A simple and efficient method for isolating small RNAs from different plant species

Background  

Small RNAs emerged over the last decade as key regulators in diverse biological processes in eukaryotic organisms. To identify and study small RNAs, good and efficient protocols are necessary to isolate them, which sometimes may be challenging due to the composition of specific tissues of certain plant species. Here we describe a simple and efficient method to isolate small RNAs from different plant species.     

Probing mode of action in plant cell cycle by the herbicide endothall, a protein phosphatase inhibitor

The mode of action of endothall, an herbicide which was reported to inhibit plant protein phosphatases 1 (PP1) and 2A (PP2A), was investigated. For initial characterization, a series of bioassays was used for comprehensive physiological profiling of endothall effects which suggested a phytotoxic mode of action similar to mitotic disrupter herbicides. Unlike known microtubule disrupters, endothall did not inhibit soybean tubulin polymerization in vitro. As shown in meristematic corn root tips, endothall distorted the orientation of cell division plane and microtubule spindle structures which led to cell cycle arrest in prometaphase. In tobacco BY-2 cells, malformed spindles together with prometaphase arrest of nuclei and abnormal perinuclear microtubule patterns were detected as early as 4 h of endothall treatment. These effects were also observed after treatment with other protein phosphatase inhibitors, cantharidin and okadaic acid, which phenocopied the mitotic changes described in tonneau1 (ton1) and tonneau2 (ton2) Arabidopsis mutants. These mutants are defective in TONNEAU2 (TON2) protein, a regulatory subunit of PP2A, which governs cell division plane and microtubule orientation. Therefore, PP2A/TON2 phosphatase complex is suggested to be an in planta molecular target of endothall. However, in BY-2 cells, additional effects of endothall, including inhibition of S-phase initiation and DNA synthesis, detected by 5-ethynyl-2′-deoxyuridine (EdU) incorporation, and condensed nuclei arrested in late mitosis were observed which were not reported in Arabidopsiston1 and ton2 mutants. This result indicates that two additional checkpoints in cell cycle were blocked by endothall which are probably not associated with TON2-pathway inhibition. Possibly, inhibition of PP1 and/or other PP2A protein phosphatases are involved in the regulation of these cell cycle phenomena.     

Dynamic plant uptake model applied for drip irrigation of an insecticide to pepper fruit plants

BACKGROUND: Drip application of insecticides is an effective way to deliver the chemical to the plant that avoids off‐site movement via spray drift and minimizes applicator exposure. The aim of this paper is to present a cascade model for the uptake of pesticide into plants following drip irrigation, its application for a soil‐applied insecticide and a sensitivity analysis of the model parameters. RESULTS: The model predicted the measured increase and decline of residues following two soil applications of an insecticide to peppers, with an absolute error between model and measurement ranging from 0.002 to 0.034 mg kg fw^?1. Maximum measured concentrations in pepper fruit were approximately 0.22 mg kg fw^?1. Temperature was the most sensitive component for predicting the peak and final concentration in pepper fruit, through its influence on soil and plant degradation rates. CONCLUSION: Repeated simulations of pulse inputs with the cascade model adequately describe soil pesticide applications to an actual cropped system and reasonably mimic it. The model has the potential to be used for the optimization of practical features, such as application rates and waiting times between applications and before harvest, through the integrated accounting of soil, plant and environmental influences. Copyright © 2011 Society of Chemical Industry     

Detection of potentially allergenic hazelnut (Corylus avellana) residues in food: a comparative study with DNA PCR-ELISA and protein sandwich-ELISA

Allergen detection is of increasing interest for food labeling purposes. A comparative study with a commercial hazelnut-specific PCR-ELISA and a sandwich-type ELISA detecting hazelnut protein was performed to investigate to what extent immunochemical and DNA-based techniques would correlate in the detection of trace amounts of potentially allergenic hazelnut residues. Both methods were highly sensitive and allowed the detection of even <10 ppm of hazelnut in complex food matrixes. The protein-ELISA was highly specific for hazelnut. However, some foods could lead to false-positive results at the 10 ppm level. The PCR-ELISA did not show any cross-reactions with non-hazelnut foods, thus reducing the probability of having false positives at the trace level. Forty-one commercial food products with and without hazelnut components on their labels were analyzed for the presence of hazelnut. Of the 27 products in which hazelnut components were detected, two samples were not identified by the protein-ELISA, and only one sample, namely one white chocolate having <1 ppm of hazelnut protein, was not detected by PCR-ELISA. The good correlation of the results of PCR-ELISA and protein-ELISA suggested that both PCR-based and immunochemical techniques are suitable for reliable detection of potentially allergenic hazelnut residues in foods at the trace level.     

Mixing of different mineral soil layers by endogeic earthworms affects carbon and nitrogen mineralization

The effect of the endogeic earthworm species Octolasion tyrtaeum (Savigny) on decomposition of uniformly ¹⁴C-labelled lignin (lignocellulose) was studied in microcosms with upper mineral soil (Ah-horizon) from two forests on limestone, representing different stages of succession, a beech- and an ash-tree-dominated forest. Microcosms with and without lower mineral soil (Bw-horizon) were set-up; one O. tyrtaeum was added to half of them. It was hypothesised that endogeic earthworms stabilise lignin and the organic matter of the upper mineral soil by mixing with lower mineral soil of low C content. Cumulative C mineralization was increased by earthworms and by the addition of lower mineral soil. Effects of the lower mineral soil were more pronounced in the beech than in the ash forest. Cumulative mineralization of lignin was strongly increased by earthworms, but only in the beech soil (+24.6%). Earthworms predominantly colonized the upper mineral soil; mixing of the upper and lower mineral soils was low. The presence of lower mineral soil did not reduce the rates of decomposition of organic matter and lignin; however, the earthworm-mediated increase in mineralization was less pronounced in treatments with (+8.6%) than in those without (+14.1%) lower mineral soil. These results indicate that the mixing of organic matter with C-unsaturated lower mineral soil by endogeic earthworms reduced microbial decomposition of organic matter in earthworm casts.     

Correlation of acrylamide generation in thermally processed model systems of asparagine and glucose with color formation, amounts of pyrazines formed, and antioxidative properties of extracts

The relations between the formation of acrylamide and color, pyrazines, or antioxidants in an asparagine/d-glucose browning model system under various conditions were investigated. The highest level of acrylamide was produced in the asparagine/glucose (1:3) system heated at 170 degrees C for 30 min (2629 microg/g asparagine). Color intensity increased with temperature and heating time. The formation of pyrazines increased steadily with an increase of temperature (140-170 degrees C) and heating time (15-60 min). Antioxidant formation varied among the samples heated under different conditions. A clear correlation between formation of acrylamide and browning color was obtained. The formation of acrylamide was linearly correlated with the formation of total pyrazines during the initial stages of the Maillard reaction. No obvious correlation between formation of acrylamide and antioxidants was observed. However, excess amounts of asparagine increased the formation of antioxidants, whereas excess amounts of glucose reduced its formation.     

Changes in the succession and diversity of protozoan and microbial populations in soil spiked with a range of copper concentrations

We studied microbial and protozoan activity, diversity and abundance as affected by Cu²⁺ amendments ranging from 0 to 1000 μg g⁻¹ over a 70-day period. At the end of the experiment the microbial population size, as indicated by substrate-induced respiration, had normalized for all Cu²⁺ concentrations, but 1000 μg g⁻¹. Protozoan abundance was negatively affected by Cu²⁺, although, only in the first few weeks. A more detailed analysis of the individual components that make up the microbial and micro-faunal populations (phospholipid fatty acid (PLFA) profile and protozoan morphotypes), however, yielded a somewhat more complex picture. For the three highest Cu²⁺ amendments (160, 400 and 1000 μg g⁻¹), there still was a significant reduction in number of differentiable protozoan morphotypes. The bacterial PLFA pattern suggested a shift from Gram-negative towards Gram-positive bacteria for the high amendments, a process where protozoan grazing most likely played a significant role. The ratio of the trans/cis isomers of the 16:1ω7 fatty acid indicated that Cu²⁺, even at low and medium concentrations, induced physiological changes in the microbial population. The relatively slight changes in total microbial and micro-faunal abundance and activity, also at the highest Cu²⁺ concentrations, probably reflected the ability of the community to compensate for loss of taxa by functional substitution.