Callose Wall Research Articles

PATTERN DETERMINATION OF THE EXINE IN CAESALPINIA JAPONICA (LEGUMINOSAE: CAESALPINIOIDEAE)

Exine development in Caesalpinia japonica Sieb. et Zucc. (Leguminosae) was studied by a combination of transmission electron microscopy (TEM) and field emission scanning electron microscopy (SEM) using a freeze‐fracture method, with special attention to the initial process of exine pattern formation. The present study confirmed that the exine pattern is determined by the plasma membrane of microspores enclosed in the callose wall at the early tetrad stage. The plasma membrane, exclusive of the future apertures, invaginates and takes the form of a reticulate pattern. The reticulate pattern corresponds to the mature exine ornamentation. Protectum is the first to be laid down on the reticulate patterned plasma membrane. Probacules are initiated under the protectum and elongate basally on protruding sites of the plasma membrane. Primexine matrix is formed in coincidence with the probacules. After the protectum and probacules are completed within the callose wall, the invaginating plasma membrane becomes smooth. After the dissolution of the callose wall, endexine is organized by the accumulation of lamellated structures, and a foot layer is formed by the deposition of nonlamellated components on the developing endexine.

Pollen wall formation in Abies concolor and a discussion on wall layer homologies

Development of the pollen wall in Abies concolor (Gord. & Glen.) Lindl. is documented using ultrastructural techniques. Pollen wall formation is initiated by the deposition of a microspore surface coat and the formation of callosic protrusions in the proximal and lateral areas, outside the plasma membrane of the microspores enclosed in tetrads. The alveolar ektexine is then elaborated on these structures. Endexine formation starts with the appearance of tripartite lamellae to the inside of the microspore surface coat around the entire microspore. Both the ektexine and endexine are elaborated while the microspores are enclosed in tetrads by the callose wall. These results are utilized in assessing homologies between various wall layers in gymnosperm and angiosperm pollen. In considering criteria of homology, pollen wall development is discussed as a causal sequence and this discussion concludes that the ektexine and endexine in gymnosperm pollen are homologous with the ektexine and endexine in angiosperm pollen.

Development of the echinate pollen wall inFarfugium japonicum (Compositae: Senecioneae)

Development of the echinate pollen grains inFarfugium (Compositae: Senecioneae) has been studied by a combination of transmission electron microscopy and field emission scanning electron microscopy with a freeze fractured method. The inner surface of the callose wall surrounding each microspore does not possess an echinate pattern before primexine deposition begins. The primexine formation coincides with the initiation of spines. The freeze fractured primexine shows probacula which form transverse rods. The developing exine has an inner spongy substructure. The endexine is formed by the accumulation of the electron dense lamellae with white lines after the dissolution of the callose wall. In the present study, it is confirmed that the developmental process of pollen formation revealed in the field emission scanning electron microscope is consistent with the results obtained using the transmission electron microscope.

Pollen tube tip growth.

This review considers pollen tube growth with regard to current information on pollen tube cytoplasm, wall structure and calcium ion interactions with pollen tubes. Pollen tubes have a marked cytoplasmic Polarity with a number of distinct zones along the tube, each with a characteristic complement of cytoplasmic and nuclear structures. The cytoplasmic structures are characteristic of secretory cells with extensive endoplasmic reticulum cisternae and numerous dictyosomes. The dictyosomes produce secretory vesicles that are mainly directed to the extending tip of the tube, where they provide new plasma membrane and wall components. The rates of secretory vesicle production and delivery have been estimated, allowing quantitative assessments of the rate of delivery of materials to the tip. Pollen tubes contain cytoskeletal components, with microtubules and microfilament strands lying axially in the main tube and diffuse microfilament strands at the tip. The tube wall consists of an outer fibrous layer containing pectins and an inner, more homogeneous layer containing callose and cellulose-like microfibrils, possessing both β-1,4 and β-1,3 linkages. Protein is also present in the wall. The tube tip lacks the inner callosic wall. This type of structure is considered to be different from that of elongating sporophyte tissue cells which are enclosed by a wall containing layers of cellulose microfibrils. Calcium ions are required for pollen tube growth and, in at least some species, act as a chemotropic agent. High concentrations of calcium ions in the external medium inhibit growth. Pollen tubes contain some calcium ions bound to the cell wall and larger amounts located intracellularly, which enter the tube at the tip. This intracellular calcium is present as ions that exist freely within the cytoplasmic Matrix and as ions bound to membrane systems. The highest concentrations in both of these pools are found at the tip and in both they decline towards the base. The structure of the tip and the activity involved in providing components for plasma membrane and Wall assembly provide a basis for considering possible mechanisms of tip growth. Two hypotheses to account for the regulation of tip extension are considered, cell wall control and cytoskeletal control. In the cell wall hypothesis, control depends on an interaction between internal turgor pressure and a plastic cell wall. The mechanical properties of the wall are assumed to be partly dependent on the availability of external calcium ions to crosslink acidic pectin chains. According to this hypothesis, high external calcium ion concentrations cause cessation of tip growth due to increased mechanical resistance of the tip wall. Various observations on plant cell-wall interactions with calcium ions and on experimentally-treated pollen tubes provide evidence that does not support this hypothesis. The cytoskeletal control hypothesis of tip growth depends on the internal tip cytoskeleton to contain the tube tip cytoplasm against the internal turgor pressure during cell wall assembly. The activities and mechanical properties of the cytoskeleton are assumed to depend on the availability of external calcium ions. High external concentrations are believed to cause a state of rigor in the cytoskeleton and hence a cessation of tip growth. Some experimental evidence is presented which suggests that the effects of excess calcium ions are on intracellular processes, and not extracellular ones. The mitochondrial zone behind the tip is believed to maintain the tip calcium ion concentration at an optimal level for growth. Some comparisons are made between tip growth in pollen tubes and that in other tip growing cells. CONTENTS Summary 323 I. Introduction 324 II. Cytoplasm 326 III. Wall structure 332 IV. Calcium 335 V. Tip growth 339 VI. Conclusions 350 References 351.

Ultrastructural localization of acid phosphatase during male meiosis and sporogenesis inDatura: Evidence for digestion of cytoplasmic structures in the vacuoles

Using a combination of electron microscopic and cytochemical techniques, the activity of the lytic enzyme acid phosphatase during male meiosis and sporogenesis inDatura innoxia Mill, has been followed. Evidence for digestion of cytoplasmic structures such as ribosomes and membranes during meiotic prophase and in the vacuolate microspores are presented. Before the onset of meiosis, acid phosphatase activity was observed chiefly in the walls of the pollen mother cell. During meiosis, due to the presence of a thick callose wall around the meiocytes, the enzyme activity could not be detected although the presence of numerous “autophagic type vacuoles” suggest lytic activity within these vacuoles. A positive reaction was, however, found in the adjacent somatic tissues. Also, low activity was observed in the small vacuoles of the young microspore, and the reaction was very intense in the large central vacuole of the old microspore which has a peripheral nucleus. In the young bicellular or mature pollen the activity was mainly observed in the small vacuoles. Other organelles showed no reaction. The significance of this cellular autophagy which is only observed at the meiotic prophase and at the vacuolate stage of the microspore is discussed, particularly in relation to the sporophyte-gametophyte transition.

Cytomixis in Meiosis During the Microsporogenesis ofOphrys Lutea: An Ultrastructural Study

SUMMARYThe ultrastructural study of microsporogenesis in Ophrys lutea revealed that the formation of cytomictic channels in the callose wall was followed by the transfer of organelles and chromatin (cytomixis) through them. The transfer of chromatin was first observed on zygo-pachytene, and throughout the other stages of prophase I. Even though restricted to some nuclear areas, cytomixis was observed at different stages of chromatin condensation. Some abnormalities were detected as a consequence of cytomixis. The significance of cytomixis as a possible meiotic event is discussed.

Tungsten and molybdenum heteropolyacids as staining and contrastingagents: Reactivity with epoxyresin-embedded cell and tissue structures

In this work, we carry out a further approach to the knowledge of the reaction mechanism of phosphotungstic and phosphomolybdic acids (PTA and PMA), as well as some derivatives, with cell structures from epoxyresin-embedded materials. Applied on thin sections from glutaraldehyde-fixed tissues, PTA and PMA induced a strong electron contrasting reaction in spermatid acrosomes, goblet cell mucin, callose and plant cell walls, endexine, intine and starch granules. In light microscopy, the localization of heteropolyacids on these structures was achieved by treatments of semithin sections with suitable reducing agents (titanous sulfate, stannous chloride, sodium borohydride, or p-phenylenediamine) to form the mixed-valence heteropolyblues, or with Schiffs's reagent. The use of PTA-dye complexes (pyronin-PTA and Mallory's PTA-hematoxylin) also showed the same staining pattern. Taking into account the chemical characteristics of the PTA- and PMA-reactive tissue elements, the present results indicate that heteropolyacids selectively enter into the highest hydrophilic structures from non-polar epoxy-embedded sections; after brief washing, they appear predominantly retained in tissue structures containing a great amount of carbohydrate components.

Ontogeny of intruding non-periplasmodial tapetum in the wild chicory,Cichorium intybus (Compositae)

The tapetal development ofCichorium intybus L. is investigated using LM and TEM and discussed in relation to the development in other species. During the second meiotic division the tapetal cells become binucleate and lose their cell walls. They intrude the loculus at the time of microspore release from the meiotic callose walls, which means that a locular cavity is never present in this species. During pollen development they tightly junct the exine, especially near the tips of the spines. During the two-celled pollen grain stage they degenerate and most of their content turns into pollenkitt. Until anther dehiscence they keep their individuality, which means that these intruding tapetal cells never fuse to form a periplasmodium. The ultrastructural cytoplasmatic changes during this development are discussed in relation to possible functions.

Tissue interactions in the developing locule ofGasteria verrucosaduring microsporogenesis

We investigated the development of the locule tissues of Gasteria verrucosa (Mill.) H. Duval during microsporogenesis using electron microscopy and summarized it in detailed pen-drawings. In the early stages of development, the epidermis, endothecium and middle layer show no remarkable processes relating to tapetum and pollen development. The tapetum regulates the disappearance of the original cell walls and the callose walls from the microspores and itself. These wall materials partly turn into starch in both tissues. The spherical shape of the meiotic callose walls and the presence of the cytomictic channels enable the pollen mother cells to divide into equally shaped microspores, which is important for random dispersal.

Cytokinesis in microspore mother cells of Impatiens sultani

Cytokinesis in Impatiens sultani microspore mother cells is simultaneous. It starts with the formation of small ingrowths of the surrounding callosic wall. Next, an incomplete cell plate is formed by fusion of small dictyosome vesicles. The cell plate consists of a network of anastomosing tubules and sacs. Aggregates of fusing vesicles are associated with bundles of microtubules, which are oriented perpendicular to the plane of the future cell walls. In the sacculate parts of the cell plate, some callose is deposited, while the associated microtubules disappear. The cell walls ultimately develop by enlargement of the previously formed wall ingrowths, which successively incorporate the elements of the cell plate. The enlargement and thickening of the walls is not accompanied by a further fusion and incorporation of dictyosome vesicles.

ONTOGENETIC DEVELOPMENT OF SPINOUS EXINE IN HIBISCUS SYRIACUS (MALVACEAE)

Pollen development in Hibiscus syriacus L. (Malvaceae) was studied with light (LM), scanning (SEM) and transmission (TEM) electron microscopes, with special attention to the formation of extremely long spines of the pollen grains. At the early tetrad stage, probacules are initiated directly on the plasma membrane and grow in coincidence with the height of primexine matrix within a callosic wall. Subsequently, a pretectum appears at the top of the probacules and then a foot layer is formed by accumulation of white line centered lamellations. Before dissolution of the callosic wall, a reticulate patterned pretectum is established around the microspores. There is not, however, any morphological indication on the initiation of the spines during the tetrad period within a callosic wall. It is after dissolution of the callosic wall that the spines of exine begin to form by the apposition of lamellated sheets. The lamellated sheets show a concentric configuration around the developing supratectal spines. The mature pollen grain is spheroidal, polycolporate, 160–170 μm in diameter, with supratectal spines 20–25 μm long. The supratectal spines of Hibiscus pollen are not homologous with the other exinous protrusions which are determined within the callosic wall during tetrad stage.

Ontogenetic Development of Spinous Exine in Hibiscus syriacus (Malvaceae)

Pollen development in Hibiscus syriacus L. (Malvaceae) was studied with light (LM), scanning (SEM) and transmission (TEM) electron microscopes, with special attention to the formation of extremely long spines of the pollen grains. At the early tetrad stage, probacules are initiated directly on the plasma membrane and grow in coincidence with the height of primexine matrix within a callosic wall. Subsequently, a pretectum appears at the top of the probacules and then a foot layer is formed by accumulation of white line centered lamellations. Before dissolution of the callosic wall, a reticulate patterned pretectum is established around the microspores. There is not, however, any morphological indication on the initiation of the spines during the tetrad period within a callosic wall. It is after dissolution of the callosic wall that the spines of exine begin to form by the apposition of lamellated sheets. The lamellated sheets show a concentric configuration around the developing supratectal spines. The mature pollen grain is spheroidal, polycolporate, 160–170 μm in diameter, with supratectal spines 20–25 μm long. The supratectal spines of Hibiscus pollen are not homologous with the other exinous protrusions which are determined within the callosic wall during tetrad stage.

Changes in intracellular localization of peroxidase during Microsporogenesis in Gymnosperms

Cytochemical investigations on peroxidase localization during microsporogenesis inLarix europaea D.C.,Taxus baccata L. andPinus sylvestris L. have revealed striking differences in the localization and activity level of this enzyme linked with the developmental stage.The localization and level of activity of peroxidase, typical of each stage, changed in the course of microsporogenesis in a strictly orderly way, giving a characteristic and stable pattern. The pattern of intracellular peroxidase localization proved to be the same for microsporogenesis of all the gymnosperms in question.It is suggested that the identity of that pattern in plants so phylogenetically distant asTaxus baccata L. andPinus sylvestris L. indicates that peroxidase activity in gymnosperms’ microsporogenesis is connected with the fundamental and genetically well stabilized processes of meiotic cytodifferentiation.Moreover, enhanced peroxidase activity has been found in the sites of callose walls synthesis of dyads and tetrads, which suggests the participation of this enzyme in callose synthesis.

Isolation of generative cells and their protoplasts from pollen ofLilium longiflorum

Methods are described for the isolation of large quantities of generative cells and their protoplasts from the pollen ofLilium longiflorum. First, large numbers of pollen protoplasts were enzymatically isolated from immature pollen grains. When they were gently disrupted mechanically, the pollen contents including spindle-shaped generative cells were released. The generative cells were separated from other structures by Percoll density gradient centrifugation. They were nearly spherical, but had a callosic cell wall. The isolated generative cells were then re-treated in enzyme solution to yield authentic protoplasts. The generative cell protoplasts, “gametoplasts”, were uniform in size and contained a condensed haploid nucleus with relatively little cytoplasm.

The Effect of Bovine Serum Albumin and Cytohelicase on Surface-Spread Synaptonemal Complexes of Rye (Secale Cereale)

Synaptonemal complexes of rye meiocytes were spread on plastic coated slides for electron microscopic observation. Two proteins generally used in synaptonemal complex spreading techniques, bovine serum albumin and cytohelicase, were applied separately or in combination in an isotonic protoplast medium at concentrations of 0.1-5%. At high concentrations these proteins proved to enhance notably the ultimate number of cells with synaptonemal complexes in the preparations. Also under this condition, centromere structures became stainable with silver nitrate in both the synaptonemal complexes of pollen mother cells and in interphase nuclei of other cell types. Since the true action of cytohelicase under appropriate spreading conditions was uncertain, the putative enzymatic digestion of cell walls was determined in a series of experiments using the fluorochrome calcofluor white as a stain of callose walls. Obvious breakdown of the cell walls was not observed before 8 min of treatment under standard conditions. This made it plausible that the prime effect of cytohelicase is that of a nonspecific protein interacting with the chromatin and improving the adhesion of synaptonemal complexes to the hydrophobic plastic film. The differential staining of the centromere structures in the presence of bovine serum albumin and cytohelicase probably reflects a reduced spreading of these structures due to preferential binding between these proteins and centromeric proteins.

Pollen wall development in Olea europaea L.

The ontogeny of the olive pollen grain (Olea europaea L.) wall is related to the structural changes which take place during the various stages of pollen grain development. The tetrad stage sees the deposition of the primexine, with probacules forming by deposition of material adjacent to the plasmalemma. Endexine development commences subsequently. The bacules and foot-layer are formed mainly from dense material found within the special callose wall. By the young free microspore stage the definitive structure of the exine has been determined. Throughout development microchannel-like structures are present in both bacules and the foot-layer. An electron-lucent zone appears between the plasmalemma and the endexine. This is the future site of intine deposition which occurs during the vacuolated microspore stage before postmeiotic mitosis. A fibrillar layer lines the arcade spaces of the exine. Between this stratum and the exine lies a thin layer of polysaccharides probably representing the remains of the primexine or glycocalyx. In the mature pollen grain the exine arcades contain numerous lipid drops (or pollenkitt), originating from the degenerating tapetum.

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.

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.

Pollen wall morphogenesis in Tragopogon porrifolius L. (Compositae: Lactuceae) and its taxonomic significance

Pollen development in Tragopogon porrifolius has been studied in the scanning electron microscope to establish the origin of taxonomically significant features. As in other members of the tribe recently investigated the echinolophate condition is shown to result from differential deposition of the tetrad callose wall and, subsequently, of primexine. Tragopogon differs from other Lactuceae in the absence of tectum and columellae in the lacunae, this also appears to be the result of differential primexine deposition. The development of onci and their extrusion at the apertures are described. The contribution of developmental studies of pollen grains to systematics is discussed.

Mechanisms of Pollen Aggregation into Pollinia inEpidendrum Ibaguense(Orchidaceae)

Abstract In Epidendrum ibaguense, pollen is aggregated into pollinia as united masses. The present study demonstrates that the mechanism of pollen aggregation is built into the various stages of pollen development. It is suggested that the pollen are held together due to a combined effect of the following features: (1) the remains of the pollen mother cell wall, (2) the fibrillar material present within the callosic cell wall during meiosis and tetrad formation, (3) the persistence of some plasmodesmata from the PMC stage of pollen development, (4) incomplete cell wall formation within tetrads, and (5) little size increase of developing microspores