Dynamic changes in global methylation and plant cell death mechanism in response to NiO nanoparticles.

MAIN CONCLUSION: This report is a first comprehensive work on the potential of engineered nickel oxide nanoparticles affecting the epigenome and modulating global methylation leading to retention of transgenerational footprints. Nickel oxide nanoparticles (NiO-NPs) are known to instigate extensive phenotypic and physiological damage to plants. In the present work, it was shown that exposure to increasing concentrations of NiO-NP-induced cell death cascades in model systems, Allium cepa and tobacco BY-2 cells. NiO-NP also generated variation in global CpG methylation; its transgenerational transmission was shown in affected cells. Plant tissues exposed to NiO-NP showed progressive replacement of essential cations, like Fe and Mg, as seen in XANES and ICP-OES data, providing earliest signs of disturbed ionic homeostasis. Fluorescent staining based confocal microscopy confirmed upsurge of H2O2 and nitric oxide after NiO-NP exposure. A NiO-NP concentration gradient-based switching-on of the cell death cascades was observed when autophagosomes were seen in samples exposed to lower and median concentrations of NiO-NP (10-125 mg L-1). The apoptotic cell death marker, caspase-3 like protein, was noted in the median to higher doses (50-500 mg L-1), and leakage of lactate dehydrogenase marking necrotic cell death was observed in samples exposed to the highest doses (125-500 mg L-1) of NiO-NP. Concomitant increase of DNA hypermethylation (quantified by ELISA-based assay) and genomic DNA damage (evaluated through Comet-based analyses) was recorded at higher doses of NiO-NP. MSAP profiles confirmed that global methylation changes incurring in the parental generation upon NiO-NP exposure were transmitted through the two subsequent generations of BY-2 cells which was supported by data from A. cepa, too. Thus, it was evident that NiO-NP exposure incited DNA hypermethylation, as an aftermath of oxidative burst, and led to induction of autophagy, apoptotic and necrotic cell death pathways. Global methylation changes induced by NiO-NP exposure can be transmitted through subsequent cell generations.

The impact of engineered nickel oxide nanoparticles on ascorbate glutathione cycle in Allium cepa L.

Engineered nickel oxide nanoparticle (NiO-NP) can inflict significant damages on exposed plants, even though very little is known about the modus operandi. The present study investigated effects of NiO-NP on the crucial stress alleviation mechanism Ascorbate-Glutathione Cycle (Asa-GSH cycle) in the model plant Allium cepa. Cellular contents of reduced glutathione (GSH) and oxidised glutathione (GSSG), was disturbed upon NiO-NP exposure. The ratio of GSH to GSSG changed from 20:1 in NC to 4:1 in roots exposed to 125 mg L-1 NiO-NP. Even the lowest treatments of NiO-NP (10 mg L-1) increased ascorbic acid (2.9-folds) and cysteine contents (1.6-folds). Enzymes like glutathione reductase, ascorbate peroxidase, glutathione peroxidase and glutathione-S-transferase also showed altered activities in the affected tissues. Further, intracellular methylglyoxal, a harbinger of ROS (Reactive oxygen species), increased significantly (~ 26 to 65-fold) across different concentrations NiO-NP. Intracellular H2O2 (hydrogen peroxide) and ROS levels increased with NiO-NP doses, as did electrolytic leakage from damaged cells. The present work indicated that multiple pathways were compromised in NiO-NP affected plants and this information can bolster our general understanding of the actual mechanism of its toxicity on living cells, and help formulate strategies to thwart ecological pollution. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01314-8.

Effect of Engineered Nickel Oxide Nanoparticle on Reactive Oxygen Species-Nitric Oxide Interplay in the Roots of Allium cepa L.

Scientists anxiously follow instances of heavy metals augmenting in the environment and undergoing bioaccumulation and trace their biomagnification across food webs, wary of their potent toxicity on biological entities. Engineered nanoparticles supplement natural pools of respective heavy metals and can mimic their effects, exerting toxicity at higher concentrations. Thus, a thorough understanding of the underlying mechanism of this precarious interaction is mandatory. Most urban and industrial environments contain considerable quantities of nickel oxide nanoparticles. These in excess can cause considerable damage to plant metabolism through a significant increase in cellular reactive oxygen species and perturbation of its cross-talk with the reactive nitrogen species. In the present work, the authors have demonstrated how the intrusion of nickel oxide nanoparticles (NiO-NP) affected the exposed roots of Allium cepa: starting with disruption of cell membranes, before being interiorized within cell organelles, effectively disrupting cellular homeostasis and survival. A major shift in the reactive oxygen species (ROS) and nitric oxide (NO) equanimity was also observed, unleashing major altercations in several crucial biochemical profiles. Altered antioxidant contents and upregulation of stress-responsive genes, namely, Catalase, Ascorbate peroxidase, Superoxide dismutase, and Rubisco activase, showing on average 50-250% rise across NiO-NP concentrations tested, also entailed increased cellular hydrogen peroxide contents, with tandem rise in cellular NO. Increased NO content was evinced from altered concentrations of nitric oxide synthase and nitrate reductase, along with NADPH oxidase, when compared with the negative control. Though initially showing a dose-dependent concomitant rise, a significant decrease of NO was observed at higher concentrations of NiO-NP, while cellular ROS continued to increase. Modified K/Na ratios, with increased proline concentrations and GABA contents, all hallmarks of cellular stress, correlated with ROS-NO perturbations. Detailed studies showed that NiO-NP concentration had a significant role in inducing toxicity, perturbing the fine balance of ROS-NO, which turned lethal for the cell at higher dosages of the ENP precipitating in the accumulation of stress markers and an inevitable shutdown of cellular mechanisms.

Silicon augments salt tolerance through modulation of polyamine and GABA metabolism in two indica rice (Oryza sativa L.) cultivars.

Polyamines (PA) have multifarious roles in plant-environment interaction and stress responses. In conjunction with GABA shunt, they regulate induction of tolerance under salinity stress in plants. Here, we tested the hypothesis that silicon improves salt tolerance through mediating vital metabolic pathways rather than acting as a mere mechanical barrier. Seedlings of two rice (Oryza sativa L.) cultivars MTU 1010 (salt-sensitive) & Nonabokra (salt-tolerant) growing in hydroponic culture were treated with NaCl (0, 25, 50 & 100 mM) combined with or without Si (2 mM). NaCl stress enhanced PA synthesizing enzymes activity and PA production in salt tolerant cultivar Nonabokra, whereas in the sensitive cultivar, MTU 1010 both declined. Enhanced activities of GABA synthesizing enzymes along with a decline in the activities of GABA degrading enzymes under NaCl exposure led to GABA accumulation in both the cultivars. The interactive effects of silicon and NaCl also induced the activities of the enzymes related to polyamine biosynthesis and inhibited polyamine degrading enzymes that enhanced PA contents in the cultivars. Supplemental Si decreased endogenous GABA levels by modulating GABA metabolising enzymes under NaCl stress. On the basis of all tested parameters cv. MTU 1010 was proven to be more responsive towards silicon application than cv. Nonabokra. Such study of silicon-induced polyamine accretion and reduced GABA accumulation may lower oxidative damage in rice cultivars under NaCl stress and thereby form a successful strategy to boost tolerance.

Exogenous silicon alters organic acid production and enzymatic activity of TCA cycle in two NaCl stressed indica rice cultivars.

The activities of TCA cycle enzymes viz., pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, succinate dehydrogenase and malate dehydrogenase as well as levels of different organic acids viz., pyruvic acid, citric acid, succinic acid and malic acid were studied in two rice cultivars viz. cv. Nonabokra and cv. MTU 1010 differing in salt tolerance grown under 25, 50 and 100 mM NaCl salinity levels. A contrasting response to salt stress on enzyme activities of TCA cycle and accumulation of organic acid was observed between two cultivars during twenty-one days period of study. Salinity caused enhanced organic acid production and increase in all five enzyme activities in cv. Nonabokra whereas in cv. MTU 1010 decrease in both organic acid production and enzymes activities were noted. Joint application of exogenous silicon along with NaCl, altered the organic acids levels and activities of enzymes in both cultivars of rice seedlings conferring tolerance against salt induced stress. Rice cv. MTU 1010 showed better response to exogenous silicon on parameters tested compared to cv. Nonabokra.

Exogenous silicon alters ascorbate-glutathione cycle in two salt-stressed indica rice cultivars (MTU 1010 and Nonabokra).

Silicon is widely available in soil and is known to mitigate both biotic and abiotic stress in plants. Very low doses of silicon are becoming increasingly essential in rice for biofortification and preventing water loss. Soil salinity is a matter of grave concern in various parts of the world, and silicon is a suitable candidate to mitigate salinity-induced stress of important plants in affected areas. The present study investigates the protective capability of exogenously applied silicon in ameliorating NaCl-induced toxicity in two rice (Oryza sativa L.) cultivars, the salt-sensitive MTU 1010, and salt-tolerant Nonabokra. Rice seedlings were treated with three doses of NaCl (25, 50, and 100 mM), initially alone and subsequently in combination with 2 mM sodium silicate (Na2SiO3, 9H2O). After 21 days, these plants were examined to determine levels of reduced glutathione, ascorbic acid, cysteine, and activities of different enzymes involved in the ascorbate-glutathione cycle, viz., glutathione reductase (GR), ascorbate peroxidase (APX), glutathione peroxidase (GPx), and glutathione S-transferase (GST). Though ROS levels increased in both the cultivars with increasing NaCl concentrations, cv. MTU 1010 accumulated comparatively higher amounts. A differential response of NaCl-induced toxicity on the two cultivars was observed with respect to the various enzymatic and non-enzymatic antioxidants. APX and GST activities, as well as, cysteine contents, increased concomitantly with salt concentrations, whereas GR activity declined at increasing salt concentrations, in both cultivars. Activity of GPx increased in cv. Nonabokra but declined in cv. MTU 1010, under similar NaCl concentrations. Reduced glutathione (GSH) contents decreased in both cultivars, whereas ascorbate contents declined in only the sensitive cultivar. Application of silicon, along with NaCl, in the test seedlings of both the cultivars, reduced ROS accumulation and boosted antioxidant defense mechanism, through enhancing ascorbate and GSH levels, and activities of ascorbate-glutathione cycle enzymes as well. However, amelioration of salt-induced damages in the sensitive cv. MTU 1010 was more pronounced upon silicon administration, than the tolerant cv. Nonabokra. Thus, cv. MTU 1010 was found to be more responsive to applied silicon. Hence, this study was instrumental in realizing a successful strategy in silicon-mediated amelioration of salinity stress in plants.

Engineered Nickel Oxide Nanoparticle Causes Substantial Physicochemical Perturbation in Plants.

Concentration of engineered nickel oxide nanoparticle (NiO-NP) in nature is on the rise, owing to large scale industrial uses, which have accreted the scope of its exposure to plants, the primary producers of the ecosystem. Though an essential micronutrient for the animal system, supported by numerous studies confirming its toxicity at higher dosages, nickel oxide is graded as a human carcinogen by WHO. A few studies do depict toxicity and bioaccumulation of nickel in plants; however, interaction of NiO-NP with plants is not well-elucidated. It is known that exposure to NiO-NP can incite stress response, leading to cytotoxicity and growth retardation in some plants, but a defined work on the intricate physicochemical cellular responses and genotoxic challenges is wanting. The present study was planned to explore cytotoxicity of NiO-NP in the model plant, Allium cepa L., its internalization in the tissue and concomitant furore created in the antioxidant enzyme system of the plant. The prospect of the NiO-NP causing genotoxicity was also investigated. Detailed assessments biochemical profiles and genotoxicity potential of NiO-NP on A. cepa L. was performed and extended to four of its closest economically important relatives, Allium sativum L., Allium schoenoprasum L., Allium porrum L., and Allium fistulosum L. Growing root tips were treated with seven different concentrations of NiO-NP suspension (10-500 mg L-1), with deionised distilled water as negative control and 0.4 mM EMS solution as positive control. Study of genotoxic endpoints, like, mitotic indices (MI), chromosomal aberrations (CAs), and chromosome breaks confirmed NiO-NP induced genotoxicity in plants, even at a very low dose (10 mg L-1). That NiO-NP also perturbs biochemical homeostasis, disrupting normal physiology of the cell, was confirmed through changes in state of lipid peroxidation malonaldehyde (MDA), as well as, in oxidation marker enzymes, like catalase (CAT), super oxide dismutase (SOD), and guiacol peroxidase (POD) activities. It was evident that increase in NiO-NP concentration led to decrease in MIs in all the study materials, concomitant with a spike of stress-alleviating, antioxidant enzymes-CAT, POD, SOD, and significant increase in MDA formation. Hence, it can be confirmed that NiO-NP should be treated as an environmental hazard.

Engineered nickel oxide nanoparticles affect genome stability in Allium cepa (L.).

Indiscriminate uses of engineered nickel oxide nanoparticles (NiO-NPs) in heavy industries have ushered their introduction into the natural environment, ensuing novel interactions with biotic components of the ecosystem. Though much is known about the toxicity of NiO-NPs on animals, their phytotoxic potential is not well elucidated. NiO-NP hinders intra-cellular homeostasis by producing ROS in excess, having profound effect on the antioxidant profile of exposed animal and plant tissues. In the present study, bulbs of the model plant Allium cepa were treated with varying concentrations of NiO-NP (10 mg L-1 - 500 mg L-1) to study changes in ROS production and potential genotoxic effect. The data generated proved a concomitant upsurge in intracellular ROS accumulation with NiO-NP dosage that could be correlated with increased genotoxicity in A. cepa. Augmented in situ ROS production was revealed through DCFH-DA assay, with highest increase in fluorescence (70% over control) in bulbs exposed to 125 mg L-1 NiO-NP. Effect of NiO-NP on genomic DNA was studied through detailed analyses of RAPD profiles which allows detection of even slightest changes in DNA sequence of treated plants. Significant differences in band intensity, loss and appearance of bands as well as genomic template stability and band sharing indices of treated plants revealed increased vulnerability of genomic DNA to NiO-NP, at even lowest concentration (10 mg L-1). This is the first report of NiO-NP induced genotoxicity on A. cepa, which confirms the nanoparticle as a potent environmental hazard.