Lorenza Bellani

Ecotoxicity of halloysite nanotube–supported palladium nanoparticles in Raphanus sativus L

Halloysite nanotubes (HNTs) are natural nanomaterials that are biocompatible and available in large amounts at low prices. They are emerging nanomaterials with appealing properties for applications like support for metal nanoparticles (NPs). The potential environmental impacts of NPs can be understood in terms of phytotoxicity. Current research has been focusing on HNT applications in cell or animal models, while their use in plants is limited so their ecotoxicological impact is poorly documented. To date there are no studies on the phytotoxic effects of functionalized halloysites (functionalized-HNTs). To develop a quantitative risk assessment model for predicting the potential impact of HNT-supported palladium nanoparticles (HNT-PdNPs) on plant life, an investigation was undertaken to explore their effects on seed germination, seedling development, and mitotic division in root tip cells of 2 lots of Raphanus sativus L. with different vigor. The results showed that exposure to 1500 mg/L of HNTs, functionalized-HNTs, and HNT-PdNPs had no significant influence on germination, seedling development, xylem differentiation, or mitotic index in both lots. Cytogenetic analyses revealed that treatments with functionalized-HNT significantly increased the number of aberrations in low-vigor seeds. These results suggest that low-vigor seeds represent a model for a stress test that would be useful to monitor the effects of NPs. Moreover the present study offers scientific evidence for the use of halloysite for environmental purposes, supporting the biological safety of HNT-PdNPs. Environ Toxicol Chem 2016;35:2503-2510.

Ecotoxicity of halloysite nanotubes supported palladium nanoparticles on Raphanus sativus L.

Halloysite nanotubes (HNTs) are natural nanomaterials that are biocompatible and available in large amounts at low prices. They are emerging nanomaterials with appealing properties for applications like support for metal nanoparticles (NPs). The potential environmental impacts of NPs can be understood in terms of phytotoxicity. Current research has been focusing on HNT applications in cell or animal models, while their use in plants is limited so their ecotoxicological impact is poorly documented. To date there are no studies on the phytotoxic effects of functionalized halloysites (functionalized-HNTs). To develop a quantitative risk assessment model for predicting the potential impact of HNT-supported palladium nanoparticles (HNT-PdNPs) on plant life, an investigation was undertaken to explore their effects on seed germination, seedling development, and mitotic division in root tip cells of 2 lots of Raphanus sativus L. with different vigor. The results showed that exposure to 1500 mg/L of HNTs, functionalized-HNTs, and HNT-PdNPs had no significant influence on germination, seedling development, xylem differentiation, or mitotic index in both lots. Cytogenetic analyses revealed that treatments with functionalized-HNT significantly increased the number of aberrations in low-vigor seeds. These results suggest that low-vigor seeds represent a model for a stress test that would be useful to monitor the effects of NPs. Moreover the present study offers scientific evidence for the use of halloysite for environmental purposes, supporting the biological safety of HNT-PdNPs. Environ Toxicol Chem 2016;35:2503-2510.

Effects of copper on germination and reserve mobilization in Vicia sativa L. seeds

The effects of different copper concentrations on percentage germination, increase in fresh weight and radicle growth of Vicia sativa L. seeds were studied. Physiological studies showed that the germination rate was not affected up to a concentration of 5 × 10⁻³ M, but already at 10⁻³ M the copper stopped root elongation. Structural and ultrastructural observations of embryo and cotyledon reserve mobilization showed that inhibition of radicle growth at 10⁻³ M Cu concentration cannot be ascribed to nutrient shortage but probably to an effect of copper on radicle cell division and elongation. In seeds treated with 5 × 10⁻³ M CuBr₂, the copper completely inhibited cotyledon protein mobilization, so that embryo protein mobilization supported normal growth of the radicle up to 30 h after imbibition. The particular protein content of adjacent cotyledon cells is also discussed.

Tocopherol, fatty acid and phytosterol content in seeds of nine wild taxa of Sicilian Brassica (Cruciferae)

Abstract The purpose of this study was to determine tocopherol (Toc), fatty acid and phytosterol content in dry seeds of nine wild taxa of Sicilian Brassica sect. Brassica. Analyses were performed by gas chromatography (GC)‐mass spectroscopy (MS). The biochemical analysis of dry seeds showed that in the nine taxa examined, α‐ and γ‐Toc were the major isomers, δ‐Toc was present in traces and β‐Toc was never found. All taxa had a similar fatty acid composition with C 16:0 (357.2 mg kg−1 DW), C 18:1 (393.94 mg kg−1 DW) and C 22:1 (618.29 mg kg−1 DW) acids present in higher amount. Unsaturated fatty acids (52.5–66.5%) were more abundant than saturated (33.5–47.5%) ones relative to the total fatty acids. Moreover, sitosterol was the most abundant (61.7–69.6%) of all phytosterols in all species and subspecies, followed by brassicasterol and campesterol at 16.0–19.3% and 11.9–20.7%, respectively. Total Toc content and the relative percentage of single isoforms were useful to discriminate seeds of B. incana, B. macrocarpa, B. rupestris group and B. villosa group. On the basis of these results, B. villosa subsp. villosa, B. villosa subsp. bivoniana, B. villosa subsp. drepanensis and B. villosa subsp. tinei can be ascribed to the B. villosa group. These taxa, having the highest T‐Toc, γ‐Toc and phytosterol contents, are potentially the most bioactive ones, and could, therefore, be exploited in breeding programmes to develop genotypes with putative positive effects on human health. Abbreviations: B, Brassica; DW, dry weight; FA, fatty acids; RH, relative humidity; T‐FA, total fatty acids; T‐Phy, total phytosterols; T‐Toc, total tocopherol; Toc, tocopherol

An ultrastructural study of the mature spermatozoid of the fern Asplenium trichomanes L. subsp. trichomanes

Abstract Asplenium trichomanes L. subsp. trichomanes spermatozoids are spirals of about five turns. Keels link the elements of the microtubular ribbon with the plates of the lamellar layer (LL) which are uninterrupted, parallel and curved with an inner angle of about 150°. Electron-opaque filaments connect the microtubules of the multilayered structure (MLS) and the osmiophilic crest, the LL and the MLS-associated mitochondrion and the latter and the plasmalemma. The nucleus occupies the 2.5–3 posterior turns and has an inner honeycomb-shaped chromatin mass and an outer highly condensed chromatin mass with randomly scattered electron-transparent areas. The basal bodies of the ca. 50 flagella are bounded by a reticulum of granular material which forms a plug inside their proximal region; the proximal region of the flagellum has a 9 + 0 pattern. The axoneme has a 9 + 2 pattern.

Analysis of extracellular vesicles produced in the biofilm by the dimorphic yeast Pichia fermentans

The yeast Pichia fermentans DISAABA 726 strain (P. fermentans) is a dimorphic yeast that under different environmental conditions may switch from a yeast‐like to pseudohyphal morphology. We hypothesize that exosomes‐like vesicles (EV) could mediate this rapid modification. EV are membrane‐derived vesicles carrying lipids, proteins, mRNAs and microRNAs and have been recognized as important mediators of intercellular communication. Although it has been assumed for a long time that fungi release EV, knowledge of their functions is still limited. In this work we analyze P. fermentans EV production during growth in two different media containing urea (YCU) or methionine (YCM) where yeast‐like or pseudohyphal morphology are produced. We developed a procedure to extract EV from the neighboring biofilm which is faster and more efficient as compared to the widely used ultracentrifugation method. Differences in morphology and RNA content of EV suggest that they might have an active role during dimorphic transition as response to the growth conditions. Our findings are coherent with a general state of hypoxic stress of the pseudohyphal cells.

Response to copper bromide exposure in Vicia sativa L. seeds: analysis of genotoxicity, nucleolar activity and mineral profile.

Copper bromide (CuBr2) effects on seed germination and plantlet development of Vicia sativa L. are evaluated through mitotic index, chromosome aberrations, nucleolar activity and mineral profile. CuBr2 induces a significant presence of micronuclei, sticky and c-metaphases, anaphase bridges and chromosome breaks. Increased number of nucleoli and scattering of AgNOR proteins from the nucleolus in the nuclear surface at CuBr2 1mM and in the cytoplasm at CuBr2 5mM, goes along with the decrease of root growth. In V. sativa embryo the content of many macro and micronutrients increases up to copper 1mM in agreement with reserve mobilization while at CuBr2 5mM some elements are present in lower amount. We hypothesize that inhibitory effects observed at 5mM are due either to a nutrient shortage or to a direct influence of copper on root cell division, evidenced by low mitotic index, high occurrence of chromosome aberrations and loss of material from the nucleolus.

Stress integrated tests and cytological analyses reveal Brassica villosa subsp. drepanensis seed quality decrease upon long-term storage

Under stress integrated germination test (SIGT), seeds undergo osmo-saline stresses, which enable to detect differences in vigour of long-term stored seeds with high germination percentage (G%). The quality of Brassica villosa subsp. drepanensis seeds stored in a genebank (at − 20°C for 16 years) was compared with seeds at harvest by standard germination tests (GT), SIGT and cytogenetic analysis. No differences were detected in G% and mean germination time under GT. Conversely, SIGT performed with NaCl − 0.9 MPa osmotic potential did not influence G% at harvest but reduced that of stored seeds, SIGT at − 1.4 MPa reduced G% of both. Cytogenetic analysis showed reduction of mitotic index, appearance of chromosomal aberrations and smaller nucleoli in stored seeds compared with harvest seeds germinated in water. SIGT at − 0.9 MPa had no effect on mitotic index, but increased chromosome aberrations and nucleoli number. SIGT at − 1.4 MPa inhibited G% of harvest and stored seeds, reduced mitoses in harvest and completely prevented it in stored seeds. The results indicate that GT does not faithfully reflect the quality of stored seeds, with misinterpretation of their vigour, whereas SIGT and cytogenetical parameters are sensitive, reliable and inexpensive methods for early prediction of genetic erosion in germplasm banks.

An integrated approach to highlight biological responses of Pisum sativum root to nano-TiO2 exposure in a biosolid-amended agricultural soil

This study focused on crop plant response to a simultaneous exposure to biosolid and TiO2 at micro- and nano-scale, being biosolid one of the major sink of TiO2 nanoparticles released into the soil environment. We settled an experimental design as much as possible realistic, at microcosm scale, using the crop Pisum sativum. This experimental design supported the hypotheses that the presence of biosolid in the farming soil might influence plant growth and metabolism and that, after TiO2 spiking, the different dimension and crystal forms of TiO2 might be otherwise bioavailable and differently interacting with the plant system. To test these hypotheses, we have considered different aspects of the response elicited by TiO2 and biosolid at cellular and organism level, focusing on the root system, with an integrative approach. In our experimental conditions, the presence of biosolid disturbed plant growth of P. sativum, causing cellular damages at root level, probably through mechanisms not only oxidative stress-dependent but also involving altered signalling processes. These disturbances could depend on non-humified compounds and/or on the presence of toxic elements and of nanoparticles in the biosolid-amended soil. The addition of TiO2 particles in the sludge-amended soil, further altered plant growth and induced oxidative and ultrastructural damages. Although non typical dose-effect response was detected, the most responsiveness treatments were found for the anatase crystal form, alone or mixed with rutile. Based on ultrastructural observations, we could hypothesise that the toxicity level of TiO2 nanoparticles may depend on the cell ability to isolate nanoparticles in subcellular compartments, avoiding their interaction with organelles and/or metabolic processes. The results of the present work suggest reflections on the promising practice of soil amendments and on the use of nanomaterials and their safety for food plants and living organisms.

Effect of treatments of different duration with NaCl on growth, differentiation and esterase activity in excised radish cotyledons

Summary Radish etiolated cotyledons were excised from the seed after 24 hours of imbibition and incubated for different times on filter paper satured with NaCl solutions or water. After the incubation of different times in NaCl, the cotyledons were thoroughly rinsed in water and grown in darkness on filter paper satured with distilled water. Continuous presence of NaCl is not a prerequisite for optimal growth. Several effects of the NaCl solutions are less intensive if the cotyledons are treated with NaCl for a relatively short time and then grown in water.