Advanced techniques to investigate the internalization mechanism of TiO2 NPs in the roots grown in a biosolid-amended agricultural soil

Plants play an important role in introducing the engineered nanoparticles (ENPs) into the food chain. The pathway of ENPs uptake from soil, their distribution in the edible plant parts, and their impact in the food production are important issues to be investigated. In the present study, Pisum sativum plants were grown at microcosm scale under medium-term TiO2 NPs exposure, to possibly mime environmental conditions in an agricultural soil amended with biosolids from a wastewater treatment plant in Pisa, Italy. TiO2 NPs were applied as pure rutile, pure anatase and a mixture of both crystalline phases in the biosolid amended-soil. Micro-XRF and μ-XANES from ID21 beamline were used for Ti elemental mapping and crystalline phase identification to indicate a relative distribution/localization of TiO2 crystalline phases within a given cross-section of roots, as well as the possible speciation and preferential crystalline phase uptake in the roots. Titanium in roots showed a main localization in the rizoderma, independently of the crystalline phase. Fewer Ti spots were found localized in the cortex or in vessel, however the roots grown in presence of a mixture of both phases showed a main presence of anatase, suggesting a preferential adsorption and translocation of this crystalline form through the roots. Our data indicated also a reduced translocation of Ti to the aerial part of the plant, confirming the chemical analysis of shoots and roots separately, which showed that Ti concentration was about 40 times lower in the upper part than in the below ground tissues. The TiO2 NPs were characterized on the basis of their size and shape by TEM analysis. Moreover, observations on cell ultrastructure of control and of anatase, rutile and mixture of both crystalline phases treated roots were performed. The root cells of plant grown in the presence of all NPs treatments shared the same alterations of ultrastructure: mitochondria with swollen cristae, nuclei with condensed chromatin, and part of the cytoplasm degraded, probably in consequence of an autophagic process. As detected by μ-XRF and μ-XANES, electron dense prismatic or round profiled particles of about 30-40 nm were observed mainly in form of aggregates in the intercellular spaces or crossing the wall of the cells next to rizoderma and in the cortex cells. Furthermore, the anatase treated cells were mostly damaged in respect to control and rutile treated roots, and more frequently internalized NPs were observed in these samples

Effects of copper on spore germination, growth and ultrastructure of Polypodium cambricum L. gametophytes

The effects of different concentrations (10-5, 5 x 10-5 and 10-4 M) of copper bromide on spore germination, growth and ultrastructure were investigated in Polypodium cambricum L. gametophytes. The inhibitory effect of Cu was observed in spores cultured on medium supplemented with 10-4 M CuBr2: germination occurred about 40 days after sowing and was only 25%. Concentrations of 5 x 10-5 and 10-4 M CuBr2 induced changes in gametophyte development, possibly by re-orientation of growth. Gametophytes treated with 10-5 and 5x 10-5 M CuBr2 took up and accumulated a large amount of copper and ultrastructural observations showed that cytoplasmic damage was limited to twisted swollen thylakoids. The ultrastructure of gametophytes treated with 10-4 M CuBr2 showed absence of a vacuolar compartment. The present observations suggest that P. cambricum gametophytes could be a suitable material for studying physiological and molecular alterations induced by excess copper

Ultrastructure, viability, and in vitro germination of the tricellular Sambucus nigra L. pollen

Mature pollen grains of Sambucus nigra L. are tricolporate, isopolar, 12.5 mm wide, 25 mm long, and tricellular. They have a tectate exine, a bilayered nexine, and a thin intine. The vegetative cell has abundant rough endoplasmic reticulum, many well-structured mitochondria, numerous apparently inactive dictyosomes, immature dividing, and starch-filled plastids and lipid bodies. The sperm cells have few and poorly structured organelles. They are linked by cytoplasmic bridges, indicating a persistent physiological unit, and are surrounded by fibrillar polysaccharide material. Freshly released pollen is 95% viable. Three- and 4-year-old pollen grains stored at 20C are 78.2% and 43% viable, respectively. In vitro germination requirements and pollen tube growth are more similar to those of bicellular pollen than to those of tricellular pollen

Effects of copper on reserve mobilization in embryo of Phaseolus vulgaris L.

The present research reports a biochemical and micro-submicroscopic analysis of copper effect on reserves mobilization during germination of Phaseolus vulgaris L. var. soisson nain hatif seeds. Dry embryonic cells are rich in protein bodies and little starch grains. In Cu-treated embryos copper inhibited 50% of albumin and globulin mobilization after 72 h imbibition. The severe alterations in treated embryo cells, observed by electron microscope, were probably the cause of the inability to utilize the amino acids freed by protein mobilization and so possibly the cause of the inhibition of P. vulgaris embryonic axes elongation

Titanium dioxide nanoparticles enhance the detrimental effect of polystyrene nanoplastics on cell and plant physiology of Vicia lens (L.) Coss. & Germ. seedlings

Polystyrene nanoplastics and titanium dioxide nanoparticles are widely spread in all environments, often coexisting within identical frameworks. Both these contaminants can induce negative effects on cell and plant physiology, giving concerns on their possible interaction which could increase each other's harmful effects on plants. Despite the urgency of this issue, there is very little literature addressing it. To evaluate the potential risk of this co-contamination, lentil seeds were treated for five days with polystyrene nanoplastics and titanium dioxide nanoparticles (anatase crystalline form), alone and in co-presence. Cytological analyses, and histochemical and biochemical evaluation of oxidative stress were carried out on isolated shoots and roots. TEM analysis seemed to indicate the absence of physical/chemical interactions between the two nanomaterials. Seedlings under cotreatment showed the greatest cytotoxic and genotoxic effects and high levels of oxidative stress markers associated with growth inhibition. Even if biochemical data did not evidence significant differences between materials treated with polystyrene nanoplastics alone or in co-presence with titanium dioxide nanoparticles, histochemical analysis highlighted a different pattern of oxidative markers, suggesting a synergistic effect by the two nanomaterials. In accordance, the fluorescence signal linked to nanoplastics in root and shoot was higher under cotreatment, perhaps due to the well-known ability of titanium dioxide nanoparticles to induce root tissue damage, in this way facilitating the uptake and translocation of polystyrene nanoplastics into the plant body. In the antioxidant machinery, peroxidase activity showed a significant increase in treated roots, in particular under cotreatment, probably more associated with stress-induced lignin synthesis than with hydrogen peroxide detoxification. Present results clearly indicate the worsening by metal nanoparticles of the negative effects of nanoplastics on plants, underlining the importance of research considering the impact of cotreatments with different nanomaterials, which may better reflect the complex environmental conditions

Nano and submicron fluorescent polystyrene particles internalization and translocation in seedlings of Cichorium endivia L

Contamination by plastics is one of the major causes of pollution of the terrestrial environment. Fragmentation of plastics into micro and nano particles may result in negative interactions between polymers and terrestrial ecosystems. The effects of nano (20 nm) and submicron (200 nm) fluorescent polystyrene (PS) particles, at different concentrations (0.01, 0.1, 1 g L−1), were analysed on chicory plant (Cichorium endivia L.), considering the following endpoints: germination percentage and seedling development after 7 and 14 days exposure; genotoxic effects; polyphenols and photosynthetic pigments content, antioxidant activity; absorption and translocation of PS particles in the seedling tissues. The results indicated no negative effects on germination of both PS particles' sizes at any concentration; seedlings elongation was affected by 1 g L−1 of 20 nm PS after 7 days exposure. Cytological analysis revealed no mitotic activity inhibition, but an uprising of chromosomal abnormalities in all treatments. Interestingly, photosynthetic pigments always increased after PS exposure. Seedlings treated with 20 nm PS showed intense fluorescence in the roots of 7 days and in the shoots of 14 days, while 200 nm PS treated seedlings exhibited low fluorescence. Electron microscopy and infrared spectroscopy confirmed 20 nm PS internalization and transport inside the plant tissue and a reduced presence of 200 nm PS. These results suggest the importance not only of particle size in plastic internalization in plant tissues, but also of cytological damages induced by particles too large to be bioaccumulated. From both aspects, consequences may arise for plant fitness, food safety and human health