AtPng1 knockout mutant of Arabidopsis thaliana shows a juvenile phenotype, morpho-functional changes, altered stress response and cell wall modifications.

In order to ascertain the role of plant transglutaminases (TGase) in growth and abiotic stress response, the AtPng1 knock out (KO) line of A. thaliana has been analyzed during plant development and under heat and wound stress. Comparing wild type (WT) and KO lines a 58-kDa band was immunodetected by anti-AtPng1p antibody in the cell wall and chloroplasts only in the WT line. A residual TGase activity, not showing correlation with development nor stress response, was still present in the KO line. The KO line was less developed, with a juvenile phenotype characterized by fewer, smaller and less differentiated cells. Chloroplast TGase activity was insensitive to mutation. Data on stressed plants showed that (i) KO plants under heat stress were more juvenile compared to WT, (ii) different responses between WT and KO lines after wounding took place. TGase activity was not completely absent in the KO line, presenting high activity in the plastidial fraction. In general, the mutation affected A. thaliana growth and development, causing less differentiated cytological and anatomical features.

Chronic heat stress affects the photosynthetic apparatus of Solanum lycopersicum L. cv Micro-Tom.

Tomato (Solanum lycopersicum L.) is one of the most widely cultivated crops in the world. Tomato is a plant model and the relationship between yield and biotic/abiotic stress has attracted increasing scientific interest. Tomato cultivation under sub-optimal conditions usually negatively impacts growth and development; in particular, heat stress affects several cellular and metabolic processes, such as respiration and photosynthesis. In this work, we studied the effects of chronic heat stress on various cytological and biochemical aspects using the Micro-Tom cultivar as a model. Photosynthetic efficiency decreased during heat stress while levels of post-photosynthetic sugars (sucrose, fructose, glucose and glucose 6-phosphate) oscillated during stress. Similarly, photosynthetic pigments (lutein, chlorophyll a, chlorophyll b and ß-carotene) showed an oscillating downward trend with partial recovery during the stress-free phase. The energetic capacity of leaves (e.g. ATP and ADP) was altered, as well as the reactive oxygen species (ROS) profile; the latter increased during stress. Important effects were also found on the accumulation of Rubisco isoforms, which decreased in number. Heat stress also resulted in a decreased accumulation of lipids (oleic and linoleic acid). Photosynthetically alterations were accompanied by cytological changes in leaf structure, particularly in the number of lipid bodies and starch granules. Prolonged heat stress progressively compromised the photosynthetic efficiency of tomato leaves. The present study reports multi-approach information on metabolic and photosynthetic injuries and responses of tomato plants to chronic heat stress, highlighting the plant's ability to adapt to stress.

Combined effects of nanoplastics and copper on the freshwater alga Raphidocelis subcapitata.

Nanoplastics are recognized as able to interact with other pollutants including heavy metals, and with natural organic matter, with implications for the potential risks to biota. We investigated the interaction of carboxylated polystyrene nanoparticles (PS-COOH NPs) with copper (Cu) and algal exudates (EPS) and how such interaction could affect Cu toxicity towards the freshwater microalga Raphidocelis subcapitata. PS-COOH NPs behavior in the presence of Cu and EPS was determined by dynamic light scattering (DLS), while PS-COOH NPs surface interaction with Cu ions and EPS was investigated by fluorimetric analysis. ICP-MS was used to test Cu ion adsorption to PS-COOH NPs in the presence and absence of algae. The interaction between PS-COOH NPs and the algal cell wall was assessed by fluorescence microscopy. Short- and long-term toxicity tests were carried out in parallel to assess the impact of PS-COOH NPs on algal growth. Results showed altered nanoparticle surface charge and hydrodynamic diameter following algal EPS exposure, supporting the hypothesis of a protein corona formation. In contrast, no absorption of Cu ions was observed on PS-COOH NPs, either in the presence or absence of algae. No differences on algal growth inhibition were observed between exposure to Cu only, and to Cu in combination with PS-COOH NPs, in short-term as well as long-term tests. However, after 72 h of exposure, the adsorption of PS-COOH NPs to algal cell walls appeared to correspond to morphological alterations, revealing potential disturbances in the mitotic cycle. Our findings confirm the ability of PS-COOH NPs to interact with EPS as shown for other nanomaterials. Environmentally realistic exposure scenarios are thus needed for evaluating nanoplastic toxicity, as nanoparticles will not maintain their pristine nature once released into natural media. Prolonged exposure and use of different end-points such as cell morphological changes and EPS production seem more reliable for the investigation of nanoplastic/algal cell interactions which can drive food chain transfer of nanoplastics and ultimately toxicity.

Cationic polystyrene nanoparticle and the sea urchin immune system: biocorona formation, cell toxicity, and multixenobiotic resistance phenotype.

In order to assess the impact of nanoplastics on marine species, polystyrene nanoparticles (PS NPs) have been largely used as model particles. Here we studied the effects of 50 nm amino-modified PS-NH2 on Mediterranean sea urchin Paracentrotus lividus immune system cells (coelomocytes) in the presence of celomic fluid (CF) and at different NP concentrations (1, 5, 10, and 25 µg mL-1) and experimental conditions (absence or presence of EDTA). PS-NH2 acquired a protein corona once incubated with CF, dominated by the toposome precursor protein (TPP). In short-term cultures, a significant concentration- and time-dependent decrease in lysosomal membrane stability and apoptotic-like nuclear alterations were observed in phagocytes upon exposure to PS-NH2 (10 and 25 µg mL-1) in CF but they resulted abolished in the presence of EDTA confirming the role of TPP in triggering PS-NH2-coelomocytes interaction and toxicity. PS-NH2 did not alter MXR phenotype but the observed dose-dependent decrease in calcein accumulation suggests the ability of PS-NH2 to affect pump's efflux activity. Overall results encourage additional studies on positively charged nanoplastics, since the observed effects on sea urchin coelomocytes as well as the TPP corona formation might represent a first step for addressing their impact on sensitive marine species.

Co-exposure to titanium dioxide nanoparticles does not affect cadmium toxicity in radish seeds (Raphanus sativus).

Recent developments on environmental fate models indicate that as nano waste, engineered nanomaterials (ENMs) could reach terrestrial ecosystems thus potentially affecting environmental and human health. Plants can be therefore exposed to ENMs but controversial data in terms of fate and toxicity are currently available. Furthermore, there is a current lack of information on complex interactions/transformations to which ENMs undergo in the natural environment as for instance interacting with existing toxic compounds. The aim of the present study was to assess the behavior and biological effects of titanium dioxide nanoparticles (n-TiO2) (Aeroxide P25, Degussa Evonik) and its interaction with cadmium (CdCl2) in plants using radish seeds (Raphanus sativus L. Parvus) as model species. Radish seeds were exposed to n-TiO2 (1-1000mg/L) and CdCl2 (1-250mg/L) alone and in combination using a seed germination and seedling growth toxicity test OECD 208. Percentage of seed germination, germination index (GI) and root elongation were calculated. Cell morphology and oxidative stress parameters as glutathione-S-transferase (GST) and catalase activities (CAT) were measured in radish seeds after 5 days of exposure. Z-Average, PdI and Z-potential of n-TiO2 in Milli-Q water as exposure medium were also determined. DLS analysis showed small aggregates of n-TiO2, negative Z-potential and stable PdI in seed's exposure media. Germination percentage, GI and root length resulted affected by n-TiO2 exposure compared to controls. In particular, n-TiO2 at 1mg/L and 100mg/L did not affect radish seeds germination (100%) while at concentration of 10mg/L, 200mg/L, 500mg/L, and 1000mg/L a slight but not significant decrease of germination % was observed. Similarly root length and GI resulted significantly higher in seeds exposed to 10mg/L and 200mg/L compared to 1mg/L, 100mg/L, 500mg/L, 1000mg/L and control (p < 0.05). On the opposite, CdCl2 significantly abolished germination % and GI compared to control seeds and a concentration dependent decrease on root elongation was observed against controls (p < 0.05). As well, significant decrease of germination %, GI and root elongation was observed in seeds co-exposed to n-TiO2 and CdCl2 at the highest concentrations (1000mg/L n-TiO2 and 250mg/L CdCl2) compared to co-exposed seeds at low concentration (1mg/L n-TiO2 and 1mg/L CdCl2) and controls (p < 0.05). Root elongation significantly increase compared to control at the lowest co-exposure concentration (p < 0.05). Similarly at intermediate concentrations of 10 and 100mg/L in co-exposure conditions, n-TiO2 did not affect CdCl2 toxicity. Concerning antioxidant enzymes, a significant increase of CAT activity in seeds exposed to single high n-TiO2 concentration (1000mg/L) was observed while n-TiO2 (1mg/L), CdCl2 (1 and 250mg/L) and co-exposure resulted significantly decreased compared to controls (p < 0.05). Regarding GST activity, a slight increase in seeds exposed to 1000mg/L n-TiO2 but no significantly was observed, however both n-TiO2 and CdCl2 alone (1 and 250mg/L, respectively) or in combinations caused a significant decrease in GST activity (p < 0.05). Therefore, overall data support the hypothesis that the presence of n-TiO2 do not affect the toxicity of CdCl2 at least at the highest concentration (100 and 250mg/L) in radish seeds. Morphological alterations in nuclei, vacuoles and shape of radish root cells were observed upon single Cd exposure and not abolished in the presence of n-TiO2. Nevertheless, although n-TiO2 seems not to reduce Cd toxicity at high concentration (up to 250mg/L), interactions cannot be excluded based on obtained results.

Comparative ecotoxicity of polystyrene nanoparticles in natural seawater and reconstituted seawater using the rotifer Brachionus plicatilis.

The impact of nanoplastics using model polystyrene nanoparticles (PS NPs), anionic (PS-COOH) and cationic (PS-NH2), has been investigated on the marine rotifer Brachionus plicatilis, a major component of marine zooplanktonic species. The role of different surface charges in affecting PS NP behaviour and toxicity has been considered in high ionic strength media. To this aim, the selected media were standardized reconstituted seawater (RSW) and natural sea water (NSW), the latter resembling more natural exposure scenarios. Hatched rotifer larvae were exposed for 24h and 48h to both PS NPs in the range of 0.5-50µg/ml using PS NP suspensions made in RSW and NSW. No effects on lethality upon exposure to anionic NPs were observed despite a clear gut retention was evident in all exposed rotifers. On the contrary, cationic NPs caused lethality to rotifer larvae but LC50 values resulted lower in rotifers exposed in RSW (LC50=2.75±0.67µg/ml) compared to those exposed in NSW (LC50=6.62±0.87µg/ml). PS NPs showed similar pattern of aggregation in both high ionic strength media (RSW and NSW) but while anionic NPs resulted in large microscale aggregates (Z-average 1109 ± 128nm and 998±67nm respectively), cationic NP aggregates were still in nano-size forms (93.99 ± 11.22nm and 108.3 ± 12.79nm). Both PDI and Z-potential of PS NPs slightly differed in the two media suggesting a role of their different surface charges in affecting their behaviour and stability. Our findings confirm the role of surface charges in nanoplastic behaviour in salt water media and provide a first evidence of a different toxicity in rotifers using artificial media (RSW) compared to natural one (NSW). Such evidence poses the question on how to select the best medium in standardized ecotoxicity assays in order to properly assess their hazard to marine life in natural environmental scenarios.

Long-term toxicity of surface-charged polystyrene nanoplastics to marine planktonic species Dunaliella tertiolecta and Artemia franciscana.

Plastic pollution has been globally recognized as a critical issue for marine ecosystems and nanoplastics constitute one of the last unexplored areas to understand the magnitude of this threat. However, current difficulties in sampling and identifying nano-sized debris make hard to assess their occurrence in marine environment. Polystyrene nanoparticles (PS NPs) are largely used as nanoplastics in ecotoxicological studies and although acute exposures have been already investigated, long-term toxicity on marine organisms is unknown. Our study aims at evaluating the effects of 40nm PS anionic carboxylated (PS-COOH) and 50nm cationic amino-modified (PS-NH2) NPs in two planktonic species, the green microalga Dunaliella tertiolecta and the brine shrimp Artemia franciscana, respectively prey and predator. PS NP behaviour in exposure media was determined through DLS, while their toxicity to microalgae and brine shrimps evaluated through 72h growth inhibition test and 14 d long-term toxicity test respectively. Moreover, the expression of target genes (i.e. clap and cstb), having a role in brine shrimp larval growth and molting, was measured in 48h brine shrimp larvae. A different behaviour of the two PS NPs in exposure media as well as diverse toxicity to the two planktonic species was observed. PS-COOH formed micro-scale aggregates (Z-Average>1µm) and did not affect the growth of microalgae up to 50µg/ml or that of brine shrimps up to 10µg/ml. However, these negatively charged NPs were adsorbed on microalgae and accumulated (and excreted) in brine shrimps, suggesting a potential trophic transfer from prey to predator. On the opposite, PS-NH2-formed nano-scale aggregates (Z-Average<200nm), caused inhibition of algal growth (EC50=12.97µg/ml) and mortality in brine shrimps at 14 d (LC50=0.83µg/ml). Moreover, 1µg/ml PS-NH2 significantly induced clap and cstb genes, explaining the physiological alterations (e.g. increase in molting) previously observed in 48h larvae, but also suggesting an apoptotic pathway triggered by cathepsin L-like protease in brine shrimps upon PS-NH2 exposure. These findings provide a first insight into long-term toxicity of nanoplastics to marine plankton, underlining the role of the surface chemistry in determining the behaviour and effects of PS NPs, in terms of adsorption, growth inhibition, accumulation, gene modulation and mortality. The use of long-term end-point has been identified as valuable tool for assessing the impact of nanoplastics on marine planktonic species, being more predictable of real exposure scenarios for risk assessment purposes.

Accumulation and embryotoxicity of polystyrene nanoparticles at early stage of development of sea urchin embryos Paracentrotus lividus.

Nanoplastic debris, resulted from runoff and weathering breakdown of macro- and microplastics, represents an emerging concern for marine ecosystems. The aim of the present study was to investigate disposition and toxicity of polystyrene nanoparticles (NPs) in early development of sea urchin embryos (Paracentrotus lividus). NPs with two different surface charges where chosen, carboxylated (PS-COOH) and amine (PS-NH2) polystyrene, the latter being a less common variant, known to induce cell death in several in vitro cell systems. NPs stability in natural seawater (NSW) was measured while disposition and embryotoxicity were monitored within 48 h of postfertilization (hpf). Modulation of genes involved in cellular stress response (cas8, 14-3-3ε, p-38 MAPK, Abcb1, Abcc5) was investigated. PS-COOH forms microaggregates (PDI > 0.4) in NSW, whereas PS-NH2 results are better dispersed (89 ± 2 nm) initially, though they also aggregated partially with time. Their respectively anionic and cationic nature was confirmed by ζ-potential measurements. No embryotoxicity was observed for PS-COOH up to 50 µg mL(-1) whereas PS-NH2 caused severe developmental defects (EC50 3.85 µg mL(-1) 24 hpf and EC50 2.61 µg mL(-1) 48 hpf). PS-COOH accumulated inside embryo's digestive tract while PS-NH2 were more dispersed. Abcb1 gene resulted up-regulated at 48 hpf by PS-COOH whereas PS-NH2 induced cas8 gene at 24 hpf, suggesting an apoptotic pathway. In line with the results obtained with the same PS NPs in several human cell lines, also in sea urchin embryos, differences in surface charges and aggregation in seawater strongly affect their embryotoxicity.

Iron-binding characterization and polysaccharide production by Klebsiella oxytoca strain isolated from mine acid drainage.

AIMS: To investigate Klebsiella oxytoca strain BAS-10 growth on ferric citrate under anaerobic conditions for exopolysaccharide (EPS) production and localization on cell followed by the purification and the EPS determination of the iron-binding stability constant to EPS or biotechnological applications. METHODS AND RESULTS: Klebsiella oxytoca ferments ferric citrate under anaerobic conditions and produces a ferric hydrogel, whereas ferrous ions were formed in solution. During growth, cells precipitate and a hydrogel formation was observed: the organic material was constituted of an EPS bound to Fe(III) ions, this was found by chemical analyses of the iron species and transmission electron microscopy of the cell cultures. Iron binding to EPS was studied by cyclic voltammetric measurements, either directly on the hydrogel or in an aqueous solutions containing Fe(III)-citrate and purified Fe(III)-EPS. From the voltammetric data, the stability constant for the Fe(III)-EPS complex can be assumed to have values of approx. 10(12)-10(13). It was estimated that this is higher than for the Fe(III)-citrate complex. CONCLUSIONS: The production of Fe(III)-EPS under anaerobic conditions is a strategy for the strain to survive in mine drainages and other acidic conditions. This physiological feature can be used to produce large amounts of valuable Fe(III)-EPS, starting from a low cost substrate such as Fe(III)-citrate. SIGNIFICANT AND IMPACT OF THE STUDY: The data herein demonstrates that an interesting metal-binding molecule can be produced as a novel catalyst for a variety of potential applications and the EPS itself is a valuable source for rhamnose purification.

Maize endosperm secretes a novel antifungal protein into adjacent maternal tissue.

A series of endosperm transfer layer-specific transcripts has been identified in maize by differential screening of a cDNA library of transcripts at 10 days after pollination. Sequence comparisons revealed among this class of cDNAs a novel, small gene family of highly diverged sequences encoding basal layer antifungal proteins (BAPs). The bap genes mapped to two loci on chromosomes 4 and 10. So far, bap-homologous sequences have been detected only in maize, teosinte and sorghum, and are not present in grasses outside the Andropogoneae tribe. BAP2 is synthesized as a pre-proprotein, and is processed by successive removal of a signal peptide and a 29-residue prodomain. The proprotein can be detected exclusively in microsomal membrane-containing fractions of kernel extracts. Immunolocalization reveals BAP2 to be predominantly located in the placentochalazal cells of the pedicel, adjacent to the basal endosperm transfer layer (BETL) cells, although the BAP2 transcript is found only in the BETL cells. The biological roles of BAP2 propeptide and mature peptide have been investigated by heterologous expression of the proprotein in Escherichia coli, and by tests of its fungistatic activity and that of the fully processed form in vitro. The mature BAP2 peptide exhibits potent broad-range activity against a range of filamentous fungi, including several plant pathogens.

Confocal imaging and immunogold electron microscopy of changes in distribution of myosin during pollen hydration, germination and pollen tube growth in Nicotiana tabacum L.

Using anti-myosin antibodies, standard immunocytochemical techniques in conjunction with confocal scanning laser microscopy and colloidal gold immunoelectron microscopy we compare changes in the distribution patterns of myosin during the early stages of pollen hydration, germination, tube growth, and myosin associated with isolated vegetative nucleus and the generative cell in Nicotiana tabacum L. Furthermore, on the Western blots of pollen tube proteins, the antimyosin antibodies crossreact only with one polypeptide of approximately 174 kDa. Confocal immunofluorescence microscopy reveals that in hydrated pollen, myosin is discretely associated with the cytoplasmic organelles and numerous punctate structures present in the center of the pollen. Within 30 min following transfer of pollen into the germination medium, that is, with the onset of germination, the centrally located punctate structures are displaced, and we find accumulation of myosin-associated organelles towards one of the germinal apertures from which the pollen tube would emerge. Subsequently, after 45 min of germination with the emergence of germination structure, few punctate structures are detected in the vegetative cytoplasm while intense immunostain is detected just below the plasma membrane of the emerging pollen tube tip. In the older parts of both short and long pollen tubes after 90 to 120 min of pollen germination, few fluorescent structures were found in the pollen tubes, however, numerous punctate fluorescent spots were concentrated in the tip region over a distance of 2 to 3 microns below the plasma membrane of the tube tip. This is further substantiated by colloidal gold immunoelectron microscopy wherein clusters of gold particles are associated with vesicle-like structures in the tip region of the pollen tubes.(ABSTRACT TRUNCATED AT 250 WORDS)