ULTRASTRUCTURAL COMPARISION OF POLLEN GRAINS FROM 2n, 3n,AND 4n PLANTS OF EUPHORBIA DULCIS

Pollen development in plants with different ploidy levels of Euphorbia dulcis is similar but some ultrastructural differences do occur. In pollen of diploid plants large aggregations of rough endoplasmic reticulum [RER] are attached to the pollen wall near the young generative cell but such aggregations are not present in other karyotypes. Plastids are detected only in young generative cells of triploid plants. In diploid plants the generative cell becomes spindle-shaped, in triploid and tetraploid plants it remains round during the movement from the pollen wall to the center of the vegetative cell. The intine surrounding the generative cell in 3n plants is thinner than that found in 2n and 4n plants. Pollen grains in tetraploid plants are twice as large as those in diploid plants. Pollen viability is 90% in 2n plants, but only 10% in 4n plants.     

Immunoelectron microscopy of myosin associated with the generative cells in pollen tubes ofNicotiana tabacum L.

In this study, polyclonal anti-myosin antibodies were used for immunogold labeling of ultrathin sections of pollen tubes ofNicotiana tabacum L. to unravel the ultrastructural localization of myosin associated with the generative cells. Clusters of immunogold particles were consistently found in association with the area of the outer surface of the vegetative cell plasma membrane present around the generative cell. Compared to the generative cell cytoplasm, the nucleoplasm showed higher numbers of gold particles. This is the first direct evidence demonstrating the presence of myosin in the nuclei of the generative cell of flowering plants. The possible implications of these findings are discussed in relation to movement of the generative cell in the pollen tube cytoplasm.     

Microfilaments (F-actin) in generative cells and sperm: an evaluation

Summary Disagreement has arisen over the presence of actin-containing microfilaments (Mfs) in angiosperm generative cells and sperm (GSP). In order to address this issue, we subjected GSP of Tradescantia virginiana, Nicotiana tabacum and Rhododendron laetum to a series of localizations using different antiactins, rhodamine phalloidin and antimyosin. Coordinate staining with antitubulin and Hoechst 33258 defined the status of the microtubule (Mt) cytoskeleton and stages of generative cell division. Additional experiments utilized cytochalasin D (CD). In no instance could Mfs be detected in GSP of the three species. Instead, Mfs seen at the periphery of GSP appear to be continuous with vegetative Mfs and thus are in the vegetative cytoplasm. Mfs are not seen in the constriction zone of dividing T. virginiana generative cells, nor are they indicated in the phragmoplast of N. tabacum and R. laetum. Myosin localizations reveal punctate staining in the vegetative cytoplasm and a thin line of fluorescence around the the outside of the generative cell. While CD seems to delay generative cell division, cytokinesis still takes place. CD-induced Mf fragments are evident in the vegetative cytoplasm but not in GSP. The weight of evidence therefore indicates that GSP do not contain Mfs. The implications of this conclusion for the behavior of GSP and the mechanism of cytokinesis in dividing generative cells are considerable.     

Brassica napus pollen development during generative cell and sperm cell formation

Summary Brassica napus pollen development during the formation of the generative cell and sperm cells is analysed with light and electron microscopy. The generative cell is formed as a small lenticular cell attached to the intine, as a result of the unequal first mitosis. After detaching itself from the intine, the generative cell becomes spherical, and its wall morphology changes. Simultaneously, the vegetative nucleus enlarges, becomes euchromatic and forms a large nucleolus. In addition, the cytoplasm of the vegetative cell develops a complex ultrastructure that is characterized by an extensive RER organized in stacks, numerous dictyosomes and Golgi vesicles and a large quantity of lipid bodies. Microbodies, which are present at the mature stage, are not yet formed. The generative cell undergoes an equal division which results in two spindle-shaped sperm cells. This cell division occurs through the concerted action of cell constriction and cell plate formation. The two sperm cells remain enveloped within one continuous vegetative plasma membrane. One sperm cell becomes anchored onto the vegetative nucleus by a long extension enclosed within a deep invagination of the vegetative nucleus. Plastid inheritance appears to be strictly maternal since the sperm cells do not contain plastids; plastids are excluded from the generative cell even in the first mitosis.     

CHANGES IN PLASTID AND MITOCHONDRION CONTENT DURING MATURATION OF GENERATIVE CELLS OF SOLANUM (SOLANACEAE)

A study was made of the number of plastids and mitochondria present in generative cells of Solanum immediately after microspore mitosis, and the fate of these organelles during development of the pollen was determined. Changes were followed via electron microscopy of anthers of S. chacoense and S. tuberosum Group Phureja × S. chacoense. In earliest stages the generative cells were oval and had one surface along the intine and other surfaces in contact with the vegetative cell. As the pollen matured the generative cells elongated, became spindle-shaped, and were completely engulfed in the vegetative cells. At the earliest stages studied, both mitochondria and plastids were present in the generative cell. Plastids of the generative cell were, in contrast to those of the vegetative cells, fewer, smaller, and lacking in starch. Through the maturation stages the content of these organelles in the vegetative cells remained unchanged. While the generative cells retained mitochondria until anthesis, their plastids disappeared completely during maturation. This selective loss during generative cell maturation could lead to transmission of those characteristics encoded in plastid DNA through the pistillate parent only. The mechanism could explain earlier genetic evidence that plastid characters of Solanum were transmitted uniparentally.     

Preliminary observations on the formation of the male germ unit in pollen tubes ofCyphomandra betacea sendt.

Summary The structure of the generative cell and its association with the vegetative nucleus in the pollen tube ofCyphomandra betacea Sendt. were observed with the electron microscope. The generative cell, bounded by its own plasma membrane and the inner plasma membrane of the vegetative cell, possesses the cytoplasmic extension which lies within the embayments of a vegetative nucleus. The generative cell contains the normal complement of organelles and, especially, microtubules which cluster into several groups adjacent to the plasma membrane, oriented along the longitudinal axis of the cell. In the pollen tube reaching the lower end of the style aftersemivivo pollination, both of the sperm cells are elongated and polyribosomes and microtubules are the outstanding feature in the cytoplasm. The two sperm cells are connected by a common transverse cell wall, while cytoplasmic channels exist in both the periplasm of the two sperm cells and the transverse wall. The leading sperm cell (S_vn) is closely associated with the vegetative nucleus. Thus the present study demonstrates the existence of the male germ unit in the pollen tube ofC. betacea. The possible cytoplasmic continuity between the sperm cells and between the gametes and vegetative cell is considered.Abbreviations S_vn sperm cell physically associated with the vegetative nucleus - S_ua sperm cell unassociated with the vegetative nucleus - RER rough endoplasmic reticulum - SER smooth endoplasmic reticulum     

Ultrastructure of Male Gametophyte in wheat I. The Formation of Generative and Vegetative Cells

The present study of the formation of the generative and vegetative cells in wheat has demonstrated some cytological details at the ultrastructural level. The phragmoplast formed in telophase of the first microsporic mitosis extended centrifugally until it connected with the intine of the pollen grain. A new cell wall was then formed to separate the generative and the vegetative cells. By unequal cytokinesis the former is small and the latter large. In early developmental stage of male gametophyte, the organelles in the cytoplasm of the generaVive cell and the vegetative cells are similar, including mitochondria, dictyosomes, rough endoplasmic retieulum, free and clustered ribosomes and plastids, but microtubules were observed only in the early cytokinesis stage. In the further developmental stage of the male gemetophyte, the generative cell gradually detached from the intine of pollen grain and grew inward to the cytoplasm of the vegetation cell. When the generative cell became round and free in the cytoplasm of the vegetative cell, the wall materials between plasma membranes of the cytoplasm of the generative and the vegetative cells disappeared completely, so that it was a naked cell with a double-layer membrane at this time. The heterogeneity between both cells was then very conspiceous. The organelles in the cytoplasm of the generative cell have hardly any changed besides the degeneration of plastids, but in vegetative cytoplasm the mitochondria and plastids increased dramatically both in number and size. The rapid deposition of starch in the plastids of the cytoplasm of the vegetative cell made the most conspicuous feature of the vegetative cell in mature pollen grain. The significance of the presence of a temporary cell wall in generative cell and heterogeneity between generative and vegetative cells are discussed.     

Generative cell division and sperm cell formation in barley

Summary Shortly before and during division, the generative cell of barley (Hordeum vulgare L.) is located near the vegetative nucleus, in the peripheral layer of the highly vacuolated vegetative cell at the aperture pole. This position is also characteristic of the two resulting sperm cells. Conventional mitosis of the generative cell is followed by cytokinesis through cell plate formation. Just after division, the two sperm cells are enclosed together within a common inner vegetative cell plasma membrane, and they gradually separate from each other only during pollen maturation. The space between the generative or sperm cell plasma membrane and the vegetative cell plasma membrane is very thin and appears to be devoid of a cell wall. Both the generative cell and the young sperm cells contain a normal set of organelles; plastids devoid of starch are only sporadically observed. Our data indicate that in Hordeum vulgare the generative cell divides after migrating inside the pollen grain. This follows the pattern of development well established for several species with tricellular pollen.     

Generative Cell Division and Sperm Cell Association in the Pollen Grain of Sambucus nigra

Generative cell division in tricellular pollen grains of Sambucusnigra L. (Caprifoliaceae) has been examined with light and electronmicroscopy. During division the generative cell is located inthe centre of the pollen grain, near to the nucleus of a surroundingvegetative cell. Conventional mitosis of the generative cellis followed by cytokinesis through centrifugal cell plate formation.Two sister sperm cells remain in spatial contact with each otherand are surrounded, as formerly their progenitor cell was, bythe vegetative cell. From the changes of shape of the generativecell during division and of the sperm cells it may be assumedthat the space between these cells and the vegetative one containsa labile, non-rigid, wall material. No plastids have been observedin the generative cell and its mitochondria appear to be unequallydistributed between the two future sperm cells during division. Sambucus nigra L., generative cell division, pollen, sperm cell association     

Origin of sperm cell association in the “male germ unit” ofBrassica pollen

The association of the two sperm cells inBrassica napus pollen following the generative cell division was investigated. The generative cell during division is located in the center of the pollen grain, within the vegetative cell. The space present between the two cells is slightly irregular as seen following standard glutaraldehyde fixation. After completion of mitosis vesicles appear in the equatorial plane, coalescing centripetally to form a cell plate which fuses with the membrane of the generative cell, dividing it in two sperm cells. They are isolated from the vegetative cell by the space between the two cell membranes and are separated from each other by a similar space resulting from the cell plate formed during cytokinesis.     

Pollen structure and germination of Crocus thomasii Ten. (Iridaceae)

Abstract

Mature pollen of C. thomasii Ten. (Iridaceae) has been studied from a morphological and physiological point of view and compared to that of C. sativus L., C. thomasii pollen is roundish, with a 83 μm diameter and a 14% of anomalous grains. Percentage of the positive alcoholdehydrogenase (ADH +) pollen grains is about 90% whereas the in vitro germinated rate reaches 55%. Exine is homogeneously thickened (2.5 μm) with randomly distributed thinner zones and 5 μm thick intine. Cytoplasm of vegetative and generative cells is very rich of small and large smooth vesicles and vesicles coated by ribosomes. The generative cell shows a thin ondulated pectocellulosic wall. Its nucleus is intensely fluorescent after treatment with the 4,6-diamidino-2-phenylindole (DAPI) fluorochrome. Although many ultrastructural aspects of the C. thomasii pollen are common in C. sativus. L., C. thomasii pollen is smaller, more regularly structured and germinates in vivo and in vitro in higher percentage than that of C. sativus.     

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.     

A study on pollen grains ofCrocus cartwrightianus (Iridaceae)

Crocus cartwrightianus andC. cartwrightianus cv.albus pollen have been studied from structural and ultrastructural points of view and the germination assayed in vivo and in vitro.C. cartwrightianus pollen is regularly shaped and sized, has a low percentage of anomalous grains and has a high germination rate in vitro, whileC. cartwrightianus cv.albus is less regularly shaped with some variation in size and has a high percentage of anomalous grains and a low germination percentage in vitro. Ultrastructural observations have revealed, in the pollen of both the taxa, the presence of a thin elongated vegetative nucleus and a generative cell surrounded by a thin membrane. However,C. cartwrightianus pollen shows a thicker intine, andC. cartwrightianus cv.albus shows numerous pollen germination anomalies which are in common withC. sativus.     

Hermodactylus tuberosus L. (Iridaceae) pollen organisation before and after anther dehiscence

ABSTRACT

The morphology, cytology and viability of Hermodactylus tuberosus L. (Iridaceae) pollen were examined from the first mitosis until maturation and after anther opening. During maturation, the pollen coat becomes modified, and the vegetative cell cytoplasm accumulates several types of reserve substances. In the vegetative cell cytoplasm, starch is quickly utilised whereas lipid inclusions of different dimensions, shape and composition occur during pollen maturation. Pollen from opened anthers have a thin pollen coat; the cytoplasm has mostly lipid reserves, and many small vesicles and vacuoles. It is similar in size or larger than pollen located inside the anther, and its viability does not decrease until one day after anther dehiscence. Large osmiophilic bodies, different from those of the vegetative cell cytoplasm, are present in the generative cell cytoplasm starting from the first stage of pollen development. The poorly developed pollen coat in pollen from opened anthers suggests that it plays a minor role in attracting insects for pollination. The size and structural and ultrastructural features of mature pollen indicate that it does not undergo dehydration and possesses sufficient vigour for immediate germination.     

Ultrastructural studies on plastids of generative and vegetative cells inLiliaceae 3. Plastid distribution during the pollen development inGasteria verrucosa (Mill.) Duval

Summary This paper describes the development of pollen grains ofGasteria verrucosa from the late microspore to the mature two-cellular pollen grain. Ultrastructural changes and the distribution of plastids as a result of the first pollen mitosis have been investigated using light and electron microscopy. The microspores as well as the generative and the vegetative cell contain mitochondria and other cytoplasmic organelles during all of the observed developmental stages. In contrast, the generative cell and the vegetative cell show a different plastid content. Plastids are randomly distributed within the microspores before pollen mitosis. During the prophase of the first pollen mitosis the plastids become clustered at the proximal pole of the microspore. The dividing nucleus of the microspore is located at the distal pole of the microspore. Therefore, the plastids are not equally distributed into both the generative and the vegetative cell. The possible reasons for the polarization of plastids within the microspore are briefly discussed. The lack of plastids in the generative cell causes a maternal inheritance of plastids inGasteria verrucosa.     

Ultrastructure and Germination Percentage of Crocus biflorus Miller subsp. biflorus (Iridaceae) Pollen

Pollen of Crocus biflorus Miller subsp. biflorus from natural habitats of Tusculum (Frascati, near Rome, Italy) has been studied in order to compare its structure and physiology to pollen of other Crocus species belonging to the Crocus sativus group. Mature pollen grains are rounded, 60 μm in diameter, in-aperturate (but with surface incisions where exine is lacking). DAPI staining reveals a spindle-shaped generative nucleus which is intensely fluorescent, and vegetative nucleus which is less fluorescent, and is elongated with numerous lobes. At anthesis the pollen is bicellular, but about 2% of tricellular grains occur among the pollen grains released from the anthers as well as on both naturally or handpollinated stigmas. Pollen germination is low in vitro, but higher in vivo. The pollen tubes are of normal shape. An electron-dense surface coat is sometimes visible on the exine, which in many cases, is detached from the exine. The vegetative cytoplasm is very rich in glycolipid bodies surrounded by endoplasmic reticulum. The generative cell has a lobed cell wall and is surrounded by the vegetative nucleus.     

Generative cells ofLilium longiflorum possess translatable mRNA and functional protein synthesis machinery

Metabolic labelling with [³⁵S]-methionine demonstrated that generative cells ofLilium longiflorum possess their own set of mRNA and are capable of synthesising proteins independently from the vegetative cell. The isolated generative cells synthesised ten proteins, of which six were unique to these specialised cells. Isolation of generative cells from pollen grains after [³⁵S]-methionine labelling resulted in an identical protein profile, therefore the synthesis of these proteins was not due to isolation shock. Addition of cycloheximide, abolished TCA-precipitable counts, whilst actinomycin D had no qualitative effect on the observed protein profile, indicating active translation of pre-existing mRNAs by the generative cells.     

Generative cell envelope in pollen grains as a secretion system,a postulate

Summary This paper describes the changes undergone by the layer dividing the vegetative from the generative cell. The abundant contents of the space inside the membrane, consisting of opaque bodies, vary considerably as the pollen grain matures, and decrease in volumepari passu with the formation of the dense bodies in the plasm of the vegetative cells. The dense bodies constitute a layer of droplets surrounding the enveloping membrane of the generative cell within the cytoplasm of the vegetative cell.Connexions have been observed between the dense bodies and the partition layer which seem to indicate that the latter is apparently responsible for their organization, and is the area in which the synthesis of their contents takes place as well. The deposition of the dense bodies on the plasmalemma of the pollen grain points to their participation in the formation of one or other of the layers in which the grain is enveloped.     

Freeze-fracture of the generative cell of Phoenix dactylifera (Arecaceae)

Summary Mature pollen of Phoenix dactylifera was freeze-fractured in germination medium. The surface of the generative cell was highly convoluted. Microtubules were not in close contact with the indentations. The vegetative cell membrane was appressed to the generative cell. Ordered ridges appeared in both fracture faces of the vegetative cell inner plasma membrane at the indentations. No ordered ridges were observed in the fracture faces of the generative cell. The nuclear envelopes of the vegetative and generative cells differed, with the generative cell having fewer and larger nuclear pores irregularly dispersed among porefree areas. These differences in plasma membrane and in nuclear envelope correlate with the subsequent differentiation of the two cells.Acknowledgements. We thank J.I. Stillman for technical assistance with the thin section preparations. This research was supported in part by NSF grant DCB-8607765 to W.W.T.     

The pollen-specific gene Ntp303 encodes a 69-kDa glycoprotein associated with the vegetative membranes and the cell wall

The pollen-specific gene Ntp303 belongs to the class of late pollen specific genes. It is first transcribed directly after pollen mitosis. Biochemical properties, appearance and precise location of the NTP303 protein during pollen development and pollen tube growth were studied by amino-acid micro-sequencing, protein gel blotting and immuno-localization. Antisera were raised against recombinant proteins, encoded by sequences of the pollen-specific Ntp303 gene. The antibodies specifically recognized a 69-kDa glycoprotein. Electron-microscopic immuno-localization of the protein revealed the presence of high concentrations of the NTP303 protein at the vegetative plasma membranes that surround the vegetative cell, the generative cell and the sperm cells of pollen and pollen tubes. The generative plasma membranes of the generative cell and the sperm cells were negative. NTP303 protein was also present in the cell walls and in callose plugs. With this method it was shown that the NTP303 protein was already present in mid-bicellular pollen, after the first, asymmetrical pollen mitosis. Possible functions for the NTP303 protein are discussed in relation to its properties and its association with the vegetative plasma membranes. Received: 9 September 1999 / Revision accepted: 4 November 1999