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PubMed1999

Pollen and anther development in Nelumbo (Nelumbonaceae).

The Nelumbonaceae are a small family of aquatic angiosperms comprising Nelumbo nucifera and Nelumbo lutea. Historically, the genus has been considered to be closely related to Nymphaeales, however new systematic work has allied Nelumbo with lower eudicots, particularly Platanus. In recent years, studies of pollen development have contributed greatly to the understanding of phylogenetic relationships, but little has been known about these events in Nelumbo. In this paper, pollen and anther development are morphologically described for the first time in N. lutea. A comprehensive ontogenetic sequence is documented, including the sporogenous tissue, microspore mother cell, tetrad, free spore, and mature pollen grain stages. The deposition of a microspore mother cell coat and callose wall, the co-occurrence of both tetrahedral and tetragonal tetrads, the formation of a primexine in tetrads, and primexine persistence into the late free spore stage are shown. The majority of exine development occurs during the free spore stage with the deposition of a tectate-columellate ectexine, a lamellate endexine, and an unusual granular layer below and intermixed with the endexine lamellae. A two-layered intine forms rapidly during the earliest mature pollen stage. Major events of anther development documented include the degradation of a secretory-type tapetum during the free spore stage and the rapid formation of U-shaped endothecial thickenings in the mature pollen grain stage. The majority of mature pollen grains are tricolpate, however less common monosulcate and diaperturate grains also develop. Co-occurring aperture types in Nelumbo have been suggested to be an important transition in angiosperm aperture number. However, aperture variability in Nelumbo may be correlated with the lateness of aperture ontogeny in the genus, which occurs in the early free spore stage. This character, as well as other details of pollen and anther ontogeny in Nelumbo, are compared to those of Nymphaeales and Platanus in an effort to provide additional insight into systematic and phylogenetic relationships. Although Nelumbo is similar to both groups in several characters, the ontogenetic sequence of the genus is different in many ways.

Journal Article↗
PubMed1997

Bnm1, a Brassica pollen-specific gene.

cDNA and genomic clones of a new pollen-specific gene, Bnm1, have been isolated from Brassica napus cv. Topas. The gene contains an open reading frame of 546 bp and a single intron of 362 bp. A comparison of the deduced amino acid sequence with sequences in data banks did not show similarity with known proteins. Northern blot analysis of developing pollen showed that Bnm1 mRNA was first detected in bicellular pollen and accumulated to higher levels in tricellular pollen. Bnm1 mRNA was not detected in leaves, stems, roots, pistils, seeds or pollen-derived embryos. RNA in situ hybridization of whole flower buds confirmed that Bnm1 was pollen-specific and expressed late in development. A promoter fragment of the Bnm1 gene fused to the gusA reporter gene yielded similar patterns of tissue specificity and developmental regulation in transgenic B. napus cv. Westar plants; however, the promoter was also active during the early stages of pollen development. The Bnm1 gene, cloned in this study, was derived from the A genome of the allotetraploid species B. napus (AACC). Southern blot analysis indicated that sequences similar to the Bnm1 gene were found in both A and C Brassica genomes. Related sequences were found in all 10 members of the Brassiceae tribe examined, but were not present in all tribes of the Brassicaceae family.

Brassica↗
PubMed1989

Gametophytic and sporophytic expression of anther-specific genes in developing tomato anthers.

The tissue localization of transcripts corresponding to five anther-specific cDNA clones isolated from tomato was determined. Transcripts specified by three of the cDNA clones were first detectable in anthers containing mitotic-stage gametophytes and were localized to the gametophyte. Transcripts specified by the two other cDNA clones were not detectable until anthers had reached a later developmental stage; these transcripts were also localized to the (now bicellular) gametophytes. Transcript levels for all of the cDNAs increased during gametogenesis and reached maximal levels in mature pollen grains. These mRNAs persisted in in vitro-grown pollen tubes, concentrating toward the tips of the growing tubes. At flower maturity, transcripts specified by each of the cDNAs were also detected in the epidermal and endothecial cell layers of the anther wall. The spatial distribution of transcripts in the anther wall was confined to that region of the anther that surrounds the locule. Transcripts were not detected in the sterile tip of the anther or in the filament. mRNA levels for these cDNA clones were markedly reduced in the anthers of several independent male-sterile mutants of tomato. Our results provide evidence that these anther-specific cDNAs represent genes expressed in both the gametophytic and sporophytic phases of the plant life cycle. The patterns of mRNA accumulation observed support the hypothesis that the proteins encoded by these genes function during pollen development and pollen tube growth.

DNA↗
PubMed2006

Integrating membrane transport with male gametophyte development and function through transcriptomics.

Male fertility depends on the proper development of the male gametophyte, successful pollen germination, tube growth, and delivery of the sperm cells to the ovule. Previous studies have shown that nutrients like boron, and ion gradients or currents of Ca2+, H+, and K+ are critical for pollen tube growth. However, the molecular identities of transporters mediating these fluxes are mostly unknown. As a first step to integrate transport with pollen development and function, a genome-wide analysis of transporter genes expressed in the male gametophyte at four developmental stages was conducted. Approximately 1,269 genes encoding classified transporters were collected from the Arabidopsis (Arabidopsis thaliana) genome. Of 757 transporter genes expressed in pollen, 16% or 124 genes, including AHA6, CNGC18, TIP1.3, and CHX08, are specifically or preferentially expressed relative to sporophytic tissues. Some genes are highly expressed in microspores and bicellular pollen (COPT3, STP2, OPT9), while others are activated only in tricellular or mature pollen (STP11, LHT7). Analyses of entire gene families showed that a subset of genes, including those expressed in sporophytic tissues, was developmentally regulated during pollen maturation. Early and late expression patterns revealed by transcriptome analysis are supported by promoter::beta-glucuronidase analyses of CHX genes and by other methods. Recent genetic studies based on a few transporters, including plasma membrane H+ pump AHA3, Ca2+ pump ACA9, and K+ channel SPIK, further support the expression patterns and the inferred functions revealed by our analyses. Thus, revealing the distinct expression patterns of specific transporters and unknown polytopic proteins during microgametogenesis provides new insights for strategic mutant analyses necessary to integrate the roles of transporters and potential receptors with male gametophyte development.

Arabidopsis↗
PubMed1981

Microspore and microgametophyte development in relation to biological activity of environmental pollutants.

The pattern of synthesis of nucleic acids and proteins in the development of the microspore and later during pollen germination and tube growth is discussed. In the pollen grain at the time of anthesis all the proteins that are required for germination and early tube growth are either already present, or if new proteins are synthesized, the messenger RNAs for their synthesis already exist in the ungerminated pollen grain. In addition, similar proteins are synthesized on new mRNAs during germination and pollen tube growth as are synthesized on premade mRNAs. The genetic program during at least the latter part of pollen maturation prior to anthesis is thus the same as that during pollen germination and tube growth. Accordingly, one cannot treat mature pollen with mutagens and expect to be able to score the pollen tubes for mutant proteins. Treatment with mutagenic compounds would have to be during pollen maturation in the anther, before the transcription for the proteins required during germination and pollen tube growth has occurred. Available evidence indicates that this is very early in pollen development, possibly soon after meiosis. The value of pollen tubes to monitor for chemicals that affect the intracellular motility systems of organisms is also discussed.

Cytoplasmic Streaming↗
PubMed1996

Complexity and Genetic Variability of Heat-Shock Protein Expression in Isolated Maize Microspores.

The expression of heat-shock proteins (HSPs) in isolated maize (Zea mays L.) microspores has been investigated using high-resolution two-dimensional electrophoresis coupled to immunodetection and fluorography of in vivo synthesized proteins. To this end, homogeneous and viable populations of microspores have been purified in sufficient amounts for molecular analysis from plants grown in controlled conditions. Appropriate conditions for thermal stress application have been defined. The analysis revealed that isolated microspores from maize display a classical heat-shock response characterized by the repression of the normal protein synthesis and the expression of a set of HSPs. A high complexity of the response was demonstrated, with numerous different HSPs being resolved in each known major HSP molecular weight class. However, the extent of this heat-shock response is limited in that some of these HSPs do not accumulate at high levels following temperature elevation. Comparative analysis of the heat-shock responses of microspores isolated from five genotypes demonstrated high levels of genetic variability. Furthermore, many HSPs were detected in microspores at control temperature, indicating a possible involvement of these proteins in pollen development at stages close to first pollen mitosis.

Journal Article↗
PubMed1997

Asymmetric cell division and cell fate in plants.

A variety of approaches has recently been employed to investigate how sister cells adopt distinct fates following asymmetric divisions during plant development. Surgical and drug studies have been used to analyze asymmetric divisions during both early embryogenesis in brown algae and pollen development in tobacco. Genetic screens have been used to identify genes in Arabidopsis thaliana that are required for specific asymmetric cell divisions during pollen and root development. These studies indicate that cell polarity and division orientation are closely tied to the process of cell fate specification, and suggest that differential inheritance of determinants and positional information may both be involved in the specification of cell fates following asymmetric cell division.

Cell Division↗
PubMed2006

Pollen-specific pectin methylesterase involved in pollen tube growth.

Pollen tube elongation in the pistil is a crucial step in the sexual reproduction of plants. Because the wall of the pollen tube tip is composed of a single layer of pectin and, unlike most other plant cell walls, does not contain cellulose or callose, pectin methylesterases (PMEs) likely play a central role in the pollen tube growth and determination of pollen tube morphology. Thus, the functional studies of pollen-specific PMEs, which are still in their infancy, are important for understanding the pollen development. We identified a new Arabidopsis pollen-specific PME, AtPPME1, characterized its native expression pattern, and used reverse genetics to demonstrate its involvement in determination of the shape of the pollen tube and the rate of its elongation.

Arabidopsis↗
PubMed1999

LeProT1, a transporter for proline, glycine betaine, and gamma-amino butyric acid in tomato pollen.

During maturation, pollen undergoes a period of dehydration accompanied by the accumulation of compatible solutes. Solute import across the pollen plasma membrane, which occurs via proteinaceous transporters, is required to support pollen development and also for subsequent germination and pollen tube growth. Analysis of the free amino acid composition of various tissues in tomato revealed that the proline content in flowers was 60 times higher than in any other organ analyzed. Within the floral organs, proline was confined predominantly to pollen, where it represented >70% of total free amino acids. Uptake experiments demonstrated that mature as well as germinated pollen rapidly take up proline. To identify proline transporters in tomato pollen, we isolated genes homologous to Arabidopsis proline transporters. LeProT1 was specifically expressed both in mature and germinating pollen, as demonstrated by RNA in situ hybridization. Expression in a yeast mutant demonstrated that LeProT1 transports proline and gamma-amino butyric acid with low affinity and glycine betaine with high affinity. Direct uptake and competition studies demonstrate that LeProT1 constitutes a general transporter for compatible solutes.

Amino Acid Sequence↗
PubMed2002

Airborne pollen: a brief life.

The transfer of pollen, whether it is transported by insects or carried by the wind, from floral anther to recipient stigma is the critical reproductive event among higher plants. In this scenario, the pollen grain functions as a fully constituted life-cycle stage, capable of growth (albeit limited) and delivery of gametes. Pollen is prepared for this role by an intricate developmental process with dual sources of structural elements and chemical constituents, including allergens. The resulting complexity relates, at least in part, to the requirements of an unforgiving recognition process at stigmatic surfaces and of active growth before the achievement of gametic union. Recently, the basic participants in pollen-stigma interactions have been defined, and they provide a striking counterpoint to human histocompatibility concerns. Pollen development offers a useful tableau in terms of which to reexamine forces affecting pollen prevalence and their interactions. Development also provides clues to the sources and significance of more minute bioaerosols now known to carry pollen allergens.

Air Pollutants↗
PubMed1997

Expression of a wheat ADP-glucose pyrophosphorylase gene during development of normal and water-stress-affected anthers.

In wheat (Triticum aestivum L.), water deficit during meiosis in the microspore mother cells (MMCs) induces pollen abortion, resulting in the failure of fertilization and a reduction in grain set. In stressed plants, meiosis in MMCs proceeds normally but subsequent pollen development is arrested. Unlike normal pollen grains, which accumulate starch during the late maturation phase, stress-affected anthers contain pollen grains with little or no starch. Stress also alters the normal distribution of starch in the anther wall and connective tissue. To determine how starch biosynthesis is regulated within the developing anthers of stressed plants, we studied the expression of ADP-glucose pyrophosphorylase (AGP), which catalyzes the rate limiting step of starch biosynthesis. Two partial-length cDNAs corresponding to the large subunit of AGP were amplified by RT-PCR from anther RNA, and used as probes to monitor AGP expression in developing anthers of normal and water-stressed plants. These clones, WAL1 and WAL2, had identical deduced amino acid sequences and shared 96% sequence identity at the nucleic acid level. In normal anthers, AGP expression was biphasic, indicating that AGP expression is required for starch biosynthesis both during meiosis and later during pollen maturation. AGP expression in stressed anthers was not affected during the first phase of starch accumulation, but was strongly inhibited during the second phase. We conclude from these results that the reduced starch deposition later in the development of stressed pollen could be the result of a lower expression of AGP. However, this inhibition of AGP expression is unlikely to be the primary cause of male sterility because anatomical symptoms of pollen abortion are observed prior to the time when AGP expression is inhibited.

Amino Acid Sequence↗
PubMed2026

A single-nucleus transcriptome atlas of soybean anthers.

Anther development is crucial for plant sexual reproduction. However, a high-resolution, cell-type-specific transcriptomic atlas of this process is lacking for the legume crop soybean (Glycine max). Here, we construct a comprehensive transcriptional atlas of developing soybean anthers using single-nucleus RNA sequencing (snRNA-seq). We identify and characterize nine distinct cell types spanning both somatic and reproductive lineages. Our analysis reveals robust transcriptional continuity across anther developmental stages and dynamic reprogramming during key transitions. Notably, the shift from diploid meiocytes to haploid unicellular microspores is marked by the induction of previously inactive genes, despite an overall reduction in transcript abundance. Subsequently, within bicellular microspores, generative and vegetative cell lineages exhibit sharply divergent transcriptional programs: generative cells specialize in mRNA export and turnover, whereas vegetative cells up-regulate translational machinery. Evolutionary analysis further indicates that generative-cell-specific genes are subject to more relaxed purifying selection compared to those specific to vegetative cells. Functional validation using mutants generated by CRISPR/Cas9-mediated genome editing and EMS mutagenesis reveals the essential roles of OSD1A and PKSA in pollen development and fertility. This high-resolution atlas provides fundamental insights into the transcriptional regulation of soybean anther development and serves as a valuable resource for manipulating male fertility to advance hybrid breeding programs. The data are available at https://databases.genedenovo.com/pollen.

Glycine max↗
PubMed1992

Identification of Embryoid-Abundant Genes That Are Temporally Expressed during Pollen Embryogenesis in Wheat Anther Cultures.

Uninucleate microspores in anther cultures of bread wheat (Triticum aestivum cv Pavon) are capable of producing haploid pollen embryoids and plants. To gain an understanding of this alternate pathway of pollen development, we constructed a cDNA library to young pollen embryoids, isolated embryoid-specific genes, and analyzed their expression patterns during morphogenesis. Two embryoid-abundant clones, pEMB4 and 94, were expressed very early during culture, suggesting that these genes are associated with development and are not simply expressed as a consequence of differentiation. The accumulation patterns of five cloned mRNAs may indicate the activation of specific genes associated with the major morphological and physiological activities connected with the differentiation of embryoids in vitro. These results suggest that embryoid-abundant gene expression is causally related to this pathway because gene expression is spatially and temporally specific and is not observed when microspores are cultured under noninductive conditions.

Journal Article↗
PubMed2001

The Arabidopsis MALE STERILITY1 (MS1) gene is a transcriptional regulator of male gametogenesis, with homology to the PHD-finger family of transcription factors.

We report here the molecular characterisation of the Arabidopsis MALE STERILITY1 gene, which is a critical sporophytic controlling factor for anther and pollen development. Homozygous ms1 mutants do not produce viable pollen, but are otherwise phenotypically normal. Degeneration of pollen occurs soon after microspore release from the tetrads, at which time the tapetum also appears abnormally vacuolated. The MS1 gene is expressed at low levels in anthers from closed buds, with expression in the tapetum at the stage of microspore release. No expression is seen in open flowers. The deduced MS1 protein sequence shows strong homology to the PHD-finger motif found in known transcription factors from humans, yeast and higher plants. Six alleles of ms1 have been identified; all result in premature termination of the MS1 protein and loss of the PHD-finger motif. MS1 is likely to play a key role in regulating transcription during specific stages of male gametogenesis and anther development. As such, MS1 provides a valuable tool for the manipulation of male sterility in higher plants.

Amino Acid Motifs↗
PubMed1981

Tradescantia micronucleus bioassay and pollen tube chromatid aberration test for in situ monitoring and mutagen screening.

The meiotic pollen mother cells (PMC) of Tradescantia (spiderwort) are highly synchronized in their prophase I and tetrad stages. Chromosomes of this stage are sensitive to physical or chemical mutagens. Thus high frequency of acentric fragments or sticky chromosomes can be induced with very low level of mutagens. These induced chromosome aberrations become micronuclei (MCN) in the synchronized tetrads and they can be easily identified and scored. Based upon these features, the Tradescantia micronucleus bioassay was established. This bioassay involves the exposure of PMC in the young inflorescences of the plant cuttings to gaseous agents through diffusion, to liquid agent through absorption and dialysis from the stem to flower buds, or to radiation. The exposed samples are fixed in aceto-alcohol (1:3) and prepared into microslides by using the aceto-carmine squash method. Frequencies of MCN in a large population of synchronized tetrads are the indications of genetic damage caused by the agents. Mature pollen grains of Tradescantia are free cells which can be cultured in lactose-agar medium. The generative cells in the cultured pollen tubes can carry out mitosis similar to the in vivo condition. The G2 interphase chromosomes of pollen mitosis are highly sensitive to gaseous or liquid chemicals and radiation. Treatments can be applied to these mitotic generative cells of the mature pollen or the mitotic generative nuclei of the developing pollen tube. The mitotic chromosomes of the generative cells are allowed to proceed through mitosis in the culture medium and slides are prepared for metaphase figures. Frequencies of various types of chromatid aberrations can be scored and used as the indices of genetic damage.

Cell Nucleus↗
PubMed1994

Characterization of a pollen-expressed gene encoding a putative pectin esterase of Petunia inflata.

From a pollen tube cDNA library of Petunia inflata, we isolated cDNA clones encoding a protein, PPE1, which exhibits sequence similarity with plant, bacterial, and fungal pectin esterases. Genomic clones containing the PPE1 gene were isolated using cDNA for PPE1 as a probe, and comparison of the cDNA and genomic sequences revealed the presence of a single intron in the PPE1 gene. During pollen development, PPE1 mRNA was first detected in anthers containing uninucleate microspores; it reached the highest level in mature pollen and persisted at a high level in in vitro germinated pollen tubes. The observed expression pattern of the PPE1 gene suggests that its product may play a role in pollen germination and/or tube growth.

Amino Acid Sequence↗
PubMed1999

Isolation of ethyl methanesulfonate-induced gametophytic mutants in Arabidopsis thaliana by a segregation distortion assay using the multimarker chromosome 1.

The life cycle of plants comprises two alternating generations, the diploid sporophyte (spore-bearing plant) and the haploid gametophyte (gamete-bearing plant). In contrast to animals, the postmeiotic cells give rise to haploid organisms whose function is to produce the gametes and to mediate fertilization. Analysis of gametophyte development and function has been hampered by the difficulty of identifying haplo-phase-specific mutants in conventional mutagenesis screens. Here we use a genetic strategy that is based on segregation distortion of nearby visible markers to screen for EMS-induced gametophytic mutants in Arabidopsis thaliana. Using the multiple marker chromosome mm1 we have isolated seven lines that displayed an altered segregation of markers. Reciprocal backcrosses of these lines showed a marked reduction of the transmission of the male and/or female gametes. Phenotypic analysis revealed that different aspects of either gametophytic development or function were affected. Three male gametophytic lines showed specific arrests during pollen development. One male gametophytic line was specifically defective in pollen tube elongation. Three gametophytic lines showed variable defects in both male and female gametophytic development.

Arabidopsis↗
PubMed2002

Natural history of hay fever and pollen sensitization, and doctors' diagnosis of hay fever and pollen asthma in German schoolchildren.

BACKGROUND: In order to prevent pollen asthma by immunotherapy it is mandatory to know the best time to initiate it. Children with hay fever complaints are at considerable risk of developing pollen asthma. Population-based data on their natural history is urgently needed. METHODS: A longitudinal cohort study was conducted over four years in six rural towns in Baden-Württemberg, Germany. A questionnaire with questions taken from the International Study of Asthma and Allergies in childhood (ISAAC) was filled in every spring and autumn. Hay fever complaints, asthma defining symptoms and new doctors' diagnosis of hay fever and asthma were recorded. Additionally a skin prick test with pollen allergens was performed every autumn. RESULTS: In 1996, 19.7% of 1101 elementary school children (age: 8.1-9.9 years (5-95%)) were found to be sensitized to pollen and 8.7% had already been diagnosed as having hay fever. In a pooled analysis of 2478 children-summers, children with positive pollen sensitization had a significantly higher risk of developing hay fever symptoms (2.63; 2.17-3.10 odds ratio (OR); 95% confidence interval (CI)) and of being diagnosed as suffering from hay fever (7.88; 4.70-13.20). Furthermore, although their OR for the development of asthma symptoms during the pollen season was 3.88 (2.48-6.07 CI), it was only 0.69 (0.24-2.01 CI) for doctors' diagnosis of pollen asthma. CONCLUSION: Children of elementary school age with pollen sensitization and a history of hay fever are at considerable risk of getting pollen asthma, but they are not quickly diagnosed as such. Specific immunotherapy might be a means of preventing asthma completely in such a situation. Our data helps to estimate the sample size for intervention studies of this kind.

Air Pollutants↗