A light and electron microscopic study of stigmas inAneilema andCommelina species (Commelinaceae)

Summary The stigmas of species inAneilema andCommelina are trifid and comprise elongate papillae. Progressive degeneration of papular cells is observed in stigmas from open flowers and at anthesis papillae may be moribund and collapsed. Fluid emanating from the hollow style flows onto the surface through ruptures in the cuticle at the interpapillar junctions into the interstices at maturity. This secretion stains positively for protein. Stigmas are of the wet type.The cuticle overlying the papillar cells is ridged and at the final stages prior to flowering this cuticle becomes detached from the underlying cellulosic wall. The sub-cuticular space so formed is filled with secretion. InAneilema species detachment of cuticle is at the papillar tip and along the lateral walls. InCommelina species the anticlinal walls of adjacent papillae are strongly attached for much of their length and thus detachment of cuticle is restricted to the papillar tip. The cell wall at the tip in both genera may proliferate forming a rudimentary transfer-cell type wall. The secretion is considered to be produced by the papillar cells. It is PAS positive but fails to stain for protein and in both the light and electron microscopes appears heterogenous.Pollen attachment, hydration, germination and early tube growth are very rapid following self-pollination, the pollen tubes entering the neck of the style within ten minutes of attachment.A unique character combination involving pollen and stigmas in these genera indicates a monophyletic origin.     

Unusual sperm cells inApiaceae

The sperm cells in the tricellular pollen grains ofCircuta virosa, Bupleurum subovatum, andApium nodiflorum differ significantly from sperm cells known so far. They are extremely destitute of plasma. Besides the sperm nucleus, no cytoplasmic organelles are observed. The wall of the sperm cell forms long, slender projections on both poles of the spindle-shaped cell.     

Some aspects of organization and histochemistry of the embryo sac ofScilla sibirica sato

Summary The present investigation deals with some of the organizational and histochemical aspects of the embryo sac ofScilla sibirica. Both the synergids and egg cell are invested by PAS-positive complete walls. The filiform apparatus comprises an elaborate system of fibrillar projections, showing extensive ramifications. The micropylar region of the embryo sac wall from where the filiform apparatus originates is composed of three distinct layers. On a histochemical basis it may be surmised that, unlike the egg cell, the synergids are metabolically very active. Two kinds of wall ingrowths (i) massive and highly branched very much akin to the filiform apparatus, and (ii) small tuberculate wall projections, are unique to the antipodal cells of S.sibirica. Small tuberculate projections have also been observed along the wall of the central cell adjacent to the nutrient-rich nucellar cells. The antipodals and the central cell show the presence of starch grains and abundant total proteins. All the cell types in the embryo sac ofS. sibirica are structurally so organized as to meet the requirements of its nutrition during pre- and postfertilization development. The presence of abundant PAS-positive granular substance in the cells of nucellar epidermis probably establishes a gradient which assists in the pollen tube growth.     

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.     

Morphological and histochemical differentiation of the pollen wall ofBetula pendula Roth,during dormancy up to anthesis

Summary Anthers ofBetula pendula were collected at regular intervals during the dormancy period until anthesis. Ultrathin sections of maturing pollen grains were especially stained for polysaccharides and proteins and examined with TEM to determine whether structural or/and chemical changes in the pollen wall occur during the dormancy period of the plant life cycle. At the beginning of the dormancy period, the microspore wall consists of a well developed tectum, columellae and a foot layer. Spinules and supratectal elements are prominent. Microchannels are present in the tectum but not obvious in the foot layer. Some of the columellae are not clearly connected with the foot layer, but some connections are evident. Pores are filled with a thick fibrillar network flocculent material. The cytoplasm is packed with starch grains and lipid globules. The stainability for acidic and neutral polysaccharides and protein was tested, and variations in the pollen wall are illustrated. As temperature increased towards the end of dormancy and before anthesis there is obvious differentiation in the morphology of the pollen wall. The granular fibrillar layer beneath the pore and the Zwischenkörper are the most variable part of the wall. Different histochemical reactions observed in different layers at the aperture sites indicate different functions of these layers.     

Structural aspects of in vitro pollen tube growth and micropylar penetration inGasteria verrucosa (Mill.) H. Duval andLilium longiflorum Thunb.

Summary In vitro penetration of the micropyle of freshly isolatedGasteria verrucosa ovules by pollen tube was monitored on agar medium. 40–60% of the micropyles were penetrated, comparable with in vivo penetration percentages. When germinated on agar,Gasteria pollen tube elongation lasts for up to 8 h while plasma streaming continues for about 20–24 h. The generative cell divides between 7 and 20 h after germination, and after 20 h the pollen tube arrives at one of the synergids. The sperm cells arrive after 22 h. The whole process takes more time in vitro than in vivo. In fast growing pollen tubes, a pulsed telescope-like growth pattern of tube elongation is observed. The formation of pollen tube wall material precedes tube elongation and probably prevents regular enlargement of the pollen tube tip-zone. Rapid stretching of the new pollen tube wall material follows, probably due to gradually increased osmotic pressure and the use of lateral wall material below the tip. The stretching ceases when the supplies of plasma membrane and excretable wall material are exhausted. Multiple pollen tube penetration of the micropyle occurs in vitro as it does in vivo. Most pollen tube growth ceases within the micropyle but, if it continues, the pollen tubes curl. Inside the micropyle the pollen tube shows haustorial growth. At the ultrastructural level, the wall thickening of in vitro pollen tubes is quite similar to that in vivo. Before transfer of pollen tube cytoplasm a small tube penetrates one of the synergids. Sperm nuclei with condensed chromatin are observed in the pollen tube and the synergid. In vivo prometaphase nuclei are found in the most chalazal part of a synergid, against the egg cell nucleus and nucleus of the central cell at a later stage. Using media forLilium ovule culture,Gasteria ovules were kept alive for at least 6 weeks. Swelling of the ovule depends on pollen tube penetration. The conditions for fertilization to occur after in vitro ovular pollination seem to be present.     

EXINE DEVELOPMENT IN GERBERA JAMESONII (ASTERACEAE: MUTISIEAE)

The development of the pollen wall in Gerbera jamesonii was studied using light and electron microscopy and histochemical stains. The primexine is patterned while the microspores are encased in the special cell wall. Bacules form at projections of the plasma membrane. Numerous ribosomes and large, single-membrane bound vesicles containing fibrillar material are observed near the developing bacule bases. The tectum and nonhomogeneous layer form simultaneously with the bacules, but do not appear to be outgrowths of them. Following dissolution of the callose cell wall, the lamellate exine-2 is laid down beginning in the apertural region. Polysaccharides are associated with the developing exine-1 and the pore regions of the exine-2. After exine-2 deposition, the exine-1 thickens by the addition of sporopollenin. When the exine is completed, a vacuole forms which displaces the nucleus, compresses the exine-2 and expands the incurved exine-1. As the vacuole shrinks, the intine and storage polysaccharides form.     

POLLEN PORE DEVELOPMENT AND ITS SPATIAL ORIENTATION DURING MICROSPOROGENESIS IN THE GRASS SORGHUM BICOLOR

The spatial relationships observed during microsporogenesis and pollen development in Sorghum bicolor indicate that a strong polarization exists in the anther locule and within individual microspores and pollen grains. During all developmental stages, each sporogenous cell and its derivatives lie continuously adjacent to the tapetum. The microspores and pollen grains form depressions in the tapetal orbicular wall. When the single pore of each microspore is initiated, as a gap in the primexine, it too lies adjacent to the tapetum and remains tightly appressed there until pollen maturity. A sequence of polar phenomena in microspores and pollen grains centers on an axis through the pore and perpendicular to the tapetal surface. These events include migrations of the microspore and vegetative nuclei, initial placement of the generative cell opposite the pore and its later migration, and a polar engorgement process whereby the pore end of the pollen grain (adjacent to the tapetum) fills with starch grains first. The tapetal cytoplasm completely degenerates at precisely the time of pollen engorgement, and its degradation products are believed to be available for pollen uptake at this time. The continuous association of the sporogenous cells or their cellular derivatives and their pores with the tapetum is thought to play an indispensible role in pollen development in sorghum and probably in all other grasses as well. The consistent position of the pore adjacent to the tapetum should be considered another common feature of microsporogenesis in the Gramineae. The characteristic exine pattern forms over the operculum and annulus of the pore, but the lamellae, which underlie the annulus, form a highly modified multilayered nexine. Membrane-like cores are observed in these lamellae and are believed to be involved in the initiation of sporopollenin deposition, but they are obliterated by pollen maturity. Neither the cores nor the lamellae are found in other parts of the pore or in the nonapertured wall.     

Immunolocalization of the cell wall components inPinus densiflora pollen

Summary In order to compare cell wall formation in gymnosperm pollen with that in angiosperm pollen, the distribution of cell wall constituents in the pollen grain and pollen tube ofPinus densiflora was studied immunocytochemically with monoclonal antibodies JIM 5 (against non- or poorly esterified pectin), JIM 7 (against highly esterified pectin), JIM 13 (against arabinogalactan proteins, AGPs), and LM 2 (against AGPs containing glucuronic acid). In the pollen grain wall, only the outer layer of the intine was labeled with JIM 5 and weakly with JIM 7. The tube wall was scarcely labeled with JIM 5 and very weakly labeled with JIM 7. In contrast, the whole of both the intine and the tube wall was strongly labeled with JIM 13 and LM 2, and the generative-cell wall was also labeled only with LM 2. The hemicellulose B fraction, which is the main polysaccharide fraction from the pollen tube wall, reacted strongly with JIM 13 and especially LM 2, but not with antipectin antibodies. These results demonstrate that the wall constituents and their localization inP. densiflora pollen are considerably different from those reported in angiosperm pollen and suggest that the main components of the cell wall ofP. densiflora pollen are arabinogalactan and AGPs containing glucuronic acid.Abbreviations AGPs arabinogalactan proteins - ELISA enzymelinked immunosorbent assay - MAbs monoclonal antibodies     

Silencing of the tobacco pollen pectin methylesterase NtPPME1 results in retarded in vivo pollen tube growth

Sperm delivery in flowering plants requires extensive pollen tube growth through the female sporophytic tissues of the pistil. The apical cell wall emerges as a central player in the control of pollen tube growth, since it provides strength to withstand the internal turgor pressure, while imparting sufficient plasticity to allow cell wall extension through the incorporation of new membrane and wall material. Within this scenario, pectin methylesterases (PMEs; EC 3.1.1.11) emerge as crucial regulators in determining the mechanical properties of pectins, the major component of the apical pollen tube wall. We previously identified NtPPME1, a pollen specific PME from Nicotiana tabacum. Here we show that silencing of NtPPME1 results in a mild but significant decrease of in vivo pollen tube growth while the overall PME activity in pollen is not significantly affected. Although the precise mechanisms responsible for the observed phenotype are not known, it seems likely that the cell must maintain a closely regulated level of PME activity in order to maintain the equilibrium between strength and plasticity in the apical cell wall. A relatively minor disturbance of this equilibrium, as caused by NtPPME1 silencing, compromises pollen tube growth.     

Wall development and its autofluorescence of sterile and fertileVicia faba L. Pollen

Summary The ultrastructural changes of the pollen wall of three types of fertile and one of sterileVicia pollen were related to the autofluorescence of the pollen wall, measured by a microspectroscopic method. Till the liberation of the microspores from the tetrad, the spectrum of the ectexine shows sometimes two maxima and has a very low intensity. After this period the endexine is formed and its spectrum has one maximum with a high intensity. The differences of the pollen wall between the sterile and fertile pollen exist of the presence of one spectral maximum during the tetrad stage, a thick endexine and the absence of the intine in the sterile pollen. The different types show much differences during the tetrad stage in the callose wall as well as the ectexine. The autofluorescence illustrates the complexity and specificity of the pollen wall development.     

Ultrastructural research on cell wall regeneration by cultured pollen protoplasts of Lilium longiflorum

Summary Protoplasts from pollen grains of Lilium longiflorum regenerate amorphous cellulosic cell walls in culture, during which some precursors of cellulose are polymerized, thus producing progressively harder cellulosic cell walls as the period of culture continues. It is presumed that the components of the cell wall regenerated during 1 week in culture differ from those of the intine of the pollen grain wall. The regenerated cell wall is formed by means of large smooth vesicles; in addition, numerous coated vesicles and pits aid in wall regeneration. The pollen tube that germinates from the 8-day-old cultured protoplast has numerous Golgi bodies and many vesicles which build the pollen tube wall. The tube wall has two layers just like a normal pollen tube wall.     

Taxonomic,evolutionary, and functional considerations ofCompositae pollen ultrastructure and sculpture

Two basic patterns of exine ultrastructure are found in theCompositae, the caveate Helianthoid pattern and the non-caveate Anthemoid pattern. TheHeliantheae, Astereae, Inuleae, Sececioneae, Calenduleae andEupatorieae all have pollen with caveate exines. TheMutisiseae, Vernonieae andCardueae have predominately Anthemoid pollen. TheAnthemideae, Arctoteae andLactuceae have pollen with exines of both patterns. Recent investigations of pollen in theVernonieae suggest that these exine ultrastructures in the family have evolved in response to mechanical stresses on the wall which are caused by changes in volume of the grain as it loses or gains water from its environment.     

Exploding pollen in Montrichardia arborescens (Araceae)

Pollen grains of Montrichardia are inaperturate with psilate ornamentation. The pollen wall is formed by a thin ectexine and an extraordinarily thick intine. In living as well as in dead pollen grains contact with water leads to a rapid swelling of the intine followed by an explosive opening of the exine. Within a few seconds a thick tube is formed, which is not the pollen tube. The pollen protoplast is situated at the tip of the tube. These intine tubes are interpreted as pollen connecting tools to keep pollen grains together and adhere them to the cuticle of the hairless pollinators.     

A functional interpretation ofLactuceae (Compositae) pollen

Pollen morphology and ultrastructure inLactuceae pollen is considered in relation to the accomodation of volume changes, pollination biology and exine-held substances. Echinate pollen grains, such as those ofCatananche, are shown to accomodate volume changes by folding along the colpi and possibly by volume changes in the cavea. The different patterns of echinolophate pollen respond in different ways. Folding along the colpi is important inScorzonera andTragopogon and to a limited extent inCichorium andEpilasia whilst inScolymus the colpi are almost immobilized. Movements of the lacunar floors take over the harmomegathic function to compensate for lack of colpus mobility. Bulging of the intine at the apertures and changes in the size of the cavea may account for part of the volume change accomodated in any pollen type. Echinolophate pollen is interpreted as being a superior means of regulating volume changes with the most economical and mechanically efficient use of wall material which has evolved independently in several tribes ofCompositae.     

Immunocytochemical and chemical analyses of Golgi vesicles isolated from the germinated pollen ofCamellia japonica

As a first step towards studying the biochemical relationship between Golgi vesicles (GVs) and tube wall components, isolation of GVs from the growing pollen tubes ofCamellia japonica was attempted using a centrifugation method with mannitol. The isolated GV was identified ultrastructurally and immunocytochemically. The main components of the GV were proteins and carbohydrates. The main monosaccharides of GV polysaccharides were galactose, arabinose and uronic acid, and pectins and arabinogalactan proteins also were detected immunochemically. An antiserum against the isolated GVs reacted with the outer layer of the pollen tube wall and the intine layers of the grain wall as well as thein situ GVs in the pollen tube and the grain cytoplasm. We have thus successfully isolated GVs and shown that they contain pectic substances and arabinogalactan proteins which contribute to formation of the pollen tube primary wall.     

Dimorphic exine sculpturing in two distylous species ofDyerophytum (Plumbaginaceae)

Light and SEM observations on the pollen ofDyerophytum africanum andD. indicum have revealed marked differences in exine features. These distylous species also have dimorphic pollen. In the short-styled individuals of both species, the sexine and nexine are of equal thickness, and the clava-like sexinous processes are short without marked projections. In the long-styled individuals, the sexine is thicker than the nexine, the clavae are higher than broad with an apical spinule. Pollen size and apertures are identical in both morphs. — Palynological evidence is presented for relationships betweenDyerophytum andCeratostigma, Plumbago andAegialitis. Moreover, the genusDyerophytum exhibits pollen morphological similarities with some species ofLinum (Linaceae).     

Ultrastructure and development of the gametophyte vaginula-sporophyte foot complex in the liverwort Targionia hypophylla L.

The development of the placental complex including the gametophyte vaginula and the bulbous foot of the sporophyte in the liverwort Targionia hypophylla L. (Marchantiales) was studied by transmission electron microscopy. The vaginula and foot are separated by an intervening space and each consist of parenchymatous cells without intercellular spaces. Transfer cells begin to differentiate at the gametophyte-sporophyte interface just prior the onset of meiosis. While a single epidermal transfer-cell layer has developed in the foot by the end of meiosis, a multilayered pattern of transfer cells is formed in the vaginula. Gametophyte transfer cells have wall labyrinths which decrease in complexity with distance from the foot, lack plasmodesmata, and show signs of degeneration in the proximity of the foot. During meiosis, amyloplasts of both vaginula and foot lack starch and develop some thylakoid grana. In the subsequent stage of spore maturation, obliteration of the wall labyrinth occurs in both gametophyte and sporophyte transfer cells. The developmental pattern of the placental complex in Targionia is discussed in relation to that of mosses.     

Two callose synthases,GSL1 and GSL5, play an essential and redundant role in plant and pollen development and in fertility

Callose, a β-1,3-glucan that is widespread in plants, is synthesized by callose synthase. Arabidopsis thaliana contains a family of 12 putative callose synthase genes (GSL1–12). The role of callose and of the individual genes in plant development is still largely uncertain. We have now used TILLING and T-DNA insertion mutants (gsl1-1, gsl5-2 and gsl5-3) to study the role of two closely related and linked genes, GSL1 and GSL5, in sporophytic development and in reproduction. Both genes are expressed in all parts of the plant. Sporophytic development was nearly normal in gsl1-1 homozygotes and only moderately defective in homozygotes for either of the two gsl5 alleles. On the other hand, plants that were gsl1-1/+ gsl5/gsl5 were severely defective, with smaller leaves, shorter roots and bolts and smaller flowers. Plants were fertile when the sporophytes had either two wild-type GSL1 alleles, or one GSL5 allele in a gsl1-1 background, but gsl1-1/+ gsl5/gsl5 plants produced an extremely reduced number of viable seeds. A chromosome with mutations in both GSL1 and GSL5 rendered pollen infertile, although such a chromosome could be transmitted via the egg. As a result, it was not possible to obtain plants that were homozygous for mutations in both the GSL genes. Pollen grain development was severely affected in double mutant plants. Many pollen grains were collapsed and inviable in the gsl1-1/gsl1-1 gsl5/+ and gsl1-1/+ gsl5/gsl5 plants. In addition, gsl1-1/+ gsl5/gsl5 plants produced abnormally large pollen with unusual pore structures, and had problems with tetrad dissociation. In this particular genotype, while the callose wall formed around the pollen mother cells, no callose wall separated the resulting tetrads. We conclude that GSL1 and GSL5 play important, but at least partially redundant roles in both sporophytic development and in the development of pollen. They are responsible for the formation of the callose wall that separates the microspores of the tetrad, and also play a gametophytic role later in pollen grain maturation. Other GSL genes may control callose formation at different steps during pollen development.     

凤仙花花药发育中多糖和脂滴组织化学研究

以不同发育时期的凤仙花花药为实验材料,采用组织化学方法,对花药发育中的结构变化及多糖和脂滴物质分布进行观察。结果表明:(1)凤仙花的花药壁由6层细胞组成,包括1层表皮细胞,2层药室内壁细胞,2层中层细胞和1层绒毡层细胞。其中绒毡层细胞的形态不明显,很难与造孢细胞区分,且在小孢子母细胞时期退化。(2)在小孢子母细胞中出现了一些淀粉粒,但减数分裂后,早期小孢子中的淀粉粒消失,又出现了一些小的脂滴;随着花粉的发育,小孢子形成大液泡,晚期小孢子中的脂滴也消失;小孢子分裂形成二胞花粉后,营养细胞中的大液泡降解、消失,二胞花粉中又开始积累淀粉;接近开花时,成熟花粉中充满细胞质,其中包含了较多的淀粉粒和脂滴。(3)在凤仙花的花药发育中,绒毡层细胞很早退化,为小孢子母细胞和四分体小孢子提供了营养物质;其后的中层细胞退化则为后期花粉发育提供了营养物质。