Air pollution relevance analysis in the bay of Algeciras (Spain).

The aim of this work is to accomplish an in-depth analysis of the air pollution in the two main cities of the Bay of Algeciras (Spain). A large database of air pollutant concentrations and weather measurements were collected using a monitoring network installed throughout the region from the period of 2010-2015. The concentration parameters contain nitrogen dioxide (NO2), sulphur dioxide (SO2) and particulate matter (PM10). The analysis was developed in two monitoring stations (Algeciras and La Línea). The higher average concentration values were obtained in Algeciras for NO2 (28.850 µg/m3) and SO2 (11.966 µg/m3), and in La Línea for PM10 (30.745 µg/m3). The analysis shows patterns that coincide with human activity. One of the goals of this work is to develop a useful virtual sensor capable of achieving a more robust monitoring network, which can be used, for instance, in the case of missing data. By means of trends analysis, groups of equivalent stations were determined, implying that the values of one station could be substituted for those in the equivalent station in case of failure (e.g., SO2 weekly trends in Algeciras and Los Barrios show equivalence). On the other hand, a calculation of relative risks was developed showing that relative humidity, wind speed and wind direction produce an increase in the risk of higher pollutant concentrations. Besides, obtained results showed that wind speed and wind direction are the most important variables in the distribution of particles. The results obtained may allow administrations or citizens to support decisions.

Morphological, histological and ultrastructural changes in the olive pistil during flowering.

Sexual reproduction is essential for the propagation of higher plants. From an agronomical point of view, this is a particularly key process because fertilization guarantees fruit formation in most cultivated fruit species. In the olive, however, in spite of its agricultural importance, little attention has been paid to the study of sexual reproduction. In order to investigate the cellular and molecular mechanisms that regulate pollen-pistil interactions in the olive during the progamic phase, it is essential to first have a good knowledge of the reproductive structures involved in such interactions. This study characterizes the anatomical and ultrastructural changes in the olive pistil, beginning from the young pistil developing within the bud until the time of petal loss and visible stigma senescence. We have correlated changes in the pistil with a series of defined floral developmental stages and determined that olive pistil structures cannot be considered completely mature and ready to be pollinated and fertilized until the onset of anthesis. Our results clearly show histological and ultrastructural variation during the diverse flowering events. We discuss whether the changes observed might influence or result from pollen-pistil interactions during the progamic phase.

Olive pollen profilin (Ole e 2 allergen) co-localizes with highly active areas of the actin cytoskeleton and is released to the culture medium during in vitro pollen germination.

Pollen allergens offer a dual perspective of study: some of them are considered key proteins for pollen physiology, but they are also able to trigger allergy symptoms in susceptible humans after coming in contact with their tissues. Profilin (Ole e 2 allergen) has been characterized, to some extent, as one of the major allergens from Olea europaea L. pollen, a highly allergenic species in the Mediterranean countries. In order to obtain clues regarding the biological role of this protein, we have analyzed both its cellular localization and the organization of actin throughout pollen hydration and early pollen tube germination. The localization of the cited proteins was visualized by confocal laser scanning microscopy immunofluorescence using different antibodies. Upon pollen hydration and pollen germination, a massive presence of profilin was detected close to the site of pollen tube emergence, forming a ring-like structure around the 'effective' apertural region. Profilin was also detected in the pollen exine of the germinating pollen grains and in the germination medium. After using a permeabilization-enhanced protocol for immunolocalization, profilin was also localized in the cytoplasm of the pollen tube, particularly at both the proximal and apical ends. Noticeable accumulations of actin were observed in the cytoplasm of the pollen tube; particularly, in both the apical region and the area immediately close to the aperture. Actin filaments were not observed, probably due to the need of further enhanced fixation procedures. The ultrastructural localization of profilin showed the presence of the protein in the cytoplasm of both the mature pollen grain and the pollen tube. The results shown here could be interpreted as signs of a massive dissociation of the actin-profilin complexes, mobilization of actin monomers, and therefore, an intense activity of the actin cytoskeleton. The extensive release of allergenic proteins from the pollen grain into the surrounding aqueous media, as described here for profilin, may help us to understand the mechanisms by which these allergens might come in contact with the human mucosa, therefore triggering the symptoms of allergy.

Pla 1 1 and Ole e 1 pollen allergens share common epitopes and similar ultrastructural localization.

BACKGROUND: English plantain (Plantago lanceolata L.) and olive (Olea europaea L.) pollens are important causes of pollinosis in large areas of North America, Australia, and the Mediterranean basin. The major pollen allergens of both plants, Pla I 1 and Ole e 1, share 38.7% of their amino acid sequences. OBJECTIVE: To analyze putative cross-reactivity between these 2 proteins. METHODS: Several antibodies and patients' sera were used in immunoblot and immunocytochemistry experiments. RESULTS: Two anti-Pla I 1 antibodies were able to bind to 3 polypeptides from olive pollen protein extracts, which correspond to the 3 glycosylation isoforms of Ole e 1 (18-22 kDa) previously described. Moreover, Pla I 1 protein was found in the cytoplasm of both the vegetative and the generative cells of P lanceolata mature pollen. On olive pollen sections, these anti-Pla I 1 antibodies displayed significant labeling in the cytoplasm of the vegetative cell and in both the exine and the material adhering to this outer layer of the pollen wall. In addition, the anti-Ole e 1 antibody 10H1 was found to cross-react with proteins of similar masses (16-20 kDa) to Pla I 1 variants. In Plantago pollen sections, the 10H1 antibody recognized proteins located in the cytoplasm of both the vegetative and generative cells. Cross-reaction was confirmed using sera from patients allergic to either plant pollen. CONCLUSION: Both allergens share common epitopes, which can be cross-recognized by different antibodies and sera from different patients, although this antigenic similarity seems to have little clinical relevance.

Differential characteristics of olive pollen from different cultivars: biological and clinical implications.

The olive tree is grown in many parts of the world. Its germplasm is very broad, with 250 varieties in Spain alone. Variations in the ability of pollen to germinate have been studied in detail and show conspicuous differences between varieties. However, commercial olive pollen from cultivars whose origin is unknown is the material that is commonly used for clinical and biological studies. We aim to assess the putative heterogeneity of olive cultivars with regard to the presence of several pollen allergens and to determine whether these differences have biological and clinical relevance. Previous studies show that most allergens isolated and characterized to date are highly polymorphic. Olive cultivars display wide differences in the expression levels of many allergens and in the number and molecular characteristics of the allergen isoforms expressed. These differences are maintained over the years, and are intrinsic to the genetics of each cultivar. Such broad polymorphism seems to be involved in the physiology of the olive reproductive system, which might include the adaptation of the plant to different environmental conditions, the establishment of the compatibility system, and pollen performance. The differences in allergen composition in cultivars, particularly in the Ole e 1 allergen, are responsible for the important differences in the allergenic potency of the extracts. These findings could have a number of implications for the diagnosis and therapy of olive pollen allergy. We discuss how cultivar differences affect extract quality, diagnostic and therapeutic efficacy and safety, and the development of new vaccines based on the use of recombinant allergens.

Nucleolar evolution and coiled bodies during meiotic prophase in Olea europaea: differential localization of nucleic acids.

We studied the ultrastructural evolution of the nucleolus during meiotic prophase in olive microsporocytes. During prophase, nuclear bodies morphologically similar to coiled bodies were observed. The nucleic acid composition of these bodies was examined in microsporocytes using electron microscopic techniques with EDTA preferential ribonucleoprotein staining, anti-DNA immunolabeling, the in situ terminal deoxynucleotidyl transferase-immunogold technique, and in situ hybridization with 18S rRNA and U3 snoRNA digoxigenin-labeled probes. The ultrastructural appearance of the meiocyte nucleolus indicated a low level of activity from the early prophase stage: the granular component was practically absent and nucleoli were constituted almost exclusively by dense fibrillar component containing large fibrillar centers that lacked chromatin inclusions. However, the appearance of reactivation vacuoles in the nucleolus during zygotene and high levels of rRNA in the nucleoplasm during pachytene support the presence of a peak in rRNA synthesis. Our results also show that the nuclear bodies that appear during prophase I are ribonucleoproteinaceous in nature; neither DNA nor ribosomal RNA were detected. The presence of U3 snoRNA, as shown by in situ hybridization in nuclear bodies from plant material, is also evidence that these structures are coiled bodies. We suggest that coiled bodies are involved not only in pre- and post-splicing events but also in the storage, transport or recycling of rRNA maturation elements.

Immunogold probes for light and electron microscopic localization of Ole e I in several Oleaceae pollens.

We investigated the immunolocalization of the olive major allergen Ole e I and Ole e I-like proteins in pollen from several Oleaceae species [olive (Olea europaea), ash (Fraxinus excelsior), privet (Ligustrum vulgaris), lilac (Syringa vulgare), and forsythia (Forsythia suspensa)]. Crossreactions among different pollens were found in enzyme immunoassays. For immunolocalization with light microscopy we used the silver enhancement technique with three monoclonal antibodies (1D8, 10H1, and 16G2) that recognize three different epitopes of the allergen Ole e I. Our findings show that the silver enhancement technique is very useful when several antibodies are to be used for rapid screening of different materials. MAb 10H1 gave the most precise results and was selected for further immunolocalization studies with transmission electron microscopy. The epitope recognized by this MAb was localized exclusively in the endoplasmic reticulum in olive pollen. In lilac, privet, and ash pollen, most of the reactivity was also seen in the endoplasmic reticulum; however, the 10H1 epitope was not detected in forsythia pollen.

Pollen wall development in Olea europaea L.

The ontogeny of the olive pollen grain (Olea europaea L.) wall is related to the structural changes which take place during the various stages of pollen grain development. The tetrad stage sees the deposition of the primexine, with probacules forming by deposition of material adjacent to the plasmalemma. Endexine development commences subsequently. The bacules and foot-layer are formed mainly from dense material found within the special callose wall. By the young free microspore stage the definitive structure of the exine has been determined. Throughout development microchannel-like structures are present in both bacules and the foot-layer. An electron-lucent zone appears between the plasmalemma and the endexine. This is the future site of intine deposition which occurs during the vacuolated microspore stage before postmeiotic mitosis. A fibrillar layer lines the arcade spaces of the exine. Between this stratum and the exine lies a thin layer of polysaccharides probably representing the remains of the primexine or glycocalyx. In the mature pollen grain the exine arcades contain numerous lipid drops (or pollenkitt), originating from the degenerating tapetum.

Developmental changes in the aperture during pollen grain ontogeny in Olea europaea L.

Changes in both size and form of the structures making up the apertural region, a modified part of the pollen grain wall, were studied during development in Olea europaea L. The very young microspores initially show thickening of the endexine around the apertural zone. This is followed by the appearance of an exinous oncus and subsequently an intinous oncus. The intinous oncus contains tubules derived from evaginations of the plasmalemma. These tubules increase in number and diameter during development.

Localization of transcripts corresponding to the major allergen from olive pollen (Ole e I) by electron microscopic non-radioactive in situ RT-PCR.

In situ reverse transcription-PCR of mRNAs corresponding to the olive major allergen (Ole e I) has been tested at the ultrastructural level in mature olive pollen. The transcripts were present in the cytoplasm of both the vegetative and the generative cells, frequently associated to ribosomes in the endoplasmic reticulum. No labeling was detected in the pollen wall, nor in vacuoles, lipid bodies, plastids or mitochondria. Localization of the major olive allergen at ultrastructural level showed the protein present mainly in the lumen of the endoplasmic reticulum vesicles or pockets scattered in the cytoplasm, and in the outer region of the pollen exine. The results confirm the rough endoplasmic reticulum as the cell system involved in both the synthesis and storage of this protein. This is the first report of in situ RT-PCR on plant material at the ultrastructural level. The method described for mRNA amplification and detection is confirmed as a valuable tool for studying gene expression in plant material.

[Proof of the subapical differential growth of the flanks in the Chara rhizoid during graviresponse]. / Nachweis des subapikalen differentiellen Flankenwachstums im Chara-Rhizoid wahrend der Graviresponse.

The displacement of resin spheres attached to the tip of the gravitropically bending Chara rhizoid was measured by means of time-lapse photography. The relative growth of the flanks which at the beginning of stimulation were opposite to each other, was calculated during the earliest response time. The physically upper subapical flank section continues growing after stimulation at a decreasing rate. The growth of the opposite lower flank section is inhibited immediately after stimulation by the position of the statoliths. As soon as the statoliths are displaced in a basal direction, the growth of this section is transiently promoted. The gravitropic downward bending of the Chara rhizoid is by bowing, not by bulging.

Fluorochromes for detection of callose in meiocytes of olive (Olea europaea L.).

The callosic wall which covers microsporocyte mother cells during meiotic division has been studied using different fluorochromes as alternatives to the widely used aniline blue. We have confirmed that both acridine orange and 4', 6' diamidino-2-phenylindole (DAPI) produce a fluorescent response to callose which is comparable in specificity and intensity to that of aniline blue; therefore, they can be used to study callose wall formation. Staining properties of these fluorochromes, as well of those of curcumin and sirofluor, reported earlier as fluorescent stains for callose, are discussed. We also discuss the efficacy of the combined use of sirofluor and DAPI to study particular aspects of the deposition of callose.

Distribution of pectins in the pollen apertures of Oenothera hookeri.velans ster/+ster.

Cell wall pectins are some of the most complex biopolymers known, and yet their functions remain largely mysterious. The aim of this paper was to deepen the study of the spatial pattern of pectin distribution in the aperture of Oenothera hookeri.velans ster/+ster fertile pollen. We used "in situ" immunocytochemical techniques at electron microscopy, involving monoclonal antibodies JIM5 and JIM7 directed against pectin epitopes in fertile pollen grains of Oenothera hookeri.velans ster/+ster. The same region was also analyzed by classical cytochemistry for polysaccharide detection. Immunogold labelling at the JIM7 epitope showed only in mature pollen labelling mainly located at the intine endo-aperture region. Cytoplasmic structures near the plasma membrane of the vegetative cell showed no labelling gold grains. In the same pollen stge the labelling at the JIM5 epitope was mostly confined to a layer located in the limit between the endexine and the ektexine at the level of the border of the oncus. Some tubuli at the base of the ektexine showed also an accumulation of gold particles. No JIM5 label was demonstrated in the aperture chamber and either in any cytoplasmic structure of the pollen grains. The immunocytochemical technique, when compared with the traditional methods for non-cellulose polysaccharide cytochemistry is fare more sensitive and allows the univocal determination of temporal and spatial location of pectins recognized by the JIM7 and JIM5 MAbs.

Transcriptional activity and distribution of splicing machinery elements during Hyacinthus orientalis pollen tube growth.

The localization of newly formed transcripts and molecules participating in pre-mRNA splicing, i.e., small nuclear ribonucleoproteins (snRNPs) and SC35 protein, in growing pollen tubes of Hyacinthus orientalis L. were analyzed in vitro and in vivo. The results indicated that the restart of RNA synthesis occurred first in the vegetative and then in the generative nucleus of both in vitro and in vivo growing pollen tubes. Changes in RNA synthesis were accompanied by the redistribution of splicing machinery elements in both vegetative and generative nuclei of the growing pollen tube. At stages of pollen tube growth when the vegetative and generative nuclei were transcriptionally active, clear differences in the distribution pattern of the splicing system components were observed in both pollen nuclei. While both small nuclear RNA with a trimethylguanosine cap on the 5' end and SC35 protein were diffusely distributed in the nucleoplasm in the vegetative nucleus, the studied antigens were only present in the areas between condensed chromatin in the generative nucleus. When the transcriptional activity of both pollen nuclei could no longer be observed at later stages of pollen tube growth, snRNPs and SC35 protein were still present in the vegetative nuclei but not in the generative nuclei. We, therefore, investigated potential differences in the spatial organization of splicing system elements during pollen tube growth. They clearly reflect differences in gene expression patterns in the vegetative and the generative cells, which may be determined by the different biological roles of angiosperm male gametophyte cells.

Immunocytochemical localization of esterified and unesterified pectins in unpollinated and pollinated styles of Petunia hybrida Hort.

Localization of pectins in the style of Petunia hybrida before and after pollination was investigated by immunocytochemistry using two primary monoclonal antibodies specific to highly (JIM7) and weakly (JIM5) methylesterified pectins. In the unpollinated style, esterified pectins occurred mainly in the cell walls of cortex tissue, while unesterified pectins were present mainly in the extracellular matrix (ECM) of the transmitting tract. After pollination no remarkable differences were found in pectin distribution in the ground tissue of the style. On the other hand, in the transmitting tract a reduction in the quantity of unesterified pectins was observed. Unesterified pectins in the extracellular regions of the transmitting tissue decreased before the penetration of the pollen tubes, indicating that pollination induces a reduction in the amount of unesterified pectins in the transmitting-tract ECM. The correlation between the degradation of strongly Ca2+-binding pectins and the growing level of those ions in the extracellular regions of the transmitting tract in the pollinated pistil of P. hybrida (M. Lenartowska et al. 1997) suggests that this process may constitute a mechanism for creating an optimum calcium medium for in vivo-growing pollen tubes. Both pectin categories were localized in pollen tubes. Esterified pectin epitopes were localized mainly in the vesicles of the tip cytoplasm. Unesterified pectin epitopes were found in the external fibrillar wall of pollen tubes.

rRNA distribution during microspore development in anthers of Beta vulgaris L. quantitative in situ hybridization analysis.

We related changes in the ultrastructural organization of the nucleoli with the results of quantitative in situ hybridizations to characterize rRNA metabolism during the development of microspore mother cells in the sugar beet anther (Beta vulgaris L.). In the course of meiotic prophase and early postmeiotic interphase the morphological characteristics of the nucleoli are typical of low or no transcriptional activity and a low rate of rRNA processing. However, we found evidence of an apparent increase in the relative numbers of 18 S rRNA transcripts in some stages of microsporogenesis. This was found in both the nucleoli and cytoplasm of pachytene meiocytes, and in later stages there was a spectacular accumulation of rRNA transcripts in nucleoli of the tetrad cells. Quantitative data are analyzed in the light of morphometric findings in the cell and their compartments to elucidate the degree to which changes in cell size are related to changes in labeling density and distribution. The results are discussed in terms of rRNA synthesis, transport and degradation as processes involved in the regulation of rRNA within microsporocytes and microspores.

Peroxisome proliferation and oxidative stress mediated by activated oxygen species in plant peroxisomes.

The existence of a relationship between clofibrate-induced peroxisome proliferation and oxidative stress mediated by activated oxygen species was studied in intact peroxisomes purified from Pisum sativum L. plants. Incubation of leaves with 1 mM clofibrate produced a remarkable increase in the peroxisomal activity of acyl-CoA oxidase and, to a lesser extent, of xanthine oxidase, whereas there was a nearly complete loss of catalase activity and a decrease in Mn-superoxide dismutase. Ultrastructural studies of intact leaves showed that clofibrate induced a five- and twofold proliferation of the peroxisomal and mitochondrial populations, respectively, in comparison with those in control leaves. Prolonged incubation with clofibrate produced considerable alterations in the ultrastructure of cells. In peroxisomal membranes, the NADH-induced generation of O2- radicals, as well as the lipid peroxidation of membranes, increased as a result of treatment of plants with clofibrate. In intact peroxisomes treated with this hypolipidemic drug, the H2O2 concentration was higher than in peroxisomes from control plants. These results demonstrate that clofibrate stimulates the production of activated oxygen species (O2- and H2O2) inside peroxisomes, as well as the lipid peroxidation of peroxisomal membranes. This effect is concomitant with a decrease of catalase and Mn-SOD activities, the main peroxisomal enzymatic defenses against H2O2 and O2-, and indicates that in the toxicity of clofibrate, at the level of peroxisomes, an oxidative stress mechanism mediated by activated oxygen species is involved.

Cytochemical features common to nucleoli and cytoplasmic nucleoloids of Olea europaea meiocytes: detection of rRNA by in situ hybridization.

We used light and electron microscopic techniques to study the composition of cytoplasmic nucleoloids during meiotic division in Olea europaea. Nucleoloids were found in two clearly distinguishable morphological varieties: one similar in morphology to the nucleolus, and composed mainly of dense fibrillar component, and another surrounded by many ribosome-like particles. Cytochemical and immunocytochemical techniques showed similar reactivities in nucleoloids and the nucleolus: both are ribonucleoproteic in nature, and possess argyrophillic, argentaffinic and highly phosphorylated proteins. Immunohistochemical techniques failed to detect DNA in either structure. In situ hybridization to a 18 S rRNA probe demonstrated the presence of ribosomal transcripts in both the nucleolus and nucleoloids. These similarities in morphology and composition may reflect similar functionalities.

Localization of the acid phosphatasic activity in plant cell nucleoli.

We have made an ultrastructural study of the nuclear acid phosphatase localization in the different tissues of Allium cepa L. anthers, after meiosis. The enzyme is preferentially located in the fibrillar component, within the nucleolus, with a greater reaction density around the nucleolar organizing region (NOR). The specificity of the reaction was proved by contrasting the results with those obtained in two different series of anthers. One of these series was incubated in Gomori's medium with an acid phosphatasic inhibitor, and the other in Gomori's medium without substrate. In both cases the acid phosphatase reaction was negative. Some authors (Hardin and Spicer, 1970; Tandler et al., 1970; Rodríguez-García and Stockert, 1979) have observed an accumulation of inorganic cations (Ca++, Mg++, ... and Na+) within the nucleolar region where the phosphatase acid activity was detected by us. The coincidence of this localization is discussed, suggesting that these cations are probably involved in the phosphatasic activity of these nucleolar zones. Our results agree with the idea that this enzyme contributes to an increase in the amount of nuclear ortophosphate ions (Harris, 1963). On the other hand this enzyme could play a part in the successive triming of the different preribosomal RNA species that occurs during their maturation (Dalgarno and Shine, 1977).