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.

Traditional Building Adaptation to New Requirements: Historic Center of Arequipa, Perú

The traditional buildings in the historic center of the city of Arequipa, Perú, recently declared of human heritage, are of volcanic tuff both in walls and in vaulted roofs on the ground floor. Having been built in the 18th century and up to the beginning of the 20th century, they have suffered many damages from the seismic movements registered in this region. Due to this, many of them have had to be rebuilt. In this presentation, the different changes to adapt the city of Spanish colonial origin to the present tertiary use are analyzed.

Similarities and Differences in Barrel Vaults of Traditional and Stately Houses in the Historic Centre of Arequipa, Perú

The traditional architecture of the centre of the city of Arequipa has been analyzed by comparing floor-plans of houses from the eighteenth and nineteenth centuries in order to explain the reasons behind the arrangement of their constructional elements and the evolution of said elements and floor-plans. The historic centre of Arequipa, a city located in the South of Perú, South America (Latitude 16°23' South, Longitude 71 °31' West), is based on a ground plan from 1540 that was set during the city's Spanish foundation. It was declared Patrimony of the Humanity by UNESCO. The manorial architecture is widely known for its decorated fronts and one-of-a-kind designs, but its differences with respect to the popular architecture are not based exclusively on decorative aspects. Peru's colonial period finished around 1825, but the barrel-vault, construction style continued in Arequipa through 1868, when an earthquake destroyed the city. Thereafter, the vaults were replaced by roofs made of rails, with cinders made out of the lava stone. The stately houses belonged to the founding families who settled around the main square on forty nine blocks that formed a square-grid, street layout. Also belonging to this category are the houses of landlords and traders from post-colonial times.

Differential in vitro and in vivo effect of barley cysteine and serine protease inhibitors on phytopathogenic microorganisms

Protease inhibitors from plants have been involved in defence mechanisms against pests and pathogens. Phytocystatins and trypsin/α-amylase inhibitors are two of the best characterized protease inhibitor families in plants. In barley, thirteen cystatins (HvCPI-1 to 13) and the BTI-CMe trypsin inhibitor have been previously studied. Their capacity to inhibit pest digestive proteases, and the negative in vivo effect caused by plants expressing these inhibitors on pests support the defence function of these proteins. Barley cystatins are also able to inhibit in vitro fungal growth. However, the antifungal effect of these inhibitors in vivo had not been previously tested. Moreover, their in vitro and in vivo effect on plant pathogenous bacteria is still unknown. In order to obtain new insights on this feature, in vitro assays were made against different bacterial and fungal pathogens of plants using the trypsin inhibitor BTI-CMe and the thirteen barley cystatins. Most barley cystatins and the BTI-CMe inhibitor were able to inhibit mycelial growth but no bacterial growth. Transgenic Arabidopsis plants independently expressing the BTI-CMe inhibitor and the cystatin HvCPI-6 were tested against the same bacterial and fungal pathogens. Neither the HvCPI-6 expressing transgenic plants nor the BTI-CMe ones were more resistant to plant pathogen fungi and bacteria than control Arabidopsis plants. The differences observed between the in vitro and in planta assays against phytopathogenic fungi are discussed

Air Traffic Management - Historic background and present situation

Ponencia invitada sobre gestion de trafico aereo en el curso de verano de la UPM Research in Decision Support Systems for future Air Traffic Management

Promotion of intangible heritage as a tool to strengthen revitalisation of historic centres: the case of de Monumenta Programme in the City of Porto Alegre (Rio Grande do Sul State) Brazil

The preservation of tangible cultural heritage does not guarantee effective revitalisation of urban historic areas as a whole. The legacy of our history consists not only of paintings, sculptures, architectural monuments and public spaces, but also the safeguarding of immaterial aspects of social life, such as oral traditions, rituals, practices, knowledge and craft skills. From 1999 to 2013, 26 Brazilian cities benefited from the Monumenta Programme - a national cultural policy that involved institutions, the private sector and the local community. The purpose of the programme was to stimulate economic growth and increase cultural and social development of the historic centres. Moreover, it sought to increase the number of residents in the benefited areas as defined in its agenda (IDB, 1999; MinC & Programa Monumenta, 2006). Using the Historic Centre of Porto Alegre as a case study, this paper examines how this cultural programme enables demographic change through the promotion of intangible cultural heritage, e.g. by supporting educational projects. The demographic flow was analysed using the microdata of the Populations Censuses (years 2000 and 2010) available from the Brazilian Institute of Geography and Statistics. The results showed an increase in low-income residents the areas that participated in the programme. This increase may have been motivated by a set of cultural-educational projects under the auspices of the Monumenta Programme. The retraining of artisans of Alfândega Square, the training of low-income youth for restoration work and the implementation of the "Black Route Museum in Porto Alegre" (Bicca, 2010) are just some examples of what was done to improve the local community's economy, to encourage social cohesion and to enhance the awareness of cultural diversity as a positive and essential value in society.