Expression of the cell cycle in sperm of Arabidopsis: implications for understanding patterns of gametogenesis and fertilization in plants and other eukaryotes

The relationship between developmental events and the cell cycle was examined in sperm of Arabidopsis thaliana. Sperm of Arabidopsis rapidly enter the S (synthesis) phase of the cell cycle after inception from mitosis of the generative cell. Sperm in pollen grains within anthers continue to synthesize DNA, and at the time of pollination, contain approximately 1.5C DNA. Following pollination, sperm continue through the S phase of the cell cycle during pollen tube growth. By the time pollen tubes reach the ovary, sperm nuclei contain approximately 1.75C DNA. Just prior to double fertilization, sperm nuclei within embryo sacs contain the 2C quantity of DNA. These data indicate that molecular programs associated with the G1-S transition and the S phase of the cell cycle are expressed in sperm cells of developing pollen grains and pollen tubes in Arabidopsis. This pattern of prefertilization S phase activity in the sperm of a flowering plant stands in marked contrast to all other non-plant eukaryotes (from ciliates to yeast to sea urchins to mammals) where sperm remain in G1 during development, prior to the initiation of gametic fusion. In addition, when patterns of cell cycle activity in sperm of Arabidopsis and other flowering plants are compared, developmental analysis reveals that heterochronic alterations (changes in the relative timing of ontogenetic events) in cell cycle activity are a central cause of the diversification of patterns of gametogenesis in higher plants. Finally, comparative analysis of the patterns of cell cycle activity in Arabidopsis and other angiosperms may be used to predict which flowering plants will be amenable to development of successful in vitro fertilization techniques.

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Prunus necrotic ringspot virus Early Invasion and Its Effects on Apricot Pollen Grain Performance

Phytopathology ◽

10.1094/phyto-97-8-0892 ◽

2007 ◽

Vol 97 (8) ◽

pp. 892-899 ◽

Cited By ~ 24

Author(s):

Khalid Amari ◽

Lorenzo Burgos ◽

Vicente Pallas ◽

María Amelia Sanchez-Pina

Keyword(s):

Developmental Stages ◽

Generative Cell ◽

Pollen Grains ◽

Growth Zone ◽

Pollen Tubes ◽

Climatic Conditions ◽

Pollen Grain ◽

Ringspot Virus ◽

Prunus Necrotic Ringspot Virus

The route of infection and the pattern of distribution of Prunus necrotic ringspot virus (PNRSV) in apricot pollen were studied. PNRSV was detected both within and on the surface of infected pollen grains. The virus invaded pollen during its early developmental stages, being detected in pollen mother cells. It was distributed uniformly within the cytoplasm of uni- and bicellular pollen grains and infected the generative cell. In mature pollen grains, characterized by their triangular shape, the virus was located mainly at the apertures, suggesting that PNRSV distribution follows the same pattern as the cellular components required for pollen tube germination and cell wall tube synthesis. PNRSV also was localized inside pollen tubes, especially in the growth zone. In vitro experiments demonstrated that infection with PNRSV decreases the germination percentage of pollen grains by more than half and delays the growth of pollen tubes by ≈24 h. However, although PNRSV infection affected apricot pollen grain performance during germination, the presence of the virus did not completely prevent fertilization, because the infected apricot pollen tubes, once germinated, were able to reach the apricot embryo sacs, which, in the climatic conditions of southeastern Spain, mature later than in other climates. Thus, infected pollen still could play an important role in the vertical transmission of PNRSV in apricot.

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Localization of actin in pollen tubes of Ornithogalum virens L.

Acta Societatis Botanicorum Poloniae ◽

10.5586/asbp.1999.014 ◽

2014 ◽

Vol 68 (2) ◽

pp. 97-102

Author(s):

Małgorzata Stępka ◽

Fabricio Ciampolini ◽

Mauro Cresti ◽

Maria Charzyńska

Keyword(s):

Pollen Tube ◽

Actin Filaments ◽

Laser Scanning ◽

Higher Plants ◽

Generative Cell ◽

Pollen Tubes ◽

Confocal Laser ◽

Vegetative Nucleus ◽

Ornithogalum Virens

The germinating pollen grain (in vivo on the stigma or in vitro in germination medium) forms a pollen tube which transports the vegetative nucleus and generative cell/two sperm cells participating in the process of double fertilization. The growth of the tube and the transport of organelles and the cells occur due to two major motor systems existing in the pollen tubes of higher plants: the tubuline-dynein/kinesin and the actin-myosin system. In pollen tubes of Ornithogalum virens the actin filaments were labelled with TRITC-phalloidin (2 µg/ml) in the PIPES buffer and the 10% sucrose, without the fixative and DMSO. Omission of the fixative and permeabilizing agent (DMSO) allowed better preservation of the structure, and the "fluorescence" of actin was observed in living pollen tubes. Observations in CLSM (confocal laser scanning microscope) showed that actin is distributed in the vicinity of the cell membrane. This could support the view that actin filaments and the plasmalemma form the pollen tube cortex along which the cytoplasmic movement of organelles, and cell transport occurs.

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Structure of the generative cell wall complex after freeze substitution in pollen tubes of Nicotiana and Impatiens

Journal of Cell Science ◽

10.1242/jcs.88.3.373 ◽

1987 ◽

Vol 88 (3) ◽

pp. 373-378

Author(s):

M. CRESTI ◽

S. A. LANCELLE ◽

P. K. HEPLER

Keyword(s):

Cell Wall ◽

Plasma Membranes ◽

Generative Cell ◽

Pollen Grains ◽

Freeze Substitution ◽

Pollen Tubes ◽

Wall Material ◽

Cell Cytoplasm ◽

Stage Of Development

The mature generative cell in pollen grains and pollen tubes is surrounded by a wall complex that includes two plasma membranes, one facing the generative cell cytoplasm and one facing the vegetative cell cytoplasm, and usually some intervening wall material. After conventional chemical fixation, the two plasma membranes are very uneven and often appear to be joined, giving the impression that numerous plasmodesmata connect the vegetative and generative cells. These areas alternate with swollen, distorted areas, which give the wall complex the appearance of being composed of a chain of vesicles. Utilizing rapid freeze fixation and freeze substitution, we have re-examined the ultrastructure of the generative cell wall complex from pollen tubes grown in vitro, and the differences are striking. The two plasma membranes are very smooth and closely appressed to a layer of wall material. Occasionally the wall complex contains swollen areas, or varicosities, and these may contain pockets of lightly stained material, but again the surrounding plasma membranes are tightly appressed to these areas. Plasmodesmata are not seen, but this does not eliminate the possibility that they may exist at an earlier stage of development.

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In vitro evidence that LHRH stimulates the recruitment of prolactin mRNA-expressing cells during the postnatal period in the rat

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0120107 ◽

1994 ◽

Vol 12 (1) ◽

pp. 107-118 ◽

Cited By ~ 21

Author(s):

A Van Bael ◽

R Huygen ◽

B Himpens ◽

C Denef

Keyword(s):

Cell Cycle ◽

Cell Population ◽

Blot Hybridization ◽

Postnatal Period ◽

S Phase ◽

Female Rats ◽

Mrna Levels ◽

Dot Blot ◽

Total Cell Population

ABSTRACT We have studied the effect of LHRH and neuropeptide Y (NPY) on prolactin (PRL) mRNA levels in pituitary reaggregate cell cultures from 14-day-old female rats, by means of in situ hybridization and Northern blot analysis. As estimated by computer-image analysis, addition of LHRH on day 5 in culture for 40 h resulted in a 37% increase in the total cytoplasmic areas of cells containing PRL mRNA, visualized using a digoxigenin-labelled PRL cRNA. The size of individual PRL-expressing cells was not influenced, nor was the content of PRL mRNA per cell. A similar effect of LHRH was found by dot blot hybridization of extracted RNA. PRL mRNA levels were not affected by NPY. LHRH induced a 29% increase in the number of PRL mRNA-expressing cells processing through the S phase of the cell cycle, visualized by the incorporation of [3H]thymidine ([3H]T) into DNA over 16 h. The fraction of [3H]T-labelled cells was 10–12% of the total cell population. NPY did not influence the number of [3H]T-positive cells expressing PRL mRNA, but completely blocked the effect of LHRH on the latter population. The present data suggest that LHRH, probably via a paracrine action of gonadotrophs, stimulates the recruitment of new lactotrophs, an action which is negatively modulated by NPY. Since the magnitude of this effect was the same in the total pituitary cell population as in cells processing through the S phase of the cell cycle and presumably mitosis, recruitment of lactotrophs seems to be based on differentiation of progenitor or immature cells into PRL-expressing cells, rather than on a mitogenic action on pre-existing lactotrophs alone.

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Transcription of the histone H5 gene is not S-phase regulated

Molecular and Cellular Biology ◽

10.1128/mcb.6.2.601-606.1986 ◽

1986 ◽

Vol 6 (2) ◽

pp. 601-606

Author(s):

S Dalton ◽

J R Coleman ◽

J R Wells

Keyword(s):

Cell Cycle ◽

Steady State ◽

Northern Blotting ◽

S Phase ◽

Erythroid Cells ◽

Dividing Cells ◽

Steady State Levels ◽

Avian Erythroblastosis Virus ◽

Histone H5

Levels of the tissue-specific linker histone H5 are elevated in mature erythroid cells as compared with levels in dividing cells of the same lineage. We examined levels of H5 mRNA in relation to the cell cycle in early erythroid cells transformed by avian erythroblastosis virus to determine whether the gene for this unusual histone is S-phase regulated. Northern blotting analyses revealed that during the cell cycle steady-state levels of H5 mRNA remained relatively constant in contrast to levels of the major core and H1 mRNAs which increased approximately 15-fold during S phase. In vitro pulse-labeling experiments involving nuclei isolated from synchronized cells at various stages of the cell cycle revealed that transcription of the H5 gene was not initiated at any particular stage of the cell cycle but was constitutive. In contrast, transcription of the H2A gene(s) initiated in early S phase, was present throughout the DNA replicative phase, and was essentially absent in G1 and G2 phases.

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Differences in cell cycle characteristics among patients with acute nonlymphocytic leukemia

Blood ◽

10.1182/blood.v69.6.1647.bloodjournal6961647 ◽

1987 ◽

Vol 69 (6) ◽

pp. 1647-1653 ◽

Cited By ~ 1

Author(s):

A Raza ◽

Y Maheshwari ◽

HD Preisler

Keyword(s):

Cell Cycle ◽

Tritiated Thymidine ◽

S Phase ◽

Total Cell ◽

Acute Nonlymphocytic Leukemia ◽

Cell Cycle Time ◽

Myeloid Leukemias ◽

History Of

The proliferative characteristics of myeloid leukemias were defined in vivo after intravenous infusions of bromodeoxyuridine (BrdU) in 40 patients. The percentage of S-phase cells obtained from the biopsies (mean, 20%) were significantly higher (P = .00003) than those determined from the bone marrow (BM) aspirates (mean, 9%). The post- BrdU infusion BM aspirates from 40 patients were incubated with tritiated thymidine in vitro. These double-labeled slides were utilized to determine the duration of S-phase (Ts) in myeloblasts and their total cell cycle time (Tc). The Ts varied from four to 49 hours (mean, 19 hours; median, 17 hours). Similarly, there were wide variations in Tc of individual patients ranging from 16 to 292 hours (mean, 93 hours; median, 76 hours). There was no relationship between Tc and the percentage of S-phase cells, but there was a good correlation between Tc and Ts (r = .8). Patients with relapsed acute nonlymphocytic leukemia (ANLL) appeared to have a longer Ts and Tc than those studied at initial diagnosis. A subgroup of patients at either extreme of Tc were identified who demonstrated clinically documented resistance in response to multiple courses of chemotherapy. We conclude that Ts and Tc provide additional biologic information that may be valuable in understanding the variations observed in the natural history of ANLL.

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Tradescantia bracteata pollen in vitro: pollen tubes development and mitosis

Acta Societatis Botanicorum Poloniae ◽

10.5586/asbp.1977.047 ◽

2015 ◽

Vol 46 (4) ◽

pp. 587-598 ◽

Cited By ~ 9

Author(s):

E. Lewandowska ◽

M. Charzyńska

Keyword(s):

Pollen Tube ◽

Generative Cell ◽

Metaphase Plate ◽

Pollen Grains ◽

Neutral Red ◽

Generative Nucleus ◽

Mitotic Spindles ◽

Germinating Pollen ◽

Vacuolar System

About 90 per cent of Tradescantia bracteata pollen germinates in vitro after 15 min. Mitosis starts in the pollen tube after about 3 h. The mitotic trans-formations of chromosomes within the generative nucleus are not synchronized. They involve succesively the linearly arranged chromosomes in the elongated generative nucleus. In metaphase the chromosomes are arranged tandem-like linearly along the pollen tube. The chromatides translocate in anaphase from various distances to the poles in a plane parallel to the metaphase plate. This suggests that chromosomes have individual mitotic spindles and that coordination of the chromosome transformations in the generative cell is much less strict than in a typical somatic mitosis. Starch is the storage material of pollen grains. In the vegetative cytoplasm of mature pollen grains minute reddish-orange vesicular structures are visible after staining with neutral red. They do not fuse with the vacuoles proper arising in germinating pollen grains to form the vacuolar system of the pollen tube.

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Stage specificity in the mesenchyme requirement of rodent lung epithelium in vitro: a matter of growth control?

Development ◽

10.1242/dev.74.1.183 ◽

1983 ◽

Vol 74 (1) ◽

pp. 183-206

Author(s):

Kirstie A. Lawson

Keyword(s):

Cell Cycle ◽

Bronchial Epithelium ◽

Branching Morphogenesis ◽

S Phase ◽

Growth Fraction ◽

Early Event ◽

Rat Lung ◽

Lung Epithelium ◽

Dividing Cells

Epithelia from lung rudiments in which secondary bronchial buds are already established (14th and 13th gestational day for rat and mouse respectively) are able to undergo branching morphogenesis and cytodifferentiation in submandibular mesenchyme in vitro, whereas lung epithelium from one day younger foetuses rarely gives a morphogenetic response to submandibular mesenchyme and usually differentiates into primary (non-budding) bronchial epithelium. The failure of 13-day rat lung epithelium to respond to submandibular mesenchyme can be prevented by peeling off the submandibular mesenchyme from the lung epithelium after 2½ days culture and replacing the same mesenchyme, or renewing it with fresh salivary mesenchyme ex vivo. Changes in the epithelial contour are visible by 10 h and buds form within 24 h; this is followed by branching morphogenesis in more than 66% of the samples. The number of cells in S-phase in the epithelium is doubled within 3 to 5 h after the operation and the number of mitotic cells (colchicine block) is increased during an 11 to 19 h period after the operation. Substituting stomach mesenchyme for submandibular mesenchyme after the operation failed to elicit morphogenesis or an increase in the number of S-phase cells in the epithelium. The proportion of epithelial cells in S-phase in unoperated recombinates does not differ from the proportion in the primary bronchial epithelium (non-budding) of homotypic lung recombinates, whereas the proportion of S-phase cells in operated recombinates approaches that found in the buds of homotypic lung recombinates. The distribution of S-phase cells in visibly responding recombinates 15 to 17 h after operation shows the same heterogeneity as in homotypic lung recombinates, newly formed buds having twice as many cells labelled with [3H]thymidine as the non-budding area. Cell cycle parameters of intact rat lung growing in vitro were estimated using the labelled mitoses method. Primary bronchial epithelium and bronchial buds both had a total cell cycle time of about 13 h and an S-phase of about 10 h. The growth fraction was 0·54 in the primary bronchus and 0·95 in the buds. It is suggested that, also in the recombinates, differences in the proportion of S-phase cells at any one time in morphogenetically active and inactive areas of the epithelium are due to differences in the growth fraction. It is concluded that an early event in the morphogenetic response of lung epithelium to submandibular mesenchyme after removing and restoring the mesenchyme is an increase in the size of the population of dividing cells and it is suggested that a high proportion of dividing cells in an epithelial population is a prerequisite for further interaction of epithelium and mesenchyme leading to branching morphogenesis.

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Comparative analyses of angiosperm secretomes identify apoplastic pollen tube functions and novel secreted peptides

Plant Reproduction ◽

10.1007/s00497-020-00399-5 ◽

2020 ◽

Author(s):

María Flores-Tornero ◽

Lele Wang ◽

David Potěšil ◽

Said Hafidh ◽

Frank Vogler ◽

...

Keyword(s):

Cell Wall ◽

Pollen Tube ◽

Turgor Pressure ◽

Pollen Grains ◽

Pollen Tubes ◽

Secreted Proteins ◽

Reproductive Tissues ◽

Small Proteins ◽

Secreted Peptides

Abstract Key message Analyses of secretomes of in vitro grown pollen tubes from Amborella, maize and tobacco identified many components of processes associated with the cell wall, signaling and metabolism as well as novel small secreted peptides. Abstract Flowering plants (angiosperms) generate pollen grains that germinate on the stigma and produce tubes to transport their sperm cells cargo deep into the maternal reproductive tissues toward the ovules for a double fertilization process. During their journey, pollen tubes secrete many proteins (secreted proteome or secretome) required, for example, for communication with the maternal reproductive tissues, to build a solid own cell wall that withstands their high turgor pressure while softening simultaneously maternal cell wall tissue. The composition and species specificity or family specificity of the pollen tube secretome is poorly understood. Here, we provide a suitable method to obtain the pollen tube secretome from in vitro grown pollen tubes of the basal angiosperm Amborella trichopoda (Amborella) and the Poaceae model maize. The previously published secretome of tobacco pollen tubes was used as an example of eudicotyledonous plants in this comparative study. The secretome of the three species is each strongly different compared to the respective protein composition of pollen grains and tubes. In Amborella and maize, about 40% proteins are secreted by the conventional “classic” pathway and 30% by unconventional pathways. The latter pathway is expanded in tobacco. Proteins enriched in the secretome are especially involved in functions associated with the cell wall, cell surface, energy and lipid metabolism, proteolysis and redox processes. Expansins, pectin methylesterase inhibitors and RALFs are enriched in maize, while tobacco secretes many proteins involved, for example, in proteolysis and signaling. While the majority of proteins detected in the secretome occur also in pollen grains and pollen tubes, and correlate in the number of mapped peptides with relative gene expression levels, some novel secreted small proteins were identified. Moreover, the identification of secreted proteins containing pro-peptides indicates that these are processed in the apoplast. In conclusion, we provide a proteome resource from three distinct angiosperm clades that can be utilized among others to study the localization, abundance and processing of known secreted proteins and help to identify novel pollen tube secreted proteins for functional studies.

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Identification of DHX9 as a cell cycle regulated nucleolar recruitment factor for CIZ1

Scientific Reports ◽

10.1038/s41598-020-75160-z ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Urvi Thacker ◽

Tekle Pauzaite ◽

James Tollitt ◽

Maria Twardowska ◽

Charlotte Harrison ◽

...

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Cell Cycle Progression ◽

Zinc Finger Protein ◽

S Phase ◽

Cycle Progression ◽

Inactive X Chromosome ◽

Functional Characterisation ◽

Interaction Partners

Abstract CIP1-interacting zinc finger protein 1 (CIZ1) is a nuclear matrix associated protein that facilitates a number of nuclear functions including initiation of DNA replication, epigenetic maintenance and associates with the inactive X-chromosome. Here, to gain more insight into the protein networks that underpin this diverse functionality, molecular panning and mass spectrometry are used to identify protein interaction partners of CIZ1, and CIZ1 replication domain (CIZ1-RD). STRING analysis of CIZ1 interaction partners identified 2 functional clusters: ribosomal subunits and nucleolar proteins including the DEAD box helicases, DHX9, DDX5 and DDX17. DHX9 shares common functions with CIZ1, including interaction with XIST long-non-coding RNA, epigenetic maintenance and regulation of DNA replication. Functional characterisation of the CIZ1-DHX9 complex showed that CIZ1-DHX9 interact in vitro and dynamically colocalise within the nucleolus from early to mid S-phase. CIZ1-DHX9 nucleolar colocalisation is dependent upon RNA polymerase I activity and is abolished by depletion of DHX9. In addition, depletion of DHX9 reduced cell cycle progression from G1 to S-phase in mouse fibroblasts. The data suggest that DHX9-CIZ1 are required for efficient cell cycle progression at the G1/S transition and that nucleolar recruitment is integral to their mechanism of action.

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