Connexin-43 prevents hematopoietic stem cell senescence through transfer of reactive oxygen species to bone marrow stromal cells

Hematopoietic stem cell (HSC) aging has become a concern in chemotherapy of older patients. Humoral and paracrine signals from the bone marrow (BM) hematopoietic microenvironment (HM) control HSC activity during regenerative hematopoiesis. Connexin-43 (Cx43), a connexin constituent of gap junctions (GJs) is expressed in HSCs, down-regulated during differentiation, and postulated to be a self-renewal gene. Our studies, however, reveal that hematopoietic-specific Cx43 deficiency does not result in significant long-term competitive repopulation deficiency. Instead, hematopoietic Cx43 (H-Cx43) deficiency delays hematopoietic recovery after myeloablation with 5-fluorouracil (5-FU). 5-FU-treated H-Cx43-deficient HSC and progenitors (HSC/P) cells display decreased survival and fail to enter the cell cycle to proliferate. Cell cycle quiescence is associated with down-regulation of cyclin D1, up-regulation of the cyclin-dependent kinase inhibitors, p21cip1. and p16INK4a, and Forkhead transcriptional factor 1 (Foxo1), and activation of p38 mitogen-activated protein kinase (MAPK), indicating that H-Cx43-deficient HSCs are prone to senescence. The mechanism of increased senescence in H-Cx43-deficient HSC/P cells depends on their inability to transfer reactive oxygen species (ROS) to the HM, leading to accumulation of ROS within HSCs. In vivo antioxidant administration prevents the defective hematopoietic regeneration, as well as exogenous expression of Cx43 in HSC/P cells. Furthermore, ROS transfer from HSC/P cells to BM stromal cells is also rescued by reexpression of Cx43 in HSC/P. Finally, the deficiency of Cx43 in the HM phenocopies the hematopoietic defect in vivo. These results indicate that Cx43 exerts a protective role and regulates the HSC/P ROS content through ROS transfer to the HM, resulting in HSC protection during stress hematopoietic regeneration.

C1A Cysteine protease-cystatin interactions in leaf senescence

Senescence-associated proteolysis in plants is a crucial process to relocalize nutrients from leaves to growing or storage tissues. The massive net degradation of proteins involves broad metabolic networks, different subcellular compartments, and several types of proteases and regulators. C1A cysteine proteases, grouped as cathepsin L-, B-, H-, and F-like according to their gene structures and phylogenetic relationships, are the most abundant enzymes responsible for the proteolytic activity during leaf senescence. Besides, cystatins as specific modulators of C1A peptidase activities exert a complex regulatory role in this physiological process. This overview article covers the most recent information on C1A proteases in leaf senescence in different plant species. Particularly, it is focussed on barley, as the unique species where the whole gene family members of C1A cysteine proteases and cystatins have been analysed.

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.

Assessment of the ozone sensitivity of 22 native plant species from mediterranean annual pastures based on visible injury

Ozone (O3) phytototoxicity has been reported on a wide range of plantspecies, inducing the appearance of specific foliar injury or increasing leaf senescence. No information regarding the sensitivity of plantspecies from dehesa Mediterranean grasslands has been provided in spite of their great biological diversity. A screening study was carried out in open-top chambers (OTCs) to assess the O3-sensitivity of 22 representative therophytes of these ecosystems based on the appearance and extent of foliar injury. A distinction was made between specific O3injury and non-specific discolorations. Three O3 treatments (charcoal-filtered air, non-filtered air and non-filtered air supplemented with 40 nl l−1 O3 during 5 days per week) and three OTCs per treatment were used. The Papilionaceae species were more sensitive to O3 than the Poaceae species involved in the experiment since ambient levels induced foliar symptoms in 67% and 27%, respectively, of both plant families. An O3-sensitivity ranking of the species involved in the assessment is provided, which could be useful for bioindication programmes in Mediterranean areas. The assessed Trifoliumspecies were particularly sensitive since foliar symptoms were apparent in association with O3 accumulated exposures well below the current critical level for the prevention of this kind of effect. The exposure indices involving lower cut-off values (i.e. 30 nl l−1) were best related with the extent of O3-induced injury on these species.

Cysteine-proteases and cystatins from barley: molecular and functional characterization in housekeeping and defense processes

Plant cysteine-proteases (CysProt) represent a well-characterized type of proteolytic enzymes that fulfill tightly regulated physiological functions (senescence and seed germination among others) and defense roles. This article is focused on the group of papain-proteases C1A (family C1, clan CA) and their inhibitors, phytocystatins (PhyCys). In particular, the protease–inhibitor interaction and their mutual participation in specific pathways throughout the plant's life are reviewed. C1A CysProt and PhyCys have been molecularly characterized, and comparative sequence analyses have identified consensus functional motifs. A correlation can be established between the number of identified CysProt and PhyCys in angiosperms. Thus, evolutionary forces may have determined a control role of cystatins on both endogenous and pest-exogenous proteases in these species. Tagging the proteases and inhibitors with fluorescence proteins revealed common patterns of subcellular localization in the endoplasmic reticulum–Golgi network in transiently transformed onion epidermal cells. Further in vivo interactions were demonstrated by bimolecular fluorescent complementation, suggesting their participation in the same physiological processes.

Seasonal evolution of soil respiration in a mixed forestry plantation of walnuts (Juglans × intermedia Carr.) and alders (Alnus cordata (Loisel.) Duby) in Domaine de Restinclières (France)

The ecological intensification of crops is proposed as a solution to the growing demand of agricultural and forest resources, in opposition to intensive monocultures. The introduction of mixed cultures as mixtures between nitrogen fixing species and non nitrogen fixing species intended to increase crop yield as a result of an improvement of the available nitrogen and phosphorus in soil. Relationship between crops have received little attention despite the wide range of advantages that confers species diversity to these systems, such as increased productivity, resilience to disruption and ecological sustainability. Forests and forestry plantations can develop an important role in storing carbon in their tissues, especially in wood which become into durable product. A simplifying parameter to analyze the amount allocated carbon by plantation is the TBCA (total belowground carbon allocation), whereby, for short periods and mature plantations, is admitted as the subtraction between soil carbon efflux and litterfall. Soil respiration depends on a wide range of factors, such as soil temperature and soil water content, soil fertility, presence and type of vegetation, among others. The studied orchard is a mixed forestry plantation of hybrid walnuts(Juglans × intermedia Carr.) for wood and alders (Alnus cordata (Loisel.) Duby.), a nitrogen fixing specie through the actinomycete Frankia alni ((Woronin, 1866) Von Tubeuf 1895). The study area is sited at Restinclières, a green area near Montpellier (South of France). In the present work, soil respiration varied greatly throughout the year, mainly influenced by soil temperature. Soil water content did not significantly influence the response of soil respiration as it was constant during the measurement period and under no water stress conditions. Distance between nearest walnut and measurement was also a highly influential factor in soil respiration. Generally there was a decreasing trend in soil respiration when the distance to the nearest tree increased. It was also analyzed the response of soil respiration according to alder presence and fertilizer management (50 kg N·ha-1·año-1 from 1999 to 2010). None of these treatments significantly influenced soil respiration, although previous studies noticed an inhibition in rates of soil respiration under fertilized conditions and high rates of available nitrogen. However, treatments without fertilization and without alder presence obtained higher respiration rates in those cases with significant differences. The lack of significant differences between treatments may be due to the high coefficient of variation experienced by soil respiration measurements. Finally an asynchronous fluctuation was observed between soil respiration and litterfall during senescence period. This is possibly due to the slowdown in the emission of exudates by roots during senescence period, which are largely related to microbial activity.

Localización óptima de una planta de bioetanol a partir de tubérculos de pataca (Helianthus tuberosus l.) en la Cuenca del Duero

El objetivo del presente trabajo es determinar la localización óptima de una planta de producción de 30.000 m3/año de bioetanol a partir de tubérculos de pataca (Helianthus tuberosus L.) cultivada en regadío, en tierras de barbecho de la Cuenca Hidrográfica del Duero (CH Duero). Inicialmente se elaboró, a partir de datos bibliográficos, un modelo de producción de pataca en base a una ecuación de regresión que relaciona datos experimentales de rendimientos de variedades tardías con variables agroclimáticas. Así se obtuvo una función de producción basada en la cantidad de agua disponible (precipitación efectiva + dosis de riego) y en la radiación global acumulada en el periodo brotación‐senescencia del cultivo. A continuación se estima la superficie potencial de cultivo de pataca en la CH Duero a partir de la superficie arable en regadío cartografiada por el Sistema de Ocupación del Suelo (SIOSE), a la cual se le aplican, en base a los requerimientos del cultivo, unas restricciones climáticas, edafológicas, topográficas y logísticas mediante el uso de Sistemas de Información Geográfica (SIG). La proporción de superficie de regadío restringida se cuantifica a escala municipal con el fin de calcular la superficie de barbecho en regadío apta para el cultivo de pataca. A partir de las bases de datos georreferenciadas de precipitación, radiación global, y la dotación de agua para el riego de cultivos no específicos establecida en el Plan Hidrológico de la Cuenca del Duero a escala comarcal, se estimó la producción potencial de tubérculos de pataca sobre la superficie de barbecho de regadío según el modelo de producción elaborado. Así, en las 53.360 ha de barbecho en regadío aptas para el cultivo de pataca se podrían producir 3,8 Mt de tubérculos al año (80 % de humedad) (761.156 t ms/año) de los que se podría obtener 304.462 m3/año de bioetanol, considerando un rendimiento en la transformación de 12,5 kg mf/l de etanol. Se estiman los costes de las labores de cultivo de pataca así como los costes de la logística de suministro a una planta de transformación considerando una distancia media de transporte de 25 km, en base a las hojas de cálculo de utilización de aperos y maquinaria agrícola oficiales del Ministerio de Agricultura, Alimentación y Medio Ambiente (MAGRAMA). Considerando el balance de costes asociados a la producción de bioetanol (costes de transformación, distribución y transporte del producto, costes estructurales de la planta, ahorro de costes por la utilización de las vinazas generadas en el proceso como fertilizante y un beneficio industrial), se ha estimado que el coste de producción de bioetanol a partir de tubérculos de pataca asciende a 61,03 c€/l. Se calculan los beneficios fiscales para el Estado por el cultivo de 5.522 ha de pataca que suministren la materia prima necesaria para una planta de bioetanol de 30.000 m3/año, en concepto de cotizaciones a la Seguridad Social de los trabajadores, impuestos sobre el valor añadido de los productos consumidos, impuesto sobre sociedades y ahorro de las prestaciones por desempleo. Se obtuvieron unos beneficios fiscales de 10,25 c€ por litro de bioetanol producido. El coste de producción de bioetanol depende del rendimiento de tubérculos por hectárea y de la distancia de transporte desde las zonas de producción de la materia prima hasta la planta. Se calculó la distancia máxima de transporte para que el precio de coste del bioetanol producido sea competitivo con el precio de mercado del bioetanol. Como resultado se determinó que el precio del bioetanol (incluido un beneficio industrial del 15%) de la planta sería igual o inferior al precio de venta en el mercado (66,35 c€/l) con una distancia máxima de transporte de 25 km y un rendimiento mínimo del cultivo de 60,1 t mf/ha. Una vez conocido el área de influencia de la planta según la distancia de transporte máxima, se determinó la localización óptima de la planta de producción de bioetanol mediante un proceso de ubicación‐asignación realizado con SIG. Para ello se analizan los puntos candidatos a la ubicación de la planta según el cumplimiento de unos requerimientos técnicos establecidos (distancia a fuentes de suministro eléctrico y de recursos hídricos, distancia a estaciones de ferrocarril, distancia a núcleos urbanos y existencia de Espacios Naturales Protegidos) que minimizan la distancia de transporte maximizando la cantidad de biomasa disponible según la producción potencial estimada anteriormente. Por último, la superficie destinada al cultivo de pataca en el área de influencia de la planta se determina en base a un patrón de distribución del cultivo alrededor de una agroindustria. Dicho patrón se ha obtenido a partir del análisis del grado de ocupación del cultivo de la remolacha en función de la distancia de transporte a la planta azucarera de Miranda de Ebro (Burgos). El patrón resultante muestra que la relación entre el grado de ocupación del suelo por el cultivo y la distancia de transporte a la planta siguen una ecuación logística. La localización óptima que se ha obtenido mediante la metodología descrita se ubica en el municipio leonés de El Burgo Ranero, donde la producción potencial de tubérculos de pataca en la superficie de barbecho situada en un radio de acción de 25 km es de 375.665 t mf/año, superando las 375.000 t mf requeridas anualmente por la planta de bioetanol. ABSTRACT Jerusalem artichoke (Helianthus tuberosus L.) is a harsh crop with a high potential for biomass production. Its main use is related to bioethanol production from the carbohydrates, inulin mainly, accumulated in its tubers at the end of the crop cycle. The aerial biomass could be used as solid biofuel to provide energy to the bioethanol production process. Therefore, Jerusalem artichoke is a promising crop as feedstock for biofuel production in order to achieve the biofuels consumption objectives established by the Government of Spain (PER 2011‐2020 and RDL 4/2013) and the European Union (Directive 2009/28/EC). This work aims at the determination of the optimal location for a 30,000 m3/year bioethanol production plant from Jerusalem artichoke tubers in the Duero river basin. With this purpose, a crop production model was developed by means of a regression equation that relates experimental yield data of late Jerusalem artichoke varieties with pedo‐climatic parameters from a bibliographic data matrix. The resulting crop production model was based on the crop water availability (including effective rainfall and irrigation water supplied) and on global radiation accumulated in the crop emergence‐senescence period. The crop potential cultivation area for Jerusalem artichoke in the Duero basin was estimated using the georeferenced irrigated arable land from the “Sistema de Ocupación del Suelo” (SIOSE) of Spain. Climatic, soil, slope and logistic restrictions were considered by means of Geographic Information Systems (GIS). The limited potential growing area was then applied to a municipality scale in order to calculate the amount of fallow land suitable for Jerusalem artichoke production. Rainfall and global radiation georeferenced layers as well as data of irrigation water supply for crop production (established within the Duero Hydrologic Plan) were use to estimate the potential production of Jerusalem artichoke tubers in the suitable fallow land according to the crop production model. As a result of this estimation, there are 53,360 ha of fallow land suitable for Jerusalem artichoke production in the Duero basin, where 3.8 M t fm/year could be produced. Considering a bioethanol processing yield of 12.5 kg mf per liter of bioethanol, the above mentioned tuber potential production could be processed in 304,462 m3/year of bioethanol. The Jerusalem crop production costs and the logistic supply costs (considering an average transport distance of 25 km) were estimated according to official agricultural machinery cost calculation sheets of the Minister of Agriculture of Spain (MAGRAMA). The bioethanol production cost from Jerusalem artichoke tubers was calculated considering bioethanol processing, transport and structural costs, industrial profits as well as plant cost savings from the use of vinasses as fertilizer. The resulting bioetanol production cost from Jerusalem artichoke tubers was 61.03 c€/l. Additionally, revenues for the state coffers regarding Social Security contributions, added value taxes of consumed raw materials, corporation tax and unemployment benefit savings due to the cultivation of 5,522 ha of Jerusalem artichoke for the 30.000 m3/year bioethanol plant supply were calculated. The calculated revenues amounted to 10.25 c€/l. Bioethanol production cost and consequently the bioethanol plant economic viability are strongly related to the crop yield as well as to road transport distance from feedstock production areas to the processing plant. The previously estimated bioethanol production cost was compared to the bioethanol market price in order to determine the maximum supply transport distance and the minimum crop yield to reach the bioethanol plant economic viability. The results showed that the proposed plant would be economically viable at a maximum transport distance of 25 km and at a crop yield not less than 60.1 t fm/ha. By means of a GIS location‐allocation analysis, the optimal bioethanol plant location was determined. Suitable candidates were detected according to several plant technical requirements (distance to power and water supply sources, distance to freight station, and distance to urban areas and to Natural Protected Areas). The optimal bioethanol plant location must minimize the supply transport distance whereas it maximizes the amount of available biomass according to the previously estimated biomass potential production. Lastly, the agricultural area around the bioethanol plant finally dedicated to Jerusalem artichoke cultivation was planned according to a crop distribution model. The crop distribution model was established from the analysis of the relation between the sugar beet (Beta vulgaris L.) cropping area and the road transport distance from the sugar processing plant of Miranda de Ebro (Burgos, North of Spain). The optimal location was situated in the municipality of ‘El Burgo Ranero’ in the province of León. The potential production of Jerusalem artichoke tubers in the fallow land within 25 km distance from the plant location was 375,665 t fm/year, which exceeds the amount of biomass yearly required by the bioethanol plant.

Iron distribution through the developmental stages of Medicago truncatula nodules.

Paramount to symbiotic nitrogen fixation (SNF) is the synthesis of a number of metalloenzymes that use iron as a critical component of their catalytical core. Since this process is carried out by endosymbiotic rhizobia living in legume root nodules, the mechanisms involved in iron delivery to the rhizobia-containing cells are critical for SNF. In order to gain insight into iron transport to the nodule, we have used synchrotron-based X-ray fluorescence to determine the spatio-temporal distribution of this metal in nodules of the legume Medicago truncatula with hitherto unattained sensitivity and resolution. The data support a model in which iron is released from the vasculature into the apoplast of the infection/differentiation zone of the nodule (zone II). The infected cell subsequently takes up this apoplastic iron and delivers it to the symbiosome and the secretory system to synthesize ferroproteins. Upon senescence, iron is relocated to the vasculature to be reused by the shoot. These observations highlight the important role of yet to be discovered metal transporters in iron compartmentalization in the nodule and in the recovery of an essential and scarce nutrient for flowering and seed production.

Characterization of a glycosylase family gene specifically expressed during winter dormancy in woody plants

Winter dormancy is the strategy used by perennial plants to survive the harsh conditions of winter in temperate and cold regions. This complex mechanism is characterized by cessation of the meristems activity, which is accompanied by the budset, the acquisition of a high tolerance to the cold temperatures and, in the case of deciduous trees, by the senescence and leaf abscission. In long-lived forest species, the length of the dormancy period limits the growing season, affecting wood production and quality. A Suppression Subtractive Hybridization (SSH) enriched in genes overexpressed during the process of winter dormancy in chesnut stems identified a DNA glycosylase gene. In order to study its role in the establishment and maintenance of the winter dormancy, a molecular characterization and seasonal expression were performed. Furthermore, we have obtained poplar transgenic plantlets overexpressing the chesnut gene.

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

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

Advanced morphometric techniques applied to the study of human brain anatomy

El desarrollo de las técnicas de imágenes por resonancia magnética han permitido el estudio y cuantificación, in vivo, de los cambios que ocurren en la morfología cerebral ligados a procesos tales como el neurodesarrollo, el envejecimiento, el aprendizaje o la enfermedad. Un gran número de métodos de morfometría han sido desarrollados con el fin de extraer la información contenida en estas imágenes y traducirla en indicadores de forma o tamaño, tales como el volumen o el grosor cortical; marcadores que son posteriormente empleados para encontrar diferencias estadísticas entre poblaciones de sujetos o realizar correlaciones entre la morfología cerebral y, por ejemplo, la edad o la severidad de determinada enfermedad. A pesar de la amplia variedad de biomarcadores y metodologías de morfometría, muchos estudios sesgan sus hipótesis, y con ello los resultados experimentales, al empleo de un número reducido de biomarcadores o a al uso de una única metodología de procesamiento. Con el presente trabajo se pretende demostrar la importancia del empleo de diversos métodos de morfometría para lograr una mejor caracterización del proceso que se desea estudiar. En el mismo se emplea el análisis de forma para detectar diferencias, tanto globales como locales, en la morfología del tálamo entre pacientes adolescentes con episodios tempranos de psicosis y adolescentes sanos. Los resultados obtenidos demuestran que la diferencia de volumen talámico entre ambas poblaciones de sujetos, previamente descrita en la literatura, se debe a una reducción del volumen de la región anterior-mediodorsal y del núcleo pulvinar del tálamo de los pacientes respecto a los sujetos sanos. Además, se describe el desarrollo de un estudio longitudinal, en sujetos sanos, que emplea simultáneamente distintos biomarcadores para la caracterización y cuantificación de los cambios que ocurren en la morfología de la corteza cerebral durante la adolescencia. A través de este estudio se revela que el proceso de “alisado” que experimenta la corteza cerebral durante la adolescencia es consecuencia de una disminución de la profundidad, ligada a un incremento en el ancho, de los surcos corticales. Finalmente, esta metodología es aplicada, en un diseño transversal, para el estudio de las causas que provocan el decrecimiento tanto del grosor cortical como del índice de girificación en adolescentes con episodios tempranos de psicosis. ABSTRACT The ever evolving sophistication of magnetic resonance image techniques continue to provide new tools to characterize and quantify, in vivo, brain morphologic changes related to neurodevelopment, senescence, learning or disease. The majority of morphometric methods extract shape or size descriptors such as volume, surface area, and cortical thickness from the MRI image. These morphological measurements are commonly entered in statistical analytic approaches for testing between-group differences or for correlations between the morphological measurement and other variables such as age, sex, or disease severity. A wide variety of morphological biomarkers are reported in the literature. Despite this wide range of potentially useful biomarkers and available morphometric methods, the hypotheses and findings of the grand majority of morphological studies are biased because reports assess only one morphometric feature and usually use only one image processing method. Throughout this dissertation biomarkers and image processing strategies are combined to provide innovative and useful morphometric tools for examining brain changes during neurodevelopment. Specifically, a shape analysis technique allowing for a fine-grained assessment of regional thalamic volume in early-onset psychosis patients and healthy comparison subjects is implemented. Results show that disease-related reductions in global thalamic volume, as previously described by other authors, could be particularly driven by a deficit in the anterior-mediodorsal and pulvinar thalamic regions in patients relative to healthy subjects. Furthermore, in healthy adolescents different cortical features are extracted and combined and their interdependency is assessed over time. This study attempts to extend current knowledge of normal brain development, specifically the largely unexplored relationship between changes of distinct cortical morphological measurements during adolescence. This study demonstrates that cortical flattening, present during adolescence, is produced by a combination of age-related increase in sulcal width and decrease in sulcal depth. Finally, this methodology is applied to a cross-sectional study, investigating the mechanisms underlying the decrease in cortical thickness and gyrification observed in psychotic patients with a disease onset during adolescence.