J.F. López-Sáez

Duration of the division cycle in binucleate and mononucleate cells

Abstract The material used consisted of meristematic cells from the root-tips of onions ( Allium cepa ) grown in aerated tap-water at a temperature of 25 °. By using caffeine to label the cells (1-hr treatment with a solution of 0.1 per cent), a population of binucleate cells was induced, through inhibition of cytokinesis in the cells which were undergoing division at the time of the treatment. The development of the labelled population showed that the binucleate cells have a division cycle of 14 hr and the mononucleate cells a cycle of 13.5 hr. In meristematic cells, interphase lasts 11.2 hr and mitosis 2.3 hr, divided into: prophase, 64 min; metaphase, 18 min; anaphase, 13 min; and telophase 42 min.

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.

Duration of the division cycle in diploid, binucleate and tetraploid cells

Abstract By means of a caffeine treatment (0.1 per cent solution, 12–15 hr, at 25 °C) a binucleate cell population is induced in root-tips of Allium cepa. In the division of these cells (bimitosis) blocks of three cells, diploid-binucleatediploid (2n; 2n + 2n; 2n), are formed and nuclear fusion in the binucleate cells give rise to blocks diploid-tetraploid-diploid (2n; 4n; 2n), which development has been studied. Knowing the duration of the diploid cycle (13.5 hr), the tetraploid cell cycle was calculated about 14.5 hr. In short, the duration of the average cycle should be: for the mononucleate diploid cells, 13.5 hr; for the binucleate cells, 14 hr; and for the tetraploid cells, 14.5 hr. Therefore, the cycle time does not only depend on the DNA amount per cell, but also on the DNA distribution in one or two nuclei and perhaps the modifications of the ratio nuclear-surface area to nuclear-volume might explain the results obtained.

Effects of caffeine, calcium and magnesium on plant cytokinesis

Abstract The study of the cytokinesis inhibition by caffeine in meristem cells of onion root tips has shown the antagonism between calcium and/or magnesium and caffeine. Moreover, the influence of chelating agents (citrate and EDTA), which potentiate the caffeine effect on cytokinesis, also suggest an essential role for both cations in this process. We propose that caffeine interferes with plant cytokinesis involving some aspect of membrane recognition and/or fusion, where calcium and magnesium are essential requirements.

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.

Analysis of the interphase accumulation induced by hydroxyurea on proliferating plant cells

Abstract Cell distribution in different compartments of the cell cycle (G1, early, middle and late S, G2 and mitosis) has been studied during continuous treatments with hydroxyurea (HU) in onion root meristems by cytophotometric and autoradiographic methods. A sublethal dosis of HU (0.75 mM) has been chosen to allow a good wave of mitotic synchrony during recovery, with a negligible level of chromosomal aberrations. Proliferating cells begin the S period in the presence of HU and are accumulated in early S, where the maximum value (60%) is reached after 8 h of treatment; at the same time middle and late S are practically empty. In the presence of the drug, residual DNA synthesis allows a slow but continuous progress of cells throughout the S period. Differential sensitivity of S cells to HU can be observed; replication is more affected in early S (85% inhibition) than in the second half of the period (70% inhibition). On the other hand, G1 cells are not apparently affected by HU, while cells in G2 show a delay in their entrance into mitosis.

Mechanism of mitotic synchronization induced by 5-aminouracil

Abstract The mechanism of mitotic synchronization induced by 5-aminouracil (5-AU) in onion root meristems has been investigated. We studied the effect of the chemical upon asynchronous meristematic cells, as well as upon a synchronous fraction of them the position of which in the cell cycle was known. The kinetic studies have shown a fairly linear relationship between the treatment time and the induced mitotic wave. Therefore, cell accumulation in a particular zone of interphase appears to be the cause of mitotic synchronization. This zone seems to be located at the end of S period. 5-AU induces maximal mitotic delay on the cells located in the accumulation zone and severely depresses the S-G2 transit while the passage through G 2 remains relatively unaffected. The possibility that preferential inhibition affects the late replicating DNA synthesis is discussed.

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.

DNA replication in the presence of protein synthesis inhibitors in higher plant cells.

Abstract The DNA synthesis in Allium cepa meristematic cells has been studied in the presence of protein synthesis inhibitors using either random or synchronous cell populations. The results suggest that protein synthesis is required specifically for initiation of the S period, but that afterwards DNA synthesis can continue in the presence of anisomycin or cycloheximide apparently at a normal rate. The cells appear to leave the S period approx. 90% of the control rate and the increase in DNA content with time, as studied in a synchronous binucleate population, is identical throughout the S period both in control and in the presence of inhibitors.

Multinucleate plant cells: II. Requirements for nuclear DNA synthesis

Abstract A synchronous population of multinucleate cells has been experimentally induced in Allium cepa root meristems by a chemical method that involves the induction of aneuploidy in individual nuclei. Whereas all multinucleate cells as a whole present a tetraploid chromosome complement, their aneuploid nuclei constitute compartments containing only part of the parental genetic material. We analysed the degree of synchrony in the course of S period progression in the unbalanced nuclei sharing a common cytoplasm (bi-, tri- and tetranucleate cells). In most of the multinucleate cells the nuclei entered DNA replication simultaneously, but a significant proportion of cells showed labelled and unlabelled nuclei which was due to the absence of DNA synthesis in some aneuploid nuclei. These nuclei lacked replicative capacity, but did not inhibit DNA synthesis of the replicating nuclei. Our findings confirm the existence of cytoplasmic factor(s) inducing the synchronous initiation of S period, and suggest strongly the occurrence of intranuclear factor(s)—likely gene products—as a requirement for the onset of DNA synthesis itself in every nucleus.