Tapetum plastids ofOlea europaea L.

Summary The plastid ontogenesis inOlea europaea tapetum has been studied. Tapetum plastids start their development as proplastids and differentiate into elaioplasts. At the end of their development the tapetal cells degenerate and are substituted by roundish lipidic masses which will later form an exine coating (Pollenkitt). During their ontogenesis the plastids are characteristically associated with membrane outlines of mostly smooth ER, which appear to be correlated with lipid accumulation inside the plastids.     

Ultrastructural evidence of endoplasmic reticulum changes during the maturation of the olive pollen grain (Olea europaea L., Oleaceae)

Characteristic features of rough endoplasmic reticulum (rER) distribution and proliferation were noted during olive pollen (Olea europaea L.) development, suggesting the physiological significance of this organelle. Initially scarce in the young microspore, ER increases as cytoplasmic vacuoles form. At the vacuolated microspore stage the cytoplasm contains numberous polysomes and elongated rER cisternae arranged preferentially in stacks, with an average intracisternal width of 0.07 µm. Stacks persist in the bicellular pollen grain but consist of fewer, shorter, dilated cisternae (mean intracisternal width 0.1 µm) containing a considerable electron-dense matrix. Cisternae in the mature grain are fragmented, leaving behind an ER of swollen pockets. Pockets of ER containing a material of greater electron density are evenly deposited along the plasmalemma, in close relation with it. A dense material is seen in the tubules of the apertural region, which was lacking in earlier stages. Our results show that ER may be involved in protein transport to the intine.     

A new approach to consider the pollen variable in forecasting yield models

Methods for forecasting harvest yields have been improved considerably in the last 20 years with the development of new data survey (remote sensing) and statistical techniques. One of these methods, based on pollen release in the atmosphere, is especially important for anemophilous species such as olive. The aim of the present work is to use a different approach to forecast the olive harvest by considering the pollen variable as “endogenous” because it is involved in the consequential processes from the formation of pollen to fruiting, the complex of which determines, more or less, the final production. Unlike models built upon a single equation (multiple linear regression analysis), the proposed estimate, based on an incomplete system of equations, recovers the consistency associated with the inference of parameters while avoiding the errors of “over-estimation.” The study, based on 17 years of data considers the quantity of olive pollen monitored and the relative annual olive production in addition to climatic, agronomic, and pathological variables associated with production. The harvest forecast provides the possibility for planning and optimizing the various stages of olive production from cultivation to distribution, including sound management of the olive supply.

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Une nouvelle approche pour considérer la variable pollen dans les modèles de prévision des rendement de récolte

Résumé  Pendant les vingt dernières années les méthodes de prévision des rendements de récolte ont été considérablement améliorées grace au développement de nouvelles techniques statistiques et d’ enquête des données (télédétection). Parmi ces méthodes celle basée sur l’émission du pollen dans l’atmosphère se révèle particulièrement importante pour les espèces anémophiles comme l’olivier. Le but de ce travail est celui d’arriver à employer une méthode différente dans la prévision des rendements de récolte de l’olivier, le tout en considérant comme “endogène” la variable pollen. Ce dernier est, en effect, impliqué dans les processus d’évolution qui vont de sa formation à la fructification, de manière à déterminer la production finale. Contrairement aux modèles établis sur une équation simple (analyse multiple de régression linéaire), l’évaluation proposée, basée sur un système inachevé d’équations, récupère la consistance connexe à l’ inférence des paramètres tout en évitant les erreurs de “surestimation”. L’étude, basée sur dix-sept ans de données, considère la quantité de pollen d’olivier détectée et conséquemment la production oléicole annuelle, outre aux variables climatiques, agronomiques et pathologiques liées à la production. Les modèles de prévision offrent la possibilité de rationaliser les différentes phases de la filière oléicole en optimisant les procédés, de la production à la distribution, y compris la gestion rationnelle des stocks.

     

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.     

Monitoring of airborne pollen and pollen calendar of Cosenza,Southern Italy

Summary From May 1986 to May 1989 surveys of airborne biological particles have been performed in the atmosphere of Cosenza, Italy with the aim of monitoring the presence of airborne pollen. The survey station is situated at Arcavacata of Rende, a hilly area 474 meters above sea level, 7 km north-west of the main town. The sampler (VPPS 2000) is located about 20 meters above ground level. The monitoring, performed in accordance with the criteria suggested by the Italian Association of Aerobiology (A.I.A.), enabled the identification of 26 different pollen types. The pollination graphics show: 1) a late winter period in which pollen from trees appears; 2) a spring period with a prevalence of Gramineae, Fagaceae, Oleaceae andParietaria pollen; 3) a late spring-summer period during which Fagaceae,Pinus and Compositae pollen grains are present; 4) a late summer-autumn and winter period characterized by a drastic reduction of airborne pollen. A study of the calendar reveals, furthermore, that: 1) Gramineae pollen is, amongst those of allergological importance, the most representative; 2)Parietaria is not perennial and is present in modest concentrations; 3)Olea reaches a high peak in June, but not as high as in other southern regions; 3) pollen from arboreal plants is prevalent compared to that of herbaceous plants.     

Cupressaceae pollen in the atmosphere of Ascoli Piceno (Central Italy) and sensitization of allergic subjects

The aim of this work is to study the incidence of pollinosis in the Health District of Ascoli Piceno, Central Italy (U.S.L.24). this being an underestimated pathology from the clinical point of view and also as a result of the recent introduction of this taxa in the National Aeroallergen Network. Since 1990, 5055 patients of both sexes with respiratory symptomatology of suspected IgE mediated aetiology have been examined in our Centre with the Skin Prick Test (SPT) using allergen panels including Cypress; 171 (3.38%) patients were found to be positive to this allergen. These results show that the subjects with symptoms in the period January–March in most cases have a sensitization toCupressus pollen and new studies will evaluate the possibility of specific immunotherapy.     

Cupressaceae pollen in the atmosphere of Ascoli Piceno (Central Italy) and sensitization of allergic subjects

The aim of this work is to study the incidence of pollinosis in the Health District of Ascoli Piceno, Central Italy (U.S.L.24), this being an underestimated pathology from the clinical point of view and also as a result of the recent introduction of this taxa in the National Aeroallergen Network. Since 1990, 5055 patients of both sexes with respiratory symptomatology of suspected IgE mediated aetiology have been examined in our Centre with the Skin Prick Test (SPT) using allergen panels including Cypress; 171 (3.38%) patients were found to be positive to this allergen. These results show that the subjects with symptoms in the period January–March in most cases have a sensitization toCupressus pollen and new studies will evaluate the possibility of specific immunotherapy.     

Identification of ancient Olea europaea L. and Cornus mas L. seeds by DNA barcoding

The analysis of ancient DNA (aDNA) provides archaeologists and anthropologists with innovative, scientific and accurate data to study and understand the past. In this work, ancient seeds, found in the "Mora Cavorso" archaeological site (Latium, Central Italy), were analyzed to increase information about Italian Neolithic populations (plant use, agriculture, diet, trades, customs and ecology). We performed morphological and genetic techniques to identify fossil botanical species. In particular, this study also suggests and emphasizes the use of DNA barcode method for ancient plant sample analysis. Scanning electron microscope (SEM) observations showed seed compact structure and irregular surface but they did not permit a precise nor empirical classification: so, a molecular approach was necessary. DNA was extracted from ancient seeds and then it was used, as template, for PCR amplifications of standardized barcode genes. Although aDNA could be highly degraded by the time, successful PCR products were obtained, sequenced and compared to nucleotide sequence databases. Positive outcomes (supported by morphological comparison with modern seeds, geographical distribution and historical data) indicated that seeds could be identified as belonging to two plant species: Olea europaea L. and Cornus mas L.     

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.     

Production of total and stainable pollen grains in Olea europaea L

The production of the total and stainable number of pollen grains per flower and per inflorescence of 13 olive cultivars (Olea europaea L.) has been investigated. Grains stainability was evaluated by using the acetic carmine staining method whereas the production of pollen grains was calculated by using a Bürker haemocytometer. All pollen characteristics taken into account varied significantly among the cultivars. The number of flowers per inflorescence ranged from a maximum of 31.3 (‘Cipressino’) to a minimum of 13.0 (‘Leccino’), the number of pollen grains per anther ranged from 123?346 (‘Arbequina’) to 40?975 (‘Sant'Agostino’), and the pollen stainability from 97.6% (‘Cipressino’) to 48.0% (‘Cellina di Nardò’). The number of stainable pollen grains per inflorescence averaged 2?559?512, ranging from a maximum of 3?913?678 (‘Nociara’) to a minimum of 940?143 (‘Sant'Agostino’). All parameters were positively correlated, whereas a linear negative correlation was found between stainability and total pollen grains both per anther and per inflorescence.     

Trend of herbaceous pollen diffusion and allergic sensitisation in Genoa, Italy

In order to investigate the relationship betweenallergenic load and allergic sensitisation prevalenceon a long period, we analysed the annual data ofherbaceous pollen airborne diffusion in Genoa(NW-Italy) for 17 years (1981–1997), with particularregard to the most important allergenic species:Poaceae, Parietaria, Artemisia, Ambrosia. Weevaluated the relative trend and compared it with theprevalence data of patients sensitisation to thesepollens in the same period.The analysis of annual pollen amount revealed asignificant rising trend (r = 0.73, p < 0.05) of Parietaria pollen (average 14.324 grains/m³/year),whereas Poaceae (2048/year) and Artemisia(170/year) pollen count did not show any significantchange. Ambrosia (average 70/year) has been rising since 1989 (r = 0.87, p < 0.05) and reached themaximum pollen count in the last three years.The prevalence of pollen sensitisation resulted to bevery high (about 75%) in our atopic patients withrespiratory symptoms and did not change significantlyin the years: Parietaria pollen allergy waspresent in about 70% of patients with at least oneSPT positive to pollens (and 55% of atopicsubjects), and this frequency, much higher than theone found in North Italy, showed low year-to-year variability.Among pollinotics patients, Poaceae cutaneoussensitisation was found at a quite stable rate in theabove mentioned period (about 51%), as well as thatto Artemisia pollen (8%). Ambrosia skinprick test positivity is very rare (about 0.5%),without any evident increase.The present study shows that, for some of theherbaceous species, we cannot demonstrate astraightforward relationship between the trend ofairborne pollen and the frequency of sensitisation inexposed people. However, in the case of Parietaria, the high level of pollen amount in theair is clearly related with the high rate of cutaneous positivity in patients, but it seems tosuggest a ``plateau' that is not modified by anyfurther increase in airborne pollen (perhaps forgenetic limiting factors). On the other hand, Ambrosia pollen count, even if increasing in Genoa'satmosphere, could still be too low, and it would takesome more time to cause allergic sensitisation in exposed people.This kind of study, pointing out the influence ofairborne allergen exposure on hypersensitivitymechanisms, should be extended in time and world-wideto obtain general conclusions.     

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.     

The effect of timing and amount of leaf removal on survival,growth and reproduction in the perennial herb Sanicula europaea L.

In Sweden the deciduous forest perennial Sanicula europaea L. (Apiaceae) is frequently subjected to leaf loss due to cattle grazing and trampling. In a two-season field experiment, the effects of the extent, timing and repetition of leaf removal on survival, growth and reproduction in S. europaea were examined. Removal of vegetative tissue in S. europaea reduced future survival, growth and flowering probabilities. The magnitude of these effects depended both on the extent and the timing of the season of the leaf losses, as greater leaf losses had more negative effects than moderate and early removals had more negative consequences than late. Moreover, the probability of an individual to regrow the same season after severe losses was higher when losses occurred early in the season than when they occurred late. And, those defoliated early that did regrow, did so to a larger extent than those defoliated later. Experimental responses were more pronounced after a second year of leaf removals, indicating that repeated herbivory exhausts resources. Thus, herbivory causing losses in vegetative tissue will affect the performance of S. europaea. However, the impact depends on the extent and timing of the leaf losses. This should be considered in managing policies and cattle should, if possible, not be introduced early in the growing season.     

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.     

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.     

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.     

Olea Europea 3-year pollen record in the area of Thessaloniki,Greece and its sensitizing significance

Summary The observations of airborne pollen ofOlea europea and the incidence of clinical manifestations in patients allergic to this pollen type have not been registered so far in the city of Thessaloniki. The purpose of this study was: 1. to assess theO. europea pollen circulation in the area of our city, and 2. to detect the percentage of sensitivity toO. europea pollen in patients with pollinosis. We collected daily pollen samples during a 3-year period (February '87-January '90), using a Burkard volumetric trap, located on a high level area in the centre of the city. The pollen counts were then registered. The O.europea pollen grains were not differentiated microscopically from the other Oleaceae, but identified through phenological criteria. The patients included in the assessment of the sensitivity toO. europea came from the out-patient clinic of bronchial asthma of the General Hospital ?G. Papanicolaou?. They had a seasonal pollinosis and they were submitted to prick test using a battery of 22 groups and an O.europea extract. Pollen ofO. europea appears first in the atmosphere of Thessaloniki at the beginning of May, shows a peak in the end of May and continues to be present till the end of June. The quantity ofO. europea pollen ranked 6th in the list of the total pollen count and its flowering period coincided with that of grasses. In a sample of 360 patients with seasonal pollinosis, we detected anO. europea pollen sensitivity combined with other alleargens in 37% of the patients and a monosensivity in 4%. We conclude that pollen ofO. europea results to be present over a relative short period of time (May–June) in the area of Thessaloniki. The percentage of patients' sensitization toO. europea pollen was a little less frequent than sensitization to grasses, even if their flowering time coincides and their presence in the air shows about the same concentration values.     

An automated system for surveying and forecasting Olea pollen dispersion

An automated system for Olea pollensurveying and forecasting is being developedfor the province of Cordoba, Spain, within theframework of the European ASTHMA project.Required input data have been split into threecategories: static data, calibration parametersand dynamic data. A 200 × 200 km potential pollenproduction map, centred on the city of Cordoba,was drawn up using digital high-resolutionsatellite data and reflecting average plantdensity, as derived from the observed spatialseparation of trees in 28 different locations.Based on archives of pollen data from 1982, thestart of the pollen season was determined byapplying both heat accumulation methods andneural network analysis including furthermeteorological parameters. The forecast andsurvey system is based on a set of twoelementary atmospheric physics modules andbiological models: emission and dispersionmodules. The system can run in different modes,allowing forecasting of the floweringstart-date and, from that date, charting theemission and dispersion of pollenconcentrations in the area. Also, wind andtemperature fields are regularly computed andupdated using deterministic numerical models tochart the spatial and temporal concentration ofOlea pollen.