Spatial Variation in Nodule Flavonoids of Soybean by Exogenous Nitrogen

Exogenous nitrogen (N) strongly affects soybean nodulation and N fixation; the regulatory mechanisms by which it modulates the flavonoid metabolic network remain largely unclear. In this study, we used a unilateral nodulation system in dual-rooted soybean plants and combined physiological measurements, quantitative real-time PCR (qPCR), and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to examine how exogenous N affects flavonoid metabolism, nodule development and N fixation activity. Exogenous N significantly decreased nodule number, dry weight and nitrogenase activity. MALDI-MSI revealed distinct tissue-specific flavonoid distribution patterns: naringenin and its downstream metabolites were enriched in the nitrogen fixation zone (NFZ), whereas isoflavonoids such as daidzein and calycosin primarily accumulated in the cortex (Ctx). Exogenous N disrupted these spatial distributions and caused a progressive reduction in metabolite abundance. qPCR analysis showed that sustained exogenous N suppressed the expression of GmCHI1A, GmCHI1B1, GmIFS1 and GmIFS2, while markedly inducing GmCHS7, GmCHS8 and GmC4H, suggesting a reallocation of metabolic flux within the flavonoid pathway. Collectively, these findings indicate that exogenous N reshapes the flavonoid metabolic network, weakens symbiotic signalling and ultimately compromises nodule development and N fixation efficiency.