Structure and function of the microtubular cytoskeleton during endosperm development in wheat: an immunofluorescence study

Summary The three-dimensional structure of the microtubular cytoskeleton of developing wheat endosperm was investigated immunocytochemically. Semi-thin sections were prepared from polyethylene glycol embedded ovaries. At the free-nuclear stage the endosperm cytoplasm with regularly distributed nuclei surrounded a large central vacuole and exhibited an extensive network of fluorescent labelled microtubular assemblies radiating from each nucleus. As was found in other coenocytes, this particular and nuclear-dependent cytoskeletal configuration functions in the arrangement of nuclei and in the stabilization of the nuclear positions. At the beginning of cellularization of the endosperm the formation of vacuoles altered the radiating networks. It is likely that the radiating microtubular arrays function in the formation of phragmoplasts, independent of nuclear divisions. The formation of anticlinal cell walls, giving rise to openended cell cylinders, coincides with the occurrence of phragmoplast microtubular arrays which were demonstrated during the period of cell wall elongation. The microtubular system radiating from the nuclei in these cell cylinders anchored the nuclei in stage- and locus-specific positions. During the development of aleurone and inner endosperm cells, cell morphogenesis was related to earlier demonstrated types of microtubular configurations in the cortical cytoplasm. This suggests that a general mechanism is involved.Abbreviations A alveolus - AL aleurone layer - CE central endosperm - CV central vacuole - DAP days after pollination - END endosperm - FITC fluorescein isothiocyanate - GAR-FITC goat anti-rabbit antibodies conjugated with FITC - I integument - IE PC inner epidermis pericarp - II inner integument - N nucleus - NC nucellus cells - NE nucellar epidermis - NUC nucellus - OI outer integument - PBS phosphate buffered saline - PC pericarp - PEG polyethylene glycol - V vacuole     

Structure and function of the microtubular cytoskeleton during megasporogenesis and embryo sac development in Gasteria verrucosa (Mill.) H. Duval

Summary Using immunocytochemical techniques, tubulin distribution in various stages of meiosis and embryo sac development was studied. In the archespore cell some microtubules appeared to be randomly oriented. During zygotene and pachytene, when the cell volume increases, a large number of microtubules in dispersed configurations and bundles were observed. During this stage the nucellar cells divide, and their parallel cortical microtubules play an important role in preparing the direction of cell enlargement. The protoderm cells show anticlinal-directed cortical microtubules. It can be concluded that the enlargement of the meiocyte during these early meiotic stages is influenced both by its own cytoskeleton and by growth of the nucellus. Thereafter, the microtubules function directly in meiosis and disappear for the greater part until the two-nucleate coenocyte is formed. In a four-nucleate coenocyte microtubules reappear around the nucleus; in a young synergid, randomly oriented microtubules are involved in cell shaping during the formation of the filiform apparatus; in the synergids of the mature embryo sac, many parallel arrays of microtubules are present. Microtubules are less abundant in other cells. It is concluded that the cytomorphogenesis of the developing coenocyte and embryo sac are due to cell growth of the nucellar cells together with vacuolation of the coenocyte.     

Rice caryopsis development Ⅰ: Dynamic changes in different cell layers

Rice caryopsis as one of the most important food sources for humans has a complex structure that is composed of maternal tissues including the pericarp and testa and filial tissues including the endosperm and embryo. Although rice caryopsis studies have been conducted previously, a systematic characterization throughout the entire developmental process is still lacking. In this study, detailed morphological examinations of caryopses were made during the entire 30-day developmental process. We observed some rapid changes in cell differentiation events and cataloged how cellular degeneration processes occurred in maternal tissues. The differentiations of tube cells and cross cells were achieved by 9 days after pollination(DAP). In the testa, the outer integument was degenerated by 3 DAP, while the outer layer of the inner integument degenerated by 7 DAP. In the nucellus, all tissues with the exception of the nucellar projection and the nucellar epidermis degenerated in the first 5 DAP. By 21 DAP, all maternal tissues, including vascular bundles, the nucellar projection and the nucellar epidermal cells were degenerated. In summary, this study provides a complete atlas of the dynamic changes in cell differentiation and degeneration for individual maternal cell layers of rice caryopsis.     

Rice caryopsis development I: Dynamic changes in different cell layers

Rice caryopsis as one of the most important food sources for humans has a complex structure that is composed of maternal tissues including the pericarp and testa and filial tissues including the endosperm and embryo. Although rice caryopsis studies have been conducted previously, a systematic characterization throughout the entire developmental process is still lacking. In this study, detailed morphological examinations of caryopses were made during the entire 30‐day developmental process. We observed some rapid changes in cell differentiation events and cataloged how cellular degeneration processes occurred in maternal tissues. The differentiations of tube cells and cross cells were achieved by 9 days after pollination (DAP). In the testa, the outer integument was degenerated by 3 DAP, while the outer layer of the inner integument degenerated by 7 DAP. In the nucellus, all tissues with the exception of the nucellar projection and the nucellar epidermis degenerated in the first 5 DAP. By 21 DAP, all maternal tissues, including vascular bundles, the nucellar projection and the nucellar epidermal cells were degenerated. In summary, this study provides a complete atlas of the dynamic changes in cell differentiation and degeneration for individual maternal cell layers of rice caryopsis.     

The Embryology of Nyssa sinensis Oliv. (Nyssaceae)

The flower develops in March and blossoms in early May in Nanjing. The cytokinesis of microsporocytes is simultaneous and most tetrads are tetrahedral. The tapetum is secretory and the nuclei become polyploid at last. The style is solid and most ovaries are unilocular, rarely bilocular. The ovule is pendulous, anatropous and unitegmic. The nucellus is pseudocrassinucellate. An obturator formed by transmitting tissue covers the micropyle. The raphe vascular strand extends into the integument when it reaches the chalaza and on a whole keeps a “U” shape. The endothelium cell is uninucleate. In most cases no nucellar cap is formed. No hypostase is found below the embryo sac. The archesporium is one-celled. The embryo sac development conforms to the Polygonum or Allium types. The degeneration of the megaspores in the linear tetrad usually occurs from the chalazal toward the micropylar end. Two synergids persist during fertilization. Three antipodal cells are uninucleate and ephemeral. Two polar nuclei fuse at the time of fertilization. The fertilization type accords with porogamy. The syngamy is premitotic. The development of endosperm is cellular. The initial four successive divisions of the primary endosperm cell are transverse-verticaltransverse-transverse subsequently, giving rise to sixteen cells of the early endosperm. The mature embryo is straight and nearly as long as the endospermous seed. The cotyledons are more or less cordate at base. The seedoat is thin and composed of 5-11 layers of compressed cells. Neither embryo nor endosperm contain the alkaloid camptothecine. The major similarities of Nyssa sinensis to the American nyssas in embryology, which may be a counted as the generic features, are the polyploid tapetum cells, the unitegmic ovule with U-shaped vascular strand, the direct enlargement of the archesporial cell to produce the megasporocyte, the pseudocrassinucellus, the usual absence of the nucellar cap, the Polygonum or Allium type of the embryo sac development, the first degeneration of the metachalazal megaspore, the ephemeral antipodal cells, a single nucleolus in the nucleus ofthe primary endosperm cell, the more or less cordate base of the cotyledons.     

Nitric oxide and hydrogen peroxide involvement during programmed cell death of Sechium edule nucellus

The nucellus is a maternal tissue that feeds the developing embryo and the secondary endosperm. During seed development the cells of the nucellus suffer a degenerative process early after fertilization as the cellular endosperm expands and accumulates reserves. Nucellar cell degeneration has been characterized as a form of developmentally programmed cell death (PCD). In this work we show that nucellus PCD is accompanied by a considerable production of both nitric oxide and hydrogen peroxide (NO and H₂O₂). Interestingly, each of the two molecules is able to induce the production of the other and to cause cell death when applied to a living nucellus. We show that the induced cell death has features of a PCD, accompanied by profound changes in the morphology of the nuclei and by a massive degradation of nuclear DNA. Moreover, we report that NO and H₂O₂ cause an induction of caspase‐like proteases previously characterized in physiological nucellar PCD.     

Caspase-Like Activities Accompany Programmed Cell Death Events in Developing Barley Grains

Programmed cell death is essential part of development and cell homeostasis of any multicellular organism. We have analyzed programmed cell death in developing barley caryopsis at histological, biochemical and molecular level. Caspase-1, -3, -4, -6 and -8-like activities increased with aging of pericarp coinciding with abundance of TUNEL positive nuclei and expression of HvVPE4 and HvPhS2 genes in the tissue. TUNEL-positive nuclei were also detected in nucellus and nucellar projection as well as in embryo surrounding region during early caryopsis development. Quantitative RT-PCR analysis of micro-dissected grain tissues revealed the expression of HvVPE2a, HvVPE2b, HvVPE2d, HvPhS2 and HvPhS3 genes exclusively in the nucellus/nucellar projection. The first increase in cascade of caspase-1, -3, -4, -6 and -8-like activities in the endosperm fraction may be related to programmed cell death in the nucellus and nucellar projection. The second increase of all above caspase-like activities including of caspase-9-like was detected in the maturating endosperm and coincided with expression of HvVPE1 and HvPhS1 genes as well as with degeneration of nuclei in starchy endosperm and transfer cells. The distribution of the TUNEL-positive nuclei, tissues-specific expression of genes encoding proteases with potential caspase activities and cascades of caspase-like activities suggest that each seed tissue follows individual pattern of development and disintegration, which however harmonizes with growth of the other tissues in order to achieve proper caryopsis development.     

ADVENTIVE EMBRYOGENESIS IN CITRUS AND ITS RELATION TO POLLINATION AND FERTILIZATION

Cytological and histological studies of seeds from three facultative apomictic Citrus cultivars show that adventive embryos develop, as a rule, from the first few cell layers of the nucellus adjacent to the embryo sac in the micropylar half and occasionally from the chalazal end. The adventive embryos initiated in nucellar tissue away from the embryo sac and most of those initiated from the chalazal end of the nucellus do not develop beyond the one-celled stage. When two or more embryos are developing in the same seed, the successful development of a given embryo depends on its location in relation to access to nutrients from the endosperm. The presence of a zygote and triploid endosperm in seeds with adventive embryos, the abortion of seed when endosperm degenerates, and the lack of seed set without pollination indicate that pollination and fertilization are essential for in vivo adventive embryogenesis.     

HISTOCHEMISTRY AND ULTRASTRUCTURE OF RICE (ORYZA SATIVA) ZYGOTIC EMBRYOGENESIS

Rice embryo development was examined, histochemically and ultrastructurally, from the time of fertilization to embryo maturity. At the time of fertilization, the megagametophyte consists of an antipodal mass of 10–15 cells, parietally positioned along the placental side of the central cell, and, at the micropylar end, two partly fused polar nuclei and the egg apparatus. Hydrolysis of adjacent nucellar tissue suggests the secretion of hydrolytic enzymes by the antipodal mass. The antipodal cells stain intensely for RNA and protein, indicating that they are metabolically active. The egg, supported by two overarching synergids, occupies a small, wall ingrowth-lined pocket of the central cell that quickly fills with cellular endosperm after fertilization. The endosperm cells, initially supplied with nutrients from wall ingrowth-derived vesicles, are digested and utilized by the embryo as a nutritive source. The developing embryo is also supplied with assimilates via the nucellus at the base of the embryo until about 8 days after fertilization. After 8 days, the embryo is no longer connected to the nucellus, and the nucellar cells at the base of the embryo are crushed. The zygote is not structurally polarized and contains a central nucleus, amyloplasts, lipid bodies, dictyosomes and extensive dilated ER. The first division of the zygote is transverse and unequal and occurs about 4 hours after fertilization. Embryo development is rapid, and within 24 hr, the embryo consists of 5–8 cells. Organ development begins with scutellum emergence in the 3-day-old embryo. The shoot apex organizes and the coleoptile develops from scutellum tissue at 4 days postfertilization, the epiblast emerges at 5 days, and the vascular bundle and root apex differentiate by 6 days after fertilization. Starch begins to accumulate in the basal cells of the 3-day-old embryo and deposition proceeds acropetally over the next 9–10 days. Lipid accumulation begins in the basal scutellum in the 6-day-old embryo and also proceeds acropetally. Storage protein synthesis is first detected in 6-day-old embryos and accumulation again proceeds acropetally, reaching the apex of the scutellum of the 25-day-old embryo. The ultrastructure of the 24-hr-old embryo is distinctive. The cells are characterized by numerous vesicles, heterochromatin and extensive nuclear evaginations.     

Development of maternal seed tissue in barley is mediated by regulated cell expansion and cell disintegration and coordinated with endosperm growth

After fertilization, filial grain organs are surrounded by the maternal nucellus embedded within the integuments and pericarp. Rapid early endosperm growth must be coordinated with maternal tissue development. Parameters of maternal tissue growth and development were analysed during early endosperm formation. In the pericarp, cell proliferation is accomplished around the time of fertilization, followed by cell elongation predominantly in longitudinal directions. The rapid cell expansion coincides with endosperm cellularization. Distribution of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling)-positive nuclei reveals distinct patterns starting in the nucellus at anthesis and followed later by the inner cell rows of the pericarp, then spreading to the whole pericarp. The pattern suggests timely and spatially regulated programmed cell death (PCD) processes in maternal seed tissues. When the endosperm is coenocytic, PCD events are only observed within the nucellus. Thereby, remobilization of nucellar storage compounds by PCD could nourish the early developing endosperm when functional interconnections are absent between maternal and filial seed organs. Specific proteases promote PCD events. Characterization of the barley vacuolar processing enzyme (VPE) gene family identified seven gene members specifically expressed in the developing grain. HvVPE2a (known as nucellain) together with closely similar HvVPE2b and HvVPE2d might be involved in nucellar PCD. HvVPE4 is strongly cell specific for pericarp parenchyma. Correlative evidence suggests that HvVPE4 plays a role in PCD events in the pericarp. Possible functions of PCD in the maternal tissues imply a potential nutritive role or the relief of a physical restraint for endosperm growth. PCD could also activate post-phloem transport functions.     

Proteomic profile of the nucellus of castor bean (Ricinus communis L.) seeds during development

In this study, we performed a proteomic analysis of nucellus from two developmental stages of Ricinus communis seeds by a GeLC-MS/MS approach, using of a high resolution orbitrap mass spectrometer, which resulted in the identification of a total of 766 proteins that were grouped into 553 protein groups. The distribution of the identified proteins in stages III and IV into different Gene Ontology categories was similar, with a remarkable abundance of proteins associated with the protein synthesis machinery of cells, as well as several classes of proteins involved in protein degradation, particularly of peptidases associated with programmed cell death. Consistent with the role of the nucellus in mediating nutrient transfer from maternal tissues to the endosperm and embryo, a significant proportion of the identified proteins are related to amino acid metabolism, but none of the identified proteins are known to have a role as storage proteins. Moreover for the first time, ricin isoforms were identified in tissues other than seed endosperm. Results are discussed in the context of the spatial and temporal distribution of the identified proteins within the nucellar cell layers.     

Seed ontogeny in Adesmia securigerifolia (Fabaceae-Adesmieae)

A study of the reproductive processes of Adesmia securigerifolia from bud to mature seed was carried out by means of field observations and the paraffin technique. Observations revealed the following new contributions to the study of legume embryology: 1) after fertilization, a small nucellar haustorium, or micropylar nucellar beak, was observed for the first time, originating from two obliterating nucellar cells that extended outwards. Their globose distal end comes in contact with the internal carpel wall, while the wedge shaped base stretches into the micropyle; a suspensor consisting of five or more cells - the two basal cells are large and falcate and fit into the micropylar pore - coexists with the undivided polar nuclei thus showing that endosperm formation begins after zygote division; 2) at the young embryo stage, a sac- shaped nuclear haustorium, formed by the endosperm, adjoins the outer integument and is not connected to the chalaza, or any vascular element; at the hilar level, a nucellar projection is formed in connection with the haustorial coenocytic endosperm. This projection persists up to the mature seed stage when it starts to degenerate, after performing another linking with the embryo nutrition system; 3) at the mature seed stage, the seed coat evolving from the outer integument has a single macrosclereid layer, though inclusions in the cell vacuoles simulate the presence of more layers and/or transverse walls. The lens, a hypodermal layer of osteosclereids (hour-glass cells), and the astrosclereids are also described.     

Nucellar projection transfer cells in the developing wheat grain

Summary Transfer cells in the nucellar projection of wheat grains at 25 ±3 days after anthesis have been examined using light and electron microscopy. Within the nucellar tissue, a sequential increase in non-polarized wall ingrowth differentiation and cytoplasmic density was evident. Cells located near the pigment strand were the least differentiated. The degree of differentiation increased progressively in cells further removed from the pigment strand and the cells bordering the endosperm cavity had degenerated. Four stages of transfer cell development were identified at the light microscope level. Wall ingrowth differentiation followed a sequence from a papillate form through increased branching (antler-shaped ingrowths) which ultimately anastomosed to form a complex labyrinth. The final stage of wall ingrowth differentiation was compression which resulted in massive ingrowths. In parallel with wall ingrowth deposition cytoplasmic density increased. During wall deposition, paramural and multivesicular bodies were prominent and were in close association with the wall ingrowths. The degeneration phase involved infilling of cytoplasmic islets within the wall ingrowths. This was accompanied by complete loss of the protoplast. The significance of this transfer cell development for sucrose efflux to the endosperm cavity was assessed by computing potential sucrose fluxes across the plasma membrane surface areas of the nucellar projection cells. Transfer cell development amplified the total plasma membrane surface area by 22 fold. The potential sucrose flux, when compared with maximal rates of facilitated membrane transport of sugars, indicated spare capacity for sucrose efflux to the endosperm cavity. Indeed, when the total flux was partitioned between the nucellar projection cells at the three stages of transfer cell development, the fully differentiated stage III cells located proximally to the endosperm cavity alone exhibited spare transport capacity. Stage II cells could accommodate the total rate of sucrose transfer, but stage I cells could not. It is concluded that the nucellar projection tissue of wheat provides a unique opportunity to study transfer cell development and the functional role of these cells in supporting sucrose transport.Abbreviations CSPMSA cross sectional plasma membrane surface area - LPMSA longitudinal plasma membrane surface area - PTS tri-sodium 3-hydroxy-5,8,10-pyrenetrisulfonate     

Contrast observation and investigation of wheat endosperm transfer cells and nucellar projection transfer cells

In cereal seed, there are no symplastic connections between the maternal tissues and the endosperm. In order to facilitate solute transport, both the nucellar projection and its opposite endosperm epithelial cells in wheat caryopsis differentiate into transfer cells. In this paper, we did contrast observation and investigation of wheat endosperm transfer cells (ETC) and nucellar projection transfer cells (NPTC). The experimental results showed that there were some similarities and differences between ETC and NPTC. ETC and NPTC almost developed synchronously. Wall ingrowths of ETC and NPTC formed firstly in the first layer nearest to the endosperm cavity, and formed later in the inner layer further from the endosperm cavity. The mature ETC were mainly three layers and the mature NPTC were mainly four layers. Wall ingrowths of ETC were flange type and wall ingrowths of NPTC were reticulate type. NPTC were not nutrient-storing cells, but the first layer of ETC had aleurone cell features, and the second layer and third layer of ETC accumulated starch granules and protein bodies.     

The nucellus degenerates by a process of programmed cell death during the early stages of wheat grain development

The nucellus, which is the maternal tissue of the wheat grain, degenerates during the early stages of development. We have investigated whether or not this degenerative process may be considered as programmed cell death (PCD). The analysis of DNA of tissues dissected from developing wheat (Triticum aestivum L. cv Chinese Spring) grains at 5-20 days post anthesis (dpa) showed the presence of DNA laddering, which is indicative of internucleosomal fragmentation of nuclear DNA, in maternal tissues but not in the endosperm. The TUNEL assay showed in-situ internucleosomal fragmentation of DNA in nuclei of parenchymal and epidermal cells of the nucellus, as well as in the pericarp, during the early stages of grain development (5 dpa). Furthermore, internucleosomal fragmentation of nuclear DNA was observed in nucellar projection cells in the middle stages of grain development (13-18 dpa), thus showing a process of PCD in these maternal tissues. Electron-transmission microscopy analysis allowed the morphology of PCD to be characterized in this plant tissue. Initially, fragmentation of the cytoplasm was observed, the nuclear envelope appeared dilated and to be forming vacuoles, and the content of heterochromatin increased. A progressive degradation of the cytosolic contents and organelles was observed, and the plasma membrane was disrupted. However, the Golgi apparatus remained intact and apparently functional even in the final stages of cell death.     

Optimization of conditions for in situ hybridization and determination of the relative number of ribosomes in the cells of the mature embryo sac of maize

Summary We have initiated experiments to understand the molecular regulation of embryo sac development in flowering plants by a study of ribosome synthesis and accumulation. Because of the very small size of the embryo sac and the large volume of ovule tissue it is embedded in, in situ hybridization with nucleic acid probes is presently the only practical method for such molecular measurements on individual cells of the embryo sac. Methods of tissue preparation, sectioning and screening of ovules for embryo sac containing sections, in situ hybridization using a ribosomal mRNA probe, and staining were optimized. Relative densities of silver grains for individual cells of the mature maize (W22) embryo sac were determined from in situ hybridizations. The silver grain counts are directly related to the numbers of ribosomes. Volumes of individual cells were determined by confocal microscope image analysis, and this permitted the calculation of the relative total numbers of ribosomes in individual cells of the embryo sac and nucellus. The central cell has a volume 260 times that of a nucellar cell at the micropylar end of the ovule, 15 times that of the egg cell, 30 times that of a synergid, and 130 times the volume of an antipodal cell. The mature maize embryo sac has 20 or more antipodal cells. The central cell has approximately 200 times the number of ribosomes as are present in a nucellar cell, about 7 times as many ribosomes as are in the egg cell, 14 times as many ribosomes as in each synergid, and about 80 times the ribosome content of individual antipodal cells. The data are discussed with respect to the utilization of the ribosomes following fertilization in the early embryo and endosperm.     

Programmed cell death of the nucellus during Sechium edule Sw. seed development is associated with activation of caspase-like proteases

The nucellus is a maternal tissue that embeds and feeds the developing embryo and secondary endosperm. During seed development, the cells of the nucellus suffer a degenerative process soon after fertilization as the cellular endosperm expands and accumulates reserves. Nucellar cell degeneration has been considered to be a form of developmentally programmed cell death (PCD). It was investigated whether or not this degenerative process is characterized by apoptotic hallmarks. Evidence showed that cell death is mostly localized in the border region of the tissue adjacent to the expanding endosperm. Cell death is accompanied by profound changes in the morphology of the nuclei and by a huge degradation of nuclear DNA. Moreover, an increase of activity of different classes of proteinases is reported, and the induction of caspase-like proteases sensitive to specific inhibitors was detected. Nucellar caspase-like proteases are characterized by an acid pH optimum suggesting a possible localization in the vacuole.