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.

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.

Phytochemical and Biological Activities in Limonium Species Collected in Different Biotopes of Tunisia.

A particular interest is nowadays given to natural antioxidants occurring in foods which can reduce the risk of several diseases through their protective effect. The genus Limonium is widely distributed in different salt regions of Tunisia and known in traditional medicine for the presence of highly effective viral and bacterial replication inhibitors. Limonium leaves have possible beneficial effects on human health for their antioxidant activities and free radical scavenging abilities. To exploit the potential of plants from extreme environments as new sources of natural antioxidants, we studied the extracts from leaves of eight Limonium species growing in extreme environments in Tunisia. Antioxidant molecules (polyphenols, flavonoids, flavonols, ascorbate, tocopherols), in vitro (DPPH, ORAC) and ex vivo antioxidant potential on human erythrocytes, antioxidant enzymes activities (superoxide dismutase, peroxidases, glutathione reductase) were evaluated to identify the species with the best antioxidant capacity. The results showed variability among the species considered in function of the environmental conditions of their natural biotopes, as for the antioxidants measured. In particular, L. vulgare from Oued Rane biotope, characterized by dryness and high temperatures, was the species with the highest enzymatic activity and antioxidant capacity, making it interesting as possible edible halophyte plant or as food complement.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Physiological and antioxidant responses of the sabkha biotope halophyte Limonium delicatulum to seasonal changes in environmental conditions.

In saline biotopes, different mechanisms may contribute to the tolerance of halophytes to high soil salinity and temperature, drought and other abiotic stresses, but their relative responses and their ecological plasticity for a given species remain unknown. In this study, we examined the responses of the salt marsh halophyte Limonium delicatulum to changing environmental conditions of its natural habitats (Sabkha "El Kalbia", Tunisia). The specific aim of the work was to check whether statistically significant correlations could be established between particular stress response mechanisms (ion uptake, activation of antioxidant systems) and soil parameters and climatic data associated with environmental stress. The results showed that the salinity of the soil increased during the months of June, July and August (dry period), concomitantly with a strong accumulation of salt ions in the aerial parts of the plant. Moreover, the highest antioxidant capacity of this halophyte (enzymes and antioxidant molecules) was reached during the same period of the year. The remaining periods (corresponding to the rainy season) did not show a significant difference in enzymes activities and level of antioxidants, with a minimum observed in the months of January and February. These results show a remarkable effect of salinity in the natural habitat on the activity of enzymes and antioxidant molecules. Other edaphic and climatic factors could also be involved to increase antioxidant capacity, such as nutrient deficiency, temperature and precipitation.

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.

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.

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.

Differences in the activity and distribution of peroxidases from three different portions of germinating Brassica oleracea seeds.

Peroxidase (POD, EC 1.11.1.7) activity, cellular localization and isozyme patterns were investigated in the seed integument, cotyledon and embryo axis of Brassica oleracea cv. Cappuccio during pregermination and seedling growth. Seeds started to germinate after 24 h of imbibition. POD activity was localized in the pigmented layer of the integument and in procambial strands of the cotyledon and embryo axis in the first 24 h of imbibition. It was localized in the integumental cells of palisade, pigmented and aleurone layers and in epidermal, meristematic, procambial cells and xylem elements of the root and hypocotyl after 48 h of imbibition. POD activity increased during germination and early seedling growth: in the integument, it reached a maximum value after 72 h of imbibition, in the embryo axis and cotyledons, it increased up to 144 h of imbibition. The increase in peroxidase activity was accompanied by the appearance of new isozymes correlated with the development of seedling tissues. The isozyme profile was characterized by nine peroxidases: isoperoxidase of 50 kDa peculiar to integuments, that of 150 kDa to cotyledons and that of 82 kDa to the embryo axis. During pregerminative phase isozymes of 84 kDa were detected in the integument and cotyledons, of 48.5 kDa in the embryo axis. After germination, peroxidase activity and the complexity of the isozyme pattern increased, suggesting that they play a relevant role after rupture of the integument.