Investigations cytochimiques,marquage immunocytochimique et effets destructeurs des U. V. sur le pollen de Dactylis glomerata L

The elucidation of the ultrastructural cytochemistry, coupled with chemical stabilizing procedures during fixation and embedding, make a significant contribution to the understanding of pollen-exine ontogenesis. A combination of the Con-A agglutination and lanthanum precipitation methods proved particular advantageous during the intine formation stage. By using immunogold techniques, it is possible to demonstrate that only the mature exine of the atmospheric pollen grains reacted positively after sectioning, not the intine. An important difference appears when immature pollen grains are treated under the same conditions: in this case, both the microsporal cytoplasm of the future pollen grain and the immature exine react, but not the intine.

In untreated pollen grains, the exine is a biological material of exceptionally high density with a very low stainability: staining for proteins and lipids is only moderate. A degradation of exine structures by U. V. radiation of 254 nm can be easily proved and the framework obtained is comparable to that induced by some chemical attacks. When sporopollenin is degraded from filamentous sub-units of the exine, stainability increases, and the cytochemical tests for acid-mucopolysaccharids are positive. It is clear that glycocalyx units within exines are chemically bound to the sporopollenin matrix. Attention can also be profitably directed to the future investigation of the function of exine frameworks, through allergen fixation in living pollen grains.     

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.     

云南松小孢子发生的超微结构研究

云南松(Pinus yunnanensis Fr.)小孢子囊发育早期,原生质体发生收缩、同时形成胼胝质壁。小孢子母细胞减数分裂前互相分离,并被胼胝质壁包围。在壁上有约0.2微米的小孔。四分体小孢子形成后,核被膜及高尔基体显得非常地活跃,他们可能与外壁的沉积有关。这个现象在早期四分体阶段出现。内壁的形成是在自由小孢子时期,开始高尔基体和核被膜仍较活跃,但随之下降。内质网和线粒体增多,许多来自内质网的泡囊通过质膜被排放到内壁中去。乌氏体与花粉外壁之间观察到了孢粉素带。     

Sporopollenin monomer biosynthesis in arabidopsis

Land plants have evolved aliphatic biopolymers that protect their cell surfaces against dehydration, pathogens, and chemical and physical damage. In flowering plants, a critical event during pollen maturation is the formation of the pollen surface structure. The pollen wall consists essentially of the microspore-derived intine and the sporophyte-derived exine. The major component of the exine is termed sporopollenin, a complex biopolymer. The chemical composition of sporopollenin remains poorlycharacterized because it is extremely resistant to chemical and biological degradation procedures. Recent characterization of Arabidopsis thaliana genes and corresponding enzymes involved in exine formation has demonstrated that the sporopollenin polymer consists of phenolic and fatty acid-derived constituents that are covalently coupled by ether and ester linkages. This review illuminates the outlines of a biosynthetic pathway involved in generating monomer constituents of the sporopollenin biopolymer component of the pollen wall.     

POLLEN WALL AND TAPETAL ORBICULAR WALL DEVELOPMENT IN SORGHUM BICOLOR (GRAMINEAE)

Pollen wall development in Sorghum bicolor is morphologically and temporally paralleled by the formation of a prominent orbicular wall on the inner tangential surface of the tapetum. In the late tetrad stage, a thin, nearly uniform primexine forms around each microspore (except at the pore site) beneath the intact callose; concurrently, small spherical bodies (pro-orbicules) appear between the undulate tapetal plasmalemma and the disappearing tapetal primary wall. Within the primexine, differentially staining loci appear, which only develop into young bacula as the callose disappears. Thus, microspore walls are devoid of a visible exine pattern when released from tetrads. Afterwards, sporopollenin accumulates simultaneously on the primexine and bacula, forming the exine, and on the pro-orbicules, forming orbicules. Channels develop in the tectum and nexine, and both layers thicken to complete the microspore exine. Channeled sporopollenin also accumulates on the orbicules. A prominent sporopollenin reticulum interconnects the individual orbicules to produce an orbicular wall; this wall persists even after the tapetal protoplasts degenerate and after anthesis. While the pollen grains become engorged with reserves, a thick intine, containing conspicuous cytoplasmic channels, forms beneath the exine. Fibrous material collects beneath the orbicular wall. The parallel development and morphological similarities between the tapetal and pollen walls are discussed.     

Conventional and novel modes of exine patterning in members of the Araceae – the consequence of ecological paradigm shifts?

Summary. In the family Araceae, the members of all subfamilies except Aroideae follow the conventional mode of exine formation pattern, which conforms with the textbook view of sporoderm stratification and chemistry (sporopollenin ektexine formed before the endexine). Only members of the subfamily Aroideae show a quite uncommon mode of exine formation pattern, with an endexine formed prior to the nonsporopollenin, polysaccharidic outer exine layer. The intine is formed simultaneously with this non-sporopollenin layer. From the differing timetable and especially from the different origin it is concluded that this outer exine layer is not homologous to the angiosperm ektexine. The fundamental question, why members of the Aroideae lack an elaborated sporopollenin ektexine, is discussed in terms of functionality of the nonsporopollenin outer exine layer. It seems that a major change in aroid evolution took place at the point when the family phylogenetically and ecologically shifted from bisexual (most subfamilies) to unisexual flowers (Aroideae only). The hypothesis is that ephemeral spathes and the absence of sporopollenin are the consequence of an adaptive syndrome for a short pollination time window in many members of the Aroideae, with short-lived pollen, an energetically not costly pollen wall, rapid germination of pollen tube, and brief receptivity of stigma. Correspondence and reprints: Institute of Botany, University of Vienna, Rennweg 14, 1030 Vienna, Austria.     

The Structure of Pollen Wall and Its Relation to Po41en Aggregation in Cymbidium goeringii (Rchb. F.) Rchb. F.

The pollen wall of tetrads located in different positions of a mature pollinium of Cymbidium goeringii was examined with the electron microscope, and the compositions of wall materials were also tested with different histochemical methods. In all tetrads of a pollinium, the pollen wall can be distingished into an exine and an intine, but the exine may be varied greatly according to the tetrad position in a pollenium. The part of the pollen wall (the outer wall) of the external tetrads, lying close, to the tapetum, is composed of two layers, i.e. the exine, and the intine. Theexine consists of tectum, granulate ectexine and endexine, without foot layer. The intine is cellulose in nature. In the outer wall between different groups of: tetrads and in the inner wall within an individual tetrad, the structure of ectexine becomes simple and the deposition of sporopollenin is roduced The degree of reduction of ectexine nicreases from the outer to inner tetrads in several external layers of a pollinium, and even the internal tetrads have a reduced ectexine or lack of it. The present study also demonstrates that the mechanism of pollen aggregation into a pollinium is built on a combined effect of the following features: (1) connected bridges formed' by intine between two pollens within a tetrad, (2) formation of cytoplasmic channels between two pollens within a tetrad, (3) incomplete cell wall formation within a tetrad, (4) little size of tetrads and compact arrangement of mature tetrads and (5) a sticky viscin material surrounded on the outside of a pollinium.     

ISOLATION OF EXINES FROM GYMNOSPERM POLLEN

Exines of certain Gymnosperms spontaneously separate from the intine during the process of hydration preceding pollen germination. Exines of pollen of Calocedrus decurrens, Chamaecyparis lawsoniana, Juniperus occidentalis, Sequoia sempervirens, and Pseudotsuga menziesii were isolated from hydrated, autoclaved pollen. Free exines were purified by centrifugation on a discontinuous sucrose gradient of densities 1.14 to 1.27 g/ml. The outer intine dissolved on autoclaving. This method may be applicable to a wide range of genera. Purified exines are of potential use in chemical analyses of sporopollenin and in production of antibodies to exine.     

ONTOGENETIC AND EVOLUTIONARY IMPLICATIONS OF A NEOTENOUS EXINE IN TAPEINOCHILOS (ZINGIBERALES: COSTACEAE) POLLEN

Tapeinochilos pollen, like that of most angiosperms, is spared by the standard acetolysis treatment because the sporoderm is impregnated with sporopollenin. This genus and its allies in the Costaceae are the only taxa in the eight families of Zingiberales that have acetolysis-resistant pollen. The sporoderm in most of the order is characterized by exine reduced to a wispy coating or layer with delicately anchored spinules and a highly elaborated intine. Ultrastructural studies on the pollen of Tapeinochilos reveal a pattern of wall development that is significantly different from the generalized angiosperm type; namely, there are no columellae, nor is there any significant accretion of sporopollenin following the dissolution of callose and release of microspores. The primexine is composed of rodlets which build up solidly between apertures and become packed into layers to form a thick, stratified exinous covering. No secondary exine develops during the free spore period and the juvenile primexine persists as the protective coat on the mature pollen grain. This pattern of pollen development is viewed as an example of neoteny in which a juvenile or immature character is retained in adulthood.     

Pollen Tetrads of Hedycarya arborea J. R. et G. Forst. (Monimiaceae)

Hedycarya has pollen in permanent tetrads. H. arborea, the New Zealand species, differs from others studied, in having a cap of more or less imperforate tectum at the distal pole of each grain. This polar region is not an aperture and the pollen tube emerges through a papillose part of the external wall of each grain. Transmission electron microscope studies of immature and mature tetrads reveal a most unusual exine structure. "Radial processes" develop by accumulation of sporopollenin around unit membranes of similar dimensions to the plasmalemma, and extend from just beyond the intine to the tectal region. The entire exine is considered ectexinous. During development, members of a tetrad are interconnected by cytoplasmic channels and the synchronous division into generative and vegetative nuclei within each tetrad is attributed to their presence. The channels become closed by the deposition of intine. Comparisons are made with exine structure in some other members of the woody Ranales and with some other plants with tetrad pollen.     

Preparation and fusion properties of protoplasts from mature pollen of Nicotiana tabacum

Summary A method to remove the exine from mature tobacco pollen and to release numerous intact pollen protoplasts has been developed. Post-anthesis binucleate pollen was treated with water, buffered with MES at pH 5.5, for two hours. Rupture of the exine was caused by the force of pollen hydration exposing the intine to subsequent enzymatic maceration. The high osmotic pressure (1000 mOsm·kg^-1 H₂O) of pollen protoplasts required a special maceration medium, 4% KCl (w/v). Action of an enzyme solution containing 1% (w/v) Macerozyme and 1% (w/v) Cellulase gave rise to viable protoplasts within 4 hours. When cultured in a tobacco mesophyll protoplast culture medium, the pollen protoplasts underwent regeneration of a cell wall, formation of various tube-shaped structures, and division of the generative nucleus into two nuclei. Using a PEG/Ca²⁺ method pollen protoplasts were fused with diploid mesophyll protoplasts. Evidence of transfer of chloroplasts into the pollen protoplasts was observed after one day of culture.Abbreviations BCP bromocresol purple - FDA fluoresceindiacetate - MES 2-(N-morpholino) ethanesulfonic acid - PEG polyethyleneglycol     

Outer Layers of the Azotobacter vinelandii Cyst

Ruthenium red stained a capsule external to the exine of the Azotobacter cyst. The central body is therefore surrounded by three layers, the intine, the exine, and the capsule, all containing acid mucopolysaccharide. Vesicles that appear to originate from the contracting cell membrane of the central body may account for the lipid content of the intine. The exine is composed of laminated sheets that tend to fragment into hexagonal pieces.     

Diffusion barriers of tripartite sporopollenin microcapsules prepared from pine pollen

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

A new look at the acetolysis method

The acetolysis method intreduced byGunnar Erdtman is still a very welcome and highly successful technique in palynology. However, acetolysis destroys all pollen material with the exception of sporopollenin that forms the outer pollen wall, the exine. Modern palynology in its application to plant systematics and phylogeny must consider all sporoderm characters, not only those of the exine. The neglect of the intine may distort some principal palynological aspects. This is illustrated by cases of total breakdown or gross modification of thin exine structures (e.g. inBeilschmiedia, Strelitzia) and by the clarification of apertures (e.g.,Polyalthia, Fissistigma, Calluna). In our view the investigation of both acetolysed and non-acetolysed pollen is obligatory for a well balanced view of pollen structure and function.     

ULTRASTRUCTURE AND DEVELOPMENT OF THE NEXINE AND INTINE IN THE POLLEN WALL OF SILENE ALBA (CARYOPHYLLACEAE)

Nexine and intine development in Silene alba (Caryophyllaceae) was investigated by electron microscopy and enzyme cytochemistry. Nexine-2 forms by deposition of sporopollenin along unit membrane lamellae closely associated with the microspore plasma membrane in the late tetrad stage. After the callose wall dissolves, electron density increases along the tangentially oriented fibers of the proximal primexine, forming nexine-1. When the exine is essentially complete, the intine begins to develop. In the nearly mature microspore, acid phosphatase activity appears in the peripheral cytoplasm just prior to its extrusion into the intine of the mature pollen grain.     

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     

云南松花粉形态研究

在云南松(Pinus yunnanensis Fr.)小孢子发生发育过程中,花粉母细胞、四分孢子及花粉粒均见有粘连现象。花粉气囊的形态、大小变化复杂多样。除一般具两个正常气囊的花粉粒外,还观察到气囊不发育、具一个气囊、二个异形气囊、三个气囊和四个气囊的花粉粒。成熟花粉壁从外至内可分为外壁外层、外壁内层、内壁外层和内壁内层,它们的构成成分及形态均有明显差别。贮存后花粉的内壁结构发生了明显变化。     

Development of the pollen grain wall: Further work withTradescantia bracteata

Summary Development of the pollen wall inTradescantia is examined between the tetrad stage and maturity, in the light of present controversy concerning the role of the tapetum in exine secretion, and the matter of the control of this process.The view that the exine in this plant is secreted entirely by the microspore protoplast is further substantiated, and is discussed in relation to sporopollenin production in general. An hypothesis is advanced to explain the mode of exine growth observed in all plants.Evidence is presented to support the contention that the sporophyte controls exine secretion through moieties inherited from the mother cell which are present in the spore cytoplasm.The differential phasing of exine development, observed to occur between species, is discussed in relation to general phase differences in pollen development, and the physiological condition of the whole plant.The plasticity of the intine inTradescantia is reported, and its importance considered in relation to the rigidity of the exine and the consequent disruption of that wall during the growth of a pollen grain.     

Re-Exposure of Tapes at High Temperature and Pressure in the Lycopodium Clavatum Spore Exine

Lamellations are visualizable through the staining commonly used in transmission electron microscopy during exine formation on Lycopodium and other spores, and the nexine of pollen grains. The lamellations so exposed consist-of dark tapes at either side of an unstained (white) line. Neither tapes nor white lines are visualizable in the exine of mature spores of Lycopodium. The continued presence of lamellations having tape-white line spacing has been demonstrated with inorganic tracers in the nexine of pollen in which lamellations otherwise appeared to be absent. Through high contrast staining methods for TEM we have observed lamellations in the residual exine following heat treatment (350[ddot]C) of mature spores of Lycopodium clavatum. The surface of these residual exines was etched by treatment with hot 2-aminoethanol and filaments were observed to protrude from the etched surfaces. The residual exine stained darkly. Lycopodium spores heated to 300[ddot]C at 1 kb pressure had long filaments exposed at the surface of the residual exine (sporopollenin). Sections of the pellet remaining after heat and pressure treatment also included bundles of closely parallel filaments and masses of isolated filaments. The filaments were stained while the exine residue, assumed to include sporopollenin, was not. Isolated filaments produce a stable metachromasia with toluidine blue indicating the presence of many closely spaced sites of negative charge. The staining of intact exines with basic dyes may result from anionic sites on filaments embedded within the exine rather than being due to sporopollenin. The results of our experiments indicate that the filaments are more resistant to heat and pressure and 2-aminoethanol than is sporopollenin. We propose that the trilamellar elements commonly called tapes and white lines might be composed of two filaments bridged by polybasic molecules.     

Towards male sterility in Pinus radiata--a stilbene synthase approach to genetically engineer nuclear male sterility

A male cone-specific promoter from Pinus radiata D. Don (radiata pine) was used to express a stilbene synthase gene (STS) in anthers of transgenic Nicotiana tabacum plants, resulting in complete male sterility in 70% of transformed plants. Three plants were 98%-99.9% male sterile, as evidenced by pollen germination. To identify the stage at which transgenic pollen first developed abnormally, tobacco anthers from six different developmental stages were assayed microscopically. Following the release of pollen grains from tetrads, transgenic pollen displayed an increasingly flake-like structure, which gradually rounded up during the maturation process. We further investigated whether STS expression may have resulted in an impaired flavonol or sporopollenin formation. A specific flavonol aglycone stain was used to demonstrate that significant amounts of these substances were produced only in late stages of normal pollen development, therefore excluding a diminished flavonol aglycone production as a reason for pollen ablation. A detailed analysis of the exine layer by transmission electron microscopy revealed minor structural changes in the exine layer of ablated pollen, and pyrolysis-gas chromatography-mass spectroscopy indicated that the biochemistry of sporopollenin production was unaffected. The promoter-STS construct may be useful for the ablation of pollen formation in coniferous gymnosperms and male sterility may potentially be viewed as a prerequisite for the commercial use of transgenic conifers.