Use of the pyrophosphatase activity as a reliable tonoplast marker in maize roots.

Membranes of maize (Zea mays L., cv LG 11) roots were fractionated by sucrose (in presence or absence of Mg2+) or dextran density gradient centrifugations and the locations of organelles were determined using marker enzymes. Latent UDPase was used as a Golgi marker, catalase for the peroxysomes, cytochrome c oxidase for the mitochondria, UDP-Gal-galactosyltransferase for the amyloplast membranes and NADH-cytochrome c reductase for the ER. Two markers were selected for the plasmalemma, the vanadate-sensitive ATPase and UDP-Glc-sterolglucosyltransferase. The distributions of the PPase and vacuolar ATPase were found to be similar after density gradient centrifugation. The PPase and vacuolar ATPase activities were clearly separated from almost all the other markers tested, however, a partial association of both activities with the ER cannot be completely ruled out. The PPase of maize roots is more active and easier to measure than the vacuolar ATPase and is therefore an excellent candidate for use as a tonoplast marker.

A Ca2+/H+ antiport system driven by the tonoplast pyrophosphate-dependent proton pump from maize roots.

Calcium uptake by tonoplast enriched membrane vesicles from maize (Zea mays L. cv. LG 11) primary roots was studied. A pH gradient, measured by the fluorescence quenching of quinacrine, was generated across sealed vesicles driven by the pyrophosphate-dependent proton pump. The fluorescence quenching was strongly inhibited by Ca2+; moreover, when increasing Ca2+ concentrations were added to vesicles at steady-state, a concomitant decrease in the proton gradient was observed. Ca2+ uptake using Ca-45(2+) was linear from 10 min when oxalate (10 mM) was present, while Ca2+ uptake was completely inhibited with proton ionophores (FCCP and monensin), indicating a Ca2+/H+ antiport. Membranes were further fractionated using a linear sucrose density gradient (10-45%) and were identified with marker enzymes. Ca2+ uptake co-migrated with the tonoplast pyrophosphate-dependent proton pumping, pyrophosphatase and ATPase activities: the Ca2+/H+ antiport is consequently located at the tonoplast.

Tonoplast localization of a calmodulin-stimulated Ca2+-pump from maize roots.

The subcellular localization of a calmodulin-stimulated calcium (Ca2+)-ATPase activity from maize roots (Zea mays L., cv LG 11) was studied. For this purpose, an efficient procedure was developed to prepare sealed plasma membrane vesicles allowing the measurement of proton and Ca2+ transport activities. Two-day-old root membranes were fractionated by sucrose and dextran density gradient centrifugation. Marker enzymes were used to study the distribution of the different membranes in the gradients and a filtration technique was developed to measure Ca-45(2+) transport in sealed vesicles. Most of the ATP-dependent Ca2+ transport activity was associated with the ER. However, a small part of this activity was associated with the tonoplast (corresponding to the activity of the H+/Ca2+ antiport) and the plasma membrane. When the Ca2+ transport was measured in the presence of exogenous calmodulin (1 muM), a 3-5-fold increase of uptake was measured. The calmodulin-stimulated activity was associated with the tonoplast vesicles only. This activity was insensitive to monensin, a proton ionophore, ruling out a direct effect of calmodulin on the H+/Ca2+ antiport. In conclusion, four different Ca2+ transporters are present in young maize root cells. A Ca2+/H+ antiport system is present on the tonoplast, whereas, the plasma membrane and the ER possess each a calmodulinin-sensitive Ca2+-ATPase. Finally, a calmodulin-stimulated Ca2+-ATPase is associated with the tonoplast.

Characterization of the tonoplast Ca2+/H+ antiport system from maize roots.

The tonoplast calcium Ca2+/H+ antiport system of maize (Zea mays L. cv LG 11) roots was characterized using the ''pH jump'' technique in order to avoid interference from the tonoplast proton and Ca2+ pumps. Ca2+ uptake was recorded in the presence of different inhibitors and divalent ions. Chemical modification of amino acid residues of the antiport was used to elucidate the amino acid residues participating in the Ca2+ transport activity. The Ca2+/H+ antiport activity was found to be strongly inhibited by ruthenium red and verapamil, whereas diethylstilbestrol was less effective. Vanadate, erythrosin B, cyclopiazonic acid, bafilomycin, thapsigargin, N,N'-dicyclohexylcarbodiimide (DCCD) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) were without effect. Lanthanum and divalent ions were strongly inhibitory (Cd2+ > Mn2+ > Sr2+ > Ba2+). While reagents modifying sulfhydryl groups (N-ethylmaleimide and 5,5'-dithio-bis(2-nitrobenzoate)) did not affect the antiport activity, modification of trytophan residues (N-bromosuccinimide) was strongly inhibitory. We conclude that ruthenium red, verapamil, lanthanum and divalent cations directly inhibit Ca2+ uptake independent of the function of the proton and Ca2+ pumps. Moreover, the results of chemically modified amino acid residues suggest that sulfhydryl groups are not involved in Ca2+ transport, while tryptophan residues seem important for this translocation.

Subcellular localization, distribution and expression of calmodulin in Zea mays roots.

The subcellular localization, distribution and the steady state level of calmodulin from maize roots (Zea mays L., cv. LG 11) were studied. To analyze the subcellular localization, 2-day old root membranes were fractionated by sucrose density gradient centrifugation and immunoblotting was done with an antibody raised against a vertebrate calmodulin (SWant) which recognized the plant calmodulin. Calmodulin was principally associated with high density fractions and particularly plasmalemma. For studying the distribution of calmodulin in various zones of Zea mays roots, a micro method of membrane preparation was developed. Most of the calmodulin was present in microsomes isolated from the root apex corresponding to the first 4 mm of a 15 +/- 2 mm root. An identical distribution was found by studying the steady state level of the protein by Northern blotting using a cDNA clone of Zea mays calmodulin.

Prevalence of obesity and overweight and associated nutritional factors in a population-based Swiss sample: an opportunity to analyze the impact of three different European cultural roots.

PURPOSE: Obesity represents a growing public health concern worldwide. The latest data in Switzerland rely on self-reported body mass index (BMI), leading to underestimation of prevalence. We reassessed the prevalence of obesity and overweight in a sample of the Swiss population using measured BMI and waist circumference (WC) and explored the association with nutritional factors and living in different linguistic-cultural regions. METHODS: Data of 1,505 participants of a cross-sectional population-based survey in the three linguistic regions of Switzerland were analyzed. BMI and WC were measured, and a 24-h urine collection was performed to evaluate dietary sodium, potassium and protein intake. RESULTS: The prevalence of overweight, obesity and abdominal obesity was 32.2, 14.2 and 33.6 %, respectively. Significant differences were observed in the regional distribution, with a lower prevalence in the Italian-speaking population. Low educational level, current smoking, scarce physical activity and being migrant were associated with an higher prevalence of obesity. Sodium, potassium and protein intake increased significantly across BMI categories. CONCLUSIONS: Obesity and overweight affect almost half of the Swiss adolescents and adults, and the prevalence appears to increase. Using BMI and WC to define obesity led to different prevalences. Differences were furthermore observed across Swiss linguistic-cultural regions, despite a common socio-economic and governmental framework. We found a positive association between obesity and salt intake, with a potential deleterious synergistic effect on cardiovascular risk.

Rapid prototyping of compliant human aortic roots for assessment of valved stents.

Adequate in-vitro training in valved stents deployment as well as testing of the latter devices requires compliant real-size models of the human aortic root. The casting methods utilized up to now are multi-step, time consuming and complicated. We pursued a goal of building a flexible 3D model in a single-step procedure. We created a precise 3D CAD model of a human aortic root using previously published anatomical and geometrical data and printed it using a novel rapid prototyping system developed by the Fab@Home project. As a material for 3D fabrication we used common house-hold silicone and afterwards dip-coated several models with dispersion silicone one or two times. To assess the production precision we compared the size of the final product with the CAD model. Compliance of the models was measured and compared with native porcine aortic root. Total fabrication time was 3 h and 20 min. Dip-coating one or two times with dispersion silicone if applied took one or two extra days, respectively. The error in dimensions of non-coated aortic root model compared to the CAD design was <3.0% along X, Y-axes and 4.1% along Z-axis. Compliance of a non-coated model as judged by the changes of radius values in the radial direction by 16.39% is significantly different (P<0.001) from native aortic tissue--23.54% at the pressure of 80-100 mmHg. Rapid prototyping of compliant, life-size anatomical models with the Fab@Home 3D printer is feasible--it is very quick compared to previous casting methods.

Igneous garnet and amphibole fractionation in the roots of island arcs: experimental constraints on andesitic liquids

To evaluate the role of garnet and amphibole fractionation at conditions relevant for the crystallization of magmas in the roots of island arcs, a series of experiments were performed on a synthetic andesite at conditions ranging from 0.8 to 1.2 GPa, 800-1,000 degrees C and variable H2O contents. At water undersaturated conditions and fO(2) established around QFM, garnet has a wide stability field. At 1.2 GPa garnet ? amphibole are the high-temperature liquidus phases followed by plagioclase at lower temperature. Clinopyroxene reaches its maximal stability at H2O-contents <= 9 wt% at 950 degrees C and is replaced by amphibole at lower temperature. The slopes of the plagioclase-in boundaries are moderately negative in T-XH2O space. At 0.8 GPa, garnet is stable at magmatic H2O contents exceeding 8 wt% and is replaced by spinel at decreasing dissolved H2O. The liquids formed by crystallization evolve through continuous silica increase from andesite to dacite and rhyolite for the 1.2 GPa series, but show substantial enrichment in FeO/MgO for the 0.8 GPa series related to the contrasting roles of garnet and amphibole in fractionating Fe-Mg in derivative liquids. Our experiments indicate that the stability of igneous garnet increases with increasing dissolved H2O in silicate liquids and is thus likely to affect trace element compositions of H2O-rich derivative arc volcanic rocks by fractionation. Garnet-controlled trace element ratios cannot be used as a proxy

Impact of Pseudomonas fluorescens strain CHA0 and a derivative with improved biocontrol activity on the culturable resident bacterial community on cucumber roots

Information on the effects of released wild-type or genetically engineered bacteria on resident bacterial communities is important to assess the potential risks associated with the introduction of these organisms into agroecosystems. The rifampicin-resistant biocontrol strain Pseudomonas fluorescens CHA0-Rif and its derivative CHA0-Rif/pME3424, which has improved biocontrol activity and enhanced production of the antibiotics 2,4-diacetylphloroglucinol (Phl) and pyoluteorin (Plt), were introduced into soil microcosms and the culturable bacterial community developing on cucumber roots was investigated 10 and 52 days later. The introduction of either of the two strains led to a transiently enhanced metabolic activity of the bacterial community on glucose dimers and polymers as measured with BIOLOG GN plates, but natural succession between the two sampling dates changed the metabolic activity of the bacterial community more than did the inoculants. The introduced strains did not significantly affect the abundance of dominant genotypic groups of culturable bacteria discriminated by restriction analysis of amplified 16S rDNA of 2500 individual isolates. About 30-50% of the resident bacteria were very sensitive to Phl and Plt, but neither the wild-type nor CHA0-Rif/pME3424 changed the proportion of sensitive and resistant bacteria in situ. In microcosms with a synthetic bacterial community, both biocontrol strains reduced the population of a strain of Pseudomonas but did not affect the abundance of four other bacterial strains including two highly antibiotic-sensitive isolates. We conclude that detectable perturbations in the metabolic activity of the resident bacterial community caused by the biocontrol strain CHA0-Rif are (i) transient, (ii) similar for the genetically improved derivative CHA0-Rif/pME3424 and (iii) less pronounced than changes in the community structure during plant growth.

Spatio-temporal transcript profiling of rice roots and shoots in response to phosphate starvation and recovery.

Using rice (Oryza sativa) as a model crop species, we performed an in-depth temporal transcriptome analysis, covering the early and late stages of Pi deprivation as well as Pi recovery in roots and shoots, using next-generation sequencing. Analyses of 126 paired-end RNA sequencing libraries, spanning nine time points, provided a comprehensive overview of the dynamic responses of rice to Pi stress. Differentially expressed genes were grouped into eight sets based on their responses to Pi starvation and recovery, enabling the complex signaling pathways involved in Pi homeostasis to be untangled. A reference annotation-based transcript assembly was also generated, identifying 438 unannotated loci that were differentially expressed under Pi starvation. Several genes also showed induction of unannotated splice isoforms under Pi starvation. Among these, PHOSPHATE2 (PHO2), a key regulator of Pi homeostasis, displayed a Pi starvation-induced isoform, which was associated with increased translation activity. In addition, microRNA (miRNA) expression profiles after long-term Pi starvation in roots and shoots were assessed, identifying 20 miRNA families that were not previously associated with Pi starvation, such as miR6250. In this article, we present a comprehensive spatio-temporal transcriptome analysis of plant responses to Pi stress, revealing a large number of potential key regulators of Pi homeostasis in plants.

Soil tillage affects the community structure of mycorrhizal fungi in maize roots

In this study we tested whether communities of arbuscular mycorrhizal fungi (AMF) colonizing the roots of maize (Zea mays L.) were affected by soil tillage practices (plowing, chiseling, and no-till) in a long-term field experiment carried out in Tanikon (Switzerland). AMF were identified in the roots using specific polymerase chain reaction (PCR) markers that had been developed for the AMF previously isolated from the soils of the studied site. A nested PCR procedure with primers of increased specificity (eukaryotic, then, fungal, then AMF species or. species-grouop specific) was used. Sequencing of amplified DNA confirmed that the DNA obtained from the maize roots was of AMF origin. Presence of particular AMF species or species-group was scored as a presence of a DNA product after PCR with specific primers. We also used single-strand conformation polymorphism analysis (SSCP), of amplified DNA samples to-check if the amplification of the DNA from maize roots matched the expected profile for a particular AMF isolate with a given specific primer pair. Presence of the genus Scutellospora, in maize roots was strongly reduced in plowed and chiseled soils. Fungi from the suborder Glomineae were more prevalent colonizers of maize roots growing in plowed soils, but were also present in the roots from other tillage treatments. These changes in community of AMF colonizing maize roots might be due to (1), the differences in tolerance to the tillage-induced disruption of the hyphae among the different AMF species, (2) changes in nutrient content of the soil, (3) changes in microbial activity, or (4) changes in weed populations in response to soil tillage. This is the first report on community composition of AMF in the roots of a field-grown crop plant (maize) as affected by soil tillage.

Detection of plant-modulated alterations in antifungal gene expression in Pseudomonas fluorescens CHA0 on roots by flow cytometry.

The biocontrol activity of the root-colonizing Pseudomonas fluorescens strain CHA0 is largely determined by the production of antifungal metabolites, especially 2,4-diacetylphloroglucinol. The expression of these metabolites depends on abiotic and biotic environmental factors, in particular, elements present in the rhizosphere. In this study, we have developed a new method for the in situ analysis of antifungal gene expression using flow cytometry combined with green fluorescent protein (GFP)-based reporter fusions to the phlA and prnA genes essential for the production of the antifungal compounds 2,4-diacetylphloroglucinol and pyrrolnitrin, respectively, in strain CHA0. Expression of phlA-gfp and prnA-gfp in CHA0 cells harvested from the rhizosphere of a set of plant species as well as from the roots of healthy, leaf pathogen-attacked, and physically stressed plants were analyzed using a FACSCalibur. After subtraction of background fluorescence emitted by plant-derived particles and CHA0 cells not carrying the gfp reporters, the average gene expression per bacterial cell could be calculated. Levels of phlA and prnA expression varied significantly in the rhizospheres of different plant species. Physical stress and leaf pathogen infection lowered phlA expression levels in the rhizosphere of cucumber. Our results demonstrate that the newly developed approach is suitable to monitor differences in levels of antifungal gene expression in response to various plant-derived factors. An advantage of the method is that it allows quantification of bacterial gene expression in rhizosphere populations at a single-cell level. To our best knowledge, this is the first study using flow cytometry for the in situ analysis of biocontrol gene expression in a plant-beneficial bacterium in the rhizosphere.