Ultrafine polystyrene nanoplastics penetrate roots and induce organelle damage and extensive transcriptomic reprogramming in Arabidopsis thaliana

Nanoplastics (NPs) are increasingly recognized as emerging environmental contaminants in terrestrial ecosystems; however, their size-dependent biological impacts and underlying mechanisms in plants remain poorly understood. Here, we provide a systematic size-gradient assessment by exposing Arabidopsis thaliana seedlings to polystyrene nanoplastics (PS-NPs) of 100, 75, 50, 25, and 15 nm, including negatively charged 25 nm particles, integrating physiological, ultrastructural, chemical, and transcriptomic analyses. Clear size-related differences were observed, with 15 nm PS-NPs inducing the most pronounced reduction in seedling growth and root elongation, whereas larger particles caused limited or non-significant effects, revealing a threshold-like increase in phytotoxicity at the ultrafine scale. Detailed analyses of 15 nm PS-NPs revealed decreased chlorophyll content, increased lipid peroxidation, and enhanced superoxide (O 2 - ) accumulation detected by nitroblue tetrazolium (NBT) staining. In contrast, 3,3′-diaminobenzidine (DAB) staining did not reveal clear differences in hydrogen peroxide (H 2 O 2 ) levels, indicating oxidative stress responses consistent with elevated membrane damage. Ultrastructural analyses further revealed marked cellular alterations, particularly affecting chloroplast organization. Electron-dense structures morphologically consistent with 15 nm PS-NPs were observed within root tissues, and their internalization was independently confirmed by pyrolysis–GC/MS detection of styrene oligomers, providing complementary ultrastructural and chemical evidence rarely combined in plant studies. Transcriptomic profiling demonstrated extensive gene-expression reprogramming, including enrichment for pathways associated with redox regulation, carbon metabolism, stress signalling, and mitochondrial-associated alternative respiratory pathways. By linking NP internalization with oxidative stress and global transcriptional responses, this study provides a mechanistic framework for size-dependent NP toxicity in plants. Together, these results demonstrate that ultrafine NPs exert disproportionate phytotoxic effects relative to larger particles and can penetrate root tissues, thereby triggering membrane damage and broad metabolic adjustments. This study advances current knowledge by identifying a critical size threshold and by integrating multi-level evidence of NP toxicity, highlighting potential risks of ultrafine plastic particles for terrestrial ecosystems. • Ultrafine 15 nm nanoplastics caused stronger phytotoxicity than larger particles. • TEM and Py-GC/MS support root internalization of 15 PS-NPs. • Exposure increased oxidative stress and altered chloroplast structure. • Ultrafine NPs induced broad transcriptional adjustments. • Results reveal pronounced size-dependent plant responses to nanoplastics.