Due to the potential for interactions between crop plants and engineered nanomaterials (ENMs), there is increasing interest in understanding the bioavailability and effects of ENMs released into soil systems. Here, we investigate the influence of root exudates on the fate of ENMs from a thermodynamic perspective. Nano isothermal titration calorimetry was applied to determine thermodynamic parameters for the interaction between nanocopper (nCu) and synthetic root exudate (SRE) and its components (including sugars, organic acids, amino acids, and phenolic acids), as well as Cu²⁺ and SRE. The measured binding constant (K_d = 5.645 × 10³ M^-1) indicated strong interactions between nCu particles and SRE, as well as with individual organic acids. The interaction between Cu²⁺ and SRE was stronger (K_d = 7.181 × 10⁴ M^-1) but varies for the individual SRE components. nCu dissolution in the presence of SRE was the predominant interaction. In addition, SRE resulted in a complex transformation of nCu, where Cu²⁺, Cu⁺, and Cu⁰ were formed via oxidation and reduction. Plant-nCu exposure experiments indicate that the binding of SRE with nCu and dissolved Cu ions can significantly decrease Cu uptake and bioaccumulation in plants. nITC provides a fundamental thermodynamic understanding of interactions between nCu and plant root exudates, providing an important tool for understanding plant NP-interactions.

Due to their unique properties, copper-based nanopesticides are emerging in the market. Thus, understanding their effect on crop plants is very important. Metabolomics can capture a snapshot of cellular metabolic responses to a stressor. We selected maize and cucumber as model plants for exposure to different doses of Cu(OH)₂ nanopesticide. GC-TOF-MS-based metabolomics was employed to determine the metabolic responses of these two species. Results revealed significant differences in metabolite profile changes between maize and cucumber. Furthermore, the Cu(OH)₂ nanopesticide induced metabolic reprogramming in both species, but in different manners. In maize, several intermediate metabolites of the glycolysis pathway and tricarboxylic acid cycle (TCA) were up-regulated, indicating the energy metabolism was activated. In addition, the levels of aromatic compounds (4-hydroxycinnamic acid and 1,2,4-benzenetriol) and their precursors (phenylalanine, tyrosine) were enhanced, indicating the activation of shikimate-phenylpropanoid biosynthesis in maize leaves, which is an antioxidant defense-related pathway. In cucumber, arginine and proline metabolic pathways were the most significantly altered pathway. Both species exhibited altered levels of fatty acids and polysaccharides, suggesting the cell membrane and cell wall composition may change in response to Cu(OH)₂ nanopesticide. Thus, metabolomics helps to deeply understand the differential response of these plants to the same nanopesticide stressor.

While the use of nanopesticides in modern agriculture continues to increase, their effects on crop plants are still poorly understood. Here, 4 week old spinach plants grown in an artificial medium were exposed via foliar spray to Cu(OH)₂ nanopesticide (0.18 and 18 mg/plant) or Cu ions (0.15 and 15 mg/plant) for 7 days. A gas chromatography-time-of-flight-mass spectrometry metabolomics approach was applied to assess metabolic alterations induced by Cu(OH)₂ nanopesticide in spinach leaves. Exposure to Cu(OH)₂ nanopesticide and copper ions induced alterations in the metabolite profiles of spinach leaves. Compared to the control, exposure to 18 mg of Cu(OH)₂ nanopesticide induced significant reduction (29-85%) in antioxidant or defense-associated metabolites including ascorbic acid, α-tocopherol, threonic acid, β-sitosterol, 4-hydroxybutyric acid, ferulic acid, and total phenolics. The metabolic pathway for ascorbate and aldarate was disturbed in all exposed spinach plants (nanopesticide and Cu²⁺). Cu²⁺ is responsible for the reduction in antioxidants and perturbation of the ascorbate and aldarate metabolism. However, nitrogen metabolism perturbation was nanopesticide-specific. Spinach biomass and photosynthetic pigments were not altered, indicating that metabolomics can be a rapid and sensitive tool for the detection og earlier nanopesticide effects. Consumption of antioxidants during the antioxidant defense process resulted in reduction of the nutritional value of exposed spinach.

Excess copper may disturb plant photosynthesis and induce leaf senescence. The underlying toxicity mechanism is not well understood. Here, 3-week-old cucumber plants were foliar exposed to different copper concentrations (10, 100, and 500 mg/L) for a final dose of 0.21, 2.1, and 10 mg/plant, using CuSO4 as the Cu ion source for 7 days, three times per day. Metabolomics quantified 149 primary and 79 secondary metabolites. A number of intermediates of the tricarboxylic acid (TCA) cycle were significantly down-regulated 1.4-2.4 fold, indicating a perturbed carbohydrate metabolism. Ascorbate and aldarate metabolism and shikimate-phenylpropanoid biosynthesis (antioxidant and defense related pathways) were perturbed by excess copper. These metabolic responses occur even at the lowest copper dose considered although no phenotype changes were observed at this dose. High copper dose resulted in a 2-fold increase in phytol, a degradation product of chlorophyll. Polyphenol metabolomics revealed that some flavonoids were down-regulated, while the nonflavonoid 4-hydroxycinnamic acid and trans-2-hydroxycinnamic acid were significantly up-regulated 4- and 26-fold compared to the control. This study enhances current understanding of copper toxicity to plants and demonstrates that metabolomics profiling provides a more comprehensive view of plant responses to stressors, which can be applied to other plant species and contaminants.

Nanobiotechnology approaches to engineering crops with enhanced stress tolerance may be a safe and sustainable strategy to increase crop yield. Under stress conditions, cellular redox homeostasis is disturbed, resulting in the over-accumulation of reactive oxygen species (ROS) that damage biomolecules (lipids, proteins and DNA) and inhibit crop growth and yield. Delivering ROS-scavenging nanomaterials to plants has been shown to alleviate abiotic stress. Here we review the current state of knowledge of using ROS-scavenging nanomaterials to enhance plant stress tolerance. When present below a threshold level, ROS can mediate redox signalling and defence pathways that foster plant acclimatization against stress. We find that ROS-triggering nanomaterials, such as nanoparticulate silver and copper oxide, have the potential to be judiciously applied to crop species to stimulate the defence system, prime stress responses and subsequently increase the stress resistance of crops.

Because copper nanoparticles are being increasingly used in agriculture as pesticides, it is important to assess their potential implications for agriculture. Concerns have been raised about the bioaccumulation of nano-Cu and their toxicity to crop plants. Here, the response of cucumber plants in hydroponic culture at early development stages to two concentrations of nano-Cu (10 and 20 mg/L) was evaluated by proton nuclear magnetic resonance spectroscopy ((1)H NMR) and gas chromatography-mass spectrometry (GC-MS) based metabolomics. Changes in mineral nutrient metabolism induced by nano-Cu were determined by inductively coupled plasma-mass spectrometry (ICP-MS). Results showed that nano-Cu at both concentrations interferes with the uptake of a number of micro- and macro-nutrients, such as Na, P, S, Mo, Zn, and Fe. Metabolomics data revealed that nano-Cu at both levels triggered significant metabolic changes in cucumber leaves and root exudates. The root exudate metabolic changes revealed an active defense mechanism against nano-Cu stress: up-regulation of amino acids to sequester/exclude Cu/nano-Cu; down-regulation of citric acid to reduce the mobilization of Cu ions; ascorbic acid up-regulation to combat reactive oxygen species; and up-regulation of phenolic compounds to improve antioxidant system. Thus, we demonstrate that nontargeted (1)H NMR and GC-MS based metabolomics can successfully identify physiological responses induced by nanoparticles. Root exudates metabolomics revealed important detoxification mechanisms.

Although toxic effects of silver nanoparticles (AgNPs) on aquatic organisms have been extensively reported, responses of nitrogen-fixing cyanobacteria to AgNPs/Ag⁺ under environmentally relevant concentrations are largely unknown. Here, cyanobacteria were exposed to different concentrations of AgNPs (0.01, 0.1, and 1 mg/L) or Ag⁺ (0.1, 1, and 10 μg/L) for 96 h. The impacts of AgNPs and Ag⁺ on photosynthesis and N₂ fixation in cyanobacteria (Nostoc sphaeroides) were evaluated. In addition, gas chromatography-mass spectrometry (GC-MS)-based metabolomics was employed to give an instantaneous snapshot of the physiological status of the cells under AgNP/Ag⁺ exposure. Exposure to high doses of AgNPs (1 mg/L) or Ag⁺ (10 μg/L) caused growth inhibition, reactive oxygen species overproduction, malondialdehyde accumulation, and decreased N₂ fixation. In contrast, low doses of AgNPs (0.01 and 0.1 mg/L) and Ag⁺ (0.1 and 1 μg/L) did not induce observable responses. However, metabolomics revealed that metabolic reprogramming occurred even at low concentrations of AgNP and Ag⁺ exposure. Levels of a number of antioxidant defense-related metabolites, especially phenolic acid and polyphenols (gallic acid, resveratrol, isochlorogenic acid, chlorogenic acid, cinnamic acid, 3-hydroxybenzoic acid, epicatechin, catechin, and ferulic acid), significantly decreased in response to AgNPs or Ag⁺. This indicates that AgNPs and Ag⁺ can disrupt the antioxidant defense system and disturb nitrogen metabolism even at low-dose exposure. Metabolomics was shown to be a powerful tool to detect "invisible" changes, not observable by typical phenotypic-based endpoints.

There has been an increasing influx of nanopesticides into agriculture in recent years. Understanding the interaction between nanopesticides and edible plants is crucial in evaluating the potential impact of nanotechnology on the environment and agriculture. Here we exposed lettuce plants to Cu(OH)2 nanopesticides (1050-2100 mg/L) through foliar spray for one month. Inductively coupled plasma-mass spectrometry (ICP-MS) results indicate that 97-99% (1353-2501 mg/kg) of copper was sequestered in the leaves and only a small percentage (1-3%) (17.5-56.9 mg/kg) was translocated to root tissues through phloem loading. Gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS) based metabolomics combined with partial least squares-discriminant analysis (PLS-DA) multivariate analysis revealed that Cu(OH)2 nanopesticides altered metabolite levels of lettuce leaves. Tricarboxylic (TCA) cycle and a number of amino acid-related biological pathways were disturbed. Some antioxidant levels (cis-caffeic acid, chlorogenic acid, 3,4-dihydroxycinnamic acid, dehydroascorbic acid) were significantly decreased compared to the control, indicating that oxidative stress and a defense response occurred. Nicotianamine, a copper chelator, increased by 12-27 fold compared to the control, which may represent a detoxification mechanism. The up-regulation of polyamines (spermidine and putrescine) and potassium may mitigate oxidative stress and enhance tolerance. The data presented here provide a molecular-scale perspective on the response of plants to copper nanopesticides.