Vegetative Nucleus Research Articles

Induced single fertilization in maize

Bicellular pollen with one vegetative nucleus and one diploid arrested generative cell (”monospermic” pollen) was induced by trifluralin treatment of diploid maize plants at 7–9 days before flowering. The arrested generative cell (seemingly a diploid sperm cell) fused with the central cell of diploid plants and produced shriveled endosperm resembling that of a 2n×4n cross in maize. Dual pollination experiments with a purple embryo marker revealed single fertilization events in which the union of one sperm cell with the egg occurs but there is no union of a second sperm cell with the central cell. Singly fertilized ovules survived at least 4 days. Furthermore, many viable triploid plants were obtained. This technique therefore appears to have the potential for manipulating ploidy level in crops and may become useful in investigating fertilization mechanisms of angiosperms.

Changes in the Extent of the Condensation of Nuclear Chromatin and the Localization of RNA During Pollen Development in <I>Nicotiana tabacum</I>

We examined the differentiation of the generative and the vegetative cells of tobacco (Nicotiana tabacum SR-1) by a newly developed cytological method using fluorescence microscopy after embedding of samples in Technovit 7100 resin. The extent of condensation of the chromatin in generative and vegetative nuclei was measured semi-quantitatively in sections of uniform thickness by microphotometry. The extent of chromatin condensation in the generative nucleus was about 5 to 10 times as high as the vegetative nucleus throughout pollen maturation which lasted about 5 days. Such a difference was clear immediately after PM I, indicating that the differentiation of the two cells had already started as soon as PM I has finished. Maturing generative cells had almost as much condensed chromatin as chromosomes of PM I. We also examined whether the generative cell has the potential for gene expression. The localization of RNA was visualized by staining with acridine orange. The results revealed the cytoplasm of the generative cell contained RNA and the density of RNA was as high as in the vegetative cell. This result suggests that the generative cell has the potential for gene expression in spite of the highly condensed chromatin in the nucleus. Furthermore, the generative nucleus in the mature pollen retained small nucleoli. Their presence implies that rRNA is synthesized consistently in generative cells.

Repetitive DNA as marker for the study of male gametogenesis in higher plants. I. Analysis in some species ofTradescantiaandNarcissus

SUMMARYThe use of heterochromatic regions, containing moderately repetitive DNA as markers, has allowed to follow the course of development of the generative and vegetative nuclei in the pollen grains of some species of Tradescantia and Narcissus. It has been found that the chromocentres, relatives to the NOR-heterochromatin, are not randomly distributed. This implies the existence of a special mechanism governing the orientation of the centromeres in the spindle-shaped generative nuclei: a predisposition which may facilitate the mitotic process in the pollen tube.

Microgametogenesis in Plumbago zeylanica (Plumbaginaceae). 1. Descriptive cytology and three‐dimensional organization

The generative cell is initiated as a small, lenticular, unpolarized cell with a cell wall traceable to two origins: the external segment originates as intine, while an inner callose positive cell wall forms de novo. As the lenticular generative cell begins its migration into the pollen cytoplasm, the generative cell becomes polarized both externally and internally, displaying a characteristic shape and patterns of organelle distribution oriented with respect to the vegetative nucleus and independent of pollen aperture location. Separation of the generative cell from the pollen wall begins at the end opposite the vegetative nucleus and results in an elongating protuberance at the opposite end of the generative cell; this becomes associated with a preformed groove located on the surface of the vegetative nucleus. The generative cell subsequently separates from the intine near the vegetative nucleus and moves progressively toward the opposite end of the cell; during this separation, the edge of the wall facing the intine becomes callose‐positive and remains so until separating from the intine. The generative cell becomes a free cell within the pollen, which is in physical association with the vegetative nucleus. Generative cell organization and organelle content become increasingly polarized during maturation, with microtubules evident both in the elongating protuberance of the generative cell and in association with organelles. The generative nucleus migrates away from the vegetative nucleus and toward the plastid‐rich end of the generative cell, whereas mitochondria are more generally distributed within the cell. Generative cell polarization is made permanent during mitotic division and cytokinesis, i.e., two sperm cells differing in morphology are formed: the larger cell associated with the vegetative nucleus (Svn) contains a majority of the mitochondria, and the smaller, unassociated sperm cell (Sua) receives the plastids.

Microtubules and the positioning of the vegetative nucleus and generative cell in the angiosperm pollen tube: a quantitative study

In germinating pollen of the liliaceous angiosperm Endymion nonscriptus, the vegetative nucleus (VN) leaves the grain first, followed by the generative cell (GC), a sequence maintained during the linear period of pollen-tube growth. The VN and GC undergo some random repositioning within the protoplast, but during the interval of extension preceding GC division they move slowly, but consistently, towards the apex. In a colchicine concentration eliminating microtubules (MTs) from the vegetative cell (VC) germination is not affected, but the sequence of departure of the VC and the GC from the grain is reversed in most instances, and the two bodies are more widely dispersed in the tube thereafter. We interpret the results as implying that the positioning of VN and GC in the VC is governed by two forces: (i) that resulting from the balancing stresses placed on their myosin-bearing surfaces in interaction with the two polarities of actin fibrils in the cytoplasm; and (ii) a second tension imposed by an endoplasmic MT system which links the bodies with microtubule organizing centres (MTOCs) at the cell membrane just proximal to the growing point. The nature of the interaction of the MT system with the VN and GC remains to be established, but it may be significant that the presence of a dynein-like molecule in the angiosperm pollen tube has recently been reported.

The actin microfilament network within elongating pollen tubes of the gymnospermPicea abies (Norway spruce)

Actin microfilaments form a dense network within pollen tubes of the gymnosperm Norway spruce (Picea abies). Microfilaments emanate from within the pollen grain and form long, branching arrays passing through the aperture and down the length of the pollen tube to the tip. Pollen tubes are densely packed with large amyloplasts, which are surrounded by branching microfilament bundles. The vegetative nucleus is suspended within the elongating pollen tube within a complex array of microfilaments oriented both parallel to and perpendicular with the growing axis. Microfilament bundles branch out along the nuclear surface, and some filaments terminate on or emanate from the surface. Microfilaments in the pollen tube tip form a 6 μm thick, dense, uniform layer beneath the plasma membrane. This layer ensheathes an actin depleted core which contains cytoplasm and organelles, including small amyloplasts, and extends back 36 μm from the tip. Behind the core region, the distinct actin layer is absent as microfilaments are present throughout the pollen tube. Organelle zonation is not always maintained in these conifer pollen tubes. Large amyloplasts will fill the pollen tube up to the growing tip, while the distinct layer of microfilaments and cytoplasm beneath the plasma membrane is maintained. The distinctive microfilament arrangement in the pollen tube tips of this conifer is similar to that seen in tip growth in fungi, ferns and mosses, but has not been reported previously in seed plants.

Actin and tubulin expression and isotype pattern during tobacco pollen tube growth

The hybridization of total RNA isolated fromNicotiana tabacum pollen tubes with probes for tubulin and actin mRNA revealed a high level of tubulin and actin transcripts in pollen tubes 1.5–18 h old. A weak signal for tubulin and a strong signal for actin mRNAs were still detected in tubes 24 h old. The results suggest that the presynthesized tubulin and actin mRNAs inN. tabacum pollen tubes are very stable or that transcription of genes encoding cytoskeletal proteins may take place during growth of the pollen tubes. The increase of the signal for α- and β-tubulin and actin in the immunoblots of one-dimensional gels between 1.5 and 6 h indicated that the transcripts observed in the northern blots were translated. The increase in the cytoskeletal protein levels correlated well with previous cytological observations which show that inN. tabacum the movement of the vegetative nucleus (VN) and generative cell (GC) from the grain into the tube is most efficient between 3 and 6 h after germination. In the two-dimensional immunoblots it was observed that the signal for three out of four α-tubulin isotypes present in pollen tubes increased between 1.5 and 3 h. The time of the increase of α-tubulins was in good agreement with the start of movement of the VN and GC from the grain into the tube, and the synthesis of the α-tubulins could be a prerequisite for the beginning of the movement. These observations lend support to the idea that the microtubule cytoskeleton, in addition to microfilaments, is involved in the transport of the VN and GC in pollen tubes.

Cytoplasmic motors and pollen tube growth

The growth of pollen tubes is characterized by an intense cytoplasmic streaming, during which the movements of smaller organelles (like secretory vesicles) and larger ones (including the generative cell and vegetative nucleus) are precisely coordinated. A well-characterized cytoskeletal apparatus is likely responsible for these intracellular movements. In recent years both microfilament and microtubule-based motor proteins have been identified and assumed to be the translocators of the several organelle categories. Their precise function during pollen tube growth is not yet clear, but apparently an actomyosin-based system is mainly responsible for pollen tube elongation. On the other hand, microtubules and microtubule-based motors have been thought to play a role in the maintenance of cell polarity. Both cytoskeletal systems (and their respective motor activities) could cooperate to ensure a precise regulation of pollen tube growth.

Henneguya malabarica sp. nov. (Myxozoa, Myxobolidae) in the Amazonian fish Hoplias malabaricus.

Henneguya malabarica sp. nov. is described from the Amazonian teleost fish Hoplias malabaricus Boch, 1794, collected from the estuarine region of the Amazon river near Belém, Brazil. Plasmodia were represented by several vegetative nuclei, generative cells, and sporogony stages contained in cyst-like plasmodia. The spore body was ellipsoidal (12.6 microns in length and 4.8 x 3.6 microns in width) and each valve presented a tapering tail (17.1 microns in length). Thin valves surrounded the binucleate sporoplasm cell and two spherical polar capsules (3.7 x 1.8 microns) that contain five to six coils of the polar filament. The spore had an iodinophilous vacuole. The basal portion of the spore body of immature and mature spores and the whole surface of the tails were surrounded by a close homogeneous sheath. The prevalence of infection was 6.7% in the fish examined. The spores differed from those of previously described species of Henneguya in their shape, size, spore, and arrangement of the polar-filament coil. The taxonomic affinities of this parasite to other species are discussed.

花柱切断受粉法によるテッポウユリ‘ジョージア’の花粉管伸長と種子形成に及ぼす予備受粉の効果

The present study was conducted to examine whether pre-pollination is effective in improving growth of pollen tubes and seed-sets of Lilium longiflorum 'Georgia' by comparing cut-style pollination (CSP) and stigmatic pollination (SP).1. Pollen tubes reached the basal end of the style by 48 hr in self-pollination of 'Georgia' and cross-pollination of 'Georgia' × 'Hinomoto' by CSP. Using the pre-pollination technique in CSP enhanced the growth of pollen tubes both in self-and cross-pollination.2. Generative cell division of pollen tubes in the style was observed 24 hr after pollination. In cross-pollination by SP, 78% of the pollen tubes had 2 sperm nuclei and 1 vegetative nucleus ('2 S + V'), while only 15% of these appeared in the self-combination. Using CSP, pollen tubes with '2 S+ V' were 32%in cross-and 9%in self-pollination. The percentages increased to 54% and 35%, respectively, when pollen of 'Hinomoto' was used in the pre-pollination treatment.3. In SP, numerous pollen tubes which penetrated the style grew in a bunch between two rows of ovules in an ovary ; they reached the ovules near the mid-region of the loculi. In CSP, pollen tubes elongated poorly in the ovary; only a few pollen tubes reached the ovules near the mid-region.4. Penetration of pollen tubes to the micropyle occurred in about 35% of the ovules in cross-pollination-1 by SP and in less than 10% of these by CSP. Pre-pollination in CSP effectively increased the percentage of ovules with a pollen tube in the micropyle.5. Only 3 full-seeds per capsule in self-and 6 in cross-pollination were obtained by CSP, whereas 11 and 23 seeds per capsule, respectively, were obtained as the result of pre-pollination.

Il citoscheletro nella crescita del tubetto pollinico

Abstract The cytoskelelon during the pollen tube growth - The process of pollen tube growth is reviewed, with a particular attention to the cytoskeleton of the pollen tube. A relevant emphasis is also given to the role of motor proteins during the translocation of organelles, generative cell and vegetative nucleus.

Light and electron microscopical observations on sperm cells of Guizotia abyssinica (Asteraceae)

The sperm cells of Guizotia abyssinica were studied during pollen development by light microscopy and at anther dehiscence by transmission electron microscopy. During development, the nuclei change shape from spherical to elongate, thread‐like and banded. They are straight or folded, and rarely spiral‐shaped when present in the pollen tube. Electron microscopy disclosed that the elongated sperm nuclei are apparently lobate. Intermittently, they are constricted and attenuated or convoluted. The major part of the sperm chromatin is condensed and peripheral, while a minor part is dispersed and central. The scanty sperm cytoplasm contains mitochondria and starch granules. The cytoplasm is mainly restricted to spaces adjoining constricted, lobed and convoluting nuclear sites. Some cytoplasmic patches become embayed in the nucleus at these sites. The periplasm bordering the sperm cells may originate from lucid dilations of the lumen between the plasma membranes of the sperm and vegetative cells. The periplasm is sometimes partially or entirely surrounded by double‐membraned endoplasmic reticulum. Folded sperm cells with less coherent periplasm possibly represent a late stage preceding discharge into the pollen tube. The sperm cells always precede the vegetative nucleus into the pollen tube.

U1 snRNP components are present in the vegetative and generative nuclei of the pollen grain

In eucaryotes, the nuclei contain small ribonucleoprotein particles (snRNPs) which mediate splicing of RNA precursors. The male gametophytes of Pinus radiata and Viburnum tinus have more than one U1 snRNA isospecies and some snRNP polypeptides detected by northern and western blotting with probes complementary to tomato U1 snRNA and by protein immunodetection. The snRNP components were localized in both vegetative and generative nuclei of the pollen grain by in situ hybridization and immunocytochemistry. This study suggests that vegetative and generative nuclei contain the splicing apparatus for the post-transcriptional RNA processing in the pollen grain.

Cellular changes during the acquisition of embryogenic potential in isolated pollen grains ofNicotiana tabacum

We used electron microscopical techniques to study ultrastructural changes during the acquisition of embryogenic competence in immature pollen grains ofNicotiana tabacum, isolated at the early- or mid-bicellular stage and cultured in vitro under starvation conditions. Cytoplasmic and nuclear changes during the starvation treatment are reported. Dedifferentiation of plastids, dilation of the wall of the generative cell, the appearance of a large vacuole, loss of nuclear pores in the vegetative nucleus, changes in chromatin and nucleolar structure, and a decrease in the size of the nucleolus were observed. We suggest that these events are the first step in the switch from generative to vegetative generation during pollen embryogenesis.

Male gametic nucleus-specific H2B and H3 histones, designated gH2B and gH3, in Lilium longiflorum

Two proteins that resemble core histones and might be specific to the male gametic (generative) nucleus within the pollen of Lilium longiflorum Thumb, (originally designated p22.5 and p18.5; K. Ueda and I. Tanaka, 1994, Planta, 192, 446–452) were characterized biochemically and immunochemically. Patterns of digestion of p22.5 and p18.5 by Staphylococcus aureus V8 protease closely resembled those of somatic histones H2B and H3, respectively. However, peptide fragments that were unique to p22.5 or p18.5 were also detected. Antibodies raised against these proteins did not cross-react with any somatic histones. These results indicate that p22.5 and p18.5 are different from somatic histones in terms of primary structure. Analysis of their amino-acid compositions revealed that p22.5 is a moderately lysine-rich protein while p18.5 is an arginine-rich protein. From these results, we conclude that p22.5 is a variant of histone H2B and p18.5 is a variant of histone H3. Immunofluorescence staining of pollen grains using the specific antibodies revealed that both p22.5 and p18.5 are only present in the generative cell nucleus and are not to be found in the vegetative cell nucleus. This study demonstrates that (i) specific histone variants are present in the male gametic nucleus of a higher plant, as they are in the sperm nucleus of animals, and (ii) distinct differences in histone composition exist between the nuclei of generative and vegetative cells in pollen. These novel histones (p22.5 and p18.5), specific to male gametic nuclei, have been designated gH2B and gH3, respectively.

Differential mechanisms of movement between a generative cell and a vegetative nucleus in pollen tubes of Nicotiana tabacum as revealed by additions of colchicine and nonanoic acid

The growth of pollen tubes of Nicotiana tabacum cultured in a petri dish was divided into five stages by the behaviors of pollen tubes, vegetative nuclei (VNs), and generative cells (GCs). Pollen tubes continued to elongate during every stage. Colchicine, at a concentration of 1 mM, preferentially inhibited the movement of the VNs but not that of GCs. Nonanoic acid preferentially inhibited the movement of GCs. These results suggest that different mechanisms of movement exist between VNs and GCs.

Identification and localization of three classes of myosins in pollen tubes of Lilium longiflorum and Nicotiana alata

The presence and localization of actin and myosin have been examined in pollen tubes of Lilium longiflorum and Nicotiana alata. Immunoblot analysis of pollen tube extracts with antibodies to actin, myosins IA and IB, myosin II, and myosin V reveals the presence of these contractile proteins. Immunofluorescence microscopy using various methods to preserve the pollen tubes; chemical fixation, rapid freeze fixation and freeze substitution (RF-FS) followed by rehydration or by embeddment in a methacrylate mixture, was performed to optimize preservation. Immunocytochemistry reaffirmed that actin is localized longitudinally in the active streaming lanes and near the cortical surface of the pollen tube. Myosin I was localized to the plasma membrane, larger organelles, the surface of the generative cell and the vegetative nucleus, whereas, myosin V was found in the vegetative cytoplasm in a punctate fashion representing smaller organelles. Myosin II subfragment 1 and light meromyosin were localized in a punctate fashion on the larger organelles throughout the vegetative cytoplasm. In addition, isolated generative cells and vegetative nuclei labeled only with the myosin I antibody. Competition studies indicated the specificity of the heterologous antibodies utilized in this study suggesting the presence of three classes of myosins in pollen. These results lead to the following hypothesis: Myosin I may move the generative cell and vegetative nucleus unidirectionally through the pollen tube to the tip, while myosin V moves the smaller organelles and myosins I and II move the larger organelles (bidirectionally) that are involved in growth.

The Appearance of Male Gamete-Specific Histones gH2B and gH3 during Pollen Development in Lilium longiflorum

The male gametic (generative) nucleus within the nearly mature pollen of Lilium longiflorum contains specific variants of histone H2B and H3, which have been designated gH2B and gH3, respectively. Using specific antibodies raised against gH2B and gH3, we examined in detail the temporal and spatial aspects of the appearance of these histone variants during male gametogenesis. Neither gH2B nor gH3 was detected in microsporocytes during meiotic division or in microspores before microspore mitosis. However, both gH2B and gH3 were abundantly present in mid bicellular pollen and these histones continued to be detectable in germinated pollen tubes after pollination. Furthermore, immunocytochemical revealed that these proteins were present not only in generative nuclei during the maturation process, but also in the two sperm nuclei formed by division of the generative nucleus. By contrast, these proteins were not found in the vegetative nuclei. It appears, therefore, that both gH2B and gH3 are specific to male gametic (generative and sperm) nuclei in L. longiflorum. The results also suggest that these male gamete-specific histones might be newly synthesized in the progenitor of sperm cells for the differentiation of male gametes, as occurs in spermatogenesis in animals.

Lithium treatment of Nicotiana tabacum microspores blocks polar nuclear migration, disrupts the partitioning of membrane-associated Ca2+, and induces symmetrical mitosis

We have found that the normal developmental pathway of Nicotiana tabacum microspores is blocked or switched when microspores are exposed to lithium, and these effects are reversible with Ca2+ and myo-inositol. Normal development was defined by the following characteristics: changes in microspore shape from spherical to oval and then ellipsoid; two nuclear displacements, first from a central location to the cell periphery, and then from the periphery to the generative pole; a localization of membrane-associated Ca2+ at the generative pole preceding nuclear division; and, finally, an asymmetrical mitosis that results in a two-celled pollen grain with well-differentiated generative and vegetative nuclei. Lithium treatment blocked the localization of membrane-associated Ca2+ at the generative pole, and instead it was evenly distributed at both poles. Lithium treatment also blocked the asymmetrical positioning of the microspore nucleus at the generative pole and resulted in an approximately four-fold increase in the frequency of symmetrical mitosis. When Ca2+ and myo-inositol were added along with lithium, the effects were substantially decreased, and there was only a small increase in the frequency of symmetrical mitosis compared with controls. The timing of treatment was important; microspores isolated before the first nuclear displacement had a low frequency of further development, while microspores isolated immediately preceding the onset of mitosis were much less sensitive to lithium, and the result was only a small increase in the frequency of symmetrical mitosis. In microspores isolated after the first nuclear displacement, a 1-day exposure to lithium was sufficient to switch the developmental pathway from an asymmetrical to a symmetrical mitosis while still allowing limited further development. However, we have not optimized culturing conditions for embryogenesis and the furthest development observed after a 1-week culture was to four- or five-celled proembryo-like structures.

Male gametophyte development inPlumbago zeylanica: cytoplasm localization and cell determination in the early generative cell

The behavior of the generative cell during male gametophyte development inPlumbago zeylanica was examined by epifluorescence microscopy and electron microscopy with organelle nucleoid as a cytoplasm marker. When the thin sections stained with 4′,6-diamidino-2-phenylindoIe (DAPI) were observed under an epifluorescence microscope, two types of fluorescence spots were detected in the cytoplasm of the pollen cells before the second mitosis. The spots emitting stronger fluorescence were confirmed as plastid nucleoids and those emitting dimmer fluorescence were mitochondrial nucleoids. Before the first mitosis, both plastid and mitochondrial nucleoids distributed randomly in the cytoplasm of the microspore. A small lenticular generative cell formed with attachment to the interior of the intine after the mitosis. Small vacuoles were found in the lenticular cell. In the cytoplasm of the lenticular cell, both plastid nucleoids and the small vacuoles were distributed randomly at the very beginning but began to migrate in opposite directions immediately. Plastid nucleoids aggregated to the side of the cell that faces the pollen center and the small vacuoles aggregated to the side of the cell that attaches to the inline. As the result, the lenticular generative cell appeared highly polarized in cytoplasm location soon after the first mitosis. In accordance with the definition of the cytoplasm polarization, the primary wall between the generative and the vegetative cells began to flex and the lenticular generative cell started to protrude towards the pollen center. When the generative cell peeled away from the inline, it was spherical in shape with the pole that aggregated plastids towards the vegetative nucleus. But the cell direction appeared to be transformed immediately. The pole that aggregated small vacuoles turned to the position towards the vegetative nucleus and the pole that aggregated plastid nucleoids turned to the position countering to the vegetative nucleus. A cellular protuberance formed at the edge of the pole that aggregated small vacuoles and elongated into a tapered end that got into contact with the vegetative nucleus. The polarization of the cytoplasm kept constant throughout the second mitosis. The small vacuoles that apportioned to the sperm cell which attached the vegetative nucleus (the leading sperm cell) disappeared during sperm cell maturation. Plastid nucleoids were apportioned to the other sperm cell (the trailing sperm cell) completely. Mitochondrial nucleoids became undetectable after the second mitosis.