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

MtNramp1 mediates iron import in rhizobia-infected Medicago truncatula cells.

Symbiotic nitrogen fixation is a process that requires relatively high quantities of iron provided by the host legume. Using synchrotron-based X-ray fluorescence, we have determined that this iron is released from the vasculature into the apoplast of zone II of M. truncatula nodules. This overlaps with the distribution of MtNramp1, a plasma membrane iron importer. The importance of MtNramp1 in iron transport for nitrogen fixation is indicated by the 60% reduction of nitrogenase activity observed in knock-down lines, most likely due to deficient incorporation of this essential metal cofactor at the necessary levels

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

Influence of graphene oxide's characteristics on the fabrication and performance of crosslinked nanofiltration membranes

Graphene oxide (GO) is emerging as an excellent next generation material for water purification membranes. Its ability to be fabricated cost-effectively in large quantities and featured characteristics, such as hydrophilicity, makes it an equitable graphene alternative in respective nanometric applications, including nanofiltration. In this study, the influence of key properties of the GO sheets, such as lateral size, surface chemistry and colloid stability, on the successful fabrication and subsequent water purification performance of crosslinked nanofiltration membranes is analysed. GO water suspensions with nanosheets of different lateral sizes and distribution of oxygenated functional groups were prepared by controlling the sonication time (from 0 to 180 min) starting from commercial GO. The variation of the physicochemical characteristics of the resulting GO sheets was comprehensively studied by means of atomic force microscopy, UV–Vis absorption spectroscopy, zeta potential measurements and X-Ray photoelectron spectroscopy. The morphology of the subsequently fabricated membranes was hereafter examined via scanning electron microscopy, while their nanofiltration performance was investigated against methylene blue solution. The influence of GO's physicochemical characteristics on membrane performance was apparent, with the average rejection values ranging from 59.8% to 98.4% at a changing lateral size and surface chemistry

Microwave heating as a novel route for obtaining carbon precursors from anthracene oil

This work describes a novel route for the preparation of pitches by oxidative polymerization of an industrial anthracene oil (AO) in a microwave semi-pilot equipment consisting in a multimode applicator having a 2.45 GHz magnetron with variable microwave power. The experimental five variables of microwave heating of AO air-blowing range between 320-380 ˚C (temperature), 0.2 - 3.9 ˚C min-1 (heating rate), 1.5 - 5 h (soaking time), 16 – 20.5 % (air/AO ratio ) and 200 – 1500 g (initial weight). Their effect on the overall microwave air-blowing process is evaluated by means of a statistical analysis. A detailed characterization of the pitches has been carried out in terms of ultimate analysis, softening point, solubility parameters (toluene insolubles (TI) and quinoline insolubles (QI)) and thermogravimetric analysis. The experiments were also carried out by using conventional heating for comparative purposes. The detailed study of the electric energy consumption of the overall microwave treatment allows estimating a significant electric energy saving of about 20 % when compared to conventional heating thus representing an excellent result in the production of carbon precursors

GPR and ERT detection and characterization of a mass burial, Spanish Civil War, Northern Spain

Around 27,000 people were killed in the province of Asturias during the Spanish Civil War, with several thousands killed after the war ended. There are currently over 2,000 known mass burial locations throughout Spain, but many more are unknown. Geophysics is a useful tool employed to help in the active attempts to document and improve knowledge about victims from this conflict. This paper details a non-invasive study of the Cementerio de El Salvador, in the city of Oviedo, Northern Spain. Part of the cemetery contains a known mass burial with approximately 1,300 individuals from the Spanish Civil War and post-war repression eras. Multi-frequency near-surface geophysical techniques were undertaken, after permission, to enhance knowledge about which, if any, techniques should be used to detect, delineate and analyse such mass graves. Multi-frequency (250 MHz and 500 MHz) ground-penetrating radar surveys were acquired together with 2D and 3D Electrical Resistivity Tomography datasets. The results have established the limits of the mass grave and improve the knowledge of the internal mass grave structure. The paper also shows the importance of considering the climatic conditions during data acquisition. This has important implications for the successful detection of recent historical mass burials using near-surface geophysics

Light regulates motility, attachment and virulence in the plant pathogen Pseudomonas syringae pv tomato DC3000.

Pseudomonas syringae pv tomato DC3000 (Pto) is the causal agent of the bacterial speck of tomato, which leads to significant economic losses in this crop. Pto inhabits the tomato phyllosphere, where the pathogen is highly exposed to light, among other environmental factors. Light represents a stressful condition and acts as a source of information associated with different plant defence levels. Here, we analysed the presence of both blue and red light photoreceptors in a group of Pseudomonas. In addition, we studied the effect of white, blue and red light on Pto features related to epiphytic fitness. While white and blue light inhibit motility, bacterial attachment to plant leaves is promoted. Moreover, these phenotypes are altered in a blue-light receptor mutant. These light-controlled changes during the epiphytic stage cause a reduction in virulence, highlighting the relevance of motility during the entry process to the plant apoplast. This study demonstrated the key role of light perception in the Pto phenotype switching and its effect on virulence

Survival of lichens and bacteria exposed to outer space conditions - Results of the Lithopanspermia experiments

n the space experiments Lithopanspermia, experimental support was provided to the likelihood of the lithopanspermia concept that considers a viable transport of microorganisms between the terrestrial planets by means of meteorites. The rock colonising lichens Rhizocarpon geographicum and Xanthoria elegans, the vagrant lichen Aspicilia fruticulosa, and endolithic and endoevaporitic communities of cyanobacteria and bacteria with their natural rock substrate were exposed to space for 10 days onboard the Biopan facility of the European Space Agency (ESA). Biopan was closed during launch and re-entry. In addition, in the Stone facility, one sample of R. geographicum on its natural granitic substrate was attached at the outer surface of the re-entry capsule close to the stagnation point, only protected by a thin cover of glass textolite. Post-flight analysis, which included determination of the photosynthetic activity, LIVE/DEAD staining, and germination capacity of the ascospores, demonstrated that all three lichen were quite resistant to outer space conditions, which include the full spectrum of solar extraterrestrial electromagnetic radiation or selected wavelength ranges. This high resistance of the lichens to space appears to be due to their symbiotic nature and protection by their upper pigmented layer, the cortex. In contrast, the rock- or halite-inhabiting bacteria were severely damaged by the same exposure. After atmospheric re-entry, the granite of the Stone sample was transformed into a glassy, nearly homogenous material, with several friction striae. None of the lichen cells survived this re-entry process. The data suggest that lichens are suitable candidates for testing the concept of lithopanspermia, because they are extremely resistant to the harsh environment of outer space. The more critical event is the atmospheric re-entry after being captured by a planet. Experiments simulating the re-entry process of a microbe-carrying meteoroid did not show any survivors

Differential Expression of Fungal Genes Determines the Lifestyle of Plectosphaerella Strains During Arabidopsis thaliana Colonization

16 Päg.The fungal genus Plectosphaerella comprises species and strains with different lifestyles on plants, such as P. cucumerina, which has served as model for the characterization of Arabidopsis thaliana basal and nonhost resistance to necrotrophic fungi. We have sequenced, annotated, and compared the genomes and transcriptomes of three Plectosphaerella strains with different lifestyles on A. thaliana, namely, PcBMM, a natural pathogen of wild-type plants (Col-0), Pc2127, a nonpathogenic strain on Col-0 but pathogenic on the immunocompromised cyp79B2 cyp79B3 mutant, and P0831, which was isolated from a natural population of A. thaliana and is shown here to be nonpathogenic and to grow epiphytically on Col-0 and cyp79B2 cyp79B3 plants. The genomes of these Plectosphaerella strains are very similar and do not differ in the number of genes with pathogenesis-related functions, with the exception of secreted carbohydrate-active enzymes (CAZymes), which are up to five times more abundant in the pathogenic strain PcBMM. Analysis of the fungal transcriptomes in inoculated Col-0 and cyp79B2 cyp79B3 plants at initial colonization stages confirm the key role of secreted CAZymes in the necrotrophic interaction, since PcBMM expresses more genes encoding secreted CAZymes than Pc2127 and P0831. We also show that P0831 epiphytic growth on A. thaliana involves the transcription of specific repertoires of fungal genes, which might be necessary for epiphytic growth adaptation. Overall, these results suggest that in-planta expression of specific sets of fungal genes at early stages of colonization determine the diverse lifestyles and pathogenicity of Plectosphaerella strains.This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) grant BIO2015-64077-R and the Spanish Research Agency (AEI) grant RTI2018-096975-B-I00 to A. Molina and by the “Severo Ochoa Programme for Centers of Excellence in R&D” grant SEV-2016-0672 (2017-2021) to the CBGP (UPM-INIA). In the frame of SEV-2016-0672 program, H. Mélida was supported with a postdoctoral contract. A. Muñoz-Barrios was financially supported by the Universidad Politécnica de Madrid (UPM) Ph.D. students PIF program, I. del Hierro was a FPU fellow (Spanish Ministry of Education, Culture and Sports grant FPU16/07118), V. Fernández-Calleja was supported by the Consejería de Educacíon e Investigacíon of Comunidad de Madrid YEI program for postdoctoral researchers (PEJD-2016/BIO-3327), and the work was further supported through a Comunidad de Madrid YEI program for laboratory technicians grant (PEJ16/BIO/TL-1570).Peer reviewe

A male sterility-associated cytotoxic protein ORF288 in Brassica juncea causes aborted pollen development

Cytoplasmic male sterility (CMS) is a widespread phenomenon in higher plants, and several studies have established that this maternally inherited defect is often associated with a mitochondrial mutant. Approximately 10 chimeric genes have been identified as being associated with corresponding CMS systems in the family Brassicaceae, but there is little direct evidence that these genes cause male sterility. In this study, a novel chimeric gene (named orf288) was found to be located downstream of the atp6 gene and co-transcribed with this gene in the hau CMS sterile line. Western blotting analysis showed that this predicted open reading frame (ORF) was translated in the mitochondria of male-sterile plants. Furthermore, the growth of Escherichia coli was significantly repressed in the presence of ORF288, which indicated that this protein is toxic to the E. coli host cells. To confirm further the function of orf288 in male sterility, the gene was fused to a mitochondrial-targeting pre-sequence under the control of the Arabidopsis APETALA3 promoter and introduced into Arabidopsis thaliana. Almost 80% of transgenic plants with orf288 failed to develop anthers. It was also found that the independent expression of orf288 caused male sterility in transgenic plants, even without the transit pre-sequence. Furthermore, transient expression of orf288 and green fluorescent protein (GFP) as a fused protein in A. thaliana protoplasts showed that ORF288 was able to anchor to mitochondria even without the external mitochondrial-targeting peptide. These observations provide important evidence that orf288 is responsible for the male sterility of hau CMS in Brassica juncea