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.

大豆作为一类共生固氮作物，在过量氮肥施用条件下，其根瘤固氮功能会受到抑制。一氧化氮（Nitric oxide, NO）是调控植物发育进程与逆境应答的关键信号分子，其生物学功能主要通过蛋白质S-亚硝基化（protein S-nitrosylation）介导，但目前该分子在大豆中的具体作用尚不明确。本研究采用单侧结瘤体系，设置氮肥、一氧化氮清除剂以及一氧化氮供体三组处理。实验结果显示：氮肥施用可提升根瘤内一氧化氮含量，同时降低固氮能力；与之相反，一氧化氮清除剂会降低根瘤内一氧化氮水平，却能增强固氮功能。外源施加的一氧化氮则通过破坏根瘤结构、减少豆血红蛋白（leghemoglobin）含量以及扰乱一氧化氮稳态，进而抑制固氮过程。本研究采用碘代TMT（iodoTMT）标记的定量蛋白质组学技术，共在238个根瘤蛋白中鉴定到287个S-亚硝基化修饰位点。受氮肥调控的蛋白主要参与固氮酶活性调控、逆境应答以及ABC转运蛋白（ABC transporters）相关通路。本研究明确了“氮肥水平-NO信号-S-亚硝基化-根瘤功能”这一调控通路，为解析S-亚硝基化在根瘤调控中的作用提供了分子层面的理论依据。