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Estimating pollen flow using SSR markers and paternity exclusion: accounting for mistyping.

Highly informative genetic markers, such as simple sequence repeats (SSRs), can be used to directly measure pollen flow by parentage analysis. However, mistyping (i.e. false inference of genotypes caused by the occurrence of null alleles, mutations, and detection errors) can lead to substantial biases in the estimates obtained. Using computer simulations, we evaluated a direct method for estimating pollen immigration using SSR markers and a paternity exclusion approach. This method accounts for mistyping and does not rely on assumptions about the distribution of male reproductive success. If ignored, even minor rates of mistyping (1.5%) resulted in overestimating pollen immigration by up to 150%. When we required at least two mismatching loci before excluding candidate fathers from paternity, the resulting pollen immigration estimates had small biases for rates of mistyping up to 4.5%. Requiring at least three mismatches for exclusion was needed to minimize the upward biases of pollen immigration caused by rates of mistyping up to 10.5%. The minimum number of highly variable SSR loci needed to minimize cryptic gene flow and obtain reliable estimates of pollen immigration varied from five to seven for a sampling scheme applicable to most conifers (i.e. when paternal haplotypes can be unambiguously determined). Between five and nine highly variable SSR loci were needed for a more general sampling scheme that is applicable to all diploid seed plants. With moderately variable SSR markers, consistently accurate estimates of pollen immigration could be obtained only for rates of mistyping up to 4.5%. We developed the POLLEN FLOW (PFL) computer program which can be used to obtain unbiased and precise estimates of pollen immigration under a wide range of conditions, including population sizes as large as 600 parents and mistyping rates as high as 10.5%.

Alleles↗

Recurrent pericarditis: a rare complication of allergen immunotherapy.

We report on a 29-year-old woman suffering from hay fever due to grass and olive tree pollens. She developed recurrent pericarditis during her first course of immunotherapy with an alum-adsorbed pollen extract. A causal relationship was established between the allergen injections and the acute pericarditis episodes on two consecutive occasions, which presented with blood eosinophilia. Blood cultures and serological tests for microorganisms were negative. There were no signs of autoimmune disease or systemic vasculitis. To the best of our knowledge, allergen immunotherapy-induced pericarditis has not been previously reported.

Acute Disease↗

Restoration of stamen development and production of functional pollen in an alloplasmic CMS tobacco line by ectopic expression of the Arabidopsis thaliana SUPERMAN gene.

The alloplasmic male-sterile tobacco line Nta(rep)S, combining the nucleus of Nicotiana tabacum with the cytoplasm of Nicotiana repanda, exhibits cadastral-type anomalies due to a fusion of several stamens with the pistil. These anomalies share similarities with Arabidopsis superman mutants. SUPERMAN (SUP) is a cadastral gene controlling the boundary between whorls 3 (androecium) and 4 (gynoecium). Thus we hypothesized that the expression of the tobacco SUP orthologue might be impaired in the alloplasmic Nta(rep)S line, and that the deficiency could be complemented by the Arabidopsis SUP gene. Here we show that the ectopic expression of SUP in the alloplasmic male-sterile tobacco line Nta(rep)S significantly increases the frequency of flowers possessing free stamens, inducing the recovery of a proper structure for whorls 3 and 4. Furthermore, flowers of transgenic plants show a significant improvement of the morphology of stamens, and more particularly of the anthers, which are able to produce few but functional pollen. The data show that ectopic expression of Arabidopsis SUP reactivates the regulatory cascade of anther development. The plausible causes of the developmental defects of anthers in the alloplasmic male-sterile tobacco line are discussed in relation to the model of regulation of the Arabidopsis SUP gene.

Arabidopsis↗

Pollen-tube growth and fertilization mode in Gymnostoma (Casuarinaceae): their characteristics and evolution.

A unique mode of fertilization called "chalazogamy", whereby the pollen tube passes through the chalaza instead of the micropyle, is known in several species of derived genera in Casuarinaceae. In this paper we report the occurrence of chalazogamy in Gymnostoma ( G. poissonianum), the most primitive genus in the family. We also show that the pollen tube grows discontinuously from the stigma to ovules in about 3 months. At the time of pollination, the ovules have not yet formed in the ovary, and require a long time to develop. The pollen tube(s) lie in a zigzag line and are branched in the upper region of the style, and their growth is arrested there until the ovary develops further. Studies of the relevant literature further revealed discontinuous pollen-tube growth in relation to a prolonged period between pollination and fertilization, as well as chalazogamy, in Betulaceae, Juglandaceae and/or Fagaceae that are closely related to Casuarinaceae. This feature may have derived early in the evolution of Fagales.

Betulaceae↗

Nifedipine-sensitive calcium channels are involved in polar growth of lily pollen tubes.

Pollen germination and tube growth of Lilium longiflorum in vitro are affected by 10(-5) M-nifedipine. Germinating 'tubes' form broad protuberances along the whole colpus. Short tubes show a high tendency to grow 'amoeboid-like' and to branch; or a second tube emerges in another region of the colpus. Longer tubes (greater than or equal to 100 micron) broaden irregularly or swell at their tips. The diameter of the tube can vary drastically within the same tube. With increasing time of treatment many tubes burst. Normal tip growth stops within 10 min, but protoplasmic streaming continues even after 15 h. More or less regularly, wall thickenings are formed along the whole tube or on the flanks of the germinating region after some hours. The internal calcium gradient, visualized by chlorotetracycline (CTC) fluorescence, is also disturbed. Nifedipine treatment results in uniform or irregular CTC fluorescence. Branching tubes temporarily show a new subapical CTC gradient. After 6-8 h of nifedipine treatment many cells are no longer stainable with CTC. The results indicate the presence of nifedipine-sensitive calcium channels in pollen tubes.

Calcium↗

Comparative analysis of the Arabidopsis pollen transcriptome.

We present a genome-wide view of the male gametophytic transcriptome in Arabidopsis based on microarray analysis. In comparison with the transcriptome of the sporophyte throughout development, the pollen transcriptome showed reduced complexity and a unique composition. We identified 992 pollen-expressed mRNAs, nearly 40% of which were detected specifically in pollen. Analysis of the functional composition of the pollen transcriptome revealed the over-representation of mRNAs encoding proteins involved in cell wall metabolism, cytoskeleton, and signaling and under-representation of mRNAs involved in transcription and protein synthesis. For several gene families, we observed a common pattern of mutually exclusive gene expression between pollen and sporophytic tissues for different gene family members. Our results provide a 50-fold increase in the knowledge of genes expressed in Arabidopsis pollen. Moreover, we also detail the extensive overlap (61%) of the pollen transcriptome with that of the sporophyte, which provides ample potential to influence sporophytic fitness through gametophytic selection.

Arabidopsis↗

The natural history of mountain cedar pollinosis.

During the winter months, pollen from the mountain cedar (MC) (Juniperus sabinoides) causes severe respiratory tract allergy in central Texas. We have been impressed with the fact that many of our MC-allergic patients had only allergic rhinitis and were only sensitive to MC pollen. We therefore studied 234 unselected MC-allergic patients at the end of the MC season. The main criteria for inclusion into the study was a bona fide history of MC pollinosis confirmed by a positive skin test. All patients completed a detailed history questionnaire, were prick skin tested by using 1:20 w/v, extracts, and had a total IgE determined with commercially available reagents (PRIST). Thirty-four percent of patients were found to be allergic only to MC and 66% were allergic to MC and other aeroallergens. As a group, patients allergic only to MC had significantly lower total IgE levels (84 IU/ml vs. 360, p < 0.001) (normal < 180), required much longer exposure intervals to cedar pollen before developing MC pollinosis (14.4 yr vs, 5.69, p < 0.001), had less of a family history of allergic disease (43% vs. 88%, p < 0.001), had less of a history of asthma or eczema (11% vs. 39%, p < 0.001), and developed allergic disease at a later age (39 yr vs. 13, p < 0.001) when compared to patients with multiple allergies. These results suggest that the MC pollen may be unique in causing allergic rhinitis in patients who have no other sensitivities. A possible explanation may lie in the carbohydrate nature of the main allergen of the MC pollen, which may facilitate allergen transport through the respiratory tract mucosa and subsequent sensitization.

Adult↗

An efficient method for flow cytometric analysis of pollen and detection of 2n nuclei in Brassica napus pollen.

A simple and reliable method was developed for isolating pollen nuclei from Brassica napus and Triticum aestivum for DNA analysis using flow cytometry. The nuclei were released from pollen by ultrasonic treatment. The isolated nuclei following filtration through nylon mesh and a purification procedure were suitable for flow cytometric analysis as well as for isolating genomic DNA. Ultrasonic treatment time was optimized for B. napus pollen at different developmental stages. The method is effective and suitable for the preparation of many samples. We analyzed the nuclear DNA levels in pollen of B. napus at three major developmental stages as well as in mature wheat pollen. Only a single 1C peak representing the haploid DNA level was detected in the nuclei isolated from Brassica uninucleate microspores as well as in mature Triticum pollen. Interestingly, diploid nuclei were detected in both binucleate and mature pollen of B. napus. The possible origins of the diploid nuclei are discussed.

Brassica napus↗

The natural history of mountain cedar pollinosis.

During the winter months, pollen from the mountain cedar (MC) (Juniperus sabinoides) causes severe respiratory tract allergy in central Texas. We have been impressed with the fact that many of our MC-allergic patients had only allergic rhinitis and were only sensitive to MC pollen. We therefore studied 234 unselected MC-allergic patients at the end of the MC season. The main criteria for inclusion into the study was a bona fide history of MC pollinosis confirmed by a positive skin test. All patients completed a detailed history questionnaire, were prick skin tested by using 1:20 w/v, extracts, and had a total IgE determined with commercially available reagents (PRIST). Thirty-four percent of patients were found to be allergic only to MC and 66% were allergic to MC and other aeroallergens. As a group, patients allergic only to MC had significantly lower total IgE levels (84 IU/ml vs. 360, p less than 0.001) (normal less than 180), required much longer exposure intervals to cedar pollen before developing MC pollinosis (14.4 yr vs. 5.69, p less than 0.001), had less of a family history of allergic disease (43% vs. 88%, p less than 0.001), had less of a history of asthma or eczema (11% vs. 39%, p less than 0.001), and developed allergic disease at a later age (39 yr vs. 13, p less than 0.001) when compared to patients with multiple allergies. These results suggest that the MC pollen may be unique in causing allergic rhinitis in patients who have no other sensitivities. A possible explanation may lie in the carbohydrate nature of the main allergen of the MC pollen, which may facilitate allergen transport through the respiratory tract mucosa and subsequent sensitization.

Adult↗

Semi-depot hyposensitization in severe hay fever: its clinical effectiveness and the therapeutic problems. (A four year study using intranasal challenge testing to monitor success).

Hyposensitization treatment for at least two years using pyridine-extracted alum precipitated (PEAP) extracts was carried out on 332 cases of severe hay fever, some of which were steroid dependent. Subjective self-assessment and weekly average use of additional medication were recorded, nasal challenge tests were conducted before and during treatment and related to the clinical picture. Successful treatment was unrelated to age, though distinctly limited where there was a history of more 15 years illness. It was however related to the pollen count from 1973 to 1976 and to the type and number of the antigens to which there was a clinical response. In spite of specific hyposensitization 4,2% of the pollinosis cases developed additional pollen asthma. In the course of treatment there were several instances of an enlargement of the individual antigen spectrum; sensitization to pollen, mould fungi, house dust and animal epithelia were recorded. In a total of 11,850 injections there were severe local reactions in 16,2% rhinoconjunctivitis in 1,6% asthma in 0,4% and urticaria in 0,4%. One female patient went into anaphylactic shock 12 hours after the administration of the antigen extract. In about 20% of the injections granulomas developed, about the size of a cherry-stone, which persisted for up to six months and which in some instances were reactivated by severe exposure to allergens. There was an unusually high percentage of organ and generalised reactions whenever the treatment was continued using a new antigen solution without reducing the dose.

Administration, Intranasal↗

A plant plasma membrane Ca2+ pump is required for normal pollen tube growth and fertilization.

Ca(2+) signals are thought to play important roles in plant growth and development, including key aspects of pollen tube growth and fertilization. The dynamics of a Ca(2+) signal are largely controlled by influx (through channels) and efflux (through pumps and antiporters). The Arabidopsis genome encodes 14 Ca(2+) pumps, 10 of which belong to a family of autoinhibited Ca(2+) ATPases (ACA) that are predicted to be activated by Ca(2+)/calmodulin. Here, we show that isoform ACA9 is expressed primarily in pollen and localized to the plasma membrane. Three independent T-DNA [portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] gene disruptions of ACA9 were found to result in partial male sterility. Complementation was observed by using a ACA9-yellow fluorescence protein (YFP) fusion that displayed plasma membrane localization. Mutant aca9 pollen displayed a reduced growth potential and a high frequency of aborted fertilization, resulting in a >80% reduction in seed set. These findings identify a plasma membrane Ca(2+) transporter as a key regulator of pollen development and fertilization in flowering plants.

Arabidopsis↗

Effects of brefeldin A on pollen germination and tube growth. Antagonistic effects on endocytosis and secretion.

We assessed the effects of brefeldin A (BFA) on pollen tube development in Picea meyeri using fluorescent marker FM4-64 as a membrane-inserted endocytic/recycling marker, together with ultrastructural studies and Fourier transform infrared analysis of cell walls. BFA inhibited pollen germination and pollen tube growth, causing morphological changes in a dose-dependent manner, and pollen tube tip growth recovered after transferring into BFA-free medium. FM4-64 labeling showed typical bright apical staining in normally growing P. meyeri pollen tubes; this apical staining pattern differed from the V-formation pattern found in angiosperm pollen tubes. Confocal microscopy revealed that exocytosis was greatly inhibited in the presence of BFA. In contrast, the overall uptake of FM4-64 dye was about 2-fold that in the control after BFA (5 microg mL(-1)) treatment, revealing that BFA stimulated endocytosis in a manner opposite to the induced changes in exocytosis. Transmission electron microscopic observation showed that the number of secretory vesicles at the apical zone dramatically decreased, together with the disappearance of paramural bodies, while the number of vacuoles and other larger organelles increased. An acid phosphatase assay confirmed that the addition of BFA significantly inhibited secretory pathways. Importantly, Fourier transform infrared microspectroscopy documented significant changes in the cell wall composition of pollen tubes growing in the presence of BFA. These results suggest that enhanced endocytosis, together with inhibited secretion, is responsible for the retarded growth of pollen tubes induced by BFA.

Acid Phosphatase↗

Essential role of the V-ATPase in male gametophyte development.

Intracellular pH homeostasis is a prerequisite for biological processes and requires the action of proton pumps. The vacuolar H(+)-ATPase (V-ATPase) is involved in regulating pH in endomembrane compartments of all eukaryotic cells. In plants, there is an additional endomembrane proton pump, H(+)-pyrophosphatase (H(+)-PPase). However, the relative roles of the two types of pumps in endomembrane acidification and energization of secondary active transport are unclear. Here, we show that a strong T-DNA insertion allele of VHA-A, the single copy gene encoding the catalytic subunit of the Arabidopsis V-ATPase, causes complete male and partial female gametophytic lethality. Severe changes in the morphology of Golgi stacks and Golgi-derived vesicles in male gametophytes are the first visible symptoms of cell degeneration leading to a failure to develop mature pollen. Similar effects on Golgi morphology were observed in pollen tubes when growth was blocked by Concanamycin A, a specific V-ATPase inhibitor. Taken together, our results suggests that V-ATPase function is essential for Golgi organization and development of the male gametophyte.

Arabidopsis↗

Response of pollen germination and tube growth to cadmium with special reference to low concentration exposure.

Cadmium is one of the most important heavy metal pollutants highly hazardous to plants. Pollen is considered to be more sensitive to pollutants than are vegetative parts of the plants. Five herb species were tested for responses in pollen germination and tube growth to Cd exposure in vitro. Pollen germination of all the species was inhibited at Cd concentrations of 2.51 microg/mL and higher, and tube growth was inhibited at concentrations of 1.58 microg/ml and higher. Cadmium, at low concentrations, stimulated pollen tube growth. The pollen response to Cd stress exhibited interspecies differences. Vicia angustifolia and V. tetrasperma were sensitive to Cd, and were inhibited in either pollen germination or tube growth by Cd at 0.01 microg/mL. Plantago depressa was less sensitive; pollen germination and tube growth were not inhibited until the Cd concentration reached 2.51 and 1.58 microg/mL, respectively, and its tube growth displayed two stimulatory peaks; the one that appeared at 1.00 microg/mL showed the highest tube length in all species tested. These results suggest that Cd, even at low concentrations, may adversely affect plant reproduction by inhibiting pollen germination and tube growth.

Cadmium↗

Determination of 2-chloroethanol in honey, beeswax, and pollen.

Procedures were developed and tested for the determination of residues of 2-chloroethanol (ethylene chlorohydrin, ECH) in honey, beeswax, and pollen. Recoveries of ECH from fortified samples averaged 91, 87, and 89%, respectively, for each substrate. The maximum amount of ECH found in substrate fumigated with ethylene oxide was 36 micrograms/g in honey, 124 micrograms/g in beeswax, and 132 micrograms/g in pollen. A tendency was noted for darker waxes, which contain larger amounts of naturally occurring chlorides than light-colored waxes, to contain the greater amounts of ECH. A gas-liquid chromatograph equipped with a Dohrmann halogen-specific detector was used for identification and quantitation.

Animals↗