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.

Polystyrene nanoplastics affect seed germination, cell biology and physiology of rice seedlings in-short term treatments: Evidence of their internalization and translocation.

Agroecosystems represent more and more a huge long-term sink for plastic compounds which inevitably undergo fragmentation, generating micro- and nano-plastics, with potential adverse effects on soil chemistry and living organisms. The present work was focused on the short-term effects of two different concentrations of polystyrene nanoplastics (PSNPs) (0.1 or 1 g L-1 suspensions) on rice seedlings starting from seed germination, hypothesizing that possible acute effects on seedlings could depend on oxidative damage trigged by PSNPs internalization. As shown by TEM analysis, PSNPs were absorbed by roots and translocated to the shoots, affected root cell ultrastructure, the germination process, seedling growth and root mitotic activity, inducing cytogenetic aberration. Treatments were not correlated with increase in oxidative stress markers, but rather with a different pattern of their localization both in roots and in shoots, impairing H2O2 homeostasis and membrane damage, despite the adequate antioxidant response recorded. The harmful effects of PSNPs on cell biology and physiology of rice seedlings could be caused not only by a direct action by the PSNPs but also by changes in the production/diffusion of ROS at the tissue/cellular level.

Synchrotron Radiation Spectroscopy and Transmission Electron Microscopy Techniques to Evaluate TiO2 NPs Incorporation, Speciation, and Impact on Root Cells Ultrastructure of Pisum sativum L. Plants.

Biosolids (Bs) for use in agriculture are an important way for introducing and transferring TiO2 nanoparticles (NPs) to plants and food chain. Roots of Pisum sativum L. plants grown in Bs-amended soils spiked with TiO2 800 mg/kg as rutile NPs, anatase NPs, mixture of both NPs and submicron particles (SMPs) were investigated by Transmission Electron Microscopy (TEM), synchrotron radiation based micro X-ray Fluorescence and micro X-ray Absorption Near-Edge Structure (µXRF/µXANES) and Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). TEM analysis showed damages in cells ultrastructure of all treated samples, although a more evident effect was observed with single anatase or rutile NPs treatments. Micro-XRF and TEM evidenced the presence of nano and SMPs mainly in the cortex cells near the rhizodermis. Micro-XRF/micro-XANES analysis revealed anatase, rutile, and ilmenite as the main TiO2 polymorphs in the original soil and Bs, and the preferential anatase uptake by the roots. For all treatments Ti concentration in the roots increased by 38-56%, however plants translocation factor (TF) increased mostly with NPs treatment (261-315%) and less with SMPs (about 85%), with respect to control. In addition, all samples showed a limited transfer of TiO2 to the shoots (very low TF value). These findings evidenced a potential toxicity of TiO2 NPs present in Bs and accumulating in soil, suggesting the necessity of appropriate regulations for the occurrence of NPs in Bs used in agriculture.

Response to Salinity in Legume Species: An Insight on the Effects of Salt Stress during Seed Germination and Seedling Growth.

The process of soil salinization and the preponderance of saline water sources all over the world represent one of the most harmful abiotic stress to plant growth. This pointed to the importance of obtaining plants which are tolerant or resistant to salt, considering that projection of climate change for the coming years indicate an increase in temperature and rain scarcity. In the current study, the effect of NaCl was investigated on germinating seeds of Lathyrus sativus L., Vicia sativa L., Vigna radiata L. R.Wilczek and Vigna unguiculata L. Walp., by combining physiological, biochemical, biostatistical and ultrastructural analyses. Our results revealed that germination was not influenced by high NaCl concentrations, while seedling growth was affected even at low NaCl concentrations, probably due to an alteration in water uptake and in organic matter biosynthesis. Nevertheless, the synthesis of antioxidant enzymes, phenolic acids and flavonoids was registered in all species, which tended to cope with the increasing salt stress, allowing a response mechanism such as cytoplasm detoxification and cellular turgor maintenance. Besides, the ultrastructural analysis evidenced plasmolyzed cells close to cells with a normal ultrastructure with no appreciable differences among the species. This research deeply investigates the mechanism of salt-stress response focusing on species never tested before for their possible tolerance to salinity.

Effect of Zinc Priming on Salt Response of Wheat Seedlings: Relieving or Worsening?

In an attempt to alleviate salt-induced damage, the application of ZnO nanoparticles has been suggested. As the use of these particles has also been associated with phytotoxicity, to better clarify the effect of zinc and its possible mitigation of salt stress, we treated wheat seedlings with ZnO (nanoparticles or their bulk-scale counterparts, amended either in the growth medium, NPs and B, or sprayed on the leaves, SPNPs and SPB) with or without subsequent treatment with salt. Growth, photosynthetic parameters, zinc and ion concentration, and in situ and biochemical determination of oxidative stress in wheat leaves and/or in roots were considered. Both Zn and NaCl significantly inhibited growth and induced severe alterations in root morphology. Oxidative stress and damage decreased or increased under ZnO treatment and in saline conditions depending on the organ and on the size and mode of application of particles. In spite of the higher stress conditions often recorded in treated leaves, neither pigment concentration nor photochemical efficiency were decreased. A large variability in the effects of ZnO treatment/priming on seedling salt response was recorded; however, the presence of a cumulative negative effect of priming and salt stress sometimes observed calls for caution in the use of ZnO in protection from saline stress.

Exploring the interaction between polystyrene nanoplastics and Allium cepa during germination: Internalization in root cells, induction of toxicity and oxidative stress.

With the aim to investigate the mechanisms of action of nano plastics (nano PS) on plants, seeds of Allium cepa were germinated for 72 h in the presence of polystyrene nano PS (50 nm size, at concentrations of 0.01, 0.1 and 1 g L-1) and, subsequently, roots were analysed by a multifaceted approach. No effect was induced by any concentration of nano PS on the percentage of seed germination while root growth was inhibited by 0.1 and 1 g L-1 nano PS. Cytological analysis of the root meristems indicated cytotoxicity (reduction of mitotic index) and genotoxicity (induction of cytogenetic anomalies and micronuclei) starting from the lowest dose. Moreover, the biochemical and histochemical analysis of oxidative stress markers gave evidence of stress induction, especially at the highest doses. Damages reported could be due to mechanical surface contact in root external layers, as evidenced by histological localization, and to the internalization of nano PS in different cellular compartments, observed under TEM. The present research underlines the hazardous nature of nano PS, that for their ability to be internalized into crop plants, can enter into different trophic levels of the food chain.

Genotoxicity of the food additive E171, titanium dioxide, in the plants Lens culinaris L. and Allium cepa L.

E171 (titanium dioxide, TiO2), an authorized foods and beverage additive, is also used in food packaging and in pharmaceutical and cosmetic preparations. E171 is considered to be an inert and non-digestible material, not storable in animal tissues, but the possible presence of TiO2 nanoparticles (NP) may present a risk to human health and the environment. We determined the presence of 15% TiO2 NP in a commercial E171 food additive product, by electron microscopy. The biological effects of E171 were assessed in Lens culinaris and Allium cepa for the following endpoints: percentage of germination, root elongation, mitotic index, presence of chromosomal abnormalities, and micronuclei. The results indicated low phytotoxicity but dose-dependent genotoxicity. We also observed internalization of TiO2 NP and ultrastructural alterations in the root systems.

TiO2 nanoparticles in a biosolid-amended soil and their implication in soil nutrients, microorganisms and Pisum sativum nutrition.

The wide use of nanoparticles (NPs), gives concern about their possible negative implications in the environment and living organisms. In particular, titanium dioxide (TiO2) NPs are accumulated in biosolids (Bs) coming from wastewater treatment plants, which in turn are used as farm soil amendments and are becoming an important way of NPs entrance in the terrestrial ecosystems. In this study, to simulate a low and cumulative load of TiO2 NPs, 80 and 800 mg TiO2per Kg of soil were spiked in the Bs prior to its addition to soil. The effects of different crystal phases of TiO2 NPs (pure anatase and pure rutile or their mixture) and their non-coated bulk counterparts (larger particles) on the availability of mineral nutrients and on the status of the bacterial communities together with the nutritional status of Pisum sativum L. plants were evaluated. Results showed the reduction, to different extents, on the availability of important soil mineral nutrients (e.g. Mn 65%, Fe 20%, P 27%, averagely), in some cases size- (e.g. P) and dose-dependent. Bacterial biodiversity was also affected by the presence of high TiO2 dose in soil. The mineral nutrition of pea plants was also altered, showing the main reduction in Mn (80% in the roots and 50% in the shoots), K, Zn, P (respectively, 80, 40, and 35% in the roots), and an increase of N in the shoots, with possible consequences on the quality of the crop. The present study gives new integrated data on the effects of TiO2 NPs in the soil-plant system, on the soil health and on the nutritional quality of crops, rising new implications for future policies and human health.

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.

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.

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 reserve 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 axis elongation.

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.

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.

Protein characterization of protein bodies from cotyledons of Mucuna pruriens (L.) DC.

Seeds of Mucuna pruriens (L.) DC. (Fabaceae) were analyzed for protein composition of protein bodies isolated from cotyledons. Protein bodies were successfully separated by Lympholyte and those of dry seeds, observed by scanning electron microscope, were elliptical or spherical in shape with a diameter of 5-12 µm. Protein content in dry seed protein bodies was 10.6 mg/g dry weight. Globulin was the largest protein fraction isolated (62.5 %), followed by albumin (18.3 %), glutelin (15.8 %) and prolamin (3.4 %). The prolamin fraction and high glutelin content are uncommon in legumes. SDS-PAGE of albumins, globulins, prolamins and glutelins provided different band numbers and molecular weights under reducing and non reducing conditions and suggested that the albumin fraction is rich in disulphide bonds.

Ultrastructural and biochemical investigations of protein mobilization of Mucuna pruriens (L.) DC. cotyledons and embryo axis.

The mobilization of storage reserves, with particular emphasis on storage proteins of Mucuna pruriens (L.) DC., cotyledons, and embryo was investigated from the ultrastructural and biochemical points of view. Proteins and starch were the two main storage substances in cotyledons, and proteins and lipids were the main ones in the embryo. Embryo protein bodies were smaller and fewer in number than those of cotyledons. Structural and ultrastructural data determined between 24 and 48 h after imbibition and between 48 and 72 h after imbibition, the end of significant embryo and cotyledon protein mobilization, respectively, indicating more precocious storage protein mobilization in the axis than cotyledons. Moreover, storage protein mobilization in embryo and cotyledons occurred before the end of germination. Water soluble proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, producing 29 bands with molecular weights from 14 to 90 KDa. Embryo extract contained more proteins than cotyledon extract, contained seven characteristic bands, and showed a higher variability of the optical density trend than cotyledon.