Antibiotic-induced oxidative stress triggers cell wall remodelling and transcriptional reprogramming in Lolium perenne L.

The presence of antibiotic residues in reclaimed irrigation water raises concerns about their potential impact on non-target plant species. This study investigated the physiological and transcriptomic responses of perennial ryegrass (Lolium perenne) to sulfamethoxazole (SMX) and trimethoprim (TMP), two frequently detected antibiotics often used in combination, applied individually and as a mixture. While seed germination was unaffected, root elongation and seedling biomass were significantly reduced under SMX and combined treatments. Antibiotic exposure induced oxidative stress, as reflected by increased H2O2 levels at the highest combined treatment, and modulation of antioxidant enzyme activities. Photosynthetic performance was impaired, evidenced by reduced electron transport rates and maximum quantum efficiency of PSII. Increased lignin deposition, together with FTIR-based evidence of cell wall modifications, indicated structural reinforcement. RNA-Seq analysis revealed transcriptional reprogramming, with most upregulated genes involved in oxidative stress responses and xenobiotic detoxification, as well as in the phenylpropanoid biosynthetic pathway and in cell wall remodelling. In contrast, genes related to photosynthesis, energy metabolism, and DNA replication and cell cycle progression were downregulated, indicating a shift from growth to defence and maintenance. Moreover, folate metabolism was altered, including enhanced folate recycling, consistent with the mode of action of the antibiotics. TMP alone induced substantially fewer transcriptional changes than SMX, whereas the combined treatment amplified these responses and additionally induced signalling pathways and membrane transport processes. Overall, these findings highlight the ecological relevance of antibiotic mixtures and their potential risks for plant performance in agroecosystems irrigated with reclaimed water.