Transcriptional dynamics of nitrogen fixation and senescence in soybean nodules: A dual perspective on host andBradyrhizobiumregulation

The Soybean–Bradyrhizobiumsymbiosis enables symbiotic nitrogen fixation (SNF) within root nodules, reducing reliance on synthetic N-fertilizers. However, nitrogen fixation is transient, peaking several weeks afterBradyrhizobiumcolonization and declining as nodules senesce in coordination with host development. To investigate the regulatory mechanisms governing SNF and senescence, we conducted a temporal transcriptomic analysis of soybean nodules inoculated withBradyrhizobium diazoefficiensUSDA110. Weekly nodule samples (2–10 weeks post-inoculation, wpi) were analyzed using RNA and small RNA sequencing, while acetylene reduction assays assessed nitrogenase activity from 4 to 7 wpi. We identified three major nodule developmental phases: early development (2–3 wpi), nitrogen fixation (3–8 wpi), and senescence (8–10 wpi). Soybean showed extensive transcriptional reprogramming during senescence, whereasBradyrhizobiumunderwent major transcriptional shifts early in development before stabilizing during nitrogen fixation. We identified seven soybean genes and several microRNAs as candidate biomarkers of nitrogen fixation, includinglipoxygenases(Lox), suggesting roles for oxylipin metabolism. Soyhemoglobin-2(Hb2), previously classified as non-symbiotic, was upregulated during senescence, implicating oxidative stress responses within aging nodules. Upregulation of theBradyrhizobium paaoperon andrpoHduring senescence suggested metabolic adaptation for survival beyond symbiosis. Additionally,Bradyrhizobium NIFgene expression showed stage-specific regulation, withnifKpeaking at 2 wpi,nifDandnifAat 2 and 10 wpi, andnifH,nifW, andnifSat 10 wpi. These findings provide insights into SNF regulation and nodule aging, revealing temporal gene expression patterns that could inform breeding or genetic engineering strategies to enhance nitrogen fixation in soybeans and other legume crops.