An uncoupling screen for autonomous embryo mutants in Arabidopsis thaliana

Simple de novo screens in Arabidopsis thaliana have previously identified mutants that affect endosperm development but viable-embryo mutants have not been identified. Our strategy to identify autonomous embryo development was to uncouple embryo and endosperm fertilisation. This involved a male-sterile mutant population being crossed with a distinct pollen parent—the pollen was needed to initiate endosperm development and because it was distinct, the maternal progeny could be selected from the hybrid population. This process was refined over three stages, resulting in a viable approach to screen for autonomous embryo mutants. From 8,000 screened plants, a mutation was isolated in which the integument cells extended from the ovule and proliferated into a second complete twinned ovule. Some embryos from the mutant were normal but others developed fused cotyledons. In addition, a proportion of the progeny lacked paternal genes.     

Early signaling through the Arabidopsis pattern recognition receptors FLS2 and EFR involves Ca2+‐associated opening of plasma membrane anion channels

The perception of microbes by plants involves highly conserved molecular signatures that are absent from the host and that are collectively referred to as microbe‐associated molecular patterns (MAMPs). The Arabidopsis pattern recognition receptors FLAGELLIN‐SENSING 2 (FLS2) and EF‐Tu receptor (EFR) represent genetically well studied paradigms that mediate defense against bacterial pathogens. Stimulation of these receptors through their cognate ligands, bacterial flagellin or bacterial elongation factor Tu, leads to a defense response and ultimately to increased resistance. However, little is known about the early signaling pathway of these receptors. Here, we characterize this early response in situ, using an electrophysiological approach. In line with a release of negatively charged molecules, voltage recordings of microelectrode‐impaled mesophyll cells and root hairs of Col‐0 Arabidopsis plants revealed rapid, dose‐dependent membrane potential depolarizations in response to either flg22 or elf18. Using ion‐selective microelectrodes, pronounced anion currents were recorded upon application of flg22 and elf18, indicating that the signaling cascades initiated by each of the two receptors converge on the same plasma membrane ion channels. Combined calcium imaging and electrophysiological measurements revealed that the depolarization was superimposed by an increase in cytosolic calcium that was indispensable for depolarization. NADPH oxidase mutants were still depolarized upon elicitor stimulation, suggesting a reactive oxygen species‐independent membrane potential response. Furthermore, electrical signaling in response to either flg22 or elf 18 critically depends on the activity of the FLS2‐associated receptor‐like kinase BAK1, suggesting that activation of FLS2 and EFR lead to BAK1‐dependent, calcium‐associated plasma membrane anion channel opening as an initial step in the pathogen defense pathway.     

The virtual ecologist approach: simulating data and observers

Ecologists carry a well‐stocked toolbox with a great variety of sampling methods, statistical analyses and modelling tools, and new methods are constantly appearing. Evaluation and optimisation of these methods is crucial to guide methodological choices. Simulating error‐free data or taking high‐quality data to qualify methods is common practice. Here, we emphasise the methodology of the ‘virtual ecologist’ (VE) approach where simulated data and observer models are used to mimic real species and how they are ‘virtually’ observed. This virtual data is then subjected to statistical analyses and modelling, and the results are evaluated against the ‘true’ simulated data. The VE approach is an intuitive and powerful evaluation framework that allows a quality assessment of sampling protocols, analyses and modelling tools. It works under controlled conditions as well as under consideration of confounding factors such as animal movement and biased observer behaviour. In this review, we promote the approach as a rigorous research tool, and demonstrate its capabilities and practical relevance. We explore past uses of VE in different ecological research fields, where it mainly has been used to test and improve sampling regimes as well as for testing and comparing models, for example species distribution models. We discuss its benefits as well as potential limitations, and provide some practical considerations for designing VE studies. Finally, research fields are identified for which the approach could be useful in the future. We conclude that VE could foster the integration of theoretical and empirical work and stimulate work that goes far beyond sampling methods, leading to new questions, theories, and better mechanistic understanding of ecological systems.     

Kohlenstoffpumpen im Ozean steuern das Klima. Globale Erwärmung

Here we describe how microscopic marine algae and animals, the plankton impact global climate and how the changing concentration of the climate relevant gas carbon dioxide (CO₂) result in shifts in the marine carbonate system, including a drop in the pH (ocean acidification). Three processes, summarily called the carbon pumps transfer carbon between the surface and the deep ocean: The solubility pump moves inorganic, dissolved carbon to depth. The biological pump transports organic, particulate carbon downward. The carbonate counter pump describes the formation and sedimentation of carbonate tests, whereby CO₂ is released into the surface ocean. On geological timescales the biological pump was strengthened during glacial times due to an increase in the iron supply, which lead to a (continued) decline in temperatures. Hence, the idea to fertilize the ocean with iron thereby strengthening the biological pump and mediating today's climate change has been discussed for the past 20 years.     

Assignment of canine MSS1 microsatellite markers to chromosomes by linkage data.

Recent advances in mapping the canine genome have led to an increase in the number of linkage studies aimed at dissecting the genetic causes of many hereditary diseases that affect the domestic dog. The first step in developing molecular tools for a whole genome scan was the characterization of a set of microsatellite markers, termed minimal screening set 1 (MSS1), that provided an estimated coverage of 10 cM. A limiting factor in use of the MSS1 is not all of the 172 MSS1 markers have been localized to specific chromosomes. Seventy-five of the markers were positioned on a total of 15 chromosomes with the original publication of the MSS1. The localization based on linkage data of 14 additional MSS1 markers to chromosomes using CRIMAP v. 2.4 to build a linkage map of 113 MSS1 markers that were polymorphic in a kindred of Dalmatians is reported here.     

Association of protein biogenesis factors at the yeast ribosomal tunnel exit is affected by the translational status and nascent polypeptide sequence

Ribosome-associated protein biogenesis factors (RPBs) act during a short but critical period of protein biogenesis. The action of RPBs starts as soon as a nascent polypeptide becomes accessible from the outside of the ribosome and ends upon termination of translation. In yeast, RPBs include the chaperones Ssb1/2 and ribosome-associated complex, signal recognition particle, nascent polypeptide-associated complex (NAC), the aminopeptidases Map1 and Map2, and the Nalpha-terminal acetyltransferase NatA. Here, we provide the first comprehensive analysis of RPB binding at the yeast ribosomal tunnel exit as a function of translational status and polypeptide sequence. We measured the ratios of RPBs to ribosomes in yeast cells and determined RPB occupation of translating and non-translating ribosomes. The combined results imply a requirement for dynamic and coordinated interactions at the tunnel exit. Exclusively, NAC was associated with the majority of ribosomes regardless of their translational status. All other RPBs occupied only ribosomal subpopulations, binding with increased apparent affinity to randomly translating ribosomes as compared with non-translating ones. Analysis of RPB interaction with homogenous ribosome populations engaged in the translation of specific nascent polypeptides revealed that the affinities of Ssb1/2, NAC, and, as expected, signal recognition particle, were influenced by the amino acid sequence of the nascent polypeptide. Complementary cross-linking data suggest that not only affinity of RPBs to the ribosome but also positioning can be influenced in a nascent polypeptide-dependent manner.     

Regulation of hepcidin in HepG2 and RINm5F cells

Despite the high impact of the antimicrobial peptide hepcidin in iron homeostasis, the regulation of this hormone is still not completely understood. Studies concerning hepcidin regulation are performed at the mRNA level. For the first time we analyzed the regulation of hepcidin not only at mRNA, but also at protein level in a hepatoma and a pancreatic beta cell line using quantitative RT-PCR and immunoblot analysis. Our data show, that hepcidin is present in HepG2 and RINm5F cells. A significant up-regulation of hepcidin was observed in both cell lines by the inflammatory cytokine interleukin-6, lipopolysaccharide, and a slight upregulation by deferoxamine. A down-regulation was detected after stimulation with erythropoietin. Hepcidin was regulated by iron in a dose dependent manner: low doses up to 3 microM increased hepcidin expression, high doses of iron (65 microM) revealed a switch-over to down-regulation of hepcidin expression. Regulation of hepcidin in HepG2 and RINm5F cells at mRNA and protein level by these substances indicates its involvement in inflammation and iron metabolism.