Ultrastructural changes associated with the induction of premature chromosome condensation in Vicia faba root meristem cells

Key message

PCC induction is regulated by several signaling pathways, and all observed effects associated with PCC induction are strongly dependent on the mechanism of action of each PCC inducer used.

Abstract

Electron microscopic observations of cells with symptoms of premature chromosome condensation (PCC) showed that the interphase chromatin and mitotic chromosomes differed with respect to a chemical compound inducing PCC. Induction of this process under the influence of hydroxyurea and caffeine as well as hydroxyurea and sodium metavanadate led to a slight decrease in interphase chromatin condensation and the formation of chromosomes with a considerably loosened structure in comparison with the control. Incubation in the mixture of hydroxyurea and 2-aminopurine brought about clear chromatin dispersion in interphase and very strong mitotic chromosome condensation. Electron microscopic examinations also revealed the characteristic features of the structural organization of cytoplasm of Vicia faba root meristems, which seemed to be dependent on the type of the PCC inducer used. The presence of the following was observed: (i) large plastids filled with starch grains (caffeine), (ii) mitochondria and plastids of electron dense matrix with dilated invaginations of their internal membranes (2-aminopurine), and (iii) large mitochondria of electron clear matrix and plastids containing protein crystals in their interior (sodium metavanadate). Moreover, since caffeine causes either the most effective loosening of chromatin fibrils (within the prematurely condensed chromosomes) or induction of starch formation (in the plastids surrounding the nuclei), this may be a proof that demonstrates the existence of a link between physical accessibility to chromatin and the effectiveness of cellular signaling (e.g., phosphothreonine-connected).     

Phosphorylation of H2AX histones in response to double-strand breaks and induction of premature chromatin condensation in hydroxyurea-treated root meristem cells of Raphanus sativus, Vicia faba, and Allium porrum

Summary. Histone H2A variant H2AX is rapidly phosphorylated on the induction of DNA double-strand breaks by ionizing radiation and hydroxyurea-mediated replication arrest, resulting in the formation of γ-H2AX foci along megabase chromatin domains nearby the sites of incurred DNA damage. In an attempt to establish a relationship between species-specific nuclear architecture and H2AX phosphorylation in S/G₂ phase-arrested root meristem cells, immunocytochemical comparisons using an antibody raised against human γ-H2AX were made among three plants differing with respect to DNA contents: Allium porrum, representing a reticulate type of DNA package, Vicia faba, having semireticulate cell nuclei, and Raphanus sativus, characterised by a chromocentric type of chromatin. Another approach was aimed at determining possible correlations between the extent of hydroxyurea-induced phosphorylation of H2AX histones and the quantities of root meristem cells induced by caffeine to enter aberrant mitotic division (premature chromosome condensation). It was concluded that the higher-order structure of chromatin may contribute to the accessibility of molecular factors engaged in the recognition and repair of genetic lesions. Consequently, in contrast to A. porrum and V. faba, a diffuse chromatin in chromocentric cell nuclei of R. sativus may become more vulnerable both to generate DNA double-strand breaks and to recruit molecular elements needed to arrange the cell cycle checkpoint functions, and thus, more resistant to factors which allow the cells to enter premature chromosome condensation spontaneously. On the other hand, however, caffeine-mediated overriding of the S-M checkpoint control system resulted in the typical appearance of premature chromosome condensation, irrespective of the genomic content of DNA. Correspondence and reprints: Department of Cytophysiology, University of Łódź, Pilarskiego 14, 90-231 Łódź, Poland.     

Uptake of tritiated thymidine in the root tip meristem of Vicia faba (L)

The use of tritium-labeled thymidine (3H-TdR) in biological research made it necessary to develop a quick and accurate method for determination of tritium activity in tissue. After 3H-TdR incorporation into the root tip meristem of Vicia faba, total 3H activity as well as 3H-DNA activity was measured by liquid scintillation counting. The incorporation rate of 3H-TdR using various parameters was examined-for example, the amount of incorporated 3H-TdR as a function of duration of treatment or as a function of thymidine concentration in the nutrient solution. The experimental results together with other data allow the calculation of the average number of incorporated thymidine molecules per labeled cell nucleus. This is necessary to interpret quantitatively the biological effects of incorporated radionuclides.     

ATM/ATR-dependent Tyr15 phosphorylation of cyclin-dependent kinases in response to hydroxyurea in Vicia faba root meristem cells

DNA damage or stalled replication forks activate cell cycle checkpoints. However, the regulation of metabolic pathways that are responsible for maintenance of genome integrity in plants is still largely unknown. Present research on Vicia faba root meristem cells indicates that inhibitory phosphorylation of cyclin-dependent kinases (Cdks) at Tyr15 plays a prominent role during blockage of cell cycle in response to genotoxic stress. Phosphorylation of P-loop in Cdks takes place in ATM/ATR-dependent manner. Although, Tyr15 phosphorylation upon hydroxyurea (HU) treatment was found in most cells classified to G1 and S phase, interestingly, the number of phoshpo-Tyr15-positive cells decreases in G2 phase. Presented data confirm much similarity in regulation of Cdks functions under genotoxic stress between plants and animals; however, they may also substantiate evolutionarily developed differences especially in regulation of G2/M transition between these two kingdoms.     

Protein phosphorylation in Vicia faba root meristem cells during the first steps of leaving principal control points after sucrose application

Before Vicia faba root meristem cells stopped by carbohydrate starvation in principal control points (PCP1 and PCP2) start sucrose induced replication and division they go through a phase of metabolic regeneration. This interval is characterised st great sensitivity to the inhibitors of cyclin-dependent protein kinases and protein phosphatases (PPs). In the present research, changes of phosphoprotein levels in the nucleolus, nucleus and cytoplasm were analysed using okadaic acid and 6-dimethylaminopurine (6-DMAP) during the first period of cell regeneration in sucrose (0–3 h). It was established that when the cells start to leave checkpoints, the balance between protein phosphorylation and dephosphorylation shifts towards the intensified activity of PPs. Furthermore, it was also established that the structures appearing during cell regeneration, which were located around cell nuclei and which contained large amounts of phosphorylated proteins, were plastids. The reactions of protein phosphorylation which took place in the plastids were directly correlated with starch synthesis and were stopped by inactivation of protein phosphatases (PP1 and/or PP2A).     

Low temperature between conditioning and challenge treatment prevents the 'adaptive response' of Vicia faba root tip meristem cells.

When the temperature during intertreatment time (2 h) between conditioning and challenge treatment of Vicia faba root tip meristems with either triethylenemelamine or maleic hydrazide was reduced from 24 degrees C to 12 degrees C no adaptive response occurred any more. The yield of metaphases with chromatid aberrations under these circumstances was similar to that observed after challenge treatment alone, i.e., no reduction occurred. This indicates that the metabolic state of the cells is of critical importance for the presence or absence of adaptive responses.     

Localization of MPM-2 recognized phosphoproteins and tubulin during cell cycle progression in synchronized Vicia faba root meristem cells.

MPM-2 antibody reacts with a subset of mitotic phosphoproteins. We followed localization of MPM-2 immunoreactive material and localization of microtubules during cell cycle progression in a highly synchronous population of Vicia faba root meristem cells and isolated nuclei. The MPM-2 antibody labelling showed significant cell cycle dependence. MPM-2 nuclear reactivity was weak and homogeneous in G1 and S phase of the cell cycle and became stronger and heterogeneous during G2, resembling staining of the nuclear matrix, with maximum staining at the G2/M interface. Similarly the staining intensity of nucleoli increased from late G1 phase to nucleoli dispersion in early prophase. During mitosis MPM-2 immunoreactivity was associated with spindle configurations and the brightest signal was localized in kinetochores from prophase to metaphase.     

Lateral root formation in Vicia faba L.

MI was found to decrease in LP with increase in cell number, and reached minimal values just before the emergence of LP to form lateral roots. These changes in MI have been correlated with the accumulation of cells in G1, 24 hours before a lateral root is formed. The durations of C and the various phases of the mitotic cycle were also investigated in LP, and compared with those in small primordia. As lateral root primordia increase in cell number, the durations of C, S and G2 become longer, while G1 becomes shorter. Also MI and GF decrease while the proportion of quiescent cells increases. Thus, there is a gradual decrease in the rate of cell proliferation as primordia increase in size. The changes which take place in these parameters during lateral root primordium development have been compared with the events which occur in seed proliferating tissues at the onset of dormancy.     

In situ activities of hexokinase and fructokinase in relation to phosphorylation status of root meristem cells of Vicia faba during reactivation from sugar starvation

The plant cell cycle is equipped with two principal control points: PCP1 in G1 and PCP2 in G2 phase. These checkpoints can arrest the cell cycle in response to carbohydrate starvation, while sugar presence can revive the replication and mitotic activity. The process of cell cycle revival is strongly repressed by okadaic acid (OA) or 6-dimethylaminopurine (6-DMAP), inhibitors of specific protein phosphatases 1 or 2A or kinases (cyclin-dependent kinases), respectively. In the present study, it was investigated whether inhibition of cell cycle revival is performed through interference of the above-mentioned inhibitors with the metabolic pathway of sucrose applied to the cells. Changes of hexokinase (HK) and fructokinase (FK) activities, key enzymes of hexose metabolism, were analyzed in Vicia faba root meristem cells arrested in G1 and G2 phase by carbohydrate starvation as well as in those recovered with glucose or sucrose in the presence of OA or 6-DMAP. It was shown that in the sugar-starved cells, the activity of both enzymes decreased significantly. During cell regeneration with carbohydrates, the activity of HK was induced more by sucrose than by glucose, while FK remained inactive after glucose addition. Moreover, in situ investigation of the activities of HK and FK showed that OA-induced and 6-DMAP-induced repression of the cell cycle revival is connected with the interference of these drugs in the metabolic pathway of sucrose. It was also indicated that stronger OA-induced and 6-DMAP-induced inhibition of the replication and mitosis revival, at the early stages of sucrose regeneration, was correlated with the stronger influence of these inhibitors on HK and FK activities.     

Effect of static electric fields in root cells of Vicia faba (Fabaceae)

Serial electron microscopic sections were prepared from half-ripened meristematic root cells of Vicia faba (Fabaceae) which had been exposed gradually to 700, 1000, 2500, 3500, and 5000 V/m static electric fields during seven days with and without Zn and Cd electrodes. At the end of five weeks, wall loosenings and very small nuclei were observed in those root cells which were exposed to static electric currents from the lower side of the medium without electrodes, while abnormalities in cell formation, e.g., two cells with one nucleus, and GER occurrence were present in an electrolytic (Cd upward and Zn downward) medium. The cells exposed to a static current from the upper side of the medium had small nuclei and abnormal cell divisions in the electrolyte, but in a non-electrolyte very large nuclei and thicker cell walls were observed, the cytoplasm was dense with GER, pinocytosis was seen filled with mitochondria, and protoplast formation with big nuclei was seen in exocytosis.