Production of low phytic acid rice by hairpin RNA- and artificial microRNA-mediated silencing of OsMIK in seeds

To produce agronomically competitive rice with nutritionally superior, environmentally safe phytic acid (PA) levels, hairpin RNA (hpRNA)- and artificial microRNA (amiRNA)-mediated gene silencing approaches were explored to reduce both myo-inositol kinase gene (OsMIK) expression and PA accumulation in rice seeds. hpRNA and amiRNA sequences targeted to OsMIK (hpMIK and amiMIK), under the control of a rice Ole18 promoter, were transformed into the rice cultivar Nippon-bare. Fourteen and 21 independent transgenic events were identified containing the hpMIK and amiMIK constructs, respectively, from which five stable homozygous transgenic lines of each were developed together with their null siblings. Southern blotting demonstrated transgene integration into the genome and quantitative real-time PCR showed that gene silencing was restricted to seeds. OsMIK transcripts were significantly reduced in both transgenic amiMIK and hpMIK seeds, which had PA levels reduced by 14.9-50.2 and 38.1-50.7 %, respectively, compared with their respective null siblings. There were no systematic significant differences in agronomic traits between the transgenic lines and their non-transgenic siblings, and no correlation between seed PA contents and decreased rates of seed germination and seedling emergence. The results of the present study suggest that Ole 18-driven OsMIK silencing via hpRNA and amiRNA could be an effective way to develop agronomically competitive low phytic acid rice.

Thy-1 binds to integrin beta(3) on astrocytes and triggers formation of focal contact sites.

BACKGROUND: Thy-1 is an abundant neuronal glycoprotein in mammals. Despite such prevalence, Thy-1 function remains largely obscure in the absence of a defined ligand. Astrocytes, ubiquitous cells of the brain, express a putative Thy-1 ligand that prevents neurite outgrowth. In this paper, a ligand molecule for Thy-1 was identified, and the consequences of Thy-1 binding for astrocyte function were investigated. RESULTS: Thy-1 has been implicated in cell adhesion and, indeed, all known Thy-1 sequences were found to contain an integrin binding, RGD-like sequence. Thy-1 interaction with beta3 integrin on astrocytes was demonstrated in an adhesion assay using a thymoma line (EL-4) expressing high levels of Thy-1. EL-4 cells bound to astrocytes five times more readily than EL-4(-f), control cells lacking Thy-1. Binding was blocked by either anti-Thy-1 or anti-beta3 antibodies, by RGD-related peptides, or by soluble Thy-1-Fc chimeras. However, neither RGE/RLE peptides nor Thy-1(RLE)-Fc fusion protein inhibited the interaction. Immobilized Thy-1-Fc, but not Thy-1(RLE)-Fc fusion protein supported the attachment and spreading of astrocytes in a Mn(2+)-dependent manner. Binding to Thy-1-Fc was inhibited by RGD peptides. Moreover, vitronectin, fibrinogen, denatured collagen (dcollagen), and a kistrin-derived peptide, but not fibronectin, also mediated Mn(2+)-dependent adhesion, suggesting the involvement of beta3 integrin. The addition of Thy-1 to matrix-bound astrocytes induced recruitment of paxillin, vinculin, and focal adhesion kinase (FAK) to focal contacts and increased tyrosine phosphorylation of proteins such as p130(Cas) and FAK. Furthermore, astrocyte binding to immobilized Thy-1-Fc alone was sufficient to promote focal adhesion formation and phosphorylation on tyrosine. CONCLUSIONS: Thy-1 binds to beta3 integrin and triggers tyrosine phosphorylation of focal adhesion proteins in astrocytes, thereby promoting focal adhesion formation, cell attachment, and spreading.

Arsenite interferes with protein folding and triggers formation of protein aggregates in yeast.

Several metals and metalloids profoundly affect biological systems, but their impact on the proteome and mechanisms of toxicity are not fully understood. Here, we demonstrate that arsenite causes protein aggregation in Saccharomyces cerevisiae. Various molecular chaperones were found to be associated with arsenite-induced aggregates indicating that this metalloid promotes protein misfolding. Using in vivo and in vitro assays, we show that proteins in the process of synthesis/folding are particularly sensitive to arsenite-induced aggregation, that arsenite interferes with protein folding by acting on unfolded polypeptides, and that arsenite directly inhibits chaperone activity. Thus, folding inhibition contributes to arsenite toxicity in two ways: by aggregate formation and by chaperone inhibition. Importantly, arsenite-induced protein aggregates can act as seeds committing other, labile proteins to misfold and aggregate. Our findings describe a novel mechanism of toxicity that may explain the suggested role of this metalloid in the etiology and pathogenesis of protein folding disorders associated with arsenic poisoning.

Reduced susceptibility to vancomycin and biofilm formation in methicillin-resistant Staphylococcus epidermidis isolated from blood cultures

This study aimed to correlate the presence of ica genes, biofilm formation and antimicrobial resistance in 107 strains of Staphylococcus epidermidis isolated from blood cultures. The isolates were analysed to determine their methicillin resistance, staphylococcal cassette chromosome mec (SCCmec) type, ica genes and biofilm formation and the vancomycin minimum inhibitory concentration (MIC) was measured for isolates and subpopulations growing on vancomycin screen agar. The mecA gene was detected in 81.3% of the S. epidermidis isolated and 48.2% carried SCCmec type III. The complete icaADBC operon was observed in 38.3% of the isolates; of these, 58.5% produced a biofilm. Furthermore, 47.7% of the isolates grew on vancomycin screen agar, with an increase in the MIC in 75.9% of the isolates. Determination of the MIC of subpopulations revealed that 64.7% had an MIC ≥ 4 μg mL-1, including 15.7% with an MIC of 8 μg mL-1 and 2% with an MIC of 16 μg mL-1. The presence of the icaADBC operon, biofilm production and reduced susceptibility to vancomycin were associated with methicillin resistance. This study reveals a high level of methicillin resistance, biofilm formation and reduced susceptibility to vancomycin in subpopulations of S. epidermidis. These findings may explain the selection of multidrug-resistant isolates in hospital settings and the consequent failure of antimicrobial treatment.

Accounting information and the prediction of farm viability

Until recently farm management made little use of accounting and agriculture has been largely excluded from the scope of accounting standards. This article examines the current use of accounting in agriculture and points theneed to establish accounting standards for agriculture. Empirical evidence shows that accounting can make a significant contribution to agricultural management and farm viability and could also be important for other agents involved in agricultural decision making. Existing literature on failureprediction models and farm viability prediction studies provide the starting point for our research, in which two dichotomous logit models were applied to subsamples of viable and unviable farms in Catalonia, Spain. The firstmodel considered only non-financial variables, while the other also considered financial ones. When accounting variables were added to the model, a significant reduction in deviance was observed.

Total lipid and fatty acid accumulation during basidiospore formation in the ectomycorrhizal fungus Pisolithus sp.

The basidiospores of Pisolithus sp. contain large amounts of lipids, indicating provision for future germination in the host rhizosphere. However, the accumulation, composition, and mobilization of lipids during formation and germination of these spores are largely unknown. In this study, lipid storage and fatty acid composition during basidiosporogenesis were analyzed in fresh basidiocarps using bright-field microscopy and gas chromatography. Abundant lipid bodies are found in the hyphae, basidia, and basidiospores of fungal basidiocarps. This evidences a considerable C transport in the basidiocarp to meet the C demand during basidiospore formation. Fatty acid composition analysis revealed the presence of 24 compounds with chains of 9 to 18 C atoms, either saturated or insaturated, with one or two insaturations. The fatty acid composition and content varied according to the developmental stage of the peridioles. In free basidiospores, the predominant compounds were 16:0, 16:1w5c, 18:1w9c, and 18:2w6,9c/18:0ante, at concentrations of 76, 46, 192, and 51 µg g-1 dry matter, respectively. Our results indicate that oleic acid is the major constituent of lipid reserves in Pisolithus sp. basidiospores. Further studies are being conducted to determine the factors that induce lipid mobilization during spore germination.

In-furrow inoculation of soybean as alternative to fungicide and micronutrient seed treatment

Soybean is a major grain crop in Brazil, and yields can be considerably improved by inoculation with selected Bradyrhizobium strains. However, the incompatibility between inoculation and seed treatments with fungicides and micronutrients represents a major barrier to the achievement of high rates of biological N2 fixation. Inoculation practices that can alleviate the negative effects of agrochemicals must therefore be found and in-furrow inoculation seems to be an attractive alternative. This study reports the results of seven field experiments conducted in three growing seasons in Brazil; three in soils previously cropped with inoculated soybean (> 10(4) cells g-1 of soil of Bradyrhizobium), and four in areas where the crop was sown for the first time (< 10² cells g-1 of soil of Bradyrhizobium). The compatibility with fungicides and micronutrients was compared in seeds inoculated with peat or liquid inoculants, or treated with different doses of liquid inoculant in-furrow. In areas with established Bradyrhizobium populations, seed-applied agrochemicals did generally not affect nodulation, but also did not increase yields, while inoculation always increased N grain accumulation or yield, and N fertilizer decreased both nodulation and yield. Where soybean was sown for the first time, the seed treatment with agrochemicals affected nodulation when applied together with peat or liquid inoculant. In-furrow inoculation alleviated the effects of seed treatment with agrochemicals; the best performance was achieved with high Bradyrhizobium cell concentrations, with up to 2.5 million cells seed-1.

Biofilm formation on bone grafts and bone graft substitutes: comparison of different materials by a standard in vitro test and microcalorimetry.

We analyzed the initial adhesion and biofilm formation of Staphylococcus aureus (ATCC 29213) and S. epidermidis RP62A (ATCC 35984) on various bone grafts and bone graft substitutes under standardized in vitro conditions. In parallel, microcalorimetry was evaluated as a real-time microbiological assay in the investigation of biofilm formation and material science research. The materials beta-tricalcium phosphate (beta-TCP), processed human spongiosa (Tutoplast) and poly(methyl methacrylate) (PMMA) were investigated and compared with polyethylene (PE). Bacterial counts (log(10) cfu per sample) were highest on beta-TCP (S. aureus 7.67 +/- 0.17; S. epidermidis 8.14 +/- 0.05) while bacterial density (log(10) cfu per surface) was highest on PMMA (S. aureus 6.12 +/- 0.2, S. epidermidis 7.65 +/- 0.13). Detection time for S. aureus biofilms was shorter for the porous materials (beta-TCP and processed human spongiosa, p < 0.001) compared to the smooth materials (PMMA and PE), with no differences between beta-TCP and processed human spongiosa (p > 0.05) or PMMA and PE (p > 0.05). In contrast, for S. epidermidis biofilms the detection time was different (p < 0.001) between all materials except between processed human spongiosa and PE (p > 0.05). The quantitative analysis by quantitative culture after washing and sonication of the material demonstrated the importance of monitoring factors like specific surface or porosity of the test materials. Isothermal microcalorimetry proved to be a suitable tool for an accurate, non-invasive and real-time microbiological assay, allowing the detection of bacterial biomass without removing the biofilm from the surface.

Iron oxidation on the surface of adventitious roots and its relation to aerenchyma formation in rice genotypes

Establishment of the water layer in an irrigated rice crop leads to consumption of free oxygen in the soil which enters in a chemical reduction process mediated by anaerobic microorganisms, changing the crop environment. To maintain optimal growth in an environment without O2, rice plants develop pore spaces (aerenchyma) that allow O2 transport from air to the roots. Carrying capacity is determined by the rice genome and it may vary among cultivars. Plants that have higher capacity for formation of aerenchyma should theoretically carry more O2 to the roots. However, part of the O2 that reaches the roots is lost due to permeability of the roots and the O2 gradient created between the soil and roots. The O2 that is lost to the outside medium can react with chemically reduced elements present in the soil; one of them is iron, which reacts with oxygen and forms an iron plaque on the outer root surface. Therefore, evaluation of the iron plaque and of the formation of pore spaces on the root can serve as a parameter to differentiate rice cultivars in regard to the volume of O2 transported via aerenchyma. An experiment was thus carried out in a greenhouse with the aim of comparing aerenchyma and iron plaque formation in 13 rice cultivars grown in flooded soils to their formation under growing conditions similar to a normal field, without free oxygen. The results indicated significant differences in the volume of pore spaces in the roots among cultivars and along the root segment in each cultivar, indicating that under flooded conditions the genetic potential of the plant is crucial in induction of cell death and formation of aerenchyma in response to lack of O2. In addition, the amount of Fe accumulated on the root surface was different among genotypes and along the roots. Thus, we concluded that the rice genotypes exhibit different responses for aerenchyma formation, oxygen release by the roots and iron plaque formation, and that there is a direct relationship between porosity and the amount of iron oxidized on the root surface.

PSD-95 promotes synaptogenesis and multiinnervated spine formation through nitric oxide signaling

Postsynaptic density 95 (PSD-95) is an important regulator of synaptic structure and plasticity. However, its contribution to synapse formation and organization remains unclear. Using a combined electron microscopic, genetic, and pharmacological approach, we uncover a new mechanism through which PSD-95 regulates synaptogenesis. We find that PSD-95 overexpression affected spine morphology but also promoted the formation of multiinnervated spines (MISs) contacted by up to seven presynaptic terminals. The formation of multiple contacts was specifically prevented by deletion of the PDZ(2) domain of PSD-95, which interacts with nitric oxide (NO) synthase (NOS). Similarly, PSD-95 overexpression combined with small interfering RNA-mediated down-regulation or the pharmacological blockade of NOS prevented axon differentiation into varicosities and multisynapse formation. Conversely, treatment of hippocampal slices with an NO donor or cyclic guanosine monophosphate analogue induced MISs. NOS blockade also reduced spine and synapse density in developing hippocampal cultures. These results indicate that the postsynaptic site, through an NOS-PSD-95 interaction and NO signaling, promotes synapse formation with nearby axons.

Self-organization mechanisms for the formation on nearshore crescentic and transverse sand bars

The formation and development of transverse and crescentic sand bars in the coastal marine environment has been investigated by means of a nonlinear numerical model based on the shallow-water equations and on a simpli ed sediment transport parameterization. By assuming normally approaching waves and a saturated surf zone, rhythmic patterns develop from a planar slope where random perturbations of small amplitude have been superimposed. Two types of bedforms appear: one is a crescentic bar pattern centred around the breakpoint and the other, herein modelled for the rst time, is a transverse bar pattern. The feedback mechanism related to the formation and development of the patterns can be explained by coupling the water and sediment conservation equations. Basically, the waves stir up the sediment and keep it in suspension with a certain cross-shore distribution of depth-averaged concentration. Then, a current flowing with (against) the gradient of sediment concentration produces erosion (deposition). It is shown that inside the surf zone, these currents may occur due to the wave refraction and to the redistribution of wave breaking produced by the growing bedforms. Numerical simulations have been performed in order to understand the sensitivity of the pattern formation to the parameterization and to relate the hydro-morphodynamic input conditions to which of the patterns develops. It is suggested that crescentic bar growth would be favoured by high-energy conditions and ne sediment while transverse bars would grow for milder waves and coarser sediment. In intermediate conditions mixed patterns may occur.

Biocontrol of seed pathogens and growth promotion of common bean seedlings by Trichoderma harzianum

The objective of this work was to evaluate isolates of Trichoderma harzianum regarding biocontrol of common bean seed-borne pathogens, plant growth promotion, and rhizosphere competence. Five isolates of T. harzianum were evaluated and compared with commercial isolate (Ecotrich), Carboxin+Thiram, and an absolute control. Bean seeds of the cultivar Jalo Precoce, contaminated with Aspergillus, Cladosporium, and Sclerotinia sclerotiorum, were microbiolized with antagonists, and seed health tests were carried out. Isolates were evaluated on autoclaved substrate and in field conditions. Ten days after sowing (DAS), plant length was measured. To test rhizosphere competence, isolates were applied in boxes containing autoclaved washed sand, and root colonization was evaluated at 10 DAS, using five plants per box. The most effective isolates in the seed health tests were: CEN287 and CEN289 to control Aspergillus; the commercial isolate to control Cladosporium; and CEN287 and CEN316 to control S. sclerotiorum. Isolates CEN289 and CEN290 promoted bean growth in greenhouse and field. Seed treatment with T. harzianum reduces the incidence of Aspergillus, Cladosporium, and S. sclerotiorum in 'Jalo Precoce' common bean seeds.

Assessing the viability of cryopreserved coconut zygotic embryos by electrolytic conductivity and potassium leaching

The objective of this work was to adapt the application of electrolytic conductivity and potassium leaching tests to assess the viability of cryopreserved embryos of 'Anão Verde do Brasil de Jiqui' (AVeJBr) coconut. The zygotic embryos were excised, sterilized and subjected to four cryoprotectant treatments combined with three incubation times (12, 16 and 20 hours), totaling 12 treatments. The pre-treatment of mature zygotic embryos of AVeJBr coconut using a cryoprotectant with 1.75 mol L-1 of sucrose + 15% glycerol for 12 and 16 hours promoted lower embryo humidity and increased viability in electrolytic conductivity and potassium leaching tests. Samples with ten embryos are sufficient for electrolytic conductivity analysis in cryopreserved or non-cryopreserved AVeJBr coconut zygotic embryos. The 4 to 8 hour imbibition period of the embryos is promising for the electrolytic conductivity analysis of non-cryopreserved mature zygotic embryos of AVeJBr coconut.

CLONAL PROPAGATION OF NEEM (Azadirachta indica A. Juss.) VIA DIRECT AND INDIRECT IN VITRO REGENERATION

ABSTRACTThe study was conducted with shoot tip explants of neem (Azadirachta indica A. Juss) to identify a viable regenerative process. Shoot tips were obtained from neem embryos cultured alternatingly in DKW medium supplemented with BAP and medium without hormones. Initial shoot development was influenced by cotyledon presence. Basal callus, excised from in vitro stem base, also presented organogenic potential. In some cases, plant lines, obtained from each seed, presented different characteristics. The most common characteristic observed in vitro was callus formation at the stem base. However, the rarest characteristics were stem callus formation and leaf senescence. The regenerated shoot tips were further subculture and rooted on a medium supplemented with IBA so that complete plants could be obtained. The rooted plants were transplanted to a greenhouse and successfully acclimatized. No significant differences in in vivo development were observed between neem plants from callus and from shoot tip propagation.

Influence of seed size and ecological factors on the germination and emergence of field bindweed (Convolvulus arvensis)

An understanding of seed germination ecology of weeds can assist in predicting their potential distribution and developing effective management strategies. Influence of environmental factors and seed size on germination and seedling emergence of Convolvulus arvensis (field bindweed) was studied in laboratory and greenhouse conditions. Germination occurred over a wide range of constant temperatures, between 15 and 40 ºC, with optimum germination between 20 and 25 ºC. Time to start germination, time to 50% germination and mean germination time increased while germination percentage and germination index decreased with an increase in temperature from 20 ºC, salinity and osmotic stress. However, germination was tolerant to low salt (25 mM) or osmotic stress (0.2 MPa), but as salinity and osmotic stress increased, germination percentage and germination index decreased. Seeds of C. arvensis placed at soil surface showed maximum emergence and decreased as seeding depth increased. Seeds of C. arvensis germinated over a wide range of pH (4 to 9) but optimum germination occurred at pH 6 to 8. Under highly alkaline and acidic pH, time to start germination, time to 50% germination and mean germination time increased while germination percentage and germination index decreased. Increase in field capacity caused decreased time to start germination, time to 50% germination and mean germination time but increased germination percentage and germination index. Bigger seeds had low time to start germination, time to 50% germination and mean germination time but high germination percentage and germination index. Smaller seeds were more sensitive to environmental factors as compared to larger or medium seeds. It can be concluded that except for pH, all environmental factors and seed sizes adversely affect C. arvensis as regards seed germination or emergence and germination or emergence traits, and larger seeds result in improved stand establishment and faster germination than small seeds, regardless of moisture stress or deeper seeding depth.