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.     

Cloning, expression and characterization of a novel four EF-hand Ca(2+)-binding protein from olive pollen with allergenic activity

A novel allergenic member of the family of Ca(2+)-binding proteins has been cloned from olive tree pollen. The isolated DNA codes for a protein of 171 amino acid residues, which displays four EF-hand sequence motifs. The encoded protein was overproduced in Escherichia coli and purified. The protein (18? omitted?795 Da), which binds Ca(2+) and IgE antibodies from patients allergic to olive pollen, undergoes Ca(2+)-dependent conformational changes. It is retained on a phenyl-Sepharose column, which indicates the existence of regulatory EF-hand domains. This fact suggests its involvement in Ca(2+)-dependent signal transduction events of the pollen grain. This allergen could be considered as a member of a new subfamily of EF-hand Ca(2+)-binding proteins since it displays a low amino acid sequence similarity with the so far known proteins.     

Effect of agrometeorological parameters on the phenology of pollen emission and production of olive trees (Olea europea L.)

The pollination period and pollen concentrationof olive trees (Oleaeuropea L.) and olive production were analysedfor Prato and Florenceusing a data set of 8 years (1991–1998).Meteorological data have been usedto obtain information about weather conditionsduring vegetative seasonsand correlations were found both for thephenology and yield.The results showed that air temperatureprevious to the onset of floweringis of great importance in determining thereproductive cycle of olive treebut the chill period in January and Februaryshould also be considered.Olive pollen collected during thepollination period was positivelycorrelated with the production level for bothsites. Weather conditionfollowing pollination were also taken intoaccount for a better assessmentof the final yield.     

The interrelationship between the accumulation of lipids,protein and the level of acyl carrier protein during the development of Brassica napus L. pollen

Lipid accumulation during pollen and tapetal development was studied using cryostat sections of unfixed anthers from Brassica napus (rapeseed). Diamidino-2-henylindole (DAPI), a DNA fluorochrome, was used to stain the pollen nuclei in order to identify ten stages of pollen development in Brassica. Storage lipids (i.e. triacylglycerides) were stained using the fluorochrome Nile red. Pollen coat lipids are formed in tapetal plastids between the mid-vacuolate and early maturation pollen stages. The pollen coat components, including lipids and a proportion of the proteins, are derived from the remnants of the tapetum, after its rupture, during the second pollen mitosis. Quantitative microfluorometric analyses demonstrated four phases of lipid body accumulation or depletion in the developing pollen cytoplasm. The majority of storage lipids found in the cytoplasm of the mature pollen grain accumulated during the late vacuolate and early maturation stages when the pollen is bicellular. The level of acyl carrier protein, a protein integrally involved in lipid synthesis, was also found to be maximal in the developing pollen during the bicellular pollen stages of development. This coincided with the most active period of lipid accumulation. These data could indicate that the lipids of the pollen are synthesized in situ, by metabolic processes regulated by expression of genes in the haploid genome.To whom correspondence should be addressed     

Phenological and aeropalynological survey in an olive orchard in Umbria (Central Italy)

The olive tree Olea europaea L. is a resource of notable value from an economic and landscape point of view in Central Italy. This study examined two cultivars (Giarraffa and Ascolana) grown in an associative stand in an experimental olive orchard, to verify the possibility of introducing new olive cultivars into Umbria. The analyses were carried out at three different altitudes in the olive orchard following criteria used for non-homogeneous samplings in order to minimize the effect due to steepness. Since the number of trees of the two cultivars is different, the sample sizes were different for the two cultivars. Phenological observations of the above mentioned cultivars and the evaluation of the pollen flow, done by using a portable pollen trap inside the olive orchard, were carried out over two seasons (1998-1999). The start of initial phenological phases differed in the two cultivars, but the differences became less evident during phases of anthesis. In aeropalynological respect, it appears that, in a hill species such as the olive tree, the flowering is correlated with altitude. Comparing the aeropalynological data from the distant pollen trap and those collected in the olive orchard, it is clear that the periods of pollen release are very similar in the two cultivars, and there is a clear synchronism in the peak pollen release. It is concluded that in Central Italy the pollen grains of Giarraffa cultivar are well compatible with the Ascolana cultivar from a phenological and aeropalynological point of view.     

Airborne olive pollen in Vigo (Northwest Spain): a survey to forecast the onset and daily concentrations of the pollen season

Temperature is one of the main factors affecting the start of flowering in tree species that flower at the beginning of spring. Knowledge of the chilling and heat needs required by plants to overcome the period of dormancy enables us to determine the onset of pollination, which is of great importance to allergy sufferers. This study attempted to obtain behaviour models with a view to determining both the onset of the olive pollen season and daily pollen concentrations during the pollination period in Vigo. Monitoring was carried out using a Lanzoni VPPS 2000 pollen trap, from 1995 to 2002 inclusive.

Olea pollen is mainly detected during the spring, principally in May. Given the geographical location, the very limited presence of this tree in the study area and the low Olea pollen concentrations detected in northern Spain as a whole, the values recorded here in the atmosphere of Vigo are particularly striking. A strong correlation was observed between total quantity olive pollen collected over the season and rainfall recorded during the second fortnight in February. According to the proposed model, an average of 680 Chilling Hours (CH) are necessary to overcome the chill period and break the state of bud dormancy, and 481 Growth Degree Days (GDD) °C are needed to induce flowering. Models for predicting daily mean pollen concentrations combine temperature and the previous days' pollen concentrations as predictor variables to provide a high level of prediction.     

The influence of relative humidity on the swelling of pollen grains in vitro

The volume of hydrated pollen grains of Petunia hybrida L. during swelling in germination medium increased three times. The volume of desiccated pollen grains increased only two times after transfer to the same medium. This difference in swelling ability is attributed to different rigidities of the pollen grain wall,ccaused by the different hydration states. The relationship between pollen grain swelling and germination metabolism with regard to relative humidity is discussed.Abbreviation RH relative humidity     

Pectin secretion and distribution in the anther during pollen development in Lilium

Using the monoclonal antibodies JIM 5 and 7, pectin was immunolocalized and quantitatively assayed in three anther compartments of Lilium hybrida during pollen development. Pectin levels in both the anther wall and the loculus increased following meiosis, were maximal during the early microspore stages and declined during the remainder of pollen ontogenesis. In the microspores/pollen grains, pectin was detectable at low levels during the microspore stages but accumulated significantly during pollen maturation. During early microspore vacuolation, esterified pectin epitopes were detected both in the tapetum cytoplasm and vacuoles. In the anther loculus, the same epitopes were located simultaneously in undulations of the plasma membrane and in the locular fluid. At the end of microspore vacuolation, esterified pectin epitopes were present within the lipids of the pollenkitt, and released in the loculus at pollen mitosis. Unesterified pectin epitopes were hardly detectable in the cytoplasm of the young microspore but were as abundant in the primexine matrix as in the loculus. During pollen maturation, both unesterified and esterified pectin labelling accumulated in the cytoplasm of the vegetative cell, concurrently with starch degradation. In the mature pollen grain, unesterified pectin epitopes were located in the proximal intine whereas esterified pectin epitopes were deposited in the distal intine. These data suggest that during early microspore development, the tapetum secretes pectin, which is transferred to the primexine matrix via the locular fluid. Further, pectin is demonstrated to constitute a significant component of the pollen carbohydrate reserves in the mature grain of Lilium. Received: 3 July 2000 / Accepted: 19 October 2000     

Immunocytochemical and chemical analyses of Golgi vesicles isolated from the germinated pollen ofCamellia japonica

As a first step towards studying the biochemical relationship between Golgi vesicles (GVs) and tube wall components, isolation of GVs from the growing pollen tubes ofCamellia japonica was attempted using a centrifugation method with mannitol. The isolated GV was identified ultrastructurally and immunocytochemically. The main components of the GV were proteins and carbohydrates. The main monosaccharides of GV polysaccharides were galactose, arabinose and uronic acid, and pectins and arabinogalactan proteins also were detected immunochemically. An antiserum against the isolated GVs reacted with the outer layer of the pollen tube wall and the intine layers of the grain wall as well as thein situ GVs in the pollen tube and the grain cytoplasm. We have thus successfully isolated GVs and shown that they contain pectic substances and arabinogalactan proteins which contribute to formation of the pollen tube primary wall.     

An integrated use of aerobiological and phenological data to analyse flowering in olive groves

Aerobiological and phenological investigation has been linked to analyse the flowering phenomena of olive (Olea europaea L.), a tree of economic importance, in Umbria, central Italy.

Olive tree flowering was analysed by phenological observations in olive groves. Aerobiological monitoring was carried out with pollen traps that captured the pollen grains in the atmosphere by remote distances. This combined study of flowering was useful in overcoming the principal limit of phenological observations caused by their geographical narrowness.

Eleven phenological stations in Perugia province were used and periodic observations were carried out during the flowering season. Two aerobiological stations located near the cities of Perugia and Spoleto were utilized to trap olive pollen grains in the atmosphere.

Graphic and statistical analyses were used in order to study flowering dynamics, and to compare the methodological approaches.

The phenological areas, according to their characteristics, were divided in two clusters each one related to a different pollen monitoring station. Statistical analyses showed the presence of a three ‐ four‐day period during which pollen released in the groves is persistent in the narrowness and resulted in a high correlation with the pollen monitored by the pollen traps. On the other hand, the phenological data considered along with the daily pollen concentrations permit the pollen peaks to be attributed to particular olive areas or olive cultivars. This demonstrates that both methodologies can be used effectively as integrated research tools in this kind of study.     

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.     

Postulated interaction between branching pollen tubes and ovules inOenothera hookeri de Vries (onagraceae)

The pollen grain germinationin vitro and progamic phase till fertilization inOenothera hookeri de Vries was observed after open and controlled pollination. The same pattern of pollen grain germination was foundin vitro and on the stigma. The pollen tubes can germinate from 1,2 or 3 poruses of the pollen grain, divide and branch during their growth in the ovary. The branches are of different length and give secondary splits. Special short branches are formed near the micropyle of the ovule. They grow into top part of integments. The pollen tubes start to branch profusely near the placental surface. In that place they are likely to react to the stimulus from mature ovules which seems to be dispersed in the exudate covering placenta.     

13. Lake Saloio (Nazaré, western Portugal)

The possible impact of altitude and the related microclimatic conditions on the total production of fruiting branches, inflorescences, flowers and pollen grains of olive trees Olea europaea was analysed. A total of 90 Picual cultivar trees, the most extensive olive cultivar in the Iberian Peninsula, were studied for a three-year period (2007–2009). The study shows that production of flowers and pollen grains in a cultivar of the olive tree varies according to the microclimate. Our study also indicates that the olive trees frequently can have up to half a million flowers per tree. Moreover, the total flower production differs between years and study areas. In the Picual cultivar, the average production of pollen grains per anther is usually more than 60?000 grains. The total production of pollen per tree is around 72?000 million on average. The most favourable microclimatic conditions for reproduction in olive trees are found in years and olive growing areas with low temperature and high precipitation records during the months prior to flowering of the olive trees. We hypothesise that olive trees tend to increase their pollen production rate as altitude increases, which can be interpreted as a reproductive strategy to ensure fertilisation.     

Effect of Controlled Crossing on the Triglyceride and Fatty Acid Composition of Virgin Olive Oils

Our study reports the triglyceride (triacylglycerol, TAG) composition of five new Tunisian virgin olive oil cultivars obtained through controlled crossings of the cultivar (cv.) Chemlali Sfax. These cultivars have been selected among a progeny of 500 olive descendants, based on an evaluation of the fatty‐acid (FA) composition of their oils. Among these samples, two were derived from the crossing with the cv. Sigoise as pollinator (SM634) or pollen acceptor (SM1110) and the others from the crossing with the cv. Meski as pollen acceptor (SM513, SM514, and SM517). The five descendants were characterized by a good fat value, a balanced FA composition, and a high content of triolein, varying between 26.9 (SM514) and 45.46% (SM1110). They had an improved FA composition as compared to that of the cv. Chemlali Sfax and their fruits were slightly bigger. The principal component analysis suggested that the TAG variables were more suitable than the total FAs for an optimum classification of the cultivar samples analyzed. The cultivars obtained through the crossing with the cv. Sigoise (as pollen acceptor or pollinator) had a more favorable composition of FAs and TAG than those obtained through the crossing with the cv. Meski, which indicated that genetic factors had the most important influence on the quality of the virgin olive oils.     

Calcium in electron-dense globoids during pollen grain maturation in Chlorophytum elatum R.Br.

The localization, elemental composition and quantitative changes in insoluble, electron-dense globoids observed in the course of pollen grain development in Chlorophytum elatum R.Br. have been investigated. During pollen maturation, small electron-dense globoids were only found within larger electron-light vesicles in the cytoplasm of the vegetative cell. The number and diameter of the electron-dense globoids increased during pollen grain maturation, and decreased after pollen germination. By energy-dispersive X-ray microanalysis, the presence of calcium, magnesium and phosphorus was detected in these globoids. The results are discussed in the light of previous findings with regard to phytate metabolism and the role of calcium in pollen germination. Received: 28 December 1996 / Accepted: 7 May 1997     

Pollen gene expression analysed by micro-isoelectric focusing of proteins from isolated pollen grains in Cucurbita pepo L.

Summary Isoelectric focusing of proteins from single pollen grains of Cucurbita pepo L. has been developed for large scale study of pollen grain populations' heterogeneity. Forty to forty-five protein bands from one pollen grain are revealed after silver staining. Applications of this technique to pollen grain populations from different genotypes are described in this paper. Possible applications and limits of this technique are discussed with respect to plant breeding especially for the measure of gene frequencies in pollen grain populations.The work was partly supported by a grant from LIMAGRAIN and AGPM     

A protein coding for a pollen-specific gene in alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis> L.) is localized mainly in the intine of the pollen wall

In previous work, we characterized several cDNA and genomic clones of genes expressed in the pollen of alfalfa (Medicago sativa L.), also known as lucerne. In this study, we have extended that work and focused on expression at the protein level of one of those genes, PO2, a low-copy gene of unknown function, but with similarities to arabinogalactan proteins. Polyclonal antibodies, specific to the PO2 protein expressed in Escherichia coli, were raised in chickens, extracted from the eggs, and used in Western blots and immunolocalization studies to identify the location of the PO2 protein in sections of mature alfalfa pollen grains and pollen tubes. Our Western blot and immunolocalization data suggest that the PO2 protein is secreted, occurs largely in the intine, and is not detectable in the pollen tube; moreover, it is not detectable on the surface of the pollen grain, nor does it diffuse out of the pollen grain into an aqueous medium.