Elio Sparvoli

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.

Comparative effects of fusicoccin and gibberellic acid on the promotion of germination and DNA synthesis initiation in Haplopappus gracilis

Abstract We have compared the effects of fusicoccin (FC) and gibberellic acid (GA 3 ) on the promotion of germination, the stimulation of cell elongation and the resumption of nuclear DNA synthesis in dormant seeds of Haplopappus gracilis . Although both FC and GA 3 promote germination and stimulate cell elongation, FC appears to act earlier and to be more effective. On the otherhand, FC has no effect on the resumption of DNA synthesis which, in contrast, is stimulated effectively by GA 3 .

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.

Interaction between abscisic acid and fusicoccin during germination and post-germinative growth in Haplopappus gracilis

Abstract Abscisic acid (ABA) 10 −5 M completely inhibits the germination of seeds of Haplopappus gracilis , and this effect can be effectively reversed by fusicoccin (FC). Nevertheless, when the analysis of reversal promoted by FC is extended to post-germinative growth, it is observed that it results only in a limited elongation of the embryos, perhaps only through cell enlargement, which does not involve any significant resumption of DNA synthesis, cell division or such developmental features as synthesis of chlorophyll and development of root hairs. It is proposed that ABA inhibition of seed germination involves at least two different mechanisms, one mechanism interfering with K + uptake at the level of the cell membrane, which is reversed by FC, promoting cell elongation, the other interfering with the synthesis of nucleic acids and/or protein synthesis and not being affected by FC.

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.

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.

Partial reversal by fusicoccin of the inhibition imposed by abscisic acid on postgerminative growth of Halopappus gracilis

Abstract On the basis of the evidence that fusicoccin (FC) can reverse the inhibition of germination induced by abscisic acid (ABA) in Haplopappus gracilis seeds, but cannot restore normal postgerminative growth, we previously suggested that ABA may act through two different mechanisms, one involving H + /K + exchange at the plasmalemma level, the other interfering with nucleic acid and/or protein synthesis. In the present work we have analysed the interaction of FC with ABA concentrations lower than the 10 −5 M previously used. In these conditions, FC is able to induce a partial but significant reversion of the inhibition of DNA and chlorophyll synthesis and, in some cases, a nearly normal post-germinative growth. We are thus concerned with a system apparently inhibited not only at a membrane level that can, in some instances, be reversed by FC, which clearly acts only at the level of an H + /K + exchange mechanism operating on the plasmalemma. The possible interpretations are discussed.

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.