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), polyamine 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 and the content of free, soluble-conjugated and insoluble-bound polyamines have been investigated. 1H NMR-profiling coupled with multivariate data treatment (PCA and PLS-DA) allowed to compare, for the first time, the metabolic patterns of non-pollinated and pollinated styles. Results clearly indicate that during the SI response TGase activity increases, resulting in the accumulation of polyamines conjugated to hydroxycinnamic acids and other small molecules. Metabolomic analysis showed a remarkable differences between pollinated and non-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 all the other metabolites, including aromatic compounds, such as flavonoids and a cynnamoil derivative.
Abstract Transglutaminases (TGases) are ubiquitous enzymes that take part in a variety of cellular functions. In the pollen tube, cytoplasmic TGases are likely to be involved in the incorporation of primary amines at selected peptide-bound glutamine residues of cytosolic proteins (including actin and tubulin), while cell wall-associated TGases are believed to regulate pollen tube growth. Using immunological probes, we identified TGases associated with different subcellular compartments (cytosol, membranes, and cell walls). Binding of cytosolic TGase to actin filaments was shown to be Ca2+ dependent. The membrane TGase is likely associated with both Golgi-derived structures and the plasma membrane, suggesting a Golgi-based exocytotic delivery of TGase. Association of TGase with the plasma membrane was also confirmed by immunogold transmission electron microscopy. Immunolocalization of TGase indicated that the enzyme was present in the growing region of pollen tubes and that the enzyme colocalizes with cell wall markers. Bidimensional electrophoresis indicated that different TGase isoforms were present in distinct subcellular compartments, suggesting either different roles or different regulatory mechanisms of enzyme activity. The application of specific inhibitors showed that the distribution of TGase in different subcellular compartments was regulated by both membrane dynamics and cytoskeleton integrity, suggesting that delivery of TGase to the cell wall requires the transport of membranes along cytoskeleton filaments. Taken together, these data indicate that a cytoplasmic TGase interacts with the cytoskeleton, while a different TGase isoform, probably delivered via a membrane/cytoskeleton-based transport system, is secreted in the cell wall of pear (Pyrus communis) pollen tubes, where it might play a role in the regulation of apical growth.
Nanoplastic debris, resulted from runoff and weathering breakdown of macro- and microplastics, represents an emerging concern for marine ecosystems. The aim of the present study was to investigate disposition and toxicity of polystyrene nanoparticles (NPs) in early development of sea urchin embryos (Paracentrotus lividus). NPs with two different surface charges where chosen, carboxylated (PS-COOH) and amine (PS-NH2) polystyrene, the latter being a less common variant, known to induce cell death in several in vitro cell systems. NPs stability in natural seawater (NSW) was measured while disposition and embryotoxicity were monitored within 48 h of postfertilization (hpf). Modulation of genes involved in cellular stress response (cas8, 14-3-3ε, p-38 MAPK, Abcb1, Abcc5) was investigated. PS-COOH forms microaggregates (PDI > 0.4) in NSW, whereas PS-NH2 results are better dispersed (89 ± 2 nm) initially, though they also aggregated partially with time. Their respectively anionic and cationic nature was confirmed by ζ-potential measurements. No embryotoxicity was observed for PS-COOH up to 50 μg mL(-1) whereas PS-NH2 caused severe developmental defects (EC50 3.85 μg mL(-1) 24 hpf and EC50 2.61 μg mL(-1) 48 hpf). PS-COOH accumulated inside embryo's digestive tract while PS-NH2 were more dispersed. Abcb1 gene resulted up-regulated at 48 hpf by PS-COOH whereas PS-NH2 induced cas8 gene at 24 hpf, suggesting an apoptotic pathway. In line with the results obtained with the same PS NPs in several human cell lines, also in sea urchin embryos, differences in surface charges and aggregation in seawater strongly affect their embryotoxicity.
An extracellular form of the calcium-dependent protein-cross-linking enzyme TGase (transglutaminase) was demonstrated to be involved in the apical growth of Malus domestica pollen tube. Apple pollen TGase and its substrates were co-localized within aggregates on the pollen tube surface, as determined by indirect immunofluorescence staining and the in situ cross-linking of fluorescently labelled substrates. TGase-specific inhibitors and an anti-TGase monoclonal antibody blocked pollen tube growth, whereas incorporation of a recombinant fluorescent mammalian TGase substrate (histidine-tagged green fluorescent protein: His6-Xpr-GFP) into the growing tube wall enhanced tube length and germination, consistent with a role of TGase as a modulator of cell wall building and strengthening. The secreted pollen TGase catalysed the cross-linking of both PAs (polyamines) into proteins (released by the pollen tube) and His6-Xpr-GFP into endogenous or exogenously added substrates. A similar distribution of TGase activity was observed in planta on pollen tubes germinating inside the style, consistent with a possible additional role for TGase in the interaction between the pollen tube and the style during fertilization.
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 microM) 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.
The author examines the way in which different countries have attempted to address the new and ever-more pressing need for privacy protection that arises from the computerized processing of personal data. She aims to identify specific elements of comparison that may then be used in the evaluation of the different regulatory techniques that have been developed. An initial distinction must be made between civil law systems and common law systems, and also between those legal systems that are subject to the requirements of European law, and those that are not. This is needed in order to be able to assess the impact of European law upon the different legal traditions of the countries involved. Following an examination of the different regulatory techniques established in these different countries, the author concludes that, in those Member States of the European Union that have civil law systems, it seems that there may now be a tendency towards regulatory models that are closer to the common law tradition.
