HvPap-1 C1A Protease Participates Differentially in the Barley Response to a Pathogen and an Herbivore

Co-evolutionary processes in plant–pathogen/herbivore systems indicate that protease inhibitors have a particular value in biotic interactions. However, little is known about the defensive role of their targets, the plant proteases. C1A cysteine proteases are the most abundant enzymes responsible for the proteolytic activity during different processes like germination, development and senescence in plants. To identify and characterize C1A cysteine proteases of barley with a potential role in defense, mRNA and protein expression patterns were analyzed in response to biotics stresses. A barley cysteine protease, HvPap-1, previously related to abiotic stresses and grain germination, was particularly induced by flagellin or chitosan elicitation, and biotic stresses such as the phytopathogenic fungus Magnaporthe oryzae or the phytophagous mite Tetranychus urticae. To elucidate the in vivo participation of this enzyme in defense, transformed barley plants overexpressing or silencing HvPap-1 encoding gene were subjected to M. oryzae infection or T. urticae infestation. Whereas overexpressing plants were less susceptible to the fungus than silencing plants, the opposite behavior occurred to the mite. This unexpected result highlights the complexity of the regulatory events leading to the response to a particular biotic stress

Evaluation of biostimulation, bioaugmentation, and organic amendments application on the bioremediation of recalcitrant hydrocarbons of soil

In the present work, the operational conditions for improving the degradation rates of Total Petroleum Hydrocarbons (TPHs) in contaminated soil from a machinery park were optimized at a microcosms scale along a 90- days incubation period. In this study, bioremediation strategies and an organic amendment have been tested to verify the remediation of soil contaminated with different hydrocarbons, mineral oils, and heavy metals. Specifically, designed biostimulation and bioaugmentation strategies were compared with and without adding vermicompost. The polluted soil harboring multiple contaminants, partially attenuated for years, was used. The initial profile showed enrichment in heavy linear alkanes, suggesting a previous moderate weathering. The application of vermicompost increased five and two times the amounts of available phosphorus (P) and exchangeable potassium (K), respectively, as a direct consequence of the organic amendment addition. The microbial activity increased due to soil acidification, which influenced the solubility of P and other micronutrients. It also impacted the predominance and variability of the different microbial groups and the incubation, as reflected by phospholipid fatty acid (PLFA) results. An increase in the alkaline phosphatases and proteases linked to bacterial growth was displayed. This stimulation of microbial metabolism correlated with the degradation rates since TPHs degradation’ efficiency after vermicompost addition reached 32.5% and 34.4% of the initial hydrocarbon levels for biostimulation and bioaugmentation, respectively. Although Polycyclic Aromatic Hydrocarbons (PAHs) were less abundant in this soil, results also decreased, especially for the most abundant, the phenanthrene. Despite improving the degradation rates, results revealed that recalcitrant and hydrophobic petroleum compounds remained unchanged, indicating that mobility, linked to bioavailability, probably represents the limiting step for further soil recovery.This work is funded by the GREENER project of the European Union’s Horizon 2020 research and innovation program (Grant Agreement No. 826312). S. Curiel pre-doctoral contract was funded by Junta de Castilla y Leon ´ (ORDEN EDU/1508/2020, de 15 de diciembre)

Senescence-associated proteolysis induced by abiotic and biotic stresses in barley leaves

Leaf senescence is a recycling process characterized by a massive degradation of macromolecules to relocalize nutrients from leaves to growing or storage tissues. Our aim is to identify and analyze the C1A Cysteine ‐Protease (CysProt) family members from barley (35 cathepsin L‐,3B‐,1Hand3F‐like) involved in leaf senescence, to study their modulation by their specific inhibitors (cystatins) and to determine their roles mediated by abiotic (darkness and N starvation) and biotic (pathogens and pest) stresses

Comparative toxicological assessment of three soils polluted with different levels of hydrocarbons and heavy metals using in vitro and in vivo approaches

The biological effects induced by the pollutants present in soils, together with the chemical and physical characterizations, are good indicators to provide a general overview of their quality. However, the existence of studies where the toxicity associated to soils contaminated with mixtures of pollutants applying both in vitro and in vivo models are scarce. In this work, three soils (namely, Soil 001, Soil 002 and Soil 013) polluted with different concentrations of hydrocarbons and heavy metals were evaluated using different organisms representative of human (HepG2 human cell line) and environmental exposure (the yeast Saccharomyces cerevisiae, the Gram-negative bacterium Pseudomonas putida and, for the in vivo evaluation, the annelid Enchytraeus crypticus). In vitro assays showed that the soluble fraction of the Soil 001, which presented the highest levels of heavy metals, represented a great impact in the viability of the HepG2 cells and S. cerevisiae, while organic extracts from Soils 002 and 013 caused a slight decrease in the viability of HepG2 cells. In addition, in vivo experiments showed that Soils 001 and 013 affected the survival and the reproduction of E. crypticus. Altogether, these results provide a general overview of the potential hazards associated to three specific contaminated sites in a variety of organisms, showing how different concentrations of similar pollutants affect them, and highlights the relevance of testing both organic and soluble extracts when in vitro safety assays of soils are performed.This work received funding from the GREENER project of the European Union’s Horizon 2020 research and innovation program (Grant Agreement No. 826312). S. Curiel-Alegre was granted with a predoctoral contract by Junta de Castilla y León and the European Social Fund (ORDEN EDU/1508/2020, de 15 de diciembre). We thank Mireya Pedrero and Andrea Martínez for their technical support. We also thank Institute of Technology Carlow and Shandong Academy of Sciences for kindly providing us with soil samples

Impact of operatoŕs experience on peri-procedural outcomes with Watchman FLX: Insights from the FLX-SPA registry

Background: The Watchman FLX is a device upgrade of the Watchman 2.5 that incorporates several design enhancements intended to simplify left atrial appendage occlusion (LAAO) and improve procedural outcomes. This study compares peri-procedural results of LAAO with Watchman FLX (Boston Scientific, Marlborough, Massachusetts) in centers with varying degrees of experience with the Watchman 2.5 and Watchman FLX. Methods: Prospective, multicenter, 'real-world' registry including consecutive patients undergoing LAAO with the Watchman FLX at 26 Spanish sites (FLX-SPA registry). Implanting centers were classified according to the center's prior experience with the Watchman 2.5. A further division of centers according to whether or not they had performed ≤ 10 or > 10Watchman FLX implants was prespecified at the beginning of the study. Procedural outcomes of institutions stratified according to their experience with the Watchman 2.5 and FLX devices were compared. Results: 359 patients [mean age 75.5 (SD8.1), CHA2DS2-VASc 4.4 (SD1.4), HAS-BLED 3.8(SD0.9)] were included. Global success rate was 98.6%, successful LAAO with the first selected device size was achieved in 95.5% patients and the device was implanted at first attempt in 78.6% cases. There were only 9(2.5%) major peri-procedural complications. No differences in efficacy or safety results according to the centeŕs previous experience with Watchman 2.5 and procedural volume with Watchman FLX existed. Conclusions: The Watchman FLX attains high procedural success rates with complete LAA sealing in unselected, real-world patients, along with a low incidence of peri-procedural complications, regardless of operatoŕs experience with its previous device iteration or the number of Watchman FLX devices implanted

White Paper 1: New foundations for a sustainable global society

Coordinación: Eduardo Moyano Estrada; Tomás García Azcárate.This volume is focused on the axis “New foundations for a sustainable global society”, and refers to the important process of global change that affects all dimensions of society, disrupting the context in which scientific work has been developed in recent decades. It is a process of change not comparable to what happened decades ago, mainly due to its breadth, multidimensionality and interdependence, and also to the fact that this process manifests itself simultaneously in many areas, territories and social groups. Its analysis therefore requires carrying out a convergence exercise between areas and lines of research, betting on a multidisciplinary approach, since both “globalization” and “sustainability” are, concepts that affect society, as a whole.Peer reviewe

Roles of C1A peptidases during barley leaf senescence mediated by abiotic stresses

Protein breakdown and mobilization from old or stressed tissues, such as leaves, to growing and sink organs, such as grains or tubers, are some of the metabolic features associated with leaf senescence, essential for nutrient recycling. Senescence may be naturally activated by endogenous signals and/or modified by the prevalence of abiotic/biotic stresses, as a survival strategy. Protein breakdown in senescing leaves involves many plastidial and nuclear proteases, regulators, different subcellular locations and a dynamic protein traffic to ensure transformation of high molecular weight proteins into transportable and useful hydrolyzed products. C1A cysteine proteases are the most abundant key players responsible for the proteolytic activity during leaf senescence. Besides, cystatins, as specific modulators of C1A protease activities, exert a regulatory role along the process. In barley (Hordeum vulgare), the whole gene family members of C1A cysteine proteases and cystatins have been identified. Elucidating the role of barley C1A proteases in response to abiotic stresses is crucial due to their impact on plant growth and grain yield and quality. Darkness and nitrogen starvation treatments were used to induce leaf senescence in barley. Both abiotic stresses strongly induced the expression of the HvPap-1 gene encoding a cathepsin F-like protease. Morphological changes presuming chloroplast dismantling designated darkness as an ideal stressor for inducing and analyzing senescence. Differences in biochemical parameters and C1A gene expression and protein accumulation among wild-type and transgenic barley plants over-expressing or silencing this gene were detected under the stress. Besides, a lifespan-delayed phenotype of HvPap-1 silenced lines was evidenced, indicating a functional role for this protease along the senescence process. Proteolysis is likewise essential throughout the mobilization of storage proteins in barley grains during germination. Manipulation of the proteolytic machinery could enhance grain yield and quality through alterations along these stages. Transgenic barley plants silencing or over-expressing HvPap-1 showed differential accumulation of starch, proteins, and free amino acids in the grain. The phenotype displayed by silencing HvPap-1 lines, showing a drastic delay in germination, was particularly striking. Alterations in the proteolytic activities associated with changes in the expression levels of several C1A proteases were also detected. Similarly, down-regulating Icy-2, encoding one of the proteinaceous inhibitors of the studied cathepsin F-like protease, also brought about important effects on grain filling. The cooperative role of cystatins and their functional relationship with cysteine proteases have been highlighted in the current study by the enhanced/reduced tolerance of plants silencing phytocystatins towards drought. Two barley phytocystatins, HvCPI-2 and HvCPI-4, were induced by this stress. Alterations in the proteolytic patterns by silencing these cystatins were concomitant with modifications in the expression of target proteases. As a result, accelerated or delayed leaf senescence, depending on the silenced cystatin, was exhibited. Results support the potential use of these plants to modulate plant responses facing abiotic stress and, at the same time, to maintain or even increase crop yields under the evidenced climate change framework According to data reported in this thesis, manipulation of C1A proteases-cystatins interactions in barley has the potential to modulate sensitivity towards specific abiotic stresses through modifications over established developmental leaf senescence programs. In addition, the in vivo implication of this proteolytic network during remobilization of stored compounds along barley grain germination is demonstrated. As a general remark, caution should be taken when designing related biotechnological tools since the plant tries to compensate the genetic modifications by modulating the expression of some other proteases or inhibitors. RESUMEN La degradación y movilización de proteínas desde tejidos maduros o sometidos a estrés, como las hojas, hasta los órganos en desarrollo o sumidero, como los granos de los cereales, son procesos metabólicos inherentes a la senescencia foliar. Los programas de senescencia se activan tanto en respuesta a señales endógenas como a estreses abióticos y bióticos como estrategia de supervivencia. La proteólisis en hojas senescentes implica multitud de proteasas de origen nuclear y plastidial, reguladores, diversas localizaciones subcelulares, así como un tráfico dinámico cuyo fin es asegurar la transformación de proteínas de alto peso molecular en productos hidrolizados que puedan transportarse y reutilizarse. La familia C1A de cisteín-proteasas engloba un buen número de enzimas responsables de la actividad proteolítica asociada a la senescencia foliar. Además, las cistatinas, inhibidores específicos de dichas proteasas, ejercen un papel regulador durante este proceso fisiológico. En cebada (Hordeum vulgare), las familias completas de proteasas C1A y cistatinas han sido identificadas. Dilucidar el papel funcional de las proteasas C1A de cebada en respuesta a estreses abióticos es esencial, debido a su impacto sobre el crecimiento de las plantas y la alteración del rendimiento y calidad del grano. Los tratamientos de oscuridad y de carencia de nitrógeno se utilizaron para inducir senescencia foliar en cebada. Ambos estreses indujeron claramente la expresión del gen HvPap-1, que codifica una proteasa tipo catepsina F. Cuando se compararon plantas control frente a líneas transgénicas de sobrexpresión y de silenciamiento para este gen en oscuridad, se observaron alteraciones significativas en parámetros bioquímicos, en patrones de expresión de genes de proteasas C1A, así como en el contenido proteico. Por otro lado, el fenotipo “stay-green” de las líneas de silenciamiento evidenció una vida útil más prolongada en estas plantas, demostrando la implicación funcional de esta proteasa a lo largo del proceso de senescencia. La proteólisis es asimismo esencial para la movilización de proteínas de reserva del grano durante la germinación. La manipulación de la maquinaria proteolítica durante este proceso fisiológico podría tener un efecto de mejora sobre la calidad del grano y el rendimiento del cultivo. Las líneas transgénicas de sobreexpresión y silenciamiento del gen HvPap-1 mostraron una acumulación diferencial de almidón, proteínas y amino ácidos en la semilla. El fenotipo de los granos de las líneas silenciadas evidenció un claro retraso en el proceso germinativo. También se observaron alteraciones en las actividades proteolíticas, asociadas a las variaciones en los niveles de expresión de genes C1A. De forma paralela, al silenciarse el gen Icy-2 que codifica uno de los inhibidores de la catepsina F estudiada, se observaron efectos en relación con el llenado y calidad del grano. La interacción y la implicación funcional de cisteín-proteasas y cistatinas en cebada se ha constatado en este estudio, tal y como se infiere de la tolerancia alterada frente a sequía en las líneas de silenciamiento de cistatinas. Dos fitocistatinas, HvCPI-2 y HvCPI-4, se indujeron específicamente por dicho estrés. Las alteraciones en los patrones proteolíticos al silenciar estas cistatinas fueron paralelas a las variaciones en la expresión de genes de sus proteasas diana. En función de la cistatina silenciada, se apreció un retraso o una aceleración en la senescencia. Estos resultados apoyan el uso de estas líneas con el objetivo de modular las respuestas a estreses diversos y mantener, o incluso incrementar, los rendimientos en el marco evidente del cambio climático. De acuerdo con los resultados obtenidos, la manipulación de las interacciones entre proteasas C1A y cistatinas en cebada permitiría modular la sensibilidad frente a estreses abióticos concretos en base a modificaciones sobre los programas de senescencia endógenos. Se confirma asimismo, la importancia in vivo de esta compleja red proteolítica durante la germinación. Como observación general, cuando se diseñen estrategias biotecnológicas basadas en estos mecanismos moleculares se han de considerar los efectos de compensación derivados de la expresión de otros inhibidores y/o proteasas de la planta