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.

<div>To compensate for their sessile existence, plants synthesize and emit a wide diversity of volatile organic compounds (VOCs) that serve important biological functions pertaining to defense, reproduction, and plant-plant signaling. In addition to their importance in plant secondary metabolism, VOCs are used as fragrances, flavoring agents, and therapeutics. Plant metabolic engineering has successfully been implemented towards the design of value-added plants with enhanced defense, improved aroma and flavor, and increased production of specialty chemicals. However, rational design requires rigorous characterization of the mechanisms controlling metabolic fluxes in a network. Thus, the major aims of this dissertation are to study biological and physical mechanisms controlling the synthesis and emission of plant VOCs. This dissertation focuses on (i) modeling 2-phenylethanol biosynthesis in Arabidopsis and (ii) characterization of the biophysical properties of flower cuticles with respect to the emission of VOCs.</div><div><br></div><div>2-Phenylethanol (2-PE) is a naturally-occurring aromatic volatile with properties that make it a candidate oxygenate for petroleum-derived gasoline. In plants, 2-PE biosynthesis competes with the phenylpropanoid pathway for the common precursor L-phenylalanine (Phe). The phenylpropanoid pathway directs up to 30% of fixed carbon towards the production of lignin, a major constituent of plant cell walls that renders biomass recalcitrant to pretreatment techniques impeding the economical production of biofuels. An initial genetic engineering approach was proposed, whereby a portion of the carbon flux towards lignin production is diverted towards the biosynthesis 2-PE. Transgenic Arabidopsis thaliana expressing enzymes catalyzing the biosynthetic steps from Phe to 2-PE were generated. Excised stems from transgenic Arabidopsis were supplied 13C6-ring labeled Phe, and isotopic enrichment of downstream metabolites were quantified to calculate fluxes. By combining flux measurements with predictions from a kinetic model of the Phe metabolic network, we hypothesized that 2-PE biosynthesis in transgenic Arabidopsis was limited by endogenous pools of cytosolic Phe. Multiple independent genetic strategies were proposed based on model-guided predictions, such as inducing Phe hyper-accumulation, reduction of the activity of the competing phenylpropanoid pathway, and sequestering the 2-PE biosynthesis pathway in plastids. Combining kinetic modeling with time-course in vivo metabolomics led to successful rational engineering of 2-PE accumulating plants.</div><div><br></div><div>The plant cuticle is the physical interface between the flower and its surrounding environment. Passage of VOCs through the cuticle is driven solely by diffusion and is thus dependent on the cuticle physicochemical properties. Wax compounds in the cuticular matrix self-assemble into a multiphase system of crystalline and amorphous regions, where their relative amounts and arrangements govern VOC diffusion. To investigate the effect of wax composition on the crystallinity and permeability of the cuticle, we characterized the cuticular waxes of Petunia hybrida petals using GC-MS, FTIR, DSC, and XRD. Petal waxes were found to be enriched with long-chain hydrocarbons forming semi-crystalline waxes localized on petal surfaces. A ternary system of wax compounds was proposed as a model for petal cuticles to investigate the effect of wax composition on cuticle crystallinity and permeability. Atomistic simulations of VOC displacement in waxes of varying chemical composition were performed at 298 K and 1 bar under NPT conditions to estimate diffusivities. Wax anisotropy was found to be highly dependent on the elongation of methylene chains, restricting the molecular diffusion path. Changes in crystalline symmetry were found to have measurable effects on VOC diffusion. Simulations of compositional variants of the model cuticle shows that changes in relative crystallinity exert differential control on the dynamics of VOC emissions.</div><div><br></div><div>To directly determine the effect of the cuticle on VOC emissions in petunia flowers, the wax exporter PhABCG12 was silenced using RNA interference, resulting in flowers with thinner cuticles. However, VOC emissions were found to have significantly decreased in transgenic flowers relative to the wild-type control. Dewaxing wild-type and transgenic petunia revealed that the cuticle serves as a site of VOC build-up during emission, and deficient coverage limits the extent to which compounds can accumulate. In addition, the cuticle was found to impart differing levels of mass transfer resistance for certain VOCs, suggesting that the cuticle controls the dynamics of VOC emissions. Taken together, petal cuticles provide an additional layer of regulation in emission of VOCs from plants.</div><div><br></div>

Flowering plants display remarkable phenotypic diversity, especially in reproductive structures, much of which is thought to be associated with pollination by animals. Pollination syndromes are collections of floral traits (e.g. flower colour, shape, odour) that are associated with a plant attracting particular functional groups of animal pollinators. We explored the extent to which traits associated with the moth pollination syndrome translated into pollination by moths in the Pacific coast dune endemic Abronia umbellata and found mixed results: in one year of study, there was no difference in seed set by day- vs. night-pollinated inflorescences, but in another year of study, night-pollinated inflorescences set significantly more seed than those pollinated during the day. We integrate this work with tests of pollen and resource limitation of seed production and with seed set surveys of natural populations to address proximate and ultimate causes of low seed set, finding low rates of pollinator visitation, high pollen limitation of seed production in all populations studied, and no evidence of endogenous resource limitation of seed production. We propose that “excess” flowers may be functionally male, serving to increase outcross siring success. The transition from self-incompatibility and obligate outcrossing to self-compatibility and predominant selfing is the most common evolutionary transition among the flowering plants and traits associated with outcrossing may become reduced across such shifts, potentially through the action of natural selection, especially if pollinators are also herbivores, or if the signals that pollinators use to locate flowers are also used by herbivores. We examined the reduction of attractive visual and olfactory floral traits in A. umbellata across a shift from outcrossing to selfing and found a reduction of all floral traits considered. We found that floral volatile emissions were reduced more strongly than flower size or floral display (number of flowers per inflorescence), but there was no evidence of an ecological cost associated with conspicuousness: we did not find reduced leaf herbivory among selfers relative to outcrossers. / Thesis (Master, Biology) -- Queen's University, 2012-08-30 19:46:23.663