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A novel class of MYB factors controls sperm-cell formation in plants.

In contrast to animals, the plant male germline is established after meiosis in distinctive haploid structures, termed pollen grains. The germline arises by a distinct asymmetric division of the meiotic products . The fates of the resulting vegetative and generative cells are distinct. In contrast to the larger vegetative cell, arrested in the G1 phase of the cell cycle, the smaller generative cell divides once to produce the two male gametes or sperm cells. Sperm cells are delivered to the female gametes by the pollen tube, which develops from the vegetative cell. In spite of recent efforts to understand pollen development , the molecular pathway controlling sperm-cell ontogenesis is unknown. Here, we present the isolation of DUO1, a novel R2R3 MYB gene of Arabidopsis, as the first gene shown to control male gamete formation in plants. DUO1 is specifically expressed in the male germline, and DUO1 protein accumulates in sperm-cell nuclei. Mutations in DUO1 produce a single larger diploid sperm cell unable to perform fertilization. DUO1 appears to be evolutionarily conserved in several plant species and defines a new subfamily of pollen-specific MYB genes.

Amino Acid Sequence↗

POLLEN GERMINATION AND TUBE GROWTH.

Many aspects of Angiosperm pollen germination and tube growth are discussed including mechanisms of dehydration and rehydration, in vitro germination, pollen coat compounds, the dynamic involvement of cytoskeletal elements (actin, microtubules), calcium ion fluxes, extracellular matrix elements (stylar arabinogalactan proteins), and control mechanisms of gene expression in dehydrating and germinating pollen. We focus on the recent developments in pollen biology that help us understand how the male gamete survives and accomplishes its successful delivery to the ovule of the sperm to effect sexual reproduction.

Journal Article↗

Pollen immunotherapy reduces the development of asthma in children with seasonal rhinoconjunctivitis (the PAT-study).

BACKGROUND: Children with allergic rhinitis are likely to develop asthma. OBJECTIVE: The purpose of this investigation was to determine whether specific immunotherapy can prevent the development of asthma and reduce bronchial hyperresponsiveness in children with seasonal allergic rhinoconjunctivitis. METHODS: From 6 pediatric allergy centers, 205 children aged 6 to 14 years (mean age, 10.7 years) with grass and/or birch pollen allergy but without any other clinically important allergy were randomized either to receive specific immunotherapy for 3 years or to an open control group. All subjects had moderate to severe hay fever symptoms, but at inclusion none reported asthma with need of daily treatment. Symptomatic treatment was limited to loratadine, levocabastine, sodium cromoglycate, and nasal budesonide. Asthma was evaluated clinically and by peak flow. Methacholine bronchial provocation tests were carried out during the season(s) and during the winter. RESULTS: Before the start of immunotherapy, 20% of the children had mild asthma symptoms during the pollen season(s). Among those without asthma, the actively treated children had significantly fewer asthma symptoms after 3 years as evaluated by clinical diagnosis (odds ratio, 2.52; P <.05). Methacholine bronchial provocation test results improved significant in the active group (P <.05). CONCLUSION: Immunotherapy can reduce the development of asthma in children with seasonal rhinoconjunctivitis.

Adolescent↗

Forecasting pollen pollution: correlation with floral development.

Variations in the time course of flowering and in its intensity were recorded for regions in Israel along with variable climatic conditions. Such variations had an effect on pollen release into the air and on pollen counts. Two approaches for forecasting allergenic pollen dispersion were compared: (1) a survey of floral development and flowering intensity and (2) a survey of airborne pollen. There was a positive correlation between the survey of flowering and airborne pollen counts, although pollen was occasionally found after the flowering season. Further, it predicted fairly accurately the beginning of the flowering time of each species in each regio, the expected flowering duration, and intensity. Conducting a survey of flowering is a simple, easy and inexpensive operation. It is highly recommended for regions with variable climates.

Air Pollution↗

Development and distribution of the major pollen allergen (Cry j I) in male flower buds of Japanese cedar (Cryptomeria japonica).

We investigated the production of the major pollen allergen [Cry j I] of Japanese cedar (Cj) in the course of male flower bud development. We found that most of the pollen was at the tetrad stage in early September, and they developed to the mature stage in mid-October (1987) or early November (1988). Large amounts of Cry j I seemed to be extractable to ABS solution from the mature stage pollen but not from the pollen at other stages, that is, the tetrad and immature stages. Mature pollen could be ruptured with ammonium bicarbonate buffer, but tetrad and immature pollen could not. By immunofluorescent technique, antigen Cry j I was detected in the pollen only at the mature stage of Cj pollen development. Therefore, we think that Cry j I is produced at the time of pollen maturation.

Allergens↗

New method for pollen identification by FT-IR spectroscopy.

A new methodology for identification of pollen was developed based on FT-IR spectroscopy. Pollen samples of twenty different plant species were collected and the diffuse reflectance infrared Fourier transform (DRIFTS) and KBr pellet spectra were recorded. Libraries of spectra were created. Spectra of unknown plant origin pollen were recorded and compared with those of the corresponding pollen library and the match value was measured automatically using the appropriate software (OMINC ver. 3.1). From the same pollen samples, microscopic slides were prepared and the photographs of the pollen grains were used as a second comparison method. Using light microscopy, the pollen identification is usually limited to the family or generic name, while FT-IR spectroscopy can distinguish species belonging to the same genus. This method is simple and fast, and when the DRIFTS technique is used the sample is not destroyed.

Algorithms↗

Microarray analysis of gene expression involved in anther development in rice (Oryza sativa L.).

In flowering plants, anthers bear male gametophytes whose development is regulated by the elaborate coordination of many genes. In addition, both gibberellic acid (GA3) and jasmonic acid (JA) play important roles in anther development and pollen fertility. To facilitate the analysis of anther development genes and how GA3 and JA regulate anther development, we performed microarray experiments using a 10-K cDNA microarray with probes derived from seedlings, meiotic anthers, mature anthers and GA3- or JA-treated suspension cells of rice. The expression level change of 2155 genes was significantly (by 2-fold or greater) detected in anthers compared with seedlings. Forty-seven genes, representing genes with potential function in cell cycle and cell structure regulation, hormone response, photosynthesis, stress resistance and metabolism, were differentially expressed in meiotic and mature anthers. Moreover, 314 genes responded to either GA3 or JA treatment, and 24 GA3- and 82 JA-responsive genes showed significant changes in expression between meiosis and the mature anther stages. RT-PCR demonstrated that gene y656d05 was not only highly expressed in meiotic anthers but also induced by GA3. Strong RNA signals of y656d05 were detected in pollen mother cells and tapetum in in situ hybridization. Further characterization of these candidate genes can contribute to the understanding of the molecular mechanism of anther development and the involvement of JA and GA3 signals in the control of anther development in rice.

Cells, Cultured↗

Development of pollinosis after ingestion of pollen.

We describe the case of a patient who developed distinct allergic reactions after the ingestion of commercially available "pollen food", while according to the history there had been no previous complaints to aero-pollen. By both in vivo as well as in vitro testing a simultaneous sensitivity to grass, tree and weed pollen, as well as to the main components of "pollen food" rock rose and viper buglows pollen could be proven. With the help of RAST-inhibition, cross-reactions between the different pollen allergens were traceable. In the pollen season after the allergic incident, the patient developed pollinosis symptoms for the first time, which intensified in the following season. On these grounds, we believe that (1) the repeated ingestion of "pollen food" resulted in a sensitization against its allergens and, due to the cross-reactions, against aero pollen allergens as well and that (2) an "allergic breakthrough" caused the manifestation of pollinosis symptoms.

Adult↗

Programmed cell death progressively models the development of anther sporophytic tissues from the tapetum and is triggered in pollen grains during maturation.

To characterize the spatial and temporal occurrence of programmed cell death (PCD) in Lilium anther tissues, we used both microscopical and molecular markers of apoptosis for developmental stages from meiosis to pollen release. The first hallmarks of PCD include cell condensation and shrinkage of the cytoplasm, separation of chromatin into delineated masses, and DNA fragmentation in the tapetum as early as the premeiosis stage. PCD then extended to other anther sporophytic tissues, leading to anther dehiscence. Although the PCD clearly affected the endothecium and the epidermis, these two cell layers remained alive until anther dehiscence. In pollen, no sign of PCD was found until pollen mitosis I, after what apoptotic features developed progressively in the vegetative cell. In addition, DNA ladders were detected in all sporophytic tissues and cell types throughout pollen development, whereas in the male gametophyte DNA ladders were only detected during pollen maturation. Our data suggest that PCD is a progressive and active process affecting all the anther tissues, first being triggered in the tapetum.

Apoptosis↗

Autoradiographs of pollen tube nuclei with calcium-45.

Autoradiography with Ca(45) has been used to obtain information about the relation between calcium and chromosomes. Labelled pollen from the Easter lily, Lilium longiflorum, was allowed to develop into pollen tubes between 5 and 6 cm. long in the styles of non-radioactive flowers. All of the nuclei, namely the tube nucleus and the two sperm nuclei, retain Ca(45) after this period of growth and development. Since the two sperm nuclei have formed during this interval by the mitotic division of the generative nucleus and growth of the tube has occurred under the influence of the tube nucleus, it is inferred that the calcium was bound in a stable nuclear component, the chromosomes.

Autoradiography↗

The S-locus specific glycoproteins of Brassica accumulate in the cell wall of developing stigma papillae.

Self-incompatibility in Brassica oleracea is now viewed as a cellular interaction between pollen and the papillar cells of the stigma surface. In this species, the inhibition of self-pollen occurs at the stigma surface under the influence of S-locus specific glycoproteins (SLSG). We used antibodies specific for a protein epitope of SLSG to study the subcellular distribution of these molecules in the stigmatic papillae. The antibodies have uncovered an interesting epitope polymorphism in SLSG encoded by subsets of S-alleles, thus providing us with useful genetic controls to directly verify the specificity of the immunolocalization data. Examination of thin sections of Brassica stigmas following indirect immunogold labeling showed that SLSG accumulate in the papillar cell wall, at the site where inhibition of self-pollen tube development has been shown to occur. In addition, the absence of gold particles over the papillar cell walls in the immature stigmas of very young buds, and the intense labeling of these walls in the stigmas of mature buds and open flowers, correlates well with the acquisition of the self-incompatibility response by the developing stigma.

Antibodies, Monoclonal↗

A pollen-specific and desiccation-associated transcript in Lilium longiflorum during development and stress.

A cDNA clone encoding a desiccation-induced protein (LLA23) has been isolated from the mature pollen of a Lilium longiflorum cDNA library. Sequence analysis revealed significant similarity between the predicted LLA23 polypeptide, particularly at the C-terminal half of the sequence and a group of water-deficit/ripening-induced proteins. The expression of LLA23 gene is pollen-specific and the transcript accumulates only at the later stage of pollen maturation prior to anthesis. Premature drying of developing pollen confirmed that the accumulation of LLA23 transcripts was associated with desiccation. The LLA23 proteins decreased their levels when pollen/pollen tubes grew in the germination buffer. Treatments of pollen with abscisic acid (ABA) and polyethylene glycol (PEG)-8000 during germination greatly retarded the disappearance of LLA23 proteins and mRNAs. The LLA23 transcripts decreased their levels in pollen tubes grown both in vitro and in vivo, but the disappearance of LLA23 transcripts in tube cells grown in vivo was slower than those grown in vitro. In situ localization using anti-chicken immunoglobulin G conjugated with gold particles confirmed that LLA23 was located in the cytoplasm of pollen grains. The protective function of the desiccation-related proteins in the cytoplasm of pollen grains is proposed.

Amino Acid Sequence↗

Recognition of pollen and other particulate aeroantigens by immunoblot microscopy.

Most grass-pollen types appear identical by normal light microscopy. Restricted antigenic cross-reactivity of Cynodon dactylon (Bermuda grass) pollen allowed development of a new method to identify antigens associated with grass-pollen grains. Pollen applied to the surface of an adhesive tape was blotted onto nitrocellulose, and the blots were identified by anti-Bermuda-grass antibodies, second antibody, and fluorescence microscopy. Of the 44 species of grass pollen studied for specificity of the method, the only species to demonstrate uniformly bright staining were Cynodon dactylon, Elymus triticoides, Elymus cinereus, and Koeleria cristata. Thirty-one species were negative, and nine other species demonstrated occasional brightly fluorescent spots, suggesting contamination. The immunoblotting method was used to study Tucson air collected continuously by a Burkard pollen and spore trap throughout April 1986. Each 2-hour transect of the adhesive tape from the trap was examined by immunoblotting and by normal light microscopy to compare antigen particle counts with grass-pollen counts. The mean antigen-particle concentration, 52.8/m3 of air, was higher than the mean grass-pollen concentration, 21.9/m3 of air, suggesting presence of amorphous Bermuda-grass antigens in air samples. Antigen-particle concentration, not grass-pollen concentration, correlated significantly with wind velocity, temperature, and time of day.

Air↗

Determination of patterning in the pollen wall of Lilium henryi.

Centrifugation of living pollen mother cells of Lilium henryi has permitted the modifications of complex walls developed on the four daughter microspores. This experimental approach has not only provided an insight into the mechanism by which patterning is established, but has also identified the stages in cell development when wall determinants are present. These determinants can be first detected late in the meiotic prophase, but are not necessarily active at the cell surface at this early stage. The fact that treatment during cytokinesis may affect subsequent wall formation, combined with the obvious interference of the colpus with the spore patterning, indicates pattern determination to be a lengthy process, commencing during late prophase and reaching completion in the young tetrad. The study of walls formed on enucleate cell fragments suggests that neither the spindle nor the cytoskeleton is intimately involved in formation of the basic reticulate patterning. It is proposed that material is inserted into the mosaic of the plasma membrane and is reorganized biophysically to form contiguous 'plates', which then modify the properties of the membrane in such a way that the cytoplasmic protrusions that constitute the first stages of wall formation only develop in the interstices between them. Changes in the plasma membrane have been detected over this period and it seems that a population of coated vesicles is closely involved in these events. Examination of the cytoskeleton of the young spore suggests that microtubules play a critical rôle in the positioning of the colpus, and reveals ways by which only small changes in this skeleton could give rise to multicolporate grains.

Cell Wall↗

The DAL10 gene from Norway spruce (Picea abies) belongs to a potentially gymnosperm-specific subclass of MADS-box genes and is specifically active in seed cones and pollen cones.

Transcription factors encoded by different members of the MADS-box gene family have evolved central roles in the regulation of reproductive organ development in the flowering plants, the angiosperms. Development of the stamens and carpels, the pollen- and seed-bearing organs, involves the B- and C-organ-identity MADS-box genes. B- and C-type gene orthologs with activities specifically in developing pollen- and seed-bearing organs are also present in the distantly related gymnosperms: the conifers and the gnetophytes. We now report on the characterization of DAL10, a novel MADS-box gene from the conifer Norway spruce, which unlike the B- and C-type conifer genes shows no distinct orthology relationship to any angiosperm gene or clade in phylogenetic analyses. Like the B- and C-type genes, it is active specifically in developing pollen cones and seed cones. In situ RNA localization experiments show DAL10 to be expressed in the cone axis, which carry the microsporophylls of the young pollen cone. In contrast, in the seed cone it is expressed both in the cone axis and in the bracts, which subtend the ovuliferous scales. Expression data and the phenotype of transgenic Arabidopsis plants expressing DAL10 suggest that the gene may act upstream to or in concert with the B- and C-type genes in the establishment of reproductive identity of developing cones.

Base Sequence↗

Morphological, cytological and BSA-based testing on limited segregation population AFLPs.

Cytoplasmic male sterility (cms) in rye (Secale cereale L.), especially cytoplasma PAMPA, is used commercially in hybrid breeding programmes. The development of molecular markers that are tightly linked to the numerous genes coding for pollen fertility is expected to have great impact in the field. Morphological and cytological analyses of plants from a three-way cross C394: [(S67P/94 x S38/94) x CHD296] indicated the presence of at least several genes acting at different stages of pollen grain development, and proved the concurrence of both approaches in plant classification. The AFLP technique combined with the Bulk Segregant Analysis (BSA) were applied to identify DNA fragments linked to the genes of interest. All the 256 possible primer pair combinations based on the MseI and EcoRI restriction sites generated distinct band patterns allowing the identification of 31143 DNA fragments, visualised using the isotopic method. On average, any given primer combination generated 122 fragments. Among 1111 and 431 potential genetic markers respectively identified in the restorer form and the maternal lines, 775 and 295 were present in the F2 population. These numbers were then reduced to 109 and 51. The identified DNA fragments were tested on a limited segregating population, C394-F2, in order to eliminate false signals and to select markers for a future marker-assisted selection programme. Twenty-five markers were selected. Four of these markers were not identified via the BSA approach, indicating that if a highly polymorphic component is used for a cross, or a polygenic trait is studied, then the use of a limited population may be required.

Breeding↗

Assessing hybrid sterility in Oryza glaberrima x O. sativa hybrid progenies by PCR marker analysis and crossing with wide compatibility varieties.

Interspecific crossing of the African indigenous rice Oryza glaberrima with Oryza sativa cultivars is hindered by crossing barriers causing 100% spikelet sterility in F(1) hybrids. Since hybrids are partially female fertile, fertility can be restored by back crossing (BC) to a recurrent male parent. Distinct genetic models on spikelet sterility have been developed predicting, e.g., the existence of a gamete eliminator and/or a pollen killer. Linkage of sterility to the waxy starch synthase gene and the chromogen gene C, both located on chromosome 6, have been demonstrated. We selected a segregating BC(2)F(3) population of semi-sterile O. glaberrima x O. sativa indica hybrid progenies for analyses with PCR markers located at the respective chromosome-6 region. These analyses revealed that semi-sterile plants were heterozygous for a marker (OSR25) located in the waxy promoter, whereas fertile progenies were homozygous for the O. glaberrima allele. Adjacent markers showed no linkage to spikelet sterility. Semi-sterility of hybrid progenies was maintained at least until the F(4) progeny generation, suggesting the existence of a pollen killer in this plant material. Monitoring of reproductive plant development showed that spikelet sterility was at least partially due to an arrest of pollen development at the microspore stage. In order to address the question whether genes responsible for F(1) sterility in intraspecific hybrids ( O. sativa indica x japonica) also cause spikelet sterility in interspecific hybrids, crossings with wide compatibility varieties (WCV) were performed. WCV accessions possess "neutral" S-loci ( S(n)) improving fertility in intraspecific hybrids. This experiment showed that the tested S(n)-loci had no fertility restoring effect in F(1) interspecific hybrids. Pollen development was completely arrested at the microspore stage and grains were never obtained after selfing. This suggests that distinct or additional S-loci are responsible for sterility of O. glaberrima x O. sativa hybrids.

Chimera↗