Integrated multi-omics reveals rye seedling responses to nanoplastic and freeze-thaw stress

Global climate change is increasing the occurrence and intensity of freeze-thaw cycles (FTCs), while industrial activities continue to contribute to nanoplastic contamination, jointly posing rising risks to plant health. Here, rye (Secale cereale L.) seedlings were grown under controlled hydroponic conditions and exposed to polystyrene nanoplastics (PSNPs;100 mg/L) and FTCs to investigate their combined effects on plant physiology, endophytic microbial communities, and molecular responses. An integrated framework combining physiological assays, 16S rRNA gene sequencing, transcriptomics, and proteomics was applied. The combined FTCs and NPs stress induced pronounced physiological stress, evidenced by elevated malondialdehyde accumulation, increased osmolytes (proline and betaine), and altered antioxidant enzyme activities. Microbial profiling revealed significant community shifts, with stress-associated taxa such as Bacillus and Paenibacillus showing differential enrichment across treatments. Notably, the combined treatment elicited the strongest transcriptomic perturbation (6073 DEGs in CK vs. FP) and highlighted marked alterations in pathways related to oxidative stress and energy metabolism; proteomic profiling further identified key proteins involved in these processes, supporting interactive effects of FTCs and PSNPs. Overall, this study provides mechanistic insights into multi-level rye seedling responses to interacting freeze-thaw and nanoplastic stress, highlighting the need to consider combined environmental drivers in agroecosystems under changing climatic conditions.