Determination of the uptake and translocation of nitrogen applied at different growth stages of a melon crop (Cucumis melo L.) using 15N isotope.

In order to establish a rational nitrogen (N) fertilisation and reduce groundwater contamination, a clearer understanding of the N distribution through the growing season and its dynamics inside the plant is crucial. In two successive years, a melon crop (Cucumis melo L. cv. Sancho) was grown under field conditions to determine the uptake of N fertiliser, applied by means of fertigation at different stages of plant growth, and to follow the translocation of N in the plant using 15N-labelled N. In 2006, two experiments were carried out. In the first experiment, labelled 15N fertiliser was supplied at the female-bloom stage and in the second, at the end of fruit ripening. Labelled 15N fertiliser was made from 15NH415NO3 (10 at.% 15N) and 9.6 kg N ha−1 were applied in each experiment over 6 days (1.6 kg N ha−1 d−1). In 2007, the 15N treatment consisted of applying 20.4 kg N ha−1 as 15NH415NO3 (10 at.% 15N) in the middle of fruit growth, over 6 days (3.4 kg N ha−1 d−1). In addition, 93 and 95 kg N ha−1 were supplied daily by fertigation as ammonium nitrate in 2006 and 2007, respectively. The results obtained in 2006 suggest that the uptake of N derived from labelled fertiliser by the above-ground parts of the plants was not affected by the time of fertiliser application. At the female-flowering and fruit-ripening stages, the N content derived from 15N-labelled fertiliser was close to 0.435 g m−2 (about 45% of the N applied), while in the middle of fruit growth it was 1.45 g m−2 (71% of the N applied). The N application time affected the amount of N derived from labelled fertiliser that was translocated to the fruits. When the N was supplied later, the N translocation was lower, ranging between 54% at female flowering and 32% at the end of fruit ripening. Approximately 85% of the N translocated came from the leaf when the N was applied at female flowering or in the middle of fruit growth. This value decreased to 72% when the 15N application was at the end of fruit ripening. The ammonium nitrate became available to the plant between 2 and 2.5 weeks after its application. Although the leaf N uptake varied during the crop cycle, the N absorption rate in the whole plant was linear, suggesting that the melon crop could be fertilised with constant daily N amounts until 2–3 weeks before the last harvest.

Validation of the SenseWear armband in circuit resistance training with different loads

The use of the SenseWear™ armband (SWA), an objective monitor of physical activity, is a relatively new device used by researchers to measure energy expenditure. These monitors are practical, relatively inexpensive and easy-to-use. The aim of the present study was to assess the validity of SWAs for the measurement of energy expenditure (EE) in circuit resistance training (CRT) at three different intensities in moderately active, healthy subjects. The study subjects (17 females, 12 males) undertook CRT at 30, 50 and 70% of the 15 repetition maximum for each exercise component wearing an SWA as well as an Oxycon Mobile (OM) portable metabolic system (a gold standard method for measuring EE). The EE rose as exercise intensity increased, but was underestimated by the SWAs. For women, Bland-Altman plots showed a bias of 1.13 ± 1.48 METs and 32.1 ± 34.0 kcal in favour of the OM system, while for men values of 2.33 ± 1.82 METs and 75.8 ± 50.8 kcal were recorded.

Estructura de una red esencial de señalización que regula la homeostasis iónica en plantas

Las plantas son organismos sésiles que han desarrollado la capacidad para detectar variaciones sutiles en su ambiente y producir respuestas adaptativas mediante rutas de señalización. Los estímulos causados por el estrés biótico y abiótico son numerosos y dependiendo del tiempo de exposición y su intensidad, pueden reducir la tasa de crecimiento de las plantas y la producción. Los cambios en la concentración del calcio citosólico libre constituyen una de las primeras reacciones intracelulares a las situaciones de estrés abiótico. En esta situación, el calcio actúa como segundo mensajero y las variaciones en su concentración son descodificadas por proteínas de unión a calcio. Las más conocidas son las manos-EF y los dominios C2. Los dominios C2 han sido descritos como dominios de unión a lípidos dependientes de calcio. Estos dominios se consideran proteínas periféricas solubles en agua que se asocian de manera reversible a los lípidos de la membrana mediante una o dos regiones funcionales: el sitio de unión a calcio y el sitio polibásico. A pesar de que se conoce la estructura molecular de algunos dominios C2, se desconocen aspectos relacionados como las reglas que dirigen su forma de interaccionar con los diferentes fosfolípidos y proteínas, la posición que ocupan en la bicapa lipídica y su papel en la transmisión de señales. En esta tesis se ha estudiado una proteína de Arabidopsis thaliana (At3g17980) representativa de una nueva familia de proteínas con dominios C2, que consiste únicamente de un dominio C2. Esta proteína, llamada AtC2.1, ha sido clonada en el vector pETM11, expresada en E. coli y purificada a homogeneidad en dos pasos cromatográficos. Se obtuvieron cristales de AtC2.1 de buena calidad mediante técnicas de difusión de vapor. La proteína fue co-cristalizada con calcio, fosfocolina (POC) y el fosfolípido 1,2-dihexanoil-sn-glicero-3-fosfo-L-serina (PSF). Se recogieron ocho conjuntos de datos de difracción de rayos X empleando radiación sincrotrón. Los cristales difractaron hasta 1.6 Å de resolución. Siete de ellos pertenecían al grupo ortorrómbico P212121, con las dimensiones de la celdilla unidad a = 35.3, b = 88.9, c = 110.6 Å, y un cristal pertenecía al grupo espacial monoclínico C2, con a = 124.84, b = 35.27, c = 92.32 Å y = 121.70º. La estructura se resolvió mediante la técnica MR-SAD utilizando el cinc como dispersor anómalo. La estructura cristalina mostró que la molécula forma un dímero en el que cada protómero se pliega como un dominio C2 típico, con la topología tipo II y presenta una inserción de 43 aminoácidos que la diferencia de los dominios C2 conocidos. El mapa de densidad electrónica mostró dos átomos de calcio por protómero. Se resolvieron las estructuras de AtC2.1 en complejo con POC o PSF. En ambos complejos, el análisis cristalográfico detectó máximos de densidad electrónica en la región correspondiente al sitio polibásico formado por las hebras 2, 3 5 y el lazo 3. Éstos se interpretaron correctamente como dos moléculas de POC y un átomo de cinc, en un complejo, y como la cabeza polar del PSF en el otro. AtC2.1 define un sitio de interacción con lípidos dependiente de cinc. En conclusión, en este trabajo se presenta la estructura tridimensional de AtC2.1, miembro representativo de una familia de proteínas de Arabidopsis thaliana, identificadas como proteínas que interaccionan con los receptores de ABA. Estas proteínas están constituidas únicamente por un dominio C2. El análisis conjunto de los datos biofísicos y cristalográficos muestra que AtC2.1 es un sensor de calcio que une lípidos usando dos sitios funcionales. Estos datos sugieren un mecanismo de inserción en membrana dependiente de calcio que trae consigo la disociación de la estructura dimérica y, por consiguiente, un cambio en las propiedades de superficie de la molécula. Este mecanismo proporciona las bases del reconocimiento y transporte de los receptores de ABA y/o otras moléculas a la membrana celular. Plants are sessile organisms that have developed the capacity to detect slight variations of their environment. They are able to perceive biotic and abiotic stress signals and to transduce them by signaling pathways in order to trigger adaptative responses. Stress factors are numerous and, depending on their exposition time and their concentration, can reduce plant growth rate, limiting the productivity of crop plants. Changes in the cytosolic free calcium concentration are observed as one of the earliest intracellular reactions to abiotic stress signals. Calcium plays a key role as a second messenger, and calcium concentration signatures, called calcium signals, are decodified by calcium binding proteins. The main calcium binding structures are the EF-hand motif and the C2 domains. C2 domain is a calcium dependent lipid-binding domain of approximately 130 amino acids. C2 domain displays two functional regions: the Ca-binding region and the polybasic cluster. Both of them can interact with the membrane phospholipids. Despite the number of C2 domain 3D structures currently available, questions about how they interact with the different target phospholipids, their precise spatial position in the lipid bilayer, interactions with other proteins and their role in transmitting signals downstream, have not yet been explored. In this work we have studied an uncharacterized protein from Arabidopsis thaliana (At3g17980) consisting of only a single C2 domain, as member of a new protein C2-domain family. This protein called AtC2.1 was cloned into the pETM11 vector and expressed in E. coli, allowing the purification to homogeneity in two chromatographic steps. Good quality diffracting crystals were obtained using vapor-diffusion techniques. Crystals were co-crystalized with calcium; phosphocholine (POC) and/or the phospholipid 1,2-dihexanoyl-sn-glycero-3-phospho-L-serine (PSF). Eight data set were collected with synchrotron radiation. Crystals diffracted up to 1.6 Å resolution and seven of them belong to the orthorhombic space group P212121, with unit-cell parameters a = 35.3, b = 88.9, c = 110.6 Å. Another crystal was monoclinic, space group C2, with a = 124.84, b = 35.27, c = 92.32 Å and = 121.70º. The structural model was solved by MR-SAD using Zn2+ as anomalous scatterer. The crystal structure shows that the molecule is a dimer. Each monomer was folded as a canonical C2 domain with the topology II with a 43 residues insertion. The electron density map reveals two calcium ions per molecule. Structures of AtC2.1, complexed with POC and PSF, have been solved. Well-defined extra electron densities were found, in both complexes, within the concave surface formed by strands 2, 3, 5 and loop 3 of AtC2.1. These densities were clearly explained by the presence of the two POC molecules, one zinc atom and head groups of PSF, occupying the cavity of the polybasic site. AtC2.1 defines a new metal dependent lipid-binding site into the polybasic site. In conclusion, in this thesis it is presented the molecular structure of AtC2.1, a representative member of a family of Arabidopsis thaliana C2 domain proteins, of unknown function, but identified as a molecular interacting unit of the ABA receptors. The joint analyses of the biophysical and crystallographic data show that AtC2.1 is a calcium sensor that binds lipids in two sites and suggest a model of calcium-dependent membrane insertion mechanism that will involve either dimer dissociation or a strong rearrangement of the dimeric structure. This mechanism may be the basis for the recognition and delivery of ABA receptors or other protein molecules to cell membranes.

Yield, nutrient utilization and soil properties in a melon crop amended with wine-distillery waste

In Spain, large quantities of wine are produced every year (3,339,700 tonnes in 2011) (FAO, 2011) with the consequent waste generation. During the winemaking process, solid residues like grape stalks are generated, as well as grape marc and wine lees as by-products. According to the Council Regulation (EC) 1493/1999 on the common organization of the wine market, by-products coming from the winery industry must be sent to alcohol-distilleries to generate exhausted grape marc and vinasses. With an adequate composting treatment, these wastes can be applied to soils as a source of nutrients and organic matter. A three-year field experiment (2011, 2012 and 2013) was carried out in Ciudad Real (central Spain) to study the effects of wine-distillery waste compost application in a melon crop (Cucumis melo L.). Melon crop has been traditionally cultivated in this area with high inputs of water and fertilizers, but no antecedents of application of winery wastes are known. In a randomized complete block design, four treatments were compared: three compost doses consisted of 6.7 (D1), 13.3 (D2) and 20 t compost ha-1 (D3), and a control treatment without compost addition (D0). The soil was a shallow sandy-loam (Petrocalcic Palexeralfs) with a depth of 0.60 m and a discontinuous petrocalcic horizon between 0.60 and 0.70 m, slightly basic (pH 8.4), poor in organic matter (0.24%), rich in potassium (410 ppm) and with a medium level of phosphorus (22.1 ppm). During each growing period four harvests were carried out and total and marketable yield (fruits weighting <1 kg or visually rotten were not considered), fruit average weight and fruit number per plant were determined. At the end of the crop cycle, four plants per treatment were sampled and the nutrient content (N, P and K) was determined. Soil samplings (0-30 cm depth) were carried before the application of compost and at the end of each growing season and available N and P, as well as exchangeable K content were analyzed. With this information, an integrated analysis was carried out with the aim to evaluate the suitability of this compost as organic amendment.