Los terpenos constituyen el mayor grupo de metabolitos secundarios, siendo componentes de las glándulas de aceites esenciales, de las flores y de las resinas defensivas de plantas aromáticas, a los que proporcionan sus aromas y sabores característicos. Los terpenos volátiles se asocian a la defensa de muchas especies de plantas, animales y microorganismos contra depredadores, patógenos y competidores. Por otra parte, estos compuestos parecen servir como señales para atraer a los polinizadores y agentes dispersores de semillas, así como a depredadores de plagas. El estudio de compuestos orgánicos volátiles emitidos durante el desarrollo del fruto y después del desafío con diferentes agentes bióticos puede ayudar a conocer las interacciones de los frutos carnosos no sólo con vertebrados dispersores y depredadores, sino también con insectos y microorganismos. Los frutos carnosos son particularmente ricos en volátiles. En los frutos cítricos, los monoterpenos son los principales componentes de las glándulas del aceite esencial de la cáscara (flavedo), siendo el D-limoneno el más abundante (hasta 95% en la naranja). Esta característica hace que los cítricos sean un buen sistema modelo para el estudio de la función de los terpenos en los frutos. La biología molecular moderna permite la realización de experimentos para comprobar la función de terpenos por medio del uso de organismos transformados genéticamente en los que se han manipulado los niveles de acumulación de dichos compuestos. En este trabajo, se ha utilizado un plásmido que alberga el cDNA completo del gen de una limoneno sintasa de cítricos (CiTMTSE1) en orientación antisentido (AS) o sentido (S) para modificar la expresión y la acumulación de D-limoneno en plantas de naranjo dulce (Citrus sinensis L. Osb.). La acumulación de D-limoneno en las frutas AS se redujo drásticamente pero la acumulación de otros terpenos también se modificó, afectando a compuestos tales como alcoholes monoterpenos, cuya concentración se incrementó en la cáscara de las frutas. Las plantas transformadas fueron morfológicamente indistinguibles de las plantas control (WT) y de las plantas transformadas con el vector vacío (EV). Los frutos transgénicos fueron desafiados con un insecto plaga y con diferentes patógenos para probar si la alteración de los niveles de acumulación de estos volátiles daba como resultado una mejora en la respuesta del flavedo frente a plagas y patógenos. Los machos de la mosca mediterránea de la fruta (Ceratitis capitata) expuestos a las frutas AS y EV en ensayos en túnel de viento fueron significativamente más atraídos por el aroma de los frutos control EV. En otros experimentos de desafío con el hongo de la podredumbre verde Penicillium digitatum y la bacteria causante de la cancrosis de los cítricos Xanthomonas axonopodis subsp. citri, las frutas transgénicas con un contenido reducido de D-limoneno mostraron elevada resistencia a estos patógenos. El alto contenido en D-limoneno en la cáscara de naranjas maduras puede ser una señal para la atracción de plagas y microorganismos que podrían estar involucrados en la facilitación del acceso a la pulpa de los frugívoros dispersores de semillas. El análisis de la expresión génica global en el flavedo de las frutas transgénicas vinculó la disminución de D-limoneno y la reducción de la expresión de genes del metabolismo de monoterpenos con la activación de la expresión de genes implicados en inmunidad innata, incluyendo factores de transcripción, genes de quinasas implicadas en la entrada de Ca2+ en la célula y genes implicados en la activación de las cascadas de MAPKs, con la consiguiente activación de la ruta de señalización de ácido jasmónico (JA), lo que provocó la activación del metabolismo de JA y un aumentó drástico de la acumulación de JA en la cáscara de la naranja tras el desafío con P. digitatum, lo que explicaría la resistencia al menos a hongos necrotrofos observada en las frutas. Estos resultados indican que la acumulación de D-limoneno en la cáscara de la naranja estaría implicada en la interacción trófica entre las frutas, insectos y microorganismos, lo cual proporciona una visión mucho más amplia de las funciones de los terpenos en la naturaleza. También representa una alternativa muy prometedora para incrementar la resistencia o tolerancia de las plantas frente a patógenos y plagas. / Terpenes, the largest group of secondary metabolites, are well known as constituents of essential oils, floral scents and defensive resins of aromatic plants, to which they impart their characteristic aromas and flavors. Terpene volatiles defend many species of plants, animals and microorganisms against predators, pathogens and competitors. Moreover, those compounds seem to serve as advertisements to attract pollinators and seed-dispersal agents as well as pest predators. The study of VOCs emitted during fruit development and after challenge with different biotic agents may help to determine the interactions of fleshy fruits not only with legitimate vertebrate dispersers and predators, but also with insects and microorganisms. Fleshy fruits are particularly rich in volatiles. In citrus fruits, monoterpenes are the main components of the essential oil glands of the peel, being D-limonene the most abundant one (up to 95% in orange fruits). This characteristic makes citrus a good model system for studying the function of terpenes in plants. Modern molecular biology now enable experiments to test terpenoid function by the use of genetically transformed organisms in which terpene levels have been manipulated. In this work, a plasmid harboring the complete cDNA of a citrus limonene synthase gene (CiTMTSE1) in antisense (AS) or sense (S) orientation was used to modify the expression and accumulation of D-limonene of sweet orange (Citrus sinensis L. Osb) plants. D-limonene accumulation in AS fruits was dramatically reduced but the accumulation of other terpenoids was also modified, such as monoterpene alcohols, whose concentration increased in the peel of fruits. Genetically transformed plants were morphologically indistinguishable from wild-type (WT) and empty vector (EV) control plants. Transgenic fruits were challenged against a pest and different pathogens to test whether volatile profile alteration results in an improvement in the response of the fruit flavedo against them. Males of the Mediterranean fruit fly (Ceratitis capitata) exposed to AS fruits versus EV in wind tunnel assays were significantly more attracted to the odor of EV control fruits. In separate experiments with the green mould rot of citrus fruits and citrus canker caused by Penicillium digitatum and Xanthomonas axonopodis subsp. citri, respectively, transgenic fruits with a reduced content in D-limonene showed resistance to both pathogens. High D-limonene content in mature orange peels may be a signal for attractiveness of pests and microorganisms which might be likely involved in facilitating the access to the pulp of seed dispersal frugivores. A global gene expression analysis of the flavedo of AS transgenic fruits linked the decrease of D-limonene and monoterpene metabolism to the up-regulation of genes involved in the innate immunity response, including transcription factors together with Ca2+ entry into the cell and activation of MAPK cascades, contributing to activation of jasmonic acid (JA) signaling, which triggered the up-regulation of JA metabolism and drastically increased the accumulation of JA in orange peels upon fungal challenge, explaining the resistance to necrotrophic fungi observed in AS fruits. These results indicate that limonene accumulation in the peel of citrus fruit appears to be involved in the successful trophic interaction between fruits, insects, and microorganisms and provide a much more comprehensive view of roles of terpenes in nature. It also represents a very promising alternative for increasing resistance or tolerance of plants to pathogens. / Rodríguez Baixauli, AM. (2013). Genetic engineering of plant volatiles in fleshy fruits: pest repellency and disease resistance through D-limonene downregulation in transgenic orange plants [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/31655 / TESIS

Plants depend on a wide variety of vectors, both biotic and abiotic, to move their seeds to locations away from parent plants. The stages between seed production and seedling establishment in the plant life cycle are under the influence of a complex set of ecological and evolutionary factors (Wang and Smith 2002). In ecological time scales, seed dispersal has been shown to influence both plant population and community dynamics (Howe and Miriti 2004, Seidler and Plotkin 2006). The evolutionary effects are seen in the way these interactions influence the fruiting season, fruit crop size, fruit size, nutritive value of fruit or seed and other strategies adopted by the plant to ensure effective dispersal of their seeds (Howe and Smallwood 1982, Schupp 1993). Most seed dispersal research has focussed on arboreal frugivores such as birds, primates and bats. In comparison, terrestrial frugivores such as ruminants, elephants and rodents are poorly researched. Large-bodied terrestrial frugivores are key to the dispersal of several large-seeded plants and are also capable of dispersing seeds to long distances (> 1 km). Given that large mammals have witnessed tremendous declines in their abundances and ranges in historical times, plants that depend upon them for long distance dispersal might have limited ability to move across fragmented landscapes under changing climates. However, very little is understood about seed dispersal by large mammals or about seed dispersal characteristics of tropical dry forests which support among the highest biomass of large terrestrial mammals. Tropical dry forests are among the most endangered tropical ecosystems (Janzen 1988). It has also been forecast that tropical dry forests in the Indian subcontinent will witness significant changes in temperature and rainfall regimes in the coming decades (Ravindranath et al. 2006). However, compared to tropical moist forests which have received most of the scientific interest, theory, methodology and base line information about seed dispersal in dry forests is poorly developed. To address some of these lacunae, I examined seed dispersal at both population and community-scales in the tropical dry forests of Mudumalai Wildlife Sanctuary from 2005 -2007. Mudumlai is located within a relatively large contiguous stretch of tropical dry forests in southern India and has an intact and abundant frugivore assemblage. In addition, the Mudumalai Forest Dynamics Plot (MFDP), a large 50 hectare plot has been monitored for recruitment, mortality and diameter growth of woody plants for the last twenty years. The plot is an invaluable resource for seed dispersal studies since it provides us an opportunity to link seed dispersal with plant population and community dynamics. In the first part of the thesis (Chapters 3 and 4), I focus on evaluating the effectiveness of ruminants as seed dispersers and then examine factors that influence frugivory by ruminants. This work was carried out at the population level, for the tree Phyllanthus emblica. The fruits of P. emblica are important non-timber forest produce from Asian dry tropics and understanding the quantity of fruit consumed by frugivores has implications for sustainable harvest practices of this resource (Shahabuddin and Prasad 2004). In the second part of the thesis (Chapters 5 and 6), I characterized seed dispersal at the community-scale for woody plants on the MFDP. Community-wide characterization of seed dispersal at sites harboring an intact faunal assemblage is critical to identify plants dependent on endangered dispersers. The second half of the thesis focuses on examining the associations between dispersal mode and fruit characteristics, and also between dispersal mode and spatial patterning of woody plants. Evaluating the effectiveness of ruminants as seed dispersers: Disperser effectiveness is defined as the contribution a disperser makes to the future reproduction of a plant. Evaluating disperser effectiveness involves addressing both quantitative aspects such as frequency of visits, quantity of fruit consumed, diversity of species dispersed, and, qualitative components such as the treatment given in mouth and gut and the suitability of seed deposition sites for germination (Schupp 1993, Dennis and Westcott 2006). Information on quantity and quality of seed dispersal services provided by ruminants is very limited. This is because techniques such as tree watches or fruit traps adapted from avian frugivore studies are ill suited to study terrestrial frugivores, and conventional camera traps provide little quantitative information on the quantity of fruit consumed by frugivores. The quantity of dispersal services provided by ruminants for P. emblica was assessed using a novel camera trap technique. Time-delay was programmed into the camera trap units (4 pictures taken 2 minutes apart) to tell apart frugivores from visitors (by comparing number of fruit remaining in the time-delay picture sequence). During a two year study using this technique, six terrestrial mammals were identified as frugivores of P. emblica. Additionally, seven mammals and one bird species visited fruiting trees but did not consume fallen fruit. Two ruminants, the Indian chevrotain Moschiola indica and chital Axis axis, were the most frequent frugivores of P. emblica and these ruminants accounted for over 95% of fruit removal, while murid rodents accounted for less than 1%. The quality of dispersal services provided by ruminants was examined using a combination of feeding trials (with captive animals) and germination experiments for large-seeded species dispersed by ruminants in tropical dry forests, including P. emblica. Ruminants regurgitated well-protected seeds larger than 5 mm, after retaining them in the rumen for 2–50 h. Though germination success was lowered after rumen retention, it was not too low (> 15%), and would still ensure dispersal if suitable micro-habitats could be found. Given the daily home-range of chital (14–20 ha in tropical dry forests (Mishra 1982)), and their seed retention times (2–50 h), movement of chital during the 1–2 d period when seeds remain in the rumen can potentially disperse seeds to distances over 1 km. Thus, our research shows that ruminants account for most of the fruit removal and are the principal primary dispersers of P. emblica. Factors influencing frugivory by ruminants: Fruit removal, the quantity of fruits consumed by frugivores, is highly variable within plant populations. Understanding factors influencing this selection operated by frugivores helps link frugivore behavior with plant demography. The outcome of such selection operated by frugivores has been examined mostly for interactions involving avian frugivores. The extension of this theory to terrestrial frugivores, which are presented with fruit on the ground, is not well understood. In addition, factors influencing fruit removal have been examined mostly with respect to changing spatial contexts such as crop size and fruiting neighborhoods. The influence of varying temporal contexts on frugivore choice, and their possible demographic and evolutionary consequences is poorly understood. We examined if temporal variation in fruit availability across a hierarchy of nested temporal levels (interannual, intraseasonal, 120 h, 24 h) altered fruit removal of P. emblica on the MFDP. The interactions between P. emblica and its primary disperser (ruminants) was mediated by another frugivore (a primate, langur), which made large quantities of fruit available on the ground to ruminants. The direction and strength of crop size and neighborhood effects on this interaction varied with changing temporal contexts. Fruit availability was higher in the first of the two study years, and at the start of the season in both years. Fruit persistence on trees, determined by primate foraging, was influenced by crop size and conspecific neighborhood densities only in the high fruit availability year. Fruit removal by ruminants was influenced by crop size in both years and neighborhood densities only in the high availability year. In both years, these effects were stronger at the start of the season. Intraseasonal reduction in fruit availability diminished inequalities in fruit removal by ruminants and the influence of crop size and fruiting neighborhoods. Temporal asymmetry in frugivore-mediated selection could reduce the potential for co-evolution between frugivores and plants by diluting selective pressures. Langurs determined the length of time fruit persisted on individual trees, and, also influenced the quantity and duration of fruit availability for the primary dispersers, ruminants. Such inter-dependencies formed between disparate animal consumers add additional levels of complexity to plant–frugivore mutualistic networks and can have potential reproductive consequences for specific individuals within populations. Seed dispersal within a tropical dry forest community: Megafauna are considered to be important seed dispersers, but their contribution has mostly been examined in isolation, with limited reference to other frugivores within communities. Community-wide characterization of seed dispersal at sites harboring an intact fauna is essential to evaluate the role of megafauna in dispersal, examine dispersal syndromes and assess the vulnerability of plant communities to disperser declines and climate change. Dispersal modes were inventoried for 92% of species and fruit traits for 84% of the woody plant community on the MFDP. Fruit–frugivore interactions were identified using a combination of camera traps, tree watches and opportunistic observations. Though 60% of the species were animal-dispersed, mechanically-dispersed species were more dominant and constituted nearly 70% of the stand. Bird-dispersed species were very rare and constituted less than 10% of the stand cumulatively. In comparison, bird-dispersed species account for ~ 50% of the stand in moist forests of tropical Asia. Three broad dispersal modes were identified – mechanical, mammal and bird-mammal, and these displayed strong associations with fruit traits. Among the 11 frequently-observed frugivore groups, the three largest frugivores, deer, bear and elephant, together dispersed 40% of the community. There was low overlap in fruit diets between these three megafaunal groups, though diets of deer and bear overlapped with different groups of small and medium-sized frugivores. Deer and bear displayed significant associations with fruit traits, while elephant did not. Among the fleshy fruits, large fruits and large-seeded fruits had fewer dispersers compared to smaller, multi-seeded fruits. Although the largest fleshy fruits and seeds were often dispersed by megafauna, few were exclusively dependent on megafauna. The three megafaunal groups were the only potential long distance dispersers for 25% of the community. For a better understanding of the impacts of megafaunal extinctions, even rare events of dispersal by megafauna should be considered since these could prove important for plants lacking other long distance dispersers. Given that these megafauna and several medium-sized frugivores are rare in degraded areas of tropical Asia (Corlett 2007, Karanth et al. 2010), one fourth of the plant community at Mudumalai could have limited ability to disperse across fragmented landscapes in response to changing climates. Seed dispersal and spatial patterns in tropical dry forests: To address the relevance of seed dispersal to plant community structure, the linkages between dispersal mode and spatial aggregation of trees were examined for the woody plant community on the MFDP. For plants, seed arrival patterns vary depending upon their dispersal mode and form the primary matrix upon which further forces shape spatial patterns. Spatial patterns were examined via the pair correlation function (statistic) and statistics derived from cluster models (Matern process). Considering only trees that had 10 individuals (48 species), it was found that tropical dry forest trees exhibited extensive spatial aggregation. The degree of spatial aggregation at Mudumalai was remarkably similar to wet forests in tropical Asia. Species with limited-dispersal (mechanically-dispersed species) were more aggregated than those with more frequent long-distance dispersal (animal-dispersed species) at smaller spatial scales (< 50 m). Animal-dispersed species had larger cluster radius, with bird-dispersed species having the largest radii. These results are in concordance with Siedler & Plotkin (2006) and Li et al. (2009) who find that mechanically-dispersed species are more aggregated than animal-dispersed species. These results demonstrate that seed dispersal has a strong influence on the spatial patterning of plant communities. Conclusion: At the population level, our results imply that P. emblica is mainly dependent on ruminants for seed dispersal. In tropical dry forest sites such as Mudumalai, up to 15% of the species are dispersed by ruminants, with langurs mediating the ruminant-plant interaction similar to P. emblica. The observed fruit removal patterns for P. emblica reiterate the fact that all fruiting individuals in a population are not equally attractive to frugivores, especially when fruits are abundant. Alterations of such complex fruit-frugivore interactions through human-mediated changes of fruit or frugivore densities (through fruit harvests or hunting of large mammalian frugivores such as ruminants), can feed back into the seed dispersal loop and affect several downstream ecological and evolutionary processes. At the community-scale, the dispersal profile of woody plants in Mudumalai’s tropical dry forests differed considerably from tropical moist sites, with a greater representation of mammal and mechanically-dispersed species. However, the linkages between seed dispersal and spatial patterns of trees at Mudumalai are similar to tropical moist forests. Extensions of the models developed from this site harbouring an intact faunal assemblage to other tropical dry forests would enable rapid characterization of dispersal profiles and identification of plant species dependent on endangered fauna for seed dispersal that should be prioritized for restoration programs.