Mercury chloride alters heterochromatin domain organization and nucleolar activity in mouse liver.

Mercury is a highly toxic element that induces severe alterations and a broad range of adverse effects on health. Its exposure is a global concern because it is widespread in the environment due to its multiple industrial, domestic, agricultural and medical usages. Among its various chemical forms, both humans and animals are mainly exposed to mercury chloride (HgCl2), methylmercury and elemental mercury. HgCl2 is metabolized primarily in the liver. We analysed the effects on the nuclear architecture of an increasing dosage of HgCl2 in mouse hepatocytes cell culture and in mouse liver, focusing specifically on the organization, on some epigenetic features of the heterochromatin domains and on the nucleolar morphology and activity. Through the combination of molecular and imaging approaches both at optical and electron microscopy, we show that mercury chloride induces modifications of the heterochromatin domains and a decrease of some histones post-translational modifications associated to heterochromatin. This is accompanied by an increase in nucleolar activity which is reflected by bigger nucleoli. We hypothesized that heterochromatin decondensation and nucleolar activation following mercury chloride exposure could be functional to express proteins necessary to counteract the harmful stimulus and reach a new equilibrium.

Molecular dynamics of seed priming at the crossroads between basic and applied research.

KEY MESSAGE: The potential of seed priming is still not fully exploited. Our limited knowledge of the molecular dynamics of seed pre-germinative metabolism is the main hindrance to more effective new-generation techniques. Climate change and other recent global crises are disrupting food security. To cope with the current demand for increased food, feed, and biofuel production, while preserving sustainability, continuous technological innovation should be provided to the agri-food sector. Seed priming, a pre-sowing technique used to increase seed vigor, has become a valuable tool due to its potential to enhance germination and stress resilience under changing environments. Successful priming protocols result from the ability to properly act on the seed pre-germinative metabolism and stimulate events that are crucial for seed quality. However, the technique still requires constant optimization, and researchers are committed to addressing some key open questions to overcome such drawbacks. In this review, an update of the current scientific and technical knowledge related to seed priming is provided. The rehydration-dehydration cycle associated with priming treatments can be described in terms of metabolic pathways that are triggered, modulated, or turned off, depending on the seed physiological stage. Understanding the ways seed priming affects, either positively or negatively, such metabolic pathways and impacts gene expression and protein/metabolite accumulation/depletion represents an essential step toward the identification of novel seed quality hallmarks. The need to expand the basic knowledge on the molecular mechanisms ruling the seed response to priming is underlined along with the strong potential of applied research on primed seeds as a source of seed quality hallmarks. This route will hasten the implementation of seed priming techniques needed to support sustainable agriculture systems.

Study of Seed Ageing in lpa1-1 Maize Mutant and Two Possible Approaches to Restore Seed Germination.

Phytic acid (PA) is a strong anti-nutritional factor with a key antioxidant role in countering reactive oxygen species. Despite the potential benefits of low phytic acid (lpa) mutants, the reduction of PA causes pleiotropic effects, e.g., reduced seed germination and viability loss related to seed ageing. The current study evaluated a historical series of naturally aged seeds and showed that lpa1-1 seeds aged faster as compared to wildtype. To mimic natural ageing, the present study set up accelerated ageing treatments at different temperatures. It was found that incubating the seeds at 57 °C for 24 h, the wildtype germinated at 82.4% and lpa1-1 at 40%. The current study also hypothesized two possible solutions to overcome these problems: (1) Classical breeding was used to constitute synthetic populations carrying the lpa1-1 mutation, with genes pushing anthocyanin accumulation in the embryo (R-navajo allele). The outcome showed that the presence of R-navajo in the lpa1-1 genotype was not able to improve the germinability (-20%), but this approach could be useful to improve the germinability in non-mutant genotypes (+17%). (2) In addition, hydropriming was tested on lpa1-1 and wildtype seeds, and germination was improved by 20% in lpa1-1, suggesting a positive role of seed priming in restoring germination. Moreover, the data highlighted metabolic differences in the metabolome before and after hydropriming treatment, suggesting that the differences in germination could also be mediated by differences in the metabolic composition induced by the mutation.

Changes in genotoxic stress response, ribogenesis and PAP (3'-phosphoadenosine 5'-phosphate) levels are associated with loss of desiccation tolerance in overprimed Medicago truncatula seeds.

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.

Physiological and molecular aspects of seed longevity: exploring intra-species variation in eight Pisum sativum L. accessions.

Conservation of plant genetic diversity is fundamental for crop improvement, increasing agricultural production and sustainability, especially in the face of climatic changes. Although seed longevity is essential for the management of seed banks, few studies have, so far, addressed differences in this trait among the accessions of a single species. Eight Pisum sativum L. (pea) accessions were investigated to study the impact of long-term (approximately 20 years) storage, aiming to reveal contrasting seed longevity and clarify the causes for these differences. The outstanding seed longevity observed in the G4 accession provided a unique experimental system. To characterize the biochemical and physical status of stored seeds, reactive oxygen species, lipid peroxidation, tocopherols, free proline and reducing sugars were measured. Thermoanalytical measurements (thermogravimetry and differential scanning calorimetry) and transmission electron microscopy combined with immunohistochemical analysis were performed. The long-lived G4 seeds neither consumed tocopherols during storage nor showed free proline accumulation, as a deterioration hallmark, whereas reducing sugars were not affected. Thermal decomposition suggested a biomass composition compatible with the presence of low molecular weight molecules. Expansion of heterochromatic areas and reduced occurrence of γH2AX foci were highlighted in the nucleus of G4 seeds. The longevity of G4 seeds correlates with the occurrence of a reducing cellular environment and a nuclear ultrastructure favourable to genome stability. This work brings novelty to the study of within-species variations in seed longevity, underlining the relevance of multidisciplinary approaches in seed longevity research.

Sodium butyrate induces genotoxic stress in function of photoperiod variations and differentially modulates the expression of genes involved in chromatin modification and DNA repair in Petunia hybrida seedlings.

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. 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.

Metabolic and gene expression hallmarks of seed germination uncovered by sodium butyrate in Medicago truncatula.

Because high-quality seeds are essential for successful crop production in challenging environments, understanding the molecular bases of seed vigour will lead to advances in seed technology. Histone deacetylase inhibitors, promoting histone hyperacetylation, are used as tools to explore aspects still uncovered of the abiotic stress response in plants. The aim of this work was to investigate novel signatures of seed germination in Medicago truncatula, using the histone deacetylase inhibitor sodium butyrate (NaB) as stress agent. NaB-treated and untreated seeds collected at 2 and 8 hr of imbibition and at the radicle protrusion stage underwent molecular phenotyping and nontargeted metabolome profiling. Quantitative enrichment analysis revealed the influence of NaB on seed nucleotide, amino acid, lipid, and carbohydrate metabolism. Up-regulation of antioxidant and polyamine biosynthesis genes occurred in response to NaB. DNA damage evidenced in NaB-treated seeds correlated with up-regulation of base-excision repair genes. Changes in N1 -methyladenosine and N1 -methylguanine were associated with up-regulation of MtALKBH1 (alkylation repair homolog) gene. N2 ,N2 -dimethylguanosine and 5-methylcytidine, tRNA modifications involved in the post-transcriptional regulation of DNA damage response, were also accumulated in NaB-treated seeds at the radicle protrusion stage. The observed changes in seed metabolism can provide novel potential metabolic hallmarks of germination.

Systems biology and genome-wide approaches to unveil the molecular players involved in the pre-germinative metabolism: implications on seed technology traits.

The pre-germinative metabolism is among the most fascinating aspects of seed biology. The early seed germination phase, or pre-germination, is characterized by rapid water uptake (imbibition), which directs a series of dynamic biochemical events. Among those are enzyme activation, DNA damage and repair, and use of reserve storage compounds, such as lipids, carbohydrates and proteins. Industrial seedling production and intensive agricultural production systems require seed stocks with high rate of synchronized germination and low dormancy. Consequently, seed dormancy, a quantitative trait related to the activation of the pre-germinative metabolism, is probably the most studied seed trait in model species and crops. Single omics, systems biology, QTLs and GWAS mapping approaches have unveiled a list of molecules and regulatory mechanisms acting at transcriptional, post-transcriptional and post-translational levels. Most of the identified candidate genes encode for regulatory proteins targeting ROS, phytohormone and primary metabolisms, corroborating the data obtained from simple molecular biology approaches. Emerging evidences show that epigenetic regulation plays a crucial role in the regulation of these mentioned processes, constituting a still unexploited strategy to modulate seed traits. The present review will provide an up-date of the current knowledge on seed pre-germinative metabolism, gathering the most relevant results from physiological, genetics, and omics studies conducted in model and crop plants. The effects exerted by the biotic and abiotic stresses and priming are also addressed. The possible implications derived from the modulation of pre-germinative metabolism will be discussed from the point of view of seed quality and technology.

Depletion of tyrosyl-DNA phosphodiesterase 1α (MtTdp1α) affects transposon expression in Medicago truncatula.

The role of plant tyrosyl-DNA phosphodiesterase 1α in genome stability is studied using a Medicago truncatula MtTdp1α-depleted line. Lack of MtTdp1α results in a 39% reduction of methylated cytosines as compared to control. RNA-Seq analyses revealed that 11 DNA transposons and 22 retrotransposons were differentially expressed in the Tdp1α-2a line. Among them all, DNA transposons (MuDR, hAT, DNA3-11_Mad) and seven retrotransposons (LTR (Long Terminal Repeat)/Gipsy, LTR/Copia, LTR and NonLTR/L1) were down-regulated, while the 15 retrotransposons were up-regulated. Results suggest that the occurrence of stress-responsive cis-elements as well as changes in the methylation pattern at the LTR promoters might be responsible for the enhanced retrotransposon transcription.

Seed priming: state of the art and new perspectives.

Priming applied to commercial seed lots is widely used by seed technologists to enhance seed vigour in terms of germination potential and increased stress tolerance. Priming can be also valuable to seed bank operators who need improved protocols of ex situ conservation of germplasm collections (crop and native species). Depending on plant species, seed morphology and physiology, different priming treatments can be applied, all of them triggering the so-called 'pre-germinative metabolism'. This physiological process takes place during early seed imbibition and includes the seed repair response (activation of DNA repair pathways and antioxidant mechanisms), essential to preserve genome integrity, ensuring proper germination and seedling development. The review provides an overview of priming technology, describing the range of physical-chemical and biological treatments currently available. Optimised priming protocols can be designed using the 'hydrotime concept' analysis which provides the theoretical bases for assessing the relationship between water potential and germination rate. Despite the efforts so far reported to further improve seed priming, novel ideas and cutting-edge investigations need to be brought into this technological sector of agri-seed industry. Multidisciplinary translational research combining digital, bioinformatic and molecular tools will significantly contribute to expand the range of priming applications to other relevant commercial sectors, e.g. the native seed market.

Copper-mediated genotoxic stress is attenuated by the overexpression of the DNA repair gene MtTdp2α (tyrosyl-DNA phosphodiesterase 2) in Medicago truncatula plants.

KEY MESSAGE: Our study highlights the use of the DNA repair gene MtTdp2α as a tool for improving the plant response to heavy metal stress. Tyrosyl-DNA phosphodiesterase 2 (Tdp2), involved in the removal of DNA topoisomerase II-mediated DNA damage and cell proliferation/differentiation signalling in animal cells, is still poorly characterised in plants. The Medicago truncatula lines Tdp2α-13c and Tdp2α-28 overexpressing the MtTdp2α gene and control (CTRL) line were exposed to 0.2 mM CuCl2. The DNA diffusion assay revealed a significant reduction in the percentage of necrosis caused by copper in the aerial parts of the Tdp2α-13c and Tdp2α-28 plants while neutral single cell gel electrophoresis highlighted a significant decrease in double strand breaks (DSBs), compared to CTRL. In the copper-treated Tdp2α-13c and Tdp2α-28 lines there was up-regulation (up to 4.0-fold) of genes encoding the α and ß isoforms of Tyrosyl-DNA phosphodiesterase 1, indicating the requirement for Tdp1 function in the response to heavy metals. As for DSB sensing, the MtMRE11, MtRAD50 and MtNBS1 genes were also significantly up-regulated (up to 2.3-fold) in the MtTdp2α-overexpressing plants grown under physiological conditions, compared to CTRL line, and then further stimulated in response to copper. The basal antioxidant machinery was always activated in all the tested lines, as indicated by the concomitant up-regulation of MtcytSOD and MtcpSOD genes (cytosolic and chloroplastic Superoxide Dismutase), and MtMT2 (type 2 metallothionein) gene. The role of MtTdp2α gene in enhancing the plant response to genotoxic injury under heavy metal stress is discussed.

Genotype-specific germination behavior induced by sustainable priming techniques in response to water deprivation stress in rice.

Water stress brought about by climate change is among the major global concerns threatening food security. Rice is an important staple food which requires high water resources. Being a semi-aquatic plant, rice is particularly susceptible to drought. The aim of this work was to develop techniques directed to promote rice resilience to water deprivation stress during germination by implementing specific seed priming treatments. Five popular Italian rice varieties were subjected to priming treatments using novel, sustainable solutions, like poly-gamma-glutamic acid (γ-PGA), denatured γ-PGA (dPGA), and iron (Fe) pulsing, alone or in combination. The effect of the developed priming methods was tested under optimal conditions as well as under water deprivation stress imposed by polyethylene glycol (PEG) treatments. The priming efficacy was phenotypically determined in terms of germination behavior by measuring a series of parameters (germinability, germination index, mean germination time, seed vigor index, root and shoot length, germination stress tolerance index). Biochemical analyses were carried out to measure the levels of iron uptake and accumulation of reactive oxygen species (ROS). Integrative data analyses revealed that the rice varieties exhibited a strong genotype- and treatment-specific germination behavior. PEG strongly inhibited germination while most of the priming treatments were able to rescue it in all varieties tested except for Unico, which can be defined as highly stress sensitive. Molecular events (DNA repair, antioxidant response, iron homeostasis) associated with the transition from seed to seedling were monitored in terms of changes in gene expression profiles in two varieties sensitive to water deprivation stress with different responses to priming. The investigated genes appeared to be differentially expressed in a genotype-, priming treatment-, stress- and stage-dependent manner. The proposed seed priming treatments can be envisioned as sustainable and versatile agricultural practices that could help in addressing the impact of climate challenges on the agri-food system.

Plant-Based Biostimulants for Seeds in the Context of Circular Economy and Sustainability.

Plant-based biostimulants (PBs), agents rich in bioactive compounds, are emerging as key players able to sustainably improve plant growth and crop productivity to address food security. PBs are generally applied as foliar spray or soil irrigation, while more recently, the application as seed priming treatments is being envisaged as a highly sustainable method to also improve seed quality and germination. Therefore, this review proposes to explore the use of PBs for the seeds industry, specifically discussing about the relevance of product market values, sustainable methods for their production, why and how PBs are used for seed priming, and pinpointing specific strengths and challenges. The collected research studies indicate that PBs applied to seeds result in improved germination, seedling growth, and stress tolerance, although the molecular mechanisms at work are still largely overlooked. The high variability of bioactive molecules and used sources point towards a huge reservoir of nature-based solutions in support of sustainable agriculture practices.

CdSe/ZnS quantum dots trigger DNA repair and antioxidant enzyme systems in Medicago sativa cells in suspension culture.

BACKGROUND: Nanoparticles appear to be promising devices for application in the agriculture and food industries, but information regarding the response of plants to contact with nano-devices is scarce. Toxic effects may be imposed depending on the type and concentration of nanoparticle as well as time of exposure. A number of mechanisms may underlie the ability of nanoparticles to cause genotoxicity, besides the activation of ROS scavenging mechanisms. In a previous study, we showed that plant cells accumulate 3-Mercaptopropanoic acid-CdSe/ZnS quantum dots (MPA-CdSe/ZnS QD) in their cytosol and nucleus and increased production of ROS in a dose dependent manner when exposed to QD and that a concentration of 10 nM should be cyto-compatible. RESULTS: When Medicago sativa cells were exposed to 10, 50 and 100 nM MPA-CdSe/ZnS QD a correspondent increase in the activity of Superoxide dismutase, Catalase and Glutathione reductase was registered. Different versions of the COMET assay were used to assess the genotoxicity of MPA-CdSe/ZnS QD. The number of DNA single and double strand breaks increased with increasing concentrations of MPA-CdSe/ZnS QD. At the highest concentrations, tested purine bases were more oxidized than the pyrimidine ones. The transcription of the DNA repair enzymes Formamidopyrimidine DNA glycosylase, Tyrosyl-DNA phosphodiesterase I and DNA Topoisomerase I was up-regulated in the presence of increasing concentrations of MPA-CdSe/ZnS QD. CONCLUSIONS: Concentrations as low as 10 nM MPA-CdSe/ZnS Quantum Dots are cytotoxic and genotoxic to plant cells, although not lethal. This sets a limit for the concentrations to be used when practical applications using nanodevices of this type on plants are being considered. This work describes for the first time the genotoxic effect of Quantum Dots in plant cells and demonstrates that both the DNA repair genes (Tdp1ß, Top1ß and Fpg) and the ROS scavenging mechanisms are activated when MPA-CdSe/ZnS QD contact M. sativa cells.

RNA-Seq analysis discloses early senescence and nucleolar dysfunction triggered by Tdp1α depletion in Medicago truncatula.

An intron-spliced hairpin RNA approach was used for the targeted silencing of the MtTdp1α gene encoding the αisoform of tyrosyl-DNA phosphodiesterase 1 in Medicago truncatula Gaertn. Tyrosyl-DNA phosphodiesterase 1, involved in the repair of DNA topoisomerase I-mediated DNA damage, has been poorly investigated in plants. RNA-Seq analysis, carried out in the MtTdp1α-depleted plants, revealed different levels of transcriptional modulation (up- and down-regulation, alternative splicing, activation of alternative promoter) in genes involved in DNA damage sensing, DNA repair, and chromatin remodelling. It is suggested that the MtTdp1α gene has new, previously undetected roles in maintaining genome integrity. Up-regulation of senescence-associated genes and telomere shortening were observed. Moreover, impaired ribosome biogenesis indicated that the MtTdp1α gene is required for the nucleolar function. In agreement with the RNA-Seq data, transmission electron microscopy detected an altered nucleolar architecture in the MtTdp1α-depleted cells. Based on the reported data, a working hypothesis related to the occurrence of a nucleolar checkpoint in plant cells is proposed.

Plant hormone signaling and modulation of DNA repair under stressful conditions.

The role played by phytohormone signaling in the modulation of DNA repair gene and the resulting effects on plant adaptation to genotoxic stress are poorly investigated. Information has been gathered using the Arabidopsis ABA (abscisic acid) overly sensitive mutant abo4-1, defective in the DNA polymerase ε function that is required for DNA repair and recombination. Similarly, phytohormone-mediated regulation of the Ku genes, encoding the Ku heterodimer protein involved in DNA repair, cell cycle control and telomere homeostasis has been demonstrated, highlighting a scenario in which hormones might affect genome stability by modulating the frequency of homologous recombination, favoring plant adaptation to genotoxic stress. Within this context, the characterisation of Arabidopsis AtKu mutants allowed disclosing novel connections between DNA repair and phytohormone networks. Another intriguing aspect deals with the emerging correlation between plant defense response and the mechanisms responsible for genome stability. There is increasing evidence that systemic acquired resistance (SAR) and homologous recombination share common elements represented by proteins involved in DNA repair and chromatin remodeling. This hypothesis is supported by the finding that volatile compounds, such as methyl salicylate (MeSA) and methyl jasmonate (MeJA), participating in the plant-to-plant communication can trigger genome instability in response to genotoxic stress agents. Phytohormone-mediated control of genome stability involves also chromatin remodeling, thus expanding the range of molecular targets. The present review describes the most significant advances in this specific research field, in the attempt to provide a better comprehension of how plant hormones modulate DNA repair proteins as a function of stress.

Noninvasive Methods to Detect Reactive Oxygen Species as a Proxy of Seed Quality.

ROS homeostasis is crucial to maintain radical levels in a dynamic equilibrium within physiological ranges. Therefore, ROS quantification in seeds with different germination performance may represent a useful tool to predict the efficiency of common methods to enhance seed vigor, such as priming treatments, which are still largely empirical. In the present study, ROS levels were investigated in an experimental system composed of hydroprimed and heat-shocked seeds, thus comparing materials with improved or damaged germination potential. A preliminary phenotypic analysis of germination parameters and seedling growth allowed the selection of the best-per-forming priming protocols for species like soybean, tomato, and wheat, having relevant agroeconomic value. ROS levels were quantified by using two noninvasive assays, namely dichloro-dihydro-fluorescein diacetate (DCFH-DA) and ferrous oxidation-xylenol orange (FOX-1). qRT-PCR was used to assess the expression of genes encoding enzymes involved in ROS production (respiratory burst oxidase homolog family, RBOH) and scavenging (catalase, superoxide dismutase, and peroxidases). The correlation analyses between ROS levels and gene expression data suggest a possible use of these indicators as noninvasive approaches to evaluate seed quality. These findings are relevant given the centrality of seed quality for crop production and the potential of seed priming in sustainable agricultural practices.

Integrative Transcriptomics Data Mining to Explore the Functions of TDP1α and TDP1ß Genes in the Arabidopsis thaliana Model Plant.

The tyrosyl-DNA phosphodiesterase 1 (TDP1) enzyme hydrolyzes the phosphodiester bond between a tyrosine residue and the 3'-phosphate of DNA in the DNA-topoisomerase I (TopI) complex, being involved in different DNA repair pathways. A small TDP1 gene subfamily is present in plants, where TDP1α has been linked to genome stability maintenance, while TDP1ß has unknown functions. This work aimed to comparatively investigate the function of the TDP1 genes by taking advantage of the rich transcriptomics databases available for the Arabidopsis thaliana model plant. A data mining approach was carried out to collect information regarding gene expression in different tissues, genetic backgrounds, and stress conditions, using platforms where RNA-seq and microarray data are deposited. The gathered data allowed us to distinguish between common and divergent functions of the two genes. Namely, TDP1ß seems to be involved in root development and associated with gibberellin and brassinosteroid phytohormones, whereas TDP1α is more responsive to light and abscisic acid. During stress conditions, both genes are highly responsive to biotic and abiotic treatments in a time- and stress-dependent manner. Data validation using gamma-ray treatments applied to Arabidopsis seedlings indicated the accumulation of DNA damage and extensive cell death associated with the observed changes in the TDP1 genes expression profiles.

Changes in Medicago truncatula seed proteome along the rehydration-dehydration cycle highlight new players in the genotoxic stress response.

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.

Identification and Characterization of SOG1 (Suppressor of Gamma Response 1) Homologues in Plants Using Data Mining Resources and Gene Expression Profiling.

SOG1 (Suppressor of the Gamma response 1) is the master-regulator of plant DNA damage response (DDR), a highly coordinated network of DNA damage sensors, transducers, mediators, and effectors, with highly coordinated activities. SOG1 transcription factor belongs to the NAC/NAM protein family, containing the well-conserved NAC domain and five serine-glutamine (SQ) motifs, preferential targets for phosphorylation by ATM and ATR. So far, the information gathered for the SOG1 function comes from studies on the model plant Arabidopsis thaliana. To expand the knowledge on plant-specific DDR, it is opportune to gather information on other SOG1 orthologues. The current study identified plants where multiple SOG1 homologues are present and evaluated their functions by leveraging the information contained in publicly available transcriptomics databases. This analysis revealed the presence of multiple SOG1 sequences in thirteen plant species, and four (Medicago truncatula, Glycine max, Kalankoe fedtschenkoi, Populus trichocarpa) were selected for gene expression data mining based on database availability. Additionally, M. truncatula seeds and seedlings exposed to treatments known to activate DDR pathways were used to evaluate the expression profiles of MtSOG1a and MtSOG1b. The experimental workflow confirmed the data retrieved from transcriptomics datasets, suggesting that the SOG1 homologues have redundant functions in different plant species.