Immunoelectron-microscopic localization of diffusible birch-pollen antigens in ultrathin sections using the protein-A/gold technique

Pollen grains from Betula pendula were fixed in a mixture of p-formaldehyde and cetylpyridinium chloride (CPC) for the precipitation of soluble pollen glycoproteins. After dehydration and embedding at low temperatures in the water-soluble resin, Lowicryl K4M, ultrathin sections of the pollen grains were incubated using specific antibodies against birch-pollen extract and protein-A/gold complexes. Antigen activity was found in the CPC-precipitated surface material and within the exine (bacular cavities) and the cytoplasm (except for starch grains and lipidic droplets). There was no labelling within the intine. The region of the germinal aperture also showed a very low degree of antigen activity. The control sections were almost completely free of background staining.     

Pollen morphology of Astragalus section Hymenostegis (Fabaceae) and evaluation of its systematic implications

Astragalus is with nearly 3000 described species the largest genus of flowering plants. So far analyses of pollen characters have only been conducted for a few species of the groups within the genus. Here we analyse pollen grains of 22 species representative for Astragalus section Hymenostegis using scanning electron microscopy. We found the basic shape of the pollen grains to be oblate-spheroidal and apertures to be tricolpate as for other eudicots. The sculpturing pattern of the exine is micro-reticulate. Pollen grains show low morphological variation among different species of this section, but differences occur between sections of the genus. We conclude that the vast morphological differentiation that occurred during the rapid radiation of section Hymenostegis was not accompanied by comparable differentiation in pollen morphology.     

Pollen size in Carex: The effect of different chemical treatments and mounting media

Pollen size and pollen aperture size for ten species of the genus Carex L., native to Estonia, have been measured using light microscopy. The species selected represent different sections of the genus, a range of habitats and different chromosome numbers. The effects of two basic chemical treatments, two mounting media and the effect of chemically induced dehydration with tertiary butyl alcohol (TBA) on the size of pollen grains were then recorded.

In general pollen size and pollen aperture size of the species examined is highly variable at both intraspecific and interspecific levels. Carex hirta has notably larger pollen grains than any of the other species investigated and, although correlations between size and chromosome number in the species examined are limited, it also has the highest chromosome number. Statistically significant size differences resulted from variations in chemical treatment, mounting media and tertiary butyl alcohol (TBA) induced dehydration. Acetolysed pollen grains are larger than potassium hydroxide (KOH) treated pollen grains. Pollen grains dehydrated after chemical treatment with TBA are larger than pollen grains not dehydrated. Pollen grains mounted in silicon oil are smaller than grains mounted in glycerine. But considering the great size variation of Carex pollen grains, the size changes caused by preparation procedures fall within the size variation range of the species examined.

All the samples contained a high number of deformed pollen grains and pollen grains with hardly distinguishable or no lateral apertures.     

Cytopathology of Anthers and Pollen From Lettuce Plants Infected by Lettuce Mosaic Virus

Thin sections of mature anthers and pollen grains from three lettuce (Lactuca sativa) plants infected with lettuce mosaic potyvirus (LMV) were studied by immunogold labelling. Labelled LMV particles were present externally on the exine of pollen grains from all plants, but were observed internally in the pollen grains from only one plant. Within mature pollen grains the virus particles were associated with the cytoplasmic bundle inclusions typical of infection by potyviruses. The tapetal plasmodium and the epidermal and endothecial layers of mature anthers from all infected plants also contained labelled virus particles, together with pinwheel and bundle inclusions.     

Barley anther culture: The effect of position on pollen development in vivo and in vitro

Locule structure and organization were studied in vivo and in vitro to determine whether the disposition of pollen within barley anthers affected the response of pollen in culture. Following release from the meiotic tetrads, juvenile barley microspores become peripherally organized around the locule, with the single pollen pore oriented towards the tapetum. Scanning electron micrographs of transverse sections from freeze fractured anthers showed that some microspores failed to locate the tapetal surface and occupied a position in the centre of the locule where they continued to develop as small, abnormal pollen grains (dimorphic pollen). Previous evidence has suggested that in some species dimorphic pollen could be the source of embryonic pollen in vitro. Cultured anthers frequently dehisced to reveal a mass of dividing pollen grains, however those anthers that remained intact retained the original locule structure and could be freeze fractured permitting examination of the developing pollen in situ. This showed that pollen embryogenesis was not restricted to dimorphic pollen, and that any grain could become Embryogenic irrespective of position.     

The role of allergenic proteins Pla a 1 and Pla a 2 in the germination of Platanus acerifolia pollen grains

Platanus acerifolia (Aiton) Willdenow is a plane tree, widely grown as an ornamental tree in many cities of the United States and Western Europe, which has become an important source of airborne allergens in our cities. The aim of the present study is to immunolocalize the major allergens in the pollen grain and to examine their potential function in the fertilization process. Observations were made in mature and hydrated, activated pollen of P. acerifolia for 5, 15, 30 min and 2 h in the germination medium. Specimens were fixed using freezing protocols for transmission electron microscopy (TEM). For immunogold labelling, cryosections and resin-embedded ultrathin sections were incubated using rabbit antisera against the purified pollen allergens Pla a 1 and Pla a 2. Elution of P. acerifolia allergens took place after 5 min of pollen incubation in buffered medium. Intense labelling of Pla a 1 and Pla a 2 was detected after pollen exudates were released. In pollen grains, Pla a 1 was predominantly localized in concentric cisternae of the endoplasmic reticulum (ER), situated between the vegetative nucleus and the generative cell, and was released from pollen grains 5 min after hydration; cytoplasmic localization decreased 15 min after hydration. In pollen grains, glycoprotein Pla a 2 was abundant in association with Golgi cisternae and vesicles situated in the apertural periphery of the mature pollen grains. Pla a 2 proteins were also detected in ER and in the generative cell wall. Immunolabelling of Pla a 2 decreased 5 min after pollen hydration but was again intense after 15–30 min in germination medium, presumably as a consequence of renewed expression and glycosylation of this protein. Pla a 1 belongs to a new class of allergens related to proteinaceous invertase and pectin methyl esterase inhibitors (PII, PMEI) which could be involved in membrane protection and pectin de-esterification control during pollen hydration. Pla a 2 has an exopolygalacturonase (PG) enzymatic activity consistent with pollen-stigma adhesion mechanisms or compatibility systems. Moreover, the expression of Pla a 2 found 15–30 min after hydration might contribute to pollen-tube growth and the modification of transmitting tissue cell walls. The abundant production and elution of Pla a 1 and Pla a 2 proteins may alter the environment in which pollen tube elongation occurs, thus promoting a potential crosstalk between the pollen and the gynoecium.     

Pollen morphology of polyscias (araliaceae): the malesian and australian species

The pollen morphology of 33 Malesian and Australian taxa of Polyscias is described, and the variation assessed by multivariate analysis. The analysis identified two main pollen types: (1) relatively small grains with a microreticulate tectum and lalongate ora (sect. Polyscias, Gelibia, Eupteron and six taxa not assignable to any of the sections as they are currently defined, (2) relatively large grains with a reticulate tectum, or which do not possess a tectum (previously unknown in the family), lalongate, lolongate or circular ora (sect. Kissodendron and Palmervandenbroekia). The systematic utility of palynologial data at various taxonomic levels within Polyscias is dicussed.     

Ultrastructural demonstration of a glycoproteinic surface coat in allergenic pollen grains by combined cetylpyridinium chloride precipitation and silver proteinate staining

Summary In allergenic birch pollen grains, highly watersoluble surface substances were precipitated by the cationic detergent cetylpyridinium chloride (CPC) during aqueous fixation. After processing the pollen for electron microscopy, ultrathin sections of pollen grains were subjected to the periodic acid — thiocarbohydrazide — silver proteinate (PA-TCH-SP) procedure according to Thiery (1967) for the detection of vicinal glycol groups. It was found that the material precipitated by CPC on the surface and within the exine cavities of the pollen wall strongly reacted with the PA-TCH-SP reagent thus indicating the presence of polysaccharides on the surface of birch pollen grains. In samples which had not been treated with the cationic detergent, PA-TCH-SP reactivity was reduced to thin linings on the surface and within the exine cavities. In both cases the exine proper did not stain whereas the intine showed moderate staining. Within the aperture region of the intine, PA-TCH-SP reactivity is preferably associated with fibrillar or reticular structures. The results are discussed with special reference to biochemical findings on allergenic birch pollen proteins.     

ORIGIN AND DEVELOPMENT OF POLLEN EMBRYOIDS AND POLLEN CALLUSES IN CULTURED ANTHER SEGMENTS OF HYOSCYAMUS NIGER (HENBANE)

The dedifferentiation of pollen grains of Hyoscyamus niger (henbane) into embryoids and calluses was examined by culturing identical segments of the same anther in a mineral salt-sucrose basal medium and in the basal medium supplemented with 2.0 mg/l 2,4-dichlorophenoxyacetic acid, respectively. Addition of auxin enhanced anther efficiency but did not affect the number of embryogenic pollen grains of an anther segment transformed into calluses. In anther segments cultured in the basal medium, the organogenetic part of the pollen embryoid was formed by the division of the generative cell alone, or by the division of both generative and vegetative cells. More or less similar pathways were followed by pollen grains of anther segments cultured in a medium containing auxin to form calluses. Culture of anther segments in a medium containing a high concentration of auxin (50.0 mg/l) led to a significant reduction in the yield of calluses which were formed almost entirely by the division of both generative and vegetative cells. The bearing of these observations on the role of auxin in determining the pathway of differentiation of embryogenic pollen grains in cultured anther segments is considered. The appearance of embryogenic pollen grains in close proximity to the tapetum as seen in longitudinal sections of cultured anther segments has suggested a role for a gradient of tapetal factors in embryogenic induction.     

Arecaceae potential for production of monofloral bee pollen

Bees often collect pollen from plants that are near their colonies or apiaries. Arecaceae are characterised by having species that flower throughout the year and produce large quantities of pollen. Pollen grains of Arecaceae have two main forms, namely monosulcate and trichotomosulcate. The study area is located in the coastal region of the state of Sergipe, Brazil, where large amounts of bee pollen are produced by Apis mellifera. This study aims to evaluate how Arecaceae contribute to the production of monofloral bee pollen. Samples of bee pollen loads were collected each month for two years (totalling 24 samples), acetolysed, and mounted on permanent slides. At least 500 grains of pollen were counted for each sample, the data was analysed, and figures were made using the Tilia software. The pollen grains of Cocos nucifera were present in 23 samples (96%), and concentrations of this pollen type varied between 1.1 and 46.8% (but were commonly above 20%). The concentration of Arecaceae pollen grains is the result of the large-scale production of coconuts along the coastline. However, the presence of other pollen types, such as Mimosa arenosa, Mimosa pudica, Mikania and Myrcia, compliments the diets of bees in the region. The results show that even when in low frequency, Cocos nucifera pollen grains represent a large proportion of pollen content compared with other pollen types, because of the large size of Arecaceae pollen grains.     

Pollen morphology of Chinese Glochidion (Phyllanthaceae) and its taxonomic implications

The pollen morphology of twenty‐five Chinese species belonging to two sections of Glochidion and one species from Phyllanthus subgen. Phyllanthodendron was examined using scanning electron microscopy (SEM). The results showed that pollen grains of Glochidion are isopolar, spheroidal to rarely subprolate in shape, (3)4–5(6)‐colporate in aperture pattern, and tectate‐reticulate to tectate‐rugulate in exine ornamentation. Fusion of colpus ends was observed in some pollen grains of G. pseudo‐obscurum and G. wrightii. The pollen grains of Phyllanthus dunnianus are similar to those of Glochidion species in their spheroidal shape and four‐colporate aperture pattern, but are slightly larger in size, and show tectate‐perforate exine ornamentation. In Glochidion, pollen morphology appears to be useful for distinguishing several macromorphologically similar. Additionally, palynological characters provide evidence for the taxonomy of the supergenus Phyllanthus s.l.     

HETEROSTYLY,POPULATION COMPOSITION,AND POLLEN FLOW IN HEDYOTIS CAERULEA

Hedyotis caerulea is a perennial, spring-flowering herb native to eastern North America with distylous flowers that differ in a number of morphological and physiological traits. However, the pin and thrum morphs produce the same numbers of buds, flowers, and fruits per plant, although it is possible that differences in these may occur in some populations at certain times of the flowering period. The two morphs are self-incompatible and cross-compatible. Most populations contained an excess of pins over thrums (anisoplethy); less commonly pins and thrums were equally represented (isoplethy). Populations change from anisoplethy to isoplethy and in the reverse direction. The spatial distribution of pin and thrum flowers in populations was random in some populations but non-random in others. There is some indication that the two morphs in some populations have somewhat different flowering periods. Pollinators seem to be chiefly bombyliid flies and perhaps thrips, but insects were rarely observed visiting flowers. In some populations, the two morphs produced equal numbers of pollen grains per flower; in others they did not. The average pollen viability varied, but on the average there was a moderate level of pollen sterility. High numbers of pollen grains remained in dehisced anthers, probably as a result of low pollinator activity. However, between 5% and 9% of the pollen produced participated in pollination. Stigmas of most pin flowers received more pin pollen grains than thrum pollen grains; on stigmas of thrum flowers pin pollen grains outnumbered thrum pollen grains. Thus, compatible pollen flow from pins to thrums was greater than in the reverse direction. Anisoplethic and isoplethic populations had the same pollen flow patterns. A plant-by-plant examination of stigmas indicated that many stigmas bore few or no pollen grains. Seed production of the two morphs was equal. Despite the inequities in pollen flow patterns, the widespread and occasionally weedy nature of H. caerulea suggests that its breeding system must be viewed as a successful one.     

Pollen morphology and localization of Ni in some Ni-hyperaccumulator taxa of Alyssum L. (Brassicaceae)

Pollen morphology of seven Alyssum L. taxa growing on serpentine soils in different places in the European Mediterranean macrobioclimate territory were studied, described and compared. Cluster analysis was performed to show similarity between species and their populations. The shape of the pollen grains varies among the species and among the grains within the same anther. The pollen grains are 3-colpate, prolate, with long and narrow colpi reaching the poles. The ornamentation of the exine varies from micro-reticulate to reticulate between the species. Pollen sterility/fertility was also calculated. The highest percentage of sterile pollen (73.76%) was calculated for Alyssum murale subsp. murale and the lowest (9.54%) for A. bertolonii subsp. bertolonii. All species are representatives of sect. Odontarrhena (C.A. Meyer) Koch well known as Ni-hyperaccumulators. Nickel and other elements present in pollen and stamen were studied by inductively coupled plasma-mass spectrometry. The stamen parts of all species were micromorphologically analyzed by scanning electron microscopy coupled to an energy-dispersive X-ray probe. Accumulation of Ni was detected in the stamens of all studied species and rarely in the pollen grains. The distribution patterns of Ni were similar among the species examined.     

Heterospecific pollen transfer between sympatric species in a midsuccessional old-field community

The cumulative (season-long) incidence of heterospecific pollen transfer (HPT) was examined using nine sympatric species in a midsuccessional old field. Inflorescences were collected weekly during the flowering season, and the proportion of foreign pollen/stigma was recorded. Flowering phenologies of sympatric species and ovule and seed counts of study species were also recorded. Heterospecific pollen was detected on some stigmas of each species. Medicago sativa (Fabaceae) received the most foreign pollen; in some cases, all of the grains on a stigma were heterospecific. Lotus corniculatus (Fabaceae) received the least amount of foreign pollen; the incidence of heterospecific pollen was near zero in most cases. The mean and range of foreign pollen received varied by as much as an order of magnitude between species. The six species with zygomorphic flowers, all Fabaceae, received more heterospecific pollen than the three species with actinomorphic flowers, Potentilla recta and P. simplex (Rosaceae) and Ranunculus acris (Ranunculaceae). This probably reflects a bias because our data were analyzed on a cumulative basis and the Fabaceae had longer flowering phenologies. HPT was not correlated with the species' relative abundance within the community. Proportion of foreign pollen received varied temporally within species, and this variation generally was not related to phenology of any sympatric taxa or the species' own phenology. Pollen grain diameter was positively related to levels of foreign pollen received by species. This might be caused by poor adhesion of large pollen grains to small stigmatic papillae or if generalist pollinators carrying large amounts of heterospecific pollen visit the large-grained species and specialists with little foreign pollen visit the small-grained species. The large proportions of heterospecific pollen on stigmas of many species indicate that HPT occurs frequently in the community we studied and the implications may include reduced seed set because of occlusion by foreign grains. As yet, however, it is unclear how important a factor HPT is in mediating pollen limitation of reproductive success.     

The seasonal variation of airborne pollen grains that cause sugipollinosis in japan in the last three years

In Japan, the problems of Sugi (Cryptomeria japonica)-pollinosis have been much discussed in recent papers and journals. The author made an investigation on the airborne pollen grains from a scientific standpoint in connection with the incidence of pollinosis. By using the Cascade Impactor the author collected 600 liters of air sample a day, at the roof of the Pharmaceutical Science Building, Toho University, Funabashi, Chiba, Japan. Each air sampling for 2 hrs was repeated three times a week since 1969. The airborne pollen grains in each sample were counted under the microscope to identify the pollen types. The pollen grains were classified into six types according to the classification of pollen grains in Japan established by lkuse. The accumulated number of pollen of each species was analysed statistically by circular plot, Weibull plot, Edwards plot and semi-logarithmic plot. In this report mainly 3B type of pollen grains (Cryptomeria, Chamaecyparis), collected in the last three years are described. The total number of pollen grains in 1988 (2820 grains) was doubled in quantity as compared with that in 1987 (1177 grains) and in 1989 (1121 grains). The 3B type of pollen grains in 1988 (1450 grains) was 3.4 times as many as in 1987 (397 grains), and 12.9 times as in 1989 (112 grains). The 3B type of pollen grains represented the major portion of total pollen, and influenced the annual amount and the dispersal period.     

Environmental risk assessment for the small tortoiseshell Aglais urticae and a stacked Bt-maize with combined resistances against Lepidoptera and Chrysomelidae in central European agrarian landscapes

The cultivation of Lepidoptera‐resistant Bt‐maize may affect nontarget butterflies. We assessed the risk posed by event MON89034 × MON88017 (expressing Cry1A.105 and Cry2Ab2 against corn borers) to nontarget Lepidoptera. Using the small tortoiseshell Aglais urticae, a butterfly species common in central Europe, as a test organism we (i) assessed the toxicity of Bt‐maize pollen on butterfly larvae; (ii) measured pollen deposition on leaves of the host plant Urtica dioica; (iii) mapped the occurrence and distribution of host plants and larvae in two arable landscapes in Germany during maize anthesis; and (iv) described the temporal occurrence of a 1‐year population of A. urticae. (i) Larvae‐fed 200 Bt‐maize pollen grains/cm² had a reduced feeding activity. Significant differences in developmental time existed at pollen densities of 300 Bt‐maize pollen grains/cm² and in survival at 400 grains/cm². (ii) The highest pollen amount found was 212 grains/cm² at the field margin. Mean densities were much lower. (iii) In one region, over 50% of A. urticae nests were located within 5 m of a maize field, while in the other, all nests were found in more than 25 m distance to a maize field. (iv) The percentage of larvae developing during maize anthesis was 19% in the study area. The amount of pollen from maize MON89034 × MON88017 found on host plants is unlikely to adversely affect a significant proportion of larvae of A. urticae. This paper concludes that the risk of event MON89034 × MON88017 to populations of this species is negligible.     

Plesiomorphic and apomorphic pollen structure characteristics of anthemideae (Asteroideae: Asteraceae)

Anthemideae (Asteroideae: Asteraceae) pollen grains have basal columellae, a structural type called “anthemoid” in earlier publications. To survey structure variation in Anthemideae pollen, we examined freeze-sectioned grains from 45 species within 23 representative genera using scanning electron microscopy (SEM). From resulting data and a literature review, we concluded that: 1) pollen of Anthemideae taxa is qualitatively identical except for Ursinia (grains essentially lack basal columellae) and the Artemisia group (branches of basal columellae are complex and interwoven); 2) the double tectum (a term introduced in this study) is a synapomorphy of Asteroideae and plesiomorphic in Anthemideae; 3) apomorphies of Anthemideae grains include large basal columellae, a thick foot layer, and absence of internal foramina; and 4) Anthemideae pollen is qualitatively different from similar pollen in Lactucoideae, a distinction we recognized by restricting “anthemoid” to Anthemideae grains. Ursinia grains have occasional basal columellae and features resembling rolled-up columellae; we consider these vestiges of a reversal to the plesiomorphic condition. To assess quantitative structural variation, 2,200 image-analysis measurements were taken from 73 SEM micrographs. Intrageneric variation was analyzed by standard deviation, and intergeneric variation by principal components analysis. Compared to other Anthemideae taxa, the structural elements of Artemisia grains have reduced dimensions and variability. Otherwise, structural radiation of Anthemideae pollen has produced a phenetic continuum.     

Sexual interference within flowers of Chamerion angustifolium

Hermaphroditism is prevalent in plants but may allow interference between male function (pollen removal and dispersal) and female function (pollen receipt and seed production) within a flower. Temporal or spatial segregation of gender within a hermaphroditic flower may evolve to reduce this interference and enhance male and female reproductive success. We tested this hypothesis using Chamerion angustifolium (Onagraceae), in which pollen removal (male) and pollen deposition (female) were measured directly on hermaphroditic and experimentally produced unisexual flowers. During a single flower visit in the field, bees deposited 159±24 (SE) pollen grains on a stigma and removed 1058±198 grains from each flower. Anther removal did not alter deposition rates. In the laboratory, bees removed 2669±273 pollen grains and deposited 209±72.3 cross-pollen and 120±28.4 facilitated self-pollen grains per visit. The presence of anthers significantly reduced cross-pollen deposition on the stigma. In contrast, pollen removal was not affected by presence of the pistil. These results suggest that within-flower interference affects female function and represents a fitness cost that can be reduced through temporal segregation of gender within the flower. Co-ordinating editor: S.-M. Chang     

The association between pollen size and Renner complex in Oenothera villaricae and O. picensis ssp. picensis and their hybrids: evidence for preanthesis pollen competition

In both Oenothera villaricae Dietrich and O. picensis ssp. picensis, chromosomes are transmitted as two Renner complexes. Reciprocal combinations of the Renner complexes produce eight different F₁ hybrids, but only seven are viable. Each species, and each F₁ hybrid, produces three sizes of pollen, approximately 50% small sterile grains, 15% medium-sized viable grains and 35% large viable grains. Medium- and large-sized grains were separated manually and subjected to random amplified polymorphic DNAs (RAPDs) analysis. A pattern of RAPD amplifications was obtained which indicates that, for each species and F₁ hybrid, one specific Renner complex characterizes the medium- and another the large-sized viable pollen. The results indicate that pollen size is determined in part by the pollen genotype and in part by the genotype of the other pollen grains developing within the same anther.     

Dynamic of polysaccharide and lipid in the developing microsporangiums of Chinese fir (Cunninghamia lanceolata)

Microsporongium development in Chinese fir (Cunninghamia lanceolata) was investigated using cytochemical methods with a special attention to the fluctuations (in amount and distribution) of polysaccharide and lipid reserves along the development of the microsporangium. Semi-thin sections of microsporangia at different developmental stages were stained with periodic acid–Schiff (PAS) reagent and Sudan Black B to detect insoluble polysaccharides and neutral lipids, respectively. In young microsporangia, microspore mother cells began to accumulate starch grains and lipids, which disappeared during microspore development. Following microspore division, the starch grains present in bicellular pollen disappeared and abundant lipid deposits were accumulated. In mature pollen, only abundant lipids accumulated as storage material. The pollen wall of C. lanceolata is predominantly composed of polysaccharidic intine, and the sporopollenin-containing exine is weakly developed and only forms a thin layer covering the intine.