Morphological anomalies in pollen tubes of Actinidia deliciosa (kiwi) exposed to 50 Hz magnetic field

The role of the pollen grain, with respect to the reproductive process of higher plants, is to deliver the spermatic cells to the embryo sac for egg fertilisation. Delivery occurs through the pollen tube, a self produced organ that is generated when the pollen grain reaches the stigma surface. The effect of magnetic fields on pollen tube growth was reported in a recent publication by Germanà et al. Pollen tube growth is an interesting candidate for the detailed study of the effects of electromagnetic fields on cytoplasmic structures and organelles. In this research Actinidia deliciosa (kiwifruit) pollen grains were germinated in the presence of an alternating magnetic field (50 Hz). Our results, although of preliminary nature, show that pollen tube growth is affected by magnetic fields. The analysis of the observed anomalies in the pollen tube appear to be the result of changes in the ionic charges within the pollen tube cytoplasm.     

Identification and characterization of plasma membrane proteins that bind to microtubules in pollen tubes and generative cells of tobacco

The organization and function of microtubules in plant cells are important in many developmental stages. Connections between microtubules and the endomembrane system of plant cells have been discovered by microscopy, but the molecular characteristics of these relationships are mostly unknown except for a few cases. Using two antibodies raised against microtubule-associated proteins (MAPs) from maize, we have identified two polypeptides that share properties of the MAP family in the pollen tube of Nicotiana tabacum. The two polypeptides (with an apparent Mr of 161 and 90 kDa) bind efficiently to animal and plant microtubules and are found in association with the cellular membranes of the pollen tube, from which they can be solubilized with a zwitterionic detergent. One of these proteins has been purified and shown to promote the assembly of tubulin and, to a lesser extent, the bundling of microtubules. Subcellular fractionation indicated that the two proteins are associated with the plasma membrane compartment. The two proteins are found to co-localize in situ with cortical microtubules in the vegetative cytoplasm of tobacco pollen tubes; co-localization is also evident in the generative cell. According to these data, both the 161 and 90 kDa polypeptides are likely to mediate the interactions between the plasma membrane and microtubules in pollen tubes. In addition, functional data indicate that these MAP-like proteins take part in the process of microtubule assembly and reorganization occurring during cell growth. The evidence that both proteins associate with different cellular compartments also suggests a broad-spectrum role in mediating the dynamic relationships between microtubules and plant cell membranes.     

Involvement of the ubiquitin/proteasome pathway in the organisation and polarised growth of kiwifruit pollen tubes

We recently reported the involvement of the ubiquitin pathway in microgametophyte development, and a direct role for the 26S proteasome in regulating pollen tube emergence in kiwifruit. Here we show that the ubiquitin/proteasome proteolytic pathway is involved not only in early kiwifruit pollen tube organisation, but also in maintaining polarised growth of tubes. By immunofluorescence analysis we show that ubiquitin and ubiquitin-protein conjugates are distributed mainly at the apex of emerging tubes, in both untreated pollen grains and pollen grains treated with MG132, an inhibitor of proteasome function. In the latter case, polysiphonous germination occurred and all the emerging areas were highly fluorescent. By adding MG132 to pollen when normal tube growth had already been established, accumulation of ubiquitin-protein conjugates, as well as a drastic reduction in tube growth and dramatic modifications of tube tip morphology were observed. Significantly, differential interference contrast microscopy analysis demonstrated that the clear zone was largely reduced or absent, and the nuclei were disconnected in their movements, reaching, in some cases, the extreme apex of the tip. These findings provide evidence that the ubiquitin- and proteasome-dependent proteolytic system could modulate the abundance and/or activity of key regulatory proteins involved in pollen tube emergence and polarised growth.     

Screening of Bifidobacterium strains isolated from human faeces for antagonistic activities against potentially bacterial pathogens

As probiotic bacteria, strains belonging to the genus Bifidobacterium colonise the gastro-intestinal tract of humans and animals at the time of birth, and they are found in young as well as in adult individuals in great numbers. Moreover, they can interact with the development of enteric infections by the production of antimicrobial metabolites. In this work 281 strains of bifidobacteria were anaerobically isolated from human faecal samples, supplied by volunteers of different ages (youngs, adults, elders), and preliminarly described by microscopic observation. All strains were screened by the fructose 6-phosphate phosphoketolase (F6PPK) test in order to confirm their classification within the genus Bifidobacterium. Selected strains were used to evaluate their antagonistic activities against Escherichia coli, Salmonella thyphimurium, Staphylococcus lentus, Enterococcus faecalis, Acinetobacter calcoaceticus, Sphingomonas paucimobilis, Listeria monocytogenes, Yersinia enterocolitica, Bacillus cereus, Clostridium sporogenes. Experiments were performed in vitro by different methods based on the observation of growth inhibition in Petri dishes. The strains that showed the highest inhibiting activities were compared by SDS-PAGE for total cell proteins, using type strains of human origin as references. Representative isolates were metabolically characterised by the BIOLOG system; a specific database was created with strains obtained from our collection and a statistical evaluation for metabolic patterns was carried out.     

In vitro pollen tube growth reveals the cytotoxic potential of the flavonols, quercetin and rutin

Flavonols are phytochemicals widely found in commonly consumed foods. In spite of their beneficial effects on human health, however, cytotoxicity and even suspected genotoxicity have also been reported for the flavonol, quercetin. This points to the need for preventive studies to identify any cytotoxic effects associated with pure flavonol intake. This work was performed with the aim of verifying whether a plant-based in vitro system, the pollen tube, could be used to evaluate the cytotoxic potential of exogenous flavonols. Increasing concentrations of the aglycone, quercetin, and its glycoside, rutin, were assayed with regard to tube growth of kiwifruit pollen, determined by applying the pollen tube growth test protocol. This test, based on the photometric quantification of pollen tube mass production in suspension cultures, has already been applied in the sensitive and reliable toxicological evaluation of a wide range of chemicals. Whereas 60-800 microM rutin promoted kiwifruit pollen tube elongation, 10-50 microM quercetin strongly inhibited growth, and also produced irreversible malformations, such as screw-like tube growth, abnormal vacuolation, alteration of organelle streaming, and nuclear positioning. Thus, the cytotoxic potentials of the two flavonols have been confirmed to differ. Pollen tubes seem to afford a promising test system for a preventive, rapid in vitro biosafety assessment of antioxidant nutritional supplements, without using laboratory animals.     

Identification and characterization of a novel microtubule-based motor associated with membranous organelles in tobacco pollen tubes

Pollen tube growth depends on the differential distribution of organelles and vesicles along the tube. The role of microtubules in organelle movement is uncertain, mainly because information at the molecular level is limited. In an effort to understand the molecular basis of microtubule-based movement, we isolated from tobacco pollen tubes polypeptides that cosediment with microtubules in an ATP-dependent manner. Major polypeptides released from microtubules by ATP (ATP-MAPs) had molecular masses of 90, 80, and 41 kD. Several findings indicate that the 90-kD ATP-MAP is a kinesin-related motor: binding of the polypeptide to microtubules was enhanced by the nonhydrolyzable ATP analog AMP-PNP; the 90-kD polypeptide reacted specifically with a peptide antibody directed against a highly conserved region in the motor domain of the kinesin superfamily; purified 90-kD ATP-MAP induced microtubules to glide in motility assays in vitro; and the 90-kD ATP-MAP cofractionated with microtubule-activated ATPase activity. Immunolocalization studies indicated that the 90-kD ATP-MAP binds to organelles associated with microtubules in the cortical region of the pollen tube. These findings suggest that the 90-kD ATP-MAP is a kinesin-related microtubule motor that moves organelles in the cortex of growing pollen tubes.     

In vitro toxicity towards kiwifruit pollen of the antimicrobial peptides magainins 1 and 2

In vitro toxicity of the antimicrobial peptides (AMPs) magainin 1 and 2 to a higher plant organism, i.e., the bicellular male gametophyte of Actinidia Deliciosa (kiwifruit), is investigated. Heavy damage to the plasma membrane, the primary cellular target of the peptides, was rapidly induced: in as few as 15 min, from 70 to nearly 100 % of pollen grains were rendered unviable by 20 microM magainin 1 or 2, respectively. Therefore, kiwifruit pollen sensitivity to natural magainins seemed to be higher if compared to the sensitivity of other pollen species towards magainin 2 amide or synthetic magainin analogues. Strong dose-dependent inhibitory effects on kiwifruit pollen performance were registered: as for magainin 1, the EC (50) at 120 min varied from 14.0 (germination) to 15.8 microM (tube elongation). The inhibitory effect was much greater when administering magainin 1 to elongating tubes rather than to ungerminated pollen grains. The two peptides differentially affected kiwifruit pollen, in line with the previously documented greater activity of magainin 2 in other cell systems. Furthermore, 20 microM magainin 1-treated pollen grains took on a shrivelled shape within 30 min of incubation, an increasingly widespread effect with higher peptide concentration. At the ultrastructural level, both protoplast shrinkage and striking organelle alterations were evident, including chromatin condensation, swelling and loss of mitochondrial cristae, dilation of rough endoplasmic reticulum cisternae, and vacuolization of cytoplasm. To our knowledge, similar alterations in animal or plant cells treated with AMPs have not been described yet.     

The cell wall of kiwifruit pollen tubes is a target for chromium toxicity: alterations to morphology, callose pattern and arabinogalactan protein distribution

Trivalent chromium has previously been found to effectively inhibit kiwifruit pollen tube emergence and elongation in vitro . In the present study, a photometric measure of increases in tube wall production during germination showed that 25 and 50 μ m CrCl₃ treatment induced a substantial reduction in levels of polysaccharides in walls over those in controls. Moreover, chromium-treated kiwifruit pollen tubes had irregular and indented cell walls. Callose, the major tube wall polysaccharide, was deposited in an anomalous punctuate pattern. Arabinogalactan proteins (AGPs), which are integral in maintaining correct tube growth and shape in kiwifruit pollen, were found to be strongly altered in their distribution after CrCl₃ treatment compared to control tube walls. Transmission electron microscopy–immunogold analysis using four monoclonal antibodies (JIM8, JIM13, JIM14 and MAC207) revealed discontinuous AGP distribution within the treated tube walls. Such clearly discernable alterations in the molecular and morphological architecture of pollen tube walls may be detrimental in vivo for the male gametophyte to accomplish its vital role in the fertilisation process.