A bHLH Complex Controls Embryonic Vascular Tissue Establishment and Indeterminate Growth in Arabidopsis

Plants have a remarkable potential for sustained (indeterminate) postembryonic growth. Following their specification in the early embryo, tissue-specific precursor cells first establish tissues and later maintain them postembryonically. The mechanisms underlying these processes are largely unknown. Here we define local control of oriented, periclinal cell division as the mechanism underlying both the establishment and maintenance of vascular tissue. We identify an auxin-regulated basic helix-loop-helix (bHLH) transcription factor dimer as a critical regulator of vascular development. Due to a loss of periclinal divisions, vascular tissue gradually disappears in bHLH-deficient mutants; conversely, ectopic expression is sufficient for triggering periclinal divisions. We show that this dimer operates independently of tissue identity but is restricted to a small vascular domain by integrating overlapping transcription patterns of the interacting bHLH proteins. Our work reveals a common mechanism for tissue establishment and indeterminate vascular development and provides a conceptual framework for developmental control of local cell divisions.

Determinants of plant establishment success in a multispecies introduction experiment with native and alien species

Determinants of plant establishment and invasion are a key issue in ecology and evolution. Although establishment success varies substantially among species, the importance of species traits and extrinsic factors as determinants of establishment in existing communities has remained difficult to prove in observational studies because they can be confounded and mask each other. Therefore, we conducted a large multispecies field experiment to disentangle the relative importance of extrinsic factors vs. species characteristics for the establishment success of plants in grasslands. We introduced 48 alien and 45 native plant species at different seed numbers into multiple grassland sites with or without experimental soil disturbance and related their establishment success to species traits assessed in five independent multispecies greenhouse experiments. High propagule pressure and high seed mass were the most important factors increasing establishment success in the very beginning of the experiment. However, after 3 y, propagule pressure became less important, and species traits related to biotic interactions (including herbivore resistance and responses to shading and competition) became the most important drivers of success or failure. The relative importance of different traits was environment-dependent and changed over time. Our approach of combining a multispecies introduction experiment in the field with trait data from independent multispecies experiments in the greenhouse allowed us to detect the relative importance of species traits for early establishment and provided evidence that species traits—fine-tuned by environmental factors—determine success or failure of alien and native plants in temperate grasslands.

Modeling Framework for the Establishment of the Apical-Basal Embryonic Axis in Plants

The apical-basal axis of the early plant embryo determines the body plan of the adult organism. To establish a polarized embryonic axis, plants evolved a unique mechanism that involves directional, cell-to-cell transport of the growth regulator auxin. Auxin transport relies on PIN auxin transporters 1], whose polar subcellular localization determines the flow directionality. PIN-mediated auxin transport mediates the spatial and temporal activity of the auxin response machinery 2-7] that contributes to embryo patterning processes, including establishment of the apical (shoot) and basal (root) embryo poles 8]. However, little is known of upstream mechanisms guiding the (re)polarization of auxin fluxes during embryogenesis 9]. Here, we developed a model of plant embryogenesis that correctly generates emergent cell polarities and auxin-mediated sequential initiation of apical-basal axis of plant embryo. The model relies on two precisely localized auxin sources and a feedback between auxin and the polar, subcellular PIN transporter localization. Simulations reproduced PIN polarity and auxin distribution, as well as previously unknown polarization events during early embryogenesis. The spectrum of validated model predictions suggests that our model corresponds to a minimal mechanistic framework for initiation and orientation of the apical-basal axis to guide both embryonic and postembryonic plant development.

Design and establishment of a biobank in a multicenter prospective cohort study of elderly patients with venous thromboembolism (SWITCO65+)

In the field of thrombosis and haemostasis, many preanalytical variables influence the results of coagulation assays and measures to limit potential results variations should be taken. To our knowledge, no paper describing the development and maintenance of a haemostasis biobank has been previously published. Our description of the biobank of the Swiss cohort of elderly patients with venous thromboembolism (SWITCO65+) is intended to facilitate the set-up of other biobanks in the field of thrombosis and haemostasis. SWITCO65+ is a multicentre cohort that prospectively enrolled consecutive patients aged ≥65 years with venous thromboembolism at nine Swiss hospitals from 09/2009 to 03/2012. Patients will be followed up until December 2013. The cohort includes a biobank with biological material from each participant taken at baseline and after 12 months of follow-up. Whole blood from all participants is assayed with a standard haematology panel, for which fresh samples are required. Two buffy coat vials, one PAXgene Blood RNA System tube and one EDTA-whole blood sample are also collected at baseline for RNA/DNA extraction. Blood samples are processed and vialed within 1 h of collection and transported in batches to a central laboratory where they are stored in ultra-low temperature archives. All analyses of the same type are performed in the same laboratory in batches. Using multiple core laboratories increased the speed of sample analyses and reduced storage time. After recruiting, processing and analyzing the blood of more than 1,000 patients, we determined that the adopted methods and technologies were fit-for-purpose and robust.

Does low phosphorus supply limit seedling establishment and tree growth in groves of ectomycorrhizal trees in a central African rainforest?

In a forest grove at Korup dominated by the ectomycorrhizal species Microberlinia bisulcata, an experiment tested whether phosphorus (P) was a limiting nutrient. P-fertilization of seven subplots 1995-97 was compared with seven controls. It led to large increases in soil P concentrations. Trees were measured in 1995 and 2000. M. bisulcata and four other species were transplanted into the treatments, and a wild cohort of M. bisulcata seedlings was followed in both. Leaf litter fall from trees and seedlings were analysed for nutrients. Growth of trees was not affected by added P. Transplanted seedlings survived better in the controls than added-P subplots: they did not grow better with added-P.M. bisulcata wildlings survived slightly better in the added-P subplots in yr 1 but not later. Litter fall and transplanted survivors had much higher concentrations of P (not N) in the added-P than control subplots. Under current conditions, it appears that P does not limit growth of trees or hinder seedling establishment, especially of M. bisculcata, in these low-P grove soils.

Endozoochory by slugs can increase bryophyte establishment and species richness

Herbivory can affect plant community composition and diversity by removing biomass and reducing light competition. Herbivory may particularly benefit low growing species such as bryophytes, which are frequently limited by light competition. Gastropods are important herbivores of seed plants and cryptogams, furthermore, they can disperse propagules such as seeds and spores via endozoochory. However, whether gastropod herbivory can reduce the dominance of vascular plants and thereby promote the germination and establishment of endozoochorously dispersed bryophyte spores has never been tested experimentally. Moreover, it is unclear whether these possible interacting effects can influence bryophyte species richness. Here, we tested for endozoochorous spore dispersal by slugs, in combination with sowing of vascular plants, in a fully factorial common garden experiment. Enclosures contained either slugs previously fed with bryophyte sporophytes, control slugs, or no slugs. After 21 days the bryophyte cover was on average 2.8 times higher (3.9 versus 1.4) and after eight months the bryophyte species richness 2.6 times higher (5.8 versus 2.2) in enclosures containing slugs previously fed with bryophyte sporophytes than in the other treatments. Furthermore, after eight months high vascular plant cover reduced bryophyte diversity. On average enclosures without seed sowing harboured 1.6 times more bryophyte species than the ones with seed sowing (4.2 versus 2.6), indicating competitive effects of vascular plants on bryophytes. Our findings suggest that slugs are important dispersal vectors for bryophytes and that they can increase bryophyte populations and maintain bryophyte diversity by reducing the dominance of vascular plants.

Hybrid ‘superswarm’ leads to rapid divergence and establishment of populations during a biological invasion

Understanding the genetic background of invading species can be crucial information clarifying why they become invasive. Intraspecific genetic admixture among lineages separated in the native ranges may promote the rate and extent of an invasion by substantially increasing standing genetic variation. Here we examine the genetic relationships among threespine stickleback that recently colonized Switzerland. This invasion results from several distinct genetic lineages that colonized multiple locations and have since undergone range expansions, where they coexist and admix in parts of their range. Using 17 microsatellites genotyped for 634 individuals collected from 17 Swiss and two non-Swiss European sites, we reconstruct the invasion of stickleback and investigate the potential and extent of admixture and hybridization among the colonizing lineages from a population genetic perspective. Specifically we test for an increase in standing genetic variation in populations where multiple lineages coexist. We find strong evidence of massive hybridization early on, followed by what appears to be recent increased genetic isolation and the formation of several new genetically distinguishable populations, consistent with a hybrid ‘superswarm’. This massive hybridization and population formation event(s) occurred over approximately 140 years and likely fuelled the successful invasion of a diverse range of habitats. The implications are that multiple colonizations coupled with hybridization can lead to the formation of new stable genetic populations potentially kick-starting speciation and adaptive radiation over a very short time.

Light and seed size affect establishment of grove-forming ectomycorrhizal rain forest tree species

• Regeneration of the dominant ectomycorrhizal tree Microberlinia bisulcata in groves in Korup, Central Africa, is very poor. The hypothesis was tested that this species is more shade intolerant than other co-occurring species. • In two 1-yr trials, each with M. bisulcata and four other species at a nursery close to Korup, growth was measured under five PAR levels, with ± added P and ± watering in the dry season. In parallel experiments the effects of PAR with two R : FR ratios were investigated. • Increasing PAR had a consistent effect on the rates of increase in plant mass and on changes in the other variables. Doubling soil P, watering and halving the R : FR ratio had almost no effect. However, across species, mass at low PAR and relative growth rate related positively and negatively, respectively, to seed mass. • One contributing factor for the poor recruitment of M. bisulcata is therefore its low survival and slow growth at low PAR, due to its small seed size. The two codominant ectomycorrhizal grove species of Tetraberlinia, with larger seeds, were less affected by low PAR.

Does proximity to conspecific adults influence the establishment of ectomycorrhizal trees in rain forest?

Three ectomycorrhizal legume trees, Microberlinia bisulcata, Tetraberlinia bifoliolata and T. moreliana, form discrete groves in the southern part of Korup National Park, in southwest Cameroon and contribute c. 45–70% of stand basal area locally in a matrix of otherwise species-rich arbuscular mycorrhizal forest. A transplant experiment was performed to assess the importance of ectomycorrhizal infection associated with proximity to parents in seedling establishment of the grove-forming species. Nonectomycorrhizal seedlings of the three species were transplanted into plots of two forest types, one of high (HEM, within-grove) and one of very low (LEM, outside the grove) abundance of all three species as adult trees. For two species (T. moreliana and M. bisulcata) there was no difference in survival over 16 months, but for the third (T. bifoliolata) survival was best in HEM forest, and correlated with the basal area of adult trees of ectomycorrhizal species. Only one species (T. moreliana) increased in biomass over the experimental period; the others declined. There was no effect of forest type on overall growth of any species, but the survivors of two (T. moreliana and M. bisulcata) had heavier stems in the HEM forest. Differences in survival and growth of transplants between the three species were in accord with the ecology of the species as inferred from the frequency distributions of adult tree size in the forest. Seedlings became infected with ectomycorrhizas in both forest types; where there was a difference in extent of infection (T. moreliana) this was not related to survival or growth; and where there was a difference in survival (T. bifoliolata) this was not related to extent of infection. These results confirm that mycorrhizal inoculum associated with conspecific adults is neither a prerequisite nor a guarantee of seedling establishment, but indicates that in some circumstances there might be benefits of being close to parents. Further research is required to unravel the complexities of ectomycorrhizal community structure in this spatially and temporally heterogeneous forest, and to clarify the extent to which the various hosts share ectomycorrhizal partners.

Establishment of a confluent monolayer model with human primary trophoblast cells: novel insights into placental glucose transport

STUDY HYPOTHESIS Using optimized conditions, primary trophoblast cells isolated from human term placenta can develop a confluent monolayer in vitro, which morphologically and functionally resembles the microvilli structure found in vivo. STUDY FINDING We report the successful establishment of a confluent human primary trophoblast monolayer using pre-coated polycarbonate inserts, where the integrity and functionality was validated by cell morphology, biophysical features, cellular marker expression and secretion, and asymmetric glucose transport. WHAT IS KNOWN ALREADY Human trophoblast cells form the initial barrier between maternal and fetal blood to regulate materno-fetal exchange processes. Although the method for isolating pure human cytotrophoblast cells was developed almost 30 years ago, a functional in vitro model with primary trophoblasts forming a confluent monolayer is still lacking. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Human term cytotrophoblasts were isolated by enzymatic digestion and density gradient separation. The purity of the primary cells was evaluated by flow cytometry using the trophoblast-specific marker cytokeratin 7, and vimentin as an indicator for potentially contaminating cells. We screened different coating matrices for high cell viability to optimize the growth conditions for primary trophoblasts on polycarbonate inserts. During culture, cell confluency and polarity were monitored daily by determining transepithelial electrical resistance (TEER) and permeability properties of florescent dyes. The time course of syncytia-related gene expression and hCG secretion during syncytialization were assessed by quantitative RT-PCR and enzyme-linked immunosorbent assay, respectively. The morphology of cultured trophoblasts after 5 days was determined by light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Membrane makers were visualized using confocal microscopy. Additionally, glucose transport studies were performed on the polarized trophoblasts in the same system. MAIN RESULTS AND THE ROLE OF CHANCE During 5-day culture, the highly pure trophoblasts were cultured on inserts coated with reconstituted basement membrane matrix . They exhibited a confluent polarized monolayer, with a modest TEER and a size-dependent apparent permeability coefficient (Papp) to fluorescently labeled compounds (MW ∼400-70 000 Da). The syncytialization progress was characterized by gradually increasing mRNA levels of fusogen genes and elevating hCG secretion. SEM analyses confirmed a confluent trophoblast layer with numerous microvilli, and TEM revealed a monolayer with tight junctions. Immunocytochemistry on the confluent trophoblasts showed positivity for the cell-cell adhesion molecule E-cadherin, the tight junction protein 1 (ZO-1) and the membrane proteins ATP-binding cassette transporter A1 (ABCA1) and glucose transporter 1 (GLUT1). Applying this model to study the bidirectional transport of a non-metabolizable glucose derivative indicated a carrier-mediated placental glucose transport mechanism with asymmetric kinetics. LIMITATIONS, REASONS FOR CAUTION The current study is only focused on primary trophoblast cells isolated from healthy placentas delivered at term. It remains to be evaluated whether this system can be extended to pathological trophoblasts isolated from diverse gestational diseases. WIDER IMPLICATIONS OF THE FINDINGS These findings confirmed the physiological properties of the newly developed human trophoblast barrier, which can be applied to study the exchange of endobiotics and xenobiotics between the maternal and fetal compartment, as well as intracellular metabolism, paracellular contributions and regulatory mechanisms influencing the vectorial transport of molecules. LARGE-SCALE DATA Not applicable. STUDY FUNDING AND COMPETING INTERESTS This study was supported by the Swiss National Center of Competence in Research, NCCR TransCure, University of Bern, Switzerland, and the Swiss National Science Foundation (grant no. 310030_149958, C.A.). All authors declare that their participation in the study did not involve factual or potential conflicts of interests.