Immunocytochemical localization of allergenic protein (Ole e I) in the endoplasmic reticulum of the developing pollen grain of olive (Olea europaea L.)

The monoclonal antibody OL-1 and transmission electron microscopy were used to locate immunologically the major allergen of olive pollen. Ole e I, during pollen grain development. Within the pollen grain, allergenic proteins are located in the cisternae of the rough endoplasmic reticulum. Our findings indicate that the synthesis of these proteins starts in the vegetative cytoplasm of olive pollen during the early maturation stage.Dedicated to Professor Andreas Sievers on the occasion of his retirement     

Profiling and functional classification of esterases in olive (Olea europaea) pollen during germination

Background and Aims

A pollen grain contains a number of esterases, many of which are released upon contact with the stigma surface. However, the identity and function of most of these esterases remain unknown. In this work, esterases from olive pollen during its germination were identifided and functionally characterized.

Methods

The esterolytic capacity of olive (Olea europaea) pollen was examined using in vitro and in-gel enzymatic assays with different enzyme substrates. The functional analysis of pollen esterases was achieved by inhibition assays by using specific inhibitors. The cellular localization of esterase activities was performed using histochemical methods.

Key Results

Olive pollen showed high levels of non-specific esterase activity, which remained steady after hydration and germination. Up to 20 esterolytic bands were identified on polyacrylamide gels. All the inhibitors decreased pollen germinability, but only diisopropyl fluorophosphate (DIFP) hampered pollen tube growth. Non-specific esterase activity is localized on the surface of oil bodies (OBs) and small vesicles, in the pollen intine and in the callose layer of the pollen tube wall. Acetylcholinesterase (AChE) activity was mostly observed in the apertures, exine and pollen coat, and attached to the pollen tube wall surface and to small cytoplasmic vesicles.

Conclusions

In this work, for the first time a systematic functional characterization of esterase enzymes in pollen from a plant species with wet stigma has been carried out. Olive pollen esterases belong to four different functional groups: carboxylesterases, acetylesterases, AChEs and lipases. The cellular localization of esterase activity indicates that the intine is a putative storage site for esterolytic enzymes in olive pollen. Based on inhibition assays and cellular localization of enzymatic activities, it can be concluded that these enzymes are likely to be involved in pollen germination, and pollen tube growth and penetration of the stigma.     

Ultrastructural changes in maturing pollen grains ofApium nodiflorum L. (Apiaceae), with special reference to the endoplasmic reticulum

Summary The ultrastructural events in 3-cellular pollen grains ofApium nodiflorum L. are investigated during pollen maturation. Three distinct developmental stages are distinguished from the formation of sperm cells up to anthesis, whereby the rough endoplasmic reticulum (RER) is mainly involved. The most conspicious form is the highly dilated RER in the vegetative cytoplasm of the youngest pollen grains, which changes to vesicular RER in the following stage. In mature pollen grains the RER has a narrow cisternal configuration and often forms stacks. Pollen activation is preceded by the accumulation of polysaccharide particles.     

Vegetative growth response of young olive trees (Olea europaea L., cv. Arbequina) to soil salinity and waterlogging

High-density olive orchards are increasing around the world, many of which may be potentially affected by salinity and waterlogging (hypoxia), two important stresses common in irrigated fields in arid and semi-arid climates. However, the response of olive to these stresses under field conditions is not well established. Therefore, our objective was to evaluate the vegetative growth response of young olive trees (Olea europaea L., cv. Arbequina) grown in a spatially variable waterlogged, saline-sodic field. We monitored the growth in trunk diameter of 341, 3-year-old olives between September 1999 and September 2000. Field contour maps were developed delineating soil salinity (ECa), relative ground elevation (RGE) and water table depth (WTD). Soil samples were also collected and analyzed for ECe and SARe in order to characterize the salinity and sodicity profiles and develop the ECa-ECe calibration equation. The infiltration rate (IR) of the crusted and uncrusted soil and the penetration resistance (PR) were also measured. The field was characterized by spatially variable ECe (2–15 dS m^–1), SARe (3–40), RGE (–4 to +4 cm) and WTD (0.5–1.9 m, with corresponding ground water EC values between 12 and 6 dS m^–1). Steady-state IR of crusted soil was only 7% of the uncrusted soil. Since the field was heavily irrigated by flooding, waterlogging conditions were related to low RGE values. Soil salinity was negatively correlated (R ² = 0.83, P<0.001) with RGE (ponded water) and WTD (upward flux), due to the evapo-concentration of water and salts at the soil surface. Thus, inverted salinity profiles developed in high salinity areas. Fifty-five percent of the olives were dead 3.5 years after planted, and most of them were located in areas of high ECe (> 10 dS m^–1), low RGE (< – 1.5 cm) and low WTD (< 1.2 m). The surviving trees had vegetative salinity tolerance values of ECe threshold = 4 dS m^–1 and slope = –12% (i.e., percent decline per unit increase in ECe above the treshold), indicating that the Arbequina olive is moderately tolerant to salinity. The RGE and WTD thresholds for olive's survival were > 0.1 cm and > 1.6 m, respectively. Thus, very small changes in ground elevation had a significant effect on olive's survival or death. The coupled effects of salinity and waterlogging (hypoxia) stresses were most detrimental for olive's growth.     

Ultrastructural and cytochemical examination of the nuclear crystalloid inclusions of Olea europaea L. mesophyll cells

Summary The nuclei of mesophyll cells of olive trees contain numerous sizeable crystalloid inclusions. Cytochemical examination using epoxy resin-embedded, semithin-sectioned tissue indicated the presence of proteins and oligoor polysaccharides in these inclusions. Their electron microscopical analysis revealed a crystalline substructure consisting of intersected subunits of high order. The spacing of the lattice fibrils and the angles of intersection were determined and used to establish a model of the unit cell of crystallization. It is suggested that the nuclear crystalloids of olive trees consist of glycoprotein molecules. They differ from the intranuclear crystalloids observed in other species predominantly in the high density of their subunit arrangement.     

Ultrastructural changes in the endoplasmic reticulum during dormancy release of apple embryos (Pyrus malus L.)

Apple embryos were treated by cold (0°C) within the fruits, to break their dormancy; the controls were treated at 12°C or at 20°C. Ultrastructural features of meristematic cells in the embryonic axis were compared for each treatment. The organization of the cells of dormant embryos was described: Endoplasmic reticulum consisted in some short rough cisternae; lipid droplets regularly arranged near the plasmalemma constituted a kind of shell; mitochondria had a few cristae; and dictyosomes were rarely observed. All these features are typical of dry seeds. After cold treatments, the only evolution observed was in the endoplasmic reticulum, where highly organized stacks appeared progressively as a function of time at 0°C. An intermediate temperature (12°C) induced similar formations in the reticulum but they were rarely observed and their degree of organization was lower than that obtained at 0°C. At 20°C, endoplasmic reticulum resembled that of the dormant embryo cells. The relation between the appearance of these structures in the reticulum and the disappearance of dormancy induced by cold is discussed.Abbreviations ER endoplasmic reticulum - RER rough endoplasmic reticulum     

Morphological and cytological development and starch accumulation in hermaphrodite and staminate flowers of olive (Olea europaea L.)

In olive (Olea europaea L.), the formation of functionally staminate flowers rather than fully functional hermaphrodites is one of the major factors limiting fruit set, as flowers with aborted pistils are incapable of producing fruit. Studies conducted on various angiosperm species have shown a correlation between flower abortion and starch content. Thus, it is important to know if starch content plays a role in regulating pistil development in olive and if so, what mechanism regulates starch distribution. Cyto-histological observations of staminate and hermaphrodite olive flowers show that pistil development in staminate flowers is interrupted after the differentiation of the megaspore mother cell. At that stage, starch grains were only detected in the ovary, style and stigma of the hermaphrodite flowers. No starch was observed in the pistils of the staminate flowers. This finding suggests a tight correlation between starch content and pistil development. The secondary origin of starch within the flower is indicated by low chlorophyll content in the gynoecium, undetectable Rubisco activity in the pistils of these two kinds of flowers and by the ultrastructure of the plastids observed by transmission electron microscope analysis. The plastids have few thylakoid membranes and grana and in the staminate flowers appeared very similar to proplastids. Considering differences in starch content between staminate and hermaphrodite flowers and the secondary origin of the starch, differences in pistil development in the staminate and hermaphrodite flowers could be related to differences in the sink strength of these two types of flowers.     

Ultrastructural aspects of the cytoplasmic origin and accumulation of oil in olive fruit (Olea europaea)

The development of oil bodies and oil droplets in fruits of olive was examined at the ultrastructural level. Both oil bodies that form in young fruits and oil droplets that develop with fruit maturation are cytoplasmic bodies. The formation of the small oil bodies occurs in localized regions of the cytoplasm. These bodies are closely associated and fuse together, forming a small oil droplet that protruded against and indented the vacuolar membrane. As the fruit matures, new oil bodies appear to form and fuse with the oil droplet, resulting in the formation of a single large oil droplet of about 30 μm in diameter in most mature mesocarp cells. The cytoplasmic region where the oil bodies formed had a granulate, ultrastructural appearance, and cytoplasmic components such as membranes and ribosomes were noticeably absent in these regions. The granulate material coated the oil bodies and oil droplets, and appeared as a thin, compressed band between the round inner surface of the droplets and the indented tonoplast. We suggest that this granulate material is involved in the synthesis of the oil and, with enlargement of the oil bodies, this coat becomes thinner in regions where they are closely associated, resulting in zones where confluence of the oil occurs.     

Aerobiological and meteorological factors’ influence on olive (Olea europaea L.) crop yield in Castilla-La Mancha (Central Spain)

Knowledge of the main biological and climate factors influencing final harvest is becoming increasingly necessary in order to obtain reliable crop estimates and, thus, ensure optimised, effective private crop management. This knowledge is also of great value to public agricultural institutions for the planning of government subsidies. Castilla-La Mancha (Central Spain) is the second largest olive-oil-producing region in Spain, the highest olive-oil-producing country in the world. This study sought to identify the main factors influencing olive fruit production in this region, including atmospheric pollen as an index of flowering intensity, and meteorological data over the flowering and fruiting seasons in two main olive-producing provinces of the region: Ciudad Real and Toledo. Statistical analysis indicated that the annual pollen index (PI) was the variable influencing most the final olive crop in both provinces. The maximum temperature in March was the meteorological variable affecting most the annual olive crop. Also, the rainfall registered in October influences the final fruit production. The integration of aerobiological and meteorological data represents an important step forward in the development of future crop forecasting models in the region of Castilla-La Mancha.     

Clinical significance of allergic sensitization toOlea europaea L. pollen in Naples area,Italy

Summary The aim of this study is the evaluation of clinical significance of the allergic sensitization toOlea europaea L. pollen allergens in the Naples area, because in the last 10 years an increasing frequency of IgE mediated sensitization to these allergens has been observed. 4124 patients of both sexes (2989 adults and 1153 children) consecutively examined in our Allergological Center from January 1, 1988 to December 30, 1989 have been considered in this study. The patients were selected on the basis of a history of rhinitis (R) and/or asthma (A) and were evaluated by clinical examination, SPT, RAST and specific or aspecific BPT. Among 2267 adults and 879 children sensitized to almost one aeroallergen we found respectively 306 (13.49%) and 75 (8.53%) subjects with an allergic sensitization toOlea europaea L. pollen. The frequency of clinical manifestation was respectively R+A (58.16%), R (39.54%), A (2.28%) in adults; R+A (61.33%), R (26.66%), A (12%) in children. A monosensitization toOlea europaea L. pollen was found in 1.33% of children and in 2.28% of adults examined. The association with other allergens especially with mites and/or other pollen was very common.     

Heat requirement for the onset of the Olea europaea L. pollen season in several sites in Andalusia and the effect of the expected future climate change

Olives are one of the largest crops in the Mediterranean region, especially in Andalusia, in southern Spain. A thermal model has been developed for forecasting the start of the olive tree pollen season at five localities in Andalusia: Cordoba, Priego, Jaen, Granada and Malaga using airborne pollen and meteorological data from 1982 to 2001. Threshold temperatures varied between 5°C and 12.5°C depending on bio-geographical characteristics. The external validity of the results was tested using the data for the year 2002 as an independent variable and it confirmed the models accuracy with only a few days difference from predicted values. All the localities had increasingly earlier start dates during the study period. This could confirm that olive flower phenology can be considered as a sensitive indicator of the effects of climate fluctuations in the Mediterranean area. The theoretical impact of the predicted climatic warming on the olives flowering phenology at the end of the century is also proposed by applying Regional Climate Model data. A general advance, from 1 to 3 weeks could be expected, although this advance will be more pronounced in mid-altitude inland areas.     

Behavior of storage lipids during development and germination of olive ( Olea europaea L.) pollen

Summary.  The presence of abundant oil bodies in the mature olive pollen grain has led us to focus on the behavior of these lipid bodies during pollen development and in vitro pollen germination. The appearance, increase, and accumulation of lipid bodies have been determined by following the sequential development of the pollen grain. Semithin slices of anthers and pollen grains were stained with Sudan Black B in order to identify neutral lipids. Ultrastructural studies were also carried out. Our results show a notable increase in lipid bodies between the young-pollen-grain stage and the mature-pollen-grain stage. Substantial polarization of lipid bodies was observed after 1 or 2 h of pollen incubation in germination medium. During pollen tube growth, the lipid bodies are located near the germinative aperture after 3 h of incubation, as well as inside the pollen tube, thus suggesting that the lipid bodies move from the pollen grain to the pollen tube. After 7 h of germination the presence of lipid bodies inside the pollen tube is no longer substantial. Our results support the idea that lipid bodies are involved in pollen germination, stigma penetration, and pollen tube growth. These results are discussed in connection with their implications for the pollen germination process. Received June 4, 2002; accepted October 29, 2002; published online April 8, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Profesor Albareda 1, 18008 Granada, Spain.     

In vitro propagation of olive (Olea europaea L.) cv. Koroneiki

Single node explants of 'Koroneiki' olive trees werecultured for one month on a modified Driver-Kuniyuki for Walnut medium, lackinggrowth regulators. The explants were subcultured once a month on a mediumsupplemented with zeatin riboside, 6-(--dimethylallylamino)purine,6-benzyladenine or thidiazuron. Zeatin riboside proved to be superior to othercytokinins in inducing shoot proliferation. The combination of olive knotextract at 25 or 50 mg l^–1 with cytokininssuppressed shoot proliferation. After two months at the proliferation stage,theexplants were cultured for one week in the dark in 1 ml liquidWoody Plant Medium supplemented with IBA, -NAA or IBA+-NAA. Theexplants were then transferred to the same solid medium lacking growthregulators, with a small layer of perlite on the surface. The combination ofthetwo auxins at 1+1 mg l^–1 resulted in almost 76%rooting. The combination of olive knot extract at 50 mgl^–1 with auxins increased the rooting percentage up toalmost 87%. Artificial infection of explants with the bacteriumPseudomonas savastanoi pv. savastanoiinhibited rhizogenesis, even in the presence of auxins. Rooted explants weresuccessfully acclimatised under a mist system, with the survival rate reachingalmost 75%.     

Concanavalin A binds to the endoplasmic reticulum and the starch grain surface of root statocytes

Using Concanavalin A (Con A) labeled with fluorescein isothiocyanate, we studied the intracellular localization of receptor molecules in the calyptra of 24-h dark-grown cress roots. Fixation in glutaraldehyde gave positive binding of the distal complex of the endoplasmic reticulum and the nucelus in the statocytes. In contrast, fixation in formaldehyde did not preserve the membrane-associated receptors, but revealed Con A affinity of the starch grain surface within the amyloplasts. Treatment of glutaraldehydefixed sections with non-ionic detergents led to partial solubilization of membrane components: the starch grain surface turned positive, though the positive binding of Con A to the endoplasmic reticulum and the nucleus remained unaffected. We therefore conclude that the Con A receptor in the membrane is a glycoprotein tightly inserted in other components of the compartment.Abbreviations Con A Concanavalin A - ER endoplasmic reticulum - FITC fluorescein isothiocyanate - NP 40 nonidet P40     

Electrophoretic profiling and immunocytochemical detection of pectins and arabinogalactan proteins in olive pollen during germination and pollen tube growth

Background and Aims

Cell wall pectins and arabinogalactan proteins (AGPs) are important for pollen tube growth. The aim of this work was to study the temporal and spatial dynamics of these compounds in olive pollen during germination.

Methods

Immunoblot profiling analyses combined with confocal and transmission electron microscopy immunocytochemical detection techniques were carried out using four anti-pectin (JIM7, JIM5, LM5 and LM6) and two anti-AGP (JIM13 and JIM14) monoclonal antibodies.

Key Results

Pectin and AGP levels increased during olive pollen in vitro germination. (1 → 4)-β-d-Galactans localized in the cytoplasm of the vegetative cell, the pollen wall and the apertural intine. After the pollen tube emerged, galactans localized in the pollen tube wall, particularly at the tip, and formed a collar-like structure around the germinative aperture. (1 → 5)-α-l-Arabinans were mainly present in the pollen tube cell wall, forming characteristic ring-shaped deposits at regular intervals in the sub-apical zone. As expected, the pollen tube wall was rich in highly esterified pectic compounds at the apex, while the cell wall mainly contained de-esterified pectins in the shank. The wall of the generative cell was specifically labelled with arabinans, highly methyl-esterified homogalacturonans and JIM13 epitopes. In addition, the extracellular material that coated the outer exine layer was rich in arabinans, de-esterified pectins and JIM13 epitopes.

Conclusions

Pectins and AGPs are newly synthesized in the pollen tube during pollen germination. The synthesis and secretion of these compounds are temporally and spatially regulated. Galactans might provide mechanical stability to the pollen tube, reinforcing those regions that are particularly sensitive to tension stress (the pollen tube–pollen grain joint site) and mechanical damage (the tip). Arabinans and AGPs might be important in recognition and adhesion phenomena of the pollen tube and the stylar transmitting cells, as well as the egg and sperm cells.     

Specialized formations of endoplasmic reticulum in parenchyma cells ofMimosa pudica L.

Summary Parenchyma cells ofMimosa pudica display close associations between two or more cisternae of the endoplasmic reticulum. These associations form simplified types of lamellar bodies in which inner paired lamellae have lost their ribonucleoprotein granules and are separated by a dense layer.     

Abnormalities induced by agricultural pesticides in the microsporogenesis of olive tree (Olea europaea L.) cultivars

The study evaluated the microsporogenesis of olive trees subjected to different agricultural pesticide applications during flowering. Inflorescences of cultivars Arbequina and MGS GRAP541 were subjected to agricultural pesticides: mineral oil, neem oil, dimethoate and deltamethrin. The floral buds were fixed in Carnoy for the microsporogenesis analysis and in Karnovsky for scanning electron microscopy. The slides were prepared by squash technique and staining with propionic carmine. The pollen viability was determined by Alexander’s stain and in vitro germination. Results show that the quantification of abnormalities in meiosis in the two cultivars caused significant effect among the treatments, being that all differed statistically from the control group. Both methods showed a higher percentage of viable pollens in the control treatment and lower percentage of viability with the agricultural pesticides. The method of pollen viability by staining presented the highest averages of viable pollens, but when compared together, both methods presented a strongly related positive linear correlation. It was concluded that the used chemical products increased the percentage of chromosomal abnormalities during microsporogenesis, which interfered in the pollen viability of the two analyzed cultivars. The product deltamethrin caused the strongest effect on meiosis and on pollen viability.