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In the present work, Petunia × hybrida leaf discs maintained on regeneration medium for 8 days were used to assess the effects of genotoxic stress induced by in vitro culture. The investigation was carried out by comparing the response of intact leaves excised from Petunia × hybrida plantlets grown in vitro and the regenerating leaf discs. In situ...
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Apoptosis, also known as programmed cell death is a highly regulated and fundamental process found in all multicellular organisms. It is not only implicated in regulatory mechanisms of cells, but has been attributed to a number of diseases, like inflammation, malignancy, autoimmunity and neuro-degeneration. A variability of pollutants can persuade...
Citations
... The single-cell gel electrophoresis (SCGE) or Comet Assay, was performed to detect and quantify DNA damage in 20-day-old A. thaliana plants as described by Ventura, Macovei, et al. (2013). The protocol consists of four steps: preparation of precoated slides, nuclei isolation, in situ lysis and microscope analysis. ...
Genotoxic stress activates the DNA‐damage response (DDR) signalling cascades responsible for maintaining genome integrity. Downstream DNA repair pathways include the tyrosyl‐DNA phosphodiesterase 1 (TDP1) enzyme that hydrolyses the phosphodiester bond between the tyrosine of topoisomerase I (TopI) and 3′‐phosphate of DNA. The plant TDP1 subfamily contains the canonical TDP1α gene and the TDP1β gene whose functions are not fully elucidated. The current study proposes to investigate the involvement of TDP1 genes in DDR‐related processes by using Arabidopsis thaliana mutants treated with genotoxic agents. The phenotypic and molecular characterization of tdp1α, tdp1β and tdp1α/β mutants treated with cisplatin (CIS), curcumin (CUR), NSC120686 (NSC), zeocin (ZEO), and camptothecin (CPT), evidenced that while tdp1β was highly sensitive to CIS and CPT, tdp1α was more sensitive to NSC. Gene expression analyses showing upregulation of the TDP2 gene in the double mutant indicate the presence of compensatory mechanisms. The downregulation of POL2A gene in the tdp1β mutant along with the upregulation of the TDP1β gene in pol2a mutants, together with its sensitivity to replication inhibitors (CIS, CTP), point towards a function of this gene in the response to replication stress. Therefore, this study brings novel information relative to the activity of TDP1 genes in plants.
... Seeds were harvested at the indicated time points, embryo axes (primed seeds) and embryos with radicle protrusion (overprimed seeds) were isolated from the cotyledons and seed coat using a razor blade. Nuclei were extracted as previously described (Ventura et al., 2014). The resulting suspension containing purified nuclei was mixed in equal volume with a solution containing 1% low melting point (Sigma-Aldrich) agarose in phosphate-buffered saline buffer (PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 ) maintained at 38°C. ...
Re‐establishment of desiccation tolerance is essential for the survival of germinated seeds facing water deficit in the soil. The molecular and ultrastructural features of desiccation tolerance maintenance and loss within the nuclear compartment are not fully resolved. In the present study, the impact of desiccation‐induced genotoxic stress on nucleolar ultrastructure and ribogenesis was explored along the rehydration−dehydration cycle applied in standard seed vigorization protocols. Primed and overprimed Medicago truncatula seeds, obtained through hydropriming followed by desiccation (dry‐back), were analysed. In contrast to desiccation‐tolerant primed seeds, overprimed seeds enter irreversible germination and do not survive dry‐back. Reactive oxygen species, DNA damage and expression profiles of antioxidant/DNA Damage Response genes were measured, as main hallmarks of the seed response to desiccation stress. Nuclear ultrastructural features were also investigated. Overprimed seeds subjected to dry‐back revealed altered rRNA accumulation profiles and up‐regulation of genes involved in ribogenesis control. The signal molecule PAP (3′‐phosphoadenosine 5′‐phosphate) accumulated during dry‐back only in primed seeds, as a distinctive feature of desiccation tolerance. The presented results show the molecular and ultrastructural landscapes of the seed desiccation response, including substantial changes in nuclear organization.
... The alkaline version of the comet assay measures mostly single-strand breaks (SSBs) formed from alkali-labile sites, but DNA-DNA or DNA-protein crosslinks may be present as well (Collins 2004). Nuclei were extracted from treated and untreated P. hybrida seedlings as previously described (Ventura et al. 2014). The resulting suspension containing purified nuclei was mixed in equal volume with a solution containing 1% low melting point (LMP) agarose in phosphate-buffered saline buffer (PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 ) maintained at 38 °C. ...
... For each oligonucleotide set, a no-template control was used. The TUBULIN (TUB) and UBIQUITIN (UBI) genes were used as reference (Donà et al. 2013;Ventura et al. 2014). The gene-specific oligonucleotide primers, designed using Primer3 (https ://prime r3.ut. ...
Main conclusion
Sodium butyrate applied to Petunia hybrida seeds under a long-day photoperiod has a negative impact (reduced seedling length, decreased production of photosynthetic pigments, and accumulation of DNA damage) on early seedling development, whereas its administration under dark/light conditions (complete dark conditions for 5 days followed by exposure to long-day photoperiod for 5 days) bypasses some of the adverse effects.
Abstract
Genotoxic stress impairs plant development. To circumvent DNA damage, plants activate DNA repair pathways in concert with chromatin dynamics. These are essential during seed germination and seedling establishment, and may be influenced by photoperiod variations. To assess this interplay, an experimental design was developed in Petunia hybrida, a relevant horticultural crop and model species. Seeds were treated with different doses of sodium butyrate (NaB, 1 mM and 5 mM) as a stress agent applied under different light/dark conditions throughout a time period of 10 days. Phenotypic (germination percentage and speed, seedling length, and photosynthetic pigments) and molecular (DNA damage and gene expression profiles) analyses were performed to monitor the response to the imposed conditions. Seed germination was not affected by the treatments. Seedling development was hampered by increasing NaB concentrations applied under a long-day photoperiod (L) as reflected by the decreased seedling length accompanied by increased DNA damage. When seedlings were grown under dark conditions for 5 days and then exposed to long-day photoperiod for the remaining 5 days (D/L), the damaging effects of NaB were circumvented. NaB exposure under L conditions resulted in enhanced expression of HAT/HDAC (HISTONE ACETYLTRANSFERASES/HISTONE DEACTEYLASES) genes along with repression of genes involved in DNA repair. Differently, under D/L conditions, the expression of DNA repair genes was increased by NaB treatment and this was associated with lower levels of DNA damage. The observed DNA damage and gene expression profiles suggest the involvement of chromatin modification- and DNA repair-associated pathways in response to NaB and dark/light exposure during seedling development.
... A DNA diffusion assay was performed on M. truncatula calli treated with the above mentioned NSC120686 concentrations to evaluate cell death events, namely programmed cell death (PCD) and necrosis. Nuclei were extracted as previously described [18,19]. Agarose precoated slides were prepared by spreading 1% agarose (1 mL) onto previously degreased slides and dried overnight at room temperature. ...
... For single cell gel electrophoresis (SCGE), also known as comet assay, nuclei were extracted from M. truncatula treated and untreated calli as previously described [18,19]. The resulting suspension containing purified nuclei was mixed in equal volume with a solution containing 1% LMP agarose in PBS buffer maintained at 38 • C. Two drops of the resulting suspension were then pipetted onto agarose-precoated slides and solidified on ice. ...
The hTdp1 (human tyrosyl-DNA phosphodiesterase 1) inhibitor NSC120686 has been used, along with topoisomerase inhibitors, as a pharmacophoric model to restrain the Tdp1 activity as part of a synergistic treatment for cancer. While this compound has an end-point application in medical research, in plants, its application has not been considered so far. The originality of our study consists in the use of hTdp1 inhibitor in Medicago truncatula cells, which, unlike human cells, contain two Tdp1 genes. Hence, the purpose of this study was to test the hTdp1 inhibitor NSC120686 as an exploratory tool to investigate the plant Tdp1 genes, since their characterization is still in incipient phases. To do so, M. truncatula calli were exposed to increasing (75, 150, 300 μM) concentrations of NSC120686. The levels of cell mortality and DNA damage, measured via diffusion assay and comet assay, respectively, were significantly increased when the highest doses were used, indicative of a cytotoxic and genotoxic threshold. In addition, the NSC120686-treated calli and untreated MtTdp1α-depleted calli shared a similar response in terms of programmed cell death (PCD)/necrosis and DNA damage. Interestingly, the expression profiles of MtTdp1α and MtTdp1β genes were differently affected by the NSC120686 treatment, as MtTdp1α was upregulated while MtTdp1β was downregulated. The NSC120686 treatment affected not only the MtTdp1 genes but also other genes with roles in alternative DNA repair pathways. Since the expression patterns of these genes were different than what was observed in the MtTdp1α-depleted plants, it could be hypothesized that the NSC120686 treatment exerts a different influence compared to that resulting from the lack of the MtTdp1α gene function.
... More importantly, we adjusted the protocol to be suitable for the use of different types of plant materials, from fresh leaves to calli and cell suspension cultures. The protocol described in this section was applied to fresh leaves from different plant species, among which 4 Medicago truncatula [15,16], Petunia hybrida [17], and Oryza sativa [18], as well as to calli (unpublished data) and cell suspension cultures [19]. ...
DNA diffusion assay is a simple, sensitive and reliable technique which allows the assessment of programmed cell death (PCD) or necrosis events based on nuclear morphology. It consists in isolating nuclei from plant material, which are then embedded in agarose and subjected to lysis in alkaline buffers. Under these conditions, and due to the presence of abundant alkali-labile sites in the DNA, small pieces of DNA diffuse in the agarose gel giving a specific halo appearance when stained with fluorescent dyes like DAPI (4′,6-diamidino-2-phenylindole). Here, we describe an optimized protocol for DNA diffusion assay applied to different types of plant cells/tissues, indicating all the critical steps required for a successful experimental procedure.
... The DNA diffusion test was subsequently used to discriminate between programmed cell death (PCD) and necrosis events in saponin-treated white poplar cells. In case of PCD, nuclei show an undefined outline, due to nucleosomal sized DNA diffusing into the agarose, while necrotic nuclei appear bigger and non homogeneous and viable nuclei were small and compact (Ventura et al., 2013 ). The percentage of viable, necrotic and PCD nuclei was calculated at 0, 6 and 24 h following saponin addition. ...
Introduction
Several molecular aspects underlying the seed response to priming and the resulting vigor profile are still poorly understood. Mechanisms involved in genome maintenance deserve attention since the balance between stimulation of germination and DNA damage accumulation versus active repair is a key determinant for designing successful seed priming protocols.
Methods
Changes in the Medicago truncatula seed proteome were investigated in this study, using discovery mass spectrometry and label-free quantification, along the rehydration-dehydration cycle of a standard vigorization treatment (hydropriming plus dry-back), and during post-priming imbibition.
Resuts and discussion
From 2056 to 2190 proteins were detected in each pairwise comparison, among which six were differentially accumulated and 36 were detected only in one condition. The following proteins were selected for further investigation: MtDRP2B (DYNAMIN-RELATED PROTEIN), MtTRXm4 (THIOREDOXIN m4), and MtASPG1 (ASPARTIC PROTEASE IN GUARD CELL 1) showing changes in seeds under dehydration stress; MtITPA (INOSINE TRIPHOSPHATE PYROPHOSPHORYLASE), MtABA2 (ABSCISIC ACID DEFICIENT 2), MtRS2Z32 (SERINE/ARGININE-RICH SPLICING FACTOR RS2Z32), and MtAQR (RNA HELICASE AQUARIUS) that were differentially regulated during post-priming imbibition. Changes in the corresponding transcript levels were assessed by qRT-PCR. In animal cells, ITPA hydrolyses 2’-deoxyinosine triphosphate and other inosine nucleotides, preventing genotoxic damage. A proof of concept was performed by imbibing primed and control M. truncatula seeds in presence/absence of 20 mM 2’-deoxyinosine (dI). Results from comet assay highlighted the ability of primed seeds to cope with dI-induced genotoxic damage. The seed repair response was assessed by monitoring the expression profiles of MtAAG (ALKYL-ADENINE DNA GLYCOSILASE) and MtEndoV (ENDONUCLEASE V) genes that participate in the repair of the mismatched I:T pair in BER (base excision repair) and AER (alternative excision repair) pathways, respectively.
Programmed cell death (PCD) is an essential component of development, biotic and abiotic responses. Hypersensitive response (HR)-associated cell death activated under pathogen attack is one of the most dramatic manifestations of PCD in plants. Signal transduction through mitogen-activated protein kinase (MAPK) cascades, a very conserved signaling pathway across eukaryotes, is a core mediator for HR-associated PCD. Therefore, monitoring MAPK activation enables the mechanisms underlying HR-associated PCD to be elucidated. Here, we describe the use of a phosphorylation-specific MAPK3/6 antibody to monitor the activation of MAPK3/6 during HR-associated PCD. The technique may be adapted for use in other types of PCD.
Aside from the great importance of Leguminosae in food and agriculture industry, legume model systems like Medicago truncatula are also essential tools to dissect complex cellular pathways and retrieve valuable information to other crops. Here, we investigated the roles played by the tyrosyl-DNA phosphodiesterase 2α (MtTdp2α) gene in cell viability and proliferation using M. truncatula suspension cultures. Our research hypothesis is that the overexpression of MtTdp2α, implicated in the removal of transient topoisomerase/DNA covalent complexes, can impact on cell suspension viability. M. truncatula suspension cultures derived from leaf explants of MtTdp2α-overexpressing lines and a control line carrying the empty vector were used. Our results showed that the control line reached the stationary growth phase by the fourth day of culture while the transgenic lines presented an extended exponential growth, reaching the stationary phase at day six following culture. The MtTdp2α-overexpressing lines also showed increased viability as compared to the control line. The transcript levels of MtSOD, MtAPX, MtMT2, MtMRE11, MtNBS1, MtRad50, MtOGG1 and MtFPG were significantly enhanced in the transgenic lines as compared to control. Overall, our results show that the MtTdp2α overexpression impacts in a positive manner on cell viability and proliferation in suspension cultures. Additionally, our study provides an insight on the suitability of M. truncatula cell suspension cultures as a promising alternative to evaluate potential protective mechanisms, with results comparable to those obtained when using whole plants.
