Elemental formula annotation of polar and lipophilic metabolites using (13) C, (15) N and (34) S isotope labelling, in combination with high-resolution mass spectrometry

The unbiased and comprehensive analysis of metabolites in any organism presents a major challenge if proper peak annotation and unambiguous assignment of the biological origin of the peaks are required. Here we provide a comprehensive multi-isotope labelling-based strategy using fully labelled (13) C, (15) N and (34) S plant tissues, in combination with a fractionated metabolite extraction protocol. The extraction procedure allows for the simultaneous extraction of polar, semi-polar and hydrophobic metabolites, as well as for the extraction of proteins and starch. After labelling and extraction, the metabolites and lipids were analysed using a high-resolution mass spectrometer providing accurate MS and all-ion fragmentation data, providing an unambiguous readout for every detectable isotope-labelled peak. The isotope labelling assisted peak annotation process employed can be applied in either an automated database-dependent or a database-independent analysis of the plant polar metabolome and lipidome. As a proof of concept, the developed methods and technologies were applied and validated using Arabidopsis thaliana leaf and root extracts. Along with a large repository of assigned elemental compositions, which is provided, we show, using selected examples, the accuracy and reliability of the developed workflow.     

N-CAM exhibits a regulatory function in pathological angiogenesis in oxygen induced retinopathy

Background

Diabetic retinopathy and retinopathy of prematurity are diseases caused by pathological angiogenesis in the retina as a consequence of local hypoxia. The underlying mechanism for epiretinal neovascularization (tuft formation), which contributes to blindness, has yet to be identified. Neural cell adhesion molecule (N-CAM) is expressed by Müller cells and astrocytes, which are in close contact with the retinal vasculature, during normal developmental angiogenesis.

Methodology/Principal Findings

Notably, during oxygen induced retinopathy (OIR) N-CAM accumulated on astrocytes surrounding the epiretinal tufts. Here, we show that N-CAM ablation results in reduced vascular tuft formation due to reduced endothelial cell proliferation despite an elevation in VEGFA mRNA expression, whereas retinal developmental angiogenesis was unaffected.

Conclusion/Significance

We conclude that N-CAM exhibits a regulatory function in pathological angiogenesis in OIR. This is a novel finding that can be of clinical relevance in diseases associated with proliferative vasculopathy.     

In vitro PPFD and media composition affect both in and ex vitro performance of Alstroemeria Butterfly-hybrids

The objective was to reduce in vitro production costs while retaining or improving plant quality, in particular the suitability for pot plant production. Plants were grown at photosynthetic photon flux densities (PPFD) of 0–40 μmol m^-2 s^-1 and sucrose concentrations of 3–7% during the multiplication phase and the effects of sucrose, BA, and NAA during root formation were investigated. Ex vitro growth were tested in both experiments. A small reduction in the rhizome multiplication rate was found with increasing PPFD and sucrose concentration. Increasing sucrose concentration reduced the number of aerial shoots. Aerial shoots were etiolated when cultured in darkness and their number increased with increasing PPFD at 3% sucrose, whereas PPFD did not affect the number of aerial shoots at 5 or 7% sucrose. During the multiplication phase a synergistic promoting effect of PPFD and sucrose was observed on root formation. Root formation after transfer to rooting medium was affected by sucrose and PPFD during the multiplication phase. PPFD did not influence root formation after propagation on 7% sucrose, whereas on 3 or 5 % sucrose root formation was gradually inhibited when PPFD was decreased below 17 μmol m^-2 s^-1. The formation of thick roots was promoted by propagation in light, and not influenced by sucrose concentration. Ex vitro growth was not affected by in vitro conditions, except for 7% sucrose during the multiplication phase that reduced flowering. Root formation on rooting medium was reduced by BA and promoted both by NAA and high levels of sucrose. The root inhibiting effect of BA could not completely be overcome by simultaneous application of NAA and high sucrose concentrations. Thick roots were only produced in the presence of NAA, and not affected by sucrose treatment. Ex vitro flowering was negatively influenced by the presence of BA during root formation and by high levels of sucrose if BA was absent in the rooting medium. High sucrose levels and NAA could partially compensate for the negative effect of BA on flowering. This revised version was published online in June 2006 with corrections to the Cover Date.     

Studying global change through investigation of the plastic responses of xylem anatomy in tree rings

Variability in xylem anatomy is of interest to plant scientists because of the role water transport plays in plant performance and survival. Insights into plant adjustments to changing environmental conditions have mainly been obtained through structural and functional comparative studies between taxa or within taxa on contrasting sites or along environmental gradients. Yet, a gap exists regarding the study of hydraulic adjustments in response to environmental changes over the lifetimes of plants. In trees, dated tree-ring series are often exploited to reconstruct dynamics in ecological conditions, and recent work in which wood-anatomical variables have been used in dendrochronology has produced promising results. Environmental signals identified in water-conducting cells carry novel information reflecting changes in regional conditions and are mostly related to short, sub-annual intervals. Although the idea of investigating environmental signals through wood anatomical time series goes back to the 1960s, it is only recently that low-cost computerized image-analysis systems have enabled increased scientific output in this field. We believe that the study of tree-ring anatomy is emerging as a promising approach in tree biology and climate change research, particularly if complemented by physiological and ecological studies. This contribution presents the rationale, the potential, and the methodological challenges of this innovative approach.     

C2 can do it, too

In this issue of Cell, report that the C2 domain of the serine/threonine protein kinase Cdelta is a phosphotyrosine binding domain and present the crystal structure of this C2 domain bound to a peptide containing phosphotyrosine. Prior to this work, C2 domains were thought to bind only to phospholipids or to unphosphorylated proteins, and the SH2 and PTB domains were the only signaling domains known to recognize phosphotyrosine. This new role for the C2 domain links phosphotyrosine recognition directly to serine/threonine kinase activity and reveals an unexpected mechanism for crosstalk between distinct signaling pathways.     

Dynamics and functional relevance of ammonia-oxidizing archaea in two agricultural soils

Crucial steps in geochemical cycles are in many cases performed by more than one group of microorganisms, but the significance of this functional redundancy with respect to ecosystem functioning is poorly understood. Ammonia-oxidizing archaea (AOA) and their bacterial counterparts (AOB) are a perfect system to address this question: although performing the same transformation step, they belong to well-separated phylogenetic groups. Using pig manure amended with different concentrations of sulfadiazine (SDZ), an antibiotic that is frequently used in veterinary medicine, it was possible to affect AOB and AOA to different degrees. Addition of manure stimulated growth of AOB in both soils and, interestingly, also growth of AOA was considerably stimulated in one of the soils. The antibiotic treatments decreased the manure effect notably on AOB, whereas AOA were affected to a lower extent. Model calculations concerning the respective proportions of AOA and AOB in ammonia oxidation indicate a substantial contribution of AOA in one of the soils that further increased under the influence of SDZ, hence indicating functional redundancy between AOA and AOB.     

Increased Abundance of IncP-1β Plasmids and Mercury Resistance Genes in Mercury-Polluted River Sediments: First Discovery of IncP-1β Plasmids with a Complex mer Transposon as the Sole Accessory Element

Although it is generally assumed that mobile genetic elements facilitate the adaptation of microbial communities to environmental stresses, environmental data supporting this assumption are rare. In this study, river sediment samples taken from two mercury-polluted (A and B) and two nonpolluted or less-polluted (C and D) areas of the river Nura (Kazakhstan) were analyzed by PCR for the presence and abundance of mercury resistance genes and of broad-host-range plasmids. PCR-based detection revealed that mercury pollution corresponded to an increased abundance of mercury resistance genes and of IncP-1β replicon-specific sequences detected in total community DNA. The isolation of IncP-1β plasmids from contaminated sediments was attempted in order to determine whether they carry mercury resistance genes and thus contribute to an adaptation of bacterial populations to Hg pollution. We failed to detect IncP-1β plasmids in the genomic DNA of the cultured Hg-resistant bacterial isolates. However, without selection for mercury resistance, three different IncP-1β plasmids (pTP6, pTP7, and pTP8) were captured directly from contaminated sediment slurry in Cupriavidus necator JMP228 based on their ability to mobilize the IncQ plasmid pIE723. These plasmids hybridized with the merRTΔP probe and conferred Hg resistance to their host. A broad host range and high stability under conditions of nonselective growth were observed for pTP6 and pTP7. The full sequence of plasmid pTP6 was determined and revealed a backbone almost identical to that of the IncP-1β plasmids R751 and pB8. However, this is the first example of an IncP-1β plasmid which had acquired only a mercury resistance transposon but no antibiotic resistance or biodegradation genes. This transposon carries a rather complex set of mer genes and is inserted between Tra1 and Tra2.