Root Meristems Of Pisum Sativum Research Articles

Histone deacetylase inhibitors and cell proliferation in pea root meristems

The histone deacetylase (HDA) inhibitors, trichostatin A (TSA) and HC toxin halt mitosis in cultured root meristems of Pisum sativum, while the anti-protozoan HDA inhibitor apicidin is ineffective. Two-dimensional PAGE of proteins from root meristems exposed to TSA and HC toxin did not show significant differences compared to controls, although a previously tested HDA inhibitor, butyrate, exhibited dramatic variations in its protein profile. Northern analysis of butyrate- and TSA-treated root meristems indicated that non-proliferating cells are expressing significant amounts of transcripts of the known cell proliferation associated genes: histone H2A, MAP kinase, cycA2:1 and cdc2. Western analysis reveals the presence of hyperacetylated nuclear proteins in HDA-inhibitor treated cells. These results suggest that the HDA inhibitors, butyrate and TSA, halt mitosis without down-regulating genes that typically have low or nonexistent expression levels in non-dividing cells.

Nuclear proteins and the onset of cell proliferation in root meristems ofPisum sativum: QP47 a novel acidic protein

AbstractA protein named QP47 has been purified from quiescent nuclei of root meristems ofPisum sativum, and used to prepare a polyclonal antibody. Immunolocalization of this protein with fluorescent probes revealed a nuclear distribution of thread-like structures. However, the relationship between the distribution of QP47 immunofluorescence and the structural organization of the chromatin required further investigation. The decrease in content of this protein in the nuclei of embryo cells seems to be correlated with the transition from quiescence to proliferation. QP47 degradation seems to depend upon an increase in the state of its phosphorylation. This protein is not present in normally proliferating cells, or in cells whose cell proliferation has been arrested by starvation or differentiation. It is hypothesized that QP47 may be required specifically during the quiescent period for specific structural organization of the chromatin.

Nuclear DNA Topoisomerases inPisum sativumL.

This paper demonstrates that in nuclei of root meristems of Pisum sativum L. there are at least two, and possibly three, different DNA topoisomerases. One enzyme is unbound by the Mono Q ion exchange column and seems to be similar to eukaryotic type I DNA topoisomerases. The other two enzymes are both retained by the Mono Q ion exchange column but they may be separated by their affinities for a single-strand DNA column. Both enzymes are difficult to classify, but it is likely that one of them is a type II topoisomerase. Prior to fractionation on Mono Q, all these different DNA topoisomerases elute in the same gel filtration peak together with other proteins, including endodeoxyribonuclease, indicating that they might form a native multiprotein complex. The nature, occurrence in vivo, and the functional role of this complex remain to be investigated. Nevertheless, the presence of different DNA topoisomerases in the same meristematic tissue might suggest that different types of topological transformation are required for different activities of DNA, such as replication, transcription and chromosome segregation.

Protein-kinases and p34 cdc2-like protein in root meristems of Pisum sativum during germination

Protein kinase activity was detected in a 0.5-M NaCl soluble protein extract obtained from nuclei purified after 24 h of germination from pea root meristematic tissue. Permeation chromatography of this extract resolved several different protein kinase activities. One was Ca 2+-dependent and was able to phosphorylate histone and casein as exogenous substrates to the incubation mixture. Another one was stimulated by the presence of Mg 2+ under Ca 2+-free condition. Two proteins (of 33.1 and 34.6 kDa) present in this extract were specifically recognised by the EGV antibody (an antibody raised against a conserved region of the p34 cdc2protein kinase). It is proposed that part of the Ca 2+-independent protien kinase activity solubilised from plant nuclei is contributed by a p34 cdc2-like protein kinase and the possible functions of nuclear p34 cdc2-like proteins during germination are discussed.

Short-chain fatty-acid-induced effects on the cell cycle in root meristems of Pisum sativum

Short-chain fatty-acid-induced effects on the cell cycle in root meristems of Pisum sativum

DNA Topoisomerase in Nuclei Purified from Root Meristems ofPisum sativum

DNA Topoisomerase in Nuclei Purified from Root Meristems of<i>Pisum sativum</i>

The effect of butyrate on the cell cycle in root meristems of Pisum sativum

The effect of butyrate on the cell cycle in root meristems of Pisum sativum

The effect of cell cycle regulators on protein profiles in cultured root meristems of Pisum sativum

The addition of various plant hormones, especially cytokinins, may influence both cell division and protein synthesis in vitro. Trigonelline, a newly proposed plant hormone, promotes cell arrest in G2 in pea meristem nuclei. Some recent evidence suggests that poly(ADP-ribose) may be implicated in trigonelline-induced G2 arrest. Results obtained herein demonstrate that the addition of trigonelline, cytokinin or poly(ADP-ribose) polymerase inhibitors to the culture medium does not alter the general polypeptide profile of the meristem. These data suggest that based on one-dimensional SDS-PAGE, root meristems which are exposed to test chemicals which alter G1/G2 distributions maintain similar general polypeptide profiles.

The induction of DNA strand breaks at specific sites by N-nitroso- N-ethylurea depends on the phases of the cell cycle

Synchronized root meristems of Pisum sativum were treated at each phase of the cell cycle with 6.25 mM N-nitroso- N-ethylurea. DNA extracted from treated cells and run in agarose gel electrophoresis exhibits a series of discrete fragments with length below 2500 bp and a significant number of unspecific single-stranded breaks (or alkali-labile sites). Experiments with micrococcal nuclease indicated that the nucleosomal organization of the chromatin is not responsible for the generation of the discrete fragments: it seems that their appearance is associated with a preferable attack of the mutagen at specific sites, characteristics for the plant genome. Moreover, a cell cycle dependent release of the discrete fragments was found with maximum at G1-S and minimum at mitosis. The model experiments designed to clarify this observation suggest that it might be determined from the cell cycle dependent fluctuation in the accessibility of the chromatin DNA and/or the process of excision-repair.

Rapid immunofluorescent determination of cells in the S phase in pea root meristems: An alternative to autoradiography

Root meristems of Pisum sativum L. cv. Lincoln were pulsed with bromodeoxyuridine, and after partial DNA denaturation the incorporated precursor was detected with an indirect immunofluorescent method by means of commercial anti‐bromodeoxyuridine monoclonal antibody and fluorescein isothiocyanate‐labeled secondary antibody. The nuclei were counterstained with the DNA‐specific fluorochrome 4′, 6‐diamidino‐2‐phenylindole (DAPI). The labeling indices (percentage of labeled calls) determined on slides prepared with the same nuclear population were very similar and in strict accordance with autoradiographic data. When DAPI fluorescence of the nuclei was measured, the unlabeled nuclei coincided with the G1 and part of the G2 regions, whereas all the labeled nuclei were in the S or G2 region. The method is reliable and, in contrast to autoradiography, data can be collected rapidly and almost immediately after the pulse. Furthermore, labeling index and the distribution of DNA contents can be obtained from the same side.

IS TRIGONELLINE A PLANT HORMONE?

Experimental results are presented so that trigonelline may be evaluated as a plant hormone. Trigonelline promotes preferential cell arrest in G2 of the cell cycle for about 40% of the cell population in root meristems of Pisum sativum. Trigonelline is present in ungerminated seeds and is transported from cotyledons to other tissues during early seedling development. These experimental results show that trigonelline satisfies all six criteria that have been used to establish whether a substance is a hormone. As the seedlings age from day 3 to 10, the concentration of trigonelline in meristems decreases and so does the proportion of cells arrested in G2. Trigonelline may be isolated from excised cotyledons and can be added back to decotyledonized seedlings or excised root meristems to have the same effect as found in intact organisms. Predominant cell arrest in G2 occurs in roots of some plant species, although other species show preponderant cell arrest in G1. Many members of the Preiss‐Handler metabolic pathway show some ability to promote cell arrest in G2 but only at concentrations 100 (10‐5 m) or 1,000 (10‐4 m) times the concentration of trigonelline (10‐7 m) necessary for function. The proportion of cells arrested in G2 is highly correlated with the concentration of trigonelline within the root meristem.

Cell Cycle Kinetics of Endoreduplication in Gamma-Irradiated Root Meristems of Pisum sativum

Cell Cycle Kinetics of Endoreduplication in Gamma-Irradiated Root Meristems of Pisum sativum

Recovery ofPisumRoot Meristems after Mitotic-inhibitory Treatments with3H-thymidine

Primary root meristems of Pisum sativum recover form a 3H-thymidine-induced reduction in mitotic activity once the roots are no longer exposed to exogenous 3H-thymidine. Cells arrested in G2 during 3H-thymidine treatment apparently do not divide for at least 16 hours after treatment, whereas cells remaining in G1 and S do divide and thereby account for recovery. Recovery occurs only when meristems are no longer exposed to exogenous (i.e. unincorporated) 3H-thymidine, suggesting that cytoplasmic irradiation from unincorporated 3H-thymidine prevents cellular recovery from 3H-thymidine-induced inhibition of cell progression through the mitotic cycle. Concentrations of 14C-thymidine which result in cytoplasmic irradiation nearly equivalent to that achieved with 3H-thymidine, but much lower levels of nuclear irradiation, also prevent recovery from 3H-thymidine-induced inhibition of mitotic activity, but do not alone produced such inhibition. These results support the contention that cytoplasmic irradiation prevents recovery from the effects of nuclear irradiation. Unincorporated 3H-thymidine also prevents recovery from sucrose deprivation in stationary phase G2 cells which have not incorporated 3H-thymidine into nuclear DNA.

The effect of ethylene on root meristems of Pisum sativum and Zea mays

Ethylene at a concentration of 100 μl l(-1) causes a slight increase in the duration of the mitotic cycle in the primary root meristems of both Pisum sativum L. and Zea mays L. This is due to a lengthening of the G 1 phase; other phases of the cycle are unaffected. Autoradiography and microdensitometry show that the rate of (3)H-thymidine incorporation into nuclei of Pisum is maximal when about half the DNA has been replicated, and that ethylene has no effect upon this rate. Ethylene causes a reduction of the number of dividing cells in the root meristem, particularly in Pisum.

Tritiated-Thymidine Induced Changes in Cell Population Kinetics in Root Meristems of Pisum sativum

The effects of 3H-thymidine on cell progression through the mitotic cycle and on cell population kinetics have been investigated. During continuous treatment there is a gradual inhibition of entry of cells from G2 into mitosis, while other stages of the cell cycle appear to be less radiosensitive. Hence there is an accumulation of cells in G2 while the proportions of cells in G1, S, and M decrease. Cells of the quiescent center are induced to synthesize DNA after inhibition of mitosis in the surrounding meristem. They do not serve to repopulate the meristem, however, since they then incorporate 3H-thymidine and are themselves arrested. It is suggested that the other slowly cycling cells of the meristem, in addition to those of the quiescent center, may also be induced to cycle more rapidly after delay in the fast-cycling population. These cells will also be arrested after a brief compensatory proliferative reaction. Thus the meristem is unable to recover from radiation damage during continuous treatment with 3H-thymidine.

Effects of Near Ultraviolet and Visible Radiations on Cell Cycle Kinetics in Excised Root Meristems of Pisum sativum

Effects of Near Ultraviolet and Visible Radiations on Cell Cycle Kinetics in Excised Root Meristems of Pisum sativum

EFFECTS OF NEAR ULTRAVIOLET AND VISIBLE RADIATIONS ON CELL CYCLE KINETICS IN EXCISED ROOT MERISTEMS OF PISUM SATIVUM

Near‐ultraviolet and visible radiations increased the duration of the mitotic cycle in excised pea root meristems primarily by lengthening the duration of the pre‐DNA synthetic period (G1). All radiations tested shortened the duration of the post‐DNA synthetic period (G2). The most pronounced effects were exhibited by green radiation, which lengthened the duration of the cell cycle, G1, DNA synthesis (S), and mitosis (M), and shortened the duration of G2. Progression of cells arrested by starvation in G1 and G2 into DNA synthesis and mitosis was also affected by light treatments. Green radiation appeared to arrest a group of cells in DNA synthesis as well as in G1 and G2. Meristems receiving green and near‐ultraviolet radiations exhibited the most rapid progression of G1 cells through S and 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...

Chromosome Endoreduplication (Endopolyploidy) in Pea Root Meristems Induced by 8-Azaguanine

SUMMARYThe effect of 8-azaguanine (5 × 10−4M) on the root meristems of Pisum sativum has been studied.The immediate effect of 8-azaguanine is an inhibition of interphase (G2) nuclei to enter mitosis ending in the absence of mitosis after a 12-hour treatment. Inhibition of DNA synthesis (H3-thymidine incorporation) starts to appear after 6 hours: 8-azaguanine does not interfere with DNA synthesis once it is initiated, but it inhibits the initiation of DNA synthesis (passage from G1 to S) at least in the majority of cells (data of the DNA cyto-photoinetry).When roots treated with 8-azaguanine for 17 hours recover in water for 24 hours, both diploid (14 monochromosomes) and endotetraploid (14 diplochromosomes = 4-chromatid chromosomes) mitoses are found in the meristem. The proportion of diplochromosome mitoses is still higher when the 24-hour water recovery follows two 8-azaguanine treatments, 17-hours each, separated by a 6-hour root immersion in 5 x 10—4M guanine. A treatment has also been found which ind...