Cadmium (Cd) induces oxidative stress and disrupts nuclear organization and chromatin-associated metabolic processes in plant cells. Therefore, identifying natural, biodegradable, non-bioaccumulative compounds that enhance plant tolerance to heavy metals is crucial. We hypothesized that Cd exposure (175 µM CdCl₂, 24 h) activates mitogen-activated protein kinases (MAPKs), triggering defined epigenetic modifications that lead to transcriptional repression, and that thyme oil (TO; 0.03% (v/v), emulsified) mitigates these effects by stabilizing chromatin organization. We analyzed nuclear MAPK (p44/42) activation, global DNA methylation (5-methylcytosine; 5-mC), and selected histone modifications as key components of early stress signaling and epigenetic regulation. We found that Cd exposure doubled global 5-mC levels and caused pronounced alterations in histone marks, including decreases in H3K4Me2 (~34%), H3T45Ph (~48%), and H4K5Ac, accompanied by strong increases in H3K9Ac (~57%) and H3K56Ac (~148%). These changes were associated with chromatin condensation and reduced transcriptional activity. In contrast, co-treatment with TO maintained MAPK activity and epigenetic parameters close to control levels, preventing chromatin compaction and transcriptional repression. Together, these findings indicate that TO stabilizes the nuclear signaling–epigenetic interface under Cd stress and represents a promising bioprotective strategy. This work provides the first demonstration that TO modulates both MAPK activation and Cd-induced histone modifications in plants.

Cadmium is considered one of the most toxic and dangerous environmental agents among heavy metals. It induces oxidative stress state, which is a source of disruption to a wide range of key cellular processes in plants. The increasing problem of heavy metal accumulation in the environment necessitates the search for strategies that enhance plant resistance to the toxic effects of these elements. Therefore, the purpose of this study was to evaluate the protective potential of thyme oil (TO) against 175 µM cadmium chloride (CdCl₂)-induced toxicity in meristematic cells of Vicia faba var. minor primary roots under in vitro conditions. To assess the impact of TO, we analyzed reactive oxygen species (ROS) production, as cadmium-induced oxidative stress can trigger cellular damage. Hydrogen peroxide (H₂O₂) was detected using 3,3′-diaminobenzidine (DAB) staining, while superoxide anion (O₂^•−) was visualized via Nitro Blue Tetrazolium (NBT) reduction. Since excessive ROS can lead to DNA damage, impair replication, and alter the proportion of interphase cells, we performed immunofluorescence labeling to examine replicating cells using a DNA synthesis marker (IdU). Additionally, we quantified disruptions in DNA replication and assessed the level of DNA damage using γ-phosphorylation of histone H2A.X at serine 139, a marker of DNA double-strand breaks (DSBs). Our results demonstrated that TO significantly mitigated cadmium-induced oxidative and genotoxic stress. Notably, TO treatment reduced the percentage of γ-H2A.X-positive cells by approximately 25% compared to cadmium-only treatment. Moreover, a 41,5% decrease in in the intensity of NBT staining (indicative of superoxide accumulation) and a 72,45% reduction in DAB staining intensity (reflecting hydrogen peroxide levels) were observed. These findings indicate that TO application may serve as a promising strategy for protecting plant tissues exposed to cadmium contamination in the future.

Environmental concerns regarding synthetic herbicides have sparked interest in plant-derived bioactive compounds as eco-friendly alternatives. This study investigated the cellular targets of sweet flag essential oil (Acorus calamus L., SEO at IC₅₀ concentration) in root meristem cells of Fabaceae (Vicia faba, Lupinus luteus) and Brassicaceae (Brassica napus, Arabidopsis thaliana), focusing on reactive oxygen species (ROS) accumulation (DAB, NBT staining), DNA replication dynamics (EdU labeling), and genome integrity (γ-H2AX immunocytochemistry, TUNEL assay, and DNA electrophoresis). SEO induced oxidative stress (200–250% of control depending on the species) and replication stress, causing DNA double-strand breaks in 50% of proliferating cells, confirmed by γ-H2AX/TUNEL. Consequently, cells were prolonged in the G₁ phase, replication activity dropped to 70% of control in Fabaceae and 80% in Brassicaceae, and EdU incorporation intensity decreased to 80% and 70% of control, respectively. An increased proportion of cells replicating heterochromatin indicated slowed S-phase progression. Despite genotoxic effects, SEO did not trigger endoreplication, apoptotic DNA fragmentation, or extensive cell death. All species exhibited a uniform stress response, although sensitivity varied, which previously enabled the establishment of selective SEO doses between Fabaceae and Brassicaceae. These findings suggest that SEO exerts phytotoxicity by disrupting S-phase progression, supporting its potential as a selective bioherbicide.

Essential oils are increasingly recognized as promising agents for sustainable weed control due to their selectivity and complex modes of action. This study evaluated the effects of Acorus calamus essential oil (SEO) on mitosis in two Fabaceae species (Vicia faba, Lupinus luteus) and two Brassicaceae species (Brassica napus, Arabidopsis thaliana) treated with species-specific IC₅₀ concentrations (0.03%, 0.025%, 0.01%, and 0.005%, respectively). Previous research showed that SEO induces oxidative stress and S-phase delay via genome instability. Here, SEO consistently disrupted mitosis across all species, reducing mitotic index by 50–60%, decreasing Cdc2 (CDKA homolog) levels, and causing chromosomal aberrations, including uneven chromatin condensation, sticky chromosomes, bridges, and micronuclei. Cells accumulated in metaphase and exhibited abnormal karyokinetic and cytokinetic spindles. Immunolabeling revealed thick, tightly packed microtubules and actin filaments, indicating excessive stabilization and impaired reorganization. Epigenetic regulation was also affected: H3T3 phosphorylation was abnormally strong, widely distributed, and persistent into anaphase/telophase, while H3S10Ph intensity was weakened. These results suggest that SEO targets multiple components of mitotic machinery and epigenetic control, regardless of species. The observed selectivity depends on dosage, not mechanism. This multi-targeted action may limit the development of plant resistance, supporting the potential of SEO as a bioherbicide in sustainable agriculture.

Cadmium (Cd) contamination, through induction of oxidative stress, severely impairs plant growth. Using primary roots of Vicia faba, we investigated how a 24 h incubation in CdCl₂ solution (175 µM) affects mitotic progression in meristems and assessed whether thyme essential oil (TO; 0.03%, v/v), as a natural antioxidant, can protect proliferating cells during simultaneous Cd exposure. Cd strongly inhibited root growth, reduced mitotic index tenfold (to 0.6%), induced chromatin condensation, decreased CDKA protein levels and CycB transcripts and proteins, caused pronounced microtubule bundling and alterations in their arrangement, disorganization of actin filaments, and disturbances in histone H3 phosphorylation (H3T3Ph, H3S10Ph). TO led to a partial recovery of mitotic index (to ~50% of the control), normalization of chromosome condensation, maintenance of cell-cycle regulators at near-control levels, preservation of proper cytoskeletal organization, and restoration of the correct H3 phosphorylation pattern. This enabled cells to progress from metaphase to anaphase and maintain phase proportions close to the control, resulting in normal root growth. These findings indicate that TO protects the mitotic cellular environment against Cd-induced disturbances. To the best of our knowledge, this is the first evidence that TO safeguards the plant mitotic apparatus under Cd stress, highlighting its potential as a natural bioprotective agent supporting plant growth.