Morphological and genetic characterisation of the root system architecture of selected barley recombinant chromosome substitution lines using an integrated phenotyping approach.

Discoveries on the genetics of resource acquisition efficiency are limited by the ability to measure plant roots in sufficient number and with adequate genotypic variability. This paper presents a root phenotyping study that explores ways to combine live imaging and computer algorithms for model-based extraction of root growth parameters. The study is based on a subset of barley Recombinant Chromosome Substitution Lines (RCSLs) and a combinatorial approach was designed for fast identification of the regions of the genome that contribute the most to variations in root system architecture (RSA). Results showed there was a strong genotypic variation in root growth parameters within the set of genotypes studied. The chromosomal regions associated with primary root growth differed from the regions of the genome associated with changes in lateral root growth. The concepts presented here are discussed in the context of identifying root QTL and its potential to assist breeding for novel crops with improved root systems.

Root morphology and seed and leaf ionomic traits in a Brassica napus L. diversity panel show wide phenotypic variation and are characteristic of crop habit.

BACKGROUND: Mineral nutrient uptake and utilisation by plants are controlled by many traits relating to root morphology, ion transport, sequestration and translocation. The aims of this study were to determine the phenotypic diversity in root morphology and leaf and seed mineral composition of a polyploid crop species, Brassica napus L., and how these traits relate to crop habit. Traits were quantified in a diversity panel of up to 387 genotypes: 163 winter, 127 spring, and seven semiwinter oilseed rape (OSR) habits, 35 swede, 15 winter fodder, and 40 exotic/unspecified habits. Root traits of 14 d old seedlings were measured in a 'pouch and wick' system (n = ~24 replicates per genotype). The mineral composition of 3-6 rosette-stage leaves, and mature seeds, was determined on compost-grown plants from a designed experiment (n = 5) by inductively coupled plasma-mass spectrometry (ICP-MS). RESULTS: Seed size explained a large proportion of the variation in root length. Winter OSR and fodder habits had longer primary and lateral roots than spring OSR habits, with generally lower mineral concentrations. A comparison of the ratios of elements in leaf and seed parts revealed differences in translocation processes between crop habits, including those likely to be associated with crop-selection for OSR seeds with lower sulphur-containing glucosinolates. Combining root, leaf and seed traits in a discriminant analysis provided the most accurate characterisation of crop habit, illustrating the interdependence of plant tissues. CONCLUSIONS: High-throughput morphological and composition phenotyping reveals complex interrelationships between mineral acquisition and accumulation linked to genetic control within and between crop types (habits) in B. napus. Despite its recent genetic ancestry (<10 ky), root morphology, and leaf and seed composition traits could potentially be used in crop improvement, if suitable markers can be identified and if these correspond with suitable agronomy and quality traits.

An in situ atomic force microscope for normal-incidence nanofocus X-ray experiments.

A compact high-speed X-ray atomic force microscope has been developed for in situ use in normal-incidence X-ray experiments on synchrotron beamlines, allowing for simultaneous characterization of samples in direct space with nanometric lateral resolution while employing nanofocused X-ray beams. In the present work the instrument is used to observe radiation damage effects produced by an intense X-ray nanobeam on a semiconducting organic thin film. The formation of micrometric holes induced by the beam occurring on a timescale of seconds is characterized.

High-throughput phenotyping (HTP) identifies seedling root traits linked to variation in seed yield and nutrient capture in field-grown oilseed rape (Brassica napus L.).

Background and Aims Root traits can be selected for crop improvement. Techniques such as soil excavations can be used to screen root traits in the field, but are limited to genotypes that are well-adapted to field conditions. The aim of this study was to compare a low-cost, high-throughput root phenotyping (HTP) technique in a controlled environment with field performance, using oilseed rape (OSR; Brassica napus) varieties. Methods Primary root length (PRL), lateral root length and lateral root density (LRD) were measured on 14-d-old seedlings of elite OSR varieties (n = 32) using a 'pouch and wick' HTP system (∼40 replicates). Six field experiments were conducted using the same varieties at two UK sites each year for 3 years. Plants were excavated at the 6- to 8-leaf stage for general vigour assessments of roots and shoots in all six experiments, and final seed yield was determined. Leaves were sampled for mineral composition from one of the field experiments. Key Results Seedling PRL in the HTP system correlated with seed yield in four out of six (r = 0·50, 0·50, 0·33, 0·49; P < 0·05) and with emergence in three out of five (r = 0·59, 0·22, 0·49; P < 0·05) field experiments. Seedling LRD correlated positively with leaf concentrations of some minerals, e.g. calcium (r = 0·46; P < 0·01) and zinc (r = 0·58; P < 0·001), but did not correlate with emergence, general early vigour or yield in the field. Conclusions Associations between PRL and field performance are generally related to early vigour. These root traits might therefore be of limited additional selection value, given that vigour can be measured easily on shoots/canopies. In contrast, LRD cannot be assessed easily in the field and, if LRD can improve nutrient uptake, then it may be possible to use HTP systems to screen this trait in both elite and more genetically diverse, non-field-adapted OSR.

Challenges and opportunities for quantifying roots and rhizosphere interactions through imaging and image analysis.

The morphology of roots and root systems influences the efficiency by which plants acquire nutrients and water, anchor themselves and provide stability to the surrounding soil. Plant genotype and the biotic and abiotic environment significantly influence root morphology, growth and ultimately crop yield. The challenge for researchers interested in phenotyping root systems is, therefore, not just to measure roots and link their phenotype to the plant genotype, but also to understand how the growth of roots is influenced by their environment. This review discusses progress in quantifying root system parameters (e.g. in terms of size, shape and dynamics) using imaging and image analysis technologies and also discusses their potential for providing a better understanding of root:soil interactions. Significant progress has been made in image acquisition techniques, however trade-offs exist between sample throughput, sample size, image resolution and information gained. All of these factors impact on downstream image analysis processes. While there have been significant advances in computation power, limitations still exist in statistical processes involved in image analysis. Utilizing and combining different imaging systems, integrating measurements and image analysis where possible, and amalgamating data will allow researchers to gain a better understanding of root:soil interactions.

Continuous, high-resolution biospeckle imaging reveals a discrete zone of activity at the root apex that responds to contact with obstacles.

BACKGROUND AND AIMS: Shining a laser onto biological material produces light speckles termed biospeckles. Patterns of biospeckle activity reflect changes in cell biochemistry, developmental processes and responses to the environment. The aim of this work was to develop methods to investigate the biospeckle activity in roots and to characterize the distribution of its intensity and response to thigmostimuli. METHODS: Biospeckle activity in roots of Zea mays, and also Jatropha curcas and Citrus limonia, was imaged live and in situ using a portable laser and a digital microscope with a spatial resolution of 10 µm per pixel and the ability to capture images every 0.080 s. A procedure incorporating a Fujii algorithm, image restoration using median and Gaussian filters, image segmentation using maximum-entropy threshold methods and the extraction of features using a tracing algorithm followed by spline fitting were developed to obtain quantitative information from images of biospeckle activity. A wavelet transform algorithm was used for spectral decomposition of biospeckle activity and generalized additive models were used to attribute statistical significance to changes in patterns of biospeckle activity. KEY RESULTS: The intensity of biospeckle activity was greatest close to the root apex. Higher frequencies (3-6 Hz) contributed most to the total intensity of biospeckle activity. When a root encountered an obstacle, the intensity of biospeckle activity decreased abruptly throughout the root system. The response became attenuated with repeated thigmostimuli. CONCLUSIONS: The data suggest that at least one component of root biospeckle activity resulted from a biological process, which is located in the zone of cell division and responds to thigmostimuli. However, neither individual cell division events nor root elongation is likely to be responsible for the patterns of biospeckle activity.

A conceptual model of root hair ideotypes for future agricultural environments: what combination of traits should be targeted to cope with limited P availability?

BACKGROUND: Phosphorus (P) often limits crop production and is frequently applied as fertilizer; however, supplies of quality rock phosphate for fertilizer production are diminishing. Plants have evolved many mechanisms to increase their P acquisition, and an understanding of these traits could result in improved long-term sustainability of agriculture. This Viewpoint focuses on the potential benefits of root hairs to sustainable production. SCOPE: First the various root-related traits that could be deployed to improve agricultural sustainability are catalogued, and their potential costs and benefits to the plant are discussed. A novel mathematical model describing the effects of length, density and longevity of root hairs on P acquisition is developed, and the relative benefits of these three root-hair traits to plant P nutrition are calculated. Insights from this model are combined with experimental data to assess the relative benefits of a range of root hair ideotypes for sustainability of agriculture. CONCLUSIONS: A cost-benefit analysis of root traits suggests that root hairs have the greatest potential for P acquisition relative to their cost of production. The novel modelling of root hair development indicates that the greatest gains in P-uptake efficiency are likely to be made through increased length and longevity of root hairs rather than by increasing their density. Synthesizing this information with that from published experiments we formulate six potential ideotypes to improve crop P acquisition. These combine appropriate root hair phenotypes with architectural, anatomical and biochemical traits, such that more root-hair zones are produced in surface soils, where P resources are found, on roots which are metabolically cheap to construct and maintain, and that release more P-mobilizing exudates. These ideotypes could be used to inform breeding programmes to enhance agricultural sustainability.

[In vitro effect of lupeol and casearin G on peripheral blood mononuclear and tumor cells]. / Efecto in vitro de los terpenos lupeol y casearina G sobre células sanguíneas y tumorales.

BACKGROUND: The rainforest is an important source of natural compounds with therapeutic properties. Although there are many anti-inflammatory and antineoplastic drugs available to the clinician, there is an ongoing need for new therapeutic drugs with fewer serious adverse effects. AIM: To evaluate the in vitro cytotoxic effects of lupeol and casearin G on tumor cells, on phagocytic activity and nitric oxide (NO) production by blood mononuclear cells. MATERIAL AND METHODS: The cytotoxic effect of these compounds on cell lines MCF-7 (human breast adenocarcinoma) and PC-3 (human prostate cancer) was measured by a colorimetric assay (MTS/PMS) and the sulphorhodamine B assay. Peripheral blood mononuclear cells were obtained from eight healthy volunteers. The effect of these compounds on nitric oxide (NO) production was measured using the Griess reaction. Their effect on phagocytic activity of PBMC was also evaluated. RESULTS: Lupeol (≥ 2 mM) resulted in a reduction of both the phagocytic index and the percentage of phagocytic monocytes and macrophages. Treatment of monocytes/macrophages with lupeol (72 µM) and casearin G (4 µM) reduced the production of NO. Neither lupeol (< 969 µM) nor casearin G (< 55 µM) had cytotoxic effects on PBMC. Casearin G showed both cytotoxic (IC50, LC50) and cytostatic (GI50) effects against tumor cells, PC-3 (IC50 = 12.5 µM; GI50 = 13.3 µM; LC50 = 51.9 µM) and MCF-7 (IC50 = 112.8 µM; GI50 = 11.8 µM; LC50 = 49.4 µM), as well as a hemolytic effect (≥ 182 µM). CONCLUSIONS: These observations indicate that lupeol and casearin G might be useful compounds in the preparation of anti-inflammatory drugs, whereas casearin G might be useful in the elaboration of antitumor drugs.

What are the implications of variation in root hair length on tolerance to phosphorus deficiency in combination with water stress in barley (Hordeum vulgare)?

BACKGROUND AND AIMS: Phosphorus commonly limits crop yield and is frequently applied as fertilizer; however, supplies of quality rock phosphate for fertilizer production are diminishing. Plants have evolved many mechanisms to increase their P-fertilizer use efficiency, and an understanding of these traits could result in improved long-term sustainability of agriculture. Here a mutant population is utilized to assess the impact of root hair length on P acquisition and yield under P-deficient conditions alone or when combined with drought. METHODS: Mutants with various root hair phenotypes were grown in the glasshouse in pots filled with soil representing sufficient and deficient P treatments and, in one experiment, a range of water availability was also imposed. Plants were variously harvested at 7 d, 8 weeks and 14 weeks, and variables including root hair length, rhizosheath weight, biomass, P accumulation and yield were measured. KEY RESULTS: The results confirmed the robustness of the root hair phenotypes in soils and their relationship to rhizosheath production. The data demonstrated that root hair length is important for shoot P accumulation and biomass, while only the presence of root hairs is critical for yield. Root hair presence was also critical for tolerance to extreme combined P deficit and drought stress, with genotypes with no root hairs suffering extreme growth retardation in comparison with those with root hairs. CONCLUSIONS: The results suggest that although root hair length is not important for maintaining yield, the presence of root hairs is implicit to sustainable yield of barley under P-deficient conditions and when combined with extreme drought. Root hairs are a trait that should be maintained in future germplasm.

Novel form of collective movement by soil bacteria.

Although migrations are essential for soil microorganisms to exploit scarce and heterogeneously distributed resources, bacterial mobility in soil remains poorly studied due to experimental limitations. In this study, time-lapse images collected using live microscopy techniques captured collective and coordinated groups of B. subtilis cells exhibiting "crowd movement". Groups of B. subtilis cells moved through transparent soil (nafion polymer with particle size resembling sand) toward plant roots and re-arranged dynamically around root tips in the form of elongating and retracting "flocks" resembling collective behaviour usually associated with higher organisms (e.g., bird flocks or fish schools). Genetic analysis reveals B. subtilis flocks are likely driven by the diffusion of extracellular signalling molecules (e.g., chemotaxis, quorum sensing) and may be impacted by the physical obstacles and hydrodynamics encountered in the soil like environment. Our findings advance understanding of bacterial migration through soil matrices and expand known behaviours for coordinated bacterial movement.

The dynamics of root meristem distribution in the soil.

Plants must develop efficient root architectures to secure access to nutrients and water in soil. This is achieved during plant development through a series of expansion and branching processes, mostly in the proximity of root apical meristems, where the plant senses the environment and explores immediate regions of the soil. We have developed a new approach to study the dynamics of root meristem distribution in soil, using the relationship between the increase in root length density and the root meristem density. Initiated at the seed, the location of root meristems in barley seedlings was shown to propagate, wave-like, through the soil, leaving behind a permanent network of roots for the plant to acquire water and nutrients. Data from observations on barley roots were used to construct mathematical models to describe the density of root meristems in space. These models suggested that the morphology of the waves of meristems was a function of specific root developmental processes. The waves of meristems observed in root systems of barley seedlings exploring the soil might represent a more general and fundamental aspect of plant rooting strategies for securing soil resources.

Live biospeckle laser imaging of root tissues.

Live imaging is now a central component for the study of plant developmental processes. Currently, most techniques are extremely constraining: they rely on the marking of specific cellular structures which generally apply to model species because they require genetic transformations. The biospeckle laser (BSL) system was evaluated as an instrument to measure biological activity in plant tissues. The system allows collecting biospeckle patterns from roots which are grown in gels. Laser illumination has been optimized to obtain the images without undesirable specular reflections from the glass tube. Data on two different plant species were obtained and the ability of three different methods to analyze the biospeckle patterns are presented. The results showed that the biospeckle could provide quantitative indicators of the molecular activity from roots which are grown in gel substrate in tissue culture. We also presented a particular experimental configuration and the optimal approach to analyze the images. This may serve as a basis to further works on live BSL in order to study root development.

Micromechanics of root development in soil.

Our understanding of how roots develop in soil may be at the eve of significant transformations. The formidable expansion of imaging technologies enables live observations of the rhizosphere micro-pore architecture at unprecedented resolution. Granular matter physics provides ways to understand the microscopic fluctuations of forces in soils, and the increasing knowledge of plant mechanobiology may shed new lights on how roots perceive soil heterogeneity. This opinion paper exposes how recent scientific achievements may contribute to refresh our views on root growth in heterogeneous environments.

[Fryns syndrome. Report on 3 new cases]. / Syndrome de Fryns. Présentation de 3 nouvelles observations.

BACKGROUND: Fryns syndrome is a lethal autosomal recessive syndrome which includes congenital diaphragmatic hernia (CDH), pulmonary hypoplasia, cranio-facial manifestations including a coarse face, a cleft palate / lip and cloudy corneae, distal limb hypoplasia and internal malformations including central nervous system, cardiovascular, gastrointestinal, urogenital and skeletal anomalies. CASES REPORT: We report on 3 patients with Fryns syndrome in the french Indian Ocean islands (La Réunion and Mayotte islands). Pregnancies were complicated by polyhydramnios. All patients had coarse face, cloudy corneae, cleft lip / palate or high-arched palate, hypoplastic nails and brachytelephalangy. They died in the neonatal period. Two had CDH and 1 did not have a diaphragmatic involvement but a severe respiratory distress syndrome with refractory hypoxemia. Cytogenetic studies of blood lymphocytes and skin fibroblasts were normal. CONCLUSIONS: Fryns syndrome is the most common multiple congenital anomaly associated with CDH. The diagnosis is strongly suggested when CHD and brachytelephalangy are associated. Phenotypes with CDH similar to Fryns syndrome have been described with cytogenetic aberrations with G-banded chromosome analysis and submicroscopic chromosome deletions detected by high resolution karyotyping or array comparative genomic hybridization (CGH). Exclusion of chromosome aberrations (including isochromosome 12p in skin fibroblasts) is needed prior to making the diagnosis of Fryns syndrome. In sporadic cases, high resolution karyotyping or array CGH should be performed for correct diagnosis and genetic counselling.

Reproducibility of the respiratory dead space measurements in mechanically ventilated children using the CO2SMO monitor.

OBJECTIVES: To assess the reproducibility of respiratory dead space measurements in ventilated children. DESIGN: Prospective study. SETTING: University pediatric intensive care unit. PATIENTS: Thirty-two mechanically ventilated children (0.13-15.4 years) who were clinically stable. METHODS: The single-breath CO(2) test (SBT-CO(2)) was recorded using the CO(2)SMO Plus from the mean of 30 ventilatory cycles during 1 h (at T0, T15, T30, T45, and T60). Airway dead space was determined automatically (Novametrix Medical Systems, USA), and manually by Bohr- Enghoff equations using data obtained by SBT-CO(2). At the end of the study period, arterial blood gas was sampled in order to calculate alveolar and physiologic dead space. Intrasubject reproducibility of measurements was evaluated by the intraclass correlation coefficient. Two-way analysis of variance was used to evaluate the relationships between time and measurements. The two methods for calculating airway dead space were compared by using two-tailed Student's t-test and Bland-Altman analysis. RESULTS: Airway dead space measurement had a good reproducibility during the 1-h period, whatever the method used (intraclass correlation coefficient: 0.84 to 0.87). No significant difference was observed with time. Airway dead space values from the SBT-CO(2) method were smaller than those from Bohr-Enghoff equations. Physiologic dead space values from the SBT-CO2 method were similar to those from Bohr-Enghoff equations. CONCLUSION: The measurement of airway dead space by the CO(2)SMO Plus was reproducible over a 1-h period in children requiring mechanical ventilation, provided ventilatory parameters were constant throughout the study. SBT-CO(2) analysis may provide a bedside non-invasive monitoring of volumetric capnography.

[Migration of a ventriculoperitoneal shunt in the liver: A rare complication]. / La perforation hépatique d'un cathéter de dérivation ventriculo-péritonéale : une complication rare.

We report on a case of migration inside the liver of the distal end of a ventriculoperitoneal shunt catheter in an adult patient. A simple laparotomy permitted the surgical removal with no haemorrhagic complication. We discuss the other cases reported in the literature and we outline the need to perform an abdominal CT scan in patients carrying a VP shunt with digestive symptoms.

Development and application of a flow cytometric potency assay for DNA vaccines.

We have developed a rapid, reliable, and sensitive quantitative flow cytometric assay to measure the in vitro potency and stability of DNA vaccines to be delivered either by particle-mediated epidermal delivery (PMED) or by electroporation. The method involves transfecting cells with test DNA and comparing the measured antigen expression to that generated with expression from known quantities of reference material DNA. The assay was adapted for performance under Good Laboratory Practice (GLP) guidelines and was successfully utilized to perform potency testing in support of a Phase I study for two hantavirus DNA vaccines delivered by gene gun. The results from the potency assays conducted over a 24-month period using this method proved to be highly reproducible with high signal-to-noise ratios. The assay was also adapted to assess the in vitro potency and stability of a DNA vaccine for Venezuelan equine encephalitis virus that will be delivered by electroporation. Our results indicate that this assay can be readily applied to support potency and stability testing of numerous DNA vaccines delivered by various methods, including multiagent vaccines.