An electron-microscope study of the intranucleolar chromatin during nucleologenesis in root meristematic cells of Allium cepa.

The various states of condensation of the chromatin material contained inside the lacunar regions of the reforming nucleolus in Allium cepa, have been investigated by means of conventional electron-microscope techniques. The observations reveal that, in the emerging early to late telophase nucleoli, the intralacunar chromatin material in question appears both in an extended and a condensed condition; from late telophase th the mid G1 period of interphase, the intralacunar chromatin material of the rapidly growing and developing nucleoli is present in an extended state only. An attempt is made to interpret these morphological findings in the light of current knowledge concerning the structural relationship of the nucleolar organizing region of the nucleolar chromosome with the interphase nucleolus in plant cells. The relevant observational evidence would be consistent with the view that the chromatin-containing lacunar regions of the reforming nucleolus in Allium cepa correspond, in fact, to cross- or oblique sections of a meandering channel through which the nucleolar organizing segment of the nucleolar chromosome passes. Assuming the applicability to intranucleolar chromatin of the general concept of condensed-inactive versus extended-active chromatin, it is concluded that gradual uncoiling and subsequent decondensation of the chromatin of the nucleolar organizing region in the form of a convoluted loop structure are key morphological and functional events associated with the process of nucleologenesis in the species investigated.     

Localization of histones and their synthesis by ammoniacal silver reaction in meristematic root tip cells of Allium cepa

Summary The ammoniacal silver reaction was used for localization of histones in meristematic root tip cells of Allium cepa. Electron microscopic observations showed that yellow or brown colour of interphase and prophase nuclei and brown nucleolar colour produced in the reaction coincides with the appearance of silver grains, about 400 Å in diameter, in the interphase and prophase chromatin and nucleoli. This together with the complete absence of staining reaction and silver grains in the cytoplasm could mean quite a specific reaction with histones and might suggest also that in these cells the site of histone synthesis is in the nucleolus.     

Sesquiterpene lactone dehydroleucodine selectively induces transient arrest in G2 in Allium cepa root meristematic cells

Dehydroleucodine is a sesquiterpene lactone recently isolated from aerial parts of a medicinal herb, Artemisia douglasiana Besser. We have previously shown that 25 and 100 microM dehydroleucodine slowed down onion root growth by 30 and 70%, respectively, affecting neither cell viability nor cell elongation. In the present study we analyze the effect of dehydroleucodine on cell cycle phases in onion (Allium cepa L.) root meristematic cells synchronized with caffeine or caffeine and hydroxyurea. Synchronized root cells treated with 100 and 200 microM dehydroleucodine present an interphase lengthening of 5.2 h and 8.2 h, respectively. The S-phase length, estimated by [3H]thymidine incorporation assay, was 6 h for both control roots and roots that had been immersed in dehydroleucodine. The peak of [3H]leucine incorporation was observed 6 h after release from synchronization in controls and in dehydroleucodine-treated roots, indicating that protein synthesis in G2 was not affected. Thus, these results show that dose-dependently dehydroleucodine selectively induces a transient arrest of meristematic cell in G2 and that dehydroleucodine can be used experimentally as a cell cycle suppressor.     

Early replicating DNA involved in the G2 to M transition in Allium cepa L meristematic cells

Summary— The involvement of genome portions replicating at different times of the S phase in the control of cell cycle events was analysed in Allium cepa L meristematic cells. 5-Azacytidine (5-azaC) was incorporated into discrete replicating DNA portions in synchronous cells. Cells treated with 5-azaC during the late S phase went through normal cell cycles while cells treated with 5-azaC during the early S phase were able to go through the immediately succeeding mitosis, as well as to begin the subsequent S phase. However, they were unable to enter a second mitosis. Thus, sequences replicating in the early S phase appear to code for a mitotic inhibitor which represses the emergence of a positive mitotic signal. This inhibition took place in the first half of the interphase (ie during G1) and resulted in a cell cycle blockage in G2.     

Soluble polypeptides from meristematic and mature cells of Allium cepa L. roots

By using electrophoresis on SDS polyacrylamide gels we have studied the soluble protein fraction from a section of the meristem, where most cells are in the division last cycle, and from the mature region of Allium cepa L. roots. In order to estimate the apparent rate of synthesis of these polypeptides we labeled a series of roots with [¹⁴C]leucine and another with [³H]leucine. Coelectrophoresis was carried out by using polypeptides from both regions, their mol.wt. being between 20,000 and 100,000 daltons. The results show that most of the polypeptides in the soluble fraction are constantly present in a cell throughout its development. These constant polypeptides are synthesized at a high rate in the meristematic region. In the mature cells these polypeptides show only a low labeling rate, while a small number of specific polypeptides appear to have a very high rate of metabolism.     

Effect of cisplatin on Allium cepa root meristem cells

Allium cepa root growth was retarded by cisplatin treatment in a dose-dependent manner. A decrease in the mitotic index (MI) and an increase in the number of interphase cells was seen in cisplatin treated root tips. An increase in the frequency of abnormal mitoses and chromosomal aberrations was also observed in cisplatin treated groups which indicates its genotoxic effect on plant cells. The endogenous glutathione (GSH) level in the root tips decreased significantly after cisplatin treatment which may favour its increased interaction with cellular DNA thereby developing enhanced chromosomal aberrations and affecting cell divisions and root growth. It is suggested that the decrease in endogenous GSH may be related to the development of cisplatin-mediated genotoxic effects in plants.     

Early and late replicating DNA involved in the G1 to S transition in Allium cepa L meristematic cells.

The involvement of portions of the genome replicated at different times of the S period in the regulation of the G1 to S transition was analyzed in Allium cepa L meristem cells. For this, DNA bromosubstitution confined to discrete portions of a previous S period followed by anoxic UVA irradiation (300-400 nm light) was performed in synchronous cells. Sequences replicated in late S appeared to be involved in the positive regulation of the initiation of replication. Hence, cells were prevented from initiating replication if irradiated at mid G1 only when the DNA sequences replicated in the last third of the previous S period were bromosubstituted. Cycloheximide-induced inhibition of protein synthesis at late G1 also prevented the G1 to S transition. Sequences replicated in mid S appeared unrelated to any control of the initiation of replication. On the other hand, sequences replicated in the first third of the S period seemed to be involved in the negative regulation of the initiation of replication, since irradiation after previous bromosubstitution of early replicating DNA sequences advanced G1 cells into the next S phase and increased the proliferative fraction of the population. Finally, the simultaneous inactivation of DNA sequences involved in both positive and negative regulation of replication allowed the cells to enter into S.     

Localization of intracellular calcium in root meristematic cells and root cap cells of maize

The localization of Ca²⁺ in cells of the periblem and dermatogen in the root meristem and the columella and peripheral cells of the root cap of maize was examined by the precipitation method of potassium pyroantimonate and EGTA-treatment. In periblem and dermatogen cells, Ca²⁺ was found to be localized in the nucleoplasm and granular zone of the nucleolus. Ca²⁺ was also found in most cell organelles: in the matrix in mitochondria, on the thylakoid membrane in proplastids, in the vacuoles and on the plasma membranes. Ca²⁺ was also distributed throughout the cytoplasmic ground matrix. Much Ca²⁺ was present in the cell wall soon after its formation during the cell division. Ca²⁺ was also conspicuous in the vesicles of Golgi in the dermatogen cells. In columella and peripheral cells, there was less Ca²⁺ in the organelles and cytoplasmic ground matrix, but Ca²⁺ was present in Golgi vesicles in the peripheral cells. Electron microscopic and X-ray microanalysis showed that Ca²⁺ was also present in the mucilaginous layer, the outermost cell wall of the peripheral cells.     

Structure and mode of formation of the nucleolus in meristematic cells of Vicia faba and Allium cepa

Interphase nucleoli from Vicia faba and Allium cepa meristematic cells are roughly classified into two categories: (a) those that commonly show a rather homogeneous texture (except for small light spaces of various sizes) and frequently contain dense particles 140 A in diameter; (b) those found more frequently in Vicia characterized by a very sharp boundary between a dense outer cortex and a much lighter central core. The dense particles are not found in such nucleoli. In Allium the boundary is more irregular and dense particles are sometimes observed in the outer layer. Many nucleoli show a structure intermediate between these two types. They are characterized by a gradient of increasing density from the center to the periphery and occasionally contain dense 140 A granules. During interphase, certain nucleoli are closely associated with segments of chromatin strands which undoubtedly represent nucleolar organizing regions. The dense 140 A granules are followed during the mitotic cycle. In Allium, they are first seen in loose clusters between arms of late anaphase chromosomes where they become more concentrated in early telophase. The substance within which they are scattered slowly increases in density during that time until finally, the particles are limited to small bodies of distinctive character. Evidence is presented suggesting that these small prenucleolar bodies fuse during telophase to give rise to the mature interphase nucleoli. Similar events are described in Vicia material except that a coating of dense substance appears around telophase chromosomes before the formation of prenucleolar bodies.     

The chromosomes and DNA of Allium cepa

Giemsa C-banded idiograms that allow the identification of all chromosomes have been prepared for Allium cepa, Ornithogalum virens, and Secale cereale. An analysis of A. cepa DNA has determined that: (1) It has the lowest GC content so far reported for an angiosperm (32%). (2) It appears to have no satellite DNA detectable by CsCl or Cs₂SO₄-Ag⁺ density gradient centrifugation. (3) Aside from fold back DNA and unreactable fragments, a C₀t curve indicates that most of the DNA can be adequately described as two major middle repetitive components (Fractions I and II) and a single copy component (Fraction III). And (4) most of the repeated DNA sequences are involved in a short period interspersion pattern with single copy and other repetitive sequences. In situ hybridization of tritiated cRNAs to fold back, long repeated, and Fraction I DNA from A. cepa to squash preparations of chromosomes and nuclei from A. cepa, O. virens, and S. cereale root tips indicates: (1) Sequences complementary to fold back DNA are scattered throughout the genome of A. cepa except for telomeric heterochromatin and nucleolus organizers while they are not detectable in the genomes of O. virens or S. cereale. (2) Although long repeated sequences are scattered throughout the genome of A. cepa, they are concentrated to some extent in telomeric heterochromatin and nucleolus organizers (NOs). Sequences complementary to long repeats of A. cepa occur primarily in chromosome three of O. virens while these sequences are more common in the genome of more distantly related S. cereale. (3) Fraction I DNA is scattered throughout the genome of A. cepa while it is hardly detectable in the genomes of O. virens and S. cereale. These results are discussed in regard to the evolutionary conservation and function of repeated DNA sequences.     

Cytotoxic effect of citrinin on relative division rate of Allium cepa root cells

Effect of different doses of citrinin, the secondary metabolite of Penicillium citrinum Thom, was investigated on Allium cepa root cells. A marked decrease in mitotic index was observed with the increased concentration of toxin. In controls the mitotic index was 16·2 which at a concentration of 400 ppb was reduced to the level of 4·7. Besides a decrease in mitotic index, several cytological abnormalities were also found, such as chromosome breakage, polyploidy, anaphase bridge, laggard, etc.     

Localization of selenium deposits in meristematic cells of Allium sativum L. roots treated with selenium salts

Ultrastructural analysis of garlic roots treated for 24 h with sodium selenate or sodium selenite at the concentrations 80, 160, 320 microM revealed the presence of selenium deposits in meristematic cells. They appeared as small and large granules or aggregates of electron-dense material. Many small granules were localised in plastids but some in mitochondria, endoplasmic reticulum as well as in Golgi apparatus, nucleus and cytoplasm. Sometimes the large granules were seen in cytoplasm but aggregates of electron-dense material only in vacuoles. It seems possible that these deposits represent a non-dissolved form of selenium, i.e. elemental selenium or its complexes with other ions.     

DNA replication stress induces deregulation of the cell cycle events in root meristems of Allium cepa

Background and Aims

Prolonged treatment of Allium cepa root meristems with changing concentrations of hydroxyurea (HU) results in either premature chromosome condensation or cell nuclei with an uncommon form of biphasic chromatin organization. The aim of the current study was to assess conditions that compromise cell cycle checkpoints and convert DNA replication stress into an abnormal course of mitosis.

Methods

Interphase-mitotic (IM) cells showing gradual changes of chromatin condensation were obtained following continuous 72 h treatment of seedlings with 0·75 mm HU (without renewal of the medium). HU-treated root meristems were analysed using histochemical stainings (DNA-DAPI/Feulgen; starch-iodide and DAB staining for H₂O₂ production), Western blotting [cyclin B-like (CBL) proteins] and immunochemistry (BrdU incorporation, detection of γ-H2AX and H3S10 phosphorylation).

Key Results

Continuous treatment of onion seedlings with a low concentration of HU results in shorter root meristems, enhanced production of H₂O₂, γ-phosphorylation of H2AX histones and accumulation of CBL proteins. HU-induced replication stress gives rise to axially elongated cells with half interphase/half mitotic structures (IM-cells) having both decondensed and condensed domains of chromatin. Long-term HU treatment results in cell nuclei resuming S phase with gradients of BrdU labelling. This suggests a polarized distribution of factors needed to re-initiate stalled replication forks. Furthermore, prolonged HU treatment extends both the relative time span and the spatial scale of H3S10 phosphorylation known in plants.

Conclusions

The minimum cell length and a threshold level of accumulated CBL proteins are both determining factors by which the nucleus attains commitment to induce an asynchronous course of chromosome condensation. Replication stress-induced alterations in an orderly route of the cell cycle events probably reflect a considerable reprogramming of metabolic functions of chromatin combined with gradients of morphological changes spread along the nucleus.