Figure 1.
Macromorphology of secondary pollen presenter in closed and open flower.
(A) Stereomicrograph of flower bud with the inferior ovary (o) and corolla (c) with some attached stamens cut away longitudinally to show the still-closed, dorsifixed anthers (a) at the same level as, and closely appressed to, the surface of the secondary pollen presenter (p) which terminates the style (s). The stigmatic surface is initially covered by the upper sterile portion of the anthers. The calyx lobes (ca) are relatively short. A prominent fringe of mainly downwardly directed hairs (h) lines the corolla tube. (B) Stereomicrograph of flower shortly after anthesis to show the spreading corolla lobes and pollen presenter carrying the pollen (yellow) above the rest of the flower (labels as in A). Shortly before anthesis, while still in bud, the anthers dehisce introrsely to release the pollen which adheres particularly to shallow longitudinal grooves in the pollen presenter. Some of the fringing hairs are directed upwards and protrude from the mouth of the corolla tube.
Figure 2.
Micromorphology of stylar head complex.
SEM micrographs illustrating surface topographical features, both in freshly-fixed, critical point dried material (A & B), and in unprocessed samples from herbarium specimens (C & D). In Vanguerieae the stigma (s) terminates the pollen presenter (p), the whole being referred to as the “stylar head complex”. In freshly-fixed pollen presenters taken from buds just prior to anther dehiscence, as in the case of A and B, the outer surfaces are largely devoid of adherent material. (A) Stylar head complex from an almost mature flower bud showing the slightly longitudinally-grooved pollen presenter and the terminal stigmatic lobes. (B) Enlarged portion of the stylar head complex illustrating the sharp boundary between the pollen presenter with its relatively large, convex, outer tangential epidermal cell walls, and the stigmatic lobes with their much smaller, rather papillate epidermal cells. (C) Surface of pollen presenter from an open flower showing secretions (initially somewhat sticky), especially in sinuses between epidermal cells. (D) Enlarged surface of pollen presenter showing secretions apparently exuded from the epidermal cells; these appear globular, or thread-like and often somewhat moniliform.
Figure 3.
Anatomy of stylar head complex.
LM transverse and longitudinal sections illustrating the anatomy of the pollen presenter (p) and associated structures; GMA-embedded, stained with periodic acid Schiff/toluidine blue. (A) Segment of a transverse section through a flower bud showing position of corolla lobes (c) and a still intact anther (a) with four pollen-filled microsporangia, two of the latter appressed to, and fitting into the longitudinal grooves of, the centrally placed pollen presenter (p). Note ring of well-developed vascular tissue (vessels without stained contents) in central parts of the presenter. Epidermal cells (e) of the pollen presenter are radially elongated (appearing multiseriate due to slightly oblique angle of section). Note darkly stained cell wall thickenings of Igersheim, some indicated with thin (on outer and inner periclinal cell walls) or thick (bands encircling anticlinal cell walls) arrows. Pollen grains (pg) have similar darkly stained protruding onci (compare Figure 12). (B) Stylar head complex in longitudinal section to show the pollen presenter with recessed style. Note prominently enlarged and radially elongated epidermal cells, as well as the terminally placed stigma (s), the latter with a single adherent pollen grain; labels as in A.
Figure 4.
Anatomy of pollen presenter epidermis.
LM transverse sections illustrating the anatomy of the pollen presenter epidermis (e); GMA-embedded, stained with periodic acid Schiff/toluidine blue. Wall thickenings of Igersheim are amorphous and stain dark purple-pink. Some of the thickenings are denoted by either single- (those of outer and inner tangential walls) or double-headed (bands of Igersheim) arrows. (A) Epidermis comprising of markedly radially-elongated cells and overlying the mesophyll with its much smaller and isodiametric parenchyma cells. Epidermal cells of longitudinal grooves are radially slightly shorter than those of the ridges. Three arrowheads (without shafts) point to epidermal cells in which are visible traces of one or more radial pectinaceous strands linking the band of Igersheim and the outer tangential cell wall thickening (most obvious in lowermost cell). These difficult-to-see radial strands are visible in sections of anticlinal walls of epidermal cells cut parallel with the surface. (B) Epidermal cells showing the wall thickenings of Igersheim. Thickenings of inner tangential walls often extend distally for a short distance along the anticlinal walls.
Figure 5.
Histochemical staining of epidermal cell walls.
Transverse sections of GMA-embedded pollen presenters treated with various histochemical stains to establish the composition of the wall thickenings of Igersheim in the epidermis; double-headed arrows indicate position of bands of Igersheim. (A) Auramine O under CLSM; this stain is a fluorescence enhancer with a strong affinity for regions containing acidic and unsaturated waxes, as well as cutin precursors and suberin. Relatively thin cuticle covering the outer anticlinal walls clearly visible because of much stronger autofluorescence. Bands of Igersheim (asterisks) show no reaction, indicating absence of cutin and suberin. (B) Tannic acid-ferric chloride under LM. Black colour of walls and thickenings indicates the presence of calcium pectate. (C) IKI under LM; starch grains stain blue-black (not illustrated). Unexpectedly, the wall thickenings of Igersheim stain prominently pale blue, most probably indicating the presence of xyloglucan. (D) Ruthenium red under LM. Cell walls, and in particular wall thickenings, stain bright red, thus confirming the presence of de-esterified pectin. Mesophyll cell walls adjacent to the epidermal cells also stain intensely.
Figure 6.
Immunolocalization of pectic homogalacturonan (HG) epitopes in epidermal cell walls.
CLSM images of transverse sections of the pollen presenter epidermis. Double-headed arrows indicate position of bands of Igersheim. (A) Wax-embedded material immunolabelled with LM19 for de-esterified HG. This form of HG is mainly confined to those thickenings of Igersheim present in the outer tangential cell walls of the epidermal cells (for better resolution see C). Fluorescence of protoplasm in mesophyll cells due mainly to autofluorescence of chloroplasts. (B) Wax-embedded material immunolabelled with LM20 for methyl-esterified HG. This form of HG is abundantly present in primary cell walls as well as all the wall thickenings of Igersheim. (C) GMA-embedded material immunolabelled with LM19 and showing enlarged portion of distal end of epidermal cells. De-esterified HG mainly confined to outer portion (derived from primary cell wall?) of those thickenings of Igersheim present in the outer tangential cell walls of the epidermal cells, but also present in primary cell walls (middle lamella?) and outer boundary of bands of Igersheim (or electron-transparent zone between plasmalemma and wall thickenings—compare Figure 8), but essentially lacking in most of the bands themselves (bands marked with asterisks). (D) Wax-embedded material immunolabelled with LM20 and showing enlarged portion of distal end of epidermal cells. Methyl-esterified HG abundantly present in the bands of Igersheim, as well as throughout thickenings of the outer tangential cell walls.
Figure 7.
Ultrastructure of outer tangential cell wall thickenings and adjacent cuticle and protoplast of epidermal cells.
TEM images of material postfixed in osmium tetroxide, embedded in SPI 812, and viewed in transverse section. (A) Outer tangential cell wall showing cuticle (cu) and slightly different electron-dense layers demarcated as the remains of the original primary cell wall (cw), thickening of Igersheim (ti), and an electron-transparent zone (et) (enlarged in C) between the wall and the plasmalemma (pl). The boundary between the primary wall and the thickening of Igersheim is not clear because the two layers merge gradually. Note extracellular deposits, probably including sticky subtances, on the cuticle. Visible in the cytoplasm are, among others, mitochondria (mi) and chloroplasts (ch) with starch grains (st). (B) Cuticle pervaded by a branched system of electron-dense fibrillar material, most probably representing microchannels. (C) Electron-transparent layer (compare A) between the plasmalemma and thickening of Igersheim showing several secretory vesicles. These vesicles are often seen to be fused to the plasmalemma and are evidently derived from the cytoplasm through exocytosis. It is suggested that these vesicles contain materials used in the formation of the cell wall thickenings; most probably also sticky secretions (compare Figure 2C, D) exuded from the cuticle surface. In young epidermal cells, and before the formation of wall thickenings (not illustrated), the cytoplasm next to the plasmalemma contains an abundance of rough endoplasmic reticulum; some of the ribosomes are still visible in the image; labels as in A.
Figure 8.
Ultrastructure of bands of Igersheim.
TEM images of material postfixed in osmium tetroxide, embedded in SPI 812, and viewed in transverse section. Shown are the distal portion of two adjacent epidermal cells (ec-1 and ec-2), primary cell walls (cw), thickenings of Igersheim (ti) affecting tangential cell walls, and bands of Igersheim (bi). (A) Distal portion of two bordering epidermal cells showing a common band of Igersheim in transverse section. These bands develop in areas of primary wall rich in plasmodesmata, the remains of which persist as electron-dense transverse areas towards the centre (compare C). Transverse section through the distal portion of a band of Igersheim between two epidermal cells. Note faintly longitudinally-layered fibrillar structure of matrix and surrounding electron-transparent zone (compare Figure 7) between plasmalemma and band; labels as in A. (C) Enlarged central portion of a band of Igersheim (bi) between two epidermal cells showing electron-dense transverse zones reflecting the prior position of plasmodesmata in the common primary cell wall.
Figure 9.
Ultrastructure of contact zone between epidermis and mesophyll.
TEM image of material postfixed in osmium tetroxide, embedded in SPI 812, and viewed in transverse section. Shown are the proximal ends of two epidermal cells (ec) bordering three mesophyll cells (mc) and associated intercellular spaces (is). Thickenings of Igersheim (ti) are restricted to the walls of the inner tangential walls of the epidermal cells; the thickenings may affect only certain parts of the wall. Note single plasmodesma (pd) (compare Figure 10) in the thickening of Igersheim. Vacuoles of mesophyll cells filled with numerous osmiophilic droplets.
Figure 10.
Ultrastructure of inner tangential wall thickenings of epidermal cells.
TEM image of material postfixed in osmium tetroxide, embedded in SPI 812, and viewed in transverse section. Enlarged thickened inner tangential cell wall of an epidermal cell (ec) bordering two mesophyll cells (mc). Position of primary cell wall is still visible as a slightly more electron-dense layer to the outside of the thickening of Igersheim (ti). Note distinct plasmodesma (pd) traversing the thickening. Such well-defined plasmodesmata have not been observed in the bands of Igersheim (compare Figure 8) and seem to be confined to thickenings in the inner tangential walls of the epidermal cells. The mesophyll cells are rich in chloroplasts (ch) and mitochondria (m; rather faintly stained in this image).
Figure 11.
Immunogold labelling of pectic homogalacturonan (HG) epitopes in epidermal cell wall thickenings.
TEM image of material embedded in LR White and examined unstained in transverse section. Short transverse lines along right-hand edge of images demarcate approximate boundaries between the cuticle (cu) and the cell wall thickening comprised of the primary cell wall (cw) and thickening of Igersheim (ti), the latter two layers intergrade and cannot be distinguished, especially in these unstained sections. (A) Outer tangential cell wall of epidermal cell immunolabelled with LM19 for de-esterified HG. This form of HG is present in most parts of the wall, but absent from the cuticle. (B) Outer tangential cell wall of epidermal cell immunolabelled with LM20 for methyl-esterified HG. This epitope is likewise absent from the cuticle, but mainly confined to the central portion of the wall thickening in this particular section. However, this distribution pattern is very variable and in other sections the methyl-esterified HG may be spread more or less throughout the wall thickening. (C) Enlarged portion of a band of Igersheim immunolabelled with LM20 for methyl-esterified HG. This epitope is abundantly and uniformly present throughout the thickening. This contrasts with de-esterified HG which is very sparsely present in these bands (not illustrated, but confirming pattern depicted in Figure 6A & C above).
Figure 12.
Morphology and contents of mature pollen grains.
Mature pollen grains are suboblate to spheroidal, isopolar, radially symmetrical and 3-porate. A prominant globular protruding oncus projects from each aperture. For most of their development, the pollen grains do not contain starch grains. However, before being shed from the anthers, large numbers of starch grains become visible inside the pollen grains, as well as in the cytoplasm towards the distal end of the epidermal cells of the pollen presenter. Following the shedding of the pollen grains, most of the starch rapidly disappears from the grains, subsequently also from most of the epidermal cells. (A) SEM micrograph of a mature pollen grain after being shed from the anther, showing the three protruding onci. The tectum is perforate with a tendency towards microreticulate, but in unacetolyzed grains exine sculpturing is usually concealed by a layer of secretions. (B) LM image of a transverse section of pollen grains still in the anther, but shortly before being shed; GMA-embedded, stained with periodic acid Schiff/toluidine blue. At this stage the pollen grains contain abundant starch grains (stained dark pink-purple to black). The pollen grains have prominent protruding onci (stained dark purple-black) and are surrounded by the remains of the tapetum (t) on the inside, followed by the endothecium (en) and then the epidermis of the microsporangium on the outside. The anther is shown in close proximity to the epidermal cells (e) of the pollen presenter, the latter also with minute starch grains concentrated in the cytoplasm below the outer tangential cell walls of the epidermal cells. The arrow denotes a band of Igersheim. (C) LM image of a transverse section of pollen grains with protruding onci shortly after having been shed from the anther; GMA-embedded, stained with periodic acid Schiff/toluidine blue. Also shown are the associated epidermal cells (e) of the pollen presenter. At this stage most of the starch grains have disappeared from the pollen grains. However, minute starch grains are still abundantly present in the cytoplasm of the epidermal cells, concentrated between the distally located bands of Igersheim (double-headed arrow) and the outer tangential cell wall.
Figure 13.
Development of the protruding onci in pollen grains.
LM transverse sections of anthers showing two stages of immature pollen grains (pg), and a mature grain, in the microsporangium; GMA-embedded, stained with periodic acid Schiff/toluidine blue. The protruding onci only make their appearance at a fairly advanced stage of pollen grain development. The microsporangium is lined with a prominent, multilayered, partly amoeboid tapetum (t). At least the inner cells of the tapetum degenerate as the pollen approaches maturity and are suspected to deposit, amongst others, sticky secretions on the grains. (A) Young pollen grains having reached more or less mature size, but still without protruding onci. A small, dark blue spot below each aperture, as well as an associated thickening of the intine (oncus), indicates the point from which the protruding oncus is about to develop. A deposit (stained pale bluish) is visible on the outer surface of the grains. (B) Later developmental stage than in A, showing the first appearance of the protruding onci. Initially the walls of the protruding onci stain less intensely than in mature grains (compare C). (C) Mature pollen grain just before it is shed, showing two protruding onci and starch grains. The intine lining the exine and the protruding onci is an essentially continuous layer.
Figure 14.
Histochemical staining and immunohistochemistry of pollen grains.
Transverse sections of GMA- or wax-embedded pollen grains treated with histochemical stains and fluorescent monoclonal antibodies to elucidate the composition of the protruding onci (marked in some images with an asterisk). (A) Auramine O in GMA-embedded material under CLSM. Exine of pollen grains show enhanced autofluorescence, but the protruding onci show no reaction, indicating absence of cutin and suberin. (B) Ruthenium red in GMA-embedded material under LM. Protruding onci stain bright red, thus confirming the presence of pectin. (C) Wax-embedded material immunolabelled with LM19 for de-esterified pectic homogalacturonan (HG) and viewed under CLSM. This form of HG is mainly confined to the outer layers of the protruding onci, and possibly also (though sparsely so) in the exine and intine. Exine visibility enhanced due to it being autofluorescent. (D) Wax-embedded material immunolabelled with LM20 for methyl-esterified HG and viewed under CLSM. This form of HG is apparently absent from the protruding onci, and seemingly also the rest of the pollen grain. Exine clearly visible due to autofluorescence.