<b>Supplementary Files: Seagrass-mediated rhizosphere redox gradients are linked with ammonium accumulation driven by diazotrophs</b>

Seagrasses can enhance nutrient mobilization in their rhizosphere via complex interactions with sediment redox conditions and microbial populations. Yet, limited knowledge exists on how seagrass-derived rhizosphere dynamics affect nitrogen cycling. Using optode and gel27 sampler based chemical imaging, we show that radial O₂ loss (ROL) from rhizomes and roots leads to formation of redox gradients around below-ground tissues of seagrass (Zostera marina), which are co-localised with regions of high ammonium concentrations in the rhizosphere. Combining such chemical imaging with fine-scale sampling for microbial community and gene expression analyses indicated that multiple biogeochemical pathways and microbial players can lead to high ammonium concentration within the oxidized regions of the seagrass rhizosphere. Symbiotic N₂-fixing bacteria (Bradyrhizobium) were particularly abundant and expressed the diazotroph functional marker gene nifH in Z. marina rhizosphere areas with high ammonium concentrations. Such association between Z. marina and Bradyrhizobium can facilitate ammonium mobilization, the preferred nitrogen source for seagrasses, enhancing seagrass productivity within nitrogen-limited environments. ROL also caused strong gradients of sulphide at anoxic/oxic interfaces in rhizosphere areas where we found enhanced <i>nifH</i> transcription by sulphate-reducing bacteria. Furthermore, we found a high abundance of methylotrophic and sulphide-oxidizing bacteria in rhizosphere areas, where O₂ was released from seagrass rhizomes and roots, and these bacteria could play a beneficial role for the plants in terms of their methane and sulphide oxidation, as well as their formation of growth factors and phytohormones. ROL from below-ground tissues of seagrass thus seems crucial for ammonium production in the rhizosphere via stimulation of multiple diazotrophic associations.