Transcriptional Activity, Chromosomal Distribution and Expression Effects of Transposable Elements in Coffea Genomes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Aplicação de extratos de algas marinhas em cafeeiro sob deficiência hídrica e estresse salino

Pós-graduação em Agronomia (Irrigação e Drenagem) - FCA

Clones de Eucalyptus sp. submetidos a estresse hídrico em dois tipos de solo

Pós-graduação em Agronomia (Irrigação e Drenagem) - FCA

Rootstocks for high fruit yield and quality of 'Tahiti' lime under rain-fed conditions

Despite considerable research conducted on 'Tahiti' lime [Citrus latifolia (Yu Tanaka) Tanaka] in several countries, few long-term studies have focused on rootstock effects on fruit production and quality under non-irrigated conditions. As for many other fruit crops, rootstock studies for 'Tahiti' lime are often based on the evaluation of several horticultural responses simultaneously, instead of considering multivariate statistical approaches which may provide with more comprehensive information. Consequently, a trial was installed to evaluate the horticultural performance of non-irrigated 'Tahiti' lime trees budded onto the following 12 rootstocks: 'HRS 801' and 'HRS 827' hybrids; 'Rubidoux', 'FCAV' and 'Flying Dragon' trifoliates; 'Sun Chu Sha Kat' and 'Sunki' mandarins; 'Cravo Limeira' and 'Cravo FCAV' 'Rangpur' limes; 'Carrizo' citrange, 'Swingle' citrumelo, and 'Orlando' tangelo. The trial was installed in 2001, in an 8 m x 5 m spacing with no supplementary irrigation. Measurements of yield, fruit quality oriented to different consuming markets, canopy volume and tree tolerance to drought, were performed. A multivariate cluster analysis identified both 'Rangpur' lime rootstocks as those inducing larger cumulative yield and higher percentage of fruits for the domestic market, with highest drought tolerance to the trees. Despite of their high susceptibility to drought stress under non-irrigated conditions, the 'Flying Dragon' and 'FCAV' trifoliate rootstocks performed outstandingly for 'Tahiti' lime, inducing higher yield efficiency, early bearing and larger percentage of high-quality fruits for foreign markets, with smaller trees more suitable for high-density plantings. (c) 2012 Elsevier B.V. All rights reserved.

Regulation of woody plant development by gibberellin catabolic and signaling genes

Gibberellin (GA) is a growth promoting hormone implicated in regulating a diversity of plant processes. This dissertation examines the role of GA metabolic and signaling genes in woody plant growth and development. Transgenic modifications, expression analysis, physiological/biochemical assays, biometric measurements and histological analysis were used to understand the regulatory roles these genes play in the model woody plant, Populus. Our results highlight the importance of GA regulatory genes in woody perennial growth, including: phenology, wood formation, phenotypic plasticity, and growth/survival under field conditions. We characterize two putative Populus orthologs of the SHORT INTERNODES (SHI) gene from Arabidopsis, a negative regulator of GA signaling. RNAi-mediated suppression of Populus SHI-like genes increased several growth-related traits, including extent of xylem proliferation, in a dose-dependent manner. Three Populus genes, sharing sequence homology to the positive regulator of GA signaling gene PHOTOPERIOD-RESPONSIVE 1 (PHOR1) from Solanum, are up-regulated in GA-deficient and insensitive plants suggesting a conserved role in GA signaling. We demonstrate that Populus PHOR1-like genes have overlapping and divergent function(s). Two PHOR1-like genes are highly expressed in roots, predominantly affect root growth (e.g., morphology, starch quantity and gravitropism), and induced by short-days (SD). The other PHOR1-like gene is ubiquitously expressed with a generalized function in root and shoot development. The effects of GA catabolic and signaling genes on important traits (e.g., adaptive and productivity traits) were studied in a multi-year field trial. Transgenics overexpressing GA 2-oxidase (GA2ox) and DELLA genes showed tremendous variation in growth, form, foliage, and phenology (i.e., vegetative and reproductive). Observed gradients in trait modifications were correlated to transgene expression levels, in a manner suggesting a dose-dependent relationship. We explore GA2ox and DELLA genes involvement in mediating growth responses to immediate short-term drought stress, and SD photoperiods, signaling prolonged periods of stress (e.g., winter bud dormancy). GA2ox and DELLA genes show substantial up-regulation in response to drought and SDs. Transgenics overexpressing homologs of these genes subjected to drought and SD photoperiods show hypersensitive growth restraint and increased stress resistances. These results suggest growth cessation (i.e., dormancy) in response to adverse conditions is mediated by GA regulatory genes.

Organic vs. Conventional Grassland Management: Do N-15 and C-13 Isotopic Signatures of Hay and Soil Samples Differ?

Distinguishing organic and conventional products is a major issue of food security and authenticity. Previous studies successfully used stable isotopes to separate organic and conventional products, but up to now, this approach was not tested for organic grassland hay and soil. Moreover, isotopic abundances could be a powerful tool to elucidate differences in ecosystem functioning and driving mechanisms of element cycling in organic and conventional management systems. Here, we studied the delta N-15 and delta C-13 isotopic composition of soil and hay samples of 21 organic and 34 conventional grasslands in two German regions. We also used Delta delta N-15 (delta N-15 plant - delta N-15 soil) to characterize nitrogen dynamics. In order to detect temporal trends, isotopic abundances in organic grasslands were related to the time since certification. Furthermore, discriminant analysis was used to test whether the respective management type can be deduced from observed isotopic abundances. Isotopic analyses revealed no significant differences in delta C-13 in hay and delta C-13 in both soil and hay between management types, but showed that delta C-13 abundances were significantly lower in soil of organic compared to conventional grasslands. delta C-15 values implied that management types did not substantially differ in nitrogen cycling. Only delta C-13 in soil and hay showed significant negative relationships with the time since certification. Thus, our result suggest that organic grasslands suffered less from drought stress compared to conventional grasslands most likely due to a benefit of higher plant species richness, as previously shown by manipulative biodiversity experiments. Finally, it was possible to correctly classify about two third of the samples according to their management using isotopic abundances in soil and hay. However, as more than half of the organic samples were incorrectly classified, we infer that more research is needed to improve this approach before it can be efficiently used in practice.

Plant-plant interactions, environmental gradients and plant diversity: A global synthesis of community-level studies

Previous syntheses on the effects of environmental conditions on the outcome of plant-plant interactions summarize results from pairwise studies. However, the upscaling to the community-level of such studies is problematic because of the existence of multiple species assemblages and species-specific responses to both the environmental conditions and the presence of neighbors. We conducted the first global synthesis of community-level studies from harsh environments, which included data from 71 alpine and 137 dryland communities to: (i) test how important are facilitative interactions as a driver of community structure, (ii) evaluate whether we can predict the frequency of positive plant-plant interactions across differing environmental conditions and habitats, and (iii) assess whether thresholds in the response of plant-plant interactions to environmental gradients exists between ``moderate'' and ``extreme'' environments. We also used those community-level studies performed across gradients of at least three points to evaluate how the average environmental conditions, the length of the gradient studied, and the number of points sampled across such gradient affect the form and strength of the facilitation-environmental conditions relationship. Over 25% of the species present were more spatially associated to nurse plants than expected by chance in both alpine and chyland areas, illustrating the high importance of positive plant-plant interactions for the maintenance of plant diversity in these environments. Facilitative interactions were more frequent, and more related to environmental conditions, in alpine than in dryland areas, perhaps because drylands are generally characterized by a larger variety of environmental stress factors and plant functional traits. The frequency of facilitative interactions in alpine communities peaked at 1000 mm of annual rainfall, and globally decreased with elevation. The frequency of positive interactions in dtyland communities decreased globally with water scarcity or temperature annual range. Positive facilitation-drought stress relationships are more likely in shorter regional gradients, but these relationships are obscured in regions with a greater species turnover or with complex environmental gradients. By showing the different climatic drivers and behaviors of plant-plant interactions in dryland and alpine areas, our results will improve predictions regarding the effect of facilitation on the assembly of plant communities and their response to changes in environmental conditions.

An underestimated record breaking event – why summer 1540 was likely warmer than 2003

The heat of summer 2003 in Western and Central Europe was claimed to be unprecedented since the Middle Ages on the basis of grape harvest data (GHD) and late wood maximum density (MXD) data from trees in the Alps. This paper shows that the authors of these studies overlooked the fact that the heat and drought in Switzerland in 1540 likely exceeded the amplitude of the previous hottest summer of 2003, because the persistent temperature and precipitation anomaly in that year, described in an abundant and coherent body of documentary evidence, severely affected the reliability of GHD and tree-rings as proxy-indicators for temperature estimates. Spring–summer (AMJJ) temperature anomalies of 4.7 °C to 6.8 °C being significantly higher than in 2003 were assessed for 1540 from a new long Swiss GHD series (1444 to 2011). During the climax of the heat wave in early August the grapes desiccated on the vine, which caused many vine-growers to interrupt or postpone the harvest despite full grape maturity until after the next spell of rain. Likewise, the leaves of many trees withered and fell to the ground under extreme drought stress as would usually be expected in late autumn. It remains to be determined by further research whether and how far this result obtained from local analyses can be spatially extrapolated. Based on the temperature estimates for Switzerland it is assumed from a great number of coherent qualitative documentary evidence about the outstanding heat drought in 1540 that AMJJ temperatures were likely more extreme in neighbouring regions of Western and Central Europe than in 2003. Considering the significance of soil moisture deficits for record breaking heat waves, these results still need to be validated with estimated seasonal precipitation. It is concluded that biological proxy data may not properly reveal record breaking heat and drought events. Such assessments thus need to be complemented with the critical study of contemporary evidence from documentary sources which provide coherent and detailed data about weather extremes and related impacts on human, ecological and social systems.

Central European vegetation response to abrupt climate change at 8.2 ka

Oxygen isotope records show a major climatic reversal at 8.2 ka in Greenland and Europe. Annually laminated sediments from two lakes in Switzerland and Germany were sampled contiguously to assess the response of European vegetation to climate change ca. 8.2 ka with time resolution and precision comparable to those of the Greenland ice cores. The pollen assemblages show pronounced and immediate responses (0–20 yr) of terrestrial vegetation to the climatic change at 8.2 ka. A sudden collapse of Corylus avellana (hazel) was accompanied by the rapid expansion of Pinus (pine), Betula (birch), and Tilia (linden), and by the invasion of Fagus silvatica (beech) and Abies alba (fir). Vegetational changes suggest that climatic cooling reduced drought stress, allowing more drought-sensitive and taller growing species to out-compete Corylus avellana by forming denser forest canopies. Climate cooling at 8.2 ka and the immediate reorganization of terrestrial ecosystems has gone unrecognized by previous pollen studies. On the basis of our data we conclude that the early Holocene high abundance of C. avellana in Europe was climatically caused, and we question the conventional opinion that postglacial expansions of F. silvatica and A. alba were controlled by low migration rates rather than by climate. The close connection between climatic change and vegetational response at a subcontinental scale implies that forecasted global warming may trigger rapid collapses, expansions, and invasions of tree species.

Rapid divergence in physiological and life-history traits between northern and southern populations of the British introduced neo-species, Senecio squalidus

Alien plants provide a unique opportunity to study evolution in novel environments, but relatively little is known about the extent to which they become locally adapted to different environments across their new range. Here, we compare northern and southern populations of the introduced species Senecio squalidus in Britain; S. squalidus has been in southern Britain for approximately 200 years and reached Scotland only about 50 years ago. We conducted common garden experiments at sites in the north and south of the species’ range in Britain. We also conducted glasshouse and growth chamber experiments to test the hypothesis that southern genotypes flower later, are more drought-tolerant, germinate and establish better at warmer temperatures, and are less sensitive to cold stress than their more northern counterparts. Results from the common garden experiments are largely consistent with the hypothesis of rapid adaptive divergence of populations of the species within the introduced range, with genotypes typically showing a home-site advantage. Results from the glasshouse and growth chamber experiments demonstrate adaptive divergence in ability to tolerate drought stress and high temperatures, as well as in phenology. In particular, southern genotypes were more tolerant of dry conditions and high temperatures and they flowered later than northern genotypes. Our results show that rapid local adaptation can occur in alien species, and they have implications for our understanding of the ecological genetics of range expansion of introduced weeds.

Genes implicados en la respuesta molecular al estrés hídrico en Pinus pinaster Ait

La presente tesis doctoral se centra en el estudio de la respuesta molecular de las coníferas mediterráneas al estrés hídrico. Para ello se ha escogido como especie modelo Pinus pinaster Ait., la conífera más abundante en España, y que habita un amplio rango de situaciones ecológicas, especialmente en lo relativo a la disponibilidad de agua. En primer lugar, se ha aplicado un estrés hídrico controlado en cultivo hidropónico y se ha generando una genoteca sustractiva con objeto de identificar los genes inducidos por el estrés, analizando su expresión en raíces, tallos y acículas. A continuación, se ha analizado, la expresión de los genes anteriormente obtenidos así como de otros seleccionados de las bases de datos disponibles, durante una sequía prolongada en tierra, similar a las que las plantas deben afrontar en la naturaleza. Se ha utilizado en este caso, además de P. pinaster, P. pinea, otra conífera mediterránea adaptada a las sequías recurrentes. Este trabajo ha permitido identificar genes candidato expresionales, presumiblemente comunes en la respuesta molecular de las coníferas al déficit hídrico. Se han detectado diferencias notables en la expresión de determinados genes, que podrían ser los responsables de las diferencias exhibidas por ambas especies en el comportamiento frente a la sequía. Entre los genes identificados como inducidos por el estrés hídrico se encuentran varios miembros de la familia de las deshidrinas. Trabajos previos han utilizado deshidrinas como genes candidato; no obstante, la falta de especificidad de ciertos fragmentos y marcadores utilizados, debido a la complejidad estructural de esta familia, resta fiabilidad a algunos de los resultados publicados. Por este motivo, se ha estudiado en detalle esta familia en P. pinaster, se han identificado y caracterizado 8 miembros y se ha analizado su patrón de expresión frente a sequía. Este estudio ha permitido describir por primera vez unos segmentos conservados en la secuencia de aminoácidos de las deshidrinas de pináceas, cuya presencia y número de repeticiones parece estar relacionado con su especificidad. Por último, se han escogido tres genes implicados en distintas fases de la respuesta al estrés hídrico para su análisis exhaustivo: una deshidrina, una nodulina y un factor de transcripción tipo AP2. Se ha caracterizado su estructura exón/intrón y secuenciado su región promotora. Además, se han obtenido líneas transformadas que sobreexpresan estos genes tanto de forma heteróloga, en la especie modelo Arabidopsis thaliana, como en el propio P. pinaster. Este material facilitará la realización de futuros estudios sobre la función y el mecanismo de actuación de estos genes en la respuesta al estrés hídrico. ABSTRACT This thesis focuses in the study of the molecular response to water stress in Mediterranean conifers. For this purpose, P. pinaster was selected as model species. It’s the most abundant conifer in Spain, living in a wide range of ecological conditions, especially regarding water availability. First, we have applied a controlled polyethylene glycol-induced water stress in hydroponic culture and obtained a suppression subtractive hybridization (SSH) library, with the aim of identifying genes induced by water stress, analysing their expression in roots, stems and needles. We have then analysed the expression patterns of the identified genes, together with other genes selected from public databases. This study was conducted throughout a prolonged drought stress in soil, similar to the ones plants have to face in nature. In this case not only P. pinaster was analysed but also P. pinea, another Mediterranean conifer well adapted to recurrent droughts. This work has enabled us to identify of reliable candidate genes, presumably shared with other conifers in the response to water stress. We observed remarkable differences in the expression of some genes, which could be involved in the differential behaviour that these species show in the water stress response. Within the genes induced by water stress, several members of the dehydrin gene family were identified. Due to the structural complexity of the family, certain ambiguities and inconsistencies have been detected in previous works that have used dehydrins as candidate genes. For this reason, we have analysed thoroughly this gene family in P. pinaster, and have identified and characterized eight different members, whose expression patterns during drought have also been assessed. This study has allowed us to identify for the first time novel conserved segments in the amino acids sequences of Pinaceae. The presence and number of repetitions of these segments could be associated with the functional specificity of these proteins. Finally, three genes involved in different steps of the water stress response were selected for an exhaustive analysis: a dehydrin, a nodulin and an AP2 transcription factor. For all of them, the exon/intron structure was established and their promoter region was sequenced. Also, transformed lines were obtained both in Arabidopsis thaliana and in P. pinaster for the constitutive overexpression of these genes. This material will facilitate the development of further studies to investigate the function of these genes during the water stress response

Estudio de la ecología de Ulmus laevis Pallas en la Península Ibérica

Esta tesis doctoral pretende profundizar en el conocimiento de la ecología de Ulmus laevis Pallas, especie autóctona en peligro de extinción en la Península Ibérica, con el fin de proponer medidas adecuadas para su conservación. Se ha estudiado la distribución natural de la especie atendiendo a aspectos edáficos. Los resultados muestran que U. laevis presenta menor capacidad de acidificación de la rizosfera, menor actividad de la reductasa férrica y menor homeostasis que U. minor Mill. cuando crecen en sustratos con una disponibilidad de hierro limitada. Estas diferencias ayudan a comprender la distribución de ambas especies en la Península Ibérica: U. laevis se ve restringido a suelos ácidos o moderadamente ácidos, mientras que U. minor es capaz de habitar tanto suelos ácidos como básicos. Se han analizado las propiedades hidráulicas y anatómicas de U. laevis, constatando que sus características son favorables en ambientes con gran disponibilidad hídrica y que se trata del olmo ibérico más vulnerable a la cavitación por estrés hídrico, por lo que la aridificación del clima y la pérdida de los freáticos supone un riesgo para sus poblaciones. Para evaluar la capacidad de recuperación de la especie se han estudiado la diversidad y estructura genética espacial de las dos mayores poblaciones españolas. Los resultados evidencian que estas poblaciones mantienen niveles de diversidad equiparables o ligeramente superiores a los europeos, pese a haber sufrido un cuello de botella prolongado durante las glaciaciones y a las reducciones poblacionales recientes. En la actualidad la endogamia no representa un riesgo para estas poblaciones. También se ha analizado la producción, dispersión y predación de semillas en Valdelatas (Madrid). Los resultados han mostrado que el viento dispersa las sámaras a corta distancia (<30 m) y que los años no veceros las probabilidades de establecimiento de regenerado son bajas. Además, la producción de sámaras vanas puede tratarse de un carácter adaptativo que aumenta la eficiencia biológica de la especie, ya que favorece la supervivencia de las semillas embrionadas disminuyendo sus tasas de predación pre- y post-dispersión. La modificación del hábitat de esta especie como consecuencia de las actividades humanas afecta de manera negativa al establecimiento del regenerado. La conservación de esta especie a largo plazo requiere la recuperación de los niveles freáticos y de regímenes hidrológicos que permitan avenidas, ya que estas crean las condiciones adecuadas para el establecimiento de regenerado al eliminar la vegetación preexistente y depositar barro. ABSTRACT Ulmus laevis Pallas is an endangered species in the Iberian Peninsula. Therefore, in order to be able to propose adequate management guidelines for its conservation, this PhD Thesis intends to advance the knowledge on the species ecology in the region. Firstly, the species natural distribution was studied in relation to soil nature. Results show that U. minor Mill. had a higher root ferric reductase activity and proton extrusion capability than U. laevis, and maintained a better nutrient homeostasis when grown under iron limiting conditions. These differences in root Fe acquisition efficiencies proved helpful to understand the distribution of these species in the Iberian Peninsula, where U. laevis is restricted to acid or moderately acid soils, whereas U. minor can grow both in acid and basic soils. Secondly, we studied Ulmus laevis’ xylem anatomy and hydraulic traits. These proved favourable for growing under high water availability, but highly susceptible to drought-stress cavitation. Therefore, this species is vulnerable to the Iberian Peninsula’s aridification. Spatial genetic structure and diversity were evaluated in two of the biggest U. laevis populations in Spain in order to evaluate their recovery capabilities. These populations maintain similar or slightly higher diversity levels than European populations, despite having undergone an ancestral genetic bottleneck and having suffered recent population size reductions. No inbreeding problems have been detected in these populations. Seed production, dispersal and predation were assessed in Valdelatas’ elm grove (Madrid). Despite U. laevis samaras being winged nuts, wind dispersed them short distances from the mother tree (<30 m). The seed shadow models show that non-mast years provide very few chances for the stand to regenerate due to their low full seed flux. Empty samaras deceive pre- and post-dispersal predators increasing full seed survival probabilities. Therefore, empty fruit production might be an adaptive trait that increases plant fitness. Finally, human-induced changes in water-table levels and river regulation may affect U. laevis seed dispersal and regeneration establishment negatively. The long-term conservation and expansion of this species in the Iberian Peninsula requires the recovery of water-tables and of natural hydrological regimes, as flooding eliminates vegetation, creating open microhabitats and deposits mud, creating the ideal conditions for seedling establishment.

Habitat management for conservation of pollinators in agro-ecosystems of Central Spain

La gestión de hábitat orientada a la conservación de polinizadores en los agro-ecosistemas requiere una selección de especies vegetales atendiendo fundamentalmente a dos criterios: i) el potencial atractivo de sus flores a los polinizadores; y ii) la simplicidad en su manejo agronómico. Además de estas premisas, es necesario considerar la capacidad invasora de estas especies vegetales, debido a que algunas de las más atractivas pueden resultar invasoras en determinados agro-ecosistemas. Por lo tanto, es preciso determinar qué especies vegetales son las más indicadas para ser implementadas en cada agro-ecosistema. En la presente tesis doctoral se plantea la búsqueda de las especies vegetales adecuadas para atraer polinizadores en los agro-ecosistemas del centro de España. En una primera aproximación, se ha evaluado la atracción y expansión espacial (potencial invasivo) de seis plantas perennes de la familia Lamiaceae (aromáticas), elegidas por ser nativas de la región mediterránea. La elección de las especies vegetales se ha llevado a cabo con el fin de crear márgenes funcionales basados en la mezcla de especies vegetales con distintos periodos de floración, de modo que prolonguen la disponibilidad de recursos florales en el tiempo. Tras un primer año dedicado al establecimiento de las especies aromáticas, en los dos años siguientes se ha estudiado la atracción individual y combinada de las especies vegetales sobre los polinizadores, y como ésta se ve afectada por la densidad y la morfología floral, utilizando para ello un diseño experimental en bloques al azar. Los resultados de este estudio han puesto de manifiesto que la morfología floral no tuvo influencia sobre la atracción de las especies vegetales, pero si la densidad floral, puesto que las especies vegetales con mayor densidad de flores (Nepeta tuberosa e Hyssopus officinalis) han mostrado mayor atracción a polinizadores. Cabe destacar que de las seis especies consideradas, dos especies de verano (Melissa officinalis y Thymbra capitata) no han contribuido de forma efectiva a la atracción de la mezcla hacia los polinizadores, mostrando una reducción significativa de este parámetro respecto a las otras especies aromáticas a lo largo del verano. Se ha observado que ninguna de las especies aromáticas evaluadas ha mostrado tendencia invasora a lo largo del estudio. En base a estos resultados, se puede concluir que entre las especies aromáticas estudiadas, N. tuberosa, H. officinalis y Salvia verbenaca son las que ofrecen mayor potencial para ser utilizadas en la conservación de polinizadores. De forma similar al caso de las plantas aromáticas, se ha llevado a cabo una segunda experimentación que incluía doce plantas anuales con floración de primavera, en la que se evaluó la atracción a polinizadores y su comportamiento agronómico. Este estudio con especies herbáceas se ha prolongado durante dos años, utilizando un diseño experimental de bloques aleatorios. Las variables analizadas fueron: el atractivo de las distintas especies vegetales a los polinizadores, su eficiencia de atracción (calculada como una combinación de la duración de la floración y las visitas de insectos), su respuesta a dos tipos de manejo agronómico (cultivo en mezcla frente a monocultivo; laboreo frente a no-laboreo) y su potencial invasivo. Los resultados de esta segunda experimentación han mostrado que las flores de Borago officinalis, Echium plantagineum, Phacelia tanacetifolia y Diplotaxis tenuifolia son atractivas a las abejas, mientras que las flores de Calendula arvensis, Coriandrum sativum, D. tenuifolia y Lobularia maritima son atractivas a los sírfidos. Con independencia del tipo de polinizadores atraídos por cada especie vegetal, se ha observado una mayor eficiencia de atracción en parcelas con monocultivo de D. tenuifolia respecto a las parcelas donde se cultivó una mezcla de especies herbáceas, si bien en estas últimas se observó mayor eficiencia de atracción que en la mayoría de parcelas mono-específicas. Respecto al potencial invasivo de las especies herbáceas, a pesar de que algunas de las más atractivas a polinizadores (P. tanacetifolia and C. arvensis) mostraron tendencia a un comportamiento invasor, su capacidad de auto-reproducción se vio reducida con el laboreo. En resumen, D. tenuifolia es la única especie que presentó una alta eficiencia de atracción a distintos tipos de polinizadores, conjuntamente con una alta capacidad de auto-reproducción pero sin mostrar carácter invasor. Comparando el atractivo de las especies vegetales utilizadas en este estudio sobre los polinizadores, D. tenuifolia es la especie más recomendable para su cultivo orientado a la atracción de polinizadores en agro-ecosistemas en el centro de España. Esta especie herbácea, conocida como rúcula, tiene la ventaja añadida de ser una especie comercializada para el consumo humano. Además de su atractivo a polinizadores, deben considerarse otros aspectos relacionados con la fisiología y el comportamiento de esta especie vegetal en los agro-ecosistemas antes de recomendar su cultivo. Dado que el cultivo en un campo agrícola de una nueva especie vegetal implica unos costes de producción, por ejemplo debidos a la utilización de agua de riego, es necesario evaluar el incremento en dichos costes en función de demanda hídrica específica de esa especie vegetal. Esta variable es especialmente importante en zonas dónde se presentan sequías recurrentes como es el caso del centro y sur-este de la península Ibérica. Este razonamiento ha motivado un estudio sobre los efectos del estrés hídrico por sequía y el estrés por déficit moderado y severo de riego sobre el crecimiento y floración de la especie D. tenuifolia, así como sobre la atracción a polinizadores. Los resultados muestran que tanto el crecimiento y floración de D. tenuifolia como su atracción a polinizadores no se ven afectados si la falta de riego se produce durante un máximo de 4 días. Sin embargo, si la falta de riego se extiende a lo largo de 8 días o más, se observa una reducción significativa en el crecimiento vegetativo, el número de flores abiertas, el área total y el diámetro de dichas flores, así como en el diámetro y longitud del tubo de la corola. Por otro lado, el estudio pone de manifiesto que un déficit hídrico regulado permite una gestión eficiente del agua, la cual, dependiendo del objetivo final del cultivo de D. tenuifolia (para consumo o solo para atracción de polinizadores), puede reducir su consumo entre un 40 y un 70% sin afectar al crecimiento vegetativo y desarrollo floral, y sin reducir significativamente el atractivo a los polinizadores. Finalmente, esta tesis aborda un estudio para determinar cómo afecta el manejo de hábitat a la producción de los cultivos. En concreto, se ha planteado una experimentación que incluye márgenes mono-específicos y márgenes con una mezcla de especies atractivas a polinizadores, con el fin de determinar su efecto sobre la producción del cultivo de cilantro (C. sativum). La elección del cultivo de cilantro se debe a que requiere la polinización de insectos para su reproducción (aunque, en menor medida, puede polinizarse también por el viento), además de la facilidad para estimar su producción en condiciones semi-controladas de campo. El diseño experimental consistía en la siembra de márgenes mono-específicos de D. tenuifolia y márgenes con mezcla de seis especies anuales situados junto al cultivo de cilantro. Estos cultivos con márgenes florales fueron comparados con controles sin margen floral. Además, un segundo grupo de plantas de cilantro situadas junto a todos los tratamientos, cuyas flores fueron cubiertas para evitar su polinización, sirvió como control para evaluar la influencia de los polinizadores en la producción del cultivo. Los resultados muestran que la presencia de cualquiera de los dos tipos de margen floral mejora el peso y el porcentaje de germinación de las semillas de cilantro frente al control sin margen. Si se comparan los dos tipos de margen, se ha observado un mayor número de semillas de cilantro junto al margen con mezcla de especies florales respecto al margen mono-específico, probablemente debido al mayor número visitas de polinizadores. Puesto que el experimento se realizó en condiciones de campo semi-controladas, esto sugiere que las visitas de polinizadores fueron el factor determinante en los resultados. Por otro lado, los resultados apuntan a que la presencia de un margen floral (ya sea mono-especifico o de mezcla) en cultivos de pequeña escala puede aumentar la producción de cilantro en más de un 200%, al tiempo que contribuyen a la conservación de los polinizadores. ABSTRACT Habitat management, aimed to conserve pollinators in agro-ecosystems, requires selection of the most suitable plant species in terms of their attractiveness to pollinators and simplicity of agronomic management. However, since all flowers are not equally attractive to pollinators and many plant species can be weedy or invasive in the particular habitat, it is important to test which plant species are the most appropriate to be implemented in specific agro-ecosystems. For that reason, this PhD dissertation has been focused on determination of the most appropriate aromatic and herbaceous plants for conservation of pollinators in agro-ecosystems of Central Spain. Therefore, in a first approximation, spatial expansion (i.e. potential weediness) and attractiveness to pollinators of six aromatic perennial plants from the Lamiaceae family, native and frequent in the Mediterranean region, were evaluated. Preliminary plant selection was based on designing a functional mixed margins consisting of plants attractive to pollinators and with different blooming periods, in order to extend the availability of floral resources in the field. After a year of vegetative growth, the next two years the plant species were studied in a randomized block design experiment in order to estimate their attractiveness to pollinators in Central Spain and to investigate whether floral morphology and density affect attractiveness to pollinators. The final aim of the study was to evaluate how their phenology and attractiveness to pollinators can affect the functionality of a flowering mixture of these plants. In addition, the spatial expansion, i.e. potential weediness, of the selected plant species was estimated under field conditions, as the final purpose of the studied plants is to be implemented within agro-ecosystems. The results of the experiment showed that floral morphology did not affect the attractiveness of plants to pollinators, but floral density did, as plant species with higher floral density (i.e. Nepeta tuberosa and Hyssopus officinalis) showed significantly higher attractiveness to pollinators. In addition, of six plant species, two summer species (Melissa officinalis and Thymbra capitata) did not efficiently contribute to the attractiveness of the mixture to pollinators, which reduced its attractiveness during the summer period. Finally, as none of the plants showed weedy behaviour under field conditions, the attractive plant species, i.e. N. tuberosa, H. officinalis and the early spring flowering Salvia verbenaca, showed good potential to conserve the pollinators. Similarly, in a second approximation, the attractiveness to pollinators and agronomic behaviour of twelve herbaceous plants blooming in spring were studied. This experiment was conducted over two years in a randomized block design in order to evaluate attractiveness of preselected plant species to pollinators, as well as their attractiveness efficiency (a combination of duration of flowering and insect visitation), their response to two different agronomic management practices (growing in mixed vs. mono-specific plots; tillage vs. no-tillage), and their potential weediness. The results of this experiment showed that the flowers of Borago officinalis, Echium plantagineum, Phacelia tanacetifolia and Diplotaxis tenuifolia were attractive to bees, while Calendula arvensis, Coriandrum sativum, D. tenuifolia and Lobularia maritima were attractive to hoverflies. In addition, floral mixture resulted in lower attractiveness efficiency to pollinators than mono-specific D. tenuifolia, but higher than most of the mono-specific stands. On the other hand, although some of the most attractive plant species (e.g. P. tanacetifolia and C. arvensis) showed potential weediness, their self-seeding was reduced by tillage. After comparing attractiveness efficiency of various herbaceous species to pollinators and their potential weediness, the results indicated that D. tenuifolia showed the highest attractiveness efficiency to pollinators and efficient self-reproduction, making it highly recommended to attract bees and hoverflies in agro-ecosystems of Central Spain. In addition, this plant, commonly known as wild rocket, has a supplementary economic value as a commercialized crop. The implementation of a new floral margin in agro-ecosystems means increased production costs, especially in regions with frequent and long droughts (as it is Central and South-East area of Iberian Peninsula), where the principal agricultural cost is irrigation. Therefore, before recommending D. tenuifolia for sustainable habitat management within agro-ecosystems, it is necessary to study the effect of drought stress and moderate and severe deficit irrigation on its growth, flower development and attractiveness to pollinators. The results of this experiment showed that in greenhouse conditions, potted D. tenuifolia could be without irrigation for 4 days without affecting its growth, flowering and attractiveness to pollinators. However, lack of irrigation for 8 days or longer significantly reduced the vegetative growth, number of open flowers, total floral area, flower diameter, corolla tube diameter and corolla tube length of D. tenuifolia. This study showed that regulated deficit irrigation can improve water use efficiency, and depending on the purpose of growing D. tenuifolia, as a crop or as a beneficial plant to attract pollinators, it can reduce water consumption by 40% to 70% without affecting its vegetative and floral development and without reducing its attractiveness to pollinators. Finally, the following experiment was developed in order to understand how habitat management can influence on the agricultural production. For this purpose, it was evaluated if the vicinity of mixed and mono-specific field margins, preselected to conserve pollinators within agro-ecosystems, can improve seed production in coriander (C. sativum). The selection of this plant species for the experiment was based on its necessity for insect pollination for production of seeds (even though some pollen can be transmitted from one flower to another by wind) and the fact that under semi-controlled field conditions established in the field it is possible to estimate its total seed production. Since D. tenuifolia is attractive for both bees and hoverflies in Central Spain, the main objective of this experiment was to estimate the impact of two different types of field margins, i.e. mono-specific margin with D. tenuifolia and mixed margin with six herbaceous species, on the seed production of potted coriander. For that reason, it was tested: i) if open pollination (control without proximate field margin and treatments with nearby mono-specific and mixed margin) increases the seed production of coriander when compared with no-pollination (covered inflorescences of coriander) under field conditions; ii) if frequency of pollinator visitation to the flowers of coriander was higher in the presence of field margins than in the control without field margin; and iii) if seed production was higher in the presence of field margins than in control plants of coriander without field margin. The results showed that the proximity of both types of floral margins (mixed and mono-specific) improved the seed quality of coriander plants, as seed weight and germination rate were higher than in control plants without field margin. Furthermore, the number of seeds produced was significantly higher in coriander plants grown near mixed margins than near mono-specific margin, probably due to an increase in pollinator visits. Since the experiment was conducted under semi-controlled field conditions, it can be concluded that pollinator visits was the main factor that biased the results, and that presence of both mixed or mono-specific (D. tenuifolia) margins can improve the production of coriander for more than 200% in small-scale gardens and, in addition, conserve the local pollinators.

Cartografía de QTL asociados a la tolerancia a estrés hídrico en frijol común (Phaseolus vulgaris L.)

Phaseolus vulgaris L. (frijol común o judía) es una leguminosa de gran demanda para la nutrición humana y un producto agrícola muy importante. Sin embargo, la producción de frijol se ve limitada por presiones ambientales como la sequía. En México, el 85% de la cosecha de frijol se produce en la temporada de primavera-verano, principalmente en las regiones del altiplano semiárido con una precipitación anual entre 250 y 400 mm. A pesar del implemento de tecnología en el campo, los factores naturales impiden al agricultor llegar a los rendimientos deseados. El Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), como instituto de investigación gubernamental en México, tiene como objetivo la mejora de cultivos estratégicos, uno de ellos, P. vulgaris. Los estudios en relación a la sequía se enfocan especialmente en la selección de genotipos tolerantes, los cuales son sometidos en condiciones de estrés y monitoreando parámetros como el rendimiento y peso de semilla, además de algunos indicadores tales como índice de cosecha. El resultado de estos trabajos ha sido la obtención de variedades con mayor tolerancia a la sequía, tales como Pinto Villa y Pinto Saltillo. En los últimos años se ha avanzado notablemente en el conocimiento de las bases moleculares en las respuestas de las plantas al estrés. De acuerdo a diversos estudios se ha demostrado que las plantas bajo estrés por sequía experimentan cambios en la expresión de genes involucrados en la señalización, regulación de la transcripción y la traducción, transporte de agua y la función directa en la protección celular. También se ha observado que el déficit de agua es causado por las temperaturas extremas y la alta concentración de sales, por lo que al nivel molecular, las respuestas al estrés tienen puntos de especificidad y puntos de entrecruzamiento. La sequía puede generar estreses secundarios, tales como el nutricional, oxidativo y osmótico. Sin embargo, es necesario identificar y caracterizar muchos de los componentes involucrados en las respuestas al déficit hídrico, la caracterización de estos genes permitirá tener una mejor comprensión de los mecanismos bioquímicos y fisiológicos involucrados en la tolerancia al estrés. Actualmente, con el apoyo de la biología molecular se han identificado algunos genes que otorgan ventajas para la adaptación a ambientes desfavorables. Por lo que el objetivo del presente trabajo es identificar marcadores genéticos asociados a rasgos fenotípicos con énfasis a la tolerancia a estrés hídrico en P. vulgaris. Una vez establecidos los marcadores asociados al estrés hídrico, es factible considerar su uso para la selección asistida por marcadores en líneas o variedades de frijol de interés para los mejoradores. Se evaluaron 282 familias F3:5 derivadas de la cruza entre los cultivares Pinto Villa y Pinto Saltillo. Las familias se sembraron bajo un diseño simple de látice 17x17, el experimento se llevo acabo en el ciclo primavera-verano del 2010 y 2011, y otoñoinvierno de 2010 en el Campo Experimental Bajío del INIFAP con dos repeticiones para cada tratamiento de humedad (riego completo y sequía terminal). En todos los genotipos se realizó el fenotipado (variables fenotípicas) y el genotipado a través de marcadores moleculares. Los análisis estadísticos se basaron en el análisis de componentes principales (Eigen Analysis Selection Index Method, ESIM), la asociación entre marcadores SNP y el fenotipado (paquete SNPassoc para R) y el análisis de varianza (ANOVA). Los valores ESIM mostraron que las variables de Rendimiento, Días a floración, Días a madurez fisiológica e Índice de cosecha fueron sobresalientes en sequía terminal, por lo que se sugieren tomarse en consideración para los estudios de sequía en P. vulgaris como monitores de evaluación a la resistencia. Se identificaron nueve familias sobresalieron por sus valores ESIM (PV/PS6, 22, 131, 137, 149, 154, 201, 236 y 273), además de presentar valores superiores para el rendimiento en comparación con los parentales. Estos genotipos son candidatos interesantes para realizar estudios de identificación de loci asociados con la respuesta al estrés, y como potenciales parentales en el desarrollo de nuevas variedades de frijol. En los análisis de asociación SNPassoc se identificaron 83 SNPs significativos (p<0,0003) asociados a los rasgos fenotípicos, obteniendo un total de 222 asociaciones, de las cuales predomina el modelo genético de codominancia para las variables Días a floración, Periodo reproductivo y Biomasa total. Treinta y siete SNPs se identificaron a diferentes funciones biológicas a través del análisis de anotación funcional, de los cuales 12 SNPs (9, 18, 28, 39, 61, 69, 80, 106, 115, 128, 136 y 142) sobresalen por su asociación al fenotipado, y cuya anotación funcional indica que se encuentran en genes relacionados a la tolerancia a la sequía, tales como la actividad kinasa, actividad metabólica del almidón, carbohidratos y prolina, respuesta al estrés oxidativo, así como en los genes LEA y posibles factores de transcripción. En el caso de los análisis ANOVA, se identificaron 72 asociaciones entre los SNPs y las variables fenotípicas (F< 3,94E-04). Las 72 asociaciones corresponden a 30 SNPs y 7 variables fenotípicas, de las que predomina Peso de 100 semillas y Periodo reproductivo. Para los rasgos de Rendimiento, Índice de cosecha y Días a madurez fisiológica se presentaron asociaciones con seis SNPs (17, 34, 37, 50, 93 y 107), de los cuales, a los SNP37 y SNP107 fueron identificados a la anotación biológica de protein binding. Por otro lado, los SNP106 y SNP128 asociados al Periodo reproductivo, son genes con actividad kinasa y actividad metabólica del almidón, respectivamente. Para los marcadores tipo AFLP, se identificaron 271 asociaciones (F<2,34E-04). Las asociaciones corresponden a 86 AFLPs con todas las variables fenotípicas evaluadas, de las que predomina peso de 100 semillas, Días a floración y Periodo reproductivo. Debido a que los en los AFLPs no es posible determinar su anotación biológica, se proponen como marcadores potenciales relacionados a la resistencia a la sequía en frijol. Los AFLPs candidatos requieren más estudios tales como la secuenciación de los alelos respectivos, así como la identificación de éstas secuencias en el genoma de referencia y su anotación biológica, entre otros análisis, de esta manera podríamos establecer aquellos marcadores candidatos a la validación para la selección asistida. El presente trabajo propone tanto genotipos como marcadores genéticos, que deben ser validados para ser utilizados en el programa de mejoramiento de P. vulgaris, con el objetivo de desarrollar nuevas líneas o variedades tolerantes a la sequía. ABSTRACT Phaseolus vulgaris L. (common bean or judia) is a legume of great demand for human consumption and an important agricultural product. However, the common bean production is limited by environmental stresses, such as drought. In Mexico, 85% of the common bean crop is produced in the spring-summer season mainly in semiarid highland regions with a rainfall between 250 and 400 mm per year. In spite of the improvement of crop technology, the natural factors hamper getting an optimal yield. The National Institute for Forestry, Agriculture and Livestock (INIFAP) is a government research institute from Mexico, whose main objective is the genetic breeding of strategic crops, like P. vulgaris L. The drought tolerance studies particularly focus on the selection of bean tolerant genotypes, which are subjected to stress conditions, by means of monitoring parameters such as yield and seed weight, plus some agronomic indicators such as harvest index. The results of these works have led to obtain cultivars with higher drought tolerance such as Pinto Villa and Pinto Saltillo. Significant achievements have been recently made in understanding the molecular basis of stress plant responses. Several studies have shown that plants under drought stress present changes in gene expression related to cell signalling, transcriptional and translational regulation, water transport and cell protection. In addition, it has been observed that the extreme temperatures and high salt concentrations can cause a water deficiency so, at the molecular level, stress responses have specific and crossover points. The drought can cause secondary stresses, such as nutritional, oxidative and osmotic stress. It is required the identification of more components involved in the response to water deficit, the characterization of these genes will allow a better understanding of the biochemical and physiological mechanisms involved in stress tolerance. Currently, with the support of molecular biology techniques, some genes that confer an advantage for the crop adaptation to unfavourable environments have been identified. The objective of this study is to identify genetic markers associated with phenotypic traits with emphasis on water stress tolerance in P. vulgaris. The establishment of molecular markers linked to drought tolerance would make possible their use for marker-assisted selection in bean breeding programs. Two hundred and eighty two F3:5 families derived from a cross between the drought resistant cultivars Pinto Villa and Pinto Saltillo were evaluated. The families were sowed under a 17x17 simple lattice design. The experiment was conducted between spring-summer seasons in 2010 and 2011, and autumn-winter seasons in 2010 at the Bajio Experimental Station of INIFAP with two treatments (full irrigation and terminal drought). All families were phenotyped and genotyped using molecular markers. Statistical analysis was based on principal component analysis (Eigen Analysis Selection Index Method, ESIM), association analysis between SNP markers and phenotype (SNPassoc package R) and analysis of variance (ANOVA). The ESIM values showed that seed yield, days to flowering, days to physiological maturity and harvest index were outstanding traits in terminal drought treatment, so they could be considered as suitable parameters for drought-tolerance evaluation in P. vulgaris. Nine outstanding families for the ESIM values were identified (PV/PS6, 22, 131, 137, 149, 154, 201, 236 and 273), in addition, these families showed higher values for seed yield compared to the parental cultivars. These families are promising candidates for studies focused on the identification of loci associated to the stress response, and as potential parental cultivars for the development of new varieties of common bean. In the SNPassoc analysis, 83 SNPs were found significantly associated (p<0.0003) with phenotypic traits, obtaining a total of 222 associations, most of which involved the traits days to flowering, reproductive period and total biomass under a codominant genetic model. The functional annotation analysis showed 37 SNPs with different biological functions, 12 of them (9, 18, 28, 39, 61, 69, 80, 106, 115, 128, 136 and 142) stand out by their association to phenotype. The functional annotation suggested a connection with genes related to drought tolerance, such as kinase activity, starch, carbohydrates and proline metabolic processes, responses to oxidative stress, as well as LEA genes and putative transcription factors. In the ANOVA analysis, 72 associations between SNPs and phenotypic traits (F<3.94E- 04) were identified. All of these associations corresponded to 30 SNPs markers and seven phenotypic traits. Weight of 100 seeds and reproductive period were the traits with more associations. Seed yield, harvest index and days to physiological maturity were associated to six SNPs (17, 34, 37, 50, 93 and 107), the SNP37 and SNP107 were identified as located in protein binding genes. The SNP106 and SNP128 were associated with the reproductive period and belonged to genes with kinase activity and genes related to starch metabolic process, respectively. In the case of AFLP markers, 271 associations (F<2.34E-04) were identified. The associations involved 86 AFLPs and all phenotypic traits, being the most frequently associated weight of 100 seeds, days to flowering and reproductive period. Even though it is not possible to perform a functional annotation for AFLP markers, they are proposed as potential markers related to drought resistance in common bean. AFLPs candidates require additional studies such as the sequencing of the respective alleles, identification of these sequences in the reference genome and gene annotation, before their use in marker assisted selection. This work, although requires further validation, proposes both genotypes and genetic markers that could be used in breeding programs of P. vulgaris in order to develop new lines or cultivars with enhanced drought-tolerance.

Nuevos mecanismos moleculares de tolerancia a sequía y otros tipos de estrés abiótico en especies arbóreas de interés económico

Las masas forestales tienen una importancia colosal para nuestra sociedad y el conjunto de la biosfera. Estudios recientes a escala mundial indican que la sequía es el factor abiótico que más afecta a su crecimiento y supervivencia, seguida por las temperaturas extremas y la salinidad. Aunque comprender los mecanismos con que las especies arbóreas toleran estas formas de estrés tiene un interés aplicado evidente, dichos mecanismos se han estudiado mucho más en especies herbáceas modelo o de interés agronómico. Existen sin embargo diferencias notables entre ellas, como se demuestra en esta tesis y en otros trabajos recientes. Nuestro estudio se centra concretamente en la respuesta molecular del chopo –el sistema modelo forestal más desarrollado– al estrés abiótico, con particular énfasis en la sequía. Utilizando una estrategia proteómica y tratamientos controlados, hemos identificado componentes mayoritarios de dicha respuesta. Su participación en la misma se ha validado mediante análisis transcripcionales detallados utilizando tecnología qRT-PCR (PCR cuantitativa en tiempo real). Hemos identificado proteínas cuyo nexo funcional con mecanismos de tolerancia ya era conocido, como chaperonas moleculares sHSP o enzimas que atenúan el estrés oxidativo, pero también proteínas cuya relación funcional con el estrés es menos clara o incluso novedosa, como polifenol oxidasas (PPO), deshidrogenasas/reductasas de cadena corta (SDR), o bicupinas (BIC), entre otras. El cuerpo central de la tesis consiste en la caracterización detallada de una PPO inusual, cuya inducción por estrés hídrico se describe por vez primera. Estas enzimas están ampliamente distribuidas en plantas, si bien su número es muy variable de unas especies a otras. Algunas, como nogal, tienen un único gen, mientras que Arabidopsis no tiene ninguno. En la última versión del genoma de chopo hemos identificado un total de 12 miembros bona fide, corrigiendo trabajos previos, y hemos caracterizado su expresión individual ante diferentes situaciones de estrés controlado y tratamientos hormonales. La isoforma antedicha es el único miembro de la familia que responde claramente a la deshidratación. También responde a salinidad y a la mayor parte de tratamientos hormonales ensayados, pero no a daño mecánico o tratamientos con metil jasmonato. Esto la diferencia de enzimas homólogas presentes en otras especies de plantas, que se han relacionado experimentalmente con estrés biótico. Los patrones de acumulación de transcritos en árboles adultos son compatibles con un papel protector frente a la sequía. La integración de nuestros estudios funcionales y filogenéticos sugiere que la familia ha sufrido un proceso reciente de diversificación y neofuncionalización, siendo la protección frente a deshidratación su papel primigenio. Aunque se conoce la actividad bioquímica in vitro de este tipo de enzimas, sus sustratos naturales son esencialmente una incógnita. Mediante expresión heteróloga en Escherichia coli BL21(DE3) hemos detectado que la enzima de chopo es capaz de oxidar L-DOPA a dopaquinona, siendo menos activa frente a otros sustratos. Por otra parte, hemos demostrado su localización cloroplástica mediante transformación transitoria de protoplastos con fusiones a la proteína fluorescente YFP. Mediante la obtención de plantas transgénicas de A. thaliana hemos demostrado que la enzima de chopo aumenta considerablemente la tolerancia in vivo frente a la deshidratación y al estrés salino. El análisis fenotípico detallado de las líneas transgénicas, combinando múltiples metodologías, nos ha permitido sustanciar que la tolerancia tiene una base compleja. Esta incluye una mayor protección del sistema fotosintético, una capacidad antioxidante muy incrementada y la acumulación de solutos osmoprotectores como la prolina. Los análisis metabolómicos nos han permitido asociar la expresión de la proteína a la síntesis de un flavano no descrito previamente en A. thaliana, vinculando la enzima de chopo con la síntesis de fenilpropanoides. También hemos observado alteraciones en los niveles hormonales que podrían subyacer a efectos pleiotrópicos con interés aplicado, como un aumento consistente del tamaño de la planta o el acortamiento del ciclo de crecimiento. Además de aportar datos novedosos sobre la funcionalidad in vivo de esta familia de oxidasas, los resultados de esta tesis demuestran que los árboles son sistemas de estudio interesantes para caracterizar nuevas estrategias de tolerancia al estrés abiótico con potencial aplicado. ABSTRACT Forests masses have an extraordinary importance for our society and the biosphere. Recent worldwide studies indicate that drought is the abiotic factor that affects more their growing and survival, followed by extreme temperatures and salinity. The understanding of how the arboreal species tolerate the stress has an evident practical interest, but their mechanisms have been studied much more in herbaceous species or with agronomic interest. However, considerable differences exist between them, as this thesis and recent studies show. Our study is focused on the molecular response of the poplar –the more developed forestry model system- to abiotic stress, specifically focused in the drought. Using a proteomic strategy and controlled treatments, we have identified main components in such response. Its participation has been validated through transcriptional analysis using qRT-PCR technology. We have identified proteins whose functional connection with tolerance mechanisms were already known, as molecular chaperones sHSP or enzymes that attenuate the oxidative stress, but also some proteins whose functional relationship with the stress is less clear or even novel, as polifenol oxidases (PPO), short chain deshidrogenases/reductases (SDR), or bicupines (BIC), among others. The central body of the thesis consists of the detailed characterization of an unsual PPO, whose induction due to drought stress is first described. These enzymes are thoroughly distributed in plants, but their number of members is very variable among species. Some of them, as the walnut tree, have a single gene, while Arabidopsis has none. We have identified a total of 12 members in the last version of the poplar genome, correcting previous works, and have characterized their individual expression against different situations of controlled stress and hormone treatments. The aforementioned isoform is the only member of the family that responds clearly to the drought. It also reacts to salinity and the majority of hormonal treatments tested, but it does not respond to mechanical damage or treatments with methyl jasmonate. This is the difference with homologue enzymes present in other plant species, which have been related experimentally with abiotic stress. The accumulation patterns of transcripts in adult trees are compatible with a protector role against drought. The integration of our functional and phylogenetic studies suggests that the family has suffered a recent process of diversification and neofunctionalization, being the protection against drought their original role. Although the in vitro biochemistry activity of this kind of enzymes is already known, their natural substracts are essentially a mystery. By means of heterologous expression of Escherichia coli BL21(DE3) we have detected that the enzyme of poplar is able to oxidize L-DOPA to dopaquinone, being less active against other substrates. Additionally, we have proven its chloroplastic location with transitory transformation of protoplasts with YFP protein fusion. By means of getting transgenic plants of A. thaliana, we have demonstrated that the poplar enzyme increases notably the in vivo tolerance against the drought and salinity stresses. The phenotypic analysis of the transgenic lines, and the use of multiple methodologies, allowed us to test the complexity of the tolerance. This includes a major protection of the photosynthetic system, a very increased antioxidant capacity and the accumulation of osmoprotectant solutes as the proline. The metabolic analysis has allowed to associate the protein expression with the synthesis of a Flavan non described previously in A. thalaiana, linking the enzyme of poplar with the synthesis of phenylpropanoids. We have observed alterations in the hormonal levels that could underlie pleiotropic effects with applied interest, as a consistent increase of the size of the plant and the reduction of the growth cycle. The results of this thesis, in addition to provide novel data about the in vivo functionality of the oxidase family, demonstrate that the trees are interesting systems of study to characterize new strategies of tolerance against abiotic stress with applied potential.