An approach to the knowledge of pollen and allergen diversity through lipid transfer protein localisation in taxonomically distant pollen grains

The aim of the present study is to localise non‐specific lipid transfer proteins (nsLTPs), obtained from Rosaceae (Prunus persica (L.) Batsch) in pollen grains of taxonomically distant plants, such as Iridaceae (Aristea latifolia G.J. Lewis), Platanaceae (Platanus acerifolia (Aiton) Willd.) and Urticaceae (Parietaria judaica L.), in order to compare pollen and nsLTP diversity. A combination of transmission electron microscopy, immunocytochemical techniques, and rabbit specific antiserum against peach nsLTPs (Pru p 3) were used. Abundant labelling to Pru p 3‐like proteins was observed in the cytoplasm, walls and pollenkitt of A. latifolia pollen grains. The presence of nsLTPs associated with the pollenkitt proves that it takes part in the defence mechanism of pollen grains. The labelling was less intense in the cytoplasm and walls of P. acerifolia. Immuno‐stained gold particles were associated with the vacuoles, lipid inclusions, endoplasmic reticulum and Golgi complex. No significant labelling was found in the P. judaica pollen grains incubated with anti‐Pru p 3 polyclonal antibodies. These results indicate important variations in the nsLTPs of different pollen species. Consequently, no taxonomic relationship between pollen grains and nsLTPs could be established.     

Validation of high-resolution DNA melting analysis for mutation scanning of the CDKL5 gene: Identification of novel mutations

Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) have been predominantly described in epileptic encephalopathies of female, including infantile spasms with Rett-like features. Up to now, detection of mutations in this gene was made by laborious, expensive and/or time consuming methods. Here, we decided to validate high-resolution melting analysis (HRMA) for mutation scanning of the CDKL5 gene. Firstly, using a large DNA bank consisting to 34 samples carrying different mutations and polymorphisms, we validated our analytical conditions to analyse the different exons and flanking intronic sequences of the CDKL5 gene by HRMA. Secondly, we screened CDKL5 by both HRMA and denaturing high performance liquid chromatography (dHPLC) in a cohort of 135 patients with early-onset seizures. Our results showed that point mutations and small insertions and deletions can be reliably detected by HRMA. Compared to dHPLC, HRMA profiles are more discriminated, thereby decreasing unnecessary sequencing. In this study, we identified eleven novel sequence variations including four pathogenic mutations (2.96% prevalence). HRMA appears cost-effective, easy to set up, highly sensitive, non-toxic and rapid for mutation screening, ideally suited for large genes with heterogeneous mutations located along the whole coding sequence, such as the CDKL5 gene.     

Biotrophic transportome in mutualistic plant–fungal interactions

Understanding the mechanisms that underlie nutrient use efficiency and carbon allocation along with mycorrhizal interactions is critical for managing croplands and forests soundly. Indeed, nutrient availability, uptake and exchange in biotrophic interactions drive plant growth and modulate biomass allocation. These parameters are crucial for plant yield, a major issue in the context of high biomass production. Transport processes across the polarized membrane interfaces are of major importance in the functioning of the established mycorrhizal association as the symbiotic relationship is based on a ‘fair trade’ between the fungus and the host plant. Nutrient and/or metabolite uptake and exchanges, at biotrophic interfaces, are controlled by membrane transporters whose regulation patterns are essential for determining the outcome of plant–fungus interactions and adapting to changes in soil nutrient quantity and/or quality. In the present review, we summarize the current state of the art regarding transport systems in the two major forms of mycorrhiza, namely ecto- and arbuscular mycorrhiza.     

Resolution of Der p1-induced allergic airway inflammation is dependent on CD4+CD25+Foxp3+ regulatory cells

Allergic airway inflammation (AAI) is characterized by airway hyperreactivity, eosinophilia, goblet cell hyperplasia, and elevated serum IgE, however, it is unclear what mediates natural resolution after cessation of allergen exposure. This is important because the outcome of subsequent allergen challenge may depend on the concurrent inflammatory milieu of the lung. Using a murine AAI model, we demonstrate that after exposure to a defined natural protein allergen, Der p1, the response in lungs and draining mediastinal lymph nodes (dMLN) peaks between 4 and 6 days then declines until resolution by 21 days. Der p1-specific serum IgE follows the same pattern while IgG1 continues to increase. Resolution of AAI is mediated by CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs), which appear in lungs and dMLN following airway challenge. Treg depletion exacerbated lung eosinophilia, increased dMLN IL-5 and IL-13 but not IL-10 secretion, and increased allergic Ab responses. Most convincingly, transfer of CD4(+)CD25(+)Foxp3(+) T cells from Ag naive mice (natural Tregs) abolished AAI, decreased dMLN IL-5 and IL-13 secretion, increased dMLN IL-10 secretion, abolished IgE, and decreased IgG1 Abs. Blocking IL-10 receptor function in vivo did not block the anti-inflammatory function of transferred natural Tregs but did restore dMLN IL-5 and IL-13 secretion. Thus natural Tregs can control AAI in an IL-10 independent manner.     

Alfalfa response to elevated atmospheric CO2 varies with the symbiotic rhizobial strain

Increased atmospheric CO₂ was shown to affect a variety of physiological processes in plants, including photosynthesis and growth with repercussions on crop yield and nutritive value. Perennial alfalfa (Medicago sativa L.) is a sustainable crop with a deep root system, living in symbiosis with rhizobium for nitrogen (N) fixation. The objective of the project was to determine the combined effects of elevated CO₂ and rhizobial strains on photosynthesis, growth, N fixation, and nutritive value of alfalfa, and on soil microflora. Alfalfa inoculated with two different strains of rhizobia (Sinorhizobium meliloti strains A2 and NRG34) was grown 2 months at day/night temperatures of 22/17°C under either 400 (near ambient) or 800 (elevated) μmol mol⁻¹ of CO₂. The photosynthetic response of alfalfa to elevated CO₂ differed according to the rhizobial strain. At the end of the experiment, elevated CO₂ stimulated photosynthetic rates by 50% in plants associated with A2 but there was no significant increase in plants nodulated with NRG34. Nitrogenase activity (+38%) and shoot growth (+60%) were stimulated under 800 μmol mol⁻¹ of CO₂ for alfalfa inoculated with both strains. Root dry weight was significantly higher at 800 μmol mol⁻¹ of CO₂ only with strain A2. Fibre concentration decreased in response to elevated atmospheric CO₂ in alfalfa inoculated with strain A2 resulting in plant material with greater nutritive value when inoculated with A2 compared to NRG34. In the soil, elevated CO₂ increased the proportion of fungi in the microbial community while decreasing Gram⁻ bacteria. For alfalfa inoculated with rhizobial strain A2, photosynthetic rates, nitrogenase activity, and growth were all stimulated by increased atmospheric CO₂ compared to less consistently positive responses to elevated CO₂ when inoculated with NRG34. Our results show that it is possible to identify rhizobial strains to improve plant performance under predicted future CO₂ concentrations with no negative effect on nutritive value. The Canadian Government’s right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Building blocks for plant gene assembly

The MultiSite Gateway cloning system, based on site-specific recombination, enables the assembly of multiple DNA fragments in predefined order, orientation, and frame register. To streamline the construction of recombinant genes for functional analysis in plants, we have built a collection of 36 reference Gateway entry clones carrying promoters, terminators, and reporter genes, as well as elements of the LhG4/LhGR two-component system. This collection obeys simple engineering rules. The genetic elements (parts) are designed in a standard format. They are interchangeable, fully documented, and can be combined at will according to the desired output. We also took advantage of the MultiSite Gateway recombination sites to create vectors in which two or three genes can be cloned simultaneously in separate expression cassettes. To illustrate the flexibility of these core resources for the construction of a wide variety of plant transformation vectors, we generated various transgenes encoding fluorescent proteins and tested their activity in plant cells. The structure and sequence of all described plasmids are accessible online at http://www.psb.ugent.be/gateway/. All accessions can be requested via the same Web site.     

Recombinant expression and characterization of a reducing-end xylose-releasing exo-oligoxylanase from Bifidobacterium adolescentis

The family 8 glycoside hydrolase (RexA) from Bifidobacterium adolescentis was expressed in Escherichia coli. The recombinant enzyme was characterized as a reducing-end xylose-releasing exo-oligoxylanase. Apart from giving insights into this new class of enzymes, knowledge of the RexA enzyme helps to postulate a mechanism for the B. adolescentis breakdown of prebiotic xylooligosaccharides.