Nuclear pores during the cell cycle in a slime mold,Physarum polycephalum

Freeze-fracture and thin sectioning techniques were used to follow in large synchronous plasmodia of Physarum polycephalum the changes in number and distribution of nuclear pores during the cell cycle. Using freeze-fracture, we determined that average pore frequency rises gradually from 14/μm² of nuclear envelope surface at early S to a value of about 22 just before prophase. Nuclear diameter averaged 3.3 γm at early S and increased to 4.3 μm at late G2. Calculating nuclear volume and average chromatin volume per nucleus with respect to time in the cell cycle leads to the conclusion that number of nuclear pores appears to be most directly related to amount of chromatin present per nucleus and to be independent of nuclear surface area.     

In vitro culture promotes partial autonomous endosperm development in unfertilized ovules of wild-type Arabidopsis thaliana var. Columbia

Partial endosperm development without paternal genome involvement was induced in unpollinated ovaries of wild-type Arabidopsis thaliana cultured in vitro. Unpollinated pistils were cultured on hormone-free Murashige and Skoog (MS) medium with addition of 6% sucrose and supplemented with: benzylaminopurine (BAP; 2 mg l^–1) combined with naphthylacetic acid (NAA; 0.1 mg l^–1), 2,4-dichlorophenoxyacetic acid (2,4-D; explants exposed to 1-h auxin shock 20 or 40 mg l^–1, and transferred to hormone-free MS medium). Initiation of autonomous endosperm (AE) development was induced on all media used in 54 ovules from 39 cultured ovaries (26%), with an average frequency of 1.4 ovules/ovary. The highest frequency of partial endosperm formation occurred on media combining the two growth regulators BAP and NAA (59% of ovaries had ovules with AE), although endosperm development was also induced on hormone-free medium (in 20.5% of ovaries). The number of AE nuclei ranged from 2 to ~50, depending on the day of culture and medium; neither cellularization nor differentiation on specific regions typical for endosperm of wild-type Arabidopsis, were noted. Fertilization independent endosperm most probably originated from the secondary nucleus, but involvement of the polar nuclei could not be excluded, as indicated by nuclear size and structure. In vitro conditions did not influence egg cell proliferation. Gynogenic embryos were observed neither in the ovules with autonomous endosperm nuclei nor in ovules without endosperm induction.     

Organic acids and cellular changes in the endosperm of Euphorbia lambii seedlings

Seedling of Euphorbia lambii Svent. were grown in the dark, and the levels of organic acids in the endosperm were monitored during the 6–7 day period in which all the reserves were depleted. Glycolate, glyoxylate, succinate, fumarate and 2-oxoglutarate occurred in traces only, the citrate concentration remained rather constant (0.4 μmol endosperm^?1), malate varied from 0.2 to 0.4 μmol endosperm^?1, but malonate appeared to be the major organic acid in the endosperm ranging from 0.75 to 1.25 μmol endosperm^?1. Radioactive malonate was easily taken up by the cotyledons of growing seedlings, and up to 11.2% of the label proceeded to the sterols, the triterpenes and triterpene esters in a 48 h incorporation period. No label from [¹⁴C]-malonate was built into the triacylglycerols in the seedling. Maximum uptake values of 0.6 μmol malonate seedling^?1 day^?1 were measured, and this value was not altered by a simultaneous uptake of sucrose. Conversely, the uptake of labeled sucrose and its subsequent conversion into sterols and (latex) triterpenes was not altered by a simultaneous uptake of low concentrations of malonate. Increased amounts (from 0.25 μmol malonate seedling^?1 and up) caused a 75–90% reduction of both uptake and conversion of sucrose into neutral lipids. To maintain a daily uptake of 4 μmol of sucrose by the cotyledons (required to maintain seedling growth) the simultaneous in vivo uptake of malonate from the endosperm was supposed not to exceed 0.2 μmol seedling^?1 day^?1. Thin sections of the endosperm revealed the morphology of the process of reserve depletion. The occurrence of vacuoles 2 days after germination, coincided with the increase in the malonate level. The protein bodies first disappeared completely from the outer layers, whereas the triacylglycerols gradually disappeared from the entire endosperm. About 80% of the endosperm cells contained a large vacuole until the stage of complete depletion, probably serving as the major site of malonate storage.     

Chromosome constitution and nucleolar behaviour in the endosperm of Cucurbita pepo L

At early stages of development the endosperm of Cucurbita pepo L. was found to be triploid with 3n=60. Higher levels of ploidy (6n, 12n, 24n, 48n) were encountered at later stages. The euploid increase in chromosome number is the result of endopolyploidy. The volume of the nucleolus increased with the increase in nuclear volume. Cells with varying numbers of nucleoli of different sizes and shapes were observed in the endosperm at all stages. Fragmentation of the nucleolus is believed to be responsible for the increase in nucleolar number. Signs of nuclear disorganisation in the endosperm were visible from the 17th day onwards. As the endosperm tissue gets absorbed by the developing embryo, the mature seed is non-endospermous.     

A stereological analysis of developing egg chambers in Ephestia kuhniella

A polytrophic ovariole of the flour moth, Ephestia kuhniella, is composed of a linear series of increasingly mature egg chambers, each consisting of an oocyte, an interconnected cluster of seven nurse cells, and a covering layer of follicle cells. This study describes changes in the volume of each component as a function of the position of the egg chamber in the ovariole. Analysis of the growth curve of the Ephestia oocyte yields two possible correlations between accelerated oocyte growth and ultrastructural events enhancing the supply of yolk materials to the oocyte: the first is the initiation of yolk synthesis by the follicle cell layer and its transfer to the oocyte, and the second is the formation of channels between the follicle cells allowing hemolymph to gain access to the oocyte. An Ephestia oocyte increases in volume from approximately 2.5 × 10³ μm³ to approximately 2.0 × 10⁷ μm³ over an average series of 58 egg chambers.     

LIGHT AND ELECTRON MICROSCOPY OF GRAZING BY POTERIOOCHROMONAS MALHAMENSIS (CHRYSOPHYCEAE) ON A RANGE OF PHYTOPLANKTON TAXA1

Grazing of fluorescent latex beads, bacteria, and various species of phytoplankton by Poterioochromonas malhamensis (Pringsheim) Peterfi (about 8.0 μm in diameter) was surveyed. The alga ingested fluorescent beads and various live or killed and nomnotile or motile organisms including bacteria, blue-green algae, green algae, diatoms, and chrysomonads. The size range of grazed prey was from 0.1 to 6.0 μm for latex beads and from 1.0 μm (bacteria) to about 21 μm (Carteria inverse) for organisms. As many as 17 latex beads (2.0 μm) or more than 10 Microcystis cells (5–6 μm) were ingested by a single P. malhamensis cell. Following such grazing, the cell increased in volume by up to about 30-fold. The range of cell volume of ingested prey was from 0.52 μm³ (bacteria) to about 3178 μm³(Carteria inversa). This study demonstrates for the first time that P. malhamensis is capable of grazing algae 2–3 times larger in diameter than its own cell and of grazing intact motile algae. Poterioochromonas malhamensis is an omnivorous grazer. Food vacuole formation and digestion processes were examined. The membrane that was derived from the plasma membrane and surrounded the prey disappeared sometime after ingestion. The food vacuole was then formed by successive fusion of numerous homogeneous vesicles accumulated around the prey. The prey was enclosed in a single membrane-bound food vacuole and then digested.     

Nucleolar DNA amplification inOrnithogalum endosperm

Summary ³H-thymidine autoradiography has revealed selective amplification of nucleolar DNA in the maturing endosperm ofOrnithogalum elatum Andr., a liliaceous plant. Apart from the unusually heavy³H-thymidine labelling in the nucleoli, there is recurrent production of labelled accessory nucleoli which migrate into the cytoplasm by dynamic blebbings of the nuclear envelope, presumably to boost protein biosynthesis in the endosperm cells which nourish the developing embryo.     

Diploid endosperm formation in Tulipa spp. and identification of a 1:1 maternal-to-paternal genome ratio in endosperms of T. gesneriana L.

Most Liliaceae plants have the tetrasporic Fritillaria-type embryo sac and normally form diploid embryos and pentaploid endosperms derived from a 4:1 maternal-to-paternal genome ratio (4m:1p) after double fertilization. Here we characterize embryo sac and endosperm formation in Tulipa spp. of Liliaceae. Chromosome analysis using seeds derived from 2x × 2x crosses of Tulipa gesneriana (2n = 2x = 24) identified diploid chromosome number in the endosperm. Similarly, flow cytometric analysis confirmed diploid endosperm formation in T. gesneriana, T. fosteriana (2n = 2x = 24) and T. greigii (2n = 2x = 24). To further study the possible mechanism of diploid endosperm formation, we made interploidy crosses of triploid (2n = 3x = 36) × diploid in which aneuploid seeds with various chromosome numbers (2n = 25–36) were produced. Again, flow cytometric analysis confirmed the same ploidy level in both embryos and endosperms at all aneuploidy levels, suggesting that only a single haploid polar nucleus contributes to endosperm formation at fertilization. Histological observation further confirmed the physical separation of two polar nuclei by a large vacuole in the Fritillaria-type embryo sac of T. gesneriana that appeared to prevent the fusion of the two polar nuclei that originated at the micropylar and chalazal ends before fertilization. Taken together, these results indicate that diploid endosperms (1m:1p) are normally formed in Tulipa spp. by fusion of the micropylar polar nucleus (n) and a spermatid (n) but not by normal triple fusion. We also show that tulip endosperm partially overcomes the triploid block mechanism that occurs in interploidy crosses. Based on these observations, the possible role of triple nuclear fusion in double fertilization is discussed.     

Transformation in Heterokaryons of <Emphasis Type="Italic">Neurospora crassa</Emphasis> Is Nuclear Rather Than Cellular Phenomenon

In Neurospora crassa, multinucleate macroconidia are used for genetic transformation. The barrier for such a transformation can be either at the cell membrane level or at the nuclear membrane level. For assessment of these possibilities, a forced heterokaryon (containing two genetically marked nuclei and auxotrophic for histidine) of Neurospora crassa was transformed with a plasmid containing his-3 ⁺ gene. The transformants, which could grow without histidine supplementation, were then resolved into component homokaryons to determine into which nucleus or nuclei the plasmid had entered. Our results suggest that the barrier for transformation in Neurospora crassa is at the nuclear level, not at the cell membrane level. In a heterokaryon containing two genetically distinct nuclei, plasmid DNA integrated into only one of the nuclear types at any instance, but never into both nuclear types. Thus, in Neurospora crassa, the competent nucleus is essential for the transformation event to take place, and at a given time only one type of nucleus is competent to take up the exogenous DNA. Genomic Southern analysis showed that the transformants harbor both ectopic and homologous integrations of the plasmid DNA. The type and number of integrations were reflected at the post-translational level, since the specific activity of histidinol dehydrogenase (the translation product of his-3 ⁺ gene) was variable among several transformants and always less than the level of the wild type. Received: 24 July 2001 / Accepted: 15 August 2001     

Instability of the maize B chromosome

Summary The B ⁹ chromosome of maize exhibits a very ordered type of instability at the second pollen mitosis, when nondisjunction may reach a level of 95%. Much less commonly the chromosome is unstable during early development of the kernel. Instability in the kernel produces recessive sectors in either the endosperm or the sporophyte, reflecting the absence of dominant markers carried by the B ⁹. The causes of B ⁹ loss in the endosperm and the sporophyte were investigated for the two observable classes of sectoring: fractional loss (single event) and multiple loss (mosaic pattern). The fractional class represents isochromosome formation by the B ⁹ (Carlson, 1970, 1971). Data presented here suggest that the isochromosome is a by-product of telocentric formation at the second pollen mitosis, and does not arise directly from the B ⁹ chromosome. The chromosomal basis for the mosaic pattern of B ⁹ loss is not completely known. However, one class of mosaic kernels displays a heritable instability of the B ⁹ chromosome which apparently results from ring chromosome formation by the B ⁹. The time of origin of the ring B ⁹ chromosome is prior to the second pollen mitosis, since the unstable chromosome generated in the male parent is transmitted to both the endosperm and the sporophyte. Finally, a genetic factor controlling B ⁹ stability in the developing endosperm has been found. A single plant (1818-1), crossed as a female parent to a B ⁹-containing stock, induced a mosaic pattern of B ⁹ loss in the endosperm at a very high rate. The characteristics of this plant are being investigated.Dedicated with much appreciation and respect to Dr. M. M. Rhoades on the occasion of his 70th birthday.     

Origin of facultative heterochromatin in the endosperm ofGagea lutea (Liliaceae)

Summary Facultative heterochromatin occurs not only in certain animals in connection with sex determination but also in members of at least one plant genus,Gagea (Liliaceae s. str.), but here in the course of embryo sac development, fertilization, and endosperm formation. The present contribution intends to provide undebatable photographic and cytometric evidence, previously not available, for the events in the course of which three whole genomes in the pentaploid endosperm nuclei ofGagea lutea become heterochroma-tinized. In this plant, embryo sac formation usually follows the Fritillaria type, i.e., the embryo sac is tetrasporic, and a 1 + 3 position of the spore nuclei is followed by a mitosis in which the three chalazal spindles fuse and two triploid nuclei are formed. A triploid chalazal polar nucleus is derived from one of these, which contributes to the pentaploid endosperm. These nuclei in the chalazal part of the embryo sac show stronger condensation compared with the micropylar ones. The pycnosis of the triploid polar nucleus is maintained and even enhanced during endosperm proliferation, while the micropylar polar nucleus and the sperm nucleus maintain their euchromatic condition. The origin of the heterochromatic masses in the endosperm nuclei from the three chalazal genomes of the central cell is unambiguously evident from the distribution of heterochromatic chromosomes in the first endosperm mitosis and the following interphase. DNA content measurements confirm a 3 2 relationship of heterochromatic and euchromatic chromosome sets, which is usually maintained up to the cellularized endosperm. Pycnotic nuclei in the chalazal part of megagametophytes are characteristic of several embryo sac types, but only forGagea spp. it is documented that such nuclei can take part in fertilization and endosperm formation.Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

ENDOSPERM DEVELOPMENT IN THE DATE PALM (PHOENIX DACTYLIFERA) (ARECACEAE)

Date seeds were sampled at regular intervals from pollination (March) to mature fruit (September) and processed for light microscopy and SDS-PAGE. Seed fresh weight rose until early June and then declined slightly through September due to a continuous decrease in water content. Cell wall formation started in May in the free nuclear endosperm and proceeded centripetally from the inner integument to the seed center. Wall thickening in each cell started in cell corners and showed a layered appearance with calcofluor white staining. It started in early June in the center of the seed and proceeded centrifugally such that the outer cells showed cell wall thickening in late June. Thickened cell walls were soft and PAS positive at inception, but staining disappeared and hardness increased during wall maturation. Cell elongation in the radial direction accompanied wall thickening. Protein body formation started after cell wall thickening and followed the same centrifugal developmental pattern. Mature protein bodies occurred in even the outermost cells by early July. No further structural changes occurred after this time. The high molecular weight storage proteins appeared in late June, which is when protein bodies had formed in all but the outer endosperm cells; however, these proteins did not appear simultaneously and minor changes in protein bands continued until maturation. α-Galactosidase activity was present in the developing endosperm and peaked at 13 wk after pollination. The data suggest that the thickened wall is deposited as a highly substituted galactomannan, but that most of the galactose side branches are clipped off presumably by α-galactosidase during cell wall polymerization.     

Endosperm response to pollen irradiation in kiwifruit

 Cytological details of endosperm development after pollination with irradiated pollen were studied in Actinidia deliciosa (kiwifruit) cultivar Hayward. Pollinations were carried out involving five different sources of pollen (Matua, Tomuri, Burt, Berryman, and fruiting male) irradiated with gamma rays at doses of 700 and 900 Gy. Non-irradiated crosses were used as controls. Irradiated pollen induced development of approximately 25–30% of the ovules. Two types of ovules were observed: (1) with both embryo and endosperm and (2) with endosperm only. No mitotic abnormalities were found in control or irradiated endosperms. Mitotic divisions were regular and nuclei spherical and evenly spaced. However, the cells of irradiated endosperm usually contained low amounts of storage products. Ploidy level of the endosperms was evaluated by nuclear size (volume) with the use of image analyzis. Mean nuclear size in control and irradiated endosperms was 1598.3 and 750.9 μm³, respectively. It is concluded that endosperm produced after pollination with irradiated pollen is autonomous and represents the 2n level. Received: 14 October 1998 / Revision accepted: 10 March 1999     

OBSERVATIONS ON THE MORPHOLOGY AND SEXUAL REPRODUCTION OF COOLIA MONOTIS (DINOPHYCEAE)1

The surface morphology of the dinoflagellate Coolia monotis Meunier was compared with the surface morphology of Ostreopsis, The apical pore of C. monotis is similar in architecture to that of Ostreopsis but considerably longer (12 μm) than in O. heptagona (8–9 μm) and O. ovata (6–7 μm). A ventral pore in C. monotis is located on the right ventral margin between apical plate l′ and precingular plate 6″ and is similar in appearance and location to the ventral pore of O. ovata. The longitudinal flagellum (20 μm) in C. monotis is longer than in O. ovata (12 μ). Although Coolia and Ostreopsis appear to be distinctly different and should remain as two separate genera, they appear to be related. Cells of C. monotis divided by binary fission. Doubling time was 3–4 days in the logarithmic phase of growth at 23°C, 12:12 h L:D, 30–90 μE-m^?2·s^?1, and a salinity of 36%. Cultures reached cell densities of 2.5 × 10³ cells·L^?1 after 15 days of growth. The sexual process in C. monotis occurred in Erdschreiber's medium when Danish soil extract was substituted with mangrove sediment extract under the culture conditions described above. Gamete fusion produced large biflagellated planozygotes (70–75 μm diam). Planozygote maturation involved cytoplasmic reorganization, loss of motility, development of a spherical shape (80–90 μm diam), and two to three orange accumulation bodies. The cells at this stage appeared to be thin-walled cysts. Further development included reorganization of cyst contents, emergence of non-motile gametes, and development of chloroplasts, sulcus, and girdle. The nucleus of the newly formed cells occupied 50% or more of the total cell volume. Meiosis occurred in the cyst, but nuclear cyclosis was not observed. Four daughter cells were produced within 36–48 h, and motile gametes developed. The gametes exhibited sexuality for 2 months and completed the sexual life cycle by going through a thin-walled cyst stage.     

Transport of metal micronutrients in the phloem of castor bean (Ricinus communis) seedlings

The metal micronutrients (MN) copper, iron, manganese, and zinc are transported via the phloem in the course of remobilization and circulation. The extent of these processes and transport species are still largely unknown. The Ricinus seedling was used to study the transport of these metal micronutrients as well as their interactions with the plant-endogenous chelator nicotianamine (NA) by daily measurements of the concentrations in the seedling parts and in the sieve tube sap obtained from a cut at the hypocotyl hook. The concentrations of these micronutrients in the phloem exudate decreased slightly from day 4 to day 8 of seedling development. Maximum values at day 4 were 65 μM for Zn, 63 μM for Fe, 27 μM for Cu, and 12 μM for Mn. The phloem transport rates reached maxima of 0.12 nmol cm^?2h^?1 for Zn and Fe at days 6 and 7, corresponding to the maximum exudation rates. The magnitude of these transport rates were in agreement with the net translocation rates estimated by analyses of the concentrations in the individual seedling parts. The NA content of the seedlings increased from day 0 (seed before sowing) until day 8, from 16 nmol to 474 nmol, which corresponds to an average net synthesis rate of about 100 nmol day^?1 between the days 4 and 8. The NA:MN ratio was constant at 0.5 in the seedlings within this period. The NA concentrations and the sum of the concentrations of all four micronutrients in the sieve tube sap showed a constant ratio of 1.25 over the entire experimental period. Thus, both complex partners were subject to a cotransport in the phloem. Removal of the supplying endosperm led to a decrease in MN and NA concentrations in the sieve tube sap to about 80% while an average excess of NA of 1.1 was maintained. Since the concentrations of other amino acids, also possible chelators of metal micronutrients, fall to about 10% after removal of the endosperm, their role seems to be negligible as vehicles of MN transport in the phloem. Thus it is suggested that the divalent micronutrients considered in this study are loaded and maybe transported as NA complexes.     

A rapid high-yield isolation method for nuclear envelope ghosts

A method is presented for the isolation of nuclear envelopes from isolated Tetrahymena macronuclei. In principle, nuclei are treated with DNase and RNase at low Ca²⁺/Mg²⁺ concentration followed by an extraction with 1 NaCl. The major advantages of this method are: (1) Unfragmented nuclear envelopes are obtained in the form of ghosts consisting of two juxtaposing nuclear membranes interrupted by pores as revealed by thin-section and freeze-etch electron microscopy. (2) The ghosts are obtained in high yield (60%) within a short period (1 h). (3) The nuclear envelopes largely retain their lipid composition. An average ghost contains about 96% of total phospholipids of an average nucleus. Nuclei and ghosts reveal an almost identical pattern of phospholipids and fatty acids as shown by thin-layer and gas-chromatography. (4) The lipids in the ghosts largely remain arranged in bilayers as probed by electron spin resonance using 5-doxylstearic acid as a spin label.     

Giemsa-based cytological assessment of area,shape and nucleus:cytoplasm ratio of goblet cells of rabbit bulbar conjunctiva

Goblet cells were visualized in impression cytology specimens from bulbar conjunctiva of the rabbit eye using Giemsa staining. Highly magnified images were used to generate outlines of the goblet cells and their characteristic eccentric nuclei. Using sets of 10 cells from 15 cytology specimens, I found that the longest dimension of the goblet cells averaged 16.7 ± 2.3 μm, the shortest dimension averaged 14.4 ± 1.8 μm and the nucleus averaged 6.3 ± 0.8 μm. The goblet cells were ellipsoid in shape and the longest:shortest cell dimension ratio averaged 1.169 ± 0.091. The goblet cell areas ranged from 108 to 338 μm² (average 193 ± 50 μm²). The area could be predicted reliably from the longest and shortest dimensions (r² = 0.903). The areas of goblet cell nuclei were 15–58 μm² (average 33 ± μm²) and the nucleus:cytoplasm area fraction was predictably greater in smaller goblet cells and less in the larger goblet cells (Spearman correlation = 0.817). The nuclei were estimated to occupy an average of 9.5% of the cell volume. The differences in size, shape and nucleus:cytoplasm ratio may reflect differences in goblet cell maturation.     

Alcuni Aspetti Citologici Dell'Endosperma di Phaseolus Coccineus L.

Abstract

Some cytological aspects of Phaseolus coccineus L. endosperm. — Cytological observations were made on the endosperm of Phaseolus coccineus, in a limited stage of development of the seed, using different dyes and labelling with ³H-thymidine and ³H-uridine. The results seem to indicate that, contrary to suspensor cells, in the endosperm of Phaseolus, at least in the considered stage, extra DNA synthesis is not present. Chromosomes in these nuclei, however, undergo several endoreduplication cycles. Nucleoli may be either one or more in a cell, and show a characteristic structure. They are often eccentric in the nucleus and extrusions of nucleolar content in the nucleoplasm or cytoplasm are seen. An apparent alternate activity of RNA synthesis in the nucleoli of the suspensor and in those of endosperm cells is discussed.     

Endosperm development in the Araceae (Alismatales) and evolution of developmental modes in monocots

The Araceae, a basal-most family of Alismatales that basally diverged subsequent to Acorales in monocot phylogeny, are known to have diverse modes of endosperm development: nuclear, helobial, and cellular. However, the occurrence of nuclear and helobial endosperm development has long been debated. Here, we report a (re-)investigation of endosperm development in Lysichiton, Orontium, and Symplocarpus of the Orontioideae (a basal Araceae), in which nuclear endosperm development was recorded more than 100 years ago. The results show that all three genera exhibit a cellular, rather than nuclear, endosperm development and suggest that the helobial endosperm development reported as an “unmistakable record” from Ariopsis is likely cellular. Thus the Araceae are very likely characterized by cellular endosperm development alone. An extensive comparison with other monocots in light of phylogenetic relationships demonstrates that a plesiomorphic cellular endosperm development is restricted to the three basal monocot orders Acorales, Alismatales, and Petrosaviales, in which evolutionary changes from cellular to nuclear endosperm development occurred twice as major events, once within Alismatales and once as a synapomorphy of the eight remaining monocot orders, including Dioscoreales, Liliales, Asparagales, and Poales, and that helobial endosperm development, which is known for many monocot families, evolved as homoplasy throughout the monocots.