Decoding self-recovery: Initial observations of radiofrequency stress responses in Nicotiana benthamiana

• Radiofrequency (RF) treatment at 360 kHz caused temporary plasma membrane disruption in plants. • Superoxide levels peaked early, signaling oxidative stress post-RF exposure. • Gene expression peaked at 6h, involving stress response and metabolism pathways. • Membrane structure recovered by 24h, showing plant adaptation to RF stress. • This study highlights RF's potential to enhance plant metabolism and stress resilience. This study investigates the cellular and molecular responses of Nicotiana benthamiana to radiofrequency (RF) exposure using a custom-built electrode–water bath system. Plants were treated with RF oscillations at 360 kHz for 20 minutes, and responses were examined from immediate to 24 hours post-treatment. Oscilloscope measurements confirmed that the applied RF waveform was transmitted through the water medium and detected in submerged leaves, albeit with reduced amplitude, validating effective exposure of leaf tissues. Transmission electron microscopy (TEM) revealed transient perturbations of the plasma membrane, including detachment and oscillation, followed by structural reorganization at 6 hours and near-complete recovery by 24 hours. These structural alterations were accompanied by a transient increase in superoxide accumulation, suggesting an acute oxidative stress response that subsided during recovery. Gene expression analysis further indicated that RF treatment transiently activated stress signaling and autophagy-related pathways during the early recovery phase. RNA sequencing additionally identified distinct temporal patterns of gene expression, with the strongest transcriptional responses occurring at 6 hours after treatment, particularly in stress-related and metabolic pathways. Our results suggest a possible link between RF exposure and changes in metabolic pathways, raising the potential for increased metabolite production; however, this possibility requires further experimental confirmation.