Oxidative replication stress induced by long-term exposure to hydroxyurea in root meristem cells of Vicia faba.

KEY MESSAGE: Low concentrations of hydroxyurea, an inhibitor of DNA replication, induced oxidative and replicative stress in root apical meristem (RAM) cells of Vicia faba. Plant cells are constantly exposed to low-level endogenous stress factors that can affect DNA replication and lead to DNA damage. Long-term treatments of Vicia faba root apical meristems (RAMs) with HU leads to the appearance of atypical cells with intranuclear asynchrony. This rare form of abnormality was manifested by a gradual condensation of chromatin, from interphase to mitosis (so-called IM cells). Moreover, HU-treated root cells revealed abnormal chromosome structure, persisting DNA replication, and elevated levels of intracellular hydrogen peroxide (H2O2) and superoxide anion (O2∙-). Immunocytochemical studies have shown an increased number of fluorescent foci of H3 histones acetylated at lysine 56 (H3K56Ac; canonically connected with the DNA replication process). We show that continuous 3-day exposure to low concentrations (0.75 mM) of hydroxyurea (HU; an inhibitor of DNA replication) induces cellular response to reactive oxygen species and to DNA replication stress conditions.

Epigenetic modifications evidenced by isolation of proteins on nascent DNA and immunofluorescence in hydroxyurea-treated root meristem cells of Vicia faba.

MAIN CONCLUSION: By implementation of the iPOND technique for plant material, changes in posttranslational modifications of histones were identified in hydroxyurea-treated root meristem cells of Vicia. Replication stress (RS) disrupts or inhibits replication forks and by altering epigenetic information of the newly formed chromatin can affect gene regulation and/or spatial organisation of DNA. Experiments on Vicia faba root meristem cells exposed to short-term treatment with 3 mM hydroxyurea (HU, an inhibitor of DNA replication) were aimed to understand epigenetic changes related to RS. To achieve this, the following histone modifications were studied using isolation of proteins on nascent DNA (iPOND) technique (for the first time on plant material) combined with immunofluorescence labeling: (i) acetylation of histone H3 at lysine 56 (H3K56Ac), (ii) acetylation of histone H4 at Lys 5 (H4K5Ac), and (iii) phosphorylation of histone H3 at threonine 45 (H3T45Ph). Certainly, the implementation of the iPOND method for plants may prove to be a key step for a more in-depth understanding of the cell's response to RS at the chromatin level.

The role of centrifugal partition chromatography in the removal of ß-asarone from Acorus calamus essential oil.

Β-asarone is a phenylpropane derivative present in the rhizomes of Acorus calamus, that was proved to exhibit toxic effects in humans. Because of its presence the whole plant that is commonly used in traditional medicine for its sedative, anti-inflammatory, neuroprotective and other properties has limited application nowadays. In the study, qualitative and quantitative analysis of a collection of nine essential oil (EO) samples of European and Asian origin was performed. The final content of ß-asarone in the tested samples ranged between 0.265 and 1.885 mg/mL. Having in mind a possible application of the EO as a biopesticide, this research aimed at the development of CPC-based purification protocol that could help remove ß-asarone from EO. It was proved that the biphasic solvent system composed of n-hexane/EtOAc/MeOH/water, 9:1:9:1 (v/v/v/v) was capable of the removal of the toxic constituent in the CPC chromatograph operated in the ascending elution mode with 2200 rpm and a flow rate of 5 mL/min. The chromatographic analysis that lasted only 144 min effectively separated ß-asarone (purity of 95.5%) and α-asarone (purity of 93.7%) directly from the crude Acorus calamus rhizome EO.

Changes in Epigenetic Patterns Related to DNA Replication in Vicia faba Root Meristem Cells under Cadmium-Induced Stress Conditions.

Experiments on Vicia faba root meristem cells exposed to 150 µM cadmium chloride (CdCl2) were undertaken to analyse epigenetic changes, mainly with respect to DNA replication stress. Histone modifications examined by means of immunofluorescence labeling included: (1) acetylation of histone H3 on lysine 56 (H3K56Ac), involved in transcription, S phase, and response to DNA damage during DNA biosynthesis; (2) dimethylation of histone H3 on lysine 79 (H3K79Me2), correlated with the replication initiation; (3) phosphorylation of histone H3 on threonine 45 (H3T45Ph), engaged in DNA synthesis and apoptosis. Moreover, immunostaining using specific antibodies against 5-MetC-modified DNA was used to determine the level of DNA methylation. A significant decrease in the level of H3K79Me2, noted in all phases of the CdCl2-treated interphase cell nuclei, was found to correspond with: (1) an increase in the mean number of intranuclear foci of H3K56Ac histones (observed mainly in S-phase), (2) a plethora of nuclear and nucleolar labeling patterns (combined with a general decrease in H3T45Ph), and (3) a decrease in DNA methylation. All these changes correlate well with a general viewpoint that DNA modifications and post-translational histone modifications play an important role in gene expression and plant development under cadmium-induced stress conditions.

Ipertrofan Revisited-The Proposal of the Complete Stereochemistry of Mepartricin A and B.

Being a methyl ester of partricin, the mepartricin complex is the active substance of a drug called Ipertrofan (Tricandil), which was proven to be useful in treatment of benign prostatic hyperplasia and chronic nonbacterial prostatitis/chronic pelvic pain syndrome. Nevertheless, no direct structural evidence on the stereochemistry of its components has been presented to date. In this contribution, we have conducted detailed, NMR-driven stereochemical studies on mepartricins A and B, aided by molecular dynamics simulations. The absolute configuration of all the stereogenic centers of mepartricin A and B was defined as 3R, 7R, 9R, 11S, 13S, 15R, 17S, 18R, 19S, 21R, 36S, 37R, and 38S, and proposed as 41R. The geometry of the heptaenic chromophore of both compounds has been established as 22E, 24E, 26E, 28Z, 30Z, 32E, and 34E. Our studies on mepartricin ultimately proved that partricins A and B are structurally identical to the previously described main components of the aureofacin complex: gedamycin and vacidin, respectively. The knowledge of the stereochemistry of this drug is a fundamental matter not only in terms of studies on its molecular mode of action, but also for potential derivatization, aiming at improvement of its pharmacological properties.

Cadmium (II)-Induced Oxidative Stress Results in Replication Stress and Epigenetic Modifications in Root Meristem Cell Nuclei of Vicia faba.

Among heavy metals, cadmium is considered one of the most toxic and dangerous environmental factors, contributing to stress by disturbing the delicate balance between production and scavenging of reactive oxygen species (ROS). To explore possible relationships and linkages between Cd(II)-induced oxidative stress and the consequent damage at the genomic level (followed by DNA replication stress), root apical meristem (RAM) cells in broad bean (V. faba) seedlings exposed to CdCl2 treatment and to post-cadmium recovery water incubations were tested with respect to H2O2 production, DNA double-strand breaks (γ-phosphorylation of H2AX histones), chromatin morphology, histone H3S10 phosphorylation on serine (a marker of chromatin condensation), mitotic activity, and EdU staining (to quantify cells typical of different stages of nuclear DNA replication). In order to evaluate Cd(II)-mediated epigenetic changes involved in transcription and in the assembly of nucleosomes during the S-phase of the cell cycle, the acetylation of histone H3 on lysine 5 (H3K56Ac) was investigated by immunofluorescence. Cellular responses to cadmium (II) toxicity seem to be composed of a series of interlinked biochemical reactions, which, via generation of ROS and DNA damage-induced replication stress, ultimately activate signal factors engaged in cell cycle control pathways, DNA repair systems, and epigenetic adaptations.

Do Plasmodesmata Play a Prominent Role in Regulation of Auxin-Dependent Genes at Early Stages of Embryogenesis?

Plasmodesmata form intercellular channels which ensure the transport of various molecules during embryogenesis and postembryonic growth. However, high permeability of plasmodesmata may interfere with the establishment of auxin maxima, which are required for cellular patterning and the development of distinct tissues. Therefore, diffusion through plasmodesmata is not always desirable and the symplastic continuum must be broken up to induce or accomplish some developmental processes. Many data show the role of auxin maxima in the regulation of auxin-responsive genes and the establishment of various cellular patterns. However, still little is known whether and how these maxima are formed in the embryo proper before 16-cell stage, that is, when there is still a nonpolar distribution of auxin efflux carriers. In this work, we focused on auxin-dependent regulation of plasmodesmata function, which may provide rapid and transient changes of their permeability, and thus take part in the regulation of gene expression.

Anti-algal activity of the 12-5-12 gemini surfactant results from its impact on the photosynthetic apparatus.

A rapid amplification of algal population has a negative impact on the environment and the global economy. Thus, control of algal proliferation is an important issue and effective procedures which reduce algal blooms and control algal fouling are highly desired. Gemini surfactants are considered to have a low environmental impact, therefore they seem to be a promising group of detergents which could reduce algal blooms in water systems. Furthermore, due to their emulsifying properties they could replace algaecides added to antifouling paints and decrease algae adhesion to various surfaces. In this study the toxic effect of the 12-5-12 gemini surfactant was investigated on Chlorella cells and close attention was paid to a potential mechanism of its action. At the high cell density (10.05 × 107 cells/mL) a dose-dependent cell death was found and the IC50 value was reached at the concentration of 19.6 µmol/L after 72-h exposure to the surfactant. The decrease in chlorophyll autofluorescence shows that the photosynthetic apparatus seems to be the target of the tested compound. The presented studies indicate that gemini surfactants could effectively reduce algal blooms in water systems, and if added to paints, they could decrease algal growth on external building walls or other water immersed surfaces.

The Role of Cutinsomes in Plant Cuticle Formation.

The cuticle commonly appears as a continuous lipophilic layer located at the outer epidermal cell walls of land plants. Cutin and waxes are its main components. Two methods for cutin synthesis are considered in plants. One that is based on enzymatic biosynthesis, in which cutin synthase (CUS) is involved, is well-known and commonly accepted. The other assumes the participation of specific nanostructures, cutinsomes, which are formed in physicochemical self-assembly processes from cutin precursors without enzyme involvement. Cutinsomes are formed in ground cytoplasm or, in some species, in specific cytoplasmic domains, lipotubuloid metabolons (LMs), and are most probably translocated via microtubules toward the cuticle-covered cell wall. Cutinsomes may additionally serve as platforms transporting cuticular enzymes. Presumably, cutinsomes enrich the cuticle in branched and cross-linked esterified polyhydroxy fatty acid oligomers, while CUS1 can provide both linear chains and branching cutin oligomers. These two systems of cuticle formation seem to co-operate on the surface of aboveground organs, as well as in the embryo and seed coat epidermis. This review focuses on the role that cutinsomes play in cuticle biosynthesis in S. lycopersicum, O. umbellatum and A. thaliana, which have been studied so far; however, these nanoparticles may be commonly involved in this process in different plants.

5-Aminouracil and other inhibitors of DNA replication induce biphasic interphase-mitotic cells in apical root meristems of Allium cepa.

KEY MESSAGE: Induction of biphasic interphase-mitotic cells and PCC is connected with an increased level of metabolism in root meristem cells of Allium cepa. Previous experiments using primary roots of Allium cepa exposed to low concentrations of hydroxyurea have shown that long-term DNA replication stress (DRS) disrupts essential links of the S-M checkpoint mechanism, leading meristem cells either to premature chromosome condensation (PCC) or to a specific form of chromatin condensation, establishing biphasic organization of cell nuclei with both interphase and mitotic domains (IM cells). The present study supplements and extends these observations by describing general conditions under which both abnormal types of M-phase cells may occur. The analysis of root apical meristem (RAM) cell proliferation after prolonged mild DRS indicates that a broad spectrum of inhibitors is capable of generating PCC and IM organization of cell nuclei. These included: 5-aminouracil (5-AU, a thymine antagonist), characterized by the highest efficiency in creating cells with the IM phenotype, aphidicolin (APH), an inhibitor of DNA polymerase α, 5-fluorodeoxyuridine (FUdR), an inhibitor of thymidylate synthetase, methotrexate (MTX), a folic acid analog that inhibits purine and pyrimidine synthesis, and cytosine arabinoside (Ara-C), which inhibits DNA replication by forming cleavage complexes with topoisomerase I. As evidenced using fluorescence-based click chemistry assays, continuous treatment of onion RAM cells with 5-AU is associated with an accelerated dynamics of the DNA replication machinery and significantly enhanced levels of transcription and translation. Furthermore, DRS conditions bring about an intensified production of hydrogen peroxide (H2O2), depletion of reduced glutathione (GSH), and some increase in DNA fragmentation, associated with only a slight increase in apoptosis-like programmed cell death events.

Irrigation-Induced Changes in Chemical Composition and Quality of Seeds of Yellow Lupine (Lupinus luteus L.).

The quality and amount of yellow lupine yield depend on water availability. Water scarcity negatively affects germination, flowering, and pod formation, and thus introduction of an artificial irrigation system is needed. The aim of this study was to evaluate the influence of irrigation on the quality of yellow lupine seeds. Raining was applied with a semi-solid device with sprinklers during periods of greatest water demand. It was shown that watered plants produced seeds of lesser quality, having smaller size and weight. To find out why seeds of irrigated plants were of poor quality, interdisciplinary research at the cellular level was carried out. DNA cytophotometry evidenced the presence of nuclei with lower polyploidy in the apical zone of mature seeds. This may lead to formation of smaller cells and reduce depositing of storage materials. The electrophoretic and Fourier transform infrared spectroscopic analyses revealed differences in protein and cuticular wax profiles, while scanning electron microscopy and energy dispersive spectroscopy revealed, among various chemical elements, decreased calcium content in one of seed zones (near plumule). Seeds from irrigated plants showed slightly higher germination dynamics but growth rate of seedlings was slightly lower. The studies showed that irrigation of lupine affected seed features and their chemical composition, an ability to germination and seedlings growth.

Irrigation affects characteristics of narrow-leaved lupin (Lupinus angustifolius L.) seeds.

MAIN CONCLUSION: While plant irrigation usually increases yield, irrigation also affects seed characteristics with respect to endoreplication level, chemical composition, number of carbonyl bands, and cuticular wax profiles. Seeds of sweet varieties of the narrow-leaved lupin have good nutritional properties; however, these plants are sensitive to water deficit. Irrigation improves lupin yield, but can affect seed characteristics. The purpose of the study was to evaluate irrigation influence on lupin seed features and their chemical composition. Morphological analyses showed worse quality of seeds from the irrigated plants, with regard to their size and weight. This was confirmed by cytophotometric analyses which revealed a lower DNA content in the nuclei of cells from the apical and basal regions of the irrigated seeds. The lower degree of polyploidy of the nuclei entails lower cell sizes and limited space for storage components. Fourier transform infrared spectroscopic analysis demonstrated that protein and cuticular wax profiles of the irrigated seeds were different from the control. The electrophoretic analyses indicated differences in protein profiles including changes in the proportion of lupin storage proteins. Among the various studied elements, only the nitrogen content decreased in the embryo axis of irrigated plants. Although germination dynamics of the irrigated seeds was higher, the seedlings' development rate was slightly lower than in the control. The hydrogen peroxide level in root meristem cells was higher during germination in the control suggesting its regulatory role in seed metabolism/signaling. Our study indicated that irrigation of lupin plant affected seed features and composition.

Endoreplication and its consequences in the suspensor of Pisum sativum.

KEY MESSAGE: DNA replication and continuous process of transcription during ongoing amitotic division accelerate the development of four-celled pea suspensor containing nuclei which create transient gradient of polyploidy necessary for correct embryo development. A suspensor, the link between embryo proper and surrounding tissues, differs significantly in size, morphology, and degree of polyploidy among the species. The suspensor of Pisum sativum consists of four polynuclear cells (two hemispherical and two elongated) formed in two layers. Their nuclei undergo endoreplication reaching, respectively, up to 256C and 128-256C DNA levels in its hemispherical and elongated parts. Our study shows that endoreplication first appears in the spherical part of the suspensor, and, subsequently, in the elongated one. At the next stages of suspensor development, the increase in DNA content takes place also in a similar order. Thus, despite simple construction of the suspensor, its development, supported by endoreplication, creates a certain gradient of polyploidy, which occurs in more extensive suspensors. Moreover, the rapid development of suspensor is supported both by the initiation of DNA replication prior to the completion of amitotic division of its polyploidal nuclei and by a continuous process of transcription, which is silenced by chromatin condensation throughout mitosis. Furthermore, the increase in DNA content correlates with the greater amount of transcripts; however, the multiplication of DNA copies does not entail an increase (but fluctuation) in the mean transcriptional activity of a particular nucleus during the next stages of suspensor development.

Mitogen-activated protein kinases concentrate in the vicinity of chromosomes and may regulate directly cellular patterning in Vicia faba embryos.

MAIN CONCLUSION: Mitogen-activated protein kinases seem to mark genes which are set up to be activated in daughter cells and thus they may play a direct role in cellular patterning during embryogenesis. Embryonic patterning starts very early and after the first division of zygote different genes are expressed in apical and basal cells. However, there is an ongoing debate about the way these different transcription patterns are established during embryogenesis. The presented data indicate that mitogen-activated protein kinases (MAPKs) concentrate in the vicinity of chromosomes and form visible foci there. Cells in the apical and basal regions differ in number of foci observed during the metaphase which suggests that cellular patterning may be determined by activation of diverse MAPK-dependent genes. Different number of foci in each group of separating chromatids and the specified direction of these mitoses in apical-basal axis indicate that the unilateral auxin accumulation in a single cell may regulate the number of foci in each group of chromatids. Thus, we put forward a hypothesis that MAPKs localized in the vicinity of chromosomes during mitosis mark those genes which are set up to be activated in daughter cells after division. It implies that the chromosomal localization of MAPKs may be one of the mechanisms involved in establishment of cellular patterns in some plant species.

Plant storage proteins - the main nourisching products - from biosynthesis to cellular storage depots.

Storage proteins of legumes are one of the main components of the human and animal diet. The substances collected in their seeds have the pro-health values, supporting the prevention of many civilization diseases. However, there are still many uncertainties about the mechanisms leading to the production of nutritious seeds. It is also difficult to identify which of their constituents and in what final form are responsible for the observed protective effects in vivo. In this work, on the background of different types of storage proteins, these deposited mainly in legumes were in the focus of interest. They were characterized on the example of pea (Pisum sativum) proteins. Mechanisms associated with their biosynthesis and transport to specific cellular compartments was presented. Ways of their post-translational processing, segregation and storage in the specific vacuoles were also discussed. Therefore, the paper presents the state-of-the-art knowledge concerning the processes making the accumulated protein deposits ready to use by plants, animals and humans.

Mitogen-activated protein kinases participate in determination of apical-basal symmetry in Pisum sativum.

Mitogen-activated protein kinases (MAPKs) are implicated in various processes in plants. Apart from response to biotic and abiotic stresses they are involved in regulation of embryo development. Although MAPKs were found to be indispensable during embryo development it has never been established at which stages of embryogenesis and in which regions of a plant embryo activated MAPKs can be observed. Here, we show that apical and basal regions display activation of the same types of MAPKs and the only difference concerns the level of their phosphorylation and cellular localization. Dually-phosphorylated MAPKs were found in nuclei of the apical region of an embryo both at the early and late cotyledonary stage while no immunofluorescence signals were detected in nuclei of the basal region. However, in this case phosphorylated MAPKs were immunodetected in cytoplasm in the apical domain of cortex cells, indicating their role in auxin transport from the basal to apical region of an embryo. Furthermore, obtained data indicate that nuclear localization of activated MAPKs may result from epigenetic modifications and polar auxin transport. The presented data and previous studies lead to the conclusion that activated MAPKs and their cellular localization define apical and basal regions during formation of an apical-basal axis.

Sanguinarine-induced oxidative stress and apoptosis-like programmed cell death(AL-PCD) in root meristem cells of Allium cepa.

A vast number of studies on plant cell systems clearly indicate that various biotic and abiotic stresses give rise to the uncontrolled increase in the level of reactive oxygen species (ROS). Excess concentrations of ROS result in damage to proteins, lipids, carbohydrates, and DNA, which may lead, in consequence, to the apoptotic cell death. The current study investigates the effects of sanguinarine (SAN), a natural alkaloid derived from the roots of Sanguinaria canadensis, on root apical meristem cells of Allium cepa. It is shown that SAN treatment generated large amounts of hydrogen peroxide (H2O2) and superoxide anion (O2·-). Oxidative stress induced in SAN-treated cells was correlated with DNA fragmentation, formation of micronuclei (MN), altered and 'degenerated' chromatin structures characteristic of apoptosis-like programmed cell death (AL-PCD). The experiments with SAN + MG132 (a proteasome inhibitor engaged in Topo II-mediated formation of cleavable complexes) and SAN + ascorbic acid (AA; H2O2 scavenger) seem to suggest, however, that the high level of H2O2 is not the only factor responsible for changes observed at the chromatin level and for the consequent cell death. Our findings imply that Topo II-DNA covalent complexes and 26S proteasomes are also involved in SAN-induced DNA damage.

PIN2-like proteins may contribute to the regulation of morphogenetic processes during spermatogenesis in Chara vulgaris.

KEY MESSAGE: PIN2-like auxin transporters are expressed, preferentially in a polarized manner, in antheridial cells of freshwater green alga Chara vulgaris , considered to be the closest relative of the present-day land plants. Chara vulgaris represents a group of advanced multicellular green algae that are considered as the closest relatives of the present-day land plants. A highly specialized structure of its male sex organs (antheridia) includes filaments consisting of generative cells, which after a series of synchronous divisions transform into mature sperm, and non-generative cells comprising outer shield cells, cylindrical manubria, and central complex of capitular cells from which antheridial filaments arise. Immunofluorescence observations indicate that PIN2-like proteins (PIN2-LPs), recognized by antibodies against PIN-FORMED2 (PIN2) auxin transporter in Arabidopsis thaliana, are expressed in both types of antheridial cells and, in most of them, preferentially accumulate in a polarized manner. The appearance of PIN2-LPs in germ-line cells is strictly confined to the proliferative period of spermatogenesis and their quantities increase steadily till antheridial filaments reach the 16-celled stage. An enhanced level of PIN2-LPs observed in the central cell walls separating two asynchronously developing parts of antheridial filaments (characterized by the plugged plasmodesmata) is correlated with an enhanced deposition of callose. Intense PIN2-LPs immunofluorescence maintained in the capitular cells and its altering polarity in manubria suggest a pivotal role of these cells in the regulation of auxin transport directionality during the whole time of antheridial ontogenesis. Immunohistochemical staining of IAA revealed a clear-cut correspondence between localization sites of auxins and PIN2-LPs. It seems probable then that a supplementary developmental mechanism has evolved in Chara, by which all antheridial elements may be integrated at the supra-cellular level via plasma membrane-targeted PIN2-LPs and auxin-mediated processes.

Cutinsomes and cuticle enzymes GPAT6 and DGAT2 seem to travel together from a lipotubuloid metabolon (LM) to extracellular matrix of O. umbellatum ovary epidermis.

In the ovary epidermis of O. umbellatum there are lipotubuloid metabolons (LMs), in which synthesis of lipids takes place. This process partly provides nourishment, and partly cuticle building blocks, transformed, among others, with the participation of cutinsomes. The cutinsomes are cutin-building structures, 40-200nm in size, which are formed as a result of self-assembly and self-esterification of hydroxy fatty acids. The cutinsomes, by binding to the cuticle, introduce into it nonlinear, amorphous and cross-linked polymers. Double-immunogold EM observations revealed that enzymes producing elements of cutin (GPAT6) and waxes (WS/DGAT) were found not only as free cytoplasmic molecules but also in many cases they were bound to carboxylate-carboxylic shell of cuntinsomes. Hence, we suppose that these enzymes can move alone or together with the cutinsomes through cytoplasm (pH 6.8-7.0), plasmalemma and the polysaccharide layer of a cell wall to the site of their functioning i.e. to the cuticle (pH 5.0).

The effects of anti-DNA topoisomerase II drugs, etoposide and ellipticine, are modified in root meristem cells of Allium cepa by MG132, an inhibitor of 26S proteasomes.

DNA topoisomerase II (Topo II), a highly specialized nuclear enzyme, resolves various entanglement problems concerning DNA that arise during chromatin remodeling, transcription, S-phase replication, meiotic recombination, chromosome condensation and segregation during mitosis. The genotoxic effects of two Topo II inhibitors known as potent anti-cancer drugs, etoposide (ETO) and ellipticine (EPC), were assayed in root apical meristem cells of Allium cepa. Despite various types of molecular interactions between these drugs and DNA-Topo II complexes at the chromatin level, which have a profound negative impact on the genome integrity (production of double-strand breaks, chromosomal bridges and constrictions, lagging fragments of chromosomes and their uneven segregation to daughter cell nuclei), most of the elicited changes were apparently similar, regarding both their intensity and time characteristics. No essential changes between ETO- and EPC-treated onion roots were noticed in the frequency of G1-, S-, G2-and M-phase cells, nuclear morphology, chromosome structures, tubulin-microtubule systems, extended distribution of mitosis-specific phosphorylation sites of histone H3, and the induction of apoptosis-like programmed cell death (AL-PCD). However, the important difference between the effects induced by the ETO and EPC concerns their catalytic activities in the presence of MG132 (proteasome inhibitor engaged in Topo II-mediated formation of cleavage complexes) and relates to the time-variable changes in chromosomal aberrations and AL-PCD rates. This result implies that proteasome-dependent mechanisms may contribute to the course of physiological effects generated by DNA lesions under conditions that affect the ability of plant cells to resolve topological problems that associated with the nuclear metabolic activities.