Sterols and Sphingolipids as New Players in Cell Wall Building and Apical Growth of Nicotiana tabacum L. Pollen Tubes.

Pollen tubes are tip-growing cells that create safe routes to convey sperm cells to the embryo sac for double fertilization. Recent studies have purified and biochemically characterized detergent-insoluble membranes from tobacco pollen tubes. These microdomains, called lipid rafts, are rich in sterols and sphingolipids and are involved in cell polarization in organisms evolutionarily distant, such as fungi and mammals. The presence of actin in tobacco pollen tube detergent-insoluble membranes and the preferential distribution of these domains on the apical plasma membrane encouraged us to formulate the intriguing hypothesis that sterols and sphingolipids could be a "trait d'union" between actin dynamics and polarized secretion at the tip. To unravel the role of sterols and sphingolipids in tobacco pollen tube growth, we used squalestatin and myriocin, inhibitors of sterol and sphingolipid biosynthesis, respectively, to determine whether lipid modifications affect actin fringe morphology and dynamics, leading to changes in clear zone organization and cell wall deposition, thus suggesting a role played by these lipids in successful fertilization.

Evaluation of Adhesive Characteristics of L. plantarum and L. reuteri Isolated from Weaned Piglets.

Limosilactobacillus reuteri and Lactiplantibacillus plantarum strains, previously isolated from weaned piglets, were considered for the evaluation of their adhesive characteristics. Lactobacilli were treated with LiCl in order to remove the surface protein layer, and probiotic activity was compared with those of untreated strains. The autoaggregation, co-aggregation to E. coli F18+, and adhesive abilities of LiCl-treated Limosilactobacillus reuteri and Lactiplantibacillus plantarum were significantly inhibited (p < 0.05) compared with the respective untreated strain. The hydrophobic and basic phenotypes were observed due to the strong affinity to chloroform and low adherence to ethyl acetate. In particular, L. plantarum showed higher hydrophobicity compared to L. reuteri, which may reflect their different colonizing ability. After treatment with LiCl to remove surface proteins, the adherence capabilities of L. reuteri and L. casei on IPEC-J2 cells decreased significantly (p < 0.001) and L. reuteri adhered more frequently. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that both L. reuteri and L. plantarum had several bands ranging from 20 to 100 kDa. Two-dimensional gel electrophoresis showed an acidic profile of the surface-layer polypeptides for both bacterial strains, and more studies are needed to characterize their profile and functions. The results confirm the pivotal role of surface proteins in the probiotic potential of L. reuteri and L. plantarum.

Protein Analysis of Pollen Tubes after the Treatments of Membrane Trafficking Inhibitors Gains Insights on Molecular Mechanism Underlying Pollen Tube Polar Growth.

Pollen tube elongation is characterized by a highly-polarized tip growth process dependent on an efficient vesicular transport system and largely mobilized by actin cytoskeleton. Pollen tubes are an ideal model system to study exocytosis, endocytosis, membrane recycling, and signaling network coordinating cellular processes, structural organization and vesicular trafficking activities required for tip growth. Proteomic analysis was applied to identify Nicotiana tabacum Differentially Abundant Proteins (DAPs) after in vitro pollen tube treatment with membrane trafficking inhibitors Brefeldin A, Ikarugamycin and Wortmannin. Among roughly 360 proteins separated in two-dimensional gel electrophoresis, a total of 40 spots visibly changing between treated and control samples were identified by MALDI-TOF MS and LC-ESI-MS/MS analysis. The identified proteins were classified according to biological processes, and most proteins were related to pollen tube energy metabolism, including ammino acid synthesis and lipid metabolism, structural features of pollen tube growth as well modification and actin cytoskeleton organization, stress response, and protein degradation. In-depth analysis of proteins corresponding to energy-related pathways revealed the male gametophyte to be a reliable model of energy reservoir and dynamics.

Heavy-Metal Phytoremediation from Livestock Wastewater and Exploitation of Exhausted Biomass.

Sustainable agriculture is aimed at long-term crop and livestock production with a minimal impact on the environment. However, agricultural practices from animal production can contribute to global pollution due to heavy metals from the feed additives that are used to ensure the nutritional requirements and also promote animal health and optimize production. The bioavailability of essential mineral sources is limited; thus, the metals are widely found in the manure. Via the manure, metallic ions can contaminate livestock wastewater, drastically reducing its potential recycling for irrigation. Phytoremediation, which is an efficient and cost-effective cleanup technique, could be implemented to reduce the wastewater pollution from livestock production, in order to maintain the water conservation. Plants use various strategies for the absorption and translocation of heavy metals, and they have been widely used to remediate livestock wastewater. In addition, the pollutants concentrated in the plants can be exhausted and used as heat to enhance plant growth and further concentrate the metals, making recycling a possible option. The biomass of the plants can also be used for biogas production in anaerobic fermentation. Combining phytoremediation and biorefinery processes would add value to both approaches and facilitate metal recovery. This review focuses on the concept of agro-ecology, specifically the excessive use of heavy metals in animal production, the various techniques and adaptations of the heavy-metal phytoremediation from livestock wastewater, and further applications of exhausted phytoremediated biomass.

The aquatic carnivorous plant Aldrovanda vesiculosa (Droseraceae) exhibits altered developmental stages in male gametophyte.

Aldrovanda vesiculosa (Droseraceae) is a rare aquatic carnivorous plant, distributed in Europe, Asia, Africa, and Australia. Aldrovanda populations can flower prolifically under favourable conditions, but seed set is very limited. We studied the structure of Aldrovanda pollen collected from flowers in different developmental stages (opened and non-opened anthers) from both European and Australian populations to elucidate pollination traits and the basis of poor seed set on the basis of microscopic observation of pollen and anther structure. Microscopic analyses of Aldrovanda pollen showed that this plant has pollen arranged in tetrads like other species in the Droseraceae family. In hydrated pollen, cytoplasmic protrusions originate from pores located along the equatorial wall of monads, and can develop into pollen tubes. Interestingly, pollen development from microspores occurs in open anthers, suggesting a delay of the developmental stages. In addition, pollen development displays altered sperm cell formation and precocious pollen germination. Precocious germination may characterize recalcitrant pollen, which naturally do not undergo dehydration before anthesis and remain partially hydrated, particularly in aquatic and wetland plants. These alterations of male gametophyte development could affect fertilization processes, and be the reason for the low reproductive capability of Aldrovanda observed both in the field and in cultures. Generally, reduced pollen longevity and very quick germination are considered an adaptation to aquatic or wet environments.

Typha latifolia and Thelypteris palustris behavior in a pilot system for the refinement of livestock wastewaters: A case of study.

In animal livestock heavy metals are widely used as feed additives to control enteric bacterial infections as well as to enhance the integrity of the immune system. As these metals are only partially adsorbed by animals, the content of heavy metals in manure and wastewaters causes soil and ground water contamination, with Zn2+ and Cu2+ being the most critical output from pig livestock. Phytoremediation is considered a valid strategy to improve the purity of wastewaters. This work studied the effect of Zn2+ and Cu2+ on the morphology and protein expression in Thelypteris palustris and Typha latifolia plants, cultured in a wetland pilot system. Despite the absence of macroscopic alterations, remodeling of cell walls and changes in carbohydrate metabolism were observed in the rhizomes of both plants and in leaves of Thelypteris palustris. However, similar modifications seemed to be determined by the alterations of different mechanisms in these plants. These data also suggested that marsh ferns are more sensitive to metals than monocots. Whereas toleration mechanisms seemed to be activated in Typha latifolia, in Thelypteris palustris the observed modifications appeared as slight toxic effects due to metal exposure. This study clearly indicates that both plants could be successfully employed in in situ phytoremediation systems, to remove Cu2+ and Zn2+ at concentrations that are ten times higher than the legal limits, without affecting plant growth.

The imbalance between dynamic and stable microtubules underlies neurodegeneration induced by 2,5-hexanedione.

Exposure to environmental toxins, including hydrocarbon solvents, increases the risk of developing Parkinson's disease. An emergent hypothesis considers microtubule dysfunction as one of the crucial events in triggering neuronal degeneration in Parkinson's disease. Here, we used 2,5-hexanedione (2,5-HD), the toxic metabolite of n-hexane, to analyse the early effects of toxin-induced neurodegeneration on the cytoskeleton in multiple model systems. In PC12 cells differentiated with nerve growth factor for 5 days, we found that 2,5-HD treatment affected all the cytoskeletal components. Moreover, we observed alterations in microtubule distribution and stability, in addition to the imbalance of post-translational modifications of α-tubulin. Similar defects were also found in vivo in 2,5-HD-intoxicated mice. Interestingly, we also found that 2,5-HD exposure induced significant changes in microtubule stability in human skin fibroblasts obtained from Parkinson's disease patients harbouring mutations in PRKN gene, whereas it was ineffective in healthy donor fibroblasts, suggesting that the genetic background may really make the difference in microtubule susceptibility to this environmental Parkinson's disease-related toxin. In conclusion, by showing the imbalance between dynamic and stable microtubules in hydrocarbon-induced parkinsonism, our data support the crucial role of microtubule defects in triggering neurodegeneration.

Bioaccumulation of heavy metals from wastewater through a Typha latifolia and Thelypteris palustris phytoremediation system.

Animal production is a source of heavy metals in livestock wastewater and also a key link in the food chain, with negative impacts on human and animal health. In intensive animal production systems, the most critical elements are zinc and copper. In order to development of innovative non-invasive strategies to reduce the environmental impact of livestock, this study assessed the ability of two plants, Typha latifolia and Thelypteris palustris, to bioaccumulate the heavy metals used in animal nutrition, from wastewater. Four mesocosms (width 2.0 m, length 2.0 m, 695 L of water, 210 kg of soil) were assembled outdoors at the Botanical Garden. Two of them were planted with T. latifolia (TL treated, n = 30; TL control, n = 30) and two with T. palustris (TP treated, n = 60; TP control, n = 60). In T0 a solution of a mineral additive premix (Zn 44.02 mg/L; Cu 8.63 mg/L) was dissolved in the treated mesocosms. At T0, d 15 (T1) and d 45 (T2) samples of roots, leaves, stems, soil and water were collected, dried, mineralized and analyzed using ICP-MS in order to obtain HMs content. We found that T. latifolia and T. palustris accumulate and translocate Zn, Cu from contaminated wastewater into plant tissues in a way that is directly related to the exposure time (T2 for Zn: 271.64 ±â€¯17.70, 409.26 ±â€¯17.70 for Cu: 47.54 ±â€¯3.56, 105.58 ±â€¯3.56 mg/kg of DM, respectively). No visual toxicity signs were observed during the experimental period. This phytoremediation approach could be used as an eco-sustainable approach to counteract the output of heavy metals.

Microtubules play a role in trafficking prevacuolar compartments to vacuoles in tobacco pollen tubes.

Fine regulation of exocytosis and endocytosis plays a basic role in pollen tube growth. Excess plasma membrane secreted during pollen tube elongation is known to be retrieved by endocytosis and partially reused in secretory pathways through the Golgi apparatus. Dissection of endocytosis has enabled distinct degradation pathways to be identified in tobacco pollen tubes and has shown that microtubules influence the transport of plasma membrane internalized in the tip region to vacuoles. Here, we used different drugs affecting the polymerization state of microtubules together with SYP21, a marker of prevacuolar compartments, to characterize trafficking of prevacuolar compartments in Nicotiana tabacum pollen tubes. Ultrastructural and biochemical analysis showed that microtubules bind SYP21-positive microsomes. Transient transformation of pollen tubes with LAT52-YFP-SYP21 revealed that microtubules play a key role in the delivery of prevacuolar compartments to tubular vacuoles.

Retarded germination of Nicotiana tabacum seeds following insertion of exogenous DNA mimics the seed persistent behavior.

Tobacco seeds show a coat-imposed dormancy in which the seed envelope tissues (testa and endosperm) impose a physical constraint on the radicle protrusion. The germination-limiting process is represented by the endosperm rupture which is induced by cell-wall weakening. Transgenic tobacco seeds, obtained by insertion of exogenous genes codifying for seed-based oral vaccines (F18 and VT2eB), showed retarded germination with respect to the wild type and modified the expression of endogenous proteins. Morphological and proteomic analyses of wild type and transgenic seeds revealed new insights into factors influencing seed germination. Our data showed that the interference of exogenous DNA influences the germination rather than the dormancy release, by modifying the maturation process. Dry seeds of F18 and VT2eB transgenic lines accumulated a higher amount of reserve and stress-related proteins with respect to the wild type. Moreover, the storage proteins accumulated in tobacco F18 and VT2eB dry seeds have structural properties that do not enable the early limited proteolysis observed in the wild type. Morphological observations by electron and light microscopy revealed a retarded mobilization of the storage material from protein and lipid bodies in transgenic seeds, thus impairing water imbibition and embryo elongation. In addition, both F18 and VT2eB dry seeds are more rounded than the wild type. Both the morphological and biochemical characteristics of transgenic seeds mimic the seed persistent profile, in which their roundness enables them to be buried in the soil, while the higher content of storage material enables the hypocotyl to elongate more and the cotyledons to emerge.

Emerging roles for microtubules in angiosperm pollen tube growth highlight new research cues.

In plants, actin filaments have an important role in organelle movement and cytoplasmic streaming. Otherwise microtubules (MTs) have a role in restricting organelles to specific areas of the cell and in maintaining organelle morphology. In somatic plant cells, MTs also participate in cell division and morphogenesis, allowing cells to take their definitive shape in order to perform specific functions. In the latter case, MTs influence assembly of the cell wall, controlling the delivery of enzymes involved in cellulose synthesis and of wall modulation material to the proper sites. In angiosperm pollen tubes, organelle movement is generally attributed to the acto-myosin system, the main role of which is in distributing organelles in the cytoplasm and in carrying secretory vesicles to the apex for polarized growth. Recent data on membrane trafficking suggests a role of MTs in fine delivery and repositioning of vesicles to sustain pollen tube growth. This review examines the role of MTs in secretion and endocytosis, highlighting new research cues regarding cell wall construction and pollen tube-pistil crosstalk, that help unravel the role of MTs in polarized growth.

Characterisation of detergent-insoluble membranes in pollen tubes of Nicotiana tabacum (L.).

Pollen tubes are the vehicle for sperm cell delivery to the embryo sac during fertilisation of Angiosperms. They provide an intriguing model for unravelling mechanisms of growing to extremes. The asymmetric distribution of lipids and proteins in the pollen tube plasma membrane modulates ion fluxes and actin dynamics and is maintained by a delicate equilibrium between exocytosis and endocytosis. The structural constraints regulating polarised secretion and asymmetric protein distribution on the plasma membrane are mostly unknown. To address this problem, we investigated whether ordered membrane microdomains, namely membrane rafts, might contribute to sperm cell delivery. Detergent insoluble membranes, rich in sterols and sphingolipids, were isolated from tobacco pollen tubes. MALDI TOF/MS analysis revealed that actin, prohibitins and proteins involved in methylation reactions and in phosphoinositide pattern regulation are specifically present in pollen tube detergent insoluble membranes. Tubulins, voltage-dependent anion channels and proteins involved in membrane trafficking and signalling were also present. This paper reports the first evidence of membrane rafts in Angiosperm pollen tubes, opening new perspectives on the coordination of signal transduction, cytoskeleton dynamics and polarised secretion.

Microtubule depolymerization affects endocytosis and exocytosis in the tip and influences endosome movement in tobacco pollen tubes.

Polarized organization of the cytoplasm of growing pollen tubes is maintained by coordinated function of actin filaments (AFs) and microtubules (MTs). AFs convey post-Golgi secretory vesicles to the tip where some fuse with specific domains of the plasma membrane (PM). Secretory activity is balanced by PM retrieval that maintains cell membrane economy and regulates the polarized composition of the PM, by dividing lipids/proteins between the shank and the tip. Although AFs play a key role in PM internalization in the shank, the role of MTs in exo-endocytosis needs to be characterized. The present results show that integrity of the MT cytoskeleton is necessary to control exo-endocytosis events in the tip. MT polymerization plays a role in promoting PM invagination in the apex of tobacco pollen tubes since nocodazole affected PM internalization in the tip and subsequent migration of endocytic vesicles from the apex for degradation. MT depolymerization in the apex and shank was associated with misallocation of a significantly greater amount of internalized PM to the Golgi apparatus and its early recycling to the secretory pathway. Fluorescence Recovery After Photobleaching (FRAP) experiments also showed that MT depolymerization in the tip region influenced the rate of exocytosis in the central domain of the apical PM.

Endocytic Pathways and Recycling in Growing Pollen Tubes.

Pollen tube growth is based on transport of secretory vesicles into the apical region where they fuse with a small area of the plasma membrane. The amount of secretion greatly exceeds the quantity of membrane required for growth. Mechanisms of membrane retrieval have recently been demonstrated and partially characterized using FM (Fei Mao) dyes or charged nanogold. Both these probes reveal that clathrin-dependent and -independent endocytosis occur in pollen tubes and are involved in distinct degradation pathways and membrane recycling. Exocytosis, internalization and sorting of PM proteins/lipids depend on the integrity of the actin cytoskeleton and are involved in actin filament organization. However, some kinds of endocytic and exocytic processes occurring in the central area of the tip still need to be characterized. Analysis of secretion dynamics and data derived from endocytosis highlight the complexity of events occurring in the tip region and suggest a new model of pollen tube growth.

Low concentration of LatB dramatically changes the microtubule organization and the timing of vegetative nucleus/generative cell entrance in tobacco pollen tubes.

Low concentration of LatB inhibits not only the actin polymerization, but also induces profound alteration of MT distribution in pollen tubes of Nicotiana tabacum. The short randomly oriented MTs in the apical and subapical regions, became organized as bundles forming subapical rings or basket-like structures, surrounding the apex. Moreover, the depolymerization of AFs in the cortical regions of the apex and subapical region affects the timing of entrance of the vegetative nucleus and generative cell into the pollen tube.

Pollen tube growth: a delicate equilibrium between secretory and endocytic pathways.

Although pollen tube growth is a prerequisite for higher plant fertilization and seed production, the processes leading to pollen tube emission and elongation are crucial for understanding the basic mechanisms of tip growth. It was generally accepted that pollen tube elongation occurs by accumulation and fusion of Golgi-derived secretory vesicles (SVs) in the apical region, or clear zone, where they were thought to fuse with a restricted area of the apical plasma membrane (PM), defining the apical growth domain. Fusion of SVs at the tip reverses outside cell wall material and provides new segments of PM. However, electron microscopy studies have clearly shown that the PM incorporated at the tip greatly exceeds elongation and a mechanism of PM retrieval was already postulated in the mid-nineteenth century. Recent studies on endocytosis during pollen tube growth showed that different endocytic pathways occurred in distinct zones of the tube, including the apex, and led to a new hypothesis to explain vesicle accumulation at the tip; namely, that endocytic vesicles contribute substantially to V-shaped vesicle accumulation in addition to SVs and that exocytosis does not involve the entire apical domain. New insights suggested the intriguing hypothesis that modulation between exo- and endocytosis in the apex contributes to maintain PM polarity in terms of lipid/protein composition and showed distinct degradation pathways that could have different functions in the physiology of the cell. Pollen tube growth in vivo is closely regulated by interaction with style molecules. The study of endocytosis and membrane recycling in pollen tubes opens new perspectives to studying pollen tube-style interactions in vivo.

Clathrin-dependent and independent endocytic pathways in tobacco protoplasts revealed by labelling with charged nanogold.

Positively charged nanogold was used as a probe to trace the internalization of plasma membrane (PM) domains carrying negatively charged residues at an ultrastructural level. The probe revealed distinct endocytic pathways within tobacco protoplasts and allowed the morphology of the organelles involved in endocytosis to be characterized in great detail. Putative early endosomes with a tubulo-vesicular structure, similar to that observed in animal cells, are described and a new compartment, characterized by interconnected vesicles, was identified as a late endosome using the Arabidopsis anti-syntaxin family Syp-21 antibody. Endocytosis dissection using Brefeldin A (BFA), pulse chase, temperature- and energy-dependent experiments combined with quantitative analysis of nanogold particles in different compartments, suggested that recycling to the PM predominated with respect to degradation. Further experiments using ikarugamycin (IKA), an inhibitor of clathrin-dependent endocytosis, and negatively charged nanogold confirmed that distinct endocytic pathways coexist in tobacco protoplasts.

Endocytic pathways in pollen tube: Implications for in vivo growth regulation.

By endocytosis eukaryotic cells can take up extracellular components and/or plasma membrane proteins for further delivering to endosomes. Although in animal cells different endocytic pathways were identified based on the requirement of a clathrin coating for vesicle internalization, endocytosis in plant cells still require to be fully characterized. The use of positively and negatively charged nanogold in combination with Ika, an inhibitor of the clathrindependent endocytosis, allowed to dissect the endocytic pathway and revealed the presence of clathrin-dependent and clathrin-independent degradative pathways.

Distinct endocytic pathways identified in tobacco pollen tubes using charged nanogold.

In an attempt to dissect endocytosis in Nicotiana tabacum L. pollen tubes, two different probes--positively or negatively charged nanogold--were employed. The destiny of internalized plasma membrane domains, carrying negatively or positively charged residues, was followed at the ultrastructural level and revealed distinct endocytic pathways. Time-course experiments and electron microscopy showed internalization of subapical plasma-membrane domains that were mainly recycled to the secretory pathway through the Golgi apparatus and a second mainly degradative pathway involving plasma membrane retrieval at the tip. In vivo time-lapse experiments using FM4-64 combined with quantitative analysis confirmed the existence of distinct internalization regions. Ikarugamycin, an inhibitor of clathrin-dependent endocytosis, allowed us to further dissect the endocytic process: electron microscopy and time-lapse studies suggested that clathrin-dependent endocytosis occurs in the tip and subapical regions, because recycling of positively charged nanogold to the Golgi bodies and the consignment of negatively charged nanogold to vacuoles were affected. However, intact positively charged-nanogold transport to vacuoles supports the idea that an endocytic pathway that does not require clathrin is also present in pollen tubes.

A methionine synthase homolog is associated with secretory vesicles in tobacco pollen tubes.

Seven isoforms of 85 kDa polypeptides (p85) were identified as methionine synthase (MetE) homologs by partial aminoacid sequencing in tobacco pollen tube extracts. Immunocytochemistry data showed a localization of the antigen on the surface of tip-focussed post-Golgi secretory vesicles (SVs), that appear to be partially associated with microtubules (Mts). The chemical dissection of pollen tube high speed supernatant (HSS) showed that two distinct pools of MetE are present in pollen tubes, one being the more acidic isoforms sedimenting at 15S and the remaining at 4S after zonal centrifugation through a sucrose density gradient. The identification of the MetE within the pollen tube and its possible participation as methyl donor in a wide range of metabolic reactions, makes it a good subject for studies on pollen tube growth regulation.