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.

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.

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.

Cell death induction and nitric oxide biosynthesis in white poplar (Populus alba) suspension cultures exposed to alfalfa saponins.

The present work reports on the biological activity of alfalfa (Medicago sativa) saponins on white poplar (Populus alba, cultivar 'Villafranca') cell suspension cultures. The extracts from alfalfa roots, aerial parts and seeds were characterized for their saponin content by means of thin layer chromatography (TLC) and electrospray ionisation coupled to mass spectrometry. The quantitative saponin composition from the different plant extracts was determined considering the aglycone moieties and determined by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) analyses. Only soyasapogenin I was detected in the seed extract while several other saponins were found in the root and leaf extracts. Actively proliferating white poplar cell cultures were challenged with the different saponin extracts. Only alfalfa root saponins, at 50 µg ml⁻¹, induced significant cell death rates (75.00 ± 4.90%). Different cell subpopulations with peculiar cell death morphologies were observed and the programmed cell death (PCD)/necrosis ratio was reduced at increasing saponin concentrations. Enhancement of nitric oxide (NO) production was observed in white poplar cells treated with root saponins (RSs) at 50 µg ml⁻¹ and release of reactive oxygen species (ROS) in the culture medium was also demonstrated. Saponin-induced NO production was sensitive to sodium azide and N(G)-monomethyl-L-arginine, two specific inhibitors of distinct pathways for NO biosynthesis in plant cells.

Genotoxic stress and DNA repair in plants: emerging functions and tools for improving crop productivity.

Crop productivity is strictly related to genome stability, an essential requisite for optimal plant growth/development. Genotoxic agents (e.g., chemical agents, radiations) can cause both chemical and structural damage to DNA. In some cases, they severely affect the integrity of plant genome by inducing base oxidation, which interferes with the basal processes of replication and transcription, eventually leading to cell death. The cell response to oxidative stress includes several DNA repair pathways, which are activated to remove the damaged bases and other lesions. Information concerning DNA repair in plants is still limited, although results from gene profiling and mutant analysis suggest possible differences in repair mechanisms between plants and other eukaryotes. The present review focuses on the base- and nucleotide excision repair (BER, NER) pathways, which operate according to the most common DNA repair rule (excision of damaged bases and replacement by the correct nucleotide), highlighting the most recent findings in plants. An update on DNA repair in organelles, chloroplasts and mitochondria is also provided. Finally, it is generally acknowledged that DNA repair plays a critical role during seed imbibition, preserving seed vigor. Despite this, only a limited number of studies, described here, dedicated to seeds are currently available.

The tyrosyl-DNA phosphodiesterase gene family in Medicago truncatula Gaertn.: bioinformatic investigation and expression profiles in response to copper- and PEG-mediated stress.

The Tdp1 gene encoding tyrosyl-DNA phosphodiesterase has been extensively investigated in animal cells, due to the role of this enzyme in the repair of topoisomerase I-DNA covalent lesions. In contrast, information in this regard is totally missing in plants. We report for the first time in plants on the Tdp1 gene family from barrel medic (Medicago truncatula Gaertn.), composed of two members, hereby named MtTdp1alpha and MtTdp1beta. The expression profiles of MtTdp1alpha and MtTdp1beta genes were evaluated in plantlets grown in vitro using copper and polyethylene glycol (PEG 6000) as stress agents. In situ detection of reactive oxygen species (ROS) was carried out by histochemical staining, while the level of oxidative DNA damage, quantified in terms of 7,8-dihydro-8-oxoguanine (8-oxo-dG), increased up to 7.4- and 6.7-fold in response to copper and PEG 6000 treatments, respectively. Quantitative real-time polymerase chain reaction revealed that both Tdp1 genes were significantly up-regulated in response to copper and PEG. The Tdp1 genes were also significantly up-regulated during seed rehydration, an aspect of seed physiology in which DNA repair is a key component. Thus, the Tdp1 genes might be used as novel tools for improving stress tolerance in crops. The expression patterns of the barrel medic top1alpha and top1beta genes, encoding distinct isoforms of DNA topoisomerase I, were also analyzed and discussed to acquire additional information on their specific functions, closely related to that of the Tdp1 gene in animal cells.

Backbone-free transformation of barrel medic (Medicago truncatula) with a Medicago-derived transfer DNA.

In the present work, Agrobacterium tumefaciens-mediated genetic transformation of the model legume Medicago truncatula Gaertn. (barrel medic) was carried out using the pSIM843 vector that contains a Medicago-derived transfer DNA, delineated by a 25-bp sequence homologous to bacterial T-DNA borders. The transfer DNA contains an expression cassette for the nptII (neomycin phosphotransferase) gene and is flanked by an expression cassette for the backbone integration marker gene ipt (isopentenyl transferase). Our results demonstrate that the Medicago-derived RB-like elements efficiently support DNA mobilization from A. tumefaciens to M. truncatula. Kanamycin-resistant shoots with normal phenotype and ipt-shooty lines were recovered at a frequency of 11.7 and 7.8%, respectively. Polymerase chain reaction (PCR) analyses demonstrated that 44.4% of the independent transgenic lines were backbone-free and evidenced the occurrence of backbone-transfer events.

Expression of the PsMTA1 gene in white poplar engineered with the MAT system is associated with heavy metal tolerance and protection against 8-hydroxy-2'-deoxyguanosine mediated-DNA damage.

Marker-free transgenic white poplar (Populus alba L., cv 'Villafranca') plants, expressing the PsMT (A1) gene from Pisum sativum for a metallothionein-like protein, were produced by Agrobacterium tumefaciens-mediated transformation. The 35SCaMV-PsMT (A1)-NosT cassette was inserted into the ipt-type vector pMAT22. The occurrence of the abnormal ipt-shooty phenotype allowed the visual selection of transformants, while the yeast site-specific recombination R/RS system was responsible for the excision of the undesired vector sequences with the consequent recovery of normal marker-free transgenic plants. Molecular analyses confirmed the presence of the 35SCaMV-PsMT (A1)-NosT cassette and transgene expression. Five selected lines were further characterized, revealing the ability to withstand heavy metal toxicity. They survived 0.1 mM CuCl(2), a concentration which strongly affected the nontransgenic plants. Moreover, root development was only slightly affected by the ectopic expression of the transgene. Reactive oxygen species were accumulated to a lower extent in leaf tissues of multi-auto-transformation (MAT)-PsMT(A1) plants exposed to copper and zinc, compared to control plants. Tolerance to photo-oxidative stress induced by paraquat was another distinctive feature of the MAT-PsMT(A1) lines. Finally, low levels of DNA damage were detected by quantifying the amounts of 8-hydroxy-2'-deoxyguanosine in leaf tissues of the transgenic plants exposed to copper.

Leaf-associated bacteria from transgenic white poplar producing resveratrol-like compounds: isolation, molecular characterization, and evaluation of oxidative stress tolerance.

The aim of this study was the isolation and characterization of the culturable bacteria inhabiting the leaves of transgenic white poplars (Populus alba L. 'Villafranca') engineered with the StSy gene for the production of resveratrol-like compounds. Resveratrol glucosides are available in small amounts from natural sources or by expensive chemical synthesis procedures. An alternative approach for the large-scale production of these relevant pharmaceuticals is the use of transgenic plants as bioreactors, although the occurrence of novel molecules in plants growing under field conditions might interfere, to some extent, with the associated microbial population. Both epiphytes and endophytes were isolated from the leaves of 2 StSy transgenic lines producing resveratrol glucosides and from an untransformed plant line grown in a greenhouse. Eleven isolates were recovered and classified as members of the genus Bacillus by 16S rDNA-based analysis. In addition, 2 isolates were classified as members of the Curtobacterium and Kocuria genera, respectively. Tolerance to hydrogen peroxide, UV-C, and paraquat was evaluated, as were the swimming and swarming motility of the leaf-associated bacteria. Interestingly, the isolates recovered from transgenic tissues showed the ability to withstand oxidative stress compared with isolates recovered from the untransformed poplar line. In vitro bioassays showed that trans-resveratrol inhibited both the swarming and swimming motilities in all the tested bacteria. The effects of trans-resveratrol on flagellin production, required for motility, were also investigated by immunoblot analysis.

DNA Diffusion Assay Applied to Plant Cells.

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.

Ultrastructural and Molecular Analyses Reveal Enhanced Nucleolar Activity in Medicago truncatula Cells Overexpressing the MtTdp2α Gene.

The role of tyrosyl-DNA phosphodiesterase 2 (Tdp2) involved in the repair of 5'-end-blocking DNA lesions is still poorly explored in plants. To gain novel insights, Medicago truncatula suspension cultures overexpressing the MtTdp2α gene (Tdp2α-13C and Tdp2α-28 lines, respectively) and a control (CTRL) line carrying the empty vector were investigated. Transmission electron microscopy (TEM) revealed enlarged nucleoli (up to 44% expansion of the area, compared to CTRL), the presence of nucleolar vacuoles, increased frequency of multinucleolate cells (up to 4.3-fold compared to CTRL) and reduced number of ring-shaped nucleoli in Tdp2α-13C and Tdp2α-28 lines. Ultrastructural data suggesting for enhanced nucleolar activity in MtTdp2α-overexpressing lines were integrated with results from bromouridine incorporation. The latter revealed an increase of labeled transcripts in both Tdp2α-13C and Tdp2α-28 cells, within the nucleolus and in the extra-nucleolar region. MtTdp2α-overexpressing cells showed tolerance to etoposide, a selective inhibitor of DNA topoisomerase II, as evidenced by DNA diffusion assay. TEM analysis revealed etoposide-induced rearrangements within the nucleolus, resembling the nucleolar caps observed in animal cells under transcription impairment. Based on these findings it is evident that MtTdp2α-overexpression enhances nucleolar activity in plant cells.

Overexpression of PDH45 or SUV3 helicases in rice leads to delayed leaf senescence-associated events.

Senescence is a very complex process characterized by a highly regulated series of degenerative events which include changes in cell structure, metabolism and gene expression. In animals, one of the indicators of senescence is telomere shortening. In plants, this aspect is more puzzling because telomere shortening is not always correlated with senescence. In some cases, there were no differences in telomere length during plant developmental stages while in other cases both shortening and lengthening have been observed. Several genes involved in telomere homeostasis have been identified in plants, including some helicases. In the present study, the salinity stress-tolerant transgenic IR64 rice plants overexpressing the PDH45 (Pea DNA Helicase 45) or SUV3 (Suppressor of Var1-3) genes were used to test their performance during natural senescence at flowering (S2) and seed maturation (S4) developmental stages. Our results reveal that both PDH45 and SUV3 transgenic rice lines present decreased levels of necrosis/apoptosis as compared to wild type plants. Additionally, in these plants, some senescence-associated genes (SAGs) were downregulated at S2 and S4 stages, while genes involved in the maintenance of genome stability and DNA repair were upregulated. More interestingly, the telomeres were up to 3.8-fold longer in the SUV3 overexpressing lines as compared to wild type plants. This was associated with an increase (2.5-fold) in telomerase (OsTERT) transcript level. This is an interesting result reporting a possible involvement of SUV3 in telomere homeostasis in plants.

Pollen Grain Preservation and Fertility in Valuable Commercial Rose Cultivars.

In the cut flower market, traditional breeding is still the best way to achieve new rose cultivars. The geographical delocalization of cultivar constitution (generally made in Europe and North America) and plant cultivation (large areas in Africa and South America) represents a limit point for crossing and selection. Rose breeders often need to overcome geographical distances, resulting in asynchrony in flowering among crossing parents, by storing and sending pollen. Hence, a key aspect in breeding programs is linked to pollen availability and conservation, jointly with the identification of parameters related to pollen fertility. In this study we present the results of three different trials. In the first, pollen diameter and pollen viability were chosen as fertility predictors of 10 Rosa hybrida commercial cultivars. In the second trial, aliquots of dried pollen grains of six R. hybrida cultivar were stored under two different temperatures (freezer at T = -20 °C and deep freezer at T = -80 °C) and after a wide range of conservation period, their viability was measured. In the third trial, the effective fertilization capacity of frozen pollen of 19 pollen donor cultivars was evaluated during 2015 crossing breeding plan, performing 44 hybridizations and correlating the number of seeds and the ratio seeds/crossing, obtained by each cultivar, with in vitro pollen germination ability.

Physical Methods for Seed Invigoration: Advantages and Challenges in Seed Technology.

In the context of seed technology, the use of physical methods for increasing plant production offers advantages over conventional treatments based on chemical substances. The effects of physical invigoration treatments in seeds can be now addressed at multiple levels, ranging from morpho-structural aspects to changes in gene expression and protein or metabolite accumulation. Among the physical methods available, "magneto-priming" and irradiation with microwaves (MWs) or ionizing radiations (IRs) are the most promising pre-sowing seed treatments. "Magneto-priming" is based on the application of magnetic fields and described as an eco-friendly, cheap, non-invasive technique with proved beneficial effects on seed germination, vigor and crop yield. IRs, as γ-rays and X-rays, have been widely regarded as a powerful tool in agricultural sciences and food technology. Gamma-rays delivered at low dose have showed to enhance germination percentage and seedling establishment, acting as an actual 'priming' treatment. Different biological effects have been observed in seeds subjected to MWs and X-rays but knowledge about their impact as seed invigoration agent or stimulatory effects on germination need to be further extended. Ultraviolet (UV) radiations, namely UV-A and UV-C have shown to stimulate positive impacts on seed health, germination, and seedling vigor. For all mentioned physical treatments, extensive fundamental and applied research is still needed to define the optimal dose, exposition time, genotype- and environment-dependent irradiation conditions. Electron paramagnetic resonance has an enormous potential in seed technology not fully explored to monitor seed invigoration treatments and/or identifying the best suitable irradiation dose or time-point to stop the treatment. The present manuscript describes the use of physical methods for seed invigoration, while providing a critical discussion on the constraints and advantages. The future perspectives related to the use of these approaches to address the need of seed technologists, producers and trade markers will be also highlighted.

Isolation and functional analysis of the 5'-flanking region of carrot top1beta gene coding for the beta isoform of DNA topoisomerase I.

We have isolated and functionally characterized the promoter region of the top1beta gene encoding carrot (Daucus carota) DNA topoisomerase Ibeta. The major transcription start site was mapped by primer extension analysis 164 nt upstream the ATG translation start codon. Sequence analysis of the 5'-upstream region of the gene revealed the presence of a canonical TATA-like box at position -35 bp and several cis-acting sequences, including a (CT)n element in the leader region of the gene, a myb-related motif and the Dof element NtBBF-1, which correlate with the inducible expression pattern of this gene. Functional reporter analysis of the top1beta 5'-flanking region was performed in both carrot and Arabidopsis thaliana transfected protoplasts. The region at -719 to +161 was sufficient to confer high expression level in both species. The transient expression assay in protoplasts induced to stop dividing confirmed that the promoter, whose activity is low in quiescent cells, is activated when protoplasts are induced to proliferate by exogenous application of growth factors.

Cell wall integrity, genotoxic injury and PCD dynamics in alfalfa saponin-treated white poplar cells highlight a complex link between molecule structure and activity.

In the present work, eleven saponins and three sapogenins purified from Medicago sativa were tested for their cytotoxicity against highly proliferating white poplar (Populus alba L.) cell suspension cultures. After preliminary screening, four saponins with different structural features in terms of aglycone moieties and sugar chains (saponin 3, a bidesmoside of hederagenin; saponins 4 and 5, monodesmoside and bidesmoside of medicagenic acid respectively, and saponin 10, a bidesmoside of zanhic acid) and different cytotoxicity were selected and used for further investigation on their structure-activity relationship. Transmission Electron Microscopy (TEM) analyses provided for the first time evidence of the effects exerted by saponins on plant cell wall integrity. Exposure to saponin 3 and saponin 10 resulted into disorganization of the outer wall layer and the effect was even more pronounced in white poplar cells treated with the two medicagenic acid derivatives, saponins 4 and 5. Oxidative burst and nitric oxide accumulation were common hallmarks of the response of white poplar cells to saponins. When DNA damage accumulation and DNA repair profiles were evaluated by Single Cell Gel Electrophoresis, induction of single and double strand breaks followed by effective repair was observed within 24h. The reported data are discussed in view of the current issues dealing with saponin structure-activity relationship.

Exploring the molecular and chemical-physical aspects of low-dose irradiation using radio-tolerant plant cells.

Animals and plants show different levels of radio-sensitivity, with safe dose values in the 0.001-1 and 1-100 Gy range, respectively. The increased radio-tolerance observed in plant cells might represent a valuable tool to investigate the events underlying the low dose (LD) response in the highly radio-sensitive animal cells. The use of radio-resistant plant systems would allow investigation of the LD effects using irradiation conditions that can be easily managed, without the technical constrains currently encountered with animal systems. The basal knowledge on the molecular mechanisms involved in the DNA damage response in plants is rapidly expanding, revealing common features with animal cells at the level of DNA damage sensing/repair, transduction pathways and antioxidant response. The present work provides a short update of the current literature dealing with the DNA damage response in animal and plant cells exposed to LD treatments (ionising radiation, particularly gamma ray) combined with the more recent advances in free radical research.

White poplar (Populus alba L.) suspension cultures as a model system to study apoptosis induced by alfalfa saponins.

In animal cells, the anticancer function played by plant saponins involves a complex network of molecular processes that still deserves investigation and apoptosis seems to be the outstanding pathway. An intriguing aspect of the biological activity of saponins is related to their effects on genome integrity. As demonstrated by the studies carried out in white poplar (Populus alba L., cv Villafranca) cell suspension cultures, plant cells can as well be used as a model system to unravel the molecular mechanisms activated by plant saponins. These recent studies have evidenced that animal and plant cells share common features in their response to saponins, paving the way for novel opportunities for both basic and applied research. Indeed, there is a certain interest in replacing the animal models for pharmacological research, at least when preliminary large-scale cytotoxicity tests are performed on wide collections of natural extracts and/or purified compounds. The review provides an up-date of the molecular pathways (signal transduction, antioxidant response, DNA repair) associated with plant saponin bioactivity, with an emphasis on apoptosis induced by alfalfa (Medicago sativa L.) saponins. The comparison between animal and plant cells as tools for the study of saponin bioactivity is also discussed in view of the most recent literature and innovative future applications.

Synergistic exposure of rice seeds to different doses of γ-ray and salinity stress resulted in increased antioxidant enzyme activities and gene-specific modulation of TC-NER pathway.

Recent reports have underlined the potential of gamma (γ)-rays as tools for seed priming, a process used in seed industry to increase seed vigor and to enhance plant tolerance to biotic/abiotic stresses. However, the impact of γ -rays on key aspects of plant metabolism still needs to be carefully evaluated. In the present study, rice seeds were challenged with different doses of γ -rays and grown in absence/presence of NaCl to assess the impact of these treatments on the early stages of plant life. Enhanced germination efficiency associated with increase in radicle and hypocotyl length was observed, while at later stages no increase in plant tolerance to salinity stress was evident. APX, CAT, and GR were enhanced at transcriptional level and in terms of enzyme activity, indicating the activation of antioxidant defence. The profiles of DNA damage accumulation were obtained using SCGE and the implication of TC-NER pathway in DNA damage sensing and repair mechanisms is discussed. OsXPB2, OsXPD, OsTFIIS, and OsTFIIS-like genes showed differential modulation in seedlings and plantlets in response to γ -irradiation and salinity stress. Altogether, the synergistic exposure to γ -rays and NaCl resulted in enhanced oxidative stress and proper activation of antioxidant mechanisms, thus being compatible with plant survival.