Plants have evolved a variety of constitutive and induced chemical defense mechanisms against biotic stress. Emission of volatile compounds from plants facilitates interactions with both beneficial and pathogenic organisms. However, knowledge of the chemical defense in roots is still limited. In this study, we have examined the root-specific biosynthesis and function of volatile terpenes in the model plant Arabidopsis. When infected with the root rot pathogen Pythium irregulare, Arabidopsis roots release the acyclic C11-homoterpene (E)-4,8-dimethylnona-1,3,7-triene (DMNT), which is a common constituent of volatile blends emitted from insect-damaged foliage. We have identified a single cytochrome P450 monooxygenase of the CYP705 family that catalyzes a root-specific oxidative degradation of the C30-triterpene precursor arabidiol thereby causing the release of DMNT and a C19-degradation product named arabidonol. We found that DMNT shows inhibitory effects on P. irregulare mycelium growth and oospore germination in vitro, and that DMNT biosynthetic mutant plants were more susceptible to P. irregulare infection. We provide evidence based on genome synteny and phylogenetic analysis that the arabidiol biosynthetic gene cluster containing the arabidiol synthase (ABDS) and CYP705A1 genes possibly emerged via local gene duplication followed by de novo neofunctionalization. Together, our studies demonstrate differences and plasticity in the metabolic organization and function of terpenes in roots in comparison to aboveground plant tissues. Additionally, we demonstrated that the arabidiol cleavage product, arabidonol, is further modified by yet unknown enzymatic reactions into three products, which are found in root exudates. We suggested a pathway for their biosynthesis based on precursor feeding experiments and NMR analysis. Although DMNT biosynthetic genes are clustered on chromosome 4 along with several potential modification genes, we did not find a possible role of these genes in the derivatization of arabidonol. Preliminary experimental results using genetic and biochemical approaches for identifying genes involved in the modification steps are also presented. In summary, this study demonstrates an alternative route for volatile terpene formation belowground different from aboveground plant tissues via triterpene degradation and provides evidence for an unexplored triterpene catabolism pathway in Arabidopsis. / Ph. D.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18104号 / 理博第3982号 / 新制||理||1574(附属図書館) / 30962 / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 森 哲, 教授 沼田 英治, 教授 疋田 努 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(理学) / 甲第24453号 / 理博第4952号 / 新制||理||1707(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 森 哲, 教授 中務 真人, 教授 中川 尚史 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Parasitic wasps are highly diverse and play a major role in suppression of herbivorous pest populations, but relatively little is known of the mechanisms driving their diversity. Molecular studies indicate that cryptic species complexes resulting from adaptations to specific hosts or host-foodplants may be common. The gregarious endoparasitoid, Cotesia congregata (Braconidae), is a model system for understanding parasitic wasp biology. It is reported to attack at least 15 species of sphingid caterpillars, most of which are plant family specialists. Molecular studies have demonstrated genetic differentiation of two host-foodplant complex sources originating from Manduca sexta on tobacco (MsT) and Ceratomia catalpae on catalpa (CcC). Response to female pheromone and elements of their courtship songs differ. Wasps from both sources mated and produced F1 hybrid offspring in the laboratory; however, 90% of hybrid females resulting from one of the reciprocal crosses failed to produce offspring. I built on this previous work by evaluating an ecological barrier, the evolution of courtship songs within the genus, and patterns of hybrid sterility among four additional host-foodplant complexes, as well as differentiation of their symbiotic bracovirus. Tests of developmental tolerance to nicotine demonstrate that MsT wasps are highly adapted to hosts feeding on tobacco, whereas CcC wasps experience high mortality. Acoustic analysis of courtship songs among host-foodplant sources of C. congregata and eleven additional species of Cotesia demonstrates that songs are species specific and appear to be correlated with genetic relatedness. Cotesia congregata from all sources mated and produced F1 hybrid offspring in the laboratory; however, hybrid females resulting from specific reciprocal crosses failed to produce progeny. Dissections of hybrid females revealed that sterile wasps lacked mature ovaries and functional bracovirus, a symbiotic virus integrated into the wasp genome and necessary to suppress the host immune system. Relative in vivo expression of wasp bracovirus genes differs between MsT and CcC host-foodplant complexes. Cumulatively, these behavioral, ecological, and genetic barriers to reproduction indicate that C. congregata is diverged into two incipient species with limited gene flow, and provides insight into the role of varied reproductive barriers in speciation of parasitic wasps.

In the fall of 1987, several areas of Florida Bay were severely affected by the sudden die-off of the seagrass Thalassia testudinum Banks ex Konig (turtle grass). Although the cause is still unknown, several factors were suggested as influencing the on-set of the die-off event including increased salinity, light stress due to self-shading, and disease. Blades of seagrass plants found in the area of die-off were infected by Labyrinthula sp, a pathogenic protist. A similar die-off occurred in another species of seagrass, Zostera marina, in the 1930s that was attributed to the pathogenic protist, Labyrinthula zosterae. Zostera marina produces inhibitory phenolic acids in response to infection by L. zosterae, a response that is diminished in plants exposed to low light and high temperature. This study examined the differences in phenolic content of healthy and infected T. testudinum leaf blades in laboratory cultures to determine if T. testudinum produces a chemical defense against pathogens similar to that of Z. marina. The possible increased susceptibility of turtle grass to Labyrinthula sp. infection under high salinity and low light was also examined. In culture, infection by Labyrinthula sp. induced a rapid, short-term production of total phenolics in Thalassia testudinum under normal, non-stressed conditions. The initial induction was followed by a sharp decline. The production of individual phenolic acids was not induced by infection. In contrast, the production of caffeic acid was inhibited by infection. Environmental stress (low salinity and low light) caused a decrease in both total phenolics and several phenolic acids. Levels of PHBA, vanillic acid, and caffeic acid decreased in low salinity (25ppt) treatments, and caffeic acid decreased in response to low light stress. There was an interaction between stress and infection that resulted in higher levels of phenolics in plants exposed to infection and stress compared to those exposed to stress alone. In culture, plants did not survive exposure to high salinity (45ppt) similar to that found in Florida Bay during the die-off event

This thesis describes the chemical investigation of three marine sponges from Antarctica and the total syntheses of natural products erebusinone (12) and its derivative, erebusinonamine (52). Investigation of the yellow Antarctic marine sponge Isodictya setifera resulted in the isolation of two secondary metabolites, purine analog (32) and 3-hydroxykynurenine (24). Chemical investigation of Isodictya setifera led to the isolation of six secondary metabolites which included 5-methyl-2-deoxycytidine (25), uridine (28), 2-deoxycytidine (31), homarine (37), hydroxyquinoline (33), 3-hydroxykynurenine (24). The latter two compounds were found to be intermediates of tryptophan catabolism in crustaceans. From the Antarctic marine sponge Isodictya antractica ceramide analog (39) was isolated and its chemical structure was assigned by a combination of spectroscopic and chemical analyses. Stereochemistry was determined by modified Mosher's method. Erebusinone (12), a yellow pigment isolated from the Antarctic marine sponge Isodictya erinacea has been implicated in molt inhibition and mortality against the Antarctic crustacean amphipod, Orchomene plebs, possibly serving as a precursor of a xanthurenic acid analog. Thought to act as a 3-hydroxykynurenine 24 mimic, erebusinone (12) may be involved chemical defense. This appears to be the first example in the marine realm of an organism utilizing tryptophan catabolism to modulate molting as a defensive mechanism. To further investigate the bioactivity and ecological role of erebusinone (12), the synthesis of this pigment was carried out in an overall yield of 44% involving seven steps which were economical and convenient. Erebusinonamine (52) was also similarly synthesized in eight steps with an overall yield of 45%.

Because organisms have limited resources to allocate to multiple life history traits, the Optimal Defense Theory (ODT) and the Growth-Differentiation Balance Hypothesis (GDBH) were developed by terrestrial plant ecologists to predict intraindividual defense allocation based on the cost of defense and these life history trade-offs. However, these theories have garnered equivocal experimental support over the years and are rarely experimentally extended from predictions of plant physiology to the palatability of the tissues an herbivore experiences. We therefore examined tissue palatability, nutritional value, and defense mechanisms in multiple Dictyotalean seaweeds in two Caribbean locations, using two herbivores. Relative palatability of tissues varied greatly with algal species, grazer species, and location. Because older bases were not consistently defended, GDBH did not predict relative palatability. We could not reject ODT without intensive measures of tissue fitness value and herbivore risk, and this theory was therefore not useful in making broad predictions of tissue palatability. In testing the physiological predictions of these theories, we found the young, growing apices of these seaweeds to be generally more nutritionally valuable than the old, anchoring bases and found organic-rich apices to be more chemically deterrent, thus supporting ODT. However, the combined chemical, nutritional, and structural traits of these algae all influenced herbivore choice. As a result, these patterns of apical value and chemical defense reflected palatability of live tissues for only one of five algal species, which rendered ODT and GDBH poor predictors of relative palatability for most algae.

Orientador: Anita Jocelyne Marsaioli / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-06T07:10:56Z (GMT). No. of bitstreams: 1 Pomini_ArmandoMateus_M.pdf: 2721194 bytes, checksum: b3d63d99fb0e61f807557af1f715e4ea (MD5) Previous issue date: 2006 / Resumo: Este trabalho abordou dois tópicos relacionados à comunicação química inter e intraespecífica. O primeiro tópico descreveu a produção de sinalizadores químicos da classe das acil-homosserina lactonas (acil-HSLs) por bactérias Gram-negativas fitopatogênicas, em mecanismos comunicativos conhecidos como quorum-sensing. Foram estudadas três espécies: Erwinia psidii RODRIGUES, que causa a principal bacteriose da goiabeira no Brasil;Pantoea ananatis SERRANO, um fitopatógeno de distribuição mundial e que causa perdas em diversas culturas agrícolas e Pantoea agglomerans EWING & FIFE, isolada da doença da ¿pinta branca do milho¿. O estudo propiciou a identificação de diversos metabólitos da classe das acil-HSLs, sendo que os produtos majoritários tiveram suas configurações absolutas estabelecidas. Avaliou-se ainda a atividade biológica de extratos, frações e produtos sintéticos com o biossensor Agrobacterium tumefaciens NTL4(pZLR4), em ensaios de expressão de enzimas ß-galactosidase. Trata-se de um trabalho pioneiro no Instituto de Química da Unicamp. Na segunda linha de pesquisas desenvolvida, o enfoque recaiu sobre alomônios produzidos por opiliões (Arachnida: Opiliones) e utilizados em XII mecanismos de defesa química contra predadores e na defesa territorial. Estudou-se as secreções de defesa das espécies Goniosoma longipes ROEWER e Camarana flavipalpi SOARES, nativas do Estado de São Paulo. Este trabalho rendeu a identificação de benzoquinonas substituídas e um fenol, inclusive com a caracterização de um produto natural inédito (2-etil-3-metil-1,4- benzoquinona) / Abstract: This work encompasses two different topics, both related to the intra and interspecific chemical communication. The first part concerns to the production of acyl-homoserine lactones (acyl-HSLs), which are signalling substances of Gram-negative, plant pathogenic bacteria. These substances are responsible for their chemical communication, in a process known as quorumsensing. Three species were studied: Erwinia psidii RODRIGUES, which causes the most important bacteriosis in Brazilian guava crops; Pantoea ananatis SERRANO, a worldwide-spread phytopathogen, which causes crop losses in different plant species and Pantoea agglomerans EWING & FIFE, isolated from the ¿white spot disease¿ of maize in Brazil. This study allowed the identification of four acyl-HSLs, and the absolute configurations of the most abundant substances were established. The activities of the extracts, fractions and synthetic products were evaluated using the bioreporter Agrobacterium tumefaciens NTL4(pZLR4), in ß-galactosidase expression assays. This is a pioneer work at the Chemistry Institute at Unicamp. The second research topic was on the opilionids allomones (Arachnida: Opiliones), employed in chemical defense mechanisms against predators and in territorial defense. The exudates of Goniosoma longipes ROEWER and Camarana flavipalpi SOARES, both native from São Paulo State (Brazil), were chemically characterized, allowing the identification of a known substituted XIV benzoquinone and a phenol, including a new natural product (2-ethyl-3- methyl-1,4-benzoquinone) / Mestrado / Quimica Organica / Mestre em Química

Orientador: Jose Roberto Trigo / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T19:03:33Z (GMT). No. of bitstreams: 1 Massuda_KamilaFerreira_M.pdf: 3824014 bytes, checksum: d4db5cb4afb22e7d40b4afc3d0677236 (MD5) Previous issue date: 2008 / Resumo: As defesas químicas em lepidópteros compreendem mecanismos que vão desde o seqüestro de compostos do metabolismo secundário de plantas até a biossíntese de novo de compostos que podem torná-los tóxicos ou impalatáveis. As larvas da borboleta Methona themisto (Nymphalidae: Ithomiinae), que apresentam coloração conspícua e se alimentam apenas da solanácea Brunfelsia uniflora, rica em compostos do metabolismo secundário, foram analisadas sob vários aspectos, para verificar se são quimicamente defendidas. O acompanhamento da sobrevivência das larvas indicou que a predação afeta significativamente a sobrevivência em seu ambiente natural. As defesas químicas dessas larvas são aparentemente ineficazes contra predadores invertebrados, como a aranha Lycosa erythrognatha e a formiga Camponotus crassus (100% dos indivíduos testados predaram as larvas), mas parecem ser eficazes contra os mantídeos Oxyopsis saussuurei (redução no tempo de manipulação da presa e predação em um segundo contato). Para predadores vertebrados como o lagarto Tropidurus itambere e a ave Gallus gallus, a defesa parece atuar em relação à palatabilidade e à coloração conspícua. Gallus gallus apresentou maior predação de larvas de 1o ínstar, sugerindo que há um incremento na impalatabilidade da larva no decorrer de seu desenvolvimento. Os testes de aprendizagem dos pintinhos demonstraram que com poucos contatos com a presa impalatável já se obtém uma resposta de rejeição visual. O emprego de uma larva de coloração semelhante a da Methona themisto indica que os pintinhos são capazes de relacionar a cor com o gosto desagradável, rejeitando assim uma presa palatável. Apenas extratos diclorometânicos das larvas testados com Gallus gallus foram significativamente rejeitados em relação a seus controles. Dessa forma, esses dados comprovam que as defesas químicas das larvas de Methona themisto atuam principalmente contra predadores vertebrados visualmente orientados / Abstract: Chemical defense in Lepidoptera involves several mechanisms such as sequestration of secondary metabolismcompounds of host plants and de novo synthesis of compounds that can provide some unpalatability or toxicity. The larvae of the butterfly Methona themisto (Nymphalidae: Ithomiinae) have a conspicuous coloration and feed exclusively on Brunfelsia uniflora (Solanaceae), that is rich in compounds of the secondary metabolismo These larvae were analyzed under several aspects to confirm if they are chemically defended. Survivorship data showed predation significantly affecting larval survival in natural habitat. Larvae chemical defenses are inefficient against invertebrate predators, such as the spider Lycosa erythr-ognatha and the ant Campónotus crassus (100% of predation), but it seems to be efficient against the mantid Oxyopsis saussuurei (reduction of prey manipulation time and predation in a second contact). To vertebrate predators, like the lizard Tropidurus itambere and the chick Gallus gallus, defense acts through conspicuous coloration and palatability. Predation by Gallus gallus was highest upon 1st ínstar larvae, suggesting an increase of unpalatability throughout development. Learning avoidance tests with Gallus gallus demonstrated that few contacts with distasteful and warning colored prey could make the predator reject visually Methona themisto larvae. Chicks visually rejected palatable larvae painted in the same color pattem of Methona themisto larvae confirming their ability to associate taste and color. Only dichloromethanic extracts tested with chicks showed significant rejection in relation to controls. These results confirm that Methona themisto larvae are chemical defended against visually oriented vertebrate predators / Mestrado / Mestre em Ecologia

Orientador: José Roberto Trigo / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T19:23:05Z (GMT). No. of bitstreams: 1 Massuda_KamilaFerreira_D.pdf: 3476211 bytes, checksum: a20550d22526cbcb5a72c290cf3e8ff1 (MD5) Previous issue date: 2015 / Resumo: As interações entre os insetos herbívoros e as plantas foram moldadas ao longo de um extenso processo coevolutivo, que gerou adaptações das plantas para evitarem a herbivoria e contra-adaptações dos herbívoros para superarem as defesas impostas pelas plantas. As folhas de Ipomoea carnea subsp. fistulosa (Convolvulaceae) são atacadas pelos besouros Cassidinae (Chrysomelidae), Chelymorpha reimoseri, Chelymorpha cribraria e Charidotella rubicunda. Esses besouros depositam seus ovos nas folhas e se alimentam das mesmas nos estágios de larva e adulto. Nesse trabalho, demonstramos a eficácia de dois mecanismos de defesa de I. carnea contra a deposição de ovos por C. reimoseri: (1) o descolamento de posturas, devido a formação de neoplasmas abaixo da postura e (2) a atração de parasitoides de ovos, por voláteis liberados de plantas induzidas pela própria postura ou por um indivíduo realmente capaz de ovipor, uma fêmea que copulou. Além disso, demonstramos duas estratégias de defesa para as três espécies cassidíneas supramencionadas contra predadores: (1) a impalatabilidade, devido ao alcaloide swainsonina, sequestrado da planta hospedeira e cuja atividade deterrente foi demonstrada para Gallus gallus e (2) a camuflagem química das larvas de C. reimoseri nas folhas da planta hospedeira, devido à alta similaridade química entre seus compostos cuticulares e os da planta hospedeira larval, que dificulta o reconhecimento por predadores quimicamente orientados / Abstract: The interactions between herbivorous insects and plants have been shaped over a co-evolutionary process that spawned adaptations of plants to avoid herbivory and counter adaptation of herbivores to overcome the defenses imposed by plants. The plant Ipomoea carnea subsp. fistulosa (Convolvulaceae) has as herbivores, beetles of Cassidinae subfamily (Chelymorpha reimoseri, Chelymorpha cribraria, and Charidotella rubicunda), which use the leaves to feed larvae and adults, as well as places for depositing their eggs. This study demonstrated the efficiency of two I. carnea defensive mechanisms against oviposition by C. reimoseri: (1) the detachment of eggs neoplasm formation, below the eggs, and (2) the attraction of eggs parasitoids by volatile released from posture-induced plants and plants induced by an individual capable of laying eggs, a mated female. Furthermore, we demonstrated the effectiveness of two defensive strategies in the three above mentioned cassidine species; (1) the unpalatability, due to swainsonine, an akaloid provided by the host plant, and its deterrent effect against predator, the chicken Gallus gallus; (2) the chemical camouflage of C. reimoseri larvae on its host plant, due to high chemical similarity among their cuticular compounds that turns difficult recognition by a chemical oriented predator / Doutorado / Doutor em Ecologia