New scenarios for crop plants: TiO2 nanoparticles in sludge-amended agricultural soil

Beyond all studies published to date on the effects of nanoparticles (NPs) on living organisms, the overall picture of their possible interactions with crop plants and with food chains are not at all clear. These emerging contaminants are becoming a worldwide problem, given that nanotechnologies are increasingly gaining ground in all sectors of the economy and innovation, with their consequent unintentional and intentional release into the environment. Poorly estimated is indeed the behavior of nanomaterials (NMs) in the different environmental matrices, especially in agricultural soils. These become a site of possible accumulation of NMs through potentially contaminated surface waters, by the use of plant protection products, and by their amendment with sludge from wastewater treatment plants. The recycle of sludge in agricultural soils is identified as one of the best environmental management practice, due to its increasing amount of production and the supply of organic matter and nutrients to the soil-plant system, but, due to the uncertainty of its contents not thoroughly tested for safety, can result a possible sink of unknown pollutants as well as of NPs. In such complex matrices the bioavailability of the different NMs often is not predictable, due to the tendency of NPs to aggregate, to adsorb/precipitate on solid phase, as well as to be coated by organic molecules. In order to investigate the environmental impact of TiO2 NPs we have used two different concentrations of the crystal forms of TiO2 (applied singularly or in a mixture as anatase and rutile NPs and as bulk material). The effects were evaluated on growth and development of the crop Pisum sativum L. at microcosm scale under long term exposure, to possibly mime environmental exposure in sludge amended agricultural soil. We were particularly interested in understanding the response elicited by NPs in tissues/organs, at cellular and organism level, taking into account different aspects. Ultrastructural studies demonstrated that the applied NPs were internalized in root cells and synchrotron studies showed that both titanium crystal forms, especially anatase, were taken up and moved to the vascular tissues. Oxidative stress was evaluated by biochemical approach and in situ histochemical techniques. The amended sludge soil was in itself an element of disturbance for plants. The presence of NPs in the sludge-amended soil pronounced oxidative damages in P. sativum, in particular that grown at the lowest NPs concentration. This result was speculatively attributed to a likely easier enter the root of more diluted NPs, having less tendency to form homo- and heteroaggregates in the complex matrix. The most responsiveness treatments seemed those conducted with anatase crystal form, alone or mixed with rutile as well as with the corresponding bulk material. The results of our work pose a reflection on the promising agronomic practices and on the use of nanomaterials and their safety, which must be carefully analyzed, in order to establish right regulation over their use, confinement, and disposal for the environmental protection and living organism health

Nanoparticles and higher plants

Abstract Nanomaterials and nanotechnology have been widely applied in the world in this last decade. Nanotechnology provides the tool and the technological platforms for the study and transformation of biological systems. Few studies have focused on the effects and mechanisms of nanomaterials on plants. The results of these studies have been reported with the aim to provide further insight into connections between plants and nanomaterials

Oil palm in vitro regeneration: microdensitometric analysis during reproduction and development

An extensive microdensitometric analysis was performed in oil palm Elaeis guineensis Jacq. during the establishment of a new embryogenic culture, on new regenerated plants at different times, and finally on seedlings obtained from adult regenerated normal and abnormal plants. Moreover nuclear DNA content was determined as well during the progression of meiosis. A variable loss of DNA content/nucleus in regenerated plants was demonstrated, in some cases very severe, depending mainly on the in vitro stationing of explants before the regeneration induction; DNA variation, but to less extent, was recorded also in seedlings from adult mother plants of the same genotype, in vivo and in vitro propagated. Considering normal and abnormal regenerated plants, DNA content variations did not seem to be necessarily connected to the abnormal phenotype. In addition stable and different variants in genome size could be obtained from in vitro culture of the same explant. Changes in the values of nuclear DNA content were determined during the meiotic prophase, confirming DNA extrusion processes also observed in other plant systems in vivo. Indeed the regeneration process may induce a quantitative DNA modulation/loss per cell in the regenerated plants but progressively DNA sequences should be regained before completion of the meiotic process. This information points to mechanisms of somaclonal variation

Female gametophyte and embryo development in Helleborus bocconei Ten. (Ranunculaceae)

In this study, we investigated cytohistochemistry, cycle progression, and relative DNA content of the female gametophyte cells of Helleborus bocconei Ten. before and after fertilization process. The early stages of embryo development were also investigated. H. bocconei possesses a monosporic seven-celled/eight-nucleate Polygonum type female gametophyte, characterized by a morpho-functional polarity. The cells of the embryo sac showed abundant reserves of polysaccharides, strongly increasing in the egg cell just before fertilization. With different timing in DNA replication during cell cycle progression, synergids, egg cells, and polar nuclei showed a haploid DNA content at the end of their differentiation, while antipodes underwent three DNA endoreduplication cycles. Programmed cell death symptoms were detectable in synergid and antipodal cells. After double fertilization, the central cell quickly underwent many mitotic cycles forming the endosperm, which exhibited a progressive increase in protein bodies and starch grains. Close to the developing embryo, the endosperm differentiated a well-defined region rich in a fibrillar carbohydrate matrix. The zygote, that does not start immediately to divide after double fertilization, developed in to an embryo that reached the heart stage at fruit maturation time. A weakly differentiated embryo at this time indicates a morpho-physiological dormancy of seeds, as a survival strategy imposed by the life cycle of this plant with seed dispersal in spring and their germination in the following winter

The intergenic spacer region of the rDNA in Haplopappus gracilis (Nutt.) Gray

In this paper we provide further information on the genome organization of Haplopappus gracilis, one of the six angiosperms showing the lowest chromosome number, i.e. 2n=4, by determining the nucleotide sequence of the Intergenic Spacer region of the rRNA genes and its cytological localization on metaphase chromosomes. DNA sequence analysis reveals the occurring of a product of 4,382 bp in length, characterized by the presence of four blocks of different repeated sequences. Our analysis also evidenced putative promoter regions with three Transcription Initiation Sites for Polymerase I, as previously reported in Artemisia absinthium, belonging to the same Asteraceae family. A fluorescent in situ hybridization with the Intergenic Spacer probe indicates the presence of rDNA genes only in the satellited chromosomes of H. gracilis; besides differences in the signal intensity between homologous chromosomes were frequently observed, so suggesting, for these chromosome sites, the presence of a variable number of rDNA gene copies, even if a divergent chromatin organization in corresponding regions can not be ruled out

Antioxidant response to cold stress in two oil plants of the genus Jatropha

Jatropha curcas and J. macrocarpa, suitable for production of biodiesel oil from their seeds, are able to live in arid and semi-arid regions, where most crops cannot survive. J. curcas is characterized by higher oil quality and seed yield, but it is not a good candidate for oil production in arid areas with freezing temperatures, due to its sensitiv- ity to chilling in comparison to J. macrocarpa. In this work, for the first time, the effects of cold stress and different mechanisms activated in these conditions have been studied in the two species. Seedlings were treated with low non-freezing temperatures with or without a previous acclimation period. Water status, pigment content, oxida- tive stress and antioxidant response were studied in acclimated and non-acclimated plants. The key features that differentiate J. macrocarpa from J. curcas were the ability to accumulate, at low temperatures, high concentrations of pigments and glutathione and significantly higher activities of ascorbate peroxidase. These data could explain the greater resistance to low temperatures of J. macrocarpa. A period of acclimation was not able to improve cold toler- ance of J. curcas and this confirms its limited adaptability to arid areas with freezing temperatures

Toxicological assays with Vicia faba L. for monitoring bioremediation processes of soils contaminated by polycyclic aromatic hydrocarbons and total petroleum hydrocarbons

The ecotoxicological assays are used both in the assessment of the toxicity of various matrices and to estimate the effectiveness of the remediation of a matrix over time. Several organisms can be employed in eco-toxicological tests in the lab, or can be observed in their natural environment representing indicators of specific environmental conditions. Higher plants can be exploited as good indicators of the quality of different matrices and used both as a standard during remediation processes of contaminated soil but also in the evaluation of soil quality. In fact, the only chemical characterization of a polluted soil does not allow to predict the real toxicity of the matrix, that may depend on the original pollutants, on degradations intermediates, often unknown, and on the synergic actions of the different toxicants. The aim of this research was to evaluate the efficacy of the process abatement of the initial concentration, in a contaminated soil, of polycyclic aromatic hydrocarbons (PAHs, 6480 ppm) and of total petroleum hydrocarbons (TPHs, 18347 ppm) by means of a biopile in terms of reduction of the final matrix toxicity. For this reason soil samples at different time intervals from the beginning of treatment in biopile were collected and chemically characterized, for the monitoring and assessment of the possible phyto-genotoxic effects on the model system Vicia faba L. The biopile treatment determined the depletion of PAHs and TPHs below 10 and 100 ppm respectively. Biological parameters such as germination and root elongation were also evaluated as well as the possible genotoxic effects by means of cytological analysis of mitotic behaviour of root meristem, based on detection of chromosomal aberrations in mitotic cells, and of micronuclei formation, detectable in interphase cells. As additionally genotoxicity endpoint, cytohistochemical detection of in situ DNA fragmentation in root apices was achieved by TUNEL reaction, that allows to recognize both single and double-strand breaks at cellular and tissue level. Comparing the obtained results it was found that the phyto-genotoxic effect of contaminated soil was completely depleted after treatment in biopile and Vicia faba was found to be a good approach for monitoring the biodegradation processes and the toxicity of these solid matrices at the end of a bio-based decontamination approach. Keywords: genotoxicity, phytotoxicity, polycyclic aromatic hydrocarbons, total petroleum hydrocarbon

Aluminum effects on embryo suspensor polytene chromosomes of Phaseolus coccineus L

Aluminum (Al) represents a widespread environmental pollutant, with severe toxic impacts on plants. In this study, we documented for the first time the structural and functional responses induced by two concentrations of AlCl3 (10−2 M and 10−1 M) in the polytene chromosomes that characterize the chromatin organization in the embryo suspensor cells of Phaseolus coccineus. Polytene chromosomes showed signs of dose-dependent genotoxicity following AlCl3 treatments with a significant increase in both chromatin stickiness and chromatin fragmentation. Polytene chromosomes specifically reacted to AlCl3 also in terms of DNA and RNA puffing activity: with respect to the control, the treatments promoted ex-novo and/or inhibited puff formation along chromosome arms, suggesting a fine modulation of the differential genome activity in response to the treatments. The nuclei of suspensors from control and treated seeds showed nucleoli mainly arranged by more than one NOR-bearing chromosome. In addition, AlCl3 treatments affected the frequency of nucleoli organized by singular organizer chromosomes, with an increase in the frequencies of nucleoli organized by chromosome II and a reduction in the frequencies of those organized by chromosomes I or V. These results confirm that, also in our system, nucleolus may react as stress response organelle. Introductio

Impact of TiO2 nanoparticles on Vicia narbonensis L.: potential toxicity effects

This work was aimed to provide further information about toxicology of TiO2 nanoparticles (NPs) on Vicia narbonensis L., considering different endpoints. After exposure to TiO2 nanoparticle suspension (mixture of rutile and anatase, size <100 nm) at four different concentrations (0.2, 1.0, 2.0 and 4.0 ‰), the seeds of V. narbonensis were let to germinate in controlled environmental conditions. After 72 h, the extent of the success of the whole process (seed germination plus root elongation) was recorded as the vigour index, an indicator of possible phytotoxicity. After the characterisation of the hydric state of different materials, oxidative stress and enzymatic and nonenzymatic antioxidant responses were considered as indicators of possible cytotoxicity and to assess if damage induced by TiO2 NPs was oxidative stress-dependent. Cytohistochemical detection of in situ DNA fragmentation as genotoxicity endpoint was monitored by TUNEL reaction. The treatments with TiO2 NPs in our system induced phytotoxic effects, ROS production and DNA fragmentation. The nonenzymatic and enzymatic antioxidant responses were gradually and differentially activated and were able to maintain the oxidative damage to levels not significantly different from the control. On the other hand, the results of DNA fragmentation suggested that the mechanisms of DNA repair were not effective enough to eliminate early genotoxicity effects

Phytoremediation for improving the quality of effluents from a conventional tannery wastewater treatment plant

In the present study, the quality of effluents from a conventional wastewater treatment plant in Italy has been analyzed. Residual level of contamination by 4-nnonylphenol, mono- and di-ethoxylated nonylphenols has been recorded in the effluents that resulted to be also phytotoxic and genotoxic. The possibility of exploiting phytoremediation as a sustainable tertiary treatment for the depletion of the priority pollutants and for the reduction in the residual toxicity has been verified at mesocosm scale. The phyto-based treatment has been performed by the exploitation of Phragmites australis by either a bacterialassisted and not assisted approach. In relation to the bacterial-assisted approach, two new bacterial strains, capable of using the nonylphenols as a sole carbon source, have been isolated. One was identified as a plant growth-promoting rhizobacteria (PGPR) belonging to the Stenotrophomonas species, and the second one was classified as a Sphingobium species strain. Both strains were independently bioaugmented in the P. australis rhizosphere. In relation to the not assisted approach, the phyto-based process determined 87, 70 and 87 % for 4-n-nonylphenol, mono-ethoxylated nonylphenols and di-ethoxylated nonylphenols, respectively. The toxicological assessment of the process evidenced the complete depletion of either the phytotoxicity or the genotoxicity of the treated effluents. With reference to the bacterial-assisted approach, the PGPR Stenotrophomonas species strain resulted to be capable of significantly increasing the efficiency of the phyto-based process in nonylphenol depletion up to 88 % for the 4-n-nonylphenol, 84 % for the mono-ethoxylated nonylphenol and 71 % for the di-ethoxylated nonylphenol