Early Physiological, Cytological and Antioxidative Responses of the Edible Halophyte Chenopodium quinoa Exposed to Salt Stress.

Quinoa (Chenopodium quinoa Willd.) is a plant of South American origin recently valorized for its nutritional and nutraceutical properties in human diet. Quinoa is cultivated in many parts of the world, with a selection of varieties with good adaptability to extreme climatic conditions and salt stress. The variety Red Faro, native to southern Chile but harvested in Tunisia, was considered for salt stress resistance, considering its seed germination and 10-day seedling growth at increasing doses of NaCl (0, 100, 200 and 300 mM). Seedlings were spectrophotometrically analyzed for antioxidant secondary metabolites (polyphenols, flavonoids, flavonols and anthocyanins), antioxidant capacity (ORAC, oxygen radical absorbance capacity, DPPH*, 2,2-diphenyl-1-pic-rylhydrazyl), antioxidant enzyme activity (superoxide dismutase (SOD), guaiacol peroxidase (GPX), ascorbate peroxidase (APX) and catalase (CAT)) and mineral nutrient content in root and shoot tissues. Cytogenetic analysis of root tip was performed to check for meristematic activity and the possible presence of chromosomal abnormalities induced by salt stress. The results indicated a general increase in antioxidant molecules and antioxidant enzymes NaCl dose-dependent, no effects on seed germination but negative effects on seedling growth, and little effect on root meristems mitotic activity. These results indicated that stress conditions can induce an increase in biologically active molecules that could be used for nutraceutical purposes.

Early evidence of the impacts of microplastic and nanoplastic pollution on the growth and physiology of the seagrass Cymodocea nodosa.

Microplastics (MPs) and nanoplastics (NPs) are ubiquitous in natural habitats and the risks their presence poses to marine environments and organisms are of increasing concern. There is evidence that seagrass meadows are particularly prone to accumulate plastic debris, including polystyrene particles, but the impacts of this pollutant on seagrass performance are currently unknown. This is a relevant knowledge gap as seagrasses provide multiple ecosystem services and are declining globally due to anthropogenic impact and climate-change-related stressors. Here, we explored the potential effects of a 12 day-exposure of seagrasses to one concentration (68 µg/L) of polystyrene MPs and NPs on the growth, oxidative status, and photosynthetic efficiency of plants using the foundation species Cymodocea nodosa as a model. Among plant organs, adventitious roots were particularly affected by MPs and NPs showing complete degeneration. The number of leaves per shoot was lower in MPs- and NPs-treated plants compared to control plants, and leaf loss exceeded new leaf production in MPs-treated plants. MPs also reduced photochemical efficiency and increased pigment content compared to control plants. Shoots of NPs-treated plants showed a greater oxidative damage and phenol content than those of control plants and MPs-treated plants. Biochemical data about oxidative stress markers were consistent with histochemical results. The effects of MPs on C. nodosa could be related to their adhesion to plant surface while those of NPs to entering tissues. Our study provides the first experimental evidence of the potential harmful effects of MPs/NPs on seagrass development. It also suggests that the exposure of seagrasses to MPs/NPs in natural environments could have negative consequences on the functioning of seagrass ecosystems. This stresses the importance of implementing cleaning programs to remove all plastics already present in marine habitats as well as of undertaking specific actions to prevent the introduction of these pollutants within seagrass meadows.

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.

Cannabis sativa L. and Brassica juncea L. grown on arsenic-contaminated industrial soil: potentiality and limitation for phytoremediation.

Phytoremediation represents a natural method to remove contaminants from soil. The goal of this study was to investigate the potential of phosphate-assisted phytoremediation by two energy crops, Cannabis sativa L. and Brassica juncea L., for the sustainable remediation of heavily arsenic-contaminated industrial soil. The two species were investigated for uptake, translocation, and physiological effects of arsenic and phosphate in a microcosm test. Although C. sativa and B. juncea were symptomless when grown in arsenic-contaminated soil, an important reduction of biomass (50 and 25%, respectively) was observed as a stress marker. Phytotoxicity and cytotoxicity effects promoted by contaminated soils were investigated in both the species and a model plant for ecotoxicity studies, Vicia faba L., which is the most developed model to test genotoxicity effects in terms of chromosomal aberration and micronuclei presence. The higher amount of arsenic was found in C. sativa and B. juncea roots (on average 1473 and 778 mg kg-1, respectively), but both species were able to uptake and translocate arsenic in leaves and stems, up to 47.0 and 189 mg kg-1, respectively. Phosphate treatment had no effect on arsenic uptake in none of the crop, but significantly improved the plant performance. Biomass production resulted similar to that of B. juncea control plants. Antioxidant enzymatic activities and photosynthetic performance responded differently in the two crops. The present investigation provides new insight for a proficient selection of the most suitable crop species for sustainable phytomanagement of a highly polluted As-contaminated site by coupled phytoremediation-bioenergy approach.

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.

Characterization of additives in plastics: From MS to MS10 multistep mass analysis and theoretical calculations of tris(2,4-di-tert-butylphenyl)phosphate.

In the analysis by electrospray (+) of an extract of hemp sprouts put in a polypropylene vial, we found a large contamination of a plastic additive. It was characterized by multiple-stage MSn experiments (MS ÷ MS10 ) and identified as tris(2,4-di-tert-butylphenyl)phosphate, also known with the synonyms F32IRS6B46, oxidized Naugard 524, and others. The MS2 ÷ MS7 spectra are characterized by consecutive eliminations of six isobutene molecules from the tert-butyl moieties, some of them also occurring in the ion source. The first three are calculated to occur preferentially from the ortho positions, whereas eliminations from the para positions are estimated to be less favored at about 5-6 kcal/mol in each step. Once the first three isobutene molecules are eliminated, the remaining three are lost from the tert-butyl moieties in para positions (MS5 ÷ MS7 ), yielding protonated triphenylphosphate, whose structure has been confirmed by the MS2 spectrum of triphenylphosphate standard: the latter spectrum is almost superimposable with the MS8 spectrum of the analyte under investigation. MS8 and MS9 spectra show main losses of water and C6 H4 molecules. The MS10 spectrum of precursor ions at m/z 215 shows the gas-phase addition of water and methanol and ions at m/z 168, attributable to the loss of a phosphorus oxide radical. Density functional theory (DFT) calculations (Becke 3LYP [B3LYP] 6-311+G(2d,2p)) have been used to evaluate structure and stability of different ionic and neutral species involved in the decomposition pathways and to calculate thermochemical data of the decomposition reactions. This multistep mass analysis combined with theoretical calculations resulted to be particularly useful and effective, yielding chemical, thermochemical, and mechanistic data of significant utility in the structural characterization and identification of the unknown analyte as well as to define its gas-phase reactivity under a multistep low-energy collision-induced dissociation regime.

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.

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.

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.

Drought stress modulates secondary metabolites in Brassica oleracea L. convar. acephala (DC) Alef, var. sabellica L.

BACKGROUND: Consumer preference today is for the consumption of functional food and the reduction of chemical preservatives. Moreover, the antimicrobial properties and health-promoting qualities of plant secondary metabolites are well known. Due to forecasted climate changes and increasing human population, agricultural practices for saving water have become a concern. In the present study, the physiological responses of curly kale Brassica oleracea L. convar. Acephala (DC) var. sabellica to drought stress and the impact of water limitation on the concentration of selected secondary metabolites were investigated under laboratory-controlled conditions. RESULTS: Results indicated that drought stress increased the content of trans-2-hexenal, phytol and δ-tocopherol, and decreased chlorophyll content. Moreover, drought stress increased antioxidant capacity and the expression of AOP2, a gene associated with the biosynthesis of aliphatic alkenyl glucosinolates, and of three genes - TGG1, TGGE and PEN2 - encoding for myrosinases, the enzymes involved in glucosinolate breakdown. CONCLUSION: The present study shows that water limitation during the growing phase might be exploited as a sustainable practice for producing curly kale with a high concentration of nutritionally important health-promoting bioactive metabolites. © 2019 Society of Chemical Industry.

Polyphenolic characterisation of plant mixture (Lisosan® Reduction) and its hypocholesterolaemic effect in high fat diet-fed mice.

Lisosan® Reduction is a plant mixture produced from powder of fermented Triticum aestivum (Lisosan® G), Desmodium adscendens, Malus domestica, Picrorhiza kurroa and Hordeum vulgare. The aim of this study was to characterise the phenolic profile of Lisosan® Reduction and to evaluate the effects of aqueous extract on mice fed a high fat diet (HFD). Syringic acid, trans sinapic acid and neochlorogenic acid were identified by HPLC-DAD to be the dominant polyphenols of Lisosan® Reduction, followed by vitexin, trans p-coumeric acid and trans ferulic acid. Mice treated with aqueous extract of Lisosan® Reduction (60 mg/kg b.w.) showed a significant decrease of serum cholesterol, glucose and triglycerides level and a significant increase of CYP7A1 gene expression, compared to HFD group.

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.

Nutraceutical potential of hemp (Cannabis sativa L.) seeds and sprouts.

In this study the antioxidant effect of Cannabis sativa L. seeds and sprouts (3 and 5 days of germination) was evaluated. Total polyphenols, flavonoids and flavonols content, when expressed on dry weight basis, were highest in sprouts; ORAC and DPPH (in vitro assays), CAA-RBC (cellular antioxidant activity in red blood cells) and hemolysis test (ex vivo assays) evidenced a good antioxidant activity higher in sprouts than in seeds. Untargeted analysis by high resolution mass spectrometry in negative ion mode allowed the identification of main polyphenols (caffeoyltyramine, cannabisin A, B, C) in seeds and of ω-6 (linoleic acid) in sprouts. Antimutagenic effect of seeds and sprouts extracts evidenced a significant decrease of mutagenesis induced by hydrogen peroxide in Saccharomyces cerevisiae D7 strain. In conclusion our results show that C. sativa seeds and sprouts exert beneficial effects on yeast and human cells and should be further investigated as a potential functional food.

Screening and identification of major phytochemical compounds in seeds, sprouts and leaves of Tuscan black kale Brassica oleracea (L.) ssp acephala (DC) var. sabellica L.

We report the spectrophotometric determination of total polyphenols, flavonoids, glucosinolates and antioxidant activity in seeds, seedlings and leaves of Tuscan black kale. The highest content of phytochemicals was observed in 10 days sprouts and antioxidant activity was maximum in 2, 4 days seedlings. Identification and characterisation of phytochemicals were performed by mass spectrometry (MS), high resolution and tandem MS with electrospray ionisation mode. Low-molecular-weight metabolites were evidenced in seeds while metabolites at high m/z range were detected in cotyledons and leaves. MS spectra evidenced different phenolic compounds (flavonoid caffeoyl glucose, hydroxycinnamic acid sinapine) and glucosinolates (glucoerucin, glucobrassicin and glucoraphanin) in function of developmental stage; galactolipids ω3 and ω6 were observed in leaves. Identification of stages with the highest phytochemicals content encourages the consumption of black kale sprouts and young leaves. Our research can support food and pharmaceutical industries for production of health promoting products from black kale.

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.

Effect of HFD/STZ on expression of genes involved in lipid, cholesterol and glucose metabolism in rats.

AIMS: The aim of the study was to evaluate lipid, cholesterol and glucose metabolism in a novel rat model of non-alcoholic fatty liver disease (NAFLD). MAIN METHODS: Rats (Wistar) were fed high fat/cholesterol diet (HFD) and a single low dose (35mg/kg) of streptozotocin (STZ). Collagen and glycogen content, oxidative stress and glucokinase activity were measured using biochemical assays. Other metabolic pathways were assessed by qRT-PCR. KEY FINDINGS: HFD/STZ treated rats, compared to control ones, showed an increase in expression of biomarkers of inflammation (TNFα, IL6), fibrosis (TGFß), mitochondrial stress (UCP2) and oxidative stress (GSH and carbonylated proteins) but not of ER stress (CHOP, XBP1). Additionally, HFD/STZ treatment caused a reduction in glycogen content, glucokinase activity (a limiting step in glycolysis) and expression of ChREBP gene (a de novo lipogenesis regulator), suggesting a modified glycolytic pathway. The cholesterol biosynthesis in HFD/STZ treated rats was inhibited (reduced expression of SREBP-2-regulated HMGCoA red and LDLr), instead the cholesterol catabolism was increased, as shown by the mRNA induction of the CYP7A1 and CYP8B1 (key genes for BA acid). A reduced gene expression of FXR-dependent SHP (a key gene for feedback inhibition of CYP7A1 and CYP8B1) and of bile acids (NTCP, OATP1A1, BSEP) and cholesterol (ABCA1) transporters was found. SIGNIFICANCE: These results widely extend the characterization of HFD/STZ rat model, which might mimic the NAFLD/NASH in diabetic humans.

Contaminant bioavailability in soil and phytotoxicity/genotoxicity tests in Vicia faba L.: a case study of boron contamination.

In this work, the model plant for genotoxicity studies Vicia faba L. was used to investigate the relation between Boron (B) content and bioavailability in soil and plant genotoxic/phytotoxic response. A total of nine soil samples were investigated: two soil samples were collected from a B-polluted industrial area in Cecina (Tuscany, Italy), the other samples were obtained by spiking control soil (from a not polluted area of the basin) with seven increased doses of B, from about 20 to 100 mg B kg-1. As expected, B availability, evaluated by chemical extraction, was higher (twofold) in spiked soils when compared with collected polluted soils with the same B total content. To analyze the phytotoxic effects of B, seed germination, root elongation, biomass production, and B accumulation in plant tissues were considered in V. faba plants grown in the various soils. Moreover, the cytotoxic/genotoxic effects of B were investigated in root meristems by mitotic index (MI) and micronuclei frequency (MCN) analysis. The results highlighted that V. faba was a B-sensitive plant and the appearance of phytotoxic effects, which altered plant growth parameters, were linearly correlated to the bioavailable B concentration in soils. Concerning the occurrence of cytotoxic/genotoxic effects induced by B, no linear correlation was observed even if MCN frequency was logarithmic correlated with the concentration of B bioavailable in soils.

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. © 2016 SETAC.

Polycyclic aromatic hydrocarbon-contaminated soils: bioaugmentation of autochthonous bacteria and toxicological assessment of the bioremediation process by means of Vicia faba L.

Two bacterial strains, Achromobacter sp. (ACH01) and Sphingomonas sp. (SPH01), were isolated from a heavily polycyclic aromatic hydrocarbon (PAH)-contaminated soil (5431.3 ± 102.3 ppm) for their capacity to use a mixture of anthracene, pyrene, phenanthrene and fluorene as sole carbon sources for growth and for the capacity to produce biosurfactants. The two strains were exploited for bioaugmentation in a biopile pilot plant to increase the bioavailability and the degradation of the residual PAH contamination (99.5 ± 7.1 ppm) reached after 9 months of treatment. The denaturing gel gradient electrophoresis (DGGE) profile of the microbial ecology of the soil during the experimentation showed that the bioaugmentation approach was successful in terms of permanence of the two strains in the soil in treatment. The bioaugmentation of the two bacterial isolates positively correlated with the PAH depletion that reached 7.9 ± 2 ppm value in 2 months of treatment. The PAH depletion was assessed by the loss of the phyto-genotoxicity of soil elutriates on the model plant Vicia faba L., toxicological assessment adopted also to determine the minimum length of the decontamination process for obtaining both the depletion of the PAH contamination and the detoxification of the soil at the end of the process. The intermediate phases of the bioremediation process were the most significant in terms of toxicity, inducing genotoxic effects and selective DNA fragmentation in the stem cell niche of the root tip. The selective DNA fragmentation can be related to the selective induction of cell death of mutant stem cells that can compromise offsprings.

Molecular aspects of zygotic embryogenesis in sunflower (Helianthus annuus L.): correlation of positive histone marks with HaWUS expression and putative link HaWUS/HaL1L.

MAIN CONCLUSION: The link HaWUS/ HaL1L , the opposite transcriptional behavior, and the decrease/increase in positive histone marks bond to both genes suggest an inhibitory effect of WUS on HaL1L in sunflower zygotic embryos. In Arabidopsis, a group of transcription factors implicated in the earliest events of embryogenesis is the WUSCHEL-RELATED HOMEOBOX (WOX) protein family including WUSCHEL (WUS) and other 14 WOX protein, some of which contain a conserved WUS-box domain in addition to the homeodomain. WUS transcripts appear very early in embryogenesis, at the 16-cell embryo stage, but gradually become restricted to the center of the developing shoot apical meristem (SAM) primordium and continues to be expressed in cells of the niche/organizing center of SAM and floral meristems to maintain stem cell population. Moreover, WUS has decisive roles in the embryonic program presumably promoting the vegetative-to-embryonic transition and/or maintaining the identity of the embryonic stem cells. However, data on the direct interaction between WUS and key genes for seed development (as LEC1 and L1L) are not collected. The novelty of this report consists in the characterization of Helianthus annuus WUS (HaWUS) gene and in its analysis regarding the pattern of the methylated lysine 4 (K4) of the Histone H3 and of the acetylated histone H3 during the zygotic embryo development. Also, a parallel investigation was performed for HaL1L gene since two copies of the WUS-binding site (WUSATA), previously identified on HaL1L nucleotide sequence, were able to be bound by the HaWUS recombinant protein suggesting a not described effect of HaWUS on HaL1L transcription.