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

Increased transcription in hydroxyurea-treated root meristem cells of Vicia faba

Hydroxyurea (HU), an inhibitor of ribonucleotide reductase, prevents cells from progressing through S phase by depletion of deoxyribonucleoside triphosphates. Concurrently, disruption of DNA replication leads to double-strand DNA breaks. In root meristems of Vicia faba, HU triggers cell cycle arrest (preferentially in G1/S phase) and changes an overall metabolism by global activation of transcription both in the nucleoplasmic and nucleolar regions. High level of transcription is accompanied by an increase in the content of RNA polymerase II large subunit (POLR2A). Changes in transcription activation and POLR2A content correlate with posttranslational modifications of histones that play a role in opening up chromatin for transcription. Increase in the level of H4 Lys5 acetylation indicates that global activation of transcription following HU treatment depends on histone modifications

Immunolocalization of dually phosphorylated MAPKs in dividing root meristem cells of Vicia faba, Pisum sativum, Lupinus luteus and Lycopersicon esculentum

Key message In plants, phosphorylated MAPKs display constitutive nuclear localization; however, not all studied plant species show co-localization of activated MAPKs to mitotic microtubules. Abstract The mitogen-activated protein kinase (MAPK) signaling pathway is involved not only in the cellular response to biotic and abiotic stress but also in the regulation of cell cycle and plant development. The role of MAPKs in the formation of a mitotic spindle has been widely studied and the MAPK signaling pathway was found to be indispensable for the unperturbed course of cell division. Here we show cellular localization of activated MAPKs (dually phosphorylated at their TXY motifs) in both interphase and mitotic root meristem cells of Lupinus luteus, Pisum sativum, Vicia faba (Fabaceae) and Lycopersicon esculentum (Solanaceae). Nuclear localization of activated MAPKs has been found in all species. Colocalization of these kinases to mitotic microtubules was most evident in L. esculentum, while only about 50 % of mitotic cells in the root meristems of P. sativum and V. faba displayed activated MAPKs localized to microtubules during mitosis. Unexpectedly, no evident immunofluorescence signals at spindle microtubules and phragmoplast were noted in L. luteus. Considering immunocytochemical analyses and studies on the impact of FR180204 (an inhibitor of animal ERK1/2) on mitotic cells, we hypothesize that MAPKs may not play prominent role in the regulation of microtubule dynamics in all plant species

The Winner Takes It All: Auxin—The Main Player during Plant Embryogenesis

In plants, the first asymmetrical division of a zygote leads to the formation of two cells with different developmental fates. The establishment of various patterns relies on spatial and temporal gene expression, however the precise mechanism responsible for embryonic patterning still needs elucidation. Auxin seems to be the main player which regulates embryo development and controls expression of various genes in a dose-dependent manner. Thus, local auxin maxima and minima which are provided by polar auxin transport underlie cell fate specification. Diverse auxin concentrations in various regions of an embryo would easily explain distinct cell identities, however the question about the mechanism of cellular patterning in cells exposed to similar auxin concentrations still remains open. Thus, specification of cell fate might result not only from the cell position within an embryo but also from events occurring before and during mitosis. This review presents the impact of auxin on the orientation of the cell division plane and discusses the mechanism of auxin-dependent cytoskeleton alignment. Furthermore, close attention is paid to auxin-induced calcium fluxes, which regulate the activity of MAPKs during postembryonic development and which possibly might also underlie cellular patterning during embryogenesis

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

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

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