A novel PGPR strain homologous to Beijerinckia fluminensis induces biochemical and molecular changes involved in Arabidopsis thaliana salt tolerance.

The use of PGPR is widely accepted as a promising tool for a more sustainable agricultural production and improved plant abiotic stress resistance. This study tested the ability of PVr_9, a novel bacterial strain, homologous to Beijerinckia fluminensis, to increase salt stress tolerance in A. thaliana. In vitro plantlets inoculated with PVr_9 and treated with 150 mM NaCl showed a reduction in primary root growth inhibition compared to uninoculated ones, and a leaf area significantly less affected by salt. Furthermore, salt-stressed PVr_9-inoculated plants had low ROS and 8-oxo-dG, osmolytes, and ABA content along with a modulation in antioxidant enzymatic activities. A significant decrease in Na+ in the leaves and a corresponding increase in the roots were also observed in salt-stressed inoculated plants. SOS1, NHX1 genes involved in plant salt tolerance, were up-regulated in PVr_9-inoculated plants, while different MYB genes involved in salt stress signal response were down-regulated in both roots and shoots. Thus, PVr_9 was able to increase salt tolerance in A. thaliana, thereby suggesting a role in ion homeostasis by reducing salt stress rather than inhibiting total Na+ uptake. These results showed a possible molecular mechanism of crosstalk between PVr_9 and plant roots to enhance salt tolerance, and highlighted this bacterium as a promising PGPR for field applications on agronomical crops.

Epigenetic Control of Plant Response to Heavy Metals.

Plants are sessile organisms that must adapt to environmental conditions, such as soil characteristics, by adjusting their development during their entire life cycle. In case of low-distance seed dispersal, the new generations are challenged with the same abiotic stress encountered by the parents. Epigenetic modification is an effective option that allows plants to face an environmental constraint and to share the same adaptative strategy with their progeny through transgenerational inheritance. This is the topic of the presented review that reports the scientific progress, up to date, gained in unravelling the epigenetic response of plants to soil contamination by heavy metals and metalloids, collectively known as potentially toxic elements. The effect of the microbial community inhabiting the rhizosphere is also considered, as the evidence of a transgenerational transfer of the epigenetic status that contributes to the activation in plants of response mechanisms to soil pollution.

The Contribution of PGPR in Salt Stress Tolerance in Crops: Unravelling the Molecular Mechanisms of Cross-Talk between Plant and Bacteria.

Soil salinity is a major abiotic stress in global agricultural productivity with an estimated 50% of arable land predicted to become salinized by 2050. Since most domesticated crops are glycophytes, they cannot be cultivated on salt soils. The use of beneficial microorganisms inhabiting the rhizosphere (PGPR) is a promising tool to alleviate salt stress in various crops and represents a strategy to increase agricultural productivity in salt soils. Increasing evidence underlines that PGPR affect plant physiological, biochemical, and molecular responses to salt stress. The mechanisms behind these phenomena include osmotic adjustment, modulation of the plant antioxidant system, ion homeostasis, modulation of the phytohormonal balance, increase in nutrient uptake, and the formation of biofilms. This review focuses on the recent literature regarding the molecular mechanisms that PGPR use to improve plant growth under salinity. In addition, very recent -OMICs approaches were reported, dissecting the role of PGPR in modulating plant genomes and epigenomes, opening up the possibility of combining the high genetic variations of plants with the action of PGPR for the selection of useful plant traits to cope with salt stress conditions.

Perenniality, more than genotypes, shapes biological and chemical rhizosphere composition of perennial wheat lines.

Perennial grains provide various ecosystem services compared to the annual counterparts thanks to their extensive root system and permanent soil cover. However, little is known about the evolution and diversification of perennial grains rhizosphere and its ecological functions over time. In this study, a suite of -OMICSs - metagenomics, enzymomics, metabolomics and lipidomics - was used to compare the rhizosphere environment of four perennial wheat lines at the first and fourth year of growth in comparison with an annual durum wheat cultivar and the parental species Thinopyrum intermedium. We hypothesized that wheat perenniality has a greater role in shaping the rhizobiome composition, biomass, diversity, and activity than plant genotypes because perenniality affects the quality and quantity of C input - mainly root exudates - hence modulating the plant-microbes crosstalk. In support of this hypothesis, the continuous supply of sugars in the rhizosphere along the years created a favorable environment for microbial growth which is reflected in a higher microbial biomass and enzymatic activity. Moreover, modification in the rhizosphere metabolome and lipidome over the years led to changes in the microbial community composition favoring the coexistence of more diverse microbial taxa, increasing plant tolerance to biotic and abiotic stresses. Despite the dominance of the perenniality effect, our data underlined that the OK72 line rhizobiome distinguished from the others by the increase in abundance of Pseudomonas spp., most of which are known as potential beneficial microorganisms, identifying this line as a suitable candidate for the study and selection of new perennial wheat lines.

DNA methylation is enhanced during Cd hyperaccumulation in Noccaea caerulescens ecotype Ganges.

In this study, we assess the DNA damage occurring in response to cadmium (Cd) in the Cd hyperaccumulator Noccaea caerulescens Ganges (GA) vs the non-accumulator and close-relative species Arabidopsis thaliana. At this purpose, the alkaline comet assay was utilized to evaluate the Cd-induced variations in nucleoids and the methy-sens comet assay, and semiquantitative real-time (qRT)-PCR were also performed to associate nucleus variations to possible DNA modifications. Cadmium induced high DNA damages in nuclei of A. thaliana while only a small increase in DNA migration was observed in N. caerulescens GA. In addition, in N. caerulescens GA, CpG DNA methylation increase upon Cd when compared to control condition, along with an increase in the expression of MET1 gene, coding for the DNA-methyltransferase. N. caerulescens GA does not show any oxidative stress under Cd treatment, while A. thaliana Cd-treated plants showed an upregulation of transcripts of the respiratory burst oxidase, accumulation of reactive oxygen species, and enhanced superoxide dismutase activity. These data suggest that epigenetic modifications occur in the N. caerulescens GA exposed to Cd to preserve genome integrity, contributing to Cd tolerance.

Protein Maps for Durum Wheat Precision Harvest and Pasta Production.

The quality traits of dough and dry pasta obtained from semolina (Triticum durum Desf. var. Biensur), harvested from a 13.6 ha field, subjected to variable-rate nitrogen (N) fertilization, were assessed to test site-specific pasta production for a short supply chain. Based on the grain quality spatial distribution, two distinct areas with protein content lower or higher than 13% were delineated and harvested selectively. The rheological properties of semolina samples obtained from those areas were evaluated. Furthermore, dry pasta was produced and characterized for its cooking behaviour and sensory characteristics. Semolina was demonstrated to have rheological characteristics (i.e., gluten aggregation time and energy, as evaluated by GlutoPeak test) positively related to the protein content as well as the related pasta, showing better cooking quality. These results are driven by the high amounts of gluten proteins, as well as by the glutenin/gliadin ratio, which are indicators of the technological quality of semolina. Overall, the results indicate that segregation of the grain with >13% of protein at harvest led to the production of semolina with a higher gluten protein content and a higher glutenin/gliadin ratio and, hence, to the production of pasta with better cooking quality. Therefore, site-specific pasta could be a potential asset for a short supply chain, aiming at improving traceability, as well as environmental and economic sustainability.

Phyto-Beneficial Traits of Rhizosphere Bacteria: In Vitro Exploration of Plant Growth Promoting and Phytopathogen Biocontrol Ability of Selected Strains Isolated from Harsh Environments.

Beneficial interactions between plants and some bacterial species have been long recognized, as they proved to exert various growth-promoting and health-protective activities on economically relevant crops. In this study, the growth promoting and antifungal activity of six bacterial strains, Paenarthrobacter ureafaciens, Beijerinckia fluminensis, Pseudomonas protegens, Arthrobacter sp., Arthrobacter defluii, and Arthrobacter nicotinovorans, were investigated. The tested strains resulted positive for some plant growth promoting (PGP) traits, such as indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylate-deaminase (ACC-deaminase), siderophore production, and solubilization of phosphates. The effect of the selected bacteria on Arabidopsis thaliana seedlings growth was assessed using different morphological parameters. Bacterial activity against the phytopathogenic fungal species Aspergillus flavus, Fusarium proliferatum, and Fusarium verticillioides was also assessed, since these cause major yield losses in cereal crops and are well-known mycotoxin producers. Strains Pvr_9 (B. fluminensis) and PHA_1 (P. protegens) showed an important growth-promoting effect on A. thaliana coupled with a high antifungal activity on all the three fungal species. The analysis of bacterial broths through ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) confirmed the presence of potential PGP-compounds, among these are desferrioxamine B, aminochelin, asperchrome B, quinolobactin siderophores, and salicylic acid.

Overexpression of ZNT1 and NRAMP4 from the Ni Hyperaccumulator Noccaea caerulescens Population Monte Prinzera in Arabidopsis thaliana Perturbs Fe, Mn, and Ni Accumulation.

Metalliferous soils are characterized by a high content of metal compounds that can hamper plant growth. The pseudometallophyte Noccaea caerulescens is able to grow on metalliferous substrates by implementing both tolerance and accumulation of usually toxic metal ions. Expression of particular transmembrane transporter proteins (e.g., members of the ZIP and NRAMP families) leads to metal tolerance and accumulation, and its comparison between hyperaccumulator N. caerulescens with non-accumulator relatives Arabidopsis thaliana and Thlaspi arvense has deepened our knowledge on mechanisms adopted by plants to survive in metalliferous soils. In this work, two transporters, ZNT1 and NRAMP4, expressed in a serpentinic population of N. caerulescens identified on the Monte Prinzera (Italy) are considered, and their expression has been induced in yeast and in A. thaliana. In the latter, single transgenic lines were crossed to test the effect of the combined over-expression of the two transporters. An enhanced iron and manganese translocation towards the shoot was induced by overexpression of NcZNT1. The combined overexpression of NcZNT1 and NcNRAMP4 did perturb the metal accumulation in plants.

Improvement of Soil Microbial Diversity through Sustainable Agricultural Practices and Its Evaluation by -Omics Approaches: A Perspective for the Environment, Food Quality and Human Safety.

Soil is one of the key elements for supporting life on Earth. It delivers multiple ecosystem services, which are provided by soil processes and functions performed by soil biodiversity. In particular, soil microbiome is one of the fundamental components in the sustainment of plant biomass production and plant health. Both targeted and untargeted management of soil microbial communities appear to be promising in the sustainable improvement of food crop yield, its nutritional quality and safety. -Omics approaches, which allow the assessment of microbial phylogenetic diversity and functional information, have increasingly been used in recent years to study changes in soil microbial diversity caused by agronomic practices and environmental factors. The application of these high-throughput technologies to the study of soil microbial diversity, plant health and the quality of derived raw materials will help strengthen the link between soil well-being, food quality, food safety and human health.

Enhancement of Zn tolerance and accumulation in plants mediated by the expression of Saccharomyces cerevisiae vacuolar transporter ZRC1.

MAIN CONCLUSION: Transgenic Arabidopsis thaliana and Populus alba plants overexpressing the zinc transporter ScZRC1 in shoots exhibit Zn tolerance. Increased Zn concentrations were observed in shoots of P. alba, a species suitable for phytoremediation. Genetic engineering of plants for phytoremediation is worth to consider if genes leading to heavy metal accumulation and tolerance are expressed in high biomass producing plants. The Saccharomyces cerevisiae ZRC1 gene encodes a zinc transporter which is primarily involved in the uptake of Zn into the vacuole. The ZRC1 gene was expressed in the model species A. thaliana and P. alba (cv. Villafranca). Both species were transformed with constructs carrying ScZRC1 under the control of either the CaMV35S promoter for constitutive expression or the active promoter region of the tobacco Rubisco small subunit (pRbcS) to limit the expression to the above-ground tissues. In hydroponic cultures, A. thaliana and poplar ScZRC1-expressing plants accumulated more Zn in vegetative tissues and were more tolerant than untransformed plants. No differences were found between plants carrying the CaMV35::ScZRC1 or pRbcS::ScZRC1 constructs. The higher Zn accumulation in transgenic plants was accompanied by an increased superoxide dismutase (SOD) activity, indicating the activation of defense mechanisms to prevent cellular damage. In the presence of cadmium in addition to Zn, plants did not show symptoms of metal toxicity, neither in hydroponic cultures nor in soil. Zn accumulation increased in shoots, while no differences were observed for Cd accumulation, in comparison to control plants. These data suggest that ectopic expression of ScZRC1 can increase the potential of poplar for the remediation of Zn-polluted soils, although further tests are required to assay its application in remediating multimetal polluted soils.

Heavy metals modulate DNA compaction and methylation at CpG sites in the metal hyperaccumulator Arabidopsis halleri.

Excess heavy metals affect plant physiology by inducing stress symptoms, however several species have evolved the ability to hyperaccumulate metals in above-ground tissues without phytotoxic effects. In this study we assume that at subcellular level, different strategies were adopted by hyperaccumulator versus the non-accumulator plant species to face the excess of heavy metals. At this purpose the comet assay was used to investigate the nucleoid structure modifications occurring in response to Zn and Cd treatments in the I16 and PL22 populations of the hyperaccumulator Arabidopsis halleri versus the nonaccumulator species Arabidopsis thaliana. Methy-sens comet assay and RT-qPCR were also performed to associate metal induced variations in nucleoids with possible epigenetic modifications. The comet assay showed that Zn induced a mild but non significant reduction in the tail moment in A. thaliana and in both I16 and PL22. Cd treatment induced an increase in DNA migration in nuclei of A. thaliana, whereas no differences in DNA migration was observed for I16, and a significant increase in nucleoid condensation was found in PL22 Cd treated samples. This last population showed higher CpG DNA methylation upon Cd treatment than in control conditions, and an up-regulation of genes involved in symmetric methylation and histone deacetylation. Our data support the hypothesis of a possible role of epigenetic modifications in the hyperaccumulation trait to cope with the high Cd shoot concentrations. In addition, the differences observed between PL22 and I16 could reinforce previous suggestions of divergent strategies for metals detoxification developing in the two metallicolous populations.

Smart agriculture for food quality: facing climate change in the 21st century.

Climate change, with increasing temperatures and atmospheric carbon dioxide levels, constitutes a severe threat to the environment and all living organisms. In particular, numerous studies suggest severe consequences for the health of crop plants, affecting both the productivity and quality of raw material destined to the food industry. Of particular concern is the reduction of proteins and essential micronutrients as iron and zinc in crops. Fighting this alarming trends is the challenge of Climate-Smart Agriculture with the double goal of reducing environmental impacts (use of pesticides, nitrogen and phosphorus leaching, soil erosion, water depletion and contamination) and improving raw material and consequently food quality. Organic farming, biofertilizers and to a lesser extent nano-carriers, improve the antioxidant properties of fruits, but the data about proteins and micronutrients are rather contradictory. On the other hand, advanced devices and Precision Agriculture allow the cultivations to be more profitable, efficient, contributing more and more to reduce pest diseases and to increase the quality of agricultural products and food safety. Thus, nowadays adoption of technologies applied to sustainable farming systems is a challenging and dynamic issue for facing negative trends due to environmental impacts and climate changes.

Is Site-Specific Pasta a Prospective Asset for a Short Supply Chain?

In the 2011-2012 season, variable-rate nitrogen (N) fertilization was applied two times during durum wheat vegetative growth in three field areas which differed in soil fertility in northern Italy. The quality traits of the mono-varietal pasta obtained from each management zone were assessed in view of site-specific pasta production for a short supply chain. To this purpose, semolina from cv. Biensur obtained from management zones with different fertility treated with N at variable rate was tested in comparison with a commercial reference (cv. Aureo) to produce short-cut pasta. Biensur semolina demonstrated to have technological characteristics positively correlated with the low-fertility zones treated with high N doses (200 and 200+15 kg/ha) and, to a lesser extent, with the high-soil-fertility zones (130 and 130 + 15 kg/ha of N). The lower quality parameters were obtained for pasta produced with wheat from medium-fertility zones, independently of the N dose applied. The derived pasta obtained from the low-fertility zones treated with high N doses had cooking and sensory properties comparable to those of pasta obtained using the reference cv. Aureo. These results are explained by the higher amounts of gluten proteins and by a higher glutenin/gliadin ratio in semolina, which are indicators of technological quality. Overall, the results indicate that segregation of the grain at harvest led to the production of semolina with higher protein content and, hence, to a higher pasta quality. Therefore, site-specific pasta could be a potential asset for a short supply chain, aiming to improve traceability and environmental and economic sustainability.

Proteins and Metabolites as Indicators of Flours Quality and Nutritional Properties of Two Durum Wheat Varieties Grown in Different Italian Locations.

Durum wheat is an important food source in Mediterranean countries, and Italy is the major producer of durum wheat in Europe. The quality of durum wheat flours depends on the type and amount of gluten proteins and starch while flour nutritional value rests on metabolite contents such as polyphenols. In this work, two Italian cultivars, Iride and Svevo, were analyzed for two years (2016-2017) in four Italian regions to explore how the environment affects: (i) reserve proteome; (ii) starch content and composition; and (iii) free, conjugated, bound phenolics and antioxidant capacity. The impact of environmental and meteorological conditions was significant for many traits. Regardless of the cultivation site, in 2017, a year with less rainfall and a higher temperature during grain filling, there was an increase in low molecular weight glutenins, in the glutenin/gliadin ratio and in the A-type starch granules size, all parameters of higher technological quality. In the same year, the cultivars showed higher amounts of polyphenols and antioxidant capacity. In conclusion, the two wheat cultivars, selected for their medium to high yield and their good quality, had higher performances in 2017 regardless of their sowing locations.

Effects of Seed-Applied Biofertilizers on Rhizosphere Biodiversity and Growth of Common Wheat (Triticum aestivum L.) in the Field.

In order to reduce chemical fertilization and improve the sustainability of common wheat (Triticum aestivum L.) cultivation, maintaining at the same time high production and quality standards, this study investigated the effects of three commercial biofertilizers on rhizosphere bacterial biomass, biodiversity and enzymatic activity, and on plant growth and grain yield in a field trial. The wheat seeds were inoculated with the following aiding microrganisms: (i) a bacterial consortium (Azospirillum spp. + Azoarcus spp. + Azorhizobium spp.); and two mycorrhizal fungal-bacterial consortia, viz. (ii) Rhizophagus irregularis + Azotobacter vinelandii, and (iii) R. irregularis + Bacillus megaterium + Frateuria aurantia, and comparisons were made with noninoculated controls. We demonstrate that all the biofertilizers significantly enhanced plant growth and nitrogen accumulation during stem elongation and heading, but this was translated into only small grain yield gains (+1%-4% vs controls). The total gluten content of the flour was not affected, but in general biofertilization significantly upregulated two high-quality protein subunits, i.e., the 81 kDa high-molecular-weight glutenin subunit and the 43.6 kDa low-molecular-weight glutenin subunit. These effects were associated with increases in the rhizosphere microbial biomass and the activity of enzymes such as ß-glucosidase, α-mannosidase, ß-mannosidase, and xylosidase, which are involved in organic matter decomposition, particularly when Rhizophagus irregularis was included as inoculant. No changes in microbial biodiversity were observed. Our results suggest that seed-applied biofertilizers may be effectively exploited in sustainable wheat cultivation without altering the biodiversity of the resident microbiome, but attention should be paid to the composition of the microbial consortia in order to maximize their benefits in crop cultivation.

Heavy Metal Pollutions: State of the Art and Innovation in Phytoremediation.

Mineral nutrition of plants greatly depends on both environmental conditions, particularly of soils, and the genetic background of the plant itself. Being sessile, plants adopted a range of strategies for sensing and responding to nutrient availability to optimize development and growth, as well as to protect their metabolisms from heavy metal toxicity. Such mechanisms, together with the soil environment, meaning the soil microorganisms and their interaction with plant roots, have been extensively studied with the goal of exploiting them to reclaim polluted lands; this approach, defined phytoremediation, will be the subject of this review. The main aspects and innovations in this field are considered, in particular with respect to the selection of efficient plant genotypes, the application of improved cultural strategies, and the symbiotic interaction with soil microorganisms, to manage heavy metal polluted soils.

Technological Quality and Nutritional Value of Two Durum Wheat Varieties Depend on Both Genetic and Environmental Factors.

Durum wheat ( Triticum turgidum L. subsp. durum (Desf.) Husn) is a major food source in Mediterranean countries since it is utilized for the production of pasta, leavened and unleavened breads, couscous, and other traditional foods. The technological and nutritional properties of durum wheat semolina depend mainly on the type of gluten proteins and on their amount, which is a genotype- and environment-dependent trait. Gluten proteins are also responsible for celiac disease (CD), an autoimmune enteropathy with a prevalence of about 0.7-2% in the human population. At this purpose, two Italian durum wheat cultivars, Saragolla and Cappelli, currently used for monovarietal pasta, were chosen to compare (i) the reserve and embryo proteome, (ii) the free and bound phenolics, antioxidant activity, and amino acid composition, and (iii) the content of immunogenic peptides produced after a simulated gastrointestinal digestion. The results obtained from 2 years of field cultivation on average showed a higher amount of gluten proteins, amino acids, and immunogenic peptides in Cappelli. Saragolla showed a higher abundance in bound phenolics, antioxidant enzymes, and stress response proteins in line with its higher antioxidant activity. However, the impact of the year of cultivation, largely depending on varying rainfall regimes through the wheat growth cycle, was significant for most of the parameters investigated. Differences in technological and nutritional characteristics observed between the two cultivars are discussed in relation to the influence of genetic and environmental factors.

A novel ß-propeller phytase from the dioxin-degrading bacterium Sphingomonas wittichii RW-1.

ß-propeller phytase-like sequences (BPP-like sequences) are widespread in the microbial world and have been found in the sequenced genomes of aquatic, soil, and plant bacteria. Exploring NCBI microbial genome database for putative genes encoding phytase, a BPP-like sequence from Sphingomonas wittichii RW-1 (Sequence ID: CP000699.1), known for its capacity of degrading polychlorinated dibenzo-p-dioxins and dibenzofurans, was recognized. The putative phytase gene (phySw) was amplified with specific primers, cloned, and overexpressed in Escherichia coli and the catalytic properties of the recombinant PhySw protein were analyzed. The results show that phySw encodes an enzyme with the properties of ß-propeller phytases: it requires the presence of Ca2+ ions, it is optimally active at 55 °C, and it has a pH optimum of 6.0 with good activity in the range 6.0-8.0. Furthermore, the enzyme exhibits a good thermostability, recovering 68% of its original activity after treatment at 80 °C for 10 min, and shows a good substrate specificity for phytic acid. These properties render this enzyme a candidate as an animal feed additive (e.g., for aquaculture industry). The isolation of phytases from a hydrocarbon-utilizing microorganism also opens new scenarios for their possible application in combating oil pollution.

16S rDNA Profiling to Reveal the Influence of Seed-Applied Biostimulants on the Rhizosphere of Young Maize Plants.

In an open field trial on two agricultural soils in NW Italy, the impact of two seed-applied biostimulants on the rhizosphere bacterial community of young maize plants was evaluated. The 16S rDNA profiling was carried out on control and treated plant rhizosphere samples collected at the 4-leaf stage and on bulk soil. In both soils, the rhizospheres were significantly enriched in Proteobacteria, Actinobacteria, and Bacteriodetes, while the abundances of Acidobacteria, Cloroflexi and Gemmatimonadetes decreased compared with bulk soil. Among the culturable bacteria genera that showed an increase by both biostimulants, most are known to be beneficial for nutrient uptake, such as Opitutus, Chryseolinea, Terrimonas, Rhodovastum, Cohnella, Pseudoduganella and the species Anaeromyxobacter dehalogenans; others are known to be involved in root growth, such as Niastella, Labrys, Chloroflexia and Thermomonas; or in plant defence, such as Ohtaekwangia, Quadrisphaera, Turneriella, and Actinoallomurus. Both biostimulants were also found to stimulate gen. Nannocystis, a potential biocompetitive agent against aflatoxigenic Aspergillus moulds. Under controlled conditions, both biostimulants enhanced the shoot and root biomass at the 4⁻5 leaf stage. We conclude that the biostimulants do not decrease the biodiversity of the microbial community rhizosphere of young maize plants, but stimulate rare bacterial taxa, some involved in plant growth and pathogen resistance, a result that may have implications in improving crop management.

Epigenetic modifications preserve the hyperaccumulator Noccaea caerulescens from Ni geno-toxicity.

The Ni hyperaccumulator Noccaea caerulescens has adapted to live in a naturally stressed environment, evolving a complex pattern of traits to cope with adverse conditions. Evidence is accumulating regarding the important role of epigenetic modifications in regulating plant responses to stress. In this study, we present data from the natural "open-field" adaptation of the Ni hyperaccumulator N. caerulescens to serpentine soil and provide the first evidence of the involvement of epigenetic changes in response to the high Ni content present in plant leaves. The alkaline comet assay revealed the integrity of the nuclei of leaf cells of N. caerulescens grown in a Ni-rich environment, while in the non-tolerant Arabidopsis thaliana exposed to Ni, the nuclei were severely damaged. DNA of N. caerulescens plants grown in situ were considerably hyper-methylated compared to A. thaliana plants exposed to Ni. In addition, qRT-PCR revealed that N. caerulescens MET1, DRM2, and HDA8 genes involved in epigenetic DNA and histone modification were up-regulated in the presence of high Ni content in leaves. Such epigenetic modifications may constitute a defense strategy that prevents genome instability and direct damage to the DNA structure by Ni ion, enabling plants to survive in an extreme environment. Further studies will be necessary to analyze in detail the involvement of DNA methylation and other epigenetic mechanisms in the complex process of metal hyperaccumulation and plants' adaptive response. Environ. Mol. Mutagen. 59:464-475, 2018. © 2018 Wiley Periodicals, Inc.