Impacts of Copper Oxide Nanoparticles on Lettuce (Lactuca sativa L.) Seedling Growth and the Potential Involvement of Nitric Oxide in Antioxidative Defense Mechanisms -

“Impacts of Copper Oxide Nanoparticles on Lettuce (Lactuca sativa L.) Seedling Growth and the Potential Involvement of Nitric Oxide in Antioxidative Defense Mechanisms."

Increasing global population, climate change, and productivity challenges necessitate sustainable agriculture practices. Nanoparticles (NPs) offer a promising solution for enhancing plant growth, seed germination, and defense against pathogens. This study focuses on the application of copper oxide nanoparticles (CuO NPs) synthesized using green tea extract on lettuce seedlings. Lettuce, a widely consumed leafy vegetable, holds significant nutritional value. The eco-friendly biogenic synthesis of CuO NPs presents a potential avenue for agricultural applications. The study investigates the dose-response of CuO NPs on seed germination and explores the seedlings’ antioxidative defense mechanisms, including reactive oxygen and nitrogen species. The findings aim to contribute to the understanding of the impact of CuO NPs on plant physiology and defense mechanisms in the context of sustainable agriculture.

Reagents: Analytical-grade reagents, including Copper chloride II (CuCl2), N-ethylmaleimide (NEM), potassium iodide (KI), 2,2-diphenyl-1-picryl-hydrazyl hydrate (DPPH), sodium nitrite (NaNO2), phosphate buffer (PBS, pH 7.4), ethylenediamine tetraacetic acid (EDTA), hydrogen peroxide (H2O2), poly(vinylpolypyrrolidone) (PVP), pyrogallol, potassium phosphate buffer, ascorbic acid, methionine, riboflavin, and nitroblue tetrazolium chloride were procured from Sigma-Aldrich, Missouri, USA. Sulfuric acid (H2SO4) was obtained from LabSynth, Diadema, SP, Brazil, and green tea powder (Camellia sinensis) from Sumioka Shokuhin Kabushikikaisha, Hiraguti, Japan.

Green Synthesis of CuO Nanoparticles: CuO nanoparticles were synthesized by adding an aqueous solution of CuCl2 (93.0 mmol L−1) dropwise into a green tea extract suspension (2.5 mg mL−1). The mixture was heated, homogenized, and then centrifuged. The resulting black precipitate of CuO NPs was freeze-dried and stored in a desiccator.

Characterization of CuO Nanoparticles:Hydrodynamic size, polydispersity index (PDI), and zeta potential of CuO NPs were analyzed by dynamic light scattering (DLS) using Zetasizer Nano ZS. Morphology of CuO NPs was examined by transmission electron microscopy (TEM) at 80 kV. Chemical surface characterization was performed using X-ray photoelectron spectroscopy (XPS) with a monochromatic argon source.

Bioassays with Lactuca sativa: Lettuce seeds (L. sativa L. cv Hanson) were utilized for bioassays to assess CuO NPs’ phytotoxicity. CuO NPs were dispersed in OECD recommended ISO test medium. Bioassays were conducted at various concentrations (0.2, 2, 20, 40, 80, 150, and 300 μg mL−1). Bioassay methodology, including sonication of the CuO NPs suspension and seed placement, followed the protocol proposed by Belo (2011) with modifications. After 5 days of incubation in the dark, seed germination, root length, and germination index (GI) were calculated.

Quantification of S-nitrosothiol and Nitrite Levels: Lettuce radicles were extracted using an aqueous solution of NEM (5 mmol L−1) and quantified for RSNO and NO2− using amperometry. WPI TBR4100/1025 amperometer and a nitric oxide specific ISO-NOP sensor were used for measurements. Data were normalized by radicle fresh weight.

Total Antioxidant Activity: Antioxidant activity (AA) of radicles was measured using the DPPH radical photometric assay. Percentage of antioxidant activity was calculated based on absorbance values and normalized by radicle weight.

Antioxidant Enzyme Activity: Enzymatic activities of ascorbate peroxidase, catalase, peroxidases, and superoxide dismutase were determined. Enzymatic activity values were normalized by radicle weight and reaction duration.

Phytotoxicity Level Classification: Phytotoxicity levels were classified based on germination index using the rating system proposed by Belo (2011).

Result and Summary:

Copper oxide nanoparticles (CuO NPs) have garnered considerable attention in agriculture for their potential applications in plant development, pest control, and soil restoration. This study focused on the effects of biosynthesized CuO NPs, derived from green tea extract, on lettuce (Lactuca sativa L.) seedling growth across concentrations ranging from 0.2 to 300 μg mL−1. The biosynthesized CuO NPs were characterized as ultra-small particles (~6.6 nm) with excellent stability in aqueous suspension.

Results from toxicity bioassays revealed that, at low concentrations (up to 40 μg mL−1), CuO NPs either had no impact on or even enhanced seed germination. However, at higher concentrations (above 40 μg mL−1), inhibition of both seed germination and radicle growth occurred, with an inhibition range of 35% to 75%. As CuO NP concentrations increased, levels of nitrite and S-nitrosothiols in radicles also increased, while superoxide dismutase and total antioxidant activities decreased. This suggests a direct dose-response relationship between CuO NP application and nitric oxide-dependent signaling pathways in plant responses.

The study demonstrated that, at low concentrations (≤20 μg mL−1) of CuO NPs, beneficial effects on seedlings were observed, enhancing plant growth and involving nitric oxide signaling. However, at higher concentrations (40 to 300 μg mL−1), these NPs exhibited phytotoxic effects, leading to a decrease in radicle growth in lettuce. Notably, the activity of antioxidant enzymes, such as superoxide dismutase, and the levels of reactive nitrogen species (RNS), including nitrite and S-nitrosothiols, were influenced by NP exposure. The research highlights the synthesis and characterization of ultra-small CuO NPs capped with green tea polyphenols, providing evidence that concentrations between 0.2 and 20 μg mL−1 are non-phytotoxic and may enhance lettuce radicle growth.

While the study sheds light on the immediate impacts of CuO NPs on lettuce seedlings, it acknowledges that the uptake, distribution, and effects at molecular and genetic levels remain unexplored in this research. The authors recommend these aspects to be considered in future investigations.

Reference:

PELEGRINO, M.T., KOHATSU, M.Y., SEABRA, A.B., MONTEIRO, L.R., GOMES, D.G., OLIVEIRA, H.C., ROLIM, W.R., DE JESUS, T.A., BATISTA, B.L. AND LANGE, C.N., 2020. Effects of copper oxide nanoparticles on growth of lettuce (Lactuca sativa L.) seedlings and possible implications of nitric oxide in their antioxidative defense. Environmental Monitoring and Assessment, 192, pp.1-14.