SECONDARY METABOLITES FROM NUDIBRANCHS Tambja stegosauriformis, Hypselodoris lajenis AND Okenia zoobotryon AND FROM BRYOZOANS Zoobotryon verticillatum AND Bugula dentata FROM THE BRAZILIAN COASTLINE

SECONDARY METABOLITES FROM NUDIBRANCHS Tambja stegosauriformis, Hypselodoris lajenis AND Okenia zoobotryon AND FROM BRYOZOANS Zoobotryon verticillatum AND Bugula dentata FROM THE BRAZILIAN COASTLINE. The chemical investigation of the MeOH extract from the bryozoan B. dentata MeOH yielded tambjamines A (1), C (3), D (4), K (6), aldehyde 8 and the new tambjamine J1(9), while the extract of its predator, the nudibranch Tambja stegosauriformis, yielded tambjamines C and K, along with aldehyde 8. Furodisinin lactone (11) was isolated from the nudibranch Hypselodoris lajensis, a compound previously isolated from Dysidea sponges. The alkaloid 2,5,6-tribromo-N-methylgramine (12) was isolated from the nudibranch Okenia zoobotryon and from its prey, the bryozoan Zoobotryon verticillatum, the only source of 12 previously known.

Towards new botanical pesticides: the toxic effect of Eremanthus goyazensis (Asteraceae) leaves essential oil against Brevipalpus phoenicis (Acari: Tenuipalpidae)

This work reports the chemical characterization of Eremanthusgoyzensis essential oil and its toxic effect over Brevipalpus phoenicis. The essential oil displayed a major composition of sesquiterpenes (61.87%) including trans-caryophillene (26.81%) and germacrene-D (13.31%). The fumigation test indicated a promising bioactivity over adult B. phoenicis individuals at 24 h (2.03 µL/L of air) and 48 h (1.08 µL/L of air) of exposition. A brief discussion of essential oils composition and their singular role on the toxic effect over B. phoenicis is provided here. Our results may contribute to a new and profitable use of a species of Brazilian flora on agribusiness.

Comparative analysis of the corps en cerise in several species of Laurencia (Ceramiales, Rhodophyta) from the Atlantic Ocean

Different species of Laurencia have proven to be a rich source of natural products yielding interesting bioactive halogenated secondary metabolites, such as terpenoids and acetogenins. It is shown that such compounds are accumulated in the spherical, reniform to claviform refractive inclusions called corps en cerise (CC), which are intensively osmiophilic and located mainly in the cortical cells of the thalli and also in trichoblast cells. Up to now, it was believed that CC were present only in these two kinds of cells. Recently, however, a species of Laurencia, L. marilzae, with CC in all cells of the thallus, i.e., cortical, medullary, including the pericentral and axial cells, as well as in the trichoblasts, was described from the Canary Islands, and subsequently also reported to Brazil and Mexico. Within the Laurencia complex, only Laurencia species produce CC. Since the species of Laurencia are targets of interest for the prospection of bioactive substances due to their potential antibacterial, antifungal, anticholinesterasic, antileishmanial, cytotoxic, and antioxidant activities, the present paper carries out a comparative analysis of the corps en cerise in several species of Laurencia from the Atlantic Ocean to obtain basic information that can support natural product bioprospection projects. Our results show that the number and size of the CC are constant within a species, independent of the geographical distribution, corroborating their use for taxonomical purposes to differentiate groups of species that present a lower number from those that have a higher number. In this regard, there was a tendency for the number of CC to be higher in some species of Laurencia from the Canary Islands. The presence of CC can also be used to distinguish species in which these organelles are present in all cells of the thallus from those in which CC are restricted to the cortical cells. Among the species analyzed, L. viridis displayed the most varied secondary metabolites composition, such as sesquiterpenes, diterpenes, triterpenes, all of which showed potent antiviral, cytotoxic, and antitumoral activities, including protein phosphatase type 2A (PP2A) inhibitory effects.

Massenspektrometrische Untersuchungen und Nachweise von organischen Hydroperoxiden und höhermolekularen Verbindungen im biogenen sekundären organischen Aerosol

Atmosphärische Aerosole beeinflussen den Strahlungshaushalt und damit das Klima der Erde. Dies geschieht sowohl direkt (Streuung und Absorption), als auch indirekt (Wolkenkondensationskeime). Das sekundäre organische Aerosol (SOA) bildet einen wichtigen Bestandteil des atmosphärischen Aerosols. Seine Bildung erfolgt durch Reaktionen von Kohlenwasserstoffen mit atmosphärischen Oxidationsmitteln (z.B. Ozon, OH-Radikalen). Eine Klasse dieser Kohlenwasserstoffe sind die Terpene. Sie werden in großen Mengen durch die Vegetation emittiert und gelten als wichtige Vorläufersubstanzen des biogenen SOAs. In den Reaktionen von Monoterpenen und Sesquiterpenen mit atmosphärischen Reaktionspartnern wird eine große Vielfalt an multifunktionellen Reaktionsprodukten gebildet, von denen bis heute nur ein Bruchteil identifiziert werden konnte. In der vorliegenden Arbeit soll im Speziellen die Bildung von organischen Peroxiden und oligomeren Verbindungen im biogenen SOA untersucht und Nachweise einzelner Moleküle erbracht werden.rnFür eine Identifizierung von organischen Peroxiden aus der Oxidation einzelner Monoterpene und Sesquiterpene mit Ozon wurden die Reaktionsprodukte direkt in eine bei Atmosphärendruck arbeitende chemische Ionisationsquelle überführt und massenspektrometrisch untersucht (online-APCI-MS). Hierdurch konnten organische Hydroperoxide in der Partikelphase nachgewiesen werden, welche sich durch eine signifikante Abspaltung von H2O2 im Tandem-Massenspektrum (MS/MS) auszeichneten. Des Weiteren sollte die Bildung von höhermolekularen Verbindungen („Dimere“) im SOA des α-Pinens untersucht werden. Hierfür wurden zunächst die Reaktionsprodukte des Cyclohexens, das als einfache Modellverbindung des α-Pinens dient, mittels online-APCI-MS und offline durch Flüssigkeitschromatographie und Elektrospray-Ionenfallenmassenspektrometrie (HPLC/ESI-MS) untersucht. Verschiedene Produkte der Cyclohexen-Ozonolyse konnten hierbei als Esterverbindungen identifiziert werden, wobei eigens synthetisierte Referenzsubstanzen für die Identifizierung verwendet wurden. In einem weiteren Experiment, indem gleichzeitig Cyclohexen und α-Pinen mit Ozon umgesetzt wurden, konnten ebenfalls eine Bildung von höhermolekularen Estern nachgewiesen werden. Es handelte sich hierbei um „Mischester“, deren Struktur aus Reaktionsprodukten der beiden VOC-Vorläufermoleküle aufgebaut war. Durch diese neuen Erkenntnisse, über die Bildung von Estern im SOA des Cyclohexens, wurden die Dimer-Bildung einer reinen α-Pinen/Ozon-Reaktion online und offline massenspektrometrisch untersucht. Hier stellten sich als Hauptprodukte die Verbindungen mit m/z 357 und m/z 367 ([M-H]--Ionen) heraus, welche zudem erstmals auf einem Filter einer Realprobe aus Hyytiälä, Finnland nachgewiesen werden konnten. Aufgrund ihrer Fragmentierung in MS/MS-Untersuchungen sowie den exakten Summenformeln aus FT-MS Messungen konnte für die Struktur der höhermolekularen Verbindung mit m/z 367 ebenfalls ein Ester und für m/z 357 ein Peroxyhemiacetal vorgeschlagen werden. Die vorgeschlagene Struktur der Verbindung m/z 367 konnte im Anschluss über eine Reaktion aus Hydroxypinonsäure mit Pinsäure bestätigt werden. Die Identifizierung der Esterverbindung des α-Pinen-SOA erfolgte ebenfalls mit Hilfe von LC-MSn-Messungen.rnDie bisher diskutierten Ergebnisse, sowie die meisten in der Literatur beschriebenen Studien befassen sich jedoch mit einzelnen Vorläuferverbindungen, im Gegensatz zu den komplexen SOA-Proben aus den Emissionen der Vegetation. Im Rahmen einer Messkampagne am Forschungszentrum Jülich erfolgte eine massenspektrometrische Charakterisierung (online-APCI-MS) des SOAs aus direkten VOC-Emissionen von Pflanzen. Durch einen Vergleich der Produktverteilung dieser erhalten online-Massenspektren mit denen aus den Reaktionen einzelner VOCs, konnten Aussagen über die in den Reaktionen umgesetzten VOCs gemacht werden. Es konnte gezeigt werden, dass in stressbedingten Situationen die untersuchten Exemplare der Betula pendula (Birke) hauptsächlich Sesquiterpene, Picea abies (Fichte) eher Monoterpene und Eucalyptus (Eukalyptus) sowohl Sesquiterpene als auch Monoterpene emittieren. Um die atmosphärischen Prozesse, die zur Bildung der Produkte im SOA führen vollständig zu verstehen, müssen jedoch noch weitere Anstrengungen unternommen werden.rn

Leaf level VOC emissions of single plants from Amazonian and Mediterranean ecosystems: Ontogeny and flooding as stress factor for VOC emissions

Die Vegetation ist die wichtigste Quelle von organischen flüchtigen Verbindungen (auf Englisch volatile organic compounds,VOCs), die einen bemerkenswerten Einfluss auf der Chemie und Physik der Atmosphäre haben. VOCs beeinflussen die oxidative Kapazität der Atmosphäre und tragen zu der Bildung und zum Wachstum von sekundären organischen Aerosolen bei, welche einerseits eine Streuung und Reflektierung der Energie verursachen und andererseits sich an der Bildung und Entwicklung von Wolken beteiligen. Ziel dieser Arbeit war die Beschreibung und der Vergleich von VOC Emissionen aus Pflanzen aus zwei verschiedenen Ökosystemen: Mediterranes Ökosystem und Tropisches Ökosystem. Für diese Aufgabe wurden gewöhnliche Pflanzen von beiden Ökosystemen untersucht. Siebzehn Pflanzenspezies aus der Mittelmeergebiet, welches bekannt ist für seine Vielfalt an VOC emittierenden Pflanzen, wurden in die Untersuchungen einbezogen. Im Gegensatz zum mediterranen Ökosystem sind nur wenig Information verfügbar über VOC Emissionen aus Blättern tropischer Baumspezies. Vor diesem Hintergrund wurden sechsundzwanzig Baumspezies aus verschiedenen Ökotypen des Amazonasbeckens (Terra firme, Várzea und Igapó) wurden auf VOC Emissionen auf Blattebene mit einem Küvetten-System untersucht. Analysen von flüchtigen organischen Verbindungen wurden online mit PTR-MS und offline mittels Sammlung auf entsprechenden Adsorbern (Kartuschen) und nachfolgender GC-FID Analyse untersucht. Die höchsten Emissionen wurden für Isoprene beobachtete, gefolgt durch Monoterpene, Methanol und Aceton. Die meisten Mittelmeer Spezies emittierten eine hohe Vielfalt an Monoterpenspezies, hingegen zeigten nur fünf tropische Pflanzenspezies eine Monoterpene mit einen sehr konservativen Emissionsprofil (α-Pinen>Limonen>Sabinen >ß-Pinen). Mittelmeerpflanzen zeigten zusätzlich Emissionen von Sesquiterpenen, während bei der Pflanzen des Amazonas Beckens keine Sesquiterpenemissionen gefunden wurden. Dieser letzte Befund könnte aber auch durch eine niedrigere Sensitivität des Messsystems während der Arbeiten im Amazonasgebiet erklärt werden. Zusätzlich zu den Isoprenoidemissionen waren Methanolemissionen als Indikator für Wachtumsvorgänge sehr verbreitet in den meisten Pflanzenspezies aus tropischen und mediterranen Gebieten. Einige Pflanzenspezies beider Ökosystemen zeigten Acetonemissionen. rnrnVOC Emissionen werde durch eine große Vielfalt an biotischen und abiotischen Faktoren wie Lichtintensität, Temperatur, CO2 und Trockenheit beeinflusst. Ein anderer, öfter übersehener Faktor, der aber sehr wichtig ist für das Amazonas Becken, ist die regelmäßige Überflutung. In dieser Untersuchung wir fanden heraus, dass am Anfang einer Wurzelanoxie, die durch die Überflutung verursacht wurde, Ethanol und Acetaldehyd emittiert werden können, vor allem in Pflanzenspezies, die schlechter an eine unzureichende Sauerstoffversorgung bei Flutung adaptiert sind, wie z.B. Vatairea guianensis. Die Spezies Hevea spruceana, welche besser an Überflutung adaptiert ist, könnte möglicherweise der gebildete Ethanol sofort remetabolisieren ohne es zu emittieren. Nach einer langen Periode einer Überflutung konnte allerdings keine Emission mehr beobachtet werden, was auf eine vollständige Adaptation mit zunehmender Dauer schließen lässt. Als Reaktion auf den ausgelösten Stress können Isoprenoidemissionen ebenfalls kurzfristig nach einigen Tage an Überflutung zunehmen, fallen dann aber dann nach einer langen Periode zusammen mit der Photosynthese, Transpiration und stomatäre Leitfähigkeit deutlich ab.rnrnPflanzen Ontogenese ist anscheinend von Bedeutung für die Qualität und Quantität von VOC Emissionen. Aus diesem Grund wurden junge und erwachsene Blätter einiger gut charakterisierten Pflanzen Spezies aus dem Mittelmeerraum auf VOC Emissionen untersucht. Standard Emissionsfaktoren von Isopren waren niedriger in jungen Blättern als in erwachsene Blätter. Hingegen wurden höhere Monoterpen- und Sesquiterpenemissionen in jungen Blätter einiger Pflanzenspezies gefunden. Dieser Befund deutet auf eine potentielle Rolle dieser VOCs als Abwehrkomponenten gegen Pflanzenfresser oder Pathogene bei jungen Blätter hin. In einigen Fällen variierte auch die Zusammensetzung der Monoterpen- und Sesquiterpenspezies bei jungen und erwachsenen Blättern. Methanolemissionen waren, wie erwartet, höher in jungen Blättern als in ausgewachsenen Blättern, was mit der Demethylierung von Pectin bei der Zellwandreifung erklärt werden kann. Diese Befunde zu Änderungen der Emissionskapazität der Vegetation können für zukünftige Modellierungen herangezogen werden. rn

Entwicklung einer Thermodesorptionseinheit für die GC/MS zur Bestimmung hochreaktiver, biogener Kohlenwasserstoffe und deren Anwendung im Rahmen von Labor- und Feldstudien

Flüchtige organische Verbindungen (volatile organic compounds, VOCs), besonders rnTerpene, gelten als Vorläufer des sekundären organischen Aerosols (secondary rnorganic aerosols, SOA). Terpene werden von Pflanzen zur Abwehr oder zur rnAttraktion von Bestäubern emittiert. Ungesättigten Verbindungen, wie Sesquiterpene, rnsind sehr ozonolyseempfindlich und weisen nur geringe Konzentrationen in der rnAtmosphäre auf. Zudem lassen sie sich mit handelsüblichen rnThermodesorptionseinheiten meist nicht ohne Artefakte nachweisen, da sie eine hohe rnReaktivität mit vielen Oberflächen zeigen. rnDiese Arbeit präsentiert Forschungsergebnisse der qualitativen und rn(semi)quantitativen Auswertungen flüchtiger organischer Verbindungen in rnLaborstudien und biogenen Emissionsproben (Feldmessungen) mittels rnThermodesorption-Gaschromatographie-Massenspektrometrie (TD-GC/MS). Speziell rnzur Analyse hochreaktiver Verbindungen wurde eine Thermodesorptionseinheit für die rnGC/MS entwickelt. Diese besteht aus einer Kryofokussierung, einem Desorptionsofen rnund einer Heizung. Die Steuerung erfolgt über eine eigens dafür geschaffene rnBedienoberf läche von Labview® an einem PC über eine nachgeschaltete SPS rn(speicherprogrammierbare Steuerung). Das komplette Desorptionsprogramm und der rnStart der GC/MS-Analyse wurden automatisiert. rnDie Quantifizierung alle Proben wurde über Diffusionsquellen und rnVergleichsmessungen durch auf Adsorptionsröhrchen aufgebrachte rnVerdünnungsreihen realisiert. Um Informationen über die mutmaßlichen Vorläufer des rnsekundären organischen Aerosols zu erhalten, wurde zudem eine Ozon-Scrubber-rnMethode basierend auf Propen entwickelt. Diese wurde anhand von Standards in einer rnReaktionskammer getestet und in Feldmessungen erfolgreich angewendet. rnQuantitative Analysen zeigen, dass die meisten Terpene so vollständig vor der rnOzonolyse bewahrt werden können. Für hochreaktive Analyte wie α-Humulen oder rnβ-Caryophyllen wurden Wiederfindungsraten von über 80 % erreicht. So konnte die rnTemperaturabhängigkeit der Terpen-Emissionen der Fichte (Picea abies) in rnFeldmessungen nachgewiesen werden. rnEine weitere Anwendungsmöglichkeit wurde mit der Unterscheidung verschiedener rnArten der gleichen Gattung anhand der Emissionsmuster und der möglichen rnAbgrenzung verschiedener Bestäubertypen am Beispiel der Gattung Salvia untersucht. rnDie Emissionsanalysen zeigen, dass eine Zuordnung der Verwandtschaftsverhältnisse rnzusätzlich zu anderen Vergleichen möglich ist. Das gleiche gilt für die Differenzierung rnvon Bestäubertypen. Die Ergebnisse der Feldmessungen wurden durch rnMethodenvergleich zwischen biogenen Emissionsmessungen mit anschließender rnTD-GC/MS-Analyse und Extraktionen der jeweiligen Blüten/Blätter mit rnanschließender GC/MS-Messung bestätigt.

Complementary mass spectrometric techniques for the characterization of the organic fraction in atmospheric aerosols

Aerosol particles are strongly related to climate, air quality, visibility and human health issues. They contribute the largest uncertainty in the assessment of the Earth´s radiative budget, directly by scattering or absorbing solar radiation or indirectly by nucleating cloud droplets. The influence of aerosol particles on cloud related climatic effects essentially depends upon their number concentration, size and chemical composition. A major part of submicron aerosol consists of secondary organic aerosol (SOA) that is formed in the atmosphere by the oxidation of volatile organic compounds. SOA can comprise a highly diverse spectrum of compounds that undergo continuous chemical transformations in the atmosphere.rnThe aim of this work was to obtain insights into the complexity of ambient SOA by the application of advanced mass spectrometric techniques. Therefore, an atmospheric pressure chemical ionization ion trap mass spectrometer (APCI-IT-MS) was applied in the field, facilitating the measurement of ions of the intact molecular organic species. Furthermore, the high measurement frequency provided insights into SOA composition and chemical transformation processes on a high temporal resolution. Within different comprehensive field campaigns, online measurements of particular biogenic organic acids were achieved by combining an online aerosol concentrator with the APCI-IT-MS. A holistic picture of the ambient organic aerosol was obtained through the co-located application of other complementary MS techniques, such as aerosol mass spectrometry (AMS) or filter sampling for the analysis by liquid chromatography / ultrahigh resolution mass spectrometry (LC/UHRMS).rnIn particular, during a summertime field study at the pristine boreal forest station in Hyytiälä, Finland, the partitioning of organic acids between gas and particle phase was quantified, based on the online APCI-IT-MS and AMS measurements. It was found that low volatile compounds reside to a large extent in the gas phase. This observation can be interpreted as a consequence of large aerosol equilibration timescales, which build up due to the continuous production of low volatile compounds in the gas phase and/or a semi-solid phase state of the ambient aerosol. Furthermore, in-situ structural informations of particular compounds were achieved by using the MS/MS mode of the ion trap. The comparison to MS/MS spectra from laboratory generated SOA of specific monoterpene precursors indicated that laboratory SOA barely depicts the complexity of ambient SOA. Moreover, it was shown that the mass spectra of the laboratory SOA more closely resemble the ambient gas phase composition, indicating that the oxidation state of the ambient organic compounds in the particle phase is underestimated by the comparison to laboratory ozonolysis. These observations suggest that the micro-scale processes, such as the chemistry of aerosol aging or the gas-to-particle partitioning, need to be better understood in order to predict SOA concentrations more reliably.rnDuring a field study at the Mt. Kleiner Feldberg, Germany, a slightly different aerosol concentrator / APCI-IT-MS setup made the online analysis of new particle formation possible. During a particular nucleation event, the online mass spectra indicated that organic compounds of approximately 300 Da are main constituents of the bulk aerosol during ambient new particle formation. Co-located filter analysis by LC/UHRMS analysis supported these findings and furthermore allowed to determine the molecular formulas of the involved organic compounds. The unambiguous identification of several oxidized C 15 compounds indicated that oxidation products of sesquiterpenes can be important compounds for the initial formation and subsequent growth of atmospheric nanoparticles.rnThe LC/UHRMS analysis furthermore revealed that considerable amounts of organosulfates and nitrooxy organosulfates were detected on the filter samples. Indeed, it was found that several nitrooxy organosulfate related APCI-IT-MS mass traces were simultaneously enhanced. Concurrent particle phase ion chromatography and AMS measurements indicated a strong bias between inorganic sulfate and total sulfate concentrations, supporting the assumption that substantial amounts of sulfate was bonded to organic molecules.rnFinally, the comprehensive chemical analysis of the aerosol composition was compared to the hygroscopicity parameter kappa, which was derived from cloud condensation nuclei (CCN) measurements. Simultaneously, organic aerosol aging was observed by the evolution of a ratio between a second and a first generation biogenic oxidation product. It was found that this aging proxy positively correlates with increasing hygroscopicity. Moreover, it was observed that the bonding of sulfate to organic molecules leads to a significant reduction of kappa, compared to an internal mixture of the same mass fractions of purely inorganic sulfate and organic molecules. Concluding, it has been shown within this thesis that the application of modern mass spectrometric techniques allows for detailed insights into chemical and physico-chemical processes of atmospheric aerosols.rn

TERPENE AND TERPENOID EMISSIONS AND SECONDARY ORGANIC AEROSOL PRODUCTION

Approximately 90% of fine aerosol in the Midwestern United States has a regional component with a sizable fraction attributed to secondary production of organic aerosol (SOA). The Ozark Forest is an important source of biogenic SOA precursors like isoprene (> 150 mg m-2 d-1), monoterpenes (10-40 mg m-2 d-1), and sesquiterpenes (10-40 mg m-2d-1). Anthropogenic sources include secondary sulfate and nitrate and biomass burning (51-60%), vehicle emissions (17-26%), and industrial emissions (16-18%). Vehicle emissions are an important source of volatile and vapor-phase, semivolatile aliphatic and aromatic hydrocarbons that are important anthropogenic sources of SOA precursors. The short lifetime of SOA precursors and the complex mixture of functionalized oxidation products make rapid sampling, quantitative processing methods, and comprehensive organic molecular analysis essential elements of a comprehensive strategy to advance understanding of SOA formation pathways. Uncertainties in forecasting SOA production on regional scales are large and related to uncertainties in biogenic emission inventories and measurement of SOA yields under ambient conditions. This work presents a bottom-up approach to develop a conifer emission inventory based on foliar and cortical oleoresin composition, development of a model to estimate terpene and terpenoid signatures of foliar and bole emissions from conifers, development of processing and analytic techniques for comprehensive organic molecular characterization of SOA precursors and oxidation products, implementation of the high-volume sampling technique to measure OA and vapor-phase organic matter, and results from a 5 day field experiment conducted to evaluate temporal and diurnal trends in SOA precursors and oxidation products. A total of 98, 115, and 87 terpene and terpenoid species were identified and quantified in commercially available essential oils of Pinus sylvestris, Picea mariana, and Thuja occidentalis, respectively, by comprehensive, two-dimensional gas chromatography with time-of-flight mass spectrometric detection (GC × GC-ToF-MS). Analysis of the literature showed that cortical oleoresin composition was similar to foliar composition of the oldest branches. Our proposed conceptual model for estimation of signatures of terpene and terpenoid emissions from foliar and cortical oleoresin showed that emission potentials of the foliar and bole release pathways are dissimilar and should be considered for conifer species that develop resin blisters or are infested with herbivores or pathogens. Average derivatization efficiencies for Methods 1 and 2 were 87.9 and 114%, respectively. Despite the lower average derivatization efficiency of Method 1, distinct advantages included a greater certainty of derivatization yield for the entire suite of multi- and poly-functional species and fewer processing steps for sequential derivatization. Detection limits for Method 1 using GC × GC- ToF-MS were 0.09-1.89 ng μL-1. A theoretical retention index diagram was developed for a hypothetical GC × 2GC analysis of the complex mixture of SOA precursors and derivatized oxidation products. In general, species eluted (relative to the alkyl diester reference compounds) from the primary column (DB-210) in bands according to n and from the secondary columns (BPX90, SolGel-WAX) according to functionality, essentially making the GC × 2GC retention diagram a Carbon number-functionality grid. The species clustered into 35 groups by functionality and species within each group exhibited good separation by n. Average recoveries of n-alkanes and polyaromatic hydrocarbons (PAHs) by Soxhlet extraction of XAD-2 resin with dichloromethane were 80.1 ± 16.1 and 76.1 ± 17.5%, respectively. Vehicle emissions were the common source for HSVOCs [i.e., resolved alkanes, the unresolved complex mixture (UCM), alkylbenzenes, and 2- and 3-ring PAHs]. An absence of monoterpenes at 0600-1000 and high concentrations of monoterpenoids during the same period was indicative of substantial losses of monoterpenes overnight and the early morning hours. Post-collection, comprehensive organic molecular characterization of SOA precursors and products by GC × GC-ToFMS in ambient air collected with ~2 hr resolution is a promising method for determining biogenic and anthropogenic SOA yields that can be used to evaluate SOA formation models.

Two enzymes responsible for the formation of herbivore-induced volatiles of maize, the methyltransferase AAMT1 and the terpene synthase TPS23, are regulated by a similar signal transduction pathway

Herbivore-induced volatiles play an important role in the indirect defense of plants. After herbivore damage, volatiles are released from the plant and can attract herbivore enemies that protect the plant from additional damage. The herbivore-induced volatile blend is complex and usually consists of mono- and sesquiterpenes, aromatic compounds, and indole. Although these classes of compounds are generally produced at different times after herbivore damage, the release of the terpene (E)-β-caryophyllene and the aromatic ester methyl anthranilate appear to be tightly coordinated. We have studied the herbivore induction patterns of two terpene synthases from Zea mays L. (Poaceae), TPS23 and TPS10, as well as S-adenosyl-L-methionine:anthranilic acid carboxyl methyltransferases (AAMT1), which are critical for the production of terpenes and anthranilate compounds, respectively. The transcript levels of tps23 and aamt1 displayed the same kinetics after damage by the larvae of Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), and showed the same organ-specific and haplotype-specific expression patterns. Despite its close functional relation to TPS23, the terpene synthase TPS10 is not expressed in roots and does not display the haplotype-specific expression pattern. The results indicate that the same JA-mediated signaling cascade maycontrol the production of both the terpene (E)-β-caryophyllene and aromatic ester methyl anthranilate.

Less is More: Treatment with BTH and Laminarin Reduces Herbivore-Induced Volatile Emissions in Maize but Increases Parasitoid Attraction

Chemical plant strengtheners find increasing use in agriculture to enhance resistance against pathogens. In an earlier study, it was found that treatment with one such resistance elicitor, BTH (benzo-(1, 2, 3)-thiadiazole-7-carbothioic acid S-methyl ester), increases the attractiveness of maize plants to a parasitic wasp. This surprising additional benefit of treating plants with BTH prompted us to conduct a series of olfactometer tests to find out if BTH and another commercially available plant strengthener, Laminarin, increase the attractiveness of maize to three important parasitic wasps, Cotesia marginventris, Campoletis sonorensis, and Microplitis rufiventris. In each case, plants that were sprayed with the plant strengtheners and subsequently induced to release volatiles by real or mimicked attack by Spodoptera littoralis caterpillars became more attractive to the parasitoids than water treated plants. The elicitors alone or in combination with plants that were not induced by herbivory were not attractive to the wasps. Interestingly, plants treated with the plant strengtheners did not show any consistent increase in volatile emissions. On the contrary, treated plants released less herbivore-induced volatiles, most notably indole, which has been reported to interfere with parasitoid attraction. The emission of the sesquiterpenes (E)-β-caryophyllene, β-bergamotene, and (E)-β-farnesene was similarly reduced by the treatment. Expression profiles of marker genes showed that BTH and Laminarin induced several pathogenesis related (PR) genes. The results support the notion that, as yet undetectable and unidentified compounds, are of major importance for parasitoid attraction, and that these attractants may be masked by some of the major compounds in the volatile blends. This study confirms that elicitors of pathogen resistance are compatible with the biological control of insect pests and may even help to improve it.

Herbivore-Induced Plant Volatiles Can Serve as Host Location Cues for a Generalist and a Specialist Egg Parasitoid

Herbivore-induced plant volatiles are important host finding cues for larval parasitoids, and similarly, insect oviposition might elicit the release of plant volatiles functioning as host finding cues for egg parasitoids. We hypothesized that egg parasitoids also might utilize HIPVs of emerging larvae to locate plants with host eggs. We, therefore, assessed the olfactory response of two egg parasitoids, a generalist, Trichogramma pretiosum (Tricogrammatidae), and a specialist, Telenomus remus (Scelionidae) to HIPVs. We used a Y-tube olfactometer to tests the wasps’ responses to volatiles released by young maize plants that were treated with regurgitant from caterpillars of the moth Spodoptera frugiperda (Noctuidae) or were directly attacked by the caterpillars. The results show that the generalist egg parasitoid Tr. pretiosum is innately attracted by volatiles from freshly-damaged plants 0–1 and 2–3 h after regurgitant treatment. During this interval, the volatile blend consisted of green leaf volatiles (GLVs) and a blend of aromatic compounds, mono- and homoterpenes, respectively. Behavioral assays with synthetic GLVs confirmed their attractiveness to Tr. pretiosum. The generalist learned the more complex volatile blends released 6–7 h after induction, which consisted mainly of sesquiterpenes. The specialist T. remus on the other hand was attracted only to volatiles emitted from fresh and old damage after associating these volatiles with oviposition. Taken together, these results strengthen the emerging pattern that egg and larval parasitoids behave in a similar way in that generalists can respond innately to HIPVs, while specialists seems to rely more on associative learning.

Isopentenyl diphosphate biosynthesis via a mevalonate-independent pathway: Isopentenyl monophosphate kinase catalyzes the terminal enzymatic step

In plants, the biosynthesis of isopentenyl diphosphate, the central precursor of all isoprenoids, proceeds via two separate pathways. The cytosolic compartment harbors the mevalonate pathway, whereas the newly discovered deoxyxylulose 5-phosphate pathway, which also operates in certain eubacteria, including Escherichia coli, is localized to plastids. Only the first two steps of the plastidial pathway, which involve the condensation of pyruvate and glyceraldehyde 3-phosphate to deoxyxylulose 5-phosphate followed by intramolecular rearrangement and reduction to 2-C-methylerythritol 4-phosphate, have been established. Here we report the cloning from peppermint (Mentha × piperita) and E. coli, and expression, of a kinase that catalyzes the phosphorylation of isopentenyl monophosphate as the last step of this biosynthetic sequence to isopentenyl diphosphate. The plant gene defines an ORF of 1,218 bp that, when the proposed plastidial targeting sequence is excluded, corresponds to ≈308 aa with a mature size of ≈33 kDa. The E. coli gene (ychB), which is located at 27.2 min of the chromosomal map, consists of 852 nt, encoding a deduced enzyme of 283 aa with a size of 31 kDa. These enzymes represent a conserved class of the GHMP family of kinases, which includes galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase, with homologues in plants and several eubacteria. Besides the preferred substrate isopentenyl monophosphate, the recombinant peppermint and E. coli kinases also phosphorylate isopentenol, and, much less efficiently, dimethylallyl alcohol, but dimethylallyl monophosphate does not serve as a substrate. Incubation of secretory cells isolated from peppermint glandular trichomes with isopentenyl monophosphate resulted in the rapid production of monoterpenes and sesquiterpenes, confirming that isopentenyl monophosphate is the physiologically relevant, terminal intermediate of the deoxyxylulose 5-phosphate pathway.

Regulation of Oleoresinosis in Grand Fir (Abies grandis)1 : Differential Transcriptional Control of Monoterpene, Sesquiterpene, and Diterpene Synthase Genes in Response to Wounding

Grand fir (Abies grandis Lindl.) has been developed as a model system for the study of wound-induced oleoresinosis in conifers as a response to insect attack. Oleoresin is a roughly equal mixture of turpentine (85% monoterpenes [C10] and 15% sesquiterpenes [C15]) and rosin (diterpene [C20] resin acids) that acts to seal wounds and is toxic to both invading insects and their pathogenic fungal symbionts. The dynamic regulation of wound-induced oleoresin formation was studied over 29 d at the enzyme level by in vitro assay of the three classes of synthases directly responsible for the formation of monoterpenes, sesquiterpenes, and diterpenes from the corresponding C10, C15, and C20 prenyl diphosphate precursors, and at the gene level by RNA-blot hybridization using terpene synthase class-directed DNA probes. In overall appearance, the shapes of the time-course curves for all classes of synthase activities are similar, suggesting coordinate formation of all of the terpenoid types. However, closer inspection indicates that the monoterpene synthases arise earlier, as shown by an abbreviated time course over 6 to 48 h. RNA-blot analyses indicated that the genes for all three classes of enzymes are transcriptionally activated in response to wounding, with the monoterpene synthases up-regulated first (transcripts detectable 2 h after wounding), in agreement with the results of cell-free assays of monoterpene synthase activity, followed by the coordinately regulated sesquiterpene synthases and diterpene synthases (transcription beginning on d 3–4). The differential timing in the production of oleoresin components of this defense response is consistent with the immediate formation of monoterpenes to act as insect toxins and their later generation at solvent levels for the mobilization of resin acids responsible for wound sealing.

Pathway of terpene excretion by the appendix of Sauromatum guttatum.

Electron microscopy of the cells of the thermogenic appendix of Sauromatum guttatum has revealed a fusion event between pocket-like structures of the rough endoplasmic reticulum (rER) and the plasma membrane. As a result of the fusion event, many regions of the plasma membrane have paired unit membranes (four leaflets instead of two). The fusion allows the transfer of osmiophilic material from the rER pockets to the plasma membrane, where the osmiophilic material is confined to bilayer, pocket-like structures. A clear correlation is found between the presence of the osmiophilic compound and sesquiterpenes. Prior to heat production, the rER- and plasma-membrane pockets are electron dense, and sesquiterpenes are detectable only in tissue extracts. On the day of heat production, electron-translucent pockets are subsequently found and the stored sesquiterpenes are released to the atmosphere. Three sesquiterpenes have been identified by gas chromatography-mass spectrometry as alpha-copaene and beta- and alpha-caryophyllene.

Estudos visando à síntese de sesquiterpenos bacanos

Nesta tese, efetuamos estudos visando à síntese de sesquiterpenos bacanos, cuja etapa chave consistiu na construção do sistema cis-hidrindânico, através de reação de contração de anel de cis-octalinas e 2-octalonas mediada por trinitrato de tálio (TTN). Apenas as cis-octalinas como, por exemplo, o cis-4a-metil-l,2,3,4,4a,5,8,8a-octahidronaftaleno e o cis-4a, 7-dimetil-l,2,3,4,4a,5,8,8a-octa-hidronaftaleno, foram passíveis de reação de contração de anel em rendimentos satisfatórios; já a cis-5,10-dimetil-l(9)-octal-2-ona levou ao produto de contração em baixo rendimento. Tentamos utilizar a reação de cis-4a-metil-l,2,3,4,4a,5,8,8a-octa-hidronaftaleno com TTN na síntese da nor-baquenolida-A, porém não conseguimos completar a síntese desta, pois não foi possível efetuar a última etapa sintética, nas várias abordagens testadas. Grandes esforços também foram empregados na preparação diastereosseletiva da cis-5,10-dimetil-l(9)-octal-2-ona através de três abordagens diferentes que foram investigadas, sendo duas delas com êxito. Contudo, o baixo rendimento (38%) da etapa de contração de anel da cis-5,10-dimetil-l(9)-octal-2-ona não permitiu a continuação da rota sintética proposta para a baquenolida-A. Também realizamos a resolução cinética de três diferentes cis-octalóis que foram preparados através da reação de Diels-Alder seguida de redução diastereosseletiva - com a lipase Novozym 435, e os produtos resolvidos foram obtidos em excelentes rendimentos isolados (≥ 40% para cada enantiômero) e excelentes excessos enantioméricos (≥ 98%).