M.E. Fernández-Gómez

Organization of argyrophilic nucleolar material throughout the division cycle of meristematic cells

SummaryThe silver impregnation of nucleolar material facilitated the study of the morphological changes which take place in the nucleolus throughout the division cycle in root tip cells ofAllium cepa. The nucleolus appears to undergo no morphological changes throughout the interphase. It undergoes disorganization during the prophase, while in the telophase it appears uniformly on the chromatin as condensing into prenucleolar bodies.The appearance of the prenucleolar bodies is unaffected by puromycin, cordycepin, or ethidium bromide. This suggests that the argyrophilic material does not undergo synthesis during the telophase, nor require RNA or protein synthesis to effect the aggregation into prenucleolar bodies. However, the organization of nucleoli from prenucleolar bodies is inhibited by both cordycepin and ethidium bromide, suggesting that RNA synthesis is involved in this proccess.In aneuploid nuclei induced by treatment with colchicine we observed the appearance of prenucleolar bodies during the telophase even in the absence of the nucleolar organizer, but in this case the formation of nucleoli fails to take place. The nucleolar organizers proved to be capable of acting only in the nucleus to which they belong, but not on other nuclei within the same cytoplasm belonging to multinucleate cells.It seems logical to assume that one of the roles of the nucleolar organizer is related with the above-mentioned RNA synthesis, which is required to the aggregation of prenucleolar bodies into nucleoli.

Effect produced by inhibitors of RNA synthesis on mitosis.

Abstract The study of mitosis in root-tip cells of Allium cepa and in populations of synchronous binucleate cells induced in this material by treatment with caffeine enabled the authors to demonstrate the detention of cells in prophase caused by treatment with 3′ deoxyadenosine (3′AdR), ethidium bromide or ribonuclease. Cells incubated at the late interphase in presence of the drugs mentioned reached the prophase but, when observed over a period of 10 h, did not succeed in initiating the metaphase. Similarly, those cells which were in prophase at the beginning of the treatments remained in prophase. When treatment was applied to cells at metaphase, mitosis followed its normal course, and the subsequent mitotic stages were not prolonged. The results obtained suggest that a synthesis of RNA, probably specific, required to enable the cells to go on from the prophase to the metaphase of mitosis, takes place during prophase.

Effects of protein synthesis inhibition during plant mitosis

Abstract The role of protein synthesis in onion root tips during mitosis has been studied, by using synchronous cell populations. Incubation in cycloheximide (CHM) or anisomycin during early or middle prophase induces the return of these cells to interphase. Therefore, it is suggested that essential proteins are synthesized, which determine the continuation of the cells in mitosis. In late prophase these treatments caused a certain delay in the entry into further stages, suggesting that a protein synthesis probably occurs which determines the duration of the transition from metaphase to anaphase. Mitotic processes which develop after metaphase do not seem morphologically dependent on protein synthesis, in spite of the fact that one of them, the nucleolar reconstruction, is markedly dependent on RNA synthesis. Unexpectedly this reorganization increases its rate in the absence of protein synthesis.

Protein synthesis requirements at specific points in the interphase of meristematic cells

Abstract A 6 h treatment with anisomycin at a concentration of 1 μg/ml enables us to modify the steady-state kinetics of a meristematic cell population of Allium cepa , and this points to a difference of sensitivity to inhibition of protein synthesis between the several periods of the cell division cycle (G1, S, G2, M). The results show that the cells are incapable of entering the S period in the presence of the inhibitor, but that, where DNA synthesis has already been initiated, the synthesis continues in the cells in question. It was found, moreover, that there is a point in the early G2 period, which has a duration of approx. 3 % of the total duration of the cycle, at which the synthesis of specific proteins appears to determine the progression of cells to mitosis.

Nucleolar segregation by adenosine 3'-deoxyriboside (cordycepin) in root-tip cells of Allium cepa.

Abstract The nucleolar segregation induced by adenosine 3′-deoxyriboside in meristematic cells of onion roots is described. The segregated nucleoli clearly showed a peripheral region of low argentophilia and high basophilia, while the central one appeared as highly silver-positive and low basophilic. Under the electron microscope the peripheral region appears as a granular-rich area and the central one showed a higher electron density. This nucleolar segregation, which is probably related to the inhibition of RNA synthesis, showed a very different rate of development according to the moment in the interphase when the cells were treated.

Interchromatin granules in plant nuclei

The interchromatin granules (IG) are well characterized subnuclear structures in animal cell nuclei which form a part of the internal nuclear matrix and are supposed to correspond to accummulation sites of snRNPs and/or maturation and transport of rRNPs; but similar structures have not yet been characterized in plant nuclei.

Cytochemical and ultrastructural studies on normal and segregated nucleoli in meristematic cells

SummaryThe location of the two nucleolar components, in meristematic root tip cells ofAllium cepa has been studied. The “segregation” of granular and fibrous components of the nucleoli was induced by adenosine-3′deoxyriboside. The relationship between the ultrastructure and the stain affinities of both nucleolar regions has been determined by using different selective nucleolar stains.The “pars fibrosa” appears positive in the silver, lead, zinc, and methylen blue stains and shows a high ATPase activity.

Effect of ethidium bromide on the fine structure of the nucleolus in plant cells

A study of the effect of ethidium bromide was carried out on root meristems of Allium cepa L. This inhibitor of nucleic acid synthesis possesses a powerfully inhibitive influence on RNA synthesis. A parallel study was also carried out on the structural effects of the drug on the nucleolus, under the light microscope with silver impregnation, and by electron microscopy. Nucleolar segregation is observable quite clearly during the first few hours of treatment, and in subsequent hours (six) a morphological change can be observed in the area known as the pars granulosa, culminating in complete dispersion after 12 hours, leaving a wide electron-clear zone around the pars fibrosa. Thus, the effect of ethidium bromide is found to be appreciably different from that of other inhibitors of RNA synthesis producing degranulation of the nucleolus in a centrifugal direction, and the failure to produce granular RNP from the fibrillar RNP points to a functional effect of the drug on the latter.

Rate of nucleologenesis as a measure of gene activity

THE nucleolus is not a permanent organelle in the life of a cell; at the initiation of a new cycle, the nucleolar formation seems to depend on RNA synthesis directed by nucleolar RNA polymerase1. We have compared the rate of nucleologenesis in meristem cells of Allium cepa L. and in those of four Vicia species with different numbers of ribosomal cistrons and different DNA contents3,4 as well as the rate of nucleologenesis in four different cell populations lying side by side in the root of Zea mays. The rate of nucleologenesis per ribosomal cistron seems to be constant in the different species growing in similar conditions, but this mean rate was modified in metabolically different subpopulations in the same root.

Further investigations on the functional role of two nuclear bodies in onion cells

Summary“Micropuffs” and coiled bodies are two nuclear bodies which have previously been described in a wide range of plant species. In this work, we have demonstrated, by means of ultrastructural cytochemical, autoradiographic and immunocytochemical methods, that the so-called “micropuffs” contain chromatin, which is organized in a supranucleosomal structure, is inactive in transcription, and has no apparent relationship with the nucleolus. These features, as well as the distribution of these structures during the interphase periods, allowed us to conclude that they represent centromeric structures, so, we propose that they be denoted by this name. Coiled bodies show the same silver-staining pattern as the nucleolus organizer (NOR), but do not appear to contain NOR chromatin nor does the early processing of rRNA precursor seem to take place in them. We postulate they are involved in later nuclear RNA processing and storage. This work clarifies some previous misunderstandings with regard to these bodies.