Pheromone lures and trap crops are appealing pest management tools that use insect and/or plant volatiles to reduce pest populations on crops of interest. Generating pheromone-emitting trap plants may allow for a continuing and highly-specific attraction of insect pests without repeated and costly application of synthetic pheromones. These trap plants may also be used to develop area-wide pest management strategies. As a proof-of-principle study we tested the possibility of producing the pheromone of the crucifer flea beetle Phyllotreta cruciferae in transgenic plants. P. cruciferae is an important pest of Brassica crops. In the presence of a host plant, males emit an aggregation pheromone, which attracts both males and females. Himachaladiene, a sesquiterpene, has been identified as a key component of the aggregation pheromone of P. cruciferae. In a close relative, Phyllotreta striolata, the compound is synthesized by a two-step pathway with an isoprenyl diphosphate synthase (PsIDS3) making (Z,E)-farnesyl diphosphate (FPP), which is converted by a terpene synthase (PsTPS1) to himachaladiene. Transient transformation of N. benthamiana with PsIDS3-TPS1 co-localized to the plastid resulted in the emission of himachaladiene and other known PsTPS1 products. Daily emissions of himachaladiene were approximately 1 µg per plant, which is six-fold higher than emissions from individual male flea beetles. Stable transformation of Arabidopsis thaliana with the same vector construct resulted in transgenic plants that expressed PsTPS1 and PsIDS3 transcripts, but no himachaladiene or other PsTPS1 products were present in volatile collections or leaf extracts of these plants. Moreover, no PsTPS1 enzyme activity was observed, indicating that post-transcriptional/translational effects prevent proper expression or targeting of functional PsIDS3 and/or PsTPS1 proteins in A. thaliana. Overall, this study demonstrates that the key component of the P. cruciferae aggregation pheromone, himachaladiene, can be transiently produced and emitted in a plant system at rates that are biologically relevant for insect attraction. However, further work is required for the stable production of the pheromone in plants. In addition, preliminary results are presented for the development of simple two-choice arenas that may allow for assessment of the movement of beetles toward host plant leaf tissue. This work can inform future efforts in developing methods for the economic production of himachaladiene in a plant system or the establishment of transgenic plants for the production and deployment of himachaladiene in a field setting. / Master of Science / The crucifer flea beetle is an important pest of vegetable and oilseed Brassica crops such as broccoli, cabbage and canola. Feeding by beetles has its greatest impact on crop health and yield in the early spring, when adult beetles emerge from overwintering sites and feed on newly- emerging Brassica seedlings. Currently these insects are controlled using broad spectrum insecticides. A general awareness of the negative aspects of insecticides drives the search for alternative pest management strategies that could diversify our management strategies and reduce reliance on insecticides. Previous work has found that the crucifer flea beetle navigates to its host plants, in part, through plant-emitted volatiles. After locating the plant host, males emit a volatile aggregation pheromone that when blended with host plant volatiles increases attraction. Here work towards the development of a specialized trap crop is presented. Plants were engineered to emit a key component of the crucifer flea beetle aggregation pheromone. In an engineered non-host plant, Nicotiana benthamiana, transient production of the aggregation pheromone was established. However, in an engineered Brassica plant, Arabidopsis thaliana, no aggregation pheromone was detected despite evidence of the presence and expression of the required biosynthetic genes for its production. A discussion on alternative engineering strategies for A. thaliana is presented. In addition, preliminary results are presented for the development of a simple behavior assay to assess the attraction of beetles toward different smells. This work can inform future efforts aimed at developing methods for the economic production of the aggregation pheromone in a plant system or the establishment of plants for the production and deployment of the aggregation pheromone in a field setting.

Plants produce secondary (or specialized) metabolites to respond to a variety of environmental changes and threats. Especially, volatile compounds released by plants facilitate short and long distance interaction with both beneficial and harmful organisms. Comparatively little is known about the organization and role of specialized metabolism in root tissues. In this study, we have investigated the root-specific formation and function of volatile terpenes in the model plant Arabidopsis. As one objective, we have characterized the two root-specific terpene synthases, TPS22 and TPS25. Both enzymes catalyze the formation of several volatile sesquiterpenes with (E)-β-farnesene as the major product. TPS22 and TPS25 are expressed in the root in distinct different cell type-specific patterns and both genes are induced by jasmonic acid. Unexpectedly, both TPS proteins are localized to mitochondria, demonstrating a subcellular localization of terpene specialized metabolism in compartments other than the cytosol and plastids. (E)-β-Farnesene is produced at low concentrations suggesting posttranslational modifications of the TPS proteins and/or limited substrate availability in mitochondria. We hypothesize that the mitochondrial localization of TPS22 and TPS25 reflects evolutionary plasticity in subcellular compartmentation of TPS proteins with emerging or declining activity. Since (E)-β-farnesene inhibits Arabidopsis root growth in vitro, mitochondrial targeting of both proteins may fine tune (E)-β-farnesene concentrations to prevent possible autotoxic or inhibitory effects of this terpene in vivo. We further investigated the role of volatile terpenes in Arabidopsis roots in interaction with the soil-borne oomycete, Pythium irregulare. Infection of roots with P. irregulare causes emission of the C11-homoterpene (or better called C4-norterpene) 4,8-dimethylnona-1,3,7-triene (DMNT), which is a common volatile induced by biotic stress in aerial parts of plants but was not previously known to be produced in plant roots. We demonstrate that DMNT is synthesized by a novel, root-specific pathway via oxidative degradation of the C30-triterpene, arabidiol. DMNT exhibits inhibitory effects on P. irregulare mycelium growth and oospore germination in vitro. Moreover, arabidiol and DMNT biosynthetic mutants were found to be more susceptible to P. irregulare infection and showed higher rates of Pythium colonization in comparison to wild type plants. Together, our studies demonstrate differences and plasticity in the metabolic organization and function of terpenes in roots in comparison to aboveground plant tissues. / Ph. D.

BACKGROUND:Microalgae hold promise for yielding a biofuel feedstock that is sustainable, carbon-neutral, distributed, and only minimally disruptive for the production of food and feed by traditional agriculture. Amongst oleaginous eukaryotic algae, the B race of Botryococcus braunii is unique in that it produces large amounts of liquid hydrocarbons of terpenoid origin. These are comparable to fossil crude oil, and are sequestered outside the cells in a communal extracellular polymeric matrix material. Biosynthetic engineering of terpenoid bio-crude production requires identification of genes and reconstruction of metabolic pathways responsible for production of both hydrocarbons and other metabolites of the alga that compete for photosynthetic carbon and energy.RESULTS:A de novo assembly of 1,334,609 next-generation pyrosequencing reads form the Showa strain of the B race of B. braunii yielded a transcriptomic database of 46,422 contigs with an average length of 756 bp. Contigs were annotated with pathway, ontology, and protein domain identifiers. Manual curation allowed the reconstruction of pathways that produce terpenoid liquid hydrocarbons from primary metabolites, and pathways that divert photosynthetic carbon into tetraterpenoid carotenoids, diterpenoids, and the prenyl chains of meroterpenoid quinones and chlorophyll. Inventories of machine-assembled contigs are also presented for reconstructed pathways for the biosynthesis of competing storage compounds including triacylglycerol and starch. Regeneration of S-adenosylmethionine, and the extracellular localization of the hydrocarbon oils by active transport and possibly autophagy are also investigated.CONCLUSIONS:The construction of an annotated transcriptomic database, publicly available in a web-based data depository and annotation tool, provides a foundation for metabolic pathway and network reconstruction, and facilitates further omics studies in the absence of a genome sequence for the Showa strain of B. braunii, race B. Further, the transcriptome database empowers future biosynthetic engineering approaches for strain improvement and the transfer of desirable traits to heterologous hosts.

In der vorliegenden Arbeit wird die Anwendung der Lewis-Säure vermittelten Cycloadditionen von Allylsilanen an alpha,beta-ungesättigte Carbonylverbindungen beschrieben. Das Sesquiterpen Isocomen wurde unter Anwendung der [3+2]-Cycloaddition an Enone synthetisch hergestellt. Die [2+2]-Cycloaddition an Acrylsäurederivate wurde angewandt für einen Einstieg zur Totalsynthese des Oleander Scale Pheromons. Im zweiten Teil der Arbeit wird die Synthese hormonell aktiver Cholesterinderivate dargestellt. Ausgehend von kommerziell erhältlichen Substanzen wurden verschiedene Substituenten in das Steroidgerüst eingeführt.

Soybean [Glycine max (L.) Merr.] is an important crop worldwide. It has been widely consumed for protein, oil and other soy products. To develop soybean cultivars with greater resistance against pests and improved meal quality, it is important to elucidate the molecular bases of these traits. This dissertation aims to investigate the biochemical and biological functions of soybean genes from four gene families, which are hypothesized to be associated with soybean defense against pests and soybean meal quality. There are three specific objectives in this dissertation. The first one is to determine the function of components in the salicylic acid (SA) signaling pathway in soybean resistance against soybean cyst nematode (Heterodera glycines, SCN). The second one is to determine whether insect herbivory induce the emission of volatiles from soybean, and if so, how these volatiles are biosynthesized. The third objective is to identify and characterize soybean mannanase genes that can be used for the improvement of soybean meal quality. The soybean genome has been fully sequenced, which provides opportunities for cross-species comparison of gene families of interest and identification of candidate genes in soybean. The cloned cDNAs of putative genes were expressed in Escherichia coli to produce recombinant enzymes. Through biochemical assays, these proteins were proved to be soybean salicylic acid methyltransferase (GmSAMT1), methyl salicylate esterase (GmSABP2-1), α[alpha]-farnesene synthase (GmTPS1) and E-β[beta]-caryophyllene synthase (GmTPS2), and endo-β[beta]-mannanase (GmMAN1). Through a transgenic hairy root system harboring overexpression of GmSAMT1 and GmSABP2-1, both of these two genes were evaluated for their biological function related to resistance against SCN. The results showed that the over-expression of GmSAMT1 and GmSABP2-1 in the susceptible soybean background lead to enhanced resistance against SCN. Among four putative soybean mannanase genes, one gene was cloned and characterized. GmMAN1 showed the endo-β[beta]-mannanase hydrolyse activity and can hydrolyze cell walls isolated from soybean seeds. In summary, using comparative and functional genomics, a number of genes involved in soybean defense and meal quality were isolated and characterized. This study provides novel knowledge and molecular tools for the genetic improvement of soybean for enhanced resistance and improved meal quality.

Transformer des produits naturels, les fonctionnaliser par hémi-synthèse ou par voie catalytique est un outil intéressant pour élargir le spectre des structures accessibles et de développer ainsi de nouvelles molécules à potentialité biologique. Nous nous sommes intéressés à la valorisation de sesquiterpènes isolés de l’huile essentielle du Cédres de l’Atlas qui représentent une famille de molécules naturelles abondantes et de faible coût. La première partie de ce travail porte sur des réactions d’hémisynthèse des himachalènes et des α-atlantones par des voies classiques d’époxydation, de dihalogéno cyclopropanation, de Grignard et de Wittig,dans le but d’accroître l’activité biologique reconnue de la molécule naturelle ou de découvrir de nouvelles activités. Les dérivés oléfiniques synthétisés ont été ensuite impliqués dans la réaction d’hydroformylation catalysée par des complexes au rhodium. Cette seconde partie décrit dans un premier temps la synthèse des ligands phospholes et biphospholes chiraux impliqués dans cette réaction. La performance des ligands a été évaluée dans la réaction d’hydroformylation de substrats modèles tels que le styrène, l’octène et l’α-méthylstyrèneet celle des ligands biphosphosles chiraux dans la version asymétrique de la réaction avecle styrène. Ensuite,la réaction a été étudiée sur les composés oléfiniques dérivés des α-atlantones. Les systèmes catalytiques [Rh(acac)(CO)2]/phospholes se montrent actifs e donnent de bonnes chimio- et régiosélectivité en faveur de l’aldéhyde linéaire. Nous avons également réalisé des réactions tandem d’hydroformylation/Wittig/hydroformylation pour obtenir des aldéhydes à long chaines aliphatiques ainsi que les réactions hydroformylation/acétalisation des alcools homo- et allyliques dérivé des α- atlantones afin d’accéder aux motifs lactones après oxydation des hemiacétals correspondants. La dernière partie est consacrée à la synthèse des nouveaux ligands pyrrol-phosphine (phosphole) (P-N) et leur évaluation dans la réaction d’arylation directe intramoléculaire catalysée des complexes du palladium sous micro-ondes. Cette réaction permet d’accéder à des lactones présentant un motif «dibenzo-pyranone».Les complexes associés avec les ligands pyrrol-phosphine montrent de bons résultats en termes de conversion et sélectivité. Un effet électronique remarquable des substituants des esters biaryles sur la chimiosélectivité de la réaction a été observé. / The functionnalisation of natural products by hemisynthesis or catalytic reactions is a useful tool to accessand develop newinteresting molecules with biological properties.We have been interested in chemical transformation of the sesquiterpenes isolated from the essential oil of Atlas Cedar, which represent a family of abundant and inexpensive natural molecules The first part of this work concerns the hemisynthesis reactions on the himachalènes and α-atlantones by classical epoxidation, dihalogénocyclopropanation, Grignard and Wittig reactions with the aim to increase the known biological activity or to investigate novel properties. Olefinic derivatives obtained were then involved in the hydroformylation reaction catalyzed by rhodium complexes. This second part describes first the synthesis of phospholeligands and chiral biphospholes involved in this reaction.The performance of these ligands has been evaluated in the hydroformylation reaction of model substrates like styrene, octene and α-methylstyrene as well as that of the chiral biphosphosles ligands in the asymmetric hydroformylation of styrene. Then the reaction has been studied with the olefin derived from α-atlantones using [Rh(acac)(CO)2]/L (L= phosphine or phosphole) catalytic systems. These systemsshow active and give good chemo- and regioselectivity in favour of the linear aldehyde. We have also carried out the tandem reactions such as hydroformylation/Wittig/hydroformylation to obtain aldehydes with long-chain aliphatic and hydroformylation/acetalization reactions of homo- and allylic alcohols derived from α-atlantones to obtain new five-and six-membered lactones after oxidation of the corresponding hemiacetals. The last part describes the synthesis of new ligands (P-N) pyrrol-phosphine (phosphole) and their evaluation in the direct intramolecular arylation reaction catalyzed by palladium complexes under microwaves.This reaction provides access to lactones with a «dibenzo-pyranone» backbone. The complexes containing pyrrol-phosphine ligands give interesting results in terms of conversion and selectivity from various biaryl esters.

Secondary metabolites are compounds that are not necessary for the survival of the organism, but which are to be related to the organism's interaction with its environment. This work studied compounds of secondary metabolism through the study of chemical and biological extracts and fractions Simaba Maiana Casar. and the involvement of candidate genes (TPS10, TPS14, and CYP71B31 CYP76C3) in the metabolism of monoterpenes in Arabidopsis thaliana. The extracts from roots and stems of Simaba Maiana were tested in antioxidant activity, molluscicide, inhibition of lymphocyte proliferation, inhibition of NO production, anti-Leishmania amazonensis and anti-Trypanosoma cruzi. The four candidate genes (CYP76C3, CYP71B31, TPS10 and TPS14) were selected with the CYPedia, which calculates the coexpression between genes based on the Arabidopsis Affymetrix ATH1 microarray. The extracts and fractions Simaba Maiana showed a lower antioxidant activity by DPPH method, low concentrations of total phenols measured by Folin-Ciocalteu, however, a good antioxidant activity by TBARS method, using three agents of oxidative damage (AAPH, FeSO4 and H2O2). The extract showed cytotoxic activity and molluscicidal concentration of 100 mg/mL. The crude extract of the stem was not active for leishmanicidal and trypanocidal. This extract did not inhibit NO production, but showed a high percentage of inhibition of lymphoproliferation. The skimmianine furoquinoline alkaloid and pellopterin furanocoumarin were isolated from chloroform fraction. The candidate genes showed a similar pattern of expression of the stamen, more specifically the top of the filaments. Heterologous expression in transiently expressed in leaves of Nicotiana benthamiana, in the volatiles of TPS10 TPS14 (alone) were found enantiomers R-(-)-linalool and S-(+)-linalool. In the extraction buffer leaf discs were found that CYP76C3 converts linalool to E-8-hydroxy-linalool and E-8-oxo-linalool, and CYP71B31 in 1,2-epoxy-linalool. The analysis of the methanol extract of the discs incubated in S-(+)-linalool showed the use of this substrate by P450s converting to lilac alcohol for both P450s. Analysis of flowers of Arabidopsis mutants showed minor differences, analyzes with extracts of fresh flowers in UPLC-MS/MS MRM mode, has been found a compound having the same signature as linalool, however with different retention time and may be is an indication of linalool bound. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Os metabólitos secundários são compostos que não são necessários para a sobrevivência do organismo, mas que apresentam-se relacionados com a interação do organismo com o seu ambiente. O presente trabalho estudou compostos do metabolismo secundário através do estudo químico e biológico dos extratos e frações Simaba maiana Casar. e a envolvimento dos genes candidatos (TPS10, TPS14, CYP76C3 e CYP71B31) no metabolismo de monoterpenóides em Arabidopsis thaliana. Os extratos das raízes e caule de Simaba maiana foram testadas em ensaios de atividade antioxidante, moluscicida, inibição da linfoproliferação, inibição da produção de NO, anti-Leishmania amazonensis e anti-Trypanosoma cruzi. Os quatro genes candidatos (CYP76C3, CYP71B31, TPS10 e TPS14) foram selecionados com o CYPedia, que calcula a co-expressão entre os genes de Arabidopsis com base no Affymetrix ATH1 microarray. Os extratos e frações de Simaba maiana mostraram uma baixa atividade antioxidante pelo método do DPPH, baixas concentrações de fenóis totais avaliados pelo método de Folin-Ciocalteu, entretanto, uma boa atividade antioxidante pelo método de TBARS, usando três agentes de danos oxidativos (AAPH, FeSO4 e H2O2). Os extratos mostraram atividade moluscicida e citotóxica na concentração de 100 mg/mL. O extrato bruto do caule não foi ativo para as atividades anti-Leishmania e anti-Trypanosoma. Este extrato não inibiu a produção de NO, mas apresentou uma alta porcentagem da inibição da linfoproliferação. O alcalóide furoquinolínico esquiamina e a furanocumarina felopterina foram isolados da fração clorofórmica. Os genes candidatos mostraram um similar padrão de expressão nos estames, mais especificamente na parte superior dos filamentos. Na expressão heteróloga transitoriamente expressa em folhas de Nicotiana benthamiana, nos voláteis de TPS10 e TPS14 (sozinho) foram encontrados os enantiômeros R-(-)-linalol e S-(+)-linalol. No tampão de extração de discos de folhas, verificou-se que CYP76C3 converte linalol em E-8-hidroxi-linalol e E-8-oxo-linalol, e CYP71B31 em 1,2-epoxilinalol. A análise do extrato metanólico dos discos foliares incubados em S-(+)-linalol mostrou a utilização deste substrato por P450s convertendo para lilac álcool para ambos os P450s. Análises de flores em plantas mutantes de Arabidopsis thaliana mostraram pequenas diferenças, em que análises com extratos de flores frescas em UPLC-MS/MS no modo MRM, foi encontrado um composto com a mesma assinatura que linalol, no entanto, com tempo de retenção diferente e pode ser uma indicação de forma ligada do linalol.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-02-01T11:42:20Z No. of bitstreams: 1 diegorodriguesmelo.pdf: 1192469 bytes, checksum: 7a17e7c77b3319206eae586410dc02e3 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-02-01T15:48:57Z (GMT) No. of bitstreams: 1 diegorodriguesmelo.pdf: 1192469 bytes, checksum: 7a17e7c77b3319206eae586410dc02e3 (MD5) / Made available in DSpace on 2016-02-01T15:48:57Z (GMT). No. of bitstreams: 1 diegorodriguesmelo.pdf: 1192469 bytes, checksum: 7a17e7c77b3319206eae586410dc02e3 (MD5) Previous issue date: 2014-02-18 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Objetivou-se avaliar a atividade inseticida do óleo essencial de Lippia sidoides e dos monoterpenos timol e carvacrol sobre larvas e pupas de Musca domestica através de testes de contato. Para a avaliação da mortalidade larval (ML) foram feitas observações 48h após o tratamento e acompanhamento das larvas sobreviventes até o estágio adulto, e estes índices foram incluídos na eficácia de tratamento larval (ETL). Para a eficácia de tratamento pupal (ETP), foram calculados a mortalidade pupal (MP) após seis dias do tratamento e o percentual de mal desenvolvimento dos adultos (PMD). Foram testadas oito concentrações do óleo essencial e das substâncias puras, variando de 0,625 até 30,0 mg/ml. A análise dos constituintes do óleo essencial mostrou o timol como seu componente majoritário (69,91%). As doses letais (DL50 e DL90) para larvas e pupas foram estimadas e o carvacrol apresentou os menores valores, seguido pelo timol e óleo de L. sidoides. Verificou-se ML de 100% a partir da concentração de 10,0 mg/ml para o carvacrol, 15,0 mg/ml para o óleo de L. sidoides e 30,0 mg/ml para o timol. Já para a ETL foi observada 100% de eficácia a partir da concentração de 5,0 mg/ml para o carvacrol e L. sidoides e 10,0 mg/ml para o timol. Com relação a MP, o óleo essencial de L. sidoides não apresentou atividade pupicida. Entretanto, para as concentrações de 20,0 e 30,0 mg/ml foi observado PMD, o que contribuiu para ETP de 44 e 46%, respectivamente. Já para o carvacrol a maior mortalidade foi alcançada na concentração de 20 mg/ml (58%) e para este terpeno não houve PMD. Para o timol observou-se maior MP (74%) para a concentração de 30,0 mg/ml com PMD de 60%, o que contribuiu para ETP de 84%. Os produtos testados apresentaram elevado potencial larvicida, o que os tornam promissores para o desenvolvimento de formulações ambientalmente seguras e não tóxicas para o controle de larvas de M. domestica e que, apesar de não terem sido totalmente efetivas no controle de pupas, seu uso para esse estágio pode ser indicado em um programa de Manejo Integrado de Pragas. / This study aimed to evaluate the insecticidal activity of the essential oil of Lippia sidoides and thymol and carvacrol monoterpenes against larvae and pupae of Musca domestica by contact tests. For the evaluation of larval mortality (LM) observations were made 48 hours after treatment and follow-up of surviving larvae to the adult stage, and these indices were included in the effectiveness of larval treatment (ELT). For the effectiveness of pupal treatment (EPT) were calculated pupal mortality (PM) after six days of treatment and the percentage of bad development of adults (PBD). Eight concentrations of the essential oil and pure substances, ranging from 0.625 to 30.0 mg/ml were tested. The analysis of the constituents of the essential oil thymol showed as its major component (69.91 %). The lethal dose (LD50 and LD90) for larvae and pupae were estimated and carvacrol showed the lowest values, followed by thymol and L. sidoides oil. It was found LM 100% at concentrations of 10.0 mg/ml for carvacrol, 15.0 mg/ml for L. sidoides oil and 30.0 mg/ml to thymol. As for the ELT 100% efficacy was observed starting at a concentration of 5.0 mg/ml for carvacrol and L. sidoides and 10.0 mg/ml to thymol. With respect to PM, the essential oil of L. sidoides showed no pupicida activity. However, for concentrations of 20.0 and 30.0 mg/ml was observed PBD, which contributed to EPT of 44 and 46%, respectively. As for carvacrol the highest mortality was achieved at a concentration of 20 mg/ml (58%) and to that terpene was not PBD. To thymol observed a higher PM (74%) for the concentration of 30.0 mg/ml with the PBD 60%, which contributed to 84% of EPT. The products tested showed high potential larvicide, which make them promising for the development of formulations environmentally safe and non-toxic for the control of larvae of M. domestica and that although they were not fully effective in the control pupae, their use for this stage can be indicated in a program of Integrated Pest Management.

Although cytoplasmic lipid droplets (LDs) are the major reserves for energy-dense neutral lipids in plants, the cellular mechanisms for packaging neutral lipids into LDs remain poorly understood. To gain insights into the cellular processes of neutral lipid accumulation and compartmentalization, a necessary step forward would be to characterize functional roles of lipogenic proteins that participate in the compartmentalization of neutral lipids in plant cells. In this study, the lipogenic proteins, Arabidopsis thaliana SEIPIN homologues and mouse (Mus Musculus) fat storage-inducing transmembrane protein 2 (FIT2), were characterized for their functional roles in the biogenesis of cytoplasmic LDs in various plant tissues. Both Arabidopsis SEIPINs and mouse FIT2 supported the accumulation of neutral lipids and cytoplasmic LDs in plants. The three Arabidopsis SEIPIN isoforms play distinct roles in compartmentalizing neutral lipids by enhancing the numbers and sizes of LDs in various plant tissues and developmental stages. Further, the potential applications of Arabidopsis SEIPINs and mouse FIT2 in engineering neutral lipids and terpenes in plant vegetative tissues were evaluated by co-expressing these and other lipogenic proteins in Nicotiana benthamiana leaves. Arabidopsis SEIPINs and mouse FIT2 represent effective tools that may complement ongoing strategies to enhance the accumulation of desired neutral lipids and terpenes in plant vegetative tissues. Collectively, our findings in this study expand our knowledge of the broader cellular mechanisms of LD biogenesis that are partially conserved in eukaryotes and distinct in plants and suggest novel targets that can be introduced into plants to collaborate with other factors in lipid metabolism and elevate oil content in plant tissues.

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.