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Roles for nitric oxide (NO) and reactive oxygen species (ROS) during pollen-tube growth have been well established in angiosperms, but there remains lack of information regarding their potential signalling roles in pollen tubes in gymnosperms. Here, the pollen-tube elongation of Arizona cypress (Cupressus arizonica Greene) was investigated. Nitric oxide, ROS, and actin were detected using their respective fluorescent probes. Both NO and ROS were observed in the nuclei of generative cells and pollen-tube cells, and in the cytoplasm in the tip region. An intracellular NO content in the pollen cells was lowered using an NO scavenger or an NO-synthase inhibitor. Similarly, an endogenous ROS content in the pollen cells was lowered using an NAD(P)H oxidase inhibitor. These treatments reduced pollen germination and pollen-tube growth, and induced severe morphological abnormalities. Inhibition of NO and ROS accumulation also severely disrupted the actin cytoskeleton in the pollen tubes. These data indicate that NO and ROS had signalling roles in pollen germination and pollen-tube formation in cypress.

Sewage sludge applications as soil amendment call for a proper ecological risk assessment due to unexpected delivery of toxic chemicals and materials. Standardized acute toxicity assays have proven to provide limited information in terms of potential hazard for soil organisms. Here, sublethal endpoints as physiological and tissue alterations were proposed as suitable tools for sewage sludge ecological risk assessment. Acute toxicity assay using earthworm Eisenia fetida (OECD n.207) was performed on sewage sludge samples from two wastewater treatment plants (A and B), and mortality, body weight, body morphology, and histology of intestinal cavity were evaluated at the end of the exposure period. Sewage sludge dilutions (1:50 and 1:100) from plant B caused a low percentage of mortality (5%), while none was observed in worms exposed to plant A. However, all specimens showed external body alterations as vesicles, swellings, constrictions, and discoloration. Histological sections highlight damages to epidermis, circular and longitudinal muscles, and chloragogenic tissue regardless of sewage sludge origin. Our findings confirm that standardized end points of acute toxicity are not sensitive enough in worms exposed to sewage sludges, while sublethal endpoints as body morphology and tissue histology are thus more suitable and appropriate endpoints for assessing potential detrimental outcomes and ecological risk for soil biota., Competing Interests: Declarations. Ethical approval: Not applicable. Consent to participate: Not applicable. Consent for publication: All authors agreed with the content, all gave explicit consent to submit, and they obtained consent from the responsible authorities at the institute where the work has been carried out. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

While UV-B radiation is beneficial to plant growth, it can also cause adverse effects. The pollen tube, a key component of plant reproduction with a tip growth mechanism, is an excellent cellular model for understanding how environmental stressors such as UV-B radiation affect plant cell growth. This research investigated the effect of UV-B on olive pollen both before and after germination. Pollen grains were hydrated and exposed to UV-B radiation for 1 h. Pollen tube germination was then evaluated 4 and 24 h after exposure. To study the effect of UV-B radiation on developing pollen tubes, pollen was germinated for 4 h prior to 1 h of UV-B exposure. Pollen tube growth was evaluated by assessing the distribution of cell wall components, the distance between callose plugs and nuclei, and the distance between the male germ unit and the pollen tube tip. We also examined the accumulation of callose synthase. The results showed that UV-B radiation significantly inhibited the growth of pollen tubes, thereby preventing successful fertilization. The effect of UV-B exposure on pollen tube growth was mainly due to the alteration of position of callose plugs and the level of callose synthase in the pollen tube, potentially affecting its growth. In addition, UV-B radiation affected the movement and integrity of the male germ unit, a critical element for successful fertilization. This research sheds light on how UV-B radiation affects the growth of pollen tubes and highlights the need for further research into the effects of UV-B radiation on plant cells and plant reproduction., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Understanding the responses of olive trees to drought stress is crucial for improving cultivation and developing drought-tolerant varieties. Water transport and storage within the plant is a key factor in drought-tolerance strategies. Water management can be based on a variety of factors such as stomatal control, osmoprotectant molecules, proteins and wood properties. The aim of the study was to evaluate the water management strategy under drought stress from an anatomical and biochemical point of view in three young Italian olive cultivars (Giarraffa, Leccino and Maurino) previously distinguished for their physiological and metabolomic responses. For each cultivar, 15 individuals in pots were exposed or not to 28 days of water withholding. Every 7 days, the content of sugars (including mannitol), proline, aquaporins, osmotins, and dehydrins, in leaves and stems, as well as the chemical and anatomical characteristics of the wood of the three cultivars, were analyzed. 'Giarraffa' reduced glucose levels and increased mannitol production, while 'Leccino' accumulated more proline. Both 'Leccino' and 'Maurino' increased sucrose and aquaporin levels, possibly due to their ability to remove embolisms. 'Maurino' and 'Leccino' accumulated more dehydrins and osmotins. While neither genotype nor stress affected wood chemistry, 'Maurino' had a higher vessel-to-xylem area ratio and a larger hydraulic diameter, which allows it to maintain a high transpiration rate but may make it more susceptible to cavitation. The results emphasized the need for an integrated approach, highlighting the importance of the relative timing and sequence of each parameter analyzed, allowing, overall, to define a "strategy" rather than a "response" to drought of each cultivar.

Durum wheat ( Triticum turgidum L. ssp. durum ) landraces, traditional local varieties representing an intermediate stage in domestication, are gaining attention due to their high genetic variability and performance in challenging environments. While major kernel metabolites have been examined, limited research has been conducted on minor bioactive components like lipids, despite their nutritional benefits. To address this, we analyzed twenty-two tetraploid accessions, comprising modern elite cultivars and landraces, to (i) verify if the selection process for yield-related traits carried out during the Green Revolution has influenced lipid amount and composition; (ii) uncover the extent of lipid compositional variability, giving evidence that lipid fingerprinting effectively identifies evolutionary signatures; and (iii) identify genotypes interesting for breeding programs to improve yield and nutrition. Interestingly, total fat did not correlate with kernel weight, indicating lipid composition as a promising trait for selection. Tri- and di-acylglycerol were the major lipid components along with free fatty acids, and their relative content varied significantly among genotypes. In particular, landraces belonging to T. turanicum and carthlicum ecotypes differed significantly in total lipid and fatty acid profiles. Our findings provide evidence that landraces can be a genetically relevant source of lipid variability, with potential to be exploited for improving wheat nutritional quality.

Introduction: In the last decade, it has been discovered that allergen-bearing extracellular nanovesicles, termed "pollensomes", are released by pollen during germination. These extracellular vesicles (EVs) may play an important role in pollen-pistil interaction during fertilization, stabilizing the secreted bioactive molecules and allowing long-distance signaling. However, the molecular composition and the biological role of these EVs are still unclear. The present study had two main aims: (I) to clarify whether pollen germination is needed to release pollensomes, or if they can be secreted also in high humidity conditions; and (II) to investigate the molecular features of pollensomes following the most recent guidelines for EVs isolation and identification., Methods: To do so, pollensomes were isolated from hydrated and germinated kiwi ( Actinidia chinensis Planch.) pollen, and characterized using imaging techniques, immunoblotting, and proteomics., Results: These analyses revealed that only germinated kiwi pollen released detectable concentrations of nanoparticles compatible with small EVs for shape and protein content. Moreover, a plant homolog of ALIX, which is a well-recognized and accepted marker of small EVs and exosomes in mammals, was found in pollensomes., Discussion: The presence of this protein, along with other proteins involved in endocytosis, is consistent with the hypothesis that pollensomes could comprehend a prominent subpopulation of plant exosome-like vesicles., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Suanno, Tonoli, Fornari, Savoca, Aloisi, Parrotta, Faleri, Cai, Coveney, Boocock, Verderio and Del Duca.)

Nanosilver applications, including sensing and water treatment, have significantly increased in recent years, although safety for humans and the environment is still under debate. Here, we tested the environmental safety of a novel formulation of silver nanoparticles functionalized with citrate and L-cysteine (AgNPcitLcys) on freshwater cnidarian Hydra vulgaris as an emerging ecotoxicological model for the safety of engineered nanomaterials. AgNPcitLcys behavior was characterized by dynamic light scattering (DLS), while Ag release was measured by inductively coupled plasma mass spectrometry (ICP-MS). H. vulgaris ( n = 12) subjects were evaluated for morphological aberration after 96 h of exposure and regeneration ability after 96 h and 7 days of exposure, after which the predatory ability was also assessed. The results show a low dissolution of AgNPcitLcys in Hydra medium (max 0.146% of nominal AgNPcitLcys concentration) and highlight a lack of ecotoxicological effects, both on morphology and regeneration, confirming the protective role of the double coating against AgNP biological effects. Predatory ability evaluation suggests a mild impairment of the entangling capacity or of the functionality of the tentacles, as the number of preys killed but not ingested was higher than the controls in all exposed animals. While their long-term sub-lethal effects still need to be further evaluated on H. vulgaris , AgNPcitLcys appears to be a promising tool for environmental applications, for instance, for water treatment and sensing.

Low-molecular-weight, aspartic-acid-rich proteins (ASP-RICH) have been assumed to be involved in the self-incompatibility process of clementine. The role of ASP-RICH is not known, but hypothetically they could sequester calcium ions (Ca 2+ ) and affect Ca 2+ -dependent mechanisms. In this article, we analyzed the effects induced by clementine ASP-RICH proteins (CcASP-RICH) when expressed in the tobacco heterologous system, focusing on the male gametophyte. The aim was to gain insight into the mechanism of action of ASP-RICH in a well-known cellular system, i.e., the pollen tube. Pollen tubes of tobacco transgenic lines expressing CcASP-RICH were analyzed for Ca 2+ distribution, ROS, proton gradient, as well as cytoskeleton and cell wall. CcASP-RICH modulated Ca 2+ content and consequently affected cytoskeleton organization and the deposition of cell wall components. In turn, this affected the growth pattern of pollen tubes. Although the expression of CcASP-RICH did not exert a remarkable effect on the growth rate of pollen tubes, effects at the level of growth pattern suggest that the expression of ASP-RICH may exert a regulatory action on the mechanism of plant cell growth.

Key Message: The article concerns the association between callose synthase and cytoskeleton by biochemical and ultrastructural analyses in the pollen tube. Results confirmed this association and immunogold labeling showed a colocalization. Callose is a cell wall polysaccharide involved in fundamental biological processes, from plant development to the response to abiotic and biotic stress. To gain insight into the deposition pattern of callose, it is important to know how the enzyme callose synthase is regulated through the interaction with the vesicle-cytoskeletal system. Actin filaments likely determine the long-range distribution of callose synthase through transport vesicles but the spatial/biochemical relationships between callose synthase and microtubules are poorly understood, although experimental evidence supports the association between callose synthase and tubulin. In this manuscript, we further investigated the association between callose synthase and microtubules through biochemical and ultrastructural analyses in the pollen tube model system, where callose is an essential component of the cell wall. Results by native 2-D electrophoresis, isolation of callose synthase complex and far-western blot confirmed that callose synthase is associated with tubulin and can therefore interface with cortical microtubules. In contrast, actin and sucrose synthase were not permanently associated with callose synthase. Immunogold labeling showed colocalization between the enzyme and microtubules, occasionally mediated by vesicles. Overall, the data indicate that pollen tube callose synthase exerts its activity in cooperation with the microtubular cytoskeleton., (© 2022. The Author(s).)

Stinging nettle is a perennial herbaceous species holding value as a multi-purpose plant. Indeed, its leaves and roots are phytofactories providing functional ingredients of medicinal interest and its stems produce silky and resistant extraxylary fibers (a.k.a. bast fibers) valued in the biocomposite sector. Similarly to what is reported in other fiber crops, the stem of nettle contains both lignified and hypolignified fibers in the core and cortex, respectively, and it is therefore a useful model for cell wall research. Indeed, data on nettle stem tissues can be compared to those obtained in other models, such as hemp and flax, to support hypotheses on the differentiation and development of bast fibers. The suitability of the nettle stem as model for cell wall-related research was already validated using a transcriptomics and biochemical approach focused on internodes at different developmental stages sampled at the top, middle, and bottom of the stem. We here sought to complement and enrich these data by providing immunohistochemical and ultrastructural details on young and older stem internodes. Antibodies recognizing non-cellulosic polysaccharides (galactans, arabinans, rhamnogalacturonans) and arabinogalactan proteins were here investigated with the goal of understanding whether their distribution changes in the stem tissues in relation to the bast fiber and vascular tissue development. The results obtained indicate that the occurrence and distribution of cell wall polysaccharides and proteins differ between young and older internodes and that these changes are particularly evident in the bast fibers., (© 2021. The Author(s).)

In recent decades, atmospheric pollution led to a progressive reduction of the ozone layer with a consequent increase in UV-B radiation. Despite the high adaptation of olive trees to the Mediterranean environment, the progressive increase of UV-B radiation is a risk factor for olive tree cultivation. It is therefore necessary to understand how high levels of UV-B radiation affect olive plants and to identify olive varieties which are better adapted. In this study we analyzed two Italian olive varieties subjected to chronic UV-B stress. We focused on the effects of UV-B radiation on RubisCO, in terms of quantity, enzymatic activity and isoform composition. In addition, we also analyzed changes in the activity of antioxidant enzymes (SOD, CAT, GPox) to get a comprehensive picture of the antioxidant system. We also evaluated the effects of UV-B on the enzyme sucrose synthase. The overall damage at biochemical level was also assessed by analyzing changes in Hsp70, a protein triggered under stress conditions. The results of this work indicate that the varieties (Giarraffa and Olivastra Seggianese) differ significantly in the use of specific antioxidant defense systems, as well as in the activity and isoform composition of RubisCO. Combined with a different use of sucrose synthase, the overall picture shows that Giarraffa optimized the use of GPox and opted for a targeted choice of RubisCO isoforms, in addition to managing the content of sucrose synthase, thereby saving energy during critical stress points.

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., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Cysteine proteases, belonging to the C1-papain family, play a major role in plant growth and development, senescence, and immunity. There is evidence to suggest that pollen cysteine protease (CP) (ZmCP03) is involved in regulating the anther development and pollen formation in maize. However, there is no report on the genome-wide identification and comparison of CPs in the pollen coat and other tissues in maize. In this study, a total of 38 homologous genes of ZmCP03 in maize were identified. Subsequently, protein motifs, conserved domains, gene structures, and duplication patterns of 39 CPs are analyzed to explore their evolutionary relationship and potential functions. The cis -elements were identified in the upstream sequence of 39 CPs , especially those that are related to regulating growth and development and responding to environmental stresses and hormones. The expression patterns of these genes displayed remarked difference at a tissue or organ level in maize based on the available transcriptome data in the public database. Quantitative reverse transcription PCR (RT-qPCR) analysis showed that ZmCP03 was preferably expressed at a high level in maize pollen. Analyses by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot, immunofluorescence and immunogold electron microscopy all validated the cellular localization of ZmCP03 in both the pollen coat and pollen cytoplasm. In addition, 142 CP genes from Arabidopsis ( Arabidopsis thaliana ), rice ( Oryza sativa ) and cotton ( Gossypium hirsutum ), together with 39 maize CPs , were retrieved to analyze their evolution by comparing with orthologous genes. The results suggested that ZmCP03 was relatively conservative and stable during evolution. This study may provide a referential evidence on the function of ZmCP03 in pollen development and germination in maize., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Li, Niu, Wu, Faleri, Tai, Zhang, Liu, Wang and Cai.)

Global warming leads to a progressive rise in environmental temperature. Plants, as sessile organisms, are threatened by these changes; the male gametophyte is extremely sensitive to high temperature and its ability to preserve its physiological status under heat stress is known as acquired thermotolerance. This latter can be achieved by exposing plant to a sub-lethal temperature (priming) or to a progressive increase in temperature. The present research aims to investigate the effects of heat priming on the functioning of tobacco pollen grains. In addition to evaluating basic physiological parameters (e.g., pollen viability, germination and pollen tube length), several aspects related to a correct pollen functioning were considered. Calcium (Ca 2+ ) level, reactive oxygen species (ROS) and related antioxidant systems were investigated, also to the organization of actin filaments and cytoskeletal protein such as tubulin (including tyrosinated and acetylated isoforms) and actin. We also focused on sucrose synthase (Sus), a key metabolic enzyme and on the content of main soluble sugars, including UDP-glucose. Results here obtained showed that a pre-exposure to sub-lethal temperatures can positively enhance pollen performance by altering its metabolism. This can have a considerable impact, especially from the point of view of breeding strategies aimed at improving crop species.

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 β-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., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

The occurrence of nanoplastics in oceans' surface waters is no more a hypothesis and it could severely affect marine organisms from different trophic levels. Nanoscale particles interaction with dissolved natural organic matter (NOM) significantly influence their behaviour and consequently bioavailability and toxicity to marine species. Extracellular polymeric substances (EPS) are among the main components of the NOM pool in seawater yet have been so far little investigated for their effect in altering the physical-chemical properties of nanosized objects. Here we employed EPS from marine diatom Phaeodactylum tricornutum to study the evolution of an eco-corona formation upon incubation with 60 nm carboxylated polystyrene nanoparticles (PS-COOH NPs), as proxy for nanoplastics in seawater. EPS significantly reduced PS-COOH NPs aggregation rate compared to biomolecule free natural seawater (NSW) and caused the formation of complexes constituted by both carbohydrate and protein components. Size Exclusion Chromatography (SEC) revealed four main distinct groups of peaks, spanning from high (>100 kDa) to low molecular weight (20 kDa) molecules, characterized by a high chemical heterogeneity. The lowering of the chromatographic signals detected after EPS incubation with PS-COOH NPs, mainly in the eluates at high molecular weight, suggests that an important fraction of EPS remained adsorbed on PS-COOH NPs. In agreement, SDS-PAGE analysis of proteins adsorbed on PS-COOH showed the occurrence of an eco-corona formed by proteins in the range of molecular weight 30-100 kDa. No toxicity to diatoms was observed upon PS-COOH exposure (72 h, 1-100 mg L -1 ) even by adding a further source of exogenous EPS during exposure. Moreover, the addition of EPS reduced ROS production, even when cells were incubated with PS-COOH NPs at 10 and 50 mg L -1 , suggesting an antioxidant scavenging activity of EPS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In clementine, failure of fertilization can result in parthenocarpic fruit development, which has several advantages, such as seedless fruit, longer shelf-life, and greater consumer appeal. Recently, S-RNases have been identified in Citrus grandis , thus revealing that the self-incompatibility (SI) reaction relies on the S-RNase gametophytic mechanism. The fundamental role of environmental factors, mostly temperature, in determining the numbers of pollen tubes reaching the ovary is also well established in Citrus . In the present work, temperature-dependent pollen-pistil interactions in C. clementina were analyzed, focusing on several morphological aspects, as well as on polyamine (PA) content and the activity and distribution of transglutaminase (TGase), both reported to be involved in the SI response in pear and in pummelo. Results clearly indicate that temperature contributed to a different activation of the SI response, which occurs at optimal temperature of 25°C but was by-passed at 15°C. TGase activity was stimulated during the SI response, and it localized differently in the compatible and incompatible interaction: in compatible pollinated styles, TGase localized inside the style canal, while it was detected all around it in incompatible crosses. TGase localization and activity were congruent with the levels of soluble and insoluble conjugated PAs and with morphological evidences, which highlighted cell wall modification occurring as a result of SI., (Copyright © 2020 Aloisi, Distefano, Antognoni, Potente, Parrotta, Faleri, Gentile, Bennici, Mareri, Cai and Del Duca.)

Marine diatoms have been identified among the most abundant taxa of microorganisms associated with plastic waste collected at sea. However, the impact of nano-sized plastic fragments (nanoplastics) at single cell and population level is almost unknown. We exposed the marine diatom Skeletonema marinoi to model polystyrene nanoparticles with carboxylic acid groups (PS-COOH NPs, 90 nm) for 15 days (1, 10, 50 μg/mL). Growth, reactive oxygen species (ROS) production, and nano-bio-interactions were investigated. No effect on diatom growth was observed, however Dynamic light scattering (DLS) demonstrated the formation of large PS aggregates which were localized at the diatoms' fultoportula process (FPP), as shown by TEM images. Increase production of ROS and reduction in chain length were also observed upon PS NPs exposure (p < 0.005). The observed PS-diatom interaction could have serious consequences on diatoms ecological role on the biogeochemical cycle of carbon, by impairing the formation of fast-sinking aggregates responsible for atmospheric carbon fixation and sequestration in the ocean sea floor. S. marinoi exposure to PS NPs caused an increase of intracellular and extracellular oxidative stress, the reduction of diatom's chain length and the adhesion of PS NPs onto the algal surface., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

To encourage the applicability of nano-adsorbent materials for heavy metal ion removal from seawater and limit any potential side effects for marine organisms, an ecotoxicological evaluation based on a biological effect-based approach is presented. ZnCl 2 (10 mg L -1 ) contaminated artificial seawater (ASW) was treated with newly developed eco-friendly cellulose-based nanosponges (CNS) (1.25 g L -1 for 2 h), and the cellular and tissue responses of marine mussel Mytilus galloprovincialis were measured before and after CNS treatment. A control group (ASW only) and a negative control group (CNS in ASW) were also tested. Methods: A significant recovery of Zn-induced damages in circulating immune and gill cells and mantle edges was observed in mussels exposed after CNS treatment. Genetic and chromosomal damages reversed to control levels in mussels' gill cells (DNA integrity level, nuclear abnormalities and apoptotic cells) and hemocytes (micronuclei), in which a recovery of lysosomal membrane stability (LMS) was also observed. Damage to syphons, loss of cilia by mantle edge epithelial cells and an increase in mucous cells in ZnCl 2 -exposed mussels were absent in specimens after CNS treatment, in which the mantle histology resembled that of the controls. No effects were observed in mussels exposed to CNS alone. As further proof of CNS' ability to remove Zn(II) from ASW, a significant reduction of >90% of Zn levels in ASW after CNS treatment was observed (from 6.006 to 0.510 mg L -1 ). Ecotoxicological evaluation confirmed the ability of CNS to remove Zn from ASW by showing a full recovery of Zn-induced toxicological responses to the levels of mussels exposed to ASW only (controls). An effect-based approach was thus proven to be useful in order to further support the environmentally safe (ecosafety) application of CNS for heavy metal removal from seawater.

Main Conclusion: The immuno-ultrastructural investigation localized cell-wall polysaccharides of bast fibers during hemp hypocotyl growth. Moreover, for the first time, the localization of a peroxidase and laccase is provided in textile hemp. In the hypocotyl of textile hemp, elongation and girth increase are separated in time. This organ is therefore ideal for time-course analyses. Here, we follow the ultrastructural rearrangement of cell-wall components during the development of the hemp hypocotyl. An expression analysis of genes involved in the biosynthesis of cellulose, the chief polysaccharide of bast fiber cell walls and xylan, the main hemicellulose of secondary cell walls, is also provided. The analysis shows a higher expression of cellulose and xylan-related genes at 15 and 20 days after sowing, as compared to 9 days. In the young hypocotyl, the cell walls of bast fibers show cellulose microfibrils that are not yet compacted to form a mature G-layer. Crystalline cellulose is detected abundantly in the S1-layer, together with unsubstituted/low-substituted xylan and, to a lesser extent, in the G-layer. The LM5 galactan epitope is confined to the walls of parenchymatic cells. LM6-specific arabinans are detected at the interface between the cytoplasm and the gelatinous cell wall of bast fibers. The class III peroxidase antibody shows localization in the G-layer only at older developmental stages. The laccase antibody shows a distinctive labelling of the G-layer region closest to the S1-layer; the signal becomes more homogeneous as the hypocotyl matures. The data provide important insights on the cell wall distribution of polysaccharide and protein components in bast fibers during the hypocotyl growth of textile hemp.

Cyclophilins (CyPs) are ubiquitous proteins involved in a wide variety of processes including protein maturation and trafficking, receptor complex stabilization, apoptosis, receptor signaling, RNA processing, and spliceosome assembly. The ubiquitous presence is justified by their peptidyl-prolyl cis-trans isomerase (PPIase) activity, catalyzing the rotation of X-Pro peptide bonds from a cis to a trans conformation, a critical rate-limiting step in protein folding, as over 90% of proteins contain trans prolyl imide bonds. In Arabidopsis 35 CyPs involved in plant development have been reported, showing different subcellular localizations and tissue- and stage-specific expression. In the present work, we focused on the localization of CyPs in pear (Pyrus communis) pollen, a model system for studies on pollen tube elongation and on pollen-pistil self-incompatibility response. Fluorescent, confocal and immuno-electron microscopy showed that this protein is present in the cytoplasm, organelles and cell wall, as confirmed by protein fractionation. Moreover, an 18-kDa CyP isoform was specifically released extracellularly when pear pollen was incubated with the Ca 2+ chelator EGTA., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

In this work, hydrophilic silver nanoparticles (AgNPs), bifunctionalized with citrate (Cit) and L-cysteine (L-cys), were synthesized. The typical local surface plasmon resonance (LSPR) at λ max = 400 nm together with Dynamic Light Scattering (DLS) measurements (<2R H > = 8 ± 1 nm) and TEM studies (Ø = 5 ± 2 nm) confirmed the system nanodimension and the stability in water. Molecular and electronic structures of AgNPs were investigated by FTIR, SR-XPS, and NEXAFS techniques. We tested the system as plasmonic sensor in water with 16 different metal ions, finding sensitivity to Hg 2+ in the range 1-10 ppm. After this first screening, the molecular and electronic structure of the AgNPs-Hg 2+ conjugated system was deeply investigated by SR-XPS. Moreover, in view of AgNPs application as sensors in real water systems, environmental safety assessment (ecosafety) was performed by using standardized ecotoxicity bioassay as algal growth inhibition tests (OECD 201, ISO 10253:2006), coupled with determination of Ag + release from the nanoparticles in fresh and marine aqueous exposure media, by means of ICP-MS. These latest studies confirmed low toxicity and low Ag + release. Therefore, these ecosafe AgNPs demonstrate a great potential in selective detection of environmental Hg 2+ , which may attract a great interest for several biological research fields.

Drought stress is one of the most severe conditions for plants, especially in the face of the emerging problem of global warming. This issue is important when considering economically relevant crops, including the tomato. For these plants, a promising solution is the valorization of local agrobiodiversity as a source of genetic variability. In this paper we investigated how six Italian tomato varieties react to a prolonged period of water depletion. We used a multidisciplinary approach, from genetics to plant physiology and cytology, to provide a detailed overview of the response of plants to stress. The varieties analyzed, each characterized by a specific genetic profile, showed a genotype-specific response with the variety 'Fragola' being the most resistant and the variety 'Pisanello' the most susceptible. For all the parameters evaluated, 'Fragola' performed in a manner comparable to that of control plants. On the contrary, 'Pisanello' appeared to be more affected and showed an increase in the number of stomata and a drastic increase in antioxidants, a symptom of acute oxidative stress. Our work suggests the existence of a valuable reservoir of genetic biodiversity with more drought-tolerant tomato genotypes opening the way to further exploitation and use of local germplasm in breeding programs.

Stinging nettle ( Urtica dioica L.) produces silky cellulosic fibres, as well as bioactive molecules. To improve the knowledge on nettle and enhance its opportunities of exploitation, a draft transcriptome of the "clone 13" (a fibre clone) is here presented. The transcriptome of whole internodes sampled at the top and middle of the stem is then compared with the core and cortical tissues sampled at the bottom. Young internodes show an enrichment in genes involved in the biosynthesis of phytohormones (auxins and jasmonic acid) and secondary metabolites (flavonoids). The core of internodes collected at the bottom of the stem is enriched in genes partaking in different aspects of secondary cell wall formation (cellulose, hemicellulose, lignin biosynthesis), while the cortical tissues reveal the presence of a C starvation signal probably due to the UDP-glucose demand necessary for the thickening phase of bast fibres. Cell wall analysis indicates a difference in rhamnogalacturonan structure/composition of mature bast fibres, as evidenced by the higher levels of galactose measured, as well as the occurrence of more water-soluble pectins in elongating internodes. The targeted quantification of phenolics shows that the middle internode and the cortical tissues at the bottom have higher contents than top internodes. Ultrastructural analyses reveal the presence of a gelatinous layer in bast fibres with a lamellar structure. The data presented will be an important resource and reference for future molecular studies on a neglected fibre crop., Competing Interests: The authors declare no conflict of interest associated with the work described in this manuscript.

Pollen-stigma interaction is a highly selective process, which leads to compatible or incompatible pollination, in the latter case, affecting quantitative and qualitative aspects of productivity in species of agronomic interest. While the genes and the corresponding protein partners involved in this highly specific pollen-stigma recognition have been studied, providing important insights into pollen-stigma recognition in self-incompatible (SI), many other factors involved in the SI response are not understood yet. This work concerns the study of transglutaminase (TGase), polyamines (PAs) pattern and metabolomic profiles following the pollination of Pyrus communis L. pistils with compatible and SI pollen in order to deepen their possible involvement in the reproduction of plants. Immunolocalization, abundance and activity of TGase as well as the content of free, soluble-conjugated and insoluble-bound PAs have been investigated. 1 H NMR-profiling coupled with multivariate data treatment (PCA and PLS-DA) allowed to compare, for the first time, the metabolic patterns of not-pollinated and pollinated styles. Results clearly indicate that during the SI response TGase activity increases, resulting in the accumulation of PAs conjugated to hydroxycinnamic acids and other small molecules. Metabolomic analysis showed a remarkable differences between pollinated and not-pollinated styles, where, except for glucose, all the other metabolites where less concentrated. Moreover, styles pollinated with compatible pollen showed the highest amount of sucrose than SI pollinated ones, which, in turn, contained highest amount of all the other metabolites, including aromatic compounds, such as flavonoids and a cynnamoil derivative.

Cold is an abiotic stress seriously threatening crop productivity by decreasing biomass production. The pollen tube is a target of cold stress, but also a useful model to address questions on cell wall biosynthesis. We here provide (immuno)cytological data relative to the impact of cold on the pollen tube cell wall. We clearly show that the growth pattern is severely affected by the stress, since the typical pulsed-growth mechanism accompanied by the periodic deposition of pectin rings is absent/severely reduced. Additionally, pectins and cellulose accumulate in bulges provoked by the stress, while callose, which colocalizes with pectins in the periodic rings formed during pulsed growth, accumulates randomly in the stressed samples. The altered distribution of the cell wall components is accompanied by differences in the localization of glucan synthases: cellulose synthase shows a more diffuse localization, while callose synthase shows a more frequent cytoplasmic accumulation, thereby denoting a failure in plasma membrane insertion. The cell wall observations are complemented by the analysis of intracellular Ca 2+ , pH and reactive oxygen species (ROS): while in the case of pH no major differences are observed, a less focused Ca 2+ and ROS gradients are present in the stressed samples. The standard oscillatory growth of pollen tubes is recovered by transient changes of turgor pressure induced by hypoosmotic media. Overall our data contribute to the understanding of the impact that cold stress has on the normal development of the pollen tube and unveil the cell wall-related aberrant features accompanying the observed alterations., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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-NH 2 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-NH 2 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-NH 2 (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-NH 2 -coelomocytes interaction and toxicity. PS-NH 2 did not alter MXR phenotype but the observed dose-dependent decrease in calcein accumulation suggests the ability of PS-NH 2 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.

Background and Aims: Pollen tubes are rapidly growing, photosynthetically inactive cells that need high rates of energy to support growth. Energy can derive from internal and external storage sources. The lack of carbon sources can cause various problems during pollen tube growth, which in turn could affect the reproduction of plants., Methods: We analysed the effects of energy deficiency on the development of Nicotiana tabacum pollen tubes by replacing sucrose with glycerol in the growth medium. We focused on cell growth and related processes, such as metabolite composition and cell wall synthesis., Key Results: We found that the lack of sucrose affects pollen germination and pollen tube length during a specific growth period. Both sugar metabolism and ATP concentration were affected by sucrose shortage when pollen tubes were grown in glycerol-based media; this was related to decreases in the concentrations of glucose, fructose and UDP-glucose. The intracellular pH and ROS levels also showed a different distribution in pollen tubes grown in sucrose-depleted media. Changes were also observed at the cell wall level, particularly in the content and distribution of two enzymes related to cell wall synthesis (sucrose synthase and callose synthase). Furthermore, both callose and newly secreted cell wall material (mainly pectins) showed an altered distribution corresponding to the lack of oscillatory growth in pollen tubes. Growth in glycerol-based media also temporarily affected the movement of generative cells and, in parallel, the deposition of callose plugs., Conclusion: Pollen tubes represent an ideal model system for studying metabolic pathways during the growth of plant cells. In our study, we found evidence that glycerol, a less energetic source for cell growth than sucrose, causes critical changes in cell wall deposition. The evidence that different aspects of pollen tube growth are affected is an indication that pollen tubes adapt to metabolic stress.

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-TiO 2 ) (Aeroxide P25, Degussa Evonik) and its interaction with cadmium (CdCl 2 ) in plants using radish seeds (Raphanus sativus L. Parvus) as model species. Radish seeds were exposed to n-TiO 2 (1-1000mg/L) and CdCl 2 (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-TiO 2 in Milli-Q water as exposure medium were also determined. DLS analysis showed small aggregates of n-TiO 2 , negative Z-potential and stable PdI in seed's exposure media. Germination percentage, GI and root length resulted affected by n-TiO 2 exposure compared to controls. In particular, n-TiO 2 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, CdCl 2 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-TiO 2 and CdCl 2 at the highest concentrations (1000mg/L n-TiO 2 and 250mg/L CdCl 2 ) compared to co-exposed seeds at low concentration (1mg/L n-TiO 2 and 1mg/L CdCl 2 ) 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-TiO 2 did not affect CdCl 2 toxicity. Concerning antioxidant enzymes, a significant increase of CAT activity in seeds exposed to single high n-TiO 2 concentration (1000mg/L) was observed while n-TiO 2 (1mg/L), CdCl 2 (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-TiO 2 but no significantly was observed, however both n-TiO 2 and CdCl 2 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-TiO 2 do not affect the toxicity of CdCl 2 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-TiO 2 . Nevertheless, although n-TiO 2 seems not to reduce Cd toxicity at high concentration (up to 250mg/L), interactions cannot be excluded based on obtained results., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Silver nanoparticles (AgNPs), embedded into a specific polysaccharide (EPS), were biogenerated by Klebsiella oxytoca DSM 29614 under aerobic (AgNPs-EPS aer ) and anaerobic conditions (AgNPs-EPS anaer ). Both AgNPs-EPS matrices were tested by MTT assay for cytotoxic activity against human breast (SKBR3 and 8701-BC) and colon (HT-29, HCT 116 and Caco-2) cancer cell lines, revealing AgNPs-EPS aer as the most active, in terms of IC50, with a more pronounced efficacy against breast cancer cell lines. Therefore, colony forming capability, morphological changes, generation of reactive oxygen species (ROS), induction of apoptosis and autophagy, inhibition of migratory and invasive capabilities and proteomic changes were investigated using SKBR3 breast cancer cells with the aim to elucidate AgNPs-EPS aer mode of action. In particular, AgNPs-EPS aer induced a significant decrease of cell motility and MMP-2 and MMP-9 activity and a significant increase of ROS generation, which, in turn, supported cell death mainly through autophagy and in a minor extend through apoptosis. Consistently, TEM micrographs and the determination of total silver in subcellular fractions indicated that the Ag + accumulated preferentially in mitochondria and in smaller concentrations in nucleus, where interact with DNA. Interestingly, these evidences were confirmed by a differential proteomic analysis that highlighted important pathways involved in AgNPs-EPS aer toxicity, including endoplasmic reticulum stress, oxidative stress and mitochondrial impairment triggering cell death trough apoptosis and/or autophagy activation., Competing Interests: CONFLICTS OF INTEREST Authors declare no conflicts of interest.

The impact of nanoplastics using model polystyrene nanoparticles (PS NPs), anionic (PS-COOH) and cationic (PS-NH 2 ), 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 LC 50 values resulted lower in rotifers exposed in RSW (LC 50 =2.75±0.67µg/ml) compared to those exposed in NSW (LC 50 =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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Proper growth of the pollen tube depends on an elaborate mechanism that integrates several molecular and cytological sub-processes and ensures a cell shape adapted to the transport of gametes. This growth mechanism is controlled by several molecules among which cytoplasmic and apoplastic polyamines. Spermine (Spm) has been correlated with various physiological processes in pollen, including structuring of the cell wall and modulation of protein (mainly cytoskeletal) assembly. In this work, the effects of Spm on the growth of pear pollen tubes were analyzed. When exogenous Spm (100 μM) was supplied to germinating pollen, it temporarily blocked tube growth, followed by the induction of apical swelling. This reshaping of the pollen tube was maintained also after growth recovery, leading to a 30-40% increase of tube diameter. Apical swelling was also accompanied by a transient increase in cytosolic calcium concentration and alteration of pH values, which were the likely cause for major reorganization of actin filaments and cytoplasmic organelle movement. Morphological alterations of the apical and subapical region also involved changes in the deposition of pectin, cellulose, and callose in the cell wall. Thus, results point to the involvement of Spm in cell wall construction as well as cytoskeleton organization during pear pollen tube growth.

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-NH 2 ) 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-NH 2 -formed nano-scale aggregates (Z-Average<200nm), caused inhibition of algal growth (EC 50 =12.97μg/ml) and mortality in brine shrimps at 14 d (LC 50 =0.83μg/ml). Moreover, 1μg/ml PS-NH 2 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-NH 2 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Various arrays of microtubules are present throughout the plant cell cycle and are involved in distinct functions. Microtubule-associated proteins (MAPs) regulate microtubule dynamics by acting as stabilizers, destabilizers, and promoters of microtubule dynamics. The MAP65 family is a specific group of cross-linkers required for structural maintenance of microtubules. In plants, different isoforms of MAP65 are differentially expressed according to their developmental program. In this work, we analyzed the differential distribution of proteins immunologically related to MAP65-1 during bud development in grapevine (Vitis vinifera L.). First, we annotated the MAP65 genes present in the Vitis genome in order to compare the number and sequence of genes to other species. Subsequently, we focused on a specific isoform (MAP65-1) by characterizing its accumulation and distribution. Proteins were extracted from different organs of Vitis (buds, leaves, flowers, and tendrils), were separated by two-dimensional electrophoresis (2-DE), and were probed by immunoblot with a specific antiserum. We found seven spots immunologically related to MAP65-1, grouped in two distinct clusters, which accumulate differentially according to the developmental stage. In addition, we analyzed the localization of MAP65-1 during three different stages of bud development. Implication of data on the use of different isotypes of MAP65-1 during Vitis development is discussed.

A strain of Klebsiella oxytoca DSM 29614 is grown on sodium citrate in the presence of 50 mg l -1 of Hg as Hg(NO 3 ) 2 . During growth, the strain produces an extracellular polymeric substance (EPS), constituted by a mixture of proteins and a specific exopolysaccharide. The protein components, derived from the outer membrane of cells, are co-extracted with the extracellular exopolysaccharide using ethanol. The extracted EPS contains 7.5% of Hg (total amount). This indicates that EPS is an excellent material for the biosorption of Hg 2+ , through chemical complexation with the EPS components. The binding capacity of these species towards Hg 2+ is studied by cyclic voltammetry, and Hg L 3 -edge XANES and EXAFS spectroscopy. The results found indicate that Hg 2+ is mainly bound to the nitrogen of the imidazole ring or other N-heterocycle compounds. The hydroxyl moities of sugars and/or the carboxyl groups of two glucuronic acids in the polysaccharide can also play an important role in sequestring Hg 2+ ions. However, N-heterocyclic groups of proteins bind Hg 2+ faster than hydroxyl and carboxyl groups of the polysaccharide., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In the late Roman Republic period (2nd-1st century BC), in the area of San Giovanni on Elba Island, previously subject to intense extraction of iron ore, a rustic villa was established by Marco Valerio Messalla, a supreme Roman magistrate. The foundations of the walls were discovered and excavated by an archaeological mission. Palaeobotanical analysis of a set of stratigraphic layers was performed. Palynological slides showed remains of palynomorphic and non-pollen objects, while data combined with anthracological investigations confirmed the hypothesis that in the 1st century AD the villa was destroyed by a fire that created a compact crust under which were discovered four broken Roman amphorae containing about five hundred apple seeds. Comparisons of archaeological and fresh seeds from reference collections showed discontinuous morphology except for one group of archaeological samples. DNA was isolated from seeds that had well-preserved embryos in all groups. DNA extracts from archaeological, wild and modern domestic seeds (controls) were amplified by PCR and tested with SSR molecular markers, followed by genome analysis., (Copyright © 2016 Académie des sciences. Published by Elsevier SAS. All rights reserved.)

Silver nanoparticles (AgNPs), embedded into a specific exopolysaccharide (EPS), were produced by Klebsiella oxytoca DSM 29614 by adding AgNO3 to the cultures during exponential growth phase. In particular, under aerobic or anaerobic conditions, two types of silver nanoparticles, named AgNPs-EPS(aer) and the AgNPs-EPS(anaer), were produced respectively. The effects on bacterial cells was demonstrated by using Escherichia coli K12 and Kocuria rhizophila ATCC 9341 (ex Micrococcus luteus) as Gram-negative and Gram-positive tester strains, respectively. The best antimicrobial activity was observed for AgNPs-EPS(aer), in terms of minimum inhibitory concentrations and minimum bactericidal concentrations. Observations by transmission electron microscopy showed that the cell morphology of both tester strains changed during the exposition to AgNPs-EPS(aer). In particular, an electron-dense wrapped filament was observed in E. coli cytoplasm after 3 h of AgNPs-EPS(aer) exposition, apparently due to silver accumulation in DNA, and both E. coli and K. rhizophila cells were lysed after 18 h of exposure to AgNPs-EPS(aer). The DNA breakage in E. coli cells was confirmed by the comparison of 3-D fluorescence spectra fingerprints of DNA. Finally the accumulation of silver on DNA of E. coli was confirmed directly by a significant Ag(+) release from DNA, using the scanning electrochemical microscopy and the voltammetric determinations.

Main Conclusion: Heat stress changes isoform content and distribution of cytoskeletal subunits in pollen tubes affecting accumulation of secretory vesicles and distribution of sucrose synthase, an enzyme involved in cell wall synthesis. Plants are sessile organisms and are therefore exposed to damages caused by the predictable increase in temperature. We have analyzed the effects of temperatures on the development of pollen tubes by focusing on the cytoskeleton and related processes, such as vesicular transport and cell wall synthesis. First, we show that heat stress affects pollen germination and, to a lesser extent, pollen tube growth. Both, microtubules and actin filaments, are damaged by heat treatment and changes of actin and tubulin isoforms were observed in both cases. Damages to actin filaments mainly concern the actin array present in the subapex, a region critical for determining organelle and vesicle content in the pollen tube apex. In support of this, green fluorescent protein-labeled vesicles are arranged differently between heat-stressed and control samples. In addition, newly secreted cell wall material (labeled by propidium iodide) shows an altered distribution. Damage induced by heat stress also extends to proteins that bind actin and participate in cell wall synthesis, such as sucrose synthase. Ultimately, heat stress affects the cytoskeleton thereby causing alterations in the process of vesicular transport and cell wall deposition.

The role of spermine (SM) was studied to verify if SM supplied to Nicotiana tabacum flower can modulate programmed cell death (PCD) of the corolla. SM has strong effects on the development and senescence of excised flowers despite its low physiological levels. The timing and duration of SM treatment is a key factor; SM counteracts PCD (verified by morphological observations, pigment contents and DNA laddering) only in the narrow developmental window of corolla expansion. Before and after, SM promotes PCD. SM exerts its pro-survival role by delaying fresh weight loss, by inhibiting reduction of pigments and finally by preventing DNA degradation. Moreover, SM deeply alters the distribution of the PA-conjugating enzyme transglutaminase (TGase). TGase is present in the epidermis during development, but it sprays also in the cell walls of inner parenchyma at senescence. After SM treatment, parenchyma cells accumulate TGase, increase in size and their cell walls do not undergo stiffening contrarily to control cells. The subcellular localization of TGase has been validated by biolistic-transformation of onion epidermal cells. Results indicated that SM is a critical factor in the senescence of N. tabacum corolla by controlling biochemical and morphological parameters; the lasts are probably interconnected with the action of TGase., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)