Harz ist ein klebriges Pflanzenprodukt mit einem oft intensiven aromatischen Geruch. Es wird von Bäumen produziert, um Wunden zu verschließen und schädliche Besucher abzuwehren. Einige Insektenarten haben jedoch die erstaunliche Fähigkeit entwickelt, mit der klebrigen Substanz umzugehen und sie sich gar zu Nutzen zu machen. So verwenden Bienen Harz beispielsweise zum Nestbau und zur Verteidigung ihrer Kolonien. Während allgemein bekannt ist, dass Bienen Pollen und Nektar sammeln, wird der Tatsache, dass sie auch Harz sammlen, allerdings sehr viel weniger Beachtung geschenkt. Ziel meiner Dissertation war es daher, herauszufinden, warum, wie und wo stachellose Bienen in Borneo (sieben untersuchte Bienenarten), Australien (acht Arten) und Costa Rica (27 Arten) Pflanzenharze sammeln und verwerten. Diese Arbeit behandelt somit die enge Beziehung zwischen einer eusozialen Insektengattung und einem chemisch und physiologisch hoch komplexen Pflanzenprodukt, das Bienen nicht nur als Nestmaterial und zur Verteidigung dient, sondern auch eine wesentliche Bedeutung für deren chemische Diversität hat. Stachellose Bienen verhalten sich hochgradig opportunistisch, wenn sie Harz sammeln, d.h. verschiedene Bienenarten sammeln Harz von denselben Baumarten, wobei sie nahezu jede verfügbare Harzquelle nutzen. Dabei finden und erkennen sie Harzquellen anhand einiger charakteristischer Mono- und Sesquiterpene, nutzen jedoch nicht das gesamte Harz-Bouquet. Die Menge an eingetragenem Harz unterscheidet sich zwischen verschiedenen Bienenarten und kolonien und varriert mit verschiedenen Umweltbedingungen. Insbesondere eine Bedrohung durch Fressfeinde (z. B. Ameisen) führt zu einer massiven Steigerung des Harzeintrages; eine manuelle Zerstörung des Nesteinganges hat dagegen relativ wenig Einfluss. Das eingetragene Harz wird zum Nestbau und zur Verteidigung gegen Fressfeinde und Mikroben genutzt. Darüber hinaus dient es als Quelle für Terpene, die von den Bienen in ihre chemischen Oberflächenprofile eingebaut werden (kutikuläre Terpene). Dabei übertragen sie nur einen Bruchteil (8 %) der gewaltigen Menge (>> 1000) an Terpenen, die man im Harz von Bäumen findet, auf ihre Oberfläche. Die übertragenen Terpene bleiben in ihrer Struktur unverändert, allerdings unterscheiden sich die Bienenarten in der Zusammensetzung der Terpenprofile auf ihrer Oberfläche, obwohl alle untersuchten Arten Harz von denselben Bäumen sammeln. Die unterschiedlichen Terpenprofile sowie die Tatsache, dass nur wenige Terpene aus dem Harz aufgenommen werden, deuten auf einen artspezifischen und bisher unbekannten Filterungsmechanismus bei stachellosen Bienen hin. Auch übersteigt durch die Aufnahme von Terpenen die chemische Diversität der Oberflächenprofile von stachellosen Bienen die zahlreicher anderer Hymenopteren. Da Bienen die Terpene aus dem Harz nur „filtern“, sie dabei aber nicht verändern, sind sämtliche Bienenarten aus Borneo, Australien und Costa den charakteristischen Harzprofilen von Bäumen aus ihren Ursprungsgebieten chemisch sehr ähnlich. Da in jeder tropischen Region andere Baumarten vorkommen, varriert die chemische Zusammensetzung der vorkommenden Harze und damit der kutikulären Terpene von dort vorkommenden Bienen. Die meisten Bienenarten mit kutikulären Terpenen findet man in Borneo, wo nahezu 100 % der untersuchten Arten aus Baumharzen gewonnene Terpene in ihre chemischen Profilen einbauen. Im Gegensatz dazu sind es in Costa Rica nur 40 % der untersuchten Arten. Auch sammeln in Borneo gelegentlich 9 von 10 Arbeiterinnen einer Tetragonilla collina Kolonie Harz, wohingegen in Australien maximal 10 % und in Costa Rica maximal 40 % der Arbeiterinnen einer Kolonie Harz sammeln. Das Vorherrschen von Harz und aus Harz gewonnenen Terpenen in der chemischen Ökologie von Bienen auf Borneo spiegelt das Vorherrschen einer bestimmten südostasiatischen Baumfamilie wieder: der Dipterocarpaceen, deren Holz ungewöhnlich harzig ist. Ein solch enger Zusammenhang zwischen der Chemie von Bienen und der von Baumharzen verdeutlicht die enge Beziehung zwischen stachellosen Bienen und den Bäumen in ihrem Habitat. Die kutikulären Terpene schützen ihre Träger vor Angreifern (z.B. Ameisen) und Mikrobenbefall. Dabei variiert eine bestimmte Gruppe – Sesquiterpene – am meisten zwischen den Arten. Diese Terpengruppe manipuliert die natürlichweise auftretende zwischen-artliche Aggression, indem sie letztere bei jenen Arten verringert, die selbst keine Sesquiterpene in ihrem Profil haben. Aggressionsminderung durch chemische Komponenten, welche aus der Umwelt aufgenommen werden, stellt somit einen bisher unbekannten Mechanismus dar, um Toleranz zwischen sonst aggressiven Arten zu erreichen. Eine derarte Herabsetzung von aggressiven Verhalten bei stachellosen Bienen kann darüber hinaus ein entscheidender Faktor für das Entstehen sogenannter Nestaggregationen sein. Dabei nisten Kolonien von Bienenarten mit und Bienenarten ohne Sesquiterpene in ihrem chemischen Profil in unmittelbarer Nachbarschaft, ohne gegeneinander aggressiv zu sein. Im Hinblick auf die zahlreichen Funktionen, die Harze und/oder aus dem Harz gewonnene Substanzen für stachellose Bienen haben, stellt Harz zweifelsohne eine bedeutende Ressource in der Welt der Bienen dar – eine Ressource, die einen direkten Einfluss auf deren chemische Ökologie, Verteidigungsmechanismen und zwischen-artliche Kommunikation ausübt. Wie genau die Bienen ihre artspezifischen Terpenprofile erzeugen, insbesondere, wie es ihnen gelingt, dabei ganze Terpengruppen auszuschließen, muss in zukünftigen Studien genauer untersucht werden. Auch stellt sich die Frage, wie wichtig eine hohe Diversität an Harzquellen und damit Baumarten für die Bienen ist! Es ist durchaus möglich, dass neben einer Vielfalt an Blütenpflanzenarten auch der „Harzreichtum“ für das Wohlergehen der Bienen eine entscheidende Rolle spielt. / Resin, a sticky sap emitting terpenoids and other volatiles, is produced by various plant species to seal wounds and protect themselves against herbivores and microbes. Among several other insects, bees have evolved the surprising ability to handle the repellent plant sap and use it to construct and defend their nests. Whereas the collection of pollen and nectar has been intensively studied in bees, resin collection has received only little attention. The aim of this dissertation was to better understand how the physiological and chemical properties of resin and resin-derived compounds (terpenes) affect the ecology of stingless bees. I therefore asked why, where and how stingless bees of Borneo (seven study-species), Australia (eight) and Costa Rica (27) collect and process plant resins, addressing the importance of a largely neglected resource not only for building and defensive properties, but also for the bees’ chemical diversity. Stingless bees are highly opportunistic resin foragers with all species collecting resin from a similar set of tree species. They locate and/or recognize resin sources on the basis of several volatile mono- and sesquiterpenes. I found that different bee species and even colonies significantly varied in the amount of resin collected. Predator attack (e.g., by ants) had the strongest affect on resin intake, whereas manual nest destruction only slightly increased the number of resin foragers. Resin is used to build, maintain and defend nests, but also as source for chemical compounds (terpenes) which stingless bees include in their surface profiles (chemical profiles). They directly transfer resin-derived compounds to their body surfaces (cuticular terpenes), but only include a subset (8 %) of the large number (>> 1000) of terpenes found in tree resins. This phenomenon can only be explained by a hitherto unknown ability to filter environmentally derived compounds which results in species-specific terpene profiles and thus in an increased chemical heterogeneity among species. Moreover, due to the addition of resin-derived substances the diversity of compounds on the bees’ body surfaces by far exceeds the chemical diversity of profiles in other hymenopterans. Because stingless bees filter but do not modify resin-derived compounds, species from Borneo, Australia and Costa Rica all resemble the characteristic resin of typical trees in their regions of origin. This chemical similarity reveals a strong correlation between the diversity of tree resins and the diversity of cuticular terpenes among stingless bees in a given habitat. Because different tree species are found in different tropical regions, the chemical composition of tree resins varies between tropical regions as does the composition of cuticular terpenes in bee species from these regions. Cuticular terpenes are however most common among stingless from Borneo, with 100 % of species studied having resin-derived terpenes in their chemical profiles. They are least common in Costa Rica, with only 40 % of species having terpenes. Likewise, resin collection was found to be highest in Tetragonilla collina colonies of Borneo where occasionally up to 90 % of foragers collected resin. By contrast, resin collection was only performed by 10 % of foragers of a given colony in Australia and by a maximum of 40 % in Costa Rica. The dominance of resin and resin-derived compounds in the chemical ecology of bees from Borneo may mirror the dominance of a particular Southeast Asian tree family: the highly resinous dipterocarps. Such a correlation between the chemistry of bees and the chemistry of tree resins therefore underlines the close relationship between stingless bees and the trees of their habitat. Cuticular terpenes are assumed to protect bees against predators and/or microbes. Sesquiterpenes, a specific group of terpenes, most vary between species and impair inter-specific aggression by reducing aggressive behavior in species without sesquiterpenes, thereby providing a novel mechanism to achieve interspecific tolerance among insects. Reduced interspecific aggression may also be an important factor enabling the non-aggressive aggregation of nests from stingless bee colonies of up to four different species, because such aggregations frequently comprise both species with and species without sesquiterpenes. Given its various functions, resin represents a highly important resource for stingless bees which directly affects their chemical ecology, defensive properties and inter-specific communication. It remains to be investigated how the bees influence the resin-derived terpene profiles on their body surface and in their nests, particularly how they manage to exclude entire groups of terpenes. Whether bees actually need a high diversity of different resin sources and therefore tree species to maintain the homeostasis of their colonies or whether they would do equally well with a limited amount of resin sources available, should also be addressed in future studies. Answers to this question will directly impair bee and forest management in (sub)tropical regions.

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.

Stuttgart, Univ., Diss., 2007.

München, Techn. Univ., Diss., 2002.

Universiẗat, Diss., 2002--Bonn.

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

Plants produce a large number of specialized or secondary compounds that aid in their reproduction and protection against biotic and abiotic stress. In this work I investigated the metabolism and function of terpenes, the largest class of specialized metabolites, in switchgrass and carrot. Switchgrass (Panicum virgatum L.), a perennial C4 grass of the Tallgrass Prairie, represents an important species in natural and anthropogenic grasslands of North America. Its natural resilience to abiotic and biotic stress has made switchgrass a preferred bioenergy crop. I have investigated the metabolism of terpenes in switchgrass leaves and roots in response to herbivory or defense hormone treatments and the application of drought. With a focus on volatile terpene metabolites, I functionally characterized over thirty genes (terpene synthases, TPSs), of which one third could be correlated with the production and release of volatile monoterpenes and sesquiterpenes that likely function in direct chemical defense or in the attraction of insect predators or parasitoids. Drought stress application caused switchgrass roots to accumulate a larger amount of oxygenated terpenes and presumably non-volatile terpenes, the function of which in direct or indirect drought stress protection requires further investigation. I also examined the metabolic dynamics and role of the monoterpene borneol, which accumulates at high concentrations in the roots of switchgrass and to a lower extent in the roots of the close relative Setaria viridis, in root microbe interactions. Although we demonstrated a successful RNAi based knock down of the borneol terpene synthase TPS04, we found no immediate evidence that borneol significantly modifies bacterial communities in the root. Further studies on Setaria and equivalent RNAi lines in switchgrass will provide more detailed and needed insight to decipher the role of monoterpene accumulation in grasses interactions with mutualists, pathogens, and pests. In an applied project, I investigated terpene specialized metabolism in carrot (Daucus carota L.) to identify genetic determinants of carrot aroma and flavor. To determine central enzymes which contribute to the terpene component of carrot volatile blends, we first analyzed tissue specific expression patterns of carrot terpene synthase genes (TPS) in the genomic model carrot (cv. DH1) and in roots of four aromatically unique colored carrot genotypes (orange-4943B, red-R6637, yellow-Y9244A and purple-P7262). We selected nineteen key biosynthetic enzymes involved in terpene formation and compared in vitro products from recombinant proteins with native volatile profiles obtained from DH1 and colored carrot genotypes. We biochemically characterized several highly expressed TPSs with direct correlations to major compounds of carrot flavor and aroma including germacrene-D (DcTPS11), (DcTPS30) and -terpinolene (DcTPS03). Random forest analysis of colored carrot volatiles revealed that nine terpene compounds are sufficient for distinguishing the flavor and aroma of raw colored carrots. Interestingly, accumulation of specific terpene compounds rather than chemical diversity is responsible for differences in sensory quality traits in colored genotypes. As accumulations of specific terpene compounds can contribute to the undesired flavor in carrot, our report provides a detailed roadmap for future breeding efforts to enhance carrot flavor and aroma. / Doctor of Philosophy / Plants produce a large number of chemicals that are important for growth, defense, flavor, and aroma. While chemical production has been studied in some major food crops (corn, tomato, rice), knowledge of the formation and function of chemicals in switchgrass and carrot is still limited. Switchgrass (Panicum virgatum L.), a grass of the Tallgrass Prairie, represents an important species grasslands of North America. Its natural resilience to stress has made switchgrass a preferred bioenergy crop. I found that switchgrass produces many compounds in the chemical class of terpenoids in roots and leaves that likely serve as a defense against damage from pests. In addition, I found that drought stress leads to the production of terpenoid compounds that may have roles in protection when water is limited. My research also demonstrates that roots of switchgrass and the related grass Setaria maintain substantial levels of the essential oil compound borneol. This terpenoid compound can act as a nutrient source for specific bacteria and/or an antimicrobial agent. Therefore, I proposed that switchgrass and Setaria roots produce borneol to establish a distinct root microbiome by recruitment of beneficial bacteria and deterrence of harmful microorganisms. To test this hypothesis, we genetically engineered plants to reduce borneol formation and accumulation in roots. Using these plants, we evaluated changes in the root microbiome in response to altered borneol levels. We found that interfering with borneol production in Setaria roots has limited influence on the microbiome inside roots. Although a similar approach was used for switchgrass, we were unable to significantly reduce borneol V formation in roots. Results from this study provide a better understanding of belowground plant-microbe interactions, and potential for enhancing resistance traits into other crop species. I also investigated the flavor and aroma compounds produced in carrots, which are considered a key supplemental vegetable due to high nutritional value and pleasant taste. Surprisingly, little has been known about the genetic factors that control flavor and aroma traits in colored carrot varieties. Therefore, I performed a robust characterization of the biosynthesis of terpenoids, which are the predominant aroma and flavor compounds in carrot. I identified several enzymes in carrot that can produce a diverse blend of terpenoids which are associated with sweet, spicy and bitter tastes. In addition, I discovered that carrot stems and leaves also maintain a rich chemistry of terpenoids similar to that in roots. Results from this work provide a baseline for engineering enhanced flavor in carrot and provide a deeper insight into essential oil formation in root crops.

Southern Magnolia (Magnolia grandiflora) is a primitive tree species that has attracted attention because of its horticultural distinctiveness, the wealth of natural products associated with it, and its evolutionary position as a basal angiosperm. Terpenoid constituents were determined from Magnolia leaves and flowers. Magnolia leaves constitutively produced two major terpenoids, andamp;acirc;-cubebene and germacrene A. However, upon wounding Magnolia leaves biosynthesized a significant array of monoand sesquiterpenoids, including andamp;acirc;-pinene, trans-andamp;acirc;-ocimene, andamp;aacute;-gurjunene, andamp;acirc;-caryophyllene and andamp;acirc;-cubebene, along with fatty acid derivatives such as cis-jasmone, for up to 19 hours after treatment. Flowers were also examined for their emission of terpene volatiles prior to and after opening, and also in response to challenge by Japanese beetles. Opened and un-opened flowers constitutively emitted a blend of monoterpenes dominated by andamp;acirc;-pinene and cis-andamp;acirc;-ocimene. However, the emission levels of monoterpenes such as verbenone, geraniol, and citral, and sesquiterpenes such as andamp;acirc;-cubebene, andamp;aacute;-farnesene, and andamp;acirc;-caryophyllene were significantly elevated in the emissions of the beetle-challenged flowers. Three cDNAs corresponding to terpene synthase (TPS) genes expressed in young Magnolia leaves were isolated and the corresponding enzymes were functionally characterized in vitro. Recombinant Mg25 converted FPP (C15) predominantly to andamp;acirc;-cubebene, while Mg17 converted GPP (C5) to andamp;aacute;-terpineol. Efforts to functionally characterize Mg11 were unsuccessful. Transcript levels for all 3 genes were prominent in young leaf tissue and significantly elevated for Mg25 and Mg11 mRNAs in stamens. A putative N-terminal signal peptide of Mg17 targeted the reporter GFP protein to both chloroplasts and mitochondria when transiently expressed in epidermal cells of Nicotiana tabacum leaves. Phylogenetic analyses indicated that Mg25 and Mg11 belonged to the angiosperm sesquiterpene synthase subclass TPS-a, while Mg17 aligned more closely to the angiosperm monoterpene synthase subclass TPS-b. Unexpectedly, intron/exon organizations for the three Magnolia TPS genes were different from one another and from other well characterized terpene synthase gene sets. The Mg17 gene consists of 6 introns arranged in a manner similar to many other angiosperm sesquiterpene synthases, but Mg11 contains only 4 introns, and Mg25 has only a single intron near the 5 terminus of the gene. Our results suggest that much of the structural diversity observed in the Magnolia TPS genes may have occurred by means other than intron-loss from a common ancestor TPS gene. Costunolide is a sesquiterpene lactone widely recognized for its diverse biological activities, including its bitter taste in lettuces, and as a precursor to the more potent pharmacological agent parthenolide. A lettuce EST database was screened for cytochrome P450 genes that might be associated with sesquiterpene hydroxylation. Five ESTs were selected based on sequence similarity to known sesquiterpene hydroxylases and three of them (Ls7108, Ls3597 and Ls2101) were successfully amplified as fulllength cDNAs. To functionally characterize these cDNAs, they were co-expressed along with a germacrene A synthase and a cytochrome P450 reductase in yeast. Based on product profile comparisons between the three different lines to the control line, only the Ls7108-harboring line produced unique compounds. Neither of the other lines showed a new product peak. The more abundant, polar product generated by the Ls7108-containing line was purified and identified as a 12-acetoxy-germacrene by NMR analysis. In vitro studies using Ls7108 microsomal proteins did not yield the 12-acetoxy-germacrene A, but the putative germacra-1(10),4,11(13)-trien-12-ol intermediate. Catalytic activity of the Ls7108 microsomal enzyme was NADPH, pH and time dependent. Our results demonstrate that Ls7108 is a lettuce cytochrome P450 which catalyzes the hydroxylation of a methyl group of the isopropenyl substituent of germacrene A, generating germacra-1(10),4,11(13)-trien-12-ol, and that when this mono-hydroxylated sesquiterpene is synthesized in yeast, an endogenous yeast enzyme further modifies the germacrenol compound by acetylation of the alcohol group at the C-12 position.

Cette thèse s’est focalisée sur les lavandes et en particulier sur trois lavandes retrouvées en France : la lavande fine (Lavandula angustifolia Miller), la lavande aspic (Lavandula latifolia Medik) et leur hybride le lavandin (Lavandula x intermedia). Capables de synthétiser de grandes quantités de composés organiques volatils (COV) et plus particulièrement des terpènes volatils tel les mono- et sesquiterpènes, les lavandes sont utilisées depuis l’antiquité par l’Homme pour les propriétés médicinales et aromatiques de ces terpènes, composants de l’huile essentielle de lavande. De par l’importance économique et écologique des terpènes volatils, ce travail de thèse présente différents aspects d’étude. Dans un premier temps la relation entre la diversité chimique, géographique et génétique de la lavande fine a été analysée sur l’ensemble de son aire de répartition. Les résultats ont mis en évidence des populations de lavande fine très différentes autant chimiquement que génétiquement au bord de leur aire de répartition. Dans un deuxième temps, l’impact du stress hydrique au cours du temps sur les contenus en terpènes volatils a été évalué sur la lavande aspic, le lavandin et six populations de lavande fine. Les résultats ont montré des tolérances différentielles en fonction des espèces et des populations ; ainsi le lavandin est plus rapidement affecté par le stress hydrique que la lavande fine. Les contenus terpéniques n’ont été que faiblement impactés par le stress hydrique et cela à des états hydriques différents selon les espèces et les populations. Malgré la grande diversité de réponse selon les composés, l’intensité du stress hydrique et les plantes ; les terpènes de la voie du camphre (bornéol, camphène et camphre) sont ceux qui présentent les plus grandes variations entre les plantes stressées et témoins. Il apparaissait alors intéressant d’étudier la voie de biosynthèse du camphre. Dans ce cadre-là, nous avons identifié et caractérisé la bornyl diphosphate synthase capable de former le bornéol à partir du bornyl diphosphate. L’ensemble de ces travaux permettent de mieux appréhender les relations entre production de terpènes volatils et environnement ainsi que de donner des outils génétiques afin de poursuivre ces investigations / The PhD was focused on lavenders and precisely on lavenders present in France: the fine lavender (Lavandula angustifolia Miller), the spike lavender (Lavandula latifolia Medik) and their hybrid the lavandin (Lavandula x intermedia). Skilled to synthetize huge organic volatils coumpounds (COV) amount and in particular volatils terpene such mono- and sesquiterpenes, lavenders are used by human from antiquity for medicinal and aromatic properties of these volatils terpenes, lavender essential oil is composed of. Due to economical and ecological volatils terpenes importance, several study aspects is considered in the PhD. In a first hand, on all the fine lavender’s repartition area, relationship between chemical, geographical and genetical diversities was assessed. Results showed chemical and genetical significant different populations, at the border of fine lavender repartition area. In a second hand, hydric stress impact over time on volatiles terpenes content was assessed on the spike lavender, lavandin and six fine lavender populations. Results put in evidence differential tolerances by species and populations; thus lavandin is more quickly affected by hydric stress than the fine lavender. Terpenes contents were slightly impacted by hydric stress and with different states amoung species and populations. Despite huge answer diversities amoung compounds, hydric stress intensity and plants; camphre pathway terpenes (borneol, comphene and camphre) are those which have the more important variations among stressed and controlled plants. Therefore study camphre biosynthesis pathway emerged. In this context, we have identified and characterized the bornyl diphosphate synthase able to produce the borneol from the bornyl diphosphate. These works allow a better understanding of relationships between volatils terpenes production and environment as well as give genetical tools to proceed to further investigations