Nitrogen Fertilization Modulates Soybean Nodulation and Nitrogen Fixation via NO-Mediated S‑Nitrosylation

Soybean, a symbiotic nitrogen-fixing crop, experiences suppressed nodule nitrogen fixation under excessive nitrogen fertilizer. Nitric oxide (NO) is a key signaling molecule regulating development and stress, primarily via protein S-nitrosylation, although its role in soybeans is unclear. Using a unilateral nodulation system, treatments with nitrogen, an NO scavenger, and an NO donor were applied. Results showed that nitrogen application increased the NO content in the nodules and reduced the nitrogen fixation capacity. Conversely, the NO scavenger lowered the NO levels but enhanced fixation. Exogenous NO inhibited fixation by damaging the nodule structure, reducing leghemoglobin, and disrupting NO homeostasis. Quantitative proteomics with iodoTMT labeling identified 287 S-nitrosylation sites on 238 nodule proteins. Nitrogen-altered proteins were involved in nitrogenase activity, stress response, and ABC transporters. This study establishes the ’nitrogen level-NO signal-S-nitrosylation-nodule function’ pathway, offering molecular insights into S-nitrosylation’s role in nodule regulation.