Pollen movement in the micropylar canal ofLarix and its simulation

InLarix pollen captured by the ovule and rested at the distal end of the micropylar canal is transferred upward to the nucellus before it develops a pollen tube. This upward movement occurs after the canal is filled with secreted fluid, despite the fact that the pollen sinks in the fluid. We examined the mechanism of the movement based on the morphology of the canal and its simulation using pipettes. When a water column moves upward in a waxed pipette, suspended particles also move upward carried by the meniscus. InL. x eurolepis the inner surface of the integument lining the micropylar canal is coated by a cuticle layer. This layer is further coated by an integumentary membrane before the fluid is secreted. This membrane, however, becomes distorted or disappears during fluid secretion. The exposed cuticle and the degenerated hydrophilic nucellar apex may facilitate the movement of the meniscus toward the nucellus as in the simulated pipette. Pollen is interpreted to move by being carried by the meniscus when the fluid recedes.     

Significance of exine shedding in Cupressaceae-type pollen

In conifers, which have non-saccate Cupressaceae-type pollen, the pollen must land on a pollination drop or be picked up by the pollination drop from the surface of the cone near the ovule before it can be taken into the ovule. After contact with the drop, the pollen intine absorbs moisture from the drop, expands and the exine is shed. In this study the significance of the shedding of the exine is interpreted from experiments in which simulated pollination drops and micropyles were used to determine the movement of pollen and other particles in suspension. The non-expanded pollen, which can be observed upon contact with the pollination drop, sheds the exine, which then functions as a non-elastic particle, while the pollen from which the exine was shed swells and functions as an elastic particle because it is enclosed by the flexible intine. Non-elastic particles are not easily transferred through narrow passages (the micropyle and micropylar canal) and tend to plug these passages. However, elastic particles, such as the swollen pollen, are easily transferred along narrow passages even when non-elastic particles are present. The simulated experiments demonstrate that exine shedding is an important feature in getting pollen through the narrow micropyle and micropylar canal to the nucellus of the ovule.     

Effects of ovular secretions on pollen in Pseudotsuga menziesii (Pinaceae)

Effects of ovular secretions on pollen grains were examined in Pseudotsuga menziesii. The exine is cast off in the micropylar canal. A membranelike structure covers parts of pollen grains and appears to protect them. The outer intine consists of fibrous materials, but it also shows a thicker filamentous appearance in some ovules during pollen elongation. The inner intine is electron-dense. Its fibrous nature is occasionally visible. Dissolution of the outer intine varies in amount and manner in ovules from different trees. The plasma membrane near the pollen wall alternatively appears normal and distorted. These different morphologies of the outer intine and of the plasma membrane are considered to result from secretions from the ovule. The outer intine may contain electron-dense globules that are formed in the tube cell and traverse the inner intine. Pollen tube formation appears to be triggered by a secretion from the ovule. Cross-pollinated grains are less distorted compared with self-pollinated grains.     

The wall ofPinus sylvestris L. pollen tubes

Summary The wall ofPinus sylvestris pollen and pollen tubes was studied by electron microscopy after both rapid-freeze fixation and freeze-substitution (RF-FS) and chemical fixation. Fluorescent probes and antibodies (JIM7 and JIM5) were used to study the distribution of esterified pectin, acidic pectin and callose. The wall texture was studied on shadow-casted whole mounts of pollen tubes after extraction of the wall matrix. The results were compared to current data of angiosperms. TheP. sylvestris pollen wall consists of a sculptured and a nonsculptured exine. The intine consists of a striated outer layer, that stretches partly over the pollen tube wall at the germination side, and a striated inner layer, which is continuous with the pollen tube wall and is likely to be partly deposited after germination. Variable amounts of callose are present in the entire intine. No esterified pectin is detected in the intine and acidic pectin is present in the outer intine layer only. The wall of the antheridial cell contains callose, but no pectin is detectable. The wall between antheridial and tube cell contains numerous plasmodesmata and is bordered by coated pits, indicating intensive communication with the tube cell. Callose and esterified pectin are present in the tip and the younger parts of the pollen tubes, but both ultimately disappear from the tube. Sometimes traces in the form of bands remain present. No acidic pectin is detected in either tip or tube. The wall of the pollen tube tip has a homogenous appearance, but gradually attains a fibrillar character at aging, perhaps because of the disappearance of callose and pectin. No secondary wall formation or callose lining can be seen wilh the electron microscope. The densily of the cellulose microfibrils (CMF) is much lower in the tip than in the tube. Both show CMF in all but axial and nontransverse orientations. In conclusion,P. sylvestris and angiosperm pollen tubes share the presence of esterified pectin in the tip, the oblique orientations of the CMF, and the gradual differentiation of the pollen tube wall, indicating a possible relation to tip growth. The presence of acidic pectin and the deposition of a secondary-wall or callose layer in angiosperms but not inP. sylvestris indicales that these characteristics are not related to tip growth, but probably represent adaptations to the fast and intrastylar growth of angiosperms.Abbreviations CMF cellulose microfibrils - II inner intine - NE nonsculptured exine - OI outer intine - RF-FS rapid-freeze fixation freeze-substitution - SE sculptured exine - SER smooth endoplasmic reliculum - SV secretory vesicles     

The role of the intine and cytoplasm in the activation and germination processes of Poaceae pollen grains

Ultrastructural modifications of the intine and cytoplasm, during the maturation, activation and germination processes are described for several Poaceae pollen grains. Allergenic and antigenic proteins were found in the non apertural intine during the times of activation and germination, using TEM immunolabelling. This fact may be related to the function of the non apertural intine during the processes of pollen activation and pollen tube formation prior to fecundation. Changes in the granular particles of the cytoplasm are described and their role in pollen wall development is suggested. The pectic‐cellulosic and callosic layers of the pollen tube were formed on the degraded intine, and a relationship between pollen tube wall development and the substances expelled from the fibrillar particles was observed. The immunolabelling of the starch granules may be in agreement with their role in the allergenic process.     

鹅掌楸属植物花粉萌发前后壁的超微结构

观察描述了在电镜下中国鹅掌楸（Ｌｉｒｉｏｄｅｎｄｒｏｎｃｈｉｎｅｎｓｅ）和北美鹅掌楸（Ｌ．ｔｕｌｉｐｉｆｅｒａ）２种植物花粉壁的超微结构及其水合后的变化。（１）成熟花粉壁由６层组成,即外壁３层──外层,中层１和中层２,内壁３层──内壁１,内壁２和内壁３。（２）花粉水合时,在内壁３与质膜之间由Ｐ一粒子（多糖－粒子）和被膜小泡参与形成新层。（３）花粉萌发时,由内壁３的一部分和新层突出萌发孔共同形成花粉管壁。（４）新层于花粉管形成早期分成２层──外染色深的果胶层和内电子透明的胼胝质层。     

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.     

Polarity, aperture condition and germination in pollen grains ofEphedra (Gnetales)

Pollen grain polarity, aperture condition and pollen tube formation were examined inEphedra americana, E. foliata, E. rupestris, E. distachya, andE. fragilis using LM, SEM and TEM. In the characteristic oblate pollen, as seen in situ in the tetrad configuration, the polar axis is the minor one and the equatorial plane runs between the two narrow ends of the microspore. The intine is thick in fresh fixed mature pollen but we have seen no indication of regions having an exceptionally thick intine that could be considered associated with an aperture or apertures. About three minutes after transferring fresh pollen to the germinating medium the ridged exine splits and twists away from the intine and its enclosed protoplast. The shed exine spreads out and curls into a scroll-like configuration that is as distinctive as that of the pollen shape had been but now having the ridges and valleys perpendicular to the long axis. The pollen tube develops, in our experience with more than a hundred germinating pollen grains, near one of the narrow tips of the pollen grain's equatorial plane. The location of the pollen tube initiation probably is related to the position of the tube cell nucleus. The pollen tube starts to grow about one hour after the exine was shed. The pollen tube emerges close to the narrow end (equator) of the gametophyte. This end emerged first as the exine is shed and is opposite to the prothallial cells. The stout pollen tube is c. 10µm in diameter grown in vitro on agar. In our germination medium the stout tube continued to elongate for about 24 hours reaching a length of c. 100 µm. With respect to exine morphology the aperture condition could be considered as inaperturate. The pollen tube, however, is formed in a germination area near one end of the exineless gametophyte.     

The Apertural Wall in Pollen of Linum grandiflorum

The apertural wall in tricolpate pollen of Linum grandiflorumwas investigated in order to understand its functioning duringdesiccation and rchydration. Whole and sectioned pollen grainswere studied with light or electron microscopy and by cytochemicalmeans. The areas of the apertures were examined in fresh drypollen, in grains moistened on agar gel or removed from compatiblestigmas, and in pollen from mature undehisced anthers The intine was found to consist of an inner ß-glucanlayer and an outer pectic layer. At the apertures the pecticlayer is thickened and overlaid by a ß-glucan layer.The pectinaceous intine stains red with basic fuchsin. The presenceof a third wall layer, the medine, was not confirmed. The aperturalintine thickenings possess considerable imbibitional capacityand at rehydration they appear as swollen lenticular bodies A procedure is described for obtaining intact exine free grains(EFG's) and whole, separated exines of L. grandiflorum. Invariably,the released EFG's consisted of protoplasts encased in the cellulosicintine. In most grains the outer intine remained attached tothe separated exine In L. grandiflorum the outer wall of the aperture expands whilethe protoplast and endintine are still infolded. Apparently,the exintine becomes detached from the endintine during desiccationand re-attaches at rehydration. It is suggested that the transientdetachment controls the influx of water into the vegetativecell Except for morph-specific exine processes no differences instructure of the aperture wall or its functioning at rehydrationwere observed between pin and thrum grains Pollen wallM, apertures, exintine, exine free grains, rehydration, desiccation, Linum grandiflorum     

CYTOLOGICAL BASIS FOR CYTOPLASMIC INHERITANCE IN PSEUDOTSUGA MENZIESII. I. POLLEN TUBE AND ARCHEGONIAL DEVELOPMENT

Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) ovules were used to study the method of pollen tube formation and penetration of the nucellus, the movement of the body cell down the pollen tube and development of the archegonia. No pollination drop forms but nucellar tip cells produce a minute secretion that may initiate pollen tube formation. Pollen tubes penetrate the nucellus causing degeneration of nucellar cells in contact with the pollen tube tip. The body cell becomes highly lobed and the tube cytoplasm forms thin sheets between the lobes. This may be the mechanism by which the large body cell is pulled down the narrow pollen tube. Body cell plastids and mitochondria remain unaltered during pollen tube growth, whereas tube cell organelles show signs of degeneration. The pollen tube penetrates the megaspore wall and settles in the archegonial chamber. During pollen elongation and pollen tube growth the egg matured. Egg cell plastids were transformed into large inclusions which filled the periphery of the egg while mitochondria migrated to the perinuclear zone. The neck cells, ventral canal cell and archegonial jacket cells are described. The significance of the body cell and egg cell ultrastructure is discussed in light of recent restriction fragment length polymorphism studies of plastid and mitochondrial inheritance in the Pinaceae.     

Postpollination-prezygotic ovular secretions into the micropylar canal inPseudotsuga menziesii (Pinaceae)

Ovular morphology was examined ultrastructurally inPseudotsuga menziesii to determine the effects of the ovule on pollen development. Vesicles containing lipid-like substances traverse cell walls of the inner epidemis of the integument and release their contents at the integument surface to form the integumentary membrane. A major aqueous secretion from the integument into the micropylar canal is proposed to occur by the movement of the integumentary membrane and its invaginations towards the center of the micropylar canal. The cellular degeneration of the nucellar apex results from the breakdown of vacuoles. After this degeneration, electron-dense substances move from the prothallial cells of the female gametophyte towards the nucellus, and many morphological changes in the nucellus, prothallial cells, and micropylar canal take place simultaneously. We interpret these changes to result from another major secretion from the prothallial cells. Egg cytoplasm appears to disorganize for a short time. Simultaneously, substantial amounts of electron dense-substances in the prothallial cells and lipid-like substances in surface cell walls of the female gametophyte move towards the nucellus as components of the third major secretion.     

Immunolocalisation of arabinogalactan proteins and pectins in Actinidia deliciosa pollen

Summary. The cell wall composition of germinating pollen grains of Actinidia deliciosa was studied by immunolocalization with monoclonal antibodies against arabinogalactan proteins (AGPs) and pectins. In ungerminated pollen, the JIM8 epitope (against a subset of AGPs) was located in the intine and in the cytoplasm, while the MAC207 epitope (against AGPs) was only located in the exine. After germination, the JIM8 and MAC 207 epitopes were located in the cytoplasm and in the pollen tube wall. The Yariv reagent that binds to AGPs was added to the germination medium inducing a reduction or inhibition in pollen germination. This indicates that AGPs are present in the growing pollen tube and play an important role in pollen germination. To identify the nature of the pectins found in pollen grains and tubes, four monoclonal antibodies were used. The JIM5 epitope (against unesterified pectins) was located in the intine, more intensely in the pore region, and along the pollen tube wall, and the JIM7 epitope (against methyl-esterified pectins) was also observed in the cytoplasm. After germination, the JIM5 epitope was located in the pollen tube wall; although, the tube tip was not labelled. The JIM7 epitope was located in the entire pollen tube wall. LM5 (against galactans) showed a labelling pattern similar to that of JIM5 and the pattern of LM6 (against arabinans) was similar to that of JIM7. Pectins show different distribution patterns when the degree of esterification is considered. Pollen tube wall pectins are less esterified than those of the pollen tube tip. The association of AGPs with pectins in the cell wall of the pollen grain and the pollen tube may play an important role in the maintenance of cell shape during pollen growth and development.Correspondence and reprints: Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal.     

DEVELOPMENT OF OMNIAPERTURATE POLLEN IN TRILLIUM KAMTSCHATICUM (LILIACEAE)

Ultrastructural changes during omniaperturate pollen development in Trillium kamtschaticum Pall, was examined using transmission electron microscopy. The pollen mother cells are not enveloped within a thick callosic wall. The microspores resulting from successive meiosis are divided by scanty deposition of callosic wall in the tetrad. A primexine/exine template is not recognizable within the tetrad during formation of exinous components. Preexinous globules, originating from vesicles in the callosic wall, accumulate electron-dense materials and develop into exinous globules. The preexinous globules have ca 10 nm wide contacts with tilted and invaginated plasma membrane of the microspore within the callosic wall. After dissolution of the callosic wall, the microspores separate and mitosis subsequently leads to the formation of a generative cell and vegetative cell encased in a loose aggregation of developing exinous globules. When the generative cell is at the pollen grain surface, the channeled zone is initiated at the opposite side of the microspore on the surface of the vegetative cell. Just before pollen maturity, a new layer develops under the channeled zone. Thus, development of the omniaperturate pollen grains of T. kamtschaticum involves some processes that are distinct from those of Canna and Heliconia and some that are similar.     

Nucellar beak structure and pollen tube growth in Cucurbitaceae

The nucellar beak is a proboscis-like outgrowth of the nucellus at the micropylar end, being the obligatory path for the pollen tube entering the ovule. Among the few angiosperm families with nucellar beak, Cucurbitaceae is remarkable because the pollen tube may develop at least two types of growth within the nucellar beak: tubular and ampulliform. Wondering about the possibility that Cucurbitaceae ovules may express some histological variation that could be related to pollen tube growth within the nucellar beak, we performed a compared anatomical and histochemical study of the nucellar beak and the pollen tube growth of ten species of Cucurbitaceae. Results show that Cucurbitaceae ovules are diverse in size and proportions (of integuments, nucellar body, and nucellar beak), and they have at least four types of nucellar beak histology: pectic-tracked, secretory-like, amylaceous, and mixed. Amylaceous and mixed nucellar beaks are related to the ampulliform growth of the pollen tube, which could have appeared independently in most derived tribes of Cucurbitaceae, although information about nucellar beak structure in the basal tribes is still needed. In addition, the understanding of the relation between amylaceous nucellar beaks and the ampulliform growth of the pollen tube, whose function is still to be discovered, might open the possibility of a unique model of pollen tube-ovule co-evolution in angiosperms.     

LP28, a lily pollen-specific LEA-like protein, is located in the callosic cell wall during male gametogenesis

. LP28, a pollen-specific LEA-like protein identified in Lilium longiflorum purportedly related to the desiccation tolerance of pollen, was localized during male gametogenesis using immuno-electron microscopy. At premeiotic interphase, LP28 label is absent from the microsporocyte. LP28 label was first detected in the cell wall of the microsporocyte at meiotic prophase I. LP28 gradually increased as the cell wall thickened. In the dyad, after the first meiotic division, LP28 label also appeared in the septum. In the tetrad, after the second meiotic division, LP28 was detected throughout the cell wall, including the septa. Immunolabeling of callose during meiosis indicated that the appearance and localization of LP28 was very similar to that of callose. After the microspores were released from the tetrad by digesting the callosic cell wall, LP28 was not found in the microspores. In bicellular pollen, just after microspore mitosis, LP28 appeared in the generative cell wall, which also consisted of callose. After pollen germination, LP28 also accumulated in the callosic layer of the elongated pollen tube wall and the callose plug. Thus, LP28 colocalized with the callosic cell wall during male gametogenesis. The possible role of LP28 with respect to wall formation during meiosis and pollen development is discussed.     

An Ultrastructural Study on Pollen Protoplasts and Pollen Tubes Germinated From Them in Gladiolus gandavensis

Large quantities of protoplasts were isolated enzymatically from the mature pollen grains in Gladiolus gandavensis. Regeneration of cell wall and germination of pollen tubes were performed during culture of purified pollen protoplasts in Ks medium supplemented with 32% sucrose, 0.1 mg/1 2,4-D, 1 mg/1 NAA and 0.2 mg/1 6-BA, with a germination rate up to 47.7%. The materials were fixed gently with gradually increasing concentration of glutaraldehyde, followed by osmium, then preembedded in a thin layer of agar and surveyed under an inverted microscope so as to select desired specimens for subsequent procedure. Small agar blocks containing specimens were dehydrated through ethanal-propylene oxide series, embedded in Araldite and ultratomed. Electron microscopic observations show that the pollen protoplasts are surrounded by a smooth plasma membrane and with ultrastructurally intact cytoplasm, a vegetative nucleus and a generative cell. After 8h of culture, wall regeneration commences resulting in a multilayered, fibrillar wall structure which is different from the intine. No exine is formed. Numerous vesicles participate actively in the wall formation. The wall is uneven in thickness around its periphery; a thickened area somewhat resembling to germ furrow is formed, from which pollen tube emerges. The tubes contain abundant plastids, mitochondria and dictyosomes. Vesicles are released out of the plasma membrane and involved in tube wall formation. After 18h of culture, the vegetative nucleus and generative cell have migrated into the tube. Technical points of preparing pollen protoplast specimens for ultastructural studies and the fearnres of wall regeneration in pollen protoplast culture are discussed.     

A cytochemical study on the role of ATPases during pollen germination in Agapanthus umbelatus L'Her.

Summary Cytochemical detection of ATPase activity in the pollen grain (PG) and pollen tube (PT) of Agapanthus umbelatus showed that the enzymes concerned presented specific patterns of membrane distribution according to their ionic dependencies and to the timecourse of germination and tube growth. In the pollen tubes Ca²⁺-ATPases were mainly localized in mitochondria and ER membranes, while Mg²⁺-ATPases were found especially in the tonoplast and in the membrane of the P-particles. K⁺-ATPases showed a high activity at the plasma membrane. In the pollen grain similar patterns of ATPase activity were observed. The highest activity of all three types was observed at the plasma membrane of the grain and at the intine and inner exine layers of the cell wall. The activity observed in the pollen grain cell wall decreased with germination time. In vivo germination studies in the presence of specific inhibitors of the ATPases showed patterns of inhibition that could be correlated with the corresponding ATPase putative role.The results are discussed in terms of the ultrastructural organization of the PG and PT, especially those correlated with (1) formation and maintenance of ionic gradients throughout the PT, (2) polarized growth and (3) hydrodynamics of PT elongation.Abbreviations PT Pollen tube - PG pollen grain - PTW pollentube wall - PGW pollen-grain wall - ER endoplasmic reticulum - NEM N-ethylmaleimide     

THE POLLEN WALL OF CANNA AND ITS SIMILARITY TO THE GERMINAL APERTURES OF OTHER POLLEN

The pollen wall of Canna generalis Bailey is exceptionally thick, but only a minor part of it contains detectable amounts of sporopollenin. The sporopollenin is in isolated spinules at the exine surface and in the intine near the plasma membrane. There is no sporopollenin in the > 10 μ thick channeled region between spinules and intine. We suggest that the entire pollen wall of C. generalis is similar to the thick intine and thin exine typical for germinal apertures in many pollen grain types. Considered functionally, the Canna pollen wall may offer an infinite number of sites for pollen tube initiation and would differ significantly from grains that are inaperturate in the sense of an exine lacking definite germinal apertures.     

Microspore ofSecale cereale as a transfer cell type

Summary In the mature microspore ofSecale cereale, a set of wall ingrowths deposited as the first (outer) intine layer between exine and the microspore plasma membrane, are revealed by electron microscopy. The wall ingrowths form a girdle in the vicinity of the apertural region at the external pole of microspore which is in contact with the tapetum, so the microspore can be considered as a transfer cell which is polarized. After microspore division the second (inner) intine layer is deposited by the vegetative cell and forms a labyrinth of branched wall ingrowths. As a result, the periphery of a vegetative cell is also irregular and appears as very thin plasmatubules or evaginations delimited by plasma membrane and penetrating the pollen wall.The possible functions of the microspore as a transfer cell and the wall-membrane system of the vegetative cell are discussed.     

Pectin distribution pattern in the apertural intine of Euphorbia peplus L. (Euphorbiaceae) pollen

The apertural inner layer (intine) of Euphorbia L. pollen grains has a characteristic but original structure that has paired thickenings, one on either side of the colpus. To determine the nature and role of this intine layer, pollen grains of Euphorbia peplus L. were germinated in vivo and in vitro. The germination process involves wall changes that facilitate formation of the pollen tube and its subsequent growth. In the thickenings of the intine of E. peplus, the unesterified pectin epitopes are more densely localised in the inner part of the middle intine. No such epitopes are located in the intine portion adjacent to the plasma membrane (cellulosic endintine). Unesterified pectin epitopes are also localised in the outer part of the intine but are restricted to the centre of the aperture, around and in the pore. The de-esterification of pectins is very advanced at the time of dehiscence and pollen germination. The stratification of the aperture intine may take the following pathway at the time of germination: the thin outer zone of the intine in the pore region becomes disorganised and undergoes dissolution with liberation of unesterified and esterified pectins; the middle intine thickenings undergo an important elastic modification, but without liberation of unesterified pectins; the cellulosic inner intine is the progenitor of the pollen tube wall. This special intine of E. peplus is an adaptation to the hydration process preceding germination, increasing intine and pollen grain wall elasticity.