Sexual reproduction, clonal diversity and genetic differentiation in patchily distributed populations of the temperate forest herb Paris quadrifolia (Trilliaceae)

Clonal plant species have been shown to adopt different strategies to persist in heterogeneous environments by changing relative investments in sexual reproduction and clonal propagation. As a result, clonal diversity and genetic variation may be different along environmental gradients. We examined the regional and local population structure of the clonal rhizomatous forest herb Paris quadrifolia in a complex of forest fragments in Voeren (Belgium). Relationships between population size (the number of shoots), shoot density (the number of shoots per m²) and local growth conditions were investigated for 47 populations. Clonal diversity and genetic variation within and among 19 populations were investigated using amplified fragment length polymorphism markers. To assess the importance of sexual reproduction, seed set, seed weight and germination success were determined in 18 populations. As predicted, local growth conditions largely affected population distribution, size and density of P. quadrifolia. Populations occurring in moist and relatively productive sites contained significantly more shoots. Here, shoots were also much more sparsely distributed compared to populations occurring in dry and relatively unproductive sites, where shoots showed a strongly aggregated distribution pattern. Clonal diversity was relatively high, compared with other clonal species (G/N ratio = 0.43 and Simpson’s D=0.81). Clonal diversity significantly (P<0.01) decreased with increasing shoot density while molecular genetic variation was significantly (P<0.01) affected by population size and local environmental conditions. Lack of recruitment and out-competition of less-adapted genotypes may explain the decreased genetic variation in dry sites. Analysis of molecular variance revealed significant genetic variation among populations (Φ _ST=0.42, P<0.001), whereas pairwise genetic distances were not correlated to geographic distances, suggesting that gene flow among populations is limited. Finally, the number of generative shoots, the number of seeds per fruit and seed weight were significantly and positively related to population size and local growth conditions. We conclude that under stressful conditions populations of clonal forest plant species can slowly evolve into remnant populations characterized by low levels of genetic variation and limited sexual reproduction. Conservation of suitable habitat conditions is therefore a prerequisite for effective long-term conservation of clonal forest plant species.     

Leaf yellowing and anthocyanin accumulation are two genetically independent strategies in response to nitrogen limitation in Arabidopsis thaliana

For the first time in Arabidopsis thaliana, this work proposes the identification of quantitative trait loci (QTLs) associated with leaf senescence and stress response symptoms such as yellowing and anthocyanin-associated redness. When Arabidopsis plants were cultivated under low nitrogen conditions, we observed that both yellowing of the old leaves of the rosette and whole rosette redness were promoted. Leaf yellowing is a senescence symptom related to chlorophyll breakdown. Redness is a symptom of anthocyanin accumulation related to whole plant ageing and nutrient limitation. In this work, Arabidopsis is used as a model system to dissect the genetic variation of these parameters by QTL mapping in the 415 recombinant inbred lines of the Bay-0xShahdara population. Fifteen new QTLs and two epistatic interactions were described in this study. The yellowing of the rosette, estimated by visual notation and image processing, was controlled by four and five QTLs, respectively. The visual estimation of redness allowed us to detect six QTLs among which the major one explained 33% of the total variation. Two main QTLs were confirmed in near-isogenic lines (heterogenous inbred family; HIF), thus confirming the relevance of the visual notation of these traits. Co-localizations between QTLs for leaf yellowing, redness and nitrogen use efficiency described in a previous publication indicate complex interconnected pathways involved in both nitrogen management and senescence- and stress-related processes. No co-localization between QTLs for leaf yellowing and redness has been found, suggesting that the two characters are genetically independent.     

Fine-scale genetic structure and gene dispersal inferences in 10 neotropical tree species

The extent of gene dispersal is a fundamental factor of the population and evolutionary dynamics of tropical tree species, but directly monitoring seed and pollen movement is a difficult task. However, indirect estimates of historical gene dispersal can be obtained from the fine-scale spatial genetic structure of populations at drift-dispersal equilibrium. Using an approach that is based on the slope of the regression of pairwise kinship coefficients on spatial distance and estimates of the effective population density, we compare indirect gene dispersal estimates of sympatric populations of 10 tropical tree species. We re-analysed 26 data sets consisting of mapped allozyme, SSR (simple sequence repeat), RAPD (random amplified polymorphic DNA) or AFLP (amplified fragment length polymorphism) genotypes from two rainforest sites in French Guiana. Gene dispersal estimates were obtained for at least one marker in each species, although the estimation procedure failed under insufficient marker polymorphism, limited sample size, or inappropriate sampling area. Estimates generally suffered low precision and were affected by assumptions regarding the effective population density. Averaging estimates over data sets, the extent of gene dispersal ranged from 150 m to 1200 m according to species. Smaller gene dispersal estimates were obtained in species with heavy diaspores, which are presumably not well dispersed, and in populations with high local adult density. We suggest that limited seed dispersal could indirectly limit effective pollen dispersal by creating higher local tree densities, thereby increasing the positive correlation between pollen and seed dispersal distances. We discuss the potential and limitations of our indirect estimation procedure and suggest guidelines for future studies.     

The impact of mechanical compression on cortical microtubules in Arabidopsis: a quantitative pipeline

Exogenous mechanical perturbations on living tissues are commonly used to investigate whether cell effectors can respond to mechanical cues. However, in most of these experiments, the applied mechanical stress and/or the biological response are described only qualitatively. We developed a quantitative pipeline based on microindentation and image analysis to investigate the impact of a controlled and prolonged compression on microtubule behaviour in the Arabidopsis shoot apical meristem, using microtubule fluorescent marker lines. We found that a compressive stress, in the order of magnitude of turgor pressure, induced apparent microtubule bundling. Importantly, that response could be reversed several hours after the release of compression. Next, we tested the contribution of microtubule severing to compression‐induced bundling: microtubule bundling seemed less pronounced in the katanin mutant, in which microtubule severing is dramatically reduced. Conversely, some microtubule bundles could still be observed 16 h after the release of compression in the spiral2 mutant, in which severing rate is instead increased. To quantify the impact of mechanical stress on anisotropy and orientation of microtubule arrays, we used the nematic tensor based FibrilTool ImageJ/Fiji plugin. To assess the degree of apparent bundling of the network, we developed several methods, some of which were borrowed from geostatistics. The final microtubule bundling response could notably be related to tissue growth velocity that was recorded by the indenter during compression. Because both input and output are quantified, this pipeline is an initial step towards correlating more precisely the cytoskeleton response to mechanical stress in living tissues.     

Structure Determination of Feline Calicivirus Virus-Like Particles in the Context of a Pseudo-Octahedral Arrangement

The vesivirus feline calicivirus (FCV) is a positive strand RNA virus encapsidated by an icosahedral T=3 shell formed by the viral VP1 protein. Upon its expression in the insect cell - baculovirus system in the context of vaccine development, two types of virus-like particles (VLPs) were formed, a majority built of 60 subunits (T=1) and a minority probably built of 180 subunits (T=3). The structure of the small particles was determined by x-ray crystallography at 0.8 nm resolution helped by cryo-electron microscopy in order to understand their formation. Cubic crystals belonged to space group P2₁3. Their self-rotation function showed the presence of an octahedral pseudo-symmetry similar to the one described previously by Agerbandje and co-workers for human parvovirus VLPs. The crystal structure could be solved starting from the published VP1 structure in the context of the T=3 viral capsid. In contrast to viral capsids, where the capsomers are interlocked by the exchange of the N-terminal arm (NTA) domain, this domain is disordered in the T=1 capsid of the VLPs. Furthermore it is prone to proteolytic cleavage. The relative orientation of P (protrusion) and S (shell) domains is alerted so as to fit VP1 to the smaller T=1 particle whereas the intermolecular contacts around 2-fold, 3-fold and 5-fold axes are conserved. By consequence the surface of the VLP is very similar compared to the viral capsid and suggests a similar antigenicity. The knowledge of the structure of the VLPs will help to improve their stability, in respect to a use for vaccination.     

Genetic portrait of Lisboa immigrant population from Cabo Verde with mitochondrial DNA analysis

Journal of Genetics -     

Cardiac effects of postconditioning depend critically on the duration of index ischemia

Postconditioning (POC) is known as the phenomenon whereby brief intermittent ischemia applied at the onset of reperfusion following index ischemia limits myocardial infarct size. Whereas there is evidence that the algorithm of the POC stimulus is an important determinant of the protective efficacy, the importance of the duration of index ischemia on the outcome of the effects of POC has received little attention. Pentobarbital sodium-anesthetized Wistar rats were therefore subjected to index ischemia produced by coronary artery occlusions (CAO) of varying duration (15-120 min) followed by reperfusion, without or with postconditioning produced by three cycles of 30-s reperfusion and reocclusion (3POC30). 3POC30 limited infarct size produced by 45-min CAO (CAO45) from 45 +/- 3% to 31 +/- 5%, and CAO60 from 60 +/- 3% to 47 +/- 6% (both P < or = 0.05). In contrast, 3POC30 increased infarct size produced by CAO15 from 3 +/- 1% to 19 +/- 6% and CAO30 from 36 +/- 6 to 48 +/- 4% (both P < or = 0.05). This deleterious effect of 3POC30 was not stimulus sensitive because postconditioning with 3POC5 and 3POC15 after CAO30 also increased infarct size. The cardioprotection by 3POC30 after CAO60 was accompanied by an increased stimulation of Akt phosphorylation at 7 min of reperfusion and a 36% lower superoxide production, measured by dihydroethidium fluorescence, after 2 h of reperfusion. Consistent with these results, cardioprotection by 3POC30 was abolished by phosphatidylinositol-3-OH-kinase inhibition, as well as nitric oxide (NO) synthase inhibition. The deleterious effect of 3POC30 after CAO15 was accompanied by an increased superoxide production with no change in Akt phosphorylation and was not affected by NO synthase inhibition. In conclusion, the effect of cardiac POC depends critically on the duration of the index ischemia and can be either beneficial or detrimental. These paradoxical effects of POC may be related to the divergent effects on Akt phosphorylation and superoxide production.     

New noncovalent inhibitors of penicillin-binding proteins from penicillin-resistant bacteria

Background

Penicillin-binding proteins (PBPs) are well known and validated targets for antibacterial therapy. The most important clinically used inhibitors of PBPs β-lactams inhibit transpeptidase activity of PBPs by forming a covalent penicilloyl-enzyme complex that blocks the normal transpeptidation reaction; this finally results in bacterial death. In some resistant bacteria the resistance is acquired by active-site distortion of PBPs, which lowers their acylation efficiency for β-lactams. To address this problem we focused our attention to discovery of novel noncovalent inhibitors of PBPs.

Methodology/Principal Findings

Our in-house bank of compounds was screened for inhibition of three PBPs from resistant bacteria: PBP2a from Methicillin-resistant Staphylococcus aureus (MRSA), PBP2x from Streptococcus pneumoniae strain 5204, and PBP5fm from Enterococcus faecium strain D63r. Initial hit inhibitor obtained by screening was then used as a starting point for computational similarity searching for structurally related compounds and several new noncovalent inhibitors were discovered. Two compounds had promising inhibitory activities of both PBP2a and PBP2x 5204, and good in-vitro antibacterial activities against a panel of Gram-positive bacterial strains.

Conclusions

We found new noncovalent inhibitors of PBPs which represent important starting points for development of more potent inhibitors of PBPs that can target penicillin-resistant bacteria.