Genetic redundancy among durum wheat accessions as assessed with SSRs and endosperm proteins

Reducing duplication in ex-situ collections is complicated and requires good quality genetic markers. This study was conducted to assess the value of endosperm proteins and SSRs for validation of potential duplicates and monitoring intra-accession variability. Fifty durum wheat (Triticum turgidum ssp. durum) accessions grouped in 23 potential duplicates, and previously characterised for 30 agro-morphological traits, were analysed for gliadin and high molecular weight glutenin (HMWG) subunit alleles, total protein, and 24 SSRs, covering a wide genome area. Similarity and dissimilarity matrices were generated based on protein and SSRs alleles. For heterogeneous accessions at gliadins the percent pattern homology (PH) between gliadin patterns and the Nei’s coefficient of genetic identity (I) were computed. Eighteen duplicates identical for proteins showed none or less than 3 unshared SSRs alleles. For heterogeneous accessions PH and I values lower than 80 identified clearly off-types with more than 3 SSRs unshared. Only those biotypes differing in no more than one protein-coding locus were confirmed with SSRs. A good concordance among proteins, morphological traits, and SSR were detected. However, the discrepancy in similarity detected in some cases showed that it is advisable to evaluate redundancy through distinct approaches. The analysis in proteins together with SSRs data are very useful to identify duplicates, biotypes, close related genotypes, and contaminations

Molecular analysis of endo-β-mannanase genes upon seed imbibition suggest a cross-talk between radicle and micropylar endosperm during germination of Arabidopsis thaliana.

The endo-β-mannanase (MAN) family is represented in the Arabidopsis genome by eight members, all with canonical signal peptides and only half of them being expressed in germinating seeds. The transcripts of these genes were localized in the radicle and micropylar endosperm (ME) before radicle protrusion and this expression disappears as soon as the endosperm is broken by the emerging radicle tip. However, only three of these MAN genes, AtMAN5, AtMAN7 and especially AtMAN6 influence the germination time (t50) as assessed by the analysis of the corresponding knock-out lines. The data suggest a possible interaction between embryo and ME regarding the role of MAN during the Arabidopsis germination process.

Arabidopsis thaliana bZIP44: a transcription factor affecting seed germination and expression of the mannanase-encoding gene AtMAN7.

Endo-β-mannanases (MAN; EC. 3.2.1.78) catalyze the cleavage of β1[RIGHTWARDS ARROW]4 bonds in mannan polymers and have been associated with the process of weakening the tissues surrounding the embryo during seed germination. In germinating Arabidopsis thaliana seeds, the most highly expressed MAN gene is AtMAN7 and its transcripts are restricted to the micropylar endosperm and to the radicle tip just before radicle emergence. Mutants with a T-DNA insertion in AtMAN7 have a slower germination than the wild type. To gain insight into the transcriptional regulation of the AtMAN7 gene, a bioinformatic search for conserved non-coding cis-elements (phylogenetic shadowing) within the Brassicaceae MAN7 gene promoters has been done, and these conserved motifs have been used as bait to look for their interacting transcription factors (TFs), using as a prey an arrayed yeast library from A. thaliana. The basic-leucine zipper TF AtbZIP44, but not the closely related AtbZIP11, has thus been identified and its transcriptional activation upon AtMAN7 has been validated at the molecular level. In the knock-out lines of AtbZIP44, not only is the expression of the AtMAN7 gene drastically reduced, but these mutants have a significantly slower germination than the wild type, being affected in the two phases of the germination process, both in the rupture of the seed coat and in the breakage of the micropylar endosperm cell walls. In the over-expression lines the opposite phenotype is observed.

Transcription factors of the bzip factors of the BZIP family affecting arabidopsis thaliana germination sensu stricto

During seed germination, the endosperm cell walls (CWs) suffer an important weakening process mainly driven by hydrolytic enzymes, such are endo-?- mannanases (MAN; EC. 3.2.1.78) that catalyze the cleavage of ?1?4 bonds in the mannan-polymers. In Arabidopsis thaliana seeds, endo-?-mannanase activity increases during seed imbibition, decreasing after radicle emergence1. AtMAN7 is the most highly expressed MAN gene in seeds upon germination and their transcripts are restricted to the micropylar endosperm and to the radicle tip just before radicle emergence. Mutants with a T-DNA insertion in this gene (K.O. MAN7) have a slower germination rate than the wild type (t50=34 h versus t50=25 h). To gain insight into the transcriptional regulation of the AtMAN7 gene, a bioinformatic search for conserved non-coding cis-elements (phylogenetic shadowing) within the Brassicaceae orthologous MAN7 gene promoters has been done and these conserved motives have been used as baits to look for their interacting transcription factors (TFs), using as a prey an arrayed yeast library of circa 1,200 TFs from A. thaliana. The basic leucine zipper AtbZIP44, but not its closely related ortholog AtbZIP11, has been thus identified and its regulatory function upon AtMAN7 during seed germination validated by different molecular and physiological techniques, such are RT-qPCR analyses, mRNA Fluorescence in situ Hybridization (FISH) experiments, and by the establishment of the germination kinetics of both over-expression (oex) lines and TDNA insertion mutants in AtbZIP44. The transcriptional combinatorial network through which AtbZIP44 regulates AtMAN7 gene expression during seed germination has been further explored through protein-protein interactions between AtbZIP44 and other bZIP members. In such a way, AtbZIP9 has been identified by yeast two-hybrid experiments and its physiological implication in the control of AtMAN7 expression similarly established.

Function of C1A barley peptidases and their inhibitors (cystatins) in barley seed germination : Funcion de las peptidasas C1A de cebada y sus inhibidores (cistatinas) en la germinacion de la semilla

Plant proteolysis is a metabolic process where specific enzymes called peptidases degrade proteins. In plants, this complex process involves broad metabolic networks and different sub-cellular compartments. Several types of peptidases take part in the proteolytic process, mainly cysteine-, serine-, aspartyl- and metallo- peptidases. Among the cysteine-peptidases, the papain-like or C1A peptidases (family C1, clan CA) are extensively present in land plants and are classified into catepsins L-, B-, H- and Flike. The catalytic mechanism of these C1A peptidases is highly conserved and involves the three amino acids Cys, His and Asn in the catalytic triad, and a Gln residue which seems essential for maintaining an active enzyme conformation. These proteins are synthesized as inactive precursors, which comprise an N-terminal signal peptide, a propeptide, and the mature protein. In barley, we have identified 33 cysteine-peptidases from the papain-like family, classifying them into 8 different groups. Five of them corresponded to cathepsins L-like (5 subgroups), 1 cathepsin B-like group, 1 cathepsin F-like group and 1 cathepsin H-like group. Besides, C1A peptidases are the specific targets of the plant proteinaceous inhibitors known as phytocystatins (PhyCys). The cystatin inhibitory mechanism is produced by a tight and reversible interaction with their target enzymes. In barley, the cystatin gene family is comprised by 13 members. In this work we have tried to elucidate the role of the C1A cysteine-peptidases and their specific inhibitors (cystatins) in the germination process of the barley grain. Therefore, we selected a representative member of each group/subgroup of C1A peptidases (1 cathepsin B-like, 1 cathepsin F-like, 1 cathepsin H-like and 5 cathepsins L-like). The molecular characterization of the cysteine-peptidases was done and the peptidase-inhibitor interaction was analyzed in vitro and in vivo. A study in the structural basis for specificity of pro-peptide/enzyme interaction in barley C1A cysteine-peptidases has been also carried out by inhibitory assays and the modeling of the three-dimensional structures. The barley grain maturation produces the accumulation of storage proteins (prolamins) in the endosperm which are mobilized during germination to supply the required nutrients until the photosynthesis is fully established. In this work, we have demonstrated the participation of the cysteine-peptidases and their inhibitors in the degradation of the different storage protein fractions (hordeins, albumins and globulins) present in the barley grain. Besides, transgenic barley plants overexpressing or silencing cysteine-peptidases or cystatins were obtained by Agrobacterium-mediated transformation of barley immature embryos to analyze their physiological function in vivo. Preliminary assays were carried out with the T1 grains of several transgenic lines. Comparing the knock-out and the overexpressing lines with the WT, alterations in the germination process were detected and were correlated with their grain hordein content. These data will be validated with the homozygous grains that are being produced through the double haploid technique by microspore culture. Resumen La proteólisis es un proceso metabólico por el cual se lleva a cabo la degradación de las proteínas de un organismo a través de enzimas específicas llamadas proteasas. En plantas, este complejo proceso comprende un entramado de rutas metabólicas que implican, además, diferentes compartimentos subcelulares. En la proteólisis participan numerosas proteasas, principalmente cisteín-, serín-, aspartil-, y metalo-proteasas. Dentro de las cisteín-proteasas, las proteasas tipo papaína o C1A (familia C1, clan CA) están extensamente representadas en plantas terrestres, y se clasifican en catepsinas tipo L, B, H y F. El mecanismo catalítico de estas proteasas está altamente conservado y la triada catalítica formada por los aminoácidos Cys, His y Asn, y a un aminoácido Gln, que parece esencial para el mantenimiento de la conformación activa de la proteína. Las proteasas C1A se sintetizan como precursores inactivos y comprenden un péptido señal en el extremo N-terminal, un pro-péptido y la proteína madura. En cebada hemos identificado 33 cisteín-proteasas de tipo papaína y las hemos clasificado filogenéticamente en 8 grupos diferentes. Cinco de ellos pertenecen a las catepsinas tipo L (5 subgrupos), un grupo a las catepsinas tipo-B, otro a las catepsinas tipo-F y un último a las catepsinas tipo-H. Las proteasas C1A son además las dianas específicas de los inhibidores protéicos de plantas denominados fitocistatinas. El mecanismo de inhibición de las cistatinas está basado en una fuerte interacción reversible. En cebada, se conoce la familia génica completa de las cistatinas, que está formada por 13 miembros. En el presente trabajo se ha investigado el papel de las cisteín-proteasas de cebada y sus inhibidores específicos en el proceso de la germinación de la semilla. Para ello, se seleccionó una proteasa representante de cada grupo/subgrupo (1 catepsina tipo- B, 1 tipo-F, 1 tipo-H, y 5 tipo-L, una por cada subgrupo). Se ha llevado a cabo su caracterización molecular y se ha analizado la interacción enzima-inhibidor tanto in vivo como in vitro. También se han realizado estudios sobre las bases estructurales que demuestran la especificidad en la interacción enzima/propéptido en las proteasas C1A de cebada, mediante ensayos de inhibición y la predicción de modelos estructurales de la interacción. Finalmente, y dado que durante la maduración de la semilla se almacenan proteínas de reserva (prolaminas) en el endospermo que son movilizadas durante la germinación para suministrar los nutrientes necesarios hasta que la nueva planta pueda realizar la fotosíntesis, en este trabajo se ha demostrado la participación de las cisteínproteasas y sus inhibidores en la degradación de las diferentes tipos de proteínas de reserva (hordeinas, albúmins y globulinas) presentes en el grano de cebada. Además, se han obtenido plantas transgénicas de cebada que sobre-expresan o silencian cistatinas y cisteín-proteasas con el fin de analizar la función fisiológica in vivo. Se han realizado análisis preliminares en las semillas T1 de varias líneas tránsgenicas de cebada y al comparar las líneas knock-out y las líneas de sobre-expresión con las silvestres, se han detectado alteraciones en la germinación que están además correlacionadas con el contenido de hordeinas de las semillas. Estos datos serán validados en las semillas homocigotas que se están generando mediante la técnica de dobles haploides a partir del cultivo de microesporas.

Flow and fracture behaviour of high performance alloys

Based on our needs, that is to say, through precise simulation of the impact phenomena that may occur inside a jet engine turbine with an explicit non-linear finite element code, four new material models are postulated. Each one of is calibrated for four high-performance alloys that can be encountered in a modern jet engine. A new uncoupled material model for high strain and ballistic is proposed. Based on a Johnson-Cook type model, the proposed formulation introduces the effect of the third deviatoric invariant by means of three different Lode angle dependent functions. The Lode dependent functions are added to both plasticity and failure models. The postulated model is calibrated for a 6061-T651 aluminium alloy with data taken from the literature. The fracture pattern predictability of the JCX material model is shown performing numerical simulations of various quasi-static and dynamic tests. As an extension of the above-mentioned model, a modification in the thermal softening behaviour due to phase transformation temperatures is developed (JCXt). Additionally, a Lode angle dependent flow stress is defined. Analysing the phase diagram and high temperature tests performed, phase transformation temperatures of the FV535 stainless steel are determined. The postulated material model constants for the FV535 stainless steel are calibrated. A coupled elastoplastic-damage material model for high strain and ballistic applications is presented (JCXd). A Lode angle dependent function is added to the equivalent plastic strain to failure definition of the Johnson-Cook failure criterion. The weakening in the elastic law and in the Johnson-Cook type constitutive relation implicitly introduces the Lode angle dependency in the elastoplastic behaviour. The material model is calibrated for precipitation hardened Inconel 718 nickel-base superalloy. The combination of a Lode angle dependent failure criterion with weakened constitutive equations is proven to predict fracture patterns of the mechanical tests performed and provide reliable results. A transversely isotropic material model for directionally solidified alloys is presented. The proposed yield function is based a single linear transformation of the stress tensor. The linear operator weighs the degree of anisotropy of the yield function. The elastic behaviour, as well as the hardening, are considered isotropic. To model the hardening, a Johnson-Cook type relation is adopted. A material vector is included in the model implementation. The failure is modelled with the Cockroft-Latham failure criterion. The material vector allows orienting the reference orientation in any other that the user may need. The model is calibrated for the MAR-M 247 directionally solidified nickel-base superalloy.

Contradictio in terminis: dialéctica de la ambigüedad en la arquitectura contemporánea

The twentieth century brought a new sensibility characterized by the discredit of cartesian rationality and the weakening of universal truths, related with aesthetic values as order, proportion and harmony. In the middle of the century, theorists such as Theodor Adorno, Rudolf Arnheim and Anton Ehrenzweig warned about the transformation developed by the artistic field. Contemporary aesthetics seemed to have a new goal: to deny the idea of art as an organized, finished and coherent structure. The order had lost its privileged position. Disorder, probability, arbitrariness, accidentality, randomness, chaos, fragmentation, indeterminacy... Gradually new terms were coined by aesthetic criticism to explain what had been happening since the beginning of the century. The first essays on the matter sought to provide new interpretative models based on, among other arguments, the phenomenology of perception, the recent discoveries of quantum mechanics, the deeper layers of the psyche or the information theories. Overall, were worthy attempts to give theoretical content to a situation as obvious as devoid of founding charter. Finally, in 1962, Umberto Eco brought together all this efforts by proposing a single theoretical frame in his book Opera Aperta. According to his point of view, all of the aesthetic production of twentieth century had a characteristic in common: its capacity to express multiplicity. For this reason, he considered that the nature of contemporary art was, above all, ambiguous. The aim of this research is to clarify the consequences of the incorporation of ambiguity in architectural theoretical discourse. We should start making an accurate analysis of this concept. However, this task is quite difficult because ambiguity does not allow itself to be clearly defined. This concept has the disadvantage that its signifier is as imprecise as its signified. In addition, the negative connotations that ambiguity still has outside the aesthetic field, stigmatizes this term and makes its use problematic. Another problem of ambiguity is that the contemporary subject is able to locate it in all situations. This means that in addition to distinguish ambiguity in contemporary productions, so does in works belonging to remote ages and styles. For that reason, it could be said that everything is ambiguous. And that’s correct, because somehow ambiguity is present in any creation of the imperfect human being. However, as Eco, Arnheim and Ehrenzweig pointed out, there are two major differences between current and past contexts. One affects the subject and the other the object. First, it’s the contemporary subject, and no other, who has acquired the ability to value and assimilate ambiguity. Secondly, ambiguity was an unexpected aesthetic result in former periods, while in contemporary object it has been codified and is deliberately present. In any case, as Eco did, we consider appropriate the use of the term ambiguity to refer to the contemporary aesthetic field. Any other term with more specific meaning would only show partial and limited aspects of a situation quite complex and difficult to diagnose. Opposed to what normally might be expected, in this case ambiguity is the term that fits better due to its particular lack of specificity. In fact, this lack of specificity is what allows to assign a dynamic condition to the idea of ambiguity that in other terms would hardly be operative. Thus, instead of trying to define the idea of ambiguity, we will analyze how it has evolved and its consequences in architectural discipline. Instead of trying to define what it is, we will examine what its presence has supposed in each moment. We will deal with ambiguity as a constant presence that has always been latent in architectural production but whose nature has been modified over time. Eco, in the mid-twentieth century, discerned between classical ambiguity and contemporary ambiguity. Currently, half a century later, the challenge is to discern whether the idea of ambiguity has remained unchanged or have suffered a new transformation. What this research will demonstrate is that it’s possible to detect a new transformation that has much to do with the cultural and aesthetic context of last decades: the transition from modernism to postmodernism. This assumption leads us to establish two different levels of contemporary ambiguity: each one related to one these periods. The first level of ambiguity is widely well-known since many years. Its main characteristics are a codified multiplicity, an interpretative freedom and an active subject who gives conclusion to an object that is incomplete or indefinite. This level of ambiguity is related to the idea of indeterminacy, concept successfully introduced into contemporary aesthetic language. The second level of ambiguity has been almost unnoticed for architectural criticism, although it has been identified and studied in other theoretical disciplines. Much of the work of Fredric Jameson and François Lyotard shows reasonable evidences that the aesthetic production of postmodernism has transcended modern ambiguity to reach a new level in which, despite of the existence of multiplicity, the interpretative freedom and the active subject have been questioned, and at last denied. In this period ambiguity seems to have reached a new level in which it’s no longer possible to obtain a conclusive and complete interpretation of the object because it has became an unreadable device. The postmodern production offers a kind of inaccessible multiplicity and its nature is deeply contradictory. This hypothetical transformation of the idea of ambiguity has an outstanding analogy with that shown in the poetic analysis made by William Empson, published in 1936 in his Seven Types of Ambiguity. Empson established different levels of ambiguity and classified them according to their poetic effect. This layout had an ascendant logic towards incoherence. In seventh level, where ambiguity is higher, he located the contradiction between irreconcilable opposites. It could be said that contradiction, once it undermines the coherence of the object, was the better way that contemporary aesthetics found to confirm the Hegelian judgment, according to which art would ultimately reject its capacity to express truth. Much of the transformation of architecture throughout last century is related to the active involvement of ambiguity in its theoretical discourse. In modern architecture ambiguity is present afterwards, in its critical review made by theoreticians like Colin Rowe, Manfredo Tafuri and Bruno Zevi. The publication of several studies about Mannerism in the forties and fifties rescued certain virtues of an historical style that had been undervalued due to its deviation from Renacentist canon. Rowe, Tafuri and Zevi, among others, pointed out the similarities between Mannerism and certain qualities of modern architecture, both devoted to break previous dogmas. The recovery of Mannerism allowed joining ambiguity and modernity for first time in the same sentence. In postmodernism, on the other hand, ambiguity is present ex-professo, developing a prominent role in the theoretical discourse of this period. The distance between its analytical identification and its operational use quickly disappeared because of structuralism, an analytical methodology with the aspiration of becoming a modus operandi. Under its influence, architecture began to be identified and studied as a language. Thus, postmodern theoretical project discerned between the components of architectural language and developed them separately. Consequently, there is not only one, but three projects related to postmodern contradiction: semantic project, syntactic project and pragmatic project. Leading these projects are those prominent architects whose work manifested an especial interest in exploring and developing the potential of the use of contradiction in architecture. Thus, Robert Venturi, Peter Eisenman and Rem Koolhaas were who established the main features through which architecture developed the dialectics of ambiguity, in its last and extreme level, as a theoretical project in each component of architectural language. Robert Venturi developed a new interpretation of architecture based on its semantic component, Peter Eisenman did the same with its syntactic component, and also did Rem Koolhaas with its pragmatic component. With this approach this research aims to establish a new reflection on the architectural transformation from modernity to postmodernity. Also, it can serve to light certain aspects still unaware that have shaped the architectural heritage of past decades, consequence of a fruitful relationship between architecture and ambiguity and its provocative consummation in a contradictio in terminis. Esta investigación centra su atención fundamentalmente sobre las repercusiones de la incorporación de la ambigüedad en forma de contradicción en el discurso arquitectónico postmoderno, a través de cada uno de sus tres proyectos teóricos. Está estructurada, por tanto, en torno a un capítulo principal titulado Dialéctica de la ambigüedad como proyecto teórico postmoderno, que se desglosa en tres, de títulos: Proyecto semántico. Robert Venturi; Proyecto sintáctico. Peter Eisenman; y Proyecto pragmático. Rem Koolhaas. El capítulo central se complementa con otros dos situados al inicio. El primero, titulado Dialéctica de la ambigüedad contemporánea. Una aproximación realiza un análisis cronológico de la evolución que ha experimentado la idea de la ambigüedad en la teoría estética del siglo XX, sin entrar aún en cuestiones arquitectónicas. El segundo, titulado Dialéctica de la ambigüedad como crítica del proyecto moderno se ocupa de examinar la paulatina incorporación de la ambigüedad en la revisión crítica de la modernidad, que sería de vital importancia para posibilitar su posterior introducción operativa en la postmodernidad. Un último capítulo, situado al final del texto, propone una serie de Proyecciones que, a tenor de lo analizado en los capítulos anteriores, tratan de establecer una relectura del contexto arquitectónico actual y su evolución posible, considerando, en todo momento, que la reflexión en torno a la ambigüedad todavía hoy permite vislumbrar nuevos horizontes discursivos. Cada doble página de la Tesis sintetiza la estructura tripartita del capítulo central y, a grandes rasgos, la principal herramienta metodológica utilizada en la investigación. De este modo, la triple vertiente semántica, sintáctica y pragmática con que se ha identificado al proyecto teórico postmoderno se reproduce aquí en una distribución específica de imágenes, notas a pie de página y cuerpo principal del texto. En la columna de la izquierda están colocadas las imágenes que acompañan al texto principal. Su distribución atiende a criterios estéticos y compositivos, cualificando, en la medida de lo posible, su condición semántica. A continuación, a su derecha, están colocadas las notas a pie de página. Su disposición es en columna y cada nota está colocada a la misma altura que su correspondiente llamada en el texto principal. Su distribución reglada, su valor como notación y su posible equiparación con una estructura profunda aluden a su condición sintáctica. Finalmente, el cuerpo principal del texto ocupa por completo la mitad derecha de cada doble página. Concebido como un relato continuo, sin apenas interrupciones, su papel como responsable de satisfacer las demandas discursivas que plantea una investigación doctoral está en correspondencia con su condición pragmática.

Uso de marcadores moleculares para la racionalización y evaluación de la calidad en las colecciones de Recursos Fitogenéticos de trigo

Esta tesis tiene dos objetivos generales: el primero, analizar el uso de proteínas del endospermo y SSRs para la racionalización de las colecciones de trigo, y el segundo, estudiar la influencia de las proteínas del endospermo, del año de cultivo y del abonado nitrogenado en la calidad en un grupo de variedades locales españolas. Dentro del primer objetivo, se estudió la diversidad genética de la colección de Triticum monococcum L. (escaña menor), y de una muestra de la colección de Triticum turgidum L. (trigo duro) del CRF-INIA, con 2 y 6 loci de gliadinas, y 6 y 24 SSRs, para la escaña menor y el trigo duro, respectivamente. Ambas colecciones presentaron una gran diversidad genética, con una gran diferenciación entre las variedades y pequeña dentro de ellas. Los loci de gliadinas mostraron una gran variabilidad, siendo los loci Gli-2 los más útiles para distinguir variedades. En la escaña menor, las gliadinas presentaron mayor poder de discriminación que los SSRs; aunque en trigo duro los SSRs identificaron más genotipos. El número de alelos encontrado fue alto; 24 y 38 en gliadinas, y 29 y 203 en SSRs, en escaña menor y trigo duro, respectivamente. En trigo duro, se identificaron 17 alelos nuevos de gliadinas lo que demuestra que el germoplasma español es muy singular. En ambas especies, se detectaron asociaciones entre la variación alélica en prolaminas y el origen geográfico y filogenético de las variedades. La utilidad de las proteínas (6 loci de gliadinas, 2 loci de gluteninas y proteína total) y de los SSRs (24 loci) para verificar duplicados, y analizar la variabilidad intraaccesión, se estudió en 23 casos de duplicados potenciales de trigo duro. Los resultados indicaron que tanto los biotipos como las accesiones duplicadas mostraban el mismo genotipo en gliadinas, pocas diferencias o ninguna en las subunidades de gluteninas HMW y proteína total, y diferencias en menos de tres loci de SSRs. El mismo resultado se obtuvo para los biotipos de la colección de T. monococcum. Sin embargo, las discrepancias observadas en algunos casos entre proteínas y SSRs demostraron la utilidad del uso conjunto de ambos tipos de marcadores. Tanto las proteínas como los SSRs mostraron gran concordancia con los caracteres agro-morfológicos, especialmente cuando las diferencias entre los genotipos eran grandes. Sin embargo, los caracteres agro-morfológicos fueron menos discriminantes que los marcadores moleculares. Para el segundo objetivo de la tesis, se analizó la variación alélica en siete loci de prolaminas relacionados con la calidad en trigo duro: Glu-A1 y Glu-B1 de gluteninas HMW, Glu-A3, Glu-B3 y Glu-B2 de gluteninas B-LMW, y Gli-A1 y Gli-B1 de gliadinas. La submuestra analizada incluía variedades locales de todas las provincias españolas donde se ha cultivado tradicionalmente el trigo duro. Todos los loci, excepto el Glu-B2, mostraron gran variabilidad genética, siendo los Glu-3 los más polimórficos. En total, se identificaron 65 alelos, de los que 29 eran nuevos, que representan una fuente importante de variabilidad genética para la mejora de la calidad. Se detectaron diferencias en la composición en prolaminas entre la convar. turgidum y la zona norte, y la convar. durum y la zona sur; el genotipo Glu-B3new-1 - Gli-B1new-1 fue muy común en la convar. turgidum, mientras que el Glu-B3a - Gli-B1c, asociado con mejor calidad, fue más frecuente en la convar. durum. En la convar. turgidum, se observó mayor variabilidad que en la convar. durum, principalmente en los loci Glu-B1 y Glu-B3, lo que indica que esta convariedad puede ser una fuente valiosa de nuevos alelos de gluteninas. Esta submuestra fue evaluada para calidad (contenido en proteína, P, y test de sedimentación, SDSS) con dos dosis de abonado nitrogenado (N), y en dos años diferentes. No se detectaron interacciones Variedad × Año, ni Variedad × N en la calidad. Para la P, los efectos ambientales (año y N) fueron mayores que el efecto de la variedad, siendo, en general, mayor la P con dosis altas de N. La variedad influyó más en el test SDSS, que no se vio afectado por el año ni el N. El aumento del contenido en proteína no influyó significativamente sobre la fuerza del gluten estimada con el SDSS. Respecto a la influencia de las prolaminas en la fuerza del gluten, se confirmó la superioridad del Glu-B3a; aunque también se detectó una influencia alta y positiva de los alelos nuevos Glu-A3new-1, y Glu-B3new-6 y new-9. La no correlación entre el rendimiento (evaluado en un trabajo anterior) y la P, en las variedades adaptadas a bajo N, permitió seleccionar cuatro variedades locales con alto rendimiento y buena fuerza del gluten para producción con bajo N. SUMMARY There are two main objectives in this thesis: The first, to analyse the use of endosperm proteins and SSRs to rationalize the wheat collections, and the second, to study the influence on quality of endosperm proteins, year and nitrogen fertilization in a group of Spanish landraces. For the first objective, we studied the genetic diversity of the collection of Triticum monococcum L. (cultivated einkorn), and of a sample of the collection of Triticum turgidum L. (durum wheat) maintained at the CRF-INIA. Two and 6 gliadin loci, and 6 and 24 SSRs, were used for einkorn and durum wheat, respectively. Both collections possessed a high genetic diversity, being the differentiation large between varieties and small within them. Gliadin loci showed great variability, being the loci Gli-2 the most useful for distinguish among varieties. In einkorn, the gliadins showed higher discrimination power than SSRs; although SSRs identified more genotypes in durum wheat. Large number of alleles were found; 24 and 38 in gliadins, and 29 and 203 in SSRs, for einkorn and durum wheat, respectively. In durum wheat, 17 new alleles of gliadins were identified, which indicate that Spanish durum wheat germplasm is rather unique. Some associations between prolamin alleles and geographical and phylogenetic origin of varieties were found in both species. The value of endosperm proteins (6 gliadin loci, 2 glutenin loci and total protein) and SSRs (24 loci) for validation of duplicates, and monitoring the intra-accession variability, was studied in 23 potential duplicates of durum wheat. The results indicated that biotypes and duplicated accessions showed identical gliadin genotype, few or none differences in HMW glutenin subunits and total protein, and less than three different SSR loci. A similar result was obtained for biotypes of T. monococcum. However, the discrepancies in some cases support the convenience to use together both marker systems. A good concordance among endosperm proteins, agro-morphological traits and SSRs were also found, mainly when differences between genotypes were high. However, agro-morphological traits discriminated less between accessions than molecular markers. For the second objective of the thesis, we analysed the allelic variation at seven prolamin loci, involved in durum wheat quality: Glu-A1 and Glu-B1 of HMW glutenin, Glu-A3, Glu-B3 and Glu-B2 of B-LMW glutenin, and Gli-A1 and Gli-B1 of gliadin. The subsample analysed included landraces from all the Spanish provinces where the crop was traditionally cultivated. All the loci, except for Glu-B2, showed high genetic variability, being Glu-3 the most polymorphic. A total of 65 alleles were studied, 29 of them being new, which represent an important source of variability for quality improvement. Differences in prolamin composition were detected between convar. turgidum and the North zone, and the convar. durum and the South zone; the genotype Glu-B3new-1 - Gli-B1new-1 was very common in the convar. turgidum, while the Glu- B3a - Gli-B1c, associated with better quality, was more frequent in the convar. durum. Higher variability was detected in the convar. turgidum than in the convar. durum, mainly at the Glu-B1 and Glu-B3, showing that this convariety could be a valuable source of new glutenin alleles. The subsample was evaluated for quality (protein content, P, and sedimentation test, SDSS) with two doses of nitrogen fertiliser (N), and in two different years. No significant Variety x Year or Variety x Nitrogen interactions were detected. For P, environmental (year and N) effects were higher than variety effect, being P values , in general, larger with high dose of N. The variety exhibited a strong influence on SDSS test, which was not affected by year and N. Increasing values of P did not significantly influence on gluten strength, estimated with the SDSS. Respect to the prolamin effects on gluten strength, the superiority of Glu-B3a was confirmed; although a high positive effect of the new alleles Glu-A3new-1, and Glu-B3new-6 and new-9 was also detected. The no correlation between yield (evaluated in a previous research) and P, in the landraces adapted to low N, allowed to select four landraces with high yield and high gluten strength for low N production.

Caracterización molecular del mutante riso 1508 de cebada : modificaciones transcripcionales y posttranscripcionales en el desarrollo de la semilla

La semilla es el principal órgano reproductivo de las plantas espermatofitas, permitiendo la dispersión de las poblaciones y asegurando su supervivencia gracias a su tolerancia a la desecación y a su capacidad para germinar bajo condiciones ambientales óptimas. El rendimiento y valor económico de los cereales, que constituyen la primera cosecha mundial, depende, en buena medida, de la eficacia con que se acumulan en la semilla sustancias de reserva: proteínas, carbohidratos y lípidos. El principal carbohidrato acumulado en la semilla de cebada es el almidón y la fracción mayoritaria de proteínas es la de las prolaminas (solubles en etanol al 70%); estas proteínas tienen muy bajo contenido en lisina, un aminoácido esencial en la dieta de animales monogástricos. Con el fin de mejorar el valor nutricional de la semilla de cebada, se han obtenido diferentes mutantes con un mayor contenido en este aminoácido. Riso 1508 es un mutante de cebada rico en lisina cuya mutación lys3a, de efectos pleiotrópicos, segrega como un único gen mendeliano. Entre otros, presenta una reducción drástica de la expresión de algunos genes que codifican proteínas de reserva de tipo prolamina, en concreto, presenta reducida la expresión de los genes que codifican B-, C- y ϒ-Hordeínas y del inhibidor de tripsina CMe, pero no tiene alterada la expresión del gen que codifica las D-Hordeínas. Este último gen carece en su promotor del motivo GLM (5’‐(G/A)TGA(G/C)TCA(T/C)‐3’), que es reconocido por factores transcripcionales bZIP. En este trabajo, el mutante de cebada Riso 1508 se ha utilizado como herramienta para profundizar en el conocimiento de la regulación génica en semillas durante las fases de la maduración y la germinación. Para ello, en una primera aproximación, se llevó a cabo un análisis transcriptómico comparando el genotipo mutante con el silvestre durante la maduración de la semilla. Además de confirmar variaciones en los genes que codifican proteínas de reserva, este análisis indicó que también estaban afectados los genes relacionados con metabolismo de carbohidratos. Por ello se decidió caracterizar la familia multigénica de sacarosas sintasa (SUSy) en cebada. Se anotaron dos nuevos genes, HvSs3 y HvSs4, cuya expresión se comparó con la de los genes HvSs1 y HvSs2, previamente descritos en el laboratorio. La expresión de los cuatro genes en tejidos diferentes y su respuesta a estreses abióticos se analizó mediante RT-qPCR. HvSs1 y HvSs2 se expresaron preferencialmente durante el desarrollo del endospermo, y HvSs1 también fue un tránscrito abundante durante la germinación. HvSs1 se indujo en hojas en condiciones de anoxia y HvSs3 por estrés hídrico, y ambos genes se indujeron por tratamientos de frío. La localización subcelular de las cuatro isoformas no fue sólo citoplásmica, sino que también se localizaron en zonas próximas a retículo endoplásmico y en la cara interna de la membrana plasmática; además, se observó una co-localización de HvSS1 con el marcador de mitocondrias. Estos datos sugieren un papel distinto aunque parcialmente solapante de las cuatro Sacarosa Sintasas de cebada, descritas hasta la fecha. Las cinéticas de expresión de los genes que codifican los TFs más importantes implicados en la regulación génica durante el desarrollo del endospermo de cebada, se analizaron por RT-qPCR en ambos genotipos, demostrando que los TFs de la clase DOF aparecieron desregulados durante todo el proceso en Riso 1508 comparado con el cv. Bomi, aunque también se observaron diferencias significativas en algunos de los que codifican bZIPs. Estudios previos indicaban que el ortólogo de BLZ2 en maíz, O2, se regula post-traduccionalmente mediante un mecanismo de fosforilación/defosforilación reversible, y que la forma defosforilada es la fisiológicamente activa. En este trabajo se demostró que BLZ2 está sujeto a este tipo de regulación y que la proteín-fosfatasa HvPP2C2 está implicada en el proceso. La interacción de HvPP2C2 y BLZ2 tiene lugar en el núcleo celular únicamente en presencia de 100 μM ABA. En el mutante Riso 1508, BLZ2 se encuentra en un estado hiperfosforilado tanto durante la maduración como durante la germinación de la semilla, lo que dificultaría la unión de BLZ2 a las secuencias GLM en los promotores de los genes que codifican B-, C-,y ϒ- Hordeínas y CMe. Summary The seed is the main reproductive organ of spermatophyte plants allowing the spread of populations and ensuring their survival through its desiccation tolerance and because of their ability to germinate under optimum environmental conditions. Yield and economic value of cereal crops, that constitute the first world crop, depend largely on the efficiency with which they accumulate in the seed reserve substances: proteins, carbohydrates and lipids. The main carbohydrate accumulated in the barley seed is starch and the major protein fraction is that of prolamins (soluble in 70% ethanol); these proteins have a very low lysine content, an essential amino-acid for the diet of monogastric animals. In order to improve the nutritional value of the barley seed, different mutants have been obtained with a higher content of this amino-acid. Riso 1508 is one lysine-rich mutant whose mutation (lys3a) segregates as a single Mendelian gene with pleiotropic effects, such as a drastic reduction of genes encoding the trypsin inhibitor CMe and the B-, C-and ϒ-hordeins, but has not altered the expression of the gene encoding the D-hordeins. This latter gene lacks in its promotor the GLM motif (5’‐(G/A)TGA(G/C)TCA(T/C)‐3’), that is recognised by bZIP transcription factors In this work we have used the barley mutant Riso 1508 as a tool for better understanding gene regulation in seeds during the maturation and germination phases. To this aim, a transcriptomic analysis was performed comparing wild and mutant genotypes during seed maturation. Besides confirming variations in the expression of genes encoding reserve proteins, this analysis indicated that some genes related with carbohydrate metabolism were also affected. It was therefore decided to characterize the multigene family of sucrose synthases (SUSy) in barley. Two new genes were annotated, HvSs3 and HvSs4, and its expression was compared with that of genes HvSs1 and HvSs2, previously described in our laboratory. The expression of the four genes in different tissues and in response to abiotic stresses was analyzed by RTqPCR. HvSs1 and HvSs2 were preferentially expressed during the development of the endosperm, and the HvSs1 transcript was also abundant upon germination. HvSs1 was induced in leaves by anoxic conditions, HvSs3 by water stress, and both genes were induced by cold treatments. The subcellular localization of all four isoforms was not only cytoplasmic, but they could be found along the endoplasmic reticulum and at the inner side of the cell membrane; HvSS1, was also associated with the mitochondrial marker. These data suggest a distinct but partially overlapping roles for the barley sucrose synthases, described so far. The expression kinetics of the genes encoding the most important TFs involved in gene regulation during barley endosperm development was analyzed by RT-qPCR in both genotypes. These data show that the genes encoding DOF TFs were mis-regulated throughout the process in Riso 1508, although significant differences were also found among some of those encoding bZIPs. Previous studies indicated that the BLZ2 orthologue in maize, O2, was post-translationally regulated by reversible phosphorylation/dephosphorylation and that the dephosphorylated protein is the physiologically active form. In this work we demostrate that BLZ2 is under a similar regulation and that the proteinphosphatase HvPP2C2 is implicated in the process. The interaction between HvPP2C2 and BLZ2 takes place in the cell nucleus only in the presence of 100 μM ABA. In the Riso 1508 mutant, BLZ2 is found in a hyperphosphorylated state in the maturation phase and upon seed germination; because of this, the BLZ2 binding to the GLM promoter sequences of genes encoding B-, C- y ϒ- Hordeins and CMe would be decreased in the mutant.

Softening-up mannan-rich cell walls

The softening and degradation of the cell wall (CW), often mannan enriched, is involved in several processes during development of higher plants, such as meristematic growth, fruit ripening, programmed cell death, and endosperm rupture upon germination. Mannans are also the predominant hemicellulosic CW polymers in many genera of green algae. The endosperm CWs of dry seeds often contain mannan polymers, sometimes in the form of galactomannans (Gal-mannans). The endo-beta-mannanases (MANs) that catalyse the random hydrolysis of the beta-linkage in the mannan backbone are one of the main hydrolytic enzymes involved in the loosening and remodelling of CWs. In germinating seeds, the softening of the endosperm seed CWs facilitates the emergence of the elongating radicle. Hydrolysis and mobilization of endosperm Gal-mannans by MANs also provides a source of nutrients for early seedling growth, since Gal-mannan, besides its structural role, serves as a storage polysaccharide. Therefore, the role of mannans and of their hydrolytic enzymes is decisive in the life cycle of seeds. This review updates and discusses the significance of mannans and MANs in seeds and explores the increasing biotechnological potential of MAN enzymes.

A molecular dynamics study of swift heavy ion irradiation of amorphous silica: the role of thermal effects

Irradiation with swift heavy ions (SHI), roughly defined as those having atomic masses larger than 15 and energies exceeding 1 MeV/amu, may lead to significant modification of the irradiated material in a nanometric region around the (straight) ion trajectory (latent tracks). In the case of amorphous silica, SHI irradiation originates nano-tracks of higher density than the virgin material (densification). As a result, the refractive index is increased with respect to that of the surroundings. Moreover, track overlapping leads to continuous amorphous layers that present a significant contrast with respect to the pristine substrate. We have recently demonstrated that SHI irradiation produces a large number of point defects, easily detectable by a number of experimental techniques (work presented in the parallel conference ICDIM). The mechanisms of energy transfer from SHI to the target material have their origin in the high electronic excitation induced in the solid. A number of phenomenological approaches have been employed to describe these mechanisms: coulomb explosion, thermal spike, non-radiative exciton decay, bond weakening. However, a detailed microscopic description is missing due to the difficulty of modeling the time evolution of the electronic excitation. In this work we have employed molecular dynamics (MD) calculations to determine whether the irradiation effects are related to the thermal phenomena described by MD (in the ps domain) or to electronic phenomena (sub-ps domain), e.g., exciton localization. We have carried out simulations of up to 100 ps with large boxes (30x30x8 nm3) using a home-modified version of MDCASK that allows us to define a central hot cylinder (ion track) from which heat flows to the surrounding cold bath (unirradiated sample). We observed that once the cylinder has cooled down, the Si and O coordination numbers are 4 and 2, respectively, as in virgin silica. On the other hand, the density of the (cold) cylinder increases with respect to that of silica and, furthermore, the silica network ring size decreases. Both effects are in agreement with the observed densification. In conclusion, purely thermal effects do not explain the generation of point defects upon irradiation, but they do account for the silica densification.

Nitrate-induced early transcriptional changes during imbibition in non-after-ripened Sisymbrium officinale seeds.

We have here demonstrated for the first time that nitrate not only accelerates testa rupture of non- AR seeds but also modifies expression pattern of the cell-wall remodeling proteins (mannanases; SoMAN6 and SoMAN7) and key genes belonging to metabolism and signaling of ABA (SoNCED6, SoNCED9, SoCYP707A2 and SoABI5) and GAs (SoGA3ox, SoGA20ox, SoGA2ox and SoRGL2). These results were obtained during Sisymbrium officinale seed imbibition in the absence of endosperm rupture. Exogenous ABA induced a notable inhibition of testa rupture in both absence and presence of nitrate being this effect sharply reversed by GA4+7. However, nitrate was capable to provoke testa rupture in absence of ABA synthesis. The expression of SoMAN6 and SoMAN7 were positively altered by nitrate. Although ABA synthesis seems apparent at the start of non-AR seed imbibition, taken together the results of SoNCED6, SoNCED9 and SoCYP707A2 expression seem to suggest that nitrate leads to a strong net ABA decrease. Likewise, nitrate positively affected the SoABI5 expression when the SoNCED9 expression was also stimulated. By contrast, at the early and final of imbibition, nitrate clearly inhibited the SoABI5 expression. The expression of SoGA2ox6 and SoGA3ox2 are strongly inhibited by nitrate whereas of SoGA20ox6 was stimulated. On the other hand, SoRGL2 transcript level decreased in the presence of nitrate. Taken together, the results presented here suggest that the nitrate signaling is already operative during the non-AR S. officinale seeds imbibition. The nitrate, in cross-talk with the AR network likely increases the favorable molecular conditions that trigger germination.

Factores transcripcionales de la clase DOF en Brachypodium distachyon: caracterización molecular de BdDOF24 durante la germinación de las semillas

La semilla es el órgano que garantiza la propagación y continuidad evolutiva de las plantas espermatofitas y constituye un elemento indispensable en la alimentación humana y animal. La semilla de cereales acumula en el endospermo durante la maduración, mayoritariamente, almidón y proteínas de reserva. Estas reservas son hidrolizadas en la germinación por hidrolasas sintetizadas en la aleurona en respuesta a giberelinas (GA), siendo la principal fuente de energía hasta que la plántula emergente es fotosintéticamente activa. Ambas fases del desarrollo de la semilla, están reguladas por una red de factores de transcripción (TF) que unen motivos conservados en cis- en los promotores de sus genes diana. Los TFs son proteínas que han desempeñado un papel central en la evolución y en el proceso de domesticación, siendo uno de los principales mecanismos de regulación génica; en torno al 7% de los genes de plantas codifican TFs. Atendiendo al motivo de unión a DNA, éstos, se han clasificado en familias. La familia DOF (DNA binding with One Finger) participa en procesos vitales exclusivos de plantas superiores y sus ancestros cercanos (algas, musgos y helechos). En las semillas de las Triticeae (subfamilia Pooideae), se han identificado varias proteínas DOF que desempeñan un papel fundamental en la regulación de la expresión génica. Brachypodium distachyon es la primera especie de la subfamilia Pooideae cuyo genoma (272 Mbp) ha sido secuenciado. Su pequeño tamaño, ciclo de vida corto, y la posibilidad de ser transformado por Agrobacterium tumefaciens (plásmido Ti), hacen que sea el sistema modelo para el estudio de cereales de la tribu Triticeae con gran importancia agronómica mundial, como son el trigo y la cebada. En este trabajo, se han identificado 27 genes Dof en el genoma de B. distachyon y se han establecido las relaciones evolutivas entre estos genes Dof y los de cebada (subfamilia Pooideae) y de arroz (subfamilia Oryzoideae), construyendo un árbol filogenético en base al alineamiento múltiple del dominio DOF. La cebada contiene 26 genes Dof y en arroz se han anotado 30. El análisis filogenético establece cuatro grupos de genes ortólogos (MCOGs: Major Clusters of Orthologous Genes), que están validados por motivos conservados adicionales, además del dominio DOF, entre las secuencias de las proteínas de un mismo MCOG. El estudio global de expresión en diferentes órganos establece un grupo de nueve genes BdDof expresados abundantemente y/o preferencialmente en semillas. El estudio detallado de expresión de estos genes durante la maduración y germinación muestra que BdDof24, ortólogo putativo a BPBF-HvDOF24 de cebada, es el gen más abundante en las semillas en germinación de B. distachyon. La regulación transcripcional de los genes que codifican hidrolasas en la aleurona de las semillas de cereales durante la post‐germinación ha puesto de manifiesto la existencia en sus promotores de un motivo tripartito en cis- conservado GARC (GA-Responsive Complex), que unen TFs de la clase MYB-R2R3, DOF y MYBR1-SHAQKYF. En esta tesis, se ha caracterizado el gen BdCathB de Brachypodium que codifica una proteasa tipo catepsina B y es ortólogo a los genes Al21 de trigo y HvCathB de cebada, así como los TFs responsables de su regulación transcripcional BdDOF24 y BdGAMYB (ortólogo a HvGAMYB). El análisis in silico del promotor BdCathB ha identificado un motivo GARC conservado, en posición y secuencia, con sus ortólogos en trigo y cebada. La expresión de BdCathB se induce durante la germinación, así como la de los genes BdDof24 y BdGamyb. Además, los TFs BdDOF24 y BdGAMYB interaccionan en el sistema de dos híbridos de levadura e in planta en experimentos de complementación bimolecular fluorescente. En capas de aleurona de cebada, BdGAMYB activa el promotor BdCathB, mientras que BdDOF24 lo reprime; este resultado es similar al obtenido con los TFs ortólogos de cebada BPBF-HvDOF24 y HvGAMYB. Sin embargo, cuando las células de aleurona se transforman simultáneamente con los dos TFs, BdDOF24 tiene un efecto aditivo sobre la trans-activación mediada por BdGAMYB, mientras que su ortólogo BPBF-HvDOF24 produce el efecto contrario, revirtiendo el efecto de HvGAMYB sobre el promotor BdCathB. Las diferencias entre las secuencias deducidas de las proteínas BdDOF24 y BPBF-HvDOF24 podrían explicar las funciones opuestas que desempeñan en su interacción con GAMYB. Resultados preliminares con líneas de inserción de T-DNA y de sobre-expresión estable de BdGamyb, apoyan los resultados obtenidos en expresión transitoria. Además las líneas homocigotas knock-out para el gen BdGamyb presentan alteraciones en anteras y polen y no producen semillas viables. ABSTRACT The seed is the plant organ of the spermatophytes responsible for the dispersion and survival in the course of evolution. In addition, it constitutes one of the most importan elements of human food and animal feed. The main reserves accumulated in the endosperm of cereal seeds through the maturation phase of development are starch and proteins. Its degradation by hydrolases synthetized in aleurone cells in response to GA upon germination provides energy, carbon and nitrogen to the emerging seedling before it acquires complete photosynthetic capacity. Both phases of seed development are controlled by a network of transcription factors (TFs) that interact with specific cis- elements in the promoters of their target genes. TFs are proteins that have played a central role during evolution and domestication, being one of the most important regulatory mechanisms of gene expression. Around 7% of genes in plant genomes encode TFs. Based on the DNA binding motif, TFs are classified into families. The DOF (DNA binding with One Finger) family is involved in specific processes of plants and its ancestors (algae, mosses and ferns). Several DOF proteins have been described to play important roles in the regulation of genes in seeds of the Triticeae tribe (Pooideae subfamily). Brachypodium distachyon is the first member of the Pooideae subfamily to be sequenced. Its small size and compact structured genome (272 Mbp), the short life cycle, small plant size and the possibility of being transformed with Agrobacterium tumefaciens (Ti-plasmid) make Brachypodium the model system for comparative studies within cereals of the Triticeae tribe that have big economic value such as wheat and barley. In this study, 27 Dof genes have been identified in the genome of B. distachyon and the evolutionary relationships among these Dof genes and those frome barley (Pooideae subfamily) and those from rice (Oryzoideae subfamily) have been established by building a phylogenetic tree based on the multiple alignment of the DOF DNA binding domains. The barley genome (Hordeum vulgare) contains 26 Dof genes and in rice (Oryza sativa) 30 genes have been annotated. The phylogenetic analysis establishes four Major Clusters of Orthologous Genes (MCOGs) that are supported by additional conserved motives out of the DOF domain, between proteins of the same MCOG. The global expression study of BdDof genes in different organs and tissues classifies BdDof genes into two groups; nine of the 27 BdDof genes are abundantly or preferentially expressed in seeds. A more detailed expression analysis of these genes during seed maturation and germination shows that BdDof24, orholog to barley BPBF-HvDof24, is the most abundantly expressed gene in germinating seeds. Transcriptional regulation studies of genes that encode hydrolases in aleurone cells during post-germination of cereal seeds, have identified in their promoters a tripartite conserved cis- motif GARC (GA-Responsive Complex) that binds TFs of the MYB-R2R3, DOF and MYBR1-SHAQKYF families. In this thesis, the characterization of the BdCathB gene, encoding a Cathepsin B-like protease and that is ortholog to the wheat Al21 and the barley HvCathB genes, has been done and its transcriptional regulation by the TFs BdDOF24 and BdGAMYB (ortholog to HvGAMYB) studied. The in silico analysis of the BdCathB promoter sequence has identified a GARC motif. BdCathB expression is induced upon germination, as well as, those of BdDof24 and BdGamyb genes. Moreover, BdDOF24 and BdGAMYB interact in yeast (Yeast 2 Hybrid System, Y2HS) and in planta (Bimolecular Fluorecence Complementation, BiFC). In transient assays in aleurone cells, BdGAMYB activates the BdCathB promoter, whereas BdDOF24 is a transcriptional repressor, this result is similar to that obtained with the barley orthologous genes BPBF-HvDOF24 and HvGAMYB. However, when aleurone cells are simultaneously transformed with both TFs, BdDOF24 has an additive effect to the trans-activation mediated by BdGAMYB, while its ortholog BPBF-HvDOF24 produces an opposite effect by reducing the HvGAMYB activation of the BdCathB promoter. The differences among the deduced protein sequences between BdDOF24 and BPBF-HvDOF24 could explain their opposite functions in the interaction with GAMYB protein. Preliminary results of T-DNA insertion (K.O.) and stable over-expression lines of BdGamyb support the data obtained in transient expression assays. In addition, the BdGamyb homozygous T-DNA insertion (K.O.) lines have anther and pollen alterations and they do not produce viable seeds.

Identificación de áreas prioritarias para la conservación de razas de maíz en la sierra de Ecuador

En Ecuador el maíz es el cultivo más importante en superficie y es base de la alimentación para la población rural que vive en los Andes. A diferencia de lo que sucede en la Costa, en la región Sierra todavía se cultivan numerosas variedades tradicionales que se agrupan en veinticuatro razas. Mantener esta diversidad es, pues, de gran importancia no solo para la seguridad alimentaria, sino también como fuente de genes para tolerancia a factores abióticos que podrían ser incorporados a las variedades modernas. Si bien parte de esta diversidad fue recolectada a mediados del siglo pasado y está siendo conservada en distintos bancos de germoplasma, es deseable que su conservación in situ también esté asegurada, entre otras razones, porque de esta manera el cultivo puede seguir evolucionando. Para poder implementar un plan de conservación en finca que contribuya a preservar este patrimonio, resulta imprescindible identificar áreas idóneas donde concentrar los recursos y conocer las características y tipologías de los agricultores que manejan la diversidad actual. Generar esta información es el objetivo principal de esta investigación y para lograrlo se han llevado a cabo cuatro estudios: (1) Análisis de la diversidad a nivel de razas e identificación de áreas de alta riqueza de razas, alta diversidad morfológica y/o alta diversidad ecogeográfica en la Sierra de Ecuador, (2) Identificación del perfil y las características de los agricultores que conservan y manejan las variedades tradicionales de maíz en la Sierra de Ecuador, (3) Análisis del conocimiento local, manejo y usos de variedades tradicionales de maíz en la Sierra de Ecuador, y (4) Identificación de áreas de alta diversidad y bajo riesgo de pérdida para la conservación en finca de maíz en la Sierra de Ecuador. Para el primer estudio se visitaron 303 fincas distribuidas a lo largo de la Sierra y se recolectaron 636 muestras que fueron caracterizadas morfológicamente mediante 14 variables: 8 relacionadas con la mazorca (forma, longitud y diámetro de la mazorca, color y diámetro de olote y número y disposición de hileras) y 7 referidas el grano (número total de granos, color, forma, longitud, anchura y grosor de grano y tipo de endospermo). Adicionalmente, las fincas donde se tomaron las muestras fueron caracterizadas ecogeográficamente mediante 5 variables climáticas (temperatura media estacional, rango de temperatura media anual, temperatura mínima de diciembre, precipitación estacional y precipitación de octubre), 2 geofísicas (altitud y pendiente) y 5 edáficas (textura principal del suelo, profundidad a roca, pH, contenido en materia orgánica y fertilidad). A partir de esta información y mediante técnicas de sistemas de información geográfica (SIG), se generaron mapas de distribución por raza en formato vectorial y un mapa de riqueza de razas, un mapa de diversidad morfológica y un mapa de diversidad ecogeográfica en formato ráster con celdas de 10 km x 10 km. Los resultados permitieron constatar que, en los últimos 60 años, no se ha perdido ninguna raza. Sin embargo, Canguil, Chaucho y Clavito han dejado de cultivarse en algunas provincias con la consiguiente erosión genética del cultivo. La caracterización morfológica detectó diferencias en el grado de variabilidad intra-raza, siendo Patillo Ecuatoriano, Racimo de Uva y Uchima las razas más heterogéneas tanto para los caracteres cualitativos como cuantitativos. A nivel climático y geofísico, también se detectaron diferencias en el grado de variación intra-raza; Cuzco Ecuatoriano, Kcello Ecuatoriano y Montaña Ecuatoriana fueron las razas que en promedio presentaron mayores rangos y coeficientes de variación para estas variables ecogeográficas. En cuanto a las condiciones edáficas todas las razas, excepto Cónico Dentado, presentaron una gran heterogeneidad, pudiendo crecer tanto en suelos ricos como pobres, con valores de pH entre ácido y moderadamente alcalino. La comparación entre razas reveló diferencias significativas en los rangos ambientales de algunas razas como Cónico Dentado, que tiende a cultivarse a menor altitud y, por tanto, en ambientes menos fríos y de mayor precipitación que Blanco Blandito, Patillo Ecuatoriano, Sabanero Ecuatoriano, Uchima y Zhima. Para la mayoría de las razas se encontraron materiales potencialmente adaptados a condiciones de estrés (precipitación estacional inferior a 500 mm y suelos con pH entre 4.5 y 5.5). Finalmente, los mapas de riqueza, de diversidad morfológica y de diversidad ecogeográfica mostraron 36 celdas de alta diversidad repartidas en las 10 provincias de la Sierra: 11 celdas en las provincias del norte, 11 en las provincias del centro y 14 en las provincias del sur. Para la caracterización e identificación de las tipologías de los agricultores que cultivan maíz en la Sierra de Ecuador y el análisis de los posibles factores de riesgo de pérdida de diversidad, se realizaron entrevistas individuales y semiestructuradas a los agricultores dueños de las fincas donde se recolectaron las muestras para el estudio de diversidad (254 en total). Las preguntas que se formularon (11 abiertas y 5 cerradas) estuvieron organizadas en seis bloques: datos del agricultor, características de la finca, diversidad y conocimiento del cultivo, manejo del cultivo, usos y flujo de semillas. Los resultados indicaron que la diversidad de maíz que hay en la Sierra de Ecuador es manejada mayoritariamente por agricultores mestizos, de entre 30 y 55 años, que cultivan una o dos variedades tradicionales para autoconsumo, en parcelas de menos de 0.5 ha y en asocio con fréjol. El análisis de segmentación mediante el algoritmo Chi-square automatic interaction detection (CHAID) permitió identificar un pequeño grupo de agricultores indígenas con parcelas medianas (entre 0.5 ha y 1.5 ha) que conservan un mayor número de variedades tradicionales por finca que el agricultor promedio. Los análisis estadísticos no detectaron diferencias significativas entre etnias (mestizo vs. indígena), géneros (hombre vs. mujer) y grupos de edad (jóvenes menores de 30 años, adultos entre 30 y 55 años y adultos mayores de 55 años) en lo que respecta al conocimiento del cultivo (criterios de reconocimiento y razones de preferencia) y manejo (tipo de cultivo), pero sí detectaron diferencias entre regiones, principalmente en el modo de cultivar el maíz; mientras que en el norte y sur tienden a sembrarlo en asocio y con un mayor número de especies, en el centro acostumbran a cultivarlo preferentemente solo. En cuanto a los usos, se recopilaron hasta 39 modos diferentes de consumir maíz, siendo Kcello Ecuatoriano y Zhima las razas para las que se registró un mayor número de usos. La comparación del número medio de usos por variedad entre etnias evidenció que los agricultores mestizos utilizan sus variedades tradicionales de forma más variada que los indígenas. Entre los factores de riesgo que se analizaron, el bajo porcentaje de jóvenes agricultores que se ocupan de las fincas podría suponer una amenaza a medio plazo por falta de relevo generacional. Adicionalmente, las numerosas sinonimias y homonimias que se detectaron y el bajo intercambio de semillas también podrían ser causa de pérdida de diversidad, bien por reemplazo o por envejecimiento de la semilla. Finalmente, se concluyó que las razas Chaucho, Complejo Chillo-Huandango, Complejo Mishca-Huandango, Cónico Dentado, Montaña Ecuatoriana y Sabanero Ecuatoriano son particularmente vulnerables, no solo por su baja presencia, sino también por el color de grano que tienen (los mismos que la mayoría de las razas más comunes) y carecer de nombres y usos específicos. Finalmente, para la priorización de áreas de conservación en finca para maíz en la Sierra de Ecuador, se utilizaron 13 criterios de diferente naturaleza: 2 ecogeográficos (precipitación, diversidad ecogeográfica), 6 biológicos (grado de presencia del cultivo, riqueza de razas, diversidad morfológica, presencia de mezclas, presencia de razas locales y riesgo de erosión genética), 3 culturales (abundancia de variedades por finca, diversidad de usos y frecuencia de intercambio) y 2 demográficos (tamaño de la población y distancia a núcleos urbanos). Mediante técnicas SIG y de evaluación multicriterio, los valores originales de las capas-criterio fueron transformados a una escala de 0 a 100. Posteriormente, las capas-criterio normalizadas fueron sumadas utilizando tres métodos de ponderación: (1) mismo peso, (2) diferente peso según la puntuación otorgada por 72 expertos, y (3) diferente peso según el método de comparación entre pares de criterios. Los resultados permitieron identificar ocho celdas de 10 km x 10 km con alta puntuación (> 65): tres celdas en el norte (una en cada una de las provincias), una celda en el centro (en la provincia de Cotopaxi), y cuatro celdas en la región sur (dos en Azuay y otras dos en Loja). ABSTRACT In Ecuador, the maize is the most important cultivation in surface and it is a base of the feeding for the rural population who lives in the Andes. In contrast to what it happens on the Coast, in the Sierra region still there are cultivated numerous traditional varieties that are grouped into twenty-four races. Maintaining this diversity is, therefore, of great importance not only for food security, but also as a source of genes for tolerance to abiotic factors could be incorporated into modern varieties. Although part of this diversity was collected in the middle of the last century and is still preserved in various germplasm banks, it is desirable for the in situ conservation also is assured, among other reasons, because in this way the crop can continue to evolve. To be able to implement a conservation plan on farm that contribute to preserving this heritage, it is essential to identify suitable areas where to concentrate resources and know the characteristics and typology of farmer who managed the current diversity. To generate this information is the main target of this investigation and to achieve this, four studies have been carried out: (1) Analysis of the diversity at races and identification of areas of high richness of races, high morphological diversity and / or ecogeographical high diversity in the Sierra of Ecuador, (2) Identification of the profile and characteristics of farmers who conserve and manage traditional varieties of maize in the Sierra of Ecuador, (3) Analysis of local knowledge, management and use of traditional varieties of maize in the Sierra of Ecuador, and (4) Identification of areas of high diversity and low risk of loss for the conservation of maize in the Sierra of Ecuador. For the first study were visited 303 farms distributed along the Sierra and collected 636 samples that were characterized morphologically by 14 variables: 8 related to the ear (shape, length and diameter of the cob, colour, and diameter of cob and number and arrangement of rows) and 7 referred to the grain (total number of grain, colour, shape, length, width, and thickness and type of grain endosperm). In addition, the farms where the samples were taken were characterized ecogeographically through 5 climatic variables (seasonal average temperature, range of average annual temperature, minimum temperature for December, seasonal precipitation and precipitation of October), 2 geophysical (altitude and slope) and edaphic 5 (main texture of the soil, deep rock, pH, content of organic matter and fertility). From this information and techniques of geographic information systems (GIS), maps were generated for distribution by race in vector format and a map of richness of races, a map of morphological diversity and a map of ecogeographical diversity in raster format with cells of 10 km x 10 km. The results allowed observing that, over the past 60 years, it has not lost any race. Nevertheless, Canguil, Chaucho and Clavito have stopped being cultivated in some provinces with the consequent genetic erosion of the cultivation. The morphological characterization detected differences in the degree of variability intra-race, being Patillo Ecuatoriano, Racimo de Uva and Uchima races more heterogeneous both for the qualitative and quantitative characters. At climate and geophysical level, also detected differences in the degree of variation intra-race; Cuzco Ecuatoriano, Kcello Ecuatoriano and Montaña Ecuatoriana were races that, on average, showed higher ranges and coefficients of variation for these geographical characters. In terms of the edaphic conditions, all races, except Cónico Dentado, showed a great heterogeneity, and can grow both in rich and poor soils, with pH values between acid and moderately alkaline. The comparison between races revealed significant differences in the environmental ranges in some races as Cónico Dentado, which tends to be grown at lower elevations and, therefore, in environments less cold and greater precipitation than Blanco Blandito, Patillo Ecuatoriano, Sabanero Ecuatoriano, Uchima and Zhima. For most of the races were found materials potentially adapted to stress conditions (seasonal precipitation less than 500 mm and soil with a pH between 4.5 and 5.5). Finally, the maps of richness, morphologic diversity and ecogeographical diversity showed 36 cells high diversity distributed in 10 provinces of the Sierra: 11 cells in the northern provinces, 11 in the central provinces and 14 in the southern provinces. For the characterization and identification of the typology of the farmers who cultivate corn in the Sierra of Ecuador and the analysis of the possible factors of risk of loss of diversity, there were realized interviews individual and semistructured to the farmers’ owners of the farms where the samples were gathered for the study of diversity (254 in whole). The questions that were formulated (11 opened ones and 5 closed ones) were organized in six blocks: data of the farmer, characteristics of the farm, diversity and knowledge of the crop, crop management, uses and seed flow. The results indicated that the maize diversity that exist in the Sierra of Ecuador is managed mainly by mestizo farmers, aged between 30 and 55, who cultivate one or two traditional varieties for self-consumption, on plots of less than 0.5 has and in associated with beans. The segmentation analysis algorithm using the Chi-square automatic interaction detection (CHAID technique), allowed to identify a small group of indigenous farmers with medium-sized plots (between 0.5 there is and 1.5 it is) that a major number of traditional varieties preserves for farm that the average farmer. The statistical analysis did not detect significant differences between ethnic groups (mestizos vs. indigenous), genres (man vs. women) and age groups (young people under 30 years of age, adults between 30 and 55 years and adults over 55 years old) in regards to the knowledge of the cultivation (recognition criteria and reasons of preference) and management (type of crop), but if detected differences between regions, mainly on the mode of cultivating the maize; while in the north and south they tend to sow in associate and with a greater number of species, in the center accustomed to cultivate it preferably only. In regards to the uses, they were compiled up to 39 different ways of consuming maize, being Kcello Ecuatoriano and Zhima the races for which a major number of uses registered. The comparison of the average number of uses per variety between ethnic groups showed that the mestizo farmers used their traditional varieties of form more varied than the indigenous people. Between the factors of risk that were analyzed, the low percentage of young farmers who deal with the farms might suppose a medium-term threat for lack of generational relief. In addition, the numerous synonyms and homonyms that were detected and the low seed exchange could also be a cause of loss of diversity, either by replacement or by aging of the seed. Finally, it was concluded that the races Chaucho, Complex Chillo-Huandango, Complex Mishca-Huandango, Cónico Dentado, Montaña Ecuatoriana and Sabanero Ecuatoriano are particularly vulnerable, not only because of their low presence, but also by the grain color they have (the same as the majority of races more common) and lack of names and specific uses. Finally, for the prioritization of maize conservation areas on farm in the Sierra of Ecuador, used 13 criteria of different nature: 2 ecogeographic (precipitation, diversity ecogeographical), 6 biological (degree of presence of the crop, races richness, morphological diversity, the presence of mixtures, presence of local races and risk of genetic erosion), 3 cultural (abundance of varieties per farm, diversity of uses and frequency of exchange) and 2 demographic (population size and distance to urban centers). Using GIS techniques and multicriteria evaluation, the original values of the layers-criterion were transformed to a scale of 0 to 100. Later, the normalized layers - criteria were added using three weighting methods: (1) the same weight, (2) different weight according to the score given by 72 experts, and (3) different weight according to the method of comparison between pairs of criteria. The results allowed to identify eight 10 km cells x 10 km with high punctuation (> 65): three cells in the north (one in each of the provinces), a cell in the center (in the Cotopaxi province), and four cells in the south region (two in Azuay and other two in Loja).

A coupled elastoplastic-damage constitutive model with Lode angle dependent failure criterion

A coupled elastoplastic-damage constitutive model with Lode angle dependent failure criterion for high strain and ballistic applications is presented. A Lode angle dependent function is added to the equivalent plastic strain to failure definition of the Johnson–Cook failure criterion. The weakening in the elastic law and in the Johnson–Cook-like constitutive relation implicitly introduces the Lode angle dependency in the elastoplastic behaviour. The material model is calibrated for precipitation hardened Inconel 718 nickel-base superalloy. The combination of a Lode angle dependent failure criterion with weakened constitutive equations is proven to predict fracture patterns of the mechanical tests performed and provide reliable results. Additionally, the mesh size dependency on the prediction of the fracture patterns was studied, showing that was crucial to predict such patterns