Microscale Spatiotemporal Variation and Generation Mechanisms of Reactive Oxygen Species in the Rhizosphere of Ryegrass: Coupled Biotic–Abiotic Processes

Reactive oxygen species (ROS) play key roles in soil biogeochemical processes, yet the occurrence and accumulation of ROS in the rhizosphere are poorly documented. Herein, we first developed a ROS-trapping membrane to in situ determine ROS in the ryegrass rhizosphere and then quantified the temporal and spatial variations of representative ROS (i.e., O₂^•─, H₂O₂, and ^•OH). Fluorescence imaging clearly visualized the production of ROS in the rhizosphere. Both O₂^•─ and H₂O₂ content increased first and then declined throughout the life cycle of ryegrass, while ^•OH concentration decreased continuously. Spatially, ROS contents remained at a relatively high level at 0-5 mm and then descended with increasing distance. The concentrations of ROS in different soils followed the order of black soil > latosol soil > yellow-brown soil > tier soil ∼ red soil. Analysis of soil properties suggested that both biotic factors (microbial community) and abiotic factors (Fe(II) and water-soluble phenols) played critical roles in ROS production. The combined processes, including Fe(II) and water-soluble phenol-mediated electron transfer, microbial community-driven extracellular O₂^•─ release, and Fe(II)/Fe(III) cycling, may be responsible for ROS production. These findings provide insights into ROS-associated rhizosphere effects and inspiration for the phytoremediation of pollutants and element cycling.