J. Van't Hof

Relationships between mitotic cycle duration, S period duration and the average rate of DNA synthesis in the root meristem cells of several plants.

The average mitotic cycle duration of root meristem cells of several plants was measured with 3H-thymidine. The results were in full accord with the statement that the cycle duration increases with an increase in the DNA content per cell. The results also showed that the average mitotic cycle duration for root meristem cells is the sum of the duration of DNA synthesis (S period) and a constant of 5.25–6.5 hr. The S period duration and the average mitotic cycle duration increase linearly with either the amount of DNA per cell or the average rate of DNA synthesis. The average rate of DNA synthesis also increased with the amount of DNA to be replicated per cell per cycle.

The action of IAA and kinetin on the mitotic cycle of proliferative and stationary phase excised root meristems.

Abstract The influence of kinetin and IAA on the G1 (presynthetic), S (DNA synthetic) and G2 (postsynthetic) periods of the mitotic cycle of actively dividing (proliferative phase) and initially nondividing (stationary phase) root meristem cells was determined with the use of tritiated thymidine ( 3 H-T ) and autoradiography. Proliferative phase meristems were cultured for 72 h in Whites medium with 2 per cent sucrose and kinetin or IAA or both. Pulse labeling with 3 H-T was performed at 72 h. The stationary phase meristems were induced by a 24 h deprivation of sucrose. These meristems were almost devoid of dividing and DNA synthesizing cells. After establishment of the stationary phase the roots were transferred to medium containing sucrose, 3H-T, and kinetin or IAA or both. Results obtained with proliferative phase cells suggested that kinetin slightly increased G2 and certainly the (G1 durations. IAA appeared to act primarily on the duration of S. The combination of kinetin and IAA produced all three results. The effect on stationary phase cells, i.e. cells that are retained in either G1 or G2 until provided sucrose, indicated that kinetin and IAA impaired the initiation of DNA synthesis of most but not all of the G1 cells. The combination of kinetin and IAA reduced the initiation of DNA synthesis by G1 cells and further impaired the progress of cells through S; G2 cells also displayed sensitivity to the combination of kinetin and IAA in that they entered mitosis at a low rate. The concentrations of kinetin and IAA used in these experiments were similar to those used to induce cell proliferation in differentiated areas of the pea root; therefore, it is probable that controlling sites or moieties are activated or derepressed in cells of differentiated tissue and inactivated or repressed in meristematic cells by the direct or indirect action of the chemicals.

DNA fiber replication in chromosomes of a higher plant (Pisum sativum)

Abstract Techniques were developed for the autoradiographic analysis of chromosomal DNA fiber replication of higher plant cells. The DNA fibers of Pisum root meristem cells replicate via many tandemly arranged replicons. Each replicon has an initiation point and the average distance between them is about 54 μm. Most of the replicons of Pisum showed bidirectional DNA chain growth that proceeded from a commonly shared initiation point. The average rate of replication per single fork is approx. 29 μm/h at 23 °C.

Measurements of wet metaphase chromosomes in the scanning transmission X-ray microscope

Radiation damage to Vicia faba chromosome structure, as measured by the mass loss, was determined in the scanning transmission X‐ray microscope for unstained specimens in both the wet and dry states. Dried specimens remain undamaged after either single or multiple images at doses up to 2400 Mrad at wavelengths of 3·15 or 3·64nm. In contrast, wet specimens are damaged irrespective of the imaging protocol. The damage induced by multiple exposures is greater than that seen in a single exposure of the same total dose. Thus, the rate of data collection is greater than or equal to the rate of damage. The damage during multiple exposures of wet chromosomes is influenced by several factors. First, the fixative used influences the extent of radiation damage. Wet chromosomes fixed with glutaraldehyde are more resistant than those fixed with formaldehyde or osmium tetroxide. A second factor is ionic strength. Damage to wet chromosomes increases if the ionic strength decreases below that at which chromatin undergoes a conformational transition. The mass of wet and dry chromosomes is the same, and consequently quantitative measurements can be made on wet specimens. Such measurements give a DNA mass fraction of 39 ± 8% for V. faba chromosomes.

Dependence on energy and aerobic metabolism of initiation of DNA synthesis and mitosis by G1 and G2 cells

Abstract Following establishment by carbohydrate starvation of a stationary phase in pea root tip meristems, the requirements for initiation of cell proliferation were investigated. Entry of G1 cells into S (DNA synthesis), G2 cells into M (mitosis) and progression of G1 cells through S and G2 into mitosis were prevented by carbohydrate starvation, anaerobiosis or treatment with 2,4-dinitrophenol. The data show that (a) both DNA synthesis and mitosis require aerobic conditions and are energy-dependent and (b) events prior to the initiation of these processes have relatively greater energy requirements than the rest of the mitotic cycle.

The size and number of replicon families of chromosomal DNA of Arabidopsis thaliana

Analysis of the replicon properties and the cell cycle ofArabidopsis thaliana (col.) at 22° C were performed via autoradiography of isolated chromosomal DNA fibers and single cells of seedlings. The cell cycle was 8.5 h and G1, S, and G2+1/2 M were 1.7, 2.8, and 4 h respectively. The average single fork rate was 5.8 μm/h and the average replicon size was 24 μm. The data best support the hypothesis that A. thaliana has two replicon families, one with approximately 687 and another with 1888 members per genome and that the families initiate replication in sequence separated by a 36 min interval. Replication of an average single replicon required a little more than 2 h or 74% of S and the 36 min interval between the initiation of replication by the two families constituted 21% of S.

Cell arrest in G2 in root meristems: A control factor from the cotyledons

Abstract A substance promoting cell cycle arrest in G 2 in the root meristem is demonstrated. This substance is produced in the cotyledons and is transported to the root.

Similar replicon properties of higher plant cells with different S periods and genome sizes

Abstract Root meristematic cells of nine unrelated diploid higher plants with genome sizes that differ 82-fold and with S periods that differ 4-fold have similar replicon sizes and single replication fork rates that average 22 μm and 8 μm/h respectively. The average replicon size of 22 μm is near the 18 μm obtained by extrapolation of measurements, taken from DNA fiber autoradiograms, to zero pulse time with [3H]thymidine. The data suggest that the duration of S is determined by the minimal number of replicon families that function sequentially during DNA replication.

Promotion of cell arrest in G2 in root and shoot meristems in Pisum by a factor from the cotyledons

Abstract Characterization of a factor within the cotyledons of Pisum that promotes cell arrest in G2 in mature root tissue and stationary phase root and shoot meristems is presented. Diffusion of the G2 factor into aseptic liquid and solid agar media enabled us to perform experiments focused on its cellular effect. The factor promotes cell arrest in G2 in shoots and roots of Pisum and roots of Vicia indicating a lack of species and organ specificity. In seedling roots of Pisum the factor promotes arrest in G2 in a large portion of the cell population. However, because pea cotyledons have a limited supply which is depleted 8–10 days after seed germination, cells previously responsive to the G2 factor in Pisum root meristems eventually assume preponderant arrest in G1. Once these cells arrest in G1 they are no longer influenced to arrest in G2. The G2 factor doss not promote arrest in G2 in meristem root cells of Helianthus and Triticum which normally show preponderant arrest in G1.

Replicon size and rate of fork movement in early S of higher plant cells (Pisum sativum).

Abstract Measurements of chromosomal DNA fiber replication of cells of cultured pea root meristems in early S via autoradiography showed a 3-fold increase in rate of fork movement in the first 2 h. The initial rate was 4.5–6 μm h−1 but forks active after 90 min moved at nearly 18 μm h−1. The faster movement was not characteristic of all replicons. Certain fibers consisted of replicons of a smaller mean size (38–42 μm) with slowly moving forks (4.5–6 μm h−1 fork−1) and others had replicons almost 50 μm long with forks that moved more rapidly.