Solid-phase synthesis and purification of a set of uniformly 13C, 15N labelled de novo designed membrane fusogenic peptides.

The transmembrane segments of soluble N-ethylmaleimide-sensitive factor (SNARE) proteins or viral envelope proteins drive membrane fusion, which suggests that simple synthetic biology constructs for fusion exist and can be evaluated. We describe the high-yield synthesis of a set of de novo designed fusogenic peptides for use in functional investigations, which are highly enriched in 13C and 15N using three equivalents of labelled amino acids and optimized reaction conditions minimizing aggregation. The biomimetic peptides have a high purity >90% and show reproducible and fusogenic activity that correlates well with the intended functional design characteristics, from strongly fusogenic to almost non-fusogenic.     

A universally applicable process for preparing stoichiometrically 1:1 labelled functional proteins

A universally applicable labelling and purification process was established to prepare biologically active proteins with a stoichiometric 1:1 ratio of attached dye-label. The dye-label is linked to a specific DNA sequence, which acts as a barcode-like tag for affinity purification. The DNA-dye tag is covalently bound to the target protein, which is present in excess to assure the binding of not more than one dye per molecule. Affinity purification occurs at magnetic beads that are functionalized with oligonucleotides that are complementary to the DNA-tag of the labelled proteins but for one or two mismatches. Washing removes all unbound, unlabelled molecules. The labelled protein is subsequently released by the addition of a fully complementary oligonucleotide. This process allows a gentle purification of a protein fraction that has exactly one label attached to each molecule under conditions that preserve protein structure.     

Uptake of ant-derived nitrogen in the myrmecophytic orchid Caularthron bilamellatum

Background and Aims Mutualistic ant-plant associations are common in a variety of plant families. Some myrmecophytic plants, such as the epiphytic orchid Caularthron bilamellatum, actively form hollow structures that provide nesting space for ants (myrmecodomatia), despite a substantial loss of water-storage tissue. This study aimed at assessing the ability of the orchid to take up nitrogen from ant-inhabited domatia as possible trade-off for the sacrifice of potential water storage capacity. Methods Nitrogen uptake capabilities and uptake kinetics of (15)N-labelled compounds (NH(4)(+), urea and l -glutamine) were studied in field-grown Caularthron bilamellatum plants in a tropical moist forest in Panama. Plants were either labelled directly, by injecting substrates into the hollow pseudobulbs or indirectly, by labelling of the associated ants in situ. Key Results Caularthron bilamellatum plants were able to take up all tested inorganic and organic nitrogen forms through the inner surface of the pseudobulbs. Uptake of NH(4)(+) and glutamine followed Michaelis-Menten kinetics, but urea uptake was not saturable up to 2 mm. (15)N-labelled compounds were rapidly translocated and incorporated into vegetative and reproductive structures. By labelling ants with (15)N in situ, we were able to prove that ants transfer N to the plants under field conditions. Conclusions Based on (15)N labelling experiments we were able to demonstrate, for the first time, that a myrmecophytic orchid is capable of actively acquiring different forms of nitrogen from its domatia and that nutrient flux from ants to plants does indeed occur under natural conditions. This suggests that beyond anti-herbivore protection host plants benefit from ants by taking up nitrogen derived from ant debris.     

Changes in spatial point patterns of pioneer woody plants across a large tropical landslide

We assessed whether the relative importance of positive and negative interactions in early successional communities varied across a large landslide on Casita Volcano (Nicaragua). We tested several hypotheses concerning the signatures of these processes in the spatial patterns of woody pioneer plants, as well as those of mortality and recruitment events, in several zones of the landslide differing in substrate stability and fertility, over a period of two years (2001 and 2002). We identified all woody individuals with a diameter >1 cm and mapped them in 28 plots measuring 10 × 10-m. On these maps, we performed a spatial point pattern analysis using univariate and bivariate pair-correlation functions; g (r) and g₁₂ (r), and pairwise differences of univariate and bivariate functions. Spatial signatures of positive and negative interactions among woody plants were more prevalent in the most and least stressful zones of the landslide, respectively. Natural and human-induced disturbances such as the occurrence of fire, removal of newly colonizing plants through erosion and clearcutting of pioneer trees were also identified as potentially important pattern-creating processes. These results are in agreement with the stress-gradient hypothesis, which states that the relative importance of facilitation and competition varies inversely across gradients of abiotic stress. Our findings also indicate that the assembly of early successional plant communities in large heterogeneous landslides might be driven by a much larger array of processes than previously thought.     

Characterization of 27 microsatellite loci in the European flat oyster Ostrea edulis

The flat oyster Ostrea edulis is native to Europe and populations have been severely depleted by the parasite Bonamia ostreae since the 1980s. Additional genetic markers are required to improve population genetics study and linkage map development for selection for B. ostrea-resistance in this species. Here, we characterized 27 novel microsatellite loci for O. edulis. Number of alleles per locus ranged from 6 to 25 and observed heterozygosity between 0.375 and 1. Null alleles were suggested at a few loci but most loci were in Hardy-Weinberg agreement enabling their reliable use in further population and mapping genetics approaches.     

The host plant Pinus pinaster exerts specific effects on phosphate efflux and polyphosphate metabolism of the ectomycorrhizal fungus Hebeloma cylindrosporum: a radiotracer,cytological staining and 31P NMR spectroscopy study

Ectomycorrhizal (ECM) association can improve plant phosphorus (P) nutrition. Polyphosphates (polyP) synthesized in distant fungal cells after P uptake may contribute to P supply from the fungus to the host plant if they are hydrolyzed to phosphate in ECM roots then transferred to the host plant when required. In this study, we addressed this hypothesis for the ECM fungus Hebeloma cylindrosporum grown in vitro and incubated without plant or with host (Pinus pinaster) and non‐host (Zea mays) plants, using an experimental system simulating the symbiotic interface. We used ³²P labelling to quantify P accumulation and P efflux and in vivo and in vitro nuclear magnetic resonance (NMR) spectroscopy and cytological staining to follow the fate of fungal polyP. Phosphate supply triggered a massive P accumulation as newly synthesized long‐chain polyP in H. cylindrosporum if previously grown under P‐deficient conditions. P efflux from H. cylindrosporum towards the roots was stimulated by both host and non‐host plants. However, the host plant enhanced ³²P release compared with the non‐host plant and specifically increased the proportion of short‐chain polyP in the interacting mycelia. These results support the existence of specific host plant effects on fungal P metabolism able to provide P in the apoplast of ectomycorrhizal roots.     

Temporal development of the barley leaf metabolic response to Pi limitation

The response of plants to P_i limitation involves interplay between root uptake of P_i, adjustment of resource allocation to different plant organs and increased metabolic P_i use efficiency. To identify potentially novel, early‐responding, metabolic hallmarks of P_i limitation in crop plants, we studied the metabolic response of barley leaves over the first 7 d of P_i stress, and the relationship of primary metabolites with leaf P_i levels and leaf biomass. The abundance of leaf P_i, Tyr and shikimate were significantly different between low Pi and control plants 1 h after transfer of the plants to low P_i. Combining these data with ¹⁵N metabolic labelling, we show that over the first 48 h of P_i limitation, metabolic flux through the N assimilation and aromatic amino acid pathways is increased. We propose that together with a shift in amino acid metabolism in the chloroplast a transient restoration of the energetic and redox state of the leaf is achieved. Correlation analysis of metabolite abundances revealed a central role for major amino acids in P_i stress, appearing to modulate partitioning of soluble sugars between amino acid and carboxylate synthesis, thereby limiting leaf biomass accumulation when external P_i is low.