Anther Tapetum Research Articles

Pollen and Tapetum Development in Male FertileRosmarinus OfficinalisL. (Lamiaceae)

Abstract The development of microspores/pollen grains and tapetum was studied in fertile Rosmarinus officinalis L. (Lamiaceae). Most parts of the cell walls of the secretory anther tapetum undergo modifications before and during meiosis: the inner tangential and radial cell walls, and often also the outer tangential and radial wall, acquire a fibrous appearance; these walls become later transformed into a thin poly-saccharidic film, which is finally dissolved after microspore mitosis. Electron opaque granules found within the fibrous/lamellated tapetal walls consist of sporopollenin-like material, but cannot be interpreted as Ubisch bodies. The middle lamella and the primary wall of the outer tangential and radial tapetal walls remain unmodified, but get covered by an electron opaque, sporopollenin-like layer. Pollenkitt is formed only by lipid droplets from the ground plasma and/or ER profiles, the plastids do not form pollenkitt precursor lipids. Tapetum maturation (“degeneration”) does not take place before late vacuolate stage. The apertures are determined during meiosis by vesicles or membrane stacks on the surface of the plasma membrane. The procolumellae are conical, but at maturity the columellae are more cylindrical in shape. The columellar bases often fuse, but a genuine foot layer is lacking. The formation of the endexine starts with sporopollenin-accumulating white lines adjacent to the columellar bases. Later, the endexine grows more irregularly by the accumulation of sporopollenin globules. In mature pollen the intine is clearly bilayered. Generative cells (GCs) and sperm cells contain a comparatively large amount of cytoplasm, and organelles like mitochondria, dictyosomes, ER, and multi-vesicular bodies, but no plastids; GCs and sperms are separated from the vegetative cell only by two plasma membranes.

An investigation of the role of the anther tapetum during microspore development using genetic cell ablation

The effects on anther development of a fusion of the Arabidopsis anther-specific apg gene promoter to a ribonuclease (barnase) in transgenic tobacco plants were examined. Contrary to expectations, viable pollen grains were produced by these plants despite the demonstration that ribonuclease expression in the microspores and tapetum caused targeted cell ablation. Transformed plants were reduced in male fertility due to ablation of a proportion of pollen dependent on apg-barnase locus number. Plants were otherwise phenotypically normal and fully female fertile, confirming the anther-specific nature of the apg promoter. In microspores inheriting an apg-barnase locus following meiosis, loss of cell viability, as judged by fluorescein diacetate staining, occurred during mid to late microspore development. Microspores not inheriting a transgene went on to mature into viable pollen grains. Premature degeneration of the tapetum was also observed as a result of apg-barnase expression, but this did not appear to disrupt the subsequent microspore and pollen developmental programmes. This was substantiated by observations of microspore development in plants in which the tapetum was rescued from ablation by crossing in a second transgene encoding a tapetum-specific inhibitor of the ribonuclease. It was determined that tapetum cell disruption occurs at the early to mid uninucleate microspore stage in apg-barnase transformants. The data presented show that after this point in microspore development the tapetum is no longer essential for the production of viable pollen in tobacco.

Tapetum-Specific Expression of the Gene for an Endo-β-1,3-glucanase Causes Male Sterility in Transgenic Tobacco

A cDNA for a pathogenesis-related endo-beta-1,3-glucanase isolated from soybean, was fused to an anther tapetum-specific promoter (Osg6B promoter) isolated from rice and the resulting chimeric gene was introduced into tobacco. The Osg6B promoter became active in the anther tapetum during formation of tetrads and the tapetal glucanase activity in the transgenic plants caused in a significant reduction in the number of fertile pollen grains. Most of the pollen grains were aberrant in shape, lacked germinal apertures and aggregate of the pollen grains. Granules of beta-1,3-glucan, which have not previously been reported, were often observed to adhere to the surface of the pollen grains. Further observations revealed that the callose wall was almost absent in the pollen tetrads of transgenic plants. In wild-type plants, by contrast, the tetrads were surrounded by callose that was degraded soon after the tetrad stage to release free microspores. Thus, the introduced gene for endo-beta-1,3-endoglucanase under the control of the Osg6B promoter caused digestion of the callose wall at the beginning of the tetrad stage, a time that was just a little earlier than the time at which endogenous glucanase activity normal appears. These results demonstrate that premature dissolution of the callose wall in pollen tetrads causes male sterility and suggest that the time at which tapetally produced glucanase is activate is critical for the normal development of microspores.

Molecular characterization of rice genes specifically expressed in the anther tapetum.

In situ localization of mRNA was carried out on two cDNAs (Osc4 and Osc6) that had been isolated from rice anthers at the microspore stage. The mRNA corresponding to each cDNA was shown to be localized only in the tapetal cells of the rice immature anthers, but not in the microspores or the mature pollen. The corresponding genomic clone, Osg6B, was isolated, and its 5'-upstream region was found to regulate beta-glucuronidase expression in the tapetum of transgenic tobacco. A set of 5' deletions was also generated and a 1095 bp 5' region was revealed to be necessary for activation of the Osg6B promoter in transgenic tobacco.

Embryology of Plagiopteron suaveolens Griffith (Plagiopteraceae) and its systematic implications

A study of the embryology of Plagiopteron suaveolens Griffith is presented in order to evaluate the affinities of the genus. Anther wall formation is of the dicotyledonous type. The anther tapetum is glandular and its cells become two to four nucleate. Cytokineses in the microspore mother cell is simultaneous and the resultant tetrad is tetrahedral. The mature pollen grains are two-celled. The anther has 4-locules at anthesis. The ovule is anatropous, bitegmic and weakly crassinucellate with only one cell-layered parietal tissue. The micropyle is zig-zag in section and formed by both inner and outer integuments which are three and three to four cell layers thick respectively. The megaspore archesporium is one-celled and the embryo sac development is of the Polygonum type. An endothelium is formed with early disintegration of nucellar tissue. The antipodals multiply to 15 to 20 cells. Embryological evidence supports the placement of Plagiopteron in its own family, Plagiopteraceae, with closest relationship with Celastraceae and close relationship with Elacocarpaceae, Rhizophoraceae and some families of the Geraniales.

Embryology of Plagiopteron suaveolens Griffith (Plagiopteraceae) and its systematic implications

A study of the embryology of Plagiopteron suaveolens Griffith is presented in order to evaluate the affinities of the genus. Anther wall formation is of the dicotyledonous type. The anther tapetum is glandular and its cells become two to four nucleate. Cytokineses in the microspore mother cell is simultaneous and the resultant tetrad is tetrahedral. The mature pollen grains are two-celled. The anther has 4-locules at anthesis. The ovule is anatropous, bitegmic and weakly crassinucellate with only one cell-layered parietal tissue. The micropyle is zig-zag in section and formed by both inner and outer integuments which are three and three to four cell layers thick respectively. The megaspore archesporium is one-celled and the embryo sac development is of the Polygonum type. An endothelium is formed with early disintegration of nucellar tissue. The antipodals multiply to 15 to 20 cells. Embryological evidence supports the placement of Plagiopteron in its own family, Plagiopteraceae, with closest relationship with Celastraceae and close relationship with Elacocarpaceae, Rhizophoraceae and some families of the Geraniales.

The Polytene Chromosomes of Anther Tapetum of Some Phaseolus Species.

The chromatin organization of tapetal cells of some Phaseolus species was analysed with Giemsa staining. They presented endoreduplication cycles which may result in large endochromocentres or more or less condensed chromatid bundles, visualized as polytene chromosomes, properly. P. coccineus and P. vulgaris showed the best developed polytenics, whereas P. acutifolius, P. lunatus, P. microcarpus and P. peduncularis, showed endochromocentres eventually associated with small chromatid bundles. The tetraploid hybrid P. acutifolius×P. vulgaris showed tapetal nuclei with the characteristics of both parents. The ability to bundling the sister chromatids was intrinsic to each species and was similar to that previously reported in embryo suspensor cells. The polytene chromosome morphology and the change from dispersed state to a more condensed one suggest the occurrence of a coiling cycle which seemed to advance in the endoprophase more often and deeper in P. coccineus and P. vulgaris than in the remainder species.

Differentiation of the Tapetum During Microsporogenesis in Tomato (Lycopersicon esculentum Mill.), with Special Reference to the Tapetal Cell Wall

During microsporogenesis and pollen maturation, the tapetum in anthers of tomato (Lycopersicon esculentum) underwent several ultrastructural changes and ultimately degenerated. The changes observed related to the secretory function of the tapetum and to the transfer of materials from the cytoplasm to the surface of tapetal cells. Electron dense deposits, initially in the vacuoles, disappeared coincident with the appearance of orbicules on the cell wall. The fibrillar wall of the tapetal cells loosened, presumably to facilitate transfer of materials through the wall. In Addition, membranous fragments were a consistent feature in the tapetum wall and may play a role in transport of materials. The cells of the inner tapetum (towards the connective) and outer tapetum (towards the epidermis) had different ultrastructural features. The cytoplasm of the outer tapetum was more electron dense and had a higher proportion of dictyosomes and mitochondria than the inner tapetum, indicating the greater secretory nature of the outer tapetum. The plastids and mitochondria also differed in morphology between the two regions. Degenerations of the tapetal cytoplasm began by the vacuolate microspore stage. At anthesis, cytoplasm was absent but the orbicular wall of the tapetum remained appressed to the wall of the middle layer of the anther.Copyright 1993, 1999 Academic Press

Ultrastructural expression of cytoplasmic male sterility in sugar beet (Beta vulgaris L.)

The development of microspore mother cells (MMC) and tapetum in male-fertile and male-sterile anthers of Beta vulgaris L. was compared at the electron microscope level. These studies were complemented by morphometric analyses of mitochondria in both tissues through successive stages of microsporogenesis. The earliest irregularities in the ultrastructure of male-sterile anthers were noted within the tapetum at the tetrad stage. These disturbances were initially expressed by a slight reduction in mitochondrial size and the appearance of concentric configurations of endoplasmic reticulum. As development proceeded, a further decrease in mitochondrial size become more conspicuous and was accompanied by a reduction in ribosome population and a failure of the tapetum to produce Ubisch bodies. This failure to produce Ubisch bodies is reflected in the underdevelopment of sterile microspore exine.

Evolutionary trends in the reproductive biology of angiosperms

Which embryologic characters are primitive, and which are advanced, has long been a controversial issue? The comparative embryology of angiosperms and gymnosperms does not provide any useful clue. Single embryological characters are not as useful for phylogenetic and evolutionary considerations as are a group of characters. Moreover, some of the characters may be merely adaptive without any evolutionary significance. The numerous variations cause further complication.
 This article is devoted to (a) distribution and phylogeny of anther tapetum, (b) affinities of megagametophyte, (c) can the angiosperm embryo sac be derived from the archegonium in gymnosperms?, (d) primitive and advanced embryological features, and (e) embryological features in Magnoliiflorae s.1. and certain Liliiflorae.

Microsporogenesis and tapetal development in fertile and cytoplasmic male-sterile sugar beet (Beta vulgaris L.)

The development of sporogenous and tapetal cells in the anthers of male-fertile and cytoplasmic male-sterile sugar beet (Beta vulgaris L.) plants was studied using light and transmission electron microscopy. In general, male-sterile anthers showed a much greater variability in developmental pattern than male-fertile anthers. The earliest deviation from normal anther development was observed to occur in sterile anthers at meiotic early prophase: there was a degeneration or irregular proliferation of the tapetal cells. Other early aberrant events were the occurrence of numerous small vesicles in the microspore mother cells (MMC) and a disorganized chromatin condensation. Deviations that occurred in sterile anthers at later developmental stages included: (1) less distinct inner structures in the mitochondria of both MMC and tapetal cells from middle prophase onwards. (2) dilated ER and nuclear membranes at MMC prophase, in some cases associated with the formation of protein bodies. (3) breakdown of cell walls in MMCs and tapetal cells at late meiotic prophase. (4) no massive increase in tapetal ER at the tetrad stage. (5) a general dissolution of membranes, first in the MMC, then in the tapetum. (6) abortion of microspores and the occurrence of a plasmodial tapetum in anthers reaching the microspore stage. (7) no distinct degeneration of tapetal cells after microspore formation. Thus, it seems that the factors that lead to abortive microsporogenesis are structurally expressed at widely different times during anther development. Aberrant patterns are not restricted to the tetrad stage but occur at early prophase.

Microsporogenesis in a normal line and in the ogu cytoplasmic male-sterile line of Brassica napus

Under an intermediate temperature regime (23° C/18° C; day/night), microsporogenesis in stamens of the ogu cytoplasmic male-sterile (CMS) line of Brassica napus terminated by the tetrad stage, although in some cases degeneration of the sporogenous tissue occurred prior to meiosis. In most cases the tetrads were collapsed and bounded by a sparse exine, but contained many organelles. Also, the tapetum in CMS anthers was abnormal and often highly vacuolated by the tetrad stage. Under low temperature conditions (18° C/15° C; day/night), neither microsporogenous nor tapetal tissues were observed. In the normal stamens, no differences were observed under different temperature regimes. In conjunction with the adjoining paper, this study demonstrates that temperature conditions strongly affect the cytological processes associated with microsporogenesis in the CMS anthers.

Invasive tapetum and tricelled pollen inAmbrosia trifida (Asteraceae, tribeHeliantheae)

Staminate flowers of giant ragweed,Ambrosia trifida L. (Asteraceae, tribeHeliantheae, subtribeAmbrosiinae) were processed into resin and sectioned 1–2 µm thick. The invasive (amoeboid) anther tapetum remains parietal until microspores are released from tetrads, then it swells and invades the locule, merging gradually into a single protoplast that flows among the microspores. After the tapetal membrane ruptures at late microspore stage, tapetal debris fills the locule, then disappears as pollen matures. Pollen becomes tricelled before anthesis. The two sperm cell nuclei are slender and wormlike. The present report supports the two generalizations that invasive tapetum and tricelled pollen are attributes of theAsteraceae.

Flavanone 3-hydroxylase and Dihydroflavonol Oxygenase Activities in anthers of Tulipa: The Significance of the Tapetum Fraction in Flavonoid Metabolism

Summary Flavanone 3-hydroxylase and dihydroflavonol oxygenase activities were shown in anthers of Tulipa c.v. Apeldoorn. Both activities are tapetum-bound as demonstrated by a separation of the loculus material (i.e. anther contents) into a pollen and a tapetum fraction. Thus, the tapetum fraction has been shown to possess an almost complete sequence of enzyme reactions involved in the biosynthesis of flavonol glycosides. It is therefore evident that the anther tapetum plays an important role in phenylpropanoid metabolism on pollen. Investigations of phenylalanine ammonia-lyase and chalcone synthase activities in anthers of various systems indicated that the observations made for Tulipa c.v. Apeldoorn do not represent a unique but a widespread phenomenon.

Nuclear DNA content in differentiated tissues of sunflower (Helianthus annuus L.)

Scanning cytophotometry following Feulgen-staining was used to determine nuclear DNA content in many differentiated tissues of nine cultivars, hybrids or selfed lines ofHelianthus annuus. Apart from such ephemeral tissues as endosperm and anther tapetum, it was found that tissue differentiation in sunflower occurs in the diploid condition, cells being arrested in the DNA presynthetic phase (G1). In certain cases, however, the nuclear DNA content of differentiated G1 cells does not exactly match the 2C DNA content found in meristematic cells, but may be either higher or lower. In endosperm and anther tapetum cells, nuclear DNA content may be as high as 24 C and 32 C, respectively. Cytological and autoradiographic analyses after3H-thymidine incorporation reveal that polyploidy in the tapetal cells is due to chromosome endoreduplication. No detectable difference between male-fertile and male-sterile plants exists as far as occurrence and level of cell polyploidy are concerned. The results are discussed in the context of previous investigations on the nuclear condition of differentiatedHelianthus annuus tissue.

Embryological investigations inCentipeda minima (Asteraceae)

Anthers are tetrasporangiate. Anther tapetum is of the periplasmodial type and its cells become polyploid. Microspore tetrads are tetrahedral. Pollen grains are shed at 3-celled stage. Ovule is anatropous, unitegmic and tenuinucellate. Embryo sac development is of the Polygonum type. Antipodals are either 2 or 3 in number. They become polyploid and are persistent. Synergids are hooked. Endosperm development is of the cellular type and embryo development conforms to the Grand period I, Megarchetype II, series A and sub series A2 in the first embryonic group and it keys out to the Calendula subtype.

Colchicine inhibits plasmodium formation and disrupts pathways of sporopollenin secretion in the anther tapetum ofTradescantia virginiana L.

During an earlier investigation, microtubules were observed at the periphery of invasion processes in the developing syncytial tapetum ofTradescantia virginiana L. They were also associated with membranous sacs that accumulate adjacent to tetrads, with putative fusion sites where the tapetal plasmodium is initiated, and, in postmeiotic stages, with the perispore membrane that encloses the developing spore cells. Colchicine was administered to developing flower buds to investigate the roles of these microtubules. The results indicate that microtubules neither initiate nor guide the tapetal invasion of the loculus. The treatments, however, resulted in absence of cell coat from invasion processes and prevention of cell fusion. They also inhibited polarized migration of membrane sacs and removed the associated microtubules. The development of an organized secretory apparatus at the perispore membrane was disrupted, with subsequent disordered deposition of sporopollenin in the extracellular spaces of the partially-fused plasmodium. The results suggest that microtubules participate in the formation and internal spatial organization of the tapetal plasmodium, and establishment of a secretory surface that normally produces sporopollenin at the tapetum-microspore interface.

Cytoskeleton, cell surface and the development of invasive plasmodial tapetum inTradescantia virginiana L.

The anther tapetum inTradescantia virginiana L. is of the invasive plasmodial type: the cells lose their walls during early spore meiosis and develop long invasion processes which invade the loculus to penetrate spaces between the sporogenous cells. Fusion to form a syncytium is delayed and conventional ultra-thin sections and the Thiery reaction reveal the presence of a loose fibrillar extracellular cell coat on the free surfaces of tapetal cells and their invasion processes. Cell fusion involves formation of apposition areas characterized by an absence of cell coat and the local appearance of microtubular arrays. Conspicuous membrane sacs, associated closely with microtubules, were found to migrate to and accumulate at the plasma membranes near the fusion sites and sporogenous cells. Microtubules are always present in the cortical regions of the tapetal cells and their invasion processes. It is surmised that microtubules are not responsible either for initiating or guiding tapetal invasion of the loculus; instead they may help to sustain the form of the invasion processes, help in the migration of membrane sacs, and participate in cell fusion. The cell coat disappears with syncytium formation towards the end of meiosis, and the developing spore cells become surrounded by a “perispore membrane”, which, derived from the original plasma membranes and augmented by membrane sacs, forms labyrinthine membrane reservoirs that are described further in the accompanying paper.

A comparative light and electron microscopic study of microspore and tapetal development in male fertile and cytoplasmic male sterile oilseed rape (Brassica napus)

The cytological development of male cells and the tapetum of male fertile and combined cytoplasmic triazine-resistant cyto-plasmic-genetic male sterile (ctr) lines of B. napus L. was studied using light, scanning electron, and transmission electron microscopy. Development of the cytoplasmic-genetic male sterile anther was similar to the normal anther up to and including meiotic prophase I. After this stage, degeneration of the microspore mother cells occurs within the callose walls, and tetrads of microspores are not formed. These degenerating microspore mother cells appear to develop numerous endoplasmic reticulum derived vesiculated structures, which may be involved in lysis of organelles. Degeneration occurs simultaneously with a proliferation of the tapetum, which eventually fills the anther locule. It is not clear whether the abortion of the microspore mother cells during meiosis stimulates proliferation of the tapetum or whether the proliferating tapetum actually interferes with microspore development thereby causing degeneration. Dilated endoplasmic reticulum cisternae containing crystalline-like deposits, and plastids with osmiophilic bodies, are frequent in cells of the proliferated tapetum of cytoplasmic-genetic male sterile anthers.

Orbicules and the ektexine are homologous sporopollenin concretions inSpermatophyta

During microsporogenesis sporopollenin becomes accumulated independently by the only two sporopollenin-producing cell types in the higher plants—the anther tapetum and the microspores—not only within the exine, but also within the orbicules. In Spermatophytes usually only a single orbicule type is found, but sometimes, e.g., inEuphorbia palustris, two different types exist within a single species. Very interestingly, most orbicules are strikingly similar in their structure, sculpture, shape and dimension to the respective ektexine: e.g., smooth sporopollenin globules are found near the totally smooth exines ofEupomatia laurina andPentaphragma sinense respectively, while inDelonix elata andEuphorbia palustris the orbicules often look like some “incomplete” tectum pieces. All these parallelisms can be attributed to the homologous and therefore highly similar genetic information to form sporopollenin in the sporogeneous tissue and the anther tapetum. The orbicules should be seen neither as sporopollenin storage, as sporopollenin transfer vehicle, nor as sporopollenin surplus.