The present study was to assess the effect of heavy metal stress on the DNA methylation of a metal-sensitive plant, Trifolium repens L. and of a metal-tolerant plant, Cannabis sativa L. The changes in the level of 5-methylcytosine (5mC) in the root DNA of plants grown on soils contaminated with different concentrations of Ni 21 , Cd 21 and Cr 61 compared with that of untreated plants, were determined by immunolabelling with a monoclonal antibody, using the Slot-Blot technique. Results showed that DNA of hemp control plants was about three times more methylated than clover DNA, for the same amount of root DNA. Heavy metal treatments induced a global dose-dependent decrease of 5mC content, both in hemp and clover, ranging from 20 to 40%. Changes in methylation pattern of 5 0 -CCGG-3 0 containing sequences were investigated by methylation-sensitive amplification polymorphism (MSAP) technique. Control plants of the same species showed a very similar pattern, suggesting that, in normal condition, methylation involves precise sites. Heavy metals induced DNA methylation changes mainly related to hypomethylation events. These variations were not randomly directed but involved specific DNA sequences, since the detected polymorphisms were the same in all the plants analysed for each treatment.Abbreviations -5mC, 5-methylcytosine; AFLP, amplified fragment length polymorphism; MSAP, methylation-sensitive amplification polymorphism; oxo8dG, 8-oxo-2 0 -deoxyguanosine; ROS, reactive oxygen species.

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Background: Pollutants may affect pollen allergenicity and thus the prevalence of allergies. Although a few studies are available in literature, the connection between pollution and the allergenic potential of pollen has yet to be clearly defined. The objective of this study was to evaluate the effect of traffic-related pollution on the allergenicity of ragweed (Ambrosia artemisiifolia L.) pollen through a field-based experiment. Methods: Mature pollen grains were collected from ragweed plants grown along main roadsides and in vegetated areas of Po river plain. The percentage of subpollen particle-releasing grains (SPPGs) was evaluated immediately after sampling by microscope and image analysis. Immunochemistry and LC-MS/MS were applied to assess the whole allergenicity and the allergen pattern characterizing the different pollen samples. Results: No statistical difference was detected in the percentage of SPPGs among pollen samples. Specifically, after hydration, the mean percentage was very low (<4%) in all the samples, regardless of the site of origin. On the contrary, pollen collected along high-traffic roads showed a higher whole allergenicity than pollen from low-traffic roads and vegetated areas which showed a reactivity similar to that of the commercial pollen 'Allergon', used as a standard. The detected higher allergenicity levels were attributed to both quantitative and qualitative differences in allergen pattern. Conclusion: Our findings show that pollen collected at different sites contains different amount and number of allergens and suggest that traffic-related pollution enhances ragweed pollen allergenicity, which may contribute to the increasing prevalence of ragweed allergy in Lombardy plain.

Peach softening is usually attributed to the dismantling of the cell wall in which endo-polygalacturonase (endo-PG)-catalysed depolymerization of pectins plays a central role. In this study, the hypothesis that the function of endo-PG is critical for achieving a melting flesh fruit texture but not for reducing fruit firmness was tested by comparing pericarp morphology and endo-PG expression and localization in melting (MF) and non-melting flesh (NMF) fruit at successive stages of ripening. MF Bolero, Springbelle, and Springcrest, and NMF Oro-A and Jonia cultivars were analysed. Both MF and NMF fruit were left to ripen on the tree and reached a firmness of <10 Newtons (N). The image analysis of pericarp tissues revealed that during softening the loss of cell turgidity was a process common to mesocarp cells of all MF and NMF fruit and was clearly visible in peaches with a firmness of less than ∼20 N. In contrast, the loss of cell adhesion was a feature exclusively observed in ripe MF fruit pericarp. In this ripe fruit, large numbers of endo-PG isoforms were highly expressed and the enzyme localization corresponded to the middle lamella. As a consequence, wide apoplastic spaces characterized the pericarp of ripe MF peaches. In contrast, no loss of cell adhesion was observed in any NMF fruit or in unripe MF peaches. Accordingly, no endo-PG was detected in unripe NMF fruit, whereas few and poorly expressed enzyme isoforms were revealed in ripe NMF and in unripe MF peaches. In this fruit, the poorly expressed endo-PG localized mainly in vesicles within the cytoplasm and inner primary cell wall. On the whole the results suggested that endo-PG function was needed to achieve melting flesh texture, which was characterized by wide apoplastic spaces and partially deflated mesocarp cells. Conversely, endo-PG activity had no critical influence on the reduction of fruit firmness given the capacity of NMF peaches to soften, reaching values of 5-10 N. As in tomato, the change of symplast/apoplast water status seems to be the main process through which peach fruit regulates its firmness.

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