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El olivo es una planta de gran importancia agronómica en los países mediterráneos, y muy especialmente en nuestra región. Nuestro grupo viene tradicionalmente estudiando numerosos aspectos de la reproducción sexual de dicha especie, tales como el desarrollo del polen, sus características (viabilidad y capacidad de germinación), el desarrollo del gineceo, los procesos de fertilización, la formación y maduración del fruto, etc. Para estos estudios utilizamos distintos métodos que incluyen desde la observación morfológica mediante técnicas de microscopía óptica, microscopía electrónica de transmisión, barrido y microscopía confocal, hasta técnicas de aislamiento y caracterización de proteínas y genes implicados en dichos procesos. Todos estos estudios nos están ayudando a comprender el sistema de reproducción sexual del olivo, una planta en la que la presencia de mecanismos de autoincompatibilidad polen-pistilo con bases aparentemente genéticas y ambientales, está empezando a ser estudiada. Otra línea de investigación en curso en nuestro grupo es aquella que se refiere al estudio de determinadas proteínas del grano de polen con funciones clave en su desarrollo y en la germinación y crecimiento del tubo polínico, y que además exhiben carácter alergénico para algunos individuos. Las características moleculares y el contenido de estos alérgenos varían significativamente entre cultivares de olivo, lo cual hace especialmente interesante su estudio en la fisiología del grano de polen, además de las implicaciones clínicas que de ello se derivan.

Ole e 1 is a well-characterized allergenic protein from olive pollen. This paper examines its presence and that of its transcripts during in vitro pollen germination and pollen tube growth. A significant increase of the protein was detected after the emergence of the pollen tube, whereas part of the protein was released into the culture medium throughout pollen germination. A slight increase in the number of Ole e 1 transcripts was also detected prior to the described rise in the protein level. Within the pollen tube, the allergen was localized in the subapical region, mainly in the lumen of endoplasmic reticulum cisternae. Ole e 1 was also localized extracellularly in the vicinity of the pollen tube cell wall. These findings are discussed regarding the biological role attributed to the protein during pollen hydration and pollen tube growth and in terms of their importance for the understanding of the allergenic response in humans. On the basis of recent findings for the LAT52 protein in tomato, we propose that the homologous Ole e 1 protein might participate in a similar signal transduction pathway in olive, to control pregermination and pollen tube emergence and guidance.

This paper describes the distribution of Ole e 3 allergen and its transcripts in the developing anther and in mature olive pollen. Northern blot and RT-PCR analyses over the course of pollen development show that Ole e 3 transcripts accumulate exclusively in mature pollen. This gene therefore corresponds to a "late gene." The sequences amplified by RT-PCR display high identity with those already reported for Ole e 3, including two Ca2+-binding motifs. Immunoblot analysis of the allergen shows that Ole e 3 accumulates during the same stage as the corresponding transcripts, suggesting a transcriptional regulation mechanism for the expression of the gene. Finally, the use of transmission electron microscopy techniques has shown that (a) the allergen is located mainly in the vicinity of membrane systems and in the aperture regions of the mature pollen grain, and (b) Ole e 3 transcripts are detectable after using in situ RT-PCR. These data are significant clues to the biological roles of the protein in olive pollen biology. Such putative functions are discussed.

[Background]: Commercial olive pollen from uncertain cultivar origin is the common material used for clinical and biological studies. We aimed to assess the putative heterogeneity of olive cultivars with regard to the presence of the major pollen allergen Ole e 1 and to determine whether these differences have clinical relevance. [Methods]: The Ole e 1 content of several cultivars was determined by immunoblotting and ultrastructural immunocytochemistry and compared to that of a commercially available olive pollen extract designed for diagnosis. Reverse transcription-polymerase chain reaction analysis of Ole e 1 transcripts was also performed. Crude protein extracts were used to carry out skin prick tests (SPTs) on 30 allergic patients in order to evaluate the clinical importance of such differences. [Results]: Ole e 1 was present in all cultivars, although significant quantitative differences were detected. Ole e 1 transcripts positively correlated with the amount of the allergen. Significant variations in the average reactivity of allergic patients to SPTs were observed depending on the cultivar considered. [Conclusions]: The presence of the Ole e 1 allergen in all the cultivars suggests that this allergen may play an essential biological role. The expression of the allergen is controlled at the transcriptional level. The significant differences in the Ole e 1 content are likely responsible for the different average reactivity exhibited by patients to the cultivars studied, although the role of other allergens cannot be excluded. Our results suggest that the use of the commercial pollen mixtures currently available may lead to mistakes in allergy diagnosis and to limited success in immunotherapy. Therefore, further standardization is strongly recommended.

The presence of the protein Ole e 1 (the olive pollen major allergen) and its transcripts during in-vitro pollen germination have been analyzed. The results obtained showed that: a) a large amount of the protein (with three variants) was found in both the mature and the germinating pollen grain, b) the protein was also secreted to the aqueous germination medium, c) Ole e 1 gene was highly expressed in both the mature and the germinating pollen grain, and d) the allergen was immunolocalized in association with dilated rough endoplasmic reticulum cisternae. These findings clearly support the idea of Ole e 1 playing a role of paramount importance in pollen germination and pollen tube growth.

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.

A study of free ubiquitin and ubiquitinated proteins has been carried out in olive (Olea europaea L.) during the late stages of pollen development and during in-vitro pollen hydration. As described in relatively few plant species, immunoblot experiments carried out using crude protein extracts have shown a reduction in the free ubiquitin level taking place during the mature pollen stage. This reduction does not coincide with a parallel reduction in the level of the corresponding transcripts, as detected by RT-PCR analysis, suggesting the existence of a post-transcriptional regulatory mechanism. Relatively high levels of free ubiquitin are quickly restored upon in-vitro pollen hydration. TEM immunocytochemical localisation of ubiquitin-conjugated compounds in the mature pollen grain showed the antibodies bound to the cytoplasm, the chromatin and the nucleolus of the vegetative and generative cells. Within the cytoplasm, labelling was concentrated in the cytosol, the endoplasmic reticulum membranes and minuscule vesicles associated to the Golgi apparatus. The fibrillar material adhering to the exine, and probably derived from the tapetum, also showed gold particles. Lipid bodies, aperture regions, mitochondria, the intine and the cell wall were devoid of labelling. The changes observed throughout pollen development, as well as the specific localisation, reflect the involvement of the ubiquitin-mediated degradative pathway in regulation of pollen metabolism.

The distribution of Ole e I (the major olive pollen allergen) and its transcripts was investigated in the anther from premeiotic stages until the dehiscent pollen stage. Crude protein extracts were analyzed by immunoblotting and probed with a monoclonal antibody to Ole e I. The protein, with three variants, was found to accumulate from the early microspore stage onwards. In addition to the previously reported localization of the protein, Ole e I has been immunolocalized for the first time within the pollen wall and in the tapetum. Reverse transcription-polymerase chain reaction analysis using specific oligonucleotides and RNA extracted from whole anthers revealed that the Ole e I gene is expressed from the late tetrad stage onwards. No expression was found in control tissues such as petals, roots or leaves. Light microscopy in situ hybridization on developing flower buds and dehiscent pollen confirmed the transcripts to be present in both the microspores and the sporophytic tissue (tapetum). Labeling was found primarily in the tapetum, reaching the highest concentration in the cytoplasm of the developing and mature pollen, once tapetum started to degenerate. In situ hybridization at the transmission electron microscope level showed the transcripts to accumulate on ribosomes of the rough endoplasmic reticulum. These studies, together with others carried out previously by us, indicated that both synthesis and storage of Ole e I take place in the endoplasmic reticulum, coincidentally with the conspicuous changes suffered by this membrane system during pollen development. This process is most likely controlled at the transcriptional level. The localization of the protein in the pollen ectexine bring new insights into the function of the allergen, which are discussed.