Hydroxyurea and Caffeine Impact pRb-like Protein-Dependent Chromatin Architecture Profiles in Interphase Cells of Vicia faba.

The survival of cells depends on their ability to replicate correctly genetic material. Cells exposed to replication stress can experience a number of problems that may lead to deregulated proliferation, the development of cancer, and/or programmed cell death. In this article, we have induced prolonged replication arrest via hydroxyurea (HU) treatment and also premature chromosome condensation (PCC) by co-treatment with HU and caffeine (CF) in the root meristem cells of Vicia faba. We have analyzed the changes in the activities of retinoblastoma-like protein (RbS807/811ph). Results obtained from the immunocytochemical detection of RbS807/811ph allowed us to distinguish five unique activity profiles of pRb. We have also performed detailed 3D modeling using Blender 2.9.1., based on the original data and some final conclusions. 3D models helped us to visualize better the events occurring within the nuclei and acted as a high-resolution aid for presenting the results. We have found that, despite the decrease in pRb activity, its activity profiles were mostly intact and clearly recognizable, with some local alterations that may correspond to the increased demand in transcriptional activity. Our findings suggest that Vicia faba's ability to withstand harsh environments may come from its well-developed and highly effective response to replication stress.

Cadmium Stress Reprograms ROS/RNS Homeostasis in Phytophthora infestans (Mont.) de Bary.

Heavy metal pollution causes many soils to become a toxic environment not only for plants, but also microorganisms; however, little is known how heavy metal contaminated environment affects metabolism of phytopathogens and their capability of infecting host plants. In this study the oomycete Phytophthora infestans (Mont.) de Bary, the most harmful pathogen of potato, growing under moderate cadmium stress (Cd, 5 mg/L) showed nitro-oxidative imbalance associated with an enhanced antioxidant response. Cadmium notably elevated the level of nitric oxide, superoxide and peroxynitrite that stimulated nitrative modifications within the RNA and DNA pools in the phytopathogen structures. In contrast, the protein pool undergoing nitration was diminished confirming that protein tyrosine nitration is a flexible element of the oomycete adaptive strategy to heavy metal stress. Finally, to verify whether Cd is able to modify P. infestans pathogenicity, a disease index and molecular assessment of disease progress were analysed indicating that Cd stress enhanced aggressiveness of vr P. infestans towards various potato cultivars. Taken together, Cd not only affected hyphal growth rate and caused biochemical changes in P. infestans structures, but accelerated the pathogenicity as well. The nitro-oxidative homeostasis imbalance underlies the phytopathogen adaptive strategy and survival in the heavy metal contaminated environment.

In search of the mRNA modification landscape in plants.

BACKGROUND: Precise regulation of gene expression is indispensable for the proper functioning of organisms in both optimal and challenging conditions. The most commonly known regulative mechanisms include the modulation of transcription, translation and adjustment of the transcript, and protein half-life. New players have recently emerged in the arena of gene expression regulators - chemical modifications of mRNAs. MAIN TEXT: The latest studies show that modified ribonucleotides affect transcript splicing, localization, secondary structures, interaction with other molecules and translation efficiency. Thus far, attention has been focused mostly on the most widespread mRNA modification - adenosine methylation at the N6 position (m6A). However, initial reports on the formation and possible functions of other modified ribonucleotides, such as cytosine methylated at the 5' position (m5C), 8-hydroxyguanosine (8-OHG) and 8-nitroguanosine (8-NO2G), have started to appear in the literature. Additionally, some reports indicate that pseudouridine (Ψ) is present in mRNAs and might perform important regulatory functions in eukaryotic cells. The present review summarizes current knowledge regarding the above-mentioned modified ribonucleotides (m6A, m5C, 8-OHG, 8-NO2G) in transcripts across various plant species, including Arabidopsis, rice, sunflower, wheat, soybean and potato. CONCLUSIONS: Chemical modifications of ribonucleotides affect mRNA stability and translation efficiency. They thus constitute a newly discovered layer of gene expression regulation and have a profound effect on the development and functioning of various organisms, including plants.

Phenylpropanoid pathway metabolites promote tolerance response of lupine roots to lead stress.

Over the past decade, there has been increasing interest in the role of phenolic compounds, especially flavonoids in plants in response to heavy metal stress. In this study, it was found that treatment of yellow lupine (Lupinus luteus L.) with Pb (150mg/l Pb(NO3)2) increased flavonoid contents in both cotyledons (by ca. 67%) and roots (by ca. 54%). Moreover, seedling roots preincubated with flavonoid extracts, derived from Pb-treated lupine cotyledons, exhibited enhanced tolerance to the heavy metal. Flavonoid preincubated lupine seedlings, growing for 48h in the presence of Pb(NO3)2, showed mitigated symptoms of lead stress, which was manifested by a significant increase in the root length and its biomass. Additionally, in seedlings pretreated with the natural flavonoid preparations an impressive rise of the antioxidant capacity was observed. Simultaneously, root cells exhibited reduced accumulation of both H2O2 and O2(-), which was associated with the decreased TBARS content and the number of dying cells under Pb stress. Taken together, accumulation of flavonoids could be an effective event in the plant׳s spectrum of defense responses to heavy metal stress, and the protective role of flavonoids against heavy metals might be associated with their ability to scavenge reactive oxygen species overproduced under lead stress.

Nitric oxide-mediated stress imprint in potato as an effect of exposure to a priming agent.

We investigated how potato exposed to a chemical agent could activate nitric oxide (NO)-dependent events facilitating more potent defense responses to a subsequent pathogen attack. Obtained data revealed that all applied inducers, i.e., ß-aminobutyric acid (BABA), γ-aminobutyric acid (GABA), laminarin, or 2,6-dichloroisonicotinic acid (INA), were active stimuli in potentiating NO synthesis in the primed potato. It is assumed, for the mechanism proposed in this paper, that priming involves reversible S-nitrosylated protein (S-nitrosothiols [SNO]) storage as one of the short-term stress imprint components, apart from epigenetic changes sensitized by NO. Based on BABA- and GABA-induced events, it should be stated that a rise in NO generation and coding the NO message in SNO storage at a relatively low threshold together with histone H2B upregulation might create short-term imprint activation, facilitating acquisition of a competence to react faster after challenge inoculation. Laminarin elicited strong NO upregulation with an enhanced SNO pool-altered biochemical imprint in the form of less effective local recall, nevertheless being fully protective in distal responses against P. infestans. In turn, INA showed the most intensified NO generation and abundant formation of SNO, both after the inducer treatment and challenge inoculation abolishing potato resistance against the pathogen. Our results indicate, for the first time, that a precise control of synthesized NO in cooperation with reversible SNO storage and epigenetic modifications might play an important role in integrating and coordinating defense potato responses in the priming phenomenon.

Estimation of the Level of Abasic Sites in Plant mRNA Using Aldehyde Reactive Probe.

Oxidation of RNA is associated with the development of numerous disorders including Alzheimer's and Parkinson's diseases, amyotrophic lateral sclerosis (ALS), cancer, and diabetes. Additionally, a correlation has been found between increase in RNA oxidation and the process of aging. In plants, elevated level of oxidatively modified transcripts has been detected during alleviation of seeds dormancy and stress response. Increasing interest on the topic of RNA oxidative modifications requires elaboration of new laboratory techniques. So far, the most common method used for the assessment of RNA oxidation is quantification of 8-hydroxyguanine (8-OHG). However, reactive oxygen species (ROS) induce also numerous other changes in nucleic acids, including formation of abasic sites (AP-sites). Recently, the level of AP-sites in RNA has been measured with the use Aldehyde Reactive Probe (ARP). In the present chapter, we describe application of this technique for the evaluation of the level of AP-sites in plant transcripts.

Mitochondria Specific Antioxidant, MitoTEMPO, Modulates Cd Uptake and Oxidative Response of Soybean Seedlings.

Numerous reports find that Cd induces formation of reactive oxygen species (ROS) in plants. However, a general ROS pool is usually studied, without distinction of their production site. In the present study, we applied a mitochondria-specific antioxidant, MitoTEMPO, to elucidate the role of mitochondria-derived ROS in the response of soybean seedlings to short-term (48 h) Cd stress. The obtained results showed that Cd caused a reduction in root length and fresh weight and increase in the level of superoxide anion, hydrogen peroxide, markers of lipid peroxidation (thiobarbituric reactive substances, TBARS) and markers of RNA oxidation (8-hydroxyguanosine, 8-OHG) in seedling roots. Application of MitoTEMPO affected Cd uptake in a dose-dependent manner and diminished the Cd-dependent induction of superoxide anion and lipid peroxidation.

Plant Recovery after Metal Stress-A Review.

Contamination of the environment with metals, their adverse impact on plant performance and transmission to the human food chain through crops and vegetables are important concerns worldwide. Although the literature on metal contamination, toxicity and plant response to this stress factor is quite abundant, there are very limited reports on the phenomenon of plant recovery after metal stress. The present article reviews available literature on the recovery process examined in various plant species, in response to several metals (Al, Cd, Cu, Ni, Pb, Zn), applied at different concentrations and treatment duration. The reviewed studies have been carried out in laboratory conditions. However, it should be highlighted that although metal stress is not as transient as most of other stress factors (e.g., drought, heat, chilling), metal concentration in the soil may still decrease due to, e.g., leaching to lower soil layers or uptake by organisms. Thus, in natural conditions, plants may be subjected to post-metal-stress conditions. The review also discusses the mechanism behind efficient recovery and the impact of post metal stress on future plant performance-possible acquisition of stress memory, adaptation to unfavorable conditions and cross-tolerance towards other stress factors.

Oxidative RNA Modifications as an Early Response of Soybean (Glycine max L.) Exposed to Copper and Lead.

Plant exposure to metals is associated with the accumulation of reactive oxygen species, which mediate the oxidation of various molecules including lipids, proteins, and nucleic acids. The aim of the present study is the evaluation of the impact of short-term Cu and Pb treatment on oxidative events in the roots of soybean seedlings, with special emphasis on RNA oxidation. The results show that an increase in total RNA oxidative modification, 8-hydroxyguanosine (8-OHG), constitutes a very early response to both applied metals, observed already within the first hour of treatment. Exposure to Cu and Pb resulted also in the increase in superoxide anion and hydrogen peroxide levels and intensified lipid peroxidation. However, these responses were most prominent after longer treatment times. On the other hand, no changes were observed in the level of protein carbonylation. It can be concluded that 8-OHG enrichment in total RNA constitutes one of the earliest reactions to metals, which precedes the symptoms of oxidative stress.

The Recovery of Soybean Plants after Short-Term Cadmium Stress.

BACKGROUND: Cadmium is a non-essential heavy metal, which is toxic even in relatively low concentrations. Although the mechanisms of Cd toxicity are well documented, there is limited information concerning the recovery of plants after exposure to this metal. METHODS: The present study describes the recovery of soybean plants treated for 48 h with Cd at two concentrations: 10 and 25 mg/L. In the frame of the study the growth, cell viability, level of membrane damage makers, mineral content, photosynthesis parameters, and global methylation level have been assessed directly after Cd treatment and/or after 7 days of growth in optimal conditions. RESULTS: The results show that exposure to Cd leads to the development of toxicity symptoms such as growth inhibition, increased cell mortality, and membrane damage. After a recovery period of 7 days, the exposed plants showed no differences in relation to the control in all analyzed parameters, with an exception of a slight reduction in root length and changed content of potassium, magnesium, and manganese. CONCLUSIONS: The results indicate that soybean plants are able to efficiently recover even after relatively severe Cd stress. On the other hand, previous exposure to Cd stress modulated their mineral uptake.

BABA-Primed Histone Modifications in Potato for Intergenerational Resistance to Phytophthora infestans.

In this paper we analyzed ß-aminobutyric acid (BABA)-primed epigenetic adjustment of potato cv. "Sarpo Mira" to Phytophthora infestans. The first stress-free generation of the potato genotype obtained from BABA-primed parent plants via tubers and seeds showed pronounced resistance to the pathogen, which was tuned with the transcriptional memory of SA-responsive genes. During the early priming phase before the triggering stress, we found robust bistable deposition of histone marks (H3K4me2 and H3K27me3) on the NPR1 (Non-expressor of PR genes) and the SNI1 gene (Suppressor of NPR1, Inducible), in which transcription antagonized silencing. Switchable chromatin states of these adverse systemic acquired resistance (SAR) regulators probably reprogrammed responsiveness of the PR1 and PR2 genes and contributed to stress imprinting. The elevated levels of heritable H3K4me2 tag in the absence of transcription on SA-dependent genes in BABA-primed (F0) and its vegetative and generative progeny (F1) before pathogen challenge provided evidence for the epigenetic mark for intergenerational memory in potato. Moreover, our study revealed that histone acetylation was not critical for maintaining BABA-primed defense information until the plants were triggered with the virulent pathogen when rapid and boosted PRs gene expression probably required histone acetyltransferase (HAT) activity both in F0 and F1 progeny.

Cadmium Stress Leads to Rapid Increase in RNA Oxidative Modifications in Soybean Seedlings.

Increase in the level of reactive oxygen species (ROS) is a common response to stress factors, including exposure to metals. ROS over-production is associated with oxidation of lipids, proteins, and nucleic acids. It is suggested that the products of oxidation are not solely the markers of oxidative stress but also signaling elements. For instance, it has been shown in animal models that mRNA oxidation is a selective process engaged in post-transcriptional regulation of genes expression and that it is associated with the development of symptoms of several neurodegenerative disorders. In the present study, we examined the impact of short-term cadmium (Cd) stress on the level of two RNA oxidation markers: 8-hydroxyguanosine (8-OHG) and apurinic/apyrimidinic sites (AP-sites, abasic sites). In the case of 8-OHG, a significant increase was observed after 3 h of exposure to moderate Cd concentration (10 mg/l). In turn, high level of AP-sites, accompanied by strong ROS accumulation and lipid peroxidation, was noted only after 24 h of treatment with higher Cd concentration (25 mg/l). This is the first report showing induction of RNA oxidations in plants response to stress factors. The possible signaling and gene regulatory role of oxidatively modified transcripts is discussed.

Proteins induced by cadmium in soybean cells.

The cadmium (Cd)-induced changes in protein pattern and identification of metal-stimulated polypeptides were analyzed in soybean cell suspension culture. The cell cultures were treated with various concentrations of Cd(2+) (3-10microM) for 24, 48 and 72h. The synthesis of [(35)S]-labeled proteins and their accumulation were analyzed by SDS-PAGE, whereas the identification of selected protein bands was performed by mass spectrometry. It is shown that Cd induced the appearance of the following proteins in soybean cells: superoxide dismutase, histone H2B, chalcone synthase and glutathione transferase.

Products of lipid, protein and RNA oxidation as signals and regulators of gene expression in plants.

Reactive oxygen species (ROS) are engaged in several processes essential for normal cell functioning, such as differentiation, anti-microbial defense, stimulus sensing and signaling. Interestingly, recent studies imply that cellular signal transduction and gene regulation are mediated not only directly by ROS but also by the molecules derived from ROS-mediated oxidation. Lipid peroxidation leads to non-enzymatic formation of oxylipins. These molecules were shown to modulate expression of signaling associated genes including genes encoding phosphatases, kinases and transcription factors. Oxidized peptides derived from protein oxidation might be engaged in organelle-specific ROS signaling. In turn, oxidation of particular mRNAs leads to decrease in the level of encoded proteins and thus, contributes to the post-transcriptional regulation of gene expression. Present mini review summarizes latest findings concerning involvement of products of lipid, protein and RNA oxidation in signal transduction and gene regulation.

The new insights into cadmium sensing.

Cadmium (Cd) is non-essential heavy metal, which in excess, exhibits deleterious effects to the most of the organisms. Mobilization of defense mechanisms against this toxic agent requires rapid activation of signaling pathways. The article presents recent advances in the research concerning cadmium signal transduction in plants. New insights into the involvement of reactive oxygen species (ROS), nitric oxide (NO), plant growth regulators, and Cd-induced protein modifications are reviewed. Moreover, the role of recently recognized Cd-associated signal elements, including micro RNAs and several cis- and trans-acting elements is discussed.

Nitric oxide mediates Cd-dependent induction of signaling- associated genes.

The first reaction of plants to stress factors, including cadmium, is activation of signal transduction pathways leading to the mobilization of defense mechanisms. In a recent study we have demonstrated that cadmium causes a rapid induction of several genes associated with cellular signaling in soybean seedlings. Here we show that nitric oxide can be an important signaling molecule mediating observed increase in genes expression under short-term cadmium stress.

The toxic Doppelganger: on the ionic and molecular mimicry of cadmium.

Cadmium is a toxic heavy metal which can cause numerous alterations in cell functioning. Exposure to cadmium leads to generation of reactive oxygen species, disorders in membrane structure and functioning, inhibition of respiration, disturbances in ion homeostasis, perturbations in cell division, and initiation of apoptosis and necrosis. This heavy metal is considered a carcinogen by the Agency for Toxic Substances and Disease Registry. At least some of the described toxic effects could result from the ability of cadmium to mimic other divalent ions and alert signal transduction networks. This review describes the role of cadmium mimicry in its uptake, reactive oxygen species generation, alterations in calmodulin, Wnt/ß-catenin and estrogen signaling pathways, and modulation of neurotransmission. The last section is dedicated to the single known case of a favorable function performed by cadmium mimicry: marine diatoms, which live in zinc deficient conditions, utilize cadmium as a cofactor in carbonic anhydrase - so far the only described cadmium enzyme.

Short term signaling responses in roots of young soybean seedlings exposed to cadmium stress.

In the present study, the expression of fourteen genes involved in various signal transduction pathways was examined in young soybean (Glycine max) seedlings exposed to cadmium at two concentrations (10 mg L(-1) and 25 mg L(-1)) for short time periods (3, 6 and 24 h). The results show that cadmium causes induction of genes encoding proteins involved in ethylene and polyamines metabolism, nitric oxide generation, MAPK cascades and regulation of other genes' expression. The bioinformatic analysis of promoter sequences of Cd-inducible genes revealed that their promoters possess several regulative motifs associated with the plant response to stress factors and abscisic acid and ethylene signaling. The involvement of ethylene in the response of soybean seedlings to cadmium stress was further confirmed by the real-time analysis of ethylene production during 24 h of CdCl2 treatment. The role of the described signaling elements in transduction of the cadmium signal in young soybean seedlings is discussed.

A common response to common danger? Comparison of animal and plant signaling pathways involved in cadmium sensing.

Exposure to cadmium results in disturbances in cell homeostasis in all living organisms. The first response to stress factors, including cadmium, is activation of signal transduction pathways that mobilize cell defense mechanisms. The aim of this review is a comparison between the signaling network triggered by Cd in plants and animals. Despite differences in the structure and physiology of plant and animal cells, their cadmium signal transduction pathways share many common elements. These elements include signaling molecules such as ROS, Ca(2+) and NO, the involvement of phospholipase C, mitogen-activated protein kinase cascades, and activation of transcription factors. Undoubtedly, both animals and plants also possess specific signaling pathways. In case of animals, Wnt/ß-catenin, sonic hedgehog and oestorgen signaling are engaged in the transduction of cadmium signal. Plant specific signal transduction pathways include signaling mediated by plant hormones. The role of ethylene and jasmonic, salicylic and abscisic acid in plant response to cadmium is also discussed.

Nitric oxide implication in cadmium-induced programmed cell death in roots and signaling response of yellow lupine plants.

The sequence of events leading to the programmed cell death (PCD) induced by heavy metals in plants is still the object of extensive investigation. In this study we showed that roots of 3-day old yellow lupine (Lupinus luteus L.) seedlings exposed to cadmium (Cd, 89µM CdCl(2)) resulted in PCD starting from 24h of stress duration, which was evidenced by TUNEL-positive reaction. Cd-induced PCD was preceded by a relatively early burst of nitric oxide (NO) localized mainly in the root tips. Above changes were accompanied by the NADPH-oxidase-dependent superoxide anion (O(2)(·-)) production. However, the concomitant high level of both NO and O(2)(·-) at the 24th h of Cd exposure did not provoke an enhanced peroxynitrite formation. The treatment with the NADPH-oxidase inhibitor and NO-scavenger significantly reduced O(2)(·-) and NO production, respectively, as well as diminished the pool of cells undergoing PCD. The obtained data indicate that boosted NO and O(2)(·-) production is required for Cd-induced PCD in lupine roots. Moreover, we found that in roots of 14-day old lupine plants the NO-dependent Cd-induced PCD was correlated with the enhanced level of the post-stress signals in leaves, including distal NO cross-talk with hydrogen peroxide.