the coming years with the increasing number of studies concerning this kind of complex interactions. Higher temperatures typically elicit a direct acceleration of insect development (Bale et al. 2002, Johns & Hughes 2002, Williams et al. 2003) leading to a greater number of lifecycles (Liu et al. 2002). O 3 usually makes plant leaves more palatable to herbivores (Jones & Coleman 1988, Bolsinger et al. 1992, Jondrup et al. 2002, Percy et al. 2002, Valkama et al. 2007) but it can induce defence responses (Vuorinen et al. 2004a, Sasaki-Sekimoto et al. 2005), some of which might also protect plants from herbivory. The direct, possibly harmful effects of O 3 on herbivores are less well known. However, O 3 is a cell damaging agent, high doses of which can be used to kill pests of stored products (Hollingsworth & Armstrong 2005) Transgenic plants and climate change Climate change will create novel challenges and set new targets for plant breeding in future years (Moose & Mumm 2008). Biotechnological methods that confer crop plants resistant to changed abiotic conditions, including drought and high temperatures (Vij & Tyagi 2007), and also adapt to elevated CO 2 or O 3 concentrations (Ainsworth et al. 2008), can be fundamental for agriculture. Transgenic and traditional cultivars will both face new growth conditions as CO 2, temperature and O 3 levels rise. However, assessments of GM crop performance and responses to these changed conditions have seldom been described in science or upon their approval for commercialization. Novak & Haslberger (2000) compared glucosinolate concentrations of a GM cultivar and its parental oilseed rape line: they showed that drought increased GS concentration up to 72.6 mmol g -1 meal, the recommended standard being less than 30 mmol g -1, whereas there were no significant differences between the GM and non-gm cultivars. The physiological response to drought was also similar in both plant lines. The authors stated that environmental influence on the composition of GM crops needs more Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008) 23

24 Sari J Himanen: Bt oilseed rape and climate change attention. It is commonly predicted that the responses of GM crops to environmental changes will be similar to those of traditional cultivars. However, it is possible that differences in their performance could arise due to allocation costs, the magnitude of which depends on the trait introduced. Coviella et al. (2000, 2002) were the first to question the performance and compositional equivalence of Bt plants under CO 2 elevation. Unsurprisingly, they reported decreased concentrations of Bt Cry1Ac toxin in Bt cotton leaves under elevated CO 2 compared to Bt cotton under ambient CO 2. This was confirmed by Chen et al. (2005) and Wu et al. (2007). The magnitude of this decrease was not dramatic, and it can be questioned whether this difference has any agricultural meaning. However, in the same studies, the authors also reported some compositional differences in secondary compounds of Bt cotton and its nontransgenic isoline, revealing the potential for differences in allocation. These findings together raised interest for further studies of the effects of abiotic variations on Bt plants. 1.3 Plant secondary compounds and herbivory Glucosinolates and herbivory Glucosinolates (GSs) are amino acidderived -thioglucoside-nhydroxysulfates, a group of secondary compounds mainly specific to the order Capparales, including the genus Brassica (Fahey et al. 2001). Approximately 120 GSs have been identified, which are classified as indolic, aliphatic or aromatic GSs according to their sidechain structure (Halkier & Gerschenzon 2006). Tryptophan is the amino acid precursor of indolic GSs, phenylalanine and tyrosine are the precursors of aromatic GSs, while aliphatic GSs have six different amino acid precursors. GSs are degraded by an endogenic -thioglucosidase, the myrosinase enzyme. Myrosinase is located in the plant in specific myrosin cells, which are physically separated from intact GSs (Bones & Rossiter 1996). This enables activation upon mechanical, chemical or environmental stimulation to form various biologically active hydrolysis products, such as isothiocyanates, thiocyanates and nitriles, of which some are also volatile. GSs and their degradation products have wide biological and ecological implications for plants, animals and humans. Antiherbivoral, antipathogenic, allelopathic and recently discovered anticarcinogenic effects to humans have been described (reviewed by Halkier & Gershenzon 2006). Due to the bioactivity of these compounds, GS biosynthesis routes and their activation by defence signalling pathways has been very actively studied in the model plant Arabidopsis thaliana over the recent years (reviewed by Grubb & Abel 2006, Yan & Chen 2007). Biologically active plant-extracts and biofumigation are further applications of GSs today (Larkin & Griffin 2007). 24 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008)

25 General introduction Different Brassica species have relatively large variation in their GS profiles. Typically, a few major compounds dominate the GS profile, with several minor constituents (Porter et al. 1991). GS concentration is also variable within the vegetative and reproductive tissues (Brown et al. 2003). Highest concentrations are encountered in the growing leaves, pods and seeds: the plant parts most needed to secure good reproduction, but also roots are rich in specific GSs. With breeding, the GS profile of B. napus has been changed to yield seed that are low in aliphatic GS content compared to traditional high-gs varieties (Chavadej et al. 1994). This has been done because aliphatic GSs have negative effects on domestic animals that eat the oilseed meal. One of the main functions of GSs is to provide resistance against generalist herbivores. For this reason, GSs, especially those belonging to the indolic subgroup, are herbivore-inducible, i.e. their concentrations are increased upon herbivory and their break-down releases biologically active products that have negative effects on attacking herbivores (Bodnaryk 1992, Hopkins et al. 1998, Bartlet et al. 1999). A similar effect has been mimicked by methyl jasmonate treatment (Bodnaryk 1994, Doughty et al. 1995). GS synthesis is also subject to genetic pressure by herbivory (Agrawal et al. 2002). Interestingly, specialist herbivores have developed specific resistance to GS defence. Examples include the cabbage aphid, Brevicoryne brassicae, which has its own myrosinase (Jones et al. 2001), Pieris rapae, which synthesizes a nitrilespecifier protein that metabolizes GSs into less toxic nitriles instead of toxic isothiocyanates (Wittstock et al. 2004) and P. xylostella (diamondback moth, DBM), which has its own desulphatase enzyme that converts active products into less active desulphoglucosinolates (Ratzka et al. 2002). In addition, specialist herbivores including DBM females employ volatile GS break-down products released from damaged plants upon tissue rupture, to locate their host (Renwick & Radke 1990). GSs are also amenable targets for genetic engineering to attain plant resistance towards herbivory (Burow et al. 2006) or pathogens (Brader et al. 2006) Volatile organic compounds (VOCs) - induction by herbivory and the role of VOCs in indirect defence Biogenic volatile organic compounds (VOCs) are lipophilic liquids with high vapour pressure, and are released from plants as volatile emissions into the surrounding atmosphere. Approximately 1700 different compounds have been identified from over 90 plant families (reviewed by Dudareva et al. 2006). Vegetative and floral VOCs can generally be classified into terpenoids, phenyl propanoids and benzenoids (containing an aromatic ring), fatty acid derivatives and amino acid derivatives (Dudareva et al. 2006). Biogenic VOCs serve in numerous ecological roles, including pollinator attraction, indirect defence (attraction of Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008) 25

26 Sari J Himanen: Bt oilseed rape and climate change predators and parasitoids of herbivore prey), allelopathy, pathogen defence, plant-plant interactions and defence against environmental stresses (Holopainen 2004). Their evolutionary background has not been totally elucidated to date. This is mostly due to their highly complex variety in nature and their numerous continuously discovered new functions (Firn & Jones 2006). Terpenoids comprise the largest group of plant VOCs. They are formed either through the cytosolic mevalonate pathway or the plastidic methylerythritol phosphate pathway (reviewed by Dudareva et al. 2006), but research has still to discover the numerous terpene synthases responsible for transforming individual terpenoid compounds from their common precursors. With this knowledge, genetic engineering could be employed in developing terpenemodified plants that selectively attract biological control agents (Kappers et al. 2005). The other main class of vegetative VOCs commonly studied is the fatty acid derivatives, including the C 6 alcohols, esters and aldehydes, generally called the green leaf volatiles (GLVs). GLVs are released when fatty acids on plant cell surfaces are broken down upon mechanical damage, herbivore feeding or other physical damage-induction (Matsui 2006). Terpenoids and GLVs are typically induced by herbivores (Paré & Tumlinson 1997), and their emissions are highly related to the degree and type of feeding damage occurring on the plant (De Moraes et al. 1998, Dean & De Moraes 2006). Herbivore-inducible VOCs can be released upon tissue rupture from the plant cell surface (fatty acid derivatives, which do not show high specificity, but are rather universal in the plant kingdom) or synthesized de novo (terpenoids) and released with a delay after wounding (Turlings et al. 1998). The more specific terpenoids are typically the best indicators of herbivore presence and can even reveal the type of herbivore that feeds on the plant to host-searching insects of higher trophic levels (Turlings et al. 1990, De Moraes et al. 1998). However, the common C 6 GLVs (Z)-3-hexen-1-ol and (Z)-3-hexenyl acetate activate numerous defence genes in neighbouring plants (Arimura et al. 2000), so they also seem to have a role in plant defence and in particular in plant-plant communication. Pest herbivores can also employ volatile cues to find host plants and during the process of egg laying (Renwick & Radke 1990). The ability of higher trophic levels to use plant-emitted VOCs to locate their hosts and prey has been most clearly reported with the model tritrophic systems consisting of Lima bean (Phaseolus lunatus L.), spider mites (Tetranychus urticae Koch) and predatory mites (Phytoseiulus persimilis) (Dicke et al. 1990) and maize, caterpillar herbivores and the parasitoid wasp Cotesia marginiventris (Turlings et al. 1990). The systems of cabbage (B. oleracea) plants - P. xylostella and Cotesia plutellae (Vuorinen et al. 2004b) and aphid-aphid parasitoid interactions on variable host 26 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008)

27 General introduction plants (Du et al. 1996) are also good examples. These studies have demonstrated that predators and parasitoids are able to find host-damaged plants by induced, purely olfactory cues, with no need for visual or physical stimuli. Thereby there exists a multivariate interacting infochemical web based on the blend of plant VOCs. Comparisons of VOC emissions from Bttransgenic cotton plants and their parent lines have indicated quantitative differences (Yan et al. 2004). Herbivore feeding on Bt maize (Turlings et al. 2005, Dean & De Moraes 2006) and Bt oilseed rape (Ibrahim et al. 2008) also shows differences between emission profiles from transgenic and natural lines. VOCs were induced in Bt plants after target herbivore feeding, but the emissions were typically lower than from the susceptible parent lines correlating with the degree of feeding damage. Identical mechanical damage with larval regurgitant applied to the wound site resulted in a highly similar VOC profile in Bt and non-bt maize, which revealed that the differences in VOCs induced by herbivory were solely related to feeding damage and not to changes in biochemical plant defence pathways (Dean & De Moraes 2006). Oviposition by DBM females has been shown to be similar on non-transgenic and Bt-transgenic canola (Ramachandran et al. 1998a), cabbage (Kumar 2004), collard (Cao et al. 2005) and broccoli (Tang et al. 1999), which also suggests that the olfactory stimuli from the plants do not significantly differ. Parasitoid attraction by VOCs has been shown to be unaffected by Bt production in maize, i.e. transgenic and nontransgenic plants were equally attractive to Cotesia marginiventris and Microplitis rufiventris parasitoids (Turlings et al. 2005). However, Schuler et al. (1999) reported greater attraction of Cotesia plutellae to non-transgenic oilseed rape than to Bt-transgenic oilseed rape. Again, this was related to feeding damage, since feeding by Bt-resistant larvae elevated the attractiveness of Bt oilseed rape to the level of non-transgenic plants (Schuler et al. 1999). 1.4 Plant secondary compounds and climate change Effects of elevated CO 2 and O 3 on glucosinolates Glucosinolate concentrations vary significantly within the growing season (Rosa et al. 1996, Gols et al. 2007) or even during a single day (Rosa et al. 1994, Rosa 1997), due to fertilization and in particular due to the sulphur supply (Falk et al. 2007). GS concentrations are also affected by fluctuations in abiotic factors such as light and temperature (Gols et al. 2007). The effects of exposure to elevated CO 2 and O 3 on GS concentrations have been relatively little studied (i.e. Karowe et al. 1997, Reddy et al. 2004, Bidart-Bouzat et al. 2005, Schonhof et al on CO 2 effects; Gielen et al on O 3 effects) with the available results showing variability, with plant species and even cultivar. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008) 27

28 Sari J Himanen: Bt oilseed rape and climate change Karowe et al. (1997) reported a decrease in total foliage GSs under CO 2 doubling for mustard (B. juncea) but not for turnip (B. rapa) or radish (Raphanus sativus) leaves. Unfortunately the authors did not include an analysis of the changes in individual GSs, since it is possible that there were changes in their concentrations even though there was no net change for total GSs. Reddy et al. (2004) found only marginal changes in GSs of two turnip rape (B. rapa ssp. oleifera) cultivars at elevated CO 2, and these changes were not consistent in both cultivars. Foliar GSs in A. thaliana were not affected by elevated CO 2 alone but under combined CO 2 enhancement and herbivory, the induced GS response was % higher than under ambient CO 2 (Bidart-Bouzat et al. 2005). The long chain aliphatic GSs were not responsive to herbivory under either of the CO 2 concentrations but the indolic GS subgroup were subject to greater induction under elevated CO 2 than ambient conditions (Bidart-Bouzat et al. 2005). Schonhof et al. (2007) found an increase in the methylsulfinylalkyl group GSs (glucoraphanin and glucoiberin) and a simultaneous decrease in indolic GSs (glucobrassicin and 4- methoxyglucobrassicin) with elevated CO 2 on broccoli (B. oleracea var. italica) heads. It is predicted that these effects are caused by altered (typically lower) nitrogen content or allocation to carbonbased defences, changed availability of amino acids, dilutional effects (increasing carbon) (Karowe et al. 1997) or changes in N/S ratio or photochemical processes (Schonhof et al. 2007). Whether O 3, as an abiotic phytotoxicant, can induce or repress GS synthesis or change the overall GS profile is not well documented. Predictions are that the plant hormones controlling GS biosynthesis and also involved in O 3 responses, salicylic acid, jasmonic acid and ethylene might interact to change GS synthesis patterns (Sasaki-Sekimoto et al. 2005). Alternatively, O 3 can change protein biosynthesis and destroy O 3 -sensitive proteins (reviewed by Fiscus et al. 2005), leading to either higher or lower availability of specific amino acids for GS biosynthesis. However, in general these interactions are not well defined. Gielen et al. (2006) reported an overall decrease in GSs in B. napus, but the responses of individual compounds were not described Effects of elevated CO 2 and O 3 on VOC emissions and indirect defence Temperature, light level, and water availability affect VOC emissions dynamically (Peñuelas & Llusià 2001, Gouinguené & Turlings 2002). Climate change factors can affect VOC emissions at the biosynthesis level (Rosenstiel et al. 2003) as well as due to physiological factors affecting their release (Niinemets et al. 2004, Noe et al. 2006). Elevated CO 2 has typically led to down-regulation of VOC emissions, especially isoprene and certain monoterpenes (Rapparini et al. 2004, Vuorinen et al. 2004b,c). This is explained by reduced availability of pyruvate for isoprenoid biosynthesis (Rosenstiel et al. 2003) or down-regulation of terpene synthase activities (Loreto et al. 28 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008)

29 General introduction 2001). In certain multitrophic systems it has also led to disturbance in volatile signaling to parasitoids (Vuorinen et al. 2004b), but this phenomenon requires further research. However, the distribution of resources to primary and secondary metabolism of plants under changed environmental conditions is highly complex, and plant-specific variation is apparent (Peñuelas & Estiarte 1998). Whether elevated CO 2 in the future will change plant VOC emissions and multitrophic communication with ecological implications, is still to be determined. Many monoterpenoids and isoprene can quench oxidative radicals and protect cell membranes in the intercellular space of leaves, which has suggested a role in abiotic stress tolerance (i.e. protecting plants against oxidative and thermal stress) for VOCs (Loreto & Velikova 2001; Affek & Yakir 2002; Loreto & Fares 2007). Accordingly, some plants increase their volatile emissions in response to O 3 exposure (Heiden et al. 1999, Llusià et al. 2002, Loreto et al. 2004, Vuorinen et al. 2004a), although isoprene emissions have been mainly reported to decrease with elevated O 3 (Velikova et al. 2005, Fares et al. 2006, Calfapietra et al. 2007). The seasonal and plant species specific variation in woody Mediterranean species grown under 40 ppb O 3 enhancement compared to ambient levels, as well as the differential responses of even individual monoterpenoid emissions detected (Llusià et al. 2002), demonstrated that predicting O 3 effects on VOC emissions is not straight-forward. In field conditions, chronically elevated O 3 has only led to very small changes in terpenoid emissions in birch (Betula pendula Roth) (Vuorinen et al. 2005) and aspen (Populus tremula tremuloides) trees (Blande et al. 2007). Thus, the general mechanisms and the dynamics for O 3 effects on VOC emissions are still largely unknown. VOC emissions are not only affected by their biosynthetic rates, but complex physiological factors can affect their release from leaves (reviewed by Niinemets et al. 2004). The potential direct effects of elevated CO 2 and O 3 activity on stomatal function, may affect the release of certain oxygenated monoterpenoids, although for many other VOCs stomatal control seems to be lacking (Niinemets et al. 2002, Niinemets & Reichstein 2003, Niinemets et al. 2004). Therefore, VOC emissions from plants could show compound-specific changes under both elevated CO 2 (reduced stomatal density or conductance) (Ainsworth & Rogers 2007) and O 3 (closure of stomata to protect photosynthetic apparatus from damage) through their differential physicochemical properties. Overall, the regulation of VOC biosynthesis by defence signalling pathways, the availability of resources for VOC production and direct physiological effects all affect VOC emissions detected under CO 2 and O 3 elevations. In addition to direct effects of elevated O 3 on plant VOC emissions, O 3 reacts with VOCs in the atmosphere (Pinto et al. 2007a,b) and thereby could lead to Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008) 29

30 Sari J Himanen: Bt oilseed rape and climate change changes in VOC cues for plant signalling (Pinto et al. 2007a, c). Pinto et al. (2007a) found that the tritrophic systems of cabbage plants, P. xylostella and C. plutellae and Lima bean (P. lunatus), spider mite (T. urticae) and predatory mite P. persimilis are not significantly disturbed by elevated O 3 even though there is clear degradation of VOCs. In field conditions there were no differences in host location or parasitism rate observed for C. plutellae under background O 3 levels of 1.5 times the ambient level (Pinto et al. 2008). 1.5 Aims of the study and description of experimental plants used Bt oilseed rape (Brassica napus L.) as a model plant Brassica napus ssp. oleifera (oilseed rape) is an oilseed crop having China, Canada, India and Germany as top countries for its cultivation globally (FAO 2008). The annual global yields of rapeseed and rapeseed oil were 49 and 17 million tonnes respectively in 2006 (FAO 2008). Most oilseed rape cultivars used today are of canola double-low quality, defined by low concentrations of both aliphatic GSs and erucic acid in seeds. After oil extraction, double-low canola meal can be safely used for animal feed. The planted area of canola has recently increased due to its properties as a bioenergy crop (Tuck et al. 2006); it also has beneficial biofumigation properties (Larkin & Griffin 2007). There are spring- and winter-type cultivars of canola available, which makes its use as an annual or biannual crop possible and widens the area of cultivation usually limited by climate restrictions. B. napus was originally a hybrid of Brassica rapa and Brassica oleracea, and it has both AA and CC genomes (2n=38) as a heritage from the parents. Being a Brassica species, it has many wild weedy relative species in nature compatible for hybridization (Wilkinson et al. 2003a, Halfhill et al. 2005). In Finland, the most cultivated oilseed is currently turnip rape B. rapa ssp. oleifera, but it is predicted that the cultivation area of the higher yielding B. napus will expand in the future (Tuck et al. 2006). Bt Cry1Ac-transgenic oilseed rape was first created by Stewart et al. (1996), who transformed two canola cultivars, 'Westar' and 'Oscar', by Agrobacterium tumefaciens mediated gene transfer using truncated synthetic Cry1Ac gene constructs. 'Westar' is a traditional spring-type canola cultivar, which is amenable to genetic transformation. Bt oilseed rape was created as a model plant for studying the ecological risks of GM plants (Stewart 2004) and was highly effective at controlling several target Lepidoptera pests (Stewart et al. 1997). Since the creation of this transgenic plant type there has been extensive research on its performance (Ramachandran et al. 1998a, b, c, 2000, Halfhill et al. 2001, Mason et al. 2003) and ecological risks for hybridization with natural relatives (Halfhill et al. 2001, 2002, 2003, 2004, 2005, Warwick et al. 2003, Zhu et al. 2004a, b, Moon et al. 2007). Since the first transformations several 30 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008)

31 General introduction transgenic lines of 'Westar' have been created with green fluorescent protein (gfp) as a selective marker (Halfhill et al. 2001), enabling easy detection of transgenic plants in the field. Of these gfp- Bt lines one was selected for further study in this thesis. This line, 'GT1', is one of the most studied lines and has effective transgene expression with no apparent fitness costs (Halfhill et al. 2001, 2003) Research objectives and outline of the experiments The main objective of this study was to assess direct and indirect defence mechanisms and biotic interactions in transgenic (insect resistant) and nontransgenic plants when grown under environmental stresses. This was experimented by comparing the general performance, allocation to secondary compounds and herbivore interactions of a transgenic insect-resistant Bt oilseed rape line and its non-transgenic parent line. The thesis includes results from multiple experiments, all of which were conducted in computer-controlled growth chambers at the University of Kuopio, with different abiotic factors included as treatments: elevated CO 2, elevated O 3 with various doses and combined elevated CO 2 and temperature. The results of these experiments are described in the following chapters by topic, each representing individual original articles. The treatments used and the parameters measured in each chapter are summarized in Table 1. In chapters 2 and 3 the main hypothesis was that allocation to secondary compounds (glucosinolates in Chapter 2 and volatile terpenoids in Chapter 3) and their induction by herbivory on oilseed rape leaves under elevated CO 2 or elevated O 3 would be lower in the Bt oilseed rape line due to the cost of Bt toxin synthesis (Coviella et al. 2002), and because of reduced feeding damage by target herbivores (Dean & De Moraes 2006). Elevated CO 2 was predicted to decrease GS and terpenoid concentrations as a result of lowered allocation to N-based secondary compounds (GSs) under C enhancement (Peñuelas & Estiarte 1999) and increased competition for pyruvate for photosynthesis (competing with allocation of substrates to isoprenoid synthesis) (Rosenstiel et al. 2003). It was hypothesized that elevated O 3 would change GS and terpenoid concentrations due to investment in antioxidative defences (Fiscus et al. 2005), activation of defence signalling pathways affecting regulation of these secondary compounds (Kangasjärvi et al. 2005) and by direct effects of O 3 on volatiles in the atmosphere (Pinto et al. 2007a,b). Herbivore induction of GSs could be higher under elevated CO 2 based on a previous study on A. thaliana (Bidart-Bouzat et al. 2005), whereas the effects of the O 3 herbivory interaction was previously unrevealed. In Chapter 4, the hypothesis for the study was that Bt oilseed rape would have a reduced ability to respond to elevated CO 2 through increased vegetative growth due to investment on Bt toxin production Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008) 31

32 Sari J Himanen: Bt oilseed rape and climate change (Coviella et al. 2002). It was also predicted that Bt toxin synthesis would result in changes in primary carbon-nitrogen metabolism in leaves. It was hypothesised that herbivores not directly affected by Bt toxin would display altered responses to the potential compositional changes in Bt plants under CO 2 and/or temperature elevations (Chen et al. 2005). Bt toxin concentration was hypothesized to decline under elevated CO 2 as a result of a dilution effect of higher vegetative biomass and/or reduced allocation of nitrogen for its biosynthesis (Coviella et al. 2002, Wu et al. 2007). The aim of chapter 5 was to investigate the stability of the transgenic trait introduced into Bt oilseed rape under elevated O 3 and to assess target herbivore performance under O 3 exposure. It was hypothesized that chronic O 3 would alter the concentration of Bt toxin in foliage due to phytotoxic effects reducing the viability of plants (Fiscus et al. 2005) and differential physiological aging of the plants, as Bt toxin fluctuations have been reported in leaves upon aging (Olsen et al. 2005, Wei et al. 2005). Target herbivores were predicted to perform better on O 3 -exposed plants due to enhanced food quality of plant material and increased palatability (Jondrup et al. 2002, Valkama et al. 2007) and therefore the transgenic trait itself could be effective faster under high O Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008)

33 General introduction Table 1. Summary of the treatments used and parameters measured in different experiments forming the data presented in chapters 2-5. Treatments used Chapter Abiotic factors Biotic factors 2 1) Elevated CO 2 (720 Plutella xylostella ppm) 2) Chronic elevated P. xylostella O 3 (75 and 150 ppb 8h daily) 3) Acute elevated O 3 (250 ppb 8 h daily for two days) Parameters measured Leaf glucosinolate concentrations Leaf biomass P. xylostella mean relative growth rate 3 1) Elevated CO 2 (720 ppm) 2) Chronic elevated O 3 (100 ppb 8h daily) 4 Elevated CO 2 (720 ppm), elevated temperature (+4 C), combined elevated CO 2 and temperature P. xylostella P. xylostella Myzus persicae Brevicoryne brassicae Foliar volatile terpenoid emissions Tritrophic interactions: Cotesia vestalis orientation Above-ground plant biomass Carbon and nitrogen concentration in leaves Photosynthesis Soluble sugars and starch concentration in leaves Chlorophyll pigment concentrations in leaves Foliar Bt toxin concentration M. persicae and B. brassicae performance (reproduction, mean relative growth rate) 5 Chronic elevated O 3 (50, 75, 100 ppb 8h daily) P. xylostella Foliar Bt toxin concentration P. xylostella mean relative growth rate and feeding damage on leaves Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008) 33

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43 CHAPTER 2 CONSTITUTIVE AND HERBIVORE-INDUCIBLE GLUCOSINOLATE CONCENTRATIONS IN OILSEED RAPE (BRASSICA NAPUS) LEAVES ARE NOT AFFECTED BY BTCRY1AC INSERTION BUT CHANGE UNDER ELEVATED ATMOSPHERIC CO 2 AND O 3 Himanen SJ, Nissinen A, Auriola S, Poppy GM, Stewart CN Jr, Holopainen JK, Nerg A-M (2008) Planta 227: Copyright (2008) Reprinted with kind permission from Springer Science+Business Media.

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45 Planta (2008) 227: DOI /s ORIGINAL ARTICLE Constitutive and herbivore-inducible glucosinolate concentrations in oilseed rape (Brassica napus) leaves are not avected by Bt Cry1Ac insertion but change under elevated atmospheric CO 2 and O 3 Sari J. Himanen Anne Nissinen Seppo Auriola Guy M. Poppy C. Neal Stewart Jr. Jarmo K. Holopainen Anne-Marja Nerg Received: 13 August 2007 / Accepted: 11 September 2007 / Published online: 9 October 2007 Springer-Verlag 2007 Abstract Glucosinolates are plant secondary compounds involved in direct chemical defence by cruciferous plants against herbivores. The glucosinolate prowle can be avected by abiotic and biotic environmental stimuli. We studied changes in glucosinolate patterns in leaves of non-transgenic oilseed rape (Brassica napus ssp. oleifera) under elevated atmospheric CO 2 or ozone (O 3 ) concentrations and compared them with those from transgenic for herbivoreresistance (Bacillus thuringiensis Cry1Ac endotoxin), to assess herbivory dynamics. Both elevated CO 2 and O 3 levels decreased indolic glucosinolate concentrations in transgenic and non-transgenic lines, whereas O 3 speciwcally increased the concentration of an aromatic glucosinolate, 2- phenylethylglucosinolate. The herbivore-inducible indolic S. J. Himanen (&) A. Nissinen J. K. Holopainen A.-M. Nerg Department of Environmental Science, University of Kuopio, P.O. Box 1627, Kuopio, Finland [email protected] A. Nissinen Agrifood Research Finland, Plant Protection, Jokioinen, Finland S. Auriola Department of Pharmaceutical Chemistry, University of Kuopio, P.O. Box 1627, Kuopio, Finland G. M. Poppy School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK C. N. Stewart Jr. Department of Plant Sciences, University of Tennessee, Knoxville, TN , USA glucosinolate response was reduced in elevated O 3 whereas elevated CO 2 altered the induction dynamics of indolic and aliphatic glucosinolates. Herbivore-resistant Bt plants experienced minimal leaf damage after target herbivore Plutella xylostella feeding, but exhibited comparatively similar increase in glucosinolate concentrations after herbivory as non-transgenic plants, indicating that the endogenous glucosinolate defence was not severely compromised by transgenic modiwcations. The observed diverences in constitutive and inducible glucosinolate concentrations of oilseed rape under elevated atmospheric CO 2 and O 3 might have implications for plant herbivore interactions in Brassica crop-ecosystems in future climate scenarios. Keywords Brassica Climate change Glucosinolate defence Plant herbivore interactions Transgenic Bt plants Abbreviations Bt Bacillus thuringiensis GM Genetically modiwed GS Glucosinolate Introduction Transgenic insect-resistant plants that produce speciwc Bacillus thuringiensis (Bt)-derived endotoxins are among the Wrst transgenic plant applications to be introduced into commercial use, together with herbicide-tolerant plants (Stewart 2004; Poppy and Wilkinson 2005; James 2006). Bt cotton, maize and rice were grown on 19 million hectares globally in 2006, and cultivation of transgenic crops has increased over the years, today amounting to over

46 428 Planta (2008) 227: million hectares under cultivation (James 2006). At the same time, factors associated with global climate change (e.g., elevated atmospheric carbon dioxide (CO 2 ), and ozone (O 3 ) concentrations), are forecasted also to have impact on the way agriculture is conducted (Long et al. 2006; IPCC 2007). Elevated CO 2 typically increases plant biomass and net photosynthesis (Poorter et al. 1997), whereas O 3 is a stress factor that decreases photosynthesis rates, and leads to the formation of reactive oxygen species in the plant leaf apoplast (Fiscus et al. 2005; Kangasjärvi et al. 2005). The evects of future climate change on the metabolism and plant herbivore interactions of GM crop cultivars are still largely unexplored, but indications for altered Bt toxin concentrations in response to elevated CO 2 have been detected on Bt cotton (Coviella et al. 2000, 2002; Chen et al. 2005). Glucosinolates (β-thioglucoside-n-hydroxysulfates; GSs) are nitrogen- and sulfur-containing, amino acid-derived secondary compounds, which are found mostly from plants of the order Capparales (Fahey et al. 2001; Halkier and Gershenzon 2006). They are classiwed into aliphatic, aromatic and indolic glucosinolates according to their precursor amino acid and the side-chain structure. In response to herbivore, mechanical or chemical stimulation, GSs are broken down by myrosinase (β-thioglucoside glucohydrolase) enzyme, which coexists in the plant tissue in speciwc myrosin cells (Bones and Rossiter 1996). Damage evoked by herbivore feeding can induce GS production and change the quantities of individual GSs produced, especially those of the indolic subgroup (Koritsas et al. 1991; Bodnaryk 1992; Bartlet et al. 1999; Bidart-Bouzat et al. 2005). Adapted (i.e., specialist) herbivores are able to tolerate or detoxify GSs and their toxic break-down products (Ratzka et al. 2002), and it is unclear if the performance of these pests is avected by the GS concentration (Giamoustaris and Mithen 1995; Siemens and Mitchell-Olds 1996; Bartlet et al. 1999; Li et al. 2000). In fact, GS degradation products can serve as feeding promoters and attractants for specialist insects, and thereby convert the induced defence of the plant into a benewt for specialist herbivores (Louda and Mole 1991). If the GS synthesis and its induction are reduced under future climate scenarios, generalist pests, i.e., insects able to tolerate low GS levels, might represent a new threat for cruciferous plants and in turn, the specialist herbivores could also be avected (Li et al. 2000). Since transgenic insect-resistant plants require target herbivores to uptake Bt toxin through feeding, diverences in GS prowles that lead to altered feeding patterns by herbivores could also avect the exposure of target insects to the Bt toxin (Schuler et al. 1999a), and thereby the evectiveness of the Bt trait. It is unclear how elevated CO 2 and O 3 levels will evect the production of plant secondary metabolites, especially glucosinolates (Bezemer and Jones 1998; Penuelas and Estiarte 1998; Reddy et al. 2004). Total GSs have decreased or remained unchanged when plants were exposed to elevated CO 2, and the magnitude and patterns have varied between diverent plant species (Karowe et al. 1997; Reddy et al. 2004), and there also seems to be variations in the individual GSs and GS subgroups (Bidart-Bouzat et al. 2005; Schonhof et al. 2007). O 3 fumigation decreases the total GS concentration of B. napus (Gielen et al. 2006), but evects on levels of the individual GSs have not been reported. In this study, we address the secondary defence of Bt cry1ac- transgenic oilseed rape, Brassica napus ssp. oleifera, since it might serve as a model for transgenic Brassicas, which have a characteristic secondary metabolism (i.e., glucosinolates) and there is the capability for interspeciwc hybridization with wild relatives (Halfhill et al. 2001, 2005). We investigated the evects of elevated CO 2, elevated ozone, herbivory and the constitutive expression of a Bt cry1ac gene on the glucosinolate concentrations present in oilseed rape. Larval damage of Plutella xylostella (diamondback moth), a Brassica specialist herbivore, which is controlled by Bt Cry1Ac, is reported to induce GS production (Koritsas et al. 1991; Bidart-Bouzat et al. 2005), but a decrease in the feeding damage to Bt plants could inhibit this defence induction process (Schuler et al. 1999b; Dean and De Moraes 2006). We also hypothesized that the induction could be decreased under elevated CO 2 because of the competition for nitrogen needed for accelerated plant growth (Bryant et al. 1983; Herms and Mattson 1992), which might lead to a lower availability of amino acids, the precursors of glucosinolates (Grubb and Abel 2006). Elevated O 3 might lead to either decreased or increased GS concentrations through various evects on plant biochemistry, e.g., by diverential amino acid availability, altered protein synthesis and changed enzyme activities (Fiscus et al. 2005). Materials and methods Plants and growth conditions Oilseed rape (B. napus L. ssp. oleifera) cv. Westar was transformed with the psam12 plasmid, which contained a truncated synthetic Bt cry1ac transgene (courtesy of Mycogen, and a GFP (green Xuorescent protein) mgfp5-er (courtesy of Jim HaseloV, University of Cambridge, UK) marker gene under the control of separate CaMV 35S promoters (Halfhill et al. 2001). Westar is a spring-type canola-quality variety, which has a low aliphatic glucosinolate concentration and it has been used widely in agriculture. The transgenic event used here, GT1, represents a single insert and evective transgene 123

47 Planta (2008) 227: expression with no apparent Wtness costs (Halfhill et al. 2001; Mason et al. 2003). Westar (considered here as the non-transgenic parent line) and Bt (the transformant line) F 4 seeds (originating from the laboratories of G.M. Poppy and C.N. Stewart Jr.) were sown in 0.66 l pots in 2:1:1 fertilized soil (Kekkilä Oyj, Tuusula, Finland; N:P:K 100:30:200 mg l 1 ):B2 sphagnum peat (Kekkilä; N:P:K 110:40:220 mg l 1 ):sand ( mm) mixture. We conducted three separate experiments, the Wrst testing CO 2 and herbivore, the second testing chronic O 3 and the third studying acute O 3 and herbivore evects on glucosinolate concentrations of oilseed rape. All three experiments were independently replicated twice and their data pooled. The descriptions of single experiments follow below. In all of the experiments, Bt-transgenic and non-transgenic plants were grown together in computer-controlled growth chambers (2.6 m 3, Bioklim 2600T, Kryo-Service Oy, Helsinki, Finland) at 20/16 C temperature with a 16L:8D photoperiod (light adjusted to PAR of approximately 250 μmol m 2 s 2 ). The plants were irrigated daily and no additional fertilizer was used. To avoid any possibility of chamber-speciwc growth condition evects, the plants inside the chamber, and the treatments between the two to four similar chambers, were rotated weekly. Herbivores One target of Lepidoptera-speciWc Bt Cry1Ac toxin is the diamond-back moth (DBM) Plutella xylostella L. (Lepidoptera: Yponomeutidae), which is a cosmopolitan Brassica pest (Talekar and Shelton 1993). The highly Bt-sensitive DBM larvae were maintained on broccoli (Brassica oleracea var. italica) with a 12L:12D photoperiod, at approximately 25 C temperature and 50% RH. Descriptions of individual experiments Experiment 1 (elevated CO 2 and herbivory) In Experiment 1, Westar and Bt plants were grown inside two growth chambers, one receiving 360 μl l 1 (ambient) CO 2 and the other 720 μl l 1 (elevated) CO 2 levels containing air (supplied from gas tanks) for the duration of the experiment (24 h daily), with continuous monitoring (as in Reddy et al. 2004). At days, half of the plants of each plant type were moved into another similar growth chamber with similar CO 2 conditions in order to exclude any air-mediated volatile induction of defence responses in the control plants. Six plants per plant type from both CO 2 levels were infested by adding Wve second to third-instar DBM larvae (approximate body length 3 5 mm) to feed on each plant. Two additional larvae were transferred onto Bt plants after 24 h to replace dead larvae and to ensure consistent feeding damage. After 48 h, the larvae were removed from the plants, and subsequently the total leaf biomass from each herbivore-damaged and corresponding intact control plant was individually deep-frozen in liquid nitrogen and stored at 80 C until analysis. Glucosinolates were analyzed from six replicates per treatment (two plant types, two CO 2 levels and herbivory/no herbivory), from a total of 48 samples. For the determination of plant growth, dry weight of leaves (drying at 60 C for 48 h) from six plants per plant type at both CO 2 levels were determined at day age. The biomass gain per day was calculated by dividing the Wnal weight by the number of days from sowing to harvest. Experiment 2 (chronic O 3 ) In Experiment 2, the treatments were control (Wltered air), 75 nl l 1 (moderate O 3 ) and 150 nl l 1 (high O 3 ) with the Westar and Bt plants being grown inside three similar growth chambers (one at each O 3 treatment). O 3 fumigation was run for 8 h daily (from 8.30 am to 4.30 pm) from the third day after sowing until the end of the experiment (total of 19 days). This resulted in AOT 40 (accumulated O 3 exposure over a threshold of 40 nl l 1 ) levels of 5,320 nl l 1 h for the 75 ppb treatment and 16,720 nl l 1 h for the 150 nl l 1 treatment. O 3 was generated from pure oxygen with an O 3 generator (Fisher OZ 500 Ozone generator, Bonn, Germany), and continuously monitored with an O 3 analyzer (Environnement S.A., Model O 3 42M, Poissy, France). NO 2 and NO x concentrations in the chambers were measured with a nitrogen oxides monitor (AC30M, Environnement S.A.) to be between 3 and 7 nl l 1. Total leaf biomass from Wve to six 22-day old plants per plant type from each O 3 treatment were individually collected for glucosinolate analysis (total of samples) and the leaf dry biomass from Wve plants per treatment was measured as described in Experiment 1, but at 14 and 21 day age. In addition, to determine the functionality of the Bt trait, relative growth rate of P. xylostella larvae was measured on detached second growth leaves of both plant types (21-day age), grown at each O 3 treatment. The petioles of detached leaves were put into 2.0 ml eppendorf-tubes Wlled with tap water and sealed with parawlm. The leaves were placed into 200 ml plastic containers (Nalgene; and the lids were replaced with Wne mesh to allow evaporation to escape. Four 2nd to 3rd instar DBM larvae were group-weighed and placed on each leaf in the growth chambers under control conditions. After 24 h, the larvae were collected, their possible mortality recorded, and living larvae were group-weighed. The relative growth rate was calculated with the equation: [ln (Wnal weight of larvae) ln (initial weight of larvae)]/duration of feeding in days (van Emden 1969). 123

48 430 Planta (2008) 227: Experiment 3 (acute O 3 and herbivory) In Experiment 3, Westar and Bt plants were grown inside one growth chamber at 20/16 C temperature with a 16L:8D photoperiod. Equal amounts of both plant types were transferred to four growth chambers (using the above mentioned growth conditions) days from sowing, with two receiving 250 nl l 1 O 3 exposure for 8 h on two consecutive days (AOT 40 3,360 nl l 1 h) and the other two chambers served as controls. In one control and one O 3 treatment chamber, six Westar and Bt plants were infested with three 2nd instar and two 4th instar P. xylostella larvae. After 48 h feeding, six plants from each treatment (two plant types, two O 3 levels, herbivory/no herbivory; total of 48 samples) were individually collected for glucosinolate analysis as in Experiment 1. However, due to technical problems during the glucosinolate analysis, the number of replicates varied from three to six in the Wrst repetition. Analysis of glucosinolates GSs were extracted from approximately 300 mg of freezedried leaves as described previously (Loivamäki et al. 2004). DesulphoGSs were analyzed with a HPLC (Hewlett- Packard series 1050) chromatograph, equipped with a Sun- Fire C 18 reversed-phase 3.5 μm, mm (Waters; in experiment 1, 2 and the second repetition of Experiment 3) or HP Lichrospher 100 RP 18e, 5 μm, mm column (the Wrst repetition of Experiment 3), and quantiwed with a UV detector (wavelength 229 nm). We used a gradient from 0 to 25% acetonitrile in 45 min (30 C, Xow rate 0.2 ml min 1 with Waters column and 0.8 ml min 1 with HP column). IdentiWcation of the major GSs was veriwed by running the same samples with HPLC-MS (Finnigan LTQ, San Jose, CA, USA), using the same column and electrospray ionization MS. Here, we used a gradient from 0 to 20% acetonitrile in 40 min and the eluting buver contained also 0.5% formic acid. Full scan mass spectra were measured from 250 to 400 m/z. The data was analyzed with Xcalibur software (Finnigan), and the individual desulphogss were identiwed by the presence of a corresponding (M + H + ) ion and (M + H ) fragment ion (resulting from the loss of glucose moiety) except for 2-hydroxy-4- pentenyl-gs, which was identiwed by ions at 323 and 144 m/z (Tolrà et al. 2000). Compounds were quantiwed as μmol g 1 dry leaf weight, with sinigrin (prop-2-enyl-gs) as an internal standard. CertiWed reference seed material BCR- 367R supplied by the European Community Bureau of Reference (Linsinger et al. 2001) was also used for testing the performance of the method. The total glucosinolate concentration was determined as the sum of individual compounds identiwed and concentrations of GS subgroups (indolic, aliphatic and aromatic GSs) summed from the concentrations of individual GS with the corresponding side-chain structures. Statistical analysis All the three experiments (each with two similar repetitions in time) were analyzed separately. Mixed model analysis of variance (ANOVA) was used to test the main evects of plant type (Bt transgenic or non-transgenic; Experiment 1, 2 and 3), CO 2 level (Experiment 1), O 3 level (Experiment 2 and 3) and herbivore feeding (Experiment 1 and 3) and their interaction evects with the two replications of the experiments as a random evect on concentrations of individual GSs, subgroups, total GSs, biomass gain and P. xylostella RGR. Variables having a non-gaussian distribution were log (x + 1) transformed prior to analysis to obtain normality and Levene s test was applied to test the equality of error variances. This showed inequality for aliphatic GSs in Experiment 1, so this was analyzed with the variable causing inequality (CO 2 level) as a random evect. Pairwise comparisons were done with Tukey HSD or Dunnett s T3 test, depending on the homogeneity of the error variances. All statistical analyses were carried out with SPSS for Windows 14.0 statistical software package (SPSS Inc.; Results EVects of elevated CO 2 and O 3 on growth of plants and herbivore performance in elevated O 3 Elevated CO 2 increased the leaf biomass of both non-transgenic and Bt-transgenic plant lines, whereas elevated O 3 had a negative impact on plant growth (Fig. 1a, b). No statistically signiwcant diverences were observed between the plant types in their vegetative growth. In Experiment 2, all P. xylostella larvae feeding in Bt plants exhibited a negative relative growth rate, which was evidence of the evectiveness of Bt toxin at all O 3 treatments (mixed model ANOVA, main evect transformation, F = , P <0.001; Fig.2). O 3 did not avect the RGR of P. xylostella on non-transgenic plants. Glucosinolate prowles of non-transgenic and Bt-transgenic oilseed rape We identiwed a total of eight glucosinolates from the leaves of oilseed rape, representing aliphatic, aromatic and indolic glucosinolates (Table 1). The indolic 3-indolylmethyl-GS (3IM-GS) was the main compound detected, followed by aromatic 2-phenylethyl-GS (2PE-GS) and indolic 4-hydroxy-3-indolylmethyl-GS (4OH-3IM-GS). In the two 123

49 Planta (2008) 227: a d mg gain Biomass -1 b d mg gain Biomass ppm CO2 720 ppm CO2 P CO2 =0.036 WESTAR Control 75 ppb ozone 150 ppb ozone P ozone <0.001 after 14 d Fig. 1 Mean (+SE) daily gain of leaf dry weight (mg per day) in nontransgenic (Westar) and Bt-transgenic B. napus plants. Plants were grown in (a) control (360 μl l 1 ) or elevated 720 μl l 1 CO 2 level (n = 12) and harvested at days age or (b) ambient (control, Wltered air, 0 nl l 1 O 3 ) or elevated (75 nl l 1 or 150 nl l 1 ) O 3 level (n = 10) and harvested at days age. SigniWcant P values (mixed model ANOVA, signiwcance at P < 0.05) for main evects (CO 2 and O 3 level) are shown separate long-term experiments (Experiment 1: elevated CO 2 and Experiment 2: chronic O 3 ), 3IM-GS accounted for and % of total glucosinolates in the non-transgenic and for and % in the Bt oilseed rape, respectively. The Bt line displayed a qualitatively similar glucosinolate prowle to the non-transgenic parent line (Tables 1, 2). No diverences were observed in the concentrations of individual GSs between intact non-transgenic and Bt-transgenic plants grown under control conditions in either of the experiments. In Experiment 1, ANOVA revealed diverences in the concentrations of 4OH-3IM-GS and 2PE-GS between the plant types (F = , P = and F = 5.918, P = 0.017, respectively). However, pairwise comparisons showed that this was related to the lower concentration of 4OH-3IM-GS in Bt plants under elevated CO 2 (Tukey HSD; P = 0.04) compared to non-transgenic plants under control conditions. The 2PE-GS concentrations were BT P ozone =0.001 WESTAR BT WESTAR BT after 21 d Relative growth rate Westar Bt P transf <0.001 CONTROL 75 PPB OZONE 150 PPB OZONE Fig. 2 Relative growth rate +SE of diamond-back moth larvae on non-transgenic (Westar) and Bt-transgenic B. napus plants grown under no O 3 (control, Wltered air, 0 nl l 1 O 3 ) or elevated (75 or 150 nl l 1 ) O 3 level, n = 14. SigniWcant P values (mixed model ANOVA, signiwcance at P < 0.05) for main evects (transformation, O 3 level) are shown not signiwcantly diverent in the pairwise comparisons (Tukey HSD, P > 0.05). In Experiment 2, concentrations of individual GSs and GS structural groups were similar in Bttransgenic and non-transgenic plants (mixed model ANOVA; P >0.05). Elevated CO 2 and herbivore induction of glucosinolates Individual GSs showed variable responses to elevated CO 2 and DBM feeding (Table 1). The 3IM-GS concentration was lower in intact plants grown under elevated CO 2 than under ambient CO 2 (mixed model ANOVA, main evect CO 2, F = 5.357, P = 0.023). Also concentrations of the indolic GS group and total GSs showed a similar reduction (F = 4.668, P =0.033 and F = 3.948, P = 0.05) and elevated CO 2 led to a marginal increase in the 2-hydroxy-4- pentenyl-gs concentration in intact plants (mixed model ANOVA, main evect CO 2, F = 3.215, P = 0.076). Otherwise the concentrations of GSs were not signiwcantly responsive to elevated CO 2. Herbivore feeding resulted in a trend to increase the 3IM-GS (mixed model ANOVA, interaction CO 2 DBM, F = 3.284, P = 0.073) and 4-methoxy-3IM-GS (mixed model ANOVA, interaction CO 2 DBM, F = 3.682, P = 0.058) concentrations more in elevated CO 2 than under control CO 2 level. In contrast, 2-hydroxy-4-pentenyl-GS and aliphatic GS group showed an opposite response, with the concentration being signiwcantly or marginally signiwcantly increased in DBM-fed plants under ambient conditions but not under elevated CO 2 (mixed model ANOVA, interaction CO 2 DBM, F = 4.088, P = and F = and P = 0.067, respectively). The concentration of 2PE-GS was increased by DBM feeding independently 123

50 432 Planta (2008) 227: Table 1 Mean ( SE) concentrations of individual, subgroup and total glucosinolates (μmol g 1 dry weight) in leaves of intact and P. xylostella (DBM)-damaged cv. Westar (non-transgenic) and Bt-transgenic B. napus plants grown in ambient (360 μl l 1 ) or elevated (720 μl l 1 ) CO 2 level (n =12) Glucosinolate Westar (non-transgenic) oilseed rape Bt-transgenic oilseed rape Ambient CO 2 Ambient CO 2 + DBM Elevated CO 2 Elevated CO 2 + DBM Ambient CO 2 Ambient CO 2 + DBM Elevated CO 2 Elevated CO 2 + DBM Aliphatic 4-MethylsulWnylbutyl-GS OH 4-Pentenyl-GS c Pentenyl-GS c Total aliphatic Aromatic 2-Phenylethyl-GS c Indolic 3-Indolylmethyl-GS c OH 3-Indolylmethyl-GS c b a,b a,b a,b a,b a,b a a,b 4-Methoxy-3-indolylmethyl-GS c a a,b a b a,b a,b a a,b 1-Methoxy-3-indolylmethyl-GS c Total indolic Total glucosinolates a, b SigniWcant diverences between treatment means (Tukey HSD test, P < 0.05) marked with divering letters c IdentiWcation veriwed with mass spectrometry 123

51 Planta (2008) 227: Table 2 Mean ( SE) concentrations of individual, subgroup and total glucosinolates (μmol g 1 dry weight) in leaves of cv. Westar (non-transgenic) and Bt-transgenic B. napus plants grown under no O 3 (control, Wltered air) or elevated (75 or 150 nl l 1 ) O 3 level (n =11) Glucosinolate Westar (non-transgenic) oilseed rape Bt-transgenic oilseed rape Control 75 nl l 1 ozone 150 nl l 1 ozone Control 75 nl l 1 ozone 150 nl l 1 ozone Aliphatic 4-MethylsulWnylbutyl-GS OH 4-Pentenyl-GS f Pentenyl-GS f Total aliphatic Aromatic 2-Phenylethyl-GS f a b c a,b a,b c Indolic 3-Indolylmethyl-GS f d d e d d e 4-OH 3-Indolylmethyl-GS f Methoxy-3-indolylmethyl-GS f Methoxy-3-indolylmethyl-GS f Total indolic d d e d d e Total glucosinolates a,b,c SigniWcant diverences between treatment means (Tukey HSD test, P < 0.05) marked with diverent letters d,e SigniWcant diverences between treatment means (Dunnett s T3 test, P < 0.05) marked with diverent letters f IdentiWcation veriwed with mass spectrometry of the CO 2 level (mixed model ANOVA, main evect DBM feeding, F = 9.536, P = 0.003). The concentration of indolic GSs showed a marginally higher response to herbivory under elevated than under control CO 2 (mixed model ANOVA, interaction CO 2 DBM, F = 3.699, P = 0.058), resembling the response of the major GS, 3IM-GS. Total GS concentration responded similarly (mixed model ANOVA, interaction CO 2 DBM, F = 3.183, P = 0.078). The response of non-transgenic and Bt-transgenic plants to elevated CO 2 and herbivory was similar otherwise, but the concentration of 4-methoxy-GS was more induced by herbivory in non-transgenic than Bt-transgenic plants (interaction plant type DBM, F = 4.488, P =0.037). Chronic elevated ozone evects on glucosinolate concentrations We found a clear change in the GS prowle of oilseed rape under chronic elevated O 3. Elevated levels of O 3 decreased the concentration of 3IM-GS (mixed model ANOVA, F = , P <0.001; Table2), and the structural group of indolic GSs (mixed model ANOVA, F = , P < 0.001), whereas it strongly increased the concentration of 2PE-GS (mixed model ANOVA, F = , P < 0.001). The percentage of 2PE-GS from total GSs increased from and (control, Westar and Bt, respectively) to and under 75 nl l 1 and as high as and % under 150 nl l 1 O 3. The responses of 2PE-GS and 3IM-GS to O 3 were similar in non-transgenic and Bt-transgenic plants. O 3 did not alter the concentrations of other GSs (mixed model ANOVA, P > 0.05). EVects of combined acute ozone and herbivore feeding on glucosinolate concentrations In Experiment 3 we attempted to clarify the dynamics of GSs in response to elevated O 3 exposure (contributions of 3IM-GS and 2PE-GS) and to discover whether there are interactions with simultaneous herbivore induction and O 3. The 3IM-GS concentration was decreased by acute O 3 (mixed model ANOVA, F = 7.552, P = 0.008) and DBM damage increased the 3IM-GS concentration in control treatment but not in O 3 -treated plants (mixed model ANOVA, O 3 DBM interaction, F = 5.027, P =0.028). The 2PE-GS concentration was increased by six to sevenfold (resulting in 2.18 and 3.19 μmol g 1 DW 1 in intact Westar and Bt plants, respectively) in acute elevated O 3 (F = , P = 0.003) (Fig. 3). In pairwise comparisons, the 3IM-GS concentration of DBM-damaged non-transgenic plants was higher than in all other treatments (Tukey HSD, P < 0.05), whereas the 2PE-GS concentration was signiwcantly higher in all O 3 -treatments (including herbivory or not), in comparison with the concentration in the leaves of undamaged non-transgenic and Bt-transgenic control plants (Tukey HSD, P <0.05). 123

52 434 Planta (2008) 227: change) (fold GS concentration Fig. 3 Changes (+SE) induced by acute O 3 -treatment (250 nl l 1 8h daily over 2 days) and diamond-back moth (DBM) feeding on the concentrations of 3-indolylmethylGS and 2-phenylethylGS compared to control cv. Westar plants (scaled to 1). SigniWcant P values for main evects of ozone and DBM feeding and their interaction (mixed model ANOVA, signiwcance at P < 0.05) are shown; n = 8 11 Discussion P O3 =0.008 P DBM =0.014 P O3*DBM =0.028 WESTAR BT WESTAR BT 3-indolylmethylGS Control Ozone Glucosinolate responses of Bt-transgenic and non-transgenic oilseed rape Control+DBM Ozone+DBM P O3 < phenylethylGS The GS concentration of GT1 Bt-transgenic oilseed rape and its parent line showed similar individual GS responses to elevated CO 2 and to elevated O 3 which indicates that there were no pleiotropic evects by Bt toxin production on constitutive or inducible GS concentrations when combined with elevated CO 2 or O 3. Previously, Bt cotton and a corresponding non-transgenic line were shown to exhibit similar responses to elevated CO 2, as total non-structural carbohydrates were increased and total nitrogen was decreased in plants of both types, which is evidence of equivalence in the way these lines respond to elevated CO 2 (Chen et al. 2005). Total phenolics were lower in Bt cotton compared with the non-transgenic parent line under low nitrogen availability, but again both lines showed similar total phenolic, terpenoid aldehyde, condensed tannin, and carbonnitrogen responses to elevated CO 2 (Coviella et al. 2002). To our knowledge, the response of Bt-transgenic plants to elevated atmospheric O 3 concentrations has not been reported earlier, but our results suggest that the growth and GS response to O 3 is not avected by Bt toxin production. Elevated CO 2 and herbivore induction of glucosinolates Elevated CO 2 decreased the concentration of total GSs and the major GS, 3IM-GS, and this points to a possible decline in constitutive GS defence in oilseed rape compared to its capabilities under ambient CO 2 conditions. Our results are in accordance with the carbon/nutrient balance hypothesis of Bryant et al. (1983), stating that nitrogen-containing defence compounds may decrease when C/N ratio of plants is increased, such as under elevated CO 2. Another possibility is that indolic GS concentrations are reduced due to dilution evect by higher vegetative biomass rather than by alterations in their synthesis. In recent studies, elevated CO 2 did not signiwcantly avect concentrations of individual GSs of A. thaliana genotypes (Bidart-Bouzat et al. 2005), or those of cabbage (Reddy et al. 2004). Species-speciWcity seems to be general, as a decrease in total GSs was found for mustard leaves, but not for turnip or radish leaves in response to elevated CO 2 (Karowe et al. 1997). In the heads of broccoli (Brassica oleracea var. italica) aliphatic GSs increased and indolic GSs decreased under elevated CO 2 (Schonhof et al. 2007). Also a cultivar-speciwc GS response for individual GSs was detected with Brassica rapa ssp. oleifera in elevated CO 2 (Reddy et al. 2004). Cv. Westar responded to CO 2 mainly by changing the concentrations of the indolic GSs, which represent the predominant GSs in this cultivar. The aliphatic GS concentration was generally low and seemed to be unavected by elevated CO 2. Possible changes in GS prowles under elevated CO 2 might have larger ecological impacts with respect to plant-insect interactions in the future, since glucosinolates seem to be subject to substantially high genetic variation in nature (Agrawal et al. 2002). It is known that indolic GSs are herbivore-inducible (Bodnaryk 1992; Siemens and Mitchell-Olds 1998; Bartlet et al. 1999) and this was also clearly rexected in the elevated 3IM-GS concentration following herbivory in Experiment 3 (acute O 3 ). However, in Experiment 1 no induction was observed under ambient CO 2 conditions. The feeding damage inxicted by the larvae was higher in Experiment 3 than in Experiment 1, since more mature instar larvae were used, which might explain the observed increase in induction. Elevated CO 2 typically causes compensatory feeding by chewing herbivores due to the altered nutritional quality of leaves (Docherty et al. 1996), which could relate to the observed trend of increased induction of indolic GSs under elevated CO 2. Also in A. thaliana, only a few minor GSs have responded to DBM feeding under ambient CO 2 and the inducibility of GS responses has increased under elevated CO 2 (Bidart-Bouzat et al. 2005). The DBM larvae produced only a few small holes on Bt oilseed rape leaves in contrast with continuous feeding and extensive defoliation of Westar. However, even this minimal damage on Bt plants still resulted in clear GS induction of the major GSs. Thus, Bt production itself did not seem to cause a signiwcant reduction of the endogenous, inducible GS defence system. 4-Methoxy-3IM-GS, on the other hand, 123

53 Planta (2008) 227: showed higher induction in non-transgenic than Bt-transgenic plants, which indicates this compound to be related to the degree of feeding damage, as reported also on Arabidopsis after aphid herbivory (Kim and Jander 2007). The quantity of feeding damage avecting defence induction on insect-resistant transgenic plants has been reported before with plant volatile indirect defence (Schuler et al. 1999b; Dean and De Moraes 2006). The emission of inducible terpenoids from Bt maize was diverent from the parent line, but this was dependent on the level and type of feeding damage and not attributable to the lower initial induction capability of the transformant line (Dean and De Moraes 2006). However, indirect defence induction activates multiple signalling and biosynthetic pathways to diverent degrees (Dudareva et al. 2006), whereas the GS pattern of a plant is usually more consistent and a quantitative induction of GSs is a typical response to herbivory (Bartlet et al. 1999; Bidart-Bouzat et al. 2005). Glucosinolate response to chronic and acute ozone exposure Previously Gielen et al. (2006) observed a decrease in total GSs of oilseed rape under O 3 exposure but to our knowledge no previous reports have described O 3 responses of individual GSs in this species. We found a clear reduction in 3IM-GS and an increase in 2PE-GS concentration under chronic and acute elevated O 3 and elevated O 3 also attenuated the DBM induction of 3IM-GS. Alterations in 2PE-GS and 3IM-GS concentrations might therefore result in altered herbivore performance under elevated O 3 (Cole 1996). After myrosinase activity 2PE-GS forms volatile break-down products such as 2-phenylethylisothiocyanate, which can act as an attractant or even toxicant against certain herbivores (Buskov et al. 2002) or inhibitory against other organisms interacting with plants such as mycorrhizal fungi (Vierheilig et al. 2000). In contrast, indolic GS degradation products are unstable and decompose readily (Fahey et al. 2001). However, they also serve as attractants for certain species such as root Xies (Gouinguené and Städler 2006). Altered dynamics of these two compounds thereby have the potential to severely alter plant herbivore interactions. We hypothesize the observed shift in GS prowle by O 3 could be regulated through changes in the concentrations of the two plant hormones: salicylic acid (SA) and jasmonic acid (JA), both of which are involved in O 3 responses (Kangasjärvi et al. 2005) and avect also GS synthesis (Mewis et al. 2005) (Fig. 4). In previous studies, 2PE-GS was increased through exogenous SA treatment on B. napus (Kiddle et al. 1994; Cole 1996). In accordance, indolic GSs have been linked to the JA-dependent pathway (Brader et al. 2001; Sasaki-Sekimoto et al. 2005) and have been Ozone Indolic GSs up-regulation (Kangasjärvi et al. 2005) + - JA SA + 2-phenylethylGS Indolic GSs Fig. 4 Interactions linking O 3 stress, plant hormones involved in signal transduction and glucosinolates (GSs). O 3 is known to avect salicylic acid (SA) and jasmonic acid (JA) contents and signalling (reviewed by Kangasjärvi et al. 2005). According to previous studies, exogenous JA induces indolic GSs (Doughty et al. 1995; Loivamäki et al. 2004) whereas SA speciwcally induces 2-phenylethyl-GS (Kiddle et al. 1994; Cole 1996) (solid lines). Our current Wndings show a decrease in indolic GSs and an increase in aromatic 2-phenylethylGS after O 3 stress (double lines). This suggests that the observed GS change may be accompanied by a positive induction of SA signalling and a suppression of JA signalling (a decrease in JA, dashed line) induced by JA treatment (Doughty et al. 1995; Loivamäki et al. 2004). Simultaneous JA and SA treatment led to attenuation of JA induction on the total GS concentration on A. thaliana, indicating that SA and JA interact physiologically in GS defence (Cipollini et al. 2004). The SAoverproducing A. thaliana mutant displayed reduced 3IM-GS and 1-methoxy-3IM-GS levels (Mikkelsen et al. 2003) but A. thaliana does not seem to produce 2PE-GS in major amounts endogenously, which limits comparison of this response to GS synthesis of oilseed rape. However, Brader et al. (2006) have shown that increasing aromatic GS synthesis in A. thaliana by genetic engineering can enhance SA-mediated defences at the same time as suppressing jasmonate-dependent defences. Our results suggest that O 3 as an abiotic elicitor of plant defences also acts to trigger certain GS defence responses of oilseed rape. We conclude that as long as transgenic-based insect resistance allows plants to be slightly but suyciently damaged to upregulate inducible GS synthesis, this endogenous defence does not seem to be compromised by transgenic defences, a factor which could be considered as important for their sustainable agricultural use (Schuler et al. 1999b). The quantitatively altered GS prowle of plants resulting from elevated CO 2 and O 3 could avect the performance of various adapted and non-adapted herbivores and also involve predation eyciency of insects of higher trophic levels through altered emissions of volatile GS degradation products (Potting et al. 1999; Schuler et al. 1999b) in the future. DiVerential herbivore performance has been noted under elevated CO 2 (Reddy et al. 2004) and O 3 (Jondrup et al. 2002), but until now the role of secondary compounds such as glucosinolates in this response has not been + (Kiddle et al. 1994; Cole 1996) (Doughty et al. 1995; Loivamäki et al. 2004) cross talk? (Traw et al. 2003; Brader et al. 2006) 123

54 436 Planta (2008) 227: thoroughly studied. Here we did not detect any signiwcant diverences in the RGR of DBM larvae in elevated O 3 but longer-term trials are needed. Moreover, since all insectresistant GM plants require plant material to be ingested by the herbivore, the amount of Bt toxin ingested is directly related to feeding preferences. Thus altered feeding from the altered GS prowle might lead to altered uptake and exposure of target and non-target herbivores to Bt toxin and thereby change the evectiveness of the Bt trait (Schuler et al. 1999a). Also, comparison of the constitutive and inducible GS patterns of oilseed rape under combined elevated CO 2 and O 3 are encouraged, since their evects seem to be partly antagonistic. Acknowledgments We thank Virpi Tiihonen (University of Kuopio, Finland) for glucosinolate sample preparations and HPLC work, and Timo Oksanen (University of Kuopio, Finland) for programming and maintaining the growth chambers. We are also grateful to Maaria Loivamäki (currently at Research Center Karlsruhe, Germany) and Delia M. Pinto (University of Kuopio, Finland) for their help in sampling during the individual experiments and Marja-Leena Hännilä (University of Kuopio, Finland) for her statistical advice. We also acknowledge Ewen MacDonald for revision of the language. This work was supported by the Academy of Finland (ESGEMO programme, decision no ) (S.J.H., A.N., J.K.H. and A.-M.N.) and enabled by USDA BRAG grants to C.N.S. References Agrawal AA, Conner JK, Johnson MT, Wallsgrove R (2002) Ecological genetics of an induced plant defense against herbivores: additive genetic variance and costs of phenotypic plasticity. Evolution 56: Bartlet E, Kiddle G, Williams I, Wallsgrove R (1999) Wound-induced increases in the glucosinolate content of oilseed rape and their evect on subsequent herbivory by a crucifer specialist. 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57 CHAPTER 3 EFFECTS OF ELEVATED CARBON DIOXIDE AND OZONE ON VOLATILE TERPENOID EMISSIONS AND MULTITROPHIC COMMUNICATION OF TRANSGENIC INSECTICIDAL OILSEED RAPE (BRASSICA NAPUS.) Himanen SJ, Nerg A-M, Nissinen A, Pinto DM, Stewart CN Jr, Poppy GM, Holopainen JK New Phytologist (in press) Copyright (2008) Wiley-Blackwell Publishing Ltd. Reprinted with kind permission.

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59 Research Blackwell Publishing Ltd Effects of elevated carbon dioxide and ozone on volatile terpenoid emissions and multitrophic communication of transgenic insecticidal oilseed rape (Brassica napus) Sari J. Himanen 1, Anne-Marja Nerg 1, Anne Nissinen 1,2, Delia M. Pinto 1, C. Neal Stewart Jr 3, Guy M. Poppy 4 and Jarmo K. Holopainen 1 1 Department of Environmental Science, University of Kuopio, P.O. Box 1627, FIN Kuopio, Finland; 2 MTT Agrifood Research Finland, Plant Protection, FIN Jokioinen, Finland; 3 Department of Plant Sciences, University of Tennessee, 2431 Joe Johnson Drive, Knoxville, TN , USA; 4 School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK Summary Author for correspondence: Sari Himanen Tel: Fax: [email protected] Received: 20 May 2008 Accepted: 30 August 2008 Does transgenically incorporated insect resistance affect constitutive and herbivoreinducible terpenoid emissions and multitrophic communication under elevated atmospheric CO 2 or ozone (O 3 )? This study aimed to clarify the possible interactions between allocation to direct defences (Bacillus thuringiensis (Bt) toxin production) and that to endogenous indirect defences under future climatic conditions. Terpenoid emissions were measured from vegetative-stage non-bt and Bt Brassica napus grown in growth chambers under control or doubled CO 2, and control (filtered air) or 100 ppb O 3. The olfactometric orientation of Cotesia vestalis, an endoparasitoid of the herbivorous diamondback moth (Plutella xylostella), was assessed under the corresponding CO 2 and O 3 concentrations. The response of terpenoid emission to CO 2 or O 3 elevations was equivalent for Bt and non-bt plants, but lower target herbivory reduced herbivore-inducible emissions from Bt plants. Elevated CO 2 increased emissions of most terpenoids, whereas O 3 reduced total terpenoid emissions. Cotesia vestalis orientated to host-damaged plants independent of plant type or CO 2 concentration. Under elevated O 3, host-damaged non-bt plants attracted 75% of the parasitoids, but only 36.8% of parasitoids orientated to host-damaged Bt plants. Elevated O 3 has the potential to perturb specialized food-web communication in Bt crops. Key words: Brassica napus ssp. oleifera (oilseed rape), Cotesia vestalis, elevated CO 2, elevated ozone, plant volatiles, Plutella xylostella (diamondback moth), transgenic plants, tritrophic interactions. New Phytologist (2008) doi: /j x The Authors (2008). Journal compilation New Phytologist (2008) Introduction Climate change and the expanding use of transgenically modified crop cultivars are predicted to be among the most important issues affecting agriculture in the coming decades, and are the subjects of extensive research efforts (James, 2007; IPCC, 2007). Production of Bacillus thuringiensis (Bt) crystal endotoxin (Cry) proteins in a crop plant limits specific herbivorous insect attack without the need for chemical treatments (Romeis et al., 2006). However, Bt toxin production requires an incremental resource investment for the plant. The possibility of competition between constitutive Bt toxin production and allocation to secondary metabolism under changed abiotic conditions related to climate change (Coviella et al., 2002; Chen et al., 2005) led us to assay secondary metabolism, in the form of volatile organic compounds (VOCs), in Bt 1

60 2 Research Cry1Ac-transgenic oilseed rape (Brassica napus) and its nontransgenic parent line under atmospheric CO 2 or O 3 elevations. We extended this evaluation to investigate the ecological effects that these possible changes in VOC emissions might have through effects on multitrophic communication. The emission of inducible VOCs by plants in response to herbivore feeding attracts predators and parasitoids, that is, insects of higher trophic levels, to prey or host, limiting further herbivore attack (indirect defence) (Dicke et al., 1990; Turlings et al., 1990; Du et al., 1996). Increases in plant VOC emissions are related to both the quantity and the quality of herbivore feeding damage (e.g. De Moraes et al., 1998; Turlings et al., 1998). Bt toxin in transgenic Bt plants inhibits target herbivore feeding and it is reasonable to suppose that reduced feeding damage by Bt-sensitive herbivores can reduce the induction of VOC emissions (Schuler et al., 1999a,b). Differences in VOC emissions from intact Bt cotton (Gossypium hirsutum; Yan et al., 2004) and herbivore-damaged Bt maize (Zea mays; Turlings et al., 2005; Dean & De Moraes, 2006) and oilseed rape (Ibrahim et al., 2008) have been found, compared with their nontransgenic parent lines. Importantly, Bt plants do allow herbivore feeding initiation and it is therefore possible that this might be adequate for VOC induction. Some quantitative differences in constitutive VOC emissions between transgenic plants and their parent lines are to be expected (Yan et al., 2004), for example as a result of differences in phenotype produced by the introduced trait (Himanen et al., 2008a), whereas possible qualitative differences indicating allocation differences could be more ecologically relevant. With VOC emissions, the genetic variation among plant cultivars can be high, as shown for example in maize lines (Degen et al., 2004). Substantial equivalence analysis of transgenic crop varieties examines how the composition of the transgenic cultivar fits into the scale of variation found among traditionally bred cultivars (Kok & Kuiper, 2003). From an ecological perspective, this kind of comparative analysis of VOC profiles could also be useful in detecting possible risks attributable to alterations in multitrophic interactions (Turlings et al., 2005). The first aim of this study was to compare the herbivoreinduced volatile emission profiles of nontransgenic and Bttransgenic oilseed rape plants after target herbivory, defining the role of insect resistance for endogenous VOC defence activation in this model species. We also wanted to investigate whether a specialized tritrophic interaction among oilseed rape, the Bt-target herbivore Plutella xylostella and its endoparasitoid Cotesia vestalis (Hymenoptera: Braconidae) is affected by Bt production. Schuler et al. (1999a) showed that the parasitoid C. vestalis preferred plants damaged by Bt-resistant larvae over those on which Bt-susceptible larvae were feeding, which provides an indication of the potential of feeding damage to affect multitrophic communication in Bt oilseed rape. However, Turlings et al. (2005) demonstrated that the parasitoids Cotesia marginiventris and Microplitis rufiventris did not distinguish between equally damaged non-bt and Bt maize despite quantitative VOC differences in their profiles. Potential differences in VOC induction in Bt plants might lead to changes in multitrophic interactions in crop ecosystems and surroundings, although these effects have to be compared with those of conventional pest control practices such as the use of chemical insecticides, which often have more harmful ecological effects (Romeis et al., 2006). Chemical control is in many cases unspecific and directly affects numerous beneficial insect species negatively (Romeis et al., 2006). The second aim of our study was to determine whether changed abiotic conditions related to future climatic conditions, namely elevated atmospheric CO 2 or O 3 exposure, affect the induction of VOC emissions and tritrophic communication comparably in non-bt and Bt oilseed rape, with the latter allocating resources to constitutive Bt toxin production. Depending on the plant species and individual compounds, terpenoid emissions have been found to increase, to decrease or to remain unaffected under elevated concentrations of CO 2 (Constable et al., 1999; Loreto et al., 2001; Staudt et al., 2001; Niinemets et al., 2004; Vuorinen et al., 2004a,b, 2005). However, C 5 -isoprenoid isoprene emission has been consistently observed to decrease under elevated CO 2 (Rosenstiel et al., 2003). O 3 stress can increase VOC emissions from plants (Heiden et al., 1999; Vuorinen et al., 2004c; Beauchamp et al., 2005; Rinnan et al., 2005), through increased release or synthesis. In addition, isoprene seems to protect plants against oxidative stress by stabilizing cellular membranes (Loreto & Velikova, 2001; Loreto et al., 2001). Also, O 3 interacts with VOCs present in the atmosphere to form secondary aerosol particles (Pinto et al., 2007a), and extinction times are low especially for the most reactive sesquiterpenes (Atkinson & Arey, 2003). The net effect of these complex factors acting together could produce a reduction, an increase, or no net change in VOCs available for parasitoid olfactory detection with O 3 elevation. Considering the functioning of multitrophic interactions in future atmospheres, O 3 exposure has not been found to disturb the orientation of the specialist predatory mite Phytoseilus persimilis on prey-infested Phaseolus lunatus or the parasitoid C. vestalis on host-damaged Brassica oleracea (Vuorinen et al., 2004c; Pinto et al., 2007b, 2008). However, elevated CO 2 exposure resulted in cultivar-specific attractiveness in the orientation behaviour of C. vestalis towards host-damaged B. oleracea (Vuorinen et al., 2004a), indicating possible changes in tritrophic interactions. To our knowledge, no previous studies of VOC emissions and tritrophic interactions under elevated CO 2 or O 3 have been performed using Bt-transgenic plants. However, these factors could be important when assessing the ecological effects of insect-resistant plants and determining whether Bt crop systems might have lower efficiency in attracting natural enemies under elevated CO 2 or O 3. We hypothesized, firstly, that Bt oilseed rape plants might have reduced VOC emissions compared with the non-bt parent line. Constitutive emissions could be reduced by The Authors (2008). Journal compilation New Phytologist (2008)

61 Research 3 allocation changes as a result of investment in Bt toxin production (Coviella et al., 2002; Chen et al., 2005) and subsequent delayed growth (Himanen et al., 2008a), whereas inducible emissions could be lower as a result of reduced target herbivory (Dean & De Moraes, 2006). Secondly, we expected that, under elevated CO 2, terpenoid emissions would be decreased in both plant types because of simultaneous increasing pyruvate competition for higher photosynthesis levels (Rosenstiel et al., 2003) and this would, again, be more pronounced in the Bt-transgenic plants investing in Bt toxin production. Our third hypothesis was that O 3 exposure causing oxidative stress and allocation to synthesis of defence proteins (Fiscus et al., 2005) could lead to increases in VOC emissions (Vuorinen et al., 2004c), but the presence of O 3 in the gas phase would lead to atmospheric degradation reactions and to a net decrease in VOCs detectable to parasitoids (Pinto et al., 2007b). Bt-transgenic plants, having the extra investment cost of Bt toxin protein synthesis, but being subject to lower herbivory, were hypothesized to have lower induction of emissions in response to elevated O 3 compared with non-bt plants. In addition, the degradation of terpenoids in the gas phase could reduce the VOCs attracting parasitoids even more in the case of Bt-producing plants. Materials and Methods Plants, herbivores and parasitoids Oilseed rape (Brassica napus ssp. oleifera L.) cv. Westar transformed to contain a truncated synthetic Bt cry1ac transgene (courtesy of Mycogen, Dow AgroSciences, Indianapolis, IN, USA) and a green fluorescent protein (GFP) mgfp5-er (courtesy of Jim Haseloff) marker gene, event GT1 (Halfhill et al., 2001), was selected for use in these experiments. This event contains a single homozygous insert and produces effective transgene expression with no apparent fitness costs. F 4 seeds of Bt-transgenic and nontransgenic oilseed rape (cv. Westar parent line) sown in 0.66-l pots in a 2 : 1 : 1 fertilized compost (Kekkilä, Tuusula, Finland; nitrogen:phosphorus:potassium (N:P:K) 100 : 30 : 200 mg l 1 ): B2 Sphagnum peat (Kekkilä; N:P:K 110 : 40 : 220 mg l 1 ): sand mixture were used in all experiments. Plants were grown in computer-controlled growth chambers (2.6 m 3 ; Bioklim 2600T; Kryo-Service Oy, Helsinki, Finland) under a 16 : 8 h light:dark photoperiod (light adjusted to photosynthetically active radiation c. 250 μmol m 2 s 1 ) with 20 : 16 C day:night temperature and daily watering. CO 2 and O 3 effects were studied in separate experiments. The herbivores used were Bt Cry1Ac-susceptible diamondback moth (DBM; Plutella xylostella L. (Lepidoptera: Yponomeutidae)) larvae originating from a colony reared on broccoli (Brassica oleracea ssp. italica) under a 12 : 12 h light:dark photoperiod, c. 25 C and 50% relative humidity at the University of Kuopio. Plutella xylostella is a cosmopolitan pest and a specialist on Brassica species (Talekar & Shelton, 1993). As parasitoids we used Cotesia vestalis (Haliday) (Hymenoptera: Braconidae) (previously known as Cotesia plutellae (Kurdjumov)), a specialist solitary endoparasitoid preferably attacking P. xylostella early instar larvae. Cotesia vestalis parasitoids were reared on DBM larvae (second instar larvae offered for egg laying) on broccoli at the University of Kuopio. Emerging C. vestalis parasitoids were collected daily into plastic insect cages, of which two sides were covered with nylon mesh to allow ventilation. They were fed on water-honey solution, and 1- to 3-d-old females were used in the Y-tube assay. Expt 1: VOCs from CO 2 treatments and tritrophic interactions Equal numbers of nontransgenic and Bt-transgenic plants were sown in two growth chambers (one at 360 μl l 1 and one at 720 μl l 1 CO 2, supplied from gas tanks, 24 h daily, with concentrations monitored continuously, as in Vuorinen et al., 2004a). The experiment was repeated twice because of the limited number of growth chambers available simultaneously. The repetition was used as a blocking factor in the statistical analysis to avoid pseudoreplication issues, as individual plants served as replicates. Also, to avoid any effects of chamberspecific growth conditions, the treatments were rotated weekly between the two similar chambers used, and the plants were rotated inside the chambers at the same time. After d, half of the plants of each plant type were moved into a similar growth chamber with similar CO 2 conditions for herbivore treatment in order to avoid any air-mediated induction of VOC synthesis in the control plants. Five individual plants per plant type and CO 2 concentration were infested with five second- to third-instar DBM larvae and additional larvae were transferred to the Bt plants after 24 h to replace any dead larvae in the first repetition. Control plants were intact plants of the same age. After 48 h, the larvae were removed from plants, and subsequently the plants were used either for collecting VOCs or for Y-tube olfactometric tests. VOCs were collected from five individual vegetative-stage plants per treatment (two plant types; two CO 2 concentrations; herbivory/no herbivory), from a total of 40 plants per repetition. The collection system used is described elsewhere (Ibrahim et al., 2005). In addition, in the VOC collections we used the same controlled CO 2 concentrations as used during growth of the plants, with the pressurized air going to the VOC collection chamber supplied from gas tanks, adjusted to 360 (control) or 720 (elevated) μl l 1 CO 2. The collection time was 30 min (first repetition) or 60 min (second repetition). The samples were analysed with a gas chromatograph-mass spectrometer equipped with a mass spectral detector (Hewlett- Packard GC 6890, MSD 5973; Hewlett-Packard, Palo Alto, CA, USA) as described previously (Ibrahim et al., 2005) with an HP-5 column (length 50 m; internal diameter 0.2 mm; film thickness 0.5 μm) in repetition 1 and with an HP-5MS The Authors (2008). Journal compilation New Phytologist (2008)

62 4 Research column (length 50 m; internal diameter 0.2 mm; film thickness 0.33 μm; J&W Scientific, Agilent Technologies, Santa Clara, CA, USA) in repetition 2. Identification was based on comparing mass spectra to the spectra of pure standards (Vuorinen et al., 2004a; Ibrahim et al., 2005) and those from the Wiley library (John Wiley & Sons, Ltd., Chichester, UK). Quantification was based on known concentrations of reference compounds injected into Tenax TA (Supelco, Bellefonte, PA, USA). As we had no standard for α-thujene and δ-3- carene, their responses were quantified using limonene and the responses of β-elemene and (E,E)-α-farnesene were quantified using (E)-β-farnesene (Vuorinen et al., 2004b; Ibrahim et al., 2008). Although this method has been used in numerous previous studies (e.g. Degenhardt & Lincoln, 2006; Blande et al., 2007; Staudt & Lhoutellier, 2007), it does not allow 100% accuracy in quantification with mass spectrometer detection, and the quantitative amounts of these compounds are tentative. However, similar treatment effects would be seen with data based purely on relative peak areas, which is why tentative quantification was preferred to at least give an estimation of the quantitative scale. The emission was calculated as ng g 1 dry weight shoot biomass h 1 (oven-drying for 48 h at 60 C). Leaf area (cm 2 per plant) was also measured by scanning or photographing the leaves and the specific leaf area (SLA) was calculated by dividing the leaf area by the dry weight of the leaves. The number of feeding holes, grouped into larger and smaller than 2 mm diameter, on the herbivore-infested plant leaves was recorded in repetition 1. A more precise quantification of the amount of leaf area fed was achieved by photographing the herbivore-damaged leaves in repetition 2, and calculating the feeding area in mm 2 with Adobe Photoshop software (Adobe Systems Incorporated, Wilmington, DE, USA). Female C. vestalis parasitoids were individually introduced into a Y-tube system described previously (Ibrahim et al., 2005). Air going to the Y-tube system was adjusted to the corresponding CO 2 concentrations (supplied from gas tanks) that the plants had been grown in (i.e. control (360 μl l 1 ) or elevated (720 μl l 1 )). The maximum time for the parasitoid to make a choice was set to 5 min. Each parasitoid individual was tested only once. A total of 8 10 plant pairs were tested per CO 2 concentration and plant type, and 8 10 wasps were tested per plant pair over two consecutive days. Comparisons were made between transgenic (DBM infested vs noninfested) and nontransgenic (DBM infested vs noninfested) plants at control (360 μl l 1 ) and elevated (720 μl l 1 ) CO 2. Expt 2: VOCs from O 3 treatments and tritrophic interactions Plants were sown at two time-points (7-d intervals to enable testing of same-aged plants) with no O 3 (control) or chronic elevated O 3 (from the 3rd day after sowing; 100 nl l 1 for 8 h daily, from 08:30 to 16:30 h) in four similar growth chambers (two for control and two for elevated O 3 ). O 3 was generated from pure oxygen with an O 3 generator (OZ 500 Ozone Generator; Fisher, Bonn, Germany), and continuously monitored with an O 3 analyser (Model O 3 42M; Environnement S.A., Poissy, France). Two days before each VOC collection or Y-tube test day, plants (17 26 d old) were randomly selected from the two chambers, and infested with DBM larvae for 48 h as in Expt 1. Plants with and without herbivores were placed into different chambers (having similar conditions as before, i.e. either filtered air or 100 nl l 1 O 3 in two chambers) to avoid air-mediated induction. For collecting VOCs and testing parasitoid behaviour under elevated O 3, a different VOC collection and Y-tube testing system than used in Expt 1 was developed (described in detail in Pinto et al., 2007b). Because of the reactive properties of O 3, a larger air space and a higher plant biomass were needed to ensure maintenance of a stable O 3 concentration inside the system. This, however, led to quantitative differences in emissions of the plants in control conditions compared with emissions from slightly younger control plants in Expt 1, and therefore the results of the two experiments are to be considered independently. For the VOC collection, four vegetative-stage plants (19 28 d old), in pots that had the soil covered with Teflon covers, were simultaneously placed inside a 22-l glass desiccator. These four plants together served as one replicate for each treatment. In total, VOCs were collected from four replicates (16 plants per treatment; 64 in total), and 20 to 28 plants per treatment were used in the Y-tube behavioural assay. Separate plants were used in VOC sampling and the Y-tube assay. VOCs were collected under no O 3 or 100 nl l 1 O 3 for 90 min from each set of plants. VOCs were analysed as in Expt 1 with an HP-5MS column. We used the same criteria in the Y-tube assay for assessing C. vestalis orientation as in Expt 1. In addition, as previous experience can enhance their searching efficacy (Potting et al., 1999), the parasitoids were offered a learning period of 30 min by placing a DBM-infested cv. Westar oilseed rape plant inside the cage at the beginning of the day on which they were used in the Y-tube tests. Comparisons were made between transgenic (DBM infested vs noninfested) and nontransgenic (DBM infested vs noninfested) plants at control (0 nl l 1 ; filtered air) and elevated (100 nl l 1 ) O 3. A total of four to six sets of plants per treatment were used over 4 d. Statistical analysis Before statistical analysis, we tested the data set for normality and equality of error variances of variable residuals. As a result, some variables were log (x + 1) or square-root transformed for normality. Biomass and specific leaf area results were analysed with a linear mixed model (plant type and CO 2 concentration as fixed effects and the repetition of the experiment as a random effect). Herbivore feeding damage results were analysed with analysis of variance with plant type and CO 2 concentration as fixed factors. The Authors (2008). Journal compilation New Phytologist (2008)

63 Research 5 Table 1 Shoot biomass ± 1 SEM and specific leaf area (SLA) ± 1 SEM in nontransgenic (Non-Bt) cv. Westar and Bt-transgenic (Bt) oilseed rape (Brassica napus ssp. oleifera) plants grown under control or elevated CO 2 and under no ozone (control, filtered air) or elevated ozone and the results of the statistical analysis a Shoot biomass (mg DW) SLA (cm 2 g 1 DW) Non-Bt Bt Non-Bt Bt Control CO ± ± ± ± 20.6 Elevated CO ± ± ± ± 15.4 F 1,36 P F 1,36 P Plant type CO 2 concentration Plant type CO 2 concentration Control, no O ± ± nl l 1 O ± ± 15.2 F 1,8 P Plant type O Plant type O a Plants were grown under the following conditions. Expt 1: 360 μl l 1 (control) or 720 μl l 1 (elevated) CO 2, with plants harvested at age d (two repetitions, n = 10, individual plants used as replicates). Expt 2: no O 3 (control) or 100 nl l 1 O 3 for 8 h daily, with plants harvested at age d (n = 3, four plants per replicate). ANOVA or mixed model results for main effects of plant type, CO 2 concentration (CO 2 ) and elevated O 3 (O 3 ) and their interactions are shown (statistical significance (P < 0.05) in bold). DW, dry weight. A linear mixed model with repetition of the experiment as a random factor (CO 2 experiment) or analysis of variance (O 3 experiment) was used in further analysis. VOC results from the CO 2 experiments were analysed first using a preplanned comparison between intact non-bt and Bt plants in the control treatment. Further models included: plant type and herbivory among CO 2 or O 3 treatments (to assay herbivore induction); plant type and CO 2 or O 3 treatment excluding herbivory (to analyse CO 2 and O 3 effects on constitutive VOCs); and finally plant type, CO 2 or O 3 treatment and herbivory to compare all treatment means within experiments. The Student Newman Keuls test was used for post hoc comparisons of VOC results. Emissions of α-thujene, (E)-4,8-dimethyl- 1,3,7-nonatriene (DMNT) and (E,E)-α-farnesene in the O 3 experiment were analysed with the nonparametric Kruskal Wallis test followed by Mann Whitney U post hoc tests with Bonferroni correction, because of failure to meet the predictions of parametric tests. Behavioural tests for C. vestalis were analysed with the nonparametric binomial test. All statistical analyses were performed using the spss for Windows 14.0 statistical software package (SPSS Inc., Chicago, IL, USA). Results For plants of both types (i.e. nontransgenic and Bt-transgenic), shoot biomass was higher in plants grown under elevated CO 2 than in those grown under control CO 2 (Table 1). The SLA was reduced by elevated CO 2. Chronic 100 nl l 1 O 3 treatment reduced shoot biomass. Bt plants had a lower total vegetative biomass than non-bt plants in both of the experiments, but the response to CO 2 or O 3 elevations was similar in the two plant types. Chronic O 3 exposure caused slight visible damage on the leaves of both plant types, which appeared as interveinal chlorosis (data not shown). DBM larvae fed successfully, in a continuous manner, on the non-bt plants, whereas the larvae were able only to initiate feeding on Bt plants. The number of feeding holes > 2 mm diameter was 9.2 ± 1.0 (mean ± 1 SEM) in nontransgenic plants and zero in Bt plants (ANOVA, F 1,16 = 240.3, P < for main effect of plant type). There were 1.8 ± 1.1 and 17.2 ± 3.5 feeding holes < 2 mm diameter in the nontransgenic and Bt-transgenic plants under control CO 2 (ANOVA, F 1,16 = 45.08, P < for main effect of plant type). The leaf area eaten was 27.6 ± 5.7 and 3.6 ± 0.5 mm 2 for the nontransgenic and Bt-transgenic plants (ANOVA, F 1,16 = 55.58, P < 0.001, main effect of plant type), respectively. Elevated CO 2 did not statistically significantly affect the number of feeding holes or the amount of leaf area fed (P > 0.05 for main effect of CO 2 concentration). There were 9.8 ± 0.6 and 0.4 ± 0.4 feeding holes > 2 mm diameter and 2.8 ± 1.2 and 13.8 ± 0.9 feeding holes < 2 mm diameter (representing total feeding areas of 33.4 ± 4.7 and 2.2 ± 0.4 mm 2 ) in the non-bt and Bt plants under elevated CO 2, respectively. We quantified terpenoid emissions in the VOC profiles of nontransgenic and Bt-transgenic plant types grown with CO 2 or O 3 enhancement and in the corresponding control conditions in both experiments (Table 2). In addition to terpenoids, one green leaf volatile (GLV), (Z)-3-hexenyl acetate, was detected The Authors (2008). Journal compilation New Phytologist (2008)

64 6 Research Table 2 Terpenoid emissions from intact and Plutella xylostella (diamondback moth (DBM))-damaged nontransgenic (Non-Bt) and Bt-transgenic (Bt) oilseed rape (Brassica napus ssp. oleifera) plants grown in (a) control CO 2 or elevated CO 2 and in (b) no ozone (control, filtered air) or elevated ozone (a) Terpenoid (ng g 1 h 1 shoot DW) Control CO 2 (360 μl l 1 ) Elevated CO 2 (720 μl l 1 ) Non-Bt Non-Bt + DBM Bt Bt + DBM Non-Bt Non-Bt + DBM Bt Bt + DBM α-thujene t 2.82 ± 0.85 a 6.52 ± 0.93 b 2.60 ± 0.90 a 4.98 ± 1.24 ab 5.83 ± 2.05 a ± 2.82 b 6.61 ± 2.22 a ± 2.13 ab α-pinene 4.84 ± ± ± ± ± 0.95 a 9.48 ± 1.00 b 4.62 ± 1.67 a 7.52 ± 1.58 ab Sabinene ± 4.62 a ± 4.54 b ± 1.43 ab ± 4.81 b ± 3.66 a ± 7.86 b ± 7.43 ab ± 7.53 b β-myrcene 8.97 ± 2.14 ab ± 1.39 b 3.79 ± 1.31 a 9.40 ± 1.60 ab 8.72 ± 2.20 a ± 2.80 b 8.08 ± 2.78 a ± 2.31 ab δ-3-carene t 0.74 ± ± ± ± ± ± 4.89 nd ± 5.02 Limonene ± 2.28 a ± 4.53 b ± 2.86 a ± 3.48 b ± 6.71 a ± b ± 7.65 a ± 8.87 ab 1,8-Cineole ± ± ± ± ± ± ± ± γ-terpinene 0.62 ± ± ± ± ± ± ± ± 0.68 (E)-DMNT 1.66 ± 1.50 a ± 5.87 b 0.35 ± 0.24 a 0.92 ± 0.43 a 1.16 ± 0.69 a ± 5.17 b 1.71 ± 1.12 a 2.65 ± 1.38 a β-elemene t 2.76 ± ± 5.79 nd 9.18 ± ± 1.19 a ± 4.58 c nd a ± 0.97 b (E,E)-α-Farnesene t 4.75 ± ± ± ± ± 0.99 a ± 0.88 b 4.63 ± 1.36 a 4.41 ± 1.12 a Total ± a ± b ± a ± ab ± a ± c ± ab ± bc (b) Terpenoid (ng g 1 h 1 shoot DW) Control (filtered air, no ozone) Elevated ozone (100 nl l 1 ) Non-Bt Non-Bt + DBM Bt Bt + DBM Non-Bt Non-Bt + DBM Bt Bt + DBM α-thujene t 0.14 ± ± ± ± 0.91 nd nd nd nd α-pinene 3.65 ± ± ± ± ± ± ± ± 2.71 Sabinene 1.16 ± 0.15 a 3.40 ± 0.65 b 1.67 ± 0.28 a 3.12 ± 0.29 b 0.78 ± ± ± ± 0.62 β-myrcene 2.21 ± ± ± ± ± ± ± ± 0.59 δ-3-carene t 1.72 ± 0.17 a 2.02 ± 0.20 a 3.39 ± 0.43 b 3.75 ± 0.52 b 0.74 ± ± ± ± 0.28 Limonene 5.16 ± ± ± ± ± ± ± ± ,8-Cineole 1.43 ± ± ± ± ± ± ± ± 1.17 γ-terpinene nd nd nd nd nd nd nd nd (E)-DMNT nd 1.10 ± 0.20 nd nd nd 0.08 ± 0.08 nd nd β-elemene t nd nd nd nd nd nd nd nd (E,E)-α-Farnesene t nd 1.24 ± ± ± 0.17 nd nd nd nd Total ± ± ± ± ± ± ± ± 5.41 DBM-damaged plants were infested for 48 h before volatile organic compound (VOC) collection. VOCs were collected under the same CO 2 and O 3 concentrations under which the plants were growing. Values correspond to the average of four (O 3 experiment) and five to ten replicates (CO 2 experiment) ± 1 SEM. Different superscript letters in the results indicate statistically significant (Student Newman Keuls test, P < 0.05) differences in treatment means (non-bt and Bt plants with or without herbivory) analysed within each CO 2 or O 3 treatment. t Tentative quantification; no authentic standard compound available; quantification based on response of structurally most related terpenoid. nd, not detected (below detection limit); DMNT, (E)-4,8-dimethyl-1,3,7- nonatriene; DW, dry weight. The Authors (2008). Journal compilation New Phytologist (2008)

65 Research 7 Table 3 P-values for main effects of CO 2 concentration, elevated O 3 and herbivory (Plutella xylostella feeding) and their interactions on emissions of individual terpenoids a (a) Source α-thu α-pin Sab β-myr δ-car Lim Cin γ-ter DMNT β-ele α-far Total CO 2 < < < < < < Her < < < < < < < < Bt CO Bt Her < CO 2 Her Bt CO 2 Her (b) Source α-thu α-pin Sab β-myr δ-car Lim Cin γ-ter DMNT β-ele α-far Total O 3 na < nd na nd na Her na < nd na nd na Bt O 3 na nd na nd na Bt Her na nd na nd na O 3 Her na nd na nd na Bt O 3 Her na nd na nd na a CO 2 control (360 μl l 1 ) vs elevated (720 μl l 1 ) CO 2 concentration; Herbivory (Her), intact vs DBM damaged; Bt, non-bt vs Bt; O 3, control (filtered air) vs elevated (100 nl l 1 ) O 3. α-thu, α-thujene; α-pin, α-pinene; Sab, sabinene; β-myr, β-myrcene; δ-car, δ-3-carene; Lim, limonene; Cin, 1,8-cineole; γ-ter, γ-terpinene; DMNT, (E)-4,8-dimethyl-1,3,7-nonatriene; β-ele, β-elemene; α-far, (E,E)-α-farnesene; nd, not detected; na, not applicable (variable does not allow ANOVA). Statistical testing is based on (a) F 1,71, linear mixed model and (b) F 1,24, ANOVA; significant effects (P < 0.05) in bold. from the emission profile, but its emission was not significantly affected by plant type, elevated CO 2 or O 3, or herbivory (results not shown). We also detected small quantities of glucosinolate breakdown products, such as isothiocyanates, thiocyanates and nitriles, from the VOC profile (results not shown). However, because of their very low concentrations, we were unable to effectively quantify their emissions. In the CO 2 experiment, the first preplanned comparison between intact non-bt and Bt plants of the control treatment revealed no significant differences (P > 0.05) in constitutive emissions between the plant types (Table 2a). Total terpenoid emission was increased by herbivory in the control CO 2 treatment (mixed model including plant type and herbivory under control CO 2, F 1,35 = 12.63, P = 0.001), and under elevated CO 2 (mixed model with plant type and herbivory under elevated CO 2, F 1,35 = 29.36, P < 0.001). Elevated CO 2 increased total emission from intact plants (mixed model including intact plants of both plant types, F 1,35 = 4.43, P = 0.042). For individual terpenoids, elevated CO 2 increased emissions of α-thujene, sabinene, limonene, 1,8-cineole and γ-terpinene from intact non-bt and Bt plants (mixed model including intact plants under both CO 2 concentrations, P < 0.05, results not shown). Under control CO 2, DBM herbivory led to increased emissions of α-thujene, sabinene, β-myrcene, limonene and β-elemene in both plant types, and also of DMNT in non-bt plants (mixed model including plants grown under control CO 2, P < 0.05, results not shown). In the elevated CO 2 treatment, emissions of all the aforementioned compounds were increased by DBM herbivory. In addition, α-pinene and δ-3-carene emissions were higher after herbivory in both plant types and also DMNT and (E,E)- α-farnesene emissions in the non-bt plants (mixed model including plants grown under elevated CO 2, P < 0.05, results not shown). The statistical model including both CO 2 and herbivory treatments showed significant interactions between elevated CO 2 and herbivory as an indication of significantly higher increases in α-thujene, α-pinene, β-myrcene, limonene and β-elemene emissions under elevated CO 2 in both plant types (Table 3a). The statistically significant interactions between plant type and herbivory indicated higher induction of DMNT in particular, and also of (E,E)-α-farnesene and α-thujene, from the non-bt plants than from the resistant Bt-transgenic plants (Table 3a). No plant type CO 2 interactions were observed (Table 3a), which indicates that the response to elevated CO 2 was similar in the two plant types. In the O 3 experiment, the preplanned comparison between intact non-bt and Bt plants revealed a higher emission of δ-3-carene from the Bt plants (F 1,12 = 2.75, P = 0.011). In a comparison of plant type and O 3 in intact plants, δ-3-carene emission was found to be reduced by O 3 (ANOVA, main effect of O 3, F 1,12 = 12.98, P = 0.004), and was higher in intact elevated O 3 -grown Bt than non-bt plants (ANOVA, main effect of plant type, F 1,12 = 17.47, P = 0.002). When all the studied factors were included in the statistical model, there The Authors (2008). Journal compilation New Phytologist (2008)

66 8 Research Fig. 1 Orientation of Cotesia vestalis parasitoids in the Y-tube olfactometer to intact versus Plutella xylostella (diamondback moth (DBM))-damaged nontransgenic (Non-Bt) and Bt-transgenic (Bt) oilseed rape (Brassica napus ssp. oleifera) plants. Plants were grown and the behavioural tests conducted under the following CO 2 and O 3 concentrations: (a) CO 2 experiment: control CO 2, 360 μl l 1 CO 2 ; elevated CO 2, 720 μl l 1 CO 2 ; (b) O 3 experiment: control, filtered air, 0nll 1 ; elevated O 3, 100 nl l 1. n represents the total number of parasitoids making a choice, excluding no choices. P-values < 0.05 indicate significant difference from the 0.5 distribution (nonparametric binomial test, exact). was a significant interaction of plant type O 3 DBM feeding (ANOVA, F 1,24 = 5.27, P = 0.031) for δ-3-carene emissions (Table 3b). When individual treatments were compared, this seemed to correspond to the decrease in δ-3-carene emission observed in Bt plants after DBM feeding compared with intact Bt plants in elevated O 3 (Table 2b). The emission of the terpenoid sabinene increased after DBM feeding (ANOVA including all factors, main effect of DBM feeding, F 1,24 = 21.74, P < 0.001), and was reduced in elevated O 3 (main effect of O 3, F 1,24 = 5.39, P = 0.030) (Tables 2b, 3b). Total terpenoid emissions were also reduced by elevated O 3 (main effect of O 3, F 1,24 = 7.14, P = 0.014). Post hoc comparisons including both plant types under control conditions revealed a significant increase of sabinene emitted after herbivory in both plant types. Under elevated O 3, the intact and DBM-damage treatment groups did not differ statistically significantly (Table 2). DBM damage induced DMNT emission from individual nontransgenic plants, whereas this compound was not emitted in a quantifiable amount from Bt plants. Also, overall the emissions were low compared with DMNT emissions in the CO 2 experiment and lacked statistical significance (nonparametric Mann Whitney U test) in this experiment. This can be partly attributed to the larger air space used in the collection system with O 3, which also led to emissions of β-elemene and γ-terpinene falling below the quantification limit. In other respects, non-bt and Bt plants or plants grown under no O 3 and under elevated O 3 did not exhibit any significant differences in terpenoid emissions. Under both control CO 2 and elevated CO 2 conditions (Expt 1), host-damaged plants attracted naïve C. vestalis in both plant types (Fig. 1a). The percentage of parasitoids orientating to DBM-damaged plants was 72.4 and 72.3% for non-bt plants, and 76.5 and 71.4% for Bt-transgenic plants under control and elevated CO 2, respectively. Similarly, in the O 3 experiment, experienced C. vestalis preferred the odour of host-damaged Bt plants (P = 0.004) and non-bt plants (P < 0.001) over that of the corresponding intact plants in the absence of O 3 (Fig. 1b) (78.0% of parasitoids orientated to non-bt and 80.0% to Bt-transgenic damaged plants). Cotesia vestalis preferred host-damaged non-bt plants to intact non- Bt plants under elevated O 3 (75.0% orientated to damaged plants), whereas there was no difference in the choices of the parasitoids between intact and host-damaged Bt plants in elevated O 3 (P = 0.143; 36.8% of parasitoids orientated to damaged plants versus 63.2% to undamaged plants) (Fig. 1b). Discussion The qualitative VOC profiles of intact Bt and non-bt plants, and the physiological responses and individual terpenoid responses of the plant types to elevated CO 2 or elevated O 3 were similar, which indicated that Bt production does not interfere with allocation of resources to the major pathways leading to VOC emissions in control conditions, or under these abiotic changes, in the vegetative stage in oilseed rape. Here, the Bt-transgenic plants showed slight developmental delays in which phenology and vegetative biomass lagged behind those of the nontransgenic plants, a finding that was similar to the results of our earlier studies on these lines (Himanen et al., 2008a,b). Higher δ-3-carene emissions in the O 3 experiment in Bt versus non-bt plants might be explained by the earlier developmental stage of the Bt plants, which could lead to higher emission capacity. This observation reveals the need for a further long-term, preferably seasonal comparison of the VOC profiles of these plant types. Yan et al. (2004) found quantitative differences between Bt and non-bt cotton plants, but they also did not perform a seasonal assessment. Previously, oviposition by DBM females was found to be similar on nontransgenic versus Bt-transgenic canola (B. napus; Ramachandran et al., 1998), cabbage (B. oleracea ssp. capitata; Kumar, 2004), and broccoli (Tang et al., 1999), The Authors (2008). Journal compilation New Phytologist (2008)

67 Research 9 which suggested that intact non-bt and Bt plants yield equivalent olfactory stimuli to DBM. In these studies there were, unfortunately, no analyses of VOCs. Other factors, such as adequate contact stimuli, can also have major effects on DBM oviposition (Talekar & Shelton, 1993; Spencer et al., 1999), and therefore equality of oviposition does not directly indicate similarity in VOC emissions. Recently, Bt-transgenic varieties have been tested as potential trap crops for pests such as DBM (Shelton et al., 2008), and in this respect equal volatile emissions from intact plants are also important to ensure efficient attraction of pest females. Significant quantitative differences in herbivore-inducible emissions have been found in comparisons between Bttransgenic plants and their parent lines in maize (Turlings et al., 2005; Dean & De Moraes, 2006) and oilseed rape (Ibrahim et al., 2008), in which there were differences in herbivore feeding type or quantity. When Bt and non-bt maize plants suffered identical amounts of herbivore damage, or mechanical damage and applied larval regurgitant, there were similar VOC emissions between the plant types (Dean & De Moraes, 2006), which indicated a significant role of feeding damage in induction rather than proposed allocation differences. Turlings et al. (2005) reported that seven compounds, including the two homoterpenes DMNT and (3E,7E)-4,8,12-trimethyl- 1,3,7,11-tridecatetraene (TMTT), were emitted in lower amounts from damaged Bt maize plants compared with identically treated non-bt maize plants. Comparing herbivoreinduced emissions on non-bt and Bt oilseed rape, Ibrahim et al. (2008) found that DMNT, a compound whose emission is expected to be strongly induced by herbivory in Brassica (Vuorinen et al., 2004a,b), and (E,E)-α-farnesene were released in smaller amounts from Bt oilseed rape. Emissions of the same two compounds were also induced less strongly in Bt than non-bt plants in the current study under elevated CO 2, and in the case of DMNT also under control CO 2. The system used for O 3 exposure clearly led to lower emissions available for detection, and presumably therefore this effect was not significant in that experiment. Bt toxin produced a large reduction in herbivory in Bt oilseed rape plants, but some damage did occur, resulting in numerous small holes in Bt plants, which led to induced emissions of most of the terpenoids. Based on the current and previous (Turlings et al., 2005; Ibrahim et al., 2008) results, DMNT emission in particular requires continuous successful feeding. Considering the capabilities of Bt plants to maintain efficient indirect defences, it is highly desirable that the transgenic trait allows plants to induce terpenoid emissions, that is, minor plant damage is advantageous. The ability of Bt to protect plants directly and also allow indirect defence seems to represent the best of both worlds. Doubled atmospheric CO 2 significantly increased terpenoid emissions from intact oilseed rape plants compared with those released from the corresponding plants grown in control CO 2, when measured per dry weight or on the basis of leaf area (data not shown). As elevated CO 2 typically has both physiological effects (i.e. it increases the thickness of leaves (also shown as reduced SLA in this study), decreases stomatal density and alters stomatal function (Niinemets et al., 2004; Vuorinen et al., 2004a)) and biochemical effects (such as competition for pyruvate which is needed for higher rates of photosynthesis (Rosenstiel et al., 2003)) on VOC emissions, based on previous knowledge the emission of VOCs could have been expected to decline with CO 2 elevation (Loreto et al., 2001; Rosenstiel et al., 2003). However, it is important to note that here we used only early vegetative-stage plants to detect compounds meaningful for tritrophy. The plants grown under elevated CO 2 had higher total biomass and leaf numbers (results not shown) compared with control CO 2 - grown plants, which could lead to differences in emissions based on plant and leaf developmental stage (i.e. phenological differences). Although the intrinsic release of VOCs through increased leaf thickness and stomatal function would have actually been decreased by CO 2 elevation (Niinemets et al., 2004), the higher number of photosynthetically active leaves could result in higher emissions, as we detected under elevated CO 2. It is known that leaf age influences VOC emissions, and younger, actively photosynthesizing leaves typically have the highest VOC synthesis levels and induction capabilities (Takabayashi et al., 1994; Gouinguené & Turlings, 2002). Therefore, assessing the effects of phenological changes over a longer timescale seems reasonable in order to reveal the seasonality in VOC emission responses, as these were clearly influenced by elevated CO 2 in young oilseed rape plants. Herbivore-inducible VOC emissions were even higher from plants grown under elevated CO 2 than from those grown under control CO 2, indicating that elevated CO 2 conditions could modify herbivore-induced defences at the vegetative stage, and enhance indirect defence in the future. The degree of feeding damage by DBM larvae did not differ statistically significantly between the CO 2 treatments, so the higher induction of VOCs by herbivory in elevated CO 2 is not attributable to increased (i.e. compensatory) feeding (Docherty et al., 1996), but is a plant-mediated effect. Allocation of resources for defence under O 3 exposure is less clear in theory compared with the effects induced by elevated CO 2, as commonly explained through changes in carbon and nitrogen dynamics (Herms & Mattson, 1992). O 3 activates defence signalling and the synthesis of defence-related proteins, and at the same time it damages existing tissues and O 3 -sensitive proteins (Fiscus et al., 2005). Acute O 3 exposure has previously been found to increase emissions of methyl salicylate, sesquiterpenes and GLVs in a number of plant species (Heiden et al., 1999; Wildt et al., 2003; Beauchamp et al., 2005), as well as the emission of homoterpenes from lima bean (Phaseolus lunatus; Vuorinen et al., 2004c). In the current study, no O 3 damage-induced increases in VOC emissions were observed under chronic 100 ppb O 3, corresponding to concentrations found typically in only severely The Authors (2008). Journal compilation New Phytologist (2008)

68 10 Research polluted areas (IPCC, 2007). However, as important for ecological interactions, most former studies have not taken into account the fact that O 3 and VOCs can react in the atmosphere to form secondary aerosols, which leads to a significant loss of biogenic VOCs (McFrederick et al., 2008), and decreases cues for multitrophic signalling in elevated O 3 environments (Pinto et al., 2007a,b,c). Our set-up does not distinguish between plant-mediated effects of O 3 on VOC emission profile and gas phase reactions of VOCs with O 3. Some VOC emissions can already be induced or repressed by chronic O 3 during the growth of plants, whereas their reactivity in the atmosphere is compound-specific, which complicates summarizing the overall change in VOC emissions caused by O 3. If plant-mediated changes predominate, more dramatic and plant species-variable changes in VOC signalling could be predicted, which would be more unpredictable in natural conditions. However, if gas-phase reactions with O 3 lead to a lower VOC abundance, these changes could be more easily predicted, as they should mainly follow chemical reaction pathways (Atkinson & Arey, 2003). Parasitoids could more readily adapt to plant-mediated changes, as these would not be rapid, in contrast to O 3 reactivity, which would lead to sudden changes and might therefore severely confuse orientation of some species. There is limited knowledge of VOC responses of crop species to O 3 (Pinto et al., 2007b), but our results are in accordance with those of recent field studies on birch (Betula pendula) and aspen (Populus tremula tremuloides) trees (Vuorinen et al., 2005; Blande et al., 2007) in which it was reported that times ambient chronic O 3 elevation leads to only very small changes in terpenoid emissions. Pinto et al. (2007a,b,c) have demonstrated in laboratory conditions that most, but not all, plant-emitted terpenoids are degraded by acute O 3 targeted to ambient ozone-grown plants. The increase of 1,8- cineole emission as a percentage of total emission with O 3 elevation in our current study confirmed the previously reported tolerance of this compound to O 3, as a result of its chemical structure, in comparison to most other terpenoids vulnerable to O 3 degradation (Pinto et al., 2007a). Such O 3 -tolerant compounds might play a major role in the third trophic level, by allowing some cues to remain unaffected. Bt toxin production did not confound the tritrophic signalling system in oilseed rape, P. xylostella and C. vestalis under control or elevated CO 2. In spite of the reduced DMNT and (E,E)-α-farnesene emissions, the induction of other terpenoids seemed to be sufficient as an olfactory cue for C. vestalis compared with intact plants. Previously, parasitoid attraction by plant-emitted VOCs has been shown to be unaffected by Bt production in maize; that is, damaged nontransgenic and transgenic plants were comparably attractive to C. marginiventris and M. rufiventris parasitoids (Turlings et al., 2005). However, Schuler et al. (1999a) reported greater attraction of C. plutellae to non-bt oilseed rape compared with Bt oilseed rape with host feeding. Again, this was related to feeding damage, as feeding of Bt-resistant larvae returned the attractiveness of Bt oilseed rape to the level of nontransgenic plants (Schuler et al., 1999a). Electrophysiological studies have shown that Cotesia species respond to over 20 different VOCs (Smid et al., 2002; Gouinguené et al., 2005). The emissions measured in the two separate experiments and in the CO 2 and O 3 treatments also showed quantitative variation in the VOC emissions with changes in the ratios of the compounds (influenced by, for example, phenological effects), yet this specialist parasitoid was able to recognize host-damaged non-bt plants in all tested combinations, revealing a high olfactory capacity. Interestingly, in all tested comparisons, C. vestalis was more attracted to the treatment releasing higher total emissions, which could also indicate quantity-based preference for plant-based VOCs. Previous experience with host-damaged plants does not seem compulsory for C. vestalis, as their orientation in control conditions was equally successful in the CO 2 experiment (unexperienced wasps) and in the O 3 experiment (experienced wasps). Among individual terpenoids, the monoterpene sabinene may be the most important volatile cue for C. vestalis in the current context, as the compound clearly responded to herbivore feeding in both experiments and appeared in lower concentration under elevated O 3. The tritrophic interaction system studied here in oilseed rape, similarly to that in B. oleracea (Pinto et al., 2007b,c, 2008), did not seem to be in jeopardy even in elevated tropospheric O 3 episodes. Therefore, even if emissions of major terpenoids are reduced by O 3, C. vestalis can rely on the unaffected compounds such as 1,8-cineole or other compounds that we were not able to detect here, such as glucosinolate breakdown products (Pinto et al., 2007c). However, O 3 has the potential to affect tritrophic signalling, especially if feeding damage is low, which obviously decreases inducible VOC emissions. Bt plants damaged by Bt-sensitive insect hosts did not attract C. vestalis under elevated O 3 here. It could be speculated that this could perhaps result in decreased parasitoid abundance around Bt plant fields (Schuler et al., 2003), especially in O 3 -polluted areas, which might have agro-ecological consequences. This emphasizes the need for non-bt refugee areas not only to limit the evolution of resistance (Bates et al., 2005; Tabashnik et al., 2008) but also to maintain natural enemy populations. In addition, C. vestalis can parasitize, but not develop inside, Bt-susceptible hosts on Bt-transgenic plants, because of the detrimental effects of Bt toxin on the host larva (Schuler et al., 2003, 2004). Therefore, sustainable parasitoid populations in the long term might be negatively affected if they are attracted to Bt plants, but have no suitable host for reproduction. In addition, parasitoids might learn to connect VOC cues from Bt plants to a negative response (if the majority of host larvae present are already dead as a result of Bt toxin), and lead to reduced attractiveness of Bt plants in field conditions. However, these negative effects of reduced host abundance will be equal for chemical control and use of transgenic Bt plants. Importantly, The Authors (2008). Journal compilation New Phytologist (2008)

69 Research 11 the real ecological effects of disturbance of this individual tritrophic interaction in agroecosystems containing Bt crops, compared with chemical insecticides or Bt biopesticide applications, might be minimal. Chemical pest control methods are not 100% effective and allow VOC induction, but they do often harm nontarget insects, including parasitoids, directly (Romeis et al., 2006). Finally, even if the orientation of C. vestalis to its host using plant-emitted VOCs is not directly altered by elevated CO 2 or O 3, host-mediated effects through altered development times and biomass on parasitoid populations can also be significant (Percy et al., 2002; Holton et al., 2003). The complexity of the tritrophic systems present in natural ecosystems calls for studies to examine parasitism rate and abundance together with plant phenology and herbivore dynamics under elevated CO 2 and O 3, singly and in combination. This could help to determine the possible connections of parasitoid abundance with concurrent changes in VOC emissions in agroecosystems with traditional or transgenic plants: a challenging but important task for future research. Acknowledgements We thank P. Tiiva, J. Heinonen, J. Julkunen and V. Tiihonen for assistance in the VOC collections and behavioural assays, and T. Oksanen for programming and maintaining the growth chambers and setting up the O 3 exposures. This work was supported by the Academy of Finland (ESGEMO Programme, decision no ) (SJH, A-MN, AN and JKH), the Finnish Cultural Foundation (SJH), ISONET (MRTN-CT ) (DMP and JKH) and the Finnish Graduate School of Environmental Science and Technology (DMP) and it was enabled by USDA BRAG grants to CNS. References Atkinson R, Arey J Gas-phase tropospheric chemistry of biogenic volatile organic compounds. A review. Atmospheric Environment 37: S197 S219. 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Planta 207: Turlings TCJ, Tumlinson JH, Lewis WJ Exploitation of herbivoreinduced plant odors by host-seeking parasitic wasps. Science 250: Vuorinen T, Nerg A-M, Holopainen JK. 2004a. Ozone exposure triggers the emission of herbivore-induced plant volatiles, but does not disturb tritrophic signalling. Environmental Pollution 131: Vuorinen T, Nerg A-M, Ibrahim MA, Reddy GVP, Holopainen JK. 2004b. Emission of Plutella xylostella-induced compounds from cabbages grown at elevated CO 2 and orientation behavior of the natural enemies. Plant Physiology 135: Vuorinen T, Nerg A-M, Vapaavuori E, Holopainen JK Emission of volatile organic compounds from two silver birch (Betula pendula Roth) clones grown under ambient and elevated CO 2 and different O 3 concentrations. Atmospheric Environment 39: The Authors (2008). Journal compilation New Phytologist (2008)

71 Research 13 Vuorinen T, Reddy GVP, Nerg A-M, Holopainen JK. 2004c. Monoterpene and herbivore-induced emissions from cabbage plants grown at elevated atmospheric CO 2 concentration. Atmospheric Environment 38: Wildt J, Kobel K, Schuh-Thomas G, Heiden AC Emissions of oxygenated volatile organic compounds from plants Part II: emissions of saturated aldehydes. Journal of Atmospheric Chemistry 45: Yan F, Bengtsson M, Anderson P, Ansebo L, Xu C, Witzgall P Antennal response of cotton bollworm (Helicoverpa armigera) to volatiles in transgenic Bt cotton. Journal of Applied Entomology 128: The Authors (2008). Journal compilation New Phytologist (2008)

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73 CHAPTER 4 INTERACTIONS OF ELEVATED CARBON DIOXIDE AND TEMPERATURE WITH APHID FEEDING ON TRANSGENIC OILSEED RAPE: ARE BACILLUS THURINGIENSIS (BT) PLANTS MORE SUSCEPTIBLE TO NONTARGET HERBIVORES IN FUTURE CLIMATE? Himanen SJ, Nissinen A, Dong W-X, Nerg A-M, Stewart CN Jr, Poppy GM, Holopainen JK (2008) Global Change Biology 14: Copyright (2008) Wiley-Blackwell Publishing Ltd. Reprinted with kind permission.

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75 Global Change Biology (2008) 14, 1 18, doi: /j x Interactions of elevated carbon dioxide and temperature with aphid feeding on transgenic oilseed rape: Are Bacillus thuringiensis (Bt) plants more susceptible to nontarget herbivores in future climate? SARI J. HIMANEN*, ANNE NISSINEN*w, WEN-XIA DONGz, ANNE-MARJA NERG*, C. NEAL STEWART JR., GUY M. POPPY} andjarmo K. HOLOPAINEN* *Department of Environmental Science, University of Kuopio, PO Box 1627, FIN Kuopio, Finland, wagrifood Research Finland, Plant Protection, FIN Jokioinen, Finland, ztea Research Institute, Chinese Academy of Agricultural Sciences, No. 1 Yungi Road, Hangzhon 31008, China, Department of Plant Sciences, University of Tennessee, 2431 Joe Johnson Drive, Knoxville, TN 37996, USA, }School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK Abstract Climate change factors such as elevated carbon dioxide (CO 2 ) and temperature typically affect carbon (C) and nitrogen (N) dynamics of crop plants and the performance of insect herbivores. Insect-resistant transgenic plants invest some nutrients to the production of specific toxic proteins [i.e. endotoxins from Bacillus thuringiensis (Bt)], which could alter the C N balance of these plants, especially under changed abiotic conditions. Aphids are nonsusceptible to Lepidoptera-targeted Bt Cry1Ac toxin and they typically show response to abiotic conditions, and here we sought to discover whether they might perform differently on compositionally changed Bt oilseed rape. Bt oilseed rape had increased N content in the leaves coupled with reduced total C compared with its nontransgenic counterpart, but in general the C : N responses of both plant types to elevated CO 2 and temperature were similar. Elevated CO 2 decreased N content and increased C : N ratio of both plant types. Elevated temperature increased C and N contents, total chlorophyll and carotenoid concentrations under ambient CO 2, but decreased these under elevated CO 2. In addition, soluble sugars were increased and starch decreased by elevated temperature under ambient but not under elevated CO 2, whereas photosynthesis was decreased in plants grown under elevated temperature in both CO 2 levels. Myzus persicae, a generalist aphid species, responded directly to elevated temperature with reduced developmental time and decreased adult and progeny weights, whereas the development of the Brassica specialist Brevicoryne brassicae was less affected. Feeding by M. persicae resulted in an increase in the N content of oilseed rape leaves under ambient CO 2, indicating the potential of herbivore feeding itself to cause allocation changes. The aphids performed equally well on both plant types despite the differences between C N ratios of Bt and non-bt oilseed rape, revealing the absence of plant composition-related effects on these pests under elevated CO 2, elevated temperature or combined elevated CO 2 and temperature conditions. Keywords: aphids, Bacillus thuringiensis, Brassica napus, Brevicoryne brassicae, climate change, elevated CO 2, elevated temperature, Myzus persicae, transgenic plants Received 7 August 2007; revised version received 12 November 2007 and accepted 12 December 2007 Introduction Transgenic insect-resistant crop plants that have been genetically modified to produce Bacillus thuringiensis Correspondence: Sari Himanen, tel , fax , [email protected] (Bt) crystalline (Cry) endotoxins with specific activity towards certain herbivores are currently grown in over 30 million hectares (James, 2006). Bt plants have been successful in limiting damage by many important insect pests, such as the cotton bollworm (Helicoverpa zea), European corn borer (Ostrinia nubilalis) and tobacco budworm (Heliothis virescens). One of their benefits is r 2008 The Authors Journal compilation r 2008 Blackwell Publishing Ltd 1

76 2 S. J. HIMANEN et al. the reduced need for insecticide applications for pest control (Romeis et al., 2006). However, possible unintended pleiotropic effects of genetic modification and the potential for the evolution of resistance in target pests are of regulatory concern (Poppy & Wilkinson, 2005). In the future, global agriculture will, inevitably, face challenges caused by climate change, which might lead to both global and local alterations in agriculture (IPCC, 2007). Elevated CO 2, temperature and drought can have various effects on different trophic levels in ecosystems (plants, herbivores, predators and parasitoids) (IPCC, 2007). Under these circumstances, transgenic plants may become even more important to sustainable agriculture. However, the performance of transgenic plants, the stability of the transgenic traits and their ecological interactions have been rarely studied under atmospheres with elevated CO 2 and temperature or both factors in combination (Coviella et al., 2000, 2002; Chen et al., 2005). Carbon dioxide (CO 2 ), as the primary determinant of the photosynthetic rate in plants, affects both the physiology and the composition [carbon (C) : nitrogen (N) ratio, allocation of resources] of crop plants (Poorter et al., 1997; Long et al., 2004). Elevated temperature interacting with elevated CO 2 concentration can influence C N dynamics of plants by decreasing vegetative developmental periods and enhancing the efficiency of photosynthesis (Morison & Lawlor, 1999). Bt plants, which are modified to produce relatively high levels (up to 1%) of a specific protein constitutively throughout their life cycle and in all parts of the plant, might have altered C and N ratios leading to interactions between the use of N for growth and structural proteins vs. production of Bt toxin (Coviella et al., 2000, 2002). Moreover, if C-enriched atmospheres lead to lower N availability and competition for N in the plant (Bryant et al., 1983), then reduced allocation could lead to decreased concentrations of active Bt toxin in the plants (Coviella et al., 2000, 2002). This would be undesirable with regards to delivering a high dose of Bt toxin for efficacy and minimizing risks for resistance management in target herbivores (Bates et al., 2005). Reduced Bt toxin concentration under elevated CO 2 has been reported in Bt cotton (Coviella et al., 2000, 2002; Chen et al., 2005); however, the biological significance of this change has yet to be determined. Cruciferous plants (Brassica genus) are agriculturally important, as they include numerous crops and vegetables such as cabbage, broccoli and oilseeds. In addition, all these produce C- and N-containing secondary compounds, glucosinolates, as part of their secondary metabolism (reviewed by Halkier & Gershenzon, 2006). Thereby, it can be hypothesized that their N demand is even more pronounced, and they are excellent model species for studying the effects of enhanced CO 2 on C N metabolism. Bt oilseed rape (Brassica napus) was produced by Halfhill et al. (2001) principally for the purpose of studying ecological risks of transgene dispersal, as Brassica plants have many wild weedy relatives compatible for hybridization (Halfhill et al., 2005). In this study, we assessed whether there are differences in the physiology and C N dynamics between Bt-transgenic and nontransgenic oilseed rape, when both are grown under elevated CO 2, elevated temperature or under both factors in combination. It can be hypothesized that as the synthesis of the Bt toxin protein requires additional N inputs, this could result in competition for the nutrient, because enhanced growth at C excess (in elevated CO 2 ) also increases N demand (Bryant et al., 1983). Brassicas are hosts for many herbivorous aphid species and the generalist aphid Myzus persicae is one of the most serious pests of oilseed rape (Desneux et al., 2006). For autumn-sown winter oilseed rape, the pest population can cause significant damage (Buntin & Raymer, 1994). The specialist Brevicoryne brassicae is the other dominant aphid species attacking Brassica plants (Desneux et al., 2006). Aphids are among the sap-feeding insects that have responded, either positively or negatively, to elevated CO 2 -induced changes in plants (Bezemer et al., 1998, 1999; Hughes & Bazzaz, 2001; Holopainen, 2002; Newman et al., 2003; Flynn et al., 2006). Aphids are not directly affected by Bt Cry1Ac toxin as they lack the specific gut receptors, and Bt toxin is also largely absent in phloem sap (Raps et al., 2001). Temperature can affect the development and population biology of aphids directly by increasing their metabolism and leading to increased abundance by decreased developmental times (Bale et al., 2002). Interactions between elevated CO 2 and temperature affecting herbivory have been reported (Bezemer et al., 1998; Veteli et al., 2002; Newman, 2003; Hoover & Newman, 2004; Flynn et al., 2006), although there is variability among different plant species in their responses to these factors singly or in combination (Zvereva & Kozlov, 2006). It could be predicted that aphids will have an even greater impact as pest herbivores on agricultural plants in future atmospheres, which is why we chose aphids for screening the performance of potentially important non-bt-susceptible pests on Bt-transgenic vs. nontransgenic oilseed rape. Our goal was to assess whether future elevated CO 2 and temperature could lead to better adaptation of these pests and to discover the role of Bt toxin production in this. Would these render Bt plants even more susceptible to nontarget pests not directly affected by Bt toxin but that would respond to changes in the plant composition brought by r 2008 The Authors Journal compilation r 2008 Blackwell Publishing Ltd, Global Change Biology, doi: /j x

77 BT OILSEED RAPE, ELEVATED CO 2 AND TEMPERATURE 3 Bt production? If so, higher pesticide inputs might be necessary for nontarget pest control. In this study, we addressed whether elevated CO 2, temperature or both factors in combination: (1) change the photosynthetic rate, C and N contents or amounts of C-based (starch and soluble sugars) compounds of Bt-transgenic vs. nontransgenic oilseed rape; (2) change the production of Bt Cry1Ac toxin in Bt oilseed rape; (3) affect the onset of reproduction, number and weight of progeny produced and mean relative growth rate (MRGR) of the non-bt-susceptible generalist (M. persicae) and specialist (B. brassicae) aphid species in Bt-transgenic vs. nontransgenic oilseed rape; and finally (4) does aphid feeding itself alter the C N dynamics of Bt-transgenic and nontransgenic oilseed rape? Materials and methods Plant material and growth conditions B. napus ssp. oleifera (oilseed rape) cultivar Westar has been previously transformed to contain a synthetic Bt cry1ac gene and green fluorescent protein (GFP) mgfp5er marker gene regulated by independent cauliflower mosaic virus 35S promoters (Halfhill et al., 2001). Line GT1 used here showed stable expression of both genes in single insert with no obvious phenotypic effects. Nontransgenic cv. Westar parent line served as a control to the transgenic line. F 4 seeds of Bt-transgenic and nontransgenic plants were sown in 0.66 L pots in 2 : 1 : 1 fertilized soil (Kekkilä, Finland, NPK: 100, 30, 200 mg L 1 ) : B2 sphagnum peat (Kekkilä, Finland, NPK: 110, 40, 220 mg L 1 ) : sand ( mm) mixture. Plants were grown in four computer-controlled growth chambers (2.6 m 3 ; Bioklim 2600 T, Kryo-Service Oy, Helsinki, Finland). The limited number of growth chambers available simultaneously (one chamber per treatment) did not allow us to use chambers as replicates to ideally exclude pseudoreplication (Hurlbert, 1984). Therefore, we conducted two consecutive experiments with similar treatments in order to limit the problem of pseudoreplication (measuring responses of the same variables and using replication in time as a random factor in the statistical analysis). Owing to limited space in the chambers and available resources, all variables could not be included in both experiments [i.e. different aphid species were studied in different experiments (M. persicae in Experiment 1 and B. brassicae in Experiment 2)]. In all analyses, individual plants served as replicates. CO 2 and temperature treatments used were following: (1) ambient CO 2, control temperature (20/16 1C); (2) elevated CO 2 (720 ppm CO 2 ), control temperature (20/ 16 1C); (3) ambient CO 2, elevated temperature (24/ 20 1C, 1 4 1C increase to control temperature); and (4) elevated CO 2 (720 ppm), elevated temperature (24/ 20 1C). Ambient CO 2 was the corresponding background level of air entering the growth chamber facilities and elevated CO 2 was supplied from gas tanks. CO 2 and temperature conditions of the chambers were continuously monitored, and the mean CO 2 and temperature ( SD) values of the treatments, calculated from hourly means, were the following: (1) Experiment 1: ppm CO 2, C and Experiment 2: ppm CO 2, C; (2) Experiment 1: ppm CO 2, C and Experiment 2: ppm CO 2, C; (3) Experiment 1: ppm CO 2, C and Experiment 2: ppm CO 2, C; and (4) Experiment 1: ppm CO 2, C and Experiment 2: ppm CO 2, C. All treatments had a 16 : 8 h photoperiod and light adjusted to photosynthetically active radiation (PAR) of approximately 250 mmol m 2 s 2. The light level used was optimal for the growth of the plants in these type of controlled conditions, but it should be noted that the light intensity is not as high as in field conditions. To avoid any effects of chamber-specific growth conditions, the plants inside the chambers and the treatments among the four chambers used were rotated weekly. In both experiments, the net photosynthesis, total C and N content and C : N ratio of Bt-transgenic and nontransgenic oilseed rape plants was compared. We also assessed the effects of aphid feeding on the C N dynamics of these plants. In addition, in Experiment 1, the Bt toxin concentration, chlorophyll pigment concentrations and the reproduction parameters and MRGR of M. persicae were measured, whereas in Experiment 2, the concentrations of starch and soluble sugars were determined and similar population growth measurements were conducted with B. brassicae. Experiment 1 was started in August and it lasted for a total of 30 days and Experiment 2 (in October) was continued for an additional 3 days (33 days in total) due to the later start of offspring production by B. brassicae compared with M. persicae. This enabled evaluation of the progeny production of aphids during their exponential population growth close to peak reproduction but not after that, as the experiment had to be limited to the vegetative stage of the plants due to prevailing GM plant biosafety regulations. The details of the two experiments are described below. Experiment 1: M. persicae Total leaf biomass, total C and total N. Total leaf biomass from six Bt-transgenic and six nontransgenic plants per CO 2 /temperature treatment and six corresponding r 2008 The Authors Journal compilation r 2008 Blackwell Publishing Ltd, Global Change Biology, doi: /j x

78 4 S. J. HIMANEN et al. plants subjected to M. persicae feeding as described below (two aphids and their following progeny on the plant) from 13th day from sowing until the end of the experiment was separately collected at 30 days age and oven dried (60 1C, 72 h). Biomass gain was determined from the intact plants. Total C and N content of the dried samples was measured with a CN analyser (Leco CN-2000, St Joseph, MI, USA) and the C : N ratio was calculated. Total soluble protein and Bt toxin concentration. The third true leaf from six Bt-transgenic plants (30 days of age) per treatment was separately collected, deep-frozen in liquid N and stored at 80 1C before analysis. Total soluble protein was determined with the Bradford method (Bradford, 1976), and Bt toxin concentration was analysed with an ELISA test (Cry1Ac PathoScreen Kit, Agdia, Elkhart, IN, USA) according to the manufacturer protocol and with Cry1Ac standards for quantitative determination. Net photosynthetic rate. Net photosynthesis was measured from the third true leaf of six day-old Bt-transgenic and nontransgenic plants from each CO 2 /temperature treatment with a CI-510 Portable Photosynthesis System (Cid Inc., Vancouver, WA, USA) under saturating light intensity of 1800 PAR. CO 2 level in the cuvette during measurements was adjusted to either or ppm (supplied from gas tanks). Chlorophyll pigment concentrations. Chlorophyll (chl) a, b and total carotenoid concentrations were analysed from the total leaf biomass collected separately from six 30-day-old Bt-transgenic and nontransgenic plants from each CO 2 /temperature treatment (same plants as used for measuring net photosynthesis) deep-frozen prior analysis. Chlorophyll was extracted from 0.1 g ground leaf material with 7 ml DMSO by incubating at 65 1C for 1 h and diluting to a 25 ml total volume. The absorbance was read spectrophotometrically (UV-1201 UV VIS Spectrophotometer, Shimadzu Corporation, Koyoto, Japan) at 665, 648 and 470 nm and results were calculated with the equations proposed by Barnes et al. (1992). M. persicae reproduction and relative growth rate. M. persicae (Sulzer) (Homoptera:Aphididae) originated from the laboratory colony at the University of Kuopio and was reared on Brassica rapa cv. Valo at 12 : 12 L : D at approximately 25 1C temperature and 50% RH. Two M. persicae adults were transferred to cotyledons or the first or second true leaves of six Bttransgenic and nontransgenic plants (13 days old) per treatment to produce progeny. After 24 h, the adults were removed and two nymphs per plant were left. If the adult had not produced any progeny at all, a nymph produced by the other adult on the same plant was used, or in a few cases, a nymph produced by an adult on another plant of the same treatment was selected. This resulted in two nymphs per plant. The nymphs were checked at 1-day age and daily from days 5 to 16 to determine the start of reproduction and to calculate the number of progeny produced per aphid. These nymphs produced by the aphids were removed and weighed daily and the mean progeny weight was calculated. If the aphid-infested leaf was mechanically damaged or approached senescence, the herbivore was transferred to the next (younger) intact leaf. On day 16, when the experiment was completed, the adults were weighed and deep-frozen for Bt toxin analysis. Bt toxin was analysed as described earlier after mechanical grinding of the aphids in liquid N. The MRGR of M. persicae in the different treatments was determined by enclosing two individually weighed nymphs (produced by the aphids in the reproduction assay), o24-h old, into clip-cages on the same plants that were used for the reproduction assay described above (26-day old), but on different leaves (leaves 2 5 depending on the previous location of the aphids). The clip-cages were made of 2 ml eppendorf tubes cut at approximately 1 cm height and covered with a fine mesh from the upper side. The tubes were attached to a wire, which formed a loop on the underside of the leaf, and the loop was surrounded with plastic foam to prevent damage of the leaf while also allowing photosynthesis. After 4 days, the aphids were individually weighed again and their MRGR was calculated with the equation: (ln W 2 ln W 1 )/time, where W 1 is the initial aphid weight, W 2 is the final aphid weight and time is the duration of the experiment (days) (van Emden, 1969). Experiment 2: B. brassicae Total leaf biomass, total C and total N. Total leaf biomass from six Bt-transgenic and six nontransgenic plants per CO 2 /temperature treatment and from six corresponding B. brassicae-infested plants were collected separately at 33 days of age and oven dried (60 1C, 72 h). Total biomass gain, total C, total N content and C : N ratio was determined as in Experiment 1. Net photosynthetic rate. Net photosynthesis was measured from six 22-day-old Bt-transgenic and nontransgenic plants as in Experiment 1, but under 360 ppm CO 2 only. Soluble sugars and starch. Total leaf biomass from six 30-day-old Bt-transgenic and nontransgenic plants per treatment was separately collected, deep-frozen in r 2008 The Authors Journal compilation r 2008 Blackwell Publishing Ltd, Global Change Biology, doi: /j x

79 BT OILSEED RAPE, ELEVATED CO 2 AND TEMPERATURE 5 liquid N and stored at 80 1C before analysing. Starch, glucose, fructose and sucrose concentrations were analysed enzymatically according to kit manufacturer instructions (Glucose/D-Glucose/D-Fructose kit and Starch kit, Boehringer Mannheim/R- Biopharm, Darmstadt, Germany). B. brassicae reproduction and relative growth rate. B. brassicae (L.) (Homoptera:Aphididae) was obtained from Rothamsted Research, Hertfordshire, UK, and was maintained on Brassica pekiniensis cv. Yamiko at 12 : 12 L:D, 251C temperature and 50% RH in University of Kuopio. The aphid reproduction measurements were performed as in Experiment 1 for M. persicae, but B. brassicae aphids were enclosed into clip-cages from the start of the experiment to avoid moving of the aphids on the plant to form colonies (to enable separation of individual aphids). The MRGR measurements of B. brassicae were performed on 29-day-old plants. Statistical analysis Aphid reproduction and MRGR results were aggregated per plant in both experiments. All data were checked for normality and equality of residual error variances and appropriately transformed (log or square-root) if needed to satisfy the assumptions of analysis of variance. Mixed-model ANOVA was used for analysis of biomass gain and C N results with the plant type, CO 2 level and temperature as fixed effects and experiment as a random effect. Absolute values were transformed to fold-change values in relation to the control treatment for biomass gain and aphid-induced changes in C and N content results. A separate ANOVA model including plant type, aphid feeding and their interaction was created to study the effects of aphid induction on C N dynamics under each CO 2 /temperature treatment separately. Photosynthesis, Bt toxin and chlorophyll pigment concentrations and aphid performance parameters were tested with ANOVA for the main effects of plant type, CO 2 level and temperature and their interactions. The cumulative reproduction of aphids was analysed with repeated measures ANOVA. Owing to the experimental set-up using individual plants as replicates, the statistical power of the analysis can be higher compared with a set-up using chambers as replicates. The data were analysed with SPSS for WINDOWS 14.0 statistical package. Results Biomass gain and net photosynthesis Elevated CO 2 increased leaf biomass in nontransgenic as well as Bt-transgenic oilseed rape plants (main effect Fold change Westar Fig. 1 Biomass gain 1 SEM in Westar and Bt-transgenic (Bt) oilseed rape plants grown under ambient or elevated (720 ppm) CO 2 level and under control (CT, 20/16 1C) or elevated (ET, 24/ 20 1C) temperature indicated as fold change to nontransgenic (Westar) plants grown under ambient CO 2 level and control temperature (n 5 12). Statistically significant (Po0.05) mixedmodel ANOVA results for main effects of plant type, CO 2 level and temperature are shown. Bt, Bacillus thuringiensis. of CO 2 level, F 1, , Po0.001), whereas Bt plants had lower biomass gain during their early vegetative growth compared with the nontransgenic plants (main effect of plant type, F 1, , P ) (Fig. 1). Elevated temperature had no significant effect on biomass gain in ambient or elevated CO 2. Elevated temperature greatly reduced net photosynthesis in leaves of both plant types (Fig. 2, main effect of temperature: F 1, , Po0.001; F 1, , Po0.001; F 1, , Po0.001 in Experiment 1 under control and elevated CO 2 and Experiment 2 control CO 2 levels, respectively). The net photosynthetic rates measured under elevated CO 2 were higher than the rates under control CO 2 (main effect of measurement CO 2 level, F 1, , Po0.001). In general, photosynthesis responded to the elevation of CO 2 level and temperature similarly in nontransgenic and Bttransgenic plants (no significant main effects for plant type). Plants grown under ambient and elevated CO 2 had an equal photosynthetic rate at 22-day age, when measured at ambient CO 2 (Experiment 2) and this was also true for 30-day-old plants, when photosynthesis was measured under 720 ppm CO 2 (Experiment 1). However, when the net photosynthesis was measured under 360 ppm CO 2 at 29-day age, it was reduced for the plants that had been grown under elevated CO 2 compared with plants grown under the control CO 2 level (main effect of CO 2 level, F 1, , P ). There was also a marginally significant interaction between temperature and CO 2 level (F 1, , P ), which points to the fact that the reduction in the photosynthetic rate in plants grown under Bt r 2008 The Authors Journal compilation r 2008 Blackwell Publishing Ltd, Global Change Biology, doi: /j x

80 6 S. J. HIMANEN et al μmol CO 2 m 2 s day W 22 day Bt 29 day W 29 day Bt 30 day W 30 day Bt Measured at 360 ppm CO 2 Measured at 720 ppm CO Experiment Experiment Fig. 2 Net photosynthesis (P n ) in Bt-transgenic (Bt) and nontransgenic (W, Westar) oilseed rape plants grown under ambient or elevated (720 ppm) CO 2 level and under control (CT, 20/16 1C) or elevated (ET, 24/20 1C) temperature. Measurements were made in two separate experiments: in Experiment 1 at time points of 29 days (measured at control CO 2 ) and 30 days (measured at elevated CO 2 ), and in Experiment 2 at 22 days (n 5 6). Statistically significant (Po0.05) ANOVA results for main effects of plant type, CO 2 level and temperature are shown. Bt, Bacillus thuringiensis. enhanced CO 2 was higher under elevated temperature (plants growing faster and being at a later growth stage). Total C, total N and C : N ratio Plant N levels were slightly higher in Experiment 2 than in Experiment 1 in overall. The CO 2 and temperature treatments were applied similarly, so this difference was predicted to be caused by some internal regulation of plant growth (Sharma et al., 2005) [reproductive growth pursuing over vegetative growth at the end of summer (Experiment 1), with no similar effect later in the autumn (Experiment 2)]. Therefore, for statistical analysis of aphid-induced changes within each experiment, C and N content and C : N ratio levels of control Westar plants were scaled to 1 for both experiments and the treatment effects compared with these set levels. In the elevated temperature treatment, there was increased C content of intact plants under control CO 2 and decreased C under elevated CO 2 (interaction CO 2 temperature, Po0.001) (Table 1). The C content was lower in Bt-transgenic compared with nontransgenic plants (main effect of plant type, P ) and elevated temperature led to a marginally significantly higher C in Bt-transgenic plants under ambient CO 2 (interaction plant type temperature, P ). In the elevated CO 2 treatment, there was highly decreased N content of both plant types (main effect of CO 2 level, Po0.001). Elevated temperature increased N content under ambient CO 2 but decreased it under elevated CO 2 (interaction CO 2 temperature, P ). In addition, the N content was higher in Bt-transgenic than in the nontransgenic plants (main effect of plant type, P ) and showed a marginally significant trend for a higher decrease by elevated CO 2 in Bttransgenic than in the nontransgenic plants (interaction plant type CO 2, P ). The C : N ratio of both plant types was increased by elevated CO 2 (main effect of CO 2 level, Po0.001). Interestingly, elevated temperature decreased the C : N ratio of plants under control CO 2 level but in contrast increased it under elevated CO 2 (CO 2 temperature interaction, P ). Bt-transgenic plants had a reduced C : N ratio compared with nontransgenic plants (main effect of plant type, P ). In response to M. persicae and B. brassicae feeding, the C content of both plant types typically decreased, although this was not statistically significant in all treatments (Fig. 3, main effects of aphid feeding). As an exception to this, under elevated CO 2 and temperature, B. brassicae feeding resulted in an increase in C content (F 1, , P ). r 2008 The Authors Journal compilation r 2008 Blackwell Publishing Ltd, Global Change Biology, doi: /j x

81 BT OILSEED RAPE, ELEVATED CO 2 AND TEMPERATURE 7 Table 1 Total carbon (%), total nitrogen (%) and C : N ratio (mean 1 SEM) in intact nontransgenic (Westar) and Bt-transgenic (Bt) oilseed rape plants grown in ambient or elevated (720 ppm) CO2 level under 20/16 1C (control) or 24/20 1C (elevated) temperature (n 5 12) and mixed-model ANOVA results for effects of plant type (Bt-transgenic vs. nontransgenic), CO2 level and temperature and their interactions Ambient CO2 Elevated CO2 20/16 1C 24/20 1C 20/16 1C 24/20 1C Westar Bt Westar Bt Westar Bt Westar Bt C (%) N (%) C : N ratio Plant type CO2 Temperature Plant type CO2 Plant type temperature CO2 temperature Plant type CO2 temperature F1, 40 P F1,40 P F1,40 P F1, 40 P F1, 40 P F1,40 P F1,40 P C o N o C : N ratio o Statistically significant effects (Po0.05) are marked in bold. Bt, Bacillus thuringiensis; CO2, carbon dioxide. r 2008 The Authors Journal compilation r 2008 Blackwell Publishing Ltd, Global Change Biology, doi: /j x

82 8 S. J. HIMANEN et al. (a) Carbon (b) Nitrogen (c) C : N ratio Intact M. persicae infested Intact M. persicae infested Intact M. persicae infested Fold change Fold chavnge Fold change Fold change Intact B. brassicae infested Fold change Intact B. brassicae infested Fold change Intact B. brassicae infested Fig. 3 Carbon (C), nitrogen (N) and C : N ratio in aphid (Myzus persicae or Brevicoryne brassicae)-infested nontransgenic (Westar) and Bt-transgenic (Bt) oilseed rape plants grown under ambient or elevated (720 ppm) CO 2 level and under control (CT, 20/16 1C) or elevated (ET, 24/20 1C) temperature indicated as fold change to nontransgenic (Westar) plants grown under ambient CO 2 level and control temperature (n 5 6). Statistically significant (Po0.05) ANOVA effects of aphid feeding and interactions of plant type and aphid feeding within each CO 2 and temperature treatment are shown. Bt, Bacillus thuringiensis. r 2008 The Authors Journal compilation r 2008 Blackwell Publishing Ltd, Global Change Biology, doi: /j x

83 BT OILSEED RAPE, ELEVATED CO 2 AND TEMPERATURE 9 When plants were exposed to M. persicae feeding, there was increased N content in nontransgenic and Bt-transgenic plants under control CO 2 level but not under elevated CO 2 (Fig. 3). M. persicae feeding induced a differential response in the N content of the two plant types under elevated CO 2 and control temperature: in nontransgenic plants, the N content was slightly decreased, but in Bt plants it was rather increased (interaction plant type aphid feeding, F 1, , P ). B. brassicae feeding increased the N content of both plant types under elevated CO 2 and control temperature (F 1, , P ), whereas in other growth conditions, no statistically significant changes were observed in the N dynamics after cabbage aphid feeding. The C : N ratio was decreased in M. persicae-fed plants compared with intact plants grown under ambient CO 2, whereas B. brassicae feeding decreased the ratio only under elevated CO 2 and control temperature (Fig. 3). Corresponding to the differential response of the N contents of the two plant types under elevated CO 2 and control temperature, a similar interaction for plant type aphid feeding (F 1, , P ) was detected in C : N ratio. Chlorophyll pigments Chl a concentrations had a trend to increase in elevated CO 2 under control temperature and also to increase at elevated temperature under control CO 2 level (Table 2). In contrast, in the elevated CO 2 and temperature treatment, the concentration was reduced compared with plants from either single elevation treatments (significant CO 2 temperature interaction, P ). Chl b, total chlorophyll and total carotenoid concentrations showed a similar response (CO 2 temperature interactions: P , and 0.026, respectively). Chl a/b ratio was reduced by elevated temperature under both CO 2 levels (main effect of temperature, P ). Carotenoid concentrations were lower in Bt-transgenic plants compared with nontransgenic plants (main effect of plant type, P ), but otherwise the pigment concentrations were equal in both plant types and showed similar responses to elevated CO 2 and temperature. Starch and soluble sugars Glucose concentrations in Bt-transgenic and nontransgenic oilseed rape leaves at the end of vegetative stage were increased by elevated temperature (F 1, , P ), particularly under control CO 2 level (Fig. 4). In contrast, elevated CO 2 decreased the glucose concentration (F 1, , P ). There was a significant interaction between CO 2 level and temperature for fructose (F 1, , P ) and a similar marginally significant one for glucose (F 1, , P ). This result indicated that elevated temperature had no such increasing effect on the concentrations of the sugars under elevated CO 2 as appeared under control CO 2 conditions. Sucrose concentrations were not significantly affected by elevated CO 2 or temperature. The main effect of plant type approached statistical significance (F 1, , P ), since the sucrose levels were slightly decreased in Bt-transgenic plants compared with the nontransgenic plants in all treatments. Glucose and fructose concentrations were similar in Bt-transgenic and nontransgenic plants. Starch concentration was strongly decreased by elevated temperature treatment under control CO 2 level (F 1, , P ), whereas it was unaffected by temperature under elevated CO 2 (interaction CO 2 level temperature, F 1, , P ) (Fig. 4). Elevated CO 2 increased the starch concentration under both temperature regimes (main effect CO 2, F 1, , Po0.001). Bt-transgenic and nontransgenic plants had equivalent starch concentrations, and the starch responses of the plant types to elevated CO 2 and temperature were similar. Bt toxin concentration Bt-transgenic oilseed rape grown under control CO 2 and temperature in Experiment 1 contained mg Bt Cry1Ac g 1 leaf fresh weight, measured from the third true leaf. Elevated CO 2 or temperature did not significantly alter the Cry1Ac concentration (P40.05); Bt concentrations of leaves were mg (ambient CO 2, elevated temperature), mg (elevated CO 2, control temperature) and mg (elevated CO 2 and elevated temperature) toxin. The proportions of Cry1Ac of total soluble protein ranged from 0.016% to 0.03% (results not shown), and these were not significantly affected by the treatments either. M. persicae and B. brassicae aphids feeding on Bt oilseed rape did not contain a quantifiable concentration of Bt Cry1Ac in any of the CO 2 and temperature treatments. M. persicae and B. brassicae reproduction and relative growth rate Elevated temperature shortened the developmental time of M. persicae aphids by approximately 3 days on both plant types (Fig. 5). The developmental time of the specialist B. brassicae was days in all treatments and not affected by plant type, elevated CO 2 or temperature (Fig. 5, Tables 3 and 4). r 2008 The Authors Journal compilation r 2008 Blackwell Publishing Ltd, Global Change Biology, doi: /j x

84 10 S. J. HIMANEN et al. Table 2 Chlorophyll pigment concentrations and chl a/b ratio (mean 1 SEM) in leaves of Bt-transgenic and nontransgenic (Westar) oilseed rape plants grown in ambient or elevated (720 ppm) CO2 level under control (20/16 1C) or elevated (24/20 1C) temperature (n 5 6) and ANOVA results for effects of plant type (Bt-transgenic vs. nontransgenic), CO2 level and temperature and their interactions Ambient CO 2 Elevated CO 2 20/16 1C 24/20 1C 20/16 1C 24/20 1C Westar Bt Westar Bt Westar Bt Westar Bt Chl a (mg/100 g FW) Chl b (mg/100 g FW) Total chlorophyll (mg/100 g FW) Carotenoids (mg/100 g FW) Chl a/b ratio Plant type CO2 Temperature Plant type CO2 Plant type temperature CO2 temperature Plant type CO2 temperature F1, 40 P F1,40 P F1,40 P F1, 40 P F1,40 P F1, 40 P F1, 40 P Chl a Chl b Total chlorophyll Carotenoids Chl a/b Statistically significant effects (Po0.05) are marked in bold. Bt, Bacillus thuringiensis; FW, final weight; CO 2, carbon dioxide. r 2008 The Authors Journal compilation r 2008 Blackwell Publishing Ltd, Global Change Biology, doi: /j x

85 BT OILSEED RAPE, ELEVATED CO 2 AND TEMPERATURE mg g 1 FW mg g 1 FW Westar Bt Westar Bt Westar Bt Westar Bt ----Sucrose Glucose Fructose Starch Fig. 4 Concentration of soluble sugars (left axis) and starch (right axis) in 30-day-old Bt-transgenic (Bt) and nontransgenic (W, Westar) oilseed rape plants grown under ambient or elevated (720 ppm) CO 2 level and under control (CT, 20/16 1C) or elevated (ET, 24/20 1C) temperature (n 5 6). Statistically significant (Po0.05) ANOVA results for main effects of plant type, CO 2 level and temperature and their interactions are shown. Bt, Bacillus thuringiensis. Final weight of adult M. persicae was not affected by the plant type, but it was significantly reduced in elevated growth temperature compared with control temperature (main effect of temperature, Po0.001) (Tables 3 and 4). Elevated CO 2 also significantly lowered the M. persicae adult weight compared with aphids on plants grown in control CO 2 level (P , Table 4). Mean weight of o24-h old M. persicae nymphs was reduced on plants grown in elevated temperature (P ), but elevated CO 2 or plant type had no significant effect on progeny weight. Performance parameters of B. brassicae (developmental time, total number of progeny and final weights of adults) were not affected by plant type, elevated CO 2 or elevated temperature (Table 3, statistical results not shown). Mean progeny weights of B. brassicae were marginally significantly increased by elevated CO 2 (P ), and reduced by elevated temperature (P ). The cumulative fecundity of M. persicae was increased by elevated temperature (repeated-measures ANOVA, F 1, , Po0.001) and reduced by elevated CO 2 (repeated-measures ANOVA, F 1, , P ) (Fig. 5a,b). The total number of progeny produced by M. persicae was highest in plants grown under control CO 2 and elevated temperature, followed by elevated CO 2 combined with elevated temperature, thereafter in the control CO 2 and control temperature and finally lowest in plants grown in elevated CO 2 under control temperature. M. persicae showed an equal rate of reproduction in Bt-transgenic and nontransgenic plants. The cumulative fecundity of B. brassicae was not affected by plant type, elevated CO 2 or elevated temperature (Fig. 5c,d; statistical results not shown). The MRGR of M. persicae and B. brassicae nymphs was similar in Bttransgenic and nontransgenic plants and this was not affected by elevated CO 2 or temperature (Tables 3 and 4). Finally, Table 5 represents a summary showing the direction (increase or decrease) of the statistically significant effects of the treatments (main effects of plant type, elevated CO 2 and elevated temperature) and the presence of elevated CO 2 temperature interactions for all the measured parameters. Discussion We found significant changes in the C N dynamics of Bt-transgenic oilseed rape and its nontransgenic parent line at the end of their vegetative growth. Presumably, the extra input requirement for producing Bt toxin could have led to the observed increase in N and the reduction in C content. The Bt toxin proportion from total soluble proteins in this plant line under the growth conditions was relatively low, and still, even this low Bt toxin production resulted in changes in the C : N ratio. Bt Cry1Ac concentrations in Bt oilseed rape show r 2008 The Authors Journal compilation r 2008 Blackwell Publishing Ltd, Global Change Biology, doi: /j x

86 12 S. J. HIMANEN et al. (a) M. persicae in Westar plants 60 (b) M. persicae in Bt plants 60 Cumulative number of progeny Cumulative number of progeny Days Days (c) B. brassicae in Westar plants Cumulative number of progeny (d) B. brassicae in Bt plants Days Days Fig. 5 Cumulative reproduction 1 SEM of Myzus persicae (a and b) and Brevicoryne brassicae (c and d) on nontransgenic (Westar) and Bt-transgenic (Bt) oilseed rape plants grown under ambient or elevated (720 ppm) CO 2 level and under control (CT, 20/16 1C) or elevated (ET, 24/20 1C) temperature. Bt, Bacillus thuringiensis. Cumulative number of progeny variation by growth conditions, leaf position and growth stage both in the field (Zhu et al., 2004) and also in greenhouse conditions (Wei et al., 2005). Therefore, C : N pattern changes in more natural field conditions would be beneficial to assay to reveal whether the change in C : N relations is constitutive or rather specific for growth stage or growth condition. Here, Bt oilseed rape plants in their vegetative stage seemed to have a higher capacity of N uptake or allocation of N to leaves than their nontransgenic parent line in equal chronological age. It could be speculated that the observed difference in N and C could solely be based on the adding to the sum by the incorporated production of the new proteins: Bt toxin, GFP marker protein and nptii selectable marker protein in the leaves (Halfhill et al., 2001). However, the relatively low concentration of Bt toxin observed in these growth conditions in this line would likely not be totally responsible to cause the observed difference in C N contents, but would also require additional changes in N allocation in the basic metabolism. The observed differences could also be due to altered growth, because genetic transformation may lead to some additional pleiotropic effects (i.e. reduced growth leading to higher protein content; Rothe et al., 2004). In fact, also these GT1 Bt-transgenic oilseed rape plants had a slightly lower biomass at the end of their vegetative stage than the nontransgenic plants, which was particularly evident under elevated CO 2 in this work and also showing a similar trend in our earlier study (Himanen et al., 2008). An important factor to consider is that the C : N ratio was evaluated during the vegetative growth only and future work should address the overall profile of C : N changes during both vegetative and reproductive stages to reveal, if this difference is more pronounced at either stage. This would enable assessing whether differences in C : N dynamics could affect the reproductive fitness of Bt plants, which is highly important for both their agronomic performance and their persistence in agricultural environments after the growing season being r 2008 The Authors Journal compilation r 2008 Blackwell Publishing Ltd, Global Change Biology, doi: /j x

87 BT OILSEED RAPE, ELEVATED CO 2 AND TEMPERATURE 13 Table 3 Performance parameters (mean 1 SEM) of Myzus persicae and Brevicoryne brassicae aphids on Bt-transgenic and nontransgenic (Westar) oilseed rape plants grown in ambient or elevated (720 ppm) CO2 level under control (20/16 1C) or elevated (24/20 1C) temperature (n 5 6) Ambient CO2 Elevated CO2 20/16 1C 24/20 1C 20/16 1C 24/20 1C Westar Bt Westar Bt Westar Bt Westar Bt Experiment 1: Myzus persicae Development time (days) Adult aphid weight (mg) Progeny weight (mg) Mean relative growth rate Experiment 2: Brevicoryne brassicae Development time (days) Adult aphid weight (mg) Progeny weight (mg) Mean relative growth rate Bt, Bacillus thuringiensis; CO 2, carbon dioxide. Table 4 ANOVA results for effects of plant type, CO2 level and temperature and their interactions on Myzus persicae performance Plant type CO2 Temperature Plant type CO2 Plant type temperature CO2 temperature Plant type CO2 temperature F1,40 P F1, 40 P F1,40 P F1,40 P F1, 40 P F1,40 P F1, 40 P Experiment 1: M. persicae Development time (days) o Total progeny o Adult weight o Progeny weight Mean relative growth rate o Statistically significant effects (Po0.05) are marked in bold. r 2008 The Authors Journal compilation r 2008 Blackwell Publishing Ltd, Global Change Biology, doi: /j x

88 14 S. J. HIMANEN et al. Table 5 Summary of the treatment effects on the measured parameters Parameter studied Treatment Bt transgene Elevated CO 2 Elevated temperature Elevated CO 2 temperature Vegetative biomass # ** " *** ns ns Photosynthesis ns ns # *** ns Carbon # ** ns ns IA*** Nitrogen " * # *** ns IA** C : N ratio # * " *** ns IA* Bt toxin na ns ns ns Soluble carbohydrates Glucose ns # * " * ns Fructose ns ns ns IA* Sucrose ns ns ns ns Starch ns " *** # ** IA** Chlorophyll pigments Chl a ns ns ns IA** Chl b ns ns ns IA* Chl a/b ratio ns ns # * ns Total chlorophyll ns ns ns IA** Carotenoids # * ns ns IA* Aphid performance Developmental time ns (M) ns (B) ns (M) ns (B) # *** (M) ns (B) ns (M) ns (B) Adult weight ns (M) ns (B) # * (M) ns (B) # *** (M) ns (B) ns (M) ns (B) Progeny weight ns (M) ns (B) ns (M) ns (B) # ** (M) ns (B) ns (M) ns (B) Cumulative reproduction ns (M) ns (B) # * (M) ns (B) " *** (M) ns (B) ns (M) ns (B) Mean relative growth rate ns (M) ns (B) ns (M) ns (B) ns (M) ns (B) ns (M) ns (B) Increase ("), decrease (#) or no effect (ns) is based on statistically significant (*Po0.05, **Po0.01, ***Po0.001) main effects of plant type, CO 2 level, temperature and CO 2 level temperature interaction. IA, interaction; na, not applicable; (M), Myzus persicae; (B), Brevicoryne brassicae. related to the risks of gene flow into the environment (Halfhill et al., 2005). Between the two experiments conducted, differences in the overall status of N levels of plants were observed, which was predicted to be caused by seasonal differences, because the growth conditions were otherwise very similar. The factors leading to seasonal variation are mostly unknown, but it might be caused by internal regulation of plant growth (induction signals for vegetative vs. reproductive growth at different times of year, circannual and circadian rhythms) (Sharma et al., 2005). The occurrence of this and other types of seasonal variation even in controlled growth conditions (Ritala et al., 2001; Sharma et al., 2005), as often found in the greenhouse, is important considering whether there are certain times of year leading to higher incidence of changes in agronomic yield due to uncontrollable intrinsically regulated factors. However, in both experiments, the general patterns (i.e. plant type main effect and interacting CO 2 temperature effect on C and N contents) were similar. In general, the responses of biomass gain and photosynthetic rate to elevated CO 2 and temperature were similar in Bt-transgenic and nontransgenic oilseed rape. Elevated CO 2 increased biomass gain and net photosynthesis, total C was increased and total N decreased, which all are highly typical responses to enhanced CO 2 with increased C (Long et al., 2004). As oilseed rape has no obvious C storage structures, the positive effect of CO 2 on biomass gain typically decreases later in the development (Reekie et al., 1998). Under elevated CO 2 level, carboxylation of ribulose bisphosphate predominates over oxygenation by the Rubisco enzyme, whereas elevated temperature can increase photorespiration (Morison & Lawlor, 1999). Net photosynthesis of oilseed rape leaves was reduced under elevated growth temperature in our study, which was mostly an effect of differences in leaf development (i.e. phenology). The plants grown under elevated temperature were clearly physiologically older due to accelerated vegetative growth under elevated temperature, even though the temporal age of all plants was identical. The reduction in photosynthesis and biomass gain in oilseed rape plants under elevated temperature thereby was affected mostly by reduced leaf duration and r 2008 The Authors Journal compilation r 2008 Blackwell Publishing Ltd, Global Change Biology, doi: /j x

89 BT OILSEED RAPE, ELEVATED CO 2 AND TEMPERATURE 15 approaching senescence. Also, a decrease in total chlorophyll by elevated CO 2, which is the most typical response of chlorophyll pigments to enhanced CO 2 (Osborne et al., 1997; Sallas et al., 2003), was apparent in oilseed rape only under elevated temperature, in accordance with a more reduced net photosynthesis under elevated than control temperature with elevated CO 2 level. Elevated temperature alone increased the amount of chlorophyll pigments in oilseed rape leaves. Direct effects of elevated CO 2 atmospheres include decreases in the stomatal conductance and stomatal sensitivity in plants as an acclimation to elevated CO 2 (Lodge et al., 2001). Rubisco enzyme activity and content can also show adaptation to higher CO 2 concentration, the magnitude of this depending on e.g. leaf age and temperature (Morison & Lawlor, 1999; Pérez et al., 2007). Indications of photosynthetic acclimation to elevated CO 2 level (Martínez-Carrasco et al., 2005) were found in transgenic and nontransgenic oilseed rape as well (i.e. lowered photosynthesis in elevated CO 2 - grown plants compared with control CO 2 -grown plants). The concentrations of soluble sugars and starch in Bt-transgenic vs. nontransgenic plant leaves responded similarly to elevated CO 2 and temperature. Nonstructural carbohydrates typically increase under elevated CO 2 and the response can be mitigated (a decreasing effect) by elevated temperature (Poorter et al., 1997; Zvereva & Kozlov, 2006). We found increased starch concentration in oilseed rape under elevated CO 2 under both of our temperature regimes, similarly as Sallas et al. (2003), who studied the responses of Scots pine seedlings. The other clear effect observed, the reduction in starch and the increase in soluble sugars under ambient CO 2 with elevated temperature, could be related to a higher assimilate targeting to other parts of the plant at the late vegetative stage (i.e. leaves were starting to senescence, a phenological effect). In the elevated CO 2 treatment, the leaves were still active and functioning as sources of C with higher photosynthesis, as seen from the photosynthetic rate measurements, and such allocation from the leaves had not started yet. Aphid feeding seemed to increase N content of oilseed rape leaves under ambient CO 2, indicating that aphid damage enhances allocation of N to leaves or its uptake by the plant. M. persicae has previously been shown to be able to activate multiple genes involved in C assimilation, photosynthesis and N and C mobilization (Divol et al., 2005), to alter biomass allocation of plants at elevated CO 2 (Hughes & Bazzaz, 2001) and to increase the concentration of glucosinolates (Mewis et al., 2006). In our study, however, under elevated CO 2, aphids did not increase the N content of oilseed rape leaves, which could be a result of reaching the limit for N uptake from maximal plant growth. The feeding of the generalist aphid M. persicae had a greater effect in changing the N dynamics than that of the specialist aphid B. brassicae, although both species are piercing herbivores (i.e. they cause rather similar feeding damage). M. persicae adult weight was higher than that of B. brassicae aphids, so the intake of nutrients by M. persicae could be assumed to be also higher and could this way lead to more pronounced effects on these plants. Another thing is that M. persicae was able to move freely on the plants, whereas B. brassicae was enclosed into clip-cages to prevent its colonization behaviour. Therefore, it might also be that for effectively activating N allocation in the plant, the actual place where the feeding is occurring is important. The response of both aphid species to elevated CO 2 and temperature was highly similar in Bt-transgenic and nontransgenic plants, even though there were differences in C N ratios between the plant types. Bt Cry1Ac toxin should not affect aphids directly: firstly because aphids feed on phloem sap, which does not contain significant amounts of Bt toxin (Raps et al., 2001; Burgio et al., 2007), and secondly, because they lack specific receptors for Cry1 toxin binding. We also screened aphids for traces of Bt toxin, but the amounts were undetectable. Schuler et al. (2001) also found no indications of pleiotropic effects of Bt B. napus on M. persicae. Previously, major factors affecting aphid performance have been reported to be, e.g., the nutrient level of the plant, the C : N ratio, the composition of amino acids, the amount of soluble carbohydrates and proteins, the presence of tannins or other carbohydratebased insoluble compounds and the concentrations of secondary compounds (Cole, 1997). Therefore, the differences in C N ratios between Bt-transgenic and nontransgenic plants alone may be negligible with regards to aphid effects. There are also aphid species-specific N requirements (Newman et al., 2003), which limits making generalizations on their responses. Of the two aphid species studied, the generalist M. persicae was more responsive to both temperature and CO 2 on oilseed rape than the specialist B. brassicae and it had nearly 50% higher progeny production than B. brassicae. However, on Brassica oleracea, Stacey & Fellowes (2002) found B. brassicae aphids to be larger with greater fat gain under elevated CO 2, whereas individually reared M. persicae had higher fecundity under elevated CO 2. Differences in irradiance (greenhouse vs. growth chamber) and total soil fertility (Newman, 2003; Newman et al., 2003) have been associated with variable CO 2 effects on aphids. In addition, variation in amino acid compositions among Brassica species (Cole, 1997) could be reasons for differential performance of these aphids on Brassica plants. Temperature was the r 2008 The Authors Journal compilation r 2008 Blackwell Publishing Ltd, Global Change Biology, doi: /j x

90 16 S. J. HIMANEN et al. dominant determinant of M. persicae fecundity on oilseed rape, whereas elevated CO 2 counteracted its effect by affecting the reproduction negatively, a phenomenon previously modelled by Newman (2003), which predicted there would be no final change with these interacting factors. The decrease in aphid weights and the increase in total progeny produced under elevated temperature are presumably related and a similar response was reported by Flynn et al. (2006) on another aphid species. The role of temperature in accelerating population growth of herbivores by shortening development times has been previously described with numerous species (e.g. Bale et al., 2002; Johns & Hughes, 2002; Williams et al., 2003), but there are interaction effects between elevated CO 2 and various abiotic factors including temperature (Newman, 2003, 2006). Our results describing the exponential reproduction phase of the aphids do not differentiate between direct effects of CO 2 and temperature on aphid physiology and indirect plant-mediated effects on aphid performance. However, previous studies with aphids indicate that temperature is acting both directly, and through plants, on aphid performance (Bale et al., 2002; Newman, 2003), whereas the effects of elevated CO 2 are caused mainly by alterations in plant material as food for herbivores (Agrell et al., 2000). In nature, population interactions between different aphid species and also multitrophic interactions with higher trophic levels are involved (Bezemer et al., 1998; Awmack et al., 2004; Hoover & Newman, 2004), which makes predicting aphid performance in future climate even more challenging (Newman, 2006). Risk analysis of Bt plants towards nontarget herbivores has focused mainly on screening the potential direct toxic effects of purified and plant-expressed Bt toxin (e.g. Howald et al., 2003; Vojtech et al., 2005). Because it is well known that herbivores are affected by plant food quality, we might expect certain lines of insect resistance transgenic plants to sometimes indirectly perturb food webs (Schuler et al., 1999; Pons et al., 2005; Sisterson et al., 2007), although this has not been observed in field studies (e.g. Head et al., 2005). Even more, the evaluation of nontarget herbivore performance on transgenic plants under altered environmental conditions (elevated CO 2 and temperature) is still not well understood (Coviella et al., 2000, 2002; Chen et al., 2005). There is no doubt that in future, with the ever-expanding use of GM plants, it will be increasingly important to understand how they respond to climate change in terms of performance and pest susceptibility. Conclusions We found changes in C N ratios in transgenic oilseed rape compared with the nontransgenic parental type, but there was no evidence of effects caused by these changes on Bt Cry1Ac nonsusceptible aphid performance, which could have raised concerns about increased susceptibility of transgenic oilseed rape to nontarget herbivory in future climates. The increased N content observed in Bt plants, however, warrants further research to reveal whether this effect is growth stage-specific, limited to plants in their early vegetative growth, or whether this also occurs or becomes even more profound during the reproductive growth stage, when it could further affect plant fitness, invasion potential or herbivore dynamics. Nevertheless, studies on herbivore pest species responding to the state of N content are encouraged to be continued on Bt plants. Although Bt oilseed rape had an overall lower biomass gain during their early vegetative growth than the nontransgenic plants in this study, the photosynthetic and carbohydrate responses to elevated CO 2 and temperature, singly and in combination, were highly similar in both plant types, revealing equal performance and abilities to resource allocation under these altered abiotic conditions. Most importantly, our study emphasizes the need for further investigations, i.e. assessing the changes occurring in interactions of elevated CO 2 and temperature on Bt plants over the entire growing season with sufficient replication and a natural light environment in field conditions. Acknowledgements We thank V. Tiihonen for conducting laboratory analyses for chlorophyll, starch and soluble sugars, T. Oksanen for programming and maintaining the growth chambers, E. Häikiö for advice on photosynthetic measurements, M. A. Ibrahim, N.-L. Nuotio and M. Valkonen for help on aphid weighing and J. Blande for supplying aphids to us. This work was supported by the Academy of Finland (S. J. H., A. N., A.-M. N and J. K. H.) (ESGEMO programme, decision no ) and enabled by USDA Biotechnology Risk Assessment grants to C. N. S. References Agrell J, McDonald EP, Lindroth RL (2000) Effects of CO 2 and light on tree phytochemistry and insect performance. Oikos, 88, Awmack CS, Harrington R, Lindroth RL (2004) Aphid individual performance may not predict population responses to elevated CO 2 or O 3. Global Change Biology, 20, Bale JS, Masters GJ, Hodkinson ID et al. (2002) Herbivory in global climate change research: direct effects of rising temperature on insect herbivores. Global Change Biology, 8, Barnes JD, Balaguer L, Manrique E, Elvira S, Davison AW (1992) A reappraisal of the use of DMSO for the extraction and determination of chlorophylls a and chlorophylls b in lichens and higher plants. 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93 CHAPTER 5 ELEVATED ATMOSPHERIC OZONE INCREASES CONCENTRATION OF INSECTICIDAL BACILLUS THURINGIENSIS (BT) CRY1AC PROTEIN IN BT BRASSICA NAPUS AND REDUCES FEEDING OF A BT TARGET HERBIVORE ON THE NON-TRANSGENIC PARENT Himanen SJ, Nerg A-M, Nissinen A, Stewart CN Jr, Poppy GM, Holopainen JK Environmental Pollution (in press) Copyright (2008) Elsevier Ltd. Reprinted with kind permission.

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95 ARTICLE IN PRESS Environmental Pollution xxx (2008) 1 5 Contents lists available at ScienceDirect Environmental Pollution journal homepage: Elevated atmospheric ozone increases concentration of insecticidal Bacillus thuringiensis (Bt) Cry1Ac protein in Bt Brassica napus and reduces feeding of a Bt target herbivore on the non-transgenic parent Sari J. Himanen a, *, Anne-Marja Nerg a, Anne Nissinen a,b, C. Neal Stewart, Jr. c, Guy M. Poppy d, Jarmo K. Holopainen a a University of Kuopio, Department of Environmental Science, P.O. Box 1627, FIN Kuopio, Finland b MTT Agrifood Research Finland, Plant Protection, FIN Jokioinen, Finland c University of Tennessee, Department of Plant Sciences, Knoxville, TN , USA d University of Southampton, School of Biological Sciences, Southampton SO16 7PX, UK Elevated atmospheric ozone can induce fluctuations in insecticidal protein concentrations in transgenic plants. article info abstract Article history: Received 27 March 2008 Received in revised form 2 July 2008 Accepted 17 July 2008 Keywords: Bacillus thuringiensis Elevated ozone Herbivory Plutella xylostella L. Transgenic plants Sustained cultivation of Bacillus thuringiensis (Bt) transgenic crops requires stable transgene expression under variable abiotic conditions. We studied the interactions of Bt toxin production and chronic ozone exposure in Bt cry1ac-transgenic oilseed rape and found that the insect resistance trait is robust under ozone elevations. Bt Cry1Ac concentrations were higher in the leaves of Bt oilseed rape grown under elevated ozone compared to control treatment, measured either per leaf fresh weight or per total soluble protein of leaves. The mean relative growth rate of a Bt target herbivore, Plutella xylostella L. larvae was negative on Bt plants in all ozone treatments. On the non-transgenic plants, larval feeding damage was reduced under elevated ozone. Our results indicate the need for monitoring fluctuations in Bt toxin concentrations to reveal the potential of ozone exposure for altering dosing of Bt proteins to target and non-target herbivores in field environments experiencing increasing ozone pollution. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Transgenic crop plants expressing Bacillus thuringiensis (Bt) crystal endotoxins (Cry) have been used to control agriculturally important insect pests since their first release in 1996 (James, 2006). Specific Bt toxins limit target herbivore damage to the crop and their use has environmental and human health advantages, such as reduced use of more harmful pesticides (Romeis et al., 2006). Bt toxin resistance evolution in insect herbivores has been raised as a severe threat for the continuing success of Bt-transgenic crops (Tabashnik et al., 2008). Indeed, the frequency of resistance alleles in Helicoverpa zea to Bt Cry1Ac cotton have been reported to increase in field populations, but resistance management tactics have been successful in delaying the onset of resistance (Tabashnik et al., 2008). One factor affecting the possibility of risk of Bt resistance evolution is the fluctuations of Bt toxin concentrations present in the transgenic crops. Variability in Bt toxin concentration occurs * Corresponding author. Tel.: þ ; fax: þ address: [email protected] (S.J. Himanen). due to various factors such as leaf age (Wei et al., 2005; Le et al., 2007), growth condition (Sachs et al., 1998; Le et al., 2007), nutrient availability (Coviella et al., 2002) and CO 2 level (Coviella et al., 2002; Chen et al., 2005; Wu et al., 2007). With regard to continuing the safe use of Bt crops, the study of critical upper and lower limits and possible alterations in Bt toxin concentrations from environmental effects should be considered. In particular, as climate change increases its role for agriculture in the future (IPCC, 2007), interactions of Bt toxin production and abiotic factors such as elevated CO 2, temperature and tropospheric ozone (O 3 ) should be particularly important to study to determine whether transgenic crop plants will continue to be effective. Elevated background concentrations of tropospheric ozone (O 3 ) have the potential to affect agricultural plant composition, resource allocation and growth patterns (Ashmore, 2005; IPCC, 2007). O 3 causes variable defence reactions in plants by oxidative stress and leads to severe crop losses in sensitive plants (Ashmore, 2005; Fiscus et al., 2005). Stable transgene expression (i.e. production of Bt toxin), together with endogenous crop fitness is desirable under these future climate conditions. Yet, evidence exists that the concentration of Bt Cry1Ac toxin in transgenic cotton has been reduced by elevated CO 2 mostly through changes in altered nitrogen status (Coviella et al., 2002; /$ see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi: /j.envpol Please cite this article in press as: Sari J. Himanen et al., Elevated atmospheric ozone increases concentration of insecticidal Bacillus thuringiensis (Bt) Cry1Ac protein in Bt..., Environ. Pollut. (2008), doi: /j.envpol

96 ARTICLE IN PRESS 2 S.J. Himanen et al. / Environmental Pollution xxx (2008) 1 5 Chen et al., 2005). To our knowledge, the effects of O 3 exposure on Bt toxin concentrations in Bt-transgenic plants are unknown even though tropospheric background concentrations of O 3 and the occurrence of high O 3 episodes have increased because of anthropogenic activities (IPCC, 2007; Sitch et al., 2007). In addition, elevating temperature and CO 2 levels will substantially increase the frequency of tropospheric O 3 episodes by the end of this century (Zeng et al., 2008). High Bt endotoxin expression is needed to deliver a high dose to target herbivores, which is considered as necessary to delay the evolution of resistance (Bates et al., 2005). There are, however, upper limits of endotoxin synthesis. Another risk for consideration is non-target effects of Bt toxin on beneficial insects, which has been extensively studied in the laboratory, with most of the work using worst-case scenarios, i.e. high doses of Cry toxins to assess potential negative effects on selected organisms present in the agroecosystems (Romeis et al., 2008). It is unlikely that these high doses are ever found in Bt plants in the field, but it is important to bear in mind that Bt toxin can be metabolised or transferred to higher trophic levels (Schuler et al., 1999; Groot and Dicke, 2002; Wei et al., 2008), so the possible effects of changes in Bt toxin concentrations in crop plants could have consequences affecting, in addition, insects of higher trophic levels. Insect feeding pattern is another ecologically important factor related to insect control by transgenic plants that should be considered in future climates, since transgenic Bt plants require target herbivore feeding for Bt toxin uptake. Herbivore feeding patterns and preferences might be altered in future climatic conditions because of, e.g. allocation changes in response to oxidative stress and changes in total protein and total amino acid levels and the nutrient metabolism of plants (Trumble et al., 1987; Holton et al., 2003; Fiscus et al., 2005; Valkama et al., 2007), or direct effects of O 3 on insect physiology or behaviour (Awmack et al., 2004), e.g. attraction towards the host plant. As a continuum, these kind of changes might affect the ecology of both target and non-target herbivore insect species and alter Bt toxin uptake and dosing. Chen et al. (2005) found both Bt transformation and elevated CO 2 to negatively affect the growth parameters of Helicoverpa armigera in cotton, but to our knowledge, the only study to assess O 3 exposure effects on herbivory in Bt-transgenic plants is our previous work, where we found no effect by chronic 75 or 150 ppb O 3 elevation on short-term mean relative growth rate of Plutella xylostella L. larvae on Bt oilseed rape (Himanen et al., 2008a). Here, we describe the use of Bt Cry1Ac-transgenic Brassica napus ssp. oleifera, oilseed rape (Halfhill et al., 2001), as a model Bt plant for ozone herbivory interaction studies. Bt Cry1Ac enables the control of lepidopteran larvae, including the cosmopolitan Brassica pest P. xylostella L. (diamondback moth). P. xylostella has evolved resistance to Bt toxin in laboratory studies and in the field (Tabashnik et al., 2003). Therefore, it is essential to evaluate Bt toxin concentration changes in Bt oilseed rape since one of its target pests has a high potential to evolve resistance. Furthermore, low toxin content could hasten resistance. We have sufficient background knowledge about Bt toxin concentrations in this model crop when grown in growth chambers, greenhouse and the field (e.g. Halfhill et al., 2001; Zhu et al., 2004; Wei et al., 2005; Le et al., 2007; Himanen et al., 2008b), which facilitates comparison of effects caused by environmental factors, such as O 3 here, on the variation caused by, e.g. growth condition. The goal of this study was to predict whether the transgenic defence trait (Bt toxin production) will be effective under elevated atmospheric O 3 concentrations in Bt oilseed rape, and to assay effects of O 3 exposure on target herbivore feeding patterns and their performance on sensitive (non-transgenic) and resistant (Bt-transgenic line) oilseed rape. 2. Materials and methods Oilseed rape (B. napus ssp. oleifera L.) cv. Westar transformed to contain a truncated synthetic Bt cry1ac transgene (courtesy of Mycogen) and a GFP (green fluorescent protein) mgfp5-er (courtesy of Jim Haseloff) marker gene (Halfhill et al., 2001) event GT1 was selected for use in these experiments. It is a very well-characterized Bt oilseed rape line, which contains a single insert and has moderate level of transgene expression with no apparent fitness costs (Halfhill et al., 2001). Equal numbers of non-transgenic cv. Westar parent line and transgenic Bt Cry1Ac F 4 seeds were sown in 0.66 l pots in 2:1:1 fertilized compost (Kekkilä, Finland, N P K: mg l 1 ):B2 Sphagnum peat (Kekkilä, Finland, N P K: mg l 1 ):sand mixture and grown together in four identical computer-controlled growth chambers (2.6 m 3, Bioklim 2600T, Kryo- Service Oy, Helsinki, Finland) under 16 L: 8 D photoperiod (light adjusted to PAR of approximately 250 mmol m 2 s 2 ) and 20/16 C temperature. The plants were watered daily. Four chambers were available for use simultaneously and these were each set to one of the four O 3 regimes; filtered air control, 50, 75 or 100 nl l 1 (ppb). Chronic O 3 exposure was run for 8 h daily, from 8:30 am to 4:30 pm (from the 3rd day after sowing). O 3 was generated from pure oxygen with an O 3 generator (Fisher OZ 500 Ozone generator, Bonn, Germany), and continuously monitored with an O 3 analyzer (Environnement S.A., Model O 3 42M, Poissy, France). Two repetitions of the experiment were conducted in time to control for chamber effect by using replication as a random factor in the statistical analysis, where individual plants served as replicates. Also, to avoid any effects of chamberspecific growth conditions, the treatments were rotated weekly between the four similar chambers used, and the plants were rotated inside the chambers at the same time. Bt Cry1Ac-susceptible P. xylostella L. (Lepidoptera: Yponomeutidae), diamondback moth (DBM), larvae originating from a colony reared on broccoli (Brassica oleracea ssp. italica) at 12 L:12 D, approximately 25 C and 50% relative humidity at the University of Kuopio were used in the target herbivore growth assessments. P. xylostella is a Brassica specialist and a cosmopolitan pest (Talekar and Shelton, 1993). For determining P. xylostella mean relative growth rate (MRGR), seven 21-d old non-bt and Bt plants from control and each O 3 treatment were randomly selected. The 1st true leaf from five of these plants per treatment was collected for Bt toxin analysis, immediately deep-frozen in liquid nitrogen, and stored at 80 C for total soluble protein and Cry1Ac protein analysis. The 3rd true leaf was detached from each plant, and the petiole was put into a 2.0 ml eppendorf tube filled with tap water, and sealed with parafilm. Thereafter, the leaves were placed into 200 ml plastic containers (Nalgene) and the lids were replaced with fine mesh to allow evaporation. Four 2nd instar DBM larvae were group-weighed, and placed on each leaf. Bt and non-bt leaves were placed in the same growth chambers, with the O 3 treatment conditions described above. After 48 h, the larvae were collected, their possible mortality recorded, and living larvae were group-weighed. The leaves were photographed to determine the leaf area eaten by the larvae in pixels, which were converted to square centimeters (Adobe Photoshop Elements 2.0). We also calculated the percentage of damaged area of the total leaf area, since the treatments could also affect the size and shape of the leaves. MRGR of the DBM larvae was calculated with the equation: [ln (final weight of larvae) ln (initial weight of larvae)]/duration of feeding in days (Van Emden, 1969). The amount of total soluble protein (TSP) was measured with the Bradford (1976) method and Cry1Ac concentration with a commercial enzyme-linked immunosorbent assay (ELISA) PathoScreen kit for Cry1Ac (Agdia, Elkhart, Indiana, US) as in Vojtech et al. (2005), except that each sample was tested in duplicate and approximately 10 mg of total protein was added to sample wells. The optical density (OD) of each sample was read at 620 nm wavelength with an ELISA plate reader (Easy Reader SF Plus, SLT Labinstruments). Before the statistical analysis the normality and the equality of error variances of variable residuals were tested and some variables were log (x þ 1) or squareroot transformed for normality. Linear mixed model was used for assessing main effects of plant type and O 3 level (fixed factors) and their interaction effects on Bt Cry1Ac concentration, total soluble protein, DBM mean relative growth rate and DBM feeding area. The model included repetition of the experiment as a random factor in all analyses and initial larval weight as a covariate in MRGR and feeding area analysis. Mixed model post hoc tests based on estimated marginal means with Bonferroni correction were used for comparing treatments within plant type. 3. Results There was significantly increased Bt Cry1Ac toxin protein concentration in transgenic oilseed rape leaves in the 100 ppb elevated O 3 treatment, measured either per leaf fresh weight or per total soluble protein of leaves (mixed model, main effect of O 3 : F 3,35 ¼ 6.889, P ¼ and F 3,35 ¼ 8.331, P < 0.001, respectively) (Table 1). Ozone had no statistically significant effect on the amount of total soluble protein in the leaves (mixed model, main effect of O 3 : F 3,35 ¼ 2.457, P ¼ 0.079) (Table 1). Please cite this article in press as: Sari J. Himanen et al., Elevated atmospheric ozone increases concentration of insecticidal Bacillus thuringiensis (Bt) Cry1Ac protein in Bt..., Environ. Pollut. (2008), doi: /j.envpol

97 ARTICLE IN PRESS S.J. Himanen et al. / Environmental Pollution xxx (2008) Table 1 Mean SEM Bt Cry1Ac (Bt toxin) concentrations as mgg 1 leaf FW (fresh weight) and ng mg 1 total soluble protein (TSP), and the amount of TSP (mg g 1 leaf FW) in Bt-transgenic B. napus plants grown under filtered air (control), 50, 75 or 100 ppb of elevated O3 (8 h daily) Treatment mg Cry1Ac (g 1 FW) ng Cry1Ac (mg 1 TSP) TSP (mg g 1 FW) Control a a a 50 ppb O a a a 75 ppb O ab ab a 100 ppb O b b a Statistically significant differences (P < 0.05) between treatments are marked with different letters (linear mixed model post hoc tests based on estimated marginal means), n ¼ 10. a Cm 2 leaf area fed a ab P plant type <0.001 P O3 =0.023 P O3 x plant type =0.018 ab b Non-Bt Bt Mean relative growth rate of P. xylostella larvae feeding on Bt oilseed rape was negative in all O 3 treatments and therefore reduced compared to that of larvae feeding on the non-transgenic plants (mixed model, main effect of plant type: F 1,103 ¼ 811.7, P < 0.001). Elevated O 3 had no statistically significant effect on MRGR of P. xylostella on non-transgenic or Bt-transgenic plants (Fig. 1). Less leaf area was consumed from the non-transgenic plants by the P. xylostella larvae under 100 ppb (expressed as cm 2 and % of total leaf area, Fig. 2a and b) and under 75 ppb (% of total leaf area, Fig. 2b) elevated O 3 compared to control treatment (mixed model, main effect of O 3 : F 3,51 ¼ 3.613, P ¼ for leaf area fed in cm 2 and F 3,51 ¼ 3.587, P ¼ for % of total leaf area fed). Elevated O 3 did not have this kind of effect on larval feeding area in Bt oilseed rape leaves (mixed model, interaction plant type O 3, F 3,103 ¼ 3.489, P ¼ for leaf area fed in cm 2 and F 3,103 ¼ 3.276, P ¼ for % of total leaf area fed), where feeding damage levels were very low in all treatments due to the presence of Bt toxin (mixed model, main effect of plant type, F 1,103 ¼ , P < for leaf area fed in cm 2 and F 1,103 ¼ , P < for % of total leaf area fed). The total leaf area eaten on Bt plants was very small (range of feeding area cm 2 per leaf) and, on average, 50 times less leaf area was consumed in Bt than in non-bt plants. 4. Discussion Bt Cry1Ac concentrations were higher in Bt oilseed rape leaves under high level of chronic O 3 exposure (100 ppb) compared to plant leaves grown under no O 3. Previously, the Bt Cry1Ac concentration increased on oilseed rape leaves as they aged (Wei et al., 2005; Le et al., 2007) and correspondingly, the amount of TSP Mean relative growth rate P plant type <0.001 Non-Bt Bt Control 50 ppb 75 ppb 100 ppb Fig. 1. Mean relative growth rate SEM of diamondback moth (P. xylostella L.) larvae feeding on non-transgenic (non-bt) and Bt-transgenic (Bt) B. napus plants grown and tested under control (filtered air), 50, 75 or 100 ppb O3 treatment. Statistically significant main effects of plant type and ozone and their interactions are shown (linear mixed model), n ¼ 14. b Percent leaf area fed Control 50 ppb 75 ppb 100 ppb a ab P plant type <0.001 P O3 =0.023 P O3 x plant type =0.024 Control 50 ppb 75 ppb 100 ppb either decreased (Wei et al., 2005) or showed an increase throughout the vegetative stage followed by a decrease or a static stage during pod formation (Le et al., 2007), which demonstrates that Cry1Ac is a relatively stable protein. Since one typical symptom of chronic O 3 stress in plants is premature senescence (Fiscus et al., 2005), this observation suggests that earlier maturation could be one reason for the observed increase in Cry1Ac under these conditions. However, in a previous study with this oilseed rape line, no significant differences in Bt toxin concentrations occurred in plants grown under elevated CO 2 and temperature, singly or in combination, although the growth of the plants was highly affected by these abiotic factors (Himanen et al., 2008b). This indicates that elevated O 3 acts more strongly than these other climate change factors on Bt toxin concentration of Bt oilseed rape. In this study, Bt toxin production remained robust under O 3 elevations and accordingly increased its proportion of TSP with O 3 exposure. Interestingly, the increase in Bt toxin concentration was higher with increasing dose of O 3 as revealed by the comparison of low (50 ppb) and medium exposures (75 ppb) to high O 3 exposure (100 ppb), and the statistical significant difference compared to control treatment appeared only with the highest exposure. The TSP concentration of leaves was not significantly affected by O 3 treatment in this study, although some previous studies have reported increases in TSP after O 3 exposure because of the release of soluble nitrogenous compounds from plant structures by O 3 action (Trumble et al., 1987). b b Non-Bt Bt Fig. 2. Leaf area consumed SEM by diamondback moth (P. xylostella L.) larvae (a) as cm 2 and (b) as percent of total leaf area in non-transgenic (non-bt) and Bt-transgenic (Bt) B. napus plants grown and tested under control (filtered air), 50, 75 or 100 ppb O3 treatment. Statistically significant main effects of plant type and ozone and their interactions are shown (linear mixed model). Different letters indicate statistically significant differences between O3 treatments among plant type (mixed model post hoc tests), n ¼ 14. Please cite this article in press as: Sari J. Himanen et al., Elevated atmospheric ozone increases concentration of insecticidal Bacillus thuringiensis (Bt) Cry1Ac protein in Bt..., Environ. Pollut. (2008), doi: /j.envpol

98 ARTICLE IN PRESS 4 S.J. Himanen et al. / Environmental Pollution xxx (2008) 1 5 The variation of Bt toxin concentration in the leaves of Bt oilseed rape in this study was in the range found previously with the same transgenic line in growth chamber (Himanen et al., 2008b), greenhouse (Wei et al., 2005) and field conditions (Le et al., 2007), and variations between different field locations (Zhu et al., 2004; Le et al., 2007). However, our results emphasize the importance of O 3 studies in a wider scale, i.e. in field conditions over several years, where other abiotic factors may interact and mediate the O 3 response of Bt toxin as well. This is important for Bt-transgenic crops in general, and in our case, it would be particularly interesting to assay whether the O 3 effect observed here is rather growth condition-specific, i.e. more pronounced in either controlled or natural environment. Since the Bt toxin concentration was shown to increase both per plant fresh weight and per total soluble protein in plants grown in environment-controlled growth chambers, there is a considerable reason to believe that this might occur in field conditions and with other plant species as well. Nevertheless, the O 3 effects on Bt concentration must be placed in context to other environmental factors present in field environments to predict transgene expression stability needed in agriculture, which is also ecologically important. Previous studies have revealed elevated CO 2 as a climate change factor that reduces Bt toxin concentration in Bt cotton plants grown both in controlled and field conditions (Coviella et al., 2002; Chen et al., 2005; Wu et al., 2007), and presumably the difference is also related to nitrogen availability. It is unknown what this effect might mean for Bt cotton in agriculture. As the current climate change will induce rises in both CO 2 and O 3 levels in the troposphere, the combined effects of elevated CO 2 and O 3 on Bt toxin concentrations of Bt plants should also be considered, as their effects might be opposing. Mean RGR of P. xylostella larvae on Bt oilseed rape was negative in all treatments, indicating that the Bt trait is robust under O 3 elevation. A similar result was found previously in a 24 h assessment of P. xylostella mean growth rate under chronic 75 and 150 ppb O 3 elevation in this oilseed rape line (Himanen et al., 2008a). The MRGR of P. xylostella was not affected by O 3 elevation in the non-transgenic plants here or in our previous study, where there was no difference in the MRGR of P. xylostella larvae feeding on non-transgenic Westar parent line under control, 75 or 150 ppb O 3. In this study there was less leaf area eaten from non-transgenic plants in the elevated O 3 treatments, which suggests that O 3 as such rendered the oilseed rape plant less favourable and attractive for the herbivore, and therefore, might actually diminish crop losses caused by P. xylostella larvae. However, lower O 3 exposure (50 and 75 nl l 1 ) did not significantly reduce the larval feeding, and the overall effects of high O 3 (100 nl l 1 ) on plant fitness (i.e. lowered photosynthetic rate and biomass) (Fiscus et al., 2005; Himanen et al., 2008a) are assumed to be more harmful, and negate any benefits obtained from potential decreased herbivory. Since the herbivores were fed with leaves from O 3 -grown plants and their feeding was assayed under the same O 3 treatment, both indirect (plant-mediated) and direct effects of O 3, as present together in nature, affected herbivore performance and feeding in this study. The indirect (i.e. plant-mediated) effect of O 3 is typically to render leaves more palatable to herbivores leading to higher leaf damage levels and weight gain in insects because of enhanced nutritional value, better digestibility of plant material or alterations in secondary compounds (Trumble et al., 1987; Jones and Coleman, 1988; Bolsinger et al., 1992; Jondrup et al., 2002; Percy et al., 2002). O 3 can induce changes such as rise in TSP as evidenced by Trumble et al. (1987) on tomato (Lycopersicon esculentum) plants, but also cause fluctuations in total free amino acid content or specific amino acid composition (Trumble et al., 1987), which can be critical for the development of certain herbivores through direct effects or by changes in secondary compound allocation. However, the O 3 response is somewhat related to the insect or plant species studied, instar used and O 3 exposure length (Holopainen, 2002; Agrell et al., 2005; Valkama et al., 2007). O 3 can, on the other hand, induce defence responses (Fiscus et al., 2005) and increase concentrations of secondary compounds (Himanen et al., 2008a), some of which might also protect plants from herbivory. For example, altered feeding patterns in Brassica plants specifically could be affected by changes in the levels of glucosinolates, the amino acid-derived secondary compounds of Brassicas, concentrations of which have been altered by elevated O 3 (150 nl l 1 ) in Bt and non-bt oilseed rape (Himanen et al., 2008a). P. xylostella has a resistance mechanism against glucosinolates (Ratzka et al., 2002), which suggests, however, that other secondary compound groups, such as proteinase inhibitors present in crucifers in significant amounts, possessing deterrent effects on DBM larvae (De Leo et al., 2001), could be higher in the plants also under oxidative stress. Unfortunately, we did not analyse proteinase inhibitors in this study, but measuring the response of these to elevated O 3 together with an analysis of DBM performance in oilseed rape could reveal whether these two factors are related. Direct effects of O 3 on herbivores are predicted to have relatively low impact compared with plant-mediated effects at current O 3 levels (Awmack et al., 2004). This view is bolstered by the prevalence of studies reporting mostly positive effects of O 3 elevation on herbivores (Valkama et al., 2007). In addition, e.g. Levy et al. (1974) demonstrated that two cockroach species and a fire ant species were unaffected by prolonged elevated O 3 exposure. Ozone is, however, a cell damaging agent and it can be used to directly kill storedproduct insects using high O 3 -fumigation doses (typically over 200 ppb is needed, Hollingsworth and Armstrong, 2005). Therefore, the net effect of O 3 exposure on different insect species is still not conclusive. Importantly, the growth (MRGR) of the P. xylostella larvae in our study was similar in control and high O 3,soitis possible that the nutritional quality of plant material for larvae was better in high O 3 leaves, but the reduced feeding behaviour caused by deterrent secondary compounds or direct effects of O 3 led to the observed response of no change in the MRGR of the herbivores. The relation of insect feeding preference and exposure to Bt toxin is important since increased Bt toxin concentrations present in leaves grown under O 3 exposure combined with possible increased feeding by herbivores non-susceptible to Bt toxin, unlike P. xylostella, might result in higher doses of toxin transmitted in ecosystems by non-target herbivores to higher trophic levels (Schuler et al., 1999; Groot and Dicke, 2002; Wilkinson et al., 2003; Wei et al., 2008). Wei et al. (2008) have shown that Cry1Ac does travel through trophic levels in amounts high enough to be detected via ELISA. 5. Conclusions We found that high chronic O 3 increased Bt Cry1Ac toxin concentration in Bt-transgenic oilseed rape leaves but did not change the levels of target herbivore feeding or their performance on Bt plants. The susceptible oilseed rape parent line, on the other hand, experienced reduced feeding damage by P. xylostella larvae with high O 3, but the MRGR of the herbivores was unaffected by O 3 suggesting that food quality for the larvae was improved. This study emphasizes the importance to assay Bt toxin concentration fluctuations in response to abiotic factors, of which O 3 exposure is a good example. Field testing is needed to reveal whether environmentally realistic O 3 concentrations in polluted areas lead to similar responses as observed in this study under controlled conditions. Higher ozone might lead to altered dosing of transgenic proteins in target and non-target herbivores and insects of higher trophic levels in two ways: through altered Bt toxin concentrations in plant material and changes in herbivore feeding patterns. Please cite this article in press as: Sari J. Himanen et al., Elevated atmospheric ozone increases concentration of insecticidal Bacillus thuringiensis (Bt) Cry1Ac protein in Bt..., Environ. Pollut. (2008), doi: /j.envpol

99 ARTICLE IN PRESS S.J. Himanen et al. / Environmental Pollution xxx (2008) Acknowledgements We thank T. Oksanen for programming and maintaining the growth chambers and setting up the O 3 exposures. This work was supported by the Academy of Finland (ESGEMO programme, decision no ) (S.J.H., A-M.N., A.N. and J.K.H.), the Finnish Cultural Foundation (S.J.H.) and ISONET (MRTN-CT ) (J.K.H) and it was enabled by USDA BRAG grants to C.N.S. References Agrell, J., Kopper, B., McDonald, E.P., Lindroth, R.L., CO 2 and O 3 effects on host plant preferences of the forest tent caterpillar (Malacosoma disstria). Global Change Biology 11, Ashmore, M.R., Assessing the future global impacts of ozone on vegetation. Plant, Cell & Environment 28, Awmack, C.S., Harrington, R., Lindroth, R.L., Aphid individual performance may not predict population responses to elevated CO 2 or O 3. 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101 CHAPTER 6 GENERAL DISCUSSION

102

103 6. GENERAL DISCUSSION 6.1 Comparison of non-transgenic and Bt-transgenic oilseed rape in terms of growth and elevated CO 2, temperature and O 3 responses and was reduced with chronic O 3 exposure (Chapters 2 and 3). Also, no visible differences in O 3 sensitivity were detected between the plant types (Himanen et al., unpublished data). Correspondingly, the net photosynthesis of Bt oilseed rape was higher under elevated CO 2 (Chapter 4), lower in elevated temperature-grown plants (Chapter 4) and reduced under chronic O 3 (Himanen et al., unpublished data); all equal responses to the non-bt line plants. Previous work on Bt cotton has also shown that it responds to elevated CO 2 with increased plant height and leaf area, although direct comparison to a non- Bt line was not included (Wu et al. 2007a). The aforementioned outcomes are typical for many crop plants in response to elevated CO 2, temperature or O 3 (Poorter et al. 1997, Morison & Lawlor 1999, Ashmore 2005, Körner 2006) especially under controlled conditions. However, it has been speculated that in nature these responses will be less straightforward due to numerous interfering environmental factors (Ainsworth et al. 2008). The Bt-transgenic line GT1 selected for use in these experiments was phenotypically highly comparable to the parent line, and previous studies have shown no adverse effects by the gene insertion under laboratory (Halfhill et al. 2001), green house (Wei et al. 2005) or field conditions (Le et al. 2007). In our growth chambers, under control conditions (no CO 2 or O 3 elevations) the Bttransgenic line showed slightly delayed early vegetative growth compared to the non-transgenic parent line in most of the experiments (Chapters 3, 4). However, this retardation of early growth typically disappeared at later growth stages and the plant lines had equal plant biomass and seed set at their reproductive stage (Himanen et al., unpublished data), which mostly determines their agricultural performance. The outline of the study restricts estimation of season-long responses and The delayed early growth could be a cost therefore limits detecting phenological of producing Bt toxin, but importantly, the patterns, which appear over the entire life original study hypothesis of reduced cycle of oilseed rape (Franzaring et al. potential of Bt plants to respond to the 2008). Elevated temperature and CO 2 has studied abiotic changes was not verified. the potential to decrease development time Even though the Bt line plants had lower to reproduction (although requiring vegetative biomass, their growth responses nitrogen in amounts sufficient to support were equal to the non-bt line plants; reproduction) (Peñuelas & Filella 2001, biomass was increased under CO 2 Körner 2006), while O 3 can lead to elevation (Chapters 2, 3 and 4), remained premature senescence (Miller et al. 1999). stable at temperature elevation (Chapter 4) To reveal the equality of Bt plant 103 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008)

104 Sari J Himanen: Bt oilseed rape and climate change responses under the reproductive allocation with the influence of altered climatic factors, a seasonal study to measure growth patterns would be an optimal continuation. 6.2 Effects of climate change factors and elevated O 3 on primary and secondary compounds - is the allocation pattern different in non-transgenic and Bttransgenic oilseed rape? As compounds of primary metabolism soluble sugars, starch, chlorophyll pigments and total carbon and nitrogen were analyzed under control conditions, under elevated CO 2, with elevated temperature and in a combined elevated CO 2 and temperature treatment (Chapter 4). Bt line plants had lowered carbon and higher nitrogen content than the non-bt line as well as lower carotenoid concentration in the leaves. However, the responses to elevated CO 2 and temperature, both singly and in combination, were not significantly different in non-bt and Bt plant lines (Chapter 4). This suggested no significant effect of Bt toxin production at vegetative growth stage on these primary compound responses to the studied climate change factors. Only a few other studies have included climate change factors in their comparative analyses of Bt-transgenic and nontransgenic plants (Coviella et al. 2002, Chen et al. 2005). Higher N content and reduced C:N ratio (but no difference in C) in Bt cotton compared to a non-bt line was previously found in plants grown in low nitrogen conditions, whereas high nitrogen availability led to equal N contents (Coviella et al. 2002). In the same study, reduced total phenolics were detected under low N from Bt plants compared to the non-bt plants. Chen et al. (2005) found lower N, total non-structural carbohydrate, soluble sugar and starch concentrations from Bt cotton compared to its non-bt counterpart, but equal water, condensed tannin and gossypol concentrations and TNC:N ratios when both plant types were grown under elevated CO 2. Again, follow-up seasonal studies could help in revealing the prevalence of differences in carbon-nitrogen contents detected at certain time points of vegetative growth, or whether they occur at particular growth stages. If these were assessed throughout the complete life cycle of the crop plant, this would better enable determination of their larger ecological meaning, for example, the plant reproduction fitness, invasion potential or herbivore dynamics. Assessing performance of Bt-resistant insect herbivores, which would respond positively to N, are encouraged by the results. A field study would enable detecting whether the magnitude of the N change is agriculturally or ecologically significant. In response to elevated CO 2 reduced nitrogen content, higher C:N ratio, lowered glucose concentrations and increased starch were detected in the foliage (Chapter 4). Plants grown under elevated 104 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008)

105 General discussion temperature had decreased photosynthetic levels, increased glucose contents, decreased starch concentrations and lowered Chl a/b ratios. Numerous interactions between elevated CO 2 and temperature were found (i.e. for C, N, C:N ratio, fructose, starch, Chl a, Chl b, total chlorophyll and carotenoids). In general elevated temperature led to faster maturation in ambient CO 2 -grown plants that had senescence-approaching physiological responses. Elevated CO 2 dominated the growth and C:N responses over temperature. These responses are again typical though definitely not universal (reviewed by e.g. Morison & Lawlor 1999). A recent meta-analysis revealed that C:N responses of plants are mostly affected by elevated CO 2, whereas sugar and starch responses to elevated CO 2 are often mitigated by temperature (Zvereva & Kozlov 2006). The results once again emphasize the need for seasonal studies (Franzaring et al. 2008), since elevated temperature clearly had the potential to hasten development of oilseed rape. The O 3 experiments (Chapters 2, 3 and 5) did not include analysis of primary compounds, so the functioning of O 3 responses of the plant lines can only be assessed based on the biomass (Chapter 6.1) and secondary compound responses. Constitutive glucosinolate profiles of Bt and non-bt oilseed rape lines were qualitatively and quantitatively equal, and elevated CO 2 and O 3 (acute and chronic) affected their GS concentrations similarly (Chapter 2), indicating equal allocation patterns for GS synthesis in the plant types. Production of nitrogen-containing secondary compounds such as GSs could be predicted to be especially vulnerable to allocation differences influenced by Bt toxin production costs in Bt oilseed rape, but this clearly was not the case. The GS profile in leaves was highly dominated by indolic GSs, whose concentrations increased markedly after P. xylostella feeding, but dropped with CO 2 as well as chronic and acute O 3 elevation in both plant lines. Such a clear, yet previously unreported response to O 3 in concentrations of the two individual GSs (3-indolylmethyl-GS and 2- phenylethylgs) was surprising and warrants future studies with O 3 on Brassicas with differing initial glucosinolate profiles. It should also be noted that combined herbivore attack and O 3 elevation resulted in the absence of herbivore induced indolic GSs, which raised speculation on the interacting involvement of the jasmonic acid and salicylic acid signalling pathways (Brader et al. 2006), though these were not directly addressed in this study. These findings surely deserve attention as future targets for GS research. The most apparent effects of elevated CO 2 on GSs appeared to be the reduction in indolic GSs under elevated CO 2, and the trend for their induction by herbivory to be higher under elevated than ambient CO 2. Similar increased induction of indolic GSs has been reported in Arabidopsis thaliana, although elevated CO 2 alone failed to affect concentrations in intact plants (Bidart-Bouzat et al. 2005). In contrast, Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008) 105

106 Sari J Himanen: Bt oilseed rape and climate change aliphatic GSs were increased more by herbivory under ambient than elevated CO 2, whereas feeding increased the concentration of the aromatic GS 2- phenylethyl-gs equally at both CO 2 levels (Chapter 2). Different structural groups therefore respond uniquely to elevated CO 2 and previous studies only reporting a change in total GSs (e.g. Karowe et al. 1997) have limited value in revealing the overall changes elicited by elevated CO 2. It was important to analyze volatile terpenoid emissions in the current study, since they connect ecological tritrophic plant-herbivore-parasitoid interactions into the Bt oilseed rape system. This has been previously used as a good system for assessing GM plant-parasitoid interactions with Bt-susceptible and resistant herbivores in controlled conditions (e.g. Schuler et al. 1999). It was predicted from previous studies that the lines might exhibit quantitative differences in highly dynamic volatiles (as reported by Yan et al in Bt cotton) and that the plants having lower target herbivore feeding damage could lose their attractiveness to natural enemies (Schuler et al. 1999). Instead we proved that even slight herbivory induced numerous terpenoid emissions from Bt oilseed rape, although non-bt line plants, with many times higher leaf damage levels emitted much greater quantities (particularly DMNT and (E,E)- -farnesene emissions) (Chapter 3). As an effect of elevated CO 2, most terpenoid emissions and the degree of herbivore-inducible emissions increased, whereas elevated O 3 reduced total terpenoid emissions, and these responses were equal on both plant lines. The O 3 effect can be explained by the ability of O 3 to degrade most terpenoids (Pinto et al. 2007a, b), whereas the CO 2 response was somewhat unexpected. Most studies have reported decreased emission rates for monoterpenoids on a seasonal scale, which can be explained, for example by pyruvate competition as needed for higher photosynthesis (Rosenstiel et al. 2003) or terpene synthase activation changes (Loreto et al. 2001). Since we used vegetative stage plants grown in growth chambers, a potential influence on the response could be larger photosynthetically active leaf biomass in elevated CO 2 -grown than control CO 2 - grown plants. Physicochemical characteristics such as measuring stomatal conductance were not included in this study, but based on the data they did not seem to play a clear role in the emission increase. No differences were detected between responses of oxygenated and nonoxygenated compounds, which are known to be differentially regulated by stomata (Niinemets et al. 2004, Noe et al. 2006). 6.3 Stability of the transgenic trait and target herbivore susceptibility under elevated CO 2 or O 3 concentrations It is highly desirable that the concentration of Bt toxin in insect-resistant GM plants is high enough to kill most pest herbivores. A Bt dose killing >95 % of heterozygous resistant individuals of target herbivores is recommended, which generally 106 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008)

107 General discussion corresponds to >0.2 % of total soluble protein in plant tissue (Gatehouse 2008). The possibility to develop resistance in target insects is much higher if the dose of the toxicant is smaller (Bates et al. 2005). Therefore stable expression of the transgene in plants leading to stable Bt toxin concentration under variable abiotic conditions is desired. Based on the current study, no indications of reduced effectiveness of Bt oilseed rape against target herbivores under elevated CO 2, temperature or their combination exist (Chapter 4). Reduced concentrations of Bt toxin have been reported in Bt cotton grown under elevated CO 2 (Coviella et al. 2002, Chen et al. 2005). Coviella et al. (2002) found that this reduction was connected to nitrogen availability, so that it only prevailed under high nitrogen concentrations. However, more recently a systematic reduction in toxin, observed in field conditions with elevated CO 2 exposure, verified this decrease (Wu et al. 2007a). A medium nitrogen supply was used in the current study on oilseed rape, so this might partly explain the lack of difference in toxin concentration. However, the lack of knowledge on ecologically meaningful consequences of Bt toxin fluctuations under elevated CO 2 for target pests raises the need for combinative effect studies of fertilization and Bt toxin concentration changes and target herbivory under control and elevated CO 2. Previous work on Bt cotton has also showed a decrease in Bt Cry1Ac toxin in drought-stressed plants (Martins et al. 2008), aging plants (Olsen et al. 2005) and in lower chlorophyll-containing plant parts (Abel & Adamczyk 2004). However, in these studies no loss of effectiveness of the insect resistance trait was found despite these Bt toxin concentration fluctuations. In contrast, seasonal variation in Bt Cry1Ab concentration in Bt corn was suggested to potentially hasten Bt toxin resistance, since increased survival of a heterozygously resistant strain of the sugarcane borer was detected in reproductive stage Bt corn hybrid plants (Wu et al. 2007b). Increased concentrations of Bt Cry1Ac toxin expressed as proportions of the fresh leaf weight and the total soluble protein were detected from Bt oilseed rape grown under elevated O 3 (Chapter 5). This kind of study on O 3 -exposed Bt plants has not been previously reported, which makes this finding especially interesting. The reason for this increased concentration might be partly explained by plant phenology. The change could be associated to differential physiological aging of the plants, since Wei et al. (2005) demonstrated that the highest Bt Cry1Ac concentrations appear in the oldest basal leaves of oilseed rape. It is true that O 3 might speed up senescence (Miller et al. 1999). Also, negative effects of O 3 on photosynthesis and biomass gain and degradation of O 3 -sensitive proteins (Fiscus et al. 2005) might increase the proportion of stably expressed Bt toxin in the total soluble protein and fresh leaf weight. However, it is important to note Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008) 107

108 Sari J Himanen: Bt oilseed rape and climate change that the increased concentrations detected with elevated O 3 do fall within natural variation levels detected under different growth conditions (i.e. field, green house) (Zhu et al. 2004, Wei et al. 2005, Le et al. 2007). This sets this finding into an agriculturally more relevant perspective and without further field experimentation, including O 3 exposure as a treatment, the potential problem of encountering Bt concentrations that are too high cannot be estimated. Nevertheless, upper limits for Bt toxin concentration are important to protect nontarget organisms from consuming high doses, which could potentially travel through trophic levels (Torres & Ruberson 2008, Wei et al. 2008). Without question, more studies on the cause of fluctuation in Bt toxin concentrations through the whole life cycle of Bt plants under elevated O 3 are encouraged by this study. Feeding by larvae of the target herbivore P. xylostella was equally inhibited on Bt oilseed rape under control conditions (Chapters 2, 3 and 5), under elevated CO 2 (Chapter 2 and 3) and under elevated O 3 (Chapters 2, 3 and 5). This did not raise concern about altered effectiveness of vegetative stage Bt oilseed rape plants due to Bt toxin fluctuations, which is important when considering actual exposure doses. Since Bt toxin is only active when ingested, in theory, altered feeding preferences in response to elevated CO 2 or O 3 (e.g. Docherty et al. 1996, Percy et al. 2002) might affect dosing of Bt toxin and cause changes in the amount of days needed for Bt toxin to kill the larvae. However, in practice this was not proved to be meaningful. Larvae themselves were not assayed for Bt toxin, so differences in the amount of Bt Cry1Ac ingested by control and high O 3 leaf- fed larvae could not be revealed. Non-Bt plant results serve to give additional information of the impact of O 3 on the role of P. xylostella as a pest of oilseed rape. Under elevated O 3, P. xylostella larvae fed less on the non-bt plants, whereas equal growth rates were attained on control and O 3 -grown leaves. The most typical response of herbivores to O 3 -fumigated leaves is enhanced feeding with better palatability (e.g. Jones & Coleman 1988), but the response can vary depending on the sensitivity of the plant to O 3 and can be plant specific. As an example, on B. rapa, the time to pupation of Pieris brassicae L. (Lepidoptera: Pieridae) larvae was negatively affected by consuming O 3 -exposed leaves of an O 3 - sensitive line, whereas the herbivore did not have preference for sensitive or resistant lines under elevated O 3 (Jondrup et al. 2002). Our finding of no change in MRGR of P. xylostella indicated enhanced quality of leaf material for these herbivores under high O 3, but also a reduced ability for them to feed. The reason for this reduced feeding damage under elevated O 3 remains unknown, but direct negative effects of O 3 (Hollingsworth & Armstrong 2005) might be one potential reason. P. xylostella possesses a desulphatase enzyme capable of detoxifying toxic GS breakdown products (Ratzka et al. 2002), so the GS defences, even if significantly changed 108 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008)

109 General discussion under elevated O 3 (Chapter 2), are unlikely to be the reason for reduced feeding activity and are more likely to be efficient against non-adapted herbivores. Other potential causes for this behaviour might be changes in carbohydrate or amino acid - based primary compounds (White 1984, Jondrup et al. 2002). An equal leaf area was consumed by DBM larvae on control and elevated CO 2 -grown non-bt oilseed rape leaves (Chapter 3); there were no signs of compensative feeding resembling those of e.g. cotton bollworm (Helicoverpa armigera) that feeds more to compensate for reduced nitrogen under elevated CO 2 on Bt cotton high carbon-low nitrogen leaves (Chen et al. 2005). Our results suggest that for oilseed rape, elevated CO 2 would not have significant effects on the pest status of the diamondback moth, whereas elevated O 3 might have contradictory effects by causing reduced feeding but enhanced growth of this global herbivore. 6.4 Nontarget herbivore performance on non-bt and Bt oilseed rape under elevated CO 2 and temperature - aphid responses Studies on the effects of Bt plants on nontarget herbivores have commonly used two approaches: first-tier laboratory testing of direct toxicity to high doses of purified or plant-expressed Bt toxins (e.g. Obrist et al. 2006) and extensive field assessments of nontarget invertebrate abundance in comparison to non-bt fields (e.g. Head et al. 2005). A recent meta-analysis with data from 42 field experiments reported higher abundance of nontarget invertebrates in Bt cotton and maize fields compared to non- Bt fields managed with insecticides (Marvier et al. 2007). In the same analysis, a comparison of Bt fields to non-bt fields without any insect control, revealed that in Bt fields there were reductions in specific taxa. In addition to toxicity and biodiversity studies, experimentation on the impact of abiotic variation on Bt plants and their herbivore interactions in field exposure systems are clearly needed, since there is still very little knowledge (Wu et al. 2007a) of these responses in a wider scale. In this study, nontarget herbivore performance (i.e. development and reproduction of two aphid species) was equal on the Bt-transgenic and nontransgenic oilseed rape lines (Chapter 4). The aphids were particularly useful for assessing climate change effects, since they typically, although not unequivocally respond to elevated CO 2 and temperature (e.g. Bezemer & Jones 1998, Hughes & Bazzaz 2001, Stacey & Fellowes 2002). Aphids do not ingest significant amounts of Bt Cry1Ac toxin, this is firstly since they feed on phloem sap which contains minimal amounts of Bt toxin, and secondly, because they lack Bt Cry1Ac receptors, which excludes direct effects by this protein (Raps et al. 2001). Therefore the aim here was to assess potential plant composition-mediated effects under modified CO 2 and temperature conditions to reveal possible effects of compositionally changed feeding material Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008) 109

110 Sari J Himanen: Bt oilseed rape and climate change on aphid performance and reproduction on the non-bt and Bt lines. Neither the generalist species M. persicae nor the specialist B. brassicae showed differences in development, growth of young larvae or reproduction on Bt or non-bt lines despite the differences in C:N ratios between the plant types. B. brassicae was not significantly affected by either elevated CO 2 or temperature, whereas M. persicae had reduced developmental time, adult and progeny weight and higher cumulative reproduction under elevated temperature and reduced adult weight and cumulative reproduction with CO 2 elevation. Stacey & Fellowes (2002) previously reported larger B. brassicae individuals on elevated CO 2 - grown B. oleracea plants and lowered ratios of M. persicae:b. brassicae with CO 2 enhancement. Therefore, plant species -specific responses, potentially connected to differential composition changes are also expected. Based on the current study, the pest status of M. persicae might be predicted to increase on oilseed rape if subjected to temperature elevations. Elevated CO 2, on the other hand, could reduce their population growth. 6.5 Induction of direct and indirect defence by herbivory in non-transgenic and Bt-transgenic oilseed rape - the relation of feeding damage and defence induction with abiotic variation Both glucosinolates and volatile terpenoids have the tendency to increase quantitatively and qualitatively (for specific compounds) after herbivore damage (Paré & Tumlinson 1997, Bartlet et al. 1999). Whether target herbivory in Bt plants is sufficient to induce direct GS defences or indirect VOC-based defences, is important for the plants' ability to attract natural enemies and to defend themselves against resistant target herbivore individuals. Mechanical damage alone releases myrosinase from plant cells which hydrolyses GSs and releases volatile isothiocyanates, but herbivore damage may also induce the synthesis of indolic GSs in particular (Bartlet et al. 1999). Of the leaf VOCs, green leaf volatiles are released from leaf fatty acids nonselectively after damage, but terpenoid synthesis is more regulated and induction is dependant on the type of damage (Paré & Tumlinson 1997). Because Bt plants are less damaged by Bt-susceptible herbivores, they may also have reduced induction of emissions (Dean & De Moraes 2006, Ibrahim et al. 2008). Whether the type of feeding attempts seen on Bt plants are sufficient to increase GS concentrations has not been reported before. It was demonstrated that all GSs in this oilseed rape cultivar were equally induced in the Bt and non-bt lines except for 4-methoxy- 3-indolylmethyl-GS. This compound was a minor constituent of the GS profile, but it clearly requires extensive feeding damage for induction. Interestingly, in Arabidopsis, the concentration of this particular GS is increased after aphid feeding and its synthesis is not systematically upregulated, but the compound is converted from 3- indolylmethyl-gs (Kim & Jander 2007). 110 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008)

111 General discussion Of the volatile terpenoids the homoterpene DMNT and sesquiterpene (E,E)- farnesene were previously identified as less increased after target herbivore feeding (P. xylostella) on the Bt line than parent non-bt line plants (Ibrahim et al. 2008). This work (Chapter 3) verified this previous report. The difference in homoterpene induction is reasonable, since they have often been shown to be clearly induced by herbivory in Brassicas (Vuorinen et al. 2004, Pinto et al. 2007a, Ibrahim et al. 2008). However, (E,E)- farnesene is a sesquiterpene, which are synthesized in the cytosol and have a precursor, farnesyldifosfate, in common with the homoterpenes. This is in contrast to geranyldifosfate-originating monoterpenes synthesized in plastids (Dudareva et al. 2006). Based on the results, it can be suggested that sesqui- and homoterpenes are more dependant on sufficient feeding damage in oilseed rape for their induction than monoterpenes. 6.6 The tritrophic system oilseed rape - diamondback moth - Cotesia vestalis and its vulnerability against CO 2 or O 3 elevations Effects of Bt toxin uptake from plants by predators or parasitoids of herbivores have recently been increasingly studied (e.g. Obrist et al. 2006, Chen et al. 2008, Torres & Ruberson 2008, Wei et al. 2008). Previous studies have mostly reported effects through reduced host quality (e.g. Schuler et al. 1999, 2003, Ferry et al. 2006, Chen et al. 2008), not direct toxicity. A specialized tritrophic system much used for laboratory studies on Brassicas (P. xylostella-c. vestalis (formerly known as C. plutellae)) (Schuler et al. 1999, Vuorinen et al. 2004, Pinto et al. 2007a) was selected for the assessment of studying the functioning of multitrophic communication (Chapter 3). The parasitoids were able to orientate towards host-damaged Bt plants, in spite of reduced damage and emissions, under control conditions and under elevated CO 2 (Chapter 3). Elevated CO 2 with increased herbivore-induced VOC emissions did not negatively affect the attraction of C. vestalis to host-damaged Bt or non-bt oilseed rape tested against intact control plants. Elevated O 3 also failed to disturb orientation of the parasitoid to hostdamaged non-bt plants despite its effective degradation of certain (but not all) terpenoids. Bt plants with reduced feeding damage were not as attractive and the parasitoids did not show preference for either intact or host-damaged Bt plants under elevated O 3. Therefore, it could be stated that elevated O 3 has the potential to affect specialized tritrophic signalling in conjunction with low inducible emissions. However, in the case of non-transgenic oilseed rape, and potentially on other Brassicas as well (Pinto et al. 2007a with similar finding on cabbage), C. vestalis can probably use cues other than O 3 -sensitive terpenoids. Alternatively, the reduction in their concentrations might not be large enough to disturb the sensitive olfactory orientation of C. vestalis. In general, much is still to be revealed on the functioning of tritrophic systems in natural agroecosystems, and this work only gives Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008) 111

112 Sari J Himanen: Bt oilseed rape and climate change an approximation of the potential impact of elevated CO 2 or O 3 on the particular specialized interaction studied. Attraction of parasitoids to Bt target hosts on Bt plants is a many-sided question of benefit versus harm: it is advantageous to attract parasitoids that could control resistant larvae in particular, but the parasitoid cannot develop in Bt-sensitive larvae (Schuler et al. 2003), which means that being attracted to Bt plants might eventually be destructive for the parasitoid population. However, most pest control practices have negative effects on parasitoid populations and more harmful pesticides may also cause direct mortality (Romeis et al. 2006, Wolfenbarger et al. 2008). This has to be acknowledged when combining target herbivore-mediated effects on parasitoids and attraction of natural enemies in practice. Although the comparison of host-damaged non-bt versus Bt plants was not included in this study, it is highly likely based on previous work (Schuler et al. 1999, Dean & De Moraes 2006) that C. vestalis would also have been attracted to the plants having higher leaf damage levels in those comparisons. However, a good herbivore to use in this kind of tests with abiotic variations would be Bt-resistant P. xylostella, thus excluding the target controlling effect of the trait. In addition, a tritrophic system not directly affected by Bt toxin such as an aphid-parasitoid -based interaction study (Du et al. 1996) could help in assessing Bt versus non-bt plant effects on VOC signalling based on nontarget herbivores. In this current research project, a Bt oilseed rape - M. persicae/b. brassicae - Aphidius colemani -tritrophic system was used in an attempt to make a comparison to the target herbivore DBM. The problem was that aphids induced negligible emissions from oilseed rape and the parasitoid failed to detect host-damaged plants in our study system even in control conditions, revealing the need to further develop the study system to effectively assay that interaction. 6.7 Methodological considerations One of the main methodological considerations of the current work is that all data included in this thesis have been collected from plants grown in growth chambers. The practical reason for this is the use of genetically modified plant material of a species with a crosshybridizing wild relative Brassica species occurring in nature in Finland. This resulted in the rationale to rule out the possibility of conducting field testing even on vegetative-stage plants. It also led to the only option available for such CO 2 and O 3 exposures being a growth chamber study, and considering the nature of the plant material used (species with a fitnessenhancing transgenic trait and having wild relatives capable of hybridization) and the premiere nature of the study, this can be thought of as a good choice. However, with several abiotic factor treatments included in each of the experiments, there inevitably arises the issue of having a sufficient number of growth chambers 112 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008)

113 General discussion available simultaneously for all these treatments and the need to control for the so called "chamber effect". Attempts to eliminate potential chamber specific differences were made by rotating the treatments between the four chambers at weekly intervals, as used previously (e.g. Johns & Hughes 2002, Vuorinen et al. 2004, Bidart-Bouzat et al. 2005). As a further solution to this problem, the replication of the experiments in time was used as a random factor in statistical analyses. Even though this did help in proving the results to be accurate with similar replications, an ideal situation would have been to have a sufficient number of identical growth chambers to use as independent replicates simultaneously. Another limitation of growth chamber studies is that it is difficult to state whether similar phenomena would be seen in field conditions (Ainsworth et al. 2008). It has been seen from previous work that chamber studies having plants in pots rather than growing with unlimited soil area around them can overestimate effects of abiotic factors on their growth (e.g. Long et al. 2006). In addition, the light levels used in growth chambers cannot be as high as is typical in field conditions (Franzaring et al. 2008), and there are no interfering environmental factors such as wind and other physical constraints. Therefore, it cannot be stated whether the effects observed also persist in field conditions and how they relate to the natural variation in these parameters when measured outdoors. On the other hand, growth chambers enable good control over interfering factors, and the accurate treatment effects at physiological, biochemical and molecular levels are better seen (Ainsworth et al. 2008). Therefore growth chamber studies are also well warranted and give invaluable information about the effects of abiotic factors on plants, not replacing but supporting larger-scale field evaluations. It has to be noted that the research of this thesis could only include one transgenic line, and the basis was to study impacts of Bt toxin production itself on ecologically relevant phenomenon. Therefore, the results cannot be regarded as assessing the use of this kind of plant transformants commercially; a process where multiple independent transformation events are compared to obtain the general status of their variation in relation to the nontransgenic parent lines. This would show potential insertional effects, i.e. the position of the transgene in the genome. However, the choice of the plant line was not at all random, but based on extensive previous work with multiple transgenic Bt events (Halfhill et al. 2001). Furthermore, the line also had a gfp marker gene in addition to the Bt Cry1Ac gene, so this would have to be more emphasized if assessing the use of this kind of plant more widely in agriculture. It is also worth mentioning that all studies in this thesis were done on vegetativegrowth stage plants, which do not allow assessing the phenological effects of the abiotic factors over the complete life cycle Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008) 113

114 Sari J Himanen: Bt oilseed rape and climate change of the plants (Franzaring et al 2008). However, the main aim of the current study was to provide results useful for assessing the effects of Bt Cry1Ac production per se in transgenic oilseed rape with emphasis on herbivore dynamics, secondary metabolism and multitrophic food web effects. With regard to the specific methods used, the synthesis of the secondary compound groups studied is highly dynamic and affected by various abiotic and biotic factors (reviewed by e.g. Dudareva et al and Halkier & Gershenzon 2006). It would require extensive measurements with multiple time-point analyses to completely cover their variable responses to the studied environmental effects. Most data reported are from selected time-points and are incorporated from multiple experiments with different kinds of exposures (such as with chronic and acute O 3 for GSs). This aims at representing the most predominant and important effects of elevated CO 2 and O 3 during the vegetative stage. collect emissions under the corresponding CO 2 and O 3 levels that were used during the growth of these plants by establishing systems with controlled atmospheres (Pinto et al. 2007a). Therefore our results mimic the natural situation more efficiently. The method used in GS analysis has been widely used and the identification of the compounds was further verified with additional runs of representative samples. However, relatively high differences in the concentrations of the main GSs especially between the individual experiments still existed. Substantial variation in GS concentrations even within a day has been reported as evidence of their high variability (Rosa et al. 1994). Importantly, the treatment effects were similar in these experiments, despite the different basic state values. The VOC collection methodology included sampling from both soil-detached and potted plants, with different air spaces used and these clearly resulted in differences in emissions quantified with the two techniques. Ibrahim et al. (2008) and Gols et al. (2007) have recently documented that even small changes in environmental conditions and seasonality are factors that affect VOC emissions from plants in otherwise identical experiments. A positive thing is that we were able to 114 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008)

115 Table 2. Summary of the results. Chapter numbers are indicated in parentheses for parameters studied in several chapters. Parameters studied Chapter(s) Effect of Bt transformation Vegetative growth 2, 3, 4 Reduction in biomass gain (3, 4). Effect of elevated CO2 Effect of elevated temperature Increase in biomass gain (2, 3, 4). Effect of elevated O3 Interaction effects No effect (4). Decrease in biomass gain (2, 3). No effects. Carbon-nitrogen ratio 4 Decreased C:N ratio. Increased C:N ratio. No effect. N.a. Elevated temperature decreased under ambient CO2, but increased under elevated CO2. Photosynthesis 4 No effect. Decrease in elevated CO2- grown plants measured under ambient CO2 on day 29, but no consistent decrease. Increased levels when measured under elevated CO2. Soluble sugars and starch 4 No effect. Decrease in glucose, increase in starch Chlorophyll pigments 4 No effect on Chl a or Chl b, reduction in carotenoids. Decrease in elevated temperature -grown plants. Increase in glucose, decrease in starch concentration. concentration. No effects. Reduction in Chl a/b ratio. Bt toxin concentration 4,5 - No effect (4). No effect (4). Increased toxin concentration (5). Glucosinolates 2 No effect. Reduction in 3-IM-GS, indolic GSs and total GSs. N.a. No effects. N.a. Temperature effect more prevalent under ambient CO2. N.a. Increase by temperature under ambient CO2, reduction by temperature under elevated CO2. N.a. Reduction in 3-IM-GS and indolic GSs. Increase in 2-PE-GS. No effects. Herbivore induction of 3-IM-GS lower under elevated O3. Higher increase in 4-methoxy- 3IM-GS after herbivory in non-bt plants.

116 Table 2 continues. Parameters studied Chapter(s) Effect of Bt transformation Volatile terpenoids 3 Increased -3-carene emission in the O3 experiment, no effects in the CO2 experiment. P. xylostella performance P. xylostella feeding damage M. persicae performance B. brassicae performance 2, 5 Reduced mean relative growth rate (2, 5). 3, 5 Reduced feeding (3, 5). Effect of elevated CO2 Effect of elevated temperature Increased emissions for most individual terpenoids and total emission. 4 No effect. Reduction in adult weight and cumulative reproduction. Effect of elevated O3 Interaction effects N.a. Reduction in sabinene, - 3-carene and total terpenoid emissions. N.a. N.a. No effect (5). No effects. Lower increases in - thujene, (E)-DMNT and (E,E)- -farnesene after herbivory in Bt than non-bt plants. Higher increases in certain terpenoids after herbivory under elevated CO2. No effect (3). N.a. Reduced feeding (5). O3 reduced feeding only in non-bt plants (5). Decreased developmental time, adult and progeny weight and increase in cumulative reproduction. N.a. No effects. 4 No effect. No effect. No effect. N.a. No effects. C. vestalis attraction 3 No effect. Successful attraction to hostdamaged plants. No effect. Successful attraction to host-damaged plants. N.a. No effect on non-bt plants (successful attraction). Disturbance of attraction to hostdamaged Bt plants. O3 effect only on Bt plants (having lower feeding damage). Abbreviations: N.a., not assayed; Chl, chlorophyll; 3-IM-GS, 3-indolylmethyl-glucosinolate; 2-PE-GS, 2-phenylethyl-glucosinolate; (E)-DMNT, (E)-4,8- dimethyl-1,3,7-nonatriene.

117 General discussion 6.8 Conclusions 1. The results of this study (Table 2) demonstrated that Bt toxin production in oilseed rape did not interfere with allocation to growth or production of secondary compounds, i.e. GSs and VOCs, under elevated CO 2 or O 3 exposure. 2. Although Bt plants had reduced levels of P. xylostella feeding, this was effective at increasing concentrations of most GSs and VOCs. Thus, these endogenic defence systems were not severely compromised by the transgenic trait despite limited target herbivory. 3. Constitutive GS concentrations in leaves were reduced under elevated CO 2, which might lead to higher susceptibility of oilseed rape to generalist herbivores. In contrast, the constitutive VOC emissions as well as induction of these direct and indirect chemical defences were rather increased under CO 2 enhancement. 4. Observed changes in concentrations of individual GSs after chronic and acute O 3 and interactions of O 3 and herbivory on GS induction might affect plant-herbivore interactions especially with future O 3 episodes, and clearly deserve further study. 5. Elevated O 3 may disturb tritrophic signalling, especially in Bt plant systems, because of reduced target feeding and ability of O 3 to degrade terpenoids. However, this finding must be placed into a correct context, so that the desired effects caused by the trait are compared to other pest control methods (such as those of insecticide use) as regarding any meaningful effects in agroecosystems. 6. The higher nitrogen concentration in leaves of Bt oilseed rape plants in comparison to non-bt plants at the vegetative stage might affect its susceptibility to Bt-resistant herbivores benefiting from increased nitrogen. However, no indications for this were detected with two nontarget aphid species studied. 7. No indication of reduction in effectiveness of the insect resistance trait under elevated CO 2, elevated temperature or their combination was observed on Bt oilseed rape. 8. Elevated CO 2 and temperature have a high potential to affect the primary metabolism of oilseed rape with probable interactive effects. The generalist aphid M. persicae might increase its status as a pest under elevated temperatures more than the specialist aphid B. brassicae on oilseed rape. 9. The observed increase in Bt toxin concentration of oilseed rape leaves with elevated O 3 indicates the need for assessing fluctuations of Bt toxin proteins over the complete life cycle of oilseed rape (and also other Bt plants) under O 3 exposure. This would enable determining whether this kind of pattern is limited to the vegetative growth stage or is more pronounced at other growth stages, and whether it could affect Bt dosing to nontarget and target herbivores. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008) 117

118 Sari J Himanen: Bt oilseed rape and climate change 10. The lower feeding damage but equal mean relative growth rate of P. xylostella on O 3 -grown non-bt oilseed rape leaves suggests that the quality of oilseed rape leaves as food is better for P. xylostella under O 3 exposure. Reduced feeding might be a direct response to O 3 or caused by composition changes in plant leaves by O Future prospects The studies included in this thesis raise a wealth of interesting aspects for future studies. Therefore its aim to serve in drawing attention to the possible importance to include assessments of abiotic factor effects on transgene stability, crop performance and plant-insect interactions in future ecological risk assessments of genetically modified crop plants seems well achieved. Due to the methodological limitations of the study, this work cannot be used directly as a basis for general GM safety assessments. Follow-up studies incorporating data collection throughout the complete life cycle of oilseed rape under these abiotic variations would be essential to assess phenological patterns (and especially Bt toxin fluctuations). In addition, studying combined effects of elevated CO 2 and O 3 (since their effects were partly antagonistic) on secondary compound concentrations, performance of nontarget herbivores potentially taking advantage of increased nitrogen content of Bt plants and below-ground responses of Bt plants under elevated CO 2 or O 3 would be interesting. The ability of oilseed rape to hybridize with wild Brassicas also warrants future assessments of abiotic factors, such as O 3, potentially affecting the performance of transgenic hybrids and backcrosses (Halfhill et al. 2005) through differential O 3 sensitivity of oilseed rape and the natural Brassicas and to reveal whether the Bt trait would affect this O 3 sensitivity. Since oilseed rape plants are known to have large floral volatile emissions (Müller et al. 2002), a comparative VOC analysis of Bt and non-bt oilseed rape at flowering could be informative and serve well as a comparison for assessing potential attraction of pollinators on Bt versus non- Bt plants. Furthermore, the mechanisms behind the observed O 3 effect on concentrations of specific GSs would certainly benefit from transcriptomic studies. In addition, there are still too few studies combining secondary metabolite analysis and transcriptomic responses when addressing interactions of herbivore feeding and O 3 exposure as factors potentially affecting plant hormone signalling pathways antagonistically. B. napus could serve in discovering GS and terpenoid pathway activation and regulation because of extensive previous work on the related crucifer model plant Arabidopsis thaliana. Methodologically, a field-exposure study including multiple transgenic plant lines and treatments for elevated CO 2 and temperature, elevated O 3 and their combination together with herbivore introductions would be a highly desirable 118 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008)

119 General discussion experimental set-up in further comparisons. Simultaneous collection of herbivore performance, physiological, metabolomic, transcriptomic and proteomic data would allow a sufficiently thorough investigation of the complex interactions involved in this model system. For revealing the responses of GM plant types to abiotic variations more widely, transgenic lines with other traits such as herbicide resistance, drought tolerance or stacked properties, might be good comparisons. The work included in this thesis set a very good basis for this type of comprehensive approach on an agriculturally important crop plant to be utilized further. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 248: (2008) 119

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125 Kuopio University Publications C. Natural and Environmental Sciences C 218. Madetoja, Elina. Novel process line approach for model-based optimization in papermaking p. Acad. Diss. C 219. Hyttinen, Marko. Formation of organic compounds and subsequent emissions from ventilation filters p. Acad. Diss. C 220. Plumed-Ferrer, Carmen. Lactobacillus plantarum: from application to protein expression p. Acad. Diss. C 221. Saavalainen, Katri. Evaluation of the mechanisms of gene regulation on the chromatin level at the example of human hyaluronan synthase 2 and cyclin C genes p. Acad. Diss. C 222. Koponen, Hannu T. Production of nitrous oxide (N 2 O) and nitric oxide (NO) in boreal agricultural soils at low temperature p. Acad. Diss. C 223. Korkea-aho, Tiina. Epidermal papillomatosis in roach (Rutilus rutilus) as an indicator of environmental stressors p. Acad. Diss. C 224. Räisänen, Jouni. Fourier transform infrared (FTIR) spectroscopy for monitoring of solvent emission rates from industrial processes p. Acad. Diss. C 225. Nissinen, Anne. Towards ecological control of carrot psyllid (Trioza apicalis) p. Acad. Diss. C 226. Huttunen, Janne. Approximation and modellingerrors in nonstationary inverse problems p. Acad. Diss. C 227. Freiwald, Vera. Does elevated ozone predispose northern deciduous tree species to abiotic and biotic stresses? p. Acad. Diss. C 228. Semenov, Dmitry. Distance sensing with dynamic speckles p. Acad. Diss. C 229. Höytö, Anne. Cellular responses to mobile phone radiation: proliferation, cell death and related effects p. Acad. Diss. C 230. Hukkanen, Anne. Chemically induced resistance in strawberry (Fragaria ananassa) and arctic bramble (Rubus arcticus): biochemical responses and efficacy against powdery mildew and downy mildew diseases p. Acad. Diss. C 231. Hanhineva, Kati. Metabolic engineering of phenolic biosynthesis pathway and metabolite profiling of strawberry (Fragaria ananassa) p. Acad. Diss. C 232. Nissi, Mikko. Magnetic resonance parameters in quantitative evaluation of articular cartilage: studies on T₁ and T₂ relaxation time p. Acad. Diss.