Jack Van't Hof

INCREASED NUCLEAR DNA CONTENT IN DEVELOPING COTTON FIBER CELLS

The nuclear DNA content of developing cotton fiber cells (Gossypium hirsutum, cv. MD51ne) increases ∼24% after 2 d postanthesis (dpa). The amount of nuclear DNA at 2 dpa is 5.4 ± 0.27 pg. At 3-4 dpa it increases to 6.7 ± 0.24 pg and by 5 dpa it is 6.8 ± 0.70 pg. These values were obtained by nuclear fluorescence after staining with Hoechst 33258. Human oral squamous cell nuclei were used as a DNA standard. Nuclear DNA content increases in fibers growing on either fertilized or unfertilized ovules. The increase also is detectable in Feulgen stained nuclei using two-wavelength cytospectrophotometry. All measurements were made on isolated fiber cell nuclei using a newly developed method tailored to cotton fiber cells. The results imply that during the early stages of development fiber cell nuclei either selectively amplify certain sequences or enter S-phase replicating a portion of their genome.

The Duration of Chromosomal DNA Synthesis, of the Mitotic Cycle, and of Meiosis of Higher Plants

A relationship between DNA content per cell and the duration of the mitotic cycle in plants was first shown by Van’t Hof and Sparrow (1963) in 1963. Since that time, work on other species has provided additional confirmatory observations, and the information in the tables which follow constitutes an introduction to the literature on the topic. The compilation is limited to the mitotic cycle of cells of higher plants as measured in vivo by the Quastler-Sherman method (Quastler and Sherman, 1959) which utilizes 3H-thymidine and autoradiography. The 2C-DNA content was calculated according to Van’t Hof (1965) when necessary and separate references are given for cases where the durations and DNA content were reported independently. Table 1 lists the 2C-DNA content, the duration of the mitotic cycle (CT), and its parts: the presynthetic (G1), DNA synthetic (S), postsynthetic (G2), and mitotic (M) periods. Data on diploid, tetraploid, and hyperploid species for which no quantitative DNA values are available are also given. The durations of G1, S, G2, M, and CT at different temperatures are listed in Table 2, and those of cells in different histological loci are shown in Table 3. Unless noted otherwise, the data refer to cells of root apices.

Cotton fibers can undergo cell division.

Ovular culture was used to determine the cell cycle aspects of cotton fiber cells. Each ovule (Gossypium hirsutum, cultivar, MD51 ne) grown under the conditions used has ~10 000 fiber cells at 4 d postanthesis. About 25% of these cells divide when ovules are cultured at 34C. Mitosis occurs after fiber cells differentiate, producing multicelled fibers. The basal and tip cells of multicelled fibers have the same characteristics as the polar ends of single-celled fibers. Most cell division occurs in ovules cultured at 2-3 d postanthesis. Multicelled fibers are rare in ovules cultured at 1 d postanthesis and absent if cultured at 7 d postanthesis. No multicelled fibers are detectable on ovules sampled from the plant regardless of age. Fiber cell division occurs in the absence of exogenous hormones. The addition of IAA and GA3 to the medium lowers the frequency of multicelled fibers. IAA alone further reduces their frequency, while GA3 by itself has no effect. The number of fiber cells per cultured ovule ranges between 9462 and 11 087 and is not significantly different from the 9892 seen in the plant at 4 d postanthesis. These findings show that a subpopulation of fiber cells, fully differentiated in appearance, retain cell cycle functions up to 4 d postanthesis.

Site of initiation of replication of the ribosomal genes of pea (Pisum sativum) detected by two-dimensional gel electrophoresis

The time course of replication of the 9 kb ribosomal DNA repeats of synchronized root cells of pea was followed by two-dimensional gel electrophoresis. The temporal order of appearance of single-stranded replication intermediates shows that replication begins within the subrepeats located in the intergenic spacer region about 1.5 kb downstream from the 3′ end of the 25S gene. Hybridization to specific probes indicated that this location is identical to that established earlier by a different method.

CHANGES IN POLYSOME ACTIVITY RELATED TO RADIATION-INDUCED MITOTIC DELAY IN SYNCHRONIZED ROOT MERISTEM CELLS.

Earlier studies with synchronized plant cell populations suggested that radiation damage resulting ultimately in mitotic delay could be expressed either immediately or at some time after exposure. ...

Production of micronucleoli and onset of cotton fiber growth

Abstract. Experiments focused on the early development of fiber cells of cotton (Gossypium hirsutum L. cv. MD51 ne) ovules produced two novel findings: one biological, the other methodological. The first concerns a micronucleolus in the nucleus of fibers. This developmental marker appears at or a little before 4 days postanthesis (dpa) in about 10% of the fibers and increases thereafter to nearly 80% provided the fibers are growing on fertilized ovules. Micronucleoli are neither seen in nuclei of fibers at 0–2 dpa nor in nuclei of non-fiber cells. Consequently, it is postulated that they are the product of specific developmental genes associated with fiber growth. The second, methodological finding, involves a cytological means of directly counting the number of fibers produced on young ovules at 1–4 dpa. The method provides quantitative data unavailable in the past. We used this method to show that emasculation caused a temporary 24-h delay in the initiation of fibers, that 30% of the fibers are affected, and that at 3 dpa both fertilized and unfertilized ovules have about 14 500 fibers. These data indicate that the fibers on fertilized and unfertilized ovules represent the same cell populations, a finding heretofore unknown.

G2 arrest and differentiation in the petal of Tradescantia clone 4430

The floral organs of Tradescantia clone 4430 were used to investigate, in terms of cell cycle parameters, cellular behaviour during the maturation of a terminally differentiating system. Petals were sampled at different stages of development for (a) cell number; (b) nuclear DNA content by cytophotometry; (c) [3H]thymidine incorporation into nuclei by autoradiography; and (d) pigment production by spectrophotometry. DNA synthesis was confirmed by measurement of [3H]thymidine incorporation into TCA-insoluble material and changes in DNA content by colorimetric estimation of DNA extracts by diphenylamine. The development of the petal involved four sequential steps. First, there was an increase in cell number, an event characterized by mitoses, DNA synthesis, a few cells in G2 and a predominance in G1. Second, there was a cessation of cell division and DNA synthesis when all the cells accumulated in G1. Third, there was a shift of a large proportion of the total cell population from G1 to the G2 stage of the cell cycle and finally, there was pigment production. In addition, cytophotometric analysis of individual tissues in the mature petal revealed tissue specific differences in the proportion of cells in G2.

Cell Population Kinetics of Pisum Root Meristem Cells during and after a Mitotic-inhibitory Exposure to Protracted Gamma-irradiation

SummaryPrimary root meristems of Pisum sativum with a mitotic cycle prolonged to ∼36 hours by lowering the temperature to 10°c, exhibited complete mitotic inhibition after 3 days of exposure to gamma-radiation at a rate of 0·83 R/min. 3H-thymidine autoradiography, 3H and 14C double-labelling autoradiography combined with Feulgen microspectrophotometry, and the colchicine-induced tetraploid technique were used to measure the cell population parameters. After mitotic inhibition occurred cells accumulated in G1 and G2 with some cells passing through S, and with time under irradiation cells moved at a reduced rate out of G1, through S and into G2, where they stopped cycle progression. After termination of irradiation, cells which were in G1 and S during irradiation were capable of division, whereas cells in G2 were not. Two extra days of exposure did not reduce the rate at which cells flowed from G1 → S after irradiation, but did produce a delay in the appearance of these cells in M. The first complete post-i...

Meristematic precursors of vascular parenchyma differentiate from G2 phase after replicating DNA discontinuously

A small population of cells representing 1% or less of those in the root-tip meristem was identified as the precursor of vascular parenchyma and certain root-cap cells in carbohydrate starved cultured pea roots. Autoradiography and cytophotometric measurements of nuclei labeled with [3 H]-thymidine showed that in the absence of carbohydrate the precursor cells replicate their DNA discontinuously accumulating temporarily in late S phase prior to differentiating from the G2 phase. Besides discontinuity of DNA synthesis, the nuclei of precursor cells undergo a change in morphology. The nuclei are shaped round when replicating DNA but later on, while differentiating, they become oblong. This transformation occurs within 72 hr after the starved roots are fed sucrose. Autoradiograms of serial cross-sections of pulse-labeled roots indicate that the cells in late S phase differentiate forming a ring around the stelar cylinder and a ring around the periphery of the root. These observations suggest that during the last half of the final S phase the precursor cells modify their chromosomal DNA and that this modification is associated with the initial steps of differentiation.

Growth and mitotic potential of multicelled fibers of cotton (Malvaceae).

We tested the hypothesis that the growth of multicelled cotton fibers of Gossypium hirsutum, cultivar MD51 ne, occurs exclusively within the tip cell. Direct cellular measurements proved the hypothesis incorrect. The results show that all cells within a fiber grow and that the relative growth of the tip cell is reduced as the number of cells per fiber increases. Also, measurements of two- and three-celled fibers show that the two daughter nuclei in two-celled fibers differ. The ability to divide resides primarily, if not exclusively, with the basal cell. Thus, the fate of the tip-cell nucleus is fixed while that of the base cell is not. This rule is unaltered by the presence of IAA (indoleacetic acid) and GA3 (gibberellic acid-3) in the culture medium.