The TRANSPLANTA collection of Arabidopsis lines: a resource for functional analysis of transcription factors based on their conditional over-expression

Transcription factors (TFs) are key regulators of gene expression in all organisms. In eukaryotes, TFs are often represented by functionally redundant members of large gene families. Overexpression might prove a means to unveil the biological functions of redundant TFs; however, constitutive overexpression of TFs frequently causes severe developmental defects, preventing their functional characterization. Conditional overexpression strategies help to overcome this problem. Here, we report on the TRANSPLANTA collection of Arabidopsis lines, each expressing one of 949 TFs under the control of a β–estradiol-inducible promoter. Thus far, 1636 independent homozygous lines, representing an average of 2.6 lines for every TF, have been produced for the inducible expression of 634 TFs. Along with a GUS-GFP reporter, randomly selected TRANSPLANTA lines were tested and confirmed for conditional transgene expression upon β–estradiol treatment. As a proof of concept for the exploitation of this resource, β–estradiol-induced proliferation of root hairs, dark-induced senescence, anthocyanin accumulation and dwarfism were observed in lines conditionally expressing full-length cDNAs encoding RHD6, WRKY22, MYB123/TT2 and MYB26, respectively, in agreement with previously reported phenotypes conferred by these TFs. Further screening performed with other TRANSPLANTA lines allowed the identification of TFs involved in different plant biological processes, illustrating that the collection is a powerful resource for the functional characterization of TFs. For instance, ANAC058 and a TINY/AP2 TF were identified as modulators of ABA-mediated germination potential, and RAP2.10/DEAR4 was identified as a regulator of cell death in the hypocotyl–root transition zone. Seeds of TRANSPLANTA lines have been deposited at the Nottingham Arabidopsis Stock Centre for further distribution.

Large-scale identification of gibberellin-related transcription factor defines group VII ERFs as functional of DELLA partners

DELLA proteins are the master negative regulators in gibberellin (GA) signaling acting in the nucleus as transcriptional regulators. The current view of DELLA action indicates that their activity relies on the physical interaction with transcription factors (TFs). Therefore, the identification of TFs through which DELLAs regulate GA responses is key to understanding these responses from a mechanistic point of view. Here, we have determined the TF interactome of the Arabidopsis (Arabidopsis thaliana) DELLA protein GIBBERELLIN INSENSITIVE and screened a collection of conditional TF overexpressors in search of those that alter GA sensitivity. As a result, we have found RELATED TO APETALA2.3, an ethylene-induced TF belonging to the group VII ETHYLENE RESPONSE FACTOR of the APETALA2/ethylene responsive element binding protein superfamily, as a DELLA interactor with physiological relevance in the context of apical hook development. The combination of transactivation assays and chromatin immunoprecipitation indicates that the interaction with GIBBERELLIN INSENSITIVE impairs the activity of RELATED TO APETALA2.3 on the target promoters. This mechanism represents a unique node in the cross regulation between the GA and ethylene signaling pathways controlling differential growth during apical hook development.

Functional genomics tools to decipher the pathogenicity mechanisms of the necrotrophic fungus Plectosphaerella cucumerina in Arabidopsis thaliana

The analysis of the interaction between Arabidopsis thaliana and adapted (PcBMM) and nonadapted (Pc2127) isolates of the necrotrophic fungus Plectosphaerella cucumerina has contributed to the identification of molecular mechanisms controlling plant resistance to necrotrophs.To characterize the pathogenicity bases of the virulence of necrotrophic fungi in Arabidopsis, we developed P. cucumerina functional genomics tools using Agrobacterium tumefaciens-mediated transformation.We generated PcBMM-GFP and Pc2127-GFP transformants constitutively expressing the green fluorescence protein (GFP), and a collection of random T-DNA insertional PcBMM transformants. Confocal microscopy analyses of the initial stages of PcBMM-GFP infection revealed that this pathogen, like other necrotrophic fungi, does not form an appressorium or penetrate into plant cells, but causes successive degradation of leaf cell layers

Nematodes as indicators of the effect of soil fumigants on soil functioning

Debido al futuro incierto de la mayor parte de los fumigantes edáficos usados actualmente en la Unión Europea, que pueden implicar riesgos para la salud humana/animal y el medio ambiente, es necesario desarrollar programas de manejo integrado para el control de plagas de cultivos. Estos programas se incluyen como obligatorios en el Reglamento (EC) No. 1107/2009. De acuerdo con este Reglamento, es obligatoria la evaluación del riesgo asociado al uso de productos fitosanitarios sobre los organismos edáficos no diana y sus funciones, además de llevar a cabo ensayos con diferentes especies indicadoras para obtener datos de toxicidad que puedan ser usados posteriormente en la evaluación de riesgo. Sin embargo, la baja representatividad de algunas de estas especies indicadoras en el área Mediterránea supone una gran limitación. En esta situación, el Panel Científico de Productos Fitosanitarios y sus Residuos de la Autoridad Europea en Seguridad Alimentaria (EFSA), ha señalado la necesidad de modificar los datos ecotoxicológicos requeridos para evaluar los efectos adversos de los productos fitosanitarios de una manera más integrada, incluyendo criterios funcionales y estructurales mediante organismos como bacterias, hongos, protozoos y nematodos. De este modo, la EFSA ha recomendado el uso de los nematodos en la evaluación de la funcionalidad y estructura del suelo. Los nematodos están globalmente distribuidos y son morfológicamente diversos; esto junto con su gran abundancia y diversidad de respuestas a las perturbaciones edáficas, los convierte en indicadores adecuados del estado del suelo. Puesto que los nematodos interaccionan con muchos otros organismos que participan en diferentes eslabones de la red trófica edáfica, jugando papeles importantes en procesos edáficos esenciales en los agroescosistemas, la diversidad de nematodos es, a menudo, usada como indicador biológico de los efectos de las prácticas agrícolas en el estado del suelo. En los últimos años, diferentes índices basados en la comunidad nematológica han facilitado la interpretación de datos complejos sobre la ecología del suelo. Los índices de la red trófica edáfica, basados en la abundancia de grupos funcionales definidos como grupos C-P y grupos tróficos, permiten la evaluación de la funcionalidad de la red trófica edáfica. Por otra parte, la dificultad en la identificación taxonómica de nematodos para explicar su uso limitado como indicadores ecológicos, es ampliamente discutida, y existe cierta controversia en cuanto a la eficacia de los diferentes métodos de identificación de nematodos. Se argumenta que la identificación morfológica es difícil y puede llevar mucho tiempo debido a la falta de expertos especializados, y se afirma que las técnicas moleculares pueden resolver algunas limitaciones de las técnicas morfológicas como la identificación de juveniles. Sin embargo, los métodos de identificación molecular tienen también limitaciones; la mayoría de las bases de datos de secuencias de ADN están fuertemente orientadas hacia los nematodos fitoparásitos, los cuales representan sólo una parte de la comunidad edáfica de nematodos, mientras que hay poca información disponible de nematodos de vida libre a pesar de representar la mayoría de los nematodos edáficos. Este trabajo se centra en el estudio de los efectos de fumigantes edáficos en la funcionalidad del suelo a través del uso de diferentes indicadores basados en la comunidad de nematodos, como los índices de la red trófica, índices de diversidad, abundancia de los taxones más relevantes etc. También se han analizado otros indicadores funcionales relacionados con la supresividad edáfica, el ciclo de nutrientes o la actividad de la microfauna del suelo. En el capítulo 1, la diversidad de nematodos estudiada en una explotación comercial de fresa y sus alrededores durante dos campañas consecutivas en el suroeste español, fue baja en los suelos fumigados con fumigantes químicos ambas campañas y, aunque se observó una recuperación a lo largo de la campaña en la zona tratada, los suelos fumigados mostraron una condición perturbada permanente. La comunidad de nematodos estuvo más asociada al ciclo de nutrientes en la zona sin cultivar que en los suelos cultivados, y se observó poca relación entre la biomasa de las plantas y la estructura de la comunidad de nematodos. Los surcos sin tratar dentro de la zona de cultivo funcionaron como reservorio tanto de nematodos fitoparásitos como beneficiosos; sin embargo estas diferencias entre los surcos y los lomos de cultivo no fueron suficientes para mantener la supresividad edáfica en los surcos. Los suelos tratados fueron menos supresivos que los suelos sin tratar, y se observaron correlaciones positivas entre la supresividad edáfica y la estructura de la red trófica edáfica y la diversidad de nematodos. En el capítulo 2, se evaluaron los efectos de dos pesticidas orgánicos con efecto nematicida y dos nematicidas convencionales sobre las propiedades físico químicas del suelo, la diversidad de nematodos y la biomasa de las plantas en condiciones experimentales en dos tipos de suelo: suelos agrícolas poco diversos y suelos provenientes de una zona de vegetación natural muy diversos. El mayor efecto se observó en el tratamiento con neem, el cual indujo un gran incremento en el número de dauerlarvas en los suelos pobres en nutrientes, mientras que el mismo tratamiento indujo un incremento de poblaciones de nematodos bacterívoros, más estables y menos oportunistas, en los suelos del pinar ricos en materia orgánica. En el capítulo 3, se comparó la eficacia de métodos moleculares (TRFLP, Terminal Restriction Fragment Length Polymorphism) y morfológicos (microscopía de alta resolución) para la identificación de diferentes comunidades denematodos de España e Irlanda. Se compararon estadísticamente las diferencias y similitudes en la diversidad de nematodos, otros indicadores ecológicos y de la red trófica edáfica. Las identificaciones mediante el uso de TRFLP sólo detectó un porcentaje de los taxones presentes en las muestras de suelo identificadas morfológicamente, y los nematodos omnívoros y predadores no fueron detectados molecularmente en nuestro estudio. Los índices calculados en base a los nematodos micróboros mostraron más similitud cuando se identificaron morfológica y molecularmente que los índices basados en grupos tróficos más altos. Nuestros resultados muestran que, al menos con la técnica usada en este estudio, la identificación morfológica de nematodos es una herramienta fiable y más precisa que la identificación molecular, puesto que en general se obtiene una mayor resolución en la identificación de nematodos. En el capítulo 4, se estudiaron también los efectos de los nematicidas químicos sobre la comunidad de nematodos y la biomasa de las plantas en condiciones experimentales de campo, donde se aplicaron en una rotación de cultivo judía-col durante un ciclo de cultivo. Se aplicaron dos tipos de enmiendas orgánicas con el objetivo de mitigar el efecto negativo de los productos fitosanitarios sobre la diversidad edáfica. El efecto de los nematicidas sobre las propiedades del suelo y sobre la comunidad de nematodos fue más agudo que el efecto de las enmiendas. La incorporación de los restos de cosecha al final del ciclo de cultivo de la judía tuvo un gran efecto sobre la comunidad de nematodos, y aunque el número total de nematodos incrementó al final del experimento, se observó una condición perturbada permanente de la red trófica edáfica a lo largo del experimento. ABSTRACT Due to the uncertain future of the soil fumigants most commonly used in the EU, that might involve risks for human/animal health and the environment, there is a need to develop new integrated pest management programs, included as mandatory in the Regulation (EC) No. 1107/2009, to control crop diseases. According to this Regulation, evaluating the risk associated to the use of the plant production products (PPP) on non-target soil fauna and their function, and developing assays with different indicator species to obtain toxicity data to be used in the risk evaluation is mandatory. However, the low representativeness of some of these indicator species in the Mediterranean area is a relevant limitation. In this situation, the Scientific Panel of Plant Protection Products and their Residues of the European Food Safety Authority (EFSA) has pointed out the necessity of modifying the ecotoxicological data set required to evaluate non-target effects of PPP in a more integrated way, including structural and functional endpoints with organism such as bacteria, fungi, protists and nematodes. Thus, EFSA has recommended the use of nematodes in the assessment of the functional and structural features of the soil. Nematodes are globally distributed and morphologically diverse, and due to their high abundance and diversity of responses to soil disturbance, they are suitable indicators of the soil condition. Since nematodes interact with many other organisms as participants in several links of the soil food web, playing important roles in essential soil processes in agroecosystems, nematode diversity is often used as a biological indicator of the effects of agricultural practices on soil condition. In the last years, various indices based on soil nematode assemblages, have facilitated the interpretation of complex soil ecological data. Soil food web indices based on the abundances of functional guilds defined by C-P groups and trophic groups, permit evaluating soil food web functioning. On the other hand, the difficulty of nematode taxonomical identification is commonly argued to explain their limited used as ecological indicators, and there is a certain controversy in terms of the efficacy of various nematode identification methods. It is argued that the morphological identification is difficult and time consuming due to the lack of specialist knowledge, and it is claimed that molecular techniques can solve some limitations of morphological techniques such as the identification of juveniles. Nevertheless, molecular identification methods are limited too, since most of the DNA-based databases are strongly oriented towards plant-parasitic nematodes that represent only a fraction of the soil nematode community, while there is little information available on free-living nematodes, which represent most soil nematodes. This work focuses on the study of the effects of soil fumigants on soil functioning through the use of different indicators based on soil nematode community as soil food web indices, diversity indices, the abundance of more relevant taxa etc. Other functional indicators related to soil suppressiveness, nutrient cycling, or the activity of soil microfauna have been also studied. In chapter 1, nematode diversity assessed in a commercial strawberry farm and its surroundings for two consecutive growing seasons in southern Spain, was low in fumigated soils with chemical pesticides throughout both seasons and, although yearly recovery occurred within the treated fields, fumigated soils showed a permanent perturbed condition. The nematode community was more closely associated to nutrient cycling in the non-cropped than in the cropped soils, and the link between plant biomass and nematode community structure was weak. Non-treated furrows within the treated fields were a reservoir of both beneficial and plant-parasitic nematodes, but such difference between furrows and beds was not enough to maintain more suppressive soil assemblages in the furrows. Treated soils were less suppressive than unmanaged soils, and there was a positive and significant correlation between soil suppressiveness and soil food web structure and diversity. In chapter 2, the effects of two organic pesticides with nematicide effect and two chemical nematicides on soil physicalchemical properties, soil nematode diversity and plant biomass in experimental conditions were assessed in two types of soils: low diversity soils from an agricultural farm, and high diversity soils from a natural vegetation area. The larger effect was observed on the neem treatment, which induced a large boost of dauer juveniles in the nutrient-depleted soil, while the same treatment induced the increase of more stable, less opportunistic, populations of generalist bacterivore nematodes in the pine forest soil, rich in organic matter. In chapter 3, comparison of the efficiency of molecular (TRFLP, Terminal Restriction Fragment Length Polymorphism) and morphological (microscopy at high magnification) identification methods was carried out in different nematode communities from five sites of different land uses in Spain and Ireland. Differences and similarities on nematode diversity and other ecological and soil food web indices assessed by both methods, were statistically compared. Molecular identification with TRFLP only detected a percentage of the taxa present in the soil samples identified morphologically, and omnivores and predators were not detected molecularly in our study. Indices involving microbial feeding nematodes were more similar between identification methods than indices involving higher trophic links. Our results show that, at least with the technique used in this study, identifying nematodes morphologically is a reliable and more precise identification tool than molecular identification, since a higher taxonomic resolution is in general obtained compared to TRFLP. In chapter 4, the effect of chemical nematicides on nematode community descriptors and plant biomass was also studied in field conditions in an experimental area in which dazomet and dimethyl disulfide was applied in a bean-cabbage rotation system for a single season. Organic amendments were incorporated into the soil with the aim of mitigate the negative effect of the pesticides on soil diversity. The effect of the nematicides was much more noticeable than the effect of the amendments on soil properties and nematode community descriptors. The incorporation of bean crop residues into the soil at the end of bean crop cycle affected soil nematode community descriptors to a great extent, and although total number of nematodes increased at the end of the experiment, a permanent perturbed soil food web condition was observed along the experiment.

Negative density dependence and environmental heterogeneity effects on tree ferns across succession in a tropical montane forest.

Although tree ferns are an important component of temperate and tropical forests, very little is known about their ecology. Their peculiar biology (e.g., dispersal by spores and two-phase life cycle) makes it difficult to extrapolate current knowledge on the ecology of other tree species to tree ferns. In this paper, we studied the effects of negative density dependence (NDD) and environmental heterogeneity on populations of two abundant tree fern species, Cyathea caracasana and Alsophila engelii, and how these effects change across a successional gradient. Species patterns harbor information on processes such as competition that can be easily revealed using point pattern analysis techniques. However, its detection may be difficult due to the confounded effects of habitat heterogeneity. Here, we mapped three forest plots along a successional gradient in the montane forests of Southern Ecuador. We employed homogeneous and inhomogeneous K and pair correlation functions to quantify the change in the spatial pattern of different size classes and a case-control design to study associations between juvenile and adult tree ferns. Using spatial estimates of the biomass of four functional tree types (short- and long-lived pioneer, shade- and partial shade-tolerant) as covariates, we fitted heterogeneous Poisson models to the point pattern of juvenile and adult tree ferns and explored the existence of habitat dependencies on these patterns. Our study revealed NDD effects for C. caracasana and strong environmental filtering underlying the pattern of A. engelii. We found that adult and juvenile populations of both species responded differently to habitat heterogeneity and in most cases this heterogeneity was associated with the spatial distribution of biomass of the four functional tree types. These findings show the effectiveness of factoring out environmental heterogeneity to avoid confounding factors when studying NDD and demonstrate the usefulness of covariate maps derived from mapped communities.

Functional response of Ulmus minor Mill. to drought, flooding and dutch elm disease

Ulmus minor es una especie arbórea originaria de Europa cuyas poblaciones han sido diezmadas por el hongo patógeno causante de la enfermedad de la grafiosis. La conservación de los olmos exige plantearse su propagación a través de plantaciones y conocer mejor su ecología y biología. Ulmus minor es un árbol de ribera, pero frecuentemente se encuentra alejado del cauce de arroyos y ríos, donde la capa freática sufre fuertes oscilaciones. Por ello, nuestra hipótesis general es que esta especie es moderadamente resistente tanto a la inundación como a la sequía. El principal objetivo de esta tesis doctoral es entender desde un punto de vista funcional la respuesta de U. minor a la inundación, la sequía y la infección por O. novo-ulmi; los factores que posiblemente más influyen en la distribución actual de U. minor. Con este objetivo se persigue dar continuidad a los esfuerzos de conservación de esta especie que desde hace años se dedican en varios centros de investigación a nivel mundial, ya que, entender mejor los mecanismos que contribuyen a la resistencia de U. minor ante la inoculación con O. novo-ulmi y factores de estrés abiótico ayudará en la selección y propagación de genotipos resistentes a la grafiosis. Se han planteado tres experimentos en este sentido. Primero, se ha comparado la tolerancia de brinzales de U. minor y U. laevis – otro olmo ibérico – a una inmersión controlada con el fin de evaluar su tolerancia a la inundación y comprender los mecanismos de aclimatación. Segundo, se ha comparado la tolerancia de brinzales de U. minor y Quercus ilex – una especie típica de ambientes Mediterránea secos – a la falta de agua en el suelo con el fin de evaluar el grado de tolerancia y los mecanismos de aclimatación a la sequía. El hecho de comparar dos especies contrastadas responde al interés en entender mejor cuales son los procesos que conducen a la muerte de una planta en condiciones de sequía – asunto sobre el que hay una interesante discusión desde hace algunos años. En tercer lugar, con el fin de entender mejor la resistencia de algunos genotipos de U. minor a la grafiosis, se han estudiado las diferencias fisiológicas y químicas constitutivas e inducidas por O. novo-ulmi entre clones de U. minor seleccionados a priori por su variable grado de resistencia a esta enfermedad. En el primer experimento se observó que los brinzales de U. minor sobrevivieron 60 días inmersos en una piscina con agua no estancada hasta una altura de 2-3 cm por encima del cuello de la raíz. A los 60 días, los brinzales de U. laevis se sacaron de la piscina y, a lo largo de las siguientes semanas, fueron capaces de recuperar las funciones fisiológicas que habían sido alteradas anteriormente. La conductividad hidráulica de las raíces y la tasa de asimilación de CO2 neta disminuyeron en ambas especies. Por el contrario, la tasa de respiración de hojas, tallos y raíces aumentó en las primeras semanas de la inundación, posiblemente en relación al aumento de energía necesario para desarrollar mecanismos de aclimatación a la inundación, como la hipertrofia de las lenticelas que se observó en ambas especies. Por ello, el desequilibrio del balance de carbono de la planta podría ser un factor relevante en la mortalidad de las plantas ante inundaciones prolongadas. Las plantas de U. minor (cultivadas en envases de 16 litros a media sombra) sobrevivieron por un prolongado periodo de tiempo en verano sin riego; la mitad de las plantas murieron tras 90 días sin riego. El cierre de los estomas y la pérdida de hojas contribuyeron a ralentizar las pérdidas de agua y tolerar la sequía en U. minor. Las obvias diferencias en tolerancia a la sequía con respecto a Q. ilex se reflejaron en la distinta capacidad para ralentizar la aparición del estrés hídrico tras dejar de regar y para transportar agua en condiciones de elevada tensión en el xilema. Más relevante es que las plantas con evidentes síntomas de decaimiento previo a su muerte exhibieron pérdidas de conductividad hidráulica en las raíces del 80% en ambas especies, mientras que las reservas de carbohidratos apenas variaron y lo hicieron de forma desigual en ambas especies. Árboles de U. minor de 5 y 6 años de edad (plantados en eras con riego mantenido) exhibieron una respuesta a la inoculación con O. novo-ulmi consistente con ensayos previos de resistencia. La conductividad hidráulica del tallo, el potencial hídrico foliar y la tasa de asimilación de CO2 neta disminuyeron significativamente en relación a árboles inoculados con agua, pero solo en los clones susceptibles. Este hecho enlaza con el perfil químico “más defensivo” de los clones resistentes, es decir, con los mayores niveles de suberina, ácidos grasos y compuestos fenólicos en estos clones que en los susceptibles. Ello podría restringir la propagación del hongo en el árbol y preservar el comportamiento fisiológico de los clones resistentes al inocularlos con el patógeno. Los datos indican una respuesta fisiológica común de U. minor a la inundación, la sequía y la infección por O. novo-ulmi: pérdida de conductividad hidráulica, estrés hídrico y pérdida de ganancia neta de carbono. Pese a ello, U. minor desarrolla varios mecanismos que le confieren una capacidad moderada para vivir en suelos temporalmente anegados o secos. Por otro lado, el perfil químico es un factor relevante en la resistencia de ciertos genotipos a la grafiosis. Futuros estudios deberían examinar como este perfil químico y la resistencia a la grafiosis se ven alteradas por el estrés abiótico. ABSTRACT Ulmus minor is a native European elm species whose populations have been decimated by the Dutch elm disease (DED). An active conservation of this species requires large-scale plantations and a better understanding of its biology and ecology. U. minor generally grows close to water channels. However, of the Iberian riparian tree species, U. minor is the one that spread farther away from rivers and streams. For these reasons, we hypothesize that this species is moderately tolerant to both flooding and drought stresses. The main aim of the present PhD thesis is to better understand the functional response of U. minor to the abiotic stresses – flooding and drought – and the biotic stress – DED – that can be most influential on its distribution. The overarching goal is to aid in the conservation of this emblematic species through a better understanding of the mechanisms that contribute to resistance to abiotic and biotic stresses; an information that can help in the selection of resistant genotypes and their expansion in large-scale plantations. To this end, three experiments were set up. First, we compared the tolerance to experimental immersion between seedlings of U. minor and U. laevis – another European riparian elm species – in order to assess their degree of tolerance and understand the mechanisms of acclimation to this stress. Second, we investigated the tolerance to drought of U. minor seedlings in comparison with Quercus ilex (an oak species typical of dry Mediterranean habitats). Besides assessing and understanding U. minor tolerance to drought at the seedling stage, the aim was to shed light into the functional alterations that trigger drought-induced plant mortality – a matter of controversy in the last years. Third, we studied constitutive and induced physiological and biochemical differences among clones of variable DED resistance, before and following inoculation with Ophiostoma novo-ulmi. The goal is to shed light into the factors of DED resistance that is evident in some genotypes of U. minor, but not others. Potted seedlings of U. minor survived for 60 days immersed in a pool with running water to approximately 2-3 cm above the stem collar. By this time, U. minor seedlings died, whereas U. laevis seedlings moved out of the pool were able to recover most physiological functions that had been altered by flooding. For example, root hydraulic conductivity and leaf photosynthetic CO2 uptake decreased in both species; while respiration initially increased with flooding in leaves, stems and roots possibly to respond to energy demands associated to mechanisms of acclimation to soil oxygen deficiency; as example, a remarkable hypertrophy of lenticels was soon observed in flooded seedlings of both species. Therefore, the inability to maintain a positive carbon balance somehow compromises seedling survival under flooding, earlier in U. minor than U. laevis, partly explaining their differential habitats. Potted seedlings of U. minor survived for a remarkable long time without irrigation – half of plants dying only after 90 days of no irrigation in conditions of high vapour pressure deficit typical of summer. Some mechanisms that contributed to tolerate drought were leaf shedding and stomata closure, which reduced water loss and the risk of xylem cavitation. Obviously, U. minor was less tolerant to drought than Q. ilex, differences in drought tolerance resulting mostly from the distinct capacity to postpone water stress and conduct water under high xylem tension among species. More relevant was that plants of both species exhibited similar symptoms of root hydraulic failure (i.e. approximately 80% loss of hydraulic conductivity), but a slight and variable depletion of non-structural carbohydrate reserves preceding dieback. Five- and six-year-old trees of U. minor (planted in the field with supplementary watering) belonging to clones of contrasted susceptibility to DED exhibited a different physiological response to inoculation with O. novo-ulmi. Stem hydraulic conductivity, leaf water potential and photosynthetic CO2 uptake decreased significantly relative to control trees inoculated with water only in DED susceptible clones. This is consistent with the “more defensive” chemical profile observed in resistant clones, i.e. with higher levels of saturated hydrocarbons (suberin and fatty acids) and phenolic compounds than in susceptible clones. These compounds could restrict the spread of O. novo-ulmi and contribute to preserving the near-normal physiological function of resistant trees when exposed to the pathogen. These results evidence common physiological responses of U. minor to flooding, drought and pathogen infection leading to xylem water disruption, leaf water stress and reduced net carbon gain. Still, seedlings of U. minor develop various mechanisms of acclimation to abiotic stresses that can play a role in surviving moderate periods of flood and drought. The chemical profile appears to be an important factor for the resistance of some genotypes of U. minor to DED. How abiotic stresses such as flooding and drought affect the capacity of resistant U. minor clones to face O. novo-ulmi is a key question that must be contemplated in future research.

Estructura espacial y dinámica del bosque montano del sur del Ecuador. Interacciones bióticas y limitaciones abióticas

Muchos estudios han descrito la composición y diversidad de los bosques montanos tropicales, pero los patrones espaciales y las diferentes tipos de relaciones de estos, entre especies o entre grupos funcionales ha sido poco documentada. El presente trabajo se realizó en tres parcelas completamente censadas del bosque de la Estación Biológica “Chamusquin” (Zamora Chinchipe, Ecuador), y se plantea como objetivo principal conocer la estructura espacial y dinámica del bosque montano tropical del sur del Ecuador, así como las interacciones bióticas y limitaciones abióticas que influyen en esta dinámica, para ello se planteó cuatro objetivos de estudio que son: conocer los patrones espaciales de los gremios ecológicos dentro de la zona de estudio; conocer la influencia de la dependencia negativa de la densidad sobre dos especies de helechos arborescentes (Cyatheaceae); conocer si existen especies o grupos ecológicos acumuladoras o repulsoras de diversidad en el área de estudio; y conocer cómo influyen la diversidad filogenética y la densidad del vecindario en la incidencia de herbivoría y parasitismo sobre las fases iniciales del establecimiento forestal (brinzales) así como en la supervivencia de los mismos Como paso previo para el análisis del resto de objetivos, dentro de cada parcela se marcaron todos los individuos con DAP ≥5 cm y se tomaron sus coordenadas (X, Y). Además se tomaron datos de variables superficiales y muestras del suelo. Las especies encontradas se clasificaron de acuerdo a sus características biológicas, asignándolas a cada uno de los cuatro gremios forestales usualmente distinguidos en los bosques tropicales: tolerantes a la sombra (TS), tolerantes parciales a la sombra (TPS), pioneras de vida larga (PVL) y pioneras de vida corta (PVC). Para el estudio del primer objetivo tas se emplearon funciones K de Ripley inhomogéneas, ajustando la heterogeneidad en base a la variación espacial de las variables ambientales registradas . Los resultados demostraron que tanto la frecuencia relativa como el patrón espacial de las diferentes estrategias funcionales varían a lo largo de la sucesión y que éste además está influido por la variación ambiental. En adición, tanto el patrón espacial como la respuesta a la variación ambiental de los diferentes gremios es distinta entre adultos y juveniles. Todo ello sugiere que el ensamblaje de la diversidad en los bosque montanos andinos está controlado por procesos deterministas más que por procesos neutrales. Para responder el segundo objetivo se estudiaron los efectos de la dependencia negativa de la densidad (DND) y la heterogeneidad ambiental en las poblaciones de dos especies de helechos arborescentes abundantes, Cyathea caracasana y Alsophila engelii, y cómo estos efectos cambian a través de un gradiente sucesional. Los patrones de especies albergan información sobre procesos tales como la competencia que puede ser revelado fácilmente utilizando técnicas de análisis de patrones punto. Sin embargo, su detección puede ser difícil debido a los efectos de factores de confusión heterogeneidad del hábitat. Aquí, empleamos funciones K y funciones de correlación de par homogéneas y no homogéneas para cuantificar el cambio en el patrón espacial de diferentes clases de tamaño con un diseño de casos-controles para estudiar las asociaciones entre helechos arborescentes jóvenes y adultos. Usando estimaciones espaciales de la biomasa de los cuatro tipos de gremios ecológicos (PVC, PVL, TPS, TS) como covariables, hemos ajustado modelos de Poisson heterogéneos a los patrones de puntos de de los helechos juveniles y los adultos y hemos explorado además la existencia de dependencia del hábitat en estos patrones. Nuestro estudio reveló efectos de la DND para C. caracasana y un fuerte filtrado ambiental que subyace al patrón de A. engelii. Encontramos también que las poblaciones de adultos y juveniles de ambas especies respondieron de manera diferente a la heterogeneidad del hábitat y en la mayoría de los casos esta heterogeneidad se asoció con la distribución espacial de la biomasa de los cuatro tipos de gremios. Estos resultados muestran la eficacia de controlar los efectos de la heterogeneidad ambiental para evitar su confusión con los patrones derivados de interacciones biológicas cuando se estudia la DND y demuestran la utilidad de los mapas de covariables derivados de comunidades biológicas como resumen de la heterogeneidad ambiental. Para nuestro tercer objetivo nos centramos en explorar cómo influyen las especies más abundantes en la organización espacial de la diversidad a lo largo de un gradiente sucesional en el bosque montano del sur del Ecuador. Para ello utilizamos la función ISAR (Individual Species Area Relationship). Encontramos que la frecuencia de especies neutras, repulsoras y acumuladoras de diversidad taxonómica varía dependiendo del grado de sucesión. Además se comprobó que la mayoría de los gremios forestales se comportó de forma neutral, pero la proporción de acumuladores, aumentó al avanzar la sucesión hacia estados más maduros, lo que indica el establecimiento de fuertes procesos competitivos a medida que avanza la sucesión y la mayor importancia del papel de las especies individuales en dichos estados. Finalmente, examinamos el efecto de la vecindad taxonómica y filogenética, así como la estrategia de vida, sobre la incidencia de la herbivoría y el parasitismo en las poblaciones de brinzales de tres fragmentos forestales en una secuencia sucesional del bosque montano húmedo. Evaluamos además los efectos de herbivoría, parasitismo, estrategia de vida y diferentes indicadores de la vecindad sobre la supervivencia de los brinzales. Por último contrastamos la posible existencia de una tendencia compensatoria de la comunidad (CCT) a nivel de fragmento forestal. Nuestros análisis no consiguieron detectar una CCT pero si pusieron de manifiesto la existencia de efectos locales de dependencia negativa de la densidad. Por ejemplo, la presencia de herbivoría y parasitismo sobre los brinzales se relacionó significativamente con una menor supervivencia de estos. Por otro lado, indicadores del efecto de la vecindad como la densidad de brinzales del mismo género y el área basal de árboles vecinos del mismo género incrementaron la prevalencia de la herbivoría o el parasitismo en los brinzales. El incremento de la incidencia de la herbivoría o el parasitismo no está exclusivamente ligado a tener una vecindad taxonómicamente idéntica (vecinos de la misma especie) sino que categorías taxonómicas más laxas como el "género" o simplemente relaciones de semejanza filogenética son capaces de predecir los efectos negativos de la vecindad. Los efectos detectados variaron en los diferentes grupos funcionales distinguidos. Los resultados que hemos obtenido en este trabajo parecen indicar que el funcionamiento de las comunidades de brinzales del bosque montano tropical no difiere mucho del reportado para comunidades de plántulas en otros bosques tropicales y cumple las predicciones de la hipótesis de Janzen y Connell, aunque matizadas por la mayor resistencia de los brinzales al efecto de herbivoría y parasitismo. ABSTRACT Many studies have described the composition and diversity of tropical montane forests, but the different spatial patterns and types of relationships between species or between functional groups has been poorly documented. This work was made in three completely surveyed forest plots at Biological Station "Chamusquin" (Zamora Chinchipe, Ecuador). Our main objective was to know the spatial structure and dynamics of the tropical montane forest in southern Ecuador, as well as the biotic interactions and abiotic constraints affecting this dynamic. More specifically, we aimed to understand the spatial patterns of ecological guilds; to explore the influence of negative density dependence on two species of tree ferns (Cyatheaceae); to determine whether some species or ecological groups structure spatially plant diversity in these forests; and to test the effects of biological neighborhood on the incidence of herbivory and parasitism and on the survival of saplings. We mapped within each plot all trees with DBH ≥5 cm. Besides, surface data variables and soil samples they were taken. The species found were classified according to their biological characteristics in four forest guilds: shade-tolerant (ST), partial shade tolerant (PST), long-lived pioneer (LLP) and short-lived pioneer (SLP). To analyze the spatial patterns of the ecological guilds, we employed the inhomogeneous version of Ripley's K-function and adjusted heterogeneity surfaces based on the spatial variation of the measured environmental variables. The results showed that both the relative frequency of each functional guild as well as their spatial pattern varied throughout succession and that the spatial pattern is explained by environmental variation. In addition, both spatial pattern and the response to spatial variation of each guild varied throughout ontogeny. All in all suggest that diversity assembly in the studied forests is ruled by deterministic instead of neutral processes. We also addressed the negative effects of density dependence (NDD) and environmental heterogeneity in populations of two species of abundant tree ferns, Cyathea caracasana and Alsophila engelii, and how these effects change across a successional gradient.. Here, we used homogeneous and inhomogeneous K and pair-correlation functions to quantify the change in the spatial pattern of different size classes with a case-control design to study associations between young and adult tree ferns. Using spatial estimates of the biomass of the four types of ecological guilds (SLP, LLP, PST, ST) as co-variables, we fitted heterogeneous Poisson models to juvenile and adult tree fern point patterns and explored the existence of habitat dependence. Our study revealed NDD effects for C. caracasana and strong environmental filtering underlying the pattern of A. engelii. We found that adult and juvenile populations of both species responded differently to habitat heterogeneity and in most cases this heterogeneity was associated with the spatial distribution of biomass of the four functional tree types. These findings show the effectiveness of factoring out environmental heterogeneity to avoid confounding factors when studying NDD and demonstrate the usefulness of covariate maps derived from mapped communities. For our third objective we focused on exploring how the most abundant species influence the spatial organization of tree diversity in these forests. For this, we used the individual species-area relationship function (ISAR). We found that the proportion of accumulator, repeller and neutral species, varied depending on the degree of succession. We found also that most guilds behaved neutrally but the proportion of accumulator guilds increased as succession advanced to more mature stages. This point, to the existence of strong competitive effects mediated by individual species in these mature forests. Finally, we examined the effects of life strategies and taxonomic and phylogenetic neighborhood on the incidence of herbivory and parasitism in the communities of saplings in the same forest fragments. We evaluated also the effects of life strategies, herbivory, parasitism and some indicators of neighborhood on sapling survival. Finally we tested for the existences of a compensatory community trend at plot scale. We did not found a CCT but we found proof of local NND effects. For instance, the prevalence of herbivory and parasitism were related to lower sapling survival. On the other hand the density of con-generic saplings and the basal area of neighbor con-generic trees were related to a higher prevalence of herbivory or parasitism in the saplings. We demonstrated that the increase in the prevalence of herbivory or parasitism it s not exclusive of a conspecific neighborhood but instead larger taxonomic categories such as "genus" or simple phylogenetic relationships are also able to predict NND effects. The NND effects varied among functional guilds. Our results show that the dynamic of sapling communities in Ecuadorian montane forests is similar to seedling dynamics in other tropical forest and follows the predictions of Janzen-Connell hypothesis, although softened by the strong resilience of saplings in comparison to seedlings.

Regenerative morphological traits in a woody species community in Tumbesian tropical dry forest.

The study of functional morphological traits enables us to know fundamental aspects of the dynamics of plant communities in local and global habitats. Regenerative morphological traits play an important role in defining plant history and ecological behavior. Seed and fruit characteristics determine to a large extent the patterns for dispersal, germination, establishment and seedling recruitment a given species exhibits on its natural habitat. Despite their prominent role, seed and fruit traits have been poorly studied at the community level of woody plant species in neo-tropical dry forests. In the present study we aimed at i) evaluate the functional role of morphological traits of seeds, fruits and embryo in woody plant species; ii) determine which are the morphological patterns present in seeds collected from the community of woody species that occur in neo-tropical dry forests; and iii) compare woody plant species seed mass values comparatively between neo-tropical dry and tropical forests. To do so, mature seeds were collected from 79 plant species that occur in the Tumbesian forest of Southwest Ecuador. The studied species included the 42 and 37 most representative tree and shrubbery species of the Tumbesian forest respectively. A total of 18 morphological traits (seven quantitative and 11 qualitative) were measured and evaluated in the seeds, fruits and embryos of the selected species, and we compared the seeds mass with other forest types. Our results showed a huge heterogeneity among traits values in the studied species. Seed mass, volume and number were the traits that vary the most at the community level, i.e. seed length ranged from 1.3 to 39 mm, and seed width from 0.6 to 25 mm. Only six embryo types were found among the 79 plant species. In 40 % of the cases, fully developed inverted embryos with large and thick cotyledons to store considerable amount of nutrients were recorded. We concluded that highly variable and functionally complementary morphological traits occur among the studied woody plants of the dry Tumbesian forest. The latter favors a plethora of behavioral mechanisms to coexist among woody species of the dry forest in response to the environmental stress that is typical of arid areas.

Regulation of the human p21/WAF1/Cip1 promoter in hepatic cells by functional interactions between Sp1 and Smad family members

The cell cycle inhibitor p21/WAF1/Cip1 is expressed in many cell types and is regulated by p53-dependent and p53-independent mechanisms. p21 is an important regulator of hepatocyte cell cycle, differentiation, and liver development, but little is known about the regulation of its synthesis in hepatocytes. We report herein that the p21 gene is constitutively expressed in human hepatoma HepG2 cells. Deletion analysis of the p21 promoter showed that it contains a distal (positions −2,300/−210) and a proximal (positions −124 to −61) region that act synergistically to achieve high levels of constitutive expression. The proximal region that consists of multiple Sp1 binding sites is essential for constitutive p21 promoter activity in hepatocytes. This region also mediates the transcriptional activation of the p21 promoter by members of the Smad family of proteins, which play important role in the transduction of extracellular signals such as transforming growth factor β, activin, etc. Constitutive expression of p21 was severely reduced by a C-terminally truncated form of Smad4 that was shown previously to block signaling through Smads. Smad3/4 and to a much lesser extent Smad2/4 caused high levels of transcriptional activation of the p21 promoter. Transactivation was compromised by N- or C-terminally truncated forms of Smad3. By using Gal4-Sp1 fusion proteins, we show that Smad proteins can activate gene transcription via functional interactions with the ubiquitous factor Sp1. These data demonstrate that Smad proteins and Sp1 participate in the constitutive or inducible expression of the p21 gene in hepatic cells.

Functional consequences of NR2 subunit composition in single recombinant N-methyl-d-aspartate receptors

Single-channel recordings were obtained from Chinese hamster ovary cells transfected with the N-methyl-d-aspartate (NMDA) receptor subunit NR1 in combination with NR2A, NR2B, NR2C, or NR2A/NR2B. NMDA-activated currents were recorded under control conditions and in the presence of a thiol reductant (DTT), an oxidant (5,5′-dithio-bis[2-nitrobenzoic acid], DTNB), or the noncompetitive antagonist CP101,606 (CP). For all subunit combinations, DTT increased the frequency of channel opening when compared with DTNB. In addition, channels obtained from NR1/NR2A-transfected cells also exhibited a pronounced difference in mean open dwell-time between redox conditions. CP dramatically reduced both the open dwell-time and frequency of channel opening of NR1/NR2B-containing receptors, but only modestly inhibited NR1/NR2A and NR1/NR2C channel activity. A small number of patches obtained from cells transfected with NR1/NR2A/NR2B had channels with properties intermediate to NR1/NR2A and NR1/NR2B receptors, including insensitivity to CP block but redox properties similar to NR1/NR2B, consistent with the coassembly of NR2A with NR2B. Hence, NMDA receptors containing multiple types of NR2 subunits can have functionally distinguishable attributes.

Molecular and functional characterization of a novel low-affinity cation transporter (LCT1) in higher plants

The transport of cations across membranes in higher plants plays an essential role in many physiological processes including mineral nutrition, cell expansion, and the transduction of environmental signals. In higher plants the coordinated expression of transport mechanisms is essential for specialized cellular processes and for adaptation to variable environmental conditions. To understand the molecular basis of cation transport in plant roots, a Triticum aestivum cDNA library was used to complement a yeast mutant deficient in potassium (K+) uptake. Two genes were cloned that complemented the mutant: HKT1 and a novel cDNA described in this report encoding a cation transporter, LCT1 (low-affinity cation transporter). Analysis of the secondary structure of LCT1 suggests that the protein contains 8–10 transmembrane helices and a hydrophilic amino terminus containing sequences enriched in Pro, Ser, Thr, and Glu (PEST). The transporter activity was assayed using radioactive isotopes in yeast cells expressing the cDNA. LCT1 mediated low-affinity uptake of the cations Rb+ and Na+, and possibly allowed Ca2+ but not Zn2+ uptake. LCT1 is expressed in low abundance in wheat roots and leaves. The precise functional role of this cation transporter is not known, although the competitive inhibition of cation uptake by Ca2+ has parallels to whole plant and molecular studies that have shown the important role of Ca2+ in reducing Na+ uptake and ameliorating Na+ toxicity. The structure of this higher plant ion transport protein is unique and contains PEST sequences.

Identification and analysis of the plant peroxisomal targeting signal 1 receptor NtPEX5

Protein translocation into peroxisomes takes place via recognition of a peroxisomal targeting signal present at either the extreme C termini (PTS1) or N termini (PTS2) of matrix proteins. In mammals and yeast, the peroxisomal targeting signal receptor, Pex5p, recognizes the PTS1 consisting of -SKL or variants thereof. Although many plant peroxisomal matrix proteins are transported through the PTS1 pathway, little is known about the PTS1 receptor or any other peroxisome assembly protein from plants. We cloned tobacco (Nicotiana tabacum) cDNAs encoding Pex5p (NtPEX5) based on the protein’s interaction with a PTS1-containing protein in the yeast two-hybrid system. Nucleotide sequence analysis revealed that the tobacco Pex5p contains seven tetratricopeptide repeats and that NtPEX5 shares greater sequence similarity with its homolog from humans than from yeast. Expression of NtPEX5 fusion proteins, consisting of the N-terminal part of yeast Pex5p and the C-terminal region of NtPEX5, in a Saccharomyces cerevisiae pex5 mutant restored protein translocation into peroxisomes. These experiments confirmed the identity of the tobacco protein as a PTS1 receptor and indicated that components of the peroxisomal translocation apparatus are conserved functionally. Two-hybrid assays showed that NtPEX5 interacts with a wide range of PTS1 variants that also interact with the human Pex5p. Interestingly, the C-terminal residues of some of these peptides deviated from the established plant PTS1 consensus sequence. We conclude that there are significant sequence and functional similarities between the plant and human Pex5ps.

Functional specificity of MutL homologs in yeast: Evidence for three Mlh1-based heterocomplexes with distinct roles during meiosis in recombination and mismatch correction

The yeast genome encodes four proteins (Pms1 and Mlh1–3) homologous to the bacterial mismatch repair component, MutL. Using two hybrid-interaction and coimmunoprecipitation studies, we show that these proteins can form only three types of complexes in vivo. Mlh1 is the common component of all three complexes, interacting with Pms1, Mlh2, and Mlh3, presumptively as heterodimers. The phenotypes of single deletion mutants reveal distinct functions for the three heterodimers during meiosis: in a pms1 mutant, frequent postmeiotic segregation indicates a defect in the correction of heteroduplex DNA, whereas the frequency of crossing-over is normal. Conversely, crossing-over in the mlh3 mutant is reduced to ≈70% of wild-type levels but correction of heteroduplex is normal. In a mlh2 mutant, crossing-over is normal and postmeiotic segregation is not observed but non-Mendelian segregation is elevated and altered with respect to parity. Finally, to a first approximation, the mlh1 mutant represents the combined single mutant phenotypes. Taken together, these data imply modulation of a basic Mlh1 function via combination with the three other MutL homologs and suggest specifically that Mlh1 combines with Mlh3 to promote meiotic crossing-over.

A functional homolog of a yeast tRNA splicing enzyme is conserved in higher eukaryotes and in Escherichia coli

tRNA splicing in the yeast Saccharomyces cerevisiae requires an endonuclease to excise the intron, tRNA ligase to join the tRNA half-molecules, and 2′-phosphotransferase to transfer the splice junction 2′-phosphate from ligated tRNA to NAD, producing ADP ribose 1′′–2′′ cyclic phosphate (Appr>p). We show here that functional 2′-phosphotransferases are found throughout eukaryotes, occurring in two widely divergent yeasts (Candida albicans and Schizosaccharomyces pombe), a plant (Arabidopsis thaliana), and mammals (Mus musculus); this finding is consistent with a role for the enzyme, acting in concert with ligase, to splice tRNA or other RNA molecules. Surprisingly, functional 2′-phosphotransferase is found also in the bacterium Escherichia coli, which does not have any known introns of this class, and does not appear to have a ligase that generates junctions with a 2′-phosphate. Analysis of the database shows that likely members of the 2′-phosphotransferase family are found also in one other bacterium (Pseudomonas aeruginosa) and two archaeal species (Archaeoglobus fulgidus and Pyrococcus horikoshii). Phylogenetic analysis reveals no evidence for recent horizontal transfer of the 2′-phosphotransferase into Eubacteria, suggesting that the 2′-phosphotransferase has been present there since close to the time that the three kingdoms diverged. Although 2′-phosphotransferase is not present in all Eubacteria, and a gene disruption experiment demonstrates that the protein is not essential in E. coli, the continued presence of 2′-phosphotransferase in Eubacteria over large evolutionary times argues for an important role for the protein.

Functional domains are specified to single-cell resolution in a Drosophila epithelium

Specification of pattern is fundamental to the development of a multicellular organism. The Malpighian (renal) tubule of Drosophila melanogaster is a simple epithelium that proliferates under the direction of a single tip cell into three morphologically distinct domains. However, systematic analysis of a panel of over 700 P{GAL4} enhancer trap lines reveals unexpected richness for such an apparently simple tissue. Using numerical analysis, it was possible formally to reconcile apparently similar or complementary expression domains and thus to define at least five genetically defined domains and multiple cell types. Remarkably, the positions of domain boundaries and the numbers of both principal and secondary (“stellate”) cell types within each domain are reproducible to near single-cell precision between individual animals. Domains of physiological function were also mapped using transport or expression assays. Invariably, they respect the boundaries defined by enhancer activity. These genetic domains can also be visualized in vivo, both in transgenic and wild-type flies, providing an “identified cell” system for epithelial physiology. Building upon recent advances in Drosophila Malpighian tubule physiology, the present study confirms this tissue as a singular model for integrative physiology.