Marisa Levi

Determination of DNA Content by Static Cytofluorimetry in Nuclei Released from Fixed Plant Tissue

SummaryNuclei were extracted by crushing formaldehyde-fixed plant tissue, and smeared on slides with chick erythrocytes as internal standards, stained with the DNA-specific fluorochrome DAPI and measured by static cytofluorimetry.The ratios between the fluorescence of 2 C pea nuclei and of the internal standards corresponded to the ratio of the respective DNA contents reported in the literature; peaks with very good coefficient of variation (5–10%) were obtained and the mean fluorescence of 4 C nuclei was very close to twice that of the 2 C nuclei; percent distribution in G1 or G2 of nuclei from different tissues ofHelianthus andPisum were in full accordance with cytophotometric data in the literature.These results confirm the reliability of this method of DNA quantification, which is simpler and quicker in comparison with cytophotometric techniques.

Functional roles of the plant α‐like and γ‐like DNA polymerases

Plant cells are endowed with two distinct DNA polymerases [ 1,2] whose properties closely resemble those of the DNA polymerases cr and y present in animal cells [3,4]. The plant DNA polymerases have consequently been named o-like [ 1 ] and y-like [2]. The B-like DNA polymerase activity is the most abundant in cultured plant cells [l ] and responds to changes in the rate of cell multiplication, whereas experiments with spinach leaves have shown that the y-like DNA polymerase is present in the chloroplast 121. A DNA polymerase has also been isolated from the mitochondria of wheat embryos [S]. Spinach mitochondria may also contain a DNA polymerase whose properties are partially different from those of the y-like DNA polymerase isolated from the chloroplasts of the same cells and are similar to those of the wheat embryo enzyme (unpublished). However, no evidence is available as yet on the existence in plant cells of a DNA repair enzyme similar to the DNA polymerase p of mammalian cells ]4,6]. By analogy with animal cells, the assignment of functions to the DNA polymerases in plant cells is hampered by the lack of conditional mutants defective in DNA synthesis. Thus, we approach this problem by exploiting the properties of aphidicolin and of ethidium bromide. Aphidicolin [7] specifically inhibits the cr-like DNA polymerase purified from plant cells 181, while the

Early cellular events during induction of carrot explants with 2,4-D

SummaryThe cellular events occurring in carrot hypocotyl explants during long-term and pulse treatment with 2,4-D were followed using different techniques (light and transmission electron microscopy, flow cytometry, PCNA staining). Different morphogenetic pathways were induced under the various experimental conditions. Nevertheless, in the explants the activated cells were the same (provascular cells) and they showed very similar structural and ultrastructural changes. The long-term treatment with 2,4-D induced rapid re-activation of the cell cycle.

The effect of Cr (VI) on different inbred lines ofZea mays

SummaryFour inbred lines ofZea mays (33.16, B 68, N 7B, B 77) were grown in nutrient solution to which K2Cr2O7 was added to give final concentration of 5 mg/l Cr (VI). The most evident differences in metal tolerance were observed between the B 68 and 33.16 line: in fact, even though the level of Cr (VI) was almost the same in the root tissues of both lines after 6 d of treatment, in the B 68 line, Cr induced marked alterations of nuclear structure and a progressive arrest of the cell cycle in G 1. In the 33.16 line, on the contrary, the integrity of the nuclei was well preserved and the progression of the cell cycle was only barely affected.

Bivariate cytofluorimetric analysis of DNA and nuclear protein content in plant tissue

SummaryTechniques of static biparametric cytofluorimetry were developed to measure DNA and protein fluorescence simultaneously in the same nucleus stained with 4′,6-diamidino-2-phenylindole (DAPI) and fluorescein isothiocyanate (FITC) fluorochromes. With these cytofluorimetric procedures, we analysed DNA and nuclear protein content in root apices during the first 72 h of pea seed germination. This method allows a more reliable, rapid and less expensive measurement of DNA and proteins than cytophotometry. Nuclear protein content can be considered as a second parameter to define subcompartments of cell cycle phases; it offers the possibility of studying the progression of plant cells through cell cycle and its control in greater detail.

Arrest of the Cell Cycle Induced by Dibutyryl-Camp and Sodium Butyrate in Root Meristems of Lettuce

SUMMARYDibutyryl-cAMP was supplied to seedlings of Lactuca sativa L. cv. Great Lakes 118, and its effect on the cell cycle in root meristems was compared to that of dibutyryl-cGMP and sodium butyrate. Dibutyryl-cAMP and sodium butyrate induced a sharp decrease in the labelling index, attributable to an arrest of the cell cycle in G1 and possibly G2, whereas dibutyryl-cGMP was ineffective. The use of dibutyryl-cAMP and sodium butyrate in studies on the control of cell cycle and cell differentiation in growing roots is suggested. Various hypotheses on the mode of action of dibutyryl-cAMP are also discussed.

Cell cycle arrest induced by theophylline in root meristem of Haplopappus gracilis

Theophylline, an inhibitor of cyclic nucleotide phosphodiesterase, induced a block of the cell cycle in G1, a temporary arrest in G2 and 70% decrease in the uptake of labelled thymidine in roots of Haplopappus. These effects are compared to those previously found with aminophylline and discussed in view of the possible involvement of cAMP in the regulation of the cell cycle in plants.

Phosphorylation of nuclear proteins and proliferation in root meristems of pea (Pisum sativum)

Protein kinase(s) and related endogenous substrates are present in nuclei of meristematic and adult root tissue of pea (Pisum sativum L., cv. Lincoln). The protein kinase(s) is bound to the chromatin and is not solubilized by 0.14 M NaCl, although such a salt concentration solubilizes a considerable number of endogenous substrates. Comparison of the electrophoretic patterns of phosphorylated nuclear proteins purified from quiescnt or actively proliferating meristematic root tissue reveals the existence of variations of phosphorylation. Differences exist also between the electrophoretic patterns of phosphorylated nuclear proteins extracted from differentiated or meristematic root tissue. The hypothesis that the variations of phosphorylation of particular proteins might be correlated with the state of proliferation and differentiation of the cell is presented.

Effect of benzyladenine on cell cycle in intact roots. A cytofluorimetric approach

Summary Cytofluorimetry following DAPI-staining of the nuclei was used, in combination with autoradiography, to study the effect of benzyladenine on the cell cycle in pea root meristems. When administered to roots of intact two-day-old seedlings, 30 µM benzyladenine induced in the terminal 2 mm a decrease in the labeling and mitotic indices and an increase in the number of nuclei in G2. At the same time, a rapid inhibition of root elongation was also observed. The shift of nuclei towards G2 was particularly evident in the second mm of the root tip zone. We suggest that the primary effect of this supraoptimal benzyladenine concentration might be the promotion of G 1 arrest. The increase of nuclei in G2 might be due to the effect of the G2-factor translocated from the cotyledons, level of which within the tip might increase as a consequence of the rapid inhibition of root elongation induced by the treatment.

Inhibition of elongation and H(+) and K (+) transport by the phosphodiesterase inhibitor aminophylline in plant tissue.

Aminophylline, an inhibitor of cyclic nucleotide phosphodiesterase (EC 3.1.4.17), inhibits elongation and correlated H+ and K+ transport in embryos of Haplopappus gracilis and in pea internode segments. Moreover, the drug strongly inhibits the stimulation of these processes by fusicoccin and indole-3-acetic acid and reduces passive permeability of the membrane. The possible mechanisms of action of aminophylline are discussed.