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Investigations into the molecular evolution of plant terpene, alkaloid, and urushiol biosynthetic enzymesWeisberg, Alexandra Jamie 09 July 2014 (has links)
Plants produce a vast number of low-molecular-weight chemicals (so called secondary or specialized metabolites) that confer a selective advantage to the plant, such as defense against herbivory or protection from changing environmental conditions. Many of these specialized metabolites are used for their medicinal properties, as lead compounds in drug discovery, or to impart our food with different tastes and scents. These chemicals are produced by various pathways of enzyme-mediated reactions in plant cells. It is suspected that enzymes in plant specialized metabolism evolved from those in primary metabolism. Understanding how plants evolved to produce these diverse metabolites is of primary interest, as it can lead to the engineering of plants to be more resistant to both biotic and abiotic stress, or to produce more complex small molecule compounds that are difficult to derive.
To that end, the first objective was to develop a schema for rational protein engineering using meta-analyses of a well-characterized sesquiterpene synthase family encoding two closely-related but different types of enzymes, using quantitative measures of natural selection on amino-acid positions previously demonstrated as important for neofunctionalization between two terpene synthase gene families. The change in the nonsynonymous to synonymous mutation rate ratio (dN/dS) between these two gene families was large at the sites known to be responsible for interconversion. This led to a metric (delta dN/dS) that might have some predictive power. This natural selection-oriented approach was tested on two related enzyme families involved in either nicotine/tropane alkaloid biosynthesis (putrescine N-methyltransferase) or primary metabolism (spermidine synthase) by attempting to interconvert a spermidine synthase to encode putrescine N-methyltransferase activity based upon past patterns of natural selection. In contrast to the HPS/TEAS system, using delta dN/dS metrics between SPDS and PMT and site directed mutagenesis of SPDS did not result in the desired neofunctionalization to PMT activity.
Phylogenetic analyses were performed to investigate the molecular evolution of plant N-methyltransferases involved in three alkaloid biosynthetic pathways. The results from these studies indicated that unlike O-MTs that show monophyletic origins, plant N-MTs showed patterns indicating polyphyletic origins.
To provide the foundation for future molecular-oriented studies of urushiol production in poison ivy, the complete poison ivy root and leaf transcriptomes were sequenced, assembled, and analyzed. / Ph. D.
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Sclareol biosynthesis in clary sage and its regulation / Biosynthèse du sclaréol et sa régulation chez la sauge sclaréeChalvin, Camille 12 July 2019 (has links)
Le sclaréol est un diterpène produit par les organes floraux de la sauge sclarée (Salvia sclarea, Lamiaceae). Il est utilisé en parfumerie pour l’hémisynthèse de l’ambroxide, une substance caractérisée par une odeur ambrée et une grande capacité de fixation des parfums. L’augmentation de la demande mondiale en sclaréol stimule actuellement les tentatives d’accroître le rendement de la production de sclaréol à partir de la sauge sclarée. L’objectif du travail présenté dans ce manuscrit était d’améliorer notre compréhension de la biosynthèse du sclaréol et de sa régulation chez la sauge sclarée, afin de mettre en évidence des stratégies d’augmentation du contenu en sclaréol de la sauge sclarée. L'analyse de la surface des calices de sauge sclarée par imagerie par spectrométrie de masse suggère que le sclaréol est principalement sécrété par des structures épidermiques spécialisées appelées trichomes glandulaires. De plus, nous avons mis en évidence les contributions respectives des deux voies de biosynthèse des terpènes présentes chez les plantes, les voies MVA et MEP, à la biosynthèse de trois terpènes de la sauge sclarée. Des expériences de marquage au ¹³C indiquent que le sclaréol et l’acétate de linalyle sont tous deux issus de la voie MEP, alors que le β-caryophyllène semble être d’origine mixte. Nous avons également étudié le rôle potentiel d’une phytohormone, le méthyljasmonate, dans la régulation de la production de sclaréol chez la sauge sclarée. Enfin, nous avons exploré la diversité génétique et phénotypique de populations croates de sauge sclarée sauvage, et montrons que ces populations représentent une ressource génétique distincte par rapport aux populations de référence. L’ensemble de ces résultats met en évidence des pistes prometteuses pour l'amélioration génétique ciblée des performances de la sauge sclarée. / Sclareol is a diterpene produced by floral organs of clary sage (Salvia sclarea, Lamiaceae). It is used in perfume industry for the hemisynthesis of ambroxide, a high-valued perfume component characterized by an amber scent and a high perfume fixation capacity. The global demand for sclareol currently rises, prompting attempts at increasing the yield of sclareol production from clary sage. The purpose of the work presented in this manuscript was to improve knowledge on sclareol biosynthesis and its regulation in clary sage, in order to highlight strategies aiming at enhancing clary sage sclareol content. The analysis of the surface of clary sage calyces by mass spectrometry imaging suggests that sclareol is mainly secreted by specialized epidermal structures called glandular trichomes. Moreover, we have highlighted the respective contributions of the two terpenoid biosynthesis pathways present in plants, MVA and MEP pathways, to the biosynthesis of three terpenoids of clary sage. ¹³C-labeling experiments indicate that sclareol and linalyl acetate both originate from the MEP pathway, whereas β-caryophyllene seems to be of mixed origin. We have also investigated the potential role of a phytohormone, methyljasmonate, in the regulation of sclareol production in clary sage. Finally, we have explored the genetic and phenotypic diversity of Croatian wild clary sage populations and show that these populations represent a distinct genetic resource compared to reference populations. Taken together, these results highlight promising avenues for targeted genetic enhancement of clary sage performances.
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