A wound- and insect-inducible expression system for transgenic plants was developed. Specifically, I demonstrate wound- and insect-inducible, localized gene expression driven by the potato proteinase inhibitor II (pinII)-promoter in transformed Arabidopsis, tobacco and white spruce. As reporter and target genes driven by the pinII-promoter, I used the GUS gene and a terpenoid synthase gene, respectively. In addition, I found that the pinII-promoter drives trichome-specific, systemically-induced gene expression in tobacco and Arabidopsis. Finally, I demonstrate that the pinII–promoter, when transformed into Arabidopsis, is extremely sensitive to subtle, low-impact stress treatment. This latter finding prompted me to use, in the second part of my thesis, the pinII-promoter in conjunction with GUS reporter gene expression to test if intact Arabidopsis plants can respond to exposure to monoterpene volatiles. My experiments using the pinII–promoter GUS reporter system clearly established that Arabidopsis plants respond to the exposure of the monoterpene volatiles tested. It is thought that monoterpenes and other volatiles can act as airborne signals between plants under stress or between distant parts of the same plant. At the outset of my thesis research, and to some extent still today, the concept of plant-plant signalling with volatiles has been met with scepticism. After establishing that Arabidopsis plants do respond in a laboratory setting to certain monoterpene volatiles, I further tested the extent of the response at the transcriptome level using a 30 K microarray platform. The gene expression analysis revealed several hundred transcripts that respond with a change of abundance in response to treatment of intact Arabidopsis plants with the monoterpenes ocimene or myrcene. Many of these transcripts were annotated as stress and defense genes including genes involved in octadecanoid signaling. Real-time PCR analyses of octadecanoid mutants confirmed a role for octadecanoid signaling in the response to the monoterpene ocimene. In addition, treatment with ocimene or myrcene caused increased levels of methyl jasmonate (MeJA) in Arabidopsis rosette leaves. However, plants treated with monoterpene prior to wounding or feeding by cabbage looper did not reveal any significant priming effect for these pre-treatments.

A wound- and insect-inducible expression system for transgenic plants was developed. Specifically, I demonstrate wound- and insect-inducible, localized gene expression driven by the potato proteinase inhibitor II (pinII)-promoter in transformed Arabidopsis, tobacco and white spruce. As reporter and target genes driven by the pinII-promoter, I used the GUS gene and a terpenoid synthase gene, respectively. In addition, I found that the pinII-promoter drives trichome-specific, systemically-induced gene expression in tobacco and Arabidopsis. Finally, I demonstrate that the pinII–promoter, when transformed into Arabidopsis, is extremely sensitive to subtle, low-impact stress treatment. This latter finding prompted me to use, in the second part of my thesis, the pinII-promoter in conjunction with GUS reporter gene expression to test if intact Arabidopsis plants can respond to exposure to monoterpene volatiles. My experiments using the pinII–promoter GUS reporter system clearly established that Arabidopsis plants respond to the exposure of the monoterpene volatiles tested. It is thought that monoterpenes and other volatiles can act as airborne signals between plants under stress or between distant parts of the same plant. At the outset of my thesis research, and to some extent still today, the concept of plant-plant signalling with volatiles has been met with scepticism. After establishing that Arabidopsis plants do respond in a laboratory setting to certain monoterpene volatiles, I further tested the extent of the response at the transcriptome level using a 30 K microarray platform. The gene expression analysis revealed several hundred transcripts that respond with a change of abundance in response to treatment of intact Arabidopsis plants with the monoterpenes ocimene or myrcene. Many of these transcripts were annotated as stress and defense genes including genes involved in octadecanoid signaling. Real-time PCR analyses of octadecanoid mutants confirmed a role for octadecanoid signaling in the response to the monoterpene ocimene. In addition, treatment with ocimene or myrcene caused increased levels of methyl jasmonate (MeJA) in Arabidopsis rosette leaves. However, plants treated with monoterpene prior to wounding or feeding by cabbage looper did not reveal any significant priming effect for these pre-treatments.

A wound- and insect-inducible expression system for transgenic plants was developed. Specifically, I demonstrate wound- and insect-inducible, localized gene expression driven by the potato proteinase inhibitor II (pinII)-promoter in transformed Arabidopsis, tobacco and white spruce. As reporter and target genes driven by the pinII-promoter, I used the GUS gene and a terpenoid synthase gene, respectively. In addition, I found that the pinII-promoter drives trichome-specific, systemically-induced gene expression in tobacco and Arabidopsis. Finally, I demonstrate that the pinII–promoter, when transformed into Arabidopsis, is extremely sensitive to subtle, low-impact stress treatment. This latter finding prompted me to use, in the second part of my thesis, the pinII-promoter in conjunction with GUS reporter gene expression to test if intact Arabidopsis plants can respond to exposure to monoterpene volatiles. My experiments using the pinII–promoter GUS reporter system clearly established that Arabidopsis plants respond to the exposure of the monoterpene volatiles tested. It is thought that monoterpenes and other volatiles can act as airborne signals between plants under stress or between distant parts of the same plant. At the outset of my thesis research, and to some extent still today, the concept of plant-plant signalling with volatiles has been met with scepticism. After establishing that Arabidopsis plants do respond in a laboratory setting to certain monoterpene volatiles, I further tested the extent of the response at the transcriptome level using a 30 K microarray platform. The gene expression analysis revealed several hundred transcripts that respond with a change of abundance in response to treatment of intact Arabidopsis plants with the monoterpenes ocimene or myrcene. Many of these transcripts were annotated as stress and defense genes including genes involved in octadecanoid signaling. Real-time PCR analyses of octadecanoid mutants confirmed a role for octadecanoid signaling in the response to the monoterpene ocimene. In addition, treatment with ocimene or myrcene caused increased levels of methyl jasmonate (MeJA) in Arabidopsis rosette leaves. However, plants treated with monoterpene prior to wounding or feeding by cabbage looper did not reveal any significant priming effect for these pre-treatments. / Science, Faculty of / Botany, Department of / Graduate

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Terpenes are important specialised metabolites produced by all organisms. Plants produce the greatest diversity of terpenoid compounds, which function in a variety of crucial processes including regulation of growth and development, energy production and plant-insect communication, including pollinator attraction and prevention of herbivore damage. Isopentenyl diphosphate (IPP), the building block for all terpenoid compounds, is synthesised in plants via two unique terpene synthesis pathways located in the plastids and the cytosol, and the regulation of these pathways is still not well understood. The aim of this research was to (1) modify and study the regulation of floral volatile production in Nicotiana tabacum (tobacco) by altering the expression of various enzymes in the terpene biosynthesis pathway and (2) determine the role of specific volatile compounds in floral odour blends in feeding and oviposition behaviours of Helicoverpa armigera, a polyphagous moth of widespread agricultural importance. Expression levels of several enzymes in the terpene biosynthetic pathway were altered by genetic modification in order to modify terpene volatile emissions produced by flowers of N. tabacum. Genes chosen for overexpression were cloned from several species and RNAi hairpins were constructed from gene fragments amplified from tobacco flower cDNA. Transgenic plants were produced by Agrobacterium-mediated transformation, and lines with high levels of transgene expression selected for analysis. The flower-specific Antirrhinum majus chalcone synthase promoter was chosen to control gene expression in transgenic lines in order to avoid the potentially deleterious effects of widespread disruption to terpene biosynthesis. Floral volatiles were sampled using two methods; solid phase microextraction, a highly sensitive technique able to detect even trace levels of volatile compounds in headspace samples, and Tenax sampling, a robust and replicable method to quantify volatile emissions. All floral headspace samples were analysed by gas chromatography-mass spectrometry. Floral volatile analysis determined that wild type Ti68 tobacco flowers emit a simple blend of floral volatiles, with only linalool, a monoterpene, and β-caryophyllene, a sesquiterpene, detected by both sampling methods. Volatile emissions were not subject to temporal regulation, but changes in the floral odour blend were detected during flower development. Overexpression of the plastidic terpene biosynthesis genes 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) and geranyl diphosphate synthase did not affect volatile production, however increased farnesyl diphosphate synthase expression in the cytosol surprisingly caused an increase in linalool emissions, synthesised in the plastids. Downregulation of DXR resulted in an albino phenotype affecting all young leaves, the upper stem and the sepals in the most severely affected lines. A significant three-fold decrease in floral linalool emissions, and a nine-fold reduction of both linalool and β-caryophyllene retained within floral tissues was observed in the transgenic lines. In feeding behaviour tests, male and virgin female H. armigera moths did not discriminate between wild type and DXR knockdown flowers at close-range, despite the significant difference in linalool emissions. Expression of an (E)-β-ocimene synthase gene controlled by the CHS promoter did not result in any transgenic plants emitting the novel monoterpene, (E)-β-ocimene. Significant problems with seed germination suggested that (E)-β-ocimene may cause embryo lethality in these lines. However, overexpression of a heterologous (S)-linalool synthase under control of the constitutive cauliflower mosaic virus 35S promoter resulted in a significant two-fold increase in volatile linalool, and β-glycosidase assays confirmed sequestration of a glycosylated linalool derivative in floral tissues. Oviposition preference tests with mated female H. armigera moths indicated a significant preference for egg-laying on wild type flowers compared to flowers with increased linalool production. The results of this research, and previous studies of volatile production in transgenic tobacco, indicate that IPP precursor exchange occurs predominantly in one direction from the cytosol to the plastids, at least under the stress caused by alterations in pathway flux. Regulation of the cytosolic terpene biosynthetic pathway upstream of IPP synthesis appears to be less strictly controlled than the plastidic pathway. Insect behavioural assays support the findings of recent studies in other moth species, and suggest that close-range feeding attraction of H. armigera may be more strongly influenced by visual cues, whereas odour cues, including contact chemoreception, play a more important role in oviposition preferences. The increase in knowledge of the olfactory contribution toward insect-plant communication demonstrated here, and from future work, will lead to improved management of pest species in agricultural and ecological settings.

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