Interaction of CuO nanoparticles with plant cells: internalization, oxidative stress, electron transport chain disruption, and toxicogenomic responses

This study investigated the toxicity of CuO nanoparticles (NPs) to Nicotiana tabacum L. cv. Bright Yellow-2 (BY-2) cells. CuO NPs with concentrations higher than 2 mg L−1 began to exhibit significant inhibition of the growth of plant cells, and the 24 h median effective concentration was calculated to be 12 mg L−1. CuO NPs (12 mg L−1) showed much higher toxicity than the released Cu2+ (0.8 mg L−1) and bulk particles (BPs, 12 mg L−1) during all the exposure times (0–24 h). CuO NPs were internalized by plant cells through endocytosis, and then located in the cytoplasm, mitochondria and vacuoles. Under CuO NP exposure, the generation of total reactive oxygen species increased by 38.3% and 40.9% compared with CuO BPs and Cu2+ treatments, respectively. The main radical species were identified as H2O2 and OH˙. CuO NPs significantly reduced the activities of complexes I and III on the mitochondrial electron transport chain, blocked the electron transfer from NADH to ubiquinone, and from ubisemiquinone to ubiquinol, induced oxidative stress, and finally led to membrane damage as indicated by an increase in the contents of malondialdehyde and lactate dehydrogenase. Global gene expression analysis from RNA-sequencing showed that CuO NPs (12 mg L−1) significantly induced the expression of 2692 differentially expressed genes (P-value 2) including 1132 up-regulated and 1560 down-regulated genes in BY-2 cells. The differentially expressed genes related to oxidative stress and mitochondria were identified according to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. This work provides insights into the molecular mechanism of nanotoxicity toward plants.