Why are plastids maternally inherited in epilobium? Ultrastructural observations during microgametogenesis

We have investigated the sequential stages of microgametogenesis by electron microscopy, to determine the basis of maternal inheritance of plastids in Epilobium. The development of both the vegetative and generative cells has been followed using a semi-artificial growth system for pollen tubes. The generative cells inside the pollen grain contains numerous mitochondria, 5–8 proplastids, and, in contrast to the vegetative cytoplasm, only a few vacuoles. When the generative cell has divided into the two sperm cells inside the pollen tube, small vesicles deriving from dicytosome cisternae become abundant. These vesicles appear to form vacuoles by fusion which then contain remnants of fibrillar, globular or membranaceous material. It is suggested that this material derives from proplastids as the proplastids disappear either before or shortly after the generative cell has divided, concurrently with the appearance of the ‘remnants’ in the vacuoles. The mitochondria of the sperm cells remain intact.     

Effects of anoxia on pollen tube growth and tube wall formation of Impatiens glandulifera

Summary When pollen of Impatiens glandulifera was cultured in aerated liquid medium for 1 h, 70% of the pollen grains germinated; these attained an average tube length of 1 mm. Subsequently, these aerobic growth conditions were changed to anaerobic by substituting a nitrogen inlet for the air inlet. As a result, the pollen tubes stopped elongating and burst. The ultrastructural changes which occurred upon inducing anoxia were studied with samples taken at 0 s, 45 s, and 4 min after changing the gas. Anoxia caused rapid and considerable changes in the ultrastructure of the dictyosome vesicles involved in cell wall formation. There was an increase in the osmiophyly of the vesicle content, and the presence of fibrillar material became apparent. Simultaneously, the fusion behavior of the dictyosome vesicles changed. Instead of the normal fusion of the dictyosome vesicles with the plasma membrane, there was a premature fusion of the vesicles with each other inside the cytoplasm that resulted in the formation of aggregates. Furthermore, the cell wall precursors that were excreted were not incorporated in their usual configuration into the growing cell wall. Instead of a smooth inner cell wall surface, irregular thickenings were formed.     

荞麦水合花粉和花粉管中的被膜小泡

荞麦水合花粉粒和生长中的花粉管中内质网潴泡形成的囊袋状结构较少见,但内质网囊袋中含有丰富的被膜小泡,直径约为100－150nm。刚刚形成的花粉管中,被膜小泡主要来自于花粉粒营养细胞的细胞质。生长中的花粉管的被膜小泡可由高尔基体分泌形成。另外还观察到内质网的碎裂也是荞麦花粉管中产生被膜小泡的一种机制。花粉管的被膜小泡中含有花粉管壁的前体物质,与花粉管的壁融合参与花粉管的生长。被膜小泡可能含有与脂体和造粉质体水解有关的酶,参与此类物质的降解。荞麦花柱和柱头细胞内含物的解体物质参与花粉管的生长。     

Fast pollen tube growth in Conospermum species

BACKGROUND AND AIMS: An unusual form of pollen tube growth was observed for several Conospermum species (family Proteaceae). The rate of pollen tube growth, the number of tubes to emerge and the ultrastructure of these tubes are given here. METHODS: Pollen was germinated in vitro in different sucrose concentrations and in the presence of calcium channel blockers, and tube emergence and growth were recorded on a VCR. Measurements were taken of the number of tubes to emerge and rate of tube emergence. Pollen behaviour in vivo was also observed. The ultrastructure of germinated and ungerminated pollen was observed using TEM. RESULTS: After 10 s to 3 min in germination medium, up to three pollen tubes emerged and grew at rates of up to 55 micro m s(-1); the rate then slowed to around 2 micro m s(-1), 30 s after the initial growth spurt. Tubes were observed to grow in pulses, and the pulsed growth continued in the presence of calcium channel blockers. Optimal sugar concentration for pollen germination was 300 g L(-1), in which up to 81 % of pollen grains showed fast germination. Germination and emergence of multiple tubes were observed in sucrose concentrations of 100-800 g L(-1). The vegetative and generative nuclei moved into one of the tubes. Multiple tubes from a single grain were observed on the stigma. Under light microscopy, the cytoplasm in the tube showed a clear region at the tip. The ultrastructure of C. amoenum pollen showed a bilayered exine, with the intine being very thick at the pores, and elsewhere having large intrusions into the plasma membrane. The cytoplasm was dense with vesicles packed with inner tube cell wall material. Golgi apparatus producing secretory vesicles, and mitochondria were found throughout the tube. The tube wall was bilayered; both layers being fibrous and loosely packed. CONCLUSIONS: It is proposed that, for Conospermum, initial pollen tube wall constituents are manufactured and stored prior to pollen germination, and that tube extension occurs as described in the literature for other species, but at an exceptionally fast rate.     

宁夏枸杞柱头和萌发花粉中钙分布特征

用焦锑酸钾沉淀法对宁夏枸杞柱头和花粉中的钙离子分布进行了研究.结果显示,宁夏枸杞柱头表皮有一覆盖层,其中有许多含钙沉淀颗粒的小泡,当花粉落到柱头后从覆盖层中吸水,在萌发孔的表面上聚集了较多的钙沉淀颗粒.同时,花粉内部出现许多含钙的小液泡,使花粉体积增大,内部产生膨压,花粉萌发;生长在覆盖层中的花粉管顶端穿过覆盖层小泡时,附近聚集了较多的钙沉淀颗粒,在花粉管壁上也附着较多的细小钙沉淀颗粒.萌发的花粉粒中由大液泡占据,在其亚顶端的细胞质中,聚集较多钙沉淀颗粒的线粒体膨大形成了一些含钙沉淀颗粒的小液泡,由这些小液泡融合形成的大液泡,将花粉管细胞质挤到其顶端,使其极性生长.这是首次发现在植物柱头覆盖层中有钙离子的现象,从体内证明了钙离子在花粉萌发过程中的现象.讨论了枸杞柱头组织中钙的分布和花粉管的萌发与生长的关系.     

ULTRASTRUCTURAL CHANGES IN DIRECTLY GERMINATING SPORANGIA OF PHYTOPHTHORA PARASITICA

Flagella and cleavage vesicles form during the initial stages of direct germination in the same manner as they do in indirect germination. Later, however, the flagella degenerate and cleavage of the cytoplasm is not complete. Instead, a new wall layer is deposited onto the existing sporangial wall, and this germination wall extends with the germ tube and forms the hyphal wall. Material for the germination wall first appears around vesicular aggregates concentrated at the periphery of the sporangial cytoplasm. As the wall forms the vesicular aggregates become encased in it, and the wall eventually consists of irregular deposits of wall material interspersed with the vesicular aggregates. These are clearly cytoplasmic in nature and different from lomasomes, since they are associated with ribosomes and other small elements of the cytoplasm. Likely to contribute to the wall formation are the endoplasmic reticulum, microbodies, dictyosome-derived vesicles, and possibly polyvesicular bodies and larger vacuoles with fibrillar contents. The basal plug of the sporangium forms the same way as the germination wall and also contains numerous vesicular inclusions. Fewer vesicular inclusions are found in the sporangial wall, and none have been observed in the walls of the growing hyphae. This difference in wall structure can be explained on the basis of their different growth patterns. Penetration of the sporangial plug is concomitant with prominent lomasomal activity and the cytoplasm at the hyphal tip is characterized by the presence of numerous vesicles, which are probably derived from dictyosomes.     

Immunogold localization of arabinogalactan proteins,unesterified and esterified pectins in pollen grains and pollen tubes ofNicotiana tabacum L.

Summary The monoclonal antibodies JIM 5 (against unesterified pectin), JIM 7 (against methyl esterified pectin), MAC 207 (against arabinogalactan proteins, AGPs), and JIM 8 (against a subset of AGPs) were utilized singly or in combinations for immunogold labelling of germinated pollen grains and pollen tubes ofNicotiana tabacum. Pectins were localized in the inline of pollen grain, unesterified pectin being more abundant than the esterified one. AGPs were co-localized with pectin in the inline, but were present preferably close to the plasma membrane. In pollen tubes, AGPs, unesterified and esterified pectins were co-localized in the outer and middle layers of the cell wall. The density of the epitopes was not uniform along the length of the pollen tube, but showed alterations. In the pollen tube tip wall esterified pectin was abundantly present, but not AGPs. In the cytoplasm esterified pectin and AGPs were detected in Golgi derived vesicles, indicating their role in the pathway of the cell wall precursors. In the cell wall of generative cell only AGPs, but no pectins were localized. The co-localization of pectins and AGPs in the cell wall of pollen grain and pollen tube might play an important role, not only in maintenance of the cell shape, but also in cell-cell interaction during pollen tube growth and development.Abbreviations AGP arabinogalactan protein - BSA bovine serum albumin - GA glutaraldehyde - MAb monoclonal antibody - NGS normal goat serum - PFA paraformaldehyde     

Effect of cadmium on pollen germination and tube growth in Lilium longiflorum and Nicotiana tabacum

Cadmium had a highly toxic effect on pollen germination and tube growth, which were greatly inhibited as metal concentrations increased. Cadmium concentrations up to 10(-2) M completely stopped pollen germination and pollen showed an increasing tendency to burst within 1 h. At low concentrations, the metal caused a slight stimulation of pollen germination, growth rate and tube elongation at the initial stages of tube development. Comparing the two plants studied, cadmium was more toxic for Nicotiana tabacum than for Lilium longiflorum pollen. Pollen tubes showed a range of strong morphological abnormalities, characterized by uneven or aberrant growth, including apical branching or swelling at the tip of the pollen tube. Cell wall intrusions at or near the tip were evident on the inner side, whereas a loose network formed from fibrillar material was observed on the outer layers. After prolonged cadmium exposure, round (ball-like) aggregates were embedded in a fine fibrillar network. Increased cadmium concentrations (10(-3)-10(-2) M) decreased or completely paralyzed cytoplasmic streaming. No typical cytoplasmic zonation existed, while cell organelles (plastids, lipid droplets) were relocated toward the tip. The vesicular apical zone was drastically reduced, with vesicles dispersed into the subapical region. Mitochondria were distributed throughout the subapical region and among the vesicles of the tube apex. Visible ultrastructural changes in cell organelles were not observed.     

THE TRANSMITTING TRACT IN GLADIOLUS. I. THE STIGMA AND THE POLLEN-STIGMA INTERACTION

The stigma papillae in Gladiolus are of the “dry” type and are highly vacuolated cells with an organelle-rich peripheral cytoplasm. The cell wall of each papilla is overlain by a distinctive cuticle possessing an irregularly scalloped inner margin. Between the cell wall and cuticle is a layer of amorphous sub-cuticular material. Lipids are detected on the papilla surface. A pollen grain will hydrate and germinate only on a papilla and not on any other (non-papillate) portion of the stigma. The pollen tube penetrates the papilla cuticle, which is forced away from the papilla cell wall by sub-cuticular pollen tube growth. As the cuticle lifts away, the sub-cuticular material disperses. At the base of the papilla, the pollen tube grows onto the adaxial non-papillate surface of the stigma lobe. At this site, the cuticle has been lifted away from the underlying cells by release of a mucilaginous substance from the latter, and the pollen tube grows within this substance beneath the detached cuticle. The cytological features of Gladiolus papillae are compared with other stigma papillae described in the literature. Also, a review of the literature, as well as some of the findings of the present study, suggest that certain prevalent interpretations of dry stigma structure and function may be open to question.     

Reproduction in seagrasses: pollen wall morphogenesis in Amphibolis antarctica and wall structure in filiform grains

The pre–meiotic anther of the marine angiosperm Amphibolis antarctica contains microsporocytes and sterile cells. The microsporocytes divide conventionally to produce tetrads, but the sterile cells degenerate and contribute to the future pe–riplasmodium. Each tetrad of young microspores is contained within a vesicle defined by a membrane. After release from the tetrad, the microspores increase in length and rapidly become filiform. The microspore nucleus soon divides and partitioning of the cytoplasm delimits the generative cell from the vegetative cell of the binucleate pollen grain. The division and the early pollen growth occurs while the grains are segregated within vesicles in the periplasmodium. These compartments, established at microspore release, remain structurally intact throughout the vacuolate period of pollen development, when pollen wall assembly begins. This process is initiated as particles migrate from the inner face of the vesicle membrane into the lumen of the vesicle and microfibrillar elements form between adjacent particles. The particles and microfibrils form a loose, three–dimensional network. The vesicle membrane then disappears and the binuclate grains become immersed in the tapetal residuum. Additional wall components are now deposited upon the primary fibrillar stratum. Short lamellae, resembling fragments of membrane, frequently associated with electron–opaque globuli, are found intermixed with the surface microfibrils. Apparently, granular material originating in the degenerating periplasmodium may be the precursor of the globuli, and contact with the lamellae brings about an alteration in state. At this stage the pollen wall is resolved as two distinct fibrillar strata and the lamellae and globuli are incorporated as inclusions into the superficial zone of the outer stratum. The mature pollen wall exhibits faint stratification and the presence of the subsurface inclusions is readily demonstrated in germinating grains by section staining with phosphotungstic acid. The pollen wall in A. antarctica is compared with that in filiform grains of other seagrasses.     

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.     

A structural study on the mode of action of CHATM Chemical Hybridizing Agent in wheat

Summary This study has compared mature pollen grains while still in the anther, as well as post-pollination responses, from untreated and CHA^TM Chemical Hybridizing Agent-treated wheat plants using light (bright-field, phase-contrast, and fluorescence), scanning, and transmission electron microscopy. The chemical, azetidine-3-carboxylic acid (A3C), was applied at three treatment levels of 0.75, 1.0 and 1.5 kg/ha for the mature pollen investigations. It was found that the major effect of A3C on mature pollen is an alteration of the wall precursor vesicles (wp-vesicles), which form a high proportion of the contents of the mature grass pollen grain. The degree of deformation of the wp-vesicles is dose dependent. There is some evidence that increased aggregations of ribosomes are formed in treated pollen cytoplasm. Pollination studies (all at a treatment level of 1.0 kg/ha) show that, in most cases, the treated pollen does not germinate, and a high percentage of the pollen grains burst (60% burst grains in treated material compared to 28% in controls). In about 20% of the cases from treated plants, a short pollen tube forms, but no tubes were seen to grow far enough to enter the stigma hairs of the pistil. Thus, A3C does not act by preventing pollen formation, but by the prevention of normal pollen tube growth. There appears to be a specific targeting of the wp-vesicles such that, even in cases where the ultra-structure of the vesicles is not altered, the normal course of events leading to the incorporation of their contents into the extending tube wall is arrested. Further studies must be undertaken to determine the significance of the effect of CHA^TM Chemical Hybridizing Agent on wp-vesicle composition.     

Divergent pollination systems in the cleistogamous species,Collomia grandiflora (Polemoniaceae)

Summary A structural study of pollination in the dimorphic flowers ofCollomia grandiflora, a cleistogamous species, reveals significant differences in stigma behavior during pollination, stylar structure, the timing of generative cell division, and pollen tube growth rate patterns. The cleistogamous flower shows a loss of protandry and the stigma is receptive only after reflexing and closing of its lobes. In contrast, the chasmogamous stigma is receptive when reflexed and closes when pollen has been deposited on the lobes. Pollen tube penetration of the dry stigma papillae and entry into the style is similar in the two morphs. The chasmogamous style is solid and the cleistogamous style partly hollow. The matrix of secretion produced by the transmitting tract cells is mainly carbohydrate with a trace of lipids. It is fibrillar in nature and appears to be partly comprised of wall material from the transmitting tract cells. In the chasmogamous pollen, the generative cell enters the tube before division, which occurs between 30 and 60 min after pollination. This division correlates with an increased growth rate for the pollen tube. In the cleistogamous pollen, contact with the stigma triggers generative cell division inside the hydrated pollen grain before germination. The two resulting sperm cells exit the grain 15–30 min after pollination when the pollen tube is in the stigma lobes. The cleistogamous pollen tube shows only one phase of growth which occurs at a rate similar to that of the slow, first phase of the chasmogamous pollen.Abbreviations CH chasmogamous - CL cleistogamous - DAPI 4, 6-diamidino-2-phenylindole     

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

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

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.     

The growth of the grass pollen tube: 1. Characteristics of the polysaccharide particles (“P-particles”) associated with apical growth

Summary Numerous polysaccharide-rich particles (P-particles) occur in the tip region of growing grass pollen tubes, where they apparently contribute to the extending wall. In other families the corresponding bodies have been shown to originate from dictyosome activity during pollen tube growth. However, in the grasses the main synthesis precedes anthesis; the P-particles represent up to 30% of the reserves of the vegetative cell of the dormant grain, numbering over one million in the pollen grain of rye. Their membranes are incomplete. The polysaccharide content, which is initially coarsely granular but becomes microfibrillar with hydration, is readily extracted with ammonium oxalate, and is probably pectic in nature. Simple methods for isolating the particles in relatively pure populations are described. Hydrolysis yields principally galactose, arabinose, glucose, and rhamnose. Apart from proteins derived from the original bounding membranes, a protein fraction is tenaciously bound to the polysaccharide. Isolated P-particles move anodically in an electrical field, and the possibility that their movement from the grain to the tube tip during growth depends on a potential gradient, already demonstrated for lily pollen tubes, is considered.     

Ultrastructural localization of acid phosphatase in the pollen tube of Prunus avium L. (sweet cherry)

The pollen tube of Prunus avium (cherry) consists of a growth zone of vesicles at the tip and an assemblage of organelles typical of an actively metabolizing cell. Electron opaque globules are closely associated with the plasma membrane and fibrillar cell wall layer at the tip. Acid phosphatase (EC 3.1.3.2) activity is localized in the membranes of 120 nm vesicles and ER system, the lumen of 50 nm vesicles, the plasma membrane and the tube nucleus.     

Germination and early tube development in vitro of Lycopersicum peruvianum pollen: Ultrastructural features

Morphologic changes occurring during pollen grain activation and ultrastructural features of Lycopersicum peruvianum Mill. pollen tube during the first stages of growth in vitro have been studied. The more evident morphologic changes during activation, in comparison to those already described for mature inactive pollen, concern dictyosomes, rough endoplasmic reticulum (RER), and ribosomes. The dictyosomes are very abundant and produce large and small vesicles. Near the germinative pores both types of vesicles are present, while all along the remaining cell wall only the large type is observed. These latter react weakly to Thiéry's test and probably contain a callose precursor necessary for the deposition of a callosic layer lining at first only the inner side of the functioning pore and occasionally the other two pores, and subsequently the entire inner surface of the cell wall. The small vesicles, highly positive to Thiéry's test, are present only near the pores and could be involved in the formation of the pectocellulosic layer of the tube wall. The setting free of RER cisterns, which in the mature inactive pollen were aggregated in stacks, coinciding with polysome formation and resumption of protein synthesis, is in accord with the hypothesized role of RER cistern stacks as a reserve of synthesizing machinery. The pollen tube reaches a definitive spatial arrangement soon after the generative cell and vegetative nucleus have moved into it. At this stage four different zones that reflect a functional specialization are present. In the apical and subapical zone two types of dictysosome-originated vesicles, similar to those found in the activated pollen grain, are present. Their role in the formation of the callosic and pectocellulosic wall layers seems to be the same as in the activated pollen grain.Abbreviations ER endoplasmic reticulum - RER rough endoplasmic reticulum Research performed under CNR program Biology of Reproduction     

Immunocytochemical localization of callose in the germinated pollen ofCamellia japonica

Summary A polyclonal antibody against -1,3-glucan, callose, extracted from the pollen tube wall ofCamellia japonica was raised in mice and, using it as a probe, the localization of callose in the germinated pollen was studied. By confocal laser scanning microscopy, callose was found in the tip region of the pollen tube and the tube wall; the immuno-fluorescence in the tube wall was less toward the base of the tube. In contrast, the tip region did not fluoresce although the whole of the tube wall did strongly with aniline blue, the specific dye for callose. Immuno-electron microscopy showed that callose was also found in Golgi vesicles which concentrated in the tip region of the pollen tube, the inner layer of the tube wall, callose plugs, and Golgi vesicles in the pollen grain. Immuno-gold labeling was often detected on the fibrous structures in Golgi vesicles and callose plugs. Based on these results, the participation of Golgi vesicles in the formation of the tube wall and callose plugs was discussed.Abbreviation TBS Tris-buffered saline - Tris Tris(hydroxy-methyl)-aminomethane - PBS phosphate-buffered saline - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - CLSM confocal laser scanning microscopy - DP degree of polymerization