aphidicolin and pronamide

Plant nuclei are known to differentiate into various shapes within a single plant. However, little is known about the mechanisms of nuclear morphogenesis. We found that nuclei of tobacco BY-2 cells were highly elongated on long-term treatment with 5 mg l⁻¹ aphidicolin, an inhibitor of DNA polymerase α. In aphidicolin-treated cells, the nuclear length was correlated with the cell length. During culture in the presence of aphidicolin, the nuclei were elongated in parallel with cell elongation. Nuclear elongation was inhibited by the inhibition of cell elongation with 2,6-dichlorobenzonitrile, a cellulose synthesis inhibitor. However, cell elongation induced in the auxin-depleted medium in the absence of aphidicolin did not cause nuclear elongation, indicating that cell elongation alone is not sufficient for nuclear elongation. Treatment with either latrunculin B or propyzamide inhibited the aphidicolin-induced nuclear elongation, indicating that both actin filaments and microtubules (MTs) are required for nuclear elongation. Observations using BY-YTHCLR2 cells, in which actin filaments, MTs and nuclei were simultaneously visualized, revealed that the longitudinally arranged MT bundles associated with the nucleus play an important role in nuclear elongation, and that actin filaments affect the formation of these MT bundles. In aphidicolin-treated cells, the nuclear DNA contents of the elongated nuclei exceeded 4C, and the nuclear length was highly correlated with the nuclear DNA content. In cells treated with 50 mg l⁻¹ aphidicolin, cells were elongated and nucleus-associated longitudinal MT bundles were formed, but the nuclear DNA contents did not exceed 4C and the nuclei did not elongate. These results indicate that an increase in the nuclear DNA content above 4C is also required for nuclear elongation.

Topics: Actin Cytoskeleton; Aphidicolin; Benzamides; Bridged Bicyclo Compounds, Heterocyclic; Cell Line; Cell Nucleus; Cell Size; Cytoskeleton; DNA, Plant; Enzyme Inhibitors; G1 Phase Cell Cycle Checkpoints; Luminescent Proteins; Microscopy, Fluorescence; Microtubules; Mimosine; Nicotiana; Nitriles; Purines; Roscovitine; Thiazolidines

Totipotency is the ability of a cell to regenerate the entire organism, even after previous differentiation as a specific cell. When totipotency is coupled with active cell division, it was presumed that cell division is essential for this expression. Here, using the stress-induction system of somatic embryos in carrots, we show that cell division is not essential for the expression of totipotency in somatic/embryonic conversion. Morphological and histochemical analyses showed that the cell did not divide during embryo induction. Inhibitors of cell division did not affect the rate of somatic embryo formation. Our results indicate that the newly acquired trait of differentiation appears without cell division, but does not arise with cell division as a newborn cell.

Topics: Aphidicolin; Benzamides; Cell Differentiation; Cell Division; Daucus carota; Enzyme Inhibitors; In Situ Hybridization; Plant Somatic Embryogenesis Techniques; Stress, Physiological; Totipotent Stem Cells

Fibrillarin is known to play an important role in precursor ribosomal RNA processing and ribosome assembly. The present study describes a fibrillarin homolog gene isolated from tobacco BY-2 cells and its expression during the cell cycle. The cDNA for a fibrillarin homolog, named NtFib1, was first cloned in Nicotiana tabacum with degenerate primers. It encodes 314 amino acids and the deduced amino acid sequence has some highly conserved functional domains, such as the glycine and arginine-rich (GAR) domain for nucleolar localization and the RNA-binding motif. The C-terminal region is highly conserved and has 7 beta-sheets and 7 alpha-helices which are peculiar to fibrillarin. Thus, it is suggested that the fibrillarin homolog of this plant species functions in the same way as the fibrillarin already known from human and yeast cells. Northern blot analysis of BY-2 cells synchronized with aphidicolin or a combination of aphidicolin and propyzamide showed that the histone H4 gene was specifically expressed in the S phase but NtFib1 mRNA remained at high levels during the cell cycle. Examination of the localization of NtFib1 protein tagged with green-fluorescent protein (GFP) suggested that some persisting in the mitotic apparatus was eventually incorporated into reconstructed nucleoli in late telophase. Newly synthesized GFP-tagged NtFib1 protein in the cytoplasm was added to the recycled protein in early mitosis. Highly concentrated actinomycin D completely inhibited the transcription of genes coding for rRNA (rDNA) but did not significantly suppress the amount of either NtFib1 mRNA or protein, although the NtFib1 protein was reversibly dislocated from nucleoli. Although hypoxic shock completely prohibited rDNA transcription, NtFib1 mRNA remained at the same level as in the control experiment, even after the 4 h treatment. These results indicate that the transcription of NtFib1 mRNA is not related to rDNA transcription and NtFib1 mRNA is resistant to disrupting factors during the cell cycle.

Topics: Amino Acid Sequence; Aphidicolin; Benzamides; Blotting, Northern; Cell Cycle; Cells, Cultured; Chromosomal Proteins, Non-Histone; Cloning, Molecular; Dactinomycin; DNA, Complementary; Gene Expression Regulation, Plant; Green Fluorescent Proteins; Microscopy, Fluorescence; Molecular Sequence Data; Nicotiana; Plant Proteins; Recombinant Fusion Proteins; RNA, Messenger; Sequence Analysis, DNA; Sequence Homology, Amino Acid

In animal systems, indomethacin inhibits cAMP production via a prostaglandin-adenylyl cyclase pathway. To examine the possibility that a similar mechanism occurs in plants, the effect of indomethacin on the cell cycle of a tobacco bright yellow 2 (TBY-2) cell suspension was studied. Application of indomethacin during mitosis did not interfere with the M/G1 progression in synchronized BY-2 cells but it inhibited cAMP production at the beginning of the G1 phase and arrested the cell cycle progression at G1/S. These observations are discussed in relation to the putative involvement of cAMP biosynthesis in the cell cycle progression in TBY-2 cells.

Topics: Aphidicolin; Benzamides; Cell Cycle; Cell Line; Cyclic AMP; Cyclin A; Dinitrobenzenes; Flow Cytometry; G1 Phase; Herbicides; Indomethacin; Mitotic Index; Nicotiana; Plants, Toxic; RNA, Messenger; S Phase; Sulfanilamides

Theories on the importance of cytokinins in G1/S transition control are manifold and contradictory. By establishing a double A(phi-PZ block, maximal synchronization of a BY-2 suspension culture was obtained to investigate the effect of cytokinin depletion on G1/S transition. Lovastatin was used as a specific inhibitor of cytokinin biosynthesis. Flow cytometry showed that the G1/S transition occurred regardless of the cytokinin drop. This observation indicates an extremely low dose requiry for that stage of the cell cycle. It is very likely that precisely the downregulation of zeatin type cytokinins matters for the G1/S transition to occur, since cytokinin addition at early G1 blocked the cycle at G1/S.

Topics: Aphidicolin; Benzamides; Cell Cycle; Cell Line; Flow Cytometry; G1 Phase; Lovastatin; Mitosis; Nicotiana; Plants, Toxic; S Phase; Zeatin