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.
| Identifer | oai:union.ndltd.org:ADTP/279398 |
| Creators | Emily McCallum |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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