Graphene nanomaterials and copper co-exposure in Populus nigra L.: ultrastructural, biochemical, and nutritional impact under in vitro conditions.

The wide application of graphene nanomaterials has led to their release into the environment, raising ecological risk concerns, especially when co-existing with other pollutants like copper (Cu), one of the most ubiquitous environmental metals. The impact of co-presence of these nanomaterials and Cu on woody plants remains unstudied and, in this regard, callus culture represents a reliable tool for toxicological studies. In this work, we investigated the effects of Cu in combination with two different graphene nanomaterials, graphene oxide (GO) and graphene nanoplatelets (GNP), on the cell ultrastructure, biochemical responses and nutrient uptake in callus culture of Populus nigra L., a pioneer tree species in the riparian ecosystem. GO and GNP alone showed an adverse impact on poplar cells, causing a significant reduction in dry weight and a notable increase in MDA levels, water and Ca uptake, and protein synthesis. Co-exposure to Cu and either GO or GNP increased dry weight while decreasing water content, MDA levels, antioxidant enzyme activities, and protein content. Furthermore, GO + Cu exposure promoted greater cellular metal uptake than the GNP + Cu treatment, indicating a greater effectiveness of GO as a Cu carrier, due to the higher presence of oxygen functional groups on its surface than GNP. Transmission Electron Microscopy (TEM) observations confirmed the cellular uptake of both GO and GNP, revealing distinct impacts on cell ultrastructure. These results provide useful information on the interaction between graphene nanomaterials and Cu for risk assessment and developing sustainable management strategies in agro-forestry.