Since 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 (1H NMR) and Gas Chromatography-Mass Spectrometry (GC-MS) based metabolomics. Changes in mineral nutrient metabolism induced by nano-Cu were determined by ICP-OES. Results showed that nano-Cu at both concentrations interfere 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 acids to reduce the mobilization of Cu ions; ascorbic acid up-regulation to combat reactive oxygen species; up-regulation of phenolic compounds to improve antioxidant system. Thus, we demonstrate that non-targeted 1H NMR and GC-MS based metabolomics can successfully identify physiological responses induced by nanoparticles. Root exudates metabolomics revealed important detoxification mechanisms.

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.