Identifying the Phytotoxicity and Defense Mechanisms Associated with Graphene-Based Nanomaterials by Integrating Multiomics and Regular Analysis

The application of graphene-based nanomaterials (GBNs) has attracted global attention in various fields, and understanding defense mechanisms against the phytotoxicity of GBNs is crucial for assessing their environmental risks and safe-by-design. However, the related information is lacking, especially for edible vegetable crops. In the present study, GBNs (0.25, 2.5, and 25 mg/kg plant fresh weight) were injected into the stems of pepper plants. The results showed that the plant defense was regulated by reducing the calcium content by 21.7–48.3%, intercellular CO2 concentration by 12.0–35.2%, transpiration rate by 8.7–40.2%, and stomatal conductance by 16.9–50.5%. The defense pathways of plants in response to stress were further verified by the downregulation of endocytosis and transmembrane transport proteins, leading to a decrease in the nanomaterial uptake. The phytohormone gibberellin and abscisic acid receptor PYL8 were upregulated, indicating the activation of defense systems. However, reduced graphene oxide and graphene oxide quantum dots trigger stronger oxidative stress (e.g., H2O2 and malondialdehyde) than graphene oxide in fruits due to the breakdown of antioxidant defense systems (e.g., cytochrome P450 86A22 and P450 77A1). Both nontargeted proteomics and metabolomics consistently demonstrated that the downregulation of carbohydrate and upregulation of amino acid metabolism were the main mechanisms underlying the phytotoxicity and defense mechanisms, respectively.