Cytoplasmic DNA apportionment and plastid differentiation during male gametophyte development in Pelargonium zonale

In the male gametophyte of Pelargonium zonale, generative and sperm cells contain cytoplasmic DNA in high density compared to vegetative cells. Cytoplasmic DNA was examined using the DNA fluorochrome DAPI (4'6-diamidino-2-phenylindole) and observed with epifluorescence and electron microscopy. The microspore cell contains a prominent central vacuole before mitosis; mitochondria and plastids are randomly distributed throughout the cytoplasm. Following the first pollen grain mitosis, neither the vegetative cell nor the early generative cell display a distributional difference in cytoplasmic DNA, nor is there in organelle content at this stage. During the maturation of the male gametophyte, however, a significant discrepancy in plastid abundance develops. Plastids in the generative cell return to proplastids and do not contain large starch grains, while those in the vegetative cell develop starch grains and differentiate into large amyloplasts. Plastid nucleoids in generative and sperm cells in a mature male gametophyte are easily discriminated after DAPI staining due to their compactness, while those in vegetative cells stained only weakly. The utility of the hydrophilic, non-autofluorescent resin Technovit 7100 in observing DAPI fluorescence is also demonstrated.     

Establishment of the male germline and sperm cell movement during pollen germination and tube growth in maize

Two sperm cells are required to achieve double fertilization in flowering plants (angiosperms). In contrast to animals and lower plants such as mosses and ferns, sperm cells of flowering plants (angiosperms) are immobile and are transported to the female gametes (egg and central cell) via the pollen tube. The two sperm cells arise from the generative pollen cell either within the pollen grain or after germination inside the pollen tube. While pollen tube growth and sperm behavior has been intensively investigated in model plant species such as tobacco and lily, little is know about sperm dynamics and behavior during pollen germination, tube growth and sperm release in grasses. In the March issue of Journal of Experimental Botany, we have reported about the sporophytic and gametophytic control of pollen tube germination, growth and guidance in maize.1 Five progamic phases were distinguished involving various prezygotic crossing barriers before sperm cell delivery inside the female gametophyte takes place. Using live cell imaging and a generative cell-specific promoter driving α-tubulin-YFP expression in the male germline, we report here the formation of the male germline inside the pollen grain and the sperm behaviour during pollen germination and their movement dynamics during tube growth in maize.Key words: male gametophyte, generative cell, sperm, pollen tube, tubulin, fertilization, maize     

DEVELOPMENT OF THE POLLEN TUBE OF ZAMIA FURFURACEA (ZAMIACEAE) AND ITS EVOLUTIONARY IMPLICATIONS

Compared with pollen tubes of conifers, gnetophytes, and angiosperms, the pollen tube of cycads is exclusively a vegetative structure, uninvolved with the siphonogamous conduction of sperm to an egg. The cycad pollen tube appears to function primarily to obtain nutrients for the extensive growth and development of the male gametophyte. Previous workers have suggested that, similar to an haustorial fungus, the cycad pollen tube penetrates the reproductive tissues of the sporophyte by enzymatically destroying nucellar cells. These earlier studies did not document the precise structural relationship between the growing male gametophyte and its “host” tissue, the nucellus. Pollen tube growth, and its relation to the nucellus, was examined in Zamia furfuracea with light and transmission electron microscopy. Following germination, the pollen tube of Zamia furfuracea grows intercellularly through the subepidermal layers of the micropylar apex of the nucellus. Electron micrographs clearly show additional localized outgrowths of the pollen tube penetrating the walls of individual nucellar cells. Intracellular haustorial growth ultimately leads to the complete destruction of each penetrated cell, and appears to induce the degeneration of proximal unpenetrated nucellar cells. This pattern of intracellular penetration of the sporophyte by the male gametophyte in Zamia furfuracea is fundamentally different from what has been described in any other major group of seed plants (where intercellular growth of the male gametophyte is the rule), and suggests that the heterotrophic and tissue-specific relationships that male gametophytes of seed plants have with their host sporophytes are substantially more diverse than had previously been known.     

Arabidopsis hapless mutations define essential gametophytic functions

In flowering plants, the egg develops within a haploid embryo sac (female gametophyte) that is encased within the pistil. The haploid pollen grain (male gametophyte) extends a pollen tube that carries two sperm cells within its cytoplasm to the embryo sac. This feat requires rapid, precisely guided, and highly polarized growth through, between, and on the surface of the cells of the stigma, style, and ovary. Pollen tube migration depends on a series of long-range signals from diploid female cells as well as a short-range attractant emitted by the embryo sac that guides the final stage of tube growth. We developed a genetic screen in Arabidopsis thaliana that tags mutant pollen with a cell-autonomous marker carried on an insertion element. We found 32 haploid-disrupting (hapless) mutations that define genes required for pollen grain development, pollen tube growth in the stigma and style, or pollen tube growth and guidance in the ovary. We also identified genomic DNA flanking the insertion element for eleven hap mutants and showed that hap1 disrupts AtMago, a gene whose ortholog is important for Drosophila cell polarity.     

Diffusion barriers of tripartite sporopollenin microcapsules prepared from pine pollen

Tripartite sporopollenin microcapsules prepared from pine pollen (Pinus sylvestris L. and Pinus nigra Arnold) were analysed with respect to the permeability of the different strata of the exine which surround the gametophyte and form the sacci. The sexine at the surface of the sacci is highly permeable for polymer molecules and latex particles with a diameter of up to 200 nm, whereas the nexine covering the gametophyte is impermeable for dextran molecules, with a Stokes' radius > or =4 nm (Dextran T 70), and for the tetravalent anionic dye Evans Blue (Stokes' radius = 1.3 nm). The central capsules obtained by dissolution of the sporoplasts showed strictly membrane-controlled exchange of non-electrolytes, with half-equilibration times in the range of minutes (monosaccharides, oligosaccharides) to hours (dextran molecules with Stokes' radii up to 2.5 nm). The dependence of the permeability coefficients of the nexine for non-electrolytes on Stokes' radius or molecular weight shows that the aqueous pores through the nexine are inhomogeneous with respect to their size, and that most pores are too narrow for free diffusion of sugar molecules. To explain the barrier function of the nexine for Evans Blue, it is assumed that at least the larger pores, which enable slow permeation of dextran molecules, contain negative charges.     

Use of a nuclear-targeted β-glucuronidase fusion protein to demonstrate vegetative cell-specific gene expression in developing pollen

To examine the site of expression of the tomato anther-specific gene, LAT52, in the developing male gametophyte, the LAT52 gene promoter was fused to a nuclear-targeted version of the β-glucuronidase (GUS) gene and introduced into tobacco. Transformed plants expressing GUS activity showed nuclear localization of the GUS reaction product to the vegetative cell of the pollen grain. No staining or localization was detected in the generative cell, at pollen maturation or during pollen tube growth in vitro. These results clearly demonstrate differential gene expression within the male gametophyte, and highlight regulatory events which determine the differing fates of the vegetative and generative cells following microspore mitosis.     

Mitochondrial dynamics in plant male gametophyte visualized by fluorescent live imaging

Visualization of organelles in living cells is a powerful method for studying their dynamic behavior. Here we attempted to visualize mitochondria in angiosperm male gametophyte (pollen grain from Arabidopsis thaliana) that are composed of one vegetative cell (VC) and two sperm cells (SCs). Combination of mitochondria-targeted fluorescent proteins with VC- or SC-specific expression allowed us to observe the precise number and dynamic behavior of mitochondria in the respective cell types. Furthermore, live imaging of SC mitochondria during double fertilization confirmed previous observations, demonstrated by electron microscopy in other species, that sperm mitochondria enter into the egg and central cells. We also attempted to visualize mutant mitochondria that were elongated due to a defect in mitochondrial division. This mutant phenotype was indeed detectable in VC mitochondria of a heterozygous F(1) plant, suggesting active mitochondrial division in male gametophyte. Finally, we performed mutant screening and isolated a putative mitochondrial protein transport mutant whose phenotype was detectable only in haploid cells. The transgenic materials presented in this work are useful not only for live imaging but also for studying mitochondrial functions by mutant analysis.     

短葶飞蓬减数分裂、雄配子体发育过程及其对应的花部形态特征

运用压片-透明法对短葶飞蓬(Erigeron breviscapus)小孢子母细胞减数分裂、雄配子体的发育过程进行了观察,并探讨了它们与花部形态特征的关系。结果表明,短葶飞蓬小孢子母细胞减数分裂的胞质分裂为同时型,四分体主要为四面体型,成熟花粉为3-细胞型;花序和花蕾形态变化与减数分裂、雄配子体的发育时期具有一定相关性,其中花蕾的长度可有效确定该花蕾中减数分裂与雄配子体发育时期。     

Spatial and temporal expression of actin depolymerizing factors ADF7 and ADF10 during male gametophyte development in Arabidopsis thaliana

The actin cytoskeleton plays a crucial role in many aspects of plant cell development. During male gametophyte development, the actin arrays are conspicuously remodeled both during pollen maturation in the anther and after pollen hydration on the receptive stigma and pollen tube elongation. Remodeling of actin arrays results from the highly orchestrated activities of numerous actin binding proteins (ABPs). A key player in actin remodeling is the actin depolymerizing factor (ADF), which increases actin filament treadmilling rates. We prepared fluorescent protein fusions of two Arabidopsis pollen-specific ADFs, ADF7 and ADF10. We monitored the expression and subcellular localization of these proteins during male gametophyte development, pollen germination and pollen tube growth. ADF7 and ADF10 were differentially expressed with the ADF7 signal appearing in the microspore stage and that of ADF10 only during the polarized microspore stage. ADF7 was associated with the microspore nucleus and the vegetative nucleus of the mature grain during less metabolically active stages, but in germinating pollen grains and elongating pollen tubes, it was associated with the subapical actin fringe. On the other hand, ADF10 was associated with filamentous actin in the developing gametophyte, in particular with the arrays surrounding the apertures of the mature pollen grain. In the shank of elongating pollen tubes, ADF10 was associated with thick actin cables. We propose possible specific functions of these two ADFs based on their differences in expression and localization.     

The evolutionary history of the seed plant male gametophyte

The role of the male gametophyte in the early history of seed plants remains an underappreciated but critical part of the evolution of a suite of characters that ultimately came to define seed plants. Recent paleobotanical discoveries and studies of extant primitive seed plant male gametophytes, when coupled with phylogenetic analyses of seed plants, provide insight into many hey events that occurred during the early evolution of seed plants. These discoveries are changing our ideas concerning the multiple origins of the sulcus (pollen grain germinal aperture) and pollen tube, the structural and physiological relationships of the male gametophyte with the host sporophyte tissues in primitive seed plants, and the evolution of siphonogamy (conduction of non-motile sperm via a pollen tube) from a zooidogamous (swimming sperm) condition.     

STUDIES OF SEED FERN POLLEN: DEVELOPMENT OF THE EXINE IN MONOLETES (MEDULLOSALES)

Monoletes pollen extracted from the seed fern synangium Dolerotheca sclerotica Baxter illustrate four stages in the development of the sporoderm. In the first stage the grains are up to 100 μm long and possess an apparent homogeneous exine in which there is little differentiation between the nexine and sexine. Numerous nexine lamellae and the initiation of sexine expansion mark stage 2 in exine ontogeny. Further expansion of the sexine continues in the third stage until the ratio between the nexine and sexine is approximately 1:5. The final stage in maturation of the sporoderm shows an expanded alveolate sexine with some of the sporopollenin units broken and disorganized. It is at this stage of development that nexine lamellae are most prominent. The formation of sporoderm layers in the fossil grains is compared with pollen grain development in living cycads (Cycadophyta) and a model proposed to account for the apparent early formation of nexine lamellae in Monoletes. The evolution of exine components in early pollen types is discussed.     

Synergid cell death in Arabidopsis is triggered following direct interaction with the pollen tube

During angiosperm reproduction, one of the two synergid cells within the female gametophyte undergoes cell death prior to fertilization. The pollen tube enters the female gametophyte by growing into the synergid cell that undergoes cell death and releases its two sperm cells within the degenerating synergid cytoplasm to effect double fertilization. In Arabidopsis (Arabidopsis thaliana) and many other species, synergid cell death is dependent upon pollination. However, the mechanism by which the pollen tube causes synergid cell death is not understood. As a first step toward understanding this mechanism, we defined the temporal relationship between pollen tube arrival at the female gametophyte and synergid cell death in Arabidopsis. Using confocal laser scanning microscopy, light microscopy, transmission electron microscopy, and real-time observation of these two events in vitro, we demonstrate that synergid cell death initiates after the pollen tube arrives at the female gametophyte but before pollen tube discharge. Our results support a model in which a signaling cascade triggered by pollen tube-synergid cell contact induces synergid cell death in Arabidopsis.     

Immunocytochemical localization of the allergenic proteins in the pollen of Cryptomeria japonica

Applying an immunocytochemical method, a localization of the protein Cry j I in the Cryptomeria japonica pollen, which is the major allergen responsible for Japanese cedar pollinosis, is investigated with the monoclonal and polyclonal antibodies produced from the protein. The protein that reacts to the polyclonal antibody localizes on the sexine, nexine, between nexine and intine layers, orbicles, cell wall of a generative cell, Golgi body and Golgi vesicles. The allergenic protein contained in the exine and orbicles of Japanese cedar pollen can diffuse or dissolve easily from there into the mucus covering of the eye and nose, causing a response in less than 1 min after exposure. Since the orbicles have a diameter of about 430 nm, they can pass easily through the pores of most protective masks to reach the sensitive tissues of the patient. The proteins react to the monoclonal antibodies (J1BO1 and J1BO7) and localize on the Golgi body, sexine, nexine and orbicles (but not between the nexine and intine layers), and on the generative cell wall. In the young pollen grain, numerous allergenic protein particles contained in the orbicles and sexine layer, but there is only a small amount of the protein between the nexine and intine layers, since the intine layer is not yet complete at this stage. More will be accumulated there during developmental maturation. The allergenic protein is also found on the tapetal materials remaining in the young anther. Since the materials forming the exine layer and orbicles come from tapetal tissue, it is assumed that some of the allergenic protein is produced in the tapetum and localized in the orbicles and pollen wall during maturation, and that the rest of the allergenic protein is produced in the Golgi body in the mature pollen grain.     

She's the boss: signaling in pollen tube reception

In angiosperms, the sperm cells are carried within the pollen tubes (male gametophytes) to the female gametophyte so that double fertilization can occur. The female gametophyte exerts control over the male, with specialized cells known as synergids guiding the pollen tubes and controlling their behavior when they enter the female gametophyte so that the sperm cells can be delivered to the egg and central cell. Upon pollen tube arrival at the ovule, signal transduction cascades mediated by receptor-like kinases are initiated in both the synergid and the tip of the pollen tube, leading to synergid cell death and pollen tube rupture. In this review, we discuss the role of these receptors and of newly discovered members of the pollen tube reception pathway.     

The role of H+/- ATPase and alternative oxidase in the regulation of intracellular pH at the different stages of development of the tobacco male gametophyte

In order to determine the role of the plasma membrane H(+)-ATPase and alternative oxidase (alternative pathway of respiration) in the regulation of intracellular pH during development of the tobacco male gametophyte, we studied the changes in pH due to the inhibition of these enzymes by orthovadanate and benzhydroxamic acid, respectively. The inhibition of these enzymes decreased the intracellular pH at all three studied stages of the male gametophyte development: middle and late binuclear pollen grains and activated mature pollen grain. The data obtained suggest that H(+)-ATPase and alternative oxidase are involved in the regulation of intracellular pH of the pollen grain during its differentiation and activation that precede germination. At the same time, during the recovery of intracellular pH after its acidification by propionic acid, it was found that other mechanisms, not related to the above mentioned, greatly contribute to the regulation of pH.     

Attractive and repulsive interactions between female and male gametophytes in Arabidopsis pollen tube guidance

Sexual reproduction in plants, unlike that of animals, requires the action of multicellular haploid gametophytes. The male gametophyte (pollen tube) is guided to a female gametophyte through diploid sporophytic cells in the pistil. While interactions between the pollen tube and diploid cells have been described, little is known about the intercellular recognition systems between the pollen tube and the female gametophyte. In particular, the mechanisms that enable only one pollen tube to interact with each female gametophyte, thereby preventing polysperm, are not understood. We isolated female gametophyte mutants named magatama (maa) from Arabidopsis thaliana by screening for siliques containing half the normal number of mature seeds. In maa1 and maa3 mutants, in which the development of the female gametophyte was delayed, pollen tube guidance was affected. Pollen tubes were directed to mutant female gametophytes, but they lost their way just before entering the micropyle and elongated in random directions. Moreover, the mutant female gametophytes attracted two pollen tubes at a high frequency. To explain the interaction between gametophytes, we propose a monogamy model in which a female gametophyte emits two attractants and prevents polyspermy. This prevention process by the female gametophyte could increase a plant's inclusive fitness by facilitating the fertilization of sibling female gametophytes. In addition, repulsion between pollen tubes might help prevent polyspermy. The reproductive isolations observed in interspecific crosses in Brassicaceae are also consistent with the monogamy model.     

YAO is a nucleolar WD40-repeat protein critical for embryogenesis and gametogenesis in Arabidopsis

Background  

In flowering plants, gametogenesis generates multicellular male and female gametophytes. In the model system Arabidopsis, the male gametophyte or pollen grain contains two sperm cells and a vegetative cell. The female gametophyte or embryo sac contains seven cells, namely one egg, two synergids, one central cell and three antipodal cells. Double fertilization of the central cell and egg produces respectively a triploid endosperm and a diploid zygote that develops further into an embryo. The genetic control of the early embryo patterning, especially the initiation of the first zygotic division and the positioning of the cell plate, is largely unknown.     

Study of some characteristics of morphogenesis and germination of Pinus pallasiana D. Don. pollen

Investigation of Pinus pallasiana D. Don. pollen morphogenesis in natural forest plantations of the southern slope of the Main ridge of Crimean mountains has been carried out. Some abnormalities of pollen grain and pollen tube formation in the course of pollen germination in vitro are described. The dynamics of developmental abnormalities of P. allasiana male gametophyte is characterized.     

The Arabidopsis mutant feronia disrupts the female gametophytic control of pollen tube reception

Reproduction in angiosperms depends on communication processes of the male gametophyte (pollen) with the female floral organs (pistil, transmitting tissue) and the female gametophyte (embryo sac). Pollen-pistil interactions control pollen hydration, germination and growth through the stylar tissue. The female gametophyte is involved in guiding the growing pollen tube towards the micropyle and embryo sac. One of the two synergids flanking the egg cell starts to degenerate and becomes receptive for pollen tube entry. Pollen tube growth arrests and the tip of the pollen tube ruptures to release the sperm cells. Failures in the mutual interaction between the synergid and the pollen tube necessarily impair fertility. But the control of pollen tube reception is not understood. We isolated a semisterile, female gametophytic mutant from Arabidopsis thaliana, named feronia after the Etruscan goddess of fertility, which impairs this process. In the feronia mutant, embryo sac development and pollen tube guidance were unaffected in all ovules, although one half of the ovules bore mutant female gametophytes. However, when the pollen tube entered the receptive synergid of a feronia mutant female gametophyte, it continued to grow, failed to rupture and release the sperm cells, and invaded the embryo sac. Thus, the feronia mutation disrupts the interaction between the male and female gametophyte required to elicit these processes. Frequently, mutant embryo sacs received supernumerary pollen tubes. We analysed feronia with synergid-specific GUS marker lines, which demonstrated that the specification and differentiation of the synergids was normal. However, GUS expression in mutant gametophytes persisted after pollen tube entry, in contrast to wild-type embryo sacs where it rapidly decreased. Apparently, the failure in pollen tube reception results in the continued expression of synergid-specific genes, probably leading to an extended expression of a potential pollen tube attractant.